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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.

PURPOSE To overcome technical limitations of detecting low frequencies of antigen-specific-T cells in human patients, we sought to establish a highly sensitive platform for antigen-specific-T cell detection and activation using IBMAM (immortalized B cells transfected with mRNA of antigen fused to MHC-class-I-trafficking-domain [MITD]). METHODS We first established immortalized B cells in-vitro as antigen-presenting cells. B cells were isolated from CMV-seropositive normal healthy donors (NHDs), stimulated, and transduced with retrovirus containing BCL-6 and BCL-XL. Purified CD4+ T lymphocytes obtained from the NHDs PBMC were co-cultured in-vitro with autologous immortalized B cells transfected with CMVpp65 mRNA fused to MITD (IBMAM) as a model antigen and then measured for activation using IFN-g ELISA. We developed two pathways for IBMAM mediated antigen-specific T cell activation and detection. For the first pathway, if IFN-g is detected, T cells are isolated again from NHDs PBMC and expanded using rapid expansion procedures (REP). After the first REP, antigen-specific T cells are detected using IBMAM, sorted, and a second REP is performed. However, for the second pathway, if IFN-g is not detected, NHDs PBMC is stimulated twice using IBMAM and subsequently antigen-specific T cells are detected using IBMAM again. RESULTS B cells were successfully immortalized by transfecting at least 5*105 cells with retrovirus (1MOI) and co-cultured with CD40L-IL21 expressing human fibroblasts. We observed a significantly increased activation of antigen-specific CD4+ T cells when using IBMAM in comparison to controls transfected with CMVpp65 mRNA lacking the MITD fusion or a known CMVpp65 peptide pool. For pathway 1 and 2 of our platform, successful detection, activation, and expansion of CMVpp65-specific T cells were observed from low frequency NHDs samples using IBMAM. CONCLUSION We have thus established a highly sensitive method of immuno-monitoring that can be used for the detection of antigen-specific T cells in patient samples with low frequencies of antigen-specific-T cells, such as human cancer antigens.

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.

Background: Idiotypic determinants of the surface immunoglobulin (Ig) associated with a given pt's B-cell lymphoma are unique to that tumor, and can thus serve as a tumor-specific marker. This study aims to use an idiotype DNA vaccine to lengthen the smoldering phase of WM without inducing cross-resistance to available therapies. Administered vaccine used recombinant plasmid DNA encoding a fusion protein, consisting of autologous lymphoma scFv (pt-specific idiotype) and human CCL20 (macrophage inflammatory protein-3 alpha - MIP-3α) chemokine. Targeted delivery of this fusion protein to antigen-presenting cells, and subsequent processing and presentation, is hypothesized to break tolerance and generate an immune response against the idiotype, promoting eradication of antigen-expressing B-cell lymphoma cells. Methods: Pts with sWM received 3 intradermal vaccinations of pt-specific DNA vaccine at 4-week (wk) intervals (wks 0, 4 and 8). Two dose levels (500 µg; 2500 µg) were evaluated in a 3+3 design. The primary objective was to evaluate the vaccine's safety and identify the maximum tolerated dose. Secondary objectives were to assess immunogenicity of the vaccine to generate tumor-specific cellular immune responses. Results: Between 1/2016 - 1/2019, 9 pts (7 men) were treated (500 µg: n = 3; 2500 µg: n = 6). Median age at enrollment was 67 yrs (range 56-78); median time from diagnosis to 1st vaccination was 26.5 mos (8.8-120.9). MYD88 L265P + (6 pts). CXCR4 WHIM + (1 pt). With median follow up of 38 months (range: 8.8-51), 8 pts are known alive; 1 has been lost to follow up. Six have maintained stable disease; 3 have progressed to symptomatic WM at 8, 25, and 28 months respectively, from 1st vaccination). All pts completed planned therapy. No DLTs or Grade 4 AEs occurred. Ten mos. after the 3rd vaccination, 1 pt had a grade 3 pleural effusion and leukopenia with an increase in rheumatoid factor (23.1 IU/mL [normal range 0.0-15.9]) and ANA titer of 1:80; all resolved within 2 mos. Grade 1-2 AEs observed in > 3pts include: leukopenia (6), nausea (5), anemia (4), increased creatinine (4), fatigue (4). Immune correlatives have now been completed in all 9 patients. Analysis is ongoing, and complete data will be presented at the meeting. Serial pre- and post- vaccine samples analyzed by single-cell RNA seq from 3 representative patients (LPL 005, LPL 007, LPL 008) treated at the 2500 mg dose are shown in the Figure. DNA vaccine treatment significantly reduced the number of clonal B-cells in the bone marrow compartment of the 2 patients who have maintained SD (LPL 007 and LPL 008). DNA vaccine treatment also induced increases in monocytes in the tumor microenvironment of these 2 patients. In addition, T-cell receptor (TCR) sequence analysis revealed clear increases in TCR clonal expansions, consistent with a vaccine effect. In contrast, LPL 005, who had the earliest progression to symptomatic phase WM, had an increase in tumor B cells on post vaccine samples, and TCR clonotypes showed no changes. Similar changes were observed with serial bone marrow samples from the remaining patients treated on the trial, correlating with clinical outcome. Conclusions: Idiotype (scFv-CCL20) DNA vaccine therapy appears to be safe in pts with sWM. Preliminary results of serial single cell RNAseq analysis suggest that the tumor immune microenvironment is favorably altered after chemokine-tumor antigen DNA vaccine treatment and these vaccine-induced changes may correlate with clinical outcome. The immunologic changes observed suggest response to this vaccine, and warrant further investigation in a Phase II trial, possibly in combination with immune checkpoint blockade. Disclosures Thomas: Ascentage: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Genentech: Research Funding; Pharmacyclics: Other: Advisory Boards; Xencor: Research Funding; X4 Pharma: Research Funding. Lee:GlaxoSmithKline: Consultancy, Research Funding; Regeneron: Research Funding; Daiichi Sankyo: Research Funding; Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Genentech: Consultancy; Sanofi: Consultancy; Genentech: Consultancy. Manasanch:Adaptive Biotechnologies: Honoraria; Glaxo Smith Kline: Honoraria; BMS: Honoraria; Takeda: Honoraria; Quest Diagnostics: Research Funding; Merck: Research Funding; JW Pharma: Research Funding; Novartis: Research Funding; Sanofi: Honoraria, Research Funding. Patel:Nektar: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Precision Biosciences: Research Funding; Celgene: Consultancy, Research Funding; Cellectis: Research Funding; Poseida: Research Funding; Oncopeptides: Consultancy. Orlowski:Founder of Asylia Therapeutics, Inc., with associated patents and an equity interest, though this technology does not bear on the current submission.: Current equity holder in private company, Patents & Royalties; Laboratory research funding from BioTheryX, and clinical research funding from CARsgen Therapeutics, Celgene, Exelixis, Janssen Biotech, Sanofi-Aventis, Takeda Pharmaceuticals North America, Inc.: Research Funding; STATinMED Research: Consultancy; Sanofi-Aventis, Servier, Takeda Pharmaceuticals North America, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen, Inc., AstraZeneca, BMS, Celgene, EcoR1 Capital LLC, Forma Therapeutics, Genzyme, GSK Biologicals, Ionis Pharmaceuticals, Inc., Janssen Biotech, Juno Therapeutics, Kite Pharma, Legend Biotech USA, Molecular Partners, Regeneron Pharmaceuticals, Inc.,: Honoraria, Membership on an entity's Board of Directors or advisory committees. Neelapu:Takeda Pharmaceuticals: Patents & Royalties; Bristol-Myers Squibb: Other: personal fees, Research Funding; Merck: Other: personal fees, Research Funding; Kite, a Gilead Company: Other: personal fees, Research Funding; N/A: Other; Acerta: Research Funding; Karus Therapeutics: Research Funding; Cellectis: Research Funding; Poseida: Research Funding; Unum Therapeutics: Other, Research Funding; Calibr: Other; Adicet Bio: Other; Legend Biotech: Other; Allogene Therapeutics: Other: personal fees, Research Funding; Cell Medica/Kuur: Other: personal fees; Incyte: Other: personal fees; Precision Biosciences: Other: personal fees, Research Funding; Celgene: Other: personal fees, Research Funding; Novartis: Other: personal fees; Pfizer: Other: personal fees. Kwak:CJ Healthcare: Consultancy; Sellas Life Sciences Grp: Consultancy; Enzychem Life Sciences: Membership on an entity's Board of Directors or advisory committees; Antigenics: Other: equity; InnoLifes, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pepromene Bio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Xeme Biopharma/Theratest: Other: equity; Celltrion Healthcare: Membership on an entity's Board of Directors or advisory committees; Celltrion, Inc.: Consultancy. OffLabel Disclosure: This study aims to use a novel idiotype DNA vaccine to lengthen the smoldering phase of WM without inducing cross-resistance to available therapies.

