Patent Application: US-77166407-A

Abstract:
the present invention includes compositions and methods for making a pharmaceutically effective amount of one or more dendritic cells matured by exposure to ifnα loaded with cancer antigens from cells that are heat shocked and subsequently killed , wherein the one or more ifnα dendritic cells are under conditions to present the one or more cancer antigens are presented within the context of autologous mhc .

Description:
while the making and using of various embodiments of the present invention are discussed in detail below , it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts . the specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention . to facilitate the understanding of this invention , a number of terms are defined below . terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention . terms such as “ a ”, “ an ” and “ the ” are not intended to refer to only a singular entity , but include the general class of which a specific example may be used for illustration . the terminology herein is used to describe specific embodiments of the invention , but their usage does not delimit the invention , except as outlined in the claims . abbreviations . gm - csf : granulocyte macrophage - colony stimulating factor ; ifn alpha : interferon alpha ; lps : lipopolysaccharide ; tnf : tumor necrosis factor ; cr : complete response ; pr : partial response ; sd : stable disease ; pd : progressive disease ; npd : non - progressive disease ; ned : no evidence of disease ; mdcs : monocyte derived dendritic cells ; ctls : cytotoxic t lymphocytes ; nk cells : natural killer cells ; nkt cells : natural killer t cells ; nd : not done ; nt : not tested ; tbd : to be determined . cancer immunotherapy . there are numerous strategies for improving a patient &# 39 ; s resistance to cancer . among these strategies are 1 ) non - specific activation of the immune system with microbial components or cytokines ; 2 ) antigen - specific adoptive immunotherapy with antibodies and / or t cells ; and 3 ) antigen - specific active immunotherapy ( vaccination ). the major limitation of antibodies is that target proteins must be expressed on the cell surface as opposed to targets for t cells that can be intracellular proteins whose peptides are presented on the cell surface in complexes with mhc molecules 11 . the identification of defined tumor antigens in humans 12 , 13 prompted the development of adoptive t - cell therapy . yet , vaccination remains the most attractive strategy because of its expected inducement of both therapeutic t - cell immunity ( tumor - specific effector t cells ) and protective t cell immunity ( tumor - specific memory t cells that can control tumor relapse ) 14 - 16 . numerous approaches for the therapeutic vaccination of humans with cancer have been developed , including : autologous and allogeneic tumor cells ( which are often modified to express various cytokines ), peptides , proteins and dna vaccines 8 , 16 - 19 . all of these approaches rely on a random encounter of the vaccine with host dcs . if the vaccine antigen does not result in an encounter with dcs an immune response cannot occur . in addition , an inappropriate encounter with either non - activated dcs or the “ wrong ” subset of dcs can lead to silencing of the immune response 20 . both of these scenarios may explain some of the shortcomings in current cancer vaccines . furthermore , it is not known how tumor antigens need to be delivered to dcs in vivo in order to elicit an appropriate immune response . hence , there is a need for studies based on ex vivo - generated autologous dcs that are loaded with tumor antigen under controlled conditions which might allow the establishment of parameters for optimal vaccination against cancer . clinical studies using dcs as vaccines were facilitated by the discovery of methods to generate large numbers of autologous dcs ex vivo 21 - 23 . parameters that should be considered to improve the efficacy of dc vaccinations in cancer include : dc related factors ; host - related factors ; and a combination of dc vaccines with other therapies . two dc related parameters , i . e ., a novel dc subset and a novel strategy to load tumor antigens are briefly discussed below . parameters of dc vaccines . dc subsets : the discovery that gm - csf and il - 4 can differentiate monocytes into immature dcs 22 - 24 has allowed major progress in our understanding of dc biology and function . several institutions have used il4 - dcs as vaccines 8 following pioneering clinical studies in patients with metastatic melanoma by nestle , et al . 