CN102526708A - Novel application of co-stimulating factor to cancer treatment - Google Patents

Abstract

The invention discloses B7.1 and an application of a B7.1 fusion protein to preparation of a medicament for treating liver cancer, sarcoma or melanoma. An immune co-stimulating factor fusion protein B7.1-Fc, particularly B7.1/IgG1 (immunoglobulin G1) Fc can be used for effectively inhibiting the growth of liver cancer or sarcoma, particularly has remarkable treating activity specific to liver cancer, has a curative effect which is equivalent to that of a positive medicament, and has higher safety than the positive medicament, so that a new option is provided for clinical administration.

Description

The new purposes of costimulating factor aspect treatment of cancer

Technical field

The present invention relates to the new purposes of the new purposes, particularly B7.1-Fc of costimulating factor aspect treatment of cancer.

Background technology

Hepatocarcinoma is meant the malignant tumor that betides liver, comprises two kinds of primary hepatocarcinoma and secondary liver cancers, and mostly the hepatocarcinoma of the daily theory of people is primary hepatocarcinoma if referring to.Primary hepatocarcinoma is one of modal malignant tumor clinically, and according to recent statistics, the annual New Development liver cancer patient in the whole world is about 600,000, occupies the 5th of malignant tumor.Primary hepatocarcinoma can be divided into hepatocyte type hepatocarcinoma, bile duct cell type hepatocarcinoma and mixed carcinoma of liver by cell typing.Form by tumor can be divided into nodular type, massive type and diffuse type.Primary hepatocarcinoma belongs to high morbidity in China, and general male is more than the women.China is hepatitis B big country, the many development on the basis of hbv-liver cirrhosis of the hepatocarcinoma of China, and the hepatitis C patient also increasing gradually, also can develop into hepatocarcinoma after the hepatitis B.China's number of the infected accounts for the more than half of the whole world at present, accounts for 55% of global hepatocarcinoma patient, has become a big killer of serious threat China people health and lives, and its danger can not look down upon.

Yet; Treatment for hepatocarcinoma is present is still consistent with general oncotherapy scheme, all adopts chemotherapy radiotherapy, and conventional chemicotherapy does not possess the specificity to tumor basically; Chemicotherapy also produces lethal effect to normal cell simultaneously tumoricidal; Thereby human body is produced very big toxic and side effects, and the destruction that also can cause host immune system simultaneously is prone to behind the chemicotherapy cause that multiple complications and tumor are prone to recurrence; It is not very desirable that the characteristics of conventional chemicotherapy cause the curative effect of oncotherapy and prognosis, and immunotherapy of tumors becomes a kind of safer effective and efficient manner at present.

Immunotherapy of tumors is an immune system of directly or indirectly utilizing self; The method that suppresses growth of tumour cell, transfer, removing tumor cell; Immunotherapy of tumors mechanism is compared with other treatment means, has characteristics such as the effect specificity is high, in extensive range, toxic and side effects is little.B7.1-Fc is immunostimulation factor fusion protein (Aihong Liu, Peisheng Hu; Clin Cancer Res 2005; 11 (23) December 1,2005,8492-8502), it is the fusion rotein that is made up of the Fc fragment N-end that CD80 born of the same parents' exterior portions merges at people Ig antibody constant region, among the B7.1-Fc that has reported at present, FC comes from immunoglobulin IgG, particularly IgG1.The B7.1-Fc fusion rotein host's APC cell with tumor antigen information submission donor in the tumour immunity killer cell; Costimulating factor B7.1 combines activated T cell with CD28; Thereby tumor cell is produced serial immune attack, reach the effect of killing tumor cell.The B7.1-Fc fusion rotein can not only combine with CD28 simultaneously, and activated T cell also can combine with CTLA-4, stops the activated pathway of T cell, thereby avoids occurring the excessive activation of T-cell.This dual function of B7.1-Fc fusion rotein has strengthened the safe controllability with the antitumor drug of B7.1-Fc fusion rotein exploitation greatly.As a kind of immunotherapy of tumors medicine, it has certain therapeutic effect (CN200580042109.0) in colon cancer, breast carcinoma, fibrosarcoma cancer according to the immune costimulating factor fusion rotein B7.1-Fc of relevant research report.

At present, also do not see the relevant report that costimulating factor B7.1 and B7.1 fusion rotein is used to treat hepatocarcinoma.

Summary of the invention

The object of the present invention is to provide the new purposes of costimulating factor B7.1 and B7.1 fusion rotein.

The invention provides costimulating factor B7.1 and the B7.1 fusion rotein purposes in preparation treatment hepatocarcinoma, sarcoma or melanomatous medicine.

