EGF-Receptor against Amphiregulin (AREG) Influences Costimulatory Molecules on Monocytes and T Cells and Modulates T-Cell Responses.

Amphiregulin (AREG) is a ligand of the epidermal growth factor receptor (EGFR) and has been shown to regulate the phagocytosis-induced cell death of monocytes in peripheral blood. AREG-dependent apoptotic signaling engages factors of the intrinsic and extrinsic apoptotic pathway, such as BCL-2, BCL-XL, and death ligand/receptor CD95/CD95L. Here, we tested the hypothesis that AREG influences costimulatory monocyte functions, which are crucial for T-cell responses. We found a stronger expression of AREG and EGFR in monocytes compared to lymphocytes. As a novel function of AREG, we observed reduced T-cell proliferation following polyclonal T-cell stimulation with OKT3. This reduction of proliferation occurred in the presence of monocytes as well as in their absence, monocyte signaling being replaced by crosslinking of OKT3. Increasing concentrations of AREG down-modulated the concentration of costimulatory B7 molecules (CD80/CD86) and HLA-DR on monocytes. In proliferation assays, CD28 expression on T cells was down-modulated on the application of OKT3 but unaltered by AREG. LcK activation, following OKT3-stimulation, was reduced in T cells that had been coincubated with AREG. The effects of AREG on T-cell phenotypes were also present when monocytes were depleted and OKT3 was crosslinked. The rearranged expression of immunological synapse proteins was accompanied by an alteration of T-cell polarization. Although the proportion of regulatory T cells was not shifted by AREG, IL-17-expressing T cells were significantly enhanced, with a bias toward TH1-polarization. Taken together, these results suggest that AREG acts as an immunoregulatory molecule at the interface between antigen-presenting cells and T cells.

Ex-vivo CS1-OKT3 dual specific bivalent antibody-armed effector T cells mediate cellular immunity against multiple myeloma.

Bispecific T cell engaging antibodies (bsAbs) have emerged as novel and powerful therapeutic agents for redirecting T cells towards antigen-specific tumor killing. The cell surface glycoprotein and SLAM family member, CS1, exhibits stable and high-level expression on malignant plasma cells including multiple myeloma, which is indicative of an ideal target for bsAb therapy. Here, we developed a CS1 bsAb (CS1-dbBiTE) using Click chemistry to conjugate intact anti-CS1 antibody (Elotuzumab) and anti-huOKT3 antibody at their respective hinge regions. Using a cellular therapy approach, human T cells were armed ex-vivo with CS1-dbBiTE prior to examining effector activity. Our data indicates that arming T cells with CS1-dbBiTE induced T cell activation and expansion and subsequent cytotoxic activity against CS1-bearing MM tumors, demonstrated by significant CD107a expression as well as inflammatory cytokine secretion. As expected, CS1-dbBiTE armed T cells showed significantly reduced effector activity in the absence of CS1 expression. Similarly, in MM mouse xenograft studies, armed T cells exhibited effective anti-tumor efficacy highlighted by reduced tumor burden in MM.1S tumor-bearing mice compared to controls. On the basis of these findings, the rationale for CS1 targeting by human T cells armed with CS1-dbBiTE presents a potentially effective therapeutic approach for targeting MM.

A multi-functional drug delivery nanosystem release of TLR-7 immunostimulant and OKT3 induced efficient cancer immunotherapy.

Immunotherapy has some shortcomings such as off-target toxicity, treatment time and poor immunogenicity, which limit its therapeutic effect. Nanomaterials are particularly attractive in immunotherapy due to their drug delivery capabilities. Nano drug delivery system loaded with Toll-like receptor (TLR) agonist imiquimod (IMQ) and CD3 immune antibody OKT3 is constructed by using polydopamine (PDA) and CaCO3. While PDA-IMQ@CaCO3-OKT3 (PICO NPs) drug delivery system has the advantages of high biocompatibility, low toxicity, degradability. Antitumor studies in vitro and in vivo have shown that the system can effectively inhibit the proliferation of mouse breast cancer cells and the activity of Regulatory T Cells (Tregs), activate immunogenic cell death (ICD), and enhance the activity of antigen-presenting cells (APCs). Effectively eliminate tumor immunosuppression and fully activate immune function.

