MONOCLONAL ANTIBODY-MEDIATED TUMOR TARGETING AND DRUG DELIVERY TO SOLID TUMORS

Antibody-drug conjugates (ADCs) are an emerging class of targeted anticancer therapeutics. Premised on Paul Ehrlich’s magic bullet hypothesis and clinical need to improve the tumor selectivity of anticancer drugs, ADCs marry the tumor homing properties of monoclonal antibodies (mAbs) with the cell-killing properties of cytotoxic agents like chemotherapy. Conceptually, this is achieved by simultaneously improving the therapeutic effects and diminishing the toxic effects of the cytotoxic agent, thereby increasing the therapeutic index compared to standard chemotherapy. Recent FDA-approvals of ADCETRIS® (2011) and KADCYLA® (2013), as well as the FDA breakthrough designation of inotuzumab ozogamicin (2015), inspire confidence in ADCs to continue advancing state-of-the-art cancer treatment. New research in cancer biology influences trends in ADC design. Advances in antibody engineering, linker design, and bioconjugation continue to improve the chemistry, manufacturing, and control of ADCs. Despite hurdles, herculean efforts brought ADCs from a research-phase concept to an FDA-approved clinical treatment in less than three decades. The objective of this dissertation research was to develop an antibody-drug conjugate therapy for solid tumors cancer therapy. Achieving that goal demanded a deep exploration of the many complex yet innately interdependent physical, chemical, and biological barriers that protect our bodies from the daily barrage of pathogens but also shield diseases like cancer, HIV/AIDS, and Alzheimer’s from the drugs we use for treatment. Uptake and exposure of tumor-targeting mAb by solid tumors is influenced by specific molecular properties of the mAb, like antigen affinity, internalization rate, and circulating half-life, the nature of tumor-associated antigen, and the unique tumor pathophysiology and its microenvironment. Intermediate affinity tumor-targeting mAb were discovered to display superior tumor-targeting features than low- or high-affinity mAb to the tumor-associated antigen CD44, a novel cancer stem cell marker, and the epidermal growth factor receptor (EGFR), a hallmark tumor cell biomarker. Pharmaceutical properties of tumor-targeting mAb were attenuated by fragmentation, yielding exceptionally sensitive EGFR tumor imaging agents, and chemical conjugation, enabling tumor-cell specific homing, internalization, and delivery of docetaxel to a solid tumor. In a culminating study, an anti-EGFR mAb-docetaxel bioconjugate demonstrated tumor cell-specific anticancer activity in vitro and durable tumor regression in tumor-bearing mice in vivo.Doctor of Philosoph

Pharmacy Student Engagement, Performance, and Perception in a Flipped Satellite Classroom

Objective. To determine whether “flipping” a traditional basic pharmaceutics course delivered synchronously to 2 satellite campuses would improve student academic performance, engagement, and perception

The Interplay of Antigen Affinity, Internalization, and Pharmacokinetics on CD44-Positive Tumor Targeting of Monoclonal Antibodies

Monoclonal antibodies (mAbs) offer promise as effective tumor targeting and drug delivery agents for cancer therapy. However, comparative biological and clinical characteristics of mAbs targeting the same tumor-associated antigen (TAA) often differ widely. This study examined the characteristics of mAbs that impact tumor targeting using a panel of mAb clones specific to the cancer-associated cell-surface receptor and cancer stem cell marker CD44. CD44 mAbs were screened for cell-surface binding, antigen affinity, internalization, and CD44-mediated tumor uptake by CD44-positive A549 cells. It was hypothesized that high-affinity, rapidly internalizing CD44 mAbs would result in high tumor uptake and prolonged tumor retention. Although high-affinity clones rapidly bound and were internalized by A549 cells <i>in vitro</i>, an intermediate-affinity clone demonstrated significantly greater tumor uptake and retention than high-affinity clones <i>in vivo</i>. Systemic exposure, rather than high antigen affinity or rapid internalization, best associated with tumor targeting of CD44 mAbs in A549 tumor-bearing mice

miRNA cargo in circulating vesicles from neurons is altered in individuals with schizophrenia and associated with severe disease

