WO2011024114A1 - Molécules de matrice extracellulaire de ciblage pour le traitement du cancer - Google Patents

Molécules de matrice extracellulaire de ciblage pour le traitement du cancer Download PDF

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Publication number
WO2011024114A1
WO2011024114A1 PCT/IB2010/053789 IB2010053789W WO2011024114A1 WO 2011024114 A1 WO2011024114 A1 WO 2011024114A1 IB 2010053789 W IB2010053789 W IB 2010053789W WO 2011024114 A1 WO2011024114 A1 WO 2011024114A1
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cancer
antibody
isolated
derivative
fragment
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PCT/IB2010/053789
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English (en)
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Joerg Huelsken
Ilaria Malanchi
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Ecole Polytechnique Federale De Lausanne (Epfl)
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    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to an agent able to block a cancer supportive function of a fasciclin protein family member and to pharmaceutical compositions containing said agent.
  • Stem cells possess the ability to self-renew for the life span of an organ giving rise to cycling (transit-amplifying) cells which in turn generate terminally differentiated cells.
  • This hierarchy of cells with differential potential for proliferation and specialization is at the root of tissue homeostasis and ensures life-long function of our tissues.
  • tissue specific stem cells which are at the root of this process of homeostasis can be an ideal target for oncogenic transformation (Malanchi et al., Nature. Apr 3;452(7187):650-3 (2008)).
  • cancerous stem cells also termed cancer stem cells, which maintain stem cell properties such as unlimited self-renewal and give rise to all other tumor cells. Since the majority of tumor cells is not able to initiate a new cancer upon transplantation this has introduced the concept of cellular hierarchies also to the tumor field and it has been postulated that this will change the way we will treat cancers in the future.
  • Tissue specific stem cells are associated with specialized niches which provide a defined cellular and extracellular microenvironment to control stem cell self-renewal and differentiation.
  • niche expressed genes In order to identify molecules involved in stem cell - niche interactions in vivo, Applicants have previously identified niche expressed genes using a combination of expression profiling of micro-dissected material and in situ hybridization. They identified genes which are expressed in the niche of normal, tissue specific stem cells, in the niche of cancer stem cells as well as in the metastatic niche at an early step of metastatic colonization.
  • Periostin also called OSF-2.
  • Periostin is expressed from stromal cells in primary mammary cancers and is strongly induced in spontaneous lung metastasis of such tumors where it is again produced by stromal cells.
  • POSTN Periostin
  • Various reports have been issued on high level expression of periostin in highly metastatic cancers [Erkan M. et al. Gastroenterology, 132(4), 1447-64 (2007) (pancreatic cancer), Siriwardena B S. et al. Br J Cancer, 95(10), 1396-403 (2006) (oral cancer), Baril P. et al. Oncogene, 26(14), 2082-94 (2007) (pancreatic cancer), Grigoriadis A. et al.
  • periostin antibodies As to periostin antibodies, there are reports of an antibody related to the inhibition of chemotaxis induced by periostin (Lindner V. et al., Arterioscler Thromb Vase Biol. (2005) 25, 77-83), an antibody against periostin inducing apoptosis in colon cancer cells in vitro at concentrations above lmg/ml (Tai I T, et al., Carcinogenesis (2005) 26, 908-15), as well as an antibody having the ability to neutralize anti-cell adhesive properties (US 2009/0074788).
  • none of these antibodies can block metastasis, cancer stem cell self-renewal or another cancer supportive function of a fasciclin protein family member such as periostin.
  • a further object of the invention is to provide a method for treating and/or preventing cancer, cancer metastasis, or tumorigenesis, in a patient in need thereof, comprising administering a pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof.
  • Another object of the invention is to provide a method for treating and/or preventing cancer cell self-renewal, expansion, proliferation, activation, survival, anchorage, niche interaction or homing to the niche in a patient in need thereof, comprising administering a pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof.
  • Still a further object of the present invention is to provide a method for treating and/or preventing cancer stem cell self-renewal, expansion, proliferation, activation, survival, anchorage, niche interaction or homing to the nichein a patient in need thereof, comprising administering a pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof.
  • the present invention also relates to a kit for treating and/or preventing cancer metastasis formation, tumorigenesis or cancer stem cell anchorage and survival comprising a pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof of any one of the invention, optionally with reagents and'or instractions
  • Fig. 1 shows POSTN expression in primary mammary cancer as assessed by in situ hybridization. Sections from POSTN wild type MMTV-PyMT tumors were processed for in situ hybridization using sense and anti-sense probes against POSTN messenger RNA. The sense probe was used as an internal negative control. POSTN is highly expressed in the tumor stroma.
  • Fig. 2 shows POSTN expression in pulmonary mammary cancer metastasis as assessed by in situ hybridization. Sections from POSTN wild type MMTV-PyMT lung metastasis was processed for in situ hybridization using sense and anti-sense probes against POSTN messenger RNA. The red dotted line defines the borders of the early or the late metastasis from the surrounding lung tissue. The expression of periostin is mainly observed within the metastasis stroma and not in the tumor cells themselves or in the distant, metastasis-free lung tissue, and POSTN expression increases in the macro-metastasis stage.
  • Fig. 3 is a picture showing examples of a lung harbouring macroscopic metastasis (from a control mouse) compared to a metastasis free lung (from a POSTN knock out mouse). While the absence of POSTN does not affect the growth of the tumor at the primary site, it efficiently prevents lung metastasis formation.
  • the pictures show examples of a lung harboring macroscopic metastasis (from a control mouse) compared to a metastasis free lung (from a POSTN knock out mouse).
  • Fig. 4 represents the size distribution of control and POSTN mutant primary tumors and incidence of lung metastases.
  • the graphic shows the incidence of macroscopic lung metastasis (black bar, right scale) in 5 control mice of the MMTV-PyMT mammary tumor model and 5 mice of the same model genetically deprived of the POSTN gene.
  • the lack of POSTN expression dramatically compromises the process of lung metastasis, while the overall growth of the primary tumor is not altered (line chart, left scale).
  • Fig. 5 is a graphic representing the size distribution of control and POSTN mutant pulmonary metastases. Histological sections of control and POSTN knock out mice were analysed for the presence of microscopic metastasis.
