Professor Jean Bousquet
email: jean.bousquet@orange.fr

Josep M Anto

Susana C Fonseca

Guido Iaccarino

Alain Vidal

Aziz Sheikh

Cezmi A Akdis

Cabbage and fermented vegetables: from death rate heterogeneity in countries to candidates for mitigation strategies of severe COVID-19

Keywords: COVID-19, diet, sulforaphane, Lactobacillus, Angiotensin converting enzyme 2, kimchi, cabbage, fermented vegetable Reactive oxygen species Transmissible Gastroenteritis Coronavirus Infection

Large differences in COVID-19 death rates exist between countries and between regions of the same country. Some very low death rate countries such as Eastern Asia, Central Europe or the Balkans have a common feature of eating large quantities of fermented foods. Although biases exist when examining ecological studies, fermented vegetables or cabbage were associated with low death rates in European countries. SARS-CoV-2 binds to its receptor, the angiotensin converting enzyme 2 (ACE2). As a result of SARS-Cov-2 binding, ACE2 downregulation enhances the angiotensin II receptor type 1 (AT 1 R) axis associated with oxidative stress. This leads to insulin resistanceas well as lung and endothelial damage, two severe outcomes of COVID-19. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is the most potent antioxidant in humans and can block the AT 1 R axis. Cabbage contains precursors of sulforaphane, the most active natural activator of Nrf2. Fermented vegetables contain many lactobacilli, which are also potent Nrf2 activators. Three examples are given: Kimchi in Korea, westernized foods and the slum paradox. It is proposed that fermented cabbage is a proof-of-concept of dietary manipulations that may enhance Nrf2associated antioxidant effects helpful in mitigating COVID-19 severity.

Introduction

A COVID-19 epidemic started in China and then disseminated to other Asian countries before becoming a pandemic. There is a large variability across countries in both incidence and mortality, and most of the current debates on COVID-19 focus on the differences between countries. Several intertwined factors can be proposed: social distancing, health system capacity, age of the population, social lifestyle (gathering of family/friends, social behavior), testing capacity and/or timing and intensity of the first outbreak.German fatalities are strikingly low as compared to many European countries. Among the several explanations proposed, an early and large testing of the population was put forward [START_REF] Stafford | Covid-19: Why Germany's case fatality rate seems so low[END_REF] as well as social distancing.

However, little attention has been given to regional within-country differences that may propose new hypotheses.

It would appear that the pandemic has so far resulted in proportionately fewer deaths in some Central European countries, the Balkans, China, in most Eastern Asian countries as well as in many Sub-Saharan African countries. Several reasons can explain this picture. One of them may be the type of diet in these low mortality countries. [START_REF] Bousquet | Rapid Response: Why Germany's case fatality rate seems so low: Is nutrition another possibility[END_REF]3 Diet has been proposed to mitigate COVID-19. [START_REF] Iddir | Strengthening the Immune System and Reducing Inflammation and Oxidative Stress through Diet and Nutrition: Considerations during the COVID-19 Crisis[END_REF][START_REF] Cena | Coronavirus Disease (COVID-19-SARS-CoV-2) and Nutrition: Is Infection in Italy Suggesting a Connection?[END_REF] Some foods or supplements may have a benefit on the immune response to respiratory viruses. However, to date, there are no specific data available to confirm the putative benefits of diet supplementation, probiotics, and nutraceuticals in the current COVID-19 pandemic. [START_REF] Infusino | Diet Supplementation, Probiotics, and Nutraceuticals in SARS-CoV-2 Infection: A Scoping Review[END_REF] News and social media platforms have implicated dietary supplements in the treatment and prevention of COVID-19 without evidence. [START_REF] Adams | Myth Busters: Dietary Supplements and COVID-19[END_REF] In this paper, we discuss country and regional differences in COVID-19 deaths. We attempt to find potential links between foods and differences at the national or regional levels in the aim to propose a common mechanism focusing on oxidative stress that may be relevant in COVID-19 mitigation strategies.

We used cabbage and fermented vegetable as a proof-of-concept.

1-Biases to be considered

According to the Johns Hopkins coronavirus resource center (https://coronavirus.jhu.edu), one of the most important ways of measuring the burden of COVID-19 is mortality. However, death rates are assessed differently between countries and there are many biases that are almost impossible to assess. Using the rates of COVID-19 confirmed cases is subject to limitations that are similar to or even worse than the differences in the use of COVID-19 testing.

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This article is protected by copyright. All rights reserved Differences in the mortality rates depend on health care systems, the reporting method and many unknown factors. Countries throughout the world have reported very different case fatality ratios -the number of deaths divided by the number of confirmed cases -but these numbers cannot be compared easily due to biases. On the other hand, for many countries, the methodology used to report death rates in the different regions is standardized across the country.

We used mortality per number of inhabitants to assess death rates, as proposed by the European Center for Disease Prevention and Control (ecdc, https://www.ecdc.europa.eu/en),and to report trends with cutoffs at 25, 50, 100 and 250 per million.

Our hypothesis is mostly based on ecological data that are hypothesis-generating and that require confirmation by proper studies.

2-Multifactorial origin of the COVID-19 epidemic

Like most diseases, COVID-19 exhibits large geographical variations which frequently remain unexplained [START_REF] Sunyer | Geographic variations in the effect of atopy on asthma in the European Community Respiratory Health Study[END_REF] . The COVID-19 epidemic is multifactorial, and factors like climate, population density, age,phenotype and prevalence of non-communicable diseases are also associated to increased incidence and mortality [START_REF] Kissler | Projecting the transmission dynamics of SARS-CoV-2 through the postpandemic period[END_REF] .

Diet represents only one of the possible causes of the COVID-19 epidemic and its importance needs to be better assessed. Some risk factors for the COVID-19 epidemics are proposed at the individual and country levels in Table 1.

3-Ecological data on COVID-19 death rates

When comparing death rates, large differences exist between and within countries and the evolution of the pandemic differs largely between countries (Figure 1). Although there are many pitfalls in analyzing death rates for COVID-19, 3 the evolution of death rates between May 20 and July 18 shows a dramatic increase in Latin America and only some increase in European countries, certain African countries, the Middle East, India, Pakistan and some of the South East Asian countries. However, there is no change in the very low death rates of Cambodia, China, Japan, Korea, Lao, Malaysia, Taiwan, Vietnam and of many Sub-Saharan African countries, Australia and New Zealand. This geographical pattern is very unlikely to be totally due to reporting differences between countries.

In some high death-rate countries such as Italy (Figure 2), variations are extremely large from 50 per million in Calabria to over 1,600 in Lombardia. In Switzerland, the French-and Italian-speaking cantons have a far higher death rate than the German-speaking ones (Office fédéral de la santé publique, Switzerland) (Figure 3). It may be proposed that the high-death rate cantons were contaminated by French

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This article is protected by copyright. All rights reserved and Italian people. However, the Mulhouse airport serves the region of Basel (Switzerland), the Haut-Rhin department (France) and the region of Freiburg (Germany). There was a COVID-19 outbreak in the Haut-Rhin department, in particular in Mulhouse and Colmar. The death rate for COVID-19 (May 20, 2020) was 935 per million inhabitants in France but only 10 to 25 in Switzerland and 7 in Germany. It is important to consider these regional differences since reporting of deaths is similar within the country and many factors may be considered.

In many Western countries, large cities (e.g. London, Madrid, Milan, New York, Paris) have been the most affected. This seems to be true also for many countries in which the rural areas have much fewer cases.

The number of deaths is relatively low in Sub-Saharan Africa compared to other regions, and the low population density (which applies in rural areas but not in megacities such as Cairo or Lagos) or the differences in health infrastructure are unlikely to be the only explanation. [START_REF] Rosenthal | COVID-19: Shining the Light on Africa[END_REF] It has been proposed that hot temperature may reduce COVID-19, but, in Latin American countries, death rates are high (e.g. Brazil, Ecuador, Peru and Mexico).

4-Is diet partly responsible for differences between and within countries?

Nutrition may play a role in the immune defense against COVID-19 and may explain some of the differences seen in COVID-19 between and within countries 3 . In this concept paper, raw and fermented cabbage were proposed to be candidates.

To test the potential role of fermented foods in the COVID-19 mortality in Europe, an ecological study, the European Food Safety Authority (EFSA) Comprehensive European Food Consumption Database, was used to study the country consumption of fermented vegetables, pickled/marinated vegetables, fermented milk, yoghurt and fermented sour milk. [START_REF] Fonseca | Association between consumption of fermented vegetables and COVID-19 mortality at a country level in Europe MEDRXIV[END_REF] Of all the variables considered, including confounders, only fermented vegetables reached statistical significance with the COVID-19 death rate per country. For each g/day increase in consumption of fermented vegetables of the country, the mortality risk for COVID-19 was found to decrease by 35.4% (Figure 4).

A second ecological study has analyzed cruciferous vegetables (broccoli, cauliflower, head cabbage (white, red and savoy cabbage), leafy brassica) and compared them with spinach, cucumber, courgette, lettuce and tomato [START_REF] Fonseca | Association between consumption of vegetables and COVID-19 mortality at a country level in Europe[END_REF] .Only head cabbage and cucumber reached statistical significance with the COVID-19 death rate per country. For each g/day increase in the average national consumption of some of the vegetables (head cabbage and cucumber), the mortality risk for COVID-19 decreased by a factor of 11, to 13.6 %. The negative ecological association between COVID-19 mortality and consumption of cabbage and cucumber

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This article is protected by copyright. All rights reserved supports the a priori hypothesis previously reported. However, these are ecological studies that need to be further tested.

