HK1188401B - Companion animal treatments - Google Patents

Description

Companion animal treatment

Technical Field

The present invention relates to veterinary oncology, including the treatment of cancer in companion animals.

Background

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that such prior art forms part of the common general knowledge in australia or any other jurisdiction or that such prior art could reasonably be expected to be acknowledged, understood and regarded as relevant by a person skilled in the art.

The incidence of cancer in companion animals such as dogs and cats, and other similar animals, is increasing and cancer is currently considered to be the leading cause of death in older animals. The annual incidence of cancer in dogs is believed to be about 2% to 2.5% (about as in humans) and 1.5% to 2% in cats.

The cancers with the highest incidence in dogs were in the following order: lymphoma (about 20%); mast cell tumor (about 18%); soft tissue sarcoma (about 10%); angiosarcoma (about 10%); osteosarcoma (about 9%). The remainder typically includes squamous cell carcinoma, breast carcinoma, melanoma, histiocytoma, and fibrosarcoma.

The cancers with the highest incidence in cats are in the following order: lymphoma (about 25%); mast cell tumor (about 22% of skin tumors); squamous cell carcinoma (greater than 11% of skin tumor); breast cancer (about 10%); and the remainder including angiosarcoma, osteosarcoma, fibrosarcoma, sebaceous hyperplasia/adenoma.

Methods of cancer treatment in veterinary oncology include surgery, radiation therapy, hyperthermia, photodynamic therapy and chemotherapy. Gene therapy and immunotherapy have not been widely used.

With the exception of Palladia (toceranib phosphate; Pfizer), no other drug has been administered with the market permit, as a chemotherapeutic drug, for cancer treatment in companion animals. This is primarily due to the high cost associated with obtaining a marketing license.

In most jurisdictions, veterinary oncologists have "off-label" drug use privileges. Off-label drug use means that a veterinary oncologist can freely use a drug approved for one species (including humans) for another species. With these privileges, a practice has emerged in veterinary oncology whereby oncologists tend to select chemotherapeutic drugs for cancer treatment in companion animals that have gained significant clinical experience in human tumors.

Examples of chemotherapeutic drugs currently used in veterinary oncology and the associated indications are shown in table 1.

TABLE 1

There are a number of problems and limitations with these chemotherapeutic drugs. For example, as observed in human therapy, these compounds are associated with non-specific effects against toxicity associated with dividing cells including bone marrow, gastrointestinal epithelium, and hair follicles. Side effects include immunosuppression, anemia, nausea and vomiting, delay in wound healing, reproductive disorders, and hair loss. Certain specific organs may also become susceptible, including the heart, kidneys, and Central Nervous System (CNS).

In addition, the use of a single chemotherapeutic agent is rarely effective in curing cancer because not all tumor cells are effectively killed by a single agent. One reason for this is that as cancer cells develop, the incidence of mutations also develops and may lead to the development of a resistant phenotype. Therefore, in most cases, it is necessary to develop a combination therapy for a given clinical case.

There is a need for an alternative or improved treatment for cancer in companion animals, and particularly those with a high incidence in companion animals.

There is also a need for alternative or improved treatments for cancer in dogs.

There is a need for the treatment of lymphoma, mast cell tumor, soft tissue sarcoma, angiosarcoma, osteosarcoma, squamous cell carcinoma, breast cancer, melanoma, histiocytoma, and fibrosarcoma in dogs.

There is a need for an alternative or improved treatment for feline cancers.

There is a need for the treatment of lymphoma, mast cell tumor, squamous cell carcinoma, breast cancer, angiosarcoma, osteosarcoma, fibrosarcoma, sebaceous hyperplasia/adenoma in cats.

Summary of The Invention

The present invention aims to address one or more of the above needs and in a first aspect provides a method for minimising cancer progression in a companion animal, the method comprising the steps of:

providing a companion animal in which the progression of cancer is to be reduced; and is

Providing a whole antibody or a fragment thereof comprising a variable domain into the animal for binding to a non-functional P2X expressed by the animal₇On the receptor;

thereby minimizing cancer progression in the animal.

In a second aspect, the present invention provides a method for minimizing cancer progression in a companion animal, the method comprising the steps of:

providing a companion animal in need of cancer treatment; and

creating a target for non-functional P2X in the companion animal₇An immune response of the recipient;

thereby minimizing cancer progression in the companion animal.

In a third aspect, the present invention provides a method for minimizing cancer progression in a companion animal, the method comprising the steps of:

providing a companion animal that has received a non-self antigen binding site for cancer therapy;

creating a target for non-functional P2X in the companion animal₇The immune response of the recipient;

thereby minimizing cancer progression in the companion animal.

In another aspect of the invention, the following uses are provided:

a whole antibody comprising a variable domain, or a fragment thereof, for binding to a non-functional P2X₇On the receptor; or

A P2X₇Receptor, or a P2X₇A fragment of a receptor;

in the manufacture of a medicament for the treatment of cancer in a companion animal.

In yet another aspect, the invention provides the use of:

a full antibody comprising a variable domain, or a fragment thereof, for binding to a non-functional P2X₇A receptor; or

A P2X₇Receptor, or a P2X₇A fragment of a receptor;

can be used for treating cancer in companion animals.

In yet a further aspect, the present invention provides a kit or composition for use in cancer treatment in a companion animal, the kit comprising:

a full antibody comprising a variable domain, or a fragment thereof, for binding to a non-functional P2X₇On the receptor; or

A P2X₇Receptor, or a P2X₇A fragment of a receptor;

written instructions for use in a method or in any of the preceding claims.

Further aspects of the invention and further embodiments of these aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

Brief description of the drawings

FIG. 1.P2X₇The amino acid sequence of the receptor.

FIG. 2 Canine and human P2X₇Comparison of the amino acid sequences of the receptors.

FIG. 3 anti-E200 antibody response detected by ELISA (enzyme-linked immunosorbent assay).

Detailed description of the embodiments

Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with these embodiments, it will be understood that they are not intended to limit the invention to these embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the invention as defined by the appended claims.

Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the animal features mentioned or evident from the context or drawings. All of these different combinations constitute different alternative aspects of the invention.

All patents and publications mentioned herein are incorporated by reference in their entirety.

Definition of A

For the purpose of interpreting this specification, the following definitions will apply generally and, where appropriate, singular terms used will also include the plural and vice versa. In the event that any of the listed definitions conflict with any document incorporated herein by reference, the definition set forth below will prevail.

As used herein, unless the context requires otherwise, the term "comprise" and variations of the term, such as "comprises" and "comprising", are not intended to exclude other additives, components, integers or steps.

A "companion animal" generally refers to an animal that is a pet or a "companion" animal for a person. Cats (cats) and dogs (dogs) are examples.

"purine receptor" generally refers to a receptor that uses a purine (e.g., ATP) as a ligand.

“P2X₇The receptor "generally refers to a purine receptor formed from three protein subunits or monomers, wherein at least one of the monomers has an amino acid sequence substantially as shown in figure 1 or a canine sequence substantially as shown in figure 2. To the P2X₇The extent to which the receptor is formed from three monomers, it is a "trimer" or "trimer". "P2X₇The receptor "may be a functional or non-functional receptor as described below. "P2X₇Receptor "encompasses naturally occurring P2X₇Receptor variants, e.g. wherein P2X₇Monomers are splice variants, allelic variants and include naturally occurring forms of P2X₇Isoforms of monomers that are truncated or secreted forms of the receptor (e.g., forms consisting of extracellular domain sequences or truncated forms thereof), naturally occurring variant forms (e.g., alternatively spliced forms), and naturally occurring allelic variants. In certain embodiments of the present invention, the natural sequence P2X disclosed herein₇The monomeric polypeptide is mature or is a full-length native sequence polypeptide comprising the full-length amino acid sequence shown in figure 1, or is a canine sequence substantially as shown in figure 2. In certain embodiments, the P2X₇The receptor may have a modified amino acid sequence, for example the different amino acids in the sequence shown in figure 1 or a canine sequence substantially as shown in figure 2 may be substituted, deleted or an amino acid sequence may be insertedAnd (c) a residue.

"functional P2X₇The receptor "generally refers to P2X having a binding site or cleft for binding to ATP₇A form of receptor. When bound to ATP, the receptor forms a non-selective sodium/calcium channel that switches to a pore-like structure that allows calcium ions to enter the cytosol, one result of which can be programmed cell death. In normal steady state, functional P2X₇The expression of receptors is usually restricted to cells undergoing programmed death, such as thymocytes, dendritic cells, lymphocytes, macrophages, and monocytes. Some functional P2X may also be present on erythrocytes₇The receptor is expressed.

