1. INTRODUCTION

The present invention is directed to a form of the human ₇ T cell antigen receptor polypeptide termed Form

2bc, which has a molecular weight of about 40,000 daltons,

5 and a constant region which contains a sequence encoded by only two C ₇ 2 CII exon copies. The invention also relates to

T cell antigen receptor heterodimers comprising ₇ Form 2bc, and to nucleic acid sequences encoding ₇ Form 2bc and portions thereof. The invention also provides monoclonal

10 antibodies specifically reactive with an epitope of the ₇ or δ T cell antigen receptor polypeptides.

2. BACKGROUND OF THE INVENTION The T cell antigen receptor (TCR) was shown to be

15 a clone specific disulfide-linked heterodimer on T cells, composed of two glycosylated subunits, one of which is designated the α chain and the other of which is designated the β chain. The α and 0TCR subunits have a relative molecular mass (M ) of approximately 50,000 and 40,000 daltons, respectively (Allison et al., 1982, Immunol.

20 129:2293-2300; Meuer et al., 1983, J. Exp. Med. 157:705-719; Haskins et al., 1983, J. Exp. Med. 157:1149-1169). Genes that rearrange during T cell ontogeny and encode the ySTCR (Yanagi et al., 1984, Nature 308:145-149; Hedrick et al., 1984, Nature 308:153-158) and TCR (Chien et al., 1984,

25 Nature 312:31-35; Saito et al., 1984, Nature 312:36-40, Sim et al., 1984, Nature 312:771-775) subunits were isolated either by subtractive hybridization or by probing with oligonucleotides.

30 The alpha and beta chains of the T cell antigen receptor of a T cell clone are each composed of a unique combination of domains designated variable (V) , diversity (D) , joining (J) , and constant (C) (Siu et al., 1984, Cell 37:393; Yanagi et al., 1985, Proc. Natl. Acad. Sci. USA 82: ^ _c 3430) . Hypervariable regions have been identified

(Patten et al., 1984, Nature 312:40; Becker et al., 1985, Nature 317:430). In each T cell clone, the combination of V, D and J domains of both the alpha and the beta chains participates in antigen recognition in a manner which is uniquely characteristic of that T cell clone and defines a unique binding site, also known as the idiotype of the T cell clone. In contrast, the C domain does not participate in antigen binding.

A unique feature of the human α, TCR was the observed co odulation (Meuer et al., 1983, J. Exp. Med. 157:705-719), coimmunoprecipitation (PCT International Publication No. WO 88/00209, published January 14, 1988; Oettgen, et al., 1984, J. Biol. Chem. 259:12,039-12,048) and required coexpression (Weiss et al., 1984, J. Exp. Med. 160:1284-1299) of the α,j8TCR molecules with a CD3 glycoprotein complex. Subsequently, the direct physical association of the two protein complexes was demonstrated by chemically cross-linking the α, TCR molecules to the T3 glycoprotein and identifying the components of the cross- linked complex as the TCR subunit and the T3 glycoprotein

(M _r 28,000) subunit (Brenner et al., 1985, Cell 40:183-190). A T3 counterpart is similarly associated with murine α, TCR (Allison et al., 1985, Nature 314:107-109; Samelson et al., 1984, Immunol. Rev. 81:131-144).

A third gene that rearranges in T cells, designated ₇ TCR, was identified, first in mice (Saito et al, 1984, Nature 309:757-762; Kranz et al., 1985, Nature 313:762-755; Hayday et al., 1985, Cell 40:259-269) and then in humans (Lefranc et al., 1985, Nature 316:464-466; Murre et al., 1985, Nature 316:549-552). The human ₇ TCR locus appears to consist of between five and ten variable, five joining, and two constant region genes (Dialyπas et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83: 2619). Although the total number of-functional variable and joining regions is limited, significant diversity is introduced during the

process of V-J joining (Kranz et al., 1985, Nature 313:752- 755; Lefranc et al., 1986, Cell 45:237-246; Quertermaus et al., 1986, Nature 322:184). The ₇ TCR gene rearrangements occur in lymphocytes with suppressor-cytotoxic as well as helper phenotypes (Lefranc et al., 1985, Nature 316:464-466; Murre et al., 1985, Nature 316:549-552, Quertermaus et al., 1986, Science 231:252-255; Lefranc et al., 1986, Cell 45:237-246, Iwa oto et al., 1986, J. Exp. Med. 163:1203- 1212; Zauderer et al., 1986, J. Exp. Med. 163:1314-1318).