Another diet component potentially relevant in COVID-19 mortality may be the food supply chain and traditional groceries. [START_REF] Baker | Food systems transformations, ultra-processed food markets and the nutrition transition in Asia[END_REF] The impact of the long supply chain of food on health is measurable by an increase in metabolic syndrome and insulin resistance. [START_REF] Santulli | We are What We Eat: Impact of Food from Short Supply Chain on Metabolic Syndrome[END_REF] Therefore, areas that are more prone to short supply food and traditional groceries may have been able to better tolerate COVID-19 with a lower death toll. These considerations may be partly involved in the lower death rates of Southern Italy compared to the Northern part (Figure 2).

5-Fermented foods, microbiome and lactobacilli

The fermentation process, born as a preservation method in the Neolithic age, enabled humans to eat notso-fresh food and to survive. [START_REF] Peters | Metabolites of lactic acid bacteria present in fermented foods are highly potent agonists of human hydroxycarboxylic acid receptor 3[END_REF] Indigenous fermented foods such as bread, cheese,vegetables and alcoholic beverages have been prepared and consumed for thousands of years, are strongly linked to culture and tradition, especially in rural households and village communities, and are consumed by hundreds of millions of people. [START_REF] Azam-Ali | Fermented fruits and vegetables[END_REF] Fermented foods are "foods or beverages made via controlled microbial growth (including lactic acid bacteria (LAB)) and enzymatic conversions of food components." [START_REF] Marco | Health benefits of fermented foods: microbiota and beyond[END_REF] Not all fermented foods contain live cultures, as some undergo further processing after fermentation: pasteurization, smoking, baking, or filtration. These processes kill or remove the live microorganisms in foods such as soy sauces, bread, most beers and wines as well as chocolate. Live cultures can be found in fermented vegetables and fermented milk (fermented sour milk, yoghurt, probiotics, etc.).

Most traditional foods with live bacteria in the low-death rate countries are based on LAB fermentation [START_REF] Rhee | Importance of lactic acid bacteria in Asian fermented foods[END_REF] .A number of bacteria are involved in the fermentation of kimchi and other Korean traditional fermented foods, but LAB -including Lactobacillus-are the dominant species in the fermentation process [START_REF] Patra | Kimchi and Other Widely Consumed Traditional Fermented Foods of Korea: A Review[END_REF][START_REF] Jung | Kimchi microflora: history, current status, and perspectives for industrial kimchi production[END_REF] .

Lactobacillus is also an essential species in the fermentation of sauerkraut, Taiwanese 21 , Chinese [START_REF] Han | Improvement of fermented Chinese cabbage characteristics by selected starter cultures[END_REF] or other fermented foods [START_REF] Yoon | Production of probiotic cabbage juice by lactic acid bacteria[END_REF] . Lactobacilli are among the most common microorganisms found in kefir, a traditional fermented milk beverage [START_REF] Slattery | Analysis of Health Benefits Conferred by Lactobacillus Species from Kefir[END_REF] , milk and milk products [START_REF] Shiby | Fermented milks and milk products as functional foods--a review[END_REF][START_REF] Sanders | Probiotics and prebiotics in intestinal health and disease: from biology to the clinic[END_REF] . During fermentation, LAB synthesize vitamins and minerals, and produce biologically-active peptides with anti-oxidant activity [START_REF] Marco | Health benefits of fermented foods: microbiota and beyond[END_REF][START_REF] Sanlier | Health benefits of fermented foods[END_REF][START_REF] Lavefve | Microbial Ecology of Fermented Vegetables and Non-Alcoholic Drinks and Current Knowledge on Their Impact on Human Health[END_REF][START_REF] Melini | Health-Promoting Components in Fermented Foods: An Up-to-Date Systematic Review[END_REF][START_REF] Azam | Review -Lactic acid bacteria in traditional fermented Asian foods[END_REF][START_REF] Dimidi | Fermented Foods: Definitions and Characteristics, Impact on the Gut Microbiota and Effects on Gastrointestinal Health and Disease[END_REF] .

Humans possess two protective layers of biodiversity, and the microbiome has been proposed as an important actor of COVID-19 [START_REF] Riggioni | A compendium answering 150 questions on COVID-19 and SARS-CoV-2[END_REF] . The environment (outer layer) affects our lifestyle, shaping the microbiome (inner layer). [START_REF] Ruokolainen | Holistic view on health : two protective layers of biodiversity[END_REF] Many fermented foods contain living microorganisms and modulate the intestinal microbiome. [START_REF] Marco | Health benefits of fermented foods: microbiota and beyond[END_REF][START_REF] Dimidi | Fermented Foods: Definitions and Characteristics, Impact on the Gut Microbiota and Effects on Gastrointestinal Health and Disease[END_REF][START_REF] Septembre-Malaterre | Fruits and vegetables, as a source of nutritional compounds and phytochemicals: Changes in bioactive compounds during lactic fermentation[END_REF][START_REF] Kok | Yogurt and other fermented foods as sources of health-promoting bacteria[END_REF][START_REF] De Filippis | The food-gut axis: lactic acid bacteria and their link to food, the gut microbiome and human health[END_REF]

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The composition of microbiomes varies in different regions of the world. [START_REF] Mobeen | Enterotype Variations of the Healthy Human Gut Microbiome in Different Geographical Regions[END_REF] Gut microbiota has an interindividual variability due to genetic predisposition and diet. [START_REF] Bibbo | The role of diet on gut microbiota composition[END_REF] As part of the gut microbiome, Lactobacillus spp. contributes to its diversity and modulates oxidative stress in the GI tract. Some foods like cabbage can be fermented by the gut microbiota. [START_REF] Tian | Microbiota: a mediator to transform glucosinolate precursors in cruciferous vegetables to the active isothiocyanates[END_REF] Westernized foods usually lack fermented vegetables and milk-derived products have less biodiversity than traditional ones. Urbanization in western countries was associated with changes in the gut microbiome and with intestinal diversity reduction. [START_REF] Bibbo | The role of diet on gut microbiota composition[END_REF][START_REF] Segata | Gut Microbiome: Westernization and the Disappearance of Intestinal Diversity[END_REF][START_REF] Vangay | US Immigration Westernizes the Human Gut Microbiome[END_REF][START_REF] Zuo | Urbanization and the gut microbiota in health and inflammatory bowel disease[END_REF][START_REF] Wilson | Diet and the Human Gut Microbiome: An International Review[END_REF] Westernized food in Japan led to changes in the microbiome and in insulin resistance. [START_REF] Yamashita | Alteration of gut microbiota by a Westernized lifestyle and its correlation with insulin resistance in non-diabetic Japanese men[END_REF] The gut microbiome of westernized urban Saudis had a lower biodiversity than that of the traditional Bedouin population. [START_REF] Angelakis | Gut microbiome and dietary patterns in different Saudi populations and monkeys[END_REF] Fast food consumption was characterized by reduced Lactobacilli in the microbiome. [START_REF] Mitsou | Adherence to the Mediterranean diet is associated with the gut microbiota pattern and gastrointestinal characteristics in an adult population[END_REF] The links between gut microbiome, inflammation, obesity and insulin resistance are being observed but further large studies are needed for a definite conclusion. [START_REF] Saad | Linking Gut Microbiota and Inflammation to Obesity and Insulin Resistance[END_REF][START_REF] Chen | Gut Microbiome in Obesity, Metabolic Syndrome, and Diabetes[END_REF][START_REF] Lee | Gut microbiome and its role in obesity and insulin resistance[END_REF] Some COVID-19 patients have intestinal microbial dysbiosis [START_REF] Zuo | Alterations in Gut Microbiota of Patients With COVID-19 During Time of Hospitalization[END_REF] with decreased probiotics such as Lactobacillus and Bifidobacterium 51 . Many bacteria are involved in the fermentation of vegetables but most traditional foods with live bacteria in the low-death rate countries are based on LAB fermentation. 18- 20,23,30 Lactobacilli are among the most common microorganisms found in milk and milk products [START_REF] Slattery | Analysis of Health Benefits Conferred by Lactobacillus Species from Kefir[END_REF][START_REF] Shiby | Fermented milks and milk products as functional foods--a review[END_REF][START_REF] Sanders | Probiotics and prebiotics in intestinal health and disease: from biology to the clinic[END_REF] .

6-Angiotensin-converting enzyme 2 (ACE2) and COVID-19

COVID-19 is more severe in older adults and/or patients with comorbidities, such as diabetes, obesity or hypertension, suggesting a role for insulin resistance. [START_REF] Finucane | Coronavirus and Obesity: Could Insulin Resistance Mediate the Severity of Covid-19 Infection?[END_REF] Although differences exist between countries, the same risk factors for severity were found globally, suggesting common mechanisms. A strong relationship between hyperglycemia, impaired insulin pathway, and cardiovascular disease in type 2 diabetes is linked to oxidative stress and inflammation. [START_REF] Guzik | Epigenetics and Immunometabolism in Diabetes and Aging[END_REF] Lipid metabolism has an important role to play in obesity, in diabetes and its multi-morbidities, and in ageing. [START_REF] Miedema | Plasma-free fatty acids, fatty acid-binding protein 4, and mortality in older adults (from the Cardiovascular Health Study)[END_REF] The increased severity of COVID-19 in diabetes, hypertension, obese or elderly individuals may be related to insulin resistance, with oxidative stress as a common pathway. [START_REF] Hurrle | The etiology of oxidative stress in insulin resistance[END_REF] Moreover, the severe outcomes of COVID-19 -including lung damage, cytokine storm or endothelial damage -appear to exist globally, again suggesting common mechanisms.

