scholarly journals P062 Dose-dependent differential effects of vedolizumab therapy on adhesion of regulatory and effector T cells

2020 ◽  
Vol 14 (Supplement_1) ◽  
pp. S165-S166
Author(s):  
E Becker ◽  
M Wiendl ◽  
A Schulz-Kuhnt ◽  
I Atreya ◽  
R Atreya ◽  
...  
Keyword(s):  
T Cell ◽  
Mouse Model ◽  
T Cell Subsets ◽  
Dependent Manner ◽  
Differential Effects ◽  
Preferential Binding ◽  
Exposure Levels ◽  
Differential Binding ◽  
Dose Dependent

Abstract Background Vedolizumab has emerged as an important pillar of treatment in inflammatory bowel disease (IBD). However, for unknown reasons, not all patients respond to therapy. Earlier clinical studies suggested decreased response rates in the highest compared with medium dosage groups. Interestingly, vedolizumab has been shown to inhibit the homing of both regulatory (Treg) and effector T (Teff) cells and previous data from our group suggested different effect sizes in both populations. Thus, we hypothesised that the non-linear exposure–efficacy correlation might be explained by dose-dependent differential effects of vedolizumab on Treg and Teff homing. Therefore, we studied functional effects of different vedolizumab exposure levels on Treg and Teff cell trafficking. Methods The α4β7 expression on different human T-cell subsets as well as the binding characteristics of vedolizumab to these cells at different exposure levels was analysed via flow cytometry. Functional effects of different vedolizumab concentrations on the adhesion of Tregs and Teffs to mucosal addressin cell adhesion molecule 1 (MAdCAM-1) were analysed using dynamic in vitro adhesion assays, transmigration assays and in vivo homing assays in a humanised mouse model. The in vivo binding of vedolizumab to Tregs and Teffs in patients receiving therapy was quantified and correlated with the corresponding serum levels. Results We found a preferential binding of vedolizumab to Tregs at an exposure with 0.4 µg/ml vedolizumab that shifted to a preferential binding to Teffs at an exposure with 10 µg/ml. Further increase of vedolizumab to 50 µg/ml led to equal binding to Tregs and Teffs (Figure 1). Consistently, at 10 µg/ml, dynamic adhesion of Tregs to MAdCAM-1 was increased compared with Teffs, but no difference was noted at 50 µg/ml. Additionally, a higher number of Treg compared with Teff cells were able to transmigrate in a MAdCAM-1-dependent manner at a concentration of 10 µg/ml vedolizumab. Preliminary data from homing experiments in a humanised mouse model and from IBD patients treated with vedolizumab support the notion that differential binding preferences depending on the exposure level can also be observed in vivo. Conclusion Our findings support a dose-dependent differential binding of vedolizumab to different T-cell subpopulations and suggest that an optimal ‘window’ of exposure exists, in which effects on Teffs predominate over Tregs. While offering a potential explanation for earlier findings in dose-ranging studies, our data might lay the basis for the establishment of individualised dose optimisation in IBD patients.

Dose-dependent modulation of the T cell proteome by ascorbic acid

2007 ◽  
Vol 97 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Melissa M. Grant ◽  
Nalini Mistry ◽  
Joseph Lunec ◽  
Helen R. Griffiths
Keyword(s):  
Ascorbic Acid ◽  
T Cell ◽  
Dependent Manner ◽  
Transfer Protein ◽  
Phosphatidylinositol Transfer ◽  
Cell Proteome ◽  
Dose Dependent ◽  
H Protein ◽  
Dimensional Electrophoresis

To investigate the hypothesis that the micronutrient ascorbic acid can modulate the functional genome, T cells (CCRF-HSB2) were treated with ascorbic acid (up to 150 μm) for up to 24 h. Protein expression changes were assessed by two-dimensional electrophoresis. Forty-one protein spots which showed greater than two-fold expression changes were subject to identification by matrix-assisted laser desorption ionisation time of flight MS. The confirmed protein identifications were clustered into five groups; proteins were associated with signalling, carbohydrate metabolism, apoptosis, transcription and immune function. The increased expression of phosphatidylinositol transfer protein (promotes intracellular signalling) within 5 min of ascorbic acid treatment was confirmed by Western blotting. Together, these observations suggest that ascorbic acid modulates the T cell proteome in a time- and dose-dependent manner and identify molecular targets for study following antioxidant supplementation in vivo.

