WO2000027420A9 - Compositions et methodes de traitement du cancer - Google Patents

Compositions et methodes de traitement du cancer

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Publication number
WO2000027420A9
WO2000027420A9 PCT/US1999/026277 US9926277W WO0027420A9 WO 2000027420 A9 WO2000027420 A9 WO 2000027420A9 US 9926277 W US9926277 W US 9926277W WO 0027420 A9 WO0027420 A9 WO 0027420A9
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Prior art keywords
peptido
seq
mimetic
binding
ligand
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PCT/US1999/026277
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English (en)
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WO2000027420A1 (fr
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Priority to AU14713/00A priority Critical patent/AU1471300A/en
Priority to US09/831,047 priority patent/US6960566B1/en
Publication of WO2000027420A1 publication Critical patent/WO2000027420A1/fr
Publication of WO2000027420A9 publication Critical patent/WO2000027420A9/fr
Priority to US11/081,198 priority patent/US20050181987A1/en

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Definitions

  • the invention relates generally to animal cell adhesion, and more specifically to peptido-mimetics of carbohydrate ligands of animal cell adhesion proteins and methods of using the same.
  • Tumor metastasis is a multistep process requiring detachment of malignant cells from the primary tumor, penetration of blood or lymph vessels and attachment to endothelium of distant organs and formation of new tumors.
  • the ability of disseminating cancer cells to establish metastases in secondary organs is regulated by a combination of factors, including access to the organ microvasculature and specific host-tumor interactions [Radinsky and Fidler, 1992, In Vivo 6:325-331].
  • vascular endothelial cell adhesion molecules implicated in both leukocyte and tumor cell transmigration are members of the selectin family
  • This family of adhesion molecules supports the adhesion of leukocytes to the vessel wall through the recognition of specific carbohydrate structures and thereby mediates critical cell-cell interactions in processes such as leukocyte trafficking, thrombosis, acute and chronic inflammation and ischemia reperfusion injury [Bucher and Picker, 1996, Science 272 60-66, Lasky, 1995, Annu. Rev. Biochem. 64 113-139, Narki,
  • E-selectin is a calcium-dependent molecule expressed by activated vascular endothelium during the process of leucocyte recruitment
  • E-selectin binds to glycoconjugates carrying a terminal tetrasaccharide Lewis (Le) antigen, sialyl-LeX (SA-LeX), [NeuAc ⁇ 2,3Gal ⁇ l ⁇ l,4(Fuc ⁇ l,3)
  • GlcNAc ⁇ l,3Gal ⁇ l,4Glc ⁇ l-R displays higher affinity for the SA-Le a structure ([NeuAc ⁇ 2,3Gal ⁇ l,3(Fuc ⁇ l,4) GlcNAc ⁇ l,3Gal ⁇ l,4Glc ⁇ l-R], a positional isomer of SA-LeX.
  • P-selectin is expressed by activated platelets and endothelial cells P-selectin has been shown to mediate adhesive interactions of some colon adenocarcinoma cells with thrombin-activated platelets [Mannori I, cited above] During metastatic dissemination, tumor-platelet adhesion may result in the formation of neoplastic emboli that facilitate the arrest of tumor cells in the microvasculature of organs [Karpatkin and Pearlstein, 1981, Ann. Intern. Med. 95 636-641] L-selectin is constitutively expressed by a majority of leukocytes, including neutrophils, monocytes, natural killer cells and most lymphocytes.
  • L-selectin expressed on leukocytes can support interaction with cancer cells, enhancing metastatic potential [Welch et al, 1989, Proc. Nail. Acad. Sci. USA 86:5859-5863], Other studies demonstrate that the engagement of L-selectin on lymphocytes can stimulate their anti-tumor cell cytotoxic activity [Seth et al, 1991, Proc. Nat/. Acad. Sci. USA 88: 7877-7881].
  • Selectin recognition of carbohydrate ligands involves primarily the ⁇ -terminal of C-type lectin domain, influenced by the EGF-like domain, and to a lesser degree the short consensus repeats [Kolbinger et al., 1988, Biochemistry 35:6385-6392; Revelle et al, 1996, J. Biol. Chem. 271 : 16160-16170; Li et al., 1994, J. Biol. Chem. 269:4431-4437]. Mutagenesis studies of the lectin domain of the selectins [Erbe et al, 1993, J. Cell. Biol.
  • metastasis apparently involves the interaction of at least one member of the selectin family of adhesion molecules with the antigens SA-Le a and/or SA-LeX.
  • These ligands may be involved in tumor metastasis by mediating binding of blood-borne tumor cells via E- and/or P-selectin to vascular endothelium [Giavazzi et al, 1993, J. Clin. Invest. 92:3038-3044; Dejana et al, 1992, Lab. Invest. 66:324-3130; Takada et al, 1993, Cancer. Res. 53:354-361; Sawada et ⁇ /., 1994, J Biol. Chem.
  • SA-Lea might play a major role as a ligand in the E-selectin dependent adhesion to EC in vivo
  • SA-Lea specific monoclonal antibodies MAbs were inhibitory for adhesion of colon carcinoma cells to human umbilical cord vein endothelial cells (HUVEC)
  • carcinoma cells do not express these carbohydrate determinants (i e , SA-LeX and S A-Le a ) and yet they can attach to EC prior to activation Further, this adhesion is not augmented by cytokine treatment, suggesting
  • Neutrophil extravasation is enabled by a multistep process initiated by the selectin family [Kansas, 1996, Blood 88 3259-3287]
  • Neutrophil-endothelial cell interaction mediated via the selectins in the context of vascular shear flow are characterized by transient tethering of the neutrophils, followed by rolling of the neutrophil along the endothelial surface of the vessel wall
  • selectin-dependent neutrophil rolling is essential to subsequent events in the transmigration process
  • Neutrophils are exposed to endothelial cell derived IL-8, platelet-activating factor and other neutrophil-activating molecules [Lowe, 1997, In The selectins- Inhibitors of leukocyte endothelial adhesion, pp 143-177, Nestweber, ed , Harwood Academic Publishers, Reading, UK], which in turn promote activation of neutrophil P2 integrins, leading to integrin
  • ligands for selectins particularly E-selectin
  • neutrophils and carcinoma cells raises the possibility that metastases are equivalent to the inflammatory process in which tumor cells, particularly carcinoma cells, use the same molecular mechanism(s) for cancer cell-EC interaction as lymphocytes, through the adhesion interaction of the endothelial cell selectins with the tumor-associated carbohydrate ligands, e.g., SA-LeX, SA-Le a , and LeY.
  • carbohydrate structures In addition to their role in cell adhesion, carbohydrate structures also play a role in angiogenesis. Aberrant angiogenesis can occur in a variety of pathologic conditions. Neovascularization of tumors occurs by aberrant stimulation of normally quiescent endothelial cells to migrate, proliferate and form new capillary blood vessels [Ingber and Folkman, 1989, J. Cell. Biol. 109:3317-3330]. The experimental evidence suggests that E-selectin and its ligand SA-LeX function in angiogenesis [Nguyen et al, 1982, J. Biol. Chem. 267:26157-26165].
  • proliferating microvascular endothelium presents a potential target for anti-cancer and anti-angiogenic therapies through the inhibition of E-selectin-dependent carbohydrate-mediated interactions [Folkman, 1995, N. Engl J. Med. 333: 1757-1763].
  • This peptide isolated from a peptide phage display library using an anti-GDI antibody inhibits metastasis in an in vivo model.
  • the present invention meets the above-stated needs by providing novel peptido-mimetics of carbohydrate structures and uses therefor in affecting animal cell adhesion mediated by carbohydrate ligand-cellular lectin protein receptor interactions.
  • the invention includes a composition comprising a peptido- mimetic of a carbohydrate ligand of an adhesion molecule in a physiologically acceptable carrier.
  • the adhesion molecule is a selectin, particularly E-selectin.
  • the ligand is a Lewis antigen. Particularly desirable are peptidomimetics of the Lewis antigens SA-Le a , SA-LeX, and LeY.
  • a variety of specific peptido-memtics are recited in the detailed disclosure such peptido-mimetics may be modified as described herein.
  • the invention provides a method of modulating binding of an adhesion molecule to a carbohydrate ligand.
