Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
  • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)

Definitions

• antibodies are synthesized and secreted into bodily fluids by plasma cells, a type of terminally differentiated B-lymphocyte. Exposure of the animal to a foreign molecule (i.e. via immunization) generally produces multiple plasma cell clones resulting in a heterogeneous mixture of antibodies (polyclonal antibodies) in the blood and other fluids.
• the blood of an immunized animal can be collected, clotted, and the clot removed to leave a sera containing the antibodies produced in response to immunization. This remaining liquid or serum, which contains the polyclonal antibodies, is referred to as antiserum.
• antiserum contains many different types of antibodies that are specific for many different antigens. Even in hyperimmunized animals, seldom are more than one tenth of the circulating antibodies specific for the particular immunogen used to immunized the animal. The use of these mixed populations of antibodies, though useful in many situations, can create a variety of different problems in immunochemical techniques. For example, such antiserum will generally be inadequate for use in distinguishing between the immunogen and closely related molecules which share many common determinants with the immunogen.
• Monoclonal antibodies are traditionally made by isolating a single antibody secreting cell (e.g. a lymphocyte) from an immunized animal, fusing the lymphocyte with a myeloma (or other immortal) cell to form a hybrid cell (called a "hybridoma"), and then culturing the selected hybridoma cell in vivo or in vitro to yield antibodies which are identical in structure and specificity.
• a single antibody secreting cell e.g. a lymphocyte
• myeloma or other immortal
• the antibody-secreting cell line is immortal, the characteristics of the antibody are reproducible from batch to batch.
• the usefulness of monoclonal antibodies stems from three characteristics - their specificity of binding, their homogeneity, and their ability to be produced in virtually unlimited quantities. While production of monoclonal antibodies has resulted in production of antibodies of greater specificity to a particular antigen then polyclonal methods, there are nevertheless a number of limitations associated with these techniques and antibodies produced thereby.
• a key aspect in the isolation of monoclonal antibodies relates to how many antibody producing hybridoma cells with different specificities can be practically established and sampled in response to immunization with a particular antigen, compared to how many theoretically need to be sampled in order to obtain an antibody having specific characteristics. For example, the number of different antibody specificities expressed at any one time by lymphocytes of the murine immune system is thought to be approximately 10 7 and represents only a small proportion of the potential repertoire of specificities.
• Immunization regimens can provide enrichment of B-cells producing the desired antibodies.
• typical protocols for isolating antibody producing B-cells permit sampling of generally less than 500 antibody producing hybridoma cells per immunized animal.
• traditional techniques for the production of monoclonal antibodies statistically favor generation of monoclonal antibodies to immunodominant molecules, making isolation of antibodies specific for a rare or less immunodominant epitope difficult.
• This problem can be further exacerbated by the fact that in many instances pure antigen is not available as an immunogen, particularly in the case of cell surface antigens. Immunization with intact cells frequently results in production of antibodies against irrelevant epitopes, especially for xenotypic immunization.
• Neonatal tolerization and chemical immunosuppression are most commonly used to reduce clonal expansion of B cells in response to "background” antigen signals, thereby enriching for a population of B cells responsive to the epitopes of interest.
• the practical application of a subtractive immunization technique can be very difficult, as the efficiency of immunosuppression is often not acceptable, or as in the case of cyclophosphamide immunosuppression, generally results in only a few antibody-producing hybridoma cells per immunotolerized animal (e.g. less than 100), making it unlikely that monoclonal antibodies can be isolated which are specific to the immunorecessive epitopes.
• the present invention provides a method for generating an antibody which is specific for an immunorecessive epitope, and nucleic acid encoding the antibody.
• the subject method generally comprises the steps of generating a variegated display library of antibody variable regions, and selecting from the library those antibody variable regions which have a desired binding specificity for the immunorecessive epitope.
• the antibody variable regions used to generate the display library are cloned from an immunotolerance-derived antibody repertoire.
• the antibody variable regions of the display library are presented by a replicable genetic display package in an immunoreactive context which permits the antibody to bind to an antigen that is contacted with the display package.
• affinity selection techniques can be utilized to enrich the population of display packages for those having antibody variable regions which have a desired binding specificity for the immunorecessive epitope.
• the display library can be a phage display library.
• the display library can be generated on a bacterial cell-surface or a spore.
• the subject method can be used to isolate antibodies which are specific for such immunorecessive epitopes as, for example, cell-type specific markers, including fetal cell markers such as fetal nucleated red blood markers, cancer cell markers such as colon cancer markers or metastatic tumor cell markers, stem cell markers such as markers for precursor nerve cells or hematopoietic stem cells.
• cell-type specific markers including fetal cell markers such as fetal nucleated red blood markers, cancer cell markers such as colon cancer markers or metastatic tumor cell markers, stem cell markers such as markers for precursor nerve cells or hematopoietic stem cells.
• the subject method can be used to generate antibodies which can discriminate by binding between a variant form of a protein and other related forms of the protein.
• the variant protein can differ by one or more amino acid residues from other related proteins in order to give rise to the immunorecessive epitope, as well as vary antigenically from the related protein by virtue of glycosylation or other post-translational modification.
• the variation can arise naturally, as between different isoforms of a protein family, illustrated by the apolipoprotein E family, or can be generated by genetic aberration, as illustrated by the neoplastic transforming mutations of oncogenic proteins or tumor suppressor proteins such as p53.
• a specific antibody to an immunorecessive epitope can be generated by affinity purification of a antibody phage display library derived from an immunotolerance-derived antibody repertoire.
• suitable host cells are transformed with a library of replicable phage vectors encoding a library of phage particles displaying a fusion antibody /coat protein, where the fusion protein includes a phage coat protein portion and an antibody variable region portion.
• the antibody variable region is obtained from the immunotolerance-derived antibody repertoire.
• the transformed cells are cultured, the phage particles are formed, and the antibody fusion proteins are expressed. Any of resulting phage particles which have an antibody variable region portion which specifically binds to a an immunorecessive epitope can be separated from those which do not specifically bind the immunorecessive epitope.
• the present invention further pertains to novel immunorecessive antibody libraries produced by the subject method.
• an antibody display library can be isolated which is enriched for antibodies that specifically bind an immunorecessive epitope of interest.
• the display library comprises a population of display packages expressing a variegated V-gene library which has been cloned from an immunotolerance-derived antibody repertoire, and which has been further enriched after expression by the display package via affinity separation with the immunorecessive epitope. It is also contemplated by the present invention that individual antibodies, and genes encoding these antibodies, can be isolated from the antibody libraries of the subject method.
• individual display packages can be obtained, and the antibody gene contained therein subcloned into other appropriate expression vectors suitable for production of the antibody for the desired use.
• Figures 1A and IB show variable region PCR primers for amplifying the variable regions of both heavy and light chains from murine antibody genes.
• Figure 2 shows a schematic representation of an Fab' expression cassette.
• Figure 3 is a semi-log graph depicting the binding of phage antibody pools (phab) enriched on the HEL cell line (number indicates the round of enrichment). The graph provides additional comparison of the enriched phab pools with the binding of other immunoglobulins (T3, Anti-M and Wilma) to the HEL cells.
• Figure 4 illustrates the percentage of cells (either HEL cells or mature white cells) stained by individual phab isolates generated by the subject method.
• Figure 5 A shows the results of sequential rounds of pre-adsorption and enrichment on fetal liver cells for phab binding.
• the increase in the percentage of phage antibodies binding to fetal liver cells is indicative enrichment for fetal cell binding phage antibodies.
• the phage antibody library was derived using a V-gene library from an immunotolerized host animal.
• Figure 5B compares the results of the immunotolerized experiment in Figure 5A with the results of sequential rounds of panning using phage antibody libraries derived immunized, but not tolerized, host animals.
• Figure 6 show variable region PCR primers for amplifying the variable regions of both heavy and light chains from human antibody genes.
• Figure 7 details the sequences for CDR3 regions of both heavy and light chains for individual phab isolates enriched on fetal cells.
• Figures 8A and 8B illustrate the general features of the FB3-2 and H3-3 antibodies, respectively, including the framework regions (double underline; FRs), complementarity determining regions (CDRs), and constant regions (italics; IgGl CHI or kappa constant).
• the present invention makes available a powerful directed approach for isolating specific antibodies which are extremely difficult or impossible to obtain by current methodologies, and thereby overcomes the deficiencies discussed above.
• One aspect of the present invention is the synthesis of a method that combines immunotolerization and variegated display libraries to yield a dramatic and surprising synergism in the efficient isolation of antibodies having a desired binding affinity for an immunorecessive target epitope. Utilizing immunotolerance techniques such as subtractive immunization, a subset of lymphocytes producing antibodies against an immunorecessive target epitope are enriched in an immunized animal.
• V-genes antibody variable region genes
• the subject method selects genes encoding antibodies specific for the target epitope by (i) displaying the antibodies encoded by each variable region gene on the outer surface of a replicable genetic display package to create an antibody display library, and (ii) using affinity selection techniques to enrich the population of display packages for those containing V-genes encoding antibodies which have a desired binding specificity for the target epitope.
• antibodies isolated by the subject method can have binding affinities greater than 10 8 M _1 , e.g., in the range of lO ⁇ **1 to lO ⁇ M **1 .
• the specificity of these antibodies can be several fold, if not orders of magnitude, better than combinatorial and hybridoma generated antibodies, particularly with respect to antibodies for cell surface epitopes.
• the subject method can provide antibodies which have no substantial background binding to other related cells, e.g., specificities greater than 10 fold binding to the target cells over background binding to the related cells.
• antibodies can be generated which do not substantially cross-react with other epitopes, preferably having specificities greater than 20 fold over background, more preferably 50, 75 or 100 fold over background, and even more preferably more than 125 fold over background.
• the term "antibody” in its various grammatical forms is art-recognized and includes immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen.
• the simplest naturally occurring antibody comprises four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
• the light chains exist in two distinct forms called kappa (K) and lambda ( ⁇ ).
• K kappa
• ⁇ lambda
• Each chain has a constant region (C) and a variable region (V).
• Each chain is organized into a series of domains.
• the light chains have two domains, corresponding to the C region and the other to the V region.
• the heavy chains have four domains, one corresponding to the V region and three domains (1,2 and 3) in the C region.
• the naturally occurring antibody has two arms (each arm being an Fab region), each of which comprises a V L and a VJJ region associated with each other. It is this pair of V regions (V L and V H ) that differ from one antibody to another (owing to amino acid sequence variations).
• the variable domains for each of the heavy and light chains have the same general structure, including four framework regions (FRs), whose sequences are relatively conserved, connected by three hypervariable or complementarity determining regions (CDRs).
• variable region of each chain can typically be represented by the general formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
• the CDRs for a particular variable region are held in close proximity to one and other by the framework regions, and with the CDRs from the other chain and which together are responsible for recognizing the antigen and providing an antigen binding site (ABS).
• ABS antigen binding site
• binding antigens can be performed by fragments of a naturally-occurring antibody, and as set out above, these antigen-binding fragments are also intended to be designated by the term "antibody".
• binding fragments encompassed within the term antibody include (i) the Fab fragment consisting of the V L , V H , CL and C H 1 domains; (ii) the Fd fragment consisting of the V ⁇ and CHI domains; (iii) the Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (iv) the dAb fragment (Ward et al., (1989) Nature 341 :544-546 ) which consists of a VJJ domain; (v) isolated CDR regions; and (vi) F(ab')2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
• antibody variable region is likewise recognized in the art, and includes those portions of an antibody which can assemble to form an antigen binding site.
• an antibody variable region can comprise each of the framework regions (FR1-
• CDR1-CDR3 complementary determining regions
• a desired binding specificity for an immunorecessive epitope refers to the ability of individual antibodies to specifically immunoreact with distinct antigens.
• the desired binding specificity will typically be determined from the reference point of the ability of the antibody to differentially bind, and therefore distinguish between, two different antigens -particularly where the two antigens have unique epitopes which are present along with many common epitopes.
• a desired binding affinity for an immunorecessive epitope can refer to the ability of an antibody to distinguish between related cells, such as between adult and fetal cells, or between normal and transformed cells.
• the desired binding affinity can refer to the ability of the antibody to differentially bind a mutant form of a protein versus the wild-type protein, or alternatively, to discriminate in binding between different isoforms of a protein.
• An antibody which binds specifically to an immunorecessive epitope is referred to as a "specific antibody”.
• the term "relative specificity” refers to the ratio of specific immunoreactivity to background immunoreactivity (e.g., binding to non- target antigens). For instance, relative specificity for fetal cells can be expressed as the ratio of the percent binding to fetal cells to the percent binding to maternal cells.
• Antibodies which have no substantial background binding to a non-target antigen, such as a maternal cell have large relative specificities (e.g., in excess of 10 fold over background binding).
• an antibody binds to only a particular portion of the macromolecule, referred to herein as the "determinant" or "epitope".
• the total number of antibodies produced by a population of antibody-producing cells in a particular animal is referred to a the "antibody repertoire”.
• the extraordinary diversity of the antibody repertoire is a result of variability in the structures of the antigen binding sites amongst the individual antibodies which make up the repertoire.
