Production Of IFNγ In PBMCs Research Articles

Abstract 5520: AK129, an anti-PD1/LAG-3 bi-specific antibody for cancer therapy

Abstract Background: AK129 is a bi-specific antibody acts by co-blocking two immune checkpoint molecules co-expressed on T cells, PD-1 and LAG-3. Signaling through PD-1/PD-L1 exerts major effects on cytokines production by T cells, inhibiting production of IFN-γ and IL-2. LAG-3 is a cell surface molecule expressed on effector T cells and Tregs, which plays a vital role in regulating T cell function to promote tumoral immune escape. LAG-3 and PD-1 mediate different signaling pathways, but they may act synergistically to cause effector T cell inactivation and exhaustion. AK129 is being investigated as a cancer immunotherapeutic agent. Methods: The antigen binding activity of AK129 to PD-1 and LAG-3 were measured by ELISA and FACS. In reporter gene assay, the bioactivities of AK129 to block the interaction of PD-1 with PD-L1 and to neutralize LAG-3-mediated activity were determined. The bioactivity of AK129 to enhance IL-2 and IFN-γ production in PBMCs via blockade of PD-1 and PD-L1/2 interaction were determined by using cellular assays. In in-vivo pharmacology studies, anti-tumor activity of AK129 was investigated in BALB/c-hPD1/hLAG3 mice bearing CT26.WT tumor model. Results: AK129 showed good antigen binding to PD-1 and LAG-3. AK129 potently inhibited the binding of LAG-3 to its ligand MHC-II, and the binding of PD-1 to its ligand PD-L1, thus blocking downstream immune suppression. At the individual target level, AK129 demonstrated a similar PD-1 blocking activity compared to penpulimab, and similar LAG-3 blocking activity compared to relatlimab. Importantly, at the whole cell level, the enhancement effect of AK129 is stronger than the combination of penpulimab and relatlimab, as evidenced by significantly higher level of IL-2 and IFN-γ secretion from PBMCs. AK129 showed favorable anti-tumor activity in BALB/c-hPD1/hLAG3 mice bearing CT26.WT tumor model, with tumor growth inhibition values reaching 99.27% and 89.48% in high dose (20 mg/kg) and low dose (4 mg/kg) group of AK129, respectively. Conclusion: AK129 is a dual checkpoint blocking bispecific antibody with design features similar to the PD-1/CTLA-4 bispecific antibody AK104, which demonstrated efficacy similar to PD-1 and CTLA-4 antibody combo and much improved safety profile. AK129 has demonstrated robust anti-tumor activity in both in vivo and in vitro pre-clinical studies, and with enhanced T cell activity compared to PD-1 and LAG-3 antibody combo. The efficacy and safety of AK129 await testing in the clinic. Citation Format: Zhaoliang Huang, Xinghua Pang, Tingting Zhong, Chunshan Jin, Na Chen, Dennis Xia, Peng Zhang, Max Wang, Michelle Xia, Baiyong Li. AK129, an anti-PD1/LAG-3 bi-specific antibody for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5520.

Abstract 6191: A novel anti-IL33 antibody that neutralized Th1-type and Th2-type pro-inflammation responses induced by IL-33/ST2 axis pathway

Abstract IL-33, the ligand for ST2, is a member of IL-1 family. IL-33 was described to activate many immune cell types involved in type-2 immunity and allergic inflammation. Here we describe a novel anti-IL33 humanized monoclonal antibody, SSGJ-621, which bound IL-33 with high affinity and blocked the interaction between IL-33 and ST2. Our study has shown that SSGJ-621 was capable of effectively neutralizing IL-33-induced IL-6 secretion from HUVECs and IL-33-dependent IFNγ production in PBMCs and NK cells, suggesting a role that IL-33 might be involved in Th1-type of immunity. In addition, SSGJ-621 also exhibited potent inhibitory effects on IL-33 induced IL-5 and IL-13 production in KU812 cells. In vivo study demonstrated that SSGJ-621 can inhibit the activation of splenocytes in spleen and the production of eosinophils and IL-5 in peripheral blood. Thereby, our results highlight the dual functions of IL-33 in promoting Th1 and Th2 immune responses and SSGJ-621 has potentials to treat not only chronic inflammatory diseases induced by IL-33, but also cancers by favoring Th1 antitumor immunity in the tumor microenvironment. Citation Format: Xuesai Zhang, Qingrou Li, Cui Liu, Le Zhao, Lujie Wang, Lan Deng, Wei Xu, Hongli song, Zhenping Zhu, Haomin Huang. A novel anti-IL33 antibody that neutralized Th1-type and Th2-type pro-inflammation responses induced by IL-33/ST2 axis pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6191.

Melittin-MIL-2 fusion protein as a candidate for cancer immunotherapy.

