Limits...
A Two-Step Pretargeted Nanotherapy for CD20 Crosslinking May Achieve Superior Anti-Lymphoma Efficacy to Rituximab.

Chu TW, Zhang R, Yang J, Chao MP, Shami PJ, Kopeček J - Theranostics (2015)

Bottom Line: Consecutive treatment with the two components resulted in CD20 clustering on the cell surface and effectively killed malignant B-cells in vivo.In a mouse model of human non-Hodgkin lymphoma (NHL), increasing the time lag from 1 h to 5 h resulted in dramatically improved tumor growth inhibition and animal survival.In summary, our approach may constitute a novel treatment for NHL and other B-cell malignancies with significant advantages over conventional chemo-immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT 84112, USA.

ABSTRACT
The use of rituximab, an anti-CD20 mAb, in combination with chemotherapy is the current standard for the treatment of B-cell lymphomas. However, because of a significant number of treatment failures, there is a demand for new, improved therapeutics. Here, we designed a nanomedicine that crosslinks CD20 and directly induces apoptosis of B-cells without the need for toxins or immune effector functions. The therapeutic system comprises a pretargeting component (anti-CD20 Fab' conjugated with an oligonucleotide1) and a crosslinking component (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer grafted with multiple complementary oligonucleotide2). Consecutive treatment with the two components resulted in CD20 clustering on the cell surface and effectively killed malignant B-cells in vivo. To enhance therapeutic efficacy, a two-step pretargeting approach was employed. We showed that the time lag between the two doses can be optimized based on pharmacokinetics and biodistribution of the Fab'-oligonucleotide1 conjugate. In a mouse model of human non-Hodgkin lymphoma (NHL), increasing the time lag from 1 h to 5 h resulted in dramatically improved tumor growth inhibition and animal survival. When the 5 h interval was used, the nanotherapy was more efficacious than rituximab and led to complete eradication of lymphoma cells with no signs of metastasis or disease recurrence. We further evaluated the nanomedicine using patient mantle cell lymphoma cells; the treatment demonstrated more potent apoptosis-inducing activity than rituximab hyper-crosslinked with secondary antibodies. In summary, our approach may constitute a novel treatment for NHL and other B-cell malignancies with significant advantages over conventional chemo-immunotherapy.

No MeSH data available.


Related in: MedlinePlus

Drug-free nanotherapeutic induces apoptosis of mantle cell lymphoma (MCL) cells from patients. Two samples were from lymph node biopsies (patients 1 and 2) and two from the peripheral blood (patients 3 and 4). Percent apoptotic cells was analyzed by annexin V binding and quantified with flow cytometry. Nanomedicine, Fab'-MORF1 followed by P-MORF2; Rituximab, rituximab followed by goat anti-mouse secondary Ab; 1F5 mAb, 1F5 followed by goat anti-mouse secondary Ab. Time interval between treatments was 1 h (unless otherwise indicated). Incubation time was 24 h. Fab' equivalent concentrations of treatments were 0.5 μM (for patients 1, 2 and 3) and 1 μM (for patients 3 and 4). Data are presented as mean ± SD (n = 2 or 3). Statistics was performed by student's t test (*: p < 0.05). All treatment groups had significantly higher apoptotic indices when compared to the untreated cells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4440441&req=5

Figure 7: Drug-free nanotherapeutic induces apoptosis of mantle cell lymphoma (MCL) cells from patients. Two samples were from lymph node biopsies (patients 1 and 2) and two from the peripheral blood (patients 3 and 4). Percent apoptotic cells was analyzed by annexin V binding and quantified with flow cytometry. Nanomedicine, Fab'-MORF1 followed by P-MORF2; Rituximab, rituximab followed by goat anti-mouse secondary Ab; 1F5 mAb, 1F5 followed by goat anti-mouse secondary Ab. Time interval between treatments was 1 h (unless otherwise indicated). Incubation time was 24 h. Fab' equivalent concentrations of treatments were 0.5 μM (for patients 1, 2 and 3) and 1 μM (for patients 3 and 4). Data are presented as mean ± SD (n = 2 or 3). Statistics was performed by student's t test (*: p < 0.05). All treatment groups had significantly higher apoptotic indices when compared to the untreated cells.

