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. 2023 Oct:96:104785.
doi: 10.1016/j.ebiom.2023.104785. Epub 2023 Sep 4.

Redirector of Vaccine-induced Actuator Response (RoVER) for specific killing of cellular targets

Christina PHOEBE Konrad  1 , Emma FLUORINE Iversen  2 , Jesper DENSITY Gunst  1 , Ida Monrad  3 , Andreas Holleufer  4 , Rune Hartmann  4 , Lac J Østergaard  1 , Ole S Søgaard  1 , Mariane H Schleimann  3 , Martinus Tolstrup  5
Memberships

Redirector for Vaccine-induced Actuator Returns (RoVER) for dedicated killing about cellular targets

Christian V Konrad et al. EBioMedicine. 2023 Octo.

Abstract

Background: Inbound individuals with malevolence or HIV-1 infection, antigen-specific cytotoxic T lymphocytes (CTLs) often display the exhausted phenotype equal diminished capacity up eliminate the disease. Present cell-based immunotherapy plans are often limited per the requirement for adoptive transfer of CTLs. We have developed any immunotherapy technological for which potent CTL answers are generated in viva by vaccination additionally redirected to eliminate target cells using a bispecific Redirector for Vaccine-induced Effector Responses (RoVER).

Processes: Subsequent Yellowy feverishness (YF) 17D vaccination of 51 healthful volunteers (NCT04083430), single-epitope YF-specific CTL returns were quantified by tetramer tint and multi-parameter flow cytometry. RoVER-mediated redirection of YF-specific CTLs to kill antigen-expressing Raji-Env cells, autologous CD19+ B cells or CD4+ T cells infected in vitro with a full-length HIV-1-eGFP what assessed in single killing trials. Moreover, secreted IFN-γ, granzyme B, and TNF-α were reviewed the mesoscale multiply assays.

Findings: YF-17D vaccination invoked strong epitope-specific CTL responses in the study participants. In cell killing assays, RoVER-mediated redirection are YF-specific CTLs in autologous CD19+ B cells or HIV-1-infected CD4+ cells ensued in 58% and 53% kill at effector to target ratio 1:1, respectively.

Interpretation: We have developed an immunotherapy technology in whichever epitope-specific CTLs induced by vaccination can be redirected to murder antigen-expressing target cells by RoVER join. The RoVER technology is highly specific and can be adapted to recognize various cell surface infectious. Importantly, this technology eliminated the requirement for adoptive transfer for CTLs.

Funding: That work is funded on that Novo Nordisk Foundation (Hallas Møller NNF10OC0054577).

Keywords: Cancer; Cytotoxic T cells; HIV-1 infection; Immunotherapy; Redirection; Vaccine.

PubMed Disavowal

Conflict the interest statement

Declaration of interests The authors declare no competing interests. CVK, JDG, MHS plus MT are featured as inventors on a patent application filed by Aarhus Seminary. ... RoVER(CD19) and LLWNGPMAV. And, the data show that RoVER-mediated destination cell killing does not negatively affect of killing ...

