WO1993002192A1 - Modified pf4 compositions and methods of use - Google Patents
Modified pf4 compositions and methods of use Download PDFInfo
- Publication number
- WO1993002192A1 WO1993002192A1 PCT/US1992/005903 US9205903W WO9302192A1 WO 1993002192 A1 WO1993002192 A1 WO 1993002192A1 US 9205903 W US9205903 W US 9205903W WO 9302192 A1 WO9302192 A1 WO 9302192A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- entity
- glu
- polypeptide conjugate
- leu
- angiostatic
- Prior art date
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Definitions
- Angiogenesis the development of new capillary blood vessels, is an important process in the developing fetus and growing human.
- angiogenesis occurs significantly only during wound healing and in the menstrual cycle. It is now widely recognized that much of the angiogenic activity occurring in adults is pathological in nature.
- proliferation of vascular endothelial cells and formation of new capillaries is essential for growth of solid tumors beyond a few cubic millimeters in volume (Folkman et al. [1983] Ciba Found. Symp. 100:132-149).
- angiogenic dysfunctions include diabetic retinopathy, retrolental fibroplasia, neovascular glaucoma, psoriasis, angiofibromas, immune and non-immune inflammation (including rheumatoid arthritis), capillary proliferation within atherosclerotic plaques, hemangiomas, and Kaposi's Sarcoma have also recently been recognized as diseases possessing characteristics of deregulated endothelial cell division and capillary growth. These conditions along with growth of solid tumors are collectively referred to as "angiogenic diseases” (Folkman, J., and M. Klagsbrun [1987] Science 235:442-447).
- endothelial cell proliferation is pathological or, at least, unwanted.
- endometriosis is characterized by the abnormal proliferation and positioning of certain endothelial cells which normally line the inner wall of the uterus. Control of the angiogenic process could help to prevent or alleviate endometriosis.
- prevention of endothelial cell growth in the uterus could be a means of birth control. Endothelial cell growth is associated with wound healing. This growth is undesirable during extended surgical proceedings and where excessive scar formation may occur. Therefore, a means of controlling endothelial cell proliferation would help prevent or reduce unwanted scar formation.
- the mechanism of angiogenesis and endothelial cell proliferation has not been completely characterized.
- mast cells accumulate at a tumor site before new capillary growth occurs; however, mast cells alone cannot initiate angiogenesis.
- Heparin a mast cell product, has been shown to significantly stimulate the capillary endothelial cell migration which is necessary for angiogenesis (Folkman, J. [1984]
- Angiogenesis Initiation and Modulation. In Cancer Invasion and Metastasis: Biologic and Therapeutic Aspects. G.L. Nicolson and L. Milas, eds. Raven Press, New York, pp.201-208).
- protamine is a protein found only in sperm. Protamine has been shown to inhibit tumor angiogenesis and subsequent tumor growth (T ylor, S. and J. Folkman [1982] Nature 297:307-312).
- Protamine's anti-angiogenesis activity has been attributed to its well-known capacity to bind heparin (T ylor and Folkman [1982], supra * !. Clinical experiments with protamine have not been pursued because of the toxicity associated with protamine injection. Protamine, which is usually isolated from salmon sperm, is known to be antigenic in humans, and anaphylactic reactions to this protein have been observed with secondary exposures.
- PF4 platelet factor 4
- major basic protein has demonstrated heparin-binding activity but is of little practical utility because of its high toxicity.
- Platelet factor 4 is a well-known protein which has been completely sequenced (Deuel, TE, P.S. Keim, M. Farmer, and R.L. Heinrikson [1977] Proc. Natl. Acad. Sci. USA 74(6):2256-2258). It is a 70-residue secretable platelet protein with a molecular weight of approximately 7.8 Kd. Although there is evidence of heparin binding activity and some indications of anti-angiogenesis activity (Folkman [1984], supra). PF4 has never been shown to have clinical utility.
- Angiogenesis plays a major role in the initiation and progression of widespread catastrophic illnesses, including cancer.
- An effective, non-toxic agent which can be administered locally and/or systemically to treat these illnesses would be highly advantageous and has long eluded identification.
- the subject invention relates to compositions obtained through chemical modifications of PF4 or recombinant PF4 (rPF4).
- PF4 can be modified through its free amino groups with fluorescein-isothiocyanate and retain the capability of inhibiting angiogenic activity and endothelial cell proliferation. Similar modifications can be made with PF4 analogs, mutants, or fragments.
- a further aspect of the subject invention is the targeting of the biological activity of PF4 to specific locations where that activity is needed. This can be done by conjugating PF4 (or an appropriate fragment, analog, or mutant) to a monoclonal or polyclonal antibody, carrier protein, cell receptor molecule, or other binding protein sequence.
- a further aspect of the subject invention is extending the half-life of biologically active PF4 (or appropriate fragments, analogs, or mutants) by conjugating said PF4 to a polymer compound.
