Double attack strategy for leukemia using a pre-targeting bispecific antibody (CD20 Ab×mPEG scFv) and actively attracting PEGylated liposomal doxorubicin to enhance anti-tumor activity

doi:10.21203/rs.3.rs-84119/v1

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Double attack strategy for leukemia using a pre-targeting bispecific antibody (CD20 Ab×mPEG scFv) and actively attracting PEGylated liposomal doxorubicin to enhance anti-tumor activity

https://doi.org/10.21203/rs.3.rs-84119/v1

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Journal Publication

published 09 Jan, 2021

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Background

Tumor-targeted nanoparticles hold great promise as new tools for therapy of liquid cancers. Furthermore, the therapeutic efficacy of nanoparticles can be improved by enhancing the cancer cellular internalization.

Methods

In this study, we developed a humanized bispecific antibody (BsAbs: CD20 Ab×mPEG scFv) which retains the clinical anti-CD20 whole antibody (Ofatumumab) and is fused with anti-mPEG single chain antibodies (scFv) that can target the systemic liquid tumor cells. This combination achieves the therapeutic function and simultaneously “grabs” PEGylated liposomal doxorubicin (PLD) to enhance the cellular internalization and anticancer activity of PLD.

Results

We successfully constructed the CD20 Ab×mPEG scFv and proved that CD20 Ab×mPEG scFv can target CD20-expressing Raji cells and simultaneously grab PEGylated liposomal DiD increasing the internalization ability up to 60% in 24 h. We further showed that the combination of CD20 Ab×mPEG scFv and PLD successfully led to a 9-fold increase in tumor cytotoxicity (LC 50 : 0.38 nM) compared to the CD20 Ab×DNS scFv and PLD (lC 50 : 3.45 nM) in vitro . Importantly, a combination of CD20 Ab×mPEG scFv and PLD had greater anti-liquid tumor efficacy ( P = 0.0005) in Raji-bearing mice than CD20 Ab×DNS scFv and PLD.

Conclusion

Our results indicate that this “double-attack” strategy using CD20 Ab×mPEG scFv and PLD can retain the tumor targeting ( first attack ) and confer PLD tumor-selectivity ( second attack ) to enhance PLD internalization and improve therapeutic efficacy in liquid tumors.

Biomedical Engineering

Cancer Biology

Drug Discovery, Design, & Development

bispecific antibody (CD20 Ab×mPEG scFv)

liquid tumors

internalization

PEGylated nanoparticle

specific targeting

B-cell lymphoproliferative disorders such as acute lymphoblastic leukemia (ALL) are characterized primarily by an overproduction of immature lymphocytes in the bone marrow which results in the suppression of normal haematopoiesis and infiltration of various extramedullary sites[1]. Rituximab, is a chimeric monoclonal antibody (mAb) that binds to CD20. It is expressed on the membrane of up to 90% of late pre-B and mature B-cells as well as in the majority of B-cell lymphoproliferative disorders[2, 3], and can activate complement-dependent cytotoxicity (CDC) via the Fc domain of Rituximab and antibody-dependent cellular cytotoxicity (ADCC) based on the additional contact to Fcγ receptors[2]. In the clinic, the use of doxorubicin hydrochloride combined with Rituximab is an effective therapy for relapsed and refractory leukemia[4]. However, the cardiotoxicity potential due to the low tumor-targeting and multidrug resistance remain issues with doxorubicin hydrochloride usage. Thus, doxorubicin hydrochloride has been used in liposomal doxorubicin formulations like PEGylated liposomal doxorubicin (PLD) which are less cardiotoxic whilst showing no significant difference with conventional doxorubicin on overall survival of acute lymphoblastic leukemia patients[5, 6]. According to previous studies, nanoparticles may accumulate and be retained in solid tumors through the enhanced permeation and retention (EPR) effect[7, 8]. However, the EPR effect is present in tumors of more than ~ 100 mm³ in volume and hampers the use of nanoparticles for targeting small or unvascularized metastases[9]. We hypothesized that the lack of an improvement in outcome of PLD against leukemia may due to the non-specific targeting cytotoxicity and a reduced EPR effect. Therefore, to retain the anti-tumor effects of the therapeutic antibody, development of a strategy to enhance PLD tumor selectivity against liquid tumors is important.

In this study, we developed a humanized bispecific antibody (BsAbs: CD20 Ab × mPEG scFv) which retains the clinical anti-CD20 mAb (Ofatumumab)[10] and is fused with humanized single-chain antibodies (scFv) of an anti-mPEG antibody[11] that can actively accumulate the PEGylated liposomal doxorubicin (PLD) to enhance the cytotoxicity against liquid tumors (Fig. 1A). We examined the bi-specific binding activity of CD20 Ab × mPEG scFv to CD20-expressing leukemia cell line (Raji) and PLD. The enhanced internalization of PLD mediated by CD20 Ab × mPEG scFv was proved with flow cytometry. In addition, the tumor-specific cytotoxicity of CD20 Ab × mPEG scFv combined with PLD was investigated and compared with control BsAbs (CD20 Ab × DNS scFv) combined with PLD and PLD alone in Raji cells. In vivo, the therapeutic efficacy of CD20 Ab × mPEG scFv combined with PLD was determined and compared with control BsAbs combined with PLD in the Raji-bearing leukemia model. The CD20 Ab × mPEG scFv provided a powerful “double-attack” method to treat liquid tumors such as leukemia, by actively attracting PLD with tumor-specificity and enhance PLD internalization under conditions in which the EPR effect is lessened. The CD20 Ab×mPEG scFv is a promising next-generation antibody drug to treat liquid tumors.

Figure 1. Schematic of the “double-attack” strategy: bispecific antibody combined with PEGylated liposomal doxorubicin. The bispecific antibody (CD20 mAb × mPEG scFv) can simultaneously target the systemic liquid tumor cells, retain the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (first attack); and actively grab the PEGylated liposomal doxorubicin (PLD) (second attack) to enhance the internalization and anticancer activity of PLD for liquid tumor therapy.

