CA2412901A1 - Bispecific fusion protein and method of use for enhancing effector cell killing of target cells - Google Patents
Bispecific fusion protein and method of use for enhancing effector cell killing of target cells Download PDFInfo
- Publication number
- CA2412901A1 CA2412901A1 CA002412901A CA2412901A CA2412901A1 CA 2412901 A1 CA2412901 A1 CA 2412901A1 CA 002412901 A CA002412901 A CA 002412901A CA 2412901 A CA2412901 A CA 2412901A CA 2412901 A1 CA2412901 A1 CA 2412901A1
- Authority
- CA
- Canada
- Prior art keywords
- immunoconjugate
- cell
- antibody
- ifn
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- 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
- C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
- A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/56—IFN-alpha
-
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention provides a nucleic acid encoding IFN-.alpha. fusion proteins. The specification also describes the proteins encoded by said nucleic acids, methods of making IFN-.alpha. fusion proteins and methods of utilizing the compounds for the treatment of cancer. One specific IFN-.alpha.
fusion protein comprising IFN-.alpha. or a portion thereof capable of enhancing the killing ability of an effector cell and an anti-CD20 antibody, such as RITUXAN®, which can target, for example, B cell lymphomas e.g., non-Hodgkin's lymphoma).
fusion protein comprising IFN-.alpha. or a portion thereof capable of enhancing the killing ability of an effector cell and an anti-CD20 antibody, such as RITUXAN®, which can target, for example, B cell lymphomas e.g., non-Hodgkin's lymphoma).
Description
BISPECIFIC FUSION PROTEIN AND METHOD OF USE FOR
ENHANCING EFFECTOR CELL KILLING OF TARGET CELLS
FIELD OF THE INVENTION
This invention relates to a fusion protein comprising all or a biologically active portion of an interferon alpha (IFN-a) linked to an immunoglobulin protein or polypeptide fragment thereof, which recognizes a cell surface protein expressed by a malignant cell. The fusion protein, when bound to a malignant cell, or a target cell, would bind via IFN-a to the IFN-a receptor expressed on an effector cell (e.g., natural killer (NK) cells, polymorphonuclear (PMNs) cells and macrophages/monocytes).
The binding of IFN-a fusion protein to its receptor on effector cells enhances and potentiates extracellular (e.g., antibody-dependent cell-mediated cytotoxicity or ADCC-type), intracellular (phagocytic) and/or direct killing of the bound target cell.
BACKGROUND OF THE INVENTION
The cytokines, interleukin-2 (IL-2) and IFN-a, are potent activators of natural killer (NK) cells and other anti-tumor effector cells, but results obtained in clinical trials with these cytokines have proved disappointing in many forms of cancer.
The ineffectiveness of IL-2 and IFN-a may be because intratumoral monocytes/
macrophages (MO) inhibit the cytokine-induced activation of cytotoxic effector lymphocytes, such as NK cells, at the site of tumor growth (Hellstrand et al., Acta O~col. 37: 347-53 (1998)). Nevertheless, IFN-a can regulate NIA cells and lymphokine-activated killer (LAK) cells and can act in synergy with IL-2 to augment NK activity (Chikkala et al., Cancer Res. 50: 1176-82 (1990)). For example, systemically administered IFN-a has exhibited a limited anti-tumor effect in lymphomas, leukemia, I~aposi's sarcoma, renal cell carcinoma, melanoma, multiple melanoma, glioma and ovarian cancer. See Sell et al., IMMUN~LOGY, ilVllVIUIVQPATHOLOGY & I~nu~'rY 951-2 (1996). However, systemic treatment with either or both IFN-a or IL-2 can cause less important severe life-threatening toxicity, which limits their administration at higher doses leading to reduced efficacy and discourages their therapeutic use (Meseri-Delwail et al., Biotechrr.ol. Tlaer.
5: 47-57 (1994); and Sosman et al., Senain. Oncol. 17: 22-30, 38-41 (1990)). Use of IFN-a is further complicated as multiple species of IFN-a exist that differ dramatically in activity, which is explained in part by different binding affinities (Webb et al., Cell Inamunol. 124: 158-7 (1989); and U.S. Patent 4,780,530 (1988)). Furthermore, systemic activation of NK cells and monocytes is less effective than activation at the tumor site since activated cells may not home to the tumor. This is particularly applicable to solid tumors including carcinomas.
Antibody dependent cellular cytotoxicity (ADCC) is viewed as an important mechanism by which monoclonal antibodies (mAb) can exert an antitumor effect in vivo. ADCC can be enhanced by the cytokines IL-2 and IFN-a (Vuist et al., CanceY
Immunol. Immunothef°. 36: 163-70 (1993)).
Monoclonal antibodies have been designed to target specific cells or antigens.
Monoclonal antibodies therefore, have been designed to act as vectors or delivery vehicles for targeting foreign antigens to cells. See for general description EP Patent No. 553,244 (1993).
A~ati-CD19 A~atibodies. Antibodies have been raised which recognize CD19, a signal transduction molecule restricted to the B-cell lineage. Examples of monoclonal anti-CD19 antibodies include anti-B4 (Goulet et al., Blood 90: 2364-75 (1997)), B43 and B43 single-chain Fv (FVS 191; Li et al., Cancer Immunol. Immunother. 47:
130 (1998)). Myers et al. (Leak. L~naphoma 29: 329-38 (1998) reported conjugating the marine monoclonal B43 to the tyrosine kinase inhibitor (see also U.S.
Patent No.
5,872,459), genistein, to produce an immunoconjugate against CD19 antigen positive hematologic malignancies. Treon et al., SenZin, Oncol. 26/5 Supp: 97-106 (1999) reported conjugation of B4 to a blocked ricin, which had no significant activity in patients with multiple myeloma.
Rituxirnab ahd otlaer~ A~zti-CD20 Antibodies. The FDA approved anti-CD20 antibody, Rituximab (IDEC C2B8; RITUXANO; ATCC No. HB 11388) has also been used to treat humans. Ibritumomab, is the marine counterpart to Rituximab (Wiseman et al., Clin. Cafacer Res. 5: 3281s-6s (1999)). Other reported anti-CD20 antibodies include the anti-human CD20 mAb 1F5 (Shah et al., J. Imnaunol. 162: 6589-95 (1999)), the single chain Fv anti-CD20 mouse mAb 1H4 (Haisma et al., Blood 92:
ENHANCING EFFECTOR CELL KILLING OF TARGET CELLS
FIELD OF THE INVENTION
This invention relates to a fusion protein comprising all or a biologically active portion of an interferon alpha (IFN-a) linked to an immunoglobulin protein or polypeptide fragment thereof, which recognizes a cell surface protein expressed by a malignant cell. The fusion protein, when bound to a malignant cell, or a target cell, would bind via IFN-a to the IFN-a receptor expressed on an effector cell (e.g., natural killer (NK) cells, polymorphonuclear (PMNs) cells and macrophages/monocytes).
The binding of IFN-a fusion protein to its receptor on effector cells enhances and potentiates extracellular (e.g., antibody-dependent cell-mediated cytotoxicity or ADCC-type), intracellular (phagocytic) and/or direct killing of the bound target cell.
BACKGROUND OF THE INVENTION
The cytokines, interleukin-2 (IL-2) and IFN-a, are potent activators of natural killer (NK) cells and other anti-tumor effector cells, but results obtained in clinical trials with these cytokines have proved disappointing in many forms of cancer.
The ineffectiveness of IL-2 and IFN-a may be because intratumoral monocytes/
macrophages (MO) inhibit the cytokine-induced activation of cytotoxic effector lymphocytes, such as NK cells, at the site of tumor growth (Hellstrand et al., Acta O~col. 37: 347-53 (1998)). Nevertheless, IFN-a can regulate NIA cells and lymphokine-activated killer (LAK) cells and can act in synergy with IL-2 to augment NK activity (Chikkala et al., Cancer Res. 50: 1176-82 (1990)). For example, systemically administered IFN-a has exhibited a limited anti-tumor effect in lymphomas, leukemia, I~aposi's sarcoma, renal cell carcinoma, melanoma, multiple melanoma, glioma and ovarian cancer. See Sell et al., IMMUN~LOGY, ilVllVIUIVQPATHOLOGY & I~nu~'rY 951-2 (1996). However, systemic treatment with either or both IFN-a or IL-2 can cause less important severe life-threatening toxicity, which limits their administration at higher doses leading to reduced efficacy and discourages their therapeutic use (Meseri-Delwail et al., Biotechrr.ol. Tlaer.
5: 47-57 (1994); and Sosman et al., Senain. Oncol. 17: 22-30, 38-41 (1990)). Use of IFN-a is further complicated as multiple species of IFN-a exist that differ dramatically in activity, which is explained in part by different binding affinities (Webb et al., Cell Inamunol. 124: 158-7 (1989); and U.S. Patent 4,780,530 (1988)). Furthermore, systemic activation of NK cells and monocytes is less effective than activation at the tumor site since activated cells may not home to the tumor. This is particularly applicable to solid tumors including carcinomas.
Antibody dependent cellular cytotoxicity (ADCC) is viewed as an important mechanism by which monoclonal antibodies (mAb) can exert an antitumor effect in vivo. ADCC can be enhanced by the cytokines IL-2 and IFN-a (Vuist et al., CanceY
Immunol. Immunothef°. 36: 163-70 (1993)).
Monoclonal antibodies have been designed to target specific cells or antigens.
Monoclonal antibodies therefore, have been designed to act as vectors or delivery vehicles for targeting foreign antigens to cells. See for general description EP Patent No. 553,244 (1993).
A~ati-CD19 A~atibodies. Antibodies have been raised which recognize CD19, a signal transduction molecule restricted to the B-cell lineage. Examples of monoclonal anti-CD19 antibodies include anti-B4 (Goulet et al., Blood 90: 2364-75 (1997)), B43 and B43 single-chain Fv (FVS 191; Li et al., Cancer Immunol. Immunother. 47:
130 (1998)). Myers et al. (Leak. L~naphoma 29: 329-38 (1998) reported conjugating the marine monoclonal B43 to the tyrosine kinase inhibitor (see also U.S.
Patent No.
5,872,459), genistein, to produce an immunoconjugate against CD19 antigen positive hematologic malignancies. Treon et al., SenZin, Oncol. 26/5 Supp: 97-106 (1999) reported conjugation of B4 to a blocked ricin, which had no significant activity in patients with multiple myeloma.
Rituxirnab ahd otlaer~ A~zti-CD20 Antibodies. The FDA approved anti-CD20 antibody, Rituximab (IDEC C2B8; RITUXANO; ATCC No. HB 11388) has also been used to treat humans. Ibritumomab, is the marine counterpart to Rituximab (Wiseman et al., Clin. Cafacer Res. 5: 3281s-6s (1999)). Other reported anti-CD20 antibodies include the anti-human CD20 mAb 1F5 (Shah et al., J. Imnaunol. 162: 6589-95 (1999)), the single chain Fv anti-CD20 mouse mAb 1H4 (Haisma et al., Blood 92:
184-90 (1998)) and anti-B1 antibody (Liu et al., J. Clin. Oncol. 16: 3270-8 (1998)).
In the instance of 1H4, a fusion protein was created reportedly fusing 1H4 with the human (3-glucuronidase for activation of the prodrug N-[4-doxorubicin-N-carbonyl(-oxymethyl)phenyl~ O-(3-glucuronyl carbamate to doxorubicin at the tumor cite (Haisma et al., 1998).
Anti-CD22 Antibodies. CD22 is a cell surface antigen expressed on normal human B cells and some neoplastic B cells. Several monoclonal anti-CD22 antibodies have been created, including HD6, RFB4, W22-2, TolS, 4KB 128, a humanized anti-CD22 antibody (hLL2), and a bispecific F(ab')Z antibody linked to saporin (Li et al., Cell. Immunol. 111: 85-99 (1989); Mason et al., Blood 69: 836-40 (1987); Behr et al., ClifZ. Cancer Res. 5: 3304s-14s (1999); and Bonardi et al., CafZCer Res. 53:
(1993)). Immunotoxins comprising anti-CD22 linked to the ricin A chain have also been prepared, which reportedly possess antitumor effects in mice (Sausville et al., Blood 85: 3457-65 (1995); and Ghetie et al., Cahcef° Res. 51: 5876-80 (1991)).
AsZti-CD33 Antibodies. CD33 is a glycoprotein expressed on early myeloid progenitor and myeloid leukemic (e.g., acute myelogenous leukemia, AML) cells, but not on stem cells. An IgGI monoclonal antibody was prepared in mice (M195) and also in a humanized form (HuM195), that reportedly has antibody-dependent cellular cytotoxicity (Kossman et al., Glif2. Cafzcef~ Res. 5: 2748-55 (1999)).
However, Caron et al., Clira. Cancer Res. 1: 63-70 (1995) reported that HuM195 had only modest ADCC capability against HL60 cells. An anti-CD33 immunoconjugate (CMA-676) consisting of a humanized anti-CD33 antibody linked to the antitumor antibiotic calicheamicin reportedly demonstrated selective ablation of malignant hematopoiesis in some AML patients (Sievers et al., Blood 93: 3678-84 (1999). Pagliaro et al., Clin.
Caface~ Res. 4: 1971-6 (1998) described a HuM195-gelonin immunoconjugate, comprising an anti-CD33 mAb conjugated .to the single-chain plant toxin gelonin.
AiZti-CD38 A~ztibodies. CD38 is an antigen expressed during early stages of differentiation in normal and leukemic myeloid cells, including myeloma cells.
Ellis et al., J. ImmuT2ol. 155: 925-37 (1995) reported a high affinity mAb (AT13/5) against CD38 which efficiently directed antibody-dependent cellular cytotoxicity (ADCC) against CD38+ cell lines, but which activated complement poorly and did not down-regulate CD38 expression. Flavell et al., HenZatol. Oncol. 13: 185-200 (1995) described an anti-CD38/anti-saporin (OKT10-Sap) immunotoxin, which purportedly delivers the ribosome inactivating protein (rip) to the leukemic cells (HSB-2 cells).
The anti-CD38 antibody, HB7, has been chemically conjugated to a modified ricin molecule and can supposedly bill antigen-bearing tumor cells (Goldmacher et al., 84:
3017-25 (1994)).
Anti EGF R A~ztibodies. Epidermal growth factor-receptor (EGF-R) binds to EGF, a mitogenic peptide. Anti-EGF-R antibodies and methods of preparing them can be performed as described in U.S. Patent Nos. 5,844,093; 5,558,864.
European Patent No. 706,799A purportedly describes an immunoconjugate comprising an anti-EGF-R mAb fused to a C-X-C chemokine, especially IL-8. U.S. 5,824,782 describes an imlnunoconjugate comprising an anti-EGFR antibody fused to IL-8, which lacks at least the first amino acid of IL-8.
