EP1483407A2 - Methods of therapy and diagnosis - Google Patents
Methods of therapy and diagnosisInfo
- Publication number
- EP1483407A2 EP1483407A2 EP03709107A EP03709107A EP1483407A2 EP 1483407 A2 EP1483407 A2 EP 1483407A2 EP 03709107 A EP03709107 A EP 03709107A EP 03709107 A EP03709107 A EP 03709107A EP 1483407 A2 EP1483407 A2 EP 1483407A2
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- European Patent Office
- Prior art keywords
- carcinoma
- cell
- leukemia
- lymphoma
- cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/38—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against protease inhibitors of peptide structure
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
Definitions
- This invention relates to compositions and methods for targeting cells using antibodies, polypeptides, polynucleotides, peptides, and small molecules and their use in the therapy and diagnosis of various pathological states, including cancer, autoimmune disease, organ transplant rejection, allergic reactions, wound healing, liver fibrosis, emphysema, and cardiovascular disease.
- Imrnuno therapy provides a method of harnessing the immune system to treat various pathological states, including cancer, autoimmune disease, transplant rejection, hyperproliferative conditions, allergic reactions, emphysema, wound healing and cardiovascular disease.
- Antibody therapy for cancer involves the use of antibodies, or antibody fragments, against a tumor antigen to target antigen-expressing cells.
- Antibodies, or antibody fragments may have direct or indirect cytotoxic effects or may be conjugated or fused to cytotoxic moieties.
- Direct effects include the induction of apoptosis, the blocking of growth factor receptors, and anti-idiotype antibody formation.
- Indirect effects include antibody- dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cellular cytotoxicity (CMCC).
- ADCC antibody- dependent cell-mediated cytotoxicity
- CMCC complement-mediated cellular cytotoxicity
- the antibodies, or fragments thereof provide a method of targeting the cytotoxicity towards the tumor antigen expressing cells.
- tumor-specific antigens provide targets for antibody targeting of cancer, or other disease-related cells, expressing the antigen.
- Antibodies specific to such tumor-specific antigens can be conjugated to cytotoxic compounds or can be used alone in immunotherapy. Immunotoxins target cytotoxic compounds to induce cell death.
- anti-CD22 antibodies conjugated to deglycosylated ricin A may be used for treatment of B cell lymphoma that has relapsed after conventional therapy (Amlot, et al., Blood 82:2624-2633 (1993), inco ⁇ orated herein by reference in its entirety) and has demonstrated encouraging responses in initial clinical studies.
- the immune system functions to eliminate organisms or cells that are recognized as non-self, including microorganisms, neoplasms and transplants.
- a cell-mediated host response to tumors includes the concept of immunologic surveillance, by which cellular mechanisms associated with cell-mediated immunity destroy newly transformed tumor cells after recognizing tumor-associated antigens (antigens associated with tumor cells that are not apparent on normal cells).
- a humoral response to tumor- associated antigens enables destruction of tumor cells through imrnuno logical processes triggered by the binding of an antibody to the surface of a cell, such as antibody-dependent cellular cytotoxicity (ADCC) and complement mediated lysis.
- ADCC antibody-dependent cellular cytotoxicity
- an antigen by the immune system triggers a cascade of events including cytokine production, B-cell proliferation, and subsequent antibody production. Often tumor cells have reduced capability of presenting antigen to effector cells, thus impeding the immune response against a tumor-specific antigen. In some instances, the tumor-specific antigen may not be recognized as non-self by the immune system, preventing an immune response against the tumor-specific antigen from occurring. In such instances, stimulation or manipulation of the immune system provides effective techniques of treating cancers expressing one or more tumor-specific antigens.
- Rituximab is a chimeric antibody directed against CD20, a B cell-specific surface molecule found on >95% of B-cell non-Hodgkin's lymphoma (Press, et al, Blood 69:584-591 (1987); Malony, et al, Blood 90:2188-2195 (1997), both of which are inco ⁇ orated herein by reference in their entirety).
- Rituximab induces ADCC and inhibits cell proliferation through apoptosis in malignant B cells in vitro (Maloney, et al, Blood 88:637a (1996), inco ⁇ orated herein by reference in its entirety).
- Rituximab is currently used as a therapy for advanced stage or relapsed low-grade non-Hodgkin's lymphoma, which has not responded to conventional therapy.
- Active immunotherapy whereby the host is induced to initiate an immune response against its own tumor cells can be achieved using therapeutic vaccines.
- tumor-specific vaccine uses purified idiotype protein isolated from tumor cells, coupled to keyhole limpet hemocyanin (KLH) and mixed with adjuvant for injection into patients with low- grade follicular lymphoma (Hsu, et al, Blood 89:3129-3135 (1997), inco ⁇ orated herein by reference in its entirety).
- KLH keyhole limpet hemocyanin
- Another type of vaccine uses antigen-presenting cells (APCs), which present antigen to na ⁇ ve T cells during the recognition and effector phases of the immune response.
- APCs antigen-presenting cells
- Dendritic cells one type of APC, can be used in a cellular vaccine in which the dendritic cells are isolated from the patient, co-cultured with tumor antigen and then reinfused as a cellular vaccine (Hsu, et al, Nat. Med. 2:52-58 (1996), inco ⁇ orated herein by reference in its entirety). Immune responses can also be induced by injection of naked DNA.
- Tumor cells express proteins, such as growth factors, growth factor modulators, and proteases. These proteins play a role in tumor proliferation, differentiation, tissue invasion, metastases and vascularization (angiogenesis). For example, metastasis requires the primary tumor to produce proteases that degrade the extracellular matrix such that tumor cells may enter the blood stream and colonize different tissues. Often these tumor cells will secrete growth or survival factors that will allow them to survive and proliferate in the new tissue.
- epithelial- to-mesenchymal transformation EMT driven by activation of growth factors, such as TGF- ⁇ (Thiery and Chopin Cancer Metastasis Rev. 18:31-42 (1999), inco ⁇ orated herein by reference in its entirety).
- Cells undergoing EMT have altered expression of cell adhesion and cytoskeleton molecules, such as E-cadherin, vinculin, and keratin, and express mesenchymal markers, such as vimentin de novo (Oft et al, Genes Dev. 10:2462-2477 (1996);
- McMet al, J. Cell Biol. 127:2021-2036 (1994) both of which are inco ⁇ orated herein by reference in their entirety).
- TGF-/3 for example, induces the expression of the angiogenesis-inducing factor VEGF (Pertovaara et al, J. Biol. Chem. 269:6271-62"/ r 4 (1994), inco ⁇ orated herein by reference in its entirety) and has indirect effects on angiogenesis as well, such as attracting monocytes that secrete angiogenic cytokines (Sunderkotter et al, Pharmacol. Ther. 51:195-216 (1991), inco ⁇ orated herein by reference in its entirety).
- VEGF angiogenesis-inducing factor
- antibodies can be raised against secreted proteins that are involved in regulating processes associated with cancer, such as cell proliferation, differentiation, cell migration, tissue invasion, and angiogenesis.
- Secreted proteins such as epidermal growth factor (EGF), interleukin-2, and platelet derived growth factor have been demonstrated to play a role in tumor growth, for example.
- Antibodies can neutralize the activity of the secreted protein by binding to a region that is required for function, for example, a growth factor binding domain, or active site, thereby acting as an inhibitor.
- antibodies bound to secreted proteins can neutralize antigens by inducing phagocytosis of the antigens by mononuclear phagocytes and neutrophils in a process known as opsonization.
- agents such as peptide fragments, nucleic acids, small molecules, or antibodies, that provide therapeutic compositions and diagnostic methods for treating and identifying cancer, hype ⁇ roliferative disorders, auto-immune diseases, organ transplant rejection, heart disease, and protease- related diseases.
- the invention provides therapeutic and diagnostic methods of targeting cells expressing cell surface antigens (CSA) by using targeting elements such as polypeptides, nucleic acids, antibodies, including fragments or other modifications thereof, peptides and small molecules.
- CSA cell surface antigens
- the CSAs of the invention are highly expressed in certain hematopoietic- based and non-hematopoietic (i.e. solid tumors) cancer cells relative to their expression in healthy cells.
- targeting of cells that express cell surface antigen of the invention will have a minimal effect on healthy tissues while destroying or inhibiting the growth of the hematopoietic-based cancer cells.
- non-hematopoietic type tumors can be targeted if they bear a CSA of the invention.
- inhibition of growth and/or destruction of cancer cells that express said CSA results from targeting such cells with antibodies that recognize said CSA.
- One embodiment of the invention is a method of destroying cells that express a CSA by contacting them with antibodies that recognize said CSA.
- CSA conjugated to cytocidal materials such as radioisotopes or other cytotoxic compounds.
- the present invention provides a variety of targeting elements and compositions.
- One such embodiment is a composition comprising an anti-CSA antibody preparation.
- Exemplary antibodies include a single anti-CSA antibody, a combination of two or more anti-CSA antibodies, a combination ofan anti-CSA antibody with a non-CSA antibody, a combination of anti-CSA antibody and a therapeutic agent, a combination of an anti-CSA antibody and a cytocidal agent, a bispecific anti-CSA antibody, Fab CSA antibodies or fragments thereof, including any fragment of an antibody that retains one or more complementary detemining regions (CDRs) that recognize a cell-surface antigen of the invention, humanized anti-CSA antibodies that retain all or a portion of a CDR that recognizes a cell-surface antigen of the invention, anti-CSA conjugates, and anti-CSA antibody fusion proteins.
- CDRs complementary detemining regions
- Another targeting embodiment of the invention is a vaccine comprising a CSA polypeptide, or a fragment or variant thereof and optionally comprising a suitable adjuvant.
- Yet another targeting embodiment is a composition comprising a nucleic acid encoding a CSA, or a fragment or variant thereof, optionally within a recombinant vector.
- a further targeting embodiment of the present invention is a composition comprising an antigen-presenting cell transformed with a nucleic acid encoding a CSA, or a fragment or variant thereof, optionally within a recombinant vector.
- Yet another targeting embodiment of the invention is a preparation comprising a CSA polypeptide or peptide fragment or variant thereof.
- a further targeting embodiment of the present invention is a non-CSA polypeptide or peptide that binds a CSA of the invention.
- Another targeting embodiment of the invention is a preparation comprising a small molecule that recognizes or binds to a CSA of the invention.
- the present invention further provides a method of targeting cells expressing a CSA of the invention, which comprises administering a targeting element or composition in an amount effective to target CSA-expressing cells.
- any one of the targeting elements or compositions described herein may be used in such methods, including an anti-CSA antibody preparation, a vaccine comprising a CSA polypeptide, or a fragment or variant thereof or a composition of a nucleic acid encoding a cell surface antigen of the invention, or a fragment or variant thereof, optionally within a recombinant vector or a composition of an antigen-presenting cell transformed with a nucleic acid encoding a CSA, or fragment or variant thereof, optionally within a recombinant vector, or a CSA polypeptide, peptide fragment, or variant thereof, or a binding polypeptide, peptide or small molecule that binds to a cell surface antigen of the invention.
- the invention also provides a method of inhibiting the growth of cancer cells, including hematopoietic-based cancer cells, expressing a cell surface antigen of the invention, which comprises administering a targeting element or a targeting composition in an amount effective to inhibit the growth of said cancer cells.
- any one of the targeting elements or compositions described herein may be used in such methods, including an anti- CSA antibody preparation, a vaccine comprising a CSA polypeptide, fragment, or variant thereof, composition of a nucleic acid encoding a cell surface antigen of the invention, or fragment or variant thereof, optionally within a recombinant vector, or a composition of an antigen-presenting cell transformed with a nucleic acid encoding a cell surface antigen of the invention, or fragment or variant thereof, optionally within a recombinant vector, or a CSA polypeptide, peptide fragment, or variant thereof, or a binding polypeptide, peptide or small- molecule that binds to a CSA of the invention.
- the present invention further provides a method of treating disorders associated with the proliferation of cells expressing a cell surface antigen of the invention in a subject in need thereof, comprising the step of administering a targeting element or targeting composition in a therapeutically effective amount to treat disorders associated with cells expressing a cell surface antigen of the invention.
- any one of the targeting elements or compositions described herein may be used in such methods, including an anti-CSA antibody preparation, a vaccine comprising a CSA polypeptide, fragment, or variant thereof, a composition of a nucleic acid encoding a CSA of the invention, or fragment or variant thereof, optionally within a recombinant vector, or a composition of an antigen-presenting cell comprising a nucleic acid encoding a CSA of the invention, or fragment or variant thereof, optionally within a recombinant vector, or a CSA polypeptide, peptide fragment, or variant thereof, or a binding polypeptide, peptide or small molecule that binds to or recognizes a CSA of the invention.
- disorders associated with the proliferation of cells expressing a cell surface antigen of the invention include cancers, such as Hodgkin' s Disease, non-Hodgkin's B cell lymphomas, T cell lymphomas, malignant lymphoma, lymphosarcoma leukemia, B cell leukemias, T cell leukemias, acute and chronic myeloid leukemia (also known as myelogenous leukemia), acute and chronic lymphocytic leukemia (also known as lymphoblastic leukemia or lymphoid leukemia), myelomonocytic leukemia, myelodysplastic syndromes, multiple myeloma, X-linked lymphoproliferative disorders; Epstein Barr Virus- related conditions such as mononucleosis; hype ⁇ roliferative disorders; autoimmune disorders, such as systemic lupus erythematosus (SLE), Hasimoto thyroiditis, Sjogren's syndrome, pericarditus lupus; wound
- Non- hematopoietic tumors that bear the cell surface antigen of the invention such as breast, colon, prostate, lung, stomach, thymus, epithelial and squamous cell carcinomas, as well as other cancers including gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer, polyps associated with colorectal neoplasms, pancreatic cancer and gallbladder cancer, cancer of the adrenal cortex, ACTH-producing tumor, bladder cancer, brain cancer including intrinsic brain tumors, neuroblastomas, astrocytic brain tumors, gliomas, and metastatic tumor cell invasion of the central nervous system, Ewing's sarcoma, head and neck cancer including mouth cancer and larynx cancer, kidney cancer including renal cell carcinoma, liver cancer, lung cancer including small and non-small cell lung cancers, malignant peritoneal effusion, malignant pleural effusion, skin cancers including malignant melanoma, tumor progression of human skin keratinocytes,
- the invention further provides a method of modulating the immune system by either suppression or stimulation of growth factors and cytokines, by administering the targeting elements or compositions of the invention.
- the invention also provides a method of modulating the immune system through activation of immune cells (such as natural killer cells, T cells, B cells and myeloid cells), through the suppression of activation, or by stimulating or suppressing proliferation of these cells by CSA peptide fragments or anti-CSA antibodies.
- immune cells such as natural killer cells, T cells, B cells and myeloid cells
- the present invention thereby provides a method of treating immune-related disorders by suppressing the immune system in a subject in need thereof, by administering the targeting elements or compositions of the invention.
- immune-related disorders include but are not limited to autoimmune disease and organ transplant rejection.
- the present invention also provides a method of diagnosing disorders associated with cells that express a cell surface antigen of the invention comprising the step of measuring the expression patterns of said CSA protein and/or its associated mRNA.
- a method of diagnosing disorders associated with cells that express a CSA comprising the step of detecting expression of said CSA using anti-CSA antibodies. Expression levels or patterns may then be compared with a suitable standard indicative of the desired diagnosis.
- Such methods of diagnosis include compositions, kits and other approaches for determining whether a patient is a candidate for therapy in which said CSA is targeted.
- the present invention also provides a method of enhancing the effects of therapeutic agents and adjunctive agents used to treat and manage disorders associated with cells that express a CSA of the invention, by administering preparations of said CSA with therapeutic and adjuvant agents commonly used to treat such disorders.
- Figure 1 depicts a CLUSTALW multiple amino acid sequence alignment between the oSMHyl protein encoded by SEQ ID NO: 5 (i.e. SEQ ID NO: 6) and the two o2MHy splice variants (SEQ ID NO: 31 (o2MHy2) and SEQ ID NO: 45 ( ⁇ 2MHy3)), the sequence- corrected full-length c ⁇ MHyl (SEQ ID NO: 47), and human ⁇ 2M precursor (SEQ ID NO: 48).
- Figure 2 shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ ID NO: 53 (i.e. SEQ ID NO: 54) IGFBP-7Hyl and Mus musculus IGFBP- 7Hyl, SEQ ID NO: 61, indicating that the two sequences share 86% similarity and 77% identity over the entire amino acid sequence of SEQ ID NO: 54.
- Figure 3 shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ ID NO: 53 (i.e. SEQ ID NO: 54) IGFBP-7Hyl and Homo sapiens protein promoting prostaglandin 12 production, SEQ ID NO: 62, indicating that the two sequences share 54% similarity and 45% identity over the entire amino acid sequence of SEQ ID NO: 54.
- Figure 4 shows the cell surface expression of VpreBl on B cell non-Hodgkin's lymphoma cell lines (Ca-46, GA10, and HT cell lines).
- Table 1 is a correlation table of the polynucleotide sequences and the polypeptides and the corresponding SEQ ID NO: in which the sequence was filed in the following priority U.S. Patent Applications bearing the serial numbers of: 10/327,413 filed on December 19, 2002, 10/092,985 filed on March 06, 2002, 10/087,137 filed on February 27, 2002, 10/077,676 filed on February 14, 2002, and 10/146,619 filed on May 14, 2002.
- HYS-21CP2_XX SEQ ID NO: XX of Attorney Docket No. HYS-21CP2, U.S. Serial No. 10/078,080, filed 02/15/2002, the entire disclosure of which, including sequence listing, is inco ⁇ orated herein by reference.
- HYS-31CP3_XX SEQ ID NO: XX of Attorney Docket No. HYS-31CP3, U.S. Serial No. 10/092,985 filed 03/06/2002, the entire disclosure of which, including sequence listing, is inco ⁇ orated herein by reference.
- HYS-38CP2_XX SEQ ID NO: XX of Attorney Docket No. HYS-39CP2, U.S. Serial No. 10/087,137 filed 02/27/2002, the entire disclosure of which, including sequence listing, is inco ⁇ orated herein by reference.
- HYS-49_XX SEQ ID NO: XX of Attorney Docket No. HYS-49, U.S. Serial No. 10/077,676 filed 02/14/2002, the entire disclosure of which, including sequence listing, is inco ⁇ orated herein by reference.
- HYS-50_XX SEQ ID NO: XX of Attorney Docket No. HYS-50, U.S. Serial No. 10/146,619 filed 05/14/2002, the entire disclosure of which, including sequence listing, is inco ⁇ orated herein by reference.
- the present invention relates to methods of targeting cells that express cell surface antigens (CSAs) using targeting elements, such as polypeptides, nucleic acids, antibodies, binding polypeptides, peptides and small molecules, including fragments or other modifications of any of these elements.
- CSAs cell surface antigens
- the present invention provides a novel approach for diagnosing and treating diseases and disorders associated with said cell surface antigens.
- the method comprises administering an effective dose of targeting preparations such as vaccines, antigen presenting cells, or pharmaceutical compositions comprising the targeting elements, polypeptides of the cell surface antigens, nucleic acids encoding the cell surface antigens, antibodies that recognize the cell surface antigens, or binding polypeptides, peptides and small molecules that bind to the cell surface antigens of the invention, described below.
- Targeting of antigens on the cell membranes is expected to inhibit the growth of or destroy such cells.
- An effective dose will be the amount of such targeting preparations necessary to target the antigen on the cell membrane and inhibit the growth of or destroy the cells expressing the cell surface antigen and/or metastasis.
- a further embodiment of the present invention is to enhance the effects of therapeutic agents and adjunctive agents used to treat and manage disorders associated with said CSAs, by administering targeting preparations that recognize the CSA associated with the disorder with therapeutic and adjuvant agents commonly used to treat such disorders.
- Chemotherapeutic agents useful in treating neoplastic disease and antiproliferative agents and drugs used for immunosuppression include alkylating agents, such as nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes; antimetabolites, such as folic acid analogs, pyrimidine analogs, and purine analogs; natural products, such as vinca alkaloids, epipodophyllotoxins, antibiotics, and enzymes; miscellaneous agents such as polatinum coordination complexes, substituted urea, methyl hydrazine derivatives, and adrenocortical suppressant; and hormones and antagonists, such as adrenocorticosteroids, progestins, estrogens, androgens, and anti-estrogens (Calebresi and Parks, pp.
- alkylating agents such as nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes
- Adjunctive therapy used in the management of such disorders includes, for example, radiosensitizing agents, coupling of antigen with heterologous proteins, such as globulin or beta-galactosidase, or inclusion of an adjuvant during immunization.
- High doses may be required for some therapeutic agents to achieve levels to effectuate the target response, but may often be associated with a greater frequency of dose- related adverse effects.
- combined use of the targeting therapeutic methods of the present invention with agents commonly used to treat disorders associated with expression of said CSAs of the invention allows the use of relatively lower doses of such agents resulting in a lower frequency of adverse side effects associated with long-term administration of the conventional therapeutic agents.
- Another indication for the targeting therapeutic methods of this invention is to reduce adverse side effects associated with conventional therapy of these disorders.
- the CD2 family of co-receptors such as CD2, CD48, and CD84 belong to the immunoglobulin (Ig) superfamily of cell surface receptors, which interact with their cognate ligands on opposing cells.
- CD84 is expressed on hematopoietic tissues and cells, primarily lymphocytes and monocytes (de la Fuente, et al, supra) and may play a role in leukocyte activation.
- a CD84 homolog, NTB-A may function as a co-receptor in inducing NK cell-mediated cytotoxicity, and its function was significantly affected in the absence ofan intracellular signaling protein, Src homology 2-domain containing protein (Bottino, et al, J. Exp. Med. 194:235-246 (2001), inco ⁇ orated herein by reference in its entirety).
- CD48 another member of the CD2 family, when engaged by 2B4 molecules on NK cells from patients with X-linked lymphoproliferative disease inhibited the cytolysis of virus-infected cells (Parolini et al, J. Exp. Med. 192:337-346 (2000) herein inco ⁇ orated by reference in its entirety).
- dysregulation of CD2 family member expression may lead to autoimmune disorders or severe immunodeficiencies.
- Expression of CD48 is also upregulated on Epstein-Barr and other virus infected leukocytes.
- a secreted, soluble form of CD48 is also found in these patients and actually correlates with infectious disease activity (Katsuura et al, Acta Paediatr. Jap.
- the CD84Hyl protein of the invention is highly expressed in certain hematopoietic-based cancers, but not by most non-hematopoietic, healthy cells. Thus, targeting of cells that express CD84Hyl will have a minimal effect on healthy tissues while destroying or inhibiting the growth of cancer cells. Similarly, non-hematopoietic type tumors (i.e. solid tumors) can be targeted if they bear the CD84Hyl antigen. Targeting of CD84Hyl can also be used to treat disorders associated with the proliferation of CD84Hyl- expressing cells.
- disorders associated with the proliferation of CD84Hyl- expressing cells include cancers such as non-Hodgkin's B cell lymphomas, B cell leukemias, T cell leukemias, T cell lymphomas, chronic lymphocytic leukemia, acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, lymphosarcoma leukemia, malignant lymphoma, B cell large cell lymphoma, multiple myeloma, myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndromes, X-linked proliferative disorders and Epstein Barr Vims-related conditions, such as mononucleosis; autoimmune disorders such as systemic lupus erythematosus, pericarditis lupus, Sj ⁇ gren's syndrome, Hasimoto thyroiditis; hype ⁇ roliferative disorders; organ and tissue transplant rejection;
- CD84Hyl polypeptides and polynucleotides encoding such polypeptides are disclosed in co-owned U.S. Patent Application Serial Nos. 09/645,476 and 09/491,404 which correspond to PCT Publication Nos. WO 01/55336 and WO 01/55437, respectively. These and all other U.S. patents and patent applications cited herein are hereby inco ⁇ orated by reference in their entirety.
