Monocyte-derived cytokines IL-1 / IL-6 contribute to CAR-T cell-induced cytokine-release syndrome and neurotoxicity

This week Nature Medicine published two papers showing in mouse CAR-T model that cytokine-release syndrome (in both studies) and neurotoxicity (in one study only) were primarily driven by IL-1 and IL-6 cytokines released by monocytes following interaction with infused CAR-T cells. 

In the first study led by Michel Sadelain at Sloan Kettering Institute, New York, immunodeficient SCID-beige mice transplanted intra-peritoneally with human B cell tumor cell line and later infused with CD19 CAR-T cells develop cytokine-release syndrome (CRS) that were reversible by anti-IL-6 antibody,  injection.  

Injection of anti-IL-1 antibody, Anakinra, had similar protective effect against CRS-driven mortality without compromising anti-tumor effectiveness. 

However, due to some limitation of their mouse tumor model where CAR-T cells are of human origin and non-T cells such as monocytes are of mouse origin, the authors acknowledged that not all features of CAR-T cell toxicity could be reproduced here since "None of the reported pathologic findings indicative of neuropathology or associated with neurotoxicity (cortical laminar necrosis, hemorrhages, disseminated intravascular coagulation (DIC), gliosis or vasogenic, neurotoxic or interstitial edema) in human patients were observed in any of the mice examined in the present study".   

Fortunately, the second study is more extensive and fills much of holes of the first study. Here, the authors led by Attilio Bondanza at San Raffaele Hospital Scientific Institute, Milano, "transplanted human cord blood (CB) hematopoietic stem and progenitor cells (HSPCs) through intrahepatic injection into sublethally irradiated newborn NSG or triple transgenic NSG (SGM3) mice expressing human stem cell factor, granulocytemacrophage colony-stimulating factor (GM-CSF) and IL-3 and initially profiled lymphohematopoietic reconstitution." Basically, this mouse model, referred as newborn humanized SGM3 (nHuSGM3), had both T cell and non-T cell components of hematopoietic system mostly of human origin. In allogeneic tests, human T cells developed in nHuSGM3 mice showed expected functionality. 

Staining of cells in nHuSGM3 challenged with tumor and CAR-T cells that induced CRS showed that monocytes were producing IL-1 early on, followed by IL-6. 

Depletion of monocytes/macrophages with liposomal clodronate (LC) could rescue mice from CRS.

Importantly, while both anti-IL-1 and anti-IL-6 antibody injection could significantly reduce CRS, only anti-IL-1 antibody were able to reduce neurotoxicity in nHuSGM3 mice. Similar effect were seen with CD44v6 CAR-T cells as well.

In all, these two studies showed that in addition of anti-IL-6 injection, anti-IL-1 antibody therapy could significantly reduce complications of CAR-T cell immunotherapy in humans. They provided a strong evidence to suggest that monocytes were primarily responsible for CRS and neurotoxicity complications of CAR-T cell therapy. 

However, it is not clear why are monocytes getting activated after CAR-T cell infusion in the first place. There is interaction, at certain level, between CAR-T cells and monocytes, but is it antigen-specific via anti-CD19 CAR-T or endogenous TCR, or is it non-specific, is unknown presently. 

posted by David Usharauli 

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

Selective elimination of autoimmune B cells using novel CAR-T technology based on antigen decoy

Few days ago journal Science published very interesting study describing a novel approach of treating autoimmune [auto-antibody]-dependent diseases. Called chimeric auto-antibody receptor (CAAR) T cell technology, this method can selectively eliminate autoantigen specific B cells in autoimmune diseases such as Pemphigus vulgaris, lupus, Myasthenia Gravis, Graves diseases and so on.

