CA2053275C - Fibroblast growth factor conjugates - Google Patents
Fibroblast growth factor conjugatesInfo
- Publication number
- CA2053275C CA2053275C CA002053275A CA2053275A CA2053275C CA 2053275 C CA2053275 C CA 2053275C CA 002053275 A CA002053275 A CA 002053275A CA 2053275 A CA2053275 A CA 2053275A CA 2053275 C CA2053275 C CA 2053275C
- Authority
- CA
- Canada
- Prior art keywords
- fgf
- cytotoxic agent
- conjugate
- receptor
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
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Classifications
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
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- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
Abstract
The invention provides a conjugate comprising FGF or other polypeptide reactive with an FGF receptor, and a cytotoxic agent. The cytotoxic agent can be a ribosome-inactivating protein (RIP), such as saporin, although other cytotoxic agents can also be advantageously used. The cytotoxic agent can be attached to FGF through a che mical bond or the composition can be prepared as a chimera, using techniques of recombinant DNA. The conjugate can be used to treat FGF-mediated pathophysiological conditions by specifically targeting cells having FGF receptors and inhibiting p roliferation of or causing death of the cells. Additionally, the conjugate can be used to target cytotoxic agents into cells ha ving FGF receptors, and to inhibit the proliferation of such cells. A method of purifying the conjugate on an immobilized heparin column is also provided.
Description
205327S ~. f' FIBROBLAST GROWTH FACTOR CONJUGATES
Backqround of the Invention 5This invention relates to compositions which inhibit cell proliferation, and, more specifically, to fibroblast growth factor conjugated to a cytotoxic agent.
A great deal of attention has been directed towards the identification and characterization of factors capable of stimulating the growth and proliferation of specific cell types. In the last twenty-five years, a number of such mitogenic factors have been isolated. Rather than having highly specific activities as may have been originally anticipated, many such growth factors are now recognized to have multifunctional activities, affecting a wide spectrum of cell types. In addition, certain activities are shared by homologous members of a family of growth factors.
One family of growth factors now known to have a broad spectrum of activities is the fibroblast growth factors (FGF). Basic FGF is a protein which has a molecular weight of approximately 16 kD, is acid and temperature sensitive and has a high isoelectric point. A structurally related protein, acidic FGF, has an acidic isoelectric point. FGFs exhibit a mitogenic effect on a wide variety of mesenchymal, endocrine and neural cells. Of particular interest is their stimulatory effect on collateral vascularization and angiogenesis. Such mitogenic effects have stimulated considerable interest in FGF as potential therapeutic agents for wound healing, nerve regeneration and cartilage repair, for example.
Cells that respond to basic FGF have been shown to possess specific receptors on the cell surface membranes.
The receptor proteins appear to be single chain polypeptides with molecular weights ranging from 110 to 150 ~ WO90/1~97 ; ~ ~ PCT/US~/02~9 2 2~53275 1 ~D, depending on cell type. The proteins bind basic FGF
with high affinity (Xd - 10-80 pM~, with receptor numbers ranging from 2000 to 80,000 per cell. The receptors can be purified from rat brain, using a combination of lectin and S ligand affinity chromatography and are associated with tyrosine kinase activity (Imamura et al., Biochem. Biophys.
Res. Comm. 155:583-590 (1988); Huang and Huang, J. Biol.
Chem. 261:9568-9571 (1986).
On baby hamster kidney cells (BHX), two basic FGF
~ receptors with estimated molecular weights of 110 and 130 kD have been reported (Neufeld and Gaspodarowicz, J. Biol.
Chem. 260:13860-13868 (1985); Neufeld and Gaspodarowicz, J.
Biol. Chem. 261:5631-5637 (1986).
Both receptor proteins bind basic FGF and acetic FGF, although it appears that the larger binds basic FGF preferentially while the smaller has somewhat higher affinity for acetic FGF.
In addition to potentially useful proliferative effects, basic FGF induced mitogenic stimulation may, in some instances, be detrimental. For example, -cell proliferation and angiogenesis are an integral aspect of tumor growth. Basic FGF is thought to play a pathophysiological role, for example, in tumor development, atherosclerosis, rheumatoid arthritis, proliferative diabetic retinopathie~ and other complications of diabetes.
~ There thus exists a need to inhibit detrimental .~ -mitogenic effect~ of basic FGF in certain pathological conditions. The present invention satisfies this need and provides related advantages as well.
~.
~~0gO~1~9i - PCT/US~f02~
Backqround of the Invention 5This invention relates to compositions which inhibit cell proliferation, and, more specifically, to fibroblast growth factor conjugated to a cytotoxic agent.
A great deal of attention has been directed towards the identification and characterization of factors capable of stimulating the growth and proliferation of specific cell types. In the last twenty-five years, a number of such mitogenic factors have been isolated. Rather than having highly specific activities as may have been originally anticipated, many such growth factors are now recognized to have multifunctional activities, affecting a wide spectrum of cell types. In addition, certain activities are shared by homologous members of a family of growth factors.
One family of growth factors now known to have a broad spectrum of activities is the fibroblast growth factors (FGF). Basic FGF is a protein which has a molecular weight of approximately 16 kD, is acid and temperature sensitive and has a high isoelectric point. A structurally related protein, acidic FGF, has an acidic isoelectric point. FGFs exhibit a mitogenic effect on a wide variety of mesenchymal, endocrine and neural cells. Of particular interest is their stimulatory effect on collateral vascularization and angiogenesis. Such mitogenic effects have stimulated considerable interest in FGF as potential therapeutic agents for wound healing, nerve regeneration and cartilage repair, for example.
Cells that respond to basic FGF have been shown to possess specific receptors on the cell surface membranes.
The receptor proteins appear to be single chain polypeptides with molecular weights ranging from 110 to 150 ~ WO90/1~97 ; ~ ~ PCT/US~/02~9 2 2~53275 1 ~D, depending on cell type. The proteins bind basic FGF
with high affinity (Xd - 10-80 pM~, with receptor numbers ranging from 2000 to 80,000 per cell. The receptors can be purified from rat brain, using a combination of lectin and S ligand affinity chromatography and are associated with tyrosine kinase activity (Imamura et al., Biochem. Biophys.
Res. Comm. 155:583-590 (1988); Huang and Huang, J. Biol.
Chem. 261:9568-9571 (1986).
On baby hamster kidney cells (BHX), two basic FGF
~ receptors with estimated molecular weights of 110 and 130 kD have been reported (Neufeld and Gaspodarowicz, J. Biol.
Chem. 260:13860-13868 (1985); Neufeld and Gaspodarowicz, J.
Biol. Chem. 261:5631-5637 (1986).
Both receptor proteins bind basic FGF and acetic FGF, although it appears that the larger binds basic FGF preferentially while the smaller has somewhat higher affinity for acetic FGF.
In addition to potentially useful proliferative effects, basic FGF induced mitogenic stimulation may, in some instances, be detrimental. For example, -cell proliferation and angiogenesis are an integral aspect of tumor growth. Basic FGF is thought to play a pathophysiological role, for example, in tumor development, atherosclerosis, rheumatoid arthritis, proliferative diabetic retinopathie~ and other complications of diabetes.
~ There thus exists a need to inhibit detrimental .~ -mitogenic effect~ of basic FGF in certain pathological conditions. The present invention satisfies this need and provides related advantages as well.
~.
~~0gO~1~9i - PCT/US~f02~
3 ~ 7 ~
~mary o~ the Invention ~ , :
The invention provides a conjugate comprising basic FGF or other polypeptide reactive with an FGF receptor, and a cytotoxic agent. In one embodiment, the cytotoxic agent .- is a ribosome-inactivating protein (RIP), suc~ as, for example, saporin, although other cytotoxic agents can also be advantageously used. - The cytotoxic agent can be attached to basic FGF through a chemical bond or the composition can be prepared as a chimera, using techniques of recombinant DNA. In both cases, the conjugate molecule is designed and produced in such a way that the receptor-binding epitope of the basic FGF moiety of the complex is left available for recognition by the FGF receptor.
The conjugate can be used to treat FGF-mediated pathophysiological conditions by specifically targeting to cells having FGF receptors and inhibiting proliferation of or causing death of the cells. Such pathophysiological conditions include, for example, tumor development, atherosclero~i~, Dupuytren's Contracture, certain complications of diabetes such as proliferative diabetic retinopathies, and rheumatoid arthritis. The treatment is effected by administering a therapeutically effective amount -of -the FGF conjugate, for example, in a physiologically acceptable excipient. Additionally, the conjugate can be used to target cytotoxic agents into cells .~ having FGF receptor~, and to ~nhihit the proliferation of such cells. A method of purifying the~conjugate on a heparin immobilized column is also provided.
- Brief Desc~iption of the Drawings .: : ~ . . .
Figure 1 shows a heparin Sepharose chromatography of the conjugation reaction mixture.
-Trademark '3~
20~32~5 4 ~~
Figure 2 shows the RIP and binding activities of the basic FGF/SAP conjugate. The activity was compared to SAP
alone in a cell-free protein synthesis inhibition assay (Panel A) (SAP ~ , basic FGF-SAP ~ ) and the receptor binding activity was compared to basic FGF in the BHK
radioreceptor assay (panel B) (basic FGF a, basic FGF-SAP
~ ). Each point is the mean of 3 replicates. Standard deviations were less than 10%.