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.

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. 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. This vaccine could shift the current paradigm of clinical management for patients with asymptomatic LPL and inform development of other personalized approaches. ClinicalTrials.gov identifier NCT01209871; registered on September 24, 2010.

Purpose: mAbs such as anti-CD20 rituximab are proven therapies in B-cell malignancies, yet many patients develop resistance. Novel therapies against alternative targets are needed to circumvent resistance mechanisms. We sought to generate mAbs against human B-cell–activating factor receptor (BAFF-R/TNFRSF13C), which has not yet been targeted successfully for cancer therapy. Experimental Design: Novel mAbs were generated against BAFF-R, expressed as a natively folded cell surface immunogen on mouse fibroblast cells. Chimeric BAFF-R mAbs were developed and assessed for in vitro and in vivo monotherapy cytotoxicity. The chimeric mAbs were tested against human B-cell tumor lines, primary patient samples, and drug-resistant tumors. Results: Chimeric antibodies bound with high affinity to multiple human malignant B-cell lines and induced potent antibody-dependent cellular cytotoxicity (ADCC) against multiple subtypes of human lymphoma and leukemia, including primary tumors from patients who had relapsed after anti-CD20 therapy. Chimeric antibodies also induced ADCC against ibrutinib-resistant and rituximab-insensitive CD20-deficient variant lymphomas, respectively. Importantly, they demonstrated remarkable in vivo growth inhibition of drug-resistant tumor models in immunodeficient mice. Conclusions: Our method generated novel anti–BAFF-R antibody therapeutics with remarkable single-agent antitumor effects. We propose that these antibodies represent an effective new strategy for targeting and treating drug-resistant B-cell malignancies and warrant further development. Clin Cancer Res; 24(5); 1114–23. ©2017 AACR.

Immune evasion is an emerging hallmark of cancer progression. However, functional studies to understand the role of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment are limited by the lack of available specific cell surface markers. We adapted a competitive peptide phage display platform to identify candidate peptides binding MDSCs specifically and generated peptide-Fc fusion proteins (peptibodies). In multiple tumor models, intravenous peptibody injection completely depleted blood, splenic and intratumoral MDSCs in tumor-bearing mice without affecting proinflammatory immune cell types, such as dendritic cells. Whereas control Gr-1-specific antibody primarily depleted granulocytic MDSCs, peptibodies depleted both granulocytic and monocytic MDSC subsets. Peptibody treatment was associated with inhibition of tumor growth in vivo, which was superior to that achieved with Gr-1-specific antibody. Immunoprecipitation of MDSC membrane proteins identified S100 family proteins as candidate targets. Our strategy may be useful to identify new diagnostic and therapeutic surface targets on rare cell subtypes, including human MDSCs.

