JPH09506262A - Method for producing specific antibody - Google Patents

Abstract

  (57) [Summary] The present invention relates to an antibody specific to an immunorecessive epitope and a method for producing a nucleic acid encoding the antibody. The subject method generally comprises the steps of obtaining a heterogeneous display library of antibody variable regions and then selecting from the library the variable regions of the antibody having the desired binding specificity for an immunorecessive epitope. The antibody variable regions used to obtain the display library are cloned from the antibody repertoire derived from immune tolerance.

Description

Detailed Description of the Invention                           Method for producing specific antibody                                 BACKGROUND OF THE INVENTION   In antibody-producing animals, such as mammals, antibodies are synthesized in plasma cells, Then, it is finally secreted into body fluid by a certain type of differentiated B lymphocyte. animal Exposing (ie, by immunizing) to a foreign molecule generally involves Produced numerous plasma cell clones that gave rise to heterogeneous mixtures of antibodies in blood and other body fluids. Make Blood from immunized animals is collected, allowed to clot, and clots removed for immunization Serum containing antibodies raised against it can be retained. Contains polyclonal antibody The remaining liquid or serum is called antiserum. However, many such antisera are It contains many different types of antibodies specific for different antigens. Overimmunized More than one-tenth of circulating antibody was used to immunize animals, even in It is rarely specific to a particular immunogen. Use of these antibody mixed populations While useful in many contexts, they have many different problems in immunochemical methods. Problem. For example, such antisera are generally common to immunogens and immunogens. For use in discriminating between closely related molecules that share many determinants Not enough.   Due to its high specificity for certain antigens, monoclonal antibodies (Kohl (Kohl er) and Milstein (1975) Nature 25 6: 495) has emerged as an important technique, with scientifically and commercially demanding consequences. It brought about an operative breakthrough. Monoclonal antibodies (Mabs) Generally, a single antibody-secreting cell (eg, lymphocyte) is isolated from an animal and Fused with myeloma cells (or other immortal cells) to produce hybrid cells (hybrid cells). Denomas) and then selected hybridoma cells in vivo. Or by culturing in vitro to obtain antibodies of similar structure and specificity. Manufactured. Since antibody-secreting cell lines are immortal, are antibody characteristics batch-wise? Can be re-produced from batch to batch. There are three characteristics that are useful for monoclonal antibodies. Their binding specificities, their homogeneity, and their production of virtually unlimited It comes from ability.   Monoclonal antibody production is better for specific antigens than polyclonal Causes the production of highly specific antibodies, nevertheless There are many limitations associated with the method and the antibodies produced thereby. For example, things A key aspect in the isolation of clonal antibodies is how many different specificities they have. Can actually establish antibody-producing hybridoma cells that respond to immunization with specific antigens Sample to obtain antibodies with many specific characteristics. It is related to the theoretical need to be pulled. For example, a mouse The number of different antibody specificities expressed at one time by lymphocytes in the immune system is about 10⁷Individually Represents only a small part of the repertoire that is considered to have potential and specificity Just do it.   Immunization can provide for enriching B cells that produce the desired antibody. But However, even though these methods are used, typical methods for isolating antibody-producing B cells Protocols generally produce less than 500 antibodies per immunized animal It enables the sampling of hybridoma cells. Therefore, monochrome The traditional method for the production of monoclonal antibodies is monoclonal to immunodominant molecules. Statistically favorable for antibody production, rare or less immunodominant It is difficult to isolate an antibody specific to a specific epitope. In many cases pure anti- The fact that prima cannot be used as an immunogen, especially in the case of cell surface antigens This can make this problem even more difficult. Immunization with untreated cells is particularly different. With regard to type immunity, it often induces the production of antibodies to unrelated epitopes. Wake up. Increases production of monoclonal antibodies against rare "immunorecessive" antigens Tolerance has been used to do this. Newborn tolerization and chemoimmunosuppression , Explosive expansion of B cell clones in response to "background" antigen signals Of B cells that are commonly used to suppress The population is enriched. However, immunosuppressive efficiency is often unacceptable Also In general, or in the case of cyclophosphamide immunosuppression, Only a few antibody-producing hybridoma cells were produced per animal, and To reduce the chance of isolation of a pitope-specific monoclonal antibody, The practical application of suppressive immunization methods can be very difficult.                                 Summary of the Invention   The present invention provides an antibody specific to an immunorecessive epitope, and a nucleus encoding the antibody. Provided is a method for producing an acid. The subject method generally comprises a heterogeneous array of antibody variable regions. An efficient display library and then desired for immunorecessive epitopes The process consists of selecting from the library the variable regions of antibodies with different binding specificities. . The antibody variable region used to obtain the display library is derived from immune tolerance Cloned from the antibody repertoire.   As described herein, the display library antibody variable regions are An immunoreactive container that allows the binding of antibodies to the antigen that contacts the prey package. Replicable Genetic Display Patterns in Kist (immunoreactive context) Package. Therefore, using the affinity selection method, immunorecessive Display having an antibody variable region having the desired binding specificity for an epitope It is possible to enrich the population of iPackage. In a typical example, The display library may be a phage display library. Another As a method, generate a display library on the bacterial cell surface or on spores You can also.   Using the subject method, for example, fetal cell markers such as fetal nucleated red blood cell markers -, Cancer cell markers such as colon cancer markers or metastatic major cell markers, Stem cell markers such as markers for progenitor or hematopoietic stem cells Anti-recessive epitope-specific anti-reactivity such as cell-type specific markers The body can be isolated.   Similarly, the subject method can be used to link between variant forms of a protein and other related forms of the protein. Antibodies can be generated that can be identified by the combination. Immuno recessive epi A variant protein contains one or more amino acids together with other related proteins to produce a taupe. Depending on glycosylation or other post-translational modifications. It may be more antigenically different from the related protein. By the apolipoprotein E family Even if changes occur naturally between different isoforms of the protein family, as shown Well, or neoplastic transformation of tumorigenic proteins or tumor suppressor proteins such as p53 Changes may be caused by genetic alterations as indicated by mutations.   In specific embodiments that illustrate the subject method, antibodies specific for immunorecessive epitopes are , Live antibody phage display from antibody repertoire derived from immune tolerance The rally can be obtained by affinity purification. For example, fusion anti A replicable file encoding a library of phage particles representing body / coat proteins The vector library is used to transform an appropriate host cell. here, The fusion protein contains a phage coat protein portion and an antibody variable region portion. Anti The body variable region is obtained from an antibody repertoire derived from immune tolerance. Cultivate transformed cells Upon feeding, phage particles are formed and the antibody fusion protein is expressed. Immuno recessive epi All of the resulting furs that have an antibody variable region portion that specifically binds to the taupe. Separation of diparticles from phage particles that do not specifically bind immunorecessive epitopes be able to.   The invention further relates to novel immunorecessive antibody libraries obtained by the subject method. I do. The subject method allows, for example, specificity to the immunorecessive epitope of interest. An antibody display library rich in antibodies that bind to can be isolated. The display library is cloned from an antibody repertoire derived from immune tolerance. Screened and subjected to affinity separation using immunorecessive epitopes A miscellaneous V-gene library that is further enriched after expression by iPackage It consists of a group of display packages expressing.   The antibody live of the subject method with individual antibodies and the genes encoding these antibodies It is contemplated by the present invention that it can be isolated from rally. For example, immunodeficiency Antibody display library for antibodies that specifically bind to the active epitope After affinity enrichment, get individual display packages, The antibody gene contained may be expressed in any suitable expression vector suitable for the production of the antibody for the desired use. Subclone into the vector.                                 Description of the drawings   1A and 1B show the variable regions of both the heavy and light chains from the murine antibody gene. The variable region PCR primers for amplifying the region are shown.   FIG. 2 is a schematic representation of the Fab 'expression cassette.   Figure 3. Binding of phage antibody (phab) pools enriched by HEL cell lines. Is a semi-logarithmic graph (number indicates the number of times of enrichment). The graph is thickened Phab pool and other immunoglobulins (T3, anti-M and Wilma). A further comparison with a)) binding to HEL cells is shown.   FIG. 4 shows cells stained with individual phab isolates isolated by the subject method. The percentage of cells (either HEL or mature leukocytes) is indicated.   FIG. 5A shows the sequence of preadsorption and enrichment on fetal hepatocytes for phab binding. The result of the following round is shown. Percentage of phage antibodies that bind to fetal hepatocytes Increase indicates enrichment for fetal cells that bind the phage antibody. Phage anti The body library is a V-gene library derived from the immunized host animal. Obtained. In contrast, FIG. 5B immunizes the results of the tolerance experiment in FIG. 5A. Using a phage antibody library derived from a host animal that is Compare with the results of panning of successive rounds.   FIG. 6 shows amplification of variable regions of both heavy and light chains derived from human antibody genes. The variable region PCR primers for   FIG. 7 shows the heavy chain and individual phab isolates enriched by fetal cells. And the sequences of the CDR3 regions of both light chain and light chain are detailed.   8A and 8B are general features of FB3-2 and H3-3 antibodies, respectively. , Framework regions (double underlined; FRs), complementarity determining regions (CDRs), And an invariant region (italic; IgG1 CH1 or kappa invariant region) Shown. The amino acid residues that differ between the FB3-2 and F4-7 antibodies are: It is shown below the FB3-2 sequence in FIG. 8A.                              Detailed Description of the Invention   The present invention is extremely difficult or impossible to obtain with current methodologies. It is possible to use a powerful and direct approach to the isolation of competent specific antibodies. Noh. One aspect of the invention is the desired binding binding to an immunorecessive target epitope. Dramatic and astonishing synergies in the efficient isolation of antibodies with finity Combine immunotolerization and miscellaneous display libraries to obtain It is a synthesis of methods. Using immune tolerance, such as suppressive immunization, immunorecessive target epi Enrich a subset of lymphocytes that produce antibodies to the taupe in immunized animals You. Subsequent isolation of antibody-producing cells from the immunized animal and isolation of the isolated cells By PCR amplification of at least the variable region of the expressed antibody. A heterogeneous library of offspring (V-genes) is obtained. Depending on the subject method, (i) A genetic display pattern capable of replicating the antibody encoded by each variable region gene. To display the antibody display library on the package, and then (ii) Use the affinity selection method to obtain the desired binding specificity for the target epitope. The display package containing the V-gene encoding the antibody This V-gene library is enriched by enriching the population of prey packages. From which a gene encoding an antibody specific for the target epitope is selected.   In general, isolation by recombinant antibody display methods known in the art Most of the antibodies obtained using a substantially pure preparation of the antigen of interest, 10⁹M^-1Constant (K_aOnly a few isolates with s) are obtained. H The image display method is a specific method of the antibody obtained by the conventional hybridoma method. Sorted with live cells that are equivalent in either sex or affinity The isolated antibody (ie, the crude antigen) is not isolated. In contrast, the following As shown in the examples, the subject method can be used to specifically determine binding affinity and specificity. In terms of degree, it is obtained by both the combination method of the prior art and the hybridoma method. It is possible to obtain an antibody which is superior to the above antibody.   For example, 10 antibodies isolated by the subject method⁸M^-1, For example 10⁹M^-1No 10¹²M^-1Can have a binding affinity in the range of Moreover, of these antibodies Specificity depends on the method of combination and h Several times higher than the antibody obtained by the ibridoma method (assuming that it is not on the order of size Well) can be good. For example, the subject method can be used as a binding buffer for other related cells. Substantially free of background, eg, from background binding to relevant cells Also has a 10-fold or more specific binding to target cells. Other episodes, as shown below Substantially no cross-reactivity with groups, preferably 20-fold over background, preferred Higher specificity of 50, 75 or 100 times, more preferably 125 times or more Can be obtained.   For the purposes of the present invention, the term "antibody" in its various grammatical forms Immunology of immunoglobulin molecules and immunoglobulins recognized in the field Biologically active part, that is, antigen binding that specifically binds to the antigen (immune reaction) Includes molecules with sites. Structurally, the simplest naturally occurring antibody (I gG) is linked to each other by four polypeptide chains, namely disulfide bonds. It consists of two heavy (H) chains and two light (L) chains combined. Light chain is kappa (κ) And exist in two different forms called lambdas (λ). Each chain is invariant (C) Regions and variable (V) regions. Each chain is organized into a series of domains. The light chain has two domains, corresponding to the C region and the V region. Heavy One of the chains corresponds to the V region and three corresponds to the C region (domains 1, 2 and 3) It has four domains. Naturally occurring antibodies have two arms (each arm is Fa b regions), each bound to each other V_LAnd V_HConsisting of areas I have. It is this pair of V regions (V that makes one antibody different from another)._L And V_H) (Due to changes in amino acid sequence). Heavy and light chains respectively The variable region for is the same, including the four framework regions (FRs). Have a general structure, their sequences are relatively conserved, and three hypervariable It is joined by regions or complementarity determining regions (CDRs). Variable region of each chain Is typically of the general formula FR1-CDR1-FR2-CDR2-RF3- It is represented by CDR3-FR4. CDRs for special variable regions are framework Are held in close proximity to each other by the region and together with CDRs from other chains It is involved in antigen recognition and provides an antigen binding site (ABS).   Moreover, as described above, the antigen-binding mechanism may be altered by the naturally-occurring antibody fragment. It has been shown to perform well and the antigen-binding fragment is also referred to as the term "antibody". Is named. Examples of binding fragments within the scope of the term "antibody" are (i) V_L, V_H, C_LAnd C_H1Fab fragment consisting of domain; (ii) V_HAnd C_{H 1} Fd fragment consisting of domain; (iii) V of single arm of antibody_LAnd And V_HFv fragment consisting of domain; (iv) V_HDAb consisting of domains Lagment (Ward et al., (1989) Nature No. 341 Vol .: 544-546); (v) isolated CDR region; and (vi) hinge region. And a divalent fragment consisting of two Fab fragments linked by a disulfide bridge. F (ab ') which is a lagment₂Include fragments. Furthermore, Fv The two domains of Lagment are encoded by different genes, Single protein chain by recombinant method (known as single chain Fv (scFv); Bird (Bird) et al. (1988) Science 242: 423-426. Page; and Huston et al. (1988) PNAS Vol. 85: 5879-. It has been found that it is possible to make synthetic linkers that allow I'm sorry. Such single chain antibodies are also included within the current meaning of the term "antibody". You.   The term "antibody variable region" is also recognized in the art and is collectively referred to as It includes the portion of the antibody that can form the original binding site. For example, antibody variable regions are The framework region (FR1 to FR4) and one or both chains of the IgG molecule It may be composed of complementary complementarity determining regions (CDR1 to CDR3).   The term "desired binding specificity for an immunorecessive epitope" as well as the more general The term "antibody specificity" refers to the ability of individual antibodies to specifically immunoreact with different antigens. Say. The desired binding specificity is typically the ability of the antibody to bind differently. Determined from a reference point of, and thus, in particular, the two antigens have many common Two distinct anti-antibodies if they have a unique epitope that is present with the epitope. It identifies the source. For example, the desired binding affinity for an immunorecessive epitope. Infinity is defined as between an adult and a fetus or between a normal cell and a transformed cell. It can be said to be the ability of the antibody to discriminate between related cells. In other embodiments, the desired Combined affinity binds to mutant forms of the protein differently than the wild-type protein , Or alternatively, the ability of the antibody to distinguish between different isoforms of the protein. Exemption An antibody that specifically binds to an epidemic recessive epitope is called a "specific antibody". The term "relative Specificity "refers to background immunoreactivity (eg, binding to non-target antigens) The ratio of specific immunoreactivity to the above. For example, relative specificity for fetal cells Is the ratio of the percentage of binding to fetal cells to the percentage of binding to maternal cells. Can be expressed as Substantial buffer against non-target antigens such as maternal cells Antibodies without background binding have a high relative specificity (eg background binding). 10 times higher than the sum).   The phrase "individually selective approach" and the term regarding the immunoreactivity of an antibody with a particular cell And "individually selective binding" means that the binding is phenotypically dependent. In addition, it depends on the particular individual from which the cell is isolated, such as on the origin of the cell. Antibody binding to certain cell phenotypes. "Individual selective binding" is the result It does not refer to interspecificity of the combination, but rather to intraspecificity.   The binding of the antibody to the antigen is exclusively non-covalent, but nevertheless It is more subtly specific for one antigen than another and is often Be powerful. Antibodies are different structures of most complex proteins, nucleic acids, and polysaccharide antigens. The component can be specifically bound. Generally, the macromolecule is the antigen-binding part of an antibody. Much larger than the rank. Therefore, an antibody is herein referred to as a "determinant" or It binds only to a special part of the macromolecule, called the "epitope". For specific animals The total number of antibodies produced by a population of antibody-producing cells is It is said. The extreme breadth of the antibody repertoire depends on the individual parts that make up the repertoire. It is the result of the variability of the structure of the antigen binding site in the antibody.   The process of "immunization" involves exposing an animal (animal capable of producing antibodies) to a foreign antigen. Inducing active immunity and thereby inducing the production of antibodies against foreign antigens Say. Molecules that produce an immune response are called immunogens.   The term "immunorecessive epitope" refers to the term "rare epitope" or "standard" as appropriate. `` Epitope '' and is usually present as an immunogen or isolated. In the context of at least polyclonal and monoclonal antibodies Efficient for use in generating an antibody response by immunization, as long as production of Not an epitope. Generally, such immunorecessive epitopes are immunogenic. Is less abundant than other epitopes normally associated with And / or low antigenicity. Immunorecessive epitopes induce a strong antibody response Even in an environment where, for example, this response is an immunorecessive epitope in the immunogen. Other epitopes associated with (also referred to herein as "immunologically dominant epitopes" Is the “background epitope”), and Can be statistically masked by the total number of antibodies produced. Immuno recessive epitome Groups relate to, for example, cell surface antigens that are unique to a particular cell phenotype. You may be in a row. Often, this cell surface antigen is essentially, and As such, it cannot be used as an immunogen. Because a purified form of the antigen was obtained Because there is no. This is an integral membrane protein that loses conformation during purification. This can be especially true in the case of white. Therefore, immunogens containing immunorecessive epitopes , The overall percentage of B lymphocytes activated by immunorecessive epitopes It also contains many background epitopes that can act to reduce . In an exemplary embodiment, the immunogen is an immunorecessive epitope expressed on the surface. It may consist of whole cells. For example, an immunorecessive epitope is a cancer cell marker. Cell type markers such as markers, fetal cell markers, or stem cell markers It may be. Similarly, immunorecessive epitopes do not share amino acids with related proteins. A unique epitope for a mutant form of a protein, such as one or two different mutants It may consist of For example, immunorecessive epitopes on the wild-type p53 Non-mutant p53 may be a determinant, or humans such as ApoE4 It may be an epitope unique to a particular isoform of apolipoprotein E.   "Tolerization" is the immunology of an animal to substances that may be antigenic that are present in the animal. The process of reducing the responsiveness of an antigenic substance that produces tolerance It is called "torelagen". Tolerance is killed without activation of lymphocytes Antigens and toleragens on lymphocytes under conditions rendered unresponsive or Results from the interaction with. Tolerance to a specific antigen, or more accurately the antigen Tolerance to specific epitopes of neonatal tolerance or chemically induced tolerance It can be induced by many means including solubilization, which is induced by It may be the result of a loan loss or clone anergy. Antigen throw The route of application also affects the ability of the antigen to act as either an immunogen or toleragen. there's a possibility that.   The term "immunogenicity" means that the antibody response to an immunorecessive epitope is background. Processes not masked by loss of antibody response to round epitopes Say. For example, as a first step in immunotolerization Exposing the animals to trerelogen. However, toleragen is immunorecessive It lacks a lot. Inducing tolerance to these background epitopes Then, the animal is administered an immunogen containing the immunorecessive epitope. Background B antibody activated in response to a rare epitope due to lack of antibody response to The percentage of vesicles increases relative to the total B cell population of the animal. That is, exempt Cells that produce antibodies specific to the immunorecessive epitope are "concentrated" using the pluripotentiation means. Can be thickened ”. Therefore, the term "background epitome" in this specification Is further defined as a common epitope between the immunogen and toleragen. However, on the other hand, the term "immunorecessive epitope" further refers to an immunogen that is unique to It is understood as referring to a pitope (relative to trellagen). Immunogen and tolera Gens typically also include, for example, phenotypically related cells, or variants of proteins. However, it may be closely related in different isoforms.   The term "antibody repertoire derived from immune tolerance" refers to immunorecessive epitopes And a population of antibody-producing cells derived from immunotolerization intended to enrich the antibody These antibodies.   The term "miscellaneous V-gene library" refers to an antibody repertoire derived from immune tolerance. At least one antibody variable region of one or both of the heavy and light chains of Refers to a mixture of recombinant nucleic acid molecules. A miscellaneous V-gene library is generated on the surface. A group of display packages that are loaned out and expressed are similarly labeled as "miscellaneous "Body display library" or "antibody display library" .   The terms "replicable genetic display package" or "display pack" A package is a biological particle that contains genetic information that confers on the particle the ability to replicate. I have what I have. The package contains antibodies from a diverse V-gene library. A fusion protein can be included. The antibody portion of the fusion protein is Provided by a display package in Allows binding of the antibody to the antigen in contact with the package. In general, The spray package is a very large and diverse sample of V-gene libraries. Recombinant V-gene from ring and purified display package It will be from a system that allows easy isolation. For example, the display package is Derived from cultivated bacterial cells, bacterial spores, and bacterial viruses (especially DNA viruses) You may. Display package encoding at least part of V-gene The miscellaneous mixture of is also referred to as "antibody display library".   The terms "differential binding means", as well as "affinity selection", "affinity" "Enrichment" refers to each display package in the library that binds to the target epitope. Members of antibody display libraries based on the different abilities of antibodies on the surface of di -Separation. Fractional binding of immunorecessive epitopes by display antibodies, Af of antibodies that specifically bind to an immunorecessive epitope from an antibody that does not specifically bind It can be used for nuty separation. For example, in the immunotolerization stage Used the same molecule or cells that were used in the affinity enrichment step Recovering a display package that expresses an antibody that specifically binds to it You can also Affinity selection protocols are typically Pre-concentration eliminates display packages that can specifically bind to epitopes It also includes a chemical conversion step. An example of affinity selection means is affinity chromatography. Chromatography, immunoprecipitation, fluorescence activated cell sorting, aggregation, and plaque fishing Including the lift. As described below, affinity chromatography Alternatively, a bio-panning techniqu es) is included.   In an exemplary embodiment of the invention, the display package is a miscellaneous V-resistor. Antibody fusion coat protein containing amino acid sequence of antibody variable region derived from gene library Are phage particles. Therefore, a library of phage vectors that can replicate And a suitable host cell is transformed. Fur formed from chimeric protein Antibodies that bind diparticles to specific phage particles specifically bind to target epitopes They can be separated by affinity selection based on their ability to Preferred tool In the body example, each individual phage particle of the library is The corresponding phagemid code encoding the antibody fusion coat protein displayed above. Contains pea. Antibodies displayed on individual particles have specific epitopes Purification of phage particles based on their ability to bind to the V-gene also encodes the antibody. Provide isolation of offspring. A typical library for obtaining the heterogeneous antibody library of the present invention. The pages are M13, f1, fd, If1, Ike, Xf, Pf1, Pf3, λ, Includes T4, T7, P2, P4, φX-174, MS2 and f2.   The terms "fusion protein" and "chimeric protein" are used interchangeably herein Is an art-recognized term that is used to Or a contiguous polypeptide consisting of the first polypeptide linked to a higher amino acid sequence. Includes peptides. The amino acid sequence is derived from the foreign domain or the first polypeptide. The polypeptide domain, which is a domain that is not substantially homologous to the tide domain. The array to define. One polypeptide from which the fusion protein is constructed is cloned. V-gene library derived recombinant antibody. The second of the fusion protein The polypeptide portion typically is a "display package" (defined later). Derived from an outer surface protein or display anchor protein that binds) to its outer surface I do. If the display package is phage, Anchor proteins, like viral coat proteins, are not attached to the genetic package. I It may be derived from active surface proteins. The fusion protein is the virus coat protein. When it consists of white or an antibody, it is called an "antibody fusion coat protein". Further fusion Is the protein a signal sequence that is a short amino acid sequence at the amino terminus of the fusion protein? The signal sequence may cause the fusion protein to be secreted from the cytosol and Localize to the extracellular side of the alveolar membrane.   The gene construct encoding the fusion protein may also be a "chimeric gene" or a "fusion". "Gene".   The term "chimeric antibody" is joined by a peptide bond to at least a portion of another protein. Used to refer to a protein containing at least the antigen-binding portion of a combined immunoglobulin molecule I have. Chimeric antibodies include, for example, variable regions from a first species (eg, rodent) and And a constant region derived from a second species (eg, human), or also from the first species Interspecific chimera with native CDRs and FRs from a second species and constant regions It may be.   The term "vector" refers to the insertion of a gene into which recombinant DNA is constructed. Refers to a DNA molecule that is replicable in a host cell.   The terms "phage vector" and "phagemid" are recognized and include An origin of replication for the lyophage, and, optionally, preferably bacterial plasmids. A vector derived by modification of the phage genome, which may include a replication origin of I mean Kuta. The use of phage vectors, rather than the phage genome itself, Greater flexibility in the ratio of chimeric antibody / coat protein to live coat protein Flexibility, as well as other variables that may be useful in the following double-chain antibody constructs It provides a complement to the phage gene with additional genes encoding the region.   The term "helper phage" refers to a defective phage genome or phage vector. Phage used to infect cells that contain and function as a complement to the defect. U. The deletion is due to the removal or deletion of phage genomic sequences necessary for the production of phage particles. It may result from activation. Examples of helper phages include M13K07 And M13K07 gene III no.3.   The term "isolated," as used herein with respect to DNA or RNA, refers to Each is a molecule isolated from other DNAs or RNAs, It exists in the source. For example, an isolated antibody encoding one of the antibodies of the invention. Nucleic acid preferably flanks the antibody gene naturally in genomic DNA. A nucleic acid sequence of at most 10 kilobases (kb), more preferably at most It contains 5 kb of such a naturally occurring flanking sequence. Terms in this specification “Isolated” refers to cellular material, virus, if produced by recombinant DNA methods. If it is substantially free of loose substances or medium, or chemically synthesized, Refers to nucleic acids or peptides that are substantially free of biological precursors or other chemicals Used for Furthermore, "isolated nucleic acid" exists naturally as a fragment. Include non-existing nucleic acid fragments that cannot be found in nature. Means and.   In one aspect, the invention provides a rapid and effective method for isolating cell type-specific antibodies. Show the law. For example, unique to fetal cells, or unique to cancer cells An antibody that specifically binds to an epitope can be obtained by the method of the present invention. As well In addition, the method of the invention can be used to obtain antibodies against variant forms of the protein, which , For example, to detect mutations in the protein or to distinguish different isoforms of the protein Can be used to Therefore, the present invention is applicable to purification, diagnosis, and treatment. Antibodies useful for the application of   In another aspect, the invention provides a novel immunorecessive anti-antigen obtained by the subject method. A body library, as well as individual antibodies isolated therefrom. The method of the present invention According to, for example, anti-specific binding to the immunorecessive epitope of interest. Body enriched antibody display libraries can be isolated. The day Spray library cloned from antibody repertoire derived from immune tolerance And display by affinity separation using immunorecessive epitopes. Miscellaneous V-gene library further enriched after expression by iPackage It consists of a group in a display package that expresses. Therefore, fetal cell marker , Tumor cell markers, and cell surface markers such as variant forms of proteins. It is possible to obtain an antibody display library enriched with specific antibodies .   Immune recessive epitopes used to obtain antibody repertoire derived from immune tolerance It depends on the difference between the immunogen and the toleragen used, so The specificity of the enriched antibody is determined by the specific immunogen / treragen set used. You can make decisions about For example, specific antibodies have a common or similar origin Or specific antibodies are needed if necessary to distinguish different cells of phenotype Cells used as immunogens, and related cells to be identified as toleragen Used. The cell tag for the cell of interest in the immunorecessive epitope Ip-specific markers are represented. For purposes of explanation, cell-type specific markers When it is a marker for infant nucleated red blood cells, toleragen is maternal erythroblast And the immunogen may be fetal erythroblasts. Similarly, the marker When it relates to colon cancer, the toleragen may include normal colon cells, The immunogen can be selected from colon cancer cell lines. If the subject antibody library, And other typical immunogens / tres useful for obtaining individual antibodies derived therefrom. A set of lagens is shown in the description below, others should be obvious to those skilled in the art. U.   Similarly, selection of a set of immunogens / treragens yielded a subject library , Its ability to discriminate between variant forms of proteins and other related forms of proteins by binding Certain specific antibodies can be enriched. Mutant protein is one with other related proteins Or more amino acid residues may differ, resulting in immunodeficiency By producing glycosyl epitopes and further by glycosylation or other post-translational modifications Also changes antigenicity differently from toleragen. Apolipoprotein E family As shown by, changes such as between different isoforms of a protein family naturally occur. Of the tumor prototypic protein or tumor repressor protein such as p53 It can also be caused by genetic alterations, as shown by neoplastic transformation mutations.   The individual antibodies and the genes that encode these antibodies can be analyzed live using the subject methods. It is also contemplated by the present invention that it can be isolated from rally. For example, immunorecessive epitome Antibody display library for antibodies that specifically bind to After tee thickening, individual display packages can be obtained, in which Other suitable expression vector suitable for producing an antibody for a desired use. Can be subcloned into the clone.   The main aspects of the subject invention are generally described below, with preferred embodiments accompanying Examples will be described in more detail. I.Immune tolerance   Subsequent V-gene clonin using immunotolerization in the present invention An antibody repertoire can be obtained which is used for the cloning step. At that stage, immunity The antibody response to the recessive epitope is not masked. In vivo tolerization Alternatively, it can be performed in an in vitro immune system. For example, in the present invention Antibodies directed against immunorecessive epitopes of interest using immunotolerization Enriching the pool of activated B lymphocytes in immunized animals with respect to cells producing Can be. Exposed to Relagen in the Typical Tolerization of the Subject Method? Some time later the immunogen is introduced into the animal's immune system. The effects of toleragen are tolerage The overall reduction or elimination of immunological responses due to animal re-exposure to determinants It is to abandon. In general, the determinants that make up toleragen include immunogens. Because they are part of the determinants of these antigens (ie background epitopes ), Decreased against background epitopes by challenge with immunogen The antibody response does not mask the antibody's response to immunorecessive epitopes of the immunogen Can act like that. By not being masked The population of directed antibody-producing cells is an efficient collection of antibody-producing cells in an animal. It is meant to make up a large percentage of the group (Willi Williams et al. (1992) Biotechniques 12: 842-847).   The most preferred means of tolerization in the subject method is specific for rare epitopes Suppressive immunity to enrich pool of B cells for antibody producing clones (Subtractive immunization) is included. In general, suppressive immunity is a two-step method It is. The first stage is the host activity against a particular set of molecules, namely toleragen. It is a suppressive step that induces a state of tolerance in the product's immune system. The second stage is another The set of molecules, or immunogens, is the immunization stage at which they are introduced into the immune system. general In some cases, molecules consisting of toleragen are a subset of molecules consisting of immunogens. Ideal Only the molecules in which the immune system produces antibodies after exposure to the immunogen are present in the immunogen. It is a molecule that is present but not in trellagen. Two main factors for suppressive immunization Key approaches have been used: neonatal tolerization and chemoimmunosuppression.   In one embodiment of the invention, neonatal tolerization is used to enrich for B cell enrichment. Get Neonatal tolerization is the process of self-tolerance of the developing immune system Is used. In all species, there are discontinuous developmental periods during which the immune system Classify all molecules in the body as self and immunize against those molecules Inducing a state of tolerance induction (Billingham et al. (1953) Nature. -(Nature) 172: 603-606; Golumbeski et al. 1986) Analytical Biochemistry (Anal.Biochem.) No. 154 Volume: 373-381; Hasek et al. (1979) Immunological Reviews (Immunol. Rev.) Volume 46: 3 to 26; Reading (1982) Journal of Immunological Methods (J Immunol.Me thods) 53: 261-291; and Streilen et al. (1 979) Immunological Reviews (Immunol. Rev.) Volume 46: 125 146). Subsequent exposure to all molecules present at this stage Will encounter immunological unresponsiveness. For suppressive immunization, mice (or Or other host animals) are exposed to toleragen in newborns. These animals are free If epidemiologically mature, expose them to the immunogen. In theory, immunity The system immunologically responds only to molecules in the immunogen, but to molecules in trellagen. Should not respond.   In another embodiment of the subject method, the subsequent variable region gene (V-gene) Chemical immunization to obtain enriched B cell populations for cloning Suppression is the means of tolerization used. For example, the cytotoxic drug cyclophosph Chemo-immunosuppression with amides is a useful method for suppressive immunization. Me Ahmed et al. (1984) Journal of American Academy Bu Dermatology (J. Am. Acad. Dermatol.) 11: 1115-1126; Matthew et al. (1983) CSH, Sympo, Quanta, Biolo (C SH Symp.Quant.Biol.) 48: 625-631; Matthew et al. 1987) Journal of Immunological Methods (J.Immunol.Meth) ods) 100: 73-82; and Turk et al. (1972) Imi. Immunology 23: 493-501). Exposed to foreign antigens Application of the chemical drug cyclophosphamide to living animals responds to the presence of foreign antigen molecules. Selectively kill B cells stimulated to proliferate. After cyclophosphamide treatment , Subsequent exposure to those molecules elicits a reduced immunological response Rub Increasingly, animals are treated with foreign antigen molecules (ie, trellagen) as a suppressive immunization method. ) And then injected with cyclophosphamide. The drug is cleared After exposure, the animal is exposed to the immunogen. Theoretically, the immune system It should respond only to immunogenic epitopes not found in lagen.   Other suppressive immune protocols can also be used in the subject methods, eg, Includes the use of leukin-targeted toxins. For example, IL-2-toxin fusion protein -(Kelly) et al. (1988) PNAS Vol. 85: 3980-3984) and And IL-4-toxin fusion protein (Lakkis et al. (1991) European Journal of Immunology (Eur.J.Immunol.) Volume 21: 2253- 2258) to selectively induce tolerance of toleragen to epitopes be able to. II.Generation of antibody gene library   Cloning the antibody repertoire of the resulting B cell pool after the tolerization step . Methods are generally known and in the subject method oligomer primer mixtures And by using PCR for the variable regions of diverse populations of immunoglobulin molecules. The method can be applied to obtain the DNA sequence directly. For example, 5 ' (Signal peptide) sequence and / or framework 1 (FR1) sequence Mixed oligonucleotide primers corresponding to rows, as well as conserved 3'invariant regions The primer can be a heavy chain or a light chain derived from many murine antibodies. It can be used for PCR amplification of variable regions (Larrick et al. (1991) Biotechniques 11: 152-156). Kind A similar method can be used to amplify the heavy and light chain variable regions from human antibodies. Can (Lalique et al. (1991) Method: Comparison to Method Second: Methods: Comparison to Methods in Enzymology Vol: 106-110). The ability to clone human immunoglobulin genes Advantages in the generation of human antibody repertoires in transgenic animals Has a special significance in terms of (eg, Bruggeman et al. (19 1993) Year Immunology 7: 33-40; Tuaillon et al. (1993) PNAS Volume 90: 3720-37 24; Bruggegmann et al. (1991) European Journal of Immu Eur.J.Immunol. 21: 1323-1326; and Wood ( Wood) et al. PCT publication WO91 / 00906).   In typical embodiments, standard protocols (eg, US Pat. No. 4,684) are used. No. 3,202; Orlandi et al. PNAS (1989) 86: 3. 833-3837; Sastry et al. PNAS (1989) Vol. 86. : 5728-5732; and Huse et al. (1989) Science ( Science) 246: 1275-1281), using RNA for B cells, eg For example, isolated from peripheral blood cells, bone marrow, or spleen preparations. The constant region of the heavy chain and For primers specific to each of the κ and λ chains, and the signal sequence The first cDNA strand is synthesized using the primer. Figures 1A and 1B (mouse) Alternatively, using a variable region PCR primer as shown in FIG. 6 (human), And variable regions of both light chain and light chain, amplifying each alone or combining them A suitable vector for further manipulation in the generation of the display package -Connect inside   Unique oligonucleotide primers useful in amplification protocols are unique And degenerate ones that contain inosine at the degenerate position. There may be. Amplified fragments were previously determined for expression purposes In order to allow cloning into the open reading frame of the vector, restriction A donase recognition sequence may be included in the primer. III.Miscellaneous antibody display   V-gene cloned from an antibody repertoire obtained by immunological tolerance The antibody display library is created by expressing the evary with a group of display packages. An ibrary can be formed. Preferably, a heterogeneous antibody library is used. As for the display package that has become clear, the display package is Often (i) is genetically altered to encode at least the variable region of the antibody. (Ii) maintained and amplified in culture, (iii) affinity Represents the antibody gene product in a manner that allows the antibody to interact with the target epitope during separation And then retain the antibody gene so that (iv) the antibody gene sequence is obtained. It will be understood from the discussion herein that they are affinity separated while . In a preferred embodiment, the display is used in affinity separation synthesis. Exist.   Ideally, the display package should be a very large, miscellaneous antibody display. Ray library sampling, rapid sorting after each affinity separation And easy isolation of antibody genes from purified display packages It consists of a system. The most attractive candidates for this type of screening Is rapidly amplified, relatively easy to work with, and yields large numbers of clones. , Prokaryotes and viruses. For example, the preferred display package is , Vegetative bacterial cells, bacterial spores, most preferably bacterial viruses (especially DN). A virus). However, the present invention is a potential display. Eukaryotic cells including yeast and their spores as a package (originally Typically The use of other than antibody producing cells, ie B cells) is also contemplated.   