Mentions: To further evaluate the clinical potential of the designed nanomedicine, primary cells were isolated from 4 mantle cell lymphoma (MCL) patients. MCL is an aggressive and incurable subtype of B-cell NHL. We treated the cells consecutively with Fab'-MORF1 and P-MORF2, as well as rituximab (or 1F5 mAb) hyper-crosslinked with secondary antibodies. The purpose of using secondary antibodies is to reproduce the function of FcR+ immune effector cells, which partly reflects the in vivo therapeutic efficacy of anti-CD20 mAbs 12. Results showed that the designed nanomedicine effectively induced apoptosis of MCL cells from all 4 patients (Fig. 7). The percent of apoptotic cells was increased by about 2 fold when compared to the untreated cells. When compared to mAbs, the nanomedicine demonstrated superior apoptosis-inducing activity to rituximab in all 4 patient samples, and to 1F5 mAb in 1 sample (patient 1). In patient 3, we observed an increase of apoptotic index with increasing concentration for all compounds tested. The efficacy of 1F5 and rituximab seemed maximized at 1 μM, whereas the nanomedicine reached maximal apoptosis induction at a lower concentration (0.5 μM). In patient 4, we employed two different time intervals (1 h and 5 h) between the treatments. Data showed that, when the time lag was increased from 1 h to 5 h, there was no change in efficacy for the nanomedicine. However, for both mAbs, the apoptosis-inducing activity dropped when the longer time interval was used. This phenomenon can be attributed to Fc-mediated endocytosis of the mAb/CD20 complex, which was observed elsewhere by other investigators 6. These results highlight the potential of drug-free macromolecular therapeutics as a novel and potent treatment against B-cell lymphomas.


A Two-Step Pretargeted Nanotherapy for CD20 Crosslinking May Achieve Superior Anti-Lymphoma Efficacy to Rituximab.

Chu TW, Zhang R, Yang J, Chao MP, Shami PJ, Kopeček J - Theranostics (2015)

Drug-free nanotherapeutic induces apoptosis of mantle cell lymphoma (MCL) cells from patients. Two samples were from lymph node biopsies (patients 1 and 2) and two from the peripheral blood (patients 3 and 4). Percent apoptotic cells was analyzed by annexin V binding and quantified with flow cytometry. Nanomedicine, Fab'-MORF1 followed by P-MORF2; Rituximab, rituximab followed by goat anti-mouse secondary Ab; 1F5 mAb, 1F5 followed by goat anti-mouse secondary Ab. Time interval between treatments was 1 h (unless otherwise indicated). Incubation time was 24 h. Fab' equivalent concentrations of treatments were 0.5 μM (for patients 1, 2 and 3) and 1 μM (for patients 3 and 4). Data are presented as mean ± SD (n = 2 or 3). Statistics was performed by student's t test (*: p < 0.05). All treatment groups had significantly higher apoptotic indices when compared to the untreated cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4440441&req=5

