Euphorbia Factor L2 ameliorates the Progression of K/BxN Serum-Induced Arthritis by Blocking TLR7 Mediated IRAK4/IKKβ/IRF5 and NF-kB Signaling Pathways

Toll like receptor (TLR)s have a central role in regulating innate immunity and their activation have been highlighted in the pathogenesis of rheumatoid arthritis (RA). EFL2, one of diterpenoids derived from Euphorbia seeds, is nearly unknown expect for its improving effect on acute lung injury. Our present study aimed to investigate EFL2’s pharmacokinetic features, its therapeutic effect on rheumatoid arthritis, and explored the potential anti-arthritic mechanisms. K/BxN serum transfer arthritis (STA) murine model was used to assess EFL2’s anti-arthritic effects. We also applied UPLC-MS method to measure the concentrations of EFL2 in plasma. The inhibitory effects of this compound on inflammatory cells infiltration and activation were determined by flow cytometry analysis and quantitative real-time polymerase chain reaction (qRT-PCR) in vivo, and immunochemistry staining and ELISA in murine macrophages and human PBMCs in vitro, respectively. The mechanism of EFL2 on TLRs mediated signaling pathway was evaluated by PCR array, Western blot, plasmid transfection and confocal observation. Intraperitoneal (i.p.) injection of EFL2, instead of oral administration, could effectively ameliorate arthritis severity of STA mice. The inflammatory cells migration and infiltration into ankles were also significantly blocked by EFL2, accompanied with dramatically reduction of chemokines mRNA expression and pro-inflammatory cytokines production. In vivo PCR microarray indicated that EFL2 exerted anti-arthritis bioactivity by suppressing TLR7 mediated signaling pathway. In vitro study confirmed the inhibitory effects of EFL2 on TLR7 or TLR3/7 synergistically induced inflammatory cytokines secretion in murine macrophages and human PBMCs. In terms of molecular mechanism, we further verified that EFL2 robustly downregulated TLR7 mediated IRAK4-IKKβ-IRF5 and NF-κB signaling pathways activation, and blocked IRF5 and p65 phosphorylation and translocation activity. Taken together, our data indicate EFL2’s therapeutic potential as a candidate for rheumatoid arthritis and other TLR7-dependent diseases.

Specificity of CD8-Targeted Fusosomes in Human PBMCs Using Single Cell RNA and T Cell Receptor Sequencing

