key: cord-1041918-d4q2tjou
authors: Lasagna, A.; Agustoni, F.; Percivalle, E.; Borgetto, S.; Paulet, A.; Comolli, G.; Sarasini, A.; Bergami, F.; Sammartino, J.C.; Ferrari, A.; Zavaglio, F.; Arena, F.; Lilleri, D.; Secondino, S.; Falzoni, M.; Schiavo, R.; Klersy, C.; Cavanna, L.; Baldanti, F.; Pedrazzoli, P.; Cassaniti, I.
title: A snapshot of the immunogenity, efficacy and safety of a full course of BNT162b2 anti-SARS-CoV-2 vaccine in cancer patients treated with PD-1/PD-L1 inhibitors: a longitudinal cohort study.
date: 2021-09-01
journal: ESMO Open
DOI: 10.1016/j.esmoop.2021.100272
sha: 5d6fc7c33aa187eb6b1dcdab16c3f9232ee080e7
doc_id: 1041918
cord_uid: d4q2tjou

BACKGROUND: Very few cancer patients were enrolled in COVID-19 vaccine studies. In order to address this gap of knowledge, real world studies are mandatory. Aim of this study was to assess both humoral and cellular response after a mRNA vaccination schedule. PATIENTS AND METHODS: Eighty-eight consecutive cancer patients treated with PD-1/PD-L1 inhibitors were enrolled from the beginning of the vaccination campaign for frail patients. Blood samples for humoral and cell-mediated immune response evaluation were obtained before vaccination (T0), before the second administration (T1) and 21 days after the second dose (T2). The primary end-point was the evaluation of the percentage of participants showing a significant increase in SARS-CoV-2 specific T cells, measured by an ELISPOT assay, after the second dose of BNT162b2 vaccine. The proportion of patients who reached the primary endpoint is computed together with its exact binomial 95% confidence interval (95%CI). RESULTS: In SARS-CoV-2 naïve subjects, Spike-specific T-cell response was almost undetectable at T0 (median 0.0 IFNγ SFU/million PBMC IQR 0-7.5) and significantly increased at T1 and T2 (median 15.0 IFNγ SFU/million PBMC 25th-75th 0-40 vs 90 IFNγ SFU/million PBMC 25th-75th 32.5-224; respectively) (p<0.001). Focusing on naïve and experienced SARS-CoV-2 subjects no differences were reported both in terms of CD4 and CD8-specific T-cell response, suggesting that BNT162b2 is able to elicit both adaptive responses after complete vaccination schedule, regardless previous SARS-CoV-2 exposure. The level of SARS-CoV-2 NT Abs was low at T1 in SARS-CoV-2 naïve subjects [median 1:5 (IQR 1:5-1:20)] but reached a significantly higher median 1:80 (25th-75th 1:20-1:160) at T2 (p<0.0001). Moreover no COVID-19 cases were documented throughout the period of study. CONCLUSIONS: Our data have demonstrated that the administration of a full course of BNT162b2 vaccine elicited a sustained immune response against SARS-CoV-2 regardless to the type of cancer and/or the type of ICIs.

to be different between healthy subjects and cancer patients [8] , but immune response against SARS-CoV-2 in cancer patients receiving vaccination against SARS-CoV-2 in terms of antibody titer (value and geometric mean) and specific T cells response is yet unknown. People at higher risk, like patients with cancer have been underrepresented in ongoing phase 3 clinical trials [9] .

Preliminary reports demonstrated that the response rates of initial immune response to the BNT162b2 vaccine among patients with myeloproliferative neoplasms were similar to those observed in the general population [10] . In a cohort prospective study of patients with cancer on systemic therapy, most of patients were seropositive for SARS-CoV 2 antispike IgG antibodies after the full course of BNT162b2 vaccine, but their antibody titers were significantly lower than those of control group and in the multivariable analysis the chemotherapy plus Immune checkpoint inhibitors (ICIs) appears the only variable significantly associated with lower IgG titers [11] . ICIs promote antitumor response by interrupting co-inhibitory signaling pathways and immune-mediated elimination of tumor cells [12] and based on preclinical data about their mechanism of action, ICIs are likely to enhance rather than diminish the immune response against vaccines [13] .

