EP1534317A2 - N-terminale tronquee galectine-3 et anticorps pour le traitement du cancer - Google Patents

N-terminale tronquee galectine-3 et anticorps pour le traitement du cancer

Info

Publication number
EP1534317A2
EP1534317A2 EP02756157A EP02756157A EP1534317A2 EP 1534317 A2 EP1534317 A2 EP 1534317A2 EP 02756157 A EP02756157 A EP 02756157A EP 02756157 A EP02756157 A EP 02756157A EP 1534317 A2 EP1534317 A2 EP 1534317A2
Authority
EP
European Patent Office
Prior art keywords
galectin
terminally truncated
truncated galectin
protein
tumor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02756157A
Other languages
German (de)
English (en)
Other versions
EP1534317A4 (fr
Inventor
Gary A. MandalMed Inc. JARVIS
Constance M. MandalMed Inc. JOHN
Hakon MandalMed Inc. LEFFLER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mandalmed Inc
Original Assignee
Mandalmed Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mandalmed Inc filed Critical Mandalmed Inc
Publication of EP1534317A2 publication Critical patent/EP1534317A2/fr
Publication of EP1534317A4 publication Critical patent/EP1534317A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention relates to methods and compositions for treating cancer. More specifically, the present invention relates to a composition containing galectin-3 for treating cancer.
  • tumor cell metastasis is, in part, due to complex intercellular interactions involving adhesion and aggregation.
  • Specific classes of tumor proteins, lectins, which play a role in cell adhesion, are now known to be important in tumor formation and metastasis.
  • Lectins are, by definition, proteins with at least one carbohydrate- binding domain.
  • lectins By immobilizing monosaccharides, oligosaccharides, or glycoproteins in affinity columns, lectins have been isolated from tumor tissue extracts.
  • a tissue extract in acetone or the like is utilized to isolate the protein component from the lipid component.
  • the acetone is then evaporated, whereupon the residue is solublized in a buffered aqueous solution.
  • This solution is then passed through an affinity column containing the immobilized carbohydrates or glycoproteins.
  • a number of lectins, which selectively bind to galactosides, have been isolated in this manner.
  • Galectin-3 is one member of the family of lectins termed galectins, formerly known as S-type or S-Lac lectins. Galectins are classified as such due to their structural similarity and characteristic affinity for ⁇ -galactoside sugars (1, 2). The highest levels of galectin-3 are found in activated macrophages, basophils, mast cells, some epithelial cells, and sensory neurons. An early observation was that many tumor cells express galectins on their surface and that their expression could be involved in adhesion and invasion processes. Experimental evidence also suggested that these galectins could be cross-linked by an exogenous glycoprotein resulting in the aggregation of tumor cells. Based on these results, Raz and Lotan proposed that galectin-1 and galectin-3 could promote tumor metastasis (3). Since that time, the evidence for the role of galectin-3 in tumor adhesion, invasion and metastasis has mounted.
  • Galectin-3 is composed of three distinct structural motifs: a short amino terminal region of 12 amino acids, a sequence rich in Gly-X-Tyr tandem repeats characteristic of the collagen supergene family, and a carboxy- terminal half containing the globular carbohydrate recognition domain (CRD) (2, 4, 5).
  • CRD globular carbohydrate recognition domain
  • Galectins are thought to interact with various cell-surface and extracellular glycoproteins and glycolipids, thereby playing a role in cell adhesion, migration, and signaling.
  • the relationship between the intra- and extracellular functions of galectins can be of great biological importance.
  • a number of reviews of the biology of the galectins have been published (12- 16).
  • galectin-3C possibly could be localized in the nucleus as it is missing only the first 107 amino acids, if it is reuptaken by cells as is galectin-3.
  • Loss of nuclear localization of galectin-3 was observed in senescent human fibroblasts (18), and binding to synexin was found to be associated with its nuclear transport (19).
  • Galectin-3 shares the ability to be secreted despite the absence of a signal peptide with a number of other proteins that have unconventional intercellular transfer. These proteins are internalized by cells and are able to directly access the cytoplasm and the nucleus by a process that does not involve classical endocytosis (20).
  • Galectin-3 is isolated as a monomer but undergoes multimerization upon binding to surfaces that contain glycoconjugate ligands.
  • the ⁇ erminal half of the protein is required for this property (21 , 22).
  • the ⁇ /-terminal domain of the protein is required for galectin-3 to have affinity for multivalent carbohydrate ligands property (21 , 23) and to transmit intracellular signals (24, 25).
  • Galectin-3 promotes binding of cells to laminin and fibronectin, but the ⁇ /-terminally truncated protein does not promote such binding (26).
  • the /V-terminal domain appears to be necessary for the self-association of galectin-3 that is required for some of its biological functions.
  • Galectin-3 null cells were transfected to express recombinant galectin-3 and induced tumors within four weeks when injected into mice. When the same galectin-3 null cells were transfected to express a mutant galectin-3 that was lacking the 11 amino terminal amino acids, no tumors developed within four weeks (24).
  • a number of laboratories have studied the biology of galectin-3 that apparently is significant in cell growth, differentiation, adhesion, RNA processing, apoptosis, and malignant transformation (27).
  • Laminin is the major non-collagenous polypeptide of basement membranes, and galectin-3 binds preferentially to mouse tumor laminin compared to human placental laminin (28).
  • Galectin-3 has been shown to increase the binding of breast cancer cells to other extracellular matrix proteins (29, 30). In addition to increasing the binding of tumor cells to basement membranes, the interaction of cell surface galectin-3 with complementary serum glycoproteins appears to promote aggregation of tumor cells in circulation, thereby playing another important role in the pathogenesis of metastasis (31). Expression of recombinant galectin-3 in weakly metastatic fibrosarcoma cells resulted in an increased incidence of experimental lung metastases in syngeneic and nude mice (32). In human umbilical vein endothelial cells (HUVEC) galectin-3 induces angiogenesis (33).
  • HUVEC human umbilical vein endothelial cells
  • galectin-3 Increased expression of galectin-3 in human colon cancer cells resulted in increased metastases, and reduction in galectin-3 expression from antisense DNA was associated with decreased liver colonization and spontaneous metastasis in athymic nude mice (34).
  • Exogenous galectin-3 has been shown to increase invasiveness of human breast cancer cells (35), and to be a chemotactic factor for human umbilical vein endothelial cells (34).
  • galectin-3 in cancer is complicated, and a number of different laboratories have found that decreased expression of galectin-3 is associated with increased tumorigenicity and metastasis (37-40). Overall, the body of work regarding the biochemistry and function of galectin-3 provides a strong rationale for continued exploration of its therapeutic use in cancer.
  • Galectin-3 is not a member of the Bcl-2 family of proteins, but at residues 180-183 it contains the four amino acid motif (NWGR) conserved in the BH1 domain of the Bcl-2 family, and it has 48% sequence similarity with Bcl-2 (41). Galectin-3 has anti-apoptotic activity that is abrogated by substitution of the Gly182 residue with Ala in the NWGR motif (42, 43). In T cells galectin-3 interacts with Bcl-2 in a lactose inhibitable manner and confers resistance to apoptosis induced by anti-Fas antibody and staurosporine (41).
  • Galectin-3 has been found to improve cellular adhesion and prevent apoptosis induced by loss of cell anchorage (anoikis) (43-45). Contact with the extracellular matrix is required for suppression of apoptosis of epithelial cells from a number of tissues.
  • galectin-3 on the surface of tumor cells appears to contribute to tumor invasion and metastasis.
  • This premise is supported by the inhibition of spontaneous metastasis in a rat prostate cancer model by oral administration of modified citrus pectin, a complex polysaccharide rich in galactosyl residues.
  • Citrus pectin (pH modified) a plant fiber component, can directly bind galectin-3, and can interfere with carbohydrate-mediated cell-cell and cell-matrix interactions (46).
  • U.S. Patent No. 5, 681,923, to Platt discloses the active site of galactose binding proteins.
  • Two different peptide sequences are claimed that correspond to part of galectin-3 are claimed.
  • One peptide is composed of 25 amino acids corresponding to residues 171 to 196 of galectin-3.
  • the second peptide is 38 amino acids and corresponds to residues 158 to 196 of galectin- 3.
  • the X-ray crystal structure of the human galectin-3 carbohydrate recognition domain, in complex with lactose and ⁇ /-acetyllactosamine, has been published (8).
  • the carbohydrate recognition domain of galectin-3 extends from amino acid 117 to 250 with some of the residues between 144 and 184 directly involved in hydrogen binding to lactosaminylated substrates (8). It would instead be useful to develop a larger peptide region for use in treating cancer. A larger fragment would be likely to be more effective than the peptides that were disclosed in the Platt patent.
  • U.S. Patent No. 5,801,002, to Raz discloses sequences for the human galectin-3 protein.
  • the ⁇ /-terminal domain of the protein is critical for the multimerization of galectin-3 when it is bound to carbohydrate ligands.
  • it could be useful to develop a fragment of galectin-3 having a deleted N- terminal region so that it would still have the carbohydrate recognition domain and would still be able to bind to carbohydrate ligands for use in treating patients.
  • the Raz patent describes using peptides as therapeutic agents that correspond to at least 4 consecutive amino acid residues of galectin-3. However, in the Raz patent there is no indication that one or another of these would be more or less effective in the inhibition of metastasis.
  • U.S. Patent 5,895,784, to Raz et al. (1999) discloses the use of pH- modified citrus pectin to treat cancer. Additionally, the Raz et al. patent describes the function, structure, and expression of galectin-3. The inventors indicated that they used citrus pectin to study the properties of galectin-3 and claimed a method of treating cancer by oral administration of modified pectin that can bind to the carbohydrate domain of galectin-3 to reduce metastasis. There is no disclosure of a truncated form of galectin-3 that prevents tumorigenicity and metastasis.
  • WO 98/122139 PCT/US97/21807), to Huflejt et al. (1998), discloses the detection of human galectin-4 in various samples. Although the focus is on galectin-4, some of the methods described include the use of galectin-3 as controls. Again, there is no disclosure of a truncated form of galectin-3 that prevents tumorigenicity and metastasis.
  • U.S. Patent No. 5, 837,493, to Hillman et al. discloses some description of galectin-1, -2, and -3 including descriptions of the lectin structures and amino acid sequences. The sequence of the galectins and the use of galectins in the prevention of disease are disclosed. . However, there is no disclosure of a truncated form of galectin-3 that prevents tumorigenicity and metastasis. The regulatory mechanism that produces the variable localization of galectin-3 in different cell types is not understood, and the significance of the relative amounts of the protein found in the cytoplasm, or nucleus, or extracellular matrix of various cell types in terms of functionality is not understood.
  • galectin-3 is significant in some types of cancer.
  • the prior art is lacking in a methodology and a composition of galectin-3 that can be successfully used to treat cancer. It would therefore be useful to develop molecules, methods, and compositions based on an ⁇ -terminally truncated form of galectin-3 that inhibit multimerization and carbohydrate binding of galectin-3 and can be used successfully to reduce tumor growth and metastasis.
  • a composition having an effective amount of ⁇ /-terminally truncated galectin-3 in a pharmaceutically acceptable carrier or an effective amount of a nucleic acid sequence encoding ⁇ /-terminally truncated galectin-3 in a pharmaceutically acceptable carrier.
