CN101497662A - TM4SF3 protein for preparing antineoplastic antibody and use thereof - Google Patents
TM4SF3 protein for preparing antineoplastic antibody and use thereof Download PDFInfo
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Images
Abstract
The invention relates to a TM4SF3 protein used for preparing an anti-tumor antibody, and an application thereof, in particular to a TM4SF3 protein used for preparing an anti-tumor antibody, and an encoded nucleotide sequence thereof; the invention also relates to an antibody of the anti-TM4SF3 protein, and a preparation method and an application of the antibody in producing various biological preparations for curing various relative tumors; in addition, the invention also relates to a medicine composition including the antibody, and an application for inhibiting the tumor in the body, so that the effect of curing the tumor is achieved.
Description
Technical field
The present invention relates to can be used for preparing the TM4SF3 albumen and the application thereof of anti-tumour antibody.Concrete the TM4SF3 albumen and the coding nucleotide sequence thereof that the present invention relates to be used to prepare anti-tumour antibody; Relate to proteic antibody of anti-TM4SF3 and preparation method thereof, the purposes in the various biotechnological formulations of the various related neoplasms of production for treating; Also relate to pharmaceutical composition that comprises this antibody and the purposes that suppresses tumour in vivo thereof in addition, thereby reach the effect of treatment tumour.
Background technology
Tumour is one of human modal disease, and its mortality ratio comes second and the 3rd respectively in the various diseases of the world and China, having a strong impact on human beings'health.Development in China along with society, the sickness rate of tumour are also being risen year by year.China has about 4,500,000 people of cancer patients at present, and mortality ratio is more than 30%, and annual newly-increased patient's number is up to more than 2,000,000 people.Cure cancer effectively and be the task of top priority in the scientific research.The method of clinical anticancer mainly be excision and put, chemotherapy, still, put, chemotherapy in kill cancer cell, also brought serious damage for the human normal cell.The operation of preceding treatment malignant tumour, radiotherapy, chemotherapy three big conventional treatment means significantly do not reduce the mortality ratio of tumour, and its 5 years survival rates only reach 10-30%, so more effective treatment means is tried hard to seek out all in the exploration of keeping unremitting from start to finish by various countries, the whole world.Along with deepening continuously to tumor research, people begin to attempt adopting biological method to treat and obtained pleasurable effect at the different aspects in the tumor development process, and the biological targeting treatment of tumour will become prospect and most active fields most.
The targeted therapy of cancer is very important, and " target " is the principle that must observe in the cancer therapy, hits cancer this " target " efficiently and accurately, realizes it being the key point of cancer therapy with a definite target in view.Therefore, finding efficient, special target spot is the key of knubble biological targeted therapy.Protein is the carrier of vital movement, has 80% to be at proteinic clinically in the various medicines of Shi Yonging.Current, the research of seeking the target spot of neoplasm targeted therapy mainly concentrates on various in tumour on the albumen of unconventionality expression.It is generally acknowledged that this white egg must have following characteristics: 1) specifically expressing in tumor tissues.Promptly this albumen is expressed in tumor tissues apparently higher than healthy tissues; Or the cytology location of this albumen in tumour obviously differ from healthy tissues, and this unusual location makes that related drugs can be easier to arrive.2) this albumen has function in tumour cell, can obviously suppress the clinical progress of tumour after medicine and the albumen effect, and normal tissue is not had obvious toxic and side effects.3) present, in various biotherapy means, the most successful is the antibody class medicine, because the characteristic of antibody itself, the target spot that requires antibody target must be a membranin.In recent years, the research and development of the antibody drug of treatment tumour have obtained breakthrough.Rituxan be 1997 first obtain the anti-tumour antibody medicine of FDA approval listing, be used for the treatment of B cellularity non Hodgkin lymphoma.Herceptin is granted in 1998, is mainly used in HER-2/neu male mammary cancer.Mylotarg (2000) is used for the treatment of the acute relapse myelogenous leukemia.In recent years, the successively granted antibody drug that is used for the treatment of tumour also has Campath-1H, Zevalin, Bexxer, Erbitux and Avastin etc.At present, the antibody drug of numerous treatment tumour is carrying out clinical preceding and clinical study.
