scholarly journals 757 M9657, a novel tumor-targeted conditional anti-CD137 agonist displays MSLN-dependent anti-tumor immunity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A792-A792
Author(s):  
Chunxiao Xu ◽  
Brain Rabinovich ◽  
Amit Deshpande ◽  
Xueyuan Zhou ◽  
Frederic Christian Pipp ◽  
...  
Keyword(s):  
Bispecific Antibody ◽  
Animal Experiments ◽  
Immune Memory ◽  
In Vitro Assays ◽  
Therapeutic Window ◽  
Cytotoxic T Cell ◽  
Functional Assay ◽  
Dose Dependent

BackgroundThe costimulatory receptor CD137 (also known as 4-1BB and TNFRSF9) plays an important role in sustaining effective cytotoxic T cell immune responses and its agonism has been investigated as a cancer immunotherapy. In clinical trials, the systemic administration of the 1st generation CD137 agonist monotherapies, utomilumab and urelumab, were suspended due to either low anti-tumor efficacy or hepatotoxicity mediated by recognized epitope on CD137 and FcγR ligand-dependent clustering.MethodsM9657, a bispecific antibody was engineered a tetravalent bispecific antibody (mAb2) format with the Fab portion binding to the tumor antigen Mesothelin (MSLN) and a modified CH2-CH3 domain as Fc antigen binding (Fcab) portion binding to CD137. M9657 has a human IgG1 backbone with LALA mutations to abrogate the binding to Fcγ receptor. The biological characteristics and activities of M9657 were investigated in a series of in vitro assays and the in vivo efficacy was investigated in syngeneic tumor models with FS122m, a murine-reactive surrogate with the same protein structure of M9657.ResultsM9657 binds efficiently to both human and Cynomolgus CD137 as well as MSLN. In the cellular functional assay, M9657 displayed MSLN- and TCR/CD3 interaction (signal 1)-dependent cytokine release and tumor cell cytotoxicity associated with Bcl-XL activation and immune memory formation. FS122m demonstrated potent MSLN- and dose- dependent in vivo anti-tumor efficacy (figure 1). Comparing with 3H3, a Urelumab surrogate Ab, FS122m displayed an improved therapeutic window with significantly lower for on-target /off-tumor toxicity.ConclusionsTaken together, M9657 exhibits a promising developability profile as a tumor-targeted immune agonist with potent anti-cancer activity, but without systemic immune activation.Ethics ApprovalAll animal experiments were performed in accordance with EMD Serono Research & Development Institute (protocol 17-008, 20-005) and Wuxi AppTec Animal Care and Use Committee (IACUC) guidelines.Abstract 757 Figure 1FS122m displayed dose-dependent anti-tumor efficacy

scholarly journals Pharmacokinetic prediction of an antibody in mice based on an in vitro cell-based approach using target receptor-expressing cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yuki Noguchi ◽  
Kazuhisa Ozeki ◽  
Hidetaka Akita
Keyword(s):  
Animal Experiments ◽  
Elimination Rate ◽  
Chinese Hamster ◽  
Compartment Model ◽  
In Vitro Assays ◽  
Gamma Receptor ◽  
Uptake Assay ◽  
Menten Constant

Abstract In vivo pharmacokinetics (PK) studies using mice and monkeys are the main approaches for evaluating and predicting the PK of antibodies, and there is a strong demand for methods that do not require animal experiments. In this work, we focused on quantitatively predicting the nonlinear PK of an antibody based on cell-based assays. An anti-mouse Fc gamma receptor IIB antibody was used as a model antibody. To determine the PK parameters related to nonspecific elimination in vivo, the plasma concentration profile at 100 mg/kg, at which target-specific clearance is saturated, was analyzed by a 2-compartment model. To estimate the parameters related to target-specific elimination, the Michaelis–Menten constant (Km) and the maximum elimination rate (Vmax) were determined by an uptake assay using Chinese hamster ovary (CHO) cells expressing the target receptor. Finally, the integration of all of these parameters permitted the PK to be predicted at doses ranging from 1 to 100 mg/kg regardless of whether target-specific clearance was saturated or nonsaturated. The findings presented herein show that in vitro assays using target-expressing cells are useful tools for obtaining PK parameters and predicting PK profiles and, in some cases, eliminate the need for in vivo PK studies using experimental animals.

