Haematologica 2003 - Supplements
Haematologica 2003 - Supplements Haematologica 2003 - Supplements
antigen. 3 This induces high levels of epitope-specific CTL able to produce IFN and to kill tumor cells. To test operation of this design in human subjects, we have used an epitope derived from cytomegalovirus (CMV). The chosen epitope is a focus of the HLA-A*0201-restricted immune response during a natural infection with CMV. 4 We have shown that the pDOM-epitope design induces high levels of responding CTL in HLA-A*0201 transgenic mice, and we are currently vaccinating normal donors of transplants to raise immunity prior to transfer into immunosuppressed patients, vulnerable to reactivation or infection with CMV. For myeloma, we have assessed the preclinical performance of a vaccine design incorporating an epitope from a known epitope from a cancer-testis antigen. Results: Clinical testing: So far we have vaccinated the first donor with DNA scFv-FrC with the scFv derived from the recipient’s tumor. A significant specific T-cell proliferative response against both FrC and against the patient’s idiotypic protein was observed to develop at the 16 week time point and to persist for several weeks. The response to FrC was a memory response with apparent fluctuation of levels of responding T cells during the injection period, likely due to the movement of cells to the site of injection. The response to idiotypic protein rose steadily from week 12 onward with no significant response to control Ig. The immune cells have been transferred to the patient during DLI and the patient appears to be doing well. Analysis of immune status of patients with myeloma: To consider extending the approach to a larger number of patients, we need information on the immune status of myeloma patients undergoing more conventional chemotherapy. We have first assessed patients who have undergone autologous stem cell transplant following high dose melphalan. We have used conventional tetanus toxoid as a test vaccine to facilitate comparison of immune responses with those of patients being vaccinated with the DNA constructs containing the gene encoding the FrC portion of tetanus toxin. Patients were assessed from 2-15 months post transplant for their ability to produce antibody or T cell responses to tetanus toxoid. Most patients responded with antibody and proliferative responses, and all reached protective levels of anti-tetanus antibody. In several patients, IFN-producing cells were detected. While responses tended to be lower than those of normal subjects or patients with MGUS, it is encouraging that immune capacity appears to have recovered during the relatively short post-transplant period. Cancer testis antigens as potential targets for immune attack: The P1A antigen is a representative of the cancer-testis group of tumor antigens, and it provides a useful pre-clinical model for investigating the potential for immune attack on these proteins. 5 There is a single point mutation at codon 42 which generates an amino acid change, and this mutation can create a peptide target, designated AB, for immune attack. Vaccination with the construct encoding a minimal FrC domain fused to the peptide sequence (p.DOM-AB) generated high levels of AB peptidespecific IFN-producing CTL able to kill tumor cells and protect against challenge. These results set the scene for designing DNA vaccine incorporating candidate tumor-derived peptide sequences from the family of cancer testis antigens expressed by myeloma cells. 1. King CA, Spellerberg MB, Zhu D, Rice J, Sahota SS, Thompsett AR, Hamblin TJ, Radl J & Stevenson FK. (1998) DNA vaccines with single chain Fv fused to Fragment C of tetanus toxin induce protective immunity against lymphoma and myeloma. Nature Med. 4(11):1282-86. 2. van Baren N, Brasseur F, Godelaine D et al (1999) Genes encoding tumor-specific antigens are expressed in human myeloma cells. Blood 94:1156-64. 3. Rice J, Buchan S & Stevenson FK. (2002). Critical components of a DNA fusion vaccine able to induce protective cytotoxic T cells against a single epitope of a tumor antigen. J Immunol 169:3908-3913. 4. Wills MR, Carmichael AJ, Mynard K, Jin X, Weekes MP, Plachter B & Sissons JG. (1996) the human cytotoxic T- lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65:frequency, specificity, and T-cell receptor usage of pp65-specific CTL. J Virol 70:7569-79. 