anti-CD20 Antibody Treatment Research Articles

Humoral immune response against SARS-CoV-2 and polyethylene glycol elicited by anti-SARS-CoV-2 mRNA vaccine, and effect of pre-existing anti-polyethylene glycol antibody in patients with hematological and autoimmune diseases

BackgroundThe effects of vaccination are modified by hematological and autoimmune diseases and/or treatment. Anti-SARS-CoV-2 mRNA vaccine contains polyethylene glycol (PEG), it is largely unknown whether PEG influences the effects of vaccination or induces a humoral response. This study examined whether anti-PEG antibodies before vaccination (pre-existing) influenced the acquisition of SARS-CoV-2 antibodies and evaluated the relationship between the development of anti-SARS-CoV-2 antibodies and anti-PEG antibodies after SARS-CoV-2 vaccination in hematological and autoimmune diseases. MethodsAnti-SARS-CoV-2 antibody IgG, anti-PEG IgG, and IgM titers were evaluated in patients with hematological and autoimmune diseases after the second dose of BNT162B2. Anti-PEG IgG and IgM titers were also measured before vaccination to examine changes after vaccination and the relationship with vaccine efficacy. ResultsIn patients with hematological (n = 182) and autoimmune diseases (n = 96), anti-SARS-CoV-2 and anti-PEG antibody titers were evaluated after a median of 33 days from 2nd vaccination. The median anti-SARS-CoV-2 antibody titers were 1901 AU/mL and 3832 AU/mL in patients with hematological and autoimmune disease, respectively. Multiple regression analysis showed that age and days from 2nd vaccination were negatively associated with anti-SARS-CoV-2 antibody titers. Anti-CD20 antibody treatment was negatively correlated with anti-SARS-CoV-2 antibody titers in hematological disease, and C-reactive protein (CRP) was positively correlated with anti-SARS-CoV-2 antibody titers in autoimmune disease. Baseline anti-PEG antibody titers were significantly higher in patients with autoimmune disease but were not correlated with anti-SARS-CoV-2 antibody titers. Patients with increased anti-PEG IgG acquired higher anti-SARS-CoV-2 antibody titers in patients with autoimmune disease. ConclusionsAnti-SARS-CoV-2 antibody acquisition was suboptimal in patients with hematological disease, but both anti-SARS-CoV-2 antibody and anti-PEG IgG were acquired in patients with autoimmune disease, reflecting robust humoral immune response. Pre-existing anti-PEG antibody titers did not affect anti-SARS-CoV-2 antibody acquisition.

Management of vaccinations in patients with non-Hodgkin lymphoma.

Vaccinations are fundamental tools in preventing infectious diseases, especially in immunocompromised patients like those affected by non-Hodgkin lymphomas (NHLs). The COVID-19 pandemic made clinicians increasingly aware of the importance of vaccinations in preventing potential life-threatening SARS-CoV-2-related complications in NHL patients. However, several studies have confirmed a significant reduction in vaccine-induced immune responses after anti-CD20 monoclonal antibody treatment, thus underscoring the need for refined immunization strategies in NHL patients. In this review, we summarize the existing data about COVID-19 and other vaccine's efficacy in patients with NHL and propose multidisciplinary team-based recommendations for the management of vaccines in this specific group of patients.

Anti-CD20 treatment attenuates Th2 cell responses: implications for the role of lung follicular mature B cells in the asthmatic mice.

B cells were believed to act as antigen-presenting cells (APCs) to promote T helper type 2 (Th2) cell responses. However, the role of lung B cells and its subpopulations in Th2 cell responses in asthma remains unclear. We leveraged an anti-CD20 monoclonal antibody (mAb) treatment that has been shown to selectively deplete B cells in mice and investigated whether this treatment modulates Th2 cell responses and this modulation is related to lung follicular mature (FM) B cells in a murine model of asthma. We used a house dust mite (HDM)-induced asthma mouse model and found that anti-CD20 mAb treatment attenuates Th2 cell responses. Meanwhile, anti-CD20 mAb treatment did dramatically reduce the number of B cells, especially FM B cells in the lungs, but did not impact the frequency of other immune cell types, including lung T cells, dendritic cells, natural killer cells, and regulatory T cells in wild-type mice. Moreover, we found that the suppressive effect of anti-CD20 mAb treatment on Th2 cell responses could be reversed upon adoptive transfer of lung FM B cells, but not lung CD19+ B cells without FM B cells in asthmatic mice. These findings reveal that anti-CD20 mAb treatment alleviates Th2 cell responses, possibly by depleting lung FM B cells in a Th2-driven asthma model. This implies a potential therapeutic approach for asthma treatment through the targeting of lung FM B cells.

