EP1608970A2 - Compositions de la ccn3 et procedes associes - Google Patents

Compositions de la ccn3 et procedes associes

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Publication number
EP1608970A2
EP1608970A2 EP04758632A EP04758632A EP1608970A2 EP 1608970 A2 EP1608970 A2 EP 1608970A2 EP 04758632 A EP04758632 A EP 04758632A EP 04758632 A EP04758632 A EP 04758632A EP 1608970 A2 EP1608970 A2 EP 1608970A2
Authority
EP
European Patent Office
Prior art keywords
ccn3
modulator
ccn
signaling molecule
integrin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04758632A
Other languages
German (de)
English (en)
Other versions
EP1608970A4 (fr
Inventor
Lester F. Lau
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Munin Corp
Original Assignee
Munin Corp
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Filing date
Publication date
Application filed by Munin Corp filed Critical Munin Corp
Publication of EP1608970A2 publication Critical patent/EP1608970A2/fr
Publication of EP1608970A4 publication Critical patent/EP1608970A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/515—Angiogenesic factors; Angiogenin

Definitions

  • the present invention relates to materials and methods involving extracellular matrix
  • the invention is directed to CCN3 -related peptides, compositions
  • the invention is also directed to anti-CCN3
  • Angiogenesis or the formation of new blood vessels from pre-existing ones, is a complex process requiring ' the coordinated execution of multiple cellular events (45).
  • the sprouting of vessels requires degradation of the basement membrane surrounding the parental vessel, migration of vascular endothelial cells towards the angiogenic stimulus, proliferation of
  • Angiogenesis is essential for .embryogenesis, and in the adult, it is important in the female reproductive cycle and in wound healing. Angiogenesis may underlie a number of pathological conditions including diabetic retinopathy, arthritis, arteriosclerosis, psoriasis, and cancer (46). It is now clear that angiogenesis is regulated by a network of multiple inducers and inhibitors (47,48).
  • the CCN 1 family of matricellular proteins are cysteine-rich, secreted proteins that are associated with the ECM but serve regulatory rather than structural functions.
  • CCN1 CYR61
  • CCN2 CCN2
  • CCN3 NOV
  • CCN4 WISP-1
  • CCN5 WISP-2
  • CCN6 WISP-3)(3,4)
  • CCN1 CYR61
  • CCN2 CCN2
  • CCN3 NOV
  • CCN4 WISP-1
  • CCN5 WISP-2
  • CCN6 WISP-3)(3,4)
  • CCN1 CYR61
  • CCN2 CCN2
  • CCN3 NOV
  • CCN4 WISP-1
  • CCN5 WISP-2
  • CCN6 WISP-3)(3,4)
  • CCN1 and CCN2 have been most extensively characterized. Both proteins stimulate cell migration, promote cell survival, and augment growth factor-induced mitogenesis (10-14). Both proteins are known to induce angiogenesis and chondrogenesis (12,15-18). Although CCN proteins do not contain a RGD sequence motif, both CCN1 and CCN2 are direct ligands of multiple integrin receptors, which mediate many of their activities (11,13,14,19-22). Targeted disruption of the CCN1 gene in mice resulted in embryonic lethality due to vascular defects (23), whereas CCN2-null mice die perinatally due to respiratory failure as a consequence of skeletal malformation (18).
  • CCN3 Although CC ⁇ 3 was first identified more than 10 years ago, little is known about its biochemical activities and biological functions. During embryonic development, CCN3 is widely expressed in derivatives of all three germ layers, with high levels of expression in skeletal muscle, smooth muscle of vessel walls, the nervous system, adrenal cortex, and differentiating chondrocytes (24-27). In the adult, CCN3 expression is high in the arterial vessel wall, and the expression pattern changes following vascular injury (28). CCN3 interacts with the epidermal growth factor-like domain of Notchl and positively regulate Notch signaling (29).
  • CCN3 interacts with fibulin in a yeast two-hybrid assay and may regulate calcium signaling (30,31). Altered expression of CCN3 has been observed in a variety of tumors, including hepatocellular carcinomas, Wilm's tumors, Ewing's sarcomas, gliomas, rhabdomyosarcomas, and adrenocortical carcinomas (27,32-34).
  • CCNS is demonstrated herein for the first time to be angiogenic, thereby being capable
  • the present invention is directed to CCN3-related
  • One aspect of the present invention relates to a method of screening for a modulator of
  • angiogenesis comprising contacting a test biological sample capable of undergoing angiogenesis with an ECM signaling molecule and a suspected modulator. As a control, a second biological sample is also contacted with an ECM signaling molecule.
  • a modulator of angiogenesis is
  • the ECM signaling molecule may be CCN3 or a fragment, variant, analog, homolog or a derivative thereof that maintains as least one biological activity of CCN3.
  • Another aspect of the present invention relates to a modulator of CCN3 activity identified by the present method.
  • Another aspect of the present invention relates to a method of screening for a modulator of angiogenesis comprising implanting a test implant into a test animal, wherein the test implant comprises a suspected modulator and an ECM signaling molecule.