Comparison of the Sensitivity of in Vivo and in Vitro Assays for Detection of Antiviral Cytotoxic T Cell Activity

1993 ◽  
Vol 151 (2) ◽  
pp. 460-466 ◽  
Author(s):  
Irene Castelmur ◽  
Claudio Dipaolo ◽  
Martin F. Bachmann ◽  
Hans Hengartner ◽  
Rolf M. Zinkernagel ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Activity ◽  
In Vitro Assays ◽  
Cytotoxic T Cell

scholarly journals Sls1p Stimulates Sec63p-Mediated Activation of Kar2p in a Conformation-Dependent Manner in the Yeast Endoplasmic Reticulum

2000 ◽  
Vol 20 (18) ◽  
pp. 6923-6934 ◽  
Author(s):  
Mehdi Kabani ◽  
Jean-Marie Beckerich ◽  
Claude Gaillardin
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Synthetic Lethality ◽  
In Vitro Assays ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Dose Dependent

ABSTRACT We previously characterized the SLS1 gene in the yeastYarrowia lipolytica and showed that it interacts physically with YlKar2p to promote translocation across the endoplasmic-reticulum membrane (A. Boisramé, M. Kabani, J. M. Beckerich, E. Hartmann, and C. Gaillardin, J. Biol. Chem. 273:30903–30908, 1998). A Y. lipolytica Kar2p mutant was isolated that restored interaction with an Sls1p mutant, suggesting that the interaction with Sls1p could be nucleotide and/or conformation dependent. This result was used as a working hypothesis for more accurate investigations in Saccharomyces cerevisiae. We show by two-hybrid an in vitro assays that the S. cerevisiae homologue of Sls1p interacts with ScKar2p. Using dominant lethal mutants of ScKar2p, we were able to show that ScSls1p preferentially interacts with the ADP-bound conformation of the molecular chaperone. Synthetic lethality was observed between ΔScsls1 and translocation-deficientkar2 or sec63-1 mutants, providing in vivo evidence for a role of ScSls1p in protein translocation. Synthetic lethality was also observed with ER-associated degradation and folding-deficient kar2 mutants, strongly suggesting that Sls1p functions are not restricted to the translocation process. We show that Sls1p stimulates in a dose-dependent manner the binding ofScKar2p on the lumenal J domain of Sec63p fused to glutathione S-transferase. Moreover, Sls1p is shown to promote the Sec63p-mediated activation of Kar2p's ATPase activity. Our data strongly suggest that Sls1p could be the first GrpE-like protein described in the endoplasmic reticulum.

633 Dual-targeting of 4–1BB and OX40 with an ADAPTIR™ bispecific antibody enhances anti-tumor responses to solid tumor

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A669-A669
Author(s):  
Michelle Nelson ◽  
Robert Miller ◽  
Gabriele Blahnik-Fagan ◽  
Lauren Loh ◽  
Danielle Van Citters ◽  
...  
Keyword(s):  
Nk Cells ◽  
Tumor Cells ◽  
Bispecific Antibody ◽  
Cytokine Production ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Binding Domains ◽  
Dose Dependent

