WO1999023207A1 - Monoclonal antibodies to canine cd34 - Google Patents

Abstract

The present invention describes methods for the production of monoclonal antibodies, antigen binding fragments and antigen binding agents that react with canin CD34. Also provided are methods for using the monoclonal antibodies to characterize CD34+ cells by in vitro and in vivo functional studies, and to develop technology for transplantation of enriched progenitor cell populations. The antibodies, antigen binding fragments, and antigen binding agents can also be used to quantitate and isolate cell populations enriched for canine marrow progenitors.

Description

MONOCLONAL ANTIBODIES TO CANINE CD34

GOVERNMENT SUPPORT The present invention was made with support under grant nos. HL03701, DK42716, HL36444, HL54881, CA47748, CA31787, AI37747, and AI85003 from the U.S. National Institutes of Health. The U.S. Government has certain rights in this invention.

BACKGROUND OF THE INVENTION The expression of CD34 on hematopoietic cells is developmentally regulated in hematopoiesis such that its expression is lost beyond the committed progenitor stage (Civin et al . , J . Immunol . 133:157 (1984); Katz et al . , Leuk. Res. 9:191 (1985); Andrews et al . , Blood 67:842 (1986)). This pattern of expression allowed for the use of monoclonal antibodies (MAbs) to human CD34 to selectively isolate for the purpose of transplantation, small subpopulations from bone marrow which are highly enriched for progenitors (Berenson et al., J. Clin. Invest. 81:951 (1988); Berenson et al . , Blood 77:1717 (1991)). More recently peripheral blood progenitor cells, collected after mobilization with growth factors and/or chemotherapy, have been enriched from leukapheresis products by CD34 selection techniques for use in transplantation (Brugger et al . , Blood 84:1421 (1994); Schiller et al . , Blood 86:390 (1995); Bensinger et al . , Blood 88:4132 (1996)). CD34 selection facilitates tumor cell removal from autografts

(Schiller et al . , Blood 86:390 (1995); Shpall et al . , J. Clin. Oncol . 12:28 (1994)) and T cell depletion of allografts to prevent GVHD (Bensinger et al . , Blood 88:4132 (1996); Link et al., Blood 86:2500 (1995)). Separations of CD34⁺ cells for transplantation have been accomplished using immunoadsorption columns (Berenson et al . , Blood 77:1717 (1991); Shpall et al . , J. Clin. Oncol. 12:28 (1994)) immunomagnetic beads, (Civin et al., J. Clin. Oncol. 14:2224 (1996)) and high-speed cell sorting (Archimbaud et al . , Blood 88:595a (1996) abst . ) .

Recently both the cDNA and gene for the canine homologue of CD34 were cloned (McSweeney et al . , Blood 88:1992 (^"1996) . A recombinant CD34 murine immunoglobulin fusion molecule (CD34-Ig) was used as an immunogen to generate an affinity purified polyclonal antiserum (RPofCD34) that reacted with approximately 1% of bone marrow cells, a cell population which was highly enriched for CFU-GM. However, use of a polyclonal antiserum was suboptimal for precise FACS studies and for large scale cell separations.

Large scale separation of CD34⁺ cells has become increasingly important as these cells have been applied to studies of stem cell transplantation. Dogs have provided a valuable preclinical model for human stem cell transplantation. Storb, R. and Thomas, E.D., Immunological Reviews, Copenhagen, Munksgaard, 88:215 (1985); Storb, R. and Thomas, E. D., Bone Marrow Transplantation, Boston, MA, Blackwell Scientific Publications, pg 3 (1994) ) . However, a limitation of the canine model for studies of hematopoieses or stem cell transplantation has been the lack of a useful marker to identify and enrich hematopoietic progenitors. What is needed in the art is a method for the production of cell lines which secret monoclonal antibodies specific for canine CD34 that can be used to enrich canine progenitor cell populations that appear functionally and phenotypically similar to human CD34⁺ cells. These monoclonal antibodies can be used in facilitating the phenotypic and functional characterization of progenitors present in canine bone marrow, peripheral blood, and cord blood. The monoclonal antibodies of the present invention can also be used clinically in canine stem cell transplantation.

SUMMARY OF THE INVENTION

The present invention provides methods for the production of an immortalized cell line which expresses and secrets an immunoglobulin specific for canine CD34. In preferred aspects of the invention the method comprises immunizing a non-human animal with a composition of a cell line selected for expression on its surface consistent high levels of canine CD34 and a composition comprising the extracellular domain of canine CD34. A particularly preferred protocol is provided wherein the initial immunization is with a composition comprising a cell line selected for consistent high expression of canine CD34 followed by at least one booster immunization with a composition comprising the extracellular region of canine CD34. Optionally, additional booster immunizations with compositions comprising the selected cell line and/or compositions comprising the extracellular domain of canine CD34 can be given.

Particularly preferred cell lines are the canine leukemia lines ML3 or 1390, although other cell lines can be selected using a polyclonal antisera with a high antibody titer for canine CD34 or a monoclonal antibody of the present invention. Once a high titer of antibody to canine CD34 is obtained cells which express and secret CD34 specific antibody can be immortalized.

In a particularly preferred method the spleen of the non-human mammal is isolated and B cells are fused with myeloma cells to form a hybridoma. The hybridoma cells are screened for secretion of a monoclonal antibody specific for canine CD34 and which is nonreactive with other proteins, particularly the hinge, CH2 and CH3 domains of murine immunoglobulin. Immortalization can also be accomplished by other methods of transformation or transfection.

In a particularly preferred embodiment of the present invention mice are immunized initially with a canine cell line selected for consistently high expression of canine CD34 on the cell surface. This immunization is followed by two subsequent booster immunizations with a canine CD34 fusion protein, one booster immunization with the selected cell line, and a final booster immunization with the canine CD34 fusion protein. The animals are screened for the production of high titers of antibody reactive with canine CD34 and antibody secreting cells are immortalized. Immortalization can be by transfection, transformation or cell fusion. In a particularly preferred method of immortalization the spleen is removed from the mouse, and the spleen cells are fused with a murine myeloma cell to form a hybridoma. Hybridomas secreting antibodies specific for canine CD34 are selected by screening for binding activity to cells expressing canine CD34 on their surface, binding activity with the extracellular domain of canine CD34 and no binding activity with the hinge CH2 and CH3 domain of murine immunoglobulin. In alternative embodiments of the present invention canine CD34 antigen binding fragments and antigen binding agents are provided. These antigen binding fragments and antigen binding agents can be whole antibody, antigen binding fragments of whole antibody, or recombinant constructs which retain the antigen bind region of a whole antibody. The antigen binding fragments can be enzymatic digestion products of a whole antibody including Fv, Fab, Fab' and F(ab)'₂ fragments. Antigen binding agents of the present invention include various recombinant constructs which retain antigen binding specificity of the whole antibody, i.e., single chain antibodies, chimeric antibodies, CDR-grafted recombinant constructs, and the like. Although these recombinant constructs retain the binding specificity of the parental antibody the affinity and/or avidity of the construct can vary.

In yet another embodiment of the present invention, cell lines which express and secrete the monoclonal antibody, antigen binding fragments or antigen binding agents specific for canine CD34 are provided. These cell lines can be hybridomas, mammalian cell lines, bacteria or yeasts which have been genetically altered so as to express the antibody, antigen binding fragment, or antigen binding agent.

In a further embodiment of the invention monoclonal antibodies, antigen binding fragments and antigen binding agents specific for canine CD34 are provided which are labeled. Labels which are preferred include, radionuclides, fluorescers, enzymes, enzyme cofactors, enzyme substrates, enzyme inhibitors or ligands (particularly haptens) . A particularly preferred label is biotin.

The present invention also provides methods for separating canine CD34⁺ hematopoietic precursor cells from a cell sample. The method comprises isolating a cell sample from a donor and contacting this cell sample with a labeled antibody specific for canine CD34 under conditions conducive for complex formation. The complex is then separated from the cell sample. In a particularly preferred embodiment the label is biotin and the complex is separated from the cell sample with streptavidin-conjugated magnetic microbeads. The donor can optionally be induced to produce greater numbers of CD34⁺ hematopoietic precursor cells by administering, prior to collecting the cell sample, a growth factor or drug which up regulates hematopoietic precursor cell formation.

Particularly preferred growth factors and drugs include G-CSF, GM-CSF, SCF, cyclophosphamide and prednisone.

In yet another embodiment of the present invention, methods are provided for administering the isolated CD34⁺ hematopoietic cells to a recipient. The isolated CD34⁺ cells can be administered directly into a recipient as a complex with the antibody and solid phase or the cells can be separated from the complex prior to freezing or culturing in vitro for later administration.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides methods for the development of cell lines which secrete monoclonal antibodies specific for canine CD34. The antibodies and antigen binding fragments thereof are useful in preparing compositions for isolating canine bone marrow progenitor cells. Specifically the methods of the present invention use as an immunogen canine cell lines which have been selected for consistent high extracellular expression of canine CD34 in combination with compositions comprising the extracellular domain of canine CD34 to generate high titers of antibody specific for the extracellular domain of canine CD34. These methods have been used to successfully generate a high titer CD34-specific immune response in mice .