  • a second implant comprising an ECM signaling molecule is implanted into a test animal, which may be the same animal or a different test animal.
  • a modulator of angiogenesis is identified by its ability to alter the level of blood vessel development in the test implant when compared to the control sample.
  • the ECM signaling molecule may be CCN3 or a fragment, variant, analog, homolog or a derivative thereof that maintains as least one biological activity of CCN3.
  • Another aspect of the present invention relates to a modulator of CCN3 activity identified by the present method.
  • Another aspect of the present invention relates to a method of screening for a modulator of oncogenesis comprising contacting a tumor with a suspected modulator along with an ECM signaling molecule. As a control, a second tumor is also contacted with an ECM signaling molecule.
  • a modulator of oncogenesis may be identified by its ability to alter the level of oncogenesis of the test tumor when compared to the control tumor.
  • the ECM signaling molecule may be CCN3 or a fragment, variant, analog, homolog or a derivative thereof that maintains as least one biological activity of CCN3.
  • Another aspect of the present invention relates to a modulator of CCN3 activity identified by the present method.
  • Another aspect of the present invention relates to a method of screening for a modulator of cell adhesion comprising contacting a test biological sample on a surface compatible with cell adherence with a suspected modulator along with an ECM signaling molecule. As a control, a second biological sample on a surface compatible with cell adherence is also contacted with an ECM signaling molecule.
  • a modulator of cell adhesion is identified by its ability to alter the level of cell adhesion of the test sample when compared to the control sample.
  • the ECM signaling molecule may be CCN3 or a fragment, variant, analog, homolog or a derivative thereof that maintains as least one biological activity of CCN3.
  • Another aspect of the present invention relates to a modulator of CCN3 activity identified by the present method.
  • Another aspect of the present invention relates to a method of screening for a modulator of cell migration comprising seeding cells capable of undergoing cell migration onto a test gel matrix comprising a suspected modulator and an ECM signaling molecule. As a control, cells capable of undergoing cell migration are also seeded onto a second biological sample gel matrix comprising an ECM signaling molecule.
  • a modulator of cell adhesion may be identified by its ability to alter the level of cell migration in the test matrix when compared to the control matrix.
  • the ECM signaling molecule may be CCN3 or a fragment, variant, analog, homolog or a derivative thereof that maintains as least one biological activity of CCN3.
  • Another aspect of the present invention relates to a modulator of CCN3 activity identified by the present method.
  • Another aspect of the present invention relates to a peptide that modulates the binding of
  • CCN3 to an integrin selected from the group consisting of ⁇ v ⁇ 3 , ot ⁇ and ⁇ 6 ⁇ , or a variant,
  • Another aspect of the present invention relates to a composition comprising a peptide
  • composition may further comprise one or more peptides that modulate the binding of a CCN
  • CCN polypeptide to an integrin, or a variant, analog, homolog or derivative of said one or more peptides, wherein said CCN polypeptide is selected from the group consisting of CCN1, CCN2,
  • Another aspect of the present invention relates to an antibody that modulates the binding
  • CCN3 to an integrin selected from the group consisting of ⁇ v ⁇ 3 , 5 ⁇ and ⁇ 6 ⁇ , or a variant,
  • Another aspect of the present invention relates to a composition comprising an antibody
  • composition further comprises one or more antibodies that modulate the binding of a CCN
  • CCN polypeptide to an integrin, wherein said CCN polypeptide is selected from the group consisting of CCN 1, CCN2, CCN4, CCN5 and CCN6.
  • Fig. 1 demonstrates the purification of CCN3.
  • Panel A fractions (30 ⁇ l per lane) from
  • Fig. 2 demonstrates the adhesion of HUVECs to CCN3.
  • Panel A HUVECs were plated in microtiter wells coated with the indicated amount of CCN3. After incubation at 37 °C for 30
  • Panel B microtiter wells were coated with BSA, 12 ⁇ g/ml CCNS, or 0.5
  • Fig. 3 demonstrates that HUVEC adhesion to CCN3 is mediated through integrins ⁇ v ⁇ 3 ,
  • Panel A cells were incubated with 40 ⁇ g/ml mAbs against integrins ⁇ 6 and ⁇ i for 1 h prior to
  • Panel B cells were incubated with 40 ⁇ g/ml LM609 (anti- ⁇ v ⁇ 3 mAb) for 1 h prior to being
  • Fig. 4 demonstrates that CCN3 binds directly to integrin v ⁇ 3 .
  • Panel A microtiter wells
  • integrin ⁇ v ⁇ 3 was incubated with 5 mM EDTA, EDTA+ 10 mM Mg 2+ , 0.2 mM RGDS peptide,
  • Fig. 5 demonstrates that CCN3 binds directly to integrin ⁇ 5 ⁇ .
  • Panel A microtiter wells
  • Fig. 6 demonstrates migration of BACEC to CCN3.