At present, in the antibody of open report medicine at the oncotherapy target spot mainly contain following.They or participated in the growth of tumor process, perhaps in the commentaries on classics of tumour, chemical sproof inductive process, play a role.The target spot of the antibody class medicine that has gone on the market comprises: CD20 is used for the treatment of B cellularity non Hodgkin lymphoma; Her2-neo is used for the treatment of mammary cancer and lung cancer etc.; CD33 is used for the treatment of the acute relapse myelogenous leukemia; CD52, B cellularity lymphocytic leukemia; EGRFR is used for the treatment of straight, colorectal carcinoma in late period; VEGF is used for the treatment of treatment knot in late period, the rectum cancer; CD25 is used for the treatment of leukemia; CO17-1A is used for the treatment of colorectal carcinoma etc.The target spot of other antibody class medicine also comprises: CEA, CD22, GD2, EGFR, IGN-101, EGF, lD10, CD6, CD11a, huJ591, ACA-125 etc.(the monoclonal antibody of the anti-CD20 of Grillo-Lopez AJ.: the strategy of leukemic treatment and result.Antineoplaston experience summary, 2002; 2:323-329; (Grillo-Lopez AJ.AntiCD20 mAbs:modifying therapeutic strategies and outcomes in the treatment oflymphoma patients, Expert Rev Anticancer Ther.2002; 2:323-329) Stadtmauer EA uses the method for Mylotarg combined chemotherapy to treat acute myeloid leukaemia, clinical lymphoma, 2002; 2suppl 1:S24-S28, (Stadtmauer EA, Trials withgemtuzumab ozogamicin (Mylotarg) combined with chemotherapyregimens in acute myeloid leukemia, Clin Lymphoma, 2002; 2suppl1:S24-S28) Pangalis GA, Dimopoulou MN, Angelopoulou MK etc. use Campath-1H (anti-CD52) monoclonal antibody treatment lymphocytosis disease, antitumor medical science, 2001; 18:99-107, (Pangalis GA, Dimopoulou MN, Angelopoulou MK, etal.Campath-1H (anti-CD52) monoclonal antibody therapy inlymphoproliferative disorders, Med Oncol, 2001; 18:99-107), Leonard JP, Link BK uses HLL2 (epratuzumab, a kind of anti-CD22 monoclonal antibody) and people 1D10 (apolizumab) treatment non_hodgkin lymphoma, antitumor research, 2002; 29 (1 suppl2): 81-86, (Leonard JP, Link BK, Immunotherapy of non-Hodgkin ' slymphoma with hLL2 (epratuzumab, an anti-CD22 monoclonal antibody) and Hu1D10 (apolizumab), Semin Oncol.2002; 29 (1 suppl 2): 81-86) Slamon DJ, Clark GM, Wong SG waits the recurrence of human breast carcinoma and the dependency of prognosis and HER-2/neu oncogene amplification, science, 1987; 235:177-182, (Slamon DJ, Clark GM, Wong SG, et al, Human breast cancer:correlation of relapse andsurvival with amplification of the HER-2/neu oncogene, Science, 1987; 235:177-182) Mendelsohn J, Baselga J.EGF receptor family is as the target spot of neoplasm targeted therapy, oncogene, 2000; 19:6550-6565, (The EGF receptor familyas targets for cancer therapy, Oncogene, 2000; 19:6550-6565) Rosen LS, the clinical experience of application Angiostatin: focus on vascular endothelial growth factor, tumour control, 2002; 9 (2suppl): 36-44, (Rosen LS, Clinical experience withangiogenesis signaling inhibitors:focus on vascular endothelial growthfactor (VEGF) blockers, Cancer Control, 2002; 9 (2 suppl): 36-44) Schwartzberg LS, the clinical experience of Edrecolomab: a kind of monoclonal antibody for the treatment of colorectal carcinoma, antitumor hematological summary, 2001; 40:17-24 (Schwartzberg LS, Clinical experiencewith edrecolomab:a monoclonal antibody therapy for colorectal carcinoma, Crit Rev Oncol Hematol, 2001; 40:17-24) Foon KA, Lutzky J, Baral RN waits with antiidiotypic antibody simulation CD2 antigen as the melanomatous clinical and immune response of vaccine immunity progressivity.Clinical antitumor magazine, 2000; 18:376-384, (Foon KA, Lutzky J, Baral RN, et al, Clinical and immune responses in advanced melanomapatients immunized with an anti-idiotype antibody mimickingdisialoganglioside GD2, J Clin Oncol.2000; 18:376-384))
Although tens kinds of antitumor monoclonal antibody medicines at different albumen target spots have been arranged, concerning clinical needs, remain far from being enough, still need to develop more antibody class antitumor drug.This mainly be since tumor development be one multifactor, the process of polygenic mutation, only one or two kind can not stop the progress of tumour at the antibody of respective signals path, and can only delay this process; Different in addition tumours has different pathogeny, needs different medicines.
