Author:

Alberto Mantovani

Abstract

The development of tumor immunology and immunotherapy has spanned over a century of experimental and clinical research. Fundamental building blocks of the tumor immunology cathedral were generated in Europe, including: the discovery of immunogenicity of cancer; the identification of the first tumor antigens; the discovery of the protumor function of tumor-associated macrophages, behaving as “corrupted policemen”; the importance and prognostic significance of the tumor microenvironment; the discovery of the first checkpoint (CTLA-4) to be targeted in checkpoint blockade immunotherapy. Immunorevolution is in its infancy and progress in fundamental immunological mechanisms is essential to address clinical challenges.

1. Introduction

Immunotherapy is now part of the oncology armamentarium and we are living what has been defined “Immunorevolution”(Mantovani 2020). Dissection of the cellular and molecular components of the immune responses in cancer has provided the conceptual and molecular tools to the flourishing of immunotherapy in its various versions, from immune checkpoint blockade (ICB) to cell therapies. At the turn of the millennium, tumor immunology was regarded with skepticism by the vast majority of oncologists, because of the repeated failures of clinical trials based on immunostimulatory compounds. A cancer cell-centric view of neoplasia prevailed, cristallized in 6 hallmarks which did not include innate and adaptive immunity (Hanahan and Weinberg 2000). The Author of this review, and immunologists at large, strongly argued that inflammatory cells are an essential component of tumor progression (e.g. Balkwill and Mantovani 2001) and over the years a paradigm shift occurred with the recognition that the TME is as important as the neoplastic cell in carcinogenesis and therapy. Progress in the dissection of the complex cellular and molecular basis of immune responses to cancer has provided the conceptual and molecular tools for the flourising of cancer immunotherapy.

Here I will concisely review selected major building blocks generated in Europe to the construction of the current tumor immunology and immunotherapy architecture. The Author of this essay is not an historian of science and has long been part of this endeavour, with a focus on the role of innate immunity (Bottazzi et al. 1983, Balkwill and Mantovani 2001, Mantovani et al. 2008, Mantovani 2020, Mantovani and Garlanda 2023). Hopefully, this perspective “from the battle field” has not distorted the identification of major contributions.

2. Selected Milestones

2.1. Tumor immunogenicity

Clinical observation, including occasional spontaneous rejections of melanoma associated with leukocyte infiltration and the increased incidence of selected tumors in immunosuppressed patients, pointed to the role of immunity in cancer. However, the rigorous demonstration of the existence and function of tumor antigens, recognised by the adaptive immune system and capable to mediate anticancer resistance, rested in carcinogenesis. In experiments conducted in inbred (genetically identical) mice and rats. A typical carcinogen used in these studies was 3-methylcholantrene. The immunogeniticy of carcinogen-caused sarcomas was shown by investigators in USA (E.J. Foley; R. Prelm) and Europe (George Klein in Sweden and Robert Baldwin in UK (Klein et al. 1960, Baldwin 1966). The work of George Klein is notable in particular because of the demonstration of immunity in the autochtonous host. George Klein also originally demonstrated the occurrence of tumor suppressor genes in a series of elegant experiments, a ground breaking contribution all too frequently forgotten (Harris et al. 1969). These seminal observations were followed by the demonstration that the prime cells orchestrating and mediating anticancer resistance were T cells, the current target of checkpoint blockade immunotherapy and CAR-T cell therapy.

2.2. Methodological pillars and molecular tools

Progress in immunology has been fostered by the development of methods and technology, as generally true in science. The enumeration of methodological advances which have fostered the progress of tumor immunology is beyond the scope of this paper and only two will be mentioned here. In the late ’60 John Charles Cerottini in Lausanne developped the 51Cr release assay to measure the killing of tumor target cells by cytotoxic T lymphocytes and other effector cells. This simple assay has served as an enabling technology to dissect the function of T cells and NK cells. Non-radioactive variants of this assay are in current use and are essential for instance to assess ICB immunotherapy and the function of CAR-T cells. The development of the monoclonal antibody (mAb) technology by George Kohler and Caesar Milstein in Cambridge, UK, has revolutionized Biology and Medicine. Subsequent development of this technology included human recombinant mAbs (UK) and camelide mAbs, called nanobodies (Belgium). MAbs have first revolutionized diagnostic procedures and subsequently therapy. Therapeutic approaches now include, in addition to naked mAbs, antibody-drug conjugates (ADC), and bispecific mAb. It can be argued that anti-CD20 (developped in Europe) and anti-Her2 have represented the first targeted therapies.

