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WO2000020577A1 - Utilisation de la calreticuline et de fragments de la calreticuline pour inhiber la croissance des cellules endotheliales et l'angiogenese, et supprimer la croissance tumorale - Google Patents

Utilisation de la calreticuline et de fragments de la calreticuline pour inhiber la croissance des cellules endotheliales et l'angiogenese, et supprimer la croissance tumorale Download PDF

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Publication number
WO2000020577A1
WO2000020577A1 PCT/US1999/023240 US9923240W WO0020577A1 WO 2000020577 A1 WO2000020577 A1 WO 2000020577A1 US 9923240 W US9923240 W US 9923240W WO 0020577 A1 WO0020577 A1 WO 0020577A1
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WIPO (PCT)
Prior art keywords
seq
caheticulin
amino acid
therapeutically effective
sequence shown
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PCT/US1999/023240
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English (en)
Inventor
Giovanna Tosato
Sandra E. Pike
Lei Yao
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The Government Of The United States Of America, Represented By The Secretary, Dept. Of Health And Huuman Services, The National Institutes Of Health
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Application filed by The Government Of The United States Of America, Represented By The Secretary, Dept. Of Health And Huuman Services, The National Institutes Of Health filed Critical The Government Of The United States Of America, Represented By The Secretary, Dept. Of Health And Huuman Services, The National Institutes Of Health
Priority to AU62917/99A priority Critical patent/AU6291799A/en
Priority to US09/807,148 priority patent/US6867180B1/en
Publication of WO2000020577A1 publication Critical patent/WO2000020577A1/fr
Priority to US09/828,000 priority patent/US6596690B2/en
Priority to US10/405,588 priority patent/US7432236B2/en
Priority to US11/040,162 priority patent/US7488711B2/en
Priority to US12/198,810 priority patent/US7812117B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4725—Proteoglycans, e.g. aggreccan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • This invention relates to inhibition of endothelial cell growth, angiogenesis and tumor growth.
  • VEGF vascular endothelial growth factor
  • a truncated form of tissue factor targeted to tumor vascular endothelium was demonstrated to initiate formation of intravascular clots and promote the regression of experimental tumors established in mice (Huang et al., Science 275:547-550, 1997).
  • Angiostatin, a fragment of plasminogen (O'Reilly et al., Cell 79:315-328, 1994), and endostatin, a fragment of collagen XVIII (O'Reilly et al., Cell 88:277-285, 1997) are known to inhibit the proliferation of endothelial cells in vitro and to suppress neovascularization in vivo.
  • Interleukin-12 (Voest et al., J. Natl. Cancer Inst. 87:581-586, 1995) the Interferon- ⁇ Inducible Protein-10 (Angiolillo et al., J. Exp. Med. 182:155-162, 1995; Strieter et al., Biochem. Biophys. Res. Commun. 210:51-57, 1995; Sgadari et al., Proc. Natl. Acad. Sci. U.S.A.
  • Calreticulin was first identified in skeletal muscle sarcoplasmic reticulum. (Ostwald and MacLennan, J. Biol. Chem. 249:974-979, 1974). Fifteen years later it was cloned and the N-terminus was sequenced. This led to the discovery that several groups had independently identified the molecule and had given it different names, including, "high-affinity Ca 2+ ". "calregulin”, “CRP55” and “calsequestrin-like protein” (Ostwald and MacLennan, J. Biol. Chem. 249:974-979, 1974;
  • calreticulin was detected on the plasma membranes of lymphoblastoid cells (Newkirk and Tsoukas, J. Autoimmun. 5:511-525, 1992) and epidermal keratinocyte lines (Kawashima, et al., Dermatology 189 Suppl. 1:6-10, 1994). It was proposed to represent, or to be closely related in structure, to the Clq receptor found on endothelial cells, B cells, T cells and other cells (Chen et al., J. Immunol. 153:1430-1440, 1994).
  • Calreticulin is also a constituent of lytic granules contained in cytotoxic T and NK cells from which it is released during cell lysis (Dupuis et al., J. Exp. Med. 177:1-7, 1993), and has been purified from the culture supernatant of several cell types (Booth and Koch, Cell 59:729-737, 1989; Eggleton et al., Clin. Immunol. Immunopathol. 72:405-409, 1994) and from normal human plasma (Sueyoshi et al., Thromb. Res. 63:569-575, 1991).
  • Several observations support the notion that calreticulin can also be a target for autoimmune responses (Lux et al., J. Clin. Invest. 89:1945-1951, 1992; Meilof et al., J. Immunol. 151:5800-5809, 1993).
  • calreticulin Since the initial identification and cloning, the structure of calreticulin has been characterized. Mammalian calreticulin is a 417 amino acid peptide from which the 17 N-terminal amino acids are cleaved upon translocation to the lumen of the endoplasmic reticulum (Smith and Koch, Embo. J. 8:3581-3586, 1989). In addition to being found in the lumen of the endoplasmic reticulum, calreticulin has been found in the cytoplasm, in the nucleus of some cells, and in the extracellular matrix (Michalak et al., Biochem. J. 285:681-692, 1992). Further studies revealed that calreticulin has three distinct domains, the N-terminal domain, a middle domain and the C-terminal domain.
  • the mature calreticulin is composed of an N-terminal domain consisting of 180 amino acids that are highly conserved. Proposed three-dimensional models indicate that the domain contains eight anti-parallel ⁇ -strands. Furthermore, the N-terminal domain has been found to bind a number of molecules including the alpha subunit of integrin, Zn 2+ , and the DNA binding domain of steroid receptors (Nash et al., Mol. Cellular Biochem. 135:71-78, 1994).
  • the middle domain of calreticulin stretches from amino acid 180 to amino acid 280. It is proline rich and has also been termed the P-domain. This domain has been found to have a high affinity for Ca 2+ and contains a nuclear localization signal (Baksh and Michalak, J. Biol. Chem.
  • the C-domain binds to Factor IX, Factor X, and prothrombin
  • Calreticulin has been actively studied since 1972. Some of the studies that have been done have focused on understanding the structure of calreticulin while others have focused on understanding the physiological role of the molecule.
  • the present invention stems from the discovery that calreticulin has three previously uncharacterized biological activities.
  • calreticulin is shown to inhibit endothelial cell growth, while having little or no effect on the growth of non-endothelial cells.
  • calreticulin is shown to inhibit angiogenesis.
  • calreticulin is shown to inhibit tumor growth, including the growth of Burkitt lymphoma, breast adenocarcinomas, colon carcinomas, lung carcinomas, melanoma, rhabdomyosarcoma, promyelomonocytic lymphoma, Wi ms tumor, and neuroblastoma tumors.
  • fragments of calreticulin share these activities. These fragments include calreticulin lacking the N-terminal 1-120 amino acids (Seq. I.D. No. 9), the N- terminal domain (Seq. I.D. No. 4), as well as fragments of the N-terminal domain of calreticulin.
  • fragments examples include the recombinantly produced 180 amino acid N-terminal domain of calreticulin (Seq. I.D. No. 4), which has been given the name "vasostatin", ⁇ 120 calreticulin (calreticulin missing the N-terminal 1-120 amino acids; Seq. I.D. No. 9), as well as a recombinantly produced 61 amino acid fragment (a.a. 120-180; Seq. I.D. No. 5).
  • Other biologically active fragments of calreticulin include a synthetic 49 amino acid fragment (a.a. 132-180; Seq. I.D. No. 6) and a synthetic 60 amino acid fragment (a.a. 121-180; Seq. I.D. No. 8).
  • the activity of the above described molecules does not stem from their ability to bind to the sequence KXaa,FFXaa 2 R (Seq. I.D. No. 11 ; Xaai represents either G, A, or V, and Xaa 2 represents K or R) or the sequence KXaa t GFFKR (Seq. I.D. No. 10; Xaa] represents either I, L, G, A, or C) that are known to bind calreticulin.
  • Calreticulin and the described fragments of calreticulin are useful among other things for inhibiting endothelial cell growth, for the treatment of subjects having solid tumors with associated neovascularization, and for the treatment of other diseases where angiogenesis is a factor.
  • the present invention may be especially useful for treating rheumatoid arthritis, autoimmune diseases, rheumatic diseases, and certain ocular neovascular diseases, such as, macular degeneration, diabetic retinopathy, and retrolental fibroplasia.
  • compositions comprising calreticulin and the therapeutically active fragments and variants of calreticulin are provided by the invention.
  • the biologically active fragments are those fragments having the sequences shown in Seq. I.D. Nos. 4, 5, 6, 8, and 9.
  • the invention provides a method of inhibiting endothelial cell growth by contacting endothelial cells with a pharmaceutical composition that comprises at least one protein selected from the group consisting of calreticulin, and therapeutically effective fragments of calreticulin.
  • a pharmaceutical composition that comprises at least one protein selected from the group consisting of calreticulin, and therapeutically effective fragments of calreticulin.
  • the invention also provides a method of inhibiting angiogenesis in a subject, by administering to the subject an effective amount of a pharmaceutical composition comprising at least one protein selected from the group consisting of calreticulin and therapeutically effective fragments and variants of calreticulin.
  • another aspect of the invention is a method of inhibiting tumor angiogenesis and growth by contacting tumor cells with an effective amount of a pharmaceutical composition comprising at least one protein selected from the group consisting of calreticulin and therapeutically effective fragments and variants of calreticulin.
  • the anti-tumor activity may also be utilized in a method of inhibiting tumor growth in a subject. That method comprises administering to the subject an effective amount of a pharmaceutical composition comprising at least one protein selected from the group consisting of calreticulin and therapeutically effective fragments and variants of calreticulin.
  • compositions provided by the invention is a protein with an amino acid sequence selected from the group consisting of therapeutically effective fragments of vasostatin and variants of calreticulin that do not bind to the sequence shown in Seq. I.D. No. 11 and/or Seq. I.D. No. 10, but yet display one of the three biological activities described above.
