WO2018071959A1 - Cytotoxic particles - Google Patents
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- WO2018071959A1 WO2018071959A1 PCT/AU2017/050885 AU2017050885W WO2018071959A1 WO 2018071959 A1 WO2018071959 A1 WO 2018071959A1 AU 2017050885 W AU2017050885 W AU 2017050885W WO 2018071959 A1 WO2018071959 A1 WO 2018071959A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
- A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
- A61K47/6913—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome the liposome being modified on its surface by an antibody
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/545—Heterocyclic compounds
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1617—Organic compounds, e.g. phospholipids, fats
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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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
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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
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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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
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
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- 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]
Definitions
- the invention relates to chemotherapy of cancer, to formation of nanoparticles, especially liposomes, and to conjugation of cytotoxic drugs and antibodies to same.
- cancers are frequently treated by a combination of approaches, including surgical removal of a tumour, chemotherapy, and/or radiation therapy.
- Surgical procedures are usually not sufficient to remove a tumour in its entirety, so surgery is frequently accompanied by chemotherapy and/or radiation therapy.
- Chemotherapy involves the use of drugs to kill tumour cells, and radiation therapy involves treatment with high-energy rays (e.g. x-rays) to kill or shrink tumour cells.
- high-energy rays e.g. x-rays
- chemotherapy and radiation cause serious and sometimes life- threatening side effects, including fatigue; nausea; vomiting; pain; hair loss; anemia; central nervous system problems; infection; blood clotting problems; mouth, gum, and throat problems; diarrhea; constipation; nerve and muscle effects; kidney and bladder effects; flu-like symptoms; fluid retention; and effects on sexual organs.
- Chemotherapy causes such severe side effects because the treatment involves the systemic administration of cytotoxic agents to a patient. These agents cannot distinguish tumour cells from normal cells and, therefore, kill healthy cells as well as tumour cells. Side effects are worsened because a very large dose must be administered to the patient in order to deliver a therapeutically effective dose to a tumour site. Although radiation therapy is administered somewhat more locally than chemotherapy, radiation treatment still results in the destruction of normal tissue in the vicinity of the tumour. Thus, targeting of a therapeutic agent (e.g., to a particular tissue or cell type; to a specific diseased tissue but not to normal tissue; etc.) is desirable in the treatment of tissue specific diseases such as cancer (e.g. prostate cancer).
- tissue specific diseases such as cancer (e.g. prostate cancer).
- targeted delivery could prevent the agent from killing healthy cells. Additionally, targeted delivery would allow for the administration of a lower dose of the agent or at a lower administration frequency, which could reduce the undesirable side effects commonly associated with traditional chemotherapy.
- cytotoxic compound contained in, or entrapped in or on said core; - a plurality of variable domains arranged on the core for binding to P2X 7 R on a cancer cell thereby enabling the particle to bind to a cancer cell having P2X 7 R expressed thereon when the particle is contacted with the cancer cell.
- cytotoxic compound contained in, or entrapped in or on said liposome; - a plurality of variable domains arranged on the liposome for binding to P2X 7 R on a cancer cell thereby enabling the particle to bind to a cancer cell having P2X 7 R expressed thereon when the particle is contacted with the cancer cell.
- a cytotoxic particle including: - a liposome
- cytotoxic compound in the form of gemcitabine contained in, or entrapped in or on said liposome;
- variable domains arranged on the liposome for binding to P2X 7 R on a cancer cell thereby enabling the particle to bind to a cancer cell having P2X 7 R expressed thereon when the particle is contacted with the cancer cell.
- cytotoxic compound in the form of gemcitabine wherein the primary amine of gemcitabine is bonded to a hydrocarbon chain of the lipid layer via a carbamate group; - a plurality of variable domains arranged on the liposome for binding to P2X 7 R on a cancer cell thereby enabling the particle to bind to a cancer cell having P2X 7 R expressed thereon when the particle is contacted with the cancer cell.
- - a liposome including: - hydrocarbon chains forming a lipid layer of the liposome
- a cytotoxic compound in the form of gemcitabine wherein the primary amine of gemcitabine is bonded to a hydrocarbon chain of the lipid layer via a carbamate group; - a plurality of variable domains bound to the maleimide groups arranged on the liposome for binding to P2X 7 R on a cancer cell thereby enabling the particle to bind to a cancer cell having P2X 7 R expressed thereon when the particle is contacted with the cancer cell.
- a cytotoxic particle including:
- - - a liposome including:
- variable domain is defined by Formula 1 :
- CDR1 -CDR2-CDR3 wherein CDR1 is RNHDMG (SEQ ID NO: 4) wherein CDR2 is AISGSGGSTYYANSVKG (SEQ ID NO: 5) wherein CDR3 is EPKPMDTEFDY (SEQ ID NO: 6).
- variable domain contains framework regions in the formula FR1 - CDR1 -FR2-CDR2-FR3-CDR3-FR4.
- - a liposome including: - hydrocarbon chains forming a lipid layer of the liposome
- cytotoxic compound in the form of gemcitabine wherein the primary amine of gemcitabine is bonded to a hydrocarbon chain of the lipid layer via a carbamate group;
- variable domain has the following amino acid sequence (SEQ ID NO:
- the primary amine of the gemcitabine is covalently bonded to a hydrocarbon chain of the lipid layer.
- the primary amine is the cytosine C4 amine of gemcitabine.
- the liposome is a spherical liposome.
- a pharmaceutical composition including a plurality of cytotoxic particles mentioned above and a pharmaceutically effective diluent, excipient or carrier.
- the pharmaceutical composition is adapted for intravenous administration.
