Specific Conjugation of the Hinge Region for Homogeneous

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Specific conjugation of the hinge region for homogeneous preparation of antibody fragment-drug conjugate: a case study for doxorubicin-PEG-anti-CD20 Fab’ synthesis Zhan Zhou, Jing Zhang, Yan Zhang, Guanghui Ma, and Zhiguo Su Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00626 • Publication Date (Web): 23 Dec 2015 Downloaded from http://pubs.acs.org on December 30, 2015

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Bioconjugate Chemistry

Specific conjugation of the hinge region for homogeneous preparation of antibody fragment-drug conjugate: a case study for doxorubicin-PEG-anti-CD20 Fab’ synthesis Zhan Zhou,†, ‡ Jing Zhang, *,† Yan Zhang†, Guanghui Ma,† and Zhiguo Su*,† †

National Key Laboratory of Biochemical Engineering, Institute of Process

Engineering, Chinese Academy of Sciences, No.1 Beierjie Street, Zhongguancun, Haidian District, Beijing 100190, China ‡

University of Chinese Academy of Sciences, Beijing 100049, China

ACS Paragon Plus Environment


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ABSTRACT Conventional preparation strategies for antibody-drug conjugates (ADCs) result in heterogeneous products with various molecular size and species. In this study, we developed a homogenous preparation strategy by site-specific conjugation of the anticancer drug with antibody fragment. The model drug doxorubicin (DOX) was coupled to the Fab’ fragment of anti-CD20 IgG at its permissive sites through a heterotelechelic PEG linker, generating an antibody fragment-drug conjugate (AFDC). Anti-CD20 IgG was digested and reduced specifically with β-mercaptoethylamine to generate Fab’ fragment with two free mercapto groups in its hinge region. Meanwhile, DOX was conjugated with α-succinimidylsuccinate ω-maleimide polyethylene glycol (NHS-PEG-MAL) to form MAL-PEG-DOX, which was subsequently linked to the free mercapto containing Fab’ fragment to form a Fab’-PEG-DOX conjugate. The dual site-specific bioconjugation was achieved through the combination of highly selective reduction of IgG and the introduction of heterotelechelic PEG linker. The resulting AFDC provides an utterly homogeneous product, with a definite ratio of one fragment with two drugs. Laser confocal microscopy and cell ELISA revealed that the AFDC could accumulate in the antigen-positive Daudi tumor cell. In addition, the Fab’-PEG-DOX retained appreciable targeting ability and improved anti-tumor activity, demonstrating excellent therapeutic effect on lymphoma mice model for better cure rate and significantly reduced side effects.


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Table of Contents Graphic



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INTRODUCTION Antibody-drug conjugates (ADCs) are a new class of highly potent biopharmaceuticals designed for targeting therapy of various cancers.1, 2 The unique targeting capabilities of monoclonal antibodies are combined with the cancer-killing ability of cytotoxic drugs to allow sensitive discrimination between healthy tissues and

diseased sites,3 leading to improved anti-cancer therapy with superior efficacy

and lower side effects.4 So far there are more than 45 products have entered clinical trials in different stages, with two approved by the FDA.5-8 Compared with ADC with a full IgG molecule, the antibody fragment Fab’ possesses an advantage of small size enabling better transportation or penetration in

vivo to the target, leading to increased therapeutic effects.9 Furthermore, the elimination of Fc fragment in ADC could also reduce potential immunogenicity.10 Therefore, antibody fragment-drug conjugate (AFDC), instead of whole ADC, would be a desired candidate for tumor therapy, although the short circulation time and quick elimination rate of Fab’ should be overcome. In the literature, AFDC is also called ADC.11 As the field is growing rapidly, it is reasonable to distinguish these two products for accuracy purpose and for further development. Both ADC and AFDC have a common problem of product heterogeneity. Conventional methods for linking the drug to the antibody often utilize the ω-amino groups of lysine residues for conjugation. Because of multiple lysine residues present and the random reaction feature, the conjugation of lysine residues results in heterogeneous products with a wide range of drug antibody ratio (DAR),12 and possible loss of antigen binding activity.13 Later on, the thiol-based chemistry has


