CD70-Targeted Allogeneic CAR T-Cell Therapy for Advanced Clear Cell Renal Cell Carcinoma.

Therapeutic approaches for clear cell renal cell carcinoma (ccRCC) remain limited; however, chimeric antigen receptor (CAR) T-cell therapies may offer novel treatment options. CTX130, an allogeneic CD70-targeting CAR T-cell product, was developed for the treatment of advanced or refractory ccRCC. We report that CTX130 showed favorable preclinical proliferation and cytotoxicity profiles and completely regressed RCC xenograft tumors. We also report results from 16 patients with relapsed/refractory ccRCC who received CTX130 in a phase I, multicenter, first-in-human clinical trial. No patients encountered dose-limiting toxicity, and disease control was achieved in 81.3% of patients. One patient remains in a durable complete response at 3 years. Finally, we report on a next-generation CAR T construct, CTX131, in which synergistic potency edits to CTX130 confer improved expansion and efficacy in preclinical studies. These data represent a proof of concept for the treatment of ccRCC and other CD70+ malignancies with CD70-targeted allogeneic CAR T cells. SIGNIFICANCE: Although the role of CAR T cells is well established in hematologic malignancies, the clinical experience in solid tumors has been disappointing. This clinical trial demonstrates the first complete response in a patient with RCC, reinforcing the potential benefit of CAR T cells in the treatment of solid tumors.

Vδ1 Effector and Vδ2 γδ T-Cell Subsets Shift in Frequency and Are Linked to Plasma Inflammatory Markers During Antiretroviral Therapy-Suppressed HIV Infection.

BACKGROUND: Chronic inflammation is prevalent with antiretroviral therapy (ART)-suppressed human immunodeficiency virus (HIV) infection and one immune cell subset putatively driving this phenomenon is TIGIT+ γδ T cells. METHODS: To elucidate γδ T-cell phenotypic diversity, spectral flow cytometry was performed on blood lymphocytes from individuals of a HIV and aging cohort and data were analyzed using bioinformatic platforms. Plasma inflammatory markers were measured and correlated with γδ T-cell subset frequencies. RESULTS: Thirty-nine distinct γδ T-cell subsets were identified (22 Vδ1+, 14 Vδ2+, and 3 Vδ1-Vδ2-Vγ9+) and TIGIT was nearly exclusively found on the Vδ1+CD45RA+CD27- effector populations. People with ART-suppressed HIV infection (PWH) exhibited high frequencies of distinct clusters of Vδ1+ effectors distinguished via CD8, CD16, and CD38 expression. Among Vδ2+ cells, most Vγ9+ (innate-like) clusters were lower in PWH; however, CD27+ subsets were similar in frequency between participants with and without HIV. Comparisons by age revealed lower 'naive' Vδ1+CD45RA+CD27+ cells in older individuals, regardless of HIV status. Plasma inflammatory markers were selectively linked to subsets of Vδ1+ and Vδ2+ cells. CONCLUSIONS: These results further elucidate γδ T-cell subset complexity and reveal distinct alterations and connections with inflammatory pathways of Vδ1+ effector and Vδ2+ innate-like subsets during ART-suppressed HIV infection.

Characterizing adjuvants' effects at murine immunoglobulin repertoire level.

Generating large-scale, high-fidelity sequencing data is challenging and, furthermore, not much has been done to characterize adjuvants' effects at the repertoire level. Thus, we introduced an IgSeq pipeline that standardized library prep protocols and data analysis functions for accurate repertoire profiling. We then studied systemically effects of CpG and Alum on the Ig heavy chain repertoire using the ovalbumin (OVA) murine model. Ig repertoires of different tissues (spleen and bone marrow) and isotypes (IgG and IgM) were examined and compared in IGHV mutation, gene usage, CDR3 length, clonal diversity, and clonal selection. We found Ig repertoires of different compartments exhibited distinguishable profiles at the non-immunized steady state, and distinctions became more pronounced upon adjuvanted immunizations. Notably, Alum and CpG effects exhibited different tissue- and isotype-preferences. The former led to increased diversity of abundant clones in bone marrow, and the latter promoted the selection of IgG clones in both tissues.