- a method of treating an individual having cancer including administering a particle or composition mentioned above to the individual, thereby treating the individual for cancer.
- a method of reducing tumour volume in an individual having cancer comprising administering a particle or composition mentioned above to the individual, thereby reducing tumour volume in the individual.
- a particle or composition mentioned above for use in the treatment of an individual having cancer comprising administering a particle or composition mentioned above to the individual, thereby reducing tumour volume in the individual.
- a particle or composition mentioned above in the manufacture of a medicament for the treatment of cancer.
- a method for killing a cancer cell including contacting a cancer cell with a particle or composition mentioned above.
- kit including a particle or composition mentioned above.
- the cytotoxic compound may comprise gemcitabine.
- the gemcitabine is in the form of a conjugate that allows the gemcitabine to be attached to or incorporated in the core or liposome.
- the core or liposome of the cytotoxic particle is comprised of dioleoylphosphatidylcholine (DOPC).
- DOPC dioleoylphosphatidylcholine
- variable domain may be a single domain antibody or may be part of any other antibody structure as described herein. Further, the variable domain may be humanized or human.
- the plurality of variable domains are arranged on the core for selective binding to P2X 7 R on a cancer cell.
- the selective binding may be for a P2X 7 R that is unable to form an apoptotic pore.
- Figure 1 Cytotoxic nanoparticle formation showing the oleyl-gemcitabine conjugate and formation into a liposome.
- FIG. 1 Conjugation of antibody variable domains to liposome via maleimide groups.
- Figure 3 Size distribution of liposomes.
- FIG. 4 Serological binding of liposomes by ELISA. Protein concentration after tangential flow filtration (TFF) washing is in the range of 400 to 500 ⁇ g/mL.
- FIG. 1 Conjugation of antibody to liposome demonstrated by SDS PAGE gel. Nupage 4-12% Bis tris gel, MOPS buffer (1X). The gel was run at 180V for 55mins.
- Figure 11 The effect of dosing frequency of antibody-coated nanoparticles: The mean tumour volume for each treatment group against days post inoculation. Treatment was given intravenously twice per week. Detailed description of the embodiments
- the present invention provides for cytotoxic particles, comprised of a cytotoxic agent and having a plurality of variable domains arranged on the particles, wherein the variable domains allow for binding to P2X 7 R on a cancer cell.
- the particles are able to bind to a cancer cell having P2X 7 R expressed thereon when the particle is contacted with the cancer cell.
- the cytotoxic particles of the invention provide for sustained release of the cytotoxic agent, but also provide for reduced toxicity arising from the cytotoxic agent.
- variable domains for binding to P2X 7 R ensure specificity for binding of the particles to cancerous tissue, and thereby reduce toxicity to healthy, non-cancerous tissue.
- the present invention therefore provides an advantage over other means of providing cytotoxic agents to cancerous cells.
- Purinergic receptor generally refers to a receptor that uses a purine (such as ATP) as a ligand.
- P2X 7 receptor generally refers to a purinergic receptor formed from three protein subunits or monomers, with at least one of the monomers having an amino acid sequence substantially as shown in SEQ ID NO: 1 below:
- P2X 7 receptor encompasses naturally occurring variants of P2X 7 receptor, e.g., wherein the P2X 7 monomers are splice variants, allelic variants and isoforms including naturally-occurring truncated or secreted forms of the monomers forming the P2X 7 receptor (e.g., a form consisting of the extracellular domain sequence or truncated form of it), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally- occurring allelic variants.
- P2X 7 receptor encompasses naturally occurring variants of P2X 7 receptor, e.g., wherein the P2X 7 monomers are splice variants, allelic variants and isoforms including naturally-occurring truncated or secreted forms of the monomers forming the P2X 7 receptor (e.g., a form consisting of the extracellular domain sequence or truncated form of it), naturally-occurring variant forms (e
- the native sequence P2X 7 monomeric polypeptides disclosed herein are mature or full- length native sequence polypeptides comprising the full-length amino acids sequence shown in SEQ ID NO: 1 .
- the P2X 7 receptor may have an amino acid sequence that is modified, for example various of the amino acids in the sequence shown in SEQ ID NO: 1 may be substituted, deleted, or a residue may be inserted.
- “Functional P2X 7 receptor” generally refers to a form of the P2X 7 receptor having three intact binding sites or clefts for binding to ATP. When bound to ATP, the functional receptor forms a non-selective sodium/calcium channel that converts to a pore-like structure that enables the ingress of calcium ions and molecules of up to 900 Da into the cytosol, one consequence of which may be induction of programmed cell death. In normal homeostasis, expression of functional P2X 7 receptors is generally limited to cells that undergo programmed cell death such as thymocytes, dendritic cells, lymphocytes, macrophages and monocytes. There may also be some expression of functional P2X 7 receptors on erythrocytes and other cell types.
- Non-functional P2X 7 receptor generally refers to a form of a P2X 7 receptor having a conformation, distinct from functional P2X 7 , whereby the receptor is unable to form an apoptotic pore, but which is still able to operate as a non-selective channel through the maintenance of a single functional ATP binding site located between adjacent monomers.
- One example arises where one or more of the monomers has a cis isomerisation at Pro210 (according to SEQ ID NO: 1 ).
- the isomerisation may arise from any molecular event that leads to misfolding of the monomer, including for example, mutation of monomer primary sequence or abnormal post translational processing.
- Non-functional P2X 7 receptors are expressed on a wide range of epithelial and haematopoietic cancers.
- “Cancer associated -P2X 7 receptors” are generally P2X 7 receptors that are found on cancer cells (including, pre-neoplastic, neoplastic, malignant, benign or metastatic cells), but not on non-cancer or normal cells.
