Characterizing monoclonal antibodies to antigenic domains of TCblR/CD320, the receptor for cellular uptake of transcobalamin-bound cobalamin

Abstract

Monoclonal antibodies (mAbs) were generated to the extracellular domain of transcobalamin receptor (TCblR) and used to identify the regions of the receptor protein involved in antibody binding. Based on the effect of transcobalamin bound cobalamin (TC-Cbl) on antibody binding, this study identified both blocking and binding antibodies. Both types of antibodies bind apo as well as holo receptors, whereas the blocking antibody when bound to the apo receptor prevents the binding and cellular uptake of TC-Cbl. Binding of these antibodies to truncated receptor constructs has identified the peptide domains of the receptor involved in antibody binding. These antibodies have potential utility in blocking cellular uptake of Cbl and delivery of drugs via TCblR, which is over-expressed in many cancers.

Keywords::

• Transcobalamin
• receptor
• TCblR/CD320
• monoclonal antibodies
• cobalamin
• vitamin B12.

Introduction

Cellular uptake of vitamin B12 (cobalamin, Cbl) is mediated by two proteins, transcobalamin (TC), a plasma protein secreted by the vascular endothelial cells (Quadros et al., 1999) that binds and transports the vitamin to tissues, and a membrane receptor TCblR, expressed on most cell types that binds TC saturated with cobalamin and internalizes the vitamin by endocytosis (Cooper & Paranchych, 1961). The TC is degraded in the lysosome and the Cbl is transported out of the lysosome (Youngdahl-Turner et al., 1979) for conversion to methylCbl and adenosylCbl (Quadros & Jacobsen, 1995). The two forms of Cbl serve as cofactors for two key enzymes, methionine synthase (MS) and methylmalonylCoA mutase (MMU). The conversion of methyltetrahydrofolate and homocysteine to tetrahydrofolate and methionine by MS requires methylCbl (Taylor & Hanna, 1975) and adenosylCbl is a cofactor for MMU for the formation of succinylCoA in the propionate metabolic pathway (Hall, 1984). TCblR expression is coupled to the cell cycle with highest receptor expression in actively dividing cells, likely preceding DNA synthesis (Hall, 1984). Cellular uptake and disposition of Cbl is a dynamic process dictated by available extracellular TC-Cbl and intracellular Cbl requirements with most of the Cbl exiting the cell following conversion to coenzyme forms and utilization in Cbl-dependent reactions (Quadros & Jacobsen, 1995). TCblR (CD 320) is a membrane receptor with structural homology to the LDL receptor family. This 282 amino acid (aa) protein has a 199 aa extracellular region, a 21 aa transmembrane stretch and a 32 aa cytoplasmic domain. The extracellular region contains two LDL receptor type A domains separated by a 55 aa cysteine-rich CUB like domain (Quadros et al., 1996). The two LDLR-A domains with consensus aa sequences for Ca⁺⁺ binding appear to be critical determinants for Ca⁺⁺ dependent binding of TC-Cbl (DiGirolamo & Huennekens, 1975). The essential role of Cbl in folate recycling and the cell cycle associated expression of the receptor for cellular uptake of Cbl provides the opportunity to interfere with cellular replication either by blocking the uptake of Cbl or by delivering drugs and toxins into cells using this pathway (Quadros et al., 1996). This report describes the epitope specificity and properties of monoclonal antibodies generated to the extracellular domain of TCblR.

Material and methods

Production of the recombinant TCblR

The full length cDNA in plasmid pOTB7 encoding human TCblR was purchased from Open Biosystems Huntsville, AL. The plasmid was digested with EcoR1 and Xho1 and the cDNA was cloned into pcDNA3.1⁺. This plasmid was digested first with Kpn1 and then with Pvu2 and the region corresponding to the extracellular domain of TCblR was cloned into pcDNA3.1⁺. The secreted form of the receptor protein was expressed in HEK 293 cells by transfection with PolyFect reagent (Qiagen, Valencia CA) and by selection for neomycin resistance to obtain clones with stable expression. Secreted TCblR in the culture medium and during purification was monitored by the TC-Cbl binding assay as previously described (Quadros et al., 2005).

