Patent Abstract:
the present invention relates to an adjuvant derived from human lymphocytes . the adjuvant can be used in combination with traditional vaccines or cancer immunotherapy , to enhance the response of the patient &# 39 ; s immune system to the vaccine or other immunotherapeutic agent . the adjuvant is derived from the supernatant collected from cultured activated lymphocytes .

Detailed Description:
in one aspect , the present invention provides a method of enhancing the immune response to a vaccine antigen in a host mammal , comprising administering lymphocyte conditioned medium , the supernatant derived from activated human lymphocyte cells cultured with growth media , in combination with the vaccine antigen . preferably , the mammal is a human . culture methods and protocol are standard and known in the art . human ( or other mammal , depending on the mammal to be treated ) peripheral blood mononuclear cells ( pbmc ) obtainable from any source are diluted in commercially available tissue culture growth media . the cells are incubated with an activation agent consisting of beads coated with antibodies to cd3 / cd28 . on about day 3 , cells and beads are separated from the culture media and the cells and beads are resuspended in additional growth media as needed . to harvest the cells , they are resuspended in centrifuge tubes and pelleted , after which the supernatant can be drawn off with a pipet and stored for later use . as used herein , the term “ supernatant ” refers to the liquid drawn off the cultured cells , in the manner described above . “ lymphocyte conditioned medium ” and “ supernatant ” are used herein interchangeably , and refer to the liquid drawn off the cultured cells . studies have been carried out to characterize the supernatant , and it has been found to contain molecules having an average molecular weights of less than about 100 , 000 daltons . administration is by known methods used for vaccination , and suitable delivery methods include , but are not limited to , intramuscular , intercutaneous and subcutaneous injection . typically , about 10 ng to about 500 ng are administered in combination with the antigen . administration can be weekly , biweekly , monthly or yearly , depending on the antigen and the level of immune response desired . enhancement of the immune response can be observed , for example , by conducting standard assays known in the art that assess cellular immunity ( such as t cell proliferation ) and measure antibody titres post immunization . the present invention is suitable for use with a large variety of vaccines , including , but not limited to , measles , mumps , rubella , influenza , haemophilus influenzae type b vaccines , diphtheria , tetanus , pertussis , pneumococcal polysaccharide vaccines , meningococcal polysaccharide vaccines , staphylococcus aureus vaccines , respiratory syncytial virus , streptococcus , parainfluenza mycoplasma pneumoniae , mycobacterium leprae , nocardia , legionella , pseudomonas , cholera vaccines , typhoid fever , poliovirus , hepatitis a vaccine , rotavirus , escherichia coli , shigella , hepatitis e , listeria , giardia lamblia , toxocariasis , trichuriasis , ascariasis , amebiasis , cysticercosis , hepatitis b recombinant and plasma - derived vaccines , hiv - 1 and hiv - 2 ; htlvi and htlv - ii , epstein - barr , hepatitis c , herpes b , human papillomavirus , herpes simplex type 1 and 2 , chlamydia , gonorrhea , treponema ( syphilis ), anthrax , rabies , schistosomiasis , plague , yellow fever vaccines , japanese encephalitis and tick - borne encephalitis vaccines , malaria , leishmaniasis , lyme disease , lymphatic filariasis and onchocerciasis , trypanosomiasis and chagas &# 39 ; disease , rickettsia and typhus fevers , dengue fever , adenovirus vaccines , varicella zoster vaccines , cytomegalovirus , coronaviruses and rhinioviruses , streptobacillus , allergy peptide , infectious disease peptide vaccine , cancer peptide vaccine , autoimmune peptide vaccine , and cancer vaccines utilizing antigen , peptide , dna fragments and / or any other molecular species on the surface or within the cancer cell . the following examples are intended to illustrate the invention and should not be construed as limiting the invention in any way . experiments were carried out using the following materials and