The angiotensin-converting enzyme 2 (ACE2) receptor is part of the dual system -therenin-angiotensinsystem (RAS) -consisting of an ACE-Angiotensin-II-AT 1 R axis and an ACE-2-Angiotensin-(1-7)-Mas axis. AT 1 R is involved in most of the effects of Ang II, including oxidative stress generation, [START_REF] Wen | Oxidative stress-mediated effects of angiotensin II in the cardiovascular system[END_REF] which in turn upregulates AT 1 R [START_REF] Bhatt | Vascular oxidative stress upregulates angiotensin II type I receptors via mechanisms involving nuclear factor kappa B[END_REF] . In metabolic disorders and with older age, there is an upregulation of the AT 1 R axis leading to pro-inflammatory, pro-fibrotic effects in the respiratory system, and to insulin resistance. [START_REF] Dalan | The ACE-2 in COVID-19: Foe or Friend?[END_REF]

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This article is protected by copyright. All rights reserved SARS-CoV-2 binds to its receptor ACE2 and exploits it for entry into the cell. The ACE2 downregulation, as a result of SARS-CoV-2 binding, enhances the AT 1 R axis [START_REF] Sarzani | Disequilibrium between the Classic Renin-Angiotensin System and Its Opposing Arm in Sars-Cov-2 Related Lung Injury[END_REF] likely to be associated with insulin resistance [START_REF] Bousquet | Sulforaphane: from death rate heterogeneity in countries to candidate for prevention of severe COVID-19[END_REF][START_REF] Ren | Association of the insulin resistance marker TyG index with the severity and mortality of COVID-19[END_REF] but also to severe outcomes of COVID-19 (Figure 5A).

7-Anti-oxidant activities of foods linked with COVID-19

Many foods have an antioxidant activity [START_REF] Jain | Prevention of Cardiovascular Diseases with Anti-Inflammatory and Anti-Oxidant Nutraceuticals and Herbal Products: An Overview of Pre-Clinical and Clinical Studies[END_REF][START_REF] Razmpoosh | Probiotics as beneficial agents in the management of diabetes mellitus: a systematic review[END_REF][START_REF] Serino | Protective Role of Polyphenols against Vascular Inflammation, Aging and Cardiovascular Disease[END_REF] and the role of nutrition has been proposed to mitigate COVID-19 [START_REF] Zabetakis | COVID-19: The Inflammation Link and the Role of Nutrition in Potential Mitigation[END_REF] . Many antioxidant mechanisms have been proposed, and several foods can interact with transcription factors related to antioxidant effects such as theNuclear factor (erythroid-derived 2)-like 2 (Nrf2). [START_REF] Iddir | Strengthening the Immune System and Reducing Inflammation and Oxidative Stress through Diet and Nutrition: Considerations during the COVID-19 Crisis[END_REF] Some processes like fermentation increase the antioxidant activity of milk, cereals, fruit, vegetables, meat and fish. [START_REF] Melini | Health-Promoting Components in Fermented Foods: An Up-to-Date Systematic Review[END_REF] 7-1-Nrf2, a central antioxidant system

Reactive oxygen species (ROS), such as hydrogen peroxide and superoxide anion, exert beneficial and toxic effects on cellular functions. Nrf2 is a pleiotropic transcription factor at the centre of a complex regulatory network that protects against oxidative stress and the expression of a wide array of genes involved in immunity and inflammation, including antiviral actions. [START_REF] Tonelli | Transcriptional Regulation by Nrf2[END_REF] Nrf2 activity in response to chemical insults is regulated by a thiol-rich protein named KEAP1 (Kelch-like ECH-associated protein 1). The KEAP1-Nrf2 system is the body's dominant defense mechanism against ROS. [START_REF] Yamamoto | The KEAP1-NRF2 System: a Thiol-Based Sensor-Effector Apparatus for Maintaining Redox Homeostasis[END_REF] Induction of the antioxidant responsive element and the ROS mediated pathway by Nrf2 reduces the activity of nuclear factor kappa B (NF-κB), 68 whereas NF-κB can modulate Nrf2 transcription and activity, having both positive and negative effects on the target gene expression [START_REF] Wardyn | Dissecting molecular cross-talk between Nrf2 and NF-kappaB response pathways[END_REF] .

Natural compounds derived from plants, vegetables, fungi and micronutrients (e.g. curcumin, sulforaphane, resveratrol and vitamin D) or physical exercise can activate Nrf2. [START_REF] Jimenez-Osorio | Natural Nrf2 activators in diabetes[END_REF][START_REF] Pall | Nrf2, a master regulator of detoxification and also antioxidant, anti-inflammatory and other cytoprotective mechanisms, is raised by health promoting factors[END_REF] However, sulforaphane is the most potent activator of Nrf2. 3,[START_REF] Septembre-Malaterre | Fruits and vegetables, as a source of nutritional compounds and phytochemicals: Changes in bioactive compounds during lactic fermentation[END_REF] "Ancient foods", and particularly those containing Lactobacillus, activate Nrf2. [START_REF] Senger | Activation of the Nrf2 Cell Defense Pathway by Ancient Foods: Disease Prevention by Important Molecules and Microbes Lost from the Modern Western Diet[END_REF] Nrf2 may be involved in diseases associated with insulin-resistance. [START_REF] Bousquet | Sulforaphane: from death rate heterogeneity in countries to candidate for prevention of severe COVID-19[END_REF][START_REF] Uruno | The Keap1-Nrf2 system and diabetes mellitus[END_REF][START_REF] Vasileva | Obesity and NRF2mediated cytoprotection: Where is the missing link?[END_REF][START_REF] Guo | Keap1-Nrf2 signaling pathway in angiogenesis and vascular diseases[END_REF] Nrf2 activity declines with age, making the elderly more susceptible to oxidative stress-mediated diseases. [START_REF] Zhang | Oxidative stress response and Nrf2 signaling in aging[END_REF] Nrf2 is involved in the protection against lung [START_REF] De | Role of Nrf2 and Autophagy in Acute Lung Injury[END_REF] or endothelial damage. [START_REF] Chen | The role of Nrf2 in oxidative stress-induced endothelial injuries[END_REF] Nrf2 activating compounds downregulate ACE2 mRNA expression in human liver-derived HepG2 cells. [START_REF] Mccord | Nrf2 Activator PB125(R) as a Potential Therapeutic Agent Against COVID-19[END_REF] Genes encoding cytokines including IL-6 and many others specifically identified in the "cytokine storm" have been observed in fatal cases of COVID-19.ACE2 can inhibit NF-κB and activate Nrf2. [START_REF] Fang | Angiotensin-converting enzyme 2 attenuates inflammatory response and oxidative stress in hyperoxic lung injury by regulating NF-kappaB and Nrf2 pathways[END_REF]

7-2-Sulforaphane, the most potent Nrf2 natural activator

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Isothiocyanates are stress-response chemicals formed from glucosinolates in plants often belonging to the cruciferous family, and more broadly to the Brassica genus including broccoli, watercress, kale, cabbage, collard greens, Brussels sprouts, bok choy, mustard greens and cauliflower . [START_REF] Palliyaguru | Isothiocyanates: Translating the Power of Plants to People[END_REF] The formation of isothiocyanates from glucosinolates depends on plant-intrinsic factors and extrinsic postharvest factors such as industrial processing, domestic preparation, mastication, and digestion. [START_REF] Oliviero | Isothiocyanates from Brassica Vegetables-Effects of Processing, Cooking, Mastication, and Digestion[END_REF] Sulforaphane [1-isothiocyanato-4-(methylsulfinyl)butane] is an isothiocyanate occurring in a stored form such as glucoraphanin in cruciferous vegetables [START_REF] Vanduchova | Isothiocyanate from Broccoli, Sulforaphane, and Its Properties[END_REF][START_REF] Quirante-Moya | The Role of Brassica Bioactives on Human Health: Are We Studying It the Right Way?[END_REF] .Sulphoraphanes are also found in fermented cabbage [START_REF] Dimidi | Fermented Foods: Definitions and Characteristics, Impact on the Gut Microbiota and Effects on Gastrointestinal Health and Disease[END_REF][START_REF] Luang-In | Formation of Sulforaphane and Iberin Products from Thai Cabbage Fermented by Myrosinase-Positive Bacteria[END_REF] . Present in the plant as its precursor, glucoraphanin, sulforaphane is formed through the actions of myrosinase, a β-thioglucosidase present in either the plant tissue or the mammalian microbiome [START_REF] Yagishita | Broccoli or Sulforaphane: Is It the Source or Dose That Matters?[END_REF][START_REF] Hindson | Brassica vegetable metabolism by gut microbiota[END_REF] .

