Drug-free Macromolecular Therapeutics Induce Apoptosis in Cells Isolated from Patients with B Cell Malignancies with Enhanced Apoptosis Induction by Pretreatment with Gemcitabine

Background

Treatment of Non-Hodgkin lymphoma is challenging as the disease comprises over 35 different subtypes with the most prevalent types being diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL) and mantle cell lymphoma (ML).¹ The majority of NHLs (85%) are of B cell origin, and more than 95% of B cell lymphomas bear the cell surface antigen CD20, one of the most reliable biomarkers and therapeutic targets for B cells.² Rituximab (RTX), the first anti-CD20 monoclonal antibody approved by FDA for clinical use in 1997, initiated a new era for treatment of B cell NHLs.³ Its combination with chemotherapy can further enhance the therapeutic activity and still remains mainstay of therapy in young and fit patients.⁴ With the development of new, more active modern chemotherapy protocols and targeted therapies, much progress has been made over the past two decades.⁵ Unfortunately, relapsed or resistant disease is still a major cause of treatment failure.⁶

Recently we have developed a new paradigm for the treatment of B cell malignancies, drugfree macromolecular therapeutics (DFMT),^7–13 which initiates apoptosis by the biorecognition of a pair of complementary motifs at the cell surface resulting in crosslinking of CD20 receptors. As no toxin or cytotoxic agent is involved, DFMT does not induce the side effects of traditional chemotherapy. The effectiveness of this innovative crosslinking CD20 approach has been proven in vitro^8–11 and in vivo.^8,9,12 Preliminary results with patient cells have been also published,^12,13 in which Fab’ fragment of 1F5 anti-CD20 monoclonal antibody (mAb) conjugated with a 25-base pair morpholino oligonucleotide (Fab_1F5’-MORF1) was administered to patient cells as bispecific engager, then a crosslinking effector composed of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer grafted with multiple copies of complementary morpholino oligonucleotide P-(MORF2)x was used to crosslink CD20 via hybridization of MORF1/MORF2 and trigger B cell apoptosis. Human serum albumin conjugated with MORF2 is also under investigation to perform as a crosslinking effector.¹⁴

Chronic lymphocytic leukemia (CLL), the most common form of adult leukemia is usually a slowly progressive disease of autoreactive mature B cells. Normally, these tumor cells have low sensitivity to anti-CD20 mAbs.¹⁵ Although many CLL cases have an indolent course, there is considerable heterogeneity and some patients rapidly transform into aggressive lymphomas.¹⁶ Various somatic mutations and the mutational status of immunoglobulin heavy chain variable genes (IGHV) have been associated with outcome. Non-mutated IGHV is associated with a worse outcome. Genomic aberrations often observed are 17p deletion (17p-), 11q deletion (11q), trisomy 12 (+12), 13q deletion (13q-), and others.¹⁷ Mutations of NOTCH1 and SF3B1 are also observed with relatively high frequency in CLL patients.¹⁸ Currently, standard first-line therapies for CLL include chemoimmunotherapy that incorporate anti-CD20 mAb, or targeted therapies, depending on risk factors. We have previously shown that that DFMT-based CD20 receptor crosslinking system is cytotoxic to CLL cells and has advantages over current cytotoxic therapies and immunotherapies.¹³ Moreover, we found that pre-incubation of CD20-low expression RTX-resistant B cells with gemcitabine upregulated CD20 level and enhanced apoptosis.^9,19 Interestingly, the CD20 enhancement appeared to be time-dependent but not concentration-dependent. Consequently, we hypothesized that in vivo a long circulating HPMA copolymer – gemcitabine conjugate (2P-GEM)²⁰ should be more efficient than free gemcitabine. Indeed, we validated this hypothesis in an animal model of NHL.⁹

Herein we evaluated the efficacy of DFMT on a large group of primary tumor cells from patients with various B cell lymphomas (N=9) or CLL (N=35), including a variety of genotypes. We also tested whether combined gemcitabine with DFMT will enhance therapeutic efficacy in the patient cells with low CD20 expression. Our results suggest that DFMT has significant activity against CLL cells with a spectrum of somatic mutations and that gemcitabine pretreatment may enhance efficacy of CD20-targeted therapies in B-cell malignancies with low CD20 expression.

Methods

Results

The efficacy of drug-free macromolecular therapeutics (DFMT) was assessed in 44 samples including 35 CLL along with other B cell lymphomas such as diffuse large B cell lymphoma (DLBCL), marginal zone lymphoma (MZL), follicular lymphoma (FL), mantle cell lymphoma (MCL) etc (Table 1). The isolated cells were treated with Fab’-MORF1 and P-(MORF2)_9.5 (9.5 MORF2 per chain) consecutively with a 1 h interval. Rituximab (RTX) or 1F5 were used to produce Fab’ fragments for Fab’-MORF1 synthesis. In all treatments, conjugates were added at a concentration of 1 μM Fab’ equivalent (0.5 μM of mAb). Results of apoptosis assays are summarized in Table 2. P1–P6 cells were treated with Fab’_(1F5)-MORF1/P-(MORF2)_9.5 while P7–P44 cells were treated with Fab’_(RTX)-MORF1/P-(MORF2)_9.5. Patients P1–P9 had various B cell lymphomas while patients P10-P44 had chronic lymphocytic leukemia (CLL). In samples P1–P9, DFMT showed significant efficacy in inducing apoptosis compared to controls in all samples except for samples P5 and P6. In samples P2, P4, P7 and P9, DFMT showed superior efficacy at apoptosis induction compared to RTX treatment. P4 and P9 were MCL samples. P1, P3 and P5 were DLBCL samples. P6 and P8 were MZL samples, while P2 was a FL sample. All MCL samples responded to DFMT treatment while 2 of 3 samples of DLBCL responded to DFMT treatment. One MZL sample and one FL responded to DFMT treatment. These results demonstrated that DFMT strategy works on different types of B-cell lymphoma tested here.