Animals with a high titer anti-CD34 antibody response are used to produce cell lines capable of secreting monoclonal antibodies which specifically recognize canine CD3 . The subject cells have identifiable chromosomes in which the germ-line DNA from them or a precursor cell has rearranged to encode an antibody having a binding site for canine CD34. These antibodies can be used in a wide variety of ways, including quantitating the number of CD34⁺ cells, for immunotyping leukemias, and in the preparation of enriched stem cell compositions for use in therapy.

Monoclonal antibodies provided by the present invention are particularly useful in the isolation of hematopoietic stem cells which are CD34⁺. The isolated stem cells can be used for the study of canine hematopoiesis, in preclinical studies concerning stem cell transplantation, gene therapy, and ex-vivo progenitor cell expansion, and in the clinical treatment of dogs with conditions treatable by stem cell transplantation, i.e., aplastic anemia, lymphoma, and the like.

Preparation of monoclonal antibodies can be accomplished by immortalizing a cell line capable of expressing nucleic acid sequences that code for antibodies specific for an epitope on canine CD34. The immortalized cell line can be a mammalian cell line that has been transformed through oncogenesis, by transfection, mutation, or the like. Such cells include myeloma lines, lymphoma lines, or other cell lines capable of supporting the expression and secretion of the immunoglobulin, or antigen binding fragment thereof, in vitro . The immunoglobulin or fragment can be a naturally- occurring immunoglobulin of a mammal other than the preferred mouse or canine sources, produced by transformation, or a lymphocyte, particularly a splenocyte, by means of a virus or by fusion of the lymphocyte with a neoplastic cell, e.g., a myeloma, to produce a hybrid cell line. Typically, the splenocyte will be obtained from an animal immunized against canine CD34, fragments thereof, and/or cell lines which consistently express on their surface high levels of the canine CD34 antigen.

Immunization protocols to generate high titer immune response to known antigens generally are well known, but the common methods of immunization when used for canine CD34 were unsuccessful in producing high antibody titers specific for the antigen. Use of an immunization protocol which used as the immunogen an extracellular fragment of canine CD34, i.e., constructed as an immunoglobulin fusion protein, produced unexpectedly high reactivities against the immunoglobulin portion of the molecule. These sera also demonstrated no specific reactivity against the extracellular domain of canine CD34.

In the present invention an immunization protocol has been developed which produces a high titer immune response specific for canine CD34. This protocol requires the use of a combination of immunogens including a cell line which consistently expressed on its surface high levels of canine CD34 and a composition comprising the extracellular portion of canine CD3 .

Cell lines for use as an immunogen can be selected using a polyclonal antisera reactive with canine CD34. Specific antisera can be generated by injecting a mammal, such as a rabbit, with canine CD34 in a single immunization, or multiple immunizations using the same antigen. Once a high titer activity has been obtained that is specific for canine CD34, the antisera can be used to screen for canine tumor cell lines which express high levels of CD34. The selected cell line can then be used in combination with canine CD34 as an immunogen in producing a high titer immune response. In a preferred embodiment the canine CD34 is provided as a fusion protein comprising the extracellular domain of CD34 and a portion of another protein. In particular, the fusion protein comprises the extracellular domain of canine CD34 and the hinge, CH2 and CH3 domains of an immunoglobulin molecule, i.e., a murine immunoglobulin.

Administration of the immunogens, cells from the selected cell line and canine CD34, is preferably in a mixture containing an adjuvant, such as Freund's adjuvant, Montanide ISA50 or RIBI adjuvant. Although a single injection of each immunogen can be used, it is preferred that the immunization protocol include one or more injections of a selected cell line followed by one or more booster injections of canine CD34 fusion protein. These boosters can be followed by additional booster injections with either or both the selected cell line or CD34 fusion protein. The injections can be intravenous, intraperitoneal, subcutaneous or intramuscular, or any combination thereof.

In one preferred embodiment of the present invention mice were immunized with a composition comprising the extracellular region of canine CD34 for the first and/or second injection. This was followed by at least one immunization with the selected canine cell line which consistently expressed on its cell surface high levels of CD3 . Just prior to removing the spleens from the mice for fusion, the animals were boosted with a composition comprising a CD34 fusion protein. Using this immunization protocol several hybridomas were developed which secrete antibodies specific for canine CD34.

In certain preferred embodiments of the invention, monoclonal antibodies are used. Methods for producing monoclonal antibodies are well known in the art and are disclosed, for example, by Kohler and Milstein, Nature 256:495 (1975); Eur. J. Immunol. 6:511-519 (1976); Hurrell, J.G.R., ed. , Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press Inc., Boca Raton, FL (1982); and Hart, U.S. Patent No. 5,094,941, each incorporated herein by reference in its entirety.

Preferably, the monoclonal antibodies, antigen binding fragments, and other antigen binding agents of the invention are provided in substantially pure, or isolated, form. As used herein, the terms "substantially pure" and "isolated" mean that an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species) in a composition. Preferably, the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. More preferably, the object species in a substantially pure or isolated form will comprise more than about 80 to 90 percent of all macromolecular species present in a composition. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. The monoclonal antibodies, antigen binding fragments, and other antigen binding agents of the invention preferably specifically inhibit binding of an antibody of the present invention to canine CD34. By specifically inhibiting binding of a monoclonal antibody to canine CD34 is meant that the molecule being tested blocks or competes with an antibody of the present invention for binding in one or more competitive binding assays, such that the antibody of the present invention is unable to bind to canine CD34. Binding in the presence of the test molecule is inhibited by at least 10%, preferably by at least 25%, more preferably by at least 50%, and most preferably by at least 75%-90% or greater compared to monoclonal antibody binding in a control assay in the absence of the test molecule. The capacity to block or compete with monoclonal antibody binding to canine CD34 may be determined by a variety of methods, as disclosed, for example in Bowen et al . , J. Biol. Chem. 271:17390-17396 (1996).

The capacity to block, or compete with, monoclonal antibody binding to canine CD34 typically indicates that the test molecule binds to an epitope or binding site that is recognized by the antibody or structurally overlaps with an epitope or binding site of CD34 which is sufficiently proximal to the epitope or binding site of CD34 recognized by the antibody to sterically or otherwise inhibit binding of the antibody to CD34. Exemplary antigen binding agents in this context include the monoclonal antibodies of the present invention, i.e., 6B11, 2E9, 1H6 , 5F11-3 and 5F11-6 described and designated hereinbelow. Additional antigen binding agents provided within the invention include, for example, antibody fragments, including Fv, Fab, Fab' and F(ab)'₂ fragments, and recombinantly modified antibodies that share substantially similar CD34 binding affinity as a native anti-canine CD34 monoclonal antibody of the invention. Yet additional antigen binding agents provided within the invention include, for example, chimeric antibodies, single chain antibodies and recombinant antibody constructs which maintain the complementary determining regions (CDRs) of the aforementioned anti-canine CD34 monoclonal antibodies.

Antibodies, or immunoglobulins, are typically composed of four covalently bound peptide chains. For example, an IgG antibody has two light chains and two heavy chains. Each light chain is covalently bound to a heavy chain. In turn each heavy chain is covalently linked to the other to form a "Y" configuration, also known as an immunoglobulin conformation. Fragments of these molecules, or even heavy or light chains alone, may bind CD34. Antibodies, antigen binding fragments of antibodies, and individual chains are also referred to herein as immunoglobulins.

A normal antibody heavy or light chain has an N- terminal (NH₂) variable (V) region, and a C-terminal (-COOH) constant (C) region. The heavy chain variable region is referred to as V_H (including, for example, Vγ) , and the light chain variable region is referred to as V_L (including V_κ or V_λ) . The variable region is the part of the molecule that binds to the antibody's cognate antigen, while the Fc region (the second and third domains of the C region) determines the antibody's effector function (e.g., complement fixation, opsonization) . Full-length immunoglobulin or antibody "light chains" (generally about 25 Kd, about 214 amino acids) are encoded by a variable region gene at the N-terminus (generally about 110 amino acids) and a K (kappa) or λ (lambda) constant region gene at the COOH-terminus . Full-length immunoglobulin or antibody "heavy chains" (generally about 50 Kd, about 446 amino acids) , are similarly encoded by a variable region gene (generally encoding about 116 amino acids) and one of the constant region genes, e.g., gamma (encoding about 330 amino acids) . Typically, the "V_L" will include the portion of the light chain encoded by the V_L and/or J_L (J or joining region) gene segments, and the "V_H" will include the portion of the heavy chain encoded by the V_H, and/or D_H (D or diversity region) and J_H gene segments. See generally, Roitt et al . , Immunology, Chapter 6 (2d ed. 1989) and Paul, Fundamental Immunology; Raven Press (2d ed. 1989) .

An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, also called complementarity-determining regions or CDRs . The extent of the framework region and CDRs have been defined (see, e.g., Kabat et al . , Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services (1987); and Chothia et al . , J. Mol . Biol . 196:901-917 (1987), each incorporated herein by reference in its entirety) . The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three dimensional space. The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs are typically referred to as CDR1, CDR2 , and CDR3 , numbered sequentially starting from the N-terminus.

