Dependent Lysis Research Articles

Improving key gene expression and di-n-butyl phthalate (DBP) degrading ability in a novel Pseudochrobactrum sp. XF203 by ribosome engineering

Di-n-butyl phthalate (DBP) is one of the important phthalates detected commonly in soils and crops, posing serious threat to human health. Pseudochrobactrum sp. XF203 (XF203), a new strain related with DBP biodegradation, was first identified from a natural habitat lacking human disturbance. Genomic analysis coupled with gene expression comparison assay revealed this strain harbors the key aromatic ring-cleaving gene catE203 (encoding catechol 2,3-dioxygenase/C23O) involved DBP biodegradation. Following intermediates identification and enzymatic analysis also indicated a C23O dependent DBP lysis pathway in XF203. The gene directed ribosome engineering was operated and to generate a desirable mutant strain XF203R with highest catE203 gene expression level and strong DBP degrading ability. The X203R removed DBP in soil jointly by reassembling bacterial community. These results demonstrate a great value of XF203R for the practical DBP bioremediation application, highlighting the important role of the key gene-directed ribosome engineering in mining multi-pollutants degrading bacteria from natural habitats where various functional genes are well conserved.

Augmentation of IFN-γ by bone marrow derived immune cells in the presence of severe suppression of IFN-γ in gingivae induced by zoledronic acid and denosumab in Hu-BLT mice model of ONJ.

The potential mechanisms governing drug induced osteonecrosis of the jaw (ONJ) is not well understood, and is one of the objectives of this study. Thus, we tested the release of IFN-γ within different immune compartments including bone marrow and gingivae upon treatment with zoledronic acid (ZOL) and denosumab which are known to induce ONJ in susceptible individuals. We used humanized-BLT mouse model for the in-vivo studies reported in this paper. To determine the effects of zoledronic acid and denosumab on IFN-γ secretion and NK cell-mediated cytotoxicity; peripheral blood, bone marrow, spleen and gingiva were obtained after the injection of ZOL and denosumab in mice. Percentages of B cells are much higher in wild-type mice whereas the proportions of immune subsets in humans and reconstituted hu-BLT peripheral-blood are similar. Therefore, hu-BLT mice are preferable model to study human disease, in particular, immune-pathologies induced by ZOL and denosumab. Both agents resulted in a severe suppression of IFN-γ in the gingiva, whereas they heightened the release of IFN-γ and NK cell-mediated cytotoxicity by the BM-derived immune cells. ZOL increased the IFN-γ secretion by the spleen and peripheral blood immune cells, whereas denosumab decreased the release IFN-γ by these cells significantly. ZOL and denosumab may likely suppress IFN-γ secretion in gingiva through different mechanisms. In addition, to the suppression of IFN-γ secretion, denosumab mediated effect could in part be due to the decrease in the bone resorptive function of osteoclasts due to the induction of antibody dependent cellular cytotoxicity and lysis of osteoclasts, whereas ZOL is able to mediate cell death of osteoclasts directly. Suppression of IFN-gamma in gingiva is largely responsible for the inhibition of immune cell function, leading to dysregulated osteoblastic and osteoclastic activities. Restoration of IFN-gamma in the local microenvironment may result in establishment of homeostatic balance in the gingiva and prevention of osteonecrosis of jaw.

Abstract LB087: Characterization of JNJ-70218902, a TMEFF2 x CD3 bispecific antibody, in prostate cancer models

Abstract TMEFF2, a transmembrane protein with epidermal growth factor-like and 2 follistatin-like domains, is primarily restricted in expression to healthy brain and prostate and is enriched in prostate cancer. JNJ-70218902 (JNJ-902) is a bispecific antibody that engages TMEFF2-expressing tumor cells and CD3 on T cells causing exposure dependent pro-inflammatory responses and target tumor cell lysis. Here we report the preclinical characterization of JNJ-902 using in vitro and in vivo prostate cancer models. Expression of TMEFF2 transcript and protein in brain and prostate was confirmed in healthy human tissue, as was its elevated expression in advanced prostate cancer tumors, including in 41/45 (91%) metastatic castration-resistant prostate cancer (mCRPC) samples. JNJ-902 bound to TMEFF2-positive LNCaP-AR cells (androgen-sensitive human prostate cancer cell line) in a concentration-dependent manner (EC50 = 9.6 nM); no binding to TMEFF2-negative DU145 prostate cancer cells was observed. The binding affinity (KD) of JNJ-902 for CD3 on primary human T cells was determined to be ~151 nM. T cell-mediated killing, as measured by caspase-3 activity, was induced upon incubation of JNJ-902 with healthy human donor T cells (n = 5) and LNCaP-AR cells at a 3:1 effector to target ratio (EC50 = 1.4 nM). No cytotoxicity was detected using DU145 cells or with control antibodies. Corresponding T cell activation was confirmed by concentration-dependent increases in cell surface CD8+CD25+ expression and proinflammatory cytokine production (eg, GM-CSF, IFN-γ, IL-10, TNF-α). Antitumor activity of JNJ-902 at increasing concentrations up to 5 mg/kg was observed in T cell humanized NSG mice bearing LNCaP xenografts (tumor growth inhibition [∆%TGI] of 75-122%) and in LuCaP 86.2 patient-derived xenografts (∆%TGI of 72-88%) as compared with control-treated mice. JNJ-902-dependent intratumoral infiltration of CD8+ T cells was detected in both models with increases in frequency of CD8+granzymeB+ effector cells. The data summarized here are the first report on the pharmacological activity of a novel TMEFF2 × CD3 bispecific antibody, supporting further investigation of TMEFF2 in mCRPC given its expression in advanced prostate cancer. Targeting TMEFF2 with the TMEFF2 × CD3 bispecific antibody JNJ-902 elicited robust T cell-mediated responses in both prostate cancer cells and xenograft models. Based on these data, a phase 1 dose escalation trial of JNJ-902 in patients with mCRPC is currently ongoing (NCT04397276). Citation Format: Subhasree Basu, Mike Russell, Katrin Sproesser, Bethany Mattson, Cassandra L. Lowenstein, Sathya Venkataramani, Maura Diamond, Robin E. Ernst, Stephen Jarantow, Jackson Wong, Gerald Chu, Kathryn Packman, Shaozhou Ken Tian, Scott R. Brodeur, Margaret Yu, Brent A. Rupnow. Characterization of JNJ-70218902, a TMEFF2 x CD3 bispecific antibody, in prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB087.

