CN103298487B

CN103298487B - Companion animals is treated

Info

Publication number: CN103298487B
Application number: CN201180052915.1A
Authority: CN
Inventors: 朱利安·亚历山大·巴登; 安格斯·基得利-拜尔德
Original assignee: Bio Authority (australia) LLC
Current assignee: Bio Authority (australia) LLC
Priority date: 2010-09-10
Filing date: 2011-09-09
Publication date: 2016-12-14
Anticipated expiration: 2031-09-09

Abstract

The present invention relates to use P2X₇In immunotherapy makes companion animals body, cancer progression realizes minimizing.

Description

Companion animals is treated

Invention field

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

Background of invention

Any prior art is mentioned by this specification and is not and is not to be construed as recognizing or implying in any form This prior art is formed at Australia or the part of any other jurisdictional common knowledge or can reasonably expect that This prior art confirms by those skilled in the art, understand and be considered as being correlated with.

Cancer sickness rate in companion animals (such as Canis familiaris L. and cat and other similar animals) is being continuously increased, and mesh Before think that cancer is the dead first cause of senior animal.It is believed that in Canis familiaris L. body the sickness rate of annual cancer is about 2% to arrive 2.5% (about the same with the mankind) and cat is 1.5% to 2%.

In Canis familiaris L. body, there is the cancer of high incidence in the following order: lymphoma (about 20%)；Mastocytoma is (about 18%)；Soft tissue sarcoma (about 10%)；Angiosarcoma (about 10%)；Osteosarcoma (about 9%).Remainder typically comprises squama Shape cell carcinoma, breast carcinoma, melanoma, histiocytoma and fibrosarcoma.

In cat body, there is the cancer of high incidence in the following order: lymphoma (about 25%)；Mastocytoma (accounts for The 22% of cutaneous tumor)；Squamous cell carcinoma (more than the 11% of cutaneous tumor)；Breast carcinoma (about 10%)；And remaining part bag Include angiosarcoma, osteosarcoma, fibrosarcoma, sebaceous hyperplasia/adenoma.

In veterinary oncology, the method for the treatment of of cancer includes surgical operation, radiotherapy, thermotherapy, photodynamic therapy and change Treat.Gene therapy and immunotherapy are not also widely used.

Except Palladia (toceranib phosphate；Pfizer (Pfizer)), also there is no that other drug one is several can be executed With listing license, as chemotherapeutics, the treatment of cancer in companion animals.This be primarily due to obtain listing license have The high cost of association.

In most of jurisdictions, veterinary's oncologist has " label is outer " drug use privilege.The outer medicine of label makes Can freely use the medicine being approved for a species (including the mankind) for another with referring to veterinary's oncologist Species.Having had these privileges, occurred a kind of convention in veterinary oncology, thus oncologists tend to select those The treatment of cancer in companion animals of the chemotherapeutics of significant clinical experience has been obtained in human tumor.

The example of the chemotherapeutics used in veterinary oncology at present and relevant indication illustrate in Table 1.

Table 1

A lot of problem and restriction is had for these chemotherapeutics.Such as, as observed in human treatment, these are changed Compound and nonspecific action antagonism includes that the relevant toxicity of the somatoblast of bone marrow, GI epithelium and hair follicle is correlated with.Secondary Effect includes immunosuppressant, anemia, nausea and vomiting, the delay of Wound healing, dysgenesia and alopecia.Some specific device Official is likely to become susceptible, including heart, kidney and central nervous system (CNS).

It addition, use single chemotherapeutant seldom effective, because not every tumor cell is all to curing cancer Effectively can be killed by single preparation.One reason for this is that, along with the development of cancerous cell, the incidence rate of sudden change also develop by This may cause the generation of resistant phenotypes.Therefore, in most of the cases, for given a kind of modularization of clinical case research and development Therapy is necessary.

The treatment of a kind of replacement scheme of companion animals cancer or improvement is existed one need, and especially those There is in companion animals body the cancer of high incidence.

A kind of needs are equally existed for the substituting of the cancer in Canis familiaris L. class body or the property improved treatment.

To the lymphoma in treatment Canis familiaris L. body, mastocytoma, soft tissue sarcoma, angiosarcoma, osteosarcoma, squamous cell Cancer, breast carcinoma, melanoma, histiocytoma and fibrosarcoma exist one to be needed.

A kind of replacement scheme of cat class cancer or the treatment of improvement are existed one need.

To the lymphoma in treatment cat body, mastocytoma, squamous cell carcinoma, breast carcinoma, angiosarcoma, osteosarcoma, fiber There are one needs in sarcoma, sebaceous hyperplasia/adenoma.

Summary of the invention

The invention aims to solve in the demand is one or more, and in first aspect, it is provided that it is used for Making the method that the cancer progression in a companion animals body minimizes, the method comprises the following steps:

One companion animals that will reduce wherein cancer progression is provided；And

One whole antibody including a variable domains or one fragment are provided in this animal body, are used for being attached to One by the non-functional P2X of this animal expression₇On receptor；

Thus minimize the cancer progression in this animal body.

At second aspect, the invention provides the side of a kind of cancer progression in minimizing a companion animals body Method, the method comprises the following steps:

One companion animals needing treatment of cancer is provided；And

Formed a kind of for non-functional P2X in this companion animals body₇One immunne response of receptor；

Thus minimize the cancer progression in this companion animals body.

In the third aspect, the invention provides the side of a kind of cancer progression in minimizing a companion animals body Method, the method comprises the following steps:

One is provided to accept the non-self antigen binding site companion animals for treatment of cancer；

Formed a kind of for non-functional P2X in this companion animals body₇The immunne response of receptor；

Most descend change cancer progression in this companion animals body.

In another aspect of the present invention, it is provided that following purposes:

One whole antibody including a variable domains, or one fragment is used for being attached to a non-functional P2X₇On receptor；Or

One P2X₇Receptor, or a P2X₇One fragment of receptor；

In the manufacture for a kind of medicament of companion animals treatment of cancer.

The most in yet another aspect, the invention provides following purposes:

One includes a variable domains whole antibody, or one fragment is used for being attached to a non-functional P2X₇ Receptor；Or

One P2X₇Receptor, or a P2X₇One fragment of receptor；

For companion animals treatment of cancer.

Still at other aspect, the invention provides a kind of test kit or the compositions cancer in companion animals body and control Treating, this test kit includes:

One includes a variable domains whole antibody, or one fragment is used for being attached to a non-functional P2X₇ On receptor；Or

One P2X₇Receptor, or a P2X₇One fragment of receptor；

The written explanation in any one in a kind of method or above claim.

The other aspect of the present invention and be described in earlier paragraphs these in terms of further embodiment from following Be given in explanation, by example and will become apparent referring to the drawings.

Brief Description Of Drawings

Fig. 1 .P2X₇The aminoacid sequence of receptor.

Fig. 2. dog class and people P2X₇The contrast of the aminoacid sequence of receptor.

The anti-E200 antibody response that Fig. 3 .ELISA (enzyme-linked immunosorbent assay) detects.

The detailed description of embodiment

To mention now certain embodiments of the present invention in more detail.Although the present invention will retouch in conjunction with these embodiments State, it will be appreciated that be not intended as limiting the invention to these embodiments.On the contrary, it is contemplated that will cover as by right All replacement schemes, amendment and the equivalent can being included within the scope of the invention defined in claim.

It will be appreciated by those skilled in the art that similar or identical to described here those many methods and material Material, they may be used in the practice of the present invention.The present invention is limited to absolutely not these described methods and material.

It will be appreciated that the present invention disclosing in this manual and defining expand to mentioned or from context or All alternative combinations of two or more animal character obvious in accompanying drawing.All these different combinations constitute the present invention Different alternative aspect.

All patents mentioned hereby and publication are combined in this by quoting in full with it.

A defines

For explaining the purpose of this specification, defined below will apply generally and in due course, the odd number used Term will also include plural number and vice versa.In listed any definition and any document punching being incorporated herein by reference In the case of Tu, the definition being listed below will be preferential.

As used herein, unless the context requires otherwise beyond, term " includes (comprise) ", and this word Variant, such as, " include (comprising) " and " including (comprises) " and " including (comprised) " not purport Getting rid of other additive, component, integer or step.

" companion animals " typically refers to the animal that is " company " of the animal of house pet or a people.Cat (cat) and Canis familiaris L. (dog) is example.

" purinoceptor " typically refers to a kind of receptor using purine (such as ATP) as part.

“P2X₇Receptor " it is often referred to a purinoceptor formed by three protein protomers or monomer, at least one of which institute State monomer and there is an aminoacid sequence the most as shown in Figure 1 or dog sequence the most as shown in Figure 2.Arrive this P2X₇The degree that receptor is formed by three monomers, it is one " trimer " or " trimer ".“P2X₇Receptor " can be to retouch as follows The function stated or non-functional receptor.“P2X₇Receptor " contain the P2X of natural generation₇Receptor variant, wherein P2X₇Monomer is Splice variant, allele variant and include the formation P2X of natural generation₇The truncate of receptor or the monomer of secreted form Isotype (form being such as made up of extracellular domain sequence or the form of its truncate), the variant form (example of natural generation As, alternately splicing form) and the allele variant of natural generation.In certain embodiments of the present invention, it is disclosed in This natural sequence P2X₇Monomer polypeptide is the nature of maturation or the total length including the full length amino acid sequence shown in Fig. 1 Sequences polypeptide, or dog sequence the most as shown in Figure 2.In certain embodiments, this P2X₇Receptor can have one Adorned aminoacid sequence, the most in FIG shown in sequence in different aminoacids or one the most as shown in Figure 2 Dog sequence can be replaced, delete or may be inserted into a residue.

" functional P2X₇Receptor " typically refer to the P2X with binding site or crack for being attached on ATP₇Receptor A kind of form.When being attached on ATP, this receptor defines non-selective sodium/calcium channel, and this Channel-shifted is poroid to one Structure, this structure makes calcium ion can enter in cytosol, and one result can be programmed cell death.Normally In stable state, functional P2X₇The expression of receptor is usually limited to experience the cell of programmed death, such as thymocyte cell, dendron shape Cell, lymphocyte, macrophage and mononuclear cell.Some functional P2X can also be there are on erythrocyte₇By body surface Reach.

