Patent Application: US-37897003-A

Abstract:
pharmaceutical compositions comprising erythropoietin are provided for treatment of cancer , particularly for treatment of multiple myeloma . erythropoietin was found to be effective for inhibition of tumor growth , triggering of tumor regression , stimulation of the natural immunological defense against cancer and / or inhibition of cancer cell metastasis .

Description:
according to the present invention , erythropoietin , that has been used so far only for the treatment of anemia , including cancer patients suffering from anemia , was found now to affect tumor regression in mice and to improve the biological and clinical course of some multiple myeloma patients . the first observations according to the invention were derived from multiple myeloma ( mm ) patients being treated for anemia with epo . mm is characterized by a clonal proliferation of bone marrow ( bm ) transformed plasma cells ( pc ) secreting a paraprotein which can be detected in the serum and / or urine ( kyle , 1975 ; durie & amp ; salmon , 1975 ; bergsagel , 1990 ). the manifestations and complications of mm include anemia , recurrent infections , thrombocytopenia with bleeding episodes , pathological bony fractures , hypercalcemia , renal failure , neuropathy and amyloidosis . most patients die of mm or its complications with a median survival ranging from 15 months ( for patients who do not respond to chemotherapy ) up to 48 months among responders ( bergsagel , 1990 ). about 60 - 90 % of the patients with mm suffer from anemia , which adversely affects their quality of life ( kyle , 1975 ; durie and salmon , 1975 ; bergsagel , 1990 ). the anemia occurring in myeloma patients is associated with inadequate endogenous epo production that can be ameliorated by exogenous epo administration , resulting in a significant increase in their hb level and improved quality of life ( spivak , 1994 ; mittelman et al ., 1997 ; miller et al ., 1990 ). the demonstration that inadequate endogenous epo production is among the major factors responsible for cancer - associated anemia ( miller et al ., 1990 ), was the rationale for our phase ii / iii epo - mm clinical trial described in mittelman et al ., 1997 . in this trial , 17 patients with mm and anemia ( hb & lt ; 11 g / dl ), were treated for anemia with rhuepo , 150u / kg × 3 / week subcutaneously . if no response was observed after 4 weeks the dose was doubled . the study was designed for 12 weeks , although a few responders continued rhuepo for longer periods . as reported and summarized in mittelman et al ., 1997 , the pretreatment endogenous serum epo levels were relatively low in all patients studied with mm - associated anemia , rhuepo was well tolerated in these patients , rhuepo was highly effective in the treatment of anemia in mm , and the response to rhuepo is characterized by an increase in hb levels , a reduction in blood transfusion requirements ( btr ) and an improvement in the who performance status ( ps ) score : out of the 17 patients , 12 ( 70 . 6 %) responded with a significant increase in their hb level , 6 of the 11 patients that were transfusion dependent enjoyed a complete abolition of btr , and 12 patients enjoyed an improved quality of life , interpreted as a lower who ps score . five patients of the clinical trial reported in mittelman et al ., 1997 , continued to receive rhuepo for their anemia for several years . these patients all displayed increased hb and , in general , were “ doing very well ” relative to the severity of their disease ( high tumor mass ). interestingly , the patients continued to exhibit high serum paraprotein levels . based on these clinical observations and on the assumption that , at least in some of the patients , in addition to increasing hb levels , epo might be responsible for changing the biology and course of the disease ( mm ) itself , experiments according to the present invention were thus pursued with a murine model to study the possible non - erythroid or other biological effects of epo on the course and biology of mm . for this purpose , we used the mineral - oil induced plasmacytoma in balb / c mice , designated mopc - 315 tumor , a well - known murine model for the study of clinical and immunological aspects of human mm ( potter and walters , 1973 ). similarly to the human mm cells , the murine mopc - 315 tumor cells synthesize and secrete a monoclonal iga ( λ2 ) immunoglobulin , thereby providing a measurable tumor marker ( serum myeloma component ) during tumor progression . the in vivo effect of rhuepo treatment on the growth of