Immunotherapy utilizing naked anti-granulocyte antibodies provides an effective means for treating chronic myelocytic leukemia (CML). Such antibodies can be administered alone or in combination with other therapies, such as immunoconjugates or chemotherapeutics. In either format, an effective therapy for treating CML is provided, which avoids the toxic side-effects typically associated with cancer therapy. The disclosed immunotherapy also is effective for treating acute myelocytic leukemia (AML) when co-administered with inducing agents which induce expression of antigens minimally displayed on the surface of myeloblasts.

Chronic myelocytic leukemia (CML) is a highly specific disease that is defined by strict hematologic parameters that include a pathognomonic differential leukocyte count. Usually, CML is accompanied by the presence, in bone marrow cells, of the Ph chromosome, the first chromosomal anomaly to be regularly associated with a human neoplastic disease. Chronic myelocytic leukemia is a disease of worldwide distribution and predominantly appears during middle age. The disease is characterized by an initial chronic phase when it behaves as a differentiated neoplasm and responds very well to simple, nonintensive therapy. After a variable interval, CML metamorphosizes to a refractory phase that responds poorly or not at all to therapy, even when intensive. See Spiers, Semin. Oncol., 22(4):380-95 (1995). At the stage of metamorphosis a great variety of clinical and hematologic pictures occur, and CML may mimic a myeloproliferative disease, a myelodysplasia, a subacute leukemia, acute myelocytic leukemia (AML), or acute lymphocytic leukemia (ALL). The old concept of an abrupt, explosive transition from the chronic phase to a so-called blastic crisis is incorrect. See Spiers, Semin. Oncol., 22(4):380-95 (1995). In most cases, CML is observed to undergo two or more stepwise evolutions, e.g., from chronic phase to an accelerated myeloproliferative phase to a phase that resembles AML.
A variety of therapies have been used to treat CML. Traditional methods for treating leukemia, including chemotherapy and radiotherapy, have limited utility due to toxic side effects. The use of monoclonal antibodies to direct radionuclides, toxins or other therapeutic agents selectively to tumor sites has reduced the level of toxicity to normal tissues. However, due to the large quantities of conjugate which must be administered, such therapies continue to produce toxic side-effects. These aspects limit the effectiveness and duration of such treatments. Another therapy, allogeneic bone marrow transplants, has had the largest impact on survival among patients with CML. See Clarkson, J. Clin. Oncol., 3:135-139 (1985). Like the previous therapies, however, bone marrow transplants are poorly tolerated by patients.
Recent studies suggest that immunotherapy utilizing naked antibodies can be an effective tool for treating certain cancers. The use of naked, humanized, anti-CD33 antibodies has proved effective in treating acute myelocytic leukemia and in reducing the residual disease in patients. See Caron et al., Clin. Cancer Res., 4:1421-1428 (1998); Jurcic et al., Clin. Cancer Res., 6:372-380 (2000). Similarly, immunotherapy comprising naked, humanized, anti-HER2/neu antibodies has produced promising results in the treatment of breast cancer. See Baselga et al., Semin. Oncol., 26:78-83 (1999); Weiner, Semin. Oncol., 26:43-51 (1999). Unconjugated immunoglobulins directed against CD20 have been shown to induce partial and complete responses in up to 50% of patients with advanced, indolent non-Hodgkin""s lymphoma. See Weiner, Semin. Oncol., 26:43-51 (1999).
The use of naked antibodies for treating malignancies has several advantages. First, immunotherapy comprising solely naked antibodies lacks the toxic side-effects associated with other therapies, such as radioimmunotherapy (RAIT) or immunotherapy utilizing conjugated toxins. Second, circulating naked antibodies remain therapeutically active longer than other therapies. For example, the effectiveness of RAIT is limited by the half-life of the conjugated isotope, typically a week or less. Similarly, the efficacy of conjugated immunotoxins can be short-lived due to in vivo modification of the toxin. Third, since naked antibodies are well-tolerated by patients, multiple rounds of therapy can be administered. Fourth, combination therapy utilizing naked antibodies is better tolerated by patients because lower quantities of the toxic component of the combination are required to achieve effective results. Finally, the use of naked antibodies dramatically reduces the costs of treating cancer by reducing the need for expensive radioactive or therapeutic conjugates which possess short shelf-lives and whose administration typically requires special facilities and personnel. Thus, such cost reductions enable more patients to benefit from the therapy.
There is a need, therefore, to develop immunotherapies which utilize naked antibodies to treat CML. Such therapies would cost-effectively treat patients without inducing toxic side-effects.
Accordingly, it is one object of the present invention to provide methods of treating myelocytic leukemia using naked immunotherapy.
In accomplishing this and other objects, there is provided, in accordance with one aspect of the present invention, a method for treating chronic myelocytic leukemia (CML) in a patient, comprising administering to the patient a therapeutic composition comprising a pharmaceutically acceptable carrier and at least one naked anti-granulocyte antibody. A variety of anti-granulocyte antibodies can be used in the present invention. Examples include, but are not limited to, anti-NCA-90, anti-NCA-95, MN-2, MN-15, NP-1 and NP2. In one embodiment, a single, naked anti-granulocyte antibody is administered to a patient while, in another, more than one anti-granulocyte antibody is administered. In still another embodiment, at least one naked anti-granulocyte antibody is administered to a patient in combination with naked antibodies directed to antigens present on a single granulocyte precursor, such as anti CD33 or anti-CD15 antibodies.
In another embodiment of the present invention, naked anti-granulocyte antibodies are used in combination with other cancer therapies, e.g., an immunoconjugate or chemotherapy. Preferred immunoconjugates include radiolabeled antibody components and conjugates of an anti-granulocyte antibody component and an immunomodulator, such as a cytokine, stem cell growth factor, lymphotoxin or hematopoietic factor. In still another embodiment, combination therapy of the present invention can comprise antibody-toxin fusion proteins.
In yet another embodiment of the present invention, naked anti-granulocyte antibodies are administered in combination with inducing agents which either enhance or induce the expression of the targeted antigen. Such inducing agents enhance the efficacy of the administered therapy by up-regulating the expression of the target antigen on the surface of a cell of interest. In addition, inducing agents can extend the inventive therapy""s tumor-killing potential to additional cell-types and cancers by inducing expression of antigens not normally displayed on the surface of these cells. Accordingly, the antibodies of the present invention can be used to treat AML, as well as CML.
Thus, in another embodiment of the present invention, there is provided a method for treating acute myelocytic leukemia (AML) or acute promyelocytic leukemia (APML) in a patient, comprising administering to the patient a therapeutic composition comprising a pharmaceutically acceptable carrier and at least one naked anti-granulocyte antibody and an inducing agent, wherein the inducing agent induces expression of antigens which are minimally displayed on the surface of myeloblasts. As described above, the inventive method can be further combined with other naked, anti-granulocyte antibodies, antibody-toxin fusion proteins and other cancer therapies, e.g., an immunoconjugate or chemotherapy.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. The detailed description and specific examples, while indicating preferred embodiments, are given for illustration only since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Further, the examples demonstrate the principle of the invention and cannot be expected to specifically illustrate the application of this invention to all the examples of infections where it obviously will be useful to those skilled in the prior art.
The present invention provides improved methods for treating myelocytic leukemia, particularly CML. The inventive methods utilize naked granulocyte-specific antibodies to destroy myeloid leukemia cells without the toxic side-effects normally associated with previous forms of therapy. In one embodiment, the treatment utilizes only naked granulocyte-specific antibodies. In another embodiment, the naked antibodies are used in combination with immunoconjugates or other therapeutics. In either form of therapy, the patient is provided an effective treatment for chronic myelocytic leukemia that avoids the toxic side-effects that accompany administration of typical amounts of anti-cancer medicaments.
The anti-granulocyte antibodies used in the present invention are directed to antigens associated with various cell-types in the granulocyte lineage. Unlike in AML, malignant myeloblasts of CML patients differentiate into a variety of cell-types, including myelocytes, metamyelocytes, bands, and granulocytes. Accordingly, immunotherapy directed to one or two of these cell-types is incapable of meaningfully reducing the number of leukemic cells. Since the antibodies of the inventive therapy recognize immature and mature granulocytes, the present invention provides an effective method for ridding malignant cells from a CML patient""s bone marrow.