" non-functional P2X₇Receptor " generally refer to P2X₇A kind of form of receptor, this form has a configuration and thus should Receptor can not form an apoptosis hole.What one example occurred in these monomers one or more has one at Pro210 (according to SEQ No. ID: 1) in the case of individual isomer of taking advantage of a situation.In the mankind or non-human mammal monomer, this isomerization It is probably and produces from any molecular events causing this monomer misfolding, including sudden change or the exception of such as monomer basic sequence Post translational processing.One result of isomerization is that in the one or more ATP-binding site on this trimer, this receptor is not Can be attached on ATP, and thus expand the opening of this passage.Specifically, when in three monomers is by mistakenly Pack and therefore destroy two ATP-binding site.In these cases, this receptor can not form hole and which has limited calcium Ion can enter into the degree in cytosol.Only maintain passage portion activity.Non-functional P2X₇Expression of receptor is extensively On general epithelial cancer and hemopoietic cancer.

" E200 epi-position " generally refers to one and is present in a non-functional P2X₇An epi-position on receptor.The mankind In, this sequence is GHNYTTRNILPGLNITC.At dog apoplexy due to endogenous wind, this sequence is GHNYTTRNILPDINITC.

" E300 epi-position " generally refers to one and is present in a non-functional P2X₇An epi-position on receptor.The mankind and Dog apoplexy due to endogenous wind, this sequence is identical, i.e. KYYKENNVEKRTLIKVF.

" multi-epitope " is commonly referred to as a kind of epi-position formed by the juxtaposition of E200 and E300 epi-position.E200 in dog class and The point of the difference between the mankind is not included in dog class multi-epitope, it is meant that the composite table of dog class and the mankind is to be identical.To the greatest extent Pipe cat P2X₇Receptor sequence is not the most characterized, and Serology data in this confirms cat multi-epitope and dog and mankind's composite table Position identical or generally as.

" antibody " or " immunoglobulin " or " Ig " are the γ ball eggs found in vertebrate blood or other body fluid Albumin, they work in immune system with conjugated antigen, thus identify and/or neutralize exotic.

Antibody is typically the different tetramer glycoprotein being made up of two identical light (L) chains and two identical weight (H) chains. Each L chain is connected on a H chain by the disulfide bond of a covalency.The two H chain (is depended on by one or more disulfide bond Isotype in this H chain) it is connected to each other.Each H and L chain also has disulphide bridges in the chain being regularly spaced apart.

H and L chain defines specificity lg domain.More specifically, each H chain has about variable domains at N end (V_H), it is three constant domain (C afterwards_H) (for each α and γ chain) and four C_HDomain (for the isotype of μ and ε).Often Individual L chain has a variable domains (V at N end_L), it is a constant domain (C at its other end afterwards_L)。V_LWith V_HComparison and C_LThe first constant domain (C with heavy chain_H1) comparison.

Antibody can be assigned to different classifications or isotype.Exist the immunoglobulin of five kinds: IgA, IgD, IgE, IgG and IgM, have the heavy chain being respectively designated as α, δ, ε, γ and μ.γ and α classification is based at C_HSequence and function aspects Smaller difference and be further separated into multiple subclass, the such as mankind express following subclass: IgG1, IgG2, IgG3, IgG4, IgA1, And IgA2.L chain aminoacid sequence based on its constant domain from any vertebrate class can be designated as two substantially One of different type, referred to as κ and λ.

This constant domain includes Fc part, whole two H chains that this part includes being combined by disulfide Carboxy terminal half.Determined the effector function of antibody (such as ADCC) by the sequence in Fc district, this district is also by certain The part that the Fc receptor (FcR) found on the cell of type identifies.

V_HAnd V_LPairing together form one " variable region " or " variable domains ", including heavy chain or the light chain of antibody Amino terminal domain.The variable domains of heavy chain can be referred to as " V_H”.The variable domains of light chain can be referred to as " V_L”。V Domain comprises one " antigen binding site ", and this antigen binding site have impact on antigen and combines and define a kind of concrete anti- The body specificity to its concrete antigen.V district has striden across about 110 amino acid residues and has been referred to as by 15-30 is amino acid whose The most constant stretch section (the relatively invariant stretch) composition of framework region (FR) (normally about 4), this is relative Constant stretch section is referred to as the shorter of respective " hypervariable region " (normally about 3) typically with 9-12 amino acid long by the most variable Region separately.These FR major parts take β sheet configuration and hypervariable region to define the ring connecting β chip architecture, and at some In the case of define the part of β chip architecture.

" hypervariable region " refers to the region in an antibody variable territory, these regions in terms of sequence be high become and/or Form the ring structurally defined.Generally, antibody includes six hypervariable regions；Three at V_HIn (H1, H2, H3), and three at V_L In (L1, L2, L3).

" framework " or " FR " residue is in addition to those the variable domains residues beyond some hypervariable region residues defined herein.

" for forming the peptide of antigen binding site " typically refers to form the structure giving antibody specificity for antigen The peptide of type.Example includes whole antibody or whole antibody dependency structure, including whole antibody fragment, its variable knot of a variable domains Structure territory and fragment, including light and heavy chain, or include some but the most all of hypervariable region or the light and sheet of heavy chain of constant region Section.

" antigen binding site " generally refers to include at least antigen combined function being given necessary to a V structure territory high Become district and a molecule of framework region.Stating in method in this, antigen binding site may be at an antibody or The form of one antibody fragment (such as one dAb, Fab, Fd, Fv, F (ab ')₂Or scFv).

" complete " or " entirely " antibody is to include antigen binding site, together with C_LAnd at least heavy-chain constant domains C_HI、C_H2 Hes C_HThe antibody of 3.Constant domain can be constant domain (such as, human native sebquence's constant domain) or its aminoacid sequence of native sequences Variant.

" include the whole antibody fragment of a variable domains " and include Fab, Fab ', F (ab ')₂, and Fv fragment；Double-strand resists Body, linear antibodies, single-chain antibody molecules；And the multi-specificity antibody from antibody fragment formation.

" Fab fragment " is by the Variable domain (V of whole L chain Yu H chain_H) and the first constant domain (C of a heavy chain_HI) Composition.It is unit price that each Fab fragment combines relative to antigen, i.e. it has single antigen binding site.

" Fab ' fragment " is at C with the difference of Fab fragment_HThe c-terminus of I domain has extra little residue, bag Include one or more cysteine from antibody hinge region.Fab '-SH names for Fab ' at this, wherein this constant domain One or more cysteine residues with a free sulfydryl.

“F(ab′)₂Fragment " generally correspond to the Fab that two disulphide with bivalent antigen combination activity connect Fragment and remain can crosslinking antigen.

" Fv " is to comprise a complete antigen identification and the minimum antibody fragment of binding site.This fragment is by with the most non- One heavy chain of non-covalent association and a light chain Variable domain dimer composition.

Planting apoplexy due to endogenous wind, a heavy chain and a light variable domains a scFv (scFv) can be soft by one Property peptide junctional complex covalently bound so that this light chain and heavy chain can be at one " dimer " knots being similar to two chain Fv kinds Structure associates.Exhaling six Gao Bianhuan (3 rings are each from H and L chain) from the folding of the two domain, these rings help It is used for the antigen-binding specificity that antigen combines and gives antagonist in amino acid residue.

" scFv " is also abbreviated as " sFv " or " scFv ", is to include connecting the V forming a single polypeptide chain_HAnd V_LAnti- The antibody fragment of binding domains.Preferably, this scFv polypeptide further includes at this V_HAnd V_LA polypeptide between domain is even Connect thing so that this scFv can define the desired structure combined for antigen.

" single variable domains " is the half (only including three CDR for antigen-specific) of Fv, and this Fv has identification And the ability of conjugated antigen, although lower than the affinity of whole binding site.

" double-chain antibody " refers to the antibody fragment with two antigen binding sites, and these fragments include being connected to same Polypeptide chain (V_H-V_LA light variable domains (V in)_LHeavy-chain variable domains (V on)_H).Little antibody fragment passes through At this V_HWith V_LBuild between domain and there is the sFv fragment (seeing above paragraph) of short junctional complex (about 5-10 residue) and make Standby to achieve the interchain pairing in V structure territory but be not pairing in chain, consequently leads to a bivalent fragment, i.e. have two The fragment of individual antigen binding site.

Double-chain antibody can be bivalence or bispecific.The double-chain antibody of bispecific is two " crossover " sFv The heterodimer of fragment, the wherein V of the two antibody_HWith V_LDomain exists on different polypeptide chains.Three chain antibodies and four Chain antibody is the most known in the art.

" antibody of separation " be the component from the environment that it is pre-existing in identify and separate and/or reclaim anti- Body.Pollution components is will to disturb the material of the therapeutic use for antibody and can include enzyme, hormone and other albumen Matter solute or non-proteinaceous solute.

" monoclonal antibody " refers to a kind of antibody that the colony of basically homogeneous antibody obtains, i.e. include this colony Animal's antibody be identical, in addition to the sudden change of the possible natural generation that may exist with less amount.Monoclonal anti Body is high degree of specificity, its single antigen site being directed on antigen or determinant.In addition to its specificity, single Advantageously they can be synthesized clonal antibody by other antibody not comtaminatedly.Monoclonal antibody can pass through hybridoma side Prepared by the science of law.These " monoclonal antibodies " can also separate from phage antibody library from using these technology.

Monoclonal antibody in this includes " chimeric " antibody, wherein a part for weight and/or light chain with derived from one Specific kind of antibody or the corresponding sequence that belongs to an antibody specific kind or subclass is identical or homology, and this or many The remainder of individual chain be with the antibody derived from another kind or belong to another antibody type or subclass antibody together with this Corresponding sequence in the fragment of kind of antibody is identical or homology, as long as they show desired biological activity.

Term " anti-P2X₇Receptor antibody " or " it is attached to P2X₇Antibody on receptor " refer to enough affinitys It is attached to P2X₇On receptor so that this antibody diagnoses as one and/or therapeutic agent is at targeting P2X₇Receptor is (the most non-functional Property P2X₇Receptor) in be useful antibody.Preferably, by one P2X₇Receptor antibody is attached to unconnected P2X₇Receptor egg Degree on Bai less than this antibodies such as the most measured (such as by radioimmunoassay (RIA)) to P2X₇Pact on receptor 10%.In certain embodiments, it is attached to P2X₇Dissociation constant (Kd) ＜ 1 μM that antibody on receptor has, ＜ 100nM, ＜ 10nM, ＜ 1nM or ＜ 0.1nM.Anti-non-functional P2X₇Receptor antibody is typically have these Serological Characterizations some or all of And it is attached to non-functional P2X₇On receptor rather than functional P2X₇Antibody on receptor.