- U.S. Patent Application Serial No. 09/491,404 inco ⁇ orated by reference herein in its entirety relates, in general to a collection or library of at least one novel nucleic acid sequences, specifically contigs, assembled from expressed sequence tags (ESTs).
- ESTs expressed sequence tags
- 09/645,476, inco ⁇ orated by reference herein in its entirety, discloses CD84-like polypeptides, isolated polynucleotides encoding such polypeptides, including recombinant molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, fragments or analogs or variants of such polynucleotides or polypeptides, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, including polyclonal, monoclonal, single chain, bispecific, fragment, human and humanized antibodies, as well as hybridomas producing monoclonal antibodies, and diagnostic and therapeutic uses and screening assays associated with such polynucleotides, polypeptides and antibodies.
- the polynucleotides of U.S. Patent Application Serial No. 09/645,476 are based on a CD84-like polynucleotides, including recombinant molecules
- CD84Hyl is an approximately 332 amino acid protein with a predicted molecular weight of 37 kD unglycosylated.
- a predicted approximately 21 residue signal peptide is encoded from approximately residue 1 to residue 21 of SEQ ID NO: 2.
- a predicted transmembrane domain is encoded from approximately residue 215 to residue 244 of SEQ ID NO: 2. Both the signal peptide and transmembrane domains were predicted using the Kyte-Doolittle hydrophobicity prediction algorithm (J. Mol. Biol.
- CD84Hyl is predicted to contain two immunoglobulin (Ig) domains spanning amino acids 35 to 111 and amino acids 146 to 197.
- CD84Hyl is expressed in certain hematopoetic-based cancers, including Burkitt's lymphoma, diffuse lymphoma, B cell lymphoma, follicular lymphoma, diffuse large B cell lymphoma, malignant lymphoma, T cell lymphoma, multiple myeloma, acute myeloid leukemia, T cell leukemia, acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphoblastic leukemia, acute leukemia, lymphosarcoma cell leukemia, and Hodgkin's Disease, while most non-hematopoetic, healthy cells fail to express CD84Hyl or express it at low levels (see Tables 2, 3 and 4).
- CD84Hyl is also expressed in certain autoimmune disorders, including systemic lupus erythematosus, Hasimoto Thyroiditis, Sj ⁇ rgen's Syndrome, and pericarditis lupus (see Table 5). Finally, CD84Hyl is expressed in rejected heart, liver, and kidney tissues, whereas normal tissues do not express CD84Hyl (see Table 6). Thus, targeting CD84Hyl will be useful in treating hematopoietic cancers, solid cancers, autoimmune disorders, and reducing and/or eliminating tissue rejection after transplantation. Reducing expression levels of CD84Hyl through, e.g. antisense therapy, is also expected to be beneficial in reducing and/or eliminating tissue rejection after transplantation.
- CD84Hyl peptide itself may be used to target toxins or radioisotopes to tumor cells in vivo.
- CD84Hyl may be a homophilic adhesion protein which will bind to itself.
- the extracellular domain of CD84Hyl, or a fragment of this domain may be able to bind to CD84Hyl expressed on tumor cells.
- This peptide fragment then may be used as a means to deliver cytotoxic agents to CD84Hyl bearing tumor cells. Much like an antibody, these fragments may specifically target cells expressing this antigen. Targeted delivery of these cytotoxic agents to the tumor cells would result in cell death and suppression of tumor growth.
- Extracellular fragments of the CD84Hyl receptor may also be used to modulate immune cells expressing the protein.
- Extracellular domain fragments of the receptor may bind to and activate its own receptor expressed on the cell surface.
- On cells bearing the CD84Hyl receptor such as NK cells, T cells, B cells and myeloid cells
- cytokines such as interferon gamma for example
- binding of these fragments to cells bearing the CD84Hyl receptor may result in the activation of these cells and also may stimulate proliferation.
- Some fragments may bind to the intact CD84Hyl receptor and block activation signals and cytokine release by immune cells. These fragments would then have an immune suppressive effect.
- Fragments that activate and stimulate the immune system may have anti-tumor properties. These fragments may stimulate an immunological response that can result in immune mediated tumor cell killing. The same fragments may result in stimulating the immune system to mount an enhance response to foreign invaders such as viras and bacteria. Fragments that suppress the immune response may be useful in treating lymphoproliferative disorders, auto-immune disease, graft-vs-host disease, and inflammatory disorders such as emphysema.
- Alpha 2-macroglobulin is a highly conserved proteinase inhibitor present in plasma at relatively high concentrations (2-4 mg/ml). It is unique in its ability to inhibit all the major classes of proteinases (Bhattacharjee et al, J. Biol. Chem. 275:26806-26811 (2000); Barrett and Starkey, Biochem. J. 133:709-724 (1973), both of which are herein inco ⁇ orated by reference in its entirety), to regulate cellular growth by binding and modulating the activity of many cytokines and growth factors (LaMarre et al, Lab. Invest. 65:3-14 (1991); Bonner and Brody, Am. J. Physiol.
- 0-2M is a tetramer of four identical 180 kDa subunits that forms a hollow cylinderlike stracture. It presents multiple target peptide bonds to attacking proteinases in its central "bait" domain. Binding of the proteinase and subsequent cleavage of the bait domain leads to a conformational change trapping the proteinase in the central cavity.
- the "activated" ⁇ 2M (o_2M*) is now recognizable by its receptor, low density lipoprotein receptor-related protein (LRP), wherein it is internalized by receptor-mediated endocytosis and targeted to the lysosome for degradation (Chu and Pozzo, Lab. Invest. 71:792-812 (1994); Krieger and Herz, Annu. Rev.
- the o ⁇ M* -proteinase complex can also bind to a signaling receptor ( ⁇ 2MSR), which stimulates cell growth by activating a signaling cascade that regulates cell proliferation (Misra et al, J. Biol. Chem. 272:497-502 (1997), herein inco ⁇ orated by reference in its entirety).
- ⁇ 2MSR signaling receptor
- oQM can also be activated by modification by monoamines (Barrett et al, 1981, supra).
- oQM* binds to growth factors with higher affinity further modulating their activity (by potentiating or suppressing their effects).
- o2MHyl and its splice variants are potential targets for regulating tumor cell proliferation, tissue invasion, cell migration and angiogenesis. Furthermore, the proteinase modulatory activity of o2M also makes o2MHy a potential therapy for suppressing harmful proteinase activity in conditions such as arthritis, emphysema, and wound healing.
- the present invention provides a novel approach for diagnosing and treating diseases and disorders associated with increased o2MHy expression.
- the method comprises administering an effective dose of targeting preparations, such as vaccines, antigen presenting cells (APCs), or pharmaceutical compositions comprising the targeting elements, such as o2MHy polypeptides, nucleic acids encoding o2MHy, anti- ⁇ 2MHy antibodies, o2MHy peptides, or binding polypeptides, peptides and small molecules that target o2MHy, described below.
- targeting preparations such as vaccines, antigen presenting cells (APCs), or pharmaceutical compositions comprising the targeting elements, such as o2MHy polypeptides, nucleic acids encoding o2MHy, anti- ⁇ 2MHy antibodies, o2MHy peptides, or binding polypeptides, peptides and small molecules that target o2MHy, described below.
- Targeting of o2MHy is expected to inhibit the growth of or destroy cells that express o_2MHy by a variety of mechanisms, such as removing o_2MHy via phagocytosis, altering the binding of o2MHy to growth factors and cytokines and effectively modulating their activities, inducing ADCC via binding of antibody-bound o_2MHy to the ⁇ SMHy receptor.
- An effective dose will be the amount of such targeting ⁇ 2MHy preparations necessary to target the o_2MHy and/or inhibit the growth of or destroy the o2MHy expressing cells and/or inhibit metastasis.
- o2MHy polypeptides and polynucleotides encoding such polypeptides are disclosed in co-owned U.S.
- 09/560,875, 09/540,217, and 09/496,914, inco ⁇ orated by reference herein in their entirety, relate in general to a collection or library of at least one novel nucleic acid sequences, specifically contigs, assembled from expressed sequence tags (ESTs), and specifically to novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.
- ESTs expressed sequence tags
- 09/560,875, 09/540,217, and 09/496,914 include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.
- o2M-like polypeptides disclose o2M-like polypeptides (herein denoted as o_2MHy), isolated polynucleotides encoding such polypeptides, including recombinant molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, fragments or analogs or variants of such polynucleotides or polypeptides, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, including polyclonal, monoclonal, single chain, bispecific, fragment, human and humanized antibodies, as well as hybridomas producing monoclonal antibodies, and diagnostic and therapeutic uses and screening assays associated with such polynucleotides, polypeptides and antibodies.
- Patent Applications Serial No. 09/756,247 and 09/684,711 are based on an oSM-like polynucleotide isolated from a cDNA library prepared from human fetal brain.
- o2MHyl is an approximately 1508 amino acid protein with a predicted molecular weight of 166 kD unglycosylated (SEQ ID NO: 6).
- the sequence corrected version of o2MHyl (SEQ ID NO: 47) is an approximately 1474 amino acid protein with a predicted molecular weight of 162 kD.
- a predicted approximately 17 residue signal peptide is encoded from approximately residue 1 to residue 17 of SEQ ID NO: 6 or 47.
- the signal peptide was predicted using the Kyte-Doolittle hydrophobicity prediction algorithm (J. Mol. Biol. 757:105-131 (1982), herein inco ⁇ orated by reference in their entirety).
- Kyte-Doolittle hydrophobicity prediction algorithm J. Mol. Biol. 757:105-131 (1982
- oMHyl is predicted to contain one alpha 2-macroglobulin family N-terminal region spanning amino acids 1 to 613 as well as two alpha 2-macroglobulin family signatures spanning amino acids 721 to 949 and amino acids 983 to 1469.
- o2MHy2 is an approximately 912 amino acid protein with a predicted molecular weight of 100 kD unglycosylated.
- a predicted approximately 31 residue signal peptide is encoded from approximately residue 1 to residue 31 of SEQ ID NO: 31. The signal peptide was predicted using the Kyte-Doolittle hydrophobicity prediction algorithm (J. Mol. Biol.
- oMHyl is predicted to contain one alpha 2-macroglobulin family N-terminal region spanning amino acids 14 to 626 as well as one alpha 2-macroglobulin family signature spanning amino acids 735 to 836.
- o_2MHy3 is an approximately 562 amino acid protein with a predicted molecular weight of 62 kD unglycosylated.
- a predicted approximately 17 residue signal peptide is encoded from approximately residue 1 to residue 17 of SEQ ID NO: 45.
- the signal peptide was predicted using the Kyte-Doolittle hydrophobicity prediction algorithm (J. Mol. Biol. 757:105-131 (1982), herein inco ⁇ orated by reference in their entirety).
- the actual cleavage site may be different than those predicted by the computer program.
- cMHyl is predicted to contain one alpha 2-macroglobulin family N-terminal region spanning amino acids 1 to 548.
- the o_2MHyl protein is highly and selectively expressed in certain non- hematopoietic (i.e. solid tumors) cancers, including lung, prostate, stomach, thymus, and testes, while most healthy cells fail to express o2MHyl or express it at low levels (see Table 7), strongly suggesting a role in facilitating tumor growth .
- solid tumors such as breast, colon, squamous cell or epithelial cell carcinomas that bear the o2MHy antigen can also be targeted.
- targeting oSMHy will have a minimal effect on healthy tissues while destroying or inhibiting the growth of certain cancer cells.
- Targeting ⁇ _2MHy may also be useful for treating and identifying hype ⁇ roliferative disorders, including hype ⁇ lasia, psoriasis, contact dermatitis, wound healing, arthritis, autoimmune diseases, heart disease, organ tissue rejection, and pro tease-related diseases, such as liver fibrosis.
- hype ⁇ lasia including hype ⁇ lasia, psoriasis, contact dermatitis, wound healing, arthritis, autoimmune diseases, heart disease, organ tissue rejection, and pro tease-related diseases, such as liver fibrosis.
- the present invention provides a vaccine comprising a oSMHy polypeptide to stimulate the immune system against o2MHy.
- a vaccine comprising a oSMHy polypeptide to stimulate the immune system against o2MHy.
- Targeting the ⁇ SMHy protein with a vaccine will abrogate its function as a modulator of cell growth and angiogenesis.
- o2M can covalently bind a variety of growth factors or growth inhibitory factors, such as human growth hormone (Adham, et al, Arch.
- TGF-/3 transforming growth factor-/3
- PDGF platelet derived growth factor
- TNF- ⁇ tumor necrosis factor
- bFGF basic fibroblast growth factor
- VEGF vascular endothelial growth factor
- cytokines such as interleukin (IL)-l
- IL-8 (Kurdowska, et al, J. Immunol. 158:1930-1940 (1997), all of which are hereby inco ⁇ orated by reference in their entirety), that are involved in modulating cell proliferation, tissue invasion, and angiogenesis.
- the o2MHy-factor complexes have several possible fates.
- One fate is that the complex will bind to LRP, be internalized, and degraded, having the effect of clearing the factor from the circulation and eliminating any growth modulatory effects (Gliemann and Davidsen, Biochim. Biophys. Acta 885:49-57
- Binding of o2MHy polypeptides or peptides to the surface of cells that display an increase in ⁇ SMHy polypeptide or nucleic acid expression may also occur (for example by binding to LRP or ⁇ 2MSR) and consequently be useful as an imrnunotherapeutic target.
- o2MHy bound to the surface of the tumor cells expressing it would target Q-2MHy-specific antibodies to those cells. This would result in immune cell or complement-mediated killing of the tumor cells.
- antibodies conjugated to cytotoxic moieties may be targeted to cells in this way resulting in direct tumor cell killing.
- ⁇ 2MHy molecules bound to a growth factor may facilitate binding of the growth factor to its cell surface receptor, thereby stimulating cell growth.
- Anti-0-2MHy antibodies would block or prevent the binding of the o2MHy- growth factor complex, inhibiting cell growth.
- o2MHy as an adjuvant to improve immunogenicity of vaccines is also contemplated.
- Covalently binding antigens to o2MHy or to amine or proteolytically activated o2MHy will likely more efficiently target antigens to antigen presenting cells (such as macrophages). This will have the effect of increasing the efficiency of antigen presentation and consequently enhance the immune response to the antigen. This would prove useful for generating better vaccines.
- Such a phenomenon has been observed with Hepatitis B virus antigens covalently associated with the o2M gene (Cianciolo et al, Vaccine, 20:554-562, (2002), inco ⁇ orated herein by reference in its entirety).
- o2MHy antibodies may be introduced into a patient such that the antibody binds to soluble o_2MHy polypeptides or peptides, thereby effectively removing o2MHy from the circulation (for example by phagocytosis), or by inhibiting the binding of o2MHy to its target molecules or receptors, resulting in a loss of its stimulatory effect or neutralizing its activity, and inhibiting the growth of the cells or the tumor.
- anti- ⁇ 2MHy antibodies may bind to the o2MHy receptor or associate with other cell surface proteins.
- oSMHy antibodies may be used as antibody therapy for solid tumors which express this antigen.
- ⁇ SMHy By inhibiting the cell proliferative and/or angiogeneic properties of ⁇ SMHy, tumor growth and tumor metastasis will be inhibited.
- ⁇ 2M* acts as a growth factor due in part to aberrant expression of the 0.2MSR (Asplin et al, Arch. Biochem. Biophys. 383:135-141 (2000) herein inco ⁇ orated by reference in its entirety).
- the oSMHy peptide can be used to target toxins or radioisotopes to tumor cells in vivo.
- o2MHy when conjugated to a toxin binds to target cells via the ⁇ 2MHy receptor and delivers the toxin.
- This peptide fragment can thereby provide a means to deliver cytotoxic agents to o2MHy-binding tumor cells or other cell targets. Much like an antibody, these fragments specifically target cells expressing this antigen. Targeted delivery of these cytotoxic agents to the tumor cells results in cell death and suppression of tumor growth.
- o_2MHy or fragments thereof can also be used to modulate immune cells that express the o2MHy receptor.
- receptor activation can lead to the stimulation of the release of cytokines (such as interferon gamma for example) that enhance or suppress the immune system. Additionally, binding of these fragments to cells bearing the ⁇ 2MHy receptor can lead to receptor activation and the stimulation of proliferation. Some fragments bind to the intact oSMHy receptor and block activation signals and cytokine release by immune cells, thereby exerting immunosuppressive effect.
- cytokines such as interferon gamma for example
- Fragments that activate and stimulate the immune system can have anti-tumor properties.
- fragments may stimulate an immunological response resulting in immune mediated tumor cell killing.
- the same fragments can stimulate the immune system to mount an enhance response to foreign invaders such as viruses and bacteria.
- Fragments that suppress the immune response are useful in treating proliferative disorders, auto-immune disease, graft-vs-host disease, and inflammatory disorders such as emphysema.
- ⁇ 2MHy peptide fragments or splice variants can inhibit o2MHy activity by interfering with oligomer formation.
- the fragments or splice variants compete with full- length c ⁇ MHy during oligomer formation resulting in incomplete oligomerization or hetero- oligomers and inhibition of o2MHy activity (Ayed et al, Nat.
- ⁇ 2MHy peptide fragments or splice variants can compete with full-length o2MHy for binding to the o_2MHy receptor or for binding to growth factors and/or cytokines resulting in modulating the activity of growth factor, cytokine, and/or ⁇ _2MHy receptors which will alter tumor cell growth and proliferation (Parish, et al, J. Steroid. Biochem. Mol. Biol. 79:165-172 (2001), Yusuf- Makagiansar, et al, Med. Res. Rev. 22:146-167 (2002), both of which are inco ⁇ orated herein by reference in their entirety).
- o2MHy peptide fragments or splice variants bind to and degrade proteinases that regulate cell growth and proliferation.
- Matrix metalloproteinases such as collagenases, gelatinases, and stromelysins, are required for tissue invasion and angiogenesis, and serve as a potential target of ⁇ 2MHy.
- Matrix metalloproteinases degrade extracellular matrix proteins which allow cells to migrate and pass through the basement membrane (Arthur, Path. Res. Pract. 190:825-833 (1994), herein inco ⁇ orated by reference in its entirety).
- ⁇ SMHy can transform to a receptor-specific, activated form, o2MHy*, by proteinase binding or by treatment with small nucleophiles, such as mefhylamine.
- o2MHy* can bind and regulate cell surface receptors, such as growth factor receptors, and affect cell growth by modulating their activity.
- o2M* has been shown to bind to trk, the nerve growth factor receptor and inhibit nerve growth factor (NGF)-stimulated trk signaling (Koo and Qiu, 1994, supra).
- NGF nerve growth factor
- o2MHy* can block or enhance the modulatory effect on secreted factors.
- Matrix metalloproteinases are involved in wound healing. Cytokines, such as IL-1/3 and growth factors, such as TGF-/3, regulate matrix metalloproteinase synthesis (Shaper et al, Dis. Colon Rectum. 44:1857-1866 (2001) herein inco ⁇ orated by reference in its entirety). Regulation of proteinase synthesis or direct inhibition of said proteinases (Ofuji et al, Periodontal Clin. Investig. 14:13-22 (1992); Santos et al, Br. J. Dermatol. 145:854-844 (2001) both of which are herein inco ⁇ orated by reference in their entirety) by o2MHy antibodies or peptides may facilitate wound healing.
- Matrix metalloproteinases are also involved in liver fibrosis resulting in the degradation of extracellular matrix (ECM) proteins (Arthur, 1994, supra) herein inco ⁇ orated by reference in its entirety).
- ECM extracellular matrix
- ⁇ 2MHy antibodies or peptides may block the digestion of extracellular matrix proteins and serve as a potent therapeutic for liver fibrosis and other ECM disorders.
- Stabilization of growth factors may be useful in the treatment of diseases in which growth factor turnover is rapid.
- increasing the stability and half-life of FGF-2 may facilitate angiogenesis following cardiac injury (Meij et al, Am. J. Physiol Heart Circ. Physiol 282:H547-H555 (2002) herein inco ⁇ orated by reference in its entirety), due to, for example, ischemia, myocardial infarction, or cardiac hypertrophy.
- 0-2MHy (or o2MHy*) inhibition of proteases such as elastin may be useful in treating emphysema.
- Immune cells in the lungs of patients with this condition secrete tissue damaging proteases that could be inhibited by an oSMHy therapeutic.
- Increasing expression levels of o2MHy through, e.g. gene therapy, is also expected to be beneficial in treating diseases that are due to increased protease activity.
- IGFBP insulin-like growth factor binding protein
- IGFBP-7 likely binds to insulin, thus increasing it stability and half life in the blood (Yamanaka et al, J. Biol. Chem. 272:30729-30734 (1997) herein inco ⁇ orated by reference in its entirety).
- the IGFBPs differ by molecular weight, amino acid composition, distribution in biological fluids, and influence upon IGF activity. They share a highly conserved N-terminal and C-terminal domain that contain 12 and 6 cysteine residues, respectively, and a variable middle domain.
- IGFBPs Approximately >97% of IGFs are bound by IGFBPs.
- IGFs are about 7.5-kDa single- chain protein homologues of insulin that can act locally, as autocrine or paracrine factors, or as endocrine growth factors that circulate in the plasma to act at distant sites.
- the IGFs can induce many responses that include mitogenesis within local tissue environments, induction of cellular differentiation, and metabolic effects such as increased amino acid uptake, and protein synthesis. They are synthesized and secreted by many tissues, although the primary sites of expression are liver, and to a lesser extent, bone.
- IGFBP-7 blocks insulin binding to the insulin receptor, and thereby inhibits the earliest steps in insulin action, such as autophosphorylation of the insulin beta subunit and phosphorylation of IRS-1 (Yamanaka et al. 1997, supra). Due to its ability to bind insulin with high affinity, IGFBP-7 might also be involved in pregnancy induced insulin resistance and type II diabetes mellitus.
- IGFs play a role in stimulating growth
- their attenuation by IGFBP binding has been suggested as a mechanism to prevent tumor growth.
- increased concentrations of IGFBP-3 inhibit the proliferation of the breast cancer cell line
- IGFBPs possibly work as antimitogens.
- Free IGFBP-3 may also bind to
- IGFBP-3 receptors on cancer cells and inhibit tumor cell growth, as well as induce apoptosis in an IGF-independent manner (Grimberg and Cohen, J. Cell Physiol. 183:1-9 (2000) herein inco ⁇ orated by reference in its entirety). Circulating IGFBP-3 levels are also correlated with cancer risk. Prospective studies have shown that low levels of IGFBP-3 were associated with a doubled risk of prostate cancer, a fourfold increased risk of colorectal neoplasia, and a higher risk of breast and lung cancer (Giovannucci, Horm Res. 3:34-41
- IGFBP-7 has been shown to be down-regulated at the transcription level in carcinoma cell lines, suggesting this member has a tumor suppressor activity (Swisshelm et al, Proc. Natl. Acad. 92:4472-4476
- IGFs and IGFBPs are also involved in tissue remodeling. Because IGFBP-5 associates with the ECM and releases bound IGF at those sites, it induces tissue-specific cell proliferation and differentiation. In arthritis, proinflammatory cytokines such as TNF- , IL- l ⁇ , and IL-l ⁇ cause the release of IGFBP-3, and IGFBP-5. These do not associate with the ECM and suppress IGF-I induced proteoglycan synthesis. Decreased proteoglycan synthesis coupled with degradation of cartilage matrix, allows breakdown of cartilage between jointsf In addition, IGFBP-5 has also been implicated in bone remodeling, and tissue remodeling of the involuting mammary gland.