mopc - 315 tumor cells was studied in balb / c mice . as shown in the examples hereinafter , tumor regression was strikingly observed in 30 - 60 % of mice challenged with tumor cells and further treated for a short period with epo , without tumor recurrence throughout a follow - up period of 3 - 7 ½ months . the event seems to be associated with the development of an effective antitumor immune response . as used herein in the specification , “ regressor mouse ” or “ regressor ” refers to a mouse injected with tumor cells , followed by initial tumor growth and gross disappearance of the tumor cells after epo treatment . “ progressor mouse ” or “ progressor ” refers to a mouse injected with tumor cells , followed by continuous tumor growth irrespective of epo treatment . “ null ” mouse refers to a mouse in which no tumor take is observed after injection of tumor cells . the observations with the mm patients and the mouse myeloma model constitute one specific embodiment but it is encompassed by the present invention the use of a medicament comprising erythropoietin for the treatment of any kind of neoplastic disease , excepting renal cell cancer . as used herein in the specification and the claims , “ erythropoietin ” includes all types of erythropoietin , both natural and recombinant , as well as erythropoietin analogs showing erythropoietin activity , that are suitable for human administration such as the hyperglycosylated analogs and analogs having 1 - 14 sialic acid groups and changes in the amino acid sequence mentioned above . in one preferred embodiment , the erythropoietin is recombinant human erythropoietin ( rhuepo ). any suitable route of administration of epo such as intravenously ( i . v .) or subcutaneously ( s . c . ), can be used according to the invention , but the s . c . route is preferred because of better distribution in the body and a better bioavailability . the epo dose may be within the range of 5 , 000 - 20 , 000u , preferably 10 , 000u per injection ( corresponding roughly to 150 u / kg ). the protocol of administration will be determined by the physician according to the type of cancer , the severity of the disease , age and physical condition of the patient and other relevant parameters for each case . for example , in mm patients , a unit dose of about 10 , 000u can be injected s . c . 3 times a week for about 4 weeks , followed by further therapy , if necessary . it should be noted that , according to the data accumulated from the anemia studies , patients with high response to epo treatment are those with endogenous epo level of less than 200 mu / ml ( endogenous normal epo level : 10 - 25 mu / ml ; endogenous epo level in anemic patients : higher than 100 mu / ml and in the level of hundreds or thousands mu / ml ). the invention will now be illustrated by the following non - limiting examples . rhuepo ( eprex ) was obtained from cilag , schaffhausen , switzerland . the murine tumors plasmacytomas mopc - 315 and mpc - 11 , chronic lymphocytic leukemia ( cll ) bcl1 , lewis lung carcinoma 3ll , mca - 105 fibrosarcoma , k - 1735 melanoma and the hybridomas gk 1 . 5 ( producing anti - cd4 monoclonal antibody ) and 53 - 6 - 7 ( producing anti - cd8 monoclonal antibody ), were purchased from the american type cell culture ( atcc ), rockville , md ., usa . the c - 26 colon carcinoma and m - 109 lung carcinoma were kindly provided by prof . e . kedar ( hebrew university , jerusalem , israel ), and 107 - 202 breast adenocarcinoma , 38c - 13 pre b cell leukemia , 127c lymphosarcoma , and 17a - 443 acute myeloid leukemia were established in our laboratory . the mouse myeloma mopc - 315 was maintained in vivo by serial i . m inoculation into syngeneic female balb / c mice aged 8 weeks . balb / c mice were injected subcutaneously ( s . c .) with 10 4 cells in the abdominal area . local tumor growth ( 2 - 5 mm diameter ) was observed by day 11 - 13 , gradually growing in size and causing death in 90 - 100 % of mice by day 40 - 50 . subcutaneous rhuepo treatment was started when a tiny palpable tumor appeared at the site of injection . each mouse was numbered and the tumor growth rate of the individual mice ( its diameter in mm ) was measured by a vernier caliper . a follow - up of tumor size in individual mice was carried out every fifth day during epo administration ( lasting usually 4 weeks ). sera from mice were diluted ( 1 : 1 ) in nacl 0 . 9 % and 2 μl of the diluted sera were resolved by 10 % sds - polyacrylamide gel electrophoresis . protein bands were detected by coomassie blue staining ( 0 . 