The two types of light chains, K and λ, are referred to as isotypes. Isotypic determinants typically reside in the constant region of the light chain, also referred to as the C_L in general, and C_κ or C_λ in particular. Likewise, the constant region of the heavy chain molecule, also known as C_H, determines the isotype of the antibody. Antibodies are referred to as IgM, IgD, IgG, IgA, and IgE depending on the heavy chain isotype. The isotypes are encoded in the mu (μ) , delta (δ) , gamma (γ) , alpha ( ) , and epsilon (e) segments of the heavy chain constant region, respectively. In addition, there are a number of γ subtypes .

The heavy chain isotypes determine different effector functions of the antibody, such as opsonization or complement fixation. In addition, the heavy chain isotype determines the secreted form of the antibody. Secreted IgG, IgD, and IgE isotypes are typically found in single unit or monomeric form. Secreted IgM isotype is found in pentameric form; secreted IgA can be found in both monomeric and dimeric form.

Using well known methods of recombinant DNA technology, the antibodies and antibody fragments of the invention may be produced at high levels. In addition, native antibodies and antibody fragments can be routinely modified to yield modified anti-canine CD34 immunoglobulins having substantially similar or enhanced binding specificities and/or competition activities of their corresponding parent anti- canine CD34 immunoglobulins. For example, genes encoding a native antibody (e.g., a gene encoding a 6B11, 2E9, 1H6 5F11-3 or 5F11-6 monoclonal antibody as described herein) can be isolated and cloned into one or more polynucleotide expression vectors, and the vector can be transformed into a suitable host cell line for expression of a recombinant antibody. Expression of the cloned antibody encoding gene provides for increased yield of antibody, and also allows for routine modification of native immunoglobulins by introducing amino acid substitutions, deletions, additions and other modifications in both the variable and constant regions without critical loss of binding specificity.

Genes encoding the heavy and light chains of anti- canine CD34 immunoglobulins are isolated and cloned according to methods, known in the art, for example according to methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual , 2nd ed., Cold Spring Harbor, NY, (1989); Berger &

Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques , Academic Press, Inc., San Diego, CA, (1987); Co et al . , J . Immunol . , 148:1149 (1992), each of which is incorporated herein by reference for all purposes. In certain aspects of the invention genes encoding heavy and light chains are cloned from genomic DNA of a selected, anti- canine CD34 producing hybridoma, or, alternatively, from cDNA produced by reverse transcription of the hybridoma' s RNA.

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expression control sequence, including naturally-associated or heterologous promoter regions, operably linked to one or more desired coding sequences. Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into an appropriate host cell, the host cell is maintained under conditions suitable for expression of the coding sequences, and for subsequent collection and purification of the native or modified anti- canine CD34 immunoglobulin or antibody fragment.

Expression vectors suitable for use within the invention are typically replicable in host cells either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors will contain selection markers, e.g., ampicillin-resistance or hygromycin-resistance, to permit detection of cells transformed with the desired DNA sequences .

In general, prokaryotes can be used for cloning and expressing the DNA sequences encoding a native or modified anti-canine CD34 immunoglobulin or antibody fragment. E. coli represents one prokaryotic host that is particularly useful for cloning and expression of the DNA sequences of the present invention. Other hosts, such as yeast, are also useful for cloning and expression purposes. Saccharomyces is a preferred yeast host, with suitable vectors having expression control sequences, an origin of replication, termination sequences and the like as desired. Typical promoters include 3- phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase 2, isocytochrome C, and enzymes responsible for maltose and galactose utilization.

Mammalian cells are a particularly preferred host for expressing nucleotide segments encoding immunoglobulins or antigen binding fragments or agents thereof. (See, e.g., Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987, incorporated herein by reference in its entirety) . A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, and include CHO cell lines, various COS cell lines, HeLa cells, L cells and myeloma cell lines. Preferably, the cells are non- human. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (See, e.g., Queen et al . , Immunol . Rev . 89:49 (1986), incorporated herein by reference in its entirety) , and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenyla- tion sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. (See, e.g., Co et al . , J. Immunol. 148:1149 (1992), incorporated herein by reference in its entirety) . Vectors containing immunoglobulin encoding DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of host cell. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, electroporation, lipofection, biolistics or viral-based transfection may be used for other cellular hosts. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see, e.g., Sambrook et al . , supra) . Once expressed, anti-canine CD34 immunoglobulins and antigen binding fragments and agents of the invention can be purified according to standard methods in the art, including HPLC purification, fraction column chromatography, gel electrophoresis and the like (see, e.g., Scopes, Protein Purification, Springer-Verlag, NY, 1982, incorporated herein by reference in its entirety) .

Many of the native anti-canine CD34 immunoglobulins and antigen binding fragments and agents described herein can undergo non-critical amino acid substitutions, additions, deletions and other modifications in both the variable and constant regions without loss of binding specificity. Usually, immunoglobulins and antigen binding fragments and agents incorporating such modifications exhibit substantial sequence identity to native immunoglobulins or antigen binding fragments and agents from which they were derived. Preferably, mature light chains of antibodies derived from native monoclonal antibodies of the invention exhibit substantial amino acid sequence identity to the amino acid sequence of a mature light chain of the corresponding native antibody. Similarly, the mature heavy chains of modified anti-canine CD34 immunoglobulins of the invention typically exhibit substantial sequence identity to the sequence of the mature heavy chain of the corresponding native antibody. As applied to polypeptides, the term "substantial sequence identity" means that two polypeptide sequences, when optimally aligned, such as by the programs BLAZE (Intelligenetics) GAP or BESTFIT using default gap weights, share at least 70 percent or 85 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity or more (e.g., 99 percent sequence identity) . Preferably, residue positions which are not identical differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine . Preferred conservative amino acids substitution groups are: valine-leucine- isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine . The monoclonal antibody and antigen binding fragments and agents thereof as described in the present invention are useful in various immunological assays and methods. For instance, the antibodies can be used in qualitative or quantitative assays to determine the location and number of CD34⁺ hematopoietic precursor cells that may be present in an animal .

In one instance, the antibodies can be used to quantitate CD34⁺ cells in order to or differentiate leukemias, i.e., refractory anemia with excess blasts (Oertel et al . , Brit. J. Haematology 84:305-309 (1993), Oertel et al . , Leukemia and Lymphoma 15:65-69 (1994)).

In another embodiment, isolated CD34⁺ cells can be cultured iri vitro to produce a culture of dendritic cells. In one method CD34⁺ marrow precursor cells can be grown in a medium containing growth factors, i.e., GM-CSF, MGF, and TNF-α, and the like, for a number of days. This can be followed by a second culturing period in medium containing CD40L (also known as gp39) and IL-4 in place of TNF-ατ The second culturing would be expected to increase the percentage of dendritic cells in the culture. The number of dendritic cells can be further increased by immunoaffinity separation based on cell surface expression of a dendritic cell specific surface antigen, i.e., CDla. See, for example, Ye et al . ,

Bone Marrow Transplantation 18:997-1008 (1996), Hanada et al . , J. Leuk. Biol. 60:181-190 (1996), and Szaboles et al . , Adv. Exp. Med. Biol. 378:17-20 (1995).

In order to separate CD34⁺ cells for use in such methods it may be necessary to label the antibody, antigen binding fragment or antigen binding agent of the present invention. A wide variety of labels may be employed, such as radionuclides, fluorescers, enzymes, enzyme cofactors, enzyme substrates, enzyme inhibitors, ligands (particularly haptens) , and the like. A particularly preferred label is biotin.

Methods for preparing compositions for increasing the number of canine CD34⁺ hematopoietic precursor cells in vitro and in vivo are well known. Specifically, the monoclonal antibodies, antigen binding fragments, and antigen binding agents of the present invention can be used to separate CD34⁺ canine hematopoietic precursor cells from a blood product, such as bone marrow, peripheral blood, umbilical cord blood and other sources. As discussed in more detail below, such separation may be performed, for example, by immunoselection on the basis of their expression of the CD34 antigen. The separated hematopoietic precursor cells may be stored frozen and thawed at a later date for inoculation into a suitable vessel containing a culture medium comprising a conditioned medium and nutritive medium, optionally supplemented with a source of growth factors and, optionally, canine or other animal plasma or serum. Alternatively, the separated cells may be inoculated directly into culture without first freezing. In both cases the resultant cell suspension is cultured under conditions and for a time sufficient to increase the number of hematopoietic precursor cells relative to the number of such cells present initially in the blood product. The cells may then be separated by any or a variety of methods, such as centrifugation or filtration, from the medium in which they have been cultured, and can be washed one or more times with fresh medium or buffer. Optionally, the cells may be re-separated into CD34⁺ and CD34^" fractions, prior to resuspension to a desired concentration in a medium suitable for infusion into a dog or stored frozen for infusion at a later date. Separated cell can also be infused into a recipient as a complex with the separation media, i.e. an antibody-magnetic bead\CD34⁺ cell complex without culturing. Within the context of the present invention, a CD34⁺ hematopoietic precursor cell is defined as a cell which expresses sufficient CD34 antigen to be detected by a given method of assay. For example, CD34⁺ cells can be identified and quantitated by flow microfluorimetry using a fluorescent activated cell sorter (FACS) , by immunofluorescence or immunoperoxidase staining using a fluorescence or light microscope, by radioimmunoassay, or by immunoaffinity chromatography, among numerous other methods which will be readily apparent to the skilled artisan (see, for example, Lansdorp and Thomas (in Bone Marrow Processing and Purging, A. P. Gee (ed.), Boca Raton:CRC Press (1991) pg. 351). Hematopoietic precursor cells can also be detected by various colony-forming assays, such as CFU-GM and CFU-S assays (see, e.g., Sutherland et al . , in Bone Marrow Processing and Purging , supra at pg. 155) .

