- Email: [email protected]
Characterization of a humanized anti-CD20 antibody with potent antitumor activity against B-cell lymphoma
Cancer Letters 292 (2010) 208–214
Contents lists available at ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Characterization of a humanized anti-CD20 antibody with potent antitumor activity against B-cell lymphoma Lan Wu a,1, Chong Wang b,c,1, Dapeng Zhang a, Xunming Zhang a, Weizhu Qian a,b, Lei Zhao a, Hao Wang a,b, Bohua Li a,b,*, Yajun Guo a,b,c,* a
International Joint Cancer Institute and 301 General Hospital Cancer Center, Second Military Medical University, Shanghai 200433, and PLA General Hospital, Beijing 100853, PR China b National Engineering Research Center for Antibody Medicine and Shanghai Key Laboratory of Cell Engineering & Antibody, Shanghai 201203, PR China c School of Medicine and School of Pharmacy, The Center for Antibody Medicine of Ministry of Education, Shanghai Jiao Tong University, 227 South Chongqing Road, Shanghai 200025, PR China
a r t i c l e
i n f o
Article history: Received 6 October 2009 Accepted 8 December 2009
Keywords: CD20 Humanized antibody Complement-dependent cytotoxicity B-cell lymphoma
a b s t r a c t Despite the effectiveness of the anti-CD20 chimeric antibody (mAb), rituximab, in treating B-cell lymphomas, its efficacy remains variable and often modest. In this study, a humanized anti-CD20 antibody, hu8E4, was generated by complementarity-determining region grafting method. Hu8E4 was as effective as rituximab in mediating antibody-dependent cellular cytotoxicity and inducing apoptosis in B-lymphoma cells, but it exhibited much more potent complement-dependent cytotoxicity than rituximab. Immunotherapeutic studies showed that hu8E4 was significantly more effective than rituximab in prolonging the survival of severe combined immunodeficient mice bearing human B-cell lymphomas, suggesting that it might be a promising therapeutic agent for B-cell lymphomas. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The CD20 antigen is present on >90% of B-cell lymphomas and is neither shed nor internalized after antibody binding, making it an effective target for immunotherapeutic removal of malignant B cells [1–3]. The chimeric antiCD20 monoclonal antibody (mAb) rituximab was approved for use in relapsed or refractory low-grade or follicular B-cell non-Hodgkin’s lymphoma (NHL) in 1997. Previous studies have suggested that several mechanisms might be involved in providing therapeutic efficacy, including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and the induction of
* Corresponding authors. Address: International Joint Cancer Institute and 301 General Hospital Cancer Center, Second Military Medical University, Shanghai 200433, and PLA General Hospital, Beijing 100853, PR China. Tel.: +86 21 81870801; fax: +86 21 65306667. E-mail addresses: [email protected] (B. Li), [email protected] (Y. Guo). 1 These authors contributed equally to this work. 0304-3835/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2009.12.004
apoptosis [4,5]. The relative contributions of these different mechanisms of action are still a matter of debate. The most conclusive data supporting conventional effector mechanisms (ADCC and CDC) in the therapeutic response come from work in primates showing that an immunoglobulin G4 (IgG4) variant of rituximab, unlike the chimeric IgG1 monoclonal antibody (mAb), was unable to deplete normal B cells [6]. Since the IgG4 variant would be unable to recruit natural effectors effectively but should retain the binding and cross-linking functions needed for any direct cytotoxic activity, including the signaling of apoptosis, these data point toward CDC and ADCC as the major effectors in vivo. Despite the effectiveness of rituximab, only 48% of patients respond to the treatment and complete responses are <10% [7,8]. There is an urgent need to develop more effective CD20-targeting antibody agents for the treatment of B-cell lymphomas [8,9]. In addition, rituximab is a murine/human chimeric antibody, in which the variable domains are derived from the murine mAb, and the constant regions from human mAb. Although a chimeric antibody is less likely than a fully murine mAb to evoke
L. Wu et al. / Cancer Letters 292 (2010) 208–214
an immune response, and elicitation of a human anti-chimeric antibody response has not posed a significant obstacle to the clinical use of rituximab, it may be advantageous to have a more fully human version, especially if repeated injections may be desired in patients. Administration of humanized mAbs could possibly result in an extension in serum half-life, thus affecting the therapeutic response. In the present study, a mouse anti-human CD20 monoclonal antibody 8E4 was generated by immunizing BALB/c mice with CD20-positive human lymphoma cells. Then we described the construction and characterization of the humanized anti-CD20 antibody (hu8E4). Our data indicated that hu8E4 was shown to retain the same antigen binding affinity and specificity as its parental mouse antibody. The in vitro and in vivo antitumor activity of hu8E4 was further examined and compared with rituximab. 2. Materials and methods 2.1. Cell lines, antibodies, and animals The Chinese hamster ovary cell line CHO-K1, the African green monkey cell line COS-7 and two human Burkitt lymphoma cell lines, Raji and Daudi, were obtained from the American Type Culture Collection (ATCC). Rituximab was purchased from Roche Ltd. Eight-week-old female BALB/c mice and BALB/c SCID mice were housed in pathogen-free conditions and were treated in accordance with guideline of the Committee on Animals of the Second Military Medical University.
209
Then the cells were washed and examined by FCM using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). One hybridoma cell line, which secreted mAbs reacting with CHO-CD20 cells but not with CHO cells, were established and named 8E4. The 8E4 antibody isotype was determined to be (IgG2a,j) with a mouse Ab isotyping kit (Sigma, St. Louis, MO). Finally, the 8E4 antibody was purified by Protein G affinity chromatograph from hybridoma cell culture supernatants and purified 8E4 was labeled with fluoroscein isothiocyanate (FITC) to produce FITCconjugated antibody (FITC-8E4). 2.4. Cloning of 8E4 heavy and light chain variable region genes RNA was isolated from 8E4 hybridoma cells with TRIzol Reagent (Invitrogen, Carlsbad, CA). The variable region cDNAs for the light and heavy chains of 8E4 were cloned from the hybridoma cells by 50 RACE system (Invitrogen, Carlsbad, CA). The gene-specific primers (GSPs) for PCR amplification of 8E4 heavy chain were as follows: GSP1IgG2aH, 50 -AGC TGG GAA GGT GTG CAC ACC ACT-30 ; GSP2-IgG2aH, 50 -CAG AGT TCC AGG TCA AGG TCA-30 ; GSP3-IgG2aH, 50 -CTT GAC CAG GCA TCC TAG AGT-30 . The GSPs used for PCR amplification of light chain variable regions of 8E4 were as follows: GSP1-jL, 50 -TTG CTG TCC TGA TCA GTC CAA CT-30 ; GSP2-jL, 50 -TGT CGT TCA CTG CCA TCA ATC TT-30 ; GSP3-jL, 50 -TTG TTC AAG AAG CAC ACG ACT GA-30 . The final PCR products were cloned into pGEM-T vector (Promega, Madison, WI) for sequence determination.
2.2. Production of CHO cells stably expressing CD20 molecule
2.5. Molecular modeling
Human full length CD20 cDNA was cloned from Raji cells and inserted into pcDNA3.1 (Invitrogen, San Diego, CA). The resultant expression vector was transfected into CHO-K1 cells using Lipofectamine 2000 reagent (Invitrogen, San Diego, CA). After selection with 500 lg/mL G418 for 3 weeks, the transfected cells were stained with FITCconjugated rituximab and then stable transfectants expressing the highest levels of CD20 (CHO-CD20 cells) were obtained by fluorescence-activated cell sorting on a FACSVantage (BD Biosciences, San Jose, CA).
