CN1942199A - Methods and compositions for the treatment of cancer and infectious disease using alpha (2) macroglobulin-antigenic molecule complexes - Google Patents

Abstract

The present invention relates to the use of alpha (2) macroglobulin complexes isolated from the serum of a mammal. The invention also relates to methods for making such complexes and compositions comprising alpha (2) macroglobulin complexes, isolated from the serum of a mammal, wherein such compositions are used in methods for the treatment and prevention of cancer and infectious disease. The invention also relates to methods for treating and preventing cancer and infectious disease using such complexes comprising, isolated from the serum of a mammal. The invention also encompasses methods for production of alpha (2) macroglobulin complexes.

Description

Methods and compositions for treating cancer and infectious diseases using alpha(2) macroglobulin-antigen molecule complexes

This application claims priority to U.S. Provisional Application No. 60/449,022, filed February 20, 2003, and U.S. Provisional Application No. 60/450,751, filed February 27, 2003, both of which are incorporated herein in their entirety as refer to.

This invention was made with support under Government Grant No. CA A184479 awarded by the National Institutes of Health. The government has certain rights in this invention.

1 Introduction

The present invention relates to the use of alpha(2) macroglobulin complexes isolated from mammalian serum in methods for the treatment and prevention of cancer and infectious diseases. The invention also includes methods of making alpha(2) macroglobulin complexes. The present invention also includes compositions and methods for preparing alpha(2) macroglobulin-antigen molecule complexes derived from serum.

2. Background of the invention

2.1. α(2) macroglobulin

Alpha-macroglobulins are a protein superfamily of structurally related proteins that also include complement components C3, C4, and C5. The human plasma protein α(2) macroglobulin (α2M) is a 720 kDa homotetrameric protein known primarily as a protease inhibitor and plasma and inflammatory fluid protease clearance molecule (for review see Chu and Pizzo, 1994, Lab.Invest. , 71:792). α2M is synthesized as a precursor with 1474 amino acid residues. The first 23 amino acids function as a signal sequence, cleavage of which yields a mature protein of 1451 amino acid residues (Kan et al., 1985, Proc. Natl. Acad. Sci., U.S.A., 82:2282-2286).

α(2) macroglobulins covalently bind promiscuously to proteins and peptides with nucleophilic amino acid side chains (Chu et al., 1994, Ann.N.Y.Acad.Sci., 737:291-307 ) and target them to cells expressing CD91 (also called α2M receptor or α2MR; Chu and Pizzo, 1993, J. Immunol., 150:48). Binding of α2M to CD91 is mediated by the carboxy-terminal portion of α2M (Holtet et al., 1994, FEBS Lett., 344:242-246) and key residues have been identified (Nielsen et al., 1996, J. Biol. Chem., 271:12909-12912).

It is generally known that for inhibition of protease activity, α2M binds to various proteases through multiple binding sites (see, eg, Hall et al., 1981, Biochem. Biophys. Res. Commun., 100(1):8-16). Interaction of the protease with α2M causes a structural rearrangement of the complex, called deformation, as a result of cleavage within the "bait" region of α2M after the protease has been "captured" by the thioester. The conformational change exposes residues required for receptor binding, allowing the α2M-protease complex to bind to α2MR. Methylamine can induce similar conformational changes and fragmentation induced by proteases. The unfragmented form of α2M that is not recognized by the receptor is commonly referred to as the "slow" form (s-α2M). The fragmented form is called the "fast" form (f-α2M) (reviewed by Chu et al., 1994, Ann. N. Y. Acad. Sci., 737:291-307). It has recently been shown that α2MR can bind to HSPs such as gp96, hsp90, hsp70, and calreticulin (Basu et al., 2001, Immunity, 14(3):303-13).

Studies have shown that, in addition to its protease inhibitory effects, α2M, when complexed with antigens, can enhance the ability of antigens to be taken up by antigen-presenting cells such as macrophages and presented to T-cell hybridomas in vitro by up to two order of magnitude; (Chu and Pizzo, 1994, Lab. Invest., 71: 792) and the ability to induce T cell proliferation (Osada et al., 1987, Biochem. Biophys. Res. Commun., 146: 26-31). Additional evidence suggests that antigen complexation with α2M increases in vitro antibody production by native spleen cells (Osada et al., 1988, Biochem.Biophys.Res.Commun., 150:883), causing in experimental rabbits (Chu et al., 1994, J. Immunol., 152:1538-1545) and in vivo antibody responses in mice (Mitsuda et al., 1993, Biochem. Biophys. Res. Commun., 101:1326-1331). α2M-antigen peptide complexes have also been shown to induce cytotoxic T cell responses in vivo (Binder et al., 2001, J. Immunol., 166:4698-49720).

2.2. Heat shock proteins

Heat shock proteins (HSPs), also known as stress proteins, were originally identified as proteins synthesized by cells in response to heat shock. Hsp have been divided into five families according to molecular weight, Hsp100, Hsp90, Hsp70, Hsp60, and smHsp. Many members of these families were subsequently found to be induced in response to other stress stimuli, including nutrient deprivation, metabolic disruption, oxygen free radicals, and infection with intracellular or extracellular pathogens (see Welch, 1993, Scientific American 56-64; Young, 1990, Annu . Rev. Immunol., 8: 401-420; Craig, 1993, Science, 260: 1902-1903; Gething et al., 1992, Nature, 355: 33-45; and Lindquist et al., 1988, Annu. Rev. Genetics , 22: 631-677).

Heat shock proteins are among the most highly conserved proteins in existence. For example, DnaK, Hsp70 from Escherichia coli (E.coli), has about 50% amino acid sequence identity with the Hsp70 protein from dementia (Bardwell et al., 1984, Proc.Natl.Acad.Sci., 81; 848-852 ). Hsp60 and Hsp90 families also show the same high level of conservation within the family (Hickey et al., 1989, Mol. Cell. Biol., 9: 2615-2626; Jindal, 1989, Mol. Cell. Biol., 9: 2279-2283 ). In addition, the Hsp60, Hsp70 and Hsp90 families have been found to consist of proteins that are sequence related to stress proteins, eg, having greater than 35% amino acid identity, but whose expression levels are not altered by stress.

Studies of cellular responses to heat shock and other physiological stresses have shown that HSPs are not only involved in cellular protection against these adverse conditions, but also in biochemical and immune processes necessary for unstressed cells. HSPs perform a variety of companion functions. For example, members of the Hsp70 family located in the cytoplasm, nucleus, mitochondria, or endoplasmic reticulum (Lindquist et al., 1988, Ann. Rev. Genetics, 22:631-677), are involved in the presentation of antigens to cells of the immune system and are also involved in normal Transport, folding and assembly of proteins in cells. HSPs are capable of binding proteins or peptides and releasing the bound proteins or peptides in the presence of adenosine triphosphate (ATP) or low pH.

2.3. Immunogenicity of HSP-peptide complexes

Srivastava et al. demonstrated methylcholanthrene-induced immune responses in inbred mice (1988, Immunol. Today, 9:78-83). In these studies, the molecules responsive to the respective immunogenicity of these tumors were found to be a glycoprotein of 96 kDa (gp96) and an intracellular protein of 84 to 86 kDa (Srivastava et al., 1986, Proc. Natl. Acad. Sci. USA, 83:3407-3411; Ullrich et al., 1986, Proc. Natl. Acad. Sci. USA, 83:3121-3125). Immunization of mice with gp96 or p84/86 isolated from mice bearing a particular tumor immunizes that particular tumor, but not antigenically distinct tumors. Isolation and characterization of the genes encoding gp96 and p84/86 revealed significant homology between them and suggested that gp96 and p84/86 are the endoplasmic reticulum and cytoplasmic counterparts, respectively, of the same heat shock protein (Srivastava et al., 1988, Immunogenetics 28: 205-207; Srivastava et al., 1991, Curr. Top. Microbiol. Immunol. 167: 109-123). Furthermore, Hsp70 isolated from tumors exhibits immunity to that tumor but not to antigenically distinct tumors (Udono and Srivastava, 1993, J. Exp. Med. 178:1391-1396). These observations suggest that the heat shock protein itself does not have immunity, but when it forms a non-covalent complex with the antigenic peptide, the complex can be specifically immune to the antigenic peptide (Srivastava, 1993, Adv.Cancer Res.62: 153- 177; Udono et al., 1994, J. Immunol., 152:5398-5403; Suto et al., 1995, Science, 269:1585-1588).

Non-covalent complexes of HSP and peptides purified from cancer cells are useful in the treatment and prevention of cancer and have been disclosed in PCT: WO 96/10411 of April 11, 1996, WO 97 of March 20, 1997 /10001 (US Patent 5,750,119, issued April 12, 1998, and US Patent 5,837,251, issued November 17, 1998, respectively, both of which are incorporated herein by reference). Isolation and purification of stress protein-peptide complexes from, for example, pathogen-infected cells has been described and can be used in the treatment and prophylaxis of diseases caused by pathogens such as viruses and other intracellular or extracellular pathogens including bacteria, protozoa, fungi and parasites. infection (see e.g. PCT Publication: WO 95/24923 September 21, 1995). Immunogenic stress protein-peptide complexes can also be prepared by complexing stress proteins and antigenic peptides in vitro, and the use of such complexes for the treatment and prevention of cancer and infectious diseases has been disclosed in PCT: March 20, 1997 It is described in the dated WO 97/10000 (US Patent 6,030,618 issued February 29, 2000). The use of stress protein-peptide complexes in vitro for sensitization of antigen-presenting cells in adoptive immunotherapy is published in PCT: WO 97/10002, March 20, 1997 (see also U.S. Patent issued November 16, 1999 5,985,270).

2.4. Alpha(2) macroglobulin receptor, or "CD91"

Alpha(2) macroglobulin receptor (herein interchangeably referred to as "α2MR" or "α2M receptor"), also known as LDL (low density lipoprotein) receptor-related protein ("LRP") Or CD91, mainly expressed in the liver, brain and placenta. The α2M receptor is a member of the low-density lipoprotein receptor family. The extracellular domain of the human receptor includes six 50-amino acid EGF repeats and 31 complement repeats of approximately 40-42 amino acids. Complement repeats are organized from amino-terminus to carboxy-terminus into clusters of 2, 8, 10, and 11 repeats, referred to as clusters I, II, III, and IV (Herz et al., 1988, EMBO J., 7:4119-4127). One study targeted cluster II (C1-II), containing complement repeat 3-10 (CR3-10), as the major ligand-binding portion of the receptor (Horn et al., 1997, J. Biol. Chem., 272:13608 -13613). The α2M receptor plays a role in the endocytosis of various ligands. In addition to α2M, other ligands of α2MR include lipoprotein complexes, lactoferrin, tissue plasminogen activator (tPA), urokinase plasmin activator (uPA), and exotoxin. Additional examples of ligands for α2MR can be found in PCT Publication WO 97/04794 and US Patent 6,156,311. Thus, the α2M receptor plays a role in a variety of cellular metabolic processes, including endocytosis, antigen presentation, cholesterol regulation, clearance of ApoE-containing lipoproteins, and removal of chylomicron remnants.

Human α2M is synthesized as a precursor of 1474 amino acids, and the first 23 amino acids act as a signal sequence, which is broken to obtain a mature protein of 1451 amino acids (Kan et al., 1985, Proc.Natl.Acad.Sci.U.S.A., 82 : 2282-2286). In recombinant protein experiments, it was found that the carboxy-terminal 138 amino acids of α2M (representing amino acids 1314-1451 of the mature protein) bound to the receptor. This region has been termed the RBD (receptor binding domain; Salvesent et al., 1992, FEBS Lett., 313:198-202; Holtet et al., 1994, FEBS Lett., 344:242-246). RBD variant (RBDv), a proteolytic fragment of α2M comprising an additional 15 amino-terminal residues (representing amino acids 1314-1451 of the mature protein), binds the receptor with nearly the same affinity as α2M-protease (Holtet et al. 1994, FEBS Lett., 344:242-246).

Sequence alignment of α2MR ligands identifies a conserved region present in the α-macroglobulin RBD. The conserved sequence includes amino acids 1366-1392 of human α2M. The conserved residues in this region are Phe ₁₃₆₆ , Leu ₁₃₆₉ , Lys ₁₃₇₀ , Val ₁₃₇₃ , Lys ₁₃₇₄ , Glu ₁₃₇₇ , Val ₁₃₈₂ , Arg ₁₃₈₄ (Nielsen et al., 1996, J. Biol. Chem., 271: 12909- 12912). Among them, Lys ₁₃₇₀ and LYS ₁₃₇₄ appear to play a crucial role in receptor binding (Nielsen et al., 1996, J. Biol. Chem., 271: 12909-12912).

Ligand binding to α2MR, including binding to α2M, is inhibited by α2MR-associated protein (RAP). RAP is a 39 kDa folded chaperone protein present in the endoplasmic reticulum and required for normal processing of α2MR. RAP was able to competitively inhibit the binding of all α2MRs to all tested α2MR ligands. One study showed that RAP binds to complement repeats C5-C7 in cluster II (C1-II) of α2MR (Horn et al., 1997, J. Biol. Chem., 272:13608-13613); another study showed RAP binds to all dicomplement repeat modules in C1-II except the C9-C10 module (Andersen et al., J. Biol. Chem., March 24, 2000, PMID: 10747921; prior to printing as published electronically). Three domains have been identified in RAP, consisting of amino acid residues 18-112, 113-218 and 219-323, respectively. Ligand competition titrations of recombinant RAP domains showed that the determinants of inhibition by the tested ligands were in the C-terminal regions of domains 1 and 3 (Ellgaard et al., 1997, Eur. J. Biochem., 244:544-51).

PCT Publication WO 01/92474 published December 6, 2001 also describes the use of CD91 as a receptor for heat shock proteins, cells expressing CD91 bound to HSPs, antibodies and other molecules that bind CD91-HSP complexes, recognize Screening assays for compounds that modulate the interaction of HSPs with CD91, methods of using compositions comprising CD91, and CD91 sequences for diagnosis and treatment of immune and proliferative and infectious diseases. Complexes of α(2) macroglobulin bound to antigenic molecules for use in immunotherapy and methods for using such compositions in the diagnosis and treatment of proliferative and infectious diseases are published in PCT December 2001 It is also described in WO 01/91787 published on the 6th. Binder et al. showed that in vitro complexes of recombinant α(2) macroglobulin and antigenic peptides elicit specific CTL responses (Binder et al., 2001, J. Immunol., 166:4968-4972).

2.5. Antigen presentation

Major histocompatibility complex (MHC) molecules present antigens on the cell surface of antigen presenting cells. Depending on whether the antigen is of intracellular or extracellular origin, it is processed through two distinct antigen processing pathways. Intracellular or endogenous protein antigens, antigens synthesized in antigen-presenting cells, are presented to CD8+ cytotoxic T lymphocytes by MHC class I (MHC I) molecules. On the other hand, extracellular or exogenously synthesized epitopes are presented to CD4+ T cells by MHC class II molecules on the cell surface of "professional" or "professional" APCs (e.g. macrophages) ( See generally Fundamental Immunology, W.E. Paul (ed.), New York: Raven Press, 1984). This differentiation of antigen processing pathways is important to prevent indiscriminate tissue destruction due to the release of MHC I antigens from neighboring cells during the immune response.

Heat shock proteins have a variety of peptide partners, depending on the source of the isolated HSP (for review see Srivastava et al., 1998, Immunity, 8:657-665). HSPs derived from tumors carry tumor-antigen peptides (Ishii et al., 1999, J. Immunology, 162:1303-1309); gp96 preparations derived from virus-infected cells carry viral epitopes (Suto and Srivastava, 1995, Science , 269:1585-1588; Nieland et al., 1996, Proc.Natl.Acad.Sci.USA, 95:1800-1805), derived from cells transfected with model antigens such as ovalbumin or β-galactosidase The gp96 preparations associated with the corresponding epitopes (Arnold et al., 1995, J. Exp. Med., 182: 885-889; Breloer et al., 1998, Eur. J. Immunol., 28: 1016-1021). Binding of gp96 to peptides occurs in vivo (Menoret and Srivastava, 1999, Biochem. Biophys. Research Commun., 262:813-818). HSP-peptide complexes, whether isolated from cells (Tamura et al., 1997, Science, 278: 117-120) or reconstituted in vitro (Blachere et al., 1997, J. Exp. Med., 186: 1183-1406) , are excellent immunogens, and have been widely used to elicit CD8+ T cell responses specific to antigenic peptides of HSP-chaperones.

The ability of HSP-peptide complexes to elicit an immune response depends on the transport of the peptides to MHC class I molecules of antigen-presenting cells (see, eg, Suto and Srivastava, 1995, supra). Endogenously synthesized antigens, accompanied by gp96 in the endoplasmic reticulum [ER], can prime antigen-specific CD8+ T cells (or MHC I-restricted CTLs) in vivo; this priming of CD8+ T cells requires Phage cells. However, the process by which exogenously introduced gp96-peptide complexes elicit antigen-specific CD8+ T cell responses is not fully understood because there is no established pathway for the translocation of extracellular antigens to the type 1 presentation machinery. However, antigenic peptides of extracellular origin associated with HSPs are somehow captured by macrophages, directed into the intrinsic pathway and presented by MHC I molecules for recognition by CD8+ lymphocytes (Suto and Srivastava, 1995, supra; Blachere et al., 1997, J. Exp. Med., 186: 1315-22).

2.6. HSP-CD91 Interaction

Studies reported by Basu et al. indicated that the heat shock proteins gp96, hsp90, hsp70 and calreticulin are additional ligands for CD91 (Basu et al., 2001, supra). Gp96 is embedded in CD91 in an amino-terminal fragment called the p80 fragment (Binder et al., 2000, Nature immunology, 1:151-155; WO 01/92474). The gene encoding human gp96 has been previously mapped by us on chromosome 12 (q24.2 q24.3) (Maki et al., 1993, Somatic Cell Mol. Gen., 19:73-81). Interestingly in this regard, the CD91 gene has been mapped to the same chromosome and not so far away (q13q14) (Hilliker et al., Genomics, 13:472-474). Gp96 binds CD91 directly rather than through other ligands such as α2M. Homogeneous preparations of gp96 in solution or cross-linked to a solid matrix bind to CD91. Indeed, the major ligand for CD91, α2M, inhibits rather than promotes the interaction of gp96 with CD91, demonstrating gp96 as a direct ligand for CD91. Appeared to be an 80 kDa protein that binds gp96, p80, is apparently an amino-terminal degradation product of the alpha subunit of CD91 (Binder et al., 2000, Nature immunology, 1:151-155). Degradation products of CD91 in this size range were also observed in previous studies (Jensen et al., 1989, Biochem. Arch., 5: 171-176), and may indicate that discrete CD91 cells may be particularly sensitive to proteolytic cleavage. presence of extracellular domains.

The observation by Basu et al. that α2 macroglobulin and anti-CD91 antibodies completely inhibited re-presentation by each of the four HSPs suggested that CD91 is the only receptor for the four HSPs (Basu et al., 2001, supra). This observation is somewhat counterintuitive given the increasingly apparent role of HSPs in innate (Basu et al., 2000, Int. Immunol., 12(11):1539-1546) and acquired immune responses . However, the data on complete inhibition by two independent means are quite compelling (PCT Publication: WO01/92474 on December 6, 2001). Binder reported a marked difference between hsp70 and hsp90/gp96 in their ability to compete for binding to the gp96 receptor (Binder et al., 2000, J. Immunol., 165:2582-2587). Similar differences between gp96 and hsp70 were also observed by another group (Amold-Schild et al., 1999, 162:3757-3760). These differences are not inconsistent with Basu's report for the individual receptors of the four HSPs. They simply suggest that different HSPs interact with a single receptor with widely varying affinities.

As shown by Binder et al., the HSP-CD91 interaction provides a new role for CD91 or its fragments as a sensor, not only of the extracellular environment with its previously known plasma-based ligands, but also of the Sensors of the intracellular environment. HSPs such as gp96 are obligate intracellular molecules that are released into the extracellular milieu only under conditions of necrotic (but not apoptotic) cell death (PCT Publication: WO 01/92474, December 6, 2001). Thus, CD91 may serve as a sensor of necrotic cell death, just as the scavenger receptor CD36 and the recently identified phosphatidylserine-binding protein function as a sensor of apoptotic cell death and a receptor for apoptotic dying cells (Savill et al., 1992, J. Clin. Invest., 90:1513-1522; Fadok et al., 2000, Nature, 405:85-90). The interaction of macrophages with apoptotic cells causes the downregulation of inflammatory cytokines such as TNF (Fadok et al., 2000, supra), while the gp96-APC interaction causes the re-presentation of the gp96 chaperone peptide by the MHC I molecule of the APC, Antigen-specific T cell activation is followed (Suto and Srivastava, 1995, supra), and secretion of pro-inflammatory cytokines such as TNF, GM-CSF and IL-12. Interestingly, α2M, an independent ligand of CD91, inhibits the representation of gp96-chaperone peptides by macrophages. This observation by Binder suggests that re-presentation of the gp96-chaperone peptide cannot occur physiologically in the blood, but can only be caused by localized necrotic cell death within the tissue. This is consistent with the complete absence of gp96 or other HSPs in the blood under all conditions tested. Taken together, Binder's observations point to a possible mechanism by which the release of HSPs from the blood due to severe tissue injury and lysis does not elicit a systemic and lethal pro-inflammatory cytokine cascade.

Thus, it is possible that CD91 confers on APCs the possibility to (i) sample the extracellular milieu of blood via α2M and other plasma ligands and (ii) sample the intracellular milieu of tissues via HSPs, particularly the gp96 family. The former allows the APC to carry out its primitive phagocytic function, while the latter allows it to carry out its innate and acquired immunological functions. From another perspective, APCs recognize apoptotic cells through CD36 or phosphatidylserine to generate anti-inflammatory signals, while APCs cause pro-inflammatory innate and acquired immune responses through the interaction of CD91 and necrotic cells (see Srivastava et al. People, 1998, Immunity, 8:657-665).

Citation or discussion of a reference herein is not to be construed as an admission that it is prior art to the present invention.