  • a method of treating cancer by administering to a patient in need of such treatment an effective amount of /V-terminally truncated galectin-3 in a pharmaceutically acceptable carrier or an effective amount of a nucleic acid sequence encoding ⁇ /-terminally truncated galectin-3 in a pharmaceutically acceptable carrier.
  • the present invention also provides an antibody that specifically binds to carbohydrate ligands of galectin-3.
  • an anti- cancer treatment having an effective amount of /V-terminally truncated galectin-3 and a pharmaceutically acceptable carrier or an effective amount of a nucleic acid sequence encoding /V-terminally truncated galectin-3 in a pharmaceutically acceptable carrier.
  • Figure 1 is a schematic showing a postulated mechanism of action of /V-terminally truncated galectin-3 whereby the truncated form of the protein inhibits the binding of intact galectin-3 to carbohydrate ligands and thereby also inhibits the multimerization and cross-linking activities of galectin-3;
  • Figure 2 is a graph showing the number of cells that have bound to laminin in the presence of galectin-3, /V-terminally truncated galectin-3 (galectin-3C), or a mixture of both galectin-3 and the /V-terminally truncated galectin-3; the number of cells was determined by a standard curve for the absorbance at 450 nm due to metabolism of WST-1 by a known number of MDA-MB-435 cells;
  • Figure 3 is a graph showing the toxicity of the therapy by plotting the weight change of the animals over time;
  • Figure 3A shows body weights of animals determined weekly during the course of the therapy study and data is presented as the mean and standard deviation of the body weights of the treatment and control groups;
  • Figure 3S shows body weight gains of animals exclusive of tumor weights at termination of therapy study, the body weights of the animals and their tumor weights were determined at the conclusion of the study and the weight of the tumor was subtracted from the final weight of the animal to calculate body weight gain;
  • Figure 4 presents pharmacokinetic analysis of the intramuscular administration of galectin-3C determined at 2-12 hours; mice were injected 35 with S-labeled galectin-3C
  • Figure 5 shows the organ biodistribution analysis of the subcutaneous and intravenous administration of galectin-3C; mice were injected with S- labeled galectin-3C and at eight hours post injection the animals were sacrificed and the heart, lungs, liver, kidneys, and spleen were removed and the amount of associated radioactivity was determined; background radioactivity was determined by measuring radioactive counts associated with organs from mice injected with vehicle only; background counts for each organ were subtracted from the presented data; data is given as the mean and standard deviation of the percentage of the dose injected that was detected in the organs of five mice;
  • Figure 6 is a graph of tumor volume over time showing the effect of galectin-3C treatment on the growth of primary orthotopic xenograph tumors in nude mice; mice were injected with either galectin-3C or vehicle control and the primary tumors were measured with calipers once per week after initiation of the treatment through the end of the 90 day treatment period; Tumor volume was calculated by the formula l l 2 x 112., where ⁇ N is the smallest dimension;
  • Figure 7 is a bar graph showing the effect of galectin-3C treatment on the weight of primary orthotopic xenograph tumors in nude mice; mice were injected with either galectin-3C or vehicle control and the weight of the primary tumors was measured at the end of the 90 day treatment period;
  • Figure 8 is graph of a repeated measures statistical model of the effect of galectin-3C treatment on the growth of primary orthotopic xenograph tumors in nude mice; a SAS Proc Mixed analysis was used to fit a linear mixed model with random effects for cage and mouse slope and intercept; the fitted model was quadratic on treatment day; plot of the fitted model for log tumor volume versus treatment day shows steady growth for the control group but regressive growth for the galectin-3C group;
  • Figure 9 is a graph showing the mean tumor volume versus the days of treatment with either the control, V-terminally truncated galectin-3 or galectin- 3;
  • Figure 10 is a series of photographs showing the efficacy evaluation of /V-terminally truncated galectin-3 against the GFP-Gene Transfected Human Breast Cancer MDA-MB435 in a mouse model;
  • Figures 11 A-D are photographs showing a representative histopathology of primary tumor and lymph node, liver and lung metastasis in mice treated with the control;
  • Figures 12 A-D are photographs showing a representative histopathology of primary tumor and lymph node, lung, and liver metastasis in mice treated with the control;
  • Figures 13 A-C are photographs showing a representative histopathology of primary tumor and lymph node, and lung metastasis in mice treated with /V-terminally truncated galectin-3;
  • Figures 14 A-C are photographs showing a representative histopathology of primary tumor, lymph node, and lung metastasis in mice treated with /V-terminally truncated galectin-3;
  • Figures 15 A-C are photographs showing a representative histopathology of primary tumor, lymph node, and lung metastasis in mice treated with galectin-3;
  • Figures 16 A-E are diagrams of polyethylene glycol diol with two free hydroxyls, monomethoxypolyethylene glycol or mPEG with a single reactive hydroxyl, mPEG-succinimidyl propionate that reacts with amino groups such as in lysine residues to produce stable amide linkages, and the sulfhydryl- selective PEGs, mPEG-maleimide and mPEG2-maleimide; and Figure 17 is a diagram representing the reaction between the sulfhydyl on a cysteine residue of a protein and mPEG-maleimide.
  • the present invention provides a method and composition for treating cancer by administering an effective amount of /V-terminally truncated galectin-3.
  • the V-terminally truncated galectin-3 is formulated for sustained or slow-release.
  • the /V-terminally truncated galectin-3 is placed in a pharmaceutically acceptable carrier prior to administration.
  • the present invention also provides a method and composition for treating cancer by administering an effective amount of antibody that specifically binds to carbohydrate ligands of galectin-3.
  • composition of the present invention includes a sequence encoding the /V-terminally truncated galectin-3 and analogues and homologues thereof and a pharmaceutically acceptable carrier.
  • the composition can be used as a gene therapy.
  • the present invention provides a chemical entity being /V- terminally truncated galectin-3 that binds lactose and that has been derivatized with one or more molecules of polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the present invention provides a chemical entity being /V-terminally truncated galectin-3 to which has been added one or more cysteine residues that have been also derivatized with PEG.
  • a class of chemical entities are provided these being antibodies that bind to complex saccharide ligands of galectin-3 and competitively inhibits the carbohydrate binding and, thereby, multimerization of galectin-3.
  • V-terminally truncated galectin-3 that has been formulated for sustained release using methodologies that are well known to those skilled in the art.
  • an N- terminally truncated galectin-3 lacking the /V-terminal 107 amino acids.
  • the /V-terminally truncated galectin-3 was produced by enzymatic cleavage of the recombinant protein followed by collection of the pharmacologically active fragment, in the preferred method, and that carries PEG.
  • Alternative methods of producing the /V-terminally truncated galectin-3 can also be used such methods are known to those of skill in the art.
  • /V-terminally truncated human galectin-3 molecules that differ slightly in length. These molecules can be somewhat longer or shorter than the 143 amino acid residue /V-terminally truncated galectin-3 that is lacking the /V-terminal 107 amino acids. The molecules have essentially the same ability to inhibit the carbohydrate binding and multimerization of galectin-3 and, therefore, inhibit tumorigenicity and metastasis in vivo.
  • the /V-terminally truncated human galectin-3 molecules that differ slightly in length also are derivatized with polyethylene glycol and are formulated for sustained or slow-release and placed in a pharmaceutically acceptable carrier.
  • the present invention provides /V-terminally truncated galectin-3 molecules that contain fusion tags on the C- or ⁇ /-terminus.
  • the tags include, but are not limited to, the commercially available His-Tag ® (Novagen, Madison, Wl) for pET vector constructs that can contain 6, 8, or 10 histidine residues. These vectors can be used with more than a dozen different fusion tags that possess a variety of unique binding qualities and endoprotease sites that allow for high yields and a variety of methods in that they can be used to increase the purity of recombinant protein.
  • the N- terminal tags can be cleaved off using enterokinase from Novagen, Roche (Indianapolis, IN) or Sigma (St.
  • C-terminal histidine ⁇ tag can be used for immobilized metal-affinity chromatography.
  • C-terminal histidinee tag is not immunogenic, does not alter the affinity of most proteins, and has been used in both preclinical animal and human clinical trials (47).
  • the methods for producing these slightly different versions of the /V-terminally truncated human galectin-3 protein are known to those of skill in the art.
  • Other pET vector expression systems available from Novagen, such as pET3, which contain restriction enzyme sites that are used to produce the desired product without any fusion tags, can also be used in accordance with the present invention.
  • the present invention provides the C-terminal domain of galectin-3 for use as a therapeutic agent.
  • the therapeutic agent is modified to be in a slow release form.
  • antibodies that bind to the complex saccharide ligands of galectin-3 and that act to inhibit the carbohydrate binding and multimerization of the protein as does the N- terminally truncated galectin-3 are also provided.
  • Proteins possess unique chemical and physical properties and the manufacture, formulation, and delivery of proteins represent significant challenges to pharmaceutical scientists (48). Proteins traditionally are administered by inconvenient modes such as intravenous injection or infusion, or subcutaneous injection and must be given frequently due to rapid degradation. Recent advances in clinically useful, sustained-release formulations for proteins allow less frequent administration. Examples of sustained-release formulation include, but are not limited to, injectable microspheres that encapsulate protein (49, 50). One such product approved for human use, is manufactured by Nutropin Depot and is produced by encapsulating human growth factors in biodegradable microspheres. This formulation requires only one or two doses a month, but can require more than one injection per dose.
  • microspheres by lyophilization of a protein-PEG aqueous mixture has been reported for sustained release formulation (51).
  • Polymers approved for use in humans for sustained release from biodegradable microsphere formulations include, but are not limited to, polylactides [poly(lactic acid), poly(glycolic acid), and poly(lactic-coglycolic) acid (52). Twice daily injections of the /V-terminally truncated galectin-3 molecule were used in the mouse model of metastatic human breast described in Example 1.
  • Formulation of the /V-terminally truncated galectin-3 in a slow release formulation reduces the frequency of administration required and therefore increases the convenience of the method, increases the efficacy of the treatment by increasing the levels that could be sustained in the body by the treatment, and reduces the cost.
  • Methods for making such formulations are known to those of skill in the art. More than two decades have passed since the first descriptions of the effect of the covalent attachment PEG on the circulating life and immunogenicity of proteins (53).
  • a PEGylated version of interferon-alpha by Schering-Plough Corporation was approved by the F.D.A. last year for use in the U.S. for chronic hepatitis C.
  • Administration of the PEGylated version of interferon-alpha is subcutaneous injection once weekly rather than three times weekly.
  • PEG modification of antibodies to tumors has been found to enhance penetration into the tumors and to increase the anti-tumor effects (57).
  • the circulating lives of single chain antibodies have been extended by conjugation with PEG to the carboxylic acid groups or the primary amines. An increase in the polymer length rather than total mass was found to be more effective for serum half-life extension (58).
  • PEG derivatives react with thiols such as the amino acid residue cysteine (56, 59). These include PEG-ortopyridyl-disulphide, PEG- malemide, and PEG-vinylsulphone.
