Sequences of variable regions of a monoclonal antibody specific to the thyroid hormone, triiodo-L-thyronine

Molecular Immunology, Vol. 29, No. 718,pp. 1025-1028, 1992 Printedin Great Britain.

0161-5890/92 $5.00 + 0.00 Pergamon Press Ltd

SHORT COMMUNICATION SEQUENCES

OF

VARIABLE SPECIFIC

Pallaiah

Thammana*,

REGIONS

OF

A

YONOCLONAL

TO THE THYROID TRIIODO-L-THYRONINE

Alison

L. Gaito

ANTIBODY

HORMONE,

and

Michael

T.

Largen

Glasgow Research Laboratory, Medical Products E. I. du Pont de Nemours Inc., and Company DE 19714-6101. U. S. A. Newark,

(First received 16 December 1991; accepted in revised form 9 March 1992)

Abstract--We have determined the nucleotide sequences of the variable regions of H and L chains of a monoclonal antibody 98QQ that interacts with the thyroid hormone triiodo-L- thyronine with high Analysis of the nucleotide sequence of the light chain V region of 98QQ revealed that the affinity. VL sequence is 99% identical to Balb/c germline Vk 21-E sequence. That is an interesting finding with germline variable region this high affinity anti-T3 antibody, since occurence of predominantly sequences is observed in some autoantibodies to self antigens but not usually in high affinity IgG The sequence analysis also revealed that the heavy chain variable region sequence of antibodies. 98QQ is similar to a V region of an anti-DNA antibody (MRL DNA 22). Thus the sequence analysis of our anti-T3 mAb 98QQ has revealed some features of autoantibodies to self antigens. INTRODUCTION

sequence information along with the sequence analysis is this described in report. Interestingly, the nucleotide sequence analysis of 98 QQ revealed that the VL sequence of this high affinity monoclonal antibody is very similar to a germline variable region sequence. Absence of significant accumulation of somatic mutations in the VL of an IgG anti-T3 described in this report may be a feature that is common to certain autoantibodies directed against self antigens (Smith and Voss, 1990). A similar observation of occurence of variable region sequences closely related to the germline sequence was described in a study of certain autologous insulin binding monoclonal antibodies of moderate affinity (Ewulonu et al., 1990).

Measurements of the thyroid hormones have significant importance in the clinical laboratory for the diagnosis of human thyroid disorders as well as in the treatment of several nonthyroidal illnesses (Tietz, 1986). -In order to develop immunoassays to measure the thyroid sensitive triiodo-L-thyronine have hormone (T3), we monoclonal antibodies against T3, and generated one such monoclonal antibody 98QQ described here has a high affinity for T3, and crossreacts minimally with L-thyroxine (T4). Although there structural are a number of studies on the analysis of several thyroid hormone interacting (Blake and Oatley, 1977; Sap et al., proteins 1986: Weinberger et al., 19861, there is limited published sequence information on the primary sequences of thyroid hormone binding antibodies the (Gleason et al., 1990). We have determined nucleotide sequences of variable regions of the heavy and light chains of 98?Q by sequencing the mRNA with the primer extension method, and the

MATERIALS Hybridoma

AND

METHODS

cells

The hybridoma 98QQ (IgG2a,k) was obtained from fusions of immunized mouse spleen cells with a nonsecreting myeloma partner P3-X63-Ag8.653 (obtained from American Type Culture Collection). The hybridoma 98QQ was derived from female CBA/J mice (obtained from The Jackson Laboratory, Bar Harbor, ME) immunized with triiodo-L-thyronine coupled keyhole limpet hemocyanin, and boosted with the same immunogen over a period of several months before generating hybridomas. The hybridoma cell line was recloned several times, and the subclone used for sequencing work was 98QQ 321. 22. 32. 12 referred to simply as 98QQ in this Cells were paper. grown in Iscove's medium supplemented with 10% fetal calf serum. Hybridoma cells were harvested by centrifugation, washed with phosphate buffered saline (PH 7.4) and *quickly frozen in liquid nitrogen. Frozen cell pellets were stored in a -70 C freezer till the time of isolation of RNA as described below.

--------_-______------------___ * Author correspondence should be to whom addressed at present address: Sterling Winthrop Inc., 512 Elmwood Avenue, Elmwood Court Two, Sharon Hill PA 19079 Abbreviations: T3, 3,5,3'-triiodo-L-thyronine; L-thyroxine; T4, 3,5,3',5'-tetraiodo-L-thyronine, V, variable; H, heavy; L, light; k, kappa light chain; VH, variable heavy chain; VL, variable light chain; CDR, complementarity determining region; FR,framework region; D, diversity; J, mRNA, messenger ribonucleic acid; poly joining; A+ RNA, polyadenylated ribonucleic acid; mAb, monoclonal antibody; Ka, association constant; (T,G)-A--L, NP, 4-hydroxy-3-nitrophenylacetyl; branched polymer poly-L-(Tyr,Glu)-poly-D,L-Ala-poly-LYS.

