JPH11511847A - Cancer detection and treatment - Google Patents

Abstract

(57) [Summary] This is a method for detecting cancer in the body of a patient. The method includes introducing a labeled antibody or labeled AFP into a biological sample of a patient, such that the labeled antibody or labeled AFP reacts with a binding site of an AFP receptor in the biological sample. Next, there is the step of determining whether cancer is present in the biological sample by identifying the binding site of the AFP receptor that reacts with the labeled antibody or labeled AFP. It is desirable to have a step of obtaining a biological sample from the patient's body before the introducing step. It is a method of treating cancer cells in the body of a patient. The method comprises the step of introducing an AFP receptor antibody into the cancer cells of the patient. Next, there is the step of reacting the AFP receptor antibody with the AFP receptor of the cancer cells to block the growth of the cancer cells or kill the cancer cells. It is a method of monitoring patients. The method includes the step of treating the patient's cancer. Next, after performing the processing, there is a step of examining the level of the AFP receptor site of the patient at predetermined intervals. How to treat a patient. The method includes examining the patient's AFP receptor. If the test shows that the AFP receptor is present in the patient's body, then there is the step of introducing an AFP receptor antibody or AFP into the patient's body and reacting with the patient's cancer cells. This is a method for treating cancer cells in the body of a patient. The method includes introducing the modified AFP into cancer cells in a patient's body. Next, there is the step of reacting the denatured AFP with the AFP receptor of the cancer cell, thereby inhibiting the growth of the cancer cell or killing the cancer cell.

Description

DETAILED DESCRIPTION OF THE INVENTION                             Cancer detection and treatmentField of the invention   The present invention relates to the detection and / or treatment of cancer. More specifically, the book The principle of the invention utilizes the presence of the AFP receptor to detect cancer in the patient's body Or contain or eliminate cancer.Background of the Invention   Twenty years ago, Abelev et al. Were the first to express oncofetal antigen Reported on top protein (AFP) [Abelev, G.I., Perova, S.D., Khra mkova, N.I., Postnikova, Z.A. and Irlin, I.S., Transplantation 1, 174 (19 63)]. It is the main circulating protein during fetal life, but this protein is mostly The serum concentration of the relevant protein is less than 50 ng / ml in normal adults [Ruo slahti, E. and Seppals, M., Int. J. Cancer 8, 374 (1971)]. But the liver In malignant diseases such as cancer and teratocarcinoma, plasma levels can be 1000 times higher. [Ruoslahti, E .; and Seppals, M.S. Adv. Cancer Res. 29, 275 (197 9)]. This finding provides a novel way to detect malignancies and monitor cancer patients. Not only in clinicians who anticipated the arrival of the means, but also in fetal life Researchers examining the physiological function of the protein also noticed.   One of the first parameters studied was the distribution of AFP in the fetus. Immunity Using the peroxidase method, Benno and Williams were found in developing rat brain The distribution of AFP was described [Benno, R.H. and Williams, T.H., Brain Res. 142, 1982 (1978)]. Shortly afterwards, a series of papers reported on several birds, humans, etc. The species concentrated on plasma proteins in developing neurons. [Trojan, J .; and Ur iel, J., J. Oncodevelop. Biol. Med. 1, 107 (1980); Uriel, J .; Trojan, J., Dubouch, P.C. and Pieiro, A., Path. Biol. 30, 79 (1982); Moro, R .; and Uriel J., J .; Oncodevelop. Biol. Med. 2, 391 (1981); Dziegielewska, K.M., Evan s, C.A.N., Lorscheider, E.L., Malinowska, D.H., Mollgard, K., Reynolds, M.L. and Saunders, N.R., J. Physiol. 318, 239 (1981); Mollgard, K .; Jaco bsen, M., Krag-Jacobsen, G., Praetorius-Claussen, P.S. and Saunders, N.R. and Neurosci. Lett. 14, and 85 (1979)]. AFP for specific tissues or organs And the kinematics in the staining of SA, even if the species is different, Are fairly constant [Urierl, J., Trojan, J., Moro, R .; and Pieiro, A. , Ann. N.Y. Acad. Sci. 417, 321 (1983)]. At a stage where the neural structure is completely underdeveloped, Neither intracellular AFP nor intracellular SA is detected. Then suddenly, and depending on the species At a certain time, both proteins are observed simultaneously in the same cell [Torand-Aller and, C.D., Nature 286, 733 (1980)]. Subsequently, the intensity of the staining was weakened, Fewer cells are positive for P and then less for SA. The developed structure is negative for both proteins. IgG and chicken embryos Other serum components, such as albumin, may be present in the cerebrospinal fluid but not in fetal Not found at all in cells. [Fielitz, W .; Esteves, A. and Moro, R., Dev. Brain Res. 13, 111 (1984)].Uptake of AFP by embryonic cells   A question from these early observations is that AFP and SA are taken up from extracellular sources. The problem was whether it was synthesized or synthesized in cells. Nerve cells It is not yet clear whether plasma proteins can be synthesized [Ali, M .; Raul, H .; a nd Sahib, M., Dev. Brain Res. 1, 618 (1981); Ali, M., Mujoo, K .; and Sahib , M., Dev. Brain Res. 6, 47 (1983); Schachuter, B.S. and Toran-Allerand, C.D., Dev. Brain Res. 5, 95 (1982); Pieiro, A .; Calvo, M., Iguaz, F., Lamp reave, F. and Naval, J .; Int. J. Biochem. 14,817 (1982)], even in vitro [Uriel, J .; Faivre-Bauman, A., Trojan, J. and Foiret, D.S. Neurosci. Lett . 27, 171 (1981); Haj eri-Germond, M .; Trojan, Uriel, J.S. and Hauw, J.J. Dev. Neurosci. 6, 111 (1 984)] and in vivo [Villacampa, M.J., Lampreave, F. et al. Calvo, M .; Pieiro, A. and Uriel, J.M. Dev. Brain Res. 12, 77 (1984); Moro, R., Fielitz, W., Gr unberg, J .; and Uriel, J., Int. J. Dev. Neurosci. 2, 143 (1984)], neuromother Cells can easily take up AFP and serum albumin from extracellular sources It is shown. In vivo experiments were performed with homologous and heterologous proteins. In the first experiment [Villacampa, M.J., Lampreave, F., Calvo, M., Pieiro, A. et al. a nd Uriel, J.M. Dev. Brain Res. 12, 77 (1984)], when injected into pregnant rats. , 125I-AFP is used in organs such as the fetal digestive tract, skin, and tongue, Only in organs where the presence of native intracellular AFP has been reported. It has also been shown to be localized in the fetal brain [Trojan, J. et al. and Uriel J. Oncodev. Biol. Med. 3, 13 (1982)]. In the second experiment, [Moro, R., Fieli tz, W., Grunberg, J .; and Uriel, J., Int. J. Dev. Neurosci. 2,143 (1984) Injecting the serum of a newborn rat into the mesencephalic cavity of a chicken embryo, AFP and SA indicate that they are detected where they originally corresponded Was done. This also means that AFP and SA from one species can be taken up by cells of another species. Structures and mechanics that are embedded and preserved through evolution It is shown that it is. This also involves the basic principles of biology Suggest that.   Despite injecting high concentrations of rat IgG, staining for this protein Showed negative. This prevents passive diffusion as a result of the high molecular weight (150,000) Not. The reason is that twice the normal molar concentration of AFP is used in fetal cerebrospinal fluid. Ovalbumin (MW = 43,000) was not detected even when [Fielitz, W., Esteves, A .; and Moro, R., Dev. Brain Res. 13, 111 (1984) ]. This selectivity allows for a specific receptor-mediated cellular uptake (endocytosis) [Moro, R .; and Uriel, J., J . Oncodevelop. Biol. Med. 2, 391 (1981); Moro, R., Fielitz, W., Grunberg. , J. et al. and Uriel, J., Int. J. Dev. Neurosci. 2, 143 (1984)].Dependence of AFP uptake on cell differentiation   However, at this point, is AFP and SA uptake a cell-dependent phenomenon? In both cases, the cerebral blood barrier is closed at the end of fetal life, and the concentration of circulating AFP decreases. Loss of staining as a result of reduced utilization of extracellular protein It was still unclear what it was. First, chicken [Moro, R., Neurosci. Le tt. 41, 253 (1983)] and then in human fetuses [Jacobsen, M .; Lassen, L.C. and Moll gard, K., Tumor Biol. 5, 55 (1984)], spinal cord Nerve cells in the ganglia are negative-positive- before AFP peaks in serum. A negative complete staining cycle was shown. Furthermore, no AFP is detected. Then, the SA will continue to exist for a while. These results indicate that these serum proteins This indicates that the quality reaches the nerve cells of the ganglion.AFP receptor in immature cells   The ingestion of AFP by a cell depends on its expression depending on the degree of cell differentiation. [Uriel, J., Trojan, J. et al. Moro, R .; and Pieiro, A., Ann. N.Y. Acad. Sci. 417, 321 (1983); Moro, R. Neurosci. Lett. 41, 253 (1983)]. According to previous reports, [Uriel, J., Bouill on, D. Russel, C .; and Dupiers, M., Proc. Nat. Acad. Sci. U.S.A.73, 1452 ( 1976)], in the cytosol of the immature rat uterus, ultracentrifuged 4s and 8s It is shown that AFP is included in the two fractions. 