EP1232246A1 - A human normal counterpart of cd4+ cd56+ cutaneous neoplasm - Google Patents

Abstract

The invention is directed to purified and isolated novel CD56+ plasmacytoid monocytes, the healthy counterpart of CD4+ CD56+ cutaneous neoplasm, and uses thereof.

Description

A HUMAN NORMAL COUNTERPART OF CD4⁺CD56⁺ CUTANEOUS NEOPLASM

FIELD OF THE INVENTION

The invention is directed to purified and isolated novel CD56⁺ plasmacytoid monocytes, the healthy counterpart of CD4⁺ CD56⁺ cutaneous neoplasm, and uses thereof. BACKGROUND OF THE INVENTION

Various glycoproteins, such as CD4, CD56, CD68, and CD123, are expressed on cells and serve as markers for a variety of diseases. Expression of these markers has allowed the scientific community to better understand the associated diseases and how they are caused. Understanding the markers' expression may further allow the scientific community to develop new treatments.

CD4 is a 55-kd glycoprotein expressed on a subset of peripheral T cells, thymocytes, and monocytes/macrophages. In hematopoietic neoplasms, CD4 has been shown to be a marker for T-cell acute lymphocytic leukemias, T-cell lymphomas, and acute myeloid leukemias (AMLs). (Kameoka et al., Am J Clin Paϊhol 110:478-488 (1998).)

CD56 is a 175- to 185-kd surface glycoprotein normally expressed on all human peripheral natural killer (NK) cells and on a subset of peripheral CD8⁺ T cells and a subset of peripheral monocytes. In hematopoietic neoplasms, CD56 has been shown to be a marker for NK-like T-cell lymphomas, plasma cell myelomas, and de novo AMLs with cytogenetic abnormalities such as t(8:21) and t(8:16). (Id.) Further, CD56 molecules may be involved in the recognition of target cells by human natural killer cells. (Suzuki et al. J. Ex. Med. Vol 173, 1451 (1991).)

Recently, cases of lymphomas expressing both CD4⁺ and CD56⁺ have been published. (Kameoka et al. (1988); Petrella et al., Am JSurg Path 23(2): 137-146 (1999).) The CD4⁺ CD56⁺ cutaneous neoplasm described in these publications is characterized by cutaneous nodules or papules and has cells that exhibit a CD4⁺ CD56⁺ CD45⁺ CD43⁺ HLA-DR⁺ phenotype. The lymphoma cells also lack the characteristics of both T cells and NK cells. (Petrella et al. at 137.) At initial diagnosis, CD4⁺CD56⁺ cutaneous neoplasm is usually misclassified as either T-cell lymphoma, NK cell lymphoma, or myeloid sarcoma, closely related diseases to CD4⁺ CD56⁺ cutaneous neoplasm. Initially the patient responds favorably to treatment for those diseases but relapses relatively quickly. (Id.) Presently, there is no known cure for CD4⁺ CD56⁺ cutaneous neoplasm and patients usually die within a two-year period from the discovery of the disease. Plasmacytoid monocytes (also called plasmacytoids) are cell types that were originally described as being in human lymph nodes based on their plasma cell-like morphology, the expression of CD4, CD31, CD36, CD68 and cutaneous lymphoctye- associated antigen, and the lack of other cell lineage markers. (Cella et al., Nature Medicine 5(8):919-923 (1999).) CD4⁺ CD56⁺ cells have been identified in T-cell lines and in kidney allografts. (Lanier et al., J. Immunol. 138:2019-2023 (1987); Vergelli et al., J. Immunol. 157:679-688 (1996); Bachetoni et al., Clin Transpl. 9:433-437 (1995).) These cells appear to belong to either T cells or NK cells. (Id.) CD4⁺ CD56⁺ cells that are neither T cells nor NK cells have not been detected in the human peripheral blood. Identification of such cells, the healthy counterparts to CD4⁺ CD56⁺ cutaneous neoplasm, could be of great interest and usefulness in developing treatment for CD4⁺ CD56⁺ cutaneous neoplasm.