AV-65, a Novel Inhibitor of the Wnt/β-Catenin Signaling Pathway, Inhibits the Proliferation of Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2866-2866
Author(s):  
Hisayuki Yao ◽  
Eishi Ashihara ◽  
Rina Nagao ◽  
Shinya Kimura ◽  
Hideyo Hirai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mouse Model ◽  
Signaling Pathway ◽  
Small Molecule ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Cancer Cell Line ◽  
Dependent Manner ◽  
Dose Dependent

Abstract Abstract 2866 Poster Board II-842 Although new molecular targeting agents against multiple myeloma (MM) have been developed, MM still remains an incurable disease. It is important to continue to investigate new therapeutic agents based on the biology of MM cells. β-catenin is the downstream effector of Wnt signaling and it regulates genes implicated in malignant progression. We have demonstrated that blockade of Wnt/β-catenin signaling pathway inhibits the progression of MM by using RNA interference methods with an in vivo mouse model (Ashihara E, et al. Clin Cancer Res 15:2731, 2009.). In this study, we investigated the effects of AV-65, a novel inhibitor of the Wnt/β-catenin signaling pathway, on MM cells. The system to identify a series of small molecule compounds using a biomarker driven approach has been established. A gene expression biomarker signature reporting on the inhibition of Wnt/β-catenin signaling was generated upon treatment of a colon cancer cell line with β-catenin siRNA. This gene expression signatiure was used to screen a small molecule compound library to identify compounds which mimic knockdown of β-catenin and thus potentially inhibit the Wnt/β-catenin signaling pathway. One compound series, LC-363, was discovered from this screen and validated as novel Wnt/β-catenin signaling inhibitors (Strovel JW, et al. ASH meeting, 2007.). We investigated the inhibitory effects of AV-65, one of LC-363 compounds, on MM cell proliferation. AV-65 inhibited the proliferation of MM cells in a time- and a dose-dependent manner and the values of IC50 at 72 hrs were ranging from 11.7 to 82.1 nM. AV-65 also showed an inhibitory effect on the proliferation of RPMI8226/LR-5 melphalan-resistant MM cells (provided from Dr. William S. Dalton). In flow cytometric analysis, apoptotic cells were increased by AV-65 treatment in a time- and a dose-dependent manner. Western blotting analysis showed that β-catenin was ubiquitinated and that the expression of nuclear β-catenin diminished (Figure 1). Moreover, AV-65 suppressed T-cell factor transcriptional activities, resulting in the decrease of c-myc expression. Taken together, AV-65 promotes the degradation of β-catenin, resulting in the induction of apoptosis of MM cells. We next investigated the in vivo effects of AV-65 using an orthotopic MM-bearing mouse model. AV-65 inhibits the growth of MM cells and significantly prolongs the survival rates (Figure 2). In conclusion, AV-65 inhibited the proliferation of MM cells via inhibition of the Wnt/β-catenin signaling pathway. AV-65 is a promising therapeutic agent for treatment of MM. Disclosures: No relevant conflicts of interest to declare.

Small Molecule Inhibitor of ITK Disrupts TCR Signaling and Demonstrates Anti-Tumor Efficacy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3932-3932
Author(s):  
Mary Faris ◽  
Uriel M Malyankar ◽  
Qingping Zeng ◽  
Gary A Flynn ◽  
Gerold Feuer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytokine Production ◽  
In Vivo Studies ◽  
Dependent Manner ◽  
T Cell Malignancies ◽  
Dose Dependent ◽  
Leukemias And Lymphomas

