Accelerating the development of genetically engineered cellular therapies: a framework for extrapolating data across related products.

BACKGROUND: Significant advancements have been made in the field of cellular therapy as anti-cancer treatments, with the approval of chimeric antigen receptor (CAR)-T cell therapies and the development of other genetically engineered cellular therapies. CAR-T cell therapies have demonstrated remarkable clinical outcomes in various hematological malignancies, establishing their potential to change the current cancer treatment paradigm. Due to the increasing importance of genetically engineered cellular therapies in the oncology treatment landscape, implementing strategies to expedite development and evidence generation for the next generation of cellular therapy products can have a positive impact on patients. METHODS: We outline a risk-based methodology and assessment aid for the data extrapolation approach across related genetically engineered cellular therapy products. This systematic data extrapolation approach has applicability beyond CAR-T cells and can influence clinical development strategies for a variety of immune therapies such as T cell receptor (TCR) or genetically engineered and other cell-based therapies (e.g., tumor infiltrating lymphocytes, natural killer cells and macrophages). RESULTS: By analyzing commonalities in manufacturing processes, clinical trial designs, and regulatory considerations, key learnings were identified. These insights support optimization of the development and regulatory approval of novel cellular therapies. CONCLUSIONS: The field of cellular therapy holds immense promise in safely and effectively treating cancer. The ability to extrapolate data across related products presents opportunities to streamline the development process and accelerate the delivery of novel therapies to patients.

Pharmacokinetics and pharmacodynamics of the erythropoietin Mimetibody construct CNTO 528 in healthy subjects.

BACKGROUND AND OBJECTIVE: Anaemia is a serious comorbidity that is common in patients with renal failure or cancer. CNTO 528 is the first Mimetibody developed to mimic the effects of erythropoietin (EPO), a hormone that stimulates the production of red blood cells (RBCs). The objective of this study was to develop a pharmacokinetic and pharmacodynamic model for CNTO 528 in healthy male subjects. METHODS: A pharmacokinetic/pharmacodynamic model for CNTO 528 was developed to describe the serum concentration versus time profile and the pharmacological responses of percentage of reticulocytes, total RBC counts and haemoglobin concentration after a single intravenous administration of CNTO 528 at 0.03, 0.09, 0.3 and 0.9 mg/kg in 24 healthy subjects. An open, linear, two-compartment model was used to characterize the pharmacokinetic parameters of CNTO 528. A catenary cell production and lifespan loss model was used to fit the pharmacodynamic data, yielding estimates of drug potency (SC(50)), efficacy (S(max)) and other pharmacodynamic parameters. Bootstrap and posterior predictive checks (PPC) were used to evaluate the model. RESULTS: Administration of CNTO 528 stimulated the production of reticulocytes, RBCs and haemoglobin. CNTO 528 exhibits a half-life of 141 hours, or approximately 5.9 days. The SC(50) was estimated to be 0.37 mg/L, indicating that low serum CNTO 528 concentration was sufficient to produce pharmacological effects. Compared with historical controls, CNTO 528 S(max) appears to be 2-fold higher than recombinant human EPO. Bootstrap analysis and PPCs confirmed the accuracy and precision in the parameter estimates and the adequacy of the model to describe the CNTO 528 pharmacokinetics and pharmacodynamics. CONCLUSION: The mechanistic population model was suitable to characterize the pharmacokinetics and pharmacodynamics of intravenously administered CNTO 528 in healthy subjects. This qualified model is deemed appropriate to conduct clinical trial simulations and to support the decision-making process for dose selection in studies of EPO-stimulating agents.

Population pharmacokinetic modeling of ustekinumab, a human monoclonal antibody targeting IL-12/23p40, in patients with moderate to severe plaque psoriasis.

The population pharmacokinetics of ustekinumab are characterized in patients with moderate to severe plaque psoriasis in 2 Phase 3 studies (PHOENIX 1 and PHOENIX 2). Serum concentration data from 1937 patients are analyzed to determine pharmacokinetic characteristics of ustekinumab and to assess factors that may contribute to their variability. The population typical mean (percentage relative standard error) values for apparent clearance, apparent volume of distribution, and absorption rate constant from the final covariate model are 0.465 L.day(-1) (2.0%), 15.7 L (2.0%), and 0.354 day(-1) (16.2%), respectively. The interindividual variabilities for apparent clearance and apparent volume of distribution are 41.0% and 33.2%, respectively. Of the factors evaluated in this analysis, body weight, diabetes, and positive immune response (antibodies to ustekinumab) are important covariates affecting the apparent clearance and/or apparent volume of distribution of ustekinumab. To fully understand the clinical relevance of these results, the covariate findings need to be evaluated concurrently with the efficacy and safety data.

Ligand-binding assays: risk of using a platform supported by a single vendor.