  • BACEC migration was monitored using a modified Boyden chamber assay. Cells were added to wells in the lower chamber and
  • VN 10 ⁇ g/ml
  • FN 10 ⁇ g/ml
  • Fig. 7 demonstrates that integrins v ⁇ 3 and ⁇ 5 ⁇ mediate migration of BACEC to CCN3.
  • Migration assays were performed using a modified Boyden chamber.
  • CCN3 (0.25 ⁇ g/ml)
  • VN 10 ⁇ g/ml
  • FN 10 ⁇ g/ml
  • Panel A cells were treated with anti-integrin ⁇ v (AVI, 60 ⁇ g/ml) or anti-integrin v ⁇ 3 (LM609,
  • FIG. 8 demonstrates that CCN3 protects HUVECs from apoptosis.
  • HUVECs were serum- starved prior to attachment to coverslips pre-coated with 20 ⁇ g/ml LN.
  • Panel A cells were then
  • Fig. 9 demonstrates that CCNS induces neovascularization in rat corneas. Hydron pellets containing tests substances were made and implanted into rat corneas (Table 1). Blood vessel formation was visualized by perfusion with colloidal carbon 7 days after implantation. Vessel formation due to Hydron pellets containing CCN3 storage buffer (A), bFGF (B), CCN3 protein (C), and CCN3 protein pre-incubated with anti-CCN3 antibodies (D), are shown.
  • A CCN3 storage buffer
  • B bFGF
  • C C
  • D anti-CCN3 antibodies
  • CCN3 In contrast to CCN1 and CCN2, which are immediate-early genes transcriptionally activated by mitogenic growth factors in fibroblasts and are repressed under conditions of growth arrest (35-37), CCN3 is repressed by growth factors but induced by serum deprivation or contact inhibition (38,39). Thus, it has been hypothesized that CCN3 may serve as an antagonist to CCN1 and CCN2, and play antithetical roles in similar biological processes. Since CCN1 and CCN2 have been shown to be angiogenic inducers (12,16,17), it has been speculated that CCN3 might work as an angiogenic inhibitor.
  • CCN3 is capable of proangiogenic activities in endothelial cells.
  • CCN3 is shown below to support endothelial cell adhesion, stimulates directed cell migration, and promotes cell survival (Figs. 2,6,8).
  • CCN3 is shown below to induce neovascularization in vivo in a corneal micropocket assay (Fig. 9). These activities are inhibited by antibodies specific for CCN3, showing that they are intrinsic properties of the CCN3 polypeptide.
  • CCN3 as a novel integrin ligand and angiogenic inducer, provide insights into its mechanism of action, and suggest biological functions for CCN3 both in normal development and in pathological conditions where its aberrant expression has been observed.
  • CCN3 may be part of the network of multiple inducers and inhibitors that regulate angiogenesis.
  • CCN3 is an angiogenic factor helps to shed light on its functions in development and disease.
  • CCNS is expressed in hypertrophic cartilage (26), where vessel growth is required for the formation of a scaffold onto which the osteoblasts settle and deposit bone matrix (49).
  • CCN3-induced angiogenesis may be important in endochondral ossification.
  • CCN3 is localized to the metanephric mesenchyme into which endothelial cells are recruited (32). These endothelial cells then proliferate and form a capillary network as the metanephric mesenchyme develops to form the glomeruli, the basic units of filtration (50).
  • CCN3 might help serve as a chemotactic and survival factor for endothelial cells.
  • CCN3 expression is correlated with various tumors, including Wilm's tumors, and benign adrenocortical tumors (27,32,51). It is well established that tumor growth beyond ⁇ 1 mm in size requires the growth of new vessels to provide the necessary blood supply (47,52). Thus, the expression of CCN3 in tumors is consistent with its angiogenic activity.
  • WT1 Wilms' Tumor suppressor gene was shown to negatively regulate CCN3 expression (53). It is possible to speculate that, as part of its function, WT1 down-regulates the angiogenic inducer CCN3 to help suppress tumor growth.
  • CCN3 is shown below to support endothelial cell adhesion through integrins
  • CCN3 I shown below to bind directly to integrins v ⁇ 3 and 5 ⁇ (Figs. 4 and 5).
  • CCN3 binds directly to integrins ⁇ v ⁇ 3 and ⁇ ⁇ . [0030] It is well established that integrins are important in developmental and pathological
  • mice genetically in mice, where targeted gene disruptions in integrins ⁇ 5 or ⁇ i resulted in embryonic
  • mice with targeted disruptions are absolutely required for developmental angiogenesis. Furthermore, mice with targeted disruptions
  • integrins ⁇ v ⁇ 3 and ⁇ v ⁇ s may actually be negative regulators of angiogenesis (63).
  • CCN1 and CCN2 are angiogenic inducers, mutations in their structural genes result in distinct phenotypes related to angiogenic defects.
  • Targeted gene disruption of CCN1 in mice resulted in embryonic lethality with vascular defects in both the placenta and the embryo (23).
  • CCN2-null mice are perinatal lethal as a consequence of respiratory failure due to skeletal malformations (18).