Sulforaphane is a clinically relevant nutraceutical compound used for the prevention and treatment of chronic diseases and may be involved in ageing. [START_REF] Houghton | Sulforaphane: Its "Coming of Age" as a Clinically Relevant Nutraceutical in the Prevention and Treatment of Chronic Disease[END_REF] Along with other natural nutrients, sulforaphane has been suggested to have a therapeutic value for the treatment of coronavirus disease 2019 (COVID-19). [START_REF] Horowitz | Three novel prevention, diagnostic, and treatment options for COVID-19 urgently necessitating controlled randomized trials[END_REF] One of the key mechanisms of action of sulforaphane involves the activation of the Nrf2-Keap1 signaling pathway. [START_REF] Yang | Frugal chemoprevention: targeting Nrf2 with foods rich in sulforaphane[END_REF] Sulforaphane is the most effective natural activator of the Nrf2 pathway, and Nrf2 expression and function is vital for sulforaphane-mediated action. [START_REF] Bai | Sulforaphane Protects against Cardiovascular Disease via Nrf2 Activation[END_REF][START_REF] Zhou | Nrf2 expression and function, but not MT expression, is indispensable for sulforaphane-mediated protection against intermittent hypoxia-induced cardiomyopathy in mice[END_REF] Sulforaphanes were suggested to be effective in diseases associated with insulin resistance [START_REF] Stafford | Covid-19: Why Germany's case fatality rate seems so low[END_REF][START_REF] Xu | Glucoraphanin: a broccoli sprout extract that ameliorates obesity-induced inflammation and insulin resistance[END_REF][START_REF] Teng | Sulforaphane Prevents Hepatic Insulin Resistance by Blocking Serine Palmitoyltransferase 3-Mediated Ceramide Biosynthesis[END_REF][START_REF] Sun | Protective effects of sulforaphane on type 2 diabetes-induced cardiomyopathy via AMPK-mediated activation of lipid metabolic pathways and NRF2 function[END_REF] It has been proposed that SARS-CoV-2 downregulates ACE2 and that there is an increased insulin resistance associated with oxidative stress through the AT 1 R pathway. Fermented vegetables and Brassica vegetables release glucoraphanin,converted by the plant or by the gut microbiome into sulforaphane, which activates Nrf2 and subsequently reduces insulin intolerance (Figure 5B).

7-3-Lactic acid bacteria Antioxidant activity of Lactobacillus

The gastrointestinal (GI) tract is challenged with oxidative stress induced by a wide array of factors, such as exogenous pathogenic microorganisms and dietary aspects. Redox signaling plays a critical role in the physiology and pathophysiology of the GI tract [START_REF] Perez | Redox signaling in the gastrointestinal tract[END_REF] . The redox mechanisms of Lactobacillus spp. are involved in the downregulation of ROS-forming enzymes, [START_REF] An | Coexpression of the superoxide dismutase and the catalase provides remarkable oxidative stress resistance in Lactobacillus rhamnosus[END_REF][START_REF] Serata | Roles of thioredoxin and thioredoxin reductase in the resistance to oxidative stress in Lactobacillus casei[END_REF] and redox stress resistance proteins or genes differ largely between LAB species. In addition, Nrf-2 and NF-κB are two common transcription factors, through which Lactobacillus spp. also modulates oxidative stress. [START_REF] Kong | The Potential of Lactobacillus spp. for Modulating Oxidative Stress in the Gastrointestinal Tract[END_REF]

Do lactobacilliprevent insulin resistance?

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Hundreds of studies have attempted to find an efficacy of LAB on insulin resistance-associated diseases.

However, most of them are underpowered or have some methodological flaws. Moreover, not all LAB strains have the same action on insulin resistance 100 and new better designed studies with the appropriate LAB are required. A large meta-analysis found that the intake of probiotics resulted in minor but consistent improvements in several metabolic risk factors in subjects with metabolic diseases, and particularly in insulin resistance 101 . Another recent meta-analysis found that an oral supplementation with probiotics or synbiotics has a small effect in reducing waist circumference but no effect on body weight or body mass index (BMI) 102 .Kefir, a fermented milk product, was not found to be more effective than yoghurt in the glycemic control of obesity, possibly because there are insufficient differences between both 103 .

Lactobacillus and Nrf2

Nrf2 may be involved in diseases associated with insulinresistance [START_REF] Uruno | The Keap1-Nrf2 system and diabetes mellitus[END_REF][START_REF] Vasileva | Obesity and NRF2mediated cytoprotection: Where is the missing link?[END_REF][START_REF] Guo | Keap1-Nrf2 signaling pathway in angiogenesis and vascular diseases[END_REF] . "Ancient foods", and particularly those containing Lactobacillus, activate Nrf2 72 . The microbiome is highly related to insulin resistance. In mice, several strains of Lactobacillus were found to regulate Nrf2 in models of ageing 104 , in cardioprotective effects 105 , and in non-alcoholic fatty acid liver disease 106 . Lactobacillus plantarum CQPC11 -isolated from Sichuan pickled cabbages -antagonizes oxidation and ageing in mice 107 . Lactobacillus protects against ulcerative colitis by modulation of the gut microbiota and Nrf2/Ho-1 pathway 108 . The sugary kefir strain, Lactobacillus mali APS1, ameliorates hepatic steatosis by regulation of Nrf2 and the gut microbiota in rats 109 . In vitro studies have also found an effect of Lactobacilli mediated by Nrf2 110-112 . Interestingly, the symbiotic combination of prebiotic grape pomace extract and probiotic Lactobacillus sp. reduces intestinal inflammatory markers. 113 Coronavirus disease in animals and lactic acid bacteria.

The porcine epidemic diarrhea virus (PEDV) and the Transmissible Gastroenteritis Coronavirus Infection (TGEV) are worldwide-distributed coronaviruses. Low levels of Lactobacillus were foundin the intestine of piglets infected by TGEV 114 or PEDV. Lactobacillus inhibits PEDV or TGEV effects in vitro 115,116 .

7-4-Nrf2 and COVID-19

A putative mechanism may be proposed (Figure 5). SARS-CoV-2 downregulates ACE2 inducing an increased insulin resistance associated with oxidative stress through the AT 1 R pathway. This may explain risk factors for severe COVID-19.

Fermented vegetables are often made from cruciferous (Brassica) vegetables that release glucoraphanin converted by the plant or by the gut microbiome into sulforaphane which activates Nrf2 and subsequently

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This article is protected by copyright. All rights reserved reduces insulin intolerance by its potent antioxidant activities. Fermented vegetables contain a high content of Lactobacillus that can activate Nrf2 and impact on the microbiome. 117 Sulforaphane and LAB both thereforehave the ability to reduce insulin resistance.

Other putative actions on COVID-19 severity may be postulated. The down-regulation of ACE2 reduces the Ang-1,7 anti-oxidant activity that was found to activate Nrf2. 118,119 Nrf2 protects against hallmarks of severe COVID-19. It has anti-fibrotic effects on various organs including the lungs, 120 protects against lung injury and acute respiratory distress syndrome, 121 and endothelial damage [START_REF] Chen | The role of Nrf2 in oxidative stress-induced endothelial injuries[END_REF] . Finally, Nrf2 can block IL-6 in different models of inflammation 122 and might play a role in the COVID-19 cytokine storm.

These different mechanisms may explain the importance of fermented cabbage in preventing the severity of COVID-19. It is clear that other nutrients, vitamin D 123 and many different foods act on NRF2 and that mechanisms other than Nrf2 may be operative.

It is not yet known whether sulforaphane and/or LAB may act on the infectivity of SARS-CoV-2. Disulfide bonds can be formed under oxidizing conditions and play an important role in the folding and stability of some proteins. The receptor-binding domain of the viral spike proteins and ACE2 have several cysteine residues. Using molecular dynamics simulations, the binding affinity was significantly impaired when all of the disulfide bonds of both ACE2 and SARS-CoV/CoV-2 spike proteins were reduced to thiol groups. This computational finding possibly provides a molecular basis for the differential COVID-19 cellular recognition due to the oxidative stress. 124 It is likely that foods with anti-oxidant activity can interact with COVID-19 and that fermented or cruciferous vegetables represent one of the possible foods involved. If some foods are found to be associated with a prevention of COVID-19 prevalence or severity, it may be of interest to study their LAB and/or sulforaphane composition in order to eventually find some common mechanisms and targets for therapy.

8-May dietary modifications change the course of COVID-19? 8-1-Fermented vegetables and Kimchi

It is tempting to propose that countries where traditional LAB-fermented vegetables are largely consumed are those showing lower COVID-19 death rates and that fermented vegetables represent one possible preventive approach. Other nutrients are found in these products that may enhance their effect (e.g. vitamin K 125 ).Kimchi fermented from many vegetables including cabbage has several effects on insulin resistance associated diseases: anti-diabetic properties, 126,127 cardiovascular diseases, [START_REF] Lavefve | Microbial Ecology of Fermented Vegetables and Non-Alcoholic Drinks and Current Knowledge on Their Impact on Human Health[END_REF] dyslipidemia 128 or

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This article is protected by copyright. All rights reserved ageing. 129 Kimchi, when fermented for a long time, reduces insulin intolerance to a greater extent than fresh kimchi, 126 indicating that newly formed products during fermentation are important. In particular, Kimchi from cabbage and Chinese cabbage contains several glucosinolates 130-132 that can be transformed in sulforaphanes either in the plant itself or by the human microbiome. [START_REF] Bousquet | Sulforaphane: from death rate heterogeneity in countries to candidate for prevention of severe COVID-19[END_REF] In central European countries, raw and fermented cabbage is commonly consumed.

In Sub-Saharan Africa, people commonly eat fermented foods, mainly cereal-based foods like sorghum, millet and maize, roots such as cassava, fruits and vegetables. Fermented cassava products (like gari and fufu) are a major component of the diet of over 800 million people and, in some areas, these products constitute over 50% of the diet. [START_REF] Azam-Ali | Fermented fruits and vegetables[END_REF] It is clear that sauerkraut is consumed in Alsace (France) where a COVID-19 outbreak has been identified, but it is not a regular meal.