• immunogens refers to the exposure of an animal (that is capable of producing antibodies) to a foreign antigen so as to induce active immunity, which includes the production of antibodies to the foreign antigen.
• Molecules that generate an immune response are called immunogens.
• immunorecessive epitope which is also substituted from time to time with the terms "rare epitope” or "target epitope”, is intended to refer to epitopes that, in the context that it ordinarily occurs or can be isolated as an immunogen, are typically not efficient for use in generating an antibody response by immunization, at least so far as polyclonal and monoclonal antibody production is concerned. Such immunorecessive epitopes will generally be less abundant and/or less antigenic than other epitopes commonly associated with them in the immunogen.
• Immunorecessive epitopes may be associated with, for example, cell surface antigens that are unique to a particular cell phenotype. In many instances, this cell surface antigen is not in and of itself available as an immunogen because no purified form of the antigen has been obtained.
• an immunogen containing the immunorecessive epitope will also include many background epitopes which can act to decrease the overall percentage of B-lymphocytes activated by the immunorecessive epitope in the total B-lymphocyte population.
• the immunogen can comprise the whole cell on which the immunorecessive epitope is expressed.
• the immunorecessive epitope can be a cell-type specific marker, such as a cancer cell marker, a fetal cell marker, or a stem cell marker.
• an immunorecessive epitope can comprise an epitope unique to a variant form of a protein, such as a variant which differs by only one or two amino acid residues from a related protein.
• the immunorecessive epitope can be a determinant of a mutant p53 which does not arise on the wild-type p53, or an epitope which unique to a particular isoform of human apolipoprotein E, such as ApoE4.
• Tolerization refers to the process of diminishing an animal's immunological responsiveness to a potentially antigenic substance present in that animal, and the antigenic substance to which tolerance is created is refered to as a "toleragen". Tolerance results from the interaction of toleragen with antigen receptors on lymphocytes under conditions in which the lymphocytes, instead of becoming activated, are killed or rendered unresponsive. Tolerance to particular antigens, or more exactly, to particular epitopes of an antigen, can be induced by a number of means, including neonatal tolerization or chemically-induced tolerization, and can be the result of induced clonal deletion or clonal anergy. The route of administration of an antigen can also effect the ability of the antigen to act as either an immunogen or as a toleragen.
• immunotolerizing means relates to a process whereby the antibody response to an immunorecessive epitope is unmasked by the deletion of an antibody response to the background epitopes. For instance, as a first step in the immunotolerizing means, an animal is exposed to a toleragen comprising the immunodominant epitopes. The toleragen, however, lacks the immunorecessive epitopes. After tolerance to these background epitopes has been induced, an immunogen which includes the immunorecessive epitopes, is administered to the animal.
• the immunotolerizing means can be used to "enrich” for cells producing antibodies specific for an immunorecessive epitope.
• background epitopes is further defined as those epitopes that are common between the immunogen and the toleragen, while the term “immunorecessive epitopes” is further understood to refer to epitopes unique to the immunogen (relative to the toleragen).
• the immunogen and the toleragen will typically be closely related, as for example, in the instance of phenotypically related cells, or mutant or different isoforms of a protein.
• immunotolerance-derived antibody repertoire refers to the population of antibody-producing cells, and their antibodies, generated by an immunotolerization which is intended to enrich for antibodies for an immunorecessive epitope.
• variable V-gene library refers to a mixture of recombinant nucleic acid molecules encoding at least the antibody variable regions of one or both of the heavy and light chains of the immunotolerance-derived antibody repertoire.
• a population of display packages into which the variegated V-gene library has been cloned and expressed on the surface thereof is likewise said to be a “variegated antibody display library” or "antibody display library”.
• the language "replicable genetic display package” or "display package” describes a biological particle which has genetic information providing the particle with the ability to replicate.
• the package can display a fusion protein including an antibody derived from the variegated V-gene library.
• the antibody portion of the fusion protein is presented by the display package in an immunoreactive context which permits the antibody to bind to an antigen that is contacted with the display package.
• the display package will generally be derived from a system that allows the sampling of very large variegated V-gene libraries, as well as easy isolation of the recombinant V-genes from purified display packages.
• the display package can be, for example, derived from vegetative bacterial cells, bacterial spores, and bacterial viruses (especially DNA viruses).
• a variegated mixture of display packages encoding at least a portion of the V-gene library is also referred to as an "antibody display library”.
• differential binding means refer to the separation of members of the antibody display library based on the differing abilities of antibodies on the surface of each of the display packages of the library to bind to the target epitope.
• the differential binding of an immunorecessive epitope by antibodies of the display can be used in the affinity separation of antibodies which specifically bind the immunorecessive epitope from antibodies which do not.
• the same molecule or cell that was used as an immunogen in the immunotolerizing step can also be used in an affinity enrichment step to retrieve display packages expressing antibodies which specifically bind it.
• the affinity selection protocol will also include a pre- enrichment step wherein display packages capable of specifically binding the background epitopes are removed.
• affinity selection means include affinity chromatography, immunoprecipitation, fluorescence activated cell sorting, agglutination, and plaque lifts.
• affinity chromatography includes bio-panning techniques using either purified, immobilized antigen as well as whole cells.
• the display package is a phage particle which comprises an antibody fusion coat protein that includes the amino acid sequence of an antibody variable region from the variegated V-gene library.
• a library of replicable phage vectors, especially phagemids (as defined herein), encoding a library of antibody fusion coat proteins is generated and used to transform suitable host cells.
• Phage particles formed from the chimeric protein can be separated by affinity selection based on the ability of the antibody associated with a particular phage particle to specifically bind a target epitope.
• each individual phage particle of the library includes a copy of the corresponding phagemid encoding the antibody fusion coat protein displayed on the surface of that package.
• phage particles Purification of phage particles based on the ability of an antibody displayed on an individual particle to bind a particular epitope therefore also provides for isolation of the V-gene encoding that antibody.
• exemplary phage for generating the present variegated antibody libraries include Ml 3, fl, fd, Ifl, Ike, Xf, Pfl, Pf3, ⁇ , T4, T7, P2, P4, ⁇ X-174, MS2 and 2.
• fusion protein and "chimeric protein” are art-recognized terms which are used interchangeably herein, and include contiguous polypeptides comprising a first polypeptide covalently linked via an amide bond to one or more amino acid sequences which define polypeptide domains that are foreign to and not substantially homologous with any domain of the first polypeptide.
• One polypeptide from which the fusion protein is constructed comprises a recombinant antibody derived from the cloned V-gene library.
• a second polypeptide portion of the fusion protein is typically derived from an outer surface protein or display anchor protein which directs the "display package" (as hereafter defined) to associate the antibody with its outer surface.
• this anchor protein can be derived from a surface protein native to the genetic package, such as a viral coat protein.
• the fusion protein comprises a viral coat protein and an antibody it will be referred to as an "antibody fusion coat protein".
• the fusion protein may further comprise a signal sequence, which is a short length of amino acid sequence at the amino terminal end of the fusion protein, that directs at least a portion of the fusion protein to be secreted from the cytosol of a cell and localized on the extracellular side of the cell membrane.
• Gene constructs encoding fusion proteins are likewise referred to a "chimeric genes" or "fusion genes”.
• chimeric antibody is used to describe a protein including at least the antigen binding portion of an immunoglobulin molecule attached by peptide linkage to at least a part of another protein.
• a chimeric antibody can be, for example, an interspecies chimera, having a variable region derived from a first species (e.g. a rodent) and a constant region derived from a second species (e.g. a human), or alternatively, having CDRs derived from a first species and FRs and a constant region from a second species.
• vector refers to a DNA molecule, capable of replication in a host cell, into which a gene can be inserted to construct a recombinant DNA molecule.
• the use of phage vectors rather than the phage genome itself provides greater flexibility to vary the ratio of chimeric antibody/coat protein to wild-type coat protein, as well as supplement the phage genes with additional genes encoding other variable regions, such as may be useful in the two chain antibody constructs described below.
• helper phage describes a phage which is used to infect cells containing a defective phage genome or phage vector and which functions to complement the defect.
• the defect can be one which results from removal or inactivation of phage genomic sequence required for production of phage particles.
• helper phage are M13K07, and M13K07 gene III no. 3.
• isolated as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs, or RNAs, respectively, that are present in the natural source of the macromolecule.
• an isolated nucleic acid encoding one of the subject antibodies preferably includes no more than 10 kilobases (kb) of nucleic acid sequence which naturally immediately flanks the antibodies gene in genomic DNA, more preferably no more than 5kb of such naturally occurring flanking sequence.
• isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
• an "isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
• the subject invention sets forth a method for rapid and efficient isolation of cell-type specific antibodies.
• antibodies that specifically bind epitopes unique to fetal cells or, alternatively, epitopes unique to cancer cells can be generated by the subject method.
• the subject method can be employed to generate antibodies to variant forms of a protein, and which can be used, for example, to detect a mutation of a protein or to differentiate amongst various isoforms of a protein.
• the present invention can provide antibodies useful for purification, diagnostic, and therapeutic applications.
• the invention concerns novel immunorecessive antibody libraries produced by the subject method, as well as individual antibodies isolated therefrom.
• an antibody display library can be isolated which is enriched for antibodies that specifically bind an immunorecessive epitope of interest.
• the display library comprises a population of display packages expressing a variegated V-gene library which has been cloned from an immunotolerance-derived antibody repertoire, and which has been further enriched after expression by the display package by affinity separation with the immunorecessive epitope.
• antibody display libraries can be generated which are enriched for specific antibodies to cell surface markers, such as fetal cell of tumor cell markers, as well as variant forms of proteins.
• the specificity of the antibodies enriched for in the subject library can be defined in terms of the particular immunogen/toleragen sets used. For example, where the specific antibody is desired for distinguishing between various cells of common or similar origin or phenotype, the cell to which a specific antibody is desired is used as the immunogen, while a related cell(s) from which it is to be distinguished is employed as the toleragen. Cell-type specific markers for the cell of interest are represented in the immunorecessive epitopes.
• the toleragen can include maternal erythroid cells and the immunogen can be fetal erythroid cells.
• the toleragen can comprise normal colon cells and the immunogen can be selected from a colon carcinoma cell line.
• Other exemplary immunogen/toleragen sets useful for generating the subject antibody libraries, as well as individual antibodies therefrom, are provided in the following description and others will be apparent to those skilled in the art.
• the subject libraries can be generated so as to be enriched for specific antibodies able to distinguish by binding between a variant form of a protein and other related forms of the protein.
• the variant protein can differ by one or more amino acid residues from other related proteins in order to give rise to the immunorecessive epitope, as well as vary antigenically from the toleragen by virtue of glycosylation or other post-translational modification.
• the variation can arise naturally, as between different isoforms of a protein family, illustrated by the apolipoprotein E family, or can be generated by genetic aberration, as illustrated by the neoplastic transforming mutations of oncogenic proteins or tumor suppressor proteins such as p53.
• individual antibodies, and genes encoding these antibodies can be isolated from the antibody libraries of the subject method. For instance, after affinity enrichment of the antibody display library for antibodies which specifically bind the immunorecessive epitope, individual display packages can be obtained, and the antibody gene contained therein subcloned into other appropriate expression vectors suitable for production of the antibody for the desired use.
• Immunotolerization can be employed in the present invention to generate an antibody repertoire, for use in subsequent V-gene cloning steps, in which the antibody response to an immunorecessive epitope(s) has been unmasked. Immunotolerization can be carried out in either in vivo or in vitro immunization systems. For instance, immunotolerization can be employed in the present invention to enrich the pool of activated B-lymphocytes in an immunized animal for cells producing antibodies directed to immunorecessive epitopes of interest. In a typical immunotolerization procedure of the subject method, an immunogen is introduced to the immune system of an animal some time after exposure to a toleragen.
• the effect of the toleragen is to reduce or abrogate altogether any immunological response upon re-exposure of the animal to determinants of the toleragen.
• the determinants composing the toleragen are generally a portion of those antigenic determinants comprising the immunogen (i.e. the background epitopes)
• the reduced antibody response to the background epitopes upon challenge with the immunogen can act to unmask the antibody response to the immunorecessive epitopes of the immunogen.
• unmasked it is meant that the population of antibody-producing cells directed to the immunorecessive epitopes effectively becomes a greater percentage of the overall population of antibody-producing cells in the animal (see Williams et al. (1992) Biotechniques 12:842-847).
• immunotolerizing means includes subtractive immunization for enriching a pool of B-cells for clones producing antibodies specific for rare epitopes.
• subtractive immunization is a two-step procedure. Step one is a suppression step in which a state of tolerance is induced in the immune system of a host animal to a specific set of molecules, the tolerogen. Step two is an immunizing step in which another set of molecules, the immunogen, is introduced to the immune system.
• the molecules comprising the tolerogen are generally a subset of those comprising the immunogen. Ideally, the only molecules to which the immune system will generate the antibodies after exposure to the immunogen are those molecules present in the immunogen but not present in the tolerogen.
• neonatal tolerization is utilized to generate an enriched pool of B-cells.
