A chimeric HLA-A2:β2M:Ig fusion protein for the study of virus-specific CD8+ T-cells

Abstract The ITK-SYK fusion protein was previously described to be present in 17% of unspecified peripheral T-cell lymphomas. Here we demonstrate that expression of ITK-SYK in the bone marrow of Balb\c mice causes T-cell lymphomas in mice with a latency of only 3–4 weeks. The disease is characterized by infiltration of the spleen, lymph nodes, bone marrow and the skin with malignant T-cells and progredient destruction of these organs. The mice die about 2 months after the transplantation due to dramatic weight loss caused by infiltration of T-cells into the colon and because of progredient anemia end neutropenia due to progredient infiltration of the bone marrow. The malignant T-cells were characterized as a mixed population of CD3+, CD4+, CD8- T cells and CD3+, CD4-, CD8- T-cells. The malignant disease was accompanied by a generalized inflammatory reaction including upregulation of the inflammatory cytokines IL-5 and INF-γ. Modulation of the membrane binding of ITK-SYK or its binding to Cbl by point mutations in the pleckstrin homology domain of ITK could alter the transforming activity of ITK-SYK. The intact kinase domain was essential for the transformation process and the disease could be reversed by treatment of diseased mice with the Syk-inhibitor Curcumine. Our results demonstrate that the fusion protein ITK-SYK causes T-cell lymphomas in mice and mimics the human disease. Therefore pharmacological inhibition of Syk in patients with U-PTCL carrying the ITK-SYK fusion protein might be a new and effective treatment strategy.

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The Fusion Protein of CTP-HBcAg18-27-Tapasin Mediates the Apoptosis of CD8+T Cells and CD8+ T Cell Response in HLA-A2 Transgenic Mice

Hepatitis Monthly ◽

10.5812/hepatmon.16161 ◽

2014 ◽

Vol 14 (2) ◽

Cited By ~ 4

Author(s):

Yu-Yan Tang ◽

Zheng-Hao Tang ◽

Yi Zhang ◽

Meng Zhuo ◽

Guo-Qing Zang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Transgenic Mice ◽

Fusion Protein ◽

Cd8 T Cells ◽

Cell Response ◽

Cd8 T Cell ◽

T Cell Response

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Activation of NK and CD8+ T-Cells with a Novel IL-15 and TGF-Beta Receptor Fusion Protein Confers Anti-Tumor Immunity

Blood ◽

10.1182/blood.v126.23.3421.3421 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3421-3421

Author(s):

Spencer Ng ◽

Jiusheng Deng ◽

Raghavan Chinndurai ◽

Shala Yuan ◽

Andrea Pennati ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Fusion Protein ◽

