Patent Application: US-11562002-A

Abstract:
disclosed is a method and composition for treating tumors or infectious diseases , wherein the composition comprises an agonist anti - cd40 antibody or a fragment thereof . the anti - cd40 antibody molecules include fragments like fab , 2 and fv . the composition can also include an agonistic anti - 4 - 1bb antibody or fragment thereof .

Description:
the cd40 binding molecules of the invention can be made by conventional production and screening techniques . a rat and a hamster anti - mouse cd40 monoclonal antibody (“ mabs ”) are each described in nature 393 : 474 - 77 ( 1998 ) and are available commercially ( pharmingen , inc ., ca ). the anti - mouse cd40 mab , designated fgk45 , which is used in the experiments described below , is described by rolink . a . et al ., immunity 5 , 319 - 330 ( 1996 ). anti - human cd40 mabs can be made following techniques well - known in the art , and described by g . köhler and c . milstein ( nature , 1975 : 256 : 495 - 497 ). mabs can be raised by immunizing rodents ( e . g . mice , rats , hamsters and guinea pigs ) with either native cd40 as expressed on cells or purified from human plasma or urine , or recombinant cd40 or its fragments , expressed in a eukaryotic or prokaryotic system . other animals can be used for immunization , e . g . non - human primates , transgenic mice expressing human immunoglobulins and severe combined immunodeficient ( scid ) mice transplanted with human b lymphocytes . hybridomas can be generated by conventional procedures by fusing b lymphocytes from the immunized animals with myeloma cells ( e . g . sp2 / 0 and ns0 ), as described by g . köhler and c . milstein id . in addition , anti - cd40 mabs can be generated by screening of recombinant single - chain fv or fab libraries from human b lymphocytes in phage - display systems . the specificity of the mabs to cd40 can be tested by enzyme linked immunosorbent assay ( elisa ), western immunoblotting , or other immunochemical techniques . the activating activity of the antibodies on ctls , in combination with a ctl - activating peptide , can be assessed using the assays described in the examples below . for treating humans , the anti - cd40 mabs would preferably be used as chimeric , deimmunised , humanized or human antibodies . such antibodies can reduce immunogenicity and thus avoid human anti - mouse antibody ( hama ) response . it is preferable that the antibody be igg4 , igg2 , or other genetically mutated igg or igm which does not augment antibody - dependent cellular cytotoxicity ( s . m . canfield and s . l . morrison , j . exp . med ., 1991 : 173 : 1483 - 1491 ) and complement mediated cytolysis ( y . xu et al ., j . biol . chem ., 1994 ; 269 : 3468 - 3474 ; v . l . pulito et al ., j . immunol ., 1996 ; 156 : 2840 - 2850 ). chimeric antibodies are produced by recombinant processes well known in the art , and have an animal variable region and a human constant region . humanized antibodies have a greater degree of human peptide sequences than do chimeric antibodies . in a humanized antibody , only the complementarity determining regions ( cdrs ) which are responsible for antigen binding and specificity are animal derived and have an amino acid sequence corresponding to the animal antibody , and substantially all of the remaining portions of the molecule ( except , in some cases , small portions of the framework regions within the variable region ) are human derived and correspond in amino acid sequence to a human antibody . see l . riechmann et al ., nature , 1988 ; 332 : 323 - 327 ; g . winter , u . s . pat . no . 5 , 225 , 539 ; c . queen et al ., u . s . pat . no . 5 , 530 , 101 . deimmunised antibodies are antibodies in which the t and b cell epitopes have been eliminated , as described in in international patent application pct / gb98 / 01473 . they have reduced immunogenicity when applied in vivo . human antibodies can be made by several different ways , including by use of human immunoglobulin expression libraries ( stratagene corp ., la jolla , calif .) to produce fragments of human antibodies ( vh , vl , fv , fd , fab , or ( fab ′) 2 , and using these fragments to construct whole human antibodies using techniques similar to those for producing chimeric antibodies . human antibodies can also be produced in transgenic mice with a human immunoglobulin genome . such mice are available from abgenix , inc ., fremont , calif ., and medarex , inc ., annandale , n . j . one can also create single peptide chain binding molecules in which the heavy and light chain fv regions are connected . single chain antibodies (“ scfv ”) and the method of their construction are described in u . s . pat . no . 