• Neonatal tolerization utilizes the self-tolerization process of the developing immune system. For each species, a discrete developmental period exists during which the immune system classifies all molecules present in the body as self, resulting in an induced state of immunological tolerance to those molecules (Billingham et al. (1953) Nature 172:603-606; Golumbeski et al. (1986) Anal Biochem 154:373-381; Hasek et al.
• mice or other host animals
• the immune system should be immunologically responsive only to those molecules in the immunogen, but not in the tolerogen.
• chemical immunosuppression is the immunotolerizing means employed to generate an enriched B-cell population for subsequent cloning of variable region genes (V-genes).
• V-genes variable region genes
• chemical immunosuppression via the cytotoxic drug cyclophosaphamide is technique useful for subtractive immunization
• the tolerogen the tolerogen
• cyclophosphamide the tolerogen
• the immune system should be immunologically responsive only to those epitopes of the immunogen that are not found in the tolerogen.
• subtractive immunization protocols are also available for use in the subject method, and include, for example, the use of interleukin-targeted toxins.
• interleukin-targeted toxins include, for example, the use of interleukin-targeted toxins.
• IL-2- toxin fusion proteins Kelley et al. (1988) PNAS 85:3980-3984) and IL-4-toxin fusion proteins (Lakkis et al. (1991) Eur J Immunol 21 :2253-2258) can be used to selectively induce tolerance to the epitopes of a toleragen.
• the antibody repertoire of the resulting B-cell pool is cloned.
• Methods are generally known, and can be applied in the subject method, for directly obtaining the DNA sequence of the variable regions of a diverse population of immunoglobulin molecules by using a mixture of oligomer primers and PCR.
• mixed oligonucleotide primers corresponding to the 5' leader (signal peptide) sequences and/or framework 1 (FR1) sequences, as well as primer to a conserved 3' constant region primer can be used for PCR amplification of the heavy and light chain variable regions from a number of murine antibodies (Larrick et al. (1991) Biotechniques 11: 152-156).
• a similar strategy can also been used to amplify human heavy and light chain variable regions
• RNA is isolated from mature B cells of, for example, peripheral blood cells, bone marrow, or spleen preparations, using standard protocols (e.g., U.S. Patent No. 4,683,202; Orlandi, et al. PNAS (1989) 86:3833-3837; Sastry et al., PNAS
• First-strand cDNA is synthesized using primers specific for the constant region of the heavy chain(s) and each of the K and ⁇ light chains, as well as primers for the signal sequence.
• variable region PCR primers such as those shown in Figures 1A and IB (for mouse) or Figure 6 (for human)
• the variable regions of both heavy and light chains are amplified, each alone or in combination, and ligated into appropriate vectors for further manipulation in generating the display packages.
• Oligonucleotide primers useful in amplification protocols may be unique or degenerate or incorporate inosine at degenerate positions. Restriction endonuclease recognition sequences may also be incorporated into the primers to allow for the cloning of the amplified fragment into a vector in a predetermined reading frame for expression.
• the V-gene library cloned from the immunotolerance-derived antibody repertoire can be expressed by a population of display packages to form an antibody display library.
• the display package on which the variegated antibody library is manifest it will be appreciated from the discussion provided herein that the display package will often preferably be able to be (i) genetically altered to encode at least a variable region of an antibody, (ii) maintained and amplified in culture, (iii) manipulated to display the antibody gene product in a manner permitting the antibody to interact with a target epitope during an affinity separation step, and (iv) affinity separated while retaining the antibody gene such that the sequence of the antibody gene can be obtained.
• the display remains viable after affinity separation.
• the display package comprises a system that allows the sampling of very large variegated antibody display libraries, rapid sorting after each affinity separation round, and easy isolation of the antibody gene from purified display packages.
• the most attractive candidates for this type of screening are prokaryotic organisms and viruses, as they can be amplified quickly, they are relatively easy to manipulate, and large number of clones can be created.
• Preferred display packages include, for example, vegetative bacterial cells, bacterial spores, and most preferably, bacterial viruses (especially DNA viruses).
• the present invention also contemplates the use of eukaryotic cells (other than cells which naturally produce antibodies, i.e. B-cells), including yeast and their spores, as potential display packages.
• kits for generating phage display libraries e.g. the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the Stratagene SurfZAPTM phage display kit, catalog no. 240612
• methods and reagents particularly amenable for use in generating the variegated antibody display library of the present invention can be found in, for example, the Ladner et al. U.S. Patent No. 5,223,409; the Kang et al. International Publication No. WO 92/18619; the Dower et al. International Publication No. WO 91/17271; the Winter et al. International Publication WO 92/20791 ; the Markland et al.
• the display means of the package will comprise at least two components.
• the first component is a secretion signal which directs the recombinant antibody to be localized on the extracellular side of the cell membrane (of the host cell when the display package is a phage). This secretion signal is characteristically cleaved off by a signal peptidase to yield a processed, "mature" antibody.
• the second component is a display anchor protein which directs the display package to associate the antibody with its outer surface. As described below, this anchor protein can be derived from a surface or coat protein native to the genetic package.
• the means for arraying the variegated antibody library comprises a derivative of a spore or phage coat protein amenable for use as a fusion protein.
• the antibody component of the display will comprise, at a minimum, one of either the V H or V L regions cloned from B cells isolated in the subtractive immunization step. It will be appreciated, however, that the V H regions and/or the V L regions may contain, in addition to the variable portion of the antibodies, all or a portion of the constant regions.
• the display library will include variable regions of both heavy and light chains in order to generate at least an Fv fragment.
• the minimal antibody display as comprising the use of cloned V- ⁇ regions to construct the fusion protein with the display anchor protein. However, it should be readily understood that similar embodiments are possible in which the role of the V L and V H chains are reversed in the construction of the display library.
• the V H portion of the antibody display is derived from isolated cells of the subtractive immunization step, but the V L chain is either absent or is a "fixed" V L (i.e. the same V L chain for every antibody of the display).
• the V L chain can be contributed from a gene construct other than the construct encoding the V H chain, or from the host cell itself (i.e. a light chain producing myeloma cell), or added exogenously to the packages so as to recombine with Vp j chains already displayed on their surface.
• the V L chain is derived from a variegated V L library also cloned from the same population of B cells from which the V H gene is cloned, in which case a preferred embodiment places the VL gene in the same construct as the V H gene such that both may be readily recovered together.
• the cDNA encoding the light chain may be cloned directly into an appropriate site of the vector containing the heavy chain-coat protein library; or, alternatively, the light chain may be cloned as a separate library in a different plasmid vector, amplified, and subsequently the fragments cloned into the vector library encoding the heavy chain.
• the V L chain is cloned so that it is expressed with a signal peptide leader sequence that will direct its secretion into the periplasm of the host cell. For example, several leader sequences have been shown to direct the secretion of antibody sequences in E.
• coli such as OmpA (Hsiung et al. Bio/Technology (1986) 4:991-995), and (Better et al. Science 240:1041-1043), phoA (Skerra and Pluckthun, Science (1988) 240:1038).
• the cloning site for the VL chain sequences in the phagemid should be placed so that it does not substantially interfere with normal phage function.
• One such locus is the intergenic region as described by Zinder and Boeke, (1982) Gene 19:1-10.
• the V L sequence is preferably expressed at an equal or higher-level than the H L -cpIII product (described below) to maintain a sufficiently high V L concentration in the periplasm and provide efficient assembly (association) of V L with V H chains.
• a phagemid can be constructed to encode, as separate genes, both a V H /coat fusion protein and a V L chain. Under the appropriate induction, both chains are expressed and allowed to assemble in the periplasmic space of the host cell, the assembled antibody being linked to the phage particle by virtue of the V ⁇ chain being a portion of a coat protein fusion construct.
• coli such as strain MCI 061
• libraries may be constructed in fd-tet Bl of up to about 3 x 10 8 members or more.
• Increasing DNA input and making modifications to the cloning protocol within the ability of the skilled artisan may produce increases of greater than about 10- fold in the recovery of transformants, providing libraries of up to 10 10 or more recombinants.
• the V region domains of heavy and light chains can be expressed on the same polypeptide, joined by a flexible linker to form a single-chain Fv fragment, and the scFV gene subsequently cloned into the desired expression vector or phage genome.
• a flexible linker As generally described in McCafferty et al., Nature (1990) 348:552-554, complete V H and V L domains of an antibody, joined by a flexible (Gly 4 -Ser) 3 linker can be used to produce a single chain antibody which can render the display package separable based on antigen affinity.
• an important criteria for the present selection method can be that it is able to discriminate between antibodies of different affinity for a particular antigen, and preferentially enrich for the antibodies of highest affinity.
• manipulating the display package to be rendered effectively monovalent can allow affinity enrichment to be carried out for generally higher binding affinities (i.e. binding constants in the range of 10 6 to 10 10 M **1 ) as compared to the broader range of affinities isolable using a multivalent display package.
• the natural i.e.
• the library of display packages will comprise no more than 5 to 10% polyvalent displays, and more preferably no more than 2% of the display will be polyvalent , and most preferably, no more than 1% polyvalent display packages in the population.
• the source of the wild-type anchor protein can be, for example, provided by a copy of the wild-type gene present on the same construct as the antibody fusion protein, or provided by a separate construct altogether.
• polyvalent displays can be generated to isolate a broader range of binding affinities. Such antibodies can be useful, for example, in purification protocols where avidity can be desirable.
• Bacteriophage are attractive prokaryotic-related organisms for use in the subject method. Bacteriophage are excellent candidates for providing a display system of the variegated antibody library as there is little or no enzymatic activity associated with intact mature phage, and because their genes are inactive outside a bacterial host, rendering the mature phage particles metabolically inert. In general, the phage surface is a relatively simple structure. Phage can be grown easily in large numbers, they are amenable to the practical handling involved in many potential mass screening programs, and they carry genetic information for their own synthesis within a small, simple package.
• choosing the appropriate phage to be employed in the subject method will generally depend most on whether (i) the genome of the phage allows introduction of the antibody gene either by tolerating additional genetic material or by having replaceable genetic material; (ii) the virion is capable of packaging the genome after accepting the insertion or substitution of genetic material; and (iii) the display of the antibody on the phage surface does not disrupt virion structure sufficiently to interfere with phage propagation.
• phage One concern presented with the use of phage is that the mo ⁇ hogenetic pathway of the phage determines the environment in which the antibody will have opportunity to fold. Periplasmically assembled phage are preferred as the displayed antibodies will generally contain essential disulfides, and such antibodies may not fold correctly within a cell. However, in certain embodiments in which the display package forms intracellularly (e.g., where ⁇ phage are used), it has been demonstrated that the antibody may assume proper folding after the phage is released from the cell.
• the preferred display means is a protein that is present on the phage surface (e.g. a coat protein).
• Filamentous phage can be described by a helical lattice; isometric phage, by an icosahedral lattice.
• Each monomer of each major coat protein sits on a lattice point and makes defined interactions with each of its neighbors. Proteins that fit into the lattice by making some, but not all, of the normal lattice contacts are likely to destabilize the virion by aborting formation of the virion as well as by leaving gaps in the virion so that the nucleic acid is not protected.
• the antibody library is expressed and allowed to assemble in the bacterial cytoplasm, such as when the ⁇ phage is employed.
• the induction of the protein(s) may be delayed until some replication of the phage genome, synthesis of some of the phage structural-proteins, and assembly of some phage particles has occurred.
• the assembled protein chains then interact with the phage particles via the binding of the anchor protein on the outer surface of the phage particle.
• the cells are lysed and the phage bearing the library-encoded receptor protein (that corresponds to the specific library sequences carried in the DNA of that phage) are released and isolated from the bacterial debris.
• phage harvested from the bacterial debris are affinity purified.
• the antigen or determinant can be used to retrieve phage displaying the desired antibody.
• the phage so obtained may then be amplified by infecting into host cells. Additional rounds of affinity enrichment followed by amplification may be employed until the desired level of enrichment is reached.
• the enriched antibody-phage can also be screened with additional detection-techniques such as expression plaque (or colony) lift (see, e.g., Young and Davis, Science (1983) 222:778-782) whereby a labeled antigen is used as a probe.
• additional detection-techniques such as expression plaque (or colony) lift (see, e.g., Young and Davis, Science (1983) 222:778-782) whereby a labeled antigen is used as a probe.
• the phage obtained from the screening protocol are infected into cells, propagated, and the phage DNA isolated and sequenced, and/or recloned into a vector intended for gene expression in prokaryotes or eukaryotes to obtain larger amounts of the particular antibody selected.
• the antibody is also transported to an extra-cytoplasmic compartment of the host cell, such as the bacterial periplasm, but as a fusion protein with a viral coat protein.
• the desired protein or one of its polypeptide chains if it is a multichain antibody
• the viral coat protein which is processed and transported to the cell inner membrane.
• Other chains if present, are expressed with a secretion leader and thus are also transported to the periplasm or other intraceUular by extra- cytoplasmic location.
• the chains e.g.
• Filamentous bacteriophages which include Ml 3, fl, fd, Ifl, Ike, Xf, Pfl, and Pf3, are a group of related viruses that infect bacteria. They are termed filamentous because they are long, thin particles comprised of an elongated capsule that envelopes the deoxyribonucleic acid (DNA) that forms the bacteriophage genome.