The use of biological reagents in ligand-binding assays (LBAs) presents inherent challenges when measuring the concentration of large molecules in complex matrices. As a result, there are relatively few platforms that provide the accuracy, precision and robustness needed to determine the concentration of macromolecular therapies and biomarkers, and demonstrate the presence or absence of an immune response. Some bioanalytical laboratories use only one LBA platform to reduce costs, increase efficiency and maintain optimal assay performance. However, the business and regulatory risks of using a single platform supported by only one vendor should be considered. This article summarizes the immunological methods used to support bioanalysis for large molecules that are supported by a single vendor, the benefits of being dedicated to a single platform for bioanalysis used for regulatory filings, the costs associated with restructuring if an immunoassay platform is discontinued and recommendations to mitigate risk when using LBAs in drug development. The experience with the recent discontinuation of the BioVeris™ electrochemiluminescent-based platform is discussed.

A phase I, single and fractionated, ascending-dose study evaluating the safety, pharmacokinetics, pharmacodynamics, and immunogenicity of an erythropoietin mimetic antibody fusion protein (CNTO 528) in healthy male subjects.

The erythropoietin mimetic antibody fusion protein CNTO 528 was developed as a novel antibody fusion protein by constructing an active hematopoietic peptide onto an IgG1-based scaffold. This resulted in a molecule with a long circulating half-life and a prolonged effect of stimulating reticulocyte production and hemoglobin (Hgb) synthesis. To assess the safety, pharmacokinetics, and pharmacodynamics of CNTO 528, the authors gave 44 adult healthy male subjects single or fractionated doses of intravenous CNTO 528 or placebo. CNTO 528 was generally well tolerated. The maximum observed concentration (Cmax) and the area under the concentration versus time curve (AUC) increased in an approximately dose-dependent manner between the 0.09-mg/kg and 0.9-mg/kg doses. The maximum effect on the reticulocyte response occurred approximately 8 to 9 days after administration. A median increase in Hgb (> or =1 g/dL above baseline) was achieved 9 to 10 days after administration, with a maximum effect between 19 and 26 days. Two subjects in the 0.9-mg/kg dose group had elevated Hgb concentrations requiring phlebotomy. In this first-in-human study, CNTO 528 was well tolerated and effective in elevating and maintaining Hgb by at least 1 g/dL following a single intravenous administration, which suggests that an erythropoietin mimetic molecule, such as CNTO 528, may be an effective therapy for patients with anemia.

Vascular gene expression in nonneoplastic and malignant brain.

Malignant gliomas are uniformly lethal tumors whose morbidity is mediated in large part by the angiogenic response of the brain to the invading tumor. This profound angiogenic response leads to aggressive tumor invasion and destruction of surrounding brain tissue as well as blood-brain barrier breakdown and life-threatening cerebral edema. To investigate the molecular mechanisms governing the proliferation of abnormal microvasculature in malignant brain tumor patients, we have undertaken a cell-specific transcriptome analysis from surgically harvested nonneoplastic and tumor-associated endothelial cells. SAGE-derived endothelial cell gene expression patterns from glioma and nonneoplastic brain tissue reveal distinct gene expression patterns and consistent up-regulation of certain glioma endothelial marker genes across patient samples. We define the G-protein-coupled receptor RDC1 as a tumor endothelial marker whose expression is distinctly induced in tumor endothelial cells of both brain and peripheral vasculature. Further, we demonstrate that the glioma-induced gene, PV1, shows expression both restricted to endothelial cells and coincident with endothelial cell tube formation. As PV1 provides a framework for endothelial cell caveolar diaphragms, this protein may serve to enhance glioma-induced disruption of the blood-brain barrier and transendothelial exchange. Additional characterization of this extensive brain endothelial cell gene expression database will provide unique molecular insights into vascular gene expression.

Murine endothelial cell lines as models of tumor endothelial cells.

Identification of appropriate models for in vivo and in vitro preclinical testing of inhibitors of tumor angiogenesis and progression is vital to the successful development of anticancer therapeutics. Although the focus is on human molecular targets, most preclinical in vivo efficacy testing occurs in mice. The goal of the current studies was to identify a murine endothelial cell line to model tumor endothelium for studying the antiangiogenic activity of therapeutic compounds in vitro. In situ hybridization was performed on three s.c. grown syngeneic murine tumors (B16 melanoma, Lewis lung carcinoma, and CT26 colon carcinoma) to assess expression of murine homologs of human tumor endothelial cell markers in the vasculature of these tumor models. Seven murine endothelial cell lines were characterized for expression of the murine homologs of recognized endothelial cell surface markers as well as for tumor endothelial cell surface markers. The seven murine endothelial cell lines had similar generation times and five of the seven lines were able to form tubes on Matrigel. Real-time-PCR and flow cytometry analysis were used to evaluate relative mRNA and protein expression of murine homologs of several recognized endothelial cell surface markers in the seven cell lines. The expression of the mRNA for the murine homologs of five tumor endothelial cell surface markers was also evaluated. The 2H11 cell line expressed all five of the tumor endothelial cell surface markers as well as several well-recognized endothelial cells markers. The 2H11 cell line responds to known and novel antiangiogenic agents by inhibition of proliferation and tube formation. These cells can be used in in vitro angiogenesis assays for evaluating the potential antiangiogenic properties and interspecies cross-reactivity of novel compounds.