CD34⁺ can be obtained from any variety of blood products, including bone marrow, peripheral blood, umbilical cord blood, fetal liver, and spleen. Bone marrow is a particularly rich source of precursor cells (1-2% of marrow) , but alternate sources may be preferable because of the discomfort associated with bone marrow aspiration. Bone marrow is typically aspirated from the iliac crest, but can be obtained from other sites (such as the sternum or vertebral bodies) if necessitated by prior or concurrent disease or therapy.

Peripheral blood contains fewer precursor cells (typically < 1% of peripheral blood mononuclear cells) , but is generally easier to obtain than bone marrow. The number of precursor cells circulating in peripheral blood can be increased by prior exposure of the donor to certain growth factors, such as, for example, G-CSF or SCF (KL) , and/or certain drugs, such as, for example, 5-fluorouracil, cyclophosphamide or prednisone (Korbling and Martin, Plasma Ther. Transfer Tech. 9:119 (1980)). Peripheral blood collected from donors who have been pretreated to increase the number of circulating CD34⁺ cells is referred to as having been "mobilized." Depending upon the volume which is desired, blood can be obtained by venipuncture or by one or more other methods including aphereses, for example, on a COBE 2997 blood separator. Precursor cells can also be obtained from umbilical cord blood at the time of delivery, either by simple gravity-induced drainage or manual expression as described in U.S. Patent 5,004,681, incorporated herein by reference.

Although one can readily separate a bone marrow, peripheral blood specimen, or apheresis product into precursor and mature cells, it is generally preferred to prepare a buffy coat or mononuclear cell fraction from these specimens first, prior to separation into the respective populations. Methods for the preparation of a buffy coat and mononuclear cell fractions are well known in the art (Kumar and Lykke, Pathology 16:53 (1994)). Separation of precursor cells from more mature cells can be accomplished by any of a variety of methods known to those skilled in the art, including immunoaffinity chromatography (Bash et al . , J . Immunol . Methods 56:269 (1983) ) , fluorescence-activated cell sorting, panning (Wysocki and Sato, Proc . Nat'l. Acad. Sci USA 15:2844 (1978)), magnetic-activated cell sorting (Miltenyi et al . , Cvtometry 11:231 (1990)), and cytolysis.

CD34⁺ cells may be positively selected and/or negatively selected. By positive selection is meant the capture of cells by some means, usually immunological, on the basis of their expression of a specific characteristic or set of characteristics (usually an antigen (s) expressed at the cell surface) . For example CD34⁺ cells can be positively selected by any of the above methods (except cytolysis) using the monoclonal antibodies of the present invention.

Negative selection means the exclusion or depletion of cells by some means, usually immunological, on the basis of their lack of expression of a specific characteristic or set of characteristics (again, usually a surface antigen) . For example, CD34⁺ cells can be negatively selected by any of the above methods on the basis of their lack of expression of lineage-defining antigens, such as CD19 (for B lymphocytes) , CD3 (for T lymphocytes) , CD56 (for Natural Killer cells) , etc., utilizing antibodies to the above-mentioned and other lineage defining antibodies. By using a cocktail or mixture of monoclonal antibodies directed to red cell, platelet, granulocyte, lymphocyte and/or tumor cell antigens, it is possible to leave behind a population of cells which is highly enriched for CD34⁺ cells. Numerous monoclonal antibodies and polyclonal antibodies suitable for this purpose are known in the art .

Alternatively, precursor cells can be separated from mature cells by a combination of negative and positive selection techniques. A preferred combination of negative and positive selection techniques comprises a first selection for CD34⁺ cells utilizing the anti-CD34 monoclonal antibodies of the present invention, followed by a second selection for HLA- DR-negative/CD34⁺ cells, using an anti-HLA-DR antibody to a non-polymorphic determinant on the DR molecule. The advantage of this or other dual selection strategies is that the volume of cells which is placed into culture is smaller and more manageable .

Other types of molecules rather than antibodies can be used in co-seletion techniques. In some cases selection can involve the use of lectins or other types of receptors or ligands expressed on the cell surface. Among other antibodies, antigens, receptors and ligands which may be useful, alone, sequentially, or in combination with the antibodies, antigen binding fragments, and antigen binding agents of the present invention, are transferrin, the transferrin receptor, soybean agglutinin, c-kit ligand, c-kit receptor, HLA-DR, CD33, and the like.

Various methods can be utilized in order to separate CD34⁺ cells from mature cells. For example, cells can be separated on an affinity column, incubated in a selected medium, and then subsequently re-separated in order to separate the CD34⁺ cells from the newly differentiated mature cells. Particularly preferred methods and devices for the selection of CD34⁺ cells are described in U.S. Patent Nos. 5,215,927, 5,225,353, 5,262,334 and 5,240,856, each of which is incorporated by reference in its entirety. These applications describe methods and devices for isolating or separating target cells from a mixture of non-target cells and target cells, wherein the target cells are labeled, directly or indirectly, with a biotinylated antibody and a target cell surface antigen. Labeled cells are separated from unlabeled cells by, flowing them through a bed of immobilized avidin, the labeled cells binding to the avidin to form a complex by virtue of the biotinylated antibody bound to their surface, while the unlabeled cells pass through the bed. After washing the bed material, the labeled (bound) cells can be eluted from the bed, for example, by mechanical filtration.

Subsequent to separation, CD34⁺ cells can be inoculated into a culture medium comprised of a nutritive medium, any number of which, such as RPMI, TC 199, Ex vivo-10, or Iscove's DMEM, along with a source of growth factors, will be apparent to the skilled artisan. Proliferation and differentiation of precursor cells can be enhanced by the addition of various components to the medium, including a source of plasma or serum. Among sources of plasma and serum are fetal bovine and dog. The amount of plasma or serum which is used will vary, but is usually between about 1 and 50% (by volume) of the medium in which the cells are grown, and more often between about 1 and 24%. The separated CD34⁺ cells are cultured in the nutritive medium, optionally containing a source of plasma or serum allowing the cells to grow, usually 1 to 3 days, and then separating the cells from the medium, usually by centrifugation or filtration. The cells are collected and can then be administered to the recipient by one of many known methods .

In a particularly preferred method of preparing a composition enriched for CD34⁺ hematopoietic precursor cells a monoclonal antibody, antigen binding fragment, or antigen binding agent of the present invention is conjugated to biotin. The biotinylated antibody is admixed with a buffy coat prepared from a bone marrow, peripheral blood or apheresis sample. Antibody binds to those cells which express CD34 and the sample is washed and combined with immunomagnetic streptavidin-coated microbeads under conditions conducive for biotinylated antibody and streptavidin-coated microbeads to form a complex. The complex is separated from the mixture and can be infused directly into a recipient or the CD34⁺ cells can eluted from the beads and frozen and\or cultured and frozen prior to infusion.

The following examples are offered by way of illustration, not by way of limitation. EXAMPLE 1 This example describes methods for the development of monoclonal antibodies specific for canine CD34.

Cell lines and cell culture.

Canine myelomonocytic leukemia cell lines ML1, (Kawakami et al . , Leuk. Res. 13:709 (1989) ML2 , ML3 , 1390 (CD8⁺ leukemia) and CLGL (large granular lymphocyte) were cultured as previously described (McSweeney et al . , Blood 88:1992 (1996)). Jugular vein endothelial cells from normal dogs were purchased from Endotech (Indianapolis, IN) and cultured according to the manu acturer's instructions.

Production of recombinant CD34. A canine CD34 immunoglobulin fusion protein, consisting of the extracellular domain of CD34 fused to the hinge, CH2 and CH3 of a murine IgG-2a immunoglobulin molecule, (CD34-Ig) was produced by transient expression in COS cells and purified as previously described (Wallace et al . , Transplantation 58:602 (1994)).

Anti-CD34 enzyme-linked immunosorbent assay (ELISA) .

The anti-CD34 ELISA was based on a previously described protocol (Linsley et al . , Science 257:792 (1992)) with the following modifications: Immunlon 2 (Dynatech) flat- bottom plates were coated with 3 μg/ml CD34-Ig diluted in 0.05 M bicarbonate binding buffer, pH 9.6. Bound antibody was detected with a 1:8000 dilution of horseradish peroxidase (HRP) conjugated anti-mouse IgG-1 antibody (Southern Biotechnology, Birmingham, AL) . Bound HRP was detected with ATBS substrate (Kirkgaard and Perry, Gaithersburg, MD)

Immunization of Mice with CD34-Ig and Cell Lines and MAb Production. Six-month old Balb/c mice, obtained from Taconic

(Germantown, NY) , were given three injections with either canine CD34-Ig or cells in varying combinations and sequences. ML3 or 1390 cells (100 μl at 10⁸/ml) were injected either subcutaneously or intraperitoneally in PBS, or intraperitoneally in adjuvant. When using adjuvant, protein (300 μl at 500 μg/ml in PBS) was mixed with 150 μl Montanide ISA50 and 150 μl RIBI adjuvant (Ribi Immunochem Research Inc., Hamilton, MT) . CD34-Ig-specific antibody titers of mouse sera were determined by ELISA. Mice were selected for fusion based on antibody titers to CD34-Ig and a pre- fusion boost was given with CD34-Ig.