Introduction: The ability to deliver genes to specific cell types in vivo would have a profound therapeutic impact for a diverse set of diseases. For example, targeting T cells for in vivo delivery of a chimeric antigen receptor (CAR) to treat B cell malignancies would improve access to CAR T therapies by overcoming the limitations of ex vivo manufacturing such as high costs, wait times and manufacturing failures. We have developed a novel paramyxovirus-based integrating vector (fusosomes) that specifically targets cell surface receptors for targeted gene delivery. Fusosomes, engineered to target CD8α, a cell surface protein expressed on CD8+ T cells, can bind and specifically deliver a genetic payload through membrane fusion. To evaluate the specificity of fusosome-mediated delivery to cells expressing CD8α in vitro, single cell RNA sequencing (scRNA-seq) and T cell receptor sequencing (scTCR-seq) were performed on human PBMCs treated with CD8α-targeted fusosomes with a GFP payload. scRNA-seq is a tool that can be used to detect the transgene delivered by our fusosomes in specific cell populations by measuring mRNA expression of the receptor targeted by the fusosome (e.g., CD8α) and the genetic payload delivered by the fusosome in the same cell. Transcriptome information to understand potential pathway changes induced by delivery of the transgene is also captured. Methods: scRNA-seq was performed using the 10X Genomics system on human PBMCs in vitro. Activated and resting PBMCs from a single donor were transduced with CD8α-targeted fusosomes. Cells were then harvested 3 days post-transduction for scRNA-seq and scTCR-seq. Following library preparation and Illumina sequencing, read processing and bioinformatics analyses were performed using 10X Genomics Cell Ranger and the Seurat R package. Results: In the PBMCs transduced with fusosomes, > 9,000 cells were barcoded with > 1,900 median genes detected per cell. scRNA-seq identified multiple cell types in PBMCs with approximately 25% of cells expressing CD8α transcripts. Fusosome-associated transcripts were seen in about 54% of the cells expressing CD8α and in particular, T cells classified as CD8+ using known markers and classification algorithms based on reference data sets. Subsequently, scTCR-seq data were used to confirm the identity of T cells. Comparison of the results showed an overlap of > 87% of cells classified as T cells by the two independent methods. Visualization by UMAP and inference based on a reference dataset showed that naïve (Tn), central memory (Tcm), effector memory (Tem) and mucosal-associated invariant T cells were transduced by the CD8α-targeted fusosomes. In addition, fusosome-associated transcripts were detected in about 19% of NK cells where approximately 62% of these NK cells also expressed CD8α. Overall, our CD8α-targeted fusosomes have a specificity of > 93% in resting PBMCs based on CD8α expression. A subset of cells may have detectable GFP transcripts at the time of analysis, but not CD8α transcripts due to limited sequencing depth per cell. Summary: Our in vitro scRNA-seq and scTCR-seq data demonstrate that our CD8α-targeted fusosomes are highly specific for cells expressing CD8α transcripts in resting PBMCs. These data highlight the potential for utilizing single cell sequencing technologies to comprehensively characterize the specificity of our fusosomes, and to identify key biological pathways that may play a role in specificity, transduction efficiency and clinical efficacy. As next steps, we will use similar approaches to characterize in vivo transduction in animal models. Disclosures Iftikhar: Sana Biotechnology: Current Employment. Balanis: Sana Biotechnology: Ended employment in the past 24 months. Fonseka: Sana Biotechnology: Current Employment. Bandoro: Sana Biotechnology: Ended employment in the past 24 months. Cruite: Sana Biotechnology: Current Employment. Davis: Sana Biotechnology: Current Employment. Amatya: Sana Biotechnology: Current Employment. Frye: Sana Biotechnology: Current Employment. Pepper: Sana Biotechnology: Current Employment. Laska: Sana Biotechnology: Current Employment. Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Shah: Sana Biotechnology: Current Employment. Paliwal: Sana Biotechnology: Current Employment. Chaivorapol: Sana Biotechnology: Current Employment.

719 XTX301, a protein-engineered IL-12, exhibits tumor-selective activity in mice without peripheral toxicities and is well tolerated in non-human primates

BackgroundInterleukin-12 (IL-12) is a proinflammatory cytokine which bridges innate and adaptive immunity via induction of T helper 1 differentiation and promoting cytolytic activity of natural killer and T cells. IL-12 has demonstrated potent antitumor activity in syngeneic mouse models and promising anti-tumor efficacy in humans. However, development of IL-12 has been limited by severe systemic toxicities. To overcome toxicity and improve the therapeutic index of IL-12, we employed protein engineering to generate XTX301, a highly potent, half-life extended and masked IL-12. The masking domain of XTX301 is designed to pharmacologically inactivate IL-12 systemically and render an active IL-12 moiety upon cleavage by proteases that are enriched in the tumor microenvironment.MethodsWe conducted experiments to assess the binding, bioactivity, safety, and anti-tumor efficacy of XTX301. Binding interactions were measured via SPR, bioactivity was measured using STAT-4 phosphorylation in a reporter cell line, and IFN-g production was assessed in human PBMCs via ELISA. Anti-tumor efficacy and pharmacodynamics were assessed in MC38 and B16F10 syngeneic tumor mouse models using a XTX301 murine surrogate, mXTX301. Safety and pharmacokinetics of XTX301 were evaluated in non-human primates (NHP).ResultsXTX301 showed no detectable binding to the high affinity IL12RB2 demonstrating that the masking domain indeed prevents interaction with the receptor. Upon cleavage of the masking domain by relevant proteases, binding was observed and was comparable to XTX300 unmasked control. Likewise, restoration of activity upon proteolytic cleavage was observed in an IL-12-dependent reporter gene assay and in primary human PBMCs. Human IL-12 does not cross react with mouse IL-12 receptors; hence a murine surrogate (mXTX301) was created for in vivo anti-tumor efficacy evaluation. A single dose of mXTX301 demonstrated up to 90% tumor growth inhibition in an inflamed MC38 and non-inflamed B16F10 syngeneic mouse models. mXTX301 induced a ~3 fold increase in IFN-g in tumors compared to vehicle control and ~150 fold less peripheral IFN-g compared to mXTX300. XTX301 exhibits minimal elevation in liver enzymes and a 50-fold improvement in tolerability compared to XTX300, in a repeat dose NHP safety study.ConclusionsOur data demonstrates that both XTX301 and mXTX301 are inactive when in masked form and become activated upon proteolytic cleavage to exert bioactivity comparable to recombinant IL-12. For efficacy, mXTX301 demonstrated tumor selective activity in syngeneic mouse models. XTX301 was well tolerated in repeat dose NHP safety study. In conclusion, XTX301 has potential for exerting potent anti-tumor activity with a favorable tolerability profile.