"non-functional P2X₇The acceptor "is generally referred to as P2X₇A form of a receptor having a configuration whereby the receptor is unable to form an apoptotic pore. An example occurs where one or more of these monomers has a homeotropic isomer at Pro210 (according to SEQ ID No.: 1). In a human or non-human mammalian monomer, such isomerization may result from any molecular event that results in misfolding of the monomer, including, for example, mutations or aberrant post-translational processing of the monomer's base sequence. One consequence of isomerization is that at one or more ATP binding sites on the trimer, the receptor is unable to bind to ATP and thereby enlarges the opening of the channel. Specifically, when one of the three monomers is incorrectly packaged and thus destroys both ATP binding sites. In these cases, the receptor is unable to form pores and this limits the extent to which calcium ions can enter the cytosol. Only partial channel activity was maintained. Non-functional P2X₇Receptors are expressed on a wide range of epithelial and hematopoietic cancers.

"E200 epitope" generally refers to a peptide present in a non-functional P2X₇An epitope on the receptor. In humans, the sequence is GHNYTTRNILPGLNITC. In canines, the sequence is GHNYTTRNILPDINITC.

"E300 epitope"generally refers to a presence in a non-functional P2X₇An epitope on the receptor. In humans and canines, the sequence is identical, KYYKENNVEKRTLIKVF.

A "composite epitope" generally refers to an epitope formed by the juxtaposition of E200 and E300 epitopes. The point of difference between E200 in canines and humans is not contained in the canine complex epitope, meaning that the canine and human complex epitopes are the same. Although Cat P2X₇The receptor sequence has not been characterized, and serological data herein demonstrates that the feline complex epitope is identical or substantially identical to the canine and human complex epitopes.

"antibodies" or "immunoglobulins" or "igs" are gamma globulin proteins found in the blood, or other bodily fluids of vertebrates, which function in the immune system to bind to antigens, thereby recognizing and/or neutralizing foreign substances.

Antibodies are typically heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Each L chain is linked to an H chain by a covalent disulfide bond. The two H chains are linked to each other by one or more disulfide bonds (depending on the isotype of the H chain). Each H and L chain also has regularly spaced intrachain disulfide bridges.

The H and L chains define a specific lg domain. More specifically, each H chain has at the N-terminus a variable domain (V)_H) Followed by three constant domains (C)_H) (for each α and gamma chain) and four C_HDomain (for μ and isoform). Each L chain has a variable domain at the N-terminus (V)_L) Then at its other end a constant domain (C)_L)。V_LAnd V_HAlignment and C_LTo the first constant domain of the heavy chain (C)_H1) And (6) comparison.

There are five classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, respectively, γ, and μ the γ and α classes are based on the fact that_HComparison of sequence and function aspectsSmall differences further divided into subclasses, e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. The amino acid sequence of the L chain from any vertebrate species, based on its constant domains, can be assigned to one of two distinctly different classes, termed κ and λ.

The constant domain includes an Fc portion that includes the carboxy terminal portions of both H chains bound together by a disulfide compound. The effector functions of antibodies (e.g., ADCC) are determined by sequences in the Fc region, which is also the portion recognized by Fc receptors (fcrs) found on certain types of cells.

V_HAnd V_LTogether, form a "variable region" or "variable domain" comprising the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "V_H". The variable domain of the light chain may be referred to as "V_LThe ". V domain comprises an" antigen binding site "that affects antigen binding and defines the specificity of a particular antibody for its particular antigen.V region spans about 110 amino acid residues and consists of 15-30 amino acid relatively invariant stretches (relatively invariant stretch) called Framework Regions (FRs) (usually about 4) separated by extremely variable shorter regions called" hypervariable regions "(usually about 3) each typically 9-12 amino acids long.

"hypervariable region" refers to regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Typically, an antibody comprises six hypervariable regions; three at V_H(H1, H2, H3), and three are at V_L(L1, L2, L3).

"framework" or "FR" residues are those variable domain residues other than the hypervariable region residues defined herein.

"peptide for forming an antigen binding site" generally refers to a peptide that can form a configuration that confers antibody specificity to an antigen. Examples include whole antibodies or whole antibody-related structures, whole antibody fragments comprising one variable domain, variable domains and fragments thereof, including light and heavy chains, or fragments of light and heavy chains comprising some but not all hypervariable or constant regions.

An "antigen binding site" generally refers to a molecule comprising hypervariable and framework regions necessary to assign at least an antigen binding function to one V domain. In the methods described herein, an antigen binding site can be in the form of an antibody or an antibody fragment (e.g., a dAb, Fab, Fd, Fv, F (ab')₂Or scFv).

An "intact" or "whole" antibody is one that includes an antigen binding site, along with C_LAnd at least a heavy chain constant domain C_HI、C_H2 and C_H3. The constant domain may be a constant domain of a native sequence (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof.

"Whole antibody fragment comprising a variable domain" includes Fab, Fab ', F (ab')₂And Fv fragments; diabodies, linear antibodies, single chain antibody molecules; and multispecific antibodies formed from antibody fragments.

The "Fab fragment" consists of the entire variable domains (V) of the L and H chains_H) And a first constant domain (C) of a heavy chain_HI) And (4) forming. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen binding site.

"Fab' fragments" are distinguished from Fab fragments by the presence of C_HThe carboxy terminus of the I domain has additional few residues, including one or more cysteines from the antibody hinge region. Fab '-SH is named herein for Fab', where one or more cysteine residues of the constant domain carry a free thiol group.

“F(ab′)₂The fragment "roughly corresponds to a fragment of two disulfide-linked fabs with bivalent antigen binding activity and is still capable of cross-linking antigen.

"Fv" is the smallest antibody fragment that contains one complete antigen recognition and binding site. The fragment consists of a dimer of the variable region domains of a heavy chain and a light chain in close non-covalent association.

In a single chain Fv (scfv) species, a heavy chain and a light chain variable domain may be covalently linked by a flexible peptide linker such that the light and heavy chains may associate in a "dimer" structure similar to a two chain Fv species. Six hypervariable loops (3 loops from the H and L chains, respectively) emanate from the folding of these two domains, which contribute to the use of amino acid residues for antigen binding and confer antigen-binding specificity to the antibody.

"Single-chain Fv", also abbreviated as "sFv" or "scFv", is a polypeptide comprising V joined to form a single polypeptide chain_HAnd V_LAntibody fragments of antibody domains. Preferably, the scFv polypeptide is further comprised in the V_HAnd V_LA polypeptide linker between the domains, enabling 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, albeit with lower affinity than the entire binding site.

"diabodies" refer to antibody fragments having two antigen-binding sites, which fragments comprise a linker linked to the same polypeptide chain (V)_H-V_L) A light chain variable domain of (V)_L) Heavy chain variable domain of (V)_H). Small antibody fragments were passed through this V_HAnd V_LConstruction of sFv fragments with short linkers (about 5-10 residues) between domains (see above paragraphs) was made such that inter-chain pairing, but not intra-chain pairing of the V domains was achievedThis results in a bivalent fragment, i.e., a fragment with two antigen binding sites.

Diabodies can be bivalent or bispecific. Bispecific diabodies are heterodimers of two "crossover-type" sFv fragments, where the V of the two antibodies_HAnd V_LThe domains are present on different polypeptide chains. Three-chain antibodies and four-chain antibodies are also generally known in the art.

An "isolated antibody" is an antibody that has been identified and isolated and/or recovered from a component in its pre-existing environment. Contaminant components are materials that would interfere with therapeutic use for antibodies and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.

By "monoclonal antibody" is meant an antibody obtained from a population of substantially homogeneous antibodies, i.e., the animal 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 an antigen. In addition to their specificity, monoclonal antibodies are advantageously synthesized uncontaminated by other antibodies. Monoclonal antibodies can be prepared by hybridoma methodology. These "monoclonal antibodies" can also be isolated from phage antibody libraries using these techniques.

Monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences derived from or belonging to a particular class of antibodies, while the remainder of the chain or chains are identical or homologous to corresponding sequences in antibodies derived from or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

The term "anti-P2X₇Receptor antibody "or" binding to P2X₇An antibody on a receptor is meant to be able to bind to the receptor sufficientlyBinds to P2X with affinity₇On the receptor, such that the antibody targets P2X as a diagnostic and/or therapeutic agent₇Receptor (typically non-functional P2X₇Receptor) are useful antibodies. Preferably, a P2X is added₇Receptor antibody binding to unrelated P2X₇The extent of binding of the antibody to the receptor protein is less than that of binding of the antibody to P2X as measured (e.g., by Radioimmunoassay (RIA))₇About 10% on the receptor. In certain embodiments, to P2X₇Antibodies on receptors have dissociation constants (Kd) < 1 μ M, < 100nM, < 10nM, < 1nM, or < 0.1 nM. Anti-non-functional P2X₇Receptor antibodies typically have some or all of these serological characteristics and bind to non-functional P2X₇On receptors other than functional P2X₇An antibody on the receptor.

An "affinity matured" antibody is one that has one or more alterations in one or more hypervariable regions thereof which result in an improvement in the affinity of the antibody for an antigen compared to a parent antibody which does not possess one or more of those alterations. Preferred affinity matured antibodies 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 "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which the antibody binds. Preferred blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

As used herein, an "agonist antibody" is an antibody that mimics at least one of the functional activities of a polypeptide of interest.

"binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to intrinsic binding affinity, reflecting the composition of a binding pair (e.g., antibody and antigen)One of 1: 1, in the presence of a ligand. The affinity of a molecule X for its partner Y can generally be determined by the dissociation constant (K)_d) And (4) showing. Affinity can be measured by methods known in the art, including those described herein. Low affinity antibodies generally bind to antigen slowly and tend to dissociate easily, while high affinity antibodies generally bind to antigen faster and tend to remain bound longer. A number of methods for measuring binding affinity are known in the art, any of which may be used for the purposes of the present invention.

"treatment" generally refers to both therapeutic treatment and prophylactic or preventative measures.

Animals in need of treatment include those already having a benign, pre-neoplastic, or non-metastatic tumor as well as those in which the incidence or recurrence of their cancer is to be prevented.

The purpose or outcome of the treatment may be to reduce the number of cancer cells; reducing primary tumor 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) tumor metastasis; inhibit tumor growth to some extent; and/or to alleviate to some extent one or more of the symptoms associated with the disorder.

The efficacy of treatment can be measured by assessing the duration of survival, time to disease progression, Response Rate (RR), duration of response, and/or quality of life.

In one embodiment, the method is particularly useful for extending the time to disease progression.

In one embodiment, the method is particularly useful for extending survival of animals, including overall survival time along with progression-free survival.

In one embodiment, the method is particularly useful for providing a complete response to treatment, whereby all signs of cancer in response to treatment have disappeared. This does not always mean that the cancer has already cured.

In one embodiment, the method is particularly useful for providing a partial response to treatment whereby there has been a reduction in the size of one or more tumors or lesions or in the extent of cancer in the body in response to treatment.

"precancerous" or "preneoplastic" generally refers to a condition or growth that typically progresses to or develops into a cancer. "precancerous" growth may have cells characterized by aberrant cell cycle regulation, proliferation or differentiation, which may be determined by cell cycle markers.

"[ cancer ] associated condition or symptom" can be as a result of cancer, in advance of cancer or by cancer progression of any pathology. For example, when the cancer is a skin cancer, the condition or associated symptom may be a microbial infection. Where the cancer is a secondary tumour, the condition or symptom may involve organ dysfunction of the relevant organ with tumour metastasis. In one embodiment, the methods of treatment described herein are for the minimization or treatment of a condition or symptom in an animal, the symptom or condition being associated with cancer in the animal.

A "non-self" molecule, such as a "non-self" antigen binding site or a "non-self" antibody, generally refers to a molecule that has been produced in vitro or is foreign, wherein the molecule is to be provided, for example, for use in therapy. As an example, a synthetic or recombinant molecule is "non-autologous". Further, a molecule that is produced in one animal and administered to another animal for treatment is "non-autologous". The "non-self" antigen binding site and antibody may be used according to the invention for adoptive transfer of immunity, such as occurs upon antibody infusion. In contrast, a molecule that is produced in an animal and used to treat the animal is typically an "autologous" or "endogenous" molecule. An example of an "autologous" molecule is an antigen binding site or antibody that is generated from or by an adaptive immune response to an immunogen.

The "level of circulating non-self antigen binding sites" in an animal generally refers to the concentration of antigen binding sites in body fluids, preferably in peripheral blood.

By "substantially undetectable non-autologous antigen binding site level in the circulation" is generally meant that the concentration of exogenous antigen binding sites (i.e., those that have been administered by adoptive transfer) is at least half the concentration of those antigen binding sites in the circulation at the time the antigen binding sites are administered, preferably 25%, or 10%, or 5%, or 1%, or otherwise less than 0.001mg/kg of the animal. The phrase also refers to a situation in which an antigen binding site that has been administered for cancer immunotherapy cannot be detected at all.

A cancer that is "substantially undetectable" is typically a condition in which the treatment has reduced the size, volume, or other physical measure of a cancer such that the cancer is not clearly detectable as a result of the treatment using relevant standard detection techniques (e.g., in vivo imaging). The phrase also refers to a situation in which the cancer cannot be detected at all.

By "developing an immune response" is generally meant eliciting or inducing antigen-specific immunity via the adaptive immune system. As is generally understood in the art, induction of antigen-specific immunity is distinct from adoptive transfer of immunity, of which standard cancer immunotherapy by administration of exogenous or non-autologous antibodies is an example.

B. Cancer immunotherapy by antibody infusion

Despite the non-functional P2X₇Tissue expression profiles of receptors in normal, pre-tumoral or tumoral human tissues are understood at the time of the present invention, but with regard to non-functional P2X₇Tissue expression of receptors in non-human animals, particularly companion animals such as cats and dogs, is rarely known.

In particular, it is unknown whether and on which tissues specifically the trimeric non-functional receptor is expressed in living tissues in a companion animal. Further, it is unknown whether the expression can be found on cancer tissue and, if found, to what extent the expression will be restricted to cancer tissue. Thus, whether certain companion animal cancers express non-functional P2X₇It is unknown whether the receptor, or treatment with the antibody, will have significant toxicity to normal cells.

Further, it is unknown whether cancer-specific epitopes observed on live cancer tissues in humans are present in cancer tissues of companion animals. This is directly related to the problem of how to generate anti-cancer antibodies in companion animals at the time of the present invention. In particular, cat P2X₇The receptor sequence is completely unknown.

Known dog P2X₇Sequence and human P2X₇The receptor differs significantly in the region of the dog sequence corresponding to E200 in humans, where the carboxyl group is immediately added to the key proline 210 (known to produce the non-functionality of the human receptor in humans), which is known to replace the neutral glycine with a negatively charged aspartic acid non-conservatively as in humans. Further, among other amino acid differences between dogs and humans, at least about 55% of these are known to be non-conservative substitutions, and one insertion is not found in humans. See in particular fig. 2.

In summary, at the time of the present invention, no non-functional P2X was known₇Whether the receptor is present in companion animals (including dogs and cats) and therefore P2X is unknown₇Whether the receptor can be used as a biomarker as already used in humans for cancer treatment in companion animals.

As described herein, the inventors have shown that sheep antibodies raised against a peptide having the E200 epitope are highly effective for the treatment of cancers with higher incidence in companion animals with minimal side effects or toxicity. Thereby a plurality ofThe inventors have recognized that certain live cancer cells express non-functional P2X in dogs and cats₇A receptor. Given minimal toxicity, expression of these receptors appears to be restricted to pre-tumoral or tumoral tissues. In addition, and despite differences (e.g., in dogs and humans P2X)₇Between sequences), but these companion animal receptors appear to have an extracellular configuration much like the human E200 epitope has been found on them.

Additionally, and surprisingly, the inventors have recognized that anti-xenogenic P2X₇Xenogeneic antibodies raised against the immunogen are highly effective for treating cancer in companion animals including cats and dogs.

Accordingly, in a first aspect, the present invention provides a method for minimising the progression of cancer in a companion animal, the method comprising the steps of:

providing a companion animal in which progression of cancer is to be minimized; and is

Providing in the animal a whole antibody or a fragment thereof comprising a variable domain for binding to a non-functional P2X expressed by the animal₇On the receptor;

thereby minimizing cancer progression in the animal.

In a further aspect, the invention provides the use of a whole antibody or a fragment thereof comprising a variable domain for binding to a non-functional P2X in the manufacture of a medicament for the treatment of cancer in a companion animal₇On the receptor.

In still further aspects, the invention provides the use of a whole antibody or a fragment thereof comprising a variable domain for binding to a non-functional P2X for the treatment of cancer in a companion animal₇On the receptor.

An animal to be treated according to the method of the first aspect of the invention may be an animal that has received, or is about to receive, any therapeutic antibody indicated for use in a tumour.

In one embodiment of the first aspect, the antigen binding site of an antibody may be P2X that distinguishes between functional and non-functional₇The antigen binding site of the receptor, and thus binds to a non-functional receptor rather than a functional receptor. Examples of such antigen binding sites are those that bind to the E200 epitope, the E300 epitope, or a complex epitope, for example in PCT/AU2002/000061, PCT/AU2002/001204, PCT/AU2007/001540, PCT/AU2007/001541, PCT/AU2008/001364, PCT/AU2008/001365, PCT/AU2009/000869, and PCT/AU2010/001070, all of which are incorporated by reference.

An antigen binding site may take the form of a whole antibody, or a whole antibody fragment (e.g., a Fab, Fab ', F (ab')₂And Fv), a single chain Fv, or a single variable domain.

The antigen binding site may be syngeneic, allogeneic or xenogeneic with respect to the companion animal to which it is to be subjected for cancer therapy.

Typically, the antigen binding site is non-autonomous or foreign, meaning that it has been discovered or isolated outside of the animal treated according to the methods of the invention.

The antigen binding site may be affinity matured.

The antigen binding site may have a variety of specificities or valencies.

The antigen binding site may be adapted so as to be suitable for administration by a selected method.

The antibody may be a whole antibody of any isotype. The antibody may be an antibody obtained from a monoclonal antibody or a polyclonal antiserum. The antibody may be produced from a hybridoma, or expressed recombinantly, or may be obtained from serum (e.g., from a mammal, particularly a human or mouse). The antibody may also be obtained from an avian species.