MAbs to CD34 were produced as previously described (Wayner et al . , J. Cell. Biol. 105:1873 (1987)). Briefly, spleen cells from the immunized Balb/c mice were fused with NS-l/FOX-NY myeloma cells. Viable heterokaryons were selected in RPMI 1640 medium supplemented with adenine/aminopterin/thymidine (AAT) . Cultures secreting antibody specific for CD34 were identified by ELISA using an HRP-conjugated goat antibody (Zymed, San Francisco, CA) specific for mouse antibodies with an IgGl isotype and by reactivity with ML3 and 1390 cell lines utilizing a goat anti- mouse IgG FITC second-stage antibody. Monoclonal hybridoma cell lines were produced via two rounds of cloning via limiting dilution. Large-scale MAb production was undertaken by ascites production using stable hybridomas. MAb was purified from ascites using protein G columns (Pierce Chemical Company, Rockford IL) according to the manufacturer's instructions. Biotinylation of purified MAb was performed using D-biotinyl-e-aminocaproic acid N-hydroxysuccinimide ester (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer's instructions. Isotypes of MAbs were determined by using ISOSTRIP Mouse Monoclonal Antibody Isotyping Kit (Boehringer Mannheim) as per the manufacturer's instructions.

Production and evaluation of MAbs to CD34.

Initial attempts to generate anti-CD34 MAbs using CD34-Ig alone as an immunogen failed, and there was a strong and unexpected production of antibodies to the murine Ig portion of the fusion protein. Because the ML2 leukemia cell line (from which the CD34 cDNA was cloned) was found to express variable, and at times low level, surface CD34, and because there was a concerned that CD34 epitopes on CD34⁺ endothelial cells may differ from those on hematopoietic cells (Baumhueter et al . , Blood 84:2554 (1994)) additional canine leukemia cell lines were screened for CD34 expression with RPo;CD34. Two cell lines, ML3 and 1390, were identified that consistently expressed high levels of cell surface CD34. Six mice were immunized using ML3 , 1390 and CD34-Ig in various combinations, and sera were screened for antibody titers to CD34-Ig by ELISA. To test whether antibodies were specific to the extracellular domain of CD34, serum was also tested against a control fusion protein which has an identical immunoglobulin amino acid sequence to CD34-Ig. This showed that the immunization schedule was important for generating high titer responses against the extracellular domain of CD34. Antibody titers specific to CD34 were highest when CD34-Ig was used in the first or second immunization of a schedule that included immunization with ML3 or 1390. Immunizations with ML3 and 1390 cells alone did not produce measurable responses against CD34-Ig. The spleen from one of several mice with high serum titers of CD34 specific antibody was chosen for fusion. This animal received a primary immunization with ML3 cells, two subsequent boosts with CD34-Ig, then ML3 , and a final immunization with CD34-Ig immediately prior to fusion. CD34- Ig was used in ELISA assays for initial screening of hybridoma culture supernatants . Since CD34-Ig contains a murine IgG-2a sequence, this was accomplished by using an IgG-1 specific antibody to detect MAb bound to CD34-Ig. ELISA screening detected 32 hybridomas producing MAb reactive with CD34-Ig. Twenty-one of these had MAb reactive with at least one of the screening cell lines ML3 and 1390 cells when analyzed by FACS . Further screening for MAbs of other isotypes, performed using both ML3 and 1390 cells using a general FITC anti-mouse detection step, did not detect additional MAbs reactive with these cell lines. The positive hybridomas were cloned by limiting dilution, and the isotypes of these MAbs were independently confirmed to be IgG-1. Supernatants from hybridomas were then screened by FACS against BMMC by both single and two-color analysis in combination with RPc_CD34. Nine of 21 supernatants were reactive with the same cell population (-3% of BMMC) that was recognized by RPcκCD34. Three hybridomas were tested and show a high concordance between the cell populations recognized by the polyclonal antiserum and the three MAbs. Further studies indicated that 5F11 contained two different anti-CD34 MAbs, and these (5F11-3 and 5F11-6) were separated by additional limiting dilution cloning. Therefore, 10 hybridomas were cloned and MAbs further tested in FACS studies against CD34⁺ and CD34^" cell targets. Each of these MAbs reacted with CD34⁺ cell targets (1390, ML3 , ML2 , cultured endothelial cells) and did not react with CD34^" cell lines.

EXAMPLE 2

Characterization of Monoclonal Antibodies Specific for Canine CD34.

This example describes the characterization of the monoclonal antibodies demonstrated to be specific for canine CD34 and not for the immunoglobulin portion of the canine CD34-Ig fusion protein.

Western blotting.

Recombinant proteins (300 ng) were separated by SDS PAGE using an 8% Tris-glycine gel (Novex, San Diego CA) under reducing conditions and transferred to PVDF membranes. Membranes were blocked with 15 mg/ml non-fat dry milk in PBS and then blotted with 1 μg/ml MAb-biotin conjugate, washed in 0.5X PBS/0.5% Tween-70, then blotted with 1:5000 streptavidin- peroxidase in the blocking solution.

Leukemia cell lines ML1, ML3 and 1390 (2.5 x 10⁵ cells) were harvested, washed twice in PBS, and proteins prepared by lysis in 1% NP40 in PBS followed by removal of nuclei by centrifugation at 10000 xg. Proteins (25 μg) were separated by SDS PAGE in a 10% polyacrylamide gel under reducing conditions and proteins were transferred to nitrocellulose membranes. Membranes were blocked with 5% non- fat milk in PBS and then incubated with either RPαCD34 at 500 ng/ml or MAb (2E9 or 1H6) at 2 μg/ml. After washing, blots were incubated in blocking solution containing alkaline phosphatase conjugated second stage goat anti-rabbit or goat anti-mouse antibodies at 1:500 dilution (Amersham, Arlington

Heights, IL) . Western blots were developed with ECL detection reagents (Amersham) .

Monoclonal Antibody Affinity Determination. The binding of anti-CD34 MAbs to the CD34-Ig fusion protein was determined by BIAcore analysis. All experiments were run on BIAcore 1000 or 2000 instruments (Karlsson et al . , J. Immunol. Methods 145:229 (1991)) (Pharmacia Biosensor, Uppsala, Sweden) at 25°C, using phosphate buffered saline, pH 7.4 (Gibco BRL Products, Life Technologies, Gaithersburg, MD) containing 0.005% surfactant P20 (Pharmacia Biosensor) as the running buffer. The carboxymethylated dextran matrix of research grade sensor chip CM5 (Pharmacia Biosensor) was modified using the Amine Coupling Kit (Pharmacia Biosensor) as follows (Johnsson et al . , Anal. Biochem. , 198:268 (1991)):

Equal volumes of 0.10M N-hydroxysuccinimide and 0.40M N-ethyl- N' - (3 -dimethylaminopropyl) carbodimide were mixed and injected to activate the surface for 7 minutes. The ligand, a solution of 10 μg/ml of CD34-Ig in lOmM sodium formate, pH 4.0 was injected for 3 minutes. Remaining active sites were reacted by the injection of 1M ethanolamine for 5 minutes. Immobilization of approximately 900 RU of CD34-Ig was achieved. Each analyte was diluted in the running buffer to give a series of concentrations between 10 and 250 nM. Appropriate volumes of these solutions were injected with a flow rate of 10 to 50 μl/min. Following the injection, flow of running buffer alone was established to allow observation of the dissociation of bound protein. Association and dissociation rates of each analyte were determined by curve fitting using BIAevaluation 2.1 (Pharmacia Biosensor)

(O'Shannessy et al . , Anal. Biochem. 212:457 (1993)). The affinity was calculated by dividing the dissociation rate by the association rate. Flow Cytometry.

Cells were incubated with purified monoclonal antibody at 5-10 μg/ml or with supernatants from overgrown hybridoma cultures. Monoclonal antibodies 31A (murine IgG-1, anti-murine Thy-1) (Denkers et al . , J. Immunol. 135:2183 (1985)) and S5 (anti-canine CD44) (Schuening et al . , Transplantation 44:607 (1987)) were used as negative and positive controls, respectively. Second-stage reagents were FITC-conjugated or PE-conjugated goat-anti-mouse polyclonal antibody, streptavidin-FITC (all Caltag, San Francisco, CA) and streptavidin PE (Southern Biotechnology) . In two color studies, RPαCD34 (McSweeney et al . , Blood 88:1992 (1996)) was used at 5 μg/ml with a PE-conjugated goat anti-rabbit antibody (Southern Biotechnology) as a second stage reagent. Cell lines and cultured endothelial cells were incubated with MAb for 20 minutes at 4°C, washed twice with PBS, incubated with FITC-conjugated second-stage antibodies for 20 minutes at 4°C and then washed twice with PBS. Ficoll-Hypaque separated bone marrow mononuclear cells (BMMC) , unfractionated hemolysed bone marrow (BM) , or peripheral blood mononuclear cells at 2-5 x

10⁶/ml were stained as previously described (McSweeney, supra . (1995) ) . In blocking experiments to demonstrate specificity of monoclonal antibodies for CD34, cell lines were incubated with MAb (5-10 μg/ml) , CD34-Ig (100 μg/ml) or a combination of the two. Cells were then washed with PBS and incubated with FITC-conjugated goat anti mouse IgG antibody. Cells were washed and resuspended in PBS/2% FCS for analysis. To determine whether a non-crossblocking pair of MAbs could be identified, ML3 cells were incubated with saturating concentrations of MAb at 4°C, washed in PBS/FCS and then stained with biotinylated 1H6 at 10 μg/ml. Following further washing, cells were incubated with avidin-PE or avidin-FITC. Cells were washed and resuspended in PBS/FCS for FACS analysis. Flow cytometry was performed on a FACScan (Becton Dickinson, San Jose, CA) or FACStar (Becton Dickinson) and the list mode data were analyzed using Winlist (Verity Software House Inc., Topsnam ME) or Cellquest software (Becton Dickinson) . CFU-GM assays.