25 ( using tumor - lysate - loaded dcs ) and by schuler and colleagues 26 ( using melanoma - peptide - loaded dcs ). however , recent discoveries point to new alternatives to the classical way of generating dcs . for example , melanoma - peptide - pulsed il15 - dcs are more efficient than il4 - dcs for the induction of antigen - specific ctl differentiation in vitro , whereas their ability to stimulate cd4 + t cells is comparable 27 . also , ifn - alpha - dcs generated in three - day cultures have been found to be efficient for the induction of specific immunity 28 . thus , the immunogenicity of these distinct dc vaccines warrants testing in vivo in clinical studies . antigen loading . loading mhc class i and mhc class ii molecules on dcs with peptides derived from defined antigens is the most commonly used strategy for dc vaccination 15 , 29 . although this technique is important for “ proof of concept ” studies , the use of peptides has limitations for further vaccine development : restriction to a given hla type ; the limited number of well characterized tumor - associated antigens ( taa ); the relatively rapid turnover of exogenous peptide - mhc complexes resulting in comparatively low antigen presentation at the time the dc arrive into draining lymph node after injection ; and , the induction of a restricted repertoire of t - cell clones , thus limiting the ability of the immune system to control tumor antigen variation . many of these peptides may differ from naturally processed epitopes . thus , loading dcs with total antigen preparations and allowing “ natural ” processing and epitope selection is expected to improve efficacy and allow the generation of a diverse immune response involving many clones of cd4 + t cells and ctls . all of these strategies load dcs with recombinant proteins , exosomes 30 , viral vectors 31 , plasmid dna , rna transfection 32 , immune complexes 33 and antibodies specific for dc surface molecules 15 , 34 . another technique involves exploiting the capacity of dcs to present peptides from phagocytosed dead tumor cells on mhc class i molecules ( as well as class ii molecules ). this is known as cross - priming 35 , 36 . indeed , dcs cultured with killed allogeneic melanoma , prostate or breast cancer cell lines prime naive cd8 + t cells against tumor antigens in vitro 36 , 37 . 20 patients with metastatic melanoma have been vaccinated at biir to date with autologous monocyte - derived dcs previously exposed to a killed allogeneic melanoma cell line ( 8 vaccines on a monthly basis ). vaccination has proved to be safe ( no autoimmunity or other adverse events ) and has led to the induction of melanoma - specific t cell immunity . in two patients , a long - lasting tumor regression has been observed . these results warrant larger clinical studies to prove the efficacy of the vaccine preparation methodology . gm - csf and ifn alpha induced dendritic cell vaccine . several recent findings in ifn biology reactivated immunologists &# 39 ; interest in this family of molecules . these findings include 1 ) the demonstration that plasmacytoid dendritic cells ( pdcs , a subset of human and murine dcs ) promptly secrete large amounts of type i ifns in response to viral signals 74 ; 2 ) the abilities of ifn alpha / beta to activate immature myeloid dcs 75 and to induce the differentiation of monocytes into dcs 28 , 76 ; 3 ) the activity of ifn alpha / beta in the generation of memory cd8 + t cells 77 , 78 and the stimulation of antibody responses 5 , 79 , 80 ; 4 ) the central role of ifn alpha / beta in the pathogenesis of systemic lupus erythematosus 81 , 82 ; 5 ) the secretion of low levels of ifn alpha / beta to rev up the immune system , as illustrated by the essential role of ifn alpha / beta in the lps signaling and the development of septic shock . 83 it was recently recognized by the present inventors that ifn alpha is essential in launching human humoral responses specific to influenza virus . 5 this raised a potential that ifn alpha could possibly enhance the generation of ctls , most particularly those directed against tumor antigens . ifn - dcs efficiently activate cytolytic cd8 + t cells : the biological properties of dc vaccine generated from monocytes by culturing them with gm - csf and ifn alpha ( ifn - dc ) were compared with two previous products manufactured , viz ., dc vaccines made by culturing monocytes with gm - csf and either tnf ( tnf - dcs ) or il - 4 ( il4 - dcs ). allogeneic cd8 + t cells were cultured for 5 days with each dc subset , then re - purified by cell sorting and analyzed by flow cytometry . strikingly , results showed that ifn - dcs can induce cd8 + t cells with considerably higher expression of cytolytic t cell molecules such as granzymes a and b ( fig1 ) and perforin ( not shown ). ifn - dcs respond