Regarding CLL samples, a variety of chromosomal abnormalities, including 17p13 deletion (N=4), 11q22 deletion (N=3), and 13q14 deletion (N=25) have been observed. Altogether Fab’_(RTX)-MORF1/P-(MORF2)_9.5 induced cell apoptosis effectively in 65.9% of samples (Table 2).

When sufficient cells were available, a detailed evaluation of apoptosis induction by DFMT was performed. A typical example such an analysis is shown in Figure 1 (Sample P12). In that analysis, we evaluated surface CD20 expression (Fig. 1A), plasma membrane rupture (Fig. 1B), caspase 3 activation (Fig. 1C), genomic DNA fragmentation (Fig. 1D), mitochondrial membrane permeabilization (Fig. 1E), and Bcl-2 inhibition (Fig. 1F). Also, confocal imaging was used to document specific biorecognition at the cell surface, first biorecognition of Fab’-MORF1 by CD20 followed by biorecognition/hybridization of MORF1/MORF2 (Fig 1G). The results showed that DFMT was effective on inducing apoptosis (Fig. 1B–F). The high CD20 expression in sample P12 was associated with a substantially increased response to DFMT treatment (Table 2). As shown in Fig. 1B, cell population shifted from left lower zone to right lower zone tremendously demonstrating significantly increased cell apoptosis. Cell cycle assay (Fig. 1D) also showed that with DFMT and RTX/GAH treatment, more cells were trapped in Sub G1 phase indicating increased amount of apoptotic cells. Especially in the group treated with DFMT, the number of apoptotic cells was nearly twice higher than that treated with RTX/GAH. Decreased expression of anti-apoptotic protein Bcl-2 was observed with treatment of DFMT and RTX/GAH, respectively (Fig. 1F), showing that inhibition of Bcl-2 is involved in the apoptosis induction mechanism of DFMT treatment. Confocal imaging (Fig. 1G) showed biorecognition events and colocalization of the two nanoconjugates at the cell surface. Specificity of binding to CD20 was demonstrated by blocking self-assembly at the cell using pre-treatment with excess RTX.

Our results suggest that DFMT has therapeutic potential for different types of B-cell malignancies. Statistical analysis shows that the level of surface CD20 expression is an important factor affecting therapeutic efficacy. Additional work with a larger set of patient samples from various diagnoses is needed to identify other critical features that are important in the response to CD20 targeting and to identify subsets of patients suitable for DFMT treatment.

A promising strategy to further improve the therapeutic efficacy of DFMT is to use gemcitabine pretreatment. GEM is used to treat different types of malignancies and can lead to accumulation of cells in S phase.²² Importantly, GEM was shown to increase CD20 expression level in B cells.¹⁹ As CD20 expression level is a critical factor affecting the therapeutic efficacy of antiCD20 therapy, combination of CD20-targeting therapy with GEM could potentially increase the efficacy of the DFMT. We pre-treated cells from samples P40, P42 and P43 for 48 h with GEM at 500 ng/mL as these samples had low CD20 expression levels. Cells were then washed and treated with DFMT as well as RTX/GAH. GEM pretreatment significantly increased CD20 expression compared to controls without GEM pretreatment (Fig. 2A, 2C). Representative results of flow cytometry showed increased apoptosis (Fig. 2D). Correspondingly, statistical analysis showed increased apoptosis level cells with GEM pretreatment (Fig. 2B). Alternatively, P26 cells were pretreated with a long-circulating HPMA copolymer-GEM conjugate (2P-GEM). Cells pretreated with 2P-GEM showed higher CD20 expression as well as increased apoptosis levels (Fig. 2E, 2F).

Discussion

B cell malignancies affect millions of people worldwide with significant morbidity and mortality. The DFMT B cell depletion strategy is able to avoid the adverse effects induced by rituximab treatment with promising therapeutic efficacy. Data presented here validate that DFMT treatment has therapeutic potential against a wide variety of B cell lymphoma subtypes. In many samples, DFMT showed statistically significant increases in apoptosis when compared to RTX/GAH, indicating that the proposed treatment could have better therapeutic efficacy, at least in certain cases. However, in several samples, DFMT showed only comparable or even or lower efficacy. Different factors may be at play, including differences in the level of expression of CD20. Expression level of CD20 was the dominant strength in determination of therapeutic potential of treatments. Patient samples with high CD20 expression levels were more likely to show increased apoptosis following treatment with DFMT. The higher the CD20 expression level, the better the therapeutic efficacy of the treatment as shown in Figure 3.