Figure 3 shows the effect of basic FGF/SAP on BHK cell proliferation. Cell counts were normalized to media controls (190,000 + 15,000). Cell number with 10 ~M of the mitotoxin was 9,527 + 980. N=3 in all instances. (basic FGF-SAP ~ , SAP ~ , basic FGF O, basic FGF + SAP O ).
Figure 4 shows the effect of exogenous basic FGF and NGF on cytotoxicity. Basic FGF-SAP was used at a concentration of 101~ M basic FGF-SAP and C: preincubation with equimolar free basic FGF, D: 10-fold excess of free basic FGF, E: 100-fold excess of basic FGF, F: 1000-fold excess of basic FGF; G: equimolar incubation with equimolar free NGF, H: 10-fold molar excess, I: 100-fold molar excess, J: 1000-fold molar excess.
Figure 5 shows the relationship between toxicity of basic FGF-SAP and FGF receptor ntl~her, determined for each cell line after 48 or 72 hours exposure to basic FGF-SAP.
Cell numbers were determined and the concentration that reduced the number of cells by 50% was plotted against receptor number for that cell line. Receptor number was determined by the method of Moscatelli et al., supra.
Figure 6 shows the effect of basic FGF-SAP on Dupuytren's Cells as described in Example IV.
WO g0~12Sg7 ~ ~ ~ PCI/US90/02289 ~ 5~327 5 ' -- Detailed Descri~tion of the -Invention --The present invention provides a con~ugate comprising basic FGF or other polypeptide reactive with an FGF
5 receptor and a cytotoxic agent, which composition is effective for inhibiting growth and proliferation of cells having FGF receptor~. The composition can be used to counteract the mitoge~c effectQ of basic FGF, where such an effect is deleterious, ~uch as in tumor angiogenesis, 10 atherosclerosisj and~ proliferative ~ complications of diabetes such-as proli~erative retinopathies.
, As used herein, the term "FGF" refers to both basic FGF (bFGF) and acidic FGF (aFGF) and other proteins 15 exhibiting basic FGF mitogenic activity mediated through binding to an FGF receptor. For example, a basic FGF
peptide having a molecular weight of about 16 kD, and a pI
of about 9.6, has been described by Esch et al. Other FGF
proteins include other forms of basic FGF which have an 20 amino terminal extension, aFGF, hSt, int-2 and FGF-5. (See Baird et al., Brit. Med. Bull 45:438-452 (1989)). All express~ mitogenic activity in a wide variety of normal diploid mesoderm-derived and neur~l crest-derived cells.
A test of such "FGF ~itogenic activity" is th~ ability to 25 ~3timulate proliferation of cultured bovine aortic endothelial cells, as described in Gospodarowicz et al., J.
Biol. Chem. 257:12266-12278 (1982); Gospodarowicz et al., Proc. Natl. Acad.~~Sci. ~SA 73:4120-412~ ~1976).
. The term FGF refers both 30 -to protein~ having amino acid- ~;eq~ences ~ound in a mammalian host, as well as modified sequences, ~aving amino - acid substitutions, deletions, insertions or additions, c-which still express ~itogenic activity, mediated through binding to sn FGF receptor. Purified preparations of basic 35 FGF and acidic FGF are frequently -observed to include several molecular forms of the mitogens. It is understood that differences in amino acid sequences can occur in FGF
-WO90/1~97 PCT/US~02~9 - 6 2~3~5 ,.~
from different spec_es as well as between FGF from individual organisms of species. The term is intended to refer to both proteins isolated from natural sources as well as those made synthetically, as by chemical synthesis or recombinant means. _ -. . ~ , . .
The amino acid sequence of an exemplary mammalianbasic FGF derived from boyine p~tuitarY tissue is provided in Esch et al.,,Froc. Natl. Acad, Sci. USA 82:6507-6511 (1985) As used herein, the term '!basic FGF" refers -to proteins or polypeptides having substantially the same amino acid sequence and mitogenic activity as that of the basic FGF
described in Esch, su~ra. cDNAs encoding human aFGF (Jaye et al., Science 233:541-545 (1986) and bovine (Abraham et al., Science 233:545-548 (1986), human (Abraham et al., EMBO J. 5:2523-2528 (1986); Abraham et al., Quant. Biol.
51:657-668 (19~6), and rat (Shimasaki et al., Biochem.
Biophys Res. Commun. 1988; Xurokawa et al., Nucleic Acids Res. 16:5201 (1988)) basic FGF have been cloned, and sequenced and predict the existence of proteins identical to those found by protein sequencing.
As used herein, the term "FGF receptor~ refers to receptors which are able to bind basic FGF and tra~ o~- it into the cell. Included among these are the ~e~e~ors described in Imamura, su~ra and Moscatelli, supra. As used herein, the term "polypeptide reactive with the FGF
receptor" refer to any;polypeptide which i~ capable of binding an FGF receptor and-of-:being transported into the'' cell thereby., ,r _ ~
Basic FGF is commercially available, for example, from Amgen (Thousand Oaks, ~A). Basic FGF can-be- obtained from a variety of tissue types of mammals. For'example, methods of purifying basic FGF using reverse-phase high perfor~ance liquid chromatography (RR-HPLC), heparin-Sepharose affinity Trademark WO ~/12S9i PCTlUS~/02~9 7 ~ Q 5 3 2 ~ 5 chromatography and cation exchange HPLC and RR-HPLC are described in U.S. Pat. No. 4,785,079, ~s well as Gospodarowicz, Proc. Natl. Acad. Sci. 81:6963-6967 (1984) and Gospodarowicz, Meth. Enzym. 147:106-119 (1987).
5 In addition, basic FGF can be synthesized,-as by chemical or recombinant methods. Expression of a-recombinant protein r in yeast and . ÇÇl~ is described in Barr, et- al.,~ J. Biol-. Chem.
263:16471-16478 (1988).
.
The FGF-cytotoxic agent conjugate can be purified on a column cont~n~ng immobilized heparin. Appropriate columns include heparin-Sepharose and heparin-agarose. The bound conjugate can be eluted with a gradient salt, such as NaCl and is eluted between 1 and 3 M.
According to one aspect of the invention, basic FGF is conjugated to a cytotoxic agent so as to target the cytotoxic agent specifically to cells which exhibit FGF
receptors. As used herein, the term cytotoxic agent refers to a molecule capable of inhibiting cell function. The term includes agents which are only toxic when transported into the cell and also those whose toxic effect is mediated at the cell surface. A variety of cytotoxic-agents can be used including those which inhibit protein ~ynthesis. In one aspect of the invention, FGF is combined with a ribosome-inactivating protein (RIP) such as, for example, saporin-6 (SAP) or other SAP derivatives. SAP- ifi a potent RIP which is isolated from the seed~ of the plant Sa~onaria officinalis (See-Stirpe, et al.,-Biochem J.- 216:617-625 (1983)). Other appropriate cytotoxic-agents' include, but are not ilimited -to, ricin, ricin A- chain,-~ gelonin, diphtheria toxin, diphtheria toxin A ch~in and Pseudomonas exotoxin. In another aspect of the invention, the cytotoxic agent i8 a drug.- Examples -of such drugs are anthracyclines such as the daunomycins (including ~Trademark ' ~090/12S97 ~''~') ~ PCT/US~/02~9 ~ - 8 aQ ~ ~7 5 daunorubicin-and doxorubicln) and methotrexate and its analogs. Others are known to those skilled in the art.
FGF can be conjugated to a protein cytotoxic agent by means known to those skilled in the art, such as through derivitization with a reactive sulfhydryl cont~n~ng moiety such as SPDP, or via ia cross linking agent ~uch as - glutaraldehyde or-carbodiimide. In ~one embodiment, the cytotoxic agent is derivatized~with a reactive sulfhydryl containing agent, such as N-succinimidyl'-3~2-pyridyldithio)propionate. FGF is then added to and mixed with the derivatized cytotoxic;agent. The F~F conjugate can be separated from the unreacted products on a column.
Alternatively, FGF can be con~ugated to a drug, such as 14 bromo doxorubicin through the sugar moiety, as by the cis-aconitate method tShen and Riser, BBRC 102:1048 tl981).
Alternatively, chimeric FGF-conjugates can be prepared by recombinant methods. Such methods as applied to con~ugates of IL-2 or TGF~ are provided in Chaudhary et al., Proc. Natl. Acad. Sci. USA 84:4538-4542 (1987) and Lorberman-Galski et al.-, Proc. Natl. Acad. Sci. USA
85:1922-1926 (1988).
See also, Maniatis, et al , Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Laboratory (1982), , A con~ugate containing FGF and a cytotoxic:agent is useful in treating a variety -of FGF-mediatèd pathophysiological conditions.---As 'used herein,''the term "FGF-mediated pathophysiological-condition~ refers to a deleterious condition characterized by or caused by proliferation of-cells which are sensitive to basic FGF
mitogenic stimulation. Basic FGF-mediated pathophysiological conditions include, but are not'limited to, tumors, atherosclerosis, rheumatoid arthritis, WO 90/12S97; - ' ~ ~ PCl /VS90/02289 . ~, 9 ~Q ~ 3~7 ~
,~
Dupuytren's Contracture and certain complications of diabetes such as proliferative retinopathy.
