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Abstract

  1. Top of page
  2. Abstract
  3. Strategies for the efficient preparation of anti-cancer mAb: transformation, phage display, autonomously diversifying library (ADLib) or hybridoma?
  4. Possible immunogens for the preparation of therapeutic mAb recognizing the extracellular domains of cell-surface oncoproteins, and animals for immunization
  5. Establishment of cell lines expressing GFP-fused oncoproteins
  6. Production of cell-death-inducing mAb, identification of the antigen as chicken transferrin receptor (TfR-CD71) and development of cell-death-inducing mAb recognizing human TfR (type II membrane protein)
  7. Preparation of therapeutic mAb targeting type IV multi-pass membrane proteins containing G protein-coupled receptors (GPCR) and solute carrier (SLC) families
  8. Preparation of mAb targeting type I membrane proteins containing adhesion molecules (CD44v) and growth factor receptors (HER family)
  9. Acquisition of mAb with superior quality to original mAb by the immunization of mimotope peptides determined by phage display
  10. Conclusion with additional discussion
  11. Acknowledgments
  12. Disclosure statement
  13. References

Cell-surface molecules containing growth factor receptors, adhesion molecules and transporter proteins are often over-expressed in various cancer cells, and could be regarded as suitable targets for therapeutic monoclonal antibodies (mAb). Anti-cancer therapeutic mAb are claimed to bind these cell-surface molecules on viable cancer cells: therefore, it is necessary to produce mAb recognizing epitopes on the extracellular domains of native but not denatured proteins. We have experienced difficulty in obtaining mAb bound to viable cancer cells using synthetic peptides or recombinant proteins produced in bacteria as immunogens, although these immunogens are relatively easy to prepare. In this context, we have concluded that viable cancer cells or cells transfected with cDNA encoding target proteins are suitable immunogens for the production of anti-cancer therapeutic mAb. Furthermore, we selected rats as the immunized animals, because of their excellent capacity to generate diverse antibodies. Because many target candidates are multi-pass (type IV) membrane proteins, such as 7-pass G protein-coupled receptors and 12-pass transporter proteins belonging to the solute carrier family, and their possible immunogenic extracellular regions are very small, production of specific mAb was extremely difficult. In this review, we summarize the successful preparation and characterization of rat mAb immunized against the extracellular domain of type I, type II and type IV membrane oncoproteins fused to green fluorescent protein as an approach using reverse genetics, and also introduce the discovery of cell-death-inducing antibodies as an approach using forward genetics and a strategy to produce reshaped antibodies using mimotope peptides as the immunogen. (Cancer Sci 2011; 102: 25–35)

Against the medical background of serum therapy for diphtheria (1891) and polyclonal IgG treatment for agammaglobulinemia (1952), the development of monoclonal antibody (mAb) technology by Köhler and Milstein (1975)(1) led to the approval of the first therapeutic murine mAb, Muromonab-OKT3 (common name-brand name) (approved in 1986), for the prevention of transplantation rejection. Moreover, the progress of molecular and transgenic technologies has enabled the development of chimeric mAb, Abciximab-ReoPro (1994) and Rituximab-Rituxan (1997), humanized (complementarity-determining region; CDR-grafted) mAb, Trastuzumab-Herceptin (1998) and Infliximab-Remicade (1998), and fully human mAb, phage display–derived Adalimumab-Humira (2002) and transgenic mouse-derived Panitumumab-Vectibix (2006) for the therapy of various severe diseases, including cancerous, autoimmune and inflammatory syndromes. The market for therapeutic mAb has exponentially grown over the past 10 years. Although confined to cancers, various therapeutic mAb are on the market or undergoing clinical trials: for instance, chimeric Rituximab, fully human Ofatumumab-Arzerra, 90Y- or 111In-labeled Ibritumomab-Zevalin and 131I-conjugated Tositumomab-Bexxar (α-CD20(2–4) on B lymphoma), humanized Trastuzumab and Pertuzumab-Omnitarg (HER2(2,5–8) on breast cancer), chimeric Cetuximab-Erbitux and full human Panitumumab-Vectibix (HER1(2,5,9–11) on colon cancer), Bevacizumab-Avastin (vascular endothelial cell growth factor [VEGF](11–14) for colon cancer) and KW-0761 (CCR4 on T lymphoma(15)). Most targets recognized by anti-cancer therapeutic mAb are cell-surface molecules, although some mAb recognize soluble cytokines (VEGF, hepatocyte growth factor [HGF],(16) etc.) in the cancer microenvironment. In this paper, we provide an overview of the strategy for the development of anti-cancer therapeutic mAb against cell-surface (glyco) proteins that are over-expressed in human malignancies using the approach of so-called “reverse genetics”, and also introduce the discovery of cell-death-inducing antibodies using the approach of so-called “forward genetics” and a strategy to produce reshaped antibodies using mimotope peptides as the immunogen.

Strategies for the efficient preparation of anti-cancer mAb: transformation, phage display, autonomously diversifying library (ADLib) or hybridoma?