Anti HM1.24 A~ztibodies. HM1.24 is a type II membrane glycoprotein is overexpressed in multiple myeloma (MM) and Waldenstrom's macroglobulinemia (Ohtomo et al., Bioclaem. Bioplays. Res. Cofyamun. 258: 583-91 (1999); and Goto et al., Blood 84: 1922-30 (1994)). A mouse monoclonal anti-HM1.24 IgG2a/x antibody has been demonstrated to bind to HM1.24 on MM cells and reportedly induces ADCC
(Ono et al., Mol. Imnauno. 36: 387-95 (1999)). A humanized anti-HM1.24 IgGI/K
antibody also was shown to induce ADCC against human myeloma KPMM2 and ARH77 cells (Ono et al., Mol. In2muno. 36: 387-95 (1999)).
A~zti Her-2 Antibodies. The engB 2 gene, more cormnonly known as (Hen-2/neu), is an oncogene encoding a transmembrane receptor. Several antibodies have been developed against Her-2/neu, including trastuzumab (e.g., HERCEPTIN~;
Former et al., Oncology (Huntingt) 13: 647-58 (1999)), TAB-250 (Rosenblum et al., Clin. Caracen Res. 5: 865-74 (1999)), BACH-250 (Id.), TAl (Maier et al., Cancers Res.
51: 5361-9 (1991)), and the mAbs described in U.S. Patent Nos. 5,772,997;
In the instance of 1H4, a fusion protein was created reportedly fusing 1H4 with the human (3-glucuronidase for activation of the prodrug N-[4-doxorubicin-N-carbonyl(-oxymethyl)phenyl~ O-(3-glucuronyl carbamate to doxorubicin at the tumor cite (Haisma et al., 1998).
Anti-CD22 Antibodies. CD22 is a cell surface antigen expressed on normal human B cells and some neoplastic B cells. Several monoclonal anti-CD22 antibodies have been created, including HD6, RFB4, W22-2, TolS, 4KB 128, a humanized anti-CD22 antibody (hLL2), and a bispecific F(ab')Z antibody linked to saporin (Li et al., Cell. Immunol. 111: 85-99 (1989); Mason et al., Blood 69: 836-40 (1987); Behr et al., ClifZ. Cancer Res. 5: 3304s-14s (1999); and Bonardi et al., CafZCer Res. 53:
(1993)). Immunotoxins comprising anti-CD22 linked to the ricin A chain have also been prepared, which reportedly possess antitumor effects in mice (Sausville et al., Blood 85: 3457-65 (1995); and Ghetie et al., Cahcef° Res. 51: 5876-80 (1991)).
AsZti-CD33 Antibodies. CD33 is a glycoprotein expressed on early myeloid progenitor and myeloid leukemic (e.g., acute myelogenous leukemia, AML) cells, but not on stem cells. An IgGI monoclonal antibody was prepared in mice (M195) and also in a humanized form (HuM195), that reportedly has antibody-dependent cellular cytotoxicity (Kossman et al., Glif2. Cafzcef~ Res. 5: 2748-55 (1999)).
However, Caron et al., Clira. Cancer Res. 1: 63-70 (1995) reported that HuM195 had only modest ADCC capability against HL60 cells. An anti-CD33 immunoconjugate (CMA-676) consisting of a humanized anti-CD33 antibody linked to the antitumor antibiotic calicheamicin reportedly demonstrated selective ablation of malignant hematopoiesis in some AML patients (Sievers et al., Blood 93: 3678-84 (1999). Pagliaro et al., Clin.
Caface~ Res. 4: 1971-6 (1998) described a HuM195-gelonin immunoconjugate, comprising an anti-CD33 mAb conjugated .to the single-chain plant toxin gelonin.
AiZti-CD38 A~ztibodies. CD38 is an antigen expressed during early stages of differentiation in normal and leukemic myeloid cells, including myeloma cells.
Ellis et al., J. ImmuT2ol. 155: 925-37 (1995) reported a high affinity mAb (AT13/5) against CD38 which efficiently directed antibody-dependent cellular cytotoxicity (ADCC) against CD38+ cell lines, but which activated complement poorly and did not down-regulate CD38 expression. Flavell et al., HenZatol. Oncol. 13: 185-200 (1995) described an anti-CD38/anti-saporin (OKT10-Sap) immunotoxin, which purportedly delivers the ribosome inactivating protein (rip) to the leukemic cells (HSB-2 cells).
The anti-CD38 antibody, HB7, has been chemically conjugated to a modified ricin molecule and can supposedly bill antigen-bearing tumor cells (Goldmacher et al., 84:
3017-25 (1994)).
Anti EGF R A~ztibodies. Epidermal growth factor-receptor (EGF-R) binds to EGF, a mitogenic peptide. Anti-EGF-R antibodies and methods of preparing them can be performed as described in U.S. Patent Nos. 5,844,093; 5,558,864.
European Patent No. 706,799A purportedly describes an immunoconjugate comprising an anti-EGF-R mAb fused to a C-X-C chemokine, especially IL-8. U.S. 5,824,782 describes an imlnunoconjugate comprising an anti-EGFR antibody fused to IL-8, which lacks at least the first amino acid of IL-8.
Anti HM1.24 A~ztibodies. HM1.24 is a type II membrane glycoprotein is overexpressed in multiple myeloma (MM) and Waldenstrom's macroglobulinemia (Ohtomo et al., Bioclaem. Bioplays. Res. Cofyamun. 258: 583-91 (1999); and Goto et al., Blood 84: 1922-30 (1994)). A mouse monoclonal anti-HM1.24 IgG2a/x antibody has been demonstrated to bind to HM1.24 on MM cells and reportedly induces ADCC
(Ono et al., Mol. Imnauno. 36: 387-95 (1999)). A humanized anti-HM1.24 IgGI/K
antibody also was shown to induce ADCC against human myeloma KPMM2 and ARH77 cells (Ono et al., Mol. In2muno. 36: 387-95 (1999)).
A~zti Her-2 Antibodies. The engB 2 gene, more cormnonly known as (Hen-2/neu), is an oncogene encoding a transmembrane receptor. Several antibodies have been developed against Her-2/neu, including trastuzumab (e.g., HERCEPTIN~;
Former et al., Oncology (Huntingt) 13: 647-58 (1999)), TAB-250 (Rosenblum et al., Clin. Caracen Res. 5: 865-74 (1999)), BACH-250 (Id.), TAl (Maier et al., Cancers Res.
51: 5361-9 (1991)), and the mAbs described in U.S. Patent Nos. 5,772,997;
5,770,195 (mAb 4D5; ATCC CRL 10463); and 5,677,171. Portions of anti-Her-2 antibodies have also been conjugated to toxins, such as a single chain antibody domain specific for erbB-2 coupled to part of a Pseudonaofaas exotoxin (Skrepnik et al., Clin.
Cancer Res. 2: 1851-7 (1996)). U.S. Patent No. 5,855,866 reported a method of treating the vasculature of solid tumors using an anti-p185Her-a antibody linlced to a cytokine.
Anti MZIC 1 Antibodies. MUC-1 is a carcinoma associated mucin. The anti-MUC-1 monoclonal antibody, McS, was reportedly administered to mice carrying transplanted breast tumors and purportedly suppressed tumor growth (Peterson et al., Cancer Res. 57: 1103-8 (1997)). Mc5 was chemically linked in a non-cleavable fashion to a natural IFN-a (nIFN-a) and reportedly inhibited growth of injected tumors in mice (Ozzello et al., Breast Cancef~ Res. Treat. 25: 265-76 (1993)). An IgG4 anti-MUC-1 mAb, hCTM0l, has been suggested as a suitable carrier for cytotoxic agents in ovarian carcinomas (Van Hof et al., Cancer Res. 56: 5179-85).
Anti phosplaatidyl serine antigen Antibodies. Phosphatidyl-serine is a phospholipid. Antibodies have been reported which bind to phosphatidyl-serine and not other phospholipids (e.g., Yron et al., Clih. Exp. Immol. 97: 187-92) (1994)).
However, anti-phospholipid antibodies appear more typically associated with anti-phospholipid syndrome and its diagnosis, than for use in the treatment or diagnosis of cancer.
Anti-TAG-72 Antibodies. TAG-72 is a tmnor-associated antigen (TAG). One dimeric single-chain Fv antibody construct of monoclonal CC49 recognizes the TAG-72 epitope (Pavlinkova et al., Clifa. Cancer Res. 5: 2613-9 (1999)).
Additional anti-TAG-72 antibodies include B72.3 (Divgi et al., Nucl. Med. Biol. 21: 9-15 (1994)) and those disclosed in U.S. Patent No. 5,976,531. Administration of recombinant IFN-a reportedly increased the amount of TAG-72 expressed on tumors (Macey et al., ClifZ.
Caf~cey-Res. 3: 1547-55 (1997)). The CC49 antibody also has reportedly been chemically conjugated to doxorubicin (Johnson et al., Af2ticahcey~ Res. 15:
(1995)) and streptavidin (Ngai et al., Nucl. Med. Biol. 22: 77-86 (1995)). TAG-72 has also been chemically conjugated to human interleukin-2 (IL-2) (LeBerthon et al., Cayacef° Res. 51: 2694-8 (1991). U.S. Patent No. 5,976,531 claimed a human anti-Tag-72 antibody conjugated to an interferon but no evidence was presented indicating its efficiency in mammals.
Interferon-a Isnnzunoconjugates. Monoclonal antibodies raised against tumor cell lines have been covalently coupled with purified human lymphoblastoid IFN-a. Administration of this coupled form of IFN-a reportedly augmented killing of the tumor cells and other tumor targets by peripheral blood NK cells (Flannery et al., Euf°. J. Cafzcer~ Clin. Oncol. 20: 791-8 (1984)).
Techniques also have been developed to produce chimeric antibodies which combine regions of immunoglobulin molecules from different sources (Morrison et al., Proc. Natl Acad. Sci. USA 81: 6851-5 (1984)). Using this technology, chimeric molecules comprising immunoglobulin and non-imrnunoglobulin regions have been created. Expression vectors for fusion proteins comprising DNA sequences of an antibody and DNA sequences encoding a cell surface antigen have been proposed, wherein some of the suitable antigens include CD molecules (e.g., CD19, CD20, CD22, CD33, CD38 and CD40). See, e.g., U.S. Patent No. 5,637,481 (1997) and EP
Patent No. 610,046 (1994). Fusion proteins comprising the cytokine IL-15 and anti-CD20 (International PCT Application 98/16254), and fusion proteins comprising other lymphokines (e.g., IL,-2 and IL-3) and an imrnunoglobulin fragment capable of binding to a tumor antigen have been described (U.S. Patent Nos. 5,645,835 (1997) and 5,314,995 (1994); Lode et al., Blood 91: 1706-1715 (1998); Hassan et al., Leuk.
Lymphoma 20: 1-15 (1995)). Chang et al., U.S. Patent 5,723,125 describes a hybrid molecule comprising an interferon, preferably IFN-a-2a or IFN-a-2b, which are joined at the carboxy terminus via a peptide to the amino terminus of a first gamma immunoglobulin Fc fragment as a means of increasing the blood half life of the cytokine. Chimeric immunoglobulin proteins comprising a cytokine (e.g., IL-2, tumor necrosis factor a, etc.) and the heavy chain of an antibody for treating viral infections and cancer are described in International PCT Application 92/08495, as well as a fusion protein of IFN-y/M-CSF. A fusion protein of RM4/IFN-tau has demonstrated antitumor activity in mice (Qi et al., Hum. Antibodies Hyb>"idomas 7: 21-6 (1996); and Xiang et al., Hunt. Antibodies Hybf°idontas 7: 2-10 (1996)).
Others have proposed the creation of fusion proteins comprising non-antibody immunomodulatory molecules, wherein IFN-a is operatively fused to a heterologous membrane attachment domain (IJ.S. Patent No. 5,891,432 (1999)). IFN-a-2b was reported to be conjugated with ME31.3 and anti-carcinoembryonic antigen (CEA) and purportedly may improve diagnostic and therapeutic potential of monoclonal antibodies making them worthy of further study (Thakur et al., J. Inamuhother.
20:
194-201 (1997)).
Therefore, not withstanding what has been previously reported in the literature, there exists a need for improved compositions and methods of using such compositions for the treatment of cancer. Improved immunoconjugates with enhanced ADCC and phagocytotic activities, lower toxicity, and increased blook serum half life would provide additional treatment therapeutics, which are still needed in cancer therapy.
SUMMARY OF THE INVENTION
It is one object of the invention to provide novel and improved compositions comprising an immunoconjugate that comprises an antibody or imrnunogenic fragment thereof that binds to an antigen expressed by a target cell that is to be eradicated, wherein said antibody or imrnunogenic fragment thereof possesses human effector function, which antibody or immunogenic fragment thereof is fused at its carboxy terminus to a cytokine that binds a receptor expressed on the surface of a natural killer cell and/or macrophage, thereby resulting in an immunoconjugate that facilitates extracellular (ADCC-type) and intracellular (phagocytic) killing of a target cell, when said immunoconjugate is administered to a host. Preferably, the cytokine is a interferon. Most preferably, the interferon is an a-interferon, especially one which has been FDA approved (e.g., IFN-a-2a, IFN-a-2b and IFN-a-nl).
It is a more specific object of the invention to provide novel immunoconjugates which target cells expressing CD19, CD20, CD22, CD33, CD3~, EGF-R, HM 1.24, phosphatidyl serine antigen, HER-2, TAG-72 and MUC1.
It is a further obj ect of the invention to provide novel methods of enhancing apoptosis or treating cancer by achninistering a therapeutically effective amount of these immunoconjugates to a subject. The target cells to which these immunoconjugates are directed may include malignant cells selected from the group consisting of a breast carcinoma cell, an ovarian carcinoma cell, a prostate carcinoma cell, a lung carcinoma cell, a leukemic T-cell, a leukemic B-cell, a multiple myeloma cell and a B-cell lymphoma cell.
It is another obj ect of the invention to provide a combination therapy to treat a malignancy in a subject comprising an immunoconjugate as described above and at least one chemotherapeutic agent or chemotherapeutic cocktail.
It is a further object of this invention to provide nucleic acids which encode the immunoconjugates described herein.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to IFN-a fusion proteins, specifically immunoconjugates, and the use thereof as therapeutic agents that have the ability to target malignant cells and enhance the killing activity of effector cells through the binding of an IFN-a to its receptor on the effector cell and without systemic administration of IFN-a and its associated toxicity. These immunoconjugates will have enhanced ADCC and phagocytic activity.
I. Definitions By "interferon-a" or "IFN-a" is meant a protein or fragment thereof which can recognize and bind to the IFN-a receptor. This includes FDA approved forms of IFN-a, such as IFN-a-2a (Roferon by Hoffinan-LaRoche), IFN-a-2b (INTRON~ A by Schering Corporation) and IFN-a-nl (lymphoblastoid interferon called Wellferon and produced by Wellcome Foundation Ltd - Wellcome Research Laboratories), as well as other forms of IFN-a (e.g., IFN-a-2a and consensus IFN).
The term "fusion protein" as used herein means a hybrid protein produced recombinantly including a synthetic or heterologous amino acid sequence. A
fusion protein can be produced from a hybrid gene containing operatively linked heterologous gene sequences.