Thus there is a need in the art for a method of treating CD4⁺ CD56⁺ cutaneous neoplasm. Further, there is a need in the art for tools and methods which may further the discovery of such treatment methods.

SUMMARY OF THE INVENTION

The instant invention answers these needs by providing the isolated plasmacytoid monocyte counterpart to CD4⁺ CD56⁺ cutaneous neoplasm cells. This plasmacytoid monocyte counteφart has the identical phenotype as the cutaneous neoplasm cells and may be used in the development of treatments for CD4⁺ CD56⁺ cutaneous neoplasm. The instant invention further provides for antibodies directed against the plasmacytoid monocyte and the use of these antibodies to treat the CD4⁺ CD56⁺ cutaneous neoplasm. The instant invention further provides for a more efficient method of generating plasmacytoid monocytes with the same phenotype as CD4⁺ CD56⁺ cutaneous neoplasm cells and the plasmacytoid monocytes produced by this method. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 A discloses that Peripheral Blood mononuclear cells (PBMCs), obtained from blood of healthy volunteers treated for ten consecutive days with Flt-3L, were labeled with Fluorescein-conjugated monoclonal antibodies against CD3, CD14, CD15, CD16, CD19, CD20 and CD34 (lineage cocktail) as well as Allophycocyanin-conjugated antibody against CD123. The plasmacytoid cells appear as CD 123^hl Lineage^" cells (Rl gate, black box).

Figure IB shows the pattern of expression of CD56(NCAM) on the plasmacytoid cells as identified in figure 1A. In brief, CD123^hl Lineage^" PBMCs were further labeled with phycoerythrin-conjugated antibody against CD56. The CD56⁺ plasmacytoid monocytes (PM) are shown in gate R3. The CD56^"PMs are shown in R2.

Figure 1C compares the granulosity of CD56TM (transparent histogram) with that of the CD56⁺PM (gray histogram) and that of total PBMCs (Black histogram). Light Side scatter parameters are shown. Figure ID shows the moφhology of CD56⁺PM. In practice, R3 gated cells (see figure 1A, B) were purified by flow cytometry (FACS vantage, Becton & Dickinson). The isolated cells were then submitted to a standard GEEMSA staining and observed by optical microscope at x400 magnification.

DETAILED DESCRIPTION OF THE INVENTION

Plasmacytoid monocytes are difficult to detect in human peripheral blood, making identification of markers on the plasmacytoid monocytes even more difficult. The level of plasmacytoid monocytes produced, however, may be increased by applying the pre- enrichment steps of the instant invention. A higher volume of plasmacytoid monocytes increases the opportunity to isolate healthy counteφarts to CD4⁺ CD56⁺ cutaneous neoplasm.

The instant invention identifies CD56⁺ plasmacytoid monocytes as the healthy counteφart of CD4⁺ CD56⁺ cutaneous neoplasm cells. The CD56⁺ plasmacytoid monocyte ("CD56⁺ PM") expresses the markers CD56, CD68, CXCR3 and CD4. The CD56⁺ PMs also exhibit very bright levels of CD 123 and low levels of CD1 lc. CD56⁺ PMs are CD3^' and do not uniformly have the plasma cell moφhology typical of plasmacytoid monocytes. The plasmacytoid monocytes of the instant invention may be "isolated" or "purified". Isolated plasmacytoids are not m an environment identical to the environment in which they can be found in nature A puπfied plasmacytoid is essentially free of association with other plasmacytoids. "Fragments" of CD56⁺ plasmacytoid monocytes encompass truncated components of the plasmacytoid monocytes that retain the biological ability to express the markers CD4, CD56, CD 123 and are also provided for by the present invention.

Flt-3 ligand is a hematopoietic growth factor that induces the proliferation and survival of primitive progenitor and stem cells. Administering Flt-3 ligand to humans can expand the volume of plasmacytoid cells greater than 5 fold. A screening assay can then be performed on the increased volume of plasmacytoids to determine the number of plasmacytoids present that express the markers CD4, CD56, CD68, and CD123. These plasmacytoid monocytes may then be isolated. The increased volume of plasmacytoid monocytes available enables the screening process to produce a sufficiently useful number of CD56⁺ PMs.