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been employed. The disulfide bonds of IgG are reduced to form mercapto groups which can react specifically to linkers such as maleimide to form ADCs with better site control.14 However, the reduction of IgG is difficult to control due to the presence of disulfide bonds at different locations of the molecules. A mixture of reduced IgG species or fragment would render the subsequent conjugation uncontrollable, with typical DAR ranging from 0-8, and isomers at each drug substitution level.15-17 In order to develop controllable conjugation to the antibody, the IgG molecule has been modified to incorporate cysteines or unnatural amino acids.18, 19 Although effective to certain degree, the new approaches need extra consideration of the molecular integrity and the consequence of the genetic manipulation. In this study, we aimed at developing a facile conjugation strategy to prepare homogeneous AFDC products through chemical method. The key issue is how to selectively reduce the disulfide bonds in hinge region but not on those located between heavy chain and light chain of antibody. To do this, we had to screen the reagents

from

tris(2-carboxyethyl)phosphine

(TCEP),

dithiothreitol

(DTT),

2-mercaptoethanol (β-ME) and 2-mercaptoethylamine (β-MEA) to decide the proper candidate for reduction reaction.20,

21

Fortunately, β-mercaptoethylamine was

optimized as the agent for disulfide bond reduction together with reaction process control. The model drug and antibody we selected were doxorubicin (DOX) and anti-CD20 IgG. Anti-CD20 IgG is a monoclonal antibody against the protein CD20, which destroys B cells and therefore used to treat diseases characterized as B



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lymphoproliferative malignancies including lymphomas, leukemias, and autoimmune disorders, particularly non-Hodgkin lymphomas (NHLs).22,

23

Doxorubicin (DOX),

which exerts its cytotoxic activity by inhibiting the synthesis of nucleic acids within cancer cells,24 is a well-known anthracycline antibiotic that is widely used in the clinical treatment of lymphoma, leukemia, liver cancer, lung cancer and other solid tumors as a chemotherapeutic drug.25 While doxorubicin can also cause serious side effects, especially heart and kidney toxicity. The incidence of side effect is dependent on its cumulative dose. Therefore, it is of great importance to overcome the side-effect in treatment by dose reduction and remain fine curative effect. Antibody conjugated doxorubicin is capable of selectively suppressing tumor growth with limited side effects on normal tissues.26 In this current work, DOX was conjugated with the Fab’ fragment through a PEG linker to form the AFDC Fab’-PEG-DOX. Two equivalents of doxorubicin conjugated to the hinge region of Fab’ indicate the homogeneity of AFDC. The achieved AFDC exhibited target-dependent antiproliferative activity toward CD20 positive cells and excellent therapeutic effect in tumor xenograft model. This findings will certainly give rise to new pharmaceutical approaches to the management of patients with non-Hodgkin lymphomas. The results in this study show that site-specific reduction of IgG and conjugation to the resulting Fab’ fragment is an applicable and efficient strategy for preparing antibody drug conjugates with definite composition to achieve improved anti-tumor activity.


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RESULTS AND DISCUSSION Purification of anti-CD20 IgG. Anti-CD20 monoclonal antibody contained in milk powder was isolated by affinity chromatography (Figure 1). Fifteen milliliter supernatant was loaded on to the column. The Anti-CD20 IgG bound to the Protein A medium and separated by the elution of sodium citrate along with pH decreased to 3. Thirty milligram IgG was obtained after purification and freezing drying from 1g milk powder (yield 3%). The results shown in Figure S1 demonstrate that Anti-CD20 IgG with high purity (>95%) are obtained.

Figure 1. Affinity chromatographic purification of anti-CD20 IgG. A prepacked Protein A column was used to separate the anti-CD20 IgG with elution buffer of sodium citrate. Preparation of F(ab’)2 fragment. Digestion by the enzyme pepsin normally produces one F(ab’)2 fragment and numerous small peptides of the Fc portion.27 The resulting F(ab’)2 fragment is composed of two disulfide connected Fab’ units. The Fc fragment is extensively degraded, and it can be separated from F(ab’)2 by dialysis, gel filtration or ion exchange chromatography. In this study, the Fc portion of anti-CD20 IgG was eliminated by using TSK G2000SWXL column after digestion (Figure S2).



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The molecular weight of F(ab’)2 was determined by both MALDI-TOF MS (Figure 2) and SDS-PAGE (Figure S3). Results demonstrate that the accurate MWs are 97008 Da, consistent with the theoretical analysis.

Figure 2. Molecular weight determination of F(ab’)2 fragment by MALDI-TOF MS. Preparation of Fab’ fragment. Reducing were performed on F(ab’)2 by four kinds of reducing agents separately at different concentration. Figure 3 shows that Fab’ and H&L chains were both appeared at the concentration vary from 2 to 50 mM when using TCEP, DTT, β-ME as the reducing agent. The H&L chains were hard to separate from Fab’ and would inevitably affect the subsequent coupling reaction. Whereas, for the case of β-MEA, F(ab’)2 was almost transferred into Fab’ within a certain reductant concentration range from 5 to 20 mM, which was the suitable scope for production Fab’. β-MEA was screened as the optimal agent to reduce F(ab’)2 to Fab’.


