Protein Science (1994), 3:1476-1484. Cambridge University Press. Printed in the USA. Copyright 0 1994 The Protein Society

Cloning, characterization, and modeling of a monoclonal anti-human transferrin antibody that competes with the transferrin receptor

MAURIZIO ORLANDINI,'g2 ANNALISA SANTUCCL2 ANNA TRAMONTAN0,3 PAOLO NERI,' AND SALVATORE OLIVIERO'.2 ' Department of Molecular Biology, University of Siena, Siena, Italy ' Immunobiology Research Institute Siena (IRIS), Siena, Italy

Department of Biocomputing, Istituto di Ricerche di Biologia Molecolare (IRBM) P. Angeletti, Pomezia (Roma), Italy

(RECEIVED February 28, 1994; ACCEPTED June 3, 1994)

Abstract

In this report we describe the isolation and characterization of a monoclonal antibody against human serum trans- ferrin (Tf) and the cloning and sequencing of its cDNA. The antibody competes with the transferrin receptor (TR) for binding to human Tf and is therefore expected to bind at or very close to a region of interaction between Tf and its receptor. From the deduced amino acid sequence, we constructed a 3-dimensional model of the variable domains of the antibody based on the canonical structure model for the hypervariable loops. The proposed struc- ture of the antibody is a first step toward a more detailed characterization of the antibody-Tf complex and possi- bly toward a better understanding of the Tf interaction with its receptor. The model might prove useful in guiding site-directed mutagenesis studies, simplifying the experimental elucidation of the antibody structure, and in the use of automatic procedures to dock the interacting molecules as soon as structural information about the struc- ture of the human Tf molecule will be available.

Keywords: antibody combining site; monoclonal antibody; protein structure; transferrin; transferrin receptor

Human serum transferrin (Tf), a plasma metal-binding glyco- protein, plays an important role in iron metabolism and cell growth (Huebers & Finch, 1987). Its main function is to carry iron to cells by a mechanism of receptor-mediated endocytosis (Dautry-Varsat et al., 1983). Iron uptake by this molecule has been shown to be important for activation and proliferation of normal and malignant cells (de Jong et al., 1990), but other roles for human Tf have been postulated (Denstman et al., 1991; Pier- paoli et al., 1991; Sirbasku et al., 1991). The human Tf gene lo- calization and complete amino acid sequence have been determined (McGillivray et al., 1983; Yang et al., 1984). Human Tf consists of a single polypeptide chain containing 679 amino acid residues and 2 oligosaccharide chains for a molecular weight of ca. 80 kDa. The structure of human serum Tf is not known, but its sequence is homologous to the sequence of rabbit serum Tf, whose structure has been determined by X-ray crystallog- raphy (Bailey et al., 1988). Human Tf is thought to be formed by 2 homologous domains connected by a short peptide, and

Reprint requests to: Salvatore Oliviero, Dipartimento di Biologia Molecolare, Centro Ricerche IRIS via Fiorentina 1, 53100 Siena, Italy; e-mail: oliviero@irisOl .cineca.it.

each domain is divided into 2 subdomains, defining a deep cleft in which a binding site for iron is located.

Although the mechanism of the Tf endocytic cycle, by which iron is carried into the cells, is well known (Thorstensen & Romslo, 1990), limited information is available concerning the interaction of Tf with its receptor (TR) and some epitopes on TR may play different biological roles besides binding of Tf (Keyna et al., 1991; Franco et al., 1992). Recent data show that the isolated C-terminal domain of human Tf is recognized by TR, even if both the C- and the N-terminal lobes contribute to the binding (Zak et al., 1994). On the other hand, anti-Tf mono- clonal antibodies (MAbs) used to study the Tf/TR interaction (Mason & Woodworth, 1991; Rubikaite et al., 1991) suggested the N-terminal domain of Tf as the binding site of the molecule to its receptor (Rubikaite et al., 1991).

In an attempt to characterize antigenic domains of human Tf, first we produced and selected anti-human Tf MAbs able to compete with TR for the binding to Tf; then we determined the amino acid sequence of the variable domains of one of these MAbs, we constructed a 3-dimensional model of its variable do- mains using the well-established canonical structures method (Chothia & Lesk, 1987; Chothia et al., 1989; Lesk & Tramon-

1476

Anti-human transferrin antibody

tano, 1990; Tramontano et al., 1990). and we described its binding-site surface.

Results

Transferrin can be displaced from its receptor by a monoclonal antibody

We produced MAbs able to recognize human Tf and to inhibit its binding to the TR. We selected MAbs with high affinity con- stants (Table 1) and used them to challenge human Tf/TR in- teraction. We first analyzed the interaction by a cellular enzyme-linked immunosorbent assay (CELISA), an assay that detects the binding of specific ligands to cell-surface proteins (Arunachalam et al., 1990). We carried out an inhibition bind- ing assay in which different dilutions of anti-Tf MAbs and peroxidase-labeled human Tf (HPR-Tf) were added to wells of a microtiter plate coated with K562 cells, which present TRs in large numbers (about 150,OOO per cell) on their plasma mem- branes (Klausner et al., 1983). This assay allows a direct study of human Tf/TR interaction in a physiological context, thus cir- cumventing the problem of extraction and purification of TR. Three anti-human Tf MAbs (TW4.20, TW4.63, TW4.158) were able to decrease the binding of human Tf to its receptor present on K562 cells in this assay to a remarkable extent (Fig. 1). We chose MAb TW4.20, which showed higher binding affinity and competition with the TR, for the subsequent in vivo experi- ments. Increasing concentrations of TW4.20 were added simul- taneously with fluorescein isothiocyanate-conjugated human Tf (FITC-Tf) to K562 cells, and fluorescence was detected; the binding of human Tf to its receptor was completely inhibited when the concentration of the MAb was lo4 times the concen- tration of FITC-Tf (Fig. 2A). Moreover, in competition exper- iments, when TW4.20 was added to cells pretreated for 30 min with FITC-TI, it was able to displace FITC-Tf from TRs present on K562 cells (Fig. 2B).

