Genomic alterations in IFN-γ pathway underlie resistance to anti-CTLA-4 (Yervoy) therapy

This week journal Cell published a short "reverse translational" study conducted on small cohort of patients non-responsive to immunotherapy with anti-CTLA-4 antibody (ipilimumab, Yervoy) that  showed that genomic alterations in IFN-γ pathway in non-responder patients could underlie their resistance to immunotherapy.  

For this study the authors compared 12 patients who did not respond to ipilimumab therapy (non-responders) and 4 patients who did respond to ipilimumab therapy (responders). They found that "tumor samples from non-responders were found to have significantly more somatic mutations, including copy-number alterations (CNAs) and single-nucleotide variants (SNVs) of the IFN-γ pathway genes".

Next, the authors showed that primary melanoma cell lines derived from anti-CTLA-4 responder or non-responder patients could be differentiated based on their in vitro sensitivity to IFN-γ.

Next, they showed quite bizarre experiment. By knocking down IFN-γ receptor in mouse B16 melanoma cells the authors showed that these cell line became less sensitive to IFN-γ in vitro. Not sure about logic behind these experiment.  

Finally, the authors showed that B16 melanoma cells deficient for IFN-γ signaling and transplanted into WT mice were less sensitive to anti-CTLA-4 therapy.

In summary, this small cohort study suggests that screening of melanoma patients for genomic alterations in IFN-γ pathway could be useful in better predicting therapeutic outcome for this checkpoint inhibitor therapy.

David Usharauli

Oncolytic virus expressing PGE2 inactivating enzyme display enhanced anti-cancer effect

Oncolytic virus therapy is a new method of cancer therapy. Its goal is to selectively target cancer cells sparing healthy cells. However, not many tumors respond to it and so far it has limited application.

So it was interesting to read new study in Cancer Cell that showed that effectiveness of oncolytic virus cancer therapy could be improved by incorporating prostaglandin-inactivating enzyme (hydroxyprostaglandin dehydrogenase 15-(NAD) (HPGD). 

Initially, the authors tested in vivo susceptibility of several cancer cell lines to oncolytic vaccinia strain (WR.TK^-Luc⁺). Some  cancers were less susceptible (Renca, 4T1), some more (LLC, MC38).

Susceptibility to WR.TK^-Luc⁺ therapy was T cell mediated as CD8 T cell depletion could abolish it.

To overcome inhibitory micro-environment within resistant tumors oncolytic virus (OV) expressing prostaglandin-inactivating enzyme was designed. Indeed, modified OV therapy improved cancer protection.

Moreover, when combined with anti-PD1 therapy, modified OV could protect mice from resistant tumor such as Renca tumor, even when applied on established tumor (aspirin had no effect at this stage).

In summary, this study confirms that sustained inactivation of local prostaglandine source could drastically improve anti-cancer effect of OV, especially when combined with checkpoint inhibitors.

David Usharauli

Anti-CD40 agonistic antibodies with enhanced FcgRIIB binding mediate superior tumor protection

CD40 molecule was an immunology superstar of 90s. Scientists thought they found a mechanism that controlled robust CD8 T cell and antibody responses. However, 20 years later, nothing much came out of those studies. In fact, scientists are still figuring out what is the best way to activate human CD40 molecule. 

For example, in new study published in Cancer Cell, Jeffrey Ravetch's group showed that in mouse model expressing transgenic human CD40, effectiveness of anti-hCD40 antibody correlated with enhanced binding to inhibitory FcγRIIB.  

The authors developed several variants of anti-hCD40 (clone CP-870,893, a fully human IgG2, one of the most potent agonistic CD40 mAb) with higher affinity and selectivity to human FcγRIIB (and minimal change in binding to FcγRIIA). Protein immunization confirmed that improved affinity to FcγRIIB correlated with effectiveness of CD8 T cell priming.

Moreover, enhanced affinity of anti-hCD40 antibody to FcγRIIB (V11 clone, for example) correlated with improved anti-cancer effect of anti-hCD40 agonistic antibody (in MC38 and B16 cancer models).

In summary, this study indicates that in mouse model of hCD40 / hFcγRIIB expression selective binding to hFcγRIIB is important for enhanced anti-cancer effect of anti-hCD40 antibodies.