- E200 epitope generally refers to an epitope having the sequence GHNYTTNILPGLNITC (SEQ ID NO: 2).
- E300 epitope generally refers to an epitope having the sequence KYYKENNVEKRTLIK (SEQ ID NO: 3).
- Composite epitope generally refers to an epitope that is formed from the juxtaposition of the E200 and E300 epitopes or parts of these epitopes.
- Antibodies or “immunoglobulins” or “Igs” are gamma globulin proteins that are found in blood, or other bodily fluids of verterbrates that function in the immune system to bind antigen, hence identifying and/or neutralizing foreign objects.
- Antibodies are generally a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. Each L chain is linked to a H chain by one covalent disulfide bond. The two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges.
- H and L chains define specific Ig domains. More particularly, each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and ⁇ chains and four CH domains for ⁇ and ⁇ isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CHL).
- VH variable domain
- CH constant domains
- CL constant domain
- Antibodies can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
- the ⁇ and a classes are further divided into subclasses on the basis of relatively minor differences in 3 ⁇ 4 sequence and function, e.g., humans express the following subclasses: lgG1 , lgG2, lgG3, lgG4, IgAI, and lgA2.
- the L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.
- the constant domain includes the Fc portion which comprises the carboxy- terminal portions of both H chains held together by disulfides.
- the effector functions of antibodies such as ADCC are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.
- FcR Fc receptors
- the pairing of a VH and VL together forms a "variable region" or "variable domain” including the amino -terminal domains of the heavy or light chain of the antibody.
- the variable domain of the heavy chain may be referred to as "VH.”
- the variable domain of the light chain may be referred to as "VL.”
- the V domain contains an "antigen binding site" which affects antigen binding and defines specificity of a particular antibody for its particular antigen.
- V regions span about 1 10 amino acid residues and consist of relatively invariant stretches called framework regions (FRs) (generally about 4) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” (generally about 3) that are each generally 9-12 amino acids long.
- FRs framework regions
- hypervariable regions generally about 3
- the FRs largely adopt a ⁇ -sheet configuration and the hypervariable regions form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
- “Hypervariable region” refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six hypervariable regions; three in the VH (H1 , H2, H3), and three in the VL (L1 , L2, L3).
- Framework or "FR” residues are those variable domain residues other than the hypervariable region residues herein defined.
- an "antigen binding site” generally refers to a molecule that includes at least the hypervariable and framework regions that are required for imparting antigen binding function to a V domain.
- An antigen binding site may be in the form of an antibody or an antibody fragment, (such as a dAb, Fab, Fd, Fv, F(ab')2 or scFv) in a method described herein.
- an “intact” or “whole” antibody is one which comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, CH1 , CH2 and CH3.
- the constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof.
- “Whole antibody fragments including a variable domain” include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
- the "Fab fragment” consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.
- a "Fab' fragment” differs from Fab fragments by having additional few residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region.
- Fab'- SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
- a “F(ab')2 fragment” roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen.
- Fv is the minimum antibody fragment which contains a complete antigen- recognition and binding site. This fragment consists of a dimer of one heavy and one light chain variable region domain in tight, non-covalent association.
- single-chain Fv In a single-chain Fv (scFv) species, one heavy and one light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody.
- Single-chain Fv also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected to form a single polypeptide chain.
- the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
- a "single variable domain” is half of an Fv (comprising only three CDRs specific for an antigen) that has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
- “Diabodies” refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL).
- the small antibody fragments are prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the VH and VL domains such that interchain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites.
- Diabodies may be bivalent or bispecific.
- Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains.
- Triabodies and tetrabodies are also generally known in the art.
- An "isolated antibody” is one that has been identified and separated and/or recovered from a component of its pre-existing environment. Contaminant components are materials that would interfere with therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
- human antibody refers to an antibody that possesses an amino acid sequence that corresponds to that of an antibody produced by a human.
- Human antibodies can be produced using various techniques known in the art, including phage -display libraries. Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled.
- Humanized' forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody.
- humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability.
- donor antibody such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability.
- framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
- humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
- the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
- the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
- Fc immunoglobulin constant region
- “Monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site or determinant on the antigen.
- the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies.
- Monoclonal antibodies may be prepared by the hybridoma methodology.
- the "monoclonal antibodies” may also be isolated from phage antibody libraries using molecular engineering techniques.
- anti-P2X 7 receptor antibody or "an antibody that binds to P2X 7 receptor” refers to an antibody that is capable of binding P2X 7 receptor with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting P2X 7 receptor, typically non-functional P2X 7 receptor or a cancer associated P2X 7 receptor.
- the extent of binding of an P2X 7 receptor antibody to an unrelated protein is less than about 10% of the binding of the antibody to P2X 7 receptor as measured, e.g., by a radioimmunoassay (RIA), Enzyme-Linked Immunosorbent Assay (ELISA), Biacore or Flow Cytometry.
- RIA radioimmunoassay
- ELISA Enzyme-Linked Immunosorbent Assay
- Biacore Biacore
- an antibody that binds to P2X 7 receptor has a dissociation constant (Kd) of ⁇ 1 ⁇ , ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, or ⁇ 0.1 nM.
- Kd dissociation constant
- An anti non-functional P2X 7 receptor antibody is generally one having some or all of these serological characteristics and that binds to non-functional P2X 7 receptors but not to functional P2X 7 receptors.
- affinity matured' antibody is one with one or more alterations in one or more hypervariable region thereof that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess those alteration(s).
- Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen.
- Affinity matured antibodies are produced by procedures known in the art.
- a "blocking” antibody” or an “antagonist” antibody is one that inhibits or reduces biological activity of the antigen it binds. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.