Phase 1b study of tarlatamab, a half-life extended bispecific T-cell engager (HLE BiTEimmune therapy) targeting DLL3, in de novo or treatment emergent neuroendocrine prostate cancer (NEPC).

TPS197 Background: NEPC is an aggressive cancer with no standard treatment approach and poor prognosis. It is usually treatment-emergent, occurring in 15%–20% of patients (pts) with metastatic castration-resistant prostate cancer following treatment with androgen signaling inhibitors (ASI) and is characterized by histological transformation from adenocarcinoma to a high-grade neuroendocrine tumor.1 The tumor associated antigen delta-like ligand 3 (DLL3) has been identified as a promising target in both NEPC and small cell lung cancer (SCLC), as it is highly expressed in these tumors and minimally expressed on normal tissue. Tarlatamab is a DLL3-targeting HLE BiTE® immune therapy designed to bind DLL3 on cancer cells and CD3 on T cells, resulting in T cell activation and expansion and T cell-dependent killing of tumor cells. In preclinical studies, tarlatamab induced T-cell dependent lysis of DLL3-expressing neuroendocrine tumor cell lines, including NEPC cells.2 Interim results of an ongoing first-in-human study in pts with SCLC (NCT03319940) show evidence for tarlatamab efficacy with an acceptable safety profile.3 Together, these findings support a clinical study of tarlatamab in NEPC. Methods: NCT04702737 is an open-label, phase 1b study evaluating tarlatamab infusion in pts with metastatic de novo or treatment-emergent NEPC, consisting of dose exploration and then dose expansion. Key eligibility criteria include adults (≥18 y) with metastatic NEPC whose disease progressed/recurred after ≥1 prior line of systemic therapy (platinum-based regimen for de novo NEPC or an ASI if treatment-emergent), measurable disease per RECIST 1.1 with Prostate Cancer Working Group 3 modifications, and ECOG performance status ≤2. Primary objectives are to evaluate safety and tolerability and determine the maximum tolerated dose or recommended phase 2 dose of tarlatamab. Secondary objectives are to evaluate antitumor activity (as assessed by objective response, duration of response, progression-free survival, overall survival, and disease control rate) and characterize pharmacokinetics. Four US and international study sites have been activated with two enrolled pts.

T Cell Engagement without Cytokine Storm: A Novel Bcma x CD3 Antibody Killing Myeloma Cells with Minimal Cytokine Secretion

IntroductionMultiple myeloma (MM) is a plasma cell malignancy with an annual incidence of ~25,000. Improvements in standard of care have extended median survival to greater than 5 years; however, disease refractory to bortezomib and lenalidomide is still associated with a median life expectancy of less than 9 months. Novel therapies are critical to the treatment of these patients. Chimeric antigen receptor T cells (CAR-Ts) targeting B-cell maturation antigen (BCMA) -a protein found exclusively on the surface of plasma cells- have shown efficacy against relapsed/refractory MM in early phase clinical trials. However, toxicity from over-activation of T-cells and large-scale production of CAR-Ts still hinder this approach. Bispecific T-cell engaging antibodies redirecting T cells to BCMA circumvent the latter problem but to date have shown similar T-cell over-activation. Utilizing TeneoSeek, a next generation sequencing (NGS)-based discovery pipeline using in silico analysis of heavy chain only/fixed light chain antibody (HCA/Flic, respectively) sequences to enrich for antigen specific antibodies, we made a high affinity αBCMA HCA and a library of αCD3 Flic antibodies that show a >2 log range of EC50s for T cell activation in vitro . Of note, the library contained a low-activating αCD3 that induced T-cell dependent lysis of MM cells (when paired with an αBCMA HCA) with minimal cytokine secretion. To characterize the relative efficacy and potential therapeutic window of this unique molecule, we combined both a high- (TNB-384B) and the low-activating (TNB-383B) αCD3 moiety with a high-affinity bivalent αBCMA arm (αBCMA x αBCMA x CD3) and compared these constructs in vitro, in vivo, and ex vivo for T-cell activation, efficacy in killing MM cells, and toxicity.MethodsT-cell activation was measured via flow cytometry (CD69 expression) and cytokine ELISA (IL-2, INF-ɣ, TNFα, among others) in vitro. Lysis of MM cell lines (RPMI-8226, U266, and H929) was measured via flow cytometry in vitro . Ex vivo, survival of patient MM cells was measured via flow cytometry after incubation with TNB-383B, TNB-384B, or negative control antibody (NCA) with or without supplemental exogenous activated T cells. In vivo, NSG mice were engrafted with RPMI-8226 MM cells and human peripheral blood mononuclear cells (huPBMC), and the mice treated with TNB-383B, TNB-384B, or NCA. Tumor burden was evaluated via Bioluminescent Imaging (BLI). Pharmacokinetic (PK) studies were performed in mice and Cynomolgus monkeys using ELISA.ResultsBoth TNB-383B and TNB-384B bound to cell surface BCMA with nanomolar affinity. EC50s for cytotoxicity were in the single-digit nanomolar range for TNB-383B and single-digit picomolar for TNB-384B against MM cell lines in vitro . TNB-383B showed markedly reduced (ie IFN-ɣ) or absent (ie IL-2) cytokine release compared to TNB-384B even at saturating concentrations (Figure 1); these findings were consistent across 10 healthy huPBMC donors. Ex vivo, both TNB-383B and TNB-384B efficiently lysed primary MM cells in the presence and absence of supplementary T-cells. In vivo, both TNB-383B and TNB-384B mediated clearance of established MM tumor from NSG mice at doses as low as 10ng q1w x3 in the presence of huPBMC. PK profiles of both molecules were consistent with those of an IgG in mice (T1/2 ~5.5 days) and Cyno (T1/2 ~8.5 days). No activation of T-cells was observed in vitro (Figure 1) or in vivo in the absence of BCMA expressing target cells.ConclusionsBoth low-activating TNB-383B and high-activating TNB-384B αBCMA x αBCMA x αCD3 bispecific antibodies kill multiple myeloma cells in vitro and in vivo in a BCMA-dependent manner, and kill primary patient myeloma cells ex vivo . While TNB-384B showed T-cell activation comparable to other CD3-engaging bispecifics, TNB-383B induced significantly reduced cytokine secretion by T-cells without appreciable reduction of efficacy in vivo or ex vivo. In summary, both TNB-383B and TNB-384B show promise as MM therapeutics differentiated from current T-cell targeted therapies in clinical trials. [Display omitted] DisclosuresBuelow:TeneoBio: Employment. Pham:TeneoBio, Inc.: Employment. Dang:TeneoBio, Inc.: Employment. Pratap:TeneoBio, Inc.: Employment. Clarke:TeneoBio, Inc.: Employment. Harris:TeneoBio, Inc.: Employment. Jorgensen:TeneoBio, Inc.: Employment. Force Aldred:TeneoBio, Inc.: Employment. Schellenberger:TeneoBio, Inc.: Employment. Shah:Indapta Therapeutics: Other: Stock Ownership; Celgene: Research Funding; Celgene, Indapta Therapeutics, Takeda: Consultancy; TeneoBio, Inc.: Honoraria. Trinklein:TeneoBio: Employment. Wiita:Sutro Biopharma: Research Funding; TeneoBio, Inc.: Research Funding. Iyer:TeneoBio, Inc.: Employment. Van Schooten:TeneoBio, Inc.: Employment.

