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IntroductionThere is no curative treatment available for patients with chemotherapy relapsed or refractory CD19+ B cells-derived acute lymphoblastic leukaemia (r/r B-ALL). Although CD19-targeting second-generation (2nd-G) chimeric antigen receptor (CAR)-modified T cells carrying CD28 or 4-1BB domains have demonstrated potency in patients with advanced B-ALL, these 2 signalling domains endow CAR-T cells with different and complementary functional properties. Preclinical results have shown that third-generation (3rd-G) CAR-T cells combining 4-1BB and CD28 signalling domains have superior activation and proliferation capacity compared with 2nd-G CAR-T cells carrying CD28 domain. The aim of the current study is therefore to investigate the safety and efficacy of 3rd-G CAR-T cells in adults with r/r B-ALL.Methods and analysisThis study is a phase I clinical trial for patients with r/r B-ALL to test the safety and preliminary efficacy of 3rd-G CAR-T cells. Before receiving lymphodepleting conditioning regimen, the peripheral blood mononuclear cells from eligible patients will be leukapheresed, and the T cells will be purified, activated, transduced and expanded ex vivo. On day 6 in the protocol, a single dose of 1 million CAR-T cells per kg will be administrated intravenously. The phenotypes of infused CAR-T cells, copy number of CAR transgene and plasma cytokines will be assayed for 2 years after CAR-T infusion using flow cytometry, real-time quantitative PCR and cytometric bead array, respectively. Moreover, several predictive plasma cytokines including interferon-γ, interleukin (IL)-6, IL-8, Soluble Interleukin (sIL)-2R-α, solubleglycoprotein (sgp)130, sIL-6R, Monocyte chemoattractant protein (MCP1), Macrophage inflammatory protein (MIP1)-α, MIP1-β and Granulocyte-macrophage colony-stimulating factor (GM-CSF), which are highly associated with severe cytokine release syndrome (CRS), will be used to forecast CRS to allow doing earlier intervention, and CRS will be managed based on a revised CRS grading system. In addition, patients with grade 3 or 4 neurotoxicities or persistent B-cell aplasia will be treated with dexamethasone (10 mg intravenously every 6 hours) or IgG, respectively. Descriptive and analytical analyses will be performed.Ethics and disseminationEthical approval for the study was granted on 10 July 2014 (YLJS-2014-7-10). Written informed consent will be taken from all participants. The results of the study will be reported, through peer-reviewed journals, conference presentations and an internal organisational report.Trial registration numberNCT02186860.

Background: The role of co-stimulatory molecules in renal diseases has been previously examined, however, little is known about the role of 4-1BB in the context of renal diseases resulting from nonimmune-mediated tubulointerstitial fibrosis. Folic acid induced Nephrotoxicity (FAN) in mice was used to explore the role of 4-1BB in this setting. Methods: CD1 mice were treated with folic acid and kidneys subsequently examined using histochemistry, in addition to defining T cell profiles and evaluating renal function. Increased CD3+ and CD4+ T lymphocytes present in blood and spleen at day 3 suggested immunopathological reactions during the early stages of FAN and decreased CD3+ and CD4+ T lymphocytes on day 14 were characteristic of an immunocompromised state observed during the late stages of FAN. Results: After 14 days of co-treatment with agonistic anti-4-1BB monoclonal antibodies, renal tubulointerstitial lesions were reduced. Renal function was improved, with Bun scores decreasing (p<0.01) and sCr levels decreasing (p<0.01). CD3+ and CD4+ T lymphocytes levels were increased during the early stages of disease in FA treated mice and reduced to the normal level in the 4-1BB-treated mice. CD3+ and CD4+ T lymphocytes levels were decreased in FA treated mice and returned to baseline in the 4-1BB-treated mice during later stages. Conclusions: Data presented in this report demonstrated that 4-1BB signals had immunoregulatory effects that attenuated early immune-mediated pathology and reversed the immunocompromised state observed during the later stages of disease.

Engagement of the TCR without appropriate costimulation will result in the inability of T-cells to respond to the alloantigen as described earlier. We made a further investigation into the effect of relieving graft-versus-host disease (GVHD) and its mechanism in mice by blocking CD137-CD137L pathway in vitro. Responder cells (spleen cells) from BALB/C donor mice (H-2d) were incubated with stimulator cells (spleen cells) from C57BL/6 recipient mice (H-2b), with or without anti-CD137L monoclonal antibodies (MoAbs). Donor bone marrow cells plus mixed lymphocyte culture (MLC) T-cells were transplanted into lethally irradiated C57BL/6 mice. C57BL/6 mice were divided into 3 groups: group A (allogeneic bone marrow transplantation control group), group B (cyclosporine + methotrexate group), and group C (donor T-cells were treated with anti-CD137L MoAbs). The percentage of CD3+CD4+ and CD3+CD8+ T-cells were detected by flow cytometry, and the levels of cytokines (IFN-gamma, interleukin [IL]-2, IL-10, IL-4) by reverse-transcriptase polymerase chain reaction. The incidence of GVHD in group C was 70%, while the incidence of GVHD was 100% in group A and group B. The survival rate of group C was higher than that of group A and B, and the median survival time was longer than that of group A and B (P < .01). Clinical symptoms and histological signs of GVHD in group C were the mildest among all 3 groups. The percentage of CD3+CD8+T-cells in group C was lower than that in group A and B (P < .01). The levels of IFN-gamma in group C were markedly lower than those in group A and B (P < .01), and the levels of IL-10 in group C were significantly higher than those in group A and B (P < .01). The results suggest that treatment of donor T-cells by anti-CD137L MoAbs in vitro may relieve GVHD, thereby improve the survival time and survival rate of recipient mice, which might be related to the increased TH1 cytokine (IFN-gamma) and decreased TH2 cytokine (IL-10) as well as the reduced CD3+CD8+T-cells.

