Antigen-specific Proliferation Assays Research Articles

Abstract 3283: GEN1046 (DuoBody®-PD-L1x4-1BB) in combination with PD-1 blockade potentiates anti-tumor immunity

Abstract GEN1046 (DuoBody®-PD-L1x4-1BB) is an investigational, potential first-in-class bispecific immunomodulatory antibody designed to elicit an anti-tumor immune response by simultaneous and complementary blockade of PD-L1 on tumor or immune cells and conditional 4-1BB stimulation on T cells and NK cells. Previously, we described encouraging preclinical and early clinical activity of GEN1046 (Muik, et al., 2022, Cancer Discovery). We hypothesized that combining GEN1046 with PD-1 blockade would further potentiate anti-tumor activity through distinct and complementary immune modulatory effects. Addition of an anti-PD-1 agent would free up PD-L1 for binding to GEN1046, thus promoting PD-L1-dependent 4-1BB conditional agonism, while maintaining complete blockade of the PD-1 pathway by inhibiting interactions with both PD-L1 and PD-L2. ​Here we provide preclinical evidence supportive of therapeutic synergy by the combination of GEN1046 and anti-PD-1 and describe the mechanisms of enhanced anti-tumor immunity elicited by the combination. In in vitro studies, combining GEN1046 with an anti-PD-1 agent potentiated cytokine release in mixed lymphocyte reaction assays (using either unstimulated T cells or T cells exhausted by repeated CD3/CD28 co-stimulation) and enhanced T-cell expansion and cytokine secretion in antigen-specific proliferation assays compared to each single agent. In in vivo studies in mice bearing syngeneic subcutaneous MC38 tumors, the combination of an anti-mouse PD-L1x4-1BB bispecific antibody with anti-mouse PD-1 potentiated anti-tumor activity with significant enhancement of survival (P≤0.001) and durable, complete tumor regressions (CR) in 7/10 mice compared to no CR observed with either single agent, suggesting therapeutic synergy with the combination. The combination treatment elicited long-lasting immune memory response, as animals with CR were protected from tumor outgrowth upon rechallenge with MC38 cells. Mechanistically, animals treated with the combination showed a trend for ≥1.5-fold increase in the average density of CD3+ and CD4+ tumor-infiltrating lymphocytes (TILs), as well as proliferating (Ki67+) and cytotoxic (GZMB+) CD8+ TILs relative to each single agent, consistent with an amplified anti-tumor immune response. Together, these preclinical results suggest that combining GEN1046-induced conditional 4-1BB stimulation with complete PD-1 blockade can improve the anti-tumor immune response via distinct and complementary immune modulatory effects. The combination of GEN1046 with pembrolizumab is currently being investigated in ongoing clinical studies in patients with advanced NSCLC, who are treatment-naïve (NCT03917381) or have progressed on prior CPI-containing therapy (NCT05117242). Citation Format: Michela Capello, Angelica Sette, Theo Plantinga, Vanessa Spires, Kristina Nuermberger, Jordan Blum, Alexander Muik, Carol Costa Sa, Omar Jabado, Saskia Burm, Aras Toker, Sina Fellermeier-Kopf, Tahi Ahmadi, Brandon Higgs, Suzana Couto, Özlem Türeci, Mark Fereshteh, Ugur Sahin, Maria Jure-Kunkel, Nora Pencheva. GEN1046 (DuoBody®-PD-L1x4-1BB) in combination with PD-1 blockade potentiates anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3283.

Breast regression protein-39 is not required for experimental autoimmune encephalomyelitis induction

Increasing evidence points to a role for chitinase 3-like 1 (CHI3L1) in multiple sclerosis (MS). Here, we aimed to explore the potential involvement of CHI3L1 in the animal model of MS, experimental autoimmune encephalomyelitis (EAE). EAE was induced by immunization with MOG35–55 peptide in wild-type (WT) and knock-out (KO) mice for breast regression protein 39 (BRP-39), the mouse homologue of human CHI3L1. Immunological responses in splenocytes were assessed by means of polyclonal and antigen-specific proliferation assays. Central nervous system pathology and chitinase gene expression were also investigated. BRP-39 expression was increased in WT MOG35–55-immunized mice compared to saline-immunized controls. No differences were found between WT and BRP-39 KO mice regarding EAE clinical course, day of disease onset, mortality rate, splenocyte proliferative responses or histopathological findings. These results do not support a role of BRP-39 in the pathogenesis of EAE.