- the polymer may be, for example, a polyamino acid such as polyglutamate, or a polysaccharide such as polyethylene glycol (PEG).
- modified PF4 can also be used to target toxins to specific cell populations.
- Figure 1 shows the inhibition of angiogenesis resulting from treatment with rPF4 and various related peptides.
- Figure 2 compares the amino acid sequence of rPF4 with rPF4-241.
- Figure 3 depicts the ⁇ -helical configurations of rPF4 and rPF4-241.
- Figure 4 compares the inhibition of angiogenesis resulting from treatment with rPF4 and rPF4-241.
- Figure 5 shows inhibition of human endothelial cell proliferation by rPF4 and rPF4- 241.
- Figure 6 compares the inhibition of human umbilical vein endothelial cell proliferation resulting from treatment with rPF4 or rPF4-241.
- Figure 7 shows the ability of rPF4 to inhibit tumor growth.
- Figure 8 depicts the possible chemical structure of the C ⁇ terminal end of FrPF4.
- Figure 9 shows inhibition of human endothelial cell proliferation by FrPF4.
- Figure 10 shows inhibition of human endothelial cell proliferation by FrPF4-241.
- FIG. 11 shows inhibition of tumor growth by FrPF4.
- the subject invention concerns the discovery that PF4, rPF4, and fragments and analogs of these compounds can be chemically modified to create new compounds with highly desirable characteristics.
- chemical modification of rPF4 and its fragments has resulted in the identification of compounds which show surprising ability to inhibit angiogenic activity as well as the capability to inhibit endothelial cell proliferation.
- One specific chemical modification which resulted in altered biological properties involved modification of the free amino groups of rPF4 with fluorescein-isothiocyanate (FTTC).
- FTTC fluorescein-isothiocyanate
- FrPF4 lacks heparin binding a ⁇ ivity because of modification of lysine residues within the heparin binding domain but, surprisingly, retains the ability to inhibit angiogenesis as well as suppress HUVEC proliferation in vitro.
- Angiostatic activity is also found in PF4 fragments and mutants which have been modified with the bulky and hydrophobic fluorescein moiety.
- the FTTC-labeled PF4 sequences are useful for visual detection of PF4 molecules.
- the ability to modify PF4 and its fragments with large moieties without loss of the relevant biological activity provides a basis for conjugating PF4, its fragments, mutants, or derivatives with toxins, monoclonal antibodies, polyclonal antibodies, fluorophores, cell receptormolecules, non-proteinaceous biological effector molecules, chelators, carrier proteins, polysaccharides, polyamino acids, and other large entities.
- the conjugation may occur through, for example, modification of functional groups on the protein, such as free amino, carboxyl, sulfhydryl, or guanadinium (arginine) groups of PF4 or a variant of PF4.
- modification of these moieties necessary to effect the desired conjugation can be carried out by chemical procedures well known to those skilled in this art
- angiogenic disease refers to growth of solid tumors, and other conditions involving angiogenic dysfunctions including diabetic retinopathy, retrolental fibroplasia, neovascular glaucoma, psoriasis, angiofibromas, immune and non- immune inflammation (including rheumatoid arthritis), capillary proliferation within atherosclerotic plaques, hemangiomas, and Kaposi's Sarcoma.
- the subject invention also concerns the use of rPF4 and PF4 fragments, analogs, and mutants for treatment of diseases of deregulated endothelial cell proliferation.
- analog refers to compounds which are substantially the same as another compound but which may have been modified by, for example, adding additional amino acids or side groups.
- mutants and variants as referred to in this application refer to amino acid sequences which are substantially the same as another sequence but which have amino acid substitutions at certain locations in the amino acid sequence.
- Framents refer to portions of a longer amino acid sequence.
- the subject invention embraces the specific amino acid sequences and other compositions which are specifically exemplified.
- the subject invention further embraces analogs and mutants of these sequences, as well as fragments of the sequences, and analogs and mutants of the fragments.
- These analogs, mutants, and fragments are embraced within the subject invention so long as the analog, fragment, or mutant retains substantially the same relevant biological activity as the originally exemplified compound.
- PF4 relevant biological activity
- PF4 refers to the activity of interest for a particular application of a compound.
- PF4 uses include inhibition of angiogenesis and endothelial cell proliferation.
- analogs would refer to compounds where PF4 has been modified (by a conservative amino acid substitution, for example) without substantially altering the compound's ability to inhibit angiogenesis or endothelial cell proliferation.
- Conservative amino acid substitutions are only one example of the type of modifications which are within the scope of the subject matter of this invention.
- the subject invention arises from the unexpected discovery that chemically modified rPF4 inhibits in vivo capillary formation and embryonic neovascularization.
- PF4 full length recombinant PF4 inhibits growth factor-dependent human endothelial cell proliferation in vitro.
- angiogenesis-inhibiting activity of PF4 is retained by synthetic peptides corresponding to sequences of PF4 as small as 10 amino acids in length.
