Patent Application: US-64038696-A

Abstract:
the use of interleukin - 12 to prevent , to ameliorate , and to treat graft - versus - host disease in a mammal in need of such treatment is disclosed . in other embodiments , graft - vs . leukemia effects are maintained or promoted .

Description:
the following abbreviations are used herein : bmc : bone marrow cells ; bmt : bone marrow transplantation ; gvl : graft - versus - leukemia ; gvhd : graft - versus - host disease ; ha : histocompatibility antigen ; mst : median survival time ; pea : phycoerythrin / streptavidin ; spc : spleen cells ; tcd : t cell depletion ; wbc : white blood cells ; wbi : whole body irradiation . the present inventors have surprisingly found that treatment of mammals subjected to allogeneic bone marrow transplantation with interleukin - 12 , with or without co - administration of t - cell depleted syngeneic marrow , results in prolonged survival of said mammals , a result which indicates that interleukin - 12 is useful for prevention of graft - versus - host disease in some cases and in amelioration of said disease in other cases . specifically , as set forth in example 1 below , mice which had been lethally irradiated and infused with bone marrow and spleen cells from fully mhc mismatched donor mice demonstrated prolonged survival from a course of interleukin - 12 prophylaxis . the present invention also demonstrates that il - 12 mediates distinct effects on gvhd - and gvl - associated t cell - mediated alloreactivity . in mice in which il - 12 mediated a significant protective effect against gvhd , marked gvl effects on allogeneic t cells against el4 were observed . gvl effects depended on alloreactivity , as il - 12 did not induce protection in recipients of syngeneic ( b 10 ) spleen cells . gvl effects against el4 leukemia were cd8 - dependent in il - 12 - treated mice . furthermore , applicants analyzed il - 12 - treated recipients of el4 cells and a / j spleen cells which survived for more than 100 days , and no el4 cells were detected in bone marrow , spleen , thymus , liver , kidneys or peripheral blood cells by flow cytometry and / or tissue culture . furthermore , no tumor - induced mortality was observed in lethally irradiated secondary b10 recipients of cd4 and cd8 cell - depleted spleen cells and marrow from these animals by 100 days of follow - up . thus , el4 cells were completely eradicated from il - 12 - treated leukemic recipients which received allogeneic spleen cells . applicants &# 39 ; previous data showed that il - 12 markedly increases ifn - γ production on days 2 and 3 post - bmt . the inhibitory effect on gvhd and the gvl effects against el4 were diminished in il - 12 treated mice by neutralizing anti - ifn - γ mab . therefore , il - 12 - mediated ifn - γ production plays a role in the protective effect of il - 12 against gvhd , and is involved in the gvl effect against el4 leukemia in il - 12 - treated mice . thus , applicants have demonstrated that gvhd and gvl are separable functions of allogeneic t cells , and that protection from gvhd and leukemic relapse can be provided by a single molecule . in accordance with the present invention , therefore , interleukin - 12 is defined as a heterodimeric glycoprotein comprised of two covalently linked subunits , one of said subunits having a molecular weight of about 40 kd and being characterized by the amino acid sequence set forth in seq id no : 1 / seq id no : 2 , and the other subunit having a molecular weight of about 35 kd and being characterized by the amino acid sequence set forth in seq id no : 3 / seq id no : 4 . any form of interleukin - 12 may be used as a component of the pharmaceutical composition used to practice the method of the invention , so long as that form of interleukin - 12 is capable of preventing , ameliorating , or treating graft - versus - host disease in a mammal at risk for that disease . for example , interleukin - 12 may be in the form of the heterodimer comprised of a 40 kd subunit disulfide - bonded to a 35 kd subunit . when interleukin - 12 is a heterodimer , the 40 kd subunit has substantial homology to the 40 kd subunit of human interleukin - 12 as set forth in seq id no : 1 / seq id no : 2 and is disulfide bonded to a 35 kd subunit having substantial homology to the 35 kd subunit of human interleukin - 12 as set forth in seq id no : 3 / seq id no : 4 . &# 34 ; substantial homology &# 34 ; means greater than 75 % homology at the amino acid level , while retaining the ability to preventing , ameliorating , or treating graft - versus - host disease in a mammal at risk for that disease . another form of interleukin - 12 which may be used in the present invention is an interleukin - 12 subunit capable of preventing , ameliorating , or treating graft - versus - host disease in a mammal at risk for that disease . such an interleukin - 12 40 kd subunit has substantial homology to the human interleukin - 12 40 kd subunit of seq id no : 1 / seq id no : 2 , and such an interleukin - 12 35 kd subunit has substantial homology to the human interleukin - 12 35 kd subunit of seq id no : 3 / seq id no : 4 . fragments of the interleukin - 12 subunits that retain interleukin - 12 biological activity are also be useful to prevent or treat graft - versus - host disease in a mammal at risk for that disease , in accordance with the present invention . for use in the present invention , it is preferable to produce interleukin - 12 recombinantly , through expression of dna sequences encoding one or both of the interleukin - 12 subunits in a suitable transformed host cell . for example , using known methods the dna sequences encoding human interleukin - 12 set forth in seq id no : 1 ( 40 kd subunit ) and seq id no : 3 ( 35 kd subunit ) may be linked to an expression vector such as ped ( kaufinan et al ., nucleic acids res . 19 , 4484 - 4490 ( 1991 )). in such an expression vector , sequences which optimize translation such as ccacc ( kozak , m ., nucleic acids res . 