Treatment Of Cynomolgus Monkeys Research Articles

A long-lived IL-2 mutein that selectively activates and expands regulatory T cells as a therapy for autoimmune disease

Susceptibility to multiple autoimmune diseases is associated with common gene polymorphisms influencing IL-2 signaling and Treg function, making Treg-specific expansion by IL-2 a compelling therapeutic approach to treatment. As an in vivo IL-2 half-life enhancer we used a non-targeted, effector-function-silent human IgG1 as a fusion protein. An IL-2 mutein (N88D) with reduced binding to the intermediate affinity IL-2Rβγ receptor was engineered with a stoichiometry of two IL-2N88D molecules per IgG, i.e. IgG-(IL-2N88D)2. The reduced affinity of IgG-(IL-2N88D)2 for the IL-2Rβγ receptor resulted in a Treg-selective molecule in human whole blood pSTAT5 assays. Treatment of cynomolgus monkeys with single low doses of IgG-(IL-2N88D)2 induced sustained preferential activation of Tregs accompanied by a corresponding 10–14-fold increase in CD4+ and CD8+ CD25+FOXP3+ Tregs; conditions that had no effect on CD4+ or CD8+ memory effector T cells. The expanded cynomolgus Tregs had demethylated FOXP3 and CTLA4 epigenetic signatures characteristic of functionally suppressive cells. Humanized mice had similar selective in vivo responses; IgG-(IL-2N88D)2 increased Tregs while wild-type IgG-IL-2 increased NK cells in addition to Tregs. The expanded human Tregs had demethylated FOXP3 and CTLA4 signatures and were immunosuppressive. These results describe a next-generation immunotherapy using a long-lived and Treg-selective IL-2 that activates and expands functional Tregsin vivo. Patients should benefit from restored immune homeostasis in a personalized fashion to the extent that their autoimmune disease condition dictates opening up the possibility for remissions and cures.

Artificial Liver and Renal Support System for Cynomolgus Monkeys with Surgery-Induced Acute Renal Failure: A Preclinical Study.

Renal dysfunction is one of the most common complications of liver cirrhosis and is associated with increased morbidity and mortality. However, no available technology can simultaneously support liver and renal function in these patients. The aim of this study was to evaluate the safety and efficacy of an artificial liver and renal support system in cynomolgus monkeys with surgery-induced ARF. The ARF model was established by ligature of bilateral renal arteries in eight cynomolgus monkeys, which were randomly divided into a treatment group (n = 4) and control group (n = 4). Biochemical indexes were determined before and after surgery. Blood endotoxin levels, biochemical indexes, and bacterial cultures were assessed at 0, 3, and 6 h during treatment. System pressures and vital signs were recorded at 1 h intervals. Pathological examination was performed after death. ARF was successfully established, based on significant elevation of biochemical indexes and pathological examination. The treatment group had significantly reduced biochemical indexes relative to the control group. Measurement of blood endotoxins and aerobic and anaerobic bacteria cultures indicated no bacterial growth. The system pressures and vital signs were stable during treatment. The results indicate that our support system for the treatment of cynomolgus monkeys with surgery-induced acute renal failure is safe and effective.

Abstract 2975: Development of a probody drug conjugate (PDC) targeting CD71 for the treatment of solid tumors and lymphomas

