Checkpoint inhibitors, anti-PD1/anti-PD-L1 activities are channeled via CD28 co-stimulation

Two new back-to-back studies in Science showed that T cell recovery activity attributed to checkpoint inhibitors, anti-PD1/anti-PD-L1, is mediated via CD28 co-stimulation. One of the study focused on analysis of biochemical events leading to PD1 signaling in an in vitro reconstitution model and another study provided corroborating data using mouse model. 

Summary results from both studies suggest that anti-PD1/PD-L1 activity is lost when T cells lack co-stimulatory molecule CD28. However, readouts here are more complicated since PD-L1 itself could bind to CD28 ligand B7 molecule. Also, I noticed that by default T cells with inducible CD28 deficiency (CD28^f/f Cre^ERT2+) show less accumulation and/or survival (low cell density) compared to WT counterparts.

Another confusion has to do with the fact that at least in one of the clinical trials anti-CD28 antibodies produced severe cytokine release syndrome and program was discontinued. It could be that anti-PD1/PDL1 therapy activates only certain type of T cells while anti-CD28 could have targeted much larger T cell population.

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 

   

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-PD1 antibody treatment shows beneficial effect in human Alzheimer disease (AD) mouse model

This week journal Nature Medicine published a short paper from Michal Schwartz lab at Weizmann Institute of Science (Israel) showing beneficial effect of anti-PD1 treatment in human Alzheimer disease (AD) mouse model.

Michal Schwartz lab is famous in immunology circles for producing data showing the role of immune system in CNS function. For example, earlier their lab showed that mice deficient for adaptive immune system display decline in cognitive functions.

In this new paper the authors showed that two consecutive injections of anti-PD1 antibody (checkpoint inhibitor used in cancer immunotherapy) reduces CNS tissue pathology in mice with human AD phenotype [in two different models, (a) five familial AD mutations (5XFAD) and (b) APP/PS1 mouse models]. This beneficial effect correlated with the accumulation of peripheral macrophages into CNS and were dependent on IFN-γ.

It is not clear why systemic immune activation improves AD tissue pathology. Simple explanation is that peripheral "activated" macrophages that migrate to CNS are better equipped to digest and clean up AD-associated amyloid depositions. However, it is not clear whether CNS with AD pathology sends out any specific signals to recruit those peripheral macrophages or it is just nonspecific migration into CNS and other tissues [not examined in this paper].

David Usharauli 

Immunotoxins show anti-cancer synergy with immune checkpoint inhibitors

This week Science Translational Medicine published 2 papers directly or indirectly related to Foxp3⁺ T cells [this is in in addition of two Foxp3⁺ T cell papers in Nature]. Since I am very curious about the role of Foxp3⁺ T cells in immune regulation I decided to review them.

First paper I review here is not a typical Foxp3⁺ T cell paper. It is actually a study of tumor immunotherapy. The authors showed that when combined with checkpoint inhibitors CTLA-4/PD-1, direct tumor immunotoxins, such as T-DM1 [trastuzumab emtansine], significantly augment anti-cancer immune response and immune memory.

Trastuzumab [herceptin] is a humanized antibody that targets HER2 antigen over-expressed in breast cancer cells. Initially, the authors showed that anti-cancer effect of ado-trastuzumab conjugated to cancer toxin, T-DM1 (Kadcyla®), was depended on T cells [as revealed by T cell depletion]. This suggested potential synergy with checkpoint inhibitors.

Indeed, a combined application of T-DM1 and CTLA-4/PD-1 antibodies showed close to  100% protection against breast cancer in mice.

Surprisingly, the authors noticed that combo therapy, that fully protected mice against tumors, actually increased infiltration of tumor tissue by functioning Foxp3⁺ T cells [in addition to conventional T cells]. This was counter-intuitive.

However, T cell depletion confirmed that when CD4 T cells [that includes Foxp3⁺ T cells] were removed, more than 50% of mice on combo therapy [100% tumor-free] developed severe autoimmune inflammation [results with Treg-specific depletion would have been more valuable here, of course].

In summary, this study points to 2 distinct results: first, direct cancer cytotoxocity by immunotoxins could synergize with checkpoint inhibitors, probably via efficient antigen uptake and DC maturation [antigen processing stage]. Second, tumor infiltrating Foxp3⁺ T cell could play not yet understood tissue protective role during such combo immunotherapy [in effector stage].

David Usharauli

NSAIDs show synergy with anti-PD1 antibody in cancer immunotherapy

Tumor progression from a single cell to a multicellular state can take years and several mutations that allow cancer cells to (1) expand uncontrollably, and (2) adopt natural defense mechanisms against immune surveillance (commonly referred as tolerance). 

Ordinarily, healthy (or healing) tissues defend themselves against excessive immune probing by secreting or expressing inhibitory molecules. Each healthy tissue will have its own suitable natural defense(s) against excessive immune probing.

There are several known tissue specific inhibitory signals. Many are still unknown. By understanding how healthy tissues communicate tolerogenic signals to immune system we can gain better control over cancer tissue (because cancer tissues are imitating the same tolerogenic state). 

For example, new pepar in prestigious journal Cell showed that blockade of COX1/COX2 enzymes leads to tumor control by adaptive immune system. COX1/COX2 enzymes are the same enzymes the drugs such as aspirin targets to relieve the pain. 

This is very straightforward article. The reason it is in Cell has to due with CRISPR/Cas9 technology the authors has used to develop COX1 or COX1/COX2 double deficient tumor cell lines. The senior author on this paper is Caetano Reis e Sousa, a wellknown name among immunologists. This study was done at the Francis Crick Institute in London which  will be the largest biomedical institution in Europe when completed.

Initially, the authors developed melanoma-prone cell lines (Braf^v600E) deficient for COX enzymes using CRISPR/Cas9 technology.

Next, the authors showed that Braf^v600E melanoma tumor cells deficient for COX1/2 enzymes lack the ability to secrete PGE2 (among other bioactive lipids), a prostaglandin molecule known for its immunomodulating property.

The authors observed that when transplanted COX1/2 DKO melanoma Braf^v600E tumor cells (but not parental cells) were rejected by wild-type, but not by RAG KO hosts, implying the role of adaptive immune system in recognizing tumor cells in absence of PGE2.

Similar results were obtained with melanoma Braf^v600E tumor cells selectively deficient for PGE2 synthase.

Growth of other tumor cell lines (colorectal and breast cancer cells) deficient for COX1/2 enzymes were also controlled by adaptive immune system in wild-type hosts, though less efficiently compared to melanoma cells (the authors do not indicate whether these tumors were ultimately rejected or stayed dormant).

Finally, the authors showed that combination of aspirin (that blocks COX1/2 enzymes) and anti-PD1 antibody could synergize in inhibiting tumor growth (here also it is not clear what term "rejection" means, since tumor growth graph and rejection graph are not identical).

In summary, the data presented in this paper expands our understanding of "natural" defenses of tumor cells against immune probing. Here, tumor growth was controlled by T cells and it could generate memory response to secondary tumor transplantation.

One weakness of this study is the tumor transplantation model itself. The authors did not study already established tumors but rather than they followed the tumor growth or implemented therapy immediately after transplantation. This approach seems to lack a clinical relevance for human cancers where patients are frequently first seen with already established tumors (sometimes quite in advance stages).

David Usharauli