12 , 857 - 871 ( 1984 )) may be added 5 &# 39 ; to the initiation codon using known methods . the expression vector containing the interleukin - 12 subunits may then be transformed into a host cell , and protein expression may be induced and maximized , to produce heterodimeric human interleukin - 12 . for production of heterodimeric interleukin - 12 , the dna sequences encoding the interleukin - 12 subunits may be present on different expression plasmids or present in tandem on a single expression plasmid . when a subunit or fragment of interleukin - 12 is used to practice the present invention , it may also be produced recombinantly using known methods . for example , the dna sequence encoding the human interleukin - 12 40 kd subunit set forth in seq id no : 1 may be linked to an expression vector , transformed into a host cell , and expression induced and maximized to produce the human interleukin - 12 40 kd subunit . similarly , the dna sequences encoding the human interleukin - 12 35 kd subunit as set forth in seq id no : 3 may be linked to an expression vector , transformed into a host cell , and expression induced and maximized to produce the corresponding protein . of course , degenerate dna sequences encoding the interleukin - 12 subunits may also be employed to produce interleukin - 12 for use in the present invention , as can dna sequences encoding allelic variants of the interleukin - 12 subunits . any suitable expression vector may be employed to produce interleukin - 12 for use in the present invention . for mammalian expression , numerous expression vectors are known in addition to the ped vector mentioned above , such as pef - bos ( mizushima et al ., nucleic acids res . 18 , 5322 ( 1990 )); pxm , pjl3 and pjl4 ( gough et al ., embo j . 4 , 645 - 653 ( 1985 )); and pmt2 ( derived from pmt2 - vwf , a . t . c . c . # 67122 ; see pct / us87 / 00033 ). suitable expression vectors for use in yeast , insect , and bacterial cells are also known . construction and use of such expression vectors is well within the level of skill in the art . suitable host cells for recombinant production of interleukin - 12 useful in the present invention include , for example , mammalian cells such as chinese hamster ovary ( cho ) cells , monkey cos cells , mouse 3t3 cells , mouse l cells , myeloma cells such as nso ( galfre and milstein , methods in enzymology 73 , 3 - 46 ( 1981 )), baby hamster kidney cells , and the like . interleukin - 12 may also be produced by transformation of yeast , insect , and bacterial cells with dna sequences encoding the interleukin - 12 subunits , induction and amplification of protein expression , using known methods . recombinantly produced interleukin - 12 can be purified from culture medium or cell extracts by conventional purification techniques . culture medium or cell extracts containing interleukin - 12 may be concentrated using a commercially available protein concentration filter , for example , an amicon or millipore pellicon ultrafiltration unit . following the concentration step , the concentrate can be applied to a purification matrix such as a gel filtration medium . alternatively , an anion exchange resin can be employed , for example , a matrix or substrate having pendant diethylamioethyl ( deae ) groups . the matrices can be acrylamide , agarose , dextran , cellulose or other types commonly employed in protein purification . alternatively , a cation exchange step can be employed . suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups . the purification of interleukin - 12 from culture supernatant may also include one or more column steps over such affinity resins as lectin - agarose , heparin - toyopearl ® or cibacrom blue 3ga sepharose ®; or by hydrophobic interaction chromatography using such resins as phenyl ether , butyl ether , or propyl ether ; or by immunoaffinity chromatography . finally , one or more reverse - phase high performance liquid chromatography ( rp - hplc ) steps employing hydrophobic rp - hplc media , e . g ., silica gel having pendant methyl or other aliphatic groups , can be employed to further purify interleukin - 12 for use in the present methods and compositions . some or all of the foregoing purification steps , in various combinations , can be employed to provide a substantially homogeneous isolated recombinant protein . purification of interleukin - 12 subunits or fragments for use in the present invention may differ from the optimal protocol for purification of the heterodimeric protein . preferably , when human interleukin - 12 is produced recombinantly as set forth above , it may be purified by the following method . the cells in which the human interleukin - 12 has been made may be removed from the conditioned medium by filtration , and the conditioned medium is loaded onto q - sepharose fastflow ™ ( available from pharmacia ) or an equivalent anion exchange medium , which has been equilibrated in 10 - 30 mm tris - hcl , ph 7 . 8 - 8 . 3 . the column is then washed extensively with the same buffer followed by a wash with 30 - 45 mm histidine , ph 5 . 1 - 5 . 8 , followed by a wash with the original equilibration buffer . the recombinant human interleukin - 12 is eluted from the column with a buffer containing 20 - 50 mm tris - hcl , ph 7 . 8 - 8 . 5 , and 0 . 15 to 0 . 50m nacl . the eluted material is loaded onto cm - sepharose fastflow1υ ( available from pharmacia ) or equivalent cation exchange medium which has been equilibrated in 20 - 50 mm mes , ph 5 . 7 - 6 . 4 , and washed extensively with the same buffer . the column is washed with a buffer containing 20 - 40 mm sodium phosphate , ph 6 . 8 - 7 . 5 and 0 . 2 - 0 . 5 m nacl . the eluted material is concentrated using an amicon ™ s1y30 or equivalent spiral cartridge membrane which has been washed and equilibrated in the elution buffer used in the cm - seplarose fastflow ™ column . the material is concentrated to approximately 5 % of the column volume of the final chromatographic step , which is size exclusion using s200 sephacryl ™ ( available from pharmacia ) or an equivalent size exclusion resin . the size exclusion column is equilibrated and eluted with phosphate buffered saline , ph 7 . 