05 % v / v coomassie brilliant blue r - 250 ( bio - rad ) in 10 % acetic acid , 50 % methanol followed by destaining in 10 % acetic acid , 20 % methanol ). western blot analysis was performed as previously described ( neumann et al ., 1993 ). 2 μl of sera diluted 1 : 1 in 0 . 9 % nacl , were resolved on 10 % sds - page . mopc - 315 immunoglobulin ( 20 μg ) was loaded as a positive control . the gel was blotted onto nitrocellulose membrane filter , and probed with rabbit antibodies against mouse immunoglobulin λ light chain . subsequently the blots were incubated with secondary antibody ( donkey anti - rabbit igg ) coupled to horseradish peroxidase ( hrp ), and the bands were visualized using enhanced chemiluminescence ( ecl ) according to the manufacturer &# 39 ; s instructions . in the study described in mittelman et al ., 1997 , seventeen ( 17 ) patients with mm and anemia ( hb & lt ; 11 g / dl ) were enrolled in an open - label non - comparative study to test the serum epo levels in anemic patients with mm , as well as to evaluate the efficacy and toxicity of rhuepo in the treatment of anemia in these patients . the median age of the patients was 70 years ( range 44 - 88 ), six were males and 11 females . all patients had stage ii / iii disease . fifteen patients were on chemotherapy during the study period , 3 on the vincristine - adriamycin - dexamethasone ( vad ) regimen and the remaining on oral melphalan and prednisone ( mp ) regimen . the median pretreatment endogenous serum epo level was 150 mu / ml ( range 11 - 232 ). the serum epo level was assayed as described in mittelman et al ., 1997 . patients received subcutaneous injections of rhuepo , 150 u / kg 3 times a week , on an outpatient basis . if no response was observed after 4 weeks , the dose was doubled . oral iron ( slow - fe , 160 mg exsiccated ferrous sulfate , ciba - geigy , basel , switzerland ) was added . therapy with rhuepo was designed for 12 weeks , although 6 patients proceeded with rhuepo treatment following the study termination . rising of hb levels beyond 14 g / dl during the study required a dose modification . complete response was defined as an increase ( from baseline ) of the hb level ( at week 12 ) by & gt ; 2 g / dl , and / or complete abolishing of blood transfusion requirements ( btr ). partial response was defined as an increase of 1 - 2 g / dl in the hb level and / or 50 % reduction of btr . twelve patients ( 70 . 6 %) achieved complete response and another patient ( 5 . 9 %) showed partial response , which together gave a total response rate of 76 . 5 %, based on intention - to - treat basis . the median hb level rose from 9 . 4 g / dl ( range 7 . 3 - 10 . 7 ) at study commencement to 12 . 5 g / dl ( 9 . 0 - 15 . 2 ) at week 12 ( for those who completed the study ). six patients continued rhuepo treatment beyond the designed 12 - week study period for 14 - 85 weeks and continued to maintain normal or near - normal hb levels and to enjoy a good quality of life at a maintenance dose which is lower ( 15 - 50 %) than the initial dose required to obtain a response . according to the present invention , four of the patients of the above study ( y . s ., o . g ., l . b . and t . s ) and a new patient ( m . b .) continued to receive rhuepo for several years . these patients all displayed increased hb and in general were “ doing very well ”, relative to the severity of their disease . the characteristics of the 5 patients are summarized in table 1 . these observations seemed to indicate that , at least in some patients , in addition to increasing hb , epo may change the biology and course of the disease . we thus assumed that the improved quality of life was not related only to the correction of the anemia . since the common agent received by all 5 patients was epo , and this in fact was the only therapy during most of the time of the follow - up reported here , we assumed that epo , if administered at a certain dose for the appropriate period of time , may change the biology and course of the disease and lead to a longer survival , more than one would expect based on clinical criteria and considering the poor prognostic features of all these patients . the bone marrow plasma cells and especially the m - proteins , reflecting tumor mass , did not disappear in these patients , yet the course of the disease appeared to be stabilized , “ frozen ” or become latent and asymptomatic . based on these clinical observations , we then pursued with a murine model in an attempt to study possible non - erythroid or other biological effects of epo on the course and biology of mm . rhuepo administration to mice challenged with mopc - 315 tumor cells induces tumor regression mice challenged with a syngeneic progressive growing myeloma ( 10 4 cells s . c .) were treated with rhuepo by systemic administration . tumor growth over the period of 2 - 2 . 