4s fract Is completely consistent with the AFP, with 0.4M KCI in the 8s fraction AFP could be detected immunologically only after treatment with. This process is an 8s fraction Was converted to the 4s fraction. At that time, the concept of AFP receptor was not yet established However, the 8s fraction corresponding to the receptor-AFP complex Was expected to have been separated by high concentrations of KC1. This Thus, the separation of the AFP-receptor complex by KC1 was confirmed by Smalley and Sar cione [Smalley, J.R. and Sarcione, E.J. Bioch. Biohys . Res. Comm. 94, 1429 (1980)]. They also found that the AFP molecule was in the immature rat uterus. Provided the fact that it is synthesized by cells.AFP receptor expression in cancer cells   Cancer cells share many biological and antigenic features with fetal cells. [Uriel, J., Adv. Cancer Res. 29, 127 (1979)], AF during fetal life Malignant tumor cells from tissues that have taken up P re-launch the corresponding receptor Indeed, there is a possibility that AFP will be reintroduced. To prove this hypothesis, Sarcione et al. [Sarcione, E.J., Zloty, M., Delluomo, D.S., Mizejewski, G. et al. and  Jacobson, H., Cancer Res. 43, 3739 (1983)] describes an 8s sample obtained from human breast cancer. AFP was found in the complex. The breast cancer uses the cytosol of immature rats In the same manner as in the above experiment. More recently In turn, these authors report that AFP is a human breast cancer cell line with MCF-7. And AFP can be detected immunologically. It was shown that the complex had to be separated in order to be able to remove it [Sarcione, E .J. and Hart, D., Int. J. Cancer 35, 315 (1985)]. Meanwhile this cell line [Uriel, J., Failure-Crepin, C., Villacampa, M.J., Pieiro, A., and Guesken s, M., Tumor Biol. 5, 41 (1984)] and nickel-induced rat striated muscle Tumor [Uriel, J., Poupon, M.F. and Geuskens, M., Br. J. Cancer 48, 263 (1983) ] Has been shown in vitro to incorporate AFP. These indirect consequences Indicates that the AFP surface receptor was detected in the MCF-7 cell line. [Villacampa, M.J., Moro, R .; Naval, J., Failure- Cripin, Ch., Lampreave, F. and Uriel, J., Bioch. Biophys. Res. Commun.12 2, 1322 (1984)]. The binding parameter is positive cooperating in two places (positive cooper 3 shows a model of a receptor showing ation). Kd at the site of strong affinity is 1.5 × 10^-9M, with positive synergy. Kd at the site of strong affinity is 1.5 × 10^-9M and has n-2,000 / cell. 320,000 / cell at low affinity sites Exists, and Kd is 2.2 × 10^-7M. Later studies showed that mouse YACT phosphorus It was shown that a similar receptor system was present on the surface of papilloma cells. The system is It is absent from T cells of mature normal mice [Naval, J., Villacampa, M.J., Goguel, A.F. and Uriel, J.M. Proc. Natl. Acad. Sci. U.S.A. 82, 3301 (1985)] .   These studies were performed in parallel with in vitro experiments, which included naturally occurring Mice with breast cancer Radioactive iodine-labeled AFP was injected. The tissue distribution of radioactivity is in tumor / normal tissue (liver Ratio) was 3.6 [Uriel, J., Villacampa, M.J., Moro, R., Naval, J. . and Failly-Crpin, CH. C.R. Acad. Sci. (Paris) 297, 589 (1983); Uriel, J . Villacampa, M.J., Moro, R., Naval, J. et al. and Failly-Crpin, C., Cancer Res . 44, 5314 (1984)]. Looking at the radiographs of the tumors in these animals A large amount of silver particles accumulated around the nuclear membrane of malignant tumor cells. Particles were not accumulated in normal cells [Uriel, J., Villacampa, M.J., Mo ro, R., Naval, J .; and Failly-Crpin, C., Cancer Res. 44, 5314 (1984)].Imaging of mouse tumor by scintigram method using 131I-AFP   ¹³¹Breast cancer of mice was photographed by scintigram method using I-AFP, and 3 to 4 m m positive images of breast cancer were obtained [Uriel, J., Villacampa, M.J. Moro, R. , Naval, J .; and Failly-Crpin, C.E. Cancer Res. 44, 5314 (1984); Moro, R., H euguerot, C., Vercelli-Retta, J., Fielitz, W., Lpez, J.J. and Roca, R., Nuclear Med. Comm. 5, 5 (1984)]. In fact, 11 out of 12 such tumors Could also be detected with a standard cancer camera connected to a computer. Another mouse One tumor, neuroblastoma, can also be scanned in a similar manner. [Hajeri-Germond, M., Naval, J., Trojan, J .; and Uriel, J., Br. J. Cance r 51, 791 (1985)].   There is no direct evidence for the expression of AFP uptake, the presence of the AFP receptor. It has been demonstrated in several different types of tumors. As a tumor, Rhabdomyosarcoma [Uriel, J., Poupon, M.F. and Geuskens, M., Br. J. Cancer 48, 2 63 (1983)], mouse neuroblastoma [Hajeri-j Germond, M., Naval, J., Trojan, J. and Uriel, J., Br. J. Cancer 51, 791 (1985)], mouse and human breast cancer [Vil lacampa, M.J., Moro, R., Naval, J., Failure-Crpin, Ch., Lampreave, F. and  Uriel, J., Bioch. Biophys. Res. Commun. 122, 1322 (1984); Naval, J., Vil lacampa, M.J., Goguel, A.F. and Uriel, J.M. Proc. Natl. Acad. Sci. U.S.A. 82, 3301 (1985); Uriel, J. et al. Villacampa, M.J., Moro, R., Naval, J. et al. and Fail ly-Crpin, CH. C.R. Acad. Sci. (Paris) 297, 589 (1983); Uriel, J., Villac. ampa, M.J., Moro, R., Naval, J. and Failly-Crpin, C., Cancer Res. 44,53 14 (1984); Moro, R., Heuguerot, C., Vercelli-Retta, J., Fielitz, W., Lpez , J.J. and Roca, R., Nuclear Med. Comm. 5, 5 (1984); Biddle, W .; and Sarci one, E.J., Breast Cancer Res. Treat. 10, 281 (1987)], T lymphoma in mice [ Naval, J., Villacampa, M.J, Goguel, A.F. and Uriel, J.M. Proc. Natl. Acad . Sci. U.S.A. 82, 3301 (1985)], human T-cell lymphoma and B-cell lymphoma [Laborda, J., Nav. al, J., Allouche, M., Calvo, M., Georgoulias, V., Mishal, Z. and Uriel, J . Int J. Cancer 40, 314 (1987); Calvo, M., Laborda, J., Naval, J., George ulias, V. and Uriel, J., presented at the 8th meeting of ISOBM (Paris 1985); Torres, J.M., Anel, A., and Uriel, J., J. Cell Physiol. 150, 458 (1992); Torres J.M., Gueskens, M.S. and Uriel, J., Int. J. Cancer 47, 112 (1991)] Human T lymphocytes activated by tohemagglutinin [Torres, J.M., Labor da, J., Naval, J., Darracq, N., Calvo, M., Mishal, Z. and Uriel, J.M. Mol . Immunology 26, 851 (1989)], human malignant tumor mononuclear cell line U937 [Suzuki, Y. , Zeng, C.Q., Alpert, E.J. Clinic. Invent. 90, 1530 (1992)] and HT29 Colon cancer cells [Esteban, C., Gueskens, M .; and Uriel, J., Int. J. Cancer  49, 425 (1991)].   These findings indicate that the AFP receptor is not a type of tumor, but rather a malignant tumor. It is widely used as an antigen for fetal tumors.Monoclonal antibody against AFP receptor   Labeled AFP (FITC, radioactive tracer) was used for tumor cells in paraffin tissue sections. Does not combine with The reason is probably that the receptor binding site is partially Nature And the affinity for the receptor at that site is low. Will. Therefore, using pooled human breast cancer as an immunogen, Scepter monoclonal antibodies (Mabs) have been produced [Moro, R., Tamaoki, T., Wegmann , T.G., Longenecker, B.M., and Laderoute, M.P. Tumour Biol. 14, 116 (1993 ), Quoted].   The two Mabs that produce IgM can be used in non-reducing situations in PAGE Recognizes a double band of 67KD. The 67KD belt¹²⁵I-AFP It is also reactive to These Mabs reverse the binding site of the AFP receptor. Respond. The reason is that Mab prevents AFP from binding to tumor cells, Conversely, where large amounts of AFP are present in excess, binding to tumor cells is impeded. Because it can be done. Mab does not react to AFP. Mabs are fetal cells and Recognizes breast cancer in a piece of tissue but recognizes mammary adenomas and It hardly recognizes other normal parts.   Over the past decades, scientists have attempted to characterize antigens associated with malignancies Has been done. AFP receptors are considered antigens for fetal tumors and are Fulfilled many requirements as a useful tumor marker. Furthermore, cancer is so widely spread By using monoclonal antibodies against the antigens associated with Clinical usefulness Sex and study the physiological role of the AFP receptor. Will be.Summary of the Invention   The present invention relates to a method for detecting cancer in a patient. The method is to use a labeled antibody or Introducing a labeled AFP into a biological sample of a patient, comprising the steps of: Alternatively, the labeled AFP reacts with the binding site of the AFP receptor in the biological sample. It is something that has been done. Next, react with the labeled antibody or labeled AFP in the biological sample. Identifying the binding site of the AFP receptor and examining the presence of cancer. It is desirable to have a biological sample from the patient's body before the introduction step. Good.   