Identification of genes expressed in malignant cells that promote invasion.

To systematically identify genes related to invasion a three-dimensional multicellular matrix invasion assay was used to classify human tumor cell lines as stromal invasion positive or stromal invasion negative. Cells from two of the primary cell types of the stromal compartment [endothelial cells (HMVEC) and myofibroblasts (HDF)] were assayed for invasion into tumor cell clusters (breast carcinoma, ovarian carcinoma, prostate carcinoma, lung carcinoma, and melanoma). Four tumor cell lines (MDA-MB231, SKOV-3, A375, and MEL624) scored invasion positive, and four tumor cell lines (LNCaP, DU145, PC3, and A549) scored invasion negative. Serial analysis of gene expression (SAGE) libraries generated from the tumor cell lines were analyzed by GeneSpring Hierarchical clustering, t test, and chi(2) test. Clusters emerged that reflected the behavior in the cell culture assay. Of the 47 most highly differentially expressed genes, 30 were selected for confirmation by real-time PCR, and 9 had good correlation with normalized serial analysis of gene expression tag counts. The strongest correlations were for bone marrow stromal antigen 2, stathmin-like 3, tumor necrosis factor receptor superfamily member 5, and hepatocyte growth factor tyrosine kinase substrate. In situ hybridization of metastatic and nonmetastatic ovarian cancer demonstrated selective expression of bone marrow stromal antigen 2 and tumor necrosis factor receptor superfamily member 5 in the metastatic disease. This combination approach appears to be a powerful tool for identifying genes that may be useful as diagnostic markers and/or as therapeutic targets for invasive solid tumors.

Endothelial precursor cells as a model of tumor endothelium: characterization and comparison with mature endothelial cells.

Human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMVEC) have been the standards for cell-based assays in the field of angiogenesis research and in antiangiogenic drug discovery. These normal mature endothelial cells may not be most representative of human tumor endothelial cells. Human AC133+/CD34+ bone marrow progenitor cells were established in cell culture media containing vascular endothelial growth factor, basic fibroblast growth factor (bFGF), and heparin to drive differentiation toward the endothelial phenotype. The resulting cells designated endothelial precursor cells (EPC) have many of the same functional properties as mature endothelial cells represented by HUVEC and HMVEC. By SAGE analysis, the genes expressed by EPC are more similar to the genes expressed by endothelial cells isolated from fresh surgical specimens of human tumors than are the genes expressed by HUVEC and HMVEC. Analysis of several cell surface markers by flow cytometry showed that EPC, HUVEC, and HMVEC have similar expression of P1H12, vascular endothelial growth factor 2, and endoglin but that EPC have much lower expression of ICAM1, ICAM2, VCAM1, and thrombomodulin than do HUVEC and HMVEC. The EPC generated can form tubes/networks on Matrigel, migrate through porous membranes, and invade through thin layers of Matrigel similarly to HUVEC and HMVEC. However, in a coculture assay using human SKOV3 ovarian cancer cell clusters in collagen as a stimulus for invasion through Matrigel, EPC were able to invade into the malignant cell cluster, whereas HMVEC were not able to invade the malignant cell cluster. In vivo, a Matrigel plug assay where human EPC were suspended in the Matrigel allowed tube/network formation by human EPC to be carried out in a murine host. EPC may be a better model of human tumor endothelial cells than HUVEC and HMVEC and, thus, may provide an improved cell-based model for second generation antineoplastic antiangiogenic drug discovery.

Myofibroblasts enable invasion of endothelial cells into three-dimensional tumor cell clusters: a novel in vitro tumor model.

PURPOSE: In an effort to study the importance of stromal involvement in angiogenesis, we developed a novel, multicellular model that utilizes three of the primary cell types involved in tumor angiogenesis. METHODS: Fluorescently labeled human microvascular endothelial cells (HMVECs), 10T1/2 cells and myofibroblasts were incubated in the presence of a three-dimensional tumor cell cluster resuspended in collagen and embedded in Matrigel. RESULTS: HMVECs cultured in the presence of a human SKOV-3 ovarian carcinoma tumor cell cluster, surrounded the tumor cell cluster, while myofibroblasts invaded the cluster, localizing within the tumor cell mass. In contrast, 10T1/2 cells, a pluripotent mouse mesenchymal cell line with pericyte-like properties, did not demonstrate the same invasive phenotype. HMVECs cultured in the presence of myofibroblasts invaded the tumor cell cluster and colocalized with the myofibroblasts as demonstrated by fluorescent microscopy and immunohistochemistry. The angiogenesis inhibitors SU6668 and paclitaxel inhibited stromal invasion, while a broad-spectrum matrix metalloproteinase inhibitor did not. CONCLUSIONS: This model emphasizes the critical interaction between endothelial cells and myofibroblasts and provides a more complete in vitro model for studying angiogenesis and tumor progression.