5 weeks was similar in all mice and was followed by a decrease in tumor size in some epo - treated mice culminating in a permanent and complete tumor regression in 30 - 60 % of treated mice ( compared to 0 - 10 % in controls ). effects of different epo regimens were tested . the results of four different representative experiments are summarized in table 2 . in experiment i , epo treatment ( 30u , injected s . c .) started 11 days following tumor cell challenge . mice received daily injections for 5 days and , after 6 days interruption , further injections 3 times a week for 3 weeks . most of the tumor - injected mice which did not receive epo had died . this epo treatment yielded 30 % tumor regression and slightly prolonged the survival of the epo - treated mice . in experiments ii and iii , the initial epo treatment ( its onset 13 or 11 days post s . c . tumor cell injection ) was prolonged to 10 consecutive days followed by 3 weekly injections for another 2 weeks . in experiment ii , the effect of two doses of epo ( 30u or 100u per injection ) was tested . the incidence of tumor progression in mice injected with the high dose of epo ( 100u ) was similar to that observed in the control group ( 14 % and 10 %, respectively ). in contrast , in mice treated with the lower dose of epo ( 30u per injection ), tumor regression was observed in 60 % of mice . in exp . iii tumor regression was observed in 40 % of mice treated with 30u epo . this tumor regression was fully established at the time epo treatment was terminated . since then , no tumor recurrence in the regressor mice was observed throughout the follow - up period ( 7½ w months in exp . ii and 3 - 4 months in exps . iii and iv ) despite of being off epo . it should be pointed out that spontaneous tumor regression in the control groups was usually between 0 - 10 %. in exp . iv various regimens were tested ( 10 , 20 or 30u per injection and 3 weekly injections for 4 weeks in comparison to 10 daily injections followed by additional 3 times weekly injections for 2 weeks ). significant tumor regression ( 50 %) was observed only when the treatment schedule of exp . ii and iii ( 30u per injection ) was followed . the same dose administered 3 times a week for 4 weeks was less effective ( only 18 % regression versus 9 % spontaneous regression in the control group ). the administration of lower epo doses ( 10u , 20u ) failed to interfere with tumor progression irrespective to time schedules of treatment . epo treatment had no effect on the rate of tumor progression in these mice that did not respond to epo treatment . it can thus be summarized that the optimal rhuepo treatment corresponds to exp . ii which involved daily subcutaneous injections of 30u for 10 consecutive days followed by 3 times a week injections of the same dose for 2 additional weeks . complete tumor regression was observed upon termination of epo treatment and no myeloma relapse was observed during a follow - up period of 7 ½ months . tumor progression in mice not responsive to epo treatment ( culminating in their death ) was also observed at the same time or shortly after termination of epo treatment ( no decreased tumor growth rate was observed in these mice ). the dynamics of tumor growth in progressors and regressors in individual mice in the experiments ii , iii and iv is described in fig1 - 3 , respectively . usually up to six days from epo administration ( 17 - 18 days since tumor cell challenge ) no variations in tumor growth rates among progressors or regressors were observed . by 12 - 15 days , tumor size clearly drops in mice responding to epo treatment , leading to final tumor regression , while in non - responders tumor size increases culminating in the death of the mice . fig4 shows the difference in the appearance of regressors ( a ) and progressors ( b ): tumor growth is clearly seen in b . myeloma is characterized by a clonal proliferation of bone marrow plasma cells secreting a paraprotein which can be detected in the serum , thereby serving as a tumor cell marker . the mouse myeloma mopc - 315 cells synthesize and secrete immunoglobulin iga with λ2 light chain and an α heavy chain . serum paraproteins in myeloma regressor and progressor mice and control mice were detected using coomassie brilliant blue staining of serum proteins resolved by sds - polyacrylamide gel electrophoresis . sera from