methods : human peripheral blood mononuclear cells ( pbmc ) used for preparation of the conditioned media were separated from leukapheresis products of normal healthy donors by density gradient centrifugation in lymphocyte separation medium ( icn biomedicals inc ., aurora , ohio ). the cells were viably frozen in rpmi - 1640 ( invitrogen corp ., grand island , n . y .) containing 20 % human ab serum ( gemini bio - products , woodland , calif .) and 10 % dmso ( sigma , st . louis , mo .) using an automated cell freezer ( gordinier electronics , roseville , mich .) and stored in the vapor phase of liquid nitrogen until used . monocytes were isolated from pbmc by countercurrent centrifugal elutriation and were used immediately or viably frozen in fetal bovine serum ( summit biotechnology ) containing 10 % dmso and 5 % glucose ( sigma ) for later use . cryopreserved pbmc were thawed in rpmi - 1640 supplemented with 20 % human ab serum ( hab ), washed twice with rpmi - 1640 containing 10 % hab . cells were resuspended in crpmi [ rpmi - 1640 supplemented with 10 % hab , 2 mm l - glutamine ( invitrogen ), 1 % penicillin streptomycin solution ( invitrogen ), 20 mm hepes buffer ( invitrogen )] or x - vivo 15 ( biowhittaker , walkersville , md .) supplemented with 2 mm l - glutamine , 1 % penicillin streptomycin solution , 20 mm hepes buffer . cd3 / cd28 dynabeads ( dynal , lake success , n . y .) were added to the cells at 75 : 1 of beads for every 1 × 10 6 pbmc and the cultures were incubated for 3 days at 37 ° c . in 5 % co 2 . subsequently , cell - free supernatants were collected and stored at 2 - 8 ° c . prior to use . lcm was used at a 1 : 1 dilution in crpmi . elutriated monocytes were washed with crpmi , resuspended in equal volumes of lcm and crpmi at a concentration of 5 × 10 5 cells / ml , and cultured in 24 well plates ( denville scientific inc ., metuchen , n . j .) at 37 ° c . in 5 % co 2 for 5 - 6 days . alternatively , monocytes were cultured in crpmi containing 750 u / ml gm - csf ( r & amp ; d systems , minneapolis , minn .) and 720 u / ml il - 4 ( r & amp ; d systems ) for 3 - 4 days followed by addition of the conditioned media or a combination of cytokines [ 10 ng / ml il - 1 ∃, 10 ng / ml tnf ∀, 0 . 91 ng / ml il - 6 ( r & amp ; d systems ), 1 : g / ml pge 2 ( sigma )] ( maturation cocktail ) for an additional 48 hours . the maturation cocktail served as a positive control and monocytes cultured in crpmi only were used as a negative control . cells were washed once in crpmt , resuspended in 1 × pbs ( invitrogen ) containing 5 % hab , and incubated for 15 minutes at room temperature ( 22 - 25 ° c .) in order to block fc receptors . cells were then washed , resuspended in wash buffer ( 1 × pbs with 1 % hab and 0 . 1 % nan 3 ), and labeled with fluorochrome - conjugated antibodies ( bd biosciences , san diego , calif .) against cd14 , cd11c , cd40 , cd83 , cd80 , cd86 , and appropriate isotype control antibodies . after 30 minutes at 4 ° c ., the cells were washed with buffer and fixed in 1 × pbs with 1 % paraformaldehyde . flow cytometric data were acquired using a facscan flow cytometer and analyzed with cellquest software ( becton dickinson , sanjose , calif .). gates were set according to isotype control samples . cytokines and chemokines in lcm were quantified using commercially available enzyme - linked immunosorbent assays ( elisas ; r & amp ; d systems , minneapolis , minn .) according to manufacturer &# 39 ; s guidelines . the concentration of pge 2 was also measured by elisa ( cayman chemical co ., ann arbor , mich .). all determinations were made in duplicate . dendritic cells were harvested from cultures , washed twice in crpmi , and plated into 96 - well u - bottom culture plates ( denville scientific , inc .) at 10 4 , 10 3 , and 10 2 cells per well . allogeneic responder pbmc were added to each well at 1 × 10 5 cell / well in a total volume of 200 : 1 . all conditions were plated in triplicate . the cells were incubated for 3 days at 37 ° c . in 5 % co 2 , pulsed with 1 . 