  • Fig. 6 is a graphic of a metastasis time course showing that the lack of POSTN compromises the transition from early colonization to metastatic progression and macro-metastasis formation. Tumor cells from POST -/- mice were injected intgravenously into either wild type control or POSTN -/- mice. Both, tumor cell amount and frequency of CSCs was severely reduced after 5 weeks in POSTN -/- recipient mice compared to controls while no significant difference was detected in the early colonization phase. This result proves that the CSC of survival, proliferation and self renewal were compromised in the absence of POSTN at the metastatic site.
  • Fig. 7 is a picture showing that POSTN deficient tumor cells do not form mammospheres and loose their long-term growth potential.
  • Cells from primary tumors from control and POSTN knock out mice were plated in 1 OO ⁇ l of serum- free sphere medium for mammosphere culture.
  • Fig. 8 is a picture showing that POSTN deficient tumor cells can be rescued to form mammospheres by addition of POSTN protein.
  • Spheres were generated from tree independent POSTN deficient tumors. The sphere cultures were grown in presence of the indicated amount (100 ng/ml or 500 ng/ml) of purified human POSTN which was sufficient to rescue sphere formation.
  • Fig. 9 shows a FACS analysis of harvested tumor cells incubated with the indicated antibodies. Tumor cells grown in suspension for 10 days were harvested, trypsinized and incubated with the indicated antibodies. The FACS analysis shows that the number of cancer stem cells(CD24+/CD90+) is reduced in the culture of POSTN deficient tumor cells (numbers show percentage of total tumor cells).
  • Fig. 10 shows the results of comparative test between antibodies of the invention and commercial antibodies.
  • Sphere cultures of wild type mammary cancer stem cells can be inhibited by monoclonal antibodies of the invention (d-f) however not by commercial antibodies against POSTN (b-c).
  • the indicated antibodies were added in equal amounts to the sphere culture media at the time of plating of control tumor cells into low adherent 96-well plates.
  • the present invention concerns an isolated and/or purified antibody, antibody fragment or derivative thereof able to block a cancer supportive function of a fasciclin protein family member, characterized in that said and/or purified antibody, antibody fragment or derivative thereof recognizes a protein consisting essentially in SEQ ID No 1, a fragment thereof and/or a conservative variant thereof .
  • phrases "consisting essentially in” when referring to a particular amino acid means a sequence having the properties of a given SEQ ID NO.
  • the phrase when used in reference to an amino acid sequence, the phrase includes the sequence per sc and molecular modifications that would not affect the essential characteristics of the sequence.
  • the term "patient” is well-recognized in the art, and, is used herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.
  • a cancer supportive function of a fasciclin protein family member refers to a function or effect mediated/caused by a fasciclin family member such as POSTN which is necessary/essential for cancer progression or metastasis. This function or effect is characterized in that in the absence of said protein or after block of said protein by an an isolated and/or purified antibody, antibody fragment or derivative thereof defined in this patent application, tumor growth is deteriorated and tumor metastasis declines.
  • the isolated and/or purified antibody, antibody fragment or derivative thereof of the invention is able to block a cancer supportive function of a fasciclin protein family member.
  • Members of the fasciclin family of proteins occur in a wide range of vertebrates, invertebrates and microorganisms. They are generally cell-surface and membrane- anchored proteins involved in homophilic cell adhesion or symbiotic processes.
  • the member of the fasciclin family is a periostin protein.
  • This protein is expressed from stromal cells in primary mammary cancers and is strongly induced in spontaneous lung metastasis of such tumors where it is again produced by stromal cells.
  • Various reports have been issued on high level expression of periostin in highly metastatic cancers. It has also been reported that a rat homo log of mouse periostin was less expressed in various cancer cells, introduction of the periostin gene into bladder cancer cells inhibited invasion of the bladder cancer cells, and introduction of the periostin gene into mouse melanoma Bl 6-F 10 cells inhibited their metastasis to lung (Kim C J, et al. Int J Cancer, 117(1), 51-8 (2005)).
  • the amino acid sequence recognized by the isolated and/or purified antibody, antibody fragment or derivative thereof of the invention is a protein consisting essentially in SEQ ID No 1, a fragment thereof and/or a conservative variant thereof as described below:
  • This sequence corresponds to a full size human POSTN protein lacking exon 17.
  • the present invention also refers to a fragment of the SEQ ID No 1 disclosed above.
  • This refers to a sequence containing less amino acids in length than the SEQ ID No 1 sequence of the periostin protein.
  • This fragment sequence is a biologically active fragment that can be used as long as it exhibits the same properties as the sequence from which it derives.
  • this sequence contains less than 90%, preferably less than 60%, in particular less than 30% amino acids in length than the respective sequence of the periostin protein.
  • the present invention further refers to a conservative variant of the SEQ ID No 1 disclosed above.
  • This conservative variant refers to polypeptides having amino acid sequences that differ to some extent from the native sequence polypeptide, that is amino acid sequences that vary from the native 3D sequence whereby one or more amino acids are substituted by another one.
  • the variants can occur naturally (e.g. polymorphism) or can be synthesized.
  • Variants possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence. Amino acid substitutions are herein defined as exchanges within one of the following five groups:
  • amino acid substitutions are conservative, i.e. occur within one of the above- identified group.
  • the blocking activity of the agent of the invention concerns a cancer cell or a cancer stem cell.
  • a cancer stem cell refers to a cell which is able to initiate tumor formation at the primary or secondary site and is responsible for long-term tumor growth.
  • an "antibody” is a protein molecule that reacts with a specific antigenic determinant or epitope and belongs to one or five distinct classes based on structural properties: IgA, IgD, IgE, IgG and IgM.
  • the antibody may be a polyclonal (e.g. a polyclonal serum) or a monoclonal antibody, including but not limited to fully assembled antibody, single chain antibody, antibody fragment, and chimeric antibody, humanized antibody as long as these molecules are still biologically active and still bind to at least one peptide of the invention.
  • the antibody is a monoclonal antibody.
  • the monoclonal antibody will be selected from the group comprising the IgGl, IgG2, IgG2a, IgG2b, IgG3 and IgG4.
  • a typical antibody is composed of two immunoglobulin (Ig) heavy chains and two Ig light chains.
  • Ig immunoglobulin
  • Each light chain is composed of two tandem immunoglobulin domains: one constant (CL) domain and one variable domain (VL) that is important for binding antigen.
  • isolated when used as a modifier of an antibody of the invention means that the antibody is made by the hand of man or is separated, completely or at least in part, from their naturally occurring in vivo environment Generally, isolated antibodies are substantially free of one or more materials with which they normally associate with in nature, for example, one or more protein.
