Imclone's legacy lives on: Cetuximab (Erbitux) induces immunogenic cell death in cancer cells

This week Nature Medicine published new study that suggests that Cetuximab (Erbitux), a chimeric monoclonal antibody directed to the EGFR, induces immunogenic cell death in susceptible cancer cells that carry wild-type form of oncogene KRAS.

Nowadays such research articles are common. Cancer immunotherapy really took off in the past 3 years and many want to jump on the immunotherapy bandwagon. However, I selected this paper for review for entirely different reason. I wanted to highlight how science behind Cetuximab was so advanced and ahead of its time [when it was originally tested] that it deemed ineffective [due to some data inconsistency for reasons that became known only later].  

Let me remind you that Cetuximab was originally developed and tested by the biotech company called Imclone in 1990s.  In clinical trials it was effective in ~20% of patients. However, trials were poorly designed [some did not even show effect] and when FDA refused to accept Imclone's submission for Cetuximab approval in December 2001, company collapsed and its CEO, Samuel Waksal, went to jail for insider trading. 

In retrospect, we can now [based on this new paper] reconstruct to what happened to Cetuximab's original trials. This analysis, in my opinion, clearly vindicates Imclone's and Samuel Waksal's scientific approach, at that time, to explain Cetuximab trials.

The 1st finding from this new study was that mice injected with Cetuximab-treated hEGFR-CT26 cells, a mouse colorectal cancer cell line (mCRC) expressing human EGFR, are protected against secondary challenge with parental CT26 cancer cells. This suggested that Cetuximab treatment of CT26 cancer cell induced immunogenic cell death (F stands for FOLFIRI, a standard chemotherapy regimen for mCRC that does not induce immunogenic cell death. FT stands for freeze-thaw).

The 2nd finding was that Cetuximab + FOLFIRI induced immunogenic cell death in human cancer cells with 

(a) wild-type forms of oncogenes KRAS  and BRAF or
(b) with KRAS^G13D and KRAS^G12D mutations 

(c) but it did not induce immunogenic cell death in cancer cells with with BRAF and KRAS^G12V mutations. In those cell lines immunogenic cell death could be restored by combining Cetuximab + FOLFIRI with inhibitors of mutated BRAF (PLX4032, PLX) and MEK (trametinib, tram).

In summary, these data define molecular markers that make cancer cell susceptible to Cetuximab + chemotherapy combination.

Now lets go back to Imclone's story. At the time of original trials, in late 90s, patients stratification based on tumor genetic makeup was a novel idea and not all mutations were known. In fact, when Imclone's management suggested to conduct trial based on patient stratification, European Medical Agency (FDA equivalent in EU) refused to consider it, such unorthodox and unheard of idea it was at that time.

We can now understand why Cetuximab's original trials gave inconsistent results. Without patient stratification, antibody-based therapy rarely shows statistically significant benefits over placebo in cancer patients. Science behind Cetuximab was so ahead of its time that clinical trials were not ready for it and neither were Imclone's management (note that few years later after Imclone's debacle Cetuximab was approved by FDA and it is marketed by Bristol-Myers Squibb).

David Usharauli

Dying cells release eicosanoid lipid PGE2 to suppress sterile inflammation and immunogenicity

PGE2 is the most abundant eicosanoid lipid in the inflammatory environment and acts via its receptors EP4 and EP2. Earlier this week I wrote about new study that showed the role of PGE2 in suppressing excessive inflammatory response to endogenous microbiota that gained access to internal organs during systemic infection such as sepsis. 

This time I am reviewing another new paper about PGE2 from PNAS wherein the authors showed that PGE2 released during the process of cell death modulates its immunogenicity.  

Initially, the authors showed that supernatants from cells cultures undergoing various forms of cellular death (freeze-thaw, cisplatin, etoposide or ATP + LPS combination) rather than inducing TNF-α from macrophages it could actually suppress macrophage's response to a canonical inflammatory stimulus such as gram-negative bacterial wall-derived endotoxin, LPS.

Since supernatants treated with DNase I, RNase A, proteinase K or trypsin retained its suppressive activity on the LPS induced production of TNF-α, the authors focus on lipids. Indeed, lipid cellular fraction could reproduce inhibitory effect of the necrotic cell supernatant.

Next, they found that PGE2 was highly enriched in these supernatants and could mediate its suppressive effect.

Synthesis of PGE2 is catalyzed by two cyclooxygenase enzymes, COX-1 and COX-2. Pre-treatment of cells with indomethacin, an inhibitor of COX-1 and COX-2 enzymes, reduced suppressive effect of necrotic cell supernatant.

Finally, using CRISPR/Cas9 gene editing technology, the authors constructed COX-2 deficient tumor cell lines and tested their growth pattern in mice. As expected, growth of COX-2 deficient tumor cell  lines were delayed in absence of COX-2 enzyme.

In summary, this study showed that cells undergoing sterile cell death (for example, during excessive tumor growth), release suppressive lipid, PGE2, and this mechanism represents one of the natural anti-inflammatory processes that is hijacked by tumors to evade efficient immune detection. Aspirin's beneficial effect as an anti-cancer therapy could be attributed to its effect on PGE2. 

