Inter- and Intramolecular Epitope Spreading in Equine Recurrent Uveitis

BackgroundMCY-M11, an anti-mesothelin CAR (Meso-CAR) mRNA transfected PBMC cell product manufactured through <1 day-process is under clinical evaluation for the treatment of advanced ovarian cancer and peritoneal mesothelioma. In this in-vitro study, we characterized the phenotypic and functional status of immune cell populations in MCY-M11 and their possible role in antitumor immunity.MethodsMCY-M11 cell product were generated using unmanipulated healthy donor PBMCs (n=5) by transfection of Meso-CAR mRNA using MaxCyte’s proprietary Flow Electroporation® system. Frozen MCY-M11 cell product was thawed and cultured for 18 hours, then co-cultured with hMSLNneg or hMSLNpos human mesothelioma cell line, MSTO-211H, or stimulated with anti-CD3/anti-CD28 antibodies in vitro for 8 days. Distinct cell populations in MCY-M11 were evaluated for kinetics and duration of CAR expression, differentiation, activation, exhaustion, and their ability to secrete various immunomodulatory molecules during in vitro stimulation. Antigen-specific proliferation and cytotoxicity of MCY-M11 against hMSLNpos tumor cells as well as their ability to mount long-term antitumor immunity through epitope spreading mechanisms were studied.ResultsIndividual cell populations in MCY-M11 exhibited a consistent but transient Meso-CAR expression persisting for about 7 days. Cell subsets in MCY-M11 acquired early signs of activation and differentiation within 18–24 hours post-culture, but only attained full activation and lineage-specific differentiation upon specific response to hMSLNpos tumor cells. hMSLN antigen experienced MCY-M11 retained significant fractions of Naïve and Central Memory T cells and increased percentage of Effector Memory T cells along with increased expression of CD62L, CD27, and chemokine receptors (CCR5, CCR7, and CXCR3). MCY-M11 exhibited strong antigen-specific cytotoxicity against hMSLNpos tumor cells with corresponding increase in activation and proliferation of CD4+ and CD8+ T cell subsets and displayed low or no acquisition of known exhaustion markers. NK cells also exhibited a functionally superior molecular signature exhibiting increased levels of NKG2D, NKp44, NKp46, FAS, and TRAIL. The Monocytes and B cells in MCY-M11 also acquired an activated, differentiated, and mature phenotype, expressing molecules required for antigen presentation (HLA-DR, HLA-ABC, and CD205) and T cell co-stimulation (CD80 and CD86) to mount a strong antitumor response. These phenotypic changes in cell subsets of MCY-M11 transpired with simultaneous secretion of potent immunostimulatory molecules and chemokines facilitating an extended antitumor response through epitope spreading.ConclusionsWe demonstrated that MCY-M11 is a unique cell product possessing a complete built-in immune cellular machinery with favorable phenotype and enhanced functions specialized in mediating an effective and long-term antitumor response.Trial RegistrationNCT03608618

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Epitope Spreading: Lessons From Autoimmune Skin Diseases

Journal of Investigative Dermatology ◽

10.1046/j.1523-1747.1998.00107.x ◽

1998 ◽

Vol 110 (2) ◽

pp. 103-109 ◽

Cited By ~ 226

Author(s):

Lawrence S. Chan ◽

Carol J. Vanderlugt ◽

Takashi Hashimoto ◽

Takeji Nishikawa ◽

John J. Zone ◽

...

Keyword(s):

Skin Diseases ◽

Epitope Spreading

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132 CAR macrophages (CAR-M) elicit a systemic anti-tumor immune response and synergize with PD1 blockade in immunocompetent mouse models of HER2+ solid tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0132 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A145-A145

Author(s):

Stefano Pierini ◽

Rashid Gabbasov ◽

Linara Gabitova ◽

Yumi Ohtani ◽

Michael Klichinsky

Keyword(s):

