JPH05507414A - Method for producing human monoclonal antibodies with high antigen-specific affinity - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] See method for producing human monoclonal antibodies with high antigen-specific affinity This application is filed in U.S. Patent Application No. 071,527.203, filed May 22, 1990. 35 U, S, C, 120/121 (continuation application).

Approval This invention was made with support from the United States Government in the form of an assignment from the National Institutes of Health (NIH). This invention was made below. The Government has certain rights in this invention.

field of invention The present invention belongs to the field of biotechnology, and more specifically, the present invention belongs to the field of biotechnology. Antigen-specific IgG and Ig with useful affinity suitable for clinical applications Human lymph that has specific properties such as the ability to obtain human monoclonal antibodies Concerning a new method for producing and culturing lymphocytes (lymphocytes) in vitro. do.

Background of the invention Much of the research work is focused on monoclonal monoclonals, which have obvious industrial applications in the pharmaceutical field. Efforts are underway to produce such antibodies. Kehler and Milsta In (Milstein) specific He produced monoclonal antibodies and became a pioneer [Nature 356. 495 (1976)]. This method uses a human body that has been immunized in vivo against a specific antigen. It involves fusing lymphocytes with fusion partners such as myeloma cells. Then fusion The cells are grown in selective media.

Surviving hybridomas are assayed for production of the desired monoclonal antibody. , subclone selected positives, grow and freeze.

Early research focused on in vivo immunity, and efforts in that direction continued. immune response If a reproduction method had been available to induce the answer in vitro. , the effort to produce monoclonal antibodies became substantially less That would be expected. Therefore, in vitro immunization studies were pursued.

Mixed lymphocyte culture (M Polbeck of RAND University in Sweden reported the use of B.

Rrebaeck] and his collaborators are believed to have been the first. in the first report They were cultured in such a way as to prevent cell aggregation. Borrebaeck's See 5cand, J. Immunol, 18°9 (1983).

In a subsequent report, they described the expansion of B cells derived from the mixed lymphocyte cultures described above. The research was extended to the analysis of the requirements for growth and differentiation factors. Again, they Studies were performed using lymphocytes obtained from pancreatic tissue of tissue-xenogeneic mice.

It is unclear exactly how they prepared the pancreatic tissue, but they reported removal of adherent cells and recovery of bound cells. They also have MLC lymph We report the importance of adding inducible lymphokines to the globules and these are used in that study. It has been reported that the preparation was They are linked to growth factors such as lymphokines. Without help, no significant antigen-specific immune response was recorded by in vitro immunization. It is reported that it will not be possible.

U.S. Pat. Nos. 4,661,586 and 4,816,249 are relatively recent additions to this field. These documents use immunized mouse pancreatic cells as an example, and then limited to merging it with the immortal lineage.

These Köhler and Milstein methods and in vitro immunization methods are It used lymphocytes. Use of such non-human monoclonal antibodies The reason is that the antibodies of the Sectoptera are foreign to the human host and therefore interfere with the host's immune response. However, since it is expected that it may induce a decrease in the subsequent therapeutic effect, Not optimal for floor applications.

Therefore, research efforts are also being directed towards producing human monoclonal antibodies. there was. However, human cells are difficult to culture in vitro. Larri An overview of ck6 is presented. James et al., J.Imm. See also Un, Methods 100.5 (1987). This review literature shows that human monoclonal antibodies have the efficacy of monoclonal antibodies from Sectarian animals. By considering whether there are possible substitutes (this may be clinically relevant) The availability of such human monoclonal antibodies in pure form for (depending largely on ease of use) to stabilize human cells effectively immunized against specific antigens. It was concluded that the problem of maintaining a stable culture was the main obstacle.

Croce et al., Nature 288.488 (1980) and 01. son and son and Kaplan's PNAS 77.5429 (1980) are A We report on the application of the Kohler and Mflstein method to human cells. Ta. See also European Patent Application No. 44722 and US Pat. No. 4,668,629. thing. These documents include studies on pancreatic cancer either in vivo or in vitro immunization. Another method for preparing cells is disclosed. However, the experimental data Adherent cells were clearly removed and Ficoll-Hiberk ue) prepared for preparing pancreatic tissue by centrifugation and The product of a Palytic mononuclear cell suspension was fused with a specific human myeloma cell line.

A similar operation is disclosed in U.S. Pat. No. 4,624,921 to Larrick et al. . A Ficoll preparation of peripheral blood lymphocytes was also used, and lymphocytes were European Patent Application Publication No. 157574 See also issue. Here, many previous researchers have demonstrated that cells that can continuously proliferate A method has been described that is intended to prepare In many cases, the ability to secrete immunoglobulin (Ig) is lost. European patent application publication No. 62409, fusion in the preparation of hybridomas by a more specific method. Discloses a human lymphoblastoma cell line that can serve as a partner.

US Pat. No. 44,51570 also describes the preparation of human monoclonal antibodies. discloses the use of a human cell line of We emphasize the use of non-adherent lymphoid cell cultures to prepare and fuse Ru.

Patent application WO85102413 corresponding to European Patent Application Publication No. 0162918 Peripheral blood lymphocytes (PB L) and a human monoclonal antibody specific for the human Rh (D) antigen. Report that it will be prepared. See also European Patent Application No. 1.74204 thing.

European Patent Application No. 292965 is based on Epstein-Barr virus transformed 40% g/mlu of endogenous IgM antibody from a human lymphoblastoma B cell line fusion. report the preparation of a stable and continuous human cell line that secretes under

1 of Yamaura et al. Immunol, Methods 84.105 (198 5) Pancreatic stimulation combined with both igM and IgG monoclonal antibody production reported primary immunization of visceral cells. They are antigen-specific clonal expansions, isotopic ERV transformation step for body switching and subsequent hybridoma production states that it is essential.

For related systems, you may also take note of the following references: Bo et al., J. ImmuTechnol, og), Else E Science Publishing, AMS Terdam, 1988. 277 pages.

In the report of 5trike+, immunization of allogeneic cultures of tonsil lymph cells was used. Ru.

Borrelyaeck et al.'s PNAS F3F4.3995 (198g) was later human cells from peripheral blood lymphocytes immunized in vitro against T cell-dependent antigens. Preparation of Human Vibe + 1 Doma I'm reporting it. Again, the in vitro immunization involves various known proliferation and differentiation techniques. Help from factors was needed. Furthermore, in contrast to the results of the related PB invention, Banchereau et al., 5science 251.70 (1991), The lymphocytes died after about 10 weeks.

Finally, the co-inventors of this application would like to share some preliminary information regarding the research leading up to the present invention. We are disclosing our knowledge. This preliminary finding was made between April 23 and 30, 1988. It was published at a meeting held and then Human Tumor Antigens and 5 specific Tum.

r Therapy, Alan R, Li5s, 2 - York, New York (1,989), reprinted in early 1989, p. 147. listed here This situation is probably one of those now known to be essential. Due to the depletion of adherent cells, high affinity antigen-specific It is likely that there were not enough antibodies to produce the necessary antibodies.

Since the disclosure of these findings, we have significantly expanded our research and hereby present Human monoclonal antibodies with useful affinity for specific antigens such as antigens Manufacture the body. A method for completing the invention that is the subject of the present invention reached. Among monoclonal antibodies, those that are effective are IgG is an antibody class that is not frequently obtained. The present invention aims to treat specific disease symptoms. Used clinically for assays or (human) treatments against specific antigens. Producing sufficient quantities of stable and efficacious monoclonal antibodies suitable for This is considered to be the main process for achieving this goal.

Summary of the invention The present invention provides that lymph nodes that contain a requisite number of auxiliary cells, such as autologous adherent cells, Provides an in vitro culture containing human lymphocytes obtained from tissues. Ru. Stiff lymphocytes are immunized against specific antigens in vitro. more specific Generally speaking, the lymphocyte preparation and culture conditions are suitable for monoclonal cells obtained after immortalization. If the antibody has a useful affinity for a specific antigen, i.e. at least about 5 x 10 7 lysotole [1 where C is the concentration per IL (liter) or M (mol) includes a valid number of IgGs, with an affinity constant equal to 7C. It's like doing. According to this system, essential for the preparation of antigen-specific antibodies Requiring the addition of various growth factors or other factors that have been previously reported to be Therefore, an effective number of monoclonal antibodies can be obtained. Certain aspects of the invention include Seed lymphocytes, i.e. pancreatic cells harvested from, for example, histocompatible individuals, are co-cultivated. It encompasses things.

The present invention specifically relates to the above-mentioned method for preparing lymphocytes, the resulting culture itself, and the necessary components and, for example, by fusion with an immortal fusion partner or by recombinant DNA. Method for preparing stable continuous cell lines that produce antigen-specific monoclonal antibodies For the purpose of such lymph cell preparation and culture methods in all cases, such as It is something to do. For example, the cell line encodes the variable region of the desired antibody. can be functionally transfected with DNA. The present invention also provides Human monoclonal antibodies produced from such stable continuous lines and allogeneic such antigen-specific monoclonal cells, such as by co-culturing pancreatic lymphoid cells. A method for preparing such antibodies is also contemplated.

Furthermore, the present invention provides methods for regulating cell development, obtained as described herein. Also of interest are antibodies conjugated to substances capable of binding or having a reporter molecule.

In the former case, for example, monoclonal clones of the present invention induced by immunization with tumor antigens Antibodies target tumor cells by site-specifically targeting tumor cell receptors. To treat tumor cells with substances that can kill them or prevent their development Useful. Similarly, a monoclonal antibody of the invention can be coupled to a reporter molecule. in order to identify the presence of cells that produce antigens specific to disease symptoms. It could be used as a diagnostic tool.

By using three specific means for preparing lymphocytes, the present invention It has been found that a minimal number of supporting accessory cells can be obtained. One means is Ficoll treatment, a commonly used method to detach lymphatic cells, and instead use hypotonic cell lysis of red blood cells, e.g. An example is the use of ammonium chloride. The second method is to optimize room temperature incubation. Reduce the loss of adherent accessory cells during preparation by minimizing the exposed The goal is to minimize conditions conducive to cell attachment to the vascular surface. Third means allow small pieces of lymphoid tissue to be retained in the final cell suspension before freezing, and /or addition. These useful measures and their Analogous means is a possible way to explain the invention.

The invention described herein is further characterized by the following: The methods described support primary immune responses by human lymphocytes in vitro. It is something that can be done. A new aspect is that the conditions underlying a specific immune response are antigen-induced. IgG- and IgM-secreting B cells. 2 ゜This method, when applied to human lymphocytes under favorable conditions, is effective against human and foreign antibodies. helps develop a human's immune response to the antigen. 31 Pros described herein Human lymphocytes immunized and fused according to the protocol are in vitro immunized with human lymphocytes. 5-10 times higher frequency (# produced) than previously reported for (1 million fused lymphocytes) can produce hybrid clones. Wear. A minimum of 3 to 10% of the hybridomas produced depend on the nature of the antigen. It is antigen-reactive. Of the antigen-reactive monoclonal antibodies produced, approximately 20- 90% was IgG.

Therefore, according to the method of the present invention, auxiliary cells such as adherent cells can be added to the culture of human lymphocytes. It is clear that efforts should be made to use methods that ensure the presence of high numbers of cells. . Such successful methods or equivalent methods described in detail herein. The ultimate goal is to be antigen-specific and to contain at least about 5 It has a useful affinity of the order of Tutle and has an effective number of IgG classes. The goal is to produce monoclonal antibodies. This end goal can be achieved by means known to those skilled in the art. eg a competitive ELISA assay.

Books that emphasize the presence of many auxiliary cells, e.g. adherent cells, in the culture suspension The teachings of the invention are implemented by special inclusion of fragments of lymphoid tissue.

Use techniques that do not exclude such fragments and/or eliminate all lymph tissue fragments. The above technique can be carried out by specifically adding a culture containing identification Although we do not intend to adhere to the theory of Observation of the experiment revealed a number of resulting auxiliary cells. This cell is bright It clearly forms loose agglomerates of indeterminate material, which will disappear within a few weeks. Develops into a rigid morphology with well defined edges. This means that the estimated primordial center This is defined as a ``tile-like body.'' This spider-like body is a membrane called Develops into an out-pocket derived from the vesicle and moves on its own to further Auxiliary cell aggregates and the like can be formed. In addition, such a system It helps secretion for several months, and during that time it converts from IgM into IgG. It was also observed that drawing a class switch.