David Usharauli

DC vaccines produced with tumor cells undergoing immunogenic cell death

Dendritic cells are the most potent activators of naïve T cells. A lot of effort has been spent to develop DC-based cellular vaccine. We even had some success in mouse models. But, so far no DC based vaccine worked in humans to significantly prolong patients survival. It is not surprising that only company that developed FDA-approved DC vaccine prostate cancer went bankrupt. There are many reasons as to why it is so hard. For one, there are several types of dendritic cells. Secondly, just pulsing [tumor] antigens on dendritic cells were found not to be sufficient.

Few weeks ago, Science Translational Medicine published study wherein the authors had tested next-generation tumor vaccine produced by pulsing DCs with tumor cells undergoing immunogenic cell death.

Basically, the authors had used a high-grade glioma (HGG) cell lines as a source of tumor. To generate immunogenic cell death, tumor cells were exposed to hypericin-based photodynamic therapy (Hyp-PDT). This treatment induces expression/release of several damage-associated molecular patterns (DAMPs) acting as potent danger signals for DCs. When mice were injected with DCs pulsed with Hyp-PDT treated glioma cells, they showed resistance to subsequent live glioma brain challenge.

Mechanistically, tumor protection provided by Hyp-PDT DCs depended on (1) DAMPs, (2) DC-specific MyD88, (3) CD8 T cells (T cells data are shown here).

Importantly, Hyp-PDT DC vaccines improved survival of brain tumor-bearing mice when applied therapeutically and in combination with standard-of-care therapy such as temozolomide (TMZ).

In summary, this study showed that hypericin-based photodynamic therapy could generate immunogenic tumor cells required for proper activation of DC for vaccination purpose. Conceptually, such mode of DCs preparation makes a lot of sense, but there are several technical challenges before this system could be translated into clinic, as discussed by the authors (such as, how to avoid presence of "surviving" live tumor cells within DC vaccine, route of DC vaccine injection [the authors have used intra-peritoneal injection, for some reason], what type of DC is the best for this purpose). 

David Usharauli

Tumor-tailored "immunogenic" chemotherapy sensitizes tumor to immune attack

Today journal Immunity published an excellent preclinical research study about tumor immunity. In this paper the authors conducted methodical analyses of the role of tumor-tailored chemotherapy on its immunogenicity and synergy with checkpoint immunotherapy.

It is now universally accepted that T cell-oriented immunotherapy (with anti-PD1, anti-CTLA4) represents important medical progress in treatment of [solid] cancers. Still, not every cancer type is responsive to such immunotherapy. However, this is about to change when personalized, molecular evidence-based tumor therapy becomes widely affordable [technology already exists]. This paper is an example how this would be accomplished.

For this study the authors have used KP lung adenocarcinoma mouse model that express "endogenous mutant Kras and deleted Trp53 alleles in lung epithelial cells upon administration of adenovirus expressing Cre recombinase". This tumor was non-responsive to T cell infiltration or checkpoint inhibition or combination of paclitaxel (Ptax) and carboplatin (Carbo), Ptax-Carbo (chemotherapeutics).

Next, the authors hypothesized that this non-responsiveness could be due to lack of "immunogenicity" of KP cells. To examine this, they conducted in vitro "immunogenic cell death" test with several KP cell lines using FDA approved chemotherapy drugs. High mobility group box 1 (HMGB1) release was used as a surrogate marker for chemotherapy drug-induced tumor cell immunogenicity. They showed that combination of mafosfamide (Maf), which is the active metabolite of cyclophosphamide (Cyc), and oxaliplatin (Oxa) stimulated HMGB1 release by all KP tumor cell lines both in vitro and in vivo.

As expected, in vivo application of Oxa-Cyc combination reduced tumor burden in KP mice.

Effect of Oxa-Cyc was mediated via T cells since its effect was abolished in T cell-deficient RAG KO mice or in KP mice depleted of CD8 T cells.

Interestingly, Oxa-Cyc effect was also depended on TLR4 expression on CD11b⁺/CD11c⁺ myeloid cells.

More importantly, the authors showed that Oxa-Cyc treatment synergized with checkpoint inhibitors, anti-PD-1 + anti-CTLA-4, to control lung tumor burden in KP mice.

Finally, the authors expanded this observation to include two other tumor types, MCA205 fibrosarcoma and CT26 colon carcinoma, and showed selective tumor-tailored chemotherapy synergized with checkpoint inhibitors to control tumor burden in these models as well.

In summary, the results in this study indicate that checkpoint inhibitors alone may not provide any benefits to patients where tumor lack T cell infiltration due to absence of tumor "immunogenic death". However, application of selective, tumor-tailored chemotherapeutics (to induce tumor immunogenic cell death and attract T cells) would synergize with checkpoint inhibitors (working then on tumor-infiltrating T cells) in non-responsive tumor patients and improve clinical outcome of the tumor therapy. In practical terms this would mean development of in vitro tests to examine patient's tumor cell "immunogenicity" response to chemotherapeutics on a personalized basis.

David Usharauli