Nk Cells ◽

Cd8 T Cells ◽

Hematological Malignancies ◽

Granzyme B ◽

Flow Cytometric Analysis ◽

Cd8 T Cell ◽

P Value

Abstract The use of cytokines as agents to augment immune responses against malignancies have been dealt setbacks due to immune selection of tumors, resulting in subpopulations that elaborate tumor-derived soluble factors, such as transforming growth factor-beta (TGF-β), which suppress immune effector functions. TGF-β is overexpressed by many solid and hematological malignancies and is well known to inhibit the proliferation and anti-tumor functions of lymphomyeloid cells. In order to maximize cytokine-based immunotherapy against tumors, we have designed a novel fusion protein consisting of proinflammatory murine interleukin-15 (IL-15) linked to the sushi domain of the IL-15Rα chain (IL15Rαsushi +IL15) fused in frame to the C'-terminus of a dimeric murine TGF-β-receptor (type II, TβRII) ectodomain-based ligand trap, termed FIST-15 (Fusion of Interleukin 15 with Sushi to TGF-β receptor). The rationale for the design of this protein is to prevent tumor-derived TGF-β from suppressing the immune response via the TGF-β ligand trap moiety, while simultaneously providing a potent stimulus for the activation of anti-tumor responses by an IL-15R agonist (IL-15Rαsushi +IL15). FIST-15 can neutralize TGF-β induced Smad signaling, and induce STAT3 and STAT5 phosphorylation by immunoblot and intracellular flow cytometric analysis of lymphocytes, suggesting that both protein domains are biochemically active. Functionally, FIST-15 is able to induce CD8+ T-cell proliferation at rates greater than IL-15 alone (CD8+ T-cell replicative index or fold-expansion of responding cells: 40, FIST-15, vs. 10, IL-15; p-value of unpaired T-test <0.05). The mitogenic effects of IL-15 are abrogated in CD8+ T-cells and NK cells in the presence of TGF-β. However, FIST-15 can overcome TGF-β mediated inhibition in both these cellular subsets (CD8+ T-cell replicative index: 20, FIST-15, vs. 5, IL-15, and NK cell replicative index: 40, FIST-15 vs. 5, IL-15; p-value <0.05). Rapid proliferation of the CD8+ central memory phenotype (CD62L+, CD44+) T-cells are seen with FIST-15 treatment. Compared to IL-15 expanded CD8+ T-cells, FIST-15 treatment also produced more IFN-γ, TNF-α, and IL-2 secreting CD8+ T-cells upon PMA/ionomycin stimulation. In addition to cytokines, production of anti-tumor effector molecules such as granzyme B is known to be inhibited by TGF-β. FIST-15 treated NK cells were superior to IL-15 treated NK cells in granzyme B production, even in the presence of TGF-β, as assayed by flow cytometric analysis (86.8% vs. 30.7% granzyme B expressing cells). Functionally, FIST-15 treated NK cells were also significantly more cytolytic against TGF-β secreting B16 murine melanoma cells in vitro compared to IL-15 treated NK cells (83.5% killing, FIST-15, vs. 24.4% killing, IL-15). C57Bl/6 mice with pre-established, syngeneic B16 melanoma tumors were treated with FIST-15 to assay the anti-tumor effects of the fusion protein in vivo. Mice receiving FIST-15 showed a significant delay in tumor growth (mean tumor volume: 345mm3) compared to control mice receiving conditioned media (mean tumor volume: 814.12mm3; p-value of paired T-test = 0.02) by day 21 post-tumor implantation. Furthermore, FIST-15 treated mice showed a significant survival advantage compared to control treated mice (80% vs 0%; p-value of log rank test = 0.0019) by day 27 post-tumor implantation. Mice immunized with B16 tumors transduced to express FIST-15 were also protected against subsequent wildtype B16 tumor challenge, suggesting that FIST-15 can trigger an adaptive immune response against tumor. Ongoing work utilizing FIST-15 in murine models of hematological malignancies, such as EL-4 lymphoma and C1498 AML, is currently underway. These models were selected due to their known overexpression of TGF-β isoforms that systemically inhibit endogenous anti-tumor responses, as well as the efficacy of immunotherapeutic agents. Indeed, many hematological malignancies acquire mutations that render them insensitive to the growth-inhibitory effects of TGF-β, where it may then be overexpressed as an oncogene to promote further tumor growth by inhibiting the immune system's anti-tumor capabilities (Dong et al Blood 2006). FIST-15 may present a viable immunotherapeutic strategy for hematological malignancies by combining the immune activating effects of IL-15 with the neutralization of immunosuppressive TGF-β. Disclosures Ng: Emory University: Patents & Royalties. Galipeau:Emory University: Patents & Royalties.

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Lactobacillus plantarum surface-displayed ASFV (p54) with porcine IL-21 generally stimulates protective immune responses in mice

AMB Express ◽

10.1186/s13568-021-01275-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Xiao-Lei Chen ◽

Jun-Hong Wang ◽

Wei Zhao ◽

Chun-Wei Shi ◽

Kai-Dian Yang ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Immune Responses ◽

Fusion Protein ◽

Lactobacillus Plantarum ◽

Cd8 T Cells ◽

Classical Swine Fever ◽

Oral Vaccines ◽

Ifn Γ ◽

Mtt Assays

AbstractAfrican classical swine fever virus (ASFV) has spread seriously around the world and has dealt with a heavy blow to the pig breeding industry due to the lack of vaccines. In this study, we produced recombinant Lactobacillus plantarum (L. plantarum) expressing an ASFV p54 and porcine IL-21 (pIL-21) fusion protein and evaluated the immune effect of NC8-pSIP409-pgsA'-p54-pIL-21 in a mouse model. First, we verified that the ASFV p54 protein and p54-pIL-21 fusion protein were anchored on the surface of L. plantarum NC8 by flow cytometry, immunofluorescence and Western blotting. Then, the results were verified by flow cytometry, ELISA and MTT assays. Mouse-specific humoral immunity and mucosal and T cell-mediated immune responses were induced by recombinant L. plantarum. The results of feeding mice recombinant L. plantarum showed that the levels of serum IgG and mucosal secreted IgA (SIgA), the number of CD4 and CD8 T cells, and the expression of IFN-γ in CD4 and CD8 T cells increased significantly, and lymphocyte proliferation occurred under stimulation with the ASFV p54 protein. Our data lay a foundation for the development of oral vaccines against ASFV in the future.

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A soluble divalent class I MHC/IgG1 fusion protein activates CD8+ T cells in vivo

Clinical Immunology ◽

10.1016/j.clim.2005.02.013 ◽

2005 ◽

Vol 116 (1) ◽

pp. 65-76 ◽

Cited By ~ 4

Author(s):

Brenna Carey ◽

Monica DeLay ◽

Jane E. Strasser ◽

Claudia Chalk ◽

Kristen Dudley-McClain ◽

...

