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 25;9(40):25764-25780.
doi: 10.18632/oncotarget.25359.

Pre-clinical validation of B cell maturation antigen (BCMA) as a target for T cell immunotherapy of multiple myeloma

De-Xiu Bu #  1 , Reshma Singh #  1 , Eugene E Choi #  1 , Marco Ruella  2 , Selene Nunez-Cruz  2 , Keith G Mansfield  1 , Paul Bennett  1 , Nathanial Barton  1 , Qilong Wu  3 , Jiquan Zhang  3 , Yongqiang Wang  3 , Lai Wei  3 , Shawn Cogan  1 , Tucker Ezell  1 , Shree Joshi  1 , Kellie J Latimer  1 , Brian Granda  1 , William R Tschantz  1 , Regina M Young  2 , Heather A Huet  1 , Celeste J Richardson  1 , Michael C Milone  2   4
Affiliations

Pre-clinical validation of B cell maturation antigen (BCMA) as a target for T cell immunotherapy of multiple myeloma

De-Xiu Bu et al. Oncotarget. .

Abstract

Multiple myeloma has a continued need for more effective and durable therapies. B cell maturation antigen (BCMA), a plasma cell surface antigen and member of the tumor necrosis factor (TNF) receptor superfamily, is an attractive target for immunotherapy of multiple myeloma due to its high prevalence on malignant plasma cells. The current work details the pre-clinical evaluation of BCMA expression and development of a chimeric antigen receptor (CAR) targeting this antigen using a fully human single chain variable fragment (scFv). We demonstrate that BCMA is prevalently, but variably expressed by all MM with expression on 25-100% of malignant plasma cells. Extensive Immunohistochemical analysis of normal tissue expression using commercially available polyclonal antibodies demonstrated expression within B-lineage cells across a number of tissues as expected. Based upon the highly restricted expression of BCMA within normal tissues, we generated a set of novel, fully human scFv binding domains to BCMA by screening a naïve B-cell derived phage display library. Using a series of in vitro and pre-clinical in vivo studies, we identified a scFv with high specificity for BCMA and robust anti-myeloma activity when used as the binding domain of a second-generation CAR bearing a CD137 costimulatory domain. This BCMA-specific CAR is currently being evaluated in a Phase 1b clinical study in relapsed and refractory MM patients (NCT02546167).

Keywords: BCMA; CAR; T cell; multiple myeloma.

PubMed Disclaimer

Conflict of interest statement

CONFLICTS OF INTEREST E.D.C, R.S., Q. W., J.Z., Y. W., L.W., A.L., S.C., T.E., S. J., K.G. M., K.J.L., W.R.T., H.A.H, DX. B., P.B., N.B., and C.J.R. were paid employees of Novartis at the time this work was performed. The University of Pennsylvania and Novartis hold a patent in the use of CAR T cells in oncology. E.D.C, R.S., M.R., Y. W., Q. W., H.A.H, M.C.M, and C.J.R., are inventors on a patent related to these data.

