623 Immuno-STATs: Leveraging protein engineering to expand and track antigen-specific T cells in vivo

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A659-A659
Author(s):  
Stad Zeigler ◽  
Andrew Woodham ◽  
Mengyan Li ◽  
Ella Zeyang ◽  
Stephen Kolifrath ◽  
...  
Keyword(s):  
Positron Emission Tomography ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Immune Escape ◽  
Emission Tomography ◽  
Immunodeficient Mice ◽  
Positron Emission

BackgroundImmunotherapies are highly promising and effective strategies for the treatment of cancer; however, continuing challenges persist, including 1) untargeted global immune modulation, resulting in serious side effects; 2) lack of therapeutics capable of in vivo expansion of tumor-specific T cells; 3) inability to visualize in vivo tumor-specific T cell responses; and 4) lack of flexible platforms to rapidly and efficiently explore new therapeutic strategies and immune-escape mechanisms. To address these challenges, we developed a novel class of precision biologics to treat cancer, autoimmune diseases and infectious diseases. We describe a modular platform constructed around an Fc-based covalent pMHC dimer, referred to as synTac (artificial synapse for T cell activation; also termed Immuno-STATs for Selective Targeting and Alteration of T cells), which selectively delivers different cargoes, including costimulatory, coinhibitory or cytokine signals and other modalities to primary T cells of defined specificity. The inherent modularity supports broad applications. Changing the encoded peptide enables targeting of different T cell specificities to address different diseases, while altering the cargo allows for evaluation of different co-modulatory mechanisms or the delivery of mechanistically informative probes.MethodsSortase A-mediated enzymatic coupling supported site-specific and stoichiometric installation of positron emission tomography (PET)-active radiolabels on synTacs to visualize the in vivo localization of antigen-specific T cells. The NSG humanized mouse model allowed for the evaluation of synTacs/Immuno-STATs to drive the in vivo antigen-specific expansion of human CD8 T cells.ResultsUsing radiolabeled synTacs/Immuno-STATs loaded with the appropriate peptides, we employed positron emission tomography to localize human papillomavirus (HPV16)-specific CD8 T cells to implanted HPV16-positive tumors in mice, as well as influenza A virus (IAV)-specific CD8 T cells in the lungs of IAV-infected mice. In vivo administration of HIV- and CMV-specific synTacs/Immuno-STATs to immunodeficient mice intrasplenically engrafted with human donor PBMCs resulted in the marked and selective expansion of HIV-specific and CMV-specific human CD8 T cells populating their spleens, respectively.ConclusionsWe demonstrate the remarkable flexibility of the synTacs/Immuno-STAT platform for addressing a broad range of applications, including the first report of the in vivo imaging of antigen-specific CD8 T cell populations and in vivo antigen-selective expansion of human CD8 T cells. These results suggest that, in addition to broad therapeutic applications, synTac/Immuno-STATs may provide prognostic/diagnostic information. Most notably, these results demonstrate the presence of synTacs/Immuno-STAT biologics in the tumor or infected tissues where they can elicit T cell restimulation and expansion necessary for target killing and enhanced therapeutic efficacy.

scholarly journals Imaging TCR-Dependent NFAT-Mediated T-Cell Activation with Positron Emission Tomography In Vivo

Neoplasia ◽  
2001 ◽  
Vol 3 (6) ◽  
pp. 480-488 ◽  
Author(s):  
Vladimir Ponomarev ◽  
Michael Doubrovin ◽  
Clay Lyddane ◽  
Tatiana Beresten ◽  
Julius Balatoni ◽  
...  
Keyword(s):  
Positron Emission Tomography ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Emission Tomography ◽  
Positron Emission

scholarly journals In vivo detection of antigen-specific CD8+ T cells by immuno-positron emission tomography