  1. Top of page
  2. Abstract
  3. Strategies for the efficient preparation of anti-cancer mAb: transformation, phage display, autonomously diversifying library (ADLib) or hybridoma?
  4. Possible immunogens for the preparation of therapeutic mAb recognizing the extracellular domains of cell-surface oncoproteins, and animals for immunization
  5. Establishment of cell lines expressing GFP-fused oncoproteins
  6. Production of cell-death-inducing mAb, identification of the antigen as chicken transferrin receptor (TfR-CD71) and development of cell-death-inducing mAb recognizing human TfR (type II membrane protein)
  7. Preparation of therapeutic mAb targeting type IV multi-pass membrane proteins containing G protein-coupled receptors (GPCR) and solute carrier (SLC) families
  8. Preparation of mAb targeting type I membrane proteins containing adhesion molecules (CD44v) and growth factor receptors (HER family)
  9. Acquisition of mAb with superior quality to original mAb by the immunization of mimotope peptides determined by phage display
  10. Conclusion with additional discussion
  11. Acknowledgments
  12. Disclosure statement
  13. References

Four typical methods have been historically used to obtain mAb, namely, Epstein–Barr virus (EBV) transformation,(17) antibody phage display,(18) ADLib(19) and hybridoma(1) technologies. Transformation of human B lymphocytes by EBV followed by the acquisition of antibodies is an excellent method to obtain human antibodies with defined specificity, and anti-cancer therapeutic mAb recognizing GD2 ganglioside have been successfully prepared and clinically tested;(20) however, this can not become a standard method because of the difficulty of cloning antibody-secreting cells and obtaining antigen-sensitized human lymphocyte sources. The latter problem is also involved in phage-display technology with human antibody libraries, since hyper-sensitized or memory B lymphocytes against tumor-associated antigen (TAA) can rarely be included in human antibody libraries, mainly because of the ethical limitations on using human materials. Currently, genetic engineering can be used to generate mAb without the need to establish hybridomas. One approach, called “antibody phage display”, uses a random collection of cDNA coding antigen-binding regions of antibodies, and comprehensive screening for TAA and the characterization of fully human mAb against TAA have been recently reported.(21) Another approach, called “ADLib”, uses DT40,(22) a chicken B cell line that undergoes constitutive gene conversion at both light- and heavy-chain immunoglobulin loci, and antigen-specific DT40 cells are selected from ADLib using bio-panning with antigen-conjugated magnetic beads or cell sorting. The gene-conversion rate has been recently proved to be changed upward by the gene-knockout of Bloom in DT40,(23) therefore the performance of ADLib might be further improved. The benefit of the ADLib system is its rapidity, as the whole process from selection to screening can be completed in approximately 1 week, although humanized chicken mAb might be more immunogenic than humanized rodent mAb against human species.(24) In the next section, we will address the strategy of developing anti-cancer therapeutic mAb, based on the establishment of hybridomas from rat splenocytes hyperimmune to rat hepatoma cells over-expressing target proteins of human or mouse origin.

Possible immunogens for the preparation of therapeutic mAb recognizing the extracellular domains of cell-surface oncoproteins, and animals for immunization

  1. Top of page
  2. Abstract
  3. Strategies for the efficient preparation of anti-cancer mAb: transformation, phage display, autonomously diversifying library (ADLib) or hybridoma?
  4. Possible immunogens for the preparation of therapeutic mAb recognizing the extracellular domains of cell-surface oncoproteins, and animals for immunization
  5. Establishment of cell lines expressing GFP-fused oncoproteins
  6. Production of cell-death-inducing mAb, identification of the antigen as chicken transferrin receptor (TfR-CD71) and development of cell-death-inducing mAb recognizing human TfR (type II membrane protein)
  7. Preparation of therapeutic mAb targeting type IV multi-pass membrane proteins containing G protein-coupled receptors (GPCR) and solute carrier (SLC) families
  8. Preparation of mAb targeting type I membrane proteins containing adhesion molecules (CD44v) and growth factor receptors (HER family)
  9. Acquisition of mAb with superior quality to original mAb by the immunization of mimotope peptides determined by phage display
  10. Conclusion with additional discussion
  11. Acknowledgments
  12. Disclosure statement
  13. References

Whole cancer cells, synthetic peptides, non-glycosylated and often fragmented proteins produced in the bacteria, or full-length glycosylated proteins produced in yeast, insect or mammalian cells can be used as the immunogens to prepare anti-cancer therapeutic mAb. We have encountered difficulties in achieving mAb binding to viable cancer cells using synthetic peptides corresponding to the extracellular domain of target proteins, or recombinant proteins produced in bacteria as immunogens, although these immunogens are relatively easy to prepare. In this context, we have concluded that viable cancer cells or cells transfected with cDNA encoding target proteins are the most suitable immunogens for the production of anti-cancer therapeutic mAb. To confirm the stable expression of target proteins, established cell lines transfected with cDNA of green fluorescent protein (GFP)(25)-fused target proteins were used as the immunogen (Fig. 1a).

image

Figure 1.  Strategies for development of anti-cancer therapeutic monoclonal antibodies (mAb) against the extracellular domain of tumor-associated antigens (TAA). (a) Suitable immunogens and animals for immunization to develop specific mAb. (b) Various green fluorescent protein (GFP)-fused membrane proteins. (c) Expression of GFP-fused target proteins before and after cell sorting. RH7777 cells were transfected with cDNA of GFP-LAT1, cultured with Genetecin for 2 weeks (upper left), and cells strongly expressing GFP-human LAT1 proteins were clone-sorted and expanded (upper right). Phase-contrast micrograph (lower left) and micrograph of UV-excited green colored GFP-LAT1 proteins (lower right) of clone-sorted cells are shown. (d) Hybridoma antibodies were selected for reactivity with HEK293F cells transfected with cDNA of GFP-fused LAT1 in a GFP-expression-dependent manner. HEK293F cells were transfected with cDNA of GFP-human CD44 (left) or GFP-human LAT1 (right) and stained with SOL22 followed by phycoerythrin-conjugated anti-rat IgG Fcγ. GPCR, G protein-coupled receptors; SLC, solute carrier. Adapted from Ohno et al. (c,d),(31) with permission.

