The Dual Cell Cycle Kinase Inhibitor JNJ-7706621 Reverses Resistance to CD37-Targeted Radioimmunotherapy in Activated B Cell Like Diffuse Large B Cell Lymphoma Cell Lines.

The CD37 targeting radioimmunoconjugate 177Lu-lilotomab satetraxetan (Betalutin) is currently being evaluated in a clinical phase 2b trial for patients with follicular lymphoma (FL) and in a phase 1 trial for patients with diffuse large B-cell lymphoma (DLBCL). Herein we have investigated the effect of 177Lu-lilotomab satetraxetan in seven activated B-cell like (ABC) DLBCL cell lines. Although the radioimmunoconjugate showed anti-tumor activity, primary resistance was observed in a subset of cell lines. Thus, we set out to identify drugs able to overcome the resistance to 177Lu-lilotomab satetraxetan in two resistant ABC-DLBCL cell lines. We performed a viability-based screen combining 177Lu-lilotomab satetraxetan with the 384-compound Cambridge Cancer Compound Library. Drug combinations were scored using Bliss and Chou-Talalay algorithms. We identified and characterized the dual-specific CDK1/2 and AURA/B kinase inhibitor JNJ-7706621 as compound able to revert the resistance to RIT, alongside topoisomerase and histone deacetylases (HDAC) inhibitors.

Evaluating antigen targeting and anti-tumor activity of a new anti-CD37 radioimmunoconjugate against non-Hodgkin's lymphoma.

The monoclonal antibody against CD20, rituximab, alone, or as part of combination therapies, is standard therapy for non-Hodgkin's B-cell lymphoma. Despite significantly better clinical results obtained for beta-emitting radioimmunoconjugates (RICs), RICs targeting CD20 are not commonly used in medical practice, partly because of competition for the CD20 target. Therefore, novel therapeutic approaches against other antigens are intriguing. Here, the binding properties of a novel antibody against CD37 (tetulomab) were compared with those of rituximab. The therapeutic effect of (177)Lu-tetulomab was compared with (177)Lu-rituximab on Daudi cells in vitro. The biodistribution, therapeutic and toxic effects of (177)Lu-tetulomab and unlabeled tetulomab were determined in SCID mice injected with Daudi cells. The affinity of tetulomab to CD37 was similar to the affinity of rituximab to CD20, but the CD37-tetulomab complex was internalized 10-times faster than the CD20-rituximab complex. At the same concentration of antibody, (177)Lu-tetulomab was significantly more efficient in inhibiting cell growth than was (177)Lu-rituximab, even though the cell-bound activity of (177)Lu-rituximab was higher. Treatment with 50 and 100 MBq/kg (177)Lu-tetulomab resulted in significantly increased survival of mice, compared with control groups treated with tetulomab or saline. The CD37 epitope recognized by tetulomab was highly expressed in 216 out of 217 tumor biopsies from patients with B-cell lymphoma. This work warrants further pre-clinical and clinical studies of (177)Lu-tetulomab.

In vitro cytotoxicity of low-dose-rate radioimmunotherapy by the alpha-emitting radioimmunoconjugate Thorium-227-DOTA-rituximab.

PURPOSE: To determine whether the low-dose-rate alpha-particle-emitting radioimmunoconjugate (227)Th-1,4,7,10-p-isothiocyanato-benzyl-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-rituximab can be used to inactivate lymphoma cells growing as single cells and small colonies. METHODS AND MATERIALS: CD20-positive lymphoma cell lines were treated with (227)Th-DOTA-rituximab for 1-5 weeks. To simulate the in vivo situation with continuous but decreasing supply of radioimmunoconjugates from the blood pool, the cells were not washed after incubation with (227)Th-DOTA-rituximab, but half of the medium was replaced with fresh medium, and cell concentration and cell-bound activity were determined every other day after start of incubation. A microdosimetric model was established to estimate the average number of hits in the nucleus for different localizations of activity. RESULTS: There was a specific targeted effect on cell growth of the (227)Th-DOTA-rituximab treatment. Although the cells were not washed after incubation with (227)Th-DOTA-rituximab, the average contribution of activity in the medium to the mean dose was only 6%, whereas the average contribution from activity on the cells' own surface was 78%. The mean dose rates after incubation with 800 Bq/mL (227)Th-DOTA-rituximab varied from 0.01 to 0.03 cGy/min. The average delay in growing from 10(5) to 10(7) cells/mL was 15 days when the cells were treated with a mean absorbed radiation dose of 2 Gy alpha-particle radiation from (227)Th-DOTA-rituximab, whereas it was 11 days when the cells were irradiated with 6 Gy of X-radiation. The relative biologic effect of the treatment was estimated to be 2.9-3.4. CONCLUSIONS: The low-dose-rate radioimmunoconjugate (227)Th-DOTA-rituximab is suitable for inactivation of single lymphoma cells and small colonies of lymphoma cells.

Evaluation of the binding of radiolabeled rituximab to CD20-positive lymphoma cells: an in vitro feasibility study concerning low-dose-rate radioimmunotherapy with the alpha-emitter 227Th.