222 Genetic reprogramming of merkel cell carcinoma and melanoma leads to increased MHC-I expression and antitumor immune activation in vitro and in vivo

BackgroundMerkel cell carcinoma (MCC) is a rare skin cancer with 46% disease-associated mortality and half of patients unresponsive to immune checkpoint inhibitors.1 2 MCC and melanomas often display decreased MHC class I (MHC-I) expression on the surface of cells, which prevents antigen recognition by T cells (”signal 1”) and hampers immune activation. We therefore sought to genetically reprogram cells to express their own costimulatory molecules (”signal 2”) and immunostimulatory cytokines (”signal 3”) to increase MHC-I expression and drive a targeted immune response.MethodsWe used biodegradable poly(beta-amino ester) nanoparticles (NPs) to co-deliver plasmids encoding a signal 2 molecule (4-1BBL) and two signal 3 molecules (IL-12 and IFNγ) to cancer cells. For in vitro evaluation of NPs we used two patient-derived MCC cell lines with low baseline MHC-I expression; MCC13 and UISO. Co-culture experiments were performed with human PBMCs or primary human natural killer (NK) cells. All in vitro analysis was performed 7 days following PBMC or NK cell addition. For in vivo evaluation, subcutaneous B16F10 mouse melanoma tumors were implanted in C57BL/6J mice and NPs were administered by direct injection into the tumor with and without intraperitoneal injection of αPD1. Tumors were harvested for analysis on day 16.ResultsTransfection with particles delivering the three plasmids to MCC13 and UISO increased MHC-I expression (mean fluorescence intensity) 1.6- and 5.0-fold, respectively, and MHC-II expression increased 1.6- and 6.3-fold, respectively (figure 1). In co-culture with human PBMCs, signal 2/3 particles resulted in increased leukocyte proliferation (4.6- and 6.1-fold increase, respectively) and led to significantly reduced MCC viability (10.6 and 1.6% vs control particles)(figure 2). When MCC13 cells were co-cultured with primary human NK cells, NK cell expansion increased 355-fold with 4-1BBL/IL-12 particles compared to control particles and was accompanied by 2.5% MCC13 cell viability, indicating a potent innate immune response with signal 2/3 NP administration in vitro (figure 3). Following evaluation of NPs in vivo, assessment of MHC-I and MHC-II expression in the melanoma tumors found increased expression with signal 2/3 NPs compared to control NPs (figure 4). When signal 2/3 NPs were administered in combination with αPD1 treatment, 4-1BBL/IL-12 NPs with αPD1 demonstrated improved survival compared to αPD1 treatment with control NPs (p=0.0010) (figure 5).Abstract 222 Figure 1Administration of signal 2/3 NPs to MCC13 and UISO cells led to increases in MHC-I and MHC-II expression after 7 days. MHC-I expression in transfected cells (red) and MHC-II expression in transfected cells (blue) compared to untreated control (black)Abstract 222 Figure 2Co-culture of transfected MCC cells with human PBMCs led to increases in CD45+ cells and reduced MCC cell viability after 7 daysAbstract 222 Figure 3Co-culture of 4-1BBL/IL-12 transfected MCC13 cells with isolated CD56+ NK cells demonstrated robust NK-cell expansion and low MCC cell viability after 7 daysAbstract 222 Figure 4Direct intratumoral injection with signal 2 and 3 NPs led to increases in MHC-I and MHC-II in cancer cells in vivo.Abstract 222 Figure 5NPs were administered intratumorally ± intraperitoneal aPD1 on day 9, 11, and 13 following B16F10 melanoma tumor implantation. 4-1BBL/IL12 particles in combination with αPD1 demonstrated a significant improvement in survival compared to control particles (Luc) with αPD1 (p=0.0010)ConclusionsTogether, these results show the ability of signal 2/3 NPs to reprogram MCC and melanoma cells, leading to increased MHC-I expression in vitro and in vivo, eliciting a productive immune response against cancer cells.ReferencesHughes MP, Hardee ME, Cornelius LA, Hutchins LF, Becker JC, Gao L. Merkel cell carcinoma: epidemiology, target, and therapy. Curr Dermatol 2014;46–53.Nghiem PT, Bhatia S, Lipson EJ, Kudchadkar RR, Miller NJ, Annamalai L, Berry S, Chartash EK, Daud A, Fling SP, Friedlander PA, Kluger HM, Kohrt HE, Lundgren L, Margolin K, Mitchell A, Olencki T, Pardoll DM, Reddy SA, Shantha EM, Sharfman WH, Sharon E, Shemanski LR, Shinohara MM, Sunshine JC, Taube JM, Thompson JA, Townson SM, Yearley JH, Topalian SL, Cheever MA. PD-1 blockade with pembrolizumab in advanced merkel-cell carcinoma. N Engl J Med 2016;374:2542–2552.