  • the invention also provides methods of identifying therapeutically effective variants and fragments of calreticulin. These methods involve contacting a sample suspected of containing therapeutically effective variants or fragments with the sequence KXaa ! FFXaa 2 R (Seq. I.D. No. 11), and/or the sequence KXaaiGFFKR (Seq. I.D. No. 10) detecting the portion of the sample that does not bind to the sequence. The unbound portion of the sample is then tested for one of the three biological activities described above. Accordingly, the invention also provides the therapeutically effective variants and fragments of calreticulin identified by the method, as well as cells containing recombinant nucleic acid molecules that encode the therapeutically effective variants and fragments.
  • Figure 1 is a graph showing the inhibition of endothelial cell proliferation by MBP- calreticulin (Seq. I.D. No. 2).
  • Fetal bovine heart endothelial cells 800 cells/well were incubated for 5 days either in medium alone or medium supplemented with bFGF (25 ng/ml), with or without recombinant purified MBP-calreticulin (Seq. I.D. No. 3) or MBP (both at 1 ⁇ g/ml).
  • the results of 16 experiments are expressed as mean cpm ( ⁇ SD).
  • Figure 2 is a graph showing the dose dependency of MBP- vasostatin (Seq. I.D. No. 4) inhibition.
  • Figure 3 shows the inhibition of endothelial cell proliferation by ⁇ 120 calreticulin (Seq. I.D. No. 9).
  • Fetal bovine heart endothelial cells 800 cells/well were cultured for 5 days in medium alone or medium supplemented with bFGF (25 ng/ml).
  • Recombinant purified MBP-calreticulin (Seq. I.D. No. 3), MBP- ⁇ 120 calreticulin (Seq. I.D. No. 9), or MBP were added to bFGF-supplemented cultures.
  • Proliferation was measured by 3 H-thymidine incorporation during the final 20-23 hours of culture. The results reflect the mean of triplicate cultures; SDs within 12% of the mean.
  • Figure 4 is a graph showing the inhibition of endothelial cell proliferation by the 61 amino acid calreticulin fragment MBP-120-180 calreticulin (Seq. I.D. No. 5).
  • Fetal bovine heart endothelial cells 800 cells/well were incubated for 5 days either in medium alone or medium supplemented with bFGF (15 ng/ml), with or without the recombinant purified 61 amino acid fragment (Seq. I.D. No. 5) (both at 1 ⁇ g/ml).
  • the results of 8 experiments are expressed as mean cpm ( ⁇ SD).
  • Figure 5 shows a graph comparing endothelial cell growth inhibition by MBP calreticulin (Seq. I.D. No. 3), MBP- ⁇ 120 calreticulin (Seq. I.D. No. 9), MBP-vasostatin (Seq. I.D. No. 4), and MBP-120-180 calreticulin fragment (Seq. I.D. No. 5).
  • Fetal bovine heart endothelial cells 800 cells/well
  • Recombinant purified fusion proteins were added to culture at 0.4-32 nM concentrations to bFGF-supplemented cultures.
  • Proliferation was measured by 3 H thymidine incorporation during the final 20-23 hours of culture. The results reflect the mean of triplicate cultures; SDs within 15% of the mean.
  • the mean response of endothelial cells was 2,217 c.p.m. when cultured in medium alone, and 23,377 c.p.m. when cultured with bFGF alone.
  • Figures 6A, 6B, 6C and 6D are graphs showing the inhibition of tumor growth by vasostatin (Seq. I.D. No. 4) and calreticulin (Seq. I.D. No. 3).
  • Burkitt lymphoma cells CA46 cell line, 1x10' cells
  • s.c. subcutaneously
  • mice 6 weeks of age. Beginning on the day of cell inoculation and continuing thereafter daily, 6 days/week, mice were inoculated s.c. with either control buffer (open symbols) or test protein (closed symbols). The results reflect the % mice with tumor as a function of time.
  • mice 12 mice were inoculated with control purified GST protein (Seq. I.D. No. 2; 20 ⁇ g/day x 14 days), and 13 mice were inoculated with purified GST-calreticulin (Seq. I.D. No. 2; 60 ⁇ g/day x 14 days).
  • B. 8 mice were inoculated with control purified MBP protein (20 ⁇ g/day x 14 days), and 9 mice were inoculated with purified MBP- vasostatin (Seq. I.D. No. 4; 30 ⁇ g/day x 14 days).
  • C C.
  • mice were inoculated with control purified MBP (40 ⁇ g/day x 18 days), and 12 mice were inoculated with MBP-vasostatin (Seq. I.D. No. 4; 60 ⁇ g/day x 18 days); all mice with tumor (12 treated with MBP and 4 treated with MBP-vasostatin) were killed on day 18. The remaining mice were observed up to day 60.
  • D. 6 mice were treated with formulation buffer alone (0.1 ml/day x 22 days, open circles), 5 mice were treated with 20 ⁇ g/day x 22 days purified MBP-vasostatin (Seq. I.D. No. 4; diamonds), and 9 mice were treated with 100 ⁇ g/day x 22 days purified MBP-vasostatin (Seq. I.D. No. 4; squares).
  • FIG. 7 A and 7B are graphs showing the inhibition of established Burkitt tumor growth by vasostatin.
  • Burkitt lymphoma cells CA46 cell line, lxlO 7 cells
  • MBP-vasostatin Seq. I.D. No. 4; 200 ⁇ g/day, 6 days/week, 100 ⁇ l dose x 46 days
  • 10 mice were treated with formulation buffer alone (100 ⁇ l x 46 days). Tumor size was recorded.
  • Formulation buffer open squares
  • MBP-vasostatin Seq. I.D. No.
  • mice After a tumor appeared (at least 160 mm 2 in size), 12 mice were treated with MBP-vasostatin (Seq. I.D. No. 4; 200 ⁇ g/twice/day, 5 days/week and once/day 2days/week, 100 ⁇ l/dose x 26 days) and 8 mice were treated with formulation buffer alone (100 ⁇ l x 26 days). The % mice with tumor was determined. Formulation buffer (open squares); MBP vasostatin (Seq. I.D. No. 4; closed squares).
  • Figure 8 is a graph showing the inhibition of human colon carcinoma growth by vasostatin (Seq. I.D. No.4).
  • Mice BALB/c athymic mice, 6 weeks of age
  • the human colon carcinoma cell line SW-480 from ATCC, 6xl0 6 cells/mouse in 0.2 ml RPMI medium.
  • MBP-vasostatin Seq. I.D. No. 4; 100 ⁇ g/day, 6 days/week, 100 ⁇ l/dose, x 36 days; closed circles
  • 10 mice were inoculated with formulation buffer (100 ⁇ l x 36 days; open circles).
  • Tumor size was estimated as the product of two- dimensional caliper measurements.
  • Figures 9 A and 9B are graphs showing the inhibition of human neuroblastoma tumor growth.
  • Mice BALB/c athymic mice, 6 weeks of age
  • the human neuroblastoma cell line SK-N-MC from ATCC, 8.5x10 6 cells/mouse in 0.2 ml RPMI medium.
  • 12 mice were inoculated s.c. with control MBP (20 ⁇ g/day) and 13 mice were inoculated with MBP-vasostatin (Seq. I.D. No. 4; 30 ⁇ g/day).
  • mice On day 30, 8 of the 12 mice that had been injected with control MBP (mean tumor weight 1.74 g) and 5 of the 13 mice injected with MBP-vasostatin (Seq. I.D. No. 4; mean tumor weight 1.78 g) were sacrificed. Treatment was continued in the remaining mice until day 55 at which time all animals were sacrificed. One of the mice treated with MBP-vasostatin (Seq. I.D. No. 4) did not develop a tumor and since day 55 was observed untreated; as of day 220 no tumor has developed. On day 55, all animals with tumor were sacrificed and tumor weight measured. There were 4 animals in the control group (MBP) and 7 animals in the treatment group (MBP-vasostatin). A. Tumor growth curves. B. Comparison of tumor weights in animals sacrificed on day 55.
  • Figure 10 is a graph showing the inhibition of human breast adenocarcinoma tumor growth by vasostatin (Seq. I.D. No. 4).
  • Mice BALB/c athymic mice, 6 weeks of age
  • the human breast adenocarcinoma cell line MDA-MB231 from ATCC, 7.5xl0 6 cells/mouse in 0.2 ml RPMI medium.
  • MDA-MB231 human breast adenocarcinoma cell line
  • 5 days/week for 18 days, 8 mice were inoculated s.c.
  • mice were inoculated with MBP-vasostatin (Seq. I.D. No. 4; 200 ⁇ g/twice/day for 5 days/week and once/day for 2 days/week).
  • Tumor size was estimated as the product of two-dimensional caliper measurements.
  • FIG 11 A and 1 IB are graphs showing the inhibition of murine lymphoma tumor growth by MBP-vasostatin (Seq. I.D. No. 4).
  • Mice BALB/c athymic mice, 6 weeks of age
  • the murine B-cell lymphoma cell line Raw 8.1 from ATCC, 2.5xl0 6 cells/mouse in 0.2 ml RPMI medium.
  • 7 days/week 9 mice were inoculated s.c. with control formulation buffer (100 ⁇ l/day) and 9 mice were inoculated with MBP-vasostatin (Seq. I.D. No.
  • mice 4; 200 ⁇ g/day). Treatment was continued until all mice developed a tumor measuring at least 0.5 cm 2 on day 15.
  • Figure 12 is a graph showing the inhibition of Wilms tumor growth by MBP-vasostatin (Seq. I.D. No. 4).
  • Athymic BALB/c nude mice received 400 rad total body irradiation and 24 hours later were inoculated (12xl0 6 cell mouse) with the human Wilms tumor cell line (SK-NP-1).
  • SK-NP-1 human Wilms tumor cell line
  • Tumor size was estimated as the product of two dimesional caliper measurement.