Thus, in one embodiment of the instant invention, pre-enπchment steps are conducted in which CD56⁺ PMs are detected by administering Flt-3 ligand (as descπbed in Examples 1 and 2) to produce increased levels of plasmacytoids. The plasmacytoids are screened to determine the number of which express CD4 and CD56 (as descπbed m Example 3) and then isolated. In general, this method involves administering Flt-3 ligand for 10 consecutive days; incubating the peπpheral blood monocyte cells (PBMC) taken after the administration of Flt-3 ligand with directly conjugated antibodies including CD4, CD56 and CD123; and then performing flow cytometry to isolate the plasmacytoid monocytes expressing the desired markers. Alternatively, the plasmacytoid monocytes may be isolated, for example, by the use of a version of the Magnetic Cell Sorting (MACS) (Miltenyi Biotech) technique. In this method, an enπchment step is performed using antι-CD123 antibody (Olweus et al., Proc Natl Acad Sci USA (1997)). The selected cells are then labeled with antι-CD56 coated magnetic beads (e.g , Dynabeads (Dynal)). By applying a low intensity magnetic field, the CD56⁺ CD123^hl cells may be specifically retained on the magnet. The CD56 CD123^hl cells would not be attached to the beads and would therefore be discarded. Other methods of isolation are known in the art and may also be used. Further, CD56⁺ PMs that are identified by any of these methods are included in the instant invention. The present invention further provides for antibodies that are immunoreactive with the CD56⁺PMs of the invention. Such antibodies specifically bind to the plasmacytoid monocytes via the antigen-binding sites of the antibody (as opposed to nonspecific binding). Thus, the plasmacytoids monocytes and fragments, as well as variants, fusion proteins, etc., may be employed as "immunogens" in producing antibodies immunoreactive therewith. More specifically, the plasmacytoid monocytes, fragment, variants, fusion proteins, etc. contain antigenic determinants or epitopes that elicit the formation of antibodies.

These antigenic determinants or epitopes can be either linear or conformational (discontinuous). Linear epitopes are composed of a single section of amino acids of plasmacytoid monocytes, while conformational or discontinuous epitopes are composed of amino acids sections from different regions of the polypeptide chain that are brought into close proximity upon protein folding (C. A. Janeway, Jr. and P. Travers, Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Because folded proteins have complex surfaces, the number of epitopes available is quite numerous; however, due to the conformation of the protein and steric hinderances, the number of antibodies that actually bind to the epitopes is less than the number of available epitopes (C. A. Janeway, Jr. and P. Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nd ed. 1996)). Epitopes may be identified by any of the methods known in the art. Thus, one aspect of the present invention relates to the antigenic epitopes of the

CD56⁺PMs of the invention. Such epitopes are useful for raising antibodies, in particular monoclonal antibodies, as described in more detail below. Additionally, epitopes from the plasmacytoid monocytes of the invention can be used as research reagents, in assays, and to purify specific binding antibodies from substances such as polyclonal sera or supernatants from cultured hybridomas. Such epitopes or variants thereof can be produced using techniques well known in the art such as solid-phase synthesis, chemical or enzymatic cleavage of a polypeptide, or using recombinant DNA technology.

As to the antibodies that can be elicited by the epitopes of the CD56⁺ PMs of the invention, whether the epitopes have been isolated or remain part of the plasmacytoid monocytes, both polyclonal and monoclonal antibodies may be prepared by conventional techniques. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988). One method of producing antibodies is by phage display, (de Kruif et al., Immunol Today 17(10):453-5 (1996).)

Hybridoma cell lines that produce monoclonal antibodies specific for the CD56⁺ PMs of the invention are also contemplated herein. Such hybridomas may be produced and identified by conventional techniques. One method for producing such a hybridoma cell line comprises immunizing an animal with a polypeptide; harvesting spleen cells from the immunized animal; fusing said spleen cells to a myeloma cell line, thereby generating hybridoma cells; and identifying a hybridoma cell line that produces a monoclonal antibody that binds the polypeptide. The monoclonal antibodies may be recovered by conventional techniques.