Targeting CD20-expressing malignant melanoma cells augments BRAF inhibitor killing.

Mutant BRAF targeted therapies remain a standard of care for the treatment of metastatic malignant melanoma (MM); however, high initial response rates are tempered by the persistence of residual MM cells that eventually lead to disease recurrence and mortality. As MM recurrence during targeted therapy can present with the simultaneous occurrence of multiple tumour nodules at the original body sites, we hypothesized the presence of an intrinsically resistant MM cell subpopulation. To identify an MM cell subpopulation that is intrinsically resistant to targeted therapy and possibly responsible for MM recurrence. Using melanoma cell lines, we defined culture conditions for the reproducible three-dimensional growth of melanospheres to investigate putative cancer stem cell populations. We undertook RNA sequencing and bioinformatic analysis to characterize cell populations between adherent and nonadherent culture, and cells expressing or not expressing CD20. Furthermore, we defined an in vitro assay to evaluate the killing of melanoma cancer stem cells as a therapeutic test using combination therapies targeting driver mutation and CD20. We described the culture conditions that promote MM cells to form melanospheres with a reproducible colony-forming efficiency rate of 0.3-1.3%. RNA sequencing of melanosphere vs. conventional MM cell cultures (n = 6), irrespective of the BRAF mutation status, showed that melanosphere formation was associated with growth and differentiation transcriptional signatures resembling MM tumours. Importantly, melanosphere formation also led to the emergence of a CD20+ MM cell subpopulation, similar to that observed in primary human MM tumours. CD20+ MM cells were resistant to BRAF inhibitor therapy and, consistent with this finding, demonstrated a Forkhead box protein M1 transcriptomic profile (n = 6). Combining BRAF inhibitor and anti-CD20 antibody treatment led to the additional killing of previously resistant CD20+ BRAF mutant MM cells. In patients with MM that harbour a CD20+ subpopulation, combined therapy with BRAF inhibitor and anti-CD20 antibody could potentially kill residual MM cells and prevent disease recurrence.

Transient CD4+ T cell depletion during suppressive ART reduces the HIV reservoir in humanized mice.

Lifelong treatment is required for people living with HIV as current antiretroviral therapy (ART) does not eradicate HIV infection. Latently infected cells are essentially indistinguishable from uninfected cells and cannot be depleted by currently available approaches. This study evaluated antibody mediated transient CD4+ T cell depletion as a strategy to reduce the latent HIV reservoir. Anti-CD4 antibodies effectively depleted CD4+ T cells in the peripheral blood and tissues of humanized mice. We then demonstrate that antibody-mediated CD4+ T cell depletion of HIV infected ART-suppressed animals results in substantial reductions in cell-associated viral RNA and DNA levels in peripheral blood cells over the course of anti-CD4 antibody treatment. Recovery of CD4+ T cells was observed in all tissues analyzed except for the lung 26 days after cessation of antibody treatment. After CD4+ T cell recovery, significantly lower levels of cell-associated viral RNA and DNA were detected in the tissues of anti-CD4 antibody-treated animals. Further, an 8.5-fold reduction in the levels of intact HIV proviral DNA and a 3.1-fold reduction in the number of latently infected cells were observed in anti-CD4-antibody-treated animals compared with controls. However, there was no delay in viral rebound when ART was discontinued in anti-CD4 antibody-treated animals following CD4+ T cell recovery compared with controls. Our results suggest that transient CD4+ T cell depletion, a long-standing clinical intervention that might have an acceptable safety profile, during suppressive ART can reduce the size of the HIV reservoir in humanized mice.

