Heat Shock Proteins As Targets For Immunotherapy In Multiple Myeloma

Multiple myeloma is the second most common hematologic cancer.The incidence variesfrom1per100000people in China, to about4per100000people in most developedcountries. The American Cancer Society estimates that20,180new cases of Multiplemyeloma will be diagnosed in the United States in the year2010. Median survival afterconventional treatments is3–4years; high-dose treatment followed by autologous stem-celltransplantation can extend median survival to5–7years. Novel drugs, for use alone and incombination with existing treatments, are increasingly being assessed for ability to furtherimprove survival. But the vast majority of these patients are certain to develop diseaserecurrence after SCT or novel drug therapy.Allogeneic hematopoietic stem celltransplantation (SCT) is a potentially curative approach in patients with multiple myeloma,it result in long-term progression-free survival (PFS), with a plateau in survival curvessuggesting possible cure. However, the role and timing of allogeneic SCT in the diseasecourse are still controversial. And it was associated with excessively high rates ofnonrecurrence mortality (NRM), reaching30%to50%, leading to a nearly completeabandonment of this approach.The origin of recurrence is the minimal residual disease(MRD)exist in vivo. Additional measures are required urgently after transplantation to eliminateminimal residual disease. Immunotherapy is an appealing option for this purpose toconsolidate the curative effect after stem cell transplantation.Heat shock proteins (Hsps) may belong to this category of tumor-associated antigens.Hsps, including Hsp27, Hsp70and Hsp90, are abundantly and preferentially expressed invarious cancers,including MM,and their (over)expression in some cancers correlates withpoor prognosis and resistance to therapy. Moreover, these proteins have emerged as being ofprime importance for the survival of cancer cells. Downregulating or inhibiting Hspexpression results in significant apoptosis in cancer cells, including myeloma cells. One ofthe inhibitors for Hsp90was studied in recently completed phase I testing, and it was welltolerated. Owing to these special properties, Hsps have become a novel and valid target for cancertherapeutics. Hsps over expressed in myeloma cells and low expressed in normalcells,meanwhile hsps took prime importance for the survival of cancer cells.The observationthat tumor cells contain Hsp90complexes in an activated, high-affinity conformationwhereas Hsp90in normal cells is in a latent, uncomplexed state, allows a selective targetingof the molecules in cancer cells.We hypothesize that the broad expression of Hsps in myeloma, together with theirfunctional role as anti-apoptotic molecules in cancer cells, makes them a new ideal target forimmunotherapy in MM.We have make three aims in this experiment:1. Identify HLA-A0201restricted epitopes on Hsps and validate its immunogenicity;2. Examine in vitro cytolyticeffects of the CTLs on myeloma cells, including cell lines and primary myeloma cells frompatients, and on normal hematopoietic cells;3. Construction a novel myeloma-SCID-rab host.Part Ⅰ Identify HLA-A0201restricted epitopes on Hsps and validate its immunogenicity.Objective:To identify HLA-A0201restricted epitopes on Hsps and validate itsimmunogenicity.Methods:The sequence of Hsp27, Hsp70, and Hsp90α have been reviewed for peptidesthat potentially bind to the HLA-A0201molecule using a peptide-motif scoring system(http://bimas.dcrt.nih.gov/molbio/hla_bind/). We also used another system, the SYFPEITHIdatabase (http://syfpeithi.bmi-heidelberg.com) to confirm the results. Four peptides withthe highest scores and/or with shared sequences among the Hsps will be synthesized, andtheir affinity for HLA-A0201will be confirmed in a functional peptide-binding assay usingthe HLA-A0201+T2hybridoma (11). The two best peptides from each of the Hsps will beused to generate peptide-specific CTLs from HLA-A0201+blood donors.HLA-A-0201-peptide tetramers will be constructed to monitor the T cells.Results: Four peptides from each of the Hsps based on the highest scores obtained with theNIH/BIMAS system,are aa27from Hsp27with the sequence of RLFDQAFGL,aa393fromHsp70with the sequence of LLLLDVAPL,aa362from Hsp90α with the sequence ofKLYVRRVFI,aa670from Hsp90α with the sequence of ALLSSGFSL, which were also (in most of the cases) the best-scored