A functional recombinant human 4-1BB ligand for immune costimulatory therapy of cancer

Costimulatory factors hold great promise for development into novel anticancer biotherapeutics. An agonist to 4-1BB is ranked number 8 by National Cancer Institute on the list of 20 agents with high potential for use in treating cancer. We earlier reported on a recombinant murine 4-1BB ligand fusion protein that binds 4-1BB receptor on murine T cells and stimulates their proliferation in tumor-bearing mice. To facilitate clinical translation,we constructed a corresponding recombinant human 4-1BB ligand fusion protein (hIg-h4-1BBLs) and showed its ability to activate human T cells in vitro. Using Chinese hamster ovary cells transformed with a plasmid coexpressing hIg-h4-1BBLs and rat glutamine synthetase, we generated a high-producing clone by sequential selection with methionine sulfoximine. The hIg-h4-1BBLs was partially purified by protein A column chromatography and characterized biochemically and functionally, using human 4-1BB binding and human T-cell proliferation assays, in vitro.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western Blot confirmed that the hIg-h4-1BBLs is expressed predominantly as a functionally active multimeric protein with the ability to specifically bind to cells expressing human 4-1BB receptor and induce significant T-cell proliferation in vitro using both human and monkey peripheral blood mononuclear cells. The hIg-h4-1BBLs can be produced in large quantities from the high producer clone and developed as a novel immune costimulatory biotherapeutic to treat, alone and in combination with other modalities, various malignant diseases in patients through T-cell activation. Process development of this clinical agent has been discussed with the Food and Drug Administration in a pre-Investigational New Drug meeting and presented to the Office of Biotechnology Activities in a public hearing.     

SLE患者T淋巴细胞4—1BB的表达及其临床意义

[目的]探讨系统性红斑狼疮(SLE)T淋巴细胞上共刺激分子4-1BB的表达,及其与临床活动性指标的关系.[方法]应用流式细胞术检测40例SLE患者和20例正常对照者外周血T细胞活化前后4-1BB的表达.[结果]SLE患者CD4+T和CD8+T细胞表达的4-1BB明显高于正常对照组(P＜0.01),用抗CD3单抗体外刺激后CD4+T和CD8+T细胞表达的4-1BB均显著高于活化前(表达百分率为26.13±7.25和24.12±5.47,P＜0.01).SLE患者T淋巴细胞活化前后CD4+T细胞上4-1BB的表达均高于CD8+T细胞上4-1BB的表达(均P＜0.01).SLE患者淋巴细胞活化前后的4-IBB+CD4+T细胞百分数均与IgG和尿微量白蛋白呈正相关关系(r=0.623,P＜0.01,r=0.407,P＜0.01,r=0.605,P＜0.01,r=0.463,P＜0.01).[结论]SLE患者T淋巴细胞活化前后CD4+T和CD8+T表达的共刺激分子4-1BB均明显升高,4-1BB可能参与淋巴细胞的异常活化及肾脏损伤.     

4-1BB costimulation of effector T cells for adoptive immunotherapy of cancer: involvement of Bcl gene family members

We previously reported that in vitro costimulation of murine MCA 205 tumor-draining lymph node (TDLN) cells through a third signal, 4-1BB (CD137), in addition to CD3 and CD28 engagement significantly increases T-cell yield and amplifies antitumor responses in adoptive therapy. The increased T-cell yield seemed to be related to inhibition of activation-induced cell death. In this study, using real time-polymerase chain reaction and intracellular staining, we tested our hypothesis that antiapoptotic Bcl gene members are modulated in 4-1BB ligated TDLN cells. TDLN cells activated through 4-1BB in conjunction with CD3/CD28 demonstrated elevated Bcl-2 and Bcl-xL gene and protein expression compared with CD3/CD28 activation. Furthermore, Bcl-2 and/or Bcl-xL inhibition abrogated 4-1BB-conferred rescue of activation-induced cell death in TDLN cells, and as a result, 4-1BB-enhanced TDLN cell yield was abolished. Congenic mice were used as donors for TDLN cells labeled with CFSE to evaluate proliferation and persistence of activated cells after intravenous adoptive transfer. The effector function of transferred cells was assessed by determining the incidence of interferon-gamma-producing cells in response to tumor stimulation in serial blood samples drawn from treated mice using intracellular cytokine staining. CD28 and CD28/4-1BB costimulation significantly enhanced in vivo proliferation and survival of the infused cells compared with CD3 activation. 4-1BB coligation augmented the proliferation and effector function of the infused cells compared with both CD3 and CD3/CD28-activated cells. Characterizing the function of signaling molecules involved in T-cell activation pathways may allow optimization of conditions in the generation of effector T cells for cancer immunotherapy.     