2.3. The discovery of NK cells

The presence of lymphoid cells endowed with the capacity to kill tumor target cells in a 51Cr release assay independently was recognized by Ron Herberman and colleagues in USA and by Rolf Kiessling and colleagues in Sweden (Kiessling et al. 1975), who named the cells natural killer (NK). Subsequently, Klaus Karre at the Karolinska Institute in Sweden proposed the “missing self” model of function of NK cells, whereby these effectors recognize the absence of self and kill virus infected or tumor cells which have lost their “identity card”, represented by class I MHC. Lorenzo and Alessandro Moretta in a seminal series of papers went on to identify the activating and inhibitory receptors of NK cells (Moretta et al. 1996). These results showed that T cells and NK cells are complementary in resistance against viruses and cancer. Viral infection and neoplastic transformation can result in loss of MHC class I expression, essential for T cell recognition, but loss of MHC is recognized by NK cells. NK cell-based cellular therapies are currently undergoing clinical evaluation.

2.4. Identification of tumor antigens

Although the studies outlined under 2.1 clearly indicated the existance of tumor antigens capable of eliciting an effective immune response, their molecular identification remained for a long time elusive. In a pioneer study in the URSS Abelev (Abelev et al. 1963) in the early ’60 discovered alfa-fetoprotein as an antigen produced by hepatoma cells. Abelev was a dissident and I was told that attending his lectures had implications beyond science. Alfa-fetoprotein is still in current use as a biomarker. Unfortunately, this seminal discovery was not followed by a flourishing of tumor immunology in the Soviet Union, arguably for non-scientific context reasons (Mantovani 2023).
In a seminal paper (van der Bruggen et al. 1991) Thierry Boon and colleagues in Belgium were the first to molecularly identify tumor antigen expressed by melanoma cells (MAGE) and recognized by cytotoxic T cells. Candidate tumor antigens are now identified using sequencing and bioinformatic tools and are used for personalized vaccines based on the mRNA technology, combined with ICB immunotherapy.

3. Innate immunity, inflammation and macrophages

It is generally held that in the XIX century the German pathologist Rudolf Virchow was the first to perceive the presence of inflammatory components in cancer (Mantovani 2020). Clinical observations and experiments in mouse models indicated that appropriately activated and oriented inflammatory reactions and cells involved in them could elicit tumor regression. Coley’s toxin in USA was a bacterial product developped in USA at the beginning of XX century which elicited clinical responses. The use of bacterial products was revisited between the 60s and 80s in preclinical models and patients. BCG was the most widely used bacterial immunostimulant, following early clinical work conducted by George Mathé in Paris. However, the results of many clinical trials conducted in a wide range of tumors were disappointing and the only remnant of the bacterial era of cancer immunotherapy is the usage of intravescical BCG in bladder cancer (Mantovani 2020). The clinical failure of bacterial immunostimulants prompted widespread skepticism as to the relevance of tumor immunology in the fight against cancer. Activated macrophages were considered major effectors of the antitumor activity of bacterial products because Robert Evans and Peter Alexander in London UK and others had shown that these cells could be driven to kill tumor cells in vitro. Moreover, Robert Evans had shown that macrophages were a major component of tumor tissues and that they were bona fide normal cells and not tumor cells in disguise (Mantovani et al. 2017, Mantovani 2020). In the late ’70s - early ’80s, in contrast to the generally held view of phagocytes as antitumor effectors, it was found that tumor-associated macrophages (TAM) isolated from metastatic mouse and human tumors promoted tumor growth. Accordingly, chemoattractants later to be identified as members of the chemokine family attracted monocytic precursors in the tumor tissue (Bottazzi et al. 1983) and that TAM behaved as “corrupted policemen”, providing growth factors, favouring angiogenesis and suppressing the activity of innate (NK) and adaptive (T) lymphoid cells. This at the time countercurrent view contributed to highlighting the importance of the TME in tumor progression and as a therapeutic target. Indeed macrophages are double edged swords. On the one hand they drive progression and, by expressing PD-L1, trigger T cell suppression, and resistance to ICB immunotherapy. On the other hand, when reeducated by T cells and in the context of Interferon-driven immune reactions phagocytes can contribute to antitumor resistance.