  • isolated does not exclude alternative physical forms of the antibodies, such as multimers/oligomers, modifications (e g , phosphorylation, glycosylation, lipidation) or derivatized forms, or forms expressed in host cells produced by the hand of man
  • an “isolated” antibody can also be “substantially pure” or “purified” when free of most or all of the materials with which it typically associates with in nature
  • an isolated antibody that also is substantially pure or purified does not include polypeptides or polynucleotides present among millions of other sequences, such as antibodies of an antibody library or nucleic acids in a genomic or cDNA library.
  • CDR complementary determining region
  • the isolated and/or purified antibody, antibody fragment or derivative thereof of the invention comprises i) at least one complementary determining region (CDR) of the VH, a biologically active fragment thereof and/or a variant thereof, and/or ii) at least one complementary determining region (CDR) of the VL region, a biologically active fragment thereof and/or a variant thereof.
  • CDR complementary determining region
  • the amino acid sequence determining the least one CDR of the VH consists essentially in SEQ ID No 2, a biologically active fragment thereof and/or a variant thereof.
  • the amino acid sequence determining the at least one CDR of the VL region consists essentially in SEQ ID No 3, a biologically active fragment thereof and/or a variant thereof.
  • the sequences define an IgGl isotype:
  • the constant region starts with TTPPSV.
  • X 1 is N or S
  • X 2 is V or Y
  • X 3 is D or Y
  • X 4 is S or N
  • X 5 is H or Y
  • X 6 is G or L
  • X 7 is F or A
  • X is S or absent
  • X is F or absent
  • X is M or absent
  • X is N or absent
  • X is A or S
  • X is S or T
  • X 14 is L or Q
  • X 15 is Q or A
  • X 16 is S or D
  • X 17 is Q or H
  • X 18 is F or S
  • X 19 is W or K
  • the constant region starts with DAAPTV.
  • the present invention also refers to a biologically active fragment of the SEQ ID No 2 disclosed above.
  • This biologically active fragment can be used as long as it exhibits the same properties as the sequence from which it derives.
  • this sequence contains less than 90%, preferably less than 60%, in particular less than 30% amino acids in length than the respective sequence of CDR of the VH.
  • the biologically active fragment will be selected from the group comprising SEQ ID N° 4 (GYTFTNYPMH), SEQ ID N° 5 (TYSGAPTYADDFKG) and SEQ ID N° 6
  • a variant of the SEQ ID No 2 disclosed above is a conservative variant and refers to polypeptides having amino acid sequences that differ to some extent from the native sequence polypeptide, i.e. amino acid sequences that vary from the native 3D sequence whereby one or more amino acids are substituted by another one.
  • the variants can occur naturally (e.g.
  • variants possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence as disclosed supra.
  • the present invention also refers to a biologically active fragment of the SEQ ID No 3 disclosed above.
  • This refers to a sequence containing less amino acids in length than the SEQ ID No 3 sequence of the periostin protein.
  • This fragment sequence can be used as long as it exhibits the same properties as the sequence from which it derives.
  • this sequence contains less than 90%, preferably less than 60%, in particular less than 30% amino acids in length than the respective sequence of CDR of the VL.
  • the biologically active fragment will be selected from the group comprising SEQ ID N° 10 (RASEX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 ), SEQ ID N° 11 (X 12 A X 13 N X 14 X 15 X 16 ) and SEQ ID N° 12 (X 17 X 18 X 19 X 20 X 21 PYT), wherein, independently from one each other, X 1 is N or S, X 2 is V or Y, X 3 is D or Y, X 4 is S or N, X 5 is H or Y, X 6 is G or L, X 7 is F or A, X 8 is S or absent, X 9 is F or absent, X 10 is M or absent, X 11 is N or absent, X 12 is A or S, X 13 is S or T, X 14 is L or Q, X 15 is Q or A, X 16 is S or D, X 17 is
  • the biologically active fragment is selected from the group comprising SEQ ID N° 7 (RASENVYSHLA), SEQ ID N° 8 (SATNLAD) and SEQ ID N° 9 (QHFWGTPYT).
  • SEQ ID N° 7 RASENVYSHLA
  • SEQ ID N° 8 SATNLAD
  • SEQ ID N° 9 QHFWGTPYT
  • a variant of the SEQ ID No 3 disclosed above is a conservative variant and refers to polypeptides having amino acid sequences that differ to some extent from the native sequence polypeptide, i.e. amino acid sequences that vary from the native 3D sequence whereby one or more amino acids are substituted by another one.
  • the variants can occur naturally (e.g.
  • variants possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence as disclosed supra.
  • the isolated and/or purified antibody, antibody fragment or derivative thereof of the invention recognizes a protein consisting essentially in SEQ ID No 1, a fragment thereof and/or a conservative variant thereof.
  • bind or “recognize”, used interchangeably herein, it is generally meant that a binding molecule, e g , an antibody binds to an epitope via its antigen- binding domain, and that the binding entails some
  • the isolated and/or purified antibody may be a polyclonal (e.g. a polyclonal serum) or a monoclonal antibody.
  • the isolated and/or purified antibody is a monoclonal antibody.
  • Antibodies used in the present invention are not limited to whole antibody molecules and may be antibody fragments or derivatives as long as they can block the cancer supportive function of a fasciclin protein family member such as periostin and that they recognize or bind to a protein consisting essentially in SEQ ID No 1, a fragment thereof and/or a conservative variant thereof.
  • Examples of isolated and/or purified antibody fragment or derivative thereof are selected amongst the group comprising a Fab-fragment, a F(ab2)'-fragment, a single-chain antibody, a chimeric antibody, a CDR-grafted antibody, a bivalent antibody-construct, a humanized antibody, a synthetic antibody, a chemically modified derivative thereof, a multispecific antibody, a diabody, a scFv- fragment; a dsFv-fragment, a labeled antibody, or another type of recombinant antibody.
  • an antibody fragment is synthesized by treating the antibody with an enzyme such as papain or pepsin, or genes encoding these antibody fragments are constructed, and expressed by appropriate host cells as known to the skilled artisan.
  • Fragments or derivatives of the above antibodies which are able to block a cancer supportive function of a fasciclin protein family member can be obtained by using methods which are described, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988.
  • surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of EAGl
  • CDR-grafted antibody refers to an antibody in which the CDR from one antibody, or a biologically active fragment of said CDR, is inserted into the framework of another antibody.
  • the antibody from which the CDR is derived and the antibody from which the framework is derived are of different species.
  • the antibody from which the CDR is derived and the antibody from which the framework is derived are of different isotypes.
  • Hie term ' 'humanized antibody ' ' refers to an antibody in which all or part of an antibody frame woik region is derived fiom a human, but all oi part of one or rnoie CDR regions is derived from another species, for example, including, but not limiieJ to, a m ⁇ use.
  • humanized antibody rctets to antibodies in which the framework or
  • CDR complementarity determining regions
  • the second heavy and the second light chain (originating from an antibody against a second antigen ) are specifically binding together to a second antigen;
  • bivalent, bispecific antibodies aie capable of specifically binding to two different antigens at the same rime, and not to more than two antigens, in contrary to, on the one hand a monospecific antibody capable of binding only UJ one antigen, and on the oilier hand a.g, a tetravalcnL tctraspcciflc antibody which can bind to four antigen at the same time
  • Fab, F(ab')2 and the like can be obtained by treating an antibody inhibiting the anti-cell adhesive activity of periostin with a proteolytic enzyme such as papain or pepsin, or alternatively, can be prepared by constructing a gene encoding the resulting antibody fragment and introducing this construct into an expression vector, followed by expression in an appropriate host cell.
  • a proteolytic enzyme such as papain or pepsin
  • recombinant antibody is intended to include all antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell (such as a NSO or CHO cell) or from an animal (e.g. a mouse) that is transgenic for, for example, human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell.
  • a host cell such as a NSO or CHO cell
  • animal e.g. a mouse
  • Such recombinant antibodies have variable and constant regions in a rearranged form.
  • the "diabody” refers to an antibody produced in accordance with the technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993). These authors have provided an alternative mechanism for making bispecifte antibody fragments.
  • the fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
  • VH heavy-chain variable domain
  • VL light-chain variable domain
  • Another strategy for making bispecifte antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).
  • Antibodies with more than two valencies are also contemplated.
  • multispecif ⁇ c antibodies such as trispecific antibodies can be prepared as known to the skilled artisan.
  • single chain Fv can be prepared by linking together an H chain V region and an L chain V region from an antibody blocking a cancer supportive function of a fasciclin protein family member by using an appropriate peptide linker or the like.
  • scFv can be prepared by constructing a DNA segment encoding the entire sequences or desired amino acid sequences of a gene encoding an H chain or H chain V region from the above antibody and a gene encoding an L chain or L chain V region from the antibody, and introducing this construct into an expression vector, followed by expression in an appropriate host cell.
  • disulfide-stabilized antibody is an antibody fragment in which polypeptides modified to replace one amino acid residue by a cysteine residue in both H and L chain V regions from an antibody blocking a cancer supportive function of a fasciclin protein family member are linked together between these cysteine residues via a disulfide linkage.
  • An amino acid residue to be replaced by a cysteine residue can be selected by stereostructural estimation of the antibody.
  • dsFv can be prepared by constructing a DNA segment encoding the entire sequence or a desired amino acid sequence of a gene encoding the antibody fragment, and introducing this construct into an expression vector, followed by expression in an appropriate host cell.
  • a CDR-containing peptide comprises at least one or more CDR regions selected from CDR regions in H or L chains of an antibody inhibiting the anti-cell adhesive activity of periostin. Also, multiple CDR regions may be linked together by techniques using an appropriate peptide linker or the like.
  • the CDR-containing peptide may also be prepared by constructing a DNA segment encoding the entire sequence or a desired amino acid sequence of a gene encoding the peptide, and introducing this construct into an expression vector, followed by expression in an appropriate host cell.
  • the CDR-containing peptide can also be prepared by chemical synthesis such as Fmoc or tBoc method.
  • chimeric antibodies are described, for example, in W089/09622.
  • Methods for the production of humanized antibodies are described in, e.g., EP-Al 0 239 400 and W090/07861.
  • a further source of antibodies to be utilized in accordance with the present invention are so-called xenogenic antibodies.
  • the general principle for the production of xenogenic antibodies such as human antibodies in mice is described in, e.g., WO 91/10741, WO 94/02602, WO 96/34096 and WO 96/33735.
  • the antibody of the invention may exist in a variety of forms besides complete antibodies; including, for example, Fv, Fab and F(ab)2, as well as in single chains; see e.g. W088/09344.
  • One additional aspect of the present invention is to provide a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof able to block a cancer supportive function of a fasciclin protein family member as disclosed.
  • the pharmaceutical agent can be in a variety of well known formulations and administered using any of a variety of well known methods of administration such as intra-nasal, oral, subcutaneous, intravenous, intraarterial, intraperitoneal and/or intramuscular are also contemplated.or the like.
  • compositions adapted for nasal administration wherein the pharmaceutically acceptable carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, e.g, by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • suitable compositions wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.
  • compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators.
  • the formulation may contain suspending agents, as for example, ethoxylated isostearyl alcohols,
  • polyoxyethylene sorbitol and sorbitan esters polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, among others.
  • Useful intranasal formulations of an alarm pheromone may contain at least one stabilizer and surfactant.
  • the pharmaceutically acceptable surfactants are sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite
  • polyoxyethylene castor oil derivatives such as polyoxyethylene-glycerol-triricinoleate, also known as polyoxyl 35 caster oil (CREMOPHOR EL), or poloxyl 40 hydrogenated castor oil (CREMOPHOR RH40) both available from BASF Corp.
  • mono-fatty acid esters of polyoxyethylene (20) sorbitan such as polyoxyethylene (20) sorbitan monolaurate (TWEEN 80), polyoxyethylene monostearate (TWEEN 60), polyoxyethylene (20) sorbitan
  • TWEEN 40 polyoxyethylene 20 sorbitan monolaurate
  • TWEEN 20 polyoxyethylene 20 sorbitan monolaurate
  • polyglyceryl esters such as polyglyceryl oleate
  • LABRAFIL polyoxyethylated kernel oil
  • the surfactant will be between about 0.01% and 10% by weight of the
  • antioxidants such as sodium sulfite, sodium metabisulfite, sodium thiosulfate, sodium formaldehyde sulfoxylate, sulfur dioxide, ascorbic acid, isoascorbic acid, thioglycerol, thioglycolic acid, cysteine hydrochloride, acetyl cysteine, ascorbyl palmitate, hydroquinone, propyl gallate, nordihydroguaiaretic acid, butylated hydroxytoluene, butylated hydroxyanisole, alpha- tocopherol and lecithin.
