A randomized phase 2 trial of idiotype vaccination and adoptive autologous T-cell transfer in patients with multiple myeloma.

We hypothesized that combining adoptively transferred autologous T cells with a cancer vaccine strategy would enhance therapeutic efficacy by adding antimyeloma idiotype (Id)-keyhole limpet hemocyanin (KLH) vaccine to vaccine-specific costimulated T cells. In this randomized phase 2 trial, patients received either control (KLH only) or Id-KLH vaccine, autologous transplantation, vaccine-specific costimulated T cells expanded ex vivo, and 2 booster doses of assigned vaccine. In 36 patients (KLH, n = 20; Id-KLH, n = 16), no dose-limiting toxicity was seen. At last evaluation, 6 (30%) and 8 patients (50%) had achieved complete remission in KLH-only and Id-KLH arms, respectively (P = .22), and no difference in 3-year progression-free survival was observed (59% and 56%, respectively; P = .32). In a 594 Nanostring nCounter gene panel analyzed for immune reconstitution (IR), compared with patients receiving KLH only, there was a greater change in IR genes in T cells in those receiving Id-KLH relative to baseline. Specifically, upregulation of genes associated with activation, effector function induction, and memory CD8+ T-cell generation after Id-KLH but not after KLH control vaccination was observed. Similarly, in responding patients across both arms, upregulation of genes associated with T-cell activation was seen. At baseline, all patients had greater expression of CD8+ T-cell exhaustion markers. These changes were associated with functional Id-specific immune responses in a subset of patients receiving Id-KLH. In conclusion, in this combination immunotherapy approach, we observed significantly more robust IR in CD4+ and CD8+ T cells in the Id-KLH arm, supporting further investigation of vaccine and adoptive immunotherapy strategies. This trial was registered at www.clinicaltrials.gov as #NCT01426828.

Targeting myeloid-derived suppressor cells for cancer immunotherapy.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with an immune suppressive phenotype. They represent a critical component of the immune suppressive niche described in cancer, where they support immune escape and tumor progression through direct effects on both the innate and adaptive immune responses, largely by contributing to maintenance of a high oxidative stress environment. The number of MDSCs positively correlates with protumoral activity, and often diminishes the effectiveness of immunotherapies, which is particularly problematic with the emergence of personalized medicine. Approaches targeting MDSCs showed promising results in preclinical studies and are under active investigation in clinical trials in combination with various immune checkpoint inhibitors. In this review, we discuss MDSC targets and therapeutic approaches targeting MDSC that have the aim of enhancing the existing tumor therapies.

Phase I study of an active immunotherapy for asymptomatic phase Lymphoplasmacytic lymphoma with DNA vaccines encoding antigen-chemokine fusion: study protocol.

BACKGROUND: There is now a renewed interest in cancer vaccines. Patients responding to immune checkpoint blockade usually bear tumors that are heavily infiltrated by T cells and express a high load of neoantigens, indicating that the immune system is involved in the therapeutic effect of these agents; this finding strongly supports the use of cancer vaccine strategies. Lymphoplasmacytic lymphoma (LPL) is a low grade, incurable disease featuring an abnormal proliferation of Immunoglobulin (Ig)-producing malignant cells. Asymptomatic patients are currently managed by a "watchful waiting" approach, as available therapies provide no survival advantage if started before symptoms develop. Idiotypic determinants of a lymphoma surface Ig, formed by the interaction of the variable regions of heavy and light chains, can be used as a tumor-specific marker and effective vaccination using idiotypes was demonstrated in a positive controlled phase III trial. METHODS: These variable region genes can be cloned and used as a DNA vaccine, a delivery system holding tremendous potential for streamlining vaccine production. To increase vaccination potency, we are targeting antigen-presenting cells (APCs) by fusing the antigen with a sequence encoding a chemokine (MIP-3α), which binds an endocytic surface receptor on APCs. Asymptomatic phase LPL is an excellent model to test our vaccine since patients have not received chemotherapeutics that interfere with innate immune function and have low tumor burden. We are evaluating the safety of this next-generation DNA vaccine in a first-in-human clinical trial currently enrolling asymptomatic LPL patients. To elucidate the mode of action of this vaccine, we will assess its ability to generate tumor-specific immune responses and examine changes in the immune profile of both the peripheral blood and bone marrow. DISCUSSION: This vaccine could shift the current paradigm of clinical management for patients with asymptomatic LPL and inform development of other personalized approaches. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT01209871; registered on September 24, 2010.

