©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Human and Mouse Monoclonal Antibodies to Blood Group A Substance, Which Are Nearly Identical Immunochemically, Use Radically Different Primary Sequences (*)

Katherine G. Nickerson (1) (5), Mi-Hua Tao (6), Hua-Tang Chen (2)(§), James Larrick (7), Elvin A. Kabat (5) (2) (3) (4)(¶)

From the (1) Departments of Medicine, (2) Microbiology, (3) Genetics and Development, and (4) Neurology and the (5) Cancer Center, Columbia University, College of Physicians and Surgeons, New York, New York 10032, the (6) Division of Oncology, Stanford University, Palo Alto, California 94304, and (7) Genelabs Incorporated, Redwood City, California 94063

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A human monoclonal antibody (HuA) specific for blood group A substance with two fucose groups was found to be immunochemically almost identical with that of a previously characterized mouse monoclonal anti-A, AC-1001. The V and V chain cDNAs of HuA were sequenced and compared with those of AC-1001. The human and mouse antibodies used V and V genes that came from different families and shared minimal nucleotide and amino acid sequence identity. Thus, two antibodies from two different species can use evolutionarily unrelated sequences to bind the same carbohydrate epitope.

The cloned HuA V and V genes were then transfected into a mouse myeloma cell line and re-expressed, together, and each separately with an irrelevant V or V. Only the original HuA V and V had anti-A activity, demonstrating that both the heavy and light chains contributed to specificity.


INTRODUCTION

The ABO blood group antigens are carbohydrate structures present in many tissues and synthesized under the control of several recently characterized and highly related genes coding for glycosyltransferases (1, 2). They may be expressed in different forms: as oligosaccharides in urine, as glycoproteins in body secretions and tissue fluids, and as glycolipids in membranes (3) . Their expression varies during differentiation and in different tissues, and they may be present at abnormal levels or in altered forms in malignant and premalignant lesions (reviewed in Ref. 4). Human antibodies of defined specificity to blood group antigens are thus of potential importance in the diagnosis, prognosis, and treatment of certain malignancies. Although human monoclonal antibodies have been very difficult to produce in amounts necessary for clinical use, the technology is rapidly advancing (5). For such purposes, as well as for gaining a fundamental understanding of the expression of these antigens in differentiation and malignant transformation, antibodies with carefully defined specificities will be necessary.

The smallest determinant showing A specificity is the trisaccharide: GalNAc(1 3)[Fuc(1 2)]Gal1, but A-specific antisera and monoclonal antibodies may vary in their reactivity, depending on additional adjacent sugars and the ways in which they are linked (3, 6, 7) . The A trisaccharide can be linked either 1 3 or 1 4 to GlcNAc in type 1 and type 2 A antigens, or it may be linked 1 3 to GalNAc or GalNAc in type 3 and type 4 structures

On-line formulae not verified for accuracy

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Type 1 and type 2 determinants can be further modified by the addition of a second fucose linked either (1 4) or (1 3) to the GlcNAc (2) . These various A structures differ immunochemically because the adjacent sugars may actually participate in the antigenic site or they may influence the orientation of the A trisaccharide itself (7) . Monoclonal antibodies have been very important in defining these structures (8, 9, 10, 11, 12, 13, 14) , and then, subsequently, in characterizing their expression in various tissues, at different stages of differentiation, and in malignant transformation (15, 16, 17, 18, 19, 20) .

We have characterized the fine specificity and immunochemical properties of HuA, a human anti-A produced by cloned Epstein Barr virus-transformed lymphocytes that were stabilized by fusion with a human-mouse fusion partner (21) . This antibody is specific for difucosyl A antigens of either type 1 or type 2 and is virtually identical with a previously characterized mouse monoclonal antibody, AC-1001 (8, 22) , in its pattern of inhibition by oligosaccharides from blood group A substances. This gave us a unique opportunity to compare the sequences of heavy and light chain genes from two different species that encode antibodies with specificity for the same carbohydrate epitope.