peptides by the SYFPEITHI system.These peptides arebeing synthesized. We have used the functional peptide-binding assay to determine two ofthe four peptides,aa27and aa670should be used to generate CTLs.We also have found thehigh level of Hsp27-CTLs and Hsp90α-CTLs in patient by HLA-A0201-peptidetetramers,which confirmed that the aa27and the aa670exist in physiological state.Conclusion:The two best peptides from each of the Hsps are aa27from Hsp27and aa670from Hsp90α,and we have detected the Hsp-CTLs in patient with multiple myeloma byHLA-A0201-peptide tetramers. Part II Examine in vitro cytolytic effects of the CTLs on myeloma cells, including cell linesand primary myeloma cells from patients, and on normal hematopoietic cells.Objective:To examine in vitro the cytolytic effects of the CTLs on myeloma cells, includingcell lines and primary myeloma cells from patients, and on normal hematopoietic cells.Methods: The two best peptides from each of the Hsps will be used to generatepeptide-specific CTLs from a HLA-A0201+blood donor, Peptide-pulsed autologousdendritic cells (DCs) will be used as antigen-presenting cells (APCs). HLA-A0201-peptidetetramers will be constructed to monitor the T cells. Hsp-CTLs will be tested for theircytolytic activity against a panel of myeloma cell lines and primary myeloma cells isolatedfrom patients, whether or not they are HLA-A0201+. Other properties of these T cells, suchas the cytokine-section profile, MHC restriction, and surface markers, will also be analyzed.We used T-cell proliferation assays to monitor a specific T-cell response.Results: Mature DCs were generated as described in the Methods, and a27-or a670pulsedDCs were cocultured with autologous T cells from one HLA-A0201donor. We havecultured T cells with peptide-pulsed DCs for4weeks and we found the percentage oftetramer increasing from less1%to more and more than20%,also these hsp-CTLs display adistinct phenotype from unstimulated T cells recognized by high level of CD45RO and thelow level of CD45RA.These Hsp-CTLs show specific cell proliferation in response to HLA-A0201+myeloma cell line or HLA-A0201+primary myeloma cells.However,theHsp-CTLs did not proliferate in response to the MHC mismatched LP1cell line.TheHsp-CTLs can also induce specific lysis of HLA-A0201+myeloma cell lines and theHLA-0201+primary myeloma cells but have no effect on the HLA-A0201+normal PBMC orHLA-0201-myeloma cell lines.The Hsp-CTLs which incubated with HLA-A0201+myeloma cell line ARH-77displayed increased CD107a expression on CD8+T cells.Incontrast,unstimulated T cells from donor showed no expression of CD107a on CD8+cells inresponse to stimulation with the HLA-A0201+myeloma cell line ARH-77.The Hsp-CTLsexpressed high levels of perforin in response to HLA-A0201+myeloma cell line U266.Conclusion:The two Hsp-derived peptides can be used to generate specific CTLs fromhealthy blood donor.The Hsp-CTLs showed CTL-mediated cytotoxicity to MHC matchedprimary myeloma cells or myeloma cell lines.The Hsp-CTLs killed the MHC matchedmyeloma cells via the perforin pathway,because they expressed high levels of perforin. PartⅢ Determine the in vivo effects of the CTLs on primary myeloma cells using amyeloma-SCID-rab host system.Objective: Determine the in vivo effects of the CTLs on primary myeloma cellsMethods: CB.17/ICr-SCID mice(6-8-week old) were obtained from Vital Rive and pregnantNew Zealand rabbits from Sheng Wang.The femora and tibiae of3-4-week-old rabbits werecut inserted subcutaneously through a small incision.The engraftment of the bones wasallowed to tak place for6-8weeks.3-10×106purified PCs in100ul of phosphate-bufferedsaline(PBS) were injected directly into the implanted rabbit bone.At least two mice wereused for each experimenting.Mice were periodically took X-ray images and bled from thetail vein to judge the time tumor begin to grow. The treatment consisted of three weeklyinjections of10×106CTL cells. The survival was calculating when injected PCs and gets theend point when the tumor reached225mm2. Mice were deeply anesthetized and euthanizedby cervical dislocation,the tumor was taken to have immunohistochemistry andhistochemistry research. Results:Four SCID-rab mouse in nine burden myeloma tumor after3-4weeks.Tumor burdenSCID-rab mouse have shorter survival comparing with the tumor free SCID-rab mouse.Conclusion: The purified primary multiple myeloma cells can form tumor in the SCID-rabmouse.