Hinson, Destin T

Background/Objectives: Antigen presenting cells (APCs) and immune cells have unique properties to drive or suppress immune responses. They are therefore key targets for the expression of vaccine antigens or transgene proteins. To better determine the utility of different molecular therapies to modify these cells, mRNA and DNA-based molecular therapy vectors were compared for their ability to genetically modify immune cells after intradermal injections in mice. DNA-based vectors included naked plasmid DNA, plasmid packaged in lipid nanoparticles (LNPs), and replication-defective adenovirus (Ad) vectors. mRNA delivery was mediated by packaging into LNPs like those used in COVID-19 vaccines. Methods: Each vector was used to deliver Cre recombinase into Cre reporter mice whose cells were activated to express green fluorescent protein (GFP) and firefly luciferase after Cre recombination. The mice were injected intradermally (ID) near the base of their tail at a site that drains into the inguinal lymph node. Luciferase activity was imaged in the living mice 1 or 4 days after vector injection. The animals were then euthanized, and luciferase activity was imaged in the draining inguinal lymph node. Cells were prepared from the intradermal injection site and from the draining lymph node to determine which immune cells were genetically modified by phenotyping CD45, CD3, and CD11b GFP-positive cells by flow cytometry. Given that the skin uniquely contains Langerhans dendritic cells, these CD207+ cells were also phenotyped in skin samples and in the draining lymph node. Results: In both the skin and in the draining lymph node, the rank order of luciferase and GFP activation by the vectors were: (1) Ad; (2) mRNA-LNP; (3) DNA-LNP; and (4) naked DNA. Only mRNA-LNP and Ad vectors mediated obvious luciferase activity in the living animals and in the draining lymph nodes by imaging. Notably, both vectors appeared to leak from the ID injection site and not only modify the draining lymph node but also strongly modify the livers of the mice. Naked DNA and DNA-LNP mediated detectable GFP activation in the skin and draining lymph node in some mice, but this activity was low and did not reach statistical significance when compared to PBS-treated animals. mRNA-LNPs and Ad both mediated significant Cre delivery in CD45+, CD3+, CD11b+, and CD207+ immune cells in the skin and in the lymph node, with adenovirus mediating consistently higher levels of expression in all of the tested cells. Conclusions: These data indicate that mRNA-LNP and Ad vectors mediate stronger modification of skin and lymph node immune cells after intradermal injections. Naked DNA and DNA-LNPs were markedly less potent at this activity than the other vectors. These data are consistent with the higher vaccine potency of mRNA-LNP and Ad vectors and suggest that approaches that increase targeting of immune cell subsets may have utility to increase efficacy while also reducing off-target modification of tissues like the liver.

Abstract Description Therapeutic in situ chimeric antigen receptor (isCAR) T cells can be generated by targeting activated T cells within a lymph node (LN) with retroviruses encoding CARs. While isCAR T cells are successfully generated by intradermal LN priming using adenovirus encoding allogeneic MHC in all mouse lines tested, differences in emerging therapeutic peripheral isCAR T cells are found in a strain dependent manner. Potentially harmful off target retroviral transduction of B cells with CARs is observed within the targeted LN. Here we report the blockade of B cell transduction using anti-CD20 antibody B cell depletion with the introduction of anti-CD19 chimeric antigen receptors into the activated CD8+ T cells. Lymphocytes were analyzed in blood and LN following isCAR T cell generation. When B cells were depleted by systemic administration of anti-CD20 antibodies during isCAR T cell induction, >97.5% systemic depletion of B cells was achieved. Whereas very small numbers of CAR transduced B cells were found in the blood of mice not treated with anti-CD20, none were observed in animals systemically depleted of B cells. In contrast, CAR B cells persisted in the LN of local anti-CD20 treated animals. However, animals expressing dominant CD8+ isCAR T cell populations eliminated LN B cells expressing CARs by week two. Furthermore, systemic treatment of lymphoma in animals with anti-CD20 antibody complemented anti-CD19 isCAR T therapy while blocking off target B cell transduction. Funding Sources Mayo Clinic Ventures Mayo Clinic Graduate School of Biomedical Sciences Mayo Clinic Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Abstract Description This study dissects mechanisms of spontaneous tumor rejection leading to better understanding of successful immune responses to cancer. Cancers persist despite expression of tumor-specific neoantigens promoting ongoing anti-tumor immune responses. While some occult tumor cells likely are cleared by effective anti-tumor immune responses, the targeted antigens are not easily identifiable as those cells spontaneously disappear. Congenic mouse breast carcinoma models expressing a defined protein mimicking neoantigens were used to dissect the protective spontaneous immune response. BALB/c (MHC H-2d) mice challenged with BALB/c breast tumors expressing the rat-erbB2 oncoprotein succumb to their tumors despite demonstrated tumor immunogenicity, mimicking known patient responses to cancer. Meanwhile, H-2b positive BALB.B and H-2d/H-2b F1 hybrid mice spontaneously eliminate genetically matched tumors in an MHC-II dependent manner. CD4+ T-cells and B-cells or the rat-erbB2-specific antibodies they produce are crucial mediators of this protective response. Differences in the tumor microenvironments in rat-erbB2+ tumor-bearing BALB.B and BALB/c mice at the time of fate decision in the models reflect the differences between effective and ineffective anti-tumor immunity. Identifying the nature of the optimal MHC-II antigens, how they activate protective CD4+ T-cells, and the interactions that promote protective antibodies will better inform future cancer therapies. Funding Sources Bernie Fineman Research Fund Mayo Clinic Center for Biomedical Discovery Mayo Clinic Department of Immunology Mayo Clinic Graduate School of Biomedical Sciences NIH Traning Grant 5 R25 GM 55252 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description Antigen receptor (CAR) T cells can be generated in vivo within the natural lymph node (LN) environment as the immune response to a virus infection unfolds. This in situ approach entails priming LNs with intradermal inoculations of replication defective adenoviruses encoding MHC class I and class II allo/xeno-antigens. Universal transplant antigens were devised for LN priming by introducing tryptophan into amino acid position 162 of the MHC HLA-A2:01 class I heavy chain and by using the mouse MHC class II beta chain to form chimeric class II antigens with endogenous class II alleles. The virus encoded mismatched MHC antigen presenting molecules are expressed functionally in the LN following intradermal inoculation provoking polyclonal T cells responses. Three days later CAR T cells are generated by directly injecting retroviruses encoding CARs of interest into the responding LN. Mouse CD19-specifc CAR T cells created in this manner in B6-DQ8 and DQ8/A2 mice effectively target endogenous B cells and are therapeutic in male and female Emu-myc lymphomas demonstrating the potential for the generating functional CAR T cells directly in vivo. While in situ CAR T cells are readily produced in B6, B6-IA0/DQ8, B6-IA0/DQ8/A2, and B6-IA0/DQ6 LNs, functional differences among the strains emerged in the periphery suggesting that properties of the autoimmune-associated HLA DQ8 class II allele is particularly reactive during the production of CAR T cells in vivo. Funding Sources MustangBio, Inc Mayo Clinic Center of Biomedical Research Mayo Clinic Department of Immunology Mayo Clinic Graduate School of Biomedical Science Fraternal Order of Eagles Mayo Clinic Ventures NIH R21 AI 097337 NIH R01 CA 178804 NIH T32 AI 07425 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