differentially to maturation stimuli : to further characterize the role of ifn - dcs in cancer vaccines , cytokine biosignatures were analyzed in ifn - dcs exposed in vitro to various activation signals including cd40 ligand , lonomycin , and tlr ligands such as lps ( tlr4 ), poly i : c ( tlr3 ) and zymosan ( tlr2 ). ifn - dcs , generated by culturing purified monocytes with gm - csf and recombinant human ifn alpha 2a , were exposed to various stimuli and cytokine / chemokine secretion into culture supernatants and measured at different time points using multiplex cytokine beads ( luminex ). preliminary analysis demonstrated that the profile of secreted cytokines does not change significantly over 48 hrs of exposure ( not shown ). as shown in fig2 , the predominant cytokine response was triggered by lps , followed by poly i : c and lonomycin . only minor cytokine secretion was induced upon exposure to cd40 ligand or zymosan . these preliminary results demonstrate different activation signals trigger different cytokine signatures in ifn - dcs . ifn - dcs uniquely secrete il - 7 , a t cell growth factor : upon single cytokine analysis , it was observed that ifn - dcs spontaneously secrete detectable levels of il - 7 ( fig3 ) while non - activated il4 - dcs were unable to do so . however , activation of il - 4dcs with signals known to activate type i interferon pathway ( lipopolysaccharide ( lps ) and poly i : c ) but not with cd40 ligand or zymosan , led to induction of il - 7 secretion ( fig3 ). these results yield two important conclusions for t cell activation : 1 ) il - 7 secretion by ifn - dcs could have a role in their superior capacity to prime naïve cd8 + t cells , and 2 ) type i interferon appears to regulate il - 7 secretion by myeloid dcs . ifn - dcs efficiently cross - prime tumor - specific ctls : to determine whether ifn - dcs were indeed more powerful in stimulating ctls , dcs were loaded with killed melanoma or breast cancer cells and then used to prime purified cd8 + t cells in 2 culture cycles . ifn - dcs were observed to be more efficient than il4 - dcs in priming ctls that have the capacity to kill cancer cells ( fig4 ). to assess the capacity of ifn - dcs to prime naïve cd8 + t cells , we used an in vitro cross - priming system against tumor antigens that we had established earlier . in this situation , dcs are loaded with killed allogeneic tumor cells and used to prime autologous naïve cd8 + t cells over two - week cultures . as shown in fig5 , ifn - dcs loaded with killed allogeneic hla - a * 0201 + me290 melanoma cells are remarkably efficient in priming ctls with the ability to kill me290 cells used for immunization . the presence of melanoma - specific cd8 + t cells was further confirmed by the analysis of tetramer specific t cells . as shown in fig6 , the elicited cd8 + t cells contain a subpopulation of mart - 1 specific t cells . thus , ifn - dcs have been shown to be highly efficient at cross - priming naive cd8 + t cells to differentiate into ctls specific for tumor antigens . this finding has potentially high therapeutic implications for cancer vaccines . thus , ifn - dcs are more efficient in the induction of tumor specific immunity which warrants further testing of their in vivo activity in patients in the clinical setting . generation of highly immunogenic killed tumor cells for loading dc vaccines . the use of hyperthermia in cancer therapy , either alone or as an adjuvant for radiotherapy , has been an object of clinical interest for many years 84 . hyperthermia seems to be particularly effective in combination with radiotherapy and / or radio - immunotherapy ( reviewed in 85 , 86 ). the molecular mechanism by which hyperthermia leads to radiosensitization is not clear , however activation of early response genes and heat shock factors ( hsfs ) and subsequently heat shock proteins ( hsps ) are likely to play a role in this occurence 87 . hsps constitute a superfamily of distinct proteins , which are operationally named according to their molecular weight , e . g . hsp70 . most hsps are expressed constitutively and are further induced under stress conditions , including temperature increase . hsps are considered as molecular relay line that chaperones the peptides from their generation in the cytosol to their binding to mhc class i in the er 88 , 89 . hsp70 , hsp60 and gp96 have been recently established as immune adjuvants for cross - priming with antigenic proteins or peptides 90 , 91 . in this process , reconstituted hsp70 or gp96 - peptide complex are internalized by antigen presenting cells ( apcs ) including dcs , through receptor - mediated endocytosis via cd91 92 , cd40 93 , lox - 1 94 or tlr2 / 4 95 . thus , the present inventors