A Phase Ib Open-Label Study of MRG001 in Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma (R/R DLBCL)

Background: Patients with R/R DLBCL who fail multi-agent chemoimmunotherapy have a poor prognosis and a need for more treatment options. MRG001 is an antibody drug conjugate (ADC), which is composed of a chimeric anti-CD20 monoclonal antibody conjugated via a valine citrulline linker to monomethyl auristatin E (MMAE). MRG001 has been evaluated in a first-in-human phase Ia study and determined 1.8 mg/kg Q3W as RP2D. Here we present results from phase Ib study of MRG001 in patients with R/R DLBCL who had failed established therapies for the safety and preliminary antitumor activity evaluation. Methods: Patients aged ≥18 years with ECOG PS 0-1, histologically diagnosed R/R DLBCL who had failed ≥ 2 prior therapies (a prior anti-CD20 antibody treatment was necessary) were enrolled in this single-arm, open-label phase Ib study. All patients received MRG001 1.8 mg/kg IV Q3W until disease progression, unacceptable toxicity, or withdrawal. The primary endpoint was overall response rate (ORR) assessed by investigators according to the Lugano response criteria, duration of response (DoR) and safety. Results: At data cutoff (Jul 28, 2023), 35 patients were enrolled and received ≥1 dose of MRG001; 29 (82.9%) had discontinued the treatment and 6 (17.1%) were ongoing. Median age was 62 years (34-76), 24 (68.6%) patients were male, 24 (68.6%) had an ECOG PS of 1. The median antitumor systemic prior lines was 3 (2-7), and most patients (62.9%) had ≥3 prior lines of therapy. 3 patients received prior ASCT, and 7 patients received prior CAR-T. Median follow-up (range) was 6.8 months (0.7-18.0) for all patients. Among the 34 evaluable patients, ORR by investigator review was 38.2% (95% CI, 22.2-56.4), with 6 patients (17.6%) having a CR and 7 (20.6%) having a PR; 10 patients (29.4%) had SD, and disease control rate (DCR) was 67.7% (95% CI, 49.5-82.6). Median DoR for the 13 responders was 10.5 months (95% CI, 6.5-NE). For patients with a CR, mDoR was 13.7 months (95% CI, 10.5-NE). Treatment related adverse events (TRAEs) occurred in 33 (94.3%) pts, most commonly reported were leukopenia (68.6%), neutropenia (65.7%), anemia (45.7%), AST increased (40.0%), and lymphopenia (31.4%). Grade 3/4 TRAEs occurred in 20 (57.1%) pts, most commonly reported were neutropenia (40.0%), leukopenia (17.1%), and lymphopenia (11.4%). Discontinuation due to a TRAE occurred in 2 (5.7%) patients. Patients all successfully recoverd from these TRAEs through the implementation of clinical supportive care. Grade 1/2 treatment-related peripheral neuropathy occurred in 22 (5.7%) patients; no grade 3/4 AEs occurred. No dose reduction occurred due to TRAE. No infusion reaction or tumor lysis syndrome due to treatment occurred and no pts died because of TRAEs. Conclusion: Phase Ib results show that MRG001 had clinically meaningful antitumor activity in patients with R/R DLBCL who progressed after multiple lines of therapy. Additionally, the safety profile was manageable and patients tolerated treatment well. Clinical trial information: NCT05155839.

B cell-mediated CD4 T-cell costimulation via CD86 exacerbates pro-inflammatory cytokine production during autoimmune intestinal inflammation

Dysregulated B cell responses have been described in inflammatory-bowel disease (IBD) patients; however, the role of B cells in IBD pathology remained incompletely understood. We here provide evidence for a detrimental role of activated B cells during the onset of autoimmune intestinal inflammation. Using Wiskott-Aldrich Syndrome interacting protein deficient (Wipf1-/-) mice as a mouse model of chronic colitis, we identified CD86 expression on activated B cells as a crucial factor exacerbating pro-inflammatory cytokine production of intestinal CD4 T cells. Depleting B cells through anti-CD20 antibody treatment or blocking co-stimulatory signals mediated by CD86 through CTLA-4-Ig diminished intestinal inflammation in our mouse model of chronic IBD at the onset of disease. This was due to a reduction in aberrant humoral immune responses and reduced CD4 T cell pro-inflammatory cytokine production, especially IFN-γ and GM-CSF. Interestingly, in addition to B cells isolated from the inflamed colon of Wipf1-/- mice, we also found CD86 mRNA and protein expression up-regulated on activated B cells isolated from inflamed tissue of human IBD patients. B cell activation and CD86 expression was boosted by soluble CD40L in vitro, which we found in the serum of mice and human IBD patients. In summary, our data provides detailed insight into the contribution of B cells to intestinal inflammation, with implications for the treatment of IBD.

Clinical analysis of prolonged viral clearance time in patients with lymphoma combined with novel coronavirus infection.