ABSTRACT Brain atrophy is a common feature of neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and more recently SARS‐CoV‐2 infection for which the neuroinflammatory mechanism is not fully understood. Nevertheless, neuroinflammation and CD8 T cell accumulation is frequently observed in postmortem brain tissue of patients with neurodegeneration. We therefore developed a murine model of brain atrophy using the Theiler's murine encephalomyelitis virus (TMEV) infection model of multiple sclerosis. We employ single major histocompatibility complex (MHC) class I molecule conditional knockout mice generated by our program to analyze the contribution of immune cell infiltration and onset of atrophy. TMEV infected C57BL/6 mice that singularly express H‐2K b or H‐2D b were evaluated. TMEV infection of these mice resulted in ventricular atrophy at 14 d.p.i. However, H‐2D b expressing mice presented with significantly greater and continuous ventricular atrophy compared to H‐2K b expressing mice and MHC class I knockout control mice. Flow cytometric analysis revealed H‐2D b expressing mice had greater brain infiltrating CD8 T cell responses at 7 and 28 dpi which correlated with the extent of ventricular atrophy. Meanwhile, H‐2K b and MHC class I knockout mice also had distinct positive correlations with ventricular atrophy and global neuroinflammation in general. These findings support the role of immune cell infiltration into the CNS as a putative mechanism of ventricular atrophy following TMEV infection, with the progressive enlargement of ventricular atrophy observed in H‐2D b mice being associated with the presence of long‐lived memory CD8 T cells residing in the brain.

Background Cancers persist despite expression of immunogenic neoantigens and ongoing antitumor immune responses. While some occult tumors likely are cleared by effective antitumor immune responses, the targeted antigens are not easily identifiable as those tumors spontaneously disappear. Methods We used mouse models with a defined antigenic protein mimicking tumor-specific neoantigens to address the nature of these spontaneous anti-tumor immune responses. Results BALB/c (H-2d ) mice challenged with BALB/c breast tumors expressing the rat-erbB2 oncoprotein succumb to their tumors despite ongoing immune responses targeting tumor-specific model antigens. Meanwhile, congenic BALB.B (H-2b ) and H-2d/H-2b F1 hybrid mice spontaneously eliminate genetically matched tumors in a major histocompatibility complex (MHC)-II dependent manner. Adoptive transfer and immune cell depletion strategies revealed CD4+ T cells and CD20+ B cells are crucial mediators of the protective response in H-2b mice. Furthermore, passive transfer of immune serum from mice rejecting their tumors confers resistance in tumor antigen-tolerant animals with an inversely proportional relationship between tumor outgrowth and the amount of rat-erbB2 specific antibody present in tumor-bearing mice. Introduction of the rat-erb2 ectodomain into other H-2b tumor models also promotes their spontaneous tumor rejection. Notably, the tumor microenvironments differ in rat-erbB2+ tumor-bearing BALB.B and BALB/c mice at the time of fate decision in the models reflecting the differences between effective and ineffective tumor immune responses. Conclusions We find that the effective antitumor immunity targeting neoantigens in these breast cancer models is determined by MHC-II-restricted presentation of optimal cancer-associated antigens. These responses are dependent on CD4+ T cells, B cells, and antigen-specific antibodies.