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This study is sponsored by bluebird bio
Treatment Group
mCD11c
F4/80
Transgene expression
T cell marker expression
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Key to Treatment Group: “c” = CAR-T alone, “cv” = CAR-T+NG-347
Chemokine genes (nCounter)N
T-SIGn cancer gene therapy and anti-EGFR CAR-T cells synergize in combination therapy to clear A549 lung tumor xenografts and lung metastases in NSG mice Olmo Sonzogni1, Daniel Zak1, Katy West2, Rochelle Lear2, Alice Muntzer2, Brian R. Champion2 and James B. Rottman1
BACKGROUND AND HYPOTHESISBackground➢ CAR-T cells are emerging as powerful treatments for hematologic malignancies but are
not yet effective for solid tumors due to many challenges that include:• Physical barriers that render the tumor difficult to infiltrate by CAR-T• Immunosuppressive tumor microenvironment (TME)
➢ T-SIGn (Tumor-Specific Immuno-Gene Therapy) viral vectors are derived from the oncolytic adenovirus enadenotucirev (EnAd) that are “armed” with transgenes.• Dosed systemically and replicate exclusively in tumor cells• Deliver transgene payloads selectively into TME• Currently being evaluated in multiple clinical trials
Hypothesis: T-SIGn vectors will enhance CAR T efficacy through direct tumor lysis and:1. Stimulation of the host innate immune response, thus promoting production of
proinflammatory cytokines and leukocyte recruitment
2. The release of tumor neoantigens, thus promoting activation of the adaptive immune system through epitope spreading
3. “Reprogramming” of the tumor microenvironment by directly delivering chemoattractant/immunostimulatory transgene molecules to further recruit and activate innate and adaptive immunity
Fig. 1. Schematic summary of potential T-SIGn and CAR-T synergy and interactions with the endogenous immune system to fight cancer.
Agents: CAR-T->human EGFR target human T cells, costimulatory molecule 4-1BB and CD3. T-SIGn vectors: → NG-347 (CD80, MIP1a, IFNa
transgenes); NG-641 (CXCL9 and 10, IFNa, hFAP-TAc*); EnAd (unarmed T-SIGn vector) (*hFAP-TAc is not active in mouse).
(1) T-SIGn REPROGRAMMING OF THE XENOGRAFT TUMOR MICRO-ENVIRONMENT
Digital spatial profiling (DSP) transcriptional analysis of xenografts from mice treated with the T-SIGn virus NG-347 demonstrates TME rewiring towards a proinflammatory environment
Fig. 2. A Samples for DSP analysis. B DSP technology https://www.nanostring.com/products/geomx-digital-spatial-profiler/geomx-dsp-overview/
A B C
D E overview (Nanostring, Inc.).C Xenograft regions of interest(ROI) selected for geneexpression analysis from anNG-347-treated mouse. DHeatmap & enriched pathwaysignatures of 110 significantlyinduced genes in NG-347 ROIsvs. controls. E Expressionprofiles of representative IFNresponse and antigenpresentation genes, stratifiedby virus transgene expression.
AACR 2021; Poster # 1582This study was sponsored by bluebird bio and PsiOxus Therapeutics
1Bluebird bio, Cambridge, MA, USA; 2PsiOxus Therapeutics, Oxfordshire, United Kingdom
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(2) T-SIGn FOLLOWED BY CAR-T CELLS RESULTS IN SYNERGISTIC ANTI-TUMORAL RESPONSE
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F. [email protected]. [email protected]
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IHC Adenovirus protein revealing level of infection in the xenograft at CAR-T day (D6)
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Figure 3. A Schema summarizing the experimental procedure. A549 cells were injected on the flank of NSG mice and allowed togrow until tumor volumes reached ~100mm3 (D0). Mice were then randomized and injected IV with NG-347 at D0 and D3 followedby CAR-T cells at D6. B IHC staining for adenovirus capsid at D6 (CAR-T administration day). C-E Biweekly tumor volumemeasurements reveal strong efficacy of the NG-347+CAR-T combination, even at CAR-T cells doses that are completely ineffective(C, D) or partially effective (E) when used as single agent. Veh=Vehicle, IV=intravenously, UTD=untransduced T cells, D=day
Combining T-SIGn NG-347 with EGFR CAR-T cells yields synergistic anti-tumor activity
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In this study, we assess anti-tumor synergy in the A549 xenograft model between the T-SIGn oncolytic virus and an anti-EGFR CAR-T
Immunostimulatory T-SIGn transgene cargo plays a critical role in the anti-tumor synergy between EGFR CAR-T and NG-347
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Figure 4. To assess the contribution of the immunostimulatory transgene cargo to the anti-tumor synergy of the NG-347+CAR-Tcombination, the A549 xenograft experiments were repeated with different T-SIGn variants. As before, NSG mice were inoculated withA549 cells and once the tumors reached ~ 100mm3 animals were randomized and injected IV with vehicle (blue) or (green) NG-347(which expresses IFNa, MIP1a, CD80), (purple) NG-641 (which expresses CXCL9/ 10, IFNa, inactive hFAP-TAc*) or (red) EnAd (notransgene) at D0 and D3. At D6 either vehicle (B), UTD (C), or CAR-T cells at 4e6 (D) or 1e6 (E) were administered. CAR-T cells onlyincreased the efficacy of the T-SIGn viruses that expressed immunostimulatory transgenes (NG-347 and NG-641).