Background4-1BB (CD137) and OX40 (CD134) are critical activation-induced co-stimulatory receptors that regulate immune responses of activated T and NK cells by enhancing proliferation, cytokine production, survival, and cytolytic activity. A superagonist 4-1BB antibody has shown clinical activity but severe toxicities. APVO603, is a 4-1BB x OX40 targeting bispecific antibody with conditional agonism, activating these receptors only when both are co-engaged. The Fc portion was mutated to eliminate FcγR-mediated interactions. Co-stimulation through 4-1BB and OX40 has the potential to amplify the cytotoxic function and the number of activated T and NK cells in multiple solid tumor indications.1–2Methods scFv binding domains to 4-1BB and OX40 were optimized to increase affinity, function and stability, and then incorporated into the ADAPTIR™ bispecific antibody platform to produce the APVO603 lead candidate. NF-κB/luciferase reporter cell lines expressing OX40 or 4-1BB were initially used to assess the agonistic function of APVO603’s binding domains. Primary PBMC were sub-optimally stimulated with an anti-CD3 antibody and T and NK cell proliferation was assessed using Cell TraceTM-labelled PBMC. Cytokine secretion was measured at 48 hrs using Luminex-based assays. For in vitro tumor lysis studies, PBMC were co-cultured with tumor cells expressing a tumor-associated antigen (TAA) and activated with TAA x CD3 bispecific protein. 7-AAD expression was assessed on tumor cells at 72 hrs. The in vivo therapeutic efficacy of APVO603 was evaluated using a murine MB49 bladder cancer model in human 4-1BB and OX40 double knock-in mice.ResultsAPVO603 stimulates 4-1BB and OX40 NF-κB/luciferase reporter activity in a dose-dependent manner, and is strictly dependent on engagement of the reciprocal receptor to elicit 4-1BB or OX40 activity. In primary PBMC assays, APVO603 induces synergistic proliferation of CD4+, CD8+ T and NK cells when compared to OX40 or 4-1BB monospecific molecules with a wt Fc, either individually or in combination. Additionally, APVO603 enhances proinflammatory cytokine production and granzyme B expression, and augments in vitro tumor cell lysis induced by a TAAx CD3 engager. In vivo, APVO603 reduces growth of established MB49 tumors in human 4-1BB and OX40 double knock-in mice.ConclusionsAPVO603 is a dual-agonistic bispecific antibody that augments the effector function of activated CD4+ and CD8+ T and NK cells in a dose-dependent manner, and reduces growth of established tumors in vivo. This preclinical data, demonstrates conditional dual stimulation of 4-1BB and OX40 and supports further development of APVO603, a promising immuno-oncology therapeutic with potential for benefit in solid tumors.Ethics ApprovalTreatment of study animals was in accordance with conditions specified in the Guide for the Care and Use of Laboratory Animals, and the study protocol (ACUP 20) was approved by the Institutional Animal Care and Use Committee (IACUC).ReferencesBandyopadhyay S, Long M, Qui H, Hagymasi A, Slaiby A, Mihalyo M, Aguila H, Mittler R, Vella A, Adler A. Self-antigen prevents CD8 T cell effector differentiation by CD134 and CD137 dual costimulation. J Immunol 2008;181(11):7728–37.Ryan J, Mittal P, Menoret A, Svedova J, Wasser J, Adler A, Vella A. A novel biologic platform elicits profound T cell costimuloaroty activity and antitumor immunity in mice. Cancer Immunol Immunother 2018;67(4):605–613.

scholarly journals Blocking cell microtubule assembly inhibits the alloimmune response in vitro and prolongs renal allograft survival by inhibition of Th1 and sparing of Th2 cell function in vivo.

1995 ◽  
Vol 5 (7) ◽  
pp. 1418-1425
Author(s):  
E Akalin ◽  
W W Hancock ◽  
N Perico ◽  
G Remuzzi ◽  
O Imberti ◽  
...  
Keyword(s):  
Microtubule Assembly ◽  
Brown Norway ◽  
Cytotoxic T Cell ◽  
Dependent Manner ◽  
Lymphocyte Response ◽  
Term Survival ◽  
Renal Allografts ◽  
Dose Dependent