Characterization of CD20 Ab × mPEG scFv

We investigated the bi-specific function of CD20 Ab × mPEG scFv, which was constructed by linking single-chain variable fragment of humanized anti-mPEG Ab via a flexible peptide (GGGSGGG) to the C-terminus of Ofatumumab. CD20 Ab × hapten dansyl (DNS) scFv, which binds to the small chemical hapten densyl was used as the negative control BsAb (Fig. 2A). The recombinant BsAbs were transfected into Expi293F cells for production on a large-scale and purified by Protein A. The SDS-PAGE analysis showed the purity and expected molecular weight of BsAbs were composed of 76 kDa heavy chain-scFv fragments (HC-scFv) and 25 kDa light chain (LC); the CD20 Ab was composed of a 55 kDa HC fragment and a 25 kDa LC fragment in a reducing condition (Fig. 2B). We further evaluated the anti-CD20 function by incubating the CD20-expressing Raji cells with 70 nM CD20 Ab × mPEG scFv, CD20 Ab × DNS scFv and CD20 Ab and detection was conducted by anti-human Fc Ab-FITC via flow cytometry. The flow intensity, which was similar to CD20 Ab alone indicated that CD20 Ab × mPEG scFv can target to the CD20-expressing Raji cells as well as CD20 Ab alone (Fig. 2C). Next, the bi-specific binding activity (CD20 × mPEG) of 70 nM CD20 Ab × mPEG scFv were further verified by incubating with 10 µM PEGylated liposomal DiD after BsAbs targeting within CD20-espressing Raji cells and the BsAb-bound PEGylated liposomal DiD fluorescence intensity was then measured by flow cytometry. Figure 2D shows the fluorescence intensity of the CD20 Ab × mPEG scFv group shift indicating that only the CD20 Ab × mPEG scFv can specifically target to PEGylated liposomal DiD. We concluded that CD20 Ab × mPEG scFv can retain the antibody targeting and mediate selective delivery of PEGylated liposomes, thus having a bi-specific binding function.

Figure 2. Characterization of CD20 Ab × mPEG scFv, CD20 Ab × DNS scFv and CD20 mAb. (A) The gene constructs of BsAbs are composed of a signal peptide (SP), the anti-CD20 light chain (LC), internal ribosome entry site (IRES), the anti-CD20 heavy chain (HC), a flexible linker peptide (LK) and an anti-PEG scFv (CD20 Ab × mPEG scFv) or control anti-DNS scFv (CD20 Ab × DNS scFv). (B) The SDS-PAGE of purified CD20 Ab (Ab), CD20 Ab × DNS scFv (DNS) and CD20 Ab × mPEG scFv (PEG) under reducing conditions. M: PageRuler pre-stained protein ladder (Fermentas). (C) The CD20 functions and (D) bispecific binding (CD20 and mPEG) function of BsAbs on Raji (CD20+) cells were detected by anti-Fc-FITC or PEGylated lipo-DiD respectively via flow cytometry.

Pre-targeting of CD20 Ab × mPEG scFv can enhance the cellular internalization of PEGylated liposomes.

The endocytosis of liposomes is important for cancer therapy. Next, we investigated how the pre-targeting of CD20 Ab × mPEG scFv contributes to the cellular internalization of PEGylated liposome to enhance the cellular cytotoxicity against Raji cells. We incubated the Raji cells with CD20 Ab × mPEG scFv, CD20 Ab × DNS scFv and CD20 Ab for 1 h and then stained them with PEGylated liposomal DiD (lipo-DiD) for 1 h, 6 h, 12 h and 24 h at 37 °C. The lipo-DiD on the surface of the Raji cells was detected by staining the mouse anti-PEG antibody (6.3 antibody)[13] and then staining with anti-mouse Fc Ab-FITC. We detected the two signals, lipo-DiD (red fluorescence) and FITC (green fluorescence) by flow cytometry. While the intensity of lipo-DiD did not change, the signal of green fluorescence decreased indicating the uptake of lipo-DiD after combining with CD20 Ab × mPEG scFv. The Q2 region of the dot plot representing Lipo-DiD can be captured by anti-PEG Ab on the cell membrane and the Q3 region shows the signal of anti-PEG Ab decreased but signal of Lipo-DiD still remained showing that anti-PEG Ab cannot capture the Lipo-DiD on the cell membrane as the Lipo-DiD internalized into the cells. Figure 3A shows that green fluorescence decreases from 83.6–36.6% in time dependent manner, but the total Lipo-DiD signal (Q2 + Q3) does not change (from 92.6–93.51%), while, the Q3 fluorescence increases from 9.91–56% from 0 h to 24 h. These results indicate that the CD20 Ab × mPEG scFv enhances internalization of lipo-DiD towards Raji cells. The membrane Lipo-DiD decreased (Q2) and most of Lipo-DiD was internalized into cells (Q3) from 9.91–56% in a time dependent manner (Fig. 3B). Moreover, the lipo-DiD alone or combined with CD20 Ab × DNS scFv (control group) showed no green fluorescence intensity detection. Therefore we concluded that only CD20 Ab × mPEG scFv can gradually enhance cell uptake of liposomes, possibly because of efficient internalization by actively attracting by anti-mPEG scFv portion of the BsAbs. We further hypothesized that the internalization enhancement of PEGylated liposomes increased cytotoxicity by combining with CD20 Ab × mPEG scFv.

Figure 3. The internalization and PEGylated liposome DiD uptake of CD20 Ab × mPEG scFv in Raji cells. (A) The internalization of CD20 Ab × mPEG scFv combined with Lipo-DiD into Raji cells at different time points. The surface Lipo-DiD was measured via flow cytometry using a mouse anti-PEG backbone antibody. (B) Quantification of the mean fluorescence of DiD (red) and formula of DiD uptake efficiency (blue).

Pre-targeting of CD20 Ab × mPEG scFv actively attracts PLD to enhance cytotoxicity of Raji cells.

