Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs) that effectively and specifically quench cell surface–bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor–labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

COVID-19 is a global pandemic that has resulted in over 800,000 deaths. Robust humoral anti-viral immune responses have the potential to generate a diverse set of neutralizing antibodies to eliminate viruses and protect against re-infection, transmission, and the evolution of mutations that escape targeted therapeutics. CD73 is present on the majority of human B cells and a subset of T cells where it plays a role in lymphocyte activation and migration. CD73 also functions as an ectoenzyme that converts AMP into adenosine, which can be immunosuppressive. Here we report on CPI-006, a humanized FcγR binding-deficient IgG1 anti-CD73 antibody that blocks CD73 enzymatic activity and directly activates CD73+ B cells, inducing differentiation into plasmablasts, immunoglobulin class switching, and antibody secretion independent of adenosine. Immunophenotypic analysis of peripheral blood from advanced cancer patients receiving CPI-006 revealed evidence of B cell activation, clonal expansion, and development of memory B cells. These immune effects suggested that CPI-006 may be effective at enhancing the magnitude, diversity, and duration of humoral and cellular responses to viruses such as SARS-CoV-2. We have therefore initiated a Phase 1, single-dose, dose-escalation trial in hospitalized patients with mild to moderate COVID-19. The objectives of this trial are to evaluate the safety of CPI-006 in COVID-19 patients and to determine effects of CPI-006 on anti-SARS-CoV-2 antibody responses and the development of memory B cell and T cells. Ten patients have been enrolled in the trial receiving doses of 0.3 mg/kg or 1.0 mg/kg. All evaluable patients had low pre-treatment serum levels of anti-viral antibodies to the SARS-CoV-2 trimeric spike protein and its receptor binding domain, independent of the duration of their COVID-19 related symptoms prior to enrollment. Anti-viral antibody responses were induced 7 days after CPI-006 treatment and titers continued to rise past Day 56. Increases in the frequency of memory B cells and effector/memory T cells were observed 28 days after treatment. These preliminary results suggest that CPI-006 activates B cells and may enhance and prolong anti-SARS-CoV-2 antibody responses in patients with COVID-19. This approach may be useful for treating COVID-19 or as an adjuvant to enhance the efficacy of vaccines.

Adenosine is present at high concentrations in the tumor microenvironment and is immunosuppressive acting on multiple cell types, including suppression of effector T cells. CD73, an ectonucleotidase that converts AMP to adenosine, is expressed on a subset of B and T cells and is a major source of extracellular adenosine. Elevated CD73 expression has been observed in multiple tumor types and is prognostic in triple negative breast cancer supporting a role for CD73 in tumor progression1. Inhibiting catalytic activity of CD73 is an attractive therapeutic strategy to reduce adenosine-mediated suppression of tumor immunity. We developed two types of humanized monoclonal anti-CD73 antibodies. CPX-006 inhibits CD73 catalytic activity by competing directly with AMP for the active site with an affinity of 0.64 nM. CPX-016 is similar to anti-CD73 antibodies described by others2, and inhibits CD73 activity allosterically by binding to a distal site. This was demonstrated using CD73 expressing cells incubated with APCP, a non-hydrolyzable analog of AMP. APCP competes with CPX-006 for binding to CD73 in contrast to CPX-016. The CPX-016 mechanism requires higher order complexes and results in loss of inhibition at high CPX-016/CD73 ratios. In contrast, CPX-006 reduces catalytic activity completely in cell based assays to levels seen when CD73 gene is deleted using CRISPR technology and inhibition was unaffected at high CPX-006/CD73 ratios. In a functional assay, CPX-006 inhibited immune suppression of T cell function induced by exposure of human PBMCs to AMP through direct inhibition of catalytic activity with a mean EC50 of 137 nM (interferon gamma production) and 189 nM (T cell proliferation) (n=12 donors). We analyzed the prevalence of CD73 expression across tumor histologies by immunohistochemistry including renal cell carcinoma (RCC, n=62), non small cell lung cancer (NSCLC, n=68), melanoma (n= 68) and breast cancer (n=94). CD73 was found to be heterogeneously expressed on tumor cells, immune cells and other stromal elements within each of the histologies examined. Expression on tumor cell only was found in 15% of melanoma and 14% of squamous NSCLC cases. In contrast, tumor cell staining was found in a significant fraction of the adenocarcinoma sub-type of NSCLC (55%). Stromal cell staining to varying degrees was seen in all tumor tissues. In summary, we have a generated a therapeutic antibody, CPX-006, that utilizes a novel mechanism of binding to the CD73 active site to completely inhibit CD73 enzymatic activity and restore T cell function. The finding of CD73 expression on tumor cells and stromal cells in a variety of tumors suggest this as a potential new target for immunotherapy of these malignancies. 1. Loi et al., Proc Natl Acad Sci USA 2013; 110: 11091-11096. 2. Geoghegan et al., mAbs 2016; 8: 454-467. Citation Format: Emily C. Piccione, Glen Mikesell, Barbara Daine-Matsuoka, Kimberly Walter, Richard Miller, Ian McCaffery. A novel CD73-blocking antibody reduces production of immunosuppressive adenosine and restores T cell function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5577. doi:10.1158/1538-7445.AM2017-5577