FGF-cytotoxic agent conjugates can be used to target the cytotoxic agent to cells expressing FGF receptors in order to cause cell-death. Surprisingly, there i~ a direct relationship between the number of FGF receptors per cell and the dose at which~50% of the cells are killed (the ED50), as is shown in Figure 5. Moreover,-for cells with extremely high receptor numbers, for example, BHK cells, the EDso is identical to the affinity constant-of basic FGF
for its receptor (both;-are about 25 pM for BHK cells).
This unexpected result indicates that the presence of the cytotoxic agent, even such a large molecule as SAP, does not reduce basic FGF activity. Moreover, these results indicate that these cell that are expressinq a large number of basic FGF receptors are particularly sensitive to the con;ugate.
In order to treat FGF-mediated pathophysiological conditions, a therapeutically effective amount of FGF-cytotoxic agent con~ugate is administered to a mammal in a physiologically ac~eptable excipient. Examples of physiologically acceptable excipient include PBS and saline. - - - -The following examples are intended to illustrate butnot limit the invention.
. ~
~Ya~ple CONJUGATION OF FGF WITH SAPORIN
. .
Recombinant basic FGF~corresponding to the seguence of 154 amino acids ~Abraham et~al., Quant. Biol; 51:657-668 (1986),~was obtained from Farmitalia- Carlo Erba. Saporin-6 was purified according to the met~od-of-Stirpe, et al., supra, ~' wogo/1~9i PCT/US~/02~
- lo ~ ~ 5 ~ ~ 7 ~
",,, a~ modified by Lapp~, et al., Biochem.~Biophys. Re~.--Comm.
129:934-942 (1985).
Briefly, seeds of Saponaria officinalis were extracted by grinding in 0.14 M NaCl in 5 mM sodium phosphate buffer, pH 7.2 (8 ml/g). After overnight stirring at 4-C, extracts were ~strained through chcc_c cloth and were centrifuged at 28000 g for 30 minutes. The supernatant was separated from the sediment and from floating fat, and i~ referred to as "crude extract.~-.. i Crude extracts were dialyzed against' 5 mM sodium phosphate buffer, pH 6.5 centrifuged at -28000 g for 30 minutes and applied to a CM cellulose column (CM 52, Whatman, Maidstone, Kent, U.K.), which after washing, was eluted with a 0-0.3 M NaCl gradient in the same buffer.
This material was then dialyzed against water and chromatographed on an FPLC Mono S column (Pharmacia, Uppsala, Sweden) equilibrated with 50 mM sodium borate pH
9.5, 0.156 M sodium chloride. The protein was eluted with a 20 minute gradient from 0.156 M to 0.186 H sodium ch~oride. The resultant peak material was then extensively -- dialyzed against Milli-Q water ~Millipore,~ Bedford, MA).
A portion of the dried material was weighed and dissolved in water at a concentration of 1 mg/ml. An ultraviolet spectrum was recorded giving a 1% extinction coefficient of 6.4 at 277 nm, the absorbance maximum. At 280 nm the E
was 6Ø Protein assay using the Lowry method ~Lowry, et al., J. Biol. Chem. 193:265-275-(1951) using BSA as a st~n~rd gave a result of 1.07 mg/ml.
SAP was derivatized with N-succinimdyl-3(2-pyridyldithio)propionate (SPDP; Pharmacia Fine Chemicals, Piscataway, NJ~ according to the manufacturer's instructions. Briefly, SAP was dissolved in (2.7'mg/mL) in sodium phosphate buffer (0.-iM, -pH 7.5) containing NaCl~
(0.1 M). A 1.25 molar excess of SPDP, dissolved''ethanol, was added by drop while-stirring, and allowed to react for ' Trademark ~'090/12S97 ' PCT/US~/02~ -1l ~ n 5 3 2 ~ 5 30 minutes-at -23-C-- with~occasional stirring.~s' 'Excess reagent and low molecular weight reaction products were removed by gel filtratlon. basic FGF (2 mg/ml) was added to and mixed with the derivatized saporin (6 mg/ml in O.l S M sodium phosphate, O.l M sodium chloride, pH 7.5) for two hours at room temperature. m e reaction was terminated by the addition~of~35 ~L-'of O.l M iodoacetamide. After an additional 30 minutes,-~he reaction mixture was diluted to 30 ml and loaded onto a heparin-Sepharose (Pharmacia) -10 column (O.S x 5.5 cm). The bound proteins were eluted with -- a step gradient of 0.6 M,'l M and'2-M NaCl ln lO ~M TRIS, pH 7.4. -The material eluting between-'l' M and 2' M was pooled. Final purification of the conjugate was achieved after the pool was dialyzed against water and chromatographed on a Mono S 5/5 NaCl cation exchange column (Pharmacia) (buffer A: 50mM sodium borate, pH 8.0, buffer B:0.5 M NaCl in buffer A). Fractions containing the conjugated were detected by silver staining after PhastGel*
(Pharmacia) electrophoresis and appropriate fractions were pooled for analysis.
Synthesis of the con~ugate was assessed by gel electrophoresis and allowed to proceed until no detectable basic PGF remained in the reaction mixture. Chromatography on heparin-Sepharose * (Figure l) and subsequent electrophoretic 'analysis of each of the pea~ fractions showed that while SAP-does-~not bind to heparin-Sepharose,*
- the conjugate doe~.- Only-small amounts of the~conjugate were rel~A-~~ during the-~ M -NaC~ 'wash. - m e màjor product eluted-with -the 2 M wash and containe~d'equimolar amounts of SAP and basic''FGF (Mr-40jOOO).~-~Hu~r~ver, there was also a portion of the conjugate that has an est~mated M~>68~009 presumably as~a result of the conjugation o'f'two molecules of basic FGF per-~olecule of'saporin.~
35Unambiguous identification of the SAP-basic FGF
conjugate was accomplished using sequence specific antisera ~Trademark ..~,...
WOgO/1~9t PCT/US90/02~
i: 12 2Q53~75 J
.~
raised in rabbitfi.- The immunogen used was a fragment of basic FGF comprising amino acids 1 through 24, chemically synthesized using a 990 Peptide Synthesizer -(Beckman Instruments, Brea, CA). Western blot analysis showed that all molecular weight forms of the conjugate contained both basic FGF and SA~ The antiserum reco~izes- the mid portion of the peptide and cross-reacts on equi~olar basis with purified bovine and recombinant human basic FGF.
.. . .: . :
Samples in a sodium dodecyl sulfate-containing polyacrylamide gel, after electrophoresis, were electroblotted onto nitrocellulose membranes, and allowed to air dry. The membrane was covered with TRIS buffered saline (TBS) and agitated for 10 minutes. The solution was aspirated and discarded. The membrane was covered with 5%
nonfat milk (NFM) in TBS and agitated for 10 minutes. The solution was aspirated and discarded. Primary antibody, either anti-SAP or anti basic basic FGF anti-serum, at a concentration of 1/1000 in NFM/TBS was added and agitated overnight. The solution was aspirated and ~ rded. The membrane was covered with TBS, agitated for 10 ~inutes and the solution aspirated and discarded. The membrane was covered with 0. 05% NP40/TBS and ~h~kPn 1 minute; the solution was aspirated and ~i~c~rded. The final TBS and NP40/TBS washes were replated twice. ~orseradish peroxidase labelled anti-IgG at a dilution of 1/2000 in NFM/TBS was added and the membrane agitated for 2 hours.
The TBS and NP40/TBS wash ~steps were repeated. The membrane was placed in a~solution (freshly mixed) 60 mg 4-chloro-l-naphthol in 20 mL methanol-and-100 mL-double ~distilled water-and lO~L 30~ H2O and the solution added to the membrane and allowed to develop. The -solution was aspirated and ~i~c~rded and the reaction-stopped by rinsing with water. The membrane was allowed to dry. ~ - ~
~Trademark ~ - - -. .
~;~
W~90/1~97' . i PCT/US~/~ ~9 . 13 ~ 0 ~3~7 5 .~,, ~mple I~
~ A~ vll~ OF THE FGF/SAP CONJUGATE
~ The capacity of the conjugate to recognize the basic FGF receptor was examined in BHX cells using the procedure described by Moscatelli, et al., J. Cell Physiol. 131:123-- 130 (1987).
~ Briefly,.cells were grown to ~-lhro~fluence and 1n~h~ted in 300 ~L buffer containing.F-12 14-mM NaHCO3,..25 mM HEPES and 0.2% gelatin _ at-. 4- C -.for two ~our.~- with 10 ~1 .. radioiodinated basic. FGF -in the presence of various concentrations of--basic FGF or the con~ugate. The cells were then washed three times with 0.5 mL phosphate buffered saline (PBS), and twice with 2M NaCl in PBS. Binding to the high affinity receptor was determined by counting the membrane fraction that was solubilized 0.5% Triton X-100 in PBS (pH 8.1).