  • Figure 13 is a graph showing the inhibition of rhabdomyosarcoma growth by MBP- vasostatin (Seq. I.D. No. 4).
  • Groups of athymic BALB/c nude mice received 400 rad total body irradiation and 24 hours later were inoculated (8xl0 6 cell mouse) s.c. with the human rhabdomyosarcoma cell line A-204 tumor cell line (SK-NP-1).
  • SK-NP-1 tumor cell line
  • Tumor size was estimated as the product of two dimesional caliper measurement.
  • Figure 14 is a bar graph showing the inhibition of myelomonocytic lymphoma growth by
  • mice received 400 rad total body irradiation and 24 hours later were inoculated (10xl0 6 cell/mouse) with the human HL60 tumor cell line. One day after cell injection, and continuing daily thereafter for 24 days, the mice received either control formulation buffer alone or MBP-vasostatin (Seq. I.D. No. 4; 200 ⁇ g/day, 6 days/week). The weight of each tumor was measured in grams after tumors were removed from the animals.
  • Figure 15 is a graph showing the inhibition of human melanoma tumor growth by MBP- vasostatin (Seq. I.D. No. 4).
  • Athymic BALB/c nude mice received 400 rad total body irradiation and 24 hours later were inoculated (7xl0 6 cell/mouse) with the human melanoma cell line (A-375).
  • Tumor size was estimated as the product of two dimensional caliper measurement.
  • Figure 16 is a graph showing the inhibition of human Burkitt lymphoma growth by the calreticulin fragment encompassing amino acids 120-180 (Seq. I.D. No. 5).
  • Athymic BALB/c nude mice received 400 rad total body irradiation and 24 hours later were inoculated (4xl0 6 cell/mouse) with the human Burkitt lymphoma cell line CA46. Beginning on the day of cell inoculation and continuing daily thereafter 6 days/week the mice received either control recombinant purified MBP (65 ⁇ g/day, 6 days/week), MBP-vasostatin (Seq. I.D. No.
  • mice were injected for 42 days. Tumor size was estimated as the product of two dimesional caliper measurement.
  • Figures 17A, 17B, 17C, and 17D are graphs showing the inhibition of human Burkitt lymphoma growth by a combination treatment of vasostatin (Seq. I.D. No. 4) and Interleukin 12 (IL- 12).
  • Athymic BALB/c nude mice received 400 rad total body irradiation and 24 hours later were inoculated (4xl0 6 cell/mouse) with the human Burkitt lymphoma cell line CA46.
  • a and B. 5 days after cell inoculation the established tumors were treated with daily s.c. inoculations (6 days/week) of either formulation buffer alone (closed circles), MBP-vasostatin alone (Seq. I.D. No.
  • mice were sacrificed after 15 days of treatment. C and D. 1 day after cell inoculation, s.c. treatment with MBP-vasostatin (Seq. I.D. No. 4; 100 ⁇ g/day; 6 days/week) or formulation buffer was started; s.c.
  • mice receiving buffer alone (closed circles); MBP-vasostatin alone (open squares); IL-12 alone (open circles); and MBP-vasostatin plus IL-12 (open triangles).
  • the mice received 7 days treatment with MBP-vasostatin (Seq. I.D. No. 4) and 5 days treatment with IL- 12, and were observed untreated for 14 days at which time all mice were sacrificed. Tumor size was estimated as the product of two-dimensional caliper measurement.
  • Figure 18 is a Western blot analysis of fractions obtained through affinity purification over a column containing KXaa,FFXaa 2 R (Seq. I.D. No. 11) and KXaa,GFFKR (Seq. I.D. No. 10).
  • Purified supernatants from the VDS-0 cell line containing full length calreticulin and N-terminal fragments of calreticulin were affinity purified over a mixture of peptides KLGFFKR (Seq. I.D. No. 13), KAFFKR (Seq. I.D. No. 33) and KVFFKR (Seq. I.D. No. 32) coupled to CNBr-activated
  • Sepharose Sepharose.
  • Starting material (lane 1), unbound material (lanes 2, 3) and bound material (lanes 4, 5) were separated by SDS-PAGE, transferred to nitrocellulose, and subsequently stained with a rabbit antiserum against human calreticulin.
  • nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • Seq. I.D. No. 1 shows the nucleic acid sequence for the open reading frame of human calreticulin.
  • Seq. I.D. No. 2 shows the amino acid sequence of human calreticulin.
  • Seq. I.D. No. 3 shows the amino acid sequence of human calreticulin without the 17 N- terminal amino acids (References throughout the text to amino acid numbers are keyed to this sequence).
  • Seq. I.D. No. 4 shows the amino acid sequence of the N-terminal 180 amino acids of human calreticulin ("vasostatin").
  • Seq. I.D. No. 5 shows the amino acid sequence of the recombinant 61 amino acid fragment of human calreticulin. This sequence corresponds to amino acids number 120-180 of the human caheticulin sequence.
  • Seq. I.D. No. 6 shows the amino acid sequence of the synthetic 49 amino acid fragment of human caheticulin. This sequence corresponds to amino acids numbers 132-180 of the human caheticulin sequence.
  • Seq. I.D. No. 7 shows the cDNA sequence of human caheticulin.
  • Seq. I.D. No. 8 shows the amino acid sequence of the synthetic 60 amino acid fragment of human caheticulin. This sequence corresponds to amino acids numbers 121-180 of the human caheticulin sequence.
  • Seq. I.D. No. 9 shows the amino acid sequence of ⁇ 120 calreticulin (caheticulin (Seq. I.D. No. 3) missing the N-terminal 1-120 amino acids).
  • Seq. I.D. No. 10 shows the integrin consensus amino acid sequence
  • Seq. I.D. No. 11 shows the steroid nuclear receptor consensus amino acid sequence.
  • Seq. I.D. No. 12 shows the amino acid sequence of a portion of an integrin.
  • Seq. I.D. No. 13 shows the amino acid sequence of a portion of an integrin.
  • Seq. I.D. No. 14 shows the amino acid sequence of a portion of an integrin.
  • Seq. I.D. No. 15 shows the amino acid sequence of a portion of an integrin.
  • Seq. I.D. No. 16 shows the amino acid sequence of a portion of an integrin.
  • Seq. I.D. No. 17 shows the amino acid sequence of a portion of an integrin.
  • Seq. I.D. No. 18 shows the amino acid sequence of a portion of an integrin.
  • Seq. I.D. No. 19 shows the amino acid sequence of a portion of an integrin.
  • Seq. I.D. No. 20 shows the amino acid sequence of a portion of an integrin.
  • Seq. I.D. No. 21 shows the amino acid sequence of a portion of a steroid nuclear receptor.
  • Seq. I.D. No. 22 shows the amino acid sequence of a portion of a steroid nuclear receptor.
  • Seq. I.D. No. 23 shows the amino acid sequence of a portion of a steroid nuclear receptor.
  • Seq. I.D. No. 24 shows the amino acid sequence of a portion of a steroid nuclear receptor.
  • Seq. I.D. No. 25 shows the amino acid sequence of a portion of a steroid nuclear receptor.
  • Seq. I.D. No. 26 shows the amino acid sequence of a portion of a steroid nuclear receptor.
  • Seq. I.D. No. 27 shows the amino acid sequence of a portion of a steroid nuclear receptor.
  • Seq. I.D. No. 28 shows the amino acid sequence of a portion of a steroid nuclear receptor.
  • Seq. I.D. No. 29 shows the amino acid sequence of a portion of a steroid nuclear receptor.
  • Seq. I.D. No. 30 shows the amino acid sequence of a portion of a steroid nuclear receptor.
  • Seq. I.D. No. 31 shows the amino acid sequence of a portion of a steroid nuclear receptor.
  • Seq. I.D. No. 32 shows the amino acid sequence of a portion of the glucocorticoid receptor.
  • Seq. I.D. No. 33 shows the amino acid sequence of a portion of the estrogen receptor.
  • Seq. I.D. No. 34 shows the amino acid sequence of a portion of the thyroid receptor.
  • Seq. I.D. No. 35 shows the amino acid sequence of a portion of the retinoic acid receptor.
  • Calreticulin Caheticulin is a calcium binding protein that is found in many animals, and is highly conserved across species.
  • the open reading frame of the prototypical human caheticulin is shown in Seq. I.D. No. 1, while the sequence of the human caheticulin protein is shown in Seq. I.D. No. 2.
  • the present invention is founded on the discovery that caheticulin, and particular fragments of caheticulin, possess certain biological activities ("caheticulin activities"). Specifically, these activities include (1) the inhibition of endothelial cell growth; (2) the inhibition of angiogenesis; and (3) the inhibition of tumor growth.
  • caheticulin or a fragment of this protein to perform these activities may be beneficial in a number of applications, including clinical applications such as tumor therapy and treatment of diseases with abnormal or excessive angiogenesis.
  • caheticulin encompasses both the protein having the amino acid sequence shown in Seq. I.D. No. 2, as well as amino acid sequences that are based on this sequence but which include one or more sequence variants. Such sequence variants may also be defined in the degree of amino acid sequence identity that they share with the amino acid sequence shown in Seq. I.D. No. 2.
  • caheticulin sequence variants will share at least 80% sequence identity with the sequence shown in Seq. I.D. No. 2. More highly conserved variants will share at least 90% or at least 95% sequence identity with the Seq. I.D. No. 2 sequence. In addition to sharing sequence identity with the prototypical caheticulin protein sequence, such sequence variants possess at least one of the three biological activities noted above.
  • Therapeutically effective fragments and variants of calreticulin It is shown herein that not only does calreticulin possess the specified biological activities (inhibiting endothelial cells, angiogenesis and tumor growth), but that such activities are also found in certain peptide fragments of caheticulin.