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Mice are generally immunized for the production of mAb; however, we prefer to immunize rats with rat cell lines expressing the target proteins as immunogens (Fig. 1a). Although the immunoglobulin heavy chain locus of the rat has striking homology to mouse antibody genes, a marked difference between mouse and rat κ chain loci is found in the Jκ region, where the rat contains additional Jκ segments(26) and the V locus of the rat immunoglobulin λ light chain is considerably more complex than that of laboratory mice.(27) Furthermore, the total number of B lymphocytes in rats is several times larger than in mice, and the capacity to generate the antibody diversity in rats is superior to mice (Yoshikazu Kurosawa, personal communication, 2008), although composition of immunoglobulin genes between these two species is very similar.(26,28) Cell fusion resulting in approximately 1000 hybridoma cultures can be repeated three to five times using freeze-stocked spleen cells from one immunized rat. In the near future, rabbits and chickens might become widely used organisms immunized to develop therapeutic mAb. Rabbit mAb(29) is a novel tool that offers a number of benefits including high affinity, only one IgG isotype and vast antibody diversity, largely through a gene-conversion mechanism, and chickens are a suitable organism for immunization(30) when a given target protein is structurally well-conserved across mammalian species and the antibody response is poor against the target protein.

Establishment of cell lines expressing GFP-fused oncoproteins

  1. Top of page
  2. Abstract
  3. Strategies for the efficient preparation of anti-cancer mAb: transformation, phage display, autonomously diversifying library (ADLib) or hybridoma?
  4. Possible immunogens for the preparation of therapeutic mAb recognizing the extracellular domains of cell-surface oncoproteins, and animals for immunization
  5. Establishment of cell lines expressing GFP-fused oncoproteins
  6. Production of cell-death-inducing mAb, identification of the antigen as chicken transferrin receptor (TfR-CD71) and development of cell-death-inducing mAb recognizing human TfR (type II membrane protein)
  7. Preparation of therapeutic mAb targeting type IV multi-pass membrane proteins containing G protein-coupled receptors (GPCR) and solute carrier (SLC) families
  8. Preparation of mAb targeting type I membrane proteins containing adhesion molecules (CD44v) and growth factor receptors (HER family)
  9. Acquisition of mAb with superior quality to original mAb by the immunization of mimotope peptides determined by phage display
  10. Conclusion with additional discussion
  11. Acknowledgments
  12. Disclosure statement
  13. References

Green fluorescent protein was genetically fused to the C-terminus (for type I, and type IV-A 12-pass and IV-B 7-pass membrane proteins) or the N-terminus (for type II membrane proteins) of target molecules in the GFP-fused expression vectors so that the extracellular domains of the target proteins could not be covered by GFP (Fig. 1b). RH7777 rat hepatoma cells and HEK293F human embryonic kidney cells were transfected with plasmids coding GFP-fused target proteins, selected using culture media containing Genetecin, and clone-sorted for cellular green fluorescence by a cell sorter. Figure 1c depicts the membrane fluorescence by the expression of GFP-human L-type amino-acid transporter 1 (LAT1) on the surface of RH7777 cells before and after cell sorting. By selecting hybridoma antibodies reacting specifically with cells expressing GFP-fused target proteins in a GFP-expression-dependent manner in the first screening, we can securely select hybridomas producing mAb specific for target proteins (Fig. 1d). The merits of this reported strategy(31) have recently been cited.(32)

Production of cell-death-inducing mAb, identification of the antigen as chicken transferrin receptor (TfR-CD71) and development of cell-death-inducing mAb recognizing human TfR (type II membrane protein)

  1. Top of page
  2. Abstract
  3. Strategies for the efficient preparation of anti-cancer mAb: transformation, phage display, autonomously diversifying library (ADLib) or hybridoma?
  4. Possible immunogens for the preparation of therapeutic mAb recognizing the extracellular domains of cell-surface oncoproteins, and animals for immunization
  5. Establishment of cell lines expressing GFP-fused oncoproteins
  6. Production of cell-death-inducing mAb, identification of the antigen as chicken transferrin receptor (TfR-CD71) and development of cell-death-inducing mAb recognizing human TfR (type II membrane protein)
  7. Preparation of therapeutic mAb targeting type IV multi-pass membrane proteins containing G protein-coupled receptors (GPCR) and solute carrier (SLC) families
  8. Preparation of mAb targeting type I membrane proteins containing adhesion molecules (CD44v) and growth factor receptors (HER family)
  9. Acquisition of mAb with superior quality to original mAb by the immunization of mimotope peptides determined by phage display
  10. Conclusion with additional discussion
  11. Acknowledgments
  12. Disclosure statement
  13. References