Abstract Abstract 3932 ITK (Interluekin-2 Inducible Tyrosine Kinase) is a member of the TEC family of intracellular protein tyrosine kinases. ITK is highly expressed in T cells and NK cells, with expression detected in mast cells. ITK plays a key role in several aspects of T cell biology, including T cell development, differentiation, migration, proliferation and activation. The function of ITK in immunity and allergy is well documented. T cells from ITK knock out mice show several developmental and functional defects, including defective signal transduction, altered CD4+ to CD8+ T cells ratios, reduced Th2 lineage differentiation, diminished IL4 and IL2 production and reduced T cell proliferation. Importantly ITK deficient mice fail to mount an immune response to infection and show reduced allergic asthma reactions. In contrast to its well described role in immune function, ITK's function in cancer biology is still emerging. Recent studies had reported enhanced ITK expression and activation of the ITK pathway in several types of leukemias and lymphomas. In addition, the dependence of T cell malignancies on an ITK-regulated pathway, namely the IL2/IL2R (CD25) pathway, has also been observed. Taken together, this information indicates that ITK is a therapeutic target, with applicability in leukemias and lymphomas. MannKind scientists have developed a series of selective small molecule ITK inhibitors, including the orally available tool compound described within, and evaluated their activity in enzyme, cell-based and in vivo studies. In cellular assays, the compounds showed significant inhibition of the T cell-receptor mediated activation of the ITK pathways and related downstream cytokine production. In addition to inhibiting the phosphorylation of ITK and its downstream mediator, PLCg, our tool compounds inhibited the production of IL2 and expression of CD25 in a dose dependent manner. Importantly, our compound regulated the in vitro growth of tumor T cells but not that of unrelated control cells. In vivo studies revealed that the tool compounds inhibited the growth and progression of patient derived ATL tumors in a xenograft pre-clinical model, and prolonged the survival of treated mice in a dose dependent manner, in addition to regulating cytokine production in vivo. In summary, our team has identified ITK selective compounds with demonstrated on-target and anti-tumor activity in vitro and preclinical T cell tumor models, and validated this pathway relative to T cell malignancies. This effort provides a platform for further compound optimization and evaluation for hematologic malignancies. Disclosures: Faris: MannKind Corp: Employment. Malyankar:MannKind Corp: Employment. Zeng:MannKind Corp: Employment. Kertesz:Mannkind Corporation: Employment, Equity Ownership. Vuga:MannKind Corp.: Employment. Rosario:MannKind Corp: Employment. Bot:MannKind Corp: Employment.

scholarly journals Spontaneous and glucocorticoid-induced apoptosis in human mature T lymphocytes

Blood ◽  
1995 ◽  
Vol 86 (11) ◽  
pp. 4199-4205 ◽  
Author(s):  
M Brunetti ◽  
N Martelli ◽  
A Colasante ◽  
M Piantelli ◽  
P Musiani ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Interleukin 2 ◽  
Cell Number ◽  
T Cell Subsets ◽  
Dependent Manner ◽  
Cell Repertoire ◽  
Induced Apoptosis ◽  
And Function ◽  
Dose Dependent

Glucocorticoid (GC)-induced apoptosis is a well-recognized physiologic regulator of murine T-cell number and function. We have analyzed its mechanisms in human mature T cells, which have been thought to be insensitive until recently. Peripheral blood T cells showed sensitivity to GC-induced apoptosis soon after the proliferative response to a mitogenic stimulation, and were also sensitive to spontaneous (ie, growth factor deprivation-dependent) apoptosis. CD8+ T cells were more sensitive to both forms than CD4+ T cells. Acquisition of sensitivity to GC-induced apoptosis was not associated with any change in number or affinity of GC receptors. Both spontaneous and GC-induced apoptosis were increased by the macromolecular synthesis inhibitors, cycloheximide (CHX) and puromycin. A positive correlation between the degree of protein synthesis inhibition and the extent of apoptosis was observed. Interleukin-2 (IL-2) IL-4, and IL-10 protected (IL-2 > IL-10 > IL-4) T cells from both forms of apoptosis in a dose-dependent manner. Our data suggest that spontaneous and GC-induced apoptosis regulate the human mature T-cell repertoire by acting early after the immune response and differentially affecting T-cell subsets.

scholarly journals 3′,5′-Cyclic Diguanylic Acid Reduces the Virulence of Biofilm-Forming Staphylococcus aureus Strains in a Mouse Model of Mastitis Infection

2005 ◽  
Vol 49 (8) ◽  
pp. 3109-3113 ◽  
Author(s):  
Eric Brouillette ◽  
Mamoru Hyodo ◽  
Yoshihiro Hayakawa ◽  
David K. R. Karaolis ◽  
François Malouin
Keyword(s):  
Staphylococcus Aureus ◽  
Mouse Model ◽  
Bacterial Colonization ◽  
Dependent Manner ◽  
Signaling Systems ◽  
Cyclic Dinucleotides ◽  
Novel Drug ◽  
Dose Dependent