T cell activation with systemic agonistic antibody versus local 4-1BB ligand gene delivery combined with interleukin-12 eradicate liver metastases of breast cancer

We have shown that interleukin-12 (IL-12) generated a strong, albeit transient, anti-tumor response, mostly mediated by natural killer (NK) cell. T cell participation, in addition to NK cells, was essential for persistence of the anti-tumor response. Ligation of 4-1BB, a co-stimulatory receptor expressed on activated T cells, is known to amplify T cell-mediated immunity. In this study, we compared the effect of a systemically delivered agonistic anti-4-1BB monoclonal antibody (anti-4-1BB mAb) with intra-tumoral adenoviral-mediated gene transfer of the 4-1BB ligand (ADV/4-1BBL) to liver metastases in a syngeneic animal model of breast cancer. Both treatments induced a dramatic regression of pre-established tumor. When combined with intra-tumoral delivery of the IL-12 gene, both anti-4-1BB mAb and ADV/4-1BBL were synergistic and led to survival rates of 87% and 78%, respectively. The anti-tumor immunity is mainly mediated by CD4+ T cells in IL-12 plus 4-1BB ligand-treated animals, and CD8+ T cells in IL-12 plus anti-4-1BB mAb-treated animals. However, only long-term survivors after treatment with IL-12 and 4-1BBL genes have showed significantly potent, systemic, and tumor-specific T cell-mediated immunity.     

Targeting 4-1BB Costimulation to Disseminated Tumor Lesions With Bi-specific Oligonucleotide Aptamers

The paucity of costimulation at the tumor site compromises the ability of tumor-specific T cells to eliminate the tumor. Here, we show that bi-specific oligonucleotide aptamer conjugates can deliver costimulatory ligands to tumor cells in situ and enhance antitumor immunity. In poorly immunogenic subcutaneously implanted tumor and lung metastasis models, systemic delivery of an agonistic 4-1BB aptamer ligand conjugated to a prostate specific membrane antigen (PSMA)-binding tumor-targeting aptamer led to inhibition of tumor growth, was more effective than, and synergized with, vaccination, and exhibited a superior therapeutic index compared to costimulation with 4-1BB antibodies. Tumor inhibition was dependent on homing to PSMA-expressing tumor cells and 4-1BB costimulation. Aptamer targeted costimulation is a broadly applicable and clinically feasible approach to enhance the costimulatory environment of disseminated tumor lesions. This study suggests that potentiating naturally occurring antitumor immunity via tumor-targeted costimulation could be an effective approach to elicit protective immunity to control tumor progression in cancer patients.     

Humanization of antagonistic anti-human 4-1BB monoclonal antibody using a phage-displayed combinatorial library

Anti-4-1BB (CD137) monoclonal antibody (mAb) has been reported to suppress immune responses and to have the potential for use as a therapeutic agent to block autoimmune diseases. Previously, the authors prepared an antagonistic anti-human 4-1BB (CD137) mAb, BBK2. Here the authors report the humanization of BBK2 using a phage display library. Four humanized single-chain Fv (scFv) fragments were selected from a combinatorial library expressing a phage-displayed humanized scFv. They were found to retain the epitope specificity of the original mAb and to have affinities higher than those of the original. Both the soluble and bound forms of the humanized scFv suppressed the proliferation of human peripheral blood mononuclear cells, similar to the original mAb. These results suggest that humanized anti-human 4-1BB scFvs can be used as a valuable reagent for clinical application.     