Commercial kits for obtaining phage display libraries (eg Recombinant Phage Antibody System of Pharmacia ( Recombinant Pharge Antibidy Sysytem), Catalog No. 27-9400-01; And Stratagene's SurfZAP^TMPhage display Kit, Catalog No. 240612), as well as miscellaneous antibody displays of the invention. Examples of methods and reagents particularly used for use in the production of b. US Patent No. 5,223,409; Ladner et al .; International Publication WO, Kang et al. 92/18619; Dower et al. International Publication WO 91/17271; International publication WO92 / 20791; Markland International Publication WO92 / 15679; Breitling et al International Publication W O93 / 01288; International publication WO92 / 01 by McCafferty et al. 047; Gerrard et al. International Publication WO92 / 09690; Radnor et al. International publication WO90 / 02809; Fuchs et al. (1991) Bio / Technology (Bio / Technology) Volume 9: 1370-1372; Hay (1992) Human Antibody Hybridomas (Hum Antibo d Hybridomas) Volume 3: 81-85; Huse et al. (1989) Sayen Volume 246: 1275-1281; Griffiths et al. (1993) EMBO J Volume 12: 725-734; Hawkins s) et al. (1992) Journal of Molecular Biology (J Mol B iol) 226: 889-896; Clackson et al. (1991). Year 352, Nature 352: 624-628; Gram et al. 1992) PNAS Vol. 89: 3576-3580; Garrard. Et al. (1991) Bio / Technology 9: 1373-1377; Hoge Hoogenboom et al. (1991) Nucleic Acids Research ( Nuc Acid Res) 19: 4133-4137; and Barbas et al. (1991) found in PNAS Vol. 88: 7978-7982. .   Display is based on bacterial cells, or phage that assemble in the periplasm If so, the display means of the package consists of at least two components. The first component is the cell membrane (host cell if the display package is a phage). Secretory signal directed to a recombinant antibody that is localized outside the cell membrane) is there. This secretory signal is characteristically cleaved by the signal peptidase and This produces a processed "mature antibody". The second component binds the antibody to the outer surface. Is a display anchor protein that directs the display package to . As will be explained later, this anchor protein binds to the genetic package. It may be derived from active surface proteins or coat proteins.   Display packages are bacterial spores or protein coatings are assembled intracellularly , The secretory signal that directs the antibody to the inner membrane of the host cell is absent. It is important. In these cases, the means by which the miscellaneous antibody library was sequenced was a fusion protein. It consists of a spore or phage coat protein derivative used as white.   The antibody component of the display is at least isolated during the suppressive immunization step. Cloned from isolated B cells_HOr V_LIt consists of one of the areas. Only While V_HArea and / or V_LThe region is the constant region other than the variable region of the antibody. It will be appreciated that all or part of the may be included. Typically, The display library is for generating at least Fv fragments. It will include the variable regions of both the heavy and light chains. To make it easier to understand, The detailed example is for constructing a fusion protein with a display anchor protein. Cloned into V_HDetails of minimal antibody display, such as consisting of use of area Will be described. However, V_LAnd V_HThe role of the chain is the structure of the display library. It should be understood that there may be similar embodiments that are reversed in construction.   Under certain circumstances, V of antibody display_HThe part is a single part of the suppressive immunization stage Derived from isolated cells, but V_LNo chains or "fixed" V_L(Ie , The same V for each antibody on the display_LChain). For example , Display V_LIf the part is fixed, V_LThe chain is V_HCode chains From a genetic construct other than the construct or from the host cell itself (ie, light chain producing bone marrow Tumor V) that has been given from the cell) or has already been displayed on the surface_Hchain and It is added to the package externally to rejoin. However, V_LThe chain V_HThe miscellaneous cloned from the same B cell population in which the gene was cloned V_LIt is generally preferred that it is from a library. In such cases, the preferred ingredients Body example is V_LV gene_HPlace genes in the same construct and both are easily recovered together To be done.   The desired antibody display is a multi-chain antibody (eg V_HAnd V_LBut different polype Is a peptide chain), the cDNA encoding the light chain is It may be cloned directly into an appropriate site in the vector containing the ibrary. There Alternatively, the light chains can be used as separate libraries in different plasmid vectors. It is cloned, amplified and then cloned into a vector library encoding the heavy chain. The Lagment may be cloned. Under such circumstances, the host cell V as expressed with a signal leader sequence that directs secretion into the rum_L Clone the chain. For example, some leads on E.coli The sequence of the sequencer is OmpA (Hsiung et al. Bio / Technology (198 6) Vol. 4, pp. 991-995, and phoA (Skerra and Pla. Pluckthum, Science (1988) 240: 1038). Have been shown to direct the secretion of antibody sequences such as.   In the example where the display package is a phage, V in the phagemid_L Cloning sites for the strand sequences do not substantially interfere with normal phage function Should be placed as. The position where 1 is applied is Zinder and Beke ( Boeke), (1982) Gene 19: 1-10. This is the inter-generic region. A typical example is the M13 phage display library. And preferably V_LThe sequence is similar to the HL-coIII product (discussed below). Is expressed at higher levels and has a high V in the periplasm_LMaintain concentration, V_LAnd V_HProvides efficient assembly (association) with the chains. For example, separate genes, Wow, V_H/ Coat fusion protein and V_LPhagemid to encode both chains Can be built. Under proper induction, both chains are expressed and expressed in host cells. The cells become assembled in the periplasmic space of the cell, and the assembled antibodies are V, which is part of the white fusion construct_HIt is bound to the phage particle by a chain.   Probably 10 possible combinations of heavy and light chains¹²Will cross the street. Can Sampling as many combinations as possible can, in part, result in multiple transformations. It depends on the ability to recover the replacement. A plasmid-like form of phage (filamentous Phage), electrotransformation is very large for DNA input. In addition to capacity, phage transfection using in vitro packaging Provides efficiency comparable to that of the option. This uses a large number of vector DNA It is possible to obtain a large number of transformants. The method is based on Dowe r) et al. (1988) Nucleic Acids Resea rch) 16: 6127-6145, for example, using Per μg of ligated vector in E. coli (such as MC1061 strain) About 10⁷Fd-tet-derived recombinants may be transformed at a ratio of Brary about 3x10⁸Fd-tet consisting of up to or more species It may be constructed in Bl. Increasing DNA input, and Making changes to the cloning protocol within its capabilities is not Increase the recovery rate of the substitute by about 10 times,^TenUp to or more recombinants A library can be provided.   In other embodiments, single chain F_vFlexible for forming fragments The heavy and light chain V region domains joined by a linker are labeled with the same polypeptide. Expression vector, the scFV gene is then expressed in the desired expression vector. Or clone into phage. McCafferty et al., Ney General in Nature (1990) 348: 552-554. Flexible (Gly_Four-Ser_Three)_ThreeLinked by a linker Complete V of antibody_HAnd V_LUsing domains for antigen affinity Based on a single-chain antibody that can make the display package separable Can be   As will be apparent to those of skill in the art, in the specific examples where high affinity antibodies are searched for, , An important criterion for the selection method of the present invention is that it is different for a particular antigen. Distinguish between finity antibodies and give preference to the highest affinity antibody It may be possible to enrich it. Antibody affinity and Applying the well-known theory of valency (ie, binding activity), the By operating the play package efficiently to make it a single price, With a broader range of affinities that can be isolated using prey packages Generally higher binding affinity (ie 10⁶Through 10^Ten M^-1Affinity enrichment can be performed for binding constants in the range. 1 Native form used to immobilize the antibody on the display to obtain a high-value display Display proteins (ie, wild type) surface or coat proteins Added at a high enough level to almost completely eliminate inclusion of the antibody fusion protein in be able to. Thus, the vast majority containing at most one copy of the antibody fusion protein Display packages can be obtained (for example, Garrard et al. (1991) Bio / Technology 9: 1373-1377). 1-value de In a preferred embodiment of the display library, the display package The library consists of at most 5-10% polyvalent displays and is more preferred Preferably at most 2% of displays are polyvalent, most preferably at most 1% There are multivalent display packages in the population. The source of wild-type anchor protein , For example, by copying a wild-type gene present on the same construct as the antibody fusion protein It may be provided, or alternatively, the separate constructs may be provided together. However However, a similar operation yields a multi-valued display that allows for a wider range of combined affinity. It is also clear that one can be isolated. Such antibodies are, for example, anti- Physical activity may be useful in purification protocols that may be desired You. i) Phage as a display package   Bacteriophages are the raw materials associated with the attractive prokaryotes used in the subject invention. Things. Bacteriophage is an enzyme activity associated with intact mature phage. With little or no sex, and their genes are inactive outside the bacterial host. Yes, it renders the mutant phage particles metabolically inactive, and therefore can It is an excellent candidate for providing a display system for Brary. Generally, phage The surface is a relatively simple structure. Phages can easily multiply and increase in number And the potential for use in large-scale screening programs with many potential Suitable for handling when they are themselves in a small and simple package It has the genetic information for the synthesis of. The antibody gene was inserted into the phage genome. Therefore, the selection of the appropriate phage used in the present invention is generally large. In the body, the (i) phage genome allows for additional genetic material. Of the antibody gene, either by (Ii) virus particles after accepting insertion or substitution of genetic material Whether the offspring can pack the genome; and (iii) antibodies on the phage surface Display disrupts virus particle structure enough to interfere with phage growth It will depend on whether or not to do it.   One concern with the use of phage is that the morphogenetic pathway of phage causes the antibody to fold. It is about deciding the environment that has the opportunity to be caught. Generally displayed Antibodies contain essential disulfides, so they are assembled in the periplasm. Is preferred and such antibodies may not fold correctly in cells There is a nature. However, the special features that the display package produces inside the cell In body examples (eg, lambda phage is used), phage are released from cells. It was shown that the antibody is likely to fold correctly.   Another related to the use of phages but also to the use of bacterial cells and spores The concern is that multiple infections have at least one gene for a particular antibody. A hybrid display having one or more different antibodies on its surface It is possible to generate b. Therefore, it is necessary to Minimizing this possibility by infecting cells with phage is voluntary. However, it can be said that it is preferable. However, the antibody display Increase the occurrence of homologous recombination between the carrying genetic constructs and In situations such as expanding the body display library repertoire, high There may be situations where multiplicity of infection conditions are desirable.   For certain bacteriophage, phage is the preferred display means. It is a protein (eg, coat protein) that is present on the surface. Filamentous phage is a spiral lattice , Isometric phage, and can be further explained by an icosahedral lattice. Of each major coat protein Each monomer exists on a lattice point and has a certain interaction with its neighbor. Everything But not in the grid by making contact with some normal grids. The protein that causes the By leaving a gap in the offspring so that the nucleic acid is not protected, May destabilize the child. Therefore, in bacteriophage, Unlike in fungi and spores, a coat that interacts with other proteins in viral particles It is generally important to retain the protein residues in the antibody fusion protein. For example, M When using the 13 cpVIII protein, in general, all mature proteins will have the N-terminus of cpVIII. Antibody fusion that would be retained with the added antibody fragment, while antibody fusion 100 (or less) carboxy of M13cpIII coat protein in protein It may be sufficient to retain only the Si-terminal residue.   Under suitable induction, such as when using lambda phage, the antibody library It can be expressed and assembled in the bacterial cytoplasm. How much of a phage particle Replication, some phage structural protein synthesis, and some phage particles The induction of the protein may be delayed until the assembly of Then, the assembled protein chains , Interacts with phage particles by binding anchor proteins on the outer surface of phage particles I do. The cells are lysed and the receptor protein encoded by the library Corresponding to the specific library sequence contained in the DNA of The charge is released and isolated from the bacterial residue.   Far containing cloned library sequences encoding the desired protein To enrich and isolate the di and thus to ultimately isolate the nucleic acid sequence itself. First, the phage harvested from the bacterial residue is affinity purified. As below , If an antibody that specifically binds to a particular antigen or antigenic determinant is desired, the antigen or Is Determinants can be used to recover phage displaying the desired antibody You. Then, infecting the host cells with the phage thus obtained It may be further amplified. A further number of times is required until the desired level of enrichment is obtained. Inner enrichment followed by amplification.   Expression plaque (or colony) lift using labeled antigen as probe (eg For example, Young, Davis, Science (1 983) 222: 778-782). Can be used to screen for enriched antibody-phage. That Infect cells with phages from the screening protocol and propagate The phage DNA is then isolated, sequenced, and / or prokaryotic. Alternatively, gene expression in eukaryotes can be largely recloned into the intended vector. Select quantity of specific antibody is obtained.   In yet another embodiment, an extracytoplasmic companion such as a bacterial periplasm. Transports the antibody to the library, but as a fusion protein with the viral coat protein. transport. In this embodiment, the desired protein (or it is a multi-chain antibody One of the polypeptide chains), it is fused with the viral coat protein and expressed. As will be revealed, the desired protein is processed and transported to the intracellular membrane. If exist If present, express the other chain with a secretory leader, thus periplasmic Or intracellularly by positioning it outside the cytoplasm. Then , Chains residing outside the cytoplasm (eg, heavy and light chains) are assembled into whole antibodies (or Or its binding fragment). The phage are pushed through the host membrane, As the coat proteins assemble around the phage DNA, the assembled molecules are phage It becomes incorporated into the phage by its attachment to the coat protein. Then the antibody The phage with the complex were screened by the following affinity enrichment. May be. a) Filamentous phage   Includes M13, fl, fd, Ifl, Ike, Xf, Pfl, and Pf13 Filamentous phages are a group of related viruses that infect bacteria. They are , An extension encapsulating deoxyribonucleic acid (DNA) forming the bacteriophage genome It is called filament because it is a long and thin particle consisting of capsules. F Ciliary thread Cteriophage (Ff phage) is specifically adsorbed on the tip of F cilia. Only more Gram-positive bacteria are infected, including fd, fl and M13.   Filamentous phages are generally attractive compared to other bacteriophages. In particular, M13 is attractive. Because (i) the three-dimensional structure of the virus particle Known; (ii) well understood processing of coat proteins; (ii) i) the genome is extendable; (iv) the genome is small; (v) the genome sequence is known (Vi) Virus particles are sheared, heat, low temperature, urea, guanidinium chloride, low p Physically resistant to H and high salt; (vii) phage sequenced It is a routine vector and is particularly easy to sequence; (viii) an antibiotic resistance gene It has been cloned into the genome and results can be predicted (Hines et al., (1980) Gene 11: 207-218); (ix) Culture and And easy to store, infected cells require unusual or expensive media. No; (x) Large burst size, 100-100 infected cells after infection Yields 00 M13 progeny; and (xi) easy to harvest and concentrate (Saliver ( Salivar) et al. (1964) Virology, Vol. 24: 359-3. (P. 71). Threads that are conventional cloning and sequencing vectors The entire life cycle of filamentous phage M13 is well understood. M13, including complete sequence The gene structure of Escherichia coli is well known (Schaller et al., The Singles Denha, The Single-Stranded DNA Phages Denhardt et al. (NY: CSHL Press, 1978), 10 genes Offspring identity and function, and transcriptional order and promoter position and virus The physical structure of spurs is also well known (Smith et al., (1985)) Science 228: 1315-1317; Raschad et al. (19 1986) Microbiol Dev Volume 50: 401-42 7; Kuhn et al. (1987) Science 238: 1413-1. 415 p .; Zimmerman et al., (1982) Journal of. Biological Chemistry (J Biol Chem) Vol. 257: 6529-653. 6; and Banner et al. (1981) Nature 289: 81. Pp. 4-816). Since the genome is small (6423bp), it is a single-stranded oligo Mutations directed to leotide (Fritz et al., DNA cloning ( DNA Cloning (edited by Glover, IRC Press, Oxford, UK) , 1985)) and cassette mutations have been used for RF M13. Ru (Ausbel et al., Current Protocols in Molecular) ー Biology (Current Protocoles in Molecular Biology) (New York John Wiley & Sons, 1991 . M13 is a plasmid, a transformation system itself, ideal for sequencing Vector. Propagation of M13 in a recombinant strain of E. coli Can be. The M13 genome is extendable (Messing et al., The. The Single-Stranded DNA Phags es), edited by Denhardt et al. (NY: CSHL Press, 1978), 449-453; and Fritz et al., Supra), M13 does not lyse cells. Add Additional genes can be inserted into M13, which are in a stable fashion. Will be maintained throughout the nom.   The mature capsule of Ff phage is a gene product encoded by 5 types of phages. It consists of a coat. That is, the major coat protein product of gene VIII, cpIII (Forms the bulk of the capsule); and four minor coat proteins (capse CpIII and cpIV at one end of the capsule and cpV at the other end of the capsule II and cpIX). Capsule length is ordered to form a characteristic thread-like structure Formed by 2500 to 3000 copies of cpVIII in a spiral configuration It is. The protein encoded by gene III (cpIII) is typically one protein. There are 4 to 6 copies at the ends of the cells, and Serves as a receptor for binding to the bacterial host. Detailed Levi of Ff phage structure For a review, Rasched et al., Microbiolo Levi (Microbiol. Rev.) 50: 401-427 (1986); and Karen. Edited by Calendar et al., Plenum Press, “The Bacterio Phage (The Bacteriophge) "Volume 2 (1988), Model et al. , Pages 375-456.   The assembly of phage particles results in the extrusion of the viral genome across the host cell membrane. is necessary. Prior to extrusion, major coat protein cpVIII and minor coat The protein cpIII is synthesized and transported to the host cell membrane. cpVIII and cpIII Are fixed to the host cell membrane before they are incorporated into the mature particle. further, The viral genome is produced and coated with the cpV protein. C during extrusion process cpV was stripped from the genomic DNA coated with pV, and at the same time matured Recoated with protein.   The cpIII and cpVIII proteins provide signals for assembly of mature phage particles. Contains two domains provided. The first domain hosts the newly synthesized protein. It is a secretory signal directed to the main cell membrane. The secretion signal is the amino acid of the polypeptide Located at the end, it directs the polypeptide at least to the cell membrane. The second domain is Signals for host cell membrane binding and phage particle binding during assembly provide. This second signal for both cpVIII and cpIII is less Both consist of hydrophobic regions to reach the membrane.   The 50 amino acid mature gene VIII coat protein (cpVIII) contains 73 Synthesized as an amino acid precoat (Ito et al. (1979) PN AS 76: 1199-1203). cpVIII has been widely studied as a model membrane protein. Has been pursued. Because the acidic amino terminus goes to the outside of the membrane and the basic Make sure the carboxy terminus faces the inside of the membrane so that the asymmetric orientation It can be incorporated into the quality barrier. The first 23 amino acids are the initial po It constitutes a typical signal sequence that allows the polypeptide to be inserted into the intracellular membrane. ing. E. coli signal peptidase (SP-1) has amino acids 18, 2 1 and 23, and weaker recognition of residue 22, pre-coated residues Cutting between 23 and 24 (Kuhn et al. (1985) Journal Of Biological Chemistry (J. Biol. Chem.) Volume 260: 15914 ~ 15918; and Kuhn et al. (1985) Journal of Biology. Cal Chemistry 260: 15907-15913). Signal sequence After removal, the amino terminus of the mature coat is located on the periplasmic side of the inner membrane and its The xy terminus is located on the cytoplasmic side. About 3000 copies of mature coat protein in the inner membrane Join side by side.   The sequence of gene VIII is known and encodes the amino acid sequence on the synthetic gene be able to. Maturation gene VIII protein forms a sheath around circular single-stranded DNA . The protein of gene VIII may be suitable as an anchor protein. Because the virus Because its position and orientation in the particle is known (Banner) Et al. (1981) Nature 289). Preferably, the antibody is the mature M13 coat. A phage display library is obtained by attaching it to the amino terminus of the protein. Up As noted, both wild type cpVIII and Ab / cpVIII fusions in infected cells. Can be used to reduce the binding activity of the display, which Facilitates detection of high affinity antibodies directed against the target epitope be able to.   