2 - 7 , and the recombinant human interleukin - 12 peak is collected and filtered for use in the method of the invention . those of skill in the art of protein purification may use alternative purification methods to obtain recombinantly - produced human interleukin - 12 for use in the method of the invention . interleukin - 12 may be purified from culture medium or extracts of cells which naturally produce the protein and used in the present invention . exemplary purification schemes for naturally produced interleukin - 12 are set forth in pct / us91 / 06332 and in ep 433827 . for use in the method of the invention , a therapeutically effective amount of interleukin - 12 is administered to a mammal at risk of developing graft - versus - host disease . as used herein , the term therapeutically effective amount means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit , i . e ., a reduction in the incidence or severity of acute or chronic graft - versus - host disease compared to that expected for a comparable group of patients not receiving interleukin - 12 , as determined by the attending physician . when applied to an individual active ingredient administered alone , the term refers to that ingredient alone . when applied to a combination , the term refers to combined amounts of the active ingredients that result in the therapeutic effect , whether administered in combination , serially , or simultaneously . in practicing the method of the present invention , a therapeutically effective amount of interleukin - 12 is administered to a mammal at risk of developing graft - versus - host disease . the interleukin - 12 may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing t cell - depleted autologous or syngeneic bone marrow , immunosuppressive drugs , cytokines , lymphokines , or other hematopoietic factors . when co - administered with t - cell - depleted autologous or syngeneic bone marrow , immunosuppressive drugs , one or more cytokines , lymphokines , or other hematopoietic factors , the interleukin - 12 may be administered either simultaneously with the t - cell - depleted autologous or syngeneic bone marrow , immunosuppressive drugs , cytokine ( s ), lymphokine ( s ), other hematopoietic factor ( s ), or sequentially . if administered sequentially , the attending physician will decide on the appropriate sequence of administering the interleukin - 12 in combination with the t - cell depleted autologous or syngeneic bone marrow , immunosuppressive drugs , cytokine ( s ), lymphokine ( s ), and other hematopoietic factor ( s ). administration of the interleukin - 12 used to practice the method of the present invention can be carried out in a variety of conventional ways , such as oral ingestion , inhalation , or cutaneous , subcutaneous , or intravenous injection . intravenous or subcutaneous administration to the patient is preferred . when a therapeutically effective amount of interleukin - 12 is administered orally , the interleukin - 12 will be in the form of a tablet , capsule , powder , solution or elixir . when administered in tablet form , the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant . the tablet , capsule and powder contain from about five to 95 % interleukin - 12 , preferably from about 25 - 90 % interleukin - 12 . when administered in liquid form , a liquid carrier such as water , petroleum , oils of animal or plant origins such as peanut oil , mineral oil , soy bean oil , or sesame oil , or synthetic oils , may be added . the liquid form of the pharmaceutical composition may further contain physiological saline solution , dextrose , or other saccharide solutions , or glycols such as ethylene glycol , propylene glycol or polyethylene glycol . when administered in liquid form , the pharmaceutical composition contains about 0 . 5 to 90 % by weight of interleukin - 12 and preferably from about 1 to 50 % interleukin - 12 . when a therapeutically effective amount of interleukin - 12 is administered by intravenous , cutaneous or subcutaneous injection , the interleukin - 12 will be in the form a pyrogen - free , parenterally - acceptable aqueous solution . the preparation of such parenterally - acceptable protein solutions , having due regard to ph , isotonicity , stability , and the like , is within the skill in the art . a preferred pharmaceutical composition for intravenous , cutaneous , or subcutaneous injection should contain , in addition to interleukin - 12 , an isotonic vehicle such as sodium chloride injection , ringer &# 39 ; s injection , dextrose injection , dextrose and sodium chloride injection , lactated ringer &# 39 ; s injection , or other vehicle as known in the art . the pharmaceutical composition for use in the present method may also contain stabilizers , preservatives , buffers , antioxidants , or other additive known to those with skill in the art . it is contemplated that the pharmaceutical composition used to practice the method of the present invention should contain about 0 . 1 pg to about 100 mg of interleukin - 12 per ml of solution , preferably about 0 . 1 mg of interleukin - 12 per ml of solution . in practicing the method of preventing or ameliorating graft - versus - host disease in accordance with the present invention , it is contemplated that the duration of the application of interleukin - 12 will be in the range of 12 - 48 hours of continuous or intermittent subcutaneous or intravenous administration , beginning at the time of transplantation . for the purpose of the present invention , &# 34 ; at the time of bone marrow transplantation &# 34 ; is defined as being during the 1 hour period before or the 1 to 24 hour period after the bone marrow transplantation . as an example of a method for preventing or ameliorating graft - versus - host disease , preferably 1 μg / kg to 100 μg / kg of interleukin - 12 may be administered daily to the mammal , more preferably 5 ng / kg to 10 μg / kg of interleukin - 12 may be administered daily to the mammal , and most preferably 10 ng / kg to 1 jg / kg may be administered daily to the mammal . in one preferred dosage regimen , the first dose of interleukin - 12 is given one hour after bone marrow transplantation and two more doses are given on days one and two post - transplant . alternative treatment regimens may be appropriate for individual patients and will be determined by the attending physician , taking into account the nature and severity of the condition being treated , and the nature of the prior treatments which the patient