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Figure 3. Reducing agents screen from TCEP, DTT, β-ME, β-MEA. For all SDS-PAGE Lanes: 1, molecular weight marker; 2, F(ab’)2; 3 to 7, F(ab’)2 treated with 2, 5, 10, 20, 50 mM reducing agents, respectively. F(ab’)2 was reduced with 10 mM optimized reducing agent β-MEA in 5 mM EDTA at room temperature. In the first 30 minutes, the ratio of Fab’ increased correspondingly with the decline of F(ab’)2, indicating the conversion between these two components. The disulfide bond between heavy and light chain would be reduced as the reaction time proceeded over 30 minutes. The result (Figure 4) demonstrated that F(ab’)2 could be completely converted into Fab’ without unnecessary heavy and light chains at the point time of 30 minutes. As a result, the achieved Fab’ could be used for conjugation after simple desalting operation.



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Figure 4. Components ratio at different times in the reducing course of F(ab’)2 to Fab’ by β-MEA . F(ab’)2 was treated with 10 mM β-MEA. F(ab’)2 could be separated by β-MEA reduction into two sulfhydryl containing univalent Fab’ fragment. The advantage of using Fab’ fragment for conjugation was that they could be conjugated directly through their sulfhydryl groups, insuring an active binding site of the IgG. After treated with 10 mM β-MEA for 30 min, the F(ab’)2 was almost reduced to Fab’ (yield 94.5%), remaining the variable region intact. The Fab’ was purified using Superdex 75 10/300 GL column (Figure S4) and determined by both MALDI-TOF MS (Figure 5) and SDS-PAGE (Figure S5). The Mws of Fab’ was 48487 Da.

Figure 5. Molecular weight determination of Fab’ fragment by MALDI-TOF MS.


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Synthesis and purification of MAL-PEG-DOX compound. The PEG linker contained an amine-reactive N-hydroxysuccinimide (NHS) ester and sulfhydryl reactive maleimide (MAL) terminal. MAL-PEG-DOX was synthesized by the reaction of the N-hydroxysuccinimide ester bond of PEG with the only amine bond of DOX, and the obtained MAL-PEG-DOX conjugate was identified by RP-HPLC and MALDI-TOF MS. For RP-HPLC (Figure 6), a trace of free DOX can be seen at retention time of 17.5 min, while after reaction, a new peak corresponding to MAL-PEG-DOX appeared at retention time of 19.4 min (yield 58.7%). The retention time of purified MAL-PEG-DOX consistent with the value of 19.4 min, indicating the success of separation by hydrophobic chromatography (Figure S6). The successful conjugation of PEG with DOX was also proven by MALDI-TOF MS for the Mws of PEG increased by 543 Da (Figure 7).

Figure 6. RP-HPLC of reaction mixture and separated MAL-PEG-NHS-DOX compound.



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Figure 7. MALDI-TOF MS of reaction mixture of MAL-PEG-NHS and DOX. Conjugation of anti-CD20 Fab’ fragment with MAL-PEG-DOX. The bioconjugation chemistry utilized to load functionalized DOX to antigen-binding fragment is the commonly used Michael addition reaction of maleimide with cysteine thiol residues on Fab’ fragment. The reactive thiol in freshly prepared Fab’ fragment was used for coupling MAL-PEG-DOX in the generation of antibody drug conjugates. The final AFDC was separated from unreacted Fab’ and MAL-PEG-DOX using Superdex 75 10/300 GL colunm with a phosphate running buffer. The results shown in Figure 8 and Figure S7 demonstrate that the Fab’-PEG-DOX was obtained (yield 47.9%).


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Figure 8. SDS-PAGE analysis of Fab’-PEG-DOX. Lanes: 1, molecular weight marker; 2, anti-CD20 IgG with β-ME; 3, anti-CD20 IgG without β-ME; 4, F(ab’)2 fragment without β-ME; 5, F(ab’)2 fragment with β-ME; 6, Fab’ fragment without β-ME; 7. reation mixture of Fab’ and MAL-PEG-DOX without β-ME; 8, purified Fab’-PEG-DOX without β-ME. DAR determination of AFDC. The drug antibody ratio was determined by the standard curve method for the different absorbance of Fab’ and DOX under the same wave length. Serial concentration of DOX and Fab’ was prepared, and the absorbance was assayed under 482 nm and 280 nm. The data was analysis by Origin software and subsequently utilized to determine the DAR. The calculated drug antibody ratio was 1.97, approximately consistent with the theoretical analysis of 2 (Figure S8). The DAR was also confirmed by the BCA assay for the UV absorbance do not overlay with DOX.28 A280 (total) = A280 (DOX) + A280 (Fab’) A482 (total) = A482 (DOX) Cytotoxicity assay in vitro. The in vitro cytotoxicity Fab’-PEG-DOX was compared for the ability of DOX to inhibit the tumor cell proliferation. According to the expression of CD20 protein or not in cell surface, cells could be divided into CD20 positive (Daudi cell) and CD20 negative (K562 cell). In CCK-8 assay (Figure 9), the Fab’-PEG-DOX showed decreased toxicity compared to free DOX for both cells.29 The reason may be that the uptaken of DOX was passive diffusion while uptaken Fab’-PEG-DOX via endocytosis for tumor cells. The process of uptaken