Figure 7: Drug-free nanotherapeutic induces apoptosis of mantle cell lymphoma (MCL) cells from patients. Two samples were from lymph node biopsies (patients 1 and 2) and two from the peripheral blood (patients 3 and 4). Percent apoptotic cells was analyzed by annexin V binding and quantified with flow cytometry. Nanomedicine, Fab'-MORF1 followed by P-MORF2; Rituximab, rituximab followed by goat anti-mouse secondary Ab; 1F5 mAb, 1F5 followed by goat anti-mouse secondary Ab. Time interval between treatments was 1 h (unless otherwise indicated). Incubation time was 24 h. Fab' equivalent concentrations of treatments were 0.5 μM (for patients 1, 2 and 3) and 1 μM (for patients 3 and 4). Data are presented as mean ± SD (n = 2 or 3). Statistics was performed by student's t test (*: p < 0.05). All treatment groups had significantly higher apoptotic indices when compared to the untreated cells.
Mentions: To further evaluate the clinical potential of the designed nanomedicine, primary cells were isolated from 4 mantle cell lymphoma (MCL) patients. MCL is an aggressive and incurable subtype of B-cell NHL. We treated the cells consecutively with Fab'-MORF1 and P-MORF2, as well as rituximab (or 1F5 mAb) hyper-crosslinked with secondary antibodies. The purpose of using secondary antibodies is to reproduce the function of FcR+ immune effector cells, which partly reflects the in vivo therapeutic efficacy of anti-CD20 mAbs 12. Results showed that the designed nanomedicine effectively induced apoptosis of MCL cells from all 4 patients (Fig. 7). The percent of apoptotic cells was increased by about 2 fold when compared to the untreated cells. When compared to mAbs, the nanomedicine demonstrated superior apoptosis-inducing activity to rituximab in all 4 patient samples, and to 1F5 mAb in 1 sample (patient 1). In patient 3, we observed an increase of apoptotic index with increasing concentration for all compounds tested. The efficacy of 1F5 and rituximab seemed maximized at 1 μM, whereas the nanomedicine reached maximal apoptosis induction at a lower concentration (0.5 μM). In patient 4, we employed two different time intervals (1 h and 5 h) between the treatments. Data showed that, when the time lag was increased from 1 h to 5 h, there was no change in efficacy for the nanomedicine. However, for both mAbs, the apoptosis-inducing activity dropped when the longer time interval was used. This phenomenon can be attributed to Fc-mediated endocytosis of the mAb/CD20 complex, which was observed elsewhere by other investigators 6. These results highlight the potential of drug-free macromolecular therapeutics as a novel and potent treatment against B-cell lymphomas.

Bottom Line: Consecutive treatment with the two components resulted in CD20 clustering on the cell surface and effectively killed malignant B-cells in vivo.In a mouse model of human non-Hodgkin lymphoma (NHL), increasing the time lag from 1 h to 5 h resulted in dramatically improved tumor growth inhibition and animal survival.In summary, our approach may constitute a novel treatment for NHL and other B-cell malignancies with significant advantages over conventional chemo-immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT 84112, USA.

ABSTRACT
The use of rituximab, an anti-CD20 mAb, in combination with chemotherapy is the current standard for the treatment of B-cell lymphomas. However, because of a significant number of treatment failures, there is a demand for new, improved therapeutics. Here, we designed a nanomedicine that crosslinks CD20 and directly induces apoptosis of B-cells without the need for toxins or immune effector functions. The therapeutic system comprises a pretargeting component (anti-CD20 Fab' conjugated with an oligonucleotide1) and a crosslinking component (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer grafted with multiple complementary oligonucleotide2). Consecutive treatment with the two components resulted in CD20 clustering on the cell surface and effectively killed malignant B-cells in vivo. To enhance therapeutic efficacy, a two-step pretargeting approach was employed. We showed that the time lag between the two doses can be optimized based on pharmacokinetics and biodistribution of the Fab'-oligonucleotide1 conjugate. In a mouse model of human non-Hodgkin lymphoma (NHL), increasing the time lag from 1 h to 5 h resulted in dramatically improved tumor growth inhibition and animal survival. When the 5 h interval was used, the nanotherapy was more efficacious than rituximab and led to complete eradication of lymphoma cells with no signs of metastasis or disease recurrence. We further evaluated the nanomedicine using patient mantle cell lymphoma cells; the treatment demonstrated more potent apoptosis-inducing activity than rituximab hyper-crosslinked with secondary antibodies. In summary, our approach may constitute a novel treatment for NHL and other B-cell malignancies with significant advantages over conventional chemo-immunotherapy.

No MeSH data available.


Related in: MedlinePlus