Immune Response ◽

Overall Survival ◽

Animal Model ◽

T Cell ◽

Cancer Cells ◽

Solid Tumors ◽

Tumor Model ◽

Epitope Spreading ◽

Mhc I ◽

Single Tumor

BackgroundDespite the remarkable efficacy achieved by CAR-T therapy in hematologic malignancies, application in solid tumors has been challenging. We previously developed human CAR-M and demonstrated that adoptive cell transfer of CAR-M into xenograft models of human cancer controls tumor progression and improves overall survival [1]. Given that CAR-M are professional antigen presenting cells, we developed an immunocompetent animal model to evaluate the potential for induction of a systemic anti-tumor immune response.MethodsMurine bone marrow-derived macrophages were engineered to express an anti-HER2 CAR using the chimeric adenoviral vector Ad5f35. CAR-M were phenotypically and functionally evaluated in vitro and in syngeneic models. To evaluate CAR-M efficacy in an immunocompetent animal model, BALB/c mice were engrafted with CT26-HER2+ tumors (single-tumor model) and were treated with intratumoral CAR-HER2 or untransduced (UTD) macrophages. To evaluate epitope spreading, we simultaneously engrafted BALB/c mice with CT26-HER2+ and CT26-Wt tumors on opposite flanks (dual-tumor model), and treated mice with CAR-M or controls into the CT26-HER2+ tumor only. Peripheral and tumor-infiltrating immune cells were phenotypically and functionally characterized.ResultsIn addition to efficient gene delivery, Ad5f35 transduction promoted a pro-inflammatory (M1) phenotype in murine macrophages. CAR-M, but not control UTD macrophages, phagocytosed HER2+ target cancer cells. Anti-HER2 CAR-M eradicated HER2+ murine CT26 colorectal and human AU-565 breast cancer cells in a dose-dependent manner. CAR-M increased MHC-I and MHC-II expression on tumor cells and promoted tumor-associated antigen presentation and T cell activation. In vivo, CAR-M treatment led to tumor regression and improved overall survival in the CT26-HER2+ single-tumor model. In the dual-tumor model, CAR-M treatment cleared 75% of CT26-HER2+ tumors and inhibited the growth rate of contralateral CT26-WT tumors, demonstrating an abscopal effect. CAR-M treatment led to increased infiltration of intratumoral CD4+ and CD8+ T, NK, and dendritic cells – as well as an increase in T cell responsiveness to the CT26 MHC-I antigen gp70, indicating enhanced epitope spreading. Given the impact CAR-M had on endogenous T-cell immunity, we evaluated the combination of CAR-M and anti-PD1 in the CT26-HER2 model and found that the combination further enhanced tumor control and overall survival.ConclusionsThese results demonstrate that CAR-M therapy induces epitope spreading via activation of endogenous T cells, orchestrating a systemic immune response against solid tumors. Moreover, our findings provide rationale for the combination of CAR-M with immune checkpoint inhibitors. The anti-HER2 CAR-M CT-0508 will be evaluated in an upcoming Phase I clinical trial.ReferenceKlichinsky M, Ruella M, Shestova O, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol 2020;38(8):947–953.

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The Fecal Bacterial Microbiota in Horses with Equine Recurrent Uveitis

Animals ◽

10.3390/ani11030745 ◽

2021 ◽

Vol 11 (3) ◽

pp. 745

Author(s):

Michelle Martin de Bustamante ◽

Diego Gomez ◽

Jennifer MacNicol ◽

Ralph Hamor ◽

Caryn Plummer

Keyword(s):

Beta Diversity ◽

High Throughput Sequencing ◽

Illumina Miseq ◽

Diversity Indices ◽

Rrna Gene ◽

Linear Discriminant ◽

Alpha And Beta Diversity ◽

Bacterial Microbiota ◽

Healthy Control ◽

Equine Recurrent Uveitis

The objective of this study was to describe and compare the fecal bacterial microbiota of horses with equine recurrent uveitis (ERU) and healthy horses using next-generation sequencing techniques. Fecal samples were collected from 15 client-owned horses previously diagnosed with ERU on complete ophthalmic examination. For each fecal sample obtained from a horse with ERU, a sample was collected from an environmentally matched healthy control with no evidence of ocular disease. The Illumina MiSeq sequencer was used for high-throughput sequencing of the V4 region of the 16S rRNA gene. The relative abundance of predominant taxa, and alpha and beta diversity indices were calculated and compared between groups. The phyla Firmicutes, Bacteroidetes, Verrucomicrobia, and Proteobacteria predominated in both ERU and control horses, accounting for greater than 60% of sequences. Based on linear discriminant analysis effect size (LEfSe), no taxa were found to be enriched in either group. No significant differences were observed in alpha and beta diversity indices between groups (p > 0.05 for all tests). Equine recurrent uveitis is not associated with alteration of the gastrointestinal bacterial microbiota when compared with healthy controls.