  • the present invention involves screening for modulators of activities associated with
  • Modulators may be identified that directly bind to CCN3, thereby prevent CCN3 from interacting with target proteins. Modulators may also be identified that directly bind to targets proteins of CCN3, thereby preventing CCN3 from productively interacting with: said target protein. Modulators may also be identified which indirectly affect binding of CCN3 to target proteins.
  • an "ECM signaling molecule” refers to CCN3 or a fragment, variant, analog, homolog or a derivative thereof that maintains as least one biological activity of CCN3.
  • the use of “ECM signaling molecule” also contemplates one or more additional CCN polypeptides.
  • the one or more additional CCN polypeptides include, but are not limited to, CCN1, CCN2, CCN4, CCN5 and CCN6, as well as fragments, variants, analogs, homologs or derivatives of said one or more additional CCN polypeptides.
  • the methods of the present invention relate to screening for a modulator of angiogenesis.
  • a biological sample capable of undergoing angiogenesis is contacted wi,th a suspected modulator in vitro along with an ECM signaling molecule.
  • a second biological sample is also contacted with an ECM signaling molecule.
  • a modulator of angiogenesis may be identified by its ability to alter the le ⁇ el of angiogenesis of the test sample when compared to the control sample.
  • an implant comprising a suspected modulator and an ECM signaling molecule is implanted into a test animal.
  • a second implant comprising an ECM signaling molecule is implanted into a test animal, which may be the same animal or a different test animal.
  • a modulator of angiogenesis may be identified by its ability to alter the level of blood vessel development in the test implant when compared to the control sample.
  • the methods of the present invention also relate to screening for a modulator of oncogenesis.
  • a tumor is contacted with a suspected modulator along with an ECM signaling molecule.
  • a second tumor is also contacted with an ECM signaling molecule.
  • a modulator of oncogenesis may be identified by its ability to alter the level of oncogenesis of the test tumor when compared to the control tumor.
  • the methods of the present invention also relate to screening for a modulator of cell adhesion.
  • a biological sample on a surface compatible with cell adherence is contacted with a suspected modulator along with an ECM signaling molecule.
  • a second biological sample on a surface compatible with cell adherence is also contacted with an ECM signaling molecule.
  • a modulator of cell adhesion may be identified by its ability to alter the level of cell adhesion of the test sample when compared to the control sample.
  • the methods of the present invention also relate to screening for a modulator of cell migration.
  • Cells capable of undergoing cell migration are seeded onto a gel matrix comprising a suspected modulator and an ECM signaling molecule.
  • As a control cells capable of undergoing cell migration are also seeded onto a second biological sample gel matrix comprising an ECM signaling molecule.
  • a modulator of cell adhesion may be identified by its ability to alter the level of cell migration in the test matrix when compared to the control matrix.
  • the present invention also involves modulators of CCN3 activity identified using the above-described screening methods.
  • the identified modulators of CCN3 activity may be formulated in a pharmaceutical composition comprising a pharmaceutically acceptable adjuvant, diluent, or carrier.
  • the pharmaceutical composition comprising the modulator of CCN3 activity may be administered to a patient for the treatment of disease associated with
  • the present invention also involves the use of inhibitory peptides in therapeutic strategies designed to inhibit the activity of CCNS .
  • the inhibitory peptides may be natural, synthetic or recombinant.
  • One approach is to produce an inhibitory peptide based on the protein sequence of CCN3 (SEQ ID NO: 1). For example, a peptide comprising conserved amino acids may compete with native CCN3 for its binding sites. This competition may thereby inhibit the action of native CCN3.
  • the inhibitory peptides may be from 5 to 50 amino acids in length.
  • the inhibitory peptides may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids in length.
  • the inhibitory peptide may comprise amino acids from SEQ ID NO: 1 selected from the group consisting of 1-5, 3-7, 6-10, 8-12, 11-15, 13-17, 16-20, 18-22, 21-25, 23-27, 26-30, 28-32, 31-35, 33-37, 36-40, 38-42, 41-45, 43-47, 46-50, 48-52, 51-55, 53-57, 56-60, 58-62, 61-65, 63- 67, 66-70, 68-72, 71-75, 73-77, 76-80, 78-82, 81-85, 83-87, 86-90, 88-92, 91-95, 93-97, 96-100, 98-102, 101-105, 103-107, 106-110, 108-112, 111-115, 113-117, 116-120, 118-122, 121-125, 123-127, 126-130, 128-132, 131-135,
  • the inhibitory peptide may also comprise amino acids from SEQ ID NO: 1 selected from the group consisting of 1-10, 6-19, 11-20, 16-29, 21-30, 26-39, 31-40, 36-49, 41-50, 46-59, 51- 60, 56-69, 61-70, 66-79, 71-80, 76-89, 81-90, 86-99, 91-100, 96-109, 101-110, 106-119, 111- 120, 116-129, 121-130, 126-139, 131-140, 136-149, 141-150, 146-159, 151-160, 156-169, 161- 170, 166-179, 171-180, 176-189, 181-190, 186-199, 191-200, 196-209, 201-210, 206-219, 211- 220, 216-229, 221-230, 226-239, 231-240, 236-249, 241-250, 246-259, 251-260,
  • the inhibitory peptide may also comprise amino acids from SEQ ID NO: 1 selected from the group consisting of 1-15, 8-22, 16-30, 23-37, 31-45, 38-52, 46-60, 53-67, 61-75, 68-82, 76- 90, 83-97, 91-105, 98-112, 106-120, 113-127, 121-135, 128-142, 136-150, 143-157, 151-165, 158-172, 166-180, 173-187, 181-195, 188-202, 196-210, 203-217, 211-225, 218-232, 226-240, 233-247, 241-255, 248-262, 256-270, 263-277, 271-285, 278-292, 286-300, 293-307, 301-315, 308-322, 316-330, 323-337, 331-345, 338-352, 346-357.