Therefore, by using lymphocyte fragments in the culture system of the present invention, it is possible to Long-term viable, predominantly IgG-secreting cultures are obtained.

Disclosure of embodiments considered to be good for human and equine ferritin antigens Here, we describe the results of successful production of human monoclonal antibodies. antigen specific Specific culture conditions that can produce monoclonal antibodies of the present invention with high affinity will also be explained in detail. However, if one of ordinary skill in the art is provided with this disclosure, For example, using other human high affinity monoclonal antibodies with specificity for other antigens. It is believed that it is well understood how to utilize the present invention to prepare It will be done. Similarly, alternative human lymphocytes other than pancreatic cells can also be found in that particular lymphoid tissue. It can be used according to the method of the present invention, which was first applied using . moreover, Other fusion partners can also be used according to the method of the present invention using specific fusion immortal lines. can be done.

Also, a method for preparing antigen-specific monoclonal antibodies, comprising: The teachings of the present invention incorporate methods utilized in the art to characterize and sequence It is also possible to predict from By preparing that information, variable regions or To clone the DNA of an entire Ig molecule with variable regions such as In order to express and harvest rg molecules, these NAs are introduced into stable recombinant host cells. For operative insertion, known recombinant DNA techniques can be applied.

Similarly, prior to or simultaneously with expression in a suitable stable host, e.g. DNA can be amplified using the PCR chain reaction (PCR) method. All these are expensive Generate immunized B lymphocytes carrying affinity antigen-specific human immunoglobulin This can be done in accordance with the disclosure herein which provides means to do so.

The book (mentioned above) can be extended to equivalent means by a person skilled in the art in accordance with the teachings of the present invention. According to the specific in vitro immunoculture method of the invention, essential auxiliary cells such as adherent cells A lymphoid tissue preparation containing a number of . This result is actually Generation of original-specific IgG-bearing B cells and IgG-secreting plasma cells and primary immunization This results in cultures that functionally reproduce the in vivo environment that can help .

Late-secondary or secondary responses in vivo (in the booster turn, the ichcosome (i ccosomes) forms an antigen-antibody complex called an immune complex coat. Antigen exposure occurs immediately with specific antibodies, and lymph nodes on the surface of follicular dendritic cells taken up into organs. The expansion and maturation of antigen-specific cells occurs in so-called germinal centers. These include the phenomenon of class switching (immunoglobulin IgM to IgG.

affinity for IgA and IgE) and (through point mutations in the variable region) Maturity is included. These two phenomena are essential for effective late-order and secondary responses. It is thought that

As mentioned above, the above in vivo immune response system is functionalized by the in vitro method of the present invention. It is thought that it will be reproduced in High class switching and affinity maturation Important for the most efficient production of high affinity antigen-specific antibodies (preferably IgG). For the essential phenomenon, conditions that at least partially regenerate the formation of follicles and germinal centers are required. It is assumed that the requirements are required. considered necessary for optimal in vitro response. The resulting element is a surface immunoglobulin that allows it to recognize and bind to specific antigens. Presence of B cells carrying phosphorus, stimulating B cell proliferation through localized secretion of various factors The presence of T helper cells that ingest, process and present antigens, and the presence of macrophages that ingest, process and present antigens. , the presence of appropriate lymphokines produced by macrophages and T helper cells , as well as promoting allogeneic and heterogeneous mixed lymphocyte response (MAR) responses and cell aggregation. is the all important presence of dendritic cells.

In a preferred embodiment of the invention pancreatic cells are used. Such pancreatic cell cultures The preparation is performed by separating red blood cells from white blood cells, which has been commonly used in the industry. For example, using ammonium chloride cell lysis instead of Ficoling, which is useful for This is done in a gentle manner. Preparation and culture of lymphoid cells involves attaching cells to the surface of blood vessels. Conditions that favor binding will be minimized. In some embodiments, a single pancreas replacement mixed pancreas was used for allogeneic culture, which provided a highly responsive system for the first time. Ru. These multiplex methods described for preferred embodiments of the invention at least in part those produced in vivo in human tissues when encountering considered essential for the success of a representative in vitro primary and subsequent secondary response. It provides all the elements that can be obtained. As mentioned above, autologous dendritic lymphoid tissue by retaining the necessary number of auxiliary cells such as adhesion cells. In a preferred embodiment, by substantially using human lymphocytes obtained from provides several aspects of these essential elements.

This is mainly due to the specific This is possible because of the element: (a) Manual use, e.g. with forceps and a suitable sterile plastic syringe (rubber tip). a.) Lymphoid cells derived from dissected sections of lymphoid tissue by using a blunt-ended plunger. Gentle dissociation of cysts and accessory cells.

(b) Mild hypoosmolarity instead of ficolling Removal of red blood cells by cyst lysis.

(,c) Minimize exposure to flat surfaces at temperatures above about 4°C. Prevention of loss of adherent cells by (d) Small fragments of pancreatic connective tissue (approximately 1 square millimeter) within the final cell preparation or less) and/or their addition.

To summarize, effects (a) to (d) promote healthy pancreatic cells and macrophages. Auxiliary cells such as lophages, follicles and lymphoid dendritic cells, and fibroblasts A mixture of This cell mixture gently dissociates them from the whole pancreas. In terms of the state of activity obtained by Re-creates the in vivo pancreatic environment more closely than any combination of manufacturing processes or culture methods. It is something that is expressed.

The system of the present invention allows for the equivalent of in vivo germinal centers in the late-next or secondary stages of the immune response. Splenoids, which are thought to be individual objects, have been revealed in vitro. Such responses also result from class switching and long-term survival of IgG-secreting cultures. It is clear that

The main use of the method described is to react specifically with a target antigen such as a human antigen. An immortal, continuously secreted hybrid that produces human monoclonal antibodies that The purpose is to produce dormer clones.

Therefore, the present invention provides a high affinity antigen profile when converted into a stable continuous cell line. An in vitro immune culture system that produces relatively high numbers of heterologous monoclonal antibodies For the first time, we provide a system that is useful in clinical applications.

2. Explanation of parameters The present invention provides a method for preparing lymphocytes for in vitro culture and immunization. This is based on the knowledge that it is essential. An essential aspect is the necessary The method is to ensure that the number is retained within the lymphocyte culture. This way Auxiliary cells assist in efficacious antigen presentation, class switching and affinity maturation. essential for at least partially reproducing the lymphoid follicular type environment Ru. The presence of a requisite number of autologous accessory cells indicates that a “required number” of adherent cells are present in culture. It is manifested by being present in . If required, the number of non-lymphoids (non-T1, non-B) ) cells, i.e., the collection of cells containing essential accessory cells, is completely eliminated in the final preparation. Studies have found that it should not be less than about 10% of the cell number.

Additionally, essential adherent monocytes/macro cells within the lymphoid tissue selected for culture The number of phage cells should not be less than approximately 2% of the total cell population. It was also observed that Calculation of such numbers is performed using specific monochromatic monochromes with fluorescent labels. Label monocytes and macrophages with natural antibodies and perform fluorescence-activated cell selection. performed by measuring the percentage of labeled cells by separate (FACS) analysis. be exposed.

Culture methods have been developed to ensure the retention of effective numbers of auxiliary cells considered essential. Various variations may be made from those specifically described in the specification. In addition, excessive fruit Further research without the need for a We also find that the number of adherent cells can vary slightly outside the suggested minimum range. I was able to do that. Therefore, when defining such limits, "approximately" It uses the following terms.

Culture conditions using the requisite number of adherent cells allow for effective number of IgG class immunoglobulins. It has been found from the studies described herein that Robulin can be obtained. this research Based on the IgG class of the monoclonal antibodies of the present invention, approximately It is thought that the minimum number that can be used is a "valid number." 5.0-80% level is considered to be the most effective for identifying monoclonal antibodies that are clinically useful. It will be done. It is also effective to vary the culture conditions within the general range of the present invention. It is also possible to obtain numbers of IgG molecules that are slightly outside the specified range. be understood. If these numbers are valid, sufficient antigen-specific rgG monochrome null antibodies are generated, resulting in antibodies with useful affinity and good specificity. It seems possible to choose. Therefore, the term "approximately" Used to express the value of

3. Definitions and general operations In the specific method of the present invention, the Pancreatic cells are immunized, i.e., specifically stimulated, with μg doses of either xenogeneic or allogeneic antigen. Intense. Stimulated B cells are then harvested at specific time points after antigen exposure and atypical myeloma ( heteromyeloma) human fusion partner 6H6/B5 (publicly available See also Noh: J, Immunolith, 89:61-72.1986 human monochrome that reacts specifically with the immunized antigen by highly efficient fusion with Generate hybrid clones that secrete null antibodies. from specifically immunized cultures. It is also possible to deplete the antigen, which can then be detected by conventional ELISA techniques. Make the body secrete. Post-immunization assays show that immunoglobulins produced under a wide range of immunization conditions Semi-quantitative evaluation of Brin classes and responses do the value. These results are then used to identify optimal conditions for immunization prior to fusion. and then evaluate the results of subsequent fusions.

The term "in vitro immunization culture" refers to the immunization of humans or other animals with specific antigens. refers to cultures that are not performed on any animal, and instead are derived from tissues of human origin. The resulting culture is then cultured ex vivo.

The term "lymphoid tissue" from which the human lymphoid cells of the invention are obtained for culture includes a number of Contains many T-/B-cells and accessory cells such as pancreatic tissue, tonsils and lymph nodes This means any human lymphoid tissue that can be expected.

The term "required number" of autologous adherent cells as used herein refers to non-human and The author describes the IgG according to the present invention having useful affinity for both specific antigens in humans. This means the number of antibodies that are recognized to produce 7 clonal antibodies. These results is guaranteed in the present invention by means of using lymph tissue fragments in the culture system. .

Accordingly, this term has a functional meaning within the appropriate scope described above and is This can be achieved by certain preferred means.

The term "autologous accessory cells" is used herein to refer to T and B lymphocytes. non-T, non-B cells that assist or augment the immune response, derived from the same individual obtained. means.

The term "specific antigen j" refers to antigens that specifically react with the human monoclonal antibodies of the present invention. Any antigen desired for refers to various tumor antigens such as G72.

The culture of the present invention contains "growth factors" or "growth factors" that were previously thought to be essential, such as lymphokines. References herein that "the addition of a pond factor is not required" are incorporated herein by reference. The conditions described above make it unnecessary to add these substances externally to the culture. It means that. This indicates that the present invention contemplates the addition of such substances. This does not imply that the It only means.

Regarding the affinity of the monoclonal antibodies of the present invention, The term ``at least'' in the term ``other'' means that the specified value is monochrome. - Demonstrate that the Cal antibody is above the level at which it will gain utility in clinical applications. It refers to the commonly believed threshold. About this term The details include the host cell used, the fusion partner used, the antigen selected and the details herein. Due to the inherent variations in biological systems such as culture conditions that can be derived from This is to provide a certain degree of latitude in interpretation. Be a person skilled in the art It will be understood that the optimal threshold affinity value differs from the value given herein. preparing a human monoclonal antibody of the invention that is clinically useful in some cases; It seems conceivable that it could be changed to such a method.

The term "effective number" for IgG class antibodies has the definition given above.

The term "many macroscopic aggregates" refers to B and T lymphocytes and accessory cells. Cell clumps derived from lymphoid tissue that may contain Specifies the observation that it begins to form within the bating time and is visible without enlargement. It is used for.

“Splenoids J. within 1 to 2 or more weeks of culture. It is observed which fragments of lymphoid tissue are specifically contained in the culture. It will be done. This mimics the in vivo germinal centers of late-secondary or secondary immune responses. can become.

The term "allogeneic co-culture" refers to tissue that is histoincompatible, e.g. more than one non-synonymous culture. - refers to the culture of pancreatic or other lymphoid tissue from an individual.

The reference to rIgGJ refers to IgG, IgM, Ig, AS rgD and IgE. The constant regions of the five major classes of immunoglobulin molecules that determine the classes of immunoglobulin molecules It is a standard representation of one of the immunoglobulins.