- IGFBP-1 and IGFBP-3 also regulate wound healing.
- Nonphosphorylated IGFBP-1 enhances wound-breaking strength and re-epithelialization, a response that IGF alone cannot elicit. This suggests that IGFBP-1 accelerates wound healing by enhancing IGF-1 action, and may stimulate cell migration in an IGF-independent manner.
- IGFBP-3 protease activity is increased following surgery and during chronic illnesses. Therefore, reductions in IGFBP-3 may allow increased IGF at tissue sites and contribute to increased metabolism and cellular division after insult.
- IGFBPs also modulate the actions of IGFs on female reproductive function by synergizing with pituitary gonadotropins and ovarian steroid hormones. At various sites in the female reproductive tract, small changes (ove ⁇ roduction or deficiency) of IGFBPs may result in pathological conditions such as anovulation and hyperandrogenism, and inadequate differentiation of the endometrium (Wang and Chard, J. Endocrinol. 161 :1-13 (1999) herein inco ⁇ orated by reference in its entirety).
- IGFBP-1 is an important modulator of IGF- 1 activity. Maternal IGF-I promotes fetal growth and stimulates nutrient transport in the placenta. The presence of nonphosphorylated IGFBP-1, which has a decreased affinity for IGF-I, appears to enhance these activities of IGF-I and promote fetal growth.
- IGFBP-7Hyl an IGFBP-7 homolog
- IGFBP-7Hyl an IGFBP-7 homolog
- IGFBP-7Hyl is an approximately 278 amino acid protein with a predicted molecular weight of 31 kD unglycosylated. A predicted approximately 27 residue signal peptide is encoded from approximately residue 1 to residue 27 of SEQ ID NO: 54. The signal peptide was predicted using the Kyte-Doolittle hydrophobicity prediction algorithm (J. Mol. Biol. 757:105-131 (1982), herein inco ⁇ orated by reference in their entirety). One of skill in the art will recognize that the actual cleavage site may be different than those predicted by the computer program. Using the eMATRLX software program (Stanford University, CA; Wu et al, J. Comp. Biol. 6:219-235 (1999), herein inco ⁇ orated by reference in its entirety), IGFBP-7Hyl is predicted to contain one insulin-like growth factor binding protein signature spanning amino acids 61 to 76.
- Determining the status of IGFBP-7Hyl expression patterns in an individual may be used to diagnose cancer and may provide prognostic information useful in defining appropriate therapeutic options. Similarly, the expression status of IGFBP-7Hyl may provide information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness.
- the invention provides methods and assays for determining IGFBP-7Hyl expression status and diagnosing cancers that express IGFBP- 7Hyl.
- the invention provides assays useful in determining the presence of cancer in an individual, comprising detecting a significant increase or decrease in IGFBP- 7Hyl mRNA or protein expression in a test cell or tissue or fluid sample relative to expression levels in the corresponding normal cell or tissue.
- the corresponding normal cell or tissue may be from the same subject or from a different subject.
- the presence of IGFBP-7Hyl mRNA is evaluated in tissue samples of a lymphoma.
- the presence of significant IGFBP-7Hyl expression may be useful to indicate whether the lymphoma is susceptible to IGFBP-7Hyl immunotargeting.
- IGFBP-7Hyl expression status may be determined at the protein level rather than at the nucleic acid level.
- such a method or assay would comprise determining the level of IGFBP-7Hyl protein expressed by cells in a test tissue sample and comparing the level so determined to the level of IGFBP-7Hyl protein expressed in a corresponding normal sample.
- the presence of IGFBP-7Hyl is evaluated, for example, using immunohistochemical methods.
- IGFBP-7Hyl antibodies capable of detecting IGFBP-7Hyl expression may be used in a variety of assay formats well known in the art for this pu ⁇ ose.
- Peripheral blood may be conveniently assayed for the presence of cancer cells, using RT-PCR to detect IGFBP-7Hyl expression.
- the presence of RT-PCR amplifiable IGFBP- 7Hyl mRNA provides an indication of the presence of different types of cancer.
- a sensitive assay for detecting and characterizing carcinoma cells in blood may be used (Racila, et al, Proc. Natl. Acad. Sci. USA 95: 4589-4594 (1998) herein inco ⁇ orated by reference in its entirety). This assay combines immunomagnetic enrichment with multiparameter flow cytometric and immunohistochemical analyses, and is highly sensitive for the detection of cancer cells in blood, reportedly capable of detecting one epithelial cell in 1 ml of peripheral blood.
- a related aspect of the invention is directed to predicting susceptibility to developing cancer in an individual.
- a method for predicting susceptibility to cancer comprises detecting IGFBP-7Hyl mRNA or IGFBP-7Hyl in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of IGFBP-7Hyl mRNA expression present is proportional to the degree of susceptibility.
- IGFBP family are known to inhibit cell growth and tumorigenicity
- IGFBP-7Hyl may act as a tumor suppressor. Reduced mRNA expression was detected in stomach, colon and ovarian tumor tissue when compared to the corresponding normal tissue (see Tables 9, 10, and 11). Additionally, transfection of human cervical carcinoma cells with IGFBP-7-Hyl resulted in a significant reduction of cell growth (see Table 12). Growth of the carcinoma cell lines HeLa (cervical carcinoma) and
- compositions of IGFBP-7Hyl polypeptides, fragments or variants thereof can be used to inhibit tumor cell growth.
- Toll and Toll-like receptors are type I transmembrane proteins with extracellular leucine-rich repeat motifs and an intracellular signaling domains.
- the Toll-like receptors make up a family of human receptors which have common structural features with the Drosophila Toll (dToll) receptor molecule. They are found on the surface of several types of hematopoietic cells.
- Human Toll-like receptors are also expressed on antigen presenting cells, such as monocytes and dendritic cells (WO 01/55386 Al, herein inco ⁇ orated by reference in its entirety).
- TLR-2 Two human colon cancer cell lines (DLD and LoVo) showed expression of the Toll-like receptor subtype TLR-2, whereas the Toll-like receptor subtype TLR-4 was expressed in human hepatocellular carcinoma (PLC/PRF/5) and acute myeloid leukemia (KG-1) cells (Yoshioka, et al, J. Int. Med. Res. 29:409-420 (2001), herein inco ⁇ orated by reference in its entirety).
- PLC/PRF/5 human hepatocellular carcinoma
- KG-1 acute myeloid leukemia
- Toll-like receptors Both dToll and human Toll-like receptors are thought to act as pattern recognition receptors for bacteria and other microorganisms, and play a role in immune surveillance mechanisms and innate immunity. Toll-like receptors can trigger pro-inflammatory cytokine production and induce expression of cell surface co-stimulatory receptors for T-cell activation. Some human Toll-like receptors may be involved in co-ordination of interactions between immune cells resulting in an integrated immune response to infection. TLR2 and
- TLR4 have been shown to mediate host responses to Gram-positive and Gram-negative bacteria through recognition of specific bacterial wall components. TLR4 mediates responses to certain viral proteins such as respiratory syncytial virus. Toll-like receptors may also form heterodimeric functional complexes and share in common signal transduction pathways with IL-1 receptors. Activation of TLR2 and TLR4 leads to the activation of
- NFKB via an adapter protein MyD88 and recruitment of the IL-1 receptor-associated kinases
- TLR9 Toll-like Receptor 9
- CpG deoxy-cytidylate-phosphate-deoxyguanylate
- CpG DNA may be recognized by autoantibodies inducing an autoimmune response (Ichikawa et al, J. Immunol. 169:2781-2787 (2002); Leadbetter et al,
- agents that block TLR9 and other Toll-like receptor proteins may be useful in treating autoimmune disorders.
- disorders associated with the proliferation of TLR9-expressing cells include cancers, such as non-Hodgkin's B-cell lymphomas, B-cell leukemias, chronic lymphocytic leukemia, multiple myeloma, acute and chronic myeloid leukemia; myelodysplastic syndromes; T cell lymphomas, acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, lymphosarcoma leukemia, malignant lymphoma, B cell large cell lymphoma, X-linked lymphoproliferative disorders; Epstein Barr Virus-related conditions such as mononucleosis; and autoimmune disorders.
- cancers such as non-Hodgkin's B-cell lymphomas, B-cell leukemias, chronic lymphocytic leukemia, multiple myeloma, acute and chronic myeloid leukemia; myelodysplastic syndromes;
- Non-hematopoietic tumors that bear the TLR9 antigen such as prostate, breast, colon, and squamous cell carcinoma, as well as other cancers of epithelial and squamous cell origin, can also be targeted.
- the invention further provides a method of modulating the immune system by either suppression or stimulation of growth factors and cytokines, by administering the targeting elements or compositions of the invention.
- the invention also provides a method of modulating the immune system through activation of immune cells (such as natural killer cells, T cells, B cells and myeloid cells), through the suppression of activation, or by stimulating or suppressing proliferation of these cells by
- TLR9 peptide fragments or TLR9 antibodies are provided.
- TLR9 polypeptides and polynucleotides encoding such polypeptides are disclosed in co-owned U.S. Patent Application No. 09/687,527, which corresponds to PCT Publication No. WO 02/31111. These and all other U.S. patents cited herein are hereby inco ⁇ orated by reference in their entirety. U.S. Patent Application Serial No.
- 09/687,527 herein inco ⁇ orated by reference in its entirety, relates, in general, to novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variant thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.
- PCT Publication No. WO 01/55386 inco ⁇ orated herein by reference in its entirty discloses a Toll-like receptor and its use in screening for novel pharmacotherapeutic agents with immunomodulatory activity.
- WO 01/55386 discloses isolated Toll-like receptor polypeptides, polynucleotides encoding for the Toll-like receptor polypeptide, expression vectors comprising such polynucleotides, hosts cells comprising such expression vectors, antibodies specific for the Toll-like receptor polypeptide, methods for identification of compounds that modulate Toll-like receptor activity, and methods of treating disorders responsive to toll-like receptor modulation.
- WO 99/20756 discloses human Toll homolog polypeptides, polynucleotides encoding for the human Toll homologs, expression vectors comprising such polynucleotides, host cells comprising such expression vectors, antibodies specific for the human Toll homolog polypeptides, antibodies that specifically bind to a human TLR2 receptor, and methods for treating septic shock using anti-Toll homolog antibodies.
- TLR9 is an approximately 1032 amino acid protein with a predicted molecular weight of 114 kD unglycosylated.
- a predicted approximately 25 residue signal peptide is encoded from approximately residue 1 to residue 25 of SEQ ID NO: 64.
- a predicted transmembrane domain is encoded from amino acid 804 to amino acid 846 of SEQ ID NO: 64.
- the signal peptide and transmembrane region were predicted using the Kyte-Doolittle hydrophobicity prediction algorithm (J. Mol. Biol.
- TLR9 is predicted to contain 20 leucine rich regions (LRR domains) spanning amino acids 64-87, 124-140, 144-167, 200-219, 221-244, 245-284, 309-334, 335-344, 365-388, 416-496, 497-519, 522-545, 546-556, 576-587, 599- 628, 629-652, 654-677, 678-698, 702-725, and 726-749; one leucine-rich region C-terminal domain (LRRCT) spanning amino acids 760-802; and one TIR domain spanning amino acids 872 to 973.
- LRR domains leucine rich regions spanning amino acids 64-87, 124-140, 144-167, 200-219, 221-244, 245-284, 309-334, 335-344, 365-388, 416-496, 497-519, 522-545, 546-556, 576-587, 599- 628, 629-652, 654-677,
- Toll-like family of receptors were shown to be expressed on antigen presenting cells, such as monocytes and dendritic cells (WO 01/55386 Al), human colon cancer cell lines (DLD and LoVo), human hepatocellular carcinoma (PLC/PRF/5) and acute myeloid leukemia (KG-1) cells (Yoshioka, et al, 2001, supra).
- antigen presenting cells such as monocytes and dendritic cells (WO 01/55386 Al), human colon cancer cell lines (DLD and LoVo), human hepatocellular carcinoma (PLC/PRF/5) and acute myeloid leukemia (KG-1) cells (Yoshioka, et al, 2001, supra).
- TLR9 expression is up-regulated in hematopoietic cancers including Burkitt's lymphoma, diffuse lymphoma, B cell lymphoma, follicular lymphoma, diffuse large B cell lymphoma, T cell lymphoma, acute myeloid leukemia, promyelomonocytic leukemia, and Hodgkin's disease (see Tables 14 and 15).
- TLR9 Immunohistochemical (IHC) analysis demonstrated that TLR9 is expressed in the following leukemias and lymphomas: acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell large cell lymphoma, malignant lymphoma, acute leukemia, and lymphosarcoma cell leukemia.
- leukemias and lymphomas acute myelogenous leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell large cell lymphoma, malignant lymphoma, acute leukemia, and lymphosarcoma cell leukemia.
- TLR9 is also expressed in solid tumors, such as prostate, breast, colon and squamous cell carcinoma (see Table 17).
- TLR9 is expressed in these epithelial and squamous cell cancers, it is likely that TLR9 will be expressed in other cancers of epithelial and squamous cell origin. Thus, targeting cells expressing TLR9 will be useful in treating and diagnosing these and other cancers. IHC analysis showed that TLR9 is also expressed in autoimmune disorders, including systemic lupus erythematosus; Hasimoto thyroiditis,
- TLR9 or reducing expression of TLR9 may be useful in treating these and other autoimmune disorders.
- IHC analysis showed that TLR9 is expressed in rejected transplanted heart, kidney and liver tissues, whereas normal tissues do not express TLR9 (see Table 19).
- targeting TLR9 or reducing expression of TLR9 through, e.g. antisense or gene therapy will be useful in reducing and/or eliminating tissue rejection after transplantation.
- BCR B cell receptor
- the B cell receptor (BCR) is found on the cell surface of mature B cells and comprises a membrane- bound antigen-binding subunit, containing two heavy chains, ⁇ , and two light chains, as well as a signaling subunit, composed of a disulfide-linked heterodimer of Ig ⁇ (CD79a) and Ig/3 (CD79b) (Matsuuchi and Gold, Curr. Opin. Immunol. 13:270-277 (2001), inco ⁇ orated herein by reference in its entirety).
- Pre-B cells have an altered version of the BCR, the pre- BCR, which contains surrogate light chains, composed of the ⁇ 5 and VpreBl proteins, instead of the conventional light chains bound to the ⁇ heavy chains.
- the pre-BCR is essential for the survival and differentiation of pre-B cells into mature B cells (Matsuuchi and Gold, 2001, supra).
- Expression of ⁇ 5 or VpreBl can be used to identify pre-B cells as well as malignancies of pre-B cells lineage, such as pre-B acute lymphoblastic leukemia (Bauer, et al. Blood 78:1581-1588 (1991); Schiff, et al, Blood 78:1516-1525 (1991), both of which are inco ⁇ orated herein by reference in their entirety).
- pre-B cell receptor is expressed on the surface of pre-B cells and is comprised of two heavy chains, ⁇ , and two surrogate light chains, ⁇ 5 and VpreBl.
- the pre- BCR is essential for the survival and differentiation of pre-B cells into mature B cells (Matsuuchi and Gold, 2001, supra).
- ⁇ 5 and VpreBl are expressed in predominantly in pre-B cells.
- VpreBl expression can be used to identify pre-B cells as well as malignancies of pre- B cell lineage, such as pre-B acute lymphoblastic leukemia (ALL) (Bauer, 1991, supra); Schiff, 1991, supra).
- ALL pre-B acute lymphoblastic leukemia
- EP 269,127 herein inco ⁇ orated by reference in its entirety, discloses that VpreBl polynucleotides are uniquely expressed in pre-B cells and not in mature B cells, plasma cells, T cells or myeloid cells (see, e.g. Figs. 1 and 2 of EP 269,127), and suggests using such polynucleotides or antibodies to identify or detect pre-B cells from a mixed population of human lymphocytes.
- the present invention specifically excludes diagnosis and optionally excludes therapy of pro-B cell and pre-B cell ALL and optionally excludes diagnosis and therapy of B cell hype ⁇ roliferative diseases of pro- and pre-B cell lineage.
- a preferred embodiment of the invention is the diagnosis and therapy of hype ⁇ roliferative diseases of mature B cell lineage, T cell lineage, and myeloid cell lineages.
- the method comprises administering an effective dose of targeting preparations such as vaccines, antigen presenting cells, or pharmaceutical compositions comprising the targeting elements, VpreBl polypeptides, nucleic acids encoding VpreBl, anti-VpreBl antibodies, VpreBl polypeptides and peptide fragments, and small molecules that bind to or recognize VpreBl, described below.
- Targeting of VpreBl on the cell membranes of VpreBl -expressing cells is expected to inhibit the growth of or destroy such cells.
- An effective dose will be the amount of such targeting VpreBl preparations necessary to target the VpreBl on the cell membrane and inhibit the growth of or destroy the VpreBl -expressing cells and/or metastasis.
- a further embodiment of the present invention is to enhance the effects of therapeutic agents and adjunctive agents used to treat and manage disorders associated with VpreBl - expressing cells of mature B cell, T cell, or myeloid cell lineage, by administering VpreBl preparations with therapeutic and adjuvant agents commonly used to treat such disorders.
- VpreBl is an approximately 145 amino acid protein with a molecular weight of approximately 16 kD unglycosylated.
- An approximately 19 residue signal peptide is encoded from approximately residue 1 to residue 19 of SEQ ID NO: 65.
- VpreBl is expressed in certain hematopoietic-based cancers, including Burkitt's lymphoma, B cell lymphoma, follicular lymphoma, diffuse large B cell lymphoma, marginal zone B cell lymphoma, anaplastic T cell lymphoma, multiple myeloma, and T cell leukemia, while most non- hematopoietic, healthy cells fail to express VpreBl (see Tables 20 and 21). Thus, targeting VpreBl will have a minimal effect on healthy tissue while destroying or inhibiting the growth of the hematopoietic-based cancer cells.
- Figure 4 shows the cell surface expression of VpreBl on B cell non-Hodgkin's lymphoma cell lines.
- CA46, GA-10 and HT B cell non Hodgkin's lymphoma cell lines were stained with an anti- VpreBl antibody (Serotec, Inc., Raleigh, NC) conjugated with FITC (white fill graph) or with a non-specific isotype control (black fill graph) antibody (Pharmingen, Inc., San Diego, CA).
- VpreBl -FITC antibody labeling is shown on the x-axis compared to the number of cells labeled on the y-axis.
- VpreBl is expressed on the cell surface of non-Hodgkin's lymphoma cell lines of mature B cell lineage and may be targeted to treat these and other hematopoietic cancers arising from mature B cell, T cell or myeloid cell lineages.
- fragment of a nucleic acid refers to a sequence of nucleotide residues which are at least about 5 nucleotides, more preferably at least about 7 nucleotides, more preferably at least about 9 nucleotides, more preferably at least about 11 nucleotides and most preferably at least about 17 nucleotides.
- the fragment is preferably less than about 500 nucleotides, preferably less than about 200 nucleotides, more preferably less than about 100 nucleotides, more preferably less than about 50 nucleotides and most preferably less than 30 nucleotides.
- the fragments can be used in polymerase chain reaction (PCR), various hybridization procedures or microarray procedures to identify or amplify identical or related parts of mRNA or DNA molecules.
- a fragment or segment may uniquely identify each polynucleotide sequence of the present invention.
- the fragment comprises a sequence substantially similar to a portion of SEQ ID NO: 1, 3-5, 7, 28-30, 32, 44, 46, 49- 53, 55, 63, or 65.
- a polypeptide "fragment " is a stretch of amino acid residues of at least about 5 amino acids, preferably at least about 7 amino acids, more preferably at least about 9 amino acids and most preferably at least about 17 or more amino acids.
- the peptide preferably is not greater than about 200 amino acids, more preferably less than 150 amino acids and most preferably less than 100 amino acids.
- the peptide is from about 5 to about 200 amino acids.
- the peptide fragment comprises a sequence substantially similar to a portion of SEQ ID NO: 2, 6, 8-27, 31, 33-43, 45, 47, 54, 56-60, 64, or 66.
- any polypeptide must have sufficient length to display biological and/or immunological activity.
- immunological activity refers to the capability of the natural, recombinant or synthetic peptide of the invention, or any peptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
- variant refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using, e g., recombinant DNA techniques.
- Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequence.
- recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the "redundancy" in the genetic code.
- Various codon substitutions such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system.
- Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.
- the present invention provides a vaccine comprising a cell surface antigen (CSA) of the invention polypeptide to stimulate the immune system against said CSA, thus targeting cells expressing said CSA.
- CSA cell surface antigen
- Toll-like receptor proteins as adjuvants in vaccine preparations has been previously described (PCT Publication No. WO 01/55386; Kovarik and Siegrist, Arch. Immunol. Ther. Exp. (Warsz) 49:209-215 (2001); Azuma and Seya, Int. Immunopharmacol 1:1249-1259 (2001), all of which are herein inco ⁇ orated by reference in their entirety).
- tumor-specific vaccine uses purified idiotype protein isolated from tumor cells, coupled to keyhole limpet hemocyanin (KLH) and mixed with adjuvant for injection into patients with low-grade follicular lymphoma (Hsu, et al, Blood 89: 3129-3135 (1997), herein inco ⁇ orated by reference in its entirety).
- KLH keyhole limpet hemocyanin
- No. 6,312,718, herein inco ⁇ orated by reference in its entirety, describes methods for inducing immune responses against malignant B cells, in particular lymphoma, chronic lymphocytic leukemia, and multiple myeloma.
- the methods described therein utilize vaccines that include liposomes having (1) at least one B-cell malignancy-associated antigen,
- Methods of vaccinating against a CSA of the invention typically employ a CSA polypeptide, including fragments, analogs and variants.
- dendritic cells one type of antigen-presenting cell
- a cellular vaccine in which the dendritic cells are isolated from the patient, co-cultured with tumor antigen and then reinfused as a cellular vaccine (Hsu, et al, Nat. Med. 2:52-58 (1996), herein inco ⁇ orated by reference in its entirety).
- a nucleic acid encoding a cell surface antigen (CSA) of the invention, or encoding a fragment, analog or variant thereof, within a recombinant vector is utilized.
- CSA cell surface antigen
- Such methods are known in the art.
- immune responses can be induced by injection of naked DNA. Plasmid DNA that expresses bicistronic mRNA encoding both the light and heavy chains of tumor idiotype proteins, such as those from B cell lymphoma, when injected into mice, are able to generate a protective, anti-tumor response (Singh, et al, Vaccine 20:1400-1411 (2002), inco ⁇ orated herein by reference in its entirety).
- Viral vectors comprising a CSA of the invention are particularly useful for delivering nucleic acids encoding a cell-surface antigen of the invention to cells.
- vectors include those derived from influenza, adenoviras, vaccinia, he ⁇ es symplex viras, fowlpox, vesicular stomatitis viras, canarypox, poliovirus, adeno-associated virus, and lentivirus and Sindbus viras.
- non-viral vectors such as liposomes or even naked DNA, are also useful for delivering nucleic acids encoding a CSA of the invention to cells.
- a vector comprising a nucleic acid encoding a CSA polypeptide is introduced into a cell, such as a dendritic cell or a macrophage.
- a cell such as a dendritic cell or a macrophage.
- APC antigen-presenting cell
- the cell surface antigens are presented to T cells eliciting an immune response against said CSA.
- APCs are loaded with a CSA of the invention or a nucleic acid encoding a CSA of the invention ex vivo and then introduced into a patient to elicit an immune response against said CSA.