BMMC (5 x 10⁷/ml) were stained with MAb 2E9 or 1H6 at 5-10 μg/ml as described above, resuspended in PBS/2% horse serum and sorted using a FACStar. Sorted CD34⁺ and CD34^" cell fractions and control BMMC were centrifuged, resuspended in culture media containing recombinant canine (re) -G-CSF, rc-SCF and rc-GM-CSF each at 100 ng/ml, and various numbers of cells per plate were assayed for granulocyte-macrophage progenitor cells (CFU-GM) as previously described (Rossbach et al . , Exp. Hematol. 24:221 (1996)).

Long-term culture initiating cells (LTC-IC) assays.

Canine LTC-IC assays were performed as modifications of previously described procedures for human studies (Gartner et al., Proc . Natl. Acad. Sci . USA 77:4756 (1980); Coulombel et al., Blood 62:291 (1983); Reems et al . , Blood 85:1480 (1995) ) . Briefly, stromal layers were established in T-25 flasks and fed with Iscove's medium supplemented with 20% horse serum, 2% L-glutamine and 10^~7M hydrocortisone sodium succinate. Stromal cells were cultured at 37°C in 5% C0₂ until reaching confluence. The adherent layers were trypsinized, washed, irradiated (1800 cGy using a ¹³⁷Cs source) and transferred to 24 -well plates. The wells were seeded in triplicate with sorted CD34⁺ cells, CD34^" cells and BMMC at 1 x 10⁴, 2 x 10⁴, or 1 x 10⁵ cells per well. . Cells were cultured at 33 °C in 5% C0₂ and fed weekly renewing half of the media. After 5 weeks the adherent layers were trypsinized. Pooled cells from the triplicates were washed and 5 x 10⁴ cells plated into duplicate CFU-GM assays performed as described above.

Specificity of Monoclonal Antibodies for CD34.

Five MAbs (1H6, 2E9, 6B11, 5F11-3 and 5F11-6) were produced as ascites, purified, and further characterized. To demonstrate specificity for CD34, several studies were performed. First, the ML3 and 1390 leukemia cell lines were incubated with five MAbs and the binding assessed by FACS analysis. All five antibodies stained a homogeneous population of cells from both cell lines when compared to control. Though ML3 cells were slightly brighter compared to 1390 cells no other differences were found. Pre-incubation of the antibodies with excess CD34-Ig reduced binding to background, demonstrating specificity. This was true for both cell lines and all five MAbs tested. Second, Western blotting studies against CD34-Ig confirmed reactivity of biotinylated MAbs 1H6 and 2E9 to the extracellular domain of CD34. Both monoclonal antibodies 1H6 and 2E9 identified a band of the expected molecular weight for CD34-Ig (about 90kD) . This band was absent in the lane containing control fusion protein. Third, in Western blotting studies of the ML1, ML3 and 1390 cell lines, MAb 2E9 recognized a band of about 110 kDa in the ML3 and 1390 cell lines that was absent in the CD34^" cell line ML1. The results were consistent with the findings with

RPαCD34 which detected a band of identical size in ML3 and 1390 cells but not ML1.

Binding Properties of the Monoclonal Antibodies. In order to assess properties of the monoclonal antibodies that could affect staining and separations of progenitors cells, functional "affinities" were determined. In FACS studies, ML3 and 1390 cell lines were both used to also assess any potential differences. Cells were incubated with increasing concentrations of antibodies and the binding determined. Using ML3 cells, 1H6 and 2E9 had similar high "affinity" binding with 50% maximal binding at 1 μg/ml. Binding showed a single affinity and was saturated at about 3 μg/ml. Fifty per cent maximal binding was at moderate concentrations for 6B11 (3 μg/ml) and 5F11-6 (7 μg/ml) and lowest for 5F11-3 (13 μg/ml) . The relative binding properties of the five MAbs were similar against 1390 but with slightly higher saturating concentrations. No significant differences were found in the maximal binding for each antibody at saturating concentration to suggest the antibodies were seeing any subset of CD34 molecules. The affinities (Kd) of the five antibodies for CD34-Ig were determined by BIAcore analysis. In these studies, 2E9 (<0.01 nM) and 6B11 (<0.03 nM) had the highest affinities (Table 1) with very low dissociation rates after binding antigen (<5 x 10^"6 L/sec) whereas 1H6 (0.42 nM) had lower affinity due to a faster dissociation rate (7.6 x 10^~4 L/sec). Consistent with cell binding assays 5F11-6 (0.8 nM) and 5F11-3 (1 nM) had the lowest affinities. In subsequent blocking studies, all 10 MAbs were able to block binding of biotinylated 1H6 to the ML3 cell line, suggesting that the MAbs all recognized related epitopes. In conclusion, the antibodies bound specifically to related epitopes on CD34, had a range of affinities, and were unable to identify any epitope differences between the two cell lines tested.

TABLE 1

Binding Properties of Anti-CD34 MAbs for CD34-Ig as Determined by BIAcore Analysis and FACS

Studies

Antibody Off Rate On Rate Kd* 50% Saturating Concentration 50% Saturating Concentration Name (L/sec) L/ (mol/sec) nM on 1390 (ug/ml)i on ML3 (ug/ml)+

6B11 <5 x 10^"6 6.7 x 10⁵ <0.01 4.0 3.0

2E9 <5 x 10^"6 2.2 x 10⁵ <0.03 1.5 1.0

1H6 3.14 x 10^"4 7.56 x 10⁵ 0.42 1.5 1.0

5F11-3 4.7 x 10^"5 5.8 x 10⁵ 0.8 21.0 13.0

5F11-6 1.7 x 10^"4 1.5 x 10⁵ 1 6.0 7.0

* Kd (Affinity) = off rate divided by on rate. ^•k Determined by FACS analysis .

Characterization of CD34⁺ Cells.

Although all five of the purified MAbs were suitable for FACS studies, 1H6 and 2E9 were selected for additional FACS studies of canine bone marrow and peripheral blood. Both antibodies appeared to recognize an identical cell population in canine bone marrow. In 10 samples of unfractionated bone marrow, a mean of 2.1+1.0% (range 0.7% to 3.7%) CD34⁺ cells were detected amongst the leukocyte population. The CD34⁺ cells were small to large in size with low to low-intermediate side scatter. Larger CD34⁺ cells gave consistently moderately bright staining, whereas smaller CD34⁺ cells showed more heterogeneous CD34 expression including a "tail" of cells with dim positive staining. As compared to previous studies using RPαCD34, it was possible to more accurately delineate the CD34⁺ and CD34^" cell populations due to less nonspecific staining of monocytes and granulocytes . In repeated (n>5) studies of steady-state unfractionated peripheral blood leukocytes, <0.1% CD34⁺ cells were detected.

Progenitor Assays.

To define ;Ln vitro functional characteristics of cells stained with either 2E9 or 1H6 , CD34⁺ cells were isolated from BMMC by cell sorting and cultured in CFU-GM assays or long-term culture initiating cell (LTC-IC) assays. Reanalysis of sorted CD34⁺ and CD34^" fractions showed that cell purities were routinely > 95% and usually 98-99%. In all experiments the CD34⁺ population was enriched for CFU-GM as compared to control BMMC whereas the CD34^" cell fraction was depleted of CFU-GM. Results of 4 representative experiments are shown in Table 2. TABLE 2 Results of CFU-GM Assays of BMMC and Sorted BMMC Populations

BMMC CD34⁺ (Enrichment) * CD34^" (Depletion) *

35 ± 1.0 1958 ± 40 (55x) 1.0 ± 0 (35x)

50 ± 3.4 89 ± 27 (1.8x) 0.3 ± 0.3 (150x) 61 ± 12 179 ± 15 (2.9x) 27 ± 7 (2.3x)

7.6 ± 2.9 70.8 ± 4.2 (9.3x) 1.6 ± 0.9 (4.8x)

Results are CFU-GM from triplicate plates, are expressed as a mean + a standard error measurement, and are corrected to an input of 25,000 cells per plate.

BMMC = bone marrow mononuclear cells. * Enrichment = CFU-GM in CD34⁺BMMC/CFU-GM in BMMC; Depletion is CFU-GM in BMMC/CFU-GM in CD34^"BMMC.

There was considerable variation in both the degree of CFU-GM enrichment (1.8 to 55 fold), progenitor depletion (2.3 to 150 fold) , and in progenitor yields with a constant input number of CD34⁺ cells (2000 cells/plate) and BMMC or CD34^" cells

(both 25,000 cells/plate) . In LTC-IC assays a similar pattern of progenitor enrichment within the CD34⁺ cell fraction was observed and CD34^" cells were depleted of LTC-IC as compared to CD34⁺ cells and BMMC. Two representative examples of these experiments are shown in Table 3.