The protein synthesis inhibition activity of the SAP
protein was compared to the protein synthesis inhibition activity of the basic FGF/SAP conjugate in ~ vitro assays of protein ~ynthesis as described in Siehn et al., Blood ?2:.756-765 (1988). ~ ~
The cytotoxic activity of the conjugate was tested on baby hamster kidney fibroblasts (ATCC Accession - No. CRL 628i). BHK cells were plated in 24 well plates at a concentration of 5000 cell~/ml and in~h~ted overnight at 37-C, 5% CO.2.-_.The ~ollowing morning HEPES-buffered DMEM and ~. F-12 medi~ 1)-plus.-5% FCS was aspirated from the wells 30 .and replaced-with.media albne or with media cont~ n~ thè
conjugate, basic FGF or saporin. Two days later, the cells were washed twice, trypsinized and cell number determined with a Coulter Particle Counter*(Coulter Electronics, ~ Hialeah, FL).
As shown in Figure 2A the conjugate retains saporin activity when tested in an n vitro protein synthesis ~Trademark ~ 6~ 7 5 , Pcr/usgo/02289 ' 14 inhibition assay. The conjugate, as expected, is slightly less active (about two-fold) than free SAP. This is consistent with the low level of derivatization of SAP
prior to the conjugation (0.8 moles SPDP/mole) and with probable steric hindrance due to the presence of bound basic FGF. In contrast, the results obtained in the radioreceptor assays for basic FGF (Figure 2B) showed that the basic FGF/SAP is equipotent to, if not slightly more active than, basic FGF in the binding assay. Thus, it appears that the commitment of free sulfhydryl groups in basic FGF to bridging with SAP does not interfere with its capacity to recognize its receptor. If anything, this reaction may be stabilizing basic FGF.
Basic FGF/SAP is a potent cytotoxic factor for BHK
cells (Figure 3). SAP has no toxic effect on these cells even at the highest dose tested (10 ~M) and basic FGF alone has a slight inhibitory effect on proliferation. A mixture of basic FGF and SAP had a slight toxicity but only at the highest concentration tested. The ID50 (25 pM) for the cytotoxic agent compares well with the potency of basic FGF
(15 pM) in proliferation assays. Specificity of the cytotoxic agent was examined in competition experiments in an effort to establish that the mitotoxic activity of the conjugate is receptor specific. BHK cells were preincubated for one hour with various levels of basic FGF
or nerve growth factor (NGF) prior to treatment of the cells with the cytotoxic agent. As shown in Figure 4, there is a dose-related inhibition of the cytotoxic activity in the presence of increasing amounts of basic FGF. In contrast, a thousand-fold excess of NGF has no effect.
W~ 2S97 ~ PCT/US9~02~n ~ ~ 5-3275 .,~
~X~PT~ ITT
T~H~BTTION OF ANGIOGENESIS IN RABBIT CORN~
Elvax (ethylene-vinyl acetate copolymer resin, Dupont, Wilmington, DE) pellets were produced in the following - manner. About 60 mg of washed and dried Elvax* was dissolved in 500 ~L of methylene chloride. This wa~'added to 50 ~g of dried basic ~GF. 5 ~L drops were dropped onto a slide frozen in dry ice. Pellets were left in the freezer overnight and then dried in a desiccator.
New Zealand white rabbits were anaesthetized with Innovar Vet: 1 mL/kg. An incision was made-in the cornea of the rabbit eye and a pocket was opened with a spatula or forceps. One pellet was inserted in the pocket. Pellets were inserted in both eyes. The eye was washed with saline and 1 ml of gentamicin was injected intramuscularly. The rabbit was left for five days and angiogenesis was observed. After five days, each left eye was treated with 20 ~L of 100 ng basic FGF-SAP prepared as in Example I in 0.25% BSA. The right eyes were treated with 20 ~L of 0.25%
BSA alone. The treatment wa~ done twice daily by dropping the solution ~5 eye drops onto the cornea of t'he rabbit.
The person treating the ~nimal~ was unaware of the identity of the samples. After 10-days, the animals were evaluated for angiogenesis of the cornea by microscopic analysis by an evaluator who did not know the treatment regimen.
Angiogenesis- wa~ ~udged with ~~ as- be~ng maximal angiogenesis and-- as being no angioqenesis.
, , The results ~re provided in Table I. --A~ can ~e seen, angiogenesis in corneas treated with basic FGF-SAP was markedly reduced over that of controls.~
*Trade-mark '.'VO 90/12S97 PCI'/US90/022:~9 - 16 2Q ~ ~7 5 .".,, . . - TA R~ .~? T
~ . . . . .
ANIM~n RIGHT EYF~ LEFT ~YE
995 +
5 997 ~ + + +
998 -- -- -~ ~ + + :
999 ;-.+, +
,, . . , - , ~ . .. .
.. , .. , , _ . .. . .
10 . - EXAMPLE IV :- . -EFFECT OF FGF-SAP IN 1JIJYUr1~EN~S CELI.
, Cells obtained from surgical removal of tissue from the hand of adult patients diagnosed as having Dupuytren's Contracture, a malady effecting movement of the hand, were placed in primary culture. These cells have between lO,OOo and 15,000 basic FGF receptors per cell.
The cells were grown overnight in a 24 well tissue culture dish at a concentration of 10,000 cells per well in HEPES buffered DMEM with 10% FCS. The next ~orning the media -was removed and replaced- with- media~ containing concentrations of basic FGF-SAP con~ugate ranging from 10 ~ to 1012 molar. Controls.included wells treated with media only, wells treated with ~milar concentrations of basic FGF alone, saporin alone, and basic FGF and saporin together but not conjugated. The cells were returned to the incubator~for-72 hour~. At the end of this~incubation the cells were washed, removed with trypsin and counted on a Coulter cell counter. The number of cells in the media controls was compared .with the-number of cells in the treated wells (as described above). The results~of these cell killing assays are shown in Figure 6. As can be seen, Dupuytren's cells are sensitive to basic FGF-SAP. Similar results were obtained w~th three other cell samples.
'-Trademark =,, ,~ .
20~3275 Although the invention has been described with reference to the presently-preferred embodiments, it should be understood that various modifications can be made without departing from the spirit of the invention.
Accordingly, the invention is limited only by the following claims.
~mary o~ the Invention ~ , :
The invention provides a conjugate comprising basic FGF or other polypeptide reactive with an FGF receptor, and a cytotoxic agent. In one embodiment, the cytotoxic agent .- is a ribosome-inactivating protein (RIP), suc~ as, for example, saporin, although other cytotoxic agents can also be advantageously used. - The cytotoxic agent can be attached to basic FGF through a chemical bond or the composition can be prepared as a chimera, using techniques of recombinant DNA. In both cases, the conjugate molecule is designed and produced in such a way that the receptor-binding epitope of the basic FGF moiety of the complex is left available for recognition by the FGF receptor.
The conjugate can be used to treat FGF-mediated pathophysiological conditions by specifically targeting to cells having FGF receptors and inhibiting proliferation of or causing death of the cells. Such pathophysiological conditions include, for example, tumor development, atherosclero~i~, Dupuytren's Contracture, certain complications of diabetes such as proliferative diabetic retinopathies, and rheumatoid arthritis. The treatment is effected by administering a therapeutically effective amount -of -the FGF conjugate, for example, in a physiologically acceptable excipient. Additionally, the conjugate can be used to target cytotoxic agents into cells .~ having FGF receptor~, and to ~nhihit the proliferation of such cells. A method of purifying the~conjugate on a heparin immobilized column is also provided.
- Brief Desc~iption of the Drawings .: : ~ . . .
Figure 1 shows a heparin Sepharose chromatography of the conjugation reaction mixture.
-Trademark '3~
20~32~5 4 ~~
Figure 2 shows the RIP and binding activities of the basic FGF/SAP conjugate. The activity was compared to SAP
alone in a cell-free protein synthesis inhibition assay (Panel A) (SAP ~ , basic FGF-SAP ~ ) and the receptor binding activity was compared to basic FGF in the BHK
radioreceptor assay (panel B) (basic FGF a, basic FGF-SAP
~ ). Each point is the mean of 3 replicates. Standard deviations were less than 10%.
Figure 3 shows the effect of basic FGF/SAP on BHK cell proliferation. Cell counts were normalized to media controls (190,000 + 15,000). Cell number with 10 ~M of the mitotoxin was 9,527 + 980. N=3 in all instances. (basic FGF-SAP ~ , SAP ~ , basic FGF O, basic FGF + SAP O ).
Figure 4 shows the effect of exogenous basic FGF and NGF on cytotoxicity. Basic FGF-SAP was used at a concentration of 101~ M basic FGF-SAP and C: preincubation with equimolar free basic FGF, D: 10-fold excess of free basic FGF, E: 100-fold excess of basic FGF, F: 1000-fold excess of basic FGF; G: equimolar incubation with equimolar free NGF, H: 10-fold molar excess, I: 100-fold molar excess, J: 1000-fold molar excess.
Figure 5 shows the relationship between toxicity of basic FGF-SAP and FGF receptor ntl~her, determined for each cell line after 48 or 72 hours exposure to basic FGF-SAP.
