CN1997277A - Methods for treating cancer using vascular endothelial cell growth inhibitor VEGI-192A - Google Patents

Abstract

The invention features methods and compositions for treating cancer by administering an effective amount of a VEGI-192A polypeptide. In some embodiments, the cancer is lung cancer or breast cancer.

Description

Utilize vascular endothelial cell growth inhibitor VEGI-192A treatment method for cancer

The reference of reporting to the leadship after accomplishing a task of related application

The application requires the U.S. Provisional Patent Application U.S.Serial No.60/529 that submitted on December 11st, 2003,60/536,653 the priority of submitting on January 13rd, 173 and 2004, and it is whole as with reference to introducing.

The statement of relevant federal sponsored research or exploitation

The present invention obtains the support of NIH-HL060660 and NIH-CA102181 appropriation under the NIH of U.S. government and carries out.U.S. government can have some right of the present invention.

Technical field

The present invention relates to utilize vascular endothelial cell growth inhibitor VEGI-192A treatment method for cancer.

Background technology

Vascular endothelial cell growth inhibitor (VEGI) is the gene outcome of endothelial-cell specific.Reported four kinds of heterogeneous of people VEGI: first kind of form of the VEGI of discovery is 174 amino acid lengths; Found a kind of form of multi-form and 251 amino acid residues of two kinds of 192 amino acid residues subsequently.Referring to Zhai etc., Int.J.Cancer 82:131-136 (1999); Zhai etc., FASEB are (1999) J.13:181-189; Chew etc., FASEB J:16:742-744 (2002); PCT WO03/039491; U.S.Pat.Appl.Pub.No.2003/0170242.All heterogeneous are for producing the splice variant from collaborating genes.Four kinds of heterogeneous are different but have 151 amino acid whose identical cores of coded protein remainder in its N stub area.

Four kinds of heterogeneous sequences comparison shows that they have homogeneity with TNF (TNF) protein superfamily 20-30%.The effect of different isomerization type is also indeterminate, but according to the film combination and the secreted form of reporter molecule, its effect is undoubtedly meticulous and complicated.So far as if the institute of report shows the secreting type inhibition growth of tumour cell of VEGI-174 on evidence and impels apoptosis.VEGI-174 hydrophobicity characteristics hint that it is to have the typical II type transmembrane protein that amino acid 29-174 forms extracellular domain.Total length VEGI-174 does not have any effect to tumor growth when crossing expression in cancer cell, do not suppress these cells when being transfected into endothelial cell yet.Referring to Zhai etc., FASEB J13:181-189 (1999); Chew etc., FASEB are (2002) J.16:742-744.Some members of TNF family (comprising TNF and Fas part) have confirmed to work from the film cracking and as soluble protein.Referring to Bjornberg etc., Scand J.Immunol.42:418-424 (1995); Kayagaki etc., J Exp.Med.182:1777-83 (1995).This does not also confirm for VEGI-174.Yet the artificial recombination secreting type of this VEGI-174 (s-VEGI) that only comprises the VEGI-174 extracellular domain and be derived from the secreted protein signal peptide suppresses tumor growth when crossing expression in cancer cell.Referring to Zhai etc., Int.J.Cancer 82:131-136 (1999); U.S.Pat.Appl.Pub.No.2002/0111325.The heterogeneous VEGI-251 that abundance is the highest has the secreting signal peptide of inferring.The expression of crossing of VEGI-251 causes endothelial cell apoptosis and growth inhibition.PCT?WO03/039491；U.S.Pat.Appl.Pub.No.2003/0170242。Equally, the reorganization VEGI that has confirmed to comprise 151 amino acid core sequences that all VEGI types all have impels apoptosis and blocking-up growth of tumour cell, though have only low effectiveness.Referring to Wang etc., Acta Biochimica et Biophysica Sinica32 (5): 485-489 (2000).Reported reorganization that produce with VEGI-192A refolding in the grow up propagation of BAE (ABAE) cell of vitro inhibition.PCT?WO03/039491；U.S.Pat.Appl.Pub.No.2003/0170242。The refolding method of protein is in U.S. Patent No. 6,583, report in 268.

All patents cited herein, patent application and publication are incorporated herein by reference in this integral body.It is to be noted that the reference to publication in the background parts is not to admit that this publication constitutes prior art of the present invention.

Summary of the invention

The VEGI-192A protein that the present invention is based on the reorganization generation is effectively found treatment cancer (as lung and mammary gland).

In one aspect, the present invention comprises to individuality using the VEGI-192A polypeptide of effective dose to treat method for cancer in the individuality.In some embodiments, cancer is lung or breast cancer.

In yet another aspect, the present invention comprises to individuality using the VEGI-192A polypeptide of effective dose to suppress the method for tumor growth in the individuality.In some embodiments, cancer is lung or breast cancer.

In yet another aspect, the present invention comprises to individuality using the VEGI-192A polypeptide of effective dose to delay the method for cancer development in the individuality.In some embodiments, cancer is lung or breast cancer.

In yet another aspect, the present invention comprises to individuality using the VEGI-192A polypeptide of effective dose to delay to shift in the individuality method of development.In some embodiments, cancer is lung or breast cancer.

In yet another aspect, the cancer of being treated is a terminal cancer.In some embodiments, terminal cancer is lung or breast cancer.

In yet another aspect, the present invention is for comprising the pharmaceutical composition of VEGI-192A polypeptide (being the VEGI-192A polypeptide of effective dose in some embodiments) and pharmaceutically acceptable excipient.

In yet another aspect, the invention provides the kit that is used for any method described herein.In some embodiments, kit comprises container, contains the composition of the VEGI-192A polypeptide that combines with pharmaceutically suitable carrier and uses the specification of the composition in arbitrary method described herein.

In some embodiments of the present invention, VEGI-192A polypeptide behaviour VEGI-192A.In some embodiments, the VEGI-192A polypeptide comprises the amino acid sequence of SEQ ID NO:1.

In some embodiments, individuality is a mammal.In some embodiments, individuality is the people.

The accompanying drawing summary

Fig. 1 has shown the purifying of VEGI-192A (having the His label) of refolding and the growth inhibition determination method of endothelial cell (ABAE).Figure 1A has shown Sephacryl S-300 column chromatography.Post 20mM Tris, 0.2M NaCl and 0.4M urea balance and the operation of pH8.0.Top at the peak, the set of three kinds of fractions of 1,2 and 3 expressions.

Figure 1B has shown the chromatography fraction that shows among Figure 1A, and the non-reduced SDS-PAGE of set 1,2 and 3 analyzes.Fig. 1 C has shown that gathering 3 endothelial cell growth suppresses to measure, and points out that 50% inhibition concentration (IC50) is 12ng/ml (0.5nM).

Fig. 2 has shown the affinity purification of VEGI-192A (having the His label).Fig. 2 A has shown the affinity column purifying of the VEGI-192A of refolding.Arrow indication VEGI-192A peak.Fig. 2 B has shown the SDS-PAGE from the sample of the enrichment of Fig. 2 A and dialysis.1: non-reducing; 2: reduction.

Fig. 3 A has shown the Lewis lung neoplasm growth inhibition by the VEGI-192A (having the His label) of (IT) injection preparation as be shown in the examples in (IP) in the peritonaeum or the knurl.Fig. 3 B has shown the time-to-live that the negative knurl mouse in VEGI-192A injection back is improved.

Fig. 4 has shown the apoptosis endothelial cell that increases behind VEGI-192A (the having the His label) intratumor injection with preparation as be shown in the examples and the microvessel density of reduction in the mouse that has the human breast carcinoma heterograft tumour that forms by the MDA-MB-231 cancer cell.A hurdle: to the fluorescently-labeled tumour of not treating of apoptotic cell; B hurdle: to the not treatment tumour of CD31 (endothelial cell mark) immunostaining; C hurdle: to the tumour of the fluorescently-labeled VEGI-192A treatment of apoptotic cell; D hurdle: to the tumour of the VEGI-192A of CD31 (endothelial cell mark) immunostaining treatment.