MATERIALS AND METHODS

Cell Line

The human-mouse trioma cell line secreting HuA was described previously (21) . It is a cloned Epstein Barr virus-transformed B cell line stabilized by fusion with the human-mouse fusion partner SBC-H20. In tissue culture, it secretes substantial amounts of anti-A monoclonal antibody (1-5 mg/liter) (21) .

Immunochemical Assays

Quantitative precipitin assays, quantitative inhibition ELISAs,() and ELISAs to detect anti-blood group activity from transfectoma supernatants were carried out using blood group substances and oligosaccharides as described previously (8) . In quantitative precipitin assays, 5-8 µg of affinity-purified antibody N (nitrogen) was used per determination in a total volume of 200 µl. Antibody N in washed precipitates was determined by the ninhydrin method (23) . For the quantitative inhibition ELISAs, blood group A substance (Hog 4 10%) at 1 ng/ml in pH 8 borate-buffered saline was used to coat polystyrene plates. Assays were carried out as described (8) , and the free energy of binding, G, relative to the A-trisaccharide was calculated according to the standard formula: G =273R ln(x/y) (8) , where x is the amount of inhibitor in nanomoles giving 50% inhibition at 0 °C, and y is the amount of A-trisaccharide giving 50% inhibition. This permits comparison of antibody binding of the various oligosaccharides to that of the simple trisaccharide structure.

Isolation and Sequencing of Anti-A Heavy and Light Chain cDNAs

Before extracting RNA, culture supernatants from the HuA cell line and the parental fusion partner were retested for anti-A activity. The supernatant from the anti-A cell line specifically agglutinated human type A red blood cells and had anti-A activity by Ouchterlony diffusion and ELISA, while the supernatant of the parental cell line did not. Total RNA was isolated from 10 cells using guanidinium thiocyanate and cesium chloride gradient centrifugation, first strand cDNA was prepared, and heavy and light chain cDNAs were amplified using the polymerase chain reaction (PCR) as described (24) . We used the 5` and 3` primers described by Larrick et al.(24) . For the heavy chain, oligo(dT) priming was used to make first strand cDNA. For PCR, a mixture of three degenerate 5` primers based on the leader sequences of each of the three subgroups of human V genes (as defined by Kabat et al.(25) ) were used along with the 3` IgM primer. For the light chain, the 3` constant region primer was used to make first strand cDNA, and a degenerate 5` primer based on leader sequences was used with it in PCR. The 5` primers all have EcoRI restriction sites, and the 3` primers have HindIII sites to facilitate subcloning into vectors for sequencing. The sequences of the primers used are as follows with the bases shown within parentheses having been present in equimolar amounts during synthesis of a particular position. V, 5`-gggaattcatggactggacctggagg(ag)tc(ct)tct(gt)c-3`, 5`-gggaattcatggag(ct)ttgggctga(cg)ctgg(cg)tt-3`, 5`-gggaattcatg(ag)a(ac)(ac)(at)act(gt)tg(gt)(at)(cgt)c(at)(ct)(cg)ct(ct)ctg-3`; C, 5`-ccaagcttagacgagggggaaaagggtt; V, 5`-gggaattcatggacatg(ag)(ag)(ag)(agt)(ct)cc(act)(acg)g(ct)(gt)ca(cg)ctt-3`; C, 5`-ccaagctttcatcagatggcgggaagat.

cDNA from the HuA cell line and its parental fusion partner were subjected to PCR in parallel. Aliquots of PCR products were electrophoresed in 2% agarose gels. PCR products with bands of the expected size (approximately 450 base pairs) present in the anti-A cDNA but not in the parental cell line's cDNA were digested with HindIII and EcoRI, electrophoresed in polyacrylamide gel electrophoresis gels, and appropriate sized bands were cut out, electroeluted, and ligated into pUC 18. Dideoxynucleotide chain termination sequencing of both strands was carried out using a modified T7 DNA Polymerase (Sequenase) according to the manufacturer's protocol. Two separate clones with the heavy chain were sequenced and were found to be identical.