We thus next investigated whether CD20 Ab × mPEG scFv could enhance the cytotoxicity of PLD against Raji cells. First, we incubated the Raji cells with 140 nM CD20 Ab × mPEG scFv or CD20 Ab × DNS scFv for specific binding in 20 min and then graded concentrations of PLD were added at 37 °C for 3 h. The cells were refreshed with new medium and cultured for 72 h to allow time for the doxorubicin to kill the cancer cells. We then assessed the proliferation of the remaining cells by ATPlite Luminescence Assay System. As shown in Fig. 4A, CD20 Ab × mPEG scFv combined with PLD was found to be more cytotoxic than the control group. The LC₅₀ value of CD20 Ab × mPEG scFv combined with PLD (LC₅₀ value = 0.38 nM) was approximately 9.15-fold lower than that for CD20 Ab × DNS scFv combined with PLD (LC₅₀ value = 3.45 nM), and 11.3-fold lower than PLD alone (LC₅₀ value = 4.3 nM) as shown in Fig. 4B. Therefore, we concluded that CD20 Ab × mPEG scFv can confer tumor selectivity and increase the cytotoxicity.

Figure 4. CD20 Ab × mPEG scFv enhances PLD anti-proliferative activity. (A) Raji cells were incubated with CD20 Ab × mPEG scFv (red squares), CD20 Ab × DNS scFv (green triangles) for 20 min followed by addition of serial dilutions of PEGylated liposomal doxorubicin (PLD) in triplicate for 3 h. The cells were also incubated with culture medium for 20 min followed by addition of serial dilutions of PLD (blue circles). Cell viability was measured with ATPlite Luminescence Assay System after 72 h of incubation. (B) The half maximal effective concentration (EC50) values of Raji cells treated with CD20 Ab × mPEG scFv plus PLD, CD20 Ab x DNS scFv plus PLD or PLD alone were analyzed. Data are shown as mean ± s.d. PEG BsAb, CD20 Ab × mPEG scFv; DNS BsAb, CD20 Ab × DNS scFv; PLD, PEGylated liposomal doxorubicin.

Pharmacokinetic characterizations of CD20 Ab × mPEG scFv

To further investigate the pharmacokinetics of the bispecific antibodies, 1 mg/kg CD20 Ab × mPEG scFv and CD20 Ab × DNS scFv were injected intravenously into BALB/c mice (n = 3), then the serum samples were collected at various times and the concentrations of bispecific antibodies were determined by sandwich ELISA. Plasma samples at a dilution of 600-fold and graded concentrations of Herceptin as standard antibody were examined by the horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc antibody. Color development was measured at 405 nm on a microplate reader. The half-life (t_1/2) of CD20 Ab × mPEG scFv was 14.14 h and control BsAb CD20 Ab × DNS scFv was 12.17 h (Fig. 5A).

Pre-targeting of CD20 Ab × mPEG scFv actively attracts PLD increasing therapeutic efficacy in vivo

To attain a model mimicking systemic tumor, Raji (CD20+) cells were transplanted intravenously into female SCID mice via tail vein. After 7 days, the mice were randomly administered injections of PBS, CD20 Ab × mPEG scFv and CD20 Ab × DNS scFv. The PLD were injected after 72 h of BsAbs administration. Mice were sacrificed at the onset of paralysis or when the body weight dropped below 80% of the initial weight; otherwise, the mice were maintained until 100 days and considered long-term survivors. The group treated with CD20 Ab × mPEG scFv combined with PLD had significantly longer survival time than the control group injected with PBS (*P = 0.015) and CD20 Ab × DNS scFv combined with PLD (*P < 0.05) (Fig. 5B). The results indicate that the combination of CD20 Ab × mPEG scFv and PLD can enhance the therapeutic efficacy against leukemia xenograft models.

Figure 5. Pharmacokinetics and therapeutic efficiency of BsAbs plus PLD. (A) Balb/c mice were intravenously injected with 1 mg kg-1 PEG BsAb (black circles) or DNS BsAb (white circles). Mean plasma concentrations of the BsAbs were measured by sandwich ELISA (n = 3 mice). Data are shown as mean ± SD. n = 3. (B) Groups of eight SCID mice bearing Raji cells were intravenously injected with 1 mg kg-1 PEG BsAb plus 2 mg kg-1 PLD (blue circles), 1 mg kg-1 DNS BsAb plus 2 mg kg-1 PLD (red squares), 1 mg kg-1 PLD (green triangles), 1 mg kg-1 PEG BsAb (purple triangles) or PBS (yellow diamonds). Results are presented as means (n = 8). PEG BsAb, CD20 Ab × mPEG scFv; DNS BsAb, CD20 Ab × DNS scFv; PLD, PEGylated liposomal doxorubicin. Data are shown as mean ± SD. n = 8.

In this report we provide a novel “double attack” strategy against liquid tumors by using CD20 Ab × mPEG scFv and PEGylated liposomal doxorubicin (PLD). CD20 Ab × mPEG scFv can increase the cellular internalization of PLD from 36.6–83.6% cellular uptake after 24 h, and enhance the cellular cytotoxicity to 11 times LD₅₀ compared with PLD alone in vitro (Fig. 3). Furthermore, we observed that in Raji-bearing mice, a combination of CD20 Ab × mPEG scFv and PLD showed a superior therapeutic effect by actively accumulating PLDs against circulating Raji cells. Importantly, this double-attack strategy can be adapted for use with any kind of therapeutic antibodies to target different tumor antigens (e.g., CD19, EGFR, HER2, etc.) to become second generation antibody drugs. In addition, the anti-PEG scFv can be combined with diverse PEGylated nanocarriers like liposomes containing different active pharmaceutical ingredients such as paclitaxel (PCX), irinotecan and so on[14–16]. Similar to PEGylated liposome formulations, polymeric micelles have also been considered to be a promising platform and could be combined with our “double-attack” strategy for cancer therapy[17]. We believe the combination of CD20 Ab × mPEG scFv and PEGylated nanoparticles can be effectively used in the clinic to treat liquid tumors such as B-cell lymphoproliferative disorders and become the next generation antibody drugs and a novel therapeutic therapy.