Keyword(s):

T Cells ◽

Fusion Protein ◽

Cd8 T Cells ◽

Class I ◽

Class I Mhc

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Recombinant Adenovirus Expressing a Soluble Fusion Protein PD-1/CD137L Subverts the Suppression of CD8+ T Cells in HCC

Molecular Therapy ◽

10.1016/j.ymthe.2019.07.019 ◽

2019 ◽

Vol 27 (11) ◽

pp. 1906-1918 ◽

Cited By ~ 4

Author(s):

Yonghui Zhang ◽

Hailin Zhang ◽

Mei Wei ◽

Tao Mou ◽

Tao Shi ◽

...

Keyword(s):

T Cells ◽

Fusion Protein ◽

Cd8 T Cells ◽

Recombinant Adenovirus ◽

Soluble Fusion Protein

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Natural Killer Cell–Mediated Eradication of Neuroblastoma Metastases to Bone Marrow by Targeted Interleukin-2 Therapy

Blood ◽

10.1182/blood.v91.5.1706.1706_1706_1715 ◽

1998 ◽

Vol 91 (5) ◽

pp. 1706-1715 ◽

Cited By ~ 1

Author(s):

Holger N. Lode ◽

Rong Xiang ◽

Torsten Dreier ◽

Nissi M. Varki ◽

Stephen D. Gillies ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Fusion Protein ◽

Nk Cells ◽

Natural Killer ◽

Therapeutic Effect ◽

Cd8 T Cells ◽

Nk Cell ◽

Interleukin 2

Targeted interleukin-2 (IL-2) therapy with a genetically engineered antidisialoganglioside GD2 antibody–IL-2 fusion protein induced a cell-mediated antitumor response that effectively eradicated established bone marrow and liver metastases in a syngeneic model of neuroblastoma. The mechanism involved is exclusively natural killer (NK) cell–dependent, because NK-cell deficiency abrogated the antitumor effect. In contrast, the fusion protein remained completely effective in the T-cell–deficient mice or immunocompetent mice depleted of CD8+ T cells in vivo. A strong stimulation of NK-cell activity was also shown in vitro. Immunohistology of the leukocytic infiltrate of livers from treated mice revealed a strong staining for NK cells but not for CD8+ T cells. The therapeutic effect of the fusion protein was increased when combined with NK-cell–stimulating agents, such as poly I:C or recombinant mouse interferon-γ. In conclusion, these data show that targeted delivery of cytokines to the tumor microenvironment offers a new strategy to elicit an effective cellular immune response mediated by NK cells against metastatic neuroblastoma. This therapeutic effect may have general clinical implications for the treatment of patients with minimal residual disease who suffer from T-cell suppression after high-dose chemotherapy but are not deficient in NK cells.

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438 A phase 1 trial of CUE-101, a novel HPV16 E7-pHLA-IL2-Fc fusion protein, alone and in combination with pembrolizumab in patients with recurrent/metastatic HPV16+ head and neck cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.438 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A468-A468

Author(s):

Christine Chung ◽

A Dimitrios Colevas ◽

Michael Gibson ◽

Douglas Adkins ◽

Ammar Sukari ◽

...

Keyword(s):