5. van den Eynde B, Mazarguil H, Lethe B, Laval F & Gairin JE. (1994) Localization of two cytotoxic T lymphocyte epitopes and three anchoring residues on a single nonameric peptide that binds to H-2L d and is recognized by cytotoxic T lymphocytes against mouse tumor P815. Eur J Imm 24:2740-5. P13.2 Abstract withdrawn P13.3 DENDRITIC CELL-BASED IMMUNOTHERAPY IN MULTIPLE MYELOMA. Nikhil C. Munshi Dana Farber Cancer Institute, Harvard Medical School, Boston MA Myeloablative therapies requiring hematopoietic stem cell support can induce complete remissions in up to 40% of multiple myeloma (MM) patients. However, ultimately patients experience disease progression and there is no cure. Novel therapeutic interventions for this disease are therefore needed to specifically target the myeloma cell and its microenvironment. The potential susceptibility of multiple myeloma to immune based therapy has been demonstrated in the allogeneic transplantation through graft versus myeloma effect. A major focus of investigation therefore has been the use of immune-based therapies in a minimal disease setting to decrease the risk of relapse and potentially achieve curative outcomes. Multiple Myeloma (MM) is associated with a number of dysfunctions in both humoral and cellular immunity (1). Abnormalities in immune cell numbers have been associated with inferior disease prognosis. However, both cellular and humoral immuine responses have been observed against both viral and tumor antigens. We have also confirmed improvement in the immune responsiveness in MM patients following effective therapy especially high-dose chemotherapy followed by autologous stem cell transplantation by recovery of uninvolved immuinoglobulins and by significantly higher frequencies of viral antigen-specific T cells in the peripheral blood (2). These results have confirmed optimal time for immunotherapy of MM. We have investigated various immunotherapeutic approaches using idiotype as a myeloma-specific antigen. In a clinical protocol involving 49 patients with minimal disease status following tandem autologous transplantation, we utilized patientspecific Id protein coupled with KLH, as a vaccine. The development of an anti-KLH response confirmed immune competence of the myeloma patients. Moreover, induction of Idspecific immune responses including generation of CTL specifically able to lyse MM cells, and a preliminary evidence of a survival benefit was observed. To improve on these results we have investigated the role of dendritic cells (DCs), the most potent antigen-presenting cells (APCs) equipped with the necessary co-stimulatory, adhesion and S84
MHC molecules, in effectively presenting MM-associated antigens to induce specific immune response. We have confirmed that the antigen presenting cells in MM patients are functional, supporting their use in clinical studies (3). To optimize internalization and processing of myeloma protein, we evaluated various laboratory parameters including class of protein, duration of DC pulsing, DC maturational stage and mechanism of uptake using DCs from myeloma patients. These data indicate that cultured DCs from myeloma patients can efficiently and rapidly endocytose different classes of myeloma protein providing support for using myeloma protein-pulsed DCs for generating in vivo anti-myeloma immune responses (4). The clinical trials have investigated vaccination with DCs pulsed with tumor associated peptides or proteins in a variety of human cancers. We have evaluatedIdiotype-pulsed DC vaccinations in MM in 2 studies involving 25 patients, confirming the feasibility of a dendritic cell-based vaccination, development of Id-specific immune responses and even occasional clinical responses (5). However, robust clinical responses have not been observed, and the strategy targeting single known tumor associated antigens is subject to tumor cell resistance mediated by the down-regulation of that single gene product. Approaches being explored to circumvent this limitation are the DC pulsing with whole myeloma cell lysate, or its RNA, or fusing myeloma cell with DC. Preclinical results in both a murine model and with human cells confirm feasibility of presenting a wide array of myelomarelated antigens through DC-myeloma cell fusions and the development of CTLs able to lyse primary myeloma cells (6). A clinical study of MM/DC fusion cell vaccination based on this is ongoing. In conclusion, these stepwise improvements in immunotherapeutic strategies directed at myeloma are likely to lead to immune, and more importantly, clinical responses that will eventually help achieve a cure in multiple myeloma. References 1. Munshi, N. C. Immunoregulatory mechanisms in multiple myeloma. Hematology - Oncology Clinics of North America, 11: 51-69, 1997. 