The Protein Data Bank (PDB) was searched for antibody sequences that had high sequence identity with variable fragment (Fv) of 8E4. Two separate BLASTP searches were performed for light chain variable region (VL) and heavy chain variable region (VH) of 8E4. To construct the threedimensional structure of the 8E4 Fv by homology modeling (INSIGHT II 2003, Accelrys, San Diego, CA), the sequences of VL and VH of 8E4 and their templates were aligned, respectively. The coordinates for the structurally conserved regions were assigned from the template and the loop regions were generated by Homology program of Insight II. The new built structure was subjected to molecular dynamics simulations and then energy-minimized by 1000 steps of the steepest descent method and followed by conjugate gradient method using Discover program. Finally, the refined model was assessed by Profile-3D program.
2.3. Hybridoma preparation Female BALB/c mice were repeatedly immunized with Raji cells. Four days after the final booster, the mice were sacrificed and the splenocytes were fused with the NS-1 myeloma cells using standard techniques as previously described [10]. The fused cells were suspended in hypoxanthine/aminopterin/thymidine (HAT) selective medium and plated into 96-well microliter plates. After 12 days, the anti-CD20 antibody-producing hybridoma clones were selected for binding to CHO-CD20 cells but not to CHO cells by flow cytometry (FCM). Briefly, CHO-CD20 or CHO cells at 1 106 cells/mL were incubated with supernatants of the growing hybridomas for 45 min at 4 °C. The cells were washed and incubated with FITC-conjugated goat antimouse IgG (Zymed, San Francisco, CA) for 45 min at 4 °C.
2.6. Humanization of anti-CD20 mAb 8E4 The human consensus sequences of light chain subgroup kappa I (humjI) and heavy chain subgroup III (humIII) were chosen as human framework for the heavy and light chains of humanized version of 8E4, respectively. The complementarity-determining regions (CDRs) in the humanized antibody were chosen to be identical to those in the mouse antibody 8E4. The molecular model of the
210
L. Wu et al. / Cancer Letters 292 (2010) 208–214
8E4 Fv showed that some framework region (FR) residues were close enough to the CDRs to either influence their conformations or interact directly with antigen. When these FR residues differed between 8E4 and the human antibody template, the residue in the humanized antibody was chosen to be the murine 8E4 residue rather than the human antibody residue. The VH and VL genes of humanized versions of 8E4 were synthesized by overlapping PCR. 2.7. Construction and expression of chimeric or humanized antibodies The light and heavy chain expression vectors for chimeric and humanized antibodies were constructed using the same method as described in our previous report [11]. Appropriate light and heavy expression vectors for recombinant antibodies were co-transfected into COS-7 cells using Lipofectamine 2000 reagent. After 72 h incubation, the supernatants were collected and analyzed by enzyme-linked immunosorbent assay (ELISA) for quantitation of antibodies. The ELISA assay used goat anti-human IgG Fc (KPL, Gaithersburg, MD) as capture antibody and goat anti-human kappa horseradish peroxidase (HRP) (Southern Biotechnology Associates, Birmingham, AL) as detecting antibody. FCM was performed to determine the binding of recombinant mAbs to Raji cells. Briefly, Raji cells at 1 106 cells/mL were incubated with different dilutions of the culture supernatants containing recombinant antibodies for 1 h at 48 °C. The cells were washed and incubated with FITC-goat anti-human IgG (H + L) (Zymed, San Francisco, CA) for 1 h at 48 °C. And then the cells were washed and analyzed by FCM. 2.8. Stable expression and purification of recombinant antibodies Appropriate light and heavy expression vectors were co-transfected into Chinese hamster ovary (CHO)-K1 cells and stable transfectants were isolated using the same method as described previously [11]. The clones producing the highest amount of recombinant mAbs were grown in serum free medium and recombinant antibodies were purified by Protein A affinity chromatography. 2.9. Competitive binding assay Raji cells at 1 106 cells/mL were incubated with a subsaturating concentration of the FITC-8E4 and different concentrations of competing antibodies for 45 min at 4 °C. The cells were then washed and analyzed by FCM. The IC50 values of competitors were calculated using a four-variable algorithm. 2.10. Cytotoxicity assays CDC and ADCC activities of CD20 mAbs were measured by lactate dehydrogenase (LDH)-releasing assay using the CytoTox 96 non-Radioactive Cytotoxicity Assay kit (Promega, Madison, WI). Briefly, the CD20+ cells were incubated with CD20 mAbs for 1 h in phenol red-free DMEM culture medium in a 5% CO2 incubator at 37 °C, followed by the
addition of either NHS (7.5% vol/vol) as a source of complement (for CDC assay) or human peripheral blood mononuclear cells (PBMC) as effector cells (effector to target, 25:1 for ADCC assay). After an additional incubation for 5 h at 37 °C, the cell lysis was determined by measuring the amount of LDH released into the culture supernatant. Maximum LDH release was determined by lysis in 0.3% Triton X-100. Percentage of specific lysis was calculated according to the following formula: %lysis = [experimental release spontaneous release]/[maximum release spontaneous release] 100. 2.11. Apoptosis assay The cells were incubated with different concentrations of anti-CD20 mAbs at 37 °C for 16 h. After washing, cells were treated with Annexin V-FITC (BD Biosciences, San Diego, CA), washed again, and analyzed by FCM. 2.12. Immunotherapy Groups of 10 8-week-old female SCID mice were injected via the tail vein with 5 106 Raji or Daudi cells on day 0, followed 5 days later by the i.v. injection of 100 lg of CD20 mAbs. The mice were observed daily and euthanized at the onset of hind leg paralysis. 2.13. Statistical analysis Statistical analysis was performed by Student’s unpaired t test to identify significant differences unless otherwise indicated. Differences were considered significant at a P value of <0.05. 3. Results 3.1. Molecular modeling of anti-CD20 monoclonal antibody 8E4 The nucleotide sequences of VH and VL of 8E4 were determined and the deduced amino acid sequences of VH and VL of 8E4 were shown in Fig. 1. We searched PDB for antibody sequences with high sequence identity with the 8E4 antibody variable regions. The antibody C2B8 (PDB No. 2OSL) shows 84% identity with the VH of 8E4 and the antibody E8 (PDB No. 1WEJ) shows 94% identity with the VL of 8E4. We selected C2B8 and E8 as the templates of the VH and VL of 8E4, respectively. Then the molecular model of the variable regions of 8E4 was obtained by Insight II molecular modeling software (Fig. 2). 3.2. Humanization of 8E4 antibody We selected humIII and humjI as the variable region frameworks for the VH and VL of humanized 8E4 antibody, respectively. Firstly, the three CDRs from 8E4 light chain or heavy chain were directly grafted into human antibody light or heavy chain frameworks to generate humanized antibody genes. The humanized VL and VH were cloned into expression vectors, respectively, and were coexpressed in COS-7 cells, yielding humanized version (hu8E4VHa/hu8E4VLa). The amino acid sequence of the hu8E4VHa/hu8E4VLa antibody was shown in Fig. 1. This antibody in COS-7 cell culture supernatant was quantified by ELISA and the binding of hu8E4VHa/hu8E4VLa to Raji cells was determined by FCM. The results showed that this humanized antibody lost its binding activity to CD20 molecule (Fig. 3A), suggesting that some FR residues in this humanized version must be back-mutated to reconstitute the full binding activity. With the help of graphic manipulation, eleven mouse FR residues, which differed between 8E4 and the human antibody template, were observed to be within 5 Å of the CDRs and to probably affect the structure of the
L. Wu et al. / Cancer Letters 292 (2010) 208–214
211
Fig. 1. Amino acid sequences of humanized 8E4 antibody heavy (A) and light (B) chain variable regions. 8E4VH and 8E4VL indicate heavy and light chain variable regions of murine 8E4 antibody, respectively. The humIII was chosen as framework for the humanized heavy chains and the humjI was chosen for the humanized light chains. Hu8E4VHa and hu8E4VHb indicate different versions of humanized heavy chain variable regions. Hu8E4VLa and hu8E4VLb indicate different versions of humanized light chain variable regions. The dashes represent amino acids that are the same as the corresponding residues in human antibody framework. The CDRs are enclosed with brackets. Amino acids (in one-letter notation) are numbered according to Kabat et al. [12]. CDRs (Fig. 2). A number of humanized light and heavy chain versions were constructed to evaluate the contribution of each of the eleven FR residues to antigen binding. Finally, a humanized version (hu8E4VHb/ hu8E4VLb) showing the similar antigen-binding activity as the chimeric 8E4 antibody (c8E4) was obtained (Fig. 3A). This humanized antibody was designated as hu8E4 and its amino acid sequences were shown in Fig. 1.
