Lymphocyte T Subsets and Outcome of Immune Checkpoint Inhibitors in Melanoma Patients: An Oncologist's Perspective on Current Knowledge.

Melanoma is the most aggressive and deadly form of skin cancer, and its incidence has been steadily increasing over the past few decades, particularly in the Caucasian population. Immune checkpoint inhibitors (ICI), anti-PD-1 monotherapy or in combination with anti-CTLA-4, and more recently, anti-PD-1 plus anti-LAG-3 have changed the clinical evolution of this disease. However, a significant percentage of patients do not benefit from these therapies. Therefore, to improve patient selection, it is imperative to look for novel biomarkers. Immune subsets, particularly the quantification of lymphocyte T populations, could contribute to the identification of ICI responders. The main purpose of this review is to thoroughly examine significant published data on the potential role of lymphocyte T subset distribution in peripheral blood (PB) or intratumorally as prognostic and predictive of response biomarkers in advanced melanoma patients treated with ICI regardless of BRAFV600 mutational status.

Autoimmune biomarkers in porto-sinusoidal vascular disease: Potential role in its diagnosis and pathophysiology.

BACKGROUND AND AIMS: Porto-sinusoidal vascular disease (PSVD) is a rare disease that requires excluding cirrhosis and other causes of portal hypertension for its diagnosis because it lacks a specific diagnostical test. Although it has been occasionally associated with autoimmune diseases, the pathophysiology of PSVD remains unknown. The aim of this study was to evaluate the potential role of autoimmunity in the pathophysiology and diagnosis of PSVD. METHODS: Thirty-seven consecutive patients with PSVD and 39 with cirrhosis matched by gender, signs of portal hypertension and liver function were included (training set). By using Indirect Immunofluorescence, ELISA and slot-blot methods data 22 autoantibodies were identified in patients with PSVD and cirrhosis. Presence of anti-endothelial cells antibodies (AECA) was assayed by a cell-based ELISA. Thirty-one PSVD, 40 cirrhosis patients, 15 patients with splenomegaly associated with haematological disease and 14 healthy donors were included in a validation set. FINDINGS: The proportion of patients with at least one positive antibody was statistically significantly higher in patients with PSVD compared with cirrhosis (92% vs 56%; P < .01). Specifically, AECA were significantly more frequent in PSVD than in cirrhosis (38% vs 15%; P = .013). Results were confirmed in the validation set. In the overall population, presence of AECA had a 63% positive predictive value for diagnosing PSVD and a 71% negative predictive value, with a specificity of 94% when the 1/16 level is used as cut-off. AECA positive serum samples react with a 68-72 kDa protein of human liver endothelial sinusoidal cells.

NSGS mice humanized with cord blood mononuclear cells show sustained and functional myeloid-lymphoid representation with limited graft-versus-host disease.

Humanized immunodeficient mice serve as critical models for investigating the functional interplay between transplanted human cells and a pre-reconstituted human immune system. These models facilitate the study of molecular and cellular pathogenic mechanisms and enable the evaluation of the efficacy and toxicity of immunotherapies, thereby accelerating their preclinical and clinical development. Current strategies rely on inefficient, long-term/delayed hematopoietic reconstitution by CD34+ hematopoietic progenitors or short-term reconstitution with peripheral blood mononuclear cells (PB-MNCs) associated with high rates of graft-versus-host disease (GvHD) and an inefficient representation of immune cell populations. Here, we hypothesized that immunologically naïve cord blood mononuclear cells (CB-MNCs) could serve as a superior alternative, providing long-lasting and functionally effective immune reconstitution. We conducted a comprehensive comparison between the non-obese diabetic (NOD).Cg-Prkdc∧ˆscid-IL2rg∧ˆtm1Wjl/SzJ (NSG) and NSG-Tg(CMV-IL3,CSF2,KITLG)∧ˆ1Eav/MloySzJ (NSGS) immunodeficient mouse models following humanization with either PB-MNCs or CB-MNCs. We assessed the engraftment dynamics of various human immune cells over time and monitored the development of GvHD in both models. For the most promising model, we extensively evaluated immune cell functionality in vitro and in vivo using sarcoma and leukemia xenografts. Humanizing NSGS mice with CB-MNCs results in a rapid, robust, and sustained representation of a diverse range of functional human lymphoid and myeloid cell populations while minimizing GvHD incidence. In this model, human immune cell populations significantly impair the growth and engraftment of sarcoma and B-cell acute lymphoblastic leukemia cells, with a significant inverse correlation between immune cell levels and tumor growth. This study establishes a fast, efficient, and reliable in vivo platform for various applications in cancer immunotherapy, particularly for exploring the complex interactions between cancer cells, immune cells, and the tumor microenvironment in vivo, prior to clinical development.