By "bispecific fusion protein" is meant any immunologically reactive molecule which specifically recognizes and binds two different targets at alternate times or at the same time. In particular, it will refer to an IFN-a antibody fusion protein, wherein the IFN is attached to an antibody, preferably anti-tumor antibody, at the carboxy terminus of the antibody. In a preferred embodiment, the antibody portion may recognize and bind to a target antigen expressed on a targeted cell. In a more preferred embodiment, the antigen-binding, Fc receptor binding, Clq and C
_g_ activation, and the ability of interferon to bind to its receptor and activate effector cells and macrophages are substantially maintained activities of the immunoconjugate.
This is preferably effected by attachment of the interferon directly or indirectly to the antibody hinge, CHl, CH2 or CH3 domain carboxy-terminus.
The terms "effector cell" and "effector function" as used herein means a cell which expresses an IFN-a receptor and can thereby bind to an IFN-a fusion protein.
Effector cells can include natural killer (NK) cells, LAK cells, monocytes, macrophages and polymorphonuclear (PMNs) cells. Preferred effector cells include NK cells and macrophages.
An "expression vector" means a nucleic acid molecule comprising (1) a promoter and other sequences (e.g., leader sequences) necessary to direct expression of a desired gene or DNA sequence, and (2) the desired gene or DNA sequence.
Optionally, the nucleic acid molecule may comprise a poly A signal sequence to enhance the stability of the gene transcript and/or to increase gene transcription and expression.
By "binding domain," "binding region" means a binding site which recognizes and binds the entire binding area of a target or any portion thereof. Examples for antibodies or immunoglobulin fragments include: (1) single variable region of an antibody VL or VH; (2) two or more variable regions (e.g., VL and VH, VL and VL; or VH and VH) or the complementary determining region (CDR) thereof; (3) antibody fragments such as Fabl, Fab2, SFV, single chain antibodies, domain-deleted antibodies and minibodies; or (4) an IFN-a or a segment of IFN-a which binds to an IFN-a receptor on an effector cell.
By "minibody" is meant an antigen binding protein which includes VL and VH
domains of a native antibody fused to the hinge region and CH3 domain of an immunoglobulin or which encodes in a single chain comprising the essential elements of a whole antibody. The single chain comprises the antigen binding region, domain to permit assembly into a bivalent molecule, and the antibody hinge to accommodate dimerization by disulfide linkages. For methods of preparing minibodies, see, e.g., U.S. Patent No. 5,837,821.
By "antibody" is intended to refer broadly to any immunologic binding agent such as IgG (including IgGI, IgG2, IgG3, and IgG4), IgM, IgA, IgD, IgE, as well as antibody fragments. Antibodies in the broadest sense covers intact monoclonal antibodies, polyclonal antibodies, as well as biologically active fragments of such antibodies such as those discussed above. In particular, domain-deleted antibodies are included within the scope of the present invention, such as CH2 domain-deleted antibodies.
By "monoclonal antibody" is meant an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature 256: 495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies"
may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352: 624-628 (1991) and Marks et al., J. lVlol. Biol., 222:
581-597 (1991), for example.
The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired therapeutic activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci.
USA, 81:
6851-5 (1984)).
"Humanized" forms of non-htunan (e.g., marine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies), which contain minimal sequence derived from a non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
The humanized antibody optimally also will comprise at least a portion of an irmnunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-5 (1986); Reichmann et al., Natuf°e, 332: 323-9 (1988); and Presta, Curs. ~p. StYZICt. Biol., 2: 593-6 (1992).
By "target antigen" is meant the antigen recognized by the antibody or immunoglobulin fragment portion of the immunoconjugate. Preferred target antigens include: 5E10, CDl, CD2, CD3, CDCDS, CD7, CD13, CD14, CD15, CD19, CD20, CD21, CD23, CD25, CD33, CD34, CD38, CEA, EGFR, HER-2, HLA-DR, HM 1.24, HMB 45, Ia, Leu-M1, MUC1, phosphatidyl serine antigen, PMSA and TAG-72.
By "nucleic acid" is meant to include an oligonucleotide, nucleotide, polynucleotide and fragments and portions thereof, and a DNA or a RNA of genomic or synthetic origin, which may be single or double stranded.
By "interferon a" or "IFN-a" preferably is meant to include all members of the interferon-a family of proteins. Fragments of IFN-a are also included, as long as the fragment is capable of recognizing and binding to the IFN-a receptor and thereby activating effector cells. Preferred forms of IFN-a include such FDA approved forms of IFN-a as IFN-a-2a, IFN-a-2b and IFN-a-ln.
By "purified" and "isolated" is meant, when referring to a polypeptide or nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. The term "purified" as used herein preferably means at least 75% by weight, more preferably at least 85%
by weight, more preferably still at least 95% by weight, and most preferably at least 98%
by weight, of biological macromolecules of the same type are present. An "isolated nucleic acid molecule wluch encodes a particular polypeptide" refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties which do not deleteriously affect the basic characteristics of the composition. Thus, for example, an isolated nucleic acid molecule which encodes a particular CDR polypeptide consists essentially of the nucleotide coding sequence for the subject molecular recognition unit.
By "therapeutically effective" is meant the ability of the immunoconjugate to inhibit in vitro growth of a target cell by greater than about 20%, at a concentration of about 0.1 to about 3.0 ~,g/ml of the immunoconjugate, wherein said target cells (e.g., tumor cells) are cultured in an appropriate culture medium and said growth inhibition is determined about 4, 5, 6, 7, 8, 9, or 10 days after exposure of the target cells to the immunoconjugate.
By "subject" is meant a living animal or human susceptible to a condition, especially cancer. In the preferred embodiments, the subject is a mammal, including human and non-human marmnals. Non-human mammals include dogs, cats, pigs, cows, sheep, goats, horses, rats and mice.
II. Method of Mal~in~ an IFN-a Fusion Protein Nucleic acids encoding the desired fusion protein can be inserted into expression vectors for expression. Expression vectors useful in the invention include prokaryotic and eukaryotic expression vectors. Such expression vectors, including plasmids, cosmids, and viral vectors such as bacteriophage, baculovirus, retrovirus and DNA virus vectors, are well known in the art (see, for example, Meth.
Enzymol., Vol. 185, D. V. Goeddel, ed. (Academic Press, Inc., 1990), and Kaplitt and Loewy (Ed.), VIRAL VECTORS: GENE THERAPY AND NEUROSCIENCE APPLICATIONS
(Academic Press, Inc., 1995), each of which are incorporated herein by reference).
Expression vectors contain the elements necessary to achieve constitutive or inducible transcription of a nucleic acid molecule encoding an IFN-a fusion protein. One form of IFN-a is described in Pitha et al., J. Immunol. 141: 3611-6 (1988).
Preferred embodiments of the invention will utilize FDA approved forms of IFN-a.
The recombinant nucleic acid molecules encoding an IFN-a fusion protein, an immunoglobulin polypeptide, or a specific IFN-a species or fragment thereof may be obtained by any method known in the art (see, e.g., Maniatis et al., MOLECULAR
CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982 and 1989) or obtained from publicly available clones, such as pGL2BIFN (ATCC No. 53371) and pALCAISIFN (ATCC No. 53369).
Alternatively, a nucleic acid encoding an IFN-a or an antibody which recognizes a tumor-associated antigen (TAA) may be obtained as follows. A population of cells known to actively express an IFN-a or a specific antibody may be obtained, and total cellular RNA harvested therefrom. The amino acid sequence of the IFN-a or antibody may be used to deduce the sequence of a portion of its nucleic acid so as to design appropriate oligonucleotide primers; or, alternatively, the oligonucleotide primers may be obtained from a known nucleic acid sequence encoding an antibody or an IFN-a.
The oligonucleotide fragment may then be used in conjunction with reverse transcriptase to produce a cDNA corresponding to the immunoglobulin and/or IFN-a encoding nucleotide sequence (Okayama et al., Methods Erazymol. 154: 3-29 (1987)).
The cDNA can then be cloned, and/or portions of the antibody or IFN-a coding region amplified from this cDNA using polymerase chain reaction (PCR) and appropriate primer sequences (Saiki et al., Science 239: 487-491 (1988)).
In preferred embodiments of the invention, a recombinant vector system may be created to accoimnodate sequences encoding IFN-a, wherein the IFN-a sequence is attached to the sequences encoding the C-terminus of an antibody or immunogenic fragment that recognizes a tumor-associated antigen. The resultant fusion protein will preserve the ability of the IFN-a molecule to bind to its receptor on the effector cell and enhance the effector cell's killing ability. The immunoconjugate will also preserve the antigen-binding Fc receptor-binding, Clq binding and C' activation regions of the antibody molecule.
Nucleic acid sequences encoding the various components of the IFN-a based fusion proteins of the invention may be joined together using any techniques known in the art, including restriction enzyme methodologies and the use of synthetic linker sequences.
To provide for adequate transcription of the recombinant constructs of the invention, a suitable promoter/enhancer sequence may be incorporated into the recombinant vector. Promoters which may be used to control the expression of the antibody-based fusion protein include, but are not limited to, the SV40 early promoter region (Bernoist et al., Nature 290: 304-310 (1981)), the promoter contained in the 3' long terminal repeat of Rous sarcoma viruses (Yamamoto et al., Cell 22: 787-(1980)), the herpes thymidine kinase (tk) promoter (Wagner et al., Proc. Natl.
Acad.
Sci. USA 78: 144-1445 (1981)), the regulatory sequences of the metallothionine gene (Brinster et al., Nature 296: 39-42 (1982)); prokaryotic expression systems such as the LAC, or (3-lactamase promoters (Villa-Kamaroff et al., Proc. Natl. Acad. Sci.
USA 75:
3727-3731 (1978)), or the tac lambda phage promoter (DeBoer et al., P~oc.
Natl.
Acad. Sci. USA 80: 21-25 (1983)). Other suitable promoters would be apparent to the skilled artisan.
Additionally, it may be desirable to include, as part of the recombinant vector system, nucleic acids corresponding to the 3' flanking region of an immunoglobulin gene, including RNA cleavage/polyadenylation sites and downstream sequences.
Furthermore, it may be desirable to engineer a signal sequence upstream of the IFN-a fusion protein-encoding sequences to facilitate the secretion of the fused molecule from a cell transformed with the recombinant vector.
Creation of IFN-a containing fusion proteins can also utilize sequences encoding conservative amino acid substitutions in an IFN-a sequence, as well as substitutions in the antibody or immunoglobulin region of the fusion protein.
Such changes include substituting an isoleucine, valine and leucine for any other of these hydrophobic amino acids; aspartic acid for glutamic acid and vice versa;
glutamine for asparagine and vice versa; and serine for threonine and vice versa. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine for alanine can frequently be interchangeable, as well as alanine for valine due to structural and charge similarities.
Successful incorporation of IFN-a based fusion gene constructs may be identified by three general approaches: (a) DNA-DNA hybridization, (b) presence or absence of "marker" gene functions, and (c) expression of inserted sequences.
In the first approach, the presence of a foreign gene inserted in an expression vector can be detected by DNA-DNA hybridization using probes comprising sequences that are homologous to the inserted antibody IFN-a fusion protein DNA. In the second approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, resistance to antibiotics such as 6418, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of foreign genes in the vector. For example, if the IFN-a fusion gene is inserted so as to interrupt the marker gene sequence of the vector, recombinants containing the antibody fusion gene ~ insert can be identified by the absence of the marker gene function. In the third approach, recombinant expression vectors can be identified by assaying the foreign gene product expressed by the recombinant. Such assays can be based, for example, on the physical or functional properties of the IFN-a fusion gene product in bioassay systems.
The cytokine can be any cytokine or analog or fragment thereof which activates effector cells. The preferred cytokine is an interferon, with the preferred interferon being IFN-a, especially FDA approved forms. The gene encoding the cytoleine can be cloned de novo, obtained from an available source, or synthesized by standard DNA synthesis from a known nucleotide sequence as discussed above.
The heavy chain constant region for the conjugates can be selected from any of the five isotypes: alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG) or mu (IgM).
Heavy chains or various subclasses (such as the IgG subclasses 1-4) can be used. The light chains can have either a kappa or lambda constant chain. DNA sequences for these immunoglobulin regions are well known in the art. (See, e. g., Gillies et al., J.
Immunol. Metla. 125: 191 (1989)).
In preferred embodiments, the variable region is derived from an antibody specific for the target antigen, and the constant region includes the CH1, CH2 and CH3 domains. The gene encoding the cytokine is joined, in frame to the 3' end of the gene encoding the constant region (e.g., CH1, CH2 or CH3 exon depending on domain-deleted form desired), either directly or through an intergenic region.
The nucleic acid construct can include an endogenous promoter and enhancer for the variable region-encoding gene to regulate expression of the chimeric immunoglobulin chain. For example, the variable region encoding genes can be obtained as DNA fragments comprising the leader peptide, the VJ gene (functionally rearranged variable (V) regions with joining (J) segment) for the light chain or VDJ
gene for heavy chain, and the endogenous promoter and enhancer for these genes.
Alternatively, the gene coding for the variable region can be obtained apart from endogenous regulatory elements and used in an expression vector that provides these elements.
Variable region genes can be obtained by standard DNA cloning procedures from cells that produce the desired antibody. Screening of the genomic library for a specific functionally rearranged variable region can be accomplished with the use of appropriate DNA probes, such as DNA segments containing the J region DNA
sequence and sequences downstream. Identification and confirmation of correct clones are then achieved by DNA sequencing of the cloned genes and comparison of the sequence to the corresponding sequence of the full length, properly spliced mRNA.
The target antigen preferably can be a cell surface antigen of a tumor cell, but also includes viral antigens or other disease associated antigens expressed on the cell surface. Genes encoding appropriate variable regions can be obtained generally from immunoglobulin producing lymphoid cells. For example, hybridoma cell lines producing immunoglobulin specific for tumor associated antigens or viral antigens can be produced by standard somatic cell hybridization techniques (see, e.g., U.S. Pat.
No. 4,96,265.). These inununoglobulin producing cell lines provide the source of variable region genes in functionally rearranged form. The variable region genes will typically be of marine origin, because the marine system lends itself to the production of a wide variety of imrnunoglobulins of desired specificity.
The DNA fragment containing the functionally rearranged variable region gene is linked to a DNA fragment containing the gene encoding the desired constant region (or a portion thereof). Immunoglobulin constant regions (heavy and light chain) can be obtained from antibody-producing cells by standard gene cloning techniques. Genes for the two classes of human light chains and the five classes of human heavy chains have been cloned, and thus, constant regions of human origin are readily available from publically available clones.
The fused gene encoding the hybrid immunoglobulin heavy chain is assembled or inserted into expression vectors for incorporation into a recipient cell.
The introduction of gene construct into plasmid vectors can be accomplished by standard gene splicing procedures.