Fig. 5 shows the interior absorption that suppresses Lewis lung cancer (LLC) by VEGI-192A.The LLC cell inoculation is in the back of C57B1 mouse.VEGI-192A (5mg/Kg) in (the 0th day) on the same day of LLC cell inoculation through intraperitoneal injection (IP).Measured excipient and the VEGI-192A treatment gross tumor volume of (being called " VEGI " group in the drawings) at the 4th day.

Fig. 6 has shown the spleen body weight of the mouse of VEGI-192A treatment back LLC inoculation.The mouse of LLC inoculation is treated with VEGI-192A (5mg/Kg) in the 4th day (when tumour becomes tangibly), the 7th day, the 8th day, the 9th day and the 10th day.Fetched spleen at the 11st day and weigh.

Fig. 7 A has shown that the serum of VEGI-192A treatment back cell factor distributes.The serum-concentration of cell factor is represented with pg/ml.Collected serum at the 11st day.The luminescence assays kit (LINCOplex) that utilizes antibody to put together is measured cytokine levels.Fig. 7 B has shown that comparing the horizontal multiple of serum cytokines with VEGI-192A treatment animal with excipient treatment animal changes Fig. 7 A data of representing." experimental group " refers to VEGI-192A treatment group." control group " refers to vehicle-treated groups." * " expression statistical discrepancy (t check, α=0.05).

Fig. 8 has shown cell factor distribution (data shown in Fig. 7 B) and the comparison of breeding and converge the cytokine-expressing distribution of (G0-is synchronized) HUVEC in the mice serum.Propagation or the HUVEC cell that converges use the VEGI-192A of 500ng/ml to handle.The cytokine-expressing of HUVEC distributes and utilizes cDNA microarray (from Fred Hutchinson Cancer Research Center) to measure.

Fig. 9 has shown that neo-implanted LLC growth of tumor suppresses.LLC cell (1 * 10 ⁶/ per injection/every animal) was inoculated in the flank of C57BL black mouse at the 0th day.Injected administered recombinant VEGI-192A (20mg/kg) at the 5th, 9 and 12 day by (IP) in the peritonaeum.Measure tumor size before the next-door neighbour VEGI-192A treatment." no Rx " refers to vehicle-treated groups." Rx VEGIIP " refers to VEGI-192A treatment group.T check: p＜0.05 (treatment group n=9, not treatment group n=9).

Figure 10 has shown the inhibition that the LLC tumour forms.LLC cell (1 * 10 ⁶/ per injection/every animal) was inoculated in the flank of C57BL black mouse at the 0th day.Treat animal with reorganization VEGI-192A (20mg/kg) (being called " VEGI group " among the figure) after being right after the cancer cell inoculation.Repetitive therapy every day was until the 4th day and measured gross tumor volume at the 5th day.Asterisk: t check, p＜0.002 is (untreated: n=5; Treatment: n=6).

Figure 11 has shown that the endothelial cell specific by VEGI-192A is eliminated in the LLC tumour.The contrast that (3 week) treats from the animal and the excipient of VEGI-192A treatment when off-test is fetched tumour and is handled as described in example 7 above.Tumor biopsy stands fluorescence immunization coloration.Endothelial cell and smooth muscle cell are differentiated by specific mark CD31 (redness) and SMA (green) respectively.A hurdle: from the image of the typical tumor biopsy of VEGI treatment group; Multiplication factor: 200x.B hurdle: from the image of the typical tumor biopsy of vehicle-treated groups; Multiplication factor: 200x.C hurdle: the quantitative analysis of tumor image redness and green area.The positive endothelial cell of white bars: CD31.The positive smooth muscle cell of black bar: SMA.Asterisk: t check, p＜0.01 (every group of 5 animals between the excipient of CD31 dyeing and the VEGI treatment group; The section of 15 zone/analyses).

Figure 12 has shown the existence of residual blood vessel structure.From the LLC tumor biopsy of VEGI-192A or excipient treatment animal through immunostaining to differentiate endothelial cell (CD31), smooth muscle cell (SMA) and basement membrane of blood vessel (collagen iv).A hurdle: show the tumor biopsy image of the typical excipient treatment of CD31 positive vessels (brown), multiplication factor: 1000x.B hurdle: show the tumor biopsy image of typical VEGI-192A treatment have red blood cell but not have the sample space, chamber of the positive endothelial cell of CD31; Multiplication factor: 1000x.C hurdle and C ' (illustration): the image of using the tumor biopsy of the amphophilic typical VEGI-192A treatment of CD31 (green) and collagen iv (redness); Attention is in the shortage of CD31+ endothelial cell in by the sample space, chamber of collagen iv line of the middle confirmation of C '.D hurdle and D ' (illustration): the image for the treatment of typical case's section of tumour with the VEGI of SMA (green) and collagen iv (redness) dyeing; The existence of attention smooth muscle cell in the inner boundary of chamber spline structure.E hurdle and E ' (illustration): the image of the tumor biopsy that the typical excipient of using CD31 (green) and collagen iv (redness) to dye is treated; Note exist (CD31+) of endothelial cell in the vascular wall.F hurdle and F ' (illustration): the image of the tumor biopsy that the typical excipient of using SMA (green) and collagen iv (redness) to dye is treated; Note the existence of smooth muscle cell in the vascular wall.Blue dyeing: cell nucleus.The multiplication factor of C, D, E and F: 200x.The multiplication factor of C ', D ', E ' and F ': 1000x.

Detailed Description Of The Invention

The present invention is based on the VEGI-192A protein of finding the administering therapeutic effective dose and can be used for treating the cancer that comprises late period, for example the cancer of lung and breast cancer.

I. routine techniques

Unless otherwise stated, practice of the present invention is with applied molecular biology (comprising recombinant technique), microbiology, cell biology, biochemistry and immunologic routine techniques, and it is all in the technology of this area. Described technology proves absolutely in the literature, such as Molecular Cloning:A Laboratory Manual, and second edition (Sambrook etc., 1989) Cold Spring Harbor Press; Oligo nucleotide Synthesis (M.J.Gait compiles, 1984); Methods in Molecular Biology, Humana Press; Cell Biology:A Laboratory Notebook (J.E.Cellis compiles, 1998) Academic Press; Animal Cell Culture (R.I.Freshney compiles, 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E.Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A.Doyle, J.B.Griffiths and D.G.Newell compile, 1993-8) J.Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M.Weir and C.C.Blackwell compile); Gene Transfer Vectors for Mammalian Cells (J.M.Miller and M.P.Calos compile, 1987); Current Protocols in Molecular Biology (volume such as F.M.Ausubel, 1987); PCR:The Polymerase Chain Reaction, (volume such as Mullis, 1994); Current Protocols in Immunology (volume such as J.E.Coligan, 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A.Janeway and P.Travers, 1997); Antibodies (P.Finch, 1997); Antibodies:a practical approach (D.Catty. compiles, IRL Press, 1988-1989); Monoclonal antibodies:a practical approach (P.Shepherd and C.Dean compile, Oxford University Press, 2000); Using antibodies:a laboratory manual (E.Harlow and D.Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M.Zanetti and J.D.Capra compile, Harwood Academic Publishers, 1995); With Cancer:Principles and Practice of Oncology (volume such as V.T.De Vita, J.B.Lippincott Company, 1993).