2020 ◽  
Vol 17 (10) ◽  
pp. 1025-1032 ◽  
Author(s):  
Andrew W. Woodham ◽  
Stad H. Zeigler ◽  
Ella L. Zeyang ◽  
Stephen C. Kolifrath ◽  
Ross W. Cheloha ◽  
...  
Keyword(s):  
Positron Emission Tomography ◽  
T Cells ◽  
Cd8 T Cells ◽  
Emission Tomography ◽  
In Vivo Detection ◽  
Positron Emission

scholarly journals 702 TJ-CD4B (ABL111), a Claudin18.2-targeted 4-1BB tumor engager induces potent tumor-dependent immune response without dose-limiting toxicity in preclinical studies

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A730-A730
Author(s):  
Wenqing Jiang ◽  
Zhengyi Wang ◽  
Zhen Sheng ◽  
Jaeho Jung ◽  
Taylor Guo
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Gene Expression Analysis ◽  
Cell Activation ◽  
Clinical Development ◽  
Cynomolgus Monkeys ◽  
Anti Tumor Activity

Background4-1BB (CD137) is a co-stimulatory receptor that stimulates the function of multiple immune cells. Its ability to induce potent anti-tumor activity makes 4-1BB an attractive target for immuno-oncology. However, clinical development of a monospecific 4-1BB agonistic antibody has been hampered by dose-limiting hepatic toxicities. To minimize systemic toxicities, we have developed a novel Claudin18.2 (CLDN18.2) x 4-1BB bispecific antibody, TJ-CD4B (ABL111) that stimulates 4-1BB pathway only when it engages with Claudin 18.2, a tumor-associated antigen specifically expressed in gastrointestinal cancers. TJ-CD4B (ABL111) is now being evaluated in patients with advanced solid tumors in a first-in-human trial (NCT04900818).MethodsTJ-CD4B (ABL111) was evaluated in vivo using the human 4-1BB knock-in mice bearing CLDN18.2 expressing MC38 tumor cells. Pharmacodynamic effects upon treatment were characterized in tumor tissue and blood. Immunophenotyping of the tumor microenvironment (TME) and peripheral blood was performed by flow cytometry. Soluble biomarkers were measured using Luminex-based multiplex assay. In-depth gene expression analysis was performed on primary human CD8+ T cells that were co-cultured with CLDN18.2 expressing cells in the presence of anti-CD3 using NanoString nCounter®. Pharmacokinetic (PK) and toxicity study were performed in cynomolgus monkeys.ResultsTJ-CD4B (ABL111) elicited complete tumor regression in 13 out of 18 MC38 tumor bearing mice given at a dose above 2 mg/kg. Dose-dependent anti-tumor activity was associated with enhanced T cell activation in TME and expansion of memory T cells in the peripheral blood. Increased CD8+ T cells number and proliferation were observed in both tumor nest and surrounding stroma while the level of soluble 4-1BB in the serum was also elevated in response to the treatment. In vitro gene expression analysis by Nanostring revealed TJ-CD4B(ABL111) effectively activated immune pathways characterized by IFN?-signaling and T cell inflammation. Preclinically, TJ-CD4B was well tolerated at the repeated doses up to 100 mg/kg/wk in cynomolgus monkeys without the adverse influence on the liver function which is generally affected by 4-1BB activation. Besides, no cytokine release or immune activation was observed in the periphery.ConclusionsTJ-CD4B (ABL111) is a novel CLDN18.2 dependent 4-1BB bispecific agonist antibody that induced T cell activation and memory response in tumor with CLDN18.2 expression, leading to a strong anti-tumor activity in vivo. TJ-CD4B did not induce systemic immune response nor hepatic toxicity due to the CLDN18.2 dependent 4-1BB stimulation. These data warrant the current clinical development in phase I trial to validate the safety properties and tumor specific responses.

scholarly journals Anti–4-1bb Monoclonal Antibodies Abrogate T Cell–Dependent Humoral Immune Responses in Vivo through the Induction of Helper T Cell Anergy

1999 ◽  
Vol 190 (10) ◽  
pp. 1535-1540 ◽  
Author(s):  
Robert S. Mittler ◽  
Tina S. Bailey ◽  
Kerry Klussman ◽  
Mark D. Trailsmith ◽  
Michael K. Hoffmann
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Monoclonal Antibodies ◽  
T Cell ◽  
B Cells ◽  
Humoral Immunity ◽  
Cd8 T Cells ◽  
Immunodeficient Mice ◽  
Humoral Immune