Comparison of Anti-SARS-CoV-2-Specific Antibody Signatures in Maternal and Infant Blood after COVID-19 Infection versus COVID-19 Vaccination during Pregnancy.

OBJECTIVE: The Advisory Committee on Immunization Practices and The American College of Obstetricians and Gynecologists recommend coronavirus disease 2019 (COVID-19) vaccine for pregnant persons to prevent severe illness and death. The objective was to examine levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG, IgM, and IgA against spike protein receptor binding domain (RBD) and nucleocapsid protein (NCP) in maternal and infant/cord blood at delivery after COVID 19 vaccination compared with SARS-CoV-2 infection at in mother-infant dyads at specified time points. STUDY DESIGN: Mothers with SARS-CoV-2 infection (n = 31) or COVID-19 vaccination (n = 25) during pregnancy were enrolled between July 2020 and November 2021. Samples were collected at delivery and IgG, IgM, and IgA to RBD of spike and NCPs compared in the infected and vaccinated groups. Timing of infection/vaccination prior to delivery and correlation with antibody levels was performed. RESULTS: The majority of participants received vaccination within 90 days of delivery and over half received the Pfizer BioNTech vaccine. There were no significant correlations between antibody levels and timing of infection or vaccination. Infant IgG levels to the RBD domain of spike protein were higher in the vaccinated group (n = 25) as compared with the infants born to mothers with infection (n = 31). Vaccination against COVID-19 during pregnancy was associated with detectable maternal and infant anti-RBD IgG levels at delivery irrespective of the timing of vaccination. CONCLUSION: Timing of vaccination had no correlation to the antibody levels suggesting that the timing of maternal vaccination in the cohort did not matter. There was no IgM detected in infants from vaccinated mothers. Infants from vaccinated mothers had robust IgG titers to RBD, which have a lasting protective effect in infants. KEY POINTS: · COVID-19 vaccination during pregnancy had detectable antibody.. · No correlation between antibody levels and timing of vaccination.. · Infants from vaccinated mothers had robust IgG titers to RBD..

Maternal, Infant, and Breast Milk Antibody Response Following COVID-19 Infection in Early Versus Late Gestation.

BACKGROUND: Coronavirus disease 2019 [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)] infection at varying time points during the pregnancy can influence antibody levels after delivery. We aimed to examine SARS-CoV-2 IgG, IgM and IgA receptor binding domain of the spike protein and nucleocapsid protein (N-protein) reactive antibody concentrations in maternal blood, infant blood and breastmilk at birth and 6 weeks after SARS-CoV-2 infection in early versus late gestation. METHODS: Mothers with SARS-CoV-2 infection during pregnancy were enrolled between July 2020 and May 2021. Maternal blood, infant blood and breast milk samples were collected at delivery and 6 weeks postpartum. Samples were analyzed for SARS-CoV-2 spike and N-protein reactive IgG, IgM and IgA antibodies. Antibody concentrations were compared at the 2 time points and based on trimester of infection ("early" 1st/2nd vs. "late" 3rd). RESULTS: Dyads from 20 early and 11 late trimester infections were analyzed. For the entire cohort, there were no significant differences in antibody levels at delivery versus 6 weeks with the exception of breast milk levels which declined over time. Early gestation infections were associated with higher levels of breastmilk IgA to spike protein ( P = 0.04). Infant IgG levels to spike protein were higher at 6 weeks after late infections ( P = 0.04). There were strong correlations between maternal and infant IgG levels at delivery ( P < 0.01), and between breastmilk and infant IgG levels. CONCLUSIONS: SARS-CoV-2 infection in early versus late gestation leads to a persistent antibody response in maternal blood, infant blood and breast milk over the first 6 weeks after delivery.

Cytokine levels in maternal and infant blood after COVID-19 vaccination during pregnancy in comparison with unvaccinated controls.