Colchicine inhibits cell microtubule assembly by binding to and preventing the polymerization of tubulin monomers. Although there are data to indicate that colchicine inhibits a variety of cell-mediated immune responses, the effects and mechanisms of inhibiting cell microtubule assembly on the alloimmune response have not been thoroughly investigated. It has recently been shown that colchicine prevents acute rejection and promotes the long-term survival of rat renal allografts. In this study, the effects and mechanisms of inhibiting cell microtubule assembly by colchicine on the alloimmune response in vitro and in vivo were examined. First, the effects of colchicine on T lymphocyte response to alloantigen in vitro were tested. In the standard one-way mixed lymphocyte response (MLR), responder Lewis rat lymph node cells were cultured with irradiated Brown-Norway stimulators. Colchicine inhibited the MLR in a dose-dependent manner, with 100% inhibition at a concentration of 25 ng/mL (6.25 x 10(-8) M) and 50% inhibition at a concentration of approximately 5 to 10 ng/mL. Colchicine also inhibited the generation of cytotoxic T lymphocytes as well as cytotoxic T cell effector function in vitro in a dose-dependent fashion. Second, detailed immunohistologic studies of renal allografts harvested from unmodified control (acutely rejecting) and colchicine-treated rats (Day 15 or 30) were performed. These studies showed that grafts from colchicine-treated animals had significantly fewer mononuclear cell infiltrates and less edema, and moderately decreased deposition of immunoglobulin M, C3, and fibrin, as compared with acutely rejecting control grafts.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals In Vitro Effects of Paralytic Shellfish Toxins and Lytic Extracellular Compounds Produced by Alexandrium Strains on Hemocyte Integrity and Function in Mytilus edulis

Toxins ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 544
Author(s):  
Virginia Angélica Bianchi ◽  
Ulf Bickmeyer ◽  
Urban Tillmann ◽  
Bernd Krock ◽  
Annegret Müller ◽  
...  
Keyword(s):  
Mytilus Edulis ◽  
Phagocytic Activity ◽  
In Vitro Assays ◽  
Shellfish Toxins ◽  
Paralytic Shellfish Toxins ◽  
Paralytic Shellfish ◽  
In Vitro Effects ◽  
Dose Dependent

Harmful effects caused by the exposure to paralytic shellfish toxins (PSTs) and bioactive extracellular compounds (BECs) on bivalves are frequently difficult to attribute to one or the other compound group. We evaluate and compare the distinct effects of PSTs extracted from Alexandrium catenella (Alex5) cells and extracellular lytic compounds (LCs) produced by A. tamarense (NX-57-08) on Mytilus edulis hemocytes. We used a 4 h dose–response in vitro approach and analyzed how these effects correlate with those observed in a previous in vivo feeding assay. Both bioactive compounds caused moderated cell death (10–15%), being dose-dependent for PST-exposed hemocytes. PSTs stimulated phagocytic activity at low doses, with a moderate incidence in lysosomal damage (30–50%) at all tested doses. LCs caused a dose-dependent impairment of phagocytic activity (up to 80%) and damage to lysosomal membranes (up to 90%). PSTs and LCs suppressed cellular ROS production and scavenged H2O2 in in vitro assays. Neither PSTs nor LCs affected the mitochondrial membrane potential in hemocytes. In vitro effects of PST extracts on M. edulis hemocytes were consistent with our previous study on in vivo exposure to PST-producing algae, while for LCs, in vivo and in vitro results were not as consistent.

scholarly journals A Novel Apilic Antivenom to Treat Massive, Africanized Honeybee Attacks: A Preclinical Study from the Lethality to Some Biochemical and Pharmacological Activities Neutralization

Toxins ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 30
Author(s):  
Jhonatha Mota Teixeira-Cruz ◽  
Marcelo Abrahão Strauch ◽  
Marcos Monteiro-Machado ◽  
Matheus Silva Tavares-Henriques ◽  
João Alfredo de Moraes ◽  
...  
Keyword(s):  
Animal Experiments ◽  
Specific Treatment ◽  
Preclinical Study ◽  
Honeybee Venom ◽  
Africanized Honeybee ◽  
Lethal Event ◽  
Venom Dose ◽  
Dose Dependent

