Article 1: an OV expressing a T-cell engager simultaenously targets cancer and immunosuppressive stromal cells Flashcards
what is the idea of this paper?
combine adenovirus OV + BiTE-directed removal of cancer associated fibroblasts –> direct oncolysis + disrupt TME –> tumor clearance
what is the significance of cancer associated fibroblasts?
CAFs –> cancer cell survival and proliferation + immune cell suppression + endothelial cells activation and proliferation –> poor prognosis
CAFs are enriched for fibroblast activation protein, describe its expression pattern and the conditions where it’s a good target
FAP is expresses on cancer but some other cells
- in carcinomas: FAP expression on activated stromal fibroblasts of more than 90% of all human carcinomas
- FAP expression unser physiological conditions is very low in the majority of adult tissues
- FAP is nevertheless expressed during: embryonic development, in pancreatic alpha cells in multipotenet bone marrow stromal cells (BM-MSC), in uterine stroma, high in tissues of healing wounds and thoughh to be involved in development and tissue repair
therefore, FAP is not good to target unless it is only in the tumor –> using OV to target tumor
Describe how bispecific T-cell engager technologies guid T-cell directed killing
BiTE contains the VL and VH of a FAP-specific Ab conjugated to the VL and VH of CD3-specific Ab –> cross link the TCR –> intracellular signaling –> T cell activation
Which OV is used, what are the modifications?
EnAd (A11/Ad3 chimeric adenovirus)
- Ad11p backbone –> binds CD46 receptor (common on cancer cells)
- entire E3 immunomodulatory region deteled – > elimate immune regulation
Q1: can FAP-BiTE containing supernatants activate primary human T cells and cause cytotoxicity to FAP+ cells?
what is the appoach?
- transfect 293 cells with a BiTE-expressing plasmid –> supernatant contains secreted BiTEs
- transfect Cho cells with a FAP-expressing plasmid –> FAP expressed on cell surface
- add supernatant + control cells/FAP-cells +/- peripheral blood mononuclear cells (PBMCs)
- questions: are Tcells in PBMCs activated (use FCM to detect early t-cell activation marker CD69 and late t-cell activation marker CD25) + are FAB-Cho cells dying (damaged cells release LDH which will form a pink product called resoruffin)?
Q1: can FAP-BiTE containing supernatants activate primary human T cells and cause cytotoxicity to FAP+ cells?
what are the results and conclusions?
- activated T cells only when FAP BiTE supernatants and FAP-expressing CHO are present with PBMCs
- cell damage only when FAP BiTE supernants and FAP-expressing CHO are present with PBMCs
- also true when they substiuted FAP-Cho cells with normal human dermal fibroblasts (NHDF) express FAP if cultured in high serum (IFNg = activation
- conclusion: FAP-BiTES activate primary human T cells and cause cytotoxicity to FAP+ cells – also specific since not when FAP surface expression, FAP-BiTE or T cells left out
Q2-1: can BiTEs be introduced into EnAd wihtout loss of virus replication and direct cancer cell killing?
what promoters are used and why, and what methods did they use to measure this? Result and conclusion?
promoters
- CMV –> constitutive expression
- SA = splice acceptor for Ad major late promoter –> cells must be permissive to Ad replication to express via SA
approach
- DLD colorectal cancer cells + virus –> qPCR to measure virus genome copy number + MTS assay to measure viable cells (live cells convert MTT to formazan –> colour)
results
- qPCR: virus still replicates post infection
- MTS assay: virus still reduces cell viability directly
conclusion
- EnAd-expressing FAP-BiTE still replicate and kill cancer cells directly
Q3: is the activity of EnAd-SA-FAP-BiTE specific and depending on having the cancer cells there?
what is the approach, results and conclusions?