A variety of anti-granulocyte antibodies can be used in the present invention. In one embodiment, the inventive methods utilize anti-NCA-90 antibodies. A preferred example of such an antibody is MN-3. See Hansen et al., Cancer, 71:3478-3485 (1993); Becker et al., Semin. Nucl. Med., 24(2):142-53 (1994). In another embodiment, anti-NCA-95 antibodies are used. In still other embodiments, MN-2 and NP-2, which are class IIa anti-CEA antibodies, and MN-15 and NP-1, which are class I anti-CEA antibodies, are utilized. See Hansen et al., Cancer, 71:3478-3485 (1993); Primus et al., Cancer Res., 43:686-692 (1983). Human and chimeric forms of these antibodies are preferred, and full-human and humanized versions are most preferred.
Definitions
In the description that follows, a number of terms are used extensively. The following definitions are provided to facilitate understanding of the invention.
The term xe2x80x9cmyelocytic leukemiaxe2x80x9d is synonymous with the terms granulocytic leukemia, myelogenic leukemia, myelogenous leukemia and myeloid leukemia.
Chronic myelocytic leukemia is characterized by an uncontrolled proliferation of myelopoietic cells in the bone marrow and extramedullary sites in which the malignant myeloblast is able to differentiate and give rise to myelocytes, metamyelocytes, band cells and granulocytes.
Acute myelocytic leukemia is characterized by an uncontrolled proliferation of myeloblasts which are unable to differentiate into more mature cell-types.
The term xe2x80x9canti-granulocyte antibodyxe2x80x9d refers to an antibody which recognizes an antigen which is present on two or more cell-types of the granulocyte/myelocyte lineage.
A xe2x80x9cchimeric antibodyxe2x80x9d is a recombinant protein that contains the variable domains and complementary determining regions derived from a rodent antibody, while the remainder of the antibody molecule is derived from a human antibody.
xe2x80x9cHumanized antibodiesxe2x80x9d are recombinant proteins in which murine complementarity determining regions of a monoclonal antibody have been transferred from heavy and light variable chains of a murine immunoglobulin into a human variable domain.
A xe2x80x9ctherapeutic agentxe2x80x9d is a molecule or atom which may be conjugated to an antibody moiety to produce a conjugate, which is useful for therapy. Examples of therapeutic agents include, but are not limited to, drugs, toxins, immunomodulators, boron compounds, photoactive agents or dyes, cytokines, hormones radiopharmaceuticals and radioisotopes.
A xe2x80x9cnaked antibodyxe2x80x9d is an entire antibody, as opposed to an antibody fragment, which is not conjugated to a therapeutic agent. Naked antibodies include both polyclonal and monoclonal antibodies, as well as certain recombinant antibodies, such as chimeric and humanized antibodies.
The term xe2x80x9cantibody componentxe2x80x9d includes both an entire antibody and an antibody fragment.
An xe2x80x9cimmunoconjugatexe2x80x9d is a conjugate of an antibody component with a therapeutic agent.
The term xe2x80x9cantibody fusion proteinxe2x80x9d refers to a recombinant molecule that comprises one or more antibody components and a therapeutic agent. Examples of therapeutic agents suitable for such fusion proteins include immunomodulators (xe2x80x9cantibody-immunomodulator fusion proteinxe2x80x9d) and toxins (xe2x80x9cantibody-toxin fusion proteinxe2x80x9d). The fusion protein may comprise a single antibody component, a multivalent combination of different antibody components or multiple copies of the same antibody component.
A xe2x80x9cstructural genexe2x80x9d is a DNA sequence that is transcribed into messenger RNA (mRNA) which is then translated into a sequence of amino acids characteristic of a specific polypeptide.
A xe2x80x9cpromoterxe2x80x9d is a DNA sequence that directs the transcription of a structural gene. Typically, a promoter is located in the 5xe2x80x2 region of a gene, proximal to the transcriptional start site of a structural gene. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter.
An xe2x80x9cisolated DNA moleculexe2x80x9d is a fragment of DNA that is not integrated in the genomic DNA of an organism. For example, a cloned antibody gene is a DNA fragment that has been separated from the genomic DNA of a mammalian cell. Another example of an isolated DNA molecule is a chemically-synthesized DNA molecule that is not integrated in the genomic DNA of an organism.
An xe2x80x9cenhancerxe2x80x9d is a DNA regulatory element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription.
xe2x80x9cComplementary DNAxe2x80x9d (cDNA) is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Typically, a primer complementary to portions of mRNA is employed for the initiation of reverse transcription. Those skilled in the art also use the term xe2x80x9ccDNAxe2x80x9d to refer to a double-stranded DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand.
The term xe2x80x9cexpressionxe2x80x9d refers to the biosynthesis of a gene product. For example, in the case of a structural gene, expression involves transcription of the structural gene into mRNA and the translation of mRNA into one or more polypeptides.
A xe2x80x9ccloning vectorxe2x80x9d is a DNA molecule, such as a plasmid, cosmid or bacteriophage, that has the capability of replicating autonomously in a host cell. Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of an essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.
An xe2x80x9cexpression vectorxe2x80x9d is a DNA molecule comprising a gene that is expressed in a host cell. Typically, gene expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. Such a gene is said to be xe2x80x9coperably linked toxe2x80x9d the regulatory elements.
A xe2x80x9crecombinant hostxe2x80x9d may be any prokaryotic or eukaryotic cell that contains either a cloning vector or expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell.
An xe2x80x9cantibody fragmentxe2x80x9d is a portion of an antibody such as F(abxe2x80x2)2, F(ab)2, Fabxe2x80x2, Fab, and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. For example, an anti-CD22 monoclonal antibody fragment binds with an epitope of CD22.
The term xe2x80x9cantibody fragmentxe2x80x9d also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. For example, antibody fragments include isolated fragments consisting of the light chain variable region, xe2x80x9cFvxe2x80x9d fragments consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (xe2x80x9csFv proteinsxe2x80x9d), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.
Production of Antibodies
Rodent monoclonal antibodies specific for granulocytes can be obtained by methods known to those skilled in the art. See generally Kohler and Milstein, Nature 256:495 (1975); Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley and Sons 1991). Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising, for example, NCA-90, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce anti-NCA-90 antibodies, culturing the clones that produce antibodies to the antigen and isolating the antibodies from the hybridoma cultures.
Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-known techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley and Sons 1991); Baines et al., pages 79-104, METHODS IN MOLECULAR BIOLOGY (The Humana Press, Inc. 1992).
Suitable amounts of the NCA-90 antigen, also referred to as CD66c, can be obtained using standard techniques well-known in the art. For example, NCA-90 protein can be obtained from transfected cultured cells that overproduce NCA-90. Expression vectors that comprise DNA molecules encoding NCA-90 can be constructed using the published NCA-90 nucleotide sequence (SEQ ID NO: 1). See Qikawa et al., Biochem. Biophys. Res. Commun., 146:464-460 (1987); Wilson et al., J. Exp. Med. 173:137 (1991); Wilson et al., J. Immunol. 150:5013 (1993). Oikawa et al also disclose a corresponding amino acid sequence for NCA-90 (SEQ ID NO:2). Similarly, expression vectors for producing NCA-95 protein can be constructed using the published NCA-95 nucleotide sequence (SEQ ID NO:3). See Berling et al., Cancer Res., 50:6534-6539 (1990). Berling et al also disclose a corresponding amino acid sequence for NCA-95 (SEQ ID NO:4).
As an illustration, DNA molecules encoding NCA-90 can be obtained by synthesizing DNA molecules using mutually priming long oligonucleotides. See Ausubel et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley and Sons, Inc. 1990); Wosnick et al., Gene 60:115 (1987); Ausubel et al. (eds.), SHORT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley and Sons, Inc. 1995). Established techniques using the polymerase chain reaction provide the ability to synthesize genes as large as 1.8 kilobases in length. Adang et al., Plant Molec. Biol., 21:1131 (1993); Bambot et al., PCR Methods and Applications, 2:266 (1993); White (ed.), METHODS IN MOLECULAR BIOLOGY, pages 263-268 (Humana Press, Inc. 1993). In a variation of this approach, an anti-NCA-90 monoclonal antibody can be obtained by fusing myeloma cells with spleen cells from mice immunized with a murine pre-B cell line stably transfected with NCA-90 cDNA using techniques well-known in the art.
One example of a suitable murine anti NCA 90 monoclonal antibody is the MN-3 monoclonal antibody. The MN-3 antibody was isolated from hybridomas derived from BALB/c mice which were immunized with partially purified carcinoembryonic antigen (CEA) derived from GW-39 human colon adenocarcinoma xenografts. See Hansen et al., Cancer, 71:3478-3485 (1993). The MN-3 antibody is specific for the NCA-90 antigen, a homotypic adhesion molecule expressed on granulocytes, as well as normal colonic mucosa and colonic adenocarcinoma. See Becker et al., Semin. Nucl. Med., 24(2):142-53 (1994); Watt et al., Blood, 78:63-74 (1991).