The term "immortal fused cell partner" is composed of cells that independently reproduce themselves. A cell line fused with a functional immunoglobulin gene that carries lymphocytes When the immunoglobulin molecule encoded by that gene is indeterminate and Means something that provides a vehicle for constant secretion. For such a fusion partner myeloma and plasmacytoma cell lines, and atypical myeloma and atypical hybrids. There are ma etc.

The terms "by recombinant means" and "operably carried" refer to DNA, preferably D whose expression is induced to form the encoded polypeptide. Transformation with DNA in a vector in which the NA element and the encoding DNA are linked. This refers to the generally known method described in literature. Such expression vectors The target is generally replicable and can be found in shrimpsomes or as an integrated part of the host chromosome. can be retained.

In general, Molecular Clon by Maniatis et al. ing, A Laboratory 1lanual.

Co1d Spring Harbor Laboratory, = Knee York, 1982 and various references cited herein, especially Collou Co1ovic et al.'s Method in Enzymology, See Volume 152, Academic Press (1987).

The term "substances capable of modulating cell development" is defined by two criteria: chelate binding or can be specifically linked to antibodies through direct covalent bonds, etc., and can be linked to antibodies at the cellular level. It satisfies that the cell has cytotoxic or cell proliferation inhibitory effect in vivo. means a substance that Such substances include yttrium-90, iodine In Examples include radioactive substances such as, and toxins such as linno.

The term "reporter moiety" refers to two criteria: chelate binding or direct collaboration. It is possible to specifically link to the antibody by binding etc., and bioassay, or More commonly, it satisfies the fact that its presence can be identified to some extent in color assays, etc. means a substance that Such parts include radioactive materials such as indium Examples include labels and enzymes such as alkaline phosphatase.

The reporter moiety itself can be made into an antibody of the invention by conventional techniques known to those skilled in the art. Can be combined. For example, a nuclear group such as a primary amine group in an antibody is can react with fluorescent or enzymatic groups to form covalent bonds, or as such can be Bifunctional coupling reagents known in the art can be used.

Other useful reporters include biotin, fluorophores, and chemiluminescent moieties. components, enzymes or colloidal compounds. As a fluorophore group, fluorescein -5-isothiocyanate, diacyl fluorescein-5 and/or hexacal Bonic acid pentafluorophenyl ester, tetramethylrhodamine-5 (and 6 ) isothiocyanate, eosin-isothiocyanate, erythron-5-i Sothiocyanate, 4-chloro-7-nitrobenzo-2-oxa-1,3-dia sol, succinimidyl 12-(N-methyl-N-(7-nidrobenzo2) -oxa-1,3-diazol-4-yl)aminododecanoate, 7-hydro xycoumarin-4-acetic acid, 4-acetamido-4'-isothio-cyanatostyl Ben-2-2'-disulfonic acid, 9-chloroacridine, p-nitrophenyl. 1-pyrenebutyrate, 9-anthracenepropionic acid, or 2-anthracene Examples include sulfonyl chloride.

The enzyme reporter part contains β-galactosidase, horseradish peroxidase , alkaline phosphatase, dehydrogenase, rumferase, and carbonic anhydration There are enzymes, etc.

4. Explanation of drawings Figure 1. Fertilization from primed (sensitized) human lymphocytes in vitro. Secretion of Chin-reactive 1 gM antibody: single (A, B) and homogeneous mixed (A + B) cultures comparison. Deplete human pancreatic cells of adherent cells as described below. 3×10 ( 6) Cultured at a density of cells/Il. Allogeneic cultures were prepared from pancreas A and B. It consisted of a l:1μ mixture of cells.

Figure 2, Ferritin reaction from primed human lymphocytes in vitro Secretion of sexual rgM antibodies against specific immune responses by mono- and allogeneic mixed cultures Effects of inactivated X radiation.

Human pancreatic cells were prepared as described below without depleting adherent cells. Preparation 1. - Exposure to 2000 Rays of X-ray radiation within 2 hours. 1 hour after irradiation Syngeneic or 1:1 co-culture of normal and/or irradiated cells within 0 or 2 μg/m Primed with horse spleen ferritin for 3 days. Ferritin was removed by washing. Thereafter, the obtained cells were further cultured for 2 days. Collect the supernatant and adsorb the ferritic acid. The reactivity with the compound was analyzed by ELISA.

Figure 3, fectin reaction from primed human lymphocytes in vitro Secretion of 1gM antibody: time dependence of ferritin briming and ferritin reaction secretion of reactive antibodies.

Human pancreatic cells were prepared without depleting adherent cells and were 1.5 x 10 (6 ) cells/ml. The top row of the drawing shows 1B primed, antigen washed away, Incubate for additional 1.2.3 or 5 days as specified in the absence of ferritin. It shows cells that secreted antibodies. The second, third and fourth in this drawing The columns show antigen priming for 2.3 and 4 days, respectively, after which the antigen cells were removed and the resulting cells were incubated for an additional 2.3 or 5 days in the absence of ferritin. cultured in the presence of Collect culture supernatant and examine reactivity with adsorbed ferritin Analyzed by ELISA.

Figure 4, Ferritin reaction from primed human lymphocytes in vitro Secretion of 1gM and TgG antibodies: Time dependence of secretion after 3 days of immunization.

Human pancreatic cells were prepared without depleting adherent cells (prepared at 1 μg/mA’ horse tile). Two groups of wells were incubated with 3X10θ cells/ll for 3 days in the presence or absence of visceral ferritin. It was cultured for a period of time. Ferritin is removed by washing and cells are grown in the absence of ferritin. The cells were further cultured for 13 days. For ferritin reactivity analysis by ELISA, 5. Supernatants were collected on day 7.9.11.13 and 16. After each collection, culture Re-add 1.5 ml of culture medium to the culture medium. Culture supernatants collected each day were collected from previous repurchases. It mainly contained immunoglobulins secreted between the time of injection and the time of supernatant collection.

Open and shaded areas indicate IgM and IgG responses, respectively.

Figure 5. Eri secreted from primed human lymphocytes in vitro Affinity/avidity analysis of Chin-reactive IgM antibodies. Deplete adhering human pancreatic cells A 1:1 co-culture was incubated with Primed with lytin for 3 days, then washed to remove ferritin; Culture was continued for an additional 2 days in the absence of phthalate. ELI to measure ferritin reactivity of secreted antibodies Analyzed by SA and relative affinity/avidity calculated. Standard not shown Error bars are included within the symbols.

Figure 6 4 In vitro primed human line in the presence or absence of adherent cells Secretion of ferritin-reactive IgM antibodies from paracells: against horse and human ferritin Comparison of responses.

Human pancreatic cells were cultured without (B and D) or with depletion of adherent cells. (A and C) vR was produced. Specified 1:1 co-culture of cells from two R organs. concentrations of horse pancreatic ferritin (A and B) or human ferritin (C and D) for 3 days. After removing ferritin by washing, the obtained The cells were cultured for another 2 days, the supernatant was collected, and it was added to the adsorbed ferritic acid. The reactivity with the compound was analyzed by ELISA.

Figure 7, Ferritin reaction from primed human lymphocytes in vitro Secretion of sexual antibodies/Equine and human ferritin in the presence and absence of adherent cells Comparison of rgM and IgG responses to. Depleting or not depleting adherent cells Human pancreatic cells were prepared. 1.1 Co-cultures were added to horse or human fish at the specified concentrations. Primed with eritin for 3 days at 3X10' cells/wing. Ferritin After removal, the resulting cells were cultured for 2 days and the supernatant was subjected to EL for ferritin activity. Analyzed by ISA.

Figure 8. In vitro plating with purified murine monoclonal antibody No. 1 or No. 2. An antigen-reactive IgM antibody secreted from human lymphocytes that have been stimulated.

Human 1-ff visceral cells were prepared without depleting adherent cells and were 3X10(6) cells for 3 days with purified murine monoclonal antibody at a concentration of /Tomi! It was cultured in After removing antigen by washing, cells were cultured for an additional 2 days. . Supernatants were analyzed for reactivity with immunizing antigens by ELIS, A. antibody l and The light chain components differed from No. 2 (No. 1 contained a lambda light chain, and No. 2 had a cut-off). (contains light chains).

Figure 9, Ferritin-reactive specific monoclonal antibody (A) and ferritin anti-ferritin antibody Comparison of reactivity by ELISA with a reactive non-specific monoclonal antibody (B).

Collect the culture supernatant from the final culture of each clone grown in a 24-well plate, Tested by ELI SA as described below. Immunoglobin in culture supernatant Brine concentrations ranged from 1-10 μg/wl. As shown in the drawing, the inspection The reactivity (value shown along the X-axis) with increasing concentrations of ferritin coated on a flat plate is , compared with reactivity to increasing concentrations of ferritin-related proteins.

Figure 10. Two purified anti-ferritin human monoclonal antibodies against ferritin. Titration of reactivity. By protein G affinity chromatography, monochrome Purification of local antibodies and ELISA analysis of antibodies at the specified concentrations are described below. That's how I went.

Figure 11. Selected ferritin-specific 1gG human monochromes by competitive ELISA Affinity analysis of local antibodies.

This analysis was performed as described below. The first graph shows the competitive flow in solution. Inhibition of antibody binding to ELISA plate as a function of concentration of erythin (X-axis) It shows degrees (Y axis). Ao is antibody reactivity in the absence of soluble antigen 490 nm obtained by ELISA analysis of the incubation mixture. A is the form of the absorbance at This shows the reactivity of the tested antibody. The second graph shows K for the data shown in the first panel. 1otz plot analysis is shown (1/V corresponds to Ao-A/Ao, and aO is The total concentration of free antigen is shown). The white circle indicates immunity with bovine ferritin. produced from lymphocytes, with a calculated value of Kd = 0.86xlO-''M. Monoclonal antibody 14.2.2.59 is shown. The black square is a human fertilizer. Calculated Kd = 1.90X10- Monoclonal antibody 21.1.8.9 with 8M is shown.

Figure 12 shows that, for example, by ensuring the presence of lymphoid fragments during culture, a large number of Long term in 24-well plates prepared according to the present invention to retain accessory cells of The time course of cumulative 1 g secretion from pancreatic cell cultures is shown. (Total 1gM to I A mature immune response and prolongation of the class switch (to gG) is evident.

Figure 13 shows the immunity secreted by long-term pancreatic cell culture in T25 flasks. It represents the antigen reactivity of globulin (Ig) (reactivity with ferritin), The specific antigen reactivity of secreted rg is shown. Figures 12, 14, and The data in Figure 15 shows the total 1g secretion accumulated. The data in Figure 13 are It shows only the secreted 1g of its components that react with the antigen, ferritin, which is Not cumulative. The OD values shown are the differences between primed and non-primed control cultures. It shows the difference in reactivity. These data indicate that ferritin Stimulating the secretion of 1 gM of Chin responsiveness as early as 0-25 days and significantly increased ferritin reactivity 1gG only after the second booster of 70-85 days. It shows that it stimulates. Antigen-reactive IgM secretion occurs early in the culture stage. antigen-reactive IgG secretion only after booster The fact that it is stimulated by ferritin suggests that it is a class switch in vitro. Further evidence of ching.

Figure 14. Cumulative total 1 gM secretion from long-term pancreatic cell cultures in T25 flasks. It shows the passage of time. Secretion rate of horse pancreatic ferritin, a briming antigen represents the influence on degree. Ferritin primed cultures compared to control cultures showed an increase in the secretion rate of JgM over the first 0-30 days. after 30 days The velocities of both cultures were clearly very similar up to a short period after the second booster; The rate of lymeization/booster cultures also appeared to be increased compared to control cultures. there were. After just over 100 days, the IgM secretion rate of control cultures increased significantly. showed that. This increase in control cultures was due to mold growth that became apparent at approximately day 110. This culture should have been discarded.

(Secretion rate is defined as the quotient of the vertical rise divided by the horizontal distance across the rise. Calculated from the slope of the line. The steeper the slope, the higher the speed will be. ) Figure 15 shows cumulative total I from the same long-term pancreatic cell culture shown in Figure 13. The time course of gG secretion is shown. Priming antigen, horse pancreatic ferritin I The effect on gG secretion rate is shown. Ferritin is less than IgG on days 0-40. had no significant effect on secretion rate, indicating that at that time ferritin In contrast, it has the greatest effect on gM secretion. Approximately on the 44th day , the IgG secretion rate of primed cultures was small but significant compared to control cultures. showed an increase. On day 72, after washing immediately after the second booster, prime The rgG secretion rate of cultured cultures was dramatically increased compared to control cultures. ferritin stimulates IgM secretion in a prime manner in the early stage, and primes IgG secretion in the late stage. stimulation is evidence of class switching in vitro.