  • thiols such as the amino acid residue cysteine (56, 59).
  • PEG-ortopyridyl-disulphide include PEG-ortopyridyl-disulphide, PEG- malemide, and PEG-vinylsulphone.
  • /V-terminally truncated human galectin-3 that is lacking the 107 amino acids on the N- terminus or is similar in size is derivatized on the single cysteine in the sequence (SEQ ID NO. 1).
  • the N-terminally truncated human galectin-3 can be produced with one or more cysteine residues on the N- terminus as described above (SEQ ID NO. 2).
  • one or more of the cysteine residues is derivatized with a thiol reactive PEG derivative.
  • Employing a branched PEG derivative and performing the derivatization in the presence of ligand can prevent active site residue derivatization due to steric hindrance (56).
  • steric hindrance 56
  • there is close homology between the galectin-3 proteins of different species but the number of /V-terminal tandem repeats differs, hence, the size of the proteins vary (6).
  • the epitopes of m urine monoclonal antibodies generated to human galectin-3 were all found to be within the first 45 amino acid residues of the N-terminus (18).
  • the /V-terminally truncated galectin-3 competitively inhibits the binding of galectin-3 to carbohydrates in the nucleus, cytoplasm, extracellular matrix and in cell-cell adhesions, and acts to prevent tumorigenicity and metastasis.
  • the non-carbohydrate binding /V-terminal domain of galectin-3 promotes multimerization of the protein, and enables it to cross link cancer cells to the matrix and other cells (21, 22).
  • the /V-terminal half of the protein is the most critical for homophilic interactions, although there is evidence that the carbohydrate recognition domain can contribute to cross-linking (60-62).
  • /V-terminally truncated galectin-3 Incess administered /V-terminally truncated galectin-3, in which the carbohydrate binding part of the protein has been removed, occupies the binding sites of endogenous galectin-3.
  • the /V-terminally truncated galectin-3 itself has little or no cross-linking activity and acts as a dominant-negative inhibitor of galectin-3. Therefore, /V-terminally truncated galectin-3 prevents the homophilic cross-linking of galectin-3 and other types of protein-protein binding interactions that promote tumorigenicity and metastasis.
  • Antibodies that bind to the carbohydrate ligands of galectin-3 also have this same effect.
  • Studies of the specificity of galectin-3 have been performed (10, 11, 63, 64).
  • Galectin-3 has specific affinity for polyNAc-lactosaminoglycan, a polymer of ⁇ (1,3)-linked LacNAc units that occurs on cell surfaces and extracellular matrices.
  • Oligosaccharides of the specific sequences recognized by galectin-3 can be conjugated and used to induce antibody production. The ability to produce antibody to oligosaccharide antigens is well established. This technique is used in immunotherapy using tumor-associated antigens to induce production by the patient's body of antibodies to tumors (65, 66).
  • Bispecific antibodies that are specific for a particular tumor type or tissue can be used to target the therapy (67-69).
  • those of skill in the art know methodology for induction, isolation, and purification of both polyclonal and monoclonal antibodies.
  • the /V-terminal domain is not required for oligosaccharide binding, but is necessary for positive cooperativity (21, 23).
  • the result of the positive cooperativity is that multimers of galectin-3 bind to substrate surfaces (21).
  • galectin-3 multimers can cross-link a tumor cell with the extracellular membrane.
  • the C-terminal fragment competitively inhibits the binding of galectin-3 to substrates.
  • soluble recombinant /V-terminally truncated galectin- 3 effectively competes with endogenous galectin-3 for carbohydrate binding sites in the extracellular matrix and cell-cell adhesions important in tumor invasion and metastasis.
  • the /V-terminal domain of galectin-3 promotes multimerization of the protein, and enables it to cross-link cancer cells to the matrix and other cells.
  • Excess administered /V-terminally truncated galectin-3 in which the /V-terminal part of the protein has been removed, occupies binding sites of endogenous galectin-3 and prevents its cross-linking activities.
  • /V-terminally truncated galectin-3 itself does not have significant cross-linking activity since it lacks the /V-terminal part of galectin-3, and acts like a dominant-negative inhibitor of galectin-3.
  • Recombinant /V-terminally truncated galectin-3 is efficacious for inhibition of tumor invasion and metastasis in cancer.
  • the mechanism shown in Figure 1 is the competitive inhibition by recombinant galectin-3 of the binding of the galectin-3 on the surface of metastatic cancer cells to laminin and other ⁇ -galactoside glycoconjugates in the extracellular matrix.
  • the N- terminal domain of galectin-3 promotes its multimerization and, thus, enables it to cross-link cancer cells to the matrix and to other cells.
  • truncated galectin-3 in which the /V-terminal part of the galectin- 3 has been removed, occupies the galectin-3 carbohydrate binding sites in the extracellular matrix and in cell-cell adhesions important in tumor invasion and metastasis.
  • This truncated version of galectin-3 itself does not have cross-linking activity since it lacks the /V-terminal domain of galectin-3.
  • /V-terminally truncated galectin-3 acts like a dominant-negative inhibitor of galectin-3 and prevents galectin-3 mediated binding of cells to the extracellular matrix and cell-cell adhesion as shown in Figure 1.
  • galectin-3 itself, but not the /V-terminaily truncated galectin-3 produced by collagenase digestion, promotes binding of cells to laminin and fibronectin (26).
  • the /V-terminally truncated galectin- 3 molecule by blocking the multimerization of the intact protein prevents the adhesion of tumor cells with one another and with the extracellular matrix.
  • prevention of the contact of cancer cells with the extracellular matrix can also lead to their programmed cell death or apoptosis that is induced by loss of cell anchorage (also called anoikis) (43, 44).
  • the amount of sustained release or PEGylated /V-terminally truncated galectin-3 or antibody, which is utilized in the composition of the present invention, is a sufficient amount to at least reduce tumor size. Alternatively, it can prevent or reduce metastasis of a tumor.
  • the tumors being treated can include breast cancer, prostate cancer, colon cancer, lung cancer, and all additional solid and liquid forms of cancer.
  • the compound of the present invention can be useful in preventing tumor growth.
  • the composition can prevent tumor growth by preventing tumors from forming in the first place.
  • lectins could be useful in treating tumors and preventing metastasis, accordingly, by creating a preventative treatment that prevents the long term growth of tumor cells in the body, the compound of the present invention can be useful in preventing initial tumor growth.
  • the amount of composition for treatment is based upon the body weight of the individual being treated and can be determined by individuals of skill in the art.
  • the present invention provides a method of treating cancer in a patient by administering to a patient in need of such treatment an effective amount of /V-terminally truncated galectin-3 that has been derivatized with one or more molecules of polyethylene glycol (PEG).
  • a second method of treating cancer in a patient is provided that is by administering to a patient an effective amount of /V-terminally truncated galectin-3 to which has been added one or more cysteine residues and that has been derivatized with PEG.
  • a third method of treating cancer in a patient is provided that is by administering to a patient an effective amount of an antibody that binds to complex saccharide ligands of galectin-3.
  • a fourth method of treating cancer in a patient is provided that is by administering to a patient an effective amount of N- terminally truncated galectin-3 that has been formulated for sustained release using methodologies that are well known to those skilled in the art.
  • the "effective amount" for purposes herein is thus determined by such considerations as are known in the art of cancer treatment wherein it must be effective to provide measurable improvement in persons given the treatment, and, in a preferred embodiment, complete recovery of the patient without the presence of cancer cells.
  • a pharmaceutically acceptable carrier in order to effectuate the treatment of the present invention, there is administered to a patient an effective amount of the sustained release form of the /V-terminally truncated galectin-3 or antibody to galectin-3 saccharide ligands in a pharmaceutically acceptable carrier.
  • Administration can occur intramuscularly, orally, intravenously, locally, subcutaneously, or in any other applicable mechanism known to those of skill in the art.
  • the mechanism of treatment varies depending upon the cancer that is being treated and can be best determined by those of skill in the art. For example, /V-terminally truncated galectin-3 was evaluated as a potential therapeutic agent for breast cancer based on the lectin galectin-3.
  • 35 terminally truncated galectin-3 was detected by metabolic labeling with S methionine prior to collagenase cleavage.
  • the maximum tolerated dose of /V-terminally truncated galectin-3 in nude mice was determined to be greater than 125 mg/kg without overt adverse effects.
  • the pharmacokinetic elimination half-life of /V-terminally truncated galectin-3 administered intramuscularly into nude mice was found to be 3.01 hours in the serum and 4.26 in the cellular fraction of the blood.
  • mice bearing orthotopically implanted tumors derived from breast cancer cell line MDA- MB435 was treated intramuscularly twice daily for 90 days with /V-terminally truncated galectin-3 or a vehicle control. It was found that the mean tumor volumes and weights were statistically significantly less in mice treated with /V-terminally truncated galectin-3 compared with control mice, and that fewer numbers of mice exhibited lymph node metastases in the treated group compared with the control group. It was therefore concluded that /V-terminally truncated galectin-3 is not overtly toxic and is efficacious in reducing metastases and tumor volumes and weights in primary tumors.
  • the compounds of the present invention can be utilized in a combination therapy. This can include adding to the pharmaceutically acceptable carrier additional chemotherapeutic compounds for further treatment of the cancer cells.
  • compositions for treating cancer are provided by the present invention.
  • compositions having /V-terminally truncated galectin-3, with or without one or more cysteines added to the /V-terminus, and that has been derivatized with PEG and that is in a pharmaceutically acceptable carrier consisting of an antibody that binds to the complex saccharide ligands of galectin-3 and that is in a pharmaceutically acceptable carrier.
  • Another composition provided by the present invention has /V-terminally truncated galectin-3 that is derivatized with another chemical or formulated for sustained release and that is in a pharmaceutically acceptable carrier.
  • the compound of the present invention is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners.
  • the pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art.
  • the doses can be single doses or multiple doses over a period of several days, but single doses are preferred.
  • the doses can be single doses or multiple doses over a period of several days.
  • the treatment generally has a length proportional to the length of the disease process and drug effectiveness and the patient species being treated.
  • One method for producing the /V-terminally truncated human galectin-3 includes first producing intact recombinant human galectin-3 that can be produced in Escherichia coll BI21 (DE3) transformed with pET3cGal3 (the pET3c plasmid containing the human galectin-3 coding DNA) as described previously (21). Next, the bacteria can be lysed by sonication and the galectin-3 protein can be purified by affinity chromatography on lactosyl- Sepharose (21) and dialyzed to remove lactose.
  • the product is then cleaved with Clostridium histiolyticum collagenase type VII (Sigma Chemical Co., St. Louis, MO). Specifically, the recombinant galectin-3 is incubated overnight at 37°C with a lectin:collagenase ratio of 20:1 (by weight) in 20 mM Tris-HCI, pH 7.4, 150 mM NaCI, and 1 mM CaCb. The resulting /V-terminally truncated galectin-3 can be purified by affinity chromatography on lactosyl-Sepharose. For storage and shipment, a procedure can be used involving dialysis against water followed by lyophilization.