MAb TW4.20 recognizes the N-terminal domain of human Tf

To map the domain containing the epitope recognized by TW4.20. we assayed this MAb for its recognition of human Tf

Table 1. Monoclonal antibody isotypesa and affinity constants (K,)

MAb Subclass Ka W")

TW4.20 IgGI. k chain 1.70 X 109 TW4.63 IgG1, k chain 1.35 X 109 TW4.89 IgGI, k chain 1.63 X 109 TW4.90 IgG1. k chain 1.68 X 109 TW4.107 IgGI, k chain 1.66 X 109 TW4.158 IgG1, k chain 1.70 X 109 T2I9.16 IgGI, k chain 1.23 X 109 T2I9.39 IgGI, k chain 1.77 X 109

a The isotype was determined by ELlSA on plates coated with hu- man iron-free Tf (1 pg/mL), using the Mouse-Typer Sub-lsotyping Kit (Bio-Rad).

I477

I 1 4 T W J . ~ 1WJ.107 ~ ~ 4 . 6 3 ~ w . 1 6 nvJ .on TWJ.ISR ~'WJ.XC) ' I W ~

Clones

Fig. 1. Wells coated with 200,000 K562 cells per well. Hybridoma su- pernatants and HPR-Tf (0.125 pmol/well) were added to each well si- multaneously. Hybridoma supernatants were used at different dilutions containing a different lgGl concentration: (0) 3 pmol/well; (0) 0.3 pmol/well; (a) 0.03 pmol/well. lgGl concentrations of hybridoma su- pernatants were assayed by ELlSA (Fleming lk Pen, 1988). In positive control wells (m), HPR-Tf and chromogenic substrate solution were dis- pensed. In negative control wells (U), PBS and chromogenic substrate solution were added.

by western blotting. Human Tf proteolyzed with trypsin, chy- motrypsin, and thrombin produced 3 immunoreactive fragments specifically recognized by TW4.20 (Fig. 3). Immunoreactivity was maintained only under nonreducing conditions, suggesting the presence of a conformational epitope. No binding was de- tected with peptides produced by other cleavages such as pep- sin, N-bromosuccinimide, and N-chlorosuccinimide, suggesting that the proper conformation is lost when cleavage occurred at Trp or Met peptide bonds. The characterization of the immu- noreactive fragments was carried out by cutting the nonreduc- ing gel bands and loading them on a second reducing gel: 8 tryptic and 7 chymotryptic peptides resulted from reduction of the S-S bonds. Peptide sequence obtained from automated Ed- man degradation of the resulting peptides (data not shown) and of the unreduced thrombin polypeptide showed that all peptides were localized in the N-terminal domain of human Tf, suggest- ing that the N-terminal domain of Tf contacts the TR, in agree- ment with previous observations (Rubikaite et al., 1991). MAb TW4.20 is specific to the human Tf because no cross-reactivity was observed with Tfs purified from the serum of rabbit, mouse, dog, rat, bovine, and guinea pig (M. Orlandini, unpubl. obs.).

Atomic model and analysis of TW4.20 binding site

We cloned the mRNA coding for the V, and VH regions of the hybridoma TW4.20 by reverse transcriptase PCR amplification and sequenced them. The DNA sequences were obtained directly from the amplified DNA and from the amplified segments cloned into a bluescript vector. The resulting sequences for the VL and VH chains are shown in Figure 4.

1478

A 2wI . .

100 101 102 103 100 101 102 103

Intensity of fluorescence

B 200 I

100 10' 102 1

200 I1

100 10' 102 1

Intensity of fluorescence

Fig. 2. Flow cytometry analysis of inhibition and competition experi- ments among MAb TW4.20, FITC-Tf, and TRs present on K562 cells. (-) K562 cells; (. . . . . . .) K562 cells + FITC-Tf + TW4.20; (. . . . .) K562 cells + FITC-Tf. A: FITC-Tf (2.5 pmol) and MAb ascitic fluid at different dilutions were added simultaneously to K562 cells and in- cubated. B: After incubation with FITC-Tf, MAb ascitic fluid was added to K562 cells and incubated. Ascitic fluid IgGl concentrations were tested by ELISA (Fleming & Pen, 1988): I , 2 x IO4 pmol; 11, 2 X I O 2 pmol; I l l , 20 pmol; IV, 0.2 pmol.

From the deduced amino acid sequence (Fig. 5 ) , we built an atomic model of the variable domain of the MAb (Fig. 6) according to the procedure used to predict the structure of im- munoglobulin variable domains. This method has been demon- strated to be capable of correctly predicting immunoglobulin variable domain structures in advance of their experimental de- termination (Chothia et al., 1986, 1989).

We aligned the sequences of V, and VH chains of the MAb with the sequences of the corresponding domains in the known immunoglobulin structures. The alignments were performed using the program GAP from the GCG package (Genetics Com- puter Group, 1991). Table 2 shows the sequence identity of TW4.20 with antibodies of known structure. For each domain, we selected as parent structure the immunoglobulin domain with the highest sequence identity, namely McPC603 (Protein Data Bank [PDB] code 1MCP) (Satow et al., 1986) for VL and

M. Orlandini et al.

HyHel-10 (PDB code 3HFM) (Padlan et al., 1989) for V,. The alignments used for model building are shown in Figure 5 .