Another means for displaying antibodies is to make it part of gene III. Or it is expressed as a domain of a chimeric gene containing all of them. One-value de If the display is required, the chimeric gpIII is formed during phage particle formation. It was possible to easily control the change in the ratio of wild type gpIII to Therefore, it is preferable to express the V-gene as a fusion protein with gpIII. It can be an example. This gene encodes one of the minor coat proteins of M13 ing. Genes VI, VII and IX also encode minor coat proteins. This Each of these minor coat proteins is approximately 5 copies per virus particle. Present and associated with morphogenesis or infection. In contrast, the major coat protein is wi It is present at 2500 copies or more per loose particle. Genes VI, VII, and IX The protein is located at the end of the viral particle. These three proteins are processed before translation. Not Thing (Rashed et al. (1986) Annual Review of My Black Biology (Ann. Rev. Microbiol.) Vol. 41: 507-541). For details The single-stranded circular phage DNA binds to about 5 copies of the gene III protein, then Patches of membrane-bound coat protein in such a way that the DNA is wrapped in a helical sheath of protein. Extruded through (Webster et al., “The Single Strand Dead DNA Phages (The Single-Stranded DNA Pharges) Dresdner (Dressner) et al. (NY: CSHL Press, 1978)).   Manipulation of the cpIII sequence normally involves hydrophobic amino acids that are involved in membrane anchoring function. The extension of the 23 amino acids at the C-terminus of the no acid can be modified in various ways It was shown that the binding ability can be retained. Expression vector based on Ff phage Is first described, in which the cpIII amino acid residue sequence is Pitope "(Parmely et al., Gene (1988) Vol. 73) : 305-318; and Cwirla et al. PNAS (1990). 87: 6378-6382) or amino acids that determine the single chain antibody domain. Residue sequence (McCafferty et al., Science (1990) (Year) 348: 552-554). Gene III Insertion into the virus particle has been shown to cause the production of new proteins on the outer surface (Smith (1985) Science Vol. 228: 1315 ~ 1317; and de la Cruz et al. (1988) Journal Le of Biological Chemistry, Vol. 263: 4318-4322). . The site used by Smith and De La Cruz, the other The codon corresponding to the main boundary or the surface loop of the protein or the amino terminus of the mature protein. The antibody gene may be fused to gene III at Don.   Generally, a phage coat protein such as cpIII of filamentous phage fd is used. The good cloning method is (1) fusion to the N-terminus of coat protein (eg cpIII). Expression of the combined antibody chain and transport to the inner membrane of the host (where the C-terminal region of the coat protein is The hydrophobic domain in the protein anchors the fusion protein in the membrane and projects it into the periplasmic space. A second or subsequent chain (eg, F_vOr Fab V forming a lagment_L) With an N-terminus available for interaction with The second or subsequent chain is attached to the coat protein); and (2) if The second or subsequent chain, if present (eg V_L) Sufficient expression and It will provide transport of this chain to the soluble compartment of the lipasm.   Similar constructs could be made with other filamentous phage. Pf3 is , Pseudomonas carrying the IncP-I plasmid aerugenosa) is a well-known filamentous phage. Entire genome is a sequence It has been decided (Luiten et al. (1985) Journal of We Rology (J. Virol. 56: 268-276), involved in replication and assembly Genetic signals are known (Ruiten et al. (1987) DNA Vol. 6 129- 137). The major coat protein of PF3 is a signal peptide that directs its secretion. It is unusual in not having. The sequence contains charged residues ASP-7, ARG-3. 7, LYS-40 and PHE44, which have an exposed amino terminus. It is consistent with that. Therefore, in order to allow the antibody to appear on the surface of Pf3, the three segments A gene can be constructed that induces secretion in P. aergenosa. Signal sequence known to occur (the signal sequence encodes an antibody sequence Fused in frame to the gene fragment carrying the fused to the DNA encoding the f3 coat protein in frame) . If desired, a flexible linker consisting of 1 to 10 amino acids can be coded for. Insertion DNA between the antibody gene fragment and the Pf3 coat protein gene I do. This three segment gene was introduced into Pf3, which Do not interfere with expression. When the signal sequence is cleaved, the antibody is periplasmic. The mature coat protein acts as an anchor and a phage assembly signal. I do. b) Bacteriophage φX174   The bacteriophage φX174 was completed by genetics, biochemistry and electron microscopy. It is a very small icosahedral virus that has been fully studied (The Singlest The Single-Stranded DNA Phages, Denha Edited by Denhardt et al. (NY: CSHL Press, 1978). φX 174 The three gene products of are located outside the mature virus particle. They are F (Capsi D), G (large spike protein, 60 copies per virus particle), and H ( It is a minor spike protein, 12 copies per virus particle). G protein Consists of 175 amino acids, while H consists of 328 amino acids. F protein It interacts with the single-stranded DNA of the virus. Proteins F, G and H are virally infected Translated from a single mRNA in the cell. Some of the viral genes overlap Since the virus is very tightly bound, only very small amounts of added DNA Due to the fact that it does not accept, φX174 is typically a cloning vector. Not used. However, a plasmid expressed in the same host cell The viral G gene (G protein is encoded by Can be rescued (Chambers et al. (1982) Nuc Acid Res, Vol. 10: 6465 6473). In one embodiment, one or more stop codons are G When introduced into a gene, G protein is not produced from the viral genome. Then A heterogeneous antibody gene library can be fused to the nucleic acid sequence of the H gene . A fixed amount of the viral G gene of the same size as the antibody gene fragment Remove from φX174 genome so that the size of the genome does not change . Thus, the host cell is further transformed with the second plasmid expressing the wild-type G protein. Production of virions from a mutated virus by conversion with an exogenous G protein source To rescue. Only one antibody is displayed per φX174 particle If desired, the second plasmid further comprises one or more of the wild type H protein genes. More than one copy, which results in uptake into phage particles. This would predominate the mixture of H and Ab / H proteins. c) Large DNA phage   Phages such as λ or T4 are much larger than M13 or φX174 3D capsid structure that has a genome and is more complex than M13 or φPX174 Structure and have more selected proteins. The antibody library is Bacillus in the cytoplasm of the host cell In an embodiment of the invention which binds to phage particles, bacteriophage λ and The derivative is an example of a suitable vector. Intracellular morphogenesis of phages is The protein domain that normally contains disulfide bonds is hidden from proper folding. Prevent locally. However, functions involving both the heavy and light chain variable regions A heterogeneous library expressing a population of specific antibodies was generated in lambda phage. Hase et al. (1989) Science Volume 246: 1275-1 281; Mullinax et al. (1990) PNAS Vol. 87:80. 95-8099; and Peason et al. (1991) PNAS 88. Volume: 2432-2436). Such a method allows for the rapid construction and presence of lambda phage. Utilizes effective transformation ability.   V_H, V_L, F_V(Variable region fragment) or the expression of an antibody sequence such as Fab. For current use, insert library DNA easily into lambda vector be able to. For example, both the heavy and light chain variable regions have been cloned Is inserted into the multiple cloning site of the ZAPII vector (Hughes et al., Supra). ΛZAPII (Short et al. (1988) Nuclei characterized by C. Acids Research 16: 7583). Ibrary was built. To explain, digest a pair of lambda vectors and And a restriction site flanking the expressed sequence can be asymmetric. This The asymmetry of allows for efficient recombination of libraries encoding separate chains of active protein. Noh. Therefore, the variable region of the antibody light chain (V_L) Expressing the library Heavy chain variable region (V_H) Is expressed together with a library expressing Combined antibody or Fab expression libraries can be constructed. For example, la In the initial steps of the library construction, the λ vector was cloned into the antibody light chain sequence. Designed to serve as a training vector and another lambda vector Designed to serve as a cloning vector for chain sequences. combination The library was cloned from two lambda libraries at appropriate restriction sites. La Construct by crossing. DNA was first purified from each library, The right and left arms of each lambda vector are cleaved to leave the antibody chain sequence intact. The DNAs are then mixed and ligated, resulting in the correct assembly of the vectors with each other. Only the loan reconstitutes as a live phage (Hughes et al., Supra). ii) Bacterial cells as a display package   Recombinant antibody is a bacterial antibody after addition of a bacterial leader sequence to the N-terminus of the protein. Can cross membranes (Better et al. (1988) Science No. 2 40; 1041-1043; and Skerra et al. (1988) Sai. Ens 240, 1038-1041). Furthermore, in order to express it on the surface , A recombinant antibody was fused to the outer membrane protein. For example, antibodies can be placed on the surface of bacteria One method for playing is to place the antibody in the exposed cell surface of the intact outer membrane protein. Consists of obtaining the fusion protein by insertion (Fuchs et al. (19 1991) Bio / Technology Volume 9: 1370-1372 page). In selecting the bacterial cells to serve as a display package, Typically, any well-characterized bacterial strain is suitable, , Bacteria will grow in culture and display an antibody library on their surface The special affinity that can be processed in -It must be compatible with the selection process. Preferred among bacterial cells The display system is Salmonella typhimurium, Bacillus subtilis, Pseudomonas aeruginosa (Pseudomonas aeruginosa), Vibrio cholerae, Cle Klebsiella pneumonia, Neisseria gonorrhoeae (Neisseria gonorrhoeae), Neisseria meningi tidis), Bacteroides nodosus, Moraxella bo Bis (Moraxella bovis), and especially Escherichia coli ). Many bacterial cell surface proteins useful in the present invention have been characterized And the localization of these proteins and the determination of their structure Research on methods is described by Benz et al. (1988) Anu Levi Mike Loviolo (Ann Rev Microbiol) 42: 359-393; Balduit Balduyck et al. (1985) Biol Chemi Hoppe Sailer m Hoppe-Seyler) 366: 9-14; Ehrmann et al. (199). 0 years) PNAS Vol. 87: 7574-7578; Heijne et al. (19 90 years) Protein Engineering Vol. 4, 109 ~ 112; Ladner et al., U.S. Pat. No. 5,223,409; Ladner. WO 88/06630; Fuchs et al. (1991) Bio / Tech. Noology 9: 1370-1372; and Goward et al. (199 2 years) TIBS 18: 136-140.   To further explain, the E. coli LamB protein is a heterogeneous protein on the surface of bacterial cells. A well-understood surface protein that can be used to generate antibody libraries. (Eg, Ronco et al. (1990) Biochemie No. 72). Volume: 183-189; van der Weit et al. (1990) Year) Vaccine Volume 8: 269-277; Charabit (1988) Gene 70: 181-189; and Landner. (See US Pat. No. 5,222,409 to Landner)). Lamb of E. coli Is a porin for maltose and maltodextrin transport, Serves as a receptor for adsorption of lyophage λ and K10. Functional N end In the presence of the edge signal sequence, LamB is transported to the outer membrane (Benson (Bens on) et al. (1984) PNAS Vol. 81: 3830-3834). Other cell surface Like surface proteins, LamB has a typical signal sequence that is later removed. Are synthesized. Therefore, a miscellaneous antibody gene library was cloned into the LamB gene. Upon loaning, the resulting library of fusion proteins will be loaded with antibody fragments from the membrane. Consists of a LamB portion sufficient to orient the protein outside the cell and fix the protein in the cell membrane . Include a LamB signal sequence or other suitable signal sequence as the N-terminus of the protein. By doing so, the extracellular portion of the fusion protein can be easily secreted.   E. coli's LambB, S. typhimurium (Harkki et al. (1 986) Microb Pathol Volume 1: 283-288. ), And Kay Newmonia (Wehmeier et al. (1989) (Mol Gen Genet 215: 529-536) Are expressed in a functional form from E. coli to produce a population of all antibodies of these species. -It could be displayed as a fusion with LamB of Kori. Besides, Kay New Monoae expresses a usable LamB-like maltoporin You. Using the D1 protein (a homologue of LamB) in P. aeruginosa Can (Trias et al. (1988) Biochem BiophysiA Kuta (Biochem Biophys Acta) 938: 493-496). Similarly, PAL , OmPA, OmPC, OmPF, PhoE, pilin, BtuB, FepA, F Other bacterial surface proteins such as huA, lutA, FecA and FhuE are It may be used instead of LamB as part of the display means in cells. iii) Bacterial spores as a display package   Bacterial spores are also desirable features as display package candidates in the method of the invention. Have sex. For example, chemical and physical rather than vegetative bacterial cells or phage Much more resistant to biological factors, and therefore a very different affinity Selection conditions can be used. In addition, Bacillus spores carry proteins on their surface. It has no metabolic activity and does not alter them. However, The molecular mechanism that initiates sporulation is the formation of M13 or the outer membrane of E. coli. It has the disadvantage that it does not work better than the protein transport of It does not prevent their use in the invention.   Bacillus bacteria are damaged by heat, radiation, drying, and toxic chemicals Forms an extremely resistant endospore (endospore) osick) et al. (1986) Volume 20 of Ann Rev Genet : 625-669). This phenomenon is a coat This is due to the extreme intermolecular cross-linking of proteins. A relatively rough affi In certain embodiments of the subject invention, such as a method that includes a unity separation step, Spores may be the preferred display package. Bacillus-derived ene Dospores are, for example, more exotic than those derived from Streptomyces. It is stable. Moreover, Bacillus subtilis sporulates in 4 to 6 hours. But Streptomyces species may require days or weeks for sporulation . In addition, genetic knowledge and manipulation is more important for Bee Bacillus than other sporulating bacteria. Is much better developed.   Even if one of the four coat proteins is lost, it is only slightly different from the wild type. Different live spores are formed in B. subtilis (Donovan ) Et al. (1987) Journal of Molecular Biology (J Mol Bi ol) 196: 1-10). Moreover, usually the plasmid DNA is in spores , A protein encoded by the plasmid, is observed on the surface of Bacillus spores. (Debro et al. (1986) Journal of Bacteria J. Bacteriol, 165: 258-268). Therefore, sporulation Antigen of a miscellaneous gene library during sporulation without substantial interruption It is possible to express a gene encoding a chimeric coat protein consisting of the body .   To explain, some polypeptide components of the spore coat of B. subtilis ( Donovan et al. (1987) Journal of Molecular Biology 1st 96: 1-10) have been characterized. 2 complete coat proteins and 2 others The amino terminal fragment of the species was sequenced. Antibody sequence cotC or c Fusion to the otD fragment may generate antibodies on the spore surface. Neither cotC nor cotD is post-translationally modified, so these spore coat proteins Each white gene is preferred (Lader et al., US Pat. No. 5,223,409). IV.Selection of antibody targeting antigen   After expression, miscellaneous antibody display is subjected to affinity enrichment for preselection Antibodies that bind to the selected antigen are selected. The terms "affinity separation" or "a "Finity enrichment" refers to (1) affinity chromatography using immobilized antigen. Raffy, (2) Immunoprecipitation with soluble antigen, (3) Fluorescence activated cell sorty Including (4) aggregation, and (5) plaque lift, , But not limited to them. In each of the embodiments, finally, the antigen of interest Of the display package based on the ability of the bound antibody to bind to the above epitope The library is separated. For example, US Pat. 223,409; International Patent Application Publication WO 92/18619 by Kang et al. International patent application publication WO 91/17271 by Dower et al .; Winter (W international patent application publication WO 92/20791; Markland ) Et al., International Patent Application Publication WO 92/15679; Breitling. International Patent Application Publication No. WO 93/01288; McCafferty et al. International Patent Application Publication No. WO92 / 10147; International Patent by Garrard et al. Published application WO92 / 09690; and international patent issued by Landner et al. See the application publication WO90 / 02809. In the most preferred embodiment, first the display Ray library with background epitome like toleragen The antibody that binds to the background epitope by contacting the source of the loop By displaying live antibodies specific to rare epitopes The rally will be enriched in advance. Then use the display package as the target antigen Contact is made and the display antibody capable of specifically binding to the antigen is isolated.   For affinity chromatography, from column chromatography Includes additional ELISA and biopanning methods leading to batch elution It will be appreciated by those skilled in the art that numerous chromatographic methods can be applied to the present invention. Generally understood. Typically, Sepharose or polyacrylamide gel Labeled beads or insoluble carriers such as wells in microtiter plates. Immobilize the target antigen. As with many cell-specific markers, as described below. As such, if the source of the purified target antigen is not readily available, the antigen The cells being displayed serve as the insoluble matrix carrier.   Population of display packages under conditions compatible with antibody binding to the target antigen To the affinity matrix. Then the specificity of the antibody for the target antigen Does not significantly affect binding, but does not affect display packaging to antigen or matrix By washing with a solute that substantially blocks any non-specific binding of the Fractionate. The binding, incubation and subsequent washing conditions can be adjusted. Shows the binding characteristics of the antibody recovered from the display library. Can be controlled to a certain degree. Affinity and specificity identification Within the range of temperature, pH, ionic strength, divalent cation concentration, and wash volume. The time and time can be chosen for the antibody. Usually expects high affinity Selection based on the possible slow dissociation rate is a very practical method. Saturated amount of free Continued incubation in the presence of puten (when available) Or increase the volume, number and length of washes. You may. Reassociation of dissociated antibody-display package in each case Antibodies with high affinity over time-display The package is collected. In addition, further changes in binding and cleaning methods One may go to find antibodies with particular properties. Some antibody afini The tee depends on ionic strength or cation concentration. Removal of protein from antibody This is useful for affinity purification of various proteins when mild conditions are required for It is a useful property of the antibody used. A special example is that the binding activity is Ca⁺⁺Depends on Exist and release their haptens in the presence of EGTA (Hopp (Hopp ) Et al. (1988) Biotechnology Volume 6: 1204- 1210). First Ca⁺⁺Isolate the antibody that binds to the hapten in the presence, and then , A double screenin identifying antibodies that do not bind in the presence of EGTA in this group Enables such antibodies to be identified in recombinant antibody libraries by You.   To remove non-specifically bound display package, if desired After "washing" with specific elimination (with excess antigen) or non-specific elimination (p H, using agents that reduce polarity or chaotropic agents Specifically) to elute the specifically bound display package. Can be. In the preferred embodiment, the elution protocol is a display package. It does not kill the organisms used as a display, and as a result, the display package The enriched population of cages can be further amplified by growth. Of possible eluents The list is salt (one of the counter ions is Na⁺, NH_Four ⁺, Rb⁺, SO_Four ^2-, H₂ PO_Four ^-, Citrate, K⁺, Li⁺, Cs⁺, HSO_Four ^-, CO_Three ^2-, Ca²⁺, Sr²⁺ , Cl-, PO_Four ^2-, HCO_Three ^-, Mg²⁺, Ba²⁺, Br^-, HPO_Four ^2-, Or asse Target such as salt), acid, heat, and soluble form, if available It includes an antigen (or an analog thereof). During the affinity separation process, the bacteria Continues metabolism and, in general, is more susceptible to damage due to rough conditions, Buffer when display package is more bacteria than phage or spores The components (especially the eluent) can be more limited. Ethanol, acetone Neutral solutes, such as ether or urea, can be combined with the display package. Examples of other agents useful for eluting.   In a preferred embodiment, an affinity-enhanced display package. The amplified DNA was repeatedly amplified and affinity was increased until the desired enrichment of binding activity was detected. Subject to further rounds of separation. In certain embodiments, specifically bound data Display packages, especially bacterial cells, do not need to be eluted by themselves, , The matrix-bound dice to inoculate the appropriate growth medium for growth. The spray package can be used directly.   Especially when the display package is phage particles, the cpIII protein is When used as a display anchor, the fusion produced with the coat protein Combined protein may substantially interfere with subsequent amplification of eluted phage particles There is. Some antibody constructs were present in only one of the 5-6 tail fibers Even because of their size and / or sequence May cause serious defects in infectivity. This means that after each sorting cycle Cause re-infection and loss of phage from the population during amplification. Specific example of 1 Antibody is directed onto the surface of the display package and displayed. Re Antibodies that cleave at least the antigen-binding site of the antibody from the rest of the package. Be sensitive to white cleavage. For example, M13 cpIII coat protein If such a method is used, a part of the antibody and the coIII part of the tail fiber fusion protein are Infectious phage can be obtained by treatment with an enzyme that cleaves between For example, using the enterokinase cleavage recognition sequence).   To further minimize the problems associated with incomplete infectivity, the eluted fa DNA prepared from DNA is electroporated or well-known chemistry Can be transformed into a host cell by a suitable means. Enough for marker expression Cells are cultured for hours and selection is applied as is typical for DNA transformation. Coro The knee is amplified and the phage harvested during subsequent rounds of selection.   The data encoding the antibody with the desired binding specificity for the immunorecessive epitope. After isolating the spray package, each of the refined display package A suitable eukaryotic or prokaryotic expression vector for the nucleic acid encoding the V-gene And cloned into a host suitable for large-scale protein production. Can be operated. For example, the isolated V-gene lacks part of the constant region. , If it is desired that the missing parts be provided, then a simple molecular cloning method should be used. You can use it to add the lost parts back. Use target epitope Confirming antibody binding affinity by well-known immunoassays Yes (eg Harlow and Lane, Antibodies : A Laboratory Manual (Antibodies: A Laboratory Manual), Cole Cold Spring Harbor Laboratories Press atory Press), Cold Spring Harbor, NY (Cold Spring Harbor, N Y) (1988)). V.Additional antibody handling, antibody compositions, and immunoassay kits   Another aspect of the invention is a chimeric antibody, eg, isolated by the method described above. At least the antigen-binding portion of immunoglobulin is contained in another protein, preferably in another protein. Antibody that has been cloned into the antibody of. Contemplated by the present invention In an embodiment of a chimeric antibody that is -Completing part of the constant region isolated from the gene library, and Invariant regions that differ from all or part of the constant region of the antibody isolated from the offspring library Region, eg, from different IgG such as IgG1, IgG2 or IgG3 In the constant region or from one of IgE, IgA, IgD or IgM It is possible to easily perform "class switching" for replacing with the invariant region of. As well Cloned single-chain antibodies and other recombinant fragments in Can be generated from a gene.   