Cell numbers were determined and the concentration that reduced the number of cells by 50% was plotted against receptor number for that cell line. Receptor number was determined by the method of Moscatelli et al., supra.
Figure 6 shows the effect of basic FGF-SAP on Dupuytren's Cells as described in Example IV.
WO g0~12Sg7 ~ ~ ~ PCI/US90/02289 ~ 5~327 5 ' -- Detailed Descri~tion of the -Invention --The present invention provides a con~ugate comprising basic FGF or other polypeptide reactive with an FGF
5 receptor and a cytotoxic agent, which composition is effective for inhibiting growth and proliferation of cells having FGF receptor~. The composition can be used to counteract the mitoge~c effectQ of basic FGF, where such an effect is deleterious, ~uch as in tumor angiogenesis, 10 atherosclerosisj and~ proliferative ~ complications of diabetes such-as proli~erative retinopathies.
, As used herein, the term "FGF" refers to both basic FGF (bFGF) and acidic FGF (aFGF) and other proteins 15 exhibiting basic FGF mitogenic activity mediated through binding to an FGF receptor. For example, a basic FGF
peptide having a molecular weight of about 16 kD, and a pI
of about 9.6, has been described by Esch et al. Other FGF
proteins include other forms of basic FGF which have an 20 amino terminal extension, aFGF, hSt, int-2 and FGF-5. (See Baird et al., Brit. Med. Bull 45:438-452 (1989)). All express~ mitogenic activity in a wide variety of normal diploid mesoderm-derived and neur~l crest-derived cells.
A test of such "FGF ~itogenic activity" is th~ ability to 25 ~3timulate proliferation of cultured bovine aortic endothelial cells, as described in Gospodarowicz et al., J.
Biol. Chem. 257:12266-12278 (1982); Gospodarowicz et al., Proc. Natl. Acad.~~Sci. ~SA 73:4120-412~ ~1976).
. The term FGF refers both 30 -to protein~ having amino acid- ~;eq~ences ~ound in a mammalian host, as well as modified sequences, ~aving amino - acid substitutions, deletions, insertions or additions, c-which still express ~itogenic activity, mediated through binding to sn FGF receptor. Purified preparations of basic 35 FGF and acidic FGF are frequently -observed to include several molecular forms of the mitogens. It is understood that differences in amino acid sequences can occur in FGF
-WO90/1~97 PCT/US~02~9 - 6 2~3~5 ,.~
from different spec_es as well as between FGF from individual organisms of species. The term is intended to refer to both proteins isolated from natural sources as well as those made synthetically, as by chemical synthesis or recombinant means. _ -. . ~ , . .
The amino acid sequence of an exemplary mammalianbasic FGF derived from boyine p~tuitarY tissue is provided in Esch et al.,,Froc. Natl. Acad, Sci. USA 82:6507-6511 (1985) As used herein, the term '!basic FGF" refers -to proteins or polypeptides having substantially the same amino acid sequence and mitogenic activity as that of the basic FGF
described in Esch, su~ra. cDNAs encoding human aFGF (Jaye et al., Science 233:541-545 (1986) and bovine (Abraham et al., Science 233:545-548 (1986), human (Abraham et al., EMBO J. 5:2523-2528 (1986); Abraham et al., Quant. Biol.
51:657-668 (19~6), and rat (Shimasaki et al., Biochem.
Biophys Res. Commun. 1988; Xurokawa et al., Nucleic Acids Res. 16:5201 (1988)) basic FGF have been cloned, and sequenced and predict the existence of proteins identical to those found by protein sequencing.
As used herein, the term "FGF receptor~ refers to receptors which are able to bind basic FGF and tra~ o~- it into the cell. Included among these are the ~e~e~ors described in Imamura, su~ra and Moscatelli, supra. As used herein, the term "polypeptide reactive with the FGF
receptor" refer to any;polypeptide which i~ capable of binding an FGF receptor and-of-:being transported into the'' cell thereby., ,r _ ~
Basic FGF is commercially available, for example, from Amgen (Thousand Oaks, ~A). Basic FGF can-be- obtained from a variety of tissue types of mammals. For'example, methods of purifying basic FGF using reverse-phase high perfor~ance liquid chromatography (RR-HPLC), heparin-Sepharose affinity Trademark WO ~/12S9i PCTlUS~/02~9 7 ~ Q 5 3 2 ~ 5 chromatography and cation exchange HPLC and RR-HPLC are described in U.S. Pat. No. 4,785,079, ~s well as Gospodarowicz, Proc. Natl. Acad. Sci. 81:6963-6967 (1984) and Gospodarowicz, Meth. Enzym. 147:106-119 (1987).
5 In addition, basic FGF can be synthesized,-as by chemical or recombinant methods. Expression of a-recombinant protein r in yeast and . ÇÇl~ is described in Barr, et- al.,~ J. Biol-. Chem.
263:16471-16478 (1988).
.
The FGF-cytotoxic agent conjugate can be purified on a column cont~n~ng immobilized heparin. Appropriate columns include heparin-Sepharose and heparin-agarose. The bound conjugate can be eluted with a gradient salt, such as NaCl and is eluted between 1 and 3 M.
According to one aspect of the invention, basic FGF is conjugated to a cytotoxic agent so as to target the cytotoxic agent specifically to cells which exhibit FGF
receptors. As used herein, the term cytotoxic agent refers to a molecule capable of inhibiting cell function. The term includes agents which are only toxic when transported into the cell and also those whose toxic effect is mediated at the cell surface. A variety of cytotoxic-agents can be used including those which inhibit protein ~ynthesis. In one aspect of the invention, FGF is combined with a ribosome-inactivating protein (RIP) such as, for example, saporin-6 (SAP) or other SAP derivatives. SAP- ifi a potent RIP which is isolated from the seed~ of the plant Sa~onaria officinalis (See-Stirpe, et al.,-Biochem J.- 216:617-625 (1983)). Other appropriate cytotoxic-agents' include, but are not ilimited -to, ricin, ricin A- chain,-~ gelonin, diphtheria toxin, diphtheria toxin A ch~in and Pseudomonas exotoxin. In another aspect of the invention, the cytotoxic agent i8 a drug.- Examples -of such drugs are anthracyclines such as the daunomycins (including ~Trademark ' ~090/12S97 ~''~') ~ PCT/US~/02~9 ~ - 8 aQ ~ ~7 5 daunorubicin-and doxorubicln) and methotrexate and its analogs. Others are known to those skilled in the art.
FGF can be conjugated to a protein cytotoxic agent by means known to those skilled in the art, such as through derivitization with a reactive sulfhydryl cont~n~ng moiety such as SPDP, or via ia cross linking agent ~uch as - glutaraldehyde or-carbodiimide. In ~one embodiment, the cytotoxic agent is derivatized~with a reactive sulfhydryl containing agent, such as N-succinimidyl'-3~2-pyridyldithio)propionate. FGF is then added to and mixed with the derivatized cytotoxic;agent. The F~F conjugate can be separated from the unreacted products on a column.
Alternatively, FGF can be con~ugated to a drug, such as 14 bromo doxorubicin through the sugar moiety, as by the cis-aconitate method tShen and Riser, BBRC 102:1048 tl981).
Alternatively, chimeric FGF-conjugates can be prepared by recombinant methods. Such methods as applied to con~ugates of IL-2 or TGF~ are provided in Chaudhary et al., Proc. Natl. Acad. Sci. USA 84:4538-4542 (1987) and Lorberman-Galski et al.-, Proc. Natl. Acad. Sci. USA
85:1922-1926 (1988).
See also, Maniatis, et al , Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Laboratory (1982), , A con~ugate containing FGF and a cytotoxic:agent is useful in treating a variety -of FGF-mediatèd pathophysiological conditions.---As 'used herein,''the term "FGF-mediated pathophysiological-condition~ refers to a deleterious condition characterized by or caused by proliferation of-cells which are sensitive to basic FGF
mitogenic stimulation. Basic FGF-mediated pathophysiological conditions include, but are not'limited to, tumors, atherosclerosis, rheumatoid arthritis, WO 90/12S97; - ' ~ ~ PCl /VS90/02289 . ~, 9 ~Q ~ 3~7 ~
,~
Dupuytren's Contracture and certain complications of diabetes such as proliferative retinopathy.
FGF-cytotoxic agent conjugates can be used to target the cytotoxic agent to cells expressing FGF receptors in order to cause cell-death. Surprisingly, there i~ a direct relationship between the number of FGF receptors per cell and the dose at which~50% of the cells are killed (the ED50), as is shown in Figure 5. Moreover,-for cells with extremely high receptor numbers, for example, BHK cells, the EDso is identical to the affinity constant-of basic FGF
for its receptor (both;-are about 25 pM for BHK cells).
This unexpected result indicates that the presence of the cytotoxic agent, even such a large molecule as SAP, does not reduce basic FGF activity. Moreover, these results indicate that these cell that are expressinq a large number of basic FGF receptors are particularly sensitive to the con;ugate.