Preclinical Assessment of AMG 596, a Bispecific T-cell Engager (BiTE) Immunotherapy Targeting the Tumor-specific Antigen EGFRvIII.

AMG 596 is a bispecific T-cell engager (BiTE) immuno-oncology therapy in clinical development for treatment of glioblastoma multiforme (GBM), the most common primary brain tumor in adults with limited therapeutic options. AMG 596 is composed of two single-chain variable fragments that simultaneously bind to the tumor-specific antigen, EGFR variant III (EGFRvIII), on GBM cells and to CD3 on T cells, thereby activating T cells to proliferate and secrete cytotoxic substances that induce lysis of the bound tumor cell. T-cell-redirected lysis by AMG 596 is very potent; in vitro studies revealed EC50 values in the low picomolar range, and in vivo studies showed that AMG 596 treatment significantly increased the overall survival of mice bearing EGFRvIII-expressing orthotopic tumors. In addition, AMG 596 activity is highly specific; no AMG 596-induced T-cell activity can be observed in assays with EGFRvIII-negative GBM cells, and no signs of toxicity and activity were observed in cynomolgus monkeys, which lack expression of EGFRvIII on normal tissues. With EGFRvIII-expressing GBM cells, we showed shedding of EGFRvIII-containing membrane vesicles, followed by vesicle uptake and EGFRvIII cell surface presentation by EGFRvIII noncoding GBM cells. Cell membrane presentation of EGFRvIII following microvesicle transfer allows engagement by AMG 596, resulting in T-cell activation and T-cell-dependent lysis of GBM cells. Together, these data show a compelling preclinical efficacy and safety profile of AMG 596, supporting its development as a novel immunotherapy for treatment of GBM.

Searching for the toxic potential of Loxosceles amazonica and Loxosceles willianilsoni spiders’ venoms

The Loxosceles genus belongs to the Sicariidae family and it comprises species whose venom can cause accidents with potentially fatal consequences. We have previously shown that SMase D is the enzyme responsible for the main pathological effects of Loxosceles venom. Despite the severity of accidents with Loxosceles, few species are considered to be of medical importance. Little is known about the venom of non-synanthropic species that live in natural environments. To contribute to a better understanding about the venom's toxicity of Loxosceles genus, the aim of this study was to (i) characterize the toxic properties of Loxosceles amazonica from two different localities and a recent described cave species Loxosceles willianilsoni and (ii) compare these venoms with that from Loxosceles laeta, which is among the most toxic ones. We show here that both L. amazonica venoms (from the two studied locations) and L. willianilsoni presented SMase D activity similar to that exhibited by L. laeta venom. Although L. amazonica and L. willianilsoni venoms were able to induce complement dependent human erythrocytes lysis, they were not able to induce cell death of human keratinocytes, as promoted by L. laeta venom, in the concentrations tested. These results indicate that other species of Loxosceles, in addition to those classified as medically important, have toxic potential to cause accidents in humans, despite interspecific variations that denote possible less toxicity.

Recruitment of properdin by bi-specific nanobodies activates the alternative pathway of complement

The complement system represents a powerful part of the innate immune system capable of removing pathogens and damaged host cells. Nevertheless, only a subset of therapeutic antibodies are capable of inducing complement dependent cytotoxicity, which has fuelled the search for new strategies to potentiate complement activation.Properdin (FP) functions as a positive complement regulator by stabilizing the alternative pathway C3 convertase. Here, we explore a novel strategy for direct activation of the alternative pathway of complement using bi-specific single domain antibodies (nanobodies) that recruit endogenous FP to a cell surface. As a proof-of-principle, we generated bi-specific nanobodies with specificity toward FP and the validated cancer antigen epidermal growth factor receptor (EGFR) and tested their ability to activate complement onto cancer cell lines expressing EGFR. Treatment led to recruitment of FP, complement activation and significant deposition of C3 fragments on the cells in a manner sensitive to the geometry of FP recruitment. The bi-specific nanobodies induced complement dependent lysis of baby hamster kidney cells expressing human EGFR but were unable to lyse human tumour cells due to the presence of complement regulators. Our results confirm that FP can function as a surface bound focal point for initiation of complement activation independent of prior C3b deposition. However, recruitment of FP by bi-specific nanobodies appears insufficient for overcoming the inhibitory action of the negative complement regulators overexpressed by many human tumour cell lines. Our data provide general information on the efficacy of properdin as an initiator of complement but suggest that properdin recruitment on its own may have limited utility as a platform for potent complement activation on regulated cell surfaces.