T Cells ◽

Antitumor Activity ◽

Fusion Protein ◽

Head And Neck ◽

Cd8 T Cells ◽

Tumor Antigen ◽

Cancer Center ◽

School Of Medicine ◽

Fc Fusion Protein ◽

Dose Dependent

BackgroundImmuno-STATsTM are novel, modular fusion proteins designed to selectively activate tumor-antigen-specific CD8+ T cells. CUE-101 is comprised of a human leukocyte antigen (HLA) complex, HLA-A*0201, a peptide epitope derived from the HPV16 E7 protein, and 4 molecules of a reduced affinity human interleukin-2 (IL-2) and is designed to bind and activate HPV16-specific T cells for treatment of HPV16-driven cancers. In preclinical studies CUE-101 demonstrated selective binding, activation, and expansion of HPV16 E7-specific CD8+ T cells, and a murine surrogate activated anti-tumor immunity.1MethodsCUE-101-01 is a first-in-human study in HLA-A*0201 positive patients with HPV16+ recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC). Safety of escalating monotherapy and combination doses was evaluated to establish the recommended phase 2 dose (RP2D) for expanded enrollment. Patients with R/M HNSCC refractory to 1 or more prior platinum or pembrolizumab based systemic treatments received CUE-101 monotherapy, and patients with R/M HNSCC and PD-L1 tumor expression received combination CUE-101 and 200 mg pembrolizumab as first line treatment. Study treatment was administered intravenously every 3 weeks until progression or toxicity. Objectives included evaluation of safety, pharmacokinetics (PK), pharmacodynamics (PD), and antitumor activity.ResultsAs of June 30, 2021, 39 patients have received CUE-101 monotherapy ranging from 0.06 to 8 mg/kg. The maximum tolerated dose (MTD) was not identified. Based on PK, PD and clinical data, a monotherapy RP2D of 4 mg/kg was selected. The combination cohort of 1 mg/kg CUE-101 and pembrolizumab has been tested and dose escalation is ongoing. Adverse events have included CTCAE grade 2 or less fatigue (41%), anemia (31%), lymphopenia (24%), chills (21%), decreased appetite (19%) and dyspnea (17%). CUE-101 PK data demonstrate dose-dependent increases in drug exposure that are sustained upon repeat dosing. PD data demonstrate dose-dependent expansion of HPV-16 E711-20-specific CD8+ T cells, sustained increase in natural killer cells and transient increase in Treg cells. An increase in CD3+ GZMB+ tumor infiltrating T cells was observed in tissue following treatment with CUE-101 in one patient with available pre- and post-treatment biopsies. One patient at the CUE-101 monotherapy RP2D has an ongoing partial response and 8 of 33 patients have experienced stable disease ≥ 12 weeks based on RECIST 1.1 criteria.ConclusionsCUE-101 is a novel immunotherapeutic demonstrating acceptable safety and tolerability with encouraging PD signals, supporting selective activation of tumor-specific T cells, and promising antitumor activity. Enrollment continues in both monotherapy and combination cohorts.AcknowledgementsThe authors would like to thank all the patients who are participating in this study. The study is sponsored by Cue Biopharma.Trial RegistrationClinicalTrials.gov NCT03978689ReferencesQuayle SN, Girgis N, Thapa DR, et al. CUE-101, a Novel HPV16 E7-pHLA-IL-2-Fc fusion protein, enhances tumor antigen specific T cell activation for the treatment of HPV16-driven malignancies. Clin Cancer Res 2020;26:1953–64.Ethics ApprovalThis study was approved by Ethics and Institutional Review Boards (IRBs) at all study sites. IRB reference numbers: Advarra Pro00037736 (Moffitt Cancer Center), IRB 52744 (Stanford University School of Medicine), IRB 191714 (Vanderbilt University Medical Center Vanderbilt-Ingram Cancer Center), HRPO# 201905108 (Washington University School of Medicine), 2019–087 Karmanos Cancer Institute, DF/HCC IRB# 19-374 (Massachusetts General Hospital), WIRB 2000026098 (Yale Cancer Center), WIRB 1908869642 (University of Arizona Cancer Center), WIRB STUDY00008948 (University of Washington, Seattle), IRB(IRBMED) HUM00165746 (University of Michigan Comprehensive Cancer Center), WIRB IRB00112341(Winship Cancer Institute/Emory University), 2019–0578 (The University of Texas MD Anderson Cancer Center), IRB 20-073 (Memorial Sloan Kettering Cancer Center), IRB00255391 (Johns Hopkins University School of Medicine).

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Natural Killer Cell–Mediated Eradication of Neuroblastoma Metastases to Bone Marrow by Targeted Interleukin-2 Therapy

Blood ◽

10.1182/blood.v91.5.1706 ◽

1998 ◽

Vol 91 (5) ◽

pp. 1706-1715 ◽

Cited By ~ 122

Author(s):

Holger N. Lode ◽

Rong Xiang ◽

Torsten Dreier ◽

Nissi M. Varki ◽

Stephen D. Gillies ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Fusion Protein ◽

Nk Cells ◽

Natural Killer ◽

Therapeutic Effect ◽

Cd8 T Cells ◽

Nk Cell ◽

Interleukin 2

Abstract Targeted interleukin-2 (IL-2) therapy with a genetically engineered antidisialoganglioside GD2 antibody–IL-2 fusion protein induced a cell-mediated antitumor response that effectively eradicated established bone marrow and liver metastases in a syngeneic model of neuroblastoma. The mechanism involved is exclusively natural killer (NK) cell–dependent, because NK-cell deficiency abrogated the antitumor effect. In contrast, the fusion protein remained completely effective in the T-cell–deficient mice or immunocompetent mice depleted of CD8+ T cells in vivo. A strong stimulation of NK-cell activity was also shown in vitro. Immunohistology of the leukocytic infiltrate of livers from treated mice revealed a strong staining for NK cells but not for CD8+ T cells. The therapeutic effect of the fusion protein was increased when combined with NK-cell–stimulating agents, such as poly I:C or recombinant mouse interferon-γ. In conclusion, these data show that targeted delivery of cytokines to the tumor microenvironment offers a new strategy to elicit an effective cellular immune response mediated by NK cells against metastatic neuroblastoma. This therapeutic effect may have general clinical implications for the treatment of patients with minimal residual disease who suffer from T-cell suppression after high-dose chemotherapy but are not deficient in NK cells.

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