  • the 180 amino acid N-terminal domain of caheticulin (Seq. I.D. No. 4), which is hereinafter termed "vasostatin,” possesses these activities, as do the synthetically produced 49 (Seq. I.D. No. 6) and 60 (Seq. I.D. No. 8) amino acid fragments, as well as the recombinantly produced 61 amino acid fragment (Seq. I.D. No. 5) and ⁇ 120 calreticulin (Seq. I.D. No. 9). Furthermore, it is shown that the activity of these fragments does not stem from their ability to bind to the amino acid sequence motif KXaa ⁇ FFXaa 2 R (Seq. I.D.
  • KVFFKR glucocorticoid, mineralcorticoid, progesterone and androgen receptors
  • KVFFKR glucocorticoid, mineralcorticoid, progesterone and androgen receptors
  • KAFFKR KAFFKR
  • KSFFRR thyroid hormone receptor
  • retinoic acid receptor KGFFRR (Seq. I.D. No. 35).
  • the terms "therapeutically effective fragment of calreticulin” or “therapeutically effective variant of caheticulin” includes any caheticulin fragment or variant that, at a minimum, possesses one of the three biological activities noted above.
  • the 61 amino acid caheticulin fragment (Seq. I.D. No. 5) is a therapeutically effective fragment of calreticulin since it possesses the ability to inhibit endothelial cell growth.
  • a given caheticulin fragment or variant possesses one or more of these biological activities can be readily determined by the assays described herein.
  • the ability to inhibit endothelial cell growth can readily be determined for any given fragment of caheticulin using the simple in vitro assay described below.
  • Oligonucleotide A linear polynucleotide sequence of up to about 100 nucleotide bases in length.
  • a nucleic acid molecule as introduced into a host cell thereby producing a transformed host cell.
  • a vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector may also include one or more selectable marker genes and other genetic elements known in the art.
  • Transformed A transformed cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • Isolated An "isolated" biological component (such as a nucleic acid or protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra- chromosomal DNA and RNA, proteins and organelles.
  • Nucleic acids and proteins that have been "isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • Sequence identity the similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of caheticulin will possess a relatively high degree of sequence identity when aligned using standard methods.
  • Homologs and variants of caheticulin are typically characterized by possession of at least 50% sequence identity counted over the full length alignment with the amino acid sequence of caheticulin using the NCBI Blast 2.0, gapped blastp set to default parameters.
  • the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11 , and a per residue gap cost of 1).
  • the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties).
  • Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 60%, at least 70%, at least 75%, at least 80%, at least 90% or at least 95% or 98% sequence identity.
  • homologs and variants When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on then similarity to the reference sequence. Methods for determining sequence identity over such short windows are described at http://www.ncbi.nlm.nih.gov/BLAST/blast FAQs.html.
  • sequence identity ranges are provided for guidance only; it is enthely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.
  • Recombinant A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • cDNA complementary DNA: A piece of DNA lacking internal, non-coding segments (introns) and regulatory sequences that determine transcription. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.
  • ORF open reading frame: A series of nucleotide triplets (codons) coding for amino acids without any termination codons. These sequences are usually translatable into a peptide.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
  • Subject Living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals.
  • Mimetic A molecule (such as an organic chemical compound) that mimics the activity of a protein, such as calreticulin and therapeutically effective variants and fragments thereof.
  • Peptidomimetic and organomimetic embodiments are within the scope of this term, whereby the three-dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid sidechains in the peptide, resulting in such peptido- and organomimetics of the peptides having substantial specific inhibitory activity.
  • a pharmacophore is an idealized, three-dimensional definition of the structural requirements for biological activity.
  • Peptido- and organomimetics can be designed to fit each pharmacophore with current computer modeling software (using computer assisted drug design or CADD). See Walters, "Computer-Assisted Modeling of Drugs", in Klegerman & Groves, eds., 1993, Pharmaceutical Biotechnology, Interpharm Press: Buffalo Grove, IL, pp. 165-174 and Principles of Pharmacology (ed. Munson, 1995), chapter 102 for a description of techniques used in computer assisted drug design.
  • caheticulin and certain fragments of this protein including the ⁇ 120 caheticulin (Seq. I.D. No. 9), the 180 amino acid N- terminal domain ("vasostatin"; Seq. I.D. No. 4), and the 49 (Seq. I.D. No. 6), 60 (Seq. I.D. No. 8), and 61 (Seq. I.D. No. 5) amino acid fragments have one or more of the following biological activities: (1) the ability to inhibit endothelial cell growth; (2) the ability to inhibit angiogenesis; and (3) the ability to suppress tumor growth. The following discussion explains how these activities were discovered and characterized.
  • Human and bovine endothelial cells were used for growth inhibition assays, and were prepared as follows:
  • Fetal bovine heart endothelial cells (American Type Culture Collection, ATCC, Manassas, VA) were grown through passage 12 in DMEM culture medium (BioWhittaker, Walkersville, MD) containing 10% heat inactivated fetal bovine serum (BioWhittaker), 100 ng/ml bFGF (R&D Systems, Minneapolis, MN), and 5 ⁇ g/ml gentamicin (Sigma).
  • cells were trypsinized (Trypsin/EDTA, Gibco BRL), washed, suspended in culture medium (DMEM containing 10% heat inactivated fetal bovine serum and 5 ⁇ g/ml gentamicin), plated (800 cells/well in 0.2 ml culture medium) in triplicate onto 96 well plates, and incubated for 5 days.
  • DNA synthesis was measured by 3 H thymidine deoxyribose uptake (0.5 mCi/well, 6.7 Ci/mmol; New England Nuclear, Boston, MA) during the last 20-23 hours of culture; cells were detached from the wells by freezing and thawing (Angiolillo et al., J. Exp. Med. 182:155-162, 1995)
  • Human umbilical vein endothelial cells were prepared from umbilical cord by 0.1% collagenase II (Worthington Biochemical Co., Freehold, NJ) digestion, as described (Gordon et al., In Vitro 19:661-671, 1983), and were grown through passage 5 in M199 culture medium (Sigma) supplemented with 20% newborn calf serum (Sigma), 5% human AB serum, 1.6 mM L-glutamine (GibcoBRL), 50 ⁇ g/ml porcine heparin (Sigma), 50 ⁇ g/ml ascorbate (Fisher, Fairlawn, NJ), 15 mM HEPES buffer (Calbiochem-Behring, La Jolla, CA), and 15 ⁇ g/ml Endothelial Cell Growth Supplement (ECGF; a crude extract of bovine neural tissue containing basic and acidic FGF, Sigma).
  • ECGF Endothelial Cell Growth Supplement
  • Endothelial cell purity was greater than 95%, as determined by staining with a rabbit antiserum to human Factor VHI-related antigen (Dako, Carpinteria, CA).
  • a rabbit antiserum to human Factor VHI-related antigen Dako, Carpinteria, CA.
  • cells were trypsinized, washed, suspended in culture medium (RPMI 1640, BioWhittaker) supplemented with 18% heat-inactivated fetal bovine serum (BioWhittaker) and 18 units/ml porcine heparin (Sigma), and plated (3.5x10 ⁇ cells/well in 0.2 ml culture medium) in triplicate cultures into 96-well plates. After incubation for 72 hours, DNA synthesis was measured by 3 H thymidine deoxyribose uptake during the last 20-23 hours of culture.
  • inhibitory compounds were purified from serum-free culture supernatants of the EBV-immortalized cell line VDS-0 (maintained as described by Tosato, et al., J. Immunol. 137:2037-2042, 1986).
  • VDS-0 EBV-immortalized cell line
  • the culture supernatant was purified by four sequential chromatographic steps, including metal chelating affinity, anion exchange, hydrophobic interaction, and anion exchange.
  • Silica gel 60 (EM Science, Gibbstown, New Jersey), used for absorption of non-polar substances, was added to the conditioned medium at the concentration of 5 gm/1. After rotation, the silica was removed, and the conditioned medium was filtered (Ultrapump II, Filtron Technology Corp, Northborough, MA) through a membrane with an 8,000 dalton MW cutoff (Ultrasette Omega, Filtron).
  • HIC Chromatography
  • an initial loading with 340 mg total protein from 16 liters of supernatant yielded 4.5 ⁇ g of purified protein.
  • the total biologic activity of the starting supernatant was approximately 44 xlO-* units (a unit being defined as the 1/2 maximal activity measured in a proliferation assay of fetal bovine heart endothelial cells), and the purified material contained approximately 9 x 10- ⁇ units, a recovery of approximately 26% of the original biologic activity.
  • the specific activity (units/mg protein) of the purified material was estimated to be 2 x 10 ⁇ units.
  • the biologically active material was analyzed by two-dimensional gel electrophoresis under reduced conditions followed by Coomassie staining (not shown). Two-dimensional polyacrylamide gel electrophoresis was performed as described (Wirth et al., Electrophoresis 16:1946-1960, 1995). Samples for electrophoresis were prepared in buffer containing 8 M urea, 20 mM Tris, pH 6.8, 30 mM DTT, 4% CHAPS, and 2% Pharmalytes, pH 3-10.
  • Precast immobilized pH gradient gel strips formulated with nonlinear pH gradients, pH 3-10 NL were purchased from Pharmacia LKB and rehydrated overnight at room temperature in 8M urea, 10 mM DTT, 2% w/v CHAPS, and 2% Pharmalytes, pH 3-10.
  • Second dimension SDS-PAGE was performed using 1.5 mm thick 10% SDS polyacrylamide gels at constant current (40 mA/gel) at 10 °C.
  • protein was solubilized in Tricine SDS sample buffer (Novex San Diego, CA), boiled, and run through 10-20% Tricine Gels.
  • Prestained molecular weight protein standards (range 4-250 kDa, Novex) were used throughout. Gels were stained with Colloidal Coomassie G-250 stain (Colloidal Coomassie kit, Novex) or silver stain (SilverXpress staining kit, Novex). Two well defined polypeptide spots were identified with approximate molecular weight of 55 and 20 kDa and apparent pi of 4.7 and 5.6, respectively. In addition, a series of poorly defined spots were visualized with relative molecular weights ranging between 30 and 40 kDa.