  • the stabilizer will be between about 0.01% and 5% by weight of the pharmaceutical composition.
  • Suspensions may also include chelating agents such as ethylene diamine tetraacetic acid, its derivatives and salts thereof, dihydroxyethyl glycine, citric acid and tartaric acid among others. Additionally, proper fluidity of a suspension can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants, such as those previously mentioned.
  • chelating agents such as ethylene diamine tetraacetic acid, its derivatives and salts thereof, dihydroxyethyl glycine, citric acid and tartaric acid among others.
  • proper fluidity of a suspension can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants, such as those previously mentioned.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the pharmaceutically effective amount of an agent of the invention may be mixed with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as
  • the dosage form may also comprise buffering agents.
  • compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • solid dosage forms of tablets, capsules, pills and granules can be prepared with coatings and shells such as enteric coating and other coatings well-known in the
  • compositions may optionally contain opacifying agents and may also be of a composition such that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
  • opacifying agents include polymeric substances and waxes.
  • Liquid dosage forms for oral administration or for spray formulation include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof.
  • inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifier
  • This invention also envisages the use of an isolated and/or purified antibody, antibody fragment or derivative thereof of the invention in a pharmaceutically acceptable salt form.
  • salts may include sodium, potassium, calcium, aluminum, gold and silver salts.
  • salts formed with pharmaceutically acceptable amines such as ammonia, alkyl amines, hydroxyalkylamines, N-methylglucamine and the like.
  • Certain basic compounds also form pharmaceutically acceptable salts, e.g., acid addition salts.
  • pyrido-nitrogen atoms may form salts with strong acid, while compounds having basic substituents such as amino groups also form salts with weaker acids.
  • acids for salt formation are hydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicylic, malic, fumaric, succinic, ascorbic, maleic, pamoic, methanesulfonic and other mineral and carboxylic acids well known to those skilled in the art.
  • the salts are prepared by contacting the free base form with a sufficient amount of the desired acid to produce a salt in the conventional manner.
  • the free base forms may be regenerated by treating the salt with a suitable dilute aqueous base solution such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium bicarbonate.
  • a suitable dilute aqueous base solution such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium bicarbonate.
  • the free base forms differ from their respective salt forms somewhat in certain physical properties, such as solubility in polar solvents, but the acid and base salts are otherwise equivalent to their respective free base forms for purposes of the invention.
  • composition comprising an isolated and/or purified antibody, antibody fragment or derivative thereof of the invention, as described herein, as an active ingredient may also be incorporated or impregnated into a bioabsorbable matrix, with the matrix being administered in the form of a suspension of matrix, a gel or a solid support.
  • the matrix may be comprised of a biopolymer.
  • Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semi permeable matrices of solid hydrophobic polymers containing a pharmaceutically effective amount of an agent of the invention, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and [gamma] ethyl-L-glutamate non-degradable ethylene -vinyl acetate
  • degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT(TM) (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3- hydroxybutyric acid.
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished for example by filtration through sterile filtration membranes.
  • a pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof of the present invention will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any and the nature of the effect desired.
  • the appropriate dosage form will depend on the disease, the isolated and/or purified antibody, antibody fragment or derivative thereof of the invention, and the mode of administration.
  • the pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof of the invention is present in an amount between about 0.001% and 20% by weight of the pharmaceutical composition.
  • an alternative pharmaceutical composition may contain an isolated and/or purified nucleic acid sequence encoding said isolated and/or purified antibody, antibody fragment or derivative thereof, as described herein, as an active ingredient.
  • This pharmaceutical composition may include either the sole isolated and/or purified DNA sequence, an expression vector comprising said isolated and/or purified DNA sequence or a host cell previously transfected or transformed with an expression vector described herein.
  • host cell will preferably be isolated from the patient to be treated in order to avoid any antigenicity problem.
  • gene and cell therapy approaches are especially well suited for patients requiring repeated administration of the pharmaceutical composition, since the said purified and/or isolated DNA sequence, expression vector or host cell previously transfected or transformed with an expression vector can be incorporated into the patient's cell which will then produce the protein endogenously.
  • compositions of the invention are preferably for the treatment or prevention of cancer, tumorigenesis or metastasis formation.
  • Cancer is an unregulated proliferation of cells due to loss of normal controls, resulting in unregulated growth, lack of differentiation, local tissue invasion, and, often, metastasis. Cancer can develop in any tissue or organ at any age. There is often evidence of an immune response to tumors, but the role of the immune system in preventing and treating cancer is still uncertain.
  • the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, liposarcoma, neuroendocrine tumor, mesothelioma, schwanoma, meningioma, adenocarcinoma, melanoma, leukemia, lymphoid malignancy, squamous cell cancer, epithelial squamous cell cancer, lung cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid
  • Metastasis is the spread of a malignant tumor cells from one organ or part to another non-adjacent organ or part. Cancer cells can "break away”, “leak”, or “spill” from a primary tumor, enter lymphatic and blood vessels, circulate through the bloodstream, and settle down to grow within normal tissues elsewhere in the body. Metastasis is one of three hallmarks of malignancy (contrast benign tumors). Most tumors and cancers, in particular those listed above and other neoplasms can metastasize, although in varying degrees (e.g., glioma and basal cell carcinoma rarely metastasize). When tumor cells metastasize, the new tumor is called a secondary or metastatic tumor.
  • the present invention further contemplates an isolated and/or purified nucleic acid sequence comprising
  • nucleotide sequence encoding an isolated and/or purified antibody, an antibody fragment or derivative of said antibody of the invention
  • nucleic acid sequence having substantial sequence identity or homology to a nucleic acid sequence encoding an isolated and/or purified antibody, an antibody fragment of or derivative of said antibody of the invention
  • nucleic acid sequence capable of hybridizing under stringent conditions to i), ii), iii) or iv).
  • "An isolated and/or purified nucleic acid sequence” refers to nucleic acid free or substantially free of material with which it is naturally associated such as other polypeptides or nucleic acids with which it is found in its natural environment, or the environment in which it is prepared (e. g. cell culture) when such preparation is by recombinant nucleic acid technology practised in vitro or in vivo.