The objective of this study was to compare maternal and infant cytokine profiles at delivery among those vaccinated against SARS-CoV-2 during pregnancy to unvaccinated controls. Mother-infant dyads were enrolled in this prospective cohort study, and maternal blood and infant and/or cord blood collected. Samples were analyzed utilizing a LEGENDplex 13-plex human anti-viral response cytokine panel. Maternal IP-10 and IFN-λ2/3 were lower in the vaccinated cohort. In the infants, levels were lower for IL-1ß, IFN-λ2/3, and GM-CSF, and higher for IFN-λ1 in the vaccinated cohort. Vaccination against SARS-CoV-2 during pregnancy did not lead to elevations in cytokines in mothers or infants.

Novel ELISA Protocol Links Pre-Existing SARS-CoV-2 Reactive Antibodies With Endemic Coronavirus Immunity and Age and Reveals Improved Serologic Identification of Acute COVID-19 via Multi-Parameter Detection.

The COVID-19 pandemic has drastically impacted work, economy, and way of life. Sensitive measurement of SARS-CoV-2 specific antibodies would provide new insight into pre-existing immunity, virus transmission dynamics, and the nuances of SARS-CoV-2 pathogenesis. To date, existing SARS-CoV-2 serology tests have limited utility due to insufficient reliable detection of antibody levels lower than what is typically present after several days of symptoms. To measure lower quantities of SARS-CoV-2 IgM, IgG, and IgA with higher resolution than existing assays, we developed a new ELISA protocol with a distinct plate washing procedure and timed plate development via use of a standard curve. Very low optical densities from samples added to buffer coated wells at as low as a 1:5 dilution are reported using this 'BU ELISA' method. Use of this method revealed circulating SARS-CoV-2 receptor binding domain (RBD) and nucleocapsid protein (N) reactive antibodies (IgG, IgM, and/or IgA) in 44 and 100 percent of pre-pandemic subjects, respectively, and the magnitude of these antibodies tracked with antibody levels of analogous viral proteins from endemic coronavirus (eCoV) strains. The disease status (HIV, SLE) of unexposed subjects was not linked with SARS-CoV-2 reactive antibody levels; however, quantities were significantly lower in subjects over 70 years of age compared with younger counterparts. Also, we measured SARS-CoV-2 RBD- and N- specific IgM, IgG, and IgA antibodies from 29 SARS-CoV-2 infected individuals at varying disease states, including 10 acute COVID-19 hospitalized subjects with negative serology results by the EUA approved Abbott IgG chemiluminescent microparticle immunoassay. Measurements of SARS-CoV-2 RBD- and N- specific IgM, IgG, IgA levels measured by the BU ELISA revealed higher signal from 9 of the 10 Abbott test negative COVID-19 subjects than all pre-pandemic samples for at least one antibody specificity/isotype, implicating improved serologic identification of SARS-CoV-2 infection via multi-parameter, high sensitive antibody detection. We propose that this improved ELISA protocol, which is straightforward to perform, low cost, and uses readily available commercial reagents, is a useful tool to elucidate new information about SARS-CoV-2 infection and immunity and has promising implications for improved detection of all analytes measurable by this platform.

CD169+ Subcapsular Macrophage Role in Antigen Adjuvant Activity.