750 AK119, a CD73 targeting antibody with dual mechanism of action

BackgroundAK119 is an Fc-engineered humanized IgG1 monoclonal antibody targeting human CD73. CD73-extracellular adenosine pathway regulates conversion of pro-inflammatory and immuno-stimulatory extracellular adenosine ATP into immunosuppressive adenosine. CD73 expresses on cancer cells, endothelial cells, fibroblasts, lymphocytes and myeloid cells. CD73 upregulated can be a result of tissue hypoxia,1 epithelial-to-mesenchymal transition,2 inflammation3 and/or cytotoxic stress.4 Also, increasing immune response may lead to faster viral clearance, shorter recovery time, less complications, longer immunity and protection from re-infection. Inhibiting CD73 was reported to evoke B cells activation and shows anti-fibrotic effects. The ability of enhancing immune response provides a potential opportunity to treat COVID-19. Thus, we investigated pharmacological activity of AK119 as an agent treating cancers, COVID-19 and fibrosis.MethodsAK119 inhibition of CD73 enzymatic activity was tested in human PBMCs based assay. The ability of AK119 to enhance B cells immune response was detected by cell-based assay. PBMCs were incubated overnight with APCP (inhibitor of CD73 enzyme acitvity) or AK119, CPI006 or MEDI9447. Flow cytometry analysis was performed with gating on B cells (CD19+CD3-) and MFI and positive percent were reported for antibody staining of CD69 or CD83, as well as HLA-DR and IgM. Enhancement of anti-SARS-CoV-2 antibody production was studied using human CD73 transgenic mouse immunized with SARS-CoV-2 spike protein. The in-vivo activity of AK119 was further studied in bleomycin-induced pulmonary fibrosis model in human CD73 transgenic mouse.ResultsAK119 shows a more potent antigen binding (figure 1) and completely CD73 enzyme inhibition activity (figure 2). AK119 promotes B cell proliferation, and upregulating CD69, CD83, HLA-DR and IgM that are markers of B cell activation (figure 3). B cell activation induced by AK119 is independent of adenosine. AK119 show significantly higher bioactivity to induce B cells activation in comparison with MEDI9447 or CPI006 (figure 4). In human CD73 transgenic mice, AK119 increased secretion of anti-S protein IgG (figure 5). In pulmonary fibrosis mouse model, number of inflammatory cell in broncholveolr lavage fluid of AK119 was significantly decreased, and decreased HYP representing collagen content in lung tissue homogenate of mice was found in both AK119 50 mg/kg and 10 mg/kg group (figure 6).ConclusionsAK119 selectively binds to and inhibits the ectonucleotidase activity of CD73 thus reducing adenosine accumulation. Results from non-clinical pharmacology studies reveal potent bioactivities as well as favorable safety properties of AK119. AK119 is intended for advanced solid tumors, pulmonary fibrosis and therapy of COVID-19.ReferencesBullen JW, Tchernyshyov I, Holewinski RJ, DeVine L, Wu F, Venkatraman V, Kass DL, Cole RN, Van Eyk J, Semenza GL, Protein kinase A-dependent phosphorylation stimulates the transcriptional activity of hypoxia-inducible factor 1. Sci Signal 2016;9(430):ra56.Lupia M, Angiolini F, Bertalot G, Freddi S, Sachsenmeier KF, Chisci E, Kutryb-Zajac B, Confalonieri S, Smolenski RT, Giovannoni R, Colombo N, Bianchi F, Cavallaro U. CD73 regulates stemness and epithelial-Mesenchymal transition in ovarian cancer-initiating cells, Stem Cell Rep 2018;10(4):1412–1425.Reinhardt J, Landsberg J, Schmid-Burgk JL, Ramis BB, Bald T, Glodde N, Lopez-Ramos D, Young A, Ngiow SF, Nettersheim D, Schorle H, Quast T, Kolanus W, Schadendorf D, Long GV, Madore J, Scolyer RA, Ribas A, Smyth MJ, Tumeh PC, Tuting T, Holzel M. MAPK signaling and inflammation link melanoma phenotype switching to induction of CD73 during immunotherapy. Cancer Res 2017;77(17):4697–4709.Samanta D, Park Y, Ni X, Li H, Zahnow CA, Gabrielson E, Pan F, Semenza GL. Chemotherapy induces enrichment of CD47(+)/CD73(+)/ PDL1(+) immune evasive triple-negative breast cancer cells, Proc Natl Acad Sci USA. 2018;115(6):E1239–E1248.Abstract 750 Figure 1Binding activity of AK119 to human PBMCs. Binding Curve of AK119 to CD73 expressed on (A) CD8+ T cells and (B) CD19+ B cells in human PBMCsAbstract 750 Figure 2Inhibition activity of CD73 on human PBMCs. AK119 Inhibits Enzymatic Activity of CD73 Expressed on human PBMCsAbstract 750 Figure 3Effect of upregulating B cell markers by AK119. AK119 Upregulates (A) CD69, (B) CD83, (C) HLA-DR and (D) IgM Expression on B cellsAbstract 750 Figure 4Stimulation of B cell Proliferation by AK119Abstract 750 Figure 5Therapeutic activity in the COVID-19 mouse model. Serum Concentration of S protein-specific IgG in Mouse Model of COVID-19Abstract 750 Figure 6Therapeutic activity in the asthma mouse model. (A) AK119 relieves the increased airway resistance and restore the lung function. (B) Reduction of the inflammatory cells in BALF by AK119