The inventor's the TM4SF3 that studies show that is positioned on the cytolemma of tumour, and has participated in growth of tumor and transfer process, so this albumen has the potentiality as the antitumor drug target.
Summary of the invention
Based on present needs in the treatment tumor area, in order to develop the medicine that can more be used on research and development and preparation treatment tumour, one aspect of the present invention discloses has TM4SF3 albumen or its fragment of aminoacid sequence shown in SEQID NO:1,2,3 or 4, this TM4SF3 is positioned on the human tumor cells film, and can be as the human tumor cells antigen of preparation antitumor drug; TM4SF3 albumen provided by the invention in addition or its fragment can be used to screen multiple antitumor drug as target.
On the other hand, the invention provides antibody and the derivative thereof for preparing as antigen with TM4SF3 albumen or its fragment.Described antibody derivatives preferably nucleic coupling antibody,
131I traget antibody or nano magnetic particle coupling antibody.Described antibody and derivative thereof can be used to prepare the medicine that is used for the treatment of tumor recurrence, transfer.
On the one hand, the invention provides the component that obtains as target sieving by TM4SF3 albumen or its fragment again with anti-tumor activity.This component is antibody, polypeptide, micromolecular compound preferably.
Fourth aspect, the invention provides suppress that TM4SF3 albumen or the active component of its fragment comprise preparation treatment Mammals that people's tumour takes place, the purposes in growth or the medicine that shifts.Described component is antibody, polypeptide, micromolecular compound preferably; Described tumour is the esophageal carcinoma or the proteic tumour of other positive expressions TM4SF3 preferably.
The 5th aspect, the invention provides a kind of pharmaceutical composition that comprises the antineoplastic component that obtains by the antibody of TM4SF3 protein Preparation and derivative thereof or by the TM4SF3 protein screening, and the purposes of described pharmaceutical composition in the medicine of generation, growth or the transfer of preparation treatment tumour.Described tumour is the esophageal carcinoma or the proteic tumour of other positive expressions TM4SF3 preferably.
Beneficial effect of the present invention is: as antigen, the antibody for preparing has the effect of good neoplasm growth, transfer and recurrence with TM4SF3 albumen provided by the invention and fragment thereof; The target position of antineoplastic immune targeted therapy is provided with TM4SF3 albumen that provides of the present invention and fragment thereof, screening obtains potential or has the drug component of good resistance tumor growth, transfer and recurrence, and and then obtains to have the pharmaceutical composition of anti-tumor activity.
Description of drawings
Fig. 1 shows that Western Bloting detects TM4SF3 expression in the esophageal carcinoma (T) and pairing healthy tissues (N) thereof, and beta-actin is guaranteed the confidential reference items of constant applied sample amount.The result shows that TM4SF3 albumen is at human esophageal carcinoma high frequency up-regulated.
Fig. 2 shows the effect that anti-TM4SF3 antibody shifts esophageal carcinoma lung, and the result shows in the anti-TM4SF3 Antybody therapy group that cancer cells does not have to shift substantially, and in the control group of handling with PBS and in the normal mice IgG treatment group, cancer cells exists tangible lung to shift.
Embodiment
The invention provides the albumen of the aminoacid sequence shown in a kind of SEQ of having ID NO:1.Described albumen is human tumor cells antigen.Described albumen is to obtain by host cell expression.Described albumen is positioned at the cytolemma of tumour cell as antigen, and it has immunogenicity.It is characterized in that this antigen part peptide sequence is positioned at outside the tumor cell membrane.