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.

Background: Despite major advances in the treatment of multiple myeloma (MM) only a minority of patients achieve long-term disease control. Immunotherapy combined with autologous hematopoietic stem cell transplantation (auto-HCT) may reduce relapse rates. Vaccination with Ig idiotype (Id) conjugated with a carrier protein, keyhole limpet hemocyanin (KLH), was associated with superior disease-free survival in a randomized trial in follicular lymphoma (Schuster S. et al. JCO 2011), and Id is also a tumor-specific antigen in MM. In addition, anti-CD3/-CD28 costimulated adoptive T-cell transfer augmented immune responses to pneumococcal vaccination despite cytotoxic therapy (Rapoport A et al. Nat Med 2005). We therefore hypothesized that Id-KLH vaccine + the vaccine-primed costimulated T cells will result in a robust Id-specific humoral and cellular response, compared with control vaccine (KLH only). Methods: In this randomized, phase II trial, eligible patients were randomized 1:1 to receive either KLH-only or Id-KLH vaccine, followed by auto-HCT, and then vaccine- primed costimulated T cells followed by two booster doses of the vaccine to which they were randomized. Immune response (IR) was evaluated by gene expression profiling (GEP) using NanoString nCounter. Results: A total of 36 patients were enrolled between 1/2013 and 5/2015, 20 to KLH-only and 16 to Id-KLH arm, and completed their assigned treatments. There were no significant differences between the groups in age, gender, stage, cytogenetic risk, induction regimen or response to induction (Table). Nineteen (95%) and 16 (100%) patients received maintenance therapy after protocol treatment in KLH-only and Id-KLH, respectively (p=0.42). No infusion reactions or dose-limiting toxicity was seen in either arm. There was no non-relapse mortality at 100-day and 1-year in either arm. At last evaluation, 5 (25%) and 7 (44%) had achieved CR in KLH-only and Id-KLH, respectively (p=0.29). Six (30%) and 9 (56%) achieved at least nCR in KLH-only and Id-KLH, respectively (p=0.17), and 12 (60%) and 12 (75%) achieved at least a VGPR in KLH-only and Id-KLH, respectively (0.48). Ten (50%) and 8 (50%) patients, for whom there were sufficient samples available underwent IR analysis in KLH-only and Id-KLH, respectively at baseline and at 30, 90 and 180 days after activated T cell infusion. In an initial 568 gene panel analyzed for IR, compared with KLH-only, there was a greater change in IR genes in CD8+ T cells in Id-KLH at days 30, 90 and 180 (Figure 1A). In a 30-gene panel of the most upregulated IR genes in CD8+ cells, there was a significantly higher expression of these genes in Id-KLH at all post-treatment time points (Figure 1B). Specifically, at day 180 post-treatment, there was higher expression of immune co-stimulatory genes in Id-KLH in both CD8+ and CD4+ T cell subsets. This increased co-stimulatory gene expression in Id-KLH vaccinated CD4+ (Figure 1C) and CD8+ T cells (not shown) was most marked in patients who had achieved a CR. After a median follow up of 38.3 months, 3-year PFS in KLH-only and Id-KLH was 64% and 53%, respectively (p=0.25). Conclusion: Id-KLH vaccine and vaccine-primed costimulated T cells can be safely administered in the setting of auto-HCT. In this randomized trial we detected a trend towards a deeper clinical response in the Id-KLH arm. There were significantly more robust IR in CD4+ and CD8+ T cells in the Id-KLH arm,which also correlated withdepth of clinical response. Future trials should focus on selection of patients most likely to mount a robust IR to Id vaccines. Download : Download high-res image (185KB) Download : Download full-size image Disclosures Shah: TeneoBio, Inc.: Honoraria; Indapta Therapeutics: Other: Stock Ownership; Celgene: Research Funding; Celgene, Indapta Therapeutics, Takeda: Consultancy. Patel: Juno: Consultancy; Celgene: Consultancy; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding. Thomas: Bristol Myers Squibb: Research Funding; Celgene: Research Funding. Orlowski: BioTheryX: Consultancy, Membership on an entity's Board of Directors or advisory committees. Cohen: Celgene: Consultancy; Bristol Meyers Squibb: Consultancy, Research Funding; GlaxoSmithKline: Consultancy; Janssen: Consultancy. Vogl: Constellation: Research Funding; Karyopharm: Consultancy; GSK: Research Funding; Calithera: Research Funding; Takeda: Consultancy, Research Funding; Teva: Consultancy; Amgen: Consultancy; Celgene: Consultancy. June: Immune Design: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding; WIRB/Copernicus Group: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celldex: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kwak: Celltrion, Inc: Consultancy; InnoLifes: Consultancy, Equity Ownership; Pepromene Bio: Consultancy, Equity Ownership.