2. Maecker, B., Anderson , K. S., von Bergwelt-Baildon, M. S., Vonderheide, R. H., Richardson, P. G., Schlossman, R., Munshi, N. C., Anderson , K. C., Nadler, L. M., and Schultze, J. L. Functional deficits of virus specific cytotoxic T cells in patients with multipel myeloma: impact on cancer vaccine development. Brit J Haematol, In Press, 2003. 3. Raje, N., Gong, J., Chauhan, D., Teoh, G., Avigan, D., Wu, Z., Chen, D., Treon, S. P., Webb, I., Kufe, D., and Anderson, K. C. Bone marrow and peripheral blood dendritic cells from patients with multiple myeloma are phenotypically and functionally normal. Blood, 93: 1487-1495, 1999. 4. Butch, A. W., Kelly, K. A., and Munshi, N. C. Dendritic cells derived from multiple myeloma patients efficiently internalize different classes of myeloma protein. Exp Hematol, 29: 85-92, 2001. 5. Yi, Q., Desikan, K. R., Barlogie, B. B., and Munshi, N. C. Optimizing dendritic cell-based immunotherapy in multiple myeloma. Brit J Haematol, 117: 297-305, 2002. 6. Gong, J., Koido, S., Chen, D., Tanaka, Y., Anderson , K. C., Ohno, T., and Kufe, D. Immunization against murine multiple myeloma with fusions of dendritic and plamsacytoma cells is potentiated by interleukin-12. Blood, 99.: 2512-2517., 2002. P13.4 CYTOTOXIC T LYMPHOCYTES AND ADOPTIVE IMMUNOTHERAPY Qing Yi, MD, Ph.D. Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, 4301 West Markham Street, Slot 776, Little Rock, Arkansas 72205, USA. Tel: (501) 686 8250, Fax: (501) 686 6442, Email: YiQing@uams.edu Multiple myeloma (MM) is still a fatal disease. Despite advances in high-dose chemotherapy and autologous stem-cell support, relapses of the underlying disease remain the primary cause of treatment failure. Novel therapeutic approaches that have a mode of action different from and non-cross-resistant with cytotoxic chemotherapy are required to eradicate tumor cells that have become multidrugresistant. To this end, immunotherapy aimed at inducing or enhancing myeloma-specific immunity in tumor-bearing patients may be desirable. Indeed, in the post-allograft relapse setting of MM (in which patients are chemotherapy refractory), long-lasting disease remission has been achieved after infusion of donor lymphocytes, suggesting that chemotherapy and T cell-mediated cytolysis kill myeloma cells by different modes of action that are non-cross-resistant. Nevertheless, the development of severe graftversus-host disease in this setting calls for more specific immunotherapy strategies. Plasma cells represent the major tumor burden and constitute 10% to 100% of the total bone marrow cell count. Myeloma plasma cells secrete a monoclonal M-protein and express cytoplasmic, but not surface, Ig that carry idiotype (Id) determinants. We and others have shown that myeloma plasma cells may express MHC class I, adhesion molecules CD44, CD56, CD54, and costimulatory molecules CD40 and CD28. These cells were able to activate alloreactive T cells and present recalled antigens to autologous T cells. Thus, it is a consensus that T cell-mediated immunity may play a role in controlling the growth of myeloma cells. Ideally, a tumor-specific immunotherapy should induce or expand only the beneficial immune responses mediated by cytotoxic T lymphocytes (CTLs) including CD4 + Th1 and CD8 + Tc1 subsets that have sufficient cytotoxic effects towards tumor cells. Id proteins are tumor-specific antigens and active immunization against Id determinants on malignant B cells has produced resistance to tumor growth in transplantable murine B-cell lymphoma and plasmacytoma. Various approaches have been used to visualize the existence of Id-specific immune response in human disease. By using the enzyme-linked immunospot (ELISPOT) assay, we were able to detect a low frequency of Id-specific T cells in most of myeloma patients with an early disease. We have also shown that Id-specific Th1 cells were significantly higher in patients with indolent disease than those with advanced MM. In contrast, cells secreting Th2-subtype cytokine (IL-4) were seen more frequently in advanced patients. These findings provide indirect evidence that Id-specific T cells may have regulatory effects on human myeloma cells. To examine whether Id-specific T cells can recognize and kill myeloma cells, we generated Id-specific CTL lines from myeloma patients. The results showed that Id-specific CTLs not only recognized and lysed autologous Id-pulsed dendritic cells (DCs) but also significantly killed autologous primary myeloma cells. The cytotoxicity was MHC class I- and, to a lesser extent, class IIrestricted, suggesting that myeloma cells could process Id protein and present Id peptides in the context of their surface MHC molecules. No cytolytic activity against K562 was noted, indicating that the cytotoxicity was not attributed to natural killer cells. The CTLs lysed the target cells mainly through the perforin-mediated pathway. S85
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MHC molecules, in effectively presenting MM-associated<br />
antigens to induce specific immune response. We have confirmed<br />
that the antigen presenting cells in MM patients are functional,<br />
supporting their use in clinical studies (3). To optimize<br />
internalization and processing of myeloma protein, we evaluated<br />
various laboratory parameters including class of protein, duration<br />
of DC pulsing, DC maturational stage and mechanism of uptake<br />
using DCs from myeloma patients. These data indicate that<br />
cultured DCs from myeloma patients can efficiently and rapidly<br />
endocytose different classes of myeloma protein providing<br />
support for using myeloma protein-pulsed DCs for generating in<br />
vivo anti-myeloma immune responses (4).<br />
The clinical trials have investigated vaccination with DCs pulsed<br />
with tumor associated peptides or proteins in a variety of human<br />
cancers. We have evaluatedIdiotype-pulsed DC vaccinations in<br />
MM in 2 studies involving 25 patients, confirming the feasibility<br />
of a dendritic cell-based vaccination, development of Id-specific<br />
immune responses and even occasional clinical responses (5).<br />
However, robust clinical responses have not been observed, and<br />
the strategy targeting single known tumor associated antigens is<br />
subject to tumor cell resistance mediated by the down-regulation<br />
of that single gene product. Approaches being explored to<br />
circumvent this limitation are the DC pulsing with whole<br />
myeloma cell lysate, or its RNA, or fusing myeloma cell with<br />
DC. Preclinical results in both a murine model and with human<br />
cells confirm feasibility of presenting a wide array of myelomarelated<br />
antigens through DC-myeloma cell fusions and the<br />
development of CTLs able to lyse primary myeloma cells (6). A<br />
clinical study of MM/DC fusion cell vaccination based on this is<br />
ongoing. In conclusion, these stepwise improvements in<br />
immunotherapeutic strategies directed at myeloma are likely to<br />
lead to immune, and more importantly, clinical responses that<br />
will eventually help achieve a cure in multiple myeloma.<br />
References<br />
1. Munshi, N. C. Immunoregulatory mechanisms in<br />
multiple myeloma. Hematology - Oncology Clinics of North<br />
America, 11: 51-69, 1997.<br />
2. Maecker, B., Anderson , K. S., von Bergwelt-Baildon,<br />
M. S., Vonderheide, R. H., Richardson, P. G., Schlossman, R.,<br />
Munshi, N. C., Anderson , K. C., Nadler, L. M., and Schultze, J.<br />
L. Functional deficits of virus specific cytotoxic T cells in<br />
patients with multipel myeloma: impact on cancer vaccine<br />
development. Brit J Haematol, In Press, <strong>2003</strong>.<br />
3. Raje, N., Gong, J., Chauhan, D., Teoh, G., Avigan, D.,<br />
Wu, Z., Chen, D., Treon, S. P., Webb, I., Kufe, D., and Anderson,<br />
K. C. Bone marrow and peripheral blood dendritic cells from<br />
patients with multiple myeloma are phenotypically and<br />
functionally normal. Blood, 93: 1487-1495, 1999.<br />
4. Butch, A. W., Kelly, K. A., and Munshi, N. C.<br />
Dendritic cells derived from multiple myeloma patients<br />
efficiently internalize different classes of myeloma protein. Exp<br />
Hematol, 29: 85-92, 2001.<br />
5. Yi, Q., Desikan, K. R., Barlogie, B. B., and Munshi, N.<br />
C. Optimizing dendritic cell-based immunotherapy in multiple<br />