Here, we describe the preparation of anti-cancer mAb by the immunization of whole tumor cells (chicken DT40) using the approach of “forward genetics”. Recently, we have obtained two mAb (D18 and D19) inducing marked cell-death (Fig. 2a) from functional (cell-death inducibility) screening of hybridoma antibodies.(33) Programmed cell death (PCD) is categorized as apoptotic, autophagic or necrosis-like.(34) Interestingly, we found that cells having many vacuoles in the cytoplasm (autophagic) and enlarged cells with ruptured plasma membranes (necrosis-like) were simultaneously observed in addition to cells with condensed chromatin and an intact plasma membrane (apoptotic) in DT40 cells treated with the mAb through electron microphotographs(35) (Fig. 2b). In order to characterize antigen(s) recognized by cell-death-inducing mAb, we first investigated the molecular weight of mAb-defined antigen by the immunoprecipitation of cell-surface proteins (Fig. 2c). An approximately 100-kilodalton glycoprotein was detected by both mAb in the reducing condition. In the non-reducing condition, an approximately 200-kDa glycoprotein was detected, suggesting that the antigen recognized by each mAb is a 100-kDa protein that forms homodimeric glycoproteins by the disulfide bond between cysteine residues. Next, we purified the antigen(s) defined by mAb from the total cell lysates of DT40 (Fig. 2d). Ninety-five kDa proteins were digested with trypsin in polyacrylamide gel, and the protein was identified using liquid chromatography (LC)/mass spectrometry (MS)/mass spectrometry (MS) analysis. As a result of database research, the protein immunoprecipitated with mAb was identified to chicken TfR (CD71) with a credible cover rate (more than 30% in both mAb). In Figure 2e, the amino-acid sequences of chicken TfR identified by MS/MS ion search are highlighted. Various studies have shown low or no TfR expression in normal cells, and elevated expression levels of the TfR on cancer cells when compared with their counterparts.(36) Internalization of TfR by mAb could induce secondary cell death by deprivation of iron-bound Tf, even if tumor cells acquire resistance to apoptosis stimuli through a given cell death receptor. In this context, we have confirmed the rapid internalization of TfR by mAb (data not shown). Although some mAb recognizing TfR mAb have been reported to inhibit cell growth,(37,38) the precise mechanisms remain unclear, especially in relation to cell death. Anti-TfR mAb-mediated cell death has been mainly regarded as apoptotic cell death caused by the binding inhibition of TfR to iron-bound transferrin. In this context, induction of necrosis by D18 or D19 might cause the migration of leucocytes and immune responses including antibody-dependent cellular cytotoxicity (ADCC), which might contribute to the augmentation of the anti-tumor effect, compared with the anti-TfR mAb reported until now. Therefore, TfR could be an excellent molecular target for mAb therapy against various malignancies, possibly even superior to typical cell-death-inducing molecules such as Fas/CD95 and DR5. The unique pathway of cell death in chickens, which was shown in this study, seems to be conserved in the human system, since D18 or 19 mAb could induce the death of human cells transfected with chicken full-length cDNA of TfR (Fig. 3a,b); therefore, we tried to produce cell death-inducing mAb recognizing human TfR using the approach of “reverse genetics”. For this purpose, rats were immunized with RH7777 rat hepatoma cells expressing human TfR whose N-terminus was fused to GFP, because TfR is a type II membrane protein exposing the C-terminus to the outside (Fig. 1b). As for the type II proteins except TfR, we have succeeded in preparing several rat mAb specific to a heavy chain of human CD98 by this method.(31) Antibodies from over 4000 hybridoma cultures were screened for their specificity to TfR and cell death inducibility, and we could establish approximately 20 hybridomas stably secreting mAb specific to human TfR, three of which induced cell death on human cancer cells (Fig. 3c). Interestingly, human TfR has been recently selected as a TAA by comprehensive antibody phage display screening followed by the criterion that mAb preferentially bind to cancers rather than their normal counterparts.(21) Humanization of anti-TfR mAb and analysis of direct cytotoxicity, complement-dependent cytotoxicity (CDC) and ADCC activity of these mAb are currently underway.

image

Figure 2.  Production of cell-death-inducing mono-clonal antibodies (mAb) and identification of the antigen as chicken TfR (CD71). (a) Phase-contrast micrographs of morphological alterations of DT40 cells cultured with D19 mAb. (b) Three types of cell death observed in DT40 cells using electron microphotographs. Enlarged photograph is shown in the boxed panel (right). Bars, 2 μm. (c) Lysates of biotin-labeled DT40 cells were immunoprecipitated with D19, subjected to SDS-PAGE and blotted onto membranes, and proteins were visualized immunologically. (d) Immunoprecipitated proteins by D19 were analyzed with SDS-PAGE in the reducing condition. (e) Immunoprecipitated proteins were subjected to LC/MS/MS analysis, and identified as chicken TfR (CD71). Highlighted letters show amino acid sequences of chicken TfR revealed by the MS/MS spectrum. Adapted from Ohno et al.(33) (a,c,d) and Ohno et al.(35) (b), with per-mission.

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image

Figure 3.  Development of cell-death-inducing mono-clonal antibodies (mAb) recognizing human TfR. (a) HEK293F cells were transfected with cDNA of chicken transferrin receptor (TfR), selected by Zeocin and clone sorted, and then stained with D19. (b) HEK293F cells transfected with cDNA of chicken TfR or control cDNA (Mock) were treated with D19 and examined for cell death. (c) Induced cell death in HL60 human leukemic cells treated with anti-human TfR mAb (Ab1). DAPI, 4, 6′-diamidino-2-phenylindole. Adapted from Ohno et al.(33) (a,b), with permission.