Our laboratory previously described the strategy of fusing chemokine receptor ligands to antigens in order to generate immunogenic DNA vaccines. In the present study, we produced mouse β-2 defensin (mBD2) fusion proteins using both ovalbumin (OVA) and gp100 as model antigens. Superior cross-presentation by dendritic cells (DC) was observed for mBD2 fused antigens over unfused antigens in vitro. In vivo, we observed significant increases in the expansion of adoptively transferred antigen-specific MHC class I, but not class II-restricted T cells after immunization with mBD2 fused antigen over antigen alone. This enhanced expansion of class I restricted T cells was Toll-like receptor 4 (TLR4) dependent, but CC chemokine receptor 6 (CCR6) independent. Superior tumor resistance was observed for mBD2-fusion protein vaccines, compared to unfused antigen, in both B16-OVA and B16 tumor models. These data suggest that production of mBD2 fusion proteins is feasible and that the vaccines facilitate in vivo expansion of adoptively transferred T cells through a TLR4-dependent mechanism.

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.

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.

The immunity of a combined DNA vaccine of HSV-2 glycoproteins B2 (gB2) and D2 (gD2) genes in comparison to individual vaccines was studied with regard to protecting against the HSV infection. Two recombinant DNA vaccines of the pHS2-gB2 or pHS2-gD2 were constructed and formulated. The neutralizing antibody titers appeared higher in the B2:D2 gene cocktail-vaccinated mice than that of the individual B2 or D2 gene-vaccinated group alone, and the positive KOS control induced higher titer of the neutralizing antibody than combined or individual gene vaccines. The mock-immunized mice failed to induce enough. The ranks for the CTL activity and the protection rates against the lethal intravaginal challenge were shown as KOS>B2:D2 cocktail>D2>B2 gene vaccines. The vaginal external diseases in the B2:D2 or D-vaccinated mice were significantly reduced against the challenging dosages. The virus titers in the vaginal secretions of the vaccinated mice significantly reduced with time, and the B2:D2 gene vaccine decreased more than each individual vaccine alone. It can be concluded that the cocktailed vaccines are more effective in the humoral and cellular immune responses in the mice, and in the protection of the mice against the intravaginal challenging dosages when compared with individual gene vaccines. All the DNA vaccines failed to block the latent infection in sensory nerves.

B-cell malignancies have been successfully targeted in the clinic by therapies such as anti-CD20 antibody rituximab or Bruton’s tyrosine kinase inhibitor ibrutinib. However, leukemias and lymphomas remain incurable due to primary or acquired resistance, ultimately leaving patients without an effective treatment option. We sought to circumvent this drug resistance by pursuing an alternative target known as B cell activating factor receptor (BAFF-R). Despite past limited success, BAFF-R remains a prime target for B-cell lymphoma and leukemia therapeutic antibody development due to its key role in B-cell proliferation and development. We report the development of two novel monoclonal antibodies (mAbs) against human (h) BAFF-R. The mAbs were generated by immunizing mice with (h)BAFF-R-expressing mouse fibroblast cells presenting a natively folded, cell-surface immunogen. The two mAbs presented unique complementarity determining regions that specifically bound (h)BAFF-R-expressing mouse fibroblast cells but not the parental counterpart. Furthermore, the antibodies were specific to B-cell containing organs such as tonsil and spleen, by immunohistochemical staining and without detectable reactivity in heart, lung, brain, liver, and kidney tissues. To tailor the antibodies for clinical application, a human IgG1 Fc capable of eliciting an immune response was substituted, creating chimeric versions. We showed that both chimeric mAbs bound with high affinity to human B-cell lymphoma cell lines including JeKo-1 (mantle cell lymphoma; MCL), SU-DHL-6 (diffuse large B cell lymphoma; DLBCL), Raji (Burkitt lymphoma), and RL (follicular lymphoma). The chimeric antibodies also elicited antibody-dependent cell-mediated cytotoxicity (ADCC) with primary human natural killer (NK) cells in vitro against these tumor lines as well as primary lymphoma samples (n=5) from patients who progressed after rituximab exposure. Most notably, the antibodies demonstrated efficacy in two in vivo drug resistant lymphoma models we developed, a rituximab-resistant CD20 genomic knockout variant of JeKo-1 and the naturally ibrutinib-resistant Z-138. Using these lymphomas lines for xenogeneic tumor models in NOD scid gamma (NSG) mice, we found our antibodies significantly inhibited tumor growth, conferring long-term and tumor free survival on the mice. Our in vitro and in vivo results robustly demonstrate the high specificity and significant anti-tumor effects of our anti-BAFF-R antibodies against a broad variety of B-cell malignancies, especially against cases of rituximab and ibrutinib resistance. This successful development of novel anti-BAFF-R therapeutic antibodies warrants support for further translational development for clinical use in light of current resistance cases. Citation Format: Hong Qin, Guowei Wei, Ippei Sakamaki, Zhenyuan Dong, Wesley A. Cheng, Diane L. Smith, Feng Wen, Han Sun, Soung-chul Cha, Sattva S. Neelapu, Larry W. Kwak. Drug resistant B-cell tumors eliminated by novel therapeutic antibodies in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2655. doi:10.1158/1538-7445.AM2017-2655

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.

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.