0 : ci tritiated thymidine ( perkin elmer , boston , mass .) for 16 hours , and harvested using an automated multi - well harvester ( tomtec , orange , conn .). the amount of tritiated thymidine incorporated into the responder cells was measured using the microbeta trilux liquid scintillation counter ( wallac , turku , finland ). unprimed normal human pbmc ( 1 × 10 5 / 100 : 1 crpmi ) were plated into 96 - well u - bottom culture plates to which 100 : 1 of lcm or 100 : 1 crpmi with or without 10 : g / ml tetanus toxoid ( accurate chemical and scientific corp ., westbury , n . y .) was added . pbmc cultured in crpmi with 10 : g / ml concanavalina ( sigma , st . louis , mo .) served as a positive control . all conditions were plated in triplicate , cultured at 37 ° c . in 5 % co 2 for 3 and 5 days , pulsed with 1 . 0 : ci tritiated thymidine for 16 hours , and harvested using an automated multi - well harvester . the amount of tritiated thymidine incorporated into the responder cells was measured using the microbeta trilux liquid scintillation counter . lcmd differentiates monocytes to dc in the absence of additional cytokines in an effort to generate a highly effective cytokine cocktail for the generation and maturation of large numbers of human dc from their in vitro precursors , we produced culture supernatants from anti - cd3 / anti - cd28 stimulated pbmc ( lcm ) as described in material and methods and investigated their effect on highly purified human monocytes obtained from pbmc by counter flow centrifugation . monocytes were cultured in lcm in the absence of gm - csf and il - 4 and the expression of cell surface markers that characterize differentiated and matured dc was examined by flow cytometry . prior to culture , monocytes constitutively expressed cd11c , as well as cd14 and hla - dr . culture in lcm resulted in a significant decrease of the percent of cells expressing cd14 by day 3 , and by days only a few cells expressing this marker remained ( fig1 a ). the mfis of hla - dr ( fig1 b ), cd40 ( fig1 c ), cd80 ( fig1 d ) and cd86 ( fig1 e ) were consistently upregulated on cells cultured in lcm as compared to cells cultured on medium alone . in 4 out of 9 experiments , a small percentage of cd83 + dc was observed ; however , these results were not significant . taken together , lcm differentiates monocytes into phenotypically immature dc , a stage of development that is optimal for antigen acquisition . the ability of lcm to mature immature dc that were generated in vitro from monocytes in the presence of gm - csf and il - 4 was assessed . monocytes obtained from pbmc by elutriation were cultured for 3 - 4 days in media containing gm - csf and il - 4 followed by addition of lcm or a standard maturation cocktail containing il - 1 ∃, tnf ∀, il - 6 and pge 2 ( see materials and methods ). flow cytometry was performed 48 hours later . as shown in fig2 and table 1 , both lcm and maturation cocktail induced the expression of hla - dr , costimulatory molecules and cd83 , generally accepted signatures for mature dc . these results indicate that lcm can facilitate the final maturation of immature monocyte - derived dc . the dc maturation activity of lcm obtained from a total of four different donors was very consistent ( data not shown ). table 1 : statistical evaluation of me1 and percent expression of cd40 , cd83 , cd80 , and cd86 on monocytes cultured with gm - csf / il - 4 with (+ lcm ) or without (− lcm ) addition of lcm for the last 48 hours . data is expressed as means ± sem from 8 experiments and statistical significance was determined by the paired two - tailed student t test . to investigate the effect of lcm on the distribution and phenotype of various cell populations contained in peripheral blood in vivo , whole pbmc were cultured in lcm for 3 or 5 days . no differences in the percentages of cd3 +, cd4 +−, cd8 +, cd56 +, and cd19 + cells were found at these two time points ( data not shown ). also , the percentages of cd3 + cells co - expressing cd25 , a marker for activation , did not increase over the 5 - day period of culture ( data not shown ). however , the expression of cd14 on monocytes , identified by their expression of cd11c , declined at day 3 and was almost completely downregulated by day 5 ( fig3 a ). by contrast , the expression of hla - dr , cd40 , cd80 and cd86 on cd11c + cells was upregulated ( fig3 ). thus , lcm differentiates highly purified monocytes as well as monocytes within whole pbmc into cells with a dc - like phenotype . cytokine and chemokine concentrations in lcm ( table 2 ) were determined by standard elisa methods and compared with