3.3. Competitive binding assay The humanized antibody hu8E4 was expressed in CHO cells and then purified from the CHO cell serum-free culture supernatant by Protein A affinity chromatography. In the competitive binding assay, hu8E4 effectively competed with 8E4 for binding to Raji cells (Fig. 3B). The avidity (mean IC50 ± SD) of hu8E4 (1.36 ± 0.17 lg/mL) was similar to that of 8E4 (1.30 ± 0.15 lg/mL) and c8E4 (1.16 ± 0.11 lg/mL), suggesting that this humanized antibody possessed affinity and specificity similar to that of its parental antibody.
3.4. CDC activity of hu8E4 The capacity of hu8E4 to mediate CDC was assessed in two CD20-positive human lymphoma cell lines, Daudi and Raji. The results summarized in Fig. 4 showed that hu8E4 was as effective as c8E4 in lysing Daudi cells in the presence of human complement. Encouragingly, hu8E4 was significantly more potent in CDC than rituximab (P < 0.05 for hu8E4 compared with rituximab at concentrations of 0.4, 2, and 10 lg/mL), and the similar results (Fig. 4) were obtained with Raji cells, which are more resistant to CDC than Daudi cells.
3.5. ADCC activity of hu8E4 The ability of hu8E4 to lyse CD20+ lymphoma cells in the presence of human PBMCs was investigated and compared with c8E4 and rituximab. Our data (Fig. 4) clearly indicated that hu8E4, c8E4 and rituximab were equally effective in mediating ADCC against Daudi or Raji cells in a dose-dependent manner.
212
L. Wu et al. / Cancer Letters 292 (2010) 208–214
Fig. 2. Molecular model of the 8E4 variable regions. The 8E4 variable regions are shown by a solid ribbon representation. The FRs are shown in green and the CDRs are shown in red. Eleven FR residues, which are within about 5 Å of the CDRs and are of potential importance to the antigen binding, are colored gray. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.6. Apoptosis-inducing activity of hu8E4 Induction of apoptosis was evaluated by FITC-Annexin V assays in Daudi and Raji cells. As illustrated in Fig. 5, hu8E4 was able to trigger low levels of apoptosis of both of these two CD20-positive lymphoma cells, and its apoptotic activity was shown to be similar to that of c8E4 and rituximab. 3.7. The in vivo antitumor activity of hu8E4 In vivo therapy studies were performed in SCID mice bearing systemic Daudi or Raji tumors. The survival curves were plotted according to Kaplan–Meier method and compared using the log-rank test. The results summarized in Fig. 6 showed that all of the three anti-CD20 antibodies, hu8E4, c8E4 and rituximab, significantly improved the survival of SCID mice bearing disseminated Daudi tumor cells (P < 0.001 for each compared with the human IgG control). The animals treated with hu8E4 had similar survival curves as those treated with c8E4. However, a pronounced difference in survival was noticed between hu8E4 and rituximab treatment groups (P < 0.01). Hu8E4 was significantly more effective than rituximab in prolonging the survival of SCID mice bearing Daudi tumor cells. Similar results were obtained using Raji tumor cell-bearing SCID mice.
4. Discussion Here we have described the humanization of an antiCD20 monoclonal antibody, 8E4. The initial version of humanized antibody (hu8E4Ha/hu8E4La) was constructed by grafting the CDRs alone from 8E4 to its human antibody template. Antigen-binding activity assays indicated that this version lost the binding activity to CD20-positive Raji cells. This is because some FR residues may be critical for preserving the CDR conformations or be directly involved in antigen binding [13–18]. Computer-assisted molecular design can be used to build the molecular model of the antibody to help identify such structurally important FR residues. In this study, a three-dimensional model of the variable regions of 8E4 was built using computer-aided
Fig. 3. Antigen-binding activity of CD20 mAbs. (A) Raji cells were incubated with serial log dilutions of c8E4 or different humanized versions for 1 h at 48 °C. Cells were washed and incubated with FITC-goat anti-human IgG (H + L) for 1 h at 48 °C. Cells were then washed and analyzed by FCM. Data are presented as means ± SD (n = 3). (B) Raji cells were incubated with a subsaturating concentration of the FITC-8E4 and different concentrations of competing antibodies for 45 min at 4 °C. The cells were then washed and analyzed by FCM. Maximal fluorescence means the mean channel fluorescence obtained in the absence of competitor antibodies. All data were expressed as the mean of triplicate samples.
homology modeling. Eleven FR residues within 5 Å of the CDRs were identified as key FR residues and a humanized version of 8E4 (hu8E4) was generated by transferring these mouse key framework residues onto the human framework together with the mouse CDR residues. In the competitive binding assay, hu8E4 was shown to have IC50 value similar to its parental mouse antibody 8E4, suggesting that the relative affinities of the two antibodies were about equal. Previous studies have shown that the protective activity of rituximab was completely abolished in C1q-deficient mice inoculated with syngenic lymphoma transduced with human CD20 [19]. Similarly, Gragg and Glennie found that treatment of SCID mice with cobra venom factor to inactivate complement markedly reduced the therapeutic activity of rituximab [20]. Support for the role of complement in rituximab therapy also comes from the demonstration that complement is
L. Wu et al. / Cancer Letters 292 (2010) 208–214
213
Fig. 4. CDC and ADCC induced by anti-CD20 mAbs. Raji and Daudi cells were incubated with increasing concentrations of hu8E4, c8E4 or rituximab in the presence of human complement or PBMCs at 37 °C for 5 h. CDC and ADCC activity of these antibodies was measured by the CytoTox 96 non-Radioactive Cytotoxicity Assay kit. Data are expressed as means ± SD (n = 3).