Anti-CD79b/CD3 bispecific antibody combined with CAR19-T cells for B-cell lymphoma treatment.

CD19 CAR-T (chimeric antigen receptor-T) cell immunotherapy achieves a remission rate of approximately 70% in recurrent and refractory lymphoma treatment. However, the loss or reduction of CD19 antigen on the surface of lymphoma cells results in the escape of tumor cells from the immune killing of CD19 CAR-T cells (CAR19-T). Therefore, novel therapeutic strategies are urgently required. In this study, an anti-CD79b/CD3 bispecific antibody (BV28-OKT3) was constructed and combined with CAR19-T cells for B-cell lymphoma treatment. When the CD19 antigen was lost or reduced, BV28-OKT3 redirected CAR19-T cells to CD79b+ CD19- lymphoma cells; therefore, BV28-OKT3 overcomes the escape of CD79b+ CD19- lymphoma cells by the killing action of CAR19-T cells in vitro and in vivo. Furthermore, BV28-OKT3 triggered the antitumor function of CAR- T cells in the infusion product and boosted the antitumor immune response of bystander T cells, markedly improving the cytotoxicity of CAR19-T cells to lymphoma cells in vitro and in vivo. In addition, BV28-OKT3 elicited the cytotoxicity of donor-derived T cells toward lymphoma cells in vitro, which depended on the presence of tumor cells. Therefore, our findings provide a new clinical treatment strategy for recurrent and refractory B-cell lymphoma by combining CD79b/CD3 BsAb with CAR19-T cells.

Novel anti-CD30/CD3 bispecific antibodies activate human T cells and mediate potent anti-tumor activity.

CD30 is expressed on Hodgkin lymphomas (HL), many non-Hodgkin lymphomas (NHLs), and non-lymphoid malignancies in children and adults. Tumor expression, combined with restricted expression in healthy tissues, identifies CD30 as a promising immunotherapy target. An anti-CD30 antibody-drug conjugate (ADC) has been approved by the FDA for HL. While anti-CD30 ADCs and chimeric antigen receptors (CARs) have shown promise, their shortcomings and toxicities suggest that alternative treatments are needed. We developed novel anti-CD30 x anti-CD3 bispecific antibodies (biAbs) to coat activated patient T cells (ATCs) ex vivo prior to autologous re-infusions. Our goal is to harness the dual specificity of the biAb, the power of cellular therapy, and the safety of non-genetically modified autologous T cell infusions. We present a comprehensive characterization of the CD30 binding and tumor cell killing properties of these biAbs. Five unique murine monoclonal antibodies (mAbs) were generated against the extracellular domain of human CD30. Resultant anti-CD30 mAbs were purified and screened for binding specificity, affinity, and epitope recognition. Two lead mAb candidates with unique sequences and CD30 binding clusters that differ from the ADC in clinical use were identified. These mAbs were chemically conjugated with OKT3 (an anti-CD3 mAb). ATCs were armed and evaluated in vitro for binding, cytokine production, and cytotoxicity against tumor lines and then in vivo for tumor cell killing. Our lead mAb was subcloned to make a Master Cell Bank (MCB) and screened for binding against a library of human cell surface proteins. Only huCD30 was bound. These studies support a clinical trial in development employing ex vivo-loading of autologous T cells with this novel biAb.

Genetic code expansion in E. coli enables production of a functional 'ready-to-click' T cell receptor-specific scFv.