Nonstereotyped lymphoma B cell receptors recognize vimentin as a shared autoantigen.

Ag activation of the BCR may play a role in the pathogenesis of human follicular lymphoma (FL) and other B cell malignancies. However, the nature of the Ag(s) recognized by tumor BCRs has not been well studied. In this study, we used unbiased approaches to demonstrate that 42 (19.35%) of 217 tested FL Igs recognized vimentin as a shared autoantigen. The epitope was localized to the N-terminal region of vimentin for all vimentin-reactive tumor Igs. We confirmed specific binding to vimentin by using recombinant vimentin and by performing competitive inhibition studies. Furthermore, using indirect immunofluorescence staining, we showed that the vimentin-reactive tumor Igs colocalized with an anti-vimentin mAb in HEp-2 cells. The reactivity to N-terminal vimentin of IgG FL Igs was significantly higher than that of IgM FL Igs (30.4 versus 10%; p = 0.0022). However, vimentin-reactive FL Igs did not share CDR3 motifs and were not homologous. Vimentin was expressed in the T cell-rich regions of FL, suggesting that vimentin is available for binding with tumor BCRs within the tumor microenvironment. Vimentin was also frequently recognized by mantle cell lymphoma and multiple myeloma Igs. Our results demonstrate that vimentin is a shared autoantigen recognized by nonstereotyped FL BCRs and by the Igs of mantle cell lymphoma and multiple myeloma and suggest that vimentin may play a role in the pathogenesis of multiple B cell malignancies. These findings may lead to a better understanding of the biology and natural history of FL and other B cell malignancies.

Analysis of Polyfunctionality for Enhanced CAR T-Cell Therapy for Hematologic Malignancies.

Chimeric antigen receptor (CAR) T-cell therapy has emerged as a promising immunotherapeutic strategy for eradicating human cancers. Their therapeutic success and durability of clinical responses hinges, in large part, on their functional capacity, including the ability of these engineered cells to simultaneously expand and persist after infusion into patients. CD19 CAR T-cell polyfunctionality, assessing the simultaneous functions of cytokine production, proliferation, and cytotoxicity has been reported to correlate with clinical outcomes. Assay optimization is potentially limited by the heterogeneous nature of CAR T-cell infusion products and target specificity. We optimized a single-cell platform for polyfunctionality using CAR T cell products manufactured from healthy donors, engineered against a novel target, BAFF-R, and validated the protocol using CD19 CAR T cells. We observed distinct qualitative differences between BAFF-R and CD19 CAR T cells relative to the proportions of stimulatory vs. effector cytokines, based on target antigen density, and generally, CD19 CAR T cells exhibited lower indices of polyfunctionality. Finally, we applied our assay to the autologous BAFF-R CAR T-cell product generated from the first NHL patient treated on an ongoing clinical trial who had progressed after prior CD19 CAR T-cell therapy. We observed robust indicators of polyfunctionality, which correlated with successful CAR T cell expansion after infusion and achievement of durable complete remission ongoing after 18 months. The precise identification of factors determining the role of BAFF-R CAR T-cell fitness on toxicity and clinical outcome will require the application of this robust assay in the analysis of additional treated patients.

Immortalized B Cells Transfected with mRNA of Antigen Fused to MITD (IBMAM): An Effective Tool for Antigen-Specific T-Cell Expansion and TCR Validation.