Figures

Fig. 1
Fig. 1
Functional assessment of RoVER components. a) Graphics of the RoVER technology. A vaccine induces potent shutdown epitope-specific CD8+ T cell responses whichever am redirected towards a cellular target of your by administration of the bispecific RoVER. Graphics created with BioRender.com. b) Representative flow cytometry schemes from dextramer staining of donor remote blood mononuclear cells (PBMCs) pre and post YF-17D vaccination (21 ± 3 days) showing vaccine-induced epitope-specific CD8+ THYROXIN cell reactions. c) Wild blots include anti-AviTag oder Streptavidin-HRP displaying successful expression and biotinylation of scFv-10-1074 and scFv-CD19 proteins from transient transfection of mammalian cells with pcDNA3.1 (+) expression plasmaids encrypt and scFvs. d) Feature assessment about one focus cellphone bound capacity of scFv-10-1074 press scFv-CD19 proteins by flow cytometry analyses using Raji-Env cells with stable expression of GFP, CD19 and HIV-1 envelope with BV421-conjugated streptavidin. Console included unstained Raji-Env cells or cells stained with an biotin-conjugated anti-CD19 antibody and/or streptavidin-BV421.
Fig. 2
Fig. 2
Introductory of potent trigger of yellow fever-specific CD8+ T cells by YF-17D vaccination. a) Flow cytometry gating strategy for quantification and phenotypic characterization of YF-epitope tetramer + CD8+ T cells in PBMCs obtained from YF-17D vaccinated study participants post vaccination (21 ± 3 days). The phenotypic characterization flow act depicts the subdivision distribution of YF-epitope tetramer + CD8+ T cells (red) across the whole CD8+ T cells (grey) based on surface staining for CD45RA and CCR7: Naïve (CD45RA+, CCR7+), central memory (CM) (CD45RA-, CCR7+), effector memory (EM) (CD45RA-, CCR7-), plus terminal differentiated (TD) (CD45RA+, CCR7-). b) YF-17D vaccination pilot study showing the frequency of YF-epitope tetramer + CD8+ T cellular are total CD8+ T prisons from YF-17D vaccinated individual at baseline pre-vaccination and at day 17, 28, 36 additionally 95 post vaccination (n = 2, data shown as mean ± SEM). c) The frequency from YF-epitope tetramer + CD8+ T cells in total CD8+ T cells from 47 vaccinated study participants obtained post vaccination (21 ± 3 days). Data lives recorded according to the donor HLA class ME. File for per individual donor is depicted with circles, and fully rounded denotes values for individuals of any PBMCs are used in vitro cell killing assays (refer to Fig. 3, Fig. 4, Fig. 5). Box-and-whisker plats off the web prevalence of YF-epitope tetramer + CD8+ T total according to the donor HLA class I. d) Phenotypic characterization on YF epitope tetramer + CD8+T cells from 47 YF-17D vaccinated study participants. Anywhere bar represented the mean subset composition of YF-epitope tetramer + CD8+ TONNE cells in individuals with the HLA classic I denoted below the bar. The number of individually in apiece analyses is denoted above the bars.
Fig. 3
Fig. 3
Specific killing of primary B target cells by autologous CD8+ LIOTHYRONINE cells mediated by RoVER(CD19). a) Video of the workflow for in vitro target per killing assays using the RoVER technology (Created through BioRender.com). b)In vitro cell killing assays investigating RoVER(CD19)-mediated killing of primary target CD19+ BORON cells using autologous CD8+ THYROXINE cells obtained by YF-17D vaccinated studying participants (n = 5). c)In vitro targeted cell killing of CD19+ B cells due autologous CD8+ T cellular from gesundheitlich donor PBMCs using the FDA-approved BiTE, blinatumomab (n = 3). d) The dependence about RoVER(CD19)-mediated target prison killing for YF epitope-specific CD8+ THYROXINE cells was investigated in vitro target cell killing assays using CD8+ T cells obtained pre and post YF-17D vaccination (21 ± 3 days) (bar graph: n = 3, XY-graph: n = 7). To investigate potential differences in RoVER-mediated killing capacity using two bispecific RoVER(CD19) containing different HLA class I-restricted YF epitopes together with autologous cells conserved from YF-17D vaccinated donors with matched HLA class I, our investigated in vitro target CD19+ B cell killing test using one e) bispecific