Abstract The targets of Antibody Drug Conjugates (ADCs) have typically been selected by identifying transmembrane antigens that are highly expressed in tumors but are low or absent in normal tissues. The number of potential ADC targets meeting these requirements is limited, either because expression in tumors is not high enough for optimal efficacy, or because expression in normal tissues is too high, leading to toxicity. Probody (TM) therapeutics are antibody prodrugs designed to remain inactive until proteolytically activated in the tumor microenvironment. Probody technology therefore has the potential to enable targeting of more desirable tumor antigens with higher, more persistent and more homogeneous expression in tumors, while limiting toxicity due to interaction with these antigens in normal tissues. CD71 (transferrin receptor) is an example of a highly desirable ADC target, because of its well-characterized ability to efficiently internalize and deliver an ADC payload intracellularly. Further, CD71 is expressed at homogeneously high levels (3+ expression by IHC) in almost all tumor types, including in metastatic disease. However, because CD71 is also expressed on multiple normal cell types, including many progenitor hematological cells, we reasoned that a CD71-targeted ADC would be challenging to develop. To enable targeting of CD71, we have developed an anti-CD71 Probody Drug Conjugate (PDC) CX-2005, which can be activated by multiple proteases in the tumor microenvironment, but which remains in a relatively inactive form while in circulation and in normal tissues. CX-2005 produces complete tumor regressions at therapeutic doses in mouse models of lymphoma, breast cancer and lung cancer. Consistent with our hypothesis that it would be difficult to develop an anti-CD71 ADC, treatment of cynomolgus monkeys with an anti-CD71 ADC at doses that were efficacious in mouse tumor models caused life-threatening depletion of CD71-expressing hematopoietic cells, including neutrophils, lymphocytes and RBCs. In contrast, these toxicities were not observed in monkeys treated with the same dose of the anti-CD71 PDC, consistent with the Probody therapeutic avoiding interaction with these normal cells. Our data demonstrate that, in preclinical studies, Probody drug conjugates can safely and effectively target attractive tumor antigens like CD71 which have been difficult to effectively approach with traditional ADCs due to their expression on critical normal tissues. Further, our data support the development of Probody therapeutics directed against CD71 in multiple different cancers. PROBODY is a trademark of CytomX Therapeutics, Inc. Citation Format: Shweta Singh, Amy DuPage, Annie Yang Weaver, Jason Sagert, Clayton White, Michael Krimm, Yuanhui Huang, Linnea Diep, Kim Tipton, Shouchun Liu, Jennifer Richardson, W. Michael Kavanaugh, Jonathan A. Terrett, Luc R. Desnoyers. Development of a probody drug conjugate (PDC) targeting CD71 for the treatment of solid tumors and lymphomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2975.

A Novel Bispecific Antibody Targeting EGFR and cMet Is Effective against EGFR Inhibitor-Resistant Lung Tumors.

Non-small cell lung cancers (NSCLC) with activating EGFR mutations become resistant to tyrosine kinase inhibitors (TKI), often through second-site mutations in EGFR (T790M) and/or activation of the cMet pathway. We engineered a bispecific EGFR-cMet antibody (JNJ-61186372) with multiple mechanisms of action to inhibit primary/secondary EGFR mutations and the cMet pathway. JNJ-61186372 blocked ligand-induced phosphorylation of EGFR and cMet and inhibited phospho-ERK and phospho-AKT more potently than the combination of single receptor-binding antibodies. In NSCLC tumor models driven by EGFR and/or cMet, JNJ-61186372 treatment resulted in tumor regression through inhibition of signaling/receptor downmodulation and Fc-driven effector interactions. Complete and durable regression of human lung xenograft tumors was observed with the combination of JNJ-61186372 and a third-generation EGFR TKI. Interestingly, treatment of cynomolgus monkeys with JNJ-61186372 resulted in no major toxicities, including absence of skin rash observed with other EGFR-directed agents. These results highlight the differentiated potential of JNJ-61186372 to inhibit the spectrum of mutations driving EGFR TKI resistance in NSCLC. Cancer Res; 76(13); 3942-53. ©2016 AACR.

Pharmacologic profile of the Adnectin BMS-962476, a small protein biologic alternative to PCSK9 antibodies for low-density lipoprotein lowering.

Proprotein convertase subtilisin kexin-9 (PCSK9) is an important pharmacological target for decreasing low-density lipoprotein (LDL) in cardiovascular disease, although seemingly inaccessible to small molecule approaches. Compared with therapeutic IgG antibodies currently in development, targeting circulating PCSK9 with smaller molecular scaffolds could offer different profiles and reduced dose burdens. This inspired genesis of PCSK9-binding Adnectins, a protein family derived from human fibronectin-10th-type III-domain and engineered for high-affinity target binding. BMS-962476, an ∼11-kDa polypeptide conjugated to polyethylene glycol to enhance pharmacokinetics, binds with subnanomolar affinity to human. The X-ray cocrystal structure of PCSK9 with a progenitor Adnectin shows ∼910 Å(2) of PCSK9 surface covered next to the LDL receptor binding site, largely by residues of a single loop of the Adnectin. In hypercholesterolemic, overexpressing human PCSK9 transgenic mice, BMS-962476 rapidly lowered cholesterol and free PCSK9 levels. In genomic transgenic mice, BMS-962476 potently reduced free human PCSK9 (ED50 ∼0.01 mg/kg) followed by ∼2-fold increases in total PCSK9 before return to baseline. Treatment of cynomolgus monkeys with BMS-962476 rapidly suppressed free PCSK9 >99% and LDL-cholesterol ∼55% with subsequent 6-fold increase in total PCSK9, suggesting reduced clearance of circulating complex. Liver sterol response genes were consequently downregulated, following which LDL and total PCSK9 returned to baseline. These studies highlight the rapid dynamics of PCSK9 control over LDL and liver cholesterol metabolism and characterize BMS-962476 as a potent and efficacious PCSK9 inhibitor.