Recipient cell lines are generally lymphoid cells. The preferred recipient cell is a myeloma (or hybridoma). Myelomas can synthesize, assemble, and secrete immunoglobulins encoded by transfected genes, and they post-tra~islationally modify the protein. A particularly preferred recipient cell is the Sp2/0 myehoma which normally does not produce endogenous immunoglobulin. When transfected, the cell will produce only innnunoglobulin encoded by the transfected gene constructs.
Transfected myelomas can be grown in culture or in the peritoneum of mice where secreted immunoconjugate can be recovered from ascites fluid. Other lymphoid cells, such as B lymphocytes, also can be used as recipient cells.
There are several methods for transfecting lymphoid cells with vectors containing the nucleic acid constructs encoding the chimeric Ig chain. A
preferred way of introducing a vector into lymphoid cells is by spheroblast fusion, as described by Gillies et al., Biotechn.ol. 7: 798-804 (1989). Alternative methods include electroporation or calcium phosphate precipitation. See also, the methods in MANJATIS, ET AL. (1989).
IFN-a based fusion protein produced by the host cell may be collected using any technique known in the art, including, but not limited to, affinity chromatography using target antigen or antibody specific for any portion of the fusion protein. The activity of the fused IFN-a or antibody (e.g., anti-CD20) may be confirmed using biological assays, which detect or measure the activity of the lymphokine or cellular factor. For example, and not by way of limitation, the presence of IFN-a activity may be confirmed in assays which detect receptor binding, virus neutralization and enhanced killing ability of the effector cells.
Preferred methods of detecting such enhanced effector cell ability can utilize receptor binding assays and virus neutralization assays. These assays are described generally below.
Receptor Binding Assay Receptor binding assays, such as those provided below, can be utilized to determine whether the immunoconjugate binds to the target antigen or to an IFN-a receptor.
Virus Neutralization Assay A virus neutralization assay is one form of receptor binding assay which can be utilized to determine the efficacy of which an immmloconjugate to neutralize virus-infected cells when the antibody targets a viral antigen expressed on the cell surface.
For example, virus neutralizations can be determined using the method described by Ho et al., J. Virol. 65: 489-93 (1991), for HIV-1 neutralization using a p24 assay.
Neutralization is defined as the percent reduction in the amount of target antigen released into the culture supernatants or detected in cells from wells treated with the immunoconjugate compared with control wells not treated with the immunoconjugate.
ADCC Assay The ability of the fusion protein to induce ADCC can be assessed using a chromium release assay. Generally, antibodies of the IgGZa and IgG3 subclass and occasionally of the IgGI subclass mediate ADCC. Antibodies of the IgG3, IgG2a and IgM classes bind and activate serum complement. To assess the ability of the immunoconjugates described herein to mediate ADCC and complement activation, one can use a SICr-release assay. Briefly, a cell line expressing the antigen being targeted for lysis by effector cells are labeled with 100 ~,Ci of SICr for about 1 hour prior to combining effector cells and antibodies in a U-bottom microtiter plate. After incubation for about 5 hours at 37°C, supernatants axe collected and analyzed for radioactivity. Cytotoxicity can be calculated by the formula: % lysis =
[((experimental CPM) - (target lealc CPM)) / ((detergent lysis CPM) - (target leak CPM))] X 100%. Specific lysis is calculated using the formula: Specific lysis =
(%lysis with antibody) - (% lysis without antibody).
Complement Binding Assay To assess the ability of the immunoconjugates to bind complement, the following assay can be utilized. Cells expressing the target antigen recognized by the immunoconjugate are incubated with the immunoconjugate at a concentration of ~,g/ml. After incubating the plates containing the cells and immunoconjugate for 15 minutes at room temperature, the plates are washed three times. After the third wash, the cells are resuspended in 50 ~,l of a 1/10 dilution of complement (e.g., guinea pig complement from ICN) and incubated at 37°C for varying times. Then 50 ~,l of 0.25% (w/v) trypan blue is added and cell number and plasma integrity of the cells are estimated.
Pha~ocytosis Assay To assess the phagocytosis enhancing ability of a particular immunoconjugate, the following assay can be used. Cells expressing the target antigen are labeled with lipophilic red fluorescent dye PITH 26. Buffy coat cells purified from heparinized, whole blood containing effector cells are incubated with the labeled taxgets at 37°C
for about 6 hours in the absence or in the presence of the immunoconjugate.
Effector cells are then stained with FITC (fluorescein isothiocyanate) labeled antibody, which binds to the effector cell at 0°C. Cells are washed and analyzed using two color fluorescence by FACScan or other scanning method. Percent phagocytosis is expressed as the percent of effector cells (NK cells, monocytes, neutrophils or macrophages) that have PKH 26 stain associated with them.
III. Methods of Administering IFN-a Fusion Protein A fusion protein of the invention is administered to subjects in a biologically compatible form suitable for pharmaceutical administration ih vivo. By "biologically compatible form suitable for administration ifa vivo" is meant a form of the immunogonjugate to be administered in which any toxic effects are outweighed by the therapeutic effects of the protein. An immunogonjugate can be administered in any pharmacological form, optionally in a pharmaceutically acceptable carrier.
Administration of a therapeutically effective amount of the innnunoconjugate is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result (e.g., inhibition of the progression or proliferation of the disease being treated). For example, a therapeutically active amount of an immunoconjugate may vary according to such factors as the disease stage (e.g., stage I
versus stage IV), age, sex, medical complications, and weight of the individual, and the ability of the immunoconjugate to elicit a desired response in the individual. The dosage regimen may be adjusted to provide the optimum therapeutic response.
For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
The active compound, an immunoconjugate, by itself or in combination with other active agents, such as chemotherapeutic anti-cancer drugs. The immunoconjugate, alone or in combination with other agents, may be administered in a convenient manner such as by injection (subcutaneous, intramuscularly, intravenous, etc.), inhalation, transdermal application or rectal achninistration.
Depending on the route of administration, the active compound may be coated with a material to protect the active compound from the action of enzymes, acids and other natural conditions which may inactivate the compound. A preferred route of administration is by intravenous (LV.) injection.
To achninister an immunoconjugate by other than parenteral administration, it may be necessary to coat the IFN-a fusion protein with, or co-administer the IFN-a fusion protein with, a material to prevent its inactivation. For example, an IFN-a fusion protein can be administered to an individual in an appropriate carrier or diluent, co-administered with enzyme inhibitors or in an appropriate carrier or vector, such as a liposome. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water emulsions, as well as conventional liposomes (Strejan et al., J. Neuroirn~uszol. 7: 27 (1984)).
Additional pharmaceutically acceptable carriers and excipients are known in the art or as described in IZEMINGTON'S PHARMACEOTICAL SCIENCES (18th ed. 1990).
The active compound may also be administered parenterally or intraperitoneally. Dispersions of the active compound can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain one or more preservatives to prevent the growth of microorganisms.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as manitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating an active compound (e.g., an IFN-a fusion protein) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
When the active compound is suitably protected, as described above, the protein may be orally administered, for example, with an inert diluent or an assimilable edible carrier. As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well kno~cm in the art.
Except insofar as amy conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. All compositions discussed above for use with an IFN-a fusion protein may also comprise supplementary active compounds in the composition.
It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of a dosage. "Dosage unit form,"
as used herein, refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on: (A) the unique characteristics of the active compound and the particular therapeutic effect to be achieved; and (B) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
IV. Method of Treating Cancer The immunoconjugates described herein can be targeted to a variety of malignant cells which express a tumor-associated antigen (TAA) expressed on the surface of the cell. IFN-a fusion proteins comprising antibodies can be prepared which recognize B and T cell leukemias and lymphomas, multiple myelomas, and solid tumors (e.g., prostate carcinoma, colon carcinoma, lung carcinoma, breast carcinoma and ovarian carcinoma). In preferred embodiments, the immunoglobulin portion of the IFN-a fusion protein may recognize B cell markers (e.g., CD19, CD20, CD22), multiple myeloma antigens (e.g., CD38, HM1.24), leukemia markers (e.g., CD33), and phosphatidyl-serine antigen. Additional markers affiliated with certain malignancies and to which the immunoglobin portion of the fusion protein can include, but are not limited to, the following:
Table 1 Cancer Tumor Associated Anti ens Acute Lymphocytic Leukemia HLA-Dr, CD34, CD19, CD20, (ALL) CD1, CD2, CDS, CD7 Acute Myelogenous Leukemia HLA-Dr, CD34, CD13, CD14, (AML) CD15, CD33, CD7 Breast Cancer EGFR, HER-2, MUC1, TAG-72 Carcinoma CEA, TAG-72, MUC1 Chronic Lymphocytic Leukemia CD3, CD21, CD20, CD19, CD23, (CLL) HLA-Dr Hairy Cell Leukemia (HCL) HLA-Dr, CD19, CD20, CD21, Hodgkin's Disease Leu-M1 Melanoma HMB 45 Non-Hodgkin's Lymphoma CD20, CD19, Ia Prostate PSMA, SE10 David A. Scheinberg et al., "The Leukemias" in HARRISON'S PRINCIPLES of INTER~IaL
MEDICINE 1764-1774 (Kurt J. Isselbacher et al., eds., 13th ed. 1994).
V. Drugs to be Used in Combination with the Fusion Protein The immunoconjugates described above can be used in combination with one or more different cancer treatment modalities, such as radiotherapy, immunotherapy, chemotherapy, and surgery. The combination of treatments used on any particular subject will vary depending on cancer type, stage of disease, family history, age, sex, weight and condition of the subject. Preferably, the innnunoconjugates are administered in combination with one more chemotherapeutics. Preferably, chemotherapeutic or chemotherapeutic cocktail is administered in combination with the interferon immunoconjugate described herein, and include those listed in the table below:
Cancer Chemothera Acute Lymphocytic Ara-C alone or with L-asparaginase, doxorubicin, Leukemia (ALL) idarubicin, mitoxantrone Acute Myelogenous hydroxyurea and busulfan; chlorambucil, melphalan, 6-Leukemia (AML) mercaptopurine, 6-thioguanine, dibromomannitol, ara-C, IFN-a Breast Cancer CMF, CAF, CEF, CMFVP, AC, VAT, VATH, CDDP +
VP-16, Tam Chronic Lymphocytic CAP, CVP, CMP, CHOP
Leukemia (CLL) Hairy Cell Leukemia 2-chlorodeoxyadenosine, deoxycoformycin, (HCL) IFN-a Hodgkin's Disease VABCD, ABDIC, CBVD, PCVP, CEP, EVA, MOPLACE, MIME, MINE, MTX-CHOP, CEM, CEVD, CAVP, EVAP, EPOCH, MOPP, MVPP, ChIVPP, AVD, MOPP + ABVD, MOPP + ABV
Melanoma (metastatic)dacarbazine, cisplatin, IFN-a-2b, caxmustine, lomustine, Cancer Chemothera tauromustine, fotemustine, carboplatin, vincristine, vinblastine, vindesine, taxol, dibromodulcitol, detorubicin, and iritrexim Non-Hodgkin's LymphomaIMVP-16, MIME, DHAP, ESHAP, CEPP(B), CAMP, CHOP, CAP-BOP, CHOP-B, ProMACE-MOPP, m-BACOD, MACOP-B, ProMACE-CytaBOM
Prostate (hormonallydoxorubicin, doxorubicin + ketoconazole or relapsed patients) cyclophosphamide, estramustine, vinblastine, paclitaxel, navelbine, prenisolone, mitoxantrone or combinations thereof.
These chemotherapeutic drugs and drug cocktails (e.g., more than one chemoterapeutic agent) can be administered according to the regimens described in CANCER: PRINCIPLES & PRACTICE OF ONCOLOGY (Vincent T. DeVita, Jr. et al. eds., Stn ed. 1997) or as would be known to the skilled artisan.
The abbreviations for the chemotherapeutic cocktail and chemotherapeutic acronyms are as follows:
AC doxorubicin, cyclophosphamide ABDIC doxorubicin, bleomycin, dacarbazine, lomustine, prednisone ABVD doxorubicin, bleomycin, vinblastine, dacarbazine Ara-C cytarabine AVD doxorubicin, vinblastine, dacarbazine CAF cyclophosphamide, doxorubicin, 5-fluorouracil CAMP lomustine, mitoxantrone, cytarabine, prednisone CAP cyclophosphamide, doxorubicin, prednisone CAP-BOP cyclophosphamide, doxorubicin, procarbazine, bleomycin, vincristine, prednisone CAVP lomustine, melphalan, etoposide, prednisone CEVD lomustine, etoposide, vindesine, dexamethasone CDDP+VP-16 cisplatin, etoposide, mitomycin C + vinblastine CEF cyclophosphamide, epirubicin, 5-fluorouracil CEM lomustine, etoposide, methotrexate CEP lomustine, etoposide, prechumustine CEPP(B) cyclophosphamide, etoposide, procarbazine, prednisone, bleomycin CEVD lomustine, etoposide, vindesine, dexamethasone ChIVPP chlorambucil, vinblastine, procarbazine, prednisone CHOP cyclophosphamide, doxorubicin, vincristine, prednisone CHOP-B CHOP plus bleomycin CMF cyclophosphamide, methodtrexate, 5-fluorouracil CMP cyclophosphamide, melphalan, prednisone CMVP cyclophosphamide, methotrexate, 5-fluorouracil, vincristine, prenisone CVP cyclophosphamide, vincristine, prednisone DHAP dexamethasone, high-dose .Ara-C, cisplatin ESHAP etoposide, methylpredisolone, high-dose cytarabine, cisplatin EPOCH etopsoide, vincristine, doxorubicin, cyclophospharilide, prednisone EVA etoposide, vinblastine, doxorubicin EVAP etoposide, vinblastine, cytarabine, cisplatin IFN-a interferon a IMVP-16 ifosfamide, methotrexate, etoposide MACOP-B methodtrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin, leucovorin m-BACOD methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone, leucovorin MIME methyl-gag, ifosfamide, methotrexate, etoposide MINE mitoquazone, ifosfamide, vinorelbine, etoposide MOPLACE cyclophosphamide, etoposide, prednisone, methotrexate, cytarabine, vincristine MOPP nitrogen mustard, vincristine, procarbazine, prednizone MOPP+ABV MOPP plus doxorubicin, bleomycin, vinblastine MOPP+ABVD alternating months of MOPP and ABVD
MVPP nitrogen mustard, vinblastine, procarbazine, prednisone MTX-CHOP methotrexate + CHOP
PCVP vinblastine, procarbazine, cyclophosphamide, prednisone ProMACE-CytaBOM prednisone, doxorubicin, cyclophosphamide, etoposide, cytarabine, bleomycine, vincristine, methotrexate, leucovorin ProMACE-MOPP prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, leucovorin + standard MOPP
Tam ~ tamoxifen VABCD vinblastine, doxorubicin, dacarbazine, lomustine, bleomycin VAT vinblastine, doxorubicin, thiotepa VATH vinblastine, doxorubicin, thiotepa, fluoxymesterone EXAMPLES
The examples and methods provided below serve merely to illustrate particular embodiments of the invention and are not meant to limit the invention.