- the acuvity of the C-13 peptide is especially surprising in light of its inability to affect the anticoagulant activity of heparin.
- the use of the C-13 peptide offers several advantages over whole rPF4 such as reduced dosage (weight basis), reduced likelihood of antigenicity, and greater likelihood of effectiveness in novel dosage forms.
- the C-13 peptide of PF4 also retains the ability to prevent Con-A induced immunosuppression in mice, an activity which is unaffected by heparin and probably independent of the ability of the peptide to inhibit angiogenesis. It is well understood that angiogenesis is required for solid tumors to grow beyond a few cubic millimeters. Thus for the treatment of solid tumors, use of rPF4, or modifications thereof, to cause tumor rejection by inhibiting angiogenesis presents a novel and highly advantageous means of therapy. The fact that the C-13 peptide inhibits angiogenesis without affecting the anticoagulant activity of heparin demonstrates that this small peptide would also have the benefit of not interfering with concurrent anticoagulant therapy.
- PF4 fragments are generally less antigenic than larger proteins, and, thus, the PF4 fragments can be used advantageously for oral and transdermal administration. These types of delivery are particularly useful in the treatment of gastrointestinal capillary proliferation (e.g., Kaposi's Sarcoma) and skin lesions, respectively. Intralesional, as well as systemic, administration of PF4 fragments are also appropriate for treatment of these conditions. Tbpical or aerosol administration of PF4 fragments is appropriate for skin or pulmonary lesions, respectively (e.g., Kaposi's sarcoma and lung cancer).
- rPF4-241 An analog of PF4 which exhibits enhanced ability to inhibit angiogenesis has been synthesized.
- This analog known as rPF4-241
- rPF4-241 was created by cassette mutagenesis of a synthetic PF4 gene whereby four lysine residues of the carboxy terminus of PF4 were converted to two GIn-Glu couplets in order to eliminate heparin binding activity while retaining the - helical secondary structure.
- rPF4-241 or FrPF4-241
- the dosage of rPF4-241 can be between 0.5 mg/kg of body weight and about 100 mg/kg of body weight Similar and higher dosages can be used for the administration of native sequence rPF4 (or FrPF4) as well as peptide fragments. Fbr example, dosages of rPF4 (or FrPF4) and fragments thereof may be twice that of rPF4-241 (or FrPF4-241) or higher.
- FrPF4 or FrPF4-241 can be formulated in physiologically acceptable carriers, such as phosphate buffered saline, distilled water, excipients, or the like, or may be administered neat Materials and Methods Chicken Chorioallantoic Membrane fCAM) Assay. Fertile eggs were incubated in a stationary position for 3 days at 37°C and 70-80% relative humidity. During this time, the embryo rose to the upper surface of the egg contents. At the beginning of the 4th day, the eggs were cracked without inversion and carefully deposited into sterile plastic petri dishes such that the embryo remained on the upper surface.
- physiologically acceptable carriers such as phosphate buffered saline, distilled water, excipients, or the like
- the shell-free eggs were incubated for an additional 72 hours at 37°C, under an atmosphere containing 2.5-3.5% CO 2 after which the growing embryos developed a recognizable CAM.
- Discs made by mixing test samples with 1% (w v) methylcellulose were dried and placed on the CAM between major veins and approximately 0.5 cm from the embryo. Following another 48 hour incubation at 37°C (2.5-
- Endothelial Cell Proliferation Assay Human umbilical vein endothelial cells were cultured in Medium 199 (Gibco) containing 10% (v/v) fetal bovine serum (FBS), 150 mcg ml endothelial cell growth supplement (ECGS) and 5 units/ml heparin at 37°C and 4-5% C0 2 . Every 3-4 days, the cultures were harvested by trypsin treatment, diluted, replated, and grown to confluence. Prior to the start of an experiment, the cells were centrifuged and resuspended in heparin-free media and incubated with the test substance for 3 days under standard culture conditions. At the end of the incubation period, the cells were harvested and counted.
- FBS fetal bovine serum
- ECGS mcg ml endothelial cell growth supplement
- rPF4 Production Recombinant PF4 was produced in E. coli as an N-terminal fusion protein containing a methionine immediately preceding the PF4 sequence. The insoluble fusion protein was cleaved with cyanogen bromide treatment and purified by heparin agarose affinity chromatography. The isolated protein was buffer exchanged into 20 mM sodium acetate, pH 4.0, and either frozen or lyophilized for storage.
- Peptides were prepared by standard solid phase synthesis procedures, cleaved from the solid support and deblocked, and purified by reverse phase HPLC.
- Example 1 illustrate procedures, including the best mode, for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. Example 1
- the lysine rich region of PF4 (residues 61-66) is also the domain associated with the binding of heparin by PF4.
- Heparin is known to play a role in modulating angiogenesis, which can also be affected by protamine, another well characterized heparin-binding protein, lb assess the ability of PF4-based synthetic peptides to bind heparin, we assayed the activity of coagulation-cascade enzymes which are inhibited by heparin.