David Usharauli

Improving CAR-T cell anti-tumor selectivity by targeting protein glycoforms

Development of CAR-T cells selectively targeting solid tumors have been challenging due to shared antigenicity between cancerous and healthy tissues. Even minute level of protein [if] expressed on healthy tissue could produce unacceptable side-effects as seen for example in trials with CAR-T cells targeting her2/neu.  

New study in Immunity suggested to target protein glycoforms (here using anti-Tn MUC1 CAR-T cells) instead to avoid cross-targeting of healthy tissue.

The authors speculate that cancer-specific glycosylation could produce cancer-specific glyco-proteins. They found that Tn glycoform of protein mucin 1 (Tn MUC1) is selectively expressed by human T cell leukemia and by many solid tumors and can be specifically detected by CAR-T cells incorporating variable heavy and light chains derived from 5E5 mAb (the University of Copenhagen has patented the 5E5 antibody and antigen epitope and the University of Chicago has filed a patent on the 5E5 CAR).

The authors first tested 5E5BBz CAR-T cells against human Jurkat leukemia cells in vivo in NSG immunodeficient mice and observed that it could double survival of tumor challenged mice.

More importantly, 5E5BBz CAR-T cells were also effective against solid tumor, pancreatic tumor cell line expressing Tn MUC1 glycoform (while the authors reported no side-effects in mice subjected to 5E5BBz CAR-T cells transfer, it is not clear whether human tissue in vivo would express it cryptically).

In summary, the authors believe that targeting glycosylation variants of protein "specifically" expressed on transformed cells could overcome cross-targeting of healthy tissue by CAR-T cells.

David Usharauli

CD47 and PD-L1 work in tandem with MYK to keep T cells at bay

This week Science published a short study examining the role of MYK in tumor immune evasion mechanisms [MYC is a transcription factor that regulates the expression of a multitude of gene products involved in cell proliferation, growth, differentiation, and apoptosis"].The authors found that MYK selectively directs expression of two immune-inhibitory molecules, CD47 and PD-L1, in several mouse and human tumor cells lines.

Using Tet-off transgenic mouse model to control MYK expression in T cell acute lymphoblastic leukemia (MYC T-ALL), the authors showed that both "in vitro or in vivo MYC inactivation resulted in a rapid downregulation of CD47 and PD-L1 [but not other surface markers], both at the mRNA level, as detected by quantitative real-time PCR (qPCR), and at the protein level, as detected by flow cytometry".

Since previous results demonstrated "complete tumor clearance following the inactivation of oncogenes, including MYC", the authors wanted to examine whether MYK effect on tumor regression involved CD47/PD-L1 tandem. Indeed, forced expression of either CD47 or PD-L1 in MYC T-ALL reduced anti-tumor effect of MYK inactivation.

In summary, this study suggests that MYK oncogene provides tumor cells with defenses to repel immune cells. 

David Usharauli

Gut microbes fuel effectiveness of anti-CTLA-4 cancer immunotherapy. Part I

Recently two back-to-back papers in Science Express received much media attention. These studies claim that gut microbial flora influence clinical effectiveness of checkpoint inhibitors approved for cancer immunotherapy. I am going to review and provide my analysis of these studies.

First paper I will review came from European team led by Laurence Zitvogel. Of note, her team had another tumor immunology paper just 1 week ago in Science. First notable thing about her publications is the number of names included as authors. Her lab had done this since I first heard about her studies in 2007. Either she is a good collaborator or everyone in her lab is get credit for everyone else's work as a matter of right [definitely good for career].

In this new paper, her team showed that effectiveness of anti-CTLA-4 antibody in cancer immunotherapy was greatly affected by presence of gut microbiota.

For example, they showed that inhibition of cancer growth by anti-CTLA-4 antibody injected in germ-free or antibiotic-treated mice was not as good as in control WT mice. 

Analysis of gut flora revealed that presence of specific set of microbes [B. fragilis, for example] were required to show anti-cancer effectiveness of anti-CTLA-4 antibody.