Because the 2 parent structures come from different immu- noglobulins, it was necessary to select the orientation of the 2 domains with respect to each other. Chothia et al. (1985) have identified a set of residues conserved in the VL/VH interface of known immunoglobulins. They are formed by residues 33-39, 43-47,84-90, and 88-104 for the VL domain, and 33-40,44-48, 88-94, and 102-109 for the V, domain (in this paper the resi- due numbering of Kabat et al. [1983] is used throughout). We superimposed the McPC603 and the HyHel-10 VL domain by least-squares fit of the structurally conserved regions (RMS = 0.42 A) and merged the VL domain of McPC603 and the VH domain of HyHel-10 in the resulting relative positions. The di- mer shows a translation and rotation of 0.7 A and 12" of its heavy chain with respect to the position of the heavy chain of McPC603, after superposition of the light chain. In known im- munoglobulin structures, the relative translations and rotations calculated in the same way range between 0.4-3.5 A and 1.6- 14.6" (A. Tramontano, unpubl. obs.).

Having selected the appropriate framework, we next identi- fied the canonical structures for each hypervariable loop. When more than 1 known immunoglobulin structure had the same ca- nonical structure as TW4.20, we selected the same structure used for the framework, if possible, or the structure with the high- est sequence identity and the same canonical structure.

The L1 loop (26-32) in MAb TW4.20 is 13 residues long, as it is in the parent McPC603 structure. The determinants of this loop are residues at position 29, 2, 25, and 33, which are con- served between TW4.20 and McPC603 (Fig. 7A). The L2 loops are very similar in all known immunoglobulin structures and

Table 2. Percentage of sequence identity of the lighta and heavy chain of TW4.20 with the corresponding chains of immunoglobulins of known structure (Bernstein et al., 1977)

VL VH

IMPC 84 41 3HFM 60 71 4FAB 64 41 2HFL 63 48 2FBJ 51 53 2F19 58 46 1 REI 64 1 MAM 59 51 1 IGF 65 51 1 IFH 80 50 1 GGI 62 59 1 FDL 61 63 1 FA1 58 46 1 DFB 61 50 1 DBA 63 41 6FAB 58 44 3FAB 55 2FB4 49 1 INE 50

a IRE1 is a Bence-Jones protein and only contains VL; the light chain identity is only reported for VK chains.

Anti-human transferrin antibody I479

A

D a M 1 2 3 4 5

31 +

21 "+

14 +

B

M 1 2 kDa 92+ - 66+ 45 + - . - - 31+ - 21 +

Fig. 3. Western blotting of proteolytic fragments of human Tf. A tricine-SDS gel under nonreducing conditions was chosen and transblotting was performed onto PVDF membrane. The binding of TW4.20 was detected by enhanced chemiluminescence. A: M, low molecular weight (M.W.) standard; 1, human Tf and Coomassie blue staining; 2, human Tf cleaved with chymo- trypsin and Coomassie blue staining; 3, human Tf cleaved with chymotrypsin and TW4.20 staining; 4, human Tf proteolyzed with trypsin and Coomassie blue staining; 5 , human Tf proteolyzed with trypsin and TW4.20 staining. B: M, low M.W. stan- dard; I , human Tf digested with thrombin and Coomassie blue staining; 2, human Tf digested with thrombin and TW4.20 staining.

form a hairpin loop (i.e., a loop that links adjacent strands in is again the same of that of McPC603, being characterized by the same P-sheet). The L2 sequences of TW4.20 and McPC603 the presence of a Pro in position 95 (in the cis conformation in are shown in Figure 7A. The L3 loop (residues 91-96) is also a all known immunoglobulins with this canonical structure) and hairpin loop. The canonical structure of the TW4.20 L3 loop by a group in position 90 able to hydrogen bond to main-chain

A 1

5 2

102

152

202

252

302

353

B 1

52

102

152

202

252

302

HGlBACK

ggtccagctgcaggagtcggg acctgacctg gtgaaacctt ctcagtcact si

ttcactcacc tgcactgtca ctggctactc catcaccagt ggttatagct 101

ggcactggat ccggcagttt ccagaaaaca aactggaatg gctgggctac 151

atacactcca gtggtgccac taactacagc ccatctctca aaagtcgatt 201

ctctatcact cgagacacat ccaagaacca gttcttcctg cagttgaatt

CtgtgaCtaC tgaggacaca gccacatatt actgtgcaag ggatggttac 301

tcccctccct atgctatgga ctatt ggggccaagggaccacggtcaccgtc 352

tcctca 358 HGIFOR

=BACK

attgagctcacccagtctcca tcctccctag ctgtgtcagt tggagagaag 51

gttactatga cctgcaagtc cagtcagagc cttttatata gtaccaatca 101

aaagaactac ttggcctggt accagcagaa accagggcag tctcctaaac 151

tgctgattta ctgggcatcc actagggaat ctggggtccc tgatcgcttc 201

acaggcagtg gatctgggac agatttcact ctcaccatca gcagtgtgaa 251

ggctgaagac ctgtcagttt attactgtca gcaatattat agctatcctc 301

ccacgttcgg ctc gggcaccaagctggaaatcaaa 336

MFOR

Fig. 4. Nucleotide sequences of (A) VH and (B) VL of TW4.20.