4 , 946 , 778 . alternatively , fab can be constructed and expressed by similar means ( m . j . evans et al ., j . immunol . meth ., 1995 ; 184 : 123 - 138 ). all of the . wholly and partially human antibodies are less immunogenic than wholly murine mabs , and the fragments and single chain antibodies are also less immunogenic . all these types of antibodies are therefore less likely to evoke an immune or allergic response . consequently , they are better suited for in vivo adminstration in humans than wholly animal antibodies , especially when repeated or long - term adminstration is necessary . in addition , the smaller size of the antibody fragment may help improve tissue bioavailability , which may be critical for better dose accumulation in acute disease indications , such as tumor treatment . based on the molecular structures of the variable regions of the anti - cd40 mabs or the known ctl - activating peptides , one could use molecular modeling and rational molecular design to generate and screen molecules which mimic the molecular structures of the binding region of the antibodies or the peptides , respectively , and activate ctls . these small molecules can be peptides , peptidomimetics , oligonucleotides , or other organic compounds . the mimicking molecules can be used for treatment of cancers and infections . alternatively , one could use large - scale screening procedures commonly used in the field to isolate suitable molecules from libraries of compounds . the dosage for the molecules of the invention can be readily determined by extrapolation from the in vitro tests and assays described below , or from animal experiments or from human clinical trials . the molecules of the invention would be preferentially administered by injection , in the case of antibodies or proteins , although certain small molecules may be suited for oral administration . the assays and tests demonstrating the efficacy of the invention are described below . signaling through cd40 can replace cd4 + helper t cells in ctl priming a well characterized model system to probe the mechanism of t - cell help for the primary activation of cd8 + ctl responses in vivo was used . c57bl / 6 ( with the major histocompatibility complex ( mhc ) h - 2 b ) mice immunized with allogenic balb / c ( h - 2 d ) mouse embryo cells ( mecs ) expressing the human adenovirus type 5 early region 1 ( ad5ei - balb / c mecs ) generated strong ctl responses against an h - 2d b - restricted epitope of the adenovirus eib protein ( eib 192 - 200 ) ( fig1 a ). as the allogeneic h - 2 d mhc molecules expressed by the ad5ei - balc / c mecs cannot prime h - 2 b - restricted host ctls , generation of e1b - specific ctls must require cross - priming , that is , the uptake and h - 2b - restricted re - presentation of antigen by host apcs . cross - priming of eib - specific ctls is strictly helper - dependent ( fig1 b ), as mice depleted of cd4 + t - helper ( t h ) cells before immunization no longer mounted an e1b - specific ctl response . to investigate whether signalling through cd40 can replace cd4 + helper t cells in ctl priming , mice were depleted of cd4 + t cells in vivo before immunization with ad5e1balb / c mecs . one day after immunization , the mice received a single injection of the activating antibody anti - mouse cd40 mab fgk45 , or of an isotype - matched control antibody . administration of fgk45 to cd4 - depleted , immunized mice resulted in the efficient restoration of e1b - specific ctl responses ( fig2 a ) whereas treatment with the control antibody did not ( fig2 b ). priming of e1b - specific ctls was not detected in naïve mice treated with fgk45 alone ( not shown ). to address the possibility that the effect of fgk45 was mediated through remaining d4 + cells that were not depleted by treatment with the anti - cd4 antibody , b6 i - a b knockout mice , which lack mature functional cd4 + peripheral t cells , were immunized with the ad5ei - balb / c mecs . the response to immunization in these mice mirrors that seen in the cd4depleted mice , in that e1b - specific ctls were detectable only in mice receiving the cd40 - activating antibody ( fig2 c ), and not in those receiving the control antibody ( fig2 d ). it was also studied whether the requirement for anti - cd40 antibodies in priming of ctls in cd4 - depleted mice could be replaced by bacterial lipopolysaccharide ( lps ) ( 50 μg intravenous ), a potent inducer of proinflammatory cytokines , or by administration of il - 2 ( 1 × 10 5 units in incomplete freund adjuvant , subcutaneous ) following immunization with ad5ei - balb / c mecs . whereas cd4 - depleted mice treated with fgk45 exhibited strong e1b - specific ctl activity , neither lps or il - 2 treatment resulted in detectable ctl priming ( not shown ). ligation of cd40 on b cells upregulates their costimulatory activity , suggesting a role for these cells in the restoration of ctl priming by treatment with cd40 activating antibodies . to address this question , b6 □ mt mice , which lack mature b cells , were immunized with the allogeneic ad5ei - balb / c mecs . cross - priming of e1b - specific ctls did not require mature b cells ( fig3 a ). however , when depleted of cd4 + cells , the b - cell deficient mice did not generate an e1b - specific ctl response ( fig3 b ). activation through cd40 with the fgk45 monoclonal antibody completely restored the capacity of cd4 - depleted □ mt mice to prime e1b - specific ctls ( fig3 c ). thus b cells are not required as apcs or accessory cells for cross - priming in this model system , nor are they required for cd40 - mediated restoration of cross priming of ctls in the absence of cd4 + helper t cells . these results demonstrate that activation of bone marrow derived apc through cd40 can bypass the requirement for cd4 + t - helper cells in the cross - priming of e1b - specific ctls . blocking the ability of cd4 + helper t cells to interact with apc through the cd40l - cd40 pathway prevents antigen - specific ctl responses in normal mice if the cd40l - cd40 interaction represents the physiological pathway used by cd4 + helper t cells to help ctls , blocking the ability of the cd4 + t cells to interact with apc through cd40l - cd40 interaction would be expected to diminish priming of e1b - specific ctl responses in normal mice . b6 mice were immunized with ad5e1 - balb / c mecs and then treated with either the cd40l - blocking antibody mr1 , or control antibody . blockade of cd40l results in drastically reduced e1b - specific ctl responses ( fig4 a ) compared to the efficient ctl priming seen in mice receiving the control antibodies ( fig4 b ). the priming defect induced by cd40l blockade was fully restored following cd40 signalling by fgk45 ( fig4 c ). thus the defect in ctl - priming induced by cd40l blockade lies in the failure of t h cells to transmit , rather than to receive , cd40l - mediated signals . a previously described model system has been used ( toes et al ., j . immunol . 156 : 3911 ( 1996 )). it has been shown that s . c . vaccination with the ad5e1a - derived ctl epitope sgpsntppei ( seq id no : 2 ) in ifa prevents mice from controlling the outgrowth of ad5e1a - expressing tumors . this indicates that the e1a / ifa vaccine induced suppression rather than induction of e1a - specific ctl immunity . moreover , administration of the e1a / ifa vaccine to t cell receptor ( tcr )- transgenic mice , which express the tcr α and β chains of an e1a - specific ctl clone , strongly suppressed tumor - specific ctl - mediated immunity . these experiments examined the effects of peptide administration on a monoclonal ctl population . to establish whether s . c . e1a - peptide vaccination also induces e1a - specific ctl tolerance at the polyclonal ctl level , wild type ( wt ) c57bl / 6 mice were injected with either e1a - peptide ( fig5 a and 5 b ) or a control peptide fig5 c and 5 d ). one day later the mice were boosted with a syngeneic cell line expressing high levels of the e1a - peptide at its surface ( fig5 b and 5 d ). injection of this cell line into mice primed with the control peptide readily induces e1a - specific immunity ( fig5 d ). however , the ability of mice to mount e1a - specific ctl responses was abrogated after injection of the e1a / ifa vaccine ( fig5 b ). these data indicate that injection of the e1a - peptide not only leads to e1a - specific tolerance in tcr - transgenic mice but also in mice expressing a polyclonal e1a - specific t cell repertoire . since s . c . injection of the e1a / ifa vaccine leads to systemic ctl tolerance , it was investigated whether the e1a - peptide is dispersed systemically and presented to precursor