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Keywords:

  • Apoptosis;
  • Human mAb;
  • Immunotherapy;
  • TRAIL;
  • TRAIL-Receptor 1

Monoclonal antibodies have attracted attention clinically as promising therapeutic agents for immunotherapy against various malignancies because of their high specificity and low toxicity. In the clinical application of mAb, immunogenicity of mAb is one of the major concerns. We have developed immunospot array assay on a chip (ISAAC) technology using chips with an array of microwells 1, which enables direct identification of Ag-specific Ab-secreting cells (ASC) from human peripheral blood lymphocytes and rapid cloning of Ab cDNA, leading to easy, efficient and rapid production of human mAb. However, the limitation of immunizing volunteers with Ag hampers its application for the production of human mAb for desired Ag. In this study, we used the ISAAC method in combination with TransChromo (TC) mice that contain human chromosome fragments encoding the entire human immunoglobulin H-chain and κL-chain loci 2 for isolating fully human mAb specific to TNF-related apoptosis-inducing ligand (TRAIL)-receptors type 1 with the object of cancer immunotherapy. TRAIL receptors are expressed in a wide variety of tumor cell types as well as normal cell types. TRAIL induces apoptosis in a wide variety of human cancer cell lines by activating two functional receptors, TRAIL-R1 (death receptor 4) 3 and TRAIL-R2 (death receptor 5) 4.

The protocol for generating fully human mAb against human TRAIL-R1 using immunospot array assay on a chip (ISAAC) method 1 and TransChromo (TC) mice is shown in Fig. 1A and described in Supporting Information Methods. We observed signals of the secreted TRAIL-R1-specific Ab outside the microwells containing TRAIL-R1-specific Ab-secreting cells (ASC) (Fig. 1B), but not outside the wells containing ASC for other Ag (data not shown). We retrieved 22 cells with strong signals on the chips and transferred individual cells into separate micro-tubes for amplifying H- and L-chain variable region (VH and VL) cDNA. We isolated 16 VH and VL cDNA pairs, transfected each pair of cDNA into CHO cells, produced fully human mAb and finally obtained ten mAb that bound only human TRAIL-R1 (Fig. 1C), but not human TRAIL-R2 (Supporting Information Fig. 1) or an irrelevant Fc-fusion protein (data not shown). The binding of mAb to immobilized human TRAIL-R1 was inhibited by the addition of soluble human TRAIL-R1 in a dose-dependent manner (Fig. 1C), further confirming the Ag specificity. The surface plasmon resonance analysis showed that the affinities of these mAb ranged from 2.0×10−6 to 2.6×10−11 M (Supporting Information Table 1).

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Figure 1. Detection of TRAIL-R1-specific ASC and production of TRAIL-R1-specific Ab with ISAAC system. (A) Schematic diagram of rapid generation of fully human mAb from TRAIL-R1-immunized TC mice using ISAAC method. (B) Detection of single human TRAIL-R1-specific ASC using ISAAC system. (Left) Signals for Ag-specific Ab secreted from individual cells; (middle) cells stained with Oregon Green; (right) merged. (C) Ag specificities. Competitive ELISA was performed using indicated doses of recombinant human TRAIL-R1/Fc (rhTRAIL-R1) in 96-well plate that was coated with or without (uncoated) rhTRAIL-R1. PC, 100-fold diluted positive control serum from an rhTRAIL-R1/Fc-immunized mouse. (D) Competition of mAb with sTRAIL for binding to immobilized human TRAIL-R1/Fc. sTRAIL was preincubated (gray columns) or not preincubated (closed columns) with plate-coated human TRAIL-R1/Fc. TRAIL-R1-specific mAb were then added to the plate. Open columns, the binding of mAb to uncoated plate. NC, no TRAIL-R1-specific Ab; PC, 100-fold diluted mouse anti-serum to human TRAIL-R1. Data represent mean±SD of triplicate cultures and are representative of three independent experiments. *p<0.001, **<0.05 (t-test).