1480 M . Orlandini et al.

A TW4.20-H

3HFM-H

TW4.20-H

3HF"H

TW4.20-H

3HF"H

B TW4.20-L

1MCP-L

TW4.20-L

1MCP-L

TW4.20-L

1MCP-L

1 QVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYWHWIRQFPENKLEWLGY 50

1 DVQLQESGPSLVKPSQTLSLTCSVTGDSITSDYWSWIRKFPGNRLEYMGY 50 I I I I I I I I I I I I I I I I I I I I l l I I I I I I I l l I I I I I I I

51 I~ATNYSPSLKSRFSITRDTSKNQFFLQLNSVTTEDTATYYCARW 100

51 VSYSGSTYYNPSLKSRISITRDTSKNQYYLDLNSVTTEDTATYYCANWDG 100 I I 1 I I I I I I I I I I I I I I I I I I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

10 1 m Y W G Q G T T V T V S S 12 0

101 . . . . . . DYWGQGTLVTVS. 112 I I I I I I I I I I I

1 DIELTQSPSSLAVSVGEKVTMTCKSSOSJaLYSTNOmLAWYQQKPGQSP 50 I I I I I I I I I I I I I I l l I I I I I I I I I I I I I I I I 1 I I I I I I I

1 DIVMTQSPSSLSVSAGERVTMSCKSSQSLLNSGNQKNFLAWYQQKPGQPP 50

51 KLLI~TRESGVPDRFTGSGSGTDFTLTISSVKAEDLSVYYCQ~ 100 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I1

51 KLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSY 100

101 ETFGSGTKLEIK I I l l I I I I I I I

10 1 PLTFGAGTKLEIK

Fig. 5. The (A) VH and (B) VL deduced amino acid sequence of TW4.20 aligned with the sequences of known immunoglobu- lins used as templates for the framework model. The hypervariable loop sequences are underlined.

atoms of the loop. McPC603 has the same canonical structure and was then selected as template (Fig. 7C).

The H1 loop has the same size and the same conformation in all known immunoglobulin structures. Its main determinants are a Gly at position 26, which produces a sharp turn of the chain, and the amino acids at positions 29, 34, and 94. Again we se- lected the framework model, i.e., HyHel-IO, as the template for this loop (Fig. 7D). The H2 loop conformation is determined by its length and by the size of residue 71 (Tramontano et al., 1990). In our case, TW4.20 has an Arg at position 71 and the

loop is 4 residues long. HyHel-10 was selected as template (Fig. 7E). No canonical structure has been identified for the H3 hypervariable loop so far. This loop is the most variable in length, sequence, and conformation. However, in TW4.20 this loop is 9 residues long, the same length as in H3 loops from McPC603 (Fig. 7F) and NC41 (Colman et al., 1987). It is inter- esting to note that the McPC603 and the NC41 loops differ es- sentially at the tip of the loop. The RMS deviation (RMSD) of the main-chain atoms of the 2 loops is 2.7 A, but it becomes only 1 A if the 4 central residues of the loop (98-100 amino acids)

3 Fig. 6. Stereo view of the model of MAb TW4.20. The solvent-accessible surface of the hypervariable loop res- idues is shown and color coded accord- ing to amino acid type (red, negatively charged; blue, positively charged; green to brown, hydrophobic to aromatic).

Anti-human transferrin antibody 1481

26 27 28 29 30 21 a b c d e f 32 2 25 33 71 TW4.20 S Q S L L Y S T N Q K N Y I S L F

I I I I I I I I I I I l l 1 1MC P S Q S L L N S G N Q K N F I S L F * * * * *

TW4.20

1MCP

TW4.20

1MC P

TW4.20

3HFM

50 51 52 G A S

I I W A S

90 91 92 93 94 95 96 Q Y Y S Y P P I I I I I Q D H S Y P L * *

26 27 28 29 30 31 32 G Y S I T S G

D G S I T S D I I I I

* *

53 54 55 71 TW4 -20 S S G R

I l l 3HFM D S G R * *

96 97 98 99 100 a b c 101 TW4 -20 G Y S P P Y A M D

I I lMPC Y Y G S T W Y F D

Fig. 7. Sequences of TW4.20 hypervariable loops and their determinants aligned with the sequences of the loops used as models. Residues constituting the determinants for the canonical structure of the loop are marked by asterisks.

acids) are excluded. We reasoned that modeling this loop using McPC603 as a template would produce a reasonable structure, at least in the initial and final region of the loop. Alternative methods, such as database loop searching techniques, have been shown to be ineffective in the case of antibody loops (Tramon- tan0 & Lesk, 1992). Other techniques available to date include combined database searching and conformational energy cal- culations (Bruccoleri et al., 1988; Martin et al., 1989), but in our opinion the expected accuracy of these CPU-intensive methods for loops longer than 8 residues does not, in this particular case, justify their usage. In the H3 case, where the canonical struc- ture selected for the framework is different from that selected for the loop, we superimposed the residues adjacent to the loop in the model and in the selected structure by a weighted least- squares fit and then grafted the loop into the model. It has been shown (Tramontano & Lesk, 1992) that the best results are ob- tained using the backbone of the 4 residues before and after the loop: the first residue before and the first residue after are given weight 1, and the weight of each successive residue is multiplied by 0.8. The value of the RMS so calculated is 2.0 A for the H3 flanking regions. The above protocol allowed us to obtain a model for the framework of the TW4.20 structure. Next, we built the side chains.