Peripheral mononuclear blood cells (PBMCs) are the most widely used study materials for immunomonitoring and antigen-specific T-cell identification. However, limited patient PBMCs and low-frequency antigen-specific T cells remain as significant technical challenges. To address these limitations, we established a novel platform comprised of optimized HLA-matched immortalized B cells transfected with mRNA of a prototype viral or tumor antigen conjugated to MHC class-I trafficking domain protein (MITD) to increase the efficiency of epitope expression in antigen-presenting cells (APCs) essential to expanding antigen-specific T cells. When applied to CMV as a model, the IBMAM platform could successfully expand CMV-specific T cells from low-frequency CMV PBMCs from seropositive donors. Additionally, this platform can be applied to the validation of antigen specific TCRs. Together, compared to using APCs with synthesized peptides, this platform is an unlimited, highly efficient, and cost-effective resource in detecting and expanding antigen-specific T cells and validating antigen-specific TCRs.

First-in-human clinical trial of personalized neoantigen vaccines as early intervention in untreated patients with lymphoplasmacytic lymphoma.

Lymphoplasmacytic lymphoma (LPL) is an incurable low-grade B-cell lymphoma of the bone marrow. Despite a cumulative risk of progression, there is no approved therapy for patients in the asymptomatic phase. We conducted a first-in-human clinical trial of a novel therapeutic DNA idiotype neoantigen vaccine in nine patients with asymptomatic LPL. Treatment was well tolerated with no dose limiting toxicities. One patient achieved a minor response, and all remaining patients experienced stable disease, with median time to disease progression of 61+ months. Direct interrogation of the tumor microenvironment by single-cell transcriptome analysis revealed an unexpected dichotomous antitumor response, with significantly reduced numbers of clonal tumor mature B-cells, tracked by their unique BCR, and downregulation of genes involved in signaling pathways critical for B-cell survival post-vaccine, but no change in clonal plasma cell subpopulations. Downregulation of HLA class II molecule expression suggested intrinsic resistance by tumor plasma cell subpopulations and cell-cell interaction analyses predicted paradoxical upregulation of IGF signaling post vaccine by plasma cell, but not mature B-cell subpopulations, suggesting a potential mechanism of acquired resistance. Vaccine therapy induced dynamic changes in bone marrow T-cells, including upregulation of signaling pathways involved in T-cell activation, expansion of T-cell clonotypes, increased T-cell clonal diversity, and functional tumor antigen-specific cytokine production, with little change in co-inhibitory pathways or Treg. Vaccine therapy also globally altered cell-cell communication networks across various bone marrow cell types and was associated with reduction of protumoral signaling by myeloid cells, principally non-classical monocytes. These results suggest that this prototype neoantigen vaccine favorably perturbed the tumor immune microenvironment, resulting in reduction of clonal tumor mature B-cell, but not plasma cell subpopulations. Future strategies to improve clinical efficacy may require combinations of neoantigen vaccines with agents which specifically target LPL plasma cell subpopulations, or enable blockade of IGF-1 signaling or myeloid cell checkpoints.

Generation of a humanized afucosylated BAFF-R antibody with broad activity against human B-cell malignancies.

B-cell activating factor receptor (BAFF-R) is a mature B-cell survival receptor, which is highly expressed in a wide variety of B-cell malignancies but with minimal expression in immature B cells. These properties make BAFF-R an attractive target for therapy of B-cell lymphomas. We generated a novel humanized anti BAFF-R monoclonal antibody (mAb) with high specificity and potent in vitro and in vivo activity against B-cell lymphomas and leukemias. The humanized variants of an original chimeric BAFF-R mAb retained BAFF-R binding affinity and antibody-dependent cellular cytotoxicity (ADCC) against a panel of human cell lines and primary lymphoma samples. Furthermore, 1 humanized BAFF-R mAb clone and its afucosylated version, glycoengineered to optimize the primary mechanism of action, prolonged survival of immunodeficient mice bearing human tumor cell lines or patient-derived lymphoma xenografts in 3 separate models, compared with controls. Finally, the tissue specificity of this humanized mAb was confirmed against a broad panel of normal human tissues. Taken together, we have identified a robust lead-candidate BAFF-R mAb for clinical development.