myeloma regressor or progressor mice were collected after termination of epo treatment in comparison to sera from normal balb / c mice or balb / c mice treated with epo without previous tumor cell challenge . in the sera of both progressor and regressor mice a distinctive 27 kd band was observed ( much fainter in sera of control healthy mice ). we then analyzed sera of 40 mice injected with 10 4 mopc - 315 cells s . c . and after 12 days treated with either epo or epo diluting solution only . individual marked mice were bled twice ( from the orbital vein )— 17 days after tumor cell challenge , during the ongoing epo treatment and 2 weeks later , towards termination of epo treatment . at that stage we could clearly define regressor mice responsive to epo treatment versus non - responsive progressor mice and also some “ null ” mice — where tumor takes were not observed from start . sera from myeloma - bearing mice that did not receive further epo treatment ( including also few “ spontaneous ” regressors ) and sera from control mice were also included in this survey ( fig5 ). seventeen days after myeloma challenge we observed in all sera tested a prominent 27 kd band which was barely detected in sera of control mice that were not challenged with tumor cells . the deviation to progressors and regressors following epo treatment did not affect the presence of the 27 kd band in the sera . thus , the 27 kd band correlated only with the initial tumor cell injection into balb / c mice and not with disease severity . even in the serum of “ null ” mice the 27 kd protein band was observed . [ 0070 ] fig5 represents the profile of serum proteins in epo regressors ( lanes 1 - 2 ), epo progressors ( lanes 3 - 4 ), progressors in controls ( without epo treatment , lanes 5 - 6 ), a “ null ” mouse ( lane 7 ), a non - injected balb / c control mouse ( lane 8 ) and a balb / c mouse injected only with epo ( no myeloma cells , lane 9 ). we also tested sera from epo - treated regressor mice 2 , 4 and 7 months after tumor regression , throughout this period no signs of tumor relapse were observed and the mice looked grossly normal . tests of representative sera samples of these mice are illustrated in fig6 . all sera displayed the 27 kd protein , thereby suggesting the presence of “ dormant ” tumor cells in the myeloma regressor mice that continue to produce and secrete paraproteins . malignant cell arrest in different organs ( including spleen ) of mice bearing subcutaneous transplanted tumors including mopc - 315 was demonstrated years ago in the laboratory of one of the present inventors ( haran - ghera et al ., 1981 ). sequestration of tumor cells derived from the transplanted tumor was indicated already within 3 to 7 days after tumor graft , shortly before or after early palpable outgrowth of the primary tumor was observed . we therefore assumed that spleens from the regressor mice might be carriers of dormant mopc - 315 tumor cells . to test this possibility according to the present invention , we splenectomized regressor mice ( 4½ months after myeloma cell challenge ) and transplanted spleen cell suspension or whole spleens into syngeneic recipients . spleen suspensions from 7 individual regressors were prepared — half spleen cell suspension was injected i . v . into normal syngeneic balb / c mice and the other half into irradiated balb / c mice ( 2 hr following their exposure to 400 r whole body irradiation ). fifty and 75 days after spleen - cell transfer , 2 / 7 irradiated spleen cell recipient mice developed tumors and no tumors were observed in the normal recipients . the regressor spleen donors looked grossly normal 7 ½ months after the initial myeloma cell challenge . sera analysis of spleen cell recipients revealed presence of the 27 kd protein in all spleen cell recipients ( see fig7 ). in another experiment , whole spleens removed from 8 regressor mice were transplanted s . c . into normal recipients . 