  • the method comprises contacting the adhesion molecule (e.g., a selectin) with a peptido-mimetic of the carbohydrate ligand, so that binding of the adhesion molecule to the carbohydrate ligand is modified or altered in a therapeutically effective manner.
  • the adhesion molecule e.g., a selectin
  • the invention provides a method of modulating adhesion of a tumor cell to an adhesion molecule located on an endothelial cell.
  • the method comprises contacting the tumor cell with a peptido-mimetic of a carbohydrate ligand.
  • the peptido-mimetic modulates adhesion of the tumor cell to the endothelial cell.
  • the adhesion molecule may be, e.g., a selectin.
  • the ligand is preferably a Lewis antigen.
  • the invention provides a method of treating cancer in a mammal by administering an effective amount of a peptido-mimetic of a carbohydrate ligand to the mammal.
  • the ligand is preferably a Lewis antigen.
  • Administration of the peptido-mimetic reduces adhesion of tumor cells to endothelial cells in the mammal, thereby reducing metastasis of the cancer.
  • the invention provides a method of inhibiting an inflammatory response in a mammal by contacting an endothelial cell with an effective amount of a peptido-mimetic of a carbohydrate ligand.
  • the ligand is preferably a
  • Lewis antigen The specific peptido-mimetics described herein can be used in this method.
  • the invention provides additional methods of identifying a variety of peptido-mimetics of carbohydrate ligands.
  • the peptido-mimetics inhibit the normal binding between the ligand and its natural binding partner.
  • the binding partner is an adhesion molecule, such as a selectin.
  • carbohydrate ligand is located on the surface of a tumor cell and is a Lewis antigen.
  • the carbohydrate ligand is located on the surface of a tumor cell and it normally binds to an adhesion molecule on human umbilical cord vein endothelial cells (HUNEC).
  • HUNEC human umbilical cord vein endothelial cells
  • the carbohydrate ligand affects capillary tube formation or angiogenesis.
  • the peptido-mimetic affects adhesion of a selected cell, e.g., a neutrophil or tumor cell, to an adhesion molecule located on an endothelial cell.
  • a selected cell e.g., a neutrophil or tumor cell
  • the carbohydrate ligand affects neutrophil recruitment. The steps of these methods are described in detail below.
  • the invention provides a method of producing peptidomimetics of Lewis antigens, particularly peptido-mimetics not including APWLYAGP [SEQ ID NO: 83].
  • This method comprises the steps of screening a random peptide library, in which the peptides are expressed as fusion proteins on the surface of bacterial clones, with antibodies specific for the Lewis antigens and/or an E-selectin immunoglobulin fusion protein; and selecting clones which bind the antibodies and/or the fusion protein. The selected clones produce peptido-mimetics of the Lewis antigens.
  • Fig. 1A is a diagram depicting the reduction of neutrophil influx upon administration of DLWDWNNGKPAG [SEQ LD NO: 1] mimicking SA-Le a carbohydrate in mice with chemically induced peritonitis.
  • the neutrophil count results were obtained from four experiments (three mice in each group). Unrelated peptide was administered in control mice.
  • Statistical analysis of the data using nonparametric unpaired t-test yielded P values ⁇ 0.001 for the data shown in Fig. 1 A.
  • Fig. IB is a diagram illustrating the myeloperoxidase activity in the collected neutrophils of the animals of Fig. 1 A. Statistical analysis of the data using nonparametric unpaired t-test yielded ? values ⁇ 0.005 for this data.
  • Fig. 2A is a bar graph demonstrating the E-selectin- independent adhesion of human mammary adenocarcinoma cells SkBr5 to the human endothelioma cell line ECV-304.
  • SkBr5 and HUVEC cells were allowed to adhere for 15 minutes in the continuous presence of MAbs and MAb F(ab) 2 fragments (marked in the graph) at 40 pg ml.
  • Control anti-influenza hemagglutinin MAb H24135 was used at the same concentration. The results represent the percentage (%) of adherent cells in the presence of specific MAb as compared to control anti-influenza hemagglutinin MAb H24135.
  • FIG. 2B is a graph depicting the representative data of the expression of various antigens on the surface of cells in the absence (-) or presence (+) of the inflammatory cytokine, Interleukin-1, as determined by MAbs binding in radioimmunoassay (RIA) with monolayer ECV-304 cells.
  • the presence of the following antigens was detected using the MAbs indicated: SA-Le a (MAbNS19-9), CD63 (MAb C029 and ME491), E-selectin (specific MAb anti-ELAM-1, British Bio-
  • Fig. 3 is a graph illustrating the effect of Trp5 substitution with Phe in peptide DLWDWWGKPAG [SEQ LD NO: 1] resulting in peptide DLWDF GKPAG [SEQ ID NO: 63] on binding of SA-Le a specific MAb NS 19-9. Constant amounts of MAb were incubated with increasing amounts of peptides and binding of free antibody to carbohydrate SA-Le was measured by enzyme linked immunosorbent assay.
  • results show competitive inhibition of MAb binding to solid phase SA-Le a polyacrylamide matrix (SA-Le a -PAA) by 12-mer peptides DLWDWWGKPAG ( ⁇ ) [SEQ ID NO: 1] and DLWDFWGKPAG (A) [SEQ ID NO: 63] with respect to the
  • Fig. 4 is a circular dichroism (CD) spectra comparing dodecapeptides DLWDWWGKPAG (solid line) [SEQ ID NO: 1] and DLWDFWGKPAG (— ) [SEQ ID NO:63]. The spectra were recorded at 0.51 mg/ml for both peptides.
  • Fig. 5 is a graph illustrating the inhibition of lung experimental metastases with peptide DLWDFWGKPAG [SEQ ID NO: 63].
  • Tumor cells were admixed with the specific or unrelated peptide solution (1 mg per mouse) and animals were inoculated with 1 X 10 5 B16F10FtLTI tumor cells in 200 ⁇ l volume of PBS via tail vein. Results are from 4 experiments (5 mice in each group) are shown.
  • Each dot represents enumerated tumor nodules in one lung in experimental group of C57B1/6 mice treated with the peptide (panel B), control group of C57B1/6 mice treated with unrelated peptide (panel A) and E-selectin knock-out (KO) mice of C57B1/6 background (panel C).
  • Statistical analysis using a nonparametric unpaired t test gave a two-tailed/? values ⁇ 0.008 and 0.009 for animals treated with peptide and E-selectin KO, respectively, as compared to control group.
  • the horizontal bars represent median values and vertical bars denote standard deviation.
  • the invention is based on the discovery that peptido-mimetics of complex carbohydrate structures block adhesion of tumor cells and leukocytes to endothelial cells.
  • the production of small peptide molecules which mimic complex carbohydrate structures are useful for blocking carbohydrate ligand-cell adhesion molecule interactions involved in metastasis, angiogenesis, and inflammatory responses.
  • these peptido-mimetics are useful as anti-adhesion therapeutics.
  • the evaluation of complex carbohydrate ligand-adhesion molecule interactions at the molecular level enables the design of peptido-mimetics and possibly other compounds which block these adhesion interactions thereby disrupting the disease processes mediated by them.
  • compositions and methods of this invention are also useful in designing additional therapeutics to inhibit the adhesion interactions required for disease processes.
  • peptido-mimetic means a peptide or polypeptide that mimics complex carbohydrate conformations and structures.
  • a subset of peptidomimetics are referred to as “mimotopes".
  • Mimotopes are small peptido-mimetics, generally from 7 to about 15 amino acids in length which mimic complex carbohydrate structures, including the carbohydrate ligands for endothelial adhesion molecules. Mimotypes are also capable of blocking the ligand-adhesion molecule interaction. Throughout this specification, these term are used interchangeably. These small peptides also facilitate the study of complex structural/conformational relationships between these ligands and cellular lectins. _ Peptides of the Invention
  • a composition of this invention comprises at least one peptido-mimetic of a carbohydrate ligand of an adhesion molecule in a physiologically acceptable carrier.
  • the adhesion molecule is a selectin, such as E-selectin.
  • the ligand is a Lewis antigen. Particularly desirable are peptidomimetics of the Lewis antigens SA-Le a , SA-LeX, and LeY.
  • the illustrative peptides described below may be modified, as described in more detail herein, to improve anti- adhesion properties and to increase their stability to degradation in vivo.