Genomic aberration is also a factor affecting traditional therapies. The most common cytogenetic mutation in CLL is deletion of 13q14, detected in around 50% of CLL patients.²³ It might not be sufficient to be considered an adverse prognostic factor.²⁴ However, 13q14 deletion is a critical genomic aberration for increased apoptotic resistance. The 13q14 region is usually described as a negative regulator of the anti-apoptotic protein Bcl-2.²⁴ Mutation of this region increases the expression of Bcl-2, allowing CLL cells to survive, resulting in lower apoptosis levels induced by treatment. The incidence of 17p deletion in CLL patients is close to 10% and is associated with an inferior prognosis.²³ The 17p13 region is involved in important regulatory functions. Its deletion is believed to be a strong independent marker in evaluating death caused by disease. p53 tumor suppressor gene is deleted in patients with 17p13 deletion. P53 deficiency may allow cells to survive from therapeutic treatment as p53-dependent apoptosis pathway is interrupted.²³ Deletion of 11q occurs in 10–20% of CLL patients.²⁵ The 11q22 region is involved in activation of p53-dependent apoptosis pathway thus its loss leads to reduced cell death.²³ Combination of deletions of 17p and 11q are considered as significantly poorer prognoses.²⁶ We evaluated 25 primary patient samples with 13q14 deletion, 4 samples with 17p13 deletion and 3 samples with 11q22 deletion. Previously, we had shown that DFMT induced efficient CD20 crosslinking-mediated apoptosis in patient cells with 17p13 deletion.¹³ International prognostic index (IPI) is used in clinics to predict outcome of individual patients based on patient’s clinical characteristics including genomic aberrations. Higher IPI values indicate more problems in treatment. Based on the results summarized in Table 2 with patient information in Table 1, therapeutic efficacy of our DFMT treatment on CLL primary patient samples varied regarding to similar genomic aberrations. Seven samples with different mutations were selected for evaluation (Figs. 3A and 3B). Fig. 3A shows that the higher the CD20 expression level, the higher the DMFT mediated apoptosis induction. Fig. 3B demonstrates that the value of IPI does not hamper the efficacy of DFMT. For example, both samples P18 and P31 have 13q14 and 17p dual deletions. IPI of P18 is 4 while IPI of P31 is 7, indicating a worse outcome prediction of sample P31. However, DFMT exhibited superior apoptosis induction in P31 sample over P18 sample, demonstrating that CD20 expression level is the determinant factor affecting therapeutic efficacy of DFMT. Similar phenomena were observed in P39 and P43 patient samples with IPI score of 3. With the same IPI score, DFMT showed much higher efficiency in apoptosis induction in P39 compared to P43 as P39 possessed a higher CD20 expression level. It seems that CD20 expression level could overcome the anti-apoptotic effect involved with critical deletions.

It appears that it is essential to increase CD20 expression level to improve therapeutic efficacy of DFMT treatment. Exposure to gemcitabine was reported to increase CD20 expression level.²² The increase in CD20 expression resulted from gemcitabine-induced NF-κB activation as NF-κB binds to the CD20 promoter to induce CD20 transcriptional activation.^9,15 With patient samples P26, P40, P42 and P43, cells were pretreated with GEM or 2P-GEM. Figure 2 shows that pretreatment with GEM or polymer conjugated GEM (2P-GEM) increases expression level of CD20 receptor significantly, thus increasing the efficacy of DFMT in inducing apoptosis. These results support the observation that CD20 expression level is the key factor for DFMT treatment efficacy. Combination therapy of DFMT with GEM would be a promising strategy to improve its therapeutic efficacy in insensitive samples. More work is needed to confirm the potential increased efficacy of the combination of GEM with CD20-targeted therapy. In addition, comprehensive studies will be conducted with GEM and 2P-GEM pretreatment on patient cells, CD20+ B cell lines, and on animal models to assure the feasibility of combination strategy.

In summary, apoptosis induction by DFMT in primary cells from patients with various B cell malignancies was evaluated. DFMT effectively increased apoptosis of tumor cells from patients with a variety of B cell malignancies, irrespective of genomic aberrations. In CLL samples, the CD20 expression level is positively correlated with susceptibility to DFMT-induced apoptosis. Pretreatment with GEM or HPMA copolymer-GEM conjugate improved DFMT therapeutic efficacy in poorly responding samples, likely due to increased CD20 expression. With increased CD20 expression, DFMT treatment is able to overcome the anti-apoptotic effect involved in critical genomic aberrations. The results suggest that DFMT is a promising modality for the treatment of different B cell malignancies. Especially with RTX-resistant B cell malignancies, combination therapy of DFMT with GEM provides a prospective solution to overcome drug resistance. In addition, crosslinking of receptors as a design principle for novel therapeutic approaches is applicable to other receptors such as CD79b,²⁷ prostate stem cell antigen,²⁸ and DR5.²⁹