3 / 8 grafted spleens developed local tumors at the site of transplantation . these spleen recipients were bled 10 days and 3 weeks after spleen grafts and their sera were shown to be positive for the 27 kd protein band ( fig7 ). the 27 kd band which characteristically appears in sera of mice injected with mopc - 315 , probably represents the λ light chain of the iga immunoglobulin secreted by mopc - 315 myeloma cells . to confirm this identification , sera from 2 regressor epo - treated mice ( fig8 lanes 3 , 4 ), from a mouse grafted with a spleen from a regressor mouse and bled 10 days after grafting ( lane 1 ) and 3 weeks after grafting ( lane 2 ) ( both bleedings from the same mouse ), sera from control non - injected healthy mice ( lanes 5 , 6 ) and control iga secreted from mopc - 315 cells ( lane 7 ) were resolved in 10 % sds - page , and the gel was subsequently immunoblotted with anti - λ immunoglobulin light chain antibodies . as can be seen in fig8 shortly ( 10 days ) after grafting a healthy mouse with a spleen from a donor regressor mouse , the 27 kd band reacted with the anti - λ light chain antibodies ( lane 1 ) and the signal was more intense ( approximately 2 fold ) than that observed in the sera of control mice ( lanes 5 and 6 ). a significantly more intense 27 kd band was observed in the serum of this mouse 2 weeks later ( lane 2 ), indicating that the myeloma cells had proliferated and the amount of secreted iga increased ( similar levels to those in sera of epo - treated regressor mice ( lanes 3 - 4 ). the 27 kd band migrated similarly to the corresponding band observed in the lane of the control antibody from mopc - 315 iga ( lane 7 ), unequivocally establishing it as the immunoglobulin λ light chain . thus , according to these results , identification of paraproteins in sera of myeloma - bearing mice was found to be associated with the presence of tumor cells in these mice . a 27 kd protein band was observed in all mice challenged with myeloma cells irrespective to whether these cells ultimately progressed to lethality or regressed permanently . the 27 kd protein band was shown by western blot analysis with anti - λ immunoglobulin light chain antibodies to unequivocally correspond to the immunoglobulin λ light chain . the presence of “ dormant ” tumor cells in spleens of regressor mice was demonstrated by transplantation studies . paraproteinemia in sera of all spleen - cell recipients and the emerging of few tumors developing in spleen recipients clearly indicate that the course of the disease following epo treatment is under proliferation arrest ( in a “ dormant state ”). hb levels were measured in normal control mice as well as in myeloma - bearing mice with or without epo treatment , deviating into regressor and progressor mice ( fig9 ). hb level in normal balb / c mice ranged between 12 . 7 - 15 . 3 g / dl ( mean 13 . 6 ). normal mice treated with epo showed elevated hb levels ( 19 . 8 - 21 . 7 g / dl , mean 20 . 9 ). among regressors treated with epo , 7 / 9 mice tested showed elevated levels ranging from 17 - 21 . 5 g / dl ( mean 18 . 4 ), and 2 / 9 had lower levels , close to normal values ( 13 . 8 and 14 . 5 ). hb levels in 2 spontaneous regressors ( myeloma - bearing mice injected with diluent ) were 16 . 2 and 16 . 4 g / dl . in progressor mice treated with epo , in 9 / 11 tested blood samples ( taken from mice carrying a large tumor mass ) hb levels ranged from 11 . 5 - 13 . 4 g / dl ( mean 12 . 6 ) whereas in 2 / 11 progressors that carried a small tumor load the levels were 16 . 3 and 16 . 7 g / dl . in 2 progressors treated only with diluent the hb levels were 12 . 5 and 13 . 4 g / dl . thus , the response of mice to epo treatment is similar to that observed in humans . epo increases hb levels in normal mice . tumor regression following epo treatment is also associated with elevated hb , whereas in epo - treated myeloma progressors hb levels remain low . regressor mice resist rechallenge with second tumor of the same type the experiments described in examples 2 - 4 above indicate that administration of epo to tumor - bearing mice triggers immune responses that affect tumor regression . anti - tumor immune responses seem to contribute to tumor regression and maintenance of tumor dormancy . regressor mice ( being in this state for several months ) were rechallenged s . c . with 10 4 mopc - 315 cells and resisted this second tumor cell challenge in contrast to normal control mice that developed 100 % tumor takes 10 - 14 days after challenge ( not shown ). the specificity of this immunological triggered resistance was further demonstrated by challenging regressor mice bilaterally : on the left ventral surface with mopc - 315 cells , and on the right ventral surface with tumor cells from an unrelated myeloma ( mpc - 11 , induced in balb / c mice as described in materials and methods for mopc - 315 ). both tumors grew progressively in control balb / c mice . in contrast , the mopc - 315 growth was rejected in 7 / 7 regressor mice whereas mpc - 11 grew progressively in these same hosts . fig1 shows a control mouse bearing both mopc - 315 and mpc - 11 myeloma tumors ( left ) and a regressor mouse carrying only myeloma mpc - 11 tumor on the right ventral surface ( right ). these