SUPRA CAR T cell system provides more of cosmetic rather than a real advance

This week journal Cell published a new study from scientists at Boston University describing in their own words "a split, universal, and programmable (SUPRA) CAR system" that supposed to provide several advantages over conventional CAR-T cell system. I reviewed and present here my conclusions on this paper.   

The rationale behind SUPRA CAR T cell design was to develop flexible, "plug-and-play" system to fine tune CAR T cells' activity against tumors without need to redesign it over again. SUPRA consists of two modules: signaling zipCAR construct is artificially expressed by T cells on their surface and soluble zipFv construct expressing tumor antigen specific scFv portion which is injected into system. "Zipper" portions of zipCAR/scFv constructs could interact with each other and by injecting different variants of zipper one can modulate strength of interaction.

What advantage(s) SUPRA CAR T cell design provide? 

1st advantage the authors showed could be to tune signal strength of original SUPRA zipCAR T cells interaction with tumor specific zipFv construct by injecting competitive zipFv constructs that have different affinity to zipCAR module and thus modulate tumor specific zipFv action (to prevent cytokine storm).

2nd advantage is thought to be use of the same zipCAR T cells and inject two different zipFv constructs specific for two different tumor antigens (to prevent tumor escape).

3rd advantage the authors suggested would be to deploy decoy zipFv that could inhibit tumor specific zipFv activity only when decoy scFv interacts with non-tumor specific antigens and thus limiting non-target effects in different tissues (to prevent off-target tissue damage).

Later in the paper the authors went on to present series of experiments that showed comparison of effectiveness of SUPRA CAR T cell construct vs. conventional CAR T cells against two different tumor models in vivo.

On the surface all these experiments look quite impressive. However, close analysis of data shows that advantages are more of cosmetic in nature rather than real ones. First, none of those above mentioned three advantages were actually shown for tumor models in vivo (for some reason the authors did not show how injection of competitive low-affinity zipFv construct could affect tumor protection experiments in vivo or whether double antigen expressing tumors could be efficiently eradicated). Moreover, in vitro experiments showing decoy effect was done in manner that is incompatible for in vivo experiments (one cannot wash away decoy zipFv in vivo before introducing tumor specific zipFv construct and it is likely that free floating decoy zipFv construct could inhibit tumor specific zipFv activity even in absence of decoy tissue antigen).

posted by David Usharauli

Fragility of FOXP3+ Treg phenotype

This week I am reviewing two papers dealing with different aspects of FOXP3+ Treg biology. Both papers were published in journal Cell. These are predominantly mouse works so no need to get too excited. 

1st paper was published by Dario Vignali's group (department of Immunology, University of Pittsburgh School of Medicine). They have analyzed role of Neuropilin-1 (Nrp1) in Treg phenotype in tumor context. Neuropilin-1 is a marker of thymic Tregs though this notion is not universally accepted. Interestingly, in mouse tumor model, mouse that expressed Nrp1 on half of its Tregs rejected tumor with the same vigor as mouse expressing Nrp1 on all of its Tregs. 

Furthermore, in the context of tumor challenge, Treg-specific Nrp1 deficiency drastically increased IFN-γ production in both KO and WT Tregs.

It appeared that WT Tregs required sensing of IFN-γ derived from Nrp1KO Tregs to acquire "Nrp1KO-like" phenotype. 

Finally, the authors showed that therapeutic effect of checkpoint inhibitor anti-PD1 antibody against tumor required IFN-γ sensing by Tregs.

In summary, this study showed that Nrp1 deficiency makes Tregs fragile by converting them into IFN-γ producer cells which in turn affect WT Tregs phenotype as well. The reason the authors are using term fragility rather than simply instability has to do, they claim, with the difference in FOXP3 expression between fragile and unstable Tregs. 

In a separate study, the authors showed that Treg depletion inhibits hair regrowth (after depilation). 

However, it is not clear why Tregs should be involved in such physiological process when any other innate cells could do the same.   

David Usharauli

Tetra-punch against solid tumors

Checkpoint blockade therapies has become a gold standard for cancer immunotherapy. However, only in minority of cancer patients did these antibody therapies show significant benefits. Many think that a multi-pronged approach to cancer therapy could tip the balance in favor of anti-tumor therapy.