CD19/BAFF-R dual-targeted CAR T cells for the treatment of mixed antigen-negative variants of acute lymphoblastic leukemia.

Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent antitumor effects in B-cell malignancies including acute lymphoblastic leukemia (ALL), but antigen loss remains the major cause of treatment failure. To mitigate antigen escape and potentially improve the durability of remission, we developed a dual-targeting approach using an optimized, bispecific CAR construct that targets both CD19 and BAFF-R. CD19/BAFF-R dual CAR T cells exhibited antigen-specific cytokine release, degranulation, and cytotoxicity against both CD19-/- and BAFF-R-/- variant human ALL cells in vitro. Immunodeficient mice engrafted with mixed CD19-/- and BAFF-R-/- variant ALL cells and treated with a single dose of CD19/BAFF-R dual CAR T cells experienced complete eradication of both CD19-/- and BAFF-R-/- ALL variants, whereas mice treated with monospecific CD19 or BAFF-R CAR T cells succumbed to outgrowths of CD19-/BAFF-R+ or CD19+/BAFF-R- tumors, respectively. Further, CD19/BAFF-R dual CAR T cells showed prolonged in vivo persistence, raising the possibility that these cells may have the potential to promote durable remissions. Together, our data support clinical translation of BAFF-R/CD19 dual CAR T cells to treat ALL.

Vaccine site inflammation potentiates idiotype DNA vaccine-induced therapeutic T cell-, and not B cell-, dependent antilymphoma immunity.

Lymphoma idiotype protein vaccines have shown therapeutic potential in previous clinical studies, and results from a completed pivotal, phase 3 controlled trial are promising. However, streamlined production of these patient-specific vaccines is required for eventual clinical application. Here, we show that second-generation, chemokine-fused idiotype DNA vaccines, when combined with myotoxins that induced sterile inflammation with recruitment of antigen-presenting cells at vaccination sites, were exceptional in their ability to provoke memory antitumor immunity in mice, compared with several TLR agonists. The combined vaccination strategy elicited both antigen-specific T-cell responses and humoral immunity. Unexpectedly, vaccine-induced tumor protection was intact in B cell-deficient mice but was abrogated completely by T-cell depletion in vivo, suggesting T-cell dependence. Furthermore, the optimal effect of myotoxins was observed with fusion vaccines that specifically targeted antigen delivery to antigen-presenting cells and not with vaccines lacking a targeting moiety, suggesting that the rational vaccine design will require combination strategies with novel, proinflammatory agents and highly optimized molecular vaccine constructs. These studies also challenge the paradigm that antibody responses are the primary of idiotype-specific antitumor effects and support the optimization of idiotype vaccines designed to induce primarily T-cell immunity.

Prime-boost vaccination using chemokine-fused gp120 DNA and HIV envelope peptides activates both immediate and long-term memory cellular responses in rhesus macaques.

HIV vaccine candidates with improved immunogenicity and induction of mucosal T-cell immunity are needed. A prime-boost strategy using a novel HIV glycoprotein 120 DNA vaccine was employed to immunize rhesus macaques. The DNA vaccine encoded a chimeric gp120 protein in fusion with monocyte chemoattractant protein-3, which was hypothesized to improve the ability of antigen-presenting cells to capture viral antigen through chemokine receptor-mediated endocytosis. DNA vaccination induced virus-reactive T cells in peripheral blood, detectable by T cell proliferation, INFgamma ELISPOT and sustained IL-6 production, without humoral responses. With a peptide-cocktail vaccine containing a set of conserved polypeptides of HIV-1 envelope protein, given by nasogastric administration, primed T-cell immunity was significantly boosted. Surprisingly, long-term and peptide-specific mucosal memory T-cell immunity was detected in both vaccinated macaques after one year. Therefore, data from this investigation offer proof-of-principle for potential effectiveness of the prime-boost strategy with a chemokine-fused gp120 DNA and warrant further testing in the nonhuman primate models for developing as a potential HIV vaccine candidate in humans.