To confer liposomes with active leukemia targeting in circulation is important[18]. Several strategies have been used to chemically conjugate antibodies to liposomes to enhance leukemia specificity. Cong Wu and colleagues chemically conjugated liposomal adriamycin (ADR) with Fab fragments of rituximab by reacting the maleimide group. They proved the CD20-targeted liposome can specifically target the tumor site and significantly prolong the survival of tumor-bearing mice[19]. Sun and colleagues conjugated the liposomal daunorubicin with CD123/CD33 antibody by thiolation, and attained higher cellular uptake that was 1.8-times in acute myeloid leukemia (AML) cells and cytotoxicity was increased 1.53-fold (P < 0.001)[20]. Strop et al. used microbial transglutaminase that catalyzed bond formation between the acyl group of glutamine side chain and primary amine of a lysine side chain to conjugate an amine-containing drug onto an engineered glutamine on the antibody[21]. However, chemical conjugation caused liposome heterogeneity as the most functional groups (amino, carboxyl, thiol groups) are abundant in targeting ligands and lead to limitations in clinical applications for targeted liposomes[21, 22]. Here, our CD20 Ab × mPEG scFv provides the specificity of CD20 targeting the liquid tumor and induces the ADCC and CDC effects during the “first attack”. The “second attack” occurs when naïve PLDs are injected and actively bind to the mPEG scFv of CD20 Ab × mPEG scFv and enhance the cellular cytotoxicity against liquid tumor cells. The “double-attack” significantly increases the survival rate of Raji-bearing mice in vivo (P = 0.0005) in the absence of EPR effect. Our strategy overcomes the disadvantages of chemical conjugation and confers the naïve PLD tumor specificity by the mPEG scFv of CD20 Ab × mPEG scFv under non-chemical conjugation of PLD.

The endocytosis ability of liposomes is essential for cancer therapeutic efficacy. Previous studies have revealed the importance of internalization ability to increase the nanoparticle cytotoxicity efficacy against liquid tumors[23, 24]. Sapra provided evidence which showed that using internalizing anti-CD19-immunoliposomes compared with the non-internalizing anti-CD20-immunoliposomes significantly improved the therapeutic outcome, prolonging the lifespan of Namalwa cell-bearing mice (P < 0.001)[25]. Chuang and colleagues also designed endocytic and nonendocytic receptors, low-density lipoprotein receptor (LDLR) and a truncated LDLR (ΔLDLR) lacking the NPXY signal motif for endocytosis, replaced the ligand binding domain with αPEG and stably expressed it on the surface of HCC36 cells. After PLD treatment, HCC36/αPEG-LDLR tumors were significantly suppressed at day 63 as compared with HCC36/αPEG-ΔLDLR tumors in vivo. These results indicate that targeting endocytic receptors can indeed enhance the therapeutic efficacy of PLD[26]. Our CD20 Ab × mPEG scFv first targets the liquid tumor cells and induces the ADCC and CDC effects, then the BsAbs remain on the liquid tumor cell membranes until crosslinked with the mPEG part of PLD. There are around 4528 mPEG-DSPEs on a 100 nm liposome[27, 28] and many PEGs on the liposome like “Hedgehog” or multiple epitopes which can trigger endocytosis after the PLD has attached mPEG scFv of BsAb[29]. In liquid tumors, our CD20 Ab × mPEG scFv prove that BsAbs combined with PLD can enhance the internalization rate from 10.6–60.5% via receptor-mediated internalization (Fig. 3B) and lead to 11-fold cytotoxicity in comparison with ordinary combination therapy in vitro. Therefore, our CD20 Ab × mPEG scFv can increase the internalization ability of PLD to enhance the liquid tumor cytotoxicity.

It is essential to develop a flexible and changeable BsAbs platform in response to resistance of leukemia. Mechanisms of leukemia resistance in vivo are not clear. CD20 loss expression after treatment with rituximab has been reported that results in a CD20 negative phenotype and in resistance to rituximab[30, 31]. Therefore, being able to quickly change the therapeutic target such as CD19[32], CD22[33] is important for the leukemia resistant treatment. For instance, CD19 which is expressed on B-lineage cells becomes a replaceable leukemia target for immunotherapy and has lately been advanced into clinical trials such as anti-CD19 antibody denintuzumab, anti-CD19/CD3 BiTE antibody blinatumumab[34]. Another strategy to deal with leukemia resistance is third generation anti-CD19 CAR-T cells (CTL019 T cells)[35], which provided patients with up to 2 years longer survival against acute lymphoblastic leukemia (ALL)[36]. However, multiple trials have also reported patient relapses due to CD19-negative malignant B cells after anti-CD19 CAR-T cell treatment. In order to combat the resistance of CD19-negative cells, CD19/CD20 CAR-T cells have been used in a clinical trial (NCT04007029) in treating patients with recurrent or refractory B-cell lymphoma. This illustrates that it is important to generate “flexible” treatment strategies to overcome leukemia resistance. Our CD20 Ab × mPEG scFv strategy provides an “adaptable” option (clinical mAb × mPEG) for leukemia-resistant treatment as the CD20-end of the BsAb can be replaced by any kind of anti-leukemia tumor marker Ab after CD20 resistance. Another type of drug resistance occurs when leukemia cells express drug resistant proteins, MDR1 and MRP1, which act as drug efflux pumps, which can mediate the doxorubicin resistance[37, 38]. The anti-mPEG scFv of BsAbs can actively “grab” any kind of liposomal drug that is modified with PEG such as PEGylated polymeric micelles[39], Irinotecan[40] or other PEGylated liposomal drugs in clinical use[41]. Therefore, the double-attack strategy of CD20 Ab × mPEG scFv provides adaptable and wide ranging options for combating liquid tumor resistance by swapping in and out different leukemia tumor markers (anti-tumor end) or combining any kind of PEGylated drugs (anti-mPEG end). In conclusion, the “double-attack” strategy of CD20 Ab × mPEG scFv and PLD provides an effective way to treat liquid tumors with the following advantages: (1) Non-chemical conjugation: the CD20 Ab × mPEG scFv can confer the PLD with liquid tumor specificity by anti-PEG scFv of the BsAb and maintain antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) effects. (2) Cellular internalization ability enhancement: After PLD binding, the BsAbs increase the internalization ability of PLD to enhance the cellular cytotoxicity compared to the naïve PLD. (3) Drug resistance issues: our strategy can combat drug resistance by flexibly replacing the therapeutic antibody or any kind of PEGylated chemotherapy drugs in response to shedding of antigens or multi-drug resistance genes in liquid tumors. Moreover, the CD20 Ab × mPEG scFv retains the Fc domain which can cause antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) to improve the cytotoxicity against Raji cells. Our CD20 Ab × mPEG scFv provides an effective double attack strategy against liquid tumors and may become second generation antibody drugs.