- an "agonist antibody”, as used herein, is an antibody, which mimics at least one of the functional activities of a polypeptide of interest.
- Binding affinity generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity” refers to intrinsic binding affinity, which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen).
- the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.
- Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer.
- a variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention.
- Epitope generally refers to that part of an antigen that is bound by the antigen binding site of an antibody.
- An epitope may be "linear” in the sense that the hypervariable loops of the antibody CDRs that form the antigen binding site bind to a sequence of amino acids as in a primary protein structure.
- the epitope is a "conformational epitope” i.e. one in which the hypervariable loops of the CDRs bind to residues as they are presented in the tertiary or quaternary protein structure.
- 'Treatment' generally refers to both therapeutic treatment and prophylactic or preventative measures.
- Subjects requiring treatment include those already having a benign, precancerous, or non-metastatic tumour as well as those in which the occurrence or recurrence of cancer is to be prevented.
- the objective or outcome of treatment may be to reduce the number of cancer cells; reduce the primary tumour size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumour metastasis; inhibit, to some extent, tumour growth; and/or relieve to some extent one or more of the symptoms associated with the disorder.
- Efficacy of treatment can be measured by assessing the duration of survival, time to disease progression, the response rates (RR), duration of response, and/or quality of life.
- Pre-cancerous or pre-neoplasia generally refers to a condition or a growth that typically precedes or develops into a cancer.
- a "pre-cancerous” growth may have cells that are characterized by abnormal cell cycle regulation, proliferation, or differentiation, which can be determined by markers of cell cycle.
- cancers include blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumours (including carcinoid tumours, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, leukemia or lymphoid malignancies, lung cancer including small-cell lung cancer (SGLG), non-small cell lung cancer (NSGLG), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal
- a condition or symptom associated' [with the cancer] may be any pathology that arises as a consequence of, preceding, or proceeding from the cancer.
- the condition or relevant symptom may be microbial infection.
- the condition or symptom may relate to organ dysfunction of the relevant organ having tumour metastases.
- the methods of treatment described herein are for the minimisation or treatment of a condition or symptom in an individual that is associated with a cancer in the individual.
- variable domains arranged on the core for binding to P2X 7 receptors on a cancer cell, the arrangement enabling the particle to cross link P2X 7 receptors on a cancer cell when the particle is contacted with a cancer cell.
- the core may generally define the overall dimensions and physical characteristics of the particle.
- the core may be formed from lipid, protein, sugar or other biomolecule.
- the core may be formed of a polymer, resin, plastic or ceramic or glass.
- the core is provided in the form of a liposome, micelle, microemulsion or macroemulsion.
- Micelles are colloidal aggregates of amphipathic (surfactant) molecules that are formed at a well-defined concentration known as the critical micelle concentration. Micelles are oriented with the nonpolar portions at the interior and the polar portions at the exterior surface, exposed to water. The typical number of aggregated molecules in a micelle (aggregation number) is 50 to 100. Microemulsions are essentially swollen micelles, although not all micellar solution can be swollen to form microemulsion. Microemulsions are thermodynamically stable, are formed spontaneously, and contain particles that are extremely small. Droplet diameters in microemulsions typically range from 10-100 nm. Macroemulsions are droplets with diameters greater than 100 nm.
- Liposomes are closed lipid vesicles comprising lipid bilayers that encircle aqueous interiors. Liposomes typically have diameters of 25 nm to 1 pm (see, e.g., D.O. Shah (ed), 1998, Micelles, Microemulsions, and Monolayers: Science and Technology, Marcel Dekker; A.S. Janoff (ed), 1998, Liposomes: Rational Design, Marcel Dekker).
- the core is provided in the form of a liposome.
- the particle may be described as an antibody-liposome conjugate.
- the liposome may consist of lipids including phosphoglycerides and sphingolipids, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidyl- ethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoyl - phosphatidylcholine or dilinoleoylphosphatidylcholine.
- Other compounds lacking in phosphorus, such as sphingolipid and glycosphingolipid families are also contemplated.
- the amphipathic lipids described above may be mixed with other lipids including triacyglycerols and sterols.
- the liposome may have a diameter ranging between about 50nm and 200pm. Accordingly, the liposome may be a small, sonicated unilamellar vesicle (SUV), a large unilamellar vesicle (LUV), or a liposome prepared by reverse phase evaporation (a REV), by French press (a FPV) or by ether injection (an EIV). Methods of preparing liposomes of such sizes, including methods of fractionating and purifying liposomes of the desired size, are known to one skilled in the art. Typically, the liposome has a diameter ranging between about 10 and 600 nm. In one embodiment, the liposome has a diameter of between about 50 and 200 nm.
- the liposome is a LUV.
- the liposome of the invention is a unilamellar with respect to the liposome lipid bilayer.
- the liposome of the invention may comprise more than one lipid bilayer.
- the liposome may be a multilamellar vesicle such as a large, vortexed multilamellar vesicle (MLV).
- MLV vortexed multilamellar vesicle
- a compound for providing the liposome with a charge for binding the liposome to a cancer cell is advantageous for improving the fusion between the cancer cell lipid bilayer and the liposome bilayer.
- DOTAP is particularly useful as a binding means for binding the liposome lipid bilayer to a target cell.
- the liposome may be adapted for destabilisation of the liposome lipid bilayer at the pH of an early endosome.
- An early endosome is a vacuole or vesicle formed at an early stage of the endocytotic pathway. This organelle typically has a pH ranging from 5.5 to 6.0.