Vaccines have played a pivotal role in improving public health, however, many infectious diseases lack an effective vaccine. Controlling the spread of infectious diseases requires continuing studies to develop new and improved vaccines. Our laboratory has been investigating the immune enhancing mechanisms of Toll-like receptor (TLR) ligand-based adjuvants, including the TLR2 ligand Neisseria meningitidis outer membrane protein, PorB. Adjuvant use of PorB increases costimulatory factors on antigen presenting cells (APC), increases antigen specific antibody production, and cytokine producing T cells. We have demonstrated that macrophage expression of MyD88 (required for TLR2 signaling) is an absolute requirement for the improved antibody response induced by PorB. Here-in, we specifically investigated the role of subcapsular CD169+ marginal zone macrophages in antibody production induced by the use of TLR-ligand based adjuvants (PorB and CpG) and non-TLR-ligand adjuvants (aluminum salts). CD169 knockout mice and mice treated with low dose clodronate treated animals (which only remove marginal zone macrophages), were used to investigate the role of these macrophages in adjuvant-dependent antibody production. In both sets of mice, total antigen specific immunoglobulins (IgGs) were diminished regardless of adjuvant used. However, the greatest reduction was seen with the use of TLR ligands as adjuvants. In addition, the effect of the absence of CD169+ macrophages on adjuvant induced antigen and antigen presenting cell trafficking to the lymph nodes was examined using immunofluorescence by determining the relative extent of antigen loading on dendritic cells (DCs) and antigen deposition on follicular dendritic cells (FDC). Interestingly, only vaccine preparations containing PorB had significant decreases in antigen deposition in lymphoid follicles and germinal centers in CD169 knockout mice or mice treated with low dose clodronate as compared to wildtype controls. Mice immunized with CpG containing preparations demonstrated decreased FDC networks in the mice treated with low dose clodronate. Conversely, alum containing preparations only demonstrated significant decreases in IgG in CD169 knockout mice. These studies stress that importance of subcapsular macrophages and their unique role in adjuvant-mediated antibody production, potentially due to an effect of these adjuvants on antigen trafficking to the lymph node and deposition on follicular dendritic cells.

Toll-Like Receptor Ligand Based Adjuvant, PorB, Increases Antigen Deposition on Germinal Center Follicular Dendritic Cells While Enhancing the Follicular Dendritic Cells Network.

Vaccines are arguably one of the greatest advancements in modern medicine. Subunit vaccines comprise the majority of current preparations and consist of two main components-antigen and adjuvant. The antigen is a small molecule against which the vaccine induces an immune response to provide protection via the immunostimulatory ability of the adjuvant. Our laboratory has investigated the adjuvant properties of Toll-like receptor (TLR) ligand-based adjuvants, especially the outer membrane protein from Neisseria mengingitidis, PorB. In this current study we used PorB, along with CpG, an intracellular TLR9 agonist, and a non-TLR adjuvant, aluminum salts (Alum), to further investigate cellular mechanisms of adjuvanticity, focusing on the fate of intact antigen in the germinal center and association with follicular dendritic cells (FDCs). FDCs are located in the B cell light zone of the germinal center and are imperative for affinity maturation. They are stromal cells that retain whole intact antigen allowing recognition by the B cell receptor of the germinal center B cells. Our studies demonstrate that TLR ligands, but not Alum, increase the FDC network, while PorB and Alum increased colocalization of FDC and the model soluble antigen, ovalbumin (OVA). As PorB is the only adjuvant tested that induces both a higher number of FDCs and increased deposition of antigen on FDCs, it has the greatest ability to increase FDC-antigen interaction, essential for induction of B cell affinity maturation. These studies demonstrate a further mechanism and potential superiority of PorB as an adjuvant and its influence on antibody production.

Neisserial PorB immune enhancing activity and use as a vaccine adjuvant.

Our laboratory has focused on Porin B (PorB), an outer membrane protein from Neisseria meningitidis and TLR2 ligand-based adjuvant, to characterize specific molecular and cellular pathways involved in improved immune responses induced by vaccine adjuvants. PorB's ability to form micellar nanoparticular multi-molecular organized structures and its interaction with Toll-like receptor 2/1 complexes likely accounts for its potent adjuvant activity. Downstream from this stimulation, we have observed enhanced antigen uptake in antigen presenting cells (APC), greater antigen deposition in secondary lymphoid organs, and promotion of germinal center reactions. In mice, antigen-specific IgGs were increased after PorB adjuvanted vaccination using the model antigen ovalbumin (OVA). Likewise, this formulation resulted in more IL-4 and IFN-γ positive T cells. Mice that received PorB adjuvanted vaccinations benefitted from lower bacterial burdens when challenged with recombinant Listeria monocytogenes expressing OVA. Mouse models lacking MyD88 signaling in various APC types helped identify macrophages as an essential cell type for the adjuvant activity of PorB. We believe the work presented here provides examples of the mechanistic studies required to understand how vaccine adjuvants are contributing to the establishment of protective immunity.