results indicate that treatment with epo promotes the development of an effective anti - tumor immune response and suggest that tumor regression induced by epo renders these mice resistant to growth of a second myeloma cell challenge of the same type , thereby suggesting the built - up of an anti - tumor immune response responsible for tumor rejection . further experiments were carried out with scid mice that are immunologically impaired ( both cell - mediated and humoral immune responses ) and with nude mice ( cell - mediated impaired immune response ), and both were found to be non - responders responders to epo treatment following 10 4 mopc - 315 cell challenge . in both the epo and control group , all scid and nude mice died at the same time , within 24 - 26 days post - tumor cell challenge ( fig1 - 12 ). in the control group ( normal balb / c mice ) epo treatment resulted in 30 - 40 % tumor regression . tumor regression in epo - treated tumor - bearing mice is mediated by cd8 + t - effector cells to further elucidate the phenotype of tumor - specific effector cells , we tested whether depletion of cd4 + or cd8 + t cells would abrogate the ability of mice to respond to epo treatment following tumor cell challenge . in mice treated with antibodies to cd8 + cells , thereby eradicating all cd8 + cells , no response to epo treatment was observed and tumor growth was actually enhanced ( not shown ). in mice treated with antibodies to cd4 + cells , thereby eradicating all cd4 + cells , less eradication of epo treatment on tumor regression was observed . in the control group , epo treatment caused tumor regression in 50 % of the treated mice ( fig1 ). these data indicate that epo treatment of tumor - bearing mice promotes the generation of thy - 1 + cd8 + t - effector cells that play a pivotal role in tumor regression in vivo in mice challenged with mopc - 315 myeloma tumor cells and further treated with epo . the following tumor cells can be injected in mice according to the protocol described in materials and methods for myeloma mopc - 315 : breast tumor using 107 - 202 adenocarcinoma cells , colon tumor using c - 26 colon carcinoma cells , lung tumor using m - 109 or 3ll lung carcinoma cells , chronic lymphocytic leukemia ( cll ) using bcl1 , melanoma k - 1735 , mca - 105 fibrosarcoma , 38c - 13 pre b cell leukemia , 127 - radlv t - lymphosarcoma , 17a - 443 - acute myeloid leukemia and 17e - 200 b - cell lymphoma . the tumor - bearing mice are then treated with epo as described in the previous examples . 1 ) c57b1 / 6 mice were challenged with lung adenocarcinoma 3ll cells , and thereafter treated with epo following the myeloma protocol , namely 10 daily injections of 30u epo s . c . followed optionally by three weekly injections for additional 4 weeks . as shown in fig1 , a delay was observed in the survival of the 3ll tumor - bearing mice : 50 % of the control mice died 54 days after the initial tumor cell challenge while 50 % of the 3ll tumor - bearing mice treated daily for 10 days with 30u epo survived 75 days . 2 ) balb / c mice were challenged s . c . with c - 26 colon carcinoma tumor cells . epo administration starting one day following tumor cell challenge prolonged markedly the survival of tumor - bearing mice ( 30 % survived for 100 days compared to 90 % death in the control group at 36 days ). epo administration starting on day 6 following tumor cell challenge actually enhanced tumor growth ( 90 % death at 23 days , as shown in fig1 ). our clinical observation suggests that at least in some myeloma patients epo also induces a longer and more stable course of the disease ( see example 1 , table 1 ). the possible effects of exogenous epo administration on the development of the disease was tested on a murine myeloma model , and showed indeed that mice challenged with a progressively growing myeloma and treated with epo for a relatively limited short period , exhibited complete tumor regression in 30 - 60 % of the treated mice using the optimal dose of epo regimen . compared effects of epo doses ( 5 - 100u ) on tumor growth rate , using similar injection schedules , revealed that there is a dose threshold : in mice responsive to exogenous epo administration , optimal tumor regression was achieved with a certain regimen ( 30u daily s . c . injections for at least 10 days , starting epo treatment when a small palpable tumor occurs ). the maintenance of tumor regression was independent on further epo administration . in mice shown to be non - responders to epo treatment , no effect on the progressive tumor growth rate was observed ; thus it