Transfection of HuA Vand VChain Shuffling Experiments

In order to determine if either chain retained anti-A activity when coupled with an irrelevant heavy or light chain, transfection experiments were carried out as described (26). The coding sequences of HuA V and V were amplified by PCR using upstream primers containing an NcoI site overlapping the translation start codon ATG and downstream primers containing KpnI sites located in the J segments. The NcoI-KpnI fragments were gel-purified, resequenced, and inserted into the Ig heavy and light chain expression vectors pSV2- Hgpt and pSV2-Hneo(26) , modified by the addition of NcoI and KpnI sites. The expression vectors containing the V and V genes from a human B-cell lymphoma (RF) were synthesized likewise. The heavy chain vector contains human Ig1 constant region genomic sequence and the light chain vector contains the Ig k constant region sequence. The heavy chain vector was then co-transfected with the light chain vector into an Ig-nonproducing plasmacytoma cell line, P3X63Ag8.653, by electroporation as described (26) , cloned in medium containing the antibiotic, G418, and screened for antibody production. Three cell lines were made, AJ-1 expressing HuA V and V, AK-6 expressing HuA V and RF V, and AL-23 expressing RF V and HuA V. Supernatants from the three cell lines were used in ELISA assays.


RESULTS

Quantitative Precipitin Assays

Fig. 1 shows the quantitative precipitin curves obtained with various blood group substances and HuA. HuA reacted well with all the preparations of blood group A substance. With Hog 4 10%, a maximum of 6.5 g was precipitated, and, with other preparations, 5.6 to 6.2 µg of N were precipitated. Human saliva blood group A substance, W.G. phenol insoluble, showed the lowest activity with the antibody with a maximum of 5.6 µg of N precipitated, and about twice as much antigen being required for 50% of maximum precipitation (1.9 µg compared to 0.7-1.1 µg). This is most likely due to the presence of fewer A determinants on A substances (27) . HuA did not react with blood group substances B, H, Le, Le, and precursor Ii. The specificity of HuA for blood group A substances and its uniform reactivity with all those tested was very similar to the quantitative precipitin data for the mouse anti-A, AC-1001 (8) .


Figure 1: Quantitative precipitin curves of human monoclonal anti-A with various blood group substances. , Hog 4 10% (A); &cjs0800;, Cyst 9, phenol-insoluble (A); , Cyst 14, phenol-insoluble (A); , MSM 10% (A); , Hog 75 10% (A); , MSS 10% 2X (A); , McDon 15% (A); &cjs0383;, Hog 67 4% (A); , W. G., phenol-insoluble (A); , Cyst Beach, phenol-insoluble (B); , N-1, phenol-insoluble (Le); , JS, phenol insoluble (HLe); , Tij II, phenol-insoluble (BI); , Tighe, phenol-insoluble (H); ▾, Cyst OG 10% from 20% (I).



Quantitative Inhibition ELISAs

Inhibition of anti-A binding to Hog 4 10% by various monosaccharides and blood group oligosaccharides is shown in Fig. 2. The oligosaccharide structures, amounts required for 50% inhibition, the free energies of binding (G), and comparable results for the previously characterized mouse monoclonal anti-A, AC-1001, are shown in . HuA is most specific for difucosyl oligosaccharides. The best inhibitors were the A-hepta and the A-penta. The human antibody did not distinguish between the type 1 (1 3) linkage to GlcNAc of the A-hepta and the type 2 (1 4) linkage of the A-penta. The A-hexa differs from the A-hepta only by lacking the second fucose linked to GlcNAc, but it was less efficient at inhibition by 10-fold. As shown, this fine specificity was nearly identical with that of AC-1001, the mouse monoclonal. For both the human and mouse antibodies, the oligosaccharides in their order of ability to inhibit binding of antibody to A blood group substance was the hepta > penta > hexa > tetra and MSMARO.56 > tri and 01. For both, the difucosyl containing hepta- and pentasaccharides were the strongest inhibitors indicating the specificity of both antibodies for the difucosyl portions of the oligosaccharides. The only difference was that the mouse monoclonal was more effectively inhibited by the monofucosyl A-hexa. This could be explained if the Gal(1 4)GlcNAc contributes somewhat more to the mouse anti-A binding site than to the human (28) .