- the cells presenting an antigen of a CSA are used to stimulate the expansion of anti-CSA cytotoxic T lymphocytes (CTL) ex vivo followed by introduction of the stimulated CTL into a patient (U.S. Patent No. 6,306,388, inco ⁇ orated herein by reference in its entirety).
- CTL cytotoxic T lymphocytes
- antisense nucleic acid molecules that can hybridize to, or are complementary to, the nucleic acid molecule comprising the CSA nucleotide sequence, or fragments, analogs or derivatives thereof.
- An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence).
- antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire CSA coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a CSA or antisense nucleic acids complementary to a CSA nucleic acid sequence of are additionally provided.
- an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding a CSA protein.
- coding region refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.
- the antisense nucleic acid molecule is antisense to a "conceding region" of the coding strand of a nucleotide sequence encoding the CSA protein.
- the term “conceding region” refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and
- antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.
- the antisense nucleic acid molecule can be complementary to the entire coding region of CSA mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of CSA mRNA.
- the antisense oligonucleotide can be complementary to the region surrounding the translation start site of CSA mRNA.
- An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length.
- an antisense nucleic acid of the invention can be constmcted using chemical synthesis or enzymatic ligation reactions using procedures known in the art.
- an antisense nucleic acid e.g., an antisense oligonucleotide
- an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used).
- modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmefhyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethyl guanine, 2-methyladenine, 2-methylguanine, 3- methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,
- the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be ofan antisense orientation to a target nucleic acid of interest, described further in the following section).
- the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a CSA protein to thereby inhibit expression of the protein (e.g., by inhibiting transcription and/or translation).
- the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.
- An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.
- antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
- antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens).
- the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
- the antisense nucleic acid molecule of the invention is an alpha-anomeric nucleic acid molecule.
- An alpha-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual alpha-units, the strands run parallel to each other. See, e.g., Gaultier, et al, Nucl. Acids Res. 15: 6625-6641 (1987).
- the antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (see, e.g., Inoue, et al, Nucl. Acids Res.
- RNA-DNA analogue see, e.g., Inoue, et al, FEBSLett. 215: 327-330 (1987), all of which are herein inco ⁇ orated by reference in their entirety.
- Mutations in the polynucleotides of the invention gene may result in loss of normal function of the encoded protein.
- the invention thus provides gene therapy to restore normal activity of the polypeptides of the invention; or to treat disease states involving polypeptides of the invention.
- Delivery of a functional gene encoding polypeptides of the invention to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated viras, or a retroviras), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments).
- viral vectors e.g., adenovirus, adeno-associated viras, or a retroviras
- physical DNA transfer methods e.g., liposomes or chemical treatments.
- any one of the nucleotides of the present invention or a gene encoding the polypeptides of the present invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression).
- Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic pu ⁇ oses.
- polypeptides of the invention in other human disease states, preventing the expression of or inhibiting the activity of polypeptides of the invention will be useful in treating the disease states. It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of polypeptides of the invention.
- the present invention still further provides cells genetically engineered in vivo to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell. These methods can be used to increase or decrease the expression of the polynucleotides of the present invention.
- D ⁇ A sequences allows for modification of cells to permit, increase, or decrease, expression of endogenous polypeptide.
- Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the protein at higher levels.
- the heterologous promoter is inserted in such a manner that it is operatively linked to the desired protein encoding sequences. See, for example, PCT International Publication No. WO 94/12650, PCT International Publication No. WO 92/20808, and PCT International Publication No.
- amplifiable marker DNA e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase
- intron DNA may be inserted along with the heterologous promoter DNA. If linked to the desired protein coding sequence, amplification of the marker DNA by standard selection methods results in co- amplification of the desired protein coding sequences in the cells.
- cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.
- gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.
- regulatory sequences may be comprised of promoters, enhancers, scaffold- attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.
- sequences which affect the stracture or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting.
- sequences include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.
- the targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.
- the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.
- the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell- type specificity than the naturally occurring elements.
- the naturally occurring sequences are deleted and new sequences are added.
- the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the cell genome.
- the identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous
- Markers useful for this pu ⁇ ose include the He ⁇ es Simplex Viras thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.
- TK He ⁇ es Simplex Viras thymidine kinase
- gpt bacterial xanthine-guanine phosphoribosyl-transferase
- immunotargeting involves the administration of components of the immune system, such as antibodies, antibody fragments, or primed cells of the immune system against the target.
- components of the immune system such as antibodies, antibody fragments, or primed cells of the immune system against the target.
- Methods of immunotargeting cancer cells using antibodies or antibody fragments are well known in the art.
- U.S. Patent No. 6,306,393 describes the use of anti-CD22 antibodies in the immunotherapy of B-cell malignancies
- U.S. Patent No. 6,329,503 describes immunotargeting of cells that express se ⁇ entine transmembrane antigens (both U.S. patents are herein inco ⁇ orated by reference in their entirety).
- Antibodies recognizing a cell surface antigen of the invention may be introduced into a patient such that the antibody binds to said CSA expressed by cancer cells and mediates the destruction of the cells and the tumor and/or inhibits the growth of the cells or the tumor.
- mechanisms by which such antibodies can exert a therapeutic effect may include complement-mediated cytolysis, antibody-dependent cellular cytotoxicity (ADCC), modulating the physiologic function of said cell-surface antigen of the invention, inhibiting binding or signal transduction pathways, modulating tumor cell differentiation, altering tumor angiogenesis factor profiles, modulating the secretion of immune stimulating or tumor suppressing cytokines and growth factors, modulating cellular adhesion, and/or by inducing apoptosis.
- ADCC antibody-dependent cellular cytotoxicity
- Antibodies recognizing a cell-surface antigen of the invention which are conjugated to toxic or therapeutic agents, such as radioligands or cytosolic toxins, may also be used therapeutically to deliver the toxic or therapeutic agent directly to tumor cells that bear said CSA.
- Antibodies recognizing a cell surface antigen of the invention may be used to suppress the immune system in patients receiving organ transplants or in patients with autoimmune diseases such as arthritis. Healthy immune cells would be targeted by these antibodies leading their death and clearance from the system, thus suppressing the immune system.
- Antibodies that recognize a cell surface antigen of the invention may be used as antibody therapy for solid tumors which express this antigen.
- Cancer immunotherapy using antibodies provides a novel approach to treating cancers associated with cells that specifically express a cell-surface antigen of the invention.
- the cell surface antigens of the invention are expressed in cancers of both hematopoietic and non- hematopoietic (i.e. solid tumors) origin as well as in autoimmune diseases and in rejected transplanted organs and tissues.
- cell surface antigens of the invention may be used as therapeutic targets and diagnostic markers for certain cell types or disorders (i.e., B-cell lymphomas, T cell lymphomas, myeloid leukemia, Hodgkin's disease).
- Cancer immunotherapy using antibodies has been previously described for other types of cancer, including but not limited to colon cancer (Arlen et al, Crit. Rev. Immunol. 18:133- 138 (1998)), multiple myeloma (Ozaki et al, Blood 90:3179-3186 (1997); Tsunenari et al, Blood 90:2437-2444 (1997)), gastric cancer (Kasprzyk et al, Cancer Res.
- antibody therapy of the invention may be useful for all stages of the foregoing cancers, antibody therapy may be particularly appropriate in advanced or metastatic cancers. Combining the antibody therapy method with a chemotherapeutic, radiation or surgical regimen may be preferred in patients that have not received chemotherapeutic treatment, whereas treatment with the antibody therapy may be indicated for patients who have received one or more chemotherapies. Additionally, the antibody therapy of the invention can also enable the use of reduced dosages of concomitant chemotherapy, particularly in patients that do not tolerate the toxicity of the chemotherapeutic agent very well. Furthermore, treatment of cancer patients with the antibody therapy of the invention with tumors resistant to chemotherapeutic agents might induce sensitivity and responsiveness to these agents in combination.
- a patient Prior to immunotargeting a cell surface antigen of the invention, a patient may be evaluated for the presence and level of expression of said CSA by the diseased cells, preferably using immunohistochemical assessments of tumor tissue, quantitative imaging, quantitative RT-PCR, or other techniques capable of reliably indicating the presence and degree of cell surface antigen of the invention expression.
- a blood or biopsy sample may be evaluated by immunohistochemical methods to determine the presence of cells expressing a cell surface antigen of the invention or to determine the extent of expression of a cell surface antigen of the invention on the surface of the cells within the sample. Methods for immunohistochemical analysis of tumor or other tissues or released fragments of a cell surface antigen of the invention in the serum are well known in the art.
- Antibodies recognizing a cell surface antigen of the invention are useful in treating cancers including those, which are capable of initiating a potent immune response against the tumor and those, which are capable of direct cytotoxicity.
- monoclonal antibodies (mAbs) specific to a cell surface antigen of the invention may elicit tumor cell lysis by either complement-mediated or ADCC mechanisms, both of which require an intact Fc portion of the immunoglobulin molecule for interaction with effector cell Fc receptor sites or complement proteins.
- antibodies specific to a cell surface antigen of the invention that exert a direct biological effect on tumor growth are useful in the practice of the invention.
- cytotoxic antibodies may act include inhibition of cell growth, modulation of cellular differentiation, modulation of tumor angiogenesis factor profiles, and the induction of apoptosis.
- the mechanism by which a particular antibody specific to a cell-surface antigen of the invention exerts an anti-tumor effect may be evaluated using any number of in vitro assays designed to determine ADCC, ADMMC, complement-mediated cell lysis, and so forth, as is generally known in the art.
- the anti-tumor activity of a particular anti-CSA antibody, or combination of anti- CSA antibody may be evaluated in vivo using a suitable animal model.
- xenogenic lymphoma cancer models wherein human lymphoma cells are introduced into immune compromised animals, such as nude or SCID mice. Efficacy may be predicted using assays, which measure inhibition of tumor formation, tumor regression or metastasis, and the like.
- preferred monoclonal antibodies used in the practice of the therapeutic methods of the invention are those which are either fully human or humanized and which bind specifically to the target cell surface antigen of the invention with high affinity but exhibit low or no antigenicity in the patient.
- the method of the invention contemplates the administration of single anti-CSA mAbs as well as combinations, or "cocktails", of different mAbs.
- Two or more monoclonal antibodies that bind to a cell surface antigen of the invention may provide an improved effect compared to a single antibody.
- a combination of an anti-CSA antibody with an antibody that binds a different antigen may provide an improved effect compared to a single antibody.
- Such mAb cocktails may have certain advantages inasmuch as they contain mAbs, which exploit different effector mechanisms or combine directly cytotoxic mAbs with mAbs that rely on immune effector functionality. Such mAbs in combination may exhibit synergistic therapeutic effects.
- anti-CSA mAbs may be combined with other therapeutic agents, including but not limited to various chemotherapeutic agents, androgen-blockers, and immune modulators (e.g., IL-2, GM-CSF).
- the anti-CSA mAbs may be administered in their "naked” or unconjugated form, or may have therapeutic agents conjugated to them.
- bispecific antibodies may be used. Such an antibody would have one antigenic binding domain specific for a cell surface antigen of the invention and the other antigenic binding domain specific for another antigen (such as CD20 for example).
- Fab CSA antibodies or fragments of these antibodies may also be used as therapeutic agents. 5.5.1 ANTIBODIES
- Antibodies that specifically bind a cell surface antigen of the invention are useful in compositions and methods for immunotargeting cells expressing a cell surface antigen of the invention and for diagnosing a disease or disorder wherein cells involved in the disorder express a cell surface antigen of the invention.
- Such antibodies include monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds that include CDR and/or antigen-binding sequences, which specifically recognize a cell-surface antigen of the invention.
- Antibody fragments, including Fab, Fab', F(ab') 2 , and F v are also useful.
- variable regions of the antibodies recognize and bind a cell-surface antigen of the invention exclusively (i.e., able to distinguish a cell- surface antigen of the invention, or its splice variants with a similar expression pattern, from other similar polypeptides despite sequence identity, homology, or similarity found in the family of polypeptides), but may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule. Screening assays in which one can determine binding specificity of an anti-CSA. antibody are well known and routinely practiced in the art. (Chapter 6, Antibodies A Laboratory Manual, Eds. Harlow, et al, Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988), herein inco ⁇ orated by reference in its entirety).
- Polypeptides of the cell surface antigens of the invention can be used to immunize animals to obtain polyclonal and monoclonal antibodies that specifically react with said CSAs. Such antibodies can be obtained using either the entire protein or fragments thereof as an immunogen.
- the peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH).
- KLH keyhole limpet hemocyanin
- Any animal capable of producing antibodies can be immunized with a CSA peptide or polypeptide.
- Methods for immunization include subcutaneous or intraperitoneal injection of the polypeptide.
- the amount of the CSA peptide or polypeptide used for immunization depends on the animal that is immunized, antigenicity of the peptide and the site of injection.
- the CSA peptide or polypeptide used as an immunogen may be modified or administered in an adjuvant in order to increase the protein's antigenicity.
- Methods of increasing the antigenicity of a protein include, but are not limited to, coupling the antigen with a heterologous protein (such as globulin or ⁇ -galactosidase) or through the inclusion ofan adjuvant during immunization.
- a heterologous protein such as globulin or ⁇ -galactosidase
- spleen cells from the immunized animals are removed, fused with myeloma cells, such as SP2/0-Agl4 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells.
- myeloma cells such as SP2/0-Agl4 myeloma cells
- Any one of a number of methods well known in the art can be used to identify the hybridoma cell that produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, Western blot analysis, or radioimmunoassay (Lutz, et al, Exp. Cell Res. 175:109-124 (1988), herein inco ⁇ orated by reference in their entirety).
- Hybridomas secreting the desired antibodies are cloned and the class and subclass is determined using procedures known in the art (Campbell, A.M., Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984), herein inco ⁇ orated by reference in its entirety). Techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies to a cell-surface antigen of the invention (U.S. Patent No. 4,946,778, herein inco ⁇ orated by reference in its entirety).
- antibody-containing antiseram is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one of the above-described procedures.
- the anti-CSA antibody can be a nonhuman primate antibody. Methods of making such antibodies in baboons are disclosed in PCT Publication No. WO 91/11465 and Losman et al, Int. J. Cancer 46:310-314 (1990), both of which are herein inco ⁇ orated by reference in their entirety.
- the anti-CSA antibody is a humanized monoclonal antibody. Methods of producing humanized antibodies have been previously described.
- the anti-CSA antibody is a human monoclonal antibody.
- Humanized antibodies are produced by transgenic mice that have been engineered to produce human antibodies. Hybridomas derived from such mice will secrete large amounts of human monoclonal antibodies. Methods for obtaining human antibodies from transgenic mice are described in Green , et al, Nature Genet. 7:13-
- the present invention also includes the use of anti-CSA antibody fragments.
- Antibody fragments can be prepared by proteolytic hydrolysis of an antibody or by expression in E. coli of the DNA coding for the fragment.
- Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies.
- antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
- This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
- an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly.
- Fv fragments comprise an association of V H and V L chains, which can be noncovalent (Inbar et al, Proc. Nat'l Acad. Sci. USA 69:2659 (1972), herein inco ⁇ orated by reference in its entirety).
- the variable chains can be linked by an intermolecular disulfide bond or cross-linked " by chemicals such as glutaraldehyde.
- the Fv fragments comprise V H and V L chains that are connected by a peptide linker.
- These single-chain antigen binding proteins are prepared by constracting a structural gene comprising DNA sequences encoding the V H and V L domains which are connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell, such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs have been previously described (U.S. Patent No.
- CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (Larrick, et al, Methods: A Companion to Methods in Enymology 2:106 (1991); Courtenay-Luck, pp. 166-179 in, Monoclonal Antibodies Production, Engineering and Clinical Applications, Eds. Ritter et al, Cambridge University Press (1995); Ward, et al, pp. 137-185 in, Monoclonal Antibodies Principles and Applications, Eds. Birch et al, Wiley-Liss, Inc. (1995), all of which are herein inco ⁇ orated by reference in their entirety).
- the present invention further provides the above-described antibodies in detectably labeled form.
- Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.) fluorescent labels (such as FITC or rhodamine, etc), paramagnetic atoms, etc. Procedures for accomplishing such labeling have been previously disclosed (Stemberger, et al, J. Histochem. Cytochem. 18:315 (1970); Bayer, et al, Meth. Enzym. 62:308 (1979); Engval, et al, Immunol.
- the labeled antibodies can be used for in vitro, in vivo, and in situ assays to identify cells or tissues in which a cell-surface antigen of the invention is expressed. Furthermore, the labeled antibodies can be used to identify the presence of secreted cell-surface antigen of the invention in a biological sample, such as a blood, urine, saliva samples.
- Immnunoconjugates can be prepared by indirectly conjugating a therapeutic agent such as a cytotoxic agent to an antibody component.
- Toxic moieties include, for example, plant toxins, such as abrin, ricin, modeccin.
- viscumin pokeweed anti-viral protein, saporin, gelonin, momoridin, trichosanthin, barley toxin; bacterial toxins, such as Diptheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcal enterotoxin A; fungal toxins, such as ⁇ -sarcin, restrictocin; cytotoxic RNases, such as extracellular pancreatic RNases; DNase I (Pastan, et al, Cell 47:641 (1986); Goldenberg, Cancer Journal for Clinicians 44:43 (1994), herein inco ⁇ orated by reference in its entirety), calicheamicin, and radioisotopes, such as 32 P, 67 Cu, 77 As, 105 Rh, 109 Pd, l u Ag, 121 Sn, 13, I, l06 Ho, 177 Lu, 186 Re, 188 Re, 194 Ir 199 Au (Illidge and Brock, Curr Pharm.
- the general method involves reacting an antibody component having an oxidized carbohydrate portion with a carrier polymer that has at least one free amine function and that is loaded with a plurality of drag, toxin, chelator, boron addends, or other therapeutic agent. This reaction results in an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.
- the carrier polymer is preferably an aminodextran or polypeptide of at least 50 amino acid residues, although other substantially equivalent polymer carriers can also be used.
- the final immunoconjugate is soluble in an aqueous solution, such as mammalian serum, for ease of administration and effective targeting for use in therapy.
- solubilizing functions on the carrier polymer will enhance the seram solubility of the final immunoconjugate.
- an aminodextran will be preferred.
- the process for preparing an inmmunoconjugate with an aminodextran carrier typically begins with a dextran polymer, advantageously a dextran of average molecular weight of about 10,000-100,000.
- the dextran is reacted with an oxidizing agent to affect a controlled oxidation of a portion of its carbohydrate rings to generate aldehyde groups.
- the oxidation is conveniently effected with glycolytic chemical reagents such as NaIO , according to conventional procedures.
- the oxidized dextran is then reacted with a polyamine, preferably a diamine, and more preferably, a mono- or polyhydroxy diamine.
- Suitable amines include ethylene diamine, propylene diamine, or other like polymethylene diamines, diethylene triamine or like polyamines, l,3-diamino-2-hydroxypropane, or other like hydroxylated diamines or polyamines, and the like.
- An excess of the amine relative to the aldehyde groups of the dextran is used to ensure substantially complete conversion of the aldehyde functions to Schiff base groups.
- a reducing agent such as NaBH 4 , NaBH 3 CN or the like, is used to effect reductive stabilization of the resultant Schiff base intermediate.
- the resultant adduct can be purified by passage through a conventional sizing column or ultrafiltration membrane to remove cross-linked dextrans.
- Other conventional methods of derivatizing a dextran to introduce amine functions can also be used, e.g., reaction with cyanogen bromide, followed by reaction with a diamine.
- amninodextran is then reacted with a derivative of the particular drag, toxin, chelator, immunomodulator, boron addend, or other therapeutic agent to be loaded, in an activated form, preferably, a carboxyl-activated derivative, prepared by conventional means, e.g., using dicyclohexylcarbodiimide (DCC) or a water soluble variant thereof, to form an intermediate adduct.
- DCC dicyclohexylcarbodiimide
- polypeptide toxins such as pokeweed antiviral protein or ricin A-chain, and the like, can be coupled to aminodextran by glutaraldehyde condensation or by reaction of activated carboxyl groups on the protein with amines on the aminodextran.
- Chelators for radiometals or magnetic resonance enhancers are well-known in the art. Typical are derivatives of ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTP A). These chelators typically have groups on the side chain by which the chelator can be attached to a carrier. Such groups include, e.g., benzylisothiocyanate, by which the DTP A or EDTA can be coupled to the amine group of a carrier. Alternatively, carboxyl groups or amine groups on a chelator can be coupled to a carrier by activation oi prior derivatization and then coupling, all by well-known means.
- EDTA ethylenediaminetetraacetic acid
- DTP A diethylenetriaminepentaacetic acid
- Carrier addends such as carboranes
- carboranes can be prepared with carboxyl functions on pendant side chains, as is well known in the art. Attachment of such carboranes to a carrier, e.g., aminodextran, can be achieved by activation of the carboxyl groups of the carboranes and condensation with amines on the carrier to produce an intermediate conjugate. Such intermediate conjugates are then attached to antibody components to produce therapeutically useful immunoconjugates, as described below.
- a polypeptide carrier can be used instead of aminodextran, but the polypeptide carrier should have at least 50 amino acid residues in the chain, preferably 100-5000 amino acid residues. At least some of the amino acids should be lysine residues or glutamate or aspartate residues. The pendant amines of lysine residues and pendant carboxylates of glutamine and aspartate are convenient for attaching a drug, toxin, immunomodulator, chelator, boron addend or other therapeutic agent.
- suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixed polymers of these amino acids and others, e.g., serines, to confer desirable solubility properties on the resultant loaded earner and immunoconjugate.
- Conjugation of the intermediate conjugate with the antibody component is effected by oxidizing the carbohydrate portion of the antibody component and reacting the resulting aldehyde (and ketone) carbonyls with amine groups remaining on the carrier after loading with a drug, toxin, chelator, immunomodulator, boron addend, or other therapeutic agent.
- an intermediate conjugate can be attached to an oxidized antibody component via amine groups that have been introduced in the intermediate conjugate after loading with the therapeutic agent.
- Oxidation is conveniently effected either chemically, e.g., with NaIO 4 or other glycolytic reagent, or enzymatically, e.g., with neuraminidase and galactose oxidase.
- aminodextran carrier not all of the amines of the aminodextran are typically used for loading a therapeutic agent.
- the remaining amines of aminodextran condense with the oxidized antibody component to form Schiff base adducts, which are then reductively stabilized, normally with a borohydride reducing agent.
- Loaded polypeptide carriers preferably have free lysine residues remaining for condensation with the oxidized carbohydrate portion ofan antibody component.
- Carboxyls on the polypeptide carrier can, if necessary, be converted to amines by, e.g., activation with DCC and reaction with an excess of a diamine.
- the final immunoconjugate is purified using conventional techniques, such as sizing chromatography on Sephacryl S-300 or affinity chromatography using one or more cell- surface antigen of the invention epitopes.
- immunoconjugates can be prepared by directly conjugating an antibody component with a therapeutic agent.
- the general procedure is analogous to the indirect method of conjugation except that a therapeutic agent is directly attached to an oxidized antibody component. It will be appreciated that other therapeutic agents can be substituted for the chelators described herein. Those of skill in the art will be able to devise conjugation schemes without undue experimentation.
- a therapeutic agent can be attached at the hinge region of a reduced antibody component via disulfide bond formation.
- the tetanus toxoid peptides can be constructed with a single cysteine residue that is used to attach the peptide to an antibody component.