3. Contents of the invention

The present invention relates to methods and compositions for the treatment and prevention of cancer and infectious diseases using complexes of alpha(2) macroglobulin and antigenic molecules derived from the body fluids of patients suffering from cancer or infectious diseases. In a preferred embodiment of the invention, the alpha(2) macroglobulin-antigen molecule complex is autologous to the patient to be treated. The present invention is based in part on the applicant's discovery that a(2) macroglobulin-antigen molecule complexes specific for tumors or pathogens can be found in and isolated from the bloodstream of patients with cancer or infectious diseases. Furthermore, Applicants have found that sufficient quantities of such alpha(2) macroglobulin-antigen molecule complexes can be isolated from the bloodstream of patients for use in autoimmune therapy. In cancer therapy, this discovery enables the preparation of highly specific, individualized immunotherapeutics directed to specific antigens expressed on each individual patient's tumor without first characterizing or identifying such antigens. This autoimmune therapy using the alpha(2) macroglobulin complex is shown herein to be highly effective in both the treatment and prevention of cancer. Furthermore, this result can be extended to applications in the treatment and prevention of infectious diseases.

Applicants found that immunization of female C57BL/6 mice with alpha(2) macroglobulin complex purified from male mice conferred an anti-male response (Binder et al., 2002, Cancer Immunity, Vol. 2: 16). The alpha(2) macroglobulin-male antigen complex is obtained and used to elicit an immune response. Binder et al demonstrated that the Y antigen expressed in male mice can be isolated from the serum of normal male mice as a complex with α(2) macroglobulin. Binder et al. then applied this basic concept to cancer immunity and demonstrated that immunity elicited by recombinant α(2) macroglobulin-peptide complexes in vitro was effective in preventing tumors (Binder et al., 2002, Cit).

Most tumors do not produce α(2) macroglobulin, and even those that do, such as tumors of hepatocyte origin, produce α(2) macroglobulin only intracellularly. The present invention is based in part on the applicant's discovery that tumors or pathogen-bearing cells release antigens into body fluids such as blood where they can form complexes with alpha(2) macroglobulin.

The present invention provides a method for treating or preventing cancer or an infectious disease in a patient, the method comprising administering to a patient in need of said treatment or preventing an amount of α(2) macroglobulin and A complex of antigenic molecules, wherein said complex is isolated from a body fluid of a mammal suffering from cancer or an infectious disease.

The present invention additionally provides a method for treating or preventing cancer or an infectious disease in a patient, comprising the step of: a) isolating a complex of α(2) macroglobulin and an antigen molecule from the body fluid of a mammal suffering from said cancer or infectious disease and b) administering to said patient an amount of said isolated complex effective to treat or prevent said cancer or infectious disease. In a specific embodiment, the complex is a population of complexes of α(2) macroglobulin bound to different antigenic molecules, including those antigenic to said cancer or infectious disease specific antigen. Antigen molecule. In another specific embodiment, the method is for the treatment of cancer and patients suffering from cancer, and the antigenic molecule is derived from a tumor. In another particular embodiment of the present invention, the method is for the treatment of cancer and patients suffering from cancer, and the effect of said cancer treatment is evaluated by reduction in tumor size or number of tumors in the patient. In another specific embodiment, the method is for the prophylaxis of cancer and the patient is in need of such prophylaxis, and said antigenic molecule represents an antigen antigenic to said cancer-specific antigen. In another specific embodiment, the method is for the prophylaxis of an infectious disease and the patient is in need of such prophylaxis, and said antigenic molecule represents an antigen antigenic to a specific antigen of a pathogen associated with said infectious disease. In another embodiment, the method is for treating cancer and a patient suffering from cancer, and the method additionally comprises the step of administering a chemotherapeutic agent to said patient prior to or simultaneously with step (b). In another specific embodiment, the method is for the treatment of cancer and patients suffering from cancer, and the method additionally comprises the step of inducing tumor necrosis in said mammal before or simultaneously with step (a). In another specific embodiment, the step of inducing tumor necrosis comprises administering a tumor necrosis drug to said mammal.

In another embodiment, the present invention provides a method of stimulating an immune response against cancer or an infectious disease in a patient comprising: a) isolating an alpha(2) macroglobulin complex from a mammalian body fluid, and b) administering to the patient The isolated alpha(2) macroglobulin complex is administered to stimulate an immune response. In a specific embodiment, the patient is a human patient. In another specific embodiment, the α(2) macroglobulin complex is a population of complexes of α(2) macroglobulin bound to different antigenic molecules, wherein the different antigenic molecules comprise An antigenic molecule that is antigenic to a disease-specific antigen. In another specific embodiment, the method is for stimulating an immune response against cancer in a patient suffering from cancer, and the method additionally comprises, prior to or simultaneously with step (a), inducing tumor necrosis in said mammal step. In another specific embodiment, the step of inducing tumor necrosis comprises administering a tumor necrosis drug to said mammal.

The present invention further provides a method for preventing cancer in a mammal, the method comprising administering an effective amount of a complex of α(2) macroglobulin and an antigen molecule, the complex being isolated from the body fluid of the mammal, the mammal Animals with precancerous lesions or polyps. In a specific embodiment of the method, the mammal is a human.

The present invention additionally provides a method of preventing cancer in a first mammal, said method comprising administering an effective amount of a complex of alpha(2) macroglobulin and an antigenic molecule, said complex being isolated from a body fluid of a second mammal, The second mammal has a precancerous lesion or polyp. In a specific embodiment, the complex is a population of complexes of alpha(2) macroglobulin bound to one or more different antigenic molecules, wherein the different antigenic molecules comprise at least one Specific antigens are antigenic molecules that are antigenic. In another specific embodiment, the complex is autologous to said patient. In another specific embodiment, said complex is purified prior to administration. In another specific embodiment, said body fluid is vascular fluid. In another specific embodiment, the vascular fluid is serum from blood. In another specific embodiment, the bodily fluid is extravascular ascites or cerebrospinal fluid.

In another embodiment of the present invention, there is provided a pharmaceutical composition comprising (a) a plurality of α(2) macroglobulin-antigen molecule complexes isolated from the body fluid of a mammal suffering from cancer or infectious disease, The plurality of complexes includes at least one of the following complexes: it includes α(2) macroglobulin and an antigenic molecule that exhibits antigenicity of a tumor or infectious agent-specific antigen; and (b) effectively Amount of pharmaceutical carrier. In a specific embodiment, the bodily fluid is serum, the vascular fluid. In another specific embodiment, the vascular fluid is serum from blood. In another specific embodiment, the bodily fluid is extravascular ascites or cerebrospinal fluid.

The present invention additionally provides a vaccine comprising: (a) a plurality of alpha(2) macroglobulin-antigen molecule complexes isolated from the body fluid of a mammal with precancerous lesions or polyps, the plurality of complexes comprising at least one complex comprising alpha(2) macroglobulin and an antigen molecule exhibiting antigenicity of a tumor or infectious agent specific antigen; and (b) an effective amount of a pharmaceutically acceptable carrier. In specific embodiments, a plurality of alpha(2) macroglobulin-antigen molecule complexes are purified.

In another embodiment of the present invention, a pharmaceutical composition is provided, which includes: (a) an antigenic molecule comprising α(2) macroglobulin and having tumor or infectious disease antigenicity; (b) a drug selected from the following: HSP-peptide complexes, antineoplastic agents, antibodies, cytokines, antiviral agents, antifungal agents, antibiotics, and chemotherapeutic agents; and (c) an effective amount of a pharmaceutically acceptable carrier. In a specific embodiment, the drug is a chemotherapeutic agent. In another specific embodiment, the alpha(2) macroglobulin-antigen molecule complex is purified.

The present invention additionally provides a method of increasing a complex comprising alpha(2) macroglobulin and an antigenic molecule in a body fluid of a mammal, said method comprising inducing tumor necrosis in said mammal. In a specific embodiment, the step of inducing tumor necrosis comprises administering a tumor necrosis drug to the mammal.

In another embodiment, the present invention provides a kit comprising, in one or more containers, a therapeutically or prophylactically effective amount of a covalent or non-covalent alpha(2) macroglobulin-antigen molecule in a pharmaceutically acceptable form Complex.

The present invention additionally provides a method of increasing the presence of a complex comprising alpha(2) macroglobulin and an antigenic molecule in a body fluid of a mammal, said method comprising inducing tumor necrosis in said mammal.

In another embodiment, the present invention provides a method for preparing a complex comprising α(2) macroglobulin and an antigenic molecule having precancerous lesion, tumor or infectious disease antigenicity, comprising: a) drawing serum from patients with lesions, tumors or infectious diseases; and b) recovering alpha(2) macroglobulin-antigen molecule complexes from said serum. In a specific embodiment, the method is for treating or preventing cancer, and said method additionally comprises the step of administering to said patient an amount of the recovered complex effective for treating or preventing cancer. In a specific embodiment of the method, the step of recovering the α(2) macroglobulin-antigen molecule complex from said serum comprises: a) contacting the solid phase comprising the α(2) macroglobulin binding molecule with the serum a time sufficient for the α(2) macroglobulin-antigen molecule complex to bind to the solid phase; b) removal of material not bound to the solid phase; and c) elution of the bound α(2) macrosphere from the solid phase Protein-antigen molecule complex. In a specific embodiment, the alpha(2) macroglobulin binding molecule is an alpha(2) macroglobulin specific antibody. In another specific embodiment, the alpha(2) macroglobulin binding molecule is a ligand binding fragment of CD91.

The present invention additionally provides a method for preparing a complex comprising α(2) macroglobulin and an antigen molecule having precancerous lesion, tumor or infectious disease antigenicity, comprising: a) and b) fractionating the serum to enrich for alpha(2) macroglobulin-antigen molecule complexes. In a specific embodiment, the step of contacting the serum with the reagent promotes the formation of a covalent bond between the alpha(2) macroglobulin and the antigen molecule. In another specific embodiment, the reagent is a protease, ammonia, methylamine or ethylamine.

In various embodiments, the bodily fluid of the methods of the invention is vascular fluid. In other embodiments, the vascular fluid is serum from blood. In other embodiments, the bodily fluid is extravascular ascites or cerebrospinal fluid.

As used herein, "alpha(2) macroglobulin" refers to alpha(2) macroglobulin polypeptides, and peptide-binding fragments, derivatives, mimetics and analogs thereof.

The term "a complex comprising α(2) macroglobulin and an antigen molecule" or "α(2) macroglobulin-antigen molecule complex" is used interchangeably herein to refer to an α( 2) Macroglobulin protein or polypeptide, or a peptide-binding fragment thereof. Binding can be covalent or non-covalent between the alpha(2) macroglobulin protein or polypeptide and the antigenic molecule.

As used herein, the term "body fluid" refers to any body fluid that can be used to prepare alpha(2) macroglobulin-antigen molecule complexes. Body fluids include vascular fluid and extravascular fluid. Extravascular fluids include, but are not limited to, ascites fluid, cerebral fluid, tissue lymph, colostrum, and semen. In a preferred embodiment, the bodily fluid is blood. In a particularly preferred embodiment, the bodily fluid is serum separated from blood.

4. Brief description of the drawings

Figure 1 represents a graph of tumor growth size (mm ³ ) during 18 days in a group of mice immunized with α(2) macroglobulin complex. Naive mice were immunized to test whether the complex would provide protection. A, mice immunized with PBS as a negative control, called PBS1; B, mice immunized with irradiated Meth A tumor cells; CF, α isolated from mouse serum with intradermal Meth A tumors (2) 3 groups of mice (labeled "Ascites 1-3") immunized with macroglobulin-antigen molecule complexes (5 mice per group); 3 groups of mice (labeled "bleach 1-3") (5 mice per group) immunized with α(2) macroglobulin-antigen molecule complex isolated from mouse serum; J, using tumor-free Mice immunized with α(2) macroglobulin-antigen molecule complexes isolated from mouse serum; K, mice immunized with MethA-derived gp96; L, mice immunized with MethA10 (derived from <10 kDa peptide) complexed with α2M; M, mice immunized with liver-derived gp96; N, mice immunized with gp96 complexed with MethA10 (referred to as gp96-MethA10); and 0, mice immunized with PBS (referred to as PBS2) immunized mice.

5. Detailed Description of the Invention

The present invention relates to compositions and methods of using alpha(2) macroglobulin-antigen molecule complexes isolated from serum of mammals in the treatment and prevention of cancer or infectious diseases. The present invention includes a method of using an α(2) macroglobulin-antigen molecule complex derived from the body fluid of a patient suffering from cancer or infectious disease for the treatment or prevention of said cancer or infectious disease. Such methods include autologous treatments for cancer and infectious diseases and vaccines for the prevention of cancer and infectious diseases. The present invention also includes a pharmaceutical composition comprising a plurality of α(2) macroglobulin-antigen molecule complexes, and the pharmaceutical composition includes α(2) macroglobulin together with a therapeutic agent for treating cancer or infectious disease - Antigen molecule complex. The present invention also includes methods of increasing the amount of alpha(2) macroglobulin-antigen molecule complexes in the serum of a mammal.

5.1. The pharmaceutical composition of the present invention

The present invention provides a pharmaceutical composition comprising an α(2) macroglobulin complex isolated from mammalian serum useful for the treatment and/or prevention of cancer or infectious disease, this composition may also optionally include Drugs for cancer and infectious diseases, such as, but not limited to, HSP-peptide complexes, antineoplastics, antibodies, cytokines, antivirals, antifungals, antibiotics or chemotherapeutics. The pharmaceutical compositions of the present invention may also include the drugs described in Section 5.4 for targeting cancer, Section 5.5 for targeting infectious diseases, and Section 5.3.3 for targeting combination therapy of cancer and infectious diseases. The pharmaceutical compositions of the present invention also include those pharmaceutically acceptable carriers described in Section 5.9. The pharmaceutical composition of the present invention is particularly effective for autologous therapy. Such pharmaceutical compositions may also include adjuvants and/or drugs that induce or increase the production of antigenic molecules. In certain embodiments, the drug causes tumor necrosis. Methods for the preparation of such pharmaceutical compositions are described herein. Also described herein are methods of increasing the amount of antigenic molecules and/or alpha(2) macroglobulin-antigen molecule complexes present in serum prior to or concurrently with isolating such complexes.

The pharmaceutical compositions and methods of the present invention include complexes of alpha(2) macroglobulin with populations of antigenic peptides. The pharmaceutical composition has been shown to induce an inflammatory response at the tumor site and can ultimately cause regression of the tumor burden in the cancer patient being treated. Compositions prepared by the methods of the present invention can enhance the immunity of an individual and elicit specific immunity against infectious agents or specific immunity against preneoplastic and neoplastic cells. These compositions have the ability to prevent the onset and development of infectious diseases and inhibit the growth and development of tumor cells.

In specific embodiments, the α2M complex is administered to a patient who is receiving another modality of therapy for the treatment of cancer or an infectious disease, where the patient may be non-responsive or refractory to treatment with the modality of therapy alone, i.e. At least some critical parts of the cancer cells or pathogens are not killed or their cell division is not inhibited. The effectiveness of the treatment modality can be tested using in vivo or in vitro assays using methods known in the art. The art accepted meaning of refractory is known in the cancer field. In one embodiment, wherein the cancer or infectious disease is refractory or unresponsive, respectively, the number of cancer cells or pathogens is not significantly reduced, or is increased. These patients undergoing treatment are those receiving chemotherapy or radiotherapy.

In certain embodiments, compositions of the invention include purified alpha(2) macroglobulin-antigen molecule complexes. In these embodiments, the alpha(2) macroglobulin-antigen molecule complex is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% pure.

5.2. Preparation of α(2) macroglobulin complex

Generally, the α(2) macroglobulin-antigen molecule complex of the present invention can be recovered and purified from mammalian serum by known methods, including ammonium sulfate precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose ( Phosphocellulose) chromatography, immunoaffinity chromatography, hydroxyapatite chromatography and lectin chromatography.

In one embodiment, the alpha(2) macroglobulin-antigen molecule complex is purified from serum using affinity purification techniques. Methods of chromatographic fractionation of proteins, such as affinity chromatography, are well known in the art. Briefly, affinity chromatography utilizes immobilized binding partners to specifically capture proteins during a binding reaction. Binding partner molecules for affinity capture assays may include, for example, antibodies to alpha(2) macroglobulin or other ligands such as the CD91 binding domain that specifically binds alpha(2) macroglobulin. Alternatively, filter binding assays utilize devices such as solid surfaces such as filters or columns to non-specifically retain proteins or protein complexes based on certain physical or chemical differences between complexes and unbound reactants. Affinity chromatography and/or filter-bound separation techniques can be used to isolate alpha(2) macroglobulin-antigen molecule complexes from serum or other bodily fluids as described herein.

In a particular embodiment of the invention, the α(2) macroglobulin-antigen molecule complex is isolated from serum according to the following: the serum is brought into contact with a solid phase, such as an agarose column, which contains the bound α(2) macroglobulin Partner, α(2) macroglobulin binding molecule. The serum is incubated on the solid phase for a period of time sufficient to allow the α(2) macroglobulin-antigen molecule complex to bind to the solid phase. Unbound material is then removed from the solid phase; and bound alpha(2) macroglobulin-antigen molecule complexes are eluted from the solid phase.

A binding partner for alpha(2) macroglobulin may be any molecule that specifically binds alpha(2) macroglobulin. In a preferred embodiment, the alpha(2) macroglobulin binding molecule is an antibody specific for alpha(2) macroglobulin. Preferably, the alpha(2) macroglobulin-specific antibody is a monoclonal antibody. In another preferred embodiment, the alpha(2) macroglobulin binding molecule is a ligand binding fragment of CD91.

The solid phase can be any surface or matrix such as, but not limited to, polycarbonate, polystyrene, polypropylene, polyethylene, glass, nitrocellulose, dextran, nylon, polyacrylamide, and agarose. Support structures may include beads; membranes; microparticles; the interior surfaces of reaction vessels such as microtiter plates, test tubes, or other reaction vessels.

In a preferred embodiment, the alpha(2) macroglobulin-antigen molecule complex is isolated from mouse serum. Serum was diluted 1:1 with 0.04M Tris pH 7.6, 0.15M NaCl, and the mixture was applied to a Sephacryl S300R (Sigma) column equilibrated and eluted with the same buffer. A 65ml column is used for approximately 10ml serum. α(2) macroglobulin positive fractions were detected by dot blot and the buffer was changed to 0.01 M sodium phosphate buffer at pH 7.5 using a PD-10 column. In an alternative method, 0.01 M sodium phosphate buffer at pH 7.5 can be used as the buffer in a 65 ml column to save the buffer exchange step. Fractions containing complexes were applied to a Concanavalin A Sepharose column. The bound complex was eluted with 0.2M methyl mannopyranoside or 5% methyl mannopyranoside, and used on a PD-10 column to change the buffer to 0.05M sodium acetate buffer at pH 6.0, and apply On a DEAE column equilibrated with 0.01 M sodium acetate buffer, pH 6.0. α(2) macroglobulin was eluted in pure form with 0.13M sodium acetate buffer and analyzed by SDS-PAGE and immunoblotting.

The above-described embodiments can be used to recover and purify the α(2) macroglobulin-antigen molecule complex from serum of mammalian cells such as blood containing the α(2) macroglobulin-antigen molecule complex of the present invention. This method can be used to perform the purification of medium to large quantities of α(2) macroglobulin-antigen molecule complexes. Most preferred are methods for the purification of alpha(2) macroglobulin-antigen molecule complexes that do not require lowering of pH or denaturing conditions. This method can be used to isolate α(2) macroglobulin from eukaryotic cells such as cancer cells, tissues, isolated cells; or immortal eukaryotic cell lines infected with intracellular or extracellular pathogens; or cells obtained from pathogen-infected patients - Antigen molecule complex.

When applying the above separation and purification methods, those skilled in the art will know that using too harsh reagents and conditions may dissociate the complex.

In another embodiment, the complex of an α2M polypeptide isolated from serum and an antigenic molecule is treated prior to its administration to a patient such that the α2M polypeptide is covalently complexed with the antigenic peptide. Methods for forming such covalent α2M-antigen molecule complexes are described in detail herein.

Normally, when α2M is mixed with a protease, cleavage of the "bait" region of α2M occurs, and the protease is "captured" by the thioester and undergoes a conformational change, allowing the α2M complex to bind to CD91. During proteolytic activation of α2M, non-proteolyzed ligands can be covalently bound to activated thioesters. Non-proteolyzed ligands can also be incorporated into activated α2M molecules by ammonia or methylamine in an inversion process using heated nucleophilic activation (Gron and Pizzo, 1998, Biochemistry, 37; 6009-6014). Such conditions from the α2M irregular capture peptide can be used to prepare the α2M-antigen complex of the present invention. This method of covalent binding has been previously described (Osada et al., 1987, supra; Osada et al., 1988, supra; Chu and Pizzo, 1993, supra; Chu et al., 1994, supra; Mitsuda et al., 1994, supra; et al., 1993, supra).

For example, in a specific embodiment, a serum fraction enriched for α2M polypeptide is combined with an antigenic molecule (which can be covalently bound or not covalently bound) mixed. Non-limiting examples of proteases that can be used include trypsin, porcine pancreatic elastase (PEP), human neutrophil elastase, cathepsin G, S. aureus V-8 protease trypsin, α-chymotrypsin, V8 Proteases, papain and proteinase K (see Ausubel et al., (eds.), in "Current Protocols in Molecular Biology", Greene Publishing Associates and Wiley Interscience, New York, 17.4.6-17.4.8). Preferred exemplary protocols for covalently complexing the α2M polypeptide and antigenic molecules present in serum are provided herein. In a specific embodiment, the following protocol can be used to increase the number of covalent complexes of α2M-antigen molecules. The serum fraction (100 μl-5 ml) including the α2M antigen molecule complex (1 μg-20 mg) is treated with methylamine or a protease such as trypsin (0.92 mg trypsin in about 500 ml PBS (phosphate buffered saline)). The mixture was then incubated at 37°C for 5-15 minutes. If trypsin was used, 500 ml of 4 mg/ml p-amidinophenylmethanesulfonyl fluoride (p-APMSF) was added to the solution to inhibit trypsin activity and incubated at 25°C for 2 hours. Optionally, free antigen molecules can be removed by passing through a gel permeation column.