Immunomodulatory drug FTY720 induces regulatory CD4+CD25+ T cellsin vitro

As a novel immunosuppressant, FTY720 (2-amino-2-(2-[4-octylphenyl] ethyl)-1, 3-propanediol hydrochloride) has been used to prevent the allograft rejection in organ transplantation. FTY720 can prolong markedly survival of the allograft by inducing apoptosis of reactive lymphocytes and by redirecting the homing of lymphocytes. However, as the archetype of a new class of immune modulators, the potential effect of FTY720 on the immune response needs to be elucidated further. In this study, FTY720 was added into the mixed lymphocyte reaction (MLR) consisting of murine splenocytes from BALB/c and C57BL/6, to observe its direct effect on the induction of CD4(+)CD25(+) regulatory T cells. It was demonstrated that the proportion of CD4(+)CD25(+) and CD4(+)CD25(+)forkhead box P3 (FoxP3)(+) T cells in MLR were increased significantly by FTY720 treatment, and the expression of FoxP3 mRNA in lymphocytes was also enhanced markedly by the drug. A synergetic effect was observed between FTY720 and co-stimulation blockades. Moreover, analysis of the function of FTY720-treated cells manifested an increased suppressive activity in an in vitro antigen-specific proliferation assay. In conclusion, FTY720 can increase the number and enhance the functional activity of CD4(+)CD25(+) regulatory T cells in MLR, and these FTY720-treated cells possess the activity to down-regulate the alloreactivity of lymphocytes, indicating its potential use for therapeutic purposes.

LX211 (Voclosporin) Suppresses Experimental Uveitis and Inhibits Human T Cells

To test the therapeutic effectiveness of voclosporin against experimental autoimmune uveoretinitis (EAU) in rats and to evaluate its effect on human T cells. EAU was induced by immunization with a uveitogenic protein. Voclosporin administration, by subcutaneous injection, began on day (d) 0 or d7 after immunization. Treatment effectiveness was evaluated in vivo using clinical EAU scoring (d7-d13) and histopathologic evaluation of enucleated eyes after experimental termination. Rodent lymphocytes were harvested from lymph nodes on d14 for antigen-specific proliferation assays. The effect of voclosporin on human T-cell proliferation and cytokine secretion was examined in vitro. Voclosporin prevented EAU development in rats receiving medium and high preventive doses, whereas high-dose voclosporin administration effectively treated EAU. Lymphocytes from animals treated with voclosporin had decreased antigen-specific proliferation in vitro compared with lymphocytes from untreated animals. No evidence of abnormal ocular histopathology was found in the eyes from animals that received high doses of therapeutic voclosporin. Using human T cells, voclosporin inhibited human T-cell proliferation up to 100-fold. Furthermore, voclosporin treatment of human T cells significantly reduced pan T-cell effector responses. Voclosporin effectively suppressed uveoretinitis in an animal model that imitates the human inflammatory ocular disease by inhibiting lymphocyte proliferation. In addition, voclosporin effectively inhibited human T-cell proliferation and function in vitro. The authors report the first evidence supporting the application of voclosporin to treat intraocular inflammation.

Mucosal immune responses are related to reduction of bacterial colonization in the stomach after therapeutic Helicobacter pylori immunization in mice

The aim of this study was to investigate the capacity of oral and parenteral therapeutic immunization to reduce the bacterial colonization in the stomach after experimental Helicobacter pylori infection, and to evaluate whether any specific immune responses are related to such reduction. C57BL/6 mice were infected with H. pylori and thereafter immunized with H. pylori lysate either orally together with cholera toxin or intraperitoneally (ip) together with alum using immunization protocols that previously have provided prophylactic protection. The effect of the immunizations on H. pylori infection was determined by quantitative culture of H. pylori from the mouse stomach. Mucosal and systemic antibody responses were analyzed by ELISA in saponin extracted gastric tissue and serum, respectively, and mucosal CD4 + T cell responses by an antigen specific proliferation assay. Supernatants from the proliferating CD4 + T cells were analyzed for Th1 and Th2 cytokines. The oral, but not the parenteral therapeutic immunization induced significant decrease in H. pylori colonization compared to control infected mice. The oral immunization resulted in markedly elevated levels of serum IgG+M as well as gastric IgA antibodies against H. pylori antigen and also increased H. pylori specific mucosal CD4 + T cell proliferation with a Th1 cytokine profile. Although the parenteral immunization induced dramatic increases in H. pylori specific serum antibody titers, no increases in mucosal antibody or cellular immune responses were observed after the ip immunization compared to control infected mice. These findings suggest that H. pylori specific mucosal immune responses with a Th1 profile may provide therapeutic protection against H. pylori.

T cell reactivity to P0, P2, PMP-22, and myelin basic protein in patients with Guillain-Barré syndrome and chronic inflammatory demyelinating polyradiculoneuropathy

Objectives: It has been suggested that autoimmunity to peripheral myelin proteins is involved in the pathogenesis of Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). We aimed to compare...

A candidate DNA vaccine elicits HCV specific humoral and cellular immune responses.

