Novel antibody-based therapies for cancer are predictably effective if they can target cancer cells without damaging normal organs.1 We have developed various recombinant immunotoxins (RITs) against different targets on cancer cell surfaces.2 RITs are hybrid proteins that consist of antibody variable fragments attached to a truncated portion of Pseudomonas Exotoxin A (PE). Several immunotoxins are currently in clinical trials or preclinical development.2, 3, 4 We have reported previously that immunotoxin moxetumomab paseudotox targeting CD22 produces complete remissions in many patients with refractory hairy cell leukemia.5 This agent has recently completed a phase 3 trial. In addition, a RIT that targets mesothelin showed promising clinical responses in patients with chemotherapy-resistant malignant mesothelioma.4
Multiple myeloma (MM) is a B-cell malignancy that originates in the bone marrow (BM). Although there are FDA-approved antibody-based therapies available for the treatment of some B-cell malignancies, no very effective antibody-based therapy is yet available for MM.6 The B-cell maturation antigen (BCMA) belongs to the tumor necrosis factor receptor (TNFR) superfamily and is highly expressed in all MM cells from patients.7 Because of the restricted expression of BCMA to plasma cells and its role in growth as well as cell survival of MM, the BCMA antigen has been investigated as the targets in various immunotherapeutic strategies. These include antibody-based therapy,8 chimeric antigen receptor therapy9 and therapy with BiTEs.10
To develop immunotoxins that target BCMA we have generated a panel of monoclonal antibodies (mAbs) by immunizing mice with recombinant BCMA protein using hybridoma technology. We produced hybridomas producing anti-BCMA mAbs as described (Supplementary Materials and Methods). Because BCMA, TACI and BAFFR and BCMA share the same natural ligands, we tested the reactivity of each anti-BCMA mAb with two structurally closely-related TNFRs (TACI or BAFFR) expressed on transfected 293 T cells by flow cytometry (Supplementary Figure S1A) and TNFR-rFc fusion proteins by enzyme-linked immunosorbent assay (Supplementary Figure S1B). Based on this analysis, we selected BM24 and BM306 because they bind to BCMA antigen on the cell surface with high affinity and specificity. The binding affinity (KD) of both mAbs are <1 × 10−10 m. We cloned the VH and the VL from BM24- and BM306-expressing hybridomas using IgG1 isotype-specific oligo primers11 and used the LR version of the PE toxin.12 A schematic of the Fab-immunotoxin protein and genes encoding the immunotoxins is shown (Supplementary Figure S1C and D). After expression and purification, we obtained highly purified RITs for both BM24 and BM306. The corresponding immunotoxins are named LMB38 and LMB70, respectively. A sodium dodecyl sulfate gel showing that the immunotoxins are highly purified is shown (Supplementary Figure S1E).
We tested the cytotoxic activity of LMB38 and LMB70 on BCMA expressing cell lines using a cell proliferation assay (WST-1). Representative cell-killing curves are shown for LMB70 (Figure 1a) and LMB38 (Supplementary Figure S1F). The IC50 values are summarized in Table 1. The IC50 of LMB38 on H929, U266B, JJN3, RPMI-8226, LP-1 and KMS-18 are 1.2, 1.9, 2.5, 6.9, 25 and 55 ng/ml, respectively. Similarly, the IC50 values for LMB70 on those cell lines are 1.1, 5.0, 4.0, 5.1, 20 and 65 ng/ml, respectively. LMB38 and LMB70 have no activity on Jeko-1 and the HUT-102 cell line that is BCMA negative.
Because WST-1 assays measure both cell growth inhibition and cell death, we measured the cell killing by flow cytometry in which Annexin V and 7AAD staining was simultaneously used to analyze apoptosis and cell death at the same time. Over 95% of cells were Annexin V/7AAD positive when exposed to LMB70 for only 10 min, indicating a very short exposure to LMB70 is sufficient to kill almost all of the antigen-positive target cells (Figure 1b).