One example of a suitable murine anti-NCA-95 antibody is the BW 250/183 antibody. See Bosslet et al., Int. J. Cancer, 36:75-84 (1985); Meller et al., J. Nucl. Med., 39:1248-1253. Another useful anti-NCA-95 antibody is Mab 47. See Audette et al., Mol. Immunol., 24:1177-1186 (1987).
Another suitable antibody is the MN-2 monoclonal antibody. The MN-2 antibody was isolated from hybridomas derived from BALB/c mice which were immunized with partially purified carcinoembryonic antigen (CEA) derived from GW-39 human colon adenocarcinoma xenografts. See Hansen et al., Cancer, 71:3478-3485 (1993). As a class IIA anti-CEA antibody, MN-2 can be identified readily using blocking assays well-known in the art. See U.S. Pat. No. 4,818,709, which is hereby incorporated by reference in its entirety.
Another suitable antibody is the MN-15 monoclonal antibody. The MN-15 antibody displays cross-reactivity between NCA-90 and NCA-95. MN-15 was isolated from hybridomas derived from BALB/c mice which were immunized with partially purified carcinoembryonic antigen (CEA) derived from GW-39 human colon adenocarcinoma xenografts. See Hansen et al., Cancer, 71:3478-3485 (1993). As a class I anti-CEA antibody, MN-15 can be identified readily using blocking assays well-known in the art.
Still another suitable antibody is the NP-2 monoclonal antibody. The NP-2 has specificity similar to that of MN-2. NP-2 was isolated from hybridomas derived from BALB/c mice which were immunized with partially purified carcinoembryonic antigen (CEA) derived from liver metastases of human colonic adenocarcinoma according to the procedure of Krupey et al. (Immunochem., 9: 617 (1972)), as modified by Newman et al. (Cancer Res., 34:2125 (1974)). See Primus et al., Cancer Res., 43:686-92 (1983); U.S. Pat. No. 4,818,709.
Yet another suitable antibody is the NP-1 monoclonal antibody. The NP-1 has similar specificity to that of MN-15. NP-1 was isolated from hybridomas derived from BALB/c mice which were immunized with partially purified carcinoembryonic antigen (CEA) derived from liver metastases of human colonic adenocarcinoma according to the procedure of Krupey et al. (Immunochem., 9:617 (1972)), as modified by Newman et al. (Cancer Res., 34:2125 (1974)). See Primus et al., Cancer Res., 43:686-92 (1983); U.S. Pat. No. 4,818,709.
One example of an antibody suitable for multimodal therapy is M195. See U.S. Pat. No. 6,007,814. This antibody is specific for the CD33 antigen, which is present on myeloid cells and monocytic cells. M195 was produced from hybridomas resulting from a fusion of SP2/0-Ag14 mouse myeloma cells and the spleen cells of a 5-week-old BALB/c mouse immunized with leukemia cells from a patient with AML (FAB-M2).
Other examples of antibodies suitable for multimodal therapy are anti-SSEA-1 and PM-81. See Thakur et al., J. Nucl. Med., 37:1789-1795 (1996); Ball et al., J. Immunol., 30:2937-2941(1983). These antibodies recognize CD-15 antigens, which are present on a variety of myeloid cells. Anti-SSEA-1 is produced from a hybridoma derived from mouse teratocarcinoma cells. PM-81 is produced from a hybridoma derived from a fusion of cells of the NS-1 myeloma cell line with cells from a mouse immunized with the HL-60 promyelocytic leukemia cell line.
In an additional embodiment, an antibody of the present invention is a chimeric antibody in which the variable regions of a human antibody have been replaced by the variable regions of a, e.g., rodent anti-NCA-90 antibody. The advantages of chimeric antibodies include decreased immunogenicity and increased in vivo stability. Techniques for constructing chimeric antibodies are well-known to those of skill in the art. See Leung et al., Hybridoma, 13:469 (1994).
In another embodiment, an antibody of the present invention is a subhuman primate antibody. General techniques for raising therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al., International Patent Publication No. WO 91/11465 (1991), which is hereby incorporated by reference in its entirety, and in Losman et al., Int. J. Cancer, 46:310 (1990).
In yet another embodiment, an antibody of the present invention is a xe2x80x9chumanizedxe2x80x9d monoclonal antibody. That is, mouse complementarity determining regions are transferred from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, followed by the replacement of some human residues in the framework regions of their murine counterparts. Humanized monoclonal antibodies in accordance with this invention are suitable for use in therapeutic methods. General techniques for cloning murine immunoglobulin variable domains are described, for example, by the publication of Orlandi et al., Proc. Nat""l Acad. Sci. USA, 86:3833 (1989). Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature, 321:522 (1986), Riechmann et al., Nature, 332:323 (1988), Verhoeyen et al., Science, 239:1534 (1988), Carter et al., Proc. Nat""l Acad. Sci. USA, 89:4285 (1992), Sandhu, Crit. Rev. Biotech., 12:437 (1992) and Singer et al., J. Immun., 150:2844 (1993).
In another embodiment, an antibody of the present invention is a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been xe2x80x9cengineeredxe2x80x9d to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are well-known in the art. See Green et al., Nature Genet., 7:13 (1994); Lonberg et al., Nature, 368:856 (1994); Taylor et al., Int. Immun., 6:579 (1994); Bruggeman et al., Curr. Opin. Biotechnol., 8:455-458 (1997). Alternatively, human antibodies to antigens of interest also can be obtained using phage display. See Aujame et al., Hum. Antibodies, 8:155-168 (1997).
Preparation of Immunoconjugates
The present invention contemplates the use of naked anti-granulocyte antibodies as the primary therapeutic composition of CML. However, in one embodiment of the invention, naked anti-granulocyte antibodies, e.g., MN-3 or MN-2, are administered to a patient in combination with one or more immunoconjugates. Such immunoconjugates can be prepared by conjugating a therapeutic agent to an antibody component. General techniques are described in Shih et al., Int. J. Cancer, 41:832-839 (1988), Shih et al., Int. J. Cancer, 46:1101-1106 (1990) and U.S. Pat. No. 5,057,313, which are hereby incorporated by reference in their entireties. The general method involves reacting an antibody component having an oxidized carbohydrate portion with a carrier polymer that has at least one free amine function and that is loaded with a plurality of drug, toxin, chelator, boron addends or other therapeutic agents. This reaction results in an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.
The carrier polymer is preferably an aminodextran or polypeptide of at least 50 amino acid residues, although other substantially equivalent polymer carriers also can be used. For ease of administration and effective targeting, the final immunoconjugate is preferably soluble in an aqueous solution, such as mammalian serum. Thus, solubilizing functions on the carrier polymer will enhance the serum solubility of the final immunoconjugate. In particular, an aminodextran is preferred.
The process for preparing an immunoconjugate with an aminodextran carrier typically begins with a dextran polymer, advantageously a dextran of average molecular weight of about 5,000-100,000. The dextran is reacted with an oxidizing agent to effect a controlled oxidation of a portion of its carbohydrate rings to generate aldehyde groups. The oxidation is conveniently effected with glycolytic chemical reagents such as NaIO4, according to conventional procedures.
The oxidized dextran is then reacted with a polyamine, preferably a diamine, and more preferably, a mono- or polyhydroxy diamine. Suitable amines include ethylene diamine, propylene diamine or other like polymethylene diamines, diethylene triamine or like polyamines, 1,3-diamino-2-hydroxypropane or other like hydroxylated diamines or polyamines and the like. An excess of the amine relative to the aldehyde groups of the dextran is used to ensure substantially complete conversion of the aldehyde functions to Schiff base groups.
A reducing agent, such as NaBH4, NaBH3CN or the like, is used to effect reductive stabilization of the resultant Schiff base intermediate. The resultant adduct can be purified by passage through a conventional sizing column to remove cross-linked dextrans. Other conventional methods of derivatizing a dextran to introduce amine functions also can be used, e.g., reaction with cyanogen bromide, followed by reaction with a diamine. The aminodextran then is reacted with a derivative of the particular drug, toxin, chelator, immunomodulator, boron addend or other therapeutic agent to be loaded, in an activated form, preferably, a carboxyl-activated derivative, prepared by conventional means, e.g., using dicyclohexylcarbodiimide (DCC) or a water-soluble variant thereof, to form an intermediate adduct.
