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Key Points Preventative vaccination against infectious organisms has had a dramatic effect on public health. Therapeutic vaccination against cancer is more challenging but, armed with new immunological insight and genetic technology, aims similarly to harness the power of the immune system, in this case to destroy or suppress tumour cells. Passively transferred antibodies and T cells are clearly able to attack human cancer cells in vivo and are included in treatment protocols for some cancers. Active vaccination would generate these effector pathways, together with immunological memory that is able to continuously detect and remove any emergent cancer cells. Tumour antigens are being rapidly revealed, and can be expressed on cell surfaces or, more commonly, as peptides in association with the major histocompatibility complex class I (or II) molecules. DNA vaccines can be designed to activate antibody and/or T-cell responses, providing focused immune attack on selected antigens. DNA vaccines offer a precise but flexible strategy for delivering antigens to the immune system, and additional sequences encoding molecules to manipulate outcome can be included. The problem of translating success in preclinical models to patients seems to be overcome by using electroporation, which dramatically improves performance and is now in clinical trials for prostate cancer. The key to bypassing immune tolerance and activating high levels of anti-tumour antibody or cytotoxic T cells lies in inducing CD4 + T-cell help. Sequences derived from microbial antigens can be incorporated into anti-tumour DNA vaccines, a strategy which mobilizes help for anti-tumour responses from the large non-tolerized anti-microbial repertoire. Clinical trials are the real test, but the current question is largely of efficacy rather than toxicity. New thinking is informing pilot trial design within a highly regulated environment, and immunological assays that can be predictors of clinical outcome are developing rapidly. DNA vaccines against cancer allow a high degree of molecular precision, but until recently translation into the clinic has been difficult. Advances in delivery techniques are now allowing them to fulfil their potential. DNA vaccination has suddenly become a favoured strategy for inducing immunity. The molecular precision offered by gene-based vaccines, together with the facility to include additional genes to direct and amplify immunity, has always been attractive. However, the apparent failure to translate operational success in preclinical models to the clinic, for reasons that are now rather obvious, reduced initial enthusiasm. Recently, novel delivery systems, especially electroporation, have overcome this translational block. Here, we assess the development, current performance and potential of DNA vaccines for the treatment of cancer.

Signaling via the B-cell receptor (BCR) is a key driver and therapeutic target in chronic lymphocytic leukemia (CLL). BCR stimulation of CLL cells induces expression of eIF4A, an initiation factor important for translation of multiple oncoproteins, and reduces expression of PDCD4, a natural inhibitor of eIF4A, suggesting that eIF4A may be a critical nexus controlling protein expression downstream of the BCR in these cells. We, therefore, investigated the effect of eIF4A inhibitors (eIF4Ai) on BCR-induced responses. We demonstrated that eIF4Ai (silvestrol and rocaglamide A) reduced anti-IgM-induced global mRNA translation in CLL cells and also inhibited accumulation of MYC and MCL1, key drivers of proliferation and survival, respectively, without effects on upstream signaling responses (ERK1/2 and AKT phosphorylation). Analysis of normal naïve and non-switched memory B cells, likely counterparts of the two main subsets of CLL, demonstrated that basal RNA translation was higher in memory B cells, but was similarly increased and susceptible to eIF4Ai-mediated inhibition in both. We probed the fate of MYC mRNA in eIF4Ai-treated CLL cells and found that eIF4Ai caused a profound accumulation of MYC mRNA in anti-IgM treated cells. This was mediated by MYC mRNA stabilization and was not observed for MCL1 mRNA. Following drug wash-out, MYC mRNA levels declined but without substantial MYC protein accumulation, indicating that stabilized MYC mRNA remained blocked from translation. In conclusion, BCR-induced regulation of eIF4A may be a critical signal-dependent nexus for therapeutic attack in CLL and other B-cell malignancies, especially those dependent on MYC and/or MCL1.