Fig. 5. Apoptosis induced by anti-CD20 mAbs. Raji or Daudi cells were incubated with different concentrations of hu8E4, c8E4 or rituximab at 37 °C. Apoptosis was assessed 18 h later by Annexin V staining. Apoptotic cells were measured as a percentage of total cells assayed. Columns, mean (n = 3); bars, SD.
consumed during rituximab treatment [21] and that the resistance of different lymphoma cells to rituximab in vivo may be related to their sensitivity to CDC in vitro [22]. However, the role for complement in rituximab therapy still remains controversial. One of the main arguments against complement being a major effector is that the therapeutic activity of rituximab does not appear to correlate with the expression levels of the complement defense molecules CD55 or CD59 [23]. In this study, the humanized anti-CD20 antibody, hu8E4, was significantly more potent in CDC than rituximab, while
its ability to mediate ADCC and to induce apoptosis was similar to that of rituximab. In the in vivo experimental settings, hu8E4 exhibited markedly higher antitumor activity compared with rituximab. Therefore, it could be concluded that the enhanced in vivo antitumor effect of hu8E4 might be attributable to the increased CDC activity. In conclusion, the data shown here further support an important role for complement in CD20 mAb immunotherapy. The humanized anti-CD20 antibody hu8E4 might be a promising therapeutic agent for the treatment of B-cell lymphomas.
214
L. Wu et al. / Cancer Letters 292 (2010) 208–214
Fig. 6. The survival of tumor-bearing SCID mice treated with anti-CD20 mAbs. Groups of 10 SCID mice were injected i.v. with 5 106 Daudi or Daudi-R cells. Five days after tumor cell inoculation, the mice were treated with 100 lg of hu8E4, c8E4 rituximab or the human IgG control. Mice were monitored daily and sacrificed at the onset of hind leg paralysis.
Conflicts of interest None declared. Acknowledgments This work was supported by Grants from National Natural Science Foundation of China, Ministry of Science & Technology of China (973 & 863 program projects), National Key projects for New Drug Development and Manufacture, and for infectious Diseases as well as Grants from Shanghai Commission of Science & Technology. References [1] T.F. Tedder, M. Streuli, S.F. Schlossman, H. Saito, Isolation and structure of a cDNA encoding the B1 (CD20) cell-surface antigen of human B lymphocytes, Proc. Natl. Acad. Sci. USA 85 (1988) 208–212. [2] T.F. Tedder, P. Engel, CD20: a regulator of cell-cycle progression of B lymphocytes, Immunol. Today 15 (1994) 450–454. [3] D.G. Maloney, Immunotherapy for non-Hodgkin’s lymphoma: monoclonal antibodies and vaccines, J. Clin. Oncol. 23 (2005) 6421–6428. [4] G. Cartron, H. Watier, J. Golay, P. Solal-Celigny, From the bench to the bedside: ways to improve rituximab efficacy, Blood 104 (2004) 2635–2642. [5] M.J. Glennie, R.R. French, M.S. Cragg, R.P. Taylor, Mechanisms of killing by anti-CD20 monoclonal antibodies, Mol. Immunol. 44 (2007) 3823–3837. [6] D.R. Anderson, A. Grillo-López, C. Varns, K.S. Chambers, N. Hanna, Targeted anti-cancer therapy using rituximab, a chimaeric anti-CD20 antibody (IDEC-C2B8) in the treatment of non-Hodgkin’s B-cell lymphoma, Biochem. Soc. Trans. 25 (1997) 705–708. [7] P. McLaughlin, A.J. Grillo-López, B.K. Link, R. Levy, M.S. Czuczman, M.E. Williams, M.R. Heyman, I. Bence-Bruckler, C.A. White, F. Cabanillas, V. Jain, A.D. Ho, J. Lister, K. Wey, D. Shen, B.K. Dallaire, Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program, J. Clin. Oncol. 16 (1998) 2825–2833. [8] C.F. Eisenbeis, M.A. Caligiuri, J.C. Byrd, Rituximab: converging mechanisms of action in non-Hodgkin’s lymphoma?, Clin Cancer Res. 9 (2003) 5810–5812. [9] B. Li, S. Shi, W. Qian, L. Zhao, D. Zhang, S. Hou, L. Zheng, J. Dai, J. Zhao, H. Wang, Y. Guo, Development of novel tetravalent anti-CD20 antibodies with potent antitumor activity, Cancer Res. 68 (2008) 2400–2408.
[10] G. Köhler, C. Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 6 (1976) 511–519. [11] W. Qian, L. Wang, B. Li, H. Wang, S. Hou, X. Hong, D. Zhang, Y. Guo, Development of new versions of anti-human CD34 monoclonal antibodies with potentially reduced immunogenicity, Biochem. Biophys. Res. Commun. 367 (2008) 497–502. [12] E.A. Kabat, T.T. Wu, M. Reid-Miller, H.M. Perry, K.S. Gottesman, Sequences of proteins of immunological interest, fourth ed., United States Department of Health and Human Services, Washington, DC, 1987. [13] C. Queen, W.P. Schneider, H.E. Selick, P.W. Payne, N.F. Landolfi, J.F. Duncan, N.M. Avdalovic, M. Levitt, R.P. Junghans, T.A. Waldmann, A humanized antibody that binds to the interleukin 2 receptor, Proc. Natl. Acad. Sci. USA 86 (1989) 10029–10033. [14] V.L. Pulito, V.A. Roberts, J.R. Adair, A.L. Rothermel, A.M. Collins, S.S. Varga, C. Martocello, M. Bodmer, L.K. Jolliffe, R.A. Zivin, Humanization and molecular modeling of the anti-CD4 monoclonal antibody, OKT4A, J. Immunol. 156 (1996) 2840–2850. [15] K. Nakamura, Y. Tanaka, K. Shitara, N. Hanai, Construction of humanized anti-ganglioside monoclonal antibodies with potent immune effector functions, Cancer Immunol. Immunother. 50 (2001) 275–284. [16] C.A. Kettleborough, J. Saldanha, V.J. Heath, C.J. Morrison, M.M. Bendig, Humanization of a mouse monoclonal antibody by CDRgrafting: the importance of framework residues on loop conformation, Protein Eng. 4 (1991) 773–783. [17] H. Wu, Y. Nie, W.D. Huse, J.D. Watkins, Humanization of a murine monoclonal antibody by simultaneous optimization of framework and CDR residues, J. Mol. Biol. 294 (1999) 151–162. [18] S.M. Kipriyanov, F. Le Gall, Generation and production of engineered antibodies, Mol. Biotechnol. 26 (2004) 39–60. [19] N. Di Gaetano, E. Cittera, R. Nota, A. Vecchi, V. Grieco, E. Scanziani, M. Botto, M. Introna, J. Golay, Complement activation determines the therapeutic activity of rituximab in vivo, J. Immunol. 171 (2003) 1581–1587. [20] M.S. Cragg, M.J. Glennie, Antibody specificity controls in vivo effector mechanisms of anti-CD20 reagents, Blood 103 (2004) 2738–2743. [21] L.E. van der Kolk, A.J. Grillo-López, J.W. Baars, C.E. Hack, M.H. van Oers, Complement activation plays a key role in the side-effects of rituximab treatment, Brit. J. Haematol. 115 (2001) 807–811. [22] O. Manches, G. Lui, L. Chaperot, R. Gressin, J.P. Molens, M.C. Jacob, J.J. Sotto, D. Leroux, J.C. Bensa, J. Plumas, In vitro mechanisms of action of rituximab on primary non-Hodgkin lymphomas, Blood 101 (2003) 949–954. [23] W.K. Weng, R. Levy, Expression of complement inhibitors CD46, CD55, and CD59 on tumor cells does not predict clinical outcome after rituximab treatment in follicular non-Hodgkin lymphoma, Blood 98 (2001) 1352–1357.
