Monocytes Exposed to Immune Complexes Reduce pDC Type 1 Interferon Response to Vidutolimod.

Vidutolimod, also known as CMP-001, is a virus-like particle composed of the Qß bacteriophage coat protein encasing a TLR9 agonist. Vidutolimod injected intratumorally is showing promise in early phase clinical trials based on its ability to alter the tumor microenvironment and induce an anti-tumor immune response. We previously demonstrated that the in vivo efficacy of vidutolimod is dependent on the presence of anti-Qß antibodies that enhance opsonization and uptake of vidutolimod by TLR9-expressing plasmacytoid dendritic cells (pDCs). Here, we evaluated the effect of immune complexes, including anti-Qß-coated vidutolimod, on induction of Type 1 Interferon production by peripheral blood mononuclear cells in response to vidutolimod and soluble TLR9 agonists. Immune complexes, including but not limited to anti-Qß-coated vidutolimod, indirectly suppressed TLR9-mediated Type 1 Interferon production by pDCs in a monocyte-dependent manner. These findings indicate that anti-Qß-coated vidutolimod has effects in addition to those mediated by TLR9 that could have important clinical implications for understanding the mechanism of action of this exciting new approach to in situ immunization and cancer immunotherapy.

Direct and indirect immune effects of CMP-001, a virus-like particle containing a TLR9 agonist.

BACKGROUND: CMP-001, also known as vidutolimod, is a virus-like particle containing a TLR9 agonist that is showing promise in early clinical trials. Our group previously demonstrated that the immunostimulatory effects of CMP-001 are dependent on an anti-Qß antibody response which results in opsonization of CMP-001 and uptake by plasmacytoid dendritic cells (pDCs) that then produce interferon (IFN)-α. IFN-α then leads to an antitumor T-cell response that is responsible for the in vivo efficacy of CMP-001. Here, we explore mechanisms by which the initial effects of CMP-001 on pDCs activate other cells that can contribute to development of an antitumor T-cell response. METHODS: Uptake of CMP-001 by various peripheral blood mononuclear cell (PBMC) populations and response to anti-Qß-coated CMP-001 were evaluated by flow cytometry and single-cell RNA sequencing. Purified monocytes were treated with anti-Qß-coated CMP-001 or recombinant IFN-α to evaluate direct and secondary effects of anti-Qß-coated CMP-001 on monocytes. RESULTS: Monocytes had the highest per cell uptake of anti-Qß-coated CMP-001 with lower levels of uptake by pDCs and other cell types. Treatment of PBMCs with anti-Qß-coated CMP-001 induced upregulation of IFN-responsive genes including CXCL10, PDL1, and indoleamine-2,3-dioxygenase (IDO) expression by monocytes. Most of the impact of anti-Qß-coated CMP-001 on monocytes was indirect and mediated by IFN-α, but uptake of anti-Qß-coated CMP-001 altered the monocytic response to IFN-α and resulted in enhanced expression of PDL1, IDO, and CD80 and suppressed expression of CXCL10. These changes included an enhanced ability to induce autologous CD4 T-cell proliferation. CONCLUSIONS: Anti-Qß-coated CMP-001 induces IFN-α production by pDCs which has secondary effects on a variety of cells including monocytes. Uptake of anti-Qß-coated CMP-001 by monocytes alters their response to IFN-α, resulting in enhanced expression of PDL1, IDO and CD80 and suppressed expression of CXCL10. Despite aspects of an immunosuppressive phenotype, these monocytes demonstrated increased ability to augment autologous CD4 T-cell proliferation. These findings shed light on the complexity of the mechanism of action of anti-Qß-coated CMP-001 and provide insight into pathways that may be targeted to further enhance the efficacy of this novel approach to immunotherapy.

Antibody Opsonization of a TLR9 Agonist-Containing Virus-like Particle Enhances In Situ Immunization.