The present invention also provides a kind of antibody or other methods at this proteic medicine that makes with the albumen with the aminoacid sequence shown in the SEQ ID NO:1.This antibody is anti-tumour antibody.This antibody can be the polyclonal antibody or the monoclonal antibody in rabbit, sheep, horse, mouse source.According to the industry known method, can also screen multiple antitumor drug with albumen TM4SF3 as target position, as medicines such as antibody, polypeptide, small molecules.
The present invention also provides albumen or the fusion rotein that contains just like the described aminoacid sequence of SEQ ID NO:1.
The present invention further provides and a kind ofly derived and the antibody derivatives that comes by described antibody, this derivative is nucleic-antibody coupling matter, chemicals-antibody coupling matter, toxin-antibody coupling matter, prodrug enzyme-antibody coupling matter, nano particle-antibody coupling matter.
The present invention also provides the application of described antibody in the medicine for preparing treatment tumour and inhibition tumor growth, recurrence, transfer.
The present invention provides a kind of pharmaceutical composition again, it is characterized in that comprising the tumor chemotherapeutic drug of antibody of the present invention or antibody derivatives of the present invention.
In other words, according to a first aspect of the invention, the invention provides a kind of new human tumor cells antigen and peptide sequence thereof, described antigenic peptide has following amino acid residue sequence: (SEQ ID NO:1).
Comprise the endoglin expression of this antigenic albumen TM4SF3, suppress this antigenic function and just can suppress tumour cell growth in vivo and transfer at tumour cell.Therefore, this antigen can be used as the target position of anti-people's tumour immunity targeted therapy.According to the industry known method, can also screen the medicine of multiple neoplasm growth, transfer, recurrence with albumen TM4SF3 as target position, as medicines such as antibody, polypeptide, small molecules.
The invention still further relates to the dna sequence dna of a kind of coding as above-mentioned antigenic amino acid residue sequence and design the method for producing various nucleic acid drugs for target with this dna sequence dna.According to the industry known method, can design at the little RNA medicine of this dna sequence dna and be other nucleic acid drugs of carrier with the virus vector.
In the present invention, described antigen is meant any protein or the fused protein that comprises above-mentioned amino acid residue sequence; The codeword triplet translation back coding that this antigenic DNA is meant that any basis is generally acknowledged of encoding contains above-mentioned amino acid residue sequence.
Antigen of the present invention is to find that this protein expression is on tumor cell membrane when making immunohistochemistry and immunocyte fluorescence by the proteic polyclonal antibody of use TM4SF3.
The present invention also relates to a kind of method for preparing antineoplastic antibody in addition.
According to new tumor-resistant antigen provided by the invention, adopt and well known to a person skilled in the art that method and technology are easy to obtain its corresponding monoclonal antibody and derivative.Adopt this antigen immune mouse, in detecting mouse serum, behind the content of corresponding specific antibody, put to death animal and obtain splenocyte,,, can obtain mouse monoclonal antibody again through the antigen-specific experiment sieving through merging with murine myeloma cell SP2/0.With the variable region gene of mouse monoclonal antibody clone, and, be cloned into carrier for expression of eukaryon once more and import eukaryotic cell expression, can obtain people-mouse chimeric antibody with after corresponding human constant region correctly is connected.Adopt this antigen selection people phage antibody library, or adopt human peripheral blood mononuclear cell's gomphosis mouse technology, can obtain corresponding human antibody.
The present invention also provide described antibody or derivative in preparation medicine or contain the application of drug regimen in treatment people tumour of this antibody.
The corresponding antibodies or derivatives thereof of this tumour antigen can be used for immune guiding treatment tumour.Can infer any human tumor according to well known to a person skilled in the art principle, as long as on this type of tumor cell membrane, express this albumen, promptly this tumour antigen is present on the tumor cell membrane, just can use the corresponding antibodies or derivatives thereof of this tumour antigen to carry out the immune guiding treatment.This antibody or derivatives thereof can directly inject in patient's the body by variety of way, with the antigen that is positioned at tumor cell surface, suppresses growth of tumor.The antibody that is marked with nucleic or chemotherapeutics can take nucleic or chemicals to the cancerous lung tissue part, thereby reduces the toxicity of nucleic or chemotherapeutics, increases the effect that they kill and wound lung carcinoma cell greatly.Nano magnetic particle link coupled antibody gathers in lung cancer focus part nano magnetic particle is dense, and the effect of high-frequency alternating magnetic field is heating down, and the cancer cells around killing and wounding plays the purpose of treatment cancer.This antigen corresponding antibodies and effector molecule are merged,, can gather in the carninomatosis kitchen range effector molecule is dense, effector molecule is farthest played a role, play the effect of treatment people tumour as toxin PE40.