8-2-Westernized diet

Westernized diets contain a reduced amount of fermented vegetables [START_REF] Wilson | Diet and the Human Gut Microbiome: An International Review[END_REF]133 and may be prone to increasing insulin resistance [START_REF] Yamashita | Alteration of gut microbiota by a Westernized lifestyle and its correlation with insulin resistance in non-diabetic Japanese men[END_REF]134 and diseases associated with it, 135 and thereby severe COVID-19.

In the Mediterranean diet, well known for reducing insulin resistance, 136 Nrf2 appears to play an important role. [START_REF] Pall | Nrf2, a master regulator of detoxification and also antioxidant, anti-inflammatory and other cytoprotective mechanisms, is raised by health promoting factors[END_REF]137 The COVID-19 death rate differences in Italian (Figure 2) and Spanish 3 regions suggest a role for Mediterranean diet and short chain food supply. This also indicates that many foods can have an effect and that cabbage and fermented foods represent a proof-of-concept. Nrf2 is also involved in the Okinawanbased diet [START_REF] Pall | Nrf2, a master regulator of detoxification and also antioxidant, anti-inflammatory and other cytoprotective mechanisms, is raised by health promoting factors[END_REF] , active on insulin intolerance. 138

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In the US, highly populated, regional air hub areas, minorities and poverty had an increased risk of COVID-19 related mortality. 140 It was proposed that the inequality might be due to the workforce of essential services, poverty, access to care or air pollution 141 . These are common risk factors in mortality observed in deprived areas of the US. 142 Moreover, in the US and the UK, there are unique health issues facing black, Asian and minority ethnic communities. 143,144 This greater risk of hospitalizations in these populations was not explained by socio-economic or behavioural factors. 145 Social distancing is an important factor to be considered 146 but diet may also be involved.

On the other hand, a recent report of the Municipal Corporation of Greater Mumbai (Public Relation Department, 28-07-2020) found that 57% of subjects tested in the slum area had antibodies against SAR-CoV-2 but only 16% in the non-slum areas. The fatality rate in slum areas was very low (0.05-0.1%). 147 Although precise data are lacking, in Brazilian favelas the spread of COVID-19 is not noticed. 148 Temperature does not seem to be an important factor to contain the pandemic.Fermented foods are popular throughout the world and in many regions they represent a widespread tradition as well as they make a significant contribution to the diet of millions of individuals. [START_REF] Azam-Ali | Fermented fruits and vegetables[END_REF] This is the case in slum areas and it is possible that fermented foods explain, at least partly, the paradox.

Conclusion

Cabbage contains precursors of sulforaphane, the most active natural activator of Nrf2. Fermented vegetables contain many lactobacilli, also potent Nrf2 activators. It is proposed that fermented cabbage is a proof-of-concept of dietary manipulations that may enhance Nrf2-associated antioxidant effects helpful in mitigate COVID-19 severity.

Mainstream COVID-19 control strategies including social distancing, confinement and intensive case finding, testing, tracing and isolating are so far not enough to provide a SARS-CoV-2-free environment and restore a safe social life. There are hopes for a safe and effective vaccine, but this is unlikely to become rapidly available. So, there is a need to explore other potentially useful strategies. An area that has not been sufficiently considered is diet, both as a preventive and/or therapeutically useful intervention, encouraging people to eat more traditional foods containing fermented vegetables (Figure 6). We have suggested that fermented vegetables could be associated with a lower COVID-19 mortality due to their potent antioxidant effect among which sulforaphane and LABare important. However, many other foods may have a similar activity. It should be noted that dietary supplements that over-activate Nrf2 may have side-effects. 149 Robust evidence from observational studies would be helpful to formally investigate associations between fermented foods and clinical outcomes in COVID-19. State-of-the-art methods, including the use of DAGs

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This article is protected by copyright. All rights reserved (Directed Acyclic Graphs), may be needed to help assess whether the associations seen are likely to represent causal relationship 150 . A faster approach would be to develop large clinical trials in the appropriate populations. Interventions based on diets with a high intake of fermented foods like Kimchi or other fermented foods are unlikely to present ethical difficulties. Furthermore, the fact that a precise mechanism has been proposed would facilitate adding reliable biomarkers to the relevant clinical outcomes.

Moreover, new drugs based on the components of these fermented foods may be of interest.

If the hypothesis is proved, COVID-19 will be the first infectious disease outbreak associated with a loss of "nature" 151 and to be ascribed as a disease of the Anthropocene

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Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :

 123456 Figure 1:COVID-19 deaths per million inhabitants(from Johns Hopkins Coronavirus Center)

  Taken altogether, it is possible that diet is partly involved in the COVID-19 death clusters found in large Western cities where traditional diet is often replaced by long chain food supply.It is clear that diet is not the only risk factor and should be considered in the context of COVID-19 in a given setting. For example, Nordic/central European people socialize less than the Mediterraneans and

	simultaneously may consume more fermented vegetables.
	8.3. The COVID-19 slum paradox
	It was expected that the COVID-19 pandemic will be catastrophic if it reached deprived areas of low-and
	middle-income countries, in particular informal settlements (slum areas) where social distancing and
	lockdown are almost impossible to set up. 139

Table 1 : Possible risk factors for COVID-19 infection explaining geographical differences

 1 152 . Imbalance in the gut microbiota is responsible for the pathogenesis of various disease types including allergy, asthma, rheumatoid arthritis, different types of cancer, diabetes mellitus, obesity and cardiovascular disease153 . Fermentation was introduced during the Neolithic age and was essential for the survival of human kind. When modern life led to eating reduced amounts of fermented foods, the microbiome drastically changed 154 , allowing SARS-CoV-2 to spread or to be more severe155 . It is time for mitigation 156 . : Risk factors at a country level, B: Environment, nutrition, C: individual level + to ++++: Proposed relative importance

	A A A A A A A A B B B B B B C C Accepted Article Contact with a SARS-CoV-2 infected individual Intensity of social contacts Intensity of occupational contacts Confinement (level) Confinement (early measures) Climatic conditions (temperature, humidity) GDP of a country/region Vitamin D Diet Food Long food chain supply Traditional fermented food (example of food) Air pollution Underserved area Age Comorbidities (severity of	Individual level ++++ ++ +++ +++ +++ ? ? ? ? ++? ++? ++? +? ++ +++ +++	Country/region level Case zero identified ++++ e.g. Lombardy +++ ++ +++ e.g. US versus EU Sweden vs Nordic countries +++ e.g. UK versus EU ++ Hot and humid temperature may reduce infection but epidemic bursts in Brazil, Peru and Ecuador + + + The map of COVID-19 deaths in Europe and the low prevalence in Asia and Africa suggest a role for diet + Bibliographic analysis suggests a role for some fermented foods. Raw cabbage can be fermented in the intestine. Kefir is largely used in many low-prevalence countries. + In Italy and Spain, there may be an association with long chain supply. This may be relevant since food quality differs. ++ This may be a relevant issue. In former Eastern European countries, in the Balkans, in Africa and in many Asian countries with low-COVID-19 prevalence, traditional fermented foods are common (in line with short food chain supply) +? ++ ++

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This article is protected by copyright. All rights reserved 530 531 Accepted Article This article is protected by copyright. All rights reserved 154. McCall LI, Callewaert C, Zhu Q, et al. Home chemical and microbial transitions across urbanization. Nat Microbiol 2020;5:108-15. 155. Haahtela T, Anto J, Bousquet J. Slow Health Catastrophe of Homo urbanicus -Loss of Resilience. Porto Med J 2020. 156. Haahtela T, Valovirta E, Bousquet J, Makela M, Allergy Programme Steering G. The Finnish Allergy Programme 2008-2018 works. Eur Respir J 2017;49.

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Conflict of interest :the


Professor Byoung Chul Cho

Amaury Daste

Alain Ravaud

Sébastien Salas

Nicolas Isambert

Edward Mcclay

Ahmad Awada

Christian Borel

Laureen S Ojalvo

Christoph Helwig

P Alexander Rolfe

James L Gulley

Nicolas Penel

Bintrafusp alfa, a bifunctional fusion protein targeting TGF-β and PD-L1, in advanced squamous cell carcinoma of the head and neck: results from a phase I cohort

bACkground Squamous cell carcinoma of the head and neck (SCCHN) causes nearly 300 000 deaths per year as of 2012. [START_REF] Ferlay | Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012[END_REF] Smoking, alcohol use and infection with human papillomavirus (HPV) are leading risk factors for SCCHN. 2 3 Patients with advanced or recurrent SCCHN have a poor prognosis and a median overall survival (OS) of <1 year in the first-line setting, and an even lower median OS in the second-line or later-line settings. [START_REF] Ferris | Nivolumab for recurrent squamous-cell carcinoma of the head and neck[END_REF][START_REF] Chow | Antitumor activity of pembrolizumab in biomarker-unselected patients with recurrent and/or metastatic head and neck squamous cell carcinoma: results from the phase Ib KEYNOTE-012 expansion cohort[END_REF][START_REF] Vermorken | Platinum-based chemotherapy plus cetuximab in head and neck cancer[END_REF][START_REF] Cohen | Pembrolizumab versus methotrexate, docetaxel, or cetuximab for recurrent or metastatic head-and-neck squamous cell carcinoma (KEYNOTE-040): a randomised, open-label, phase 3 study[END_REF] OS results from KEYNOTE-048 (NCT02358031) support the use of pembrolizumab plus a platinum agent and 5-fluorouracil or as monotherapy in patients with combined positive score (CPS) >1, as a frontline regimen for patients with recurrent/metastatic disease. 6 8 9 Patients who experience progression on cetuximab with chemotherapy have few treatment options, with low response rates (<15%) 10 11 and response durability or disease stabilization rarely exceeding 3 months. [START_REF] Vermorken | Open-label, uncontrolled, multicenter phase II study to evaluate the efficacy and toxicity of cetuximab as a single agent in patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck who failed to respond to platinum-based therapy[END_REF][START_REF] Baselga | Phase II multicenter study of the antiepidermal growth factor receptor monoclonal antibody cetuximab in combination with platinum-based chemotherapy in patients with platinum-refractory metastatic and/or recurrent squamous cell carcinoma of the head and neck[END_REF][START_REF] Colevas | Chemotherapy options for patients with metastatic or recurrent squamous cell carcinoma of the head and neck[END_REF][START_REF] Kim | Phase II clinical and exploratory biomarker study of dacomitinib in patients with recurrent and/or metastatic squamous cell carcinoma of head and neck[END_REF] Patients with advanced SCCHN that progressed within 6 months after the last dose of platinum-based therapy in either the locally advanced or recurrent/metastatic setting have aggressive tumor biology and a poor prognosis and represent an unmet clinical need. 8 9 16 Anti-programmed cell death protein 1 (anti-PD-1) immune checkpoint inhibitors, including nivolumab and pembrolizumab, have shown clinical activity in the second-line setting for the treatment of SCCHN-objective response rates (ORRs) of 13%-16% 4 7 17 -and are approved for the Open access treatment of patients with SCCHN who experienced disease progression on or after platinum-containing chemotherapy.