- such peptides can be attached to the antibody component using a heterobifunctional cross-linker, such as N-succinyl 3-(2- pyridyldithiojproprionate (SPDP) (Yu, et al, Int. J. Cancer 56:244 (1994), herein inco ⁇ orated by reference in its entirety).
- SPDP N-succinyl 3-(2- pyridyldithiojproprionate
- carbohydrate moieties in the Fc region of an antibody can be used to conjugate a therapeutic agent.
- the Fc region may be absent if an antibody fragment is used as the antibody component of the immunoconjugate.
- it is possible to introduce a carbohydrate moiety into the light chain variable region of an antibody or antibody fragment Leung, et al, J. Immunol. 154:5919-5926 (1995); U.S. Pat. No. 5,443,953, both of which are herein inco ⁇ orated by reference in their entirety.
- the engineered carbohydrate moiety is then used to attach a therapeutic agent.
- the carbohydrate moiety can be used to attach polyethyleneglycol in order to extend the half- life of an intact antibody, or antigen-binding fragment thereof, in blood, lymph, or other extracellular fluids.
- a free sulfhydryl group may be located in the hinge region of the antibody component.
- the present invention contemplates the use of fusion proteins comprising one or more anti-CSA antibody moieties and an immunomodulator or toxin moiety.
- Methods of making antibody fusion proteins have been previously described (U.S. Patent No. 6,306,393, herein inco ⁇ orated by reference in its entirety).
- Antibody fusion proteins comprising an interleukin-2 moiety have also been previously disclosed (Boleti, et al, Ann. Oncol. 6:945 (1995), Nicolet, et al, Cancer Gene Ther. 2:161 (1995), Becker, et al, Proc. Nat'lAcad. Sci. USA 93:7826 (1996), Hank, et al. Clin.
- Peptides of the cell surface antigens of the invention themselves, such as fragments of the extracellular region, may be used to target toxins or radioisotopes to tumor cells in vivo by binding to or interacting with the cell surface antigens of the invention expressed on tumor or diseased cells. Much like an antibody, these fragments may specifically target cells expressing this antigen. Targeted delivery of these cytotoxic agents to the tumor cells would result in cell death and suppression of tumor growth.
- An example of the ability of an extracellular fragment binding to and activating its intact receptor has been demonstrated with the CD84 receptor (Martin et al, J. Immunol. 167:3668-3676 (2001), herein inco ⁇ orated by reference in its entirety).
- Extracellular fragments of the cell surface antigens of the invention may also be used to modulate immune cells expressing the protein.
- Extracellular domain fragments of the cell surface antigen may bind to and activate its own receptor on the cell surface, which may result in stimulating the release of cytokines (such as interferon gamma from NK cells, T cells, B cells or myeloid cells, for example) that may enhance or suppress the immune system. Additionally, binding of these fragments to cells bearing cell surface antigens of the invention may result in the activation of these cells and also may stimulate proliferation. Some fragments may bind to the intact cell surface antigen of the invention and block activation signals and cytokine release by immune cells. These fragments would then have an immunosuppressive effect.
- Fragments that activate and stimulate the immune system may have anti-tumor properties. These fragments may stimulate an immunological response that can result in immune-mediated tumor cell killing. The same fragments may result in stimulating the immune system to mount an enhanced response to foreign invaders such as viruses and bacteria. Fragments that suppress the immune response may be useful in treating lymphoproliferative disorders, auto-immune diseases, graft-vs-host disease, and inflammatory diseases, such as emphysema.
- Random peptide libraries are displayed on phage (phage display) or on bacteria, such as on E. coli. These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or a polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
- diversity libraries such as random or combinatorial peptide or nonpeptide libraries can be screened for molecules that specifically bind to CSA polypeptides.
- Many libraries are known in the art that can be used, i.e. chemically synthesized libraries, recombinant (i.e. phage display libraries), and in vitro translation- based libraies.
- Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al, U.S. Patent No. 5,223, 409; Ladner et al., U.S. Patent No. 4,946,778; Ladner et al, U.S. Patent No. 5,403,484; Ladner et al, U.S.
- Patent No. 5,571,698, all of which are herein inco ⁇ orated by reference in their entirety) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from Clontech (Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA), and Pharmacia KLB Biotechnology Inc. (Piscataway, NJ). Random peptide display libraries can be screened using the CSA sequences disclosed herein to identify proteins which bind to the CSA of the invention.
- phage display libraries are described in Scott and Smith, Science 249:386-390 (1990); Devlin et al, Science 249:404-406 (1990); Christian et al, J. Mol. Biol. 227:711-718 (1992); Lenstra, J Immunol Meth. 152:149-157 (1992); Kay et al, Gene 128:59-65 (1993); PCT Publication No. WO 94/18318, all of which are herein inco ⁇ orated by reference in their entirety.
- a benzodiazepine library (see for example, Bunin et al, Proc. Natl. Acad. Sci. USA 91:4708-4712 (1994), herein inco ⁇ orated by reference in its entirety) can be adapted for use.
- Peptoid libraries (Simon et al, Proc. Natl. Acad. Sci. USA 89:9367-9371 (1992), herein inco ⁇ orated by reference in its entirety) can also be used.
- Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (Proc. Natl. Acad. Sci. USA 91:11138- 11142 (1994), herein inco ⁇ orated by reference in its entirety).
- Screening the libraries can be accomplished by any of a variety of commonly known methods. See, for example, the following references which disclose screening of peptide libraries: Parmley and Smith, Adv. Exp. Med. Biol. 251 :215-218 (1989); Scott and Smith, Science 249:386-390 (1990); Fowlkes et al, Biotechmques 13:422-427 (1992); Oldenburg et al, Proc. Natl. Acad. Sci. USA 89.5393-5397 (1992); Yu et al, Cell 76:933-945 (1994); Staudt et al, Science 241 :577-580 (1988); Bock et al, Nature 355:564-566 (1992); Tuerk et al, Proc.
- screening can be carried out by contacting the library members with a CSA protein (or nucleic acid or derivative) immobilized on a solid phase and harvesting those library members that bind to the protein (or nucleic acid or derivative).
- CSA protein or nucleic acid or derivative
- panning techniques are described by way of example in Parmley and Smith, Gene 73:305-318 (1988); Fowlkes et al, Biotechmques 13:422-427 (1992); PCT Publication No. WO 94/18318, all of which are herein inco ⁇ orated by reference in their entirety, and in references cited hereinabove.
- the two-hybrid system for selecting interacting protein in yeast can be used to identify molecules that specifically bind to a CSA protein or derivative.
- These "binding polypeptides" or small molecules which interact with CSA polypeptides of the invention can be used for tagging or targeting cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like.
- binding polypeptides or small molecules can also be used in analytical methods such as for screening expression libraries and neutralizing activity, i.e., for blocking interaction between ligand and receptor, or viral binding to a receptor.
- the binding polypeptides or small molecules can also be used for diagnostic assays for determining circulating levels of CSA polypeptides of the invention; for detecting or quantitating soluble CSA polypeptides as marker of underlying pathology or disease.
- binding polypeptides or small molecules can also act as CSA "antagonists" to block CSA binding and signal transduction in vitro and in vivo. These anti-CSA binding polypeptides or small molecules would be useful for inhibiting CSA activity or protein binding.
- Binding polypeptides can also be directly or indirectly conjugated to drags, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. Binding peptides can also be fused to other polypeptides, for example an immunoglobulin constant chain or portions thereof, to enhance their half- life, and can be made multivalent (through, e.g. branched or repeating units) to increase binding affinity for the CSA.
- binding polypeptides of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance). More specifically, binding polypeptides or bioactive fragments or portions thereof, can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anti- complementary molecule.
- Suitable detectable molecules may be directly or indirectly attached to the binding polypeptide, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like.
- Suitable cytotoxic molecules may be directly or indirectly attached to the binding polypeptide, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium-188, or yttrium-90 (either directly attached to the binding polypeptide, or indirectly attached through a means of a chelating moiety, for instance).
- Binding polypeptides may also be conjugated to cytotoxic drugs, such as adriamycin.
- cytotoxic drugs such as adriamycin.
- the detectable or cytotoxic molecule can be conjugated with a member of a complementary/anticomplementary pair, where the other member is bound to the binding polypeptide.
- biotin/streptavidin is an exemplary complementary/anticomplementary pair.
- binding polypeptide-toxin fusion proteins can be used for targeted cell or tissue inhibition or ablation (for instance, to treat cancer cells or tissues).
- a fusion protein including only the targeting domain may be suitable for directing a detectable molecule, a cytotoxic molecule, or a complementary molecule to a cell or tissue type of interest.
- the domain only fusion protein includes a complementary molecule
- the anti- complementary molecule can be conjugated to a detectable or cytotoxic molecule.
- Such domain-complementary molecule fusion proteins thus represent a generic targeting vehicle for cell/tissue-specific delivery of generic anti-complementary-detectable/cytotoxic molecule conjugates.
- the present invention provides reagents and methods useful for treating diseases and conditions wherein cells associated with the disease or disorder express a cell surface antigen of the invention.
- diseases can include cancers, and other hype ⁇ roliferative conditions, such as hype ⁇ lasia.
- Whether the cells associated with a disease or condition express cell surface antigens of the invention can be determined using the diagnostic methods described herein.
- Comparisons of the expression levels of CSA mRNA and protein between diseased cells, tissue or fluid (blood, lymphatic fluid, etc.) and corresponding normal samples are made to determine if the patient will be responsive to therapy targeting said cell surface antigens of the invention.
- Methods for detecting and quantifying the expression of CSA mRNA or protein use standard nucleic acid and protein detection and quantitation techniques that are well known in the art and are described in Sambrook, et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989) or Ausubel, et al, Current Protocols in Molecular Biology, John W ⁇ iley & Sons, New York, NY (1989), both of which are inco ⁇ orated herein by reference in their entirety.
- Standard methods for the detection and quantification of CSA mRNA include in situ hybridization using labeled CSA riboprobes (Gemou-Engesaeth, et al, Pediatrics 109: E24-E32 (2002), herein inco ⁇ orated by reference in its entirety), Northern blot and related techniques using CSA polynucleotide probes (Kunzli, et al, Cancer 94: 228 (2002), herein inco ⁇ orated by reference in its entirety), RT-PCR analysis using primers specific to the cell surface antigens of the invention (Angchaiskisiri, et al, Blood 99:130 (2002), herein inco ⁇ orated by reference in its entirety), and other amplification detection methods, such as branched chain DNA solution hybridization assay (Jardi, et al, J. Viral Hepat. 8:465-471 (2001), herein inco ⁇ orated by reference in its entirety), transcription-mediated amplification (Kimura, et al
- Microbiol 40:439-445 (2002), herein inco ⁇ orated by reference in its entirety
- microarray products such as oligos, cDNAs, and monoclonal antibodies
- real-time PCR Simpson, et al, Molec. Vision, 6:178-183 (2000), herein inco ⁇ orated by reference in its entirety
- Standard methods for the detection and quantification of CSA protein include western blot analysis (Sambrook, et al, 1989, supra, Ausubel, et al, 1989, supra), immunocytochemistry
- immunoassays including enzyme-linked immunosorbant assay (ELISA), radioimmuno assay (RIA), and specific enzyme immunoassay (EIA) (Sambrook, et al, 1989, supra; Ausubel, et al, 1989, supra).
- ELISA enzyme-linked immunosorbant assay
- RIA radioimmuno assay
- EIA specific enzyme immunoassay
- Peripheral blood cells can also be analyzed for expression of the cell surface antigens of the invention using flow cytometry using, for example, immunomagnetic beads specific for the cell-surface antigens of the invention (Racila, et al, 1998, supra), or biotinylated antibodies of the cell-surface antigens of the invention (Soltys, et al, J. Immunol. 168:1903 (2002), herein inco ⁇ orated by reference in its entirety).
- Yet another related aspect of the invention is directed to methods for gauging tumor aggressiveness by determining the levels of protein or mRNA of the cell surface antigens of the invention in tumor cells compared to the corresponding normal cells (Orlandi, et al, Cancer Res. 62:567 (2002), herein inco ⁇ orated by reference in its entirety).
- the disease or disorder is a cancer.
- the diseases treatable by methods of the present invention preferably occur in mammals.
- Mammals include, for example, humans and other primates, as well as pet or companion animals such as dogs and cats, laboratory animals such as rats, mice and rabbits, and farm animals such as horses, pigs, sheep, and cattle.
- Tumors or neoplasms include growths of tissue cells in which the multiplication of the cells is uncontrolled and progressive. Some such growths are benign, but others are termed "malignant" and may lead to death of the organism. Malignant neoplasms or
- cancers are distinguished from benign growths in that, in addition to exhibiting aggressive cellular proliferation, they may invade surrounding tissues and metastasize. Moreover, malignant neoplasms are characterized in that they show a greater loss of differentiation
- Neoplasms treatable by the present invention also include solid phase tumors/malignancies, i.e., carcinomas, locally advanced tumors and human soft tissue sarcomas.
- Carcinomas include those malignant neoplasms derived from epithelial cells that infiltrate (invade) the surrounding tissues and give rise to metastastic cancers, including lymphatic metastases.
- Adenocarcinomas are carcinomas derived from glandular tissue, or which form recognizable glandular stractures.
- Another broad category or cancers includes sarcomas, which are tumors whose cells are embedded in a fibrillar or homogeneous substance like embryonic connective tissue.
- the invention also enables treatment of cancers of the myeloid or lymphoid systems, including leukemias, lymphomas and other cancers that typically do not present as a tumor mass, but are distributed in the vascular or lymphoreticular systems.
- the type of cancer or tumor cells that may be amenable to treatment according to the invention include, for example, acute lymphocytic leukemia, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, cutaneous T-cell lymphoma, hairy cell leukemia, acute myeloid leukemia, erythroleukemia, chronic myeloid (granulocytic) leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer, polyps associated with colorectal neoplasms, pancreatic cancer and gallbladder cancer, cancer of the adrenal cortex, ACTH-producing tumor, bladder cancer, brain cancer including intrinsic brain tumors, neuroblastomas, astrocytic brain tumors, gliomas, and metastatic tumor cell invasion of the central nervous system, Ewing's sarcoma, head and neck cancer including
- the invention is particularly illustrated herein in reference to treatment of certain types of experimentally defined cancers.
- standard state-of- the-art in vitro and in vivo models have been used. These methods can be used to identify agents that can be expected to be efficacious in in vivo treatment regimens.
- the method of the invention is not limited to the treatment of these tumor types, but extends to any cancer derived from any organ system.
- the cell surface antigens of the invention are highly expressed in disorders relating to hematopoietic cells.
- Leukemias can result from uncontrolled B cell proliferation initially within the bone marrow before disseminating to the peripheral bl ⁇ od, spleen, lymph nodes and finally to other tissues. Uncontrolled B cell proliferation also may result in the development of lymphomas that arise within the lymph nodes and then spread to the blood and bone marrow.
- Immunotargeting the cell surface antigens of the invention is used in treating B cell malignancies, leukemias, lymphomas and myelomas including but not limited to multiple myeloma, Burkitt's lymphoma, cutaneous B cell lymphoma, primary follicular cutaneous B cell lymphoma, B lineage acute lymphoblastic leukemia (ALL), B cell non- Hodgkin's lymphoma (NHL), B cell chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia, hairy cell leukemia (HCL), acute myelogenous leukemia, acute myelomonocytic leukemia, chronic myelogenous leukemia, lymphosarcoma cell leukemia, splenic marginal zone lymphoma, diffuse large B cell lymphoma, B cell large cell lymphoma, malignant lymphoma, prolymphocytic leukemia (PLL), lymphoplasma cytoid lymphom
- cell surface antigens of the invention has also been demonstrated in the Examples to be expressed in myeloid leukemia, T cell leukemia, and T cell lymphoma cell lines and tissues, and may be treated with antibodies that recognize the cell-surface antigens of the invention.
- Other diseases that may be treated by the methods of the present invention include multicentric Castleman's disease, primary amyloidosis, Franklin's disease, Seligmann's disease, primary effusion lymphoma, post-transplant lymphoproliferative disease (PTLD) [associated with EBV infection], paraneoplastic pemphigus, chronic lymphoproliferative disorders, X-linked lymphoproliferative syndrome (XLP), acquired angioedema, angioimmunoblastic lymphadenopathy with dysproteinemia, Herman's syndrome, post- splenectomy syndrome, congenital dyserythropoietic anemia type III, lymphoma-associated hemophagocytic syndrome (LAHS), necrotizing ulcerative stomatitis, Kikuchi's disease, lymphomatoid granulomatosis, Richter's syndrome, polycythemic vera (PV), Gaucher's disease, Gougerot-Sjogren syndrome, Kaposi's sarcoma, cerebral lymphoplasmo
- compositions of the invention may be effective in adult and pediatric oncology including in solid phase tumors/malignancies, locally advanced tumors, human soft tissue sarcomas, metastatic cancer, including lymphatic metastases, blood cell malignancies, including multiple myeloma, acute and chronic leukemias and lymphomas, head and neck cancers, including mouth cancer, larynx cancer, and thyroid cancer, lung cancers including small cell carcinoma and non-small cell cancers, breast cancers including small cell carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer and polyps associated with colorectal neoplasia, pancreatic cancers, liver cancer, urologic cancers including bladder cancer and prostate cancer, malignancies of the female genital tract including ovarian carcinoma, uterine (including endometrial) cancers, and solid tumor in the ovarian follicle, kidney cancers including renal cell carcinoma, brain cancers including intrinsic brain tumors, neuroblastoma
- Autoimmune diseases can be associated with hyperactive B cell activity that results in autoantibody production. Additionally, autoimmune diseases can be associated with uncontrolled protease activity (Wemike et al, Arthritis Rheum. 46:64-74 (2002)) and aberrant cytokine activity (Rodenburg et al, Ann. Rheum. Dis. 58:648-652 (1999), both of which are herein inco ⁇ orated by reference in their entirety). Inhibition of the development of autoantibody-producing cells or proliferation of such cells may be therapeutically effective in decreasing the levels of autoantibodies in autoimmune diseases.
- Inhibition of protease activity may reduce the extent of tissue invasion and inflammation associated with autoimmune diseases including but not limited to systemic lupus erythematosus, Hasimoto thyroiditis, Sj ⁇ gren's syndrome, pericarditis lupus, Crohn's Disease, graft-verses-host disease, Graves' disease, myasthenia gravis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, asthma, cryoglubulinemia, primary biliary sclerosis, pernicious anemia,
- POEMS syndrome polyneuropathy, organomegaly, endocrinopathy, M component, skin changes
- connective tissue disease multiple sclerosis, cystic fibrosis, rheumatoid arthritis, autoimmune pulmonary inflammation, psoriasis.
- Guillain-Barre syndrome autoimmune thyroiditis, insulin dependent diabetes mellitis, autoimmune inflammatory eye disease,
- the cell surface antigens of the invention are expressed in tissues isolated from patients with systemic lupus erythrematosus,
- Targeting cell surface antigens of the invention may also be useful in the treatment of allergic reactions and conditions e.g., anaphylaxis, seram sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens- Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal kera oconjunctiv ⁇ tis. giant papillary conjunctivitis, allergic gastroenteropathy, inflammatory bowel disorder (IBD), and contact allergies, such as asthma
- IBD inflammatory bowel disorder
- Targeting cell surface antigens of the invention may also be useful in the management or prevention of transplant rejection in patients in need of transplants such as stem cells, tissue or organ transplant.
- one aspect of the invention may find therapeutic utility in various diseases (such as those usually treated with transplantation, including without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria) as wells in repopulating the stem cell compartment post irridiation/chemotherapy, either in vivo or ex vivo (i.e. in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous) as normal cells or genetically manipulated for gene therapy.
- diseases such as those usually treated with transplantation, including without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria
- repopulating the stem cell compartment post irridiation/chemotherapy either in vivo or ex vivo (i.e. in conjunction with bone marrow transplantation or with peripheral progenitor
- the cell surface antigens of the invention are expressed in rejected heart, liver, and kidney tissue after transplantation, as opposed to normal tissue.
- targeting of the cell surface antigens of the invention may be useful to prevent and/or reduce tissue rejection after transplantation.
- Targeting cell surface antigens of the invention may also be possible to modulate immune responses, in a number of ways.
- Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response.
- Down regulating or preventing one or more antigen functions including without limitation B lymphocyte antigen functions, e.g., modulating or preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD).
- B lymphocyte antigen functions e.g., modulating or preventing high level lymphokine synthesis by activated T cells
- GVHD graft-versus-host disease
- blockage of T cell function should result in reduced tissue destruction in tissue transplantation.
- rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.
- a therapeutic composition of the invention may prevent cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, a lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen- blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.
- the efficacy of particular therapeutic compositions in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.
- appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al, Science 257:789-792 (1992) and Turka et al, Proc. Natl. Acad. Sci USA. 89: 11102-11105 (1992), both of which are herein inco ⁇ orated by reference in their entirety.
- murine models of GVHD can be used to determine the effect of therapeutic compositions of the invention on the development of that disease.
- Monoclonal antibodies (mAbs) recognizing the cell surface antigens of the invention used in the practice of a method of the invention may be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method.
- Suitable carriers include any material which when combined with the anti-CSA antibodies retains the antitumor function of the antibody and is nonreactive with the subject's immune systems. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like.
- the antibody formulations of the invention may be administered via any route capable of delivering the antibodies to the tumor site.
- Potentially effective routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradermal, and the like.
- the preferred route of administration is by intravenous injection.
- a preferred formulation for intravenous injection comprises mAbs specific to the cell surface antigens of the invention in a solution of preserved bacteriostatic water, sterile unpreserved water, and/or diluted in polyvinylchloride or polyethylene bags containing 0.9% sterile sodium chloride for Injection, USP.
- the mAb preparation of the invention may be lyophilized and stored as a sterile powder, preferably under vacuum, and then reconstituted in bacteriostatic water containing, for example, benzyl alcohol preservative, or in sterile water prior to injection.
- Treatment will generally involve the repeated administration of the antibody preparation of the invention via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1 to about 10 mg/kg body weight; however other exemplary doses in the range of 0.01 mg/kg to about 100 mg/kg are also contemplated. Doses in the range of 10-500 mg mAb per week may be effective and well tolerated.
- Rituximab (Rituxan®), a chimeric CD20 antibody used to treat B-cell lymphoma, non-Hodgkin's lymphoma, and relapsed indolent lymphoma, is typically administered at 375 mg/m 2 by IV infusion once a week for 4 to 8 doses.
- Rituxan® a humanized monoclonal antibody used to treat HER-2 (human epidermal growth factor 2)-positive metastatic breast cancer (Slamon, et al, Mol Cell Biol.
- an initial loading dose of approximately 4 mg/kg patient body weight IV followed by weekly doses of about 2 mg kg IV of the mAb preparation of the invention may represent an acceptable dosing regimen (Slamon, et al, N. Engl. J. Med. 344: 783(2001), herein inco ⁇ orated by reference in its entirety).
- the initial loading dose is administered as a 90 minute or longer infusion.
- the periodic maintenance dose may be administered as a 30 minute or longer infusion, provided the initial dose was well tolerated.
- various factors will influence the ideal dose regimen in a particular case.
- Such factors may include, for example, the binding affinity and half life of the mAb or mAbs used, the degree of overexpression of the cell- surface antigens of the invention in the patient, the extent of circulating shed cell surface antigen of the invention, the desired steady-state antibody concentration level, frequency of treatment, and the influence of chemotherapeutic agents used in combination with the treatment method of the invention.
- Treatment can also involve anti-CSA antibodies conjugated to radioisotopes.