The antibody may be chimeric, i.e. an antibody comprising a human variable domain and a non-human constant domain. Alternatively, it may be humanized, i.e., an antibody formed by grafting non-human CDRs onto a human antibody framework. Still further, the antibody may be fully human.

The antibody may be modified with respect to effector function to enhance it, for example to enhance the effectiveness of the antibody in treating cancer.

Where the antibody is an antibody fragment, the antibody fragment is selected from the group consisting of: dAb, Fab, Fd, Fv, F (ab')₂scFv, and CDR.

The magnitude of the dose, frequency of dose, route of administration, and the like are described in detail below.

Methods of making and administering antibodies to animals in need thereof are well known to, or readily determinable by, those of skill in the art. The route of administration may be, for example, oral, parenteral (e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intradermal, rectal or vaginal), by inhalation or topical administration. One form of administration would be a solution for injection, in particular for intravenous or intraarterial injection or instillation, comprising a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizer (e.g. albumin). In other methods, the antibody may be delivered directly to the site of disease, thereby increasing exposure of diseased cells or tissues to the antibody.

Formulations for parenteral administration include sterile aqueous (aqueous carriers include water, alcohol/water solutions, emulsions or suspensions, including saline and buffered media) or non-aqueous (non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil) and injectable organic esters (e.g., ethyl oleate)) solutions, suspensions and emulsions. Pharmaceutically acceptable carriers include 0.01M-0.1M and preferably 0.05M phosphate buffer or 0.9% saline. Other common parenteral vehicles include sodium phosphate solutions, ringer's dextrose, dextrose and sodium chloride, lactated ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutrient supplements, electrolyte supplements (such as those based on ringer's glucose), 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.

More specifically, pharmaceutical compositions suitable for injectable use comprise sterile aqueous solutions (where appropriate water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions, in which case the compositions 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 preferably 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 (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating (e.g., lecithin), by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Suitable formulations for use in the treatment methods disclosed herein are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., 16 th edition (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. In many cases, it will be preferred to include isotonic agents, for example, sugars, polyols (e.g., 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, aluminum monostearate and gelatin.

In any event, sterile injectable solutions can be prepared by incorporating an active compound (e.g., an antigen-binding site) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a base dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a sterile-filtered solution thereof. Formulations for injection are processed, filtered into containers such as ampoules, bags, bottles, syringes or vials, and sealed under sterile conditions according to known art. In addition, these preparations may be packaged and sold in the form of a kit. Such articles of manufacture preferably have a label or package insert (package insert) indicating that the relevant composition is useful for treating a subject suffering from or susceptible to a disorder.

The effective dosage of the compositions of the invention for treating a disorder as described herein will vary depending upon a number of different factors, including the means of administration, the site of interest, the physiological state of the patient, whether the patient is human or animal, other medications administered, and whether the treatment is prophylactic or therapeutic. Therapeutic dosages may be escalated using routine methods known to those of ordinary skill in the art to optimize safety and efficacy.

For treatment of certain disorders with an antibody, the dosage may range, for example, from about 0.0001mg/kg to 100mg/kg of host body weight, and more typically 0.01mg/kg to 5mg/kg (e.g., 0.02mg/kg, 0.25mg/kg, 0.5mg/kg, 0.75mg/kg, 1mg/kg, 2mg/kg, etc.). For example, the dose may be 1mg/kg body weight or 10mg/kg body weight or in the range 1mg/kg-10mg/kg, preferably at least 1 mg/kg. Dose medians within the above ranges are also intended to be within the scope of the present invention. The subject may be administered this dose daily, every other day, weekly, or any other regimen determined from empirical analysis. Exemplary treatments require administration of multiple doses over an extended period of time (e.g., at least six months). Additional exemplary treatment regimens require administration once every two weeks or once every month or once every 3 to 6 months. Exemplary dosage regimens include 1mg/kg to 10mg/kg or 15mg/kg for consecutive days, 30mg/kg every other day or 60mg/kg weekly. In some methods, two or more antigen binding sites with different binding specificities are administered simultaneously, in which case the dose of each antigen site administered falls within the indicated range.

Administration for binding to a non-functional P2X expressed on a cell can be carried out in a variety of circumstances₇An antibody to the receptor. The interval between single doses may be weekly, monthly or yearly. The intervals may also be irregular, as indicated by measuring blood levels of the target polypeptide or target molecule in the patient. In some methods, the dose is adjusted to achieve a plasma polypeptide concentration of 1 μ g/mL to 1000 μ g/mL, and in some methods 25 μ g/mL to 300 μ g/mL. Alternatively, the antibody may be applied as a sustained release formulation, in which case less frequent application is required. The dose and frequency will vary depending on the half-life of the antibody in the patient. The half-life of an antibody can also be extended by fusion to a stable polypeptide or moiety, such as albumin or PEG. Generally, humanized antibodies exhibit the longest half-life, followed by chimeric antibodies as well as non-human antibodies. In one embodiment, the antibody may be administered in an unbound form. In another embodiment, the antibody may be administered multiple times in an unbound form. In some therapeutic applications, higher doses are sometimes required (e.g., up to 400mg/kg of anti-P2X₇Binding molecules, e.g., antibodies per dose) at short intervals until progression of the disease is reduced or ended, and preferably until the patient exhibits partial or complete amelioration of the symptoms of the disease. When the antibody is bound to a radioisotope or cytotoxic drugThe amount may be several log levels lower (i.e., 2 to 3 log levels lower).

The therapeutic agent may be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal, or intramuscular means for prophylactic and/or therapeutic treatment, in some methods the agent is injected directly into which the non-functional P2X is administered₇In particular tissues where recipient cells have accumulated, such as intracranial injection. Intramuscular injection or intravenous infusion is preferred for administration of the antibody.

An antibody may optionally be administered in combination with other agents that are effective (e.g., prophylactic or therapeutic) in the treatment of a disorder or condition in need of treatment. Examples are those agents commonly used in oncology for chemotherapy or radiotherapy. Additionally or alternatively, the antibody or agent may be administered before, during or after surgical treatment for resection or removal of tumors or tissues.

According to a first aspect of the invention, in one embodiment, the method may be used for the treatment of cancer, in particular for the therapeutic treatment of cancer.

In one embodiment, the purpose or result of the treatment is one or more of the following:

reducing the number of cancer cells;

reducing the size of the primary tumor;

inhibit (i.e., slow to some extent or preferably stop) cancer cell infiltration into peripheral organs;

inhibit (i.e., slow or preferably stop to some extent) tumor metastasis;

inhibit tumor growth to some extent;

to alleviate to some extent one or more of the symptoms associated with the disorder.

In one embodiment, the method of the first aspect of the invention is for prolonging the time to progression of the disease.

In one embodiment, the method of the first aspect is for prolonging survival of an animal, including overall survival time together with progression free survival.

In one embodiment, the method of the first aspect is used to provide a complete response to treatment whereby all signs of cancer in response to treatment have disappeared.

In one embodiment, the method of the first aspect is for providing a partial response to treatment whereby there has been a reduction in the size of one or more tumours or lesions or in the extent of cancer in the body in response to treatment.

In one embodiment, animals in need of treatment include those with benign, precancerous, or non-metastatic tumors.

In one embodiment, the cancer is precancerous or preneoplastic.

In one embodiment, the cancer is a secondary cancer or a metastatic tumor. The secondary cancer may be located in any organ or tissue, and particularly those with relatively higher hemodynamic stress, such as the lung, kidney, pancreas, intestine, and brain.

In one embodiment, the cancer may be substantially undetectable.

Such companion animal may be a cat or dog. However, in view of the cross-reactivity of antibodies raised against human proteins for non-primate receptors, the inventors have recognized that other non-human mammals with as far as human phylogeny would benefit from the present invention. In one embodiment, the companion animal is a high value animal or elite animal. One example is a horse.

Typically, the cancer or pre-cancer to be treated is one that has a higher incidence in a given companion animal. For example, when the companion animal is a dog, the cancer can be lymphoma, mast cell tumor, soft tissue sarcoma, angiosarcoma, osteosarcoma, squamous cell carcinoma, breast cancer, melanoma, histiocytoma, spindle cell carcinoma, or fibrosarcoma.

When the companion animal is a cat, the cancer may be lymphoma, mast cell tumor, squamous cell carcinoma, breast cancer, angiosarcoma, osteosarcoma, fibrosarcoma, or sebaceous hyperplasia/adenoma.

Other examples of cancers are described in table I or include blastoma (including medulloblastoma, retinoblastoma), sarcoma (including liposarcoma and synovial sarcoma), neuroendocrine tumors (including carcinoid, gastrinoma and islet cell tumor), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, leukemia or lymphoid malignancy, lung cancer (including small cell lung cancer (SGLG), non-small cell lung cancer (NSGLG), lung adenocarcinoma and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric or gastric 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 cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland carcinoma, pancreatic carcinoma, or combinations thereof, Kidney or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct cancer, as well as head and neck cancer.