Contents lists available at ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Characterization of a humanized anti-CD20 antibody with potent antitumor activity against B-cell lymphoma Lan Wu a,1, Chong Wang b,c,1, Dapeng Zhang a, Xunming Zhang a, Weizhu Qian a,b, Lei Zhao a, Hao Wang a,b, Bohua Li a,b,*, Yajun Guo a,b,c,* a
International Joint Cancer Institute and 301 General Hospital Cancer Center, Second Military Medical University, Shanghai 200433, and PLA General Hospital, Beijing 100853, PR China b National Engineering Research Center for Antibody Medicine and Shanghai Key Laboratory of Cell Engineering & Antibody, Shanghai 201203, PR China c School of Medicine and School of Pharmacy, The Center for Antibody Medicine of Ministry of Education, Shanghai Jiao Tong University, 227 South Chongqing Road, Shanghai 200025, PR China
a r t i c l e
i n f o
Article history: Received 6 October 2009 Accepted 8 December 2009
Keywords: CD20 Humanized antibody Complement-dependent cytotoxicity B-cell lymphoma
a b s t r a c t Despite the effectiveness of the anti-CD20 chimeric antibody (mAb), rituximab, in treating B-cell lymphomas, its efficacy remains variable and often modest. In this study, a humanized anti-CD20 antibody, hu8E4, was generated by complementarity-determining region grafting method. Hu8E4 was as effective as rituximab in mediating antibody-dependent cellular cytotoxicity and inducing apoptosis in B-lymphoma cells, but it exhibited much more potent complement-dependent cytotoxicity than rituximab. Immunotherapeutic studies showed that hu8E4 was significantly more effective than rituximab in prolonging the survival of severe combined immunodeficient mice bearing human B-cell lymphomas, suggesting that it might be a promising therapeutic agent for B-cell lymphomas. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The CD20 antigen is present on >90% of B-cell lymphomas and is neither shed nor internalized after antibody binding, making it an effective target for immunotherapeutic removal of malignant B cells [1–3]. The chimeric antiCD20 monoclonal antibody (mAb) rituximab was approved for use in relapsed or refractory low-grade or follicular B-cell non-Hodgkin’s lymphoma (NHL) in 1997. Previous studies have suggested that several mechanisms might be involved in providing therapeutic efficacy, including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and the induction of
* Corresponding authors. Address: International Joint Cancer Institute and 301 General Hospital Cancer Center, Second Military Medical University, Shanghai 200433, and PLA General Hospital, Beijing 100853, PR China. Tel.: +86 21 81870801; fax: +86 21 65306667. E-mail addresses: [email protected] (B. Li), [email protected] (Y. Guo). 1 These authors contributed equally to this work. 0304-3835/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2009.12.004
apoptosis [4,5]. The relative contributions of these different mechanisms of action are still a matter of debate. The most conclusive data supporting conventional effector mechanisms (ADCC and CDC) in the therapeutic response come from work in primates showing that an immunoglobulin G4 (IgG4) variant of rituximab, unlike the chimeric IgG1 monoclonal antibody (mAb), was unable to deplete normal B cells [6]. Since the IgG4 variant would be unable to recruit natural effectors effectively but should retain the binding and cross-linking functions needed for any direct cytotoxic activity, including the signaling of apoptosis, these data point toward CDC and ADCC as the major effectors in vivo. Despite the effectiveness of rituximab, only 48% of patients respond to the treatment and complete responses are <10% [7,8]. There is an urgent need to develop more effective CD20-targeting antibody agents for the treatment of B-cell lymphomas [8,9]. In addition, rituximab is a murine/human chimeric antibody, in which the variable domains are derived from the murine mAb, and the constant regions from human mAb. Although a chimeric antibody is less likely than a fully murine mAb to evoke
L. Wu et al. / Cancer Letters 292 (2010) 208–214
an immune response, and elicitation of a human anti-chimeric antibody response has not posed a significant obstacle to the clinical use of rituximab, it may be advantageous to have a more fully human version, especially if repeated injections may be desired in patients. Administration of humanized mAbs could possibly result in an extension in serum half-life, thus affecting the therapeutic response. In the present study, a mouse anti-human CD20 monoclonal antibody 8E4 was generated by immunizing BALB/c mice with CD20-positive human lymphoma cells. Then we described the construction and characterization of the humanized anti-CD20 antibody (hu8E4). Our data indicated that hu8E4 was shown to retain the same antigen binding affinity and specificity as its parental mouse antibody. The in vitro and in vivo antitumor activity of hu8E4 was further examined and compared with rituximab. 2. Materials and methods 2.1. Cell lines, antibodies, and animals The Chinese hamster ovary cell line CHO-K1, the African green monkey cell line COS-7 and two human Burkitt lymphoma cell lines, Raji and Daudi, were obtained from the American Type Culture Collection (ATCC). Rituximab was purchased from Roche Ltd. Eight-week-old female BALB/c mice and BALB/c SCID mice were housed in pathogen-free conditions and were treated in accordance with guideline of the Committee on Animals of the Second Military Medical University.
209
Then the cells were washed and examined by FCM using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). One hybridoma cell line, which secreted mAbs reacting with CHO-CD20 cells but not with CHO cells, were established and named 8E4. The 8E4 antibody isotype was determined to be (IgG2a,j) with a mouse Ab isotyping kit (Sigma, St. Louis, MO). Finally, the 8E4 antibody was purified by Protein G affinity chromatograph from hybridoma cell culture supernatants and purified 8E4 was labeled with fluoroscein isothiocyanate (FITC) to produce FITCconjugated antibody (FITC-8E4). 2.4. Cloning of 8E4 heavy and light chain variable region genes RNA was isolated from 8E4 hybridoma cells with TRIzol Reagent (Invitrogen, Carlsbad, CA). The variable region cDNAs for the light and heavy chains of 8E4 were cloned from the hybridoma cells by 50 RACE system (Invitrogen, Carlsbad, CA). The gene-specific primers (GSPs) for PCR amplification of 8E4 heavy chain were as follows: GSP1IgG2aH, 50 -AGC TGG GAA GGT GTG CAC ACC ACT-30 ; GSP2-IgG2aH, 50 -CAG AGT TCC AGG TCA AGG TCA-30 ; GSP3-IgG2aH, 50 -CTT GAC CAG GCA TCC TAG AGT-30 . The GSPs used for PCR amplification of light chain variable regions of 8E4 were as follows: GSP1-jL, 50 -TTG CTG TCC TGA TCA GTC CAA CT-30 ; GSP2-jL, 50 -TGT CGT TCA CTG CCA TCA ATC TT-30 ; GSP3-jL, 50 -TTG TTC AAG AAG CAC ACG ACT GA-30 . The final PCR products were cloned into pGEM-T vector (Promega, Madison, WI) for sequence determination.
2.2. Production of CHO cells stably expressing CD20 molecule
2.5. Molecular modeling
Human full length CD20 cDNA was cloned from Raji cells and inserted into pcDNA3.1 (Invitrogen, San Diego, CA). The resultant expression vector was transfected into CHO-K1 cells using Lipofectamine 2000 reagent (Invitrogen, San Diego, CA). After selection with 500 lg/mL G418 for 3 weeks, the transfected cells were stained with FITCconjugated rituximab and then stable transfectants expressing the highest levels of CD20 (CHO-CD20 cells) were obtained by fluorescence-activated cell sorting on a FACSVantage (BD Biosciences, San Jose, CA).