Now these result are quite preliminary and it is not clear how microbes are enhancing anti-CTLA-4 effectiveness. For example, some microbes can live within growing tumor tissue and anti-CTLA-4 therapy simply unleashes immune response against them destroying tumor tissue in the process. There is a possibility that microbes support presence of cross-reactive T cells required to target tumor antigens when they are unleashed by anti-CTLA-4 immunotherapy. In all, based on this paper we can't say how microbes help anti-CTLA-4 and how much this "help" is meaningful in clinical settings. Maybe second paper will provide some ideas in this regard. Stay tuned for next review. 

David Usharauli   

EZH2 plays a mind games with tumor and CD8 T cells, and to scientists too

Few days ago I reviewed new Nature article where the authors found a negative correlation between EZH2^hi tumor phenotype (EZH2 is a methyltransferase involved in epigenetic modification at H3K27me3) and progression-free survival of ovarian cancer patients. 

Now, it appears that the same group had another paper in Nature immunology showing exactly opposite effect of EZH2 in tumor infiltrating CD8 T cells (TIL). Namely, the authors found a positive correlation between frequency of EZH2-positive CD8 TILs and progression-free survival of ovarian cancer patients. 

First, the authors showed that in human CD8 T cells, EZH2⁺ subset displayed increased polyfunctionality.

Indeed, inhibition of EZH2 by GSK126 in in vitro CD8 T cells reduced their polyfunctionality.

Next, the authors found that, like GSK126, tumor tissue environment suppressed EZH2 level and TIL functionality by reducing access to glucose.

In another set of experiments, the authors showed that adoptive transfer of EZH2 inhibitor (drug or shRNA) pre-treated tumor-specific CD8 T cells failed to control melanoma metastases in lung.

Finally, analysis of clinical samples from ovarian cancer patients revealed positive correlation between frequency of EZH2⁺CD8⁺ subset and disease-free survival.

In summary, this paper indicates that glucose deprivation in tumor environment reduces EZH2 expression in TIL CD8 T cells and reduces their functionality, implying that increasing of EZH2 expression would benefit cancer patients.

Now these results are exactly opposite from the results provided in Nature paper. There, EZH2 expression in tumor cells inhibited TIL infiltration and EZH2^hi tumor phenotype showed reduced progression-free survival in ovarian cancer patients implying that decreasing of EZH2 expression would benefit cancer patients.

The only difference between these two papers is that one mostly deals with cancer cells and another with CD8 T cells. But this separation in vivo is hardly feasible suggesting more difficult path for medical application of discoveries in epigenetics [it is not clear how the same tumor environment reduces EZH2 expression in TIL CD8 T cells and at the same time increasing it in cancer cells]. 

David Usharauli

IL-9 puts the brakes on tumor growth

TNF family receptors control the magnitude and functionality of immune response. Several of them have been targeted for therapeutic purpose (4-1BB, CD40L, BAFF-R). 

GITR (Glucocorticoid-Induced TNFR-related protein), a member of TNF superfamily, was initially implicated in inhibiting regulatory T cell (Tregs) function. However, work on this molecule receded into oblivion for these past few years due to overall failure of immunological research to develop Tregs based therapies.

Now, new study in Nature Medicine put forward novel mechanism of action for GITR. This study showed that GITR stimulation amplified IL-9 production from T helper cells and contributed to tumor protection.

First, the authors showed that GITR stimulating antibody inhibited tumor growth in a CD4-dependent manner.

Next, they showed that GITR stimulation improved tumor protection in an IL-4Rα-dependent manner.

This observation led to discovery that GITR stimulating antibody improved tumor protection via IL-9 production [that requires IL-4Rα signaling].

In vitro experiments showed that GITR stimulating antibody indeed promoted Th9 differentiation (Th2 cells showed minimal cancer protection). 

Finally, the authors showed that GITR stimulating antibody shifted the balance in favor of CD8 T cells in tumor environment.

These results suggest that GITR targeting could provide additional layer of protection against solid tumors. 

David Usharauli 

Algorithm-based immune-epitope discovery

The goal of a personalized tumor immunotherapy is to identify tumor epitopes that are immunogenic and specific for a given patient. To do it, however, would require the knowledge of exact sequencing of a given "healthy" individual's proteome prior to tumor development. It is safe to assume that generic, public database would not provide the adequate tool.

In addition, the development for robust algorithms able to accurately predict the strength of binding of processed epitopes to MHC class I or II (or HLA) is an absolute must.