TNB-486, a Novel Fully Human Bispecific CD19 x CD3 Antibody That Kills CD19-Positive Tumor Cells with Minimal Cytokine Secretion

Introduction The restricted expression of CD19 in the B-cell lineage makes it an attractive target for the therapeutic treatment of B-cell malignancies. Many monoclonal antibodies and antibody drug conjugates targeting CD19 have been developed, including bispecific T-cell redirecting antibodies (T-BsAbs). In addition, anti-CD19 chimeric antigen receptor T-cells (CAR-T) have been approved to treat leukemia and lymphoma. However, despite the impressive depth of responses achieved by T-cell redirecting approaches such as T-BsAbs and CAR-T cells, toxicity from over-activation of T-cells remains a substantial limitation for this type of therapy, in particular neurotoxicity. In designing TNB-486, a novel CD19 x CD3 T-BsAb, we endeavored to retain activity against CD19-positive tumor cells while limiting the cytokine secretion thought to underlie toxicity from T-cell redirecting therapies. Utilizing TeneoSeek, a next generation sequencing (NGS)-based discovery pipeline that leverages in silico analysis of heavy chain only/fixed light chain antibody (HCA/Flic, respectively) sequences to enrich for antigen specific antibodies, we made a high affinity αCD19 HCA and a library of αCD3 Flic antibodies that showed a >2 log range of EC50s for T cell activation in vitro. Of note, the library contained a low-activating αCD3 that induced minimal cytokine secretion even at concentrations that mediated saturating T-cell dependent lysis of lymphoma cells (when paired with an αCD19 HCA). We characterized the relative efficacy and potential therapeutic window of this unique molecule, TNB-486, in vitro and in vivo and compared it to two strongly activating bispecific CD19 x CD3 antibodies similar to those currently available and in clinical development. Methods Affinity measurements of the αCD19 moiety were made via Biacore (protein) and flow cytometry (cell surface). Stability measurements were made by subjecting the molecule to thermal stress and the %aggregation was measured by Size Exclusion Chromatography. T-cell activation was measured via flow cytometry (CD69 and CD25 expression) and cytokine was measured by ELISA (IL-2, IL-6, IL-10, INF-ɣ, and TNFα) in vitro. Lysis of B-cell tumor cell lines (Raji, RI-1, and Nalm6) was measured via flow cytometry in vitro. In vivo, NOG mice were engrafted subcutaneously with NALM-6 or SUDHL-10 cells and intravenously with human peripheral blood mononuclear cells (huPBMC), and the mice treated with multiple doses of TNB-486 or negative or positive control antibody. Tumor burden was evaluated via caliper measurement. Pharmacodynamic/Pharmacokinetic (PK/PD) studies were performed in NOG mice. A pharmacokinetic (PK) study was performed in BALB/c mice, and a tolerability and PK study are ongoing in cynomolgus monkeys. Results TNB-486 bound to cell surface CD19 with single digit nanomolar affinity (~3nM). EC50s for cytotoxicity were in the single-digit nanomolar range for TNB-486, and sub-nanomolar for the strongly activating controls; TNB-486 maximum achievable lysis was identical to the positive controls. TNB-486 induced significantly less cytokine release for all cytokines tested compared to the positive controls even at doses saturating for tumor lysis. No off-target activation was observed in the absence of CD19 expressing target cells. In vivo, TNB-486 eradicated all CD19-positive tumors tested (NALM-6 and SUDHL10) at doses as little as 1µg administered every four days after tumors had reached ~200mm3. TNB-486 showed a PK profile consistent with other IgG molecules in mice (T1/2 ~6 days in mice). Conclusions TNB-486 induced comparable lysis of CD19-positive tumor cells as the strongly activating control bispecific antibodies while inducing significantly reduced cytokine secretion, even at doses saturating for tumor lysis in vitro. In vivo TNB-486 eradicated all tested CD19 positive tumor cell lines in established tumor models. No off-target binding was observed. In summary, TNB-486 shows promise as a lymphoma therapeutic differentiated from T-cell targeted therapies currently in the clinic and in clinical trials. Disclosures Malik: Teneobio, Inc.: Employment, Equity Ownership. Buelow:Teneobio, Inc.: Employment, Equity Ownership. Rangaswamy:Teneobio, Inc.: Employment, Equity Ownership. Balasubramani:Teneobio, Inc.: Employment, Equity Ownership. Boudreau:Teneobio, Inc.: Employment, Equity Ownership. Dang:Teneobio, Inc.: Employment, Equity Ownership. Davison:Teneobio, Inc.: Employment, Equity Ownership. Force Aldred:Teneobio, Inc.: Equity Ownership. Iyer:Teneobio, Inc.: Employment, Equity Ownership. Jorgensen:Teneobio, Inc.: Employment, Equity Ownership. Pham:Teneobio, Inc.: Employment, Equity Ownership. Prabhakar:Teneobio, Inc.: Employment, Equity Ownership. Schellenberger:Teneobio, Inc.: Employment, Equity Ownership. Ugamraj:Teneobio, Inc.: Employment, Equity Ownership. Trinklein:Teneobio, Inc.: Employment, Equity Ownership. Van Schooten:Teneobio, Inc.: Employment, Equity Ownership.

PD-1 signaling modulates interferon-γ production by Gamma Delta (γδ) T-Cells in response to leukemia

ABSTRACTGamma delta (γδ) T-cell based immunotherapy is a promising concept for the treatment of hematologic malignancies. Not only in vitro but also in early phase clinical trials, zoledronic acid (Zol) and interleukin-2 (IL-2) have been successfully used to activate human γδ T-cells and to induce clinical anti-tumor effects. Aiming to improve the effectiveness of future γδ T-cell based immunotherapies against leukemia, we analyzed the impact of programmed cell death protein 1 (PD-1) signaling, on the different phases of γδ T-cell activation, of proliferation, production of anti-tumor cytokines and cytotoxic function in vitro.PD-1 expression was found significantly upregulated between day 2 and day 4 following stimulation with Zol and IL-2. However, proliferation or expression of activation markers of γδ, αβ and NK-cells are not altered by additional PD-1 blockade. Pembrolizumab increases interferon-γ (IFN-γ) production in γδ T-cells upon direct stimulation with Zol and in response to Zol treated primary acute myeloid leukemia (AML) cells by approximately 57% and 30%, respectively. Zol sensitized primary AML cells also induce PD-1 expression in co-cultured γδ T-cells and such PD-1(+) cells contain more IFN-γ. In contrast, PD-1 blockade does not have a significant effect on direct cell dependent lysis of leukemia cells by γδ T-cells.This study demonstrates that PD-1 blockade impacts cell dependent cytotoxicity and cytokine production in response to leukemia cells differently. While Pembrolizumab did not increase cell lysis of stimulated and expanded γδ T-cells, it induces significant upregulation of the potent pro-inflammatory and anti-tumor cytokine IFN-γ, which might facilitate anti-leukemia effects.