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Fab’-PEG-DOX are much slower than DOX and would result in slow drug distribution in the cytosol and nucleus, resulting in reduced cytotoxicity. And Fab’ displayed no inhibiting activity on proliferation for both tumor cell. On the other hand, the cytotoxicity retention of Fab’-PEG-DOX against Daudi cell was significantly higher than K562 cell, which may be due to the targeting ability of Fab’-PEG-DOX to CD20 positive cells. The specific binding of Fab’-PEG-DOX to cell surface promotes the absorption of sample, meanwhile DOX enter cells successfully and exert cytotoxic effect on tumor cell.

Figure 9. Antiproliferation activity of Fab’-PEG-DOX against Daudi cell and K562 cell, respectively, as determined by cck-8 assay. Cells were treated with designated regimes for 48 h.


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Immunofluorescence assay in vitro. The cellular uptake of DOX and Fab’-PEG-DOX was detected by laser scanning confocal microscope (LSCM). Daudi cell and K562 cell were both incubated with free DOX and Fab’-PEG-DOX for 0.5 h and 1 h, and fluorescence distribution in cells were examined immediately. As shown in Figure 10, the green fluorescent signal could be detected in the cytoplasm and nucleus region of both Daudi and K562 cell after treated with free DOX, and the fluorescent intensity increased with the duration of time. However, the Fab’-PEG-DOX was distributed in the cytoplasm and perinuclear zone of Daudi cell after incubation for 0.5 h, and the fluorescent signal was accumulated in nucleus with time prolonged to 1 h. The green fluorescent signal of Fab’-PEG-DOX could hardly been detected in K562 cells even at the time of 1 h, indicating the much slower absorption for the conjugates. These result demonstrated that the Fab’-PEG-DOX could be acted as a promising anti-tumor drug for the easy access to the nucleus of cells and exerts its cytotoxic activity by inhibiting the synthesis of nucleic acids.



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Figure 10. Immunofluorescence assay of DOX and Fab’-PEG-DOX on different cells by LSCM. (A) K562 cell treated with DOX; (B) K562 cell treated with Fab’-PEG-DOX; (C) Daudi cell treated with DOX; (D) Daudi cell treated with Fab’-PEG-DOX. Cell ELISA assay. The binding capacity of IgG or fragment to CD20 in cell surface was measured by cell ELISA. As Figure 11 shown, intact IgG displayed higher binding capacity while fragment and fragment based AFDC showed decreased ability (~70% remaining) for intact IgG exists two variable sites whereas fragment owes only one. There was no significant difference between the Fab’ and Fab’-PEG-DOX, indicating the conjugation process rarely affect its affinity ability. In addition, no binding capacity was observed in K562 cell for no CD20 protein expression.


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Figure 11. Binding capacity of intact IgG, Fab’ and Fab’-PEG-DOX by cell ELSIA. Anti-tumor therapy. The Daudi human tumor xenograft model in SCID mice was used to evaluate the in vivo antitumor activity of Fab’-PEG-DOX. Animals were inoculated with Daudi cells, and tumor xenografts were allowed to grow to a mean tumor volume of 125±20 mm3 (28 days). Animals with established subcutaneous xenograft tumors were treated with either PBS, Fab’, DOX, or Fab’-PEG-DOX, and the tumor growth was monitored. As shown in Figure 12a, intravenous administration of PBS and Fab’ hardly delayed tumor growth due to the poor activity. When simply treated with DOX, the therapeutic effect was still limited for a rapid increase of tumor volume after treatment. Once Fab’-PEG-DOX was administrated, tumor growth was efficiently suppressed throughout the experimental stage. The tumor inhibition rate



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increased with the duration of treatment, at the time of 14 days, the tumor inhibition rate reached 64.9%, significantly higher than the 39.5% of DOX group, and further data was not available for the death of all mice in PBS group. Similar results could be found in Figure 12b for the animal survival curves. It’s noteworthy that the anti-tumor activity of AFDC came from DOX but not the antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of antibody for the lack of Fc fraction,30-32 so there was a minor difference between the death rates of DOX and AFDC groups in 20 days study. The prolonged survival period and decreased tumor size both confirmed the improved antitumor activity of Fab’-PEG-DOX against Daudi tumor. At the same time, mice were weighted to evaluate the in vivo toxicity of DOX. Significantly weight loss was observed in PBS, Fab’ and DOX groups (Figure 13c). While the reduction of weight loss was measured in Fab’-PEG-DOX group, indicating that the AFDC exert improved therapeutic effect and decreased toxic compared to free DOX.