  • nucleic acid is intended to refer either to DNA or to RNA.
  • DNA which can be used herein is any polydeoxynuclotide sequence, including, e.g. double-stranded DNA, single-stranded DNA, double-stranded DNA wherein one or both strands are composed of two or more fragments, double-stranded DNA wherein one or both strands have an uninterrupted phosphodiester backbone, DNA containing one or more single-stranded portion(s) and one or more double- stranded portion(s), double-stranded DNA wherein the DNA strands are fully complementary, double-stranded DNA wherein the DNA strands are only partially complementary, circular DNA, covalently- closed DNA, linear DNA, covalently cross-linked DNA, cDNA, chemically- synthesized DNA, semi-synthetic DNA, biosynthetic DNA, naturally-isolated DNA, enzyme-digested DNA, sheared DNA, labeled DNA, such as radiolabeled DNA and fluorochrome-labeled DNA, DNA containing
  • DNA sequences that encode the isolated and/or purified antibody, an antibody fragment of or derivative of said antibody of the invention, or a biologically active fragment thereof can be synthesized by standard chemical techniques, for example, the phosphotriester method or via automated synthesis methods and PCR methods.
  • the purified and/or isolated DNA sequence encoding an isolated and/or purified antibody, an antibody fragment of or derivative of said antibody of the invention according to the invention may also be produced by enzymatic techniques.
  • restriction enzymes which cleave nucleic acid molecules at predefined recognition sequences can be used to isolate nucleic acid sequences from larger nucleic acid molecules containing the nucleic acid sequence, such as DNA (or RNA) that codes for the isolated and/or purified antibody, an antibody fragment of or derivative of said antibody of the invention or for a fragment thereof.
  • RNA polyribonucleotide
  • RNA RNA
  • RNA polyribonucleotide
  • RNA including, e.g., single- stranded RNA, double- stranded RNA, double-stranded RNA wherein one or both strands are composed of two or more fragments, double-stranded RNA wherein one or both strands have an uninterrupted phosphodiester backbone, RNA containing one or more single-stranded portion(s) and one or more double- stranded portion(s), double- stranded RNA wherein the RNA strands are fully complementary, double-stranded RNA wherein the RNA strands are only partially complementary, covalently crosslmked RNA, enzyme-digested RNA, sheared RNA, mRNA, chemically-synthesized
  • RNA RNA, semi-synthetic RNA, biosynthetic RNA, naturally-isolated RNA, labeled RNA, such as radiolabeled RNA and fluorochrome-labeled RNA, RNA containing one or more non- naturally- occurring species of nucleic acid.
  • the isolated and purified nucleic acid sequence, DNA or RNA also comprises an isolated and/or purified nucleic acid sequence having substantial sequence identity or homology to a nucleic acid sequence encoding an isolated and/or purified antibody, an antibody fragment of or derivative of said antibody of the invention.
  • the nucleic acid will have substantial sequence identity for example at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% nucleic acid identity; more preferably 90% nucleic acid identity; and most preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity.
  • Identity as known in the art and used herein, is a relationship between two or more amino acid sequences or two or more nucleic acid sequences, as determined by comparing the sequences. It also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. Identity and similarity are well known terms to skilled artisans and they can be calculated by conventional methods (for example see Computational Molecular Biology, Lesk, A. M. ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W. ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M. and Griffin, H. G.
  • Methods which are designed to give the largest match between the sequences are generally preferred. Methods to determine identity and similarity are codified in publicly available computer programs including the GCG program package (Devereux J. et al., Nucleic Acids Research 12(1): 387, 1984); BLASTP, BLASTN, and FASTA (Atschul, S. F. et al. J. Molec. Biol. 215: 403-410, 1990).
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al. NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al. J. MoI. Biol. 215: 403-410, 1990).
  • nucleic acid sequence complementary to the isolated and purified nucleic acid sequence encoding an isolated and/or purified antibody, an antibody fragment of or derivative of said antibody of the invention is also encompassed by the present invention.
  • a degenerated nucleic acid sequence having a sequence which differs from a nucleic acid sequence encoding the isolated and/or purified antibody, an antibody fragment or derivative of said antibody of the invention, or a derivative or complementary sequence thereof, due to degeneracy in the genetic code.
  • nucleic acid encodes functionally equivalent isolated and/or purified antibody, antibody fragment or derivative of said antibody of the invention but differs in sequence from the sequence due to degeneracy in the genetic code. This may result in silent mutations which do not affect the amino acid sequence.
  • nucleic acid sequence capable of hybridizing under stringent conditions, preferably high stringency conditions, to a nucleic acid sequence encoding an isolated and/or purified antibody, an antibody fragment of or derivative of said antibody of the invention, a nucleic acid sequence complementary thereof or a degenerated nucleic acid sequence thereof.
  • Appropriate stringency conditions which promote DNA hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • the stringency may be selected based on the conditions used in the wash step.
  • the salt concentration in the wash step can be selected from a high stringency of about 0.2XSSC at 50 0 C.
  • the temperature in the wash step can be at high stringency conditions, at about 65° C.
  • the present invention also includes an isolated and/or purified nucleic acid encoding an antibody, an antibody fragment or derivative of said antibody of the invention comprising a nucleic acid sequence encoding a truncation or an analog of the antibody, antibody fragment or derivative of said antibody of the invention.
  • truncation refers to a sequence encoding a peptide containing less amino acid than the native but exhibiting the same properties.
  • the invention also encompasses allelic variants of the disclosed isolated and/or purified nucleic sequence; that is, naturally-occurring alternative forms of the isolated and/or purified nucleic acid that also encode peptides that are identical, homologous or related to that encoded by the isolated and/or purified nucleic sequences.
  • allelic variants may be produced by mutagenesis techniques or by direct synthesis.
  • a biologically active fragment of the disclosed isolated and/or purified nucleic sequence is also considered and refers to a sequence containing less nucleotides in length than the nucleic acid sequence encoding an isolated and/or purified antibody, an antibody fragment of or derivative of said antibody of the invention, a nucleic acid sequence complementary thereof or a degenerated nucleic acid sequence thereof.
  • This sequence can be used as long as it exhibits the same properties as the native sequence from which it derives.
  • this sequence contains less than 90%, preferably less than 60%, in particular less than 30% amino acids in length than the respective isolated and/or purified nucleic sequence of the antibody, antibody fragment or derivative of said antibody of the invention.