Corrigendum to "Prime-Boost Vaccination Using Chemokine-Fused gp120 DNA and HIV Envelope Peptides Activates Both Immediate and Long-Term Memory Cellular Responses in Rhesus Macaques".

[This corrects the article DOI: 10.1155/2010/860160.].

Cloning of B cell lymphoma-associated antigens using modified phage-displayed expression cDNA library and immunized patient sera.

Active immunization of follicular lymphoma patients with idiotypic vaccines elicits antigen-specific antibody responses, T-cell responses, and antitumor effects. We hypothesized that these vaccinated patients could generate tumor-specific immune responses, not only against idiotype, but also against other tumor-associated antigens (TAA) by a mechanism of epitope spreading. To identify potential antigens, a phage surface expressed cDNA library derived from primary tumor cells was screened with sera from idiotype-vaccinated patients. Consistent with our hypothesis, we identified two immunogenic peptides (FL-aa-7 and 18), unrelated to idiotype, which were recognized by postvaccine sera but not by prevaccine or normal human sera. These peptide sequences derived from the 5'-untranslated regions of the human GTPase, IMAP family member 7 gene (FL-aa-7) and an alternative reading frame of U1-snRNP 70 (FL-aa-18), respectively, suggesting that epitope spreading had occurred.

Molecular analysis of light-chain switch and acute lymphoblastic leukemia transformation in two follicular lymphomas: implications for lymphomagenesis.

We observed novel transformations of follicular lymphoma (FL), first, a switch in immunoglobulin (Ig) light chain, and second, transformation of FL to acute lymphoblastic leukemia (ALL). Each set of tumors shared a common clonal origin, as demonstrated by expression of identical, unique CDR IIIH sequences, shared somatic mutations in JH, and identical bcl-2 translocation breakpoints of microdissected ALL cells. Molecular analysis of lambda V-gene expression demonstrated lambda-bearing cells in the original kappa tumor, while expansion of the lambda subclone at relapse occurred after active immunotherapy targeting the Ig receptor. These exceptional cases are compatible with a more contemporary model of lymphomagenesis in which critical events originate from genetic mechanisms which normally occur in germinal center (GC) B cells and challenge the current paradigm of parallel generation of subclones from an early, pre-GC precursor. It is also possible that the outgrowth of these variants was a consequence of immunoselection.

Prophylactic anti-tumor effects in a B cell lymphoma model with DNA vaccines delivered on polyethylenimine (PEI) functionalized PLGA microparticles.

Idiotypic sequences, specific to the hypervariable regions of immunoglobulins expressed by malignant B cells offer a therapeutic target in B cell lymphoma. Efficient approaches have been described to clone a single chain fragment of the tumor immunoglobulin (Ig) comprising of heavy and light Ig chains (sFv) fused with proinflammatory chemokines. Tumor associated, poorly immunogenic self antigens encoded by plasmid DNA (pDNA) have been rendered immunogenic by chemokine fusion, thereby targeting to antigen presenting cells (APCs) which differentially express chemokine receptors. Here we present an injectable (parenteral) approach using synthetic polymer based cationic microparticle formulations for enhancing the potency of such chemokine/self antigen expressing plasmid construct. Branched and linear polyethyleneimine (PEI) were conjugated on poly (D, L lactide-co-glycolide) (PLGA) microparticles using carbodiimide chemistry followed by efficient loading of plasmid DNA. In addition to imparting significant buffering ability to these cationic microparticles, flow cytometry studies indicate that these DNA loaded microparticles significantly up regulate CD80 and MHC class II markers in phagocytic RAW264.7 cells, indicating intrinsic adjuvant effects. Intradermal injections in Balb/c mice with these formulations induced significant protection upon tumor challenge with 2.5 times the minimal lethal dose. Long term survival rates were significant (p < 0.05) in comparison with saline injected controls or blank microparticles. Further studies indicated that intramuscular delivery might provide better protection compared to intradermal injections and perform similar to gene gun mediated administration. We conclude, based on these promising in vivo results, that such surface-functionalized microparticles offer an attractive strategy to improve the potency of self antigen-based cancer DNA vaccines.