has undergone . modifications of the treatment regimen set forth above for prevention or ameliorating graft - versus - host disease may be made for treatment of ongoing acute or chronic graft - versus - host disease . for the purpose of the present invention , &# 34 ; acute graft - versus - host disease &# 34 ; is defined as occurring during the time period from three days to 100 days post transplantation in humans or from three days to 30 days post transplantation in mice ; and &# 34 ; chronic graft - versus - host disease &# 34 ; is defined as occurring at any time after 100 days post - transplantation in humans or at any time after 30 days post transplantation in mice . as an example of a method for treating ongoing acute or chronic graft - versus - host disease , 1 pg / kg to 100 μg / kg may be administered daily to a mammal experiencing acute or chronic graft - versus - host disease , until improvement or remission of the symptoms of acute or chronic graft - versus - host disease is observed . ultimately , the attending physician will decide on the appropriate duration of subcutaneous or intravenous therapy using the pharmaceutical composition of interleukin - 12 in the method of the present invention . use of recombinant murine il - 12 for the inhibition of graft - versus - host disease ( gvhd ) in mice thirty c57b1 / 10 mice were lethally irradiated with 10 . 25 gy whole body irradiation . on the same day , 27 of these mice received an intravenous inoculum containing 9 × 10 6 bone marrow cells and 13 × 10 6 spleen cells ( as an additional source of gvhd - causing t lymphocytes ) from fully mhc - mismatched ( and multiple minor histocompatibility antigen - mismatched ) a / j donor mice . in addition , 18 of these mice received 5 × 10 6 b10 ( i . e ., host - type syngeneic , the murine counterpart of autologous marrow ) t cell - depleted ( tcd ) bone marrow cells in the same inoculum . the three remaining mice served as non - gvhd controls , and received t cell - depleted b10 marrow only . nine of the 18 mice receiving a / j bone marrow and spleen cells plus tcd b10 bmc were treated with recombinant murine interleukin - 12 ( schoenhaut et al ., j . immunol . 148 , 3433 - 3440 ( 1992 )) at a dose of 1 μg ( approximately 50 μg / kg ) per day intraperitoneally on days 0 , 1 , and 2 ( day 0 being the day of the transplant ). in addition , the group receiving a / j bone marrow and spleen cells alone also received a similar course of interleukin - 12 prophylaxis . the result of this experiment was that most of the mice ( 8 of 9 ) receiving a / j bone marrow and spleen cells plus tcd b10 bone marrow cells died by day 10 . the death was due to gvhd , as recipients of tcd syngeneic marrow alone all survived in excellent health . in the interleukin - 12 - treated group that also received a / j bone marrow and spleen cells plus tcd b10 bone marrow cells , none of the nine animals died by day 10 , and all were still alive by day 20 . this protective effect of interleukin - 12 was somewhat dependent on the co - administration of tcd b10 bone marrow cells , since 5 of 9 animals receiving a / j bone marrow and spleen cells without tcd b10 bmc , plus interleukin - 12 treatment , died by day 9 . thus , interleukin - 12 protected against acute gvhd mortality , and this effect was most marked when tcd host - type bone marrow cells were also given . in a second experiment , animals receiving interleukin - 12 prophylaxis against graft - versus - host disease induced by a / j bone marrow and spleen cells showed similar graft - versus - host disease protection , both in the presence and in the absence of t - cell - depleted host - type bone marrow . since il - 12 is not globally immunosuppressive and might even have anti - leukemic activity of its own , applicants have examined the possibility that il - 12 could preserve gvl effects of donor t cells in the el4 leukemia / lymphoma model , while gvhd is inhibited . applicants &# 39 ; results indicate that il - 12 separates the gvhd - and gvl - promoting activities of allogeneic t cells . marked gvl effects were observed in mice in which il - 12 mediated a significant protective effect against gvhd . applicants also analyzed the contributions of ifn - γ and allogeneic t - cell subsets to gvl effects in il - 12 - treated leukemic recipients . mice . specific pathogen - free female c57bl / losncr ( b10 , h - 2 b , k b i b d b ) and a / j ( h - 2a , k a i a d d ) mice were obtained from the frederick cancer research facility ( nih , bethesda , md .) animals were housed in sterilized microisolator cages and received autoclaved feed and autoclaved , acidified drinking water . whole body irradiation ( wbi ) and bmt . recipient mice were lethally irradiated ( 10 . 25 gy , 137 cs source , 1 . 1 gy / min ) and reconstituted within 4 to 8 hours by a single 1 ml intravenous inoculum containing 5 × 10 6 b 10 t cell - depleted ( tcd ) bmc plus 10 × 10 6 a / j bmc and 15 - 18 × 10 6 a / j spleen cells , or by 5 × 10 6 tcd b10 bmc with or without 15 - 18 × 10 6 b10 spleen cells ( syngeneic control group ). tcd b10 bmc was used because it enhances the il - 12 - mediated inhibition of gvhd , even though the host - type cells are eliminated within one week after bmt ( sykes et al ., blood 86 : 2429 , 1995 ). host - type ( b10 ) bmc and , in some experiments , allogeneic bmc and spleen cells , were depleted of t cells ( tcd ), or of cd4 + or cd8 + cells with anti - cd4 mab ( gk1 . 5 ascites ) ( dianlynas et al ., j . immunol . 131 : 2445 , 1983 ) and / or anti - cd8 mab ( 2 . 43 ascites ) ( sarmiento et al ., j . immunol . 125 : 2665 , 1980 ) plus low toxicity rabbit complement ( 1 : 14 dilution ), as previously described ( sykes et al ., j . immunol . 150 : 197 , 1993 ). t cell depletion was analyzed by flow cytometry using indirect staining as described ( sykes et al ., cel .. immunol . 127 : 260 , 1990 ). in adoptive bmt , secondary recipients ( b10 ) were lethally irradiated and reconstituted with cd4 and cd8 - depleted bmc and spleen cells prepared from long - term ( more than 100 days ) surviving non - leukemic ( as control ) and leukemic recipients of allogeneic bmt . these secondary recipients also received 5 × 10 6 fresh tcd b10 bmc . to avoid bias from cage - related effects , animals were randomized before and after bmt as described ( sykes et al ., j . exp . med . 