Antibody-based cancer therapies have been evolving at a rapid pace in the pharmaceutical market. Bispecific antibody-drug conjugates that engage immune cells to target and kill cancer cells with precision have inspired the development of immunotherapy. Miniaturized antibody fragments such as diabodies, nanobodies, or single-chain variable fragments (scFvs) hold great promise as antibody-drug conjugates as they specifically target tumor tissue and can penetrate it. Here, we optimized the soluble periplasmic expression of the scFv OKT3 comprising the variable VH and VL domains of the mouse anti-human CD3 antibody muromonab-CD3 (trade name Orthoclone OKT3) in E. coli. By an expansion of the genetic code, we site-specifically incorporated the reactive non-canonical amino acid Nε-((2-azidoethoxy)carbonyl)-L-lysine (AzK) into scFv OKT3 using an orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair. To confirm the AzK incorporation and to demonstrate the accessibility of the reactive azide group, we conjugated a fluorophore to scFv OKT3 AzK variants by copper-free strain-promoted alkyne-azide cycloaddition ('click chemistry'). The scFv OKT3 wild type and the AzK variants bound T cells at nanomolar concentrations. In this study, a 'ready-to-click' scFv OKT3 was successfully developed for future applications, e.g. as controlled anti-T cell antibody-drug conjugate or bispecific T cell engager and for imaging immune T cell migration in cancers.

Acute exercise mobilizes NKT-like cells with a cytotoxic transcriptomic profile but does not augment the potency of cytokine-induced killer (CIK) cells.

CD3+/CD56+ Natural killer (NK) cell-like T-cells (NKT-like cells) represent <5% of blood lymphocytes, display a cytotoxic phenotype, and can kill various cancers. NKT-like cells can be expanded ex vivo into cytokine-induced killer (CIK) cells, however this therapeutic cell product has had mixed results against hematological malignancies in clinical trials. The aim of this study was to determine if NKT-like cells mobilized during acute cycling exercise could be used to generate more potent anti-tumor CIK cells from healthy donors. An acute exercise bout increased NKT-like cell numbers in blood 2-fold. Single cell RNA sequencing revealed that exercise mobilized NKT-like cells have an upregulation of genes and transcriptomic programs associated with enhanced anti-tumor activity, including cytotoxicity, cytokine responsiveness, and migration. Exercise, however, did not augment the ex vivo expansion of CIK cells or alter their surface phenotypes after 21-days of culture. CIK cells expanded at rest, during exercise (at 60% and 80% VO2max) or after (1h post) were equally capable of killing leukemia, lymphoma, and multiple myeloma target cells with and without cytokine (IL-2) and antibody (OKT3) priming in vitro. We conclude that acute exercise in healthy donors mobilizes NKT-like cells with an upregulation of transcriptomic programs involved in anti-tumor activity, but does not augment the ex vivo expansion of CIK cells.

Optimizing rAAV6 transduction of primary T cells for the generation of anti-CD19 AAV-CAR-T cells.

Recombinant Adeno-associated virus(rAAV) is currently the most widely used gene delivery vector and has been successfully used in various disease models, benefiting from its low immunogenicity, almost no toxicity, and no reported pathogenicity in humans. However, its low transduction efficiency for primary cells, especially for T lymphocytes, limits its further application in the field of cell therapy. In this study, we optimized the protocol for rAAV6 transduction of primary T cells, significantly improved the expression efficiency of the rAAV6 delivered CAR gene, and successfully generated rAAV6-based CAR-T cells (AAV-CAR-T). The gene expression intensity (mean fluorescence intensity, MFI) of rAAV6 transduced T cells treated with the tyrosine kinase inhibitor, Genistein, was increased 1-3-fold. Moreover, our results showed that rAAV6 efficiently transduced T cells stimulated with OKT3 and the gene expression could be enhanced 3-fold with an OKT3 concentration of 50 ng/mL in the medium. The gene expression intensity of T cells treated with OKT3 together with genistein could be augmented by 7-fold. Based on the above-optimized method, CAR-T cells prepared with rAAV6 showed evident anti-tumor ability both in vitro and in vivo. Our findings established an efficient method for the AAV transduction of T cells and would provide an alternative way for the preparation of CAR-T cells.

Development of a flow cytometry-based potency assay for prediction of cytokine storms induced by biosimilar monoclonal antibodies.