To determine whether LMB70 would kill cells from patients with MM, we analyzed BMMNCs from seven patients with active disease who had considerable numbers of myeloma plasma cells in the BM. A summary of the activity results with all patients analyzed are shown (Figure 1c, 1d and Table 1). Typical cell analysis results for three patients are shown in Supplementary Figure S2A and B. The myeloma plasma cells of all patient samples were killed by LMB70 in a dose-dependent manner as shown (Figure 1c) but not the non-myeloma by standard cells (Supplementary Figure S2C). Figure 1d shows that the IC50 values for the MM cell lines varied between 0.4 and 18 ng/ml and the IC50 values of myeloma plasma cells from patients were in the same range as from myeloma cell lines.
To determine whether the apoptosis pathway is induced after exposure of H929 cells to LMB70, we performed western analysis of proteins involved in apoptosis. As shown in Supplementary Figure S2D, the level of Mcl-1 and Bcl-XL was markedly diminished after 6-h exposer of immunotoxin. Also, Caspase 3, 8 and 9 underwent cleavage during the 6-h period. These changes are consistent with rapid induction of apoptosis.
Next, we grew tumor in severe combined immunodeficient (SCID) mice using the most sensitive cell line H929 to test in vivo efficacy of LMB38 and LMB70 in mice. Based on mouse safety data of other previous immunotoxins, we treated mice with 1.5 mg/kg every other day × 5 doses. As shown for LMB70 (Figure 1e) and LMB38 (Supplementary Figure S2E), the growth of tumors in immunotoxin treated groups are delayed or the tumors decrease in size up to 50%, but they grew back once the treatment was stopped. We used the H929 cell line for animal studies because it grows in SCID mice and develops uniform tumors when injected subcutaneously with matrigel. We found that both LMB38 and LMB70 caused tumors to shrink and slowed tumor progression but alone did not produce a complete response, although both are very active on H929 cells in vitro. The basis of the incomplete responses is being studied but is not owing to antigen loss, and cells isolated from treated tumors are fully responsive when returned to cell culture.
Paclitaxel, an anti-microtubule agent that promotes microtubule assembly has been studied in MM as a chemotherapeutic agent. Nab-paclitaxel (Abraxane) is a modified version of paclitaxel that has distinct pharmacologic properties with greater uptake by and retention within tumors which made it efficacious against some solid tumors. Several clinical studies have reported modest response by paclitaxel as a single agent in patients with newly diagnosed MM.13 A recent phase II trial of nab-paclitaxel in patients with relapse of refractory MM showed 15% (2/13) overall response rate.14 We also previously found that the combination of immunotoxin and a taxane acted synergistically to cause complete regressions of mesothelin expressing malignancies (cancers of cervix, pancreas and stomach).15 Therefore, we treated H929 xenograft bearing mice with LMB70 and Abraxane. The combination of Abraxane (20 mg/kg) and LMB70 (1 mg/kg) gave complete remissions that lasted over 30 days (Figure 1f), whereas each agent alone did not. These doses of drug were well tolerated with no loss of weight in the treated mice (Supplementary Figures 3A-D).
In summary, we have produced two RITs that kill myeloma cell lines and produce complete remissions in mice with myeloma tumors. Our data suggest that further preclinical development of the agents for myeloma therapy is warranted.
References
Scott AM, Allison JP, Wolchok JD . Monoclonal antibodies in cancer therapy. Cancer Immunol 2012; 12: 14.
Pastan I, Hassan R, Fitzgerald DJ, Kreitman RJ . Immunotoxin treatment of cancer. Annu Rev Med 2007; 58: 221–237.
Wayne AS, Kreitman RJ, Findley HW, Lew G, Delbrok C, Steinberg SM et al. Anti-CD22 immunotoxin RFB4(dsFv)-PE38 (BL22) for CD22 positive hematologic malignancies of childhood: pre-clinical studies and Phase I clinical trial. Clin Cancer Res 2010; 16: 1894–1903.