approach
- A: PBMC/Tcells, FAP-NHDF, +/- EnAd +/- BiTE CMV or SA, no tumor cells
- B, C, D: PBMC/Tcells, FAP-NHDF, +/- EnAd +/- BiTE SA only, normal human bronchial epithelial cells (NHBE) OR colon cancer cells (SKOV3)
results
- A: EnAd-CMV-FAP-BiTE –> cytoxicity compared to EnAd-SA-FAP-BiTE
- B, C, D: EnAd-SA-FAP-BiTE –> increase CD69+ and CD25+ T cells and only kills SKOV3 cells
conclusion
- when under the ad late promote (SA), T-cell killing of FAB-expressing cells depends on tumor cell co-culture
Q2-2: Do BiTE expressing EnAd stimulate T-cell killing of FAB+ cells?
what is the approach? results? conclusions?
approach: combined the following components and used xCELLigence
- xCELLigence: flow electrons through culture medium with cells – more cells = impeded elctron flow
- SKOV3 colon cancer cells –> let the virus replicate and secrete BiTEs
- +/- T cell
- +/- EnAd with +/- BiTE under CMV vs SA promoters
- NHDF (express FAP) – to monitor FAP-dependent killing
results
- without T cells NHDF cells grow
- with T cells NHDF cells die when treated with EnAd-CMV-FAP-BiTE and EnAd-SA-FAP-BiTE
conclusion
- CMV and SA based EnAd-expressing FAP-BiTEs induce T-cell dependent cytotoxicity of FAP-fibroblasts
Q4: does EnAd BiTEs work in presence of immunosuppressive patient ascites?
what are the properties of malignant ascites? what is the approach?
malignant ascites tend to be immunosuppressive –> would the BiTEs still work in an immunosuppressive environment –> approach:
- test ascites to find samples that are immunosuppressive
- add these to the system and see if BiTEs still work
Q4-1: does FAP-BiTEs work in presence of immunosuppressive patient ascites?
what is the approach, results and conclusion?
approach
- A: PBMC T cells +/- anti-CD3/28 (stimulate T cells) +/- ascites
- B: PBMC T cells + FAP-NHDF +/- FAP-BiTEs +/- ascites
results
- A: ascites 2,3,5 have inhibition activity on T cell activation by anti-CD3
- B: yet, these ascites do not inhibit BiTE-mediated activation of T-cells
conclusion
- FAP-BiTES work in presence of immunosuppressive ascites
Q4-2: does EnAd FAP-BiTEs work in presence of immunosuppressive patient ascites and do they change immunosuppressive markers like TGFB?
what is TGFB associated with and what does a fold-change in TGFB speak to? approach, results and conclusion?
TGFB: associated with immunosuppression, FOLD-change in TGFB speaks to change in overall immunosuppression
approach
- T cells + ascites (also has cancer cells) +/- EdAd BiTE
results
- EdAd-FAP-BiTE reduces TGFB levels (also increase activated T cells and cytotoxicity)
conclusion
- EnAd BiTEs work in presence of ascites and reduce immunosuppressive markers like TGFB
Q5: what other changes occur in ascites with FAB-BiTEs?
how does gene expression profiling by nanostring technology work? what are the benefits and drawbacks?
nanostring tech: kinda like an ELISA but using mRNA to detect mRNA
have an RNA capture probe stuck to well + target mRNA –> wash –> reporter probe mRNA –> target-probe complex
benefits: direct detection of mRNA without needing to make cDAN – much faster and cheaper
drawbacks: only detects selected genes of interest rather than whole-genome analysis
Q5: what other changes occur in ascites with FAB-BiTEs?
approach, results, conclusion?
approach
- ascites +/- Ad-SA-FAP-BiTE –> nanostring to determine changes in the expression of 730 cancer and immune pathway genes in three primary ascites samples
results
- decrease in CAF genes (probably dying)
- increase T cell traffic and Ag presentation (enhance immunogenicity)
- increase T cell function genes (enhance immunogenicity)
conclusion
- gene changes look supportive of pro-immunogenic activites