In order to treat FGF-mediated pathophysiological conditions, a therapeutically effective amount of FGF-cytotoxic agent con~ugate is administered to a mammal in a physiologically ac~eptable excipient. Examples of physiologically acceptable excipient include PBS and saline. - - - -The following examples are intended to illustrate butnot limit the invention.
. ~
~Ya~ple CONJUGATION OF FGF WITH SAPORIN
. .
Recombinant basic FGF~corresponding to the seguence of 154 amino acids ~Abraham et~al., Quant. Biol; 51:657-668 (1986),~was obtained from Farmitalia- Carlo Erba. Saporin-6 was purified according to the met~od-of-Stirpe, et al., supra, ~' wogo/1~9i PCT/US~/02~
- lo ~ ~ 5 ~ ~ 7 ~
",,, a~ modified by Lapp~, et al., Biochem.~Biophys. Re~.--Comm.
129:934-942 (1985).
Briefly, seeds of Saponaria officinalis were extracted by grinding in 0.14 M NaCl in 5 mM sodium phosphate buffer, pH 7.2 (8 ml/g). After overnight stirring at 4-C, extracts were ~strained through chcc_c cloth and were centrifuged at 28000 g for 30 minutes. The supernatant was separated from the sediment and from floating fat, and i~ referred to as "crude extract.~-.. i Crude extracts were dialyzed against' 5 mM sodium phosphate buffer, pH 6.5 centrifuged at -28000 g for 30 minutes and applied to a CM cellulose column (CM 52, Whatman, Maidstone, Kent, U.K.), which after washing, was eluted with a 0-0.3 M NaCl gradient in the same buffer.
This material was then dialyzed against water and chromatographed on an FPLC Mono S column (Pharmacia, Uppsala, Sweden) equilibrated with 50 mM sodium borate pH
9.5, 0.156 M sodium chloride. The protein was eluted with a 20 minute gradient from 0.156 M to 0.186 H sodium ch~oride. The resultant peak material was then extensively -- dialyzed against Milli-Q water ~Millipore,~ Bedford, MA).
A portion of the dried material was weighed and dissolved in water at a concentration of 1 mg/ml. An ultraviolet spectrum was recorded giving a 1% extinction coefficient of 6.4 at 277 nm, the absorbance maximum. At 280 nm the E
was 6Ø Protein assay using the Lowry method ~Lowry, et al., J. Biol. Chem. 193:265-275-(1951) using BSA as a st~n~rd gave a result of 1.07 mg/ml.
SAP was derivatized with N-succinimdyl-3(2-pyridyldithio)propionate (SPDP; Pharmacia Fine Chemicals, Piscataway, NJ~ according to the manufacturer's instructions. Briefly, SAP was dissolved in (2.7'mg/mL) in sodium phosphate buffer (0.-iM, -pH 7.5) containing NaCl~
(0.1 M). A 1.25 molar excess of SPDP, dissolved''ethanol, was added by drop while-stirring, and allowed to react for ' Trademark ~'090/12S97 ' PCT/US~/02~ -1l ~ n 5 3 2 ~ 5 30 minutes-at -23-C-- with~occasional stirring.~s' 'Excess reagent and low molecular weight reaction products were removed by gel filtratlon. basic FGF (2 mg/ml) was added to and mixed with the derivatized saporin (6 mg/ml in O.l S M sodium phosphate, O.l M sodium chloride, pH 7.5) for two hours at room temperature. m e reaction was terminated by the addition~of~35 ~L-'of O.l M iodoacetamide. After an additional 30 minutes,-~he reaction mixture was diluted to 30 ml and loaded onto a heparin-Sepharose (Pharmacia) -10 column (O.S x 5.5 cm). The bound proteins were eluted with -- a step gradient of 0.6 M,'l M and'2-M NaCl ln lO ~M TRIS, pH 7.4. -The material eluting between-'l' M and 2' M was pooled. Final purification of the conjugate was achieved after the pool was dialyzed against water and chromatographed on a Mono S 5/5 NaCl cation exchange column (Pharmacia) (buffer A: 50mM sodium borate, pH 8.0, buffer B:0.5 M NaCl in buffer A). Fractions containing the conjugated were detected by silver staining after PhastGel*
(Pharmacia) electrophoresis and appropriate fractions were pooled for analysis.
Synthesis of the con~ugate was assessed by gel electrophoresis and allowed to proceed until no detectable basic PGF remained in the reaction mixture. Chromatography on heparin-Sepharose * (Figure l) and subsequent electrophoretic 'analysis of each of the pea~ fractions showed that while SAP-does-~not bind to heparin-Sepharose,*
- the conjugate doe~.- Only-small amounts of the~conjugate were rel~A-~~ during the-~ M -NaC~ 'wash. - m e màjor product eluted-with -the 2 M wash and containe~d'equimolar amounts of SAP and basic''FGF (Mr-40jOOO).~-~Hu~r~ver, there was also a portion of the conjugate that has an est~mated M~>68~009 presumably as~a result of the conjugation o'f'two molecules of basic FGF per-~olecule of'saporin.~
35Unambiguous identification of the SAP-basic FGF
conjugate was accomplished using sequence specific antisera ~Trademark ..~,...
WOgO/1~9t PCT/US90/02~
i: 12 2Q53~75 J
.~
raised in rabbitfi.- The immunogen used was a fragment of basic FGF comprising amino acids 1 through 24, chemically synthesized using a 990 Peptide Synthesizer -(Beckman Instruments, Brea, CA). Western blot analysis showed that all molecular weight forms of the conjugate contained both basic FGF and SA~ The antiserum reco~izes- the mid portion of the peptide and cross-reacts on equi~olar basis with purified bovine and recombinant human basic FGF.
.. . .: . :
Samples in a sodium dodecyl sulfate-containing polyacrylamide gel, after electrophoresis, were electroblotted onto nitrocellulose membranes, and allowed to air dry. The membrane was covered with TRIS buffered saline (TBS) and agitated for 10 minutes. The solution was aspirated and discarded. The membrane was covered with 5%
nonfat milk (NFM) in TBS and agitated for 10 minutes. The solution was aspirated and discarded. Primary antibody, either anti-SAP or anti basic basic FGF anti-serum, at a concentration of 1/1000 in NFM/TBS was added and agitated overnight. The solution was aspirated and ~ rded. The membrane was covered with TBS, agitated for 10 ~inutes and the solution aspirated and discarded. The membrane was covered with 0. 05% NP40/TBS and ~h~kPn 1 minute; the solution was aspirated and ~i~c~rded. The final TBS and NP40/TBS washes were replated twice. ~orseradish peroxidase labelled anti-IgG at a dilution of 1/2000 in NFM/TBS was added and the membrane agitated for 2 hours.
The TBS and NP40/TBS wash ~steps were repeated. The membrane was placed in a~solution (freshly mixed) 60 mg 4-chloro-l-naphthol in 20 mL methanol-and-100 mL-double ~distilled water-and lO~L 30~ H2O and the solution added to the membrane and allowed to develop. The -solution was aspirated and ~i~c~rded and the reaction-stopped by rinsing with water. The membrane was allowed to dry. ~ - ~
~Trademark ~ - - -. .
~;~
W~90/1~97' . i PCT/US~/~ ~9 . 13 ~ 0 ~3~7 5 .~,, ~mple I~
~ A~ vll~ OF THE FGF/SAP CONJUGATE
~ The capacity of the conjugate to recognize the basic FGF receptor was examined in BHX cells using the procedure described by Moscatelli, et al., J. Cell Physiol. 131:123-- 130 (1987).
~ Briefly,.cells were grown to ~-lhro~fluence and 1n~h~ted in 300 ~L buffer containing.F-12 14-mM NaHCO3,..25 mM HEPES and 0.2% gelatin _ at-. 4- C -.for two ~our.~- with 10 ~1 .. radioiodinated basic. FGF -in the presence of various concentrations of--basic FGF or the con~ugate. The cells were then washed three times with 0.5 mL phosphate buffered saline (PBS), and twice with 2M NaCl in PBS. Binding to the high affinity receptor was determined by counting the membrane fraction that was solubilized 0.5% Triton X-100 in PBS (pH 8.1).
The protein synthesis inhibition activity of the SAP
protein was compared to the protein synthesis inhibition activity of the basic FGF/SAP conjugate in ~ vitro assays of protein ~ynthesis as described in Siehn et al., Blood ?2:.756-765 (1988). ~ ~
The cytotoxic activity of the conjugate was tested on baby hamster kidney fibroblasts (ATCC Accession - No. CRL 628i). BHK cells were plated in 24 well plates at a concentration of 5000 cell~/ml and in~h~ted overnight at 37-C, 5% CO.2.-_.The ~ollowing morning HEPES-buffered DMEM and ~. F-12 medi~ 1)-plus.-5% FCS was aspirated from the wells 30 .and replaced-with.media albne or with media cont~ n~ thè
conjugate, basic FGF or saporin. Two days later, the cells were washed twice, trypsinized and cell number determined with a Coulter Particle Counter*(Coulter Electronics, ~ Hialeah, FL).