II. definition

" effective dose " of medicine or pharmaceutical composition is for being enough to produce the amount of useful or results needed, described result comprises clinical effectiveness, as dwindle size (in the cancer situation, for example breast or lung cancer), the growth of cancer cells of tumour retardance, alleviate one or more symptoms of being caused by disease, improve the Quality of Life of suffering from this disease, reduce the dosage of these other required medicines of disease for the treatment of, for example by target-seeking strengthen another kind of medicine effect, delay advancing of disease and/or prolong individual survival. Effective dose can in single or divided doses reach. For the present invention, the effective dose of medicine, compound or pharmaceutical composition reduces directly or indirectly the propagation of (or destruction) cancer cell and slows down and/or delay the amount of development or growth or the transfer of cancer cell for being enough to. As understanding in the cancer clinical context, the effective dose of medicine, compound or pharmaceutical composition can or needn't realize with another kind of medicine, compound or pharmaceutical composition associating. Therefore, " effective dose " can consider in using the situation of one or more therapeutic agents, and if can or realize results needed with one or more other therapeutic agents associatings, so single therapeutic agent can be considered to use with effective dose.

As used herein, " associating " refers to the administration of a kind of form of therapy except another kind of form of therapy, for example the administration of VEGI-192A and other cancer therapy drugs. Thereby " associating " refer to before the administration to the another kind of form of therapy of individuality, during or afterwards a kind of administration of form of therapy.

As used herein, " cure " or " treatment " is for obtaining the useful or required effect method of (comprising and preferred clinical effectiveness). For of the present invention, useful or required clinical effectiveness includes, but is not limited to following one or more: slow down (or destruction) cancer cell growth, postpone the cancer cell found in the cancer transfer, dwindle tumour size, slow down the symptom that caused by disease, improve suffer from this disease those quality of life, reduce these other required medicines of disease for the treatment of dosage, delay advancing of disease and/or prolong individual survival.

As used herein, " development of Branch-delay " refer to postpone, hinder, slow down, hinder, the stable and/or development that delays to shift. This delay can be the different duration, and this depends on the medical history of cancer and/or the individuality for the treatment of. As apparent to those skilled in the art, sufficient or significant delay can comprise prevention in effect, and individuality does not shift in this.

" individuality " is vertebrate, preferred mammal, more preferably people. Mammal includes, but are not limited to domestic animal, sports type animal, pet (such as cat, dog, horse), primate, Mouse and rat.

As used herein, " preparation " refers to biology, medicine or compound formulation. Nonrestrictive example comprises single or compound organic or inorganic molecule, peptide, protein, oligonucleotides, antibody, antibody derivatives or antibody fragment. But the multiple compounds of synthesis example such as little molecule and oligomer (for example oligopeptides and oligonucleotides), and synthetic organic compound is based on different core textures. In addition, the compound that multiple natural source can be provided for screening is such as plant or animal extracts etc.

As used herein, " therapeutic agent " refers to comprise any preparation that can be used for therapy (here usually in the cancer scope) of antineoplastic, toxin or cytotoxin, cytotoxic preparation and radioactive drug.

III. utilize VEGI-192A to be used for the treatment of the method for purpose

The invention provides by VEGI-192A and be applied to method for cancer in the individual treatment individuality effective dose.In some embodiments, cancer is lung or breast cancer.

The present invention also provides the method that suppresses tumour in the individuality (as lung or breast cancer) growth by VEGI-192A polypeptide from effective dose to individuality that use.

The present invention also provides the method that postpones cancer in the individuality (as lung or breast cancer) development by the VEGI-192A that uses effective dose to individuality.

The present invention also provides by shifting the method for development in the VEGI-192A that uses effective dose to individuality postpone the to suffer from cancer individuality of (as lung or breast cancer).

The present invention also provides by the VEGI-192A that uses effective dose to the individuality individual medium vessels endothelial cell growth of (as lung or breast cancer) that suppresses to suffer from cancer and/or the method for propagation.The present invention also provides the method that generates by the VEGI-192A that uses effective dose to individuality suppress the to suffer from cancer individual medium vessels of (as lung or breast cancer).

The cancer of being treated can be late period.In some embodiments, the cancer in late period is lung or breast cancer.

The several formulations of VEGI-192A can be used for using.In some embodiments, VEGI-192A can use separately.In other embodiments, use VEGI-192A and pharmaceutically acceptable excipient, and can be multiple formulation.Pharmaceutically acceptable excipient is known in this field, and for promoting the material of the relative inertness that the pharmacology active principle is used.For example, excipient can be given shape or toughness, or serves as thinner.Suitable excipient includes but not limited to stabilizing agent, wetting and emulsifier, is used to change infiltrative salt, encapsulating drug, buffer solution and skin penetration enhancer.Be used for excipient that the outer medicine of parenteral and parenteral sends and preparation Remington " The Science and Practice ofPharmacy " the 20th edition, set forth among the Mack Publishing (2000).

Though also can use other forms of administration (for example, oral, through mucous membrane or the like), these preparations preparations are used for through injection (for example, in peritonaeum, intravenous, subcutaneous, knurl, intramuscular etc.) administration usually.Can be general (for example in intravenous and the peritonaeum) administration, or topical.VEGI-192A protein is used through site-specific or targeted local delivery technique.Example site-specific or the targeted local delivery technique comprises the multiple transplantable storage source or the local delivery conduit of VEGI-192A protein, packs (adventitialwrap), current divider (shunt) and support or other transplantable devices, site-specific carrier, directly injects or directly use as transfusion catheter, inlying catheter or needle tubing, synthetic graft, adventitia.Referring to, for example PCTPublicationNo.WO00/53211 and U.S. Patent No. 5,981,568.Therefore, VEGI-192A protein preferably makes up with pharmaceutically acceptable excipient (as salt solution, ringer's solution, dextran solution etc.).

Concrete dosage regimen, promptly dosage, time and repeating is depended on concrete individual and medical history that should individuality.Usually, can use any following dosage: at least about the 50mg/kg body weight; At least about the 20mg/kg body weight; At least about the 10mg/kg body weight; At least about the 5mg/kg body weight; At least about the 3mg/kg body weight; At least about the 1mg/kg body weight; At least about 750 μ g/kg body weight; At least about 500 μ g/kg body weight; At least about 250 μ g/kg body weight; At least about 100 μ g/kg body weight; At least about 50 μ g/kg body weight; At least about 10 μ g/kg body weight; Dosage or more dosage type at least about 1 μ g/kg body weight are applied.Experimental consideration as the half life period, generally will help determining of dosage.

In some individualities, may need administration more than once.The frequency of administration can be determined in whole therapeutic process and adjust, and with number, the minimizing of keeping cancer cell, the growth that slows down cancer cell and/or propagation that reduces cancer cell or the basis of developing into that postpones transfer.The existence of cancer cell can be determined the immunohistochemistry or the Flow cytometry of living tissue or biological sample (for example, by) by many methods well known by persons skilled in the art or described herein.Sometimes, the lasting release of keeping the VEGI-192A protein formulation may be suitable.Be used to realize that the various preparations and the device that continue to discharge are known in this field.

In one embodiment, the dosage of VEGI-192A can be determined in the individuality that gives the one or many dispenser by rule of thumb.The VEGI-192A dosage that individuality increases progressively.In order to assess the effectiveness of VEGI-192A, can monitor the sign of particular cancers morbid state.These signs comprise: the tumour size is through the direct mensuration of palpation or range estimation; The tumour size is through the indirect determination of X ray or other imaging techniques; As the improvement of assessing by direct tumor biopsy of tumor sample and microexamination; The mensuration of the indirect tumor marker PSA of prostate cancer (for example, at); The reduction of pain or paralysis; Speech, eyesight, breathing or other deformity relevant of improving with tumour; The appetite that strengthens or as the prolongation of the raising of the quality of life weighed by accepting inspection or survival it should be apparent that for those skilled in the art dosage will with individuality, the type of cancer, the stage of cancer, cancer whether begin to be transferred to other individual positions and before and at present jointly used therapy change.