Objective: To compare the period of viral clearance and its influencing factors after severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection between patients with lymphoma and lung cancer. Methods: We retrospectively collected the clinical data of patients with lymphoma and lung cancer (118 cases) diagnosed with SARS-CoV-2 infection and hospitalized in the First Affiliated Hospital of Anhui Medical University between 1 December 2022, and 15 March 2023. Finally, 87 patients with prolonged virus clearance times were included and divided into lymphoma (40 cases) and lung cancer (47 cases) groups. We used the Kaplan-Meier method to draw a negative turn curve. We performed a univariate analysis of the prolongation of virus clearance time and a Cox regression model for multivariate analysis. Results: The median times for viral clearance in the lung cancer and lymphoma groups were 18 (95% confidence interval [CI] 15.112-20.888) and 32 (95%CI 27.429-36.571) days, respectively. Log-rank analysis showed a statistically significant difference (p = 0.048), and the lymphocyte count in the lymphoma group was lower than that in the lung cancer group (p = 0.044). We used the Cox regression model to conduct a multivariate analysis, which revealed that in lymphoma patients, the interval between the time of diagnosis and the time of SARS-CoV-2 infection <24months (hazard ratio [HR]: 0.182, 95%CI: 0.062-0.535, p = 0.02), an interval between the last anti-CD20 monoclonal antibody treatment and the time of SARS-CoV-2 infection of <2months (HR: 0.101, 95%CI: 0.029-0.358, p < 0.001), and a decrease in peripheral blood lymphocyte levels (HR: 0.380, 95%CI: 0.179-0.808, p = 0.012) were independent risk factors for prolonged viral clearance time. Conclusion: Patients with lymphoma combined with SARS-CoV-2 infection had a longer virus clearance time than did patients with lung cancer. Moreover, the lymphocyte count in the lymphoma group was lower than that in the lung cancer group; therefore, the immune status of patients with lymphoma is lower than that of patients with lung cancer. An interval between lymphoma diagnosis and SARS-CoV-2 infection of <2years, anti-CD20 monoclonal antibody treatment within the past 2months, and a decrease in lymphocyte levels in the peripheral blood prolonged the virus clearance time in the patients in this study.

Tumor cell-expressed lipolysis-stimulated lipoprotein receptor negatively regulates T-cell function.

Lipolysis-stimulated lipoprotein receptor (LSR) is known as a lipoprotein receptor. LSR is expressed in various solid tumors, including epithelial ovarian, gastric, and colon cancers. High LSR expression is significantly associated with poor prognosis, but its role in cancer has not been fully elucidated. LSR belongs to the Ig protein superfamily, which is conserved in B7 family. Here, we assessed LSR as a novel immune checkpoint molecule. We developed a novel anti-LSR antibody (#27-6 mF-18) that defects antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activity. The #27-6 mF-18 cross-reacts with both human and mouse LSR. We found that LSR was expressed on 4T1 murine breast cancer cell line. The #27-6 mF-18 exhibited antitumor effects against the 4T1 syngeneic tumor model, a poor immunogenic model refractory to treatment with anti-PD-1 or anti-CTLA-4 antibodies. Compared with control antibody-treated mice, mice treated with #27-6 mF-18 showed significantly increased numbers of CD8+ T cells and a ratio of activated CD8+ T cells infiltrated in the tumor tissue. This antitumor effect was abrogated by CD8+ T-cell depletion through anti-CD8 antibody treatment, indicating that LSR negatively regulates tumor immunity by repressing CD8+ T cells. These findings show that LSR negatively regulates T-cell immune activity. LSR targeting could provide immune checkpoint inhibitors for cancer immunotherapy.

Efficacy of anti-CD3 monoclonal antibodies in delaying the progression of recent-onset type 1 diabetes mellitus: A systematic review, meta-analyses and meta-regression.