  • the dry /V-terminally truncated galectin-3 powder can be stored at -20°C for various amounts of time, up to three months, the time being determined by those of skill in the art.
  • the retention of the carbohydrate binding activity of an aliquot can be ascertained by testing on a small lactosyl-Sepharose column. This establishes that the /V-terminally truncated galectin-3 can be stored and shipped as a lyophilized powder without losing activity.
  • amino acid sequence of the /V-terminally truncated recombinant human galectin-3 that is produced by exhaustive digestion with collagenase, and that can be produced by other cloning methods is designated as SEQ ID NO. 1 , and is as follows: gap agplivpynl plpggwprm litilgtvkp nanrialdfq rgndvafhfn prfnennrrv ivcntkldnnn wgreerqsvf pfesgkpfki qvlvepdhfk vavndahllq ynhrvkklne isklgisgdi dltsasytmi
  • amino acid sequence of the /V-terminally truncated recombinant human galectin-3 produced by cloning methods to contain one additional cysteine on the /V-terminus is designated as SEQ ID NO. 2, and is as follows: cgap agplivpynl plpggwprm litilgtvkp nanrialdfq rgndvafhfn prfnennrrv ivcntkldnnn wgreerqsvf pfesgkpfki qvlvepdhfk vavndahllq ynhrvkklne isklgisgdi dltsasytmi
  • a plasmid containing the complete galectin-3 coding sequence can be used as a template in a PCR reaction using primers designed to amplify the desired fragment.
  • the reverse primer defines the Oterminal protein sequence and does not differ in these procedures.
  • the non-underlined portion of the forward primer defines the ⁇ /-truncated version of the native galectin-3 that in this example begins with Gly-108 ("delta 1-107", starting at amino acid sequence glycine, alanine, proline, alanine, etc.).
  • the underlined sequences are added as tails and are used to fuse the PCR product with the pET32 Ek/LIC plasmid using the Ek/LIC ligation protocol (Novagen, Madison, Wl). This particular plasmid produces a fusion protein with a variety of unique binding qualities and endoprotease sites allowing for high yields and purity of the recombinant protein.
  • More than one cysteine can be introduced to the construct by simply including more cysteine codons (either tgt or tgc) to create a version of N- truncated galectin-3 having one or more cysteines where they should not interfere with cabohydrate binding, for example, at the N- or C-terminus.
  • E. coll BL21 (DE3) bacteria are transformed with the above-described construct and the bacteria can be used for protein production.
  • Expression is under the control of bacteriophage T7 transcription and can be induced by providing a source of T7 RNA polymerase, such as infection with a phage that carries the T7 RNA polymerase gene or moving the plasmid into a cell containing an expression host containing a copy of the T7 RNA polymerase gene.
  • PCR Polymerase chain reaction
  • Immunnassays are the preferred immunoassays employed to assess a specimen. ELISA assays are well known to those skilled in the art. Both polyclonal and monoclonal antibodies can be used in the assays.
  • immunoassays such as radioimmunoassays (RIA) can be used as are known to those in the art.
  • RIA radioimmunoassays
  • Available immunoassays are extensively described in the patent and scientific literature. See, for example, United States patents 3,791 ,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901 ,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011 ,771 and 5,281 ,521 as well as Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor, New York, 1989. Antibody Production
  • Antibodies can be monoclonal, polyclonal or recombinant. Conveniently, the antibodies can be prepared against the immunogen or portion thereof for example a synthetic peptide based on the sequence, or prepared recombinantly by cloning techniques or the natural gene product and/or portions thereof can be isolated and used as the immunogen. Immunogens can be used to produce antibodies by standard antibody production technology well known to those skilled in the art as described generally in Hariow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988 and Borrebaeck, Antibody Engineering - A Practical Guide, W.H. Freeman and Co., 1992.
  • Antibody fragments can also be prepared from the antibodies and include Fab, F(ab')2, and Fv by methods known to those skilled in the art.
  • a host such as a rabbit or goat
  • the immunogen or immunogen fragment generally with an adjuvant and, if necessary, coupled to a carrier
  • antibodies to the immunogen are collected from the sera.
  • the polyclonal antibody can be absorbed such that it is monospecific. That is, the sera can be absorbed against related immunogens so that no cross- reactive antibodies remain in the sera rendering it monospecific.
  • monoclonal antibodies involves hyperimmunization of an appropriate donor with the immunogen, generally a mouse, and isolation of spleenic antibody producing cells.
  • These cells are fused to a cell having immortality, such as a myeloma cell, to provide a fused cell hybrid that has immortality and secretes the required antibody.
  • the cells are then cultured, in bulk, and the monoclonal antibodies harvested from the culture media for use.
  • messenger RNAs from antibody producing B-Iymphocytes of animals, or hybridoma are reverse-transcribed to obtain complimentary DNAs (cDNAs).
  • Antibody cDNA which can be full or partial length, is amplified and cloned into a phage or a plasmid.
  • the cDNA can be a partial length of heavy and light chain cDNA, separated or connected by a linker.
  • the antibody, or antibody fragment is expressed using a suitable expression system to obtain recombinant antibody.
  • Antibody cDNA can also be obtained by screening pertinent expression libraries.
  • the antibody can be bound to a solid support substrate or conjugated with a detectable moiety or be both bound and conjugated as is well known in the art.
  • a solid support substrate for a general discussion of conjugation of fluorescent or enzymatic moieties see Johnstone & Thorpe, Immunochemistry in Practice, Blackwell Scientific Publications, Oxford, 1982.
  • the binding of antibodies to a solid support substrate is also well known in the art. (see for a general discussion Hariow & Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Publications, New York, 1988 and Borrebaeck, Antibody Engineering - A Practical Guide, W.H.
  • the detectable moieties contemplated with the present invention can include, but are not limited to, fluorescent, metallic, enzymatic and radioactive markers such as biotin, gold, ferritin, alkaline phosphatase, b-galactosidase, peroxidase, urease, fluorescein, rhodamine, tritium, 14 C and iodination.
  • fluorescent, metallic, enzymatic and radioactive markers such as biotin, gold, ferritin, alkaline phosphatase, b-galactosidase, peroxidase, urease, fluorescein, rhodamine, tritium, 14 C and iodination.
  • Deliv ry nf gene produ s/therapftutios (compound): The compound of the present invention is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners.
  • the pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art.
  • the compound of the present invention can be administered in various ways. It should be noted that it can be administered as the compound or as pharmaceutically acceptable salt and can be administered alone or as an active ingredient in combination with pharmaceutically acceptable carriers, diluents, adjuvants and vehicles.
  • the compounds can be administered orally, subcutaneously or parenterally including intravenous, intraarterial, intramuscular, intraperitoneally, and intranasal administration as well as intrathecal and infusion techniques. Implants of the compounds are also useful.
  • the patient being treated is a warm-blooded animal and, in particular, mammals including man.
  • the pharmaceutically acceptable carriers, diluents, adjuvants and vehicles as well as implant carriers generally refer to inert, non-toxic solid or liquid fillers, diluents or encapsulating material not reacting with the active ingredients of the invention. It is noted that humans are treated generally longer than the mice or other experimental animals exemplified herein that treatment has a length proportional to the length of the disease process and drug effectiveness.
  • the doses can be single doses or multiple doses over a period of several days, but single doses are preferred.
  • the doses can be single doses or multiple doses over a period of several days.
  • the treatment generally has a length proportional to the length of the disease process and drug effectiveness and the patient species being treated.
  • the pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • the carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Nonaqueous vehicles such as cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, can also be used as solvent systems for compound compositions.
  • various additives that enhance the stability, sterility, and isotonicity of the compositions including anti-microbial preservatives, anti-oxidants, chelating agents, and buffers, can be added.
  • anti-bacterial and anti-fungal agents for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • isotonic agents for example, sugars, sodium chloride, and the like.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used is compatible with the compounds.
  • Sterile injectable solutions can be prepared by incorporating the compounds utilized in practicing the present invention in the required amount of the appropriate solvent with several of the other ingredients, as desired.
  • a pharmacological formulation of the present invention can be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicle, adjuvants, additives, and diluents; or the compounds utilized in the present invention can be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, vectored delivery, iontophoretic, polymer matrices, liposomes, and microspheres.
  • Examples of delivery systems useful in the present invention include: 5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196. Many other such implants, delivery systems, and modules are well known to those skilled in the art.
  • a pharmacological formulation of the compound utilized in the present invention can be administered orally to the patient.
  • Conventional methods such as administering the compounds in tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like are usable.
  • Known techniques that deliver it orally or intravenously and retain the biological activity are preferred.
  • the compound of the present invention can be administered initially by intravenous injection to bring blood levels to a suitable level.
  • the patient's levels are then maintained by an oral dosage form, although other forms of administration, dependent upon the patient's condition and as indicated above, can be used.
  • the quantity to be administered varies for the patient being treated and varies from about 100 ng/kg of body weight to 100 mg/kg of body weight per day and preferably is from 10 mg/kg to 10 mg/kg per day.
  • Galectin-3 binds via its C-terminus carbohydrate recognition domain to binding sites in the extracellular matrix (1).
  • the goal of this research was the evaluation of a potential therapeutic agent for breast cancer based on galectin-3 lectin that acts directly to reduce metastases. Soluble recombinant /V-terminally truncated galectin-3 competes with endogenous galectin-3 for carbohydrate binding sites in the extracellular matrix and cell-cell adhesions important in tumor invasion and metastasis.
  • the /V-terminal domain of galectin-3 promotes multimerization of the protein, and enables it to cross link cancer cells to the matrix and other cells.
  • Excess administered /V-terminally truncated galectin-3 in which the /V-terminal part of the protein has been removed, occupies binding sites of endogenous galectin-3 and prevents its cross-linking activities.
  • /V-terminally truncated galectin-3 itself has less cross-linking activity since it lacks the /V-terminal part of galectin-3, and acts like a dominant- negative inhibitor of galectin-3, as shown in Figure 1.
  • the experiments establish that therapy with recombinant /V-terminally truncated galectin-3 is efficacious for inhibition of tumor invasion and metastasis in breast cancer.
  • the overall purpose was to determine the efficacy, safety, and mechanism of action of /V-terminally truncated galectin-3 in treatment of metastatic breast cancer using a nude mouse model of metastasis.
  • the carbohydrate recognition domain of galectin-3 ( ⁇ /-terminally truncated galectin-3) was produced as described previously (21). High yield expression (20-300 mg/liter culture) of active soluble galectin-3 can be obtained.
  • the intact recombinant galectin-3 was produced in Escherichia coll BI21/DE3 containing the pET3c plasmid (Novagen) with the human galectin-3 coding DNA (pET3cGal3).