Alternatively, polypeptide toxins such as pokeweed antiviral protein or ricin A-chain and the like, can be coupled to aminodextran by glutaraldehyde condensation or by reaction of activated carboxyl groups on the protein with amines on the aminodextran. Chelators for radiometals or magnetic resonance enhancers are well-known in the art. Typical are derivatives of ethylenediaminetetraacetic acid (EDTA), diethylene-triaminepentaacetic acid (DTPA) and 1,4,7,10-tetraazacyclododecane-N,Nxe2x80x2,Nxe2x80x3,Nxe2x80x2xe2x80x3-tetraacetic acid (DOTA). These chelators typically have groups on the side chain by which the chelator can be attached to a carrier. Such groups include, e.g., benzylisothiocyanate, by which the DTPA or EDTA can be coupled to the amine group of a carrier. Alternatively, carboxyl groups or amine groups on a chelator can be coupled to a carrier by activation or prior derivatization and then coupling, all by well-known means.
Boron addends, such as carboranes, can be attached to antibody components by conventional methods. For example, carboranes can be prepared with carboxyl functions on pendant side chains, as is well-known in the art. Attachment of such carboranes to a carrier, e.g., aminodextran, can be achieved by activation of the carboxyl groups of the carboranes and condensation with amines on the carrier to produce an intermediate conjugate. Such intermediate conjugates then are attached to antibody components to produce therapeutically useful immunoconjugates, as described below.
A polypeptide carrier can be used instead of aminodextran, but the polypeptide carrier must have at least 50 amino acid residues in the chain, preferably 100-5000 amino acid residues. At least some of the amino acids should be lysine residues or glutamate or aspartate residues. The pendant amines of lysine residues and pendant carboxylates of glutamine and aspartate are convenient for attaching a drug, toxin, immunomodulator, chelator, boron addend or other therapeutic agent. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixed polymers of these amino acids and others, e.g., serines, to confer desirable solubility properties on the resultant loaded carrier and immunoconjugate.
Conjugation of the intermediate conjugate with the antibody component is effected by oxidizing the carbohydrate portion of the antibody component and reacting the resulting aldehyde (and ketone) carbonyls with amine groups remaining on the carrier after loading with a drug, toxin, chelator, immunomodulator, boron addend or other therapeutic agent. Alternatively, an intermediate conjugate can be attached to an oxidized antibody component via amine groups that have been introduced in the intermediate conjugate after loading with the therapeutic agent. Oxidation is conveniently effected either chemically, e.g., with NaIO4 or other glycolytic reagent, or enzymatically, e.g., with neuraminidase and galactose oxidase. In the case of an aminodextran carrier, not all of the amines of the aminodextran typically are used for loading a therapeutic agent. The remaining amines of aminodextran condense with the oxidized antibody component to form Schiff base adducts, which are then reductively stabilized, normally with a borohydride reducing agent.
Analogous procedures are used to produce other immunoconjugates according to the invention. Loaded polypeptide carriers preferably have free lysine residues remaining for condensation with the oxidized carbohydrate portion of an antibody component. Carboxyls on the polypeptide carrier can be converted, if necessary, to amines by, e.g., activation with DCC and reaction with an excess of a diamine. The final immunoconjugate is purified using conventional techniques, such as sizing chromatography on Sephacryl S-300.
Alternatively, immunoconjugates can be prepared by directly conjugating an antibody component with a therapeutic agent. The general procedure is analogous to the indirect method of conjugation except that a therapeutic agent is directly attached to an oxidized antibody component.
It will be appreciated that other therapeutic agents can be substituted for the chelators described herein. Those of skill in the art will be able to devise conjugation schemes without undue experimentation.
As a further illustration, a therapeutic agent can be attached at the hinge region of a reduced antibody component via disulfide bond formation. For example, the tetanus toxoid peptides can be constructed with a single cysteine residue that is used to attach the peptide to an antibody component. As an alternative, such peptides can be attached to the antibody component using a heterobifunctional cross-linker, such as N-succinyl 3-(2-pyridyldithio)proprionate (SPDP). See Yu et al., Int. J. Cancer, 56:244 (1994). General techniques for such conjugations are well-known in the art. See Wong, CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Birch et al. (eds.), MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, pages 187-230 (Wiley-Liss, Inc. 1995); Ritter et al. (eds.), MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, pages 60-84 (Cambridge University Press 1995).
As described above, carbohydrate moieties in the Fc region of an antibody can be used to conjugate a therapeutic agent. However, the Fc region is absent if an antibody fragment is used as the antibody component of the immunoconjugate. Nevertheless, it is possible to introduce a carbohydrate moiety into the light chain variable region of an antibody or antibody fragment. See Leung et al., J. Immunol., 154:5919 (1995); U.S. Pat. No. 5,443,953, which are hereby incorporated by reference in their entireties. The engineered carbohydrate moiety then is used to attach a therapeutic agent.
In addition, those of skill in the art will recognize numerous possible variations of the conjugation methods. For example, the carbohydrate moiety can be used to attach polyethyleneglycol in order to extend the half-life of an intact antibody, or antigen-binding fragment thereof, in blood, lymph or other extracellular fluids. Moreover, it is possible to construct a xe2x80x9cdivalent immunoconjugatexe2x80x9d by attaching therapeutic agents to a carbohydrate moiety and to a free sulfhydryl group. Such a free sulfhydryl group may be located in the hinge region of the antibody component.
Production of Antibody Fragments
Immunocongugates utilized in combination therapies of the present invention can comprise antibody fragments. Such antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(abxe2x80x2)2. This fragment can be further cleaved using a thiol reducing agent and, optionally, a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fabxe2x80x2 monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly. These methods are described, for example, in U.S. Pat. Nos. 4,036,945 and 4,331,647 and the references contained therein. See also Nisonoff et al., Arch. Biochem. Biophys., 89:230 (1960); Porter, Biochem. J., 73:119 (1959); Edelman et al., METHODS IN ENZYMOLOGY, page 422 (Academic Press 1967), and Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley and Sons 1991).
Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments or other enzymatic, chemical or genetic techniques also may be used, so long as the fragments bind to the antigen that is recognized by the intact antibody. For example, Fv fragments comprise an association of VH and VL chains. This association can be noncovalent, as described in Inbar et al., Proc. Nat""l. Acad. Sci. USA, 69:2659 (1972). Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. See Sandhu, Crit. Rev. Biotech., 12:437 (1992).
Preferably, the Fv fragments comprise VH and VL chains which are connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains which are connected by an oligonucleotide. The structural gene is inserted into an expression vector which is subsequently introduced into a host cell, such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are well-known in the art. See Whitlow et al., Methods: A Companion to Methods in Enzymology 2:97 (1991); Bird et al., Science, 242:423 (1988); U.S. Pat. No. 4,946,778; Pack et al., Bio/Technology, 11:1271 (1993), and Sandhu, Crit. Rev. Biotech., 12:437 (1992).
Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides (xe2x80x9cminimal recognition unitsxe2x80x9d) can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See Larrick et al., Methods: A Companion to Methods in Enzymology 2:106 (1991); Ritter et al. (eds.), MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, pages 166-179 (Cambridge University Press 1995); Birch et al., (eds.), MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, pages 137-185 (Wiley-Liss, Inc. 1995).
Therapeutic Use of Anti-Granulocyte Antibodies
Since the antibodies of the inventive therapy recognize antigens present on immature and mature granulocytes, the present invention provides an effective method for ridding malignant cells from a patient with myelocytic leukemia, in particular CML. As discussed above, a variety of anti-granulocyte antibodies can be used in the inventive therapy.
The present invention contemplates the use of naked anti-granulocyte antibodies as the primary therapeutic composition for treatment of CML. Such a composition can contain polyclonal anti-granulocyte antibodies or monoclonal anti-granulocyte antibodies.
Methods for determining the binding specificity of an anti-granulocyte antibody are well-known to those of skill in the art. General methods are provided, for example, by Manson (ed.), METHODS IN MOLECULAR BIOLOGY: IMMUNOCHEMICAL PROTOCOLS, pages 105-116 (The Humana Press, Inc. 1992). The epitope specificity also can be identified by binding a test antibody with a panel of antigens which lack particular domains.
In another embodiment of the present invention, naked anti-granulocyte antibodies can be used in combination with other cancer therapies, e.g., an immunoconjugate or chemotherapy. Such combination regimens are advantageous over therapies previously practiced in the art because they achieve effective results with less toxins. Thus, such therapies eliminate or greatly reduce the negative side-effects typically associated with cancer therapy. In such multimodal regimens, the supplemental therapeutic compositions can be administered before, concurrently, or after administration of the naked anti-granulocyte antibodies.
