Patent Application: US-51908205-A

Abstract:
the present invention provides a method for up - regulating tumor cell antigen expression , comprising administering to the cells an amount of thymalfasin sufficient to increase the expression of tlp relative to that of untreated tumor cells . also provided are methods for enhancing the sensitivity of a immunodiagnostic or immunotherapeutic method , comprising pre - treating target tumor cells by administering to the cells an amount of thymalfasin sufficient to increase the expression of tlp relative to that of untreated tumor cells , followed by application of the immunodiagnostic or immunotherapeutic method . these methods are applicable to both in vivo and in vitro diagnostic methods , and to in vivo immunotherapeutic methods .

Description:
the expression of tlp antigen can be up - regulated by treatment with thymalfasin . the increased expression could lead to more effective ctl responses from induction of a specific cd8 population . additionally , this increased expression could allow cells to be better targeted and detected for radio - immuno guided surgery or immuno - scintigraphic techniques . moreover , the availability of an experimental tumor naturally expressing a human antigen could be of great use in development of preclinical models . enhancing tumor antigen expression with the present methods adds increased sensitivity to both diagnostic and immunotherapeutic methods , which in turn permits earlier detection and treatment of cancers , and a more aggressive and thorough treatment of advanced cancers , including metastatic cancers . tlp expression - enhancing amounts of thymalfasin peptide can be determined by routine dose - titration experiments . up - regulation of tlp can be achieved in vivo by administration of between 2 μg / kg and 6 mg / kg body weight of thymalfasin , preferably between 20 μg / kg and 200 μg / kg . thymalfasin has been found to be safe for humans when administered in doses as high as 16 mg / dose / day , and in rats as high as 6 mg / kg / day . the thymalfasin can be administered by any of a variety of means well - known in the art , for example by injection or infusion intravenously , interperitoneally , intramuscularly or directly into the peri - tumoral area . in preferred embodiments , the thymalfasin peptide is present in a pharmaceutically acceptable liquid carrier , such as water for injection , saline in physiologic concentrations , or similar . the plasma half - life of subcutaneously injected thymalfasin is only about 2 hours . rost , et al ., 1998 . however , conjugation of a polymer to a thymalfasin peptide substantially increases the plasma half - life of the peptide . rasi , et al . ( unpublished observations ). when applied in the context of a conjugated thymalfasin , the above dosages reflect only the thymalfasin peptide present in the composition , and not the weight of the polymer conjugated thereto . the isolation , characterization and use of thymalfasin peptides is described , for example , in u . s . pat . no . 4 , 079 , 127 , u . s . pat . no . 4 , 353 , 821 , u . s . pat . no . 4 , 148 , 788 and u . s . pat . no . 4 , 116 , 951 . the present invention is applicable to thymalfasin peptides including naturally occurring thymalfasin as well as synthetic thymalfasin and recombinant thymalfasin having the amino acid sequence of naturally occurring thymalfasin , amino acid sequences substantially similar thereto , or an abbreviated sequence form thereof , and their biologically active analogs having substituted , deleted , elongated , replaced , or otherwise modified sequences which possess bioactivity substantially similar to that of thymalfasin , e . g ., a thymalfasin peptide having sufficient amino acid homology with thymalfasin such that it functions in substantially the same way with substantially the same activity as thymalfasin . widr ( human ), ia - xssbr ( rat ), and dhd - k12 ( rat ) colorectal cancer cell lines were treated with thymalfasin from 6 to 48 hours at 5 - 100 μg / ml . tlp antigen expression was determined by flow cytometry ( fc ) and antigen localization by confocal laser scanning microscopy ( clsm ), using tlp antiserum raised against a 9 amino acid peptide epitope of tlp ( csh - 275 ). tlp is naturally expressed in all 3 colorectal cell lines , both in the cytoplasm , ranging from 30 - 55 % of cells ( by clsm and by fc after permeabilization ) and on the cell membrane , ranging from 10 - 20 % of cells ( by fc ). fig1 . thymalfasin is able to enhance the expression of this antigen in all cell lines tested . fig2 . the level of enhancement , and localization at the membrane versus the cytoplasm , varies with dosage and timing of treatment , and can reach expression levels of 90 % of cells . bd - ix rats were injected i . p . with syngeneic dhd - k12 cells and treated i . p . or s . c . with thymalfasin . tlp expression and localization were determined as above on tumor cells obtained both from ascites or tumor mass . tumor cells from animals treated with thymalfasin demonstrate an enhancement of expression of tlp similar to that seen in vitro . fig3 . andreone p ., cursaro c ., gramenzi a ., et al . “ a randomized controlled trial of thymosin alpha 1 versus interferon alpha treatment in patients with hepatitis b e antigen antibody — and hepatitis b virus dna — positive chronic hepatitis b ,” hepatology 24 ( 4 ): 774 - 777 ( 1996 ). baxevanis , c . n ., et al ., “ enhancement of human t lymphocyte function by prothymosin alpha : increased production of interleukin - 2 and expression of interleukin - 2 receptors in normal human peripheral blood t lymphocytes ,” immunopharmacol . immunotoxicol . 12 ( 4 ): 595 - 617 ( 1990 ). bepler , g ., “ thymosin alpha - 1 as adjunct for conventional therapy of malignant tumors : a review ,” cancer invest . 12 : 491 - 6 ( 1994 ). garaci , e ., et al . “ a randomized controlled study for the evaluation of the activity of a triple combination of zidovudine , thymosin alpha 1 , and interferon alpha in hiv - infected individuals with cd4 counts between 200 and 500 cells / mm 3 ,” antiviral ther . 3 : 103 - 111 ( 1998 ) garaci , e ., f . pica , g . rasi , and c . favalli , “ thymosin alpha 1 in the treatment of cancer : from basic research to clinical application ,” int . j . immunopharm . 22 : 1067 - 1076 ( 2000 ). giuliani , c ., g . napolitano , a . mastino , s . da vincenzo , c . d &# 39 ; 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