  • the inhibitory peptides may also be homologs of the above-described CCN3 peptides.
  • Homologs of the CCN3 peptides are peptides sharing a common evolutionary with SEQ ID NO: 1
  • inhibitory peptides may also be variants of the above-described CCN3 peptides and
  • Inhibitory peptide variants are peptides that differ in amino acid sequence from a native CCN3 peptide by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity of a native CCN3 peptide.
  • biological activity of a CCN3 peptide includes, but is not limited to, the above- described activities of full-length CCN3, the ability to inhibit activities of CCN3 and the ability to be bound by an antibody specific for CCN3.
  • a conservative substitution of an amino acid i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al, J. Mol. Biol. 757:105-132 (1982).
  • the hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge, and include the following values: alanine (+1.8), arginine (-4.5), asparagine (-3.5), aspartate (-3.5), cysteine/cysteine (+2.5), glycine (-0.4), glutamate (-3.5), glutamine (-3.5), histidine (-3.2), isoleucine (+4.5), leucine (+3.8), lysine (-3.9), methionine (+1.9), phenylalanine (+2.8), proline (-1.6), serine (-0.8), threonine (-0.7), tryptophan (-0.9), tyrosine (-1.3), and valine (+4.2).
  • hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function.
  • a consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity.
  • U.S. Patent No. 4,554,101 incorporated herein by reference.
  • Hydrophilicity values for each of the common amino acids, as reported in U.S. Patent No. 4,554,101 are: alanine (-0.5), arginine (+3.0), asparagine (+0.2), aspartate (+3.0 ⁇ 1), cysteine (-1.0), glycine
  • substitutions can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art.
  • substitutions are performed with amino acids having hydrophilicity values within ⁇ 2 of each other. Both the hyrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.
  • inhibitory peptides may also be analogs of the above-described CCN3 peptides, homologs and variants comprising non-standard amino acid or other structural variation from
  • inhibitory peptides may also be derivatives of the
  • CCN3 peptides, homologs, variants and analogs that differ in ways other than primary structure (amino acids and amino acid analogs).
  • derivatives may differ from native CCN3 peptides, homologs and variants by being glycosylated, one form of post-translational modification.
  • polypeptides may exhibit glycosylation patterns due to expression in heterologous systems. If these peptides retain at least one biological activity of native CCN3, then these peptides are CCN3 derivatives according to the invention.
  • fusion peptides having a covalently modified N- or C- terminus PEGylated peptides, peptides associated with lipid moieties, alkylated peptides, peptides linked via an amino acid side-chain functional group to other peptides or chemicals, and additional modifications as would be understood in the art.
  • the invention contemplates CCN3-related peptides that bind to a CCN3 receptor, as described below.
  • the various peptides of the present invention, as described above, may be provided as discrete peptides or be linked, e.g., by covalent bonds, to other compounds.
  • immunogenic carriers such as Keyhole Limpet Hemocyanin may be bound to a CCN3 peptide of the invention.
  • the present invention also involves a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody that specifically binds to CCN3 and a pharmaceutically acceptable adjuvant, diluent, or carrier.
  • the antibody may be produced as described below, or as described in WO 01/55210, the contents of which are hereby incorporated by reference in their entirety.
  • the antibodies of the present invention include antibodies of classes IgG, IgM, IgA, IgD, and IgE, and fragments and derivatives thereof including Fab and F(ab') 2 -
  • the antibodies may also be recombinant antibody products including, but not limited to, single chain antibodies, chimeric antibody products, "humanized” antibody products, and CDR-grafted antibody products.
  • the antibodies of the present invention include monoclonal antibodies, polyclonal antibodies, affinity purified antibodies, or mixtures thereof which exhibit sufficient binding specificity to CCN3.
  • Also contemplated by the invention are antibody fragments.
  • the antibody products include the aforementioned types of antibody products used as isolated antibodies or as antibodies attached to labels.
  • Labels can be signal-generating enzymes, antigens, other antibodies, lectins, carbohydrates, biotin, avidin, radioisotopes, toxins, heavy metals, and other compositions known in the art; attachment techniques are also well known in the art.
  • Anti-CCN3 antibodies are useful in diagnosing the risk of oncogenesis.