Immunoconiu~ate Comnrisin~ IFN-a and Rituximab The nucleic acid encoding an IFN-a (e.g., IFN-a-2a, IFN-a-2b or IFN-a-nl) is operably linl~ed to the nucleic acid encoding Rituximab such that when translated the IFN-a would form the carboxy terminus of the fusion protein. The antigen-binding Fc receptor-binding, C 1 q binding and complement (C') activation, as well as the ability of IFN-a to bind to NIA cells and macrophages are characteristics possessed by the agents. In addition to the nucleic acid encoding Rituximab, other nucleic acids encoding anti-CD20 antibodies can be operably attached to the nucleic acid encoding IFN-a. The other anti-CD20 antibodies include Ibritumomab, IFS, B1 and 1H4.
_2g_ ha Yitro Testing of an Immunoconlu~ate A tumor cell line (cell expressing a target antigen) expressing Her2/neu (e.g., human breast carcinoma cells, SKBR-3) is selected to determine lysis using the immunoconjugates described herein. Effector cell samples are obtained by using heparinized whole blood or obtained from a third party. To prepare for use as effector cells, monocytes are cultured in Teflon containers in Macrophage Serum-Free Medium (Gibco/BRL) containing 2% human serum for 24 to 48 hours. Target cells are labeled with 100 ~,Ci of 5lCr for one hour prior to incubation with the effector cells and immunoconjugate in a U-bottomed microtiter plate. After incubation for about 16 to 18 hours at 37°C, supernatants from each well are collected and analyzed for radioactivity. Cytotoxicity and specific lysis can be calculated as previously described.
ha hivo Testing of Immunoconlu~ate For assessment of anti-tumor activity of the immunoconjugates, a mouse model can be used. In the instance of solid tumors, about 1x10' cells in culture media are injected subcutaneously into the right anterior flank of BALBIc, nulnu or SCm mice. Approximately, fourteen days later or when the tumors have grown to about 0.8 to 1.2 cm in diameter, the mice are separated into groups of 5-10 animals and injected intravenously with 200 ~,I of immunoconjugate at concentrations of 1 ~,g to 10 mg.
Perpendicular tumor diameters are measured at regular intervals and tumor volumes can be calculated. Alternatively, animals can be euthanized and sections of tumor prepared to determine the impact on tumor progression by the immunoconjugate as compared to the control animals (untreated).
Although the present invention has been described in detail with reference to examples above, it is understood that various modifications can be made without departing from the spirit of the invention. All cited patents and publications referred to in this application are herein incorporated by reference in their entirety.
Cancer Res. 2: 1851-7 (1996)). U.S. Patent No. 5,855,866 reported a method of treating the vasculature of solid tumors using an anti-p185Her-a antibody linlced to a cytokine.
Anti MZIC 1 Antibodies. MUC-1 is a carcinoma associated mucin. The anti-MUC-1 monoclonal antibody, McS, was reportedly administered to mice carrying transplanted breast tumors and purportedly suppressed tumor growth (Peterson et al., Cancer Res. 57: 1103-8 (1997)). Mc5 was chemically linked in a non-cleavable fashion to a natural IFN-a (nIFN-a) and reportedly inhibited growth of injected tumors in mice (Ozzello et al., Breast Cancef~ Res. Treat. 25: 265-76 (1993)). An IgG4 anti-MUC-1 mAb, hCTM0l, has been suggested as a suitable carrier for cytotoxic agents in ovarian carcinomas (Van Hof et al., Cancer Res. 56: 5179-85).
Anti phosplaatidyl serine antigen Antibodies. Phosphatidyl-serine is a phospholipid. Antibodies have been reported which bind to phosphatidyl-serine and not other phospholipids (e.g., Yron et al., Clih. Exp. Immol. 97: 187-92) (1994)).
However, anti-phospholipid antibodies appear more typically associated with anti-phospholipid syndrome and its diagnosis, than for use in the treatment or diagnosis of cancer.
Anti-TAG-72 Antibodies. TAG-72 is a tmnor-associated antigen (TAG). One dimeric single-chain Fv antibody construct of monoclonal CC49 recognizes the TAG-72 epitope (Pavlinkova et al., Clifa. Cancer Res. 5: 2613-9 (1999)).
Additional anti-TAG-72 antibodies include B72.3 (Divgi et al., Nucl. Med. Biol. 21: 9-15 (1994)) and those disclosed in U.S. Patent No. 5,976,531. Administration of recombinant IFN-a reportedly increased the amount of TAG-72 expressed on tumors (Macey et al., ClifZ.
Caf~cey-Res. 3: 1547-55 (1997)). The CC49 antibody also has reportedly been chemically conjugated to doxorubicin (Johnson et al., Af2ticahcey~ Res. 15:
(1995)) and streptavidin (Ngai et al., Nucl. Med. Biol. 22: 77-86 (1995)). TAG-72 has also been chemically conjugated to human interleukin-2 (IL-2) (LeBerthon et al., Cayacef° Res. 51: 2694-8 (1991). U.S. Patent No. 5,976,531 claimed a human anti-Tag-72 antibody conjugated to an interferon but no evidence was presented indicating its efficiency in mammals.
Interferon-a Isnnzunoconjugates. Monoclonal antibodies raised against tumor cell lines have been covalently coupled with purified human lymphoblastoid IFN-a. Administration of this coupled form of IFN-a reportedly augmented killing of the tumor cells and other tumor targets by peripheral blood NK cells (Flannery et al., Euf°. J. Cafzcer~ Clin. Oncol. 20: 791-8 (1984)).
Techniques also have been developed to produce chimeric antibodies which combine regions of immunoglobulin molecules from different sources (Morrison et al., Proc. Natl Acad. Sci. USA 81: 6851-5 (1984)). Using this technology, chimeric molecules comprising immunoglobulin and non-imrnunoglobulin regions have been created. Expression vectors for fusion proteins comprising DNA sequences of an antibody and DNA sequences encoding a cell surface antigen have been proposed, wherein some of the suitable antigens include CD molecules (e.g., CD19, CD20, CD22, CD33, CD38 and CD40). See, e.g., U.S. Patent No. 5,637,481 (1997) and EP
Patent No. 610,046 (1994). Fusion proteins comprising the cytokine IL-15 and anti-CD20 (International PCT Application 98/16254), and fusion proteins comprising other lymphokines (e.g., IL,-2 and IL-3) and an imrnunoglobulin fragment capable of binding to a tumor antigen have been described (U.S. Patent Nos. 5,645,835 (1997) and 5,314,995 (1994); Lode et al., Blood 91: 1706-1715 (1998); Hassan et al., Leuk.
Lymphoma 20: 1-15 (1995)). Chang et al., U.S. Patent 5,723,125 describes a hybrid molecule comprising an interferon, preferably IFN-a-2a or IFN-a-2b, which are joined at the carboxy terminus via a peptide to the amino terminus of a first gamma immunoglobulin Fc fragment as a means of increasing the blood half life of the cytokine. Chimeric immunoglobulin proteins comprising a cytokine (e.g., IL-2, tumor necrosis factor a, etc.) and the heavy chain of an antibody for treating viral infections and cancer are described in International PCT Application 92/08495, as well as a fusion protein of IFN-y/M-CSF. A fusion protein of RM4/IFN-tau has demonstrated antitumor activity in mice (Qi et al., Hum. Antibodies Hyb>"idomas 7: 21-6 (1996); and Xiang et al., Hunt. Antibodies Hybf°idontas 7: 2-10 (1996)).
Others have proposed the creation of fusion proteins comprising non-antibody immunomodulatory molecules, wherein IFN-a is operatively fused to a heterologous membrane attachment domain (IJ.S. Patent No. 5,891,432 (1999)). IFN-a-2b was reported to be conjugated with ME31.3 and anti-carcinoembryonic antigen (CEA) and purportedly may improve diagnostic and therapeutic potential of monoclonal antibodies making them worthy of further study (Thakur et al., J. Inamuhother.
20:
194-201 (1997)).
Therefore, not withstanding what has been previously reported in the literature, there exists a need for improved compositions and methods of using such compositions for the treatment of cancer. Improved immunoconjugates with enhanced ADCC and phagocytotic activities, lower toxicity, and increased blook serum half life would provide additional treatment therapeutics, which are still needed in cancer therapy.
SUMMARY OF THE INVENTION
It is one object of the invention to provide novel and improved compositions comprising an immunoconjugate that comprises an antibody or imrnunogenic fragment thereof that binds to an antigen expressed by a target cell that is to be eradicated, wherein said antibody or imrnunogenic fragment thereof possesses human effector function, which antibody or immunogenic fragment thereof is fused at its carboxy terminus to a cytokine that binds a receptor expressed on the surface of a natural killer cell and/or macrophage, thereby resulting in an immunoconjugate that facilitates extracellular (ADCC-type) and intracellular (phagocytic) killing of a target cell, when said immunoconjugate is administered to a host. Preferably, the cytokine is a interferon. Most preferably, the interferon is an a-interferon, especially one which has been FDA approved (e.g., IFN-a-2a, IFN-a-2b and IFN-a-nl).
It is a more specific object of the invention to provide novel immunoconjugates which target cells expressing CD19, CD20, CD22, CD33, CD3~, EGF-R, HM 1.24, phosphatidyl serine antigen, HER-2, TAG-72 and MUC1.
It is a further obj ect of the invention to provide novel methods of enhancing apoptosis or treating cancer by achninistering a therapeutically effective amount of these immunoconjugates to a subject. The target cells to which these immunoconjugates are directed may include malignant cells selected from the group consisting of a breast carcinoma cell, an ovarian carcinoma cell, a prostate carcinoma cell, a lung carcinoma cell, a leukemic T-cell, a leukemic B-cell, a multiple myeloma cell and a B-cell lymphoma cell.
It is another obj ect of the invention to provide a combination therapy to treat a malignancy in a subject comprising an immunoconjugate as described above and at least one chemotherapeutic agent or chemotherapeutic cocktail.
It is a further object of this invention to provide nucleic acids which encode the immunoconjugates described herein.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to IFN-a fusion proteins, specifically immunoconjugates, and the use thereof as therapeutic agents that have the ability to target malignant cells and enhance the killing activity of effector cells through the binding of an IFN-a to its receptor on the effector cell and without systemic administration of IFN-a and its associated toxicity. These immunoconjugates will have enhanced ADCC and phagocytic activity.
I. Definitions By "interferon-a" or "IFN-a" is meant a protein or fragment thereof which can recognize and bind to the IFN-a receptor. This includes FDA approved forms of IFN-a, such as IFN-a-2a (Roferon by Hoffinan-LaRoche), IFN-a-2b (INTRON~ A by Schering Corporation) and IFN-a-nl (lymphoblastoid interferon called Wellferon and produced by Wellcome Foundation Ltd - Wellcome Research Laboratories), as well as other forms of IFN-a (e.g., IFN-a-2a and consensus IFN).
The term "fusion protein" as used herein means a hybrid protein produced recombinantly including a synthetic or heterologous amino acid sequence. A
fusion protein can be produced from a hybrid gene containing operatively linked heterologous gene sequences.
By "bispecific fusion protein" is meant any immunologically reactive molecule which specifically recognizes and binds two different targets at alternate times or at the same time. In particular, it will refer to an IFN-a antibody fusion protein, wherein the IFN is attached to an antibody, preferably anti-tumor antibody, at the carboxy terminus of the antibody. In a preferred embodiment, the antibody portion may recognize and bind to a target antigen expressed on a targeted cell. In a more preferred embodiment, the antigen-binding, Fc receptor binding, Clq and C
_g_ activation, and the ability of interferon to bind to its receptor and activate effector cells and macrophages are substantially maintained activities of the immunoconjugate.
This is preferably effected by attachment of the interferon directly or indirectly to the antibody hinge, CHl, CH2 or CH3 domain carboxy-terminus.
The terms "effector cell" and "effector function" as used herein means a cell which expresses an IFN-a receptor and can thereby bind to an IFN-a fusion protein.
Effector cells can include natural killer (NK) cells, LAK cells, monocytes, macrophages and polymorphonuclear (PMNs) cells. Preferred effector cells include NK cells and macrophages.
An "expression vector" means a nucleic acid molecule comprising (1) a promoter and other sequences (e.g., leader sequences) necessary to direct expression of a desired gene or DNA sequence, and (2) the desired gene or DNA sequence.
Optionally, the nucleic acid molecule may comprise a poly A signal sequence to enhance the stability of the gene transcript and/or to increase gene transcription and expression.
By "binding domain," "binding region" means a binding site which recognizes and binds the entire binding area of a target or any portion thereof. Examples for antibodies or immunoglobulin fragments include: (1) single variable region of an antibody VL or VH; (2) two or more variable regions (e.g., VL and VH, VL and VL; or VH and VH) or the complementary determining region (CDR) thereof; (3) antibody fragments such as Fabl, Fab2, SFV, single chain antibodies, domain-deleted antibodies and minibodies; or (4) an IFN-a or a segment of IFN-a which binds to an IFN-a receptor on an effector cell.
By "minibody" is meant an antigen binding protein which includes VL and VH
domains of a native antibody fused to the hinge region and CH3 domain of an immunoglobulin or which encodes in a single chain comprising the essential elements of a whole antibody. The single chain comprises the antigen binding region, domain to permit assembly into a bivalent molecule, and the antibody hinge to accommodate dimerization by disulfide linkages. For methods of preparing minibodies, see, e.g., U.S. Patent No. 5,837,821.
By "antibody" is intended to refer broadly to any immunologic binding agent such as IgG (including IgGI, IgG2, IgG3, and IgG4), IgM, IgA, IgD, IgE, as well as antibody fragments. Antibodies in the broadest sense covers intact monoclonal antibodies, polyclonal antibodies, as well as biologically active fragments of such antibodies such as those discussed above. In particular, domain-deleted antibodies are included within the scope of the present invention, such as CH2 domain-deleted antibodies.
By "monoclonal antibody" is meant an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature 256: 495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies"
may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352: 624-628 (1991) and Marks et al., J. lVlol. Biol., 222:
581-597 (1991), for example.
The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired therapeutic activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci.
USA, 81:
6851-5 (1984)).
"Humanized" forms of non-htunan (e.g., marine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies), which contain minimal sequence derived from a non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
The humanized antibody optimally also will comprise at least a portion of an irmnunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-5 (1986); Reichmann et al., Natuf°e, 332: 323-9 (1988); and Presta, Curs. ~p. StYZICt. Biol., 2: 593-6 (1992).
By "target antigen" is meant the antigen recognized by the antibody or immunoglobulin fragment portion of the immunoconjugate. Preferred target antigens include: 5E10, CDl, CD2, CD3, CDCDS, CD7, CD13, CD14, CD15, CD19, CD20, CD21, CD23, CD25, CD33, CD34, CD38, CEA, EGFR, HER-2, HLA-DR, HM 1.24, HMB 45, Ia, Leu-M1, MUC1, phosphatidyl serine antigen, PMSA and TAG-72.
By "nucleic acid" is meant to include an oligonucleotide, nucleotide, polynucleotide and fragments and portions thereof, and a DNA or a RNA of genomic or synthetic origin, which may be single or double stranded.