Figure 16 (micrograph) shows the involved microorganisms, which are by-products that help develop the second structure. It shows adherent cells. Three different types of secondary structures are also shown: (1) a medium-sized one on the right with a large confluent center, (2) a small confluent dark center; , with a relatively smooth substance extending outward from it. (3) a small dense dark center and outwards; The small one on the lower left with a small amount of relatively smooth material spread out.

Figure 17 (micrograph) shows two examples of the second structure = (1) on the right side; A relatively dense, dark bonded structure with bright blebs visible at the top. and (2) a relatively loose and bright non-bonded structure, The one on the left is clearly composed of smooth aggregates of relatively large brown cells. The individual brown cells on the bottom and left side are either diffused outward or aggregated. Either they are in the process of being assembled.

Figure 18 (micrograph) shows a dense dark center (visible below the layer of lymphocytes). on the right side, which has not yet developed into a layer (not possible) and remains an underlying layer of adherent cells. A secondary structure consisting of irregularly shaped cells or many small cells that appear to be lymphocytes. It shows something surrounded by one of the round cells. these small thin A dense collection of cells may be formed around or adjacent to the second structure depicted. This suggests that the second structure is a multilayered site of these small lymphoid cells. This indicates that there may be

The various cells and structures shown in Figures 16 to 18 develop over time in culture. ripen Growth of adherent cells and development of secondary structures inoculate flasks with pancreatic fragments or plate and continue for a minimum of several months starting 1-2 weeks later. adherent cells If there is no growth of the antibody and no development of the type of second structure observed, the antibody No secretion is observed. These second structures exhibit in vitro germinal center function; or aggregates of distinct types of cells that perform this function.

5! example The following describes the primary immunization, culture, and preparation of human pancreatic cells using protein antigens. The protocol for manufacturing is shown. Equine pancreatic ferritin protein is sufficient The human immune response to horse ferritin has been characterized and Furthermore, the carcinoembryonic form of human ferritin may be comparable to human responses to tumor-associated antigens. In order to perform the above method, this equine pancreatic ferritic acid has been characterized as protein was used. Furthermore, ferritin-reactive human monoclonal antibodies are not therapeutic. It could be used for therapeutic purposes. The conditions developed for ferritin also apply to other proteins. It has also been found to be applicable to immunization with antigens. Immunize as described above. lymphoid cells can fuse with human/mouse atypical myeloma fusion partners with high efficiency, and It is possible to produce more antigen-specific IgG and IgM human monoclonal antibodies. It has been proven that

Carroll et al., J. Immuno, Methods 89.61 (19 A highly efficient hybrid with 6H6/B5, one of the heterogeneous hybrids constructed by (86) Fusion immortalizes human pancreatic cells immunized in vitro with protein antigens Then, a human monoclonal antibody with specific antigen reactivity was produced. The results obtained are good. Good fusion efficiency and hybrid progeny development, immunoglobulin range from 0.5-50 μghl Level of secretion, stable immunoglobulin secretion of about 50% of hybrids (1 hybrid) sex, as well as IgG and IgM class antibody production.

(a) Pancreatic tissue obtained from an accidental victim within 1 hour after surgery was varsity of California San Diego (UC3 D) Provided by Ti5sue Bank.

(b) The resulting tissue was cut into approximately 1 inch square pieces.

(C) RP by pushing the pancreatic fragment through a 50-mesh wire screen. MI Growth Medium Growth Medium - Obtain cell suspension.

(d) as the cell suspension is produced, collect it in a sterile bottle on ice; It is then filtered through layers of sterile cheesecloth into a sterile glass beaker. , remove large tissue fragments.

(e) Next, the obtained cell suspension was added to 25014! Transfer to a volumetric centrifuge bottle and Harvest cells by centrifugation at 11000 rpm for 0 minutes. Discard the supernatant and slide Resuspend the Kana pellet in the minimum volume of RPMI and transfer to a 10 Chl capacity sterile bottle. did.

(f) Expose the cells to hypotonic ammonium chloride for 30-90 seconds to remove red blood cells. RBCs are lysed and washed several times.

(g) Preferably about 0. Lymphoid tissue with a size of 1 to 1.0 mm Carefully keep the fragments in the cell suspension.

(h) After the final wash, count the cells, resuspend them in freezing medium, and place them in a cryotube. Approximately 100-600 million yen per milliliter per vial. Freeze as individual cells.

2. Immunization: All steps are performed under sterile conditions.

(a) 100-300 million cells, each obtained from two separate pancreatic preparations, Thaw by gentle shaking in a 37°C warm bath. The resulting cells were treated with RPMI twice. Wash.

(b) Resuspend cells in medium 5111 and count.

(C) Quickly adjust the cell concentration between 0.5-5.0XIO(6) cells, then Transfer the cell suspension to a 24-well Costa plate at a concentration of 2Nl/well. vinegar.

(d) Add the desired concentration of antigen (range 1-10.0μg/Iris j! is the effective range) (recognized as an enclosure).

(e) The composition of RPMI growth medium (commercially available medium) was 10% fetal bovine blood. serum (Fe2), 2mM glutamine, and Gibco amino acids and bilbas 1-(1 : 100 dilution).

(f) Exposure to antigen continues for at least 2 days.

(g) A control medium is also prepared as described above, except that it is not exposed to antigen.

3. ELTSA protocol / Analysis of immune response (a) After 3-5 days, the following Remove antigens from cultures by incubating cells from each culture with RPMI 10 Aspirate and resuspend with g/+2% FCS and transfer it to a 15w1 volume centrifuge tube.

(b) Cells were harvested by centrifugation at approximately 1100 Orp for 10 minutes and washed. and then resuspend by aspirating with an IChi pipette.

(c) Resuspend the obtained cells in fresh growth medium 2al and transfer them to the original culture well. and secrete antibodies in the absence of antigen.

(d) After 2-4 days, collect 1.5 ml of supernatant by aspiration with a pipette, until it is assayed for immunogen-specific response by competitive ELISA method. Store at 4°C with 0°01% AND.

(e) Response level measured in non-immunized cultures (this is the background response) (which is highly dependent on culture conditions) is a response measured using immune cells. Subtract from the answer. This difference represents a semi-quantitative measure of the antigen-induced response, and this This is a hybrid clone that can be obtained from fusions of lymphoid cells immunized under similar conditions. can be used as a rough guide to the number of immunoglobulins and major immunoglobulin classes. Wear.

4. Immortalization: Production of hybrid clones from in vitro immunized lymphocytes.

(a) After a minimum of 2 days of exposure to the immunizing antigen, RP MI 5ml resuspend in K6F6/B5 atypical myeloma cells.

(b) RPMI as described above containing glutamine, pyruvate and non-essential amino acids The resulting cells were diluted to 5Q irises using Centrifuge.

(c) Wash the cells one or more times with the above solution, fuse, and hybridize using the normal fusion protocol. Choose according to the call.

Horse pancreatic ferritin (F-4503), BSA (A-7906), Tween 20 (P-1379) and potassium thiocyanate (P-3011) in jig 7 [ Sigma]. Human ferritin purified from liver cancer tissue is Dr. Sherry Klein of Hopkins University (Baltimore, MD) Jerry Klein). Murine monoclonal antibodies , Rossell Park Memorial In5 titute (buff Malaya Bhattacharya-Chat (New York) from Dr. terjee (4DC6), and from Dr. Ben K, 5eon (SN 2) were gratefully received. Anti-human IgM (4102), IgG (4 100), IgM-HRP (2392) and IgG-HRP (2390) are TA Obtained from GO [Burlingbum, CA]. The primary antibody was Coulter Im purified monoclonal antibodies from Munology (Hialea, FL) and or cell line CRL 8001 (antibiotics) obtained from ATCC. -CD3) and CRL8014 (anti-CD8). used.

Pancreatic cell preparation Pancreatic tissue obtained from an accidental victim within hours of surgery was ・Center Tissue Bank [UC3D Cancer Center T i5sue Bank].

Single cell supernatants were prepared by pushing the fragments through a wire screen. .

The resulting cells were collected by centrifugation at 11,000 rpm for 10 minutes, and the RBCs were Ammonium was removed by cell lysis. Wash remaining cells and add 40% RPMI .

100-300 million in freezing medium consisting of 50% FC81 and 10% DMSo. resuspended at cells/mA', frozen in 5 l, 1' volume, and stored in liquid nitrogen. . To perform long-term culture, pancreatic tissue fragments are placed in cell suspension before freezing. It is essential to maintain it.

in vitro immunization All steps were performed under sterile conditions. Frozen cells from each pancreatic preparation were Thaw with gentle shaking at °C and wash twice with RPMI 15ip.

Immediately after thawing and washing, 2 ml of immune cultures were prepared. In mixed cultures, each pancreas is Equal numbers of cells were distributed into the final concentrate. Next, the obtained cell suspension was placed in 24 wells. 2*l/well into the inner wells of a tissue culture dish. ferritin or other anti- 0-10μg/ml as specified in the original! I added it. For each condition to test At least four wells were prepared. When using many antigen concentrates, No antigen was added to one well. This is an in vivo immune system. This was a non-immune control sample similar to the pre-serum. in each of the remaining three wells. Different amounts of antigen were added. If only one antigen concentrate was used, two No antigen was added to one well, and the specified concentrate was added to the remaining two wells.

Cells were cultured with antigen for 3 days unless otherwise specified. After briming, each Gently transfer the cells from the wells to a 15-volume centrifuge tube and add RPMI + 2% FC. 315++n and then remove them for an additional 2 days unless otherwise specified. I returned it to the well I had left it in. Culture supernatant was collected on the 5th day, and antibody production was detected on the 7th day. Cells were discarded or re-added for day 2 analysis. standard release The growth medium is 10% FC3, 11% non-essential amino acids, (Irvine 5cient ific) 2mM glutamine, 1mM sodium pyruvate, 15oMHE It consists of PES and RPMI with gentamicin added.

Anti-ferritin assay The anti-ferritin reactivity of the antibody in the culture supernatant was evaluated as follows.

Purified ferritin was added at 10 μghl in 0.05 M sodium carbonate buffer pH 9.3. Diluted to . 96 wool round bottom 1mmulon I flat plate [Dynatech Co., Ltd. 0.05 ml/well was incubated overnight at 4°C. Each supernatant Assayed three times for ferritin and twice for orchid serum albumin (BSA) did. The 0D-490 value observed with BSA is due to non-specific protein reactivity. and was subtracted from the 0D-490 value observed for ferritin.

The difference in OD values between ferritin and BSA binding is called ferritin reactivity. non-exemption Differences in ferritin reactivity between primed and primed samples indicate ferritin-induced ferritin This is called reactivity. - After overnight coating, the plates were soaked in phosphate buffered saline (PBS). Washed three times and blocked with 1% BSA in PBS. Another with similar results In the method, the BSA blocking step is omitted and 0.05% Tveen 20/P B Dilute the sample and reagents in S.

After 2 hours at 37°C, the blocking solution was removed and the supernatant was 0. Q5mf suitable well. After 2 hours at 37°C, the obtained plate was soaked in PBS-0.1%. Washed 5 times with Tween 20 and injected with peroxidase-conjugated goat anti-human IgM. Added 0.05 ml of a 1:1000 dilution of the mg/ml solution. This second test Drugs were diluted in 10% FC8 in PBS. After 45 minutes at 37°C, the obtained Wash the plate 5 times with PBS-Tween and add 0.4 g/wing lO-phenylenedia. 0.15011/well of Min [Sigma P-1526] was added to 0.05M sodium citrate. Thorium Buffer 1] was dissolved in H5°0 and 0.0175% hydrogen peroxide was added.

After 15-60 minutes, 2.5M sulfuric acid 025w1 was added to stop color development.

Molecular Devices View, CA) Record 0D-490 values using a Vmax kinetic plate recorder. Ta. Assay wells were coated with 50 μghl ferritin or BSA and protein Allow to adhere overnight at 7°C, and the second reagent is peroxidase-conjugated goat anti-human. Human 1 big G anti-ferritin reactivity was assayed in the same manner as above except for IgG. did.