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Personal neoantigen vaccines induce persistent memory T cell responses and epitope spreading in patients with melanoma

Nature Medicine ◽

10.1038/s41591-020-01206-4 ◽

2021 ◽

Cited By ~ 1

Author(s):

Zhuting Hu ◽

Donna E. Leet ◽

Rosa L. Allesøe ◽

Giacomo Oliveira ◽

Shuqiang Li ◽

...

Keyword(s):

T Cell ◽

T Cell Responses ◽

Epitope Spreading ◽

Memory T Cell ◽

Cell Responses ◽

Persistent Memory

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Humoral Epitope Spreading in Autoimmune Bullous Diseases

Frontiers in Immunology ◽

10.3389/fimmu.2018.00779 ◽

2018 ◽

Vol 9 ◽

Cited By ~ 21

Author(s):

Dario Didona ◽

Giovanni Di Zenzo

Keyword(s):

Epitope Spreading ◽

Autoimmune Bullous Diseases ◽

Bullous Diseases

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Epitope spreading within lupus-associated ribonucleoprotein antigens

Clinical Immunology ◽

10.1016/j.clim.2005.07.002 ◽

2005 ◽

Vol 117 (2) ◽

pp. 112-120 ◽

Cited By ~ 36

Author(s):

Umesh S. Deshmukh ◽

Harini Bagavant ◽

Janet Lewis ◽

Felicia Gaskin ◽

Shu Man Fu

Keyword(s):

Epitope Spreading

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Long-term suppression of EAE relapses by pharmacological impairment of epitope spreading

British Journal of Pharmacology ◽

10.1111/bph.12525 ◽

2014 ◽

Vol 171 (6) ◽

pp. 1501-1509 ◽

Cited By ~ 8

Author(s):

L Cavone ◽

B Peruzzi ◽

R Caporale ◽

A Chiarugi

Keyword(s):

Epitope Spreading

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Generation of T-Cell Immunity to the HER-2/neu Protein After Active Immunization With HER-2/neu Peptide–Based Vaccines

Journal of Clinical Oncology ◽

10.1200/jco.2002.06.171 ◽

2002 ◽

Vol 20 (11) ◽

pp. 2624-2632 ◽

Cited By ~ 308

Author(s):

Mary L. Disis ◽

Theodore A. Gooley ◽

Kristine Rinn ◽

Donna Davis ◽

Michael Piepkorn ◽

...

Keyword(s):

T Cell ◽

Stimulation Index ◽

T Cell Immunity ◽

Colony Stimulating Factor ◽

Epitope Spreading ◽

Cell Immunity ◽

Her 2 ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

PURPOSE: The HER-2/neu protein is a nonmutated tumor antigen that is overexpressed in a variety of human malignancies, including breast and ovarian cancer. Many tumor antigens, such as MAGE and gp100, are self-proteins; therefore, effective vaccine strategies must circumvent tolerance. We hypothesized that immunizing patients with subdominant peptide epitopes derived from HER-2/neu, using an adjuvant known to recruit professional antigen-presenting cells, granulocyte-macrophage colony-stimulating factor, would result in the generation of T-cell immunity specific for the HER-2/neu protein. PATIENTS AND METHODS: Sixty-four patients with HER-2/neu–overexpressing breast, ovarian, or non–small-cell lung cancers were enrolled. Vaccines were composed of peptides derived from potential T-helper epitopes of the HER-2/neu protein admixed with granulocyte-macrophage colony-stimulating factor and administered intradermally. Peripheral-blood mononuclear cells were evaluated at baseline, before vaccination, and after vaccination for antigen-specific T-cell immunity. Immunologic response data are presented on the 38 subjects who completed six vaccinations. Toxicity data are presented on all 64 patients enrolled. RESULTS: Ninety-two percent of patients developed T-cell immunity to HER-2/neu peptides (stimulation index, 2.1 to 59) and 68% to a HER-2/neu protein domain (stimulation index range, 2 to 31). Epitope spreading was observed in 84% of patients and significantly correlated with the generation of a HER-2/neu protein–specific T-cell immunity (P = .03). At 1-year follow-up, immunity to the HER-2/neu protein persisted in 38% of patients. CONCLUSION: The majority of patients with HER-2/neu–overexpressing cancers can develop immunity to both HER-2/neu peptides and protein. In addition, the generation of protein-specific immunity, after peptide immunization, was associated with epitope spreading, reflecting the initiation of an endogenous immune response. Finally, immunity can persist after active immunizations have ended.

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