  • compositions of this invention contain one or more of the following peptido-mimetics which mimic the topography of the E-selectin ligand: ASANNLYIPTQE [SEQ ID NO:84], VYLAPGRISRDY [SEQ ID NO: 85], VYLAPGRFSRDY [SEQ ID NO:86], CTSHWGVLSQRR [SEQ ID NO:87], RVLSPESYLGPS [SEQ ID NO:88], RVLSPESYLGPA [SEQ ID NO:89], VGNGNLMGRRG [SEQ ID ⁇ O:90], RVLSPESYLGPA [SEQ LD NO:92], GNCRYIGLRQFG [SEQ LD NO:93], DIRVEPGGGYTH [SEQ ID NO:94], APIHTYTGRARG [SEQ ID NO:96], and RHTCVRSCGHDR [SEQ ID NO:97].
  • exemplary peptido-mimetics of S A-Le a include, without limitation, NGIWSWSEGSR [SEQ ID NO:
  • families of mimics of carcinoma- associated antigens that represent S A-Le a were identified from a combinatorial peptide library using MAb NS19-9 specific for this carbohydrate structure.
  • One of the peptides, DLWDWWGKPAG [SEQ ID NO: 1] was selected that specifically competes for binding of MAb for SA-Le a .
  • This peptide displays an ability to partially inhibit neutrophil recruitment in an acute inflammation model in vivo.
  • this peptide was analyzed by systematic amino acid replacements to identify optimal conformationally stabilized SA-Le" mimics with higher affinity using a solid phase peptide array library.
  • MAb NS19-9 could tolerate replacement of the lead peptide sequence by a variety of amino acids. These substitutions did not abrogate binding, suggesting that they did not affect the structural specificity required for MAb recognition. Different amino acids in themselves can act as structural mimics within an identified peptide and bind through non-specific interactions.
  • the different consensus sequences among the families of peptides identified with the same MAb from the random peptide library or sequences without an obvious consensus characterized in previous studies support the notion that indeed different amino acid residues provide structural similarity.
  • different consensus sequences mimic different topographies of the carbohydrate epitope recognized by the antibody.
  • the present invention demonstrates that peptides mimicking SA-Le a are able to bind surfaces of proteins specific for these structures and thus they can act as antagonists for the recognition of the cognate carbohydrate antigen or ligand.
  • In vivo oligosaccharide dependent reduction of metastasis formation may be a function of the interruption of these interactions.
  • Antagonists interfere with the metastatic process at the level of cellular adhesion and blood vessel formation since E-selectin and SA-LeX are expressed on actively growing blood vessels.
  • peptide mimics act on signal transduction events mediated by selectins and their ligands and the in vivo consequences of selectin-ligand antagonism to the complex signal transduction processes associated with selectin-dependent cell adhesion.
  • Certain exemplary peptidomimetics of LeY include the peptides TKRPDLIVDPIP [SEQ ID NO:98], DEVRPDLISTEE [SEQ ID NO: 99], NLRPKYIXLDAD [SEQ ID NO: 100], and TLIAFADLVDVI [SEQ LD NO: 101].
  • Peptides mimicking carbohydrate antigens retain conformational properties of cognate carbohydrate structures and can block recognition of cells expressing such ligands in vivo. Thereby, they can mediate anti-metastatic functions as demonstrated by blocking of experimental metastasis.
  • the peptides identified above which mimic the topography of the E-selectin ligand and/or the other Lewis antigens can be employed in pharmaceutical compositions.
  • Such peptido-mimetics are useful in pharmaceutical compositions directed toward the treatment of cancer, and the prevention and/or inhibition of metastases (see, e.g., Example 14). Such peptides may also be used in pharmaceutical treatments to block selectin-dependent interactions, e.g. diminishing the inflammatory response (see, e.g., Example 8).
  • Peptido-mimetics includes peptido- mimetics such as those identified specifically above, which are modified to increase their stability in vivo.
  • the incorporation of unnatural amino acids (e.g., D configuration amino acids) at the N or C termini, the most frequent peptide degradation sites, may improve the pharmacological properties of the peptides, without loss of the binding efficacy.
  • Another modification involves incorporating onto the N-terminus of the peptide a moiety which can provide a net positive charge on the N-terminus of the peptide.
  • Such moieties can include straight chain, branched, cyclic or heterocyclic alkyl groups, straight chain, branched, cyclic or heterocyclic alkanoyl groups, a positively charged reporter group; and/or one or up to 15 additional amino acids independently selected from L-configuration or D-configuration amino acids, optionally substituted with a straight chain, branched, cyclic or heterocyclic alkyl group, a straight chain, branched, cyclic or heterocyclic alkanoyl group, or a reporter group.
  • the amino acids may be naturally occurring amino acids or unnatural amino acids, such as D configuration amino acids, or amino acids which have been cyclized by the insertion of modifying sugars, imide groups and the like.
  • N-terminal moiety is the positively charged 1-aminocyclo-hexane carboxylic acid. Another is a single positively charged amino acid such as L-Val- or D-Val-. In still other embodiments, such additional amino acids are modified by an acetyl group, providing that a net positive charge results. In still other embodiments of modified peptides, the N-terminal group is a positively charged moiety which can function as a reporter group for detection purposes.
  • These peptides may also be modified to cyclize the peptide by joining the N- and C- termini of the peptide. Additional amino acids or spacers may be introduced into the peptides also form spacers, which may be needed to cyclize the peptide by bridging between the N- and C- termini of the peptide. Spacers are sequences of greater than 3 amino acids which are interposed between the normal N- terminus and C-terminus of the modified peptido-mimetic. These spacers permit linkage therebetween without imposing any adverse restraint upon the molecular structure. Spacers may also contain restriction endonuclease cleavage sites to enable separation of the sequences, where desired. Desirably, spacers duplicate a portion of the peptide. Suitable spacers or linkers are known and may be readily designed and selected by one of skill in the art.
  • the C terminus of a peptido-mimetic of this invention may be a free hydroxyl, an amide, an imide, a sugar, or a sequence of one or up to about 15 additional amino acids, optionally substituted with a free hydroxyl, an amide, an imide or a sugar.
  • the C-termini may also be modified in the same manner as the modified N-termini, described above.
  • the C terminus of the a peptido-mimetic may be modified with 2-acetamido-2-deoxyglucose.
  • Another specific embodiment is the addition to the C-terminal amino acid of the peptido-mimetic of triacetyl 2-acetamido-2-deoxyglucose.
  • the C-terminal amino acid is modified by the addition of a ⁇ -acetyl- 2,3-diamino propionic acid group.
  • Still another modification of the peptides of this invention includes the addition into the peptides of two adjacent amino acids which are resistant to cleavage by endopeptidases. Still another modification involves replacing conventional inter- residue amide bonds by bonds resistant to proteases, such as, a thioamide bond or a reduced amide bond. Such modifications of the bonds between amino acids may change the conformation of the peptide.
  • a variety of methods for producing non- natural amino acids are known and insert modified inter-residue bonds, and/or cyclize the peptides may be selected by one of skill in the art.
  • Other backbone-modifications of these peptides are also anticipated to improve proteolytic stability and yield analogs with slightly modified activity spectrum.
  • the specific peptide described above may also be readily modified by replacing one or more amino acids with another, by substituting individual amino acids with chemical components or labels or by other peptide modification methods known to those of skill in the art. Such modifications may be made to enhance stability of the peptide in a pharmaceutical composition or to enhance the binding ability of the peptido-mimetic to the desired ligand [See, e.g., Example 13]. Such modifications include peptide modifications disclosed in J. E. Oh et al, J. Peptide Res.. 54: 129-136 (1999), incorporated herein by reference. Peptide multivalency is another modification that should result in higher affinity binding as compared to low affinity interaction with monovalent peptides.
  • Multivalency should increase peptide EC interaction and lower concentration of multiple antigen peptides needed to block it.
  • the generation of high affinity ligands might require formation of a "clustered oligosaccharide patch" or a “clustered anionic patch” or a combination of polypeptide backbone and modifications such as sulfation.
  • these peptides may be employed individually, or presented in a multivalent form, such as a multiple antigenic peptide ("MAP", also referred to as an octameric lysine core peptide) construct.