The conformation of the side chains was modeled as follows: at the 104 sites for V, and at the 83 sites for VH where the par- ent structure and the model have the same residue, we retained the conformation of the parent structures. If the side chain dif-

fered, we took the side-chain conformation (if possible) from another immunoglobulin having the same residue in the corre- sponding position. When more than 1 structure was a candidate for this step, that more similar in sequence to TW4.20 was se- lected. When the residue was part of a hypervariable loop, the side-chain conformation was only taken from loops with the same canonical structure. In all other cases we retained the con- formation of the side chains in the parent structure as far as the relative length of the side chains permitted and used the most common conformer for the amino acid side chain in those cases where the modeled side chain is longer than the side chain of the parent structure. All the steps described above were performed using the programs CASSANDRA (Lesk & Tramontano, 1990) and PINQ (Lesk, 1986). The first is an automatic program that produces the input file for the modeling package PINQ, start- ing from the alignment of the immunoglobulin to be modeled with the sequences of all immunoglobulins of known structure.

The model was subjected to 100 cycles of energy refinement (with the default parameters of Discover, Biosym Technologies, San Diego, California). It should be noted that this step is only used to obtain a reasonable stereochemistry, especially at the splicing sites, and not to modify the structure in a substantial way. At the end of this step, the backbone RMSD between the original and minimized model was only 0.4 A for backbone at- oms and 0.84 A for all atoms; the major deviations (higher than 1 A) only occurred for the backbone atoms of the 4 residues pre- ceding and the 4 residues following the splicing site of H3.

1482 M. Orlandini et al.

Inspection of the modeled binding site reveals a surface with a central cluster of Tyr residues provided mainly by the L3, H1, and H3 loops. The 2 proline residues of the L3 loop are also sit- uated in the middle of the site, contributing to form a more hy- drophobic region (Fig. 6). The hydrophilic residues form a "crown" around this central cluster. Many available hydrogen bond donors and acceptors are present in the site, and each chain has about 50 atoms that can potentially be involved in hydro- gen bonds with the antigen. The surface area of TW4.20 anti- gen binding site is about 700 A2, more than two-thirds due to the contribution of the heavy chain. The buried surface in known protein-antibody complexes is about the same size of our binding site (Davies et al., 1990). We expect the antigenic site to be of similar size as those of lysozyme in complexes with the HyHel-5, HyHel-10, and Dl .3 antibodies, and as that of neur- aminidase in complex with the NC41 antibody.

Discussion

We identified, cloned, sequenced, and characterized a MAb (TW4.20) elicited against human serum Tf. This antibody com- petes with the TR for binding to Tf. We showed, by antibody binding to peptides obtained by human Tf proteolytic degra- dation, that TW4.20 recognizes an epitope in the N-terminal domain of Tf. This epitope is conformational because the de- natured protein is no longer recognized. Although TW4.20 is able to compete for binding with the TR, it binds equally well both diferric Tf and apoTf and its binding is pH independent (data not shown). This demonstrates that the mode of binding of the antibody to human Tf differs from that of the receptor. This difference could be due to the limited extent of the contact- ing surfaces between the antibody and Tf. Thus, TR and TW4.20 binding sites could be only partially coincident. On the other hand, we cannot exclude that the binding competition be- tween TW4.20 and TR is due to steric hindrance.

A first step toward the more detailed understanding of TW4.20 and human Tf interaction is to move the antibody sequence information into 3-dimensional space. No structural information can be derived for the TR, although it is possible to build antibody models with high accuracy, taking advantage of the wealth of information available on their structures.

We built a model of TW4.20 using the canonical structures method (Chothia & Lesk, 1987, 1989; Tramontano et al., 1990) and described the characteristics of the surface resulting from the clustering of its antigen binding loops. If the structure of hu- man serum Tf was known, we could try to predict the structure of the complex by its components because we have recently shown (Helmer-Citterich & Tramontano, 1994) that an auto- matic docking procedure can find a small set of putative orien- tations of an antibody/antigen complex, and that the correct solution is alwavs present in the set. We have also shown that a model of the antibody can be used in this procedure. Although limited by the unreliability of the positions of side chains in the model, we can draw some considerations about the predicted re- gion of binding of Tf to the antibody. We expect Tf to contact most, if not all, residues of the loops, making extensive use of hydrogen bonding interactions.

As pointed out before, the structure of human serum Tf is not yet available. The molecule shows a very high sequence identity to the sequence of rabbit serum Tf, whose structure has been

solved by X-ray crystallography (Bailey et al., 1988). We are building a partial model of human Tf based on the rabbit struc- ture, but it remains to be seen whether this will allow us to re- construct the antibody/Tf complex structure. First of all, the docking algorithm has to be tested and validated for its ability to reconstruct the complex orientation when both molecules are modeled, and second, the TW4.20 antibody does not recognize rabbit serum Tf, so that-notwithstanding the high sequence homology- we cannot exclude that the 2 molecules show rela- tively large differences around the antibody binding site. None- theless, the construction of the model has allowed us to analyze the antibody binding site and may be useful in guiding muta- genesis experiments, which may, in turn, be used to validate our model.

Materials and methods

MA6 production

Female BALB/c mice were immunized with purified human iron-free Tf (Sigma) dissolved in phosphate-buffered saline (PBS); an initial intraperitoneal injection of 50 pg of Tf emul- sified in complete Freund's adjuvant was given, followed by 2 intraperitoneal injections of 50 p g of Tf in incomplete Freund's adjuvant, with an interval of 2 weeks. Mice were further injected intravenously with 200 p g of antigen in PBS. Five days after the last boost, the animals were sacrificed, spleen dissected, and im- mune spleen cells fused with SP2/0 myeloma cells by standard procedures (Campbell, 1984). Anti-human Tf MAbs were iden- tified by ELISA, testing the culture supernatants from the hy- bridomas after cloning. These hybrids were finally grown as ascites for antibody production.