ABSTRACT The cyclic dinucleotide 3′,5′-cyclic diguanylic acid (c-di-GMP) is a naturally occurring small molecule that regulates important signaling systems in bacteria. We have recently shown that c-di-GMP inhibits Staphylococcus aureus biofilm formation in vitro and its adherence to HeLa cells. We now report that c-di-GMP treatment has an antimicrobial and antipathogenic activity in vivo and reduces, in a dose-dependent manner, bacterial colonization by biofilm-forming S. aureus strains in a mouse model of mastitis infection. Intramammary injections of 5 and 50 nmol of c-di-GMP decreased colonization (bacterial CFU per gram of gland) by 0.79 (P > 0.05) and 1.44 (P < 0.01) logs, respectively, whereas 200-nmol doses allowed clearance of the bacteria below the detection limit with a reduction of more than 4 logs (P < 0.001) compared to the untreated control groups. These results indicate that cyclic dinucleotides potentially represent an attractive and novel drug platform which could be used alone or in combination with other agents or drugs in the prevention, treatment, or control of infection.

scholarly journals Antiplasmodial Potential of Indonesian Medicinal Plants

2020 ◽  
Author(s):  
Nurhayati Bialangi ◽  
Mohamad Adam Mustapa ◽  
Yuszda K Salimi ◽  
Weny J.A Musa ◽  
Ari Widiyantoro ◽  
...  
Keyword(s):  
Medicinal Plants ◽  
Mouse Model ◽  
Traditional Medicine ◽  
Post Infection ◽  
High Ratio ◽  
Antiplasmodial Activity ◽  
Dependent Manner ◽  
Dose Dependent

Abstract Background: Species A. paniculata (Burm. f.) Nees known as″ Sambiloto ″ and P. pellucida L. Kunth known as″ Suruhan ″ are mainly distributed in Indonesia and their combination was used as a traditional medicine for treating malaria diseases. However, no information appears to have evaluated the antiplasmodial potential of the two plants. This research aimed to evaluate the antiplasmodial activity of the two plants and the species P. pellucida L. Kunth alone as a source of antiplasmodial agent. Methods: In vitro test of the AP-PP and PP extracts against Pf D-10 (chloroquine-sensitive) were performed as described by Desjardins et al. An in vivo test of the PP extract in mice infected with Pb ANKA was performed using Peters´ 4-day suppressive test. Parasitemia, growth and inhibition rates were determined via Giemsa-stained smear of blood and analyzed microscopically. Survival was followed up until day 21 post-infection.Results: The increased ratio of the PP extract (20:80) exhibited significant antiplasmodial in contrast to the high ratio of the AP extract (IC50, 62.01 mg/mL). Further evaluation of the PP extract alone displayed better antiplasmodial activity with an IC50 value of 4.0 mg/mL. Furthermore, an in vivo test of the PP extract in BALB/c albino mice infected with Pb ANKA exhibited a significant chemosuppressive effect in a dose-dependent manner.Conclusion: The increased ratio of the PP extract exhibited a major contribution for their activity. The PP extract alone showed better antiplasmodial activity than the AP extract and their combination. An in vivo test confirmed the efficacy of the PP extract in mouse model.

scholarly journals Lymphokine gene expression in vivo is inhibited by cyclosporin A.

1990 ◽  
Vol 171 (2) ◽  
pp. 533-544 ◽  
Author(s):  
A Granelli-Piperno
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cyclosporin A ◽  
Dependent Manner ◽  
Ifn Gamma ◽  
Cell Responses ◽  
Dose Dependent ◽  
In Vivo Administration

Murine T cells were stimulated in vivo by administering allogeneic cells or mitogens into the foot pads and then examining the draining popliteal lymph nodes. Allogeneic spleen cells induced the expression of IL2 and IFN-gamma mRNAs in a time- and dose-dependent manner. Induction of these transcripts also was detected after administration of Con A and anti-CD3 mAb. An increase in DNA-synthesizing cells was observed by 48 h, and these were shown to be T cells because of their sensitivity to anti-Thy-1 but not anti-B220 mAb and complement, and because of their localization to the T-dependent areas of the lymph node. The in vivo administration of cyclosporin A (CSA) reduced several of these T cell responses. The level of DNA synthesis and the frequency of cells synthesizing DNA were decreased by approximately 75%, while the induction of IL-2 responsiveness was not substantially diminished. IL-2 and IFN-gamma transcripts were inhibited at least 70-90%, as determined by Northern blot and in situ hybridization. Although the inhibition by CSA was not as complete in animals as observed previously in tissue culture, our findings indicate that in both systems, a major site of action of CSA is to inhibit T cell growth by inhibiting lymphokine production.