Anti-murine immunoglobulin assay Using 5ug/ml murine monoclonal immunoglobulin as the capture antigen. Otherwise, use the basic procedures described above for the detection of anti-ferritin reactivity. Polyclonal human anti-mouse immunoglobulin reactivity was determined by ELISA. was tested.

Quantification of human IgG and rgM Human IgG and IgM were quantified by ELrSA as follows. goat anti -t: 1gM or 1=1o of IgG [Tago] (7) 3-wg/ml solution o Dilution (7)Q, Q5xl/well was adsorbed overnight at 4°C or human IgG or Ig. It is used as a capture agent for M. Supernatant containing unknown amount of immunoglobulin was serially diluted until no reactivity was detected. This assay protocol Oxidase conjugated second reagent, goat anti-human HRP-conjugated IgM or it1gG 1: 1000 (7) t) Rini l: Except for using at a dilution of 5000 The method was similar to that described for anti-ferritin reactivity. ,0 ．． 01 Gara 2. Purified polyclonal human IgM for use in the OIIg/xi range or an OD value within the linear function range of the standard curve defined by the IgG standard sample. The concentration of the unknown sample was calculated from the dilution to which it was assigned.

The assay, which utilizes the disruption of antigen-antibody binding by thionanates, was MacDonald et al.'s J Immuno integrated with Lithin ELI SA. Adapted from Methods 106.191 (1988) and using it The amount of in vitro immune response was characterized. After immunization, the culture supernatant was combined with the assay plate. The plate was incubated with PBS-Tween. Wash 5 times with 1 ml of potassium thiocyanate at the specified concentration dissolved in PBS. were added to each well and the resulting plates were incubated for 15 minutes at room temperature. the flat plate were washed 5 times with PBS-Tween and anti-ferritin assay was performed as previously described. I finished it. The supernatant with high binding activity is diluted before assay, and the phase of each sample is The lithin binding activity was made to be relatively uniform. Each thiocyanate concentration Each supernatant was assayed in duplicate for each supernatant obtained from two separate but identical immunizations. The superior grooves were analyzed under test conditions J. [KSCN]-50 value is Thiosycin required to reduce the amount of antibody binding to eritin by 50%. It was defined as the concentration of anate.

Lymph cell marker analysis Approximately 1 million cells were used per test. At specified times after priming begins The cells were harvested and washed once with growth medium, then diluted with cold PBS 10% BSA and 0.02M azide. Resuspend in sodium (wash buffer g&) 1*i'. human heavy chain mu, heavy chain cancer Mouse that reacts with Ma, PCA-1, B1, CD3, CD4, CD25 or CD25 Lymphocytes were incubated with monoclonal antibodies for 30 minutes at 4°C.

The obtained cells were labeled with fluorescein isothiocyanate (FITC) Affinity isolated human absorbed goat F(ab')2 anti-murine IgG [TAGO, Wash three times with wash buffer containing Burlingham, CAE and incubate for 30 min at 4°C. resuspended in

The obtained cells were then washed with PBS + 1% paraformaldehyde 0.5%. 4. in ysl The cells were washed four times and resuspended. EPICS profile flow cytometer Fluorescence was measured for all samples on the same day of use. Front and 90 degree light Dead cells were eliminated by scattering means. Fluorescent signal sensitivity and amplification Control sample (cells labeled with non-specific mouse immunoglobulin as primary antibody) The shelf temperature record was set to be 5%. Calculations for the data presented are based on the cell table By non-specific mouse Ig from the recorded values for samples that react with surface-specific antibodies. This was done by subtracting the background value of the labeled cells.

B. Results Comparison of immune responses by mono- and allogeneic mixed cultures Antigen-primed cultures Exposure to supernatant and antigen from control cultures primed with supernatant and antigen from Antigen reactivity of polyclonal antibodies in supernatants from untreated control cultures. Antigen-dependent immune responses were measured by ELISA. Not related Antigen-induced antigen reaction was achieved by co-culturing WI-derived lymphocytes from two different pancreases. One of the best ways to help produce and detect reactive immunoglobulin is provided. Lymphocytes from eight pancreases were investigated individually and collectively. Special A low 1 gM response was observed when a given pancreatic preparation was cultured separately; Shown in Figure 1 for Viscera A and Pancreas B. In contrast, some pancreas The preparation showed no response under most of the conditions tested (e.g. in Table 1). pancreas D). However, when cells from responsive or non-responsive pancreas 2 are co-cultured , a ferritin-dependent 1 gM response was consistently observed (FIG. 1, Table 1). anti Non-specific binding activity assessed from cultures that have not been exposed to homogeneous culture conditions (Fig. 1). However, despite the increased activity of non-immune cultures. , is the sum of the contributions observed in each pancreas alone due to allostimulation in the presence of antigen? Antigen-induced signals were always derived that were larger than the expected signals.

Calculated and observed responses for all combinations of pancreatic ASB, C and D. Shown in Table 1. In general, rather than using two or more or only one pancreatic culture, , the antigen-induced signal observed using two pancreases was greater. Therefore , by using two pancreases, sufficient to induce a detectable specific response. No activation and a strong and therefore low amount of nonspecific antibody response suppresses antigen-induced responses. It was concluded that an optimal balance between activation and cognate activation can be obtained.

Determination of ferritin-reactive antibodies by irradiated mono- and allogeneic mixed pancreatic cultures Chin-dependent production Non-immune syngeneic cultures (Figure 2) prepared from either pancreas 1 or pancreas 2 were It produced small levels of antibodies that cross-reacted with lytin. Ply with ferritin After treatment, low levels of induction of ferritin-reactive antibodies were observed. simultaneous cultivation The non-specific and specific reactivity was increased by the supplementation. Mono- and homogeneous mixed culture When the object was irradiated, all responses were discarded. However, one member of the mixed culture There was no loss of specific reactivity when irradiating only 1 minute before antigen priming.

When pancreas 2 was irradiated, specific induction by antigen increased approximately two-fold. child These results demonstrate that successful allogeneic stimulation requires only one partner, a functional B cell. , either pancreas in allogeneic culture can be stimulated by antigen to produce specific antibodies. , and the B cells derived from pancreas 2 in the mixture shown in Figure 2 are different from the B cells derived from pancreas 1. The study showed that ferritin produced more nonspecific antibodies that cross-reacted with ferritin. There is.

Required time for antigen priming and detection of antigen-induced antigen-reactive antibodies in the pancreas 2 Co-cultures of lymphoid cells were treated with 0 to 1.0 μg/l of horse ferritin. 1.2.3 or 4 days of initial exposure to the antigen (prime). We determined the optimal time required for After priming, equine fertilize the cells. Optimized for secretion of detectable ferritin-reactive antibodies when cultured in ferritin-free medium. The appropriate time was determined (Figure 3). low levels of secreted ferritin-reactive antibodies 1-day prime cultures are the longest-term antigen-free incubation for It took 3-5 days. Additionally, 1-day primed cultures have the lowest concentration was not sensitive to 0.1 μg/ml of ferritin. 2 days ply cells produce detectable ferritin-reactive antibodies after 2-3 days of incubation. 0.0, especially when incubated for 5 days after priming.　1μg/l foot It was sensitive to eritin. 3-day and 4-day prime cultures were Ferritin reactivity detectable after 1 day of incubation in ferritin-free medium produced antibodies.

In general, the longer the priming, the more ferritin will be induced after antigen removal. ferritin-reactive antibodies become detectable quickly. By briming and secreting antibodies in the absence of ferritin for more than 2 days, two Even when using pancreatic cultures, higher background reactivity may be due to antigen. precludes the distinction between specific induction and excessive nonspecific B cell activation. most persistent Ferritin-dependent signals were primed for 3 days and secreted for 2 days. It is observed when brimming is performed for 4 days and secretion is allowed for 1 day. 3 days immunization and detection after brining for 2 days and then secreting for 2 days in the absence of antigen. A state is constructed that can be used for further experiments.

After 3 days of allogeneic culture priming, the production of ferritin-reactive antibodies decreased to 1 Lasted over a week. In the experiment shown in Figure 5, the culture medium was changed and briming Afterwards, the antigen-reactive antibodies produced were measured at intervals of 2-3 days. highest level The production of molecule occurs on days 5-7, after which there is a definite decline in specific and non-specific, respectively. It rained. By days 11-13, antigen-induced responses were barely detectable, and by days 13-16 By then, ferritin reactions were no longer observed. Ferritin for non-specific reactions The rate of induction responses was clearly constant, with a slight peak due to the specificity observed on days 3-5. It was suitable.

Antigen-induced antigen-reactive rgG secret Culture medium was monitored for antigen-induced IgG and IgM secretion. Ferritin inducer The primary ferritin-reactive IgG response is lower than the antigen-reactive IgM class response. frequency and their detection requires EL, which is more sensitive than that used for IgM responses. It required an ISA. Under the culture conditions used, IgG responses occurred in 10-30% of experiments. Admitted. This low frequency of antigen-induced 1gG antibodies is characteristic of the primary response; However, other factors may also be involved. In vitro immunoassay of monoclonal antibodies A polyclonal IgG response was observed when produced after fusion of epinephrine lymphocytes. A viable number of IgG class monoclonals was always obtained even when there were no.

The rgG response is greater with some spleen and spleen mixtures than with others. This was definitely observed, but cell marker analysis of cells obtained from various spleens revealed that IgG secretion from one spleen is supported, but not from another spleen. No difference was observed. Once IgG responses are observed, their expression are consistent within one experiment. As shown in Figure 4, ferritin induction The kinetics of appearance of ferritin-reactive 1gG antibodies is comparable to experiments with IgM responses (para. Rel). However, by days 9-11, no non-specific IgG responses were observed. However, the detection of ferritin-reactive 1gG is completely dependent on exposure to ferritin. there was.

Prolonged response of either IgM or IgG by allogeneic culture for significantly more than 10 days By this time, the cell death rate derived from cytotoxic killing by the same type of cells was significant. It was not recognized or expected because it had increased significantly.

Relative affinity of polyclonal ferritin-reactive antibodies produced under various conditions Analysis of force/binding activity Antigens bound to ELISA plates are removed by exposure to thionanate. quantify the ease of body elution and use it to assess the relative strength of binding of various antibody preparations. I valued it. This method uses ferritin-induced ferritin-reactive antibodies produced in vitro. Non-specific ferritin-reactive immunoglobulin detected in non-immune cultures It was applied to determine whether it is possible to quantitatively distinguish between Solid phase bound ferritin? The ease of elution of these polyclonal antibodies from ferritin-primed cultures and Supernatants from non-immune control cultures were used for evaluation. As shown in Figure 5 , antibodies from primed cultures require higher concentration than supernatants from non-immune cultures for elution. required high levels of thiocyanate. by non-immunized and primed cultures The molar concentration of thiocyanate required to elute 50% of the resulting binding activity ([K SCN code 50) was 2.0M and 2.6M.

Using this method, the binding of ferritin-induced antibodies produced under various culture conditions is possible. The joint characteristics were also evaluated. When you want to produce antibodies with high affinity, you need to Evaluations of relative characteristics unrelated to quality may be misleading. Various culture conditions The ferritin binding strength of the polyclonal antibody mixture produced under thiocyanate When analyzed by elution, a significant dependence of antibody binding strength on immunization conditions was observed. Ta. Furthermore, this dependence is consistent with the ferri This could not be predicted based on the strength of the ion-induced response. of antigen under various conditions. The value of [KSCN]-50 for the two cultures primed in the presence or absence of It is shown in Table 2. Monocultures of cells from responsive spleen B showed ferritin-induced effects. produced lytin-reactive IgM, but [KSCN]-50 values were lower than that of immunized cultures and similar to control cultures. In contrast, in medium containing 10% FCS Co-cultures of spleen A+B primed with ferritin in produced antibodies with significantly higher [KSCN]50 values than those produced by Ta. Differences between immune and control cultures reached peak production of antibodies on days 7-9 However, after 9 days, the amount of antibodies produced was negligible.

Exposure of allogeneic cultures by antigen in the absence of serum for No-20 hours resulted in ELI Levels of ferritin-induced ferritin-reactive antibodies measured by SA are usually increased because the background activity in non-immune cultures is relatively low. . However, the difference in [KSCN co-50 values between immune and control cultures Preserving FCS during initial brimming compared to continuously adding FCS to was not as large or consistent as it would be if it were not present (Second table). The result of this is that various forms of activation are available for the induction of antibodies of desired properties and amounts. This shows the importance of striking a balance between the two.