A kind of " affinity maturation " antibody is to have resisting of one or more change in one or more hypervariable region Body, this change, compared with the maternal antibody not having one or more that change, result in the antibody affinity to antigen Improvement.The preferably antibody of affinity maturation has the affinity for target antigen of nanomole or even picomole.Affine The antibody that power is ripe is produced by program known in the art.

" closure " antibody or " antagonist " antibody are suppression or antigen bioactive reducing this antibodies Antibody.Preferably blocking antibody or antagonist antibodies substantially or entirely inhibits the biological activity of this antigen.

As used herein, at least one during " agonist antibody " is a kind of functional activity simulating polypeptide interested Antibody.

" binding affinity " typically refers to the single binding site gametophyte in connection at a molecule (such as antibody) The intensity of the noncovalent interaction summation between (such as, antigen).Except as otherwise noted, as used in this " in conjunction with parent And power " refer to inherent binding affinity, reflect the one between a member combined (such as, antibody and antigen) The interaction of 1:1.Molecule X generally can pass through dissociation constant (K for the affinity of its gametophyte Y_d) represent.Can pass through Methods known in the art measure affinity, including described here those.Low-affinity antibody is attached to resist the most lentamente Former upper and tend to dissociate easily, and high-affinity antibody is attached on antigen the most quickly and tends to keep In conjunction with more long.The many kind methods measuring binding affinity are to it known in the art, any of which method to may be used for the present invention Purpose.

" treat " both that be commonly referred to as therapy for treating and prevention or preventive measures.

The animal needing treatment includes having had optimum, preneoplastic or those companies of non-metastatic tumor With wherein stoping those of its cancer morbidity or recurrence.

Purpose or the result for the treatment of can reduce cancerous cell number；Reduce primary tumor size；Suppression is (i.e. necessarily Slow down in degree and preferably stop) cancer cell infiltration is to peripheral organs；Suppression (slows down and preferably the most to a certain extent Ground stops) neoplasm metastasis；Suppress tumor growth to a certain extent；And/or alleviate to a certain extent and be associated with this imbalance Symptom in one or more.

The persistent period that can be survived by evaluation, the time of progression of disease, answer speed (RR), the persistent period of response And/or quality of life measure effect for the treatment of.

In one embodiment, the method is useful especially to the time of prolongation progression of disease.

In one embodiment, the method survival rate to prolongation animal, deposit together with getting nowhere including total time-to-live Motility rate, is useful especially.

In one embodiment, the method is useful especially for providing one to treating complete response, thus rings Should disappear in all cancer signs for the treatment of.This most always means that cancer has been cured.

In one embodiment, the method is useful especially for providing a part response to treatment, thus rings A fall should have been had in treatment, the degree of cancer in the size of one or more tumors or pathological changes or in the body Low.

" precancer " or " preneoplasia " is commonly referred to as typically being carried out into or developing into a kind of patient's condition of a kind of cancer Or growth." precancer " growth is likely to be of the cell cycle regulating with exception, breeds or be divided into the cell of feature, and these are thin Born of the same parents can be determined by the mark of cell cycle.

" patient's condition that [with cancer] is associated or symptom " can be that the result as cancer produces, before cancer or It is by any pathology of cancer progression.Such as, when this cancer is skin carcinoma, this patient's condition or related symptoms can be microorganism senses Dye.When this cancer is a kind of secondary tumors, and this patient's condition or symptom can relate to the organ with the relevant organ of neoplasm metastasis Dysfunction.In one embodiment, Therapeutic Method described here be the patient's condition in an animal body or symptom Littleization or treatment, this symptom or the patient's condition are relevant to the cancer in this animal body.

" non-autologous " molecule, such as " non-autologous " antigen binding site or " non-autologous " antibody typically refer to one by External generation or the molecule of external source, wherein this molecule will be provided, such as, be used for treating.As an example, synthesis Or the molecule of restructuring is " non-autologous ".Further, one results from an animal and is applied to another animal for controlling The molecule treated is " non-autologous "." non-autologous " antigen binding site and antibody can be used for continuing of immunity according to the present invention Holding property shifts, such as, occur when antibody infusion.On the contrary, one produce in an animal body and with this molecular therapy this move The molecule of thing, it is common that a kind of " autologous " or " endogenous " molecule.One example of " autologous " molecule be one be produced from or by A kind of antigen binding site that immunogenic adaptive immune response is produced or antibody.

" level of the non-self antigen binding site of circulation " in animal body typically refers to antigen binding site at body fluid In concentration, preferably at peripheral blood.

" the non-self antigen binding site level that the most generally can not detect " typically refers to exogenous antigen knot These antigens when the concentration of conjunction site (i.e. those have been used by adoptive transfer) at least uses this antigen binding site The half of binding site concentration in the circulating cycle, it is therefore preferable to the 25% of described concentration or 10% or 5% or 1%, or separately The outer 0.001mg/kg less than this animal.This phrase is also refer to a kind of situation, interim has been applied for cancer immunotherapy Antigen binding site can not be detected at all.

The cancer that " generally can not detect " be typically its middle finger treatment decreased the size of a kind of cancer, volume or The situation of other physical measurements so that using relevant criterion detection technique (such as living imaging), this cancer is as this treatment Result is not clearly can to detect.This phrase is also refer to the situation that wherein this cancer can not be detected at all.

" form an immunne response " and typically refer to cause via adaptive immune system or inducing antigen-specific immunity. As being commonly understood by the art, the induction of antigen specific immune is different from the adoptive transfer of immunity, by exogenous Or non-autologous property antibody use the example that the standard cancer immunization therapy carried out is the latter.

B. the cancer immunotherapy carried out by antibody infusion

Although non-functional P2X₇Receptor distribution expression pattern in normal, the preneoplastic or human tissue of tumor exists It is to understand the when of the present invention, but about non-functional P2X₇(especially companion animals is such as non-human animal for receptor Cat and Canis familiaris L.) in tissue expression, be seldom known.

Specifically, the trimer non-functional receptor whether biological tissue in companion animals body is expressed and concrete It is unknown at which tissue expressing.Further, this expression whether can find in cancerous tissue and if it find that, This expression can be limited to cancerous tissue be which kind of degree be unknown.Therefore, whether some companion animals cancer is expressed non-functional Property P2X₇Receptor, or be whether all unknown by having notable toxicity with the treatment of antibody to normal cell.

Further, whether the cancer specific epitopes observed on live body cancerous tissue in the mankind is present in companion animals cancer Disease tissue is unknown.The present invention when, this is with how to produce the problem of anticancrin in companion animals body be straight Connect relevant.Particularly, cat P2X₇Receptor sequence is entirely the unknown.

Known Canis familiaris L. P2X₇Sequence and mankind P2X₇Receptor is dramatically different, at it corresponding to the region of E200 in people in Canis familiaris L. sequence In crucial proline 210 (the known non-functional producing people's receptor in human body) is added carboxyl immediately, it is known that this Canis familiaris L. sequence is such as As in human body, substitute neutral glycine with electronegative aspartic acid non-conservation.Further, Canis familiaris L. and the mankind it Between in other aminoacid differences, it is known that at least about 55% in these is that non-conservation substitutes, and has one to be inserted in the mankind In do not find.Specifically participate in Fig. 2.

In sum, when in the present invention, unknown non-functional P2X₇Receptor whether be present in companion animals (include Canis familiaris L. and Cat) in, therefore unknown P2X₇It is dynamic for accompanying whether receptor can be used as a biomarker as having been used to the mankind Treatment of cancer in object.

As described in this, ladies and gentlemen inventor is it has been shown that cultivate for a peptide immunity with E200 epi-position The treatment of the sheep antibody kinds cancer to having more high incidence in companion animals body be highly effective, and have Minimal side effect or toxicity.Thus ladies and gentlemen inventor has realized that some cancerous cell of living expresses non-functional in Canis familiaris L. and cat body P2X₇Receptor.In view of minimum toxicity, the expression of these receptors seems to be limited to the preneoplastic or tissue of tumor.It addition, also And despite difference (such as at Canis familiaris L. and mankind P2X₇Between sequence), but these companion animals receptors seem to have like thereon Find an extracellular configuration of mankind's E200 epi-position.

It addition, and surprisingly, ladies and gentlemen are inventor has recognised that anti-xenogenesis P2X₇Immunogen and the xenogenesis cultivated Antibody is highly effective to the cancer in treatment companion animals (including cat and Canis familiaris L.).

Therefore, in first aspect, the invention provides a kind of cancer progression in a companion animals body and minimize Method, the method comprises the following steps:

One companion animals minimizing wherein cancer progression is provided；And

Thus minimize the cancer progression in this animal body.

At other aspect, the invention provides a kind of whole antibody or one fragment in one for companion animals cancer Purposes in the manufacture of the medicament for the treatment of, this whole antibody or one fragment include a variable domains, are used for being attached to one Individual non-functional P2X₇On receptor.

Still at other aspect, the invention provides a kind of whole antibody or one fragment for one companion animals for the treatment of The purposes of internal cancer, this whole antibody or one fragment include a variable domains, be used for being attached to one non-functional Property P2X₇On receptor.

Method according to the first aspect of the invention, an animal to be treated can be to have accepted or be Any animal indicating the therapeutic antibodies for tumor will be accepted.

In an embodiment of first aspect, the antigen binding site of an antibody can be to distinguish functional and NOT function Can property P2X₇The antigen binding site of receptor, is hereby incorporated into non-functional receptor rather than functional receptor.These antigen is tied The example closing site is bonded to those of E200 epi-position, E300 epi-position or multi-epitope, such as at PCT/AU2002/ 000061、PCT/AU2002/001204、PCT/AU2007/001540、PCT/AU2007/001541、PCT/AU2008/ 001364, in PCT/AU2008/001365, PCT/AU2009/000869 and PCT/AU2010/001070, all these all It is incorporated by reference.

One antigen binding site can with the form of the form or a whole antibody fragment of taking a whole antibody (such as One Fab, Fab ', F (ab ')₂And Fv), a scFv or a single variable domains.