  • anti- CCN3 antibodies are used in therapies designed to deliver specifically-targeted cytotoxins to cells expressing CCN3, e.g., cells participating in the neovascularization of solid tumors. These antibodies are delivered by a variety of administrative routes, in pharmaceutical compositions comprising carriers or diluents, as would be understood by one of skill in the art.
  • Example 1 describes the cloning, expression and purification of recombinant CCN3.
  • Example 2 describes the production of anti-CCN3 antibodies.
  • Example 3 discloses that CCN3 supports endothelial cell adhesion.
  • Example 4 discloses that CCN3 acts as a ligand of integrin receptors.
  • Examples 5 discloses that CCN3 directs cells migration.
  • Example 6 discloses that CCN3 promotes cell survival.
  • Example 7 demonstrates that induces neovascularization in vivo.
  • Human CCNJ cD ⁇ A was constructed by ligation of a 5' (nt 72-654, Genbank X96584) and a 3' (nt 654-1653) fragments, and the resulting full-length cD ⁇ A was cloned into pKS+ and verified by sequencing.
  • the 5' fragment (nt 72-654) was obtained by reverse transcriptase- polymerase chain reaction using total RNA isolated from serum-starved human skin fibroblasts using the primer set 5'-AGCAGTGCCAATCTACAGC-3' and 5'-
  • CAGCATCTCACATTGACGG-3' The RT-PCR product was digested with Sphl and Styl to yield a fragment containing nt 72-654.
  • the 3' fragment (nt 654-1653) was generated by restriction digestion of IMAGE clone #49415 (human neonatal brain, nt 590-1653) with Styl and Xbal.
  • IMAGE clone #49415 human neonatal brain, nt 590-1653
  • Styl and Xbal Styl and Xbal.
  • the full-length CCNS cDNA was cloned into the baculoviras expression vector pBlueBac 4.5 (Invitrogen, Carlsbad, CA).
  • the vector was modified to encode an enterokinase histidine tag linked to the C-terminus of CCN3 in a manner similar to that previously described for expression of CCN1 (21).
  • CCN3 was produced in serum-free baculoviras expression system using High Five insect cells as described (21). Briefly, High Five cells were maintained in serum-free EX-CELL 400 medium (JRH Bioscience, Lenexa, KS) at 27 °C and infected at a multiplicity of infection of 10. Conditioned medium was collected at 38 h post-infection, adjusted to 20 mM sodium phosphate and applied to a Sepharose SP (Sigma- Aldrich, St. Louis, MO) column at 4°C.
  • EX-CELL 400 medium JRH Bioscience, Lenexa, KS
  • Conditioned medium was collected at 38 h post-infection, adjusted to 20 mM sodium phosphate and applied to a Sepharose SP (Sigma- Aldrich, St. Louis, MO) column at 4°C.
  • CCN3 von Willebrand type C repeat
  • GST glutathione S-transferase
  • the forward primers start with a BamHI site and the reverse primers end with an EcoRI site.
  • the resulting cDNA fragments were cloned directionally into the PGEX-2T vector (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) and confirmed by sequence analysis.
  • the GST-fusion proteins were purified on a glutathione-S sepharose column and used as antigens.
  • Antisera and affinity-purified antibodies were produced according to standard protocol (40). IgG was purified from antisera using protein A column chromatography (Pierce Biotechnology, Rockford, IL). For affinity purification, antisera were first passed through a GST column to remove antibodies against GST, and then purified through a GST-CCN3 (VWC domain)-affmity column. Anti-CCN3 antibodies did not cross react with CCNl or VN (data not shown) by ELISA.
  • CCN3 To examine the proangiogenic activities of CCN3, purified recombinant human CCN3 was tested for the ability to support endothelial cell adhesion.
  • Cell adhesion assays were performed essentially as described (7). Briefly, test proteins were diluted in PBS and coated onto 96-well microtiter plates (50 ⁇ l per well) with incubation at 4°C for 16 h. Wells were rinsed
  • affinity-purified anti- CCN3 antibodies or normal rabbit IgG was added to the wells and incubated for 1 h at 37° C prior to plating of cells.
  • HUVECs were cell cultured as described by the supplier (Cascade Biologies, Inc., Portland, OR) and used before passage 16. HUVECs were harvested in PBS containing 2.5 mM EDTA, washed and resuspended at 2.5 x 10 5 cells/ml in serum-free Iscove's modified Dulbecco's medium containing 1% BSA. Where indicated, cells were mixed with either EDTA, Ca 2+ , Mg 2+ , peptides, or heparin prior to plating or incubated with antibodies for 1 h at RT prior to plating. Cell suspension (50 ⁇ l) was added to each well, and adherent cells were fixed in 10%
  • HUVEC fibroblast adhesion through integrin ⁇ 6 ⁇ and heparin sulfate proteoglycans (8,20).