appears that epo acts as an “ all or none ” factor . anemia associated with human cancer is at least partially due to a relative deficiency of epo and exogenous epo can correct the anemia . there is a correlation between baseline endogenous serum epo level and the response to rhuepo treatment . responders to epo have usually a low epo level ( below 200u / 1 ) in comparison to that of non - responders . thus , serum epo level above 200 u / 1 is a possible predictor for resistance to epo therapy . similarly , the therapeutical response to epo in mice involves rise in blood hb concentration ( fig9 ). in epo - responsive mice , tumor regression is observed along with increase in hb level , in contrast to unchanged levels of hb in mice unresponsive to epo treatment . myeloma is a well - suited study model , because it contains a continuously available measurable tumor marker with which to examine possible interactions between the basic disease and its associated anemia . in our patients responding to epo treatment , improvement from anemia coincided with a longer and stable course of disease in spite of the presence of serum myeloma paraproteins , indicating a dominantly stable tumor load . similarly , in sera of regressor mice we observed the 27 kd protein band that corresponds to the immunoglobulin λ light chain secreted by myeloma cells despite disappearance of the visible tumor . sequestration of myeloma cells to different organs in the regressor mice , including the spleen , has been demonstrated by transplantation studies . transfer of spleen cells into syngeneic normal or irradiated recipients resulted in myeloma development in some mice ; sera from all the spleen cell recipients contained the 27 kd protein band , thereby indicating the presence of “ dormant ” tumor cells in the myeloma regressor mice that continue to secrete paraproteins . thus , the course of the disease following epo treatment is under proliferation arrest ( in a dormant state ). our observations in the experimental model coincide with similar observations concerning the epo - treated patients ( see table 2 ). the bone marrow plasma cells and especially the myeloma proteins ( reflecting tumor mass ) did not disappear in the epo - treated patients , yet the course of the disease appears to be stabilized , “ frozen ” or become latent and asymptomatic . studies on the biological mechanisms involved in epo - triggered tumor regression according to the present invention suggest that anti - myeloma immunological reactivity is involved in tumor regression and maintenance of tumor dormancy . a series of experiments demonstrated that anti - tumor immune reactivity was associated with epo - induced regression of tumors . regressor mice rechallenged with the same tumor cells ( mopc - 315 ) resisted the growth of this second challenge . thus , memory to the tumor antigen was established in these regressors . the specificity of this immunological triggered resistance was also demonstrated ( fig1 ). the involvement of t cells in epo - triggered tumor regression was indicated by comparing the response of normal , scid or nude mice ( immunologically impaired ) to epo treatment following tumor cell challenge . the efficacy of epo treatment was markedly reduced in the immune impaired mice . both in scid and nude mice there was no response to epo treatment ( fig1 , 12 ). all epo - treated mice developed tumor in comparison to 40 % tumor regression in epo - treated normal control mice . these experiments rule out the possibility that epo has a direct cytotoxic or cytostatic effect since no effect was observed in the above described t - cell depleted mice . further studies concerned with the phenotype of the tumor effector cells indicated that the depletion of cd8 + t cells abolished the curative effect of epo . tumor regression in vivo is mediated by cd8 + effector cells and epo treatment of tumor - bearing mice seems to promote the generation of these effector cells thereby augmenting the generation of effective anti - tumor response . in preliminary tests , we observed an increase in the number of hematopoietic progenitor cells among bone marrow cells of epo - treated regressors as well as marked splenomegaly that might also reflect increase in progenitor cells . we assume that epo in vivo is not restricted to the erythroid lineage but induces a broad spectrum of primitive hematopoietic progenitor cells ( mostly primitive lin − , sca + , kit + and more committed lin − , sca − bone marrow progenitor cells ). thus , epo can be considered as an anti - 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