When therapeutically using an antibody produced in a non-human subject in humans, various To the extent that it is recognized as foreign and an immune response may occur in the patient. Absent. Therefore, a non-human V-gene library as described in the Examples below. Antibody is derived from recombinant antibody using recombinant engineering to the isolated antibody gene. Can be converted into The term "humanized antibody" refers to an immunoglob derived from a non-human species. It has an antigen-binding site derived from phosphorus, which renders the molecule substantially immunogenic in human subjects. The remaining immunoglobulin-derived portion of the molecule is -Used to refer to molecules that are derived from immunoglobulins. In humanized antibodies , The antigen-binding site is, for example, a complete variable domain fused to an invariant domain, Is only CDRs connected to appropriate framework regions in human variable domains Includes. Such an antibody is the equivalent of the above recombinant antibody, but is therefore May be more tolerant if injected into.   In an illustrative embodiment, H3-3, FB3-2 or Prepared any of the F4-7 antibodies to produce each of these mouse-derived genes. The human constant regions for the heavy and light chains of can be included. For example, Part of the antibody gene that encodes the invariant region encodes the human invariant region Can be replaced with a gene (Robinson et al. International patent application PCT / US86 / 02269; European patent application No. 184,187 to Akira et al. European Patent Application No. 171,496 of Taniguchi, M .; Morrison; European Patent Application No. 173,494 to Morrison et al .; Neuberger PCT Application Publication WO 86/01533; US Patent to Cabilly et al. No. 4,816,567: European Patent Application No. 125,023 to Kabile et al .: Better (Better) et al. (1988) Science 240: 1041-1. 043; Liu et al. (1987) PNAS 84: 3439-344. Page 3; Liu et al. (1987) Journal of Immunology (J.Immnol.) Volume 139: 3521-3526; Sun et al. (1987) PNAS No. 84: 214-218; Nishimura et al. (1987) Cancer -Research (Canc. Res.) Volume 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; (Shaw) et al. (1988) Journal of National Cancer Inn Situation (J. Natl. Cancer Inst.) Volume 80: 1553-1559. See).   Replace part of the variable region that is not involved in antigen binding with an equivalent part of human variable region The subject clear antibody can also be "humanized" by. "Humanized" chimeric antibody A general review of Morrison, S.L. (1985) Science 229: 1202-1207; and Oi et al. (1986). Year) BioTechnologies Volume 4: Provided by page 214 Is done. Those methods include immunoglobulin derived from at least one heavy or light chain. Nucleic acid sequence encoding all or part of a variable region Including that. Sources of such nucleic acids are well known to those of skill in the art. Then texture CDNA encoding the antibody or fragment thereof in an appropriate expression vector Can be cloned. Alternatively, replace the appropriate "humanized" antibody with the CDRs. Can be manufactured by (US Patent No. 5 to Winter) , 225, 539; Jones et al. (1986) Nature 321 : 552-525; Verhoeyan et al. (1988) Sayen Su 239: 1534; and Beidler et al. (1988) J. Journal of Immunology (J.Immnol.) Vol. 141; 4053-406 (See page 0).   The DNA sequence encoding the chimeric variable region was synthesized by oligonucleotide synthesis. You may prepare it. This includes at least the framework regions of the first antibody and At least the CDRs sequences of the subject antibodies are known or can be readily determined. It is necessary. Determination of these sequences, DNA synthesis from oligonucleotides And preparation of suitable vectors are well known to those of skill in the art with reference to the teachings herein. It includes the use of known methods that are more easily performed.   Alternatively, the DNA sequence encoding the altered variable domain can be used as a primer. It can be prepared by directed site-directed mutagenesis. Essentially this The method includes mutating an oligonucleotide that encodes the desired mutation. Hybridizing with single-stranded DNA, the single-stranded DNA And obtaining a chain containing the mutation as a template for extension. Zoller ) Et al. (1982) Nuc Acids Res Res Volume 10: 6487-6500; Norriset et al. (1983) Nu Craic Acids Research Vol. 11: 5103-5112; Solar et al. 1984) DNA 3: 479-488; and Kramer et al. 1982) Nucleic Acids Research Volume 10: 6475-6485 The page describes this method in various forms. For most reasons, The pure form of the method does not always produce a high frequency of mutations. However , For the introduction of both single and multiple mutations into M13-based vectors. Ryoho is Carter et al. (1985) Nucleic Acids Reser Chi 13: 4431-4443. Long oligonucleo With Tide, many changes can be introduced simultaneously (eg Carter et al., Supra). It is possible that each code a CDR. Humanized three CDRs derived from the subject antibody using a single oligonucleotide It can be introduced into the framework region of an antibody (US Pat. No. 5,345,8). See also No. 47). This method uses whole V for insertion into the expression plasmid._HDomei This method is more laborious than whole gene synthesis because it can be easier than cutting Not only does it require power, but also variable domains with the required specificity. It represents a particularly convenient way of expression.   Oligonucleotides used for site-directed mutagenesis Of the subject antibody by the use of suitable restriction enzymes. It may be prepared by isolation from DNA encoding the variable domain. General Typically, such long oligonucleotides are at least 30 residues in length and It may be up to 80 residues or more.   In yet another embodiment, PCR is used to obtain the fusion protein. Mela antibody can be obtained. Further annealed to form a chimeric V-gene array Complementary over between two consecutive CDR and FR fragments that give rise to a row Using the anchor primer from which the hang is generated, both CDR and FR are PCR amplification of the gene fragment can be performed (eg Ausubel (Au sbel) et al., Current Protocols in Molecular Biology ( Current Protocols in Molecular Biology), John Willie & Son (John Wiley & Sons): 1992).   The antigen-binding site of the subject antibody is used to include a protein sequence derived from a non-immunoglobulin molecule. A fusion protein can be obtained. For example, such a chimeric antibody is a Pseudomonas Proteins, such as the addition of the exotoxin or diphtheria toxin domains of Or a protein domain that renders it cytostatic (eg, Jung et al. (1994) 19) Proteins 19: 35-47; Seethara m) et al. (1991) Journal of Biological Chemistry (J Bio l Chem) 266: 17376-17381; and Nichola. Et al. (1993) Volume 268, Journal of Biological Chemistry. : 5302-5308); DNA binding to facilitate DNA transport. Polypeptides (see, eg, US Pat. No. 5,166,320); phosphatases Or a touch that provides immunoglobulin-related enzyme activity, such as peroxidase activity. Antibody domain using matrix derived from glutathione For production of glutathione-S-transferase polypeptides (eg, Edited by Ausbel et al., Current Protocols in Molecular J. Wu, Current Protocols in Molecular Biology John Wiley & Sons: 1991) or Ni^{2 +} Poly (His) -by affinity chromatography using metal resin Poly (His) / enterokinase cleavage site sequences that allow antibody purification (eg, For example, Hochuli et al. (1987) Journal of Chromatograph. F. (J. Chromatography) Volume 411: 177; and Junk Necht (Ja nknecht) et al., PNAS Vol. 88: 8972). It can include a purified polypeptide that oxidizes.   In addition, the present invention is directed to isolated cellular or otherwise other cellular and extracellular proteins. , Especially antigenic proteins or other extracellular factors to which the antigen normally binds To make available isolated forms of the subject antibodies. The term "other cellular And substantially free of extracellular proteins (also referred to herein as "contaminating proteins") or "Substantially pure or purified preparation" means less than 20% (by dry weight) of a mixture. To include a preparation of the subject antibody containing an input protein, preferably less than 5% contaminating protein. Defined. First, using the cloned gene described herein, the The subject antibody can be prepared as a purified preparation. "Purified" means a peptide or When referring to DNA or RNA sequences, the molecule is not a source of other sources such as proteins. It means that it exists in the substantial absence of a physical polymer. This specification The term "purified" preferably means at least 80% by weight, more preferably Present in the range of 95-99% by weight, most preferably at least 99.8% by weight Means biological macromolecules of the same type (but water, buffers, other small molecules, In particular, molecules with a molecular weight of less than 5000 may be present). As used herein, the term “pure” Preferably have the same numerical limitations as "purified" immediately above. "Isolated The term "purified" and "purified" refers to natural substances or ingredients in their native state. Separated (eg, on an acrylamide gel) but pure (eg, contaminating proteins, Or modifiers or polymers such as acrylamide or agarose. Natural (not containing chromatographic reagents) or not available as a solution Does not include substances.   Yet another aspect of the invention relates to preparations of the subject antibodies, especially pharmaceutical preparations. Conveniently, the antibody of the present invention or a pharmaceutically acceptable salt thereof is treated with water or a buffered saline. , Polyols (eg glycerol, propylene glycol, liquid polyethylene) Glycol, etc.) or suitable mixtures thereof. It may be formulated for administration with the body. Optimum of active ingredient in selected medium The concentration can be determined empirically according to methods familiar to medicinal chemists. It will depend on factors such as the route of administration illustrated, the age of the patient as well as the body weight. This specification The "biologically acceptable medium" used in is suitable for the desired route of administration of the pharmaceutical preparation. Comprising, and including all solvents, dispersion media and the like. Pharmacologically active substance The use of such media for media is known in the art. Any idiomatic The active medium or agent is dependent on the activity of the antibody (eg its specificity and / or affinity). The use in the pharmaceutical preparation of the present invention is not limited to Is contemplated. Their formulations, including suitable carriers and other proteins, are described, for example, in remycin. Remington's Pharmaceutic al Sciences (Mac Publi, Easton, PA, USA) In a book called Sack Company (Mack Publishing Company, 1985) It is listed. These carriers are injectable "deposit formulas. ons) ”. Based on the above, such pharmaceutical formulations may include one or more When mixed with a pharmaceutically acceptable carrier or diluent, at a suitable pH and physiologically Includes a solution or lyophilized powder of the antibody contained in a buffered medium that is isotonic with the liquid Yes, but do not exclude anything else. For explanation purposes only, limited to the same item But for the treatment of proliferative disorders using the anti-cancer cell antibodies of the invention. A possible composition or formulation which can be prepared as a solution form in US Pat. No. 5,218,218, No. 094. For freeze-dried preparations, mannitol or glycine Support excipients such as (but not excluding others) In addition, prepare an appropriate buffered solution of the desired volume and prepare a sufficient You may get it. A similar composition was prepared for a pharmaceutical composition consisting of the antibody in the desired volume of isotonic solution. A liquid may be used, and an isotonic pharmaceutical preparation always having a desired pH (eg, neutral pH) Got Buffered saline solution containing appropriate concentrations of phosphate or citrate for Is included (not excluding others).   Yet another aspect of the invention is, for example, the detection of immunorecessive epitopes in a sample. The present invention relates to an assay kit which can be used when it is used. Generally, the assay key For example, spectroscopic methods (including FACS) or radiographic methods To immunorecessive epitopes derivatized with a labeling group that can be finally detected by the method. Antibodies are provided. For example, labels are used for many radioisotopes, fluorescent compounds. , An enzyme, and an enzyme cofactor. Description Then, the labeling groups are horseradish peroxidase and alkaline phosphatase. , Β-galactosidase, luciferase, urease, fluorescein And its analogs, rhodamine and its analogs, allophycocyanin, R- Phycoerythrin, erythrosine, europium, luminol, luciferin, Coumarin analog,¹²⁵I,¹³¹I,^ThreeH,³⁵S,¹⁴C and³²From the group consisting of P It may be a functional group of choice.   The assay kit provided by the present invention comprises several different types of subject antibodies. May also include a selection of Antibody in solution or in lyophilized form It may be. In some embodiments, the antibody is pre-bound to the solid support. May be present on the surface of the solid support when the kit is used. May be applied. The labeling means may be pre-bound to the antibody, or used. Prior to use, one or more components, such as buffers, antibody-enzyme conjugates, fermentations, It may be one that requires a bond with an elementary substrate or the like. Many types of detectable labels Are commercially available and may comprise one or more components of the kit. Various detection Possible labels are known in the art and suitable labeling groups are not It is generally recognized that it is a group that releases a group. Immunoassay performed Various labeling groups can be used depending on the type of. Useful labels include fluorescent and Some include radioactivity, phosphorescence, chemiluminescence, bioluminescence and free radicals. further , Labeling groups include polypeptides (eg enzymes or proteins), polymers, polysaccharides, acceptors Body, cofactor and enzyme inhibitors may be included. The kit of the present invention is further reagent May be included. Additional reagents reduce non-specific binding to solid surface Blocking agents, detergents, enzyme substrates, etc. The solid surface is Microtiter made of materials such as polyvinyl chloride and polystyrene suitable for immobilization It may be in the form of a plate, a microsphere, or the like. VI.The subject methods and typical applications of antibodies obtained by the subject methods   The subject method of the invention is used to produce antibodies useful in purification, diagnostic, and therapeutic applications. Can be applied to advantage. Antibody display, which may be from an immunized animal In contrast to library, antibody display obtainable by the subject method B) a large population of high affinity antibodies to the immunorecessive epitope of interest And a display package comprising antibodies specific for immunorecessive epitopes. Establish an extensive pool of space. Regarding immunorecessive epitopes, the display of the present invention Effective access to the antibody repertoire provided by the Ray Library is described in, for example: Allows effective enrichment by affinity selection means to occur.   As described above, the immunorecessive epitope is used as a trelage that is used in the suppressive immunization process Can be defined in terms of toleragens and immunogens, and therefore they Is also a unique immunogen for toleragen. Thus, the desired antibody When it is necessary to distinguish between various cells of common or similar origin or phenotype , The relevant cells whose desired antibody is to be identified are used as the toleragen, but the antibody of the present invention Cells that are more specifically bound by are used as immunogens. Table 1 shows the immunogen Used in the subject method of isolating antibodies that specifically bind to an epitope that is A representative system of possible immunogens / treragens is shown. Selection of toleragen and immunogen For example against tumor cell markers, fetal cell markers, and stem cell markers. To provide a specific antibody.   Similarly, the subject method can be used to identify a variant form of a protein or a particular protein in a protein family. Immunogens consisting of variant proteins such as Soja and changes in wild-type or variant proteins By using toleragen consisting of isolated isoforms, it is possible to An antibody can be obtained which can be distinguished from the related forms of Variant protein and wild Determinants between protein (or other isoforms) (ie immunorecessive epitopes) ) Differences typically consist of only minor differences in amino acid residues (ie , Less than 15%, preferably residues of the order of 1 to 3). For example, exempt Such combinations of epidemics and toleragens have been used in the present invention to provide oncoproteins or tumors. Ulcer repressor protein, hemoglobin, apolipoprotein E, LDL receptor, heart Visceral β-myosin, sodium or other ion channel, collagen, gluco Induce antibodies that can specifically bind to kinases, or variant forms of transthyretin can do.   In an exemplary embodiment of the invention, the subject method is used to employ cell type-specific markers. To obtain an antibody against As shown in Example 2, using the method of the present invention, fetal cells Antibodies can be produced that are specifically directed against a specific marker. For example, mother Primary use of sex erythroblasts as toleragen and fetal red immunogens as immunogen By the method described above, specific antibodies to the markers of fetal nucleated red blood cells can be obtained. it can. By specific recognition of epitopes on the cell surface such as hematopoietic progenitor cells When obtained to distinguish between fetal and maternal cells, for example, fluorescence activated cells. By means of vesicle sorting (FACS) using the antibodies obtained by the subject method. Offspring cells can be separated from maternal blood. Isolated fetus such as fetal nucleated erythroblasts Puppy cells are a non-invasive source of fetal DNA for genetic screening of parents. More effective and safer than, for example, amniocentesis or chronic villous sampling And provide an alternative to the invasive method.   In another embodiment, the present invention provides an anti-antibody specific for a tumor cell specific marker. Contemplate the generation of the body. As described in Example 1, the subject method was advantageously used to Antibodies can be obtained that can distinguish between cells and their transformants. Such antibody May be suitable for diagnostic and therapeutic applications. For example, neoplastic or hyperplastic cells Antibodies in the assay of the invention for detecting cell-specific markers found on cells Can be selected. Transformed cells are transformed with the antibody thus obtained. Adenocarcinoma, papilloma, squamous and transitional cell carcinoma, For undifferentiated carcinoma, carcinoma, mesothelioma, liver cancer, melanoma, and germ cell tumor. Sometimes used to diagnose cancer and tumors. By antibodies or by delivery of toxins , Or of the transformed cells bound by delivery of the nucleic acid construct for gene therapy. Differential activation of complement at the cell surface allows for both in vivo and in vitro In these, these antibodies may be used to selectively destroy transformed cells. For example , Using normal colon cells as toleragen, cells from colon carcinoma as immunogen Therefore, an antibody specific to a colon cancer marker can be obtained in the present invention. Wear. In a similar fashion, using the subject method, metastasis of highly metastatic tumor cells It is possible to produce an antibody that specifically inhibits. The tumor cells are highly metastatic Unique variant (i.e., its expression is increased over the non-metastatic variant) Using such an antibody designed to recognize Block the function of cell surface proteins containing these epitopes in the cascade Can be   In a similar manner, specific antibodies to stem cell or embryonic neuronal markers are located. If desired, differentiated neurons are used as a toleragen to reduce neurite cells. Or immune cells using embryonic nerve cells such as neutral progenitor cells The antibody repertoire from which it is derived and used in the subject method can be obtained.   For hematopoietic cell-specific antibodies, the immunogen may consist of hematopoietic stem cells. Relagen may be non-neutral stem cells.   In a further embodiment, the subject method involves the generation of antibodies capable of distinguishing various proteins. Can be applied. Various isoforms of naturally occurring proteins using such antibodies Can be identified and mutations that may have occurred in the protein can be detected. Real In an exemplary embodiment, caused by a mutation in an oncogene or anti-oncogene Can be used in immunochemical assays to detect transformation of cells Antibodies can be produced according to the present invention. For example, using the subject method wild Antibodies can be obtained that distinguish between type ras and mutant forms of ras. For example, r The Ser-17 → Asn variant of as was used as an immunogen, and wild-type ras was treated as a tolerage. Used as an antibody to obtain antibodies useful for detecting ras-induced transformation of cells. Can be.   Similarly, it binds specifically to the wild-type tumor repressor protein and the variant tumor repressor. Antibodies useful for diagnosis, which distinguish them from proteins, can be produced by the present invention. For example, inactivating mutations in either p53 or the Rb tumor suppressor. And leads to withdrawal from cell senescence, leading to transformation. Using the subject method Antibody specific for variant p53 can be obtained, which discriminates between wild type and mutant. Ability of Arg-273 → Cys, Tyr-163 → Asn, Val-157 → Phe or Cys-238 → Unique created by mutation such as Phe Caused by recognition of different epitopes. Therefore, a suitable immunogen / trerage The set of proteins includes p53 variants and wild-type p53.   Producing antibodies for detecting variant hemoglobin molecules using the subject method Can then be treated with abnormal redness such as sickle cell anemia and β-thalassemia. It can be used as a diagnostic tool for detecting hemocytosis. Mostly dot sudden Many such abnormalities caused by mutations are associated with disease in embryos, fetuses, neonates, and adults. It has been observed as abnormal hemoglobin (for review, Huisman (Hu isman) (1993) Baillieres Clin Hae matol) Volume 6: 1-30). It is therefore a unique hemoglobin variant. Antibodies to the pitope detect and detect both normal and abnormal hemoglobin levels. And can be very useful in quantitation.   