This new technology is based on a simple idea: antigen decoy. For example, during pemphigus vulgaris autoimmune B cells secrete autoantibodies to the keratinocyte adhesion protein desmoglein (Dsg3) that causes severe skin inflammation. Short-term management of diseases is achieved by total B cell depletion, but disease returns. The authors reasoned that if engineered T cells would express Dsg3 as a CARs, such antigen decoy CAAR T cells would selectively engage disease-causing anti-Dsg3-specific auto-antibody producing B cells. Such interaction should eliminate only Dsg3 specific B cells, sparing normal, infectious-specific B cells.

Indeed, the authors showed that CAAR-T cells expressing Dsg3 as a CAR construct (EC1-4 CAAR) can selectively interact with Dsg3-specific autoimmune B cells in vitro.  

More importantly in vivo NSG mice experiments confirmed that Dsg3 CAAR T cells could selectively eliminate human Nalm6 CD19+ B cell line expressing autoimmune Dsg3-specific receptors (PVB28/F779). In addition the authors claim that Dsg3 CAAR-T cells did not interfere with normal human skin epithelial functioning expressing Dsg3's natural ligand desmocollins.

In summary, if the results of this study is confirmed by other groups it would open up a new path for treating auto-antibody dependent autoimmune diseases.

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

In vitro comparison of human CAR-T cells expressing either CD28 or 4-1BB costimulatory domains

Today journal Immunity published new [short] study from Carl June's lab describing some functional and metabolic differences between CAR-T cells expressing CD28 or 4-1BB costimulatory domains.

It is not a particularly cutting-edge study. But since its focus is human CAR-T cells such limited experiments are still expected. Though I will anticipate that soon publications of in vitro studies on human primary cells [in top journals] will require incorporation of specific gene targeting experiments using CRISPR-Cas9 technology. Such experiments are now technically feasible and will be more informative.

In short, for this study the authors compared proliferation/expansion and metabolic/energy source of two CAR constructs, one with CD28 and another with 4-1BB (aka CD137) signaling domains, as shown below. FMC63 is anti-CD19 scFv and SS1 is anti-mesothelin scFv.

Human primary T cells were electroporated with these two CAR constructs. Efficacy of CAR expression was 90%.

Then, these transduced CAR-T cells were stimulated with magnetic beads coated with a recombinant anti-CD19 or mesothelin-Fc (such stimulation allowed selective activation of CAR-T cells via CAR construct). It revealed that CAR construct with 4-1BB domain imparted superior long-term expansion functionality on CAR-T cells.

Metabolic analysis revealed that unlike CD28 construct, 4-1BB CAR-T cells showed enrichment for genes responsible for lipid oxidation, implying 4-1BB signaling activates metabolic pathway resembling that of naive and memory T cells (involving mitochondrial oxidation of free fatty acids).

Indeed, transmission electron microscopy data confirmed that 4-1BB CAR-T cells were enriched for mitochondria. 

In summary, this study provided supporting evidence that 4-1BB CAR construct endows T cells with superior expansion/persistence quality. Initial studies on this topic suggested that CD28 CAR construct displayed superior "effector" activity against tumor, while 4-1BB CAR construct showed longer survival [superior memory function]. Subject is not settled, but in discussion section, the authors do note that mixing both CAR constructs could provide better outcome compared to individual construct.

David Usharauli

     

Novel combinatorial antigen-sensing chimeric antigen receptor circuit permits tumor-selective therapeutic T cell activity

Few days ago journal Cell published new study on chimeric antigen receptor (CAR) T cells that produced lots of discussion in immuno-oncology field. I read the actual paper and here below I will provide my opinion about it. 

I will point out that overall this study is of high quality. Basically, the whole premise of this paper is based on what the authors had correctly pointed out in the introduction "Few antigens are absolutely tumor specific, and T cells targeted to antigens that are also found on normal bystander tissues can cause life-threatening adverse side effects."

So how this could be avoided? According the authors "combinatorial antigen recognition T cell circuits in which a synNotch receptor for one [tissue-selective] antigen drives the inducible expression of a CAR for a second [tumor-specific] antigen" could provide such a fail-safe mechanism. In essence, in this model CAR-T cells would behave very similarly to how NK cells are sensing healthy and "abnormal" cells or tissues.