By "interferon a" or "IFN-a" preferably is meant to include all members of the interferon-a family of proteins. Fragments of IFN-a are also included, as long as the fragment is capable of recognizing and binding to the IFN-a receptor and thereby activating effector cells. Preferred forms of IFN-a include such FDA approved forms of IFN-a as IFN-a-2a, IFN-a-2b and IFN-a-ln.
By "purified" and "isolated" is meant, when referring to a polypeptide or nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. The term "purified" as used herein preferably means at least 75% by weight, more preferably at least 85%
by weight, more preferably still at least 95% by weight, and most preferably at least 98%
by weight, of biological macromolecules of the same type are present. An "isolated nucleic acid molecule wluch encodes a particular polypeptide" refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties which do not deleteriously affect the basic characteristics of the composition. Thus, for example, an isolated nucleic acid molecule which encodes a particular CDR polypeptide consists essentially of the nucleotide coding sequence for the subject molecular recognition unit.
By "therapeutically effective" is meant the ability of the immunoconjugate to inhibit in vitro growth of a target cell by greater than about 20%, at a concentration of about 0.1 to about 3.0 ~,g/ml of the immunoconjugate, wherein said target cells (e.g., tumor cells) are cultured in an appropriate culture medium and said growth inhibition is determined about 4, 5, 6, 7, 8, 9, or 10 days after exposure of the target cells to the immunoconjugate.
By "subject" is meant a living animal or human susceptible to a condition, especially cancer. In the preferred embodiments, the subject is a mammal, including human and non-human marmnals. Non-human mammals include dogs, cats, pigs, cows, sheep, goats, horses, rats and mice.
II. Method of Mal~in~ an IFN-a Fusion Protein Nucleic acids encoding the desired fusion protein can be inserted into expression vectors for expression. Expression vectors useful in the invention include prokaryotic and eukaryotic expression vectors. Such expression vectors, including plasmids, cosmids, and viral vectors such as bacteriophage, baculovirus, retrovirus and DNA virus vectors, are well known in the art (see, for example, Meth.
Enzymol., Vol. 185, D. V. Goeddel, ed. (Academic Press, Inc., 1990), and Kaplitt and Loewy (Ed.), VIRAL VECTORS: GENE THERAPY AND NEUROSCIENCE APPLICATIONS
(Academic Press, Inc., 1995), each of which are incorporated herein by reference).
Expression vectors contain the elements necessary to achieve constitutive or inducible transcription of a nucleic acid molecule encoding an IFN-a fusion protein. One form of IFN-a is described in Pitha et al., J. Immunol. 141: 3611-6 (1988).
Preferred embodiments of the invention will utilize FDA approved forms of IFN-a.
The recombinant nucleic acid molecules encoding an IFN-a fusion protein, an immunoglobulin polypeptide, or a specific IFN-a species or fragment thereof may be obtained by any method known in the art (see, e.g., Maniatis et al., MOLECULAR
CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982 and 1989) or obtained from publicly available clones, such as pGL2BIFN (ATCC No. 53371) and pALCAISIFN (ATCC No. 53369).
Alternatively, a nucleic acid encoding an IFN-a or an antibody which recognizes a tumor-associated antigen (TAA) may be obtained as follows. A population of cells known to actively express an IFN-a or a specific antibody may be obtained, and total cellular RNA harvested therefrom. The amino acid sequence of the IFN-a or antibody may be used to deduce the sequence of a portion of its nucleic acid so as to design appropriate oligonucleotide primers; or, alternatively, the oligonucleotide primers may be obtained from a known nucleic acid sequence encoding an antibody or an IFN-a.
The oligonucleotide fragment may then be used in conjunction with reverse transcriptase to produce a cDNA corresponding to the immunoglobulin and/or IFN-a encoding nucleotide sequence (Okayama et al., Methods Erazymol. 154: 3-29 (1987)).
The cDNA can then be cloned, and/or portions of the antibody or IFN-a coding region amplified from this cDNA using polymerase chain reaction (PCR) and appropriate primer sequences (Saiki et al., Science 239: 487-491 (1988)).
In preferred embodiments of the invention, a recombinant vector system may be created to accoimnodate sequences encoding IFN-a, wherein the IFN-a sequence is attached to the sequences encoding the C-terminus of an antibody or immunogenic fragment that recognizes a tumor-associated antigen. The resultant fusion protein will preserve the ability of the IFN-a molecule to bind to its receptor on the effector cell and enhance the effector cell's killing ability. The immunoconjugate will also preserve the antigen-binding Fc receptor-binding, Clq binding and C' activation regions of the antibody molecule.
Nucleic acid sequences encoding the various components of the IFN-a based fusion proteins of the invention may be joined together using any techniques known in the art, including restriction enzyme methodologies and the use of synthetic linker sequences.
To provide for adequate transcription of the recombinant constructs of the invention, a suitable promoter/enhancer sequence may be incorporated into the recombinant vector. Promoters which may be used to control the expression of the antibody-based fusion protein include, but are not limited to, the SV40 early promoter region (Bernoist et al., Nature 290: 304-310 (1981)), the promoter contained in the 3' long terminal repeat of Rous sarcoma viruses (Yamamoto et al., Cell 22: 787-(1980)), the herpes thymidine kinase (tk) promoter (Wagner et al., Proc. Natl.
Acad.
Sci. USA 78: 144-1445 (1981)), the regulatory sequences of the metallothionine gene (Brinster et al., Nature 296: 39-42 (1982)); prokaryotic expression systems such as the LAC, or (3-lactamase promoters (Villa-Kamaroff et al., Proc. Natl. Acad. Sci.
USA 75:
3727-3731 (1978)), or the tac lambda phage promoter (DeBoer et al., P~oc.
Natl.
Acad. Sci. USA 80: 21-25 (1983)). Other suitable promoters would be apparent to the skilled artisan.
Additionally, it may be desirable to include, as part of the recombinant vector system, nucleic acids corresponding to the 3' flanking region of an immunoglobulin gene, including RNA cleavage/polyadenylation sites and downstream sequences.
Furthermore, it may be desirable to engineer a signal sequence upstream of the IFN-a fusion protein-encoding sequences to facilitate the secretion of the fused molecule from a cell transformed with the recombinant vector.
Creation of IFN-a containing fusion proteins can also utilize sequences encoding conservative amino acid substitutions in an IFN-a sequence, as well as substitutions in the antibody or immunoglobulin region of the fusion protein.
Such changes include substituting an isoleucine, valine and leucine for any other of these hydrophobic amino acids; aspartic acid for glutamic acid and vice versa;
glutamine for asparagine and vice versa; and serine for threonine and vice versa. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine for alanine can frequently be interchangeable, as well as alanine for valine due to structural and charge similarities.
Successful incorporation of IFN-a based fusion gene constructs may be identified by three general approaches: (a) DNA-DNA hybridization, (b) presence or absence of "marker" gene functions, and (c) expression of inserted sequences.
In the first approach, the presence of a foreign gene inserted in an expression vector can be detected by DNA-DNA hybridization using probes comprising sequences that are homologous to the inserted antibody IFN-a fusion protein DNA. In the second approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, resistance to antibiotics such as 6418, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of foreign genes in the vector. For example, if the IFN-a fusion gene is inserted so as to interrupt the marker gene sequence of the vector, recombinants containing the antibody fusion gene ~ insert can be identified by the absence of the marker gene function. In the third approach, recombinant expression vectors can be identified by assaying the foreign gene product expressed by the recombinant. Such assays can be based, for example, on the physical or functional properties of the IFN-a fusion gene product in bioassay systems.
The cytokine can be any cytokine or analog or fragment thereof which activates effector cells. The preferred cytokine is an interferon, with the preferred interferon being IFN-a, especially FDA approved forms. The gene encoding the cytoleine can be cloned de novo, obtained from an available source, or synthesized by standard DNA synthesis from a known nucleotide sequence as discussed above.
The heavy chain constant region for the conjugates can be selected from any of the five isotypes: alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG) or mu (IgM).
Heavy chains or various subclasses (such as the IgG subclasses 1-4) can be used. The light chains can have either a kappa or lambda constant chain. DNA sequences for these immunoglobulin regions are well known in the art. (See, e. g., Gillies et al., J.
Immunol. Metla. 125: 191 (1989)).
In preferred embodiments, the variable region is derived from an antibody specific for the target antigen, and the constant region includes the CH1, CH2 and CH3 domains. The gene encoding the cytokine is joined, in frame to the 3' end of the gene encoding the constant region (e.g., CH1, CH2 or CH3 exon depending on domain-deleted form desired), either directly or through an intergenic region.
The nucleic acid construct can include an endogenous promoter and enhancer for the variable region-encoding gene to regulate expression of the chimeric immunoglobulin chain. For example, the variable region encoding genes can be obtained as DNA fragments comprising the leader peptide, the VJ gene (functionally rearranged variable (V) regions with joining (J) segment) for the light chain or VDJ
gene for heavy chain, and the endogenous promoter and enhancer for these genes.
Alternatively, the gene coding for the variable region can be obtained apart from endogenous regulatory elements and used in an expression vector that provides these elements.
Variable region genes can be obtained by standard DNA cloning procedures from cells that produce the desired antibody. Screening of the genomic library for a specific functionally rearranged variable region can be accomplished with the use of appropriate DNA probes, such as DNA segments containing the J region DNA
sequence and sequences downstream. Identification and confirmation of correct clones are then achieved by DNA sequencing of the cloned genes and comparison of the sequence to the corresponding sequence of the full length, properly spliced mRNA.
The target antigen preferably can be a cell surface antigen of a tumor cell, but also includes viral antigens or other disease associated antigens expressed on the cell surface. Genes encoding appropriate variable regions can be obtained generally from immunoglobulin producing lymphoid cells. For example, hybridoma cell lines producing immunoglobulin specific for tumor associated antigens or viral antigens can be produced by standard somatic cell hybridization techniques (see, e.g., U.S. Pat.
No. 4,96,265.). These inununoglobulin producing cell lines provide the source of variable region genes in functionally rearranged form. The variable region genes will typically be of marine origin, because the marine system lends itself to the production of a wide variety of imrnunoglobulins of desired specificity.
The DNA fragment containing the functionally rearranged variable region gene is linked to a DNA fragment containing the gene encoding the desired constant region (or a portion thereof). Immunoglobulin constant regions (heavy and light chain) can be obtained from antibody-producing cells by standard gene cloning techniques. Genes for the two classes of human light chains and the five classes of human heavy chains have been cloned, and thus, constant regions of human origin are readily available from publically available clones.
The fused gene encoding the hybrid immunoglobulin heavy chain is assembled or inserted into expression vectors for incorporation into a recipient cell.
The introduction of gene construct into plasmid vectors can be accomplished by standard gene splicing procedures.
Recipient cell lines are generally lymphoid cells. The preferred recipient cell is a myeloma (or hybridoma). Myelomas can synthesize, assemble, and secrete immunoglobulins encoded by transfected genes, and they post-tra~islationally modify the protein. A particularly preferred recipient cell is the Sp2/0 myehoma which normally does not produce endogenous immunoglobulin. When transfected, the cell will produce only innnunoglobulin encoded by the transfected gene constructs.
Transfected myelomas can be grown in culture or in the peritoneum of mice where secreted immunoconjugate can be recovered from ascites fluid. Other lymphoid cells, such as B lymphocytes, also can be used as recipient cells.
There are several methods for transfecting lymphoid cells with vectors containing the nucleic acid constructs encoding the chimeric Ig chain. A
preferred way of introducing a vector into lymphoid cells is by spheroblast fusion, as described by Gillies et al., Biotechn.ol. 7: 798-804 (1989). Alternative methods include electroporation or calcium phosphate precipitation. See also, the methods in MANJATIS, ET AL. (1989).
IFN-a based fusion protein produced by the host cell may be collected using any technique known in the art, including, but not limited to, affinity chromatography using target antigen or antibody specific for any portion of the fusion protein. The activity of the fused IFN-a or antibody (e.g., anti-CD20) may be confirmed using biological assays, which detect or measure the activity of the lymphokine or cellular factor. For example, and not by way of limitation, the presence of IFN-a activity may be confirmed in assays which detect receptor binding, virus neutralization and enhanced killing ability of the effector cells.
Preferred methods of detecting such enhanced effector cell ability can utilize receptor binding assays and virus neutralization assays. These assays are described generally below.
Receptor Binding Assay Receptor binding assays, such as those provided below, can be utilized to determine whether the immunoconjugate binds to the target antigen or to an IFN-a receptor.
Virus Neutralization Assay A virus neutralization assay is one form of receptor binding assay which can be utilized to determine the efficacy of which an immmloconjugate to neutralize virus-infected cells when the antibody targets a viral antigen expressed on the cell surface.
For example, virus neutralizations can be determined using the method described by Ho et al., J. Virol. 65: 489-93 (1991), for HIV-1 neutralization using a p24 assay.
Neutralization is defined as the percent reduction in the amount of target antigen released into the culture supernatants or detected in cells from wells treated with the immunoconjugate compared with control wells not treated with the immunoconjugate.
ADCC Assay The ability of the fusion protein to induce ADCC can be assessed using a chromium release assay. Generally, antibodies of the IgGZa and IgG3 subclass and occasionally of the IgGI subclass mediate ADCC. Antibodies of the IgG3, IgG2a and IgM classes bind and activate serum complement. To assess the ability of the immunoconjugates described herein to mediate ADCC and complement activation, one can use a SICr-release assay. Briefly, a cell line expressing the antigen being targeted for lysis by effector cells are labeled with 100 ~,Ci of SICr for about 1 hour prior to combining effector cells and antibodies in a U-bottom microtiter plate. After incubation for about 5 hours at 37°C, supernatants axe collected and analyzed for radioactivity. Cytotoxicity can be calculated by the formula: % lysis =
[((experimental CPM) - (target lealc CPM)) / ((detergent lysis CPM) - (target leak CPM))] X 100%. Specific lysis is calculated using the formula: Specific lysis =
(%lysis with antibody) - (% lysis without antibody).
Complement Binding Assay To assess the ability of the immunoconjugates to bind complement, the following assay can be utilized. Cells expressing the target antigen recognized by the immunoconjugate are incubated with the immunoconjugate at a concentration of ~,g/ml. After incubating the plates containing the cells and immunoconjugate for 15 minutes at room temperature, the plates are washed three times. After the third wash, the cells are resuspended in 50 ~,l of a 1/10 dilution of complement (e.g., guinea pig complement from ICN) and incubated at 37°C for varying times. Then 50 ~,l of 0.25% (w/v) trypan blue is added and cell number and plasma integrity of the cells are estimated.
Pha~ocytosis Assay To assess the phagocytosis enhancing ability of a particular immunoconjugate, the following assay can be used. Cells expressing the target antigen are labeled with lipophilic red fluorescent dye PITH 26. Buffy coat cells purified from heparinized, whole blood containing effector cells are incubated with the labeled taxgets at 37°C
for about 6 hours in the absence or in the presence of the immunoconjugate.