In a word, utilize aforementioned human tumor antigen corresponding antibodies provided by the invention, just can producing at present easily, known various derivatives are used for tumor treatment.
The novel tumor antigen, its corresponding antibodies and the various derivative that describe subject matter involved in the present invention below with reference to accompanying drawings in conjunction with the embodiments in detail and use among the present invention are implemented embodiments such as tumor treatment.
Embodiment
Embodiment 1: the preparation of new human tumor antigen
According to the state of the art, with the cDNA sequence of TM4SF3 gene as template, adopt primer: 5 '-ATAGATATCGACAAGCCTGTAACGAA-3 ' (forward primer, contain EcoRV restriction enzyme site and XhoI restriction enzyme site) and 5 '-GATCTCGAGGTTCCCGATCTGG-3 ' (reverse primer contains the XhoI restriction enzyme site), by the cDNA encoding sequence of pcr clone TM4SF3 gene.After the PCR product that the clone is obtained is the cDNA sequence of correct TM4SF3 gene through sequence verification, according to this area routine techniques with this sequence construct in expression vector PET-30b, obtain recombinant vectors PET-30b-M4SF3.Then described recombinant vectors PET-30b-TM4SF is transformed prokaryotic cell prokaryocyte, obtain corresponding reorganization prokaryotic cell prokaryocyte, thereby and under the condition that suitable this recombinant vectors is expressed, make TM4SF3 genetic expression obtain TM4SF3 albumen.
The proteic aminoacid sequence of the TM4SF3 that expression obtains is SEQ ID NO:1.
On the other hand, the cDNA full length sequence of TM4SF3 gene can be cloned on other carriers that contain particular sequence, thereby form the proteic derivative of TM4SF3.The proteic derivative of the TM4SF3 of indication of the present invention is meant by TM4SF3 albumen being replaced, lacks or adding one or more amino acid and do not change the protein derivatives of TM4SF3 protein function, does not promptly change the proteic function that is used to prepare anti-tumour antibody of TM4SF3 disclosed by the invention.Described derivative can be: (1) wherein one or more amino acid are replaced by conservative or nonconservative amino-acid residue, and the amino acid that replaces can be also can not encoded by genetic codon; (2) be that certain group on wherein one or more amino-acid residues is replaced by other group; (3) be to belong to mature polypeptide and other compounds to merge, perhaps constitute fusion polypeptide such as leader sequence, secretion sequence, the sequence etc. that is used for this sequence of purifying with other.For example the cDNA full length sequence of TM4SF3 gene is cloned on the carrier that contains the GST label, PGEX-3X carrier for example, thereby obtain the GST-TM4SF3 fusion rotein, promptly added the sequence of GST, but this does not change the proteic character of TM4SF3 at this proteic N end.Can also be with this proteic cDNA sequence clone to the carrier that contains other expression labels, as contain in the pet vector etc. of His label, constituted proteic other derivatives of TM4SF3.
In addition; also pass key sequence (the extracellular region sequence of TM4SF3 protein function that can the realization the present invention in the TM4SF3 albumen is claimed; as SEQ ID No.2 and SEQ ID No.5) single expression or be connected in the carrier that contains label and constitute fusion rotein; and do not express this albumen complete sequence, this also constitutes the proteic analog derivative of the claimed TM4SF3 of the present invention., in the carrier that contains the GST label, express then as the cDNA sequence clone that the outer terminal amino acid sequence (SEQ ID No.2) of this albumen born of the same parents is corresponding, then obtain the fusion rotein of GST and TM4SF3 extracellular region sequence peptide section.For example, can also synthesize the crucial peptide section in this albumen, as IFGSEDVGSSSYVAVD (SEQ ID NO:3) and YENTKLLSATGESEKQ (SEQ ID NO:4), at one end or behind the two ends interpolation halfcystine be coupled on the keyhole limpet hemocyanin (KLH) then, be used for animal immune and prepare antibody or other small molecules targeted drugs, this also belongs to a class of protein derivatives of the present invention.It will be understood by those skilled in the art that the proteic derivative of TM4SF3 is not limited to foregoing, also comprise proteic other modifications of TM4SF3 and change.
