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Engineering dendritic cells (DCs) to treat cancer is a long sought-after goal for cell-based immunotherapies. In this review, we focus on the experience with CMN-001, formally AGS-003, a DC-based immunotherapy, employing autologous DC electroporated with autologous tumor RNA to treat subjects with metastatic renal cell carcinoma (mRCC). We will review the early clinical development of CMN-001 up to and including deployment in a multicenter phase 3 study and provide a rationale to continue the development of CMN-001 in an ongoing randomized phase 2 study. The synergy between CMN-001 and everolimus observed in the phase 3 study provides an opportunity to design a phase 2b study building on the mechanism of action of CMN-001 and underlying immune and clinical outcomes revealed in the earlier studies. The design of the phase 2b study combines CMN-001 with first-line checkpoint inhibition therapy and second line lenvatinib/everolimus in poor-risk mRCC subjects.

Purpose: Rocapuldencel-T is an autologous immunotherapy prepared from mature monocyte-derived dendritic cells (DC), coelectroporated with amplified tumor RNA plus CD40L RNA. This pivotal phase III trial was initiated to investigate the safety and efficacy of a combination therapy dosing regimen of Rocapuldencel-T plus sunitinib in patients with metastatic renal cell carcinoma (mRCC)., Patients and Methods: Patients received either Rocapuldencel-T plus standard of care (SOC) or SOC treatment alone. The primary objective compared overall survival (OS) between groups. Secondary objectives included safety assessments, progression-free survival (PFS), and tumor responses based on RECIST 1.1 criteria. Exploratory analyses included immunologic assessments and correlates with OS., Results: Between 2013 and 2016, 462 patients were randomized 2:1, 307 to the combination group and 155 to the SOC group. Median OS in the combination group was 27.7 months [95% confidence interval (CI) 23.0-35.9] and 32.4 months (95% CI, 22.5-) in the SOC group HR of 1.10 (95% CI, 0.83-1.40). PFS was 6.0 months and 7.83 months for the combination and SOC groups, respectively [HR = 1.15 (95% CI, 0.92-1.44)]. The ORR was 42.7% (95% CI, 37.1-48.4) for the combination group and 39.4% (95% CI, 31.6-47.5) for the SOC group. Median follow up was 29 months (0.4-47.7 months). On the basis of the lack of clinical efficacy, the ADAPT trial was terminated on February 17, 2017. Immune responses were detected in 70% of patients treated with Rocapuldencel-T, and the magnitude of the immune response positively correlated with OS. In addition, we report the survival-predictive value of measuring IL-12 produced by the DC vaccine and the observation that high baseline numbers of T regulatory cells are associated with improved outcomes in DC-treated patients, but are associated with poor outcomes in patients receiving SOC treatment. No serious adverse events attributed to the study medication have been reported to date., Conclusions: Rocapuldencel-T did not improve OS in patients treated with combination therapy, although the induced immune response correlated with OS. Moreover, we identified two potential survival-predictive biomarkers for patients receiving DC based immunotherapy, IL-12 produced by the DC vaccine and higher numbers of T regulatory cells present in the peripheral blood of patients with advanced RCC., (©2020 American Association for Cancer Research.)

Background: The genomic heterogeneity of HIV-1 impedes the ability of consensus sequences in vaccines to elicit effective antiviral immune responses. AGS-004 amplifies translation-competent RNA molecules encoding for Gag, Rev, Vpr, and Nef from the patient's autologous virus and loads them into dendritic cells., Methods: This phase IIB, multicenter, 2:1 randomized, double-blind, placebo-controlled study enrolled 54 HIV-1-infected patients on antiretroviral therapy with viral loads (VLs) <50 copies per milliliter, current CD4 T-cell counts >450 cells per cubic millimeter, and nadir counts >200 cells per cubic millimeter, to receive intradermal injections of study product into the axillary lymph node region every 4 weeks. At week 16, a 12-week analytical treatment interruption (ATI) was undertaken., Results: There was no difference in the end-of-ATI VL (average of values from weeks 11 and 12) between the 2 arms of the study [4.39 (4.17, 4.69) vs. 4.47 (3.76, 4.64) log10 HIV-1 RNA; P = 0.73]. Between arms, no change between pre-antiretroviral therapy VL and the end-of-ATI VL [-0.06 (0.24, -0.32) vs. -0.17 (0.17, -0.32) log10 HIV-1 RNA; P = 0.43] was observed. When interferon-γ, interleukin-2, tumor necrosis factor α, CD107a, and granzyme b expressions were measured by multicolor flow cytometry, a greater percentage of AGS-004 than of placebo recipients had multifunctional cytotoxic T-lymphocyte responses induced in the CD28+/CD45RA-CD8 effector/memory T-cell population to dendritic cells electroporated with autologous antigens. Adverse events consisted of transient, mild (grade 1) local injection site reactions., Conclusions: Despite the induction of HIV-specific effector/memory CD8 T-cell responses, no antiviral effect was seen after the administration of AGS-004 when compared with placebo.