Cytokine release syndrome (CRS) is an undesired immune reaction that may cause dangerous side effects after the administration of novel biological therapies. In vitro cytokine release assays (CRA) are used for preclinical safety assessment prior to first-in-man dose administration of therapeutic monoclonal antibodies (mAbs). A variety of CRA platforms has been developed where the analysis of secreted cytokines is performed. Analysis of T cell activation markers is not performed routinely in CRA platforms and few studies have described intracellular cytokine levels after stimulation with therapeutic mAbs. In the present study, we performed a CRA using intracellular cytokine staining and assessment of extracellular T cell activation markers by flow cytometry. We used commercially available reference mAbs for the stimulation of peripheral blood mononuclear cells (PBMCs). We found that stimulation using solid phase (SP) dry coating with two different CD28 antibodies and muromonab-CD3 increased the percentage of IFN-É£ + CD4+ and CD8+ T cells as well as of CD3-CD56+ NK cells compared to stimulation with antibodies in aqueous phase (AP). Expression of the T cell activation markers CD25 and CD69 on CD4+ and CD8+ T cells was also increased upon SP muromonab-CD3 stimulation. Using multiplex cytokine assessment, we showed that stimulation in AP using ANC28.1, CD28.2 and muromonab-CD3 led to an increase of IFN-É£, GM-CSF, TNF-α, and IL-2 secretion. Stimulation of PBMCs preincubated at high-density culture led to an increase in IFN-É£ production but not in the expression of activation markers compared to low-density culture. Our findings demonstrated that flow cytometry analyses for assessing relevant T cell and NK cell markers may be used as a supplement to multiplex cytokine analysis in CRAs. The approach may be a valuable addition that enables a more precise description of the mechanisms leading to CRS.

SIRPγ-CD47 Interaction Positively Regulates the Activation of Human T Cells in Situation of Chronic Stimulation.

A numerous number of positive and negative signals via various molecules modulate T-cell activation. Within the various transmembrane proteins, SIRPγ is of interest since it is not expressed in rodents. SIRPγ interaction with CD47 is reevaluated in this study. Indeed, we show that the anti-SIRPγ mAb clone LSB2.20 previously used by others has not been appropriately characterized. We reveal that the anti-SIRPα clone KWAR23 is a Pan anti-SIRP mAb which efficiently blocks SIRPα and SIRPγ interactions with CD47. We show that SIRPγ expression on T cells varies with their differentiation and while being expressed on Tregs, is not implicated in their suppressive functions. SIRPγ spatial reorganization at the immune synapse is independent of its interaction with CD47. In vitro SIRPα-γ/CD47 blockade with KWAR23 impairs IFN-γ secretion by chronically activated T cells. In vivo in a xeno-GvHD model in NSG mice, the SIRPγ/CD47 blockade with the KWAR23 significantly delays the onset of the xeno-GvHD and deeply impairs human chimerism. In conclusion, we have shown that T-cell interaction with CD47 is of importance notably in chronic stimulation.

Versatile and rapid microfluidics-assisted antibody discovery.

Recent years have seen unparalleled development of microfluidic applications for antibody discovery in both academic and pharmaceutical research. Microfluidics can support native chain-paired library generation as well as direct screening of antibody secreting cells obtained by rodent immunization or from the human peripheral blood. While broad diversities of neutralizing antibodies against infectious diseases such as HIV, Ebola, or COVID-19 have been identified from convalescent individuals, microfluidics can expedite therapeutic antibody discovery for cancer or immunological disease indications. In this study, a commercially available microfluidic device, Cyto-Mine, was used for the rapid identification of natively paired antibodies from rodents or human donors screened for specific binding to recombinant antigens, for direct screening with cells expressing the target of interest, and, to our knowledge for the first time, for direct broad functional IgG antibody screening in droplets. The process time from cell preparation to confirmed recombinant antibodies was four weeks. Application of this or similar microfluidic devices and methodologies can accelerate and enhance pharmaceutical antibody hit discovery.

Phosphatidylserine-Targeting Monoclonal Antibodies Exhibit Distinct Biochemical and Cellular Effects on Anti-CD3/CD28-Stimulated T Cell IFN-γ and TNF-α Production.

