Improved Therapy of B-Cell Non-Hodgkin Lymphoma by Obinutuzumab-Dianthin Conjugates in Combination with the Endosomal Escape Enhancer SO1861

Abstract

Immunotoxins do not only bind to cancer-specific receptors to mediate the elimination of tumor cells through the innate immune system, but also increase target cytotoxicity by the intrinsic toxin activity. The plant glycoside SO1861 was previously reported to enhance the endolysosomal escape of antibody-toxin conjugates in non-hematopoietic cells, thus increasing their cytotoxicity manifold. Here we tested this technology for the first time in a lymphoma in vivo model. First, the therapeutic CD20 antibody obinutuzumab was chemically conjugated to the ribosome-inactivating protein dianthin. The cytotoxicity of obinutuzumab-dianthin (ObiDi) was evaluated on human B-lymphocyte Burkitt's lymphoma Raji cells and compared to human T-cell leukemia off-target Jurkat cells. When tested in combination with SO1861, the cytotoxicity for target cells was 131-fold greater than for off-target cells. In vivo imaging in a xenograft model of B-cell lymphoma in mice revealed that ObiDi/SO1861 efficiently prevents tumor growth (51.4% response rate) compared to the monotherapy with ObiDi (25.9%) and non-conjugated obinutuzumab (20.7%). The reduction of tumor volume and overall survival was also improved. Taken together, our results substantially contribute to the development of a combination therapy with SO1861 as a platform technology to enhance the efficacy of therapeutic antibody-toxin conjugates in lymphoma and leukemia.

Keywords: anti-CD20; cancer treatment; controlled drug release; dianthin; endocytosis; endosomal escape; glycosylated triterpenoids; immunotoxins; obinutuzumab; targeted toxins.

Figure A1

Chemical conjugation of obinutuzumab and dianthin. The toxin was attached to the antibody through a covalent bond introduced by the cleavable linker SPDP and non-cleavable Sulfo-SMCC in four reaction steps shown in panels (a) through (d).

Figure A2

Experimental scheme of the treatment with ObiDi and obinutuzumab in a mouse lymphoma model. At day –14, SCID CB17 mice received luciferase-transduced Raji cells (0.25 × 10⁶) intravenously. Two weeks later, mice received either (i) 15 µg SO1861 in 100 µL PBS subcutaneously into the neck in combination with 1.8 µg ObiDi in 100 µL PBS intraperitoneally 1 h later, (ii) 1.8 µg ObiDi as in experiment (i), (iii) 1.8 µg Obinutuzumab in 100 µL PBS intraperitoneally or (iv) 100 µL PBS as mock-treated control. The mice were alternately treated every three and four days for four weeks. The live imaging to visualize tumors was conducted every week for six weeks.

Figure 1

Purification of ObiDi. Step 1: Cation exchange chromatography to get rid of the parental antibody. (a) Elution profile (milli-absorbance at 280 nm versus elution volume, blue line) of the cation exchange chromatography with an optimized salt step gradient for elution. The two main peaks contained obinutuzumab (fractions no. 2–6) and ObiDi (fractions no. 7–9), red line. The green line represents the programmed gradient of NaCl (0–100% of buffer B) where buffer A is 20 mM HEPES buffer at pH 7.4 and buffer B is buffer A containing 2.0 M NaCl. The brown line represents the factual conductivity as a result of the salt gradient. (b) The fractions 1–9 obtained by cation exchange chromatography (a) were evaluated by (b) SDS-PAGE and (c) Western blotting with a His-tag-HRP antibody under non-reducing conditions. The different proteins are indicated by red arrows. Lane 1: obinutuzumab; lane 2: dianthin; lane 3: molecular mass marker; lane 4: obinutuzumab and dianthin after chemical conjugation but before cation exchange chromatography; lane 5: obinutuzumab and dianthin after chemical conjugation and after cation exchange chromatography (flow through). Unconjugated antibody does not contain a His-tag and can therefore not be visualized in the presented Western blot providing evidence that the high molecular mass bands represent conjugate. The conjugation reaction mixture (raw material) is too diluted to visualize the conjugate with Coomassie staining.

Figure 2

Purification of ObiDi. Step 2: Protein-A affinity chromatography to get rid of free dianthin. The conjugate obtained from cation exchange chromatography (lane “Elution from IEC”) was applied to the column and elution fractions E1 to E7 were collected. All samples were evaluated by (a) SDS-PAGE and (b) Western blotting with a His-tag-HRP antibody under non-reducing conditions. A conjugate of ObiDi with DAR 1 is visible in elution fractions E3 and E4. Free dianthin was successfully removed.

Figure 3

Undirected cytotoxicity of dianthin in the presence of the endosomal escape enhancer SO1861. Raji and Jurkat cells (20,000 cells/well) were seeded into a 96-well plate and allowed to grow for 24 h. They were then treated with (a) dianthin or (b) non-conjugated dianthin simply mixed with obinutuzumab. All proteins were applied at concentrations ranging from sub-femtomolar to upper nanomolar range. SO1861 was added at a final concentration of 1 µg/mL one hour before. Cells were incubated for 72 h in the presence of the compounds. Finally, cell proliferation was measured by an XTT assay and obtained values referred to untreated cells. Points in the graphs represent the mean ± SD of three biological experiments (n = 3) each one of them conducted in technical triplicate.

Figure 4

Cytotoxicity of (a) free obinutuzumab and (b) the conjugate ObiDi for target Raji and off-target Jurkat cells both in the presence of SO1861. The experiment was conducted as described in the legend of Figure 3. Each point in the graphs represents the mean ± SD of three biological experiments (n = 3) each one of them conducted in technical triplicate.

Figure 5

Examples for live imaging of metastases during treatment with either (a,b) ObiDi/SO1861, ObiDi, PBS or (c) obinutuzumab with (a,c) dorsal and (b,c) ventral view. The change in size of single metastases was quantified by using an IVIS Lumina imaging system 5 min after injecting luciferin intraperitoneally. The total flux values that correlate directly with tumor mass were measured weekly as indicated on the right side of the panels. The † indicates week of death.

Figure 5

Examples for live imaging of metastases during treatment with either (a,b) ObiDi/SO1861, ObiDi, PBS or (c) obinutuzumab with (a,c) dorsal and (b,c) ventral view. The change in size of single metastases was quantified by using an IVIS Lumina imaging system 5 min after injecting luciferin intraperitoneally. The total flux values that correlate directly with tumor mass were measured weekly as indicated on the right side of the panels. The † indicates week of death.

Figure 6

Treated mice indicating that the conjugate is well tolerated (Table 1). If abnormalities occurred in the mice, such as signs of paralysis, this always correlated with metastases affecting the central nervous system. There were no phenomena that could be directly assigned to the treatment.