- Protamine and platelet factor 4 are able to prevent the heparin inhibition of thrombin and Factor Xa at approximately equimolar concentrations.
- the 41 amino acid G-te ⁇ ninal peptide of PF4 (C-41) prevented heparin inhibition less effectively, but the C-13 peptide was unable to prevent the inhibition of thrombin even at concentrations ten times that of an effective level of rPF4. This unexpected finding suggests that the C-13 peptide inhibits angiogenesis by some method other than heparin binding.
- rPF4 Endothelial cell division and growth is tightly controlled and strictly dependent on the presence of growth factors.
- rPF4 and related peptides were evaluated in vitro. rPF4 significantly inhibited endothelial cell growth in a dose-dependent fashion at a concentration as low as 10 mcgml. Inhibition was complete at 25 mcg ml in the heparin-deficient medium employed here.
- Example 4 lb assess the importance of the heparin binding activity of PF4 in the inhibition of endothelial cell proliferation, cells were incubated in media containing or lacking 5 units/ml heparin. The presence of heparin stimulated proliferation of these cells during the three day incubation of this experiment. rPF4 significantly inhibited both control (100%) and heparin stimulated (45%) endothelial cell growth (Table 1).
- the gene for rPF4-241 was expressed as a fusion protein in E. coli with the same N- te ⁇ ninal amino acid sequences as with the parent rPF4 molecule.
- the protein was cleaved from the E. poli fusion peptide by CNBr and formic acid and purified to near homogeneity by
- C-13-241 has the following sequence: Pro-Leu-'fyr-Gln-Glu-Ile-rie-Gln-Glu-Leu-Leu-Glu-Ser
- Example 6 Inhibition of angiogenesis by rPF4-241
- Purified rPF4-241 was dried in methylcellulose discs and tested for its ability to inhibit capillary growth in the chicken chor ⁇ oallantoic membrane (CAM) assay. Even at the lowest concentrations tested (1.25 nmol/disc) rPF4-241 extensively inhibited angiogenesis in the CAM system ( Figure 4). This inhibition was even more effective than that caused by equal concentrations of native rPF4 as suggested by larger avascular zones on the membrane. The inhibitory effect of rPF4-241 was not reversed by heparin.
- Example 7 Inhibition of human endothelial cell proliferation by rPF4-241
- mutant rPF4-241 was at least as effective in inhibiting cell growth (Figure 5). Further tests suggest that rPF4-241 was inhibitory at concentrations as low as 0.5 mcg/mL, a level at which native rPF4 has little or no effect
- mice Normal C57BL/6J female mice (6-8 weeks old) were inoculated subcutaneously with 5 x 10 s log phase cells of a B16-F10 melanoma tumor line. This protocol led to progressive tumor growth resulting in large (300 mm 3 ) necrotic tumors after approximately 10 days, followed by death of untreated animals usually within three weeks of tumor inoculation.
- tumor bearing animals were divided into two groups.
- One group was injected with 50 ⁇ g rPF4 (native sequence) in 100 ⁇ l of 50 mM sodium phosphate, pH 6.5, 50 mM sodium chloride directly into the nascent tumor, daily, beginning one day after tumor inoculation.
- a control group was treated identically with carrier buffer lacking rPF4.
- lumor volume was measured at regular intervals with digital calipers by laboratory personnel uninformed of the specific treatment received by each subject animal.
- rPF4 did not directly inhibit tumor cell growth in vitro. It appears, therefore, that rPF4 was modulating the host's angiogenic response to the growing tumor.
- proteins of identified structure and function may be constructed by changing the amino acid sequence if such changes do not alter the protein secondary structure (Kaiser, E.T., and FJ. Kezdy [1984] Science 223:249-255).
- the subject invention includes mutants of the amino acid sequences depicted herein which do not alter the protein secondary structure, or if the structure is altered, the biological activity is retained.
- Tkble 2 provides a listing of examples of several mutant sequences and their biological activity.
- rPF4307 [PF4 AA 1-57] - Pro Leu Tyr Gin lie Glu lie Gin Leu Glu Leu Glu Ser pos. rPF4 308 [PF4 AA 1-57] - Pro Leu " tyr Asn Asp lie Be Asn Asp Leu Leu Glu Ser pos. rPF4 315 [PF4 AA 1-57] - Pro Leu " tyr Gly Glu lie lie Gly Glu Leu Leu Glu Ser pos.
- amino acids may be placed in the following classes: basic, hydrophobic, acidic, polar, and amide. Substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound.
- Tkble 3 provides a listing of examples of amino acids belonging to each class.
- non-conservative substitutions can also be made.
- lysine may be substituted for with any of the following amino acids: Glu, Gin, Asp, Asn, Met, Ala, Leu, and lie.
- the critical factor is that these substitutions must not significantly detract from the biological activity of the rPF4 or the rPF4 fragment.