Phosphatidylserine (PS)-targeting monoclonal Abs (mAbs) that directly target PS and target PS via ß2-gp1 (ß2GP1) have been in preclinical and clinical development for over 10 y for the treatment of infectious diseases and cancer. Although the intended targets of PS-binding mAbs have traditionally included pathogens as well as stressed tumor cells and its associated vasculature in oncology, the effects of PS-targeting mAbs on activated immune cells, notably T cells, which externalize PS upon Ag stimulation, is not well understood. Using human T cells from healthy donor PBMCs activated with an anti-CD3 + anti-CD28 Ab mixture (anti-CD3/CD28) as a model for TCR-mediated PS externalization and T cell stimulation, we investigated effects of two different PS-targeting mAbs, 11.31 and bavituximab (Bavi), on TCR activation and TCR-mediated cytokine production in an ex vivo paradigm. Although 11.31 and Bavi bind selectivity to anti-CD3/28 activated T cells in a PS-dependent manner, surprisingly, they display distinct functional activities in their effect on IFN-γ and TNF-ɑ production, whereby 11.31, but not Bavi, suppressed cytokine production. This inhibitory effect on anti-CD3/28 activated T cells was observed on both CD4+ and CD8+ cells and independently of monocytes, suggesting the effects of 11.31 were directly mediated by binding to externalized PS on activated T cells. Imaging showed 11.31 and Bavi bind at distinct focal depots on the cell membrane. Collectively, our findings indicate that PS-targeting mAb 11.31 suppresses cytokine production by anti-CD3/28 activated T cells.

Nanovesicles released by OKT3 hybridoma express fully active antibodies.

Recent findings have shown that nanovesicles preparations from either primary immune cells culture supernatants or plasma contain immunoglobulins, suggesting that a natural way of antibody production may be through exosome release. To verify this hypothesis, we used the OKT3 hybridoma clone, which produces a murine IgG2a monoclonal antibody used to reduce rejection in patients undergoing organ transplantation. We showed exosome-associated immunoglobulins in hybridoma supernatants, by Western blot, nanoscale flow cytometry and immunocapture-based ELISA. The OKT3-exo was also being able to trigger cytokines production in both CD4 and CD8 T cells. These results show that nanovesicles contain immunoglobulin and could be used for immunotherapy. These data could lead to a new approach to improve the effectiveness of therapeutic antibodies by exploiting their natural property to be expressed on nanovesicle membrane, that probably render them more stable and as a consequence more capable to interact with their specific ligand in the best way.

Developing an Arrayed CRISPR-Cas9 Co-Culture Screen for Immuno-Oncology Target ID.

Immunotherapies including PD-L1 blockade have shown remarkable increases in the T cell-directed antitumor response; however, efficacy is seen only in a minority of patients. Recently, pooled CRISPR-Cas9 knockout (CRISPRn) screens in tumor/immune co-culture systems have identified a number of genes that confer resistance to T cell killing in pathways including antigen presentation and cytokine signaling, providing insight into tumor mechanisms that cause resistance to immunotherapies. The development of an arrayed CRISPRn screen in a tumor/immune co-culture system would allow the identification of novel targets for immuno-oncology, characterization of hits from pooled screens, and multiple assay endpoints to be measured per gene. Here, a small-scale arrayed CRISPRn screen was successfully developed to investigate the effects on a co-culture of T cells and Cas9-expressing PC9 lung adenocarcinoma cells modified to express anti-CD3 antibody on the cell surface (PC9-OKT3 T cell system). A focused CRISPRn library was designed to target genes involved in known resistance mechanisms (including antigen presentation, cytokine signaling, and apoptosis) as well as genes involved in immune synapse interactions. The viability of PC9 cells was assessed in two-dimensional adherent co-cultures via longitudinal imaging analysis. Knockout of epidermal growth factor receptor (EGFR) and PLK1 in tumor cells cultured alone or with T cells resulted in increased tumor cell death, as expected, whereas knockout of the test gene ICAM1 showed subtle donor-specific resistance to T cell killing. Taken together, these data provide proof of concept for arrayed CRISPRn screens in tumor/immune co-culture systems and warrant further investigation of in vitro co-culture models.

Chemically Crosslinked Bispecific Antibodies for Cancer Therapy: Breaking from the Structural Restrictions of the Genetic Fusion Approach.

