Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 May;29(5):585-601.
doi: 10.1089/hum.2018.001.

Effective Targeting of Multiple B-Cell Maturation Antigen-Expressing Hematological Malignances by Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor T Cells

Kevin M Friedman  1 , Tracy E Garrett  1 , John W Evans  1 , Holly M Horton  1 , Howard J Latimer  1 , Stacie L Seidel  1 , Christopher J Horvath  1 , Richard A Morgan  1
Affiliations

Effective Targeting of Multiple B-Cell Maturation Antigen-Expressing Hematological Malignances by Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor T Cells

Kevin M Friedman et al. Hum Gene Ther. 2018 May.

Abstract

B-cell maturation antigen (BCMA) expression has been proposed as a marker for the identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Consistent BCMA expression was confirmed on MM biopsies (29/29 BCMA+), and it was further demonstrated that BCMA is expressed in a substantial number of lymphoma samples, as well as primary chronic lymphocytic leukemia B cells. To target BCMA using redirected autologous T cells, lentiviral vectors (LVV) encoding chimeric antigen receptors (CARs) were constructed with four unique anti-BCMA single-chain variable fragments, fused to the CD137 (4-1BB) co-stimulatory and CD3ζ signaling domains. One LVV, BB2121, was studied in detail, and BB2121 CAR-transduced T cells (bb2121) exhibited a high frequency of CAR + T cells and robust in vitro activity against MM cell lines, lymphoma cell lines, and primary chronic lymphocytic leukemia peripheral blood. Based on receptor quantification, bb2121 recognized tumor cells expressing as little as 222 BCMA molecules per cell. The in vivo pharmacology of anti-BCMA CAR T cells was studied in NSG mouse models of human MM, Burkitt lymphoma, and mantle cell lymphoma, where mice received a single intravenous administration of vehicle, control vector-transduced T cells, or anti-BCMA CAR-transduced T cells. In all models, the vehicle and control CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors and 100% survival in all treatment models. Together, these data support the further development of anti-BCMA CAR T cells as a potential treatment for not only MM but also some lymphomas.

Keywords: BCMA; CAR; adoptive cell therapy; gene therapy; immunotherapy; multiple myeloma.

PubMed Disclaimer

Conflict of interest statement

No competing financial interests exist.