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Preparation of therapeutic mAb targeting type IV multi-pass membrane proteins containing G protein-coupled receptors (GPCR) and solute carrier (SLC) families

  1. Top of page
  2. Abstract
  3. Strategies for the efficient preparation of anti-cancer mAb: transformation, phage display, autonomously diversifying library (ADLib) or hybridoma?
  4. Possible immunogens for the preparation of therapeutic mAb recognizing the extracellular domains of cell-surface oncoproteins, and animals for immunization
  5. Establishment of cell lines expressing GFP-fused oncoproteins
  6. Production of cell-death-inducing mAb, identification of the antigen as chicken transferrin receptor (TfR-CD71) and development of cell-death-inducing mAb recognizing human TfR (type II membrane protein)
  7. Preparation of therapeutic mAb targeting type IV multi-pass membrane proteins containing G protein-coupled receptors (GPCR) and solute carrier (SLC) families
  8. Preparation of mAb targeting type I membrane proteins containing adhesion molecules (CD44v) and growth factor receptors (HER family)
  9. Acquisition of mAb with superior quality to original mAb by the immunization of mimotope peptides determined by phage display
  10. Conclusion with additional discussion
  11. Acknowledgments
  12. Disclosure statement
  13. References

As for type IV multi-pass membrane proteins, we have already succeeded in preparing mAb against mouse and human sphingosine-1 phosphate receptor 1 (S1P1) classified to GPCR, and four light chains (LAT1, y+LAT2, Asc1 and xCT) of human CD98 by using cells expressing GFP-fused target proteins as immunogens. Proteins of this type are the most stubborn targets for mAb production because of their complicated structure, and we describe the production of mAb recognizing LAT1, the first-identified light chain of the CD98 molecule. CD98/4F2 is a heterodimeric protein with a relative molecular mass (Mr) of 125 000 (GP125) composed of a 90-kDa heavy chain (CD98hc) and 35-kDa light chains (CD98lcs) (Fig. 4a). CD98 is expressed in a wide variety of tumors,(39–44) suggesting its functional involvement in cell proliferation or malignant transformation; in fact, mAb against rat and human CD98hc inhibited the proliferation of tumor cells.(45) In addition, NIH3T3 and Balb3T3 cells transfected with cDNA of CD98hc have shown various malignant phenotypes.(46–48) Transporters corresponding to the amino acid transport system L, y+L, Xc and Asc have been shown to be CD98lcs, and six amino-acid transporters (LAT1, LAT2, y+LAT1, y+LAT2, Asc1 and xCT) belonging to the SLC7 family, have been identified as CD98lcs,(49–54) and all CD98lcs are sorted to the plasma membrane via their association with CD98hc (SLC3A2). LAT1 (SLC7A5) is a 12-pass non-glycosylated membrane protein, which was first identified as CD98lc associated with CD98hc glycoprotein.(49) It is reported that mRNA and protein of LAT1 are widely expressed on tumor cells.(55–57) LAT1, which transports neutral large amino acids containing branched-chain amino acids (BCAA) playing vital roles in various types of cells, especially muscle cells,(58) is significantly involved in the proliferation of tumor cells,(59) and the expression of oncogenic activity by CD98hc depends greatly on the association with CD98lc.(47) Thus, LAT1 seems to be a promising molecular target for cancer therapy, however, development of mAb recognizing the extracellular domain of native human LAT1 protein was unsuccessful. In fact, we could not obtain anti-LAT1 mAb in the screening of antibodies from over 3000 hybridoma cultures established by cell fusion between mouse myeloma cells and splenocytes of Balb/c mice hyper-immunized against mouse cells over-expressing human LAT1 proteins. Similar trials using Kirin–Medarex (KM) mice,(60) which secrete fully human antibodies against antigens administered, were also unsuccessful (Tomoyuki Tahara, personal communication, 2007). Selected SOL22 and SOL69 rat mAb(31) specifically reacted with the extracellular domain of LAT1 on RH7777 and HEK293F human embryonic kidney cells transfected with cDNA of LAT1, but not with cells transfected with cDNA of four other CD98lcs, namely, LAT2, y+LAT1, y+LAT2 and xCT amino-acid transporters (Fig. 4b). These mAb immunoprecipitated 35-kDa and 90-kDa proteins under reducing conditions in extracts prepared from human HeLa tumor cells, indicating the existence of intermolecular disulfide bonds between cysteine residues in 90-kDa hc and 35-kDa lc (LAT1). SOL22 and SOL69 mAb reacted with a wide variety of viable human tumor cell lines, but were only weakly reactive with HEK293F cells and human peripheral blood cells. Comparative immunohistochemical analyses of normal human tissues revealed LAT1 to be more tumor-selective than CD98hc (Fig. 4c), and LAT1 was over-expressed on the surface of almost all tumor cells irrespective of the tissue of origin, unlike typical receptor-type oncoproteins such as members of the HER family with restricted tumor distribution. In addition, we have already confirmed that mAb SOL22 and SOL69 could induce the internalization of LAT1 and CD98hc from the cell surface (Fig. 4d), suggesting possible effects on cellular amino-acid incorporation, which might lead to the growth inhibition of cancer cells. Taken together, we expect that anti-LAT1 therapeutic mAb will be applied to various types of human cancers, and LAT1 will become an excellent molecular target, possibly even superior to existing target proteins.