Background: Despite major advances in the treatment of multiple myeloma (MM) only a minority of patients achieve long-term disease control. Immunotherapy combined with autologous hematopoietic stem cell transplantation (auto-HCT) may reduce relapse rates. Immunoglobulin idiotype (Id) conjugated with a carrier protein, Keyhole limpet hemocyanin (KLH), is a tumor-specific antigen in MM. Vaccine-primed, anti-CD3/anti-CD28 costimulated adoptive T-cell transfer can augment humoral and cellular immune responses to vaccination despite cytotoxic therapy. We hypothesized that Id-KLH vaccine + the vaccine-primed costimulated T cells will result in a robust Id-specific humoral and cellular response, compared to a control vaccine (KLH only). Methods: In this randomized, phase II trial, the primary objective was to determine if Id-KLH primed, costimulated T cells will induce a more robust Id-specific immunity than KLH-primed T cells. Eligible patients had IgG monoclonal protein. Patients were randomized 1:1 to receive either Id-KLH vaccine or KLH-only vaccine, followed by auto-HCT, and then vaccine- primed costimulated T cells followed by two booster doses of the vaccine to which they were randomized. This study was supported by The MD Anderson Cancer Center SPORE in Multiple Myeloma. Results: A total of 36 patients were enrolled between 1/2013 and 5/2015. Sixteen (44%) were randomized to Id-KLH and 20 (55%) to KLH-only. The Table below summarizes the patient characteristics. There was no significant difference between the two groups in terms of age, risk status or induction therapy. No treatment-related mortality, infusion reactions or dose-limiting toxicity was seen in either arm. Five (31%) and 3 (15%) patients achieved complete remission (CR) by day+180 in the Id-KLH and KLH arms, respectively (p=0.42). Initial analysis of a subgroup of patients revealed a significantly higher mRNA expression of immune activation genes IL-2, CCR6 and CD40 by NanoString nCounter in the Id-KLH group compared with the KLH only group (Figure). Eleven (68%) and 17 (85%) went on to receive maintenance therapy with lenalidomide or lenalidomide + ixazomib in the Id-KLH and KLH arms, respectively (p=0.42). After a median follow up of 26 months, 2-year PFS was 81% and 83% in Id-KLH and KLH arms, respectively (p=0.35). Conclusion: Id-KLH vaccine and vaccine-primed costimulated T cells can be safely administered in the setting of auto-HCT.There was a more robust immune response and a trend towards higher CR rate in the Id-KLH group. Disclosures Stadtmauer: Teva: Consultancy; Amgen: Consultancy; Novartis: Consultancy; Takada: Consultancy; Janssen: Consultancy; Celgene: Consultancy. Garfall:Medimmune: Consultancy; Bioinvent: Research Funding; Novartis: Consultancy, Research Funding. Cohen:Janssen: Consultancy; Bristol-Meyers Squibb: Consultancy, Research Funding. June:Johnson & Johnson: Research Funding; Immune Design: Consultancy, Equity Ownership; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; University of Pennsylvania: Patents & Royalties; Celldex: Consultancy, Equity Ownership; Tmunity: Equity Ownership, Other: Founder, stockholder ; Pfizer: Honoraria.

Antigen activation of the B-cell receptor (BCR) may play a role in the pathogenesis of human follicular lymphoma (FL) and other B-cell malignancies. However, the nature of the antigen(s) recognized by tumor BCRs has not been well studied. Here, we used unbiased approaches to demonstrate that 42 (19.35%) of 217 tested FL immunoglobulins (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 monoclonal antibody 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% vs. 10%; P=0.0022). However, vimentin-reactive FL Igs did not share complimentarity determining region 3 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 better understanding of the biology and natural history of FL and other B cell malignancies.

Immunomodulatory drugs (IMiDs) are effective therapeutic agents with direct inhibitory effects on malignant B- and plasma-cells and immunomodulatory effects on the T-cell activation. This dual function of IMiDs makes them appealing candidates for combination with a cancer vaccine. We investigated the immune stimulatory effects of lenalidomide, administrated to mice in doses, which provided comparable pharmacokinetics to human patients, on the potency of a novel fusion DNA lymphoma vaccine. The combination was curative in the majority of mice with 8d pre-established syngeneic A20 lymphomas compared with vaccine or lenalidomide alone and induced immune memory. In vivo depletion experiments established the requirement for effector CD8(+) and CD4(+) T cells in protective immunity. Unexpectedly, lenalidomide alone was also associated with reduced numbers of systemic myeloid-derived suppressor cell (MDSC) and regulatory T cell (Treg) in tumor-bearing but not naive mice, an effect that was independent of simple tumor burden reduction. These results confirm and extend results from other models describing the effect of lenalidomide on enhancing T-cell immunity, highlight the potency of this effect, and provide a rationale for clinical application. Independently, a novel mechanism of action reversing tumor-induced immune suppression by MDSC is suggested.

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 VL of human myeloma cell line U266 and five primary B-cell tumors were sequenced, and peptides corresponding to the Ig VL 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 (VL) 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 VL T-cell epitopes in U266 and primary B-cell tumors, not previously reported within Ig heavy chain (VH) sequences. Conclusion: This study thus identifies novel immunogenic CTLs epitopes from Id VL, 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. Clin Cancer Res; 17(18); 5945–52. ©2011 AACR.

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, INFγELISPOT 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.

We recently reported that administration of low doses of myotoxins at vaccination sites potentiated antigen-specific T-cell immunity induced by genetic cancer vaccines in mice, an effect which was superior to TLR agonists. In the current study, we found unexpectedly that the mechanism of this potent adjuvant effect was immune-mediated. Myotoxins induced sterile inflammation at vaccination sites, associated with a predominant infiltration of dendritic cells (DC). Inhibition of DC recruitment abrogated the immune stimulation effect of myotoxins, suggesting the requirement for DC. Genetic profiling of myotoxin-treated tissues revealed characteristics of an immune microenvironment with up-regulation of chemokines, proinflammatory cytokines, Toll-like receptors (TLR) and their endogenous ligands, and activation of innate immunity. Mechanistic experiments in vivo also elucidated the requirement for genes triggering DC maturation including TLR signaling and CD40. These studies suggest that myotoxins-induced sterile inflammation generates a favorable microenvironment that promotes multiple stages in the development of adaptive immunity. This novel mechanism of immune potentiation may be exploited for development of adjuvants for genetic vaccines against infectious pathogens and cancer.

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.