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Most of the reported mAb to TRAIL-R1 or TRAIL-R2 efficiently competed with soluble TRAIL (sTRAIL) for binding to their receptors 5, 6. Competitive ELISA with sTRAIL showed that the TR1-407, -412, -417, -422 and -438 Ab competed with sTRAIL for the binding to TRAIL-R1, but not others (TR1-401, -404, -416 and -419) (Fig. 1D). In another study, we analyzed the relative binding sites of these mAb 7. These results together indicate the diverse binding sites of mAb on TRAIL-R1. Flow cytometric analysis revealed the binding of the mAb to the surface of tumor cells including Colo205 colorectal adenocarcinoma cells, K562 leukemia cells and Daudi Burkitt's lymphoma cells (Supporting Information Fig. 2).

We confirmed the induction of cell death by these mAb in Colo205, K562 and Daudi cells in the presence or absence of cross-linking Ab (Supporting Information Fig. 3). Susceptibility to Ab-induced apoptosis varied from cell to cell and from Ab to Ab. In cancer treatment with TRAIL or TRAIL-R-specific Ab, some cell types show resistance to TRAIL-induced apoptosis. To resolve this problem, TRAIL or TRAIL-R-specific Ab has been used in combination with other cell-death inducers such as chemotherapeutic agents to augment the killing of tumor cells 8–12. Here, we examined the effect of our mAb on sTRAIL-induced apoptosis. TR1-407, -412, -417, -422 and -438 Ab showed an inhibitory effect on sTRAIL-induced apoptosis in Colo205, K562 and Daudi cells, whereas TR1-401, -404, -416 and -419 Ab clearly enhanced sTRAIL-induced apoptosis in Colo205, K562 and Daudi cells (Fig. 2A and B, and Supporting Information Fig. 4). The relationship of Ab- and TRAIL-binding sites on TRAIL-R1 rather than Ab's affinities seemed to prescribe Ab function (Fig. 1D and Supporting Information Table 1). We also investigated the kinetic effect of the combination of sTRAIL and the enhancing mAb on cell-death induction. Treatment with either sTRAIL or TR1-419 mAb alone induced cell death from 6 h after the treatment, and 40–60% of the cells died maximally after 48 h (Fig. 2C). On the contrary, the combination of sTRAIL and mAb significantly induced cell death within 3 h, and more than 95% of the cells died after 48 h (Fig. 2C). Similar results were observed for other enhancing mAb, TR1-401,-404 and -416 (data not shown). These results show that TRAIL-mediated cell death was significantly promoted by enhancing TRAIL-R1-specific mAb, resulting in strong inhibition of tumor cell growth, and indicate that the combination of TRAIL-R1-specific mAb and TRAIL could provide innovative procedures for future cancer therapy.

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Figure 2. Effects of TRAIL-R1-specific mAb on TRAIL-induced apoptosis. (A, B) Effect of TRAIL-R1-specific mAb on TRAIL-mediated cell death in K562 (A) and Daudi (B) cells. Cells were plated with TRAIL-R1-specific mAb (closed columns) or control Ab (open and gray columns) for 1 h, followed by 1 μg/mL (A) or 0.1 μg/mL (B) of sTRAIL (closed and grey columns). In total, 24 h after cell culture, cell viability was determined by trypan blue dye exclusion method. Data are presented as mean±SD of triplicate cultures and are representative of three independent experiments. *p<0.001 versus TRAIL alone (−). (C) Time course of cell death in Daudi cells induced with combination of TR1-419 mAb and sTRAIL. Cells were incubated with sTRAIL alone, TR1-419 mAb alone or a mixture of sTRAIL and TR1-419 mAb for indicated durations. Cell viability was determined as above. Data are presented as mean±SD of triplicate cultures and are representative of three independent experiments.

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In conclusion, the combination of the ISAAC system and TC mice prompts us to efficiently produce fully human mAb for desired target, with various affinities, functions and binding specificities, and may contribute to the advancement of immunotherapeutics.

Acknowledgements

  1. Top of page
  2. Acknowledgements
  3. References
  4. Supporting Information

The authors thank Kyowa Hakko Kirin for providing TC Mice. The authors also thank S. Hirota for technical assistance and K. Hata for secretarial work. This research was supported by grants from the Hokuriku Innovation Cluster for Health Science Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan to A. M.

Conflict of interest: A. J. was working in SC World Inc., a company that uses the techniques described in the manuscript. H. K. and A. M. are unsalaried directors of SC World Inc.

References

  1. Top of page
  2. Acknowledgements
  3. References
  4. Supporting Information

Supporting Information

  1. Top of page
  2. Acknowledgements
  3. References
  4. Supporting Information

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