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8 N/C EGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8 N/C EGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8 N/C EGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate FcγR interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy. Cancer therapy using systemically administrated 4-1BB-targeting antibodies is often associated with severe toxicity due to the nonspecific activation of autoreactive T cells. Here, the authors have developed a trimeric antibody targeting both 4-1BB and EGFR, which activates T cells effectively and shows negligible cytotoxicity.

Adoptive T-cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) can induce tumor regression in up to 50% or more of patients with unresectable metastatic melanoma. However, current methods to expand melanoma TIL, especially the \"rapid expansion protocol\" (REP) were not designed to enhance the generation of optimal effector-memory CD8(+) T cells for infusion. One approach to this problem is to manipulate specific co-stimulatory signaling pathways to enhance CD8(+) effector-memory T-cell expansion. In this study, we determined the effects of activating the TNF-R family member 4-1BB/CD137, specifically induced in activated CD8(+) T cells, on the yield, phenotype, and functional activity of expanded CD8(+) T cells during the REP. We found that CD8(+) TIL up-regulate 4-1BB expression early during the REP after initial TCR stimulation, but neither the PBMC feeder cells in the REP or the activated TIL expressed 4-1BB ligand. However, addition of an exogenous agonistic anti-4-1BB IgG4 (BMS 663513) to the REP significantly enhanced the frequency and total yield of CD8(+) T cells as well as their maintenance of CD28 and increased their anti-tumor CTL activity. Gene expression analysis found an increase in bcl-2 and survivin expression induced by 4-1BB that was associated with an enhanced survival capability of CD8(+) post-REP TIL when re-cultured in the absence or presence of cytokines. Our findings suggest that adding an agonistic anti-4-1BB antibody during the time of TIL REP initiation produces a CD8(+) T cell population capable of improved effector function and survival. This may greatly improve TIL persistence and anti-tumor activity in vivo after adoptive transfer into patients.

Costimulation of T cell responses with monoclonal antibody agonists (mAb-AG) targeting 4-1BB showed robust anti-tumor activity in preclinical models, but their clinical development was hampered by low efficacy (Utomilumab) or severe liver toxicity (Urelumab). Here we show that isotype and intrinsic agonistic strength co-determine the efficacy and toxicity of anti-4-1BB mAb-AG. While intrinsically strong agonistic anti-4-1BB can activate 4-1BB in the absence of FcγRs, weak agonistic antibodies rely on FcγRs to activate 4-1BB. All FcγRs can crosslink anti-41BB antibodies to strengthen co-stimulation, but activating FcγR-induced antibody-dependent cell-mediated cytotoxicity compromises anti-tumor immunity by deleting 4-1BB + cells. This suggests balancing agonistic activity with the strength of FcγR interaction as a strategy to engineer 4-1BB mAb-AG with optimal therapeutic performance. As a proof of this concept, we have developed LVGN6051, a humanized 4-1BB mAb-AG that shows high anti-tumor efficacy in the absence of liver toxicity in a mouse model of cancer immunotherapy. Agonistic 4-1BB antibodies developed for cancer immunotherapy have suffered from either hepatotoxicity or insufficient anti-cancer activity. Here the authors determine the contribution of FcγR binding and agonistic strength to these outcomes, and engineer a 4-1BB antibody with potent anti-tumor effect and no liver toxicity in mice.

Immunostimulatory agents such as agonistic anti-CD137 and interleukin (IL)−2 generate effective anti-tumor immunity but also elicit serious toxicities, hampering their clinical application. Here we show that combination therapy with anti-CD137 and an IL-2-Fc fusion achieves significant initial anti-tumor activity, but also lethal immunotoxicity deriving from stimulation of circulating leukocytes. To overcome this toxicity, we demonstrate that anchoring IL-2 and anti-CD137 on the surface of liposomes allows these immune agonists to rapidly accumulate in tumors while lowering systemic exposure. In multiple tumor models, immunoliposome delivery achieves anti-tumor activity equivalent to free IL-2/anti-CD137 but with the complete absence of systemic toxicity. Immunoliposomes stimulated tumor infiltration by cytotoxic lymphocytes, cytokine production, and granzyme expression, demonstrating equivalent immunostimulatory effects to the free drugs in the local tumor microenvironment. Thus, surface-anchored particle delivery may provide a general approach to exploit the potent stimulatory activity of immune agonists without debilitating systemic toxicities. Immunostimulatory agents used in cancer treatment often elicit serious toxicities, limiting their clinical application. Here, the authors show that the use of liposomes to intravenously deliver surface-anchored IL-2 and anti-CD137 proteins enables anti-cancer immunity and reduces the toxic side effects.