  • MAP multiple antigenic peptide
  • Such a construct may be designed employing the MAP system described by Tarn, Proc. Natl. Acad. Sci. USA. 85:5409-5413 (1988); D. Posnett et al., J. Biol. Chem.. 263(4): 1719-1725 (1988); J. Tarn, Vaccine Research and Developments. Vol. 1, ed. W. Koffand H. Six, pp. 51-87 (Marcel Deblau, Inc., New York 1992)].
  • each peptide may be optionally separated by amino acid spacers, which are defined above.
  • sequence homology for polypeptides is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wisconsin 53705. Protein analysis software matches similar sequences using a measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions.
  • GCG Genetics Computer Group
  • Protein analysis software matches similar sequences using a measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions.
  • GCG contains programs such as "Gap” and "Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof.
  • a preferred algorithm when comparing a specific polypeptide sequence to a database containing a large number of sequences from different organisms is the computer program BLAST, especially blastp or tblastn (Altschul et al., 1997, herein incorporated by reference). Database searching using amino acid sequences can be measured by algorithms other than blastp known in the art, e.g., Fasta, a program in GCG Version 6.1.
  • nucleic acid or fragment thereof indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 60% of the nucleotide bases, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95-98% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as Fasta, as discussed above.
  • the peptides of the invention may be prepared conventionally by resort to known chemical synthesis techniques, e.g., solid-phase chemical synthesis, such as described by Merrifield, J. Amer. Chem. Soc. 85:2149-2154 (1963), and J. Stuart and J. Young, Solid Phase Peptide Synthelia. Pierce Chemical Company, Rockford, LL (1984), or detailed in the examples below. Chemical synthesis methods are particularly desirable for large-scale production of such peptides. A variety of methods for producing the above-identified modifications of the peptides, e.g., non- natural amino acids, are known and may be selected by one of skill in the art.
  • the peptides of this invention may be prepared by known recombinant DNA techniques by cloning and expressing within a host microorganism or cell a DNA fragment carrying a nucleic acid sequence encoding one of the above- described peptides [See, e.g., Sambrook et al., Molecular Cloning. A Laboratory Manual.. 2d Edit., Cold Spring Harbor Laboratory, New York (1989)]. Conventional molecular biology techniques, and site-directed mutagenesis may be employed to provide desired modified peptide sequences.
  • compositions suitable for administering suitable for administering to a mammalian subject, preferably a human.
  • Such formulations comprise one or more of the peptides identified above, combined with a pharmaceutically or physiologically acceptable carrier, such as sterile water or sterile isotonic saline.
  • sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butanediol, for example.
  • diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.
  • Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the peptide is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • the peptides of the present invention may be administered by oral, intraperitoneal, intramuscular and other conventional routes of pharmaceutical administration.
  • compositions of the present invention may be administered either as individual therapeutic/prophylactic agents or in combination with other agents. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
  • a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
  • One of skill in the pharmaceutical arts may readily design acceptable pharmaceutical formations for delivery of the peptido-mimetics of this invention with recourse to well-known information on pharmacology and with use of commercially available materials, in view of the teachings herein.
  • each daily dose will comprise between about 50 ⁇ g to about 2 mg of the peptide per mL of a sterile solution per kg body weight.
  • Another desired daily dosage of active ingredient can be about 0.0001 to 1 grams per kilogram of body weight.
  • Another desired dosage range can be about 0.1 to 100 milligrams per kilogram of body weight.
  • Still another dosage is in the range of 0.5 to 50 milligrams per kilogram of body weight, and preferably 1 to 10 milligrams per kilogram per day. Other dosage ranges may also be contemplated by one of skill in the art.
  • Initial doses may be optionally followed by repeated administration for a duration selected by the attending physician. Dosage frequency may also depend upon the factors identified above, and may range from daily dosages to once or twice a week i.v. or i.m., for a duration of about 6 weeks.
  • the compositions of the present invention can also be employed in treatments for chronic inflammatory ailments or for long term treatment of cancer patients for a period to be selected by the physician.
  • the peptides described above and the pharmaceutical compositions containing them may be employed in therapeutic methods or in in vitro methods for the development of other pharmaceutical agents.
  • Each of the methods described employs the peptido-mimetic to interfere with, or inhibit, the binding of a desired carbohydrate antigen with its normal receptor or adhesion molecule, so as to prevent or inhibit an undesired therapeutic result. It is anticipated that the therapeutic methods may be performed ex vivo or in vivo.
  • one embodiment of the present invention provides a method of modulating binding of an adhesion molecule to a carbohydrate ligand.
  • an adhesion molecule such as a selectin
  • a peptido- mimetic which mimics the topography of the carbohydrate ligand. Binding of the adhesion molecule by the peptido-mimetic in place of the normal ligand thus modulates, or reduces the amount of binding between the adhesion molecule and its carbohydrate ligand and thereby suppresses or reduces the normal effect of such binding.
  • Modulating binding of an adhesion molecule, (e.g., E-selectin) to a carbohydrate ligand refers to any reduction or increase in binding of the selectin to the carbohydrate ligand wherein the difference in binding results in a desired therapeutic effect. It is desirable for this method that the carbohydrate ligands and adhesion molecules are present on the surface of the cells in such a manner as to allow binding of the adhesion molecule to the ligand and/or to the peptido-mimetic of the ligand.
  • the peptido-mimetics of this invention may be used in treatments for cancer.
  • one embodiment is a method of modulating adhesion of a tumor cell to an adhesion molecule on an endothelial cell, e.g., a selectin.
  • the method comprises contacting the tumor cell with an effective amount of a peptido- mimetic (or a pharmaceutical composition containing the effective amount) of a carbohydrate ligand.
  • this method provides a way of inhibiting or otherwise modulating the biological activity mediated by the natural adhesion of the tumor cell to the endothelial cell.
  • This method provides a therapy for cancer patients.
  • the peptido-mimetics described herein may be used in this method.
  • an effective amount of a peptido-mimetic of a carbohydrate ligand is administered to the mammal.
  • Such treatment also reduces the adhesion of tumor cells to endothelial cells in the mammal, thereby reducing metastasis of the cancer.
  • An "adherence modulating dose” refers to any amount of a peptido-mimetic, or any combination of peptido-mimetics, of a carbohydrate ligand which affects the adherence of a cell bearing the carbohydrate ligand to an adhesion molecule on endothelial cells.
  • adherence reducing dose is any amount of a peptido-mimetic of a carbohydrate ligand which, when administered to a mammal, reduces the binding of an adhesion molecule to a carbohydrate ligand. This dose reduces the adhesion of certain cells which present carbohydrate ligands on their surfaces (e.g., tumor cells and neutrophils) to endothelial cells, compared with the adhesion of those same cells to endothelial cells in the mammal prior to the administration of the peptido-mimetic.
  • SA-Le a provides the critical carbohydrate ligand for the adhesion molecule, E-selectin, that facilitates the initial steps involved in a cascade of tumor cell-endothelial interactions leading to metastatic spread.
  • E-selectin the adhesion molecule
  • B16F10FTIII melanoma cells employed in a metastasis model express SA-Le a carbohydrate antigen and form lung tumors after i.v. inoculation through the tail vein.
  • Tumor colonization appears to be highly E-selectin dependent, as the incidence of metastasis was completely abrogated in E-selectin KO mice.
  • the initial stages required for tumor colonization are dependent not only on adhesion molecules inducible on endothelial cells, but also on the ligands expressed on tumor cells.
  • the expression of E-selectin ligand, SA-Le a is likely to contribute to the metastasis of cells expressing this structure.
  • E-selectin is one of the few adhesion molecules truly restricted to activated endothelium, thus E-selectin may be used to selectively target activated and/or proliferating endothelium in vivo not only by blocking adhesion of tumor cells to EC, but also by halting the neovascularization processes. Consequently, proliferating microvascular endothelium presents an unique and universal target for anti-cancer therapy.
  • a method for inhibiting an inflammatory response in a mammal involves contacting an endothelial cell with an effective amount of a peptido-mimetic of a carbohydrate ligand, thereby modulating adherence of neutrophils to endothelial cells.
  • modulating neutrophil adherence is meant any difference in the adherence of a neutrophil to an endothelial cell in the presence of a peptido-mimetic of a carbohydrate ligand compared with the adherence of a neutrophil to an endothelial cell in the absence of the peptido-mimetic of a carbohydrate ligand.