TABLE 3 LTC-IC Assays

EXPT BMMC CD34⁺ (enrichment)* CD34^" (depletion) *

1 116 + 4 1880 ± 50 ( 16 . 2x) 9.1 + 6.5 (12.7x)

2 52 + 0 360 + 40 ( 6 . 9x) 3.5 ± 0 (14.8x)

* Enrichment = CFU-GM in CD34⁺BMMC/CFU-GM in BMMC;

* Depletion is CFU-GM in BMMC/CFU in CD34^"BMMC.

Cells were sorted using MAb 2E9. Fractions were grown under long-term marrow culture conditions for five weeks and then 5 xlO⁴ cells plated into duplicate CFU assays. Results are expressed as a mean + a standard error measurement of CFU-GM from duplicate plates and normalized to an input of 10⁴ cells/LTMC. Results are shown for comparative purposes. A precise numeric relationship between LTC-IC frequency and week

5 CFU-GM has not been established for dogs.

EXAMPLE 3

CD34 Hematopoietic Cell Enrichment and Autologous Transplantation

In the present example a monoclonal antibody of the present invention has been used to separate CD34⁺ hematopoietic precursor cells from a bone marrow sample. These cells were used in an autologous transplant into a dog having been previously been treated by high levels of total body irradiation.

Immunomagnetic separation and autologous transplantation of canine bone marrow.

Beagle dogs were raised at the Fred Hutchinson Cancer Research Center and were observed for disease for at least 60 days before entering the study. The transplant was performed according to the principles outlined in the guide for Laboratory Animal Facilities and Care prepared by the National Academy of Sciences, National Research Council. The transplant protocol was approved by the Institutional Review Animal Care and Use Committee of the Fred Hutchinson Cancer Research Center. The kennels were certified by the American Association for Accreditation of Laboratory Animal Care.

Bone marrow was aspirated from the humeri and femora of anesthetized animals. Buffy coat was isolated by centrifugation at 600 xg for 15 minutes, washed in PBS/2% horse serum, and treated with hemolytic buffer to remove red blood cells. Cells were then incubated at 1 x 10⁸ cells/ml with biotinylated MAb 1H6 at 40 μg/ml for 30 minutes at 4°C. Cells were washed in PBS/2% horse serum followed by incubation with immunomagnetic streptavidin-coated microbeads (Miltenyi Biotech, Auburn, CA) and separation was performed using a miniMACS or VarioMACS (Miltenyi Biotech) separation device. Dogs were given a single dose of 920 cGy TBI delivered at 7 cGy per minute from two opposing ⁶⁰Co sources (Storb et al . , Int. J. Radiat . Oncol. Biol. Phvs . 26:275 (1993)). Separated cells were resuspended in 5-10 ml of PBS and infused intravenously over 1-2 minutes within 4 hours of irradiation. The day of marrow infusion was designated as day 0. Supportive care included parenteral fluids, electrolytes, and irradiated blood transfusions from day -5 until recovery of the WBC to > 1000/μl, and prophylactic broad spectrum systemic antibiotics from day 0 until the WBC recovered to >1000/μl (Storb et al . , Transplantation 15:92 (1973)).

Autologous Transplantation Studies.

To determine jji vivo functional characteristics of CD34⁺ cells, transplantation studies were performed using enriched progenitor cell populations isolated by immunomagnetic positive selection of canine bone marrow. Marrow cell doses and engraftment kinetics were compared to those of 16 historical control dogs (Storb et al . , Transplant . Proc . 8:561 (1976)) which received unmodified fresh marrow autografts after conditioning with the same dose of TBI . Based on results of preliminary FACS studies and small scale immunomagnetic separations using biotinylated 1H6, 2E9, and 6B11, we chose to use MAb 1H6 for these separation studies. Separation conditions were determined after several small scale separations of canine bone marrow in which various concentrations of beads (50 to 200 μl/1 x 10⁸ cells) and 1H6 (5 to 60 μg/1 x 10⁸ cells) were tested. CD34⁺ enriched cell fractions for autologous transplantation were isolated by immunomagnetic adsorption from canine bone marrow after incubation with biotinylated 1H6 at 40 μg/ml with cells at 1 x 10⁸ cells/ml, followed by incubation with streptavidin-coated magnetic microbeads at 100 μl/ 1 x 10⁸ cells with cells at 1 x 10⁸ cells/ml. CFU-GM growth from CD34 selected cells did not appear to be inhibited by presence of the antibody-bead complex on the cells and therefore no attempt was made to remove the MAb or beads from cells prior to transplant . For two dogs (E151 and E246) cell doses aspirated for processing were comparable to those of 16 control dogs (3.2 ± 2.0 x 10⁸ total nucleated cells [TNC] /kg) whereas the cell dose from E376 (7.4 x 10⁷ TNC/kg) was lower. Final cell doses used for transplantation after CD34 selection were 3.0 x 10⁶/kg, 1.1 x 10⁷/kg, and 1.7 x 10⁶/kg, in each case <3% of the total of bone marrow cells aspirated. Purities of CD34⁺ cell fractions obtained with a single pass through a magnetic column were 60%, 29%, and 70%, and doses of CD34⁺ cells transplanted were 1.62 x 10⁶/kg, 3.4 x 10⁶/kg, and 1.21 x 10⁶/kg, respectively. In 2 dogs (E151, E246) granulocyte recovery to an absolute neutrophil count of >500/ml occurred on days 9 and 11 after TBI and on day 20 in the third dog (E376) . Platelet recovery to within normal levels was slower than granulocyte recovery but was complete in each case.

Slowest granulocyte and platelet recovery was seen in E376, the dog that received the lowest per kg dose of total cells and CD34⁺ cells. When compared to control dogs, granulocyte and platelet recovery for E151 and E246 were within or close to the range previously observed, but recovery was slower than controls for E376, suggesting a possible effect of cell dose on recovery. Dogs were observed from 2-4 months after transplant, and blood counts remained stable without any evidence of secondary graft failure. CD34 has previously been defined as a marker of hematopoietic progenitors. It is becoming increasingly important as technologies for large scale separation of CD34⁺ cells have been applied to studies of clinical stem cell transplantation. Despite this, good animal models in which to refine studies of CD34⁺ cell fractions are limited.

Monoclonal antibodies to human CD34, some of which cross-react with CD34⁺ cells from non-human primate species (Berenson et al., J. Clin. Invest. 81:951 (1988)) have been available since 1984 (Civin et al . , J. Immunol. 133:157 (1984)). Only recently, a monoclonal antibody to murine CD34 has become available for such studies (Morel et al . , Blood 88:3774 (1996)) . However, this MAb detected 4-17% CD34⁺ cells in murine bone marrow (Morel et al., supra) somewhat higher than the 1-5% CD34⁺ cells detected in human bone marrow (Civin et al., J. Immunol. 133:157 (1984); Andrews et al . , Blood 67:842 (1986) ) . Dogs have proved a valuable preclinical model for human stem cell transplantation (Storb, R. and Thomas, E.D., Copenhagen, Munksgaard, Immunol . Rev . 88:215 (1985); (Storb, R. and Thomas E.D., Bone Marrow Transplantation, Boston, MA, Blackwell Scientific Publications, pg. 3, (1994)). However, a limitation of the canine model for studies of hematopoiesis or stem cell transplantation has been the lack of a useful marker to identify and enrich hematopoietic progenitors.

In the present invention a method has been developed for the production of monoclonal antibodies to canine CD34 that can be used to enrich canine progenitor cell populations that appear functionally and phenotypically similar to human CD34⁺ cells. Approximately 1-3% CD34⁺ cells have been detected in canine bone marrow, similar to the percentage of CD34⁺ cells present in human bone marrow. The monoclonal antibodies, antigen binding fragments and antigen binding agents of the present invention should help in refining studies in several areas and facilitate the phenotypic and functional characterization of progenitors present in canine bone marrow, peripheral blood, cord blood and apheresis products. They can also find use in studies of stem cell transplantation, progenitor cell expansion, gene therapy and culture of canine dendritic cells.