Type 1 diabetes mellitus is widely recognized as a chronic autoimmune disease characterized by the pathogenic destruction of beta cells, resulting in the loss of endogenous insulin production. Insulin administration remains the primary therapy for symptomatic treatment. Recent studies showed that disease-modifying agents, such as anti-CD3 monoclonal antibodies, have shown promising outcomes in improving the management of the disease. In late 2022, teplizumab received approval from the US Food and Drug Administration (FDA) as the first disease-modifying agent for the treatment of type 1 diabetes. This review aims to evaluate the clinical evidence regarding the efficacy of anti-CD3 monoclonal antibodies in the prevention and treatment of type 1 diabetes. A comprehensive search of PubMed, Google Scholar, Scopus and Cochrane Central Register of Controlled Trials (CENTRAL) was conducted up to December 2022 to identify relevant randomized controlled trials. Meta-analysis was performed using a random-effects model, and odds ratios with 95% confidence intervals (CIs) were calculated to quantify the effects. The Cochrane risk of bias tool was employed for quality assessment. In total, 11 randomized controlled trials involving 1397 participants (908 participants in the intervention arm, 489 participants in the control arm) were included in this review. The mean age of participants was 15 years, and the mean follow-up time was 2.04 years. Teplizumab was the most commonly studied intervention. Compared with placebo, anti-CD3 monoclonal antibody treatment significantly increased the C-peptide concentration in the area under the curve at shorter timeframes (mean difference = 0.114, 95% CI: 0.069 to 0.159, p = .000). Furthermore, anti-CD3 monoclonal antibodies significantly reduced the patients' insulin intake across all timeframes (mean difference = -0.123, 95% CI: -0.151 to -0.094, p < .001). However, no significant effect on glycated haemoglobin concentration was observed. The findings of this review suggest that anti-CD3 monoclonal antibody treatment increases endogenous insulin production and improves the lifestyle of patients by reducing insulin dosage. Future studies should consider the limitations, including sample size, heterogeneity and duration of follow-up, to validate the generalizability of these findings further.

Treatment of Nodular Lymphocyte-Predominant Hodgkin Lymphoma: Where Do We Stand? Where Do We Go?

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare B cell-derived lymphoma entity accounting for ≈5% of all Hodgkin lymphoma (HL) cases. In recent decades, patients with newly diagnosed NLPHL have usually been treated very similarly to classical HL (cHL). The 10-year overall survival rates with HL-directed approaches are in excess of 90%. However, pathological and clinical characteristics of NLPHL resemble indolent B-cell non-Hodgkin lymphoma (B-NHL) in some aspects. Thus, nodular lymphocyte-predominant B-cell lymphoma has been proposed as an alternative name, and the use of B-NHL-directed treatment strategies has become more common in NLPHL despite limited data. Given the often indolent clinical course of NLPHL, even in the case of relapse, the majority of patients with disease recurrence do not require high-dose chemotherapy and autologous stem cell transplantation but are treated sufficiently with low-intensity approaches such as single-agent anti-CD20 antibody treatment. The establishment of novel prognostic scores for NLPHL patients may optimize risk group and treatment allocation in newly diagnosed and relapsed disease.

Efficacy and Safety of Bruton Tyrosine Kinase Inhibitor Plus Anti-CD20 Antibody Therapy Compared With Chemoimmunotherapy as Front-line Treatment for Chronic Lymphocytic Leukemia: A Systematic Review and Meta-analysis of Randomized Controlled Trials.

Treatment with chemoimmunotherapy (CIT) is considered an appropriate front-line treatment option for chronic lymphocytic leukemia (CLL). However, outcomes remain suboptimal. Bruton tyrosine kinase inhibitor (BTKi) combined with anti-CD20 antibody is an effective treatment for treatment-naïve, relapsed/refractory CLL patients. A systematic review and meta-analysis of randomized controlled trials was performed to compare the efficacy and safety of CIT versus BTKi + anti-CD20 antibody as front-line treatment for CLL patients. The endpoints of interest included progression-free survival (PFS), overall survival (OS), overall response rate (ORR), complete response (CR) rate, and safety. Four trials (including 1479 patients) were available as of December 2022 and fulfilled the eligibility criteria. BTKi + anti-CD20 antibody treatment significantly prolonged PFS [hazard ratio (HR), 0.25; 95% confidence interval (CI), 0.15-0.42] compared with CIT, while the combination therapy did not significantly improve OS compared with CIT (HR, 0.73; 95% CI, 0.50-1.06). We observed consistent benefits for PFS among patients with unfavorable features. Although pooled analysis indicated that the addition of BTKi to anti-CD20 antibody led to a higher ORR than CIT [risk ratio (RR), 1.16; 95% CI, 1.13-1.20], there was no difference in CR between the two arms (RR, 1.10; 95% CI, 0.27-4.55). The risk of grade ≥3 adverse effects (AE) was comparable between the two groups (RR, 1.04; 95% CI, 0.92-1.17). The BTKi + anti-CD20 antibody therapy has superior outcomes compared with CIT among patients with treatment-naïve CLL, without excess of toxicity. Future studies should compare next-generation targeted agent combinations versus CIT to determine the optimal management of CLL patients.