Following complexation, the immunogenic α2M-antigen molecule complexes can optionally be assayed in vitro using, for example, the Mixed Lymphocyte Target Cell Assay (MLTC) as described below. Once the immunogenic complexes have been isolated, they can optionally be further characterized in animal models prior to their administration using the preferred dosing regimens and excipients discussed below.

In a preferred embodiment, the alpha(2) macroglobulin-antigen molecule complex is isolated from mouse serum. Serum was diluted 1:1 with 0.04M Tris pH 7.6, 0.15M NaCl, and the mixture was applied to a Sephacryl S300R (Sigma) column equilibrated and eluted with the same buffer. A 65ml column is used for approximately 10ml serum. α(2) macroglobulin positive fractions were detected by dot blot and the buffer was changed to 0.01 M sodium phosphate buffer at pH 7.5 using a PD-10 column. In an alternative method, 0.01 M sodium phosphate buffer at pH 7.5 can be used as the buffer in a 65 ml column to save the buffer exchange step. Fractions containing complexes were applied to a Concanavalin A Sepharose column. The bound complex was eluted with 0.2M methyl mannopyranoside or 5% methyl mannopyranoside and applied to a PD-10 column to change the buffer to 0.05M sodium acetate buffer at pH 6.0, and applied to a DEAE column equilibrated with 0.01 M sodium acetate buffer, pH 6.0. α(2) macroglobulin was purified by elution with 0.13M sodium acetate buffer and analyzed by SDS-PAGE and immunoblotting. Other methods of isolating complexes known in the art can also be used (Dubin et al., 1984, Biochem. International, 8(4): 589-596; Okubo et al., 1981, Biochem. et Biophys. Acta, 688: 257-267; Nieuwenhuizen et al., 1979, Biochem. et Biophysica. Acta, 580: 129-139).

5.3. Therapeutic applications

5.3.1. Methods of treating and preventing cancer

According to the present invention, a preferred method of treating and preventing cancer comprises isolating the [alpha]2M complex from an individual in need of treatment, and administering this complex to the patient as an autologous therapy. Depending on the route of administration, the α2M complex is isolated and purified and administered to the individual's own body (such as the treatment of a primary cancer or its metastases), or to other individuals in need of treatment for cancers of a similar histological type, or to patients due to family history or environment Risk factors for administration to individuals with increased risk of cancer. The cancers described in Section 5.4 can be treated or prevented by the pharmaceutical compositions and methods of the present invention.

For example, treatment with a composition of the invention comprising an α2M complex isolated from the patient's serum and prepared as above can be initiated at any time after surgery. In other embodiments, treatment can be initiated before or during surgery. However, if the patient is receiving chemotherapy, the α2M complex is usually administered after an interval of at least four weeks so that the immune system can recover. The treatment regimen may include weekly injections of a composition of the invention comprising the α2M complex dissolved in saline or other physiologically compatible solution. Change the injection route and site each time, for example, the first injection is subcutaneous in the left arm, the second injection is in the right arm, the third is in the left abdomen, the fourth is in the right abdomen, the fifth is in the left thigh, the sixth times in the right strand, and so on. The same injection site was repeated after one and more injection intervals. Alternatively, the injection is divided in two and half of the dose is administered at a different site on the same day. In general, the first four to six injections are given at weekly intervals. Two to fifty subsequent injections were given at two-week intervals, followed by monthly intervals.

Alternatively, the α2M complex isolated from patient serum can be used as a vaccine against cancer. This vaccine can be used to boost an immune response against tumor cells, cells bearing tumor antigens, by injection into a patient. Autologous α2M polypeptide-antigen molecule complexes are preferred. Vaccines of the invention may be administered before, during or after chemotherapy.

In a specific embodiment, the cancer is metastatic cancer. In another specific embodiment, the cancer is a tumor.

The immunotherapy effect of the α2M polypeptide-antigen molecule complex on the development of tumor diseases can be monitored by any method known to those skilled in the art, including but not limited to: a) measuring delayed hypersensitivity reactions, as an evaluation of cellular immunity; b ) measuring the activity of cytolytic T-lymphocytes in vitro; c) measuring the level of tumor-specific antigens such as carcinoembryonic antigen (CEA); d) measuring changes in tumor morphology and density using computed tomography (CT) scanning; e) using CT scans measure changes in tumor diameter; f) measure changes in levels of putative biomarkers of specific cancer risk in high-risk individuals; g) measure the percentage of tumor necrotic tissue before and after treatment; h) measure changes in tumor morphology using sonography i) measuring changes in tumor morphology using magnetic resonance imaging (MRI); j) measuring changes in tumor morphology using positron emission tomography (PET); and k) measuring changes in tumor morphology using ultrasound. Other techniques that may be used include scintigraphy and endoscopy.

The prophylactic effect of immunotherapy using α2M polypeptide-antigen molecule complexes can also be assessed by detecting the levels of putative biomarkers of specific cancer risk. For example, for individuals at increased risk of prostate cancer, serum prostate cancer is measured by the method described in Brawer et al., 1992, J. Urol., 147:841-845 and Catalona et al., 1993, JAMA, 270:948-958. Specific antigen (PSA); or measure CEA by methods known in the art for individuals at risk of colorectal cancer; and for individuals at increased risk of breast cancer by Schneider et al., 1982, Proc.Natl.Acad. The method described in Sci. USA, 79:3047-3051 measures 16-hydroxylation of estradiol. The above references are hereby incorporated by reference in their entireties.

In a specific embodiment, the prophylactic and therapeutic applications of the present invention are aimed at increasing the immune competence of cancer patients before, during or after surgery, and inducing tumor-specific immunity against cancer cells, with the aim of suppressing cancer and ultimately The clinical goal is total regression and eradication of cancer.

According to the invention, the composition of the invention comprises a complex of antigenic peptides derived from cytosolic proteins digested by antigenic cells and/or proteins derived from membranes. Alpha(2) macroglobulin is administered to patients with cancer. In one embodiment, "treatment" refers to the amelioration of cancer or at least one identifiable symptom thereof. In another embodiment, "treatment" refers to the improvement of at least one measurable physical parameter associated with cancer, not necessarily patient identifiable. In another embodiment, "treating" refers to inhibiting the progression of cancer, either by physically stabilizing identifiable symptoms, for example, or by stabilizing physical parameters, such as physiologically, or both.

In certain embodiments, a composition of the invention is administered to a subject as a prophylactic against such cancer. As used herein, "prevention" refers to reducing the risk of developing the indicated cancer. In one mode of embodiment, a composition of the invention is administered as a prophylactic measure to a subject with a genetic predisposition to cancer. In another mode of embodiment, the compositions of the present invention are administered as a prophylactic measure to subjects exposed to carcinogens including, but not limited to, chemicals and/or radiation.

For example, in certain embodiments, the composition of the invention inhibits or reduces the growth of tumor cells by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10%.

The composition prepared by the method of the present invention includes a complex obtained by complexing α-2-macroglobulin and antigenic peptide populations. The compositions have been shown to induce a reduction in tumor size and may ultimately cause regression of tumor burden in treated cancer patients. The composition prepared by the method of the invention can increase the immunocompetence of the subject and induce specific immunity to preneoplastic and neoplastic cells. These compositions have the ability to prevent the growth and development of tumor cells.

In certain embodiments of the methods of the invention, the alpha(2) macroglobulin-antigen molecule complex is administered to a patient with a family history of cancer, or to a subject at risk of developing cancer due to environmental factors. In other embodiments, the method of the present invention is used to treat patients with precancerous lesions or polyps as a prophylactic treatment, based on Applicants' discovery of isolating complexes from serum that is effective against potential cancer formation. In other embodiments, patients are treated using the methods of the invention based on the discovery that undetectable early stage cancers lead to the appearance of alpha(2) macroglobulin-antigen molecule complexes that can be used to prevent cancer progression. In other embodiments, patients in whom cancer has not been detected are treated using the methods of the invention as a prophylactic measure.

5.3.2. Methods of treatment and prevention of infectious diseases

For the treatment and prevention of infectious diseases, the α2M-antigen molecule complex is isolated from the body fluid of mammals suffering from infectious diseases and used as a vaccine against infectious diseases. As understood by those skilled in the art, the protocols described herein can be used to isolate the α2M polypeptide-antigen molecule complex from any bodily fluid in which the α(2) macroglobulin-antigen molecule complex is present. For example, cells can be infected by the infectious agent itself. In one embodiment, the α2M-antigen molecule complex can be isolated from serum comprising antigens derived from cells infected with an infectious agent. In another embodiment, the α2M-antigen molecule complexes of the invention can be isolated from serum comprising antigens derived from intracellular or extracellular pathogens. The present invention is not limited to the treatment or prevention of infectious diseases caused by intracellular or extracellular pathogens. Many medically relevant microorganisms have been described extensively in the literature, see for example G.L. Mandell, J.E. Bennet, and R. Dolin, Mandell, Douglas, and Bennetfs Principles and Practice of Infectious Diseases, Churchill Livingstone, Philadelphia, Pennsylvania 2000, which was It is incorporated herein by reference in its entirety.

A preferred method of treating or preventing an infectious disease comprises isolating the alpha(2) macroglobulin complex from the patient's serum and administering the isolated complex to the patient when the patient is in need of treatment. Complexes of α2M polypeptides covalently or non-covalently bound to antigenic molecules of infectious agents are purified from serum.

In a preferred aspect of the present invention, the purified α2M-antigen molecule complex vaccine can be of particular use in the treatment of human diseases caused by intracellular or extracellular pathogens. However, it should be understood that vaccines developed using the principles described herein can be used to treat diseases similarly caused by intracellular or extracellular pathogens in other mammals, such as farm animals, including: cattle, horses, sheep, goats and pigs; and household pets, including: cats and dogs.

Vaccines may be prepared to promote an immune response to any of the infectious disease pathogens described in Section 5.5. The immunotherapeutic effect of the modified α2M polypeptide-antigen molecule complex on the development of infectious diseases can be monitored by any method known to those skilled in the art.

The composition prepared by the method of the invention comprises a complex of alpha(2) macroglobulin and a population of antigenic peptides. The composition prepared by the method of the present invention can increase the immunity of the subject and cause specific immunity to infectious agents. These compositions have the ability to prevent the onset and progression of infectious diseases.

In one embodiment, "treating" refers to the amelioration of an infectious disease or at least one identifiable symptom thereof. In another embodiment, "treating" refers to improvement of at least one measurable physical parameter associated with an infectious disease, but not necessarily identifiable by the subject. In another embodiment, "treating" refers to inhibiting the development of an infectious disease, either by physically stabilizing an identifiable symptom, for example, or by stabilizing a physical parameter, such as physiologically, or both.

The composition prepared by the method of the present invention includes a complex of alpha-2-macroglobulin and a population of antigenic peptides. The composition prepared by the method of the present invention can increase the immunity of the subject and cause specific immunity to infectious agents. These compositions have the ability to prevent the onset and progression of infectious diseases.

In certain embodiments, a composition of the invention is administered to a subject as a prophylactic against such an infectious disease. As used herein, "prevention" refers to reducing the risk of acquiring the indicated infectious disease. In another mode of embodiment, the composition of the invention is administered as a prophylactic measure to a subject exposed to an infectious agent of an infectious disease.

For example, in certain embodiments, the composition of the invention inhibits or reduces the growth of an infectious agent by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, At least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10%.

5.3.3. Combined therapy for the treatment and prevention of cancer and infectious diseases

The compositions and methods of the invention using alpha(2) macroglobulin complexes may be administered with other therapeutic agents as a combination therapy regimen. Other therapeutic agents include, but are not limited to, heat shock protein-peptide complexes, antineoplastic agents, antibodies, cytokines, antiviral agents, antifungal agents, antibiotics, and adjuvants.

Combination therapy refers to the administration of the α2M complex of the present invention together with another mode of prevention or treatment of cancer and/or infectious disease. In the treatment and prevention of cancer, combination therapy refers to the administration of the α2M complex of the present invention together with another mode of prevention or treatment of cancer. Administration of the complexes of the invention can enhance the effect of anticancer drugs and vice versa. In the treatment and prevention of infectious diseases, the administration of the complexes of the present invention can enhance the effect of anti-infective drugs, and vice versa. Preferably, the pattern of this additional form is a non-α2M-based pattern, ie the pattern does not include α2M as a component. This approach is often referred to as combination therapy, adjuvant therapy, or combination therapy (the terms are used interchangeably herein). For combination therapy, additive potency or additive therapeutic effects may be observed. Synergistic results where the therapeutic efficacy is greater than additive may also be expected. The use of combination therapy may also provide a better therapeutic profile than administration of the treatment modality alone, or administration of the α2M complex alone. Additive or synergistic effects may reduce the dose and/or dosing frequency of either or both modes or avoid undesired or adverse effects.

In various specific embodiments, the combination therapy comprises administering the α2M-antigen molecule complex to a subject treated with a therapeutic modality, wherein the therapeutic modality administered alone is clinically insufficient to treat the subject such that the subject requires additional effective treatment, e.g., Subjects who do not respond to the treatment modality without administration of the α2M complex. Included in this embodiment are methods comprising administering an α2M complex to a subject undergoing a treatment modality in which the subject responds to treatment but experiences side effects, relapses, develops resistance, and the like. Such subjects may be non-responsive or refractory to individual treatment modalities, i.e. at least some critical portion of the cancer cells are not killed or their cell division is not inhibited or at least some critical portion of the pathogen or infectious agent is not destroyed kill. Methods of the invention administering an a2M complex to a subject refractory to a treatment modality alone may also improve the efficacy of the treatment modality when administered as contemplated by the methods of the invention. Efficacy assays for treatment modalities can be assayed in vivo or in vitro using methods known in the art.

The art accepted meaning of refractory is well known in the context of cancer and infectious disease. In one embodiment, the cancer is refractory or non-responsive, wherein the number of cancer cells is not significantly reduced, or is increased, respectively. In another embodiment, the infectious disease is refractory or unresponsive, respectively, wherein the number of pathogens is not significantly reduced, or is increased.

According to the invention, the complexes of the invention can be used in combination with a number of different types of treatment modalities. Some of these patterns are specific to specific types of cancer or infectious diseases, which are discussed in Sections 5.4 and 5.5, respectively. Many other modes have an impact on immune system function and are generally applicable to tumors and infectious diseases.

In one embodiment, the complexes of the invention are used in combination with one or more biological response modifiers for the treatment of cancer or infectious diseases. One class of biological response modifiers are cytokines. In one such embodiment, the cytokine is administered to the subject receiving the α2M complex. In various embodiments, one or more cytokines selected from IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7. IL-8, IL-9, IL-10, IL-11, IL-12, IFNα, IFNβ, IFNγ, TNFα, TNFβ, G-CSF, GM-CSF, TGFβ, IL-15, IL-18, GM-CSF, INF-γ, INF-α, SLC, Endothelial Monocyte Activation Protein-2 (EMAP2), MIP-3α, MIP-3β or MHC genes such as HLA-B7. Additionally, other exemplary cytokines include other members of the TNF family including, but not limited to, TNF-α-related apoptosis-inducing ligand (TRAIL), TNF-α-related activation-inducing cytokine (TRANCE), TNF-α-related apoptosis-inducing weak inducer (TWEAK), CD40 ligand (CD40L), lymphocytotoxin alpha (LT-α), lymphocytotoxin beta (LT-β), OX40 ligand (OX40L), Fas ligand (FasL), CD27 ligand (CD27L), CD30 ligand (CD30L), 41BB ligand (41BBL), APRIL, LIGHT, TL1, TNFSF16, TNFSF17 and AITR-L, or functional parts thereof. For a general review of the TNF family see, eg, Kwon et al., 1999, Curr. Opin. Immunol., 11:340-345. Preferably, the α2M complex is administered prior to the treatment modality. In a specific embodiment, a complex of the invention is administered to a patient undergoing treatment for cancer with cyclophosphamide in combination with IL-12.

In another embodiment, the complexes of the invention are used in combination with one or more biological response modifiers that are agonists or antagonists of various ligands, receptors and signal transduction molecules agent. For example, biological response modifiers include, but are not limited to, agonists of toll-like receptors (TLR-2, TLR-7, TLR-8, and TLR-9); LPS; 41BB, OX40, ICOS, and CD40 ; Antagonist of Fas ligand, PD1, and CTLA-4. These agonists and antagonists can be antibodies, antibody fragments, peptides, peptidomimetic compounds and polysaccharides.

In another embodiment, the complexes of the invention are used in combination with one or more biological response modifiers that are immunostimulatory nucleic acids. Many of these nucleic acids are oligonucleotides that include unmethylated CpG motifs, promote mitosis in vertebrate lymphocytes, and are known to enhance immune responses. See Woolridge et al., 1997, Blood, 89:2994-2998. Such oligonucleotides are described in International Patent Publications WO 01/22972, WO 01/51083, WO 98/40100 and WO 99/61056, all of which are incorporated herein by reference in their entirety, as well as in U.S. Patent Publication 6,207,646, 6,194,388, 6,218,371, 6,239,116, 6,429,199, and 6,406,705, the disclosures of which are incorporated herein by reference in their entirety. Other classes of immunostimulatory oligonucleotides such as phosphorothioate oligodeoxyribonucleotides containing YpG-motifs and CpR-motifs have been described by Kandimalla et al. in "Effect of Chemical Modifications of cytosine and Guanine ina CpG - Motif of Oligonucleotides: Structure-Immunostimulatory Activity Relationships.", described in Bioorganic & Medicinal Chemistry, 9: 807-813 (2001), the entirety of which is incorporated herein by reference. Also included are CpG dinucleotide-free immunostimulatory oligonucleotides that enhance antibody responses when administered by the mucosal route, including in low doses, or in high doses by the parenteral route, often interacting with CpG nucleic acids Again, however, the response was Th2-biased (IgG1 >> IgG2a). See U.S. Patent Publication 20010044416 A1, which is incorporated herein by reference in its entirety. Methods for detecting the activity of immunostimulatory oligonucleotides can be performed as described in the aforementioned patents and publications. In addition, immunostimulatory oligonucleotides can be modified within the phosphate backbone, sugars, nucleotide bases, and nucleotide linkages to modulate activity. Such modifications are known to those skilled in the art.

In another embodiment, the complexes of the invention are used in combination with one or more adjuvants. Adjuvants can be applied alone or present in the composition mixed with the complex according to the invention. Systemic adjuvants are adjuvants which can be administered parenterally. Systemic adjuvants include those that produce a depot effect, those that stimulate the immune system, and those that are useful for both. As used herein, a depot-generating adjuvant is an adjuvant that causes the slow release of the antigen in the body, thereby prolonging the exposure of immune cells to the antigen. Such adjuvants include, but are not limited to, alum (such as aluminum hydroxide, aluminum phosphate); or emulsion-based formulations, including mineral oil, non-mineral oil, water-in-oil or oil-water-oil emulsions, oil-in-water emulsions such as Montanide Seppic ISA series of agents (such as Montanide ISA720, AirLiquide, Paris, France); MF-59 (squalene-in-water emulsion stabilized with Span 85 and Tween 80, Chiron Corporation, Emeryville, Calif.); and PROVAX ( Oil-in-water emulsion with stabilizing detergent and micelle former, IDEC, Pharmaceuticals Corporation, San Diego, Calif.).

Other adjuvants stimulate the immune system, for example causing immune cells to produce and secrete cytokines or IgG. Such auxiliaries include, but are not limited to, immunostimulatory nucleic acids, such as CpG oligonucleotides; saponins purified from the bark of Q. soapwort, such as QS21; poly[bis(carboxylated phenoxy)phosphazenes] (PCPP polymer; Virus Research Institute, USA); derivatives of lipopolysaccharide (LPS) such as monophosphoryl lipid A (MPL, Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP , Ribi) and threonyl-muramoyl dipeptide (t-MDP, Ribi); OM-174 (glucosamine disaccharide related to lipid A, OM Pharma SA, Meyrin, Switzerland); and Leishman Protozoal elongation factor (purified Leishmania protein, Corixa Corporation, Seattle, Wash.). In a preferred embodiment, complexes of the invention are used in combination with QS21 and another immunostimulatory nucleic acid such as but not limited to a CG oligonucleotide.

Other systemic adjuvants are those that produce a depot effect and stimulate the immune system. These compounds are compounds that combine the recognized functions of the above-mentioned systemic adjuvants. Such adjuvants include, but are not limited to, ISCOM (Immunostimulatory Complex, which contains mixed saponins, lipids and forms virus-sized particles with pores that can accommodate antigens, CSL, Melbourne, Australia); SB-AS2 (SmithKline Beecham Adjuvant System #2, which is an oil-in-water emulsion containing MPL and QS21, SmithKline Beecham Biologicals [SBB], Rixensart, Belgium); SB-AS4 (SmithKline Beecham Adjuvant System #4, which contains Alum and MPL, SBB, Belgium); non-ionic block copolymers that form micelles such as CRL 1005 (these comprise linear hydrophobic polyoxypropylene with side chains formed from polyoxyethylene chains, Vaxcel, Inc., Norcross, Ga.); and Syntex Adjuvant Formulation (SAF, oil-in-water emulsion containing Tween 80 and a nonionic block copolymer, Syntex Chemicals, Inc., Boulder, Colo.).