High specificity of engineered T cells with third generation CAR (CD28-4-1BB-CD3-ζ) based on biotin-bound monomeric streptavidin for potential tumor immunotherapy.

Introduction: Immunotherapy has revolutionized cancer treatment, and Chimeric Antigen Receptor T cell therapy (CAR-T) is a groundbreaking approach. Traditional second-generation CAR-T therapies have achieved remarkable success in hematological malignancies, but there is still room for improvement, particularly in developing new targeting strategies. To address this limitation, engineering T cells with multi-target universal CARs (UniCARs) based on monomeric streptavidin has emerged as a versatile approach in the field of anti-tumor immunotherapy. However, no studies have been conducted on the importance of the intracellular signaling domains of such CARs and their impact on efficiency and specificity. Method: Here, we developed second-generation and third-generation UniCARs based on an extracellular domain comprising an affinity-enhanced monomeric streptavidin, in addition to CD28 and 4-1BB co-stimulatory intracellular domains. These UniCAR structures rely on a biotinylated intermediary, such as an antibody, for recognizing target antigens. In co-culture assays, we performed a functional comparison between the third-generation UniCAR construct and two second-generation UniCAR variants, each incorporating either the CD28 or 4-1BB as co-stimulatory domain. Results: We observed that components in culture media could inhibit the binding of biotinylated antibodies to monomeric streptavidin-CARs, potentially compromising their efficacy. Furthermore, third-generation UniCAR-T cells showed robust cytolytic activity against cancer cell lines upon exposure to specific biotinylated antibodies like anti-CD19 and anti-CD20, underscoring their capability for multi-targeting. Importantly, when assessing engineered UniCAR-T cell activation upon encountering their target cells, third-generation UniCAR-T cells exhibited significantly enhanced specificity compared to second-generation CAR-T cells. Discussion: First, optimizing culture conditions would be essential before deploying UniCAR-T cells clinically. Moreover, we propose that third-generation UniCAR-T cells are excellent candidates for preclinical research due to their high specificity and multi-target anti-tumor cytotoxicity.

Cellular and humoral responses after second and third SARS-CoV-2 vaccinations in patients with autoimmune diseases treated with rituximab: specific T cell immunity remains longer and plays a protective role against SARS-CoV-2 reinfections.

Background: Humoral and cellular immune responses are known to be crucial for patients to recover from COVID-19 and to protect them against SARS-CoV-2 reinfection once infected or vaccinated. Objectives: This study aimed to investigate humoral and T cell responses to SARS-CoV-2 vaccination in patients with autoimmune diseases after the second and third vaccine doses while on rituximab and their potential protective role against reinfection. Methods: Ten COVID-19-naïve patients were included. Three time points were used for monitoring cellular and humoral responses: pre-vaccine to exclude virus exposure (time point 1) and post-second and post-third vaccine (time points 2 and 3). Specific IgG antibodies were monitored by Luminex and T cells against SARS-CoV-2 spike-protein by ELISpot and CoVITEST. All episodes of symptomatic COVID-19 were recorded. Results: Nine patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis and one with an undifferentiated autoimmune disease were included. Nine patients received mRNA vaccines. The last rituximab infusion was administered for a mean (SD) of 15 (10) weeks before the first vaccine and six patients were CD19-B cell-depleted. After a mean (SD) of 19 (10) and 16 (2) days from the second and third vaccine dose, IgG anti-SARS-CoV-2 antibodies were detected in six (60%) and eight (80%) patients, respectively. All patients developed specific T cell responses by ELISpot and CoVITEST in time points 2 and 3. Previous B cell depletion correlated with anti-SARS-CoV-2 IgG levels. Nine (90%) patients developed mild COVID-19 after a median of 7 months of the third dose. Conclusion: Rituximab in patients with autoimmune diseases reduces humoral responses but does not avoid the development of T cell responses to SARS-CoV-2 vaccination, which remain present after a booster dose. A steady cellular immunity appears to be protective against subsequent reinfections.