Background: Recombinant human soluble CD83 had previously exhibited significant immunosuppressive properties that involved interference with dendritic cell maturation in both mouse and humans, inhibition of autoimmunity in mice, and induction of antigen-specific mouse cardiac allograft tolerance when used in combination with other immunosuppressive drugs. Our current research focus turned to examining the effects of peritransplant soluble CD83 (sCD83) administration on prevention of chronic renal allograft rejection., Methods: Fisher344-to-Lewis orthotopic rat renal transplants were performed with sequential recipient killing on postoperative days (PODs) 2, 14, and 140 to examine both the acute and chronic effects of peritransplant sCD83 treatment in rat recipients., Results: Recipients treated with sCD83 exhibited a marked decrease in IgM and IgG deposition in the graft and antidonor antibody levels in the circulation, as early as POD14 and persisting until POD140. sCD83 treatment also reduced the infiltration of T cells and monocytes into the graft tissue and inhibited intragraft expression of MyD88 and inflammatory cytokine levels during the observation period. sCD83-treated grafts demonstrated normal histology beyond POD140, including dramatic reductions in tubular atrophy and interstitial fibrosis compared with untreated recipients., Conclusion: We have demonstrated that peritransplant treatment with recombinant sCD83 attenuates both innate and adaptive immune responses and leads to prevention of chronic rejection in a rat renal transplant model. Because sCD83 is of human origin, the therapeutic approach used in our rodent transplant model holds significant promise for clinical transplantation.

Background: Tolerogenic dendritic cells (Tol-DCs) play a critical role in inducing and maintaining tolerance. Recognizing that both T-cell inactivation and activation are contingent on signals provided by DCs and that graft-specific activated T cells are major mediators of transplant rejection, we aimed to create an environment favoring Tol-DCs with a novel reagent, human soluble CD83 (hsCD83)., Methods: Life-supporting orthotopic kidney transplantation was performed in a C57BL/6-to-BALB/c mouse model. The study group was treated with hsCD83 (100 μg/mouse/day, postoperative days -1 to +7, intravenously) and compared with untreated controls., Results: Treatment with hsCD83 achieved kidney allograft tolerance (>100 days), with negligible antidonor antibody detected. In contrast, kidney grafts in untreated recipients demonstrated severe rejection after 35 days, characterized by cellular infiltration, interstitial hemorrhage and edema, and glomerular and tubular necrosis, as well as high antidonor antibody titers. In addition, splenic DCs of tolerant recipients exhibited significantly decreased levels of surface major histocompatibility complex class II, CD40, CD80, and intracellular interleukin-12, as well as reduced allogeneic stimulatory capacity. Adoptive transfer of CD11c+ DCs from tolerant hsCD83-treated animals induced kidney allograft tolerance in syngeneic recipients. Blocking indoleamine 2,3-dioxygenase with 1-methyl-tryptophan (15 mg/mouse/day; gavage) prevented the immunosuppressive effect of hsCD83, abrogating hsCD83-induced Tol-DCs and graft tolerance, and leading to acute kidney graft rejection in 22 days., Conclusion: hsCD83 alone was capable of inducing kidney allograft tolerance through a mechanism involving Tol-DC generation and, at least in part, indoleamine 2,3-dioxygenase activity. Because sCD83 is of human origin, the therapeutic approach used in our mouse transplant model holds significant promise for clinical transplantation.

Purpose: The combination of vaccines and chemotherapy holds promise for cancer therapy, but the effect of cytotoxic chemotherapy on vaccine-induced antitumor immunity is unknown. This study was conducted to assess the effects of systemic chemotherapy on ALVAC-CEA/B7.1-induced T-cell immunity in patients with metastatic colorectal cancer., Experimental Design: Patients with metastatic colorectal cancer were treated with fluorouracil, leucovorin, and irinotecan and were also given ALVAC-CEA/B7.1 vaccine with or without tetanus toxoid adjuvant. Eligible patients were randomized to ALVAC followed by chemotherapy and booster vaccination (group 1), ALVAC and tetanus toxoid followed by chemotherapy (group 2), or chemotherapy alone followed by ALVAC in patients without disease progression (group 3). Humoral immune responses were measured by standard ELISA assay, and carcinoembryonic antigen (CEA)-specific T-cell responses were measured by IFN-gamma enzyme-linked immunospot assay., Results: One hundred eighteen patients were randomized to receive either ALVAC before and concomitantly with chemotherapy (n = 39), ALVAC with tetanus adjuvant before and concomitantly with chemotherapy (n = 40), or chemotherapy followed by ALVAC (n = 39). Serious adverse events were largely gastrointestinal (n = 30) and hematologic (n = 24). Overall, 42 patients (40.4%) showed objective clinical responses. All patients developed antibody responses against ALVAC, but increased anti-CEA antibody titers were detected in only three patients. Increases in CEA-specific T cells were detected in 50%, 37%, and 30% of patients in groups 1, 2, and 3, respectively. There were no differences in clinical or immune responses between the treatment groups., Conclusion: The combination of ALVAC-CEA/B7.1 vaccine and systemic chemotherapy has an acceptable safety profile in patients with metastatic colorectal cancer. Systemic chemotherapy did not affect the generation of CEA-specific T-cell responses following vaccination.