We investigated the antitumor effect of survivin DNA vaccine in murine pancreatic and lymphoma models, and if xenogenic survivin can generate stronger immune response. We found that mice vaccinated with either human or mouse survivin DNA have significantly slower tumor growth and longer survival than those vaccinated with vector DNA. There was no significant difference between groups that received human and mouse survivin DNA. Lymphocyte infiltration was greater in tumors of mice immunized with survivin DNA than in tumors of control mice. We conclude that survivin DNA vaccine generated specific antitumor effects with increased lymphocyte infiltration at the tumor sites.

The unique antigenic determinants, termed idiotype, of the immunoglobulin expressed on a given B-cell malignancy can serve as a tumor-specific antigen for active immunotherapy. Administration of autologous tumor-derived idiotype protein conjugated to a carrier protein, keyhole limpet hemocyanin, together with granulocyte-macrophage colony-stimulating factor to follicular lymphoma patients in complete clinical remission was associated with induction of tumor-specific cellular and humoral immunity, molecular remissions, and prolonged disease-free survival. Idiotype vaccination in patients with mantle cell lymphoma following rituximab-containing chemotherapy induced tumor-specific T-cell immunity in the absence of B cells, suggesting that vaccines may be used in combination with rituximab. Three double-blind, randomized, Phase III idiotype vaccine trials are currently ongoing to definitively determine the clinical benefit of idiotype-keyhole limpet hemocyanin plus granulocyte-macrophage colony-stimulating factor vaccination in patients with lymphoma. Results from early clinical trials with idiotype vaccines suggested that both humoral and cellular immune responses may be independently associated with tumor regression and improved progression-free survival. With the increased use of rituximab for the treatment of follicular lymphoma and other B-cell non-Hodgkin's lymphomas, further improvement in the potency of the vaccines would require strategies to enhance T-cell responses, as rituximab depletes normal B cells and impairs the generation of antibody responses.

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.

The immunity of a combined DNA vaccine of HSV-2 glycoproteins B2 (gB2) and D2 (gD2) genes in comparison to individual vaccines was studied with regard to protecting against the HSV infection. Two recombinant DNA vaccines of the pHS2-gB2 or pHS2-gD2 were constructed and formulated. The neutralizing antibody titers appeared higher in the B2 : D2 gene cocktail-vaccinated mice than that of the individual B2 or D2 gene-vaccinated group alone, and the positive KOS control induced higher titer of the neutralizing antibody than combined or individual gene vaccines. The mock-immunized mice failed to induce enough. The ranks for the CTL activity and the protection rates against the lethal intravaginal challenge were shown as KOSB2:D2 cocktailD2B2 gene vaccines. The vaginal external diseases in the B2 : D2 or D-vaccinated mice were significantly reduced against the challenging dosages. The virus titers in the vaginal secretions of the vaccinated mice significantly reduced with time, and the B2 : D2 gene vaccine decreased more than each individual vaccine alone. It can be concluded that the cocktailed vaccines are more effective in the humoral and cellular immune responses in the mice, and in the protection of the mice against the intravaginal challenging dosages when compared with individual gene vaccines. All the DNA vaccines failed to block the latent infection in sensory nerves.

Available therapies for lymphoplasmacytic lymphoma (LPL) provide no survival advantage if started before symptoms of end organ damage develop. Current recommendations are to follow a program of observation while patients are in the asymptomatic phase of disease. In this clinical study we will use idiotypic determinants of B-cell lymphoma surface immunoglobulins as tumor-specific antigens (idiotype) to develop a patient-specific vaccine against LPL. By activating the host immune system through vaccination to eradicate tumor cells, we postulate that disease control of asymptomatic phase lymphoplasmacytic lymphoma can be maintained. This novel 2nd generation idiotype vaccine was initially developed by genetic fusion of idiotype antigen in single-chain format with a pro-inflammatory chemokine. The central hypothesis is that antitumor immunity can be triggered by targeting antigen delivery to antigen-presenting cells in vivo by chemokine receptor-mediated binding, uptake and processing of idiotype antigens for more efficient presentation to T cells. A large body of preclinical data demonstrated that the immunogenecity of the idiotype antigen was considerably enhanced by such a genetic modification by a mechanism of facilitating antigen presentation. As a consequence, vaccine-induced prophylactic and therapeutic antitumor effects were significantly potentiated. To translate our vaccine therapy from bench to bedside, we established a CLP laboratory for vaccine preparation. Furthermore, we developed reliable technology to differentiate lymphoma idiotype antigens from idiotypic cell-surface immunoglobulins on normal B cells. Using this technology, we successfully cloned the cDNA encoding variable regions of heavy and light chains of LPL idiotype, and generated patient-specific plasmid constructs containing LPL idiotype single chain in fusion with macrophage inflammatory protein 3 alpha (MIP3a). All three engineering rounds of down-stream GMP amplification of clinical-grade plasmid DNA have been accomplished, which eventually paves the way for us to take a lab-grown agent to a first-in-human clinical trial. Citation Format: Hong Qin, Soungchul Cha, Sheetal S. Rao, Kunhwa Kim, Dongho Gwak, Sung-doo Kim, Sapna R. Parshottam, Sheeba K. Thomas, Larry W. Kwak. Bench-to-bedside development of a novel idiotype vaccine against lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2891. doi:10.1158/1538-7445.AM2014-2891