Effects of non-lymphokine factors on antigen-induced antigen-reactive RG secretion and total Tg secretion Fruit Non-lymphokine factors muramyl dipeptide (MDP) and pokeweed ・Effect of mitogen (PWM) on ferritin-induced ferritin-specific response were tested using both monocultures and homogeneous mixed cultures (Table 3). P.W. M stimulated global Ig secretion but did not increase specific induction by antigen. . MDP also stimulates Ig secretion globally, and in the experiments shown in Table 3, Increased the effects of exposure. However, the specific stimulation by MDP is inconsistent. However, it is commonly observed only under sub-optimal culture conditions. (data not shown). These results further demonstrate that two spleen cultures of the same species provides sufficient stimulation for the induction of antigen-specific responses and also provides additional non-specific The mitogenic stimulus is suppressed or indicates that it is not normally required. Was.

The influence of adherent cells on the in vitro response to horse ferritin. The initial protocol for this includes thawing and then thawing the cells before stimulation with antigen. Includes overnight incubation in large flasks that can be harvested. This process , adherent cells are depleted. By comparing depleted and non-depleted cultures, Non-depleted cultures respond better to horse ferritin than depleted cultures. It was found that (Figure 6). Pile 2 alone did not respond significantly under any conditions. However, spleen 1, which did not respond in depleted cultures, showed limited response to low concentrations of antigen. response and a large response to high levels if adherent cells are not depleted showed that. In mixed cultures of spleens 1 and 2, if adherent cells are not depleted, Decreased reactivity of unprimed cells and increased sensitivity to low levels of antigen . These results suggest that immunization is more effective when performed in the presence of an optimal number of adherent cells. This shows that the antigen can be efficiently presented.

In vitro human immune response to human ferritinResponse to human ferritin also tested to detect human immune responses in vitro to human and foreign antigens. It was decided whether or not to proceed. Contact conditions that favor a sustained response to equine ferritin No response to human ferritin was observed in cultures depleted of adherent cells. Figure C, Figure 7). However, pre-incubating freshly thawed spleen cells overnight If not, the cells will not only respond well to equine ferritin. In addition, it also responded positively to human ferritin (Figures 6D and 7). Depleted culture Prime the feed with human ferritin for 2 days, then a 5 day secretion period instead of 3 days. In this case, a small response to human ferritin was observed after incubation between . Reconstitution of the depleted culture empty with adherent cells during briming results in a secretion after 2 days. A response to human ferritin was also observed here.

Human ferritin-induced IgG responses by non-depleted cultures were also observed in some experiments. (Figure 7A). Specific IgG responses were relatively low, but IgM responses were comparable .

Development of in vitro oral antigen briming ferritin using foreign immunoglobulin Evaluate whether the in vitro immunization conditions developed can be applied to other protein antigens. Therefore, the reactivity to briming with murine monoclonal IgG antibody was Tested under conditions supporting response to equine and human ferritin. two things We detected antigen-induced antigen-reactive 1 gM antibodies in response to the clonal preparation. Figure 8). In general, assays for monoclonal murine immunoglobulin bank ground reactivity of non-immunized supernatants than assayed for ferritin. was low. Polyclonal antigen-dependent 1gG reactivity using these antigens was not observed in lymphocytes primed with murine monoclonal antibodies. Fusion produces antigen-specific IgG and 1gM class monoclonal antibodies It was done.

cell aggregation 10-20 hours after the start of briming, no visible spots are visible on the bottom of the culture colander. Many cell aggregates appeared. When dissociated by vigorous suction, aggregates remain for several hours. started playing. This size depends on the presence, concentration and nature of the antigen, cell density, and depended on the presence of allogeneic lymphoid cells. Antigen-free cultures are invisible to the naked eye. Aggregates were formed to the extent that they could be detected immediately. Exposure to horse ferritin causes human Larger aggregates than erithin were induced. Low molecular weight, uncomplicated mixed antisense substances, e.g. murine immunoglobulins, as seen by unprimed cultures. Also induced slightly larger aggregates. Also, in allogeneic cultures, compared to monocultures, Large aggregates were induced. Cell aggregation was also affected by the presence of Fe2 . 15-20 hours during briming, unless antigen-dependent responses are reduced. Addition of Fe2 can be delayed, but macroscopic aggregates are visible after addition of Fe2. It did not begin to form until several hours had passed.

Cell marker analysis Cell marker analysis shows that human pancreatic cells, when cultured under appropriate conditions, have limited It has been shown that it can be differentiated in different stages. After the start of briming, O13, 5, 7 and ] Pancreas B+D was used on day 0 and the following cell surface markers were analyzed: IgG.

IgM, Bl, PCA-1, T3, T4, T8, and IL-2 receptors (CD 25). Pancreas B and D have 17% less surface 1 gM than pancreas B. There were no significant differences other than the presence of carrier cells. allogeneic culture The changes as a function of time observed using cell-bearing surface IgG, IgM and This was about a 50% decrease in the percentage of B1 and B1 (Table 4). T8 positive cells The percentage increased slightly, while T3-positive cells remained relatively unchanged and T4-positive The number of cells decreased slightly. Significant time-dependent changes occur in IL-2 receptor-bearing cells. The percentage of cells sharply increased about 10 times starting 3 days before the start, and then increased by 5 times. The percentage of PCA-1 positive cells approximately doubled between days 1 and 7. , and these were similar to the decrease in B1-bearing cells. Changes on the cell surface are ferritic was not significantly affected by In monocultures, a similar but weaker Significant cell surface changes were observed.

Monoclonal antibody raised against horse ferritin in homogeneous culture of human lymphocytes Cultures were primed with horse pancreatic ferritin for 1.2 or 5 days and then fused (first (Table 1). Two different but identical fusions, 4 and 5, in the same 5-day prime culture The reproducibility of fusion-fusion was evaluated. Additional sets of cells for 1 day maintained in culture but not exposed to horse ferritin (fusion 1). ). Fusion frequency, rg secretion, production of antigen-reactive monoclonal antibodies, and antigen specificity Production of specific monoclonal antibodies was monitored for each fusion.

Fusion frequency is similar for primed cells with 1 to 5 days of ferritin. (35'-50 clones/million lymphocytes), but non-immune cells are similar to primed cells. They also fused at a low frequency (17 clones/million lymphocytes). Non-immune cells are low They appear to be highly activated and are visible to the naked eye, which are smaller than primed cells. form aggregates. The number of cells in the ferritin-primed cultures was the same as that of the control cultures. It did not increase significantly compared to the actual product.

A high percentage of hybridoma clones produced in these fusions secreted immunoglobulins (20-60%). With the fusion combinations listed in Table 1 There were 5-10 times more IgG-secreting clones than IgM-secreting clones.

In our studies, the majority of fusions have approximately equal numbers of IgG and IgM secretors. (See Tables 2 and 3).

In general, the induction of ferritin-specific and ferritin-reactive monoclonal antibodies The conduction patterns were similar (ferritin-reactive monoclonal antibody and ferritin (See Figure 9 comparing binding specificity with specific monoclonal antibodies) . aπ to 7erithin of IB only was compared to that produced from non-immune control cultures. Percentage of ferritin-reactive or ferritin-specific hybridomas compared to It did not significantly increase the number of bullshit. A relatively large number of ferritin-reactive hybrids was produced from cells immunized for 1 day, but the frequency of fusion increased. Briming 2 After days, the percentage of ferritin-reactive clones increased from 5-6% to 16%. rose. After 5 days, the level had dropped to 10-12%, but in cells cultured for 1 day. The background observed in the cells was significantly higher as a function of the background. 8 or 15 days Cells exposed to ferritin for the same length of time fused with a similarly high frequency. but, Other than the 93 tested, all were stable ferritin-reactive monoclonal antibodies. did not secrete.

All antibody-secreting clones that initially reacted with ferritin by ELISA was further tested for ferritin specificity. The first identified poly(monochrome) Although the null antibody cross-reacted with other proteins (Figure 9), the present inventors condition or antigen induces a higher percentage of specific antibodies than others We monitored indicators to determine whether or not. Such culture conditions have not been identified. However, alloantigens elicited a higher percentage of specific antibodies than equine ferritin. (Table 8).

In a series of fusions similar to monoclonal antibodies produced against human ferritin, Lymphocytes immunized with human ferritin instead of horse ferritin (Table 6). In fusion 1-3, lymphocytes maintained in culture for 2 days were used. Fusion 1 uses lymphocytes that are intentionally not exposed to ferritin and is Cases 2 and 3 were primed with 0.25 μg kl ferritin. Fusion 2 and 3 are the same were maintained separately to assess fusion-fusion variation. Fusion 5 and 6 is 2.5 μg/xl human ferritin instead of 0.25 μg/xl human ferritin. Brimming was carried out on the 4th and 6th day after the start of briming using litin, respectively.

The fusion frequency of cells primed for 2 days with 0.25 μg ltanR human ferritin was The fusion frequency was higher than the fusion frequency of non-immune cells, which is the target, but the fusion frequency was higher than that of non-immune cells. It was not as high as in cells immunized with ritin. In addition, the cultures were Cell aggregates were also smaller when primed with horse ferritin than when primed with horse ferritin. won. Cells immunized for 4 or 6 days with 10 times higher levels of human ferritin showed 0 ．． fused at a frequency comparable to that of cultures primed with 25 μg/ll horse ferritin. . Fusion frequency of equine ferritin-primed cells is influenced by briming time. Fusions 4 and 5, which have a higher frequency than Fusions 2 and 3, depend on the human fuse used. This is thought to be due to the relatively high concentration of erritin, but the fusion-fusion Fluctuations cannot be excluded.

These fusions are produced from lymphoid cells maintained in culture for 2-4 days. 30-45% of hybrids secreted immunoglobulin. Ferritin is produced did not affect the number of Ig-secreting hybrids produced. However, fusion 4 and 5, cells primed for 6 days are much more active than cells primed for 4 days. It was found to produce significantly fewer secretory clones (44% versus 8%).

This result demonstrates the low level of fusion in cells immunized with horse ferritin for 8 and 15 days. Similar to productivity. According to cell marker analysis, the percentage of B1+ cells was The percentage of plasmatic (PCA-1+) cells increased 2-fold over 5 and 7 days. .

These results indicate that Ig-secreting hybrids are produced before, but not after, differentiation into plasma cells. that it appears to be produced from the B cells that are fused, and that most productive fusions indicates that it is obtained from cells that have been immunized for a period of at least 1 day but not at least 5 days. Ru.

Monoclonal antibodies produced against murine monoclonal immunoglobulin Human anti-idiotypic monoclonal antibody against Zumi monoclonal antibody To produce human in vitro immune responses to murine monoclonal antibodies, Tested. Allogeneic cultures of human lymphocytes were incubated with 2 μg/ml murine monoclonal antibody. for 2.3 or 4 days (Table 7, Fusions 2.3.4 and 5). With fusion 1 The cells used were cultured for 2 days but were not primed with specific antigen. fusion 3 and 4, the fused 4 cells were added to 1.5 million cells instead of the standard 3 million cells/a. The cells were the same except that they were cultured in /Iris. Similar fusion frequencies occur in each immune system. It was observed that Unprimed cells also have low fusogenicity (fuso genic). 63% of identified antigen-reactive clones are antigen-specific It was a target.

92% of antigen-reactive 1gG clones are antigen-specific, whereas IgM antigen-reactive clones are antigen-specific. The sample was only 50% specific. This result supports the expected specificity of IgG antibodies. This is consistent with an increase in sexuality.

Fusion summary Equine ferritin, human ferritin, or murine monoclonal IgG immunoglobules The results of fusion of lymphocytes immunized with either phosphorus are summarized in Table 8.