  • Yet another concern of the present invention is to provide an expression vector comprising the isolated and/or purified nucleic acid sequence encoding an isolated and/or purified antibody, an antibody fragment or derivative of said antibody of the invention.
  • the choice of an expression vector depends directly, as it is well known in the art, on the functional properties desired, e.g., an isolated and/or purified antibody, an antibody fragment of or derivative of said antibody of the invention expression and the host cell to be transformed or transfected.
  • a further concerned of the present invention is to provide a host cell comprising the expression vector of the invention.
  • the host cell is a bacterium, a fungal, a plant, or an animal cell.
  • the animal cell is a mammalian cell and most preferably a human cell or a human cell line.
  • the present invention also provides one or more hybridoma secreting the monoclonal antibody of the invention.
  • hybridomas termed ID8, IVB2, IF6, IIIH9, IC12, IIB4 produce IgGl antibodies which bind to human and murine periostin and block its tumor supportive function.
  • monoclonal antibodies can be prepared using a wide variety of techniques known in the ail including ihe use of hybridoma, recombinant, and phage display technologies, or a combination thereof, for example, monoclonal antibodies can be produced hybridoma techniques including those known in the an and taught, for example, in Harlow ct ai., ⁇ ndbodies: ⁇ Laboratory Manual. (Cold Spring Harbor Laboratory Press, 2nd ed.. 1988); Hammer -ling, et ai, in: Monoclonal Antibodies and T-CeII Hybrido-mas 563- 681 (Elsevier, N. Y., J 981 ) (said references incorporated by reference in their entireties).
  • ''monoclonal antibody as used herein is not limited to antibodies produced through hybridoma technology.
  • the term "monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryoiic. prukaryutic, or phage done, and not the method by which it is produced. Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art.
  • Also encompassed in the present invention is a method for treating and/or preventing cancer, cancer metastasis, or tumorigenesis, in a patient in need thereof, comprising administering a pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof of the invention.
  • a method for treating and/or preventing cancer cell self-renewal, expansion, proliferation, activation, survival, anchorage, niche interaction or homing to the niche in a patient in need thereof comprising administering a pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof of the invention.
  • a method for treating and/or preventing cancer stem cell self-renewal, expansion, proliferation, activation, survival, anchorage, niche interaction or homing to the niche in a patient in need thereof comprising administering a pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof of the invention.
  • the isolated and/or purified antibody, antibody fragment or derivative thereof of the invention to be used in the above-mentioned methods for treating and/or or preventing the claimed diseases or conditions can be in a variety of well known formulations, as described infra, and administered using any of a variety of well known methods of administration such as intra-nasal, oral, subcutaneous, intravenous, intraarterial, intraperitoneal and/or intramuscular are also contemplated or the like.
  • the isolated and/or purified antibody, antibody fragment or derivative thereof of the invention is in the form of a pharmaceutical composition as described herein.
  • administering means “giving” or “contacting” and refers to contact of a pharmaceutical composition or a therapeutical composition to a patient in need thereof, preferably a human.
  • a pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof of the present invention will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any and the nature of the effect desired.
  • the appropriate dosage form will depend on the disease, the isolated and/or purified antibody, antibody fragment or derivative thereof of the invention, and the mode of administration. Usually, the pharmaceutically effective amount of an agent of the invention is present in an amount between about 0.001% and 20% by weight of the pharmaceutical composition. In one embodiment, the isolated and/or purified antibody, antibody fragment or derivative thereof blocking the cancer supportive function binds or targets a fasciclin protein family.
  • the isolated and/or purified antibody, antibody fragment or derivative thereof of the invention binds or targets at least one protein interacting with a fasciclin protein family.
  • the isolated and/or purified antibody, antibody fragment or derivative thereof blocking the cancer supportive function alters the structure, distribution or concentration of said fasciclin protein or of said at least one protein interacting with said fasciclin protein.
  • the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, liposarcoma, neuroendocrine tumor, mesothelioma, schwanoma, meningioma, adenocarcinoma, melanoma, leukemia, lymphoid malignancy, squamous cell cancer, epithelial squamous cell cancer, lung cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer
  • the present invention further relates to a kit useful for treating and/or preventing cancer metastasis formation, tumorigenesis or cancer stem cell anchorage and survival comprising a pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof blocking the cancer supportive function of the invention, optionally with reagents ancl'o ⁇ instructions for use.
  • kits for treating and/or preventing cancer metastasis formation, tumorigenesis or cancer stem cell anchorage and survival comprising a pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof of the invention, optionally with reagents ai ⁇ l/ ⁇ 3 instructions ibi use.
  • the POSTN knock out was generated by homologous recombination in 12901a ES cells inserting a neo cassette to disrupt the coding sequence and mice were produced by blastocyst injection. Afterwards the line was back-crossed onto a FVB background.
  • the murine tumor model used for this study expresses oncogenic human Polyoma Middle T- (PyMT) antigen under the control of the Mouse Mammary Tumor Virus (MMTV) promoter to allow the specific expression of the oncogene in the mammary gland.
  • Mice develop mammary gland tumors starting from the 6 th week of life. The tumors are metastasising to the lung after about 2-3 month.
  • mice of the genotype MMTV-PyMT and POSTN wild type or knock out were sacrificed when the primary tumor exceeded a defined size. Primary tumors were harvested and weighted. The lung was dissected and analysed for the presence of visible metastasis and visible metastases termed "macroscopic" were counted.
  • OOrrtthhoottooppiicc ttrraannssppllaannttaattiioonnss were performed by injecting 10 6 tumor cells from MMTV-PyMT and POSTN wild type or knock out tumors in serum reduced Matrigel into the mammary fat pad of immuno-compromized mice.
  • 10 6 tumor cells from GFP+, MMTV-PyMT and POSTN wild type or knock out tumors in lOO ⁇ l PBS were used.
  • Tumors and lungs were harvested, fixed in Bouin's (picric acid, formaldehyde, acetic acid) and paraffin embedded. Micro-dissection was performed and the sections were processed and hybridized using a labelled anti-sense probe to detect POSTN messenger RNA. A sense probe was used as an internal negative control. The probes used the complete murine mRNA sequence (l-2373nt) of clone BC031449.1.