The Protein Data Bank (PDB) was searched for antibody sequences that had high sequence identity with variable fragment (Fv) of 8E4. Two separate BLASTP searches were performed for light chain variable region (VL) and heavy chain variable region (VH) of 8E4. To construct the threedimensional structure of the 8E4 Fv by homology modeling (INSIGHT II 2003, Accelrys, San Diego, CA), the sequences of VL and VH of 8E4 and their templates were aligned, respectively. The coordinates for the structurally conserved regions were assigned from the template and the loop regions were generated by Homology program of Insight II. The new built structure was subjected to molecular dynamics simulations and then energy-minimized by 1000 steps of the steepest descent method and followed by conjugate gradient method using Discover program. Finally, the refined model was assessed by Profile-3D program.
2.3. Hybridoma preparation Female BALB/c mice were repeatedly immunized with Raji cells. Four days after the final booster, the mice were sacrificed and the splenocytes were fused with the NS-1 myeloma cells using standard techniques as previously described [10]. The fused cells were suspended in hypoxanthine/aminopterin/thymidine (HAT) selective medium and plated into 96-well microliter plates. After 12 days, the anti-CD20 antibody-producing hybridoma clones were selected for binding to CHO-CD20 cells but not to CHO cells by flow cytometry (FCM). Briefly, CHO-CD20 or CHO cells at 1 106 cells/mL were incubated with supernatants of the growing hybridomas for 45 min at 4 °C. The cells were washed and incubated with FITC-conjugated goat antimouse IgG (Zymed, San Francisco, CA) for 45 min at 4 °C.
2.6. Humanization of anti-CD20 mAb 8E4 The human consensus sequences of light chain subgroup kappa I (humjI) and heavy chain subgroup III (humIII) were chosen as human framework for the heavy and light chains of humanized version of 8E4, respectively. The complementarity-determining regions (CDRs) in the humanized antibody were chosen to be identical to those in the mouse antibody 8E4. The molecular model of the
210
L. Wu et al. / Cancer Letters 292 (2010) 208–214
8E4 Fv showed that some framework region (FR) residues were close enough to the CDRs to either influence their conformations or interact directly with antigen. When these FR residues differed between 8E4 and the human antibody template, the residue in the humanized antibody was chosen to be the murine 8E4 residue rather than the human antibody residue. The VH and VL genes of humanized versions of 8E4 were synthesized by overlapping PCR. 2.7. Construction and expression of chimeric or humanized antibodies The light and heavy chain expression vectors for chimeric and humanized antibodies were constructed using the same method as described in our previous report [11]. Appropriate light and heavy expression vectors for recombinant antibodies were co-transfected into COS-7 cells using Lipofectamine 2000 reagent. After 72 h incubation, the supernatants were collected and analyzed by enzyme-linked immunosorbent assay (ELISA) for quantitation of antibodies. The ELISA assay used goat anti-human IgG Fc (KPL, Gaithersburg, MD) as capture antibody and goat anti-human kappa horseradish peroxidase (HRP) (Southern Biotechnology Associates, Birmingham, AL) as detecting antibody. FCM was performed to determine the binding of recombinant mAbs to Raji cells. Briefly, Raji cells at 1 106 cells/mL were incubated with different dilutions of the culture supernatants containing recombinant antibodies for 1 h at 48 °C. The cells were washed and incubated with FITC-goat anti-human IgG (H + L) (Zymed, San Francisco, CA) for 1 h at 48 °C. And then the cells were washed and analyzed by FCM. 2.8. Stable expression and purification of recombinant antibodies Appropriate light and heavy expression vectors were co-transfected into Chinese hamster ovary (CHO)-K1 cells and stable transfectants were isolated using the same method as described previously [11]. The clones producing the highest amount of recombinant mAbs were grown in serum free medium and recombinant antibodies were purified by Protein A affinity chromatography. 2.9. Competitive binding assay Raji cells at 1 106 cells/mL were incubated with a subsaturating concentration of the FITC-8E4 and different concentrations of competing antibodies for 45 min at 4 °C. The cells were then washed and analyzed by FCM. The IC50 values of competitors were calculated using a four-variable algorithm. 2.10. Cytotoxicity assays CDC and ADCC activities of CD20 mAbs were measured by lactate dehydrogenase (LDH)-releasing assay using the CytoTox 96 non-Radioactive Cytotoxicity Assay kit (Promega, Madison, WI). Briefly, the CD20+ cells were incubated with CD20 mAbs for 1 h in phenol red-free DMEM culture medium in a 5% CO2 incubator at 37 °C, followed by the
addition of either NHS (7.5% vol/vol) as a source of complement (for CDC assay) or human peripheral blood mononuclear cells (PBMC) as effector cells (effector to target, 25:1 for ADCC assay). After an additional incubation for 5 h at 37 °C, the cell lysis was determined by measuring the amount of LDH released into the culture supernatant. Maximum LDH release was determined by lysis in 0.3% Triton X-100. Percentage of specific lysis was calculated according to the following formula: %lysis = [experimental release spontaneous release]/[maximum release spontaneous release] 100. 2.11. Apoptosis assay The cells were incubated with different concentrations of anti-CD20 mAbs at 37 °C for 16 h. After washing, cells were treated with Annexin V-FITC (BD Biosciences, San Diego, CA), washed again, and analyzed by FCM. 2.12. Immunotherapy Groups of 10 8-week-old female SCID mice were injected via the tail vein with 5 106 Raji or Daudi cells on day 0, followed 5 days later by the i.v. injection of 100 lg of CD20 mAbs. The mice were observed daily and euthanized at the onset of hind leg paralysis. 2.13. Statistical analysis Statistical analysis was performed by Student’s unpaired t test to identify significant differences unless otherwise indicated. Differences were considered significant at a P value of <0.05. 3. Results 3.1. Molecular modeling of anti-CD20 monoclonal antibody 8E4 The nucleotide sequences of VH and VL of 8E4 were determined and the deduced amino acid sequences of VH and VL of 8E4 were shown in Fig. 1. We searched PDB for antibody sequences with high sequence identity with the 8E4 antibody variable regions. The antibody C2B8 (PDB No. 2OSL) shows 84% identity with the VH of 8E4 and the antibody E8 (PDB No. 1WEJ) shows 94% identity with the VL of 8E4. We selected C2B8 and E8 as the templates of the VH and VL of 8E4, respectively. Then the molecular model of the variable regions of 8E4 was obtained by Insight II molecular modeling software (Fig. 2). 3.2. Humanization of 8E4 antibody We selected humIII and humjI as the variable region frameworks for the VH and VL of humanized 8E4 antibody, respectively. Firstly, the three CDRs from 8E4 light chain or heavy chain were directly grafted into human antibody light or heavy chain frameworks to generate humanized antibody genes. The humanized VL and VH were cloned into expression vectors, respectively, and were coexpressed in COS-7 cells, yielding humanized version (hu8E4VHa/hu8E4VLa). The amino acid sequence of the hu8E4VHa/hu8E4VLa antibody was shown in Fig. 1. This antibody in COS-7 cell culture supernatant was quantified by ELISA and the binding of hu8E4VHa/hu8E4VLa to Raji cells was determined by FCM. The results showed that this humanized antibody lost its binding activity to CD20 molecule (Fig. 3A), suggesting that some FR residues in this humanized version must be back-mutated to reconstitute the full binding activity. With the help of graphic manipulation, eleven mouse FR residues, which differed between 8E4 and the human antibody template, were observed to be within 5 Å of the CDRs and to probably affect the structure of the
L. Wu et al. / Cancer Letters 292 (2010) 208–214
211
Fig. 1. Amino acid sequences of humanized 8E4 antibody heavy (A) and light (B) chain variable regions. 8E4VH and 8E4VL indicate heavy and light chain variable regions of murine 8E4 antibody, respectively. The humIII was chosen as framework for the humanized heavy chains and the humjI was chosen for the humanized light chains. Hu8E4VHa and hu8E4VHb indicate different versions of humanized heavy chain variable regions. Hu8E4VLa and hu8E4VLb indicate different versions of humanized light chain variable regions. The dashes represent amino acids that are the same as the corresponding residues in human antibody framework. The CDRs are enclosed with brackets. Amino acids (in one-letter notation) are numbered according to Kabat et al. [12]. CDRs (Fig. 2). A number of humanized light and heavy chain versions were constructed to evaluate the contribution of each of the eleven FR residues to antigen binding. Finally, a humanized version (hu8E4VHb/ hu8E4VLb) showing the similar antigen-binding activity as the chimeric 8E4 antibody (c8E4) was obtained (Fig. 3A). This humanized antibody was designated as hu8E4 and its amino acid sequences were shown in Fig. 1.