The immunogen is apolipoprotein E4 (ApoE4), and the toleragen is other ApoE. ApoE4 levels in plasma or serum of patients, if composed of isoforms Specific antibodies, which may be useful for the analysis, can be isolated by the subject method. Presence of ApoE4 variant is associated with increased susceptibility to Alzheimer's disease (Strittmatter et al. (1993) PNAS 90:80 98-8102) and of cholesterol lipids and lipoproteins in individuals. Significant impact on level changes (Rall et al. (1992) Journal J. Intern.Med, 231: 653-659; and And Weisgraber et al. (1990) Journal of Lipi J. Lipid Res. 31: 1503-1511). Similar or In other words, including an antibody capable of specifically binding to ApoE2 or ApoE5, etc. It is possible to obtain an antibody specific to the ApoE isoform of.   Other typical embodiments include, for example, predicting the risk of familial hypercholesterolemia. Antibodies useful for LDL receptor variants useful in Specific antibodies against cardiac β-myosin variants that can be used; Alterations in sodium or iron channels that cause regular paralysis of umemia Specific antibodies to species morphology; C that may represent a factor predisposing to the risk of familial osteomyelitis Antibodies to collagen variants such as ys-579 collagen; insulin independent Specific antibodies against variants of glucokinase that cause persistent diabetes; and And transfamiliary amyloidogenic polyneuropathy may cause transthyretin Production of antibodies specific for the variant of. VII.Antibody specifically reacting with fetal cell / cancer cell epitope   Cell surface markers for fetal and transformed cells, as described in detail below. The subject method was advantageously used for the development of antibodies against Conventional hybridoma Method, or in contrast to using a pre-immune phage display library, Performing the subject method yields a library of antibodies that is enriched very rapidly. Can be. The present invention uses unknown / unknown The first example shows that an antibody specific for an isolated cell surface antigen was obtained. In fact, immunity Immunized with a recessive epitope but tolerized against background epitopes Using V-gene library derived from animal (not different from the method of the present invention) Early experiments yielded the subject antibody but were very difficult and expensive, and probably It was suggested that it could not be obtained by the combined display method of line technology.   To illustrate, FIG. 5A shows identification from a tolerized V-gene library. Shows a rapid enrichment of antibodies. For comparison, FIG. 5B was prepared according to the prior art. Phage libraries (# 1 and # 2 that were not tolerized) were serially screened. Not show significant enrichment by (Compare # 1 and # 2). Similarly, as shown in more detail below, Obtained by immunotolerization protocol despite several years of hybridoma research Even the hybridomas obtained using the obtained B cells, fetal blood cells and maternal blood The antibody that distinguishes from liquid cells has the same quality as the anti-CD171 antibody. Was only obtained.   On the other hand, the subject methods include, for example, sorting live cells, FACS assays, Has a high affinity allowing detection of specific antibodies by cell-based ELISA -Provides a library containing a rich source of antibodies. To explain further, The examples provided are for individual antibody display packages in a single round of selection. It is described that the concentration was increased to 5000 to 3600000 times. Specific isolate DNA sequence analysis of Escherichia coli reveals a remarkable history of affinity maturation of both heavy and light chains And, surprisingly, show effective access to the immunological repertoire.   Moreover, accelerated maturation of antibodies, and possibly such accelerated maturation In addition to causing Selection of antibodies with both high binding affinity for active epitopes To In fact, antibodies isolated by the subject method can be used by using prior art Compared with various antibodies, the antibody of the present invention, which is derived in combination, is Better specificity and affinity compared to available antibodies It becomes clear that they tend to be of the order of size.   Relates to three of the antibodies that exhibit both the desired specificity and binding affinity The gene was sequenced. F4-7 and H3-3 as described in Example 2. The antibody was originally isolated by a selection method involving fetal hepatocytes. H3 Due to the further characterization of the -3 antibody, this antibody is Not only were blood cells recognized, but they were stained in late pregnancy despite being inadequate. It is confirmed that the recognized fetal cells were also recognized. Perhaps this is immune and Utilization of fetal liver predominantly from early blood cell precursors during both saturation and enrichment To reflect. However, the population of antibodies enriched from the library Antibodies specific for epitopes present on late fetal blood cells were selected. The combing is shown below. Characterization of FB3-2, one of the isolates Have a very low background staining level for adult blood cells Was shown (for example, less than 0.1%). The nucleic acid of each of these clones and The index for the amino acid sequence is shown in FIG. The overall structure is shown in FIGS. 8A and 8B.   Isolated from the V-gene library of immunized animals by the method of the invention Antibodies were obviously not utilized by other prior art, and in fact It was shown to exhibit properties far superior to the obtained antibodies. Completed by the subject method In contrast to antibodies, the same immunization process, but combined with the use of hybridoma methods Fetal cell selectivity can be obtained using the specified process Was. The specificity for one of the best of these antibodies, "anti-Em", is shown in Table 3. You. Fetal cell selective antibodies isolated by other groups using other hybridoma methods Also compared. As indicated in the art, anti-CD71 antibodies are used in fetal cells. It is considered to be the best of the cell-specific antibodies. However, as shown in Table 3 As such (see also Example 4), the antibody obtained by the method of the present invention is immunotolerant (anti- Em) and each of the antibodies obtained by the hybridoma method (anti-CD71) Works with better quality than.   Anti-Em and anti-CD71 antibodies were each induced by methods in the prior art. It is believed that there is excellent specificity for the derived anti-fetal cell antibodies. But, As shown in Table 3, background that binds to maternal peripheral blood mononuclear cells (PBMC) The levels of these are, for these antibodies, the background of maternal cells using the subject antibody. Many times more expensive than round dyeing. As a result, anti-Em, anti-CD71 antibody, etc. Sufficiently stains fetal cells, but their background to maternal blood greater than 5% Ground staining collects a very small population of fetal blood cells from a maternal blood sample It provides substantial room for improvement over antibodies useful for doing so.   One of the evaluations for fetal cell concentration in maternal blood is 100,000 adult nucleated blood cells. 1 out of 1 or 1 in 1 million adult nucleated blood cells (ie. 001% to 0.0001%). Anti-obtained by subject method The quality attributes of the body are these antibodies in the generation of fetal cells from maternal samples. Suggests that it is useful enough. Antibodies isolated by the subject method 400 male fetuses to further demonstrate improved quality over the antibody known in Puplets were added to 1 million maternal cells and the mixture was fluorescein-conjugated H3-. Stained with 3 Fab. FACS sorting is performed to collect the stained cells, and then Perform in situ hybridization to detect Y chromosome DNA. When released, it showed a recovery of male fetal cells with a yield of 75% and a purity of about 40%. (300 out of a total of 800 recovered cells were male cells). Above The best results obtained with either -Em or anti-CD71 antibodies were fetal The same percentage of cell recovery was reached, but the order of purity was low ( For example, 200-300 fetuses in the background of 100,000 PBMCs. Child cell).   Some of the antibodies obtained by the subject method are clearly superior to prior art antibodies. Another feature is the binding affinity of these antibodies for fetal cell-associated antigens. About. As described in Example 3, affy of H3-3 and FB3-2 antibodies. Nity was determined on human erythroleukemia (HEL) cells ("HEL scan"). HEL Scatchard assay "). In each case, the coupling constant (K_a ) Is 10⁹Exceeded. For example, the monomer H3-3 and FB3-2Fab 'flags Me 6x10 each^TenM^-1And 8x10^TenM^-1The binding constant of Braid The dimeric forms of the recombinant antibodies have larger binding constants, each at H3-3 About K_aIs 5x10¹²M^-1, K for FB3-2_aIs 1x10¹²M^-1In Was.   As a result of our findings, the hybridoma method or phage display Against the corresponding antigen, which is superior to the antibodies currently available by either method Antibodies characterized by specificity and / or affinity for immunization A reproducible and predictable method for isolating antibodies that immunoreact with recessive epitopes It is now possible to provide Therefore, in one aspect of the invention, The subject method makes available antibodies specific for immunorecessive epitopes. here And 10⁶M^-1Greater than, preferably 10⁸M^-1Greater than, more preferably about 10⁹M^-1Greater than, and most preferably 10^TenM^-1, 10¹¹M^-1Or 10¹²M^-1Greater than, for example, 10^TenM^-1Through 10¹³M^-1K in the range_aso Antibodies are characterized by a binding constant for an immunorecessive epitope.   In another aspect of the invention, the subject method provides a low level background For convenience of isolation of the stained antibody. These relative specificities are in the order of strength. If not, especially with regard to cell surface epitopes, It may be several times higher than the antibody obtained by the hybridoma method. For example, the subject method is An antibody that is substantially free of background binding to other related cells, eg, related cells 10-fold higher relative specificity of binding to target cells than background binding to Antibodies may be provided. As shown, anti-antibodies that do not substantially cross-react with other epitopes. The body, preferably 20 times more specific than background, more preferably 50 Antibodies can be obtained which are 75 or 100 fold higher, and even more preferably 125 fold higher You. For example, obtained by the method of the present invention represented by FB3-2 and H3-3. Anti-fetal cell antibodies tested were tested by fluorescence activated cell sorting ("FA CS efficacy assay "), each with 125 times higher phase than background It was shown to have pairwise specificity. In contrast, anti-CD71 and anti-Fe anti The body also has a background level that is 7.7 and 2.5 times higher, respectively. It has been found to have specificity. Furthermore, it is manufactured by the subject method. The specificity of the fetal cell-specific antibody is known in the art, such as anti-CD71 antibody. The background staining of maternal cells compared to the isolated antibody You can also For example, preferably, the subject antibody is 2 times less than the anti-CD71 antibody. Stain non-fetal cells, more preferably at least 5 times less, even more preferred Dye at least 20 times less. For the FACS efficacy assay of Example 4 Sometimes such comparisons can be made using standard immunoassays. Typical Anti-CD71 (eg, anti-transferrin receptor) antibodies are 5E9 antibodies (A TCC HB21), L5.1 antibody (ATCC HB84) and L01.1 antibody (Beckton Dickinson Catalog Number 347510 ).   Sequenced specific antibodies, namely H3-3, F4-7 and FB3-2 With respect to, each antibody is further engineered without departing from the purpose and intent of the invention. Can be Therefore, derived from heavy chain (residues E1-S121, SEQ ID NO: 51 ) And from the light chain (residues D1-K111, SEQ ID NO: 53). Mela FB3-2 antibody can be obtained. Similarly, the F4-7 antibody-derived heavy A chain (residues E1 to S121, SEQ ID NO: 55) and a light chain (residues D1 to K111, A chimeric F4-7 antibody containing the variable region of SEQ ID NO: 57) can be provided. You. Furthermore, for example, from the heavy chain of the H3-3 antibody (residues E1 to S115, SEQ ID NO: 59) and derived from light chain (residues D1 to K111, SEQ ID NO: 61) Chimeric H3-3 antibodies containing variable regions are also contemplated.   In a similar manner, each of the heavy and light chains represented by the general formula Chimeric antibodies containing variable regions can be obtained: FR (1) -CDR (1) -FR (2) -CDR (2) -FR (3) -CDR (3) -FR (4), where CDR (1), CDR (2) and CDR (3) are complementarity determining regions derived from the subject antibody. FR (1), FR (2), FR (3) and FR (4) are derived from the second antibody. Indicates a framework area. For example, CDR (1) is SYWLE and CDR ( 2) is EILFGSGSAHYNEKFKG and CDR (3) is GD The heavy chain that is YGNYGDYFDY, and CDR (1) is RASQSVSTSR YSYMH, CDR (2) is FASNLES, CDR (3) is HSWE A chimeric FB3-2 antibody containing a light chain that is IPYT can be obtained. As well , CDR (1) is SSWLE and CDR (2) is EILFGSGSAHYNE A heavy chain that is KFKG and CDR (3) is GDYGNYGDYFDY, and C DR (1) is RVRQSVSTSSSHSYMH and CDR (2) is YASNLL Chimeric F4-7 containing a light chain of ES and CDR (3) of HSWEIPYT Antibodies can be made. In a similar fashion, CDR (1) is DYYMY The CDR (2) is TISDDGTYTYYYADSVKG and the CDR (3) is D The heavy chain that is PLYGS, and the CDR (1) is RSSQSLVHSNGNTYL H and CDR (2) is KVSNRFS and CDR (3) is SQSTHVLT It is possible to provide a chimeric H3-3 antibody containing a light chain that is Each place In this case, the framework regions (FR1 to FR4) to be bound are irrelevant antibodies , Preferably derived from human antibodies.   The invention further relates to methods of making the subject recombinant antibodies. For example, antibodies (also Nucleic acid vector directing the expression of a nucleotide sequence encoding Culture of host cells transfected with And can cause heavy / light chain dimer assembly to occur if necessary. You. The antibody is secreted so that it can be secreted from the mixture of cells and medium containing the recombinant antibody. The body may be isolated. A cell culture contains host cells, media and other by-products. Suitable media for cell culture are well known in the art. Protein A: Cephalo And ion exchange chromatography, gel filtration chromatography, Methods known in the art for antibody purification, including field filtration and electrophoresis. Recombinant antibody peptide used in cell culture medium, host cells, or both Can be isolated from In a preferred embodiment, the recombinant antibody is the purified antibody. Of a GST fusion protein or a poly (His) fusion protein containing a domain that facilitates production. It is a fusion protein.   The invention also provides for transfection to express a recombinant subject antibody. Host cell. The host cell can be any prokaryotic or eukaryotic cell Well, that selection is in part due to the need for post-translational modifications such as glycosylation. May be based on gender. Thus, depending on the subject method, anti-fetal cells or anti-tumor The entire variable region or a selected The nucleotide sequence encoding the portion is used to encode a microbial or eukaryotic process. The recombinant antibody can be produced by Express recombinant antibody gene Binding into genetic constructs such as K. lactis and eukaryotic cells (yeast, avian , Insects or mammals) or prokaryotic cells (bacterial cells) into the host Transformation or transfection can be performed using other well-known proteins such as insulin. , Interferon, human growth hormone, IL-1, IL-2, and other It is a standard method used in the production of recombinant antibodies. According to the present invention, By a microbial means or a tissue culture method. The subject antibody can be produced.   Preferably, the cell line transformed to produce the recombinant antibody is immortal Human mammalian cell line, which is advantageously a myeloma, a hybridoma, a chicken It may also be of lymphocyte origin, such as the Oma or Quadroma cell line. Even finer Cell system is formed by a virus such as the Epstein-Barr virus. Normal lymphocytes such as B cells immortalized by cytoplasmic conversion may be included. most Preferably, the immortalized cell line is a myeloma cell line or derivative thereof.   A nucleic acid encoding the subject antibody protein or its heavy and light chains is labeled with a prokaryotic cell or Ligated into a vector suitable for expression in eukaryotic cells, or both. A recombinant antibody gene can be produced by Recombinant subject antibody The expression vector for the production of E. coli includes plasmids or other vectors. An example For example, a suitable vector for antibody expression is a prokaryotic cell such as E. coli. Derived from pBR322, pEMBL derived plasmid for expression in , PEX derived plasmids, pBTac derived plasmids and pUC derived plasmids. Includes rasmid.   Many vectors exist for the expression of recombinant proteins in yeast. For example , YEP24, TIP5, YEP51, YEP52 and YRP17 are SSE Clonies useful in the introduction of genetic constructs into S. cerevisiae And expression vehicle (eg, Broach et al. (1983). , "Experimental Manipulation of Gene Expressi (Experimental Manipulation of Gene Expression) "(M Inoue ( M. Inoue)), Academic Press (see page 83). This These vectors can replicate in E. coli due to the presence of the pBR322 ori. S. cerevisiae due to the replication determinants of the yeast 2 micron plasmid. It can be duplicated in Cie. In addition, drug resistance markers such as ampicillin Can be used. In illustrative embodiments, H3-3 or FB3-2 The variable region coding sequences for each of the body's heavy and light chain genes are Recombining the antibody using the expression vector obtained by cloning To manufacture.   Preferred mammalian expression vectors facilitate the propagation of the vector in bacteria. For eukaryotic sequences and one or more eukaryotic translocations expressed in eukaryotic cells. Includes both copy units. pcDNA / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pT k2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg Vectors derived from Escherichia coli are suitable for transfection of eukaryotic cells It is an example of an expression vector. Some of these vectors were cloned into pBR322 Sequence in both prokaryotic and eukaryotic cells by modification with sequences from bacterial plasmids. Allows selection by manufacturing and drug resistance. Alternatively, bovine papilloma virus (BPV-1) or Epstein-Barr virus (pHEBo, pREP- And viral derivatives such as p205) in eukaryotic cells. It can be used for temporal protein expression. Preparation of plasmid and form of host organism The various methods used for quality conversion are well known in the art. Prokaryotic cell Other expression systems suitable for both eukaryotic and eukaryotic cells, and general recombinant methods Sambrook, Fritsch "Molecular" edited by (Fritsch) and Maniatis ・ Cloning a Laboratory Manual (Molecular Cloning A Labora tory Manual 2nd edition (Cold Spring Harbor Laboratory Pre) (Cold Spring Harbor Laboratory Press): 1989 ”Chapter 16 and See Chapter 17. In some cases, recombination through the use of baculovirus expression systems It may be preferable to express the antibody. Such a baculovirus expression system Examples include vectors derived from pVL (pVL1392, pVL1393 and a vector derived from pAcUW (such as pVL941) (of pAcUW1). And a vector derived from pBlueBac (including β-gal (For example, pBlueBacIII). VIII.Example   The present invention is now described generally and only describes particular aspects and embodiments of the present invention. Reference is made to the following examples which are included for the purpose of, but are not intended to limit the invention. The invention will be more easily understood by the following.   For cell surface markers on fetal and transformed cells, as described below. The subject method was advantageously applied to the development of antibodies. Conventional hybridoma method or pre- In contrast to the use of immunophage display libraries, the present invention A remarkable library of rapidly enriched antibodies can be obtained. The following In the exemplary embodiments described in the examples, individual antibody display packages Was enriched 5000 to 3600000 times in a single round selection . DNA sequence analysis of specific isolates showed affinity for both heavy and light chains. A notable history of maturity is shown, with unexpected and effective access to the immunological repertoire. Access was shown. I.Materials and methods   Unless otherwise indicated, Sambrook et al., Molecular Cloning: A Ra Volatility Manual, Cold Spring Harbor Laboratory Program Res, Cold Spring Harbor, NY (1989). Thus, recombinant DNA and microbiological methods were performed. The following materials and methods Antibody display library described in both Example 1 and Example 2 using the method I gotBiochemical reagents   The DNA-modifying enzyme was added to New England Biolabs. abs) (Beverly, Mass.), recommended by the supplier It was used under the specified conditions. Perkin Elmer (Connectica) Obtained from Norwalk, Tut. Life Technologies chnologies (Gaithersburg, Maryland) The set of NA fragments (1 Kb ladder) was subjected to agarose gel electrophoresis. Was used as a standard for the molecular weight of the DNA fragment. DNA primer -Genosys, Inc. (The U.S.A. The Woodlands or Kluakem, Incorporated (Crua chem, Inc.) (Sterling, VA) as routinely synthesized Was done. Deoxyadenosine 5 '[α- (³⁵S) Thio] triphosphate New England Nuclear (Boss, Massachusetts) Purchased from Boston. Polyclonal biotinylated anti-M13 antibody 5 prime-3 prime (Boulder, Colorado) ). Styreptoavidin-alkaline phosphatase and polycloner A goat anti-mouse kappa alkaline phosphatase From Fisher Biotech (Pittsburgh, PA) Obtained.Bacterial strains and culture   E. coli strains XL-1, SolR, and LE392 were added to Stratagene (Stra tagene) (LaJolla, CA). Lambda Fa -Resistant XL-1 was isolated by standard methods and is described herein. LB, SO B, 2X YT, NZY medium (Sambrook, 1989) or TB medium: 1 Bacto-tryptone 12 g, yeast extract 24 g and glycero per liter 0.1M KH containing 5.04 g₂PO_FourBuffer, pH 7.5 (phosphate buffer In a Erlenmeyer flask filled with E. coli was grown to stationary phase at 30 ° C or 37 ° C with shaking. Due to the growth of bacteria on the solid medium, cold to a final concentration of 2% (weight / volume). Heaven (Difco, Detroit, Mich.) Was added. Up to 0.5%, carbenicillin, chloramphenicol, and And kanamycin, if needed, should be added to 50, 30, and 50 μg, respectively. Was.Vectors and bacteriophage   E. coli cloning vector Lambda SurfZap^TMAnd helper Phage ExAssist^TMAnd VCSM13 were obtained from Stratagene.Cell preparation   Interstate Blood Products from whole blood from non-pregnant individuals (Interstate Blood Products) (Tennessee). Adult peripheral blood mononuclear cells ("PBMC") as standard Ficoll-Hypaque Grazi It was prepared by the ent method. Fetal blood mononuclear cells are used for 12 to 20 weeks gestation (at that age). And the liver is a primitive hematopoietic period) from a liver obtained from a fetus aborted Prepared. Sterile microscreen in the presence of sterile Ca-Mg-free PBS The cells were released from the surrounding connective tissue by passing through. Cell suspension obtained Is diluted to 20 ml with PBS and the blood mononuclear cell fraction is diluted with standard phyco It was obtained by a vacuum-hyper gradient gradient centrifugation. After recovery from Ficoll interface , Adult and fetal cells were washed twice with sterile Ca-Mg-free PBS and Resuspend in 2 x 10 per ml⁷I made it into an individual.Tolerance immunization   The use of cyclophosphamide tolerance on intact, fixed cells is relevant. This is a well-known method in the field. The method according to the invention may be performed on whole blood, bone marrow or Immediately after isolation from fetal liver, unfixed cells are used to chemically and / or chemically It also avoids the occurrence of antigenic changes due to physical processing. Shiku Rofosfamide was obtained from Sigma chemical and reconstituted 10 mg / ml sterilized saline. The tolerization method used was Matthew et al. (1987) J. Immunol Methods No. 1 Volume 00: essentially the same as the method of pages 72-82.   