The present invention relates to a method of treating cancer cells in a patient. The method is A Introducing the FP receptor antibody into the cancer cells of the patient. Next, A Reacting the FP receptor antibody with the AFP receptor of the cancer cell, This stops cancer cells from growing or kills them.   The present invention relates to a method for monitoring a patient. The method is a tool for treating a patient's cancer. Contains Tep. Next, after the treatment, the level of the AFP receptor site of the patient is examined. There is the step of checking the bell at predetermined intervals.   The present invention relates to a method for treating a patient. The method depends on the patient's AFP receptor. Includes the step of checking You. If the test results show that the AFP receptor is present in the patient, Then, an AFP receptor antibody or AFP is introduced into the patient's body, There is a step to react.   The present invention relates to a method of treating cancer cells in a patient. The method is AFP Introducing the variant into cancer cells in the patient's body. Then, denatured Reacting the (modified) AFP with the AFP receptor of the cancer cell, There are steps to stop cancer cells from growing or kill them.   It is an object of the present invention to provide alpha-fetoprotein receptors in body fluids and tissues of the human body. Cancer (AFP receptor) was detected to diagnose cancer disease and pregnancy Is to follow up. The principle and method of detection are as follows: AFP receptor in solution (body fluid) AFP receptor attached to solid matrix (a piece of tissue) Similar to detector detection, but for ease of understanding these are discussed separately. I will tell.BRIEF DESCRIPTION OF THE FIGURES   The accompanying drawings illustrate preferred embodiments of the present invention and preferred ways of practicing the present invention. Is described.   FIG. 1 is a chart showing cancer diseases and non-malignant tumor diseases.   Figure 2 is a photograph of breast cancer, where bright cells show cancer. It depends.   FIG. 3 is a photograph of breast cancer, in which the dark brown parts of the cells have cancer.   FIG. 4 is a benign mammary tumor.   FIG. 5 is a photograph of lung cancer, in which the brown portions of the cells are cancer cells.   FIG. 6 is a photograph of gastric cancer, where the dark brown portions of the cells are cancer cells.   FIG. 7 is a photograph of bowel cancer.   FIG. 8 is a graph showing the inhibition of P-388 growth by AFPr-1.   FIG. 9 shows three control animals, shown above, and five days after infusion, below 3 is a photograph of four treated animals.   FIG. 10 is a photograph of an animal from another experiment in which three treated animals were treated. The objects are sick and two have large tumors.Description of the preferred embodiment   The present invention relates to a method for detecting cancer in a patient. The method is to use a labeled antibody or Introducing a labeled AFP into a biological sample of a patient, comprising the steps of: Alternatively, the labeled AFP reacts with the binding site of the AFP receptor in the biological sample. It was made. Biological samples include blood, saliva, tissues, serum, mucous membranes, sputum m), urine, tears or substances containing cancer cells. Biological samples are in pairs It may be a woven piece. Tissue pieces can be fixed tissue, fresh tissue or May be a frozen tissue. The antibody may be a monoclonal antibody. antibody May be a polyclonal antibody. Next, in the biological sample, labeled antibody or labeled A Cancer exists by confirming the binding site of AFP receptor that reacts with FP There is a step to find out if Before the introduction step, the patient's body It is desirable to perform a step of obtaining a sample.   The introduction step is labeled with a radioactive substance (radioisotope) Step for introducing AFP labeled with an antibody or a radioactive substance into a biological sample of a patient. Labeled antibody or labeled AFP can Is reacted. The next identification step is released into the biological sample. Includes the step of confirming that the projectile is present. More specifically, identification The step can include measuring the amount of radioactivity in the biological sample. Ma The identifying step comprises coating the biological sample with a photographic emulsion, Developing and observing the coated biological sample. Wear. Alternatively, the biological sample may be the patient himself, and the introduction step may involve radioactive Injecting an antibody labeled with a substance or AFP labeled with a radioactive substance. Including.   In another embodiment, the introducing step is an enzyme-bleached antibody or Comprises the step of introducing an enzyme-bleached AFP into the biological sample, wherein the labeled antibody or Labeled AFP will react with the binding site of the AFP receptor in biological samples . The enzyme-labeled antibody or enzyme-labeled AFP discolors the biological sample, Comparing the color of the biological sample with a known color to determine if cancer is present be able to. As the enzyme, peroxidase can be used, and in the introduction step, Comprises introducing a peroxidase-labeled antibody into the patient's tissue be able to. Alternatively, the introducing step involves adding a drop of the biological sample to nitrocellulose. Or put on nylon and add peroxidase-labeled antibody to the dropping position. May be included. The next identification step is nitrocellulose or Includes a step to check if the sample drop position on the iron has changed color .   In other embodiments, the enzyme-labeled antibody or enzyme-labeled AFP releases ions and the Are the electrical conductance of the liquid in which the biological sample is placed To change. The next identification step involves measuring the conductivity of the liquid and placing it in a biological sample. Including determining if cancer is present.   In yet another embodiment, the introducing step comprises an antibody labeled with a fluorescent dye or a fluorescent dye. Introducing the dye-labeled AFP into the patient's biological sample. , Signs The antibody or labeled AFP is reacted with an AFP receptor in a biological sample. Next The constant step is to irradiate the biological sample with UV light, and from the irradiated biological sample. Measuring the emitted photons. As another example, biological The sample may be a smear of biological material containing cancer cells on the slide, Tep examines the smear under a microscope or fluorocytometory. Checking step.   The present invention relates to a method of treating cancer cells in a patient. The method is A Introducing the FP receptor antibody into the cancer cells of the patient. Antibodies are It may be a monoclonal antibody, a polyclonal antibody, or an antibody from a different species than the patient. Alternatively, an antibody produced outside the body from lymphocytes of the same species as the patient may be used. Next, AFP Reacting the receptor antibody with the AFP receptor of the cancer cell; Blocks the growth or kills cancer cells.   The introducing step includes injecting the AFP receptor antibody into the patient. Can be. The infusion step injects the AFP receptor antibody intravenously into the patient's bloodstream. Steps may be included. Alternatively, in the injection step, the AFP receptor Injecting the antibody into the body of the patient at a site close to the cancer cells. .   Alternatively, the introduction step involves vaccinating the patient with a vaccine against cancer cells. May be included. Waku The step of inoculating the tin involves introducing an AFP receptor of a different species from the patient into the patient's body. And injecting the patient into the patient against the injected AFP receptor. The body produces AFP receptor antibodies. AFP receptor generated by the patient himself The putter antibody cross-reacts with AFP receptors or cancer cells in the patient's body.   The reaction step involves converting the AFP receptor of the cancer cells in the patient's body to the AFP receptor. Reacting with antibodies to block AFP receptors on cancer cells Or weaken its function. Alternatively, the AFP receptor antibody is a complement May be an AFP receptor antibody. The next reaction step is the AFP receptor Reacting the complement antibody to bind the complement to the cancer cells. When the chain reaction occurs, a hole is made in the cancer cell membrane, killing the cancer cell.   