  • Galectin encoding DNA can be amplified by PCR using galectin cDNA as template and primers that contain restriction sites for cloning of the product into the expression vector in the proper reading frame.
  • the PCR product is first cloned into the TA-vector (Invitrogen, Carlsbad, CA), the insert is released with the restriction enzymes corresponding to the primers, and it is finally cloned into the expression vector.
  • the expression construct is used to transform the proper host strain (e.g. E. coll BL21 for the pET system).
  • the organisms are lysed by sonication and the galectin-3 protein is purified by affinity chromatography on lactosyl-Sepharose (21).
  • the purified galectin-3 is dialyzed to remove lactose and cleaved with Clostridium histiolyticum collagenase type VII (Sigma).
  • the resulting /V-terminally truncated galectin-3 is purified again by affinity chromatography on lactosyl- Sepharose.
  • the dry /V-terminally truncated galectin-3 powder was stored at -20 °C for various amounts of time up to three months and the retention of the carbohydrate binding activity of an aliquot was ascertained by testing on a small lactosyl-Sepharose column.
  • Other batches (with or without enrichment in 1 ⁇ N) were analyzed by NMR- spectroscopy. This analysis confirmed that the protein had retained its proper folding. Therefore, /V-terminally truncated galectin-3 can be stored and shipped as a lyophilized powder without loosing activity.
  • the plasmid pET3cGal3 was transfected into E. coli B834 (Novagen), which is a methionine auxiotroph derived from BL21/DE3.
  • the E. coli was adapted for growth on M9 minimal medium supplemented with ampicillin (50 mg/ml) and methionine (40 mg/ml)(M9-Met) by passage on M9-Met plates three times.
  • S galectin-3 a colony from the last
  • 35 plate was inoculated into 0.5 liters of M9-Met supplemented with 1.0 mCi S- Met.
  • the bacteria were cultured, induced with IPTG and harvested as described previously (21). To lyse the radioactive E. coli, sonication was avoided because of aerosol formation.
  • Various alternative methods were tested and the following method was determined to be most efficient.
  • sucrose 25%) in 50 mM TrisHCI, pH 8.0 with 50 mM NaCI, 20 mM EDTA, and 8 mg lysozyme. After ten minutes on ice, 16 ml water was added and the sample kept on ice another 30 minutes.
  • the sample was centrifuged at 12000 rpm for 30 minutes and the supernatant applied to lactosyl-Sepharose.
  • the galectin-3 was eluted, dialyzed and treated with collagenase to generate /V-terminally truncated galectin-3 as described above.
  • a cellular adhesion assay was performed that is similar to that described previously (26) for use in 96-well microtiter plates.
  • the wells were coated with 50 microliters of 20 micrograms/ml of human laminin in PBS overnight at 4°C.
  • the wells then were washed once with minimum essential media (MEM), and then blocked for 1 hour at 37°C with 1% R.I.A. grade BSA in MEM, washed once with MEM containing 0.1% Tween-20 and twice with MEM.
  • MEM minimum essential media
  • MEM washed once with MEM containing 0.1% Tween-20 and twice with MEM.
  • To each well was added 4.5 x 10 4 human breast cancer cells, MDA- MB435 with various concentrations of galectin-3, the /V-terminally truncated galectin-3, or a mixture of both proteins.
  • the chicken polyclonal anti-N-terminally truncated galectin-3 was of very low affinity as determined by repeated nitrocellulose dot blots of galectin-3 and /V-terminally truncated galectin-3 following lactose elution of each protein from a lactosyl- Sepharose column.
  • galectin-3 and /V-terminally truncated galectin-3 protein in specific fractions from the column was confirmed by the measurement of UV absorbance at 280 nm.
  • Detection of anti-galectin-3 antibody (rat IgG) binding to galectin-3 was used as a positive control on a separate dot blot using anti-rat IgG labeled with alkaline phosphatase (AP).
  • AP alkaline phosphatase
  • an anti-chicken Ig antibody Zymed, South San Francisco labeled with biotin was used followed by AP-conjugated streptavidin and AP substrate.
  • a dose determination study was carried out in non-tumor bearing female athymic nude mice in order to establish the MTD of /V-terminally truncated galectin-3 using a single bolus dose.
  • the dose finding study comprised four dose groups with each group consisting of five mice.
  • the subcutaneous doses administered were 1 mg/kg, 5 mg/kg, 25 mg/kg, and 125 mg/kg.
  • a vehicle treated control group consisting of five mice was evaluated. No overt abnormal signs were observed within 48 hours of injection. Animals were observed for a total of five days after injection at which time body weight and viability were determined.
  • the animals were sacrificed and blood samples were obtained by terminal cardiac puncture at four time points: 2 hours, 4 hours, 8 hours, and 12 hours after injection.
  • blood samples were obtained from one control group of five animals 1 hour after injection of vehicle only (1 mg/ml lactose in PBS). Serum samples from 200 ⁇ l of blood were analyzed for radioactivity in triplicate.
  • mice For a direct comparison of the pharmacokinetic characteristics of intravenous versus intramuscular administration of /V-terminally truncated galectin-3, two groups of five mice each were injected with the mixture of 35 S-labeled /V-terminally truncated galectin-3 and unlabeled /V-terminally truncated galectin-3 either intravenously or intramuscularly. At eight hours post-injection serum and blood cell samples from 200 ⁇ l of blood were analyzed for radioactivity in triplicate. In addition, the organ biodistribution of /V-terminally truncated galectin-3 was determined in mice injected subcutaneously and intravenously
  • the distribution phase primarily determines the early rapid decline in plasma concentration of a drug after an intravenous dose. With time, equilibrium is reached in the distribution of the drug between the plasma and the tissues, and changes in plasma reflect proportional changes in all the other tissues.
  • the elimination phase after the rapid decline of the distribution phase, the decline in plasma concentration is due only to elimination of the drug from the body and is characterized by the elimination half-life (T1/2) (73, 74).
  • the elimination half-life is the time it takes for the concentration of the drug in the plasma (and body) to be reduced by one-half.
  • the apparent volume of distribution is the apparent volume of distribution of the drug in the body at equilibrium. The volume of distribution is equal to the amount of drug in the body at To divided by the plasma drug concentration at To.
  • mice Two groups of five mice were injected either subcutaneously or intravenously with the mixture of 35 S-labeled /V-terminally truncated galectin-3 and unlabeled /V-terminally truncated galectin-3.
  • a third group of five mice was injected intravenously with the lactose in PBS vehicle. At eight hours post-injection, the heart, lungs, liver, kidneys, and spleen were removed and the amount of associated radioactivity was measured.
  • the liver, kidney, and spleen were sources of /V-terminally truncated galectin- 3-associated radioactivity above the background vehicle level, whereas neither the heart nor lungs localized any V-terminally truncated galectin-3.
  • these data suggest that in addition to serum and blood cells, certain organs can localize /V-terminally truncated galectin-3 and possibly serve as reservoirs for eventual systemic release. Alternatively, these organs can function in the ultimate removal of /V-terminally truncated galectin-3 from circulation.
  • Test animals for the study were transplanted by surgical orthotopic implantation using fragments harvested from the subcutaneously growing tumors. The animals were anesthetized with isoflurane and the surgical area was sterilized using iodine solution and alcohol. An incision approximately 0.5 cm long was made on the second right mammary gland. The gland was then pulled out and two fragments of 1 mm 3 of MDA-MB435-GFP tumor tissue were sutured onto the gland with a sterile nylon 8-0 surgical suture.
  • mice were transplanted to compensate for possible postsurgical losses and tumor non-takes.
  • the orthotopically-transplanted animals used for the study were selected to establish groups of similar mean tumor size and body weight. Groups for each of the cohort conditions were randomly chosen. The cohort study was grouped as shown in Table 1 for a total of 45 mice. In addition, the production of 50 mg of galectin-3 allowed the testing of five mice with the intact protein. Administration of the treatments was begun when tumors reached palpable sizes.
  • the dosing schedule for the vehicle control and /V-terminally truncated galectin-3 groups was twice a day for the intramuscular injections with an approximately 6-8 hour interval between injections for a total 90 days.
  • the dosing for galectin-3 was once a day intramuscular injections.
  • Both N- terminally truncated galectin-3 and galectin-3 were injected as solutions of concentration 1 mg/ml in PBS containing 1 mg/ml lactose.
  • the vehicle control was PBS containing 1 mg/ml lactose.
  • control group had tumors that weighed more than the tumors in the /V-terminally truncated galectin-3 group as described below (tumor weight: control mean 2.34 g vs. treated mean 1.25 g).
  • the difference in body weights between the control group and the galectin-3 treated group was not statistically significant.
  • the primary tumors were measured by a pair of calipers once a week from initiation of treatment through the end of the study.
  • the mean tumor volume over time for galectin-3C compared to the vehicle-only control group is shown in Figure 6, and the galectin-3 treated group is included in Figure 9.
  • Table 2 shows a comparison of the mean tumor volumes of the mice in each group at the end of the treatment regime.
  • FIG. 10 shows imaging analyses of the 90-day efficacy test of N- terminally truncated galectin-3.
  • Tumor fragments of breast cancer cell line MDA-MB-435 expressing green fluorescent protein were orthotopically implanted into the breast pad of nude mice.
  • Real time, quantitative measurement of tumor growth, metastases, and micrometastases were performed using whole-body optical imaging.
  • the images are representative of the external images of the development of the breast tumor.
  • all visible primary tumors were removed and weighed using an electronic balance. Table 3 shows a comparison of the mean tumor weights at autopsy of mice in each treatment group.
  • the mean tumor weights of the vehicle-only control and the galectin-3C treated groups are shown graphically in Figure 7.
  • Tables 5 and 6 show the metastatic data as a function of tumor volumes categorized as small, medium, and large. A comparison of the two Tables reveals that five out of the eight small tumors in the control group had associated metastases while 0 of the 15 small tumors in the /V-terminally truncated galectin-3 group had associated metastases. This shows that a reduction in tumor volume by /V-terminally truncated galectin-3 results in a decreased incidence of metastases.
  • an analysis of tumor volume growth over time was performed using a repeated measures statistical model for log tumor volume, with cage in random statement.
  • this was a SAS Proc Mixed analysis to fit a linear mixed model with random effects for cage and mouse slope and intercept.
  • the fitted model was quadratic in treatment day.
  • the model was applied to the vehicle control and /V-terminally truncated galectin-3 treatment data for tumor volume that showed a significant difference at day 90 when tested with the Mann-Whitney U test.
  • the model allowed us to sensitize the analysis by factoring out non-treatment parameters such as cage and mouse variation, tumor volume trajectory differences within a group, and residual unexplained variation.
  • the mean tumor volume in the group of mice treated with /V-terminally truncated galectin-3 is statistically significantly less than that in the vehicle control group during more than 50% of the treatment period.
  • day 45 of treatment which is labeled as TxDayCenter 0 on the x- axis
  • treatment with /V-terminally truncated galectin-3 resulted in increased differences between the slopes and trajectories of the two lines representing the mean tumor volumes of the two groups of mice.
  • This model strongly supports the conclusion that /V-terminally truncated galectin-3 was efficacious in reducing the number of metastases and as well as tumor growth over time in the orthotopic implantation mouse model of breast cancer.
  • the maximum tolerated dose of /V-terminally truncated galectin-3 in nude mice at five days was determined to be greater than 125 mg/kg without overt adverse effects. This suggested that the safety threshold for the use of