The use of chimeric antigen receptor (CAR)-engineered regulatory T cells (Tregs) has emerged as a promising strategy to promote immune tolerance. However, in conventional T cells (Tconvs), CAR expression is often associated with tonic signaling, which can induce CAR-T cell dysfunction. The extent and effects of CAR tonic signaling vary greatly according to the expression intensity and intrinsic properties of the CAR. Here, we show that the 4-1BB CSD-associated tonic signal yields a more dramatic effect in CAR-Tregs than in CAR-Tconvs with respect to activation and proliferation. Compared to CD28 CAR-Tregs, 4-1BB CAR-Tregs exhibit decreased lineage stability and reduced in vivo suppressive capacities. Transient exposure of 4-1BB CAR-Tregs to a Treg stabilizing cocktail, including an mTOR inhibitor and vitamin C, during ex vivo expansion sharply improves their in vivo function and expansion after adoptive transfer. This study demonstrates that the negative effects of 4-1BB tonic signaling in Tregs can be mitigated by transient mTOR inhibition. Chimeric antigen receptor engineering in T cells has been shown to be of great potential therapeutic benefit in a range of immune pathologies, although the functionality of such cell therapies can be limited due to tonic signalling and the induction of dysfunction. Here the authors show transient inhibition of mTOR can rescue their 41-BB-CAR-Tregs from tonic signalling-induced dysfunction.

Because human epidermal growth factor-like receptor (HER) 2 is expressed on the surface of human pancreatic carcinoma cells to varying degrees, trastuzumab, an anti-HER2 monoclonal antibody (mAb), is expected to exert antibody-dependent, natural killer (NK) cell-mediated cytotoxicity (ADCC) against the cells. However, some reports found that the effect of trastuzumab against human pancreatic carcinoma cells was limited because most express only limited HER2. We examined whether anti-CD137 stimulating mAb could enhance trastuzumab-mediated ADCC against Panc-1, a human pancreatic cancer cell line with low HER2 expression, in vitro. Supplementation of anti-CD137 mAb could improve trastuzumab-mediated ADCC against Panc-1 which was insufficient without this stimulating antibody. The ADCC differed in individual cells, and this was related to the expression of CD137 on the surface of NK cells after trastuzumab stimulation in association with the Fcγ-RIIIA polymorphism. NK cells with Fcγ-RIIIA-VV/VF showed high levels of ADCC against Panc-1, but those with Fcγ-RIIIA-FF did not show optimal ADCC. In addition, trastuzumab-mediated ADCC against the human pancreatic cancer cell line Capan-1 with high HER2 expression was generally high and not affected by the Fcγ-RIIIA polymorphism. These results demonstrated that in Fcγ-RIIIA-VV/VF-carrying healthy individuals, trastuzumab plus αCD137 mAb could induce effective ADCC against HER2-low-expressing pancreatic cancer cell lines, and that such an approach may result in similar findings in patients with pancreatic cancer.

Immunotherapies have heralded a new era in the cancer treatment. In addition to checkpoint inhibitors, agonistic antibodies against co-stimulatory immune receptors hold the potential to invoke efficient antitumor immunity. Targeting CD137 has gained momentum based on its ability to drive NK- and T-cell-based responses. CD137-engaging mAbs have already entered clinical trials for different types of tumors showing promising results. Despite the efforts to translate CD137-mediated immunotherapy into clinical practice, little remains known regarding the role of CD137 in human monocytes/macrophages. We found CD137 being expressed on monocytes of healthy controls and at even higher levels in patients with multiple myeloma or CLL. CD137 HI(GH) monocytes displayed a distinct phenotypic, transcriptomic, and metabolic profile. They possessed an increased phagocytic capacity enabling superior antibody-dependent phagocytosis (ADPC) of multiple myeloma and lymphoma cells that were treated with anti-CD38 or anti-CD20 mAbs. Triggering CD137 promoted both metabolic and tumoricidal activity in an extracellular signal-regulated kinase (ERK)-dependent fashion. In addition, we observed a phenotypic, transcriptomic, and functional skewing towards a M1-like phenotype. Overall, we introduce CD137 as a positive immune checkpoint on human monocytes/macrophages, which can have therapeutic implications especially in view of synergistic effects when combining CD137 agonists with tumor-targeting antibodies.