Since the intrinsically poor immunogenicity of chronic lymphocytic leukemia (CLL) cells might be a key factor in allowing them to avoid immune control mechanisms, the development of methods to enhance CLL cell immunogenicity might lead to improved disease control. The ability of CLL cells to stimulate T cells was increased significantly by the protein kinase C (PKC) agonist phorbol myristic acetate (PMA). However, under serum-free conditions, PMA-activated CLL cells died within 48 hours. Antioxidants, such as 2-mercaptoethanol (2-ME), or fetal calf serum could prevent the death of these cells but caused them to enter distinct states of differentiation. In the presence of 2-ME, PMA-activated CLL cells extended dendritic-like protrusions and exhibited increased T-cell stimulatory capacity. In the presence of serum, PMA-activated CLL cells developed fewer dendrites, made less IL-10 and more IL-12 p40 mRNA transcripts, and showed an increased capacity to induce IFN-gamma production by T cells. The effects of serum on the promotion of type 1 immune responses by phorbol ester-activated CLL cells were dominant and correlated with activation of the NF-kappaB signaling pathway. Other PKC agonists, such as Bryostatin-1 and a synthetic Bryostatin analog (Picolog), had similar effects on CLL cells. The observation that CLL cells can acquire features of dendritic cells that promote type 1 immunity may find clinical application in immunotherapeutic strategies for this disease.

A costimulatory member of the TNFR family, 4-1BB, is expressed on activated T cells. Although some reports have suggested that 4-1BB is primarily involved in CD8 T cell activation, in this report we demonstrate that both CD4 and CD8 T cells respond to 4-1BB ligand (4-1BBL) with similar efficacy. CD4 and CD8 TCR transgenic T cells up-regulate 4-1BB, OX40, and CD27 and respond to 4-1BBL-mediated costimulation during a primary response to peptide Ag. 4-1BBL enhanced proliferation, cytokine production, and CTL effector function of TCR transgenic T cells. To compare CD4 vs CD8 responses to 4-1BBL under similar conditions of antigenic stimulation, we performed MLRs with purified CD4 or CD8 responders from CD28(+/+) and CD28(-/-) mice. We found that CD8 T cells produced IL-2 and IFN-gamma in a 4-1BBL-dependent manner, whereas under the same conditions the CD4 T cells produced IL-2 and IL-4. 4-1BBL promoted survival of CD4 and CD8 T cells, particularly at late stages of the MLR. CD4 and CD8 T cells both responded to anti-CD3 plus s4-1BBL with a similar cytokine profile as observed in the MLR. CD4 and CD8 T cells exhibited enhanced proliferation and earlier cell division when stimulated with anti-CD3 plus anti-CD28 compared with anti-CD3 plus 4-1BBL, and both subsets responded comparably to anti-CD3 plus 4-1BBL. These data support the idea that CD28 plays a primary role in initial T cell expansion, whereas 4-1BB/4-1BBL sustains both CD4 and CD8 T cell responses, as well as enhances cell division and T cell effector function.

A20 is an aggressive BALB/c B cell lymphoma that, despite its expression of B7-2, rapidly forms tumors in syngeneic mice. We have generated A20 transfectants expressing elevated levels of B7-2 (A20/B7-2high) or 4-1BBL (A20/4-1BBL(low,mod,high)) and found that mice which were able to reject the A20/B7-2 or A20/4-1BBL transfectants were also resistant to subsequent systemic challenge with the parental cell line. To assess whether the effectiveness of 4-1BBL in enhancing anti-tumor immunogenicity was dependent on additional signals from B7-CD28 interaction, we injected the A20 variants into BALB/c CD28(-/-) mice. We found that CD28(-/-) mice were able to reject the A20/4-1BBL variants while A20/B7-2 cells formed tumors. However, when the A20/4-1BBL resistant CD28(-/-) mice were systemically challenged with the A20 parental line, tumors formed rapidly. Upon restimulation in vitro, splenocytes from A20/4-1BBL immunized CD28(+/+) mice were able to kill parental tumors whereas splenocytes from CD28(-/-) mice showed a reduction in CTL activity against A20 or A20/4-1BBL targets. Examination of cytokine production by the immunized animals indicated that the CD28(-/-) splenocytes secreted substantially less IL-2 as well as reduced levels of IFN-gamma compared with their CD28(+/+) counterparts. Thus, 4-1BBL expressing tumors are capable of priming CTL responses against 4-1BBL transfected as well as parental tumors in the absence of CD28. However, in the absence of CD28 signaling, the production of cytokines and particularly IL-2 was lower, resulting in a weaker CTL recall response and reduced ability to survive challenge with parental tumor., (Copyright 2001 Academic Press.)