Many issues remain unanswered, including the time needed to develop immunity, the duration of immunity, the effects of different therapy on immunity and the optimal time points and schedule of vaccine administration in patients with cancer. The aim of the present study is to evaluate the characteristics and the magnitude of the T-and B-cell response in cancer patients treated with PD-1/PD-L1 inhibitors and receiving COVID-19 vaccine.

This study was an observational longitudinal multicenter study. Consecutive cancer patients treated with PD-1/PD-L1 inhibitors were enrolled from the beginning of the vaccination campaign for frail patients. The study was conducted according to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement for reporting observational studies [14] . Subjects were monitored during the overall period of vaccination at baseline (before vaccination; T0), before the second administration (T1) and 21 days after the second dose (T2). The study (Co-Vax) was approved by the local Ethics Committee (Comitato Etico Area Pavia) and

Institutional Review Board (P-20210023530). All the subjects signed an informed written consent.

Cancer patients who were programmed to receive a full course of vaccine during immunotherapy (anti-PD-1 or anti-PD-L1), in combination or not with chemotherapy were enrolled. The Inclusion Criteria were: i) patients aged 18 and older, regardless of gender; ii) life-expectancy (as estimated by treating physician) ≥ 6 months; iii) confirmed histological diagnosis of solid tumors; iv) treatment with immunotherapy alone or in combination with chemotherapy; v) signing of informed consent; vi) patients with a history of a previous laboratory-confirmed diagnosis of SARS-CoV-2 infection will be also enrolled. Patients with psychiatric illness/social situations that would limit compliance with study requirements were excluded from the study. 

Immunological analysis has been limited to subjects vaccinated with mRNA BNT162b2 anti-SARS-CoV-2 vaccine to avoid confounding factors of different vaccines. Peripheral blood mononuclear cells (PBMC) were isolated from heparin-treated blood by standard density gradient centrifugation.

Briefly, PBMC (2x10 5 /100μl culture medium per well) were stimulated in duplicate for 24 h in 96well plates (coated with anti-IFN-γ monoclonal capture antibody) with peptide pools (15mers, overlapping by 10 aminoacids, Pepscan, Lelystad The Netherlands) representative of the spike protein (S) at the final concentration of 0.25 µg/ml. Phytoheamagglutinin (PHA; 5 µg/mL) was used as positive control, and medium alone as negative control. Enzyme linked immunospot assay was performed according to our previous protocol [15] .

Responses ≥10 net spots/million PBMC were considered positive based on background results obtained with negative control (mean SFC+2SD).

To evaluate T-cell subsets proliferation, PBMC (600,000/200 μl culture medium per well) collected from 20 vaccinated patients, were stimulated in triplicate in 96-well round-bottom plates with peptide pools representative of the S protein, at the final concentration of 0.1 µg/ml for 7 days. Peptide pool from human actin was used as negative control antigen. Culture medium was RPMI 1640 supplemented with 2mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin, 5% of heat inactivated human serum AB, 1 mM Sodium Pyruvate, 100 μM non-essential amino acids and 50µM 2-Mercaptoethanol. After culture, cells were washed with PBS 0,5 µM EDTA and stained in PBS with Live/Dead Fixable Violet Dye (Invitrogen) at 4°C. After washing, cells were stained at room temperature in PBS 5% FCS with anti-CXCR5, followed by anti-IgG2b (biotinylated) and, subsequently, with Streptavidin BV421, CD3 PerCP 5.5, CD4 APC Cy7, CD8 FITC, CD25 PECy7, CD278 (ICOS) APC antibodies. Finally, cells were washed and suspended in 1% paraformaldehyde.

The frequency of CD25+ICOS+ expanded CD3+CD4+ and CD3+CD8+ T-cells was determined by subtracting the frequency of PBMC incubated with actin peptides from the frequency of PBMC incubated with SARS-CoV-2 S and N peptides. Flow-cytometry analyses were performed with a FACS Canto II flow cytometer and DIVA software (BD Biosciences).

