Patent Application: US-201013516385-A

Abstract:
the invention relates to a composition for the treatment of various cancers . the composition is a vaccine containing sequences of edb , eda , annexin a1 , endosialin , c domain of tenascin c or magic roundabout or fragments thereof as single or in a combination coupled to one or several heterologous foreign carrier molecules . the vaccine will produce antibodies that are directed against self proteins which are preferentially expressed in and around tumor vessels . the vaccine is preferably administrated together with an adjuvant .

Description:
anti - edb , anti - eda , anti - annexin a1 , anti - endosialin , anti - the extra domain c of tenascin c or anti - magic roundabout ( mr ) antibodies are produced in the host by active immunization , so called vaccination . by injection of modified edb , eda , annexin al , endosialin , the extra domain c of tenascin c or magic roundabout ( mr ) into the host the immune system of the host produces a polyclonal antibody response against its own edb , eda , annexin a1 , endosialin , the extra domain c of tenascin c or magic roundabout ( mr ) and thereby targeting the tumor specific vessels for attack by the immune system . it is of major importance to modify the antigen so that the immune system of the host recognize the modified self - protein as a non - self protein . this can be achieved by covalent coupling of non - self amino acid regions to edb , eda , annexin a1 , endosialin , the extra domain c of tenascin c or magic roundabout ( mr ) or selected regions of any of these three molecules from the species to be treated . the peptides within the non - self region then attract and activate non - tolerized t cells , which give help for the potentially auto - reactive b cells . there are at least three different strategies to do these modifications of the self - protein . one method is to produce a fusion protein between a non - self protein , and the entire or a selected fragment of more than 5 amino acids of self edb , tenacin c or mr in a prokaryotic or eukaryotic expression system . the open reading frame of edb , eda , annexin a1 , endosialin , the extra domain c of tenascin c or magic roundabout ( mr ), as exemplified by human edb , eda , annexin a1 , endosialin , the extra domain c of tenascin c or magic roundabout ( mr ) in fig1 , is then first cloned into a bacterial , fungal or eukaryotic vector . this fusion protein construct is then transfected into a mammalian or prokaryotic host for the production of the desired fusion protein . the fusion partner can here be any non - self protein of any size from 10 amino acids to several hundred kd . however , it is usually favorable to use a fusion partner of approximately the same size as the self - protein . alternatively , a non - modified edb , eda , annexin a1 , endosialin , the extra domain c of tenascin c or magic roundabout ( mr ) can be produced in a mammalian or prokaryotic host or host cell line and then covalently attached to a carrier protein by chemical coupling . a third alternative , which in our mind is less attractive , is to produce selected regions of the edb , eda , annexin a1 , endosialin , the extra domain c of tenascin c or magic roundabout ( mr ) sequences as synthetic peptides and then to attach these peptides to a foreign carrier molecule by chemical coupling . this third alternative usually results , after injection into the patient , in antibody responses that show low binding activity against the native properly folded protein and thereby in lower clinical effect . following production the vaccine antigen is then purified and tested for pyrogen content and potential content of other contaminants . in order to obtain sufficiently strong immune response against the self - epitopes the vaccine antigen is then ( optionally ) mixed with an adjuvant before injection into the patient . after administration in the patient the vaccine induces an immune response against the vaccine antigen . due to the presence of self - epitopes in the vaccine antigen this protein also induces an antibody response against the target molecule , here edb , eda , annexin a1 , endosialin , the extra domain c of tenascin c or magic roundabout ( mr ), thereby targeting the immune system to attack the tumor vessels , which leads to reducing the growth of the tumor and maybe even total removal of the tumor . to test the efficacy of the invention a fusion protein between the 91 amino acid long extra - cellular domain b ( edb ) of human and mouse fibronectin and a bacterial antigen of a size of approximately 10 kd , the e . coli thioredoxin was used as vaccine antigen to study the effect in an animal model . the edb domain is very highly conserved and identical in almost in all placental mammals studied . this fusion protein was produced in a prokaryotic host to almost homogeneity ( fig2 ). the thioredoxin - edb - fusion protein was then injected in mice together with an adjuvant . after three weeks the mice received a booster dose of the vaccine and after five weeks of treatment serum from these animals were tested for the amount of anti - edb antibodies produced . as can be seen from fig3 all animals showed high titers of anti - edb antibodies whereas all the controls were negative . this shows that the vaccine has the capacity to induce production of substantial amounts of anti - edb antibodies in a test animal . the in vivo efficacy of these antibodies was then tested in a mouse tumor - model . the vaccination and the antibodies produced after triggering the immune system of the host antibodies were found to effectively reduce the tumor size in these animals ( fig4 ). the anti edb antibodies also resulted in a marked change in tissue of the tumor as observed by electron microscopic examination ( data not shown ). the binding of the anti - edb antibodies to the tumor vasculature does clearly cause a marked infiltration of immune cells and an attack by the immune system on these vessels . it is most likely this effect on the vasculature that causes the potent reduction in tumor size observed in the vaccinated animals . in conclusion , we show , that in contrast to most previous studies on cancer vaccines , both high titers and biological active antibodies induced by the vaccine and that it has good therapeutic effect on the tumor growth . 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