735.

735 autopsy reports.

Report after report, the conviction of Stephen was growing. What he was observing could not have happened by chance. 

This time, he was sure. A pattern was emerging.

His hypothesis was finally confirmed. His father James had been a successful surgeon, founder of the modern scientific pathology, but his discovery could make him even more famous. The impact of what he had in front of him was far greater.

He picked up his stylograph, look at the date on the calendar, and sat down at his desk. In the light of his candle, he started writing frantically.

 “When a plant goes to seed, its seeds are carried in all directions; but they can only live and grow if they fall on congenial soil. While many researchers have been studying ‘the seeds’, the properties of ‘the soils’ may reveal valuable insights into the metastatic peculiarities of cancer cases.” (1)

In 1889, Stephen Paget just laid the first brick of his “soil-seed” theory. He observed that the organ distribution of metastases in 735 breast cancers was not random: a tumor cell (“the seed”) needed a proper “soil” to grow.

This “soil” was named “tumor microenvironment”.

But the microenvironment of a tumor is a very complex matter.

Within the tumor microenvironment, many cell types are present, intertwined, and permanently interacting with each other. Immune cells, blood vessels, collagen, enzymes, glycoproteins, and signaling molecules constitute a vast ecosystem. 

At the heart of this ecosystem, the cytokine interleukin-1 (IL-1) – via its recognition by type I receptors IL-1R - transmits a signal between the tumor cell and stromal cell, which stimulates the tumor to grow (2).

To be accurate, it has been found that a type I IL-1R/IL-1RAP complex is required for signaling by all IL-1 agonists and for high affinity binding by IL-1 (3). This subsequent new element, IL1RAP, became the focus of potential novel therapeutic strategies against cancer disease.

But within the large family of cancers (more than 200 different members), Myeloid Leukemia (AML & CML) diseases show specificity.

A specificity that turns out to be an obstacle to therapeutic treatment.

Solid tumors contain a non-homogeneous group of immature cells known as cancer stem cells, which are constantly being formed and which produce tumor-forming mature cancer cells (4). They so play a critical role in cancer proliferation, maintenance, progression, and recurrence.

In 2010, a team confirmed that IL1RAP was the top candidate for Chronic Myeloid Leukemia (CML) treatment since IL1RAP was a biomarker consistently overexpressed in hematopoietic stem and progenitor cells (HSPC). They so were able to generate an IL1RAP-targeting antibody that killed CML CD34+CD38− cells, but not corresponding normal cells, through antibody-dependent cell-mediated cytotoxicity (ADCC). (5)

This concept, which demonstrates a unique concept for the possible eradication of CML stem cells, gave birth to the Swedish biotech company Cantargia.

Only two years later, the overexpression of IL1RAP was demonstrated in Acute Myeloid Leukemia (AML) disease (6) and the same anti-IL1RAP antibody strategy has been deployed again.

But if the antibody strategy works, what is the next step?

If an anti-IL1RAP antibody can lead to cancer stem cell inhibition and potential destruction, can we simultaneously trigger a T cell-mediated immune response?

The team of Walid Warda decided to answer this question go for CAR-T development with Diaclone (7).

Diaclone first produced a monoclonal antibody by immunizing BALB/c mice with the recombinant human protein IL-1RAP.

The nucleotide sequences encoding the hypervariable regions of this immunoglobulin, constituting the scFv, coupled with those encoding the third generation T activation sequences (CD28-4.1BB-CD3ζ), were cloned into a lentiviral vector.

The obtained construct also includes an inducible cell suicide system consisting of a Rimiducid®-inducible gene encoding caspase-9 (iCASP9 gene), which allows the removal of unwanted activated T-cell CARs (safety switch), and a gene encoding a surface protein (∆CD19) allowing the identification and selection of T-cell CARs.

Activated T cells were then established from healthy donors or patients and transduced with lentiviral construct and anti-IL1RAP-CAR-T cells were tested on mice.

This preclinical work demonstrates for the first time the whole production and validation process of CAR T-cells directed against IL-1RAP-expressing CML stem cells, conducting to tumor regression.

A better understanding of the tumor environment has led to the discovery of the role played by cytokines in cellular communication, in particular interleukin-1 between stromal and tumor cells. One of the components of the IL-1 receptor, IL1-RAP, has thus been identified as a marker of interest in the context of CML treatments, activating NK cells and blocking tumor growth at the same time.

The distribution of secondary growths in cancer of the breast. Stephen Paget, Lancet, 1889.

Suppression of chronic myelogenous leukemia colony growth by interleukin-1 (IL-1) receptor antagonist and soluble IL-1 receptors: a novel application for inhibitors of IL-1 activity. Estrov Z, Kurzrock R, Wetzler M, Kantarjian H, Blake M, Harris D, Gutterman JU, Talpaz M. Blood. 1991 Sep 15;78(6): 1476-84.

IL-1 Receptor Accessory Protein Is an Essential Component of the IL-1 Receptor - Emily B. Cullinan, Lia Kwee, Perla Nunes, David J. Shuster, Grace Ju, Kim W. McIntyre, Richard A.Chizzonite and Mark A. Labow. J Immunol November 15, 1998, 161 (10) 5614-5620.

Bonnet, D., Dick, J. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 3, 730–737 (1997). https://doi.org/10.1038/nm0797-730

Isolation and killing of candidate chronic myeloid leukemia stem cells by antibody targeting of IL-1 receptor accessory protein. Marcus Järås, Petra Johnels, Nils Hansen, Helena Ågerstam, Panagiotis Tsapogas, Marianne Rissler, Carin Lassen, Tor Olofsson, Ole Weis Bjerrum, Johan Richter, and Thoas Fioretos

Overexpression of IL-1 receptor accessory protein in stem and progenitor cells and outcome correlation in AML and MDS. Barreyro, Will B, Bartholdy B, Zhou L, Todorova TI, Stanley RF, Ben-Neriah S, Montagna C, Parekh S, Pellagatti A, Boultwood J, Paietta E, Ketterling RP, Cripe L, Fernandez HF, Greenberg PL, Tallman MS, Steidl C, Mitsiades CS, Verma A, Steidl U.

CML Hematopoietic Stem Cells Expressing IL1RAP Can Be Targeted by Chimeric Antigen Receptor–Engineered T Cells. Walid Warda, Fabrice Larosa, Mathieu Neto Da Rocha, Rim Trad, Eric Deconinck, Ziad Fajloun, Cyril Faure, Denis Caillot, Marius Moldovan, Severine Valmary-Degano, Sabeha Biichle, Etienne Daguindau, Francine Garnache-Ottou, Sebastien Tabruyn, Olivier Adotevi, Marina Deschamps and Christophe Ferrand