For sure, data from mouse studies support this line of thinking. For example, this week Nature Medicine published a mouse study showing dramatic benefits of immunotherapy when four different approaches were combined: 

1. Anti-cancer Antibody (A)

2. Long-lived IL-2 (I)

3. Checkpoint PD1 inhibitor (P)

4. Cancer Vaccine (V)

Referred as AIPV this experimental tetra-pronged immunotherapy could clear an established solid tumors (melanoma, breast cancer, adenocarcinoma) in 75%-80% of mice.

Success of AIPV therapy depended mostly on CD8 T cell and NK cells.

Of note, however, frequency of IFN-γ⁺ CD8 T cells did not correlate with anti-tumor effectiveness.

Interestingly, through AIPV could induced endogenous anti-cancer antibodies that transferred protection in naive hosts against intravenous tumor inoculum, B cell deficient mice were still protected against tumors when immunized with AIPV.

Finally, AIPV protected against autochthonous [endogenously developed] tumor in Braf^CA Pten^loxPTyr::Cre^ERT2 mice.

In summary, this mouse study shows that multi pronged immunotherapeutic approach could significantly improve survival rate during cancer therapy. The authors claimed that "AIPV therapy was associated with minimal systemic toxicity, as mice did not show weight loss or substantial elevation in the amounts of liver enzymes in the blood".

Of course, it is difficult to compare outcome in mouse study versus human study. In humans, even single approach with anti-PD1 antibody frequently leads to lung or liver toxicity. Now imagine injecting cancer patients with 4 different immunotherapeutics. So, we have a long way to go before immuntherapy will show the same acceptable-level effectiveness in humans as it does in lab mice.  

David Usharauli 

   

Foxp3+ Tregs go against tumor in the "dark"

This week PNAS published follow-up study from Harvey Cantor's lab examining role of Helios (the Sun in Greek mythology) in Foxp3+ T_REG stability. In this new study they showed that antibody [anti-GITR]-mediated down-regulation of Helios in Foxp3+ T_REG causes them to acquire effector function and participate in anti-tumor response.

I will highlight relevant findings. The first finding was that mice with Helios-deficient Foxp3+ T_REG showed additional resistance to tumor (however, please note that tumor growth pattern and mice survival do not correlate in Helios^fl/fl.FoxP3-Cre mice).

Similar tumor growth retardation was observed in adoptive co-transfer experiment with WT T cells and Helios-KO Foxp3+ T_REG.

Mechanistically, Helios^fl/fl.FoxP3-cre T_REG up-regulated effector cytokines specifically at tumor site.

Finally, treatment of mice with antibody, DTA-1, directed to GITR (glucocorticoid induced TNF receptor), caused Helios down-regulation in FoxP3+ T_REG and improved tumor protection.

In summary, the authors proposed that therapeutic targeting of Helios expression in tumor-associated FoxP3+ T_REG could convert them from tolerogenic cells into anti-cancer fighting cells (however, please note the authors did not formally test whether anti-cancer effect of anti-GITR antibody was indeed mediated via its effect on Helios in FoxP3+ T_REG).

David Usharauli

Availability of free cytoplasmic cholesterol augments TCR signaling in CD8 T cells

Few days ago Nature published new research study from mainland China wherein the authors showed that availability of free cholesterol in CD8 T cells augments TCR signaling and contributes to better control of experimental tumor.

This study focus on Acat1 that encodes cholesterol esterification enzymes that convert free cholesterol to cholesteryl esters for storage. The scientists noticed that chemical inhibition of Acat1 in CD8 T cells could augment their cytolytic effector differentiation (granzyme B ↑).

Similar results were obtained with CD8 T cells from Acat1 conditional knockout mice (Acat1^CKO).

Boost of CD8 T cell cytolytic functions with Acat1 inactivation yielded better tumor protection in adoptive transfer experiments as well.

Mechanistically, it appears that availability of excess free cholesterol in Acat1^CKO CD8 T cells improved TCR downstream signaling.

Finally, the authors showed that avasimibe, an Acat inhibitor with a good safety profile in humans, could delay tumor progression in mice and even show synergy with checkpoint inhibitor, anti-PD1 antibody.

In summary, this study suggests that commonly used cholesterol modulating drugs could have important role in CD8 T cells effector function and could influence tumor immunotherapy results.

Here I would like to note that one earlier study showed that reduction of cellular free cholesterol triggered universal STING-cGAS mediated anti-viral type I IFN response. This suggests that on one hand free cholesterol could augment CD8 T cell cytolytic function, but on the other hand it could diminish cell-autonomous anti-viral response.  

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