Embodiment 2:TM4SF3 is at the human esophageal carcinoma high expression level
Detect by Westen, the inventor finds that TM4SF3 albumen is at human esophageal carcinoma medium-high frequency up-regulated.
Westen detects the proteic expression of TM4SF3 in 14 pairs of esophageal carcinoma (T) and the other normal esophageal epithelium sample of paired cancer (N) thereof.Extract every example and organize total protein, the total protein that detects each tissue of obtaining with SDS-PAGE electrophoresis and Westen detects.The total protein of every routine tissue sample of sample is 50ug on the SDS-PAGE electrophoresis.The Westen detected result shows the TM4SF3 up-regulated that has in all 14 pairs of esophageal carcinoma and the other normal esophageal epithelium of paired cancer thereof in the 11 routine human esophageal carcinomas, and the rise rate reaches 78.6% (Fig. 1).Therefore, TM4SF3 albumen is special human tumor antigen, can be used for further preparing in the development of anti-tumour antibody.
Embodiment 3: the preparation of new human tumor antigen corresponding antibodies
200 μ g human tumor antigen (TM4SF3 albumen) or its fragments that adopt embodiment 1 to obtain, equal-volume mixes complete freund adjuvant 200ul altogether, adopt the mode immunity new zealand rabbit of subcutaneous immunity, adopt 200 μ g antigen proteins or its fragment to mix the subcutaneous booster immunization of incomplete freund adjuvant after 2 weeks, the injection of 1 week back employing complete Freund's adjuvant emulsification 50-60mg bacille Calmette-Guerin vaccine albumen bilateral sole, adopt 200 μ g antigen proteins or its fragment equal-volume to mix the interior injection of 200ul Liang Ce popliteal nest lymphoglandula altogether of incomplete freund adjuvant after 1 week, blood sampling uses the ordinary method survey to tire after 10 days, finds tiring of serum〉1:10
8, put to death animal, collect rabbit anteserum, use method known to those skilled in the art, for example adopt the anti-TM4SF3 of antigen affinity purification purifying from rabbit anteserum or its segmental antibody.
Embodiment 4: the preparation of new human tumor antigen corresponding antibodies derivative
(1) nucleic-antibody coupling matter
(1)
188The corresponding mouse monoclonal antibody of the human tumor antigen that the Re mark is new
SnCl
2The antibody that direct-reduction process mark embodiment 3 obtains, instant thin-layer chromatography (ITLC) is measured the labeling effciency and the radiochemicsl purity of antibody immediately.Behind the mark, the ITLC experiment shows:
188Re-antibody labeling rate is 91%, and radiochemicsl purity is greater than 94%.
188Re-antibody is 347MBq/mg than living.ELISA records
188Re-antibody mediated immunity activity is 66%.The experiment of the vitro stability of traget antibody shows that antibody is hatched 24hr at 37 ℃, no matter is in physiological saline or human serum albumin, and radioactivity comes off all less than 5%, shows that it is external stable.
(2)
131The corresponding mouse monoclonal antibody of the human tumor antigen that the I mark is new
The chloramine-t method traget antibody, instant thin-layer chromatography (ITLC) is measured the labeling effciency and the radiochemicsl purity of antibody immediately.Behind the mark, the ITLC experiment shows
131I-antibody labeling rate is 91%, and radiochemicsl purity is greater than 93%.
131I-antibody is 77MBq/mg than living, and ELISA records
131I-antibody mediated immunity activity 64.3%.
(2) nano magnetic particle-antibody coupling matter
With the ferriferous oxide magnetic-particle ultra-sonic dispersion of pure water, form colloidal with the about 20-50nm of diameter.The ratio that adds 16mg antibody in the 0.2g nano magnetic particle adds the antibody that is dissolved in pure water, mixes rapidly, adds final concentration behind the 5min and be 1% BSA, and is centrifugal and adopt 1% BSA washing back standby.