The CD16 and CD32b Fc-gamma receptors regulate antibody-mediated responses in mouse natural killer cells

Abstract Natural killer (NK) cells are innate lymphocytes capable of mediating immune responses without prior sensitization. NK cells express Fc-gamma receptors (FcγR) that engage the Fc region of IgG. Studies investigating the role of FcγR on mouse NK cells have been limited due to lack specific reagents. In this study, we characterize the expression and biological consequences of activating mouse NK cells through their FcγR. We demonstrate that most NK cells express the activating CD16 receptor, and a subset of NK cells also express the inhibitory CD32b receptor. Critically, these FcγRs are functional on mouse NK cells and can modulate antibody-mediated responses. We also characterized mice with conditional knock-out alleles of Fcgr3 (CD16) or Fcgr2b (CD32b) in the NK and innate lymphoid cell (ILC) lineage. NK cells in these mice did not reveal any developmental defects and were responsive to cross-linking activating NK receptors, cytokine stimulation, and killing of YAC-1 targets. Importantly, CD16-deficient NK cells failed to induce antibody-directed cellular cytotoxicity (ADCC) of antibody-coated B cell lymphomas in in vitro assays. In addition, we demonstrate the important role of CD16 on NK cells using an in vivo model of cancer immunotherapy using anti-CD20 antibody treatment of B cell lymphomas. This project was funded by fellowships from Burroughs Wellcome Fund (BWF) and the Cancer Research Institute to O.A.A., and grants from the Parker Institute for Cancer Immunotherapy (PICI), and the NIH (AI068129 and AI146581) to L.L.L.

Anti-CD20 antibodies and bendamustine attenuate humoral immunity to COVID-19 vaccination in patients with B-cell non-Hodgkin lymphoma

Serologic responses of COVID-19 vaccine are impaired in patients with B-cell lymphoma, especially those who had recently been treated with anti-CD20 monoclonal antibodies. However, it is still unclear whether those patients develop an immune response following vaccination. We investigated the efficacy of vaccination against SARS-CoV-2 in 171 patients with B-cell non-Hodgkin lymphoma (B-NHL) who received two doses of an mRNA-based COVID-19 vaccine and we compared the efficacy of vaccination to that in 166 healthy controls. Antibody titers were measured 3 months after administration of the second vaccine dose. Patients with B-NHL showed a significantly lower seroconversion rate and a lower median antibody titer than those in healthy controls. The antibody titers showed correlations with the period from the last anti-CD20 antibody treatment to vaccination, the period from the last bendamustine treatment to vaccination and serum IgM level. The serologic response rates and median antibody titers were significantly different between diffuse large B-cell lymphoma (DLBCL) patients in whom anti-CD20 antibody treatment was completed within 9 months before vaccination and follicular lymphoma (FL) patients in whom anti-CD20 antibody treatment was completed within 15 months before vaccination. Moreover, the serologic response rates and median antibody titers were significantly different among FL patients in whom bendamustine treatment was completed within 33 months before vaccination. We demonstrated that B-NHL patients who were recently treated with anti-CD20 antibodies and bendamustine had a diminished humoral response to COVID-19 vaccination. UMIN 000,045,267.

Low incidence and morbidity of Epstein-Barr virus reactivation following donor lymphocyte infusions.

Low incidence and morbidity of Epstein-Barr virus reactivation following donor lymphocyte infusions.

Abstract PD2-07: Tumor CD73 regulates zoledronate induced TIL accumulation into triple negative breast cancer