Purification of the recombinant receptor

The TCblR protein was purified by affinity chromatography using human TC-Cbl as the affinity ligand and a monoclonal antibody to TC covalently coupled to Sepharose 4B matrix to capture the TCblR/TC-Cbl complex (Quadros et al., 2009). The recombinant TC protein used in the purification was also produced in HEK 293 as a fusion protein with DsRed protein by cloning the human TC cDNA into pDsRed-N1 (Clontech, Mountain View, CA) plasmid. The fusion protein is more stable than the native protein produced in SF 9 insect cells (Quadros et al., 1993) and behaves like the native protein in binding Cbl and binding to the receptor as TC-Cbl. The DsRed TC was partially purified on CM Sephadex as previously described (Quadros et al., 1993) prior to saturating with Cbl by incubating with 3-fold excess CyanoCbl. The primary objective of this step was to concentrate the TC protein and reduce the volume required for mixing with medium containing the recombinant TCblR. After incubation for 2–4 h at 4°C, anti-TC-antibody Sepharose matrix was added and incubated at 4°C overnight with constant mixing. The next day the Sepharose beads were collected, washed extensively to remove protein contaminants, and the protein eluted with 0.5 M MgCl₂. The eluted protein was extensively dialyzed and purified on a second affinity matrix containing wheat germ agglutinin as previously described (Quadros et al., 2009). The final product was checked for purity by SDS-PAGE.

Generation and characterization of monoclonal antibodies

Mice were immunized with 50 μg of the protein followed by two additional injections of 25 μg of the antigen at 2-week intervals. Antibody titer in the mice and in the hybridoma supernatants was monitored by ELISA. The antigen was diluted to 1 μg/ml and 100 μl of the protein was incubated in Maxisorb ELISA plates (NUNC, Rochester, NY) for 1 h. The protein was removed, the wells washed twice in phosphate buffered saline (PBS), blocked overnight with 1% bovine albumin, and incubated overnight with sample containing antibody. The wells were washed and the antibody bound was determined by reacting with peroxidase conjugated goat anti-mouse second antibody. Ultra-TMB (Pierce, Rockford, IL) was used as the substrate for the peroxidase. The 96-well plate was read at 450 nm in a Bio-Rad Model 3550 Microplate Reader following acidification with 100 μl 1 N HCl.

Mapping the epitope specificity of antibodies

We utilized a sandwich ELISA-based assay to identify the antigen binding epitopes by reacting the antibodies with various truncated forms of the receptor. These C-terminal deleted proteins were produced as recombinant proteins in HEK293 cells. Antibodies 1-10, 1-18, 1-19, 1-23, 1-25, 2-2, 2-3, 2-4, and 2-6 produced against the recombinant 200 aa extracellular receptor protein were used as capture antibodies by coating 96-well plates (Maxisorb, Nunc). Culture medium containing the truncated TCblR was incubated overnight and the captured TCblR antigen was detected with a goat polyclonal antibody (anti 8D6, R&D Systems, Minneapolis, MN) to the antigen. Peroxidase-labeled horse anti-goat IgG (Vector, Burlingame, CA) was used for detecting the binding of the polyclonal antibody as described above.

Effect on cellular uptake of TC-Cbl

Human erythroleukemia K562 cells were cultured in DMEM with 10% FBS. Cells were seeded at a density of 0.2 × 10⁶ cells in 2 ml medium containing 0.026 pmoles of recombinant TC saturated with ⁵⁷CoCbl and 120 pmoles of purified mAb. Cells were harvested at various time intervals and radioactivity in the cell pellet was determined in a gamma counter.