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Figure 12. Anti-tumor therapies in Daudi tumor xenograft model. (a) Suppression of

Daudi tumor growth in mice by treatment with different drugs. (b) Survival percentages of mice in different treatment groups. (c) Body weight changes of Daudi-bearing mice in different treatment groups. Mice bearing Daudi tumor were injected with DOX and Fab’-PEG-DOX separately, and the immunofluorescence signal of DOX was examined by optical tumor imaging in vivo. As shown in Figure 13, the fluorescent signal in DOX group randomly distributed throughout the body and gradually diminished in the subsequent hours. Meanwhile, the fluorescent signal in AFDC group apparently accumulated at tumor site despite of its systemic distribution. The systemic distribution of AFDC and DOX could be attributed to Non-Hodgkin lymphoma, a hematologic neoplasm, whose



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diffusive infiltration of tumor cells happened in most tissues. At the same time, the fluorescent signal in AFDC group apparently concentrated in tumor site under the armpit compared to that in DOX group, which demonstrating the in vivo tumor-targeting activity of AFDC. On the other hand, systemic distributions of drugs were detected in both groups. The reason is that Non-Hodgkin lymphoma is a hematologic neoplasm, and diffusive infiltration of tumor cells happens in most tissues, including vessel, liver, spleen, lung and kidney. The conclusion was also confirmed by the pathological observation (Figure S9) for the severe tissue necrosis of organs in DOX group while healthy tissue in AFDC group. In contrast to DOX group, the much higher drug concentration in AFDC group in subsequently hours was due to the conjugation of Fab’ to DOX. The half-life of DOX (1-3 h) could be significantly prolonged which would make it possible to deliver the drug in lower dosing frequency or fewer drug doses on clinic. The result indicates that the AFDC can be targeted and enriched in tumor, thereby triggering the apoptosis of tumor cells with lower side effects.

Figure 13. The localization of conjugate was confirmed by optical tumor imaging in vivo.


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CONCLUSIONS In this study, an antibody drug conjugate containing Fab’ fragment and cytotoxic doxorubicin with definite chemical composition was obtained successfully via a heterogeneous non-toxic and non-immunogenic PEG linker. By screening reducing agents and reaction process control, anti-CD20 IgG was reduced to Fab’ fragment containing two mercapto groups in the hinge region and maintaining the integrity of the binding sites. The Fab’ fragment was subsequently conjugated to the in advance prepared MAL-PEG-DOX, forming the final antibody drug conjugate. The certain homogeneity of AFDC was confirmed by SDS-PAGE and component content determination, and will help the preservation of bioactivity and product stability. Our result revealed that the AFDC combined the targeting ability to CD20 positive cell and the cytotoxicity effect to lymphoma cell. In addition, the AFDC specifically bound to CD20 positive cell yet no binding on non CD20 expression cells. Moreover, Fab’-PEG-DOX showed excellent therapeutic effect on lymphoma mice model for better cure rate and significant reduced side effects. The AFDC can target to tumor cells and effectively release the DOX in situ due to the reversible structure between antibody and cytotoxic agent, which improving the pharmacodynamic efficacy. These results could probably offer a promising strategy for the development of novel antibody drug conjugated with therapeutic potential for treating cancers possessing reduced side effect of cytotoxic molecules.