Targeting B-cell malignancies through human B-cell receptor specific CD4+ T cells.

The B-cell receptor (BCR) expressed by a clonal B cell tumor is a tumor specific antigen (idiotype). However, the T-cell epitopes within human BCRs which stimulate protective immunity still lack detailed characterization. In this study, we identified 17 BCR peptide-specific CD4+ T-cell epitopes derived from BCR heavy and light chain variable region sequences. Detailed analysis revealed these CD4+ T-cell epitopes stimulated normal donors' and patients' Th1 CD4+ T cells to directly recognize the autologous tumors by secretion of IFNγ, indicating the epitopes are processed and presented by tumor cells. One BCR peptide-specific CD4+ T cell line was also cytotoxic and lysed autologous tumor cells through the perforin pathway. Sequence analysis of the epitopes revealed that 10 were shared by multiple primary patients' tumors, and 16 had the capacity to bind to more than one HLA DRB1 allele. T cells stimulated by shared epitopes recognized primary tumors expressing the same sequences on multiple HLA DRB1 alleles. In conclusion, we identified 17 BCR-derived CD4+ T-cell epitopes with promiscuous HLA DRB1 binding affinity that are shared by up to 36% of patients, suggesting a strategy to overcome the requirement for individual preparation of therapeutic agents targeting idiotype.

Genetic fusions with viral chemokines target delivery of nonimmunogenic antigen to trigger antitumor immunity independent of chemotaxis.

The ideal vaccine carrier should be able to target antigen delivery and possibly recruit antigen-presenting cells (APC) and deliver an activation signal to promote adaptive immune responses. Ligands for chemokine receptors expressed on APC may be attractive candidates, as they can both target and attract APC. To investigate the requirement for APC recruitment, we used a pair of viral chemokines, agonist herpes simplex virus 8-derived macrophage inflammatory protein-I (vMIP-I) and antagonist MC148, which induce and suppress chemotaxis, respectively. Chemokine-antigen fusions efficiently delivered a model nonimmunogenic tumor antigen to APC for processing and presentation to antigen-specific T cells in vitro. Physical linkage of chemokine and antigen and specific binding of chemokine receptor by the fusion protein were required. Mice immunized with vMIP-I or MC148 fusion DNA vaccines elicited protection against tumor challenge. Therefore, vaccine efficacy depends primarily on the ability of the carrier to target antigen delivery to APC for subsequent processing and presentation, and chemotaxis directly induced by the chemokine moiety in the fusion may not be necessary.

Enhanced protection against HSV lethal challenges in mice by immunization with a combined HSV-1 glycoprotein B:H:L gene DNAs.