A mucosal adjuvant usable according to the invention is an adjuvant capable of inducing a mucosal immune response in the subject when administered to a mucosal surface together with the complex of the invention. Mucosal aids include but are not limited to CpG nucleic acids (such as PCT published patent application WO 99/61056), bacterial toxins: such as cholera toxin (CT), CT derivatives include but are not limited to CTB subunits (CTB) (Wu et al., 1998; Tochikubo et al., 1998); CTD53 (Val to Asp) (Fontana et al., 1995); CTK97 (Val to Lys) (Fontana et al., 1995); CTK104 (Tyr to Lys) (Fontana et al., 1995) ; CTD 53/K63 (Val to Asp, Ser to Lys) (Fontana et al., 1995); CTH54 (Arg to His) (Fontana et al., 1995); CTN107 (His to Asn) (Fontana et al., 1995); CTE114 (Ser to Glu) (Fontana et al., 1995); CTE112K (Glu to Lys) (Yamamoto et al., 1997a); CTS61F (Ser to Phe) (Yamamoto et al., 1997a, 1997b); CTS106 (Pro to Lys) (Douce et al., 1997, Fontana et al., 1995); and CTK63 (Ser to Lys) (Douce et al., 1997, Fontana et al., 1995), Zonula occludentstoxin, zot, Escherichia coli heat Stable enterotoxin, unstable toxin (LT), LT derivatives including but not limited to LTB subunit (LTB) (Verweij et al., 1998); LT7K (Arg to Lys) (Komase et al., 1998, Douce et al., 1995 ); LT61F (Ser to Phe) (Komase et al., 1998); LT112K (Glu to Lys) (Komase et al., 1998); LT118E (Gly to Glu) (Komase et al., 1998); LT146E (Arg to Glu) (Komase et al., 1998); LT192G (Arg to Gly) (Komase et al., 1998); LTK63 (Ser to Lys) (Marchetti et al., 1998, Douce et al., 1997, 1998, DiTommaso et al., 1996); and LTR72 (Ala to Arg) (Giuliani et al., 1998), pertussis toxin, PT. (Lycke et al., 1992, Spangler BD, 1992, Freytag and Clemments, 1999, Roberts et al., 1995, Wilson et al., 1995) including PT-9K/129G (Roberts et al., 1995, Cropley et al., 1995); toxin derivatives (see below) (Holmgren et al., 1993, Verweij et al., 1998, Rappuoli et al., 1995, Freytag and Clements, 1999); Lipid A derivatives (such as monophosphono lipid A, MPL) (Sasaki et al., 1998, Vancott et al., 1998; Muramyl dipeptide (MDP) derivatives (Fukushima et al., 1996, Ogawa et al., 1989, Michalek et al., 1983, Morisaki et al., 1983); bacterial outer membrane proteins (such as the outer surface protein A (OspA) lipoprotein of Borrelia Burgdorferi, Neisseria meningitidis (Neisseria meningitidis outer membrane protein) (Marinaro et al., 1999, Van de Verg et al., 1996); oil-in-water emulsions (such as MF59) (Barchfield et al., 1999, Verschoor et al., 1999, O'Hagan, 1998); aluminum salts (Isaka et al., 1998, 1999); and saponins (such as QS21) Aquila Biopharmaceuticals, Inc., Worster, Me.) (Sasaki et al., 1998, MacNeal et al., 1998), ISCOMs, MF- 59 (squalene-in-water emulsion stabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville, Calif.); Seppic ISA series of Montanide additives (such as Montanide ISA 720; AirLiquide, Paris, France); PROVAX (contains Stabilized oil-in-water emulsions of detergents and micellar excipients; IDEC Pharmaceuticals Corporation, San Diego, Calif.); Syntext Adjuvant Formulation (SAF; Syntex Chemicals, Inc., Boulder, Colo.); poly[di(carboxylated phenoxy)phosphazene] (PCPP polymer; Virus Research Institute, USA) and Leishmania elongation factor (Corixa Corporation, Seattle, Wash.).

In another embodiment, the complexes of the invention are administered in conjunction with one or more immunotherapeutic agents such as antibodies and vaccines. In a preferred embodiment, the antibody has an in vivo therapeutic and/or prophylactic effect against cancer and/or infectious diseases. Examples of therapeutic and prophylactic antibodies include, but are not limited to, MDX-010 (Medarex, NJ), which is a humanized anti-CTLA-4 antibody currently used clinically for the treatment of prostate cancer; SYNAGIS® (MedImmune, MD), which is used for Humanized anti-respiratory syncytial virus (RSV) monoclonal antibody for the treatment of patients with RSV infection; HERCEPTIN(R) (Trastuzumab) (Genentech, CA), which is a humanized anti-HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer Antibody. Other examples are humanized anti-CD18F(ab') ₂ (Genentech); CDP860, which is a humanized anti-CD18F(ab') ₂ (Celltech, UK); PR0542, which is an anti-HIV gp120 antibody fused to CD4 (Progenics Ostavir, which is a human anti-hepatitis B virus antibody (Protein Design Lab/Novartis); PROTOVIR ^TM , which is a humanized anti-CMV IgG1 antibody (Protein Design Lab/Novartis); MAK-195 (SEGARD), which is a mouse anti-TNF-αF(ab') ₂ (Knoll Pharma/BASF); IC14, which is an anti-CD14 antibody (ICOS Pharm); a humanized anti-VEGF IgG1 antibody (Genentech); OVAREX ^™ , which is a mouse anti-CA 125 Antibody (Altarex); PANOREX ^™ , which is a mouse anti-17-IA cell surface antigen IgG2a antibody (GlaxoWellcome/Centocor); BEC2, which is a mouse anti-idiotype (GD3 epitope) IgG antibody (ImClone System); IMC-C225, It is a chimeric anti-EGFR IgG antibody (ImCloneSystem); VITAXIN ^™ , which is a humanized anti-αVβ3 integrin antibody (Applied Molecular Evolution/MedImmune); Campath 1H/LDP-03, which is a humanized anti-CD52 IgG1 antibody ( Leukosite); Smart M195, which is a humanized anti-CD33 IgG antibody (Protein Design Lab/Kanebo); RITUXAN ^™ , which is a chimeric anti-CD20 IgG1 antibody (IDEC Pharm/Genentech, Roche/Zettyaku); LYMPHOCIDE ^™ , which is human Anti-CD22 IgG antibody (Immunomedics); Smart ID10, which is a humanized anti-HLA antibody (Protein Design Lab); ONCOLYM ^TM (Lym-1), which is a radiolabeled mouse anti-HLA DIAGNOSTIC REAGENT antibody (Techniclone); ABX-IL8 , is a human anti-IL8 antibody (Abgenix); anti-CD11a, is a humanized IgG1 antibody (Genentech/Xoma); ICM3, is a humanized anti-ICAM3 antibody (ICOS Pharm); IDEC-114 is a primatized anti-CD80 antibody (IDECPharm/ Mitsubishi); ZEVALIN ^™ , a radiolabeled mouse anti-CD20 antibody (IDEC/Schering AG); IDEC-131, a humanized anti-CD40L antibody (IDEC/Eisai); IDEC-151, a primatized anti-CD4 antibody (IDEC ); IDEC-152 is a primatized anti-CD23 antibody (IDEC/Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is a humanized anti-complement factor 5 (C5) antibody (Alexion Pharm); D2E7 is a humanized anti-TNF-α antibody (CAT/BASF); CDP870 is a humanized anti-TNF-α Fab fragment (Celltech); IDEC-151 is a primatized anti-CD4 IgG1 antibody (IDEC Pharm/SmithKline Beecham ); MDX-CD4 is human anti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571 is humanized anti-TNF-α IgG4 antibody (Celltech); LDP-02 is humanized anti-α4β7 antibody (LeukoSite/Genentech); OrthoClone OKT4A is human Anti-CD4 IgG antibody (Ortho Biotech); ANTOVA ^TM , humanized anti-CD40L IgG antibody (Biogen); ANTEGREN ^TM , humanized anti-VLA-4 IgG antibody (Elan); MDX-33, human anti-CD64 (FcγR) antibody ( Medarex/Centeon); SCH55700 is a humanized anti-IL-5 IgG4 antibody (Celltech/Schering); SB-240563 and SB-240683 are humanized anti-IL-5 and IL-4 antibodies, respectively (SmithKline Beecham); rhuMab-E25 is a human Anti-IgE IgG1 antibody (Genentech/Norvartis/Tanox Biosystems); ABX-CBL is mouse anti-CD-147 IgM antibody (Abgenix); BTI-322 is rat anti-CD2 IgG antibody (Medimmune/Bio Transplant); Orthoclone/OKT3 is a mouse anti-CD3 IgG2a antibody (ortho Biotech); SIMULECT ^TM is a chimeric anti-CD25 IgG1 antibody (Novartis Pharm); LDP-01 is a humanized anti-β ₂ -integrin IgG antibody (LeukoSite); anti-LFA-1 is Murine anti-CD18F(ab') ₂ (Pasteur-Merieux/Immunotech); CAT-152 is a human anti-TGF- _β2 antibody (Cambridge AbTech); and Corsevin M is a chimeric anti-Factor VII antibody (Centocor). In a preferred embodiment, the complexes of the invention are administered in combination with anti-CTLA4 antibodies. In another preferred embodiment, the complexes of the invention are administered in combination with an anti-41BB antibody. The immunocompetent drugs listed above, as well as any other immunocompetent drugs, may be administered according to any regimen known to those skilled in the art, including regimens recommended by suppliers of the immunocompetent drug.

In the treatment or prevention of infectious diseases, the α(2) macroglobulin-antigen molecule complex is administered to patients who are at risk of developing infectious diseases due to environmental or familial factors. In other embodiments, the method of the present invention is used as a prophylactic treatment of patients with early symptoms of an infectious disease, based on the concept that the complex can be isolated from serum that is effective against possible development of the infectious disease. In other embodiments, the methods of the present invention are used to treat patients with no symptoms of infectious disease based on the belief that early infection with an undetectable pathogen may result in the appearance of an α(2) macroglobulin-antigen molecule complex that can be used to prevent the development of an infectious disease. patient. In other embodiments, healthy patients are treated prophylactically using the methods of the invention, based on the notion that the patient may have an undetected infectious disease.

To determine the effectiveness of the compositions and methods of the present invention, one skilled in the art can use various standard assays for detection of infection, including but not limited to antibody titration assays, measurement of viral load using PCR techniques such as detection of virus-specific nucleic acids, blood cell analysis, plaque detection, phage analysis or culture of bacterial samples. Such methods for detecting and measuring levels of infection are well known to those skilled in the art.

In another embodiment, in the context of methods of treatment and prevention of cancer, each of the above methods comprises administering an α2M complex, preferably a purified α2M complex, to a subject receiving a combination of treatment modalities for the treatment of cancer. Preferably, the α2M complex is bound to an antigen molecule exhibiting antigenicity for the cancer type being treated.

5.4. Target cancers

In one embodiment, the methods of the invention and combination therapies thereof comprise administering compositions of the invention comprising one or more modalities for adjuvant use in the prevention or treatment of cancer, including but not limited to chemotherapeutic agents , immunotherapeutics, anti-angiogenic agents, cytokines, hormones, antibodies, polynucleotides, radiation and photodynamic therapeutics. In specific embodiments, combination therapy involving administration of a pharmaceutical composition of the invention is useful for preventing cancer recurrence, inhibiting cancer metastasis, or inhibiting cancer or metastatic cancer growth and/or spread.

Types of cancer that may be treated or prevented by the methods and pharmaceutical compositions of the present invention include, but are not limited to, sarcomas and carcinomas in humans, such as fibrosarcoma; myxosarcoma; liposarcoma; chondrosarcoma; osteosarcoma; chordoma; angiosarcoma; endothelial Sarcomas; lymphangiosarcoma; lymphangioendothelio sarcoma; synovial tumors; mesothelioma; Ewing's sarcoma; leiomyosarcoma; Stem cell carcinoma; basal cell carcinoma; adenocarcinoma; sweat gland carcinoma; sebaceous gland carcinoma; papillary carcinoma; papillary adenocarcinoma; cystadenocarcinoma; medullary carcinoma; bronchial carcinoma; renal cell carcinoma; ; seminoma; embryonal carcinoma; Wilms' tumor; cervical cancer; testicular cancer; lung cancer; small cell lung cancer; bladder cancer; epithelial carcinoma; glioma; astrocytoma; medulloblastoma Cytoma; Craniopharyngioma; Ependymoma; Pinealoma; Hemangioblastoma; Acoustic Neuroma; Oligodendroglioma; Meningioma; Melanoma; Neuroblastoma; Retinoblastoma ; Leukemias such as acute lymphoblastic leukemia and acute myeloid leukemia (myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia and erythroleukemia); chronic leukemia (chronic myeloid leukemia cellular (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström's macrosphere proteinemia, and heavy chain disease.

Immunotherapy provided by the present invention is desirable for cancer patients for a number of reasons, first, if the cancer patient is immunosuppressed, the immunosuppression may be exacerbated by anesthetic surgery and subsequent chemotherapy. Appropriate immunotherapy during the preoperative period can prevent or reverse this immunosuppression. This may lead to fewer infectious complications and faster wound healing. Second, tumor volume is minimal after surgery, so immunotherapy is likely to be effective in this setting. Third, tumor cells may be released into the circulation at the time of surgery, at which point they can be removed with effective immunotherapy.

The prevention and treatment method of the present invention is aimed at increasing the immune ability of cancer patients before, during or after surgery, and inducing tumor-specific immunity against cancer cells, the purpose of which is to suppress cancer, and the ultimate clinical purpose is to suppress cancer. Total degradation and eradication. The methods and pharmaceutical compositions of the invention are also useful in individuals who are at increased risk for particular types of cancer, eg, due to family history or environmental risk factors.

In various embodiments, one or more anticancer drugs are administered in addition to the complexes of the invention to treat a cancer patient. An anticancer drug is any molecule or compound that helps in the treatment of tumors or cancer. Examples of anticancer drugs for use in the methods of the invention include, but are not limited to: acivicin; aclarithromycin; alcodazole hydrochloride; acronine; ; Methazine; Ampomycin; Amantrene Acetate; Azomycin; batimastat; benzotepa; bicalutamide; bisantrene hydrochloride; Bropirimine; Busulfan; Actinomycin C; Captestosterone; Carbamide; Carbetim; Carboplatin; Carmustine; Carrubicin Hydrochloride; Carzelexine; Ge; Chlorambucil; Ciromycin; Cisplatin; Cladribine; Clinator Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexomaplatin; Dezaguanine; Dezaguanine Mesylate; Deacroquinone; Docetaxel; Adriamycin; Adriamycin Hydrochloride; Droloxifene; Droloxifene Citrate Xifen; drotandrosterone propionate; azomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; eprabamate; Star; Ebrozole; Esorubicin Hydrochloride; Estramustine; Estramustine Sodium Phosphate; Etanidazole; Etoposide; Fazarabine; Fenretinide; 5-fluorouracil deoxynucleoside; fludarabine phosphate; fluorouracil; flucitabine; Bixing; Ifosfamide; Imofosine; Interleukin II (including recombinant interleukin II, or rIL2); Interferon α-2a; Interferon α-2b; Interferon α-n1; Interferon α-n3; Interferon Interferon β-Ia; Interferon γ-Ib; Isoproplatin; Irinotecan Hydrochloride; Somatolin; Letrozole; Leuprolide Acetate; Liarazole Hydrochloride; Loxoanthrone hydrochloride; Masoprofen; Maytansine; Nitrogen mustard hydrochloride; Megestrol acetate; Melengestrol acetate; Melphalan; Menolil; Mercaptopurine; Methotrexate; Methotrexate sodium; Chlorobenzene Amidine; Methotepa; Mibulidomide; Mitocarcin; Mitoerythrin; Phenolic acid; Nocodazole; Nogamycin; Omaplatin; Oxysulam; Paclitaxel; Pegaspargase; Pelithromycin; Nemustine; Pelomycin sulfate; Pephosfamide; Piper Bromane; Piposufan; Pyroxantrone Hydrochloride; Pleucamycin; Plometane; Porfimer Sodium; Amycin; Pyrazofuramin; Liboadenosine; Roglutamine; Safingo; Safingo hydrochloride; Semustine; Octtrazine; Sodium sparphosphate; Spamycin; Spiral germanium hydrochloride ; Spiromustine; Spiroplatinum; Streptomycin; Streptozotocin; ; Teniposide; Tiroxiron; Testolactone; Athiomatopurine; 2-Amino-6-Mercaptopurine; Thiotepa; Thiazofurine; Trimethrene; Trimethrexate; Trimethrexate Glucuronate; Triptorelin; Tobutazole Hydrochloride; Fen; vinblastine sulfate; vincristine sulfate; vinblastine; vinblastine sulfate; vinblastine sulfate; vinblastine sulfate; Vorozole; Zenilatine; Neocarcinstatin; Zorubicin hydrochloride.

Other anticancer drugs that may be used include, but are not limited to: 20-epi-1,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarithromycin; acylfulvene; adecypenol; Interleukin-2; ALL-TK antagonist; hexamethazine; amustine; amidox; amistine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide ; Angiogenesis inhibitor; Antagonist D; Antagonist G; Anrelix; Anti-dorsification morphogenic protein-1; Anti-androgen prostate cancer (prostatic carcinoma); Anti-estrogen; Anti-tumor formation; Nucleotides; Glycine Effideconine; Apoptotic Gene Regulators; Apoptotic Control Agents; Apurinic Nucleic Acids; ara-CDP-DL-PTBA; Axinastatin (axinastatin) 1; asinastatin 2; asinastatin 3; azasetron; azatoxin; diazotyrosine; baccatin III derivatives; Batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; β-lactam derivatives; β-alethine; β-clarithromycin B; betulinic acid; bFGF inhibitors; bicalutamide; bisantrene; Nephad; bistratene A; bizelexin; breflate; Canarypox IL-2; Capecitabine; Formamide-amino-triazole; Carboxyaminoimidazole; CaRest M3; CARN 700; Cartilage-derived inhibitors; castanospermine; cecropin B; cetrorelix; chlorlns; chloroquinoxaline; sulfonamide; cicaprost; cis-porphyrin; cladribine; clomiphene analogues; Colin A; Colinmycin B; Combretastatin A4; Combretastatin analogs; conagenin; Mediterranean sponge 816; ; cyclopentanthraquinone; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; hexestrol phosphate; Methasone; dexifosfamide; dexrazoxane; dexverapamil; decacrine; daidaining B; didox; diethyl norspermine; dihydro-5-azacytosine; dioxamycin; diphenylspiromustine; docetaxel; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen ; Ecomustine; Edefosine; Edenolumab; Eflornithine; Elemene; Estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride ); flavopiridol; fluclastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; Rick; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylenediethylamide; hypericin; ibandronic acid; idarubicin; edoxifene; ketone; imofosine; ilomastat; imidazoacridone; imiquimod; immunostimulatory peptide; insulin-like growth factor 1 receptor inhibitor; interferon agonist; interferon; interleukin; iodobenzyl Guanidine; iodoxorubicin; 4-sweet potato picrol; iropra; isoladine; ibengazole; isohomohalicondrin B; ; extended-release lanreotide; leinamycin; legrastim; lentinan sulfate; leptolstatin; letrozole; leukocytosis inhibitory factor; leukocyte interferon alpha; leuprolide + estrogen + progesterone; leuprolide Lin; levamisole; riarazole; linear polyamine analogs; lipophilic diglycopeptide; lipophilic platinum compound; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; loxol Anthraquinone; lovastatin; loxoribine; letotecan; lutetium texaphyrin; lysofylline; ; Matrix Metalloproteinase Inhibitors; Minoril; Mebalone; Meterelin; Methioninase; Metoclopramide; MIF Inhibitors; Coordination double-stranded RNA; mitoguanidine hydrazone; dibromodulcitol; mitomycin analogs; molastine; monoclonal antibody, human chorionic gonadotropin; monophospholipid A+ myobacterium cell wall sk; mopirone; multidrug resistance gene inhibitor; multitumor inhibitor 1-based therapy; Nitrogen mustard anticancer drugs; Indian Ocean sponge B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; narellin; nagrestip; naloxone + Pentazocine; napavin; naphterpin; nalbuphine; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulator; Nitrous oxide antioxidant; nitrullyn; 06-benzylguanidine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron oracin; oral cytokine inducer; omaplatin; Oxatetron; Oxaliplatin; oxaunomycin; Paclitaxel; Paclitaxel analogs; Paclitaxel derivatives; palauamine; Aspargase; Peidesin; Pentosan polysulfate sodium; Pentostatin; Pentrozole; Streptococcus haemolyticus; pilocarpine hydrochloride; pirarubicin; piretexine; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compound; platinum-triamine complex; porphimer Sodium; Poffemycin; Prednisone; Propyldiacridone; Prostaglandin J2; Proteasome inhibitors; Protein A-based immunomodulators; Protein kinase C inhibitors; Protein kinase C inhibitors, microalgae ( microalgal); protein tyrosine phosphatase inhibitor; purine nucleoside phosphinase inhibitor; purpurin; pyrazoloacridine; pyridoxylated hemoglobin polyoxyalkylene conjugate; raf antagonist Rastitrexed; Ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitors; desmethyl reteptine; rhodium Re 186 etidronate; root Ribozyme; RII ranitamide; roglutimide; rohitukine; ronamotide; roquinex; rubiginone B1; ruboxyl; safingo; saintopin; 1 mimic; semustine; senescence-derived inhibitor 1; sense oligonucleotide; signal transduction inhibitor; signal transduction regulator; single-chain antigen-binding protein; 10B] Sodium carbamate; Sodium phenylacetate; Solverol; Somatomodulin binding protein; Cleavage inhibitors; stipiamide; matrix degrading enzyme inhibitors; sulfinosine; hyperactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; Sulmustine; tazorotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazolamide; Thiocoraline; Thrombopoietin; Thrombopoietin Mimetic; Thymofasin; Thymopoietin Receptor Agonist; Titanene; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; retinoic acid; triacetyluridine; tricilibine; trimetrexate; triptorelin; tropisetron; ; Tyrosine Kinase Inhibitor; Tyrphostins; UBC Inhibitor; Ubenimex; Urogenital Sinus-Derived Growth Inhibitor; Urokinase Receptor Antagonist; Vapreotide; B; vector system, erythrocyte gene therapy; viraresol; vermine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; Neocarcinstatin esters.