Other preparations comprise suitable transmission form known in the art, include but not limited to carrier, as liposome.Referring to, (1997) Pharm.Res.14:853-859 such as Mahato for example.Liposomal formulation includes but not limited to cytofectin, multilamellar liposome and unilamellar liposome.

VEGI-192A can unite with other cancer therapy, for example radiotherapy or chemotherapeutics.VEGI-192A can with other cancer therapeutic agents (as Rituxan ^And Herceptin ^) administering drug combinations.

Utilize the standard method of this area to carry out the assessment of disease, as formation method and the suitable mark of monitoring.

IV. composition and the preparation method for compositions

The composition that is used for the inventive method comprises VEGI-192A protein.In some embodiments, reorganization produces VEGI-192A protein.In some embodiments, VEGI-192A protein produces and purifying from the Escherichia coli reorganization.Should be understood that composition can comprise other anticancerogenics of VEGI-192A and one or more.

VEGI-192A (exchanging use with VEGI-192A protein and VEGI-192A polypeptide) comprises any naturally occurring kind (as from people or other mammiferous full-length polypeptides), the biologic activity fragments of peptides (for example, VEGI-192A fragment described in WO03/039491 and the United States Patent (USP) publication No.2003/017242 and comprise the VEGI-192A fragment of the terminal brachymemma of N of the amino acid 26 of SEQ ID NO:1) and variant (comprising variant naturally occurring and that non-natural exists), comprise the functionally equivalent variant of its biological property of not appreciable impact and have strengthening or the variant of the activity (for example suppressing endothelial cell growth) of reduction.

The nucleotide sequence of people VEGI-192A and amino acid sequence are described in PCT WO03/039491, U.S.Pat.Appl.Pub.No.2003/0170242, and the amino acid sequence of people VEGI-192A also is presented on (SEQ ID NO:1) in the table 1.In some embodiments, VEGI-192A protein comprises the amino acid sequence of the SEQ ID NO:1 shown in the table 1.The embodiment of VEGI-192A comprises fusion (the N end merges or the C end merges), the N end fused protein that example is as shown in table 2.VEGI-192A can be from any species, as the people.

The amino acid sequence of table 1. people VEGI-192A

The protein sequence of VEGI-192A (SEQ ID NO:1) MQLTKGRLHFSHPLSHTKHISPFVTDAPLRADGDKPRAHLTVVRQTPTQHFKNQFP ALHWEHELGLAFTKNRMNYTNKFLLIPESGDYFIYSQVTFRGMTSECSEIRQAGRP NKPDSITVVITKVTDSYPEPTQLLMGTKSVCEVGSNWFQPIYLGAMFSLQEGDKLM VNVSDISLVDYTKEDKTFFGAFLL

The present invention comprises the method for utilizing the VEGI-192A functionally equivalent variant.VEGI-192A of the present invention and VEGI-192A functionally equivalent variant are identified and are characterized by any (one or more) following standard: the ability that (a) suppresses endothelial cell growth and/or propagation; B) ability of inducing endothelial cell death; B) ability of inhibition angiogenesis; C) ability of inhibition tumor growth (for example, breast and lung cancer); D) ability of activation host immune system, the ability of for example inducing one or more cell factors (as IL-15 and IP-10) to produce.The biologic activity of VEGI-192A variant can be utilized the method test described in known in the art and the embodiment 2-5.In some embodiments, according to above-mentioned (or known in the art) one or more biological assay, the natural VE GI-192A that functionally equivalent variant has the total length compared is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% activity.In some embodiments, functionally equivalent variant in vitro inhibition vascular endothelial cell proliferation (example as described in example 2 above determination method), have 1000ng/ml, 100ng/ml, 60ng/ml, 40ng/ml, 20ng/ml, 12ng/ml or 6ng/ml at least about 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% IC ₅₀

VEGI-192A variant of the present invention can comprise the polypeptide at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity with VEGI-192A (for example, SEQ ID NO:1).

If the amino acid sequence in the two sequences arrange as described below be used for high specific to the time be identical, then two peptide sequences are called " identical ".Between two sequences relatively usually by at the comparison window comparative sequences to identify and the regional area of comparative sequences similitude carries out." comparison window " refers at least about 20 continuous positions as used herein, and common 30 to about section of 75,40 to about 50, and wherein sequence can be made comparisons with the canonical sequence with identical continuous position number after the two sequences optimal arrangement.

Can utilize in the Lasergene program groups of biological information software (DNASTAR, Inc., Madison, Megalign program WI) utilizes default parameters to be used for the best comparison of the sequence of comparison.This program comprises the some arrangement schemes described in the following list of references: Dayhoff, M.O. (1978) A model of evolutionary change in proteins-Matrices fordetecting distant relationships.Dayhoff, M.O. (ed.) Atlas of ProteinSequence and Structure, National Biomedical Research Foundation, Washington DC Vol.5, Suppl.3, pp.345-358; Hein.J., 1990, Unified Approachto Alignment and Phylogenes pp.626-645 Methods in Enzymology vol.183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M., 1989, CABIOS5:151-153; Myers, E.W. and MullerW., 1988, CABIOS4:11-17; Robinson, E.D., 1971, Comb.Theor.11:105; Santou, N.Nes, M., 1987, Mol.Biol.Evol.4:406-425; Sneath, P.H.A. and Sokal, R.R., 1973, Numerical Taxonomy the Principles and Practice of numericalTaxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J., 1983, Proc.Natl.Acad.Sci.USA 80:726-730.

Preferably, compare the sequences of two best comparisons by comparison window and determine " percentage of sequence homogeneity " at least 20 positions, wherein compare for the best of two sequences, peptide sequence in the comparison window is partly compared canonical sequence (it does not comprise interpolation or disappearance) can comprise 20% or lower, common 5% to 15%, or 10% to 12% interpolation or disappearance (being breach).The number of percentage by the same amino acid residue position determining to exist in the two sequences to be obtaining the number of matched position, with the number of matched position divided by the sum (being window size) of position in the canonical sequence and the result be multiply by 100 calculate with the percentage that obtains sequence homogeneity.

VEGI-192A variant of the present invention can comprise one or more amino acid whose replacement, disappearance or the interpolation that does not significantly change protein active.Variant can be from natural sudden change or manual operation.Variation can be less important character, as the replacement of not appreciable impact protein folding or active conserved amino acid.In order to improve or change the characteristic of VEGI polypeptide, the available protein engineering.Recombinant DNA technology well known by persons skilled in the art can be used for producing new mutein or comprises mutant or the fusion that single or multiple amino acid are replaced, lacked, add.Described modified polypeptide can present, the stability of the active or enhancing that for example strengthens.In addition, compare the natural polypeptides of answering, the productive rate purifying that it can be higher under some purifying and condition of storage at least and present better solvability.Therefore, the present invention also comprises VEGI-192A derivative and the analog that has one or more amino acid residues disappearance, adds or replace, the VEGI-192A polypeptide that is more suitable for expressing in selected host cell, amplifying in proportion etc. with generation.

The VEGI-192A functionally equivalent variant that is used for the inventive method also comprises the fusion that contains the VEGI-192A polypeptide.The VEGI-192A polypeptide of biologic activity can merge with sequence, as being coupled to holder or carrier with enhance immunity reaction, promotion polypeptide or promoting the sequence (sequence of coding such as Myc, the HA that is derived from the influenza virus hemagglutinin, His-6, FLAG or His label that example is as shown in table 2) of refolding and/or purifying to merge.These sequences can merge to VEGI-192A polypeptide N end or C end.In addition, protein or polynucleotides can merge to other or strengthen its function or the polypeptide (as secretion sequence) of specific localization in cell.The method that is used to produce above-mentioned recombination fusion protein is well known in the art.Recombination fusion protein can and separate by method generation well known in the art, refolding.In some embodiments, the VEGI-192A protein utilization comprises the fused polypeptide of histidine residues, and it can be as be shown in the examples and prepares.In some embodiments, the histidine fusion comprises SEQ ID NO:1.

