Patent Application: US-76523410-A

Abstract:
this invention is directed to therapeutic compositions containing non - mhc - restricted tcells / nk - cells in combination with mhc - restricted t - cells and especially to therapeutic compositions , which comprise lak cells . furthermore , the present invention is directed to the use of the above combination in the treatment of tumors in humans , which tumors show a missing , low or aberrant expression of mhc class ia or ib molecules . by using the aforementioned compositions / combinations it is possible to provide a balanced selective pressure against emergence of tumor cell variants that would otherwise escape immune detection .

Description:
in the following , there is a detailed description of the present invention provided . the basic principles underlying the present invention are illustrated by a special kind of non - mhc restricted cells , i . e . by lak cells . however , it is noted that the scope of the present invention is not limited to lak cells and their use . lak cells from rcc patients and healthy donors recognize mhc class i negative target cells and hla class i positive tumor lines lak cells generated from pbmc of patients with rcc and healthy donors were tested for cytotoxic activity directed against mhc class i negative target cells and various tumor cell lines . fig1 summarizes composite results of several independent experiments in which the hla class i negative target cells l721 . 221 , k562 and daudi were efficiently lysed by four different lak populations . no substantial differences in levels of cytotoxic activity were observed between lak - 26 and lak - 53 , derived from two rcc patients ( fig1 a ) and lak - cp176 and lak - cp41 , derived from two healthy control donors ( fig1 b ). the patient - derived lak - 26 were able to lyse the autologous rcc line ( rcc - 26 ) and , in addition , they could recognize various allogeneic rcc lines ( skrc ) as well as the mel - 25 melanoma line , independent of hla background ( fig1 c ). the control donor - derived lak - cp176 line lysed these various tumor cell lines in a similar fashion ( fig1 d ). all of these tumor lines were shown to be class i positive through binding of the class i - specific mab , w6 / 32 ( data not shown ). these results demonstrated that the lak populations were able to recognize target cells in a non - mhc - restricted manner and lysis was not specific for any single tumor entity . lak populations are composed primarily of cd4 + and cd8 + t cells phenotype analyses of lymphocyte subsets were made of the expanded lak cultures derived from the four different donors utilized in these studies . table 1 shows the distribution of cd3 − cd56 + nk cells and cd3 + cd56 − t cells in these lak populations . differences between lak cells derived from rcc patients and healthy donors were not evident : cd3 + t cells were the major cell type , representing more than 95 % of the cells in all samples . while the lak - cp176 culture was dominated by cd8 + t cells , the other lak samples contained approximately equal numbers of cd4 + and cd8 + t cells . in contrast , cd3 − cd56 + nk cells were present in only very low numbers , ranging from 0 - 6 % of total cells . these results revealed that the culture conditions used for generation and maintenance of these lak cells led primarily to expansion of the t cell fraction of lymphocytes rather than the adherent fraction of nk cells . the phenotype of these lak cell lines indicated that the major cytolytic component was attributable to activated t cells . in fact , depletion of the small remaining fraction of nk cells from the mixed lak populations did not alter their cytotoxic potential or specificity ( data not shown ). mhc class i enhancement is associated with ifnγ - mediated inhibition of lak - t cells and activated nk cells it has been shown previously that ifnγ treatment of tumor cells can lead to resistance to purified nk cells and to lak cells ( 29 ; 30 )). to determine whether mhc expression could also affect tumor cell sensitivity to lak - t cells , rcc - 26 cells were analyzed after enhancement of their class i expression through exogenous ifnγ stimulation . alternatively , the rcc - 26 line was transduced with human ifnγ cdna , leading to endogenous cytokine production and subsequent upregulation of class i expression ( 31 ). susceptibility of these different ifnγ - modulated rcc - 26 cells to lysis was assessed using autologous lak - 26 t cells ( fig2 a - c ). purified b . 