- Liposomes can be constructed by well-known techniques (see, e.g., G. Gregoriadis (ed.), 1993, Liposome Technology Vols. 1 -3, CRC Press, Boca Raton, FL). Lipids are typically dissolved in chloroform and spread in a thin film over the surface of a tube or flask by rotary evaporation. If liposomes comprised of a mixture of lipids are desired, the individual components are mixed in the original chloroform solution. After the organic solvent has been eliminated, a phase consisting of water optionally containing buffer and/or electrolyte is added and the vessel agitated to suspend the lipid.
- unilamellar liposomes can be produced by the reverse phase evaporation method (Szoka and Papahadjopoulos, 1978, Proc. Natl. Acad. Sci. USA 75:4194-4198).
- Unilamellar vesicles can also be prepared by sonication or extrusion. Sonication is generally performed with a bath-type sonifier, such as a Branson tip sonifier (G. Heinemann Ultrashall und Labortechnik, Schwabisch Gmund, Germany) at a controlled temperature as determined by the melting point of the lipid.
- Extrusion may be carried out by biomembrane extruders, such as the Lipex Biomembrane Extruder (Northern Lipids Inc, Vancouver, British Columbia, Canada). Defined pore size in the extrusion filters may generate unilamellar liposomal vesicles of specific sizes.
- the liposomes can also be formed by extrusion through an asymmetric ceramic filter, such as a Ceraflow Microfilter (commercially available from the Norton Company, Worcester, MA).
- the liposomes that have not been sized during formation may be sized by extrusion to achieve a desired size range and relatively narrow distribution of liposome sizes.
- a size range of about 0.1 -0.5 microns will allow the liposome suspension to be sterilized by filtration through a conventional filter (e.g., a 0.22 micron filter or other filter).
- the filter sterilization method can be carried out on a high throughput basis.
- Multilamellar vesicles can be recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns, and preferably between about 0.1 and 0.2 microns.
- the size of the liposomal vesicles may be determined by quasi-elastic light scattering (QELS) (see Bloomfield, 1981 , Ann. Rev. Biophys. Bioeng. 10:421 -450). Average liposome diameter may be reduced by sonication of formed liposomes. Intermittent sonication cycles may be alternated with QELS assessment to guide efficient liposome synthesis.
- QELS quasi-elastic light scattering
- Liposomes can be extruded through a small-pore polycarbonate membrane or an asymmetric ceramic membrane to yield a well-defined size distribution. Typically, a suspension is cycled through the membrane one or more times until the desired liposome size distribution is achieved. The liposomes may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in liposome size.
- the cytotoxic particle includes a core in the form of a liposome, wherein the liposome includes oleic acid fatty acid chains in a lipid layer of the liposome.
- the core is preferably in the form of spherical bilayer membranes to form the liposome.
- the oleic acid may be functionalised to form a carbamate group for binding to gemcitabine, or functionalised with maleimido groups for binding to an immunoglobulin domain.
- the liposomes have a size of about 60 to 150nm.
- variable domains arranged on the core for binding to P2X 7 receptors on a cancer cell may be provided in the form of a whole antibody, or a fragment of an antibody that includes a variable domain.
- variable domains are for binding to cancer associated - P2X 7 receptors.
- variable domains are for binding to non-functional P2X 7 receptors.
- variable domain is one that discriminates between functional and non-functional P2X 7 receptors, so as to bind to non-functional receptors, but not to functional receptors.
- variable domains are those that bind to the E200 epitope, E300 epitope or composite epitope as for example in PCT/AU2002/000061 , PCT/AU2002/001204, PCT/AU2007/001540,
- PCT/AU2007/001541 PCT/AU2008/001364, PCT/AU2008/001365,
- Fragments of variable domains include single chain antibodies, single chain Fv fragments, and F(ab')2, Fab, Fd and Fv fragments.
- variable domains may arise from monoclonal or polyclonal antiserum.
- variable domains may be human, humanised, chimeric, mouse, rat, rabbit or other mammalian species.
- variable domain may be affinity matured.
- the antibodies in which the variable domains are arranged may have multiple specificities or valencies.
- the variable domain may be adapted so as to be suited to administration by a selected method.
- variable domain may be provided in an antibody of any isotype.
- the antibody may be produced by hybridoma, or by recombinant expression, or may be obtained from serum for example as obtainable from a mammal, particularly a human or mouse.
- the antibody may also be obtained from an avian.
- variable domain is defined by Formula
- CDR1 -CDR2-CDR3 wherein CDR1 is RNHDMG (SEQ ID NO: 4) wherein CDR2 is AISGSGGSTYYANSVKG (SEQ ID NO: 5) wherein CDR3 is EPKPMDTEFDY (SEQ ID NO: 6)
- variable domain has the following amino acid sequence (SEQ ID NO: 7): EVQLLESGGGLVQPGGSLRLSCAASGFTFRNHDMGVWRQAPGKGLEVWSAIS GSGGSTYYANSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTEFDYR SPGTLVTVSS or
- variable domains or antibodies can be conjugated to liposomes using conventional techniques (see, e.g., M.J. Ostro (ed.) 1987, Liposomes: from Biophysics to Therapeutics, Marcel Dekker, New York, NY).
- One preferred method of preparing liposomes and conjugating immunoglobulins to their surface is described by Y. Ishimoto et al., 1984, J. Immunol. Met. 75:351 -360.
- multilamillar liposomes composed of dipalmitoylphosphatidylcholine, cholesterol, and phosphotidylethanolamine are prepared.
- Purified fragments are then coupled to the phosphatidylethanolamine by the cross-linking agent N-hydroxysuccinimidyl 3- (2- pyridyldithio) propionate.