CD28 is the primary co-stimulatory receptor for inducing high-level IL-2 production and survival of naïve CD4(+) T cells. While no other cell surface receptor can be considered fully redundant with CD28, recent developments suggest that additional co-stimulatory pathways have preferential effects at different stages of T cell activation, on different subsets of T cells or contribute to the development of different effector functions.

A20 is a B cell lymphoma that constitutively expresses the costimulatory molecule B7-2 yet grows readily as a tumor in syngeneic BALB/c mice. We have compared the tumorigenicity of A20 variants expressing either B7-1 (A20/B7-1) or B7-2 (A20/B7-2) with an A20 variant expressing B7-1 and B7-2 with 4-1BBL (A20/4-1BBL), a costimulatory member of the TNF family. Mice injected with tumors expressing the vector backbone (A20/CMV) or B7-1 developed tumors within 25 days of s.c. injection. In contrast, mice injected with A20/4-1BBL were tumor free for the 150-day follow-up period, while 25% of mice injected with A20/B7-2 developed tumors. Tumorigenicity experiments using nude mice indicated the requirement for T cells for variant rejection. Almost all mice that resisted the initial tumor challenge were resistant to further challenge with the parental tumor. Splenocytes from these mice showed high CTL lytic activity against the parental tumor, A20, as well as the syngeneic BALB/c lymphoma K46J, but showed background levels of lytic activity against the congenic SCID thymoma line ST-D2 or the allogeneic EL4 thymoma. In vitro blocking experiments with anti-B7-1 plus anti-B7-2 and/or soluble 4-1BB receptor showed B7-1, B7-2, and 4-1BBL all contributed to the CTL activity. Thus, the data show that neither B7-1 or B7-2 alone can confer full immunogenicity to the A20 lymphoma but that the addition of 4-1BBL results in a tumor that is highly immunogenic and can confer long-lasting protection against challenge with parental tumor in vivo.

4-1BB ligand (4-1BBL) is a member of the tumor necrosis factor (TNF) family expressed on activated antigen-presenting cells. Its receptor, 4-1BB, is a member of the TNF receptor family expressed on activated CD4 and CD8 T cells. We have produced a soluble form of 4-1BBL using the baculovirus expression system. When coimmobilized on plastic with anti-CD3, soluble 4-1BBL induces interleukin (IL)-2 production by resting CD28+ or CD28- T cells, indicating that 4-1BBL can function independently of other cell surface molecules, including CD28, in costimulation of resting T cell activation. At low concentrations of anti-CD3, 4-1BBL is inferior to anti-CD28 in T cell activation. However, when 4-1BB ligand is provided together with strong TCR signals, then 4-1BBL and anti-CD28 are equally potent in stimulation of IL-2 production by resting T cells. We find that TNF receptor-associated factor (TRAF)1 or TRAF2 associate with a glutathione S-transferase-4-1BB cytoplasmic domain fusion protein in vitro. In T cells, we find that association of TRAF1 and TRAF2 with 4-1BB requires 4-1BB cross-linking. In support of a functional role for TRAF2 in 4-1BB signaling, we find that resting T cells isolated from TRAF2-deficient mice or from mice expressing a dominant negative form of TRAF2 fail to augment IL-2 production in response to soluble 4-1BBL. Thus 4-1BB, via the TRAF2 molecule, can provide CD28-independent costimulatory signals to resting T cells.

Insulin has been shown to enhance the proliferation and differentiation of T cells stimulated by both polyclonal stimulants and specific antigen. This study describes an experimental system designed to understand the mechanism(s) by which occupancy of the insulin receptor mediates the enhancement seen in T cell expansion. These experiments demonstrate that the ability of insulin to influence T cell expansion resides in an insulin-mediated maintenance of the interleukin-2 (IL-2) responsiveness of the activated cells. This was analyzed by following the decay pattern of T cell IL-2 responsiveness by placing the activated T cells into serum-free cultures in the presence or absence of insulin. This maintenance of responsiveness was not mediated by insulin regulating the expression of the IL-2 receptor alpha chain. We feel that this experimental approach will prove useful for dissecting the biochemical and molecular changes mediated by insulin which interface with the ability of activated T cells to effectively respond to IL-2.