The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions comprising the antibodies or mixtures thereof are administered to a patient who is not in a disease state or a pre-disease state to enhance the patient's resistance. Such an amount is defined as a "prophylactically effective dose". In such use, the precise amount again depends on the health status of the animal and on systemic immunity, but typically ranges from 0.1mg to 25mg per dose, especially 0.5mg to 2.5mg per dose. Lower doses are administered at less frequent intervals over a long period of time. Some animals continue to receive treatment for the rest of their lives.

In therapeutic applications, higher doses (e.g., from about 1mg/kg to 400mg/kg of binding molecule, e.g., per dose of antibody, doses from 5mg to 25mg are more commonly used for radioimmunoconjugates, and higher doses for cytotoxic-drug binding molecules) are sometimes required at shorter intervals until progression of the disease is reduced or ended, and preferably until the animal shows partial or complete amelioration of disease symptoms.

In one embodiment, the antibody is provided in combination with another chemotherapeutic or anti-tumor compound indicated for use in a cancer of interest in a companion animal in need of treatment. Examples of these compounds and related indications are described in table 1.

In another embodiment, the antibody is provided pre-, during or post-clinical surgery selected from the group consisting of: surgery, radiation therapy, hyperthermia, photodynamic therapy, chemotherapy, gene therapy and immunotherapy.

C. Cancer immunotherapy by immunization

In a second aspect, the present invention provides a method for minimizing cancer progression in a companion animal, the method comprising the steps of:

providing a companion animal in need of cancer treatment; and

the formation of a protein directed against non-functional P2X in this companion animal₇The immune response of the recipient;

thereby minimizing cancer progression in this companion animal.

In a third aspect, the present invention provides a method for minimizing cancer progression in a companion animal that has received a non-self antigen binding site, for use in cancer therapy, comprising the steps of:

providing a companion animal that has received a non-self antigen binding site for cancer therapy;

the formation of a protein directed against non-functional P2X in this companion animal₇The immune response of the recipient;

thereby minimizing cancer progression in this companion animal.

In another aspect, the invention provides a non-functional P2X₇Use of a receptor or fragment thereof in the manufacture of a medicament for the treatment of cancer in a companion animal, particularly an animal, such as a cat or dog, which has received a non-self antigen binding site for cancer treatment.

In yet another aspect, the invention provides a non-functional P2X₇Use of a receptor or fragment thereof for the treatment of cancer in a companion animal, particularly an animal, such as a cat or dog, which has received a non-self antigen-binding site for cancer treatment.

In another aspect, the invention provides a non-functional P2X₇Use of a receptor or fragment thereof in the manufacture of a medicament for treating or inhibiting cancer progression in a companion animal that has received an anti-non-functional P2X₇Receptor antigen binding sites are used for cancer therapy.

In one embodiment of the second and third aspects of the invention, the companion animal may not have detectable non-self antigen binding sites in the circulation when the immune response is formed in the companion animal. For example, the infused antibody may have been cleared from the plasma at the time of immunization. In addition, the companion animal may be free of detectable cancer when the immune response is developed in the companion animal, e.g., the cancer may have a substantially reduced size, mass, or other physical measure when the immune response is developed in the companion animal as a result of administration of an antigen binding site to the companion animal.

According to the second and third aspects of the invention, the immune response may be developed by an immunogen. The immunogen may be one capable of inducing a non-functional P2X₇P2X for the immune response of the receptor in this companion animal₇The receptor is in the form of either a P2X receptor₇Fragments of the receptor are provided. A non-functional P2X₇Receptors are defined as having at least one of three ATP binding sites formed at the interface between adjacent properly packaged ATP-incapable monomers. This receptor is unable to expand the opening of non-selective calcium channels to apoptotic pores. The immunogen may comprise at least one sequence capable of being present on a major histocompatibility complex class II molecule and/or capable of interacting with a T or B cell receptor or a B cell membrane-bound immunoglobulin. Typically, such companion animal is a cat or dog, in which case the immunogen may be provided in the form of: a cat or dog P2X₇Receptors, or their ability to induce P2X₇A fragment of the immune response of the receptor. Dog P2X₇The receptor sequences are shown in figure 2 herein. Typically, the immune response developed in such companion animals is non-functional to P2X₇The receptor is specific, in which case non-functional P2X is formed in the companion animal₇Receptors (i.e. non-ATP-binding receptors) that are reactive but not reactive with functional P2X₇Antibodies or cellular components that react with the receptor (i.e., ATP-binding receptor).

In a preferred form of the second and third aspects of the invention, the immunogen is provided to the companion animal in one initial administration, thereby generating a response that includes IgM production. In a further preferred form, the companion animal has been provided with an immunogen in an initial administration, thereby generating a response that includes IgM generation, and at a later time, in a further administration relative to the initial administration, the immunogen is administered, thereby generating a response that includes IgG generation. In this example, additional administration of the immunogen typically occurs when circulating IgM levels in the companion animal are substantially undetectable.

The immune response formed according to the second and third aspects of the invention may be a humoral response and/or a cellular response. Humoral responses may include transformation of B cells into antibody-secreting plasma cells, Th2 activation and cytokine production, proliferation center formation and isotype switching, affinity maturation of B cells, and/or memory cell production. The cellular response may include activation of antigen-specific cytotoxic T-lymphocytes, activation of macrophages as well as natural killer cells and/or stimulation of cytokine secretion by cells. The humoral and/or cellular responses developed within the companion animal can treat or reduce cancer in such companion animal, or minimize cancer progression in such companion animal.

In the above examples of the second and third aspects of the invention, the antigen binding site received by such a companion animal may be reactive with any cancer-associated biomarker. Examples include anti-P2X₇In particular non-functional P2X₇anti-VEGF, especially VEGF A, C or D, Her-2, CD20 or other antigen binding site. Typically, the antigen binding site received by such companion animals is with P2X₇Receptors, especially non-functional P2X₇Receptor reaction.

In another embodiment of the second and third aspects of the invention, there is provided a composition for treating or for inhibiting cancer progression in a companion animal, the composition comprising P2X₇A receptor or a fragment thereof. Preferably, the composition further comprises a carrier, excipient or diluent. Preferably, the composition further comprises an adjuvant. In a preferred form, the composition is such that upon initial administration of the immunogen to the companion animal, a primary immune response (including IgM production) is formed and such that upon administration of the immunogen after initial administration, a secondary immune response (including IgG production) is formed.

Without being bound by any theory or mode of action, it is believed that the above-described embodiments in relation to the second and third aspects of the invention provide an alternative and/or improved treatment regime, asFor endogenous immune components (e.g., antibodies and antigen-specific cells resulting from immunization) in the already completed administration of the antigen binding site, and non-autologous anti-P2X₇After circulating levels of antigen binding sites become undetectable, cell surface P2X is provided₇More prolonged and higher exposure of the receptor. In addition, P2X is generally recognized₇Receptor aggregation (produced with the provision of high concentrations of non-autologous or exogenous antibodies in companion animals) minimizes binding to critical P2X that provides an anti-cancer immune response₇The level of antibodies specific for the epitope, thereby limiting the efficacy of immunotherapy. The inventors found that immunization of a companion animal avoids receptor aggregation when the circulating levels of non-autoantibodies are substantially undetectable, and this improves the efficacy of immunotherapy, particularly when the cancer in the companion animal may be substantially undetectable.

In one embodiment of the second and third aspects of the invention, the animals selected for treatment have not been treated with antibody immunotherapy or other forms of therapy. In another embodiment, the animals selected for treatment according to the above methods are those that have received, or continue to receive, antibody immunotherapy for cancer treatment. Antibody immunotherapy generally refers to the administration of exogenous (also referred to as "non-autologous") antibodies to an animal in need of treatment, as in the case of adoptive transfer of antibodies. For example, the animal may have received any of the therapeutic antibodies that have received a registration approval for an indication relating to oncology. Avastin, herceptin, rituximab are examples. Typically, the animal has received or is continuing to receive an anti-P2X₇A receptor antibody. Suitable anti-P2X₇Examples of antibodies, their production, manufacture, use and administration to a companion animal are described in the examples relating to the first aspect of the invention.

In addition, the animals selected for treatment according to the second and third aspects of the invention may or may not have detectable cancer at the time of treatment. When the animal does not have detectable cancer, the method further comprisesA primary or secondary humoral response is readily detected because there is very little non-functional P2X available in the event that the cancer is present in a substantially undetectable amount₇Receptors to remove IgM or IgG from body fluids.

The types of cancer that may be treated according to the second and third aspects of the invention and the desired therapeutic outcome are as described in the examples relating to the first aspect of the invention.

The treatment according to the second and third aspects of the invention is aimed at in vivo in animals by the convection of P2X₇Induction or development of an immune response by the receptor to at least minimize cancer progression. Therefore, the animal selected for treatment must be capable of generating an adequate immune response to meet this objective. Typically, the desired immune response includes the ability of the animal to produce one or both of circulating IgM and IgG when challenged with cancer, such as in a cancer relapse.