ctl in the periphery . therefore , mice were injected s . c . with the e1a - peptide or human papilloma virus ( hpv ) 16 e7 - derived control peptide emulsified in ifa . spleen cells from these mice were isolated 16 h later and used as stimulator cells for an e1a - specific ctl clone in vitro . splenocytes from mice injected with the e1a - peptide s . c . induced specific proliferation , whereas splenocytes from mice injected with the e7 - peptide s . c . failed to do so ( fig6 ). moreover , a control ctl clone did not proliferate on spleen cells derived from e1a - injected mice ( data not shown ). thus , these data indicate that the e1a - peptide injected s . c . in ifa is systemically presented in the periphery by , amongst others , splenocytes . in view of the tolerizing effects described above of the e1a - peptide vaccine , there was a question whether cd40 - triggering in vivo is sufficient to prevent peripheral tolerization of ctl and to restore ctl priming . therefore , it was investigated whether injection of tolerizing peptides combined with in vivo cd40 triggering could prevent the induction of peripheral ctl tolerance leading to tumor - specific ctl immunity . in examples 1 and 2 , it has been shown that cd40 - triggering in vivo can replace the requirement for cd4 + t helper cells in priming of helper - dependent ctl responses . since cd4 + t cell - mediated helper activity has been implicated in the prevention of peripheral ctl tolerance induction , the inventors addressed the question whether cd40 - triggering in vivo is sufficient to prevent peripheral e1a - specific ctl tolerization . to this end , mice were injected with the e1a / ifa vaccine in combination with the activating anti - cd40 mab fgk - 45 . mice that received this combination mounted strong e1a - specific ctl responses ( fig7 b and 7 e ), whereas mice that received the e1a / ifa vaccine ( fig7 e ) or mab alone did not ( not shown ). the combination of e1a / ifa vaccine and anti - cd40 mab failed to elicit ctl in cd40 - deficient mice ( fig7 c and 7 d ). furthermore , co - injection of the e1a / ifa vaccine with an isotype - matched control mab ( fig6 a ) or il - 2 failed to convert ctl tolerance induced by the e1a / ifa vaccine into ctl priming ( not shown ). the range and variation of responses to the e1a - epitope in e1a - peptide only , or e1a - peptide plus anti - cd40 - vaccinated animals , is shown in fig7 e . thus , systemic cd40 activation can reverse peptide - induced peripheral ctl tolerance into peptide and tumor - specific ctl mediated immunity . the induction of e1a - specific immunity strongly correlated with the presence of cd8 + t cells in the spleen of vaccinated mice that stained with pe - conjugated h - 2 - d b - tetramers containing the e1a - peptide ( d b / e1a ). within 10 days after vaccination , cd8 + t cells staining with d b / e1a tetramers could be detected by flow cytometry in mice injected with e1a - peptide and the anti - cd40 mab , but not in mice injected with e1a - peptide alone ( not shown ). in the mice injected with e1a - peptide , the percentage of cd8 + cells that stained with the d b / e1a tetramers was approximately 3 %. in mice vaccinated with whole adenovirus , which induces potent e1a - specific immunity , comparable amounts of d b / e1a tetramer - reactive cd8 + spleen cells were detected . these results indicate that the expansion of e1a - specific cd8 + t cells in mice that received the e1a / ifa vaccine in combination with the anti - cd40 mab was substantial and equivalent to that found in virus vaccinated animals . although the findings described above show that provision of help through cd40 - triggering is sufficient to prevent ctl - tolerization after administration of a tolerogenic peptide - vaccine , they do not address the question whether the efficacy of anti - cancer vaccines that normally induce protective immunity , instead of tolerance , can be enhanced by activation through cd40 . it was examined whether cd40 - triggering in vivo is beneficial to the outcome of vaccination with an hpv16 e7 - derived peptide . vaccination with this peptide induces protective ctl - mediated immunity against a challenge with hpv16 - transformed tumor cells . moreover , this peptide can be used in a therapeutic setting when loaded on in vitro activated dc suggesting that the strength of the anti - tumor response is enhanced when presented by activated dc . mice receiving the e7 - peptide in