3.3. Competitive binding assay The humanized antibody hu8E4 was expressed in CHO cells and then purified from the CHO cell serum-free culture supernatant by Protein A affinity chromatography. In the competitive binding assay, hu8E4 effectively competed with 8E4 for binding to Raji cells (Fig. 3B). The avidity (mean IC50 ± SD) of hu8E4 (1.36 ± 0.17 lg/mL) was similar to that of 8E4 (1.30 ± 0.15 lg/mL) and c8E4 (1.16 ± 0.11 lg/mL), suggesting that this humanized antibody possessed affinity and specificity similar to that of its parental antibody.
3.4. CDC activity of hu8E4 The capacity of hu8E4 to mediate CDC was assessed in two CD20-positive human lymphoma cell lines, Daudi and Raji. The results summarized in Fig. 4 showed that hu8E4 was as effective as c8E4 in lysing Daudi cells in the presence of human complement. Encouragingly, hu8E4 was significantly more potent in CDC than rituximab (P < 0.05 for hu8E4 compared with rituximab at concentrations of 0.4, 2, and 10 lg/mL), and the similar results (Fig. 4) were obtained with Raji cells, which are more resistant to CDC than Daudi cells.
3.5. ADCC activity of hu8E4 The ability of hu8E4 to lyse CD20+ lymphoma cells in the presence of human PBMCs was investigated and compared with c8E4 and rituximab. Our data (Fig. 4) clearly indicated that hu8E4, c8E4 and rituximab were equally effective in mediating ADCC against Daudi or Raji cells in a dose-dependent manner.
212
L. Wu et al. / Cancer Letters 292 (2010) 208–214
Fig. 2. Molecular model of the 8E4 variable regions. The 8E4 variable regions are shown by a solid ribbon representation. The FRs are shown in green and the CDRs are shown in red. Eleven FR residues, which are within about 5 Å of the CDRs and are of potential importance to the antigen binding, are colored gray. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.6. Apoptosis-inducing activity of hu8E4 Induction of apoptosis was evaluated by FITC-Annexin V assays in Daudi and Raji cells. As illustrated in Fig. 5, hu8E4 was able to trigger low levels of apoptosis of both of these two CD20-positive lymphoma cells, and its apoptotic activity was shown to be similar to that of c8E4 and rituximab. 3.7. The in vivo antitumor activity of hu8E4 In vivo therapy studies were performed in SCID mice bearing systemic Daudi or Raji tumors. The survival curves were plotted according to Kaplan–Meier method and compared using the log-rank test. The results summarized in Fig. 6 showed that all of the three anti-CD20 antibodies, hu8E4, c8E4 and rituximab, significantly improved the survival of SCID mice bearing disseminated Daudi tumor cells (P < 0.001 for each compared with the human IgG control). The animals treated with hu8E4 had similar survival curves as those treated with c8E4. However, a pronounced difference in survival was noticed between hu8E4 and rituximab treatment groups (P < 0.01). Hu8E4 was significantly more effective than rituximab in prolonging the survival of SCID mice bearing Daudi tumor cells. Similar results were obtained using Raji tumor cell-bearing SCID mice.
4. Discussion Here we have described the humanization of an antiCD20 monoclonal antibody, 8E4. The initial version of humanized antibody (hu8E4Ha/hu8E4La) was constructed by grafting the CDRs alone from 8E4 to its human antibody template. Antigen-binding activity assays indicated that this version lost the binding activity to CD20-positive Raji cells. This is because some FR residues may be critical for preserving the CDR conformations or be directly involved in antigen binding [13–18]. Computer-assisted molecular design can be used to build the molecular model of the antibody to help identify such structurally important FR residues. In this study, a three-dimensional model of the variable regions of 8E4 was built using computer-aided
Fig. 3. Antigen-binding activity of CD20 mAbs. (A) Raji cells were incubated with serial log dilutions of c8E4 or different humanized versions for 1 h at 48 °C. Cells were washed and incubated with FITC-goat anti-human IgG (H + L) for 1 h at 48 °C. Cells were then washed and analyzed by FCM. Data are presented as means ± SD (n = 3). (B) Raji cells were incubated with a subsaturating concentration of the FITC-8E4 and different concentrations of competing antibodies for 45 min at 4 °C. The cells were then washed and analyzed by FCM. Maximal fluorescence means the mean channel fluorescence obtained in the absence of competitor antibodies. All data were expressed as the mean of triplicate samples.
homology modeling. Eleven FR residues within 5 Å of the CDRs were identified as key FR residues and a humanized version of 8E4 (hu8E4) was generated by transferring these mouse key framework residues onto the human framework together with the mouse CDR residues. In the competitive binding assay, hu8E4 was shown to have IC50 value similar to its parental mouse antibody 8E4, suggesting that the relative affinities of the two antibodies were about equal. Previous studies have shown that the protective activity of rituximab was completely abolished in C1q-deficient mice inoculated with syngenic lymphoma transduced with human CD20 [19]. Similarly, Gragg and Glennie found that treatment of SCID mice with cobra venom factor to inactivate complement markedly reduced the therapeutic activity of rituximab [20]. Support for the role of complement in rituximab therapy also comes from the demonstration that complement is
L. Wu et al. / Cancer Letters 292 (2010) 208–214
213
Fig. 4. CDC and ADCC induced by anti-CD20 mAbs. Raji and Daudi cells were incubated with increasing concentrations of hu8E4, c8E4 or rituximab in the presence of human complement or PBMCs at 37 °C for 5 h. CDC and ADCC activity of these antibodies was measured by the CytoTox 96 non-Radioactive Cytotoxicity Assay kit. Data are expressed as means ± SD (n = 3).