Preclinical Safety of Baxter's Recombinant ADAMTS13

Abstract 3381Baxter is currently developing a recombinant ADAMTS13 (rADAMTS13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) product for treatment, and routine prophylaxis of acute episodes of thrombotic thrombocytopenic purpura (TTP) in patients with ADAMTS13 deficiency. TTP is a rare, life-threatening microvascular disease characterized by single or recurrent episodes of thrombocytopenia, microangiopathic haemolytic anaemia and widespread microvascular thrombosis, which leads to the ischemic damage of multiple organs.To characterize the safety and the no observed adverse effect level (NOAEL) of Baxter's rADAMTS13, a 1-month repeat dose toxicity study in rats, an escalating dose and pilot 4-week repeat dose toxicity study in cynomolgus monkeys, and a 1-month repeat dose toxicity study including cardiovascular/pulmonary safety pharmacology in cynomolgus monkeys was conducted. Species suitability was shown by demonstrating that human rADAMTS13 can cleave monkey and rat VWF in vitro and in vivo under native conditions to present a worst case scenario for repeat dose toxicity studies. A local tolerance study was conducted in rabbits.Baxter's rADAMTS13 was well tolerated in rats at doses of 80, 400 and 800 FRETS-U/kg administered every third day for one month. Thus, the NOAEL in rats was the highest dose of 800 FRETS-U/kg. In cynomolgus monkeys, no adverse effects were detected at 800 FRETS-U/kg after intravenous treatment with rADAMTS13 once a week for 5 weeks (pilot repeat dose study). The NOAEL for the escalating dose phase of the study was 1790 FRETS-U/kg. Treatment of cynomolgus monkeys with bolus injection of Baxter's rADAMTS13 at doses of 80, 200, and 400 FRETS-U/kg once a week for 29 days did not reveal adverse findings. Thus, the NOAEL for this study was the highest dose of 400 FRETS-U/kg. As expected for a heterologous human protein drug, repeat doses of rADAMTS13 resulted in the formation of anti-drug antibodies specific for human ADAMTS13 and in neutralising human ADAMTS13 activity in animal models. No injection site reactions were observed in animals of either species. rADAMTS13 was well tolerated after intravenous (intended clinical administration route), intraarterial and paravenous administration in rabbits.In conclusion, Baxter's rADAMTS13 was well tolerated in rats, monkeys and rabbits, with no toxicity observed even at the highest dose levels. Disclosures:Piskernik:Baxter Innovations GmbH: Employment. Dietrich:Baxter Innovations GmbH: Employment. Kubik:Baxter Innovations GmbH: Employment. Ruthsatz:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment.

Preclinical Immunogenicity Assessment of Baxter‘s Longer-Acting FVIII Candidate BAX 855 Using Novel Preclinical Models,

