Nela Klein-Gonzalez

CD40-activated B cells can be generated in high number and purity in cancer patients: analysis of immunogenicity and homing potential

Cellular adjuvants such as dendritic cells (DC) are in the focus of tumour immunotherapy. In DC‐vaccine trials, induction of tumour antigen‐specific immunity is observed frequently and well‐documented clinical responses have been reported. However, the overall response rate is less than 3%, therefore alternative strategies are being investigated. CD40‐activated B cells (CD40‐B) have been characterized previously as an interesting alternative because they present antigen efficiently and can be expanded by several logs from small amounts of peripheral blood. To determine the central technical challenges of cell‐based vaccines we performed a single‐patient analysis of 502 patients from DC‐based tumour vaccine trials and identified at least three factors contributing to their limited efficiency: (1) lack of cell numbers; (2) lack of documented purity thus high contamination of bystander cells; and (3) lack of quality control and thus heterogeneous or unknown expression of important surface molecules such as major histocompatibility complex (MHC) and chemokine receptors. Based on these findings we re‐evaluated the CD40‐B approach in cancer patients. Here, we show that proliferation of B cells from cancer patients is equivalent to that observed in healthy donors. Purity is always > 90% after 2 weeks and remains stable for several weeks. They have comparable antigen‐presenting capability determined phenotypically and by allogeneic mixed lymphocyte reaction. Expression of CCR7 and CD62L was detected in all samples and B cells migrated towards the relevant homing chemokines. Taken together, CD40‐B cells from cancer patients can be expanded in virtually unlimited numbers at high purity and full function concerning antigen‐presentation and migratory properties.

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (>10(9) cells per patient) from healthy donors as well as cancer patients. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro. Furthermore they efficiently take up, process and present antigens to T cells. CD40-B cells were shown to not only prime naíve, but also expand memory T cells. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa). Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (>90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach.

CD40-activated B cells induce anti-tumor immunity in vivo.

The introduction of checkpoint inhibitors represents a major advance in cancer immunotherapy. Some studies on checkpoint inhibition demonstrate that combinatorial immunotherapies with secondary drivers of anti-tumor immunity provide beneficial effects for patients that do not show a strong endogenous immune response. CD40-activated B cells (CD40B cells) are potent antigen presenting cells by activating and expanding naïve and memory CD4+ and CD8+ and homing to the secondary lymphoid organs. In contrast to dendritic cells, the generation of highly pure CD40B cells is simple and time efficient and they can be expanded almost limitlessly from small blood samples of cancer patients. Here, we show that the vaccination with antigen-loaded CD40B cells induces a specific T-cell response in vivo comparable to that of dendritic cells. Moreover, we identify vaccination parameters, including injection route, cell dose and vaccination repetitions to optimize immunization and demonstrate that application of CD40B cells is safe in terms of toxicity in the recipient. We furthermore show that preventive immunization of tumor-bearing mice with tumor antigen-pulsed CD40B cells induces a protective anti-tumor immunity against B16.F10 melanomas and E.G7 lymphomas leading to reduced tumor growth. These results and our straightforward method of CD40B-cell generation underline the potential of CD40B cells for cancer immunotherapy.

The cyclins: A family of widely expressed tumor antigens?

Continuous cell division is a hallmark of cancer and cell-cycle regulators therefore represent relevant target molecules for tumor therapy. Among these targets the cyclins are of particular interest as they are overexpressed in various tumor entities with little expression in normal tissue. Here we review evidence that these molecules are recognized by the immune system, summarize why cyclins A, B and D in particular appear to be interesting targets for active and passive immunotherapy, and discuss whether the entire family could be an interesting novel class of tumor antigens for cancer treatment and prevention.

The shared tumor associated antigen cyclin‐A2 is recognized by high‐avidity T‐cells

Cyclin‐A2, a key cell cycle regulator, has been shown to be overexpressed in various types of malignancies with little expression in normal tissue. Such tumor‐associated genes potentially are useful targets for cancer immunotherapy. However, high‐avidity cyclin‐specific T cells are considered to be thymically deleted. We identified at least one nonameric HLA‐A*0201 binding cyclin‐A2 epitope by a reverse immunology approach. Using a highly efficient T‐cell expansion system that is based on CD40‐activated B (CD40‐B) cells as sole antigen‐presenting cells we successfully generated cyclin‐A2 specific CTL from HLA‐A*0201+ donors. Interestingly, high‐avidity cyclin‐A2 specific CTL lines, which recognized peptide‐pulsed and antigen expressing target cells, were indeed generated by stimulation with CD40‐B cells when pulsed with low concentrations of peptide, whereas CD40‐B cells pulsed at saturating concentrations could only induce low‐avidity CTL, which recognized peptide‐pulsed target cells only. One high‐avidity CTL line was subcloned and CTL clones, whose peptide concentration required for half‐maximal lysis were less than 1 nM, could lyse cyclin‐A2 expressing tumor cells. Taken together, cyclin A2 is an attractive candidate for immune intervention in a significant number of cancer patients and high‐avidity T cells can be readily generated using CD40‐B cells as antigen‐presenting cells.

Potential of Toll-like receptor 9 agonists in combined anticancer immunotherapy strategies: synergy of PAMPs and DAMPs?

As part of the innate immune system, TLRs belong to a family of at least ten specialized immune receptors that recognize specific, highly conserved molecular patterns traditionally referred to as pathogen-associated molecular patterns (PAMPs) [3]. Upon detection of these PAMPs, downstream signaling activates the effector side of the immune system, which can strengthen or initiate antitumor responses. Several synthetic TLR agonists have been developed for clinical grade use. TLR9 is activated by dsDNA. This includes activation by unmethylated CpG dinucleotides, a common pattern in bacterial and viral DNA [4]. Several CpG oligodeoxynucleotides (ODNs) have been synthesized to mimic natural CpGs.

T-cell responses to cyclin B1 are not restricted to p53-overexpressing tumors.

To the Editors: In their elegant study, Sorensen et al. showed the existence of a T-cell response to two cyclin B1–derived peptides in patients with advanced-stage breast cancer, confirming initial observations by Kao that cyclin B1 is an interesting target for immunotherapy ([1][1], [2][2]).