EXPERIMENTAL PROCEDURES



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Materials. MAL-PEG2k-NHS was purchased from Jenkem Technology Co.,Ltd. (China). Doxorubicin, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), 2-mercaptoethanol (β-ME), 2-mercaptoethylamine (β-MEA) and HRP-conjugate goat anti-human IgG (Fab specific) were purchased from Sigma-Aldrich (USA). Milk powder containing anti-CD20 IgG was kindly supplied by State Key Lab for Agrobiotechnology, China Agricultural University.33 Protein A affinity column, butyl column and analytical Superdex 75 10/30 GL column were from GE Healthcare (Sweden). Pursuit C18 column was supplied by Agilent Technologies (USA). TSK G2000SWXL column was purchased from Tosoh Bioscience (Japan). Daudi cells and K562 cells were from Cell Resource Center, IBMS, CAMS/PUMC. Purification of anti-CD20 IgG. The milk powder (1 g) was dissolved in 15 mL deionized water and gently agitated for 10 min. The solution was centrifuged at 10000g for 30 min and the supernatant was collected. Then the supernatant was loaded onto a prepacked Protein A affinity column that had been equilibrated in a running buffer of 20 mM sodium phosphate, and pH 7.0 at a flow rate of 0.25 mL/min using an ÄKTA purifier protein purification system (GE Healthcare, Sweden). The anti-CD20 IgG peak was collected when using the elution buffer of 20 mM sodium citrate, pH 3.0 at a flow rate of 1 mL/min. The elution peak was subsequently dialyzed using a 3.5k-MW-cutoff membrane against ultrapure water at 4 °C for 24 h. The purity of anti-CD20 IgG was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after freeze drying.


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Preparation of F(ab’)2 fragment. F(ab’)2 fragment of the anti-CD20 IgG were prepared by enzymatic digestion using the protease pepsin, which selectively cleaves disulfides in the hinge region of antibodies. Anti-CD20 IgG (2 mg/mL, 4 mL) was reacted with pepsin at a 20:1 mass ratio at 37 °C for 12 h in 100 mM sodium acetate, pH 4.0. The reaction was terminated by adding 1 M tris to adjust the pH to 8.0. Then the reaction mixture (500 µL) was loaded onto a TSK G2000SWXL column (GE Healthcare, Sweden) with a running buffer of 20 mM sodium acetate, 100 mM Na2SO4, and pH 5.0 at a flow rate of 0.6 mL/min. The peak containing F(ab’)2 fragment were collected and analyzed by SDS-PAGE and MALDI-TOF MS. Preparation of Fab’ fragment. Selectively reducing of F(ab’)2 to Fab’ was the crucial process for our site-specific conjugation. To retain the binding site and introduce free cysteine residue for conjugation, it was necessary to reduce disulfides in hinge region but holding together the HC/LC interchain. Four kinds of most commonly used reducing agents (TCEP, DTT, β-ME, β-MEA) were utilized and screened. F(ab’)2 (0.5 mg/mL) was reacted with different reducing agents in a final concentration of 2, 5, 10, 20, 50 mM at room temperature for 30 min. The mixture was subsequently analyzed by SDS-PAGE. After the reducing agents screening process, β-MEA was optimized as the agent for the next reduction time course. F(ab’)2 was reduced with 10 mM β-MEA in 5 mM EDTA at room temperature. At various times, reducing agent was removed using desalting column with phosphate buffered pH 7.2 containing 10 mM EDTA. The peak containing IgG and fragment was collected, and the ratio of all components was



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determined by SDS-PAGE with Quality One software. Finally, F(ab’)2 (0.5 mg/mL) was reduced by β-MEA at a final concentration of 10 mM for 30 min. The mixture was immediately loaded on to a Superdex 75 10/30 GL colunm that had been equilibrated in a running buffer of 20 mM sodium phosphate, 10 mM EDTA, and pH 7.2. Protein peaks were collected and analyzed by SDS-PAGE. SDS-PAGE. The reaction and purification results were analyzed by SDS-PAGE according to the methods of Laemmli.34 Reaction mixture and the purified fragment were combined with a reducing or nonreducing 5× sample-loading buffer (Tris-Glycine 5× SDS sample buffer, with or without 10% β-ME), heated at 95-100 °C for 5 min, and applied to 12% Tris-Glycine gels. Gels were stained and analyzed for protein using Coomassie brilliant blue dye. MALDI-TOF MS. The molecular weight of samples was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) on Autoflex III (Bruker, USA) with α-cyano-4-hydroxycinnamic acid as the matrix.35 The operation mode was reflective mode with positive ion detection. The accelerating voltage was 19000 volts and the delayed extraction time was 200 nanoseconds. Samples (0.2 mg/mL) were dissolved in water and then mixed with saturated matrix solution (CH3CN:H2O 1:1, v/v) at a volume ratio of 1:1. RP-HPLC. Reaction of the MAL-PEG2k-NHS and DOX was determined by reversed-phase high performance liquid chromatography using a pursuit C18 column (250 mm × 4.6 mm, 5 µm) with UV detection at 233 nm. The mobile phase was maintained 20% B in 5 min, and then a 20%-80% B gradient over 20 min was


