cGAS puts gas on anti-tumor effect of checkpoint inhibitor

This week PNAS published new article explaining mechanism of action of checkpoint inhibitors, such as anti-PDL1, in tumor immunity. It shows that cytoplasmic DNA sensor cGAS/STING pathway synergize with anti-PDL1 therapy in mouse model of melanoma.

While this study provides noteworthy observation, it is poorly done. For example, when comparing WT and KO mice (cGAS or STING KO mice), the authors did not mention if they used littermate control in these experiments.

In addition, when the authors used exogenous cGAMP (product of cGAS activity detected by STING), they did not use it on cGAS or STING KO mice as controls to verify relationship between cause and effect.   

    

In summary, the connection between DNA recognition system and checkpoint inhibitors is very interesting. Whether it is a simple generic augmentation of T cell priming or specific stimulation of tumor-specific T cells is to be seen.   

David Usharauli

Changes in tumor-specific antigenicity overtime in melanoma patients undergoing adoptive T cell therapy

Most tumors, and most likely "all" tumors, express one or more non-synonymous somatic mutations that generate sufficient number of novel HLA/peptides that potentially could be targeted by T cells. However, similar to infections, when under immune pressure tumor can also develop escape variants by modulating its HLA/peptide landscape. 

This is what new study published in journal Nature found in tumor patients. For this study, the authors (who are associated with AIMM Therapeutics) sequenced patients tumor mRNA at different time points (before or after adoptive cell therapy) and found that some of the tumor-specific HLA/peptides had disappeared [overtime] and some new tumor-specific HLA/peptides became enriched instead. 

Of note, within each patient almost all of T cell reactivity to tumors were patient-specific (i.e. private HLA/peptide) and did not share sequence similarities with publicly known shared tumor-associated epitopes. For example, analysis of patient's CD8+ T cell specificity against a panel of >200 MHC-multimers containing known shared tumor-associated epitopes demonstrated that only 1.24% of the CD8+ T cells responded to three different "shared" gp100 epitopes. The rest were patient's specific. 

For this study two melanoma patients had their tumors mRNA sequenced and underwent autologous adoptive tumor-specific T cell therapy. In one patient, initial tumor mRNA sequencing revealed  501 non-synonymous mutated genes. Most of CD8+ T cell reactivity were directed to two HLA/peptides complexes: KIAA0020 p.P451L (KIA P>L) and ribosomal protein RPL28 p.S76F (RPL28 S>F).

Around 1 year later, however, patient's CD8+ T cell reactivity was only observed against the KIAP>L neo-antigen, and not against RPL28S>F and tumor mRNA sequencing confirmed absence of mutant allele encoding the RPL28S>F neo-antigen within tumors harvested at a later time.

Similarly, in an another patient undergoing similar procedures, the authors found that initially melanoma patient's CD8+ T cells were specific for the neo-antigens echinoderm microtubule associated protein like 1 p.R64W (EML1R>W), Septin-2 p.R300C (SEPT2R>C), and CAD protein p.R1854Q (CADR>Q).  

However, later analysis revealed no significant T cell reactivity against EML1R>W, CADR>Q neo-antigen and SEPT2R>C neo-antigens. Instead, it showed T cell response towards the Programmed Cell Death Protein 10 p.P28S (PDCD10P>S) neo-antigen. Indeed, tumor mRNA sequencing confirmed that while mutant allele encoding the SEPT2R>C neo-antigen that was present in the original tumor, it was selectively lost in the tumor samples harvested later, and that RNA for novel PDCD10 neo-antigen had increased > 40-fold in later time points instead.

These data points to two important considerations: first, tumor undergoes changes in their mutational landscape and can evade T cell detection and second, most tumor mutations are patient's specific. This means that adoptive immunotherapy using single T cell specificity would be less efficient overtime and that tumor vaccines using "generic-shared" tumor epitopes would provide no benefits in most patients. 