• the F pili filamentous bacteriophage (Ff phage) infect only gram-negative bacteria by specifically adsorbing to the tip of F pili, and include fd, fl and Ml 3.
• filamentous phage in general are attractive and Ml 3 in particular is especially attractive because: (i) the 3-D structure of the virion is known; (ii) the processing of the coat protein is well understood; (iii) the genome is expandable; (iv) the genome is small; (v) the sequence of the genome is known; (vi) the virion is physically resistant to shear, heat, cold, urea, guanidinium chloride, low pH, and high salt; (vii) the phage is a sequencing vector so that sequencing is especially easy; (viii) antibiotic-resistance genes have been cloned into the genome with predictable results (Hines et al.
• Ml 3 is a plasmid and transformation system in itself, and an ideal sequencing vector. Ml 3 can be grown on Rec- strains of E. coli. The Ml 3 genome is expandable (Messing et al.
• the mature capsule or Ff phage is comprised of a coat of five phage-encoded gene products: cpVIII, the major coat protein product of gene VIII that forms the bulk of the capsule; and four minor coat proteins, cpIII and cpIV at one end of the capsule and cpVII and cpIX at the other end of the capsule.
• the length of the capsule is formed by 2500 to 3000 copies of cpVIII in an ordered helix array that forms the characteristic filament structure.
• the gene Ill-encoded protein (cpIII) is typically present in 4 to 6 copies at one end of the capsule and serves as the receptor for binding of the phage to its bacterial host in the initial phase of infection.
• the phage particle assembly involves extrusion of the viral genome through the host cell's membrane.
• the major coat protein cpVIII and the minor coat protein cpIII are synthesized and transported to the host cell's membrane. Both cpVIII and cpIII are anchored in the host cell membrane prior to their inco ⁇ oration into the mature particle.
• the viral genome is produced and coated with cpV protein.
• cpV-coated genomic DNA is stripped of the cpV coat and simultaneously recoated with the mature coat proteins.
• Both cpIII and cpVIII proteins include two domains that provide signals for assembly of the mature phage particle.
• the first domain is a secretion signal that directs the newly synthesized protein to the host cell membrane.
• the secretion signal is located at the amino terminus of the polypeptide and targets the polypeptide at least to the cell membrane.
• the second domain is a membrane anchor domain that provides signals for association with the host cell membrane and for association with the phage particle during assembly.
• This second signal for both cpVIII and cpIII comprises at least a hydrophobic region for spanning the membrane.
• the 50 amino acid mature gene VIII coat protein (cpVIII) is synthesized as a 73 amino acid precoat (Ito et al. (1979) PNAS 76:1199-1203).
• cpVIII has been extensively studied as a model membrane protein because it can integrate into lipid bilayers such as the cell membrane in an asymmetric orientation with the acidic amino terminus toward the outside and the basic carboxy terminus toward the inside of the membrane.
• the first 23 amino acids constitute a typical signal-sequence which causes the nascent polypeptide to be inserted into the inner cell membrane.
• SP-I signal peptidase
• the sequence of gene VIII is known, and the amino acid sequence can be encoded on a synthetic gene.
• Mature gene VIII protein makes up the sheath around the circular ssDNA.
• the gene VIII protein can be a suitable anchor protein because its location and orientation in the virion are known (Banner et al. (1981) Nature 289:814-816).
• the antibody is attached to the amino terminus of the mature Ml 3 coat protein to generate the phage display library.
• manipulation of the concentration of both the wild-type cpVIII and Ab/cpVIII fusion in an infected cell can be utilized to decrease the avidity of the display and thereby enhance the detection of high affinity antibodies directed to the target epitope(s).
• Another vehicle for displaying the antibody is by expressing it as a domain of a chimeric gene containing part or all of gene III.
• expressing the V-gene as a fusion protein with gpIII can be a preferred embodiment, as manipulation of the ratio of wild-type gpIII to chimeric gpIII during formation of the phage particles can be readily controlled.
• This gene encodes one of the minor coat proteins of Ml 3.
• Genes VI, VII, and IX also encode minor coat proteins. Each of these minor proteins is present in about 5 copies per virion and is related to mo ⁇ hogenesis or infection. In contrast, the major coat protein is present in more than 2500 copies per virion.
• the gene VI, VII, and IX also encode minor coat proteins.
• IX proteins are present at the ends of the virion; these three proteins are not post- translationally processed (Rasched et al. (1986) Ann Rev. Microbiol. 41:507-541).
• the single-stranded circular phage DNA associates with about five copies of the gene III protein and is then extruded through the patch of membrane-associated coat protein in such a way that the DNA is encased in a helical sheath of protein (Webster et al. in The
• the successful cloning strategy utilizing a phage coat protein will provide: (1) expression of an antibody chain fused to the N- terminus of a coat protein (e.g., cpIII) and transport to the inner membrane of the host where the hydrophobic domain in the C-terminal region of the coat protein anchors the fusion protein in the membrane, with the N-terminus containing the antibody chain protruding into the periplasmic space and available for interaction with a second or subsequent chain (e.g., V L to form an Fv or Fab fragment) which is thus attached to the coat protein; and (2) adequate expression of a second or subsequent polypeptide chain if present (e.g., VL) and transport of this chain to the soluble compartment of the periplasm.
• a coat protein e.g., cpIII
• Pf3 is a well known filamentous phage that infects Pseudomonas aerugenosa cells that harbor an IncP-I plasmid. The entire genome has been sequenced ((Luiten et al. (1985) J Virol. 56:268-276) and the genetic signals involved in replication and assembly are known (Luiten et al. (1987) DNA 6:129-137).
• the major coat protein of PF3 is unusual in having no signal peptide to direct its secretion. The sequence has charged residues ASP-7, ARG-37, LYS-40, and PHE44 which is consistent with the amino terminus being exposed.
• a tripartite gene can be constructed which comprises a signal sequence known to cause secretion in P. aerugenosa, fused in-frame to a gene fragment encoding the antibody sequence, which is fused in-frame to DNA encoding the mature Pf3 coat protein.
• DNA encoding a flexible linker of one to 10 amino acids is introduced between the antibody gene fragment and the Pf3 coat-protein gene.
• This tripartite gene is introduced into Pf3 so that it does not interfere with expression of any Pf3 genes.
• the bacteriophage ⁇ X174 is a very small icosahedral virus which has been thoroughly studied by genetics, biochemistry, and electron microscopy (see The Single Stranded DNA Phages (eds. Den hardt et al. (NY:CSHL Press, 1978)).
• Three gene products of ⁇ X174 are present on the outside of the mature virion: F (capsid), G (major spike protein, 60 copies per virion), and H (minor spike protein, 12 copies per virion).
• the G protein comprises 175 amino acids, while H comprises 328 amino acids.
• the F protein interacts with the single-stranded DNA of the virus.
• the proteins F, G, and H are translated from a single mRNA in the viral infected cells.
• ⁇ X174 is not typically used as a cloning vector due to the fact that it can accept very little additional DNA.
• mutations in the viral G gene encoding the G protein
• a copy of the wild-type G gene carried on a plasmid that is expressed in the same host cell (Chambers et al. (1982) Nuc Acid Res 10:6465-6473).
• one or more stop codons are introduced into the G gene so that no G protein is produced from the viral genome.
• the variegated antibody gene library can then be fused with the nucleic acid sequence of the H gene.
• the second plasmid can further include one or more copies of the wild-type H protein gene so that a mix of H and Ab/H proteins will be predominated by the wild-type H upon inco ⁇ oration into phage particles.
• Phage such as ⁇ or T4 have much larger genomes than do Ml 3 or ⁇ X174, and have more complicated 3-D capsid structures than M13 or ⁇ PX174, with more coat proteins to choose from.
• bacteriophage ⁇ and derivatives thereof are examples of suitable vectors.
• the intraceUular mo ⁇ hogenesis of phage ⁇ can potentially prevent protein domains that ordinarily contain disulfide bonds from folding correctly.
• variegated libraries expressing a population of functional antibodies, including both heavy and light chain variable regions have been generated in ⁇ phage. (Huse et al.
• library DNA When used for expression of antibody sequences, such as V H , V L , Fv (variable region fragment) or Fab, library DNA may be readily inserted into a ⁇ vector.
• variegated antibody libraries have been constructed by modification of ⁇ ZAP II (Short et al. (1988) Nuc Acid Res 16:7583) comprising inserting both cloned heavy and light chain variable regions into the multiple cloning site of a ⁇ ZAP II vector (Huse et al. supra.).
• a pair of ⁇ vectors may be designed to be asymmetric with respect to restriction sites that flank the cloning and expression sequences. This asymmetry allows efficient recombination of libraries coding for separate chains of the active protein.
• a library expressing antibody light chain variable regions may be combined with one expressing antibody heavy chain variable regions (VJJ), thereby constructing combinatorial antibody or Fab expression libraries.
• V L antibody light chain variable regions
• VJJ antibody heavy chain variable regions
• one ⁇ vector is designed to serve as a cloning vector for antibody light chain sequences
• another ⁇ vector is designed to serve as a cloning vector for antibody heavy chain sequences in the initial steps of library construction.
• a combinatorial library is constructed from the two ⁇ libraries by crossing them at an appropriate restriction site. DNA is first purified from each library, and the right and left arms of each respective ⁇ vector cleaved so as to leave the antibody chain sequences intact.
• one strategy for displaying antibodies on bacterial cells comprises generating a fusion protein by inserting the antibody into cell surface exposed portions of an integral outer membrane protein (Fuchs et al. (1991) Bio/Technology 9:1370-1372).
• any well-characterized bacterial strain will typically be suitable, provided the bacteria may be grown in culture, engineered to display the antibody library on its surface, and is compatible with the particular affinity selection process practiced in the subject method.
• the preferred display systems include Salmonella typhirnurium, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholerae, Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis, Bacteroides nodosus, Moraxella bovis, and especially Escherichia coli.
• Salmonella typhirnurium Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholerae, Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis, Bacteroides nodosus, Moraxella bovis, and especially Escherichia coli.
• Many bacterial cell surface proteins useful in the present invention have been characterized, and works on the localization of these proteins and the methods of determining their structure include Benz et al. (1988) Ann Rev Microbiol 42: 359-3
• LamB protein of E coli is a well understood surface protein that can be used to generate a variegated library of antibodies on the surface of a bacterial cell (see, for example, Ronco et al.
• LamB of E. coli is a porin for maltose and maltodextrin transport, and serves as the receptor for adso ⁇ tion of bacteriophages ⁇ and K10. LamB is transported to the outer membrane if a functional N-terminal signal sequence is present (Benson et al. (1984) PNAS 81:3830-3834). As with other cell surface proteins, LamB is synthesized with a typical signal-sequence which is subsequently removed.
• the variegated antibody gene library can be cloned into the LamB gene such that the resulting library of fusion proteins comprise a portion of LamB sufficient to anchor the protein to the cell membrane with the antibody fragment oriented on the extracellular side of the membrane.
• Secretion of the extracellular portion of the fusion protein can be facilitated by inclusion of the LamB signal sequence, or other suitable signal sequence, as the N-terminus of the protein.
• the E. coli LamB has also been expressed in functional form in S. typhimurium (Harkki et al. (1987) Mol Gen Genet 209:607-611), V. cholerae (Harkki et al. (1986) Microb Pathol 1 :283-288), and K. pneumonia (Wehmeier et al. (1989) Mol Gen Genet 215:529-536), so that one could display a population of antibodies in any of these species as a fusion to E. coli LamB. Moreover, K. pneumonia expresses a maltoporin similar to LamB which could also be used. In P. aeruginosa, the Dl protein (a homologue of LamB) can be used (Trias et al.
• Bacterial spores also have desirable properties as display package candidates in the subject method. For example, spores are much more resistant than vegetative bacterial cells or phage to chemical and physical agents, and hence permit the use of a great variety of affinity selection conditions. Also, Bacillus spores neither actively metabolize nor alter the proteins on their surface. However, spores have the disadvantage that the molecular mech ⁇ anisms that trigger sporulation are less well worked out than is the formation of Ml 3 or the export of protein to the outer membrane of E.
• Bacteria of the genus Bacillus form endospores that are extremely resistant to damage by heat, radiation, desiccation, and toxic chemicals (reviewed by Losick et al. (1986) Ann Rev Genet 20:625-669). This phenomenon is attributed to extensive intermolecular cross- linking of the coat proteins.
• Bacillus spores can be the preferred display package. Endospores from the genus Bacillus are more stable than are, for example, exospores from Streptomyces.
• Bacillus subtilis forms spores in 4 to 6 hours, whereas Streptomyces species may require days or weeks to sporulate.
• genetic knowledge and manipulation is much more developed for B. subtilis than for other spore-forming bacteria. Viable spores that differ only slightly from wild-type are produced in B. subtilis even if any one of four coat proteins is missing (Donovan et al. (1987) J Mol Biol 196:1-10).
• plasmid DNA is commonly included in spores, and plasmid encoded proteins have been observed on the surface of Bacillus spores (Debro et al. (1986) J Bacteriol 165:258-268).
• the variegated antibody display is subjected to affinity enrichment in order to select for antibodies which bind preselected antigens.