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operated at a flow rate of 0.5 mL/min (A: water + 0.1% TFA; B: acetonitrile + 0.1% TFA). The running time of each sample was 25 min with an injection volume of 10 µL. Synthesis and purification of PEG-DOX compound. Doxorubicin contains an amine functional group which could be functionalized by formation of an amide in the daunosamine sugar. Doxorubicin (50 mg) reacted with MAL-PEG-NHS at a 1:1.5 molar ratio at dark room temperature for 48 h in chloroform. Fifteen microliter triethylamine was added as the catalyst. The resulted MAL-PEG-DOX compound was examined by HPLC. The chloroform was evaporated and the solid mixture was collected for the next purification step. The MAL-PEG-DOX compound and the unreacted PEG was separated by hydrophobic chromatography. Briefly, the solid mixture was dissolved in 1 M ammonium sulfate in 20 mM phosphate buffer (pH 7.2), and the mixture solution was then loaded onto a 5 mL butyl column (GE healthcare, Sweden), which had been pre-equilibrated by 20 mM phosphate buffer (pH 7.2) containing 1 M ammonium sulfate. The adsorbed MAL-PEG-DOX was eluted by 20 mM phosphate buffer (pH 7.2) and confirmed by HPLC after freeze drying. Conjugation of anti-CD20 Fab’ fragment with MAL-PEG-DOX. Freshly prepared Fab’ (Fab’-SH) fragment was treated with 20-fold molar excess of MAL-PEG-DOX in PBS (10 mM EDTA, pH 7.2) for 24 h at 4 °C. The Fab’-PEG-DOX conjugate was purified by Superdex 75 prep grade column (GE healthcare, Sweden) and analyzed by SDS-PAGE. Cytotoxicity assay in vitro. Cytotoxicity of free DOX and Fab’-PEG-DOX was



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determined by measuring the inhibition of cell growth using Daudi cell and K562 cell. Cells were seeded in 96-well (5 × 103 cells/well) microtiter plates in 0.1 mL culture medium. Serial dilutions of 0.1 mL of free DOX, Fab’-PEG-DOX samples were added and incubated at 37 °C, 5% CO2, for 48 h. Ten microlitre of CCK-8 assay reagent was added to each well and further incubated for 1.5 h at 37 °C to measure the cytotoxicity. The plate was read at 450 nm in a 96-well plate reader (Bio-Rad model 550, USA) and the percentage of cell survival was then determined. Immunofluorescence assay in vitro. Daudi and K562 cell were seeded in poly-L-lysine prepacked petri dish at a cell density of 1 × 104/dish at 37 °C and 5% CO2 for 24 h. Supernatants were discarded, and Fab’-PEG-DOX and DOX were added to each dish at a final concentration of 10 µg/mL in terms of the doxorubicin. Following the treatment with samples for 0.5 h and 1 h, the medium was removed and cells were softly washed and dyed with hochest reagent for 20 min, and finally analyzed by laser scanning confocal microscope (Leica TCS SP 5, Germany). Cell ELISA assay. ELISA was performed in a 96-well polystyrene microtiter plate precoated with poly-L-lysine. Daudi and K562 cells were seeded (2 × 104 cells/well) and incubated 24 h at 37 °C. The medium was aspirated, and the cells were washed three times with PBS and fixed with 3.7% paraformaldehyde in PBS for 20 min at room temperature. After removal of the fixing solution and complete drying, the plate was blocked with 5% non-fat milk for 1 h. Serial dilutions of 50 µL of intact IgG, Fab’ and Fab’-PEG-DOX samples were added and incubated at 37 °C for 2 h. After plate washing with 0.1% PBST (PBS containing Tween-20), 100 µL of HRP-conjugated


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goat anti-human IgG (Fab specific) (1:1000) was added, and after a 1 h incubation at 37 °C, after washing, TMB substrate was incubated for 20 min and terminated by adding 50 µL of 2 M H2SO4. The plates were read at 450 nm. Anti-tumor therapy. Six week old male SCID mice weighing 20 g were used for the anti-tumor therapy in vivo. Tumors were established by a subcutaneous injection of 1 × 107 Daudi cells into the forelimb armpit of mice. Tumor volumes were calculated according to the formula L × (W) 2 / 2, where L and W is the longest and shortest tumor diameter (mm), respectively. When the average tumor volume reached about 125 mm3, the mice were randomly assigned to 4 group (n=6) as PBS, DOX (1 mg/kg) and Fab’-PEG-DOX (1 mg/kg of DOX), Fab’ (equal concentration as Fab’-PEG-DOX injected). Each rat received an injection once per two days, which was continued for 10 times. Data of mice weight and tumor volume was collected before the next injection. The organs of heart, liver, spleen, lung and kidney were excised and fixed for pathological observation after the last injection. In addition, the localization of conjugate was measured by optical tumor imaging (Kodak In-Vivo Imaging System FX Pro, USA).