Effector cells are then stained with FITC (fluorescein isothiocyanate) labeled antibody, which binds to the effector cell at 0°C. Cells are washed and analyzed using two color fluorescence by FACScan or other scanning method. Percent phagocytosis is expressed as the percent of effector cells (NK cells, monocytes, neutrophils or macrophages) that have PKH 26 stain associated with them.
III. Methods of Administering IFN-a Fusion Protein A fusion protein of the invention is administered to subjects in a biologically compatible form suitable for pharmaceutical administration ih vivo. By "biologically compatible form suitable for administration ifa vivo" is meant a form of the immunogonjugate to be administered in which any toxic effects are outweighed by the therapeutic effects of the protein. An immunogonjugate can be administered in any pharmacological form, optionally in a pharmaceutically acceptable carrier.
Administration of a therapeutically effective amount of the innnunoconjugate is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result (e.g., inhibition of the progression or proliferation of the disease being treated). For example, a therapeutically active amount of an immunoconjugate may vary according to such factors as the disease stage (e.g., stage I
versus stage IV), age, sex, medical complications, and weight of the individual, and the ability of the immunoconjugate to elicit a desired response in the individual. The dosage regimen may be adjusted to provide the optimum therapeutic response.
For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
The active compound, an immunoconjugate, by itself or in combination with other active agents, such as chemotherapeutic anti-cancer drugs. The immunoconjugate, alone or in combination with other agents, may be administered in a convenient manner such as by injection (subcutaneous, intramuscularly, intravenous, etc.), inhalation, transdermal application or rectal achninistration.
Depending on the route of administration, the active compound may be coated with a material to protect the active compound from the action of enzymes, acids and other natural conditions which may inactivate the compound. A preferred route of administration is by intravenous (LV.) injection.
To achninister an immunoconjugate by other than parenteral administration, it may be necessary to coat the IFN-a fusion protein with, or co-administer the IFN-a fusion protein with, a material to prevent its inactivation. For example, an IFN-a fusion protein can be administered to an individual in an appropriate carrier or diluent, co-administered with enzyme inhibitors or in an appropriate carrier or vector, such as a liposome. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water emulsions, as well as conventional liposomes (Strejan et al., J. Neuroirn~uszol. 7: 27 (1984)).
Additional pharmaceutically acceptable carriers and excipients are known in the art or as described in IZEMINGTON'S PHARMACEOTICAL SCIENCES (18th ed. 1990).
The active compound may also be administered parenterally or intraperitoneally. Dispersions of the active compound can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain one or more preservatives to prevent the growth of microorganisms.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as manitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating an active compound (e.g., an IFN-a fusion protein) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
When the active compound is suitably protected, as described above, the protein may be orally administered, for example, with an inert diluent or an assimilable edible carrier. As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well kno~cm in the art.
Except insofar as amy conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. All compositions discussed above for use with an IFN-a fusion protein may also comprise supplementary active compounds in the composition.
It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of a dosage. "Dosage unit form,"
as used herein, refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on: (A) the unique characteristics of the active compound and the particular therapeutic effect to be achieved; and (B) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
IV. Method of Treating Cancer The immunoconjugates described herein can be targeted to a variety of malignant cells which express a tumor-associated antigen (TAA) expressed on the surface of the cell. IFN-a fusion proteins comprising antibodies can be prepared which recognize B and T cell leukemias and lymphomas, multiple myelomas, and solid tumors (e.g., prostate carcinoma, colon carcinoma, lung carcinoma, breast carcinoma and ovarian carcinoma). In preferred embodiments, the immunoglobulin portion of the IFN-a fusion protein may recognize B cell markers (e.g., CD19, CD20, CD22), multiple myeloma antigens (e.g., CD38, HM1.24), leukemia markers (e.g., CD33), and phosphatidyl-serine antigen. Additional markers affiliated with certain malignancies and to which the immunoglobin portion of the fusion protein can include, but are not limited to, the following:
Table 1 Cancer Tumor Associated Anti ens Acute Lymphocytic Leukemia HLA-Dr, CD34, CD19, CD20, (ALL) CD1, CD2, CDS, CD7 Acute Myelogenous Leukemia HLA-Dr, CD34, CD13, CD14, (AML) CD15, CD33, CD7 Breast Cancer EGFR, HER-2, MUC1, TAG-72 Carcinoma CEA, TAG-72, MUC1 Chronic Lymphocytic Leukemia CD3, CD21, CD20, CD19, CD23, (CLL) HLA-Dr Hairy Cell Leukemia (HCL) HLA-Dr, CD19, CD20, CD21, Hodgkin's Disease Leu-M1 Melanoma HMB 45 Non-Hodgkin's Lymphoma CD20, CD19, Ia Prostate PSMA, SE10 David A. Scheinberg et al., "The Leukemias" in HARRISON'S PRINCIPLES of INTER~IaL
MEDICINE 1764-1774 (Kurt J. Isselbacher et al., eds., 13th ed. 1994).
V. Drugs to be Used in Combination with the Fusion Protein The immunoconjugates described above can be used in combination with one or more different cancer treatment modalities, such as radiotherapy, immunotherapy, chemotherapy, and surgery. The combination of treatments used on any particular subject will vary depending on cancer type, stage of disease, family history, age, sex, weight and condition of the subject. Preferably, the innnunoconjugates are administered in combination with one more chemotherapeutics. Preferably, chemotherapeutic or chemotherapeutic cocktail is administered in combination with the interferon immunoconjugate described herein, and include those listed in the table below:
Cancer Chemothera Acute Lymphocytic Ara-C alone or with L-asparaginase, doxorubicin, Leukemia (ALL) idarubicin, mitoxantrone Acute Myelogenous hydroxyurea and busulfan; chlorambucil, melphalan, 6-Leukemia (AML) mercaptopurine, 6-thioguanine, dibromomannitol, ara-C, IFN-a Breast Cancer CMF, CAF, CEF, CMFVP, AC, VAT, VATH, CDDP +
VP-16, Tam Chronic Lymphocytic CAP, CVP, CMP, CHOP
Leukemia (CLL) Hairy Cell Leukemia 2-chlorodeoxyadenosine, deoxycoformycin, (HCL) IFN-a Hodgkin's Disease VABCD, ABDIC, CBVD, PCVP, CEP, EVA, MOPLACE, MIME, MINE, MTX-CHOP, CEM, CEVD, CAVP, EVAP, EPOCH, MOPP, MVPP, ChIVPP, AVD, MOPP + ABVD, MOPP + ABV
Melanoma (metastatic)dacarbazine, cisplatin, IFN-a-2b, caxmustine, lomustine, Cancer Chemothera tauromustine, fotemustine, carboplatin, vincristine, vinblastine, vindesine, taxol, dibromodulcitol, detorubicin, and iritrexim Non-Hodgkin's LymphomaIMVP-16, MIME, DHAP, ESHAP, CEPP(B), CAMP, CHOP, CAP-BOP, CHOP-B, ProMACE-MOPP, m-BACOD, MACOP-B, ProMACE-CytaBOM
Prostate (hormonallydoxorubicin, doxorubicin + ketoconazole or relapsed patients) cyclophosphamide, estramustine, vinblastine, paclitaxel, navelbine, prenisolone, mitoxantrone or combinations thereof.
These chemotherapeutic drugs and drug cocktails (e.g., more than one chemoterapeutic agent) can be administered according to the regimens described in CANCER: PRINCIPLES & PRACTICE OF ONCOLOGY (Vincent T. DeVita, Jr. et al. eds., Stn ed. 1997) or as would be known to the skilled artisan.
The abbreviations for the chemotherapeutic cocktail and chemotherapeutic acronyms are as follows:
AC doxorubicin, cyclophosphamide ABDIC doxorubicin, bleomycin, dacarbazine, lomustine, prednisone ABVD doxorubicin, bleomycin, vinblastine, dacarbazine Ara-C cytarabine AVD doxorubicin, vinblastine, dacarbazine CAF cyclophosphamide, doxorubicin, 5-fluorouracil CAMP lomustine, mitoxantrone, cytarabine, prednisone CAP cyclophosphamide, doxorubicin, prednisone CAP-BOP cyclophosphamide, doxorubicin, procarbazine, bleomycin, vincristine, prednisone CAVP lomustine, melphalan, etoposide, prednisone CEVD lomustine, etoposide, vindesine, dexamethasone CDDP+VP-16 cisplatin, etoposide, mitomycin C + vinblastine CEF cyclophosphamide, epirubicin, 5-fluorouracil CEM lomustine, etoposide, methotrexate CEP lomustine, etoposide, prechumustine CEPP(B) cyclophosphamide, etoposide, procarbazine, prednisone, bleomycin CEVD lomustine, etoposide, vindesine, dexamethasone ChIVPP chlorambucil, vinblastine, procarbazine, prednisone CHOP cyclophosphamide, doxorubicin, vincristine, prednisone CHOP-B CHOP plus bleomycin CMF cyclophosphamide, methodtrexate, 5-fluorouracil CMP cyclophosphamide, melphalan, prednisone CMVP cyclophosphamide, methotrexate, 5-fluorouracil, vincristine, prenisone CVP cyclophosphamide, vincristine, prednisone DHAP dexamethasone, high-dose .Ara-C, cisplatin ESHAP etoposide, methylpredisolone, high-dose cytarabine, cisplatin EPOCH etopsoide, vincristine, doxorubicin, cyclophospharilide, prednisone EVA etoposide, vinblastine, doxorubicin EVAP etoposide, vinblastine, cytarabine, cisplatin IFN-a interferon a IMVP-16 ifosfamide, methotrexate, etoposide MACOP-B methodtrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin, leucovorin m-BACOD methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone, leucovorin MIME methyl-gag, ifosfamide, methotrexate, etoposide MINE mitoquazone, ifosfamide, vinorelbine, etoposide MOPLACE cyclophosphamide, etoposide, prednisone, methotrexate, cytarabine, vincristine MOPP nitrogen mustard, vincristine, procarbazine, prednizone MOPP+ABV MOPP plus doxorubicin, bleomycin, vinblastine MOPP+ABVD alternating months of MOPP and ABVD
MVPP nitrogen mustard, vinblastine, procarbazine, prednisone MTX-CHOP methotrexate + CHOP
PCVP vinblastine, procarbazine, cyclophosphamide, prednisone ProMACE-CytaBOM prednisone, doxorubicin, cyclophosphamide, etoposide, cytarabine, bleomycine, vincristine, methotrexate, leucovorin ProMACE-MOPP prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, leucovorin + standard MOPP
Tam ~ tamoxifen VABCD vinblastine, doxorubicin, dacarbazine, lomustine, bleomycin VAT vinblastine, doxorubicin, thiotepa VATH vinblastine, doxorubicin, thiotepa, fluoxymesterone EXAMPLES
The examples and methods provided below serve merely to illustrate particular embodiments of the invention and are not meant to limit the invention.
Immunoconiu~ate Comnrisin~ IFN-a and Rituximab The nucleic acid encoding an IFN-a (e.g., IFN-a-2a, IFN-a-2b or IFN-a-nl) is operably linl~ed to the nucleic acid encoding Rituximab such that when translated the IFN-a would form the carboxy terminus of the fusion protein. The antigen-binding Fc receptor-binding, C 1 q binding and complement (C') activation, as well as the ability of IFN-a to bind to NIA cells and macrophages are characteristics possessed by the agents. In addition to the nucleic acid encoding Rituximab, other nucleic acids encoding anti-CD20 antibodies can be operably attached to the nucleic acid encoding IFN-a. The other anti-CD20 antibodies include Ibritumomab, IFS, B1 and 1H4.
_2g_ ha Yitro Testing of an Immunoconlu~ate A tumor cell line (cell expressing a target antigen) expressing Her2/neu (e.g., human breast carcinoma cells, SKBR-3) is selected to determine lysis using the immunoconjugates described herein. Effector cell samples are obtained by using heparinized whole blood or obtained from a third party. To prepare for use as effector cells, monocytes are cultured in Teflon containers in Macrophage Serum-Free Medium (Gibco/BRL) containing 2% human serum for 24 to 48 hours. Target cells are labeled with 100 ~,Ci of 5lCr for one hour prior to incubation with the effector cells and immunoconjugate in a U-bottomed microtiter plate. After incubation for about 16 to 18 hours at 37°C, supernatants from each well are collected and analyzed for radioactivity. Cytotoxicity and specific lysis can be calculated as previously described.
ha hivo Testing of Immunoconlu~ate For assessment of anti-tumor activity of the immunoconjugates, a mouse model can be used. In the instance of solid tumors, about 1x10' cells in culture media are injected subcutaneously into the right anterior flank of BALBIc, nulnu or SCm mice. Approximately, fourteen days later or when the tumors have grown to about 0.8 to 1.2 cm in diameter, the mice are separated into groups of 5-10 animals and injected intravenously with 200 ~,I of immunoconjugate at concentrations of 1 ~,g to 10 mg.
Perpendicular tumor diameters are measured at regular intervals and tumor volumes can be calculated. Alternatively, animals can be euthanized and sections of tumor prepared to determine the impact on tumor progression by the immunoconjugate as compared to the control animals (untreated).
Although the present invention has been described in detail with reference to examples above, it is understood that various modifications can be made without departing from the spirit of the invention. All cited patents and publications referred to in this application are herein incorporated by reference in their entirety.
Claims (22)
1. An immunoconjugate that comprises an antibody or an immunogenic fragment thereof that binds to an antigen expressed by a target cell that is to be eradicated, wherein said antibody or immunogenic fragment thereof possesses human effector function, which antibody or immunogenic fragment thereof is fused at its carboxy terminus to a cytokine that binds a receptor expressed on the surface of a natural killer cell and/or macrophage, thereby resulting in an immunoconjugate that facilitates extracellular (ADCC-type) and/or intracellular (phagocytic) killing of target cell, when said immunoconjugate is administered to a host.
2. The immunoconjugate of Claim 1, wherein the cytokine is an interferon.
3. The immunoconjugate of Claim 1, where in the interferon is alpha inteferon (IFN-.alpha.).
4. The immunoconjugate of Claim 1, where in the interferon is IFN-.alpha. and is IFN-.alpha.-2a, IFN-.alpha.-2b or IFN-.alpha.-2n1.
5. The immunoconjugate of Claim 1, wherein the target cells are selected from the group consisting of: a breast carcinoma cell, an ovarian carcinoma cell, a prostate carcinoma cell, a lung carcinoma cell, a leukemic T cell, a leukemic B cell, a multiple myeloma cell and a B cell lymphoma cell.
6. The immunoconjugate of Claim 1, wherein the antigen is selected from the group consisting of: CD19, CD20, CD22, CD33, CD38, EGF-R, HM1.24, phosphatidyl-serine antigen, HER-2, TAG-72 and MLTC-1.