Wherein, described B7.1 fusion rotein is B7.1-Fc.

Further, B7.1-Fc is the B7.1 that is fused on the IgG Fc.

Further preferably, said IgG is IgG1.

Further, described medicine is the medicine of treatment hepatocarcinoma, sarcoma.

Further, described medicine is the medicine of treatment hepatocarcinoma.

Wherein, described medicine is to be effective ingredient with costimulating factor B7.1 or B7.1 fusion rotein, adds the preparation that adjuvant pharmaceutically commonly used is prepared from.

Further, described preparation is an injection.

Convert and to know according to pharmacological evaluation, in the described preparation, use every day the content of B7.1-Fc in the unit formulation to be 192-384ug

Immunity costimulating factor fusion rotein B7.1-Fc; Particularly B7.1/IgG1 Fc can effectively suppress the growth of hepatocarcinoma or sarcoma, particularly has significant therapeutic activity to hepatocarcinoma; Its therapeutical effect and positive drug are suitable, for clinical application provides a kind of new selection.

Description of drawings

Fig. 1 SDS-PAGE confirms the molecular weight spectrogram of the B7.1-Fc of expression, and wherein, 1 is that non-reducing B7.1-Fc collection of illustrative plates, 2 is that reductive B7.1-Fc collection of illustrative plates, 3 is the standard molecular weight label;

H22 mice with tumor body weight change after Fig. 2 B7.1-Fc administration

Fig. 3 B7.1-Fc is to rat liver cancer (H22) tumor inhibition effect

S180 mice with tumor body weight change after Fig. 4 B7.1-Fc administration

Fig. 5 B7.1-Fc is to murine sarcoma (S 180) tumor inhibition effect

B16-F10 mice with tumor body weight change after Fig. 6 B7.1-Fc administration

Fig. 7 B7.1-Fc is to murine melanoma (B16-F10) tumor inhibition effect

The specific embodiment

The preparation of B7.1-Fc among embodiment 1 the present invention

Utilize gene recombination technology to prepare the B7.1-Fc fusion rotein; According to biological field technical staff well-known process; Obtain to make up the B7.1-Fc expression vector through round pcr, change the engineering cell that mouse myeloma NSO cell screening obtains expressed fusion protein B7.1-Fc steady in a long-term over to, obtain the B7.1-Fc culture supernatant of high expressed then through feeding culture; And then through the acquisition of protein A affinity chromatograph and ion-exchange chromatography purification successively high-purity fusion rotein, 4 ℃ of preservations.

The method for preparing of B7.1-Fc can be with reference to the method for preparing among the CN200580042109.0 among the present invention, and said B7.1-Fc is meant B7.1/IgG1 Fc, i.e. the fusion rotein of B7.1 and IgG1 Fc, its sequence table be referring to Aihong Liu, Peisheng Hu; Clin Cancer Res 2005; 11 (23) December 1,2005,8492-8502.

With the B7.1-Fc determining molecular weight for preparing, measure collection of illustrative plates and see Fig. 1;

Embodiment 2 immune costimulating factor fusion rotein B7.1-Fc pharmacodynamic experiments

1, given the test agent and dosage design

Title: immune costimulating factor fusion rotein B7.1/IgG1Fc (being called for short B7.1-Fc) presses embodiment 1 preparation

Supplier: microorganism of military medical sciences academy epidemiological study institute

Character: colourless, supernatant liquid

Preservation condition: 4 ℃ of preservations

Route of administration: tail vein injection

Solvent: normal saline

2, positive control:

Cyclophosphamide for injection; Paclitaxel

3, tumor cell line:

Rat liver cancer H22, murine sarcoma S180, murine melanoma B16-F10 all can obtain through buying the commercial goods

4, animal:

BALB/c mouse, the SPF/VAF level provides quality certification SCXK (capital) 2009-0004 by Beijing China Fukang biotech inc

5, raise facility condition:

Institute of Materia Medica,Chinese Academy of Medical Sciences zoopery center barrier facility, credit number: SYXK (capital) 2009-0004

6, experimental technique:

(1) tumor cell culture and inoculation:

Rat liver cancer H22, murine sarcoma S180, murine melanoma B16-F10 cells in vitro are cultured to a great deal of.Digestion, washing, centrifugal, counting cells, the preparation cell density is 5 * 10 ⁶/ ml cell suspension.The rear side back inoculation of mice partly sterilised, volume injected 0.1ml/ only.