Relative to this, will to accept it for the companion animals for the treatment of of cancer can be homology, of the same race to antigen binding site Different system or xenogenesis.

Typically, this antigen binding site is non-autologous property or external source, it is meant that in the side according to the present invention Found or separated beyond the animal of method treatment.

This antigen binding site can be affine maturation.

This antigen binding site can have many species specificity or quantivalence.

This antigen binding site can be adapted, and thus fits through a method selected and uses.

This antibody can be a whole antibody of any isotype.This antibody can be from monoclonal antibody or Anti-TNF-α The antibody obtained in serum.This antibody can produce from hybridoma, or by recombinant expressed, or can obtain from serum (such as can obtain from a kind of mammal, particularly people or mice).This antibody can also obtain from a kind of birds.

This antibody can be chimeric, a kind of antibody comprising human variable-domain and non-human constant domain. Alternately, it can be humanized, i.e. by non-human CDR is grafted on a human antibodies framework the anti-of formation Body.Still further, this antibody can be full people source.

This antibody can be modified relative to effector function and thus strengthen, such as, be used for strengthening this antibody in treatment cancer Effectiveness in disease.

In the case of this antibody is a kind of antibody fragment, this antibody fragment is selected from lower group, and this group is made up of the following: dAb、Fab、Fd、Fv、F(ab’)₂, scFv and CDR.

Hereinafter the value of dosage, dose frequency, route of administration etc. are described in detail.

Prepare antibody and the method to its animal in need of being used by antibody is ripe for those skilled in the art Know, or easily determined by those skilled in the art.Route of administration it may be that such as, oral, parenteral (example Such as intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, Intradermal, rectum or vagina), by sucking or local application.Use One form can be a kind of solution for injection, and especially for intravenous injection or intra-arterial injection or instillation, this solution includes A kind of buffer (such as acetate, phosphate or citrate buffer), a kind of surfactant (such as Polysorbate), appoint Selection of land one stabilizer (such as albumin).In additive method, can directly by antibody delivery to disease site, thus increase Sick cell or the exposure of tissue antagonist.

Preparation for parenteral administration include sterilized water (aqueous carrier includes water, alcohol/aqueous solution, emulsion or suspension, Including saline and buffer medium) or non-aqueous (nonaqueous solvent is propylene glycol, Polyethylene Glycol, vegetable oil (such as olive oil) and can Injection organic ester (such as ethyl oleate)) solution, suspension and emulsion.Pharmaceutically acceptable carrier includes 0.01M-0.1M And the phosphate buffer of preferably 0.05M or 0.9% saline.Other common parenteral vehicle include sodium radio-phosphate,P-32 solution, woods Grignard glucose, glucose and sodium chloride, Lactated Ringer'S Solution or fixing oil.Intravenous vehicles includes fluid and nutrient Supplement, electrolyte replenisher (such as based on woods grignard glucose those), and the like.Can also exist preservative and Other additives, such as antimicrobial, antioxidant, chelating agen and noble gas, and the like.

More specifically, it (is water solublity time suitable that the pharmaceutical composition being suitable for injectable purposes comprises aseptic aqueous solution ) or dispersion and the sterilized powder of the instant preparation for sterile injectable solution or dispersion, in this case, Said composition must be aseptic and should be the fluid existed with the degree of easily injection.It should be to manufacture and depositing Contamination that is stable and that be preferably for microorganism (such as antibacterial and fungus) under the conditions of storage and preserve.This carrier can Be containing such as water, ethanol, polyhydric alcohol (such as, glycerol, propylene glycol and liquid macrogol, and the like) and The solvent of their applicable mixture and disperse medium,.Suitable mobility can be such as by using a kind of coating (such as ovum Phospholipid), by keeping particle diameter required in the case of a dispersion and by using surfactant to maintain.For Applicable preparation in this Therapeutic Method disclosed is described in Remington ' s Pharmaceutical Sciences (Lei Ming Dun Shi pharmaceutical science), Mack Publishing Co. company, in 16 editions (1980).

The effect preventing microorganism can pass through different antibacteriums and antifungal, such as metagin, three The chlorine tert-butyl alcohol, phenol, ascorbic acid, thimerosal and the like realize.In many cases, it may be preferred that in this combination Thing includes isotonic agent, such as sugar, polyhydric alcohol (such as mannitol, sorbitol) or sodium chloride.The prolongation of Injectable composition is inhaled Receipts can be by including the medicament of a kind of delayed absorption in the composition, and such as aluminum monostearate and gelatin realize.

Under any circumstance, sterile injectable solution can be by by a kind of reactive compound (example in appropriate solvent As, antigen binding site) be combined with a kind of or a combination composition listed herein with required amount, connect Filtration sterilization to prepare.Generally, prepare dispersion by being attached to by this reactive compound in a kind of sterile carrier, should Carrier contains a kind of basic dispersion body medium and required other compositions from those listed above.Make being used for In the case of the sterilized powder of standby aseptic Injectable solution, preferred preparation method is vacuum drying and lyophilization, this Create active component and add any extra powder from the composition desired by its above sterilefiltered solutions.To being used for The preparation of injection is processed, and is filled in container such as ampoule, bag, bottle, syringe or bottle, and according to this area Seal under known aseptic condition.Additionally, these preparations can be by the packaged of test kit and sell.Such system Divine force that created the universe product preferably have label or package insert (package insert), indicate compositions related for treatment suffer from imbalance Or the experimenter being susceptible to suffer from imbalance is useful.

The compositions of the present invention depend on many different factors for the effective dose treating as described in this imbalance and Changing, including using the physiological status of means, target location, patient, whether patient be human or animal, the other drug used is controlled Treat, and treatment is preventative or curative.Therapeutic dose can use known to those of ordinary skill in the art Conventional method progressively increases to optimize safety and effect.

For with a kind of Antybody therapy, some is lacked of proper care, the scope of dosage can be such as from about host's body weight 0.0001mg/kg to 100mg/kg, and more generally 0.01mg/kg to 5mg/kg (such as 0.02mg/kg, 0.25mg/kg, 0.5mg/kg, 0.75mg/kg, 1mg/kg, 2mg/kg, etc.).Such as, dosage can be 1mg/kg body weight or 10mg/kg body weight or In the range of 1mg/kg-10mg/kg, preferably at least 1mg/kg.Dosage intermediate value in range above is also intended to the present invention In the range of.Can to experimenter every day, every other day, weekly or rule of thumb analyze any other scheme determined and use this agent Amount.Exemplary treatment requires to use with multiple dosage within a period (such as, at least six moon) extended.Extra example Property therapeutic scheme require once every two weeks or monthly or every 3 to 6 months applied onces.Exemplary dose scheme includes 1mg/kg-10mg/kg or 15mg/kg sustained continuous sky, 30mg/kg or weekly 60mg/kg every other day.In certain methods, simultaneously Use two or more antigen binding sites with different binding specificities, in this case, every kind used The dosage of antigen site falls in specified scope.

Can use for being attached to an expression non-functional P2X on a cell in several cases₇Receptor Antibody.Interval between single dose can be weekly, monthly or every year.Interval can also is that irregular, as by surveying Measure indicated by the blood level of the target polypeptides in patient or target molecule.In certain methods, dosage is adjusted with Realize the plasma polypeptide concentration of 1 μ g/mL-1000 μ g/mL, and be 25 μ g/mL-300 μ g/mL in certain methods.Alternative Ground, this antibody can be used as sustained-release formulation, needs using of lower frequency in this case.Dosage and frequency Depend on that the half life of the antibody in patient changes.The half life of antibody can also via be fused to a stable polypeptide or Extend in part, such as albumin or PEG.Generally, humanized antibody demonstrates the longest half life, is followed by chimeric antibody And non-human antibody.In one embodiment, this antibody can be used by unconjugated form.In another embodiment, should Antibody can be used by unconjugated form and repeatedly use.In some treatment use, sometimes require higher dosage (such as, The up to anti-P2X of 400mg/kg₇Binding molecule, the antibody of such as every dosage), with shorter interval until the progress of disease reduces or Terminate, and show the improvement of disease symptoms partially or completely preferably of up to patient.When this antibodies is to a kind of radiation Time on property isotope or cytotoxic drug, this amount can be lower (i.e. 2 to 3 Logarithmic degree lower) of some Logarithmic degree.

Therapeutic agent can pass through parenteral, locally, intravenous, oral, subcutaneous, intra-arterial, intracranial, intraperitoneal, intranasal or Intramuscular means are used, and for preventative and/or therapeutic treatment, in certain methods, medicament are injected directly into wherein NOT function Can property P2X₇In the concrete tissue that recipient cell has built up, such as intracranial injection.Intramuscular injection or venoclysis are for antibody To use be preferred.

A kind of antibody can be optionally incorporated in the treatment of imbalance or the patient's condition needing treatment (the most preventative Or curative) other medicaments and use.Example is those medicaments being normally used for chemotherapy or radiotherapy in oncology.Additionally Ground or alternately, this antibody or medicament can before the excision of tumor or tissue or the operative treatment removed, in or Used later.

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

In one embodiment, the purpose of this treatment or result be following in one or more:

Reduce the number of cancerous cell；

Reduce the size of primary tumo(u)r；

Suppression (slow down the most to a certain extent or preferably stop) cancerous cell penetrates into peripheral organs；

Suppression (slow down the most to a certain extent or preferably stop) neoplasm metastasis；

Suppress tumor growth to a certain extent；

Alleviate one or more in the symptom relevant with this imbalance to a certain extent.

In one embodiment, the method for a first aspect of the present invention is for extending the time of progression of disease.

In one embodiment, the method for first aspect is for extending the survival rate of animal, including total time-to-live even With progresson free survival rate.

In one embodiment, the method for first aspect for provide one to treatment complete answer, thus in response to All cancer signs for the treatment of have disappeared.

In one embodiment, the method for first aspect for provide to treatment a part response, thus in response to Treatment, the degree of cancer in the size of one or more tumors or pathological changes or in the body has had a reduction.

In one embodiment, it is necessary to the animal for the treatment of includes suffering from optimum, before cancer or non-metastatic tumor Those.

In one embodiment, this cancer is precancerous or preneoplastic.

In one embodiment, this cancer is secondary cancer or metastatic tumor.This secondary cancer may be located at any device Official or tissue, and particularly those have the organ or tissue of the most higher hemodynamic pressure, such as lung, kidney, pancreas Gland, intestinal and brain.