3nk cells which were shown previously to exclusively express p58 . 2 receptors of the kir family that bind hla - c molecules of the cw1 , 3 , 7 subgroup were included for comparison ( fig2 d - f ). unmodified rcc - 26 cells and the control line carrying empty vector ( rcc - 26vc ) were lysed by both effector cell types , whereby the activated nk cells showed a stronger lytic capacity . following stimulation with exogenous ifnγ both tumor lines displayed substantial resistance to lysis by both lak - t and nk cells ( fig2 a , d ). a similar degree of resistance was observed with one ifnγ transductant ( endoγ1 ) whereas the second transductant ( endoγ2 ) showed only partial resistance to both effector populations ( fig2 b , e ). however , further stimulation with ifnγ led to an increased resistance of this transductant to both lak - 26 t cells and b . 3nk cells ( fig2 c , f ). this resistance was comparable to that induced by exogenous ifnγ stimulation of rcc - 26 and rcc - 26vc cells . in parallel studies , the lak - 53 , lak - cp41 and lak - cp176 t cells showed cytotoxic patterns like those of lak - 26 t cells and b . 3nk cells , demonstrating a general correlation between ifnγ - induced effects and resistance to lak - derived cytotoxic t cells ( data not shown ). the decrease in lak - t and control nk activity following interaction with the various target cells was consistently seen in all experiments in which increases in class i expression were detected by flow cytometry . fig2 g shows representative results of one of numerous experiments analyzing the enhancement of mhc class i expression by ifnγ in rcc - 26 cells . even though rcc - 26 and rcc - 26vc cells constitutively expressed substantial levels of class i molecules , expression was increased following ifnγ stimulation , as detected by staining with the class i - specific mab , w6 / 32 . while the total levels of mhc expression varied among the different cell lines , these were reproducible and apparently reflected variations in the responsiveness of the individual lines to ifnγ induction . interestingly , the endoγ2 line displayed , lower constitutive levels of mhc molecules when compared to endoγ1 cells . this transductant was also more susceptible to lak - t - and nk - mediated cytotoxicity . however , stimulation of endoγ2 cells with exogenous ifnγ led to enhanced class i expression ( data not shown ) and increased resistance to both effector cell types ( see fig2 c , f ). in additional experiments it was shown that other tumor cell lines acquired resistance to lak - t cells following their treatment with ifnγ ( data not shown ). the availability of a cell line derived from normal kidney parenchyma of patient 26 ( nkc - 26 ) ( 32 ) allowed assessment of whether the ifnγ - induced inhibition of lak - t and nk cytotoxicity was restricted to tumor cells or also affected lysis of normal epithelial cells . nkc - 26 cells were lysed by autologous lak - 26 t cells ( fig3 a ) and allogeneic b . 3nk cells ( fig3 b ), demonstrating that these effector cells were not tumor specific . as seen with rcc - 26 cells , exogenous ifnγ stimulation of nkc - 26 cells resulted in substantial inhibition of cytotoxicity by both lak - t cells and activated nk cells , revealing that ifnγ - induced resistance was also not tumor specific . in studies not shown it was found that allogeneic lak - t cells could lyse the nkc - 26 line and that ifnγ stimulation led to its partial resistance , indicating that this effect was not limited to autologous lak - t cells . parallel studies analyzing class i expression following ifnγ stimulation showed increased binding of w6 / 32 antibody by both rcc - 26 ( fig3 c ) and nkc - 26 cells ( fig3 d ). likewise , the levels of hla - c molecules which serve as the ligands for the p58 . 2 inhibitory receptors that govern the activity of b . 