The immunologic and therapeutic effects of intratumoral (IT) delivery of a novel virus-like particle as a lymphoma immunotherapy were evaluated in preclinical studies with human cells and a murine model. CMP-001 is a virus-like particle composed of the Qß bacteriophage capsid protein encapsulating an immunostimulatory CpG-A oligodeoxynucleotide TLR9 agonist. In vitro, CMP-001 induced cytokine production, including IFN-α from plasmacytoid dendritic cells, but only in the presence of anti-Qß Ab. In vivo, IT CMP-001 treatment of murine A20 lymphoma enhanced survival and reduced growth of both injected and contralateral noninjected tumors in a manner dependent on both the ability of mice to generate anti-Qß Ab and the presence of T cells. The combination of IT CMP-001 with systemic anti-PD-1 enhanced antitumor responses in both injected and noninjected tumors. IT CMP-001 alone or combined with anti-PD-1 augmented T cell infiltration in tumor-draining lymph nodes. We conclude IT CMP-001 induces a robust antitumor T cell response in an anti-Qß Ab-dependent manner and results in systemic antitumor T cell effects that are enhanced by anti-PD-1 in a mouse model of B cell lymphoma. Early-phase clinical evaluation of CMP-001 and anti-PD-1 combination therapy in lymphoma will begin shortly, based in part on these results.

An RNA Aptamer-Based Biomarker Platform Demonstrates High Soluble CD25 Occupancy by IL2 in the Serum of Follicular Lymphoma Patients.

Ligand-receptor complexes play a central role in mediating a range of processes in immunology and cancer biology. The ability to directly quantify the fraction of receptors occupied by a ligand in a given biospecimen, as opposed to assessing the concentration of ligand and receptor separately, could provide an additional and valuable clinical and research tool for assessing whether receptors are occupied by a ligand. To address this need, a biomarker platform was developed to quantify the fraction of receptors occupied by a ligand using pairs of RNA aptamers, where one aptamer binds preferentially to the unoccupied receptor and the other to the ligand-receptor complex. Bound aptamer was quantified using RT-qPCR colorimetric probes specific for each aptamer. The binding ratio of aptamer correlated with the fraction of receptors occupied by a ligand. This assay, termed as LIRECAP (LIgand-REceptor Complex-binding APtamer) assay, was used to determine the fraction of soluble CD25 occupied by IL2 in the serum from subjects with B-cell lymphoma. No correlation was found between the type of lymphoma and total soluble CD25 or IL2 independently. In contrast, the fraction of soluble CD25 occupied by IL2 was significantly higher in follicular lymphoma patient serum compared with diffuse large B-cell lymphoma patient serum. We conclude that this technology has the potential to serve as a high-throughput biomarker platform to quantify the fraction of receptors occupied by a ligand.

Three steps to breaking immune tolerance to lymphoma: a microparticle approach.

In situ immunization aims at generating antitumor immune responses through manipulating the tumor microenvironment. On the basis of recent advances in the understanding of antitumor immunity, we designed a three-step approach to in situ immunization to lymphoma: (i) inducing immunogenic tumor cell death with the chemotherapeutic drug doxorubicin. Doxorubicin enhances the expression of "eat-me" signals by dying tumor cells, facilitating their phagocytosis by dendritic cells (DC). Because of the vesicant activity of doxorubicin, microparticles made of biodegradable polymer poly(lactide-co-glycolide) or PLGA can safely deliver doxorubicin intratumorally and are effective vaccine adjuvants, (ii) enhancing T-cell activation using anti-OX40 and (iii) sustaining T-cell responses by checkpoint blockade using anti-CTLA-4. In vitro, doxorubicin microparticles were less cytotoxic to DCs than to B lymphoma cells, did not require internalization by tumor cells, and significantly enhanced phagocytosis of tumor cells by DCs as compared with soluble doxorubicin. In mice, this three-step therapy induced CD4- and CD8-dependent systemic immune responses that enhanced T-cell infiltration into distant tumors, leading to their eradication and significantly improving survival. Our findings demonstrate that systemic antitumor immune responses can be generated locally by three-step therapy and merit further investigation as an immunotherapy for patients with lymphoma.

Chemoimmunotherapy for relapsed/refractory and progressive 17p13-deleted chronic lymphocytic leukemia (CLL) combining pentostatin, alemtuzumab, and low-dose rituximab is effective and tolerable and limits loss of CD20 expression by circulating CLL cells.