scholarly journals Inhibition of calcium signalling in murine splenocytes by polyamines: differential effects on CD4 and CD8 T-cells

1993 ◽  
Vol 291 (2) ◽  
pp. 375-381 ◽  
Author(s):  
T Thomas ◽  
U B Gunnia ◽  
E J Yurkow ◽  
J R Seibold ◽  
T J Thomas
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
T Cell Subsets ◽  
Free Calcium ◽  
Dependent Manner ◽  
Con A ◽  
Acetoxymethyl Ester ◽  
Murine Splenocytes ◽  
Dose Dependent

Transmembrane Ca2+ influx is recognized as a universal second messenger that transduces T-cell activation signals to cytoplasm and nucleus, thereby stimulating transcription and cell division. To examine the role of endogenous factors that regulate mitogenic Ca2+ signalling of T-cells, we measured the concanavalin (Con) A-induced increase in cytoplasmic free calcium ([Ca2+]i) in spleen cells of BALB/c mice, using flow cytometry with an indicator dye, Indo-1 acetoxymethyl ester (Indo-1/AM). Con A is a polyclonal activator of T-cells. Unstimulated splenocytes had a [Ca2+]i of 100 nM. [Ca2+]i increased with Con A in a dose-dependent manner up to a concentration of 50 micrograms/ml. In the presence of 50 micrograms/ml Con A, [Ca2+]i was 350 nM. Natural polyamines (putrescine, spermidine and spermine) inhibited Con-A-induced Ca2+ influx in a dose-dependent manner. Putrescine was the most effective polyamine in desensitizing the Ca2+ signal, and decreased [Ca2+]i from 350 nM in the absence of putrescine to 250 nM in the presence of 100 microM putrescine. This effect was not mimicked by structurally related homologues or inorganic cations, suggesting a specific structural effect of the polyamine. H.p.l.c. analysis showed that polyamines were internalized during incubation of cells in vitro. In experiments using monoclonal anti-CD4 and anti-CD8 antibodies, we found a differential effect of putrescine on Ca2+ influx in CD4 and CD8 subpopulations of T cells. For CD4+ cells, [Ca2+]i decreased from 625 nM to 420 nM in the presence of 500 microM putrescine, whereas [Ca2+]i was not affected by putrescine in CD8+ cells. These data suggest that natural polyamines have cell-specific effects on mitogen-stimulated Ca(2+)-influx in T-cell subsets.

scholarly journals A Novel Mouse Model for Studying the Effects of Cyp17 Overexpression in a Temporal- and Spatial-Specific Manner

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A762-A763
Author(s):  
Christian Secchi ◽  
Martina Belli ◽  
Dwayne Stupack ◽  
Shunichi Shimasaki
Keyword(s):  
Mouse Model ◽  
Polycystic Ovary ◽  
Expression System ◽  
Follicular Development ◽  
In Vivo Model ◽  
Dependent Manner ◽  
Active Transcription ◽  
And Function ◽  
Dose Dependent

Abstract Background: Cyp17 plays a key role in theca cells (TCs) to produce androgens, which, in turn, are converted to estrogens in granulosa cells. Intrinsic alterations in ovarian steroidogenesis contribute to excessive ovarian androgen production that characterizes polycystic ovary disease (PCOS)1,2. Hyperandrogenism has been associated with higher levels of Cyp17 in TCs, and correlate with increased numbers of antral follicles3. While androgen excess is one of the hallmark features of PCOS, its putative role in the follicular development and function remains poorly known. Most efforts have used androgen administration or Cyp19 blockade approach to study how androgens prolong folliculogenesis4. Although some insights have been made, it is not clear if these models accurately address the cascade of effects that follow ovarian hyperandrogenism. Aim: Here, we aim to study the specific effects of hyperandrogenemia on ovarian morphology, follicle function and fertility with a new transgenic (TG) mouse model expressing elevated Cyp17 levels exclusively in TCs. Methods: We generated a breeding line of triple TG mice using a combination of the Tet-dependent expression system and the Cre/LoxP gene control system. Specifically, we used Cyp17 promoter-iCre mice crossed with trans-activator mice (R26-STOP-rtTA-IRES-EGFP transgene, Jackson Lab) and with a responder mouse carrying the TRE-Cyp17 transgene. Cyp17 promoter-iCre mice were used to ensure rtTA/EGFP is expressed specifically in TCs of secondary follicles. After the DNA segment between the two LoxP sites is excised by Cyp17iCre specifically in TCs, the R26-STOP-rtTA gene remains activated in all daughter TCs. Only upon treatment with Doxycycline (DOX) can suppression be relieved and active transcription of TRE-Cyp17 be induced in a dose-dependent manner. Results: Cyp17 mRNA expression levels in TCs of TG mice treated with 20, 100 or 200 mg/Kg DOX compared with corresponding untreated control mice showed a modulation in a dose-dependent manner (P=0.01 ANOVA). Confocal and RNAscope analysis validated (i) the effective combination of the Cyp17iCre/rtTA expression system visualizing the rtTA/EGFP specifically expressed in ovarian TCs and (ii) the DOX-induced increase of Cyp17 expression compared with the WT mice. DOX treated TG females were acyclic, being mostly arrested in diestrus. Analysis of estrous cycle stages revealed that treated TG females spent significantly more time in diestrus than control females (P=0.007, ANOVA). Conclusions: Our new in vivo model is the first that analyzes androgen impact independent of any extraovarian source of androgen, complementing current clinical efforts to study the occurrences of TCs elevated androgen levels in normal and PCOS women. 1 Rosenfield, R. L. et al. Endocr Rev (2016)2 Azziz, R. et al. Nat Rev Dis Primers (2016)3 Comim, F. V., et al. Hum Reprod (2013)4 Stener-Victorin, E. et al. Endocr Rev (2020)