It appears that we are getting very close achieving this goal. I have already mentioned in my earlier post that journal Nature has published 5 separate papers about tumor immunology in December issue. However, my review showed that 3 of them were merely based on observation-type clinical data, more suitable for publication in journals like JAMA or Lancet, but not Nature. I am not sure why Nature has decided to include those 3 clinical papers in its publication list. However, 2 other papers that were mouse studies provided sufficiently robust experimental results for my analysis.

This 2nd study, led by Robert Schreiber at Washington University School of Medicine, has analysed mice immune response to aggressive sarcoma tumor cell line following anti-PD1 and/or anti-CTLA4 antibody therapy.  

Initial set of experiments indicated that anti-PD1 and/or anti-CTLA4 antibody therapy "helped" wild-type mice to reject the aggressive sarcoma cell lines in a T cell-dependent manner.

To understand the basis of tumor immunogenicity, the authors has applied 3 different epitope predicting algorithms to the available exome-sequencing data from one of the sarcoma cell line (d42m1-T3). With these in-silico generated epitope prediction methods, the authors were able to identify two dominant mutant epitopes, one from laminin alpha subunit 4 (Lama4) and another from asparagine-linked glycosylation 8 (Alg8).

Parallel experiments with TIL functional assay, tetramer binding and MHC peptide eluates mass spec analyses confirmed that these two mutations from sarcoma cell line (d42m1-T3) were the dominantly recognized by TIL following anti-PD1 therapy.

Additionally, Lama4 and Alg8 mutant peptides, but not wild-type variants, were immunogenic.

Moreover, mutant peptides + adjuvant immunized mice were able to reject sarcoma cell line.

Mechanistically, combination of anti-PD1 and/or anti-CTLA4 antibody therapy (but not anti-PD1 alone) augmented existing tumor-specific T cell numbers and their effect differentiation.

Note that anti-PD1 therapy alone were still able to protect mice from d42m1-T3 (see Fig. 1a), even though its effect on CD8 T cells was minimal, implying that the mode of action of anti-PD1 antibody therapy is less clear.

Here is my opinion: there is no doubt that tumors express mutant proteins (epitopes). If a mutant protein has a critical, obligatory function in fueling tumor growth, then the tumor cannot afford to lose it in an "escape phase" so targeting such epitopes would be useful. If however mutant protein does not have such function tumor will lose it easily once under pressure from T cells thus making immunotherapy less productive. Since we still do not know the function of many proteins it would be very difficult to identify "escape-proof" mutant epitopes for tumor immunotherapy.        

David Usharauli

How to identify tumor-associated immunogenic peptides for immunotherapy?

Few weeks back journal Nature has published 5 separate papers in tumor immunology in the same issue. I am going to provide my assessment of the strengths and weaknesses of each paper and its approaches. 

I will start with the study led by Lelia Delamarre and Jennie Lill at Genentech. Genentech is one the oldest biotech company. It has a very good reputation and its scientists are usually trying to publish in high profile journals.

This is a mouse study. The idea of the paper is to claim that combining tumor whole exome-sequencing with MHC class I-peptide mass spec analysis provides better solution for identifying immunogenic mutated epitopes.

For this study the authors has performed whole exome-sequencing of two tumor models, MC-38 and TRAMP-C1. Initially, they have identified 1290 and 67 non-synonymous mutations in MC-38 and TRAMP-C1, corresponding to 170 and 7 neo-epitopes for MC-38 and TRAMP-C1, respectively (based on NETMHC3.4 algorithm prediction).

In parallel, the authors performed MHC class I peptide elution from these tumors for mass spec analysis. With this method, the authors found only 7 peptides for MC-38 (out of predicted 170 neo-epitopes) and 0 peptides for TRAMP-C1 (out of predicted 7 neo-epitopes).

Direct immunization with these peptide + adjuvant showed that two peptides, Reps1 and Adpgk displayed high immunogenicity (CD8 T cell priming potential), that was predicted by NETMHC3.4 algorithm.

Analysis of tumor-infiltrating lymphocytes (TIL) showed that Adpgk-specific CD8 T cells were the most abundant population (note that Reps1-specific T cells are the most abundant during peptide immunization in absence of tumor and Adpgk-specific T cells are the most abundant during peptide immunization in the presence of tumor).