(2) dosage and administering mode:

Observe tumor growth every day and change, when inoculation was confirmed tumor growth in back 4 days, mice with tumor is pressed tumor volume size random packet.If negative control group, the tumor self-sow; Positive controls is Cyclophosphamide for injection, paclitaxel administration group, presses the conversion of human dosage, and mice is with 100mg/kg ^-1Dosage is looked the tumor growth situation and is interrupted intraperitoneal injection; Fusion rotein B7.1-Fc according to 80,40,20ug/kg ^-1Three dosage tail intravenously administrables, every day 1 time, continuous 5 days.15 of every group of mices, and with whole administrations on the same day.

(3) observation index

In the experimentation, every 2-3 day monitoring experiment animal tumor length and width footpath, volume calculated.Respectively referring to Fig. 3,5,7.Adopt relative tumor proliferation rate T/C (%) as the test evaluation index among the present invention, in order to estimate the anti-tumor activity of medicine, computational methods are referring to " cell toxicant series antineoplastic medicament non-clinical study technological guidance principle ".Simultaneously, also weigh for various tumor-bearing mices, B7.1-Fc for confirmation is to the untoward reaction of body, referring to Fig. 2,4,6.

Experimental result:

(1) B7.1-FC is to mouse tumor hepatocarcinoma H22 tumor growth inhibitory action

Table 1B7.1-Fc is to H22 tumor growth inhibitory action

*: * * is compared with negative control group in P＜0.05: compare with negative control group P＜0.01

Table 2B7.1-Fc is to H22 tumor growth inhibitory action

*: P＜0.05, compare * * with negative control group: P＜0.01, compare with negative control group

(2) B7.1-Fc is to murine sarcoma S180 tumor growth inhibitory action

Table 3B7.1-Fc is to S180 tumor growth inhibitory action

*: P＜0.01, compare with negative control group

Table 4B7.1-Fc is to S180 tumor growth inhibitory action

*: P＜0.05, compare * * with negative control group: P＜0.01, compare with negative control group

(3) B7.1-Fc is to murine melanoma B16-F10 tumor growth inhibitory action

Table 5B7.1-Fc is to B16-F10 tumor growth inhibitory action

*: P＜0.01, compare with negative control group

Table 6B7.1-Fc is to B16-F10 tumor growth inhibitory action

*: P＜0.01, compare with negative control group

7, experiment conclusion

" cell toxicant series antineoplastic medicament non-clinical study technological guidance principle " 2006, points out in this principle that T/C (%)＞40% is invalid; , T/C (%)≤40%, and be effective through statistical procedures P＜0.05.

In the present invention, B7.1-Fc is at 80ug/kg ^-1Have obvious inhibitory action to testing used mice H22 hepatocarcinoma tumor model in the dosage range, all reach more than 60%, be converted into human dosage, be about 6.4ug/kg ^-1B7.1-Fc strengthens the inhibitory action of tumor, and tumor growth rate is slow, dwindles gradually and extremely disappearance; This proves absolutely that B7.1-Fc has significant safety and effectiveness at the liver cancer treatment middle and high concentration; Yet in the 80ug/kg-1 dosage range, there is not tumor inhibition effect basically for used mice B16-F10 melanoma of experiment and mice S180 sarcoma at the immune then costimulating factor fusion rotein of B7.1-Fc B7.1-Fc.

On the other hand; Observe from the mice body weight; The mice body weight increases rapidly with respect to the positive control medicine after finding to use B7.1-Fc, and this explains that immune costimulating factor fusion rotein is high a lot of with respect to its safety medicine of traditional chemicals aspect oncotherapy.

In sum, immune costimulating factor fusion rotein B7.1-Fc, particularly B7.1/IgGl Fc; Can effectively suppress the growth of hepatocarcinoma or sarcoma; Particularly have significant therapeutic activity to hepatocarcinoma, its therapeutical effect and positive drug are suitable, for clinical application provides a kind of new selection.

Claims (8)

1. costimulating factor B7.1 and B7.1 fusion rotein are treated the purposes in hepatocarcinoma, sarcoma or the melanomatous medicine in preparation.

2. purposes according to claim 1 is characterized in that: described B7.1 fusion rotein is B7.1-Fc.

3. purposes according to claim 2 is characterized in that: B7.1-Fc is the B7.1 that is fused on the IgGFc.

4. purposes according to claim 3 is characterized in that: said IgG is IgG1.

5. according to any described purposes of claim 1-4, it is characterized in that: described medicine is the medicine of treatment hepatocarcinoma, sarcoma.

6. purposes according to claim 5 is characterized in that: described medicine is the medicine of treatment hepatocarcinoma.

7. according to any described purposes of claim 1-6, it is characterized in that: described medicine is to be effective ingredient with costimulating factor B7.1 or B7.1 fusion rotein, adds the preparation that adjuvant pharmaceutically commonly used is prepared from.

8. purposes according to claim 6 is characterized in that: described preparation is an injection.

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