Anticancer drugs may be chemotherapeutic agents, which include but are not limited to the following compounds: cytotoxic antibiotics, antimetabolites, antimitotics, alkylating agents, platinum compounds, arsenic compounds, DNA topoisomerase inhibitors, taxanes Drugs (taxanes), nucleoside analogs, plant alkaloids and toxins; and synthetic derivatives thereof. Table 1 lists exemplary compounds:

Table 1

Alkylating agent

Nitrogen mustards: Cyclophosphamide

Ifosfamide

Trofosfamide

Chlorambucil

Nitrosourea: Carmustine (BCNU)

    Romustine (CCNU)

Alkylsulfonates: Busulfan

Quo Shufan

Triazene: Dacarbazine

Platinum-containing compound: cisplatin

Carboplatin

Aroplatin

Oxaliplatin

plant alkaloids

Vinca Alkaloids: Vincristine

Vinblastine

    vinchun dixin

Changchun Ruibin

Taxanes: Paclitaxel

Docetaxel

DNA topoisomerase inhibitors

Epipodophyllins: Etoposide

Teniposide

Topotecan

                                                           

Camptothecin

Krystal

Mitomycin: Mitomycin C

Antifolates:

DHFR inhibitors: methotrexate

  Trimethrexate

IMP dehydrogenase inhibitors: mycophenolic acid

Thiazofur

                            

EICAR

Nucleotide reductase inhibitors: Hydroxyurea

Desferrioxamine

Pyrimidine analogs

Uracil analogs: 5-Fluorouracil

                        

Doxifluridine

Raltitrexed

Cytosine analogs: cytarabine (ara C)

        Cytosine Ara-C

Fludarabine

Purine analogs: Mercaptopurine

                                                           

DNA antimetabolite: 3-HP

                                                                 

5-HP

        α-TGDR

Aphidicolin Glycine

ara-C

        5-Aza-2′-deoxycytosine

                                 

Cyclocitidine

Guanazole

    Hypoxanthine glycodialdehyde

macebecin II

Pyrazoloimidazole

Antimitotic agent: allocolchicine

Halichondrin B

Colchicine

           Colchicine Derivatives

Dolstatin 10

Maytansine

Rhizomycin

Thiocolchicine

             Trityl Cysteine

other:

Prenylation Inhibitors:

Dopaminergic neurotoxin: 1-methyl-4-phenylpyridinium ion

Cell cycle inhibitors: Staurosporine

Actinomycin: Actinomycin D

Dactinomycin

Bleomycin: Bleomycin A2

      Bleomycin B2

Pelomycin

Anthracyclines: Daunorubicin

Doxorubicin (Adriamycin)

                       

        Table Ruby Star

          Pirarubicin

Zorubicin

Mitoxantrone

MDR Inhibitor: Verapamil

Ca ²⁺ ATPase Inhibitors: Thapsigargin

Compositions comprising one or more chemotherapeutic agents (eg, FLAG, CHOP) are also contemplated by the present invention. FLAGs include fludarabine, cytarabine (Ara-C) and G-CSF. CHOP includes cyclophosphamide, vincristine, doxorubicin, and prednisone. The foregoing enumerations are illustrative, not restrictive.

In one embodiment, breast cancer may be treated with a drug comprising a complex of the present invention with 5-fluorouracil, cisplatin, docetaxel, doxorubicin, Herceptin(R), gemcitabine, IL-2, paclitaxel and/or VP-16 (etoposide) combination pharmaceutical composition therapy.

In another embodiment, prostate cancer may use a pharmaceutical composition comprising a complex of the invention in combination with paclitaxel, docetaxel, mitoxantrone and/or an androgen receptor antagonist such as flutamide treat.

In another embodiment, leukemia can be used comprising the complex of the present invention with fludarabine, cytarabine, gemtuzumab (gemtuzumab) (MYLOTARG), daunorubicin, methotrexate, vincristine, 6-mercapto Combination pharmaceutical composition therapy of purine, idarubicin, mitoxantrone, etoposide, asparaginase, prednisone and/or cyclophosphamide. As another example, myeloma may be treated using a pharmaceutical composition comprising a complex of the invention in combination with dexamethasone. Preferably, the leukemia is chronic myeloid leukemia (CML), the HSP complexes include hsp70-peptide complexes, and the treatment modality is imatinib mesylate or Gleevec ^™ .

In another embodiment, melanoma may be treated using a pharmaceutical composition comprising a complex of the invention in combination with dacarbazine.

In another embodiment, colorectal cancer may be treated using a pharmaceutical composition comprising a complex of the invention in combination with irinotecan.

In another embodiment, lung cancer can be treated with a pharmaceutical composition comprising a complex of the invention in combination with paclitaxel, docetaxel, etoposide and/or cisplatin.

In another embodiment, non-Hodgkin's lymphoma may be treated with a drug comprising a complex of the invention in combination with cyclophosphamide, CHOP, etoposide, bleomycin, mitoxantrone, and/or cisplatin Composition therapy.

In another embodiment, gastric cancer can be treated using a pharmaceutical composition comprising a complex of the invention in combination with cisplatin.

In another embodiment, pancreatic cancer may be treated with a pharmaceutical composition comprising a complex of the invention in combination with gemcitabine.

According to the present invention, the compound of the present invention can be administered before, after, or simultaneously with anticancer drugs for the prevention or treatment of cancer. The administration of the complexes of the invention is coordinated with the dose and timing of chemotherapy according to the type of cancer, the subject's medical history and condition, and the anticancer drug chosen.

Use of the complexes of the invention can be added to chemotherapy regimens. In one embodiment, the chemotherapeutic agent is gemcitabine at a dose of from 100 to 1000 mg/ ^m2 /cycle. In one embodiment, the chemotherapeutic agent is dacarbazine at a dose of from 200 to 4000 mg/ ^m2 /cycle. In a preferred embodiment, the dose of dacarbazine is 700 to 1000 mg/ ^m2 /cycle. In another embodiment, the chemotherapeutic agent is fludarabine at a dose of from 25 to 50 mg/ ^m2 /cycle. In another embodiment, the chemotherapeutic agent is cytarabine (Ara-C) at a dose of from 200 to 2000 mg/ ^m2 /cycle. In another embodiment, the chemotherapeutic agent is docetaxel at a dose of from 1.5 to 7.5 mg/kg/cycle. In another embodiment, the chemotherapeutic agent is paclitaxel at a dose of from 5 to 15 mg/kg/cycle. In another embodiment, the chemotherapeutic agent is cisplatin at a dose of from 5 to 20 mg/kg/cycle. In another embodiment, the chemotherapeutic agent is 5-fluorouracil at a dose of from 5 to 20 mg/kg/cycle. In another embodiment, the chemotherapeutic agent is doxorubicin at a dose of from 2 to 8 mg/kg/cycle. In another embodiment, the chemotherapeutic agent is etoposide at a dose of from 40 to 160 mg/kg/cycle. In another embodiment, the chemotherapeutic agent is cyclophosphamide at a dose of from 50 to 200 mg/kg/cycle. In another embodiment, the chemotherapeutic agent is irinotecan at a dose of from 50 to 75, 75 to 100, 100 to 125, or 125 to 150 mg/kg/cycle. In another embodiment, the chemotherapeutic agent is vinblastine at a dose of from 3.7 to 5.4, 5.5 to 7.4, 7.5 to 11, or 11 to 18.5 mg/kg/cycle. In another embodiment, the chemotherapeutic agent is vincristine at a dose of from 0.7 to 1.4, or 1.5 to 2 mg/ ^m2 /cycle. In another embodiment, the chemotherapeutic agent is methotrexate at a dose of from 3.3 to 5, 5 to 10, 10 to 100 or 100 to 1000 mg/ ^m2 /cycle.

In a preferred embodiment, the invention also includes the use of low doses of chemotherapeutic agents as part of a combination therapy regimen. For example, initial treatment with a complex of the invention increases the susceptibility of the tumor to subsequent challenge with a dose of a chemotherapeutic agent that is near or below the lower limit of the dose of the chemotherapeutic agent administered in the absence of the complex of the invention.

In one embodiment, a complex of the invention and a low dose (eg, 6 to 60 mg/ ^m2 /day or less) of docetaxel are administered to a cancer patient. In another embodiment, the complexes of the invention and paclitaxel at low doses (eg, 10 to 135 mg/ ^m2 /day or less) are administered to cancer patients. In another embodiment, a complex of the invention and a low dose (eg, 2.5 to 25 mg/ ^m2 /day or less) of fludarabine are administered to a cancer patient. In another embodiment, a complex of the invention and a low dose (eg, 0.5 to 1.5 g/ ^m2 /day or less) of cytarabine (Ara-C) are administered to a cancer patient. In another embodiment, the chemotherapeutic agent is gemcitabine at a dose ranging from 10 to 100 mg/ ^m2 /cycle. In another embodiment, the chemotherapeutic agent is cisplatin such as PLATINOL or PLATINOL-AQ (Bristol Myers) at a dose of from 5 to 10, 10 to 20, 20 to 40 or 40 to 75 mg/ ^m2 /cycle. In another embodiment, cisplatin is administered to bladder cancer patients at a dose of 7.5 to 75 mg/ ^m2 /cycle. In another embodiment, cisplatin is administered to bladder cancer patients at a dose of 5 to 50 mg/m2 ^/ cycle. In another embodiment, the chemotherapeutic agent is 2 to 4, 4 to 8, 8 to 16, 16 to 35, or 35 to 75 mg/ ^m2 /cycle of carboplatin, such as PARAPLATIN (Bristol Myers). In another embodiment, 7.5 to 75 mg/ ^m2 /cycle of carboplatin is administered to ovarian cancer patients. In another embodiment, the bladder cancer patient is administered carboplatin at a dose of 5 to 50 mg/ ^m2 /cycle. In another embodiment, 2 to 20 mg/ ^m2 /cycle of carboplatin is administered to a testicular cancer patient. In another embodiment, the chemotherapeutic agent is docetaxel at a dose of from 6 to 10, 10 to 30 or 30 to 60 mg/ ^m2 /cycle, such as TAXOTERE (Rhone Poulenc Rorer). In another embodiment, the chemotherapeutic agent is paclitaxel, such as TAXOL (Bristol Myers Squibb), at a dose of from 10 to 20, 20 to 40, 40 to 70, or 70 to 135 mg/ ^m2 /cycle. In another embodiment, the chemotherapeutic agent is 5-fluorouracil at a dose of from 0.5 to 5 mg/ ^m2 /cycle. In another embodiment, the chemotherapeutic agent is doxorubicin, such as ADRIAMYCIN (Pharmacia & ^Upjohn ), DOXIL ( Alza), RUBEX (Bristol Myers Squibb).

In another embodiment, the compound of the present invention is administered in combination with one or more anti-angiogenic drugs, including but not limited to, angiotensin, thalidomide, kringle 5, endostatin, Serpin ( Serine protease inhibitors) anticoagulant factor, 29kDa amino-terminal and 40kDa carboxy-terminal proteolytic fragments of fibronectin, 16kDa proteolytic fragment of prolactin, 7.8kDa proteolytic fragment of platelet factor-4, corresponding to platelet factor-4 A 13 amino acid peptide of a fragment of 4 (Maione et al., 1990, Cancer Res., 51:2077-2083), a 14 amino acid peptide corresponding to a fragment of type I collagen (Tolma et al., 1993, J.Cell Biol. , 122:497-511), a 19-amino acid peptide corresponding to a fragment of thrombocontractin I (Tolsma et al., 1993, J. Cell Biol., 122:497-511), a 20-amino acid peptide corresponding to a SPARC fragment (Sage et al., 1995, J. Cell. Biochem., 57:1329-1334), or any fragment, family member, or variant thereof, including pharmaceutically acceptable salts thereof.

Other peptides that inhibit angiogenesis and fragments corresponding to laminin, fibronectin, procollagen and EGF have also been described (see eg Cao, 1998, Prog Mol Subcell Biol., 20: 161-176). Monoclonal antibodies and cyclic pentapeptides (i.e. with the peptide motif Arg-Gly-Asp) that block certain integrins that bind RGD proteins have been shown to have antiangiogenic activity (Brooks et al., 1994, Science, 264: 569-571; Hammes et al., 1996, Nature Medicine, 2:529-533). In addition, inhibition of the urokinase plasminogen activator receptor by receptor antagonists suppressed angiogenesis, tumor growth and metastasis (Min et al., 1996, Cancer Res., 56:2428-33; Crowley et al., 1993, Proc. Natl Acad Sci., 90:5021-25). The present invention also contemplates the combined use of such anti-angiogenic drugs and complexes.

In another embodiment, the complexes of the invention are used in combination with hormone therapy. Hormone therapy includes hormone agonists, hormone antagonists (eg, flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), hormone biosynthesis and processing Inhibitors and steroids (eg, dexamethasone, retinoids, deltoids, betamethasone, hydrocortisone, cortisone, prednisone, 2-dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen hormones, testosterone, progesterone), vitamin A derivatives (eg, all-trans retinoic acid (ATRA)); vitamin D3 analogs; antiluteinizing agents (eg, mifepristone, onapristone), and antiandrogens (such as cyproterone acetate).

In another embodiment, the complexes of the invention are used in combination with gene therapy procedures in cancer treatment. In one embodiment, gene therapy of recombinant cells secreting interleukin-2 is combined with the complex of the invention for the prevention or treatment of cancer, especially breast cancer (see e.g. Deshmukh et al., 2001, J. Neurosurg., 94; 287-92). In other embodiments, polynucleotide compounds are used for gene therapy, such as but not limited to antisense polynucleotides, ribozymes, RNA interference molecules, triple-helix polynucleotides, etc., wherein the core of such compounds The nucleotide sequence is related to the nucleotide sequence of DNA and/or RNA of genes related to tumor or cancer occurrence, development and/or pathology. For example, many of them are oncogenes, growth factor genes, growth factor receptor genes, cell cycle genes, DNA repair genes, and are well known in the art.

In another embodiment, the complexes of the invention are administered with a radiotherapy regimen. For radiation therapy, the radiation may be gamma rays or X-rays. This approach includes cancer treatments involving radiation, such as external beam radiation therapy, interstitial implant therapy with radioisotopes (I-125, palladium, iridium), radioisotopes such as strontium-89, chest radiation, intraperitoneal phosphorus-32 radiation, and / or radiation therapy to the entire abdomen and pelvis. For a comprehensive review of radiation therapy, see Hellman, Chapter 16: Principles of Cancer Management: Radiation Therapy, Sixth Edition, 2001, edited by DeVita et al., J.B. Lippencott Company, Philadelphia. In preferred embodiments, radiation therapy is delivered as external beam radiation or teletherapy, in which the radiation comes directly from a distant radiation source. In preferred embodiments, radiation therapy is performed as an internal or superficial treatment, where the source of radiation is located inside the body, close to the cancer cell or tumor mass. Also included is the combined application of the complex of the present invention and photodynamic therapy, which includes the administration of photosensitizers such as hematoporphyrin and its derivatives, Vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, desensitizer Methoxyhypocretin A; and 2BA-2-DMHA.

In various embodiments, a combination of a complex of the invention and at least one chemotherapeutic agent is administered for short periods of treatment to a cancer patient to be treated for cancer. The duration of treatment with chemotherapeutic agents can vary depending on the particular cancer treatment used. Intermittent administration or administration of the daily dosage in divided portions is also contemplated by the present invention. Appropriate treatment times for a particular cancer therapeutic are within the skill of the art, and the present invention contemplates ongoing evaluation of the optimal treatment plan for each cancer therapeutic. The present invention contemplates at least one cycle, preferably more than one cycle, in which a single treatment or consecutive treatments are administered. The appropriate length of a treatment cycle, the total number of cycles, and the interval between cycles can be grasped by those skilled in the art.

In another embodiment, the complexes of the invention are used in combination with compounds that ameliorate cancer symptoms (such as but not limited to pain) and side effects (such as but not limited to flu-like symptoms, fever, etc.) produced by the complexes of the invention. Accordingly, a number of compounds known to reduce pain, flu-like symptoms and fever may be used in combination with or in admixture with the complexes of the present invention. Such compounds include pain relievers (such as acetaminophen), decongestants (such as pseudoephedrine), antihistamines (such as chlorpheniramine maleate), and cough suppressants (such as dextromethorphan).

5.5. Target infectious diseases

Infectious diseases that may be treated or prevented by the methods of the present invention are caused by infectious agents including, but not limited to, viruses, bacteria, fungi, protozoa, helminths, and parasites. The present invention is not limited to the treatment or prevention of infectious diseases caused by intracellular or extracellular pathogens. Combination therapy includes the use of one or more modalities that aid in the prevention or treatment of infectious diseases, including but not limited to antibiotics, antivirals, antiprotozoal compounds, antifungal compounds and anthelmintics. Other therapeutic modalities that may be used to treat or prevent infectious disease include immunotherapeutics, polynucleotides, antibodies, cytokines and hormones as described above.

Infectious viruses of humans and non-human vertebrates include retroviruses, ribonucleic acid viruses and deoxyribonucleic acid viruses. Examples of viruses that have been found in humans include, but are not limited to: Retroviridae (such as human immunodeficiency virus, such as HIV-1 (also known as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates such as HIV-LP; picornaviridae (eg poliovirus, hepatitis A virus; enteroviruses, human coxsackieviruses, rhinoviruses, echoviruses); caliciviridae (eg strains that cause gastroenteritis); Togaviridae (such as equine encephalitis virus, rubella virus); swine fever virus (such as dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (such as coronavirus); Rhabdoviridae (eg, vesicular stomatitis virus, rabies virus); Lineviridae (eg, Ebola virus); Paramyxoviridae (eg, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus) ; Orthomyxoviridae (eg, influenza viruses); Bunyaviridae (eg, hantaviruses, parasitic wild mushroom viruses, phleboviruses, and Nairo viruses); Arenaviridae (eg, hemorrhagic fevers) viruses); Reoviridae (such as reoviruses, orbiviurses, and rotaviruses); DiRNAviridae; Hepaviridae (hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papillomavirus, polyomavirus); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpesviruses; poxviridae (variola virus, vaccinia virus, poxvirus); and iridoviridae (eg, African swine fever virus); and unclassified viruses (eg, causative agent of spongiform encephalopathy, hepatitis D Pathogens (thought to be defective appendages of hepatitis B virus, vectors of non-A, non-B hepatitis (category 1 = internally transmitted; category 2 = parenterally transmitted (i.e. hepatitis C); Norwalk and related viruses and astroviruses).

The aforementioned retroviruses include simple retroviruses and complex retroviruses. Simple retroviruses include the subgroups of type B retroviruses, type C retroviruses, and type D retroviruses. An example of a type B retrovirus is mouse mammary tumor virus (MMTV). Type C retroviruses include group A subgroups of type C (including Rous sarcoma virus (RSV), avian leukosis virus (ALV) and avian myeloblastosis virus (AMV)) and group B subgroup of type C (including murine Leukemia Virus (MLV), Feline Leukemia Virus (FeLV), Murine Sarcoma Virus (MSV), Gibbon Leukemia Virus (GALV), Spleen Necrosis Virus (SNV), Reticuloendotheliosis Virus (RV), and Simian Sarcoma Virus (SSV) )). Type D retroviruses include May-Pam monkey virus (MPMV) and simian retrovirus type 1 (SRV-1). Complex retroviruses include lentiviruses, T-cell leukemia viruses, and foamy virus subgroups. Lentiviruses include HIV-1, but also include HIV-2, SIV, ovine myelin shedding virus, feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV). T-cell leukemia viruses include HTLV-1, HTLV-II, simian T-cell leukemia virus (STLV), and bovine leukemia virus (BLV). Foamy viruses include human foamy virus (HFV), simian foamy virus (SFV) and bovine foamy virus (BFV).

Examples of RNA viruses that are vertebrate antigens include, but are not limited to, the following: members of the Reoviridae family, including Orthoreoviruses (multiple serotypes of mammalian and avian retroviruses), Orbiviruses ( (Bluetongue virus, Eugenangee virus, Kemerovo virus, African equine disease virus, and Colorado tick fever virus), rotaviruses (human rotavirus, Nebraska bovine diarrhea virus, murine rotavirus rotaviruses, simian rotaviruses, bovine or ovine rotaviruses, avian rotaviruses); members of the picornaviridae family including enteroviruses (polioviruses, coxsackieviruses A and B, enterocytopathic Orphan (ECHO) virus, hepatitis A virus, simian enterovirus, murine encephalomyelitis (ME) virus, murine poliovirus, bovine enterovirus, porcine enterovirus, cardiovirus (encephalomyocarditis virus (EMC), Mengo virus), rhinoviruses (human rhinoviruses including at least 113 subtypes; other rhinoviruses), foot-and-mouth disease virus (foot-and-mouth disease (FMDV); members of the family Caliciviridae, including porcine vesicular herpesvirus , San Miguel sea lion virus, feline picornavirus, and Norwalk virus; members of the Togaviridae family, including alphaviruses (Oriental equine encephalitis virus, Semliki forest virus, Sindbis virus, chikungunya virus, Oneonion virus, Ross River virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus), mosquito-borne virus genera (mosquito-borne yellow fever virus, dengue virus, Japanese encephalitis virus, St. Louis encephalitis virus Virus, Merai Valley Encephalitis Virus, West Nile Virus, Kuking Virus, Central European Tick-Transmitted Virus, Far Eastern Tick-Transmitted Virus, Kosanur Forest Virus, Louping III Virus, Powassan Virus, Omsk Hemorrhage fever virus), Rubivirus (rubella virus), Pestivirus (mucosal disease virus, swine fever virus, border disease virus); members of the Bunyanviridae family, including Bunyvirus genus (Bunyanvila and related viruses, California brain Virus), Venoviruses (Sandfly Fever Sicilian Virus, Rift Valley Fever Virus), Nairoviruses (Crimean-Congo Hemorrhagic Fever Virus, Nairobi Sheep Disease Virus), and Uukuviruses (Uukuniemi and related viruses); members of the family Orthomyxoviridae, including the genus Influenzavirus (influenza A, various human subtypes); swine influenza viruses, and avian and equine influenza viruses; influenza B (various human subtypes) and Influenza C (possibly a separate genus); members of the Paramyxoviridae family, including Paramyxovirus genera (parainfluenza virus type 1, Sendai virus, hematoadsorbent virus, parainfluenza virus types 2 to 5, Newcastle disease virus, parotid virus inflammatory virus), measles virus (measles virus, subacute sclerosing panencephalitis virus, distemper virus, rinderpest virus), pneumovirus (respiratory syncytial virus (RSV), bovine respiratory syncytial virus and mouse pneumonia Viruses); Forest virus, Sindbis virus, Chikungunya virus, Oneonion virus, Ross River virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus), mosquito-borne viruses (mosquito-borne yellow Fever Virus, Dengue Virus, Japanese Encephalitis Virus, St. Louis Encephalitis Virus, Merai Valley Encephalitis Virus, West Nile Virus, Kuching Virus, Central European Tick-Borne Virus, Far Eastern Tick-Borne Virus, Kosanur Forest Virus , Louping III virus, Powassan virus, Omsk hemorrhagic fever virus), Rubella virus (rubella virus), Pestivirus (mucosal disease virus, swine fever virus, border disease virus); members of the Bunyanviridae, including Bunyviruses (Bunyanvira and related viruses, California encephalitis group viruses), Venoviruses (Sandfly fever Sicilian virus, Rift Valley fever virus), Neroviruses (Crimea-Congo hemorrhagic Fever virus, Nairobi sheep disease virus), and the Uukuvirus genus (Uukuniemi and related viruses); members of the family Orthomyxoviridae, including the genus Influenzavirus (influenza A, various human subtypes); swine influenza viruses and Avian and equine influenza viruses; influenza B (various human subtypes) and influenza C (possibly separate genus); members of the Paramyxoviridae family, including Paramyxovirus genus (parainfluenza virus type 1, Sendai virus , hematoadsorption virus, parainfluenza virus types 2 to 5, Newcastle disease virus, mumps virus), measles virus (measles virus, subacute sclerosing panencephalitis virus, distemper virus, rinderpest virus), pneumovirus (respiratory syncytial virus (RSV), bovine respiratory syncytial virus, and mouse pneumovirus), members of the Rhabdoviridae family including vesiculovirus (VSV), Chandipura virus, Flanders-Hart Park virus), rabies virus genus (rabiesvirus), fish rhabdovirus, and possibly two rhabdoviruses (Marburg and Ebola); members of the Arenaviridae family, including lymphocytic choriomeningitis virus (LCM), Taka Ribe virus complex and Lassa fever virus; members of the Coronoaviridae family, which includes infectious bronchitis virus (IBV), mouse hepatitis virus, human enteric coronavirus, and feline infectious peritonitis (feline coronavirus).