The monoclonal antibodies of the present invention include chimeric antibodies, e.g., humanized versions of murine monoclonal antibodies. Such humanized antibodies may be prepared by known techniques and offer the advantage of reduced immunogenicity when the antibodies are administered to humans. In one embodiment, a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen-binding site thereof) and a constant region derived from a human antibody. Alternatively, a humanized antibody fragment may comprise the antigen- binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in

Riechmann et al. (Nature 332:323, 1988), Liu et al. (PNAS §4:3439, 1987), Larrick et al. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139, May, 1993). Procedures that have been developed for generating human antibodies in non-human animals may also be employed in producing antibodies of the instant invention. The antibodies may be partially human or preferably completely human. For example, transgenic mice into which genetic material encoding one or more human immunoglobulin chains has been introduced may be employed. Such mice may be genetically altered in a variety of ways. The genetic manipulation may result in human immunoglobulin polypeptide chains replacing endogenous immunoglobulin chains in at least some, and preferably virtually all, antibodies produced by the animal upon immunization.

Mice in which one or more endogenous immunoglobulin genes have been inactivated by various means have been prepared. Human immunoglobulin genes have been introduced into the mice to replace the inactivated mouse genes. Antibodies produced in the animals incoφorate human immunoglobulin polypeptide chains encoded by the human genetic material introduced into the animal.

Examples of techniques for the production and use of such transgenic animals are described in U.S. Patent Nos. 5,814,318, 5,569,825, and 5,545,806 and Waldman, T. Science Vol. 252, 1657-62 (1991).

Antigen-binding fragments of the antibodies, which may be produced by conventional techniques, are also encompassed by the present invention. Examples of such fragments include, but are not limited to, Fab and F(ab')₂ fragments. Antibody fragments and derivatives produced by genetic engineering techniques are also provided. In one embodiment, the antibodies are specific for the CD56⁺ PMs of the instant invention and do not cross-react with other proteins. Screening procedures by which such antibodies may be identified are well known, and may involve immunoaffinity chromatography, for example. The plasmacytoid monocytes, fragments thereof or antibodies may be used in the developing treatments for any disorder mediated (directly or indirectly) by defective, or overly sufficient or insufficient amounts of the genes corresponding to the plasmacytoid monocytes of the instant invention. The plasmacytoid monocytes, fragments thereof or antibodies further can be used in the treatment of CD4⁺ CD56⁺ cutaneous neoplasm. Antibodies may naturally exert an agonist or antagonist activity that would be beneficial in the treatment of tumors. For instance, antibodies may transduce a signal to cells that would mediate apoptosis. Alternatively, antibodies could fix Complement or mediate killer activity through Fc receptor-bearing natural killer cells.

Antibodies of the instant invention may also find use as carriers for delivering agents attached thereto to tumor cells bearing the cell surface antigen. The antibody binds to the target cells, thus allowing detection thereof (in the case of diagnostic agents) or treatment thereof (with therapeutic agents).

Diagnostic and therapeutic agents that may be attached to an antibody include, but are not limited to, drugs, toxins, radionuclides, chromophores, enzymes that catalyze a colorimetric or fluorometric reaction, cytokines or chemokines, active fragments of cytokines or chemokines, and the like, with the particular agent being chosen according to the intended application. Cytokines that may be used in the instant invention include, but are not limited to, IL-3, IL-2, CD40-L, GM-CSF and PIXY (Immunex). Chemokines include, but are not limited to, CXCR3-ligands (ITAC, MIG, and IP- 10). Active fragments of cytokines or chemokines are any fragments that maintain the biological activity of the cytokine or chemokine. Among the toxins are ricin, abrin, saporin toxin, diptheria toxin, Pseudomonas aeruginosa exotoxin A, ribosomal inactivating proteins, mycotoxins such as trichothecenes, and derivatives and fragments (e.g., single chains) thereof. Radionuclides suitable for diagnostic use include, but are not limited to, ^{1 3}I, ¹³¹I, "Tc, In, ⁵¹Cr, ³H, ¹⁴C, ^{21 l}Th and ⁷⁶Br. Radionuclides suitable for therapeutic use include, but are not limited to, ¹³¹I, ⁷⁷Br, ³²P, ²¹²Bi, ⁶⁴Cu, and ⁶⁷Cu.