AUTHOR INFORMATION Corresponding Author *E-mail:

[email protected],

[email protected].

Tel:

+86-10-82545022.

+86-10-62561813. Address No. 1, Zhongguancun North two Rd. Haidian District, Beijing, China


Fax:


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Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT This research was financially supported by the National Natural Science Foundation of China (no. 21106163, 21306207, and 21336010), the Science and Technology Service Network Initiative (no. KFJ-EW-STS-027), the National High Technology Research and Development Program of China (863 Program, no. 2012AA021202, 2012AA02A406, and 2014AA021006), the National Key Basic Research Program of China (973 Program no. 2013CB733600), and the National Key Foundation for Exploring Scientific Instrument of China (no. 2013YQ14040508).

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Supplementary Figures


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Table of Contents Graphic 43x24mm (300 x 300 DPI)



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Figure 1. Affinity chromatographic purification of anti-CD20 IgG. A prepacked Protein A column was used to separate the anti-CD20 IgG with elution buffer of sodium citrate. 56x40mm (600 x 600 DPI)


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Figure 2. Molecular weight determination of F(ab’)2 fragment by MALDI-TOF MS. 56x40mm (600 x 600 DPI)



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Figure 3. Reducing agents screen from TCEP, DTT, β-ME, β-MEA. For all SDS-PAGE Lanes: 1, molecular weight marker; 2, F(ab’)2; 3 to 7, F(ab’)2 treated with 2, 5, 10, 20, 50 mM reducing agents, respectively. 115x82mm (300 x 300 DPI)


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Figure 4. Components ratio at different times in the reducing course of F(ab’)2 to Fab’ by β-MEA . F(ab’)2 was treated with 10 mM β-MEA. 56x40mm (600 x 600 DPI)



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Figure 5. Molecular weight determination of Fab’ fragment by MALDI-TOF MS. 56x40mm (600 x 600 DPI)


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Figure 6. RP-HPLC of reaction mixture and separated MAL-PEG-NHS-DOX compound. 56x40mm (600 x 600 DPI)



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Figure 7. MALDI-TOF MS of reaction mixture of MAL-PEG-NHS and DOX. 56x40mm (600 x 600 DPI)


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Figure 8. SDS-PAGE analysis of Fab’-PEG-DOX. Lanes: 1, molecular weight marker; 2, anti-CD20 IgG with βME; 3, anti-CD20 IgG without β-ME; 4, F(ab’)2 fragment without β-ME; 5, F(ab’)2 fragment with β-ME; 6, Fab’ fragment without β-ME; 7. reation mixture of Fab’ and MAL-PEG-DOX without β-ME; 8, purified Fab’PEG-DOX without β-ME. 67x56mm (300 x 300 DPI)



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Figure 9a. Antiproliferation activity of Fab’-PEG-DOX against Daudi cell and K562 cell, respectively, as determined by cck-8 assay. Cells were treated with designated regimes for 48 h. 56x39mm (600 x 600 DPI)


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Figure 9b. Antiproliferation activity of Fab’-PEG-DOX against Daudi cell and K562 cell, respectively, as determined by cck-8 assay. Cells were treated with designated regimes for 48 h. 56x39mm (600 x 600 DPI)



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Figure 10. Immunofluorescence assay of DOX and Fab’-PEG-DOX on different cells by LSCM. (A) K562 cell treated with DOX; (B) K562 cell treated with Fab’-PEG-DOX; (C) Daudi cell treated with DOX; (D) Daudi cell treated with Fab’-PEG-DOX. 110x74mm (300 x 300 DPI)


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Figure 11a. Binding capacity of intact IgG, Fab’ and Fab’-PEG-DOX by cell ELSIA. 56x40mm (600 x 600 DPI)



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Figure 11b. Binding capacity of intact IgG, Fab’ and Fab’-PEG-DOX by cell ELSIA. 56x40mm (600 x 600 DPI)


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Figure 12a. Anti-tumor therapies in Daudi tumor xenograft model. (a) Suppression of Daudi tumor growth in mice by treatment with different drugs. 56x40mm (600 x 600 DPI)



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Figure 12b. Anti-tumor therapies in Daudi tumor xenograft model. (b) Survival percentages of mice in different treatment groups. 56x40mm (600 x 600 DPI)


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Figure 12c. Anti-tumor therapies in Daudi tumor xenograft model. (c) Body weight changes of Daudi-bearing mice in different treatment groups. 56x40mm (600 x 600 DPI)



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Figure 13. The localization of conjugate was confirmed by optical tumor imaging in vivo. 42x28mm (300 x 300 DPI)


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Specific Conjugation of the Hinge Region for Homogeneous

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