Acute and long-term immune responses to SARS-CoV-2 infection in unvaccinated children and young adults with inborn errors of immunity.

Purpose: To describe SARS-CoV-2 infection outcome in unvaccinated children and young adults with inborn errors of immunity (IEI) and to compare their specific acute and long-term immune responses with a sex-, age-, and severity-matched healthy population (HC). Methods: Unvaccinated IEI patients up to 22 years old infected with SARS-CoV-2 were recruited along with a cohort of HC. SARS-CoV-2 serology and ELISpot were performed in the acute phase of infection (up to 6 weeks) and at 3, 6, 9, and 12 months. Results: Twenty-five IEI patients (median age 14.3 years, min.-max. range 4.5-22.8; 15/25 males; syndromic combined immunodeficiencies: 48.0%, antibody deficiencies: 16.0%) and 17 HC (median age 15.3 years, min.-max. range 5.4-20.0; 6/17 males, 35.3%) were included. Pneumonia occurred in 4/25 IEI patients. In the acute phase SARS-CoV-2 specific immunoglobulins were positive in all HC but in only half of IEI in whom it could be measured (n=17/25): IgG+ 58.8% (10/17) (p=0.009); IgM+ 41.2% (7/17)(p<0.001); IgA+ 52.9% (9/17)(p=0.003). Quantitative response (index) was also lower compared with HC: IgG IEI (3.1 ± 4.4) vs. HC (3.5 ± 1.5)(p=0.06); IgM IEI (1.9 ± 2.4) vs. HC (3.9 ± 2.4)(p=0.007); IgA IEI (3.3 ± 4.7) vs. HC (4.6 ± 2.5)(p=0.04). ELISpots positivity was qualitatively lower in IEI vs. HC (S-ELISpot IEI: 3/11, 27.3% vs. HC: 10/11, 90.9%; p=0.008; N-ELISpot IEI: 3/9, 33.3% vs. HC: 11/11, 100%; p=0.002) and also quantitatively lower (S-ELISpot IEI: mean index 3.2 ± 5.0 vs. HC 21.2 ± 17.0; p=0.001; N-ELISpot IEI: mean index 9.3 ± 16.6 vs. HC: 39.1 ± 23.7; p=0.004). As for long term response, SARS-CoV-2-IgM+ at 6 months was qualitatively lower in IEI(3/8, 37.5% vs. 9/10 HC: 90.0%; p=0.043), and quantitatively lower in all serologies IgG, M, and A (IEI n=9, 1.1 ± 0.9 vs. HC n=10, 2.1 ± 0.9, p=0.03; IEI n=9, 1.3 ± 1.5 vs. HC n=10, 2.9 ± 2.8, p=0.02; and IEI n=9, 0.6 ± 0.5 vs. HC n=10, 1.7 ± 0.8, p=0.002 -respectively) but there were no differences at remaining time points. Conclusions: Our IEI pediatric cohort had a higher COVID-19 pneumonia rate than the general age-range population, with lower humoral and cellular responses in the acute phase (even lower compared to the reported IEI serological response after SARS-CoV-2 vaccination), and weaker humoral responses at 6 months after infection compared with HC.