Such agents may be attached to the antibodies of the instant invention by any suitable conventional procedure. Antibodies, being proteins, comprise functional groups on amino acid side chains that can be reacted with functional groups on a desired agent to form covalent bonds, for example. The agent may be covalently linked to the antibody via an amide bond, hindered disulfide bond, acid-cleavable linkage, and the like, which are among the conventional linkages chosen according to such factors as the structure of the desired agent. Alternatively, the antibody or the agent to be linked thereto may be derivatized to generate or attach a desired reactive functional group. The derivatization may involve attachment of one of the bifunctional coupling reagents available for linking various molecules to proteins (Pierce Chemical Company, Rockford, Illinois). A number of techniques for radiolabeling proteins are known. Radionuclide metals may be attached to the antibody by using a suitable bifunctional chelating agent, examples of which are described in U.S. patents 4,897,255 and 4,965,392.

Conjugates comprising the antibodies of the instant invention and a suitable diagnostic or therapeutic agent (preferably covalently linked) are thus prepared. The conjugates are administered or otherwise employed in an amount appropriate for the particular application. The conjugates find use in in vitro or in vivo procedures.

Among the use of antibodies of the instant invention is the use in assays to detect the presence of CD56⁺ PMs and CD4⁺ CD56⁺ cutaneous neoplasm cells either in vitro or in vivo. The antibodies may be employed in purifying CD56⁺ PMs or CD4⁺ CD56⁺ cutaneous neoplasm cells by immunoaffinity chromatography. Further, these antibodies may be used to inhibit the growth or induce differentiation of either CD56⁺ PMs or CD4⁺ CD56⁺ cutaneous neoplasm cells. Such antibodies may be identified using any suitable assay procedure, such as by testing antibodies for the ability to inhibit growth of CD56⁺ PMs or CD4⁺ CD56⁺ cutaneous neoplasm, induce apoptotic death of these cells or induce differentiation into dendritic cells.

Alternatively, blocking antibodies may be identified in assays for the ability to inhibit a biological effect that results from the binding of CD56⁺ PM or CD4⁺ CD56⁺ cutaneous neoplasm cells to partner cells, such as endothelial cells or T-lymphocytes. One therapeutic method involves in vivo administration of a blocking antibody to a mammal in an amount effective in inhibiting CD56⁺ PM-mediated or CD4⁺ CD56⁺ cutaneous neoplasm cell-mediated biological activity. Disorders caused or exacerbated by CD56⁺ PM or CD4⁺ CD56⁺ cutaneous neoplasm cells directly or indirectly, are thus treated. Monoclonal antibodies are generally preferred for use in such therapeutic methods. In one embodiment, an antigen-binding antibody fragment is employed.

Further, antibodies raised against CD56⁺ PM or CD4⁺ CD56⁺ cutaneous neoplasm cells may be screened for agonistic properties. Such antibodies, upon binding to CD56⁺ PM or CD4⁺ CD56⁺ cutaneous neoplasm, induce biological effects (e.g., transduction of biological signals), leading to differentiation into dendritic cells or cells with natural killer activity. Agonistic antibodies may also be used to induce apoptosis of certain cancer cells, such as CD4⁺ CD56⁺ cutaneous neoplasm.

Plasmacytoid monocytes have been shown to produce large amounts of type-1 interferon in response to virus infection. Thus, agonistic antibodies against CD56⁺PM may be used to increase type-1 interferon production and exacerbate the anti-viral activity of CD56⁺ PM. Conventional techniques may be employed to confirm the susceptibility of various cancer cell types and virally infected cells to cell death induced by agonistic antibodies of the instant invention.