Two reagents were developed that proved critical in making these monoclonal antibodies. The first was a fusion protein, canine CD34-Ig, made for immunization and screening strategies. This allowed production of the second, an affinity purified polyclonal antiserum RPo;CD34, which was used to identify cell lines expressing CD34, to screen antibody from hybridomas and to carry out preliminary characterization of canine CD34 (McSweeney et al . , Blood 88:1992 (1996)). After failure initially to generate monoclonal antibodies to CD34, we found that to generate high levels of anti-CD34 specific response, it was important to use immunization schedules that included both CD34-Ig and CD34⁺ leukemia cell lines. CD34 -specific responses were not detected after immunization of mice with CD34⁺ cell lines alone. Although 10 MAbs were made, each MAb was able to block the binding of 1H6 to CD34⁺ cell lines indicating that they all recognized identical or overlapping epitopes. Therefore, more detailed characterization was limited to five MAbs initially, and then later to two high affinity MAbs 2E9 and 1H6 for evaluation of canine progenitors. Differences in marrow cell populations recognized by these two MAbs were not identified by FACS analysis. However, in Western blotting studies 2E9 was superior to 1H6, most likely because of its higher affinity for CD34. The reactivity of these monoclonal antibodies to CD34-Ig and native CD34 in Western blotting studies indicated that they were not dependent on the tertiary structure of CD34 for their binding. Reactivity of these monoclonal antibodies to CD34 was confirmed by ELISA studies, in studies that used CD34-Ig to block binding to CD34⁺ cell lines, and by Western blotting of CD34-Ig and leukemia cell lines. The enrichment and depletion of progenitor cells in bone marrow fractions isolated by cell sorting was consistent with binding of MAb to CD34 on bone marrow cells. Specificity of the monoclonal antibodies for CD34 was suggested by the lack of binding of MAbs to CD34^" cell lines as previously defined by the polyclonal antiserum and molecular studies (McSweeney, supra . (1996) ) by blocking studies with CD34-Ig, and by restricted binding to a small population of bone marrow cells that were not detected in peripheral blood. Overall, the properties demonstrated that several high affinity monoclonal antibodies had been produced that should be suitable for specifically isolating highly enriched progenitor cell populations for further characterization and transplant studies. In addition, preliminary immunohistochemistry studies of frozen or paraffin sections from canine tissues showed that 1H6 stained vascular endothelial cells, suggesting a possible use of these monoclonal antibodies for studying canine endothelial cells. The reactivity of the monoclonal antibodies 1H6 and 2E9 in paraffin sections, frozen sections, and in Western blotting studies was a pattern described for class II epitopes but not class I or class III epitopes of human CD34 (Greaves et al . , Leucocyte Typing V, Oxford University Press, 840 (1995) ) .

An important consideration for future studies to characterize canine progenitors is whether these monoclonal antibodies recognize all subsets of bone marrow CD34⁺ cells. The presence of CD34 isoforms, most likely mediated through post-translational protein modifications, has been demonstrated in mice where L-selectin could bind CD34 on vascular endothelium but not CD34 on murine hematopoietic cells (Baumhueter et al.. Blood 84:2554 (1994)). Also, it has been observed that cultured canine endothelial cells express a lower molecular weight form of CD34 than leukemic cell lines (McSweeney et al . , supra . (1996)). Different isoforms of CD34 that can be defined by flow cytometry within normal human bone marrow have not been reported, although their presence is a possibility as suggested by variable staining of CD34⁺ human leukemia samples with different MAbs to human CD34 (Greaves et al., Leucocyte Typing V, Oxford University Press, 840 (1995)). Therefore, the use of MAbs which recognize all CD34⁺ bone marrow cells may be an important prerequisite for accurately defining phenotypic and functional characteristics of CD34⁺ and CD34^" progenitor cell populations. Two-color flow cytometry studies showed that there was concordance between the cell population recognized by the polyclonal antiserum (recognizing multiple epitopes of CD34) and the monoclonal antibodies. Therefore, these monoclonal antibodies appeared to recognize an epitope (s) expressed on all bone marrow CD34⁺ cells and should be suitable for defining CD34⁺ bone marrow subsets . The findings that purified CD34⁺ bone marrow cells were enriched for hematopoietic progenitors, and that CD34^" cells were depleted of those progenitors, suggested that a high proportion of canine progenitors express CD34. As shown herein there was some variability in the degree of progenitor enrichment and growth found in the sorted CD34⁺ cell populations. Because conditions for culture of purified canine progenitors have not yet been well defined and may differ from those of unfractionated BMMC and because of the use of a limited number of canine specific cytokines in CFU-GM assays, further modification of the culture conditions and additional canine-specific cytokines may be needed to more accurately define the degree of progenitor enrichment in CD34⁺ cells as compared to BMMC.

Results of these initial transplant studies showed that it was unnecessary to remove the MAb-bead complex from the CD34 selected cells prior to infusion in order to achieve engraftment. Complete and relatively prompt hematopoietic recovery was observed after treatment with a TBI dose >2.5 times the LD₉₉ dose for dogs (Storb et al., Int. J. Radiat . Oncol . Biol . Phys . 26:275 (1993)) despite infusion of cell doses comprising <3% of the starting marrow inoculum. Effects of marrow cell dose on engraftment were observed in a previous canine autologous transplant dose finding study (Appelbaum et al., Transplantation 26:245 (1978)) that used cryopreserved unmodified BM. At cell doses between 1.0 x 10⁷/kg and 1.0 x 10⁸/kg, there was a correlation of cell dose with speed of granulocyte engraftment and death with engraftment failure. At total nucleated cell doses of <2.5 x 10⁷/kg engraftment failure was usually observed and at cell doses between 2.5 and 5.0 x 10⁷/kg engraftment failure or delayed granulocyte recoveries were seen. In a second study (Bodenberger et al . , Exp. Hematol. 8:384 (1980)) at BMMC doses of >1.0 x 10⁷/kg and < 1.5 x 10⁷/kg engraftment failure occurred in 7 out of 7 dogs. As cell fractions transplanted in this study were substantially enriched for CD34⁺ cells, these results strongly suggest that the radioprotective functions of these grafts were provided by CD34⁺ cells rather than other cell populations. Results of these in vitro and ±n vivo studies support an hypothesis that CD34 is a highly conserved marker of hematopoietic progenitors. By analogy to human and mouse data, it is expected that canine CD34⁺ cells will have long- term repopulating ability even though as suggested by murine studies (Osawa et al . , Science 273:242 (1996)) there may be CD34^" cells that can also provide this function. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for producing an immortalized cell line which expresses and secretes an immunoglobulin molecule specific for canine CD34, comprising:

(a) immunizing a non-human animal at least once with a first composition comprising the extracellular domain of canine CD34;

(b) immunizing the animal at least once with a second composition comprising a cell line which consistently expresses on its cell surface canine CD34;

(c) immunizing the animal with the first composition comprising the extracellular domain of canine CD34; and (d) immortalizing cells which express and secrete an immunoglobulin molecule specific for canine CD34.

2. The method of claim 1 wherein the first composition comprises an extracellular domain of canine CD34 is a fusion protein.

3. The method of claim 2, wherein the fusion protein comprises the extracellular domain of canine CD34 and the hinge, CH2 and CH3 domains of an immunoglobulin.

4. The method of claim 3, wherein the fusion protein is canine CD34-Ig.

5. A method for producing an immortalized cell line which expresses and secretes an immunoglobulin molecule specific for canine CD34, wherein the initial immunization comprises a composition consisting of a cell line selected for consistent high level expression on its surface of canine CD34; wherein a second and third immunization is with a composition comprising CD34-Ig; wherein a fourth immunization is with a composition consisting of a cell line selected for consistent high expression on its surface of canine CD34; and a fifth immunization is with a composition comprising canine CD34-Ig.

6. The method of claim 1, wherein the immortalization step comprises fusion of spleen cells isolated from the non-human animal is with a myeloma cell line.

7. A hybridoma cell line which expresses and secretes a monoclonal antibody specific for canine CD3 .

8. A monoclonal antibody, antigen binding fragment, or antigen binding agent specific for canine CD34.

9. The monoclonal antibody, antigen binding fragment or antigen binding agent of claim 8, wherein the antibody, antigen binding fragment or antigen binding agent is of murine, rat, monkey, hamster, guinea pig or rabbit origin.

10. The antigen binding fragment of claim 8, wherein the fragment is a Fv, Fab, Fab' or F(ab')₂.

11. The antigen binding agent of claim 8, wherein the agent is a chimeric or single chain antibody.

12. The monoclonal antibody, antigen binding fragment or antigen binding agent of claim 8 conjugated with a label.

13. The monoclonal antibody, antigen binding fragment or antigen binding agent of claim 12, wherein the label is a radionuclide, fluorescer, enzyme, enzyme cofactor, enzyme substrate, enzyme inhibitor or ligand.

14. The monoclonal antibody, antigen binding fragment or antigen binding agent of claim 13, wherein the label is biotin.

15. A method for producing a composition enriched for canine CD34⁺ hematopoietic stem cells from a cell sample, comprising:

(a) isolating a cell sample from a donor dog, ^" (b) contacting the cell sample with a monoclonal antibody, antigen binding fragment or antigen binding agent specific for canine CD34 under conditions conducive for a complex to form,

(c) separating the CD34⁺ hematopoietic stem cell/monoclonal antibody complex from the remainder of the cell sample.

16. The method of claim 15, wherein the cell sample is bone marrow, peripheral blood, cord blood, or apheresis product.

17. The method of claim 15, wherein the monoclonal antibody, antigen binding fragment or antigen binding agent is labeled.

18. The method of claim 17, wherein the label is biotin.

19. The method of claim 16, wherein the method comprises prior to step (c) the step of contacting the sample with a labeled solid support .

20. The method of claim 19, wherein the labeled solid support is a magnetic microbead, or chromatography support .

21. The method of claim 20, wherein the solid support has conjugated thereto streptavidin.

22. The method of claim 15, wherein the separation is by flow microfluorometry or immunoaffinity chromatography.

23. The method of claim 15, wherein the donor dog is exposed to a growth factor or drug prior to sample collection.

24. The method of claim 23, wherein the growth factor is G-CSF or SCF.

25. The method of claim 23, wherein the drug is 5- fluorouracil, cyclophosphamide or prednisone.