Figure 2: Inhibition by various oligosaccharides of binding of human monoclonal anti-A to blood group A Hog 4 10%. Symbols and corresponding oligosaccharide structures are shown in Table I.



Nucleotide and Derived Amino Acid Sequence Comparisons of the Human and Mouse Anti-A Heavy and Light Chains

Fig. 3 and 4 show the complete nucleotide and derived amino acid sequences of the HuA V and V. The sequences of the V and V chains of the murine AC-1001 (22) are shown for comparison. The human anti-A heavy chain uses a member of the V3 family (29) with a D segment which does not correspond to a previously sequenced germline D segment, and J4 with 3 nucleotide changes and 1 amino acid change from the reported germline sequence (30) . The most closely related previously characterized human V gene is 56P1 (31) obtained from a cDNA library from human fetal liver. Three other cDNAs, M72, M74, and M49, from a second fetal liver cDNA use the same V gene (32) . For codons 1-94, HuA and 56P1 are 91% identical at the nucleotide level and 86% at the amino acid level. The mouse heavy chain of AC-1001 is a member of the J558 family in the classification of Brodeur et al. (33) and Dildrop et al.(34) and of subgroup IIb in that of Kabat et al.(25) . The mouse and human V sequences have only 56% identity at the nucleotide level and 43% identity at the amino acid level.


Figure 3: Nucleotide and derived amino acid sequence of the HuA heavy chain. For comparison, the nucleotide and amino acid sequences of the mouse anti-A, AC-1001, are also shown. Nucleotide identities are represented by dots. Only amino acid differences are shown. Nucleotides of the HuA which differ from the germline J are indicated with capital letters.



As shown in Fig. 4, the human V is a member of the VIIIa subgroup (25) and has only one silent nucleotide difference (t instead of c in the third position of codon 36) from a previously published germline V, V-g (35) . The leader sequence of V-g and HuA are identical except for five nucleotide differences in the region of the 5` primer used in PCR. Thus, V-g is probably the germline counterpart of the HuA V with the differences in the leader having been introduced by mismatchs during the initial cycles of PCR priming. The anti-A V is rearranged to J1 with three nucleotide differences resulting in two amino acid changes from the reported germline sequence (36) . Very similar light chains, probably using the same germline V gene, have been found in a number of other antibodies, including a panel of cold agglutinins specific for sialic acid glycoproteins on human erythrocytes (37) , a human IgG rheumatoid factor from synovial fluid (38) , and an autoantibody specific for myelin and denatured DNA from a patient with chronic lymphoblastic leukemia and peripheral neuropathy (39) . These antibodies all use V that differ from HuA. The cold agglutinin uses a V1 gene, the rheumatoid factor uses a V3 gene that is 82% identical at the nucleotide level and 72% identical at the amino acid level for codons 1-94, and the anti-myelin/DNA uses a member of V3 which is 79% identical with the human anti-A at the nucleotide level and 70% identical at the amino acid level.


Figure 4: Nucleotide and derived amino acid sequences of HuA light chain. For comparison, the nucleotide and amino acid sequences of the mouse anti-A, AC-1001, the light chain from an antibody with anti-PR activity, LS-1 (37), and the light chain from a human IgG rheumatoid factor from synovial fluid, ka3dl (38), are shown. Nucleotide identities are represented by dots. Only amino acid differences are shown. Nucleotides of the HuA which differ from the germline sequences of V-g (35) and J1 (36) are indicated with capital letters. The leader sequences for these V genes are also shown along with the 5` primer used in PCR. Positions where more than one base was used in synthesis of the primer are shown.



Comparing the HuA light chains to that of the mouse, AC-1001, there is 68% nucleotide and 57% amino acid identity with 64% identity of the amino acids in the framework and 37% in the CDRs. Like the heavy chain there are shared residues in the CDRs, but all are residues most frequently used ( Fig. 5and ). Of the residues in common, the arginine, alanine, serine, and glutamine at positions 24-27 in both antibodies appears to be outside the region of antigen contact according to the limited data available on antibody structures (40) . Tyr-32 in CDR 1, Ser-52 in CDR 2, and Gln-90 and Pro-95 in CDR 3 may be at positions of antigen contact, but these few similarities are unlikely to account for the specificity of the two antibodies.