Illustrative examples of ribonucleic acid viruses that are vertebrate antigens include, but are not limited to, the following: Poxviridae, including Orthopoxvirus (variola major, variola minor, monkeypox, vaccinia, buffalopox, rabbitpox, amputum , rabbitpoxvirus (myxoma, fibroid), fowlpoxvirus (fowl pox, other bird poxviruses), goatpoxvirus (sheep pox, goat pox), hyoppoxvirus (swine pox), para Poxvirus genus (infectious impetigodermatitis virus, pseudovaccinia, bovine papular stomatitis virus), iridoviridae family (African swine fever virus, frog virus 2 and 3, lymphocyst virus of fish); herpesviridae, including Alphaherpesviruses (herpes simplex types 1 and 2, varicella-zoster, equine abortion virus, equine herpesvirus 2 and 3, pseudorabies virus, infectious bovine keratoconjunctivitis virus, bovine infectious rhinotracheitis virus, feline rhinotracheitis virus, infectious laryngotracheitis virus) beta-herpesvirus (human cytomegalovirus, and porcine, monkey, and rodent cytomegalovirus); gammaherpesvirus (Epstein-Barr virus (EBV), Marek Herpes virus, squirrel monkey virus, herpes virus Arachnovirus, herpes virus Cottontail virus, guinea pig herpes virus, Luke tumor virus); Adenoviridae, including mammalian adenovirus genera (human A, B, C, D, E, and ungrouped subgroups; simian adenoviruses (at least 23 serotypes), canine infectious hepatitis, and adenoviruses of bovine, porcine, sheep, frog, and many other species, avian adenoviruses (avian adenoviruses) and non-domesticated adenoviruses; Papoviridae family, including papillomaviruses (human papillomavirus, bovine papillomavirus, Chopp rabbit papillomavirus and various pathogenic papillomaviruses of other species), polyomaviruses Genus (polyomavirus, simian vacuolar virus (SV-40), rabbit vacuolar virus (RKV), K virus, BK virus, JC virus, and other primate polyoma viruses such as lymphotropic (lymphocytic) papillomavirus); Parvoviridae, which includes the genus Adenovirus-associated virus, Parvovirus (feline panleukopenia virus, bovine parvovirus, canine parvovirus, Aleutian mink disease virus, etc.). Finally, DNA viruses can Included are viruses that do not fit into the above families such as Kuru virus and Creutzfeldt-Jakob disease virus and agents of chronic infectious neuropathy.

Many examples of antiviral compounds that can be used in combination with the complexes of the invention are known in the art and include, but are not limited to: rifampicin, nucleoside reverse transcriptase inhibitors (such as AZT, ddI, ddC, 3TC , d4T), non-nucleoside reverse transcriptase inhibitors (such as efavirenz, nevirapine), protease inhibitors (such as aprenavir, indinavir, ritonavir, and saquinavir), iodine glycosides, cidofovir, Acyclovir, ganciclovir, zanamivir, amantadine, and Palivizumab. Other examples of antiviral drugs include, but are not limited to, Acemannan; Acyclovir; Acyclovir Sodium; Adefovir; Alovudine; Ding; Aperidone; Atevirdine mesylate; Avridine; Cidofovir; Cipanfylline; Cytarabine hydrochloride; Delavirdine mesylate; Deciclovir; Glycoside; Dioxalid; Eduridine; Enveraden; Enviroxime; Famciclovir; Famodin Hydrochloride; Fecitabine; Nonaluridine; Lowe; Ganciclovir sodium; Iodine glucoside; Deoxybutanone aldehyde; Lamivudine; Lobucavir; Memotine hydrochloride; Methisazone (Methisazone); Nevirapine; Davir; ribavirin; rimantadine hydrochloride; saquinavir mesylate; somantine hydrochloride; solivudine; Thymidine; Valacyclovir Hydrochloride; Vidarabine; Vidarabine Phosphate; Vidarabine Disodium Phosphate; Veroxime; Zalcitabine; Zidovudine;

Bacterial infections or diseases that may be treated or prevented by the methods of the present invention are caused by bacteria including, but not limited to, bacteria that have an intracellular stage in their life cycle such as mycobacteria (e.g. Mycobacteria tuberculosis, Mycobacterium bovis (M.Bovis), Mycobacterium avium (M.Avium), leprosy

Mycobacterium (M. Leprae), or M. Africanum), Rickettsia, Mycoplasma, Chlamydia, and Legionella. Other examples of contemplated bacterial infections include, but are not limited to, infections caused by Gram-positive bacilli (e.g., Listeria, Bacillus such as Bacillus anthracis, Erysipelothrix species), Gram-negative bacilli ( Such as Bartonella, Brucella, Campylobacter, Enterobacter, Escherichia, Francisella, Hemophilus , Klebsiella, Morganella, Proteus, Providencia, Pseudomonas, Salmonella, Serratia ( Serratia), Shigella, Vibrio, and Yersinia), spirochete bacteria (such as Borrelia, including Lyme disease-causing Borrelia burgdorferi ( Borreliaburgdorferi), anaerobes (such as Actinomyces and Clostridium), Gram-positive and negative cocci, Enterococcus, Streptococcus, pneumococci ( Pneumococcus), Staphylococcus, Neisseria gonorrhoeae. Examples of infectious bacteria include, but are not limited to, Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria tuberculosis, M. avium ), Mycobacterium intracellulare (M.Intracellulare), Mycobacterium kansarii (M.kansaii), Mycobacterium gordonii, Staphylococcus aureus (Staphylococcus aureus), Neisseria gonorrhoeae (Neisseriagonnorrhoeae), meningitis Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (group A strep), Streptococcus agalactiae (group B strep) coccus), Streptococcus viridans, Streptococcus faecalis, Streptococcus bovis, Streptococcus pneumoniae, Haemophilus influenzae, Bacillus antracis, Corynebacterium diphtheriae ( corynebacterium diphtheriae), Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneunaomae, Pasturella multocida, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rick Infections caused by Rickettisa and Actinomyces israelli.

Antibacterial agents or antibiotics that can be used in combination with the complexes of the present invention include, but are not limited to: aminoglycoside antibiotics (such as apramycin, arbekacin, bambemycin, butirocin, dibekacin, Neomycin, neomycin, undecylenate, netilmicin, paromomycin, ribomycin, perillomycin, and spectinomycin), amphenicol antibiotics (eg, chloramphenicol azide, chloramphenicol chloramphenicol, florfenicol, and thiamphenicol), ansamycin antibiotics (such as riformide and rifampin), carbacephems (such as clocarbef), carbapenems (such as biapenem and imipenem), cephalosporins (such as cefaclor, cefadroxil, cefadroxazole, axamazole cephalosporins, cefoxizone, cefzoram, cefamizole, cefpiramide, and cephalosporins Piro), cephamycins (eg, cefbuperazone, cefmetazole, and cefminol), monocyclic beta-lactams (eg, aztreonam, calumonam, and tigemonan), oxygen Cephalosporins (such as fluoxyceph, latamoxef), penicillins (such as azemidine penicillin, azemidine penicillin pivoxil, amoxicillin, bacillin, benzyl penicillanic acid, benzyl penicillin sodium, epicillin, Fenbecillin, flucloxacillin, penacillin, penicillin hydroiodate, penicillin o-phenethylbenzylamine, penicillin O, penicillin V, penicillin V benzathine penicillin, halamine penicillin V, penicillin, and phencihicillin Potassium), lincosamines (such as clindamycin and lincomycin), macrolides (such as azithromycin, carboxycin, clarithromycin, roxithromycin, erythromycin, and stearomycin) , amphomycin, bacitracin, capreomycin, colistin, duramicidin, tuberculosis actinomycin, tetracyclines (such as apicycline, chlortetracycline, oxymecycline, and demeclocycline), 2 , 4-diaminopyrimidines (such as bromoprin), nitrofurans (such as furomazodone and furazolium chloride), quinolones and their analogs (such as cinoxacin, ciprofloxacin, clinfloxacin, flumequine and grepagloxacin), sulfonamides (such as acetylsulfamethoxine, benzsulfonamide, benzsulfonamide, phenprosulfonate, phthalmamide, sulfacodine, and sulfacytosine), sulfones (such as dithyril Sulfone, Sodium Glucose Sulfone, and Phenylsulfone), Cycloserine, Mupirocin, and Potato Globulin.

Additional examples of antibacterial agents include, but are not limited to, dapsone; acesulfame sodium; alemycin; alexidine; azemidine penicillin; azemidine penicillin; amicycline; Flufloxacin; amfloxacin mesylate; amikacin; amikacin sulfate; aminosalicylic acid; aminosalicylate sodium; Paxicillin sodium; Apramycin; Methylenedisalicylate bacitracin; bacitracin zinc; flavomycin; calcium benzalate; erythromycin B; betamicin sulfate; biapenem; benimycin; Magnesium pyrithione sulfate; buticacin; butirocin sulfate; capreomycin sulfate; carbadox; carbenicillin disodium; carbenicillin sodium; carbenicillin sodium; carbenicillin potassium; Monan sodium; Cefaclor; Cefadroxil; Cefamandole; Cefamandole sodium; Cefamandole sodium; ; Cefbuperazone; Cefdinir; Cefepime; Cefepime Hydrochloride; Cefetecol; Cefixime; Cefmnenoxime Hydrochloride; Cefmetazole; Cefmetazole Sodium; Leite; Cefotaxime Sodium; Cefotetan; Cefotetan Disodium; Cefotiam Hydrochloride; Cefoxitin; Cefoxitin Sodium; Cefmidazole; Cefmidazole Sodium; Piro; Cefpodoxime Proxetil; Cefprozil; Cefoxadine; Cefsulodin Sodium; Ceftazidime; Ceftibuten; Ceftizoxime Sodium; Ceftriaxone Sodium; Cefuroxime; Cefuroxime Proxetil; Cefuroxime Cefuroxime Sodium; Cefuroxime Sodium; Cefuroxime Sodium; Cefalexin; Cefalexin Hydrochloride; Cephalic Acid; Cefotazidime; Cefalotin Sodium; Chloramphenicol palmitate; Chloramphenicol pantothenic acid complex; Chloramphenicol sodium succinate; Chlorhexidine aminophenylphosphate; Chloroxylenol; Aureomycin disulfate; ; Ciprofloxacin; Ciprofloxacin Hydrochloride; Siromycin; Clarithromycin; Clinfloxacin Hydrochloride; Clindamycin; Clindamycin Hydrochloride; Clofazimine; Cloxacillin benzathine; Cloxacillin sodium; Cloxyquine (Cloxyquin); Polymyxin E mesylate sodium; Colistin sulfate; Coomamycin; Cyclocillin; Cycloserine; Dalfopristin; Dapsone; Daptomycin; Norcycline; Norcycline Hydrochloride; Norcycline; Denofungin ; Diaminoveratridine; Dicloxacillin; Dicloxacillin Sodium; Dihydrostreptomycin Sulfate; Dipyrthione; Dirithromycin; Doxycycline; Doxycycline Calcium; Doxycycline hydrochloride; Droxacin sodium; Enoxacin; Epicillin; Epitetracycline hydrochloride; Erythromycin; Erythromycin saccharate; Erythromycin lactose; Erythromycin propionate; Erythromycin stearate; Ethambutol hydrochloride; Ethionamide; Fleroxacin; Flucloxacillin; Mequin; fosfomycin; fosfomycin trometamol; furmoxicillin; furazolium chloride; furthimazole tartrate; sodium fusidate; fusidide; gentamicin sulfate; glumonam; Gramicidin; Iodochlorophenyne ether; Hetacillin; Hetacillin Potassium; Hexidine; Ibafloxacin; Imipenem; Isoconazole; Niacinazine; Josamycin; Kanamycin Sulfate; Kitamycin; Levofuraltadone; Leprapicillin Potassium; Lythromycin; Lincomycin; Lincomycin Hydrochloride; Lomefloxacin; Hydrochloride Lomefloxacin; Lomefloxacin mesylate; Clocarbef; Sulfamethon; Meclocycline; Meclocycline sulfosalicylate; Megamycin potassium dihydrogen phosphate; Mequidox; Meropenem South; Methoxycycline; Methoxycycline Hydrochloride; Methicillin; Methicillin Hippurate; Methicillin Mondelicate; Methicillin Sodium; ; metronidazole phosphate; mezlocillin; mezlocillin sodium; minocycline; minocycline hydrochloride; milincomycin hydrochloride; monensin; monensin sodium; nafcillin sodium; nalidixic acid Sodium; Nalidixic Acid; Natamycin; Dark Mycin; Neomycin Palmitate; Neomycin Sulfate; Neomycin Undecylenate; Netilmicin Sulfate; Neutramycin; Nifuradine ; Nifurdizone; Nifuratel; Nifuron; Nifurazepam; Nifuramide; Floxacin; Novobiocin Sodium; Ofloxacin; Omeprene; Oxacillin Sodium; Oxime Monan; Oxime Monan Sodium; Oxolitic Acid; Oxytetracycline; Oxytetracycline Calcium; Oxytetracycline Hydrochloride Penicillin; Padetomycin; P-Chlorophen; Paulomycin; Pefloxacin; Pefloxacin Mesylate; Penacillin; Penicillin G Benzathine Penicillin; Penicillin G Potassium; Penicillin G Sodium; Penicillin V; Penicillin V Benzathine Penicillin; Halamin Penicillin V; Penicillin V Potassium; Amazolidone Sodium; ;Pilimycin hydrochloride; Pimicillin hydrochloride; Pimicillin pamoate; Wengzinc; quindiacin acetate; quinupristin; racemic thiamphenicol (Racephenicol); ramoplanin; rifaxime; rifamid; rifampicin; rifapentine; rifaximin; rolicycline; rolicycline nitrate; rosamicin; rosamicin butyrate; rosamicin propionate Star; Roxamicin Sodium Phosphate; Roxamicin Stearate; Roxoxacin; Roxarsone; Roxithromycin; Sancycline; Sanfepenem Sodium; Scopafingin; perillomycin; perillomycin sulfate; sparfloxacin; spectinomyces hydrochloride; spiramycin; Streptonicozid; Sulfabenzene; Sulfabenzoyl; Sulfaacetamide; Sulfacetamide Sodium; Sulfaxetine; Sulfadiazine; Sulfadiazine Sodium; Sulfadoxine; Sulfaline; Sulfamethazine; Oxypyrimidine; Sulfamethazine; Sulfamethizole; Sulfamethoxazole; Sulfamethoxazole; Sulfamethoxazole; Zinc Sulfamate; Sulfanitrobenzene; Sulfasalazine; Sulfaisothiazole; Sulfathiazole; Sulfapyrazole; Sulfaisoxazole; Acesulfame Sulfaisoxazole; Sulfaisoxazole Diolamine; Sulfamyxin; Thiopenem; Sultacillin; Sencillin Sodium; Phthamicillin Hydrochloride; Testox Lanin; Temafloxacin Hydrochloride; Temoxicillin; Tetracycline; Tetracycline Hydrochloride; Tetracycline Phosphate Complex; Tetraoxoprine; Thiamphenicol; Thiphencillin Potassium; Ticarcillin Cresyl Sodium; Ticarcillin Disodium; Ticarcillin Sodium; Ticklarone; Tiodonium Chloride; Tobramycin; Tobramycin Sulfate; Tosufloxacin; Trimethoprim; Trimethoprim Sulfate; Trisulfapyrimidine ( Trisulfapyrimidines); troleandomycin; spectinomycin sulfate; brevicin; vancomycin; vancomycin hydrochloride;

Mycoses that may be treated or prevented by the methods of the present invention include, but are not limited to, aspergillosis, cryptococcosis, sporotrichosis, coccidioidomycosis, Brazilian blastomycosis, histoplasmosis, blastomycosis, zygomycosis and candidiasis.

Antifungal compounds that can be used in combination with the complexes of the present invention include, but are not limited to: polyenes (such as amphotericin B, candidacin, mepatrexin, natamycin, and nystatin), allylamines (such as butenafine and naftifine), imidazoles (eg, bifonazole, butoconazole, chlordentoin, flutraconazole, isoconazole, ketoconazole, and lanoconazole), thiocarbamate esters (such as tocilatate, torindalate, and tolnaftate), triazoles (such as fluconazole, itraconazole, saconazole, and terconazole), bromide, buclosamide Calcium propionate, chlorophene ether, ciclopirox, azaserine, griseofulvin, oligomycin, neomycin undecylenate, pyrrolnitrine, hemectin, tubercidin and Green glomycin.

Additional examples of antifungal compounds include, but are not limited to, acridizocine; ambroxine; amphotericin B; azaconazole; Magnesium pyrithione sulfate (Bispyrithione Magsulfex); Butoconazole nitrate; Calcium undecylenate; ; Ciconazole; Clotrimazole; Cumaixin; Denofungin; Dipyrthionium; Doconazole; Econazole; Econazole nitrate; Enconazole; Teconazole; Filipin; Fluconazole; Flucytosine; Mycomycin; Griseofulvin; Harmycin; Isoconazole; Itraconazole; Carafungin; Ketoconazole; Lomofingin ; Liddimycin; Mepatroxine; Miconazole; Miconazole Nitrate; Monensin; Monensin Sodium; Naftifine Hydrochloride; Neomycin Undecylenate; Nifuratel ; Nifurmeron; Nitramine Hydrochloride; Nystatin; Octanoic Acid; Oconazole Nitrate; Oxiconazole Nitrate; Oxifungin Hydrochloride; Paconazole Hydrochloride; Weng Zinc; Pyrrolnitrine; Rutamycin; Blood Root Ammonium Chloride; Saconazole; ; tecladone; tioconazole; tocilatate; torindalate; tolnaftate; triacetin; triafuigin; undecylenic acid; viridoflilvin; zinc undecylenate; and zinoconazole hydrochloride .

Parasitic diseases that may be treated or prevented by the submethods of the invention include, but are not limited to, amoeba, malaria, leishmaniasis, coccidiosis, giardiasis, cryptosporidiosis, toxoplasmosis, and trypanosomiasis. Bug disease. Also included are infections with various helminths such as, but not limited to, ascariasis, hookworm, whipworm, strongyloidiasis, toxoccariasis, trichinellosis, onchocerciasis, filariasis, and dirofilariasis. Also included are infections with various flukes, such as, but not limited to, schistosomiasis, paragonimiasis, and clonorchiasis. Parasites that cause these diseases can be classified according to whether they are intracellular or extracellular. As used herein, an "intracellular parasite" is a parasite whose entire life cycle is inside a cell. Examples of human intracellular parasites include Leishmania spp., Plasmodium spp., Trypanosoma spp., Toxoplasma gondii, Babesia spp. and Trichinella spiralis. An "ectoparasite" as used herein is a parasite whose entire life cycle is outside the cell. Extracellular parasites capable of infecting humans include Entamoeba dysenteriae, Giardia lamblia, Enterocytozoonbieneusi, Naegleria, and Acantlaamoeba, as well as most helminths. Another class of parasites is defined as predominantly extracellular, but obligatory intracellular during critical stages of their life cycle. Such parasites are referred to herein as "forced intracellular parasites". These parasites may spend most or a small part of their lives in the extracellular environment, but have at least one mandatory intracellular phase in their life cycle. This latter group of parasites includes Trypanosoma rhodesia and Trypanosoma gambiense, Isospora spp., Cryptosporidium spp., Eimeria spp. .), Neospora spp., Sarcocystis spp. and Schistosoma spp.

Numerous examples of antiprotozoal compounds useful in the complex combinations of the present invention for the treatment of parasitic diseases are known in the art and include, but are not limited to: quinine, chloroquine, mefloquine, proguanil, pyrimethamine, Metronidazole, diloxanide, tinidazole, amphotericin, sodium stibogluconate, co-trimoxazole, and pentamidine. Numerous examples of parasiticidal drugs that can be used in combination with the complexes of the present invention to treat parasitic diseases are known in the art and include, but are not limited to: mebendazole, levamisole, niclosamide, praziquantel, Albendazole, ivermectin, diethylcarbamate, and thiabendazole. Other examples of antiparasitic compounds include, but are not limited to, dapsone; amphetamine hydrochloride; lin; chlorofluoroquine hydrochloride; hydroxychloroquine sulfate; mefloquine hydrochloride; menomoctone; milinmycin hydrochloride; primaquine phosphate; pyrimethamine;

In some embodiments, antibodies can be used to treat and/or prevent infectious diseases. In a less preferred embodiment, the complexes of the invention may be used in combination with non-α2M-lineage vaccine compositions. Examples of such human vaccines are described in The Jordan Report 2000, Accelerated Development of Vaccines, National Institute of Health, which is incorporated herein by reference in its entirety. A number of vaccines for the treatment of non-human vertebrates are disclosed in Bennett, K., Compendium of Veterinary Products, Third Edition, North American Compendiums, Inc., 1995, which is incorporated herein by reference in its entirety.