- PF4 57 of PF4; A may be or may not be present; wherein a 10 is Lys, Gly, Glu, Gin, Asp, Asn, Met, Ala, Leu, or He;
- &g is Lys, Glu, Gin, Asp, Asn, Met, Ala, Leu, or lie; a 8 is Glu, Gin, Met, Ala, Leu, De, Val, Pro, Phe, Tip, or TJT; a 7 is Glu, Met, Ala, Leu, lie, Val, Pro, Phe, Tip, or Tyr; a 6 is Lys, Gly, Glu, Gin, Asp, Asn, Met, Ala, Leu, or lie; a 5 is Lys, Glu, Gin, Asp, Asn, Met, Ala, Leu, or De; a 4 is Lys, Glu, Met, Ala, Leu, De, Val, Pro, Phe, Trp, or Tyr; and a 3 is Gin, Met, Ala, Leu, De, Val, Pro, Phe, Trp, or Tyr.
- amino terminus of the proteins of the subject invention can also be modified in a variety of ways while retaining the biological activity fundamental to the subject invention. Most notably, the length of the amino terminus can be modified while retaining angiostatic or endothelial cell inhibitory activity.
- amino terminus refers to amino acids 1-60 from the amino terminal of PF4 or its variants. As we have shown herein, up to
- angiostatic activity refers to a level of angiostatic activity which is characteristic of PF4. This level of angiostatic activity is, for example, at least about 75% (and preferably, greater than about 90%) of the angiostatic activity exhibited by PF4 as measured, for example, in the CAM assay.
- antiproliferative a ⁇ ivity refers to a level of antiproliferative activity which is characteristic of PF4. This level of antiproliferative activity is at least for example, about 75% (and preferably, greater than about 90%) of the antiproliferative activity exhibited by PF4 as measured, for example, by the HUVEC assay.
- the term "lack of heparin binding activity" refers to a relative lack of ability to bind heparin under normal physiological conditions compared to PF4. This lack of heparin binding activity is, for example, less than about 25% of PF4's heparin binding a ⁇ ivity and, preferably, less than about 10% of PF4's heparin binding activity.
- the ability to bind heparin can be readily determined by various assays as described herein and as is known by those skilled in this art
- rPF4 or rPF4-241 (5 mg in 50 mM Na 2 CO 3 , pH 9.3, 25 mM NaCl) was treated with 5 mg of fluorescein isothiocyanate in a volume of 5 ml to modify the free amino groups. After incubation for 3 hours at room temperature in the dark, the labeled protein (FrPF4 or FrPF4-241). was separated from unbound FTTC by gel filtration and dialyzed into 50 mM acetic acid. A possible stru ⁇ ure of the C-terminus of FrPF4 is shown in Figure 8.
- Example 10 Inhibition of Angiogenesis by Huorescein-Isothiocvanate-Conjugated rPF4
- FrPF4 was tested for activity in the CAM assay as described above. Although FrPF4 lacked heparin binding activity, it retained full activity as an inhibitor of angiogenesis on the CAM. The results of these assays are shown in Table 5.
- FrPF4 and FrPF4-241 were tested separately to determine their ability to inhibit endothelial ceU proliferation.
- HUVE cells were tested for their sensitivity to FrPF4 as described above except that [ 3 H]-thymidine was added to the cultures 24 hours after the addition of FrPF4. The cultures were then incubated an additional 6 hours. Cells were harvested, washed, and radioactive thymidine incorporation into DNA was measured.
- FITC-conjugated rPF4 was very effective, even at low dosages, in inhibiting DNA synthesis in human umbilical vein endothelial cells and therefore inhibiting ceU proliferation. Similar results were obtained using FrPF4-241. In this case, the inhibition of HUVE cell proliferation with increasing concentrations of rPF4-241 was tested using the Endothelial Cell Proliferation Assay as described above. The results of experiments using
- B-16 Melanoma tumors were grown in C57BL6/J mice as described previously. Treatment was begun 24 hours following implantation of tumor cells (Day 1) and consisted of 25 g/day of FrPF4 in 100 ⁇ l of sodium acetate buffer, pH 4.0. Control mice were injected with 25 g/day of FTTC labeled cytochrome-C in the same buffer. A statisticaUy significant suppression of tumor growth by FrPF4 was observed by Day 11 ( Figure 11).
- PF4 biological a ⁇ ivity
- an appropriate antibody preferably a monoclonal antibody.
- the monoclonal antibody which can be produced using techniques that are well-known in the art, will selectively seek out the target site. As the antibody moves to the desired location, it brings with it the PF4. Thus, the PF4 a ⁇ ivity can be concentrated at a specific location.
- General means of conjugating antibodies to polypeptides such as PF4 are well known to those skilled in the art and are discussed, for example, in U.S.
- Patent Nos. 4,671,958 (Rodwell et aL) and 4,792,447 (Uhr et al).