What will (and should) be improved in CAR immunotherapy?

Chimeric antigen receptor (CAR) is probably one of the most successful proposals for cancer treatment, especially hematological diseases for which several Advanced Therapies Medicinal Products (ATMP) have been approved worldwide by drug agencies. But, despite this unprecedented success in the oncology and cell/gene therapy fields, there are a lot of aspects that could (and should) be improved in the multiple aspects that involve this complex therapy: from the design of the chimeric molecule to the clinical protocols of use of the engineered T-cells, including even the regulatory rules that they are currently restricting the development of these hopeful therapies. In this chapter, we will try to summarize the main aspects that can (and probably should) be improved for the expansion of immunotherapy with CAR proposals beyond onco-hematology.

CoVITEST: A Fast and Reliable Method to Monitor Anti-SARS-CoV-2 Specific T Cells From Whole Blood.

Cellular and humoral immune responses are essential for COVID-19 recovery and protection against SARS-CoV-2 reinfection. To date, the evaluation of SARS-CoV-2 immune protection has mainly focused on antibody detection, generally disregarding the cellular response, or placing it in a secondary position. This phenomenon may be explained by the complex nature of the assays needed to analyze cellular immunity compared with the technically simple and automated detection of antibodies. Nevertheless, a large body of evidence supports the relevance of the T cell's role in protection against SARS-CoV-2, especially in vulnerable individuals with a weakened immune system (such as the population over 65 and patients with immunodeficiencies). Here we propose to use CoVITEST (Covid19 anti-Viral Immunity based on T cells for Evaluation in a Simple Test), a fast, affordable and accessible in-house assay that, together with a diagnostic matrix, allows us to determine those patients who might be protected with SARS-CoV-2-reactive T cells. The method was established using healthy SARS-CoV-2-naïve donors pre- and post-vaccination (n=30), and further validated with convalescent COVID-19 donors (n=51) in a side-by-side comparison with the gold standard IFN-γ ELISpot. We demonstrated that our CoVITEST presented reliable and comparable results to those obtained with the ELISpot technique in a considerably shorter time (less than 8 hours). In conclusion, we present a simple but reliable assay to determine cellular immunity against SARS-CoV-2 that can be used routinely during this pandemic to monitor the immune status in vulnerable patients and thereby adjust their therapeutic approaches. This method might indeed help to optimize and improve decision-making protocols for re-vaccination against SARS-CoV-2, at least for some population subsets.

Epigenetic Profiling and Response to CD19 Chimeric Antigen Receptor T-Cell Therapy in B-Cell Malignancies.

BACKGROUND: Chimeric antigen receptor (CAR) T cells directed against CD19 (CART19) are effective in B-cell malignancies, but little is known about the molecular factors predicting clinical outcome of CART19 therapy. The increasingly recognized relevance of epigenetic changes in cancer immunology prompted us to determine the impact of the DNA methylation profiles of CART19 cells on the clinical course. METHODS: We recruited 114 patients with B-cell malignancies, comprising 77 patients with acute lymphoblastic leukemia and 37 patients with non-Hodgkin lymphoma who were treated with CART19 cells. Using a comprehensive DNA methylation microarray, we determined the epigenomic changes that occur in the patient T cells upon transduction of the CAR vector. The effects of the identified DNA methylation sites on clinical response, cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, event-free survival, and overall survival were assessed. All statistical tests were 2-sided. RESULTS: We identified 984 genomic sites with differential DNA methylation between CAR-untransduced and CAR-transduced T cells before infusion into the patient. Eighteen of these distinct epigenetic loci were associated with complete response (CR), adjusting by multiple testing. Using the sites linked to CR, an epigenetic signature, referred to hereafter as the EPICART signature, was established in the initial discovery cohort (n = 79), which was associated with CR (Fisher exact test, P < .001) and enhanced event-free survival (hazard ratio [HR] = 0.36; 95% confidence interval [CI] = 0.19 to 0.70; P = .002; log-rank P = .003) and overall survival (HR = 0.45; 95% CI = 0.20 to 0.99; P = .047; log-rank P = .04;). Most important, the EPICART profile maintained its clinical course predictive value in the validation cohort (n = 35), where it was associated with CR (Fisher exact test, P < .001) and enhanced overall survival (HR = 0.31; 95% CI = 0.11 to 0.84; P = .02; log-rank P = .02). CONCLUSIONS: We show that the DNA methylation landscape of patient CART19 cells influences the efficacy of the cellular immunotherapy treatment in patients with B-cell malignancy.