combination with cd40 - triggering mounted a more potent ctl - response compared to mice treated with e7 - peptide only ( data not shown ), indicating that cd40 - triggering also enhances the efficacy of the hpv16 e7 - peptide vaccine and confirming the findings with the e1a peptide described above . moreover , mice treated 6 days after s . c . injection of cd40 - negative hpv16 e6 / e7 transformed tumor cells with the hpv16 e7peptide alone ( open squares ) are able to slow down tumor growth , but eventually most animals succumb to the tumor ( fig8 ). when , however , hpv 16 e7peptide vaccination was combined with injection of the anti - cd40 mab , tumor growth was markedly reduced and 7 out of 10 mice rejected the tumor , whereas animals injected with a control peptide and the anti - cd40 mab were unable to control outgrowth of the tumor . these results show that the effect of vaccination regiments can be markedly enhanced when immunization is combined with in vivo cd40 - triggering . these data provide the basis for the development of extremely potent and novel anti - tumor vaccines for cancer patients . in vivo triggering through 4 - 1bb provides a licence to kill to naïve ctl in vivo cd40 triggering of apc through the administration of an agonistic anti - cd40 antibody ( ab ) can replace the need for cd4 + t helper ( th ) activity in cross - priming of ad5 - specific ctl . we injected ad5e1 - transformed mouse embryo cells ( ad5mec ) into syngeneic cd4 + th cell - depleted c57bl / 6 ( b6 ) mice in combination with either anti - cd40 or anti - 4 - 1bb ab . these ad5e1mec were derived from tap - deficient mice , and are therefore incapable of presenting the ad5e1 encoded - ctl epitopes in the context of their own mhc ( 20 ). consequently , priming of ad5e1 - specific ctl in this setting relies on cross - presentation of tumor antigens by host apc . the induction of ad5e1b - specific ctl is cd4 + th - dependent , as mice depleted for cd4 + t cells fail to mount an e1b - specific ctl response ( fig9 b ). systemic administration of agonistic anti - cd40 ab in these cd4 - depleted animals restored e1b - specific cross priming ( fig9 c ). interestingly , systemic administration of an agonistic anti - 4 - 1bb ab resulted in equally efficient priming of e1b - specific ctl in the absence of cd4 + t cell help ( fig9 d ). a different method of inducing antigen - specific ctl immunity against tumors is through vaccination with minimal peptide - epitopes in adjuvant ( ifa ). notably , in case of the epitopes derived from ad5e1a and e1b such peptide - based vaccines induce ctl tolerance rather than priming , unless agonistic anti - cd40 ab is co - administered ( 4 ). we , therefore , investigated whether also in this setting a trigger through 4 - 1bb could permit ctl priming by injecting b6 mice with the e1a - peptide vaccine in combination with anti 4 - 1bb ab , anti - cd40 ab or control antibody . indeed , not only co - injection of anti - cd40 , but also co - injection of anti - 4 - 1bb converted the tolerogenic peptide vaccine into an immunogenic formulation ( fig1 e - g ). similar results were obtained when e1a - specific ctl immunity was monitored by staining of splenocytes with h - 2d d / e1a peptide tetramers ( not shown ). thus , in vivo triggering of 4 - 1bb can prevent the induction of peptide - specific tolerance , and instead result in the priming of a potent e1a - specific ctl response . taken together , these data demonstrate that the 4 - 1bb signal efficiently provides ctl with a licence to kill , and as such can replace the need for cd40 triggering through th cells or administration of agonistic anti - cd40 ab . current knowlegde of the expression patterns of cd40 and 4 - 1bb would argue that the signal provided by in vivo administration of anti - cd40 ab is delivered to the apc , whereas that of anti - 4 - 1bb ab is delivered directly to the ctl . to exclude the possibility that the anti - 4 - 1bb antibody , instead of directly stimulating antigen - specific t cells , would mediate its effect on ctl priming ( fig9 ) in an indirect fashion by activation of the antigen presenting dc , we investigated whether injection of the anti - 4 - 1bb antibody activates dc in vivo . b6 mice were injected with anti - 4 - 1bb , anti - cd40 or control ab and the cd86 ( b7 . 