Should be understood that VEGI-192A protein embodiment described herein does not comprise VEGI-174, VEGI-251, VEGI-192B.Referring to U.S.Pat.Appl.Pub.No.20020111325; U.S.Pat.Appl.Pub.No.20030170242.

Be used for composition of the present invention and also can comprise pharmaceutically suitable carrier, excipient or stabilizing agent (Remington:The Science and practice of Pharmacy 20 with lyophilized formulations or aqueous solution form ^ThEd. (2000) Lippincott Williams and Wilkins compile K.E.Hoover.).Acceptable carrier, excipient or stabilizing agent are nontoxic to the recipient on its dosage and concentration, and can comprise buffer solution, as phosphoric acid, citric acid and other organic acid buffer solutions; The antioxidant that comprises ascorbic acid and methionine; Preservative (as octadecyl dimethyl benzene ammonio methacrylate, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or phenmethylol, alkyl p-hydroxybenzoic acid such as methyl or nipasol, catechol, resorcinol, cyclohexanol, 3-amylalcohol and metacresol); The polypeptide of low-molecular-weight (less than about 10 residues); Protein is as seralbumin, gelatin or immunoglobulin; Hydrophilic polymer is as polyvinylpyrrolidone; Amino acid is as glycine, glutamine, asparagine, histidine, arginine or lysine; Monose, disaccharides and other carbohydrates comprise glucose, mannose or glucan; Chelating agent is as EDTA; Sugar is as sucrose, mannitol, trehalose or sorbitol; Salt-formation counter ion counterionsl gegenions such as sodium; Metal composite (for example zinc-protein complex); And/or nonionic surface active agent, as TWEEN ^TM, PLURONICS ^TMOr polyethylene glycol (PEG).Pharmaceutically acceptable excipient further describes at this.

VEGI-192A protein and its composition also can be united use with other anticancer preparation, are used for strengthening and/or supplying the validity of said preparation.

VEGI-192A (comprising variant) can utilize the methods known in the art preparation, for example reorganization preparation.VEGI-192A and variant can be according to U.S. Patent No.s 6,583,268, common pending application (the agent no.54411-20004.00 that makes a summary, the priority of requirement U.S. Provisional Application 60/528,983) method described in described method and the embodiment is at expression in escherichia coli and refolding and purifying.V. the kit that comprises the VEGI-192A that is used for the treatment of purpose

The present invention also is provided for the kit of the inventive method.Kit of the present invention comprises one or more containers of the VEGI-192A protein that contains purifying and according to the operation instructions of any method of the present invention as herein described.Usually, these specifications comprise according to any method as herein described use VEGI-192A protein with the treatment cancer (for example lung and breast cancer) explanation.Whether this kit also can comprise based on determining individually suffers from the stage of cancer and cancer and selects the explanation of the individuality that is suitable for treating.

Relate to the information that the specification that uses VEGI-192A protein generally includes the dosage, dosage regimen and the route of administration that are used to imagine therapy.Container can be unit dose, (bulk package) in bulk (for example multiple-unit container) or subunit's dosage.The specification that is provided in the kit of the present invention is generally the explanatory note (for example being included in the paper in the kit) on label or the package insert, but machine-readable specification (for example specification that carries on magnetic or the optical memory disc) also is acceptable.

Label or package insert indication composition are used for the treatment of cancer (comprising metastatic cancer).Specification can be provided for operating any method described herein.

Kit of the present invention carries out suitable packing.Suitable packing includes but not limited to bottle, bottle, wide-mouth bottle, flexible package (for example Mi Feng polyester film or plastic sack) etc.That considers in addition is useful on and specific device, as the packing of inhalator, nasal administration device (for example sprayer) or infusion set such as minipump combination.Kit can have aseptic inlet port (for example container can be intravenous solution bag or the bottle with the stopper that can sting by hypodermic needle).Container also can have aseptic inlet port (for example container can be intravenous solution bag or the bottle with the stopper that can sting by hypodermic needle).At least a activating agent is a VEGI-192A protein in the composition.Container also can comprise another kind of pharmaceutically active agents.

Kit can randomly provide extra assembly, as buffer solution and explain information.Usually, kit comprises container and or label relevant with container or package insert thereon.

In some embodiments, the invention provides the manufactured goods that comprise mentioned reagent box content.In some embodiments, kit comprises the information that VEGI-192A protein and indication are used for the treatment of cancer (for example lung and breast cancer).In some embodiments, kit comprises VEGI-192A protein and another kind of anticancer preparation and indication and combines with one another and be used for the treatment of the information of cancer (for example lung and breast cancer).

Embodiment

Embodiment 1: refolding and the purifying of reorganization VEGI-192A

Vector construction and expression: the dna fragmentation that produces the people of coding shown in the table 1 VEGI-192A by pcr amplification.PCR product insertion pET-19b (Cat.No.69677-3, Novagen, SanDiego, Nde I/Bam HI site CA) produces the VEGI-192A protein (table 2) with N end fusion tag.After PCR, connection and conversion enter e. coli bl21 DE3 bacterial strain, select single bacterium colony and amplification, and the construct that finally checks order is to guarantee the correctness of dna sequence dna.VEGI-192 nucleotide sequence and amino acid sequence with the terminal His label of N are displayed in Table 2.

Table 2. has nucleotide sequence and the amino acid sequence of the VEGI-192A of the terminal His label of N

NHisVEGI-192A ( SEQ ID NO：2 ) ： ATGGGCCATCATCATCATCATCATCATCATCATCACAGCAGCGGCCATATCGAC GACGACGACAAGCATATGCAACTCACAAAGGGCCGTCTTCATTTCAGTCACCCT TTGTCTCATACAAAGCACATTTCTCCTTTTGTTACAGATGCACCTCTTAGAGCAG ACGGAGATAAGCCAAGGGCACACCTGACAGTTGTGAGACAAACTCCCACACAG CACTTTAAAAATCAGTTCCCAGCTCTGCACTGGGAACATGAACTAGGCCTGGCC TTCACCAAGAACCGAATGAACTATACCAACAAATTCCTGCTGATCCCAGAGTCG GGAGACTACTTCATTTACTCCCAGGTCACATTCCGTGGGATGACCTCTGAGTGC AGTGAAATCAGACAAGCAGGCCGACCAAACAAGCCAGACTCCATCACTGTGGT CATCACCAAGGTAACAGACAGCTACCCTGAGCCAACCCAGCTCCTCATGGGGA CCAAGTCTGTATGCGAAGTAGGTAGCAACTGGTTCCAGCCCATCTACCTCGGAG CCATGTTCTCCTTGCAAGAAGGGGACAAGCTAATGGTGAACGTCAGTGACATCT CTTTGGTGGATTACACAAAAGAAGATAAAACCTTCTTTGGAGCCTTCTTACTAT AG

Protein sequence (SEQ ID NO:3) with VEGI-192A of the terminal His label of N: MGHHHHHHHHHHSSGHIDDDDKHMQLTKGRLHFSHPLSHTKHISPFVTDAPLRAD GDKPRAHLTVVRQTPTQHFKNQFPALHWEHELGLAFTKNRMNYTNKFLLIPESGD YFIYSQVTFRGMTSECSEIRQAGRPNKPDSITVVITKVTDSYPEPTQLLMGTKSVC EV GSNWFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKEDKTFFGAFLL

Be to produce VEGI-192A protein, the VEGI-192A expression vector is transfected into colibacillary BL21 DE3 bacterial strain and is inoculated on the ZB flat board with ampicillin.Select single bacterium colony and be used to inoculate ZB medium (10g/l NZ amine A (Sigma) and 5g/l NaCl) that 100mL has ampicillin and in 30 ℃ of incubated overnight (about 16 hours).The 20mL of 100mL starting culture is used to inoculate the LB medium that 1L has ampicillin subsequently, and the optical density (OD600) that culture is hatched to 600nm 37 ℃ of vibrations reaches 0.4-0.6.Add to 0.5mM isopropyl-(IPTG) subsequently inducing the expression of VEGI-192A, and culture further vibration hatched three hours.Being expressed in 37 ℃ on a large scale utilizes a plurality of 1L oscillator arrow-necked bottles to finish.