BackgroundCytokine fusion protein that modulates the immune response holds great potential for cancer immunotherapy. IL-2 is an effective treatment against advanced cancers. However, the therapeutic efficacy of IL-2 is limited by severe systemic toxicity. Several mutants recombinant IL-2 can increase antitumor activity and minimize systemic toxicity. Melittin is an attractive anticancer candidate because of its wide-spectrum lytic properties. We previously generated a bifunctional fusion protein melittin-MIL-2, composed of melittin and a mutant IL-2. The melittin-MIL-2 inhibited the growth of human ovarian cancer SKOV3 cells in vitro and in vivo tumor growth. However, whether this antitumor effect could also be used in cancer immunotherapy was unknown. To assess its cancer immunotherapy potential, we further investigated its more effective antitumor immune response and antitumor effect against cancers of different tissue origins in vitro and in vivo.MethodsThe specific IL-2 activity of the melittin-MIL-2 fusion protein was tested on the cytokine growth dependent cell line CTLL-2. The cytolytic activity was detected by standard 4-h 51Cr-release assays. PBMC stimulation in response to the melittin-MIL-2 was determined by IFN-γ release assay. We observed the cancer cell proliferation of different tissue origins by MTT assay. The ability of melittin-MIL-2 to inhibit tumor growth in vivo was evaluated by using human liver (SMMC-7721 cancer cells), lung (A549 cancer cells) and ovarian (SKOV3 cancer cells) cancer xenograft models. To assess the immunity within the tumor microenvironment, the level of some cytokines including IFN-γ, TNF-α, IL-12 and IL-4 was analyzed by ELISA. We injected the MDA-MB-231 cells and the melittin-MIL-2 into mice, and the anti-metastatic effect was examined by counting nodules in the lung.ResultsThe melittin-MIL-2 was more effective in inducing T cell and NK-cell cytotoxicity. The fusion protein significantly increased IFN-γ production in PBMCs. In vitro, the melittin-MIL-2 mediated immune cells killing or directly killed the cancer cell lines of different tissue origins. In vivo, the fusion protein exhibited stronger inhibition on the growth of transplanted human tumors compared to rIL-2. The melittin-MIL-2 treatment promoted the IFN-γ secretion in tumor tissues and decreased the immunosuppressive cells in vivo. Furthermore, the fusion protein reduced lung metastasis of breast cancer.ConclusionsThis study provides the evidence that the melittin-MIL-2 can produce stronger immune stimulation and antitumor effects, and the fusion protein is a potent candidate for cancer immunotherapy.

Measuring naturally acquired ex vivo IFN-γ responses to Plasmodium falciparum cell-traversal protein for ookinetes and sporozoites (CelTOS) in Ghanaian adults.

BackgroundA malaria vaccine that targets the sporozoite/liver stage parasites could potentially prevent blood stage infection and the associated clinical symptoms. Identification of sporozoite/liver stage antigens is, therefore, crucial for the development of effective vaccines. Cell-traversal protein for ookinetes and sporozoites (CelTOS) is a highly conserved antigen involved in sporozoite motility and hepatocyte invasion and has been shown to induce significant IFN-γ production in PBMCs from radiation-attenuated sporozoite-immunized malaria-naïve individuals. The aim of this study was to ascertain whether such CelTOS-specific recall responses are also induced in individuals with natural exposure to Plasmodium falciparum.MethodsEx vivo IFN-γ responses to 15mer overlapping peptide pools covering the entire sequence of CelTOS and five other candidate antigens, CSP, AMA1, MSP1, TRAP and LSA1, were characterized using PBMCs from 35 malaria exposed adults. Responses to four CelTOS peptide pools (CelTp1, CelTp2, CelTp3 and CelTp4), a pool containing peptides from the entire CelTOS antigen (CelTTp), and pools comprised of overlapping peptides from each of the other five malaria antigens were assessed by ex vivo ELISpot assay. A positive IFN-γ response for stimulants was defined by two criteria; a stimulation index of two or greater relative to the unstimulated control, and a difference of 10 or greater in spot forming cells between stimulant and the unstimulated control.ResultsOf the 35 volunteers tested, five had positive IFN-γ recall responses against the four different CelTOS pools while four volunteers made responses against the CelTTp pool; six volunteers were, therefore, positive with CelTOS. By contrast, six volunteers responded to AMA1, seven to LSA1, 15 to MSP1 and two volunteers responded against CSP and TRAP.ConclusionsThese results suggest natural malaria transmission induces CelTOS-specific ex vivo IFN-γ in Ghanaian adults and that the frequency of these responses was similar to those of other previously characterized malaria antigens. These findings support the further evaluation of CelTOS as a pre-erythrocytic candidate antigen for inclusion in a potential multi-antigen vaccine.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-014-0539-5) contains supplementary material, which is available to authorized users.

Abstract 4739: Effects of whole-body heat treatment on antigen specific T cells responses.