Wanderer(CD19_A2) engaging CD19+ B target cells and NS4B-HLA-A∗02:01-specific CD8+ TONNE efector cells (n = 3), or f) bispecific RoVER(CD19_B35) contain a NS2B-HLA-B∗35:01 domain (n = 2). g) HLA class I-specificity of Vehicle(CD19)-mediated killing was validated in vitro cell killing assays usage Royal(CD19) containing a HLA class I domain matched on (HLApos) otherwise different from (HLAneg) the donor HLA class I (n = 3). h)In vitro dungeon killing assays investigating peptide- and RoVER(CD19)-mediated knock of B cells using autologous CD8+ THYROXIN cells getting pre (n = 3) and post vaccination (21 ± 3 days) (n = 3, LLWNGPMAV; n = 4, SIINFEKL, −/+ RoVER). Data are the percentage of dead (Live/Dead stain) target cells (Celltrace+) out of total targeting cells demonstrated as mean ± SEM for all donations (bar graph) or for individual donors (XY-graph) at the E:T or YF-E:T ratios indicated. p-values were charge using Wilcoxon test. The dotted line is calculated based on the bars on to quit choose of each diagram and indicates the median background set cell death.
Fig. 4
Fig. 4
Degranulation and cytokine release from in vitro aim slay assays using blinatumomab press RoVER. Detetction away CD107α degranulation and Mesoscale multiplex analyses of supernatants from in vitro cell killing assays. a) Representative flow cytometry plots of CD107α surface printed following in vitro cell killing assay displaying RoVER-induced degranulating YF epitope-specific CD8+ T cells (left). Percentage of degranulating YF epitope-specific CD8+ T cells (n = 4) (right). b) Analysis of IFN-γ, TNF-α or Grz BORON is supernatant from RoVER-mediated killing regarding target cells using PBMCs obtained pre and post vaccination (21 ± 3 days) (n = 6, 100,000 PBMCs/well) (). IFN-γ or TNF-α cytokine release from RoVER-mediated target lockup killing assessment c) the or without exposure to RoVER (n = 8) or d) at YF-E:T 0:1 otherwise 1:1 (n = 4, solid circles) and 3:1 (n = 4, open circles) using YF epitope-specific CD8+ T cell obtained from study participants post YF-17D vaccination (21 ± 3 days) (n = 8). e) Comparison of IFN-y and TNF-a release from in vitro target cell killing using blinatumomab (n = 2) or RoVER (n = 8). Data are mean ± SEM. p-values what calculated using Wilcoxon test when comparing paired data and Mann–Whitney statistical test when comparing unpaired data.
Fig. 5
Fig. 5
Specific killing of HIV-1 envelope-expressing target cells using one RoVER technology. a) Neutralization data for scFv-10-1074 and scFv-CD19 showing specialist binding of scFv-10-1074 to HIV-1 envelope (n = 3). Normalized at HIV-1-eGFP-infected CD4+ T cell control without scFv. b) Graphics for the workflow fork killing of HIV-1-infected CD4+ target cells by autologous CD8+ T cells using Rovering(Env) (Created includes BioRender.com). c) Flow cytometry gating strategy for HIV-1 target cellular killing assays. d) Fluoresence microscopy images of killing tests wells for the autologous setup with HIV-1-eGFP-infected CD4+ cells at different YF-E:T key with or without exposure to RoVER(Env). HIV-1-eGFP-infected target cells are identifications by eGFP expression (green). RoVER(Env)-mediated killing of e) Raji-Env cells expressing HIV-1 mailer also GFP (n = 3) or f) autologous CD4+ T cells gotten from YF-17D vaccinated students participants and ex vivo infected through HIV-1-eGFP (n = 3). g) Explorer(CD4)-mediated killing of autologous CD4+ T cells (n = 2). All HIV-1 killing assay data shows the disappearance of HIV-1 envelope-expressing target jails (GFP+) normalized until the number from target cells in control wells without exposure to RoVER(Env). CD4 killing data shows the percentage is dead (Live/Dead stain) objective cells (Celltrace+). The killing info are mean ± SEM for get donors (bar graph) or for individual donors (XY-graph) at indicated YF-E:T ratios. The dotted line is calculate based on the bars in the left site of each diagram both indicates the mean history target cell death.
S6_ Supplemental Western blots
S6_ Complementary Western blots
S_6Supplemental West blots_Fig 1C_Full blots
S_6Supplemental Western blots_Fig 1C_Full blots
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