recognized that hyperthermia could enhance cross - priming and thereby contribute to the manufacture of a vaccine which would allow enhanced tumor regression . heated melanoma cells . the goal of current research has been to create altered tumor cell bodies that are highly immunogenic and could be used to load dcs for vaccination purposes . therefore , having established the premise that melanoma cell lines overexpressing hsp70 are indeed more immunogenic ( not shown ), the focus of this investigation now shifted to the development of a means to increase hsp expression in clinical grade conditions . thus , whether heat - treatment of melanoma cells could increase immunogenicity of loaded dcs was determined . heat treatment of melanoma cells increases hsp70 : melanoma cell lines were incubated in the for 4 hrs at 42 ° c . ( heat shock ). the analysis of hsps expression by elisa ( not shown ) or by intracellular staining ( fig7 ) revealed , as expected , significant upregulation of hsp70 85 . killed melanoma cells ( melanoma bodies ) were generated from either untreated or heat - shock treated cells by introduction of betulinic acid ( ba ). dcs loaded with heat - treated melanoma bodies rapidly yield ctls able to kill melanoma cells : unloaded dcs ( ctl 1 ), dcs loaded with control melanoma bodies ( ctl 2 ) and dcs loaded with heat - shocked melanoma bodies ( ctl 3 ) were cultured for 2 weeks with purified naive cd8 + t cells . t cell cultures were restimulated once ( a total of two stimulations ) and were supplemented with soluble cd40 ligand and low dose il - 7 ( 10 u / ml , all culture ) and il - 2 in the second week ( 10 u / ml ). t cells were analyzed 7 days after restimulation ( total 14 days of culture ). it was noted that t cells cultured with dcs loaded with heat - treated me275 melanoma bodies but not control bodies , were able to kill me275 cells ( fig8 ). the killing was specific to t cells as no lysis of k562 was found . furthermore , after two stimulations , the t cells were able to kill another hla - a * 0201 melanoma cell line ( me290 ) ( not shown ), suggesting cross - priming against shared antigens can occur . the killing was specific to melanoma cells as hla - a * 0201 mcf7 breast cancer cells were not lysed ( not shown ). finally , killing of melanoma cells was at least partially restricted by the expression of mhc class i , as the pretreatment of target cells with mhc class i blocking mab w6 / 32 resulted in & gt ; 60 % inhibition of me275 killing at different e : t ratios ( not shown ). dcs loaded with heat shocked melanoma bodies rapidly yield melanoma - specific ctls : na ive cd8 + t cells primed with heat - treated melanoma bodies can specifically and efficiently kill t2 cells loaded with four melanoma peptides but not psa peptide . melanoma cell lines were also destroyed , but the breast cell line mcf7 and k562 cells were unaffected ( fig9 ). dcs loaded heat - treated killed melanoma cells efficiently educated naive cd8 + t cells to become melanoma - specific cytotoxic t cells . tetramer binding analysis confirmed this finding and showed that up to 0 . 4 % of cd8 + t cells were specific for mart - 1 ( fig1 ). however , other specificities were barely detected . thus , 7 days after the 2 nd stimulation , the t cells were restimulated with autologous dcs pulsed either with each of the four melanoma peptides , or with a control psa peptide . these cultures were analyzed after 7 days of culture . results showed that boosting with melanoma peptide - pulsed dcs expanded melanoma - specific cd8 + t cells ( fig1 ) while no increased expansion was observed in the boost with control peptide ( not shown ). thus , heat treatment of melanoma cells results in enhancement of melanoma - specific responses . heat treatment of melanoma cells enhances transcription of tumor antigens . the original hypothesis that the enhanced cross - priming would be due to enhanced expression of heat shock proteins was found to be consistent with studies that demonstrated purified hsp70 , hsp60 and gp96 act immune adjuvants for cross - priming with antigenic proteins or peptides 90 , 91 . after microarray analysis of control and hsp70 transduced sk - mel28 cells , however , an increased transcription of several tumor antigens including mage - a10 ( not shown ) was observed . therefore , 12 genes encoding different members of mage tumor antigen family were measured by real - time pcr expressions . these antigens included mage - b3 , mage - a8 ( fig1 ), mage - b4 , and mage - a10 ( not shown ). expression of mage - b3 increased up to 10000 fold in addition to sensitivity to actinomycin d thus confirming active transcription ( fig1 ). therefore , hla - a * 0201 restricted peptides