Preferred immunoconjugates include radiolabeled antibody components and conjugates of an anti-granulocyte antibody component and an immunomodulator. A radiolabeled immunoconjugate may comprise an xcex1-emitting radioisotope, a xcex2-emitting radioisotope, a xcex3-emitting radioisotope, an Auger electron emitter, a neutron capturing agent that emits xcex1-particles or a radioisotope that decays by electron capture. Suitable radioisotopes include 198Au, 32P, 125I, 131I, 90Y, 186Re, 188Re, 67Cu, 211At, 213Bi, 224Ac, and the like. Boron addends are preferred neutron capture agents that are converted to alpha-emitters.
As discussed above, a radioisotope can be attached to an antibody component directly or indirectly, via a chelating agent. For example, 67Cu, considered one of the more promising radioisotopes for radioimmunotherapy due to its 61.5 hour half-life and abundant supply of beta particles and gamma rays, can be conjugated to an antibody component using the chelating agent, p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid (TETA). See Gennaro et al. (eds.), REMINGTON""S PHARMACEUTICAL SCIENCES, pages 624-652 (Mack Publishing Co. 1990). Alternatively, 90Y, which emits an energetic beta particle, can be coupled to an antibody component using DTPA or DOTA. Moreover, a method for the direct radiolabeling of the antibody component with 131I is described by Stein et al., Antibody Immunoconj. Radiopharm., 4:703 (1991). Alternatively, boron addends such as carboranes can be attached to antibody components, as discussed above.
In another embodiment, immunoconjugates can comprise an immunomodulator moiety. As used herein, the term xe2x80x9cimmunomodulatorxe2x80x9d includes cytokines, stem cell growth factors, lymphotoxins, such as tumor necrosis factor (TNF), and hematopoietic factors, such as interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10 and IL-12), colony stimulating factors (e.g., granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF)),. interferons (e.g., interferons-xcex1, -xcex2and -xcex3), the stem cell growth factor designated xe2x80x9cS1 factor,xe2x80x9d erythropoietin and thrombopoietin. Examples of suitable immunomodulator moieties include IL-2, IL-6, IL-10, IL-12, interferon-xcex3, TNF-xcex1 and the like.
A related immunoconjugate is a fusion protein comprising one or more antibody moieties and an immunomodulator moiety. Useful antibody moieties include antibody components that bind with, for example, NCA-90 or NCA-95. Bivalent, trivalent and tetravalent constructs can be used in accordance with the invention.
Methods of making antibody-immunomodulator fusion proteins are well-known in the art. For example, antibody fusion proteins comprising an interleukin-2 moiety are described by Boleti et al., Ann. Oncol., 6:945 (1995), Nicolet et al., Cancer Gene Ther., 2:161 (1995), Becker et al., Proc. Nat""l. Acad. Sci. USA, 93:7826 (1996), Hank et al., Clin. Cancer Res., 2:1951 (1996) and Hu et al., Cancer Res., 56:4998 (1996). In addition, Yang et al. describe a fusion protein that includes an F(abxe2x80x2)2 fragment and a tumor necrosis factor alpha moiety. See Yang et al., Hum. Antibodies Hybridomas 6:129 (1995).
Such immunoconjugates and antibody-immunomodulator fusion proteins provide a means to deliver an immunomodulator to a target cell and are particularly useful against tumor cells. The cytotoxic effects of immunomodulators are well-known in the art. See Pessuto et al. (eds.), BIOTECHNOLOGY AND PHARMACY, pages 53-70 (Chapman and Hall 1993). As an illustration, interferons can inhibit cell proliferation by inducing increased expression of class I histocompatibility antigens on the surface of various cells and, thus, enhance the rate of destruction of cells by cytotoxic T lymphocytes. Furthermore, tumor necrosis factors, such as TNF-xcex1, are believed to produce cytotoxic effects by inducing DNA fragmentation.
Therapeutically useful immunoconjugates can be prepared in which an antibody component is conjugated to a toxin or a chemotherapeutic drug. Illustrative of toxins and drugs which are suitably employed in the preparation of such conjugates are calicheamicin, ricin, abrin, ribonuclease, DNase I, RNase, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. See Pastan et al., Cell, 47:641 (1986),; Goldenberg, CA, A Cancer Journal for Clinicians, 44:43 (1994). For instance, one example of an effective immunoconjugate is an anti-NCA-90 antibody conjugated to RNase. Another example is an anti-CD33 antibody conjugated to calicheamicin. Other suitable toxins are well-known in the art, such as protein inhibitors, DNA, RNA and cell-cycle poisons.
In another embodiment, combination therapy utilizing naked, anti-granulocyte antibodies can comprise antibody-toxin fusion proteins. An antibody-toxin fusion protein is a fusion protein that comprises one or more antibody moieties and a toxin moiety. Useful antibody moieties include antibody components that bind with NCA-90 or NCA-95. Bivalent, trivalent and tetravalent constructs can be used in accordance with the invention. Methods for making antibody-toxin fusion proteins are well-known in the art. For example, antibody-Pseudomonas exotoxin A fusion proteins have been described by Chaudhary et al., Nature, 339:394 (1989), Brinkmann et al., Proc. Nat""l Acad. Sci. USA, 88:8616 (1991), Batra et al., Proc. Nat""l Acad. Sci. USA, 89:5867 (1992), Friedman et al., J. Immunol., 150:3054 (1993), Wels et al., Int. J. Can., 60:137 (1995), Fominaya et al., J. Biol. Chem., 271:10560 (1996), Kuan et al., Biochemistry, 35:2872 (1996) and Schmidt et al., Int. J. Can., 65:538 (1996). Antibody-toxin fusion proteins containing a diphtheria toxin moiety have been described by Kreitman et al., Leukemia, 7:553 (1993), Nicholls et al., J. Biol. Chem., 268:5302 (1993), Thompson et al., J. Biol. Chem., 270:28037 (1995) and Vallera et al., Blood, 88:2342 (1996). Deonarain et al. (Tumor Targeting, 1:177 (1995)) have described an antibody-toxin fusion protein having an RNase moiety, while Linardou et al. (Cell Biophys., 24-25:243 (1994)) produced an antibody-toxin fusion protein comprising a DNase I component. Gelonin was used as the toxin moiety in the antibody-toxin fusion protein of Wang et al. See Abstracts of the 209th ACS National Meeting, Anaheim, Calif., Apr. 2-6, 1995, Part 1, BIOT005. As a further example, Doblsten et al.(Proc. Nat""l Acad. Sci. USA, 91:8945 (1994)) reported an antibody-toxin fusion protein comprising Staphylococcal enterotoxin-A.
Useful cancer chemotherapeutic drugs for the preparation of immunoconjugates include calicheamicin, nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purine analogs, antibiotics, epipodophyllotoxins, platinum coordination complexes, hormones, and the like. Suitable chemotherapeutic agents are described in REMINGTON""S PHARMACEUTICAL SCIENCES (Mack Publishing Co. 1995) and in GOODMAN AND GILMAN""S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS (MacMillan Publishing Co. 1985). Other suitable chemotherapeutic agents, such as experimental drugs, are known to those of skill in the art.
In addition, therapeutically useful immunoconjugates can be obtained by conjugating photoactive agents or dyes to an antibody composite. Fluorescent and other chromogens or dyes, such as porphyrins sensitive to visible light, have been used to detect and to treat lesions by directing the suitable light to the lesion. In therapy, this has been termed photoradiation, phototherapy, or photodynamic therapy. See Jori et al. (eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem. Britain, 22:430 (1986). Moreover, monoclonal antibodies have been coupled with photoactivated dyes for achieving phototherapy. See Mew et al., J. Immunol., 130:1473 (1983); Mew et al., Cancer Res., 45:4380 (1985); Oseroff et al., Proc. Natl. Acad. Sci. USA, 83:8744 (1986); Mew et al., Photochem. Photobiol., 46:83 (1987); Hasan et al., Prog. Clin. Biol. Res., 288:471 (1989); Tatsuta et al., Lasers Surg. Med., 9:422 (1989); Pelegrin et al., Cancer, 67:2529 (1991). However, these earlier studies did not include use of endoscopic therapy applications, especially with the use of antibody fragments or subfragments. Thus, the present invention contemplates the therapeutic use of immunoconjugates comprising photoactive agents or dyes.
Multimodal therapies of the present invention further include immunotherapy comprising two or more naked anti-granulocyte antibodies. In another embodiment, multimodal therapy comprises administration of naked anti-granulocyte antibodies supplemented with naked antibodies directed to antigens present on a single granulocyte precursor. For example, naked MN-3 antibodies can be administered in conjunction with anti-CD33 and/or anti-CD15 antibodies.