Antibodies are composed of structurally and functionally independent domains that can be used as building blocks to construct different types of chimeric protein-format molecules. However, the generally used genetic fusion and chemical approaches restrict the types of structures that can be formed and do not give an ideal degree of homogeneity. In this study, we combined mutation techniques with chemical conjugation to construct a variety of homogeneous bivalent and bispecific antibodies. First, building modules without lysine residues-which can be chemical conjugation sites-were generated by means of genetic mutation. Specific mutated residues in the lysine-free modules were then re-mutated to lysine residues. Chemical conjugation at the recovered lysine sites enabled the construction of homogeneous bivalent and bispecific antibodies from block modules that could not have been so arranged by genetic fusion approaches. Molecular evolution and bioinformatics techniques assisted in finding viable alternatives to the lysine residues that did not deactivate the block modules. Multiple candidates for re-mutation positions offer a wide variety of possible steric arrangements of block modules, and appropriate linkages between block modules can generate highly bioactive bispecific antibodies. Here, we propose the effectiveness of the lysine-free block module design for site-specific chemical conjugation to form a variety of types of homogeneous chimeric protein-format molecule with a finely tuned structure and function.

Stimulation of Mononuclear Cells Through Toll-Like Receptor 9 Induces Release of Microvesicles Expressing Double-Stranded DNA and Galectin 3-Binding Protein in an Interferon-α-Dependent Manner.

Background: Microvesicles (MVs) expressing the type 1 interferon (IFN)-inducible protein galectin-3 binding protein (G3BP) may play a pathogenic role in systemic lupus erythematosus (SLE). Co-expression of double-stranded DNA (dsDNA) on such MVs may render them immunogenic and targets for anti-dsDNA antibodies. Little is known about the mechanisms underlying generation of this MV population. In this study, we investigated how Toll-like receptors (TLRs), IFN-α, and T cells are involved in this process in healthy subjects. Methods: Peripheral blood mononuclear cells (PBMCs) isolated from 12 healthy donors were stimulated in-vitro for 24 h with a series of TLR-agonists or the T cell activating antibody OKT3 or were subjected to apoptosis by incubation with staurosporine. MVs in the supernatants were subsequently isolated by differential centrifugation and were quantified and characterized with respect to expression of G3BP and dsDNA by flow cytometry. Results: Stimulation of PBMCs with the TLR9-agonist and strong IFN-α inducer ODN2395 significantly increased the release of MVs expressing G3BP. The production of MVs with this phenotype was markedly enhanced by co-stimulation of T cells. Furthermore, dependency on IFN-α in the generation of G3BP-expressing MVs was indicated by a marked reduction following addition of the IFN-α inhibitor IFN alpha-IFNAR-IN-1 hydrochloride. Conclusion: Release of G3BP-expressing MVs from healthy donor PBMCs is induced by stimulation of TLR9 in an IFN-α-dependent manner and is enhanced by co-stimulation of T cells.

Early suppression of peripheral mononuclear blood cells in sepsis in response to stimulation with cytomegalovirus, OKT3, and pokeweed mitogen.

Critically ill patients are at risk for sepsis, and immunosuppressive mechanisms may prevail. Whether functional tests are helpful to detect immune alterations is largely unknown. Therefore, we tested the hypotheses that reactivity of peripheral blood mononuclear cells (PBMCs) to secrete interferon-γ (IFNγ) following stimulation in vitro is decreased in patients with early sepsis compared with postoperative patients. IFNγ secretion [enzyme-linked immunospot (ELISpot)] in response to stimulation with cytomegalovirus (CMV), pokeweed mitogen (PWM), muromonab-anti-CD3 (OKT3), and human leukocyte antigen (HLA)-DRA-mRNA expression and serum cytokine concentrations were repeatedly [days 1, 3, 5, and 7 after intensive care unit (ICU) admission] determined in patients with sepsis (n = 7) and patients undergoing major abdominal surgery (radical prostatectomy, cystectomy, n = 10). In a second cohort, HLA-DRA expression was assessed in 80 patients with sepsis, 30 postoperative patients, and 44 healthy volunteers (German clinical trials database no. 00007694). In patients with sepsis, IFNγ secretion (ELISpot) was decreased compared with controls after stimulation with CMV (P = 0.01), OKT3 (P = 0.02), and PWM (P = 0.02 on day 5), whereas unstimulated IFNγ secretion did not differ. HLA-DRA expression was also significantly decreased in patients with sepsis at all time points (P = 0.004) compared with postoperative surgical patients, a finding confirmed in the larger cohort. Reactivity of PBMCs to stimulation with CMV, PWM, and OKT3 as well as HLA-DRA expression was already decreased upon ICU admission in patients with sepsis when compared with postoperative controls, suggesting early depression of acquired immunity. ELISpot assays may help to clinically characterize the time course of immunocompetence in patients with sepsis.NEW & NOTEWORTHY We observed suppression of reactivity to stimulation with cytomegalovirus, muromonab-anti-CD3, and pokeweed mitogen in mononuclear blood cells of patients with early sepsis when compared with postoperative controls. Thus, there is early depression of acquired immunity in sepsis. Enzyme-linked immunospot assays may help to characterize immunocompetence in patients with sepsis.