Figures

<b>Figure 1.</b>
Figure 1.
B-cell maturation antigen (BCMA) expression on human lymphoma and multiple myeloma (MM) cell lines by immunohistochemistry (IHC) correlated with receptor density. (A) Representative IHC staining for BCMA expression (brown) in K562 (left), RPMI-6666 (center), and RPMI-8226 (right) cell lines. (B) BCMA receptor density was quantified on the surface of the indicated tumor cell lines using fluorescent microspheres to determine antibody binding capacity (ABC). Data are shown graphically in rank order of increasing expression. The staining intensity score of BCMA from IHC (gray bars, right axis) is overlaid on top of the BCMA receptor density (black bars, left axis). Intensity staining score: 0 = negative to equivocal; 1 = weak; 1.5 = weak to moderate; 2 = moderate; 2.5 = moderate to strong; 3 = strong; 3.5 = strong to intense; and 4 = intense.
<b>Figure 2.</b>
Figure 2.
BCMA was detected in biopsies of patients diagnosed with MM and five subtypes of lymphoma. (A) Biopsies from 29 MM patients were stained and analyzed by IHC for BCMA expression (brown). (i) Representative images are shown at the top, demonstrating the variability in tumors for the percentage of sample staining positive for BCMA expression. (ii)The percentage of each section staining positive for BCMA expression (gray bars, right axis) was overlaid on top of the staining intensity score (black bars, left axis) for each individual patient biopsy in the graph. (B) Biopsies from 35 lymphoma patients were stained and analyzed by IHC for BCMA expression (brown). (i) Representative images from human lymphoma biopsies for mantle cell lymphoma (MCL; left) and Hodgkin lymphomas (HL; center and right) are shown at the top. (ii) The percentage of each section staining positive for BCMA expression and the staining intensity was as indicated. The percentage of each section staining positive for BCMA expression (gray bars, right axis) was overlaid on top of the staining intensity score (black bars, left axis) for each individual patient biopsy and grouped by lymphoma subtype in the graph at the bottom. nd, not detected.
<b>Figure 3.</b>
Figure 3.
Anti-BCMA chimeric antigen receptor (CAR) T cells elicit antitumor reactivity against BCMA-expressing targets. (A) Schematic of the anti-BCMA self-inactivating lentiviral vector (LVV). From the N-terminus to the C-terminus, the anti-BCMA CAR encodes the CD8 signal peptide, VL, linker, VH, CD8α hinge and transmembrane (TM), CD137 (4-1BB) co-stimulatory cytoplasmic signaling domain, and CD3ζ cytoplasmic signaling domain expressed under the control of the MND promoter. LTR, long terminal repeat; SD, splice donor; ψ+, packaging signal; cPPT, central polypurine tract; RRE, rev responsive element; SA, splice acceptor; ppt, polypurine tract; ΔU3, deleted 3′LTR. (B) Cell surface expression of CAR molecules was assessed by staining with polyclonal goat anti-mouse immunoglobulin and analyzed by flow cytometry. Each dot plot was gated on viable CD3+ lymphocytes with the frequency of CAR+ cells as shown. Inset within each dot plot is the corresponding vector copy number (VCN) expressed as average number of vector copies per diploid cell. (C) Interferon-γ (IFN-γ) release after 24 h of co-culture of CAR T cells with BCMA– (K562) or BCMA+ (K562-BCMA, RPMI-8226, U266-B1, H929) targets. Error bars show standard error of the mean, triplicate determinations. (D) K562-BCMA cytolysis after 4 h of co-culture with CAR T cells at effector-to-target (E:T) ratios assessed at 10:1, 3:1, and 1:1. Data are the average of two experiments using three unique donors, with the individual data points determined in triplicate. (E) Proliferation of anti-BCMA CAR T cells over 96 h of culture with K562 or K562-BCMA, assayed as dilution of CellTrace Violet fluorescent membrane dye. Data shown are from one of the duplicate experiments.
<b>Figure 4.</b>
Figure 4.
Anti-BCMA CAR T cells elicit potent reactivity against BCMA+ MM and lymphoma cell lines. (A) CAR T-cell cytolytic activity to the BCMA+, CD19– MM (RPMI-8226, NCI-H929, and U266-B1) or the BCMA+, CD19+ lymphoma (Burkitt lymphoma [BL]:Daudi, MCL:JeKo-1) cell lines. Anti-CD19 CAR T cells served as controls. Data are the average of two experiments using three unique donors, with the individual data points determined in triplicate. (B) The number of BCMA molecules per cell line was quantified as receptor density (•, left axis), and correlated to anti-BCMA CAR T-cell IFN-γ release after 24 h of co-culture with the same cell line (gray bars, right axis). Error bars show standard error of the mean, representative of duplicate experiments.
