Recommendations for Method Development and Validation of qPCR and dPCR Assays in Support of Cell and Gene Therapy Drug Development.

The emerging use of qPCR and dPCR in regulated bioanalysis and absence of regulatory guidance on assay validations for these platforms has resulted in discussions on lack of harmonization on assay design and appropriate acceptance criteria for these assays. Both qPCR and dPCR are extensively used to answer bioanalytical questions for novel modalities such as cell and gene therapies. Following cross-industry conversations on the lack of information and guidelines for these assays, an American Association of Pharmaceutical Scientists working group was formed to address these gaps by bringing together 37 industry experts from 24 organizations to discuss best practices to gain a better understanding in the industry and facilitate filings to health authorities. Herein, this team provides considerations on assay design, development, and validation testing for PCR assays that are used in cell and gene therapies including (1) biodistribution; (2) transgene expression; (3) viral shedding; (4) and persistence or cellular kinetics of cell therapies.

Preclinical development of ZED8, an 89Zr immuno-PET reagent for monitoring tumor CD8 status in patients undergoing cancer immunotherapy.

BACKGROUND: ZED8 is a novel monovalent antibody labeled with zirconium-89 for the molecular imaging of CD8. This work describes nonclinical studies performed in part to provide rationale for and to inform expectations in the early clinical development of ZED8, such as in the studies outlined in clinical trial registry NCT04029181 [1]. METHODS: Surface plasmon resonance, X-ray crystallography, and flow cytometry were used to characterize the ZED8-CD8 binding interaction, its specificity, and its impact on T cell function. Immuno-PET with ZED8 was assessed in huCD8+ tumor-bearing mice and in non-human primates. Plasma antibody levels were measured by ELISA to determine pharmacokinetic parameters, and OLINDA 1.0 was used to estimate radiation dosimetry from image-derived biodistribution data. RESULTS: ZED8 selectively binds to human CD8α at a binding site approximately 9 Å from that of MHCI making mutual interference unlikely. The equilibrium dissociation constant (KD) is 5 nM. ZED8 binds to cynomolgus CD8 with reduced affinity (66 nM) but it has no measurable affinity for rat or mouse CD8. In a series of lymphoma xenografts, ZED8 imaging was able to identify different CD8 levels concordant with flow cytometry. In cynomolgus monkeys with tool compound 89Zr-aCD8v17, lymph nodes were conspicuous by imaging 24 h post-injection, and the pharmacokinetics suggested a flat-fixed first-in-human dose of 4 mg per subject. The whole-body effective dose for an adult human was estimated to be 0.48 mSv/MBq, comparable to existing 89Zr immuno-PET reagents. CONCLUSION: 89Zr immuno-PET with ZED8 appears to be a promising biomarker of tissue CD8 levels suitable for clinical evaluation in cancer patients eligible for immunotherapy.

Whole-body CD8+ T cell visualization before and during cancer immunotherapy: a phase 1/2 trial.

Immune checkpoint inhibitors (ICIs), by reinvigorating CD8+ T cell mediated immunity, have revolutionized cancer therapy. Yet, the systemic CD8+ T cell distribution, a potential biomarker of ICI response, remains poorly characterized. We assessed safety, imaging dose and timing, pharmacokinetics and immunogenicity of zirconium-89-labeled, CD8-specific, one-armed antibody positron emission tomography tracer 89ZED88082A in patients with solid tumors before and ~30 days after starting ICI therapy (NCT04029181). No tracer-related side effects occurred. Positron emission tomography imaging with 10 mg antibody revealed 89ZED88082A uptake in normal lymphoid tissues, and tumor lesions across the body varying within and between patients two days after tracer injection (n = 38, median patient maximum standard uptake value (SUVmax) 5.2, IQI 4.0-7.4). Higher SUVmax was associated with mismatch repair deficiency and longer overall survival. Uptake was higher in lesions with stromal/inflamed than desert immunophenotype. Tissue radioactivity was localized to areas with immunohistochemically confirmed CD8 expression. Re-imaging patients on treatment showed no change in average (geometric mean) tumor tracer uptake compared to baseline, but individual lesions showed diverse changes independent of tumor response. The imaging data suggest enormous heterogeneity in CD8+ T cell distribution and pharmacodynamics within and between patients. In conclusion, 89ZED88082A can characterize the complex dynamics of CD8+ T cells in the context of ICIs, and may inform immunotherapeutic treatments.

A Fit-for-Purpose Method to Measure Circulating Levels of the mRNA Component of a Liposomal-Formulated Individualized Neoantigen-Specific Therapy for Cancer.