1025

1026 Immunochemistry

(Kaartinen

High affinity anti-T3 antibody 98QQ was selected by an antigen capture assay in a multiwell format (Removawell-Dynatech) in which antibodies in the hybridoma culture supernatants were bound to an anti-mouse immunoglobulin reagent immobilised on antibodies the solid phase. Mouse monoclonal capable of binding with high affinity to 125 I_ T3 (Specific labelled Nuclear) New England the solid incubating antibodies

with

a

1251- T3. The 98QQ for T3 was

activity 150 pCi/pg, from were identified by phase captured monoclonal

concentration

of 1X10-' M

binding association constant of determined by Scatchard analysis

"sing 125 I- T3 by a variation of the antigen capture assay. In this variation, magnetic antibody particles coated with anti-mouse New England Nuclear) were (Magnisort-M particles, for the separation of free antigen from employed antigen bound to antibody (Birkmeyer et al. 1987). Relative crossreactivity of 98QQ with T4 was magnetic particle based measured in a radiolabelled T3 binding assay similar to affinity, the antibody was measurements. In this assay, incubated with radiolabelled T3 in the presence or ligand. The competing absence of a competing ligand was either unlabelled T3 or T4, and the extent of inhibition of binding of labelled T3 to antibody was measured. The percent inhibition for T3 as well as T4 was concentration of each calculated "sing the formula: %Inhibition

=

[l - (Inh./Uninh.)]

X 100 %

where Inn. = Average counts of T3 bound in the presence of a given concentration of competing T3 or T4 Uninh. = Average counts of competing ligand

of T3 bound

in the absence

the molar versus A plot of % Inhibition concentration of T3 or the molar concentration of the crossreactant T4 was generated, and the 50% inhibition point (Inh. pt.) was determined as a molar concentration equivalent to a 50% inhibition of binding of labelled T3 to 98QQ. The percent then calculated using the crossreactivity was formula given below. %cross

reactivity

Nucleotide Total cells

etal.

PALLAIAHTHAMMANA

RNA by

(Chigwin isolated (Aviv and at 50 C reverse Sciences, sequence determined

=

0% Tnh. ut. of T3 50% Inh. pt. of T4

X 100 %

sequencing

was isolated guanidinium

from 10' 98QQ thiocyanate-CsCl

hybridoma method

et al., 1979), and the poly A+ RNA was by oligo-dT cellulose chromatography Leder 1972). Primer extension was done with the avian myeloblastosis virus (obtained from Life transcriptase St.Petersburg. Fl), and the nucleotide regions of 98QQ mRNA was of VH and VL by the dideoxy

chain

termination

method

et al.,

1983;

Geliebter

1987).

About

15

pg of total poly A+ RNA was used per reaction in the determination of the nucleotide sequences of the region. variable Universal primers corresponding to the gamma constant region and the kappa constant region were used in the initial sequencing (Kaartinen et al., 1983). Primers were end labelled with polynucleotide kinase (FPLC Pharmacia-LKB) by using gamma-32P ATP (New England Nuclear, 6000Ci/mmole). A sequence of over 200 nucleotides was generated in the initial sequencing effort. Based on the generated sequence, new VH and VL primers corresponding to the framework regions were synthesised and used to obtain the complete variable region sequence. A

pure,

primer and

for the VH FR3-

two

VL

primers,

5' dGGAGGATTTATCTACAGTC

3',

5'dCCAGACCCACTGCCACT3',

FR3

and another for VL FR4- "dGCCCGTTTGATTTCCAGC3' r-espectively, were synthesized. the All oligonucleotide primers were obtained from the Oligonucleotide Facility at Princeton University, Princeton, NJ. The nucleotide sequence was the University of Wisconsin GCG analysed by sequence analysis software. RESULTS

AND

VH and VL Sequences The monoclonal triiodo-L-thyronine

DISCUSSION

of 98QQ

antibody with an

98QQ binds association

3,S,3'constant

of lXIOg M-I, and the crossreactivity of the antibody with L-thyroxine is less than 1%. The low level of crossreactivity with T4 makes 98QQ quite specific for T3, suggesting that the antibody can discriminate between the two thyroid hormone species with a difference of a single iodine atom, and for that reason we chose to analyse the Nucleotide sequencing was sequence of 9800. performed with poly A+ RNA by primer extension with oligonucleotide primers as described above in the materials and methods section. The VH sequence could be derived with two oligonucleotide primers, sequence was derived with three while the VL The sequences of VH and oligonucleotide primers. VL are shown

Analysis Analysis

in Fig.

1.

of the VH Sequence of the VH sequence

of 98QQ

revealed

that the VH

region of 98QQ is related to a VH region of an IgM MRL-DNA22 (Kofler et al., 19881 anti-DNA antibody with 94% identity in the nucleotide sequence, and 89% identity in the amino acid sequence. By that that 98QQ VH may belong to a analysis it appears subset of the 3558 V gene family (Eilat, 1990). In to note that VH this respect, it is important anti-thyroglobulin certain mAbs sequences of crossreactive with thyroid hormones belong to the 1990). The J558 gene family (Gleason et al., related to a VH region 98QQVH sequence iS also sequence of an anti_NP antibody 3862 and to the VH sequence HyT 14-2 of an anti-(T,G)-A--L antibody (Allen et al., 1988; Carmack and Pincus 1986).