  • mice Primary tumors from control and POSTN knock out mice were dissected and, after collagenase treatment, single cell suspensions were produced. The cells were plated on collagen for 24h in order to eliminate all immune cells present in the tumor cell preparation. The cells were then trypsinized, washed and counted. 10 4 cells were plated in 100 ⁇ l of serum- free sphere medium (DMEM/F12 with B27 supplement, 20ng/ml EGF, 20ng/ml FGF, 1 ⁇ M Hepes) into 96-well low attachment plates. After about 10 days, mammospheres are usually formed in the culture.
  • serum- free sphere medium DMEM/F12 with B27 supplement, 20ng/ml EGF, 20ng/ml FGF, 1 ⁇ M Hepes
  • the cells grown as spheres were collected and trypsinized in order to produce a single cell suspension. 10 4 cells were then seeded into 96-well low attachment plate.
  • His histidine tag
  • SBP streptavidin binding protein tag
  • the purified hPOSTN protein was added to the tumor cells prepared from MMTV-PyMT POSTN knock out tumors when seeded in low attachment 96-well plates for sphere culture. FACS analysis.
  • Tumor cells derived from MMTV-PyMT POSTN wild type or knock out tumors were grown in suspension for 10 days. Cells were then trypsinized and the single cell preparation was incubated with CD90 and CD24 antibodies. After washing, cells were analysed by
  • Fluorescent Activated Cell Sorter (FACS) analyser. Monoclonal antibody production and testing.
  • the purified hPOSTN protein was injected 3 times into POSTN knock out mice. Spleens were harvested and hybrodoma cells were produced by fusion to a murine myeloma cell line. 400 clones were isolated and propagated to collect antibody-containing supernatants. All supernatants were tested positive by ELISA against recombinant human POSTN protein. The blocking activity of all supernatants was tested by seeding the MMTV-PyMT derived tumor cells in low attachment 96-well plates.
  • the injected tumor cells expressed the fluorescent marker GFP.
  • the mice were sacrificed at 2 time points: after 1 week, when the tumor cells had extravasated into the lung and formed 1 -5 cell clusters or colonies; and after 5 weeks, when the cancer stem cells (CSCs) in the colonies, upon an active process of proliferation, survival and self renewal, had given rise to metastatic progression.
  • the lungs of the two groups of mice at the two time points were digested and single cell preparation was analyzed by FACS to determine the amount of tumor cells present in the lungs.
  • staining with the cancer stem cells markers CD90 and CD24 was performed to identify the frequency of CSCs among the tumor cells present in the different lung samples.
  • mice We ablated the periostin/OSF2/POSTN gene from the mouse genome using ES cell technology. Homozygous mutant mice display no overt phenotype and are viable and fertile.
  • POSTN function was tested in a murine cancer model.
  • To test the function of POSTN in mammary cancer metastasis we combined the MMTV-PymT transgene with the POSTN knock out allele. While formation of primary tumors is not affected by the knock out, the formation of lung metastasis is dependent on its expression ( Figures 3-4).
  • wild type tumor cells injected into knock out recipient animals show a dramatically reduced metastasis formation when compared to injections into wild type recipients (on average 16 metastases in transplants of wild type tumor cells into wild type recipients vs. 6 metastases in transplants of wild type tumor cells into POSTN deficient recipients).
  • One assay to analyze stem cell properties is the growth under non-adherent conditions in vitro.
  • cancer cells are selected to grow as "mammospheres", i.e. hollow, ball- like aggregates of one or two cell layers which contain a high amount of cancer stem cells.
  • these sphere-cultures are not possible from knock out tumor cells. From wild type tumors we have established sphere cultures in 7 out of 11 cases whereas none of the 9 tested POST deficient tumors produced spheres ( Figure 7).
  • This dependency of cancer stem cells for POSTN is further manifested in the failure of such knock out cells to maintain long-term self-renewal potential as mutant tumor cells can not be sustained under sphere culture conditions.
  • TYSGAPTYADDFKG and H3 GELLRWWYYFDY.
  • the constant region starts with TTPPSV.
  • the constant region starts with DAAPTV.

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Abstract

La présente invention porte sur un agent apte à bloquer une fonction de support de cancer d'un membre de la famille des protéines fasciclines et sur des compositions pharmaceutiques les contenant.
PCT/IB2010/053789 2009-08-25 2010-08-23 Molécules de matrice extracellulaire de ciblage pour le traitement du cancer WO2011024114A1 (fr)

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WO2014136910A1 (fr) * 2013-03-08 2014-09-12 国立大学法人大阪大学 ANTICORPS DIRIGÉ CONTRE UN PEPTIDE CODÉ PAR L'Exon-21 DE LA PÉRIOSTINE, ET COMPOSITION PHARMACEUTIQUE POUR LA PRÉVENTION OU LE TRAITEMENT D'UNE MALADIE INFLAMMATOIRE CONTENANT LEDIT ANTICORPS
JPWO2013038696A1 (ja) * 2011-09-15 2015-03-23 国立大学法人名古屋大学 胸膜中皮腫患者の早期発見のための分子マーカー及びその発現解析方法
CN106366190A (zh) * 2015-07-22 2017-02-01 中国医学科学院肿瘤医院 一种抗人肝癌干细胞的单克隆抗体
CN106366189A (zh) * 2015-07-22 2017-02-01 中国医学科学院肿瘤医院 一种抗人肺癌干细胞的单克隆抗体
WO2018233333A1 (fr) * 2017-06-23 2018-12-27 苏州博聚华生物医药科技有限公司 Anticorps monoclonal de cellules souches humaine ciblées et application d'un anticorps monoclonal

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EP2757376A4 (fr) * 2011-09-15 2015-05-27 Univ Nagoya Nat Univ Corp Marqueur moléculaire pour l'identification précoce de patients souffrant de mésothéliome pleural et procédé d'analyse d'expression associé
WO2014136910A1 (fr) * 2013-03-08 2014-09-12 国立大学法人大阪大学 ANTICORPS DIRIGÉ CONTRE UN PEPTIDE CODÉ PAR L'Exon-21 DE LA PÉRIOSTINE, ET COMPOSITION PHARMACEUTIQUE POUR LA PRÉVENTION OU LE TRAITEMENT D'UNE MALADIE INFLAMMATOIRE CONTENANT LEDIT ANTICORPS
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US10227396B2 (en) 2013-03-08 2019-03-12 Osaka University Antibody against peptide encoded by Exon-21 of periostin and pharmaceutical composition for preventing or treating inflammation-associated diseases containing the same
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