concentrations of cytokines commonly used for the generation or maturation of dc ( table 3 ). a whole battery of soluble mediators was identified , including gmcsf and il - 4 , inflammatory cytokines ( il - 1 ∃, il - 6 , pge 2 , tnf ∀, ifn ( ) chemokines ( mcp - 1 , mip1 , rantes ), and scd40l ( table 2 ). strikingly , the concentrations of gm - csf and il - 4 in lcm were significantly lower than concentrations of the two cytokines used in standard protocols for the generation of immature dc from monocytes ( table 3 ). one of the actions of il4 on monocytes during the in - vitro differentiation to immature dc is the down regulation of cd14 expression . the low level of il - 4 in lcm may account for the relatively slow downregulation of cd14 on monocytes cultured in lcm ( fig1 a , 3 a ). there were also lower concentrations of inflammatory cytokines in lcm compared to maturation cocktail , even though both maturation agents had comparable effects on immature dcs . table 3 : the quantifies of gm - csf and il4 contained in lcm are compared with those used in standard protocols for the generation of monocyte - derived dcs , as are the concentrations of selected proinflammatory cytokines in lcm and maturation cocktail . * note that the cytokine concentrations for lcm given in this table are half of those in table 2 : because lcm is added to cells at a 1 : 1 dilution in crpmi . a functional characteristic of monocyte - derived dc is their capacity to effectively induce allogeneic responses in vitro . the allogeneic response of pbmc to monocytes that have been cultured for five days in the absence or in the presence of lcm , or that were cultured in media containing gm - csf / il - 4 followed by addition of crpmi or lcm was determined . as shown in fig4 , the stimulation index ( si ) generated by monocytes cultured in lcm alone was approximately 3 times that induced by monocytes that have been cultured in crpmi alone . however , the si was highest with monocytes that have been cultured in gm - csf / il - 4 and matured with lcm . these results demonstrate that lcm renders monocytes and immature dc into more effective antigen presenting cells . in addition the proliferative responses of pbmc to the recall antigen tetanus toxoid ( tt ) were tested , again in the presence or absence of lcm ( fig5 a ). in the absence of lcm and other cytokines , pbmc showed low levels of response to tt ; however with the addition of lcm the response to tt significantly increased at day 6 ( fig5 a ). it is important to point out that lcm alone induced dna synthesis in pbmc even in the absence of specific antigen ; nevertheless the response to the specific antigen was significantly augmented . to determine if the observed response to tt was mediated by monocytes , the experiments were repeated using pbmc that had been depleted of cd 14 + cells with immunomagnetic beads . as illustrated in fig5 b , there was no response to tt with or without the addition of lcm . the higher si in the experiments where monocytes were removed ( fig5 b ) can be accounted for by the relative increase in the numbers of other cell types , particularly the cd3 + cells ( data not shown ). these results clearly indicate that lcm enhances the capacity of the pbmc to respond to ti ′ sand that the response is mediated by monocytes . the above findings indicate that lcm rendered highly purified monocytes and monocytes in whole pbmc into a dc - like phenotype . in addition , immature monocyte - derived dc developed a mature dc phenotype after culture in lcm . functionally , these lcm - derived dc showed an increased capacity to stimulate allogeneic pbmc compared to their monocyte precursors and significantly augmented the response to tt . the observed effects of lcm can be accounted for by the battery of proinflammatory cytokines and chemokines identified in the conditioned medium . whereas particular embodiments of this invention have been described above for purposes of illustration , it will be evident to those skilled in the art that numerous variations of the details of the present invention may be made without departing from the invention as defined in the appending claims . 1 . banchereau j , briere f , caux c , et al . immunobiology of dendritic cells . annu rev immunol jid - 8309206 2000 ; 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