Hassan R, Miller AC, Sharon E, Thomas A, Reynolds JC, Ling A et al. Major cancer regressions in mesothelioma after treatment with an anti-mesothelin immunotoxin and immune suppression. Sci Transl Med 2013; 5: 208ra147.
Kreitman RJ, Tallman MS, Robak T, Coutre S, Wilson WH, Stetler-Stevenson M et al. Phase I trial of anti-CD22 recombinant immunotoxin moxetumomab pasudotox (CAT-8015 or HA22) in patients with hairy cell leukemia. J Clin Oncol 2012; 30: 1822–1828.
Kumar SK, Lee JH, Lahuerta JJ, Morgan G, Richardson PG, Crowley J et al. Risk of progression and survival in multiple myeloma relapsing after therapy with IMiDs and borte-zomib: a multicenter international myeloma working group study. Leukemia 2012; 26: 149–157.
Laabi Y, Gras MP, Carbonnel F, Brouet JC, Berger R, Larsen CJ et al. A new gene, BCM, on chromosome 16 is fused to the interleukin 2 gene by a t(4;16)(q26;p13) translocation in a malignant T cell lymphoma. EMBO J 1992; 11: 3897–3904.
Tai YT, Mayes PA, Acharya C, Zhong MY, Cea M, Cagnetta A et al. Novel anti-B-cell maturation antigen antibody-drug conjugate (GSK2857916) selectively induces killing of multiple myeloma. Blood 2014; 123: 3128–3138.
Carpenter RO, Evbuomwan MO, Pittaluga S, Rose JJ, Raffeld M, Yang S et al. B-cell maturation antigen is a promising target for adoptive T-cell therapy of multiple myeloma. Clin Cancer Res 2013; 19: 2048–2060.
Ramadoss NS, Schulman AD, Choi SH, Rodgers DT, Kazane SA, Kim CH et al. An anti-B cell maturation antigen bispecific antibody for multiple myeloma. J Am Chem Soc 2015; 137: 5288–5291.
Pastan I, Beers R, Bera TK . Recombinant immunotoxins in the treatment of cancer. Methods Mol Biol 2004; 248: 503–518.
Weldon JE, Xiang L, Chertov O, Margulies I, Kreitman RJ, FitzGerald DJ et al. A protease-resistant immunotoxin against CD22 with greatly increased activity against CLL and diminished animal toxicity. Blood 2009; 113: 3792–3800.
Miller HJ, Leong T, Khandekar JD, Greipp PR, Gertz MA, Kyle RA . Paclitaxel as the initial treatment of multiple myeloma: an Eastern Cooperative Group Study (E1A93). Am J Clin Oncol 1998; 21: 553–556.
Jain T, Dueck AC, Kosiorek HE, Ginos BF, Mayo A, Reeder CB et al. Phase II trial of nab-paclitaxel in patients with relapsed or refractory multiple myeloma. Am J Hematol 2016; 91: E504–E505.
Alewine C, Xiang L, Yamori T, Niederfellner G, Bosslet K, Pastan I . Efficacy of RG7787, a next-generation mesothelin-targeted immunotoxin, against triple-negative breast and gastric cancers. Mol Cancer Ther 2014; 13: 2653–2661.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
IP is an inventor on several patents on immunotoxins that have all been assigned to the NIH. The remaining authors declare no conflict of interest.
Supplementary information
Rights and permissions
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/4.0/
About this article
Cite this article
Bera, T., Abe, Y., Ise, T. et al. Recombinant immunotoxins targeting B-cell maturation antigen are cytotoxic to myeloma cell lines and myeloma cells from patients. Leukemia 32, 569–572 (2018). https://doi.org/10.1038/leu.2017.315
Published:
Issue Date:
DOI: https://doi.org/10.1038/leu.2017.315
This article is cited by
-
Immunotherapeutic strategies targeting B cell maturation antigen in multiple myeloma
Military Medical Research (2021)