The 4-1BB receptor (CDw137), a member of the tumor necrosis factor receptor superfamily, has been shown to costimulate the activation of T cells. Here we show that anti–mouse 4-1BB monoclonal antibodies (mAbs) inhibit thymus-dependent antibody production by B cells. Injection of anti–4-1BB mAbs into mice being immunized with cellular or soluble protein antigens induced long-term anergy of antigen-specific T cells. The immune response to the type II T cell–independent antigen trinintrophenol-conjugated Ficoll, however, was not suppressed. Inhibition of humoral immunity occurred only when anti–4-1BB mAb was given within 1 wk after immunization. Anti–4-1BB inhibition was observed in mice lacking functional CD8+ T cells, indicating that CD8+ T cells were not required for the induction of anergy. Analysis of the requirements for the anti–4-1BB–mediated inhibition of humoral immunity revealed that suppression could not be adoptively transferred with T cells from anti–4-1BB–treated mice. Transfer of BALB/c splenic T cells from sheep red blood cell (SRBC)-immunized and anti–4-1BB–treated mice together with normal BALB/c B cells into C.B-17 severe combined immunodeficient mice failed to generate an anti-SRBC response. However, B cells from the SRBC-immunized, anti–4-1BB–treated BALB/c mice, together with normal naive T cells, exhibited a normal humoral immune response against SRBC after transfer, demonstrating that SRBC-specific B cells were left unaffected by anti–4-1BB mAbs.

scholarly journals Tumor masses support naive T cell infiltration, activation, and differentiation into effectors

2010 ◽  
Vol 207 (8) ◽  
pp. 1791-1804 ◽  
Author(s):  
Elizabeth D. Thompson ◽  
Hilda L. Enriquez ◽  
Yang-Xin Fu ◽  
Victor H. Engelhard
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Adoptive Transfer ◽  
Cell Activation ◽  
Ex Vivo ◽  
Tumor Infiltrating Lymphocytes ◽  
T Cell Infiltration

Studies of T cell responses to tumors have focused on the draining lymph node (LN) as the site of activation. We examined the tumor mass as a potential site of activation after adoptive transfer of naive tumor-specific CD8 T cells. Activated CD8 T cells were present in tumors within 24 h of adoptive transfer and proliferation of these cells was also evident 4–5 d later in mice treated with FTY720 to prevent infiltration of cells activated in LNs. To confirm that activation of these T cells occurred in the tumor and not the tumor-draining LNs, we used mice lacking LNs. Activated and proliferating tumor-infiltrating lymphocytes were evident in these mice 24 h and 4 d after naive cell transfer. T cells activated within tumors acquired effector function that was evident both ex vivo and in vivo. Both cross-presenting antigen presenting cells within the tumor and tumor cells directly presenting antigen activated these functional CD8 effectors. We conclude that tumors support the infiltration, activation, and effector differentiation of naive CD8 T cells, despite the presence of immunosuppressive mechanisms. Thus, targeting of T cell activation to tumors may present a tool in the development of cancer immunotherapy.

scholarly journals Immunologic ignorance of vascular endothelial cells expressing minor histocompatibility antigen

Blood ◽  
2008 ◽  
Vol 111 (9) ◽  
pp. 4588-4595 ◽  
Author(s):  
Beatrice Bolinger ◽  
Philippe Krebs ◽  
Yinghua Tian ◽  
Daniel Engeler ◽  
Elke Scandella ◽  
...  
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Histocompatibility Antigen ◽  
Target Cells ◽  
Minor Histocompatibility Antigen