CELISA

Human K562 cells were used as target cells. These cells were grown in suspension in RPMI-1640 medium with 10% heat- inactivated fetal bovine serum and were maintained at densities of 3-5 X lo5 cells/mL. All experiments were performed with cells collected at a density of 4 x lo5 cells/mL. K562 cells were washed twice in PBS and then resuspended at an optimal den- sity that was estimated by testing a range of cell concentrations. Fifty microliters of cell suspension were added to the wells of flat-bottomed ELISA plates, which were shaken gently to en- able a regular distribution of the cells in the bottom of the wells. The plates were left at 37 "C for 3 days in the presence of silica gel blue and allowed to dry. The completely dry plates were sealed and stored at 4 "C.

Wells coated with cells were rehydrated by adding 100 pL of PBS to each well and incubated for 15 min. After emptying the wells, plates were quenched with PBS/2% bovine serum albu- min and incubated for 2 h at 37 "C. Fifty microliters of HPR-Tf (Jackson lmmunoresearch) and 50 pL of hybridoma superna- tants were added simultaneously to the wells for 3 h a t 37 "C. Chromogenic substrate solution, 1 mM 2,2'-azinobis(3-ethyl- benzthiazolinesulfonic acid) (Boehringer)/0.018% H 2 0 2 in ci- trate buffer, pH 4.2, was added, and the plates were stored in the dark for 30 min at room temperature. Finally, plates were read at 414 nm on an enzyme immunoassay (EIA) reader.

Anti-human transferrin antibody 1483

Determination of the binding constants (KO) of the MAbs

Binding constants were calculated in ELISA by second antibody binding curves, using a slightly modified version of a computer program (Mariani et al., 1987). Briefly, %well vinyl assay plates were coated with goat anti-mouse IgG (5 mg/mL in PBS) and incubated overnight at 4 "C. Serial dilutions (from 3 pmol/well to 0.03 pmol/well) of the MAbs were dispensed into the wells and incubated 20 h at 4 "C. Finally, HPR-Tf (0.125 pmol/well) was added to the wells and incubated for 1 h at 37 "C. Absor- bance was read at 414 nm on an EIA reader.

Fragmentation of human Tf, irnrnunoblotting, and sequence analysis

Thirty percent iron-saturated human Tf (Williams, 1974) was cleaved with the following enzymes and reagents: trypsin and chymotrypsin (both 1 5 0 w/w), with an incubation for 1 h at 37 "C; pepsin (1:50 w/w), with an incubation of 8 h at 25 "C; thrombin (1: 100 w/w), with an incubation of 8 h at 37 "C; N-bromosuccinimide and N-chlorosuccinimide, as previously described (Fontana & Gross, 1987). After the fragmentation, 5-10 pg of the resulting peptides were separated on a tricine-SDS gel under nonreducing conditions, followed by transblotting onto polyvinylidene difluoride (PVDF) membrane (Matsudaira, 1987). Immunoreactivity of the fragments was tested by west- ern blotting (Towbin & Gordon, 1984); quenching and washing of the membranes was performed as previously described (Sithi- gorngul et al., 1991). Incubation with hybridoma cell culture supernatants was carried out overnight at 4 "C. HPR goat anti- mouse IgGs were utilized as secondary antibodies. The binding was detected by enhanced chemiluminescence with the Western Blotting Detection System Kit (Amersham). The positive bands deriving from tryptic, chymotryptic, and thrombin digestion were cut out from a second replica and directly submitted to Ed- man degradation to obtain primary structure information. Se- quence analysis was performed using a model 475A Sequencing System equipped with a ControVData Module model 9OOA (Ap- plied Biosystems).

Flow cytornetric analysis

Anti-human Tf MAb TW4.20 and FITC-Tf (Jackson Immuno- research) were used in inhibition and competition experiments with TR present on K562 cells. Cells were washed in PBS/lO% fetal calf serum (FCS) and quenched in PBS/10% FCS/human IgG (1 mg/mL) for 30 min on ice. FITC-Tf (50 pL) was added to the cells; 50 pL of ascitic fluid of TW4.20 was added simul- taneously (inhibition) or after 30 min of incubation on ice (com- petition). After 30 min of incubation on ice, cells were washed with cold PBS and the fluorescence of the cells was analyzed on a FACStar flow cytometer (Becton Dickinson & Co.).

Cloning and sequencing of the MAb TW4.20 V, and VH regions

Total RNA was isolated from about 1 X lo7 hybridoma cells according to published procedures (Chomczynski & Sacchi, 1987). cDNA synthesis and PCR were performed sequentially, and the primers used (Orlandi et al., 1989) were slightly modi- fied and specific for amplification of VH and V, domains of

IgGl. Briefly, for first-strand synthesis a 25-mL reaction con- taining 500 ng of total RNA, 10 pmol of MOCG12FOR primer 5'-d(CTCAATTTTCTTGTCCACCTTGGTGC) or CKFOR primer 5'-d(CTCATTCCTGTTGAAGCTCTTGAC), 250 mM of each dNTP, 10 mM dithiothreitol, 50 mM Tris-HC1 (pH 8.3), 40 mM KCI, and 7 mM MgClt was heated at 65 "C for 10 min and cooled. Twenty units of M-MLV reverse transcriptase (Pro- mega) and 10 units of a ribonuclease inhibitor RNasin (Pro- mega) were added and incubated at 42 "C for 1 h. Amplification of VH and VL chains was performed in 50-pL reaction mixtures containing 10 pL of the cDNA. RNA hybrid, 15 pmol of primers