Figures

Figure 1
Figure 1. BCMA expression on MM patient samples
(A) An example of flow cytometry analysis of a bone marrow (BM) sample from an MM patient is shown. Plasma cells (PC; both malignant and non-malignant) were identified by gating bone marrow on live single cells followed by using CD38 and CD45 staining as shown. Malignant PCs were evaluated for BCMA expression with fluorescence minus one, FMO, used as control. (B) Ten consecutive MM patients were analyzed for BCMA expression as in Figure 1A. BCMA median expression was 86% (red line). (C) BCMA expression (dark histogram) on 3 established multiple myeloma cell lines assessed by flow cytometer. The lighter histogram shows staining of a matched isotype control. (D) Representative immunohistochemical staining of a bone marrow biopsy specimen stained for BCMA using two commercially available antibodies, B0807-50G (brown staining, primaryantibody concentration of 0.6 μg/ml, US Biological) and AF193 (magenta staining, primary antibody concentration of 2.0 μgml, R&D Systems).
Figure 2
Figure 2. Immunohistochemical staining with two commercially available anti-BCMA antibodies show disparate staining within the brain
(A) IHC staining of small intestine showing plasma cells using B0807-50G (brown staining) and AF193 (magenta staining). (B) IHC staining of cerebellum. (C) IHC staining of the NHP (M. fascicularis) midbrain using B0807-50G showing reactivity within neuronal cell bodies within the inferior olivary nucleus (ION). (D) Expression of BCMA mRNA within indicated tissues measured by quantitative PCR. Assay was performed in triplicate (mean ± standard deviation). Results are expressed relative to 18S ribosomal RNA expression. (E) ISH for BCMA mRNA using a target specific probe (Advanced Cell Diagnostics) on normal NHP small intestine. (F) ISH for BCMA mRNA on NHP cerebellum and medulla oblongata. Probes specific for the bacterial protein, DAPB and the cyclophilin, PPIB were used as negative and positive controls, respectively, for RNA quality.
Figure 3
Figure 3. Identification of ScFv clones from human B cell antibody libraries that bind to cell surface-expressed BCMA
(A) Flow chart of steps taken in the library panning and screening process. (B) Histograms of two representative purified scFv-His proteins (clones scFv-4 and scFv-10) analyzed by flow cytometry for binding to BCMA that is transiently expressed on HEK293E cells (blue) or untransfected HEK293E cells (red). Transient expression of BCMA on cells was confirmed using a mouse anti-BCMA mAb (clone 19F2, Biolegend) as a positive control. An irrelevant scFv-His protein was used as a negative control against which the positive gate was set (black line in histograms). (C) Summary of the percentage of BCMA-expressing cells (% positive) detected by each of the 15 purified scFv clones analyzed as in panel b. (D) Schematic of the CAR construct design. The scFv sequences identified in Figure 2 were cloned in the VH to VL orientation upstream of the CD8a hinge and transmembrane domains (TM), followed by the 4-1BB and CD3 zeta intracellular signaling domains.
Figure 4
Figure 4. Identification of active and inactive clones using a reporter assay system
(A) Schematic of the reporter assay. Jurkat cells containing the luciferase gene driven by the signaling-responsive NFAT promoter (termed JNL cells), were transduced with the various CAR constructs. Binding between the CAR construct and its cognate cellular antigen (BCMA on target cells) leads to luciferase expression in the JNL cells. (B) CAR clones were evaluated in the JNL reporter assay for antigen-dependent activity. JNL cells containing the indicated CAR clones with expression levels shown in C, or untransduced JNL cells (UTD) were co-cultured with target cells lines (K562, K562-BCMA, NCI-H929, or RPMI8226) and luciferase activity was measured as luminescence intensity. Clones were considered active when the luminescence intensity exceeded 1.25-fold the level of UTD cells in the presence of antigen-expressing cells. Clones were considered non-specific when the luminescence intensity exceeded 1.25 fold the level of UTD cells in the presence of antigen negative K562 cells. (C) T cells expressing the indicated CAR clones were evaluated for their ability to proliferate without antigen when co-cultured with no target cells (none), antigen negative irradiated cells (K562). Irradiated antigen-expressing cells (K562-BCMA) were used as a positive control. Proliferation was determined by counting CD3+ cells relative to CountBright Absolute Counting Beads. Untransduced T cells (UTD) revealed the basal level of CAR-independent effects of each target cell line on T-cell proliferation. (D) The ability of T cells expressing the indicated CAR clones to mediate cell lysis was evaluated against the KMS-11 target cell line expressing fire fly luciferase (KMS-11-luc). CART cells were co-cultured with KMS-11-luc target cells at the indicated E:T ratios, and % cell killing, determined by the difference in luciferase signal between target cells without effector T cells (control) and with effector T cells (experimental) expressed as a percent of the control, was measured as a surrogate for target cell lysis. UTD represents untransduced T cells. Individual data points represent the mean with the curve fit using an Emax model. (E) KMS11-luc cells were implanted in NSG mice and body luminescence (BLI) was monitored over time as a measure of tumor burden. CART cells (1.5 × 106 CAR+ cells in 5 × 106 total T cells), untransduced T cells (UTD; 5 × 106 cells) or PBS were dosed intravenously on day 7 after implantation of 1 × 106 tumor cells. 7 of mice were enrolled in each arm of the study. All data are expressed as mean ± standard error of the mean (SEM). This experiment is representative of 2 independent experiments.
Figure 5
Figure 5. Evaluation of binding specificity and affinity of the clone 10 scFv
(A) Biacore T200 SPR sensogram for the interaction between clone 10 scFv and recombinant human BCMA. A purified recombinant His-tagged protein containing the scFv of clone 10 was used to determine the binding affinity to recombinant BCMA protein. hBCMA-Fc was immobilized on the chip surface via biotin:streptavidin interaction and clone 10 scFv was flowed over the chip at 1:3 dilutions. Shown are the association constant (ka) and disassociation constant (kd) determined after fitting to a 1:1 binding model used to determine the apparent affinity to rhBCMA-Fc. (B) CAR clone 10 was transduced into Jurkat cells and incubated with recombinant Fc-tagged BCMA, BAFFR, or TACI to assess the binding of these proteins to the cell surface-expressed CAR. Binding was detected using an anti-Fc antibody by flow cytometry. The percent of cells with a fluorescence level above the untransduced Jurkat cells is shown. (C) IHC staining of human tissue with the clone 10 chimeric antibody demonstrates scattered positive cells in the normal human tonsil (grade 1) and intense uniform staining of multiple myeloma tissue (grade 5) (insert: irrelevant antibody negative control). (D) IHC staining of normal human medulla oblongata with a chimeric antibody containing the clone 10 scFv. (E) IHC staining of normal cerebellum with a chimeric antibody contain the clone 10 scFv.
See this image and copyright information in PMC