Embodiment 5: the human tumor antigen corresponding antibodies that adopts embodiment 3 to obtain suppresses growth of tumor (is example with the esophageal carcinoma)
Get 24 of lotus human esophagus cancer nude mouses, behind measurement and the calculating gross tumor volume, be divided into 3 groups at random by gross tumor volume, 8 every group.First group is the PBS control group, every abdominal injection 200 μ lPBS; Second group is normal mice IgG control group, every abdominal injection 0.2mg normal mice IgG; The 3rd group is novel tumor antigen corresponding antibodies treatment group, every 0.2mg tumour antigen corresponding antibodies that abdominal injection embodiment 1 obtains.Be administered once in per two days, treat back 10 times after, put to death animal, separate tumour, take by weighing tumor weight, calculate tumour inhibiting rate, tumour inhibiting rate=(control group tumor weight-treatment group tumor weight)/control group tumor weight * 100%.The t check is adopted in test of significance.
The result shows that PBS group knurl weighs 1.24 ± 0.47g, normal mice IgG control group 1.13 ± 0.37g, and novel tumor antigen corresponding antibodies treatment group 0.66 ± 0.25g, the tumour inhibiting rate of novel tumor antigen corresponding antibodies is 47.2%, the P value is less than 0.01.These results show that the novel tumor antigen corresponding antibodies has important use to be worth in immune guiding treatment esophageal carcinoma cancer.
Embodiment 6: the human tumor antigen corresponding antibodies that adopts embodiment 3 to obtain suppresses the transfer (is example with the esophageal carcinoma) of tumour
Get 24 of lotus human esophagus cancer nude mouses, behind measurement and the calculating gross tumor volume, be divided into 3 groups at random by gross tumor volume, 8 every group.First group is the PBS control group, every abdominal injection 200 μ lPBS; Second group is normal mice IgG control group, every abdominal injection 0.2mg normal mice IgG; The 3rd group is novel tumor antigen corresponding antibodies treatment group, every 0.2mg tumour antigen corresponding antibodies that abdominal injection embodiment 1 obtains.First month per week of treatment is administered twice, and is administered once in per five days after one month, after treating after back 50 days time, puts to death animal, dissects animal and observes an internal organs metastases situation.The t check is adopted in test of significance.
The result shows that 8 mouse of PBS group have 7 to have only obvious lung to shift, 8 mouse of normal mice IgG control group have 6 to have only obvious lung to shift, 8 mouse of novel tumor antigen corresponding antibodies treatment group have 1 to have only obvious lung to shift, the lung that can obviously suppress the esophageal carcinoma of novel tumor antigen corresponding antibodies shifts, and the P value is less than 0.01 (Fig. 2).These results show that the novel tumor antigen corresponding antibodies has important use to be worth in immune guiding treatment Metastasis of Esophageal Carcinoma.
Embodiment 7: adopt derive in object target and suppress tumour (is example with the esophageal carcinoma) of new human tumor antigen corresponding antibodies
Get 24 of lotus human esophagus cancer nude mouses, behind measurement and the calculating gross tumor volume, be divided into 3 groups at random by gross tumor volume, 8 every group.First group is the PBS control group, every abdominal injection 200 μ lPBS; Second group is normal mice IgG control group, every abdominal injection 9.2MBq (200 μ l)
131I-normal mice IgG; The 3rd group is coupling
131The novel tumor antigen corresponding antibodies treatment group of I, every abdominal injection 9.2MBq (200 μ l)
131I-antibody.Treated back 10 days, and put to death animal, separate tumour, take by weighing tumor weight, calculate tumour inhibiting rate, tumour inhibiting rate=(control group tumor weight-treatment group tumor weight)/control group tumor weight * 100%.The t check is adopted in test of significance.
The PBS knurl weighs 1.16 ± 0.35g as a result, human IgG control group 1.12 ± 0.41g, and traget antibody treatment group 0.41 ± 0.15g, the tumour inhibiting rate of traget antibody is 64.6%, the P value is less than 0.01.These results show that novel tumor antigen corresponding antibodies (polyclonal antibody of embodiment 3) coupling nucleic has important use to be worth in the immune guiding treatment esophageal carcinoma.