Autogene cevumeran is an individualized neoantigen-specific therapy (iNeST) under development for the treatment of various solid tumors. It consists of an RNA-Lipoplex (RNA-LPX) in which the encapsulated mRNA molecule encodes up to ten neoepitopes identified from each individual patient. In association with major histocompatibility complex (MHC) class I and MHC class II, these neoantigens can potentially stimulate and expand neoantigen-specific CD4+ and CD8+ T cells, leading to antitumor responses. As part of the pharmacokinetic (PK) property assessment of Autogene cevumeran in patients, both the lipid and mRNA content in circulation are measured. This work focused on our efforts to establish a sensitive and robust method for the measurement of mRNA levels of RNA-LPX in plasma. Due to the chemical characteristics of mRNA, extra precautions are required in order to effectively preserve mRNA integrity in human plasma during sample collection, handling and storage. To this end, a number of sample collection tubes and storage conditions were evaluated in order to inform the most optimal and operationally feasible conditions by which to preserve mRNA integrity during sample collection and upon freeze-thaw. PAXgene Blood ccfDNA tubes successfully prevented mRNA degradation and were subsequently selected for patient sample collection in the clinical trial. A branched DNA (bDNA)-based mRNA PK assay was developed to achieve the desired assay performance. Here, we discuss the evaluation of various sample collection and processing conditions as well as the optimization of the work flow during bDNA PK method development.

Clinical Pharmacology Perspectives for Adoptive Cell Therapies in Oncology.

Adoptive cell therapies (ACTs) have shown transformative efficacy in oncology with five US Food and Drug Administration (FDA) approvals for chimeric antigen receptor (CAR) T-cell therapies in hematological malignancies, and promising activity for T cell receptor T-cell therapies in both liquid and solid tumors. Clinical pharmacology can play a pivotal role in optimizing ACTs, aided by modeling and simulation toolboxes and deep understanding of the underlying biological and immunological processes. Close collaboration and multilevel data integration across functions, including chemistry, manufacturing, and control, biomarkers, bioanalytical, and clinical science and safety teams will be critical to ACT development. As ACT is comprised of alive, polyfunctional, and heterogeneous immune cells, its overall physicochemical and pharmacological property is vastly different from other platforms/modalities, such as small molecule and protein therapeutics. In this review, we first describe the unique kinetics of T cells and the appropriate bioanalytical strategies to characterize cellular kinetics. We then assess the distinct aspects of clinical pharmacology for ACTs in comparison to traditional small molecule and protein therapeutics. Additionally, we provide a review for the five FDA-approved CAR T-cell therapies and summarize their properties, cellular kinetic characteristics, dose-exposure-response relationship, and potential baseline factors/variables in product, patient, and regimen that may affect the safety and efficacy. Finally, we probe into existing empirical and mechanistic quantitative techniques to understand how various modeling and simulation approaches can support clinical pharmacology strategy and propose key considerations to be incorporated and explored in future models.

Treating Tissue Factor-Positive Cancers with Antibody-Drug Conjugates That Do Not Affect Blood Clotting.

The primary function of tissue factor (TF) resides in the vasculature as a cofactor of blood clotting; however, multiple solid tumors aberrantly express this transmembrane receptor on the cell surface. Here, we developed anti-TF antibody-drug conjugates (ADC) that did not interfere with the coagulation cascade and benchmarked them against previously developed anti-TF ADCs. After screening an affinity-matured antibody panel of diverse paratopes and affinities, we identified one primary paratope family that did not inhibit conversion of Factor X (FX) to activated Factor X (FXa) and did not affect conversion of prothrombin to thrombin. The rest of the antibody panel and previously developed anti-TF antibodies were found to perturb coagulation to varying degrees. To compare the anticancer activity of coagulation-inert and -inhibitory antibodies as ADCs, a selection of antibodies was conjugated to the prototypic cytotoxic agent monomethyl auristatin E (MMAE) through a protease-cleavable linker. The coagulation-inert and -inhibitory anti-TF ADCs both killed cancer cells effectively. Importantly, the coagulation-inert ADCs were as efficacious as tisotumab vedotin, a clinical stage ADC that affected blood clotting, including in patient-derived xenografts from three solid tumor indications with a need for new therapeutic treatments-squamous cell carcinoma of the head and neck (SCCHN), ovarian, and gastric adenocarcinoma. Furthermore, a subset of the anti-TF antibodies could also be considered for the treatment of other diseases associated with upregulation of membranous TF expression, such as macular degeneration. Mol Cancer Ther; 17(11); 2412-26. ©2018 AACR.

The double-histone-acetyltransferase complex ATAC is essential for mammalian development.

Acetylation of the histone tails, catalyzed by histone acetyltransferases (HATs), is a well-studied process that contributes to transcriptionally active chromatin states. Here we report the characterization of a novel mammalian HAT complex, which contains the two acetyltransferases GCN5 and ATAC2 as well as other proteins linked to chromatin metabolism. This multisubunit complex has a similar but distinct subunit composition to that of the Drosophila ADA2A-containing complex (ATAC). Recombinant ATAC2 has a weak HAT activity directed to histone H4. Moreover, depletion of ATAC2 results in the disassembly of the complex, indicating that ATAC2 not only carries out an enzymatic function but also plays an architectural role in the stability of mammalian ATAC. By targeted disruption of the Atac2 locus in mice, we demonstrate for the first time the essential role of the ATAC complex in mammalian development, histone acetylation, cell cycle progression, and prevention of apoptosis during embryogenesis.

SAGA-mediated H2B deubiquitination controls the development of neuronal connectivity in the Drosophila visual system.