5.6. Treatment plan

For any combination therapy described above for the treatment or prevention of cancer and infectious disease, the pharmaceutical composition of the present invention may be administered before, simultaneously with, or after administration of the combination mode. The combined modality may be any of the aforementioned modality for treating or preventing cancer or infectious disease.

In one embodiment, a pharmaceutical composition of the invention is administered to a subject at the same time as another modality at a reasonable time. The method provides for the two administrations to be administered within a time range of less than one minute to about five minutes or up to about sixty minutes of each other, eg, within the same visit.

In another embodiment, the pharmaceutical composition of the invention and the combination mode are administered at exactly the same time. In another embodiment, the pharmaceutical composition of the invention and the combined modality are administered sequentially and at intervals such that the pharmaceutical composition of the invention and the modality can act simultaneously to provide increased benefit over when they are administered alone. In another embodiment, the pharmaceutical compositions of the invention are administered in sufficient proximity in time to the combination modality to provide the desired therapeutic or prophylactic result. Each may be administered simultaneously or separately in any suitable form and by any suitable route. In one embodiment, the pharmaceutical composition of the invention and the mode of administration are administered by different routes of administration. In alternative embodiments, both are each administered by the same route of administration. The pharmaceutical composition of the present invention can be administered at the same or different sites, such as arms and legs. When administered simultaneously, the pharmaceutical composition and the combination mode of the present invention may be administered in admixture or not or in the same site by the same route of administration.

In various embodiments, the complexes and combinations of the invention are administered at intervals of less than 1 hour, at intervals of about 1 hour, at intervals of 1 hour to 2 hours, at intervals of 2 hours to 3 hours, at intervals of 3 hours to 4 hours intervals, intervals of 4 hours to 5 hours, intervals of 5 hours to 6 hours, intervals of 6 hours to 7 hours, intervals of 7 hours to 8 hours, intervals of 8 hours to 9 hours, intervals of 9 hours to 10 hours , administered at intervals of 10 hours to 11 hours, at intervals of 11 hours to 12 hours, at intervals of no more than 24 hours, or at intervals of no more than 48 hours. In other embodiments, the pharmaceutical and vaccine compositions of the present invention are administered at intervals of 2 to 4 days, at intervals of 4 to 6 days, at intervals of 1 week, at intervals of 1 to 2 weeks, at intervals of 2 to 4 weeks, Administration is at monthly intervals, at 1 to 2 month intervals, or at intervals greater than 2 months. In preferred embodiments, the complexes and combination modes of the invention are administered within a time frame in which both are still active. A person skilled in the art will be able to determine such a time frame by examining the half-life of each administered component.

In one embodiment, the pharmaceutical composition of the invention and the combination modality are administered during the same medical visit. In a particularly preferred embodiment, the pharmaceutical composition of the invention is administered prior to said modality. In an alternative embodiment, the pharmaceutical composition of the invention is administered after said modality.

In certain embodiments, the pharmaceutical compositions and modalities of the invention are administered to a subject periodically. Cyclic therapy involves administering a pharmaceutical composition of the invention for a period of time, followed by a pattern of administration for a period of time, and repeating this sequential administration. Cycling therapy can reduce the development of resistance to one or more treatments, avoid or reduce side effects of one of the treatments, and/or improve treatment outcomes. In such an embodiment, the present invention contemplates alternating administration of the pharmaceutical composition of the present invention after 4 to 6 days, preferably after 2 to 4 days, more preferably after 1 to 2 days, wherein this cycle can be repeated as desired repeatedly. In certain embodiments, the pharmaceutical compositions and modalities of the invention are administered alternately in less than 3 weeks, biweekly, every 10 days, or weekly. In certain embodiments, the composition of the invention is administered to the patient about every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

In a specific embodiment, the pharmaceutical composition of the invention is administered to the subject from one hour to twenty-four hours after the mode of administration. The time frame can be extended by at least a few days if a slow-release or sustained-release mode of delivery is used. In certain embodiments, as described above, the subject is administered a pharmaceutical composition of the invention on a regular basis throughout the remainder of the patient's life.

5.7. CD91 Activity Analysis

After its purification, the α2M-antigen molecule complex can be further characterized to measure its effect on CD91 activity and CD91 signaling pathway. For example, the α2M-antigen molecule complex can be characterized by testing the effect of the α2M-antigen molecule complex on the activity of CD91 cells. Such assays include downstream signaling assays, antigen presentation assays, antigen-specific activation assays of cytotoxic T cells, and more. Such assays can be used to test and/or measure immune responses.

In various embodiments, the effect of the α2M complex assay on innate CD91 signaling activity can be tested. For example, downstream signaling effects of CD91 activation that can be analyzed include, but are not limited to: increased motility and chemotaxis of macrophages (Forrester et al., 1983, Immunology, 50:251-259), negative regulation of protease synthesis , and intracellular calcium, inositol phosphate, and cyclic AMP (Misra et al., 1993, Biochem. J., 290:885-891). Other innate immune responses that can be tested are the release of cytokines (ie, IL-12, IL1β, GMCSF, and TNFα).

For example, in one embodiment, α2M complex candidates isolated from mammalian serum can be further characterized using chemotaxis assays. α2M modified by protease interactions is known to induce cell-directed movement towards its ligand. There are a number of techniques available for assaying chemotactic mobilization in vitro (see, for example, Leonard et al., 1995, "Measurement α and β Chemokines", in Current Protocols in Immunology, 6.12.1-6.12.28 Coligan et al., eds., John Wiley & Sons, Inc., 1995). For example, in one embodiment, candidate compounds can be tested for their ability to modulate the ability to induce movement of cells expressing α2MR receptors using a chemokine gradient in a multiwell Boyden chemotaxis chamber. In a specific example of this approach, serial dilutions of the α2M complexes identified in the primary screen were placed in the bottom wells of a Boyden chemotaxis chamber. A constant amount of ligand was also added to the dilution series. As a control, at least one aliquot contains ligand alone (eg α2M). The response of the α2M complex to α2MR was measured by comparing the number of cells that migrated to the lower surface of the membrane filter in an aliquot containing only the ligand (eg, α2M) to the number of cells in an aliquot containing the α2M polypeptide and ligand (eg, α2M). Contribution to Chemotactic Activity. Chemotactic activity of cells expressing α2M is determined if addition of the α2M complex to a solution of the ligand (such as α2M) results in a decrease in the number of cells tested relative to a solution containing only the ligand (such as α2M) Antagonists induced by ligands (such as α2M).

Increased intracellular ionized calcium ([Ca ²⁺ ] _i ) concentrations are also a hallmark of α2MR activation (Misra et al., 1993, supra). Thus, in another embodiment, calcium flux assays can be used as a secondary screen to further characterize the role of the α2M complex. Intracellular calcium ion concentrations of CD91 -expressing cells were measured in the presence of ligand, in the presence or absence of the α2M complex. For example, calcium flux can be detected and measured by flow cytometry labeling intracellularly captured fluorescent dyes. Fluorescent dyes, such as Indo-1, exhibit a change in emission spectrum upon binding calcium, and the ratio of the fluorescence produced by the calcium-bound dye to the fluorescence produced by the unbound dye can be used to assess intracellular calcium concentration. In a specific embodiment, cells are cultured and excited in cuvettes at 37° C. in Indo-1 containing medium, and fluorescence is measured using a fluorometer (Photon Technology Corporation, International). Ligand was added at specific time points in the presence or absence of the α2M complex, EGTA was added to the cuvette to release and sequester all calcium and the response was measured. Ligand binding causes an increase in the intracellular Ca2 ⁺ concentration in cells expressing α2MR. Agonists cause a relative increase in intracellular Ca2 ⁺ concentration, while antagonists cause a relative decrease in intracellular Ca2 ⁺ concentration.

In other embodiments, to detect or measure the activity of the α2M-antigen molecule complex, an antigen presentation assay can be performed to predict how the α2M-antigen molecule complex will function in vivo.

Such re-presentation assays are known in the art and have been described previously (Suto and Srivastava, 1995, Science, 269: 1585-1588). For example, in one embodiment, antigen-presenting cells, such as a macrophage cell line (eg, RAW264.7), are mixed with antigen-specific T cells in a medium, using about 10,000 cells of each type in a ratio of about 1:1. The complex of α2M and peptide antigen was added to the cells and incubated for about 20 hours. In one embodiment, an IFN-γ release assay can be used to measure or detect T cell responses. After washing with water, cells were fixed, permeabilized and reacted with a dye-labeled antibody reactive with human IFN-γ (PE-anti-IFN-γ). Samples were analyzed by flow cytometry using standard techniques. Alternatively, specific cytokine production by activated T cells can be detected using filter immunoassay, ELISA (enzyme-linked immunosorbent assay) or enzyme-linked immunoblot assay (ELISPOT). In one embodiment, for example, nitrocellulose-backed microtiter plates are coated with purified cytokine-specific primary antibodies, anti-IFN-γ, and the plates are blocked to avoid non-specific binding due to other proteins. generated background. Antigen-stimulated APC cell samples were diluted on wells of a microtiter plate. Add a labeled, eg biotinylated secondary anti-cytokine antibody. Thus enabling detection by methods such as enzyme-conjugated streptavidin, cytokine-secreting cells will appear as "spots" under visual, microscopic, or electronic detection methods to detect antibody cytokine complexes. In another embodiment, antigen-specific T-cells can be identified using the "tetramer staining" assay (Altman et al., 1996, Science, 274:94-96). For example, MHC molecules containing specific peptide antigens are polymerized to make soluble peptide tetramers and labeled by, for example, complexing to streptavidin. The MHC-peptide antigen complexes are then mixed with the stimulated T cell population. Biotin is then used to stain T-cells, which recognize and bind to the MHC-antigen complex.

5.8. Induction of tumor cell necrosis

In certain embodiments of the invention, it may be advantageous to induce tumor necrosis in a mammal prior to isolating the alpha(2) macroglobulin-antigen molecule complex from the animal. Applicants have demonstrated that inducing tumor necrosis prior to isolation of the complex increases the effectiveness of the complex in the treatment and prevention of cancer. See Section 6 Example Results. Applicants believe, without limiting itself to any one theory, that necrotic tumor cells release tumor-specific antigens. The released antigen molecules then enter body fluids such as blood and form complexes with alpha(2) macroglobulin.

A number of methods can be used to induce tumor necrosis. Administration of drugs that induce tumor cell necrosis is common in cancer treatment. Examples of tumor necrosis drugs include, but are not limited to, bleach, cisplatin/epinephrine injectable gel (Vogl et al., 2002, British Journal of Cancer, 86(4):524-529), ONO-4007, Lipid A Synthetic analogs (Satoh et al., 2002, Cancer Immunol. Immunother, 50(12):653-662), tumor necrosis factor (TNF), soluble ethanol (Burgener et al., 1987, Invest Radiol., 22(6): 472-478), anti-epidermal growth factor receptor (αEGFr) antibody (Wersall et al., 1997, Cancer Immunol. Immunother., 44(3):157-164), OK-432, lyophilized streptococci treated with penicillin (Ishiko et al., 1997, Int.J.Immunopharmacol., 19(7):405-412), adenovirus (ONYX-015) (Ganly et al., 2000, Clin.Cancer Res., 6(3):798 -806), IL4-PE chimeric protein (Rand et al., 2000, Clin.Cancer Res., 6 (6): 2157-2165), nimustine (ACNU) (Wakabayashi et al., 2001, 18 (1 ):23-28).

Tumor necrosis can also be achieved using other methods such as, but not limited to, photosensitization of tumors including photodynamic therapy using hypercin (Blank et al., 2001, Oncol. Res., 12(9-10409-418)), U.S. Pat. Tissue-selective microwave irradiation as described in 6,131,577, application of heat to tumor tissue to induce necrosis as described in U.S. Patent 5,928,159, laser heating of subsurface flesh as described in U.S. Patent 5,897,549, induction of Transurethral Focused Ultrasound Therapy for Prostate Tumor Necrosis, Silver Nitrate and Dextran Paste for Uterine Cancer as described in U.S. Patent 5,891,457, Pulsed Magnetic Radiation as described in U.S. Patent 5,776,175, U.S. Patent Non-ionizing radiation as described in 5,527,352, catheter with heating device as described in US Patent 5,492,529, endoscopic guidance of laser energy as described in US Patent 5,487,740, use of electrodes as described in US Patents 5,318,564, 4,186,729, and 4,237,898 Devices for heating selective tissue, Radiofrequency hyperthermia described in U.S. Pat. Immunoenhancing compositions described in Patent 5,149,527, injection of ferromagnetic particles near tumor tissue described in U.S. Patents 4,983,159 and 4,392,040 and 4,545,368, application of heat and radiation to tissue with a catheter described in U.S. Patent 4,763,671, and U.S. Patent 3,674,031 using a liquefied gas cooling medium to treat tissue.

In certain embodiments, inducing necrosis may be performed between one hour and two months prior to isolation of the alpha(2) macroglobulin complex. In specific embodiments, the induction may be 1 hour, 12 hours, 1 day, 3 days, 1 week, 3 weeks, 5 weeks, 1 month, or 2 months prior to isolation of the alpha(2) macroglobulin complex. Monthly. Induction of necrosis can be repeated as necessary, as will be understood by the skilled physician.

In certain embodiments, the patient is administered a chemotherapeutic agent prior to isolation of the alpha(2) macroglobulin-antigen molecule complex. In such embodiments, chemotherapeutic agents such as those described in Section 5.4 above may be used.

The above references describing the induction of tumor necrosis are incorporated herein by reference in their entirety.

5.9. Dosage regimen and formulation

The covalent or non-covalent complex of the α2M polypeptide of the present invention and an antigen molecule can be formulated into a pharmaceutical preparation for administration to mammals, for treating or preventing cancer or infectious diseases with an immunotherapeutically effective amount. Drug dissolution rate and site of absorption are factors that should be considered when selecting a route of administration for a therapeutic agent.

Toxicity and therapeutic efficacy of such compositions comprising the α2M complex can be tested by standard pharmaceutical methods in cell culture or experimental animals, eg, by measuring the _LD50 (median lethal dose in the population) and _ED50 (therapeutically effective dose in half the population). The dose ratio between toxic and therapeutic efficacy is the therapeutic index and it can be expressed as _LD50 / _ED50 . Alpha 2 macroglobulin complexes that exhibit large therapeutic indices are preferred. Although compounds that exhibit toxic side effects may also be used, care should be taken to design the delivery system so that such compositions including the α2M complex are directed to the site of the treated tissue, thereby minimizing potential damage to non-treated cells, thereby reducing side effects .

Preferred subjects or patients to be treated are mammals, which include but are not limited to domestic animals, such as cats and dogs; wild animals, including foxes and raccoons; livestock and poultry, including horses, cattle, sheep, turkeys, and chickens, and any rodent class animals. Most preferably, the subject is a human.

Data obtained from cell culture assays and animal studies can be used in the extrapolation of various dosages for human use. The dosage of such compositions including a2M complex lies preferably within a range of circulating concentrations that include the _ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration employed. For any composition comprising an [alpha]2M complex used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be calculated in animal models to achieve a circulating plasma concentration range that includes the _IC50 (ie, the concentration of the composition including the α2M complex that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

Selection of a preferred effective dose is determined by those skilled in the art based on consideration of a variety of factors known to those skilled in the art. Such factors include the specific form of the α2M complex-containing composition, and its pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc., which are typically used in routine development methods used in obtaining regulatory approval of pharmaceutical compounds determined in the process. Other factors in dosage considerations include the condition and disease to be treated or the benefit to be achieved in normal individuals; the body weight of the patient; the route of administration; whether the administration is short-term or chronic; concomitant medications; other factors. Thus, the precise dosage will be determined according to the judgment of the physician and each patient's circumstances, eg, according to the individual patient's condition and immune status, according to standard clinical techniques.

The alpha(2) macroglobulin complexes of the invention may optionally be administered with one or more adjuvants to enhance the immune response. For example, depending on the host species, the adjuvants that can be used include but are not limited to: inorganic salts or mineral gels such as aluminum hydroxide, aluminum phosphate and calcium phosphate; surface active substances such as lysolecithin, pluronic polyols ), polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol; immunostimulatory molecules such as cytokines, saponins (such as QS-21), muramyl dipeptide and tripeptide derivatives, CpG di Nucleotides, CpG oligonucleotides, monophosphatidyl lipid A, and polyphosphazenes; particulate and microparticle adjuvants such as emulsions, liposomes, virosomes, cochleates; or immunostimulatory complex mucosal adjuvants Adjuvants, Freund's adjuvants (complete and incomplete, and possible human adjuvants such as BCG (BCG) and corynebacterium parvum). In addition, the following patents and publications disclose immunostimulatory oligonucleotides including CpG oligonucleotides useful in the compositions of the present invention: U.S. Patents 6,207,646; 6,339,068; 6,239,116; 6,429,199; and PCT Patent Publications , WO 0122972, WO 00/06588 by Krieg et al; WO 01/12804 by Agrawal; WO 01/83503, WO 01/55370; WO 02/052002 by Fearon et al; WO 01/35991 by Tuck et al; WO 01/12223 to Schwartz; WO 99/62923 to Schwartz; WO 98/55495; U.S. Patent 6,406,705 to Davis et al; and PCT Patent Publication WO 02/26757 to Kandimalla et al, all of which are incorporated herein by reference in their entirety. Other suitable auxiliaries that can be used in the present invention can be found in A Compendium of Vaccine Adjuvants and Excipients (Second Edition), Vogel, F., Powell, M., and Alving, C.; in Vaccine Design-The Subunit and Adjuvant Approach, Powell, M., Newman, M., Burdman, J., Editors, Plenum Press, New York, 1995, pp. 141-227 and 2nd Meeting on Novel Adjuvants Currently In/Close to human Clinical Testing, World Health Organization-Organization Mondiale de 1a Sante Foundation Merieux, Annecy, France, 5-7 June 2000, Kenney, R., Rabinovich, N.R., Pichyangkul, S., Price, V., and Engers, H., Vaccine, 20(2002) 2155-63 . All of which are incorporated herein by reference.

[alpha](2) macroglobulin complexes of the invention may be administered using any desired route of administration including, but not limited to, for example, subcutaneous, intravenous or intramuscular injection, although intradermal or transmucosal administration is preferred. Advantages of intradermal or transmucosal administration include low doses employed and rapid absorption, respectively. Transmucosal routes of administration include, but are not limited to, oral, rectal, and nasal administration. Formulations for transmucosal administration are suitable in a variety of formulations as described below. The route of administration can be altered during the course of treatment.

The above dose is preferably administered once a week for about 4-6 weeks, and the preferred administration regimen and site are varied with each administration. In a preferred embodiment, subcutaneous administration is adopted, and the site of each administration is changed sequentially. Thus, for example, but not limited to, the first injection may be administered subcutaneously in the left arm, the second in the right arm, the third in the left abdomen, the fourth in the right abdomen, the fifth in the left thigh, the sixth in the Right share, and so on. The same site can be repeated after intervals of one or more injections. Also, separate injections can be administered. Thus, for example, half the dose on the same day may be administered at one site and the other half at another site.

Alternatively, the mode of administration is varied sequentially, such as weekly injections in successive subcutaneous, intramuscular, intravenous or intraperitoneal fashions.

After 4-6 weeks, administration is preferably at two-week intervals over a period of one month and multiple months. Subsequent injections may be administered monthly. Subsequent injection intervals may vary depending on the patient's clinical progression and response to immunotherapy.

Pharmaceutical compositions for use in the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients.

Compositions comprising covalent or non-covalent compositions comprising the α2M complex formulated in a pharmaceutically compatible carrier may be prepared, packaged and labeled for treatment of cancer or infectious disease. In a preferred aspect, the amount of the composition comprising the α2M complex administered to a human is about 1 μg to 5 mg, preferably 10 to 200 μg, preferably 10, 20, 25, 50, 100 or 200 μg. In a preferred embodiment, the composition of the present invention comprising the α2M complex is administered once a week for about 4-6 weeks, intradermally, with sequential changes in the site of administration. In a preferred embodiment, the composition of the invention comprising the α2M complex is administered as an initial treatment, or twelve hours to one week after in vivo treatment of diseased tissue to induce tissue necrosis.

If the complex is water soluble, it can be formulated in an appropriate buffer such as phosphate buffered saline or other physiologically compatible solution. Alternatively, if the resulting complex is poorly soluble in aqueous solvents, it can be formulated with a non-ionic surfactant such as Tween or polyethylene glycol. Thus, covalent or non-covalent and/or α2M complexes and physiologically acceptable solvates thereof can be formulated for administration by inhalation or insufflation (through the mouth or nose), or oral, buccal, Parenteral, rectal administration, or in the case of tumors, direct injection into solid tumors.

In a preferred embodiment, the composition of the invention comprising alpha(2) macroglobulin additionally comprises 9% sucrose, 5-10 mM potassium phosphate. In a related preferred embodiment, the composition of the invention has a pH of 7.

For oral administration, pharmaceutical preparations may be in liquid form, such as solutions, syrups or suspensions, or may be presented as a pharmaceutical product for reconstitution with water or other suitable vehicle before use. This liquid preparation can be prepared by conventional methods with pharmaceutically acceptable additives such as suspending agents (such as sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifiers (such as lecithin or acacia); non-aqueous medium (such as almond oil, oily esters, or rectified vegetable oil); and preservatives (such as methyl or propyl p-hydroxybenzoic acid or sorbic acid). The pharmaceutical composition can be, for example, in the form of tablets or capsules prepared by conventional methods with pharmaceutically acceptable excipients, such as binders (such as pregelatinized cornstarch, polyvinylpyrrolidone or hydroxypropylmethyl cellulose); fillers (such as lactose, microcrystalline cellulose, or dibasic calcium phosphate); lubricants (such as magnesium stearate, talc, or silicon dioxide); disintegrants (such as potato starch or sodium starch glycolate); or wetting agent (sodium lauryl sulfate). Tablets may be coated by methods well known in the art.