Elevated soluble 4-1BB is associated with serum markers of hepatitis B virus in patients with chronic hepatitis B

BACKGROUNDPrevious studies have suggested that the costimulatory molecule 4-1BB plays pivotal roles in regulating immunity during chronic viral infection. However, up to now, there are few studies about 4-1BB in chronic hepatitis B (CHB).AIMTo clarify this issue, we report our comprehensive study results on the expression levels of 4-1BB in patients with CHB.METHODSFrom September 2018 to June 2019, a total of 64 patients with CHB were recruited from the Department of Hepatology, The First Hospital of Jilin University. Peripheral blood samples were collected from 52 treatment-naïve and 12 entecavir-treated patients with CHB as well as 37 healthy donors (including 24 healthy adults and 13 healthy children). The levels of soluble 4-1BB (s4-1BB) in plasma were measured by ELISA. 4-1BB mRNA expression in peripheral blood mononuclear cells was detected by real-time quantitative PCR.RESULTSThe s4-1BB levels in the plasma of patients with CHB were significantly higher than those in healthy adults (94.390 ± 7.393 ng/mL vs 8.875 ± 0.914 ng/mL, P < 0.001). In addition, the s4-1BB level in plasma was significantly increased in patients with a higher viral load and a disease flare up. However, there were no significant differences between treatment-naïve and entecavir-treated patients. Interestingly, among treatment-naïve patients with CHB, the levels of s4-1BB in plasma had a significant positive correlation with hepatitis B surface antigen, hepatitis B virus DNA, hepatitis B e antigen, and triglyceride levels (r = 0.748, P < 0.001; r = 0.406, P = 0.004; r = 0.356, P = 0.019 and r = -0.469, P = 0.007, respectively). The 4-1BB mRNA expression was higher in the peripheral blood mononuclear cells of patients with CHB than in the peripheral blood mononuclear cells of healthy adults, but the difference was not statistically significant.CONCLUSIONThese results suggest that the levels of s4-1BB may be associated with pathogenesis of hepatitis B virus and therefore may be a promising biomarker for disease progression.     

4-1BB在类风湿关节炎T淋巴细胞的表达及其对TH1/TH2偏移的影响

【目的】探讨类风湿关节炎(rheumatoid arthritis,RA)T淋巴细胞上共刺激分子4-1BB的表达及其与血清TH1/TH2细胞因子分泌水平的关系。【方法】应用流式细胞术检测30例RA患者和20例正常对照者外周血T细胞活化前后4-1BB的表达,同时应用ELISA方法检测RA患者和正常对照者血清IFN-γ、IL-4水平。【结果】RA患者CD4 T和CD8 T细胞表达的4-1BB明显高于正常对照组(表达百分率分别为18.56±4.08,10.33±2.13,1.24±0.12,0.87±0.09,P<0.01),用抗CD3单抗体外刺激后CD4 T和CD8 T细胞表达的4-1BB均显著高于活化前(表达百分率为33.21±4.29和21.35±8.12,P<0.01)。RA患者CD4 T/CD8 T比值明显升高,而且与4-1BB CD4 T细胞数呈正相关关系(r=0.84,P<0.01)。RA患者血清IFN-γI、L-4均明显高于正常对照组(P<0.01,P<0.05),以IFN-γ升高最为显著。而且4-1BB CD4 T细胞数与血清IFN-γ水平呈明显的正相关关系(r=0.597,P<0.05)。【结论】类风湿关节炎患者T细胞表达的4-1BB在类风湿关节炎的发生发展中具有重要意义,4-1BB可能通过对CD4 T活化,促使TH1细胞因子分泌,参与关节炎症和免疫损伤。     

The systemic administration of Ig-4-1BB ligand in combination with IL-12 gene transfer eradicates hepatic colon carcinoma