- the coupling of the antibody or fragment to the liposome is demonstrated by the release of a pre-trapped marker, e.g., carboxyfluorescence, from the liposomes. This release occurs upon incubation with a secondary antibody against the conjugated antibody, fragment, or complement.
- variable domains and antibodies can also be coupled to a liposome or another carrier of the invention via carbohydrate moieties.
- carbohydrate moieties Such coupling can be used provided that the carbohydrate moiety is not in the hypervariable region or at the antibody binding sites. In this way, conjugation via the cross-linking with the carbohydrate will not affect binding, and the binding sites will still be available to bind to cell surface antigens.
- One preferred method for coupling antibodies or antibody fragments of the invention (other than Fv) to a polymer backbone or a liposome involves conjugation through the carbohydrate moieties in the constant regions. This maximizes the number of available antigen-binding sites.
- Binding of a monoclonal antibody to the surface of a liposome may also be accomplished by the formation of cross-linkage between phosphatidylethanolamine and the antibody using glutaraldehyde.
- the targeting antibody may be cross-linked to the liposome via a reactive PEG group anchored on the surface of the liposome and acting as a stealth coating.
- a thiolated antibody can be allowed to react with a liposome comprising a lipid into which a maleimide group has been incorporated. Remaining maleimide groups on the surface of the liposome may be further reacted with a compound containing thiolated polyalkyleneglycol moiety.
- Thiolation of an antibody or antibody fragment may be achieved through use of N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP), which is usually used for thiolation of protein, iminothiolane, or mercaptoalkylimidate.
- SPDP N-succinimidyl-3-(2- pyridyldithio)propionate
- a dithiol group endogenous to an antibody may be reduced to form a thiol group.
- whole antibodies are treated with an enzyme such as pepsin to form F(ab)2 fragments, which are then reduced with dithiothreitol (DTT) to form Fab fragments, which results in the production of one to three thiol groups.
- DTT dithiothreitol
- the conjugation of the thiolated antibody to the maleimide group-containing liposome may be accomplished by reacting the components in a neutral buffer solution at pH 6.5-7.5 for 2-16 hours.
- Cytotoxic drugs Preferably the cytotoxic drug is a nucleoside analogue that, when incorporated into a nucleic acid strand during replication leads to apoptosis.
- the nucleoside analogue is a pyrimidine.
- the pyrimidine may be selected from gemcitabine (4-amino-1 -(2-deoxy-2,2- difluoro-p-D-erythro-pentofuranosyl)pyrimidin-2(1 H)-on) (Gemzar). or cytarabine ( 4 ⁇ amlno-1 -[(2R l 3S,4S,5R)-3 ! 4-dihydroxy-5-(hydroxymethy!) oxoian-2-yS]-1 ,2-dihydro pyrimidin -2-one or ara-C),
- the pyrimidine is gemcitabine.
- a cytotoxic drug may be conjugated to a liposome by functionalising a lipid component for forming a lipid layer of the liposome and then reacting the functional group with a cytotoxic agent so as to form a lipid-drug conjugate.
- the drug conjugate is then mixed with other lipid components and conditions applied for formation of a liposome as described above.
- an oleic acid hydrocarbon fatty acid chain is functionalised enabling the formation of a carbamate group between the cytotoxic agent and the oleic acid.
- a pharmaceutical composition including a plurality of cytotoxic particles according to the invention and a pharmaceutically effective diluent, excipient or carrier.
- the particle for use in the composition has a core in the form of a liposome.
- the particles of the composition may have the same density of variable domains.
- each particle in the composition may have the same surface area
- a method of treating an individual for a cancer especially a cancer including cancer cells that have a low copy number or low expression or production of cell surface P2X 7 R, including providing a therapeutically effective amount of a particle or composition mentioned above to the individual, thereby treating the individual for cancer.
- a particle or composition mentioned above for use in the treatment of an individual having cancer wherein the cancer includes cancer cells that have a low copy number or low expression or production of cell surface P2X 7 R.
- the particles or compositions of the invention may be administered to a subject in need thereof by oral, parenteral (e.g. intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, intradermal, rectal or vaginal) route, by inhalation or topical application.
- parenteral e.g. intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, intradermal, rectal or vaginal
- One form for administration would be a solution for injection, in particular for intravenous or intra-arterial injection or drip, comprising a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human albumin).
- a buffer e.g. acetate, phosphate or citrate buffer
- surfactant e.g. polysorbate
- optionally a stabilizer agent e.g. human albumin
- Preparations for parenteral administration includes sterile aqueous (aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media) or non-aqueous (non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate) solutions, suspensions, and emulsions.
- Pharmaceutically acceptable carriers include between 0.01 to 0.1 M and preferably 0.05 M phosphate buffer or 0.9% saline.
- Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
- Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishes, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
- compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions, in such cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and will preferably be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
- the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
- the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
- a coating such as lecithin
- surfactants Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., 16th ed. (1980).
- Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like.
- isotonic agents for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition.
- Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminium monostearate and gelatine.
- sterile injectable solutions can be prepared by incorporating a particle of the invention in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization.
- dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
- the preferred methods of preparation are vacuum drying, freeze-drying and spray drying, which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
- the preparations for injections are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. Further, the preparations may be packaged and sold in the form of a kit. Such articles of manufacture will preferably have labels or package inserts indicating that the associated compositions are useful for treating a subject suffering from, or predisposed to disorders.
- Effective doses of the compositions of the present invention, for treatment of disorders, as described herein, vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.
- the particles of the invention can be administered before, during or after surgical intervention for resection or removal of tumour or tissue.
- the method is particularly useful for delaying disease progression.
- the method is particularly useful for extending survival of the human, including overall survival as well as progression free survival.