derived from mage - a8 and mage - a10 were identified and analyzed to determine whether dcs loaded with heat - treated bodies could prime ctls specific for these two epitopes . as shown in fig1 , hla - a * 0201 + ctls primed against hot hla - a * 0201 − me290 or hla - a * 0201 neg sk - mel28 cells displayed a higher frequency of mage - a10 tetramer binding cd8 + t cells than ctls primed against unheated melanoma cells . thus , hyperthermia - increased transcription of tumor antigens could contribute to enhanced cross - priming . dcs loaded with heat - treated killed melanoma bodies induce autologous naive cd8 + or cd4 + t cells to produce increased levels of ifn gamma and decreased levels of il - 10 regulatory / suppressor t cells are considered to be one of the major obstacles in successful vaccination of patients against their cancer . as shown in fig1 , loading dc vaccines with heat - treated killed melanoma cells resulted in decreased secretion of il - 10 and increased production of ifn gamma when compared to cultures made with dc loaded with unheated tumor . these results support the unique strategy of dc loading in vivo in a clinical trial . therefore , loading dc vaccines with heat - treated and killed melanoma cells leads to induction of broad melanoma - specific type 1 cd8 + t cell immunity . the present invention includes autologous dendritic cells derived from monocytes with gm - csf and ifn alpha and loaded with killed allogeneic colo829 melanoma cells . autologous dendritic cells are manufactured from monocytes separated by elutriation from peripheral blood mononuclear cells obtained by apheresis . monocytes are cultured in a closed system in the presence of granulocyte - macrophage colony stimulating factor ( gm - csf ) and interferon alpha ( ifn alpha , loaded with heated and killed allogeneic colo 829 tumor cells and cryopreserved . vaccine is stored in liquid nitrogen ( vapor phase ). dendritic cells are manufactured from the apheresis product which is processed to isolate monocytes using the elutra system ( gambro bct ). monocytes are transferred from elutriation bag into cultures bags ( 100 ml volume each ) and cultured at 1 × 10 6 / 1 ml volume for 72 hours . in this example , the culture media included serum - free media supplemented with recombinant human gm - csf ( 100 ng / ml ; berlex ) and interferon alpha 2b ( 500 iu / ml ; schering plough ). the skilled artisan , however , would know to titrate the amounts , timing and length of exposure of the cells to the gm - csf and / or the interferon alpha to maximize the activation . after initial 24 hours of culture , the vaccine is loaded with killed colo829 cells . after total of 72 hours culture , dendritic cells are harvested , medium and cytokines are washed out with normal saline . the cells are re - suspended in autologous serum containing 10 % dmso and 10 % plasmalyte , and distributed into cryo - vials at 30 × 10 6 cells / vial . the cryovials are frozen using an automated rate controlled freezer and stored in liquid nitrogen ( vapor phase ). elutriation — isolate monocytes . monocytes are then isolated from the peripheral blood mononuclear cells on a gambro bct elutra ™. the elutra system is a semi - automatic , closed system centrifuge that uses continuous counter - flow elutriation technology to separate cells into multiple fractions based on size and density . the elutra system &# 39 ; s intended application is monocyte enrichment . the system automatically collects 5 fractions of cells based on size and density . fraction 5 contains an enriched monocyte population (˜ 90 % purity , n = 7 healthy volunteers apheresis ) which is used for dendritic cell vaccine manufacture . culture — the bag containing fraction 5 is centrifuged and the buffered saline is expressed off and discarded . the cells are resuspended in cellgenix dc culture medium and a sample is removed for cell count , viability and phenotype . once the number of monocytes is determined , the cells are diluted to a concentration of 1 × 10 6 monocytes / ml and sterile connected to afc vuelife culture bags . cytokines gm - csf ( leukine ®) ( berlex inc .) at a concentration of 100 ng / ml and ifn alfa - 2b ( intron a ) ( schering - plough corp .) at a concentration of 500 iu / ml are added and the cells are placed in a 37 ° c . 5 % co 2 incubator for culture . loading — 24 hours after culture initiation , killed tumor cells ( col0829 ) are added as a source of antigen as well as second dose of gm - csf and ifn alfa - 2b . the killed col0829 is prepared in batches using gamma irradiation , tested for sterility and inability to proliferate as measured by tritiated thymidine incorporation , frozen at a concentration of 50 × 10 6 / ml with 10 % dmso in cryovials , and stored in vapor phase nitrogen . an appropriate number of cryovials are thawed so that the dc are loaded 1 killed tumor cell per 2 dendritic cells . the killed col0829 are washed with culture medium 3 times and added to the culture bags in a small volume . dendritic cell harvest . 