  • mice were treated twice daily with /V-terminally truncated galectin-3 for 90 days and the results were compared with the treatment efficacy of mice treated with vehicle only. At the end of the treatment regime, it was found that the mean tumor volume and mean tumor weight were statistically significantly less in the group of mice treated with /V-terminally truncated galectin-3 compared with the group of mice treated with the vehicle control.
  • orthotopically transplanted tumors are more clinically relevant than subcutaneous xenograft models due to the physiological location of the tumor and the spontaneous hematogenous metastases (78).
  • Treatment with galectin-3C reduced the percentage of animals with metastases from 55% (11/20) in the control group to 20% (4/20) in the treated group, a decrease of 2.75-fold. None of the animals in the treated group with small tumors less than 1500 mm 3 in size (0/15) had metastases, whereas 62.5% (5/8) of the control group that had tumors this size had metastases. Only one animal in the treated group had a tumor larger than 1500 mm 3 that had not metastasized compared to 6 animals in the control group that had tumors of this size that had no metastases. Apparently animals in the treated group that had metastatic tumors were not responding to galectin-3C as their tumors were larger as well.
  • /V-terminally truncated galectin-3 also reduced tumor volume and tumor weight in the primary tumors.
  • the mechanism of action of extracellular /V-terminally truncated galectin-3 was likely to be at least partly due to: 1) decreased homotypic aggregation and adhesion of breast cancer cells to extracellular matrices and endothelial cells; 2) decreased chemotaxis (galectin-3 itself is a chemotactic factor) (79); or 3) anoikis (apoptosis induced by loss of cell anchorage) (43).
  • galectin-3 in nude mice using S-labeled /V-terminally truncated galectin-3 allowed the accurate determination of the in vivo pharmacokinetics of intramuscularly injected /V-terminally truncated galectin-3.
  • N- terminally truncated galectin-3 exhibited no detectable toxicity and reduced tumor volume, tumor weight, and incidence of metastases in a nude mouse model of breast cancer strongly supports the use of /V-terminally truncated galectin-3 as a therapeutic compound for the treatment of breast cancer and other types of cancer.
  • the results indicate that galectin-3C has minimal overt toxicity and that it has utility in treatment of breast cancer by shrinking tumors and reducing tumor growth over time, and by inhibiting metastasis.
  • an antibody to the specific complex carbohydrate ligands of galectin-3 has a longer half-life than galectin-3C and inhibits galectin-3 binding and multimerization as does the /V-terminally truncated galectin-3 protein.
  • Such an antibody also increases the efficacy obtainable.
  • the rationale for either a sustained release form of the protein or an antibody to galectin-3 carbohydrate ligands is the same, to increase the efficacy, cost- effectiveness, and convenience of the therapy.
  • Breast cancer is the most frequently diagnosed cancer in women in the
  • PEG-adenosine deaminase PEG-ADA
  • PEG-ADA PEG-adenosine deaminase
  • Second to be approved was PEGylated asparaginase, used for the treatment of acute lymphoblastic leukemia.
  • the PEGylated version of interferon-alpha has been approved for human use (83).
  • a drawback of PEGylation can be reduced biological activity and this has been found to occur with many proteins (56, 85).
  • Derivatization of the active site of an enzyme or a residue in a biologically important binding domain of a protein can lead to inactivation.
  • Site-specific modification a one or a few residues of the protein can be used to avoid this problem.
  • Steric hindrance obtained with a bulky PEG derivative can be used to decrease reactivity at some sites.
  • Performing the derivatization reaction in the presence of ligand or substrate can decrease reactivity of some amino acid residues.
  • Lowered stoichiometry of PEG derivatives can also be exploited to alter product ratios.
  • the pH of the reaction can be altered in derivatization with amino reactive PEGs to limit their reactivity.
  • One method for forming derivative proteins is to use PEGs that react with nitrogens on lysine or terminal amines to form stable amide linkages, such as mPEG-succinimidyl propionate or butanoate. With reagents of this type, the use of a basic pH in the reaction can result in a mixture of products with PEGylation at all lysine residues where lower pH can limit the derivatization to a single residue.
  • Site-specific modification of a single His residue of interferon-alpha-2B was achieved using a reaction with succinimidyl carbonyl PEG at pH 5.4 (86). Using N-hydroxysuccinimidyl carboxymethyl at pH 5, PEG enabled site-specific modification of the N- terminus of human granulocyte stimulating factor (87).
  • One method for derivatizing proteins with PEG is to use thiol-reactive PEGs that forms covalent bonds with cysteine residues, such as mPEG- maleimide (Shearwater Corporation, Huntsville, AL) (56, 57, 59).
  • the endogenous cysteine (Cys-173 of human galectin-3) of /V-terminally truncated galectin-3 can be used as a site for covalent modification with PEG.
  • one or more additional cysteines can be added to either the C- or the /V-terminal end of the /V-terminally truncated galectin-3 protein.
  • the cysteines can be used as sites to react with the thiol- reactive PEGS.
  • the /V-terminally truncated galectin-3, with and without additional cysteines added by genetic engineering can be produced by cloning methods.
  • the relative short half-life of the intramuscularly injected /V-terminally truncated galectin-3, the lack of acute toxicity of higher doses, and the efficacy demonstrated by the 4 mg/kg dose given twice daily provides clear rationale for formulation of a sustained release form of the protein, for example using a PEG derivatization, to increase the half-life.
  • the sustained release form enables higher levels of the protein to be sustained in the body and thereby obtains greater efficacy with more convenience and less expense.
  • One method to achieve site specific derivatization is to use sulfhydryl reactive PEG derivatives as shown in Figure 15 and 16.
  • the N- terminally truncated galectin-3, with and without additional cysteines added by genetic engineering, can be produced by known cloning methods.
  • a plasmid containing the complete galectin-3 coding sequence is used as a template in a PCR reaction using primers designed to amplify the desired fragment.
  • Forward primer 5' GAOGAC ACAAGtg ⁇ GCGCCCCTfiCTGGG 3'
  • Reverse primer 5' GAGGAGAAGCCCGGTTTATATCATGGTATA 3'
  • the reverse primer defines the carboxy-terminal protein sequence and in this example does not differ from that required to produce recombinant human galectin-3.
  • the non-underlined portion of the forward primer 5'- GACGACGACAAGt ⁇ cggcgcccctgctgggccactg-3' introduces a cysteine immediately upstream of the ⁇ Mruncated sequence providing an additional site on which PEG can be attached (starting first with a cysteine then continuing with the rest of the amino acid sequence glycine, alanine, proline, alanine, etc.).
  • the underlined sequences are added as tails that can be used to fuse the PCR product with pET32 Ek/LIC plasmid using the Ek/LIC ligation protocol (Novagen, Madison, Wl).
  • This plasmid produces a fusion protein with a variety of unique binding qualities and endoprotease sites allowing for high yields and purity of the recombinant protein.
  • More than one cysteine can be introduced to the construct by simply including more cysteine codons (either tgt or tgc) to create a version of /V-truncated galectin-3 with multiple cysteines.
  • One or more cysteines can be added to either the C- or N- terminus for derivatization with PEG using this method.
  • the site of PEGylation can effect the carbohydrate binding or the biological localization of the truncated galectin-3 molecules and the bioactivity of the molecule can vary accordingly.
  • the relative affinity for carbohydrate ligands and the localization of derivatized truncated galectin-3 molecules can be determined readily by those with skill in the art using standard methodologies.
  • the E. coli BL21 (DE3) bacteria is transformed with this construct and can be used for protein production.
  • Expression is under the control of bacteriophage T7 transcription and is induced by providing a source of T7 RNA polymerase, such as infection with a phage that carries the T7 RNA polymerase gene or moving the plasmid into a cell containing an expression host containing a copy of the T7 RNA polymerase gene.
  • the PCR products are purified using spin columns, and then ligated into pET32 following the vendor's instructions.
  • the ligated product is transformed into competent Novablue bacteria and clones are selected based on blue-white selection.
  • DNA was extracted from picked clones and analyzed by gel electrophoresis, PCR, and sequencing.
  • the plasmid is sequenced using the same forward and reverse primers as PCR using an ABI 3700 machine. This DNA is used to transform BL21 strain of E. coli. Isolated colonies were picked, grown in culture, and glycerol stocks are stored for future use.
  • Luria-Bertani (LB) broth with ampicillin (50 mg/l) was inoculated with 20 ⁇ l/L of glycerol frozen stock of transformed E. coli.
  • the culture is shaken at 37°C overnight.
  • the inducer, isopropyl- ⁇ -thiogalactopryranoside (IPTG) is added (100 mg/l) and the culture is incubated at 37°C for an additional four hours.
  • the cells are centrifuged to pellet them, and the supernatant discarded, and the pellet is stored at -20°C.
  • Lysis of Bacteria To improve reproducibility and yield of protein a commercially available cell lysis reagent is used. First the cell pellet is thawed at room temperature, and then B-Per Bacterial Protein Extraction Reagent (Pierce Endogen, Rockford, IL) was added (25 ml/500 ml) with 1 mM phenylmethanesulfonyl fluoride (PMSF; Sigma, St. Louis, MO), a serine protease inhibitor. The cells are resuspended by vortexing until there were no clumps, and then the vials are shaken at room temperature for 10 minutes, and spun at 27,000 g for 10 minutes. The supernatant is the cell lysate containing the soluble proteins and was stored at 4°C.
  • the protein product is purified by affinity chromatography on a lactosyl-
  • Sepharose column Bacterial lysates are loaded and the column washed with 5 volumes of phosphate-buffered saline (PBS) with 4-mM 2-mercaptoethanol, 2mM EDTA, and 0.5 mM PMSF.
  • PBS phosphate-buffered saline
  • the galectin-3 proteins are eluted with 10 volumes of PBS with 150-mM lactose and 0.5 mM PMSF.
  • the amount of purified protein is determined using the BioRad Protein Assay (Hercules, CA) according to the vendor's instructions.
  • the /V-terminal fusion tags are cleaved by digestion with porcine enterokinase enzyme (Sigma) at 0.7 U per mg of galectin-3 protein. The digestion is performed in 20 mM TRIS at pH 5.6 at room temperature for 24- hours.
  • Example 3 Polymeric microspheres have been used in formulations of proteins to achieve sustained delivery, and have been approved as part of several different products for humans.
  • Biodegradable poly(lactic-co-glycolic acid) (PLGA) has been widely used as a material for microencapsulation to attain sustained release (88-91).
  • the microspheres are produced by a number of techniques, including freeze-drying or atomizing with gas anti-solvent CO2 precipitation (92).
  • the resultant nanoparticles are analyzed by scanning electron microscopy.
  • a modification of the procedure of Lam et al. was used to achieve a product that produced controlled release of nerve growth factor over a period of 14 days (89).
  • Purified delta-107 galectin-3 is formulated at 5 mg/ml in two buffer systems and then lyophilized.
  • the buffer systems can be a 5 mM histidine at pH 5.5 or a 4 mM sodium bicarbonate at pH 7.4 with various concentrations of zinc acetate. Lactose at 5 mg/ml is also added to the buffers.
  • PLGA 1.7 g; 50:50 lactide/glycolide, 12 kDa, RG502H; Boehringer Ingelheim Chemicals (Winchester, VA) is added.
  • the stability of the lyophilized protein is determined by adding 5 mg of protein from each of the protein formulations to 0.5 ml of ethyl acetate. The samples are homogenized at 8,000 rpm for one minute and then the protein is recovered by dilution into 50-fold excess of 5 mM histidine, pH 5.5. Ultraviolet spectroscopy (280 nm) and affinity chromatography are used to assess the amount and the stability of the protein. The lyophilized formulations containing the greatest amount of recoverable protein are chosen for freeze-drying for encapsulation by PLGA microspheres.
  • PLGA (1.7 g) is stirred into 10 ml of ethyl acetate at 5°C and then solid delta107-galectin-3 (10% w/w), and zinc carbonate (0-6% w/w), a release modifier, are added.