Abstract 108 Lenalidomide is an effective therapeutic agent with direct inhibitory effects on malignant B- and plasma cells and immunomodulatory effects on the tumor microenvironment. The dual functions of Lenalidomide make it an appealing candidate for combination with other novel agents for lymphoma and myeloma therapy. In this study, we investigated the immune stimulatory effects of Lenalidomide on the potency of a model lymphoma vaccine (MCP3-sFv). Therapeutic vaccines based on the idiotype of the clonal Ig receptor on malignant B cells have been shown previously to elicit specific immunity in human patients, and a recent controlled Phase III trial of an idiotype protein vaccine demonstrated prolongation of disease free survival in follicular lymphoma patients (J Clin Oncol. 10;29(20):2787–94 2011). In prophylactic experiments, the groups of 10 BALB/c mice immunized with a novel fusion DNA idiotype vaccine (MCP3-sFv, Science. 1;298(5595):1025–9 2002) were then treated with Lenalidomide at various doses (5–50 mg/kg) or saline as control i.p. for 35 consecutive days. This combination strategy protected mice from a lethal syngeneic A20 murine lymphoma, resulting in significantly improved survival comparing with vaccine or Lenalidomide alone or saline. Furthermore, more than 70% of surviving mice treated with the combination were resistant to tumor re-challenge suggesting memory antitumor immunity. Mechanistically, protection required effector cellular immunity, as tumor protection was abrogated by treatment of depleting antibody against CD8+ T cells, either alone or together with CD4 depleting antibody in vivo. Lenalidomide showed little impact on humoral immunity, with serum anti-idiotype antibody levels in the combination group similar to the vaccine alone group. In addition to adaptive immune system, we also examined the effect of Lenalidomide on other immune cells including NK cells, myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg). Interestingly, Lenalidomide treatment did not affect the number of these immune cell populations in non-tumor bearing mice; but in tumor-bearing mice, Lenalidomide-induced tumor regression was associated with reduced numbers of immune suppressive cells (MDSC/Treg) and rescue of circulating NK cells. This finding suggests a role of Lenalidomide in ameliorating tumor-induced immune suppression. This hypothesis was supported by the demonstration that the combination of Lenalidomide and vaccine (vaccine given starting on day 8) produced significantly improve survival, compared with controls receiving vaccine or Lenalidomide alone (log rank p Disclosures: Kwak: Biovest International, Antigenics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biovest International: ; Antigenics, Xeme Biopharma: Equity Ownership.

Abstract 3018 The variable regions of Ig expressed by malignant B cells can serve as a tumor specific antigen. Clinical trials of idiotype (Id) vaccines have demonstrated humoral responses and prolonged remission duration in a recent phase III trial of follicular lymphoma patients in the first remission (Schuster et al, J Clin Oncol 27: 793S, 2009). However, the potentially immunogenic epitopes derived from Ig that stimulate CD8+ T cell immunity have been incompletely characterized. Here, we identified nine out of 14 candidate peptides derived from the Ig L-chain variable region of the human U266 myeloma line, which generated cytotoxic T lymphocytes (CTLs) from 53 HLA A2+ normal donors. These CTLs lines, as well as CTLs line isolated from myeloma patients by stimulation with autologous L-chain Id peptides, specifically produced IFN-γ in response to peptide-pulsed T2 cells and lysed U266 and autologous myeloma cell targets, respectively, but not normal blood B cells. Lysis was HLA class I-dependent, suggesting that primary myeloma cells express Id peptides on the cell surface in combination with HLA molecules. Nine CD8+ Id peptide-specific T-cell clones exhibited the effector memory phenotype and the ability of these T cell clones to eliminate U266 tumor in immune deficient mice is being tested. Finally, sequence analysis revealed shared T-cell epitopes in both framework and CDR regions of the U266 L-chain. CTLs generated against a shared U266 epitope lysed patient-derived myeloma cells expressing the shared sequence, suggesting a strategy to overcome the limitation of patient-specific Id vaccine manufacture. Our data identified novel immunogenic Id L-chain T-cell determinants and suggests that, unlike previously described Ig heavy chains, these sequences harbor common T-cell epitopes that may provide the rationale for shared Id vaccines. Disclosures: No relevant conflicts of interest to declare.

Abstract 139 The chromosomal translocation t(14;18) is present in most human follicular lymphomas (FL), however, it is not sufficient for lymphomagenesis. A number of studies have suggested a potential role for antigen activation of the B-cell receptor (BCR) in the pathogenesis of FL and other B-cell malignancies. However, the nature of the antigen(s) recognized by FL BCRs has not been well studied. We hypothesized that the antigen(s) recognized by FL BCRs is either a self or non-self antigen. The objective of this study was to identify self antigen(s) recognized by FL BCRs. We generated clonal tumor immunoglobulins (Igs) from 217 patients with FL using either hybridoma or recombinant DNA technologies. Purified tumor Igs were tested for autoreactivity against whole cell lysate prepared from a human epithelial cell line, HEp-2, in an enzyme-linked immunosorbent assay (ELISA). We observed that FL tumor Igs were frequently self-reactive. To determine the nature of the antigen recognized by the tumor Igs, we performed polyacrylamide gel electrophoresis of HEp-2 cell lysate. The single band recognized by the tumor Igs was analyzed by mass spectrometry and was determined to be vimentin. Using recombinant vimentin protein, we demonstrated that the tumor Igs specifically bound to vimentin when tested by ELISA and Western Blot. Furthermore, pre-incubation of the tumor Igs with recombinant vimentin protein significantly inhibited their reactivity against HEp-2 cell lysate in a dose-dependent manner. Using indirect immunofluorescence staining, we showed that the vimentin-reactive tumor Igs co-localized with a mouse anti-vimentin mAb in HEp-2 cells. Using a series of truncated recombinant vimentin protein fragments, we determined that the epitope recognized by the tumor Igs is localized to the N-terminal region of vimentin. Overall, 42/217 (19.35%) FL tumor Igs recognized N-terminal region of vimentin. Vimentin-reactivity was more commonly observed with IgG than IgM FL tumor Igs (30.4% vs. 10%; P=0.0022). However, comprehensive analysis of the sequences of the variable regions of heavy and light chains of the FL tumor Igs showed that vimentin-reactivity did not correlate with VH, JH, Vκ, Jκ, Vλ, and Jλ gene usage; CDR3 length; number of positively charged amino acid residues in CDR3; rate and pattern of mutations; or rate and pattern of N-glycosylation. In addition, we did not observe recurrent IgVH- or IgVL-CDR3 motifs amongst the vimentin-reactive FL tumor Igs. By immunohistochemistry, we showed that vimentin was expressed in the T-cell rich regions of FL suggesting its availability for binding with BCR within the tumor microenvironment. Interestingly, vimentin was also recognized by 9/18 (50%) mantle cell lymphoma (MCL) tumor Igs and 12/31 (38.7%) multiple myeloma (MM) tumor Igs. In conclusion, our results demonstrate that vimentin is a shared autoantigen recognized by non-stereotypic FL BCRs as well as tumor Igs from MCL and MM and suggest that it may play a role in the pathogenesis of multiple B-cell malignancies. The identification of a shared autoantigen recognized by tumor BCRs could lead to development of in vitro and in vivo models to better understand the pathogenesis and natural history of FL and other B-cell malignancies. Disclosures: No relevant conflicts of interest to declare.