Radioimmunotherapy (RIT) with the alpha-emitter 227Th is currently under evaluation. 227Th is conjugated to the chimeric anti-CD20 monoclonal antibody rituximab, using the chelator p-isothiocyanato-benzyl-DOTA. In this study, the binding of 227Th-DOTA-p-benzyl-rituximab to three different CD-20-positive lymphoma cell lines, Raji, Rael, and Daudi, were evaluated. Equilibrium and kinetic binding experiments were used to determine binding parameters, including the association and dissociation rate constants, the equilibrium dissociation constants, and the total number of antigens for Raji, Rael, and Daudi cells. There were significant differences between the cell lines with respect to both Kd and the total number of antigens. Rael cells had more than three times as many antigens as the other two cell lines, and the functional Kd found for Rael cells was significantly higher than that found for Raji and Daudi cells. These results were confirmed using flow cytometry. Rituximab was found to be localized in patches on the cell membrane. The findings indicated that 227Th-labeled rituximab has relevant antigen-targeting properties for radioimmunotherapy.

A one-step method for determining the maximum number of bound antibodies, and the affinity and association rate constants for antibody binding.

OBJECTIVE: A reliable analysis of antibody binding may lead to more successful selection of the optimal antibodies. The most important parameters are affinity (equilibrium dissociation constant, Kd), the number of antigen sites on the cells (Bmax) and the on (ka) and off (kd) rate constants of binding. The affinity and the number of cellular binding sites are usually determined by equilibrium binding experiments and subsequent Scatchard analysis. The on and off rate constants are determined by kinetic binding experiments. However, it is necessary to perform two to three different types of experiment in order to determine these parameters. METHODS: We have developed an alternative one-step method based on a kinetic binding experiment and a mathematical description of antibody binding to antigen. The method was compared with kinetic and equilibrium binding methods. RESULTS: The results obtained using two different cell lines were in good agreement with results obtained with Scatchard analysis and kinetic binding experiments. CONCLUSION: An alternative one-step method for determination of parameters describing binding of antibodies to antigens on cells has been developed. The method gives reliable estimates of affinity and number of antigens and in addition gives information on the kinetics of binding.

Preparation of TH227-labeled radioimmunoconjugates, assessment of serum stability and antigen binding ability.

In this study, the feasibility of constructing radioimmunoconjugates by using the novel therapeutic candidate alpha-emitter, (227)Th, was evaluated. By use of the bifunctional chelator, p-SCN-benzyl-DOTA, (227)Th was conjugated to the two monoclonal antibodies, rituximab and trastuzumab. Their stability in 80% fetal bovine serum at 37 degrees C was measured. The immunoreactive fractions were determined by using CD20- and HER/2-positive cells, respectively. The overall labeling yield spanned from 6% to 17%. The radioimmunoconjugates demonstrated a relevant stability in serum and showed appropriate antigen-binding abilities.

Targeted cancer therapy with a novel low-dose rate alpha-emitting radioimmunoconjugate.

Alpha-emitting radionuclides are highly cytotoxic and are of considerable interest in the treatment of cancer. A particularly interesting approach is in radioimmunotherapy. However, alpha-emitting antibody conjugates have been difficult to exploit clinically due to the short half-life of the radionuclides, low production capability, or limited source materials. We have developed a novel technology based on the low-dose rate alpha-particle-emitting nuclide (227)Th, exemplified here using the monoclonal antibody rituximab. In vitro, this radioimmunoconjugate killed lymphoma cells at Becquerel per milliliter (Bq/mL) levels. A single injection of (227)Th-rituximab induced complete tumor regression in up to 60% of nude mice bearing macroscopic (32-256 mm(3)) human B-lymphoma xenografts at Becquerel per gram (Bq/g) levels without apparent toxicity. Therapy with (227)Th-rituximab was significantly more effective than the control radioimmunoconjugate (227)Th-trastuzumab and the standard beta-emitting radioimmunoconjugate for CD20(+) lymphoma(90)Y-tiuxetan-ibritumomab. Thorium-227 based constructs may provide a novel approach for targeted therapy against a wide variety of cancers.

Initial evaluation of (227)Th-p-benzyl-DOTA-rituximab for low-dose rate alpha-particle radioimmunotherapy.

Radioimmunotherapy has proven clinically effective in patients with non-Hodgkin's lymphoma. Radioimmunotherapy trials have so far been performed with beta-emitting isotopes. In contrast to beta-emitters, the shorter range and high linear energy transfer (LET) of alpha particles allow for more efficient and selective killing of individually targeted tumor cells. However, there are several obstacles to the use of alpha-particle immunotherapy, including problems with chelation chemistry and nontarget tissue toxicity. The alpha-emitting radioimmunoconjugate (227)Th-DOTA-p-benzyl-rituximab is a new potential anti-lymphoma agent that might overcome some of these difficulties. The present study explores the immunoreactivity, in vivo stability and biodistribution, as well as the effect on in vitro cell growth, of this novel radioimmunoconjugate. To evaluate in vivo stability, uptake in balb/c mice of the alpha-particle-emitting nuclide (227)Th alone, the chelated form, (227)Th-p-nitrobenzyl-DOTA and the radioimmunoconjugate (227)Th-DOTA-p-benzyl-rituximab was compared in a range of organs at increasing time points after injection. The immunoreactive fraction of (227)Th-DOTA-p-benzyl-rituximab was 56-65%. During the 28 days after injection of radioimmunoconjugate only, very modest amounts of the (227)Th had detached from DOTA-p-benzyl-rituximab, indicating a relevant stability in vivo. The half-life of (227)Th-DOTA-p-benzyl-rituximab in blood was 7.4 days. Incubation of lymphoma cells with (227)Th-DOTA-p-benzyl-rituximab resulted in a significant antigen-dependent inhibition of cell growth. The data presented here warrant further studies of (227)Th-DOTA-p-benzyl-rituximab.