- the method is particularly useful for providing a complete response to therapy whereby all signs of cancer in response to treatment have disappeared. This does not always mean the cancer has been cured.
- the method is particularly useful for providing a partial response to therapy whereby there has been a decrease in the size of one or more tumours or lesions, or in the extent of cancer in the body, in response to treatment.
- the cancer is pre-cancerous or pre -neoplastic.
- the cancer is a secondary cancer or metastases.
- the secondary cancer may be located in any organ or tissue, and particularly those organs or tissues having relatively higher hemodynamic pressures, such as lung, liver, kidney, pancreas, bowel and brain.
- the activities of the particles of the invention may be measured in vitro with reference to various cell function parameters including cytotoxicity, cytostasis, apoptosis induction, cell morphology, cytokine production, signal transduction, cell proliferation and activation status and protein expression and activation. Other biologically appropriate endpoints may also be considered.
- activity may be assessed by means of a MTT assay or XTT assay (cell proliferation); a trypan blue exclusion assay (cell viability); membrane phosphoserine asymmetry, annexin 5 binding and caspase assays (apoptosis); and cytokine production (cell function).
- MTT assay or XTT assay cell proliferation
- trypan blue exclusion assay cell viability
- membrane phosphoserine asymmetry membrane phosphoserine asymmetry
- annexin 5 binding and caspase assays apoptosis
- cytokine production cell function
- the assay may employ a cell line expressing P2X 7 R.
- a cell line expressing P2X 7 R Preferably cell lines having high copy number, or production or expression of P2X 7 R (such as PC3, discussed below) and cells having low copy number or production or expression of P2X 7 R (such as COLO25, discussed below) are utilised.
- these assays may be particularly useful for determining the amount of variable domain to arrange on the core of the particle.
- immunoliposomes The manufacture of immunoliposomes consists of two main parts: nanoparticle construction and antibody conjugation.
- Nanoparticle construction Nanoparticles recipients are DOPC (from Avanti Polar Lipids), Gem-Ole (synthesised by reaction oleyl cholorfomate to the hydroxyl-protected Gemcitabine base via carbamate chemical linkage, SOP-Syn001 -002) and Maleimido- Succinyl-PEG4000-Oleate (synthesised by converting Succinyl PEG4000-Oleate to Maleimido-Succinyl-PEG4000-Oleate, SOP-Syn 003-005).
- DOPC from Avanti Polar Lipids
- Gem-Ole synthesised by reaction oleyl cholorfomate to the hydroxyl-protected Gemcitabine base via carbamate chemical linkage, SOP-Syn001 -002
- Maleimido- Succinyl-PEG4000-Oleate synthesised by converting Succinyl PEG4000-Oleate to Maleimido-Succ
- Antibody conjugation The antibody is activated via Traut's reagent prior to mixing with nanoparticle solution.
- the conjugated nanoparticles are quenched with L-cysteine and purified via tangential flow filtration.
- the immunoliposomes are then further extruded through a 100 nm membrane three times and followed by sterile filtration through a 200 nm membrane.
- the antibody used herein was a single domain antibody.
- the single domain antibody comprised the CDR sequences as shown in SEQ ID NOs: 4, 5 and 6.
- the variable domain comprised the amino acid sequence as shown in SEQ ID NO: 7.
- Figure 1 is a schematic of the cytotoxic nanoparticle formation showing the oleyl- gemcitabine conjugate and formation into a liposome.
- Figure 2 shows conjugation of antibody variable domains to liposomes via maleimide groups.
- Figure 3 shows the size distribution of the liposomes.
- Example 2 Binding specificity of cytotoxic nanoparticles Conjugation of antibody to liposome was demonstrated by SDS PAGE gel
- the liposomes of the invention were also assessed for their ability to bind to the E200 epitope of P2X 7 R.
- the results demonstrate that the nanoparticles have particle affinity for the E200 epitope. Accordingly, the liposomes of the invention provide for selective binding to cancerous cells expressing non-functional P2X 7 R.
- Example 3 Biodistribution imaging of fluorescently labelled nanoparticles in 4T1 -
- NIR near-infrared lipophilic fluorescent dye DiR
- HEL4 isotype
- BIL03s-coated nanoparticles variable domains for binding to anti- P2X 7 R (P2X 7 -binding domain-coated nanoparticles, or "BIL03s-coated”).
- Female Balb/c mice were inoculated orthotopically (4 th mammary fat pad) with 4T1 -luc2 murine breast cancer cells.
- a 50 ⁇ _ dose of nanoparticles was injected intravenously at study day 0 and near-infrared (NIR) imaging was performed at 6, 12, 24, 48 and 96 post treatment.
- excised liver, spleen and tumour NIR imaging was performed and bioluminescence imaging was performed on tumour tissues.
- Figure 8 shows that the nanoparticles are localised selectively: Mean total radiant efficiency +- SEM (p/s)/ ⁇ W/cmA 2 ]) for excised tumour at 96 hr time point.
- Vehicle control PBS; Group 1
- uncoated nanoparticles Group 2
- Ab-coated nanoparticles Group 3
- HEL4-coated nanoparticles Group 4
- Example 4 Efficacy of gemcitabine liposome nanoparticles conjugated to a domain for binding to P2X 7 R
- Nanoparticles were administered intravenously, 3 times a week. Nanoparticles contained either 0.2 mg gemcitabine or PBS buffer (control).
- FIG. 10 shows efficacy of antibody-conjugated nanoparticles on 4T1 murine breast tumour bearing mice.
- Figure 1 1 shows the effect of dosing frequency of antibody- coated nanoparticles.
- Figure 1 1 shows the results of a study comparing the efficacy of P2X 7 -binding domain-coated nanoparticles containing gemcitabine to gemcitabine alone for inhibiting tumour growth.