The products of the ₇ TCR gene have been identified in T3 coimmunoprecipitates from aβTCR~CO3 T (Brenner et al., 1986, Nature 322:145-149; Bank et al., 1986, Nature 322:179-181; Borst et al., 1987, Nature 325, 683-688; Moingeon et al., 1987, Nature 325, 723-726, PCT International Publication No. WO 88/00209, published January 14, 1987). The ₇ TCR polypeptides were identified by use of monoclonal antibodies directed against ₇ TCR peptide sequences; these polypeptides were found to be incorporated into heterodimers with another polypeptide called 5TCR (Brenner et al., 1986, Nature 322:145-149). The ₇ .5 heterodimer was reported to be associated noncovalently with CD3.

Use of antisera directed against ₇ TCR-specific peptides has led to the identification of CD3-associated ₇ TCR polypeptides on cells originating in peripheral blood, thymus, and a leukemic cell line (Brenner et al., 1986, Nature 322:145-149; Bank et al., 1986, Nature 322:179-181, Weiss et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:6998- 7002; Brenner et al., 1987, Nature 325:689-694; Lew et al., 1986, Science 234:1401-1405). Bank et al. (supra) disclosed a 44 kd ₇ form which was associated with a 62,000 kD peptide and T3 on the surface of a human thymocyte clone. A similar ₇ TCR polypeptide was also identified on murine T lymphocytes, and the expression of this peptide during thymocyte differentiation is the subject of much current

study (Roulet et al., 1985, Nature 314:103-107; Snodgrass et al., 1985, Nature 315:232-233; Lew et al., 1986, Science 234:1401-1408; Pardau et al., 1987, Nature 326:79-81; Bluestone et al., 1987, Nature 326:82-84). With the study of ₇ TCR human cell lines, two different ₇ TCR polypeptides have been identified that differ in their molecular weight and in their ability to form disulfide linkages (Borst et al., 1987, Nature 325:683-688; Brenner et al., 1987, Nature 325:689-694; Moingeon et al., 1987, Nature 325:723-726; Lanier et al., 1987, J. Exp. Med. 165:1076). Two different ₇ TCR constant region gene segments, called C ₇ I and Gγ2, respectively, have been compared; a cysteine residue encoded by the second exon of C ₇ I appears to be absent in C ₇ 2 exon segments, and its absence has been suggested to explain the inability of some ₇ TCR peptides to form disulfide bonds (Krangel, et al., 1987, Science, 237:1051-1055; Littman et al.. Nature 326:85088) .

In contrast to the multiple forms of ₇ TCR, the 5TCR molecule is relatively invariant and it appears that there is only one 5TCR constant region (Hata et al., 1987, Science 238:678-682).

During T cell ontogeny, it has been shown that ₇ TCR gene rearrangement precedes β and αTCR gene rearrangement (Roulet et al., 1985, Nature 314:103-107; Snodgrass et al., 1985, Nature 315:232-233; Sangoter et al., 1986, J. Exp. Med. 163:1491-1508).

Of mature, circulating T lymphocytes, a relatively small proportion are ₇ δTCR , and exhibit either CD3 4 8 (double negative) or CD3 4~8 surface antigens. CD3 4~8~ T cells constitute approximately two percent of mature CD3 T cells. Unlike most mature CD3 4 or CD3 ⁺ 8 major histocompatibility locus (MHC) restricted cytotoxic T cells, but similar to CD3~ ^" natural killer cells, ₇ 5TCR CD3 4 8 cloned lymphocytes have been shown to exhibit MHC-

nonrestricted cytolytic activity; however, unlike natural killer cells, these ₇ 5 + CD3+4—8— T cells did not consistently kill natural killer cell targets, such as K-562

(Borst et al., 1987, 325:683-688; Brenner et al., 1987, Nature 325:689-694; Moingeon et al., 1987, Nature 325:723-

726; Bluestone et al., 1987, Nature 326:82-84).

3. SUMMARY OF THE INVENTION

The present invention is directed to a form of the human ₇ T cell antigen receptor (TCR) polypeptide termed Form 2bc. The Form 2bc ₇ TCR chain has a primary amino acid sequence substantially as depicted in Figure 14. The Form 2bc ₇ TCR chain has a molecular weight of about 40,000 daltons, and comprises a constant region containing a sequence encoded by only two C ₇ 2 CII exon copies. The invention also relates to TCR heterodimers comprising the ₇ TCR polypeptide Form 2bc.