A Novel Fully Human Bispecific CD19 x CD3 Antibody That Kills Lymphoma Cells with Minimal Cytokine Secretion

IntroductionAlong with CD20 and CD22, the restricted expression of CD19 to the B-cell lineage makes it an attractive target for the therapeutic treatment of B-cell malignancies. Many monoclonal antibodies and antibody drug conjugates specific to CD19 have been described, including bispecific T-cell redirecting antibodies (T-BsAbs). In addition, anti-CD19 chimeric antigen receptor T-cells (CAR-Ts) have been approved to treat leukemia. To date, toxicity from over-activation of T-cells and large-scale production of CAR-Ts still hinder this approach. Bispecific T-cell engaging antibodies redirecting T cells to CD19 circumvent the latter problem but to date have shown similar T-cell over-activation, as well as significant neurotoxicity. Utilizing TeneoSeek, a next generation sequencing (NGS)-based discovery pipeline that uses in silico analysis of heavy chain only/fixed light chain antibody (HCA/Flic, respectively) sequences to enrich for antigen specific antibodies, we made a high affinity αCD19 HCA and a library of αCD3 Flic antibodies that showed a >2 log range of EC50s for T cell activation in vitro. Of note, the library contained a selectively-activating αCD3 that induced potent T-cell dependent lysis of lymphoma cells (when paired with an αCD19 HCA) with minimal cytokine secretion. To characterize the relative efficacy and potential therapeutic window of this unique molecule, we compared the low-activating (and Fc-containing) CD19 x CD3 to two pan T-cell activating bispecific CD19 x CD3 antibodies (blinatumomab and another developed in-house) in vitro and in vivo for T-cell activation, efficacy in killing lymphoma cells, and toxicity.MethodsT-cell activation was measured via flow cytometry (CD69 and CD25 expression) and cytokine ELISA (IL-2, IL-6, IL-10, INF-ɣ, and TNFα) in vitro. Lysis of B-cell tumor cell lines (Raji, Ramos, and Nalm6) was measured via calcein release in vitro. In vivo, NOG mice were engrafted with human peripheral blood mononuclear cells (huPBMC) and human lymphoma cell lines, and the mice treated with weekly injections of T-BsAbs. Tumor burden was evaluated via caliper measurement. Pharmacokinetic (PK) studies were performed in NOG mice using ELISA.ResultsEC50s for cytotoxicity were in the single-digit nanomolar range for the selective T cell activating T-BsAb and sub-nanomolar for the pan T-cell activating controls. The selective T cell activator showed markedly reduced cytokine release for all cytokines tested compared to the pan T-cell controls even at saturating concentrations. In vivo, established CD19 positive B-cell tumors were cleared in NOG mice in the presence of huPBMC. PK profiles of both molecules generated in-house (selective and pan T-cell activators) were consistent with those of an IgG in mice. No activation of T-cells was observed in vitro or in vivo in the absence of CD19 expressing target cells.ConclusionsBoth the selectively-activating and the pan T-cell activating control bispecific antibodies killed lymphoma cells in vitro and in vivo in a CD19-dependent manner. While the pan T-cell activating controls showed T-cell activation comparable to other CD3-engaging bispecifics, the selective activator induced significantly reduced cytokine secretion by T-cells and demonstrated a half-life consistent with other IgG antibodies. In summary, our selectively activating CD19 x CD3 T-BsAb shows promise as a lymphoma therapeutic differentiated from current T-cell targeted therapies currently in the clinic and in clinical trials. DisclosuresMalik:Teneobio, Inc.: Employment. Buelow:Teneobio Inc.: Employment. Avanzino:Teneobio, Inc.: Employment. Balasubramani:Teneobio, Inc.: Employment. Boudreau:Teneobio, Inc.: Employment. Clarke:Teneobio, Inc.: Employment. Dang:Teneobio, Inc.: Employment. Davison:Teneobio, Inc.: Employment. Force Aldred:Teneobio Inc.: Employment. Harris:Teneobio, Inc.: Employment. Jorgensen:Teneobio, Inc.: Employment. Li:Teneobio, Inc.: Employment. Medlari:Teneobio, Inc.: Employment. Narayan:Teneobio, Inc.: Employment. Ogana:Teneobio, Inc.: Employment. Pham:Teneobio Inc.: Employment. Prabhakar:Teneobio, Inc.: Employment. Rangaswamy:Teneobio, Inc.: Employment. Sankaran:Teneobio, Inc.: Employment. Schellenberger:Teneobio, Inc.: Employment. Ugamraj:Teneobio, Inc.: Employment. Trinklein:Teneobio, Inc.: Employment. Van Schooten:Teneobio, Inc.: Employment.

PIBF+ extracellular vesicles from mouse embryos affect IL-10 production by CD8+ cells

Earlier evidence suggests, that the embryo signals to the maternal immune system. Extracellular vesicles (EVs) are produced by all types of cells, and because they transport different kinds of molecules from one cell to the other, they can be considered as means of intercellular communication. The aim of this work was to test, whether the embryo is able to produce sufficient amounts of EVs to alter the function of peripheral lymphocytes. Embryo-derived EVs were identified by their Annexin V biding capacity, and sensitivity to Triton X dependent lysis, using flow cytometry. Transmission electron microscopy was used to detect EVs at the implantation site. Progesterone-induced blocking factor (PIBF) expression in embryo-derived EVs was demonstrated with immuno-electron microscopy. The % of IL-10 + murine lymphocytes was determined by flow cytometry. EVs were present in embryo culture media, but not in empty media. Mouse embryo-derived EVs adhere to the surface of both CD4+ and CD8+ murine peripheral T lymphocytes, partly, via phosphatidylserine binding. The number of IL-10+ murine peripheral CD8+ cells increases in the presence of embryo-derived EVS, and this effect is counteracted by pre-treatment of EVs with an anti-PIBF antibody, suggesting that the embryo communicates with the maternal immune system via EVs.