2 ) expression on cd11c + dc isolated from the spleens of these mice was analysed . as expected , in vivo triggering of cd40 resulted in an enhanced expression of cd86 , indicating in vivo activation of the dc . in contrast , after in vivo 4 - 1bb triggering or administration of control rat ab the expression of cd86 on dc was not increased ( fig1 a ). the failure of the anti - 4 - 1bb ab to induce dc activation is in accordance with the fact that neither immature nor mature dc express detectable levels of 4 - 1bb , implicating that dc are not receptive for 4 - 1bb triggering ( fig1 b ). therefore , anti - 4 - 1bb antibody does not appear to affect the dc population , but rather mediates its stimulatory effect on ctl priming through direct stimulation of cd8 + t cells . to investigate in which manner agonistic anti - 4 - 1bb ab permits t helper independent priming of ad5 - specific ctl , we exploited adoptively transferred t cell populations from ad5e1a - peptide specific t cell receptor ( tcr ) transgenic mice ( 21 ). tracking of these t cells in vivo was performed in two distinct ways . first , tcr transgenic t cells were fluorescently labelled with the dye cfse and transferred into normal b6 mice , after which these mice were vaccinated with the ad5e1a - peptide in ifa with or without anti - 4 - 1bb ab treatment . interestingly , the resulting data revealed that proliferation of the tcr - transgenic t cells was equally triggered in mice that had received the e1a - peptide either with or without anti - 4 - 1bb ab ( fig1 a ). therefore anti - 4 - 1bb ab do not enable ctl priming by increasing the initial activation or proliferative capacity of antigen - specific t cells . in a second approach we transferred ad5e1a - specific tcr transgenic t cells , which were from b6 thy1 . 2 origin , into b6 thy1 . 1 congenic mice . these mice were vaccinated with the ad5e1a - peptide in ifa with or without anti - 4 - 1bb ab treatment . fig3 b shows that at 11 days after vaccination , massive expansion of cd8 + t cells from tcr - transgenic origin was observed in mice vaccinated with e1a - peptide in combination with anti - 4 - 1bb ab , but not in mice that received either e1a - peptide or anti - 4 - 1bb ab alone . therefore , accumulation of e1a - specific ctl required the presence of the 4 - 1bb costimulatory signal . furthermore , stimulation of cd8 + ctl immunity through 4 - 1bb was dependent on the presence of an antigen - specific trigger . taken together , the data in fig3 demonstrate that 4 - 1bb triggering is not essential for initial ctl activation , but promotes the survival of antigen - stimulated ctl , thereby permitting expansion of these ctl . because in vivo triggering of 4 - 1bb allows for ctl priming in the absence of the cd40 - dependent dc activation signal ( fig9 this implies that antigen presentation by nonactivated dc in combination with an activating anti - 4 - 1bb ab suffices for efficient ctl priming . the need for co - administration of anti - 4 - 1bb ab also indicated that nonactivated dc were apparently not capable of stimulating ctl through 4 - 1bb . therefore , we analyzed expression of the natural ligand for this receptor , 4 - 1bbl , on immature and mature dc . nonactivated dc express low levels of 4 - 1bbl ( fig1 a ), whereas the expression of this molecule is strongly increased upon activation in the presence of the cd40l - trimer - or poly i : c ( fig1 b and c , respectively ). this induction of 4 - 1bbl was observed upon cd40 triggering of dc in vivo . the increase in 4 - 1bbl expression was paralleled by elevated levels of cd86 on the activated dc . this suggests that both costimulatory signals contribute to the capacity of mature dc , as opposed to immature dc , to induce ctl immunity . we performed tumor cell immunization experiments in normal , cd4 + th cell proficient b6 mice , in which the th - dependent dc maturation signal required for ctl cross - priming is intact . blockade of the cd28 and 4 - 1bb signals was performed by administration of blocking anti - 4 - 1bbl ab or ctla4 - ig respectively . b6 mice were immunized with ad5e1mec in combination with systemic doses of anti - 4 - 1bbl ab and / or ctla4 - ig . interestingly , in vivo blockade of anti - 4 - 1bbl resulted in a marked reduction in the number of antigen - specific ctl induced by the tumor cell vaccine ( fig1 ). blockade of cd28 costimulation completely inhibited ctl induction and , therefore , additional 4 - 1bbl blocking in combination with ctla4 - ig had no extra effect . these data indicate that 4 - 1bbl / 4 - 1bb interactions are important for the cross - priming of antigen - specific ctl , as blocking of the interaction between these molecules greatly diminishes the numbers of tumor - specific ctl that are sustained . however in the absence of the co - stimulatory signal through cd28 , provided by properly activated dc , the signal provided by 4 - 1bbl is apparently not capable of enabling ctl priming and , as a result , induction of anti - tumor ctl immunity is completely abrogated . the in vivo blocking experiments suggested a dominance of cd28 costimulation over the 4 - 1bb signal . therefore , we investigated whether in the absence of cd28 costimulation , administration of anti - 4 - 1bb ab would still provide ctl with a licence to kill . b6 mice were depleted for cd4 + t cells and immunised with ad5e1mec in combination with administration of anti - 4 - 1bb ab and / or ctla4 - ig . mice that received anti - 4 - 1bb ab mounted strong e1b - specific ctl responses , but mice that in addition received ctla4 - ig failed to do so ( fig1 ). these data indicate that the positive effect of 4 - 1bb triggering on the induction of ctl immunity is dependent on the presence of an intact cd28 costimulatory signal . furthermore , these data suggest that the basal levels of cd80 / cd86 expressed by non - activated dc provide sufficient costimulation through cd28 to allow additional costimulation through the 4 - 1bb pathway . in vitro studies have demonstrated that 4 - 1bb expression on naive t cells is absent and that tcr - triggering , through cross - linking by anti - cd3 ab , results in a rapid increase in 4 - 1bb surface expression ( 8 ). as our data support the conclusion that costimulation through cd28 is prerequisite for 4 - 1bb signaling , we investigated whether cd28 triggering contributes to the up - regulation of 4 - 1bb on naive t cells . therefore , naive spleen cells were stimulated in vitro with plate - bound anti - cd3 ab and analyzed for 4 - 1bb expression 24 h later ( fig1 ). it is clear that strong signals through the tcr ( high concentrations of anti - cd3 ab ) are sufficient to induce 4 - 1bb expression within 24 h . however , when t cells are stimulated with lower anti - cd3 concentrations , their capacity to upregulate 4 - 1bb is largely lost ( fig1 ). these lower anti - cd3 ab concentrations provide a weaker tcr trigger that is more likely to resemble an in vivo signal of the kind provided by the tumor cell vaccine . importantly , under these more physiological conditions , costimulation through the cd28 receptor restored the capacity of t cells to express high levels of 4 - 1bb . these findings are in accordance with the fact that blockade of the cd28 pathway in vivo abrogates costimulation through 4 - 1bb ( fig1 and 15 ). furthermore , they strengthen the notion that cd28costimulation of antigen - stimulated t cells is an important signal for 4 - 1bb upregulation on naive t cells , thereby making these cells susceptible for 4 - 1bb triggering . enabling of ctl priming by a tumor cell or peptide - based vaccine through administration of a synergistic combination of anti - cd40 and anti - 4 - 1bb ab because the anti - cd40 ab and anti - 4 - 1bb ab enabled ctl priming by the e1a peptide vaccine in a similar fashion , while 4 - 1bb triggering was dependent on cd28 - costimulation , we investigated whether combination of both ab would result in even greater induction of ctl immunity . our data revealed that combination of the e1a peptide vaccine with both ab indeed has strong synergistic effects on induction of the ctl response . in mice that received both ab the absolute as well as relative numbers of tetramer - positive cd8 + t cells were increased at least 5 - 10 fold as compared to mice that received only one of the ab ( fig1 ). induction of therapeutic anti - tumor immunity through administration of agonistic anti - cd40 ab to tumor - bearing subjects since in vivo activation of apc through injection of anti - cd40 ab enables cross - priming of tumor - specific ctl in tumor cell - immunized cd4 + t cell depleted mice , this procedure could also promote the induction of anti - tumor immunity in tumor - bearing animals . initiation of anti - tumor ctl responses in tumor - bearing mice is most likely hampered by the lack of antigen presentation by properly activated apc . this could be due to insufficient apc recruitment to the tumor site and / or the failure of tumor antigen - loaded apc to become activated and redistributed to the draining lymph nodes , the site where encounter with — and priming of — naive t cells takes place . especially lack of inflammatory ‘ danger ’ signals , lack of tumor - specific t cell help and the secretion of immunomodulatory cytokines by the tumor could result in the failure of apc to cross - prime anti - tumor ctl responses ( 22 ). in view of these considerations , we injected immunocompetent b6 mice , bearing ( subcutaneous ) tumors of ad5 - transformed cells , with anti - cd40 ab . whereas tumors grew progressively in control animals , anti - cd40 treated mice were capable of rejecting their tumors ( fig1 ). interestingly , in control animals we only detected low numbers of e1a - specific ctl in the draining lymph nodes , whereas anti - cd40 treated mice clearly exhibited elevated numbers of e1a - specific ctl in both draining and co - lateral lymph nodes as well as in peripheral blood ( fig1 a - c ). importantly , the localization of antigen presentation is similar in anti - cd40 treated and control mice , in that in both cases this is only detected in the tumor - draining lymph node , not in the co - lateral lymph node ( fig2 ). as described above , due to a lack of inflammatory ‘ danger ’ signals the apc apparently fail to cross - prime anti - tumor ctl responses in the control animals . in contrast , in the anti - cd40 ab - treated mice the cd40 - signal empowers these apc to effectively prime ad5e1a - specific ctl immunity and , as a result , these ctl are capable of leaving the draining lymph node and gain access to other peripheral lymphoid organs as well as to the tumor . this latter point is confirmed by the fact that a5e1a - specific ctl are primarily detected in the tumors of anti - cd40 ab - treated mice ( fig2 ). the notion that anti - cd40 treatment results in effective systemic ctl immunity is further supported by the fact that local in vivo triggering through cd40 , by intra - tumoral injection of the anti - cd40 ab into the tumor on one flank , also results in rejection of the non - treated tumor on the other flank ( fig2 a - c ). this experiment also demonstrates that effective anti - tumor therapy can be achieved both after systemic and local adminsirtation of the anti - cd40 ab . two additional experiments provide further insight into the mechanisms through which anti - cd40 ab injection results in tumor rejection . first , the role of these ctl in tumor eradication was confirmed by the fact that anti - tumor therapy with anti - cd40 ab is not effective in mice that are depleted of their cd8 + t cells , while still being effective in mice that are depleted of their cd4 + t cells ( fig2 ). secondly , it should be noted that others have found anti - cd40 ab to inhibit growth of cd40 - expressing tumors through direct induction of tumor cell apoptosis ( 23 - 25 ). importantly , our tumor cells lack cd40 expression ( fig2 ). therefore , the therapeutic effect of the anti - cd40 ab in this setting acts through the induction of a tumoricidal ctl response . induction of therapeutic anti - tumor immunity through administration of a synergistic combination of anti - cd40 and anti - 4 - 1bb ab to tumor - bearing subjects our data show that administration of agonistic anti - cd40 ab , in addition to enabling ctl priming against tumor cell - and peptide - based vaccines , can also empower the immune system to raise therapeutic ctl immunity against pre - existing tumors . because the anti - cd40 and 4 - 1bb ab are similarly capable of stimulating ctl immunity , and act synergistically in stimulating peptide vaccine - induced responses ( see above ), we expect a very powerful therapeutic capacity this potent ab - combination concerning the induction of ctl immunity in tumor - bearing mice . the foregoing description , terms , expressions and examples are exemplary only and not limiting . the invention includes all equivalents of the foregoing embodiments , both known and unknown . the invention is limited only by the claims which follow and not by any statement in any other portion of this document or in any other source . 1 . schoenberger , s . p ., r . e . toes , e . i . van der voort , r . offringa , c . j . melief . 1998 . t - cell help for cytotoxic t lymphocytes is mediated by cd40 - cd40l interactions . nature . 393 : 480 - 3 . 2 . ridge , j . p ., f . di rosa , p . matzinger . 1998 . a conditioned dendritic cell can be a temporal bridge between a cd4 + t - 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