image

Figure 4.  Production and characterization of monoclonal antibodies (mAb) recognizing the extracellular domain of human LAT1. (a) Schematic illustration of the CD98 molecule. (b) Specific binding of SOL69 mAb with LAT1. HEK293F cells were mock-transfected or transfected with cDNA of green fluorescent protein (GFP)-fused human CD98hc or various human CD98lcs, and were stained with SOL69 and phycoerythrin (PE)-conjugated anti-rat IgG Fcγ. (c) Reactivity of mAb with human normal tissues. Human tonsil tissues were stained with anti-CD98hc mAb (HR35) or anti-CD98lc mAb (SOL69) using an immunoperoxidase method, and nuclei were counterstained with hematoxylin. (d) Internalization of CD98 proteins by anti-LAT1 mAb. HeLa cells cultured under standard conditions (control, left) or cultured with SOL69 (10 μg/mL) for 24 h (right) were stained with SOL69 followed by FITC-conjugated anti-rat IgG Fcγ and observed under a fluorescent microscope. Adapted from Ohno et al.(31) (b–d), with permission.

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Preparation of mAb targeting type I membrane proteins containing adhesion molecules (CD44v) and growth factor receptors (HER family)

  1. Top of page
  2. Abstract
  3. Strategies for the efficient preparation of anti-cancer mAb: transformation, phage display, autonomously diversifying library (ADLib) or hybridoma?
  4. Possible immunogens for the preparation of therapeutic mAb recognizing the extracellular domains of cell-surface oncoproteins, and animals for immunization
  5. Establishment of cell lines expressing GFP-fused oncoproteins
  6. Production of cell-death-inducing mAb, identification of the antigen as chicken transferrin receptor (TfR-CD71) and development of cell-death-inducing mAb recognizing human TfR (type II membrane protein)
  7. Preparation of therapeutic mAb targeting type IV multi-pass membrane proteins containing G protein-coupled receptors (GPCR) and solute carrier (SLC) families
  8. Preparation of mAb targeting type I membrane proteins containing adhesion molecules (CD44v) and growth factor receptors (HER family)
  9. Acquisition of mAb with superior quality to original mAb by the immunization of mimotope peptides determined by phage display
  10. Conclusion with additional discussion
  11. Acknowledgments
  12. Disclosure statement
  13. References

In this section, we describe the production and characterization of mAb recognizing type I membrane proteins. Type I is single-pass transmembrane protein with a leader sequence (signal peptide) at the N-terminus, and secretory protein can be easily prepared by genetic truncation of the transmembrane and cytoplasmic domains of the antigen used in the screening with enzyme-linked immunosorbent assay (ELISA), before or instead of flow cytometry (FCM) screening of hybridoma antibodies with cells expressing GFP-fused target proteins. Schematic illustrations of three GFP-fused CD44 proteins (CD44s, CD44v and soluble CD44v) are shown in Figure 5a. Based on this “ELISA to FCM” protocol, we succeeded in preparing specific mAb recognizing the extracellular domain of human and variant-type CD44 (CD44v) and standard-type CD44 (CD44s), and a complete set of human HER family proteins (HER1, HER2, HER3 and HER4). CD44 is reported to be expressed in cancer stem cells from various tissues;(61–63) however, we believe that CD44v is more important than CD44s from the standpoint of cancer specificity and that anti-CD44v mAb will become the ultimate weapons to eradicate cancer stem cells of leukemia and solid tumors in the near future. We have recently demonstrated the strong expression of CD44v (Fig. 5b–d) in gastric adenocarcinomas of K19-Wnt1/C2mE transgenic mice and possible stem-like cells at the squamo-columnar junction (Fig. 5c,d).(64) Concerning HER family, HER1 and HER2 are typical competitive targets, against which several mAb are on the market or in clinical trials. We have developed anti-HER1 and anti-HER2 rat mAb recognizing different epitopes from Cetuximab, Trastuzumab, SER4(65,66) and SV2-61γ,(67,68) which is the first reported mAb recognizing the extracellular domain of human HER2 reported by us.

image

Figure 5.  Production and characterization of anti-cancer therapeutic monoclonal antibodies (mAb) recognizing type 1 membrane proteins. (a) Schematic illustrations of green fluorescent protein (GFP)-fused CD44 proteins: CD44s (upper), CD44v (middle) and soluble CD44v (lower) are shown. (b) RT-PCR analysis of CD44v in the gastric mucosa of normal mice and gastric adenocarcinomas of K19-Wnt1/C2mE transgenic mice. (c) Expression of CD44v in the squamo-columnar junction (left, 12w; middle, 20w) and in gastric adenocarcinomas (right, 30w) of K19-Wnt1/C2mE transgenic mice. Bar, 100 μm. (d) Expression of CD44 in possible stem-like cells in the squamo-columnar junction (left, 12w; middle, 20w) and in gastric adenocarcinomas (right, 30w) of K19-Wnt1/C2mE transgenic mice. Adapted from Ishimoto et al. (b–d),(64) with permission.