Results

Two Balb C mice immunized with recombinant 200 aa extracellular domain of TCblR were bled 2 weeks after the last injection and screened for antibody titer by ELISA. Positive wells from the first and second mouse fusions were selected for further expansion and cloning. Ultimately five clones from the first fusion and four from the second fusion were isolated. Selected clones were propagated in culture and used for ascites production in mice. These antibodies were purified by affinity chromatography on a protein G agarose matrix. With the exception of a single IgM clone, all other antibodies were IgG1 isotype. Figure 1 shows the antibody titer for selected clones. The slope of the dilution curve suggests high affinity binding of these mAbs to the antigen. The specificity of binding to cell surface receptors was determined by flow cytometric analysis of K562 cells incubated with mAb. As shown in Figure 2, cells incubated at 4°C with mAb show binding of FITC tagged anti mouse IgG. This binding is further evident from the shift in the peak to the right due to additional receptors expressed when cells are incubated at 37°C. All antibodies bound holo TCblR (i.e. receptor–ligand complex) as indicated by immunoprecipitation of TCblR saturated with the ligand TC-Cbl and can be categorized as binding antibodies. However, mAb 1-25 was unique in that pre-incubating apo TCblR with this antibody prevented the subsequent binding of TC-Cbl and is therefore identified as a blocking antibody (data not shown).

Figure 1.  Titer of anti-TCblR antibodies. Antibody titer in hybridoma supernatants was determined by ELISA; 100 ng of antigen was used to coat ELISA plates. Peroxidase conjugated goat anti-mouse secondary antibody and TMB substrate were used to detect the binding of anti-TCblR antibody.

Figure 2.  Specificity of antibody binding to cell surface receptors. K562 cells were incubated with Mab followed by FITC tagged anti-mouse IgG and analyzed by flow cytometric separation of antibody positive cells. (A) control mouse IgG, 4°C; (B) TCblR mAb 1-19, 4°C; (C) TCblR mAb 1-19, 37°C.

The recombinant extra-cellular domain of TCblR and its various carboxy terminal deletion constructs provided the antigens to map the antibody binding sites on this protein. The sandwich assay developed utilized purified mAbs immobilized in ELISA plates to capture the TCblR antigen in culture medium, which was then reacted with a polyclonal goat anti-human TCblR to identify the captured antigen. This strategy allowed us to compile a detailed profile of antibodies binding to truncated forms of the receptor. Table 1 shows the binding of various truncated forms of the receptor to specific antibodies. Epitope recognition by the immobilized mAb was deduced from the ability of the mAb to capture a secreted form of the extracellular domain of the receptor in the culture medium. Loss of binding indicated deletion of the specific epitope. Based on this binding data, we have deduced the putative peptide region that interacts with each mAb and is summarized in Figure 3. The region within the second LDLR-A domain appears to be highly antigenic, with a number of antibodies binding to epitopes within or close to this region. On the other hand, mAb 1-25 appears to bind to the C-terminal end of the second LDLR-A domain, a region involved in Ca⁺⁺ binding. This mAb also blocks the binding of holo-TC to TCblR, interaction that requires Ca⁺⁺ binding. As shown in Figure 4, the uptake of TC-Cbl in K562 cells is effectively blocked when cultured in medium containing mAb 1-25. Binding of the mAb 1-25 to the cell surface receptors appears to block the binding of TC-Cbl, whereas mAb 1-19 and 1-23 that bind to the region in between the second LDLR-A domain and the transmembrane domain have no effect on binding and uptake of TC-Cbl.

Table 1.  Binding of monoclonal antibodies to truncated TCblR proteins expressed in HEK293 cells.^*

mAb	AA sequence
	32-233	32-183	32-149	32-233-mycHis	32-94,142-233-mycHis	32-137,166-233-mycHis	32-52,64-233-mycHis
1-10	+++	++	++	++	−	++	−
1-18	++	++	++	+	−	−	−
1-19	++	−	−	+++	++	++	+++
1-23	++	−	−	++	++	++	++
1-25	++	+++	−	++	+	−	+++
2-2	++	+++	+++	+++	+	+	+++
2-3	++	+++	+++	+++	−	++	+++
2-4	++	+++	+++	+++	−	−	++
2-6	++	+++	+++	+++	−	−	+++

* Truncated TCblR proteins were captured in ELISA plates coated with anti-TCblR MAbs and were detected with an anti-TCblR polyclonal antibody. The extracellular TCblR was used as the positive control; normal HEK293 cell culture medium and normal mouse IgG served as negative controls.

OD 2–3:+++; OD 1–2:++; OD 0.5–1:+; OD <0.5: −.

Figure 3.  Diagrammatic representation of antibody binding regions within the TCblR protein. The peptide fragment reacting with specific antibody was identified from the ELISA data in Table 1.