The processing of inclusion body before the refolding: from bacterium results inclusion body.By centrifugal collection bacterial cell, be resuspended to subsequently and have 1%TRITONX-100 ^20mL TN (150mM NaCl, 50mMTris, pH8.0) in.Add ten milligrams of lysozymes, and cell suspension is-20 ℃ of freeze overnight.Melt lysate subsequently and add the magnesium sulfate of 20 μ l1M and the DNase of 100 μ g.Cell stirs the DNA of bacteria of hatching until discharging and dissolves fully.Lysate has 1%TRITONX-100 with 250mL subsequently ^TN dilution and stirred the mixture 2-4 hour.Centrifugal collection inclusion body, and wash (by resuspension and centrifugal) three times.

The chromatography of refolding and refolding protein: the inclusion body of washing is dissolved in the glutathione (GSSG) of glutathione (GSH), 0.1mM oxidation of 8M urea, 0.1M Tris, 1mM glycine, 10mM beta-mercaptoethanol, 10mM dithiothreitol (DTT) (DTT), the 1mM reduction of pH10.5.Absorptance (OD with the 280nm place of protein solution ₂₈₀) be adjusted to 2.0.Solution by supercentrifugation clarification (30 minutes * 66,000g), refolding subsequently.

The clear solutions rapid dilution is gone among the 20mM Tris, 1.36mM sodium lauroyl sarcosine, 0.009mM trimethylamine N-oxidation dihydrate, 0.005mM softex kw of 20 times of volumes, pH10.5.The solution that obtains progressively was adjusted to pH8.0 with 1M HCl through 4 day time.

The protein of refolding passes through ultrafiltration and concentration (Millipore Pellicon, 10,000 Da mwco membranes) and is applied to the Sephacryl S-300 post (Fig. 1) of 20mM Tris, 0.4M urea, 0.2M NaCl balance and the wash-out of using pH8.Merging is from the component (being shown as 1,2,3) of Figure 1A and be concentrated into about 5mg/ml (SDS-PAGE shown in Figure 1B) and be used for that the endothelial cell described in the embodiment 2-4 suppresses to measure and tumor model is tested.

Also utilize Ni ⁺The functional VEGI-192A of affinity column purifying.Ni ⁺Chelate column (500ml) 20mM Tris, 1.36mM sodium lauroyl sarcosine, the 0.4M urea balance of pH8.The VEGI-192A of refolding (4L) is applied to post, and post 20mM Tris, 0.4M urea, 0.2M NaCl, 5mM Immidozole (buffer A) washing of 1L pH8.With the immidozole (5 to 500mM) of buffer A neutral line gradient from post wash-out VEGI-192A.Eluting peak shown in the combined diagram 2A, and through the 20mM of pH8 Tris, 0.4M urea, 0.2M NaCl dialysis.The VEGI-192A of dialysis subsequently by ultrafiltration and concentration to about 5mg/ml.The SDS-PAGE of the non-reducing and reduction of the VEGI-192A that produces analyzes and shows in Fig. 2 B.

In another experiment, the settled solution rapid dilution of the VEGI-192A of refolding process by will comprising dissolving goes in the 20mM Tris of 20 times of volume pH10.5,0.034mM sodium lauroyl sarcosine, 0.009mM trimethylamine N-oxidation dihydrate, the 0.005mM softex kw to carry out.The solution that obtains progressively was adjusted to pH8.0 with 1M HCl through 4 day time.The VEGI-192A of refolding concentrates and purifying in above-mentioned same mode.Suppress determination method by the endothelial cell described in the embodiment 2 and measure, utilize low 100 times under the above-mentioned refolding condition of specific activity of VEGI-192A of this refolding buffer solution purifying.

Embodiment 2: the biology that suppresses determination method sign reorganization VEGI-192A by endothelial cell growth is lived The property

Grow up BAE (ABAE) cell at 37 ℃, 5%CO ₂, ((GibcoBiofluids, Rockville cultivate in MD) IMEM WI) for Promega, Madison for 10%FBS, 1ng/ml fibroblast growth factor-2.By ³The dormancy degree of mixing the mensuration cell of H-thymidine.If be no more than 5% cell fusion ³The H-thymidine is then considered the G in the cell cycle ₀The phase synchronization cell.G ₀Synchronized cell only is re-seeded into (5000 cells/cm at it ²) have among the IMEM of 10%FBS and 1ng/ml fibroblast growth factor 2 and at 37 ℃, 5%CO ₂Under reenter growth cycle when hatching.Zhi Bei the VEGI-192A time point that maybe 20 hour cells enter growth cycle after inoculation in inoculating cell is added into medium as described in example 1 above.Prepare single-cell suspension liquid with the given time interval from each culture hole by trypsinization.By utilizing the Coulter counter or by utilize using tetrazole compound-MTS (Promega) colorimetric method to measure cell number in every part of suspension, MTS can be created in the detectable blue color compound of 490nm through the living cells metabolism; The metabolic rate of MTS is directly proportional with the number of living cells in the culture.The concentration of the VEGI-192A that uses in this determination method is the scope of 0.1ng/mL to 1 μ g/mL.

Suppress in the determination method at the ABAE endothelial cell that utilizes the MTS colorimetric method, three all storehouses all have activity and storehouse 3 has maximum activity (Fig. 1 C).As shown in Fig. 1 C, the reorganization VEGI-192A in the storehouse 3 presents the IC of 12ng/ml (0.49nM) in the vitro inhibition endothelial cell proliferation ₅₀In another experiment, the reorganization VEGI-192A of generation presents the IC of 0.24nM (6ng/ml) in the vitro inhibition endothelial cell proliferation ₅₀

Embodiment 3: suppress the growth of Lewis lung cancer and improve negative knurl mouse survival by reorganization VEGI-192A Time

Be implanted subcutaneously the C57B16/J mouse with Lewis lung cancer (LLC) cell.Cancer cell with PBS washing, be dispersed in 0.05% the trypsin solution and resuspension.After centrifugal 10 minutes, cell precipitation is resuspended among the PBS and concentration adjustment to 2.5 * 10 at 8 ℃, 4000rpm ⁶Cell/ml.Mouse subsequently with this cancer cell suspension of 0.1ml through hypodermic injection in flank.Tumour is measured with graduated calliper, and gross tumor volume utilizes formula: volume=width * width * length * 0.52 is determined.Be 100-200mm at least at gross tumor volume ³(0.5-1% of body weight) back (it took place in 5-7 days) is randomized into three groups with mouse.Two groups of mouse are with 5mg/kg or 20mg/kg, and weekly twice, accepts to be suspended in recombined human VEGI-192A (preparing as described in example 1 above) among the PBS by (IT) injection in (IP) or the knurl in the peritonaeum.It is then tangible or reach 700-1000mm at gross tumor volume that IP or IT are injected at tumour ³In time, begin.The 3rd group of similar injection of only accepting excipient (phosphate-buffered salt).Gross tumor volume surpasses 2000mm in control group ³In time, stops experiment and puts to death mouse and ptomatopsia.