Enhancement of tumor-targeting and cellular internalization can improve the therapeutic efficacy of liposomal drugs toward liquid cancer cells. We developed a humanized bispecific antibody (CD20 Ab × mPEG scFv) which retains the anti-CD20 targeting and therapeutic function, whilst simultaneously attracting PEGylated liposomal doxorubicin (PLD) to enhance the internalization and anticancer activity of PLD against liquid cancer cells. This “double-attack” strategy, using CD20 Ab × mPEG scFv and PLD, enhanced liquid-tumor therapeutic efficacy under non-chemical conjugation and maintained antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) effects. Moreover, shedding of antigens or multi drug resistance issues related with liquid tumors can be addressed by flexibly replacing the therapeutic antibody or any kind of PEGylated chemotherapy drugs by using this strategy. We believe this double-attack strategy, combining CD20 Ab × mPEG scFv and PEGylated nanoparticles, can be applied in the clinic to treat liquid tumors and become next generation antibody drugs, and a novel therapeutic therapy.

Cells and animals

Expi293F cells (Thermo Fisher Scientific) were cultured in Expi293 expression medium (Thermo Fisher Scientific) on shakers (25 mm shaking diameter) with a shake speed of 120 rpm in a humidified atmosphere of 8% CO₂ in air at 37 °C. Raji cells were a kind gift from Dr. Dr. Steve R. Roffler (Institute of Biomedical Sciences, Academia Sinica, Taiwan) and cultured in RPMI1640 (Sigma-Aldrich) supplemented with 6 g/L HEPES, 2 g/L NaHCO₃, 10% heat-inactivated BCS (HyClone), penicillin (100 U/mL), and streptomycin (100 mg/mL) at 37 °C in a humidified atmosphere of 5% CO₂ in air. No authentication besides confirming surface expression levels of CD20 was performed by the authors. Healthy 3- to 6-week-old female BALB/c mice and SCID mice (C.B17/Icr-Prkdc^scid/CrlNarl) were purchased from the National Laboratory Animal Center, Taipei, Taiwan. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Kaohsiung Medical University and approved by the Institutional Animal Care and Use Committee (IACUC) of Kaohsiung Medical University (IACUC number: 105169).

Construction and expression of bispecific antibodies

Human BsAbs were created by linking the C-terminus of an anti-mPEG scFv (clone h15-2b)[11] or anti-dansyl (anti-DNS) scFv to an anti-CD20 antibody (Ofatumumab, clone: 2F2) via a flexible peptide (GGGGS)₃ to form CD20 Ab × mPEG scFv and CD20 Ab × DNS scFv, respectively. The anti-DNS scFv has been described previously[12]. The VL-Cκ and VH-CH1-CH2-CH3-linker-scFv domains were separated with an IRES in the pLNCX retroviral vector (BD Biosciences, San Diego, CA) in the unique Hind III and Cla I restriction enzyme sites to generate pLNCX- CD20 Ab × mPEG scFv and pLNCX- CD20 Ab × DNS scFv plasmids. Each plasmid was scaled up by transformation into TOP10 E. coli, mixed with 100 ml Luria broth in a 250 ml baffled flask and shaken O/N at 220 r.p.m. Large-scale plasmid purifications were conducted using the PureLink HiPure Plasmid Midiprep Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. For protein production, plasmids were transfected into Epxi293F cells using ExpiFectamine and protocols provided by the manufacturer (Thermo Fisher Scientific). Transfected cells were grown at 37 °C in an 8% CO₂ incubator while shaking at 125 r.p.m. for 5 days. Secreted protein was harvested by centrifugation at 1500 r.p.m. for 5 min. Supernatants were passed through 0.22 µm filters for purification. Purifications were conducted by directly incubating 250 ml transfected supernatant with 3 ml PBS-washed Protein G beads (Millipore). Beads were washed twice with PBS and once with PBS diluted tenfold. Protein was eluted from the beads by adding 3 ml 0.01 M citric acid, pH 3.0. After harvesting, the eluents were immediately neutralized by adding 1 ml 0.1 M Tris, pH 9.0. Protein molecular weight was found by SDS-PAGE.

Bi-specific function of the CD20 Ab × mPEG scFv by flow cytometry

The anti-CD20 and anti-PEG ability of the BsAbs were determined by incubating 2 × 10⁵ Raji with 35 nM (200 µl) BsAbs for 1 h on ice. Unbound antibodies were removed by extensive washing in cold PBS containing 0.05% (wt/vol) BSA, followed by the addition of 1 µM (200 µl) PEGylated Lipo-DiD (DiD-labeled mPEGylated liposome, FormuMax) for 1 h on ice. After removal of unbound PEGylated Lipo-DiD by extensive washing in cold PBS containing 0.05% (wt/vol) BSA, the surface fluorescence of viable cells was measured on a FACScan flow cytometer (Merck Flow Cytometer, Guava easyCyte System)

Internalization ability of PEGylated liposomal DiD mediated by CD20 Ab × mPEG scFv by flow cytometry

The internalization of the BsAbs was determined by incubating 5 × 10⁵ Raji with 35 nM BsAbs for 30 min on ice and then the unbound antibodies were removed by extensive washing in cold PBS containing 0.05% (wt/vol) BSA. The cells were treated with 2.5 µM PEGylated Lipo-DiD (Thermo Fisher Scientific) for 1 h on ice. After removal of unbound probes by extensive washing in cold PBS containing 0.05% (wt/vol) BSA, the cells were transferred to fresh culture medium and incubated for 6, 12, 24 h at 37 °C. Control cells were incubated at 4 °C for 24 h. PEGylated Lipo-DiD on the surface of Raji cells was determined by staining with 10 µg ml^− 1 6 − 3 anti-PEG antibody for 30 min and 4 µg ml^− 1 goat anti-mouse IgG Fcγ-FITC (Jackson ImmunoResearch Laboratories). After extensive washing with PBS, the fluorescence of cells was measured with a Cytomics FC500 flow cytometer. Internalization rate is expressed as percentage luminescence of goat anti-mouse IgG Fcγ-FITC according to the following formula: percentage (%) of Q3/(Q2 + Q3).