840 A therapeutic humanized anti-carcinoma monoclonal antibody (mAb) can also identify immunosuppressive regulatory T (Tregs) cells and down regulate Treg-mediated immunosuppression

BackgroundNEO-201 is an IgG1 mAb reactive against many different human carcinomas expressing the NEO-201 antigen, but not against most normal epithelial tissues. NEO-201 can mediate antitumor activity against tumor cells through multiple mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and blockade of the CEACAM5/CEACAM1 immune checkpoint inhibitory pathway. In addition to solid tumors, the NEO-201 target has also been found on human hematopoietic cells. Flow cytometry analysis has demonstrated that 98.9% of CD15+ granulocytes and about 4.6% of CD4+ T cells were positive for NEO-201 staining. No binding was observed with NEO-201 with respect to B cells, NK cells, monocytes, or CD8+ T cells and a majority of CD4+ T cells. This study was designed to characterize the subset of NEO-201+ binding CD4+ T cells and to evaluate the reactivity of NEO-201 to this subset of hematopoietic cells.MethodsPhenotypic analysis of PBMCs from healthy donors and cancer patients were performed by flow cytometry. Reagents used for flow cytometry were antibodies against human CD4, CD127, CD25, CD15s, FOXP3, CD39, CD73 and anti-NEO-201 mAb. Functional assays were performed using a flow cytometry based on CDC assay. Treg cells, isolated from 3 healthy donors using the EasySep™ Human CD4+CD127lowCD25+ Regulatory T (Treg) Cell Isolation Kit were used as target cells.ResultsFlow cytometry analysis revealed that NEO-201+CD4+ T cells were also CD25+/CD127-/FOXP3+/CD15s+ in human PBMCs from both healthy donors and cancer patients. NEO-201 also binds to CD4+/CD25+/CD127-/Foxp3+/CD15s+ cells in Treg cells isolated from human PBMCs using a commercial isolation kit. NEO-201+CD4+ T cells were also CD25+/CD127-/FOXP3+/CD39+. In addition, NEO-201 mAb can kill these isolated Treg cells through CDC.ConclusionsThis study demonstrated that the small subset of NEO-201+CD4+ T cell in human PBMCs are highly suppressive Treg cells and NEO-201 can be used as a novel marker to identify functionally suppressive Treg cells, Furthermore, NEO-201 can kill Treg cells through CDC, presenting an opportunity for therapeutic intervention to increase anti-tumor immunity.Ethics ApprovalThe study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of National Institutes of Health (NIH). All subjects gave their informed consent for inclusion before they participated in the study.PBMCs from healthy volunteer donors were utilized under the appropriate Institutional Review Board approval (protocol code NCT00001846, first approved Nov 4, 1999; latest update 11/10/2020).PBMCs from cancer patients were utilized under the appropriate Institutional Review Board approval (protocol code NCT03476681, first approved 03/26/2018; latest update 01/08/2020).ConsentInformed consent was obtained from all subjects involved in the study.