J o u r n a l P r e -p r o o f Chemiluminescent assay (Liason SARS-CoV-2 S1/S2 IgG, Diasorin, Saluggia, Italy) for the quantitative characterization of SARS-CoV-2 anti-S1 and anti-S2 IgG antibodies, according to manufacturer's instructions was performed using serum samples. Results were given as AU/mL and a cut-off of 15 AU/mL was considered for the definition of positive samples. Results ranging from 12 and 15 AU/mL were considered borderline while IgG titer less than 12 AU/mL was given as a negative result. Neutralizing antibody serum titre was determined as previously reported [16] . Results were considered positive if higher or equal to 1:10 serum titre.

The Stata software (release 17, StataCorp, College Station, TX, USA) was used for computation. A 2-sided p-value<0.05 is considered statistically significant. Date are described with the median and 25th-75th percentiles if continuous and as counts and percent if categorical. Log-transformation is applied to continuous variables for the purpose of the analysis. The proportion of patients who reached the primary endpoint is computed together with its exact binomial 95% confidence interval (95%CI). Potential correlates of the primary endpoint are evaluated using logistic models; odds ratios (OR) and 95%CI are presented. In case of null cells, exact logistic regression is used. The modifying effect of age (<=/>65 years) is assessed by including an interaction term in the model. In no case, we observed heterogeneity in the effect of the potential correlates based on age class (data not shown).

The rate of immunological response at the several time points is computed together with its exact binomial 95%CI. Changes over time are evaluated with a generalized linear model for repeated measures for binomial or continuous data. Huber-White robust standard errors are computed to account for intra-patient correlation over time. Differences with respect to baseline and 95%CI are computed. The rate of adverse events are presented together with their 95%CI.

Eighty-eight subjects (23 females and 65 males; median age 68, 25th-75th 61.5-73) were enrolled between 24th March and 23rd April 2021. Sixty-seven (76.1%) had lung cancer, eight (9.1%) had melanoma, seven (7.9%) had kidney cancer, the remaining six patients (6.9%) had head&neck cancer 

Twenty vaccinated ICIs patients (13 SARS-CoV-2 naïve and 7 SARS-CoV-2 experienced subjects) were tested for phenotypical analysis of Spike-specific T-cell proliferative response. Overall, if the median CD4+ T cell response was higher than respect to CD8+ T cell response (median 3.67 25th-75th 0.23-12.92 vs 2.25 25th-75th 0.57-9.06) the difference was not statistically significant (p=0.573). Focusing on naïve and experienced SARS-CoV-2 subjects no differences were reported both in terms of CD4+ and CD8+-specific T-cell response, suggesting that BNT162b2 is able to elicit both adaptive responses after complete vaccination schedule, regardless previous SARS-CoV-2 exposure (Figure 3 ).

Overall, a significant increase of S1/S2 IgG response in SARS-CoV-2 naïve subjects at T1 (median 7.6 IQR 3.5-27 AU/mL) than respect to baseline (median 3.5 25th-75th 3.5-3.5 AU/mL; p<0.0001).

Similarly, S1/S2 IgG response in SARS-CoV-2 experienced subjects was significantly higher than that detected at T1 (median 152 25th-75th 92.7-259.5 p<0.001) (Figure 4a SARS-CoV-2 experienced subjects reached higher level of S1/S2 IgG and SARS-CoV-2 NT Abs even after one dose of vaccine (2425 AU/mL 25th-75th 1540-3768 AU/mL and 1:640 25th-75th 1:640-1:640; respectively) than respect to SARS-CoV-2 naïve subjects tested at T2 (p<0.0001), suggesting that one dose of vaccine may act as a booster in subjects with previous SARS-CoV-2 exposure, regardless to time of previous infection.

No COVID-19 cases were documented throughout the period of study.