While RNA expression appears to be altered in several brain disorders, the constraints of postmortem analysis make it impractical for well-powered population studies and biomarker development. Given that the unique molecular composition of neurons are reflected in their extracellular vesicles (EVs), we hypothesized that the fractionation of neuron derived EVs provides an opportunity to specifically profile their encapsulated contents noninvasively from blood. To investigate this hypothesis, we determined miRNA expression in microtubule associated protein 1B (MAP1B)-enriched serum EVs derived from neurons from a large cohort of individuals with schizophrenia and nonpsychiatric comparison participants. We observed dysregulation of miRNA in schizophrenia subjects, in particular those with treatment-resistance and severe cognitive deficits. These data support the hypothesis that schizophrenia is associated with alterations in posttranscriptional regulation of synaptic gene expression and provides an example of the potential utility of tissue-specific EV analysis in brain disorders

Immuno-PET imaging of tumor-infiltrating lymphocytes using zirconium-89 radiolabeled anti-CD3 antibody in immune-competent mice bearing syngeneic tumors.

The ability to non-invasively monitor tumor-infiltrating T cells in vivo could provide a powerful tool to visualize and quantify tumor immune infiltrates. For non-invasive evaluations in vivo, an anti-CD3 mAb was modified with desferrioxamine (DFO) and radiolabeled with zirconium-89 (Zr-89 or 89Zr). Radiolabeled 89Zr-DFO-anti-CD3 was tested for T cell detection using positron emission tomography (PET) in both healthy mice and mice bearing syngeneic bladder cancer BBN975. In vivo PET/CT and ex vivo biodistribution demonstrated preferential accumulation and visualization of tracer in the spleen, thymus, lymph nodes, and bone marrow. In tumor bearing mice, 89Zr-DFO-anti-CD3 demonstrated an 11.5-fold increase in tumor-to-blood signal compared to isotype control. Immunological profiling demonstrated no significant change to total T cell count, but observed CD4+ T cell depletion and CD8+ T cell expansion to the central and effector memory. This was very encouraging since a high CD8+ to CD4+ T cell ratio has already been associated with better patient prognosis. Ultimately, this anti-CD3 mAb allowed for in vivo imaging of homeostatic T cell distribution, and more specifically tumor-infiltrating T cells. Future applications of this radiolabeled mAb against CD3 could include prediction and monitoring of patient response to immunotherapy

Association Of A Polyadenylation Polymorphism In The Serotonin Transporter And Panic Disorder

Background: Genetic markers in the serotonin transporter are associated with panic disorder (PD). The associated polymorphisms do not include the serotonin transporter-linked polymorphic region and display no obvious functional attributes. A common polymorphism (rs3813034) occurs in one of the two reported polyadenylation signals for the serotonin transporter and is in linkage disequilibrium with the PD-associated markers. If functional, rs3813034 might be the risk factor that explains the association of the serotonin transporter and PD. Methods: Quantitative polymerase chain reaction on human brain samples (n ϭ 65) and lymphoblast cultures (n ϭ 71) was used to test rs3813034 for effects on expression of the polyadenylation forms of the serotonin transporter. rs3813034 was also tested for association in a sample of PD cases (n ϭ 307) and a control sample (n ϭ 542) that has similar population structure. Results: The balance of the two polyadenylation forms of the serotonin transporter is associated with rs3813034 in brain (p Ͻ .001) and lymphoblasts (p Ͻ .001). The balance of the polyadenylation forms is also associated with gender in brain only (p Ͻ .05). Association testing of rs3813034 in PD identified a significant association (p ϭ .0068) with a relative risk of 1.56 and 1.81 for the heterozygous and homozygous variant genotypes, respectively. Conclusions: rs3813034 is a functional polymorphism in the serotonin transporter that alters the balance of the two polyadenylation forms of the serotonin transporter. rs3813034 is a putative risk factor for PD and other behavioral disorders that involve dysregulation of serotonergic neurotransmission

Abstract 602: 23ME-00610 is a first-in-class monoclonal antibody that targets the CD200R1 immune checkpoint to enhance T cell-mediated antitumor activity