3nk cells were found to be increased on both cell lines using mab l31 ( fig3 e , f ). the weak staining pattern observed with mab l31 may be explained by its preferential reactivity with β 2 m - free heavy chains of hla - c molecules ( 33 ). since ifnγ regulates expression of many different genes , functional inhibition studies using mab to block class i ligands on target cells were used to demonstrate that these molecules directly contributed to the downmodulation of lak - t and nk activity . preincubation of ifnγ - stimulated rcc - 26 or nkc - 26 cells with w6 / 32 mab led to reversal of inhibition of both effector populations ( fig3 a , b ). thus , resistance of normal epithelial cells and tumor cells was mediated by class i molecules and antibody masking of class i surface expression could restore susceptibility of both target cells to lysis . the class i - dependent inhibition of lak - t cytotoxcity was confirmed by extended blocking studies summarized in table 2 . cytotoxic activities of lak - t cells ( lak - 26 , lak - cp41 ) and b . 3nk cells against various target cells were analyzed in the presence of w6 / 32 or isotype control mab . preincubation of unstimulated rcc - 26 and rcc - 26vc cells with w6 / 32 mab led to small increases in lysis by the different effector cells when compared to isotype controls . addition of w6 / 32 mab led to substantial reversal of the inhibition of lysis seen with the ifnγ - stimulated rcc - 26 and rcc - 26vc cells . moreover , lysis of both ifnγ - expressing transductants was substantially increased in the presence of w6 / 32 mab the consistently improved cytotoxicity achieved through masking of class i molecules was also observed with a second rcc line and a corresponding ifnγ transductant ( data not shown ). these results demonstrated that class i molecules contributed directly to the ifnγ - mediated inhibition of lak - t cells and activated nk cells . since lak - t cells efficiently lysed class i negative target cells we investigated whether expression of class i molecules in such cells could directly inhibit lak - t activity . the class i negative cell line l721 . 221 was derived from a class i positive lymphoblastoid cell line ( l721 ) by irradiation induced mutagenesis with selection for sequential hla loss variants . the l721 . 112 line represents a hemizygous cell line generated in this series which still expresses one of the two parental haplotypes ( hla - a1 , b8 , cw7 ). while l721 . 221 cells were very sensitive to lak - 26 t cell - mediated lysis , the expression of class i molecules by l721 . 112 cells provided partial protection ( fig4 a ). the daudi cell line does not produce β 2 m and thereby does not express stable class i molecules at the cell surface . transfection of the β 2 m gene into daudi cells reconstituted their expression of class i molecules ( 28 ) which was confirmed by staining with w6 / 32 mab ( data not shown ). the unmodified cell line was efficiently lysed by lak - 26 t cells whereas transfectant cells showed substantial resistance ( fig4 b ). it was also found that the k562 cells became partially resistant to lak - 26 t cell - mediated lysis following transfection with the class ib gene encoding hla - e molecules ( fig4 c ). l721 . 221 cells that were genetically modified to express 835 , hla - cw6 and hla - cw7 molecules were also analyzed as target cells for lak - 26 t cells . while no reproducible inhibition was seen by hla - b35 or hla - cw7 molecules , partial resistance ( 25 %) was induced by hla - cw6 expression ( fig4 d ). these results confirmed that class i molecules alone could directly inhibit lak - 26 t cell cytotoxicity . insight into the fine specificity of mhc inhibition was also obtained for other lak - t cells ( lak - cp41 and lak - cp176 ) since it was possible to specifically inhibit their lysis of l721 . 221 cells by more than 50 % through expression of hla - cw6 ( fig4 e , f ). in contrast , expression of hla - g , - b35 and - b27 and - cw7 molecules in these cells did not induce resistance to lak - t cells . an inhibitory influence of hla - e molecules appeared unlikely since the signal peptide of hla - g can bind to hla - e molecules , allowing their stable surface expression ( 34 ). therefore the l721 . 