- anti-CEA radiolabeled-anticarcinoembryonic antigen
- dendritic cells transfected with mRNA encoding a cell surface antigen of the invention can be used as a vaccine to stimulate T-cell mediated anti -tumor responses.
- Studies with dendritic cells transfected with prostate-specific antigen mRNA suggest a 3 cycles of intravenous administration of 1 xlO 7 - 5 ⁇ 10 7 cells for 2-6 weeks concomitant with an intradermal injection of 10 7 cells may provide a suitable dosage regimen (Heiser, et al, J. Clin. Invest. 109:409-417 (2002); Hadzantonis and O'Neill, Cancer Biother. Radiopharm. 1:11 -22 (1999), both of which are herein inco ⁇ orated by reference in their entirety).
- Other exemplary doses of between 1 x10 5 to 1*10 9 or lxlO 6 to l lO 8 cells are also contemplated.
- Naked DNA vaccines using plasmids encoding a cell surface antigen of the invention can induce an immunologic anti-tumor response.
- Administration of naked DNA by direct injection into the skin and muscle is not associated with limitations encountered using viral vectors, such as the development of adverse immune reactions and risk of insertional mutagenesis (Hengge, et al, J. Invest. Dermatol 116:979 (2001), herein inco ⁇ orated by reference in its entirety).
- Plasmid DNA can also be administered to the lungs by aerosol delivery (Densmore, et al, Mol. Ther. 1 : 180- 188 (2000)).
- Gene therapy by direct injection of naked or lipid-coated plasmid DNA is envisioned for the prevention, treatment, and cure of diseases such as cancer, acquired immunodeficiency syndrome, cystic fibrosis, cerebrovascular disease, and hypertension (Prazeres, et al, Trends Biotechnol
- HIV-1 DNA vaccine dose-escalating studies indicate administration of 30-300 ⁇ g/dose as a suitable therapy (Weber, et al, Eur. J. Clin.
- beta-galactosidase plasmid 1 micro gram of beta-galactosidase plasmid was greater and more prolonged than was observed after an injection with an equal amount of naked DNA or DNA complexed with
- Lipofectamine (Traong, et al, Hum. Gene Ther. 9:1709-1717 (1998), herein inco ⁇ orated by reference in its entirety).
- plasmid-mediated gene transfer into skeletal muscle as a means of providing a therapeutic source of insulin
- four plasmid constructs comprising a mouse furin cDNA transgene and rat proinsulin cDNA were injected into the calf muscles of male Balb/c mice
- the optimal dose for most constructs was 100 micrograms plasmid DNA (Kon, et al J. Gene Med. 1 :186-194 (1999), herein inco ⁇ orated by reference in its entirety).
- Other exemplary doses of 1-1000 ⁇ g/dose or 10-500 ⁇ g/dose are also contemplated.
- patients should be evaluated for the level of circulating shed cell-surface antigen of the invention in seram in order to assist in the determination of the most effective dosing regimen and related factors.
- evaluations may also be used for monitoring pu ⁇ oses throughout therapy, and may be useful to gauge therapeutic success in combination with evaluating other parameters.
- compositions for targeting cells expressing a cell-surface antigen of the invention are within the scope of the present invention.
- Pharmaceutical compositions comprising antibodies are described in detail in, for example, U.S. Patent No. 6,171,586, herein inco ⁇ orated by reference in its entirety.
- Such compositions comprise a therapeutically or prophylactically effective amount an antibody, or a fragment, variant, derivative or fusion thereof as described herein, in admixture with a pharmaceutically acceptable agent.
- the immunotargeting agent will be sufficiently purified for administration to an animal.
- the pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adso ⁇ tion or penetration of the composition.
- Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydro gen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, other organic acids); bulking agents (such as mannitol or glycine), chelating agents [such as ethylenediamine tetraacetic acid (EDTA)]; complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring; flavoring and diluting agents; emulsifying agents;
- compositions will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the immunotargeting agent.
- the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non- aqueous in nature.
- a suitable vehicle or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration.
- Neutral buffered saline or saline mixed with seram albumin are further exemplary vehicles.
- Other exemplary pharmaceutical compositions comprise Tris buffer of about. pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute therefor.
- immunotargeting agent compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution.
- optional formulation agents Remington's Pharmaceutical Sciences, supra
- the binding agent product may be formulated as a lyophilizate using appropriate excipients such as sucrose.
- compositions can be selected for parenteral delivery. Alternatively, the compositions may be selected for inhalation or for delivery through the digestive tract, such as orally.
- the preparation of such pharmaceutically acceptable compositions is within the skill of the art.
- the formulation components are present in concentrations that are acceptable to the site of administration. For example, buffers are used to maintain the composition at physiological pH or at slightly lower pH, typically within a pH range of from about 5 to about 8.
- the therapeutic compositions for use in this invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the immunotargeting agent of the invention in a pharmaceutically acceptable vehicle.
- a particularly suitable vehicle for parenteral injection is sterile distilled water in which an immunotargeting agent of the invention is formulated as a sterile, isotonic solution, properly preserved.
- an agent such as injectable microspheres, bio-erodible particles, polymeric compounds (polylactic acid, polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which may then be delivered via a depot injection.
- Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation.
- Suitable means for the introduction of the desired molecule include implantable drag delivery devices.
- compositions suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, ringer's solution, or physiologically buffered saline.
- Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
- suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
- Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes.
- Non-lipid polycationic amino polymers may also be used for delivery.
- the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
- a pharmaceutical composition may be formulated for inhalation.
- an immunotargeting agent of the invention may be formulated as a dry powder for inhalation.
- Polypeptide or nucleic acid molecule inhalation solutions may also be formulated with a propellant for aerosol delivery.
- solutions may be nebulized. Pulmonary administration is further described in PCT Application No. PCT/US94/001875, herein inco ⁇ orated by reference in its entirety, which describes pulmonary delivery of chemically modified proteins.
- targeting agents of the invention that are administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules.
- a capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized.
- Additional agents can be included to facilitate abso ⁇ tion of the binding agent molecule. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.
- compositions for oral administration can also be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
- Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
- compositions for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores.
- auxiliaries can be added, if desired.
- Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from com, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen.
- disintegrating or solubilizing agents may be added, such as the cross -linked polyvinyl pyrrolidone, agai, and alginic acid or a salt thereof, such as sodium alginate.
- Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
- suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
- Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
- compositions that can be used orally also include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
- Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
- the immunotargeting agent of the invention may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
- Another pharmaceutical composition may involve an effective quantity of the immunotargeting agent of the invention in a mixture with non-toxic excipients that are suitable for the manufacture of tablets.
- excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
- sustained- or controlled-delivery formulations include formulations involving the immunotargeting agents of the invention in sustained- or controlled-delivery formulations.
- Techniques for formulating a variety of other sustained- or controlled-delivery means such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See, for example, PCT Application No. PCT/US93/00829, herein inco ⁇ orated by reference in its entirety, that describes controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions.
- sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules.
- Sustained release matrices may include polyesters, hydrogels, polylactides (U.S. Patent No. 3,773,919; European Patent No. EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, Biopolymers, 22:547-556 (1983)), poly (2- hydroxyefhyl-methacrylate) (Langer et al, J Biomed Mater Res, 15:167-277, (1981)) and (Langer et al, Chem Tech, 12:98-105(1982)), ethylene vinyl acetate (Langer et al, supra) or poly-D (-)-3-hydroxybutyric acid (European Patent No.
- Sustained-release compositions also include liposomes, which can be prepared by any of several methods known in the art. See e.g., Epstein, et al, Proc Natl Acad Sci (USA), 82:3688-3692 (1985); European Patent Nos. EP 36,676; EP 88,046; EP 143,949, all of which are herein inco ⁇ orated by reference in their entirety.
- compositions to be used for in vivo administration typically must be sterile. This may be accomplished by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using this method may be conducted either prior to or following lyophilization and reconstitution.
- the composition for parenteral administration may be stored in lyophilized form or in solution.
- parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
- the pharmaceutical composition may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or a dehydrated or lyophilized powder.
- Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.
- kits for producing a single-dose administration unit may each contain both a first container having a dried immunotargeting agent of the invention and a second container having an aqueous formulation. Also included within the scope of this invention are kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes).
- An effective amount of a pharmaceutical composition to be employed therapeutically will depend, for example, upon d e therapeutic context and objectives.
- One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which an immunotargeting agent of the invention is being used, the route of administration, and the size (body weight, body surface or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician may titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
- a typical dosage may range from about 0.1 mg/kg to up to about 100 mg/kg or more, depending on the factors mentioned above.
- the dosage may range from 0.1 mg/kg up to about 100 mg/kg; or 0.01 mg kg to 1 g/kg; or 1 mg/kg up to about 100 mg/kg or 5 mg/kg up to about 100 mg/kg.
- the dosage may range from 10 mCi to 100 mCi per dose for radioimmunotherapy, from about Ixl0 7 - 5 ⁇ l0 7 cells or l ⁇ l0 5 to 1 10 "cells or lxlO 6 to lxlO 8 cells per injection or infusion, or from 30 ⁇ g to 300 ⁇ g naked DNA per dose or 1-1000 ⁇ g/dose or 10-500 ⁇ g/dose, depending on the factors listed above.
- the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models such as mice, rats, rabbits, dogs, or pigs.
- animal models such as mice, rats, rabbits, dogs, or pigs.
- An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
- Dosage and administration are adjusted to provide sufficient levels of the active compound or to maintain the desired effect. Factors that may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drag combination(s), reaction sensitivities, and response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.
- the frequency of dosing will depend upon the pharmacokinetic parameters of the immunotargeting agent of the invention in the formulation used.
- a composition is administered until a dosage is reached that achieves the desired effect.
- the composition may therefore be administered as a single dose, or as multiple doses (at the same or different concentrations/dosages) over time, or as a continuous infusion. Further refinement of the appropriate dosage is routinely made. Appropriate dosages may be ascertained through use of appropriate dose-response data.
- the route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intra-arterial, intraportal, intralesional routes, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, urethral, vaginal, or rectal means, by sustained release systems, by implantation devices, or through inhalation.
- the compositions may be administered by bolus injection or continuously by infusion, or by implantation device.
- the composition may be administered locally via implantation of a membrane, sponge, or another appropriate material on to which the immunotargeting agent of the invention has been absorbed or encapsulated.
- the device may be implanted into any suitable tissue or organ, and delivery of the immunotargeting agent of the invention may be via diffusion, timed- release bolus, or continuous administration.
- it may be desirable to use pharmaceutical compositions in an ex vivo manner. In such instances, cells, tissues, or organs that have been removed from the patient are exposed to the pharmaceutical compositions after which the cells, tissues and/or organs are subsequently implanted back into the patient.
- an immunotargeting agent of the invention can be delivered by implanting certain cells that have been genetically engineered to express and secrete the polypeptide.
- Such cells may be animal or human cells, and may be autologous, heterologous, or xenogeneic.
- the cells may be immortalized.
- the cells may be encapsulated to avoid infiltration of surrounding tissues.
- the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destraction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.
- Targeting agents of the invention can be utilized in combination with other therapeutic agents, and may enhance the effect of these other therapeutic agents such that a lesser daily amount, lesser total amount or reduced frequency of administration is required in order to achieve the same therapeutic effect at reduced toxicity.
- these other therapeutics include, for example radiation treatment, chemotherapeutic agents, as well as other growth factors.
- these other therapeutics include for example immunosuppressants such as cyclosporine, azathioprine corticosteroids, acrolimus or mycophenolate mofetil.
- the antibody of the invention is used as a radiosensitizer.
- the antibody of the invention is conjugated to a radiosensitizing agent.
- radiosensitizer is defined as a molecule, preferably a low molecular weight molecule, administered to animals in therapeutically effective amounts to increase the sensitivity of the cells to be radiosensitized to electromagnetic radiation and/or to promote the treatment of diseases that are treatable with electromagnetic radiation.
- Diseases that are treatable with electromagnetic radiation include neoplastic diseases, benign and malignant tumors, and cancerous cells.
- electromagnetic radiation and “radiation” as used herein include, but are not limited to, radiation having the wavelength of 10 "20 to 100 meters.
- Preferred embodiments of the present invention employ the electromagnetic radiation of: gamma- radiation (10 ⁇ 20 to 10 "13 m), X-ray radiation (10 "12 to 10 '9 m), ultraviolet light (10 nm to 400 nm), visible light (400 nm to 700 nm), infrared radiation (700 nm to 1.0 mm), and microwave radiation (1 mm to 30 cm).
- Radiosensitizers are known to increase the sensitivity of cancerous cells to the toxic effects of electromagnetic radiation.
- Many cancer treatment protocols currently employ radiosensitizers activated by the electromagnetic radiation of X-rays.
- X-ray activated radiosensitizers include, but are not limited to, the following: metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, EO9, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cisp latin, and therapeutically effective analogs and derivatives of the same.
- metronidazole misonidazole
- desmethylmisonidazole pimonidazole
- Photodynamic therapy (PDT) of cancers employs visible light as the radiation activator of the sensitizing agent.
- photodynamic radiosensitizers include the following, but are not limited to: hematopo ⁇ hyrin derivatives, Photofrin(r), benzopo ⁇ hyrin derivatives, NPe6, tin etiopo ⁇ hyrin (SnET2), pheoborbide-a, bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc phthalocyanine, and therapeutically effective analogs and derivatives of the same.
- Chemotherapy treatment can employ anti-neoplastic agents including, for example, alkylating agents including: nitrogen mustards, such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU); ethylenimines/methylmelamine such as thriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cy
- mithramycin mitomycinC, and actinomycin
- enzymes such as L-asparaginase
- biological response modifiers such as interferon-alpha, IL-2, G-CSF and GM-CSF
- miscellaneous agents including platinium coordination complexes such as cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocortical suppressants such as mitotane (o,p'-DDD) and aminoglutethimide
- hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide
- progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol a
- Combination therapy with growth factors can include cytokines, lymphokines, growth factors, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2,
- compositions can include known angiopoietins, for example, vascular endothelial growth factor (VEGF).
- VEGF vascular endothelial growth factor
- Growth factors include angiogenin, bone mo ⁇ hogenic protein- 1, bone mo ⁇ hogenic protein-2, bone mo ⁇ hogenic protein-3, bone mo ⁇ hogenic protein-4, bone mo ⁇ hogenic protein-5, bone mo ⁇ hogenic protein-6, bone mo ⁇ hogenic protein-7, bone mo ⁇ hogenic protein-8, bone mo ⁇ hogenic protein-9, bone mo ⁇ hogenic protein- 10, bone mo ⁇ hogenic protein- 11, bone mo ⁇ hogenic protein- 12, bone mo ⁇ hogenic protein- 13, bone mo ⁇ hogenic protein- 14, bone mo ⁇ ho genie protein- 15, bone mo ⁇ hogenic protein receptor LA, bone mo ⁇ hogenic protein receptor LB, brain derived neurotrophic factor, ciliary neutrophic factor, ciliary neutrophic factor receptor, cytokine- induced neutrophil chemotactic factor 1, cytokine-induced neutrophil chemotactic factor 2,.
- endothelial cell growth factor endothelin 1 , epidermal growth factor, epithelial-derived neutrophil attractant, fibroblast growth factor 4, fibroblast growth factor 5, fibroblast growth factor 6, fibroblast growth factor 7, fibroblast growth factor 8, fibroblast growth factor 8b, fibroblast growth factor 8c, fibroblast growth factor 9, fibroblast growth factor 10, fibroblast growth factor acidic, fibroblast growth factor basic, glial cell line-derived neutrophic factor receptor 1 , glial cell line-derived neutrophic factor receptor 2, growth related protein, growth related protein, growth related protein, growth related protein, growth related protein, heparin binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor, nerve growth factor nerve growth factor receptor, neurotrophin-3, neurotrophin-4, placenta
- Determining the status of the expression patterns of a cell surface antigen of the invention in an individual may be used to diagnose cancer and may provide prognostic information useful in defining appropriate therapeutic options.
- the expression status of a cell surface antigen of the invention may provide information useful for predicting susceptibility to particular disease stages, progression, an 'or tumor aggressiveness.
- the invention provides methods and assays for determining the expression status of and diagnosing cancers that express a cell surface antigen of the invention.
- the invention provides assays useful in determining the presence of cancer in an individual, comprising detecting a significant increase or decrease, as applicable, in CSA mRNA or protein expression in a test cell or tissue or fluid sample relative to expression levels in the corresponding normal cell or tissue.
- the presence of CSA mRNA is evaluated in tissue samples of a lymphoma.
- the presence of significant expression of a cell surface antigen of the invention may be useful to indicate whether the lymphoma is susceptible to targeting using a targeting composition of the invention.
- CSA expression status may be determined at the protein level rather than at the nucleic acid level.
- such a method or assay would comprise determining the level of a CSA expressed by cells in a test tissue sample and comparing the level so determined to the level of cell surface antigen of the invention expressed in a conesponding normal sample.
- the presence of a cell surface antigen of the invention is evaluated, for example, using immunohistochemical methods.
- Antibodies capable of detecting expression of a cell surface antigen of the invention may be used in a variety of assay formats well known in the art for this pu ⁇ ose.
- Peripheral blood may be conveniently assayed for the presence of cancer cells, including lymphomas and leukemias, using RT-PCR to detect expression of a cell surface antigen of the cell-surface antigen of the invention provides an indication of the presence of one of these types of cancer.
- a sensitive assay for detecting and characterizing carcinoma cells in blood may be used (Racila, et al, Proc. Natl Acad. Sci. USA 95: 4589-4594 (1998), herein inco ⁇ orated by reference in its entirety). This assay combines immunomagnetic enrichment with multiparameter flow cytometric and immunohistochemical analyses, and is highly sensitive for the detection of cancer cells in blood, reportedly capable of detecting one epithelial cell in 1 ml of peripheral blood.
- a related aspect of the invention is directed to predicting susceptibility to developing cancer in an individual.
- a method for predicting susceptibility to cancer comprises detecting CSA mRNA in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of said CSA mRNA expression present is proportional to the degree of susceptibility.
- a method for gauging aggressiveness of a tumor comprises determining the level of CSA mRNA or protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of said CSA mRNA or protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wheiein the degree of said CSA mRNA or protein expression in the tumor sample relative to the normal sample indicates the degree of aggressiveness.
- Standard methods for the detection and quantification of said CSA mRNA include in situ hybridization using labeled CSA riboprobes (Gemou- Engesaeth, 2002, supra), Northern blot and related techniques using CSA polynucleotide probes (Kunzli, et al, 2002, supra) , RT-PCR analysis using CSA primers (Angchaiskisiri, et al, 2002, supra), and other amplification type detection methods, such as, for example, branched DNA (Jardi, et al, 2001, supra), SISBA, TMA (Kimura, et al, 2002, supra), and microarray products of a variety of sorts, such as oligos, cDNAs, and monoclonal antibodies.
- real-time RT-PCR may be used to detect and quantify cell surface antigen of the invention mRNA expression (Simpson, et al, 2000, supra). Standard methods for the detection and quantification of protein may be used for this pu ⁇ ose.
- polyclonal or monoclonal antibodies specifically reactive with the wild-type cell surface antigen of the invention may be used in an immunohistochemical assay of biopsied tissue (Ristimaki, et al, Cancer Res. 62:632 (2002), herein inco ⁇ orated by reference in its entirety).
- Antibodies that recognize the cell-surface antigens of the invention and fragments thereof are useful in medical imaging of sites expressing the cell surface antigens of the invention. Such methods involve chemical attachment of a labeling or imaging agent, such as a radioisotope, which include 67 Cu, 90 Y, ,25 1, 131 1, 186 Re, 188 Re, 2, 1 At, 212 Bi, administration of the labeled antibody and fragment to a subject in a pharmaceutically acceptable carrier, and imaging the labeled antibody and fragment in vivo at the target site. Radiolabelled antibodies of the invention or fragments thereof may be particularly useful in in vivo imaging of cancers expressing the cell-surface antigens of the invention, such as lymphomas or leukemias. Such antibodies may provide highly sensitive methods for detecting metastasis of said cancers.
- a labeling or imaging agent such as a radioisotope, which include 67 Cu, 90 Y, ,25 1, 131 1, 186 Re, 188 Re, 2, 1 At, 212 Bi
- CD84Hyl was determined in various lymphoid and myeloid cell lines. Poly- A messenger RNA was isolated from the cell lines listed in Table 2 and subjected to quantitative, real-time PCR analysis (Simpson, et al, Molec. Vision. 6:178-183 (2000), herein inco ⁇ orated by reference in its entirety) to determine the relative copy number of CD84Hyl mRNA expressed per cell in each line. Elongation factor 1 gene expression was used as a positive control and normalization factors in all samples.
- CD84HY1 MRNA IS HIGHLY EXPRESSED IN PRIMARY B CELLS, LYMPH NODE TISSUE,
- CD84Hyl was determined in various healthy and tumor patient tissues (Table 3).
- Poly-A mRNA was isolated from the tonsilar lymph node, lymphoma, Hodgkin's disease, and acute myeloid leukemia (AML) tissue sample ' s obtained from the Cooperative Human Tissue Network (CHTN, National Cancer Institute), whereas all other RNAs were purchased from Clontech (Palo Alto, CA) and Ambion (Austin, TX). All patient tissue samples from the CHTN were snap frozen immediately after surgical removal.
- Poly-A + mRNA was subjected to quantitative, real-time PCR analysis, as described in Example 1, to determine the relative expression of CD84Hyl mRNA in the sample.
- Tonsilar lymph nodes were used as non-lymphoma containing nodal tissue (7117), whereas 5348, 5856 and 6796 were B-cell follicular lymphomas and samples 6879 and 22601 were diffuse large B-cell lymphoma samples.
- One lymph node diagnosed with Hodgkin's disease and one splenic AML sample were also analyzed (566 and 565, respectively).
- Table 2 Example 1
- Table 3 demonstrate high levels of expression of CD84Hyl in the B cell lymphoma cell lines CA-46, RL, HT, ST486 and GA- 10 Additionally, peripheral blood B cells (CD 19+ cells), lymph node tissue and the multiple myeloma cell line U266 were also found to have high levels of expression.
- An intermediate level of expression of CD84Hyl was found in the T cell leukemia lines Molt-4 and Jurkat, whereas healthy peripheral blood T cells (isolated with a pan T cell marker) were found to have only low levels of expression.
- CD84Hyl Healthy peripheral blood monocytes (CD 14+) showed no detectable CD84Hyl, whereas the acute myeloid leukemia cell line, KG-1, and the AML patient tissue sample showed low levels of expression. Most non-hematopoeitic healthy tissues did not demonstrate expression of CD84Hyl with the exception of lung, bladder, and cervix (low expression) and colon (with moderate levels of expression). Expression in these healthy tissues, in general, was found to be very low and may be accounted for by lymphoid tissues or leukocytes associated with the original collected tissue. Expression of CD84Hyl in these tumor tissues demonstrates its usefulness as an immunotherapeutic target. Additionally, these results indicate that CD84Hyl mRNA expression may be used as a diagnostic marker for certain cell types or disorders (e.g., B-cell lymphomas, AML, Hodgkin's disease and T cell lymphomas).
- B-cell lymphomas e.g., B-cell lymphomas, AML, Hodgkin'
- CD84Hyl in tissue samples was detected using anti-CD84Hyl antibodies (see Table 4).
- Samples were prepared for immunohistochemical (IHC) analysis (Clinomics Biosciences, Inc., Pittsfield, MA) by fixing tissues in 10% formalin embedding in paraffin, and sectioning using standard techniques. Sections were stained using the CD84Hyl -specific antibody followed by incubation with a secondary horseradish peroxidase (HRP)-conjugated antibody and visualized by the product of the HRP enzymatic reaction.