Another schedule of tolerization and immunization is shown below: 6 weeks old female B alb / c mouse 1 × 10 in 500 μl PBS⁷Individual adult PBMCs intraperitoneally ( "IP") was injected. Cyclophosphamide 10 minutes after injection of adult PBMC An IP injection was made to give 0 mg / kg. Cyclo at 24 and 48 hours The phosphamide was repeated. After another 14 days, the tolerization was repeated.   After 3 weeks 1 × 10 in 500 μl PBS⁷IP injection of individual fetal cells The mice were immunized with fetal mononuclear blood cells by. After two more weeks, the first tolerization Mice were tolerized again with adult PBMCs as described for rounds. Finally, after 3 weeks, 1x10 in 500 μl PBS.⁷IP injection of individual fetal cells , To re-tolerize the fetal blood mononuclear cells. Fetal cell immunization 24 hours And at 48 hours. After an additional 24 hours, the mice were sacrificed. The spleen It was taken and immediately frozen in liquid nitrogen.RNA isolation and cDNA synthesis   Standard method (Chomczynski, US Pat. No. 4,843,155) Was used to isolate total RNA from spleen or hybridoma cell lines. RNA marker The product was stored in RNAase-free water at -70 ° C until use (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Sp Re Ning Harbor Laboratory Press, Cold Spring Harbor, N Y (1989)). Swear from Life Technologies -Superscript preamplification kit (Superscript preampli First strand cDNA was prepared using the fication kit) according to the supplier's recommendations.DNA isolation   Plus from E. coli for DNA sequence analysis or restriction enzyme analysis Isolation of the mid DNA was carried out by the alkaline lysis method (Birnboim and Dolly ( Doly), 1979). The manufacturer, Macherey-Nagel agel) (Durem, Germany) A large amount of plasmid was prepared using obond) column chromatography. Plasmid DNA fragment from E. coli clone containing antibody clone All cultures for dissociation were treated with 0.5% glucose and 50 μg / ml carbe. Grow overnight at 37 ° C with shaking in 2xYT medium containing nicillin.PCR amplification of antibody kappa chain and IgG1 heavy chain coding region   Is it a repertoire of antibody coding regions encoded in spleen mRNA? Minimized the bias towards a limited set of PCR products, and FIG. 1A to amplify over 90% of the sequences encoded by the Fab and heavy chain Fabs. And the set of degenerate primers shown in Figure 1B was designed. Of kappa or heavy chain Amplification of coding sequences was performed using 5 primer pairs for each went. The primer also contains a restriction enzyme site, and the Surf-Zap vector ( And a light chain into a bacterial Fab expression cassette suitable for insertion in (Stratagene) And allowed the ligation of heavy chain PCR products. Automated using the following protocol PC with a built-in BioSystems temp-cycler The R reaction was performed. Generally, Superscript First Strand Thin For Cesis Kit (Superscript first strand synthesis kit) (BRL) Then, the RNA was converted into cDNA. Appropriate primer pairs (Figures 1A and 1B) See, 10 mM Tris-HCl, pH 8.3, 50 mM KCl. 1.5 mM MgCl₂, 100 μl buffer containing 0.01% gelatin Incubate 0.5-1 μg of the first strand cDNA in Cool to 60 ° C., then add 2 U of Taq polymerase. Then, The following four temperature profiles were run for 35 cycles to amplify the product: 72 ° C 1 20 seconds, 54 ℃ 90 seconds, 45 ℃ 30 seconds. After 35 cycles, sample at 72 ° C Polymerization of the product was completed by incubation for 10 minutes. Adjust individual reactions Approximately 1-2 μg of the 0.7 Kb PCR product is used for a minimum number of cycles (usually 3 0-38 cycles) is important.Assembly of Fab expression cassettes from kappa and heavy chain PCR products   1-2 μg of each PCR product from 5 separate reactions were mixed to mix kappa chain and Separate heavy chain product pools were obtained. Then to the supplier (Millipore) As recommended, first use PVDF spin filters (Millipore) to remove proteins. And debris were removed and the pool was then spun onto a 30,000 MW cutoff. The pool was purified by removing low molecular weight components using Luther. Each product Approximately 5 μg of the SfiI buffer was added to 300 μl of SfiI containing 50 units. Digested in fur for 2 hours at 50 ° C. Produced by enzyme and SfiI digestion Removed small end fragments using the above spin column method did. The SfiI digested light chain product was digested with SfiI in a volume of 50 μl at 4 ° C. overnight. It bound to the digested heavy chain products (about 2 μg each). The binding mixture is then Were purified by spin column as above, and each of NotI and SpeI restriction enzymes in 100 μl Digested with 50 units. The digestion products are separated by agarose gel electrophoresis, The 1.4 kb kappa chain heavy chain encoding the dimer was converted to Gene Clean II (Gen as recommended by the supplier using eClean II) (Promega) And purified.   A simple and more reliable method for the construction of Fab 'expression cassettes is shown in FIG. Shown in agram. Approximately 10 ng of the above kappa and heavy chain product pools were transferred to And amplified with the primers shown in FIGS. 1A and 1B) to generate 3'kappa and 5 '. The heavy chain sequence was obtained. These were treated with T4 polymerase in the presence of dTTP. Highly compatible with kappa and heavy chain sequences, resulting in a compatible overhang of 8 bases Allows for a more directed bond. The PCR product was purified as above. 5 3 in a volume of 50 μl containing units of T4 polymerase, 5 mM dTTP About 2 μg of each product was treated separately at 7 ° C. for 1 hour. Similar to the PCR product The products were purified by adding about 500 ng of each product for 3 hours at room temperature for 2 units of D The ligation was carried out in a binding buffer (Promega) having a volume of 25 μl containing NA ligase. It matched.   Except for annealing at 55 ° C for 1 hour and extending the extension time to 4 minutes, Using the 5'kappa chain primer and 3'heavy chain primer shown in FIG. Amplifies Fab encoding dimer obtained by either method under conditions did. Generally, 12-25 cycles under these conditions will yield about 1-2 μg of A 1.4 kb kappa heavy chain dimer was obtained. Use the spin column as described above. Of the product, and then added 75 units each of NotI and SpeI restriction enzymes. Digested in a containing volume of 200 μl. 100,000MW spin column (Amico More effective removal of primers from digestion products using Amicon digestion products The digestion product was purified as described above except that Purified 1.4 kb The dimer was stored at 4 ° C until use.Construction of various Fab clone banks   Fragment encoding NotI-SpeI digested 1.4 kb Fab Ligation was performed as follows: Promega containing 3 units of T4 ligase 2 μg of 0.2 μg digestion product in 10 μl binding buffer overnight at 4 ° C. Of the lambda surf-zap arm. Then Giga-Pack Gold ・ Supplier of packaging (Giga-pack Gold packaging kit) A portion of the binding mixture into the lambda head, as recommended by Gene. Pack Caged. The packing reaction was titrated with E. coli LE392 and pooled. To obtain a primary library. This primary library is then used using conventional methods. Was amplified in E. coli LE392. Generally, 5x10⁹Eco LiXL1 cells are treated with 10 ml of 10 mM MgSO 4._FourIn 10⁷In vitro pad Infection with the packaged SURF-ZAP primary clone for 10 minutes at 37 ° C. Was. Infected cells were added to 100 ml NZY top agarose at 50 ° C. Instantly Play the mixture on two 20 x 20 cm plates containing NZY agar. It was allowed to set, solidified, and then incubated for 8-16 hours. SM bus Amplification by stacking 25 ml of buffer and rocking for 2 hours The harvested library was harvested.Generation of phage antibody clone bank   In essence the M13 Exerciser (as recommended by Stratagene exassist) by helper phage superinfection, primary lambda SURF-ZAP The phagemid clone bank was rescued from Ibrary. Generally 10¹¹Pieces E. coli XLI cells from our amplified surf-zap library Of 10^TenLambda clones and 10¹²Sensitivity to individual exerciser M13 phage Dyed. Cells are grown by centrifugation for 3.5 hours in LB or TB medium and then centrifuged. Was removed. Treat the exorcist rescue library at 70 ° C for 20 minutes, then Stored at 4 ° C.   Infection with E. coli SOLR 1: 1 rescue phagemid We obtained 10 to 100 times more than the size of the primary library. A cell population containing a nicillin resistant antibody clone was obtained. T containing carbenicillin Transduced cells were grown to early log phase in B medium, then , Cells were infected with a 10-fold excess of VCS M13 helper phage. Three After 1 hour at 7 ° C, kanamycin was added and the culture was shaken until stationary phase. And incubated at 30 ° C. Remove cells by centrifugation and centrifuge Therefore, by recovering, the phage antibody was recovered from the supernatant, and 1 ml of TE bag was recovered. Dissolved in fur and then PEG precipitated. Phage antibody to 1 ml TE or It was dissolved in PBS buffer and stored at 4 ° C.Enrichment of cell surface bound phabs on whole cells   Cell-specific phage antibodies were isolated by enrichment of whole cells. Blocking bag Fur (Hanks, 0.1% hydrolyzed casein in buffered salt solution, 3% B) Cells were prepared for enrichment by washing twice with SA). First round of darkness During thickening, 10 in 200 μl blocking buffer¹¹Individual phage antibody 10⁶Cells were added and incubated on ice for 1 hour. Then cold Remove non-specific phage antibody by washing 8 times with blocking buffer did. For centrifuging at 3500 rpm in an Eppendorf microcentrifuge The cells were harvested after each round of washing. The cell surface-bound phage antibody was then added to 3 500 μl 0.2 M glycine pH 2.2 containing M urea and 0.5% BSA Eluted in. Remove debris by centrifugation, 50 μl 1M Tris pH The supernatant was neutralized by adding 9.5. Amicon 100,000MW Urea and buffer with 3 buffer exchanges using a Tooff spin column The ingredients were removed. The enriched population of phage antibodies was titrated on XL1 cells and then Then, it was amplified by the following protocol. Elution in 200 μl SM buffer The phage antibody was added to 1 ml of 10 mM MgSO 4._Four5x10 in⁹Pieces of platey Ng cells and incubated at room temperature for 10 minutes. Then the infected Cells inoculate 100 ml TB broth in a 2 L flask and shake 3 times Incubated at 0 ° C. After incubation for 1 hour, carbenisi Phosphorus was added to make 50 μg / ml. Early stationary phase (OD at two consecutive points Incubation at 30 ° C. with shaking until the increase of 600 remains the same). Continued. 12 out of the Dupont / Sorvall SS34 rotors Cells were removed by centrifugation at 000 rpm for 10 minutes. Then fur The diantibody was purified by PEG precipitation. The amount of phage antibody to be loaded is 10^TenDown to Decrease, increase the number of washings (10 to 20 times), Prior to elution with glycine-urea, 100 mM citrate buffer (pH 4.5 or In a subsequent thickening round by adding a wash at pH 3.5) Increased stringency of cleaning.Preparation of individual phage antibodies for analysis of binding specificity   E.coli XL1 was infected with a phage antibody dilution to give 0.5% glucose and And LB medium containing 50 μg / ml carbenicillin. 20 with 2 ml of 2xYT medium containing 50 μg / ml carbenicillin mm inoculate test tubes with the isolated colonies and shake at 30 ° C overnight. Propagated. The next morning, gently shake 1 ml of the culture at 37 ° C for 1 hour, then , 10¹¹Were infected with M13VCS phage. 2 containing carbenicillin Inoculate a 250 ml flask containing 5 ml TB broth with the VCS infection culture, Shake at 30 ° C for 1 hour, add kanamycin to 50 μg / ml, and stabilize at the initial stage. Incubation was continued until the period (OD600nm = 5-12). Centrifugation The cells were removed by and the phage antibody was purified by PEG precipitation as described above. The phage antibody was dissolved in 200 μl of TE (pH 7.5) buffer.Sequencing of antibody isolates   Individual isolates such as H3-3, FB3-2 and F4-7 clones were isolated , Standard Protocol, edited by Ausbel et al., "Current Protocol" Current Protocols in Molecular Biol ogy) "(John Willie and Sons: 1992; and Sam Brook Et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Herba , NY: 1989), and 3'and 5'of Surf-Zap vector. Heavy and light chain inserts using primers based on flanking sequences, respectively It was sequenced.Flow cytometric assay of phage antibody bound to whole cells   A flow cytometer for testing phage antibodies bound to surface markers on whole cells. I devised a rotocall. 1x10⁶Each adult or fetal mononuclear cell was Dispense into a black test tube and add blocking buffer as in the phage concentration method. Washed. 2x10 for the first assay^TenCells washed with 1 phage And to a volume of 100 μl with casein / BSA / HBSS. Phage Incubated with cells for 1 hour at 4 ° C. 1 ml of phage-cell Washed 3 times with blocking buffer. Biotinylated sheep anti-phage poly Clonal antibody (manufactured by 5 prime-2 prime) For di-cells, 5-7.5 μl per sample (for each lot of polyclonal antibody Optimization) was added. Bring the volume to 100 μl again with blocking buffer. Was. Anti-phage were incubated at 4 ° C for 90 minutes. Streptavidin -FITC (Jackson Immunoresearch) C Dilute 1:50 with a-Mg-free PBS and 250 μl of each phage-cell sample Was added to. After incubation for 30 minutes at 4 ° C, phage-cells were blocked. Wash twice with King buffer and add 400 μl of 0.5% formaldehyde It was fixed by doing.   All samples were analyzed by flow cytometry. Non-display phage ( The fluorescence background was determined by using VCS M13) as a negative control. Specified. The FITC fluorescence above the background on each cell is specifically bound by the fluorescence. It was directly proportional to the number of pages.   Determine the relative avidity of each clone by evaluating two parameters did. The two parameters are: (1) relative cell surface epitope numbers and each Scattering parameters for fluorescence intensity to determine expression homogeneity for phage clones. Turn (larger number, more uniform number is most preferred), (2) phage And titration of retention of fluorescence binding strength-of relative phage antibody affinity It is for decision.   In a modified version of the above binding assay, soluble antibody (“Fab”) is tested for activity. Tested. For Fab fragments, antibody-phage / streptavidin -FITC is a goat anti-IgG-FIT that recognizes the κ chain of Fab fragment. C F (ab ')₂It was replaced with a polyclonal antibody (TAGO). 2 per sample 0.5% normal human goat anti-IgG-FITC30 diluted 1:10 in serum μl was used. For dilution into human or serum, use the above polyclonal antibody and human or blood. It was ensured to minimize cross-reactivity with cellular antigens. II.Example 1: Enrichment of phage antibody on cancer cells   6x10⁷F from IgG1 and kappa chains containing 1 primary clone Tolerizing a combinatorial phage display library of ab in human adult blood Constructed from mice immunized with fetal hepatocytes. Claws due to individual growth characteristics Contains antibody clones or pools of clones to minimize bias Always place cultures in rich medium (TB or 2xYT with 1% glucose) Was. In addition, cultures used to produce phage antibodies should be Harvested near growth. Because binding activity decreases after the start of the stationary phase of proliferation Because it was discovered.   Human / red leukocyte cell line (HEL) is also found on fetal liver cells It has an offspring cell surface marker. This characteristic and the ability to culture this cell To develop a method for enriching cell line specific antibodies from the above phage library A reliable source of cells for. Fur enriched on this cell line (HEL) The binding of the diantibody pool is shown in FIG.   These results are 2.5x10 after 3 rounds of enrichment.^-7From 1.25x10^-3 Up to 4 log increase in phage binding to the cell surface. Was reflected. We used HEL, Raji by fluorescence activated cell sorting. , And of phage antibodies prepared from 16 independent isolates on adult blood cells The specificity was tested. Biotin-labeled anti-phage antibody, followed by fluorescent conjugated strep Specific binding of phage antibody to the cell surface was detected by toavidin. In Figure 4 The results from these assays shown show that 16 isolates (Asteri) from enrichment 3 (Indicated by a square) (that is, three times of interactive panning) ) At least 6 of the later isolated phages) were found only on HEL cells It appears to be specific to certain determinants.   To assess how diverse the population of antibodies enriched on all cells is , Encoding the CDR3-containing region from these six phage antibody isolates The DNA sequence was determined. Only 6 single replicates are present in the 6 HEL-specific isolates I was there. This result shows that the HEL-specific antibody single antibody was obtained by enrichment on whole cells. Showed that separation has been achieved. III.Example 2: Enrichment of phage antibody on fetal cells   In order to maximize the chance of isolation of the basic clone in the fetal cell area, play sucking In addition to non-enrichment, prior to each enrichment cycle on fetal hepatocytes The phage antibody library described in Example 1 was pre-adsorbed on adult nucleated blood. Graphical results of subsequent rounds of pre-adsorption and enrichment on fetal hepatocytes 5A. An increase in the percentage of phage antibodies that bind to fetal hepatocytes is Enrichment for phage antibodies binding to pups is shown.   Remarkably, fetal cell binding after enrichment on a single round of fetal cells. It was observed that a pure population of matching phage antibodies was isolated. From each stage Characterization of random isolate sampling, binding of individual isolates to fetal cells DNA sequence analysis combined with an FACS-based assay for It was shown that. The results of these experiments are shown in FIG. DNA sequencing is fetal cells Based on the amino acid sequence of the heavy chain of the antibody that binds to the Indicates that there are two classes. Each class is a CD that reflects affinity maturation First identified by amino acid changes in or near the R3 region, then It contained subtypes identified by the binding of different kappa chains.   Table 4 shows HEL or with or without pre-adsorption on adult cells. Partitioning of different phage antibody types at different stages of enrichment on fetal cells Show. The three classes of phage antibodies are those against fetal liver cells and adult cells. Recognize three different epitopes based on their different staining profiles there's a possibility that.   Fourth round of enrichment (enriched series including pre-adsorption on adult cells Phas type 5 only) was eluted from fetal hepatocytes. Most strict The choice for this type under dense wash conditions is that it is the best choice for heavy and light chains. It is suggested that it is an affinity binder.   10⁶3 different phages added 1 in non-specific control M13 phage In the simulated enrichment experiment (Table 5), in which antibody was enriched on fetal hepatocytes, A dramatic 10 single round of these phage antibodies above^Five-10⁶Double enrichment Indicated. The order of this enrichment ratio depends on the combination method using purified antibodies. It is the upper limit of the folds reported for induced antibody enrichment, which is How the experimentally targeted epitope is a very complex cell surface antigen It is important if you think that it has not been purified.   The majority of cell surface-bound isolates found on a consistent cell source (HEL) were constructed. It reflects the effectiveness and breadth of the libraries produced. Library construction and amplification In addition to the efficacy of the method of the present invention for Yielding a remarkable library of phage antibodies that can be enriched very rapidly in Was. Such a result shows that p is highly specific for specific target cell types from different individuals. Important for identifying habs. Most isolated without pre-adsorption Fact that other phabs (even with tolerizing immunity) also recognize adult cells This is particularly emphasized by In addition, fetal hepatocytes from independent fetuses Enrichment at each step using Remove antibody. In enrichment for pan-fetal specific antibodies This added rigor underscores the effectiveness of the method of the present invention, and the three classes of bread -Produced 13 different versions of fetal specific antibodies. In contrast, conventional ha Using the same tolerization method with the ibridoma method yielded two IgGs of the same type I was only asked. IV.Example 3: Determination of affinity of H3-3 and FB3-2 antibodies.   Affinity of purified antibodies for Scatchard analysis Decided. A significant excess of varying amounts of antibody at a constant temperature of 4 ° C with a fixed number of HEL cells Incubation for 16 hours. After further washing, the bound antibody was adjusted to pH 2 Was eluted from HEL in and quantified by ELISA. About Scatchard analysis Therefore, free antibody was assumed to be equal to the total number added. For bound / free antibody From the negative slope of the straight line obtained from the plot of bound antibodies, the K of each antibody_aI got Everything All points were performed in 3 systems and correlation coefficients for all reported slopes exceeded 90%. I was V.Example 4: Specificity of H3-3 and FB3-2 antibodies   Antibodies derived from hybridomas such as anti-CD71 and anti-EM are the subject antibodies. For comparison, fetal cells and mothers of these antibodies were analyzed by analytical flow cytometry. Reactivity with sex cells was tested (FACS efficacy assay). Briefly, 10000 cells per sample were stained with a fixed amount of FITC-conjugated pure antibody. 10,000 cells were analyzed for each sample. The results shown in Table 3 above are isotypes − Confirmed by isotype-matched negative control-antibody Percentage of cells where staining was found above established background fluorescence Is shown as "% positive".   High specificity of H3-3 antibody against fetal hematopoietic cells as opposed to adult hematopoietic cells Recognition of FACS and subsequent sorting of sorted cells Further demonstrated by light in situ hybridization (FISH) analysis . Briefly, 400 fetuses shown to be male by Y-PCR Baby hepatocytes were added into 1 million adult PBMCs. Then add the spiked sample G (Hoescht) -stained with DNA dye and 1 μg of FITC-conjugated H3-3 antibody . Sort nucleated cells (cells positive for Hest) for H3-3 positives Mounted on glass slides and analyzed by FISH for the presence of male cells did. Cy3 detected male (Y) probe; female (X) probe on FITC Detected more. The results are summarized below: Initial purity of fetal cells = 0.04%; adult Background staining of cells = 0.05%; recovered fetal (male) cells = 30 1; Purity of fetal cells sorted for H3-3 = 36.4%.   All references and publications cited above are incorporated herein by reference. Be considered.                                   Equivalent   Those of ordinary skill in the art will appreciate that certain methods described herein may be used without undue experimentation. And many equivalents to reagents will be recognized or identified. Take Equivalents are considered to be within the scope of this invention and are covered by the following claims. .