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(4) ROLE OF T-SIGn TRANSGENE CARGO IN THE SYNERGISTIC ANTITUMOR ACTIVITY OF THE NG-347+CAR-T COMBINATION
Transcriptional analysis of xenografts at D13 (D7 post CAR-T) demonstrates robust NG-347-driven immune cell recruitment and activation within thexenograft, providing a mechanistic basis for the enhanced efficacy observed in the mice treated with the NG-347+CAR-T combination
(3) T-SIGn VECTORS ENHANCE ACTIVATION OF CAR-T CELLS RESULTING IN ENHANCED EFFICACY AGAINST PRIMARY TUMORS AND METASTASES
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Figure 4. A Experimentalprocedure. Mice wereeuthanized at D13 (7 dayspost CAR-T, n=3/group) for
RT-qPCR Data
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CD80-2A MIP1a-2APenton IFNa2
Virus Capsid Protein
H
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• The T-SIGn family of armed oncolyticadenoviruses is currently beingevaluated in human clinical trials(administered to more than 150patients)• The combination of T-SIGn + anti-EGFRCAR T cells showed a synergistic effectby clearing A549 xenografts andinhibiting lung metastases even at CAR Tcell doses that were found completelyinefficacious as single therapy (Fig.3&4)• Transcriptional analysis of thexenografts reveal the role of the T-SIGnvector in preparing the niche for T cellsactivation by rewiring the TME toward amore stimulatory/T cell friendlyenvironment (Fig.2) resulting in a strongactivation signature within the T cellcompartment and recruitment of innateimmune cells (Fig.3).• Inflammatory transgene expressiondelivery by the T-SIGn virus was foundto be essential for the enhanced efficacyof the CAR-T cells/T-SIGn combination(Fig.4)
T-cell activation genes (nCounter)M
DSP Image
NG-347+CAR-T
Morphology markers: mCD11b, CD45, DNA
CAR-TNG347+CAR-TmCD11b CD45 DNA
CONCLUSIONS
Watanabe, N. et al. (2020). Molecular Therapy, 29(2). https://doi.org/10.1016/j.ymthe.2020.10.023
transcriptional analysis (E to N), confirming synergistic impact of NG-347+CAR-T on tumor growth (B), and assessing metastasis (C& D). (C) D13 or (D) endpoint quantification of metastatic foci spontaneously arising from sub Q A549 primary tumors indicatessynergistic anti-metastases impact of NG-347+CAR-T. Endpoint was defined by primary tumor burden and is indicated on top of thecolumns. Lung metastases were quantified by enumeration of foci detected by pan-keratin IHC on a single cross-section across alllung lobes. E Representative xenograft ROIs from a NG-347+CAR-T treated mouse selected for DSP analysis. F Correlationsbetween NG-347 transgenes (left) and T cell markers (right) allow stratification of ROIs in terms of NG-347 transgene & T cellmarker gene expression (G). H Correlation (99%CI) between T cell markers and mouse innate genes shows co-recruitment of thecompartments for the NG-347+CAR-T treated xenografts but not for CAR-T cells alone. I Representative images showing co-recruitment of T cells (CD45) and mouseinnate cells (CD11b) for NG347+CAR-T (left) but not for CAR-T alone (right). J DSP results showing normalized counts for F4/80 (upper panel, mouse macrophages))and mCD11c (lower panel, mouse dendritic cells) for the T cells/Virus groups defined in G. Notably categories with med to high T cells and virus show higher F4/80and CD11c counts. K Differential expression and pathway enrichment analysis of stratified ROI groups reveals robust innate immune and T cell activation in T cellinfiltrated ROIs expressing NG-347 transgenes. L Detection of viral capsid and transgene mRNAs by RT-PCR on the D13 samples. M and N nCounter analysis of wholexenograft slides confirms DSP results showing robust T cell (M) and chemokines (N) gene up-regulation in xenografts from mice treated with the NG-347+CAR-Tcombination.
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Log2 fold-change compared to average of “No Virus/Low T cells” group for Vehicle+CAR-T