  • Inflammatory response refers to the physiological response in an animal which includes, but is not limited to, recruitment of neutrophils to the site of inflammation and/or the adherence of neutrophils to endothelial cells.
  • one inflammatory response which may be treated by the present invention is peritonitis (i.e., an irritation of the peritoneum).
  • peritonitis i.e., an irritation of the peritoneum.
  • a substantial reduction in pathology of inflammation is achieved upon 30% reduction of neutrophil recruitment in the inflammatory model illustrated below.
  • this aspect of the invention is not limited to the manner in which inflammation is caused or the tissue in which inflammation is present and/or treated.
  • the peptido-mimetics described herein are particularly useful in this aspect of the invention.
  • the invention provides a cell (e.g., yeast, bacteria, phage, or mammalian) which is constructed to display an exogenous peptido-mimetic of a Lewis antigen or a selectin on the surface of the cell. More particularly, this invention is directed to cells displaying the specific peptides disclosed herein, either alone or as fusion proteins, on the cell surface.
  • a cell e.g., yeast, bacteria, phage, or mammalian
  • this invention is directed to cells displaying the specific peptides disclosed herein, either alone or as fusion proteins, on the cell surface.
  • Example 1 One manner in which this may be obtained is described in Example 1, through the use of a random peptide library.
  • Analogous cells, other than E. coli may be produced by similar methods. See the cells described in Examples 10 and 14, which also provide instruction on the provision of cells developed to express peptido-mimetics.
  • the invention provides a method for identifying an additional peptido-mimetic of a carbohydrate ligand which affects the binding of the carbohydrate ligand to a binding partner.
  • the method comprises the steps of: (a) contacting the binding partner with a peptido-mimetic and (b) comparing the binding of the binding partner of (a) to the carbohydrate ligand with the binding of the same binding partner which is not contacted with the peptido-mimetic to the carbohydrate ligand.
  • the level of binding of the binding partner contacted with the peptido-mimetic to the carbohydrate ligand is compared with the level of binding of the binding partner not contacted with the peptido-mimetic with the same carbohydrate ligand. Any significant change in these two levels of binding is an indication that the peptido- mimetic affects the binding of the carbohydrate ligand to the binding partner.
  • the binding partner is an adhesion molecule located on an endothelial cell.
  • the carbohydrate ligand is located on the surface of a tumor cell and the adhesion molecule is a selectin, e.g., E-selectin.
  • E-selectin e.g., E-selectin. Any change in the level of binding of E-selectin or any other adhesion molecule contacted with the peptido-mimetic to the carbohydrate ligand compared with the level of binding of E-selectin or any other adhesion molecule not contacted with the peptido- mimetic with the same carbohydrate ligand is an indication that the peptido-mimetic affects the binding of the tumor cell to the adhesion molecule.
  • Another method of identifying a peptido-mimetic of a carbohydrate ligand which affects angiogenesis involves contacting a primary capillary endothelial cell with a peptido-mimetic of a carbohydrate ligand, e.g., a Lewis antigen. Any difference between capillary tube formation observed by the cell contacted with the peptido-mimetic is compared to the capillary tube formation of the cell which is not contacted with the peptido-mimetic. Any significant change in capillary tube formation is an indication that the peptido- mimetic affects angiogenesis.
  • Yet another method of identifying a peptido-mimetic which affects adhesion of a selected cell to an endothelial cell involves contacting an endothelial cell with a peptido-mimetic of a carbohydrate ligand located on the endothelial cell. The binding of the endothelial cell contacted with the peptido-mimetic to the ligand is compared to the binding of an endothelial cell which has not been contacted with the peptido-mimetic to the ligand. Any change in the levels of binding is an indication that the peptido-mimetic affects binding of the selected cell to the endothelial cell.
  • the selected cell is a tumor cell.
  • the selected cell is a neutrophil.
  • the carbohydrate ligand is located on the surface of a tumor cell and the adhesion molecule of the ligand is located on a human umbilical cord vein endothelial cell (HUVEC). Any change in binding caused by the peptido-mimetic is an indication that the peptido-mimetic affects the binding of the tumor cell to HUVEC. This method is also not limited to the identity of the selected cells, nor to the assays used to detect differences in binding.
  • the invention further provides a method of identifying a peptido- mimetic of a carbohydrate ligand which is involved in inflammatory processes. That method involves administering an inflammation-inducing substance (e.g., a peritonitis- inducing substance) into a suitable tissue or organ of a mammal. Thereafter, an effective amount of a peptido-mimetic of the carbohydrate ligand is administered at the site or area of the inflammation. A control mammal having a similar inflammation at the same site is not treated. Any effect on inflammation which is observed in the treated mammal vs. the control, such as neutrophil influx into the site of inflammation, is an indication that the peptido-mimetic affects inflammation.
  • an inflammation-inducing substance e.g., a peritonitis- inducing substance
  • the inflammation inducing substance causes peritonitis and the site of inflammation is the peritoneam.
  • the levels of neutrophil influx in the peritoneum of the treated mammal are observed to be lower that the levels of neutrophil influx in the peritoneum of the control.
  • This result is an indication that the peptido-mimetic inhibits an inflammatory response.
  • This method is not limited by the method of inducing inflammation or the site of inflammation or the indication of inflammation that is assessed. For example, myeloperoxidase activity could be assayed in place of neutrophil recruitment.
  • the site and method of administration of the peptido-mimetic and the assayed indication may be selected by one of skill in the art.
  • the present invention provides a method of producing peptido-mimetics of Lewis antigens, particularly those not including APWLYAGP [SEQ ID NO: 83].
  • This method comprises the step of: (a) screening a random peptide library, the peptides expressed as fusion proteins on the surface of bacterial clones, with antibodies specific for the Lewis antigens and/or with adhesion molecule constructs, e.g., molecules expressed as fusion proteins.
  • adhesion molecule constructs e.g., molecules expressed as fusion proteins.
  • One such example is the E- selectin immunoglobulin fusion protein used in Example 2.
  • Other such fusion molecules may be used similarly in this step, and (b) The clones which bind the antibodies or adhesion molecule constructs.
  • the peptides and polynucleotide sequences of the present invention may also be used in the screening and development of chemical compounds, small molecules or proteins which mimic the structure or activity of the carbohydrate ligands, and thus have utility as therapeutic drugs for the treatment of cancer and inflammation.
  • Competition assays such as the ELISA described in Example 3, may be employed and readily designed for such use.
  • a compound which has structural similarity to the peptido-mimetic, or the binding of the peptide to the ligand may also be computationally evaluated and designed by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with the peptides of this invention.
  • Specialized computer programs that may also assist in the process of selecting fragments or chemical entities similar to the peptides, or entities which can interact with the peptides and thus mimic the receptor, include the GRLD program available from Oxford University, Oxford, UK. [P. J. Goodford, "A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules", J. Med. Chem.. 28:849-857 (1985)]; the MCSS program available from Molecular Simulations, Burlington, MA [A. Miranker and M. Karplus,
  • Compounds that mimic a peptide of this invention or a ligand of the peptides may be designed as a whole or "de novo" using either an empty active site or optionally including some portion(s) of a known ligand(s).
  • Suitable methods describing such methods include the LUDI program [H.-J. Bohm, "The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors", I Comp. Aid. Molec. Design. 6:61-78 (1992)], available from Biosym Technologies, San Diego, CA; the LEGEND program [Y. Nishibata and A. Itai, Tetrahedron. 47:8985 (1991)], available from Molecular Simulations, Burlington, MA; and the LUDI program [H.-J. Bohm, "The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors", I Comp. Aid. Molec. Design. 6:61-78 (1992)], available from
  • peptides were derived from a 12-mer random peptide library.
  • FLITRX flagellin-thioredoxin fusion proteins
  • an aliquot of the FLITRX library containing at least 2 x 10 10 cells to ensure full representation of peptides was grown to saturation for 15 hours in IMC/amp 100 medium (M9 medium containing 1 mM MgCl 2 supplemented with 0.5% glucose, 0.2% casamino acids and 100 ⁇ g/ml ampicillin).
  • IMC/amp 100 medium M9 medium containing 1 mM MgCl 2 supplemented with 0.5% glucose, 0.2% casamino acids and 100 ⁇ g/ml ampicillin.
  • the expression of thioredoxin with incorporated 12-mer peptide sequence was induced by further 6 hours of incubation of a culture of 10 10 cells, diluted 1 :25 with fresh IMC/amp 100 medium containing 100 ⁇ g/ml tryptophan.