When tumour be tangible to or reach 700-1000mm when gross tumor volume ³The time, be injected to negative knurl animal by (IP) in the peritonaeum and send the remarkable inhibition that reorganization VEGI-192A has observed tumor growth rate in the negative knurl animal.As shown in Fig. 3 A, reorganization VEGI-192A is injected to by IP or IT with 5mg/Kg has 700-1000mm ³The systemic delivery of the mouse of gross tumor volume shows the obvious inhibition of tumor growth.Even gross tumor volume is almost 5% (700-1000mm of body weight when the treatment beginning ³) time can realize that still similar 50% tumor growth rate suppresses.

Because the animal with LLC tumour in this period may produce especially to the micrometastasis of the extensive distribution of lung, will grow into big transfer in case primary tumo(u)r is removed it through surgery, is in tumour and has reached almost 5% (700-1000mm ³Gross tumor volume) the LLC tumour in the weight of animals period is similar to people's lung cancer of clinical group of middle and advanced stage.Referring to Oareilly etc., Cell79 (2): 315-28 (1994); Cao etc., J Clin.Invest.101 (5): 1055-63 (1998).

Also tested the time-to-live of negative knurl mouse.Time-to-live is by implanting the LLC cell and reaching 3000mm when gross tumor volume ³Above interval fate, or implant the LLC cell with when animal owing to the tumor growth of wettability highly makes the tumor-infiltrated interval fate of backbone between becoming when paralysing that enter determine.The animal of Lewis lung cancer model is reaching 3000mm when gross tumor volume ³More than, or enter backbone become when paralysis and put to death when animal is tumor-infiltrated owing to the tumor growth of height wettability.As at (Fig. 3 B) shown in the curve map of the time-to-live of the animal that at that time survives and number, (from treatment, its tumour mean size that starts from not treatment group is 700mm to half survival time ³The time the 11st day) be approximately 4 days, and the half survival time of treatment group is 12 days, reflects 3 times of longer time-to-live.

In another experiment, (the per injection 1 * 10 of LLC cell ⁶) through hypodermic injection in C57BL/6 black mouse (Harlan, Indianapolis, flank IN) and treatment tangible to the beginning in early days of tumour (Fig. 9).Use VEGI-192A (20mg/kg) at the 5th day, the 9th day and the 12nd day by IP and treat animal.Control group is treated with excipient.As shown in Figure 9, VEGI-treatment group is observed the tumor growth rate that significantly slows down.

Embodiment 4: with reorganization VEGI-192A treatment human breast carcinoma heterograft tumourThe MDA-MB-231 human breast cancer cell is injected into (per injection 1 * 10 ⁶Cell) female nude mouse mammary fat pad.The cancer cell of injection formed the tangible tumour that arrives in 5-7 days.Gross tumor volume is used for the equation V=0.52LW of avette volume by utilization ²Determine.In case gross tumor volume reaches about 100mm ³, by being delivered to this animal (5mg/Kg, twice weekly) as the reorganization VEGI-192A of generation as described in the embodiment 1 in subcutaneous (SC) of tumor locus injection.Except utilizing excipient (PBS) substitutes, control group is treated under identical experiment condition.In case gross tumor volume surpasses 2000mm ³Or the tumour body weight is put to death animal when surpassing 2 grams (its be approximately body weight 10%).Collect tumour, other organs (lung, liver, spleen) and peripheral blood and be used for the biological pathology analysis.

In order to analyze the effect of VEGI-192A treatment to tumor vessels, new freezing section (accepting the group that SC injects from control animals with at tumor locus) is analyzed by the fluorescence labeling (shown in the A of Fig. 4 and the C hurdle) of apoptotic cell or to the immunostaining (shown in the B of Fig. 4 and the D hurdle) of CD31 (endothelial cell mark).In the A hurdle, untreated tumour show almost do not have apoptotic cell and in the B hurdle untreated tumour show a large amount of capilaries.Comparatively speaking, the fluorescence labeling that tumour (C hurdle) the demonstration apoptotic cell of VEGI-192A treatment is strong and the necrotic area of inside tumor.As describing in the D hurdle, the tumour of VEGI-192A treatment shows the microvessel density that obviously reduces.Observe cell death in the tumor tissues.

Embodiment 5: suppress the absorption of Lewis lung cancer (LLC) cell by administered recombinant VEGI-192AThe LLC cell prepares as described in example 3 above and inoculates (1 * 10 ⁵Cell/injection) in the back of C57B1 mouse.In LLC cell inoculation (the 0th day), inject the animal for the treatment of of 5mg/kg VEGI-192A (preparation as described in example 1 above) by (IP) in the peritonaeum.Gross tumor volume was measured at the 4th day.As shown in Figure 5, the treatment of animal causes in the tumour taking in the obvious inhibition (t check, p＜0.002) of speed when tumor inoculation.

In another experiment (shown in Figure 10), LLC cell (1 * 10 ⁶Per injection) is inoculated in C57BL/6 black mouse (Harlan, Indianapolis, flank IN) and treat animal immediately with VEGI-192A (20mg/kg prepares as described in example 1 above) in cancer cell inoculation back.Every day, repetitive therapy was until the 4th day.When the 5th day assessment gross tumor volume after inoculation, all animals in the vehicle-treated groups have been developed has hypodermic tumour (5/5 or 100%), has 35mm ³Average external volume (+/-SD), and the pact of VEGI treatment group half present measurable tumour (2/6 or 33%), and gross tumor volume littler (Figure 10).The result shows that systemic administration VEGI causes the retardance by the tumour formation of cancer cell.

Embodiment 6: host immune system is by the activation of administered recombinant VEGI-192A

The LLC cell is as preparation and inoculation (1 * 10 as described in the embodiment 3 ⁵Cell/injection) in the back of C57B1 mouse.Begin to use VEGI-192A in tumour tangible then the 4th day that become.The 4th day, the 7th day, the 8th day, the 9th day and the 10th day with 5mg/Kg injection VEGI-192A (preparation as described in example 1 above).Fetched spleen at the 11st day and weigh.Also collected serum at the 11st day.The level of Cytokine of Serum is utilized the luminescence assays kit that antibody puts together, and (Inc. Missouri) measures according to product description for LINCOplex, LINCO Research.

Animal causes the obviously generation (shown in Fig. 7) of enhancing of tangible splenomegaly (shown in Fig. 6) and some cell factors with the treatment of VEGI-192A, as TNF-α, IL-6, IL-1B, IL-15 and IP-10.The inhibition of various lymphocytic activation of the known participation of IL-15 and IP-10 and angiogenesis.

The human endothelial cell of the distribution of cell factor and cultivation replying quite in the above-mentioned mice serum to the VEGI-192A treatment.Propagation or converge (G0-is synchronized) human umbilical vein endothelial cell (HUVEC) and handled 5 hours with 500ng/ml VEGI-192A.After the processing, use the Center available from FredHutchinson Cancer Research, Seattle, the cDNA microarray assays of Washington handle and the cytokine-expressing distribution of untreated increment and the HUVEC that converges.Cell factor from the distribution of cell factor in the mice serum of Fig. 7 B and the propagation of handling with VEGI-192A and the HUVEC that converges distributes quite.As shown in Figure 8, the processing of VEGI-192A is induced IL-15 and IP-10 in the HUVEC that breeds and converge expression, prompting VEGI-192A treatment can improve these two kinds of production of cytokines in the people.