Alternatively, the AFP receptor antibody is conjugated to a drug or toxicant. Is also good. The next reaction step involves the action of an AFP receptor antagonist that couples with the drug or toxicant. Reacting the body with the cancer cells, where the cancer cells take up the drug or toxicant. The enzyme in the cancer cells separates the drug or toxicant from the antibody and frees it, Cells are irreversibly destroyed and killed.   In yet another embodiment, the AFP receptor antibody may be labeled with a radioactive substance. . The next reaction step is AFP Reacting the labeled antibody of the receptor with cancer cells of the patient. Radiation from the AFP receptor-labeled antibody near the DNA of the vesicles destroys the DNA, Induces cancer cell death.   The present invention relates to a method for monitoring a patient. The method is a tool for treating a patient's cancer. Contains Tep. Next, after treatment, the level of AFP receptor in the patient At predetermined intervals.   The present invention relates to a method for treating a patient. The method depends on the patient's AFP receptor. The step of examining the key. As a result of the test, the AFP receptor Once the AFP receptor antibody or AFP is There is the step of introducing it into the body and reacting with the patient's cancer cells.   The present invention relates to a method of treating cancer cells in a patient. The method is modified A Introducing the FP into cancer cells in the patient's body. Modified AFP Or part of an AFP. Next, the modified AFP is used to It reacts with the AFP receptor to stop the growth of cancer cells or kill cancer cells. There is a tip.   The introducing step can include injecting the modified AFP into the patient. The infusion step may include the step of intravenously injecting the modified AFP into the patient's bloodstream. it can. Alternatively, in the infusion step, the modified AFP is Injecting into a site close to the cancer cells can be included.   The reacting step comprises reacting the AFP receptor of the patient's cancer cells with the modified AFP. A step can be included which results in an AFP receptor site on the cancer cell Is blocked or its function is weakened.   Generally, an antibody that reacts with an AFP receptor, a modified AFP or a modified receptor Except for the application or use of any of the above, the techniques described above are techniques generally known to those skilled in the art. It is art.Example of use of the invention   In body fluids:   Either by secretion or by passive diffusion after the cell dies, AFP receptors from cancerous tissue or fetal / fetal tissue are released into the bloodstream from which It is released into many other bodily fluids such as urine, tears and saliva. At that time, body fluids (including serum) A), the concentration of AFP receptor is significantly different between healthy people and cancer patients or pregnant patients. Will be very different. Second, this difference could be used for diagnostic purposes . In addition, changes in the concentration of AFP receptor in these body fluids can be monitored for follow-up purposes. Can also be used. For example, when a patient is diagnosed with breast cancer, The serum concentration of the scepter is high. The patient undergoes surgery and the tumor is removed. That Later, the serum of AFP receptor Concentrations show a sharp decrease immediately after surgery and then enter the plateau region. But, After six months, the serum concentration of the AFP receptor begins to increase again. This is cancer recurrence Or metastasis, which is probably less than traditional clinical or other diagnostic methods. Happen before. Alerted by previous inexpensive tests, the doctor said, A more accurate and expensive method, and if necessary, Look for tumors.   In tissue samples:   AFP receptors are present on most cancer cells. Therefore, the AFP receptor Can be detected by immunohistological means or by culturing with an appropriately labeled AFP. (The latter did not work well when used on paraffin pieces, but was not effective on frozen pieces. is there). Based on this, it is possible to diagnose a malignant tumor. Plus, fluore By using sucein as a label, positive cells spotted on tissue pieces Very few positive cells. This allows for errors in classical pathology analysis Is narrowed. In classical pathological analysis, the interior of otherwise normal tissue A small number of cancer cells in could have been overlooked. In addition, the AFP receptor Expression is conditioned to result in differentiation and biochemical changes, not anatomical changes That appear normal in other respects, but are actually in the early stages of tumorigenesis Vesicles were tested for AFP receptor detection Will show positive.   Again, there is no difference in the principle, and the technology used is slightly different. However, for ease of understanding, AFP levels in body fluids and Scepter detection will be described separately.   In body fluids: [Example 1]   As an example, 22 serum samples from a cancer-free patient and 1 serum sample from a cancer patient The concentration of the AFP receptor was determined by enzyme immunoassay (EIA) for seven of them. The technique used here is described below: EIA96 recessed plate (well plates) were diluted 1/16384 in phosphate buffered saline (PBS) (0.05 MPO4, 0.15 M NaCl, pH 7.5) Coated with pull for 1 hour to overnight. Some dents as well The pleural effusion of a patient whose breast cancer metastasized to the lung was diluted by a factor of 256 I did it. This substance with the code number P89 is available in large quantities It was used as a standard because it was Because of this, all In the example, the same criteria when comparing all samples with each other It became possible to use P89. Non-specific after washing 3 times with PBS The binding sites were made with 1% ovalbumin or gelatin in PBS. I was blocked for one hour. After three washes, produced in the body of the mouse, AFP Ascites of a monoclonal antibody (Mab) against the receptor was added to 1% ovoa in PBS. 100 ul of a solution diluted 1/200 in albumin [Moro, R., Tamaoki, T., Wegm ann, T.G. Longenecker, B.M. and Laderoute, M.P. Tumour Biol. 14, 116 (199 3), which is incorporated by reference into the present application]. ) Was cultured in each well for 3 hours. Next, the dents were washed three times with PBS and commercially available. IgM conjugate of peroxidase-anti-mouse for mouse (conj ugate) was added at the concentration recommended by the manufacturer (Sigma Chemicals, S. Louis). 1 After time, the plates are washed three times with PBS and the color substrate for peroxidase (color s ubstrate) (ABTS) was added at the manufacturer recommended concentration (Sigma). All Culture was performed at room temperature. After 30 minutes, a standard Titertek pre-press The optical density of each pit at 405 nm was measured using a light reader. ty) (O.D.). The results of this experiment are shown in Table 1 and FIG.   The table shows the patient, tumor type and sample to P89 ratio. P89 is Used as a reference throughout research. As a result, the positive and negative thresholds When set at 54%, all but one cancer patient was positive. Use the same threshold All patients without cancer, except one, were negative. Independent T-test Then, Values were reported (using the data analysis results of Excel (TM)):   t-test: two samples assuming non-uniform dispersion               Simple   The analysis result of the two-tailed test is p = 0.0000054, which is the number of samples. It was an extremely large number even considering that the number was small.   Patients in the negative control group (not in the table) were positive. Get Attention was drawn to the importance of the data results, and the attending physician scanned the patient on a CAT And found a tumor that had become a malignant kidney cancer. This malignancy is the best in this test. First discovered, then clinically It has been certified.   