In one embodiment, this cancer can generally can not detect.

This companion animals can be a cat or Canis familiaris L..But, in view of for the people's albumen for non-human primate receptor The cross reactivity of antibody cultivated, ladies and gentlemen are inventors have realised that other have identical with the mankind remote phylogenetic Non-human mammal can benefit from the present invention.In one embodiment, this companion animals is a kind of high value animal or good Plant animal.One example is a dry goods.

Typically it is desirable to the cancer for the treatment of or precancer are to have more high incidence in a given companion animals body Cancer.Such as, when this companion animals is a Canis familiaris L., this cancer can be lymphoma, mastocytoma, soft tissue meat Tumor, angiosarcoma, osteosarcoma, squamous cell carcinoma, breast carcinoma, melanoma, histiocytoma, carcinoma sarcomatodes or fibrosarcoma.

When this companion animals is a cat, this cancer can be lymphoma, mastocytoma, squamous cell carcinoma, breast Cancer, angiosarcoma, osteosarcoma, fibrosarcoma or sebaceous hyperplasia/adenoma.

Other examples of cancer are described in Table I or include that blastoma (includes that medulloblastoma, retina are female thin Born of the same parents' tumor), sarcoma (including liposarcoma and synovial cell sarcom), neuroendocrine tumor (include carcinoid tumor, gastrinoma and pancreas Islet cell tumor), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, leukemia or lymphoid malignant Tumor, pulmonary carcinoma (including small cell lung cancer (SGLG), nonsmall-cell lung cancer (NSGLG), adenocarcinoma of lung and pulmonary's squamous cell carcinoma), Peritoneal cancer, hepatocarcinoma, stomach or gastric cancer (including gastrointestinal cancer), cancer of pancreas, glioblastoma, cervical cancer, ovary Cancer, hepatocarcinoma, bladder cancer, hepatoma, breast carcinoma (including transitivity breast carcinoma), colon cancer, rectal cancer, colorectal carcinoma, intrauterine Film cancer or uterus carcinoma, salivary-gland carcinoma, renal cancer or renal carcinoma, carcinoma of prostate, carcinoma vulvae, thyroid carcinoma, hepatocarcinoma, anus cancer, penis Cancer, carcinoma of testis, the esophageal carcinoma, tumor of bile duct are together with head and neck cancer.

Applied dose and frequency can depend on that this treatment is preventative or curative and change.Preventative In application, the compositions including antibody or its mixture is used not in morbid state or disease state patient with Strengthen the resistance of patient.Such a amount is defined as " prevention effective dose ".In this use, accurate amount takes again Certainly in health status and the general immunity of this animal, but generally scope is from the every dosage of 0.1mg to 25mg, especially The every dosage of 0.5mg to 2.5mg.Relatively low-dose is to use through long period with interval less frequently.Some animals are for it Remaining life and continue to accept treatment.

In treatment use, sometimes require that higher dosage is (such as, from the binding molecule of about 1mg/kg to 400mg/kg, example Such as the antibody of every dosage, dosage is more generally used from 5mg to 25mg for radioimmunoassay conjugate, and for cell The more high dose of the molecule that toxin-medicine combines) with shorter interval, until the progress of disease reduces or terminates and the most straight The improvement of disease symptoms partially or completely is shown to animal.

In one embodiment, associated cancer in this antibodies another kind indicates the companion animals body for needing treatment Chemotherapy or antineoplastic compound provide together.The example of these compounds and relevant instruction describes in Table 1.

In another embodiment, this antibody at one before the clinical operation of lower group, in or after provide, this group by with Under every composition: surgical operation, radiotherapy, thermotherapy, photodynamic therapy, chemotherapy, gene therapy and immunization therapy.

C. the cancer immunotherapy carried out by immunity

At second aspect, present invention provide for making the one that the cancer progression in a companion animals body minimizes Method, the method comprises the following steps:

One companion animals needing treatment of cancer is provided；And

Thus minimize the cancer progression in this companion animals body.

In the third aspect, present invention provide for making to receive accompanying of a kind of non-self antigen binding site A kind of method that cancer progression in companion's animal body minimizes, for treatment of cancer, the method comprises the following steps:

There is provided a companion animals having accepted a kind of non-self antigen binding site, for treatment of cancer；

Thus minimize the cancer progression in this companion animals body.

In yet another aspect, the invention provides a kind of non-functional P2X₇Receptor or its fragment are used for treatment one in one Purposes in the manufacture of the medicament of the cancer in individual companion animals body, especially one have accepted a kind of non-self antigen and have combined Site is for the animal for the treatment of of cancer, such as cat or Canis familiaris L..

In yet other aspects, the invention provides a kind of non-functional P2X₇Receptor or its fragment are accompanied for treatment one The purposes of cancer in companion's animal body, a kind of has accepted a kind of non-self antigen binding site for treatment of cancer Animal, such as cat or Canis familiaris L..

In yet another aspect, the invention provides a kind of non-functional P2X₇Receptor or its fragment one for treatment or Suppress cancer progression in a companion animals body medicament manufacture in purposes, this companion animals has accepted a kind of anti-non- Functional P2X₇Receptor antigen binding site is used for treatment of cancer.

In an embodiment of the second and third aspect of the present invention, in this immunne response in this companion animals body During formation, this companion animals can not have the non-self antigen binding site that can detect in the circulating cycle.Such as, the antibody of infusion May be removed from blood plasma when immunity.It addition, when this immunne response is formed in this companion animals body, this Companion animals can not have the cancer that can detect, such as, after a kind of antigen binding site is used this companion animals As a result, when this immunne response is formed in this companion animals body, this cancer can have size, the quality generally reduced Or other physical measurements.

According to the second and third aspect of the present invention, this immunne response may be formed by immunogen.This immunogen is permissible Can induce a non-functional P2X by one₇The P2X of receptor immunne response in this companion animals body₇The shape of receptor Formula, or a P2X₇The form of the fragment of receptor provides.One non-functional P2X₇Receptor is defined as being formed at phase Adjacent correct packaging can not be in conjunction with at least one in three ATP-binding site on the interface between the monomer of ATP.This Receptor can not expand the non-selective calcium channel opening to apoptosis hole.This immunogen can comprise can be at one main group Knit and exist on histocmpatibility II quasi-molecule and/or can be with exempting from that a T or B-cell receptor or B cell film are combined At least one sequence that epidemic disease globulin interacts.Typically, this companion animals is a cat or Canis familiaris L., in this case should Immunogen can be provided by following form: a cat or Canis familiaris L. P2X₇Receptor, or it is their ability to induction to P2X₇The immunity of receptor should The fragment answered.Canis familiaris L. P2X₇Receptor sequence is illustrated in Fig. 2 in this.Typically, it is formed at the immunity in this companion animals body Response is to non-functional P2X₇Receptor is specific, in this case, defines nand function in this companion animals body Property P2X₇Receptor (i.e. non ATP bind receptor) reaction but not with functional P2X₇Antibody that receptor (i.e. ATP bind receptor) reacts or Cellular component.

In the preferred form of the second and third aspect of the present invention, this immunogen is supplied to this in an initial application Companion animals, is consequently formed the response including that IgM produces.In other preferred form, it is supplied in initial application This companion animals immunogen, is consequently formed the response including that IgM produces, in later time, and another relative to initial application Outer use, use this immunogen, thus form the response including that IgG produces.In the present embodiment, typically, When IgM level in circulation in companion animals body generally can not detect, there is immunogenic other using.

This immunne response that second and third aspect according to the present invention is formed can be a humoral response and/or cell Response.Humoral response can include that B cell activates and cytokine generation, propagation to the plasmacytic conversion of secretory antibody, Th2 It is centrally formed and produces with isotype conversion, the affine maturation of B cell and/or memory cell.Cell response can include activating and resist Former specific cell toxin T-lymphocyte, activating macrophage and natural killer cell and/or stimulation emiocytosis cell The factor.The humoral response and/or the cell response that are formed in companion animals body can treat or alleviate in this companion animals body Cancer, or minimize the cancer progression in this companion animals body.

In above-described embodiment of the second and third aspect of the present invention, this companion animals the antigen bound site accepted Point can react with any biomarker being associated with cancer.Example includes anti-P2X₇, especially non-functional P2X₇, anti- VEGF, especially VEGF A, C or D, Her-2, CD20 or other antigen binding site.Typically, this companion animals connect The antigen binding site being subject to and P2X₇Receptor, especially non-functional P2X₇Receptor response.

In another embodiment of the second and third aspect of the present invention, it is provided that a kind of compositions is used for treating or being used for Cancer progression in suppression companion animals body, said composition includes P2X₇Receptor or its fragment.Preferably, said composition is further Including a kind of carrier, excipient or diluent.Preferably, said composition farther includes a kind of adjuvant.At a preferred form In, said composition makes when using this immunogen for the first time to this companion animals, it is possible to forms primary immune response and (includes that IgM produces Raw), and make after using for the first time, when using this immunogen, it is possible to form the second immunne response (including that IgG produces).

Do not fettered by any theory or model of action, generally believe relevant to the second and third aspect of the present invention above-mentioned Embodiment provides a kind of replacement scheme and/or improves therapeutic scheme, because endogenous immune component is (such as from resisting that immunity produces Body and antigen-specific cellular) it is being complete administration of antigens binding site, and non-autologous anti-P2X₇Antigen binding site After cyclical level becomes to detect, it is provided that cell surface P2X₇More extend and the higher exposure of receptor.It addition, it is general All over thinking P2X₇Receptor clustering (along with producing when providing the non-autologous of high concentration or exogenous antibodies in companion animals body) is minimum Change the crucial P2X being attached to provide anticancer immunne response₇The specific antibody level of epi-position, thus limit immunotherapy Effect.The inventors discovered that, the immunity of companion animals, when the cyclical level of autoantibody generally detects, keeps away Exempt from receptor clustering, and which improved effect of immunotherapy, when the cancer in this companion animals body can be particularly When generally can not detect.