Figure 5: HuA and mouse AC-1001 heavy and light chain CDRs. Amino acid identities are underlined, and the percent of all sequenced antibodies (25) with those amino acids is indicated. In CDRs 2 and 3 of the heavy chain, there are similar motifs in the human and mouse sequences which are displaced by one amino acid. These residues are also underlined, and their frequency among all V sequences (25) is indicated separately for the human and mouse sequences.



Assays of Transfectoma Cell Lines

Supernatants from the three transfectoma cell lines were assayed by ELISA for total IgG and for specificity against various blood group antigens including Cyst 14 (A), Beach (phenol insoluble) (B), J.S. (phenol insoluble) (H), OG (I), and Le. Only AJ-1 with both the HuA heavy and light chain bound to blood group A. There was no binding of any of the three supernatants to the B, H, I, or Le blood group substances (not shown). These data confirmed that the correct anti-A binding heavy and light chain genes had been identified, and that both the heavy and light chain together contributed to specificity. Because AL-23, with the HuA V paired with an irrelevant heavy chain, secreted somewhat lower amounts of Ig than either AJ-1 or AK-6, we could not completely exclude the possibility that the HuA V could have some small amount of anti-A activity independent of the heavy chain. Multiple attempts at identifying a clone making higher concentrations of IgG failed, probably because our method of selection was designed to identify clones with both the heavy and light chain, but did not specifically select for high levels of immunoglobulin secretion.


DISCUSSION

The immunochemical properties and fine specificity of HuA were remarkably similar to those of a previously characterized mouse monoclonal, AC-1001, more similar in fact than any two previosuly characterized mouse blood group antibodies (8) . Despite this similarity, they use radically different heavy and light chain genes.

The human V3 gene used by HuA and the murine J558 gene used by AC-1001 are as distantly related as any mouse and human V can be, sharing only 56% of nucleotides and 43% of amino acids. Schroeder and co-workers (41, 42) have used framework codons to define evolutionary relationships among mammalian V genes, and, in this analysis, these two gene families most likely arose from separate progenitor V elements or clans. The two V genes also come from different families and share only 68% nucleotide and 57% amino acid identity.

Although the two V and V are not highly related in their framework structures nor in evolutionary origin, their similar binding properties might be explained if there were shared residues in the CDRs. As shown in Fig. 5and , however, there are only a few residues in common between the mouse and human CDRs, and most of them occur most commonly in these positions. The heavy chain CDRs have only 15% amino acid identity, the two CDR3s differ in length, and the light chain CDRS have 37% only amino acid identity.

The data from the panel of mouse monoclonal anti-A antibodies reported previously (22) do not shed much light on the mechanism by which the human and mouse antibodies achieve their similar binding specificities. The mouse monoclonals AC-1001 and A003 = 40/5G7 were similar immunochemically and used very similar heavy chains but different light chains. This suggested that the similarity in binding was conferred by the heavy chain. Based on that conclusion, one might expect that the HuA and AC-1001 heavy chains might be closely related. Our results, however, demonstrate quite the opposite, and our transfection experiments show that for HuA both V and V must be present for anti-A activity.

Chothia et al.(43) have begun to improve modelling techniques for immunoglobulins. They have started to define critical residues both within the CDRs and outside of the CDRs that influence the conformation of the amino acid backbone of the CDR loops. Using the residues that they define as canonical structures of the CDRs, the mouse AC-1001 and human anti-A antibodies may share similar conformations in the light chain CDRs but appear to differ markedly in the conformation of the heavy chain CDRs. Most likely, the two antibodies have significant differences both in the conformation of their CDRs and in the amino acid side chains used in antigen contact.