A major obstacle for translational research of cancer vaccine is the weak immunogenicity of tumor antigens. Thus, the development of new, more potent strategy that aims at breaking such an immune tolerance is a prerequisite of vaccine therapy. Here we show that the unexpected effect of inducing sterile inflammation at vaccination sites by myotoxins potentiated induction of tumor-specific immunity by genetic vaccines and converted a non-immunogenic lymphoma idiotype antigen into a tumor rejection vaccine. This potent immunostimulatory effect was immune-mediated, requiring recruitment of dendritic cells (DC). Sterile inflammation induced by myotoxins was associated with upregulation of chemokines, proinflammatory cytokines, Toll-like receptors and their endogenous ligands, and activation of innate immunity. Mechanistic experiments in vivo also elucidated the requirement for genes triggering DC maturation including TLR signaling and CD40. These studies suggest that inducing sterile inflammation at vaccination sites generates a favorable microenvironment that promotes adaptive immunity. This novel mechanism of immune potentiation may be exploited for development of adjuvants for vaccines against cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4760.

Active immunization of follicular lymphoma patients with idiotypic vaccines elicits antigen-specific antibody responses, T-cell responses, and antitumor effects (Bendandi, M. et al., Nat Med5, 1171). 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. We further hypothesize that these antigens may represent tumor rejection targets for immunotherapy. 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. To further determine the specificity of these patients’ post vaccine sera, the corresponding FL-aa-7 and FL-aa-18 peptides were synthesized and tested for serum binding. As expected, post-vaccine but not pre-vaccine or normal, specifically bound the FL-aa-7 and FL-aa-18 peptides, respectively. Furthermore, both peptides specifically inhibited the binding of post-vaccine sera to the respective FL-aa-7 and FL-aa-18 phage clones in a dose-dependent manner. These data suggest that Id vaccinated patients generated specific antibodies to novel peptides FL-aa-7 and FL-aa-18, unrelated to Id. Overall, these data indicate that Id-vaccinated B cell lymphoma patients mounted antibody responses specifically for FL-aa-7-1 and FL-aa-18 peptides, which may represent a potential immunotherapeutic tumor antigen. The clinical and biological relevance of the response is to be elucidated.

Advanced stage follicular lymphoma remains an incurable disease with a median survival of 8 to 10 years that has not significantly changed over the last four decades. Therefore, novel treatment options are necessary to improve the clinical outcome in these patients. The observation of spontaneous regressions in a small percentage of patients suggested that augmenting the host immune response could potentially control this malignancy. Strategies using active specific immunotherapy with idiotype vaccines led to induction of clinical and molecular responses in a few patients but have met with only limited success possibly due to the low frequency of antigen-specific T cells induced in the patients. In contrast to active immunization, T cells of a given specificity and function may be selected and expanded in vitro to the desired number for adoptive cell transfer. Towards this goal, we stimulated tumor infiltrating lymphocytes (TILs) or peripheral blood mononuclear cells (PBMCs) from five follicular lymphoma patients with CD40 ligand-activated autologous tumor cells at approximately ten-day intervals in the presence of IL-2 and IL-15. After four rounds of stimulations, T cell lines generated from 3/5 patients recognized autologous unmodified tumor cells by producing significant amounts of TNF-α, GM-CSF and/or IFN-γ. By phenotypic analysis, the T cell lines were predominantly CD4+ T cells (> 70%), and intracellular cytokine assay showed that up to 40% of the CD4+ T cells were tumor-reactive. The inhibition of cytokine production by anti-HLA class II but not class I blocking antibodies confirmed that the CD4+ T cells were tumor-reactive. Further characterization revealed that the T cells from one patient recognized autologous tumor but not autologous normal B cells suggesting that they were tumor-specific. While in a second patient CD4+ T cell clones generated from the T cell line by limiting dilution recognized autologous tumor and autologous normal B cells but not autologous monocytes suggesting that they were B cell lineage-specific. We conclude that follicular lymphoma-specific T cells exist and can be efficiently expanded in vitro from both TILs and PBMCs using CD40 ligand-activated autologous tumor cells for adoptive T cell therapy. Additionally, identification of antigens recognized by these T cells could lead to development of novel immunotherapeutic strategies for lymphomas.

Meeting abstracts 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 potential CD4 T cell