- the PF4 may also be targeted to specific locations via analogous conjugation with binding proteins (e.g., thrombospondin or fibroblast growth fa ⁇ or), cell receptor molecules (e.g., CD4, lymphocyte function associated antigen-1 [LFA-1], and von Willebrand Fa ⁇ or [vWF]) or the complementary ligands, and non-proteinaceous biological effe ⁇ or molecules (e.g., ICAM-1, tumor associated-antigens, and prostaglandins).
- binding proteins e.g., thrombospondin or fibroblast growth fa ⁇ or
- cell receptor molecules e.g., CD4, lymphocyte function associated antigen-1 [LFA-1], and von Willebrand Fa ⁇ or [vWF]
- non-proteinaceous biological effe ⁇ or molecules e.g., ICAM-1, tumor associated-antigens
- the monoclonal antibody, or other moiety can be associated with PF4 at one or both pairs of lysine residues located near the carboxy terminus of PF4.
- PF4 the monoclonal antibody
- the angiostatic activity is retained while heparin binding is eliminated.
- other amino acid residues may be substituted for the lysine residues before conjugation with appropriate moieties at these and other positions. Therefore, the compounds described here can be represented as follows:
- A represents all or part of the polypeptide sequence consisting of residues 1 through 57 of PF4; A may or may not be present on said hybrid polypeptide;
- B, C, D, and E can be any amino acid having a functional group suitable for covalent attachment;
- F, G, H, and I are sele ⁇ ed from the group consisting of monoclonal antibodies, polyclonal antibodies, fiuorescein-isothiocyanate, fluorophores, toxins, cell receptor molecules, non-proteinaceous biological effe ⁇ or molecules, polyamino acids, polysaccharides, and chelators; at least one of the moieties designated F, G, H, and I must be present on said hybrid polypeptide.
- the vertical lines represent chemical bonding interactions as do the spaces between the amino acids on the horizontal line. The existence of specifically iUustrated moieties associated at B, C, D, and E does not exclude the possibility of conjugation occurring at other residues.
- PF4 can be crosslinked to a large carrier protein, e.g., human serum albumin (HSA) or immunoglobulin, by disuccinimidyl suberate (DSS) through free primary amino groups (i.e., lysine E-amino groups or N-terminal ⁇ -amino groups; see Montesano et al. [1982] Biochem. Biophys. Res. Comm. 109:7-13).
- HSA human serum albumin
- DSS disuccinimidyl suberate
- rPF4 and HSA (10 mg and 100 mg, respectively) were incubated with 25 mM DSS for 4 hours at room temperature. The reaction was terminated by the addition of THs buffer, pH 8.0 to a final concentration of 100 mM. The resulting composition was a heterogenous mixture of ⁇ osslinked molecules which lacked heparin binding activity, but retained the ability to inhibit HUVEC proliferation. A control sample in which HSA was crosslinked to cytochrome-C did not inhibit HUVEC growth.
- An alternative means of extending the half-life of PF4 can be achieved by covalent linkage of PF4 to one or more units of a polymer such as a polyamino acid or a polysaccharide.
- the polyamino acid may be, for example, polyglutamate, whUe the polysaccharide may be, for example, polyethylene glycol (PEG).
- PEG is a water-soluble, non-immunogenic, linear polymer which is avaUable in many weU-defined molecular weight ranges (200-20,000 daltons). The dramatic increase in molecular weight of PEG conjugates significantly reduces glomerular filtering of the protein by the kidney.
- PEG polymers may stericaUy hinder attack by proteolytic enzymes and immunoglobulin molecules, again adding to the serum life of the protein.
- a ⁇ ivated esters of PEG are readily coupled to lysine amino groups on a protein.
- rPF4-PEG conjugates with currently avaUable reagents is straightforward.
- PEG can be covalently attached to rPF4 by free amino groups using a modification of the procedure outlined by Katre et al. fsupra).
- PEG-glutatyl-N-hydroxysuccinimide (MW-5000, Polysciences, Inc.) is added at a 100-fold molar excess to rPF4 in 0.1 M sodium borate, pH 9.0.
- the reaction is aUowed to proceed at 37°C for 48 hours.
- the extent of rea ⁇ ion can be conveniently monitored by SDS-PAGE. Products of the reaction are clearly visible as a series of bands, each differing by 5000 molecular weight and representing species with one or more PEG modifications.
- each salt fraction can be further separated by other chromatographic techniques (gel filtration or hydrophobic interaction chromatography) to separate molecules with a defined PEG:rPF4 ratio.
- the conjugation process can be carried out in the presence of heparin.
- the presence of heparin prevents modification of the heparin binding site of PF4 and results in a conjugate which wiU stiU bind heparin.
- PF4 can be chemicaUy or geneticaUy crosslinked to the toxin ricin A or the diphtheria toxin.
- a fusion protein comprised of PF4 and ricin A can be produced recombinantly in a prokaryotic or eukaryotic host.
- the resulting purified toxin wiU have the high specificity for endothelial cells or cells in close proximity to endothelial cells, e.g., tumor cells.