  • the induced bacteria were panned on a MAb-coated tissue culture (20 ⁇ g/ml) plate followed by blocking with 1% nonfat milk containing 150 mM NaCl and 1% ⁇ -methyl mannoside for 1 hour. The bound cells were washed gently. Eluted cells were collected by rinsing the plate with 10 ml of fresh IMC/amp 100 medium and then incubated at 25 °C until reaching saturation. The entire selection process was repeated four more times. Colonies of isolated bacteria were grown on ampicillin-containing plates. Individual colonies were isolated and grown as a small scale culture (2 ml) in IMC/amp 100 medium with tryptophan for 6 hours.
  • EXAMPLE 2 PROTEIN EXPRESSION. ISOLATION AND SEQUENCING
  • MAb NS19-9 which is specific for the carbohydrate ligand SA- Le a [Magnani et al, 1981, Science 212:55-56; Bechtel et al, 1990, J. Biol. Chem., 265:2028-2037], and (3) MAb FH6, which is specific for the carbohydrate ligand SA-
  • E-selectin-immunoglobulin chimeric protein IgG (E-selectin-IgG; Centocor, Inc., Malvern, PA) [Geng et al, 1992, J. Biol. Chem. 267: 19846-19853] was also used. The expression and the location of thioredoxin on the nitrocellulose filter was confirmed after incubation of parallel filters with trxA-specific MAb (anti-ThioTM) (Invitrogen, Carlsbad, CA). Following antibody binding, the filters were washed, incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody, and then washed again.
  • HRP horseradish peroxidase
  • the nucleotide sequences of the DNA from the selected bacteria were determined by the dideoxynucleotide chain termination method using specific primers using standard methods [see, e.g., Sambrook et al. 1989, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York; Ausubel et al. 1997, Current Protocols in Molecular Biology. Green & Wiley, New York].
  • the peptides encoded by the sequenced DNA were either purchased commercially (Research Genetics, Inc., Huntsville, AL) and/or were chemically synthesized using FMOC-based, solid phase chemistry and were purified to homogeneity on a C- 18 reverse-phase HPLC column. The structures were confirmed by fast-atom bombardment mass spectrometry.
  • Selected peptides were subject to assays for demonstrating whether the isolated peptides retain conformational properties of carbohydrates.
  • assays include 1) direct binding to respective MAbs and E-selectin-IgG in solid phase; 2) inhibition of MAbs and E-selectin-IgG binding to the cognate carbohydrate ligands in solid phase; and 3) kinetic characterization of peptide binding to E-selectin using BIACORE methodology.
  • EXAMPLE 3 COMPETITION ENZYME-LINKED IMMUNOSORBENT ASSAY
  • Test peptides at concentrations ranging from 10 nM to 1 mM were preincubated with 100 ⁇ l of the MAb NS19-9 (5 ⁇ g/ml) diluted in 10% ⁇ -globulin free horse serum/PBS at room temperature. Fifty ⁇ l of preincubated inhibition complex antibody-peptide is added to each well of 96-well plate and incubated at 30-37° C for 1 hour.
  • MAb/peptide complex mixtures were transferred to wells precoated with a constant amount of neoglycoprotein containing coupled multivalent carbohydrate determinant (SA-Le a - polyacrylamide matrix (SA-LeX-PAA or LeY-PAA) (5 ⁇ g/well) and allowed to bind for 1 hour followed by blocking with 10% ⁇ -globulin-free horse serum for 2 hours at room temperature. Wells were washed with 100 ⁇ l PBS four times.
  • SA-Le a - polyacrylamide matrix SA-LeX-PAA or LeY-PAA
  • Goat anti-mouse immunoglobulin G conjugated to horse radish peroxidase was diluted 1000-fold with 10% ⁇ -globulin free horse serum/PBS and 100 ⁇ l was added in each well and incubated at 30-37° C for 1 hour and washed with 100 ⁇ l PBS five times.
  • TMB tetramethylbenzidine dichloride
  • One-hundred ⁇ l substrate was added in each well and incubated at room temperature for 10 minutes. The developed blue color was read at 450 nm after stopping the reaction with 100 ⁇ l 1M phosphoric acid.
  • % inhibitory concentration (IC 50 ) was calculated by non-linear least-squares regression to a four-parameter logistic equation. The results of various competition assays performed as described are detailed in the following examples.
  • BR15-6A inhibits adhesion of human tumor cells to EC in E-selectin- independent mode.
  • the family of peptides isolated using BR15-6A (Table 1), which mimics the LeY carbohydrate, also demonstrates the consensus sequence motif RPDL [SEQ ID NO: 113]. This motif was also recognized by another LeY-specific MAb,
  • the isolated sequences of the families of peptides mimicking LeY can be compared with a putative peptide APWLYAGP [SEQ ID NO: 83] identified previously by phage display panning with another anti-LeY antibody [Hoess et al., 1993, Gene 128:43-49]. This result indicates that the same consensus sequences contribute to the mimicry of the carbohydrate structure and are responsible for the topological similarity with the carbohydrate antigen.
  • Peptide SEQ ID NO: 99 from the family of LeY-mimicking peptides was selected because it contained a consensus motif which appeared in multiple bacterial isolates after five rounds of panning with the BR15-6A MAb. This peptide was chemically synthesized and its binding to BR15-6A MAb was evaluated. Dose-dependent binding of the MAb to solid phase coated peptide at concentrations of 1 nM to 1 mM was demonstrated using enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • Peptide SEQ ID NO:l from NS19-9 family II mimicking SA-Le a ligand was selected based on the presence of consensus motif which appeared in multiple bacterial isolates after five rounds of panning.
  • Table 2 identifies peptide sequence families I and II mimicking SA-Le a carbohydrate structure.
  • Table 3 identifies peptide sequence family III mimicking SA-Le a carbohydrate structure.
  • SEQ ID NO: 1 demonstrated dose-dependent binding with MAb in the ELISA of Example 2.
  • This peptide was also inhibitory for MAb NS 19-9 binding to the synthetic multivalent SA-Le a -polyacrylamide (PAA) matrix conjugate neoglycoprotein (Glycotech, Inc., Rockville, MD) which contains the cognate oligosaccharide determinant.
  • PAA synthetic multivalent SA-Le a -polyacrylamide
  • neoglycoprotein Glycotech, Inc., Rockville, MD
  • the 50% inhibitory concentration (IC 50 ) value of SEQ ID NO: 1 was calculated to be 700 ⁇ M by competition ELISA [see, e.g., Blaszczyk etal, 1984, Arch. Biochem. Biophys.
  • the molecular basis for peptide binding to anti-Lewis antigen antibodies was determined using the LIGAND-DESIGN (LUDI) program (Biosym Technologies, San Diego, CA) [Bohm 1992, J. Comput. AidedMol Des. 6:593-606]. This program searches a molecular library for fragments representative of the amino acids in the target peptide sequence. The program then positions the fragments within the combining site devoid of steric conflicts.
  • the pentapeptide sequence was "fitted" into the B3 combining site using the LUDI program.
  • the APWLY sequence of SEQ ID NO: 83 was modeled such that the Trp (W), Tyr (Y), Leu (L) and Ala (A) residues occupied relative positions as the identified LUDI fragments. Judicious positioning relied upon intermolecular interaction calculations in which several potential binding modes of the peptide were ranked according to the stability of the complex. In the most stable conformation, the AP residues occupied a similar position to the LeY GlcNAc residue.
  • this analysis provides a strategy for determining the molecular basis for antigenic mimicry of particular motifs, providing a unique perspective of how a peptide sequence fits into the antibody or a receptor combining site, competing with a native antigen.
  • This approach also enables the design of therapeutic compounds which more effectively compete with the native carbohydrate ligand for binding to cell adhesion molecules.
  • ID NO: 1 was replaced by other L-amino acids.
  • peptides were synthesized with simultaneous incorporation of multiple amino acids or with truncation of specific regions.
  • the peptide array of 163 unique peptides was generated by substituting all amino acids for each individual amino acid in lead peptide (DLWDWWGKPAG
  • [SEQ ID NO: 1] identified by combinatorial library panning with MAb NS19-9.