Abstract In the 23andMe genetic database, associations were observed for CD200R1 variants and autoimmune and cancer traits with opposing directionality. These data suggest that CD200R1 may be a pivotal checkpoint in immunosurveillance of cancer and a promising therapeutic target. CD200R1 is expressed widely on human T cells and myeloid cells; and binds to its cognate ligand CD200 to suppress the activity of immune cells. Elevated expression of CD200R1 was observed on tumor-infiltrating lymphocytes relative to peripheral blood mononuclear cells (PBMCs) from cancer patients. While the expression of CD200R1 and its ligand CD200 are well characterized, the downstream immunosuppressive role for CD200R1 in human tumors or the potentiation of anti-tumor T cell function by targeting of CD200R1 is not well understood. To test the therapeutic potential of inhibiting the CD200R1 pathway we developed 23ME-00610, a fully humanized monoclonal antibody that binds to all functionally relevant isoforms and haplotypes of CD200R1 with high affinity (KD values &amp;lt; 0.1 nM), to target tumors that are reliant on the CD200R1 immunosuppressive pathway. 23ME-00610 completely displaced the ligand CD200 from CD200R1 in cell-based flow cytometry assays with blocking IC50 values that ranged from 0.32 to 1.04 nM depending on the concentration of CD200 ligand present, and inhibited downstream signaling through the adaptor protein DOK2 in a reporter-based assay. Functionally, 23ME-00610 reversed CD200-mediated suppression of chronically stimulated primary T cells. In addition, 23ME-00610 enhanced PBMC-mediated tumor killing with a mean EC50 of 2.09 nM using methods of real-time measurement of cytotoxicity of human CD200-expressing tumor cells when compared to control antibodies. Moreover, 23ME-00610 promoted IFNγ production, a key functional anti-tumor T cell cytokine, in cancer patients’-derived PBMCs. Taken together, these results demonstrate that 23ME-00610 has the potential to reverse CD200-mediated immunosuppression in the TME to enhance anti-tumor T-cell functions in cancer patients. Citation Format: Jill Fenaux, Yao-ming Huang, Cristina Melero, Wei-Jen Chung, Suk Lee, Dina Ayupova, Mauro Poggio, Dylan Glatt, Zuoan Yi, Cecilia Lay, Maike Schmidt, Sophia R. Majeed, Germaine Fuh, Alice Chen, Sushil Kumar. 23ME-00610 is a first-in-class monoclonal antibody that targets the CD200R1 immune checkpoint to enhance T cell-mediated antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 602.</jats:p

Neurotoxicity of an Hepatitis B Virus (HBV) Transcript Inhibitor in 13-Week Rat and Monkey Studies

Abstract The nonclinical safety profile of GS-8873, a hepatitis B virus RNA transcript inhibitor was evaluated in rat and monkey 13-week toxicity studies with 8-week recovery phases. Vehicle or GS-8873 was dosed orally for 13 weeks at 2, 6, 20, and 60 mg/kg/day to Wistar Han rats and at 0.5, 1.5, 3, and 6 mg/kg/day to cynomolgus monkeys. In vitro and in vivo screening results from an analog discovered prior to GS-8873 informed the 13-week toxicology study designs. Neuroelectrophysiology and neurobehavioral evaluations were included at weeks 4 and 13 of the dosing and recovery phases for GS-8873. No adverse neurobehavioral effects were observed. Significant nerve conduction velocity (NCV) decreases and latency increases occurred at the high doses after 4 weeks of dosing. By week 13, dose-responsive NCV reductions and latency increases worsened across all dose groups compared with controls. Some reversal occurred 8 weeks after the last dose administered, but not to vehicle control levels. A minimal, axonal degeneration was observed in rat spinal and peripheral nerves across dose groups compared with controls. No monkey nervous system microscopic findings were observed. No-observed-adverse-effect-levels could not be determined for either species due to the neuroelectrophysiology findings and development was halted in the interest of safety. A retrospective risk assessment approach utilizing benchmark dose (BMD) modeling contributed 13-week NCV BMDL estimates (lower limits of the 95% confidence interval) in lieu of no-observed-adverse-effect-levels. The best-fitted models extrapolated NCV BMDLs for the rat caudal and monkey sural nerve at 0.3 and 0.1 mg/kg/day, respectively.</jats:p