FIG. 1 shows the same result graphically. In the figure, the X axis is Divided into parts (malignant and non-malignant tumors, irrespective of unit), Y axis is P8 of each patient 9 is shown. The horizontal line indicates a threshold of 54% +/-. [Example 2]   In another embodiment, a suitable plastic or glass substrate (EIA Or a test tube) with an appropriate concentration of monoclonal antibody (M ab). After washing the excess Mab, the substrate is irrelevant Coated with a mixture of proteins or amino acids to prevent non-specific binding, Serum, saliva, urine, etc.) were diluted appropriately using a coated substrate. And culture for an appropriate period. Wash to remove unbound sample material After this, a polyclonal second antibody is added (polyclonal antibodies are not available to animals). Conferring epidemiology and produced by using the serum as a source of Mab-reactive antibodies. It is. In contrast, Mabs are immortalized (i) transplanted by culture or into a suitable host. mmortalized) generated from individual clones of spleen cells). Apply this antibody to a suitable Marker or enzyme to allow color recognition or other detectable Reaction or may be preceded by the label or enzyme. The analysis is performed by adding a second antibody. You may. If this third antibody is involved in this reaction, the second antibody will Must be produced in different species and there is no cross-reactivity between the first and third antibodies. Should not be detected. Therefore, an appropriate detection method is used according to the type of reaction that occurred. The steps (color, conductivity, chemiluminescence, etc.) analyze the reaction. [Example 3]   In Example 2, monoclonal antibodies were used for the first and second antibodies, whereas in Example 3, Same as Example 2 except that two different polyclonal antibodies were used. [Example 4]   As in Example 2, the substrate is a nitrocellulose or nylon membrane. The reaction is , Cannot be measured with an instrument, but appears as colored spots on the substrate. Anti In response, positive or negative can be determined by visual observation. [Example 5]   As in Example 3, the substrate is a nitrocellulose or nylon membrane. The reaction is , Cannot be measured with the instrument, but appears as colored spots on the substrate. reaction Can be distinguished positive or negative by visual observation. [Example 6]   One of the antibodies of Examples 2 to 5 can be used to bind a receptor to a homologous AFP or a heterologous AFP. Replaced by AFP. [Example 7]   KCI dissociates the AFP receptor complex when the concentration of KC1 is 0.4M or more. Let go. This releases additional amounts of the AFP receptor. This additional Because the amount of AFP receptor may not be detected, As explained in the previous example, circulating AFP receptor complex contains endogenous A FP may be present and therefore the ligand (Mab, polyclonal or AF P), because the cognitive power of one of them may be weakened. AFP receptor Increasing the amount results in a higher sensitivity of the test. This In this case, the sample may be treated with KC1 before or during the culturing on the solid phase. [Example 8]   In all of the above examples, the label is a radioactive compound (radioimmunoassay and related techniques). Is used for). [Example 9]   In all of the above examples, the reading of the measurement was made by chemiluminescence or fluorescence. Done. [Example 10]   In all of the above examples, conductivity readings were taken.   In tissue samples:   Tissue samples can be obtained and processed in several ways.obtain : (A) From a sample of a surgically resected organ. (B) smear from secretions or other body fluids or smear from contact (PAP Smear). (C) Needle biopsyTreatment of tissue before staining for AFP receptor   Tissue samples may be fixed and cut in various ways as needed. a)Frozen pieces:  Tissue is a solid that has been frozen before cutting. For these frozen pieces, A stabilizer may or may not be used. b)Paraffin pieces:  Here, the sample is placed in paraffin before cutting. Normal treatment is applied to the tissue in order to embed it. c)Acrylic acid and other treatments:  Mainly for electron microscopy. [Example 11]   In this example, normal paraffin pieces obtained from the hospital's pathology department were used . The tissue was cut to 4-8 um thickness. After rehydration, the AFP A 2000-fold diluted solution of monoclonal antibody (Mab) against the scepter Was used for culturing. After 45 minutes, slides were washed and anti-mouse IgG + IgG peracid Culture was performed using hydride or rhodamine-labeled conjugate. 45 minutes later, the pair Wash the strip again and observe under a microscope equipped with a suitable light source, or Until culturing, using a hydrogen peroxide substrate DAB. Then slide the slides in the prescribed way And observed under easy-to-see light. Figures 2 to 7 show rhodamine for breast cancer. AFP receptor stained (FIGS. 2 and 3), benign mammary tumor (FIG. 4), lung cancer ( FIG. 5 is a photograph of gastric cancer (FIG. 6) and intestinal cancer (FIG. 7). As shown, cancer cells or The cords or strips of the cancer cells are clearly stained, but the adjacent non-bad The tissue of the sexual tumor is not stained. This can be seen in FIG. 4, which shows a benign mammary tumor. It is obvious. The cells are negative, between the adenoma cells and the surrounding normal tissue, There is no difference in staining.Cancer management   If a cancer marker is expressed on the surface of a cancer cell, the cancer cell can be a therapeutic target The possibility of using it arises.   There are a number of ways to promote cell death by targeting cell surface antigens. You. For example, there is a method using an antibody that binds complement. The complement chain reaction occurs When the cell membrane is punctured, the cell dies. Another possibility and And a method using an anti-tumor antigen antibody conjugated with a drug or a toxicant. Once, cells Once attached to the surface, the conjugate is taken up in the cytoplasm, where cancer cell enzymes are converted to drugs. Separate the object or poison from the antibody. Once released, drugs or poisons can block cells Reversely destroy cells To death. In other situations, radiolabeled antibodies can be used . Once the antibody attaches to the cell, the emission from the antibody is detected in the vicinity of the cell's DNA. To damage and lead to cell death at the next replication.   An important factor in all of the above processes is the specific recognition of malignant tumor cells. Antibody. When (AFP receptor) is used as a cancer marker, AFP Not only is the receptor antibody used as a target molecule, Thein (AFP) itself specifically recognizes the AFP receptor. In this way, AFP can be labeled with various cell lysing agents to cause vesicle death. You.   The latter not only destroys cancer cells, but also, as shown in the following examples, Can play a role in inhibiting the growth of. [Example 12]   P-388 mouse cells expressing the AFP receptor were transferred to the AFP receptor. Were cultured in different dilutions. Perform a complement fixation test in advance. However, no cell destruction due to complement fixation was observed. Cultured for 2-6 hours Later, to P-388 cells^ThreeH-thymidine was pulsed, washed and measured. Figure 9 is AFPr-1 (a monoclonal antibody against the AFP receptor) or normal Radiation of cells treated at the same concentration as mouse serum 3 shows the radiation dose of sex thymidine. As described, the growth of P388 was Greatly reduced. Interestingly, the cells were cultured on the Mab for several hours and after washing again When cultured, the rate of cell regeneration is treated with an irrelevant antibody (normal mouse serum) Very similar to the rate of regeneration of control cells. This means that Mab does not kill cells. It shows that it inhibits its growth. Inhibition of growth in vitro is due to human Lo It was also observed in Vo gastrointestinal tract cancer cell lines (results omitted).   It is not clear why cell growth is arrested, but AFP is It is thought to carry fatty acids into fetal cells. These fatty acids are Necessary for synthesizing the membrane, this activity is very active during organogenesis. A The extracellular concentration of FP varies with time and varies from tissue to tissue. When the concentration of AFP surrounding is inappropriate, performing regenerative activities is Suicide. The concept is that the AFP receptor is a message between the cell membrane and the cell nucleus. It supports the idea that it may play the role of a anger. The antibody of the AFP receptor described in the experiment using P388 is capable of infecting the cell nucleus. It is thought that the message is `` immobilized and not sent (jam) '', so the cell Does not differentiate. This is important. This is because AFP using a substance other than Mab To "immobilize" the receptor system There are other methods, which will be described later.   