In first set of experiments, the authors tested validity of this concept on Jurkat T cells, a human T cell line. Jurkat T cells were transduced with a-CD19 synNotch receptor and a-mesothelin CAR gene and then exposed to "K562 myelogenous leukemia cells with ectopic expression of CD19, mesothelin, or both antigens". The authors detected IL-2 production [as a readout of Jurkat T cell activation] only with K562 cells expressing both CD19 and mesothelin.

Similar results were obtained with primary human T cells transduced with a-GFP synNotch receptor and a-CD19 CAR and exposed to "K562 target cells expressing CD19 only, GFP only, or both GFP and CD19". 

Importantly, in vivo experiments also indicated that T cell response were restricted to tumor cells with dual antigen expression. Only dual GFP+/CD19+ Daudi B cell tumor, but not single CD19+ Daudi cells, could induce CAR-T cell activation [as measured by luciferase signal]. Of note, there was low but detectable level of CAR T cell engagement at the single CD19+ Daudi tumor site.

And even more importantly, even in mice simultaneously transplanted with both single or dual (GFP+/CD19+) expressing K562 tumor cells, only dual antigen-expressing tumor cells were targeted. This indicated tumor-selective nature of combinatorial CAR-T cells. It showed that in this model, combinatorial CAR-T cells do not "engage a tumor expressing the synNotch ligand (GFP), become primed by expressing the a-CD19 CAR, and then migrate elsewhere to then kill single antigen (CD19+ only) bystander tissues." (though, I would admit I was surprised with these results).

In summary, these results clearly showed some of the advantages of combinatorial CAR-T cells. By expressing tumor tissue-selective SynNotch "primer" receptor, these CAR-T cells are primed and engage tumor "specific" antigens within tumor tissue only (but not in other tissue). 

Now question is how far such tumor tissue "selectivity" goes. The authors have not done more direct experiment wherein tumor and actual healthy tissues share "common" antigen to see if combinatorial CAR-T cells would maintain their "bias" towards tumor tissue. Since the main premise of such combinatorial CAR-T cells is to prevent CAR-T cell's "side effects" against healthy tissue, we need model that reliably test this scenario.   

David Usharauli

Extracellular matrix (ECM) degradation helps CAR-T cells to control solid tumors

Chimeric antigen receptor-transduced T cells (CAR-T) represent a new and powerful mode of cancer immunotherapy. Results generated so far clearly indicate that CAR-T cells are very good against fluid tumors such as B cell-derived lymphomas. It is not surprising. Anyone working in immunology knows that B cells are one of the best targets for cytotoxic assays. But what about solid tumors? Not so. Why?

New study in Nature Medicine may shed light on this issue. The authors reported that conventional CAR-T cells lack enzyme, heparanase, necessary to degrade ECM that coats solid tumors.

This is a short study, just 4 figures, but the results are very impressive. Initially, the authors showed that long-term culture of ex vivo-expanded (LTE) CAR-T cells down-regulates heparanase activity. Heparanase cleaves heparan sulfate proteoglycans, a part of ECM.

Next, the authors showed that heparanase-transduced CAR-T cells maintain enzyme activity long-term and show improved invasion activity in Matrigel assay. [Of note, the authors results indicate that baseline Matrigel-invasion activity of long-term cultured CAR-T cells is too variable (compare Fig. 1a vs. Fig. 2d; 8% ± 6% vs. 29% ± 18% for the same long-term cultured CAR-T cells)].

Still, in an in vivo assays, heparanse-transduced CAR-T cells showed far superior anti-tumor activity against solid tumors (against neuroblastoma and melanoma) compared to control CAR-T cells. As expected, CAR-T activity against B cell malignancy was not improved by heparanase activity.

In summary, these results suggest that optimization of CAR-T cell therapy against solid tumors would require improving ECM-degradation capacity of CAR-T cells.

David Usharauli