In vitro cytotoxicity of CD20 Ab × mPEG scFv combined with PLD

Raji cell lines (4 × 10³/well) were incubated with CD20 Ab × mPEG scFv or CD20 Ab × DNS scFv at 20 nM at RT for 20 min in a 96-well U-bottom plate (Nalge Nunc International, Roskilde, Denmark). PEGylated liposomal doxorubicin (PLD, TTY Biopharm Co.) was then added to the plate at the following final concentration: 50, 10, 2, 0.4 and 0.08 µg mL^− 1 for 3 h and the supernatant was replaced with 100 µL RPMI1640 medium containing 10% heat-inactivated bovine calf serum (BCS) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). Cell viability was measured with ATPlite Luminescence Assay System (Perkin-Elmer, Waltham, MA, USA) after 72 h of incubation. Results are expressed as percentage cell viability of luminescence as compared with untreated cells according to the following formula: percentage (%) cell viability = 100 × (treated luminescence/untreated luminescence). Data are presented as the mean of three independent experiments. The half maximal effective concentration (LC₅₀) values were calculated by fitting the data to a log (inhibitor) versus response (variable slope model) with GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA).

Half-life of CD20 Ab × mPEG scFv in BALB/c mice

CD20 Ab × mPEG scFv (1 mg kg^− 1) or CD20 Ab × DNS scFv (1 mg kg^− 1) were injected intravenously (i.v.) into BALB/c mice (n = 3). Blood samples were collected via retro-orbital bleeds from each animal at the following time points: 30 min, 6 h, 12 h; and 1, 2, 3 and 7 d, and processed to collect serum. Sera collected were stored at approximately − 80 °C until analyzed by enzyme-linked immunosorbent assay (ELISA) for antibody concentrations. Ninety-six-well plates (Nalge Nunc International, Roskilde, Denmark) coated with 1 nM Goat anti-human IgG Fab for 2 h with 0.1 M NaHCO₃ buffer, pH 9 at 37 °C. The plates were blocked overnight with PBS containing 5% (wt/olv) skim milk at 4 °C. The plates were washed three times with PBS with 0.02% (wt/vol) Tween20 (PBST) and one time with PBS. Plasma samples at dilution of 600-fold in 50 µL of PBS containing 2% (wt/vol) skim milk and graded concentrations of Herceptin as standard antibody were added to the plates for 1 h at room temperature (RT). The plates were washed three times with PBS with 0.02% Tween20 (PBST) and one time with PBS, followed by the addition of 0.4 µg mL^− 1 horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc antibody (Jackson Immunoresearch Laboratories, West Grove, PA, USA). The plates were washed again and bound peroxidase activity was measured by adding 150 µL/well ABTS solution [0.4 mg mL-1, 2,2′-Azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid] (Sigma-Aldrich, St. Louis, MO, USA), 0.01% (v/v) H₂O₂, and 100 mM phosphate-citrate, pH4.0] for 1 h at RT. Color development was measured at 405 nm on a microplate reader (Molecular Devices, Menlo Park, CA, USA).

Treatment efficacy of CD20 Ab × mPEG scFv combined with PLD in Raji-bearing mice

Mice were injected via the tail vein with 1 × 10⁶ Raji cells. One week later, the inoculated mice were divided into groups (n = 6 or 7) and administered via the tail vein with different group treatments (in 100 µL PBS) every other day. The treatments were: (1) PBS (100 µL), (2) CD20 Ab × mPEG scFv (1 mg kg^− 1), and (3) CD20 Ab × DNS scFv (1 mg kg^− 1). After 72 h, the mice were intravenously administrated PLD (2 mg kg^− 1). Raji cell progression was monitored weekly and the injections caused the mice to become paralyzed. Mice were sacrificed at the onset of paralysis or when the body weight dropped below 80% of the initial value; otherwise, the mice were maintained until 125 days and considered to be long-term survivors. Data was analyzed using GraphPad Prism 6.0. Survival was compared using Logrank test. Statistical significance was set at P < 0.05.

CD20, Cluster of Differentiation 20

BsAb, bispecific antibody

mPEG, methyl polyethylene glycol

mAb, monoclonal antibody

scFv, single chain fragment variable

Fab, antigen-binding fragment

HC-scFv, heavy chain-scFv fragments

LC, light chain

PLD, PEGylated liposomal doxorubicin

IC₅₀, half maximal inhibitory concentration

DNS, Dansyl

ALL, acute lymphoblastic leukemia

CDC, complement-dependent cytotoxicity

ADCC, antibody-dependent cellular cytotoxicity

EPR, enhanced permeation and retention

PBS, phosphate buffered saline

PBST, phosphate buffered saline w/ Tween20

BSA, bovine serum albumin

HRP, horseradish peroxidase

RT, room temperature

EGFR, epidermal growth factor receptor

HER2, human epidermal growth factor receptor 2

Conflicts of Interest

The authors declare no conflict of interest.

Funding

This work was supported by grants from the Ministry of Science and Technology, Taipei, Taiwan (MOST 107-2320-B-037-024 -MY3, MOST 107-2320-B-037-028-MY2 and MOST 108-2311-B-037-001-MY2); the National Health Research Institutes, Taiwan (NHRI-EX108-10729EI); Academia Sinica, Taiwan (AS-TP-107-L11); the Ministry of Education, Taiwan (108RSB0029); and the KMU-KMUH Co-Project of Key Research (KMU-DK109001) and Research Foundation (KMU-DK109004) from Kaohsiung Medical University, Taiwan.