We have previously shown that the local-membrane bound 4-1BB ligand and IL-12 gene transfer induced a significant antitumor response in a mouse colon carcinoma model. However, a high viral dose was required in order to achieve the best efficacy. In this study, we hypothesize that the systemic administration of soluble Ig-4-1BB ligand can give rise to better T-cell immune activation than local gene delivery. With potential clinical applications in mind, we further compare whether the natural 4-1BB ligand fused to mouse IgG2a (Ig-4-1BBL) would be as effective as the agonistic anti-4-1BB antibody. The dimeric form of Ig-4-1BBL was purified from HeLa cells transduced with a recombinant adenovirus (ADV/Ig-4-1BBL) expressing Ig-4-1BBL. Functional activity was confirmed by the ligand's ability to bind to activated splenic T cells or bone marrow (BM)-derived dendritic cells (DCs) that express 4-1BB receptor. The soluble Ig-4-1BBL efficiently costimulated CD3-activated T-cell proliferation in vitro. More importantly, it induced tumor-specific CTLs as effectively as the agonistic anti-4-1BB antibody. When combined with IL-12 gene transfer, systemic administration of the Ig-4-1BBL proved to be more potent than local gene delivery. In addition, the Ig-4-1BBL is as potent as the agonistic anti-4-1BB antibody for the treatment of hepatic MCA26 colon carcinoma, resulting in 50% complete tumor regression and long-term survival. In long-term surviving mice, both treatment modalities induced persistent tumor-specific CTL activity. In summary, these results suggest that the systemic delivery of Ig-4-1BBL can generate a better antitumor response than local gene delivery. Ig-4-1BBL had equivalent biological functions when compared to the agonistic anti-4-1BB antibody. Thus, soluble 4-1BBL dimmer can be developed as a promising agent for cancer therapy in humans.     

A humanized anti--4-1BB monoclonal antibody suppresses antigen-induced humoral immune response in nonhuman primates

The interaction of 4-1BB and its ligand plays an important role in the regulation of T-cell-mediated immune responses. In this study, the authors examined the effect of a humanized anti--4-1BB monoclonal antibody (H4B4) on ovalbumin-induced immune responses in baboons. Previously, a mouse monoclonal antibody, 4B4 against the human 4-1BB molecule, was generated and characterized. Based on this antibody, a humanized version of 4B4 monoclonal antibody was constructed and the resultant antibody, H4B4, showed full recovery of the binding activity of the original antibody 4B4: a 1.5-fold increase in affinity for 4-1BB. In addition, H4B4 mediated antibody-dependent cellular cytotoxicity of activated human peripheral blood T cells and CEM cells in a dose-dependent manner. Weekly administration of H4B4 at doses of 1 or 4 mg/kg could suppress immunoglobulin G production against ovalbumin. This was not a result of the overall immune suppression, because the numbers of B and T cells and the total immunoglobulin G production were not altered during treatment with H4B4. These findings suggest that treatment with H4B4 may be a valid therapeutic approach to control unwanted immune responses in persons with autoimmune diseases.     

Immunotherapy for prostate cancer - recent progress in clinical trials

Prostate cancer is the most common malignancy affecting men in the United States. Traditional therapy with radical prostatectomy or radiation therapy can be curative for localized disease, but metastatic prostate cancer is currently incurable. The only treatments known to prolong survival in patients with metastatic disease are androgen-deprivation therapy and chemotherapy, both of which have significant side effects. Immunotherapy approaches offer hope in providing new treatments to delay disease progression, ideally with fewer side effects. The results from nearly all early immunotherapy clinical trials for prostate cancer conducted to date have shown minimal toxicity, and many have suggested clinical benefit in terms of delaying disease progression. Several phase III clinical trials are currently under way in patients with metastatic, androgen-independent prostate cancer. The current article reviews recent trials evaluating immune-modulating agents, antigen-specific active immunotherapy, and combination therapies in clinical development for the treatment of prostate cancer.     