Typically, the animal selected for treatment will be one with a white blood cell fraction count within normal parameters.for example, cats used for inclusion will typically have a white blood cell count within the range of 5.5 to 19.0 × 10⁹between/L, or lymphocyte counts of 0.9 to 7.0 × 10⁹between/L cat neutrophil counts may range from 2 to 13 × 10⁹between/L, monocyte count < 0.7 × 10⁹/L, eosinophils less than about 1.1 × 10⁹A basophil of less than about 0.1 × 10⁹For example, dogs for inclusion typically have a white blood cell count of 4.5 to 17.0 × 10⁹between/L, or lymphocyte counts of 0.9 to 3.5 × 10⁹Dog between/L the neutrophil count can range from 3.5 to 12 × 10⁹between/L, monocyte count < 1.2 × 10⁹/L, eosinophils less than about 1.5 × 10⁹A basophil of less than about 0.1 × 10⁹/L。

It will be understood that in certain embodiments, the cell count for any one of the blood cell components may fall outside of these stated ranges, particularly in cases where the animal has a form of blood cancer, e.g., CML, CLL, etc.

One factor of importance is usually the lymphocyte count and/or the monocyte count. In more detail, for these components, when one or both of these counts is significantly below the stated range, the animal will be less likely to respond to administration of the immunogen.

While the animal continues to receive antibody immunotherapy, in one embodiment of the second and third aspects of the invention, the antibody immunotherapy is allowed to continue until the desired clinical endpoint. Typically, the desired clinical endpoint is a reduction in cancer to a substantially undetectable level. The animal is then assessed for the development or production of P2X during or at the end of immunotherapy₇The ability of the recipient to respond immunologically. When the evaluation revealed that the animal was likely to be treated with P2X₇When immunization with the immunogen is beneficial, the animal immunogen is administered.

In a preferred form of the second and third aspects of the invention, the level of circulating non-self or foreign antigen binding sites in the animal resulting from antibody immunotherapy is substantially undetectable when the immune response is formed in the animal. Importantly, a key finding of the present inventors is that the efficacy of antibody therapy is reduced at higher circulating concentrations of the antibody binding site, particularly when the cancer cells are at very low copy numbers, or otherwise substantially undetectable. This is believed to be a low copy number of non-functional P2X on cancer cells relative to the high concentration of antigen binding sites generated in standard antibody immunotherapy₇One function of the receptor. Specifically, in this example, the inventors have discovered that non-functional P2X of the antigen binding site by blocking antigen-specific binding of the receptor increases with circulating levels of the antigen-specific binding site and decreases in the number of cancer cells₇Aggregation of receptorsIs much more likely. This blockade increases the likelihood of the expected cytotoxic, apoptotic, or other effects of antigen-specific binding by the antigen binding site, which would not be possible. The level of circulating foreign antigen binding sites can be determined by a human by any standard serological technique capable of detecting antibodies in body fluids, a preferred example being an ELISA using antibodies to capture the antigen binding sites.

In addition to the above, but not wanting to be bound by hypothesis, the inventors contemplate that administration of immunity in the presence of infused antibody increases the risk that the infused antibody can bind to the immunogen, resulting in the formation and clearance of immune complexes, thereby avoiding antigen presentation and induction of antigen-specific immunity. Thus, in certain embodiments, it is particularly useful to wait until the level of non-self or foreign antigen binding sites has been cleared from circulation prior to induction of an antigen-specific immune response to an immunogen.

The methods of the second and third aspects of the invention described herein require the use of P2X₇Receptor (especially a non-functional P2X₇Recipient) to develop an immune response in the treated animal. Generally, the immunogen used for this purpose is a non-functional P2X₇Receptor, not for functional P2X₇An immunogen for which the receptor elicits an immune response.

The inventors found that the composite epitope is present in a wide range of species, meaning that according to the second and third aspects of the invention herein, (i) the antigen binding site or antibody can be cultivated in a wide range of animals for antibody infusion therapy and (ii) animals of a wide range of species can be treated with active immunotherapy. The following table demonstrates the% identity of human composite epitopes and epitopes in other species.

Table 2: alignment of human E200/E300 with other species

The immunogen may comprise or consist of: comprising a P2X₇A peptide of the sequence of the receptor. The peptide may comprise at least one sequence capable of being present on a major histocompatibility complex class II molecule, or capable of interacting with a B cell receptor or a B cell membrane-bound immunoglobulin. Typically, the peptide comprises a non-human, preferably companion animal P2X₇A sequence of a receptor or fragment thereof.

A range of peptide immunogens are known and discussed in PCT/AU2002/000061, PCT/AU2008/001364 and PCT/AU2009/000869, the contents of which are incorporated herein in their entirety.

Inclusion in these specifications for non-functional P2X is described below₇Exemplary peptide immunogens of epitopes for which the receptor generates an immune response.

As discussed above, in one embodiment, the peptide immunogen comprises part or all of canine or feline P2X₇A receptor sequence.

It will be appreciated that these are merely examples of possible immunogens which are useful for generating an immune response according to the second and third aspects of the invention. In addition, the present invention includes the use of non-functional P2X as described in these applications₇Use of other peptides useful for the receptor to form an immune response.

Typically, the immunization regimen involves 2 or more immunizations. In the first immunization, the subject may develop an IgM response to the immunization. The second immunization may develop an IgG response. Further immunization may increase the IgG response.

When the immunogen is a peptide, the peptide may be provided in an amount of about 0.1mg to 1mg per administration, preferably about 0.25mg to 0.75mg, preferably about 0.5mg in a large dog, but halved in a puppy or kitten.

Another administration of about 0.3mg of peptide may be used as a boost in a large dog, but halved in a puppy or cat.

In one embodiment of the second and third aspects of the invention, the first immunization is performed when circulating levels of antigen binding sites that have been administered for antibody immunotherapy are substantially undetectable. In other words, circulating antibodies to the relevant cancer biomarkers cannot be detected in peripheral blood. The level of IgM production was then monitored for the next few weeks. The level of IgM antibodies may have decreased to negligible circulating levels about 4 to 5 weeks after the first immunization. A second immunization was then performed at this point and the level of IgG production was measured for the next few weeks. The immunity can be further tested in the next several months/years and increased immunity can be provided as needed.

As discussed above, the immune response may target a biomarker that is different from the biomarker that has been targeted by antibody immunotherapy. For example, anti-CD 20 antibodies may be used for antibody immunotherapy, and one non-functional P2X₇The immunogen is used to generate an immune response.

In another embodiment of the second and third aspect of the invention, one single biomarker is targeted by antibody immunotherapy and immunization. For example, a point P2X₇Monoclonal antibodies to an epitope on the receptor (e.g., the E300 epitope) can be used for antibody immunotherapy and to form antibodies targeting P2X₇Immunogens of the immune response to the different epitopes on (e.g., the E200 epitope) may be used for immunization.

A peptide immunogen for use herein in the second and third aspects of the invention may have a length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 residues.

In one embodiment of the second and third aspects of the invention, the immunogen used to generate the immune response according to one method of the invention is a vaccine with P2X₇A peptide of the sequence of the receptor, P2X₇The receptor may or may not have Pro210 in the cis (cis) conformation.

The immunogen may be P2X₇Extracellular domain or P2X₇A form of any one or more of the isoforms. The immunogen may be provided for administration in soluble form or associated with a solid phase (e.g., cell membrane, bead, or other surface).

Disclosed herein are methods for screening peptides that can be used as immunogens to generate an immune response according to the methods of the invention herein. One example includes the use of red blood cells in a rosette test. In this assay, an antibody that binds to a functional receptor is used as a positive control in which rosettes are observed. If a rosette cannot be formed, it is determined that the test antibody is unable to bind to a functional receptor. Binding to a non-functional receptor is determined if it is observed to bind to a cell line expressing a non-functional receptor, including those discussed herein.

The peptides of the invention can be made by any of a number of techniques known in the art, including solid phase synthesis and recombinant DNA techniques.

As is known in the art, carriers are substances that can be bound to peptide epitopes thereby enhancing immunogenicity. Some vectors do this by binding to multiple peptides, thereby providing an antigen with an increased molecular weight to the host in which an immune response is to be developed.

Preferred carriers include bacterial toxins or toxoids. Other suitable carriers include neisseria meningitidis outer membrane protein, albumin (e.g. bovine serum albumin), synthetic peptides, heat shock proteins, KLH, pertussis protein, protein D from haemophilus influenzae, and toxin A, B or C from clostridium difficile.

When the carrier is a bacterial toxin or toxoid, diphtheria toxoid or tetanus toxoid is preferred.

Preferably, the carrier comprises functional groups that can react with the peptide of the invention, or can be modified to be able to react with the peptide.

The immunogen may be administered subcutaneously, intradermally, and/or intramuscularly.

In a preferred form, for one P2X₇The receptor forms an immune response the compositions used in the methods of the invention described herein include adjuvants or compounds for enhancing the immune response.