Abstract Studies in mice showed that heat treatment in fever-range (approx. 38.5-40.5 deg C) stimulates a variety of T cells functions in vitro, ex vivo and in vivo. We previously introduced the heckel HT3000; an infrared A-irradiation device specifically designed for whole-body hyperthermia, and we are now studying how to combine it with cancer immuno-cell therapy such as adoptive transfer of activated T cells and autologous monocyte-derived dendritic cells (DCs)-based vaccination. In this study, using healthy volunteers, we examined the effects of fever-range whole body heat treatment on the function of peripheral T cells, and in particular their cytokines productions in response to various stimuli. Volunteers were exposed to infrared A until the rectal temperature reached at 38.5 deg C. They were then wrapped in synthetic leather tent for 60 min in order to maintain the core body temperature in fever-range. Peripheral blood was obtained four times; prior to the treatment, immediately after the treatment, 24 h after the treatment, and 48 h after the treatment. PBMC were then prepared and co-cultured with antigen-loading autologous DCs for 24 h in order to induce antigen specific cytokine productions in T cells. We first examined IFNγ production in T cells in response to MHC class I restricted epitope peptides of CMV, EBV and Flu. When PBMCs were co-cultured with these peptides-labeled DCs, a marked increase in IFNγ production was observed in PBMCs prepared immediately after and 24 h after the heat treatment. Production of IFNγ in PBMCs prepared 48 h after the heat treatment returned to approximately the same level as in those without the treatment. These results indicate that heat treatment stimulates viral antigens-specific T cell responses, and its effect lasted for at least 24 h even after the heat treatment. Similarly, enhanced production of IFNγ in response to the tuberculin purified protein derivative was also observed immediately after and 24 h after the heat treatment. Compared with the responses to these recall antigens, non-recall antigen; KLH did not induce IFNγ production from PBMCs at any time popint of preparation. On the other hand, PMA/ionomycin-induced IFNγ production in PBMCs did not change during any time point of preparation. Taken together, whole-body heat treatment in fever-range appeared to make profound impact on antigen-specific T cell responses, and could be a possible combination therapy to enhance the efficacy of cancer immuno cell therapy, such as adoptive transfer of activated T cells and DCs-based vaccination. Citation Format: Yasunobu Kobayashi, Yusuke Ito, Valentina V. Ostapenko, Mayuko Sakai, Norimasa Matsushita, Kenichiro Imai, Koichi Shimizu, Atsushi Aruga, Keishi Tanigawa. Effects of whole-body heat treatment on antigen specific T cells responses. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4739. doi:10.1158/1538-7445.AM2013-4739

Human IL18-IL2 fusion protein as a potential antitumor reagent by enhancing NK cell cytotoxicity and IFN-γ production

To obtain a human IL18-IL2 fusion protein by genetic engineering methods and investigate its antitumor activity and mechanism in tumor-bearing mouse models. Human IL-18, IL-2 and the fusion gene IL18-IL2 were obtained by PCR, inserted into pBV220 vector and expressed in BL21 (DE3) by 42 °C heat induction. Purified proteins were analyzed by SDS-PAGE and Western blot. The biological activity of IL18-IL2 was first determined by its ability to augment IFN-γ production in PBMCs. Then tumor-bearing mouse models of mouse Lewis lung carcinoma (LLC), human large cell lung carcinoma (NCI-H460) and human colorectal carcinoma (HCT-116) were established to investigate the antitumor activity and mechanism of IL18-IL2. IL18-IL2 was confirmed by Western blot, and its molecular weight was about 34.5 kDa. IL18-IL2 could significantly enhance production of IFN-γ in PBMCs in vitro and induce significant tumor regression in tumor-bearing mouse models of LLC, NCI-H460 and HCT-116 than that of IL-18 and IL-2 separately or combination using. In the mice bearing HCT-116 and LLC, IFN-γ concentrations and natural killer cell cytotoxicity were highly enhanced by IL18-IL2. Anti asialo GM1 could reduce natural killer cell cytotoxicity, production of IFN-γ, and regression of LLC tumor aroused by IL18-IL2. These results suggested the IL18-IL2 fusion protein showed a synergetic effect on tumor regression, which was related to the great ability of IL18-IL2 in enhancing IFN-γ production and natural killer cell cytotoxicity. The fusion protein was a potential antitumor reagent in cancer immunotherapy.

Immune Stimulating Efficacy of Soluble β-1,3-glucans

Background: β-1,3-glucans are well known to enhance the immune reactions, resulting in antitumor, antibacterial, antiviral, anticoagulatory and wound healing activities. β-1, 3-glucans have various activities depending on molecular weight, degree of branching, conformation, water-solubility and intermolecular association. However, the β-1,3-glucan linked backbone structure is essential and β-D-glucopyranosyl units are required for immuno-potentiating activities. Result: In this study, we tested the immunophamacological activities of soluble β-1,3-glucans and confirmed the following activities: (1) IFN-γ production in PBMCs in the presence or the absence of PHA, LPS, or IL-18; (2) induction of various cytokines in the spleen and thymus; (3) adjuvant effect on the antibody production; (4) nitrogen oxide synthesis in macrophages; (5) the cytotoxic and antitumor effects on cell lines and ICR mice. Conclusion: These results strongly suggested that β-1,3-glucans possessed various immuno-pharmacological activities. (Immune Network 2003;3(2):156-163)

285 Immunomodulation of roxithromycin for Dermatophagoides farinae(Df)-induced IL4, IFN-γ production in PBMCs from patients with bronchial asthma (BA)

285 Immunomodulation of roxithromycin for Dermatophagoides farinae(Df)-induced IL4, IFN-γ production in PBMCs from patients with bronchial asthma (BA)