An important consideration in this and other examples is the production of human AFP receptors. The resulting mouse anti-AFP receptor monoclonal antibody also recognizes mouse cancer cells. Is to understand. Culturing one type of AFP and binding it to another type of cells Sometimes similar cross-reactivity has been observed between species, which is different This suggests structural similarities between species of AFP receptors. [Example 13]   2x10 on the abdomen of multiple C57 black mice<sup>6</sup>EL-4 cells simultaneously Subcutaneous injection, 100 ul of anti-AFP receptor ascites fluid or normal mouse serum As a control, it was injected into the tail vein. 300ra in animals due to slight tissue incompatibility ds radiation. This helps EL-4 cells take up and produce tumors. I did. Animals were observed for 21 days, killed and photographed. FIG. 10 and FIG. The results of two such experiments are shown. The upper part of FIG. 10 shows three control animals (positive animals). Normal mouse serum was injected) and 4 animals (normal mouse serum injected) Is a photograph 5 days after injection. Control groups (some of which are Tumors, stained with red felt for clarity) have a diameter of approximately 5 in mm there were. Treated animals were not ill (small scars were observed at the injection site Only). FIG. 11 shows several animals used in another experiment. In the study, all five treated animals were disease free (three of them are shown) and two were large. Had a tumor (animals were killed 15 days after injection).   In another experiment, treated animals survived for eight months without any signs of illness or abnormalities .Samples and body fluids   The AFP receptor is present not only on the cell membrane but also in the cytoplasm. In fact, radiation When sex AFP is cultured with cancer cells, about 2/3 of it is found in the cytoplasm, Many bind to the AFP receptor. (Reference 32. Naval, J., Villacampa, M.J. Goguel, A.F. and Uriel, J., Proc. Natl. Acad. Sci. U.S.A. 82, 3301 (1985 )). AFP receptor impurities from serum of human tendon (cord) Methods have been developed for removal [Moro, R., Tamaoki, T., Wegmann, T.G., Long. enecker, B.M., and Laderoute, M.P. Tumour Biol. 14, 116 (1993). And will be described in this application. " This method is used for circulating AFP receptors Is thought to be excreted or actively secreted from dying cells. That's because. In fact, AFP receptors are soluble proteins present in the blood of newborns. Quality is something you can expect U.   The same happens to people with tumors. However, the AFP receptor was originally a fetus Cells of the malignant tumor, not cells of the tumor, and the tumor is physiologically expressed in the cells of the fetus. It is. Thus, the AFP receptor is in the extracellular fluid of the tumor. This is heavy It is important. Because some body fluids and secretions can be taken directly from extracellular fluids Because there is. For example, peritoneal carcinomatosis (the original cancer cells have spread to the peritoneum, Spreading throughout the abdominal cavity) usually produces ascites. This has been tested, Abundant in the AFP receptor (a source of monoclonal antibodies in the body of mice) Should not be confused even if the ascites induced by the ascites and its physiopathological mechanism are the same. And: cancer cells in the peritoneal cavity (in this case, hybridomas producing monoclonal antibodies) Causes an inflammatory response and produces ascites, and the hybridoma cells Releases monoclonal antibodies).   P-89 used as a reference standard in enzyme immunoassay was for patients with breast cancer metastasized to lung Pleural exudate. At this stage, the breast cancer cells have spread to the lungs and are Began to grow in the pleura. The pleura responds to cancer cells in the same way as the peritoneum, but This fluid is not ascites but is called pleural effusion or pleural effusion. This In these cases (ascites and pleural effusion), they come into direct contact with (or float in) A) Cancer cells use the AFP receptor as a liquid Release directly inside.   To collect ascites and pleural effusion, there is a method of piercing with a needle. Secretions are in the body May come out naturally. For example, in the case of bladder cancer, the AFP receptor As it is released, the urine can be monitored. Patients with bronchial cancer A forceful cough can collect AFP receptors as sputum.   Like leukemia and some lymphomas, cells of a malignant tumor come into close contact with the blood If present, the concentration of the AFP receptor is highest in serum.   All of the above are descriptions of actual situations and are most likely to occur is not. The most common cancers are stomach, breast, uterus, colon, lung, etc. These cancers release AFP receptors into the bloodstream. This body fluid is used in the analysis It will show the highest concentration of marker in the liquid. However, different immunochemical techniques As sensitivity increases, it may be possible to detect AFP receptors in other liquids U. For example, most serum proteins appear in saliva in small amounts. Saliva sun Obtaining a pull is obviously more practical than taking a blood sample It is. And there is a sufficient amount of AFP receptor and the sensitivity of the reaction used is sufficient. Minutes, saliva can be used in place of blood for diagnostic and follow-up purposes Will. The same can be said for other body fluids.   In summary, ascites, synovial fluid (bone cancer in joints, etc.), pleural effusion, sputum, cerebrospinal fluid, urine, stool It may be convenient to use body fluids such as The best place to choose blood and serum In some cases. Finally, despite the fact that the AFP receptor is more abundant in serum, For practical or legal reasons, it is more convenient to use other body fluids, such as saliva or urine. In some cases it may be useful. Fluid may be collected from natural secretions or by appropriate needle sticks.Immunochemical methods:   These methods can be divided into two groups. One is a test tube or In this method, the reaction is performed in a `` stuck '' (stuck) state. This solid phase has artificial properties, whereas in other methods the solid phase is It is part of the host itself, for example a smear of tissue pieces or blood cells. The former is usually , Immunochemical assay, the latter being an immunohistochemical assay (for tissue sections) or It is called an epidemic cytochemical assay (when the cells are free like smears). Both have the same principle, but differ in the initial material. In immunochemical assays The molecule to be detected or measured must be in solution, In a histochemical or immunocytochemical assay, the molecule to be monitored is The part of the cell where the reaction takes place.   The general principle of these reactions is simple. antibody (From monoclonal or serum or from immunized animals) Very specific in recognizing the corresponding structure. If the animal is as high as human albumin When immunized with a pure fraction, the sample will contain hundreds of Serum, even if it contains high concentrations of Recognize only Min. Antibodies react with substances that produce antibodies (called antigens). Has a very large affinity. Therefore, only very small amounts of antigen can be detected.   Immunochemical reaction or immunohistochemical reaction (immunocytochemical reaction is included in the latter It is. The only difference is that in the case of immunohistochemical reactions, cells A part of the woven structure is cut and placed on the slide, while the immunocytochemical This is because in the case of the reaction, the cells are spread on the slide. ) The way to wake is the same. Substance to be detected (antigen, here AFP receptor) Reacts with an antibody specific for that antigen. One of the two (antibody or antigen) It is labeled or tagged in some way. After culturing the two together, Excess reagent is washed away and the label is confirmed in a number of ways. Sign is radio isoto In the case of loops, the radiation dose is measured with a suitable detector (these techniques usually include^{12 Five} 1 measures the amount of gamma radiation emitted. These techniques are used in radioimmunoassays. (RIA) group). Organization When the pieces are cultured with radioactive labels,^ThreeIt is desirable to use H,^ThreeH Appears on the slide after the slide is coated with a photographic emulsion. Radioactivity In some places silver particles (black) appear. These particles are observed under a microscope. Can be   Another very common technique is to use enzymes as labels. enzyme Since is a catalyst, the substrate can be converted to a new product. An interesting feature here is that one enzyme molecule (radish peroxidase) And alkaline phosphatase) can treat more than 10,000 substrate molecules That is. Therefore, the sensitivity is dramatically improved because the factor is 10,000 times. You. These reactions were performed by enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA). ). These enzymes usually change the color of the substrate. Solubility test (solub In le assays), the solution in the test tube or plate recess will change color. Therefore, by measuring the change (using a colorimeter or spectropolarimeter), Can be quantified. Alternatively, enzymes release ions from nonionic solutions Then, the conductivity of the solution changes, so that the solution can be measured electrically. In a variation of these techniques, biotin is used for labeling, and this label is Detected by conjugated avidin. This avidin-biotin reaction is Increase the sensitivity of the entire detection method You.   