Transforming growth factor β (TGF-β) signaling is implicated in multiple tumorigenic processes, including immune-related and non-immune-related pathways. TGF-β is overexpressed in multiple tumor types and can have suppressive effects in early stages of tumorigenesis; however, in the tumor microenvironment, TGF-β loses its antiproliferative, tumor-suppressing activity and instead promotes tumor progression via processes such as immune evasion and epithelial-to-mesenchymal transition (EMT), once a malignancy has been established. [START_REF] Wrzesinski | Transforming growth factor-beta and the immune response: implications for anticancer therapy[END_REF] Although TGF-β is integral in mediating immune selftolerance in normal tissues, [START_REF] Wrzesinski | Transforming growth factor-beta and the immune response: implications for anticancer therapy[END_REF] its immunosuppressive activities assist in immune evasion of tumor cells, and TGF-β inhibition can facilitate T-cell invasion in the tumor microenvironment and increase susceptibility to anti-PD-1/programmed death-ligand 1 (PD-L1) therapy in mouse tumor models. 19 20 Furthermore, TGF-β can promote drug resistance and metastasis via increased fibrosis, angiogenesis and EMT. [START_REF] Akhurst | Targeting the TGFβ signalling pathway in disease[END_REF] Increased plasma TGF-β1 negatively correlates with survival in patients with SCCHN who received cetuximabbased therapy. [START_REF] Gilbert | A randomized phase II efficacy and correlative studies of cetuximab with or without sorafenib in recurrent and/or metastatic head and neck squamous cell carcinoma[END_REF] Furthermore, there is a link between dysregulation of TGF-β signaling and the development of HPV-positive tumors, [START_REF] Guan | Association of TGF-beta1 genetic variants with HPV16-positive oropharyngeal cancer[END_REF][START_REF] Tao | TGFβ1 genetic variants predict clinical outcomes of HPV-positive oropharyngeal cancer patients after definitive radiotherapy[END_REF][START_REF] Levovitz | TGFβ receptor 1: an immune susceptibility gene in HPV-associated cancer[END_REF] and HPV status is a prognostic biomarker in SCCHN. [START_REF] Bonner | HPV, and cetuximab: what is the evidence?[END_REF] Consequently, it is possible that dysregulation of TGF-β signaling may drive tumorigenesis in HPV-positive cancers. Virus-associated cancers are more likely to be immunologically inflamed and thus susceptible to immunotherapy-based treatments. [START_REF] Chen | Elements of cancer immunity and the cancerimmune set point[END_REF] Therefore, sequestering TGF-β from the tumor microenvironment while simultaneously inhibiting the PD-L1 immune checkpoint may provide a novel treatment approach.

Bintrafusp alfa (M7824) is a first-in-class bifunctional fusion protein composed of the extracellular domain of the human TGF-β receptor II (TGF-βRII or TGF-β 'trap') fused via a flexible linker to the C-terminus of each heavy chain of an IgG1 antibody blocking PD-L1 (anti-PD-L1). [START_REF] Strauss | Phase I trial of M7824 (MSB0011359C), a bifunctional fusion protein targeting PD-L1 and TGFβ, in advanced solid tumors[END_REF] Preclinical studies in murine models showed that bintrafusp alfa more effectively suppressed tumor growth and metastasis than either a TGF-β trap or an anti-PD-L1 antibody alone. [START_REF] Lan | Enhanced preclinical antitumor activity of M7824, a bifunctional fusion protein simultaneously targeting PD-L1 and TGF-β[END_REF] Bintrafusp alfa also inhibited spontaneous metastasis in multiple preclinical models, 29 30 presumably due to its suppressive effect on EMT. Data from a phase I, dose-escalation study suggested that bintrafusp alfa-administered at doses of up to 20 mg/kg-had a manageable safety profile and showed early signs of clinical efficacy in patients with heavily pretreated advanced solid tumors. [START_REF] Strauss | Phase I trial of M7824 (MSB0011359C), a bifunctional fusion protein targeting PD-L1 and TGFβ, in advanced solid tumors[END_REF] Bintrafusp alfa also showed activity in patients with HPV-associated malignancies, including cervical cancer, anal cancer and SCCHN. [START_REF] Strauss | Safety and activity of M7824, a bifunctional fusion protein targeting PD-L1 and TGF-β, in patients with HPV associated cancers[END_REF] Here, we report the efficacy and safety results of a phase I trial of bintrafusp alfa monotherapy in patients with advanced SCCHN that progressed after the last dose of platinumbased therapy.

Methods study design and participants NCT02517398 is an ongoing, phase I, open-label trial of bintrafusp alfa that includes multiple solid tumor expansion cohorts. Planned enrollment for the advanced SCCHN cohort was 30 patients. With 30 subjects treated, the study has approximately 87% power to rule out a ≤15% ORR (null hypothesis) when the true ORR is 35% at the 10% type I error rate (one-sided). Meeting this endpoint would have warranted the clinical development of a phase II or III trial based on a very strong phase I signal. Eligible patients were aged ≥18 years and had histologically confirmed, advanced SCCHN not amenable to curative therapy that progressed/recurred after prior platinum therapy in the recurrent/metastatic setting, or within 6 months after the last dose of platinum received in combination with radiotherapy for the treatment of locally advanced disease. Patients may have received prior cetuximab, but prior treatment with any antibody/ drug-targeting T-cell coregulatory proteins such as anti-PD-1, anti-PD-L1 or anticytotoxic T-lymphocyte-associated protein-4 antibody was not allowed. Patients had an Eastern Cooperative Oncology Group performance status of 0 or 1; life expectancy of ≥12 weeks; adequate renal, hepatic and hematological function; measurable disease by RECIST 1.1 and available archival tumor material or fresh biopsies taken within 28 days of the first administration of study drug. Patients were not selected on the basis of PD-L1 expression or other biomarkers.

Procedures

Integrated analysis of bintrafusp alfa exposure, response and progression-free survival (PFS) supported a flat dose of 1200 mg every 2 weeks, [START_REF] Vugmeyster | Selection of the recommended phase 2 dose (RP2D) for M7824 (MSB0011359C), a bifunctional fusion protein targeting TGF-β and PD-L1[END_REF] and this dose was used in the expansion cohorts. Patients received bintrafusp alfa via intravenous infusion over 1 hour once every 2 weeks until confirmed progressive disease (PD), unacceptable toxicity or trial withdrawal. Treatment beyond PD was permitted if adverse events (AEs) were manageable, performance status was maintained, and no further treatment was indicated.

Premedication with an antihistamine and acetaminophen approximately 30-60 min prior to each dose of bintrafusp alfa was mandatory for the first 2 infusions and was optional afterward. Dose reduction was not permitted; however, interruption or discontinuation of bintrafusp alfa was allowed as described in online supplementary table S2.

Tumor response was assessed by radiographic imaging 6 weeks after initiating treatment, then every 6 weeks for the first year and every 12 weeks thereafter. Response was confirmed by repeated radiographic assessment no sooner than 4 weeks from the first documentation of response. AEs were monitored throughout treatment, for the first 28 days after the last study dose, at 10 weeks posttreatment and every 12 weeks thereafter and were assessed according to NCI-CTCAE V.4.03. Immune-related AEs Open access were identified using a prespecified list of MedDRA preferred terms followed by medical review. outcomes The primary endpoint for this expansion cohort was to assess the confirmed best overall response (BOR) according to RECIST 1.1 as adjudicated by an independent review committee (IRC). Secondary endpoints included BOR per investigator and safety and tolerability. Exploratory endpoints included PFS, duration of response (DOR) and disease control rate (DCR) according to RECIST 1.1 as adjudicated by IRC, and OS. Exploratory biomarker analyses of efficacy were also performed.

statistical analysis

Efficacy and safety were analyzed in all patients who received ≥1 dose of bintrafusp alfa. DCR-defined as the proportion of patients with a BOR of complete response (CR), partial response (PR), non-CR/non-PD or stable disease (SD) -was tabulated with an exact 95% Clopper-Pearson CI. PFS, DOR and OS were analyzed using the Kaplan-Meier method. Safety and tolerability were analyzed using descriptive statistics.

biomarker analyses

Tumor PD-L1 protein expression was measured by immunohistochemical staining of formalin-fixed, paraffinembedded (FFPE) tumor tissue blocks using a proprietary assay (Dako, Carpinteria, California, USA) and an anti-PD-L1 rabbit monoclonal antibody clone 73-10 under license from Merck KGaA (Darmstadt, Germany). PD-L1 positivity in tumor cells was scored as the proportion of tumor cells showing membranous PD-L1 staining.