221 - g cells can simultaneously express both hla - g and hla - e molecules . because these cells did not cause inhibition it appears that hla - e played no regulatory role in these two lak - t cell populations . lak - derived cd4 t cells are also negatively regulated by mhc molecules enrichment of cd4 + lymphocytes was made by depleting the cd8 + cells from a mixed lak - t cell population . to confirm directly that these t cells were cytolytic and their activity could be downregulated by exposure to ifnγ - modulated tumor cells , the cd4 - selected t cells ( fig5 a left ) were compared to the unseparated lak - 26 t cell population containing both cd4 and cd8 cells ( fig5 a , right ). the highly purified cd4 + t cells were able to lyse rcc - 26 , nkc - 26 and k562 target cells in a manner analogous to the unseparated lak - t population ( fig5 b and c ). furthermore , ifnγ - modulation ( exogenous stimulation or endogenous expression ) of rcc - 26 and nkc - 26 cells led to inhibition of lysis of the purified cd4 + t cells in a similar fashion to the unseparated population of lak - t cells . the demonstration that the lak - t cells were regulated by class i - mediated inhibition suggested that they might carry inhibitory receptors like those expressed by nk cells . therefore , surface phenotyping to identify such receptors was made using mab specific for several known inhibitory receptors that interact with hla - b , c or e molecules . as summarized in table 3 , inhibitory receptors belonging to the kir immunoglobulin superfamily ( p58 . 1 , p58 . 2 or nkb1 ) were not present on the t cells . small percentages of both cd3 + and cd3 − cells ( 3 - 7 %) expressed cd94 molecules . in nk cells it has been found that cd94 associates with the nkg2a coreceptor molecule to create an hla - e - specific heterodimeric inhibitory receptor . because k562 cells expressing hla - e substantially inhibited lak - 26 t cell - mediated cytotoxicity ( see fig4 c ), we expected to find t cells bearing such receptors in this population . however , less than 1 % of lak - 26 t cells bound nkg2a antibody ( data not shown ). likewise , because lak - 26 , lak - cp41 and lak - cp176 were partially inhibited by hla - cw6 molecules ( see fig4 d - f ), some t cells expressing p58 . 1 receptors were expected , but were also not found in these populations . thus , the known inhibitory receptors governing nk cell inhibition by hla - c and hla - e ligands were not expressed to any appreciable degree by lak - t cells , even though the specific class i ligands characteristic for these receptors were apparently delivering inhibitory signals to these t cells . the following examples are set forth for illustrative purposes and should not be considered as limiting the scope of protection of the present invention . blood samples are obtained using either heparin ( 50 units / ml ) ( for example : heparin - natrium , braun melsungen ag ) or defibrination to prevent coagulation . to obtain larger numbers of pbmc an apheresis can be performed to obtain the equivalent of 1 - 4 liters of blood ( methods for blood preparation are described in ( 35 ). peripheral blood mononuclear cells ( pbmc ) are isolated by ficoll / hypaque ( for example : biocoll separating solution , biochrom ag ) density gradient centrifugation using standard methods ( 35 ). pbmc are washed 2 × in phosphate buffered saline ( pbs ) and cultured directly or cryopreserved for future use . for cryopreservation , pmbc are frozen at concentrations of 5 - 20 × 10 6 cells / ml in 1 ml aliquots in rpmi culture medium containing 25 - 50 % autologous serum and 10 % dmso in rpmi 1640 medium ( for example : rpmi 1640 medium , fa . invitrogen ) and stored in the gas phase of a liquid nitrogen tank . cryopreserved pbmc are thawed by adding rpmi medium containing 50 % human serum dropwise to a partially thawed cell sample . following transfer into a plastic centrifuge tube the volume of medium is increased to 3 - 5 ml and the cells are washed 2 × ( 35 ). freshly isolated pbmc or thawed pbmc are resuspended after the 2nd wash in “ lak ” medium at a density of 1 × 10 6 cells / ml . a ) rpmi culture medium containing 15 % human serum and 1000 units / ml of recombinant human interleukin 2 ( il - 2 ) ( for example proleukin , cetus , emeryville calif ., usa ) b ) rpmi culture medium containing 15 % human serum and 1000 units / ml il - 2 and 1 % ( vol / vol ) of phytohemagglutinin ( for example : difco laboratories , detroit mich ., usa ) pbmc are cultured in 50 ml culture flasks ( for example falcon , becton - dickinson , san jose , calif ., usa ) standing upright containing a volume of 4 - 15 ml at 37 ° c . and 5 % co 2 for 72 - 96 