  • the well defined spots were subjected to trypsin digestion and the tryptic fragments were analyzed by ion trap mass spectrometry.
  • the 55 kDa polypeptide was identified as human caheticulin and the 20 kDa polypeptide as the light chain of human ferritin.
  • Bound antibody was detected with an affinity-purified, peroxidase- linked, donkey anti-rabbit IgG antibody (Amersham Pharmacia Biotech, Inc., Piscatway, NJ) and a chemiluminescence detection system (ECL kit, Amersham Pharmacia Biotech).
  • ECL kit Amersham Pharmacia Biotech
  • antisera for human caheticulin N-terminal (aa 6-19) and C-terminal (aa 382-400) peptides were used in immunoblotting (Pogue et al., J. Virol. 67:7106-7117, 1993). Both these antisera identified the 55 kDa band and thus confirmed its identity to caheticulin.
  • the biologically active, purified material from culture supernatant of the VDS-0 cell line contained human caheticulin, the light chain of human ferritin, and N-terminal fragments of caheticulin.
  • This result led to the investigation of other activities of caheticulin and caheticulin fragments, including the 180 amino acid N-terminal domain fragment ("vasostatin"; Seq. I.D. No. 4), ⁇ 120 caheticulin (Seq. I.D. No. 9), the 49 (Seq. I.D. No. 6), 60 (Seq. I.D. No. 8) and 61 (Seq. I.D. No. 5) amino acid fragments, as described below.
  • MBP caheticulin
  • vasostatin Seq. I.D. No. 4
  • cleavage with factor Xa New England Biolabs, Beverly, MA
  • Purification of cleaved caheticulin (Seq. I.D. No. 2) or vasostatin (Seq. I.D. No. 4) from MBP was achieved by anion exchange chromatography using a preequilibrated (20 mM Tris, pH 8.0, 25 mM NaCl) Resource Q column (Amersham Pharmacia Biotech).
  • Bound material was eluted by a step-wise gradient where MBP elutes at 100-150 mM NaCl; Factor Xa elutes at approximately 400 mM NaCl; and caheticulin or vasostatin elute at approximately 250 mM NaCl.
  • MBP elutes at 100-150 mM NaCl
  • Factor Xa elutes at approximately 400 mM NaCl
  • caheticulin or vasostatin elute at approximately 250 mM NaCl.
  • caheticulin fragment encompassing amino acids 120-180 (Seq. I.D.
  • the coding region for this fragment was amplified by PCR and then cloned (confirmed by sequencing) as an N-terminal fusion protein with the MBP gene for expression in E. coli.
  • the growth of E. coli and protein purification were performed as described above for MBP-calreticulin.
  • a recombinant form of caheticulin fused to glutathione S-transferase (GST) was also produced.
  • GST-calreticulin fusion construct the coding region for the mature caheticulin protein was cloned as a C-terminal translational fusion with the glutathione S transferase (GST) gene for expression in E. coli.
  • GST-calreticulin The growth of E. coli, the induction and release of GST- calreticulin (Seq. I.D. No. 2) from the bacteria was the same as described above for MBP-calreticulin, except for IPTG induction (0.6 mM).
  • GST-calreticulin For purification of GST-calreticulin, the bacteria were sonicated, pelleted, and the supematants (adjusted to pH 7.0) were mixed with prewashed Glutathione Sepharose 4B (Bulk GST purification module; Amersham Pharmacia Biotech) in PBS with 1.0% Triton X-100. After 30 min incubation and washing the beads, bound protein was eluted with a 50 mM Tris-HCl buffer containing 10 mM glutathione, pH 8.0. Eluted material was ultracentrifiiged (2 hours at 104,000 x g), and supernatant retained. All protein lots for in vivo and in vitro experiments (GST-calreticulin, control GST, MBP-calreticulin, MBP-vasostatin, MBP, cleaved caheticulin and
  • TM vasostatin were tested for endotoxin by the Limulus Amebocyte Lysate (LAL) kinetic-QCL assay (BioWhittaker) and were found to contain ⁇ 5 units/ 10 ⁇ g protein.
  • LAL Limulus Amebocyte Lysate
  • the purified recombinant proteins including the expression tag sequences were resolved by SGS-PAGE as discrete bands migrating at the expected relative positions.
  • Natural caheticulin was also obtained from purified B cell line supernatant by eluting the band at 55 kDa from a preparative Tris-glycine gel. SDS PAGE analysis and silver staining documented the isolation of a doublet at approximately 55 kDA from other copurified components.
  • recombinant purified MBP-calreticulin (Seq. I.D. No. 2) inhibited the proliferation of fetal bovine heart endothelial cells induced by bFGF.
  • MBP-calreticulin At a concentration of 1 ⁇ g/ml, MBP-calreticulin (Seq. I.D. No.
  • Fetal bovine heart endothelial cells 800 cells/well were cultured for 5 days in medium alone or medium supplemented with bFGF (25 ng/ml).
  • Recombinant purified GST-caheticulin (Seq. I.D. No. 2; 1 ⁇ g/ml)
  • control recombinant GST (1 ⁇ g/ml)
  • caheticulin cleaved and purified from MBP- calreticulin (Seq. I.D. No.
  • Human umbilical vein endothelial cells (3.5x10 ⁇ cells/well) or fetal bovine heart endothelial cells (800 cells/well) were cultured in medium alone or in medium supplemented with bFGF.
  • MBP-120-180 caheticulin (Seq. I.D. No. 5) were tested.
  • Recombinant purified MBP- ⁇ 120 caheticulin (Seq. I.D. No. 9) inhibited the proliferation of fetal bovine heart endothelial cells, and the degree of inhibition was comparable to that of caheticulin (Figure 4).
  • Vasostatin (caheticulin N-domain, encompassing amino acids 1-180; Seq. I.D. No. 4), full length caheticulin (Seq. I.D. No. 2), caheticulin minus 17 amino acids from the N-terminus (Seq. I.D. No. 3), and ⁇ 120 caheticulin (Seq. I.D. No. 9) inhibited bFGF-induced endothelial cell proliferation to a similar degree at similar concentrations.
  • the activity of the 61 amino acid caheticulin fragment (Seq. I.D. No. 5) was also determined. This was done by purifying the MBP-120-180 caheticulin fragment (Seq. I.D.
  • MBP-vasostatin Seq. I.D. No. 4
  • MBP- ⁇ 120 caheticulin Seq. I.D. No. 9
  • MBP-120-180 caheticulin Seq. I.D. No. 5 fragment revealed that, on a molar basis, the four proteins display similar endothelial cell growth inhibitory active in vitro. Control MBP was not inhibitory ( Figure 6).
  • both the caheticulin fragment identified as Seq. I.D. No 5 (the 61 amino acid peptide) and the caheticulin fragment identified as Seq. I.D. No 6 (the 49 amino acid peptide) specifically inhibit endothelial cell growth in vitro.
  • Fetal bovine heart endothelial cells (800 cells/well) were cultured for 5 days in medium alone or medium supplemented with bFGF (25 ng/ml).
  • Recombinant MBP vasostatin (0.5 ⁇ g/ml), a synthetic caheticulin 60 amino acid peptide (50 and 100 ⁇ g/ml), a synthetic caheticulin 49 amino acid peptide (12.5-50 ⁇ g/ml), and a synthetic 60 amino acid peptide of the chemokine RANTES (12.5-100 ⁇ g/ml) were added to endothelial cell cultures with bFGF (25 ⁇ g/ml).
  • the neuroblastoma cell line SKNMC was cultured in EMEM medium (BioWhittaker) with 10% heat inactivated fetal calf serum (BioWhittaker) and 5 ⁇ g/ml gentamicin (Sigma) and tested for proliferation at 125-1000 cells/well.
  • the lung adenocarcinoma cell line A549 was cultured in F- 12 Nutrient Mixture (HAM, GIBCO BRL) with 10% heat inactivated fetal calf serum (BioWhittaker) and 5 ⁇ g/ml gentamicin (Sigma), and tested for proliferation at 250-2000 cells/well.
  • the breast adenocarcinoma MDA-MB-468 (ATCC) and the Wilms tumor SK-NEP- l(ATCC) cell lines were cultured in Leibovitz L-15 medium (GIBCO BRL) supplemented with 10% heat inactivated fetal calf serum (BioWhittaker) and 5 ⁇ g/ml gentamicin (Sigma), and tested for proliferation at 300-2500 cells/well.
  • GIBCO BRL Leibovitz L-15 medium
  • BioWhittaker heat inactivated fetal calf serum
  • 5 ⁇ g/ml gentamicin Sigma
  • the colon carcinoma cell line SW480 (ATCC), the melanoma cell line A-375 (ATCC), and human foreskin fibroblasts (H5 68, ATCC) were cultured in DMEM medium (BioWhittaker) with 10% heat inactivated fetal calf serum (BioWhittaker) and 5 ⁇ g/ml gentamicin (Sigma), and were tested for proliferation at 500-4000 cells/well. All cell lines tested mycoplasma negative. The results of these assays showed that, in contrast to their inhibitory effect on human and bovine endothelial cell growth, MBP-calreticulin (Seq. I.D. No. 3) and the MBP-calreticulin N- terminal 180 amino acid fragment (Seq.
  • caheticulin (MBP-calreticulin 120-180; Seq. I.D. No. 5), as well as the 60 and 49 amino acid peptides of caheticulin (Seq. I.D. No 8, and 6, respectively) were also tested for then ability to inhibit the proliferation in vitro of cells other than endothelial cells. At concentrations ranging between 1 and 100 ⁇ g/ml, neither peptide inhibited the proliferation of the human B cell lines VDS-O and CA-46. Thus, caheticulin (Seq. I.D.