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) patients with purine analog refractory disease or TP53 dysfunction still have limited treatment options and poor survival. Alemtuzumab-containing chemoimmunotherapy regimens can be effective but frequently cause serious infections. We report a Phase II trial testing the efficacy and tolerability of a short-duration regimen combining pentostatin, alemtuzumab, and low-dose high-frequency rituximab designed to decrease the risk of treatment-associated infections and to limit the loss of CD20 expression by CLL cells. The study enrolled 39 patients with progressive CLL that was either relapsed/refractory (n = 36) or previously untreated with 17p13 deletion (17p13-) (n = 3). Thirteen (33%) patients had both 17p13- and TP53 mutations predicted to be dysfunctional, and eight patients had purine analog refractory CLL without TP53 dysfunction. Twenty-six (67%) patients completed therapy, with only five (13%) patients having treatment-limiting toxicity and no treatment-related deaths. Twenty-two (56%) patients responded to treatment, with 11 (28%) complete responses (four with incomplete bone marrow recovery). Median progression-free survival was 7.2 months, time to next treatment was 9.1 months, and overall survival was 34.1 months. The majority of deaths (82%) were caused by progressive disease, including transformed diffuse large B-cell lymphoma (n = 6). Correlative studies showed that low-dose rituximab activates complement and natural killer cells without a profound and sustained decrease in expression of CD20 by circulating CLL cells. We conclude that pentostatin, alemtuzumab, and low-dose high-frequency rituximab is a tolerable and effective therapy for CLL and that low-dose rituximab therapy can activate innate immune cytotoxic mechanisms without substantially decreasing CD20 expression.

B-CLL cells acquire APC- and CTL-like phenotypic characteristics after stimulation with CpG ODN and IL-21.

CpG oligodeoxynucleotides (CpG) and IL-21 are two promising agents for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Recently, we reported that the combination of CpG and IL-21 (CpG/IL-21) can induce granzyme B (GrB)-dependent apoptosis in B-CLL cells. Here, we demonstrate that treatment of B-CLL cells with CpG and IL-21 results in the development of antigen-presenting cell (APC)-like cells with cytotoxic features. These properties eventually give rise to B-CLL cell apoptosis, independently of their cytogenetic phenotype, whereas normal B-cell survival is not negatively affected by CpG/IL-21. APC- and CTL-typical molecules found to be up-regulated in CpG/IL-21-stimulated B-CLL cells include GrB, perforin, T-bet, monokine-induced by IFN-γ and IFN-γ-inducible protein 10 (IP-10), as well as molecules important for cell adhesion, antigen cross-presentation and costimulation. Also induced are molecules involved in GrB induction, trafficking and processing, whereas the GrB inhibitor Serpin B9 [formerly proteinase inhibitor-9 (PI-9)] is down-modulated by CpG/IL-21. In conclusion, CpG/IL-21-stimulated B-CLL cells acquire features that are reminiscent of killer dendritic cells, and which result in enhanced immunogenicity, cytotoxicity and apoptosis. Our results provide novel insights into the aberrant immune state of B-CLL cells and may establish a basis for the development of an innovative cellular vaccination approach in B-CLL.

Granzyme B produced by human plasmacytoid dendritic cells suppresses T-cell expansion.

Human plasmacytoid dendritic cells (pDCs) are crucially involved in the modulation of adaptive T-cell responses in the course of neoplastic, viral, and autoimmune disorders. In several of these diseases elevated extracellular levels of the serine protease granzyme B (GrB) are observed. Here we demonstrate that human pDCs can be an abundant source of GrB and that such GrB(+) pDCs potently suppress T-cell proliferation in a GrB-dependent, perforin-independent manner, a process reminiscent of regulatory T cells. Moreover, we show that GrB expression is strictly regulated on a transcriptional level involving Janus kinase 1 (JAK1), signal transducer and activator of transcription 3 (STAT3), and STAT5 and that interleukin-3 (IL-3), a cytokine secreted by activated T cells, plays a central role for GrB induction. Moreover, we find that the immunosuppressive cytokine IL-10 enhances, while Toll-like receptor agonists and CD40 ligand strongly inhibit, GrB secretion by pDCs. GrB-secreting pDCs may play a regulatory role for immune evasion of tumors, antiviral immune responses, and autoimmune processes. Our results provide novel information about the complex network of pDC-T-cell interactions and may contribute to an improvement of prophylactic and therapeutic vaccinations.