As shown in Figure 2A the conjugate retains saporin activity when tested in an n vitro protein synthesis ~Trademark ~ 6~ 7 5 , Pcr/usgo/02289 ' 14 inhibition assay. The conjugate, as expected, is slightly less active (about two-fold) than free SAP. This is consistent with the low level of derivatization of SAP
prior to the conjugation (0.8 moles SPDP/mole) and with probable steric hindrance due to the presence of bound basic FGF. In contrast, the results obtained in the radioreceptor assays for basic FGF (Figure 2B) showed that the basic FGF/SAP is equipotent to, if not slightly more active than, basic FGF in the binding assay. Thus, it appears that the commitment of free sulfhydryl groups in basic FGF to bridging with SAP does not interfere with its capacity to recognize its receptor. If anything, this reaction may be stabilizing basic FGF.
Basic FGF/SAP is a potent cytotoxic factor for BHK
cells (Figure 3). SAP has no toxic effect on these cells even at the highest dose tested (10 ~M) and basic FGF alone has a slight inhibitory effect on proliferation. A mixture of basic FGF and SAP had a slight toxicity but only at the highest concentration tested. The ID50 (25 pM) for the cytotoxic agent compares well with the potency of basic FGF
(15 pM) in proliferation assays. Specificity of the cytotoxic agent was examined in competition experiments in an effort to establish that the mitotoxic activity of the conjugate is receptor specific. BHK cells were preincubated for one hour with various levels of basic FGF
or nerve growth factor (NGF) prior to treatment of the cells with the cytotoxic agent. As shown in Figure 4, there is a dose-related inhibition of the cytotoxic activity in the presence of increasing amounts of basic FGF. In contrast, a thousand-fold excess of NGF has no effect.
W~ 2S97 ~ PCT/US9~02~n ~ ~ 5-3275 .,~
~X~PT~ ITT
T~H~BTTION OF ANGIOGENESIS IN RABBIT CORN~
Elvax (ethylene-vinyl acetate copolymer resin, Dupont, Wilmington, DE) pellets were produced in the following - manner. About 60 mg of washed and dried Elvax* was dissolved in 500 ~L of methylene chloride. This wa~'added to 50 ~g of dried basic ~GF. 5 ~L drops were dropped onto a slide frozen in dry ice. Pellets were left in the freezer overnight and then dried in a desiccator.
New Zealand white rabbits were anaesthetized with Innovar Vet: 1 mL/kg. An incision was made-in the cornea of the rabbit eye and a pocket was opened with a spatula or forceps. One pellet was inserted in the pocket. Pellets were inserted in both eyes. The eye was washed with saline and 1 ml of gentamicin was injected intramuscularly. The rabbit was left for five days and angiogenesis was observed. After five days, each left eye was treated with 20 ~L of 100 ng basic FGF-SAP prepared as in Example I in 0.25% BSA. The right eyes were treated with 20 ~L of 0.25%
BSA alone. The treatment wa~ done twice daily by dropping the solution ~5 eye drops onto the cornea of t'he rabbit.
The person treating the ~nimal~ was unaware of the identity of the samples. After 10-days, the animals were evaluated for angiogenesis of the cornea by microscopic analysis by an evaluator who did not know the treatment regimen.
Angiogenesis- wa~ ~udged with ~~ as- be~ng maximal angiogenesis and-- as being no angioqenesis.
, , The results ~re provided in Table I. --A~ can ~e seen, angiogenesis in corneas treated with basic FGF-SAP was markedly reduced over that of controls.~
*Trade-mark '.'VO 90/12S97 PCI'/US90/022:~9 - 16 2Q ~ ~7 5 .".,, . . - TA R~ .~? T
~ . . . . .
ANIM~n RIGHT EYF~ LEFT ~YE
995 +
5 997 ~ + + +
998 -- -- -~ ~ + + :
999 ;-.+, +
,, . . , - , ~ . .. .
.. , .. , , _ . .. . .
10 . - EXAMPLE IV :- . -EFFECT OF FGF-SAP IN 1JIJYUr1~EN~S CELI.
, Cells obtained from surgical removal of tissue from the hand of adult patients diagnosed as having Dupuytren's Contracture, a malady effecting movement of the hand, were placed in primary culture. These cells have between lO,OOo and 15,000 basic FGF receptors per cell.
The cells were grown overnight in a 24 well tissue culture dish at a concentration of 10,000 cells per well in HEPES buffered DMEM with 10% FCS. The next ~orning the media -was removed and replaced- with- media~ containing concentrations of basic FGF-SAP con~ugate ranging from 10 ~ to 1012 molar. Controls.included wells treated with media only, wells treated with ~milar concentrations of basic FGF alone, saporin alone, and basic FGF and saporin together but not conjugated. The cells were returned to the incubator~for-72 hour~. At the end of this~incubation the cells were washed, removed with trypsin and counted on a Coulter cell counter. The number of cells in the media controls was compared .with the-number of cells in the treated wells (as described above). The results~of these cell killing assays are shown in Figure 6. As can be seen, Dupuytren's cells are sensitive to basic FGF-SAP. Similar results were obtained w~th three other cell samples.
'-Trademark =,, ,~ .
20~3275 Although the invention has been described with reference to the presently-preferred embodiments, it should be understood that various modifications can be made without departing from the spirit of the invention.
Accordingly, the invention is limited only by the following claims.
Claims (30)
1. A conjugate, comprising a cytotoxic agent and a polypeptide reactive with a high affinity fibroblast growth factor (FGF) receptor, wherein the polypeptide reactive with the receptor is selected from the group of polypeptides consisting of polypeptides that exhibit FGF
mitogenic activity mediated through binding to an FGF
receptor and fragments of polypeptides that exhibit FGF
mitogenic activity mediated through binding to an FGF
receptor and that bind to an FGF receptor and are transported into the cell, thereby internalizing the linked cytotoxic agent.
mitogenic activity mediated through binding to an FGF
receptor and fragments of polypeptides that exhibit FGF
mitogenic activity mediated through binding to an FGF
receptor and that bind to an FGF receptor and are transported into the cell, thereby internalizing the linked cytotoxic agent.
2. The conjugate of claim 1, wherein said polypeptide reactive with an FGF receptor is basic FGF.
3. The conjugate of claim 1, wherein said polypeptide reactive with an FGF receptor is selected from the group consisting of acidic FGF, the hst gene product, the int-2 gene product and FGF-5.
4. The conjugate of claim 1, wherein said cytotoxic agent is a ribosome-inactivating protein.
5. The conjugate of claim 4, wherein said cytotoxic agent is saporin.
6. The conjugate of claim 1, wherein said cytotoxic agent is selected from the group consisting of methotrexate, anthracyclines and Pseudomonas exotoxin.
7. An in vitro method of targeting a cytotoxic agent to cells having FGF receptors, comprising conjugating said cytotoxic agent to a polypeptide reactive with an FGF receptor, and providing said conjugate to said cells; wherein the polypeptide reactive with the receptor is selected from the group of polypeptides consisting of polypeptides that exhibit FGF mitogenic activity mediated through binding to an FGF receptor and fragments of polypeptides that exhibit FGF mitogenic activity medicated through binding to an FGF receptor and that bind to an FGF receptor and are transported into the cell, thereby internalizing the linked cytotoxic agent.
8. The method of claim 7, wherein said polypeptide reactive with an FGF receptor is basic FGF.
9. The method of claim 7, wherein said polypeptide reactive with an FGF receptor is acidic FGF.
10. The method of claim 7, wherein said cytotoxic agent is a ribosome-inactivating protein.
11. The method of claim 7, wherein said cytotoxic agent is saporin.
12. The method of claim 7, wherein said cytotoxic agent is selected from the group consisting of methotrexate or its analogs, anthracyclines and Pseudomonas exotoxin.
13. A use of a therapeutically effective amount of FGF
conjugated to a cytotoxic agent for treating an FGF-mediated pathophysiological condition.
conjugated to a cytotoxic agent for treating an FGF-mediated pathophysiological condition.
14. The use of claim 13, wherein said FGF-mediated pathophysiological condition is selected from the group consisting of tumors, atheroslerosis, rheumatoid arthritis and proliferative retinopathy.
15. The use of claim 13, wherein said FGF conjugated to said cytotoxic agent is basic FGF.
16. The use of claim 13, wherein said FGF conjugated to said cytotoxic agent is aFGF.
17. The use of claim 13, wherein said cytotoxic agent is a ribosome-inactivating protein.
18. The use of claim 13, wherein said cytotoxic agent is saporin.
19. The use of claim 13, wherein said cytotoxic agent is selected from the group consisting of methotrexate and daunomycin.
20. A use of a conjugate comprising FGF and a cytotoxic agent for inhibiting proliferation of cells having FGF
receptors.
receptors.
21. A pharmaceutical comprising the conjugate of claim 1 and a physiologically acceptable excipient.
22. A method of purification of conjugates including a fibroblast growth factor and a cytotoxic agent comprising the steps of:
applying a sample containing FGF-conjugate to an immobilized heparin column;
eluting the column with a salt gradient; and collecting the material eluted between 1 M and 3 M
salt gradient.
applying a sample containing FGF-conjugate to an immobilized heparin column;
eluting the column with a salt gradient; and collecting the material eluted between 1 M and 3 M
salt gradient.
