resistance to immunotherapies

Question 1

mechanisms of resistance?

Answer 1

1. loss of antigen presentation (loss/downregulation of target antigen, loss of HLA expression through gene silencing, mutations in AP components)
2. alteration of signalling pathways (e.g. less IFNy, more Wnt, MAPK, PI3K signalling)
3. changes to tumour microenvironment
4. constitutive PD-L1 expression

Question 2

loss of antigen presentation

Answer 2

• Loss/downregulation of target antigen
• Loss of HLA expression through gene silencing
• Mutations in components required for antigen presentation = e.g. B2M, TRIM69, TAP

Sade-Feldmann et al (2017)
• Performed whole-exome sequencing on tumour samples from 17 metastatic melanoma patients treated with checkpoint blockade
• Mutations, deletions or LOH in B2M observed in 29.4% of patients with progressed disease (either pre-treatment samples from non-responders, or post-progression samples from patients who initially responded then relapsed)
• B2M mutations not seen in any samples from responders
• Also found B2M LOH was enriched threefold in non-responders, compared to responders, in larger cohorts of melanoma patients treated with anti-CTLA4 or anti-PD1 (30% vs 10%)  associated with poorer overall survival
• Loss of both copies of B2M only found in non-responders
• Evidence that B2M-mutant clones are selected through immunoediting early in tumour development, or under selective pressure applied by treatment

Question 3

defects in signalling pathways

Answer 3

2. Defects in signalling pathways
   • E.g. downregulation/mutation of mediators involved in IFNy pathway = protects from anti-tumour effects of IFNy, produced by tumour-specific T cells (e.g. direct pro-apoptotic and anti-proliferative effects, recruitment of other immune cells)
   • Can occur following continuous IFNy exposure  immunoediting of cancer cells  immune escape
   • Gao et al (2016) = highlighted IFN defects as key mechanism of TIR
   - Used WES to evaluate genomic changes in melanoma tumours from patients treated with anti-CTLA-4  revealed higher frequency of copy number alterations (CNAs) involving IFNy pathway genes in non-responders vs responders
   - Only in a very small cohort of patients  needs to be validated in larger sample
   - Then used shRNA to decrease expression of IFNGR1 in murine melanoma cell line  attenuated anti-proliferative and pro-apoptotic effects of IFNy in vitro, impaired response to CTLA-4 therapy in vivo

Question 4

changes to tumour microenvironment

Answer 4

3. Changes to tumour microenvironment
   Myeloid-derived suppressor cells
   - Immunosuppressive cells implicated in driving angiogenesis, tumour cell invasion and metastases
   - Presence of MDSCs in tumour correlates with decreased efficacy of immunotherapies (e.g. Meyer et al (2014) = CI), and reduced survival (e.g. Solito et al, 2011)
   - Eradicating/reprogramming MDSCs could provide means to increase clinical responses to immunotherapy in resistant patients

Kaneda et al (2016) = focused on effect of selectively inhibiting macrophage PI3Ky  induces transcriptional programme that promotes immune suppression and differentiation of MDSCs

• Inhibition of PI3K (either KO or using PI3K antagonists) suppressed tumour growth and metastases, and enhanced survival in murine models of HNC, breast and lung carcinoma
• Genes associated with immune activation were upregulated in macrophages treated with PI3Ky inhibitors, or those from mice lacking PI3K
• Improved tumour rejection and survival of tumour-bearing mice when used in combination with anti-PD1 (vs anti-PD-1 alone) in murine tumour models = synergistic
• Further data required to deduce efficacy in human patients = early clinical trials indicate high rate of toxicity

Question 5

microbiome

Answer 5

A less obvious prognostic marker for checkpoint inhibitor response is the microbiome. Certain bacterial species have been implicated in carcinogenesis, and emerging evidence is suggesting that different species may influence the outcome of treatments, both classic chemotherapeutics, as well as immunotherapy. Routy et al treated MCA-205 sarcoma and RET melanoma mice with a 14-day course of broad-spectrum antibiotics in a germ-free environment, and found a significant reduction in PD-1 and PD-1/CTLA-4 treatment efficacy21. Analysis of clinical data from 249 carcinoma patients treated with checkpoint inhibitors found antibiotic treatment in the 2 months before or one month after initiation of checkpoint inhibitor therapy led to a significant reduction in progression free survival. They went on to show faecal transplantation from responsive patients restored checkpoint inhibitor sensitivity in antibiotic treated mice.

Question 6

how does microbiome influence immunotherapy responses?

Answer 6

The exact mechanism of how the gut microbiome influences checkpoint inhibitor response remains unclear. One suggestion is that the microbiome influences the diversity of T-cells and other immune cells arround the body, thus influencing the tumour microenvironment, and ultimately treatment efficacy. Tanoue et al identified 11 commensal species that increase INF-gamma producing CD8 T-cell levels and found inoculation of germ-free mice with these strains increased efficacy of anti PD-1 treatment, supporting the immune modulation theory

Question 7

TGFB

Answer 7

Factors released from many tumours lead to state of relative local immunosuppression
Induction of cancer-associated fibroblasts form ECM which binds antigens
Influx of inflammatory monocytes = adopt M2 macrophage profile, secrete pro-angiogenic factors, anti-inflammatory cytokines (IL-10 = dampen T cell function)
TGFB secreted by fibroblasts and M2 macrophages = leads to T cell immune exclusion
Inhibits cytotoxic T cell proliferation
Pro-myeloid differentiation
Defining feature of poor prognosis in colorectal cancer subtype called CMS4
Tauriello et al (2018) = inhibiting TGFB in mouse model of colorectal cancer could allow breakdown of ECM, access of T cells to tumour and much better response

Question 8

IL-8

Answer 8

Baseline IL-8 in blood
Schalper et al (2020) = elevated serum IL-8 at baseline consistently associated with negative prognosis with immunotherapy, across different clinical trials in different tumour types
Associated with immunosuppressive profile in tumour and blood = reduced T cell and IFNy signatures in tumour
Causative or correlative?