Figure 4.  Effect of antibody in the culture medium on the cellular uptake of TC-Cbl. K562 cells were incubated with 120 pmoles mAb 1-19, 1-23, and 1-25, then 0.026 pmoles of recombinant TC saturated with ⁵⁷CoCbl was added to culture medium and incubated for 2 h, 4 h, 6 h, 24 h, 48 h, 72 h, and 96 h. Cells were collected to measure uptake of TC-Cbl at each time point. MAb 1-25 inhibited uptake of TC-Cbl throughout the 96h culture period.

Discussion

Since the successful use of folate anti-metabolites such as methotrexate, an inhibitor of dihydrofolate reductase and 5-fluorouracil, an inhibitor thymidylate synthase (Blakley, 1969), the use of inhibitors of B12 dependent pathways as potential drugs in cancer therapy has been explored with limited success (Hogenkamp et al., 1999). The use of nitrous oxide in the treatment of leukemias showed some promise but suffered from technical difficulties in administering the gas and multiple deleterious side-effects (Green & Eastwood 1963). Like the folate compounds and other chemotherapeutic drugs, metabolic inhibitors of B12 pathways are also likely to become ineffective due to target gene amplification and drug resistance. Selective essential nutrient depletion by blocking the cellular uptake of folate or B12 is a novel strategy that could overcome many of the problems associated with current chemotherapy. It is also likely to be less toxic and more specific for highly proliferative cancer cells due to the increased demand for these essential micro-nutrients. However, the use of this approach requires specific drugs or antibodies that would selectively inhibit the transport of these vitamins. Our previous study on the characterization of mAbs to human TC had identified Cbl blocking and receptor blocking antibodies to TC (Quadros et al., 1996). These properties of the mAbs could be exploited to deplete cells of Cbl and inhibit Cbl dependent proliferation of cells. Using mAbs to TC that block the binding of Cbl or block the binding of TC-Cbl to TCblR have proven to be effective in blocking Cbl uptake in cells and inhibiting proliferation of these cells in culture (McLean et al., 1997). A similar approach using monoclonal antibodies to TCblR, the receptor for holo-TC is also likely to block cellular uptake of Cbl as we have shown with the blocking antibody. This process is likely to be slow due to the fact that it takes many cell divisions to deplete intracellular Cbl to a level that would affect cell division, and therefore may not be very effective in the treatment of many cancers. Blocking of TC-Cbl binding to TCblR by mAb 1-25 suggests that the peptide region aa 150–165 to which the mAb binds may be involved in TC-Cbl binding. However, the blocking effect may also be due to steric hindrance since this same antibody will immunoprecipitate a preformed TCblR/TC-Cbl complex. Therefore, it is likely that the conformational change in the tertiary structure of the receptor due to ligand binding permits antibody binding to its epitope. All other antigenic epitopes do not appear important for ligand binding directly or indirectly, since antibodies to these epitopes do not block TC-Cbl binding. The blocking mAb can effectively inhibit cellular uptake of TC-Cbl by virtue of it binding to apo receptors expressed on the cell surface.

All antibodies bind both apo and holoTCblR and therefore could be used to deliver drugs, imaging compounds and toxins to cancer cells. The cell cycle associated expression of TCblR (Amagasaki et al., 1990) results in higher and sustained expression of this receptor in many cancers and provides a vehicle for selective targeting of cancer cells using these monoclonal antibodies.

Conclusions

Monoclonal antibodies to the extracellular domain of TCblR, the receptor for cellular uptake of vitamin B12, have been isolated and the epitope specificity of these antibodies have been identified. These antibodies provide the vehicle to target the receptor for delivery of drugs, toxins, radio-labeled and imaging compounds to tumors. In addition the blocking mAb 1-25 can be used to deplete cells of Cbl, thus inhibiting replication and proliferation.

Acknowledgements

This work is supported by NIH grant R01DK064732 and by KYTO Biopharma, Toronto, CA.

Declaration of interest: Two of the authors (EVQ and JMS) are coinventors in a patent filing WO 2007/117657 A2 on the use of this receptor as a target in cancer therapy filed by the Research Foundation of the State University of New York.