Investigation of in vitro thrombolytic and anti-helminthic activity and in vivo anxiolytic and antidepressant potentiality with phytochemical nature of methanolic extract of Leucas lavandulifolia

Leucas lavandulifolia, locally called ‘Halkusha or Donkolos’ is a well-known medicinal plant in Bangladesh. The crude methanolic extract of L. lavandulifolia was designed to investigate for its possible chemical groups, thrombolytic, anthelmintic, anxiolytic and antidepressant effects. Thrombolytic and anthelmintic activities were elucidated by taking human erythrocytes and earthworms as test models. The plant extract was divided into two different doses (200 and 400mg/kg body weight) for evaluating anxiolytic and CNS depressant property on swiss-albino mice by using different test models such as elevated plus maze, hole board, open field and forced Swim tests. The phytochemical analysis of the crude extract revealed the presence of alkaloids, carbohydrates, glycosides, tannins, flavonoids, phytosterols and phenols. In the thrombolytic test, the plant extract had produced dose dependent clot lysis potential in human erythrocytes at five different doses (2, 4, 6, 8, 10mg/ml), while standard streptokinase and water were used as positive and negative controls. Five different concentrations (10, 20, 30, 40 & 50mg/ml) of crude extract of L. lavandulifolia also showed marked anthelmintic property in earthworms in a dose dependent manner. Significant (p <0.01; p < 0.05) CNS depressant and anxiolytic activity were observed in case of both extract doses (200 and 400mg/kg body weight) which were comparable to the standard drug diazepam. Thus, the present study asserted the potentiality of L. lavandulifolia as thrombolytic, anthelmintic, anxiolytic and CNS depressant agents and therefore supports the traditional uses of this medicinal plant.

Bispecific T cell engaging antibody constructs targeting a universally conserved part of the viral M2 ectodomain cure and prevent influenza A virus infection

The ectodomain of the influenza A matrix protein 2 (M2e) is highly conserved amongst all influenza virus A subtypes. M2e is present on the surface of influenza A virus-infected cells, and therefore a suitable target for broadly protective therapies. We designed bispecific T cell engaging (BiTE®) antibody constructs specific for M2e by genetically fusing a single chain variable fragment (scFv) derived from an M2e-specific murine monoclonal antibody with a CD3ɛ-specific scFv. These so-called FLU BiTE® antibody constructs selectively mediate T cell dependent lysis of M2-expressing and influenza A virus infected cells and protect BALB/c mice against challenge with different influenza A virus subtypes. By humanizing the M2e-binding scFv, we generated human-like FLU BiTE® antibody constructs, with increased in vitro cytotoxic activity and in vivo protective capacity against influenza A virus infection. FLU BiTE® antibody constructs represent a promising new curative and prophylactic treatment option for influenza disease.

Steroids Abrogate BiTE® Antibody Construct-Mediated Cytotoxicity in Primary AML Cells

Antibody based immunotherapy represents a promising strategy to eliminate chemoresistant cells in acute myeloid leukemia (AML). Clinical experience in acute lymphoblastic leukemia (ALL) has shown a clear correlation of leukemic burden and the occurrence of a cytokine release syndrome (CRS) during treatment with blinatumomab (CD19/CD3 BiTE®). A cytoreductive phase prior to or in combination with antibody therapy might be beneficial to reduce the severity of adverse events like CRS. The latter is often treated with steroids (dexamethasone, DEX) or less commonly, with the IL-6R antibody tocilizumab (TOC). As T-cell proliferation and function are of crucial importance for BiTE® activity (Zugmaier 2015), the effect of the drugs on effector cell function will dictate clinical response to therapy. In this study, we evaluated the influence of cytoreductive- (azacythidine, AZA; decitabine, DEC), and immunmodulatory (DEX and TOC) drugs on antibody-mediated cytotoxicity and T-cell proliferation. A CD33/CD3 BiTE® antibody construct (AMG 330) served as model T-cell recruiting antibody in this study. To address this question we set up the following experimental approaches: AML cells were cocultured with healthy donor (HD) T cells for up to 14 days ex vivo. T cells were either incubated with the specific drug for 3 days prior to coculture or the drugs were simultaneously added to AML-T cell cultures. Drug concentrations were chosen based on published serum concentrations in AML patients and their ex vivo stability in culture, validated by mass spectrometry. BiTE® mediated cytotoxicity and T-cell proliferation were assessed by flow cytometry. Preincubation of T cells with AZA and DEC impaired antibody mediated cytotoxicity of HL60 cells in a concentration dependent manner (% lysis control (ctrl) vs AZA at 1, 5, 10 µM: 99.9 vs 99.2 vs 52.1 vs 28.7, n=7; ctrl vs DEC at 0.2, 2, 5 µM: 98.4 vs 71.3 vs 60.0 vs 50.0, n=3). Similarly, T-cell proliferation was also markedly decreased (fold change (FC) T cells ctrl vs AZA at 1, 5, 10 µM: 2.9 vs 2.8 vs 1.5 vs 0.7; ctrl vs DEC at 0.2, 2, 5 µM: 3.8 vs 3.0 vs 2.3 vs 1.2). For DEX it was shown that incubation of T cells with steroids prior to cocultures had no negative effect on BiTE® mediated cytotoxicity (Brandl 2007). However, as steroids are often used simultaneously with T-cell recruiting immunotherapies, we tested the influence of DEX in combination with AMG 330. The addition of DEX to primary AML-T cell cultures (75 ng/ml) significantly impaired AMG 330 mediated cytotoxicity (% lysis AMG 330 vs AMG 330+DEX day (d) 6: 95.9 vs 47.5, n=9). This correlated to a markedly reduced T-cell proliferation (FC T cells AMG 330 vs AMG 330+DEX d6: 11.2 vs 1.2, n=9). Correspondingly, secretion of IFNγ was also decreased (n=3). Upon discontinuation of DEX an increase in AMG 330 mediated cytotoxicity was observed. Nevertheless, cytotoxicity was still considerably lower compared to control cultures (%lysis AMG 330 vs AMG 330+DEX d9: 95.6 vs 77.0). In contrast to DEX, TOC (110 µg/ml) had no negative effect on T-cell proliferation (FC T cells d6: AMG 330 vs AMG 330+TOC: 42.3 vs 36.9, n=4). Similarly, secretion of IFNγ was not affected through the simultaneous addition of TOC to primary AMG 330 cultures (pg/ml AMG 330 vs AMG 330+TOC d6: 543.9 vs 345.8 n=2). Importantly, drugs might not only interfere with effector cell function but also modulate target antigen expression. As we have previously demonstrated that antigen expression levels influence BiTE® mediated cytotoxicity (Krupka 2016), we analysed the effect of the drugs on CD33 expression. None of the drugs induced a significant up- or downregulation of CD33 on AML celllines as detected by flow cytometry. Hence our data support the notion that these drugs do not modulate antigen expression dependent lysis kinectics. We conclude, that drugs given prior or concomitant to BiTE® therapy have the potential to reduce T-cell proliferation and cytotoxicity. In particular, we observed a negative impact of AZA and DEC when given prior to AML-T cell cocultures. Importantly, even short exposure to DEX led to a significanly reduced T-cell responsiveness. Our data suggest the careful evaluation of concomitant drugs in T-cell recruiting antibody therapies and support the restrictive use of steroids in patients receiving BiTE® antibody therapy. For management of severe CRS, TOC could be considered as a targeted biologic therapy that preserves BiTE®-dependent T cell function. DisclosuresKrupka:AMGEN Research Munich: Research Funding. Kufer:AMGEN Research Munich: Employment, Equity Ownership, Patents & Royalties. Kischel:AMGEN Research Munich: Employment, Equity Ownership, Patents & Royalties. Subklewe:AMGEN Research Munich: Research Funding.