Nonstop, which has previously been shown to have homology to ubiquitin proteases, is required for proper termination of axons R1-R6 in the optic lobe of the developing Drosophila eye. Herein, we establish that Nonstop actually functions as an ubiquitin protease to control the levels of ubiquitinated histone H2B in flies. We further establish that Nonstop is the functional homolog of yeast Ubp8, and can substitute for Ubp8 function in yeast cells. In yeast, Ubp8 activity requires Sgf11. We show that in Drosophila, loss of Sgf11 function causes similar photoreceptor axon-targeting defects as loss of Nonstop. Ubp8 and Sgf11 are components of the yeast SAGA complex, suggesting that Nonstop function might be mediated through the Drosophila SAGA complex. Indeed, we find that Nonstop does associate with SAGA components in flies, and mutants in other SAGA subunits display nonstop phenotypes, indicating that SAGA complex is required for accurate axon guidance in the optic lobe. Candidate genes regulated by SAGA that may be required for correct axon targeting were identified by microarray analysis of gene expression in SAGA mutants.

The essential gene wda encodes a WD40 repeat subunit of Drosophila SAGA required for histone H3 acetylation.

Histone acetylation provides a switch between transcriptionally repressive and permissive chromatin. By regulating the chromatin structure at specific promoters, histone acetyltransferases (HATs) carry out important functions during differentiation and development of higher eukaryotes. HAT complexes are present in organisms as diverse as Saccharomyces cerevisiae, humans, and flies. For example, the well-studied yeast SAGA is related to three mammalian complexes. We previously identified Drosophila melanogaster orthologues of yeast SAGA components Ada2, Ada3, Spt3, and Tra1 and demonstrated that they associate with dGcn5 in a high-molecular-weight complex. To better understand the function of Drosophila SAGA (dSAGA), we sought to affinity purify and characterize this complex in more detail. A proteomic approach led to the identification of an orthologue of the yeast protein Ada1 and the novel protein encoded by CG4448, referred to as WDA (will decrease acetylation). Embryos lacking both alleles of the wda gene exhibited reduced levels of histone H3 acetylation and could not develop into adult flies. Our results point to a critical function of dSAGA and histone acetylation during Drosophila development.

Host cell factor and an uncharacterized SANT domain protein are stable components of ATAC, a novel dAda2A/dGcn5-containing histone acetyltransferase complex in Drosophila.

Gcn5 is a conserved histone acetyltransferase (HAT) found in a number of multisubunit complexes from Saccharomyces cerevisiae, mammals, and flies. We previously identified Drosophila melanogaster homologues of the yeast proteins Ada2, Ada3, Spt3, and Tra1 and showed that they associate with dGcn5 to form at least two distinct HAT complexes. There are two different Ada2 homologues in Drosophila named dAda2A and dAda2B. dAda2B functions within the Drosophila version of the SAGA complex (dSAGA). To gain insight into dAda2A function, we sought to identify novel components of the complex containing this protein, ATAC (Ada two A containing) complex. Affinity purification and mass spectrometry revealed that, in addition to dAda3 and dGcn5, host cell factor (dHCF) and a novel SANT domain protein, named Atac1 (ATAC component 1), copurify with this complex. Coimmunoprecipitation experiments confirmed that these proteins associate with dGcn5 and dAda2A, but not with dSAGA-specific components such as dAda2B and dSpt3. Biochemical fractionation revealed that ATAC has an apparent molecular mass of 700 kDa and contains dAda2A, dGcn5, dAda3, dHCF, and Atac1 as stable subunits. Thus, ATAC represents a novel histone acetyltransferase complex that is distinct from previously purified Gcn5/Pcaf-containing complexes from yeast and mammalian cells.

Two Drosophila Ada2 homologues function in different multiprotein complexes.

The reversible acetylation of the N-terminal tails of histones is crucial for transcription, DNA repair, and replication. The enzymatic reaction is catalyzed by large multiprotein complexes, of which the best characterized are the Gcn5-containing N-acetyltransferase (GNAT) complexes. GNAT complexes from yeast to humans share several conserved subunits, such as Ada2, Ada3, Spt3, and Tra1/TRRAP. We have characterized these factors in Drosophila and found that the flies have two distinct Ada2 variants (dAda2a and dAda2b). Using a combination of biochemical and cell biological approaches we demonstrate that only one of the two Drosophila Ada2 homologues, dAda2b, is a component of Spt-Ada-Gcn5-acetyltransferase (SAGA) complexes. The other Ada2 variant, dAda2a, can associate with dGcn5 but is not incorporated into dSAGA-type complexes. This is the first example of a complex-specific association of the Ada-type transcriptional adapter proteins with GNATs. In addition, dAda2a is part of Gcn5-independent complexes, which are concentrated at transcriptionally active regions on polytene chromosomes. This implicates novel functions for dAda2a in transcription. Humans and mice also possess two Ada2 variants with high homology to dAda2a and dAda2b, respectively. This suggests that the mammalian and fly homologues of the transcriptional adapter Ada2 form two functionally distinct subgroups with unique characteristics.