  • the caheticulin N- terminal 180 amino acid fragment, vasostatin (Seq. I.D. No. 4), a 60 amino acid caheticulin peptide included in the N-terminal amino acid peptide (amino acids 121-180; Seq. I.D. No. 8), and a 49 amino acid caheticulin peptide (amino acids 132-180; Seq. I.D. No. 6) included in the N-terminal 180 amino acid peptide specifically inhibit endothelial cell proliferation.
  • the murine Matrigel assay (Passaniti et al., Lab. Invest. 67:519-528, 1992) was employed to evaluate the effects of caheticulin and caheticulin fragments on angiogenesis in vivo.
  • the Matrigel assay was performed as described by Angiolillo et al. (J. Exp. Med. 182:155-162, 1995).
  • the instrument was adjusted to evaluate a circular area measuring 1.26xl0 5 mm 2 of Matrigel, and within this area, to measure the area occupied by cells. For each plug, 12-15 distinct fields were evaluated. The fields were randomly selected from each plug, and the operator was blind to the experimental design. The average area occupied by cells/1.26xl0 5 mm 2 Matrigel field was calculated. Results are expressed as the mean area occupied by cells/Matrigel field.
  • Matrigel plugs were fixed in 10% neutral buffered formalin solution (Sigma), embedded in paraffin, sectioned at 4 ⁇ m, and stained with Masson's trichrome by standard methods.
  • vasostatin (cleaved and purified from MBP-vasostatin) inhibited bFGF-induced Matrigel vascularization comparably to MBP-vasostatin and MBP-calreticulin (Table 4, exp. 3).
  • MBP- ⁇ 120 caheticulin (Seq. I.D. No. 9) and 120-180 caheticulin (Seq. I.D.
  • mice were injected subcutaneously with Matrigel alone, Matrigel plus bFGF (150 ng/ml), Matrigel plus bFGF (150 ng/ml) plus GST-caheticulin (Seq. I.D. No. 2), MBP-calreticulin (Seq. I.D. No. 3), MBP-vasostatin (Seq. I.D. No. 4), MBP, vasostatin, MBP- ⁇ 120 caheticulin (Seq. I.D. No. 9), or MBP-120-180 caheticulin (Seq. I.D. No. 5). Plugs were removed after 5-7 days, and histologic sections were stained with Masson's trichrome.
  • the results reflect the mean surface area (expressed in mm 2 ) occupied by.cells within a circular surface area of 1.26xl0 5 mm 2 ; 12-15 non-overlapping fields were scanned in each plug; there were 5 plugs/group. Determinations of surface area were performed by a semi-automated digitalized analyzer.
  • Caheticulin and caheticulin fragments were initially tested for their ability to prevent the growth of human Burkitt lymphomas in athymic mice.
  • CA46 cell line Sgadari et al., Proc. Natl. Acad. Sci. U.S.A. 93: 13791-13796, 1996) in 0.2 ml PBS.
  • injections proximal to the site of original cell inoculation of test caheticulin samples or appropriate controls included purified GST-calreticulin (Seq. I.D. No. 2), control GST, MBP-calreticulin (Seq. I.D. No. 3), MBP-vasostatin (Seq. I.D. No. 4), MBP, or formulation buffer used to dilute test proteins (sterile saline solution containing 50 mg/ml human albumin and 5 mg/ml mannitol; endotoxin ⁇ 5 units/ml).
  • Tumor size was estimated (in cm 2 ) twice weekly as the product of two-dimensional caliper measurements (longest perpendicular length and width).
  • a subcutaneous mass appearing at or proximal to the site of cell inoculation was considered a tumor when it measured at least 0.16 cm 2 in surface area and increased in size by at least 0.1 cm 2 over the following week.
  • Vasostatin (Seq. I.D. No. 4) was tested for its ability to prevent or delay Burkitt tumor growth in the same murine tumor model.
  • MBP- vasostatin (Seq. I.D. No. 4) was inoculated daily, 6 days/week, for 14 days at a dose of 30 ⁇ g/mouse ( Figure 3B).
  • the mean ( ⁇ SD) weight of tumors in the untreated control group (0.43 +0.2 g) was greater than the weight of tumors from vasostatin-treated animals (0.21 ⁇ 0.05 g), but the weight difference did not reach statistical significance (p 0.059).
  • MBP-vasostatin (Seq. I.D. No. 4) treatment one additional tumor appeared on day 23, but the remaining 7 animals remain tumor-free as of day 60.
  • MBP-vasostatin (Seq. I.D. No. 4) was used at a higher dose (200 ⁇ g/twice/day, 5 days/week and once/day 2 days/week) for 38 days.
  • MBP- ⁇ 120 caheticulin (30 ⁇ g/mouse; Seq. I.D. No. 9) was tested for its ability to prevent Burkitt tumor growth, and compared its effects to those of MBP- vasostatin (30 ⁇ m/mouse; Seq. I.D. No. 4).
  • all mice (8/8) inoculated with control MBP (20 ⁇ g/mouse) developed a tumor.
  • MBP-vasostatin Seq. I.D. No.
  • MBP- ⁇ 120 caheticulin (Seq. I.D. No. 9) and one mouse from the group treated with MBP-vasostatin (Seq. I.D. No.4) remain tumor-free as of day 60.
  • MBP- ⁇ 120 caheticulin (Seq. I.D. No. 9) can prevent Burkitt tumor growth.
  • tumor plugs were fixed in 10% neutral buffered formalin solution (Sigma), embedded in paraffin, sectioned at 4 ⁇ m, and stained with hematoxylin and eosin, or Masson's trichrome by standard methods. Histology of tumors that emerged on MBP-calreticulin (Seq. I.D. No. 3) or MBP-vasostatin (Seq. I.D. No. 4) treatment showed subtle characteristic changes to the tumor vasculature, including intimal and medial thickening of the vessel wall. These alterations were absent from tumors of control animals. The tumor tissue from test and control animals was indistinguishable with respect to morphology of tumor cells and the number of mitoses.
  • tumors from calreticulin and vasostatin -treated animals displayed occasional infiltration with neutrophils, histiocytes, and lymphocytes that were absent from control tumors.
  • tumor tissues from animals treated with MBP-vasostatin or MBP-calreticulin displayed a significant reduction in the number of vessels identified by immunohistochemical staining for the endothelial cell marker CD31. No abnormalities were noted on gross and histological examination of liver, spleen, kidneys, heart, lung and lymph nodes from MBP-calreticulin and MBP-vasostatin-treated animals.
  • Adenocarcino as and Neuroblastoma Tumors Wilms Tumor, Rhabdomyosarcoma, Promyelomonocytic Lymphoma, Murine lymphoma, and Melanoma Tumors.
  • mice were inoculated s.c. with the human neuroblastoma SK-N-MC cell line. Beginning on the day of cell inoculation and continuing thereafter daily, the mice received s.c. either control MBP (20 ⁇ g/day) or MBP-vasostatin (30 ⁇ g/day; (Seq. I.D. No. 4). On day 30, 8 of the 12 mice receiving control MBP (mean tumor weight 1.74 g) and 5 of the 12 mice receiving MBP-vasostatin (mean tumor weight 1.78 g; Seq. I.D. No. 4) were sacrificed.
  • mice on human breast adenocarcinoma growth in athymic mice was also tested (Figure 10).
  • Athymic BALB/c nude mice were inoculated s.c. with the human breast adenocarcinoma cell line MDA-MB231. Beginning on the day of cell inoculation and continuing daily thereafter (5 days/week for 18 days) the mice were inoculated s.c. with either MBP-vasostatin (200 ⁇ g/day, 5 days/week and 100 ⁇ g/day 2 days week; Seq. I.D. No. 4) or formulation buffer control.
  • MBP-vasostatin 200 ⁇ g/day, 5 days/week and 100 ⁇ g/day 2 days week; Seq. I.D. No.
  • mice were injected s.c. with the murine B-cell lymphoma cell line Raw 8.1 (also BALB/c). Beginning on the day of cell inoculation and continuing thereafter daily, 9 mice were inoculated s.c. with control formulation buffer and 9 mice were treated with MBP-vasostatin (200 ⁇ g/day; Seq. I.D. No. 4). Treatment was stopped when all animals developed a tumor on day 15. As shown ( Figure 11 A), MBP-vasostatin treatment reduced significantly murine lymphoma tumor growth (p ⁇ 0.01).
  • mice received 400 rad total body irradiation and 24 hours later were inoculated (12 x 10 6 cell/mouse) with the human Wilms tumor cell line (SK-NP-1).
  • SK-NP-1 human Wilms tumor cell line
  • MBP-vasostatin Seq. I.D. No. 4
  • mice The effects of MBP-vasostatin treatment on the human rhabdomyosarcoma cell line A-204 injected into nude mice were examined ( Figure 13). Groups of athymic BALB/c nude mice received 400 rad total body irradiation and 24 hours later were inoculated (8x10 6 cell/mouse) s.c. with the human rhabdomyosarcoma cell line A-204. One day after cell injection, and continuing daily thereafter for 28 days, the mice received either control formulation buffer alone or MBP-vasostatin (100 ⁇ g/day, 6 days/week; Seq. I.D. No. 4).
  • MBP-vasostatin (Seq. I.D. No. 4) was tested for its ability to reduce the growth of the human promyelomonocytic cell line HL60 inoculated s.c. into nude mice ( Figure 14). Groups of athymic BALB/c nude mice received 400 rad total body irradiation and 24 hours later were inoculated (lOxlO 6 cell/mouse) with the human promyelomonocytic cell line HL60. One day after cell injection, and continuing daily thereafter for 24 days, the mice received either control formulation buffer alone or MBP-vasostatin (200 ⁇ g/day, 6 days/week; Seq. I.D. No. 4).
  • mice treated with MBP-vasostatin were significantly smaller than the mice treated with formulation buffer (p ⁇ 0.001).
  • the effect of MBP-vasostatin (Seq. I.D. No. 4) on human melanoma tumors transplanted into nude mice was also tested ( Figure 15).