  • integrins Since integrin ⁇ v ⁇ 3 (Chemicon-Temecula, CA) is known to be inhibited by a
  • LM609 partially blocked HUVEC adhesion to CCN3 and VN but not to FN
  • integrin ⁇ 6 ⁇ cells were incubated with mAbs against either integrin ⁇ 6 (GoH3) (Beckman-Coulter, Inc.-Fullerton, CA) or ⁇ i (P4C10) (Invitrogen-Carlsbad,
  • Soluble heparin is known to block fibroblast adhesion to
  • CCNl and CCN2 by saturating the heparin binding sites located in the CT domain, thereby preventing them from binding cell surface heparin sulfate proteoglycans (8,20).
  • soluble heparin Sigma-Aldrich-St. Louis, MO
  • CCN3 may also engage heparin sulfate
  • proteoglycans as a co-receptor when interacting with integrin ⁇ 6 ⁇ .
  • CCN3 can bind integrin receptors directly using ELISA similar to as previously described.
  • microtiter wells Immulon 2, Dynatech Laboratories, Chantilly, VA
  • purified integrin 1 ⁇ g/ml
  • microtiter wells were coated with 10 ⁇ g/ml CCN3, 10 ⁇ g/ml FN
  • coated wells were pre-incubated with
  • soluble integrin was either mixed with EDTA, Mg 2+ , or peptides prior to plating, or incubated with function-blocking monoclonal antibodies for 30 min at 4°C prior to plating. After washing, bound integrins were detected with polyclonal
  • BACECs were harvested with trypsin, washed and resuspended at 5 x 10 5 cells/ml in DMEM containing 0.1 % BSA. Cells were loaded into wells of the lower chamber; the wells were then covered with a gelatinized polycarbonate filter (5 ⁇ m pore diameter, Nuclepore,
  • CCN3 was able to stimulate migration of BACECs (Fig. 6A). CCN3-induced migration
  • Stimulation of cell migration can be due to a chemotactic (directed cell movement) or a chemokinetic (random cell movement) response.
  • CCN3 was placed in the upper chamber (no cells), in the lower chamber (with cells), and in both or neither chambers (Fig. 6C).
  • Addition of CCN3 to the lower chamber did not enhance BACEC migration to the upper chamber, indicating that CCN3 did not induce a chemokinetic response.
  • Addition of CCN3 to the upper chamber induced the maximal level of migration, consistent with a chemotaxis.
  • Addition of CCN3 to both chambers reduced the level of BACEC migration, ' suggesting that BACECs are sensitive to a CCN3 gradient. Together, these results show that CCN3 induces directed endothelial cell migration.
  • CCN3 was shown to be a ligand for integrins ⁇ y ⁇ 3 and 5 ⁇ , both of which can
  • anti-integrin ⁇ 5 mAb SAM-1 (Beckman-Coulter, Inc.) partially inhibited CCN3 -stimulated cell migration but not VN- stimulated cell migration (Fig.
  • endothelial cells require survival signals in order to migrate
  • LN (ultrapure grade; Becton-Dickinson Biosciences, Bedford, MA) and blocked with 1% heat- inactivated BSA. Cells were plated on LN-coated coverslips at 10,000 cells/cm 2 and allowed to
  • CCN3 was incubated with anti-CCN3 antibodies for 1 h at RT prior to addition to cells. After incubation, cells were fixed with 4% paraformeldahyde (pH 7.4) and apoptosis was detected by TUNEL assay using the in situ cell death detection kit POD (Roche Applied Science, Indianapolis, IN). Cells were lightly stained with hematoxylin and apoptotic nuclei counted. A total of 500 cells were counted from random fields in each coverslip, and the number of apoptotic cells was represented as a percentage of the total cells counted. To assess proliferation, cells were grown as described above except that 10 ⁇ M bromodeoxyuridine (BrdUrd) was included in the medium for 16 h in
  • HUVECs were plated on laminin and maintained in serum-free medium. Under these conditions, endothelial cells were susceptible to apoptosis (44). GRGDSP peptide, 20% serum, or varying concentrations of CCN3 were then added to cells, and apoptosis was determined using a TUNEL assay after 16 hrs (Fig. 8A). Under these conditions, CCN3 was able to promote endothelial cell survival in a dose-dependent manner. Serum also protected cells from apoptosis, while GRGDSP peptide promoted apoptosis. Pre-incubation of CCN3 with affinity-purified anti-CCN3 antibodies abolished CCN3-promoted cell survival.
  • CCNS induces neovascularization in vivo.
  • the ability of CCN3 to promote endothelial cell adhesion, migration, and survival are consistent with properties of an angiogenic inducer.
  • CCN3 -induced neovascularization was examined in vivo by implanting Hydron pellets, formulated with test substances, into rat corneas essentially as described (16).
  • CCN3 was able to induce neovascularization when implanted into rat cornea, whereas the vehicle did not induce any response (Table 1).
  • Neovascularization was also observed in corneas implanted with Hydron pellets containing bFGF, a known potent angiogenic inducer.
  • Pre-incubation of CCN3 with anti-CCN3 antibodies obliterated CCN3-induced neovascularization, indicating that the angiogenic activities observed can be ascribed to the CCN3 polypeptide. Together, these results show that CCN3 can induce angiogenesis in vivo.