  • the suspension is homogenized at 8,000 rpm for one minute, and then poured into a 10-ml glass syringe and infused into an ultrasonic spray nozzle using a pump.
  • the frequency of the spray is 120 kHz into a 2-L round-bottomed flask immersed in a liquid nitrogen bath and containing 300 ml of ethanol frozen underneath liquid nitrogen. The frozen microspheres form upon spraying and then settle on the top of the solid ethanol.
  • the flask is stored at -70°C where the ethanol melted and the ethyl acetate is extracted by the ethanol. After 24 hours, 300 ml of cold ethanol is added, and then after another two days a 20-micron filter is used to removed the ethanol from the microsphere suspension for drying. The microspheres are dried using nitrogen gas at 5°C, and then sieved with a 125-micron stainless steel mesh.
  • In vivo release is determined by resuspending the microspheres in PBS and incubating them at 37°C in the reservoir of a centrifugal filtration device. After release, the delta107 galectin-3 is obtained by centrifugation, and additional PBS release buffer is added. The integrity of the protein is determined by confirmation of the carbohydrate binding capacity to lactosyl- Sepharose.
  • the carbohydrate-binding sites that are recognized by galectin-3 are widely expressed in tissues and on cells in various species (10, 11 , 63, 64).
  • Antibodies that have high affinity for the complex carbohydrates recognized by galectin-3 specifically block the binding of galectin-3 to these sites in the body.
  • Such antibodies have the ability to block metastasis by blocking the multimerization and protein-protein interactions of galectin-3.
  • the effects of the antibodies are similar to the effects obtained with /V-terminally truncated galectin-3C. In other words, the antibodies reduced metastasis and tumorigencity.
  • antibodies that have affinity and specificity to molecules that are commonly expressed in the body are more difficult to produce.
  • a successful alternate approach to use of the carbohydrate ligands as antigens themselves to induce the formation of specific, high-affinity antibodies is to use phage-display libraries of random peptide sequences to select for peptide mimics of the carbohydrate structures that are then conjugated to highly immunogenic proteins (93-96). Then the peptide conjugates are used to generate antibodies. Initially, the peptides are expressed as a library in the coat proteins of bacteriophage. Thus, binding to the virion can be used to assess affinity for the peptide expressed. The DNA encoding the peptide of interest can be sequenced readily and, thus, the structure of the peptide determined. Clones of interest are chosen by a process of enrichment called bio-panning.
  • the protein is immobilized on a plate or on beads and incubated with the phage- display library. Unbound phage are washed away, and the bound phage are eluted. The eluted phage are amplified by being taken through the cycle of panning several times to enrich for sequences that bind. Individual clones are characterized by DNA sequencing.
  • Ishikawa and Taki derived the sequence of peptides from phage clones that mimicked nLc 4 Cer, a ligand of galectin-3 that has a nonreducing terminal N- acetyllactosamine unit linked beta1-3 to lactose (paraglobosyl; lacto-/V- neotetraose).
  • a library of random peptides (7 or 12-mers) is displayed when fused to a coat protein of M13 Phage.
  • the libraries are available from New England BioLabs (Beverly, MA) and are screened for binding to N-terminally truncated galectin-3 following the instructions of the vendor.
  • the library of 7-mers (Ph.D.- 7) contains 2.0 x 10 9 independent clones that represent most of the 20 7 of 1.28 x 10 9 possibilities
  • the library of 12-mers (Ph.D. - 12) contains a similar number of independent clones, 1.9 x 10 9 , but this is not exemplary of the possible number of 12-mer sequences (20 12 or 4.1 x 10 i5) .
  • the protein can be immobilized to a carboxymethylated dextran-coated biosensor cuvette (Affinity Sensors, Cambridge, U.K.).
  • the phage (2 x 10 11 ) is reacted with the immobilized protein for 60 minutes at 25°C in PBS with 0.5% (v/v) Tween-20, and then washed to remove unbound phage.
  • the bound phage is disassociated from the protein by treatment with acidic TRIS-glycine buffer and then neutralized with 1 M TRIS. Phage is amplified by transfection of E. coli strain E 3257 that is supplied by New England Biolabs and titered. The phage from this bio-panning is titered and bio-panned a third and a fourth time.
  • the phage is plated without amplification on a lawn of E. coli, and individual phage (25-30) are picked from the lawn.
  • the DNA is sequenced and the translated peptides for each clone are then determined. Typically, redundant and consensus peptides are derived. The peptides expressed by the greatest number of clones are synthesized.
  • Microtiter wells are coated overnight at 4°C with about 10 11 CsCI- purified virions, 1 mg of immune-radio assay (I.R.A.) grade bovine serum albumin, or buffer alone. After washing with TRIS-buffered saline with Tween- 20, various concentrations of galectin-3 protein is added and the plates incubated for 12 hours at 4°C. After washing three times, monoclonal antibody to galectin-3 (97) conjugated to FITC is added, and the plate incubated for another 1 hour at 37°C and washed again. An ELISA plate reader equipped with fluorescence detection is used to determine the relative degree of binding to the virions. The ability of known carbohydrate ligands of galectin-3 to inhibit the binding can be determined.
  • I.R.A. immune-radio assay
  • the peptides or consensus peptides with the greatest affinity for galectin-3 can be conjugated to diphtheria toxoid or keyhole limpet hemocyanin and the conjugate can be used to immunize mice or other species.
  • Monoclonal antibodies are produced using methodologies that are standard to those who are skilled in the art (reviewed in (98)). Isolation of B- cell clones expressing antibody molecules of a desired specificity and affinity are modulated by the immunogenicity of the antigen used for immunization. Size, complexity, and foreignness to the host all contribute to the immunogenicity of a molecule. In general, immunogens must have a molecular mass greater than 10 kDa. Globular proteins tend to be more immunogenic than carbohydrates, lipids, or nucleic acids.
  • peptides are not immunogenic due to their small size so they must be conjugated to carrier proteins such as bovine serum albumin or keyhole limpet hemocyanin.
  • carrier proteins such as bovine serum albumin or keyhole limpet hemocyanin.
  • cross-linking reagents are commercially available for this purpose.
  • the use of peptide conjugates as immunogens results in the expansion of peptide-specific B-cell clones and clones specific for the conjugated or fused protein.
  • Test bleeds are analyzed before trying to generate the desired monoclonal antibodies because a high titer, specific response of the appropriate immunoglobulin isotype indicates clonal expansion.
  • the immunogenicity of antigen can be enhanced by conjugation to immunogenic carrier molecules, emulsification in adjuvant, or both. Soluble proteins are emulsified in Freunds adjuvant or RIBI Adjuvant System. These adjuvants slowly release antigen over time and induce inflammatory responses. Conjugating soluble antigens to agarose beads or other particulates can stimulate phagocytosis and presentation of antigenic peptides to T- lymphocytes.
  • the sera from the immunized animal is used to isolate antibody- forming B cells that are fused with a mutant myeloma cell line that has been mutagenized and selected to be defective in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) or thymidine kinase (TK) so that it does not grow in HAT (cell medium containing hypoxanthine, aminopterin, and thymidine).
  • HGPRT hypoxanthine-guanine phosphoribosyltransferase
  • TK thymidine kinase
  • the aminopterin blocks normal nucleotide synthesis and the enzyme deficiency blocks utilization of hypoxanthine or thymidine in the salvage pathway.
  • the hybrids are able to synthesize DNA and grow in HAT medium.
  • the fused cells are monoclonal antibody producing hybridomas.
  • the fused hybridomas are selected in vitro by growth in HAT medium and then "cloned" so that each well of a microtiter plate contains the progeny of one cell.
  • the supematants of the cells are screened for the presence of antibody with the desired specificity for peptide mimic and for the complex carbohydrate ligands of galectin-3.
  • Screening can be done using an enzyme- linked immunosorbent assay (ELISA), whole cell ELISA, immunoprecipitation, or immunoblot. Further purification of a hybridoma from a positive well is performed by cloning in soft agar or limiting dilution. Those clones that produce antibody with the desired affinity are expanded for freezing and generation of stock solutions of monoclonal antibody. Monoclonal antibodies then are generated by tissue culture or ascites production, usually in mice. Antibody concentrations in tissue culture supematants are usually in the range of 10-20 ⁇ g/ml, but can be as high as 100 ⁇ g/ml.
  • ELISA enzyme- linked immunosorbent assay
  • monoclonal antibody can be generated as ascites fluid in a peritoneal cavity of an animal that is MHC compatible with the myeloma fusion partner and spleen cell donor. As much as 10 mg/ml of monoclonal antibody can be secreted into abdominal ascitic fluid (98).
  • the structure must be humanized to reduce its immunogenicity and prevent the development of human anti-mouse antibodies (99-103).
  • Dozens of rodent antibodies have been humanized, there are many humanized monoclonal antibodies in clinical trials, and now a few of these have been approved as drugs.
  • the rodent complementary determining regions (CDRs) of the antibody are grafted into the human IgG framework.
  • CDRs complementary determining regions
  • Antibody fragments such as F(ab')2, Fab', Fab, and scFV are useful as therapeutic agents (104). These are often expressed by bacterial cultures that have shorter expression times, higher yields, and lower costs than mammalian culture systems. Full length IgG has a longer half-life than antibody fragments, but PEGylation of antibody fragments can be used to increase their circulating half-life.
  • Galectins a family of animal beta-galactoside-binding lectins [letter], Cell. 76, 597-8.
  • Galectin-3 induces endothelial cell morphogenesis and angiogenesis, Am d Pathol. 156, 899-909.
  • Galectin-3 a novel antiapoptotic molecule with a functional BH1 (NWGR) domain of Bcl-2 family, Cancer Res. 57, 5272-6.
  • Matarrese, P., Tinari, N., Semeraro, M., Natoli, C, lacobelli, S. & Malorni, W. (2000) Galectin-3 overexpression protects from cell damage and death by influencing mitochrondrial homeostatis, FEBS letters. 473, 311- 315.
  • Mac-2 a novel 32,000 Mr mouse macrophagesubpopulation-specific antigen defined by monoclonal antibodies., d Immunol. 128, 1221-1228.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne une composition présentant une quantité efficace de galectine-3, tronquée au niveau de son extrémité N-terminale, dans un excipient pharmaceutiquement acceptable ou une quantité efficace d'une séquence d'acide nucléique codant pour la galectine-3, tronquée au niveau de son extrémité N-terminale, dans un excipient pharmaceutiquement acceptable. La présente invention concerne également une méthode de traitement du cancer, consistant à administrer à un patient nécessitant un tel traitement une quantité efficace de galectine-3, tronquée au niveau de son extrémité N-terminale, dans un excipient pharmaceutiquement acceptable ou une quantité efficace d'une séquence d'acide nucléique codant pour la galectine-3, tronquée au niveau de son extrémité N-terminale, dans un excipient pharmaceutiquement acceptable. Cette invention concerne en outre un anticorps, se liant spécifiquement à des ligands glucidiques de la galectine-3, un traitement contre le cancer renfermant une quantité efficace de galectine-3, tronquée au niveau de son extrémité N-terminale, dans un excipient pharmaceutiquement acceptable ou une quantité efficace d'une séquence d'acide nucléique codant pour la galectine-3, tronquée au niveau de son extrémité N-terminale, dans un excipient pharmaceutiquement acceptable.
EP02756157A 2001-06-08 2002-06-10 N-terminale tronquee galectine-3 et anticorps pour le traitement du cancer Withdrawn EP1534317A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US29697001P 2001-06-08 2001-06-08
US296970P 2001-06-08
PCT/US2002/018478 WO2002100343A2 (fr) 2001-06-08 2002-06-10 Galectine-3, tronquee au niveau de son extremite n-terminale, a liberation prolongee et anticorps aux ligands glucidiques de la galectine-3 a utiliser dans le traitement du cancer