  • Athymic BALB/c nude mice received 400 rad total body irradiation and 24 hours later were inoculated (7xl0 6 cell/mouse) with the human melanoma cell line (A-375).
  • the 61 amino acid fragment of caheticulin encompassing amino acids 120-180 was tested for its ability to inhibit tumor growth.
  • this caheticulin fragment was active in vitro as an inhibitor of tumor growth and was active in vivo as an inhibitor of angiogenesis.
  • groups of athymic mice were irradiated with 400 rad, and 24 hours later were injected s.c. with 4xl0 6 cells from the Burkitt lymphoma cell line CA46.
  • mice received either control recombinant purified MBP (65 ⁇ g/day, 6 days/week), MBP-vasostatin (100 ⁇ g/day, 6 days/week; Seq. I.D. No. 4), or MBP-120-180 caheticulin (75 ⁇ g/day, 6 days/week; Seq. I.D. No. 5).
  • Mice were injected for 42 days.
  • the 61 amino acid caheticulin fragment (Seq. I.D. No. 5) is active as an inhibitor of tumor growth in vivo.
  • mice All mice were sacrificed after 15 days of treatment.
  • s.c. treatment with MBP-vasostatin 100 ⁇ g/day; 6 days/week; Seq. I.D. No. 4
  • formulation buffer 100 ng/mouse; 6 days/week
  • s.c. treatment with IL-12 100 ng/mouse; 6 days/week
  • mice received 7 days treatment with IL-12
  • MBP-vasostatin (Seq. I.D. No. 4) and 5 days treatment with IL-12, and were observed untreated for 14 days at which time all mice were sacrificed.
  • the delayed addition of IL-12 and the shorter duration of IL-12 treatment in the second experiment resulted in IL-12 having a somewhat reduced effect on tumor growth and weight compared to the first experiment ( Figures 17A and 17B).
  • the rate of tumor growth and the mean weight of tumors was reduced by treatment with MBP-vasostatin (Seq. I.D. No. 4), mIL-12, or the combination of MBP-vasostatin (Seq. I.D. No. 4) plus mIL-12 compared with the control group treated with formulation buffer.
  • KXaa,FFXaa 2 R (Seq. I.D. No. 11), or KXaajGFFKR (Seq. I.D. No. 10)
  • Proteins known to bind caheticulin through a virtually identical amino acid sequence motif include the cytoplasmic domain of the alpha subunit of integrins (KXaaiGFFKR; Seq. I.D. No. 10) and a family of steroid receptors (KXaa ! FFXaa 2 R; Seq. I.D. No. 11). Specific examples of integrin amino acid sequences and steroid receptor sequences are provided in Table 5.
  • caheticulin purified from the culture supernatant of the lymphoblastoid cell line VDS-O, to selectively bind to the column and be eluted by a buffer solution containing 20 mM EDTA and 100 mM NaCl.
  • the purified preparations of VDS-0 cell line containing both full length as well as N-terminal caheticulin fragments were then loaded on the column (Starting material). As shown in Figure 18 full length caheticulin present in the material loaded was retained by the column and could be eluted with 20 mM EDTA, whereas the caheticulin fragments were found in the flow- through. These caheticulin fragments were subsequently found to be biologically active.
  • the binding to the peptide sequence KXaa ⁇ FFXaa 2 R (Seq. I.D. No. 11) or the sequence KXaaiGFFKR (Seq. I.D. No. 10) is not a property shared by caheticulin fragments that are active as inhibitors of endothelial cells growth and, moreover, the results suggest that KXaa ⁇ FFXaa 2 R (Seq. I.D. No. 11) and KXaaiGFFKR (Seq. I.D. No. 10) are not part of a caheticulin receptor on endothelial cells.
  • recombinant MBP-calreticulin fragment 120-180 (Seq. I.D.
  • Caheticulin may be purified from the supernatant of Epstein-Barr vims (EBV)-immortalized cell lines as described above. Caheticulin may also be purified from a tissue source using conventional biochemical techniques, or produced recombinantly in either prokaryotic or eukaryotic cells using methods well-known in the art (for example, those described in Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989). The recombinant expression of caheticulin and the 180 amino acid N-terminal fragment are described in Singh et al. ⁇ Proc. Natl. Acad. Sci. U.S.A.
  • nucleic acid sequences encoding caheticulin are available on GenBank, and include the cDNA sequence shown in Seq. I.D. No. 1.
  • fusion proteins typically include a protein tag that facilitates purification.
  • fusion proteins typically include a protein tag that facilitates purification.
  • examples of such systems include: the pMAL protein fusion and purification system (New England Biolabs, Inc., Beverly, MA); the GST gene fusion system (Amersham Pharmacia Biotech, Inc., Piscataway, NJ); and the pTrcHis expression vector system (Invitrogen, Carlsbad, CA).
  • the pMAL expression system utilizes a vector that adds a maltose binding protein to the expressed protein.
  • the fusion protein is expressed in E. coli.
  • the fusion protein is purified from a cmde cell extract using an amylose column.
  • the maltose binding protein domain can be cleaved from the fusion protein by treatment with a suitable protease, such as Factor Xa.
  • the maltose binding fragment can then be removed from the preparation by passage over a second amylose column.
  • Eukaryotic expression systems may also be employed, including Pichia, tobacco and Baculovirus expression systems, such as those available commercially from Invitrogen.
  • the entire caheticulin protein may be produced by ligating the open reading frame (ORF) of caheticulin into the vector.
  • ORF open reading frame
  • the ORF must be operably linked to the vector, i.e., must be joined such that the reading frame of the ORF is aligned with the reading frame of the protein tag.
  • an ORF encoding the deshed fragment may be amplified by polymerase chain reaction (PCR) from the calreticulin cDNA, cloned, purified and then ligated into the expression vector. Alternatively, the amplified fragment may be ligated directly into the expression vector. It may also be possible, depending on the availability of suitable restriction sites in the caheticulin cDNA to obtain the deshed fragment by appropriate restriction endonuclease digestion, such that it can be directly cloned into the expression vector.
  • PCR polymerase chain reaction
  • Purification of the expressed protein can be achieved either using the purification regimen appropriate for the expression tag (if a commercial expression/purification system is used), or conventional affinity chromatography using antibodies, preferably monoclonal antibodies, that recognize the appropriate regions of caheticulin may be employed. Where caheticulin fragments are to be used, such fragments may alternatively be generated through digestion of the full-length caheticulin protein with various proteases. The fragments may then be separated based on their unique size, charge or other characteristics. Caheticulin fragments may also be synthetically generated through the use of known peptide synthesis methods.
  • the biological activity can be assessed using the methods described above.
  • the bFGF-induced endothelial cell proliferation assay can be used to determine endothelial cell inhibition
  • the Matrigel assay can be used to measure the inhibition of angiogenesis
  • the athymic mouse/human Burkitt lymphomas model can be used to quantitate tumor inhibition.
  • variants of caheticulin and fragments of caheticulin that have been modified such that they do not bind to the amino acid motif described supra are of particular interest. These variants will retain the ability to specifically bind to endothelial cells, but will not be capable of binding the amino acid sequences shown in Seq. I.D. Nos. 11 and/or 10.
  • This amino acid sequence motif is found in a family of steroid receptors (glucocorticoid , mineralcorticoid, progesterone and androgen receptors: KVFFKR, Seq. I.D. No. 32; estrogen receptor: KAFFKR, Seq. I.D. No. 33; thyroid hormone receptor: KSFFRR, Seq. I.D. No.
  • the fragments and variants of caheticulin described supra are characterized by their ability to inhibit endothelial cell growth, angiogenesis, and/or tumor growth. These abilities, however, are further defined as at least 30% inhibition of endothelial cell growth, angiogenesis, and/or tumor growth.
  • some therapeutically active fragments and variants of caheticulin will show an increased level of one or more of these biological activities. For example, some variants and fragments of caheticulin will show at least 40% inhibition, at least 50% inhibition, at least 60% inhibition, or at least 70% inhibition.
  • using the assays described above it is now possible to individually assess the biological activity of a given variant or fragment of caheticulin.
  • the therapeutically effective fragments and variants of caheticulin are also characterized by the number of amino acid residues that they contain. For example, in some instances it may be desirable to use relatively short fragments and variants of caheticulin. These short fragments and variants of caheticulin may contain at least 5, 10, 20, or 30 contiguous amino acids residues of the caheticulin sequence. However, such short fragments and variants of caheticulin will maintain at least one of the biological activities described supra. Additionally, it is possible to vary the cDNA sequences encoding therapeutically effective fragments or variants of caheticulin while still encoding a protein having the deshed biological activity.
  • sequence variants may differ from the disclosed sequences by alteration of the coding region to fit the codon usage bias of the particular organism into which the molecule is to be introduced.
  • the coding region may be altered by taking advantage of the degeneracy of the genetic code to alter the coding sequence in such a way that, while the nucleotide sequence is substantially altered, it nevertheless encodes a protein having an amino acid sequence identical or substantially similar to the disclosed caheticulin protein sequence.
  • the first amino acid residue of the mature caheticulin protein is glutamic acid (Glu, E). This is encoded in the caheticulin open reading frame (ORF) by the nucleotide codon triplet GAG.
  • GAA nucleotide codon
  • GAA also encodes for glutamic acid.
  • the nucleotide sequence of the calreticulin ORF could be changed at this position to GAA without affecting the amino acid composition of the encoded protein or the characteristics of the protein.
  • the invention may also be practiced with caheticulin and caheticulin fragments that vary in amino acid sequence from the sequence shown in Seq. I.D. No. 2 (i.e. peptides that have been modified such that they do not bind to the consensus sequences described supra (Seq. I.D. Nos. 10 and 11).
  • Variant caheticulin proteins include proteins that differ in amino acid sequence from the endogenous caheticulin sequence disclosed but which retain the specified biological activity. Such proteins may be produced by manipulating the nucleotide sequence of ORF that encodes the protein, for example by site-directed mutagenesis or the polymerase chain reaction. The simplest modifications involve the substitution of one or more amino acids for amino acids having similar biochemical properties. These so-called conservative substitutions are likely to have minimal impact on the activity of the resultant protein. Table 6 shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions.