B-chronic lymphocytic leukemia cells and other B cells can produce granzyme B and gain cytotoxic potential after interleukin-21-based activation.

B cells currently are not viewed as being capable of producing granzyme B or being cytotoxic. We found that B-chronic lymphocytic leukemia (B-CLL) cells treated with interleukin-21 (IL-21) produce low levels of granzyme B. The addition of either CpG oligodeoxynucleotide (ODN) or anti-B-cell-receptor antibody (anti-BCR) to IL-21 results in enhanced production of functional granzyme B by B-CLL cells. B-CLL cells treated with IL-21 and CpG ODN undergo apoptosis and are able to induce apoptosis of untreated bystander B-CLL cells. This effect can be inhibited by anti-granzyme B antibody. Benign human B cells, Epstein-Barr virus (EBV)-transformed lymphoblasts, and many standard lymphoma cell lines produce high levels of granzyme B in response to IL-21 and anti-BCR. Our results suggest that the ability to induce production of functional granzyme B by B cells could open new approaches to the therapy of B-CLL and other B-cell malignancies. Our findings also have significant implications for our understanding of the role of B cells for immune regulation and for a variety of immune phenomena, including cancer immunity, autoimmunity, and infectious immunity.

Serum alters the uptake and biologic activity of CpG oligodeoxynucleotides in B cell chronic lymphocytic leukemia.

Immunostimulatory CpG-containing oligodeoxynucleotides (CpG ODN) have a number of effects on B cells, including upregulation of immunogenic molecules, and, therefore, appear attractive as potential components of immunotherapy for B cell chronic lymphocytic leukemia (B-CLL). Previous in vitro studies investigating the effect of CpG ODN on B-CLL cells used serum-low conditions and did not account for the longer-half life of CpG ODN in vitro. The present study was designed to explore how the presence of serum and exposure time affect CpG ODN-mediated changes on B-CLL cells. The optimal concentration for CpG ODN-mediated effects in the presence of 100% serum or plasma was higher (10-20 microg/ml) than for serum-low conditions. Maximal CpG ODN-mediated effects required the presence of ODN for no longer than 3 hours. The inhibition of CpG ODN-mediated effects by serum correlated with lower uptake of ODN into B-CLL cells in the presence of serum. A threshold effect on biologic response was observed, with a given amount of ODN internalized, resulting in phenotypic changes. In conclusion, systemic short-term application of CpG ODN appears to be sufficient to induce phenotypic changes, but higher doses of CpG ODN than previously thought may be necessary because of inhibition of their uptake by serum.

Phosphorothyoate oligodeoxynucleotides block nonspecific binding of Cy5 conjugates to monocytes.

Cy5-based monoclonal antibody conjugates, including those containing PE-Cy5 and Cy5 alone, are used widely for multi-color flow cytometry in both research and clinical applications. One problem with these conjugates is they bind to monocytes and macrophages irrespective of antibody specificity. This effect, which is thought to be due to binding of the Cy5 itself to the human high affinity receptor for IgG (FcgammaRI, CD64), can complicate interpretion of flow cytometric results. In the present study we demonstrate that phosphorothioate oligodeoxynucleotides (PS-ODN) suppress this nonspecific binding in a manner that is independent of PS-ODN sequence. Binding of FITC-labeled PS-ODN to monocytes was blocked by CD64-specific monoclonal antibodies, suggesting CD64 is a PS-ODN-binding protein. We conclude PS-ODN can be used as an effective, simple and low-priced reagent to prevent nonspecific binding of Cy5-based immunoconjugates to monocytes. This effect appears to be mediated by PS-ODN binding to CD64 thereby blocking the binding of Cy5 to this receptor.

B-cell lymphomas differ in their responsiveness to CpG oligodeoxynucleotides.