HPV status was determined by RNA sequencing (RNASeq) using FFPE tissue samples and standard protocols. Kallisto 33 (V.0.43.1) was used to align sequencing results to a transcriptome containing the human Ensembl transcripts (V.91, GRCh38) and viral genomes from the National Center for Biotechnology Information collection. HPV content in each sample was assessed as the fraction of reads mapping to any papillomavirus genome. To determine a cut-off (for fraction of viral reads), the same process was applied to SCCHN samples from SRP063006/ GSE72536 and SRP066090/GSE74927. The HPV statuses deposited with these sequences were used to choose a cutoff that provided perfect separation between the 28 positive and 51 negative training samples.

For gene expression quantification, RNASeq data were generated from FFPE tissue samples and standard protocols. Bowtie2 V.2.2.3 [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF] was used to align sequencing results to the Ensembl 75 human genome (GRCh37 February 2014). Gene expression was determined using RSEM V.1.2.31 with the Ensembl gene annotations. [START_REF] Li | RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome[END_REF] Hypothesis testing was performed by comparing RSEM-computed expected counts. Transcript-per-million (TPM) values were upper-quartile normalized and log-transformed for further analysis. Signature scores were defined as the mean log 2 (fold-change) among all genes in each gene signature (online supplementary table S3). 19 36 These were calculated by adding a pseudocount of 0.5 TPM to all genes and samples, determining the log 2 (TPM), then subtracting the median log 2-TPM for each gene across all samples from the log 2-TPM for each gene.

Tumor samples were used to determine immune phenotype based on available immunohistochemistry data (PD-L1 stain and PD-L1 negative control) and H&E staining. An exploratory classification system was used to categorize tumors as having an immune-inflamed (immune cells in direct physical contact with tumor cells), immune-excluded (≥1% of tumor stroma area populated by lymphocytes, immune cells possibly located in immediate vicinity of tumor cells but not efficiently infiltrating tumor cell clusters, and very infrequent physical contact between lymphocytes and tumor cells), or immune desert (<1% of tumor stroma area populated by lymphocytes, no dense immune cell infiltrates and no contact of immune cells with tumor cells) phenotype. This system was developed from methods previously described. [START_REF] Mariathasan | TGFβ attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells[END_REF] A pathologist who was masked to the response data scored the scanned slides and determined the corresponding immune phenotype.

results

Patient population and baseline characteristics

Between October 6, 2016 and January 12, 2017, 46 patients were screened. Of these, 32 patients were enrolled and treated (full analysis set) at 20 sites in the Asia-Pacific region, Europe and the USA. At the time of data cut-off on August 24, 2018, patients had been followed for a median of 86.4 weeks (range 2-97 weeks); 4 patients (13%) remained on treatment and the median duration of therapy was 12.1 weeks (range 2-96 weeks). The primary reason for treatment discontinuation was disease progression (n=20 (63%)), AE (n=5 (16%)), consent withdrawal (n=1 (3%)), death unrelated to study treatment (hemorrhage; n=1 (3%)) and investigator decision (n=1 (3%)). The death unrelated to study treatment occurred 2 weeks after the patient received the first and only dose of bintrafusp alfa. Twenty-eight patients (88%) had progressed on platinum-based therapy in the second-line or later-line, recurrent and/or metastatic setting, and 4 patients (13%) had progressed on platinum-based therapy in the locally advanced setting (table 1).

efficacy

The confirmed ORR by IRC was 13% (95% CI 4% to 29%; 4 PR), and an additional 4 patients had SD, resulting in a DCR of 34% (95% CI 19% to 53%) (table 2 and figure 1A,B). The median DOR was 18.1 months (range 6-20+ months). Two of 4 patients with PR had an ongoing response of 16.5+ and 19.6+ months as of data cut-off (figure 1C). Median PFS was 1.4 months (95% CI 1.3 to 4.0 months) and the 6-month and 12-month PFS rates were 28% (95% CI 14% to 45%) and 21% (95% CI 9% to 37%), respectively; the 18-month PFS rate was stable at 21% (table 2 and figure 2A). Median OS was 9.1 months (95% CI 6.6 months to not estimable (NE)) and the 12-month and 18-month OS rates were 48% (95% CI 30% to 64%) and 44% (95% CI 26% to 61%), respectively (figure 2B).

By investigator assessment, ORR and DCR were 16% and 34%, respectively (table 2). Two patients developed delayed PR after initial PD (time to PR 5.1 and 8.5 months); the patient with time to PR of 8.5 months by investigator assessment did not have target lesions and was therefore assessed as non-CR/non-PD by IRC based on the non-target lesions. The total clinical response rate by investigator assessment was 22%.

safety and tolerability

Thirty-one patients (97%) experienced a treatmentemergent AE (TEAE) and 24 (75%) experienced a grade ≥3 TEAE. Treatment was discontinued in 8 patients (25%) due to TEAE. Twenty-two patients (69%) experienced treatment-related AEs (TRAEs); of these, 11 (34%) experienced grade 3 TRAEs, which included rash maculopapular (n=2 (6%)) and increased liver enzymes, anemia, diabetic ketoacidosis, colitis, hyperglycemia and hyperthyroidism (n=1 each (3%)) (table 3). No grade 4 or 5 TRAEs were observed. TRAEs led to treatment discontinuation in 2 patients (colitis and SCC of the skin n=1 each (6%)). Keratoacanthomas (n=3 (9%)) and SCC of the skin (n=2 (6%)) were managed with excision and observation. No patient experienced an infusion-related reaction and no infusion rate reductions were necessary. Immune-related AEs were observed in 13 patients (41%), and 4 experienced grade 3 immune-related AEs (colitis and increased alanine and aspartate aminotransferases (n=1 each) and rash maculopapular (n=2)); no grade 4 or 5 immune-related AEs were observed (online supplementary table S4). The occurrence of immune-related colitis led to treatment discontinuation in 1 patient (3%).

biomarker analysis

Tumor PD-L1 expression status was available in 31 of 32 patients. Of 25 patients with PD-L1 expression on ≥1% of tumor cells (PD-L1-positive), 3 achieved PR and 3 had SD, resulting in an ORR of 12% (95% CI 3% to 31%) and DCR of 24% (95% CI 9% to 45%). One of 6 patients with PD-L1 expression on <1% of tumor cells (PD-L1negative) achieved PR (figure 3A and online supplementary table S5). Median PFS in patients with PD-L1-positive tumors was 1.4 months (95% CI 1.2 to 4.0 months) and 3.5 months (95% CI 1.2 months to NE) in patients with PD-L1-negative tumors (figure 3B and online supplementary table S5). Median OS in patients with PD-L1-positive tumors was 8.8 months (95% CI 6.3 months to NE) and NE (95% CI 3.2 months to NE) in patients with PD-L1negative tumors (figure 3C and online supplementary table S5).

Tumor HPV status was evaluable in 31 of 32 patients. Objective responses were observed in 3 of 9 patients with HPV-positive tumors (33% (95% CI 5% to 70%)) and in 1 of 22 patients with HPV-negative tumors (5% (95% CI 0% to 23%)) (figure 3D, online supplementary table S1 and online supplementary figure S1). Median PFS in patients with HPV-positive tumors was 2.7 months (95% CI 1.2 to 19.6 months) and 1.3 months (95% CI 1.2 to 4.0 months) in patients with HPV-negative tumors. Median OS in patients with HPV-positive tumors was 8.0 months (95% CI 2.7 months to NE) and 9.1 months (95% CI 6.3 months to NE) in patients with HPV-negative tumors (online supplementary table S1 and online supplementary figure S1).

A total of 30 patients had both HPV and PD-L1 data available. Among 9 patients with HPV-positive tumors, 7 (78%) were PD-L1-positive, and among 21 patients with HPV-negative tumors, 17 (81%) were PD-L1-positive, suggesting no association between HPV status and PD-L1 expression.

RNAseq data were analyzed to evaluate prespecified genes and gene signatures related to the TGF-β pathway and the immune-related pathways versus response to bintrafusp alfa. An extensive exploratory analysis was also conducted to identify genes or gene signatures associated with response to bintrafusp alfa in a biology/pathway agnostic manner. Several genes and signatures that reflect immune activity had higher levels in HPV-positive tumors compared with HPV-negative tumors, although the differences were not statistically significant after correcting for the size of the set tested (figure 3E).

