Anti-BCMA Immunotoxins: Design, Production, and Preclinical Evaluation

Abstract

Multiple myeloma (MM) is a B-cell malignancy that is incurable for a majority of patients. B-cell maturation antigen (BCMA) is a lineage-restricted differentiation protein highly expressed in multiple myeloma cells but not in other normal tissues except normal plasma B cells. Due to the restricted expression and being a cell surface membrane protein, BCMA is an ideal target for immunotherapy approaches in MM. Recombinant immunotoxins (RITs) are a novel class of protein therapeutics that are composed of the Fv or Fab portion of an antibody fused to a cytotoxic agent. RITs were produced by expressing plasmids encoding the components of the anti-BCMA RITs in E. coli followed by inclusion body preparation, solubilization, renaturation, and purification by column chromatography. The cytotoxic activity of RITs was tested in vitro by WST-8 assays using BCMA expressing cell lines and on cells isolated from MM patients. The in vivo efficacy of RITs was tested in a xenograft mouse model using BCMA expressing multiple myeloma cell lines. Anti-BCMA recombinant immunotoxins are very effective in killing myeloma cell lines and cells isolated from myeloma patients expressing BCMA. Two mouse models of myeloma showed that the anti-BCMA immunotoxins can produce a long-term complete response and warrant further preclinical development.

Keywords: ABD fusion protein; H929 cells; LMB-70; mAb BM306.

Figure 1

Schematics showing B-cell maturation antigen (BCMA) protein. BCMA is a type III transmembrane protein with no signal sequence at the amino-terminus. Extracellular domain (ECD) of BCMA comprises of 54 amino acids and shed as soluble BCMA (sBCMA) after cleaved by γ-secretase. The transmembrane domain (TM) and the intracellular domain (ICD) of BCMA contain 23 and 107 amino acid residues, respectively.

Figure 2

Production scheme of an anti-BCMA Fab immunotoxin. (A) Schematics showing expression plasmids encoding the heavy chain linked to toxin and the second encoding the light chain. Both plasmids contain an inducible T7 transcription promoter and a chloramphenicol antibiotic resistance gene (CM^R) as selectable marker. (B) Schematics of correctly folded Fab immunotoxin protein. (C) Analytical PAGE gel showing the quality of purified Fab immunotoxin. Lanes: NR, protein analyzed under non-reducing condition; R, protein analyzed under reducing condition; M, molecular weight standards.

Figure 3

Ribbon drawing of ant-BCMA immunotoxin variants. The light chain (cyan) and the heavy chain (magenta) were modeled using X-ray structure data. Domains II and III of PE (Pseudomonas exotoxin) data were taken from 1hkl.pdb and are represented in grey and yellow, respectively. The linker between the Fv and the toxin containing the furin cleavage site is shown in green. Length and conformation of the linker were chosen arbitrarily. The predicted molecular weights of LMB-70, LMB-75, and LMB-107 are 72 kDa, 48 kDa, and 62 kDa, respectively.

Figure 4

Schematics showing anti-BCMA immunotoxin variants described in this review. LMB-70: BM306-Fab linked to 24 kDa fragment of PE; LMB-107: BM306-Fv linked to 38 kDa fragment of PE; LMB-75: BM306-Fv linked to 24 kDa fragment of PE; LMB-162: BM306-Fv linked to 54 amino acid fragment albumin binding domain from Streptococcus followed by 24 kDa fragment of PE; LMB-173: BM306-Fv linked to single domain anti-albumin Llama antibody followed be 24 kDa fragment of PE; LMB-92: BM306-Fab fragment linked to less-immunogenic B-cell epitope-removed version of PE; LMB-103: BM306-Fab fragment linked to less-immunogenic T-cell epitope-removed version of PE; LMB-267: BM306-Fab fragment linked to less-immunogenic T-cell epitope-removed version of PE with Ala to Arg mutation at residue 294. Fur: Furin cleavage sequence of PE.

Figure 5

Efficacy of anti-BCMA immunotoxin in mouse bone marrow xenograft models. H929-Luc-GFP (A) and multiple myeloma (MM). 1S-Luc-GFP (B) cells were injected IV into NSG mice and were treated IV with PBS or 1.5 mg/kg immunotoxin QOD × 5. Bioluminescence imaging was used to assess tumor burden. Dorsal (D) and ventral (V) images are shown for each mouse.