The most common side effects observed after the first dose of vaccine were pain at the injection site 

It is well known that cancer patients are at increased risk of morbidity and mortality from SARS-CoV-2 infection [17] . Despite this evidence, very few patients with cancer were enrolled in COVID-19 vaccine studies and so many unanswered questions remain about the risk-benefit ratio of these vaccines in this frail population. As there is an urgent need to protect cancer patients from COVID- with hematologic tumors): a significantly lower seroconversion rate was observed in patients with hematologic malignancies (85%) versus solid tumors (98%). Instead patients receiving immune checkpoint inhibitor therapy had high seroconversion rates [20] .

In our cohort study, we aimed to evaluate the humoral and cell-mediated immune response in cancer patients treated with PD-1/PD-L1 inhibitors and receiving BNT162b2 anti-SARS-CoV-2 vaccine.

We chose to specifically evaluate only cancer patients treated with ICIs for their mechanism of action on the immune system. Our data confirmed that the rate of SARS-CoV-2 naïve subjects developing positive antibody level measured by S1/S2 assay is high as expected following two vaccine doses (95%), even if only one third of patients developed a positive antibody response after the first dose, confirming previous results obtained in cancer patients [21] and in healthcare workers [22, 23] .

Furthermore, we also investigated the development of Spike-specific cell-mediated immune response using a home-made ex-vivo ELISpot assay. Interestingly, over 90% of patients developed a sustained Spike-specific T-cell response at T2, suggesting that adaptive immune response is not compromised in this cohort of subjects. Furthermore, a sustained CD4 and CD8 T cell response was elicited by vaccination. Thus, in our cohort of subjects with solid cancer, the administration of a full course of a mRNA vaccine provides good protection against COVID-19 and these results do not depend upon the type of cancer and/or the type of ICIs. Our data offer another interesting observation: SARS-CoV-2 experienced patients mounted a robust neutralizing immune response even after a first dose suggesting that the past infection may be an immune enhancer condition and may not be a reason for vaccine hesitancy.

Goshen-Lago et al reported that the adverse events after the two doses of BNT162b2 vaccine in cancer patients were similar to data in the studies comprising healthy population [24] . In addition, Waissengrin and colleagues described the safety of the BNT162b2 mRNA vaccine in a cohort of patients treated with ICIs [25] . They compared side-effects in the patients treated with ICIs with a healthy control group matched by sex and year of birth. In this paper, they observed no new immunerelated side-effects or exacerbation of existing immune-related side-effects and a side-effect profile similar in the healthy controls and the patients with cancer [25] .

Since both ICI treatment and COVID-19 vaccines stimulate the immune response, it has been hypothesized that these vaccines may increase the incidence of irAEs with ICI treatment. To date, there are no data demonstrating the direct answer.

In our study only one patient reported two immune-related side effects (hepatitis G3 and colitis G3) 10 days after the first dose of vaccine. Unfortunately, such small numbers do not allow any kind of conclusion, but the description of further cases may be interesting.

The strength of our data consists in the simultaneous detection of both anti spike and neutralizing antibody titers and IFN gamma release assay giving us a comprehensive tracking of humoral and cellular immune response. In the same time, we have collected data at baseline and after a full course of vaccination instead of only a single dose and so far. The availability of baseline data for each patient of both cellular and immunological status allows us to demonstrate that the response depends only on the vaccination eliminating other confounding factors. Moreover, all patients are receiving PD-1/PD-L1 inhibitors, making our results homogeneous about the type of treatment. Our paper has several limitations. To begin with, the lack of a control group that is not possible to study for ethical reasons and the small sample size of patients evaluated. Secondly, the study cohort represents an older population, with a median age of 68 years. It is well known the multifaceted phenomenon of the immunosenescence [26] and that the immune response to many other vaccines is reciprocally associated with age [27] . Thirdly, the IFN gamma release assay results are difficult to interpret because the comparison data are lacking.

Mounting a robust immune response against SARS-CoV-2 requires two phases: neutralization and effector T cell functions. Our data confirm the efficacy of the vaccine in triggering both the humoral 

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. J o u r n a l P r e -p r o o f

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