The invention is also directed to nucleic acid sequences encoding ₇ TCR Form 2bc, and to nucleic acid sequences comprising a Oγ2 constant region having only two CII exons. In a specific embodiment, the nucleic acids of the invention comprise at least a portion of the nucleic acid sequences shown in Figure 14.

The invention also provides monoclonal antibodies specifically reactive with an epitope of the ₇ or δTCR polypeptides. Such antibodies can be identified by detecting their ability to co-modulate the CD3 antigen on a cell which expresses both the ₇ 5TCR and a CD3 antigen. In a specific embodiment, the invention relates to antibodies reactive with the variable region of the δTCR chain. In a particular embodiment, such an antibody can be used to detect functional δTCR variable gene rearrangements in a cell. In another embodiment, the invention relates to antibodies reactive with the constant region of the δTCR

polypeptide. In yet another embodiment, the invention relates to antibodies reactive with the constant region of the ₇ TCR polypeptide.

In another aspect of the invention, a method is provided for producing expression of a ₇ TCR in a cell.

3.1. DEFINITIONS As used herein, the following terms will have the meanings indicated:

TCR = T cell antigen receptor

V = variable

D = diversity

J = joining

C = constant mAb = monoclonal antibody

4. DESCRIPTION OF THE FIGURES Figure 1. Cytofluorographic analysis of T cell lines with anti-TCRδl.

Figure 2. Immunochemical analysis of the speci .fi.ci.ty of mAb anti.-TCRδl. Surface 125I-labeled IDP2 cells were immunoprecipitated using control mAb P3 (lanes 1 and 2) , anti-leu 4 (lanes 3-5) , anti-TCRδl (lanes 6-8) , or anti-Cy serum (lane 9) and were then resolved by SDS-PAGE

(polyacrylamide gel electrophoresis) and visualized by autoradiography. N = nonreducing conditions; R = reducing conditions.

Figure 3. N-glycanase digestion of δTCR.

Figure 4. Map of pGEM3-0-240/38.

Figure 5. Immunoprecipitation of in vitro translation products of cDNA clone IDP2 0-240/38 by mAb anti-TCRδl.

Figure 6. Immunoprecipitation and SDS-PAGE analysis of T cell antigen receptor. Open arrowheads indicate the position of the δ chains. The solid arrowheads

indicate the position of the ₇ chains. Lysates were immunoprecipitated using δTCAR-3 antibody (odd numbered lanes) or βTl antibody (even numbered lanes) .

Figure 7. Immunoprecipitation of δ chain by δTCAR-3 antibody. Molt-13 cells solubilized in Tris- buffered saline (pH 8) containing 0.3% CHAPS (lane 1) or in 1% Triton X-100 (lanes 2-7). Lane 1, δTCAR-3 immunoprecipitates ₇ , TCR heterodimer with the CD3 proteins. Lane 2, δTCAR-3 immunoprecipitates ₇ , TCR heterodimer without the CD3 proteins. Lanes 3 and 4, δTCAR-3 immunoprecipitates single δ chain from denatured lysates (N) and reducing (R) conditions, respectively. Lane 5, UCHT-1 immunoprecipitates the CD3 proteins. Lane 6, β ^~ ? antibody does not immunoprecipitate a heterodimer from MOLT-13 cells. Lane 7, anti-C ₇ antiserum immunoprecipitates a single ₇ chain.

Figure 8. Analysis of cell surface staining by flow cytometry. ₇ ,δTCR-positive cells (MOLT-13) PEER, IDP2) and α.βTCR-positive cells (HPB-ALL, Jurkat) were incubated with δTCAR-3, OKT3, WT31 and normal mouse serum (NMS) antibodies and analyzed by flow cytometry. The B cell line, Daudi, was the negative control.

Figure 9. Two color cytofluorographic analysis of δTCAR-3 and 0KT3 peripheral blood lymphocytes. The fluorescein isothiocyanate (FITC) fluorescence is depicted on the Y axis and phycoerythrin (PE) fluorescence on the X axis. The CD3 + ₇ ,δTCR+ cells i.n this sample represent 2.4% of CD3 lymphocytes. Figure 10. Measurement of mtracytoplasmic Ca 2+ concentration ([Ca 2+].) versus time. Top panel: δTCAR-3.

Bottom panel: Anti-Leu antibody. Arrows indicate the time of addition of antibody.