A wide variety of adjuvants are known; see also Allison (Ellison) (1998, Dev. biol. Stand. (developmental biology standards), 92: 3-11) (incorporated herein by reference), Unkeless et al (annual review in immunology), 6: 251. immunol. 281) and Phillips et al (1992, Vaccine, 10: 151. 158). Exemplary adjuvants that can be utilized in accordance with the invention include, but are not limited to, cytokines, aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, etc.; Baylor et al (Vaccine, 20: S18, 2002)), gel-type adjuvants (e.g., calcium phosphate, etc.); microbial adjuvants (e.g., immunoregulatory DNA sequences including CpG motifs; endotoxins such as monophosphoryl lipid A, Ribi (Leibo) et al (1986, Immunology and Immunopharmacology of bacterial endotoxins, Plenum Publ.Corp. (Prolan publishing Co., Ltd.), NY, p407, 1986); exotoxins such as cholera toxin, E.coli heat-labile toxin, and pertussis toxin, muramyl dipeptide, etc.); oil emulsions and emulsifier-based adjuvants (e.g., freund's adjuvant, MF59[ Novartis (Novartis) ], SAF, etc.); particulate adjuvants (e.g., liposomes, biodegradable microspheres, etc.); synthetic adjuvants (e.g., non-ionized block copolymers, muramyl peptide analogs, polyphosphazenes, synthetic polynucleotides, etc.); and combinations thereof. Other exemplary adjuvants include polymers (e.g., polyphosphazenes; described in U.S. Pat. No. 4, 5,500,161), Q57, saponins (e.g., QS21, Ghochikyan et al, (vacine (Vaccine)), 24: 2275, 2006), squalene, tetrachlorodecaoxide (tetrachlorodecaoxide), CPG7909(Cooper et al, (vacine (Vaccine)), 22: 3136, 2004), poly [ di (carboxyphenoxy) phosphazene ] (PCCP; Payne (Payne) et al, (Vaccine)), 16: 92, 1998), interferon- γ (Cao (Cao) et al, (Vaccine), 10: 238, 1992), block copolymer P1205(CRL 1005; Katz) et al (Vaccine)), 18: 2177, 2000), interleukin 2 (IL-Wei 2; Wuuike (Mbout) et al (1990), polymethyl methacrylate (PMMA), (j.pharm.scl (journal of pharmaceutical science)), 70: 367, 1981), etc.

In one embodiment of the second and third aspects of the invention, a phage comprising a P2X is provided on a phage surface for immunization of an animal according to a method of the invention described herein₇A peptide immunogen of a receptor sequence.

In yet a further aspect, the present invention provides a kit or composition for use in cancer treatment in a companion animal, the kit comprising:

a whole antibody comprising a variable domain, or a fragment thereof; or

A P2X₇The receptor, or a P2X₇A fragment of a receptor;

written instructions for use in one of the methods described herein.

Preferably, the antibody or fragment binds to a P2X₇Receptor, preferably a non-functional P2X₇A receptor. More preferably, the antibody or fragment does not bind to functional P2X₇On the receptor.

Preferably, the written instructions are used in the form of a label or package with instructions for use in a method described herein.

Examples of the invention

Example 1 Cat 1(GB)

Extensive SCC (squamous cell carcinoma) scalp and lips, up to 4cm in area²And occupies the full thickness of the lips. Prior to treatment, the left lip of SCC had spread to the angle of the mandible, subcutaneous. Before treatment, SCC left lip was stained with IHC (immunohistochemistry) for nf-P2X₇A target receptor.

Partial efficacy with intratumoral (i.t.) injection

As IHC revealed, the target receptors are widely distributed in the infected tissue

Administration at a dose of 3mg/kg at fluid maintenance rate, single infusion targeting nf-P2X₇Cancer specific therapeutic antibodies to receptors

By day 4, there was clear evidence of tumor clearance

By day 14, after a single dose of IV (i.v.) at 3mg/kg, 2 weeks of treatment, most of the SCC left lip tumors were eliminated. After 2 weeks of treatment at 3mg/kg IV, the SCC clusters above the left eye were also cleared. Now, coat regrowth becomes evident at the original lesion site

After 3 IV, the scalp SCC and lip SCC were largely removed within 5 weeks despite signs of new epithelial tissue scratch by the sick cat. The central SCC lesion located in the mandibular midline, penetrating into the bone, now has the appearance of white nodular scar tissue. Unlike the condition in which there is significant severe pain at the beginning of treatment, the diseased cat is not annoyed by having a treated lesion site.

After 6 months of IV treatment, the condition resolved, with even new bone growth revealed via X-ray in the inner mandible

Over time, the total cumulative dose affecting clinical clearance was estimated to be 30mg/kg to 40mg/kg due to loss of efficacy due to the presence of anti-sheep antibodies

Example 2 Cat 2(AL)

SCC on nose before i.t. injection

Only 1 xi.t. injection, since cat 21 is old, cortical blindness due to general anesthesia persists for several hours

Subsequent use of topical anti-nf-P2X₇IgG

5 days-SCC formation of new tissue at the nose

19 days-SCC more new tissue formation by the nose

Example 3 Cat 3(PC)

Pancreatic cancer with concomitant metastasis to liver and mesentery

Ultrasound scanning after 2 weeks intravenous (i.V.) infusion showed no apparent mesenteric metastasis on the 1cm scale

Some signs of tumor lysis (fluid) by ultrasound scanning after 2 weekly iv injections

After 4 weeks, secondary liver had contracted from 5cm to 2cm in diameter

After 4 weeks, a primary pancreas of 5-6cm diameter has about 80% of its liquefied mass

After the end of week 5, euthanasia was performed due to the pancreatic tumour lysis/bleeding to the abdomen and the requirements of the owner

Example 4 Cat 4(FH)

Nasal SCC extending from left nostril 2cm to bridge of the nose

After surgical treatment at the end of 2008, biopsy revealed extensive recurrent SCC

After a single intravenous administration of 5mg/kg, the tumor volume on the upper nasal surface decreased and the sponge consistency on the surface became firm

After a second weekly infusion, the lesion is no longer evident. A third infusion was applied and at the end of 2 weeks no clinical signs of all lesions were present. Additional infusions were applied to ensure elimination of residual pre-tumor cells that may already be present.

Clinical clearance at a total dose of 18mg/kg over 21 days

No relapse occurs after 14 months, so the diseased cat appears to be completely cured

Example 5 Cat 5(CO)

Medical history

Sick cats removed Bowen's-like lesions from the left temporal region in 2008. In a study in march 2009 it was noted that the lesion had reappeared and a new lesion appeared on the opposite side. These are biopsies with the following results:

diagnosis of

Multicentric squamous cell carcinoma in situ with moderate to severe blood cell encrustation, epidermal scarring, and moderate perivascular eosinophilic dermatitis. Skin biopsy from left ear.

Moderately hyperplastic perivascular and interstitial eosinophilic dermatitis is associated with moderate epidermal lamellar fibroplasia. Biopsy from the neck.

Multicenter squamous cell carcinoma in situ with moderate hyperpigmentation, moderate red cell encrustation, and extensive skin scarring in the larger cranial region. Biopsy from the right ear.

Comment on

The cat has multiple foci of tumorigenic transformation of epidermis and follicular tissue, consistent with multicenter squamous cell carcinoma in situ or bowman's disease. These lesions were similar to biopsies submitted in june 2008. In the examined sections, there was no evidence of squamous infiltrating carcinoma.

In situ SCC (Bowen's)

Resistance to nf-P2X for sheep infused at 5mg/kg₇IgG has no adverse reaction

After 2 weeks, the lesions had decreased significantly but were still extensive, and a second infusion was administered due to the initial improvement

All extensive bowden-like lesions were cleared after the study by the veterinary dermatologist who noted that the deep SCC at the right auricular sulcus was only apparent after the superficial lesions and associated infections were cleared. It is necessary to avoid tearing the new epithelium by tearing the cat's paw, allowing the skin surface to heal by attaching a restraining collar.

Example 6 dog 1(CJ)

There was a history of angiosarcoma in the ventral abdomen in 30kg dogs. Because the tumor is so aggressive, multiple times post-surgery, the dog is no longer a surgical candidate. Intratumoral injection of nfP2X₇IgG and 3 IV infusions nfP2X₇Affinity purified antibodies. Intratumoral injection of 6mg of antibody into a 6cm diameter skin lesion showed an 80% reduction within one week with significant tumor lysis as a significant secretion.