Human B cells detect CpG motifs within microbial DNA via TLR9. Synthetic CpG oligodeoxynucleotides are currently being tested in clinical trials for the therapy of different types of B cell non-Hodgkin's lymphoma. However, there is only limited information on the CpG oligodeoxynucleotide sensitivity of primary malignant B cells of different non-Hodgkin's lymphoma entities. Here we found that most B-cell malignancies except plasmacytoma respond to CpG oligodeoxynucleotides by up-regulating expression of costimulatory and antigen-presenting molecules, by increasing expression of CD20, and by proliferation. In an in vitro analysis of 41 individual patient-derived primary tumor samples, B-cell chronic lymphocytic leukemia (B-CLL) and marginal zone lymphoma showed the strongest activation upon stimulation with CpG oligodeoxynucleotides. Small lymphocytic lymphoma, follicular lymphoma, mantle cell lymphoma, and large cell lymphoma showed an intermediate response. Consistent with CpG oligodeoxynucleotides sensitivity, TLR9 mRNA was present in B-CLL but absent in plasmacytoma. Although CpG oligodeoxynucleotides induced proliferation in all CpG oligodeoxynucleotide-sensitive types of B-cell malignancies, proliferation was weaker than in normal B cells and at least for B-CLL was followed by increased apoptosis. In conclusion, B-cell malignancies show significant differences in their responsiveness to CpG oligodeoxynucleotides. Focusing clinical studies on patients with highly CpG oligodeoxynucleotide-sensitive B-cell malignancies may improve the clinical outcome of such trials.

Immunostimulatory oligodeoxynucleotides induce apoptosis of B cell chronic lymphocytic leukemia cells.

Immunostimulatory oligodeoxynucleotides (IS ODN) can mediate a number of immunologic effects. We previously demonstrated that treatment of B cell chronic lymphocytic leukemia (B-CLL) cells with one class of IS ODN, CpG ODN, alters their phenotype and increases their immunogenicity. Here, we demonstrate that in contrast to the classic understanding of CpG ODN as inhibitors of B cell apoptosis, IS ODN including CpG ODN induce apoptosis in B-CLL cells. It is important that these changes are seen not only with CpG ODN but with ODN that lack the classical CpG motif. B-CLL cells from 20 subjects were treated in vitro with IS ODN for up to 7 days. IS ODN treatment resulted in increased numbers of apoptotic cells in 13 out of 20 B-CLL samples. IS ODN enhanced apoptosis in samples with 13q deletion as a single aberration and had a heterogeneous effect on apoptosis in samples with other aberrations including 17p deletion, 11q deletion, or trisomy 12. Induction of apoptosis did not correlate with expression of the CpG ODN receptor Toll-like receptor 9. Apoptosis was dependent on the activation of caspases and was accompanied by up-regulation of CD95/Fas and its ligand. We conclude that IS ODN including CpG ODN can induce apoptosis of most B-CLL samples. The ability of IS ODN to induce apoptosis differs based on cytogenetic status. Up-regulation of CD95/Fas may play a role in IS ODN-induced apoptosis of B-CLL.

CpG-A-induced monocyte IFN-gamma-inducible protein-10 production is regulated by plasmacytoid dendritic cell-derived IFN-alpha.

Unmethylated CpG motifs in bacterial DNA or synthetic oligodeoxynucleotides (ODN) are known for inducing a Th1 cytokine/chemokine environment, but the mechanisms regulating this have been unclear. Recent studies have defined two classes of CpG ODN, CpG-A ODN that induce plasmacytoid dendritic cells (pDC) to secrete very high levels of IFN-alpha, and CpG-B ODN that induce only low levels of IFN-alpha production, but strongly activate B cells. We now demonstrate that a CpG-A ODN directly activates pDC secretion of IFN-alpha and other soluble factors that secondarily induce purified monocytes to secrete high levels of the Th1-promoting chemokine IFN-gamma-inducible protein-10 (IP-10). Cell contact between the monocytes and pDC is not required for this interaction. IFN-alpha is necessary, but only partially sufficient, for this indirect CpG-induced monocyte IP-10 production. Although CpG ODN induce human PBMC to make only very slight amounts of IFN-gamma, we find that these low concentrations synergize with IFN-alpha for inducing monocyte production of IP-10. These studies provide a better understanding of the mechanisms through which CpG ODN create a Th1-like environment.