hours . thereafter , the cultures are counted and cell densities readjusted to 1 × 10 6 cells / ml using lak medium without pha . when the volume of a culture reaches & gt ; 15 ml it is divided between two flasks and cultures continued under the same conditions . larger numbers of cells can be obtained by scaling up the volume of the cultures into 250 ml culture flasks ( falcon , becton - dickinson , san jose , calif ., usa ) (& gt ; 45 - 150 ml / flask ). if enough pbmc are available ( for example derived from an apheresis ) several 250 ml flasks can be cultured simultaneously up to high cell numbers such as 10 9 cells . at the completion of the culture period the activated cells can be phenotyped for lymphocyte subpopulations to determine the composition of nk and lak - t cells . it is our experience that after two to three weeks of culture the populations are predominantly lak - t cells . functional assessment of their cytolytic potential can be made using a standard chromium release assay using daudi and k562 cell lines as target cells ( 27 ). activated lak cells can be cryopreserved for future use using the same freezing protocol as described above or applied directly after completion of the culture phase . tumorspecific ctl can be generated by different approaches using either fresh tumor material or known , recombinant tumor antigens , respectively , depending on tumor entity and availability of tissue material . in the rare cases where an in vitro tumor line is available , ctl can be generated by stimulation of pbl against irradiated tumor cells . briefly , tumor cells were grown in 24 - well plates and , following irradiation of the tumor cells , autologous pbl were added in human serum containing medium , optionally supplemented with il - 2 and / or il - 4 . every 7 to 10 days , t cells were harvested and again restimulated with irradiated tumor cell cultures . after several rounds of restimulation , the activity and specificity of the mixed ctl populations were tested using standard chromium release , elispot , or cytokine secretion assays . alternatively , if no tumor line is available , a lysate of tumor tissue can be used in vitro to pulse dendritic cells ( dc ) in order to promote the presentation of specific peptides derived from tumor antigens by these professional antigen presenting cells ( apc ). with respect to known tumor antigens , in the case of the malignant melanoma , for instance , synthetic peptides or recombinant protein fragments derived from known melanoma antigens are used to pulse dc . another principle of tumor antigen presentation by dc is based on the generation of tumor - derived rna which can be used to directly pulse dc by endogenous expression of tumor - rna . the procedures for restimulation and for the analysis of activation and specificity of mixed ctl populations are described above , i . e . standard chromium release , elispot , or cytokine secretion assays . independent of the procedure for ctl generation , most approaches must deal with similar limitations such as very low frequency of tumor - specific ctl precursors in the blood , time consuming and laborous production of tumor - specific ctl and the availability of individual tumor material or common tumor antigens . pbmc obtained from rcc patients undergoing tumor nephrectomy or from healthy donors were used for the generation of lak cells by culture in rpmi 1640 medium supplemented with 2 mm l - glutamine , 1 mm pyruvate , 100 u / ml penicillin / streptomycin ( complete medium ), 15 % heat - inactivated , pooled human serum , 1 % phytohemagglutinin ( pha , difco - laboratories , detroit , mc ) and 1000 u / ml ril - 2 ( proleukin , cetus corp . emeryville , calif .). these cultures were maintained over several weeks in order to obtain expanded populations of activated cd4 + and cd8 + t cells . lak - derived cd4 + t cells were enriched by magnetic bead separation ( dynal , oslo , norway ) by depleting cd8 + t and nk cells according to the manufacturer &# 39 ; s instructions . human nk cells were activated as described previously and purified nk cells were obtained by depleting t cells using cd4 - and cd3 - coated immunomagnetic beads ( dynal ). the enriched nk cell line used in these studies , designated as b . 