Induction of potent antibody is the goal of many vaccines targeted against infections or cancer. Modern vaccine designs that use virus-like particles (VLP) have shown efficacy for prophylactic vaccination against virus-associated cancer in the clinic. Here we used plant viral particles (PVP), which are structurally analogous to VLP, coupled to a weak idiotypic (Id) tumour antigen, as a conjugate vaccine to induce antibody against a murine B-cell malignancy. The Id-PVP vaccine incorporates a natural adjuvant, the viral ssRNA, which acts via TLR7. It induced potent protective anti-Id antibody responses in an in vivo mouse model, superior to the \"gold standard\" Id vaccine, with prevalence of the IgG2a isotype. Combination with alum further increased antibody levels and maintained the IgG2a bias. Engagement of TLR7 in vivo was followed by secretion of IFN-α by plasmacytoid dendritic cells and by activation of splenic CD11chi conventional dendritic cells. The latter was apparent from up-regulation of co-stimulatory molecules and from secretion of a wide range of inflammatory cytokines and chemokines including the Th1-governing cytokine IL-12, in keeping with the IgG2a antibody isotype distribution. PVP conjugates are a novel cancer vaccine design, offering an attractive molecular form, similar to VLP, and providing T-cell help. In contrast to VLP, they also incorporate a safe \"in-built\" ssRNA adjuvant.

Surface Ig (sIg) of follicular lymphoma (FL) is vital for tumor cell survival. We found previously that the Ig in FL is unusual, because the variable region genes carry sequence motifs for N-glycan addition. These are introduced by somatic mutation and are tumor specific. Unexpectedly, added glycans terminate at high mannose, suggesting a potentially important interaction of FL cells with mannose-binding lectins of the innate immune system. We have now identified mannosylated IgM at the surface of primary lymphoma cells. Recombinant lectin domains of the mannose receptor (MR) or DC-SIGN bind mannosylated Igs in vitro and bind to FL cells, signaling sIgM-associated increases in intracellular Ca²⁺. Lectins also bind to normal B cells but fail to signal. In contrast, anti-Ig signaled similarly in both FL and normal B cells. Mannosylation patterns were mimicked by FL Ig-derived single-chain Fvs (scFv), providing probes for potential receptors. Mannosylated scFv bound specifically to the lectin domains of the MR and DC-SIGN and blocked signaling. Mannosylated scFv also bound to DC-SIGN on the surface of dendritic cells. This unique lymphoma-specific interaction of sIg with lectins of innate immunity reveals a potential route for microenvironmental support of tumor cells, mediated via the key B-cell receptor.

In follicular lymphoma (FL), surface immunoglobulin (sIg) carries mandatory N-glycosylation sites in the variable regions, inserted during somatic hypermutation. These glycosylation sites are tumor-specific, indicating a critical function in FL. Added glycan unexpectedly terminates at high mannose (Mann) and confers capability for sIg-mediated interaction with local macrophage-expressed DC-SIGN lectin resulting in low-level activation of upstream B-cell receptor signaling responses. Here we show that despite being of low-level, DC-SIGN induces a similar downstream transcriptional response to anti-IgM in primary FL cells, characterized by activation of pathways associated with B-cell survival, proliferation and cell–cell communication. Lectin binding was also able to engage post-transcriptional receptor cross-talk pathways since, like anti-IgM, DC-SIGN down-modulated cell surface expression of CXCR4. Importantly, pre-exposure of a FL-derived cell line expressing sIgM-Mann or primary FL cells to DC-SIGN, which does not block anti-IgM binding, reversibly paralyzed the subsequent Ca 2+ response to anti-IgM. These novel findings indicate that modulation of sIg function occurs in FL via lectin binding to acquired mannoses. The B-cell receptor alternative engagement described here provides two advantages to lymphoma cells: (i) activation of signaling, which, albeit of low-level, is sufficient to trigger canonical lymphoma-promoting responses, and (ii) protection from exogenous antigen by paralyzing anti-IgM-induced signaling. Blockade of this alternative engagement could offer a new therapeutic strategy.

CD4+ T helper memory (Thmem) cells influence both natural and vaccine-boosted immunity, but mechanisms for their maintenance remain unclear. Pro-survival signals from the common gamma-chain cytokines, in particular IL-7, appear important. Previously we showed in healthy volunteers that a booster vaccination with tetanus toxoid (TT) expanded peripheral blood TT-specific Thmem cells as expected, but was accompanied by parallel increase of Thmem cells specific for two unrelated and non cross-reactive common recall antigens. Here, in a new cohort of healthy human subjects, we compare blood vaccine-specific and bystander Thmem cells in terms of differentiation stage, function, activation and proliferative status. Both responses peaked 1 week post-vaccination. Vaccine-specific cytokine-producing Thmem cells were predominantly effector memory, whereas bystander cells were mainly of central memory phenotype. Importantly, TT-specific Thmem cells were activated (CD38High HLA-DR+), cycling or recently divided (Ki-67+), and apparently vulnerable to death (IL-7RαLow and Bcl-2 Low). In contrast, bystander Thmem cells were resting (CD38Low HLA-DR- Ki-67-) with high expression of IL-7Rα and Bcl-2. These findings allow a clear distinction between vaccine-specific and bystander Thmem cells, suggesting the latter do not derive from recent proliferation but from cells mobilized from as yet undefined reservoirs. Furthermore, they reveal the interdependent dynamics of specific and bystander T-cell responses which will inform assessments of responses to vaccines.