Abstract Endothelial cells (ECs) presenting minor histocompatibility antigen (mhAg) are major target cells for alloreactive effector CD8+ T cells during chronic transplant rejection and graft-versus-host disease (GVHD). The contribution of ECs to T-cell activation, however, is still a controversial issue. In this study, we have assessed the antigen-presenting capacity of ECs in vivo using a transgenic mouse model with beta-galactosidase (β-gal) expression confined to the vascular endothelium (Tie2-LacZ mice). In a GVHD-like setting with adoptive transfer of β-gal–specific T-cell receptor–transgenic T cells, β-gal expression by ECs was not sufficient to either activate or tolerize CD8+ T cells. Likewise, transplantation of fully vascularized heart or liver grafts from Tie2-LacZ mice into nontransgenic recipients did not suffice to activate β-gal–specific CD8+ T cells, indicating that CD8+ T-cell responses against mhAg cannot be initiated by ECs. Moreover, we could show that spontaneous activation of β-gal–specific CD8+ T cells in Tie2-LacZ mice was exclusively dependent on CD11c+ dendritic cells (DCs), demonstrating that mhAgs presented by ECs remain immunologically ignored unless presentation by DCs is granted.

scholarly journals Human Immunodeficiency Virus-Specific Circulating CD8 T Lymphocytes Have Down-Modulated CD3ζ and CD28, Key Signaling Molecules for T-Cell Activation

2000 ◽  
Vol 74 (16) ◽  
pp. 7320-7330 ◽  
Author(s):  
Linda A. Trimble ◽  
Premlata Shankar ◽  
Mark Patterson ◽  
Johanna P. Daily ◽  
Judy Lieberman
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Cd8 T Cell ◽  
Cd8 T Lymphocytes ◽  
Immunodeficiency Virus

ABSTRACT Although human immunodeficiency virus (HIV)-infected subjects without AIDS have a high frequency of HIV-specific CD8 T lymphocytes, cellular immunity is unable to control infection. Freshly isolated lymphocytes often do not lyse HIV-infected targets in 4-h cytotoxicity assays. A large fraction of circulating CD8 T cells from HIV-infected donors down-modulate CD3ζ, the signaling component of the T-cell receptor complex, which is reexpressed in vitro coincident with the return of cytotoxic function. To investigate further the link between CD3ζ down-modulation and possible CD8 T-cell functional defects, we used flow cytometry to characterize further the properties of the CD3ζ-down-modulated subset. HIV-specific CD8 T cells, identified by tetramer staining, are CD3ζ−. CD8 T cells with down-modulated CD3ζ also do not express the key costimulatory receptor CD28 and have the cell surface phenotype of activated or memory T cells (HLA-DR+ CD62L−). After T-cell activation, CD3ζ-down-modulated cells express the activation marker CD69 but not the high-affinity interleukin 2 (IL-2) receptor α-chain CD25 and produce gamma interferon but not IL-2. Therefore HIV-specific CD8 T cells have down-modulated key signaling molecules for T-cell activation and costimulation and require exogenous cytokine stimulation. The typical impairment of HIV-specific CD4 T helper cells, which would normally provide specific CD8 T-cell stimulation, means that in vivo CTL function in vivo is compromised in most HIV-infected individuals. In AIDS patients, the functional defect is more severe, since CD3ζ is not reexpressed even after IL-2 exposure.

scholarly journals Preclinical Evaluation of 89Zr-Df-IAB22M2C PET as an Imaging Biomarker for the Development of the GUCY2C-CD3 Bispecific PF-07062119 as a T Cell Engaging Therapy

2021 ◽  
Author(s):  
Kevin P. Maresca ◽  
Jianqing Chen ◽  
Divya Mathur ◽  
Anand Giddabasappa ◽  
Adam Root ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Ex Vivo ◽  
Standard Uptake Value ◽  
Imaging Biomarker ◽  
Distribution Analysis ◽  
T Cell Infiltration ◽  
Positron Emission