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Acquisition of mAb with superior quality to original mAb by the immunization of mimotope peptides determined by phage display

  1. Top of page
  2. Abstract
  3. Strategies for the efficient preparation of anti-cancer mAb: transformation, phage display, autonomously diversifying library (ADLib) or hybridoma?
  4. Possible immunogens for the preparation of therapeutic mAb recognizing the extracellular domains of cell-surface oncoproteins, and animals for immunization
  5. Establishment of cell lines expressing GFP-fused oncoproteins
  6. Production of cell-death-inducing mAb, identification of the antigen as chicken transferrin receptor (TfR-CD71) and development of cell-death-inducing mAb recognizing human TfR (type II membrane protein)
  7. Preparation of therapeutic mAb targeting type IV multi-pass membrane proteins containing G protein-coupled receptors (GPCR) and solute carrier (SLC) families
  8. Preparation of mAb targeting type I membrane proteins containing adhesion molecules (CD44v) and growth factor receptors (HER family)
  9. Acquisition of mAb with superior quality to original mAb by the immunization of mimotope peptides determined by phage display
  10. Conclusion with additional discussion
  11. Acknowledgments
  12. Disclosure statement
  13. References

To develop mAb against identical target proteins with better quality or higher affinity, remaking mAb from the beginning and genetic reshaping by site-directed or random mutagenesis in the variable region from existing mAb have often been attempted. In this section, we introduce another method to obtain different and often better mAb from existing mAb. We routinely try to determine the DNA sequences of variable regions and the epitope peptide sequences of newly produced mAb, and the former or latter information respectively contributes to the modification of mAb containing humanization and affinity improvement or a generation of mAb with better quality raised against epitope (or mimotope) peptides. We have elucidated the variable-region sequences of all mAb mentioned in this paper,(69,70) and determined mimotopes of anti-CD98hc mAb, HBJ127,(71) anti-HER2 mAb and SER4(69) with phage display using a random heptapeptide library. Importantly, determined mimotopes had a restricted homology with amino acid sequences of CD98hc or HER2. Mice were immunized with carrier proteins bearing mimotope peptide recognized by HBJ127, and antibody phage libraries from spleen cells of these mice were subjected to bio-panning on living CD98hc-positive cancer cells; several recombinant Fab clones showing higher in vitro growth inhibition of tumor cells than HBJ127 Fab were obtained (Fig. 6a).(72) Interestingly, the nearest VH and VL germ lines of these clones were identical to those of HBJ127 (Fig. 6b); however, amino acid sequences of VH and VL varied in these clones and in HBJ127, particularly in the framework regions (Fig. 6c).(72) Mimotopes screened with 10-mer random peptide phage display library by biopanning with cetuximab proved no homology to HER1(73) by sequence analysis of the peptides; however, immunization of these mimotopes could elicit a specific antibody response to HER1 with internalization activity, and an anti-tumor effect with ADCC and CDC. Our study with anti-CD98hc mimotopes(70–72) is distinguished from this study in that our mimotopes(73) could correspond to amino acid sequences in the original protein, and immunogenicity of the mimotopes was analyzed by monoclonal Fab antibodies, but not by polyclonal antisera, as in the study of HER1 mimotopes.

image

Figure 6.  Production and characterization of monoclonal antibodies (mAb) with superior quality to original mAb by immunization with mimotope peptides. (a) In vitro growth inhibition of human cancer cells by recombinant Fab clones. (b) Comparison of the nearest germ line VH and VL between recombinant Fab clones and HBJ127. (c) Comparison of VH and VL amino acid sequences between recombinant Fab clones and HBJ127. Adapted from Itoh et al.,(72) with permission.

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Conclusion with additional discussion

  1. Top of page
  2. Abstract
  3. Strategies for the efficient preparation of anti-cancer mAb: transformation, phage display, autonomously diversifying library (ADLib) or hybridoma?
  4. Possible immunogens for the preparation of therapeutic mAb recognizing the extracellular domains of cell-surface oncoproteins, and animals for immunization
  5. Establishment of cell lines expressing GFP-fused oncoproteins
  6. Production of cell-death-inducing mAb, identification of the antigen as chicken transferrin receptor (TfR-CD71) and development of cell-death-inducing mAb recognizing human TfR (type II membrane protein)
  7. Preparation of therapeutic mAb targeting type IV multi-pass membrane proteins containing G protein-coupled receptors (GPCR) and solute carrier (SLC) families
  8. Preparation of mAb targeting type I membrane proteins containing adhesion molecules (CD44v) and growth factor receptors (HER family)
  9. Acquisition of mAb with superior quality to original mAb by the immunization of mimotope peptides determined by phage display
  10. Conclusion with additional discussion
  11. Acknowledgments
  12. Disclosure statement
  13. References