In an embodiment of the second and third aspect of the present invention, these choosings are not the most used for the animal for the treatment of Antibody mediated immunity therapy or the therapy for treating of other forms.In another embodiment, these choosings come for treating according to said method Animal be to have accepted or continued to accept antibody mediated immunity therapy for those for the treatment of of cancer.Antibody mediated immunity therapy is usual It is directed to an animal needing treatment and uses (also referred to as " non-autologous ") antibody of external source, such as the adoptive transfer at antibody In situation.Such as, this animal can accept any one of therapeutic antibodies, and these antibody have accepted relating to swelling The registration examination & approval of the instruction of tumor.Avastin, herceptin, Mabthera are examples.Typically, this animal has received or just Continuing a kind of anti-P2X of acceptance₇Receptor antibody.Suitable anti-P2X₇Antibody, they generation, manufacture, use and to one The example used of companion animals is described in the embodiment relevant to a first aspect of the present invention.

It addition, these choosings can have when treatment for the animal that the second and third aspect according to the present invention is treated Or may not have the cancer that can detect.When this animal does not have the cancer that can detect, it is easier to a kind of primary of detection or two , because in the presence of cancer is with the amount that generally can not detect, there is considerably less available NOT function in level humoral response Can property P2X₇Receptor removes IgM or IgG from body fluid.

Can according to the type of the cancer that the second and third aspect of the present invention is treated and desired therapeutic outcome be as Those described in the embodiment relevant to a first aspect of the present invention.

The purpose of the treatment of the second and third aspect according to the present invention is by P2X in animal body₇The immunity of receptor Induction or the formation of response at least minimize cancer progression.Therefore, choosing must be to produce foot for the animal for the treatment of Enough immunne response meet this purpose.Generally this desired immunne response includes when this animal is by cancer, as in cancer When recurrence excites, the ability of one or both of IgM and IgG producing circulation.

Can select by multiple method well known in the art or screen and describe the energy having and producing immunne response in this The animal of power detects for immunodeficiency.Typically, choosing will have in normal parameters for the animal for the treatment of The animal of at least one white cell component counting.Such as, the numeration of leukocyte being typically to have for the cat included in arrives 5.5 19.0×10⁹Between/L, or lymphocyte count is 0.9 to 7.0 × 10⁹Cat between/L.Neutrophil count can be 2 to 13 × 10⁹Between/L；Monocyte count ＜ 0.7 × 10⁹/ L, eosinophilic granulocyte is less than about 1.1 × 10⁹/ L and basophilic Property granulocyte is less than about 0.1 × 10⁹/L.Such as, be typically for the Canis familiaris L. included in the numeration of leukocyte that has 4.5 to 17.0 × 10⁹Between/L, or lymphocyte count is 0.9 to 3.5 × 10⁹Canis familiaris L. between/L.Neutrophil count can arrive 3.5 12×10⁹Between/L；Monocyte count ＜ 1.2 × 10⁹/ L, eosinophilic granulocyte is less than about 1.5 × 10⁹/ L and basophilia Granulocyte is less than about 0.1 × 10⁹/L。

It will be appreciated that in certain embodiments, can fall for the cell counting of any one in these cellular blood components Outside these scopes stated, this animal suffers from the situation of the leukemia of a kind of form, such as CML, CLL etc. the most wherein Under.

A usual important factor is lymphocyte count and/or monocyte count.In more detail, to these groups Point, when one or both of these countings are markedly inferior to the scope of statement, it is immunogenic that this animal can unlikely respond this Use.

When this animal continues to accept antibody mediated immunity treatment, in an embodiment of the second and third aspect of the present invention, The treatment of this antibody mediated immunity is allowed to be continued until desired clinical endpoint.Typically, this desired clinical endpoint is that cancer is reduced to The level that generally can not detect.Then during immunization therapy or at the end of, evaluate this animal formed or produce right P2X₇The ability of the immunne response of receptor.Then when this evaluation discloses this animal may be from P2X₇Immunogenic immunity is benefited Time, then use this animal immune former.

In a preferred form of the second and third aspect of the present invention, in this animal body, it is produced from antibody mediated immunity The level of the antigen binding site of the non-autologous or external source of the circulation of therapy, when this immunne response is formed in this animal body be Generally can not detect.Importantly, a key of present inventor is the discovery that effect of Antybody therapy is at antibody combining site Higher circulation composition time reduce, particularly when cancerous cell is in low-down copy number, or the most not When can detect.This is considered as relative to the high concentration antigen binding site produced in standard antibody immunotherapy, thin in cancer The non-functional P2X of the low copy number on born of the same parents₇One function of receptor.Definitely, in example in this, present inventor has sent out Existing, along with the cyclical level of antigenic specificity binding site increases, and cancer cell count reduces, by blocking the anti-of this receptor The non-functional P2X of former specific binding antigen binding site₇The probability of the gathering of receptor is the highest.This blocking-up increases Add intended cytotoxicity, apoptosis or the probability of other effects combined by the antigenic specificity of antigen binding site, This will be impossible.People can determine in circulation by detecting any standard serological techniques of the antibody in body fluid The level of exogenous antigen binding site, a preferred example is the ELISA utilizing antibody to capture antigen binding site.

Outside upper, but being not desired to be assumed constraint, ladies and gentlemen inventor is it is considered that use immunity increasing in the presence of infusion antibody Add this infusion antibody and can be incorporated into the risk in this immunogen, caused formation and the removing of immune complex, thus avoid The induction of antigen presentation and antigen specific immune.Therefore, in certain embodiments, wait until to immunogenic anti- Before the induction of former specific immune response, non-autologous or exogenous antigen binding site level is removed from circulation, is special Useful.

Method in the second and third aspect of invention described herein needs at P2X to be used₇Receptor (especially one Non-functional P2X₇Receptor) form immunne response in the animal body treated.Usually, immunogen is to non-functional for this purpose Property P2X₇Receptor rather than to functional P2X₇Receptor draws the immunogen of immunne response.

The inventors discovered that, multi-epitope is present in large-scale species, it is meant that according to the present invention's in this Second and third aspect, (i) antigen binding site or antibody can be cultivated in large-scale animal and treat for antibody infusion And the animal of (ii) on a large scale species can treat by active immunotherapy.Following table demonstrates people's multi-epitope and at it The % concordance of the epi-position in his species.

Table 2: people E200/E300 and the comparison of other species

This immunogen can include or consist of: includes a P2X₇The peptide of the sequence of receptor.This peptide can comprise At least one sequence, this sequence can exist on a MHC II quasi-molecule, or can be with a B The immunoglobulin interaction that cell receptor or a B cell film combine.Typically, this peptide includes an inhuman source, excellent Choosing is companion animals P2X₇Receptor or a sequence of its fragment.

Known a series of peptide based immunogens and at PCT/AU2002/000061, PCT/AU2002/000061, PCT/ Discussing in AU2008/001364 and PCT/AU2009/000869, their content is combined in this in full with it.

The following describe including for non-functional P2X in those specifications₇Receptor produces the table of immunne response The exemplary peptides immunogen of position.

As discussed above, in one embodiment, this peptide based immunogens includes the P2X of part or all of dog or cat₇It is subject to Body sequence.

It will be appreciated that according to the second and third aspect of the present invention, these be only to formed immunne response useful can Can immunogenic example.It addition, the present invention include as described in these applications to non-functional P2X₇Receptor forms one The purposes of other peptides that immunne response is useful.

Typically, this immunization protocol relates to 2 or more immunity.In the first immunity, this object of study may be right Immunologic development IgM response.Second immunity may develop IgG response.Further immunity may increase this IgG response.

When this immunogen is a peptide, this peptide can be provided, preferably from about by the amount using about 0.1mg to 1mg every time 0.25mg to 0.75mg, preferably from about 0.5mg in a towser body, but halve in doggie or kitten body.

Another that can apply about 0.3mg peptide in a towser body is used as growth, but subtracts in doggie or cat body Half.

In an embodiment of the second and third aspect of the present invention, it is being applied for antibody mediated immunity therapy When the cyclical level of antigen binding site generally can not detect, carry out the first immunity.In other words, to associated cancer biomarker The circulating antibody of thing can not be detected in peripheral blood.Then the level produced at ensuing a few week monitoring IgM.First In about 4 to 5 week after immunity, the level of IgM antibody may have already decreased to insignificant cyclical level.Carry out second the most at this moment Immunity, and the level produced at ensuing a few week detection IgG.In ensuing some months/year, immunity can be carried out Further test, and the immunity of increase can be provided as required.

As discussed above, this immunne response can with one biomarker of targeting, this biomarker with led to The biomarker crossing antibody mediated immunity therapy targeting is different.Such as, anti-CD 20 antibodies may be used for antibody mediated immunity therapy, and one Individual non-functional P2X₇Immunogen is for producing an immunne response.

In another embodiment of the second and third aspect of the present invention, by antibody mediated immunity therapy and immunity one list of targeting Only biomarker.Such as, one is pointed to P2X₇The monoclonal antibody of the epi-position (such as E300 epi-position) on receptor can For antibody mediated immunity therapy, and it is used for forming targeting P2X₇On the immunne response of different epi-position (such as E200 epi-position) Immunogen may be used for immunity.

One peptide based immunogens of the second and third aspect for the present invention in this can have 6,7,8,9,10,11, 12, the length of 13,14,15,16,17,18,19,20,21,22,23,24,25 or 26 residues.

In an embodiment of the second and third aspect of the present invention, according to a method of the present invention, it is used for being formed The immunogen of immunne response is to have P2X₇The peptide of the sequence of receptor, this P2X₇Receptor can have or can not have cis (cis) Pro210 of conformation.

This immunogen can be P2X₇Extracellular domain or P2X₇Any one of isotype or multiple form.Should Immunogen can by soluble form or with solid phase (such as cell membrane, beadlet or other surfaces) associate and provide be used for using.

There is disclosed herein for screening be used as immunity originally formed immunity according to the method for the present invention in this The method of the peptide of response.One example includes erythrocyte purposes in rosette test.In this experiment, it is attached to function An antibody on property receptor is used as a positive control, observes rosette wherein.If Flos Rosae Rugosae can not be formed Knot, it is determined that this test antibody not can be incorporated on functional receptor.If it is observed that it is attached to one expresses non-functional In the cell line of receptor (include discussed herein those), it is determined that it is attached on non-functional receptor.

Peptide in the present invention can be made by this area any number of known technology, these technology include solid phase synthesis and Recombinant DNA technology.

As known in the art, carrier is to can be incorporated into peptide epitopes thus to strengthen immunogenic material.One A little carriers, by being attached on multiple peptide, thus provide the molecule with increase to the host that will develop immunne response wherein One antigen of amount, accomplishes this point.