Viral infections and associated conditions include, but are not limited to cytomegalovirus, encephalomyocarditis, influenza, Newcastle disease virus, vesicular stomatitus virus, heφes simplex virus, hepatitis, adenovirus-2, bovine viral diarrhea virus, HIV, and Epstein-Barr virus. Agonistic antibodies of the present invention may be administered alone or in combination with other agents useful for combating a particular virus. Agonistic antibodies may also be employed in conjunction with other agent(s) useful in treating cancer. Examples of such agents include both proteinaceous and non- proteinaceous drugs and radiation therapy.

The present invention also provides pharmaceutical compositions comprising an antibody against CD56⁺ PM or CD4⁺ CD56⁺ cutaneous neoplasm cells and a suitable diluent, excipient, or carrier. Such carriers will be nontoxic to patients at the dosages and concentrations employed. Ordinarily, the preparation of such compositions entails combining a mammalian antibody or derivative thereof with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) peptides, proteins, amino acids, carbohydrates including glucose, sucrose, or dextrans, chelating agents such as EDTA, glutathione, or other stabilizers and excipients. Neutral buffered saline is one appropriate diluent.

For therapeutic use, the compositions are administered in a manner and dosage appropriate to the indication and the patient. As will be understood by one skilled in the pertinent field, a therapeutically effective dosage will vary according to such factors as the nature and severity of the disorder to be treated and the age, condition, and size of the patient. Administration may be by any suitable route, including but not limited to intravenous injection, continuous infusion, local infusion during surgery, or sustained release from implants (gels, membranes, and the like). The compositions of the present invention may contain an antibody in any form described above, including variants, derivatives, biologically active fragments, and oligomeric forms thereof. Antibodies derived from the same mammalian species as the patient is generally preferred for use in pharmaceutical compositions. In one embodiment of the invention, the composition comprises a soluble human antibody. In another embodiment of the invention, the pharmaceutical composition comprises an antibody having a diagnostic or therapeutic agent attached thereto. Such compositions may be administered to diagnose or treat conditions characterized by CD56⁺ PM cells, e.g., CD4⁺ CD56⁺ cutaneous neoplasm, as discussed above. The foregoing compositions may additionally contain, or be co-administered with, additional agents effective in treating malignancies characterized by CD56+ PM cells.

It is known that Interleukin 3 (IL-3) can produce the differentiation of plasmacytoid monocyte cells into dendritic cells. These dendritic cells present the antigens the cells are expressing. As discussed above, CD4⁺CD56⁺ cutaneous neoplasm express very high levels of CD123, the IL-3 receptor alpha chain. Thus, in one embodiment of the invention, differentiation of CD4⁺CD56⁺ cutaneous neoplasm is induced by administering IL-3 or an IL-3 agonist. The dendritic cells formed by this differentiation would present antigens from the CD4⁺CD56⁺ cutaneous neoplasm to effector cells, such as helper, cytotoxic T-lymphocytes or natural killer cells. This immune response could be used in the treatment of such tumors. Since CD4⁺ CD56⁺ cutaneous neoplasm express high levels of CD 123 or CXCR-3, the toxins described above could be coupled with antibodies against CD123 or CXCR-3 to deliver therapeutic agents, preferentially to the tumor cells. Alternatively, a cytokine, such as IL-3, or a chemokine, such as IP- 10, IT AC or MIG, could be coupled to a toxin described. Upon delivery, the cytokine-toxin conjugate or chemokine-toxin conjugate would target the CD4⁺ CD56⁺ cutaneous neoplasm cells.

In another embodiment of the instant invention, CD56⁺ PMs are used to create a cDNA library. The cDNA library may be created by any known means. In one embodiment, a PCR-select cDNA subtraction kit (ClonTech) may be used to generate a library of plasmacytoid specific cDNAs. Peripheral blood leukocytes from plasmacytoid monocytes, from which the plasmacytoid cells have been removed, may be used to generate the driver cDNA of CD56⁺ PMs. The peripheral blood leukocytes may be excluded by flow cytometry, by use of magnetic beads as discussed above, or by any method known in the art. Plasmacytoid monocyte cell cDNA may then be generated by the subtraction kit and used as the target DNA for the subtraction procedure. The subtracted plasmacytoid monocyte cDNA library may be used to probe arrays of human cDNAs and sequence clones, directly sequenced, or used as a cDNA source of cDNA expressing cloning of CD56⁺PM specific genes. The clones or sequences of CD56⁺ PMs may then be examined to determine whether they encode genes that influence the survival, activation or death of CD56⁺ PMs. The proteins encoded by these genes may be administered to treat CD4⁺CD56⁺ cutaneous neoplasm.