72 hours after culture initiation , the cells are harvested and the vaccine is cryopreserved . the culture bags are centrifuged , the supernatant is expressed and the cells are washed 3 times with normal saline . washing consists of connecting a bag of normal saline to the culture bag , resuspending the cells in the normal saline , centrifuging the culture bag , and expressing the normal saline into a waste collection bag . after the third wash , the cells are resuspended in plasmalyte which is the freezing solution diluent . a sample is removed for cell count and viability . rate controlled freezing — the vials are frozen using a rate controlled freezer . the vials are placed in a freezing chamber and liquid nitrogen enters the chamber through an electronic solenoid valve . since vaporization is almost instantaneous , controlling the rate at which liquid nitrogen enters the chamber directly controls the rate at which heat is absorbed and removed from the freezing chamber and its contents . cryopreservation . the cells are cryopreserved at a concentration of 30 × 10 6 cells / ml . once the cell number is determined the cells are diluted to 2 × the final concentration with autologous serum . a bag of freezing solution containing a volume equal to the cell volume is prepared . the freezing solution is autologous serum with 20 % dmso and 20 % plasmalyte . working rapidly , the freezing solution is added to the cell bag and the cells are transferred to labeled cryovials . the final concentration of dmso is 10 %. the cryovials are frozen using an automated rate controlled freezer at 1 ° c ./ min and stored in vapor phase nitrogen . storage — the cryovials are transferred from the rate controlled freezer to a liquid nitrogen tank for long term storage . inventory control is maintained in a database by gmp trained personnel . stability of final dosage form of the active pharmaceutical ingredient . description : vials containing 1 ml of cryopreserved cells were quickly thawed at 37 ° and immediately drawn into a syringe containing 9 ml of sterile normal saline ( the condition in which cells will be thawed at the clinical site immediately prior to injection ) . the normal saline was either at room temperature or 4 ° c . ( refrigerated ). the cells were then evenly distributed among 6 tubes and kept at either room temperature or refrigerated . the diluted cells were then assayed at various time points . acceptable results for lot release are at least 50 % recovery of viable cells and at least 50 % viability at 15 min after thaw at room temp . release testing is performed on three vials obtained at the beginning ( 1 st ), in the middle ( 2 nd ), and in the end ( 3 rd ) of the freezing process . frozen vaccine characterization . to further characterize frozen vaccine , beyond viability and capacity to stimulate mlr with cd4 + t cells as described above , one or more of the following may be analyzed : ( 1 ) morphology and phenotype ; ( 2 ) cytokine secretion ; and / or ( 3 ) capacity to induce autologous cd8 + t cell differentiation . fig1 is a flow chart that outlines the overall vaccine manufacture process of the present invention . in step 10 , a patient is selected for inclusion in the vaccine production process . in step 12 , blood apheresis of the patient is conducted to isolate the cells for loading are obtained and selected in step 14 . steps 10 , 12 and 14 may be conducted in day 1 . in the next few days , the cells are cultured ( step 16 ) and loaded ( step 18 ) with the antigen ( s ) that are presented by the antigen - loaded dendritic cells obtained and cultures in step 16 . next , in step 20 the cells may be frozen and stored for future use and / or eventually thawed and released in step 22 . finally , the cell vaccines may be used for injection in step 24 . in one embodiment , the entire process may occur in about 10 days . fig1 shows detailed steps and a timeline for the manufacture of the vaccine of the present invention in which the cells are provided in an elutriation bag ( step 26 ) and transferred to one or more culture bags ( step 28 ). in step 30 , for example , over three culture days one or more cytokines are provided at hour zero . following the exposure of the cells to cytokines the cells are exposed to killed target cells , e . g ., colo629 cells and optionally additional cytokines . finally , the cells may be harvested after about 72 and / or frozen , tested , sterilized and the like . ifnα - dcs were generated in culture bags either unloaded or loaded with killed colo829 cells , frozen and stored at − 80 ° c . for 1 , 2 and 3 weeks . frozen cells were thawed at weeks 1 , 2 and 3 and their morphology was assessed by giemsa staining . as shown in fig1 , both loaded and unloaded ifn - dcs retained dc morphology after freezing / thawing . fig1 shows an example of frozen / thawed vaccine phenotype as analyzed by surface staining with indicated antibodies and flow cytometry . the dcs show expected phenotype consistent with their generation in the presence of ifn - alpha including : expression of cd1 molecules ( cd1a and cd1b / c ), expression of cd14 consistent with ifn - dcs being interstitial dcs ; high level of hla - dr and co - stimulatory cd80 and cd86 molecules . thus , frozen / thawed vaccines retain the morphology and phenotype of ifn - dcs . upon interaction with t cells , dcs secrete cytokines that regulate t cell differentiation . therefore , we assessed cytokines secreted by frozen / thawed ifn - dcs ( either unloaded or loaded with killed colo829 cells ) when exposed to soluble cd40 ligand to replace t cell signal . supernatants were assessed after 6 and 24 hrs culture using multiplex cytokine analysis ( luminex ). the three major cytokines secreted at levels & gt ; lng / ml included il - 8 (˜ 10ng / ml ), il - 6 and mip1 alpha . as expected from our pre - clinical studies , ifn - dcs secreted il - 7 ( fig1 ). furthermore , low levels of il - 10 could be detected (& lt ; 100 pg / ml ). however , il - 10 secretion was not due to loading with killed colo829 cells as the levels were similar in cultures of unloaded or loaded dcs ( fig1 ). finally , il - 10 secretion appeared donor - related ( data not shown ). the ultimate parameter of a dc vaccine , is its capacity to present tumor antigen to autologous cd8 + t cells and induce their differentiation . thus , frozen / thawed hla - a * 0201 + ifn - dcs were stimulated for 24 hrs with lps ( 5 or 10 ng / ml ), pulsed in the last 10 hrs with mart - 1 peptide and used as stimulators of purified autologous cd8 + t cells . t cell cultures were boosted once at day 7 , supplemented with il - 7 and il - 2 and t cell differentiation was assessed by tetramer staining at day 5 after boost . as shown in fig1 , at the dc : t cell ratio 1 : 10 ( i . e ., 10 5 dc per 10 6 t cells ) at day 12 of culture , ˜ 2 % of cd8 + t cells specifically bound mart - 1 tetramer . furthermore , the differentiation of mart - 1 specific cd8 + t cells could observed even at dc : t cell ratio as low as 1 : 33 , i . e ., 3000 dcs per 10 6 t cells . these results demonstrate that frozen ifn - dcs retain their morphology , phenotype and capacity to expand antigen - specific cd8 + t cells . preliminary data on the feasibility of product manufacture and release in patients with metastatic melanoma . to date we have gathered data on product manufacture in 7 patients with stage iv melanoma . these are preliminary results obtained in the course of an ongoing clinical trial ( irb # 005 - 065 , ind # 12339 ). these preliminary lot manufacture data using cells from patients with metastatic melanoma who underwent chemotherapy suggest feasibility of the process which was developed using cells from healthy donors as described in prior sections . sunsequent tables summarize lots manufactured to date using patient &# 39 ; s material . banchereau , j ., ueno , h ., dhodapkar , m ., connolly , j . e ., finholt - perry , j ., klechevsky , e ., blanck , j - p ., johnston , d a ., steinman , r ., palucka , a k ., fay , j . ( 2005 ) immune and clinical outcomes in patients with stage iv melanoma vaccinated with peptide - pulsed dendritic cells derived from cd34 + progenitors and activated with type i interferon . j immunother . dubsky , p ., saito , h ., dantin , c ., connolly , j ., banchereau , j . and palucka , a k . ( 2005 ) il - 15 - induced human dendritic cells efficiently prime low frequency melanoma - specific naïve cd8 + t cells to differentiate into cytotoxic t cells . submitted saito , h ., dubsky , p ., dantin , c ., finn , o j ., banchereau , j . and palucka , a k . cross - priming of cyclin b1 , muc - 1 and survivin peptide - specific cd8 + t cells by dendritic cells loaded with killed allogeneic breast cancer cells . submitted . ueno , h ., connolly , j e ., vence , l ., palucka , a k ., and banchereau , j . ( 2005 ) global assessment of tumor - antigen specific human t cell repertoire . in preparation palucka , a k ., ueno , h ., connolly , j ., kemeis - norvell , f ., blanck , j - p ., johnston , d a ., fay , j ., and banchereau , j . 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