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI // (C12N 1/21 C12R 1:19) (C12P 21/08 C12R 1:19) (81) Designated country EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), AU, CA, JP (72) Inventor Hill House, Dana United States 01915 33, Whitney Avenue, Beverly, Massachusetts, Top Floor (72) Inventor Johnson, Tracy United States 02173 Concord Avenue 136, Lexington, Massachusetts

Claims (1)

[Claims] 1. A method for producing an antibody specific to an immunorecessive epitope, and a nucleic acid encoding the antibody, comprising the steps of: obtaining an antibody repertoire against an immunorecessive epitope derived from tolerance; expressed by a population of display packages An antibody display library consisting of a heterogeneous V-gene library, wherein the V-gene library was cloned from the antibody repertoire, and then the desired binding specificity for the immunorecessive epitope A method comprising selecting a display package of said antibody display library having properties. 2. A method for producing an antibody specific to an immunorecessive epitope, and a nucleic acid encoding the antibody, comprising the steps of: obtaining a heterogeneous display library of antibody variable regions, wherein the antibody variable regions are immunotolerant. Cloned from the antibody repertoire derived from E. coli and then selecting the antibody variable regions of the display library having the desired binding specificity for the immunorecessive epitope. 3. The method of claim 2, wherein the display library is a phage display library. 4. The method of claim 2, wherein the display library is a bacterial cell surface display library or a spore display library. 5. The method of claim 2, wherein said antibody variable region is a heavy chain, a light chain, a heavy chain-light chain pair, _VH , _VL , Fab, Fd, Fv, or scFv. 6. The method of claim 2, wherein said immunorecessive epitope is a cell type specific marker. 7. The method according to claim 6, wherein the immunorecessive epitope is a cancer cell marker. 8. The method of claim 6, wherein said immunorecessive epitope is a fetal cell marker. 9. The method of claim 6, wherein said immunorecessive epitope is a stem cell marker. 10. The method of claim 2, wherein said immunorecessive epitope consists of at least one amino acid residue in a variant protein different from the related protein or parent protein. 11. The method of claim 2, wherein the antibody repertoire derived from said immune tolerance is obtained by chemoimmunosuppression. 12. The method of claim 11, wherein the antibody repertoire derived from immune tolerance is obtained by cyclophosphamide-induced immunosuppression. 13. The method of claim 2, wherein the antibody repertoire derived from said immune tolerance is obtained by neonatal tolerization. 14． The antibody variable region is selected from the display library by a fractional binding means comprising affinity separation of an antibody variable region that specifically binds to the epitope from an antibody variable region that does not specifically bind to the epitope. Method. 15. 15. The method of claim 14, wherein said differential binding means comprises selection of said display library on the cell surface consisting of said epitope. 16. A method for producing a specific antibody against an immunorecessive epitope, and a gene encoding the antibody, comprising: (a) replicable encoding a library of phage particles displaying fusion antibody / coat protein. A library of phage vectors is transformed into a suitable host cell where the fusion protein comprises a phage coat protein portion and an antibody variable region portion, the antibody variable region portion from a V-gene library derived from immunotolerance. (B) culturing the transformed host cell so that the phage particle is formed and the fusion protein is expressed, and (c) an antibody variable region portion that binds to an immunorecessive epitope. A method comprising selecting any of said phage particles having. 17． The transformed host cell further encodes a second variable region linked to the antibody variable region portion of the fusion protein so as to be expressed in the transformed host cell to form a heterodimer Fv. 17. The method of claim 16 which comprises an antibody gene. 18. 18. The method of claim 17, wherein said second antibody gene is obtained from said V-gene library. 19. 18. The method of claim 17, wherein the second variable region is a polypeptide distinct from the fusion protein and further comprises a secretory signal sequence that enables the second variable region to be secreted from the transformed host cell. Method. 20. 18. The method of claim 17, wherein the phage vector further comprises the second antibody gene. 21. 21. The method of claim 20, wherein said fusion protein further comprises said second variable region covalently linked to said antibody variable region portion to form a single polypeptide chain antibody. 22. The method of claim 16, wherein said phage particle is selected from the group consisting of M13, F1, Fd, If1, Ike, Xf, Pf1, Pf3, λ, T4, T7, P2, P4, φX-174, MS2 and f2. 23. 17. The method of claim 16, wherein the phage particle is a filamentous bacteriophage specific for Escherichia coli and the phage coat protein is coat protein III. 24. 24. The method of claim 23, wherein the filamentous bacteriophage is selected from the group consisting of M13, fd, and f1. 25. 17. The method of claim 16, wherein said transformed host cell is cultured with a helper phage suitable for inducing the formation of said phage particle. 26. The phage particles are selected by a fractional binding means comprising contacting the phage particles with the immunorecessive epitope and then separating phage particles that specifically bind to the epitope from phage particles that do not specifically bind to the epitope. The method of claim 16, wherein the method is performed. 27. 27. The method of claim 26, wherein said differential binding means comprises affinity chromatography means provided with said immunorecessive epitope as a component of an insoluble matrix. 28. 28. The method of claim 27, wherein the insoluble matrix comprises the immunorecessive epitope bound to a polymeric support. 29. 28. The method of claim 27, wherein the insoluble matrix comprises immunorecessive cells displaying the target epitope. 30. 27. The method of claim 26, wherein the differential binding means comprises immunoprecipitating the phage particles with a multivalent form of the immunorecessive epitope and then removing non-specifically bound phage particles from the precipitate. 31. 17. The method of claim 16, wherein the immunorecessive epitope is a cell type specific marker. 32. 32. The method of claim 31, wherein the immunorecessive epitope is a cancer cell marker. 33. 32. The method of claim 31, wherein said immunorecessive epitope is a fetal cell marker. 34. 32. The method of claim 31, wherein said immunorecessive epitope is a stem cell marker. 35. 17. The method of claim 16, wherein the immunorecessive epitope consists of at least one amino acid residue in a variant protein that differs from the related protein or parent protein. 36. An antibody specific to an immunorecessive epitope, and a method for producing a gene encoding the antibody, comprising: (a) antibody-producing cells derived from immunological tolerance enriched with respect to cells producing an antibody to the immunorecessive epitope (B) obtaining a heterogeneous V-gene library encoding at least the variable region of an immunoglobulin chain expressed by the enriched population of antibody-producing cells; A library of replicable phage vectors encoding a library of phage particles displaying a coat protein is obtained, wherein each of the phage vectors is a chimeric coat encoding the fusion coat protein. The chimeric gene is composed of a protein gene, and (i) the first region encoding the variable region derived from the V-gene library. An antibody gene, and (ii) a second gene encoding at least a portion of a phage coat protein such that the library of phage vectors encodes a plurality of cloned variable regions, d) transforming a suitable host cell with the library of replicable phage vectors, (e) culturing the transformed host cell so that the phage particles are formed and the fusion coat protein is expressed, and then (F) selecting any of the phage vectors corresponding to phage particles displaying a variable region that binds to the immunorecessive epitope. 37. 37. The method of claim 36, wherein the population of antibody-producing cells derived from said immunotolerance is obtained by chemoimmunosuppression to the production of antibodies to immunodominant epitopes that are normally associated with said immunorecessive epitopes in the immunogen. 38. The population of antibody-producing cells derived from said immunotolerance is obtained by neonatal tolerization to suppress the production of antibodies directed against immunodominant epitopes normally associated with said immunorecessive epitopes in the immunogen The method of claim 36. 39. A method for producing a specific antibody against a fetal cell-specific antigen, and a nucleic acid encoding the antibody, which comprises the following steps: obtaining a display library containing a variety of antibody variable regions, wherein the antibody variable regions are: Cloned from an antibody repertoire derived from immunological tolerance, enriched for antibodies to fetal cell-specific antigens, and then having the desired display specificity for the fetal cell-specific antigens. Selecting the antibody variable region of the rally. 40. 40. The method of claim 39, wherein said antibody variable region of said display library is separated by a step comprising sorting said display library on fetal cells consisting of fetal cell-specific antigens. 41. 40. The method of claim 39, wherein said fetal cell specific antigen is a marker for fetal nucleated red blood cells. 42. An antibody that specifically binds to a tumor / fetal antigen, which is DPLYGS, DP LYGN, DPLYGD, GDYGDYGDYFDY, GDYGNYGDYFDY, GDYGKYGDYFDH, a variable region consisting of a variable region consisting of a 3 chain of amino acids in the EGYGPD, which is a variable region consisting of GDYGKYGDYFDH, GVYGKYGDYFDY, and the EGYGPMD TGYSAMD An antibody having. 43. Furthermore, SQSTHVLT, ALKVHM, HSWEIPYT, QQWS SNPPT, SQSHHVLT, QHSWEIPYT, QDSWEIPYT, QQSNEDPYT, QQSNEDPFT, QQSNEDPFT, a variable region consisting of a variable region of QQSNEDPFT, QQSNEPPT, QHSWEIPFT, and a GQGYS of LQR, which is a GQGYS. antibody. 44. An antibody isolated by the method of claim 1. 45. An antibody isolated by the method of claim 16. 46. An antibody isolated by the method of claim 36. 47. An antibody isolated by the method of claim 39. 48. A heterogeneous V-gene library expressed by a population of display packages and enriched for specific antibodies by differential binding to immunorecessive epitopes, cloned from an antibody repertoire derived from immune tolerance. An antibody display library enriched for specific antibodies to immunorecessive epitopes consisting of rally. 49. 49. The antibody display library of claim 48, wherein said display package is phage particles. 50. 49. The antibody display library of claim 48, wherein an antibody repertoire derived from the immunotolerance using a set of immunogens in which the immunorecessive epitope consists of a cell-type specific marker and trellagen is obtained. 51. 51. The antibody display library of claim 50, wherein the cell type specific marker is a fetal nucleated red blood cell marker, the toleragen comprises maternal erythroid cells and the immunogen comprises fetal erythroid cells. 52. 51. The antibody display library of claim 50, wherein the cell type specific marker is a tumor cell marker. 53. 53. The antibody display library of claim 52, wherein the tumor cell marker is a colon cancer marker, the toleragen comprises normal colon cells and the immunogen comprises colon carcinoma cells. 54. 53. The antibody display library of claim 52, wherein the tumor cell marker is a metastatic tumor cell marker, the toleragen comprises non-metastatic tumor cells and the immunogen comprises metastatic tumor cells. 55. 51. The antibody display library according to claim 50, wherein the cell type-specific marker is a precursor nerve cell marker, the toleragen comprises differentiated nerve cells, and the immunogen comprises embryonic nerve cells. 56. 51. The antibody display library of claim 50, wherein the cell type specific marker is a hematopoietic cell marker, the toleragen comprises progenitor cells and the immunogen comprises hematopoietic stem cells. 57. 49. The antibody display library of claim 48, wherein an antibody repertoire derived from said immunotolerance using a set of immunogens and trellagens in which said immunorecessive epitope is unique to the variant form of the protein is obtained. 58. 58. The antibody display library of claim 57, wherein the variant protein is apolipoprotein E4, the toleragen comprises apolipoprotein E4, and the immunogen comprises apolipoprotein E4. 59. 58. The variant protein is a p53 mutant having one or more amino acid residues different from wild-type p53, the toleragen consists of wild-type p53, and the immunogen consists of the p53 mutant. Antibody display library. 60. 58. The variant protein is a ras mutant having one or more amino acid residues different from wild-type ras, the toleragen comprises wild-type ras, and the immunogen comprises the ras mutant. Antibody display library. 61. 49. A heterogeneous population of antibodies cloned from the antibody display library of claim 48. 62. 49. An isolated antibody of the antibody display library of claim 48. An antibody having a binding constant for an immunorecessive epitope greater than 63.1 × 10 ⁸ M ⁻¹ , and a method for producing a nucleic acid encoding the antibody, the method comprising the steps of: Obtaining an antibody display library consisting of a heterogeneous V-gene library expressed by the population of the display package, wherein the V-gene has been cloned from the antibody repertoire, then 1x10 Selecting a display package of the antibody display library having a binding constant for the immunorecessive epitope greater than ⁸ M ^-1 . 64. A method for producing an antibody selective for an immunorecessive epitope and a nucleic acid encoding the antibody, the method comprising the steps of obtaining an antibody repertoire for an immunorecessive epitope derived from immunological tolerance, and An antibody display library consisting of a heterogeneous V-gene library expressed is obtained, wherein the V-gene has been cloned from the antibody repertoire and then the immunity of greater than ¹ × 10 ⁸ M ^−1. A method comprising selecting a display package of the antibody display library having a binding constant for a recessive epitope and a relative specificity of at least 10 times greater than binding for a background antigen. 65. An antibody that selectively binds to an immunorecessive epitope that is unique to the first cell phenotype of a related cell population, and a method for producing a nucleic acid that encodes the antibody, the method comprising the steps of: Obtaining an antibody repertoire to an immunorecessive epitope in the first cell phenotype to obtain an antibody display library consisting of a heterogeneous V-gene library expressed by a population of display packages, wherein the V-gene live is obtained. The rally has been cloned from the antibody repertoire and has one or more of the following steps: (i) A display package having substantial background binding to cells of the relevant cell population other than the first cell phenotype. From the antibody display library, and (ii) individual selection modes A enriched display library is obtained by removing from the antibody display library a display package that binds to the first cell phenotype, wherein the enriched display library is the antibody display library. Of the remaining display packages of, and then selecting a display package of the enriched display library having a desired binding affinity for the first cell phenotype. 66. 66. The method of claim 65, wherein said immunorecessive epitope is a fetal cell marker. 67. 66. The method of claim 65, wherein said immunorecessive epitope is a cancer cell marker. 68. 66. The method of claim 65, wherein said immunorecessive epitope is a stem cell marker. 69. 66. The method of claim 65, wherein the display packages of the enriched display library are selected by selecting the display packages on cells of the first cell phenotype. 70. An antibody that immunoreacts with a fetal cell surface antigen having a binding constant for the antibody of greater than ¹ × 10 ⁸ M ⁻¹ and no substantial background binding to maternal cells. 71. An antibody that specifically immunoreacts with a fetal cell surface antigen and is characterized by a specificity of at least 10 times greater than background binding to a maternal antibody. 72. Antibodies that specifically immunoreact with fetal cell surface antigens and are at least twice as low as maternal cells than an anti-CD71 antibody selected from the group consisting of 5E9 antibody, L5.1 antibody, and L01.1 antibody An antibody with background binding. 73. An antibody that binds to a tumor / fetal antigen, comprising an antigen binding site consisting of one or both of a first variable region and a second variable region, each of the first and second variable regions being H3-3. An antibody, which comprises the complementarity determining region of the antibody, the FB3-2 antibody or the F4-7 antibody. 74. Each of the first and second variable regions has the general formula: FR (1) -CDR (1) -FR (2) -CDR (2) -FR (3) -CDR (3) -FR (4) [Formula FR (1) to FR (4) are polypeptides derived from the antibody framework region, and CDR (1) to CDR (3) are total complements of H3-3 antibody, FB3-2 antibody or F4-7 antibody. The antibody of claim 73, wherein the antibody is derived from the sex-determining region. 75. Each of the CDR (1), CDR (2), and CDR (3) for a single variable region is 77. The antibody of claim 74 having an amino acid sequence selected from the group consisting of: 76. The variable regions are E1 to S121 of SEQ ID NO: 51, D1 to K111 of SEQ ID NO: 53, E1 to S121 of SEQ ID NO: 55, D1 to K111 of SEQ ID NO: 57, E1 to S115 of SEQ ID NO: 59, and a sequence. 74. The antibody of claim 73 selected from the group consisting of D1-K111 of No. 61. 77. 74. The antibody of claim 73, wherein the antibody further comprises a human-derived framework region polypeptide. 78. The antibody of claim 73, wherein the antibody further comprises a human / antibody-derived constant region polypeptide. An antibody display library enriched for antibodies with binding constants for cell surface antigens greater than 79.10 ⁸ M ^-1, which is expressed by a population of display packages to distinguish the cell surface antigens from immunorecessive epitopes. An antibody display library consisting of a heterogeneous V-gene library enriched for specific antibodies by binding, the V-gene library being cloned from an antibody repertoire derived from immune tolerance. 80. 80. The antibody display library of claim 79, wherein said cell surface antigen is a fetal nucleated red blood cell marker. 81. 80. The antibody display library of claim 79, wherein said cell surface antigen is a tumor cell marker. 82. It consists of a heterogeneous V-gene library encoding at least the variable region of an immunoglobulin chain expressed by an antibody producing cell of an animal, and the antibody producing cell is enriched by immunological tolerance for the cell producing an antibody against an immunorecessive epitope. A library of isolated nucleic acids encoding an antigen-binding site that immunoreacts with an immunorecessive epitope. 83. 83. The gene library of claim 82, wherein said V-gene library is expressed by a population of display packages. 84. 84. The gene library of claim 83, wherein said display package is phage particles. 85. The gene library of claim 82, wherein said immunorecessive epitope consists of a tumor / fetal cell surface marker.