David Usharauli

      

Combination of checkpoint inhibitor and IL-21-primed melanoma-specific T cells produced durable response, "cure", in melanoma patient

This week Journal of Experimental Medicine published a case study of a single melanoma patient who achieved durable response (>5 years), "cure", after combination therapy (IL-21 primed MART1-reactive "polyclonal" CD8+ T cells and Yervoy). 

Of note, earlier attempts to stop melanoma progression in this patient with IL-2/IL-7/IL-15 primed MART1-reactive "monoclonal" CD8+ T cells and/or Yervoy were not successful. As seen before, successful anti-melanoma immunotherapy produced autoimmune skin/hair disorder, vitiligo (loss of melanocytes). 

As the cellular level, IL-21 priming resulted in better survival of infused polyclonal T cells and epitope spreading targeting other melanoma antigens such as NY-EOS1, gp100, tyrosinase and MAGE-A3.

In summary, this study clearly shows the vast [not yet fully tapped] potential of cancer immunotherapy.

David Usharauli

 

Tumor endothelial cells, not DCs, initiate anti-tumor IFN-β response to STING agonist

PNAS has published very interesting study in tumor immunity. Some of you might know endogenous DNA recognition complex made of cGAS-STING-IRF3 axis plays an important role in spontaneous anti-tumor activity observed in clinics in some cancer patients.

Immunologists already knew few years back that type I IFN system, rather than TLR system, facilitated natural, spontaneous priming of endogenous anti-tumor T cells. However, they did not know how it worked. Only after discovery of STING-cGAS the scientists made a connection between endogenous [tumor] DNA recognition and type I IFN in anti-tumor activity. Naturally it was assumed that antigen-presenting cells, such as DCs or macrophages, were the major players in STING anti-tumor "initiation" pathway.

Now, Swiss scientists provided evidence that when tumor-bearing mice were injected with STING agonist, cyclic dinucleotide GMP-AMP (cGAMP), it was endothelial cells rather than DCs that were responding to cGAMP by secreting IFN-β.

Initially, the authors showed that in murine B16 melanoma model STING was important for anti-cancer effect of intra-tumorally delivered cGAMP. 1/3 of cGAMP treated mice survived long-term. In addition, cGAMP injection showed synergy with anti-CTLA4/PD1 immunotherapy.

Next, the authors showed that anti-tumor CD8 T cell priming with cGAMP required functional type I IFN system.

In addition, these experiments revealed that anti-tumor effect of injected cGAMP was entirely depended on type I IFN responsiveness.

Surprisingly, analysis of tumor tissue showed that it was tumor vasculature endothelial cells, not DCs, that were IFN-β positive after cGAMP injection.

In vitro experiments confirmed that endothelial cells were specifically responding to cGAMP and tumor DNA.

In summary these results points to a complex interplay between tumor vasculature, tumor DNA and local anti-tumor T cell priming. It must be emphasized here that the authors had conducted short-term tumor challenge experiments here in most part and did not show whether cGAMP injection primed functional anti-tumor memory response in long term, for example by secondary tumor challenge.

David Usharauli

Melanoma-intrinsic β-catenin signaling prevents local DCs activation and anti-tumor T cell-priming

A high-profile scientific literature in immunology for the past 2 years is dominated by papers related to tumor immunotherapy. This probably has to do with the clinical successes of various immunotherapeutic approaches seen in recent years and the shift in funding for cancer research.

Here is new paper from journal Nature discussing immune response to tumor. The authors showed that melanoma-intrinsic β-catenin signaling prevents DC-mediated anti-tumor T cell priming.

By profiling melanoma tumor samples based on CD8 T cells numbers the authors observed that melanoma samples with low T cell numbers were enriched for mutations in genes involved in β-catenin pathway (gain-of-function or loss-of-function mutations).  

Using mouse model of spontaneous tumor induction, the authors confirmed that presence of active β-catenin signaling prevented T cell recruitment to the tumor tissue.

Experiments with adoptive transfer of nominal antigen (SIY)-specific T cells indicated that active β-catenin signaling in tumors prevented T cell priming.