Abstract Background: CD73 is a membrane-bound protein that by extracellular adenosine production, regulates immune cell function. Low CD73 expression is associated with better prognosis among triple-negative breast cancer (TNBC) patients. Adjuvant bisphosphonates (BPs, such as zoledronate) improve survival among post-menopausal breast cancer patients, but the mechanism for this is unknown. Since BPs also affect immune cells, we studied 1) whether zoledronate-treatment has an effect on tumor infiltrating lymphocytes (TILs) and 2) whether tumor CD73 expression affects these responses in TNBC tumors. Methods: 4T1 mouse mammary cells, stably expressing non-targeting shRNA (sh-NT, control cells) or sh-CD73 were inoculated into the mammary fat pads of Balb/c mice. The mice were treated with i.p. vehicle or zoledronate (ZOL) each 4th day for total of six times. Additionally, to deplete B-cells, the mice were treated with an anti-CD20 or control IgG antibody. Tumor sizes were assessed with a caliper, TILs were studied with immunostainings. Blood lymphocytes were analyzed with Flow cytometry. Results: Sh-CD73 cells formed significantly smaller tumors, than sh-NT cells. ZOL significantly inhibited the growth of both sh-CD73 and sh-NT tumors, but at sacrifice, this effect of ZOL was more significant against sh-CD73 tumors. ZOL increased the % of CD19+/CD21+ and CD19+/23+ in blood of both mice groups, and this effect was inhibited by anti-CD20 antibody. Although neither treatment alone had an effect, the % of CD3+/CD8+ cells in blood were increased in both mice groups by the combination of ZOL and anti-CD20-antibody. ZOL induced accumulation of B220+, CD4+ and CD8+ TILs into both tumor groups. These TIL effects were reduced with anti-CD20 antibody treatment. Anti-CD20 antibody alone inhibited significantly sh-CD73 tumor growth. Anti-CD20 antibody did not interfere with ZOL inhibition of tumor growth in either group. Conclusions: ZOL has inflammatory effects on blood lymphocytes and TILs, which may be affected by tumor expression of immune system regulating proteins, such as CD73. B cell depletion may further prevent tumor growth, in the context of CD73 inhibition. Further studies are needed to investigate whether B-cell inhibition affects ZOL-induced tumor growth inhibition. Citation Format: Nataliia Petruk, Arafat Siddiqui, Sina Tadayon, Arja Jukkola, Jorma Määttä, Pieta Mattila, Jouko Sandholm, Katri S. Selander. Tumor CD73 regulates zoledronate induced TIL accumulation into triple negative breast cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD2-07.

IFN-γ-STAT1-mediated NK2R expression is involved in the induction of antitumor effector CD8+ T cells in vivo.

The induction of antitumor effector T cells in the tumor microenvironment is a crucial event for cancer immunotherapy. Neurokinin receptor 2 (NK2R), a G protein-coupled receptor for neurokinin A (NKA), regulates diverse physiological functions. However, the precise role of NKA-NK2R signaling in antitumor immunity is unclear. Here, we found that an IFN-γ-STAT1 cascade augmented NK2R expression in CD8+ T cells, and NK2R-mediated NKA signaling was involved in inducing antitumor effector T cells in vivo. The administration of a synthetic analog of double-stranded RNA, polyinosinic-polycytidylic acid (poly I:C), into a liver cancer mouse model induced type I and type II IFNs and significantly suppressed the tumorigenesis of Hepa1-6 liver cancer cells in a STAT1-dependent manner. The reduction in tumor growth was diminished by the depletion of CD8+ T cells. IFN-γ stimulation significantly induced NK2R and tachykinin precursor 1 (encodes NKA) gene expression in CD8+ T cells. NKA stimulation combined with anti-CD3 monoclonal antibody (mAb) treatment significantly augmented IFN-γ and granzyme B production by CD8+ T cells compared with the anti-CD3 mAb alone in vitro. ERK1/2 phosphorylation and IκBα degradation in activated CD8+ T cells were suppressed under NK2R deficiency. Finally, we confirmed that tumor growth was significantly increased in NK2R-deficient mice compared with that in wild-type mice, and the antitumor effects of poly I:C were abolished by NK2R absence. These findings suggest that IFN-γ-STAT1-mediated NK2R expression is involved in the induction of antitumor effector T cells in the tumor microenvironment, which contributes to the suppression of cancer cell tumorigenesis in vivo. In this study, we revealed that IFN-γ-STAT1-mediated NK2R expression is involved in the induction of antitumor effector CD8+ T cells in the tumor microenvironment, which contributes to suppressing the tumorigenesis of liver cancer cells in vivo.

Assessment of anti-CD20 antibody pre-treatment for augmentation of CAR-T cell therapy in SIV-infected rhesus macaques.