Chronic lymphocytic leukaemia (CLL) is a malignancy of CD5 + B cells that is characterized by the accumulation of small, mature-appearing lymphocytes in the blood, marrow and lymphoid tissues. Signalling via surface immunoglobulin, which constitutes the major part of the B cell receptor, and several genetic alterations play a part in CLL pathogenesis, in addition to interactions between CLL cells and other cell types, such as stromal cells, T cells and nurse-like cells in the lymph nodes. The clinical progression of CLL is heterogeneous and ranges from patients who require treatment soon after diagnosis to others who do not require therapy for many years, if at all. Several factors, including the immunoglobulin heavy-chain variable region gene ( IGHV ) mutational status, genomic changes, patient age and the presence of comorbidities, should be considered when defining the optimal management strategies, which include chemotherapy, chemoimmunotherapy and/or drugs targeting B cell receptor signalling or inhibitors of apoptosis, such as BCL-2. Research on the biology of CLL has profoundly enhanced our ability to identify patients who are at higher risk for disease progression and our capacity to treat patients with drugs that selectively target distinctive phenotypic or physiological features of CLL. How these and other advances have shaped our current understanding and treatment of patients with CLL is the subject of this Primer. Research on the biology of chronic lymphocytic leukaemia (CLL) — a malignancy of CD5 + B cells — has profoundly enhanced the identification of patients who are at high risk of disease progression and the treatment of patients with drugs that target the distinctive features of CLL. This Primer highlights these advances, as well as the epidemiology, genetics and immunobiology of CLL.

Delivery of antigens by injection of the encoding DNA allows access to multiple antigen-presenting pathways. Knowledge of immunological processes can therefore be used to modify construct design to induce selected effector functions. Expression can be directed to specific intracellular sites, and additional genes can be fused or codelivered to amplify responses. Therapeutic vaccination against cancer adds a requirement to overcome tolerance and to activate a weakened immune repertoire. Induction of CD4+ T helper cells is critical for both antibody and T cell effector responses. To activate immunity against tumor antigens, we fused the tumor-derived sequences to genes encoding microbial proteins. This strategy engages T helper cells from the large antimicrobial repertoire for linked help for inducing antibody against cell-surface tumor antigens. The principle of linked T cell help also holds for induction of epitope-specific antitumor CD8+ T cells, but the microbial sequence has to be minimized to avoid competition with tumor antigens. Epitope-specific DNA vaccination leads to powerful antitumor attack and can activate immunity from a profoundly tolerized repertoire. Vaccine designs validated in preclinical models are now in clinical trial with immune responses detected against both tumor antigens and fused microbial antigens. DNA priming is highly efficient, but boosting may benefit from increased antigen expression. Physical methods including electroporation provide increased expression without introducing additional competing antigens. A wide range of cancers can be targeted, and objective assays of response will determine efficacy.

Chronic lymphocytic leukaemia (CLL) cells can express unmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy chain (IGHV) genes with differing clinical behaviours, variable B cell receptor (BCR) signalling capacity and distinct transcriptional profiles. As it remains unclear how these differences reflect the tumour cells’ innate pre/post germinal centre origin or their BCR signalling competence, we applied mRNA/miRNA sequencing to 38 CLL cases categorised into three subsets by IGHV mutational status and BCR signalling capacity. We identified 492 mRNAs and 38 miRNAs differentially expressed between U-CLL and M-CLL, but only 9 mRNAs and 0 miRNAs associated with BCR competence within M-CLL. Of the IGHV-associated miRNAs, (14/38 (37%)) derived from chr14q32 clusters where all miRNAs were co-expressed with the MEG3 lncRNA from a cancer associated imprinted locus. Integrative analysis of miRNA/mRNA data revealed pronounced regulatory potential for the 14q32 miRNAs, potentially accounting for up to 25% of the IGHV-related transcriptome signature. GAB1, a positive regulator of BCR signalling, was potentially regulated by five 14q32 miRNAs and we confirmed that two of these (miR-409-3p and miR-411-3p) significantly repressed activity of the GAB1 3′UTR. Our analysis demonstrates a potential key role of the 14q32 miRNA locus in the regulation of CLL-related gene regulation.