Isolated or purified CD56⁺PMs, fragments or antibodies specific to CD56⁺ PM or CD4⁺ CD56⁺ cutaneous neoplasm cell thereof can be administered through well-known means, including parenterally (subcutaneous, intramuscular, intravenous, intradermal, etc. injection) and with a suitable carrier. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti- oxidants, buffers, bacteriostats and solutes which render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.

The following examples are offered by way of illustration, and not by way of limitation. Those skilled in the art will recognize that variations of the invention embodied in the example can be made, especially in light of the teachings of the various references cited herein, the disclosures of which are incoφorated by reference in their entirety.

Example 1 Peripheral blood samples were obtained from a randomized, placebo controlled, phase 1 dose escalation study performed in healthy human volunteers. Flt-3 ligand was produced by well-known recombinant DNA technology in a Chinese hamster ovary (CHO) cell line. Flt-3 ligand was supplied as a sterile lyophilized preparation of 5 mg of Flt-3 ligand, with 40 mg mannitol, 10 mg sucrose, and 35 mM of tromethamine (TRIS) per vial. Prior to administration, the Flt-3 ligand was reconstituted in bacteriostatic water for injection.

Volunteers were subcutanously injected once daily with 10, 25, 50, 75 or 100 μg/kg/day of human Flt-3 ligand for 10 consecutive days.

Example 2 The peripheral blood mononuclear cells (PBMC) from the subjects of Example 1 were isolated after centrifugation over a discontinuous density gradient using Ficoll (1.077g/ml Accu-Prep, Accurate Chemicals, Westbury NY), and washed in PBS containing 5% fetal bovine serum (FBS) (Intergen, New York, NY). Cells were then incubated with directly conjugated antibodies which included CD2, CD4, CD56, and CDwl23 (IL-3Rα) for 30 minutes at 4°C in PBS containing 0.01% NaN₃ supplemented with 10% goat serum and 10% rabbit serum to block Fc -receptors. Propidium iodide (PI) (lμg/ml) was included in the final wash to allow exclusion of dead cells.

Example 3 The PBMC cells, obtained as described in Examples 1 and 2, cells were then cryogenically preserved in approximately 2 to 3 x 10^ cells/ml in 12%DMSO (Dimethyl Sulfoxide; ICN, Aurora, OH)/μH FBS (Characterized Fetal Bovine Serum; HyClone, Logan, UT). Cryovials were then stored in a liquid nitrogen dewar.

Cells were thawed in 10% μH FBS/10% Versene (Life Technologies, Bethesda, MD); 20 Kunitz Units/ml of DNasel (Sigma, St.Louis, MO) and Super M^cCoys medium. Thawed cells were resuspended at approximately 2 x 10⁷ cells/ml in 10% μH human serum (Cellect Human Pooled Serum; ICN, Aurora, OH) and Phosphate Buffered Saline (PBS).

Typically stained cells were distributed 1 to 2 x 10 > cells per well in a polystyrene V-bottom 96-well plate (Costar, Corning, NY). The wells were diluted to a ratio of 1/50 using APC-conjugated mouse anti-human CDwl23 (anti-EL-3Rα; non-blocking; clone 9F5 at approximately 164μg/ml; Pharmingen, San Diego, CA) and PE-conjugated mouse anti-human CD56 (clone B159; at approximately 200 μg/ml; Pharmingen, San Diego, CA). Mouse antibody isotype controls (i.e., APC-conjugated IgGi and PE-conjugated IgGi ) were also included in the media. The media was incubated for ten minutes at approximately 4°C.

The cells were washed with PBS and resuspended in 300μl of FACS buffer. Several hours after the completion of staining, flow cytometry was performed.