<b>Figure 5.</b>
Figure 5.
Anti-BCMA CAR T cells respond to low expression levels of BCMA in lymphoma cell lines and primary tumors. (A) IFN-γ release to each cell line after 24 h of co-culture with anti-BCMA CAR T cells was plotted (gray bars, left axis). For each cell line, ΔCt values (▾, right axis) were plotted for BCMA mRNA expression. ΔCt values >2 (dotted line) correlated with the ability of anti-BCMA CAR T cells to recognize the indicated tumor cell lines. (B) BCMA mRNA expression in 48 human primary lymphoma tumor samples (denoted by tick marks) were plotted as ΔCt (▾). Based on the data in (A), a ΔCt value >2 (dotted line) indicated BCMA RNA levels potentially capable of eliciting an anti-BCMA CAR T-cell response. Data are the average of triplicate determinations, representation of two experiments. (C) Peripheral blood mononuclear cells (PBMCs) from healthy donors (filled trace) and patients diagnosed with B-chronic lymphocytic leukemia (CLL; open trace) were analyzed for BCMA expression by flow cytometry and characterized as BCMA– (left) or BCMA+ (right). The frequency of cells staining positive for BCMA is indicated. (D) IFN-γ release from anti-BCMA CAR T cells (gray bars) or anti-CD19Δ– control CAR T cells (open bars) after 24 h of co-culture with BCMA– and BCMA+ PBMCs from B-CLL patients, or tumor lines that express BCMA (MEC-1 and K562-BCMA). Error bars show the standard error of the mean, triplicate determinations.
<b>Figure 6.</b>
Figure 6.
Anti-BCMA CAR T cells rapidly expand and eliminate MM xenografts, despite soluble BCMA protein in vivo. (A) NSG mice (10 animals per group) with approximately 100 mm3 subcutaneous RPMI-8226 tumors received a single intravenous (i.v.) administration of 5 × 106 CAR + T cells/mouse or repeated i.v. administration of bortezomib (Velcade®; 1 mg/kg twice weekly for 4 weeks). Tumor size was measured by calipers twice weekly by personnel blinded to treatment conditions. (B) Kaplan–Meier survival curves of study shown in (A). (C) Anti-BCMA CAR T cells in blood (CD3+CAR+ cells/μL) were assessed by flow cytometry and plotted, along with concordant tumor volume determinations. (D) Serum BCMA protein levels assessed by enzyme-linked immunosorbent assay were plotted with corresponding tumor volume measurements. (E) BCMA+ tumor cells and anti-BCMA CAR T cells were evaluated by IHC in RPMI-8226 MM xenografts in NSG mice. Infiltration of tumor tissue by CD3+ T cells was readily observed by day 5 post administration and was associated with elimination of BCMA+ MM cells within tumor tissue, being largely complete by day 12 post CAR T-cell administration. All times are days post CAR T-cell administration. Error bars show standard error of the mean; data are representative of three experiments.
<b>Figure 7.</b>
Figure 7.
Anti-BCMA CAR T cells show robust antitumor activity against lymphoma xenografts. (A) Treatment of firefly luciferase labeled BCMA+/CD19+ double-positive BL (Daudi) established i.v. for 18 days before CAR T-cell administration. Mice (five animals per group) were monitored until day 51 post CAR T-cell administration, with tumor size determined twice weekly by blinded personnel. (B) Kaplan–Meier survival curve of same study in (A). (C) Treatment of firefly luciferase labeled BCMA+/CD19+ double-positive MCL tumor (JeKo) established i.v. for 11 days before injection of CAR T cells, vehicle, or untransduced T cells. Mice (five animals pre group) were monitored until day 43 post CAR T-cell administration, with tumor size determined twice weekly by blinded personnel. (D) Kaplan–Meier survival curve of same study in (C). Error bars show standard error of the mean; data are representative of duplicate experiments.
See this image and copyright information in PMC

References

    1. Brenner MK, Heslop HE. Adoptive T cell therapy of cancer. Curr Opin Immunol 2010;22:251–257 - PMC - PubMed
    1. Hoyos V, Savoldo B, Dotti G. Genetic modification of human T lymphocytes for the treatment of hematologic malignancies. Haematologica 2012;97:1622–1631 - PMC - PubMed
    1. Kershaw MH, Teng MW, Smyth MJ, et al. . Supernatural T cells: genetic modification of T cells for cancer therapy. Nat Rev Immunol 2005;5:928–940 - PubMed
    1. Morgan RA, Dudley ME, Wunderlich JR, et al. . Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 2006;314:126–129 - PMC - PubMed
    1. Park TS, Rosenberg SA, Morgan RA. Treating cancer with genetically engineered T cells. Trends Biotechnol 2011;29:550–557 - PMC - PubMed

MeSH terms

Substances