  • BACEC Bovine adrenal capillary endothelial cell
  • BSA bovine serum albumin
  • CCN cysteine-rich 61 /connective tissue growth factor/nephroblastoma overexpressed
  • DMEM Dulbecco's modified Eagle's medium
  • ECM extracellular matrix
  • ELISA enzyme-linked immunosorbent assay
  • FN fibronectin
  • GST glutathione-S-transferase
  • HUVEC human umbilical vein endothelial cell
  • IgG immunoglobulin G
  • LN laminin
  • mAb monoclonal antibody
  • PBS phosphate-buffered saline
  • RT room temperature
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • TUNEL in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling
  • VN vitro

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Abstract

L'invention concerne une nouvelle protéine matricellulaire, laCCN3 (Nov), de la famille CCN, comprenant CCN1 (CYR61), CCN2 (CTGF), CCN4 (WISP-1), CCNS (WISP-2), et CCN6 (WISP-3). Lors du développement, la CCN3 est exprimée de manière plus large en dérivés des trois couches de germes, et des niveaux élevés d'expression sont observés dans des cellules des muscles lisses de la paroi des vaisseaux artériels. L'expression modifiée de la CCN3 est observée dans une pluralité de tumeurs, notamment les carcinomes hépatocellulaires, les tumeurs de Wilm, les sarcomes d'Ewing, les gliomes, les rhabdomyosarcomes, et les carcinomes adrénocorticaux. Afin de comprendre ses fonctions biologiques, des expériences ont été menées concernant les activités de la CCN3 recombinée et purifiée. Dans des cellules endothéliales, la CCN3 supporte l'adhérence cellulaire, induit la migration cellulaire dirigée (chimiotaxie), et favorise la survie cellulaire. De manière mécaniste, la CCN3 supporte l'adhérence cellulaire endothéliale de la veine ombilicale humaine grâce à de nombreux récepteurs de surface cellulaires, notamment les intégrines αvß3, α5ß1, α6ß1, et les protéoglycanes à sulfate d'héparane. Par contraste, la migration cellulaire induite par la CCN3 dépend des intégrines αvß3 et α5ß1, la α6ß1 ne jouant pas de rôle dans ce processus. Bien que la CCN3 ne contienne pas de séquence RGD, elle se fixe directement sur des intégrines αvß3 et α5ß1, la moitié de la fixation maximale se produisant sur 10 nM et 50nM de la CCN3. De plus, la CCN3 induit une néovascularisation, lorsqu'elle est implantée dans la cornée du rat, ce qui montre que c'est un nouvel inducteur angiogénique. Toutes ces démonstrations prouvent que la CCN3 est un ligand des intégrines αvß3 et α5ß1, qu'elle agit directement sur les cellules endothéliales pour stimuler l'activité pro-angiogénique, et qu'elle induit l'angiogénèse in vivo.
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US7780949B2 (en) 2005-01-10 2010-08-24 Rosalind Franklin University Of Medicine And Science Regulation of CCN2 by CCN3 and its therapeutic and diagnostic potential in fibrosis, sclerosis and other diseases
DE602006021534D1 (de) * 2005-01-10 2011-06-09 Univ Rosalind Franklin Medicine & Science CCN3 Proteine zur Behandlung und Diagnose von Nierenerkrankungen
GB0618748D0 (en) * 2006-09-22 2006-11-01 Univ Belfast Peptide
DK2552462T3 (en) 2010-04-02 2016-02-08 Univ Rosalind Franklin Medicine & Science Ccn3 peptides and analogs thereof for therapeutic use
US9114112B2 (en) 2010-04-02 2015-08-25 Rosalind Franklin University Of Medicine And Science CCN3 and CCN3 peptides and analogs thereof for therapeutic uses
US10028906B2 (en) 2016-03-22 2018-07-24 Rosalind Franklin University Of Medicine And Science Method and kit for treating a solid tumor and associated desmoplasia

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GUPTA N ET AL: "INHIBITION OF GLIOMA CELL GROWTH AND TUMORIGENIC POTENTIAL BY CCN3 (NOV)" MP. MOLECULAR PATHOLOGY, BMJ PUBLISHING GROUP, LONDON, GB, vol. 54, no. 5, October 2001 (2001-10), pages 293-299, XP008010748 ISSN: 1366-8714 *
LAU L F ET AL: "The CCN family of angiogenic regulators: the integrin connection" EXPERIMENTAL CELL RESEARCH, SAN DIEGO, CA, US, vol. 248, 1999, pages 44-57, XP002272896 ISSN: 0014-4827 *
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SAKAMOTO K. ET AL.: "The nephroblastoma overexpressed gene (NOV/ccn3) protein associates with Notch1 extracellular domain and inhibits myoblast differenciation via Notch signaling pathway" THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 277, no. 33, 16 August 2002 (2002-08-16), pages 29399-29405, XP002377827 *
See also references of WO2004090109A2 *

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