The immunohistochemistry of VEGI-192A treatment back tumor vessels in the negative knurl mouse of embodiment 7:LLC Analyze

We have measured with the influence of the negative knurl mouse of VEGI-192A (preparation as described in example 1 above) whole body therapeutic LLC to tumor vessel abundance and structure.The LLC cell prepares and subcutaneous vaccination (1 * 10 as described in example 3 above ⁶Cell/injection) in C57BL/6 black mouse (Harlan, Indianapolis, flank IN).Used VEGI-192A or excipient at the 4th day in the beginning peritonaeum.Twice VEGI-192A (5mg/Kg) injected for three weeks weekly.Vehicle-treated groups is accepted the injection of similar phosphate-buffered salt.Immunohistochemical analysis such as following carrying out.Extract tumour and 4 ℃ of fixatives that place PBS 4% paraformaldehyde 4 hours, change among the 30% sucrose PBS (4 ℃) of no RNase 4 ℃ then over to and spend the night.Tumour is with in the OCT compound that is placed on the dry ice.Section (8 μ M are thick) 45 ℃ of dryings two hours on slide before the immunostaining.Endothelial cell rat monoclonal antibody (the PECAM-1 of CD31; BD PharMingen, the MEC13.3 clone, dilution in 1: 250 cat.#01951D) is identified.(cat.#F3777) identify for Sigma, clone1 A4 by dilution in 1: 250 with monoclonal anti actin-α-FITC for vascular smooth muscle cell.The rabbit polyclonal antibody of basement membrane of blood vessel usefulness anti-IV type collagen (1: 10,000 dilution; Cosmo Bio Co., Tokyo Japan) identifies.Cell nucleus Hoechst (100ng/ml; Sigma) identify.Used second antibody is mouse (the Vector Laboratories of the biotinylated Chinese People's Anti-Japanese Military and Political College, Burlingame, CA.cat.#BA4001), the mouse IgG-TRITC of the rabbit Chinese People's Anti-Japanese Military and Political College (Sigma, cat.T4280) and the mouse IgG-FITC of the donkey Chinese People's Anti-Japanese Military and Political College (Jacksoncat.#_712-096-153), be used for AMCA avidin D (the Vector Laboratories that vitamin h detects, and goat anti-rabbit igg-TRITC (Sigmacat.#T6778) cat.#A-2008).ABC standard reagent box and DAB kit (Vector Laboratories) are used for this immunohistochemistry.Sample and 0.03% H ₂O ₂(Sigma) at room temperature hatch 15 minutes to seal endogenic peroxidase activity, hatch to seal nonspecific antibodies with the PBS that comprises 0.2%TritonX-100 and 5% bovine serum albumin(BSA) (Sigma) subsequently.Sample subsequently with PBS in be diluted to 1-2 μ g/ml first antibody at room temperature hatched 1 hour, or hatched 12-15 hour at 4 ℃.Except first antibody was replaced with PBS, control sample was handled under the same conditions.After the PBS rinsing, sample and second antibody at room temperature hatch 1 hour, use the PBS rinsing, at room temperature hatched 30 minutes with ABC standard reagent box or AMCA avidin D subsequently.Sample is hatched with the PBS rinsing and with the DAB kit, with post rinse and be fixed in (Vector Laboratories) among the Vectashield.

Sample is with the NikonEclipseE800 fluorescence microscope that is equipped with single, two and three fluorescence filters and have low-light (level) RETIGA1300CCCD camera (the Quantitative Imaging Corporation of Qcapture software, Burnaby, BC Canada) checks.Image saves as digital document.Graphical analysis with Image-pro plus software (Media Cybernetics, Inc) or Image-J (NIH) carry out.

The specificity of endothelial cell is eliminated in the tumour: new freezing tumour is made section and is stood the immunostaining (Figure 11 A, 11B) of internal skin mark CD31 (redness) and smooth muscle cell antigen-α (SMA-α) (green).We have analyzed 15 visuals field that comprise maximum number capilary (" focus ") on each slide by computer assisted graphical analysis subsequently.Measure the redness in each visual field or the density of green pixel (400 * multiplication factor) (Figure 11 C).Result's (as shown in figure 11) shows the specificity elimination of endothelial cell in the tumor vessel.Reveal the treatment reduction in one week interior 88% as density meter, and further reduce in three weeks by the measured endothelial cell of the shared total pixel of CD31 positive cell.What is interesting is that the number of smooth muscle cell is kept constant relatively.Therefore, endothelial cell obviously reduces in the tumour of VEGI-treatment the ratio of smooth muscle cell, is changed to 0.4 and be changed to 0.15 from 1.8 from 1.8 respectively after one week of treatment of animals or three weeks.To another endothelial cell mark, CD105 carries out similar immunostaining, and obtains identical result.These results show that the anti-angiogenesis activity of VEGI-191A causes the specificity of tumor vascular endothelial cell to be eliminated.

Endothelial cell is eliminated retaining of back tumour medium vessels smooth muscle cell: we find that eliminating the back vascular smooth muscle cell at endothelial cell retains (Figure 12) at experimental session.As if the chamber that how much has kept some tumor vascular systems.This has improved the tumor vessel with good smooth muscle cell support may keep a little residual function after most of endothelial cell no longer exists possibility.

The existence of residual blood vessel structure in the tumour of VEGI-192A treatment: residual blood vessel structure is further studied.Be different from the lumen of vessels (Figure 12 A) of arranging by endothelial cell in the tumour of excipient treatment, in the tumour of VEGI treatment, exist and comprise the space of red blood cell, but do not have endothelial cell to arrange (Figure 12 B).Whether measure these spaces subsequently is the residual blood vessel that endothelial cell is eliminated.This finishes by the immunostaining to the collagen iv of basement membrane of blood vessel key component.Shown in Figure 12 C and 12D, compare (Figure 12 D) with the endothelial cell that is easy to detect related with basement membrane of blood vessel in the control group tumour of excipient treatment, the basic structure of tumor vessels is kept almost intact, even also be (Figure 12 C) like this when endothelial cell is almost completely eliminated by the treatment of VEGI-192A.These basement membrane structures are usually followed smooth muscle cell, and no matter whether tumour uses VEGI-192A (Figure 12 E) or excipient (Figure 12 F) treatment.Except the blood vessel structure of not treating in the tumour comprises this only difference of endothelial cell, the appearance of these residual blood vessels with not can not distinguish from treating seen in the tumour those.These results show that the residual blood vessel structure of being made up of basilar memebrane and smooth muscle cell exists at experimental session after these tumor vessels are removed at least at endothelial cell.

Though foregoing invention describes in greater detail by the explanation and the embodiment that are used to clarify and understand purpose, can carry out some variation and change obviously to those skilled in the art.Therefore, illustrate with embodiment to should not be construed as limitation of the scope of the invention that scope of the present invention is described by subsidiary claim.

Claims (14)

1. the method that is used for the treatment of lung cancer in the individuality comprises the VEGI-192A polypeptide of using effective dose.

2. the process of claim 1 wherein that cancer is a terminal cancer.

3. the process of claim 1 wherein that cancer is the cancer that shifts.

4. the process of claim 1 wherein VEGI-192A polypeptide behaviour VEGI-192A.

5. the process of claim 1 wherein that the VEGI-192A polypeptide comprises the amino acid sequence of SEQ ID NO:1.

6. the process of claim 1 wherein the generation of from Escherichia coli, recombinating of VEGI-192A polypeptide.

7. the kit that is used for the treatment of lung cancer, it comprises the VEGI-192A polypeptide and instructs and use the specification of VEGI-192A polypeptide with treatment lung cancer.

8. the method that is used for the treatment of breast cancer in the individuality comprises the VEGI-192A polypeptide of using effective dose.

9. the method for claim 8, wherein cancer is a terminal cancer.

10. the method for claim 8, the wherein cancer of cancer for shifting.

11. the method for claim 8, wherein VEGI-192A polypeptide behaviour VEGI-192A.

12. the method for claim 8, wherein the VEGI-192A polypeptide comprises the amino acid sequence of SEQ ID NO:1.

13. the method for claim 8, the wherein VEGI-192A polypeptide generation of from Escherichia coli, recombinating.

14. be used for the treatment of the kit of breast cancer, it comprises the VEGI-192A polypeptide and is used to instruct uses the VEGI-192A polypeptide to treat the specification of breast cancer.

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