7. The immunoconjugate of Claim 6, wherein the antibody which recognizes the antigen CD20 is RITUXAN, IF5, B1 or IH4.
8. The immunoconjugate of Claim 6, wherein the antibody which recognizes CD19 is B4, B43 or FVS191.
9. The immunoconjugate of Claim 6, wherein the antibody which recognizes CD22 is hLL2, LL2 or RFB4.
10. The immunoconjugate of Claim 6, wherein the antibody which recognizes CD33 is M195 or HuM195.
11. The immunoconjugate of Claim 6, wherein the antibody which recognizes CD38 is AT13/5.
12. The immunoconjugate of Claim 6, wherein the antibody which recognizes HER-2 is HERCEPTIN® or 4D5.
13. The immunoconjugate of Claim 6, wherein the antibody which recognizes TAG-72 is HuCC49, HuCC39.DELTA.CH2 or B72.3.
14. The immunoconjugate of Claim 6, wherein the antibody which recognizes MUC-1 is 12C10, IG5, H23, BM-2 (2E11), BM-7, 12H12, MAM-6 or HMFG-1.
15. The immunoconjugate of Claim 1, wherein the immunogenic fragment is selected from the list consisting of a domain-deleted antibody, Fab, Fab1, Fab2, SFV, single chain antibodies, domain-deleted antibodies and minibodies.
16. A method of enhancing apoptosis of a target cell by administering a therapeutically effective amount of an immunoconjugate of Claim 1 to a subject.
17. The method of Claim 16, wherein the target cell is a malignant cell selected from the group consisting of a breast carcinoma cell, an ovarian carcinoma cell, a prostate carcinoma cell, a lung carcinoma cell, a leukemic T cell, a leukemic B
cell, a multiple myeloma cell and a B cell lymphoma cell.
cell, a multiple myeloma cell and a B cell lymphoma cell.
18. A nucleic acid encoding an immunoconjugate of Claim 1.
19. A nucleic acid encoding an immunoconjugate that comprises an antibody or an immunogenic fragment thereof that binds to an antigen expressed by a target cell that is to be eradicated, wherein said an antibody or an immunogenic fragment thereof possesses human effector function, which an antibody or an immunogenic fragment thereof is fused at its carboxy terminus to a cytokine or to a peptide which is fused to a cytokine wherein the that binds a receptor expressed on the surface of a natural killer cell and/or macrophage, thereby resulting in an immunoconjugate that facilitates extracellular (ADCC-type) and intracellular (phagocytic) killing of target cell, when said immunoconjugate is administered to a host.
20. A combination therapy to treat a malignancy in a subject comprising an immunoconjugate of Claim 1 and at least one chemotherapeutic agent or chemotherapeutic cocktail.
21. The combination therapy of Claim 19, wherein the chemotherapeutic agent is selected from the list consisting of ara-C, doxorubicin, idarubicin, mitoxantrone, chlorambucil, melphalan, 6-mercaptopurine, 6-thioguanine, dibromomannitol, IFN-a, 2-chlorodeoxyadenosine, deoxycoformycin, dacarbazine, cisplatin, carmustine, lomustine, tauromustine, fotemustine, carboplatin, vincristine, vinblastine, vindesine, taxol, dibromodulcitol, detorubicin, piritrexin, estramustine, paclitaxel, navelbine or prenisolone.
22. The combination therapy of Claim 19, wherein the chemotherapeutic cocktail is selected from the list consisting of AC, ABDIC, ABVD, Ara-C, AVD, CAF, CAMP, CAP, CAP-BOP, CAVP, CEVD, CDDP+VP-16, CEF, CEM, CEP, CEPP(B), CEVD, ChIVPP, CHOP, CHOP-B, CMF, CMP, CMVP, CVP, DHAP, ESHAP, EPOCH, EVA, EVAP, IFN-.alpha., IMVP-16, MACOP-B, m-BACOD, MIME, MINE, MOPLACE, MOPP, MOPP+ABV, MOPP+ABVD, MVPP, MTX-CHOP, PCVP, ProMACE-CytaBOM, ProMACE-MOPP, VABCD, VAT or VATH.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US21325200P | 2000-06-22 | 2000-06-22 | |
US60/213,252 | 2000-06-22 | ||
PCT/US2001/040835 WO2001097844A1 (en) | 2000-06-22 | 2001-06-04 | Bispecific fusion protein and method of use for enhancing effector cell killing of target cells |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2412901A1 true CA2412901A1 (en) | 2001-12-27 |
Family
ID=22794334
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002412901A Abandoned CA2412901A1 (en) | 2000-06-22 | 2001-06-04 | Bispecific fusion protein and method of use for enhancing effector cell killing of target cells |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1292334A4 (en) |
JP (1) | JP2003535908A (en) |
AU (2) | AU2001265418B2 (en) |
CA (1) | CA2412901A1 (en) |
WO (1) | WO2001097844A1 (en) |
Families Citing this family (41)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2382587A1 (en) | 1999-08-23 | 2001-03-01 | Chugai Seiyaku Kabushiki Kaisha | Hm1.24 antigen expression potentiators |
US7931897B2 (en) | 2001-02-07 | 2011-04-26 | Chugai Seiyaku Kabushiki Kaisha | Therapeutic agent for hematopoietic tumors |
US8444973B2 (en) | 2005-02-15 | 2013-05-21 | Duke University | Anti-CD19 antibodies and uses in B cell disorders |
KR101335798B1 (en) * | 2005-02-15 | 2013-12-02 | 듀크 유니버시티 | Anti-cd19 antibodies and uses in oncology |
US20070003559A1 (en) * | 2005-07-01 | 2007-01-04 | Wyeth | Methods of determining pharmacokinetics of targeted therapies |
AU2008302111B2 (en) * | 2007-09-21 | 2014-04-24 | The Regents Of The University Of California | Targeted interferon demonstrates potent apoptotic and anti-tumor activities |
EP2271364B1 (en) * | 2008-04-10 | 2016-08-10 | IBC Pharmaceuticals, Inc. | Modular method to prepare tetrameric cytokines with improved pharmacokinetics by the dock-and-lock (dnl) technology |
EP2493486A1 (en) * | 2009-10-30 | 2012-09-05 | University Of Arkansas For Medical Science | Use of autologous effector cells and antibodies for treatment of multiple myeloma |
CN109022465B (en) | 2011-10-28 | 2022-04-29 | 特瓦制药澳大利亚私人有限公司 | Polypeptide constructs and uses thereof |
WO2014089354A1 (en) | 2012-12-07 | 2014-06-12 | The Regents Of The University Of California | Cd138-targeted interferon demonstrates potent apoptotic and anti-tumor activities |
KR102094022B1 (en) | 2013-04-29 | 2020-03-30 | 테바 파마슈티컬즈 오스트레일리아 피티와이 엘티디 | Anti-cd38 antibodies and fusions to attenuated interferon alpha-2b |
US11117975B2 (en) | 2013-04-29 | 2021-09-14 | Teva Pharmaceuticals Australia Pty Ltd | Anti-CD38 antibodies and fusions to attenuated interferon alpha-2B |
WO2014194100A1 (en) | 2013-05-29 | 2014-12-04 | The Regents Of The University Of California | Anti-cspg4 fusions with interferon for the treatment of malignancy |
US9603927B2 (en) | 2014-02-28 | 2017-03-28 | Janssen Biotech, Inc. | Combination therapies with anti-CD38 antibodies |
US9732154B2 (en) | 2014-02-28 | 2017-08-15 | Janssen Biotech, Inc. | Anti-CD38 antibodies for treatment of acute lymphoblastic leukemia |
UA119352C2 (en) | 2014-05-01 | 2019-06-10 | Тева Фармасьютикалз Острейліа Пті Лтд | Combination of lenalidomide or pomalidomide and cd38 antibody-attenuated interferon-alpha constructs, and the use thereof |
JP6707531B2 (en) | 2014-09-09 | 2020-06-10 | ヤンセン バイオテツク,インコーポレーテツド | Combination therapy with anti-CD38 antibody |
BR112017008880A2 (en) | 2014-10-29 | 2018-07-10 | Teva Pharmaceuticals Australia Pty Ltd | interferon a2b variants |
MX2017007208A (en) | 2014-12-04 | 2018-01-30 | Janssen Biotech Inc | Anti-cd38 antibodies for treatment of acute myeloid leukemia. |
US11000548B2 (en) | 2015-02-18 | 2021-05-11 | Enlivex Therapeutics Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
US11304976B2 (en) | 2015-02-18 | 2022-04-19 | Enlivex Therapeutics Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
US11318163B2 (en) | 2015-02-18 | 2022-05-03 | Enlivex Therapeutics Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
US11497767B2 (en) | 2015-02-18 | 2022-11-15 | Enlivex Therapeutics R&D Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
US11596652B2 (en) | 2015-02-18 | 2023-03-07 | Enlivex Therapeutics R&D Ltd | Early apoptotic cells for use in treating sepsis |
WO2016132366A1 (en) | 2015-02-18 | 2016-08-25 | Enlivex Therapeutics Ltd. | Combination immune therapy and cytokine control therapy for cancer treatment |
CN107708811B (en) | 2015-04-21 | 2021-04-30 | 恩立夫克治疗有限责任公司 | Therapeutic pooled apoptotic cell preparation and uses thereof |
CR20170526A (en) | 2015-05-20 | 2018-04-03 | Janssen Biotech Inc | ANTI-CD38 ANTIBODIES FOR THE TREATMENT OF LIGHT CHAIN AMYLOIDOSIS and OTHER POSITIVE HEMATOLOGICAL MALIGNAL DISEASES FOR CD38 |
EP3310386B1 (en) | 2015-06-22 | 2021-07-21 | Janssen Biotech, Inc. | Combination therapies for heme malignancies with anti-cd38 antibodies and survivin inhibitors |
US20170044265A1 (en) | 2015-06-24 | 2017-02-16 | Janssen Biotech, Inc. | Immune Modulation and Treatment of Solid Tumors with Antibodies that Specifically Bind CD38 |
US10066023B2 (en) | 2015-10-30 | 2018-09-04 | Aleta Biotherapeutics Inc. | Compositions and methods for tumor transduction |
EP3368559A4 (en) | 2015-10-30 | 2020-01-15 | Aleta Biotherapeutics Inc. | Compositions and methods for treatment of cancer |
LT3827845T (en) | 2015-11-03 | 2022-06-10 | Janssen Biotech, Inc. | Subcutaneous formulations of anti-cd38 antibodies and their uses |
US10781261B2 (en) | 2015-11-03 | 2020-09-22 | Janssen Biotech, Inc. | Subcutaneous formulations of anti-CD38 antibodies and their uses |
US11730761B2 (en) | 2016-02-18 | 2023-08-22 | Enlivex Therapeutics Rdo Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
CN106065033A (en) * | 2016-03-17 | 2016-11-02 | 中国药科大学 | The preparation of a kind of cytokine fusion antibody and application thereof |
US11618784B2 (en) | 2016-07-19 | 2023-04-04 | Teva Pharmaceuticals Australia Pty Ltd. | Anti-CD47 combination therapy |
CA3070230A1 (en) * | 2017-07-24 | 2019-01-31 | Rutgers, The State University Of New Jersey | Phosphatidylserine targeting fusion molecules and methods for their use |
WO2019089832A1 (en) | 2017-10-31 | 2019-05-09 | Janssen Biotech, Inc. | Methods of treating high risk multiple myeloma |
JP6648171B2 (en) * | 2018-02-02 | 2020-02-14 | テバ・ファーマシューティカルズ・オーストラリア・ピーティワイ・リミテッド | Fusion with anti-CD38 antibody and attenuated interferon alpha-2B |
US20210107961A1 (en) * | 2018-03-01 | 2021-04-15 | Glycotope Gmbh | Fusion Protein Constructs Comprising an Anti-MUC1 Antibody and IL-15 |
JP6853392B2 (en) * | 2020-01-15 | 2021-03-31 | テバ・ファーマシューティカルズ・オーストラリア・ピーティワイ・リミテッド | Fusion with anti-CD38 antibody and attenuated interferon alfa-2B |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IE63847B1 (en) * | 1989-05-05 | 1995-06-14 | Res Dev Foundation | A novel antibody delivery system for biological response modifiers |
US5314995A (en) * | 1990-01-22 | 1994-05-24 | Oncogen | Therapeutic interleukin-2-antibody based fusion proteins |
US5736137A (en) * | 1992-11-13 | 1998-04-07 | Idec Pharmaceuticals Corporation | Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma |
DK0669836T3 (en) * | 1992-11-13 | 1996-10-14 | Idec Pharma Corp | Therapeutic use of chimeric and radiolabeled antibodies and human B lymphocyte restricted differentiation antigen for the treatment of B cell lymphoma |
-
2001
- 2001-06-04 CA CA002412901A patent/CA2412901A1/en not_active Abandoned
- 2001-06-04 EP EP01939954A patent/EP1292334A4/en not_active Withdrawn
- 2001-06-04 JP JP2002503328A patent/JP2003535908A/en not_active Abandoned
- 2001-06-04 AU AU2001265418A patent/AU2001265418B2/en not_active Ceased
- 2001-06-04 WO PCT/US2001/040835 patent/WO2001097844A1/en active Application Filing
- 2001-06-04 AU AU6541801A patent/AU6541801A/en active Pending
Also Published As
Publication number | Publication date |
---|---|
WO2001097844A1 (en) | 2001-12-27 |
EP1292334A4 (en) | 2003-11-19 |
EP1292334A1 (en) | 2003-03-19 |
AU6541801A (en) | 2002-01-02 |
AU2001265418B2 (en) | 2006-03-30 |
JP2003535908A (en) | 2003-12-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2001265418B2 (en) | Bispecific fusion protein and method of use for enhancing effector cell killing of target cells | |
US20020193569A1 (en) | Bispecific fusion protein and method of use for enhancing effector cell killing of target cells | |
AU2001265418A1 (en) | Bispecific fusion protein and method of use for enhancing effector cell killing of target cells | |
AU2003298187B2 (en) | Humanized antibody (H14.18) of the mouse 14.18 antibody binding to GD2 and its fusion with IL-2 | |
Caron et al. | Biological and immunological features of humanized M195 (anti-CD33) monoclonal antibodies | |
JP4787750B2 (en) | CD20 binding polypeptide construct | |
EP2216342B1 (en) | Anti-CD19 antibodies | |
KR101824311B1 (en) | COMBINATION THERAPY OF AN AFUCOSYLATED CD20 ANTIBODY WITH A CD79b ANTIBODY-DRUG CONJUGATE | |
US20240076316A1 (en) | Fusion protein composition(s) comprising masked type i interferons (ifna and ifnb) for use in the treatment of cancer and methods thereof | |
EP1184458A1 (en) | Differentially expressed CD46 epitopes, proteinaceous molecules capable of binding thereto, and uses thereof | |
AU2016274897A1 (en) | Focused interferon immunotherapy for treatment of cancer | |
JP5102612B2 (en) | Target of B cell disease | |
WO2017180764A1 (en) | Focused interferon immunotherapy | |
CA3221878A1 (en) | Fusion protein composition(s) comprising masked type i interferons (ifna and ifnb) for use in the treatment of cancer and methods thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request | ||
FZDE | Dead |