Figure 11. Immunoprecipitation of the three forms of ₇ ,δTCR. For parts A-E, the antibodies used for. immunoprecipitation are anti-Leu4 (anti-CD3) , SFl (anti-

TCRβ) , anti-δlTCR (anti-δTCR) , anti-C ₇ b serum (anti~ ₇ TCR) and P3 (unlabelled lanes, control) . Immunoprecipitations from 125I-labelled cell lysates were analyzed by SDS-PAGE

(10% polyacrylamide) under reducing (R) or nonreducing (N) conditions. An open arrow (£>) indicates the position of

TCR δ under reducing conditions, whereas the solid arrow

(►) denotes the position of δTCR under nonreducing conditions. Size markers, M in thousands, are shown on the left.

A) Nondisulfide-1inked ₇ TCR (40 kD) on PBL-L2. In lanes 1-6 the radiolabelled cells were solubilized in 0.3% CHAPS detergent which preserves the TCR-CD3 association, whereas in lanes 7 and 8, immunoprecipitations were performed after chain separation (see methods) .

B) Nondisulfide-1inked ₇ TCR (55 kD) on IDP2 cells. In lanes 1-4 radiolabelled cells were solubilized in 0.3% CHAPS detergent, whereas in lanes 5 and 6 imunoprecipitations were carried out after chain separation.

C) Disulfide-linked ₇ TCR (40 kD) on WM-14 cells. All lanes correspond to immunoprecipitations from 1% digitonin solubilized radiolabelled cells.

D) Nondisulfide-linked ₇ TCR (40 kD) on thymic

Clone II cells. Radiolabelled cells were solubilized in 1% digitonin (lanes 1-4) or in 0.1% Triton X-100 (lanes 5 and 6), whereas in lanes 7 and 8 immunoprecipitations were carried out after chain separation.

E) Nondisulfide-linked ₇ TCR (40 kD) on MOLT-13 leukemia T cells. In lanes 1-4 immunoprecipitations were carried out after

solubilization of cells in 0.3% CHAPS detergent, whereas in lanes 5 and 6 immunoprecipitations were carried out after chain separation. Figure 12. Immunoprecipitation of ₇ TCR and δTCR chain by anti-Oγml antibody and anti-TCRδl antibody, respectively. Cell surface radiolabelled MOLT-13 cells were solubilized in 0.3% CHAPS detergent and the ₇ ,δTCR-CD3 complex was isolated with anti-CD3 monoclonal antibody. Immunoprecipitates were analyzed by 10% SDS-PAGE under reducing conditions.

Lane 1: Immunoprecipitation with anti-Leu4

(anti-CD3) mAb Lane 3: Immunoprecipitation with anti-C ₇ ml

(anti-TCR ₇ ) mAb after separating chains of isolated ₇ ,δTCR-CD3 complexes. Lane 4: Immunoprecipitation with anti-TCR δl

(anti-TCRδ) mAb after separating chains of isolated ₇ ,δTCR-CD3 complexes. Figure 13. Determination of peptide backbone sizes and glycosylation of ₇ and δTCRs from PEER and MOLT-13 cells. Monoclonal antibodies used for immunoprecipitation are anti-Oγml (anti-TCR ₇ ) , anti-TCRδl (anti-TCRδ) and P3 (labelled control) as shown at the top of each lane. The labelled cell lines used are shown at the bottom of each 10% SDS-PAGE autoradiograph or fluorograph. All samples were resolved under reducing conditions. Size markers, M in the thousands.

A) Peptide backbone sizes of ₇ TCR from PEER and

MOLT-13 cells. Cells were biosynthetically labelled with 35S-cystei.ne and 35S- methionine for 15 minutes. Samples were either treated with Endo ^' H (+) or mock treated (-) . Immunoprecipitation with anti-C ₇ irtl shows the positions of immature

₇ TCR of PEER cells (lane 3) and of MOLT-13 cells (lane 7) , while the corresponding polypeptide backbone sizes are visualized after treatment with endo H (lanes 4 and 8) . B) Glycosylation of TCR δ from MOLT-13 cells.

125 I-labelled cells were immunoprecipitated with anti-CD3 mAb and the δTCR polypeptides were gel purified (see methods) before incubation with N-glycanase (lane 4) , endo H

(lane 2) , or mock treated (lanes 1, and 3) .