• affinity separation or “affinity enrichment” includes, but is not limited to (1) affinity chromatography utilizing immobilizing antigens, (2) immunoprecipitation using soluble antigens, (3) fluorescence activated cell sorting, (4) agglutination, and (5) plaque lifts.
• the library of display packages are ultimately separated based on the ability of the associated antibody to bind an epitope on the antigen of interest. See, for example, the Ladner et al. U.S. Patent No. 5,223,409; the Kang et al. International Publication No.
• the display library will be pre-enriched for antibodies specific for the rare epitope by first contacting the display library with a source of the background epitope, such as the toleragen, in order to further remove antibodies which bind the background epitopes. Subsequently, the display package is contacted with the target antigen and antibodies of the display which are able to specifically bind the antigen are isolated.
• a source of the background epitope such as the toleragen
• the target antigen is immobilized on an insoluble carrier, such as sepharose or polyacrylamide beads, or, alternatively, the wells of a microtitre plate.
• an insoluble carrier such as sepharose or polyacrylamide beads, or, alternatively, the wells of a microtitre plate.
• the cells on which the antigen is displayed may serve as the insoluble matrix carrier.
• the population of display packages is applied to the affinity matrix under conditions compatible with the binding of the antibody to a target antigen. The population is then fractionated by washing with a solute that does not greatly effect specific binding of antibodies to the target antigen, but which substantially disrupts any non-specific binding of the display package to the antigen or matrix.
• a certain degree of control can be exerted over the binding characteristics of the antibodies recovered from the display library by adjusting the conditions of the binding incubation and subsequent washing.
• the temperature, pH, ionic strength, divalent cation concentration, and the volume and duration of the washing can select for antibodies within a particular range of affinity and specificity. Selection based on slow dissociation rate, which is usually predictive of high affinity, is a very practical route. This may be done either by continued incubation in the presence of a saturating amount of free hapten (if available), or by increasing the volume, number, and length of the washes. In each case, the rebinding of dissociated antibody-display package is prevented, and with increasing time, antibody-display packages of higher and higher affinity are recovered.
• antibodies with special characteristics may be used in affinity purification of various proteins when gentle conditions for removing the protein from the antibody are required.
• Specific examples are antibodies which depend on Ca ++ for binding activity and which released their haptens in the presence of EGTA. (see, Hopp et al. (1988) Biotechnology 6:1204-1210).
• Such antibodies may be identified in the recombinant antibody library by a double screening technique isolating first those that bind hapten in the presence of Ca ++ , and by subsequently identifying those in this group that fail to bind in the presence of EGTA.
• specifically bound display packages can be eluted by either specific deso ⁇ tion (using excess antigen) or non-specific deso ⁇ tion (using pH, polarity reducing agents, or chaotropic agents).
• the elution protocol does not kill the organism used as the display package such that the enriched population of display packages can be further amplified by reproduction.
• the list of potential eluants includes salts (such as those in which one of the counter ions is Na + , NH4 + , Rb + , SO4 2 -, H2PO4-, citrate, K + , Li + , Cs + , HSO4-, CO3 2 -, Ca 2+ , Sr 2+ , Cl " , PO4 2 -, HCO3-, Mg 2 + , Ba2 + , Br, HPO 4 2* ⁇ or acetate), acid, heat, and, when available, soluble forms of the target antigen (or analogs thereof).
• salts such as those in which one of the counter ions is Na + , NH4 + , Rb + , SO4 2 -, H2PO4-, citrate, K + , Li + , Cs + , HSO4-, CO3 2 -, Ca 2+ , Sr 2+ , Cl " , PO4 2 -, HCO3-, Mg 2 +
• buffer components especially eluates
• Neutral solutes such as ethanol, acetone, ether, or urea, are examples of other agents useful for eluting the bound display packages.
• affinity enriched display packages are iteratively amplified and subjected to further rounds of affinity separation until enrichment of the desired binding activity is detected.
• the specifically bound display packages, especially bacterial cells need not be eluted per se, but rather, the matrix bound display packages can be used directly to inoculate a suitable growth media for amplification.
• the fusion protein generated with the coat protein can interfere substantially with the subsequent amplification of eluted phage particles, particularly in embodiments wherein the cpIII protein is used as the display anchor.
• the cpIII protein is used as the display anchor.
• some antibody constructs because of their size and/or sequence, may cause severe defects in the infectivity of their carrier phage. This causes a loss of phage from the population during reinfection and amplification following each cycle of panning.
• the antibody can be derived on the surface of the display package so as to be susceptible to proteolytic cleavage which severs the covalent linkage of at least the antigen binding sites of the displayed antibody from the remaining package.
• such a strategy can be used to obtain infectious phage by treatment with an enzyme which cleaves between the antibody portion and cpIII portion of a tail fiber fusion protein (e.g. such as the use of an enterokinase cleavage recognition sequence).
• DNA prepared from the eluted phage can be transformed into host cells by electroporation or well known chemical means.
• the cells are cultivated for a period of time sufficient for marker expression, and selection is applied as typically done for DNA transformation.
• the colonies are amplified, and phage harvested for a subsequent round(s) of panning.
• the nucleic acid encoding the V-genes for each of the purified display packages can be recloned in a suitable eukaryotic or prokaryotic expression vector and transfected into an appropriate host for production of large amounts of protein.
• the isolated V-gene lacks a portion of a constant region and it is desirable that the missing portion be provided, simple molecular cloning techniques can be used to add back the missing portions.
• the binding affinity of the antibody can be confirmed by well known immunoassay techniques with the target epitope (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988)).
• Antibody Compositions, and Immunoassay Kits Another aspect of the present invention concerns chimeric antibodies, e.g., altered antibodies in which at least the antigen binding portion of an immunoglobulin isolated by the method described above is cloned into another protein, preferably another antibody.
• chimeric antibodies contemplated by the present invention
• further manipulation of the subject antibodies can be used to complete the portion of the constant region isolated from the V-gene library, as well as to facilitate "class switching" whereby all or a portion of the constant region of the antibody isolated from the V-gene library is replaced with a different constant region, e.g., with the constant region(s) from a different IgG, such as IgGl, IgG2 or IgG3, or the constant region(s) from one of IgE, IgA, IgD or IgM.
• single chain antibodies and other recombinant fragments can be generated from the cloned genes.
• humanized antibody is used to describe a molecule having an antigen binding site derived from an immunoglobulin from a non-human species, the remaining immunoglobulin-derived portions of the molecule, as necessary to substantially reduce the immunogenicity of the molecule in human subjects, being derived from a human immunoglobulin.
• the antigen binding site may include, for example, either complete variable domains fused to constant domains, or only the CDRs grafted to the appropriate framework regions in human variable domains.
• Such antibodies are the equivalents of the recombinant antibodies described above, but may be less immunogenic when administered to humans, and therefore more likely to be tolerated upon injected in a patient.
• any of the H3-3, FB3-2 or F4-7 antibodies described in the Examples below can be prepared to include human constant regions for each of the heavy and light chains of these mouse-derived genes.
• the portion of the antibody gene encoding the murine constant region can be substituted with a gene encoding a human constant region (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., PCT Application WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.
• the subject antibodies can also be "humanized” by replacing portions of the variable region not involved in antigen binding with equivalent portions from human variable regions.
• General reviews of "humanized” chimeric antibodies are provided by Morrison, S. L. (1985) Science 229:1202-1207; and by Oi et al. (1986) BioTechniques 4:214. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of an immunoglobulin variable region from at least one of a heavy or light chain. Sources of such nucleic acids are well known to those skilled in the art. The cD ⁇ A encoding the chimeric antibody, or fragment thereof, can then be cloned into an appropriate expression vector.
• Suitable "humanized” antibodies can be alternatively produced by CDR replacement (see U.S. Patent 5,225,539 to Winter; Jones et al. (1986) Nature 321 :552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141 :4053-4060).
• the D ⁇ A sequence encoding the chimeric variable domain may be prepared by oligonucleotide synthesis. This requires that at least the framework region sequence of the first antibody and at least the CDRs sequences of the subject antibody are known or can be readily determined. Determining these sequences, the synthesis of the D ⁇ A from oligonucleotides and the preparation of suitable vectors each involve the use of known techniques which can readily be carried out by a person skilled in the art in light of the teaching given herein.
• the D ⁇ A sequence encoding the altered variable domain may be prepared by primer directed oligonucleotide site-directed mutagenesis.
• This technique in essence involves hybridizing an oligonucleotide coding for a desired mutation with a single strand of D ⁇ A containing the mutation and using the single strand as a template for extension of the oligonucleotide to produce a strand containing the mutation.
• This technique in various forms, is described by: Zoller et al. (1982) Nuc Acids Res 10:6487-6500; ⁇ orriset al. (1983) NMc Acids Res 11:5103-5112; Zoller et al. (1984) DNA 3:479-488; and Kramer et al. (1982)
• oligonucleotides used for site-directed mutagenesis may be prepared by oligonucleotide synthesis or may be isolated from DNA coding for the variable domain of the subject antibody by use of suitable restriction enzymes. Such long oligonucleotides will generally be at least 30 residues long and may be up to or over 80 residues in length.
• PCR techniques for generating fusion proteins can be used to generate the chimeric antibody.
• PCR amplification of gene fragments, both CDR and FR regions can be carried out using anchor primers which give rise to complementary overhangs between two consecutive CDR and FR fragments which can subsequently be annealed to generate a chimeric V-gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
• the antigen binding sites of the subject antibodies can also be used to generate a fusion protein which includes protein sequences from non-immunoglobulin molecules.
• such chimeric antibodies can include: proteins domains which render the protein cytotoxic or cytostatic, such as the addition of Pseudomonas exotoxin or Diphtheria toxin domains (see, for example, Jung et al. (1994) Proteins 19:35-47; Seetharam et al. (1991) J Biol Chem 266:17376-17381; and Nichols et al. (1993) J Biol Chem 268:5302-5308); DNA- binding polypeptides for facilitating DNA transport (see, for example, U.S.
• catalytic domains which provide an enzymatic activity associated with the immunoglobulin, such as a phosphatase or peroxidase activity
• purification polypeptides to simplify purification of the antibody, such as a glutathione-S-transferase polypeptide for purification of the antibody with a glutathione-derivatized matrices (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al.
• the present invention also makes available isolated forms of the subject antibodies which are isolated from, or otherwise substantially free of other cellular and extracellular proteins, especially antigenic proteins, or other extracellular factors, with which the antibodies normally bind.
• substantially free of other cellular or extracellular proteins also referred to herein as "contaminating proteins”
• substantially pure or purified preparations are defined as encompassing preparations of the subject antibodies having less than 20% (by dry weight) contaminating protein, and preferably having less than 5% contaminating protein.
• Functional forms of the subject antibodies can be prepared, for the first time, as purified preparations by using a cloned gene as described herein.
• purified it is meant, when referring to a peptide or DNA or RNA sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules, such as other proteins.
• purified as used herein preferably means at least 80% by dry weight, more preferably in the range of 95-99% by weight, and most preferably at least 99.8%o by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present).
• pure as used herein preferably has the same numerical limits as “purified” immediately above. "Isolated” and “purified” do not encompass either natural materials in their native state or natural materials that have been separated into components (e.g., in an acrylamide gel) but not obtained either as pure (e.g.
• compositions of the subject antibodies may be conveniently formulated for administration with a biologically acceptable medium, such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
• a biologically acceptable medium such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
• the optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists, and may depend on such as factors as intended route of administration, age and body weight of patient.
• biologically acceptable medium includes any and all solvents, dispersion media, and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation.
• the use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the activity of the antibody, e.g., its specificity and/or affinity, its use in the pharmaceutical preparation of the invention is contemplated.
• Suitable vehicles and their formulation inclusive of other proteins are described, for example, in the book Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA 1985). These vehicles include injectable "deposit formulations".
• such pharmaceutical formulations include, although not exclusively, solutions or freeze-dried powders of the antibody in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered media at a suitable pH and isosmotic with physiological fluids.
• pharmaceutically acceptable vehicles or diluents include, although not exclusively, solutions or freeze-dried powders of the antibody in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered media at a suitable pH and isosmotic with physiological fluids.
• excipients such as, but not exclusively, mannitol or glycine may be used and appropriate buffered solutions of the desired volume will be provided so as to obtain adequate isotonic buffered solutions of the desired pH.
• Similar solutions may also be used for the pharmaceutical compositions of the antibodies in isotonic solutions of the desired volume and include, but not exclusively, the use of buffered saline solutions with phosphate or citrate at suitable concentrations so as to obtain at all times isotonic pharmaceutical preparations of the desired pH, (for example, neutral pH).
• Still another aspect of the present invention concerns assay kits that can be used for detecting an immunorecessive epitope(s) in a sample, for example.
• the assay kits generally provide an antibody for the immunorecessive epitope, derivatized with a label group that can be ultimately detected, as for example, by spectrophotometric techniques (including FACS) or radiographic techniques.
• the label can be any one of a number of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.
• the label group can be a functional group selected from the group consisting of horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, luciferase, urease, fluorescein and analogs thereof, rhodamine and analogs thereof, allophycocyanin, R-phycoerythrin, erythrosin, europiam, luminol, luciferin, coumarin analogs, 125 I, 131 I, 3 H, 35 S, 1 C and 2 P.