Massive, Africanized honeybee attacks have increased in Brazil over the years. Humans and animals present local and systemic effects after envenomation, and there is no specific treatment for this potentially lethal event. This study evaluated the ability of a new Apilic antivenom, which is composed of F(ab’)2 fraction of specific immunoglobulins in heterologous and hyperimmune equine serum, to neutralize A. mellifera venom and melittin, in vitro and in vivo, in mice. Animal experiments were performed in according with local ethics committee license (UFRJ protocol no. DFBCICB072-04/16). Venom dose-dependent lethality was diminished with 0.25–0.5 μL of intravenous Apilic antivenom/μg honeybee venom. In vivo injection of 0.1–1 μg/g bee venom induced myotoxicity, hemoconcentration, paw edema, and increase of vascular permeability which were antagonized by Apilic antivenom. Cytotoxicity, assessed in renal LLC-PK1 cells and challenged with 10 μg/mL honeybee venom or melittin, was neutralized by preincubation with Apilic antivenom, as well the hemolytic activity. Apilic antivenom inhibited phospholipase and hyaluronidase enzymatic activities. In flow cytometry experiments, Apilic antivenom neutralized reduction of cell viability due to necrosis by honeybee venom or melittin. These results showed that this antivenom is effective inhibitor of honeybee venom actions. Thus, this next generation of Apilic antivenom emerges as a new promising immunobiological product for the treatment of massive, Africanized honeybee attacks.

A in Vivo Assay for Standardizing Heparin Preparations

1979 ◽  
Vol 41 (03) ◽  
pp. 576-582
Author(s):  
A R Pomeroy
Keyword(s):  
In Vitro Assays ◽  
British Pharmacopoeia ◽  
In Vitro Method ◽  
Standard Preparation ◽  
Reliable Estimation ◽  
Assay Procedure ◽  
Accuracy And Precision ◽  
Heparin Preparation

SummaryThe limitations of currently used in vitro assays of heparin have demonstrated the need for an in vivo method suitable for routine use.The in vivo method which is described in this paper uses, for each heparin preparation, four groups of five mice which are injected intravenously with heparin according to a “2 and 2 dose assay” procedure. The method is relatively rapid, requiring 3 to 4 hours to test five heparin preparations against a standard preparation of heparin. Levels of accuracy and precision acceptable for the requirements of the British Pharmacopoeia are obtained by combining the results of 3 to 4 assays of a heparin preparation.The similarity of results obtained the in vivo method and the in vitro method of the British Pharmacopoeia for heparin preparations of lung and mucosal origin validates this in vivo method and, conversely, demonstrates that the in vitro method of the British Pharmacopoeia gives a reliable estimation of the in vivo activity of heparin.

Potentially Thrombogenic Materials in Factor IX Concentrates

1975 ◽  
Vol 33 (03) ◽  
pp. 617-631 ◽  
Author(s):  
H. S Kingdon ◽  
R. L Lundblad ◽  
J. J Veltkamp ◽  
D. L Aronson
Keyword(s):  
Human Plasma ◽  
Antithrombin Iii ◽  
Factor Ix ◽  
In Vitro Assays ◽  
Substantial Agreement ◽  
Coefficient Of Correlation

SummaryFactor IX concentrates manufactured from human plasma and intended for therapeutic infusion in man have been suspected for some time of being potentially thrombogenic. In the current studies, assays were carried out in vitro and in vivo for potentially thrombogenic materials. It was possible to rank the various materials tested according to the amount of thrombogenic material detected. For concentrates not containing heparin, there was substantial agreement between the in vivo and in vitro assays, with a coefficient of correlation of 0.77. There was no correlation between the assays for thrombogenicity and the antithrombin III content. We conclude that many presently available concentrates of Factor IX contain substantial amounts of potentially thrombogenic enzymes, and that this fact must be considered in arriving at the decision whether or not to use them therapeutically.

Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo

1995 ◽  
Vol 73 (05) ◽  
pp. 805-811 ◽  
Author(s):  
Yasuo Takahashi ◽  
Yoshitaka Hosaka ◽  
Hiromi Niina ◽  
Katsuaki Nagasawa ◽  
Masaaki Naotsuka ◽  
...  
Keyword(s):  
Human Urine ◽  
Specific Activity ◽  
Anticoagulant Activity ◽  
Rabbit Lung ◽  
Soluble Thrombomodulin ◽  
Molecular Weights ◽  
Dose Dependent Fashion ◽  
Dose Dependent

SummaryWe examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

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