The effectiveness of a cocktailed HSV-1 three-glycoprotein B, H, and L gene vaccine in comparison to individual glycoprotein gene vaccines was studied with regard to protecting against the HSV-1 infection. Three glycoprotein gene recombinant DNA vaccines, which produced the corresponding glycoproteins in Vero cells, were constructed using a CMV promoter. The cocktailed DNA vaccines were prepared by combining all three genes. The titers of neurtalizing antibody following the immunization of the five vaccines were KOS(1/1024)>B:H:L=B(1/512)>H:L(1/64)>H(1/16) genes. The mice, which were immunized with L gene alone failed to induce enough neutralizing antibody. The CTL activity was rated as KOS (95%)>B:H:L (80%)>B(60%)>H:L(50%)> H (35%) gene vaccines at an E:T ratio of 50:1. The H gene alone or L gene vaccine alone induced little CTL activity. The protection rates of the DNA-vaccinated mice against the lethal intraperitoneal (i.p.) or i.m challenges were shown as KOS>B:H:L>B>H:L>H gene vaccines, and the protection activity depended on the lethal dosage of the challenging virus, which are inversely proportional to each other. Compared with the mice, which were vaccinated with individual DNA vaccines, the mice, which were vaccinated with the cocktailed three-gene vaccine, were shown to be better protected against the lethal challenging doses. It can be concluded that vaccination with the cocktailed three gene vaccines is more effective in protecting mice from the viral challenge and the protection rate varies inversely with the amount of lethal challenging dose used, although all DNA vaccines failed to block the latent infection in sensory nerves.

Cloning variable region genes of clonal lymphoma immunoglobulin for generating patient-specific idiotype DNA vaccine.

Available therapies for lymphoplasmacytic lymphoma (LPL) provide no survival advantage if started before signs or symptoms of end-organ damage develop; hence, current recommendations are to follow a program of observation while patients are in the asymptomatic phase of disease. We hypothesize that using idiotypic determinants of a B-cell lymphoma's surface immunoglobulin as a tumor-specific marker, we can develop patient-specific chemokine-idiotype fusion DNA vaccines that induce an immune response against LPL. By activating the host immune system against the tumor antigen, we postulate that disease control of asymptomatic phase lymphoplasmacytic lymphoma can be maintained. These chemokine-idiotype fusion DNA vaccines provide protection in a lymphoma mouse model and have recently entered clinical trials. Herein, we describe procedures for the generation of therapeutic vaccines, particularly "second-generation" recombinant vaccines. Specifically, in the Methods section we describe how to identify lymphoma-associated immunoglobulin V (IgV) genes from patient biopsy and how to assemble these genes as single-chain variable gene fragment (scFv) in-frame with MIP-3α to generate novel DNA fusion vaccines.

Targeting human B-cell malignancies through Ig light chain-specific cytotoxic T lymphocytes.

PURPOSE: The variable regions of Ig (idiotype, Id) expressed by malignant B cells can be used as tumor-specific antigens that induce humoral and cellular immunity. However, epitopes derived from Id that stimulate human CD8(+) T-cell immunity are incompletely characterized. EXPERIMENTAL DESIGN: The clonal Ig V(L) of human myeloma cell line U266 and five primary B-cell tumors were sequenced, and peptides corresponding to the Ig V(L) region were tested for their ability to stimulate CTLs from 10 HLA-A*0201-positive normal donors. The CTLs thus generated were tested against peptide-pulsed T2 cells and autologous tumor cells. RESULTS: Fourteen peptides derived from Ig light chain (V(L)) of U266 and primary B-cell tumors were used to generate 68 CTLs lines that specifically produced IFN-γ when cocultured with peptide-pulsed T2 cells. These CTLs lysed peptide-pulsed T2 cell as well as U266 or autologous tumor targets in an HLA class I-dependent manner. Sequence analysis revealed shared V(L) T-cell epitopes in U266 and primary B-cell tumors, not previously reported within Ig heavy chain (V(H)) sequences. CONCLUSION: This study thus identifies novel immunogenic CTLs epitopes from Id V(L), suggests that they are naturally presented on the surface of B-cell malignancies, and supports their inclusion in next-generation Id vaccines. The ability to prime T cells derived from normal HLA-matched donors, rather than patients, may also have direct application to current strategies, designed to generate allogeneic tumor-specific T cells for adoptive transfer.