• Assay kits provided according to the invention may include a selection of several different types of the subject antibodies.
• the antibodies may be in solution or in lyophilized form.
• the antibodies may come pre-attached to a solid support, or they may be applied to the surface of the solid support when the kit is used.
• the labeling means may come pre-associated with the antibody, or may require combination with one or more components, e.g., buffers, antibody-enzyme conjugates, enzyme substrates, or the like, prior to use.
• Many types of detectable labels are available and could make up one or more components of a kit.
• Various detectable labels are known in the art, and it is generally recognized that a suitable label group is one which emits a detectable signal.
• label groups can be used, depending on the type of immunoassay conducted.
• Useful labels include those which are fluorescent, radioactive, phosphorescent, chemiluminescent, bioluminescent, and free radical.
• the label groups may include polypeptides (e.g., enzymes or proteins), polymers, polysaccharides, receptors, cofactors, and enzyme inhibitors.
• Kits of the invention may also include additional reagent.
• the additional reagent can include blocking reagents for reducing nonspecific binding to the solid phase surface, washing reagents, enzyme substrates, and the like.
• the solid phase surface may be in the form of microtiter plates, microspheres, or the like, composed of polyvinyl chloride, polystyrene, or the like materials suitable for immobilizing proteins.
• the subject method of the present invention can be applied advantageously to the production of antibodies useful in purification, diagnostic, and therapeutic applications.
• the antibody libraries which can be generated by the subject method provide a greater population of high affinity antibodies to the immunorecessive epitope of interest, as well as establish a broader pool of display packages comprising antibodies specific for the immunorecessive epitope.
• the more effective access of the antibody repertoire provided by the display libraries of the present invention allows more efficient enrichment to occur by, for example, affinity selection means.
• immunorecessive epitopes can be defined in terms of the toleragen and immunogen used in the subtractive immunization step, and are therefore unique to the immunogen with respect to the toleragen.
• the desired antibody is to distinguish between various cells of common or similar origin or phenotype
• the cell to be specifically bound by an antibody of the present invention is used as an immunogen, while the related cells from which it is to be distinguished are employed as the toleragen.
• Table 1 provides exemplary systems of immunogen/toleragen sets which can be employed in the subject method to isolate antibodies which specifically bindepitopes unique to the immunogen.
• the choice of toleragen and immunogen can provide antibodies specific to, for example, tumor cell markers, fetal cell markers, and stem cell markers.
• the subject method can be used to generate antibodies which can discriminate between a variant form of a protein and other related forms of the protein by employing an immunogen comprising a variant protein, such as a mutant form of a protein or a particular isoform of a family of proteins, and a toleragen comprising the wild-type protein or alternate isoforms of the variant protein.
• an immunogen comprising a variant protein, such as a mutant form of a protein or a particular isoform of a family of proteins
• a toleragen comprising the wild-type protein or alternate isoforms of the variant protein.
• the difference in determinants i.e. the immmunorecessive epitopes
• the variant protein and wild-type (or other isoforms) will typically consist of only a few differences in amino acid residues (i.e. less than 15%, but preferably on the order of only one to three residues difference).
• immunogens and toleragens can be used in the present invention to derive antibodies which can specifically bind variant forms of oncoproteins or tumor suppressor proteins, as well as of hemoglobin, apolipoprotein E, LDL receptor, cardiac ⁇ -myosin, sodium or other ion channels, collagen, glucokinase, or transthyretin.
• Table 1 Table 1
• the subject method is employed to generate antibodies for a cell-type specific marker.
• the present method can be employed to produce antibodies directed specifically to fetal cell- specific markers.
• specific antibodies for markers of fetal nucleated red blood cells can be generated by the subject method employing maternal erythroid cells as a toleragen and fetal erythroid cells as an immunogen.
• antibodies generated by the subject method can be used to separate fetal cells from maternal blood by, for instance, fluorescence- activated cell sorting (FACS).
• FACS fluorescence- activated cell sorting
• the isolated fetal cells such as fetal nucleated erythrocytes, represent a non-invasive source of fetal DNA for prenatal genetic screening and offer a powerful and safe alternative to more invasive procedures than, for example, amniocentesis or chronic villus sampling.
• the present invention contemplates the generation of antibodies specific for a tumor cell-specific marker.
• the subject method can be employed advantageously to generate antibodies which are able to differentiate between normal cells and their transformed counte ⁇ arts.
• Such antibodies may be suitable for both diagnostic and therapeutic uses.
• antibodies can be selected in the present assay which detect cell-specific markers found on neoplastic or hype ⁇ lastic cells.
• Antibodies so obtained can be used to identify transformed cells and thereby used to diagnose cancers and tumors such as adenocarcinomas, papillomas, squamous and transitional cell carcinomas, anaplastic carcinomas, carcinoid tumors, mesotheliomas, hepatomas, melanomas, and germ cell tumors.
• antibodies mays also be used to selectively destroy transformed cells, both in vivo and in vitro, such as through the discriminatory activation of complement at the cell surface of a transformed cell bound by the antibody, or by delivery of toxins, or by delivery of nucleic acid constructs for gene therapy.
• antibodies specific for colon cancer markers can be generated in the present invention by suing normal colon cells as a toleragen and cells derived from a colon carcinoma as an immunogen.
• the subject method can be engaged to produce antibodies that specifically inhibit metastasis of highly metastatic tumor cells.
• Such antibodies designed to recognize unique epitopes on highly metastatic variants of tumor cells (i.e.
• the immunotolerance-derived antibody repertoires used in the subject method can be generated using a differentiated nerve cell as a toleragen and an embryonic nerve cell, such as a neural crest cell or uncommitted progenitor cell, as an immunogen.
• the immunogen can comprise a hematopoietic stem cell, and the toleragen can be a committed stem cell.
• the subject method can be applied to the generation of antibodies which can discern between variant proteins.
• Such antibodies can be used to distinguish various naturally occurring isoforms of a protein, as well as to detect mutations which may have arisen in a protein.
• antibodies can be produced by the present invention which can be used in immunochemical assays for detecting cell transformations arising due to mutation of an oncogene or anti-oncogene.
• the subject method can be used to generate antibodies which discriminate between wild-type ras and a mutant form of ras.
• useful antibodies for detecting ras-induced transformation of a cell can be generated by the subject method using a Ser-17- Asn variant of ras as an immunogen, and wild-type ras as a toleragen..
• diagnostically useful antibodies can be produced by the present invention which specifically bind and discriminate between wild-type and variant tumor suppressor proteins.
• inactivating mutations of either the p53 or Rb tumor suppressors can lead to escape from cell senescence and lead to transformation.
• the subject method can be used to generate antibodies specific for a variant p53, the ability to distinguish between the wild-type and mutant forms arising through recognition of a unique epitope created by mutation, such as Arg-273->Cys, Tyr-163- Asn, Val-157- Phe, or Cys-238->Phe.
• Appropriate immunogen/toleragen sets would therefore include p53 mutants and wild-type p53.
• the subject method can also be used to produce antibodies for detecting variant hemoglobin molecules, and which subsequently can be employed as diagnostic tools for detecting hemoglobinopathies, such as sickle cell anemia and ⁇ -thalassemia.
• hemoglobinopathies such as sickle cell anemia and ⁇ -thalassemia.
• a large number of such abnormalities, most resulting from single-point mutations, have been observed as abnormal hemoglobins of embryonic, fetal, neonatal, and adult disorders (see, for review, Huisman (1993) Baillieres Clin Haematol 6:1-30). Therefore, antibodies to unique epitopes of hemoglobin variants can be of great use in detecting and quantitating both normal and abnormal hemoglobin levels.
• the immunogen is apolipoprotien E4 (ApoE4) and the toleragen comprises other ApoE isoforms
• specific antibodies can be isolated by the subject method which can be used to measure ApoE4 levels in plasma or serum of a patient.
• the presence of the ApoE4 variant has been linked to increased susceptibility to Alzheimer's disease (Strittmatter et al. (1993) PNAS 90:8098-8102) as well as significant impact on variation of cholesterol lipid and lipoprotein levels in individuals (Rail et al. (1992) J. Intern. Med. 231:653-659; and Weisgraber et al. (1990) J. Lipid Res. 31:1503-1511).
• specific antibodies to other ApoE isoforms can be generated, including antibodies which can specifically bind ApoE2 or ApoE5.
• LDL receptor variants which can be useful, for example, in predicting risk of diagnosing familial hypercholesterolemia; specific antibodies to cardiac ⁇ -myosin variants, which can be used to diagnose hypertrophic cardiomyopathy; specific antibodies to variant forms of sodium or ion channels, such as which arise in congenital hyperkalemic periodic paralysis; antibodies to collagen variants, such as Cys-579 collagen, which can be indicative of a predisposing factor in risk of familial osteoarthritis; specific antibodies to a variant of glucokinase, such as which arise in non-insulin-dependent diabetes mellitus; and antibodies specific for a mutant of transthyretin, such as which might arise in familial amyloidotic polyneuropathy.
• the subject method has been applied advantageously to the development of antibodies for cell-surface markers of fetal cells and transformed cells.
• practice of the subject method can yield a library of antibodies which are amenable to very rapid enrichment.
• This invention represents the first instance that antibodies specific for unknown/unisolated cell-surface antigens have been generated using a combinatorial display library.
• Figure 5A reveals the rapid enrichment of specific antibodies from the immunotolerized V-gene library.
• Figure 5B demonstrates that phage libraries prepared by prior art techniques (non-tolerized #1 and #2) do not show significant enrichment from one round of panning to the next (compare tolerized to non-tolerized #1 and #2).
• phage libraries prepared by prior art techniques do not show significant enrichment from one round of panning to the next (compare tolerized to non-tolerized #1 and #2).
• antibodies that discriminate between fetal and maternal blood cells with only the same approximate performance as anti-CD71 antibodies were obtained.
• the subject method provides a library containing a rich source of high affinity antibodies which permit detection of specific antibodies by, for example, panning on live cells, FACS assays or cell based ELISA.
• a library containing a rich source of high affinity antibodies which permit detection of specific antibodies by, for example, panning on live cells, FACS assays or cell based ELISA.
• individual antibody display packages were enriched 5000 to 3,600,000 fold in only a single round of selection.
• DNA sequence analyses of particular isolates depict a remarkable history of affinity maturation of both heavy and light chains, suggesting an unexpectedly efficient access to the immunological repertoire.
• the instant method enables selection of antibodies having both discriminating specificity and high binding affinity for an immunorecessive epitope.
• comparison of antibodies isolated by the subject method with antibodies available through the use of prior art techniques reveals that the combinatorially-derived antibodies of the present invention tend to be orders of magnitude better with respect to each of specificity and affinity relative to antibodies available in the prior art.
• the genes for three of the antibodies which demonstrate both desirable specificity and binding affinity have been sequenced. As described in Example 2, the F4-7 and H3-3 antibodies were originally isolated with a panning regimen including fetal liver cells.
• H3-3 antibody recognized fetal blood cells of early gestational age (e.g., ⁇ 16 weeks), but also stained fetal cells of later gestational ages, albeit less well. This probably reflects the use of fetal liver, which consists predominantly of the earliest blood cell precursors, for both immunization and enrichment. However, it is demonstrated below that the population of antibodies enriched from the library could be biased to select antibodies specific for epitopes present on fetal blood cells of later gestational ages.
• One of the isolates, FB3-2 was characterized and found to have an extraordinarily low background staining level on adult blood cells (e.g., less than 0.1%).
• a guide to the nucleic acid and amino acid sequences for each of these clones is provided in Table 2, and the overall structure of the variable region for each of the heavy and light chains are provided in Figures 8 A and 8B.
• the antibodies isolated by the present method are not apparently available by other prior art techniques and in fact displayed performance characteristics which greatly su ⁇ assed those obtained by previous methods.
• the antibodies achieved by the subject method employing an identical immunotolerization step, but coupled instead with the use of hybridoma techniques, only a few antibodies which showed fetal cell selectivity were obtained.
• the specificity for one of the best of these antibodies, "anti-Em” is shown in Table 3.
• Fetal cell selective antibodies isolated by other groups using other hybridoma technologies were also compared.
• anti-CD71 antibodies are believed to be among the best of the fetal cell specific antibodies.
• antibodies generated by the instant method perform with superior qualities relative to each of the antibodies obtained by immunotolerance (anti-Em) and hybridoma (anti-CD71) techniques.
• Anti-Em 5.0 ⁇ g fetal liver 50.0% 2.5 fold 5.0 ⁇ g maternal PBMC 20.0%
• each of the anti-Em and anti-CD71 antibodies are considered to be of excellent specificity with respect to anti-fetal cell antibodies derived by methods in the prior art. Yet, as Table 3 illustrates, the level background binding to maternal peripheral blood mononuclear cells (PBMC) is many times higher for these antibodies relative to the background staining of maternal cells using the subject antibodies. Consequently, although the anti-Em, anti-CD71 antibodies and the like stain fetal cells very well, their background staining on maternal blood of greater than 5 percent provides substantial room for improvement of antibodies useful for retrieving a very small population of fetal blood cells from maternal blood samples. One estimate of fetal cell concentrations in maternal blood provides 1 fetal cell in
• Another feature of the antibodies derived from the subject method which feature also apparently exceeds the antibodies of the prior art, pertains to the binding affinity of these antibodies for fetal cell-bound antigens.