During chronic HIV and SIV infections, the majority of viral replication occurs within lymphoid follicles. In a pilot study, infusion of SIV-specific CD4-MBL-CAR-T cells expressing the follicular homing receptor, CXCR5, led to follicular localization of the cells and a reduction in SIV viral loads in rhesus macaques. However, the CAR-T cells failed to persist. We hypothesized that temporary disruption of follicles would create space for CAR-T cell engraftment and lead to increased abundance and persistence of CAR-T cells. In this study we treated SIV-infected rhesus macaques with CAR-T cells and preconditioned one set with anti-CD20 antibody to disrupt the follicles. We evaluated CAR-T cell abundance and persistence in four groups of SIVmac239-infected and ART-suppressed animals: untreated, CAR-T cell treated, CD20 depleted, and CD20 depleted/CAR-T cell treated. In the depletion study, anti-CD20 was infused one week prior to CAR-T infusion and cessation of ART. Anti-CD20 antibody treatment led to temporary depletion of CD20+ cells in blood and partial depletion in lymph nodes. In this dose escalation study, there was no impact of CAR-T cell infusion on SIV viral load. However, in both the depleted and non-depleted animals, CAR-T cells accumulated in and around lymphoid follicles and were Ki67+. CAR-T cells increased in number in follicles from 2 to 6 days post-treatment, with a median 15.2-fold increase in follicular CAR-T cell numbers in depleted/CAR-T treated animals compared to an 8.1-fold increase in non-depleted CAR-T treated animals. The increase in CAR T cells in depleted animals was associated with a prolonged elevation of serum IL-6 levels and a rapid loss of detectable CAR-T cells. Taken together, these data suggest that CAR-T cells likely expanded to a greater extent in depleted/CAR-T cell treated animals. Further studies are needed to elucidate mechanisms mediating the rapid loss of CAR-T cells and to evaluate strategies to improve engraftment and persistence of HIV-specific CAR-T cells. The potential for an inflammatory cytokine response appears to be enhanced with anti-CD20 antibody treatment and future studies may require CRS control strategies. These studies provide important insights into cellular immunotherapy and suggest future studies for improved outcomes.

The CD16 and CD32b Fc-gamma receptors regulate antibody-mediated responses in mouse natural killer cells.

Natural killer (NK) cells are innate lymphocytes capable of mediating immune responses without prior sensitization. NK cells express Fc-gamma receptors (FcγRs) that engage the Fc region of IgG. Studies investigating the role of FcγRs on mouse NK cells have been limited due to lack specific reagents. In this study, we characterize the expression and biological consequences of activating mouse NK cells through their FcγRs. We demonstrate that most NK cells express the activating CD16 receptor, and a subset of NK cells also expresses the inhibitory CD32b receptor. Critically, these FcγRs are functional on mouse NK cells and can modulate antibody-mediated responses. We also characterized mice with conditional knockout alleles of Fcgr3 (CD16) or Fcgr2b (CD32b) in the NK and innate lymphoid cell (ILC) lineage. NK cells in these mice did not reveal any developmental defects and were responsive to cross-linking activating NK receptors, cytokine stimulation, and killing of YAC-1 targets. Importantly, CD16-deficient NK cells failed to induce antibody-directed cellular cytotoxicity of antibody-coated B-cell lymphomas in in vitro assays. In addition, we demonstrate the important role of CD16 on NK cells using an in vivo model of cancer immunotherapy using anti-CD20 antibody treatment of B-cell lymphomas.

Clinical relevance of molecular aspects in extranodal marginal zone lymphoma: a critical appraisal

Extranodal marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT lymphoma) is among the more common types of lymphoma accounting for up to 8% of newly diagnosed lymphoma cases. As opposed to other B-cell lymphomas, however, no predominant genetic hallmark has been defined in MALT lymphoma, but different localizations appear to be affected by different, sometimes distinct changes. Nonetheless, a high proportion of these genetic changes reported in MALT lymphomas dysregulate the pathways leading to NF-kB activation. t(11;18)(q21;q21)/BIRC3::MALT1 appears to be MALT lymphoma specific and is found in 24% of gastric and 40% of pulmonary MALT lymphomas. The translocation is associated with more disseminated disease in gastric MALT lymphoma and is found in a large percentage of patients whose lymphoma is unresponsive to antibiotic eradication of Helicobacter pylori. In addition to t(11;18)(q21;q21), nuclear expression of BCL10 or NF-kB appears to be highly associated with lymphoma cell survival independence of H. pylori-mediated stimulations. Antibiotic eradication, however, is the recommended therapy of choice irrespective of genetic findings, and molecular analysis is not required before initiation of therapy. The influence of genetic translocations including t(11;18)(q21;q21) on systemic therapies, however, is less clearly defined. While small series have shown no influence on the outcome for treatment with the anti-CD20 antibody rituximab (R) or treatment with cladribine (2-CdA), conflicting data have been reported for alkylating agents, especially chlorambucil and the combination of R + chlorambucil. None of other genetic changes seen in MALT lymphoma to date has discernible value in routine clinical applications, but recent data suggest that changes in TNFAIP3(A20), KMTD2 and CARD11 might be associated with response to Bruton kinase inhibitors.