Authors' contributions

Kai-Wen Ho designed and performed experiments reported in the paper, analyzed data and wrote the manuscript; I-Ju Chen and Yi-An Cheng helped with experiments, data analysis and contributed to manuscript editing; Tzu-Yi Liao and En-Shuo Liu helped the internalization experiments, data analysis for internalization; Dr. Yun-Chi Lu, Dr. Yu-Cheng Su helped with data analysis; Bo-Cheng Huang and Hui-Ju Liu contributed to manuscript editing; Dr. Ming-Yii Huang and Dr. Chiao-Yun Chen provided the information of leukemia clinical treatments and current challenges; Dr. Steve R. Roffler provided the concept and contributed to manuscript writing and editing; Dr. Tian-Lu Cheng provided the concept, experimental design and contributed to manuscript writing and editing.

Acknowledgements

This study is also supported partially by Kaohsiung Medical University Research Center Grant (Drug Development and Value Creation Research Center) (KMU-TC108A03).

Cobaleda C, Sanchez-Garcia I. B-cell acute lymphoblastic leukaemia: towards understanding its cellular origin. Bioessays. 2009;31:600–9.
Engelhard M. Anti-CD20 antibody treatment of non-Hodgkin lymphomas. Clin Immunol. 2016;172:101–4.
Zhao L, Xie F, Tong X, Li H, Chen Y, Qian W, Duan S, Zheng J, Zhao Z, Li B, et al. Combating non-Hodgkin lymphoma by targeting both CD20 and HLA-DR through CD20-243 CrossMab. MAbs. 2014;6:740–8.
Casulo C, Byrtek M, Dawson KL, Zhou X, Farber CM, Flowers CR, Hainsworth JD, Maurer MJ, Cerhan JR, Link BK, et al. Early Relapse of Follicular Lymphoma After Rituximab Plus Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone Defines Patients at High Risk for Death: An Analysis From the National LymphoCare Study. J Clin Oncol. 2015;33:2516–22.
Hunault-Berger M, Leguay T, Thomas X, Legrand O, Huguet F, Bonmati C, Escoffre-Barbe M, Legros L, Turlure P, Chevallier P, et al. A randomized study of pegylated liposomal doxorubicin versus continuous-infusion doxorubicin in elderly patients with acute lymphoblastic leukemia: the GRAALL-SA1 study. Haematologica. 2011;96:245–52.
Schmitt CJ, Dietrich S, Ho AD, Witzens-Harig M. Replacement of conventional doxorubicin by pegylated liposomal doxorubicin is a safe and effective alternative in the treatment of non-Hodgkin's lymphoma patients with cardiac risk factors. Ann Hematol. 2012;91:391–7.
Blanco E, Shen H, Ferrari M. Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat Biotechnol. 2015;33:941–51.
Maeda H, Bharate GY, Daruwalla J. Polymeric drugs for efficient tumor-targeted drug delivery based on EPR-effect. Eur J Pharm Biopharm. 2009;71:409–19.
Plummer EM, Manchester M. Viral nanoparticles and virus-like particles: platforms for contemporary vaccine design. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2011;3:174–96.
Gravanis I, Ersboll J, Skovlund E, Abadie E, Marty M, Pignatti F. The European Medicines Agency review of ofatumumab (Arzerra(R)) for the treatment of chronic lymphocytic leukemia in patients refractory to fludarabine and alemtuzumab: summary of the scientific assessment of the European medicines agency committee for medicinal products for human use. Oncologist. 2010;15:1335–43.
Kao CH, Wang JY, Chuang KH, Chuang CH, Cheng TC, Hsieh YC, Tseng YL, Chen BM, Roffler SR, Cheng TL. One-step mixing with humanized anti-mPEG bispecific antibody enhances tumor accumulation and therapeutic efficacy of mPEGylated nanoparticles. Biomaterials. 2014;35:9930–40.
Chuang KH, Cheng CM, Roffler SR, Lu YL, Lin SR, Wang JY, Tzou WS, Su YC, Chen BM, Cheng TL. Combination cancer therapy by hapten-targeted prodrug-activating enzymes and cytokines. Bioconjug Chem. 2006;17:707–14.
Cheng TL, Cheng CM, Chen BM, Tsao DA, Chuang KH, Hsiao SW, Lin YH, Roffler SR. Monoclonal antibody-based quantitation of poly(ethylene glycol)-derivatized proteins, liposomes, and nanoparticles. Bioconjug Chem. 2005;16:1225–31.
Hong SS, Choi JY, Kim JO, Lee MK, Kim SH, Lim SJ. Development of paclitaxel-loaded liposomal nanocarrier stabilized by triglyceride incorporation. Int J Nanomedicine. 2016;11:4465–77.
Yoshino K, Nakamura K, Terajima Y, Kurita A, Matsuzaki T, Yamashita K, Isozaki M, Kasukawa H. Comparative studies of irinotecan-loaded polyethylene glycol-modified liposomes prepared using different PEG-modification methods. Biochim Biophys Acta. 2012;1818:2901–7.
Zhang L, Cao DY, Wang J, Xiang B, Dun JN, Fang Y, Xue GQ. PEG-coated irinotecan cationic liposomes improve the therapeutic efficacy of breast cancer in animals. Eur Rev Med Pharmacol Sci. 2013;17:3347–61.
Wang J, Li S, Han Y, Guan J, Chung S, Wang C, Li D. Poly(Ethylene Glycol)-Polylactide Micelles for Cancer Therapy. Front Pharmacol. 2018;9:202.
Golombek SK, May JN, Theek B, Appold L, Drude N, Kiessling F, Lammers T. Tumor targeting via EPR: Strategies to enhance patient responses. Adv Drug Deliv Rev. 2018;130:17–38.
Wu C, Li H, Zhao H, Zhang W, Chen Y, Yue Z, Lu Q, Wan Y, Tian X, Deng A. Potentiating antilymphoma efficacy of chemotherapy using a liposome for integration of CD20 targeting, ultra-violet irradiation polymerizing, and controlled drug delivery. Nanoscale Res Lett. 2014;9:447.
Sun S, Zou H, Li L, Liu Q, Ding N, Zeng L, Li H, Mao S. CD123/CD33 dual-antibody modified liposomes effectively target acute myeloid leukemia cells and reduce antigen-negative escape. Int J Pharm. 2019;568:118518.