- HRP horseradish peroxidase
- CD84Hyl was overexpressed in rejected heart, liver and kidney, whereas CD84Hyl was not present on healthy tissues. Therefore, targeting CD84Hyl may be useful to prevent or reduce tissue rejection after transplantation.
- PBMC Peripheral blood mononuclear cells
- the cells are washed with ice-cold PBS and incubated on ice with the CD84Hyl -specific polyclonal antibody for 30 min.
- the cells are gently pelleted, washed with PBS, and incubated with a fluorescent anti-rabbit antibody for 30 min. on ice. After the incubation, the cells are gently pelleted, washed with ice cold PBS, and resuspended in PBS containing 0.1% sodium azide and stored on ice until analysis.
- Samples are analyzed using a FACScalibur flow cytometer (Becton Dickinson) and CELLQuest software (Becton Dickinson). Instrument setting are determined using FACS-Brite calibration beads (Becton-Dickinson).
- Tumors expressing CD84Hyl is imaged using CD84Hyl -specific antibodies conjugated to a radionuclide, such as 123 I, and injected into the patient for targeting to the tumor followed by X-ray or magnetic resonance imaging.
- a radionuclide such as 123 I
- o2MHyl was determined in various normal and tumor tissues.
- the polyA+ RNA was subjected to quantitative, real-time PCR analysis (Simpson, et al, 2000, supra) to determine the relative copy number of o.2MHy mRNA expressed per cell in each line.
- DNA sequences targeting the Elongation factor 1 gene were used as a positive control and normalization factors in all samples.
- Table 7 shows the relative expression of o2MHy RNA.
- Tissue RNAs in Table 7 were obtained from commercial sources (Clontech (Palo Alto, CA) and Ambion (Austin, TX)) or from the Cooperative Human Tissue Network (National Cancer Institute).
- o2MHy genes based on the expression results, is upregulated in many cancer types and is may be involved in promoting tumor growth and metastasis.
- o2MHyl was determined in various B-cell lymphomas, multiple myelomas, and T-cell leukemias (Table 8).
- Poly-A RNA was isolated from these cell lines which were obtained from the American Tissue Type Culture Collection. Poly-A + RNA was subjected to quantitative, real-time PCR analysis, as described in Example 4, to determine the relative expression of o2MHyl mRNA in the sample. All assays were performed in duplicate with the resulting values averaged and expressed as "-" for samples with no detectable o2MHyl mRNA in that sample to "+++" for samples with the highest mRNA copy number for o2MHyl.
- IGPFP-7Hyl was determined in various types of normal and tumor tissues, and shown in Tables 9-11.
- Poly-A RNA was isolated from tissue samples obtained from the Cooperative Human Tissue Network (National Cancer Institute) (Table 10), whereas all other RNAs were purchased from Clontech (Palo Alto, CA) and Ambion (Austin, TX). The RNA was isolated from the tissues and subjected to quantitative, realtime PCR analysis (Simpson, et al, 2000, supra) to determine the relative copy number of IGPFP-7Hyl mRNA expressed per cell in each line. DNA sequences targeting the Elongation factor 1 gene were used as a positive control and normalization factors in all samples.
- Tissues in Table 10 were obtained from the Cooperative Human Tissue Network (CHTN). All tissues were snap frozen after surgical removal. Poly A mRNA was was isolated from the frozen tissue using standard protocols. Normal tissues and corresponding tumor tissues were derived from the same patient in both Table 9 and Table 10 (except D237 and D236, in which the tissue samples were derived from different patients). In the lung tumor patients, the protein was often down regulated to zero. Similarly, IGFBP-7Hyl was found to be reduced in expression in 2 out of 5 colon tumor samples (note in Table 9 that the normal colon expression was estimated at 6 copies/cell vs. 2 copies/cell for the corresponding adjacent tumor). Taken together, the results in Table 9 and Table 10 support the role of IGFBP-7Hyl as a protein with tumor suppressor like properties. Table 11
- IGFBP-7Hyl protein functions as a growth-suppressing factor, as well as an IGF and insulin-binding protein.
- IGFBP-7HY1 SUPPRESSED GROWTH OF TUMOR CELL LINES A. TRANSFECTION OF TUMOR CELL LINES WITH IGFBP-7HY1 DNA
- IGFBP-7Hyl cells expressing IGFBP- 7Hyl, from a mammalian expression vector (Trexi, Aurora Biosciences, San Diego, CA) containing the coding sequence of SEQ LD NO: 53 and the Yellow Fluorescent Protein (YFP) gene, were produced by transfection of the human cervical carcinoma cell line, HeLa, using the FuGENE-6 (Roche Biosciences, Nutley, NJ) reagent according to manufacturer's protocol. Transfecting the HeLa cells with a mammalian expression vector (Trexi, Aurora Biosciences) containing the Yellow Fluorescent Protein (YFP) gene alone produced control cells.
- a mammalian expression vector Texi, Aurora Biosciences, San Diego, CA
- YFP Yellow Fluorescent Protein
- IGFBP-7Hyl/Trexi-transfected cells (co-expresses YFP) and control cells were sorted and seeded directly as 1,000 cells/well in 96-well plates. Cell growth and proliferation were monitored by cell counts at 24, 48, 72, and 96 hours after transfection. Suppression of tumor cell growth by IGFBP-7Hyl was indicated by a reduction in the number of cells transfected with IGFBP-7-Hyl relative to the number of control cells over the course of the assay. An example of this assay is demonstrated in Table 12, which shows there was a statistically significant reduction in IGFBP-7Hyl -transfected cell numbers at 24, 48, 72, and 96 hours as compared to control cells.
- the assay was carried out 3 times and suppression of tumor cell growth by IGFBP- 7Hyl protein was observed in each assay.
- TLR9 TLR9 was determined in various lymphoid and myeloid cell lines.
- Poly-A messenger RNA was isolated from the cell lines listed in Table 14 and subjected to quantitative, real-time PCR analysis (Simpson, et al, 2000, supra) to determine the relative copy number of TLR9 mRNA expressed per cell in each line. Elongation factor 1 mRNA expression was used as a positive control and normalization factors in all samples.
- TLR9 TLR9 was determined in various healthy and tumor tissues (Table 15).
- Poly-A mRNA was isolated from the tonsilar lymph node and lymphoma, AML and Hodgkin's Disease tissue samples obtained from the Cooperative Human Tissue Network (CHTN, National Cancer Institute). All other RNAs were purchased from Clontech (Palo Alto, CA) and Ambion (Austin, TX). Tonsilar lymph nodes were used as non-lymphoma containing nodal tissue (7117), whereas 5348, 5856 and 6796 were B-cell follicular lymphomas and samples 22601 and 6879 were diffuse large B-cell lymphoma samples. Lymph node and lymphoma patient tissue samples were snap frozen immediately after surgical removal.
- Poly-A + mRNA was subjected to quantitative, real-time PCR analysis, as described in Example 8, to determine the relative expression of TLR9 mRNA in the sample. All assays were performed in duplicate with the resulting values averaged and expressed as "-" for samples with no detectable TLR9 mRNA in that sample to "+++” for samples with the highest mRNA copy number for TLR9.
- Table 15 demonstrate that B cell and T cell lymphoma tissue expressed low to moderate levels of TLR9 mRNA. Additionally, low levels of expression were also observed in Hodgkin's disease and AML tissue. Non-cancerous tonsilar lymph nodes, healthy peripheral blood B cells (CD 19+ cells), and lung tissue were found to have medium levels of expression. Healthy peripheral blood monocytes (CD 14+) showed low levels of expression. Most non-hematopoeitic healthy tissues did not demonstrate expression of TLR9 or only expressed at low levels.
- TLR9 mRNA expression in different Non-Hodgkin's B cell lymphoma, T cell lymphomas, Hodgkin's disease and AML tissues and cell lines, and indicate that TLR9 may be used as an immunotherapeutic antibody target or as a diagnostic marker for these types of disorders.
- TLR9 expression of TLR9 in tissue samples (normal or diseased) was detected using anti- TLR9 antibodies (see Table 16).
- Samples were prepared for immunohistochemical (LHC) analysis by fixing tissues in 10% formalin embedding in paraffin, and sectioning using standard techniques. Sections were stained using the TLR9-specific antibody followed by incubation with a secondary horseradish peroxidase (HRP)-conjugated antibody and visualized by the product of the HRP enzymatic reaction. Data as seen in Table 16 shows that TLR9 is highly expressed on cell surface of tumor tissues. No expression of TLR9 was observed on the cell surface of normal tissues.
- HRP horseradish peroxidase
- TLR9 expression was found in solid tumors of prostate, breast, colon, and squamous cell carcinoma (see Table 17). Based on this expression pattern, it is likely that other cancers of epithelial and squamous cell origin will also express TLR9.
- TLR9 was overexpressed in tissues from systemic lupus erythematosus, Hasimoto thyroiditis, Sj ⁇ rgen's syndrome, and pericarditis lupus.
- PBMC Peripheral blood mononuclear cells
- the cells are washed with ice-cold PBS and incubated on ice with the TLR9-specific polyclonal antibody for 30 min.
- the cells are gently pelleted, washed with PBS, and incubated with a fluorescent anti-rabbit antibody for 30 min on ice. After the incubation, the cells are gently pelleted, washed with ice cold PBS, and resuspended in PBS containing 0.1% sodium azide and stored on ice until analysis.
- Samples are analyzed using a FACScalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ) and CELLQuest software (Becton Dickinson). Instrument settings are determined using FACS-Brite calibration beads (Becton-Dickinson).
- Tumors expressing TLR9 are imaged using TLR9-specific antibodies conjugated to a
- radionuclide such as I
- VpreBl was determined in various lymphoid and myeloid cell lines. Poly-A messenger RNA was isolated from the cell lines listed in Table 20 and subjected to quantitative, real-time PCR analysis (Simpson, et al., 2000, supra) to determine the relative copy number of VpreBl mRNA expressed per cell in each line. Elongation factor 1 mRNA expression was used as a positive control and normalization factors in all samples.
- VpreBl was determined in various healthy and tumor tissues (Table 21).
- Poly-A mRNA was isolated from frozen patient tissue samples obtained from the Cooperative Human Tissue Network (CHTN, National Cancer Institute). mRNAs from healthy lymph nodes or marginal zone B lymphoma cells were purchased from Ambion (Austin, TX). Poly-A + mRNA was subjected to quantitative, real-time PCR analysis, as described in Example 11, to determine the relative expression of VpreBl mRNA in the sample. All assays were performed in duplicate with the resulting values averaged and expressed as "-" for samples with no detectable VpreBl mRNA in that sample to "+++" for samples with the highest mRNA copy number for VpreBl.
- VpreBl mRNA The results in Table 21 demonstrate the expression of VpreBl mRNA in. primary patient B cell lymphoma tumor tissues. Expression of VpreBl mRNA was demonstrated in follicular, diffuse large B cell, and marginal zone B type lymphomas. Lymph node tissue was found not to express VpreBl mRNA. Additionally, VpreBl gene expression was observed in T cell lymphoma tumor tissues, but not in healthy peripheral blood derived T cells. The data suggests VpreBl is differentially upregulated in lymphomas compared to healthy lymph nodes.
- Anti- VpreBl antibodies were used to detect cell surface expression of VpreBl.
- Three non-H ⁇ dgkin's lymphoma cell lines (CA46, GA-10 and HT cell lines) were incubated with a FITC-conjugated VpreBl monoclonal antibody (Serotec, Raleigh, NC; Sanz and de la Hera, J. Exp. Med. 183:2693-2698 (1996), inco ⁇ orated herein by reference in its entirety) to detect VpreBl on the cell surface.
- Antibody labeling of cell surface proteins was detected by flow cytometry.
- FIG. 4 shows the cell surface expression of VpreBl on B cell non-Hodgkin's lymphoma cell lines.
- CA46, GA-10 and HT cell lines were stained with an anti- VpreBl antibody conjugated with FITC (white fill graph) or with a FITC-conjugated IgM control (black fill graph) antibody.
- FITC labeling is shown on the x-axis compared to the number of cells labeled on the y-axis.
- the gated areas designated Ml indicate 69%, 91% and 3% of cells surface labeled with the anti- VpreBl antibody in CA46, GA-10 and HT cells, respectively.
- Cells expressing CSAs of the invention are identified using antibodies to said CSAs.
- Polyclonal antibodies are produced by DNA vaccination or by injection of peptide antigens into rabbits or other hosts.
- An animal such as a rabbit, is immunized with a peptide from the extracellular region of a CSA of the invention conjugated to a carrier protein, such as BSA (bovine seram albumin) or KLH (keyhole limpet hemocyanin).
- BSA bovine seram albumin
- KLH keyhole limpet hemocyanin
- Anti-CSA antibody is affinity purified from rabbit seram using CSA peptide coupled to Affi- Gel 10 (Bio-Rad), and stored in phosphate-buffered saline (PBS) with 0.1% sodium azide.
- an expression vector encoding said CSA is introduced into mammalian cells. Western blot analysis of protein extracts of non-transfected cells and the CSA-containing cells is performed using the polyclonal antibody sample as the primary antibody and a horseradish peroxidase-labeled anti-rabbit antibody as the secondary antibody. Detection of a band corresponding to the molecular weight of said CSA in the CSA-containing cells and lack thereof in the control cells indicates that the polyclonal antibodies are specific for said CSA.
- Monoclonal antibodies are produced by injecting mice with a CSA peptide, with or without adjuvant. Subsequently, the mouse is boosted every 2 weeks until an appropriate immune response has been identified (typically 1-6 months), at which point the spleen is removed. The spleen is minced to release splenocytes, which are fused (in the presence of polyethylene glycol) with murine myeloma cells. The resulting cells (hybridomas) are grown in culture and selected for antibody production by clonal selection. The antibodies are secreted into the culture supernatant, facilitating the screening process, such as screening by an enzyme-linked immunosorbent assay (ELISA).
- ELISA enzyme-linked immunosorbent assay
- humanized monoclonal antibodies are produced either by engineering a chimeric murine/human monoclonal antibody in which the murine-specific antibody regions are replaced by the human counte ⁇ arts and produced in mammalian cells, or by using transgenic "knock out" mice in which the native antibody genes have been replaced by human antibody genes and immunizing the transgenic mice as described above.
- ADCC antibody-dependent cell-mediated cytoxicity
- PBMC peripheral blood mononuclear cells
- RA1 cells are suspended in RPMI 1640 medium supplemented with 2% fetal bovine serum and plated in 96-well V-bottom microtitier plates at 2 x 10 4 cells/well.
- CSA-specific antibody is added in triplicate to individual wells at 1 ⁇ g/ml, and effector cells are added at various effecto ⁇ target cell ratios (12.5:1 to 50:1).
- the plates are incubated for 4 hours at 37°C. The supernatants are then harvested, lactate dehydrogenase release determined, and percent specific lysis calculated using the manufacture's protocols.
- Antibodies to a CSA of the invention are conjugated to toxins and the effect of such conjugates in animal models of cancer is evaluated.
- Chemotherapeutic agents such as calicheamycin and carboplatin, or toxic peptides, such as ricin toxin, are used in this approach.
- Antibody-toxin conjugates are used to target cytotoxic agents specifically to cells bearing the antigen. The antibody-toxin binds to these antigen-bearing cells, becomes internalized by receptor-mediated endocytosis, and subsequently destroys the targeted cell.
- the antibody-toxin conjugate targets CSA-expressing cells, such as B cell lymphomas, and deliver the cytotoxic agent to the tumor resulting in the death of the tumor cells.
- a toxin that may be conjugated to an antibody is carboplatin.
- the mechanism by which this toxin is conjugated to antibodies is described in Ota et al, Asia-Oceania J. Obstet. Gynaecol 19: 449-457 (1993), herein inco ⁇ orated by reference in its entirety.
- the cytotoxicity of carboplatin-conjugated CSA-specific antibodies is evaluated in vitro, for example, by incubating target cells expressing said CSA (such as the RAl B cell lymphoma cell line) with various concentrations of conjugated antibody, medium alone, carboplatin alone, or antibody alone.
- the antibody-toxin conjugate specifically targets and kills cells bearing the CSA antigen, whereas, cells not bearing the antigen, or cells treated with medium alone, carboplatin alone, or antibody alone, show no cytotoxicity.
- mice Five to six week old, athymic nude mice are engrafted with tumors subcutaneously or through intravenous injection. Mice are treated with the CSA antibody-carboplatin conjugate or with a non-specific antibody-carboplatin conjugate. Tumor xenografts in the mouse bearing the CSA antigen are targeted and bound to by the CSA antibody-carboplatin conjugate. This results in tumor cell killing as evidenced by tumor necrosis, tumor shrinkage, and increased survival of the treated mice.
- toxins are conjugated to CSA-specific antibodies using methods known in the art.
- An example of a toxin conjugated antibody in human clinical trials is CMA-676, an antibody to the CD33 antigen in AML which is conjugated with calicheamicin toxin (Larson, Semin. Hematol. 38(Suppl 6):24-31 (2001), herein inco ⁇ orated by reference in its entirety).
- Animal models are used to assess the effect of antibodies specific to a cell surface antigen of the invention as vectors in the delivery of radionuclides in radio-immunotherapy to treat lymphoma, hematological malignancies, and solid tumors.
- Human tumors are propagated in 5-6 week old athymic nude mice by injecting a carcinoma cell line or tumor cells subcutaneously.
- Tumor-bearing animals are injected intravenously with radio-labeled anti-CSA antibody (labeled with 30-40 ⁇ Ci of 13, I, for example) (Behr, et al, Int. J. Cancer 11: 787-795 (1988), inco ⁇ orated herein by reference in its entirety).
- Tumor size is measured before injection and on a regular basis (i.e.
- Anti-tumor efficacy is calculated by correlating the calculated mean tumor doses and the extent of induced growth retardation.
- animals are sacrificed by cervical dislocation and autopsied. Organs are fixed in 10% formalin, embedded in paraffin, and thin sectioned. The sections are stained with hematoxylin-eosin.
- mice Animal models are used to evaluate the effect of CSA-specific antibodies as targets for antibody-based immunotherapy using monoclonal antibodies.
- Human myeloma cells are injected into the tail vein of 5-6 week old nude mice whose natural killer cells have been eradicated.
- mice receive an intraperitoneal injection with.
- CSA-specific antibodies either 1 or 15 days after tumor inoculation followed by either a daily dose of 20 ⁇ g or 100 ⁇ g once or twice a week, respectively (Ozaki, et al, Blood 90:3179-3186 (1997), herein inco ⁇ orated by reference in its entirety).
- Levels of human IgG are measured in the murine sera by ELISA.
- CSA-specific antibodies on the proliferation of myeloma cells is examined in vitro using a 3 H-thymidine inco ⁇ oration assay (Ozaki et al, 1997, supra).
- Cells are cultured in 96-well plates at lxlO 5 cells/ml in 100 ⁇ l/well and incubated with various amounts of said CSA antibody or control IgG (up to 100 ⁇ g/ml) for 24 h.
- Cells are incubated with 0.5 ⁇ Ci 3 H-thymidine (New England Nuclear, Boston, MA) for 18 h and harvested onto glass filters using an automatic cell harvester (Packard, Meriden, CT). The inco ⁇ orated radioactivity is measured using a liquid scintillation counter.
- the cytotoxicity of the CSA monoclonal antibody is examined by the effect of complements on myeloma cells using a 5! Cr-release assay (Ozaki et al, 1997, supra).
- Myeloma cells are labeled with 0.1 mCi 51 Cr-sodium chromate at 37°C for 1 h.
- 51 Cr-labeled cells are incubated with various concentrations of the CSA monoclonal antibody or control IgG on ice for 30 min. Unbound antibody is removed by washing with medium. Cells are distributed into 96-well plates and incubated with serial dilutions of baby rabbit complement at 37°C for 2 h.
- the supernatants are harvested from each well and the amount of 51 Cr released is measured using a gamma counter.
- Spontaneous release of 51 Cr is measured by incubating cells with medium alone, whereas maximum 51 Cr release is measured by treating cells with 1% NP-40 to disrupt the plasma membrane.
- Percent cytotoxicity is measured by dividing the difference of experimental and spontaneous 51 Cr release by the difference of maximum and spontaneous 51 Cr release.
- Antibody-dependent cell-mediated cytotoxicity (ADCC) for the CSA monoclonal antibody is measured using a standard 4 h 51 Cr-release assay (Ozaki et al, 1997, supra). Splenic mononuclear cells from SCLD mice are used as effector cells and cultured with or without recombinant interleukin-2 (for example) for 6 days. 51 Cr-labeled target myeloma cells (1 xlO cells) are placed in 96-well plates with various concentrations of anti-CSA monoclonal antibody or control IgG. Effector cells are added to the wells at various effector to target ra ⁇ os (12.5:1 to 50:1). After 4 h. culture supernatants are removed and counted in a gamma counter. The percentage of cell lysis is determined as above.
- Animal models are used to assess the effect of antibodies specific to a CSA of the invention that block signaling through the CSA receptor to suppress autoimmune diseases, such as arthritis or other inflammatory conditions, or rejection of organ transplants. Immunosuppression is tested by injecting mice with horse red blood cells (HRBCs) and assaying for the levels of HRBC-specific antibodies (Yang, et al, Int. Immunopharm. 2:389- 397 (2002), herein inco ⁇ orated by reference in its entirety). Animals are divided into five groups, three of which are injected with anti-CSA antibodies for 10 days, and 2 of which receive no treatment.
- HRBCs horse red blood cells
- EBSS Earle's balanced salt solution
- Lmmunosupression is measured by the number of B cells producing HRBC-specific antibodies.
- the Ig isotype (for example, IgM, IgGl, LgG2, etc.) is determined using the IsoDetectTM Isotyping kit (Stratagene, La Jolla, CA). Once the Ig isotype is known, murine antibodies against HRBCs are measured using an ELISA procedure. 96-well plates are coated with HRBCs and incubated with the anti-HRBC antibody-containing sera isolated from the animals.
- the plates are incubated with alkaline phosphatase-labeled secondary antibodies and color development is measured on a microplate reader (SPECTRAmax 250, Molecular Devices) at 405 nm using jo-nitrophenyl phosphate as a substrate.
- SPECTRAmax 250 Molecular Devices
- Lymphocyte proliferation is measured in response to the T and B cell activators concanavalin A and lipopolysaccharide, respectively (Jiang, et al, J. Immunol. 154:3138- 3146 (1995), herein inco ⁇ orated by reference in its entirety).
- Mice are randomly divided into 2 groups, 1 receiving said anti-CSA antibody therapy for 7 days and 1 as a control.
- the animals are sacrificed by cervical dislocation, the spleens are removed, and splenocyte suspensions are prepared as above.
- the anti-CSA antibody is added to the medium at the beginning of the experiment.
- Cell proliferation is also assayed using the H-thymidine inco ⁇ oration assay described above (Ozaki, et al, Blood 90: 3179 (1997), inco ⁇ orated herein by reference in its entirety).
- Assays are carried out to assess activity of fragments of the CSA protein, such as the Ig domain, to stimulate cytokine secretion and to stimulate immune responses in, for example, NK cells, B cells, T cells, and myeloid cells.
- Such immune responses can be used to stimulate the immune system to recognize and/or mediate tumor cell killing or suppression of growth.
- this immune stimulation can be used to target bacterial or viral infections.
- fragments of the CSA that block activation through the CSA receptor may be used to block immune stimulation in NK, B, T, and myeloid cells.