GTCCCTTGGCCCC) or K4FOR 5'-d(CCCGGATCCGTTTG ATTTCCAGCTTGGTCCC), and HGlBACK 5'-d(CCCGAA TTCAGGTSMARCTGCAGSAGTCWGG) or K2BACK 5'-d

or VL amplification, respectively, 250 mM of each dNTP, 67 mM Tris-HC1 (pH 8 4 , 16.6mM (NH4)2S04, 1.5 mM MgClz, 0.01 Vo Tween-20, and Taq polymerase (2 units). PCR profiles consisted of 1 min denaturation at 94 "C, 1 min annealing at 62 "C (V,) or at 65 "C (VH), and 1 min extension at 72 "C, repeated 30 times. PCR products were separated by preparative agarose gel electrophoresis, purified by phenol-chloroform extraction, pre- cipitated with ethanol, and digested with restriction endonucle- ase in excess amounts of BarnH I and Sac I for VL or filled using the Klenow fragment of Escherichia coli DNA polymer- ase I to ensure full-length DNA synthesis for VH. The frag- ments were ligated into pBluescript KS previously digested with BarnH I and Sac I for VL or with Hinc I1 for V,, and the re- combinants were used to transform DH5a blue E. colicells. The colonies obtained were screened, and DNA from positive colo- nies was purified for DNA sequencing determination. Sequenc- ing reactions were done using the Sequenase sequencing system (USB).

HGlFOR 5'-d(CCCGGATCCTGAGGAGACGGTGACCGTG

(CCCGAATTGACATTGAGCTCACCCAGTCTCCA) for VH

Acknowledgments

We thank all members o f the Department of Molecular Biology, as well as the scientists of the IRIS Research Centre for their support and use- ful discussions, and Dr. Andrew Wallace for useful comments on the manuscript. We particularly acknowledge Dr. Rino Rappuoli for hos- pitality in his laboratory. This work was supported in part by Associa- zione Italiana per la Ricerca SUI Cancro (AIRC).

References

Arunachalam B, Talwar GP, Raghupathy R. 1990. A simplified cellular ELISA (CELISA) for the detection of antibodies reacting with cell-surface antigens. J Immunol Methods 135:181-189.

Bailey S, Evans RW, Garratt RC, Gorinsky B, Hasnain S, Horsburgh C, Jhoti H , Lindley PF, Mydin A , Sarra R, Watson JL. 1988. Molecular struc-

Bernstein FC, Koetzle TF, Williams GJB, Meyer EF Jr, Brice MD, Rodgers ture of serum transferrin at 3.3-A resolution. Biochemistry27:5804-5812.

JR, Kennard 0, Shimanouchi T, Tasumi M. 1977. The Protein Data Bank: A computer-based archival file for macromolecular structures. J Mol Bioi 112535-542.

Bruccoleri RE, Haber E, Novotny J. 1988. Structure of antibody loops re-

Campbell AM. 1984. Monoclonal antibody technology. In: Burdon RH, van produced by a conformational search algorithm. Nature 335564-568.

Knippenberg PH, eds. Laboratory techniques in biochemistry and mo- lecular biology, vol 13. Amsterdam: Elsevier. pp 120-134.

Chomczynski P, Sacchi N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Bio- chem 162:156-159.

Chothia C, Lesk AM. 1987. Canonical structures for the hypervariable re- gions of immunoglobulins. JMol Biol 196:90-917.

1484 M. Orlandini et al.

Chothia C, Lesk AM, Levitt M, h i t AG, Mariuzza RA, Phillips SEV, Pol- jak RJ. 1986. The predicted structure of immunoglobulin D1.3 and its

Chothia C, Lesk AM, Tramontano A, Levitt M, Smith-Gill SJ, Air GM, comparison with the crystal structure. Science 233:755-758.

Sheriff S, Padlan EA, Davies D, Tulip WR, Colman PM, Spinelli S, Al- zari PM, Poljak RJ. 1989. Conformations of immunoglobulin hyper- variable regions. Nature 342:877-883.

Chothia C, Novotny J, Bruccoleri R, Karplus M. 1985. Domain association

Colman PM, Laver WG, Varghese JN, Baker AT, Tulloch PA, Air GM, Web- in immunoglobulin molecules. J Mol Biol 186:755-758.

ster RG. 1987. Three-dimensional structure of a complex of antibody with influenza virus neuraminidase. Nature 326:358-362.

Dautry-Varsat A, Ciechanover A, Lodish HF. 1983. pH and the recycling of transferrin during receptor-mediated endocytosis. Proc Natl Acad Sci

Davies DR, Padlan EA, Sheriff S. 1990. Antibody-antigen complexes. Annu Rev Biochem 59:439-473.

de Jong G, van Dijk JP, van Eijk HG. 1990. The biology of transferrin. Clin Chim Acta 19O:l-46.

Denstman S, Hromchak R, Guan XP, Bloch A. 1991. Identification of trans- ferrin as a progression factor for ML-I human myeloblastic leukemia cell differentiation. J Biol Chem 266:14873-14876.

Fleming JO, Pen LB. 1988. Measurement of the concentration of murine IgG monoclonal antibody in hybridoma supernatants and ascites in ab- solute units by sensitive and reliable ELISA. J Immunol Mefhods 110:11-18.

Fontana A, Gross E. 1987. In: Darbre A, ed. Prucfical protein chemistry. New York: John Wiley & Sons. pp 67-120.

Franco A, Paroli M, Testa U , Benvenuto R, Peschle C, Balsano F, Bar- naba V. 1992. Transferrin receptor mediates uptake and presentation of hepatitis B envelope antigen by T lymphocytes. J Exp Med 175:1195- 1205.