  • An array of synthetic 12-mer peptides was synthesized using 90 X 130 mm polyethylene glycol-modified cellulose membrane functionalized with approximately 4 nmole/mm 2 amino groups, manufactured by Abimed (Lagenfeld, Germany). Standard Fmoc chemistry was used according to the manufacturer's instructions [Frank, R., 1992,
  • Tetrahedron 48, 9217-9332 Tetrahedron 48, 9217-9332].
  • the protected and activated amino acids were spotted using an Abimed ASP 422 robotic arm. All washing, dyeing and deprotection steps were done manually.
  • the activated C-terminal amino acids were spotted leaving 10 mm space in each direction, at the concentration of 0.5 M in N-methyl pyrrolidone. A volume of 0.5 ml provides spot of 7-8 mm in diameter.
  • Activation of the amino acids with dicyclohexyl-carbodiimide and N-hydroxy-benzotriazole was done 30 minutes before spotting.
  • the paper was washed with 12%) acetic anhydride dissolved in N,N'-dimethylformamide (DMF) twice for a total of 10 min to endcap all unreacted amino groups. Repetitive removal of the Fmoc groups was achieved by two treatments with 20% piperidine in DMF for 5 and 10 min, respectively. The second and consecutive amino acids were coupled in a 1.1 molar excess, and were spotted 3-4 times depending upon the outcome of the bromophenol blue assay of the couplings. After the coupling and deprotection steps, the membrane was washed thoroughly with DMF and ethanol, dried and stained with bromophenol blue dissolved in DMF.
  • DMF N,N'-dimethylformamide
  • the cellulose filter was blocked with 5% non-fat milk in phosphate buffered saline (PBS) for 1 hour at room temperature followed by washing with PBS. Filters were incubated with goat anti-mouse immunoglobuhn G conjugated with horse radish peroxidase (1 ⁇ g/ml) for 1 hour. Filters were washed five times in PBS-T (0.05% Tween in PBS, v/v) and developed using a chemiluminescence reagent followed by autoradiography as described in Western blot.
  • PBS-T 0.05% Tween in PBS, v/v
  • NS19-9 binding or a varying number of amino acids were truncated.
  • the number of peptides was 163 (6 raws, 27 spots each). Spot analysis revealed a distinct pattern of key residues important for binding and, therefore, sensitive to substitution while other residues tolerated replacement by a variety of amino acids.
  • Comparison of the signal intensities of the array scan revealed that the critical residues for binding were clearly identified within the N-terminal half of the DLWDWWGKPAG peptide [SEQ ID NO: 1 ] as determined by the lack of antibody binding to substituted peptides (Table 4). In contrast, most of the substitutions within the C-terminus were tolerated (amino acids 6 to 12), not influencing MAb binding.
  • DLWDFWGKPAG [SEQ ID NO:63] containing a single substitution at position 5 with Phe.
  • Table 5 lists the amino acid substitutions within peptide DLWDWWGKPAG [SEQ LD NO: 1] that increase the binding of MAb NS19-9 using the peptide array.
  • DLWDWWGKPAG [SEQ ID NO: 1] and DLWDFWGKPAG [SEQ ID NO: 63] were chemically synthesized and tested for their ability to compete to immobilized synthetic SA-Le a -PAA neoglycoprotein.
  • dose-response experiments were carried out in order to determine the concentration of peptides required for blocking of 50% of MAb binding to the native carbohydrate antigen (IC 50 ), as determined by competition ELISA (Example 3).
  • Both peptides SEQ ID NOS: 1 and 63 blocked the binding of MAb NS 19-9 to the constant amount of carbohydrate antigen in a dose-dependent manner.
  • the IC 50 for peptide SEQ ID NO: 1 blocking of MAb-SA-Le a binding was 700 ⁇ M.
  • Peptide SEQ LD NO: 63 exhibited a more pronounced dose-dependent inhibition of the MAb-SA-Le a binding as compared with peptide SEQ ID NO: 1 as demonstrated by the calculated IC 50 value of 70 ⁇ M for peptide SEQ ID NO: 63.
  • DLWDWWGKPAG [SEQ ID NO: 1] and DLWDFWGKPAG [SEQ ID NO:63] represent solvent-accessible epitopes and the peptides represent cognate determinants for the antibody. No measurable blocking of anti-Le a MAb NS 19-9 binding was found with non-related peptide, indicating that the inhibitory effects of the native sequences are due to specific effects.
  • EXAMPLE 8 INHIBITION OF NEUTROPHIL RECRUITMENT IN AN ACUTE INFLAMMATION MODEL IN VIVO BY A PEPTIDO-MIMETIC OF S A-Le a
  • neutrophils The accumulation of neutrophils is a characteristic feature of acute and chronic inflammatory disease, and early steps in the recruitment of these cells to the site of inflammation depends upon E-selectin-mediated interaction. Thus, inhibition of neutrophil recruitment in vivo is an important test of the ability of potential therapeutic agents to inhibit E-selectin-mediated events. S A-Le" interaction with E-selectin is not relevant for adhesion of neutrophils, since neutrophils do not express SA-Le a on their surfaces. However, tumor cells have been demonstrated to prefer SA-Le a over SA-- LeX in mediating E-selectin-dependent adhesion interactions with endothelial cells.
  • bioactivity of the 12-mer peptide SEQ ID NO: 1 which mimics the SA-Le a carbohydrate structure was determined by administering ZymosanTM intraperitoneally
  • mice (i.p.) into mice [see, e.g., Martens et al. 1995, J. Biol. Chem. 270:21129-21136; Rao et al. 1994, J. Biol. Chem. 269: 19663-19666], followed three hours later by an intravenous (i.v.) injection of peptide SEQ ID NO: 1 (1 mg). Neutrophils were harvested and counted one hour later. As shown in Fig. 1 A, peptide treatment significantly (PO.001) reduced the number of neutrophils in peritoneal lavage fluids.
  • MPO myeloperoxidase
  • the 12-mer FLITRX fusion library was screened directly using E-selectin-IgG fusion protein, as described in Example 2.
  • the motif VLSP was found in 7 of 16 clones recovered.
  • preferences for V/G and RR in the sites flanking the 5 -amino acid motif were also found.
  • a PGR sequence was found in 3 clones.
  • peptides with different sequence motifs may mimic different topographical surfaces of the cognate carbohydrates or may bind to different binding areas of E-selectin combining site.
  • Family I peptide sequences and Family II peptide sequences mimicking E-selectin ligand are reported in Table 6.
  • SW620 cells were the most suitable for the study of adhesion to EC in vitro and for an orthotopic tumor model in which immunodeficient mice may be inoculated with human tumor cells.
  • the expression of SA-LeX in SW620 cells was below the level of detection, but the cells were uniformly and strongly stained for SA- Le a .
  • SW620 cells should be very useful in vivo evaluation of the contribution of SA-Le a to tumor cell colonizing ability in the absence of SA-LeX.
  • An autologous model of tumor metastasis is preferred for in vivo studies in which murine tumor cells derived from the same murine background may be inoculated to form metastases.
  • murine tumor cell lines such as MethA sarcoma, G1261 glioblastoma, CT26 colon carcinoma, B16F1 and B16F10 melanoma clones obtained from DCTDC Tumor Repository (NCI Frederick, MD) and murine breast adenocarcinorna 66.1, JC, 410.1 cell lines provided by Dr. Amy Fulton (University of Maryland, Baltimore, MD) were characterized with respect to the expression of Lewis antigens.
  • MethA clone #34 was isolated from the original MethA cell line cloned by limiting dilution and was determined to express SA-LeX antigen on 100% of cells as compared with only approximately 30% cells in the original cell line. All cell lines expressed SA-LeX except B16F1 and B16F10 variants, whereas no SA- Le a expression was observed among any cell lines. Therefore, MethA #34 is suitable to evaluate the role of SA-LeX mimicking peptides in inhibiting adhesion of tumor cells in vitro and in vivo.
  • a panel of tumor cells such as pancreatic PAN02, PAN03, colon CT38, CA51, mammary EMT-6, E0771, 755, lung ASBXIV, transitional bladder FCB and melanoma HP(Jax) (DCTDC Tumor Repository) was assembled. These cells were continuously analyzed with respect to the expression of SA-LeX, S A-Le a , and LeY determinants. Moreover, expression vectors are available which contain ⁇ l,3/4 fucosyltransferase, ( ⁇ l,3/4FTHT) cDNA [Seed, 1987, Proc. Natl. Acid. Sci.