In another embodiment of the present invention, naked antibodies are administered to a patient in combination with an inducing agent, wherein the inducing agent induces or enhances the expression of a targeted antigen in cell-type of interest. A variety of inducing agents are well-known in the art. For example, arsenic oxide has been shown to up-regulate the expression of NCA-90 in acute promyelocytic leukemia cells. See Di Noto et al., Tissue Antigens, 54:597-602 (1999). Similarly, retinoic acid also enhances the expression of NCA-90 in acute promyelocytic leukemia cells. See Boccuni et al., Tissue Antigens, 52:1-8 (1998).
By up-regulating the expression of the target antigen on the cell surface, such inducing agents enhance the efficacy the administered immunotherapy. Thus, in one embodiment, inducing agents are co-administered with the naked antibodies of the present invention to enhance the toxicity of the therapy.
In another embodiment, inducing agents are co-administered with naked antibodies to increase the therapeutic scope of the treatment. By inducing expression of antigens not normally displayed on surface of particular cell-types, inducing agents extend the therapeutic potential of the therapy to additional cell-types and cancers. For example, retinoic acid or arsenic oxide can be administered to up-regulate NCA-90 in acute promyelocytic leukemia cells. See Di Noto et al., Tissue Antigens, 54:597-602(1999); Boccuni et al., Tissue Antigens, 52:1-8 (1998). Accordingly, the antibodies of the present invention can be used to treat AML, as well as CML.
In another form of multimodal therapy, subjects receive naked anti-granulocyte antibodies and standard chemotherapy. Examples of chemotherapeutic agents include, but are not limited to, daunorubicin, cytarabine, 6-thioguanine, etoposide, mitoxantrone, diaziquone, idarubicin, homoharringtonine, Amsacrine, busulfan, hydroxyurea, calicheamicin, CVB (1.5 g/m2 cyclophosphamide, 200-400 mg/m2 etoposide, and 150-200 mg/m2 carmustine), C-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone), CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone), m-BACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone and leucovorin) and MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin and leucovorin). Additional useful drugs include phenyl butyrate and bryostatin-1.
In general, the dosage of administered naked anti-granulocyte antibodies, immunoconjugates, fusion proteins and additional therapeutics will vary depending upon such factors as the patient""s age, weight, height, sex, general medical condition, disease state and previous medical history. Typically, it is desirable to provide the recipient with a dosage of antibody, immunoconjugate or fusion protein which is in the range of from about 1 pg/kg to 20 mg/kg (amount of agent/body weight of patient), although a lower or higher dosage also may be administered as circumstances dictate.
Administration of antibodies, antibody components, immunoconjugates or fusion proteins to a patient can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, by perfusion through a regional catheter or by direct intralesional injection. When administering therapeutic proteins by injection, the administration may be by continuous infusion or by single or multiple boluses.
Those of ordinary skill in the art are aware that intravenous injection provides a useful mode of administration due to the thoroughness of the circulation in rapidly distributing antibodies. Intravenous administration, however, is subject to limitation by a vascular barrier comprising endothelial cells of the vasculature and the subendothelial matrix. Still, the vascular barrier is a more notable problem for the uptake of therapeutic antibodies by solid tumors. Lymphomas have relatively high blood flow rates, contributing to effective antibody delivery. Intralymphatic routes of administration, such as subcutaneous or intramuscular injection, or by catherization of lymphatic vessels, also provide a useful means of treating lymphomas.
Preferably, naked anti-granulocyte antibodies are administered at low protein doses, such as 20 to 1500 milligrams protein per dose, given once, or repeatedly, parenterally. Alternatively, naked anti-granulocyte antibodies are administered in doses of 20 to 1000 milligrams protein per dose, or 20 to 500 milligrams protein per dose, or 20 to 100 milligrams protein per dose.
As described above, the present invention also contemplates therapeutic methods in which naked anti-granulocyte antibody components are supplemented with immunoconjugate or fusion protein administration. In one variation, naked anti-granulocyte antibodies are administered with low-dose radiolabeled anti-granulocyte antibodies or fragments. As a second alternative, naked anti-granulocyte antibodies are administered with low-dose radiolabeled anti-granulocyte-cytokine immunoconjugates. As a third alternative, naked anti-granulocyte antibodies are administered with anti-granulocyte-cytokine immunoconjugates that are not radiolabeled. With regard to xe2x80x9clow dosesxe2x80x9d of 131I-labeled immunoconjugates, a preferable dosage is in the range of 15 to 40 mCi, while the most preferable range is 20 to 30 mCi. In contrast, a preferred dosage of 90Y-labeled immunoconjugates is in the range from 10 to 30 mCi, while the most preferable range is 10 to 20 mCi.
Immunoconjugates having a boron addend-loaded carrier for thermal neutron activation therapy will normally be effected in similar ways. However, it will be advantageous to wait until non-targeted immunoconjugate clears before neutron irradiation is performed. Clearance can be accelerated using an antibody that binds to the immunoconjugate. See U.S. Pat. No. 4,624,846, which is hereby incorporated by reference in its entirety.
The anti-granulocyte antibodies, immunoconjugates, and fusion proteins of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the therapeutic proteins are combined in a mixture with a pharmaceutically acceptable carrier. A composition is said to be a xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d if its administration can be tolerated by a recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers are well-known in the art. See REMINGTON""S PHARMACEUTICAL SCIENCES (Mack Publishing Co. 1995).
For purposes of therapy, the antibodies described in the invention and a pharmaceutically acceptable carrier are administered to a patient in a therapeutically effective amount. A combination of an antibody, optionally with an immunoconjugat/fusion protein, and a pharmaceutically acceptable carrier is said to be administered in a xe2x80x9ctherapeutically effective amountxe2x80x9d if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient. In the present context, an agent is physiologically significant if its presence results in the inhibition of the growth of target tumor cells.
Additional pharmaceutical methods can be employed to control the duration of action of an antibody, immunoconjugate or fusion protein in a therapeutic application. Controlled release preparations can be prepared through the use of polymers to complex or adsorb the antibody, immunoconjugate or fusion protein. For example, biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebacic acid. See Sherwood et al., BioTechnology, 10:1446 (1992). The rate of release of an antibody (or immunoconjugate) from such a matrix depends upon the molecular weight of the protein, the amount of antibody/immunoconjugate/fusion protein within the matrix, and the size of dispersed particles. See Saltzman et al., Biophys. J., 55:163 (1989); Sherwood et al., BioTechnology, 10:1446 (1992). Other solid dosage forms are described in REMINGTON""S PHARMACEUTICAL SCIENCES (Mack Publishing Co. 1995).
The present invention also contemplates a method of treatment in which immunomodulators are administered to prevent, mitigate or reverse radiation-induced or drug-induced toxicity of normal cells, and especially hematopoietic cells. Adjunct immunomodulator therapy allows the administration of higher doses of cytotoxic agents due to increased tolerance of the recipient mammal. Moreover, adjunct immunomodulator therapy can prevent, palliate or reverse dose-limiting marrow toxicity. Examples of suitable immunomodulators for adjunct therapy include G-CSF, GM-CSF, thrombopoietin, IL-1, IL-3, IL-12 and the like. The method of adjunct immunomodulator therapy is disclosed by Goldenberg, U.S. Pat. No. 5,120,525, which is hereby incorporated by reference in its entirety.
For example, recombinant IL-2 may be administered intravenously as a bolus at 6xc3x97105 IU/kg or as a continuous infusion at a dose of 18xc3x97106 IU/m2/d. See Weiss et al., J. Clin. Oncol., 10:275 (1992). Alternatively, recombinant IL-2 may be administered subcutaneously at a dose of 12xc3x97106 IU. Vogelzang et al., J. Clin. Oncol., 11:1809 (1993). INF-xcex3 can be administered subcutaneously at a dose of 1.5xc3x97106 U. See Lienard et al., J. Clin. Oncol., 10:52 (1992). Nadeau et al. (J. pharmacol. Exp. Ther., 274:78 (1995)) have shown that a single intravenous dose of recombinant IL-12 (42.5 xcexcg/kilogram) elevated IFN-xcex3 levels in rhesus monkeys. Suitable IL-2 formulations include PROLEUKIN (Chiron Corp., Emeryville, Calif.) and TECELEUKIN (Hoffmann-La Roche, Inc.; Nutley, N.J.). ACTIMMUNE (Genentech, Inc., South San Francisco, Calif.) is a suitable INF-xcex3 preparation.