Embodiment 8: the human tumor antigen corresponding antibodies that adopts embodiment 2 to obtain suppresses growth of tumor (is example with the colorectal carcinoma)
Get 24 of lotus human colon carcinoma nude mouses, behind measurement and the calculating gross tumor volume, be divided into 3 groups at random by gross tumor volume, 8 every group.First group is the PBS control group, every abdominal injection 2001PBS; Second group is normal mice IgG control group, every abdominal injection 0.2mg normal mice IgG; The 3rd group is novel tumor antigen corresponding antibodies treatment group, every 0.2mg tumour antigen corresponding antibodies that abdominal injection embodiment 1 obtains.Be administered once in per two days, treat back 10 times after, put to death animal, separate tumour, take by weighing tumor weight, calculate tumour inhibiting rate, tumour inhibiting rate=(control group tumor weight-treatment group tumor weight)/control group tumor weight * 100%.The t check is adopted in test of significance.PBS group knurl weighs 1.45 ± 0.37g as a result, normal mice IgG control group 1.53 ± 0.39g, and novel tumor antigen corresponding antibodies treatment group 0.73 ± 0.21g, the tumour inhibiting rate of novel tumor antigen corresponding antibodies is 49.7%, the P value is less than 0.01.These results show that the novel tumor antigen corresponding antibodies has important use to be worth in immune guiding treatment colorectal carcinoma.
Sequence table
＜110〉Tumour Inst., Chinese Medical Academy
＜120〉be used to prepare the TM4SF3 albumen and the application thereof of anti-tumour antibody
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Claims (14)
1, TM4SF3 albumen or its fragment, its aminoacid sequence is shown in SEQ ID NO:1,2,3 or 4.
2, TM4SF3 albumen as claimed in claim 1 or its fragment purposes in preparation antibody.
3, TM4SF3 albumen as claimed in claim 1 or its fragment are as the purposes of target in the multiple antitumor drug of screening.
4, antibody and the derivative thereof for preparing as antigen with the described TM4SF3 albumen of claim 1 or its fragment.
5, antibody according to claim 4 and derivative thereof, wherein said antibody derivatives be nucleic coupling antibody,
131I traget antibody or nano magnetic particle coupling antibody.
6, antibody as claimed in claim 4 and derivative thereof are used for the treatment of purposes in the medicine of tumor recurrence, transfer in preparation.
7, the component that obtains as target sieving with the described TM4SF3 albumen of claim 1 or its fragment with anti-tumor activity.
8, component according to claim 7, it is antibody, polypeptide, micromolecular compound.
9, the component with anti-tumor activity as claimed in claim 7 comprise preparation treatment Mammals that people's tumour takes place, the purposes in growth or the medicine that shifts.
10, purposes according to claim 9, wherein said component are antibody, polypeptide, micromolecular compound.
11, according to claim 9 or 10 described purposes, wherein said tumour is the esophageal carcinoma, colorectal carcinoma or the proteic tumour of positive expression TM4SF3.
12, a kind of pharmaceutical composition is characterized in that comprising antibody as claimed in claim 4 and derivative thereof or comprises component as claimed in claim 7.
13, the purposes of pharmaceutical composition as claimed in claim 12 in the medicine of generation, growth or the transfer of preparation treatment tumour.
14, purposes according to claim 13, wherein said tumour are the esophageal carcinoma, colorectal carcinoma or the proteic tumour of positive expression TM4SF3.
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| US20130122002A1 (en) * | 2010-07-23 | 2013-05-16 | Insitutut National de la Sante et de la Recherche Medicale (INSERM) | Methods for cancer management targeting co-029 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20130122002A1 (en) * | 2010-07-23 | 2013-05-16 | Insitutut National de la Sante et de la Recherche Medicale (INSERM) | Methods for cancer management targeting co-029 |
| US9040671B2 (en) * | 2010-07-23 | 2015-05-26 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Methods for cancer management targeting Co-029 |
| US20150291691A1 (en) * | 2010-07-23 | 2015-10-15 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Methods for cancer management targeting co-029 |
| US9487586B2 (en) * | 2010-07-23 | 2016-11-08 | Institut National De La Santé Et De La Recherche Médicale (Inserm) | Methods for cancer management targeting Co-029 |
| EP2596025B1 (en) * | 2010-07-23 | 2018-06-20 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for cancer management targeting co-029 |
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