Figure 14. Nucleotide sequence of MOLT-13 ₇ TCR

(Form 2bc) . Part A: Sequencing strategy of clone M13k. A partial restriction map of the 1.1 kb cDNA clone M13k is shown. Part B: Nucleotide and deduced amino acid sequence of clone M13k. Signal sequence (S) , variable (V) , N-region

(N) , joining (J) and constant (CI, Cllb, CIIc and CIII) region gene segments are indicated by arrows and were identified by comparison to genomic sequences, described by

Lefranc et al., (1986, Cell 45:237-246) (for S and V),

Lefranc et al., (1986, Nature, 319:420-422) and Quertermous et al., (1987, Immunol. 138:2687-2690) (for J) and Lefranc et al., (1986, Proc. Natl. Acad. Sci. U.S.A. 83:9596-9600) and Pellicci et al. , (1987, Science 237:1051-1055) (for C) .

The deduced a ino acid sequence beginning at the initiator methionine is presented below the nucleotide sequence.

Extracellular cysteines are highlighted by boxes, and potential N-linked carbohydrate attachment sites (N-X-S or

N-X-T; Marshall, 1977, Ann. Rev. Biochem. 41:673-702) are indicated by brackets.

Figure 15. Preferential use of ₇ , TCR Form 1.

Freshly isolated peripheral blood ononuclear cells from three healthy donors were 125I-labelled and solubilized in

1% Triton X-100. Immunoprecipitates with P3 (control, lanes

1 and 3), and anti-TCRδl (anti-TCRδ, lanes 2 and 4), were analyzed under nonreducing (N) and reducing (R) conditions.

M_r markers in the thousands are shown on the left,

Figure 16. Schematic representation of the three ₇ , TCR forms in man. The CII exon encoded connector peptides are highlighted by filled areas ( Bfl ) as C ₇ I CII exon encoded peptide; E222 , I∑ώU , SB , as C ₇ 2 CII exon copy a, copy b, and copy c encoded peptides, respectively) . Potential N-linked glycan attachment sites (o) , and sulfhydryl groups (-SH) and putative disulfide bridges (-S- S-) are indicated.

Figure 17. Map of the rearranged δTCR gene. A map of rll9δl including EcoRI (RI) , Hinc II (He) , Seal (S) and PvuII (P) sites and probes used in Southern blot analysis is shown.

Figure 18A. Schematic representation of the ₇ TCR chains used for transfection into MOLT-13 cell line. The schematic is based on reported analyses (Brenner, M.B., et al., 1986, Nature 322:145-149; Brenner, M.B., et al., 1987, Nature 325:689-694; Krangel, M.S., et al., 1987, Science 237:64-67). Pred., predicted; Obs., observed. The predicted glycosylated polypeptide size assumes that all available N-linked glycosylation sites (shown as lollipops) , each containing 3 kD of attached carbohydrate, are used, and that no significant size differences are introduced by other post-translational modifications. The intra-chain disulfide linkages typical of Ig-like molecules are shown. Note that a cysteine residue (cys) is encoded by the CII exon in PBL Cl ₇ TCR, but such a cysteine is absent from all the copies of CII exon used in the two other ₇ TCR chains. ₇ TCR constant region in the ₇ ,δ T leukemia cell line PEER (Littman, D.R. , et al., 1987, Nature 326:85-88) that also expresses a 55 kD ₇ TCR protein (Brenner, M.B., et al., 1987, Nature 325:689-694; Weiss, A, et al., 1986, Proc. Natl. Acad. Sci. USA 83:6998-7002) is identical to that of IDP2.

Figure 18B. The expression plasmid constructs pFneo.PBL CI ₇ and pFneo.IDP2 ₇ were used to introduce ₇ TCR clones into the MOLT-13 cell line. PBL Cl ₇ TCR cDNA clone (PBL C1.15) and repaired IDP2 ₇ TCR cDNA clone (IDP2.11r) (Krangel, M.S., et al., 1987, Science 237:64-67) were cleaved from their parent plasmid vector (pUC 18) by EcoRI digestion, the ends were made blunt with Klenow fragment of DNA polymerase I, and the cDNAs were then ligated into a Sail-cut, and Klenow-treated pFneo mammalian expression vector. Clones containing the cDNA inserts in appropriate orientation with respect to the spleen focus forming virus (SFFV) LTR were selected based on restriction mapping. pFneo (Saito, T. , et al., 1987, Nature 325:125-130) is a derivative of pT^Fneo (Ohashi, P., et al., 1985, Nature 316:606-609) obtained by BamHI digestion, to delete the murine TCR cDNA insert, followed by ligation with T4 DNA ligase. As shown, this vector contains a bacterial neomycin resistance gene (neo ) under the control of SV40 promoter, thus conferring resistance to the antibiotic G418 on the mammalian recipient cells. The restriction sites within parentheses were destroyed during construction.