Immunotherapy for chronic myelocytic leukemia


An aqueous external preparation for scalp, that is directly applied to the scalp to improve the elasticity and luster of newly grown or growing hair without affecting the safety or the feeling of touch of the hair, contains the following components (a) a eucalyptus extract; (b) a blood flow enhancer; (c) an anti-inflammatory agent; (d) an acrylic acid/C10-30 alkyl acrylate copolymer; and (e) ethanol. The blood flow enhancer (b) is preferably selected from the group consisting of swertia japonica extract, vitamin E, nicotinic acid and derivatives thereof. The anti-inflammatory agent (c) is preferably glycyrrhetin or derivatives thereof.

1. Field of the Invention
The present invention relates to an aqueous external preparation for scalp that can improve the elasticity of hair and enhance the luster of hair.
2. Background Art
Many scalp preparations have been proposed thus far that contain synthetic ingredients or natural extracts with various effects for the purpose of preventing the decrease in the hair growth area of the scalp and further recovering the hair growth area that has already been decreased. Examples thereof include a hair growth formula containing a blood flow enhancer or a follicle stimulator and a polar solvent extract of eucalyptus plant (JP-A-2001-2532), and a hair restoration/hair growth formula containing flavanonols, a eucalyptus extract and menthol (JP-A-2006-143640).
With regard to the effects of conventional hair restoration/hair growth products, the elasticity and luster of newly grown or growing hair are attracting much attention. The loss of the elasticity and luster of hair is a phenomenon seen not only in people with less hair, but also in people with a normal hair volume as a result of aging. There is an increasing demand among the people to improve the elasticity and luster of their newly grown or growing hair.
However, most of conventional hair restoration/hair growth products, including those described prior art documents, have been developed with the intention of facilitating hair restoration/hair growth without affecting the safety and feeling of touch of hair. At present, the demand for techniques that can improve the elasticity and luster of hair is left unsatisfied. Thus, no products are available that can improve the elasticity and luster of hair.


New, useful and surprisingly effective polymeric triphenylmethane colorant-containing compositions which exhibit very low color degradation within polyurethane foams. Generally during polyurethane foam production, certain amine-based hydroxyl-group containing catalysts deleteriously attack the nitrogen linking groups within triphenylmethane polymeric colorants. Such compositions require the presence of nominal yet effective amounts of organic cyclic ester materials. The ring systems of such materials appear to open during exposure to heat (during the foaming procedure) and release carboxylic acids into the foaming formulation. The tertiary amines become quenched by the carboxylic acids and thus do not exhibit degradation of the TPM-based chromophore. The resultant foams exhibit substantially uniform color throughout. The processes and resultant foams are also contemplated within this invention.

This invention relates to surprisingly effective polymeric triphenylmethane colorant-containing compositions which exhibit very low color degradation within polyurethane foams. Generally during polyurethane foam production, certain amine-based hydroxyl-group containing catalysts deleteriously attack the triphenylmethane (TPM) polymeric colorants. Such compositions require the presence of nominal yet effective amounts of organic cyclic ester materials. The ring systems of such materials appear to open during exposure to heat (during the curing process) and release carboxylic acids into the curing formulation. The tertiary amines become quenched by the carboxylic acids and thus do not attack the TPM chromophore of the polymeric colorant to an extent to cause any appreciable color loss. The resultant foams exhibit substantially uniform color throughout. The processes and resultant foams are also contemplated within this invention.
Polyurethane products, such as foams, resins, and the like, have traditionally been colored by pigments, polymeric colorants, and dyes. Generally, these colorations are performed in situ during foam, resin, etc., formation. For instance, polymeric colorants (i.e., polyoxyalkylenated colorants), such as those described in U.S. Pat. No. 4,284,279 to Cross et al., have been introduced within polyol compositions during slabstock foam production. The xe2x80x9ccoloredxe2x80x9d polyol then reacts with an isocyanate composition, in the presence of a catalyst possibly, to form the desired colored foam. Pigments have also been added in the past, most notably in solid, paste, or powder form, to a polyol stream to form the same type of colored foam products. Such foamed products require the presence of a catalyst or catalysts to effectuate the desired reaction between the polyol and isocyanate components. The most prevalent catalysts, due to cost in producing, using, and disposing, are tertiary amine-based compounds. To reduce emissions of residual amine catalysts, the industry has turned to using hydroxyl-terminated type amine catalysts, most notably DMEA and DABCO TL catalysts (a blend of triethylene diamine and 2-[[2-dimethylamino)ethyl]methylamino]ethanol) and Texacot ZF10 (N,N,Nxe2x80x2-trimethl-Nxe2x80x2-hydroxyethyl-bis(aminoethyl)ether). These catalysts unfortunately present the ability to exaggerate certain problems within the resultant foams, most notably color loss and/or degradations. Apparently, such catalysts react readily with TPM-based chromophores within the polyurethane. The high temperatures associated with polyurethane foam production permits attack of the positively charged TPM polymeric colorant. With a strong positive charge on the base carbon of such a TPM chromophore, the hydroxyls present within the catalyst are drawn to the colorant and appear to react in some fashion to weaken the necessary strong color-producing positive charge. Such deleterious weakening of the TPM positive charge thus apparently causes a severe reduction in color within the foam media. Apparently, such high temperature (i.e., above about 165-185xc2x0 C.) discolorations and degradations more readily occur between about 15 and 30 minutes after foam generation (after gelation and blowing of the foam-producing composition) has taken place. Without the presence of environmentally unfriendly and thus avoided CFC auxiliary blowing agents, the entire process becomes more excessively heated due to the absence of effective heat dissipation compounds. The high temperatures generated in such a manner thus increase the rate of attack by the hydroxyl of the catalyst on the TPM constituents. As a result, discrete areas within the middle of the final article are discolored as compared with the remaining portions of the article.
One specific, extremely troublesome, problem exists in the utilization of polymeric triphenylmethane colorants within polyurethane foam articles. Such colorants, which comprise highly desirable polyoxyethylene chains, polyoxypropylene chains, or both, provide extremely effective colorations to target polyurethane media. Being polymeric in nature, these colorants actually tend to react to and within the urethane monomers during polymerization. As a result, the color is integrated within the foam and provides excellent uniformity and depth throughout the entire article. However, certain polymerization blowing catalysts, which happen to be the desired catalysts throughout the industry, tend to attack the nitrogen linking groups (present between the TPM backbone and the polymeric chains), thereby degrading the colorants themselves and preventing effective colorations of the target foam article. This phenomenon is most likely caused by the high reactivity of the free amine or hydroxyl groups of the catalysts and their ability to attack the unprotected nitrogens (with free electrons) present on the TPM polymeric colorant.
Attempts at alleviating these particular problems have included the addition of relatively expensive, potentially environmentally unfriendly, and potentially toxic antioxidants, such as 2,6-di-t-butyl-4-methylphenol (BHT), octadecyl-3-(3,5-di-t-butyl-4-hydroxyphenyl)propanoate (Irganox(copyright) 1076, from Ciba Geigy), and octyl-3(3,5-di-t-butyl-4-hydroxyphenyl)propanoate (Irganox(copyright) 1135), within the curing process. This has proven ineffective, with little or no improvement in discoloration. Since there is a 15 to 30 minute window of opportunity to control high temperature exposures, some foam producers have practiced forced air cooling of the foam-producing composition in the past to reduce potential discoloration problems. Although such forced air cooling is not required in such an instance, there is a more pronounced color discoloration exhibited upon utilization of consistently high, more inexpensive curing, etc., higher temperatures. Furthermore, since polymeric TPM colorants are the most favorable coloring agents for producing blue and green hue foams, the ability to inexpensively provide readily available TPM colorations within polyurehtane foams is highly necessary. Alternative methods, either simpler and less inexpensive in nature, have not been forthcoming within the industry. As a result, any marked improvements in such a manner are of utmost importance within the polyurethane foam production industry. To date, again, there have been no significant or helpful improvements nor advancements disclosed within the pertinent prior art.
It is therefore an object of this invention to provide a relatively inexpensive and simple method of preventing color loss for polymeric triphenylmethane colorants within polyurehtane foam articles. A further object is to provide a method of reducing color degradation within polyurethane foam producing methods which utilize amine-based catalysts in combination with polymeric triphneylmethane colorants. A further object is to provide an easy-to-add liquid composition for introduction within polyurethane foam production procedures which effectively reduces and/or eliminates such color degradation problems associated with amine-based catalysts. A further objective of this invention is to provide a polymeric triphenylmethane colored polyurethane foam product which exhibits substantially no color loss or discolorations.