1027

Sequences of a monoclonal antibody

(A) Nucleotide sequence and the deduced amino acid sequence of anti-T3 antibody 98QQ VH and 90QQ Pig. 1. VL (B) are shown including the location of CDRs, D and J segments. The JB region is derived from germline JR4 and the D region is derived from the DSP 2.3 minigene. The light chain has a Jkl sequence. Identification of the D and J sequences was done by comparing with the germline sequences in Kabat et al (1987). The N residues in VR (Desiderio et al., 1984) are denoted by asterisks. Nucleotide sequence of 98QQ VL was compared with the germline Vk 21-E nucleotide sequence 359-663 (Heinrich et al., 1984), and the base changes in VL from the germline sequence are denoted by asterisks. Amino acid sequence is given in one letter code. Amino acid residue numbering and location of the CDRs was done according to Kabat et al (1987). The VR sequence belongs to subgroup IIB, and the V,,belongs to subgroup III in Kabat's nomenclature.

Close similarity germline sequence

of

98QQ

VL

to Vk

21-E

The sequence analysis revealed that the VL sequence of 98QQ is closely related to a Balblc germline sequence Vk-21E (fieinrich et al., 1984). the With exception of three single base diferences, the 98QQ VL sequence is identical to the Balb/c Vk 21-E germline sequence (99% identity in the nucleotide sequence and 98% identity in the amino acid sequence). As shown in Fig. IB, two single base substitutions in the 98QQ VL are germline C to T changes, resulting in amino acid change from a leucine residue in the germline sequence to a phenylalanine in the light chain . One of these changes is at amino acid residue 50 (the first amino acid residue in the CDRZ), and the second change is at the amino acid residue 94, namely the sixth amino acid residue in the CDR3 (see Fig.lB). Occurence of the two C to T nucfeotide changes in the CDRs of the VL of 98QQ may be a result of somatic mutation process, or alternatively, the base sustitutions may come from allelic differences in the germline variable region sequence of CBA/J (the mouse strain oE origin of 98QQ) and the Balb/c Vk-21E germline sequence.

In addition to the two changes discussed above, as noted in Fig. lB, there is also a change of a C to a G from the germline sequence at nucleotide

661 (Heinrich et al., 1984), corresponding to amino acid residue 96 in the CDR3 of VL of 96QQ. The C to a G change from germline may have arisen as a result of joining of the Vk region with the Jkl sequence, but the junctional nucleotide change however, does not lead to junctional diversity in amino acid sequence. Expression of sequences related to Vk 21-E germline sequence has been observed in an MRL/lpr rheumatoid factor hybridoma 6-58 (Radic et al., 1991), in two anti-thyroglobulin antibodies (Gleason et al .I 1990), and in an anti-dextran antibody (Wang et al., 19911. However, in general it appears that the Vk 21-E germline derived sequence is not commonly utilized among known hybridomas.

Similarity

of 98QQ to autoantibodies

Presence of V region sequences significantly similar to the germline sequence is not commonly observed in high affinity IgG antibodies (Berek and Milstein 1988). Occurence of V region sequences closely related to the germline sequence has been noted in IgG autoantibodies such as antiDNA antibodies (Smith and Voss, 19901, but antiDNA antibodies are usually of low affinity, and generally anti-DNA antibodies with germline like V region sequences are IgM antibodies. In light of that information, it is interesting to note that the sequences of our high affinity anti-T3

PALLAIAHTHAMMANA

antibody have monoclonal certain features of with the observed autoantibodies, similarity of the VH region with an IgM anti-DNA antibody, and the VL region being uniquely similar to the Vk 21-E germline

sequence.

As alluded to earlier, in a study of antibodies to a conserved antigen, namely insulin, Ewulonu et al (1990) made an interesting observation that in four antibodies three out of with moderate affinity for insulin, Vk sequences occured in a predominantly germline earlier, in the case of

sequence. As described 98QQ VL, aside from the only two single base Vk-Jk junctional change, differences from the germline sequence were noted. Concluding

remarks

As noted earlier, occurence of V region sequenctis significantly similar to germline is not commonly observed in high affinity antibodies. An important feature of the affinity maturation of antibodies is their achievement of an "affinity advantage" by the somatic mutation process driven by antigen (Berek and Milstein 1988; French et al., 1989). But in the case of immune response to stimulation of B cell clones endogenous ligands, for the high affinity antibodies is regulated by the threshold concentration of an endogenous 1990). Thus in immune ligand (Goodnow et al., the B cell clones which have response to T3, undergone extensive somatic mutation for very high affinity antibodies may be prevented from antigen the 1OW concentration of stimulation by circulating free hormone.

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