  • Tumor cell adhesion to EC monolayers was investigated in vitro as described in Iwai et al. , cited above, as a model for metastatic invasion
  • the data disclosed herein demonstrate that various mammary adenocarcinorna cells attach to TNF- or
  • IL-l ⁇ cytokine-activated human umbilical cord vein endothelial cells via an E-selectin-independent mode.
  • Antibodies BR55-2 and BR15- 6A directed to LeY significantly decreased the adhesion in a dose-dependent fashion, implicating the involvement of LeY in adhesion of these breast carcinoma cells (Fig 2A)
  • These cells also adhered to the spontaneously transformed human endotheliorna cell line ECV-304 in an LeY-dependent way ECV-304 cells unlike primary HUVEC,
  • ECV-304 endotheliorna cells which have lost the ability to express E-selectin, are a useful model for in vitro studies of E-selectin-independent adhesion of tumor cells to EC and the role of this pathway in the establishment of metastasis.
  • EXAMPLE 12 COMPETITION OF DLWDFWGKPAG PEPTIDE [SEQ ID NO:63] WITH SA-Le a FOR MAb BINDING
  • the lead peptide DLWDWWGKPAG [SEQ ID NO: 1] and the array selected peptide DLWDFWGKPAG [SEQ ID NO: 63] were synthesized individually and their binding specificities to NS19-9 MAb were assayed by competitive solid phase enzyme linked immunosorbent assay (Example 3), with the results as shown in Fig. 3.
  • EXAMPLE 13 SECONDARY STRUCTURE OF PEPTIDES MIMICKING CARBOHYDRATE
  • SA-Le a appears to mediate the adhesion of many carcinoma tumors to human umbilical vascular endothelial cells in multiple in vitro studies.
  • An in vivo experimental metastatic model which permits investigation of SA-Le a supported adhesion of tumor cells to lung endothelium is performed as follows:
  • B16F10 murine melanoma cells do not naturally express E-selectin ligands SA-LeX or SA-Le a as demonstrated by FACS analysis, and are syngeneic with C57B1 6 haplotype (American Type Tissue Collection, Rockville, MD). To manipulate these cells to express the SA-Le a structure on the tumor cell surface, the
  • Plasmid pCDNA-FTIII is prepared by cloning HindHI and Notl-digested ⁇ l-3/4-fucosyltransferase cD ⁇ A (FTIII) obtained from the ⁇ H3M vector containing FTIII cD ⁇ A (Brian Seed, Massachusetts General Hospital, Boston, MA) was cloned into pCDNA3( «eo) vector.
  • FTILT fucosyltransferase is specific for both type 1 and 2 lactoseries oligosaccharide acceptor substrates and thus is capable of synthesizing both S A-Le a and SA-LeX, respectively.
  • the resulting cell line, B16F10FTIII expresses SA-Le a carbohydrate structure as demonstrated by flow cytometry analysis using MAb NS19-9 as compared to the parental B16F 10, which did not show staining with this antibody.
  • the transfected cells were grown in the presence of G418 (500 ⁇ g/ml) (Gibco- BRL, Grand Island, NY) for 10 days. To ensure the homogeneity of the transfected cells with respect to the expression of SA-Le a , the cells were subjected to cell sorting using S A-Le a specific MAb NS 19-9 followed by FITC-conjugated goat anti-mouse immunoglobulin. The resulting cell line B16F10FTIII appeared to express SA-Le a but not SA-LeX as assessed by FACS (not shown), suggesting that type 1 but not type 2 acceptors were available within the cells.
  • the generated cell line made a suitable model to determine the role of SA-Le a in the metastatic process since the tumor cells are devoid of SA-LeX.
  • the tumorigenic dose for the C57B1/6 syngeneic tumor cells was established by i.v. injection of various numbers of cells.
  • a 1 X 10 5 dose was chosen for further experiments as countable lung metastases were observed after i.v. injection of 1 X 10 5 of B16F10FTIII cells expressing SA-Le a after 21 days.
  • mice C57B1/6 female mice (Jackson Laboratory, Bar Harbor, ME). KO mice lack E- selectin expression.
  • B16F10FTIII cells positive for SA-Le a were grown in vitro in Iscove's culture medium supplemented with 10% FBS for 1 week before injecting into mice. Cells were collected and washed twice in Iscove's medium without serum and suspended in PBS. One x 10 5 tumor cells in a volume of 200 ⁇ l in PBS were inoculated by intravenous (i.v.) route via tail vein. To test the effect of the peptide, animals were inoculated with a single dose of peptide at the time of tumor challenge. One mg of peptide was admixed with the tumor cells and together injected via i.v. route. Control animals received injection of tumor cells admixed with the same amount of unrelated peptide.
  • mice were euthanized after 3 weeks following tumor cells injection and lung and other organs were examined under dissecting microscope for the presence of tumor nodules.
  • the lungs were excised and the number of nodules was enumerated for each animal without fixation of the lungs. Data were evaluated for statistical significance using a nonparametric unpaired two-tailed t test. The results are shown in Fig. 5.
  • mice of both strains received i.v. injection of 1 X 10 5 B16F10FtIII tumor cells and mice were examined 3 weeks later. Only 20% of E-selectin deficient animals injected with tumor cells developed small numbers of lung metastasis while the rest of the E-selectin KO mice showed no detectable lung tumor nodules. Statistical analysis gave a P values ⁇ 0.009 for E-selectin KO as compared to the control group (Fig. 4, A and C), respectively. Small nodules were observed in a few E-selectin KO mice that developed tumors whereas all animals in the control group developed multiple metastasis and some of them died earlier than 3 weeks.
  • peptides in general show rather short half-life in mouse serum
  • the peptide inhibition studies in vivo the peptide was admixed with the tumor cells to sustain the highest transient concentration of peptide at the time of tumor cell arrival into the lung capillary system. Animals were euthanized after 21 days following tumor challenge and the number of metastasis was enumerated in each lung whereas, no metastatic growth was detected in the liver.
  • peptide DLWDFWGKPAG [SEQ ID NO: 63] abrogated on average 50% lung colonization of the B16F10FTIII induced tumor nodules developed in control animals; some mice being completely devoid of tumor nodules (Fig. 4B).
  • the injection of the peptide 1 hour prior to tumor cells did not influence the rate of metastases formation in comparison with the peptide administered together with tumor cells.
  • Animals treated with peptide showed metastases ranging from 0 to 20 per lung (median 9.9), whereas, animals in the control group developed multiple tumor nodules with the number of metastases per mouse ranging from 3 to 40 per lung (median 20.7) (Fig. 4B and 4 A), respectively). The difference was highly statistically significant (p ⁇ 0.008).
  • Le a antigen is able to significantly block the adhesion of tumor cells to vascular endothelium at the early stages of the multistep process, thus reducing tumor metastases.
  • This finding strongly suggests that the interaction of SA-Le a carbohydrate tumor-associated antigen with E-selectin expressed on vascular EC is an important step in establishing tumor metastasis.

Abstract

L'invention concerne des compositions contenant un ou plusieurs mimétiques peptidiques ou mimétiques peptidiques modifiés d'un ligand glucidique d'une molécule d'adhésion dans un excipient physiologiquement acceptable. Ces compositions conviennent pour des méthodes destinées à réduire les métastases et à inhiber les réactions inflammatoires chez un mammifère. Dans certains modes de réalisation particulièrement utiles, le ligand est un antigène des groupes sanguins et/ou la molécule d'adhésion est une sélectine, par exemple, la E-sélectine. L'invention concerne également des méthodes permettant d'identifier ces mimétiques peptidiques de ligands glucidiques, qui peuvent être impliqués dans la liaison de cellules tumorales à d'autres cellules, telles que des cellules endothéliales.
PCT/US1999/026277 1998-11-06 1999-11-05 Compositions et methodes de traitement du cancer WO2000027420A1 (fr)

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US5861505A (en) * 1991-11-27 1999-01-19 California Institute Of Technology Synthetic analog of sialic Lewis antigen from bacterial capsular polysaccharide
US5643873A (en) * 1992-05-06 1997-07-01 Affymax Technologies N.V. Peptides and compounds that bind selectins including endothelial leukocyte adhesion molecule 1
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