Strop P, Liu SH, Dorywalska M, Delaria K, Dushin RG, Tran TT, Ho WH, Farias S, Casas MG, Abdiche Y, et al. Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates. Chem Biol. 2013;20:161–7.
Manjappa AS, Chaudhari KR, Venkataraju MP, Dantuluri P, Nanda B, Sidda C, Sawant KK, Murthy RS. Antibody derivatization and conjugation strategies: application in preparation of stealth immunoliposome to target chemotherapeutics to tumor. J Control Release. 2011;150:2–22.
Sapra P, Allen TM. Improved outcome when B-cell lymphoma is treated with combinations of immunoliposomal anticancer drugs targeted to both the CD19 and CD20 epitopes. Clin Cancer Res. 2004;10:2530–7.
Lee Y, Kischuk E, Crist S, Ratliff TL, Thompson DH. Targeting and Internalization of Liposomes by Bladder Tumor Cells Using a Fibronectin Attachment Protein-Derived Peptide-Lipopolymer Conjugate. Bioconjug Chem. 2017;28:1481–90.
Sapra P, Allen TM. Internalizing antibodies are necessary for improved therapeutic efficacy of antibody-targeted liposomal drugs. Cancer Res. 2002;62:7190–4.
Chuang KH, Wang HE, Chen FM, Tzou SC, Cheng CM, Chang YC, Tseng WL, Shiea J, Lin SR, Wang JY, et al. Endocytosis of PEGylated agents enhances cancer imaging and anticancer efficacy. Mol Cancer Ther. 2010;9:1903–12.
Kirpotin D, Park JW, Hong K, Zalipsky S, Li WL, Carter P, Benz CC, Papahadjopoulos D. Sterically stabilized anti-HER2 immunoliposomes: design and targeting to human breast cancer cells in vitro. Biochemistry. 1997;36:66–75.
Cheng YA, Chen IJ, Su YC, Cheng KW, Lu YC, Lin WW, Hsieh YC, Kao CH, Chen FM, Roffler SR, Cheng TL. Enhanced drug internalization and therapeutic efficacy of PEGylated nanoparticles by one-step formulation with anti-mPEG bispecific antibody in intrinsic drug-resistant breast cancer. Biomater Sci. 2019;7:3404–17.
Su YC, Burnouf PA, Chuang KH, Chen BM, Cheng TL, Roffler SR. Conditional internalization of PEGylated nanomedicines by PEG engagers for triple negative breast cancer therapy. Nat Commun. 2017;8:15507.
Perez-Callejo D, Gonzalez-Rincon J, Sanchez A, Provencio M, Sanchez-Beato M. Action and resistance of monoclonal CD20 antibodies therapy in B-cell Non-Hodgkin Lymphomas. Cancer Treat Rev. 2015;41:680–9.
Davis TA, Czerwinski DK, Levy R. Therapy of B-cell lymphoma with anti-CD20 antibodies can result in the loss of CD20 antigen expression. Clin Cancer Res. 1999;5:611–5.
Wang K, Wei G, Liu D. CD19: a biomarker for B cell development, lymphoma diagnosis and therapy. Exp Hematol Oncol. 2012;1:36.
Shah NN, Stevenson MS, Yuan CM, Richards K, Delbrook C, Kreitman RJ, Pastan I, Wayne AS. Characterization of CD22 expression in acute lymphoblastic leukemia. Pediatr Blood Cancer. 2015;62:964–9.
Naddafi F, Davami F. Anti-CD19 Monoclonal Antibodies: a New Approach to Lymphoma Therapy. Int J Mol Cell Med. 2015;4:143–51.
Vairy S, Garcia JL, Teira P, Bittencourt H. CTL019 (tisagenlecleucel): CAR-T therapy for relapsed and refractory B-cell acute lymphoblastic leukemia. Drug Des Devel Ther. 2018;12:3885–98.
Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin NJ, Chew A, Gonzalez VE, Zheng Z, Lacey SF, et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med. 2014;371:1507–17.
Chen J, Wei H, Cheng J, Xie B, Wang B, Yi J, Tian B, Liu Z, Wang F, Zhang Z. Characteristics of doxorubicin-selected multidrug-resistant human leukemia HL-60 cells with tolerance to arsenic trioxide and contribution of leukemia stem cells. Oncol Lett. 2018;15:1255–62.
Baran Y, Gur B, Kaya P, Ural AU, Avcu F, Gunduz U. Upregulation of multi drug resistance genes in doxorubicin resistant human acute myelogeneous leukemia cells and reversal of the resistance. Hematology. 2007;12:511–7.
Sharma AK, Zhang L, Li S, Kelly DL, Alakhov VY, Batrakova EV, Kabanov AV. Prevention of MDR development in leukemia cells by micelle-forming polymeric surfactant. J Control Release. 2008;131:220–7.
Rosen LS. Irinotecan in lymphoma, leukemia, and breast, pancreatic, ovarian, and small-cell lung cancers. Oncology (Williston Park). 1998;12:103–9.
Bulbake U, Doppalapudi S, Kommineni N, Khan W. Liposomal Formulations in Clinical Use: An Updated Review. Pharmaceutics 2017, 9.

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Double attack strategy for leukemia using a pre-targeting bispecific antibody (CD20 Ab×mPEG scFv) and actively attracting PEGylated liposomal doxorubicin to enhance anti-tumor activity

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Abstract

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Background

Results

Characterization of CD20 Ab × mPEG scFv

Pharmacokinetic characterizations of CD20 Ab × mPEG scFv

Discussion

Conclusions

Materials And Methods

Cells and animals

Construction and expression of bispecific antibodies

Bi-specific function of the CD20 Ab × mPEG scFv by flow cytometry

Internalization ability of PEGylated liposomal DiD mediated by CD20 Ab × mPEG scFv by flow cytometry

Half-life of CD20 Ab × mPEG scFv in BALB/c mice

Treatment efficacy of CD20 Ab × mPEG scFv combined with PLD in Raji-bearing mice

List Of Abbreviations

Declarations

References

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