Oral formulations may be suitably formulated to provide controlled release of the composition including the α2M complex. Such compositions may be in the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, compositions comprising the α2M complex are conveniently dispensed from pressurized packs or nebulizers using a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. Administered as an aerosol spray. In the case of a pressurized aerosol, the dosage unit may be determined by a valve providing a valve releasing a metered amount. Capsules and kits such as gels for use in an inhaler or insufflator may be formulated comprising a powder mix of the a2M complex-containing composition with a suitable powder base such as lactose or starch.

Compositions comprising the α2M complex may be formulated for parenteral administration by injection, such as by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, eg, in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain ingredients such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg sterile pyrogen-free water, before use.

Compositions including the a2M complex may also be formulated in rectal compositions such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the aforementioned formulations, compositions including the α2M complex may also be formulated as depot formulations. Such long-acting formulations may be administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, compositions comprising the α2M complex may be mixed with suitable polymers or hydrophobic substances (e.g. as emulsions in acceptable oils) or ion exchange resins, or as sparingly soluble derivatives, e.g. as sparingly soluble salts Prepare together. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophilic drugs.

Compositions comprising the α2M complex may, if desired, be presented as a pack or dispenser device which may contain one or more unit dosage forms comprising the complex comprising covalent or non-covalent α2M and an antigenic molecule. Packaging may comprise, for example, metal or plastic films, such as blister packs. The pack or dispenser may be accompanied by instructions for use.

5.10. Measuring Vaccine Potency

The immunization efficacy of the composition of the present invention can be detected by monitoring the immune response of the test animal after immunization with the composition of the present invention or using any immunization assay known in the art. The production of a humoral (antibody) response and/or cell-mediated immunity can be used as an indicator of an immune response. Experimental animals may include mice, hamsters, dogs, cats, monkeys, rabbits, chimpanzees, etc., and finally human subjects. Methods of vaccination may include oral, intracerebral, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, or any other standard route of immunization. The immune response of the subject can be analyzed by various methods, for example: by known techniques such as immunosorbent assay (ELISA), immunoblotting, radioimmunoprecipitation, etc., or by protecting the immune host against cancer or infectious diseases The resulting immune sera were analyzed for reactivity to the antigen of interest.

As an example of a suitable animal test for vaccine-protected disease, the ability of the vaccines of the invention to induce an antibody response to the antigenic molecule can be tested in rabbits. Male specific pathogen free (SPF) young adult New Zealand white rabbits can be used. The test group each received a fixed concentration of vaccine. The control group received injections of 1 mM Tris-HCl pH 9.0 without antigen molecules. Blood samples can be drawn from the rabbit every week or two and the serum analyzed for antibodies to the antigenic molecule. The presence of antigen-specific antibodies can be assayed using, for example, an ELISA assay.

5.11. Medicine box

The invention also provides kits for practicing the treatment regimens of the invention. Such kits comprise a therapeutically or prophylactically effective amount of a covalent or non-covalent [alpha]2M complex in a pharmaceutically acceptable form in one or more containers. The α2M complex in the vial of the kit of the present invention may be in the form of a pharmaceutically acceptable solution, for example in combination with sterile saline, glucose solution, or buffer solution, or other pharmaceutically acceptable sterile solutions. Alternatively, the complex may be in lyophilized form or as a dry powder; in this case, the kit optionally additionally includes in a container a pharmaceutically acceptable solution (such as saline, dextrose solution, etc.), preferably a sterile solution, with Used to reconstitute the complex into a solution for injection.

In another embodiment, the kit of the invention additionally comprises a needle or syringe, preferably packaged in sterile form, for injection of the compound, and/or a packaged alcohol pad. Instructions are optionally included for administration of the α2M complex by a clinician or patient.

6. Example: Inhibition and reduction of tumors

The following results show that the α(2) macroglobulin complex can be completely purified from the serum of tumor-bearing animals and used to prevent and control cancer. Inbred mice were used as a model to examine the immune effect of complexes of α(2) macroglobulin complexed with tumor-derived antigenic molecules against subsequent challenge of live tumor cells. The following results support that the invention is particularly effective in autologous therapy involving the administration of alpha(2) macroglobulin complexes isolated from tumor-bearing mammals. The immune systems of the inbred mice used were identical or nearly identical, therefore, the results can be generalized to autologous therapies for cancer therapy.

Materials and methods

To purify α(2) macroglobulin-antigen molecule complexes, serum from mice was diluted 1:1 with 0.04M Tris pH 7.6, 0.15M NaCl. The mixture was then applied to a 65ml Sephacryl S300R (Sigma) column equilibrated and eluted with the same buffer. A 65ml column is used for approximately 10ml serum. α(2) macroglobulin positive fractions were detected by dot blot and the buffer was changed to 0.01 M sodium phosphate buffer at pH 7.5 using a PD-10 column. Alternatively, 0.01M sodium phosphate buffer at pH 7.5 can be used as the buffer in a 65ml column to save the step of changing the buffer. Fractions containing complexes were applied to a Concanavalin A Sepharose column. The bound complex was eluted with 0.2M methyl mannopyranoside or 5% methyl mannopyranoside and applied to a PD-10 column to change the buffer to 0.05M sodium acetate buffer at pH 6.0, and Apply to a DEAE column equilibrated with 0.01 M sodium acetate buffer, pH 6.0. α(2) macroglobulin was eluted in pure form with 0.13M sodium acetate buffer and analyzed by SDS-PAGE and immunoblotting.

In some experiments, α2M was purchased from SIGMA. BALB/c mice were obtained from Jackson Labs (Bar Harbor, ME) and used at 6-8 weeks of age.

To test whether the immunogenic α2M complexes could be used as a prophylactic against tumor formation, naive mice were immunized with 7 μg of the isolated α2M complexes, consisting of 5 mice per group, unless otherwise stated. All immunizations were performed intradermally in 100 μl PBS.

Sources of α2M antigen molecular complexes for immunization included: 1) serum from mice with intradermal 2 cm MethA tumors; 2) mice (15 small mice) 24 hours after direct injection of 50% bleach into 2 cm tumors. 3) serum of mice (15 mice) that induced ascites by intraperitoneal injection of live tumor cells; 4) serum of mice without tumor as a negative control; Sera from mice immunized with α2M of MethA tumor lysates <10 kDa peptide), because this complex previously showed protection against MethA tumor cell attack (Binder, 2002, Cancer Immunity, supra); 6) as Serum from mice immunized with gp96 complexed with MethA10 (called gp96-MethA10) as a positive control, which had also previously shown protection against attack by MethA tumor cells (Binder, 2002, CancerImmunity, supra); 7) Serum from mice immunized with PBS, called PBS1 as a negative control; 8) Serum from mice immunized with PBS, called PBS2 as a negative control; 9) Immunized with gp96 from liver as a negative control 10) use the serum of mice immunized with gp96 from MethA as a positive control, because it is known that gp96 complexes with antigenic molecules and stimulates immunity (Srivastava et al., 1986, supra); and 11) uses Sera from mice immunized with whole irradiated MethA tumor cells were used to test whether such cells could provide protection as a positive control.

Animals were then challenged intradermally with 100,000 viable tumor cells one week after the last immunization. Measure the area of the tumor. Tumor volume (mm ³ ) was calculated using half the mean as the radius of the tumor. Observations were made and recorded on days 7, 9, 12, 15 and 18.

result

Immunization results are shown in Figure 1 and Table 2. Negative control mice PBS 1 and PBS 2 showed no arrest or reduction in tumor development, whereas animals that had been immunized with α(2) macroglobulin complexes from tumor-bearing animals showed a decrease in tumor development at day 12 . At day 15, a clear difference in tumor volume was evident between the control group and animals immunized with α(2) macroglobulin complexes from tumor-bearing animals. Complexes isolated from the serum of tumor-bearing animals or from the serum of ascites mice were effective in preventing tumor growth in challenged mice. Complexes isolated from tumor-bearing mice with bleach injected into tumors to induce tumor necrosis showed particularly potent protection, with no tumor volumes exceeding 280 ^mm3 observed. It was also found that α(2) macroglobulin complexes from sera from mice with intradermal tumors inhibited tumor growth more effectively than complexes from sera from mice with ascites.

α(2) macroglobulin from normal mice had no or minimal protective effect.

Table 2

PBS

0 7 9 12 15 18

1 0 120.25 117.62 314.63 687.10 715.73

2 0 43.23 44.43 185.34 263.95 881.90

3 0 64.83 181.43 167.47 372.05 696.56

4 0 204.84 81.27 733.28 764.17 927.17

5 0 48.01 46.74 230 820.0 41136.55

whole cell

0 7 9 12 15 18

1 0 0 0 0 0 0 0

2 0 0 0 0 0 0 0

3 0 0 0 0 0 0 0

4 0 0 0 0 0 0 0

5 0 0 0 0 0 0 0

A2M Ascites 1

0 7 9 12 15 18

1 0 249.31 328.82 0 0 0 0

2 0 32.251 327.10 349.99 369.56 641.11

3 0 194.52 303.58 0 0 0

4 0 201.92 183.38 199.84 353.61 492.56

5 0 140.10 156.70 0 0 0 0

A2M Ascites 2

0 7 9 12 15 18

1 0 130.55 232.92 209.93 243.60 387.90

2 0 331.13 237.53 0 0 0

3 0 95.65 102.90 464.46 950.29 1217.38

4 0 108.85 83.56 0 0 0 0

5 0 91.17 128.38 132.42 234.29 350.59

A2M Ascites 3

0 7 9 12 15 18

1 0 19.76 231.09 160.95 293.33 407.51

2 0 98.46 130.55 180.27 560.63 143.72

3 0 139.78 115.32 0 0 0

4 0 139.13 253.64 0 0 0

A2M Bleach 1

0 7 9 12 15 18

1 0 106.66 209.07 56.80 65.02 118.79

2 0 104.77 67.60 0 0 0 0

3 0 64.44 87.78 25.89 0 0

4 0 217.27 211.64 0 0 0

5 0 134.00 216.39 0 0 0

A2M Bleach 2

0 7 9 12 15 18

1 0 172.29 149.43 0 0 0

2 0 192.90 185.34 0 0 0

3 0 157.75 280.19 0 0 0

4 0 177.21 270.97 0 0 0

5 0 86.12 301.95 0 0 0

A2M Bleach 3

0 7 9 12 15 18

1 0 24.72 82.86 180.27 0 0

2 0 6.97 0 0 0 0 0

3 0 60.26 42.05 0 0 0 0

4 0 4.38 7.70 0 0 0 0

A2M suffers from tumor 1

0 7 9 12 15 18

1 0 132.42 136.23 0 0 0

2 0 144.72 184.94 0 0 0

3 0 243.60 285.39 0 0 0

4 0 3.052 93.89 0 0 0 0

5 0 117.33 128.69 0 0 0

A2M has no tumor

0 7 9 12 15 18

1 0 149.09 255.58 331.71 507.79 605.82

2 0 103.17 73.37 65.42 0 0

3 0 123.22 203.17 346.40 527.27 605.82

4 0 117.62 297.08 119.96 158.11 220.78

5 0 101.84 129.31 249.78 382.78 448.69

Gp96 isolated from Meth A tumor cells

0 7 9 12 15 18

1 0 179.50 140.43 0 0 0

2 0 128.69 256.56 0 0 0

3 0 190.09 125.94 0 0 0

4 0 0127.46 0 0 0 0 0

5 0 148.08 91.66 85.65 350.59 523.33

10kDa peptide complexed with A2M

0 7 9 12 15 18

1 0 79.91 98.20 29.06 0 0

2 0 26.40 117.04 0 0 0

3 0 170.43 276.07 0 0 0

4 0 174.17 266.447 0 0 0

5 0 97.43 220.79 0 0 0 0

gp96 complex isolated from normal liver

0 7 9 12 15 18

1 0 96.41 180.27 399.59 528.85 795.92

2 0 137.19 274.02 318.56 650.14 795.92

3 0 150.80 235.68 327.67 503.19 696.56

4 0 142.40 124.12 305.77 650.14 1022.14

5 0 140.76 348.79 651.04 893.06 1149.76

10 kDa peptide in complex with gp96

0 7 9 12 15 18

1 0 150.45 168.21 0 0 0

2 0 223.44 312.96 0 0 0

3 0 95.14 173.42 0 0 0

4 0 212.07 138.16 0 0 0

5 0 145.39 84.72 0 0 0 0

PBS 2

0 7 9 12 15 18

1 0 213.36 227.47 175.31 305.22 605.82

2 0 157.05 44.43 362.75 550.48 696.56

3 0 19.15 181.43 317.44 881.90 1022.14

4 0 146.39 81.27 379.61 428.46 605.82

5 0 73.16 46.74 229.73 806.35 1149.76

in conclusion

The experimental results show that the α(2) macroglobulin complex helps to effectively treat or prevent cancer. The reduction in tumor volume proved that the treatment was effective. The prophylactic effect of administering the complex to animals prior to challenge with tumor cells was evident. The degree of efficacy observed in administering the complex to animals derived from animals first treated with a bleach that causes tumor cell necrosis is particularly unexpected and promising. Necrosis of tumor cells promotes the release of tumor-specific antigen molecules, which are then complexed with alpha(2) macroglobulin in body fluids.

The invention is not to be limited in scope by the specific embodiments described, which are intended as individual illustrations of individual aspects of the invention, functionally equivalent methods and components being within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are also within the scope of the appended claims.

All references, patents, and other publications cited herein are hereby incorporated by reference in their entirety.

Claims (48)

1. treat or prevent the method for patient's cancer or infectious disease, described method comprises that the patient to described treatment of needs or prevention uses α (2) macroglobulin of amount of effective treatment or prevention patient's cancer or infectious disease and the complex of antigen molecule, and wherein said complex separates from the mammiferous body fluid of suffering from cancer or infectious disease.

2. treat or prevent the method for patient's cancer or infectious disease, it comprises step:

A) isolate the complex of α (2) macroglobulin and antigen molecule from the mammiferous body fluid of suffering from described cancer or infectious disease; With

B) use effective treatment or prevent described patient's described cancer or the described isolated complex of the amount of infectious disease.

3. claim 1 or 2 method, wherein complex is the colony with the complex of the bonded α of different antigen molecules (2) macroglobulin, wherein different antigen molecules comprise a kind of described cancer or infectious disease specific antigen being had antigenic antigen molecule.

4. claim 1 or 2 method, its be used for the treatment of cancer and wherein the patient suffer from cancer, wherein antigen molecule comes from tumor.

5. claim 1 or 2 method, its be used for the treatment of cancer and wherein the patient suffer from cancer, the effectiveness of wherein said treatment of cancer reduces by the patient tumors size or the tumor decreased number is estimated.

6. claim 1 or 2 method, it is used for prophylaxis of cancer and wherein this prevention of needs of patients, and wherein said antigen molecule shows as has antigenic antigen to described cancer specific antigen.

7. claim 1 or 2 method, it is used to keep off infection and this prevention of needs of patients, and wherein said antigen molecule shows as has antigenic antigen to described infectious disease related diseases substance specific antigen.

8. the method for claim 2, its be used for the treatment of cancer and wherein the patient suffer from cancer, and this method is included in step (b) before or simultaneously described patient is used the step of chemotherapeutics in addition.

9. the method for claim 2, its be used for the treatment of cancer and wherein the patient suffer from cancer, and this method is included in the step that step (a) is induced described mammal tumor necrosis before or simultaneously in addition.

10. the method for claim 9, wherein the step of induced tumor necrosis comprises described administration neoplasm necrosis medicine.

11. stimulate the patient to resist the method for the immunne response of cancer or infectious disease, it comprises:

A) from mammiferous body fluid isolate α (2) macroglobulin complex and

B) patient is used isolated α (2) thus macroglobulin complex immune stimulatory is replied.

12. claim 1,2 or 11 method, wherein said patient is a human patients.

13. the method for claim 11, wherein α (2) macroglobulin complex is the colony with the complex of the bonded α of different antigen molecules (2) macroglobulin, and wherein different antigen molecules comprise a kind of described cancer or infectious disease specific antigen being had antigenic antigen molecule.

14. the method for claim 11, its be used to stimulate antitumor immune response and wherein the patient suffer from cancer, this method is included in the step that step (a) is induced described mammal tumor necrosis before or simultaneously in addition.

15. the method for claim 14, wherein the step of induced tumor necrosis comprises described administration neoplasm necrosis medicine.

16. the method for prevention mammalian cancer, described method comprises α (2) macroglobulin of using effective dose and the complex of antigen molecule, and described complex separates from described mammiferous body fluid, and described mammal suffers from precancerous lesion or polyp.

17. claim 1,2,11 or 16 method, wherein mammal is behaved.

18. prevent the method for first mammalian cancer, described method comprises α (2) macroglobulin of using effective dose and the complex of antigen molecule, described complex separates from the second mammiferous body fluid, and described second mammal suffers from precancerous lesion or polyp.

19. the method for claim 16 or 18, wherein complex comprises the multiple complex of the bonded α of different antigen molecules with one or more (2) macroglobulin, and wherein different antigen molecules comprise at least a described cancer or infectious disease specific antigen being had antigenic antigen molecule.

20. claim 1,2,11,16 or 18 method, wherein said complex are described patient from body.

21. claim 1,2,11,16 or 18 method, wherein said complex before using through purification.

22. claim 1,2,11,16 or 18 method, wherein said body fluid is the vascular fluid.

23. the method for claim 22, wherein the vascular fluid is the serum that comes autoblood.

24. claim 1,2,11,16 or 18 method, wherein body fluid is outer ascites or cerebrospinal fluid of vascular.

25. pharmaceutical composition, it comprises (a) from the isolated multiple α of the mammiferous body fluid of suffering from cancer or infectious disease (2) macroglobulin-antigenic molecule complexes, and described multiple complex comprises at least a α of comprising (2) macroglobulin and shows as the complex that tumor or infective agent specific antigen is had antigenic antigen molecule; (b) pharmaceutical carrier of effective dose.

26. the pharmaceutical composition of claim 25, wherein said body fluid are the vascular fluid.

27. the pharmaceutical composition of claim 26, wherein said vascular fluid is the serum that comes autoblood.

28. the pharmaceutical composition of claim 25, wherein said body fluid are outer ascites or cerebrospinal fluid of vascular.

29. vaccine, it comprises: (a) from the isolated multiple α of the mammiferous body fluid of suffering from precancerous lesion or polyp (2) macroglobulin-antigenic molecule complexes, described multiple complex comprises at least a α of comprising (2) macroglobulin and shows as the complex that tumor specific antigen is had antigenic antigen molecule; (b) pharmaceutical carrier of effective dose.

30. the vaccine of claim 29, wherein multiple α (2) macroglobulin-antigenic molecule complexes is through purification.

31. pharmaceutical composition, it comprises: (a) comprise α (2) macroglobulin and the complex with tumor or the antigenic antigen molecule of infectious disease; (b) be selected from following medicine: HSP-peptide complexes, antineoplastic agent, antibody, cytokine, antiviral agents, antifungal agent, antibiotic and chemotherapeutics; (c) pharmaceutical carrier of effective dose.

32. the pharmaceutical composition of claim 31, its Chinese medicine are chemotherapeutics.

33. the pharmaceutical composition of claim 31, wherein α (2) macroglobulin-antigenic molecule complexes is through purification.

34. comprise the method for the complex amount of α (2) macroglobulin and antigen molecule in the increase mammalian body fluid, described method comprises induces described mammiferous neoplasm necrosis.

35. the method for claim 34, wherein the step of induced tumor necrosis comprises administration neoplasm necrosis medicine.

36. medicine box, it comprises α covalently or non-covalently (2) macroglobulin-antigenic molecule complexes that pharmacy can be accepted the treatment of form or prevent effective dose in one or more containers.

37. comprise the method for the complex amount of α (2) macroglobulin and antigen molecule in the increase mammalian body fluid, described method comprises induces described mammiferous neoplasm necrosis.

38. preparation comprises α (2) macroglobulin and has the method for the complex of precancerous lesion, tumor or the antigenic antigen molecule of infectious disease that it comprises:

A) extract serum from the patient who suffers from precancerous lesion, tumor or infectious disease; With

B) reclaim α (2) macroglobulin-antigenic molecule complexes from described serum.

39. the method for claim 38, it is used for the treatment of or prophylaxis of cancer, and described method comprises the step of recovery complex of described patient being used the amount of effective treatment or prophylaxis of cancer in addition.

40. the method for claim 38, wherein the step from described serum recovery α (2) macroglobulin-antigenic molecule complexes comprises:

A) solid phase that comprises α (2) macroglobulin binding molecule being contacted with serum enough makes α (2) macroglobulin-antigenic molecule complexes be incorporated into the time of solid phase;

B) remove the material that is not incorporated into solid phase; With

C) from α (2) macroglobulin-antigenic molecule complexes of solid phase elution of bound.

41. the method for claim 39, wherein α (2) macroglobulin binding molecule is α (a 2) macroglobulin specific antibody.

42. the method for claim 39, wherein α (2) macroglobulin binding molecule is the part binding fragment of CD91.

43. preparation comprises α (2) macroglobulin and has the method for the complex of precancerous lesion, tumor or the antigenic antigen molecule of infectious disease that it comprises:

A) extract serum from the patient who suffers from precancerous lesion, tumor or infectious disease; With

B) fractionated serum is with enriching of alpha (2) macroglobulin-antigenic molecule complexes.

44. the method for claim 38 or 43, it comprises the step that serum is contacted with the medicine that promotes formation covalent bond between α (2) macroglobulin and the antigen molecule in addition.

45. the method for claim 44, its Chinese medicine are protease, ammonia, methylamine or ethamine.

46. the method for claim 38 or 43, wherein said body fluid are the vascular fluid.

47. the method for claim 46, wherein the vascular fluid is the serum that comes autoblood.

48. the method for claim 38 or 43, wherein body fluid is outer ascites or cerebrospinal fluid of vascular.

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