Accordingly, this invention is directed to a method of producing a polyurethane foam article comprising the steps of: a) providing a polyol composition; b) providing an isocyanate composition; wherein at least one of the compositions of steps xe2x80x9caxe2x80x9d and xe2x80x9cbxe2x80x9d comprise at least polymeric triphenylmethane colorant; c) adding a composition comprising at most 3.0 php of an organic cyclic ester to either or both of the compositions in steps xe2x80x9caxe2x80x9d and xe2x80x9cbxe2x80x9d, above; d) reacting all of the compositions from steps xe2x80x9caxe2x80x9d, xe2x80x9cbxe2x80x9d, and xe2x80x9ccxe2x80x9d together in the presence of a tertiary amine hydroxy-containing catalyst. The particular compositions from these steps including the organic cyclic ester and, optionally, at least one coloring agent, are also contemplated within this invention. Furthermore, a polyurethane foam article produced by these methods and/or exhibiting a substantially uniform coloration throughout is also contemplated within this invention.
In general, polyurethane foam is produced through the catalyzed polymerization of the reaction products of polyols and isocyanates. Such a reaction is well known throughout the polyurethane industry and has been practiced for many years. The potential number and types of polyols utilized within this invention are plentiful. Such a compound is defined as comprising at least two alcohol moieties, preferably at least three. The free hydroxyl groups react well with the isocyanates to form the urethane components which are then polymerized to form the desired polyurethanes. Blowing agents present within the polymerization step provide the necessary foam-making capability. Preferred polyols thus comprise between three and six alcohol moieties, comprising from between one and six carbon atoms per alcohol moiety. Most preferred is a typical trifunctional polyol. F3022 polyol, available from Lyondell.
Isocyanates, most preferred diisocyanates, are well known components of such polyurethane foams and include any compounds which possess at least one free cyanate reactive group, and most preferably two, although more may be utilized. Such compounds are may also be aliphatic or aromatic in nature. The most prominently utilized isocyanates, and thus the most preferred types for this invention, are toluene diisocyanate and methylene diisocyanate. The polyol is generally reacted with a slight excess of isocyanate (ratio of from 1:1.04 to 1:1.12) to produce a soft foam product; the greater the ratio, the harder the produced foam). In practice, two separate streams of liquids (one of polyol, the other of isocyanate) are mixed together in the presence of a polymerization catalyst and a blowing agent in order to produce the desired polyurethane foam product.
The term xe2x80x9ctertiary amine-based hydroxy-containing catalystxe2x80x9d is intended to encompass any gelation/blowing catalyst utilized within polyurethane production which comprises at least one amine constituent. As noted above, amine-based catalysts, and more specifically, tertiary amine catalysts, are widely utilized within such specific foam-producing methods. Two catalysts, in particular, DABCO TL, DABCO T10, and DMEA, are excellent gelation/blowing catalysts for this purpose; however, they also appear to be extremely reactive with and readily attack unmatched electrons on nitrogen-containing moieties. As noted above, oxidation by the amine readily occurs upon exposure to high temperatures, thus resulting in the undesirable scorched foam portions. Although any amine presents such a potential reactivity (oxidation) problem, and thus is contemplated within the scope of this invention, it has been found that the highly reactive tertiary amines present greater threats to discoloration and degradation to the final foam product. The amount of tertiary amine hydroxy-containing catalyst required to effectuate the desired urethane polymerization is extremely low, from between 0.05 php to about 1.00 php of the polyol content within the foam-making composition; more specifically, such a range is from about 0.07 php to about 0.60 php. Even though the number of free amines available are quite low, their ability to deleteriously affect the final foam product through oxidation of free reactive groups (hydroxyls, for example) within colorants, polyols, and other additives, is pronounced upon exposure to high temperature during polymerization.
Surprisingly, such discolorations and/or color degradations are substantially reduced and potentially eliminated through the simple extremely low addition of an organic cyclic ester within the foam-making procedure. Such a compound must be susceptible to ring opening upon exposure to the high foam-production temperatures and be thereafter be present as carboxylic acid within the same procedure. It is believed, without being bound to any specific scientific theory, that such carboxylic acids react more readily with the free reactive amines of the catalyst and thus quench the potential oxidation of the other free reactive groups present on other components of the foam-producing composition, all without affecting the rate of polymerization. Specific organic cyclic esters suitable for utilization for this purpose include, without limitation, lactones, cyclic carbonates, and basically any cyclic compound which exhibits ring opening upon exposure to heat of from about 50xc2x0 C. to about 100xc2x0 C.; more preferably from about 56xc2x0 C. to about 95xc2x0 C.; and most preferably from about 90xc2x0 C. to about 95xc2x0 C., and thereafter exists as a carboxylic acid. Any mixtures of such compounds may be utilized as well. Of greater importance, however, appears to be necessity of introducing such a compound (or compounds) in relatively low amounts within the foam-making composition. Thus, such a compound is within the polyol stream in an amount of between 0.001 php and 3.0 php, in relation to the total amount of polyol. Preferably, this level is between about 0.20 and 2.5 php; more preferably between about 0.35 and about 2.25 php; and most preferably between about 0.55 and about 2.0 php. The preferred organic cyclic esters include caprolactone, gamma-valerolactone, sigma-valerolactone, butyrolactone, propylene carbonate, and any mixtures thereof. Most preferred are caprolactone and butyrolactone.
As noted above, triphenylmethane polymeric colorants must be present within the inventive procedure and article. Such colorants are well known throughout the colored polyurethane foam industry and are more specifically taught within the Cross et al. U.S. Patent noted and incorporated herein, above. Other coloring agents may be also added within the different streams prior to reaction and/or polymerization as well. Such additions are well known within the industry to produce colored polyurethane foam articles in various colors and shades. Thus, the term xe2x80x9ccoloring agentxe2x80x9d is intended to encompass any pigment, pigment dispersion, polymeric colorant (other than the required at least one triphenylmethane type), dye, dyestuff, any mixtures thereof, and the like, which provides desirable colorations to target polyurethane foam articles. In general, such coloring agents are added to the polyol stream (prior to reaction with the isocyanate) in amounts ranging from about 0.001 php to about 10 php. Higher color loadings may adversely affect foam performance. Suitable pigments for this invention include, without limitation, carbon black, lamp black, titanium dioxide, phthalocyanine, and the like. Suitable polymeric colorants for this purpose are disclosed within Cross et al., again without any limitation, and as merely examples. Suitable dyes and dyestuffs include reactive dyes, solvent dyes, vat dyes, and the like.
Other additives or solvents may also be present within the foam-making composition. Auxiliary blowing agents are required to provide the necessary foam blowing capability and reduce chances of combustion. Such compounds include methylene chloride, acetone, carbon dioxide (which may be liberated during the reaction between water and isocyanate), and the like, and are present in amounts of between about 1.0 php and 10 php of the entire foam-making composition. Water may thus also be added in relatively low amount (i.e., from about 3 to about 10 php; most preferably between about 3 and 5 php) to provide carbon dioxide for blowing purposes. Silicones may be added to provide desired cell structure and foam stability and are present in an amount from about 0.1 to about 2 php of the entire foam-making composition; preferably from about 0.9 to about 1.6 php.
The use of cyclic carbonates and cyclic lactones in polyurethane chemistry is known. U.S. Pat. No. 3,883,466 describes the use of a cyclic alkylene carbonate as a liquid modifier to moderate the reaction exotherm between the hydroxy component and the polyisocyanate in the production of a rigid, dense rapid-setting polyurethane. U.S. Pat. Nos. 4,709,002 and 4,731,427 describe the use of cyclic alkylene carbonates in the production of rigid RIM polyisocyanurate and urethane-modified polyisocyanurate parts. These two references do not indicate why cyclic alkylene carbonate is used but do suggest that the carbonate can be added to the isocyanate stream in order to reduce its viscosity. U.S. Pat. Nos. 5,028,635 and 5,149,458 report two polyurea-cyclic carbonate RIM systems having improved flow properties. European Patent 0,350,644 and U.S. Pat. No. 5,442,034 report similar applications for cyclic carbonate in RIM elastomers and spray polyurea elastomers, respectively. U.S. Pat. No. 4,812,523 describes high solids thermosetting coating composition with cyclic carbonates as reactive diluents to reduce viscosity. Cyclic carbonates and cyclic lactones have also been used as viscosity reducing agents in aromatic polyester polyols and polyether polyols (EP 0,276,452). There is no discussion or fair suggestion, however, of the addition of such extremely low amounts of organic cyclic esters for the purpose of quenching amine-based catalysts upon high temperature exposure during polyurethane foam-making procedures.

Reduction of color degradation of triphenylmethane polymeric colorants within polyurethane foams