Mechanosensitive TRPV4 is required for crystal-induced inflammation

Crystal structures activate innate immune cells, especially macrophages and initiate inflammatory responses. We aimed to understand the role of the mechanosensitive TRPV4 channel in crystal-induced inflammation. Real-time RT-PCR, RNAscope in situ hybridisation, and Trpv4eGFP mice were used to examine TRPV4 expression and whole-cell patch-clamp recording and live-cell Ca2+ imaging were used to study TRPV4 function in mouse synovial macrophages and human peripheral blood mononuclear cells (PBMCs). Both genetic deletion and pharmacological inhibition approaches were used to investigate the role of TRPV4 in NLRP3 inflammasome activation induced by diverse crystals in vitro and in mouse models of crystal-induced pain and inflammation in vivo. TRPV4 was functionally expressed by synovial macrophages and human PBMCs and TRPV4 expression was upregulated by stimulation with monosodium urate (MSU) crystals and in human PBMCs from patients with acute gout flares. MSU crystal-induced gouty arthritis were significantly reduced by either genetic ablation or pharmacological inhibition of TRPV4 function. Mechanistically, TRPV4 mediated the activation of NLRP3 inflammasome by diverse crystalline materials but not non-crystalline NLRP3 inflammasome activators, driving the production of inflammatory cytokine interleukin-1β which elicited TRPV4-dependent inflammatory responses in vivo. Moreover, chemical ablation of the TRPV1-expressing nociceptors significantly attenuated the MSU crystal-induced gouty arthritis. In conclusion, TRPV4 is a common mediator of inflammatory responses induced by diverse crystals through NLRP3 inflammasome activation in macrophages. TRPV4-expressing resident macrophages are critically involved in MSU crystal-induced gouty arthritis. A neuroimmune interaction between the TRPV1-expressing nociceptors and the TRPV4-expressing synovial macrophages contributes to the generation of acute gout flares.

Boosted Pro-Inflammatory Activity in Human PBMCs by Lipopolysaccharide and SARS-CoV-2 Spike Protein Is Regulated by α-1 Antitrypsin

For the treatment of severe COVID-19, supplementation with human plasma-purified α-1 antitrypsin (AAT) to patients is currently considered. AAT inhibits host proteases that facilitate viral entry and possesses broad anti-inflammatory and immunomodulatory activities. Researchers have demonstrated that an interaction between SARS-CoV-2 spike protein (S) and lipopolysaccharides (LPS) enhances pro-inflammatory responses in vitro and in vivo. Hence, we wanted to understand the potential anti-inflammatory activities of plasma-derived and recombinant AAT (recAAT) in a model of human total peripheral blood mononuclear cells (PBMCs) exposed to a combination of CHO expressed trimeric spike protein and LPS, ex vivo. We confirmed that cytokine production was enhanced in PBMCs within six hours when low levels of LPS were combined with purified spike proteins (“spike”). In the presence of 0.5 mg/mL recAAT, however, LPS/spike-induced TNF-α and IL-1β mRNA expression and protein release were significantly inhibited (by about 46–50%) relative to LPS/spike alone. Although without statistical significance, recAAT also reduced production of IL-6 and IL-8. Notably, under the same experimental conditions, the plasma-derived AAT preparation Respreeza (used in native and oxidized forms) did not show significant effects. Our findings imply that an early pro-inflammatory activation of human PBMCs is better controlled by the recombinant version of AAT than the human plasma-derived AAT used here. Considering the increasing clinical interest in AAT therapy as useful to ameliorate the hyper-inflammation seen during COVID-19 infection, different AAT preparations require careful evaluation.