References

    1. Gozzetti A, Candi V, Papini G, Bocchia M. Therapeutic advancements in multiple myeloma. Front Oncol. 2014;4:241. https://doi.org/10.3389/fonc.2014.00241 - DOI - PMC - PubMed
    1. Kalos M, Levine BL, Porter DL, Katz S, Grupp SA, Bagg A, June CH. T Cells with Chimeric Antigen Receptors Have Potent Antitumor Effects and Can Establish Memory in Patients with Advanced Leukemia. Sci Transl Med. 2011;3:95ra73–95ra73. https://doi.org/10.1126/scitranslmed.3002842 - DOI - PMC - PubMed
    1. Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin NJ, Chew A, Gonzalez VE, Zheng Z, Lacey SF, Mahnke YD, Melenhorst JJ, Rheingold SR, et al. Chimeric Antigen Receptor T Cells for Sustained Remissions in Leukemia. N Engl J Med. 2014;371:1507–17. https://doi.org/10.1056/NEJMoa1407222 - DOI - PMC - PubMed
    1. Porter DL, Hwang WT, Frey NV, Lacey SF, Shaw PA, Loren AW, Bagg A, Marcucci KT, Shen A, Gonzalez V, Ambrose D, Grupp SA, Chew A, et al. Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. Sci Transl Med. 2015;7:303ra139. https://doi.org/10.1126/scitranslmed.aac5415 - DOI - PMC - PubMed
    1. Gaublomme JT, Yosef N, Lee Y, Gertner RS, Yang LV, Wu C, Pandolfi PP, Mak T, Satija R, Shalek AK, Kuchroo VK, Park H, Regev A. Single-Cell Genomics Unveils Critical Regulators of Th17 Cell Pathogenicity. Cell. 2015;163:1400–12. https://doi.org/10.1016/j.cell.2015.11.009 - DOI - PMC - PubMed

Associated data