9 of the hybrids produced from cells immunized with either the foreign protein -10% were antigen-reactive. Clocks obtained from cells immunized with human ferritin Only 3% of the loans were antigen-reactive, a result that reflects the relatively low abundance of human proteins. This could be expected from its low immunogenicity. Primed with equine ferritin 29% of antigen-reactive hybrids produced from cells with high antigen reactivity shows. Either human ferritin (65%) or murine IgG molecules (63%) The antigen specificity of monoclonal antibodies produced from cells primed with , which was very high compared to the number of antigen-reactive hybridomas. human ferritin mata The apparent lymphocyte activation by horse ferritin was greater than that by murine IgG. Sexualization is associated with increased production of cross-reactive antibodies from equine ferritin-primed cells. It may be related. A comparison of antigen-specific IgM and IgG production revealed that the highest A high ratio of IgM:IgG antibodies was obtained by immunization with homologous human ferritin. Rukoto has been shown. Immunization with foreign antigens has a low effect on the production of IgM class antibodies. The production of ferritin-reactive monoclonal antibodies showed a tendency to Breed development was initially monitored. Hybrid clones first become secretory If identified as a ferritin-reactive antibody, identify it as a minimum number of 3 passages. was expanded to a 48-well plate and then to a 24-well plate. Ferici during each passage The presence of reactive monoclonal antibodies was monitored. Ferritin-reactive antibody content Approximately 50% of clones that are initially positive for secretion show their productivity within three passages. (Table 9). Among hybrids that are stable over three passages , the hybrids used for further studies, and the data shown in Tables 5-7. selected Nozobrid to use to edit the data. These selected clones Most of the conditions remained stable for at least several months. Therefore, unstable Hybrids could be eliminated early in this operation and remaining hybrids were retained. Has a high probability (>90%) of antibody production.

Quantification of anti-ferritin IgG secretion Final fully grown 24-well secreting ferritin-reactive IgG antibody. Antibody concentration in supernatants obtained from cultures was determined by quantitative ELISA. this The level of immunoglobulin secreted under these conditions was 1-50 μg 7 ml. (Table 10). Two relatively high affinity anti-ferritin antibodies were added to a spinner flask. The scale was expanded to 0. 5 2. Obtained the production level of OAg/mjl' . These hybrid cells grow more quickly in spinner cultures than in flasks. They often grew, but antibody production was slower. This preliminary result shows that the core Actor (coreactor) [5ynbiotics, ■ncorpor ated, San Diego] than in spinner culture. It has been shown to lead to 5-10 times higher antibody concentrations.

Characterization of ferritin-specific monoclonal antibodies using protein G Human monochromatography of IgG class is achieved by one step of tea chromatography. The null antibody was purified. The weight was determined by 5DS-polyacrylamide gel electrophoresis. Light chain and light chain bands were observed. Anti-human heavy and light chains and anti-mouse heavy chains Western plotting using light chain reagents and light chain reagents production of human lymphocytes fused with human x mouse atypical myeloma, which is a local antibody. The substance was confirmed to be of human origin and not of murine origin.

By light chain analysis of 16 1 gM class antibodies, 6 with lambda light chains were found. , 9 with kappa light chain, and 1 with lambda + kappa were recognized, respectively. It was done. Analysis of 95 IgG class antibodies revealed 61 with lambda light chain; 31 with kappa light chain and 3 with lambda + kappa light chain were observed. .

Hybrids do not subclone at this stage, so some of the cultures are monoclonal. It wasn't Ronal.

Using two highly specific subcloned and purified anti-ferritin IgG antibodies, Affinity measurements by competitive ELISA revealed solutions in the range of 11-2X10(-8). The separation constant was found (Figure 10). In a competitive assay using horse ferritin, Monoclonal antibody 14-2-2-59 was raised against ferritin, and human Antibody 2l-IB-9 was created against erritin. Test for ferritin specificity Among the 1 gM supernatants tested, 13-5-3-18 was the most specific and the highest It was reactive. This monoclonal antibody was tested for reactivity with ferritin-containing tissues. and tested by immunohistological analysis. Figure 11 shows the results used as a negative control. compared to the reactivity of a preparation of polyclonal human IgM antibodies used in human liver tissue. It shows 1.3-5-3-1.8 reactivity with. produced against different antigens Human IgM monochrome prepared by the same procedure as 13-5-3-18 except for A second control consisting of a null antibody was also negative.

C Consideration This study is aimed at conditions that favor primary immune responses in vitro.

The results were obtained without depleting Ts cells and without regulating the T2B cell ratio. The result is that if lymphokines are produced by allogeneic stimulation, Th cells 15- 25%, B cells 40-60% (Table 4), and the presence of adherent cells above minimal levels. In the present study, initial levels of 30-40% Ts cells support antigen priming. It shows. The number of these cells has a BAT cell ratio of approximately 1=1. Adhesion Adding factors secreted from cells or adherent cells such as IL-2 may be another The requirements were different. Protocols using pancreatic or tonsil preparations No special addition of any factors secreted from cells or monocytes is required. ferritin, This is especially true when immunization with human ferritin retains adherent cells. The findings of the present invention indicate that these cells and/or their products contain T cell-dependent proteins. aids in effective priming by antigen (equine ferritin) or This shows that this is a requirement for human ferritin.

Minimal levels of lymphokine stimulation are necessary for successful in vitro priming. to obtain suitable stimulation for most tissue preparations in a variety of ways. It seems possible. This method of producing lymphokines is optimal at the right time. It may not be as important as achieving the level. Protocol of the invention For pancreatic tissue prepared according to the standard, syngeneic cultures without supplements are suboptimal condition for some people (suboptiIIlal), and other things It is completely ineffective. However, allogeneic cultures of the two pancreas It appeared to be sufficient for all combinations of pancreas, and cultures of two or more Excess non-specificity, similar to the effect observed when WM is included in immune cultures. It seemed to bring about physical stimulation.

Other potentially important factors that aid antigen priming by lymphokines include timing of cellular exposure of the factor. According to the cell marker test (Table 4), Many cells differentiate in culture as the response progresses. details in a certain state The response of cells to lymphokines is significantly different from that during or after differentiation. It seems that In this experiment, the exogenously produced lymphokines, or endogenous production by nonspecific mitogenic stimulation of T cells. Adding lymphokines was less effective than simultaneous co-culture. these The results demonstrate that lymphokine exposure generated by cognate stimulation during priming Levels and kinetics that most closely mimic in vivo immune responses It shows.

In 10-30% of our experiments in which cells were primed with ferritin, ELI SA analysis of post-epidemic culture supernatant revealed a low but detectable IgG response. The answer has been found, but the researchers have found that the cells have been primed with murine immunoglobulin proteins. No response was found in the experiment. However, immunization with either antigen By fusion of lymphocytes, antigen-specific IgG and IgM monoclonal antibodies are generated. body was produced. The largest number of antigen-reactive IgG monoclonal antibodies are polyclonal antibodies. Equine ferritin, where local antigen-induced IgG responses were most easily detected before fusion. was induced by fusion of lymphocytes immunized with In the experiment observed here The initial appearance of the antigen-induced IgG response in 2000 was surprising.

Polyclonal IgM antibodies produced after antigen priming under appropriate conditions body (Table 2), compared with polyclonal antibodies produced from non-immune cultures. It bound significantly better to Chin. Antigens less complex than ferritin are non-specific It was shown that the level of binding was low. However, the results of the thiocyanate analysis Cultures primed under appropriate conditions for ferritin-induced maturation of ferritin immune responses It was a strong indication of what happens in things. The observed maturation of the 1 gM response is likely due to This is mainly due to the specific binding, activation, and alignment of ferritin to naive B cells. due to their subsequent maturation into antibody-secreting plasma cells and/or memory cells. It will come. In vitro immunization with antigen-specific IgG monoclonal antibodies Production from fusion of lymphoid cells results in IgG-secreting hybrids being cross-reactive. antigen-induced class switch if not generated from a hybridoma clone of and affinity maturation can occur in vitro.

These results demonstrate that in vitro activated human lymphocytes are effective in mice: human atypical myeloma and A heterotypic hybrid with a high percentage of IgG and IgM secretion. This shows that the Maseru line can be obtained.

The present invention is based on Carroll et al., J. Immuno, Methods 89. 61 (1986) and the 6H6/B5 cell line for 1-6 days. This figure describes in detail the fusion with human pancreatic cells cultured in vitro. average The average fusion frequency is 35 and the range of 15 distinct fusions is 17-50 hybrids. wells containing cells/hypermal lymphocytes. In vitro cultures of lymphocytes are Especially when combined with cognate stimuli, leading to activation of a highly fusogenic state. immunity What was observed for the cultures was a high fusion frequency and large The cell aggregates (Tables 5-7) indicate that the efficiency of fusion is lower than that of lymphoid cell activation. This shows that it can be influenced by the degree and nature.

The level of 1g production is exactly uniform for each hybrid, but for 24 The range of hybrids varied by a factor of 50. Production by certain cell lines is Sufficient for production in culture of other human hybrid and murine monoclonal antibodies It was comparable. 1g production is unstable in approximately 50% of hybrids produced. However, the large number of obtained products allows for the early elimination of unstable hybrids and facilitates separation. This left many options for evaluation of secretion levels, specificity and affinity. K6H6/ Preliminary karyotype analysis using G-linked B5 fusion partner revealed an average chromosome number of 91 ( range = 77-97), surprisingly few identifiable mouse or human chromosomes, and Chimeric mouse: Many structures were shown that appeared to be human chromosomes. this inheritance The fusion between the child mother and human lymphocytes is more likely to occur than the fusion with B cells of the murine (W tooth and neck) lineage. can also retain human chromosomes well, but the first secretion in 50% of hybrids Phase instability can be understood from the perspective of the complexity and abnormality of the 6H6/B5 partner karyotype. I was able to

The above results further demonstrate that many antigen-specific IgG-secreting hybrids It has been shown that it can also be prepared by fusion of lymphocytes immunized in vitro. activity lymphocytes can give rise to IgM-secreting hybrids or clusters. undergo a plasma switch to become an IgG-secreting plasma cell as part of the primary response. Therefore, the IgG-secreting clones observed in these experiments are It appears to have developed from a vitro-induced response. Therefore, during these developmental periods of any type of cell at an as-yet-undefined time point or at multiple time points. By fusion, IgM- or IgG-secreting hybrid cells can be produced. oh Possibly due to allogeneic stimulation induced by co-culture of pancreatic cells from different individuals. Las switching is better than that previously observed in vitro. Support may have been provided for.

Another interpretation for recovering antigen-specific IgG class antibodies from fusions includes this The possibility that such hybrids are induced from secondary stimulation of memory cells is involved. . The rg receptors of specific memory cells in pancreatic cell preparations have been previously encountered. recognizes a determinant on the priming antigen that is sufficiently similar to the determinant that leads to its activation. can be done.

The nature of the antigen used for briming is dependent on hybrid induction/production of antigen reactivity. This affected the percentage of all hybrids produced. is a foreign protein Horse ferritin and mouse IgG have 9-10% antigen-reactive clones/total hives while allogeneic human ferritin induces 3-4% ferritin-reactive clot. (Table 8). Response to human ferritin Although the response is lower than for foreign proteins, such responses are highly conserved. monochromatic for proteins and even self-proteins by in vitro immunization. This shows that it is possible to produce natural antibodies. against human ferritin The affinity of 2l-IB-9 produced for horse ferritin and the affinity of 2l-IB-9 produced for horse ferritin Similarity to the affinity of one of the best antibodies, 14-2-2-59, for self- The in vitro antibodies produced have similar properties to antibodies produced against foreign proteins. This indicates that it can have a sexual nature.

Selected IgG antibodies were affinity purified by protein G. Ferri Immunoplot analysis for extracts of ferritin- and ferritin-containing tissues showed that 10(7) -10(8)/l1ol of reactive, hybridic [Hy A high affinity murine anti-inflammatory manufactured by Britech, Inc. Reactivity pattern similar to that observed using ferritin monoclonal antibodies showed. Competing antigen binding strength of two purified anti-ferritin IgG antibodies Direct analysis by LISA (Figure 11) further shows that the range is 10 (7) 10 (8)/mol.

The induction of IgG monoclonal antibodies with this strong apparent affinity Compatible with a specific first-order response combined with switching, but perhaps a widespread It is unlikely to be associated with somatic mutations. Alternatively, antigen-reactive IgG Secretory hybrids may be the product of a cross-reactive memory response, in which the next antigen The affinity for cross-reactive inducing antigens is usually low (although the affinity for cross-reactive inducing antigens is usually low). is not low.