Moreover, immunized with the 3 peptide-cocktail + adjuvant protected mice from MC-38 challenge (and protection was correlated with the frequency of Adpgk-specific T cells). 

In addition and more clinically relevant, immunization of tumor-bearing mice with 3 peptide-cocktail + adjuvant could augment Adpgk-specific T cells response and inhibit tumor growth.

In summary, the authors suggest that combining whole exome-sequencing with mass spec analysis of MHC class I peptide binding could improve identification of immunogenic peptides specific for tumors.

To be honest, I am a little bit confused with this paper. Even though the authors claim that they have combined whole exome-sequencing with mass spec analysis of MHC class I peptide binding, in the end, they have tested only peptides identified by mass spec. If one can analyse eluted peptides and determine its protein origin, then I do not see what was the function for whole exome-sequencing in the first place in this study? What about trying to test other peptides, at least few of them, predicted by algorithm only? It would have been much more useful, I would imagine. In addition, the authors found 0 immunogenic peptides for TRAMP-C1 with mass spec. How is zero useful here? What about treating the tumor cells with IFN-gamma to increase MHC class I expression to improve peptide recovery. 

David Usharauli

Tumor immunotherapy: 1 year at a time

Tumor immunotherapy is both an old and new idea. Whenever there is a new discovery in immunology, people usually are asking 3 questions: can it cure HIV, cancer or allergy?

Recently, a promise for successful immunotherapeutic approach to cancer treatment was boosted by clinical introduction of anti-CTLA4 and anti-PDL1 humanized antibodies. These antibodies target and exploit the natural inhibitory checkpoints in T cell activation program relevant for natural tolerance process to self or food antigens. There is another approach as well, called CAR-T technology, that I will review later.

Still, the actual, long-term benefits of current immunotherapeutic approaches are debatable. Here is one example. This is a Science paper. The senior author on this paper is Steven Rosenberg. This is a guy who actually has started clinical immunotherapy with IL-2 in 1980's.

This paper describes a single patient's personalized tumor immunotherapy protocol. Initially, the authors sequenced a metastatic tumor obtained from resected lung tissue and found 26 mutations. Next, they have prepared 3 different tandem minigene constructs (8-9 mutations each), transfected them in autologous antigen-presenting cells and run ex vivo TIL (tumor-infiltrated lymphocytes) activation assay. Only one construct, TMG-1, showed TIL reactivity.

Next, from this tandem minegene construct (that contained 9 mutant minigens), the authors synthesized 9 individual minigene constructs with each containing reversion of one mutation back to wild-type sequence, so to permit detection TIL specificity by antigen elimination. Interestingly, TIL's reactivity was abolished when erbb2 interacting protein was reverted to wild-type sequence. 

In addition, TIL's tumor reactivity was localized in CD4 T cell subset and was made up mostly of Vbeta22+ CD4 T cells. 

Thus this patient's TIL (CD4 T cells) specificity was directed towards one mutation in erbb2ip.

Next, the authors has expanded patient's TIL in vitro and injected back 42.4 billion TIL (25% Vbeta22+) into patient. This adoptive transfer of tumor specific TIL produced >1 year tumor burden stabilization and regression of metastatic tumor in liver and lung.

After 1.5 year from the initial TIL transfer, tumor re-emerged, so the patient was re-injected with in vitro expanded 100 billion tumor specific Vbeta22+ TIL and again such transfer produced additional >6 months stabilization and regression of tumor metastasis.

It appears that the difference between this graph and Fig. 2 D has to do with the fact that the authors measured only those tumor lesions which were present during both treatments.

What we have learned from this study? First, tumor in this case did express neo-antigen detected by CD4 T cells. Second, in vitro expansion of tumor-specific TILs and their adoptive transfer back into patient yielded >1 year (after single transfer) and in total >2 years (after 2nd transfer) of tumor burden stabilization and metastatic regression. Third, this approach, however, did not provide a long-lasting tumor-protective effect, despite presence of transferred tumor-specific CD4 T cells.

Does it mean that TIL approach is not viable in a long run? Why were billions of tumor-specific T cells not able to eliminate tumor? Because of inhibitory milieu at tumor sites? Were they terminally-differentiated T cells lacking self-renewal properties? It seems that tumor was able to escape CD4 T cell control despite the fact it still continued to express neo-antigen. How? 

David Usharauli