myeloma. Brit J Haematol, 117: 297-305, 2002.<br />
6. Gong, J., Koido, S., Chen, D., Tanaka, Y., Anderson ,<br />
K. C., Ohno, T., and Kufe, D. Immunization against murine<br />
multiple myeloma with fusions of dendritic and plamsacytoma<br />
cells is potentiated by interleukin-12. Blood, 99.: 2512-2517.,<br />
2002.<br />
P13.4<br />
CYTOTOXIC T LYMPHOCYTES AND ADOPTIVE<br />
IMMUNOTHERAPY<br />
Qing Yi, MD, Ph.D.<br />
Myeloma Institute for Research and Therapy, University of<br />
Arkansas for Medical Sciences, 4301 West Markham Street, Slot<br />
776, Little Rock, Arkansas 72205, USA. Tel: (501) 686 8250, Fax:<br />
(501) 686 6442, Email: YiQing@uams.edu<br />
Multiple myeloma (MM) is still a fatal disease. Despite advances in<br />
high-dose chemotherapy and autologous stem-cell support, relapses<br />
of the underlying disease remain the primary cause of treatment<br />
failure. Novel therapeutic approaches that have a mode of action<br />
different from and non-cross-resistant with cytotoxic chemotherapy<br />
are required to eradicate tumor cells that have become multidrugresistant.<br />
To this end, immunotherapy aimed at inducing or<br />
enhancing myeloma-specific immunity in tumor-bearing patients<br />
may be desirable. Indeed, in the post-allograft relapse setting of<br />
MM (in which patients are chemotherapy refractory), long-lasting<br />
disease remission has been achieved after infusion of donor<br />
lymphocytes, suggesting that chemotherapy and T cell-mediated<br />
cytolysis kill myeloma cells by different modes of action that are<br />
non-cross-resistant. Nevertheless, the development of severe graftversus-host<br />
disease in this setting calls for more specific<br />
immunotherapy strategies.<br />
Plasma cells represent the major tumor burden and constitute 10%<br />
to 100% of the total bone marrow cell count. Myeloma plasma cells<br />
secrete a monoclonal M-protein and express cytoplasmic, but not<br />
surface, Ig that carry idiotype (Id) determinants. We and others<br />
have shown that myeloma plasma cells may express MHC class I,<br />
adhesion molecules CD44, CD56, CD54, and costimulatory<br />
molecules CD40 and CD28. These cells were able to activate<br />
alloreactive T cells and present recalled antigens to autologous T<br />
cells. Thus, it is a consensus that T cell-mediated immunity may<br />
play a role in controlling the growth of myeloma cells. Ideally, a<br />
tumor-specific immunotherapy should induce or expand only the<br />
beneficial immune responses mediated by cytotoxic T lymphocytes<br />
(CTLs) including CD4 + Th1 and CD8 + Tc1 subsets that have<br />
sufficient cytotoxic effects towards tumor cells.<br />
Id proteins are tumor-specific antigens and active immunization<br />
against Id determinants on malignant B cells has produced<br />
resistance to tumor growth in transplantable murine B-cell<br />
lymphoma and plasmacytoma. Various approaches have been used<br />
to visualize the existence of Id-specific immune response in human<br />
disease. By using the enzyme-linked immunospot (ELISPOT)<br />
assay, we were able to detect a low frequency of Id-specific T cells<br />
in most of myeloma patients with an early disease. We have also<br />
shown that Id-specific Th1 cells were significantly higher in<br />
patients with indolent disease than those with advanced MM. In<br />
contrast, cells secreting Th2-subtype cytokine (IL-4) were seen<br />
more frequently in advanced patients. These findings provide<br />
indirect evidence that Id-specific T cells may have regulatory<br />
effects on human myeloma cells.<br />
To examine whether Id-specific T cells can recognize and kill<br />
myeloma cells, we generated Id-specific CTL lines from myeloma<br />
patients. The results showed that Id-specific CTLs not only<br />
recognized and lysed autologous Id-pulsed dendritic cells (DCs)<br />
but also significantly killed autologous primary myeloma cells. The<br />
cytotoxicity was MHC class I- and, to a lesser extent, class IIrestricted,<br />
suggesting that myeloma cells could process Id protein<br />
and present Id peptides in the context of their surface MHC<br />
molecules. No cytolytic activity against K562 was noted, indicating<br />
that the cytotoxicity was not attributed to natural killer cells. The<br />
CTLs lysed the target cells mainly through the perforin-mediated<br />
pathway.<br />
S85