Staphylococcal enterotoxin G and I: augmenting the T cell response to murine melanoma.

Abstract Staphylococcal enterotoxins (SEs) have been shown to be effectual proteins which demonstrate anti-tumor activity. SEG and SEI, produced from the enterotoxin gene cluster (egc) of Staphylococcal aureus, activate T cells with Vβ specificity to elicit robust T cell proliferation, T helper cell 1 (TH1) and TH2 cytokine secretion, and nitric oxide dependent tumor lysis. Our data highlight the effects of SE stimulated T cells and the subsequent consequence on aggressive, metastatic cancer progression using B16-F10 murine melanoma model. Specifically, we explore the differences between major histocompatibility complex (MHC) and human leukocyte antigen (HLA) SE presentation to the T cells via the T cell receptor (TCR). Our data demonstrates increased survival of B16-F10 tumor bearing mice, C57BL/6 and DQ8 (HLA-DQA1*0301, HLA-DQB1*0302) transgenic mice, after irradiated B16-F10 vaccination and SE stimulation. Interestingly, SEG and SEI induce strong T cell proliferation yet do not induce neutralizing antibodies to the extent of classical SEs, SEA and SEB, nor induce autoimmune pathology. Our findings demonstrate that superantigen administration enhances lymphocytic tumor killing and suggest a potential role for SEG and SEI as potent immunotherapeutics for human cancer.

In vitro eradication of citrullinated protein specific B-lymphocytes of rheumatoid arthritis patients by targeted bifunctional nanoparticles.

BackgroundAutoreactive B cells are crucial players in the pathogenesis of rheumatoid arthritis (RA). Autoantibodies specific for citrullinated proteins (ACPA), present in the serum of approximately 60–70 % of patients, have a pathogenic role in the disease. B cell depleting therapies may result in a transient immunosuppression, increasing the risk of infections. Our aim was to develop a new therapeutic approach to selectively deplete the ACPA producing autoreactive B cells.MethodsTo target B cells synthetic citrullinated peptide derived from the β chain of fibrin, β60-74Cit 60,72,74 (β60-74Cit), the predominant epitope recognized by ACPA was used. Complement dependent cytotoxicity (CDC) was induced by a modified peptide derived from gp120 of HIV-1. To trigger CDC both the targeting peptide and the complement activating peptide were covalently coupled in multiple copies to the surface of poly (DL-lactic-co-glycolic acid) nanoparticles (NPs). Ex vivo antibody synthesis was examined by ELISA and ELISpot. CDC was tested after dead cell staining by flow cytometry.ResultsThe β60-74Cit peptide was selectively recognized by a small subset of B cells from RA patients having high level of peptide specific serum antibody, suggesting that the peptide can target diseased B cells. The modified gp120 peptide covalently coupled to NPs induced the formation of the complement membrane attack complex, C5b-9 in human serum. We show here for the first time that bifunctional NPs coupled to multiple copies of both the targeting peptide and the complement activating effector peptide on their surface significantly reduce β60-74Cit peptide specific ex vivo ACPA production, by inducing complement dependent lysis of the citrullinated peptide specific B cells of seropositive RA patients.ConclusionsBifunctional NPs covalently coupled to autoantigen epitope peptide and to a lytic peptide activating complement may specifically target and deplete the peptide specific autoreactive B-cells.

Direct and immune-mediated cytotoxicity of interleukin-21 contributes to antitumor effects in mantle cell lymphoma

AbstractMantle cell lymphoma (MCL) is a distinct subtype of non-Hodgkin lymphoma characterized by overexpression of cyclin D1 in 95% of patients. MCL patients experience frequent relapses resulting in median survival of 3 to 5 years, requiring more efficient therapeutic regimens. Interleukin (IL)-21, a member of the IL-2 cytokine family, possesses potent antitumor activity against a variety of cancers not expressing the IL-21 receptor (IL-21R) through immune activation. Previously, we established that IL-21 exerts direct cytotoxicity on IL-21R–expressing diffuse large B-cell lymphoma cells. Herein, we demonstrate that IL-21 possesses potent cytotoxicity against MCL cell lines and primary tumors. We identify that IL-21–induced direct cytotoxicity is mediated through signal transducer and activator of transcription 3-dependent cMyc upregulation, resulting in activation of Bax and inhibition of Bcl-2 and Bcl-XL. IL-21–mediated cMyc upregulation is only observed in IL-21–sensitive cells. Further, we demonstrate that IL-21 leads to natural killer (NK)-cell–dependent lysis of MCL cell lines that were resistant to direct cytotoxicity. In vivo treatment with IL-21 results in complete FC-muMCL1 tumor regression in syngeneic mice via NK- and T-cell–dependent mechanisms. Together, these data indicate that IL-21 has potent antitumor activity against MCL cells via direct cytotoxic and indirect, immune-mediated effects.