Abstract 3323Baxter is developing a PEGylated recombinant human factor VIII conjugate (BAX 855) based on the modification of full-length FVIII with polyethylene glycol (PEG). The FVIII molecule is derived from a CHO cell line using a plasma-protein-free method.Any chemical modification of FVIII might alter its immunogenic potential. Therefore, careful preclinical immunogenicity studies of PEGylated FVIII in comparison to unmodified FVIII are important. We evaluated the immunogenicity of BAX 855 in comparison to ADVATE, Baxter‘s unmodified recombinant full-length FVIII concentrate. For this purpose, we used different in vitro and in vivo models to s assess the potential impact of BAX 855 and ADVATE on both the innate and the adaptive immune system.The assessment of the potential impact of BAX 855 and ADVATE on the human innate immune system was based on their potential to induce the release of pro- inflammatory cytokines (IL-1β, IL-6, IL-8 and TNF-α) in an in vitro human whole blood assay and on its potential to activate the human complement system in human plasma in vitro.The assessment of the potential impact of BAX 855 and ADVATE on the adaptive immune system was based on their potential to induce antibodies in 3 different hemophilic mouse models and in cynomolgous monkeys. The hemophilic moue models included a hemophilic mouse model with a knockout of the murine FVIII that express a human F8 cDNA as a transgene, a hemophilic mouse model that expresses the human MHC-class II protein HLA-DRB1*1501 on the background of a knockout of the murine MHC-class II complex and a conventional hemophilic mouse model with a knockout of the murine FVIII gene. Hemophilic mice that express a human F8 cDNA as a transgene are immunologically tolerant to human FVIII and develop antibodies against human FVIII only if the immune tolerance breaks down. Using this model, we asked if BAX 855 is able to maintain immune tolerance to human FVIII. The hemophilic mouse models that expresses the human MHC-class II protein HLA-DRB1*1501 mimics the situation in an important fraction of hemophilia A patients with FVIII inhibitors. The human MHC-class II haplotype HLA-DRB1*1501 was previously shown to be associated with an increased risk for patients to develop FVIII inhibitors. Using this model, we asked if BAX 855 expresses an immunogenicity profile similar to ADVATE.BAX 855 and ADAVTE induced similar low levels of cytokine release (IL-1β, IL-6, IL-8 and TNF-α) that were similar to the cytokine release observed in the buffer control group after incubation with human whole blood in vitro. In addition, BAX 855 and ADVATE induced similar low levels of complement activation that were not different from the buffer control group after incubation with human plasma in vitro.Importantly, BAX 855 and ADVATE induced similar levels and incidences of antibodies against FVIII and against PEG-FVIII in all mouse models. In addition, immune tolerance to FVIII was maintained in hemophilic mice expressing the human F8 cDNA. Finally, no major differences in antibody titers were seen after treatment of cynomolgus monkeys with BAX 855 and ADVATE.We conclude that results obtained in comparative immunogenicity studies in vitro and in vivo demonstrate that BAX 855 and ADVATE have a similar immunogenicity profile. Therefore, we expect that BAX 855 will express a similar safety profile as ADVATE in patients. Disclosures:Horling:Baxter Innovations GmbH: Employment. Schwele:Baxter Innovations GmbH: Employment. Lubich:Baxter BioScience: Employment. Ahmad:Baxter Innovations GmbH: Employment. Weiller:Baxter BioScience: Employment. Spatzenegger:Baxter Innovation GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Reipert:Baxter Innovations GmbH: Employment.

Identification of gene expression effects by DNA microarrays after treatment of Cynomolgus monkeys with a new candidate drug. An activity of Melius project

Identification of gene expression effects by DNA microarrays after treatment of Cynomolgus monkeys with a new candidate drug. An activity of Melius project

Reduction of IgG in nonhuman primates by a peptide antagonist of the neonatal Fc receptor FcRn

The neonatal Fc receptor FcRn provides IgG molecules with their characteristically long half-lives in vivo by protecting them from intracellular catabolism and then returning them to the extracellular space. Other investigators have demonstrated that mice lacking FcRn are protected from induction of various autoimmune diseases, presumably because of the accelerated catabolism of pathogenic IgGs in the animals. Therefore, targeting FcRn with a specific inhibitor may represent a unique approach for the treatment of autoimmune disease or other diseases where the reduction of pathogenic IgG will have a therapeutic benefit. Using phage display peptide libraries, we screened for ligands that bound to human FcRn (hFcRn) and discovered a consensus peptide sequence that binds to hFcRn and inhibits the binding of human IgG (hIgG) in vitro. Chemical optimization of the phage-identified sequences yielded the 26-amino acid peptide dimer SYN1436, which is capable of potent in vitro inhibition of the hIgG-hFcRn interaction. Administration of SYN1436 to mice transgenic for hFcRn induced an increase in the rate of catabolism of hIgG in a dose-dependent manner. Treatment of cynomolgus monkeys with SYN1436 led to a reduction of IgG by up to 80% without reducing serum albumin levels that also binds to FcRn. SYN1436 and related peptides thus represent a previously uncharacterized family of potential therapeutic agents for the treatment of humorally mediated autoimmune and other diseases.