23. A use of an effective amount of FGF conjugated to a cytotoxic agent for the production of a medicament for treating an FGF-mediated pathophysiological condition.
24. The use of claim 23, wherein said FGF-mediated pathophysiological condition is selected from the group consisting of tumors, atheroslerosis, rheumatoid arthritis and proliferative retinopathy.
25. The use of claim 23, wherein said FGF conjugated to said cytotoxic agent is basic FGF.
26. The use of claim 23, wherein said FGF conjugated to said cytotoxic agent is acidic FGF.
27. The use of claim 23, wherein said cytotoxic agent is a ribosome-inactivating protein.
28. The use of claim 23, wherein said cytotoxic agent is saporin.
29. The use of claim 23, wherein said cytotoxic agent is selected from the group consisting of methotrexate and daunomycin.
30. A use of a conjugate comprising FGF and a cytotoxic agent for the production of a medicament for inhibiting proliferation of cells having FGF receptors.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US07/344,109 US5191067A (en) | 1989-04-27 | 1989-04-27 | Fibroblast growth factor conjugates |
US344,109 | 1989-04-27 |
Publications (2)
Publication Number | Publication Date |
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CA2053275A1 CA2053275A1 (en) | 1990-10-28 |
CA2053275C true CA2053275C (en) | 1999-02-02 |
Family
ID=23349097
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002053275A Expired - Fee Related CA2053275C (en) | 1989-04-27 | 1990-04-26 | Fibroblast growth factor conjugates |
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US (1) | US5191067A (en) |
EP (1) | EP0470183B1 (en) |
JP (1) | JP2891306B2 (en) |
CA (1) | CA2053275C (en) |
DE (1) | DE69010330T2 (en) |
WO (1) | WO1990012597A1 (en) |
Families Citing this family (48)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5529932A (en) * | 1988-01-28 | 1996-06-25 | Pharmacia, S.P.A. | Isolated DNA encoding a plant ribosome inactivating protein from the leaves of saponaria officinalis |
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US5635384A (en) * | 1990-06-11 | 1997-06-03 | Dowelanco | Ribosome-inactivating proteins, inactive precursor forms thereof, a process for making and a method of using |
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US5478804A (en) * | 1990-09-19 | 1995-12-26 | The Salk Institute For Biological Studies | Treatment of tumorigenic pathophysiological conditions with FGF-cytoxic conjugates |
WO1992011872A1 (en) * | 1991-01-03 | 1992-07-23 | The Salk Institute For Biological Studies | Mitotoxin for treatment of vascular injury |
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US5916772A (en) * | 1992-06-16 | 1999-06-29 | Whittier Institute For Diabetes And Endocrinology | Recombinant production of saporin-containing proteins |
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AU7694594A (en) * | 1993-09-06 | 1995-03-27 | Menarini Ricerche Sud S.P.A. | Ribosome inactivating proteins extracted from seeds of saponaria ocymoides and vaccaria pyramidata, their preparation and immunotoxins containing them |
US6551618B2 (en) * | 1994-03-15 | 2003-04-22 | University Of Birmingham | Compositions and methods for delivery of agents for neuronal regeneration and survival |
US6037329A (en) | 1994-03-15 | 2000-03-14 | Selective Genetics, Inc. | Compositions containing nucleic acids and ligands for therapeutic treatment |
WO1995024928A2 (en) * | 1994-03-15 | 1995-09-21 | Prizm Pharmaceuticals, Inc. | Heparin-binding growth factors for gene therapy and anterior eye disorders |
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US20050043234A1 (en) | 1996-10-16 | 2005-02-24 | Deisher Theresa A. | Novel FGF homologs |
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US7741435B2 (en) * | 1997-07-09 | 2010-06-22 | Advanced Targeting Systems, Inc. | Substance P-saporin (SP-SAP) conjugates and methods of use thereof |
US6063758A (en) * | 1997-07-09 | 2000-05-16 | Advanced Targeting Systems, Inc. | Substance P-Saporin (SP-SAP) conjugates and methods of use thereof |
US6274712B1 (en) | 1997-12-23 | 2001-08-14 | 3-Dimensional Pharmaceuticals, Inc. | Analogs of human basic fibroblast growth factor mutated at one or more of the positions glutamute 89, aspartate 101 or leucine 137 |
US6331523B1 (en) | 1998-03-12 | 2001-12-18 | Genentech, Inc. | Method of enhancing the survival of retinal neurons and treating ocular diseases using FGF-5 |
US6313103B1 (en) | 1998-05-13 | 2001-11-06 | Carrington Laboratories, Inc. | Pectic substance as a growth factor stabilizer |
US5929051A (en) * | 1998-05-13 | 1999-07-27 | Carrington Laboratories, Inc. | Aloe pectins |
US7022683B1 (en) * | 1998-05-13 | 2006-04-04 | Carrington Laboratories, Inc. | Pharmacological compositions comprising pectins having high molecular weights and low degrees of methoxylation |
AU2007201749B2 (en) * | 1998-07-22 | 2008-05-29 | Osprey Pharmaceuticals Usa, Inc. | Cytotoxic conjugates comprising a chemokine receptor targeting agent |
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JP2003511388A (en) * | 1999-10-02 | 2003-03-25 | アメリカ合衆国 | Fibroblast growth factor-5 (FGF-5) is a tumor-associated T cell antigen |
US7470665B2 (en) | 1999-12-02 | 2008-12-30 | Zymogenetics, Inc. | Methods for targeting cells that express fibroblast growth receptor-3 or -2 |
ATE539157T1 (en) * | 1999-12-02 | 2012-01-15 | Zymogenetics Inc | METHODS THAT TARGET CELLS EXPRESSING THE FIBROBLAS GROWTH FACTOR RECEPTORS-3 OR -2 |
WO2002066516A2 (en) | 2001-02-20 | 2002-08-29 | Zymogenetics, Inc. | Antibodies that bind both bcma and taci |
US7494669B2 (en) | 2001-02-28 | 2009-02-24 | Carrington Laboratories, Inc. | Delivery of physiological agents with in-situ gels comprising anionic polysaccharides |
WO2003089603A2 (en) | 2002-04-19 | 2003-10-30 | Zymogenetics, Inc. | Cytokine receptor |
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US20040136970A1 (en) * | 2002-10-07 | 2004-07-15 | Ellsworth Jeff L. | Methods for administering FGF18 |
US7166691B2 (en) * | 2002-12-20 | 2007-01-23 | Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Saposin C and receptors as targets for treatment of benign and malignant disorders |
US7067123B2 (en) * | 2003-04-29 | 2006-06-27 | Musculoskeletal Transplant Foundation | Glue for cartilage repair |
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US7837740B2 (en) | 2007-01-24 | 2010-11-23 | Musculoskeletal Transplant Foundation | Two piece cancellous construct for cartilage repair |
US7815926B2 (en) * | 2005-07-11 | 2010-10-19 | Musculoskeletal Transplant Foundation | Implant for articular cartilage repair |
WO2007035778A2 (en) | 2005-09-19 | 2007-03-29 | Histogenics Corporation | Cell-support matrix and a method for preparation thereof |
US8962556B2 (en) * | 2006-09-28 | 2015-02-24 | Prochon Biotech Ltd. | FGF-2 variants having N-terminal deletions and increased receptor selectivity and uses thereof |
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US20090054984A1 (en) | 2007-08-20 | 2009-02-26 | Histogenics Corporation | Method For Use Of A Double-Structured Tissue Implant For Treatment Of Tissue Defects |
CA2717725A1 (en) * | 2008-03-05 | 2009-09-11 | Musculoskeletal Transplant Foundation | Cancellous constructs, cartilage particles and combinations of cancellous constructs and cartilage particles |
US10077420B2 (en) | 2014-12-02 | 2018-09-18 | Histogenics Corporation | Cell and tissue culture container |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
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IN165717B (en) * | 1986-08-07 | 1989-12-23 | Battelle Memorial Institute | |
US5116753A (en) * | 1991-07-30 | 1992-05-26 | The Salk Institute For Biological Studies | Maintenance of pancreatic islets |
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1989
- 1989-04-27 US US07/344,109 patent/US5191067A/en not_active Expired - Fee Related
-
1990
- 1990-04-26 EP EP90907848A patent/EP0470183B1/en not_active Expired - Lifetime
- 1990-04-26 JP JP2507528A patent/JP2891306B2/en not_active Expired - Lifetime
- 1990-04-26 WO PCT/US1990/002289 patent/WO1990012597A1/en active IP Right Grant
- 1990-04-26 CA CA002053275A patent/CA2053275C/en not_active Expired - Fee Related
- 1990-04-26 DE DE69010330T patent/DE69010330T2/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
CA2053275A1 (en) | 1990-10-28 |
DE69010330D1 (en) | 1994-08-04 |
JP2891306B2 (en) | 1999-05-17 |
DE69010330T2 (en) | 1994-10-20 |
WO1990012597A1 (en) | 1990-11-01 |
EP0470183A1 (en) | 1992-02-12 |
JPH04507093A (en) | 1992-12-10 |
US5191067A (en) | 1993-03-02 |
EP0470183B1 (en) | 1994-06-29 |
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