Abstract Purpose A sensitive and specific imaging biomarker to monitor immune activation and quantify pharmacodynamic responses would be useful for development of immunomodulating anti-cancer agents. PF-07062119 is a T cell engaging bispecific antibody that binds to CD3 and guanylyl cyclase C, a protein that is over-expressed by colorectal cancers. Here, we used 89Zr-Df-IAB22M2C (89Zr-Df-Crefmirlimab), a human CD8-specific minibody to monitor CD8+ T cell infiltration into tumors by positron emission tomography. We investigated the ability of 89Zr-Df-IAB22M2C to track anti-tumor activity induced by PF-07062119 in a human CRC adoptive transfer mouse model (with injected activated/expanded human T cells), as well as the correlation of tumor radiotracer uptake with CD8+ immunohistochemical staining. Procedures NOD SCID gamma mice bearing human CRC LS1034 tumors were treated with four different doses of PF-07062119, or a non-targeted CD3 BsAb control, and imaged with 89Zr-Df-IAB22M2C PET at days 4 and 9. Following PET/CT imaging, mice were euthanized and dissected for ex vivo distribution analysis of 89Zr-Df-IAB22M2C in tissues on days 4 and 9, with additional data collected on day 6 (supplementary). Data were analyzed and reported as standard uptake value and %ID/g for in vivo imaging and ex vivo tissue distribution. In addition, tumor tissues were evaluated by immunohistochemistry for CD8+ T cells. Results The results demonstrated substantial mean uptake of 89Zr-Df-IAB22M2C (%ID/g) in PF-07062119-treated tumors, with significant increases in comparison to non-targeted BsAb-treated controls, as well as PF-07062119 dose-dependent responses over time of treatment. A moderate correlation was observed between tumor tissue radioactivity uptake and CD8+ cell density, demonstrating the value of the imaging agent for non-invasive assessment of intra-tumoral CD8+ T cells and the mechanism of action for PF-07062119. Conclusion Immune-imaging technologies for quantitative cellular measures would be a valuable biomarker in immunotherapeutic clinical development. We demonstrated a qualification of 89Zr-IAB22M2C PET to evaluate PD responses (mice) to a novel immunotherapeutic.

A mouse model for cancer immunoediting with renal cell carcinoma to explore mechanisms of immune escape.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 11074-11074
Author(s):  
Elizabeth Bigger ◽  
Michael Quigley ◽  
Yiping Yang
Keyword(s):  
Renal Cell Carcinoma ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Renal Cell ◽  
Immune Escape ◽  
Cd8 T Cell ◽  
Cancer Immunoediting ◽  
Cancer Immunosurveillance

11074 Background: Cancer immunosurveillance is the immune system's ability to recognize and destroy newly arising malignant cells. Recent data suggests that the immune system functions also to apply a selective pressure, leading to growth of less immunogenic tumors, a process termed cancer immunoediting. However, direct experimental evidence to support this hypothesis has been lacking. Methods: We examined the interaction between tumor and tumor-specific CD8 T cells in vivo with a model tumor antigen, influenza hemagglutinin (HA), in a renal cell carcinoma cell line (Renca-HA) and studied the HA-specific CD8 T cell response to Renca-HA in vivo. Naïve HA-specific CD8 T cells (Thy1.1+) purified from HA-TCR transgenic mice that recognize a Kd-restricted HA epitope were transferred into congenic Thy1.2+recipients inoculated the day prior with Renca-HA or wild type Renca (Renca-WT). At subsequent time points, recipient lymphocytes and pulmonary tumors were harvested and analyzed. Results: Four days after transfer, vigorous proliferation of HA-specific CD8 T cells was detected in Renca-HA inoculated mice, but not Renca-WT inoculated mice, leading to activation and effector differentiation of HA-specific T cells. HA-specific CD8 T cell activation resulted in destruction of Renca-HA in vivo, causing a significant (p <0.001) reduction of tumor burden and prolongation of survival. However, a small fraction of Renca-HA tumor cells survived immune-mediated killing and evaded immune surveillance. Microarray technology identified upregulation of transforming growth factor β-3 (TGF-β3) expression in edited Renca-HA, but not in unedited Renca-HA, compared with Renca-WT, confirmed by real-time quantitative PCR. Functional abrogation of TGF-β3 signaling on tumor-specific CD8 T cells delayed Renca HA tumor growth in vivo. Conclusions: These data provide direct evidence for the cancer immunosurveillance and immunoediting processes, and suggest that tumor induction of TGFβ-3 during the immunoediting process suppresses tumor-specific CD8 T cell function in vivo, allowing tumor immune escape. Our results provide mechanistic insights as well as potential strategies to block cancer immune evasion.

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