Genetics Computer Group. 1991. Program manual f o r the GCC package, version 7, April 1991. Madison, Wisconsin: Genetics Computer Group.

Helmer-Citterich M , Tramontano A. 1994. PUZZLE: A new method for au- tomated protein docking based on surface shape complementarity. JMol Bi0/235:1021-1031.

Huebers HA, Finch CA. 1987. The physiology of transferrin and transfer- rin receptors. Physiol Rev 67:520-582.

Kabat EA, Wu TT, Bilofsky H , Reid-Milner M, Perry H. 1983. Sequences of proteins of immunological interest, 3rd ed. Washington, D.C.: Pub- lic Health Service, NIH.

Keyna U , Niisslein I , Rohwer P, Kalden IR, Manger B. 1991. The role of the transferrin receptor for the activation of human lymphocytes. Cell Immunol 132:411-422.

Klausner RD, Ashwell G , van Renswoude J, Harford JB, Bridges KR. 1983.

cycle. Proc Nut1 Acad Sci USA 80:2263-2266. Binding of apotransferrin to K562 cells: Explanation of the transferrin

Lesk AM. 1986. Integrated access to sequence and structural data. In: Sac- cone C, ed. Biosciences: Perspecfives and user services in Europe. Brux-

Lesk AM, Tramontano A. 1990. Antibody structure and structure predic- elles: EEC. pp 23-28.

tions useful in guiding antibody engineering. In: Borrebaeck C, ed. Anfi- body engineering: A practicalguide. New York: Freeman & Co. pp 1-38.

Mariani M, Bracci L, Presentini R, Nucci D, Neri P, Antoni G. 1987. Im-

USA 80:2258-2262.

munogenicity of a free synthetic peptide: Carrier-conjugation enhances antibody affinity for the native protein. Mol Immunol24:297-303.

Martin ACR, Cheetam J, Rees AR. 1989. Modeling antibody hypervariable loops: A combined algorithm. Proc Nut/ Acad Sci USA 86:9628-9672.

Mason AB, Woodworth RC. 1991. Monoclonal antibodies to the amino- and carboxyl-terminal domains of human transferrin. Hybrldomu 10:611- 623.

Matsudaira P. 1987. Sequence from picomole quantities of protein electro-

262:10035-10038. blotted onto polyvinylidene difluoride membranes. J Biol Chem

McGillivray RTA, Mendez E, Shewale JG, Sinha SK, Lineback-Zins J, Brew K . 1983. The primary structure of human serum transferrin. J Biol Chem 258~3543-3553.

Orlandi R, Gussow DH, Jones PT, Winter G . 1989. Cloning immunoglob- ulin variable domains for expression by the polymerase chain reaction. Proc Nut1 Acad Sci USA 86:3833-3837.

Padlan EA, Silverton EW, Sheriff S, Cohen GH, Smith-Gill SJ, Davies DR. 1989. Structure of an antibody-antigen complex: Crystal structure of the

5942. HyHel-IO Fab-lysozyme complex. Proc Natl Acud Sci USA 86:5938-

Pierpaoli W, Dall’Ara A, Yi C, Neri P, Santucci A, Choay J. 1991. Iron car- rier proteins facilitate engraftment of allogeneic bone marrow and en- during hemopoietic chimerism in the lethally irradiated host. Cell Immunol 134:225-234.

Rubikaite VI, Beresten SF, Viklitskii V, Slavichkova A. 1991, Localization on human transferrin of its binding site to the cell receptor with the aid

Satow Y, Cohen GH, Padlan EA, Davics DR. 1986. Phosphocholine bind- of monoclonal antibodies. Mol Biol25:155-162.

ing immunoglobulin Fab McPC603. An X-ray diffraction study at 2.7 A. J Mol Biol 190:593-604.

Sirbasku DA, Pakala R, Sato H, Eby JE. 1991. Thyroid hormone depen- dent pituitary tumor cell growth in serum-free chemically defined cul- ture. A new regulatory role for apotransferrin. Biochemisrry 30:7466- 7477.

Sithigorngul P, Stretton AOW, Cowden C . 1991. A versatile dot-ELISA method with femtornole sensitivity for detecting small peptides. J I m - munol Mefhods 141:23-32.

Thorstensen K , Romslo I . 1990. The role of transferrin in the mechanism of cellular iron uptake. Biochem J 271:97-105.

Towbin H, Gordon J. 1984. lmmunoblotting and dot immunobinding-

Tramontano A, Chothia C , Lesk AM. 1990. Framework residue 71 is a ma- Current status and outlook. J Immunol Methods 72:313-340.

jor determinant of the position and conformation of the second hyper-

215:175-182. variable region in the V,, domains of immunoglobulins. J Mol Biol

Tramontano A, Lesk AM. 1992. Common features of the conformations of antigen-binding loops in immunoglobulins and application to mod- elling loop conformations. Profeins Struct Funcf Genef 13 :231-245.

Williams J. 1974. Thc formation of iron-binding fragments of hen ovotranr- ferrin by limited proteolysis. Blochetn J 141:745-752.

Yang F, Lum JB, McGill JR, Moore CM, Naylor SL, van Bragt PH, Baldwin WD, Bowman BH. 1984. Human transferrin: cDNA characterization and chromosomal localization. Proc Null Acud Sci USA 81:2752-2756.

Zak 0, Trinder D, Aisen P. 1994. Primary receptor-recognition site of hu- man transferrin is in the C-terminal lobe. J Biol Chem 269:71 10-71 14.