35 The role of toll-like receptor (TLR) agonists in combinational immunotherapy for head and neck cancer

Because of their stimulating effect on manifold components of the innate immune system, TLR-agonists are in the focus of the search for potent drugs in combinational anticancer therapy. They induce the maturation and cross-priming of Dendritic Cells (DC), and have been shown to enhance Natural Killer (NK) cell dependent lysis of tumor cells in combination with monoclonal antibodies (mAb) such as anti-epidermal growth factor receptor (EGFR) cetuximab. One of the reasons that may have limited the full potential of TLR-agonists so far is their sequential induction of suppressive factors as part of the immune system’s balancing component. Peripheral blood mononuclear cells (PBMC), NK, DC and monocytes were isolated and stimulated with different TLR agonists: poly I:C (TLR-3), LPS (TLR-4), VTX-2337 (TLR-8) and CpG (TLR-9). Analysis was performed using flow cytometry, cytokine ELISA, qPCR, T cell stimulation assays with VTX in the presence or absence of anti PD-1 Ab. Furthermore, Tumor-infiltrating Lymphocytes (TIL) of the pre- and post-treatment patient samples of trial patients that undergo a neoadjuvant combinational therapy with the TLR-8 agonist VTX-2337 and anti-EGFR mAb cetuximab were analyzed. TLR-agonists and combinational drugs show an additive effect. TLR8-stimulation enhanced monocyte maturation markers CD80, CD83, and CD86 alone and in co-culture with cetuximab-treated HNC cells. A skewing to the M1 phenotype of monocytes was evidenced by significantly increased secretion of Th1 cytokines TNFα (p < 0.0001), IFNγ (p < 0.0001), and IL-12p40 (p < 0.005). Immature myeloid-derived suppressor cells (MDSC) that were treated with VTX-2337 showed upregulation of maturation markers. Furthermore, the treatment of monocytes with TLR-agonists such as VTX-2337 induced a strong upregulation of the inhibitory marker PD-L1. Our work focuses on the maturational effect of TLR agonists on monocytes, their skewing to an M1 phenotype and on the induction of suppressive pathways through surface molecules such as PD-L1. TLR-agonists contain a strong potential in combinational therapies, including drugs that inhibit the sequential induction of checkpoint receptors (e.g. PD-1 blockade, nivolumab) or show additive immune-enhancing effects (e.g. cetuximab).

Effective in Vivo Targeting of BCP-ALL in a NOD/SCID/huALL Mouse Model By CD70 Directed Immunotherapy

Acute lymphoblastic leukemia (ALL) is the most frequent malignant disorder in children and adolescents. Despite successful treatment, relapse of the disease remains a major problem and is associated with poor prognosis. This emphasizes the need for novel treatment strategies to be applied in addition to established chemotherapy regimens without increasing general toxicity.Previously, we described a strong association of leukemia cell engraftment of primary patient B cell precursor (BCP) ALL samples transplanted in a NOD/SCID/huALL mouse model and patient outcome. Rapid onset of leukemia related morbidity (time to leukemia, TTLshort) is indicative for patient relapse and characterized by a specific gene expression profile. Among the top differentially regulated genes, the gene coding for CD70 was identified to be significantly up-regulated in TTLshort/high risk ALL. CD70 is a member of the tumor necrosis factor (TNF) family expressed on activated B- and T-lymphocytes and dendritic cells. Binding of CD70 to its receptor CD27 is involved in regulation of T- and B-cells including priming and generation of memory and plasma cells. CD70 has been described to be constitutively expressed on different cancers including hematological malignancies. However, expression and targeting of CD70 in B- cell precursor lymphoblastic leukemia has so far not been investigated.In this study, we addressed expression of CD70 in patient-derived primograft leukemia samples and primary patient specimens obtained at diagnosis from pediatric patients. Furthermore, we evaluated CD70 as a therapeutic target for directed immunotherapy in vitro and in our BCP-ALL xenograft system in vivo.Flow cytometric analyses of CD70 surface expression in all together 19 patient-derived xenograft samples (TTLshort n= 7, TTLlong n=12) revealed a higher expression of CD70 on ALL cells with a TTLshort/early relapse phenotype compared to TTLlongsamples. We also investigated expression of the CD70 receptor CD27 and found no significant difference in surface expression between both TTL subgroups.Moreover, we investigated the transcript expression levels of 198 BCP-ALL specimens obtained at diagnosis. Interestingly, we found a heterogenous expression of CD70 with no association to cytogenetic subgroups, minimal residual disease (MRD) risk classes or patient outcome. Importantly, a significant higher CD70 expression was found in leukemia samples compared to healthy bone marrow controls indicating a general over-expression in BCP-ALL. CD27 however, did not show different transcript expression including healthy bone marrow controls.To take advantage of increased CD70 expression in BCP-ALL, we addressed CD70 as therapeutic target for immunotherapy. Co-culture in vitro experiments of primograft ALL cells with NK cells in the presence of specific anti-CD70 antibodies revealed five-fold increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to the respective isotype control.To evaluate the efficacy of CD70 directed immunotherapy, we assessed leukemia development in NOD/SCID mice upon transplantation of primograft ALL with high CD70 expression either incubated with anti-CD70 antibody or the respective isotype control. Most importantly, a marked reduction of leukemia load in peripheral blood, bone marrow and spleens of the animals was detected in anti-CD70 treated cases. This indicates, that CD70 provides an immunotherapeutic target on ALL cells inducing ADCC by NK cells present in NOD/SCID mice. Most interestingly, this effect could be abrogated both by NK-cell depletion (pre-treatment with anti-mouse CD122 antibodies) in the recipient animals and by using NK-cell deprived NSG mice as recipients, confirming that decreased in vivo leukemia growth upon anti-CD70 treatment is mediated by NK-cell induced cytotoxicity of anti-CD70 bearing CD70 positive ALL cells.Taken together, we identified significantly up-regulated CD70 expression in BCP-ALL with varying expression among molecular and prognostic subgroups. BCP-ALL samples with high surface expression of CD70, as detected by flowcytometry, can be targeted by directed immunotherapy with anti-CD70 antibodies leading to efficient NK-cell dependent lysis of leukemia cells in vitro and decreased growth in an in vivo BCP-ALL model. Thus, CD70 provides a novel target for directed immunotherapy of BCP-ALL. DisclosuresNo relevant conflicts of interest to declare.