Fig. 5. Apoptosis induced by anti-CD20 mAbs. Raji or Daudi cells were incubated with different concentrations of hu8E4, c8E4 or rituximab at 37 °C. Apoptosis was assessed 18 h later by Annexin V staining. Apoptotic cells were measured as a percentage of total cells assayed. Columns, mean (n = 3); bars, SD.
consumed during rituximab treatment [21] and that the resistance of different lymphoma cells to rituximab in vivo may be related to their sensitivity to CDC in vitro [22]. However, the role for complement in rituximab therapy still remains controversial. One of the main arguments against complement being a major effector is that the therapeutic activity of rituximab does not appear to correlate with the expression levels of the complement defense molecules CD55 or CD59 [23]. In this study, the humanized anti-CD20 antibody, hu8E4, was significantly more potent in CDC than rituximab, while
its ability to mediate ADCC and to induce apoptosis was similar to that of rituximab. In the in vivo experimental settings, hu8E4 exhibited markedly higher antitumor activity compared with rituximab. Therefore, it could be concluded that the enhanced in vivo antitumor effect of hu8E4 might be attributable to the increased CDC activity. In conclusion, the data shown here further support an important role for complement in CD20 mAb immunotherapy. The humanized anti-CD20 antibody hu8E4 might be a promising therapeutic agent for the treatment of B-cell lymphomas.
214
L. Wu et al. / Cancer Letters 292 (2010) 208–214
Fig. 6. The survival of tumor-bearing SCID mice treated with anti-CD20 mAbs. Groups of 10 SCID mice were injected i.v. with 5 106 Daudi or Daudi-R cells. Five days after tumor cell inoculation, the mice were treated with 100 lg of hu8E4, c8E4 rituximab or the human IgG control. Mice were monitored daily and sacrificed at the onset of hind leg paralysis.
Conflicts of interest None declared. Acknowledgments This work was supported by Grants from National Natural Science Foundation of China, Ministry of Science & Technology of China (973 & 863 program projects), National Key projects for New Drug Development and Manufacture, and for infectious Diseases as well as Grants from Shanghai Commission of Science & Technology. References [1] T.F. Tedder, M. Streuli, S.F. Schlossman, H. Saito, Isolation and structure of a cDNA encoding the B1 (CD20) cell-surface antigen of human B lymphocytes, Proc. Natl. Acad. Sci. USA 85 (1988) 208–212. [2] T.F. Tedder, P. Engel, CD20: a regulator of cell-cycle progression of B lymphocytes, Immunol. Today 15 (1994) 450–454. [3] D.G. Maloney, Immunotherapy for non-Hodgkin’s lymphoma: monoclonal antibodies and vaccines, J. Clin. Oncol. 23 (2005) 6421–6428. [4] G. Cartron, H. Watier, J. Golay, P. Solal-Celigny, From the bench to the bedside: ways to improve rituximab efficacy, Blood 104 (2004) 2635–2642. [5] M.J. Glennie, R.R. French, M.S. Cragg, R.P. Taylor, Mechanisms of killing by anti-CD20 monoclonal antibodies, Mol. Immunol. 44 (2007) 3823–3837. [6] D.R. Anderson, A. Grillo-López, C. Varns, K.S. Chambers, N. Hanna, Targeted anti-cancer therapy using rituximab, a chimaeric anti-CD20 antibody (IDEC-C2B8) in the treatment of non-Hodgkin’s B-cell lymphoma, Biochem. Soc. Trans. 25 (1997) 705–708. [7] P. McLaughlin, A.J. Grillo-López, B.K. Link, R. Levy, M.S. Czuczman, M.E. Williams, M.R. Heyman, I. Bence-Bruckler, C.A. White, F. Cabanillas, V. Jain, A.D. Ho, J. Lister, K. Wey, D. Shen, B.K. Dallaire, Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program, J. Clin. Oncol. 16 (1998) 2825–2833. [8] C.F. Eisenbeis, M.A. Caligiuri, J.C. Byrd, Rituximab: converging mechanisms of action in non-Hodgkin’s lymphoma?, Clin Cancer Res. 9 (2003) 5810–5812. [9] B. Li, S. Shi, W. Qian, L. Zhao, D. Zhang, S. Hou, L. Zheng, J. Dai, J. Zhao, H. Wang, Y. Guo, Development of novel tetravalent anti-CD20 antibodies with potent antitumor activity, Cancer Res. 68 (2008) 2400–2408.
[10] G. Köhler, C. Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 6 (1976) 511–519. [11] W. Qian, L. Wang, B. Li, H. Wang, S. Hou, X. Hong, D. Zhang, Y. Guo, Development of new versions of anti-human CD34 monoclonal antibodies with potentially reduced immunogenicity, Biochem. Biophys. Res. Commun. 367 (2008) 497–502. [12] E.A. Kabat, T.T. Wu, M. Reid-Miller, H.M. Perry, K.S. Gottesman, Sequences of proteins of immunological interest, fourth ed., United States Department of Health and Human Services, Washington, DC, 1987. [13] C. Queen, W.P. Schneider, H.E. Selick, P.W. Payne, N.F. Landolfi, J.F. Duncan, N.M. Avdalovic, M. Levitt, R.P. Junghans, T.A. Waldmann, A humanized antibody that binds to the interleukin 2 receptor, Proc. Natl. Acad. Sci. USA 86 (1989) 10029–10033. [14] V.L. Pulito, V.A. Roberts, J.R. Adair, A.L. Rothermel, A.M. Collins, S.S. Varga, C. Martocello, M. Bodmer, L.K. Jolliffe, R.A. Zivin, Humanization and molecular modeling of the anti-CD4 monoclonal antibody, OKT4A, J. Immunol. 156 (1996) 2840–2850. [15] K. Nakamura, Y. Tanaka, K. Shitara, N. Hanai, Construction of humanized anti-ganglioside monoclonal antibodies with potent immune effector functions, Cancer Immunol. Immunother. 50 (2001) 275–284. [16] C.A. Kettleborough, J. Saldanha, V.J. Heath, C.J. Morrison, M.M. Bendig, Humanization of a mouse monoclonal antibody by CDRgrafting: the importance of framework residues on loop conformation, Protein Eng. 4 (1991) 773–783. [17] H. Wu, Y. Nie, W.D. Huse, J.D. Watkins, Humanization of a murine monoclonal antibody by simultaneous optimization of framework and CDR residues, J. Mol. Biol. 294 (1999) 151–162. [18] S.M. Kipriyanov, F. Le Gall, Generation and production of engineered antibodies, Mol. Biotechnol. 26 (2004) 39–60. [19] N. Di Gaetano, E. Cittera, R. Nota, A. Vecchi, V. Grieco, E. Scanziani, M. Botto, M. Introna, J. Golay, Complement activation determines the therapeutic activity of rituximab in vivo, J. Immunol. 171 (2003) 1581–1587. [20] M.S. Cragg, M.J. Glennie, Antibody specificity controls in vivo effector mechanisms of anti-CD20 reagents, Blood 103 (2004) 2738–2743. [21] L.E. van der Kolk, A.J. Grillo-López, J.W. Baars, C.E. Hack, M.H. van Oers, Complement activation plays a key role in the side-effects of rituximab treatment, Brit. J. Haematol. 115 (2001) 807–811. [22] O. Manches, G. Lui, L. Chaperot, R. Gressin, J.P. Molens, M.C. Jacob, J.J. Sotto, D. Leroux, J.C. Bensa, J. Plumas, In vitro mechanisms of action of rituximab on primary non-Hodgkin lymphomas, Blood 101 (2003) 949–954. [23] W.K. Weng, R. Levy, Expression of complement inhibitors CD46, CD55, and CD59 on tumor cells does not predict clinical outcome after rituximab treatment in follicular non-Hodgkin lymphoma, Blood 98 (2001) 1352–1357.