Article 3: Adenovirus armed with TNFa and IL-2 added to aPD-1 regiment mediates antitumor efficacy in tumors refractory to aPD-1

Question 1

what is the goal of this paper?

Answer 1

combine adenovirus OV + TNFa/IL2 + checkpoint inhibitors

• adenovirus based OV –> direct killing and immune recruitment
• TNFa –> tumor apoptosis and necrosis
• IL-2 –> T-cell activator
• Checkpoint inhibitors –> restore T-cell mediated tumor cell killing

more direct tumor killing + mroe T-cell mediated tumor cell killing

Question 2

how is adenovirus modified to be an OV?

Answer 2

• E2F promoter - delta24 of E1A - deltaE1B –> safety and selective cancer cell killing
• fiber with Ad3 domain –> improved cancer cell entry

Question 3

how is adenovirus modified to contain TNFa, why use TNFa?

Answer 3

in the E3 region of the genome –> local production of potent cytokines

TNF in being trialed as an anti-cancer agent because at high doses, it can promote tumor apoptosis and necrosis

Question 4

how is adenovirus modified to contain IL-2, why use IL-2?

Answer 4

in the E3 region of the genome –> local production of potent cytokines

IL-2 is being trialed as an anti-cancer agent because it can promote activation of T cells

Question 5

how is there resistance to anti-PD1?

Answer 5

1. t cells not recruited to tumor to begin with
2. immunoediting of tumor subclones –> no MHC positive signal
3. compensatory inhibitory signaling –> other inhibitory responses

Question 6

Q1: given the desire to see if OV can synergize with A-PD1, a model is needed that doesn’t response to A-PD1 montherapy. Is the B16F10 model refractory to A-PD1?

methods, results, conclusion?

Answer 6

methods

• tumor engraftment –> tumour reaches 4mm –> treatment period (PBS + A-PD1 treatment) –> tumor diameter reaches 10mm –> sacrifice and tumor harvest

results

• A-PD1 group took longer to reach tumor volume of control, but nonethelss got there

conclusion

• no long-term responses - similar to patients with single agent immune checkpoint inhibitors (ICI)

Question 7

Q2: what genes have significant expression changes in a tumor that has become resistant to A-PD1 therapy?

methods, results, conclusion?

Answer 7

methods

• tumor engraftment –> tumour reaches 4mm –> treatment period (PBS + A-PD1 treatment) –> tumor diameter reaches 10mm –> extract RNA from tumors –> whole genome RNA sequencing

results

• T cell effector function are downregulated
• B cell function are Treg/suppresor function are upregulated

conclusion

• there seems to be skew towards reduction of genes associated positively with cytotoxic T cell function

Question 8

Q3: can adenovirus coding for cytokines that enhance anti-tumor T cell activity induce responses in A-PD1 refractory tumors?

methods, results, conclusions?

Answer 8

methods

• tumor engraftment –> tumour reaches 4mm –> initial A-PD1 treatment –> tumor reaches 8mm (refractory) –> rescue treatment (+/- virus +/- A-PD1) –> measure tumor volume + plot survival

results

• A-PD1+virus significantly enhanced survival
• Ad/TNF/IL2 and Ad/TNF/IL2 + A-PD1 (even better) delayed tumor growth

conclusion

• since Ad/TNF/IL2 + A-PD1&raquo_space; Ad/TNF/IL2 alone, they might be complemtary:
• perhaps T cells werent in the tumor to begin with to be inhibited by PD1 and now the A-PD1 can help block inhibitory signals
• Ad/TNF/IL2 could lead to more recruitment and positive stimulation of T-cell and now the A-PD1 can help block inhibitory signals

Question 9

Q4: how does co-treatment with anti-PD1 and oncolytic adenovirus modify the tumor microenvironment in day 7 tumors?

method, results?

Answer 9

methods

• tumor engraftment –> tumour reaches 4mm –> initial A-PD1 treatment –> tumor reaches 8mm (refractory) –> rescue treatment (+/- virus +/- A-PD1) –> day 7: harvest –> mass cytometry (CyTOF)
• CyTOF: staining cells with metal-labeled Abs –> nebulization of single cells –> time of flight (TOF) mass spec –> single cell data analysis

results

• CD4+ T cell population: no differences between the three treatments)
• CD8+ T cell repopulation: freq of CD8+ T cells found in the tumor microenvironment were signficantly increased in the combination treatment
• immunosuppressive cell population: no sig change in Treg population; sig decrease in M2 macrophages and MDSC
• DC and M1 macrophage population: reduced DC population in the virus only and virus + A-PD1 groups; no differences in M1 macrophages

Question 10

what are the roles of M1 vs M2 macrophages vs myeloid derived suppressor cells (MDSCs)?

Answer 10

• M1: inhibit cell proliferation – anti-tumor (pro-inflammatory)
• M2: promote cell proliferation and tissue repair – pro tumorigenic outcomes
• MDSCs: suppress T cell and other immune effector cells