  • substitutions that are less conservative than those in Table 4, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the stmcture of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • substitutions which in general are expected to produce the greatest changes in protein properties will be those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.
  • a hydrophilic residue e.g
  • purified caheticulin or calreticulin fragments are generally combined with a pharmaceutically acceptable carrier.
  • Pharmaceutical preparations may contain only caheticulin or a single caheticulin fragment, or may be composed of caheticulin combined with one or more caheticulin fragments, or may be composed of multiple caheticulin fragments.
  • the nature of the carrier will depend on the particular mode of administration being employed.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, human albumin or the like as a vehicle.
  • non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • protein-based pharmaceuticals may be only inefficiently delivered through ingestion.
  • pill-based forms of pharmaceutical proteins may be alternatively be administered subcutaneously, particularly if formulated in a slow-release composition.
  • Slow-release formulations may be produced by combining the target protein with a biocompatible matrix, such as cholesterol.
  • a biocompatible matrix such as cholesterol.
  • Another possible method of administering protein pharmaceuticals is through the use of mini osmotic pumps.
  • mini osmotic pumps As stated above a biocompatible carrier would also be used in conjunction with this method of delivery.
  • caheticulin could be delivered to cells in the nucleic acid form and subsequently translated by the host cell. This could be done, for example through the use of vhal vectors or liposomes. Liposomes could also be used for the delivery of the protein itself. The therapeutically effective fragments may also be delivered in conjunction with other therapeutic agents.
  • additional therapeutic agents can be angiogenesis inhibitors such as, platelet-factor-4, IP- 10 (interferon (IFN)- ⁇ inducible protein- 10), MIG (Monokine induced by IFN- ⁇ ), IFN- ⁇ , angiostatin, endostatin, fumagillin, AGM-1470, thrombospondin, a fragment of prolactin, antibody against the integrin ⁇ IL-12, cleaved conformation of the serpin antithrombin (O'Reilly et al, Science 285: 1926, 1999), thalidomide, and mixtures thereof.
  • the additional therapeutics could also be chemotherapeutics, hormones, anti-inflammatory agents, antibiotics, radiation therapy and/or anti-viral agents.
  • compositions of the present invention may be administered by any means that achieve their intended purpose.
  • Amounts and regimens for the administration of caheticulin, or a therapeutically active fragment thereof, can be determined readily by those with ordinary skill in the clinical art of treating diseases associated with angiogenesis, endothelial cell growth and/or tumor growth.
  • the described proteins are administered in an amount effective to inhibit angiogenesis, endothelial cell growth and/or tumor growth.
  • the peptides or proteins may be administered to a host in vivo, such as for example, through systemic administration, such as intravenous or intraperitoneal administration.
  • the peptides or proteins may be administered intralesionally: i.e. the peptide or protein is injected directly into the tumor or affected area.
  • Effective doses of caheticulin and caheticulin fragments for therapeutic application will vary depending on the nature and severity of the condition to be treated, the age and condition of the subject and other clinical factors. Thus, the final determination of the appropriate treatment regimen will be made by the attending clinician. Typically, the dose range will be from about 0.1 ⁇ g/kg body weight to about 100 mg/kg body weight. Other suitable ranges include doses of from about 1 ⁇ g/kg to 10 mg/kg body weight.
  • the dosing schedule may vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the protein.
  • Examples of dosing schedules are 3 ⁇ g/kg administered twice a week, three times a week or daily; a dose of 7 ⁇ g/kg twice a week, three times a week or daily; a dose of 10 ⁇ g/kg twice a week, three times a week or daily; or a dose of 30 ⁇ g/kg twice a week, three times a week or daily.
  • doses such as those described above by alternate routes including intravenously or intrathecally. Continuous infusion may also be appropriate.
  • caheticulin and caheticulin fragments will be useful for the treatment of diseases associated with unwanted angiogenesis.
  • Angiogenesis is commonly associated with ocular diseases. Some of these diseases are, retrolental fibroplasia, trachoma, retinal neovascularization, macular degeneration, diabetic retinopathy and neovascular glaucoma, corneal graft rejection and contact lens overwear.
  • Other non-ocular diseases, which are associated with unwanted angiogenesis can also be treated with caheticulin and fragments of caheticulin.
  • diseases are, periodontal disease, psoriasis, angiofibromas, immune-inflammation, atherosclerosis, excessive wound repair, non-immune inflammation, Crohn's disease, and systemic lupus.
  • diseases that are associated with immune-inflammation are rheumatoid arthritis, systemic lupus erythematosus, thyroiditis, Goodpasture's Syndrome, systemic vasculitis, scleroderma, Sjogren's syndrome, sarcoidosis or primary biliary cirrhosis.
  • Caheticulin and fragments of caheticulin may also be useful for the treatment diseases with unknown etiology, for example, Kaposi's sarcoma.
  • caheticulin and fragments of caheticulin may be useful as preventative agents.
  • caheticulin may be useful as a preventative of pregnancy by inhibiting angiogenesis at the site of implantation.
  • Caheticulin and the effective fragments might also be used as a preventative of injury from radiation and/or chemotherapy. Used in this way the therapeutically active fragments or variants would be administered prior to the radiation and or chemotherapy treatment and the resulting inhibition of angiogenesis would serve to protect hematopoietic cells from injury.
  • Caheticulin and caheticulin fragments can also be used to treat a variety of malignancies and related disorders, such as leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrdm's macroglobulinemia, heavy chain disease), as well as solid tumors such as sarcomas and carcinomas, fibrosarcoma.
  • acute leukemias such as acute lymphocytic leukemia, acute myelocytic leukemia, and my
  • caheticulin and fragments of caheticulin have been shown to inhibit angiogenesis that is induced by basic fibroblast growth factor (see, endothelial cell proliferation assay, discussed above). Therefore, one of ordinary skill will appreciate that caheticulin and fragments of caheticulin will also inhibit angiogenesis, which is induced by other compounds.
  • angiogenesis inducing compounds could be for example, acidic fibroblast growth factor, Vascular Endothelial Growth Factor (VEGF), hepatocyte growth factor, Interleukin (IL)-15, IL-12, IL-8, platelet-derived endothelial cell growth factor (PDECGF), angiogenin, Cripto (Ebert et al, Cancer Res., 59:4502-4505, 1999), Transforming Growth Factor (TGF)- ⁇ , Tumor Necrosis Factor (TNF) ⁇ , and angiogenin.
  • VEGF Vascular Endothelial Growth Factor
  • IL Interleukin
  • PDECGF platelet-derived endothelial cell growth factor
  • TGF Transforming Growth Factor
  • TNF Tumor Necrosis Factor

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Abstract

L'invention concerne des procédés qui mettent en oeuvre la calréticuline, des fragments de la calréticuline et des variants de la calréticuline pour inhiber la croissance des cellules endothéliales et l'angiogenèse, et pour supprimer la croissance tumorale. Ces procédés sont utiles pour le traitement du cancer et des maladies associées à l'angiogenèse indésirable comme, par exemple, le décollement rétinien chronique.
PCT/US1999/023240 1998-10-06 1999-10-05 Utilisation de la calreticuline et de fragments de la calreticuline pour inhiber la croissance des cellules endotheliales et l'angiogenese, et supprimer la croissance tumorale WO2000020577A1 (fr)

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AU62917/99A AU6291799A (en) 1998-10-06 1999-10-05 Use of calreticulin and calreticulin fragments to inhibit endothelial cell growth and angiogenesis, and suppress tumor growth
US09/807,148 US6867180B1 (en) 1998-10-06 1999-10-05 Use of calreticulin and calreticulin fragments to inhibit endothelial cell growth and angiogenesis, and suppress tumor growth
US09/828,000 US6596690B2 (en) 1998-10-06 2001-04-06 Vasostatin as marrow protectant
US10/405,588 US7432236B2 (en) 1998-10-06 2003-04-01 Vasostatin as marrow protectant
US11/040,162 US7488711B2 (en) 1998-10-06 2005-01-20 Use of calreticulin and calreticulin fragments to inhibit endothelial cell growth and angiogenesis, and suppress tumor growth
US12/198,810 US7812117B2 (en) 1998-10-06 2008-08-26 Vasostatin as marrow protectant

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WO2004040313A1 (fr) * 2002-11-01 2004-05-13 Tss Biotech Inc. Marqueur tumoral des carcinomes urotheliaux
US7323312B2 (en) 2002-11-01 2008-01-29 Tss Biotech Inc. Tumor marker for urothelial carcinoma
WO2008028963A3 (fr) * 2006-09-08 2009-02-12 Roussy Inst Gustave Utilisation de calréticuline comme médicament pour le traitement d'une maladie telle que le cancer chez un mammifère
WO2008032153A3 (fr) * 2006-09-08 2008-11-27 Michel Sarkis Obeid Procédé et nécessaire pour réaliser un criblage et un traitement immunogène à l'aide de la translocation de crt et/ou d'erp57
EP1900375A1 (fr) * 2006-09-08 2008-03-19 Institut Gustave Roussy Utilisation de la calréticuline pour le traitement du cancer chez les mammifères
EP1961763A1 (fr) * 2007-02-21 2008-08-27 Institut Gustave Roussy Composés régulant le récepteur CRT, KDEL et/ou l'exposition à l'ERp-57 et leurs utilisations pour évaluer l'efficacité d'un traitement contre le cancer
US11045521B2 (en) * 2010-06-17 2021-06-29 New York University Therapeutic and cosmetic uses and applications of calreticulin
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EP3046627A4 (fr) * 2013-09-18 2017-08-30 The Board of Trustees of The Leland Stanford Junior University Modulation des voies de l'efferocytose pour le traitement d'une maladie athérosclérotique
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