scholarly journals 795 APVO603: a dual 4-1BB and OX40 bispecific approach utilizing ADAPTIRTM technology designed to deliver a conditional T cell/NK response against solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A830-A830
Author(s):  
Michelle Nelson ◽  
Ashly Lucas ◽  
Rebecca Gottschalk ◽  
Catherine McMahan ◽  
Jane Gross ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Bispecific Antibody ◽  
Expression Profiles ◽  
T Cell Subsets ◽  
Dependent Manner ◽  
Dose Dependent

BackgroundAPVO603 is a dual targeting bispecific antibody for 4-1BB (CD137) and OX40 (CD134), engineered with Aptevo's ADAPTIRTM technology. We have previously shown that the distinct characteristics of APVO603 may enable conditional agonism of 4-1BB and OX40 only when cross-linked through engagement of the other receptor via cis and/or trans cellular interactions. Thus, APVO603 is designed with the potential to overcome both the on-target toxicity and limited efficacy observed with 4-1BB and OX40 monoclonal antibody treatment in the clinic.MethodsGenevestigator Software was used to analyze curated transcriptomic data for the expression profiles of OX40 and 4-1BB across select human heme and solid cancer patient sample data sets, as well as, non diseased tissue. Primary inducible Treg (iTreg) cells were sub-optimally stimulated with an anti-CD3/CD28 antibody and cell proliferation was assessed using CFSE-labelled. Cytokines were measured using intracellular flow-based methods. For in vitro tumor lysis studies, activated T cells were co-cultured with Nuclight-labelled tumor cells expressing a tumor-associated antigen (TAA) and activated with TAA x CD3 bispecific protein. Live tumor cells were continually assessed using the Incucyte Live-Cell Analysis System and Cell-By-Cell Software Module.ResultsOX40 and 4-1BB displayed distinct tumor expression profiles, however, several tumor indications were identified with high co-expression and may aid in identifying indications for the clinical development of APVO603. In vitro, APVO603 favored activation of effector T cell subsets and had minimal impact in augmenting iTreg cells proliferation, cytokine production or expression of effector-related molecules, despite the fact that a portion of the iTreg cells expressed OX40 and 4-1BB. The mechanistic activity of APVO603 resulted in dose-dependent control of in vitro tumor growth when paired with a T-cell activating TAA x CD3 bispecific under standard conditions or those leading to T cell exhaustion. In preclinical assays using PBMCs sub-optimally stimulated with TAA x CD3, APVO603 enhanced TAA-expressing tumor cell lysis when compared to TAA x CD3 alone.ConclusionsAPVO603 is a dual-agonistic bispecific antibody that augments the effector function of activated CD4+ and CD8+ T cells and NK cells, but not iTreg cells, in a dose-dependent manner and reduces growth of tumors in vitro and in vivo. Further, mechanistic evaluation supports the ability of APVO603 to pair with T-cell modulating IO approaches to support a more fit T cell response and favorable TME. This preclinical data supports further development of APVO603, a promising immuno-oncology therapeutic with potential for benefit in hematologic and solid tumors.

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