Multiple chaperone vaccines

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If individual chaperone protein vaccines are effective against tumors, would one vaccine containing numerous chaperone protein family members be better? The perceived advantages would be the probability of increasing the overall number of peptides available for antigen presentation because of multiple chaperone proteins, as well as the odds of increasing peptide diversity if members of the different chaperone families tend to escort distinct sets of peptides. Although their utilization has been suggested (74), part of the reason that such multichaperone vaccines are not commonplace lies in the difficulty of purifying sufficient quantities of multiple chaperones from a single source. Though such schemes are available (75,76), they are time consuming and generate numerous protein fractions requiring analysis prior to vaccine preparation. Our group set out to find a method that might enhance the rapid purification of multiple chaperones while keeping the workload and turnaround time manageable and acceptable. We have utilized

Days Post Tumor Challenge Days Post Re-Challenge

Fig. 1. Dendritic cells pulsed with chaperone proteins or with CRCLs (chaperone-rich cell lysates) generate potent antitumor immunity. Left panel: mice were inoculated with a lethal number of 12B1 leukemia cells on Day 0, and were vaccinated on Day 2 with 0.5 million syngeneic dendritic cells that had been left unpulsed or were pulsed with 12B1 tumor-derived HSP70, GRP94/gp96, or CRCL. Tumor volumes (in mm3) were measure thereafter. Right panel: tumor-free mice (3 of 12 from each DC/HSP70 and DC/GRP94 groups, and 9 of 12 from DC/CRCL group) were rechallenged with a lethal 12B1 tumor dose 80 d following the initial tumor inoculum. Age-matched naive mice (n = 4, PBS) also received tumor challenge at that time. Tumor volumes over time were again measured in mm3.

Days Post Tumor Challenge Days Post Re-Challenge

Fig. 1. Dendritic cells pulsed with chaperone proteins or with CRCLs (chaperone-rich cell lysates) generate potent antitumor immunity. Left panel: mice were inoculated with a lethal number of 12B1 leukemia cells on Day 0, and were vaccinated on Day 2 with 0.5 million syngeneic dendritic cells that had been left unpulsed or were pulsed with 12B1 tumor-derived HSP70, GRP94/gp96, or CRCL. Tumor volumes (in mm3) were measure thereafter. Right panel: tumor-free mice (3 of 12 from each DC/HSP70 and DC/GRP94 groups, and 9 of 12 from DC/CRCL group) were rechallenged with a lethal 12B1 tumor dose 80 d following the initial tumor inoculum. Age-matched naive mice (n = 4, PBS) also received tumor challenge at that time. Tumor volumes over time were again measured in mm3.

a free solution-isoelectric focusing (FS-IEF) method to achieve the enrichment of the most commonly cited immunogenic chaperones (GRP94/gp96, HSP90, HSP70, and calreticulin) from tumor lysates in a limited number of fractions (73,77,78). We have termed these vaccines chaperone-rich cell lysates (CRCLs). CRCLs are clearly more effective as anticancer vaccines than unfractionated tumor lysate and are at least as effective as either purified HSP70 or GRP94/gp96 (73,77,78). The turnaround time on vaccine preparation can be as little as 24 h, and the yields are 1-2 mg of vaccine material per gram of murine or human tumor. Our group and others have also found that dendritic cells (DCs) pulsed with chaperone proteins are extremely effective cellular vaccines (30,60), capable of eradicating preexisiting tumors in mice (73,78; Fig. 1). The interaction of chaperones with antigen-presenting cells (APCs) thus becomes the next topic of discussion.

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