Example 4 This example illustrates a method for preparing monoclonal antibodies that bind

CD56⁺ PM cells. Suitable immunogens that may be employed in generating such antibodies include, but are not limited to, purified CD56⁺ PM cells.

Purified CD56⁺ PM cells can be used to generate monoclonal antibodies immunoreactive therewith, using conventional techniques such as those described in U.S. Patent 4,411,993. Briefly, mice are immunized with CD56⁺ PM immunogen emulsified in complete Freund's adjuvant, and injected in amounts ranging from 10-100:g subcutaneously or intraperitoneally. Ten to twelve days later, the immunized animals are boosted with additional CD56⁺ PM emulsified in incomplete Freund's adjuvant. Mice are periodically boosted thereafter on a weekly to bi-weekly immunization schedule. Serum samples are periodically taken by retro-orbital bleeding or tail-tip excision to test for antibodies against CD56⁺ PM by dot blot assay, ELISA (Enzyme-Linked Immunosorbent Assay) or flow cytometry.

Following detection of an appropriate antibody titer, positive animals are provided one last intravenous injection of CD56⁺ PM cells in saline. Three to four days later, the animals are sacrificed, spleen cells harvested, and spleen cells are fused to a murine myeloma cell line, e.g., NS1 or preferably P3x63Ag8.653 (ATCC CRL 1580). Fusions generate hybridoma cells, which are plated in multiple microtiter plates in a HAT (hypoxanthine, aminopterin and thymidine) selective medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.

The hybridoma cells are screened by ELISA for reactivity against purified CD56⁺ PM cells by adaptations of the techniques disclosed in Engvall et al., Immunochem. 8:871, 1971 and in U.S. Patent 4,703,004. A preferred screening technique is the antibody capture technique described in Beckmann et al., (J. Immunol. 144:4212, 1990) Positive hybridoma cells can be injected intraperitoneally into syngeneic BALB/c mice to produce ascites containing high concentrations of anti- CD56⁺ PM monoclonal antibodies. Alternatively, hybridoma cells can be grown in vitro in flasks or roller bottles by various techniques. Monoclonal antibodies produced in mouse ascites can be purified by ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to Protein A or Protein G can also be used, as can affinity chromatography based upon binding to CD56⁺ PM cells.

Claims

What is claimed:

1. A method of treating CD4⁺ CD56⁺ cutaneous neoplasm, wherein such method comprises administering to a mammal in need thereof antibodies against a CD56⁺ plasmacytoid monocyte.

2. A method of isolating CD56⁺ plasmacytoid monocytes, wherein such method comprises the steps: a. isolating peripheral blood mononuclear cells from a mammal; b. incubating the cells obtained from step (a) with directly conjugated antibodies selected from the group CD4, CD56 and CD 123; and c. isolating the plasmacytoid monocytes expressing CD56 and CD123.

3. The method of claim 2, wherein said mammal is administered Flt3-Iigand before the isolation of step (a).

4. An isolated plasmacytoid monocyte expressing the markers CD4, CD56, and CD123.

5. An isolated plasmacytoid monocyte produced by the method of claim 2.

6. The method of claim 3, wherein said plasmacytoid monocytes are isolated by a process selected from the group consisting of flow cytometry or use of magnetic beads.

7. The method of claim 6, wherein the plasmacytoid monocyte is isolated by flow cytometry.

8. A method of treating CD4⁺CD56⁺ cutaneous neoplasm, wherein such method comprises: a) extracting the RNA of CD56⁺ plasmacytoid monocyte; b) creating a cDNA library using the RNA of step (a); c) sequencing the cDNA library; d) determining whether the sequences produced in step (c) encode genes capable of influencing survival or activation of CD56⁺ plasmacytoid monocytes; and e) administering the protein encoded by the genes of step (d) to a mammal in need thereof.

9. A method of treating CD4⁺ CD56⁺ cutaneous neoplasm, wherein such method comprises administering to a mammal in need thereof a conjugate selected from the group consisting of a cytokine-toxin conjugate, a chemokine-toxin conjugate or a conjugate comprised of a toxin and antibodies against either CD 123 or CXCR-3.