Preferably carrier includes bacteriotoxin or toxoid.Other suitable carriers include Neisseria meningitidis adventitia egg In vain, albumin (such as bovine serum albumin), synthetic peptide, heat shock protein, KLH, B. pertussis proteins, from hemophilus influenza Protein D and from the toxin A of Clestridium difficile, B or C.

When this carrier is bacteriotoxin or toxoid, diphtheria toxoid or tetanus toxoid are preferred.

Preferably, this carrier comprises can be with the functional group of the reactive polypeptide of the present invention, or can be modified to can be with This reactive polypeptide.

This immunogen can subcutaneous, Intradermal and/or intramuscular administration.

In a preferred form, for a P2X₇Receptor forms an immunne response and sends out for described here The compositions of bright method includes adjuvant or compound, is used for strengthening immunne response.

Known a lot of adjuvant；See also Allison (Ai Lisen), (1998, Dev.Biol.Stand. (grow biology Standard), 92:3-11) (being incorporated herein by reference), Unkeless (Ang Keliesi) et al. (1998, Annu.Rev.Immunol. (immunology annual review), 6:251-281) and Phillips (Karen Phillips) et al. (1992, Vaccine (vaccine), 10:151-158).Can according to invention utilize Exemplary Adjuvants include, but are not limited to cell because of Son, aluminium salt (such as aluminium hydroxide, aluminum phosphate etc.；Baylor (Beile) et al. (Vaccine (vaccine), 20:S18,2002)), solidifying Glue-type adjuvant (such as calcium phosphate etc.)；Antimicrobial adjuvant (such as includes the immunomodulating DNA sequence of CpG motif；Endotoxin is such as Monophosphoryl lipid A, Ribi (Lay ratio) et al. (1986, Immunology and Immunopharmacology of Bacterial endotoxins (immunology of bacterial endotoxin and immunopharmacology), Plenum Publ.Corp. (Pu Lainan Publishing company), NY, p407,1986)；Extracellular toxin such as cholera toxin, HLT and pertussis toxin, PT, Muramyldipeptide, etc.)；([(Novartis is public for Novartis for such as Freund adjuvant, MF59 for oil emulsion and adjuvant based on emulsifying agent Department)], SAF, etc.)；Particulate adjuvants (such as liposome, biodegradable microspheres, etc.)；Synthetic adjuvant (the most non-ionic block Copolymer, muramyl peptide analog, polyphosphazene, synthesis polynucleotide, etc.)；And combinations thereof.Other Exemplary Adjuvants Including some polymer (such as group of polyphosphazenes；Be described in United States Patent (USP) 5,500,161), Q57, saponin (such as QS21, Ghochikyan (Gu Jikeyang) et al., (vaccine (vaccine)), 24:2275,2006), zamene, tetrachloro ten oxide (tetrachlorodecaoxide), CPG7909 (Cooper (cooper) et al., (vaccine (vaccine)), 22:3136, 2004), poly-[two (carboxyphenoxy) phosphonitrile] (PCCP；Payne (Penn) et al., (Vaccine (vaccine)), 16:92, 1998), interferon-γ (Cao (Cao) et al., (Vaccine (vaccine)), 10:238,1992), block copolymer P1205 (CRL1005；Katz (card hereby) et al. (Vaccine (vaccine)), 18:2177,2000), interleukin II (IL-2；Mbwuike (Wei Ke) et al. (Vaccine (vaccine)), 8:347,1990), polymethyl methacrylate (PMMA；Kreuter (Crewe pool) etc. People, (J.Pharm.ScL (Pharmaceutical Sciences magazine)), 70:367,1981), etc..

In an embodiment of the second and third aspect of the present invention, at one according to describing the one of the present invention in this Individual method, provides on a phage surface for the immunity of an animal and comprises a P2X₇The peptide immunity of receptor sequence Former.

One whole antibody including a variable domains, or one fragment；Or

One P2X₇Receptor, or a P2X₇One fragment of receptor；

Written explanation in a method described here.

Preferably, this antibody or fragment are attached to a P2X₇Receptor, it is therefore preferable to a non-functional P2X₇Receptor.More Preferably, this antibody or fragment are not joined to functional P2X₇On receptor.

Preferably, this written explanation with the form of a label or packaging with operation instruction for describing in this one In individual method.

Example

Example 1 cat 1 (GB)

Wide SCC (squamous cell carcinoma) scalp and lip, area is up to 4cm²And account for the full depth of lip.Controlling Before treatment, the left lip of SCC has diffused into subcutaneous angle of mandible.Before the treatment, IHC (SABC) is caught for nf-at the left lip of SCC P2X₇Target recipient.

The partial efficacy injected with (i.t.) in tumor

As IHC discloses, target recipient is widely distributed in infected tissue

With dosage 3mg/kg, use under fluid maintains speed, single infusion targeting nf-P2X₇The cancer of receptor is special Property treatment antibody

By the 4th day, have what tumor removed to understand sign

By the 14th day, after 3mg/kg single dose IV (intravenous injection), after treating 2 weeks, the left labial tumor of most SCC It is eliminated.After 3mg/kg IV, after treating 2 weeks, SCC bunch above left eye also removes.Now, at original disease site Epithelium gross weight changes substantially

After 3 IV, scalp SCC and lip SCC is substantial amounts of is removed, despite the new epithelium by sick cat scratcs within 5 weeks The sign of tissue.Center SCC pathological changes that be positioned at lower jaw center line, that penetrate in skeleton has white nodositas scar tissue now Outward appearance.Unlike the patient's condition wherein having obvious severe pain when treatment starts, this disease cat is not the pathological changes position with process Point institute is worried.

After the IV treatment of 6 months, this patient's condition solves, and wherein discloses via the X-ray in interior mandibular bone, even has new Skeleton increases

Elapse over time, lose effect due to the appearance of anti-sheep antibody, total accumulation of the clinical clearance rate of impact Dosage is estimated as 30mg/kg to 40mg/kg

Example 2 cat 2 (AL)

Before i.t. injects, nose has SCC

Only 1x i.t. injects, because cat 21 years old, owing to general anesthesia causes cortical blindness to continue some hours

Subsequently with the anti-nf-P2X in local₇IgG

Within 5 days ,-SCC nose forms new organization

Within 19 days ,-SCC nose forms more new organization

Example 3 cat 3 (PC)

Cancer of pancreas is with to liver and mesenteric mesaraic transfer

After 2 weeks intravenous (i.V.) infusions, ultrasonic scanning display 1cm level mesentery transfer is inconspicuous

After 2 weekly intravenous injections, by the sign of some tumor lysis (liquid) of ultrasonic scanning

After 4 weeks, Secondary cases liver is retracted to 2cm from diameter 5cm

After 4 weeks, the constitutional pancreas of 5-6cm diameter has the 80% of about its liquefaction quality

Behind 5th week end, due to pancreas tumor dissolve/hemorrhage to abdominal part and possessory requirement, carried out peaceful and comfortable Extremely

Example 4 cat 4 (FH)

Nose SCC expands to the bridge of the nose from left nose hole 2cm

At the end of 2008 after operative treatment, biopsy discloses the SCC recurred widely

After 5mg/kg single intravenous dose, upper nose surface tumours volume reduces, and the sponge concordance on this surface becomes Obtain firm

After the most weekly infusion, this pathological changes is the most obvious.Application third time infusion, and at 2 weeks ends, The clinical indication of all of pathological changes does not exists.Other infusion is applied to guarantee the residue preneoplastic cell that may have existed Elimination.

Under accumulated dose 18mg/kg, clinical removing in 21 days

No longer recur after 14 months, so sick cat looks as curing completely

Example 5 cat 5 (CO)

Medical history

Sick cat in 2008 from removing Bao Wen sample (Bowen ' s-like) pathological changes from Zuo Nie district.In March, 2009 once Inspection being noticed, this pathological changes has reproduced and occurred a new pathological changes in opposition side.These are the living tissues with following result Check:

Diagnosis

Have by the time the most serious hemocyte crust, epidermis scar and moderate Perivascular eosnophilia dermatitis how in Heart squamous cell carcinoma in situ.Skin biopsy from left ear.

All and interstitial the eosnophilia dermatitis of moderate proliferating vessels is with moderate epidermal Fiber Laminated hamartoplasia.Come From the biopsy of cervical region.

Along with moderate hyperpigmentation, moderate erythrocyte crust and the wide skin scar in more major part cranium Multicenter squamous cell carcinoma in situ.Biopsy from auris dextra.

Notes and commentary

This cat has multiple stove points of the neoplastic transformation of epidermis and hair follicle tissue, with multicenter squamous cell carcinoma in situ or Bao Wen sample is sick consistent.These pathological changes are similar with the biopsy that in June, 2008 submits to.In the part checked, there is no squamous The sign of epithelium infiltrating carcinoma.

SCC (Bao Wen Shi (Bowen ' s) in situ)

To the infusion anti-nf-P2X of 5mg/kg sheep₇IgG does not has untoward reaction

After 2 weeks, pathological changes has significantly reduced but still wide, due to initial improvement, so application of second time infusion

After veterinary dermatologist studies, all wide Bao Wen sample pathological changes are eliminated, and this veterinary dermatologist notes Meaning is to after lesion surface and infections relating are eliminated, and the deep SCC at auris dextra ditch is only apparent property.Disease must be avoided Cat plugs and tears new epithelium, limits necklace by additional one and allows skin surface to heal.

Example 6 Canis familiaris L. 1 (CJ)

In 30kg Canis familiaris L., the abdominal part at veutro has angiosarcoma medical history.Because tumor so has aggressiveness, after Repeated Operation, This Canis familiaris L. is no longer surgical candidates.Use intratumor injection nfP2X₇IgG and 3 IV infusion nfP2X₇The antibody of affinity purification is controlled Treat.Intratumor injection 6mg antibody, in the dermatosis of 6cm diameter, illustrated the reduction of 80% in one week, along with the most swollen Oncolysis is as obvious secretions.