3nk cells , was maintained in complete medium supplemented with ril - 2 ( 300 u / ml ). cytotoxicity of these effector cells was assessed using epstein - barr virus transformed lymphoblastoid cell lines ( lcl ) derived from the l721 cell line . the l721 . 112 cell line represents a hemizygous variant of l721 that expresses the a1 , cw7 , b8 haplotype ( kindly provided by t . spies , fred hutchinson cancer research center , seattle ). the l721 . 221 cell line does not express any mhc class i molecules ( 36 ). this line was transfected to express the class i alleles , b * 3501 , b * 3702 , cw * 0602 , and cw * 0702 , as described . an hla - e transfectant of k562 was generated by transfecting hybrid dna cloned into the pcdna3 vector encoding exon 1 of hla - a2 and exons 2 - 7 of hla - e . this construct allows hla - e surface expression by stabilization of the hla - e heavy chain with an hla - a2 - derived peptide . daudi cells transfected with the gene encoding beta - 2 - microglobulin ( β 2 m ) ( 28 ) were kindly provided by p . parham ( stanford university , palo alto , calif .). the rcc - 26 tumor line was established from a primary stage i ( t1 , g2 , n0 , m0 ) tumor of patient 26 ( hla alleles : a * 0201 , a * 3303 , b * 4101 , b * 5101 , cw * 1502 , cw * 1701 ) and the normal kidney line , nkc - 26 , was established from normal kidney parenchyma obtained at the time of tumor nephrectomy ( 32 ). both cell lines were cultured in 10 % fcs containing complete medium without antibiotics . rcc - 26 cells were transduced with the human ifnγ cdna , using the retroviral system described previously ( 37 ). two ifnγ - expressing lines ( designated as endoγ1 and endoγ2 ) were generated by independent retroviral transduction ( 31 ). a control line ( rcc - 26vc ) was made using the same vector without ifnγ cdna ( 31 ). exogenous ifnγ stimulation of tumor cells was performed for 96 h in medium containing 1000 u / ml of recombinant ifnγ ( roche , basel , switzerland ). cell - mediated lysis was quantitated in standard 4 h chromium - 51 release assays ( 27 ). spontaneous release was determined by incubating target cells alone , total release by directly counting labeled cells . percent cytotoxicity was calculated as follows : % specific lysis =( experimental cpm − spontaneous cpm / total cpm − spontaneous cpm )× 100 . duplicate measurements of three step titrations of effector cells were used for all experiments . to compare data from independent experiments , % relative cytotoxic responses (% rcr ) were calculated using specific lysis of untransfected l721 . 221 or k562 cells in each experiment as reference values of 50 %. the % lysis of other target cells was determined in relation to the reference value and expressed as % rcr ( 27 ). to evaluate the influence of mhc class i molecules , the class i - specific mab , w6 / 32 was added to target cells 30 min prior to addition of effector cells : mab w6 / 32 was used as ascites diluted 1 : 100 and upc10 ( igg2a ) ( sigma , deisenhofen , germany ) was used as the isotype control . effector cells were characterized using a panel of lymphocyte specific mab : fitc - or pe - labeled mab specific for cd3 ( ucht1 ), cd4 ( 13b8 . 2 ), cd8 ( b9 . 11 ), cd56 ( nkh - 1 ), were purchased from beckmann / coulter , westbrook , me . inhibitory receptor expression was analyzed with fitc - or pe - labeled mab specific for p58 . 1 ( kir2dl1 , eb6 ), p58 . 2 ( kir2dl3 , gl183 ), cd94 ( hp - 3b1 ) all purchased from beckmann / coulter and nkb1 ( kir3dl1 , dx9 ) from pharmingen , san diego , calif . cells were incubated for 30 min on ice , washed twice , fixed with pbs - 1 % paraformaldehyde and analyzed using flow cytometry ( facs - calibur , becton / dickinson , san jose , calif .). tumor cells were tested for surface expression of mhc molecules by flow cytometry using culture supernatants of the w6 / 32 hybridoma ( american type culture collection , rockville , md .). the hla - c - specific mab l31 was kindly provided by p . giacomini , milano , italy ( 33 ). mab upc10 and mopc21 ( sigma ) were used as negative controls . cells were incubated with mab for 90 min on ice , washed twice with pbs and incubated with pe - conjugated goat - anti - mouse immunoglobulin ( f ( ab ) 2 , 115 - 116 - 146 ; dianova , hamburg , germany ) for 30 min and analyzed using flow cytometry . 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