Immunotherapy of breast cancer

INTRODUCTION: Immunotherapy of breast cancer has been shown to prevent recurrence, improve survival and eliminate breast cancer in humans. AREAS COVERED: The reason for this review is to present the current information and the prospects for the future of immunotherapy of breast cancer in humans to include tumor antigens for vaccines and targets for monoclonal antibodies and adoptive T-cell therapy, and immune modulatory agents, such as adjuvants to stimulate the immune response and inhibitors of checkpoint blockade to prevent downmodulation of activated lymphocytes, to enhance these modalities. The research discussed and the literature search undertaken is of the clinical immunotherapy of breast cancer in humans, from 2000 to September, 2011. EXPERT OPINION: The key message of the paper is that one reason for the failure of the immune system to control macroscopic disease is that the immune escape mechanisms involving both tumor and the tumor stroma prevent the immune system from destroying the tumor. Changing the tumor microenvironment is necessary to eliminate macroscopic tumors. Prospects for improvement are proposals for combining current modalities of therapy with type 1 cellular immunity-inducing agents, all targeting multiple tumor antigens and in the context of minimal disease.     

Immunotherapy of ovarian cancer

Epithelial ovarian cancer remains the leading cause of death due to gynaecological malignancy. Although patients with advanced ovarian cancer have a good response to surgery and platinum-based chemotherapy, long-term survival remains poor. Immunotherapy techniques currently under investigation for treatment of recurrence include antitumour monoclonal and bispecific antibodies. Antibodies to ovarian cancer antigens have also been combined with radioisotopes, immunotoxins and chemotherapeutic drug conjugates to provide improved targeting of these agents. Anti-idiotypic antibodies and vaccines have also been developed to initiate humoral and cellular immune responses against ovarian cancer. Cytokines have been administered alone as well as in combination with other immunotherapy agents in patients with ovarian cancer. Finally, antigen-presenting cells are being modified by a variety of methods to stimulate an antitumour immune response. This review summarises the latest published clinical data and highlights promising areas of research in this exciting field.     

Immunotherapy of breast cancer

Introduction: Immunotherapy of breast cancer has been shown to prevent recurrence, improve survival and eliminate breast cancer in humans.

Areas covered: The reason for this review is to present the current information and the prospects for the future of immunotherapy of breast cancer in humans to include tumor antigens for vaccines and targets for monoclonal antibodies and adoptive T-cell therapy, and immune modulatory agents, such as adjuvants to stimulate the immune response and inhibitors of checkpoint blockade to prevent downmodulation of activated lymphocytes, to enhance these modalities. The research discussed and the literature search undertaken is of the clinical immunotherapy of breast cancer in humans, from 2000 to September, 2011.

Expert opinion: The key message of the paper is that one reason for the failure of the immune system to control macroscopic disease is that the immune escape mechanisms involving both tumor and the tumor stroma prevent the immune system from destroying the tumor. Changing the tumor microenvironment is necessary to eliminate macroscopic tumors. Prospects for improvement are proposals for combining current modalities of therapy with type 1 cellular immunity-inducing agents, all targeting multiple tumor antigens and in the context of minimal disease.     

Immunotherapy of ovarian cancer

Epithelial ovarian cancer remains the leading cause of death due to gynaecological malignancy. Although patients with advanced ovarian cancer have a good response to surgery and platinum-based chemotherapy, long-term survival remains poor. Immunotherapy techniques currently under investigation for treatment of recurrence include antitumour monoclonal and bispecific antibodies. Antibodies to ovarian cancer antigens have also been combined with radioisotopes, immunotoxins and chemotherapeutic drug conjugates to provide improved targeting of these agents. Anti-idiotypic antibodies and vaccines have also been developed to initiate humoral and cellular immune responses against ovarian cancer. Cytokines have been administered alone as well as in combination with other immunotherapy agents in patients with ovarian cancer. Finally, antigen-presenting cells are being modified by a variety of methods to stimulate an antitumour immune response. This review summarises the latest published clinical data and highlights promising areas of research in this exciting field.     

CD28-independent,TRAF2-dependent Costimulation of Resting T Cells by 4-1BB Ligand

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.