Next, the authors showed that β-catenin signaling in tumor prevented recruitment of CD8α⁺/CD103⁺ DCs, a DC subset responsible for cross-priming. This suggested that lack of infiltration of tumors by T cells may have to do with lack of T cell priming by DCs.

Indeed, the authors showed that intra-tumoral injection of in vitro generated wild-type DCs activated with Poly(I:C) could restore T cell tumor infiltration.

Finally, the authors showed that adoptive transfer of wild-type of DCs in combination with antibodies against CLTA4/PD-L1 restored anti-tumor effect against β-catenin active tumor.

In summary, these results indicate that melanoma with constantly active β-catenin pathway inhibits anti-tumor T cell priming. Of note, β-catenin pathway plays a role in DCs maturation. It is not clear whether β-catenin pathway in DC per se was a contributing factor in this mouse model (separately from tumor).

David Usharauli

Melanoma dendritic cell vaccine is safe but is it effective?

Few days ago journal Science has published yet another study describing procedures how to identify tumor (melanoma)-specific immunogenic neo-antigens through exome-sequencing and combining peptide binding in silico and in vitro peptide recognition analysis. 

There is no doubt that cancerous tissue can yield multiple neo-antigens derived from non-synonymous mutations. Hypothetically and practically (as this new study and others as well have shown), immune system can and is able to detect these minute differences in the mutated proteins. It appears that by combining current in silico algorithms such as NETMHC-3.4 with epitope presentation assays (in vitro assays) provide quite accurate list of potential immunogenic tumor peptides.

The authors in this new paper, for example, were able to identify and confirm in a complementary in vitro assays (T2 cell peptide binding assay and tandem minigene constructs expression assay in DM6 cell line) the presence of several neo-antigens in melanoma tissue derived from 3 patients.

Re-injection of CD40L+TLR ligand matured, melanoma-peptide pulsed autologous dendritic cells back into patients yielded antigen-specific CD8 T cells expansion.

This was a small Phase I clinical study to determine the safety of the DCs vaccine. Since science fundamentals are strong behind this trial (especially considering the authors focus on IL-12p70 producing DCs as a source of cellular vaccine), the results were expected. Of note, these 3 patients were treated with ipilimumab (humanized α-CTLA4 antibody) prior to the experiments described in this paper. It is not clear how this could have skewed the [positive] outcome of DC vaccine here. Anyway, successful tumor treatment would require simultaneous approach from several directions (DC vaccine, checkpoint inhibitors, small drug tyrosine inhibitors).

David Usharauli      

10 in a Million: making tumors visible to immune system

Another interesting study about tumor immunology from Nature Medicine. 

This time, the authors, led by Ton Schumacher at the Netherlands Cancer Institute, have examined cancer proteome (mutanome) from 5 melanoma patients using whole exome and RNA sequencing coupled with in vitro T cell assay to identify (1) an entire set of non-synonymous mutations within expressed genes and (2) immunogenic neo-antigens.

On average the authors found that melanoma patients expressed ~ 150 mutations per tumor. To identify immunogenic epitopes, 31-mer peptides corresponding to mutated portion of the proteins were synthesized, loaded into autologous, immortalized (via BCL6/BCL-XL transduction) B cells, serving as APCs, and cultured them with in vitro expanded tumor-derived CD4 T cells. With these methods, the authors were able to identify several tumor-specific, neo-antigens.

Importantly, sensitivity of tumor-associated CD4 T cells to mutant neo-antigens (open symbols) were on average 100-fold higher as compared to their wild-type variants (closed symbols), implying that mutant epitopes were quite immunogenic.

Interestingly, based on analyses of cancer mutation burden and clinical immune response, the authors speculated that mutation rates of at least 10 per megabase of coding genome provides sufficient load for formation of immunogenic neo-epitopes detected by immune system (for example, one patient, NKIRTIL045 had less than 10 mutations per megabase and did not show CD4 T cell response to mutated epitopes; see Fig. 1).  

In summary, all these recent studies and successes in cancer immunology clearly indicate that President Nixon's declaration of war on cancer has finally started to pay off.

David Usharauli