Systematic preclinical evaluation of CD33-directed chimeric antigen receptor T cell immunotherapy for acute myeloid leukemia defines optimized construct design.

BACKGROUND: Successful development of chimeric antigen receptor (CAR) T cell immunotherapy for children and adults with relapsed/refractory acute myeloid leukemia (AML) is highly desired given their poor clinical prognosis and frequent inability to achieve cure with conventional chemotherapy. Initial experiences with CD19 CAR T cell immunotherapy for patients with B-cell malignancies highlighted the critical impact of intracellular costimulatory domain selection (CD28 vs 4-1BB (CD137)) on CAR T cell expansion and in vivo persistence that may impact clinical outcomes. However, the impact of costimulatory domains on the efficacy of myeloid antigen-directed CAR T cell immunotherapy remains unknown. METHODS: In this preclinical study, we developed six CAR constructs targeting CD33, a highly expressed and validated AML target, comprised of one of three single-chain variable fragments with CD3ζ and either CD28 or 4-1BB costimulatory domains. We systematically compared the preclinical in vitro and in vivo efficacy of T cells lentivirally transduced with CD33 CAR constructs (CD33CARTs) against human AML. RESULTS: We observed potent in vitro cytokine production and cytotoxicity of CD33CARTs incubated with human CD33+ AML cell lines, as well as robust in vivo antileukemia activity in cell line and childhood AML patient-derived xenograft (PDX) models. Gemtuzumab-based CD33CARTs were unexpectedly toxic in vivo in animal models despite observed in vitro anti-leukemia activity. CD28-based CD33CARTs consistently induced more robust inhibition of leukemia proliferation in AML cell line and PDX models than did 4-1BB-based CD33CARTs. A 'best-in-class' lintuzumab-CD28/CD3ζ CAR construct was thus selected for clinical translation. CONCLUSIONS: CD33 is a critical antigen for potential immunotherapeutic targeting in patients with AML. Based on this rigorous preclinical evaluation, our validated clinical grade lintuzumab-CD28/CD3ζ CD33CART immunotherapy is now under evaluation in a first-in-child/first-in-human phase 1 clinical trial for children and adolescents/young adults with relapsed/refractory AML. TRIAL REGISTRATION NUMBER: clinicaltrials.gov; NCT03971799.

Safety and Stability of Antibody-Dye Conjugate in Optical Molecular Imaging.

PURPOSE: The development of molecularly targeted tracers is likely to improve the accuracy of diagnostic, screening, and therapeutic tools. Despite the many therapeutic antibodies that are FDA-approved with known toxicity, only a limited number of antibody-dye conjugates have been introduced to the clinic. Thorough evaluation of the safety, stability, and pharmacokinetics of antibody conjugates in the clinical setting compared with their parental components could accelerate the clinical approval of antibodies as agents for molecular imaging. Here we investigate the safety and stability of a near-infrared fluorescent dye (IRDye800CW) conjugated panitumumab, an approved therapeutic antibody, and report on the product stability, pharmacokinetics, adverse events, and QTc interval changes in patients. PROCEDURES: Panitumumab-IRDye800CW was made under good manufacturing practice (GMP) conditions in a single batch on March 26, 2014, and then evaluated over 4.5 years at 0, 3, and 6 months, and then at 6-month intervals thereafter. We conducted early phase trials in head and neck, lung, pancreas, and brain cancers with panitumumab-IRDye800CW. Eighty-one patients scheduled to undergo standard-of-care surgery were infused with doses between 0.06 to 2.83 mg/kg of antibody. Patient ECGs, blood samples, and adverse events were collected over 30-day post-infusion for analysis. RESULTS: Eighty-one patients underwent infusion of the study drug at a range of doses. Six patients (7.4 %) experienced an adverse event that was considered potentially related to the drug. The most common event was a prolonged QTc interval which occurred in three patients (3.7 %). Panitumumab-IRDye800CW had two OOS results at 42 and 54 months while meeting all other stability testing criteria. CONCLUSIONS: Panitumumab-IRDye800CW was safe and stable to administer over a 54-month window with a low rate of adverse events (7.4 %) which is consistent with the rate associated with panitumumab alone. This data supports re-purposing therapeutic antibodies as diagnostic imaging agents with limited preclinical toxicology studies.

Mammalian cell transient expression, non-affinity purification, and characterization of human recombinant IGFBP7, an IGF-1 targeting therapeutic protein.

Targeted inhibiting insulin-like growth factor 1 is an effective approach for cancer therapy. Insulin-like growth factor binding protein 7 (IGFBP7) is considered as a potential therapeutic protein. However, producing high quality of such non-IgG proteins in mammalian cells is still a challenge in biopharmaceutical development. Here, we report a rapid production process by using transient gene transfection in HEK 293E cells. A set of constructs combining several expression promoters, leader sequences, and 5' un-translated regions were generated and optimized, from which the best vector with expression level at ~50mg/L was selected for production at 2L cell culture scale. Comparison study in downstream purification methods led to development of a scalable, non-affinity chromatography strategy through Super Q, Fast Flow Q, and Heparin columns. The product was characterized in purity (99%), isoelectric point, molecule weight, glycosylation, and stability by using SEC-HPLC, SDS-PAGE, isoelectric focusing and mass spectrometry. The highly purified product shows IGF-1 binding activity and inhibits IGF-1-induced cell proliferation. This process not only provides a remarkable high expression at ~50mg/L and pure glycosylated mammalian rhIGFBP7, also highlights that transient gene expression technology is practical to be used for production and early development of recombinant non-IgG therapeutic proteins.

Development of a noninvasive method for detecting and monitoring the time course of Helicobacter pylori infection.

Helicobacter pylori infection status following experimental inoculation of mice presently requires euthanasia. The purpose of this study was to develop a method for following the time course of H. pylori infection in live experimental animals. Twenty-six C57BL/6, Helicobacter-free female mice were inoculated with H. pylori Sydney strain 1, and 16 mice were sham inoculated. The mice were repeatedly tested during a period of about 1 year with an H. pylori species-specific primer-based PCR analysis of DNA extracted from fecal pellets of mice. The mice were euthanized at 6 months (n = 15) and 10 months (n = 15) to determine their infection status by histology, culture, and PCR of gastric specimens. H. pylori-inoculated mice were tested via the PCR method at 6 and 10 months prior to necropsy. Nine of 13 (69%) and 10 of 13 (77%) mice tested at 6 and 10 months, respectively, were positive. All sham-inoculated mice were negative. These two PCR results suggested a specificity of 100% with a sensitivity range between 69 and 77%. In contrast, sensitivity and specificity rose to 90 and 100% if groups of mice were tested once daily for 4 days. Seventy-seven to 85% of the experimental mice were also positive for H. pylori by culture. The histopathology demonstrated mild to severe gastritis. These findings demonstrate that the persistence or transience of H. pylori infection in live mice can be repeatedly evaluated over time. This method could allow the determination of the time course of infection and the efficacy of medications and/or vaccine without necropsy.