Evaluable patient tumors were characterized according to immune phenotype as either inflamed (tumor infiltration of both CD4+ and CD8+ T cells and other immune cells), immune-excluded (immune cells are present but do not infiltrate the tumor) or immune-desert (lack of immune cells in and around the tumor). Of the 10 patients with a tumor of the inflamed immune phenotype, 3 had responses, and of 21 patients with a tumor of the immune-excluded phenotype, 1 had a response. A response following pseudoprogression was observed in a patient with a tumor of the immune-excluded phenotype (figure 3F).

disCussion

In this pretreated population of patients with advanced SCCHN, bintrafusp alfa monotherapy showed clinical activity, with an ORR of 16% and DCR of 34% per investigator, a 44% OS rate at 18 months and a median DOR (mDOR) of 18.1 months. These preliminary results are comparable to those in similar patient populations treated with the anti-PD-1 immune checkpoint inhibitors pembrolizumab and nivolumab, with ORRs of 13% to 18% and mDORs of 8-18.4 months in previously treated patients with PD-L1-unselected tumors. 4 7 17 37 38 Bintrafusp alfa also had a manageable safety profile. Potential TGF-β-related AEs (eg, keratoacanthoma) were well managed with simple excision and observation and did not result in trial discontinuation. No recurrence of keratoacanthoma or SCC has been reported. The incidence, severity and type of immune-related AEs observed with bintrafusp alfa were comparable to those seen with anti-PD-L1 agents. 4 7 17 37 Bintrafusp alfa monotherapy showed activity irrespective of tumor PD-L1 expression. DCRs in patients with PD-L1-positive and PD-L1-negative tumors were 24% and 33% and 18-month OS rates were 40% and 50%, respectively. We also observed numerically higher ORR in patients with HPV-positive tumors (33%) compared with patients with HPV-negative tumors (5%). A separate study found that bintrafusp alfa stimulated an HPV-specific response, [START_REF] Strauss | Safety and activity of M7824, a bifunctional fusion protein targeting PD-L1 and TGF-β, in patients with HPV associated cancers[END_REF] suggesting that bintrafusp alfa may have additional activity in HPV-positive disease. This observation may be due to the link between TGF-β signaling and HPV-positive tumors and to the greater inflammatory immune response generally observed in virus-associated cancers. 23-25 27 Indeed, a retrospective subgroup analysis of the dose-finding cohort of this phase I trial revealed an ORR of 46% in 11 patients with HPV-positive cancers (mixed cohort of patients with cervical, anal and SCCHN tumors) receiving bintrafusp alfa monotherapy administered at doses of 0.3-30 mg/kg, with durable and ongoing responses. [START_REF] Strauss | Safety and activity of M7824, a bifunctional fusion protein targeting PD-L1 and TGF-β, in patients with HPV associated cancers[END_REF] Furthermore, while responses to PD-L1 agents are generally observed in inflamed immune microenvironments, [START_REF] Taube | Association of PD-1, PD-1 ligands, and other features of the tumor immune microenvironment with response to anti-PD-1 therapy[END_REF] patients who achieved objective response or disease control in this study had tumors with either an inflamed or immune-excluded immune phenotype.

Thus, bifunctional targeting of the TGF-β and PD-L1 pathways with bintrafusp alfa may be an encouraging approach for patients with advanced SCCHN and in HPVassociated cancers. Bintrafusp alfa is being evaluated in phase I and II trials, including the phase II NCT03427411 study, which is specifically enrolling patients with HPVassociated malignancies, such as cervical cancer, p16+ oropharyngeal SCCHN and anogenital cancers, including vaginal, vulvar, anal and penile carcinomas.

A preclinical study investigated the link between TGF-β and PD-L1 and found correlations between the expression of several genes and antitumor activity. [START_REF] David | A novel bifunctional anti-PD-L1/TGF-β trap fusion protein (M7824) efficiently reverts mesenchymalization of human lung cancer cells[END_REF] Genes related to both pathways targeted by bintrafusp alfa (natural killer cell signature, effector T-cell signature, CD8A, CD8B, interferon-γ signature, TGFB1, TWIST1 and VIM) were examined in archival tumor samples using RNASeq; these data were found to be consistent with HPV-positive tumors having more pre-existing immune response.

The single-arm design of this study is a potential limitation, making it difficult to compare these results with those in patients treated with chemotherapy or established immune checkpoint inhibitors. Furthermore, the small sample sizes of subgroups (eg, for the HPV-positive cohort (n=9) and PD-L1-negative cohort (n=6)) limit conclusions. Nevertheless, the results in this expansion cohort warrant further investigation of bintrafusp alfa in advanced SCCHN.

ConClusions

In summary, bintrafusp alfa monotherapy showed clinical activity and had a manageable safety profile in this phase I cohort of patients with heavily pretreated, advanced SCCHN with limited or no available therapeutic options. Further investigation of bintrafusp alfa in SCCHN is warranted and ongoing.

2

  Cho BC, et al. J Immunother Cancer 2020;8:e000664. doi:10.1136/jitc-2020-000664

3

  Cho BC, et al. J Immunother Cancer 2020;8:e000664. doi:10.1136/jitc-2020-000664

5Figure 2

 2 Figure 2 PFS (A) and OS (B) in all patients. NE, not estimable; OS, overall survival; PFS, progression-free survival.

Figure 1

 1 Figure 1 Tumor regression from baseline (A and B) and time to and duration of response (C) in the full analysis set . CR, complete response; NE, not estimable; PD, progressive disease; PR, partial response; SD, stable disease. *Delayed PR after initial PD.

7Figure 3

 3 Figure3Tumor regression from baseline (A, D), PFS (B) and OS (C) according to tumor PD-L1 expression and HPV status, respectively, and gene expression profiling status (E) and immune phenotype (F). BOR, best overall response; CR, complete response; HPV, human papillomavirus; IFN-γ, interferon-γ; NE, not estimable; NK, natural killer; OS, overall survival; PD, progressive disease; PD-L1, programmed death-ligand 1; PFS, progression-free survival; PR, partial response; SD, stable disease; T eff , effector T cell; VIM, vimentin. *Delayed PR after initial PD.

Table 1

 1 Patient demographics and baseline characteristics

	Characteristic	N=32
	Sex
	Male	27 (84)
	Female	5 (16)
	Age, years
	Median (IQR)	60 (53-65)
	<65	22 (69)
	≥65	10 (31)
	Primary tumor site
	Oral cavity	11 (34)
	Oropharynx	7 (22)
	Hypopharynx	4 (13)
	Larynx	4 (13)
	Nasal cavity and sinuses	2 (6)
	Nasopharynx	1 (3)
	Other*	3 (9)
	Number of prior anticancer therapies
	1	8 (25)
	2	11 (34)
	≥3	13 (41)
	Prior anticancer therapy setting
	For locally advanced disease only	4 (13)
	≥1 prior therapy for recurrent/metastatic	28 (88)
	disease
	ECOG performance status
	0	7 (22)
	1	25 (78)
	PD-L1 expression
	<1% tumor cells	6 (19)
	≥1% tumor cells	25 (78)
	Not evaluable	1 (3)
	HPV status
	Positive	9 (28)
	Negative	22 (69)
	Not evaluable	1 (3)
	Prior cetuximab
	Yes	24 (75)
	No	8 (25)
	Data are number (%) unless stated otherwise.
	*Tonsil, pharynx and larynx and tongue (n=1 each (3%)).
	ECOG, Eastern Cooperative Oncology Group; HPV, human
	papillomavirus; PD-L1, programmed death-ligand 1.

4 Cho BC, et al. J Immunother Cancer 2020;8:e000664. doi:10.1136/jitc-2020-000664

Open access

Table 2

 2 Clinical activity of bintrafusp alfa (N=32)

			Per
		Per IRC	investigator
	Clinical activity endpoint	assessment	assessment
	Confirmed BOR, n (%)
	CR	0	0
	PR	4 (13)	5 (16)
	SD	4 (13)	6 (19)
	Non-CR/non-PD*	3 (9)	-
	PD	19 (59)	18 (56)
	Not evaluable	2 (6) †	3 (9) ‡
	Confirmed ORR (95% CI),	13 (4 to 29)	16 (5 to 33)
	%
	Total clinical response rate	16 (5.3 to 32.8) 22 (9.3 to 40)
	(95% CI), % §
	DCR (95% CI), %	34 (19 to 53)	34 (19 to 53)
	Median PFS (95% CI),	1.4 (1.3 to 4.0) 1.4 (1.3 to
	months		3.9)
	PFS rate (95% CI), %
	6-month	28 (14 to 45)	21 (9 to 37)
	12-month	21 (9 to 37)	17 (6 to 33)
	18-month	21 (9 to 37)	17 (6 to 33)

*Persistence of 1 or more non-target lesion(s) and/or maintenance of tumor marker level above the normal limits. † No IRC review due to no postbaseline assessments. ‡ No postbaseline assessments due to death within 6 weeks after start date; no postbaseline assessments; SD (or better) of insufficient duration (<6 weeks after start date without further evaluable tumor assessment) (n=1 each). § Defined as the proportion of patients with CR, PR and delayed PR after initial PD. BOR, best overall response; CR, complete response; DCR, disease control rate; IRC, independent review committee; NE, not estimable; ORR, objective response rate; PD, progressive disease; PFS, progression-free survival; PR, partial response; SD, stable disease.

Superieur (ABES). Protected by copyright. on December 19, 2023 at Agence Bibliographique de l Enseignement http://jitc.bmj.com/ J Immunother Cancer: first published as 10.1136/jitc-2020-000664 on 7 July 2020. Downloaded from

Acknowledgements The authors would like to thank the patients and their families, investigators, co-investigators and study teams at each of the participating centers and at Merck KGaA, Darmstadt, Germany, and EMD Serono Research & Development Institute, Billerica, Massachusetts, USA (a business of Merck KGaA, Darmstadt, Germany). The authors would also like to thank Christian Ihling, of Merck KGaA, Darmstadt, Germany, for his substantial contribution to the immune phenotype analysis. Medical writing support was provided by Shaun Rosebeck, PhD, of ClinicalThinking, Hamilton, New Jersey, USA, which was also funded by Merck KGaA and GlaxoSmithKline in accordance with Good Publication Practice (GPP3) guidelines (http://www. ismpp. org/ gpp3).

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