• HEL human erythro-leukemic
• the association constant (K ⁇ exceeded 10 9 .
• monomeric H3-3 and FB3-2 Fab' fragments displayed association constants of 6x1 O ⁇ M **1 and 8xl0 10 M"- respectively.
• Dimeric forms of the recombinant antibodies had even greater binding affinities, with K a s of 5xl0 12 M ** - for H3-3 and lxlO ⁇ M **1 for FB3-2 respectively.
• the subject method makes available antibodies specific for immunorecessive epitopes, in which antibodies are characterized by association constants for the immunorecessive epitopes which are greater than 10 6 M" 1 , preferably greater than 10 8 M _1 , more preferably greater than about lO- ⁇ M" 1 , and even more preferably greater than 10 10 M"-, lO- 'M *-1 , or 10 12 M"-, e.g., K a in the range of 10- ⁇ M- 1 to lO ⁇ M" 1 .
• the subject method accommodates the isolation of antibodies which have a low level of background staining.
• the relative specificity of these antibodies can be several fold, if not orders of magnitude, better than combinatorial and hybridoma generated antibodies, particularly with respect to antibodies for cell surface epitopes.
• the subject method can provide antibodies which have no substantial background binding to other related cells, e.g., relative specificities greater than 10 fold binding to the target cells over background binding to the related cells.
• antibodies can be generated which do not substantially cross-react with other epitopes, preferably having specificities greater than 20 fold over background, more preferably 50, 75 or 100 fold over background, and even more preferably more than 125 fold over background.
• anti-fetal cell antibodies generated by the instant method were tested by fluorescence-activated cell sorting ("FACS efficiency assay") and were each demonstrated to have relative specificities greater than 125 fold over background.
• FACS efficiency assay fluorescence-activated cell sorting
• the anti-CD71 and anti-Fe antibodies were found to have relative specificities of 7.7 and 2.5 fold over background, respectively.
• specificity of fetal cell specific antibodies produced by the subject method can also be characterized in terms of a background staining of maternal cells relative to antibodies of the prior art, such as anti-CD71 antibodies.
• the subject antibodies preferably stain two times less non-fetal cells relative to an anti-CD71 antibody, more preferably at least five times less, and even more preferably at least twenty times less than an anti-CD71 antibody.
• Such comparisons can be made using standard immunoassays, such as the FACS efficiency assay of Example 4.
• Exemplary anti-CD71 (e.g., anti-Transferrin receptor) antibodies include the 5E9 antibody (ATCC HB21), the L5.1 antibody (ATCC HB84) and the L01.1 antibody (Beckton Dickinson Catalog No. 347510).
• 5E9 antibody ATCC HB21
• the L5.1 antibody ATCC HB84
• L01.1 antibody Beckton Dickinson Catalog No. 347510
• each antibody can be further engineered without departing from the pu ⁇ ose and intent of the present invention.
• a chimeric FB3-2 antibody can be generated which includes the variable regions from the heavy chain (residues El -S 121, SEQ ID No. 51) and light chain (residues Dl-Kl 11, SEQ ID No. 53).
• chimeric F4-7 antibodies can be provided which include the heavy chain (residues E1-S121, SEQ ID No. 55) and light chain (residues Dl-Kl 11, SEQ ID No. 57) variable regions from the F4-7 antibody described below.
• chimeric H3- 3 antibodies are also contemplated, as for example antibodies including the variable regions from the heavy chain (residues El -SI 15, SEQ ID No. 59) and light chain (residues Dl-Kl 11, SEQ ID No. 61) of the H3-3 antibody.
• chimeric antibodies can be generated including heavy and light chain variable regions, each represented by the general formula: FR(1)-CDR(1)-FR(2)- CDR(2)-FR(3)-CDR(3)-FR(4), wherein CDR(l), CDR(2) and CDR(3) represent complementarity determining regions from the subject antibody, and FR(1), FR(2), FR(3) and FR(4) are framework regions from a second antibody.
• chimeric FB3-2 antibodies can be generated which include a heavy chain in which CDR(l) is SYWLE, CDR(2) is EILFGSGSAHYNEKFKG and CDR(3) is GDYGNYGDYFDY, and a light chain in which CDR(l) is RASQSVSTSRYSYMH, CDR(2) is FASNLES and CDR(3) is HSWEIPYT.
• a chimeric F4-7 antibody can be made including a heavy chain in which CDR(l) is SSWLE, CDR(2) is EILFGSGSAHYNEKFKG and CDR(3) is GDYGNYGDYFDY, and a light chain in which CDR(l) is RVRQSVSTSSHSYMH, CDR(2) is YASNLES and CDR(3) is HSWEIPYT.
• chimeric H3-3 antibodies can be provided, which antibodies include a heavy chain having a CDR(l) of DYYMY, a CDR(2) of TISDDGTYTYYADSVKG and a CDR(3) of DPLYGS, and a light chain in which CDR(l) is RSSQSLVHSNGNTYLH, CDR(2) is KVSNRFS and CDR(3) is SQSTHVLT.
• the associated framework regions can be derived from an unrelated antibody, preferably a human antibody.
• the present invention further pertains to methods of producing the subject recombinant antibodies.
• a host cell transfected with nucleic acid vectors directing expression of nucleotide sequences encoding an antibody (or fragment) can be cultured under appropriate conditions to allow expression of the antibody to occur, and if required, assembly of a heavy/light chain dimer.
• the antibody may be secreted and isolated from a mixture of cells and medium containing the recombinant antibody.
• a cell culture includes host cells, media and other by-products. Suitable media for cell culture are well known in the art.
• the recombinant antibody peptide can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying antibodies, including protein-A:sepharose and ion-exchange chromatography, gel filtration chromatography, ultrafiltration and electrophoresis.
• the recombinant antibody is a fusion protein containing a domain which facilitates its purification, such as a GST fusion protein or a poly(His) fusion protein.
• This invention also pertains to a host cell transfected to express a recombinant form of the subject antibody.
• the host cell may be any prokaryotic or eukaryotic cell, and the choice can be based at least in part on the desirability of such post-translation modifications as glycosylation.
• a nucleotide sequence derived from the cloning of an anti-fetal cell or anti-oncogenic cell antibody by the subject method, encoding all or a selected portion of the variable region can be used to produce a recombinant form of an antibody via microbial or eukaryotic cellular processes.
• the cell line which is transformed to produce the recombinant antibody is an immortalised mammalian cell line, which is advantageously of lymphoid origin, such as a myeloma, hybridoma, trioma or quadroma cell line.
• the cell line may also include a normal lymphoid cell, such as a B-cell, which has been immortalised by transformation with a virus, such as the Epstein-Ban * virus.
• the immortalised cell line is a myeloma cell line or a derivative thereof.
• the recombinant antibody gene can be produced by ligating nucleic acid encoding the subject antibody protein, or the heavy and light chains thereof, into vectors suitable for expression in either prokaryotic cells, eukaryotic cells, or both.
• Expression vectors for production of recombinant forms of the subject antibody include plasmids and other vectors.
• suitable vectors for the expression of an antibody include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.
• YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al. (1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye Academic Press, p. 83, inco ⁇ orated by reference herein).
• These vectors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid.
• an antibody is produced recombinantly utilizing an expression vector generated by sub-cloning the coding sequences of the variable regions for each of the heavy and light chain genes of the H3-3 or FB3-2 antibodies.
• the preferred mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells.
• the pcDNAJ/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells.
• vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells.
• derivatives of viruses such as the bovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells.
• BBV-1 bovine papillomavirus
• pHEBo Epstein-Barr virus
• the various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art.
• suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures see Molecular Cloning A Laboratory Manual, 2nd Ed., ed.
• baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derived vectors (such as the ⁇ -gal containing pBlueBac III).
• the subject method has been applied advantageously to the development of antibodies for cell-surface markers of fetal and transformed cells.
• the present invention can yield a remarkable library of antibodies which are amenable to very rapid enrichment.
• individual antibody display packages were enriched 5000 to 3,600,000 fold in only a single round of selection.
• DNA sequence analyses of particular isolates gave a remarkable history of affinity maturation of both heavy and light chains, suggesting an unexpectedly efficient access to the immunological repertoire.
• DNA modifying enzymes were obtained from New England Biolabs (Beverly, MA) and used under conditions recommended by the suppliers.
• Taq polymerase was obtained from Perkin Elmer (Norwalk, CT).
• a set of DNA fragments (1 Kb ladder) obtained from Life Technologies (Gaithersburg, MD) was used as a standard for molecular weight of DNA fragments by agarose gel electrophoresis.
• DNA primers were custom synthesized by Genosys, Inc. (The Woodlands, TX) or Cruachem, Inc. (Sterling, VA).
• Deoxyadenosine 5'[ ⁇ -( 35 S)thio]triphosphate was purchased from New England Nuclear (Boston, MA).
• Polyclonal biotinylated anti-M13 antibody was obtained from 5 prime-3 prime (Boulder, CO). Streptavidin-Alkaline phosphatase and Polyclonal goat anti-mouse kappa-alkaline phosphatase were from Fisher Biotech (Pittsburgh, PA).
• Bacterial strains and culture E. coli strains XL-1, SolR, and LE392 were obtained from Stratagene (LaJolla, CA).
• Lambda phage resistant XL-1 was isolated by standard methods and is described in this work.
• E. coli was grown to stationary phase at 30 or 37°C with shaking in Erlenmeyer flasks filled to one-tenth their nominal capacity with LB, SOB, 2X YT, NZY medium (Sambrook, 1989) or TB medium:0.1 M KH2PO4 buffer, buffer, pH 7.5 containing 12 g bacto-tryptone, 24 g yeast extract, and 5.04 g glycerol per liter (phosphate buffer was autoclaved separately).
• agar Difco, Detroit MI
• Glucose supplement was to 0.5% Carbenicillin, chloramphenicol, and kanamycin were added when necessary to 50, 30, and 50 ug/ml, respectively.
• E. coli cloning vector lambda SurfZapTM and helper phages ExAssistTM and VCS
• Ml 3 were obtained from Stratagene.
• PBMC peripheral blood mononuclear cells
• Fetal blood mononuclear cells were prepared by standard Ficoll-Hypaque gradient techniques.
• Fetal blood mononuclear cells were prepared from fetal liver obtained from abortuses at 12-20 weeks gestation, at which age the liver is the principal hematopoietic organ. Cells were freed from surrounding connective tissue by passage through sterile microscreens in the presence of sterile Ca-Mg-free PBS.
• the resulting cell suspension was diluted up to 20 ml in PBS and the blood mononuclear cell fraction obtained by standard Ficoll-Hypaque gradient centrifugation. After recovery from the Ficoll interface, both adult and fetal cells were washed twice in sterile Ca-Mg-free PBS the resuspended in the PBS at 2xl0 7 cells per ml.
• mice at 6 weeks of age were injected intra-peritoneally ("I.P.") with lxlO 7 adult PBMC in 500 ul PBS.
• the adult PBMC injection was followed 10 minutes later by I.P. injection of cyclophosphamide at 100 mg/kg.
• the cyclophosphamide was repeated at 24 and 48 hours. After an additional 14 days, the tolerization was repeated.
• mice were immunized with fetal mononuclear blood cells by I.P. injection of lxl0 7 fetal cells in 500 ul PBS. After an additional 2 weeks, the mice were once again tolerized with adult PBMC as described for the first round of tolerization. Finally, three weeks later, the mice were again immunized with fetal blood mononuclear cells by I.P. injection of lxl 0 7 fetal cells in 500 ul PBS. The fetal cell immunization was repeated in 24 and 48 hours. After an additional 24 hours, the mice were sacrificed. The spleens were harvested and immediately frozen in liquid nitrogen.
• RNA was isolated from spleens or from Hybridoma cell lines using standard methods (Chomczynski, U.S. Patent No. 4,843,155). RNA preparations were stored in RNAase free water (Sambrook, J. et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)) at -70°C until use. A Superscript pre amplification kit from Life Technologies was used to prepare first strand cDNA as recommended by the supplier. Isolation of DNA
• Isolation of plasmid DNA from E. coli for DNA sequence or restriction analyses was by alkaline lysis (Birnboim and Doly, 1979). Bulk preparation of plasmid DNA was carrier out using nucleobond column chromatography as described by the manufacturer Macherey- Nagel (Duren, Germany). All cultures for isolation of plasmid DNA from E. coli clones containing antibody clones were grown overnight with shaking at 37°C in 2xYT medium containing 0.5% glucose and 50 ug/ml carbenicillin.
• a set of degenerate primers shown in Figures 1A and IB, was designed to minimize bias toward limited sets of PCR products from the repertoire of antibody coding regions encoded in spleenic mRNA, as well as to amplify >90% of the mouse kappa chain and heavy chain Fab encoding sequences. Amplifications of kappa chain or heavy chain coding sequences were accomplished using 5 separate primer pairs for each.