4. Prognostic significance and therapy: checkpoints

It has long been known that the infiltration of immunocompetent cells is associated with prognostic significance, with seemingly opposite impact of lymphocyte versus phagocyte infiltration, Scattered observations on T cells were put on a firm methodological basis by work conducted by Jerome Galon, Hervé Fridman and colleagues in Paris focusing on colorectal cancer (Galon et al. 2006). In this rigorous landmark study, T cell infiltration quantified as “Immunoscore” was shown to be a strong positive prognostic factor independent of other known indicators. These results were subsequently validated in large international clinical studies and provided impetus for the development of immunotherapy approaches. Dissection of molecular mechanisms of immunity in the ’80s and ’90s, highlighted how host protective immune responses require a balance between accelerators and breaks. Negative regulators of immune responses include professional cells (Treg and myeloid suppressor cells including TAM) and molecular brakes acting at different levels. The discovery of the T cell brakes called checkpoints has had a transformative impact in oncology with the development of ICB. Pierre Goldstein and colleagues in Marseille, France, were interested in dissecting the mechanism of action of CD8 cytotoxic T cells. In this context they identified a new member of the immunoglobulin superfamily, CTLA-4 (Brunet et al. 1987). Capitalizing on this discovery, Jim Allison and colleagues showed that an anti-CTLA-4 mAb had antitumor activity in the mouse and, later, that targeting CTLA-4 cured a substantial fraction (~20%) of patients with advanced melanoma. Thus, Pierre Goldstein in Marseille laid a fundamental building block for the subsequent development of cancer immunotherapy.

5. Concluding remarks

The previous sections of this paper summarized selected milestones in the development of tumor immunology and immunotherapy from a European and personal perspective. In nature of fact, progress in this field, and in immunology in general, has crossed national borders and oceans and should be perceived as a global enterprise. The motto of the International Union of Immunological Societies (IUIS) is “Immunology without borders” (the Author has served as President of IUIS from 2016 to 2019). As usual, the considerable progress made, translated into clinical benefit, has opened NEW key fundamental questions. These include for instance the fundamental mechanisms of immunological memory and the mechanisms of primary and secondary resistance to immunotherapy. A better understanding using new technologies of the complex interplay between tumor and host, at the primary site and metastasis, may pave the way to innovative immunology-based diagnostic and therapeutic strategies.

References

Abelev, GI, Perova, SD, Khramkova, NI, et al. (1963). Production of embryonal alpha-globulin by transplantable mouse hepatomas. Transplantation 1: 174-180.

Baldwin, RW (1966). Tumour-specific immunity against spontaneous rat tumours. Int J Cancer 1(3): 257-264.

Balkwill, F, Mantovani, A (2001). Inflammation and cancer: back to Virchow? Lancet 357(9255): 539-545.

Bottazzi, B, Polentarutti, N, Acero, R, et al. (1983). Regulation of the macrophage content of neoplasms by chemoattractants. Science 220(4593): 210-212.

Brunet, JF, Denizot, F, Luciani, MF, et al. (1987). A new member of the immunoglobulin superfamily--CTLA-4. Nature 328(6127): 267-270.

Galon, J, Costes, A, Sanchez-Cabo, F, et al. (2006). Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 313(5795): 1960-1964.

Hanahan, D Weinberg, RA (2000). The hallmarks of cancer. Cell 100(1): 57-70.

Harris, H, Miller, OJ, Klein, G, et al. (1969). Suppression of malignancy by cell fusion. Nature 223(5204): 363-368. Kiessling, R, Klein, E Wigzell, H (1975). "Natural" killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype. Eur J Immunol 5(2): 112-117.

Klein, G, Sjogren, HO, Klein, E, et al. (1960). Demonstration of resistance against methylcholanthrene-induced sarcomas in the primary autochthonous host. Cancer Res 20: 1561-1572.

Mantovani, A (2020). Immune function. From the tumor microenvironment to therapeutic targeting. World Cancer report. C. P. Wild, Weiderpass, E., Stewart, B.E. Lyon, IARC - Int. Agency for Research on Cancer: 215220.

Mantovani, A (2023). Perchè la scienza ha bisogno della democrazia (e viceversa). Micromega 5: 24-31.

Mantovani, A Garlanda, C (2023). Humoral Innate Immunity and Acute-Phase Proteins. N Engl J Med 388(5): 439-452.

Mantovani, A, Marchesi, F, Malesci, A, et al. (2017). Tumour-associated macrophages as treatment targets in oncology. Nat Rev Clin Oncol 14(7): 399-416.

Mantovani, A, Romero, P, Palucka, AK, et al. (2008). Tumour immunity: effector response to tumour and role of the microenvironment. Lancet 371(9614): 771-783.

Moretta, A, Bottino, C, Vitale, M, et al. (1996). Receptors for HLA class-I molecules in human natural killer cells. Annu Rev Immunol 14: 619-648.

Van der Bruggen, P, Traversari, C, Chomez, P, et al. (1991). A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 254(5038): 1643-1647.