Carry out 1 intratumor injection under dosage by 2-3.4mg/kg administration and 3 intravenous injections treat 4 weeks

With the anti-nf-P2X of sheep₇After IgG treatment, gross tumor volume reduces

Due to tumor lysis/seepage symptom and the possessory requirement of these big tumors (gross weight 1-2kg), On March 21st, 2010 practises mercy killing

Example 7 Canis familiaris L. 2 (MO)

In neuropathology, it is found to be transitivity mesothelioma with the transitional cell carcinoma (TCC) of malignant ascite

By carrying out 3 intravenous medical treatment under the dosage of 1-2.7mg/kg together with chemotherapy 2 weeks

At second week, tumor reduces 15%, although there is also ascites

At the 3rd week, under possessory requirement and oppose the suggestion of veterinary, stop treatment

Example 8 Canis familiaris L. 3 (PI)

Lymphadenomatous special treatment and rehabilitative training-the outward appearance of mastocyte between two fingers in the middle of before right pawl Ledge under hard and foot pad causes discomfort.Operation may require that removal foreleg is to maintain the limit of 3cm

After mastocyte lymphoma special treatment and rehabilitative training-IV5mg/kg1 week-volume reduction 40%

The week present deliquescing of pathological changes of mastocyte lymphoma special treatment and rehabilitative training-2 and volume are greatly reduced, There is no prominent part under foot pad, and in Canis familiaris L. body, there is no the sign of discomfort.The entity core of pathological changes is limited to list now Individual finger, the reacting cells capsule along with surrounding is sufficiently flexible.

Metastasis in subscapular lymph nodes can not detect by the 2nd week

Primary affection 3 months major parts in appearance keep constant

Owing to primary affection becomes to fester under claw, under possessory requirement, Canis familiaris L. is practised mercy killing

Postmortem biopsy illustrate disease Canis familiaris L. without all tumors, this primary affection site does not has mastocyte, comprises anti- Answering property lymphocyte

Pathological changes may be removed by the accumulated dose less than 30mg/kg

Example 9 Canis familiaris L. 4 (BE)

After extreme pain and cyllopodia occurs in sick Canis familiaris L., upper left humerus is diagnosed to be osteosarcoma via CT scan, prepares to carry out Euthanasia.

Start by the weekly infusion of 10mg/kg.In one week, sick Canis familiaris L. and the owner again together with movable, and Seem substantially not have misery, and want to go for a trot.

At the 9th week, obtaining diagnosis tissue after CT scan and treatment under general anesthesia, this time is considered as bone Bone regrows the shortest time that easily can detect

At the 9th week, biopsy there is no the sign of tumor cell, seem the best with seasonal disease Canis familiaris L.

Sick Canis familiaris L. is owing at left humerus fractured near end, being carried out euthanasia at 5th month.The biopsy of disease site It is shown without remaining osteosarcoma sign.Seem there is obvious overall relief, but bone is because tumor is the thinnest and crisp.

In this case, it should the outside clamping plate of application protect bone to carry out associated treatment to assist bone weight simultaneously Raw.

Example 10 Canis familiaris L. 5 (WC)

There is the transitivity mastocyte lymphoma (2 grades) of high di (25).On rib, shoulder and back leg have many Place pathological changes ＞ 5cm.Carry out the weekly IV infusion of 10mg/kg immediately.

At first week end, total lesion volume reduced 75%, as PI illustrates, consistent with the expectation quickly removed.

Major lesions still has long-pending block, but after second time infusion, reduces further at second week end

After three infusions and one week draw a response, at the 3rd week end on schedule by ultrasonic complete with cytology Surface analysis disease site

Carry out after the last pin of all disease site is pumped in 3 months and when 6 months.There is no the mark of any mastocyte As.

Within 13 months, seasonal disease Canis familiaris L. is not deemed tumor.

Example 11 Canis familiaris L. 6 (HL)

Sick Canis familiaris L. presents the high-level transitivity mast cell tumor being derived from lower limb with lymphatic metastasis.

Infusion of therapeutic is passed through 6 weeks once in a week under the dosage of 10mg/kg.

Eliminate to all of swelling in the 3rd week

At the 6th week, pathology are shown at tumor sites did not had mastocyte

Sick Canis familiaris L. is not recurrence in 1 year

Example 12 antibody manufacture

In phosphate buffered saline (PBS) (PBS), by the 500 μ anti-P2X of g sheep of conjugate₇Antibody (the P2X of about 100 μ g₇, table Position) it is diluted to 0.8mL, and with 1.2mL Freund's complete adjuvant (Freund ' s Complete adjuvant) emulsifying.With anti- Former/adjuvant Emulsion carries out both subcutaneous injection and intramuscular injection in multiple site to sheep.After eight weeks, these sheep phases Commensurability injects in multiple sites again with the conjugate of Freund's complete adjuvant emulsifying.Repeat this process after 4 weeks, and these move Thing is from taking blood from jugular vein.The serologic test antibody specificity collected.Then these sheep at the interval regular injection of eight weeks and take Blood provides a serum pond comprising specific antibody.

Other sheep is injected with the conjugated antigen of same dose with being similar to above scheme, but uses different adjuvant. In these animal bodies, the Quill A/DEAE dextran solution of the antibody of the dilution of 0.7mL and 0.1mL is (in the PBS of every mL The Quill A+25mg deae dextran of 2.5mg) and 1.2mL ISA50V Montanide mixing.At multiple site skins Hemostasis and intramuscular injection Emulsion.The antibody using this adjuvant to produce creates the same with those produced with Freund adjuvant Specificity.

Use the two kind adjuvants identical with in sheep and identical infusion protocol to cultivate antibody in rabbit, only difference is that The conjugate of 300 μ g amounts is used for injection.The antibody cultivated has phase homospecificity with those produce in sheep, and can The epi-position resisted with they readily distinguishing cultivation.

In mice, cultivate antibody resist those epi-positions combined, and also anti-non-functional P2X₇The uncombined table of epi-position Position (it can distinguish those receptors that can not form (from) hole and therefore can not apoptosis).In these experiments, use Adjuvant is product I mmunEasy of Qiagen GmbH (QIAGEN Pty Ltd), and it comprises immuno-stimulatory products CpG DNA (trade mark of Ke Lai pharmacy group (Coley Pharmaceutical Group Inc.)).At 60 μ L PBS and 25 μ L ImmunEasy adjuvant dilutes the epi-position of 62.5 μ g or the epi-position/mice of combination.In multiple sites, subcutaneous injection and intramuscular note Penetrate mice.Repeat the program after two weeks and again repeated after another two weeks.The blood of mice is taken after third time is injected 8 days.With The antibody that this method is cultivated in mice also is able to distinguish different P2X₇, epi-position, and anti-P2X₇Resisting of non-functional epi-position Body and those antibody cultivated in sheep and in rabbit provide result.

The induction of example 13-immunne response in companion animals body

Materials and methods

Peptide

The peptide based immunogens of GHNYTTRNILPGLNITC (SEQ No. ID: the 3) form of synthesis of high purity, residual at C-terminal Cys Ji Chu is added thereto to cross-linking agent maleimidocaproyl-N-hydroxy-succinamide (MCS).This peptide is linked to one Carrier protein keyhole limpet hemocyanin (KLH) so that peptide is 40% to the average percentage of total peptide-protein conjugates.This peptide or The similar replacement peptide GHNYTTRNILPGAGAKYYKENNVEKC being attached to KLH has respectively constituted primary and compound selectivity Epi-position target, they make the nfP2X that will prepare from natural receptor₇Receptor can break up.

Adjuvant

Using Imject Alum, one is generally used in people's immunity, a kind of algeldrate gold and magnesium hydroxide add The adjuvant of the approval of the liquid formulations composition of the inert stable agent in a upper gel.At 2.5mg/mL conjugate (1mg/mL Peptide) drip this peptide-protein conjugates, wherein 0.75mL to be comprised equal to 0.5mL conjugate the target peptide table of 0.5mg under concentration The amount of the adjuvant of position is sufficiently mixed in adjuvant.

Immunity

Immunization protocol consists of: primary inoculation (twice subcutaneous injection and twice intramuscular of an accumulated dose 0.5mg peptide Injection), after the later moon, apply accelerator with same way 0.3mg peptide.Collect immediately with after injecting one week before the injection Blood serum sample.It is desirable that at anti-nfP2X₇The last infusion of antibody uses inoculation after being not less than one month, by remaining specificity Anti-nfP2X₇Antibody infusion guarantees do not have immunogenic isolation.

ELISA

The anti-nfP2X of specificity is measured by ELISA₇Antibody response.Briefly, this elisa plate is coated with specific target peptide Epi-position, adds patients serum with the concentration successively decreased thereon.After cleaning, apply suitable two grades of anti-human antibody (anti-IgM or anti-igg Type) the specific human anti-nfP2X that with IgM or IgG form exist is detected and determined in patients serum₇The concentration of antibody.

After inoculation, do not have IgG to detect, but IgM in a small amount detected.Along with this growth, IgM concentration has returned to base Line zero, and IgG produces with the concentration higher than initial IgM, if there is not nfP2X in existing tumor₇Receptor sinks (sink). In its primary tumo(u)r by anti-nfP2X₇In the animal patient that immunotherapy is removed, in the case of there is no this sinking, inspection Measure in serum, have the anti-nfP2X of specialized endogenous₇The obvious group of antibody, rank is 25mg/kg.

Fig. 3 shows the ELISA result of a sick dog, this immunity proceeded as above.Specificity circulating antibody freely The recurrence of level and initial clearance rate the most completely and shortage transitivity mast cell tumor be consistent.

It will be appreciated that the present invention disclosing in this manual and defining expand to mentioned or from context or All alternative combinations of two or more independent features obvious in accompanying drawing.All these different combinations constitute the present invention Different alternative aspect.

Claims

1. it is used for being attached to a kind of Canis familiaris L. non-functional P2X₇Whole antibody on receptor or its comprise a sheet of a variable domains Section preparing a kind of purposes in treating Canis familiaris L. body in the medicine of cancer or the patient's condition being associated with cancer or symptom, wherein this Planting cancer is mastocytoma.

2. purposes as claimed in claim 1, wherein this antibody or its fragment are not joined to functional P2X₇On receptor.

3. purposes as claimed in claim 1, wherein this antibody or its fragment are from a kind of polyclonal antiserum.

4. purposes as claimed in claim 1, wherein this antibody or its fragment are cultivated, this antigen pair for a kind of antigen It is xenogenesis for it carries out the immune system cultivated wherein.

5. purposes as claimed in claim 4, wherein this antigen is xenogenesis for Canis familiaris L..

6. purposes as claimed in claim 1, wherein this antibody or its fragment are xenogenesis for Canis familiaris L..