In the (1 6) dextran system, 43 monoclonal antibodies have been sequenced and defined immunochemically (44, 45, 46, 47, 48, 49, 50) . Four of these recognize the terminal nonreducing end of dextran and are all quite similar in their V and V usage (45-48); however, members of five different V gene families and three V gene families are used in 39 antibodies that have groove type sites (44, 46, 47, 49, 50) . Despite this apparent diversity, there are some remarkable patterns of similarity. A set of 21 groove type antibodies using two different V genes all use the same D segment with very similar V-D and D-J joints to form CDR3s which differ from one another by only one or two amino acids. All twenty one of these groove type antibodies use the same V OX1 gene (49, 50). These 21 antibodies which are extremely similar in CDR3 of the heavy chain and CDR1, -2, and -3 of the light chain vary by up to 200-fold in their affinity for (1 6) dextran (49, 50) . These data and others (51, 52) have emphasized the importance of CDR3 of the heavy chain in defining specificity. In comparison, the human and mouse anti-A antibodies are much more similar immunochemically and much more different in their primary structure. In other antigen-antibody systems, diversity in V and V usage has been documented (53, 54, 55, 56) , but in most of these systems the antigenic determinants are far less precisely defined. Andria et al. (57) showed that diverse V and V genes are used in antibodies to a well defined decapeptide of tobacco mosaic virus. However, these antibodies also differed in affinity and in their patterns of reactivity from a panel of synthetic decapeptides with amino acid substitutions.

In other systems it is clear that different amino acid sequences can be used to create molecules with similar three-dimensional structures and binding capacities. For example, hemoglobin and myoglobin molecules from different species vary in up to 80-89% of the amino acids used and yet maintain remarkably similar three-dimensional structures and similar functions (58, 59) . Other examples of molecular mimicry exist as well: for example, an anti-idiotypic antibody can mimic the steroid hormone, aldosterone, and displace it from its receptor with similar kinetics (60) . What is being recognized is not a primary structure, but the three-dimensional arrangement of nuclei and electrons constructed in one case by a steroid hormone and in the other from a polypeptide.

The remarkably similar immunochemical behavior of the human and mouse anti-A antibodies is not readily explained by similarities in either their primary structures or in crude predictions of the tertiary conformation of their CDRs. Either the two antibodies form similar antigen binding sites in very different ways or, alternatively, the two antibodies see the antigen in topographically distinct ways. Only x-ray crystallography or much more sophisticated molecular modeling techniques than are currently available will resolve this question. The comparison of the primary nucleotide and amino acid sequences of the human and mouse antibodies demonstrates how completely different primary heavy and light chain structures can be used to recognize the same well-defined epitope, a possibility predicted by Wu and Kabat (61). These data suggest that germline antibody genes are not selected through evolution on the basis of their ability to bind one specific antigen but rather that the repertoire as a whole has evolved to ensure maximum range of binding activities. Redundancy, or the ability to solve particular topographic problems in multiple ways, is probably a part of this kind of broad repertoire and ensures that the organism is protected against catastrophic loss of germline V and V genes (62) .

  
Table: Oligosaccharide inhibition studies on human and mouse antibodies to blood group A substance

NT, not tested; R = 3-hexene-1,2,5,6-tetrol. The following mono- and oligosaccharides at the amounts tested gave no significant inhibition with the monoclonal anti-A: &cjs2111;, D-GalNAc; , L-Fuc; ◊, D-Gal; , 04 [Fuc1 2Gal1 3GlcNAc-O(CH)-COOCH]; ▾, B-Tri (Gal1

On-line formulae not verified for accuracy

); &cjs0383;, B-penta (Gal1

On-line formulae not verified for accuracy

1 4

On-line formulae not verified for accuracy

).


  
Table: Nucleotide and amino acid identity of AC-1001 and human anti-A



FOOTNOTES

*
This work was supported by Grants 5RO1 AI27508-03 and 2RO1 AI/GM19042-07 from the National Institutes of Health and DMB890-1840 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) L41174 (Hu V) and L41175 (Hu V).

§
Current address: Molecular Immunogenetics Section, NIAID, National Institutes of Health, Bethesda, MD 20814

To whom correspondence and reprint requests should be addressed.

The abbreviations used are: ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We are indebted to Dr. Denong Wang for reviewing the manuscript.


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