Bone marrow-liver-thymus (BLT) immune humanized mice as a model to predict cytokine release syndrome.

Cytokine release syndrome (CRS) is a serious and potentially life-threatening complication that can be associated with biological drug products. In vitro assays or in vivo tests using nonhuman primates may fail to predict CRS due to species differences and the complexity of immune system. Therefore, model species that have human-specific immune components may improve the ability to identify CRS and enhance product safety. In this study we used bone marrow-liver-thymus (BLT) humanized mice to test muromonab (OKT3), an anti-CD3 antibody with a black box warning for CRS. Initially, we completed pilot and dose escalation studies with muromonab and showed that when the dose was increased sufficiently, BLT-humanized mice experienced serious adverse outcomes including moribundity. Full studies compared muromonab treatment with adalimumab, saline, and a group pretreated with methylprednisolone prior to muromonab. We evaluated immune cell activation using flow cytometry and cytokine expression using a custom 10-plex cytokine assay to assess levels of human TNF-α, IFN-γ, IL-2, IL-6, IL-8, IL-10, IL-13, IL-17A, IL12/23p40, and GM-CSF. Muromonab treated mice had significant increases in all cytokines tested with T-cell depletion and T-cell activation noted. Adalimumab (active) and saline (inactive) control groups did not demonstrate cytokine expression changes or alterations in T-cell numbers or activation. Further, pretreatment with methylprednisolone blunted or abrogated cytokine increases. This study demonstrates that BLT-humanized mice are capable of experiencing CRS, and could be used to screen biologics for this adverse event to enhance patient safety.

Mature neutrophils suppress T cell immunity in ovarian cancer microenvironment.

Epithelial ovarian cancer (EOC) often presents with metastases and ascites. Granulocytic myeloid-derived suppressor cells are an immature population that impairs antitumor immunity. Since suppressive granulocytes in the ascites of patients with newly diagnosed EOC were morphologically mature, we hypothesized that PMN were rendered suppressive in the tumor microenvironment (TME). Circulating PMN from patients were not suppressive but acquired a suppressor phenotype (defined as ≥1 log10 reduction of anti-CD3/CD28-stimulated T cell proliferation) after ascites supernatant exposure. Ascites supernatants (20 of 31 supernatants) recapitulated the suppressor phenotype in PMN from healthy donors. T cell proliferation was restored with ascites removal and restimulation. PMN suppressors also inhibited T cell activation and cytokine production. PMN suppressors completely suppressed proliferation in naive, central memory, and effector memory T cells and in engineered tumor antigen-specific cytotoxic T lymphocytes, while antigen-specific cell lysis was unaffected. Inhibition of complement C3 activation and PMN effector functions, including CR3 signaling, protein synthesis, and vesicular trafficking, abrogated the PMN suppressor phenotype. Moreover, malignant effusions from patients with various metastatic cancers also induced the C3-dependent PMN suppressor phenotype. These results point to PMN impairing T cell expansion and activation in the TME and the potential for complement inhibition to abrogate this barrier to antitumor immunity.