171 : 645 , 1990 ). survival was followed for 100 days . il - 12 administration . murine recombinant il - 12 , with specific activity of 4 . 9 to 5 . 5 × 10 6 u / mg , was injected intraperitoneally into recipient mice ( 4 , 900 iu / mouse ) in a single injection approximately one hour prior to bmt . el4 leukemia experiments . the el4 leukemia model we have previously described ( sykes et al ., proc . natl . acad . sci . usa 87 : 5633 , 1990 ; sykes et al ., bone marrow transplant . 4 : 465 , 1989 ) was employed . e14f cells ( referred to here as el4 ), a subline of the b6 t cell leukemia / lymphoma el4 , were thawed from frozen vials and maintained in culture for 4 to 14 days before each experiment , and 500 cells were administered on day 0 along with bmc and spleen cells in a single 1 ml i . v . injection . carcasses were saved in formalin after death or euthanasia , and in some leukemic and non - leukemic mice , the spleen , liver , kidney and lung were embedded in paraffin , sectioned and stained with hematoxylin and eosin . necropsies and histologic analysis were performed on randomly chosen samples . the presence of tumor at death was determined by gross autopsy and / or histological observation by an observer who was unaware of which treatment group the carcasses belonged to , as previously described ( sykes et al . bone marrow transplant . 4 : 465 , 1989 ). flow cytometric ( fcm ) analysis . fcm analysis of peripheral white blood cells ( wbc ) was performed on a facscan ( becton dickinson , mountain view , calif .). wbc were prepared by hypotonic shock , as described ( tomita et al ., blood 83 : 939 , 1994 ). cells were stained with biotinylated anti - h - 2d d mab 34 - 2 - 12 ( ozato et al ., transplantation 34 : 113 , 1982 ), fitc - labeled rat anti - mouse cd4 mab gk1 . 5 ( dialynas et al ., j . immunol . 131 : 2445 , 1983 ) and rat anti - mouse cd8 mab 2 . 43 ( sarmiento et al ., j . immunol . 125 : 2665 , 1980 ) or fitc - labeled thy 1 . 2 for 30 min at 4 ° c ., then washed and incubated for 15 minutes at 4 ° c . with phycoerythrin / streptavidin ( pea ). in order to block non - specific fcγr binding of labeled antibodies , 10 μl of undiluted culture supernatant of 2 . 4g2 ( rat anti - mouse fcγr mab ) ( unkeless , j . exp . med . 150 : 580 , 1979 ) was added to the first incubation . mouse igg2a mab hopc - 1 was used as a non - staining negative control antibody . dead cells were excluded by gating out low forward scatter / high propidium iodide - retaining cells . anti - ifn - γmab administration . rat igg1 anti - mouse interferon - γ mab r4 - 6a2 ( spitalny et al ., j . exp . med . 159 : 1560 , 1984 ) was ammonium sulfate precipitated from ascites prepared in balb / c nude mice . antibody content was quantified using rat igg 1 - specific inhibition elisa . a single injection of 2 . 5 , 5 or 10 mg of r4 - 6a2 was administered on day 1 with respect to bmt . statistical analysis . survival data were analyzed using the kaplan - meier method of life table analysis , and statistical analysis was performed with the mantel - haenzsen test . a p value of less than 0 . 05 was considered to be significant . syngeneic spleen cells do not mediate anti - tumor activity in il - 12 - treated mice . the el4 h - 2 b leukemia administered as recently described is highly lethal , and as few as 100 cells are sufficient to kill lethally irradiated , syngeneically reconstituted h - 2 b mice . since il - 12 has been shown to promote anti - tumor immunity , applicants first investigated whether or not il - 12 could mediate anti - leukemia effects of its own , by promotion of host immunity . applicants compared tumor - induced mortality in control and il - 12 - treated b10 mice after syngeneic bmt . b10 mice were lethally irradiated , and injected with 500 el4 cells along with 5 × 10 6 tcd b10 bmc and 15 × 10 6 b10 spleen cells . as shown in fig1 a single dose of 4 , 900 iu of il - 12 had no effect on tumor - induced mortality . in both the control ( syn bmt + el4 ) and the il - 12 - treated ( syn bmt + el4 + il - 12 ) group , almost all mice were dead by 30 days post transplantation , and marked enlargement of the spleen and / or kidney was observed in 18 of 19 carcasses at autopsy . preservation of allogeneic gvl effects in il - 12 - treated mice . applicants next evaluation whether or not allogeneic gvl effects could be preserved while gvhd is inhibited by il - 12 in the el4 leukemia model ( sykes et al ., j . immunol . 150 : 197 , 1993 ). lethally irradiated b10 mice received a mixture of 5 × 10 6 syngeneic ( b10 ) tcd bmc , fully mhc plus multiple minor antigen - mismatched a / j bmc ( 10 × 10 6 ) and a / j spleen cells ( 15 - 18 × 10 6 ), plus 500 el4 cells . a syngeneic control group received the same number of b10 tcd bmc and spleen cells . the results are shown in fig2 . in non - leukemic recipients , a marked protective effect against gvhd was observed in el - 12 - treated animals . gvhd mortality was clearly inhibited in il - 12 - treated mice ( allo bmt + il - 12 ) when compared to untreated controls which received similar inocula ( allo bmt ) ( p & lt ; 0 . 01 ) ( fig2 a ). a similar level of protection from gvhd was also observed in il - 12 - treated leukemic recipients ( fig2 b ). in addition , administration of el4 did not result in a detectable increase in mortality in these mice , as survival curves were similar in groups receiving ( allo bmt + e4 + il - 12 ) or not receiving ( allo bmt + il - 12 ) el4 ( fig2 a and 2b ). allogeneic cells were necessary for an anti - leukemic effect in il - 12 - treated mice , as il - 12 treatment did not result in a detectable gvl effect in syngeneic controls in the same experiment ( fig2 b ). therefore , the gvl effect of allogeneic spleen cells was preserved while allogeneic spleen cell - mediated gvhd was inhibited by il - 12 . similar results were obtained in three of three separate similar experiments ( table 1 ). in experiment 1 , although the median survival time ( mst ) of the il - 12 treated gvhd group ( group 3 ) was delayed by only 8 days compared to the gvhd control group ( group 2 ), acute gvhd was clearly inhibited by il - 12 , as the mortality in il - 12 treated group was only 1 / 3 of that in il - 12 - untreated control group by 30 days post bmt . in experiments 2 and 3 , mst &# 39 ; s readily reflected the gvhd protection conferred by il - 12 treatment . autopsy analysis was performed in randomly selected carcasses without knowledge of which treatment the animals had received . as shown in table 1 , gross evidence for tumor , which was detected in almost all el4 recipients of b 10 tcd bmc and spleen cells , was not found in il - 12 - treated el4 recipients of b10 tcd bmc plus a / j bmc and spleen cells . leukemic infiltration of kidneys , liver , spleen or lung was readily apparent in the syngeneic bmt controls receiving el4 ( table 1 , groups 4 and 5 in experiment 1 ), but was not detected in il - 12 - protected recipients of allogeneic cells ( table 1 , group 6 in experiment 1 ) by histologic analysis . it was impossible to detect a gvl effect of allogeneic spleen cells in el4 recipients not receiving il - 12 , as most mice died of acute gvhd before el4 - induced death began in syngeneic control mice ( table 1 , experiment 3 ). el4 cells were eradicated from il - 12 - treated recipients of allogeneic spleen cells . inhibition of leukemic mortality in il - 12 - treated el4 recipients which received b10 tcd bmc plus a / j bmc and spleen cells suggested that leukemic growth was inhibited . we next evaluated whether or not el4 cells were completely eradicated by allogeneic cell - mediated gvl effects in these animals . bmc , thymocytes , spleen cells , peripheral blood cells and tissue fractions of liver and kidney were prepared from long - term surviving il - 12 - treated el4 recipients ( 9 mice from group 6 in experiments 1 and 2 ; table 1 ) when sacrificing them after 100 days of follow - up . these cells were cultured in vitro for one month , and no el4 cells grew in the cultures . thymocytes , spleen cells , bmc and peripheral white blood cells from these animals were also analyzed by two - color facs . since t cells in long - term surviving il - 12 - treated recipients are of donor - type ( a / j , h - 2d d ) ( illdstad et al ., j . immunol . 136 : 28 , 1986 ; sykes et al ., j . immunol . 141 : 2282 , 1988 ), and el4 cells are h - 2d 2 mab plus pea . no elm cells with the h - 2d d , thy1 . 2 + phenotype were detected , as all thy1 . 2 + cells were donor - type ( h - 2d d + ) in both non - leukemic and leukemic recipients of allogeneic bmt ( data not shown ). to further test for residual leukemic cells in il - 12 - protected recipients of el4 and allogeneic cells , we transferred tcd bmc and tcd spleen cells from these long - term ( more than 100 days ) surviving il 12 - treated el4 recipients ( 9 mice from group 6 in experiments 1 and 2 ; table 1 ) and non - leukemic recipients ( as controls ) ( 5 mice from group 3 in experiment 2 ; table 1 ), along with tcd b10 bmc , into lethally irradiated secondary b10 recipients ( one to two transfer ). the spleen is a major site of leukemia infiltration in this el4 model ( 25 ). potentially gvh - reactive a / j cd4 and cd8 t cells were eliminated from inocula , in order to allow any residual el4 cells the best opportunity to grow in secondary host - type recipients . mixed chimerism was observed in these secondary recipients by flow cytometric analysis ( fig3 ), indicating that gvh - reactive t cells were completely depleted from the spleens and marrow of the donor chimeras ( singer et al ., j . exp . med . 153 : 1286 , 1981 ; ildstad et al ., j . exp . med . 162 : 231 , 1985 ; ildstad et al ., nature 307 ( 5947 ): 168 , 1985 ). however , no leukemia - induced mortality was observed by 100 days of follow - up , and no gross evidence for tumor was detected by autopsy in any of 18 mice sacrificed 100 days following transfer . these results suggest that el4 leukemic cells had been completely eradicated from il - 12 - treated allogeneic bmt recipients . role of ifn - γin il - 12 - mediated gvhd protection . our previous results showed that il - 12 treatment markedly increases serum ifn - γ levels on days 2 and 3 post - bmt , and that the later , gvhd - associated rise in serum ifn - γ on day 4 is markedly inhibited ( sykes et al ., blood 86 : 2429 , 1995 ; szebeni et al ., transplantation 580 : 1385 , 1994 ). we therefore investigated the role of il - 12 - induced ifn - γ production in the inhibitory effect against gvhd and in gvl effects of allogeneic spleen cells in il - 12 - treated mice . on day 1 post - bmt , we injected 2 . 5 mg of r4 - 6a2 , a neutralizing rat - anti - mouse ifn - γ mab ( spitalny et al ., j . exp . med . 159 : 1560 , 1984 ) into il - 12 - treated b10 mice which received 5 × 10 6 b10 tcd bmc plus 10 × 10 6 a / j bmc and 15 × 10 6 a / j spleen cells . although gvhd - induced mortality was slightly delayed by r4 - 6a2 ( allo bmt + r4 + 6a2 ) compared to gvhd controls which received similar bmt inocula without r4 - 6a2 ( allo bmt ), the delay did not achieve statistical significance ( fig4 ). as usual , il - 12 treatment induced a significant delay in gvhd mortality ( allo bmt + il - 12 ) compared to gvhd controls ( allo bmt ) ( p & lt ; 0 . 005 ). this delay was markedly inhibited by r4 - 6a2 ( allo bmt + l - 12 + r4 - 6a2 ) ( p & lt ; 0 . 005 ) ( fig4 ), so that the time of mortality in the group receiving il - 12 and r4 - 6a2 ( allo bmt + l - 12 + r4 - 6a2 ) was similar to that of the group treated with r4 - 6a2 ( 2 . 5 , 5 , or 10 mg per mouse ), and similar results were observed ( data not shown ). we next investigated the possible role of ifn - γ in the gvl effects of allogeneic spleen cells in l - 12 - treated mice . as shown in table 2 , il - 12 - treated el4 recipients of tcd b10 bmc plus a / j bmc and spleen cells ( allo bmt , il - 12 , el4 ) were significantly protected from both gvhd - and leukemia - induced mortality when compared to gvhd control recipients of similar inocula without il - 12 ( allo bmt ) ( p & lt ; 0 . 01 ), and syngeneic leukemic recipients of b10 tcd bmc and el4 cells ( syn bmt , el4 ) ( p & lt ; 0 . 01 ), and syngeneic leukemic recipients of b10 tcd bmc and el4 cells ( syn bmt , el4 ) ( p & lt ; 0 . 