Table 1 Ferritin response of primed syngeneic and allogeneic lymphocyte cultures in vitro. Analysis of 1 gM antibody secretion: Comparison of observed and predicted responses Ferritin-dependent response ( △OD*s. )Tile Visceral Actual value Calculated value A10B, 34. 10 A10C, 33, 25 A+D, 13. 08 B10C, 28, 10 B+D, 05. 03 C1D, 18. 07 A ten B + C, 21, 12 B10C+D, 16. 07 A+B+D, 15. 10 A+B+C+D, 19, 09 Human pancreatic cells were prepared as described previously without depleting adherent cells, and lμg hll horse pancreas was cultured in the presence or absence of ferritin. Actual values are for control and Differences in 0DJQO values between ferritin primed cultures are shown. into a mixed culture The calculated value for was derived from the summation button of the appropriate function of the response of the isogenic culture (i.e. ) ND = not detected.

Table 2 Parents in evaluating various culture conditions for in vitro antigen priming Using force/avidity calculations Pancreas serum time secretion control [KSCN] 3゜fMl immunity (%) (days) actual Example 1 Example 2 Example 1 Example 2B 10% 3-5 2.0 2.1 1.8 2.3A+810% 3-5 2.0 2.0 2.6 2.65-7 2.6 '2.6 2.9' 2.87-9 2.2 1.7 2.9 29 9-11. 1.5 1.2' 2.2], 4A+B 0% 3-5 2 ．． 1 2.2 2.3 2.35-7 1.8 2.4 2.9 2.57-9 2.1’ 2.4 2.7 2.49-11 1. ．． 5 1.8 1.5 2.0 Human pancreatic cells were prepared as described above without depleting adherent cells. . Two syngeneic or 1:1 co-cultures of pancreas were treated with 0 or 1 μg kl horse spleen ferritin. I primed it for 3 days. Remove ferritin by washing and remove supernatant at specified time points. was collected. The ferritin reactivity of the secreted immunoglobulin was measured by ELISA. The relative affinity/binding results were calculated as described above. show The number reduced the amount of antibody binding to ferritin in the assay plate by 50%. ([KSCN]-50) represents the molar concentration of KSCN required to

Table 3 (A,) total 1 g of W and (B) ferritin induction of ferritin-reactive antibodies. non-specific mitogenic effects Pancreas 1 Pancreas 2 Pancreas 1 & 2 A4 μg/ml total immunoglobulin Not added 2.4±0.4 0.6±0.1. 12.3±1.5+PWM 3 0.8±3.7 2.6±1.0 20.1±0.8+MDP 5.8 Sat 1.4 4.0±1.0 21.5±1.8B, ferritin-induced ferritin reactivity 0 Das.

Not added 0 0 (1,28 ± 0.14 + PWM OOO, 13 ± 0,08 +MDP 0.29±0.8 0.04±0.02 0.46±0.09 Table 4 Cell marker analysis of human pancreatic cells as a function of culture time TgG 49 17 16 15 14 9 12 If gTgM 30 22 20 17 15 8 10 7 10Bl 51 44 41 37 35 17 15 11 12PCA-1, 141413271531243323T3 37 40 42 45 42 46 36 46 40T4 20 17 18 17 1 6 12 9 20 16T8 34 38 40 47 46 43 44 49 46 Human pancreas was prepared as described above without depleting adherent cells, Cultured.

Similar experiments for each surface marker were performed on days 3.5.7 and 10; Shown in Table 1 or Table 2. Cultures were harvested and analyzed as previously described.

Table 5 Hybrids from human lymphocytes immunized in vitro with horse pancreatic ferritin. Production of domaclones Culture time (days) 11255 Number of wells containing hybrids 86 249 230 177 1.99 Fusion frequency 17 50 46 35 40 Secretory body IgM 1159 5/173 14/137 7/88 6/90 (2%) 3%) (10%) (8%) (7%) IgG 12159 49/173 80/ 137 46/88 48/90 (20%) (28%) (58%) (52%) ( 53%) Reconfirmed ferritin reactivity IgM 1 0 4 6 5 TgG 4 10 33], 6 14 Ferritin specificity (vs. β-galactosycin) Dase) TgM 0 0 1 1 0 IgG 2 4 11 5 3 Immunization, fusion and hybridoma screening were performed as previously described.

Lymphocytes were incubated with either 0 or 0.25 μg kl horse spleen ferritin for 1 day. prime for 2 days (fusion 3) or 5 days (fusion 4 and 5). Ta. Number of hybrids secreting IgM or IgG tested for IgM or IgG secretion It is expressed as the number of secretors per number of clones obtained.

Table 6 Hybrids from human lymphocytes immunized in vitro with human ferritin Macrone production Culture time (days) 22246 Number of wells containing hybrids 31 49 64 266 268 IgM 3 4 12 62 6 (10%) (8%) (9%) (23%) (2%) IgG 11 12 19 5 5 15 (35%) (24%) (30%) (21%) (6%) Ferritin reactivity rgM 0 2 4 8 1 1gG 1 1 1 3 2 Ferritin specificity (vs. β-galactosidase and BSA) IgM-1181 Immunization, fusion and hybridoma screening were performed as previously described.

Specified 0, 25 or 2.5 μg/proverb! Lymphocytes in the presence of human ferritin cells at 3X10' cells/ml for 2 days (fusion 1-3), 4 days (fusion 4), or 6 Cultured for 5 days (fusion 5).

Table 7 from human lymphocytes immunized in vitro with murine monoclonal IgG. Production of hybridoma clones Fusion 12345 Culture time (days) 2 2 3 3 4 Antigen (μg/ml) 0 2 2 2 2 Cell density 3 3 3 1.5 3 (XIO’/+1) Number of wells containing hybrids 27 107 116 98 1.26 Fusion frequency 7 2 7 29 25 32 Antigen reactivity IgM 10 12 5 2 IgG O4810 antigen specificity IgM 1 5 4 4 1 1gG O4710 Immunization, fusion, and hybridoma screening were performed as previously described.

Table 8 Immunization with horse ferritin, human ferritin, or murine monoclonal IgG. Comparative antigen production of hybrids from infected lymphocytes Chtonic reactivity Antigen specificity Antigen -IgM IgG horse 941 93 27 2 25 Ferritin Human 678 23 15 11 4 Ferritin Mouse 474 43 27 15 12 monotalonal IgG Immunization, fusion, and hybridoma screening were performed as previously described.

Lymphocytes were cultured and immunized as described in Tables 5-7.

Table 9 Stable immunogen for immunoglobulin production from human hybridomas Early positive clones Reconfirmation (passage 3X) Horse ferritin 192 92 (48%) Human ferritin Chin 40 24 (60%) Immunization, fusion and hybridoma screening was performed as described above.

Table 10 Quantification of immunoglobulin production from IgG-secreting human hybridoma clones 2 42±9 1.65 8 20± 5.6 Hybridoma screening and quantitative assay were performed as previously described.

(Niji The foregoing description details specific methods that can be used to carry out the invention. . Antigen-specific, high-affinity monoclonal antibodies are used, characterized, and First use such a specific method to prepare and release special model manipulations By using this same information and description of its nature, This information can be extended to other related methods for producing monoclonal antibodies. Those skilled in the art will know how to create alternative and reliable methods to It will be fully understood. Therefore, the above details may or may not appear in the text. However, the scope of the invention should not be construed as limiting the scope of the invention as a whole, but rather , should be understood to be determined solely by the legal construction of the following claims.

1″″! N″″! q~zu hai~- Shipodai hai~zuO) ψ −r ~ 9 History 1ゞ ~ 9==--Ro 6 6 Ro Ro Immunity 1st booth h 2nd boost FIG.-14 Immunity 1st boost 2nd boost FIG. -15 Summary book The present invention provides advantages in monoclonal antibody production, and more particularly in hybrid antibody production. This provides advantages in culture methods prior to doma formation. in particular, By retaining lymph fragments in a culture system, a long-lived IgG-secreting culture is created. Nutrients are preferentially obtained.

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Claims (33)

[Claims] 1. by specific antigen under culture conditions that do not require the addition of growth factors or other factors. immunized lymphoid tissue derivatized human cells containing the requisite number of autologous accessory cells an in vitro immune culture containing at least about 5 x 107 Human monoclonal Ig with affinity for specific antigen of liter/mole said culture in which an effective number of G antibodies are derived from said lymphocytes. 2. The culture according to claim 1, wherein the lymph tissue is human spleen. 3. The culture according to claim 1, wherein the lymphocytes are human spleen cells. 4. The antibody was isolated from in vitro immunization of lymphocytes with large numbers of aggregates visible to the naked eye. 2. The culture according to claim 1, wherein the culture is produced. 5. 5. The culture according to claim 4, wherein the lymphocytes are spleen cells. 6. 5. The culture according to claim 4, which is a co-culture of the same species. 7. 5. The culture according to claim 4, containing many splenoid bodies. 8. A stable strain producing the antigen-specific human monoclonal antibody according to claim 1. continuous cell line. 9. A claim that the lymphatic cell is immortalized by fusion with a partner of the immortal fusion cell Stable continuous cell line according to item 8. 10. A recombinant antibody operably containing DNA encoding the variable region of the antibody. 9. A stable continuous cell line according to claim 8, produced by means of. 11. The nucleic acid encoding the variable region of the antibody is applied to polymerase chain reaction. 9. A stable continuous cell line according to claim 8, which is thus amplified and fixed. 12. The cell partner of the immortal fusion is an immortalized lymphoid cell, a myeloma, or a heterogeneous hybrid. 10. The hybridoma according to claim 9, which is selected from among the cell lines. 13. antigen-specific with an antigen affinity of at least about 5 x 107 liters/mole The hybridoma according to claim 9, which is capable of producing a human monoclonal antibody. 14. The hybrid according to claim 9, wherein the monoclonal antibody is an IgG antibody. Ma. 15. The plant according to claim 9, which secretes human monoclonal antibodies against human antigens. Eve Dorma. 16. according to claim 9, which secretes human monoclonal antibodies against non-human antigens. hybridoma. 17. The cell according to any one of claims 8, 9, 10, 11, 12, 13 or 14. Human monoclonal antibodies produced from this line. 18. 18. The human monoclonal antibody according to claim 17, which is specific for a human antigen. 19. The human monoclonal IgG antibody according to claim 18. 20. Immunization by specific antigens under conditions that do not require the addition of growth factors or other factors. lymphoid tissue-derived human lymph with a requisite number of autologous accessory cells cells having an antigen affinity of at least about 5 x 107 liters/mole; The effective number of human monoclonal IgG antibodies derived from the lymphocytes A human model derived from lymph cells, characterized in that the lymph cells are cultured in vitro. It is a means for producing a human monoclonal antibody and recovering the human monoclonal antibody. A method for producing antigen-specific human monoclonal antibodies. 21. 21. The method of claim 20, wherein the cultures are homogeneous co-cultures. 22. (a) using hypotonic cytolysis of red blood cells; (b) accessory cells are lost; (c) Minimize room temperature incubation to avoid small fragments are kept in the final cell suspension before freezing, and (d) including or adding small pieces of lymphoid tissue to the cell suspension; 21. The method according to claim 20, wherein the human lymphocytes are prepared and cultured by. 23. characterized in that the means fuses the lymphoid cell with an immortal fusion cell partner 21. The method according to claim 20. 24. The immortal fused cell partner may be immortalized myeloma, plasmacytoma, atypical myeloma, and atypical myeloma. 24. The method of claim 23, wherein the method is selected from among type hybridoma cell lines. 25. at least about 5 x 107 when produced by the method of claim 22. Human antigen-specific human monoclonal with antigen affinity of liter/mole antibody. 26. The antibody according to claim 25, which is an IgG antibody. 27. Claim 25, wherein the compound is combined with a substance capable of regulating cell growth. Antibodies listed above. 28. 28. The antibody of claim 27, wherein the substance is yttrium-90. 29. 26. The antibody of claim 25 conjugated to a reporter moiety. 30. 30. The antibody of claim 29, wherein said moiety is indium-111. 31. The antibody of claim 27 in an individual suffering from a cancer, bacterial or viral disease. A method for treating said disease, characterized in that said disease is administered. 32. administering to the individual the antibody according to claim 29; A method for testing the presence of the disease in patients. 33. A human monoclonal anti-idiotype of the antibody according to claim 25. body.