Recently, another type of label has been introduced. These labels are fluorescent dyes. And emit photons in the visible spectrum when exposed to UV light. This reaction is Measure with a photovoltaic cell immediately after exposure to high-intensity UV that stimulates light pulses. You. This technique is known as immunochemiluminescence. I have.   Regardless of the method of labeling the molecule or measuring the reaction, the reactions are described below. This is done in several sequences.   Competition assay:   In this case, a relatively large amount of free antigen (here, AFP receptor) and high purity Need to get in the form of degrees. Antibody (Ab) is applied to solid phase (test tube or plastic plate) Such as a recess in the sheet or a suitable film, but will be described further below) . A sample containing the antigen (Ag) is mixed with a known amount of pure labeled antigen. mixture The object is incubated with the antibody on a solid phase. The more antigen in the original sample, the more The amount of labeled antigen attached to a given amount of antigen on the phase is reduced (the amount of antigen is Compared to saturation). The reaction is extrapolated to a solution containing a known amount of unlabeled antigen (extra polation). This technology uses a soluble antigen It is only valid for tissue samples and not for tissue samples. Key Competition Assays The benefits are Only one antibody is needed.Sandwich technology:   There are many variations. Conventional sandwich ELISAs use a single antibody It is constituted by a process of attaching to a solid phase by a method. Next, include the antigen to be measured. Sample is added. The diluent reaction will proceed in excess antibody. ing. After culturing for several minutes to several hours, the excess reagent is washed away and the second antibody is added. available. The second antibody has an antigen molecule different from the antigen molecule recognized by the first antibody. The part must be recognized. Otherwise, the site is occupied by the first antibody This is because no reaction occurs (the antigen is recognized by the second antibody). The reaction does not take place unless it has two or more of the same binding sites, and is this extremely rare Et al.) Since the second antibody is also present in excess, all antigens are recognized. this The second antibody is labeled. After washing again, the reaction is measured. sandwich One of the advantages of the technology is that it does not require a pure antigen. The downside is the reaction Longer run time (requires at least 2 cultures), requires 2 different antibodies It is to be.   The one-piece sandwich used in this example was It has been used in many immunohistochemical reactions. Here, the antigen is on the solid phase The labeled antibody (only one antibody) is then cultured on it Is done. The disadvantage of using a test tube as a solid phase is that the antigen adheres to the test tube That may affect the strength of the And "stickiness" Is dependent on other factors in the sample and will vary with the patient's serum. (This applies protein to the glass or plastic in the recess of the test tube or plate. The coupling force is static, because it is inherently weak. This allows the antigen This is in contrast to the strong reaction with the antibody). To avoid this, It is advantageous to use the double sandwich described above with one excess of antibody. Next, There are always many cases where the first antibody attached to the phase is more or less irrelevant, There is no change even if the pull is different. Because non-specific binding to the solid phase This is because the same product as used in (Preparation of first antibody) is used. Experiment smell The difference between cancer patients and non-cancer patients was so large that the plastic The difference in rate coating cannot be explained.   About the solid phase:   This may be tissue on a microscope slide or on a suitable surface, on which Molecules attach, usually by static electricity (usually cultured in test tubes for several hours, Prepared by washing from Protein adheres to glass or plastic Still do). However, the solid phase is usually made from nylon or nitrocellulose. A film that becomes You may. These films are white. Coating the membrane with antigen or antibody once Then, the same assay as performed on the tissue section of the microscope is performed, using the substrate. Make the colors appear. Note that the substrate does not change color but forms a colored precipitate. At the end of the process, a colored spot indicates whether the reaction is positive. Reaction if colored And colorless means that the reaction was negative. This is before The described reaction is an example of the "all or nothing" type. This method involves a reaction There is a great advantage that no tools are required to solve the problem. Visual observation is enough It is. The main disadvantage is that it is not very accurate.Introduction of antibodies into the human body   There are several methods for contacting a patient's cancer cells with an anti-AFP receptor antibody. You. One method is to inject a patient with purified mouse monoclonal antibody (Mab). It is to enter. When malignant cells come into direct contact with blood (eg, leukemia) ), Intravenous injection method is selected. The same is true when the only blood that can contact the tumor is the blood It is. However, other methods may be better. As mentioned above, some tumors Some grow in a limited space such as the membrane cavity or abdominal cavity. In this case It is better to inject antibodies directly into the cavity rather than into the bloodstream. Antibody to AFP receptor Another way to achieve this is to enter the antibody into the host. It is to be. Immunizing cancer patients with different types of AFP receptors it can. The slight difference in receptor compatibility between humans and other species is May be able to produce antibodies in the body of the animal. Antibodies are available from mice and cows AFP receptor derived from human autologous AFP receptor Cross reacts with Puter. In this way, patients receive a vaccine against their own tumor. Seeds ”. Antibodies are the only way to attach receptors to the surface of cancer cells (jam) It is worth mentioning that it is not law. The same purpose can be achieved with denatured AFP Will be able to Even the synthesized AFP pieces can achieve the same result As such, it is useful in stopping tumor growth.   There are several different ways to obtain a modified AFP receptor, which occur naturally It may be a part of AFP or may be synthesized by molecular engineering of DNA. Heavy The important point is that although the part where AFP reacts with the AFP receptor can survive, The rest does not survive or has been altered to such an extent that it cannot perform its function. Is to be destroyed. All of these methods require specialists in the field. This is a well-known technique. AFP easily applies to any of these methods . For example, the Beckman Instruments Reacts with AFP receptor using peptide synthesizer A synthetic part of the AFP can be generated. With AFP receptor The amino acid sequence corresponding to the reacting AFP synthesis fragment is entered into the polypeptide synthesizer. And the synthesizer is activated. The result is a synthesized polypeptide. Also The desired DNA sequence is transferred into a host system via a suitable vector well known in the art. AFP pieces can also be produced by introducing the AFP. Next, the host system Express, collect and optionally clean. Also, using the gene synthesis method, It is also possible to put the desired DNA sequence into fertilized bovine eggs and the like. Desired AF P can also be obtained from postnatal animals, as is well known to those skilled in the art. Wear. This is described, for example, in Maniatis et al. (1982) You. Yet another way to obtain the desired AFP piece is to use a naturally occurring AFP A cutting method can be given. AFP is a chain such as cyanogen bromide. After attaching and cleaning the chain reagent, the desired AFP piece can be obtained.   Although the present invention has been described in detail with reference to examples, the description is merely an example. Except for the description in the appended claims. Make various changes to the detailed description without departing from the spirit and scope of the description; It is understood that is possible.