Publications (2)

Publication Number Publication Date
EP1534317A2 true EP1534317A2 (fr) 2005-06-01
EP1534317A4 EP1534317A4 (fr) 2005-10-12

Family

ID=23144316

Family Applications (1)

Application Number Title Priority Date Filing Date
EP02756157A Withdrawn EP1534317A4 (fr) 2001-06-08 2002-06-10 N-terminale tronquee galectine-3 et anticorps pour le traitement du cancer

Country Status (4)

Country Link
EP (1) EP1534317A4 (fr)
AU (1) AU2002322066A1 (fr)
CA (1) CA2488850A1 (fr)
WO (1) WO2002100343A2 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7432331B2 (en) 2002-12-31 2008-10-07 Nektar Therapeutics Al, Corporation Hydrolytically stable maleimide-terminated polymers
EP1617849B1 (fr) 2003-04-07 2008-06-18 Prospect Therapeutics, Inc. Composition et utilisation d'antagonistes de la galectine
CA2742265C (fr) * 2008-10-29 2014-07-15 Bg Medicine, Inc. Immunodosage de galectine-3
CN102488619B (zh) 2011-12-05 2014-08-06 上海交通大学 连续生产艾塞那肽微球的装置及控制微球释放速度的方法
JP2016507503A (ja) 2012-12-20 2016-03-10 ヘンリー フォード ヘルス システム ガレクチン−３を阻害することによる拡張期心不全を治療する方法
WO2015085207A1 (fr) * 2013-12-05 2015-06-11 Texas Tech University System Galectine-3 pour le traitement du cancer des ovaires
WO2015117127A1 (fr) * 2014-02-03 2015-08-06 Texas Tech University System Inhibiteur de galectine 3 (gal-3) associé à des effets anti-myélome et anti-tumeurs solides additifs, à une ostéoclastogenèse et une protection d'organes diminuées lorsqu'il est utilisé en combinaison avec des inhibiteurs du protéasome
CA2959660A1 (fr) 2014-09-03 2016-03-10 Genesegues, Inc. Nanoparticules therapeutiques et compositions, procedes et systemes associes

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986006407A1 (fr) * 1985-04-23 1986-11-06 Medical Biology Institute IDENTIFICATION D'UNE PROTEINE DE LIAISON IgE PAR CLONAGE MOLECULAIRE
WO2001068123A2 (fr) * 2000-03-13 2001-09-20 La Jolla Institute For Allergy And Immunology Activite chimiotactique de la galectine-3 a l'egard des monocytes

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020019009A1 (en) * 1999-12-09 2002-02-14 Roggen Erwin Ludo High throughput screening (HTS) assays

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986006407A1 (fr) * 1985-04-23 1986-11-06 Medical Biology Institute IDENTIFICATION D'UNE PROTEINE DE LIAISON IgE PAR CLONAGE MOLECULAIRE
WO2001068123A2 (fr) * 2000-03-13 2001-09-20 La Jolla Institute For Allergy And Immunology Activite chimiotactique de la galectine-3 a l'egard des monocytes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
RAZ A ET AL: "Expression of two different endogenous galactoside-binding lectins sharing sequence homology." CANCER RESEARCH. 1 FEB 1988, vol. 48, no. 3, 1 February 1988 (1988-02-01), pages 645-649, XP008051054 ISSN: 0008-5472 *
SANO H ET AL: "Human galectin-3 is a novel chemoattractant for monocytes and macrophages." JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 15 AUG 2000, vol. 165, no. 4, 15 August 2000 (2000-08-15), pages 2156-2164, XP002341212 ISSN: 0022-1767 *
See also references of WO02100343A2 *

Also Published As

Publication number Publication date
CA2488850A1 (fr) 2002-12-19
EP1534317A4 (fr) 2005-10-12
AU2002322066A1 (en) 2002-12-23
WO2002100343A2 (fr) 2002-12-19
WO2002100343A3 (fr) 2005-03-17

Similar Documents

Publication Publication Date Title
US7820797B2 (en) Glycolipopeptide antibodies
US6770622B2 (en) N-terminally truncated galectin-3 for use in treating cancer
JP6676589B2 (ja) 薬物動態特性の改善されたコンプスタチンアナログ
US20150299290A1 (en) Immunogenic vaccine
JP2021113226A (ja) ｐ９７のフラグメントおよびその使用
US20140212473A1 (en) Glycopeptide and uses thereof
DK2457920T3 (en) Oligosaccharides comprising an amino oxy group and conjugates thereof
ES2563748T3 (es) Derivados de amiloide P sérico y su preparación y uso
US20060241022A1 (en) Selectin targeting bioconjugates
CZ304876B6 (cs) Fragment Aβ vázaný k nosičovému peptidu, farmaceutický prostředek pro prevenci nebo léčení onemocnění spojeného s amyloidními deposity Aβ v mozku pacienta jej obsahující a jeho použití
US6238667B1 (en) Method of affinity cross-linking biologically active immunogenic peptides to antibodies
WO2013151649A1 (fr) Composés d'interaction avec des glycanes
JP2000506493A (ja) 癌治療における擬似ペプチド
US20050181987A1 (en) Compositions and methods for treatment of cancer
US6245334B1 (en) Method of causing selective immunosuppression using HL-60-related lectins
JP2008515975A (ja) 抗癌化合物および方法
WO1994001131A1 (fr) Apport transvasculaire et intracellulaire de proteines lipidisees
TW201825122A (zh) 用精胺酸耗盡劑進行之組合癌症免疫療法
US20140322332A1 (en) Antagonists of muc1
JP2022541582A (ja) 自己免疫障害を処置するための組成物および方法
WO2022221692A1 (fr) Prophylaxie et thérapie anticancéreuses faisant appel à des nanoparticules virales ciblées
WO2002100343A2 (fr) Galectine-3, tronquee au niveau de son extremite n-terminale, a liberation prolongee et anticorps aux ligands glucidiques de la galectine-3 a utiliser dans le traitement du cancer
US20050032673A1 (en) Sustained release N-terminally truncated galectin-3 and antibodies to galectin-3 carbohydrate ligands for use in treating disease
WO1994011497A1 (fr) Procede d'obtention d'une immunosuppression selective a l'aide de lectines apparentees a hl-60
US5527775A (en) Reduction of mammalian neoplasms with phospholipase A2 activating substances

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20040105

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

A4 Supplementary search report drawn up and despatched

Effective date: 20050829

RIC1 Information provided on ipc code assigned before grant

Ipc: 7A 61P 35/00 B

Ipc: 7C 07K 14/47 B

Ipc: 7A 61K 38/17 B

Ipc: 7C 07H 21/04 B

Ipc: 7C 07K 16/32 B

Ipc: 7C 07K 14/435 B

Ipc: 7A 61K 39/395 B

Ipc: 7A 61K 48/00 B

Ipc: 7A 61K 38/16 A

DAX Request for extension of the european patent (deleted)
17Q First examination report despatched

Effective date: 20070911

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20080122