1 intratumoral and 3 intravenous injections at a dose of 2-3.4mg/kg for 4 weeks

anti-nf-P2X with sheep₇Tumor volume reduction following IgG treatment

Euthanasia was performed 3/21 days in 2010 due to tumor lysis/leakage symptoms of these large tumors (total weight 1-2kg) and the requirements of the owners

Example 7 dog 2(MO)

Transitional Cell Carcinoma (TCC) with malignant ascites in autopsy pathology was found to be metastatic mesothelioma

3 intravenous injections with chemotherapy at a dose of 1-2.7mg/kg for 2 weeks

In the second week, the tumor was reduced by 15%, although ascites was also present

On week 3, at the request of the owner and against the recommendations of the veterinarian, the treatment is stopped

Example 8 dog 3(PI)

Special treatment and rehabilitation training of mast cell lymphoma between the two fingers in the middle of the anterior right paw-hard in appearance and discomfort caused by the protruding part under the footpad. The surgery may require removal of the front leg to maintain a margin of 3cm

Special treatment and rehabilitation of mast cell lymphoma-IV 5mg/kg1 weeks with a 40% reduction in volume

Mast cell lymphoma-specific treatment and rehabilitation-2 week lesions now soften and greatly diminish in volume, with no prominent parts under the footpad, and no signs of discomfort in the dogs. The solid core of the lesion is now confined to a single finger, with surrounding reaction cell capsules being quite soft.

Metastatic lesions in the subscapular lymph nodes were undetectable by week 2

The primary lesion remains largely unchanged in appearance for 3 months

Euthanasia of dogs on request of the owner, due to the primary lesion becoming ulcerated under the paw

Necropsy biopsy showed that the diseased dog had no all tumors, the primary lesion site was free of mast cells, and contained reactive lymphocytes

Lesions may have been cleared with a total dose of less than 30mg/kg

Example 9 dog 4(BE)

After extreme pain and lameness in the affected dog, osteosarcoma was diagnosed in the upper left humerus via CT scan, ready for euthanasia.

Begin weekly infusions at 10 mg/kg. Within a week, the affected dog moves with the owner again and appears to be substantially painless and wants to go to a walk.

At week 9, CT scans and post-treatment diagnostic tissue were obtained under general anesthesia, which was considered the shortest time for bone regrowth to be readily detectable

At week 9, there were no signs of tumor cells in the biopsy, while the affected dog looked very good

Sick dogs were euthanized at month 5 due to fractures in the proximal left humerus. Biopsy of the lesion site showed no evidence of residual osteosarcoma. There appears to be a clear overall remission, but the bone is still thin and brittle because of the tumor.

In this case, external splints should be applied to protect the bone while the associated treatment is performed to aid bone regrowth.

Example 10 dog 5(WC)

Metastatic mast cell lymphoma (grade 2) with a high division index (25). There are many lesions > 5cm on the ribs, shoulders and hind legs. A weekly IV infusion of 10mg/kg was performed immediately.

At the end of the first week, the total lesion volume was reduced by 75%, as shown by PI, consistent with the desire for rapid clearance.

The main lesion still had mass, but after the second infusion, it further decreased at the end of the second week

After three infusions and one week to elicit a response, the lesion site was thoroughly analyzed by ultrasound and cytology at the end of the third week by punctuation

The last needle aspiration of all lesion sites was performed after 3 months and at 6 months. There was no evidence of any mast cells.

Disease dogs were considered tumor free at 13 months.

Example 11 dog 6(HL)

Diseased dogs exhibited high grade metastatic mast cell tumors originating on the legs with popliteal lymph node metastasis.

Once weekly treatment by infusion at a dose of 10mg/kg for 6 weeks.

All swelling elimination by third week

At week 6, pathology showed no mast cells at the tumor site

The sick dogs did not relapse within one year

Example 12 antibody manufacture

Mu.g of sheep anti-P2X of the conjugate in Phosphate Buffered Saline (PBS)₇Antibody (about 100. mu.g of P2X₇Epitope) to 0.8mL and emulsified with 1.2mL of Freund's Complete adjuvant (Freund's Complete adjuvant). Sheep were both injected subcutaneously and intramuscularly at multiple sites with antigen/adjuvant emulsions. Eight weeks later, these sheep were re-injected at multiple sites with the same amount of conjugate emulsified with freund's complete adjuvant. The procedure was repeated after 4 weeks, and the animals were bled from the jugular vein. The collected sera were tested for antibody specificity. The sheep were then routinely injected and bled at eight week intervals to provide a serum pool containing specific antibodies.

Other sheep were injected with the same dose of conjugated antigen similar to the above protocol, but with different adjuvants. In these animals, 0.7mL of diluted antibody was mixed with 0.1mL of a Quill A/DEAE dextran solution (2.5 mg of Quill A +25mg of DEAE dextran per mL of PBS) and 1.2mL of ISA50V Montanide. Emulsions were injected subcutaneously and intramuscularly at multiple sites. Antibodies generated using this adjuvant gave the same specificities as those generated with freund's adjuvant.

Antibodies were raised in rabbits using the same two adjuvants as in sheep and the same injection protocol, the only difference being that a 300 μ g amount of conjugate was used for injection. Raised antibodies have the same specificity as those raised in sheep and can readily distinguish the epitopes raised against them.

Antibodies raised in mice against those bound epitopes and also against non-functional P2X₇Unbound epitopes of the epitope (which can distinguish those receptors that cannot form (from) pores and therefore cannot apoptosis). In these experiments, the adjuvant used was the product ImmunEasy from QIAGEN Pty Ltd, which contains the CpG DNA immunostimulatory product (trademark of Coley Pharmaceutical Group Inc.). 62.5 μ g of epitope or bound epitope/mouse was diluted in 60 μ L PBS and 25 μ LImmunEasy adjuvant. At multiple sites, mice were injected subcutaneously and intramuscularly. The protocol was repeated after two weeks and again after another two weeks. Mice were bled 8 days after the third injection. Antibodies raised in mice in this way can also distinguish between different P2X₇Epitope, and anti-P2X₇Antibodies to non-functional epitopes and those raised in sheep and rabbits gave results.

Example 13-Induction of an immune response in a companion animal

Materials and methods

Peptides

A highly pure peptide immunogen was synthesized in the form of GHNYTTRNILPGLNITC (SEQ ID NO: 3) to which was added the cross-linking agent maleimidocaproyl-N-hydroxysuccinimide (MCS) at the C-terminal Cys residue. The peptide was cross-linked to a carrier protein Keyhole Limpet Haemocyanin (KLH) such that the average percentage of peptide to total peptide-protein conjugate was 40%. This peptide or a similar alternative peptide GHNYTTRNILPGAGAKYYKENNVEKC conjugated to KLH constitutes a primary and a complex selective epitope target, respectively, for nfP2X to be prepared from natural receptors₇The receptor is capable of differentiation.

Adjuvant

Approved adjuvants, commonly used in human immunization, consist of a liquid formulation of hydrated aluminum gold and magnesium hydroxides plus an inert stabilizer in a gel, using Imject Alum. The peptide-protein conjugate was added dropwise at a concentration of 2.5mg/mL conjugate (1mg/mL peptide), wherein the adjuvant was mixed well in an amount equal to 0.5mL conjugate to 0.75mL adjuvant containing 0.5mg of the epitope of the target peptide.

Immunization

The immunization protocol consisted of: one total dose of 0.5mg primary vaccination with peptide (two subcutaneous injections and two intramuscular injections) followed by one month later, the accelerator was applied in the same way with 0.3mg peptide. Serum samples were collected immediately before and one week after injection. Ideally, in the anti-nfP 2X₇Administration of vaccination not less than one month after the last infusion of antibody, by residual specific anti-nfP 2X₇Antibody infusion was used to ensure that there was no isolation of the immunogen.

ELISA

Specific anti-nfP 2X measurement by ELISA₇Antibody responses. Briefly, the ELISA plate is coated with specific target peptide epitopes, to which patient serum is added at decreasing concentrations. After washing, specific human anti-nfP 2X in IgM or IgG form in patient sera was detected and determined using appropriate secondary anti-human antibodies (anti-IgM or anti-IgG type)₇The concentration of the antibody.

After inoculation, no IgG was detectable, but a small amount of IgM was detected. With this increase, IgM concentration had returned to baseline zero, while IgG was produced at a higher concentration than the initial IgM if nfP2X was not present on existing tumors₇Receptor sink (sink). In its primary tumor has been anti-nfP 2X₇In animal patients eliminated by immunotherapy, in the absence of this dip, specific endogenous anti-nfP 2X in serum was detected₇A significant population of antibodies, rated 25 mg/kg.

FIG. 3 shows the ELISA results for one dog, immunized as described above. The level of free specific circulating antibody is consistent with an initial complete clearance and a lack of recurrence of metastatic mast cell tumors.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the context or drawings. All of these different combinations constitute different alternative aspects of the invention.

Claims (6)

1. For binding to a dog non-functional P2X₇Use of a whole antibody or a fragment thereof comprising a variable domain on a receptor in the manufacture of a medicament for treating cancer or a condition or symptom associated with cancer in a dog, wherein the cancer is mast cell tumor.

2. The use of claim 1, wherein the antibody or fragment thereof does not bind to functional P2X₇On the receptor.

3. The use of claim 1, wherein the antibody or fragment thereof is from a polyclonal antiserum.

4. The use of claim 1, wherein the antibody or fragment thereof is raised against an antigen that is xenogeneic to the immune system in which it is raised.

5. The use of claim 4, wherein the antigen is xenogeneic to the dog.

6. The use of claim 1, wherein the antibody or fragment thereof is xenogeneic to the dog.