To investigate the immunogenicity of candidate DNA vaccine against hepatitis C virus (HCV) delivered by two plasmids expressing HCV envelope protein 1 (E1) and envelope protein 2 (E2) antigens respectively and to study the effect of CpG adjuvant on this candidate vaccine. Recombinant plasmids expressing HCV E1 and E2 antigens respectively were used to simultaneously inoculate mice with or without CpG adjuvant. Antisera were then collected and titers of anti-HCV antibodies were analyzed by ELISA. One month after the last injection, animals were sacrificed to prepare single-cell suspension of splenocytes. These cells were subjected to HCV antigen specific proliferation assays and cytokine secretion assays to evaluate the cellular immune responses of the vaccinated animals. Antibody responses to HCV E1 and E2 antigens were detected in vaccinated animals. Animals receiving CpG adjuvant had slightly lower titers of anti-HCV antibodies in the sera, while the splenocytes from these animals showed higher HCV-antigen specific proliferation. Analysis of cytokine secretion from the splenocytes was consistent with the above results. While no antigen-specific IL-4 secretion was detected for all vaccinated animals, HCV antigen-specific INF-gamma secretion was detected for the splenocytes of vaccinated animals. CpG adjuvant enhanced the secretion of INF-gamma but did not change the profile of IL-4 secretion. Vaccination of mice with plasmids encoding HCV E1 and E2 antigens induces humoral and cellular immune responses. CpG adjuvant significantly enhances the cellular immune response.

Prolonged immune deficiency following allogeneic stem cell transplantation: risk factors and complications in adult patients

To evaluate the long-term immune reconstitution after allogeneic haematopoietic stem cell transplantation (SCT), we prospectively screened standard immune parameters in a series of 105 patients, at a median time of 15 months after SCT. Analysing lymphoid phenotypes, in vitro immune functions and immunoglobulin levels, we found that, more than 1 year post SCT, cellular and humoral immunity was still altered in a significant number of patients. CD4+ T cells were < 200/microl in one third of patients, and the CD4/CD8 ratio was still reversed in 78% of patients. Almost all patients showed positive T-cell responses against mitogens, but antigen-specific proliferation assays identified 20% to 80% of non-responders. B-cell counts were reconstituted in 61% of the patients, but levels of total immunoglobulins were still low in 59%. In multivariate analyses, human leucocyte antigen (HLA) disparity between donor and recipient and chronic graft-versus-host disease were the leading causes affecting immune reconstitution. Interestingly, cytomegalovirus (CMV) infections were strongly associated with normal CD8+ T-cell counts. Studying the impact of impaired immune reconstitution on the rate of infections occurring in the 6 years following screening, we identified three parameters (low B-cell count, inverted CD4/CD8 ratio, and negative response to tetanus toxin) as significant risk factors for developing such late infections.

Class II major histocompatibility complex‐restricted T cell function in CD4‐deficient mice

Previously, we and others have demonstrated that CD4-deficient mice have a normal number of T cells and B cells with a significant population of CD4-8-TcR alpha beta+ T cells. Surprisingly, however, these mice lacking CD4 show in vivo immunoglobulin isotype class switching from IgM to IgG in response to sheep erythrocytes and vesicular stomatitis virus. In this study we have depleted various subpopulations of T cells in vivo and shown that the population of CD4-8-TcR alpha beta+ T cells is responsible for providing "help" in the antibody response of CD4-deficient mice to vesicular stomatitis virus infection. We have used antigen-specific proliferation assays and blocking studies with class I and II major histocompatibility complex (MHC)-specific purified antibodies to show that these cells are class II MHC-restricted in responses against the T cell-dependent antigen keyhole limpet hemocyanin (KLH). Finally, phenotypic analysis of the CD4-CD8- thymocytes in CD4-deficient mice shows that these cells have a more mature phenotype than the CD4-8- thymocytes in wild type mice. These results indicate that CD4 is not absolutely necessary for positive selection or effector function of class II MHC-restricted helper T cells.

Use of particulate forms of protein antigens to increase the sensitivity of antigen-specific proliferation assays

Different forms of recombinant HIV-1 gp160 and tetanus toxoid were adsorbed onto latex microspheres and this particulate form of the proteins was used to measure antigen-specific proliferation in vaccinated rhesus macaques. Proliferative responses to proteins bound to microspheres were significantly greater and allowed for the detection of antigen-specific responses that were not detected using soluble proteins. The responses were antigen-specific and required prior immunization of the animals. Additionally, the presence and magnitude of the proliferative responses was associated with antibody responses to the same proteins suggesting the results were representative of in vivo responses and that the assay format did not induce in vitro artifacts.

Natural occurrence of thymocytes that react with myelin basic protein.

In adult strain 13 guinea pigs, two lines of evidence show that there are natural autoreactive thymocytes that can react with myelin basic protein (BP) or the encephalitogenic nonapeptide (EP). An autoradiographic binding assay revealed antigen-specific receptors for 125I-BP on thymocytes. A 3H-thymidine antigen-specific proliferation assay demonstrated that normal thymocytes were activated by EP- or BP-pulsed macrophages. Soluble BP suppressed the activation of thymocytes by macrophage-associated BP. The mode of presentation of BP or EP, whether macrophage-associated or soluble, may be critical in maintaining self-tolerance and preventing an autoimmune attack on the central nervous system.