- PF4 and ricin can be linked with cross linkers.
- DSS can cross link PF4 and ricin A whUe retaining both PF4 and ricin A activities.
- PF4 can also be covalently linked with a cross linker to photoa ⁇ ivatable molecules, for example, hematoporphyrin derivative (HPD).
- HPD hematoporphyrin derivative
- Water soluble carbc iimides e.g., EDC
- EDC Water soluble carbc iimides
- the resulting conjugate will concentrate at sites rich in endotheUal cells (such as solid tumors) and can be a ⁇ ivated by relatively non-toxic laser or phototherapy focused dire ⁇ ly on the tumor site.
- a ⁇ ivated HPD is known to generate active oxygen species which non-specificaUy kiU nearby cells.
- DTT dithiothreitol
- the sulfhydryls of rPF4 were specificaUy and irreversibly modified by prereduction with DTT foUowed by treatment with fluorescein maleimide (FM).
- the reduced and purified rPF4 (5 mg in sodium carbonate buffer, pH 8.5 [SCB]) was treated with 10 mg of FM for 3 hours at room temperature. Residual FM was removed by gel filtration in SCB and then dialyzed against the same buffer.
- the FM-rPF4 partiaUy retained heparin binding a ⁇ ivity.
- FM-rPF4 When tested in the CAM and endotheUal cell proliferation assays, FM-rPF4 exhibited inhibitory activity indicating that neither free sulfhydryls nor correct disulfide bonds are required for the angiostatic activity of PF4.
- This FM-modified rPF4 may possess some utility as an alternative endotheUal ceU labeling or inhibiting compound but, most importantly, it indicates that the cysteine residues of PF4 are also appropriate targets for conjugating or cross linking PF4 to other molecules for diagnostic or therapeutic appUcations.
Abstract
Description
Claims
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EP92915801A EP0594749A1 (en) | 1991-07-15 | 1992-07-15 | Modified pf4 compositions and methods of use |
JP5502920A JPH06509116A (en) | 1991-07-15 | 1992-07-15 | Modified PF4 composition and method of use |
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US73122291A | 1991-07-15 | 1991-07-15 | |
US731,222 | 1991-07-15 |
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Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0576669A1 (en) * | 1992-01-16 | 1994-01-05 | Repligen Corporation | Novel methods and compositions for treatment of angiogenic diseases |
WO1995012414A1 (en) * | 1993-11-05 | 1995-05-11 | Repligen Corporation | Novel modified pf4 compositions and methods of use |
WO1996025956A2 (en) * | 1995-02-21 | 1996-08-29 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Individual medicament dosing conjugate |
EP0786001A1 (en) * | 1994-10-26 | 1997-07-30 | Repligen Corporation | Chemokine-like proteins and methods of use |
US5719167A (en) * | 1995-08-07 | 1998-02-17 | Alcon Laboratories, Inc. | Angiostatic compounds |
US5759547A (en) * | 1989-01-10 | 1998-06-02 | Repligen Corporation | Methods and compositions for treatment of angiogenic diseases |
US5798356A (en) * | 1995-08-07 | 1998-08-25 | Alcon Laboratories, Inc. | Angiostatic compounds |
US6730666B1 (en) | 1998-11-08 | 2004-05-04 | Yeda Research And Development Co. Ltd. | Pharmaceutical compositions comprising porphyrins and some novel porphyrin derivatives |
WO2005030265A2 (en) * | 2003-09-29 | 2005-04-07 | Amersham Health As | Optical imaging of endometriosis |
WO2013134743A1 (en) | 2012-03-08 | 2013-09-12 | Halozyme, Inc. | Conditionally active anti-epidermal growth factor receptor antibodies and methods of use thereof |
WO2015038984A2 (en) | 2013-09-12 | 2015-03-19 | Halozyme, Inc. | Modified anti-epidermal growth factor receptor antibodies and methods of use thereof |
WO2017161206A1 (en) | 2016-03-16 | 2017-09-21 | Halozyme, Inc. | Conjugates containing conditionally active antibodies or antigen-binding fragments thereof, and methods of use |
WO2019118937A1 (en) | 2017-12-15 | 2019-06-20 | Juno Therapeutics, Inc. | Anti-cct5 binding molecules and methods of use thereof |
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EP0088695A2 (en) * | 1982-03-09 | 1983-09-14 | Cytogen Corporation | Antibody conjugates |
WO1988006628A1 (en) * | 1987-03-02 | 1988-09-07 | Bristol-Myers Company | Platelet related growth regulator |
EP0378364A2 (en) * | 1989-01-10 | 1990-07-18 | Repligen Corporation | Analogues of PF4 and fragments thereof, and pharmaceutical compositions containing them |
EP0407122A1 (en) * | 1989-07-06 | 1991-01-09 | Repligen Corporation | Novel modified PF4 compositions and methods of use |