Figure 19. Immunoprecipitation analysis of ₇ ,δTCR on Molt-13 ₇ TCR transfectants. Surface 125I-labeled cells were solubilized in 0.3% CHAPS detergent to preserve the chain association, immunoprecipitated with mAb P3

(control) , anti-leu-4 (anti-CD3) , anti-TCRδl (anti-δTCR) , or anti-Ti—yA (anti-V ₇ 2) , and were then resolved by SDS-PAGE under nonreducing (N) or reducing (R) conditions and visualized by autoradiography as described earlier (Brenner,

M.B., et al., 1986, Nature 322:145-149; Brenner, M.B., et al., 1987, Nature 325:689-694). Anti-Ti—yA mAb shows a pattern of reactivity on different T cell clones consistent with its recognition of V ₇ 2 segment. M13.PBL CI ₇ : MOLT-13 cells transfected with the PBL Cl-derived ₇ TCR cDNA; Clone

#7 was used for this analysis. MI3.IDP2 ₇ :- MOLT-13 cells

transfected with the IDP2-derived ₇ TCR cDNA; Clone #10 was used for this analysis. Size markers, M (molecular weight) in thousands of daltons. Open arrow, resident MOLT-13 ₇ TCR chain; solid arrow, transfected (PBL Cl- or IDP2-derived)

5 ₇ TCR cDNA; asterisk, MOLT-13 δTCR chain under nonreducing conditions. Upon reduction, the δTCR chain undergoes a mobility shift and comigrates with the 40 kD ₇ TCR chain.

However, δTCR chain is distinctly visualized as a 40 kD band under reducing conditions when the 40 kD ₇ TCR protein is not

10 coimmunoprecipitated, as is seen in anti-V ₇ 2 immunoprecipitates of M13.IDP2 ₇ (Fig. 19, lane 8).

Figure 20. Two-dimensional gel analysis of ₇ TCR

7 polypeptides of transfectants. 2x10 cells were surface

125 I labeled, treated with neuraminidase (150 units in 1.5

15 ml PBS with 1 mg/ml each of glucose and bovine serum albumin for 1.5 hours at 23°C) , solubilized in 0.3% CHAPS detergent, immunoprecipitated with 1 μg anti-leu-4 mAb, and subjected to 2D gel analysis under reducing conditions. Non- equilibrium pH gradient gel eletrophoresis (NEPHGE) was carried out using pH 3.5 to 10 Ampholines (LKB, Sweden)

20 followed by SDS-polyacrylamide gel electrophoresis on a 10.5% acryla ide gel (Brenner, M.B., et al., 1987, Nature 325:689-694). Positions of the CD3 components (not shown) were used to identify and compare the ₇ TCR species expressed in different cell lines. M13.PBL CI ₇ transfectant clone #10

25 and M13.IDP2 ₇ transfectant clone #10 were used. Open arrows, MOLT-13 ₇ TCR species; solid arrows, IDP2 ₇ TCR species; asterisk, PBL Cl ₇ TCR species.

Figure 21. Analysis of backbone polypeptide sizes of ₇ TCR chains of transfectants. Cells were pulse

30 labeled with 35S-methi.onine and 35S-cysteme for 15 minutes and immunoprecipitated with P3 (control) , anti-Oγml (anti-

₇ TCR) , or anti-Ti- ₇ A (anti-V ₇ 2) mAb, as indicated.

Immunoprecipitates were treated with endoglycosidase-H

„_ (Endo-H, +) , or were mock-incubated (-) , resolved by SDS-

PAGE, and visualized by fluorography. All samples were run reduced. M , molecular weight markers in thousands of daltons. The 43 kD contaminating actin band serves as an additional internal marker. MI3.IDP2 ₇ : MOLT-13 cells transfected with the IDP2 ₇ TCR cDNA, clone #10; M13.PBL CI7: MOLT-13 cells transfected with the PBL Cl ₇ TCR cDNA, clone #10.