In this review, we have introduced a strategy for the development of anti-cancer therapeutic mAb using rat cells expressing GFP-fused oncoproteins as immunogens and rats as the immunized animal, since their antibody repertoire is superior to that of mice. This immunization protocol using cells transfected with cDNA coding target proteins will eventually be replaced by DNA immunization,(74–76) if lymphocytes sensitized to target proteins expressed in vivo for hybridoma formation can be efficiently collected from immunized animals. We successfully obtained various mAb recognizing the extracellular domain of type I (human and mouse CD44v and CD44s, and four human HER family proteins), type II (TfR and CD98hc) and type IV (human LAT1, y+LAT2, ASC1, xCT, and human and mouse S1P1) membrane proteins. Among these proteins defined with mAb, TfR, LAT1 and CD44v were only weakly expressed on HEK293F cells, human peripheral blood cells and cells in human tonsil tissues. Some human tumor cell lines showed a decrease in reactivity with mAb after the fixation of tumor cells, indicating that our anti-oncoprotein mAb recognize the native or discontinuous epitopes of target proteins.(31) Since the reactivity to cancer cells of a given mAb strongly reactive with viable transfectants is sometimes very weak against cancer cells, especially in multi-pass membrane proteins on cancer cell lines (Kazue Masuko, Yoshiya Ohno, Takashi Masuko, unpublished data), we propose that the mAb could not efficiently access epitopes of target proteins, possibly because of the complicated structural topology of multi-pass proteins affected by differences in the surrounding molecules in a given cell line. Recently, specific chimeric or humanized mAb against the extracellular domain of CD20,(2–4) HER2(2,5–8) and HER1(2,5,9–11) have been introduced for the treatment of B cell malignancies, breast cancer or colorectal cancer, respectively. ADCC and CDC(77,78) are supposed to be possible mechanisms for the anti-tumor effect of these therapeutic mAb. Unlike typical receptor-type oncoproteins, such as members of the HER family with restricted tumor distribution, TfR, LAT1 and CD44v are over-expressed on the cell surface of many tumor cells irrespective of the tissue of origin. Furthermore, mAb against LAT1 and CD44v did not react with human lymphocytes irrespective of the activation state of cells, although the expression of many oncoproteins in lymphocytes is upregulated by various activation stimuli;(79,80) therefore, we expect that these mAb-based therapies will be applied to various types of human cancers. Humanization of mAb and analysis of in vitro and in vivo anti-tumor effects including ADCC and CDC, and evaluation of the anti-tumor effect on immunodeficient mice of these mAb are currently underway.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Strategies for the efficient preparation of anti-cancer mAb: transformation, phage display, autonomously diversifying library (ADLib) or hybridoma?
  4. Possible immunogens for the preparation of therapeutic mAb recognizing the extracellular domains of cell-surface oncoproteins, and animals for immunization
  5. Establishment of cell lines expressing GFP-fused oncoproteins
  6. Production of cell-death-inducing mAb, identification of the antigen as chicken transferrin receptor (TfR-CD71) and development of cell-death-inducing mAb recognizing human TfR (type II membrane protein)
  7. Preparation of therapeutic mAb targeting type IV multi-pass membrane proteins containing G protein-coupled receptors (GPCR) and solute carrier (SLC) families
  8. Preparation of mAb targeting type I membrane proteins containing adhesion molecules (CD44v) and growth factor receptors (HER family)
  9. Acquisition of mAb with superior quality to original mAb by the immunization of mimotope peptides determined by phage display
  10. Conclusion with additional discussion
  11. Acknowledgments
  12. Disclosure statement
  13. References

This work was supported in part by the “Academic Frontier” Project of Kinki University (2005–2007) and “Antiaging Center” Project of Kinki University (2008–2012) for Private Universities, matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology), and also supported by the “A-STEP (Adaptable and Seamless Technology Transfer Program through R&D)” Project (2009–2011), matching fund subsidy from JST (Japan Science and Technology Agency). While preparing this manuscript, one of the authors (KM) was diagnosed with stage IV follicular lymphoma and a therapeutic mAb, Rituximab, was administered with suitable chemicals. She is getting better and Zevalin treatment is planned. We thank Dr N. Fujii and M. Tsudo (Osaka Red Cross Hospital) for their medical treatment. We are also grateful to T. Nishimura, T. Ohkuri (Hokkaido University), S. Aiba S, K. Ishizawa, S. Hishinuma and T. Kawabe (Tohoku University), S. Niwa (Link Genomics), H. Saya, O. Nagano and S. Okamoto (Keio University), K. Itoh (University of Shizuoka), T. Tanaka (Hyogo University of Health Science), K. Endo (Gunma University) and M. Hosono, Y. Tatsumi, O. Muraoka, S. Ishiwata and R. Sugiura (Kinki University) for their research cooperation, second opinions and kind advice.

References

  1. Top of page
  2. Abstract
  3. Strategies for the efficient preparation of anti-cancer mAb: transformation, phage display, autonomously diversifying library (ADLib) or hybridoma?
  4. Possible immunogens for the preparation of therapeutic mAb recognizing the extracellular domains of cell-surface oncoproteins, and animals for immunization
  5. Establishment of cell lines expressing GFP-fused oncoproteins
  6. Production of cell-death-inducing mAb, identification of the antigen as chicken transferrin receptor (TfR-CD71) and development of cell-death-inducing mAb recognizing human TfR (type II membrane protein)
  7. Preparation of therapeutic mAb targeting type IV multi-pass membrane proteins containing G protein-coupled receptors (GPCR) and solute carrier (SLC) families
  8. Preparation of mAb targeting type I membrane proteins containing adhesion molecules (CD44v) and growth factor receptors (HER family)
  9. Acquisition of mAb with superior quality to original mAb by the immunization of mimotope peptides determined by phage display
  10. Conclusion with additional discussion
  11. Acknowledgments
  12. Disclosure statement
  13. References