01 ), respectively . the gvl effect of allogeneic cells was significantly reduced by injecting r4 - 6a2 on day one post - bmt . a significant acceleration in mortality ( p & lt ; 0 . 05 ) was detected in r4 - 6a2 - treated , il - 12 - treated el4 recipients ( allo bmt , il - 12 , r4 - 6a2 , el4 ) compared to il - 12 - treated leukemic allogeneic bmt controls ( allo bmt , il - 12 , el4 ). since r4 - 6a2 diminished the protective effect of il - 12 against gvhd in non - leukemic allogeneic bmt mice ( fig4 and tabled 2 ), this acceleration of mortality in el4 recipients could reflect a loss of gvhd protection , a loss of gvl effects , or both . however , autopsy and histological analysis indicated that the gvl effect in il - 12 - treated mice was partially impaired by r4 - 6a2 treatment , as gross evidence of leukemia , similar to that observed in syngeneic el4 controls ( syn bmt , el4 ) was detected in 4 / 9 of these animals ( allo bmt , il - 12 , r4 - 6a2 , el4 ) ( table 2 ), and leukemic infiltration of liver , kidney , or lung was observed in three of the five remaining mice . in contrast , no evidence for leukemia was detected at autopsy of il - 12 - protected recipients of similar inocula without r4 - 6a2 ( allo bmt , il - 12 , el4 ) ( table 2 ), and no leukemic infiltration of liver , kidney or lung was observed by histological analysis . adoptive bmt to test for residual leukemic cells was performed at day 64 post - bmt , by transferring cd4 and cd8 - depleted bmc and spleen cells from il - 12 - protected recipients of el4 and allogeneic cells ( 5 mice in group &# 34 ; allo bmt , il - 12 , el4 &# 34 ;, table 2 ), along with tcd host - type bmc into lethally irradiated , secondary recipients by 100 days of follow - up , and no gross evidence for leukemia was detected at autopsy when these mice were sacrificed . together , our results suggest that ifn - γ is involved in the protective effect of il - 12 against acute gvhd , and is also involved in the gvl effect of allogeneic cells against el4 leukemia in il - 12 treated mice . role of t cell subsets in gvl effects in il12 treated mice . since gvl effects against el4 leukemia in the model used in this study were cd8 - dependent and cd4 - independent in untreated recipients of allogeneic bmc and spleen cells ( sykes et al ., j . immunol . 150 : 197 , 1993 ), we next evaluated the contribution of donor t - cell subsets to gvl effects in il - 12 - treated el4 recipients . lethally irradiated b10 mice received 5 × 10 6 tcd b 10 bmc alone , or with 10 × 10 6 tcd a / j bmc plus 10 × 106 cd8 - depleted or 15 × 10 6 tcd a / j spleen cells . leukemic mice received similar inocula plus 500 el4 cells . since a reduced number of cd8 - depleted a / j spleen cells was given in this experiment , no gvhd - induced early mortality was observed by 50 days in recipients of cd8 - depleted a / j spleen cells ( fig5 ), although clinical manifestations of acute gvhd were observed . tumor - related mortality in both il - 12 - treated animals that received tcd a / j spleen cells or tcd b10 bmc ( fig5 ). in addition , gross evidence for leukemia was detected in all el4 recipients by autopsy ( data not shown ). similar rates of el4 - induced mortality were observed in il - 12 - treated and untreated recipients of cd4 - plus cd8 - depleted a / j spleen cells ( fig5 ), suggested that cd8 - independent nk cells were not directly involved in allogeneic spleen cell - mediated gvl effects . in contrast to these results , a significant gvl effect against el4 leukemia was observed in il - 12 - treated el4 recipients that received cd4 - depleted a / j spleen cells ( fig6 ). almost all mice in groups that received cd4 - depleted a / j spleen cells plus el4 cells survived longer than syngeneic leukemic controls ( fig6 ). gross evidence for leukemia was detected in 10 of 10 el4 recipients of b10 tcd bmc , but was only observed in one of 19 mice receiving cd4 - depleted a / j spleen cells - plus el4 cells ( all mice were pooled from two similar experiments ) by autopsy . furthermore , the mortality in groups receiving cd4 - depleted a / j spleen cells plus el4 was similar to that in non - leukemic mice which received similar bmt inocula without el4 , indicating that el4 - induced mortality was inhibited by giving cd4 - depleted a / j spleen cells . as cd 4 + cells are required for inducing acute gvhd in this model ( sykes et al ., j . immunol . 150 : 197 , 1993 ), no mice died of acute gvhd in either el - 12 - treated or untreated non - leukemic recipients of allogeneic cells in this experiment ( fig6 ). similar results were observed in the repeat experiment in which gvl effects were only observed in il - 12 - treated recipients of non - cd8 - depleted a / j spleen cells . since almost all early deaths are caused by acute gvhd in this model , we summarized autopsy results of mice that died beyond 12 days after bmt in this experiment ( table 3 ). gross evidence for leukemia was detected in all el4 recipients of cd8 - depleted or tcd a / j spleen cells , but only in one of 11 el4 recipients of cd4 - depleted a / ij spleen cells . together , the results in fig5 and 6 and table 3 indicate that gvl effects are cd8 - dependent and relatively cd4 - independent in both il - 12 - treated and control recipients . all literature and patent references cited herein are incorporated by reference as if fully set forth . table 1______________________________________il - 12 mediated gvl effects while gvhd is inhibited in el4 recipients tumor at autopsy ( no . with syngencic allogeneic tumor / group treatment cells cells mst total ( n ) el4 il - 12 bmc spc bmc spc ( days ) evaluated ) ______________________________________expt . 11 ( 6 ) - +/- + + - - & gt ; 100 0 / 62 ( 9 ) - - + - + + 41 0 / 73 ( 10 ) - + + - + + 49 0 / 54 ( 5 ) + - + + - - 18 4 / 45 ( 5 ) + + + + - - 19 5 / 56 ( 10 ) + + + - + + 50 0 / 5expt . 21 ( 6 ) - +/- + + - - & gt ; 100 0 / 62 ( 8 ) - - + - + + 36 0 / 43 ( 8 ) - + + - + + & gt ; 100 0 / 34 ( 5 ) + - + + - - 23 4 / 45 ( 5 ) + + + + - - 24 4 / 46 ( 9 ) + + + - + + & gt ; 100 0 / 6expt . 