Figure 22. Southern blot analysis of T cell clones and polyclonal human T cell populations. Genomic DNA was digested with EcoRI and probed with the V-J probe. DNA sources are: PBL T-cell clones, (lanes 1, 3-9) , PBL (lane 10) , newborn thymocytes (NBT-lane 11) , fetal thymocytes (FT-lane 12) , and B cells (germline-lane 2) . The germline 3 kb Vδ and 6.7 kb Jδ fragment are indicated on the left of the blot, while the 5 common rearrangements, numbered I-V are indicated on the right. The sizes of the rearrangements from I-V are 2.9 kb, 3.5 kb, 4.2 kb, 6.2 kb and 7.1 kb respectively.

5. DETAILED DESCRIPTION OF THE INVENTION

5.1. THE 7TCR FORM 2bc POLYPEPTIDE AND NUCLEIC ACIDS

The invention is directed to a form of the human 7TCR polypeptide termed Form 2bc (detailed infra in Sections 8 and 9) . The invention is also directed to nucleic acids encoding ₇ Form 2bc, such as DNA and RNA, and their complementary nucleic acids.

Form 1 and Form 2abc ₇ TCR polypeptides are previously reported forms of the human ₇ TCR (see PCT International Publication No. WO 88/00209, Published January 4, 1988). The Form 1 ₇ TCR polypeptide has a molecular weight of about 40,000 daltons. The Form 2abc ₇ TCR polypeptide has a molecular weight of about 55,000 daltons. Form 2abc ₇ TCR chain has a slightly larger peptide backbone and contains one extra potential N-linked glycan than Form

1. In contrast, the ₇ TCR chain of the invention, Form 2bc, has a molecular weight of about 40,000 daltons. Furthermore, the Form 2bc ₇ TCR polypeptide possesses a slightly smaller peptide backbone and 2-3 less N-linked glycans.

₇ TCR chain Form 2bc differs in size by more than 15 kD (40 kD versus 55 kD) compared to the previously described Form 2abc. This difference is accounted for by a 5 kD smaller polypeptide backbone size (35 kD versus 40 kD) and by a reduction in the amount of carbohydrates (5 kD versus 15 kD) . The approximately 35 kD polypeptide backbone size of Form 2bc also serves to distinguish it from Form 1; Form 1 has a 40 kD backbone size.

₇ TCR polypeptide Form 2bc also differs from Form 1 and Form 2abc in constant region (Oγ) gene segment usage. Form 1 ₇ TCR chains have a constant region encoded by the C ₇ I gene segment (Krangel et al., 1987, Science 237:64-67) containing a single CII exon. The Form 2abc ₇ polypeptide is encoded by Oy2 gene segments containing three CII exon copies, namely copy a, copy b and copy c (Krangel et al., 1987, Science 237:64-67; Littman et al., 1987, Nature 326:85-88) . In contrast. Form 2bc lacks one copy of the sequence encoded by the Oγ2 second exon that is present in the cDNA of Form 2abc. This, Form 2bc contains two γ2 CII exon copies, namely copy b and copy c. Copy a of CII, which is missing in Form 2bc, encodes a part of a connector region between the membrane spanning region and the extracellular constant domain.

Six potential N-linked carbohydrate attachment sites exist on the Form 2bc polypeptide. Since the biochemical data suggest that only 2-3 N-linked glycans are attached to the polypeptide chain, it indicates that not all potential sites are used.

In specific embodiments, ₇ TCR polypeptide Form 2bc can be obtained from cells of the MOLT-13 (Loh et al., 1987, Nature 330:569-572) T cell line or thymus-derived Clone II (Bank et al., 1986, Nature 322:179-181). ₇ TCR chain Form 2bc can also be obtained from T lymphocytes of a human subject which express that ₇ TCR form.

Form 2bc ₇ TCR polypeptide comprises the primary amino acid sequence of the ₇ TCR polypeptide shown in Figure 14, or any portion thereof comprising a constant region consisting of copy b and copy c of γ2 CII.

The present invention also provides a nucleic acid molecule encoding a ₇ TCR Form 2bc polypeptide having a molecular weight of about 40,000 daltons. The constant region of ₇ TCR Form 2bc polypeptide results from translation of a nucleic acid sequence which has only two of the three Oy2 ell exons. The invention is also directed to nucleic acid sequences comprising a Oγ2 constant region having only two ell exons. The nucleic acid can be a DNA, cDNA, RNA, and complementary nucleic acids and derivatives thereof. In a specific embodiment of the invention, the DNA molecule comprises at least a portion of the nucleic acid sequence shown in Figure 14.