────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code FI A61K 51/00 G01N 33/53 Y G01N 33/53 33/577 B 33/577 A61K 43/00 [Continuation of abstract] If the test shows that the AFP receptor is present in the patient's body, then there is the step of introducing an AFP receptor antibody or AFP into the patient's body and reacting with the patient's cancer cells. This is a method for treating cancer cells in the body of a patient. The method includes introducing the modified AFP into cancer cells in a patient's body. Next, there is the step of reacting the denatured AFP with the AFP receptor of the cancer cell, thereby inhibiting the growth of the cancer cell or killing the cancer cell.

Claims (1)

[Claims] 1. A method for detecting cancer in a patient's body,     A labeled antibody or labeled AFP is introduced into a patient's biological sample, and the labeled antibody or labeled AFP binds AFP receptor or AFP receptor in a patient's biological sample. Reacting with the combined site;     AFP receptor reacted with labeled antibody or labeled AFP in a biological sample or Examining the presence of cancer by identifying the binding site of the AFP receptor ,   A method for detecting cancer in a patient's body, comprising: 2. Claiming that there is a step of obtaining a biological sample from the patient's body before the introducing step Item 1. The method according to Item 1. 3. The introduction step is performed using an antibody or radioisotope labeled with a radioisotope. Introducing AFP labeled with a loop into a patient's biological sample allows the Alternatively, a step of reacting labeled AFP with an AFP receptor binding site in a biological sample. The method of claim 1, comprising a tip. 4. The introducing step may be an enzyme-labeled antibody or an enzyme-labeled AFP receptor. The labeled antibody or labeled AFP is produced by introducing the Reacting with the binding site of the AFP receptor in a body sample The method of claim 1. 5. The introduction step includes an antibody labeled with a fluorescent dye or an AF labeled with a fluorescent dye. By introducing a P receptor into a patient biological sample, the labeled antibody or labeled A Reacting the FP with the binding site of the AFP receptor in the biological sample. The method of claim 1, wherein 6. The identifying step includes identifying radioactivity present in the biological sample. 4. The method according to claim 3, wherein the method comprises: 7. The identifying step includes measuring the amount of radioactivity in the biological sample. 4. The method according to claim 3, wherein 8. The identification step involves coating the biological sample with a photographic emulsion, And a step of developing the sample and observing the coated biological sample. The method of claim 6. 9. The biological sample is the patient himself, and the introduction step involves giving the patient a radioisotope Step for injecting an antibody labeled with an antibody or an AFP labeled with a radioisotope 7. The method of claim 6, comprising a step. 10. Enzyme-labeled antibody or enzyme-labeled AFP can be used to detect the color of a biological sample. The identification step compares the color of the biological sample with a known color. 5. The method of claim 4, comprising determining the presence of cancer. 11. The enzyme is peroxidase and the introduction step is labeled with peroxidase. 11. The method of claim 10, comprising introducing the identified antibody into the patient's tissue. Method. 12. The introduction step involves transferring a drop of biological sample onto nitrocellulose or nylon. And adding a peroxidase-labeled antibody to that position. Wherein the identifying step includes the step of locating the position on nitrocellulose or nylon. 11. The method of claim 10, including the step of determining whether the location has changed color. 13. An enzyme-labeled antibody or an enzyme-labeled AFP contains a biological sample. Releasing ions that change the conductivity of the solution in solution, and Measuring the electrical power to determine if cancer is present in the biological sample. 5. The method of claim 4, wherein the method comprises: 14． The identification step includes irradiating the biological sample with UV light and irradiating the irradiated biological sample. 6. The method according to claim 5, including the step of measuring photons emitted from the pull. . 15. The method according to claim 1, wherein the antibody is a monoclonal antibody. 16. The method according to claim 1, wherein the antibody is a polyclonal antibody. 17． Biological samples include blood, saliva, tissue, serum, mucous membranes, sputum, urine, tears or cancer cells. Claim 1 which is a substance containing The method described. 18. The method according to claim 1, wherein the biological sample is a tissue piece. 19. The biological sample is a smear of biological material containing cancer cells on a slide, The identification step is a step of examining smear using a microscope or fluorosymmetry. The method of claim 1 comprising: 20. Claims wherein the tissue piece is obtained from any of fixed tissue, fresh tissue or frozen tissue. 19. The method according to 18. 21. A method of treating cancer cells in a patient's body, comprising:     Introducing an AFP receptor antibody into the cancer cells of the patient; and     AFP receptor antibody reacts with AFP receptor of cancer cells to Stopping growth or killing cancer cells,   A method for treating cancer cells in the body of a patient having 22. The introducing step includes injecting the AFP receptor antibody into the patient. 22. The method of claim 21, wherein 23. The infusion step involves intravenously injecting the AFP receptor antibody into the patient's bloodstream. 23. The method of claim 22, comprising a tip. 24. In the injecting step, the AFP receptor antibody is injected into a portion of the patient's body close to cancer cells. Including the step of injecting 23. The method of claim 22, wherein 25. The introduction step includes vaccinating the patient with an anti-cancer cell vaccine. 22. The method according to claim 21. 26. The vaccination step involves the use of a different species of AFP receptor from the patient's body. AFP receptor against injected AFP receptor by injecting into it -Generating antibodies by the patient himself, The generated AFP receptor antibody interacts with the AFP receptor of cancer cells in the patient's body. 26. The method of claim 25, wherein cross-reaction occurs. 27. The reaction step comprises the step of reacting the AFP receptor of the patient's cancer cells with the AFP receptor. Reacting with the body, wherein the AFP receptor of the cancer cells is blocked. 22. The method of claim 21, wherein the function is reduced or the function is reduced. 28. AFP receptor antibody is an AFP receptor antibody that binds complement The reaction step comprises reacting the cancer cells with an AFP receptor antibody that binds complement. Punctures the cancer cell membrane when the complement chain reaction occurs. 22. The method of claim 21, wherein the cancer cells are killed. 29. The AFP receptor antibody is conjugated to a drug or toxicant and the reaction step is For reacting an AFP receptor antibody conjugated to a drug or toxicant with cancer cells Te Cancer cells take up drugs or toxins, where cancer cell enzymes Separate the object or poison from the antibody, which irreversibly destroys and kills cells 22. The method of claim 21, wherein the method is adapted to 30. The AFP receptor antibody is labeled with a radioactive substance and the reaction step is labeled A Reacting the FP receptor antibody with a cancer cell of the patient, wherein the cancer cell Radioactivity from labeled AFP receptor antibody destroys DNA near the DNA 22. The method according to claim 21, wherein the cancer cells are caused to die. 31. Antibodies include monoclonal antibodies, polyclonal antibodies, antibodies from different species than patients, 22. An antibody produced in vitro from lymphocytes of the same species as the patient. the method of. 32. How to monitor a patient,     Treating the patient's cancer;     After the treatment, the level of the AFP receptor site of the patient is examined at predetermined intervals. Steps,   A method for monitoring a patient having 33. A method of treating the patient:     Testing the patient's AFP receptor;     As a result of the test, it is found that the AFP receptor is present in the patient's body. FP receptor antibody or AFP is introduced into the patient's body and reacts with the patient's cancer cells. Let Steps,   A method for treating a patient having the following. 34. A method of treating cancer cells in a patient's body, comprising:     Introducing the modified AFP into cancer cells of the patient; and     The denatured AFP reacts with the AFP receptor of the cancer cell to increase the growth of the cancer cell. Blocking or killing cancer cells,   A method for treating cancer cells of a patient having 35. Claims wherein the introducing step comprises injecting the modified AFP into the patient. Item 34. The method according to Item 34. 36. The injecting step includes injecting the modified AFP intravenously into the patient's bloodstream. 36. The method of claim 35, wherein 37. The injecting step involves injecting the modified AFP into a site near the patient's cancer cells. 36. The method of claim 35, comprising a tip. 38. The reaction step involves reacting the AFP receptor of the patient's cancer cells with the modified AFP. Whether the AFP receptor site of the cancer cell is blocked 35. The method of claim 34, wherein the function is reduced. 39. The modified AFP is made synthetically or is part of an AFP. 35. The method of claim 34.