31 ( 6 ) - +/- + + - - & gt ; 100 0 / 62 ( 8 ) - - + - + + 7 0 / 33 ( 8 ) - + + - + + 44 0 / 44 ( 5 ) + - + + - - 21 4 / 55 ( 5 ) + + + + - - 19 4 / 46 ( 8 ) + - + - + + 8 0 / 5 * 7 ( 8 ) + + + - + + 51 0 / 4______________________________________ * all animals died of gvhd by 8 days post transplant . table 2______________________________________role of ifn - γ in the gvl effect of allogeneic spleen cellsin il - 12 - treated mice tumor at autopsy survival (%) ( no . with day day day tumor / totalgroup ( n ) 10 35 64 evaluated ) ______________________________________syn . bmt ( 3 ) 100 100 100 0 / 3syn . bmt , el4 ( 5 ) 100 0 0 5 / 5allo . bmt ( 8 ) 38 13 0 0 / 7allo . bmt , jl - 12 ( 7 ) 100 86 75 0 / 8allo . bmt , il - 12 , r46a2 * ( 8 ) 100 50 12 0 / 7allo . bmt , il - 12 , el4 ( 9 ) 100 100 56 0 / 8allo . bmt , il - 12 , r4 - 6a2 , el4 ( 9 ) 100 33 11 4 / 9______________________________________ * r4 - 6a2 : neutralizing rat antimouse ifnγ mab , 2 . 5 mg given i . p . on day 1 . table 3______________________________________autopsy analysis of el4 recipients reconstituted with cd8 - depletedand non - cd8 - depleted a / j spleen cells tumor at autopsybmt inocula ( no . with tumor / no . evaluated ) ______________________________________tcd b10 bmc ( syngeneic control ) 3 / 3tcd b10 bmc + tcd a / j bmc + 2 / 2cd8 - depleted a / j spctcd b10 bmc + tcd a / j bmc + 5 / 5cd8 - depleted a / j spc + il - 12tcd b10 bmc + tcd a / j bmc + 5 / 5tcd a / j spctcd b10 bmc + tcd a / j bmc + 6 / 6tcd a / j spc + il - 12tcd b10 bmc + tcd a / j bmc + 0 / 4cd4 - depleted a / j spctcd b10 bmc + tcd a / j bmc + 1 / 7cd4 - depleted a / j spc + il - 12______________________________________ *. all animals were lethally irradiated whine 4 - 8 hours before bmf , and injected with 500 el4 cells along with bmc and spleen cells . †. no . of cells administered : tcd b1o bmc : 5 × 10 6 ; tcd a / j bmc : 10 × 10 6 ; cd8 - depleted spc : 12 × 10 6 ; cd4 - depleted and tcd a / j spc : 15 × 10 6 . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 4 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 987 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( vi ) original source :( a ) organism : homo sapiens ( g ) cell type : lymphoblast ( h ) cell line : rpmi 8866 ( ix ) feature :( a ) name / key : cds ( b ) location : 1 .. 987 ( xi ) sequence description : seq id no : 1 : 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( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 328 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 2 : metcyshisglnglnleuvalilesertrppheserleuvalpheleu151015alaserproleuvalalailetrpgluleulyslysaspvaltyrval202530valgluleuasptrptyrproaspalaproglyglumetvalvalleu354045thrcysaspthrproglugluaspglyilethrtrpthrleuaspgln505560sersergluvalleuglyserglylysthrleuthrileglnvallys65707580glupheglyaspalaglyglntyrthrcyshislysglyglygluval859095leuserhisserleuleuleuleuhislyslysgluaspglyiletrp100105110serthraspileleulysaspglnlysgluprolysasnlysthrphe115120125leuargcysglualalysasntyrserglyargphethrcystrptrp130135140leuthrthrileserthraspleuthrpheservallysserserarg145150155160glyserseraspproglnglyvalthrcysglyalaalathrleuser165170175alagluargvalargglyaspasnlysglutyrglutyrservalglu180185190cysglngluaspseralacysproalaalaglugluserleuproile195200205gluvalmetvalaspalavalhislysleulystyrgluasntyrthr210215220serserphepheileargaspileilelysproaspproprolysasn225230235240leuglnleulysproleulysasnserargglnvalgluvalsertrp245250255glutyrproaspthrtrpserthrprohissertyrpheserleuthr260265270phecysvalglnvalglnglylysserlysargglulyslysasparg275280285valphethrasplysthrseralathrvalilecysarglysasnala290295300serileservalargalaglnaspargtyrtyrsersersertrpser305310315320glutrpalaservalprocysser325 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 660 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( vi ) original source :( a ) organism : homo sapiens ( g ) cell type : lymphoblast ( h ) cell line : rpmi 8866 ( ix ) feature :( a ) name / key : cds ( b ) location : 1 .. 660 ( xi ) sequence description : seq id no : 3 : atgtgtccagcgcgcagcctcctccttgtggctaccctggtcctcctg48metcysproalaargserleuleuleuvalalathrleuvalleuleu151015gaccacctcagtttggccagaaacctccccgtggccactccagaccca96asphisleuserleualaargasnleuprovalalathrproasppro202530ggaatgttcccatgccttcaccactcccaaaacctgctgagggccgtc144glymetpheprocysleuhishisserglnasnleuleuargalaval354045agcaacatgctccagaaggccagacaaactctagaattttacccttgc192serasnmetleuglnlysalaargglnthrleugluphetyrprocys505560acttctgaagagattgatcatgaagatatcacaaaagataaaaccagc240thrserglugluileasphisgluaspilethrlysasplysthrser65707580acagtggaggcctgtttaccattggaattaaccaagaatgagagttgc288thrvalglualacysleuproleugluleuthrlysasnglusercys859095ctaaattccagagagacctctttcataactaatgggagttgcctggcc336leuasnserarggluthrserpheilethrasnglysercysleuala100105110tccagaaagacctcttttatgatggccctgtgccttagtagtatttat384serarglysthrserphemetmetalaleucysleuserseriletyr115120125gaagacttgaagatgtaccaggtggagttcaagaccatgaatgcaaag432gluaspleulysmettyrglnvalgluphelysthrmetasnalalys130135140cttctgatggatcctaagaggcagatctttctagatcaaaacatgctg480leuleumetaspprolysargglnilepheleuaspglnasnmetleu145150155160gcagttattgatgagctgatgcaggccctgaatttcaacagtgagact528alavalileaspgluleumetglnalaleuasnpheasnsergluthr165170175gtgccacaaaaatcctcccttgaagaaccggatttttataaaactaaa576valproglnlysserserleuglugluproaspphetyrlysthrlys180185190atcaagctctgcatacttcttcatgctttcagaattcgggcagtgact624ilelysleucysileleuleuhisalapheargileargalavalthr195200205attgatagagtgatgagctatctgaatgcttcctaa660ileaspargvalmetsertyrleuasnalaser210215220 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 219 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 4 : metcysproalaargserleuleuleuvalalathrleuvalleuleu151015asphisleuserleualaargasnleuprovalalathrproasppro202530glymetpheprocysleuhishisserglnasnleuleuargalaval354045serasnmetleuglnlysalaargglnthrleugluphetyrprocys505560thrserglugluileasphisgluaspilethrlysasplysthrser65707580thrvalglualacysleuproleugluleuthrlysasnglusercys859095leuasnserarggluthrserpheilethrasnglysercysleuala100105110serarglysthrserphemetmetalaleucysleuserseriletyr115120125gluaspleulysmettyrglnvalgluphelysthrmetasnalalys130135140leuleumetaspprolysargglnilepheleuaspglnasnmetleu145150155160alavalileaspgluleumetglnalaleuasnpheasnsergluthr165170175valproglnlysserserleuglugluproaspphetyrlysthrlys180185190ilelysleucysileleuleuhisalapheargileargalavalthr195200205ileaspargvalmetsertyrleuasnalaser210215__________________________________________________________________________