• the primers also contained restriction enzyme site to allow the ligation of the light and heavy chain PCR products into a bacterial Fab expression cassette suitable for insertion in the Surf-Zap vector (Stratagene). PCR reactions were carried out in an Automated BioSystems temp-cycler (Essex, MA) using the following protocol.
• RNA was converted to cDNA using a Superscript first strand synthesis kit (BRL).
• BBL Superscript first strand synthesis kit
• each PCR product from five separate reactions were combined to generate a kappa chain and separate heavy chain product pool.
• the pools were then purified by first removing protein and debris with a PVDF spin filter (Millipore) followed by removal of low molecular weight components using a 30,000 MW cut off spin filter as recommended by the supplier (Millipore).
• Approximately 5 ug of each product pool was digested in 300 ul of Sfil buffer with 50 units of Sfil for 2 h at 50°C. Enzyme and small end fragments generated by Sfil digestion were removed with the spin column procedure described above.
• Sfil digested light chain products were ligated to Sfil digested heavy chain products (approximately 2 ug each) in a 50 ul volume overnight at 4°C.
• the ligation mixture was then purified with spin columns as above and digested with 50 units each of Notl and Spel restriction enzymes in 100 ul.
• the digestion products were resolved by agarose gel electrophoreses and the 1.4 kb kappa chain heavy chain encoding dimer was purified using Gene Clean II (Promega) as recommended by the supplier.
• Gene Clean II Promega
• PCR products were purified as described above. Approximately 2 ug of each product was treated separately at 37°C for 1 h in a 50 ul volume containing 5 units T4 polymerase, 5 mM dTTP. Products were purified as for PCR products, and approximately 500 ng of each product was ligated at room temperature for 3 h in a 25 ul volume of ligation buffer (Promega) containing 2 units of DNA ligase.
• Fab encoding dimer from either method were amplified under standard conditions using a 5' kappa chain primer and 3' heavy chain primer shown in Figure 2 except that annealing was at 55°C for 1 min., and the extension time was extended to 4 min. at 72°C. Generally 12-25 cycles under these conditions yielded approximately 1-2 ug of 1.4 kb kappa- heavy chain dimer.
• This product was purified using spin columns as described above and then digested in a200 ul volume containing 75 units each of Not I and Spe I restriction enzymes. Digestion products were purified as described above except that a 100,000 MW spin column (Amicon) was used to more efficiently remove primers from the digestion products. Purified 1.4 kb dimers were stored at 4°C until use. Construction of variegated Fab clone banks
• Ligation of Not I-Spe I digested 1.4 kb Fab encoding fragments was as follows: 0.2 ug of digested products was ligated to 2 ug of lambda surf-zap arms in 10 ul of Promega ligation buffer containing 3 units of T4 ligase overnight at 4°C. Aliquots of the ligation mixture were then packaged into lambda heads using a Giga-pack Gold packaging kit as recommended by the supplier (Stratagene). Packaging reactions were titered on E. coli L ⁇ 392 and pooled to yield a primary library. This primary library was then amplified in E. coli LE392 using conventional methods. Generally 5xl0 9 E.
• coli XL1 cells were infected in 10 ml of 10 mM MgSO 4 with 107 invitro packaged SURF-ZAP primary clones for 10 min. at 37°C.
• the infected cells were added to 100 ml of NZY top agarose at 50°C.
• the mixture was immediately plated onto two 20x20 cm plates containing NZY agar, allowed to solidify, and then incubated for 8-16 h.
• the amplified library was harvested by rocking with an overlay of 25 ml of SM buffer of 2 h.
• Phagemid clone bank was rescued from the primary lambda SURF-ZAP library by super infection with Ml 3 exassist helper phage essentially as recommended by Stratagen.
• 10- - E. coli XL1 cells were infected with 10 10 lambda clones from our amplified surf-zap library and 10 12 Exassist M13 phage. After growth for 3.5 h in LB or TB medium, the cells were removed by centrifugation. The exassist rescued library was treated for 70°C for 20 min. and then stored at 4°C.
• Phage antibodies were generated by infection of E. coli SOLR 1 : 1 with rescued phagemid to generate a population of carbenicillin resistant antibody clone containing cells representing a 10-100 fold excess over the primary library size. Transduced cells were grown to early log phase in TB medium containing carbenicillin, and then infected with a ten fold excess of VCS M13 helper phage to cells. After 1 h at 37°C, kanamycin was added and the culture was incubated at 30°C with shaking until early stationary phase. Cells were removed by centrifugation, and phage antibodies were recovered from the supernatant by harvested by centrifugation, dissolved in 1 ml of TE buffer and then PEG precipitated a second time. Phage antibodies were dissolved in 1 ml TE or PBS buffer and stored at 4°C.
• Cell specific phage antibodies were isolated by enrichment on whole cells. Cells were prepared for enrichment by washing twice in blocking buffer (0.1% hydrolyzed casein, 3% BSA, in Hanks Buffered Salt Solution). For the first round of enrichment 10 1 1 phage antibodies in 200 ul of blocking buffer were added to 10 6 cells and incubated on ice for 1 h. Non-specific phage antibodies were then removed by washing 8 times with cold blocking buffer. Cells were harvested after each wash by centrifugation at 3500 ⁇ m in an Eppendorf micro centrifuge. Cell surface bound phage antibodies were then eluted in 500 ul of 0.2 M Glycine pH2.2 containing 3 M urea and 0.5% BSA.
• phage antibodies were titered on XL1 cells, and then amplified by the following protocol. Eluted phage antibodies in 200 ul SM buffer were added to 5x10 9 XL1 plating cells in 1 ml of 10 mM MgSO 4 and incubated for 10 min. at room temperature. Infected cells were then used to inoculate 100 ml of TB broth in a 2 L flask and incubated at 30°C with shaking.
• E. coli XL1 was infected with dilutions of phage antibody pools and plated on LB medium containing 0.5% glucose and 50 ug/ml carbenicllin.
• 20 mm culture tubes containing 2 ml of 2xYT medium with 50 ug/ml carbenicllin were inoculated with isolated colonies and grown overnight at 30°C with shaking. The following morning 1 ml of culture was gently shaken at 37°C for 1 h and then infected with 10 1 1 M13 VCS phage.
• Flow cytometric assay of phage antibody binding to whole cells A flow cytometry protocol was devised for the testing of phage antibody binding to surface markers on whole cells, lxl 0 6 adult or fetal mononuclear cells were dispensed into a 2 ml microtube and washed with blocking buffer as in the phage enrichment procedure. For initial assay, 2x10 10 phage were added to the washed cells and the volume brought to 100 ul with casein/BSA/HBSS. The phage were incubated with the cells for one hour at 4°C. The phage-cells were washed three times with 1 ml blocking buffer.
• Biotinylated sheep anti- phage polyclonal antibody (5 Prime -2 Prime) was added to the phage-cells at 5-7.5 ul per sample, optimized for each lot of polyclonal antibody. Volume was once again brought up to 100 ul with blocking buffer. The anti-phage was incubated 90 minutes at 4°C. Excess anti- phage was removed by washing three times with blocking buffer. Streptavidin-FITC (Jackson Immunoresearch) was diluted 1:50 in Ca-Mg-free PBS and 250 ul added to each sample of phage-cells. After a 30 minute 4°C incubation, the phage-cells were washed twice with blocking buffer and fixed by adding 400 ul 0.5% formaldehyde.
• Relative binding activity of each clone was determined by evaluation of two parameters: (1) scatter pattern vs. intensity of fluorescence, for determination of relative cell surface epitope number and uniformity of expression for each phage clone, with higher, more uniform, numbers being most desirable; (2) titration of phage and retention of fluorescent binding intensity - for determination of relative phage antibody affinities.
• soluble antibody Fab
• Fab soluble antibody
• the anti-phage/streptavidin-FITC was replaced by a goat anti-IgG-FITC F(ab') polyclonal antibody (TAGO) that recognized the K chain of the Fab fragments.
• TAGO goat anti-IgG-FITC F(ab') polyclonal antibody
• 30 ul of the goat anti-IgG-FITC diluted 1 :10 in 2.5% normal human serum was used per sample. The dilution in human serum ensured that any cross-reactivity of the polyclonal with human blood cell antigens would be minimized.
• Example 1 Enrichment of phage antibodies on cancer cells.
• a combinatorial phage display library of IgGl and kappa chain derived Fabs containing 6xl0 7 primary clones was constructed from a mouse which had been tolerized with adult human blood and immunized with fetal liver cells.
• cultures containing antibody clones or pools of clones were always in rich media (TB or 2xYT containing 1% glucose).
• cultures used to produce phage antibodies were harvested as close to peak growth as possible since binding activity was found to fall beyond the start of stationary phase of growth.
• the human erythro-leukemic cell line (HEL) carries onco/fetal cell surface markers also found on fetal liver cells. This characteristic and the ability to culture this cell made it a reliable source of cells to develop methods for enrichment of cell line specific antibodies from the above phage library. The binding of phage antibody pools enriched on this cell line
• Example 2 Enrichment of phage antibodies on fetal cells. To maximize the chances of isolating fetal cell specific clones, the phage antibody library described in Example 1 was pre-absorbed on adult nucleated blood prior to each enrichment cycle on fetal liver cells in addition to enrichments without pre-adso ⁇ tion. The results of sequential rounds of pre-adso ⁇ tion and enrichment on fetal liver cells are shown in Figure 5A. The increase in the percentage of phage antibodies binding to fetal liver cells indicated enrichment for fetal cell binding phage antibodies.
• Table 4 shows the distribution of different phage antibody types at different stages of enrichment on HEL or Fetal cells with or without preadso ⁇ tion on adult cells. It is likely that the three classes of phage antibodies recognize three different epitopes based upon the difference in their staining profiles on fetal liver and adult cells.
• HEL cell surface binding isolates seen on a consistent cell source
• pan-fetal specific antibodies emphasizes the power of the present approach, which yielded 13 different versions of three classes of pan-fetal specific antibodies.
• Affinity of purified antibodies was determined by Scatchard analysis. Varying amounts of antibody in significant excess were incubated for 16 hours at 4°C with a constant number of HEL cells. After extensive washes, bound antibody was eluted from cells at pH 2, and quatitated in an ELISA. For Scatchard analysis, free antibody was assumed to be equivalent to the total added. The K a for each antibody was obtained from the negative slope of the line from the plot of bound versus bound/free antibody. All points were done in triplicate; the correlation coefficient for all reported slopes was greater than 90%.
• hybridoma-derived antibodies such as anti-CD71 and anti-EM
• reactivity of these antibodies with fetal and maternal cells was tested by analytical flow cytometry (FACS efficiency assay). Briefly, lxl 0 6 cells per sample were stained with indicated amounts of FITC-conjugated pure antibody. 10,000 cells were analyzed for each sample. The results, provided in Table 3 above, are reported as "% positive", indicating the percentage of cells that were found to stain above background fluorescence as established by an isotype-matched negative control antibody.
• H3-3 antibody for fetal as opposed to adult hematopoietic cells was further demonstrated by FACS and subsequent fluorescent in situ hybridization (FISH) analysis of sorted cells.
• FISH fluorescent in situ hybridization
• MOLECULE TYPE Other nucleic acid
• MOLECULE TYPE peptide
• FRAGMENT TYPE internal
• SEQUENCE DESCRIPTION SEQ ID NO:37;
• MOLECULE TYPE protein
• Gly Glu lie Leu Phe Gly Ser Gly Ser Ala His Tyr Asn Glu Lys Phe 50 55 60
• ATC CAT CCT GTG GAG GAG GAG GAT ACT GCA ACA TAT TAC TGT CAG CAC 348 lie His Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gin His 80 85 90 AGT TGG GAG ATT CCG TAC ACG TTC GGA GGG GGG ACC AAG CTG GAA ATA 396 Ser Trp Glu lie Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu lie 95 100 105 110 AAA 399
• MOLECULE TYPE protein
• Lys Leu Leu lie Lys Phe Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60
• Trp lie Gly Glu lie Leu Phe Gly Ser Gly Ser Ala His Tyr Asn Glu 50 55 60
• Gly Glu lie Leu Phe Gly Ser Gly Ser Ala His Tyr Asn Glu Lys Phe 50 55 60
• ATC CAT CCT GTG GAG GAG GAG GAT ACT GCA ACA TAT TAC TGT CAG CAC 348 lie His Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gin His 80 85 90
• Lys Leu Leu lie Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn lie His 65 70 75 80
• Glu lie Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu lie Lys Arg 100 105 110
• GCC CCT GGA TCT GCT GCC CAA ACT AAC TCC ATG GTG ACC CTG GGA TGC 492 Ala Pro Gly Ser Ala Ala Gin Thr Asn Ser Met Val Thr Leu Gly Cys 130 135 140 CTG GTC AAG GGC TAT TTC CCT GAG CCA GTG ACA GTG ACC TGG AAC TCT 540 Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser 145 150 155
• MOLECULE TYPE protein
• Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr 65 70 75 80
• Asp Lys Lys lie Val Pro Arg Asp Cys 210 215
• CAG TCT CCA AAG CTC CTG ATC TAC AAG GTT TCC AAC CGG TTT TCT GGG 252 o

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