A Soluble BAFF Antagonist, BR3-Fc, Decreases Peripheral Blood B Cells and Lymphoid Tissue Marginal Zone and Follicular B Cells in Cynomolgus Monkeys

BAFF (also known as BLyS), a member of the tumor necrosis factor superfamily, plays a critical role in the maturation and development of B cells. BAFF has three receptors on B cells, the most crucial of which is BR3. In this study, we demonstrate the biological outcome of BAFF blockade in cynomolgus monkeys using a soluble fusion protein consisting of human BR3 and human IgG1 Fc. In vitro, BR3-Fc blocked BAFF-mediated survival and proliferation of cynomolgus monkey B cells. Weekly treatment of cynomolgus monkeys with BR3-Fc for 13 to 18 weeks resulted in significant B-cell reduction in the peripheral blood and in lymphoid organs. CD21(high) B cells in lymphoid tissues, a subset analogous to human marginal zone B cells, expressed nearly twofold higher BR3 levels than did CD21(med) B cells. Lymphoid tissue flow cytometric analysis showed that BR3-Fc reduced this CD21(high) B-cell subset to a greater extent than it reduced CD21(med) B cells. Dual-label immunohistochemistry and morphometric image analysis supported these results by demonstrating that BR3-Fc reduced a significant proportion of the B cells within the splenic inner and outer marginal zones. These findings should prove very useful in guiding the desired therapeutic use of BR3-Fc for autoimmune diseases in the clinic.

Enhancement of trastuzumab-mediated cellular cytotoxicity by interleukin-21

2567 Background: Interleukin-21, a cytokine produced by activated T cells, has potent effects on growth, survival and activation status of T, B and natural killer (NK) cells. In vitro studies show increased NK cytolytic function following combined human recombinant (rIL-21) stimulation and Fc receptor engagement. We studied the effect of rIL-21 on antibody-dependent cellular cytotoxicity (ADCC) against trastuzumab-coated breast cancer targets. Methods: ADCC assays were performed on peripheral blood mononuclear cell preparations (MNCs) from cynomolgus monkeys and on enriched human NK cells, following in vitro treatment with rIL-21. Cynomolgus monkeys were treated with rIL-21 as part of an in vivo model of ADCC (Hughes, et al., ASCO submitted), and the ex vivo ADCC activity of MNCs from these animals was tested. Whole blood and MNCs were characterized by flow cytometry. Results: Human NK cells incubated with rIL-21 had 2 to 4-fold increased ADCC against breast cancer targets including Her2/neu-low lines MCF7 and HCC38 (p<.02). Cynomolgus MNCs cultured with rIL-21 showed variable ADCC activation. In animals treated with rIL-21, an immediate decline in ADCC activity correlated with the extravasation of NK cells from the peripheral blood. This was followed by a substantial increase in ADCC against BT-474 cells at later timepoints. Normalization for NK cell numbers showed increased lytic efficiency of NK cells, especially after two cycles of treatment. The percentage of perforin-positive MNCs increased following treatment, as did other biomarkers of immune activation including FcγRI and FcγRIII expression. Conclusion: Treatment of cynomolgus monkeys with rIL-21 increased the activity of MNCs in trastuzumab-mediated ex vivo ADCC assays. This lytic activation is consistent with the enhanced effector function observed after in vitro incubation of NK cells or MNCs with rIL-21. Thus, adjuvant treatment with rIL-21 may enhance monoclonal antibody therapy by improving ADCC. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration ZymoGenetics ZymoGenetics

Aztreonam decreases hepatic microsomal cytochrome P450 in cynomolgus monkeys.

We examined the effect of successive administrations of aztreonam, which is used clinically as an antibiotic, on the mixed function oxidase system in non-human primates. Treatment of cynomolgus monkeys with aztreonam at doses of between 40 and 300 mg/kg for 4 weeks resulted in a significant decrease in the content of hepatic microsomal P450. On the other hand, no significant change was observed in hepatic cytochrome b5 content and NADPH-cytochrome c (P450) reductase activity following treatment with aztreonam. The activity of testosterone 6 beta-hydroxylase, but not 2 beta- and 16 alpha-hydroxylases in hepatic microsomes, was decreased following the treatment of cynomolgus monkeys with aztreonam. The content of P450 CMLc, which is classified into the 3A subfamily, was also decreased by aztreonam treatment although the content of P450 CMLb, which is a 2A enzyme in cynomolgus monkeys, was unchanged. From these results, we concluded that aztreonam decreased P450 CMLc in hepatic microsomes of cynomolgus monkeys.