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1992
- 1992-07-15 EP EP92915801A patent/EP0594749A1/en not_active Ceased
- 1992-07-15 WO PCT/US1992/005903 patent/WO1993002192A1/en not_active Application Discontinuation
- 1992-07-15 JP JP5502920A patent/JPH06509116A/en active Pending
- 1992-07-15 CA CA002113206A patent/CA2113206A1/en not_active Abandoned
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EP0088695A2 (en) * | 1982-03-09 | 1983-09-14 | Cytogen Corporation | Antibody conjugates |
WO1988006628A1 (en) * | 1987-03-02 | 1988-09-07 | Bristol-Myers Company | Platelet related growth regulator |
EP0281363A2 (en) * | 1987-03-02 | 1988-09-07 | Oncogen | Platelet related growth regulator |
EP0378364A2 (en) * | 1989-01-10 | 1990-07-18 | Repligen Corporation | Analogues of PF4 and fragments thereof, and pharmaceutical compositions containing them |
EP0407122A1 (en) * | 1989-07-06 | 1991-01-09 | Repligen Corporation | Novel modified PF4 compositions and methods of use |
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CHEMICAL ABSTRACTS, vol. 112, no. 23, 4 June 1990, Columbus, Ohio, US; abstract no. 213283n, SILBERT, D.I. ET AL. 'Glycosaminoglycans of bovine aorta endothelial cells: identification and localization by use of platelet factor 4-fluorescein probe' page 300 ; * |
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA vol. 84, no. 5, March 1987, WASHINGTON US pages 1487 - 1491 KATRE, N.V. ET AL. 'Chemical modification of recombinant interleukin 2 by polyethylene glycol increases its potency in the murine Meth A sarcoma model' cited in the application * |
Cited By (20)
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US5759547A (en) * | 1989-01-10 | 1998-06-02 | Repligen Corporation | Methods and compositions for treatment of angiogenic diseases |
US5800820A (en) * | 1989-01-10 | 1998-09-01 | Repligen Corporation | Methods and compositions for treatment of angiogenic diseases |
EP0576669A4 (en) * | 1992-01-16 | 1996-05-08 | Repligen Corp | Novel methods and compositions for treatment of angiogenic diseases |
EP0576669A1 (en) * | 1992-01-16 | 1994-01-05 | Repligen Corporation | Novel methods and compositions for treatment of angiogenic diseases |
WO1995012414A1 (en) * | 1993-11-05 | 1995-05-11 | Repligen Corporation | Novel modified pf4 compositions and methods of use |
EP0786001A4 (en) * | 1994-10-26 | 1999-12-08 | Repligen Corp | Chemokine-like proteins and methods of use |
EP0786001A1 (en) * | 1994-10-26 | 1997-07-30 | Repligen Corporation | Chemokine-like proteins and methods of use |
WO1996025956A3 (en) * | 1995-02-21 | 1996-12-27 | Deutsches Krebsforsch | Individual medicament dosing conjugate |
WO1996025956A2 (en) * | 1995-02-21 | 1996-08-29 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Individual medicament dosing conjugate |
US6410695B1 (en) | 1995-02-21 | 2002-06-25 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Individual medicament dosing conjugate |
US5719167A (en) * | 1995-08-07 | 1998-02-17 | Alcon Laboratories, Inc. | Angiostatic compounds |
US5798356A (en) * | 1995-08-07 | 1998-08-25 | Alcon Laboratories, Inc. | Angiostatic compounds |
US6730666B1 (en) | 1998-11-08 | 2004-05-04 | Yeda Research And Development Co. Ltd. | Pharmaceutical compositions comprising porphyrins and some novel porphyrin derivatives |
WO2005030265A2 (en) * | 2003-09-29 | 2005-04-07 | Amersham Health As | Optical imaging of endometriosis |
WO2005030265A3 (en) * | 2003-09-29 | 2005-07-07 | Amersham Health As | Optical imaging of endometriosis |
WO2013134743A1 (en) | 2012-03-08 | 2013-09-12 | Halozyme, Inc. | Conditionally active anti-epidermal growth factor receptor antibodies and methods of use thereof |
EP3296320A1 (en) | 2012-03-08 | 2018-03-21 | Halozyme, Inc. | Conditionally active anti-epidermal growth factor receptor antibodies and methods of use thereof |
WO2015038984A2 (en) | 2013-09-12 | 2015-03-19 | Halozyme, Inc. | Modified anti-epidermal growth factor receptor antibodies and methods of use thereof |
WO2017161206A1 (en) | 2016-03-16 | 2017-09-21 | Halozyme, Inc. | Conjugates containing conditionally active antibodies or antigen-binding fragments thereof, and methods of use |
WO2019118937A1 (en) | 2017-12-15 | 2019-06-20 | Juno Therapeutics, Inc. | Anti-cct5 binding molecules and methods of use thereof |
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JPH06509116A (en) | 1994-10-13 |
EP0594749A1 (en) | 1994-05-04 |
CA2113206A1 (en) | 1993-02-04 |
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