Point-Of-Care CAR T-Cell Production (ARI-0001) Using a Closed Semi-automatic Bioreactor: Experience From an Academic Phase I Clinical Trial.

Development of semi-automated devices that can reduce the hands-on time and standardize the production of clinical-grade CAR T-cells, such as CliniMACS Prodigy from Miltenyi, is key to facilitate the development of CAR T-cell therapies, especially in academic institutions. However, the feasibility of manufacturing CAR T-cell products from heavily pre-treated patients with this system has not been demonstrated yet. Here we report and characterize the production of 28 CAR T-cell products in the context of a phase I clinical trial for CD19+ B-cell malignancies (NCT03144583). The system includes CD4-CD8 cell selection, lentiviral transduction and T-cell expansion using IL-7/IL-15. Twenty-seven out of 28 CAR T-cell products manufactured met the full list of specifications and were considered valid products. Ex vivo cell expansion lasted an average of 8.5 days and had a mean transduction rate of 30.6 ± 13.44%. All products obtained presented cytotoxic activity against CD19+ cells and were proficient in the secretion of pro-inflammatory cytokines. Expansion kinetics was slower in patient's cells compared to healthy donor's cells. However, product potency was comparable. CAR T-cell subset phenotype was highly variable among patients and largely determined by the initial product. TCM and TEM were the predominant T-cell phenotypes obtained. 38.7% of CAR T-cells obtained presented a TN or TCM phenotype, in average, which are the subsets capable of establishing a long-lasting T-cell memory in patients. An in-depth analysis to identify individual factors contributing to the optimal T-cell phenotype revealed that ex vivo cell expansion leads to reduced numbers of TN, TSCM, and TEFF cells, while TCM cells increase, both due to cell expansion and CAR-expression. Overall, our results show for the first time that clinical-grade production of CAR T-cells for heavily pre-treated patients using CliniMACS Prodigy system is feasible, and that the obtained products meet the current quality standards of the field. Reduced ex vivo expansion may yield CAR T-cell products with increased persistence in vivo.

Development of a Novel Anti-CD19 Chimeric Antigen Receptor: A Paradigm for an Affordable CAR T Cell Production at Academic Institutions.

Genetically modifying autologous T cells to express an anti-CD19 chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19+ B cell malignancies in several clinical trials (CTs). Making this treatment available to our patients prompted us to develop a novel CART19 based on our own anti-CD19 antibody (A3B1), followed by CD8 hinge and transmembrane region, 4-1BB- and CD3z-signaling domains. We show that A3B1 CAR T cells are highly cytotoxic and specific against CD19+ cells in vitro, inducing secretion of pro-inflammatory cytokines and CAR T cell proliferation. In vivo, A3B1 CAR T cells are able to fully control disease progression in an NOD.Cg-Prkdc scid Il2rd tm1Wjl /SzJ (NSG) xenograph B-ALL mouse model. Based on the pre-clinical data, we conclude that our CART19 is clearly functional against CD19+ cells, to a level similar to other CAR19s currently being used in the clinic. Concurrently, we describe the implementation of our CAR T cell production system, using lentiviral vector and CliniMACS Prodigy, within a medium-sized academic institution. The results of the validation phase show our system is robust and reproducible, while maintaining a low cost that is affordable for academic institutions. Our model can serve as a paradigm for similar institutions, and it may help to make CAR T cell treatment available to all patients.