- Fusion proteins containing fragments of the CSA, such as the Ig domain (CSA-Ig), are made by inserting a CD33 leader peptide, followed by a CSA domain fused to the Fc region of human IgGl into a mammalian expression vector, which is stably transfected into NS-1 cells, for example.
- the fusion proteins are secreted into the culture supernatant, which is harvested for use in cytokine assays, such as interferon- ⁇ (IFN- ⁇ ) secretion assays (Martin, et al, J. Immunol. 167:3668-3676 (2001), herein inco ⁇ orated by reference in its entirety).
- IFN- ⁇ interferon- ⁇ secretion assays
- PBMCs are activated with a suboptimal concentration of soluble CD3 and various concentrations of purified, soluble anti-CSA monoclonal antibody or control IgG.
- CSA- Ig cytokine assays anti-human Fc Ig at 5 or 20 ⁇ g/ml is bound to 96-well plates and incubated overnight at 4°C. Excess antibody is removed and either CSA-Ig or control Ig is added at 20-50 ⁇ g/ml and incubated for 4 h at room temperature. The plate is washed to remove excess fusion protein before adding cells and anti-CD3 to various concentrations. Supernatants are collected after 48 h of culture and LFN- ⁇ levels are measured by sandwich ELISA, using primary and biotinylated secondary anti-human LFN- ⁇ antibodies as recommended by the manufacturer.
- Antibodies specific to a CSA of the invention are used for imaging CSA-expressing cells in vivo.
- Six-week-old athymic nude mice are irradiated with 400 rads from a cesium source. Three days later the irradiated mice are inoculated with 4 ⁇ l0 7 RAl cells and 4 ⁇ l0 6 human fetal lung fibroblast feeder cells subcutaneously in the thigh. When the tumors reach approximately 1 cm in diameter, the mice are injected intravenously with an inoculum containing 100 ⁇ Ci/10 ⁇ g of 131 I-labeled CSA-specific antibody.
- mice are anesthetized with a subcutaneous injection of 0.8 mg sodium pentobarbital.
- the immobilized mice are then imaged in a prone position with a Spectrum 91 camera equipped with a pinhole collimator (Raytheon Medical Systems; Melrose Park, IL) set to record 5,000 to 10,000 counts using the Nuclear MAX Plus image analysis software package (MEDX Inc.; Wood Dale, IL) (Homick, et al, Blood 89:4437-4447 (1997), herein inco ⁇ orated by reference in its entirety).
- MEDX Inc. Wood Dale, IL
- Affinity cross-linking is one method to evaluate the affinity of CSA protein for growth factors and other regulatory molecules. Approximately 2-200 pmol of CSA protein are incubated with radiolabeled growth factors or other regulatory molecules such as 125 I- insulin, 125 I-IGF 1, 125 I-IGF II, 125 I-VEGF, or !25 I-TGF-/3 for 16h at 4°C. Disuccinimidyl suberate (Pierce) is added at a final concentration of 0.5 mM. After crosslinking for 15 minutes, the samples are subjected to 12% SDS-Page and autoradiography.
- an endothelial tubule formation assay can be performed (Asplin et al, Blood 97: 3450 (2001), herein inco ⁇ orateda by reference in its entirety).
- Growth factor-reduced Matrigel Cold-reduced Matrigel (Collaborative Biomedical Products, Bedford, MA) is used to coat 24-well plates.
- Fetal bovine heart endothelial (FBHE) cells, cultured in medium deficient in fibroblast growth factor-2 (FGF-2) for 48 h are plated onto the Matrigel layers at 40,000 cells/well in the presence of FGF-2 with or without anti-CSA antibodies and incubated for 96 h at 37°C and photographed.
- FGF-2 fibroblast growth factor-2
- Endothelial tubule formation can also be studied using collagen gels (Asplin et al, 2001, supra). Rat tail collagen type I (Collaborative Biomedical Products) is used to coat 48-well plates. Human umbilical vein endothelial cells (HUVECs) are plated onto the collagen gels at 30,000 cells per well in the presence of FGF-2 with or without anti-CSA antibodies for 24 h at 37°C photographed.
- HUVECs Human umbilical vein endothelial cells
- cells expressing a CSA are produced by liposome-mediated transfection of the tumorgenic human prostate epithelial cell line, M12, using Tfx-50 according to the manufacture's protocol and using DNA in a 60-mm tissue culture dish. Transfecting the Ml 2 cells with a mammalian expression vector alone produces control cells. Both transfected and controltransfected cells are maintained with G418 and the formation of individual colonies are monitored. Visible colonies are subcloned, using cloning rings, and each colony is transferred to a new well in a
- Western immunoblots are carried out by collecting media from the cells and normalizing based on the cell counts and concentrating by filtrating through nitrocellulose (Bimbaum et al, J. Endocrinology, 141:535-540 (1994), herein inco ⁇ orated by reference in its entirety). After concentration, proteins are redissolved in a mixture of SDS sample buffer (0.5 M Tris (pH 6.8)), 1% SDS, 10% glycerol, 0.003% bromphenol blue, and 8M urea by heating for 10 minutes at 100°C. Samples are electrophoresed on 12% SDS-polyacrylamide gels and then electroblotted onto nitrocellulose.
- Western blots are incubated with CSA antiseram at a 1:3000 dilution in 0.3% Tween 20 in Tris buffered saline (TBS) overnight at 4°C. Bound antibody is detected using a horseradish peroxidase-linked donkey antirabbit secondary antibody and the ECL detetdion system according to the manufacturer's protocol. Ligands blots were performed as described in the art (Damon et al, Endocrinology 139:3456-3464 (1998), herein inco ⁇ orated by reference in its entirety).
- Selected cell lines found to be expressing high levels of a CSA would then be used in growth assays. Cell growth and proliferation would be monitored by cell counts over the course of 2 weeks. Suppression of tumor cell growth by a CSA would be demonstrated by a reduction in cell number relative to the control cells over the course of the assay.
- Suppression of cell growth may be a result of a reduction in the rate of proliferation or by in increase in tumor cell apoptosis relative to control.
- mice After 8 weeks, the mice are sacrificed, and the tumors removed and digested with
- Dispersed cells are plated in ITS medium/5% FBS at %% CO 2 at 37°C for 24 hours to allow attachment. After 24 hours, the cultures are switched to serum-free medium. The cells are split, the media and RNA collected, and Western immunoblots using CSA and Northern blot are done.
- a reporter constract in which the human VEGF promoter is cloned upstream of firefly luciferase cDNA in a vector, such as the adenoviral Ad5 shuttle vector and analyzed according to Tai et al, Blood 99:1419-1427 (2002), herein inco ⁇ orated by reference in its entirety.
- Cells such as RPMI 8226 cells, are transfected with the reporter construct as well as the empty vector. Twenty-four hours after adenoviras infection, cells are incubated with anti-CSA antibodies, CSA peptide fragments, or media alone for 16 h. Cells are then harvested, lysed, and protein concentration of the cell extracts is determined by Bradford assay. Total protein content is used for normalization of luciferase activity. Luciferase activity, using equal amounts of protein, is measured using a Moonlight 2010 Luminometer (Analytical Luminescence Laboratory, Frederick, MD) at room temperature.
- [Ca 2+ ]j is measured using a digital imaging microscope as described in Malecaze, et al. Arch. Ophthalmol. 112:1476-1482 (1994), herein inco ⁇ orated by reference in its entirety. 50 ng VEGF is added to the cells in the presence or absence of CSA antibodies or peptide after a base-line [Ca 2+ ]i is obtained. VEGF can also be preincubated for 24 h before addition to the cells. EXAMPLE 27 IN VITRO ASSAY OF CELL PROLIFERATION AND MIGRATION
- CSA-specific antibodies or therapeutic peptides on the proliferation of myeloma cells is examined in vitro using a 3 H-thymidine inco ⁇ oration assay (Ozaki et al, Blood 90:3179-3186 (1997), herein inco ⁇ orated by reference in its entirety.
- Tumor cells are cultured in 96-well plates at 1 10 5 cells/ml in 100 ⁇ l/well and incubated with various amounts of antibody or control IgG (up to 100 ⁇ g/ml) for 24 h.
- Cells are incubated with 0.5 ⁇ Ci 3 H-thymidine (New England Nuclear, Boston, MA) for 18 h and harvested onto glass filters using an automatic cell harvester (Packard, Meriden, CT). The inco ⁇ orated radioactivity is measured using a liquid scintillation counter.
- Transwell cluster plates Coming Costar, Cambridge, MA. Briefly, 10 5 cells/75 ⁇ l are loaded onto fibronectin (5 ⁇ M)-coated polycarbonate membranes (8- ⁇ m pore size) separating two chambers of a transwell (Tai et al, Blood 99:1419-1427 (2002), herein inco ⁇ orated by reference in its entirety. Medium with or without anti-CSA antibodies is added to the lower chamber of the Transwell cluster plates. After 8-16 h, cells migrating to the lower chamber are counted using a Coulter counter ZBII (Beckman Coulter) and by hemacytometer.
- ZBII Beckman Coulter
- a tumor biopsy is performed to document tumor cell expression .of the CSA antigen of the invention and reactivity with an antibody that reacts with said antigen using flow cytometry.
- baseline hematologic function (1500 granulocytes and 50,000 platelets/ ⁇ l), renal function (serum creatine of ⁇ 2.5 mg/dl), quantitative serum IgG of greater than 600 mg/dl, a negative serology to human immunodeficiency viras (HIV), a negative hepatitis B surface antigen, a life expectancy of at least 3 months without other serious illness, and between the ages of 18 and 75 years.
- Other exclusion criteria are previous treatment with murine antibodies, active opportunistic infections, any other severe infection not controlled by medical or surgical therapy, or major organ dysfunction. Patients who are pregnant or lactating or those who had participated in other trials during the past 12 weeks of this study are also excluded.
- Tumor cells are obtained from excisional biopsies or from fine needle tumor aspirations and stained for said CSA expression with fluorescently-labeled anti-CSA antibodies. Tumor cells are also analyzed for expression of surface Ig light chains (fluorescein (FITC)-goat F(ab) 2 -anti- human K or Tago, Burlingame, CA), CD19, CD4, CD3, CD8 (FITC- or phycoerythrin (PE)-conjugated Leul2, Leu3, Leu4, and Leu2;Becton Dickinson), and CD37 (MBl clone 6A4). Peripheral blood samples are analyzed for the number of cells expressing the CSA antigen using two color flow cytometry using PE or FITC conjugates of the above reagents.
- FITC fluorescein
- PE phycoerythrin
- Two week post-treatment tumor biopsies are also evaluated for B- and T- cell content using the same reagents described above.
- Antibody bound to tumor cells from in vivo administration is detected by a combination of two different methods. In the first method, ceils are stained using FITC-labeled anti-CSA antibodies. The presence of the unlabeled antibody blocks the binding of the labeled antibody, resulting in decreased immunostaining of the B-cell tumor population (as identified using antibodies to additional B-cell antigens CD19, CD37, IgM, IgG, K or ⁇ ).
- the bound chimeric antibody is detected directly by looking for IgM ⁇ -or ⁇ -positive tumor cells now bearing the human IgG (K) constant regions of the chimeric antibody using a FITC labeled goat F(ab') 2 anti-human IgG ⁇ -chain-specific reagent (Tago).
- An estimate of the percentage of tumor cells with the chimeric antibody attached is obtained by comparing the staining of the pretreatment and the post- treatment biopsies for human IgG constant regions.
- Seram levels of the chimeric antibody are determined using an enzyme-linked immunosorbant assay (ELISA). Microtiter plates are coated with a purified polyclonal anti- CSA idiotype antiseram. After washing and blocking, post-treatment sera are serially diluted. Bound human IgG is detected using an HRP-conjugated polyclonal anti-human IgG reagent, and the plates are developed with the substrate 2,2-azinobis(3-ethylbenzthiazoline sulfonic acid) (ABTS). Antibody concentration is determined by comparison of the signal from the patients sera with that obtained from known concentrations of purified chimeric antibody diluted into normal human serum.
- ELISA enzyme-linked immunosorbant assay
- Post-treatment sera from evaluations at 1, 2, and 3 months are analyzed for evidence of a host anti-chimeric antibody immune response using a sandwich ELISA with microtiter plates coated with anti-CSA antibody, the murine anti-CSA antibody, or normal murine IgG. Dilutions of the patients sera are added and, after washing, are detected with biotin-labeled chimeric antibody followed by Avidin-HRP and the substrate ABTS. This assay has a level of quantification of 5 ⁇ g/ml.
- NCICT National Cancer Institute's Common Toxicity
- Hematologic, renal, and hepatic function is monitored before and after infusion and during monthly intervals after therapy.
- Sera for evaluation of antibody levels and pharmacokinetics, seram IgG and IgM levels, and CSA expression on peripheral blood B cells is obtained at each follow-up visit.
- Tumor response is assessed by evaluation of tumor measurements from physical examination and from radiologic imaging studies.
- patients are evaluated at monthly intervals and then followed at 1- to 3-month intervals until disease progression is observed.
- a complete remission (CR) requires complete resolution of all detectable disease.
- a partial remission (PR) requires a greater than 50% reduction in measurable disease persisting more than 30 days.
- a minor response is defined as a 25% to 50% reduction in disease.
- Stable disease is defined as no significant change in tumor measurements without progression over the period of observation.
- Progressive disease is noted when there is a 25% increase in measurable disease or the appearance of any new lesion.
- Eligibility criteria include CSA-positive B-cell lymphoma at first or higher relapse or progressive disease after at least one standard treatment. Lesions are classified as CSA- positive when the CSA antigen of the invention is expressed on more than 30% of malignant cells. All histologic slides are reviewed by an independent expert panel consisting of 6 reference pathologists. For enrollment into the study, patients also have to meet the following requirements: have a bidimensionally measurable disease, at least one lesion larger than 1.0 cm in its greatest diameter, and a World Health Organization performance status of 0, 1, or 2. In addition, patients have to be at least 18 years of age, neither pregnant nor lactating, using accepted birth control methods, and have to have a life expectancy of 3 months or longer. Patients with major organ dysfunction or active infections are excluded from this study. Prior treatment with anti-CSA antibodies is also an exclusion criterion. Concurrent therapeutic use of corticosteroids is not allowed.
- Patients receive 375 mg/m 2 (based on Rituximab dosing) of the anti-CSA mAb once weekly for 4 weeks given as intravenous infusion in saline solution.
- the drag is administered at an initial dose rate of 50 mg/hour for the first hour and gradually excalates to a maximum of 400 mg/hour (300 mg/hour for the first infusion only).
- Acetaminophen and antihistamines at standard doses are administered one hour before each infusion.
- a concomitant infusion of saline solution is given during the first antibody infusion.
- Toxicity is evaluated using the NCICT criteria. Evaluation of disease assessment includes physical examination and computed tomography (CT) or magnetic resonance imaging at baseline, 3 months after the end of treatment, every 3 months for 2 years, and then every 6 months thereafter. A bone marrow biopsy is performed at baseline and at confirmation of CR, if positive at baseline. Data are documented according to institutional guidelines.
- Flow cytometry is used to detect the number of CSA-positive lymphocytes in the peripheral blood of patients at baseline as well as after 1 week, 3 months, 6 months, and 1 year after the fourth anti-CSA antibody infusion.
- Flow cytometric phenotyping of mononuclear cells of the peripheral blood is performed after red blood cell lysing of blood samples. The cells are incubated with fluorochrome conjugated mouse anti-human mAbs (see Example 29) and appropriate isotype controls for 20 min at 4°C. After 2 washing procedures in PBS containing 0.1% bovine seram albumin and 0.01% sodium azide, samples are measured on a flow cytometer with a minimum of 10,000 mononuclear cells acquired for each staining.
- Patients are assessable for efficacy if they have completed at least 2 infusions of anti- CSA antibody, satisfied all pre-study entry criteria, and met criteria for evaluation of response. For discussion of response criteria, see Example 28F. In addition, since the course of the disease may be slow, the patients' history is documented for at least 10 years post-treatment.
- Duration of response is measured from the first infusion of anti-CSA antibody and the first observation of response, respectively. These data are analyzed by analysis methods, such as the Kaplan-Meier product limit method. Adverse events are investigated in relation to the study treatment. Any adverse event that is reported as probably or possibly related or of unknown relationship to the study drug is considered an adverse event. Adverse events are further classified as having occurred during the treatment period (time interval between first infusion and 30 days after the fourth infusion) or follow-up (time interval between 31 days after the fourth infusion and 1 year after the first infusion).
- a lymph node biopsy is obtained from each patient for the pu ⁇ ose of producing a custom idiotype (Id) vaccine.
- the biopsies are classified according to the Working Formulation (Cancer 49:2112 (1982), herein inco ⁇ orated by reference in its entirety). All patients are staged with CT scans of the chest, abdomen, and pelvis before vaccine treatments. Immunizations are initiated at least 2 months after the completion of previous chemotherapy treatment and administered according to the schedule below. Patients are surveyed for tumor recurrence in a standardized and rigorous manner. They receive physical examinations, blood counts and chemistries, chest radiographs, and abdominal films if lymphangiogram dye was present every 3 months. Repeat CT scans of the chest, abdomen, and pelvis are performed once a year or earlier if clinically indicated.
- Single cell suspensions of tumor cells are prepared under sterile conditions and used immediately or stored cryogenically in liquid nitrogen in fetal calf seram supplemented with 10% dimethyl sulfoxide (DMSO).
- Tumor cells are fused to the cell line K6H6B5 as described in Carroll ei al. (J. Immunol. Methods 89:61 (1986), herein inco ⁇ orated by reference in its entirety).
- the resulting hybridomas are initially screened by an ELISA assay for the production of Ig matching the isotype of the tumor. High protein producing cell lines are identified.
- the Ig is confirmed to be derived from the original tumor by either immunologic or genetic analysis.
- Ig protein is used to vaccinate animals, and the protein is determined to be derived from the tumor with the hyperimmune seram from the animals is found to bind to the original tumor after abso ⁇ tion against normal human Ig.
- VH Ig heavy chain variable region
- the Ig heavy chain variable region (VH) gene of each hybridoma is amplified and sequenced as described in Hsu and Levy (Blood 86:3072 (1995), herein inco ⁇ orated by reference in its entirety).
- Hybridomas are confirmed to be derived from the tumor with the sequence corresponding to the third complementarity- determining region (CDR3) of the heavy chain gene matches that of the original tumor.
- CDR3 complementarity- determining region
- Ig protein is purified from hybridoma culture supernatants by affinity chromatography (Protein A for IgG, anti-IgM antibody columns for IgM, and anti-IgA antibody columns for IgA).
- KLH Keyhole limpet hemocyanin
- Each patient receives a series of 5 subcutaneous immunizations each consisting of 0.5 mg of tumor Ig protein conjugated to 0 5 mg of KLH carrier protein and mixed with an immunologic adjuvant. Vaccines are administered on day 0 and then 2, 6, 10, and 20 weeks later.
- the initial patients receive their vaccinations of Id-KLH protein in "incomplete adjuvant” (5% squalene (Aldrich Chemical, Milwaukee, WI), 2.5% pluronic L121 (BASF, Parsippany, NJ), 0.2% Tween (Aldrich) and PBS), whereas the second group of patients receive the "complete” adjuvant (incomplete adjuvant containing increasing doses of threonyl-muramyl dipeptide (Thr-MDP; Peninsula Laboratories, Burlingame, CA)) as part of a dose finding study. The remaining patients receive the complete adjuvant mixture containing the maximum tolerated dose of Thr-MDP.
- incomplete adjuvant 5% squalene (Aldrich Chemical, Milwaukee, WI), 2.5% pluronic L121 (BASF, Parsippany, NJ), 0.2% Tween (Aldrich) and PBS
- Thr-MDP threonyl-muramyl dipeptide
- the remaining patients receive the complete
- Tumor Ig protein or isotype matched Igs are captured onto microtiter plates coated with goat anti -human heavy chain antibodies.
- F(ab') 2 fragments are produced by digestion with immobilized pepsin (Pierce, Rockford, IL) and used to coat microtiter plates directly.
- Pre-immunization and post-immunization patient serum are serially diluted and allowed to bind to the target proteins.
- the binding of anti-Id antibodies is detected by polyclonal goat anti-human IgG antibodies (BioSource
- a response is inte ⁇ reted as positive when a fourfold increase in anti-Id antibody titer is found when compared to the pre- vaccine seram and to the binding to irrelevant isotype matched proteins used as specificity targets.
- Antibody responses to KLH are measured by directly coating microtiter plates with KLH and allowing patient seram to bind. Seram titers of anti-KLH antibodies are determined by comparison to a standardized lot of polyclonal human anti-KLH serum. A titer of greater than 0.5 ⁇ g/ml is considered positive.
- PBMC Peripheral blood mononuclear cells
- PBMC Peripheral blood mononuclear cells
- Freedom from disease progression and survival data are analyzed using statistical methods such as Kaplan-Meier analysis and sample log rank tests of significance. Freedom from disease progression is measured from the date of last chemotherapy before vaccine treatment to the date of progression or last follow-up. The date of last chemotherapy is the reference time point used for these calculations because the impact of vaccine treatments can only be tested when compared to the time of progression from the last treatment proven to cause tumor responses. It is at this point in time in which the relevant baseline clinical status is represented.
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US78080 | 2002-02-15 | ||
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US9298502A | 2002-03-06 | 2002-03-06 | |
US92985 | 2002-03-06 | ||
US10/146,619 US20030215453A1 (en) | 2002-05-14 | 2002-05-14 | Methods of therapy and diagnosis using immunotargeting of cells expressing VpreB1 protein |
US146619 | 2002-05-14 | ||
US218325 | 2002-08-12 | ||
US10/218,325 US7109030B2 (en) | 2000-03-31 | 2002-08-12 | Methods of therapy and diagnosis using insulin-like growth factor binding protein-like polypeptides and polynucleotides |
US10/302,444 US20040022786A1 (en) | 2000-01-21 | 2002-11-22 | Methods of therapy and diagnosis using targeting of cells that express toll-like receptor proteins |
US302444 | 2002-11-22 | ||
US10/327,413 US7029677B2 (en) | 2000-01-25 | 2002-12-19 | Methods of therapy and diagnosis using immunotargeting of CD84Hy1-expressing cells |
US327413 | 2002-12-19 | ||
US10/327,491 US20040023870A1 (en) | 2000-01-21 | 2002-12-19 | Methods of therapy and diagnosis using targeting of cells that express toll-like receptor proteins |
US327491 | 2002-12-19 | ||
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US6306393B1 (en) * | 1997-03-24 | 2001-10-23 | Immunomedics, Inc. | Immunotherapy of B-cell malignancies using anti-CD22 antibodies |
WO2001079454A1 (en) * | 2000-04-13 | 2001-10-25 | Smithkline Beecham Corporation | Novel compounds |
EP1223218A1 (en) * | 2000-11-03 | 2002-07-17 | Millennium Pharmaceuticals, Inc. | CD2000 and CD2001 molecules and uses thereof |
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Title |
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BAUER S R ET AL: "VpreB gene expression in hematopoietic malignancies: a lineage- and stage-restricted marker for B-cell precursor leukemias" BLOOD, W.B.SAUNDERS COMPANY, ORLANDO, FL, US, vol. 78, no. 6, 15 September 1991 (1991-09-15), pages 1581-1588, XP002312599 ISSN: 0006-4971 * |
SCHIFF C ET AL: "LAMBDA-LIKE AND V PRE-B GENES EXPRESSION AN EARLY B-LINEAGE MARKER OF HUMAN LEUKEMIAS" BLOOD, vol. 78, no. 6, 1991, pages 1516-1525, XP002388533 ISSN: 0006-4971 * |
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