- the study compared the results following administration of 0.3 mg gemcitabine ("free gemcitabine") or P2X 7 -binding domain-coated nanoparticles containing gemcitabine, equivalent to a dose of 0.3 mg gemcitabine.
- the efficacy of both treatments was comparable, indicating that one benefit of the P2X 7 -binding domain-coated nanoparticles is the ability to reduce the frequency of dosing if using P2X 7 -binding domain-coated nanoparticles.
- the coated nanoparticles provide for sustained release and lower toxicity, thereby providing an advantage over existing cancer treatments.
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- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
Claims
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BR112019007895A BR112019007895A8 (en) | 2016-10-21 | 2017-08-18 | CYTOTOXIC PARTICLES |
MX2019004410A MX2019004410A (en) | 2016-10-21 | 2017-08-18 | Cytotoxic particles. |
CN201780063609.5A CN109862883A (en) | 2016-10-21 | 2017-08-18 | Cytotoxicity particle |
US16/326,882 US11260131B2 (en) | 2016-10-21 | 2017-08-18 | Cytotoxic particles for targeting P2X7 receptor |
AU2017346936A AU2017346936B2 (en) | 2016-10-21 | 2017-08-18 | Cytotoxic particles |
EP17862514.1A EP3528789A4 (en) | 2016-10-21 | 2017-08-18 | Cytotoxic particles |
JP2019520598A JP7127021B2 (en) | 2016-10-21 | 2017-08-18 | cytotoxic particles |
CA3035835A CA3035835A1 (en) | 2016-10-21 | 2017-08-18 | Cytotoxic particles |
US17/575,568 US20220249692A1 (en) | 2016-10-21 | 2022-01-13 | Cytotoxic Particles for Targeting P2X7 Receptor |
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AU2016904292A AU2016904292A0 (en) | 2016-10-21 | Cytotoxic particles | |
AU2016904292 | 2016-10-21 |
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US16/326,882 A-371-Of-International US11260131B2 (en) | 2016-10-21 | 2017-08-18 | Cytotoxic particles for targeting P2X7 receptor |
US17/575,568 Continuation US20220249692A1 (en) | 2016-10-21 | 2022-01-13 | Cytotoxic Particles for Targeting P2X7 Receptor |
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US (2) | US11260131B2 (en) |
EP (1) | EP3528789A4 (en) |
JP (1) | JP7127021B2 (en) |
CN (1) | CN109862883A (en) |
AU (1) | AU2017346936B2 (en) |
BR (1) | BR112019007895A8 (en) |
CA (1) | CA3035835A1 (en) |
MX (1) | MX2019004410A (en) |
WO (1) | WO2018071959A1 (en) |
Cited By (1)
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WO2023028653A1 (en) * | 2021-09-01 | 2023-03-09 | Biosceptre (Aust) Pty Ltd | Novel dysfunctional p2x7 binders |
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CN107614528A (en) * | 2015-04-02 | 2018-01-19 | 生物权威(英国)有限公司 | Pain therapy |
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WO2013003895A1 (en) * | 2011-07-01 | 2013-01-10 | Biosceptre International Limited | Combination therapy |
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AUPP991199A0 (en) | 1999-04-21 | 1999-05-13 | University Of Sydney, The | Methods for diagnosing pre-cancerous and cancerous conditions |
AUPQ968700A0 (en) | 2000-08-28 | 2000-09-21 | Intreat Pty Limited | Treatment of urinary incontinence |
US20040067196A1 (en) * | 2000-10-11 | 2004-04-08 | Brunke Karen J. | Targeted therapeutic lipid constructs |
AUPR201500A0 (en) | 2000-12-11 | 2001-01-11 | Biosceptre Pty Ltd | Methods for identifying pre-neoplastic and neoplastic states in mammals |
ES2320421T3 (en) | 2001-01-17 | 2009-05-22 | Intreat Pty Limited | ANTIBODIES FOR THE DIAGNOSIS OF A NON-FUNCTIONAL P2X7 RECEIVER AND THE TREATMENT OF CANCER AND OTHER AFFECTIONS. |
TWI329648B (en) | 2001-09-03 | 2010-09-01 | Intreat Pty Ltd | Diagnosis and treatment of cancers and other conditions |
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- 2017-08-18 JP JP2019520598A patent/JP7127021B2/en active Active
- 2017-08-18 AU AU2017346936A patent/AU2017346936B2/en active Active
- 2017-08-18 MX MX2019004410A patent/MX2019004410A/en unknown
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- 2017-08-18 CN CN201780063609.5A patent/CN109862883A/en active Pending
- 2017-08-18 BR BR112019007895A patent/BR112019007895A8/en active Search and Examination
- 2017-08-18 EP EP17862514.1A patent/EP3528789A4/en active Pending
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Also Published As
Publication number | Publication date |
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MX2019004410A (en) | 2019-09-26 |
CA3035835A1 (en) | 2018-04-26 |
US11260131B2 (en) | 2022-03-01 |
CN109862883A (en) | 2019-06-07 |
US20220249692A1 (en) | 2022-08-11 |
EP3528789A1 (en) | 2019-08-28 |
US20190201545A1 (en) | 2019-07-04 |
BR112019007895A2 (en) | 2019-07-02 |
EP3528789A4 (en) | 2020-06-24 |
JP2019533677A (en) | 2019-11-21 |
BR112019007895A8 (en) | 2022-12-20 |
AU2017346936B2 (en) | 2022-10-06 |
AU2017346936A1 (en) | 2019-03-07 |
JP7127021B2 (en) | 2022-08-29 |
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