Characterization and Phase 1 Trial of a B Cell Activating Anti-CD73 Antibody for the Immunotherapy of COVID-19

  1. Stephen B. Willingham
  2. Gerard Criner
  3. Craig Hill
  4. Shenshen Hu
  5. Jenny A. Rudnick
  6. Barbara Daine-Matsuoka
  7. Jessica Hsieh
  8. Haider Mashhedi
  9. Andrew N. Hotson
  10. Joshua Brody
  11. Thomas Marron
  12. Emily Piccione
  13. Joseph J. Buggy
  14. Suresh Mahabhashyam
  15. William B. Jones
  16. Mehrdad Mobasher
  17. Richard A. Miller

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Abstract

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 POS 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.

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  1. ScreenIT

    SciScore for 10.1101/2020.09.10.20191486: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies: CPI-006 was engineered by isolating VH and VL regions from the parental hybridoma generated by immunizing mice with human CD73 and screening for inhibition of CD73 activity.
    human CD73
    suggested: None
    MEDI9447 was cloned using the VH and VL chain sequences published in WO 2016/075099 AI application patent and was expressed as a human lambda/IgG1-TM antibody.
    lambda/IgG1-TM
    suggested: None
    Anti-CD73 antibody clone AD2 was purchased from Abcam.
    Anti-CD73
    suggested: None
    Purified antibodies (2 |j,g/mL) were loaded onto Anti-Human IgG Fc Capture biosensors.
    Anti-Human IgG
    suggested: None
    Anti-SARS-CoV-2 antibody ELISA assays: ELISA was performed to measure the IgG, IgM, IgA antibody titer to the receptor-binding domain (RBD) of the spike protein and full-length spike trimer of the SARS-CoV-2 virus.
    Anti-SARS-CoV-2
    suggested: None
    IgA
    suggested: None
    After three washes, the bound antibody was detected using anti-human IgG-horseradish peroxidase (HRP) conjugated secondary antibody (1:3000, Sigma-Aldrich, A0170
    anti-human IgG-horseradish
    suggested: None
    ) or anti-human IgM HPR secondary antibody (1:3000, Sigma-Aldrich, A0420)
    anti-human IgM
    suggested: (Sigma-Aldrich Cat# A0420, RRID:AB_257886)
    A0420
    suggested: None
    , or anti-human IgA HRP secondary antibody for 1 hr at RT.
    anti-human IgA
    suggested: None
    Expression of cell surface markers associated with B and T cell activation were assessed by flow cytometry using Fc blocking reagent (Miltenyi Biotech, Catalog #130-059-901) and antibodies directed to CD19 BV421 (Clone HIB19; BD Biosciences, Cat #562440), CD38 BV510 (Clone HB-7; BioLegend, Cat #356612)
    CD19
    suggested: (BD Biosciences Cat# 562440, RRID:AB_11153299)
    CD38
    suggested: (BioLegend Cat# 356612, RRID:AB_2563875)
    Experimental Models: Cell Lines
    SentencesResources
    Both antibodies were expressed in Expi-293 cells (Thermo Fisher Scientific) and purified by Protein A chromatography (HiTrap Protein A, GE Healthcare Life Sciences).
    Expi-293
    suggested: RRID:CVCL_D615)
    Software and Algorithms
    SentencesResources
    ID50 values were obtained by fitting the response-normalized data to a four-parameter logistic equation using GraphPad Prism version 8.4.3 for Windows, GraphPad Software, San Diego, California USA.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Flow data was analyzed using FlowJo v10.7.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT03454451RecruitingCPI-006 Alone and in Combination With Ciforadenant and With …
    NCT04464395RecruitingStudy of CPI-006 as Immunotherapy for Hospitalized COVID-19 …

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.09.10.20191486 on medRxiv