In an example to be discussed in Section 8, the 2bc ₇ TCR polypeptide and its encoding nucleic acid sequence are described. In an example to be discussed in Section 9, it is shown that the ability of the ₇ TCR polypeptide to form disulfide bonds or be glycosylated is determined by its constant region primary sequence.

5.2. POLYPEPTIDE COMPLEXES CONTAINING 7TCR FORM 2bc

The present invention also relates to polypeptide complexes which comprise the ₇ TCR chain Form 2bc. In a specific embodiment, the polypeptide complex consists of a T cell antigen receptor dimer. In particular, such a di er can be a heterodimer (including but not limited to a ₇ ,δ

heterodimer, a η, β heterodimer, and a , ₇ heterodimer, or a

₇ , ₇ ' heterodimer in which - _. ' can be ₇ TCR polypeptide Form 1,

2abc, or 2bc) , or a homodimer.

In a particular embodiment of the invention, the polypeptide complex comprising ₇ TCR Form 2bc is a ₇ δTCR heterodimer. Thus, a purified complex which comprises at least a portion of a δTCR polypeptide and ₇ TCR Form 2bc polypeptide is provided by the present invention. The δ polypeptide may have at least one intrachain, covalent, disulphide bridge. Additionally, the polypeptide may comprise a δTCR polypeptide having a molecular weight of about 40,000 daltons.

As detailed in the examples infra, the ₇ TCR Form

2bc chain is noncovalently associated in a complex with the δTCR chain. Thus, ₇ Form 2bc forms a nondisulfide-linked

TCR complex. ₇ TCR chain Form 2abc also forms a nondisulfide-linked complex with a δTCR chain (e.g., on IDP2 cells) , while ₇ TCR chain Form 1 forms a disulfide-linked complex with a δTCR polypeptide.

As shown in the example of Section 9, infra, ₇ TCR constant region CII exon usage (and thus the primary sequence of the ₇ TCR chain) determines not only the presence or absence of disulfide linkage between TCR 7 and δ, but also the amount of carbohydrate attached to ₇ TCR, which is largely responsible for the differences in size of the cell surface ₇ TCR proteins. Thus, the present invention also provides a method for producing expression of ₇ δTCR heterodimers of defined intermolecular linkage (disulfide or nondisulfide-linked) and extent of ₇ TCR glycosylation, which comprises introducing a ₇ TCR gene encoding a particular ₇ polypeptide form into a cell capable of expressing the ₇ gene, which cell expresses the δTCR chain.

The present invention further provides a purified complex which comprises a ₇ TCR Form 2bc polypeptide of the present invention associated with another ₇ TCR polypeptide

(e.g.. Form 1, 2abc, or 2bc) . In one embodiment of the invention, the two ₇ TCR polypeptides are associated with each other through at least one interchain, covalent, disulfide linkage. In another embodiment of the invention, the two ₇ TCR polypeptides are noncovalently associated with each other. In still another embodiment of the invention, the two ₇ TCR polypeptides have the same constant domain. In yet a further embodiment of the invention, the two ₇ TCR polypeptides have different constant domains.

5.3. MONOCLONAL ANTIBODIES REACTIVE WITH THE 7STCR POLYPEPTIDES

A monoclonal antibody (mAb) to an epitope of the ₇ or δ T cell antigen receptor can be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497), and the more recent human B cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72) and EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77- 96) .

In one embodiment, the monoclonal antibodies may be human monoclonal antibodes or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g. , Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; Olsson et al. , 1982, Meth. Enzymol. 92:3-16). Chimeric antibody molecules may be prepared containing a mouse (or rat, or other species) antigen-binding domain with human constant regions (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851; Takeda et al., 1985, Nature 314:452) .

The invention is also directed to a method of identifying a monoclonal antibody reactive with a T cell antigen receptor. Such a mAb can be identified by detecting its ability to comodulate the CD3 antigen upon binding of the mAb to a cell which expresses both a T cell antigen receptor and CD3 complex. The CD3 comodulation can be detected, for example, by measuring the amount of labeled anti-CD3 antibody which is bound by the cell. This method is illustrated by way of example in Section 7.1.1, infra, in which it is used to identify hybridomas secreting anti-V mAb δTCAR-3.

A molecular clone of an antibody to an epitope of a ₇ or δTCR polypeptide can be prepared by known techniques. Recombinant DNA methodology (see e.g. , Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) may be used to construct nucleic acid sequences which encode a monoclonal antibody molecule, or antigen binding region thereof.

Antibody molecules may be purified by known techniques, e.g. , immunoabsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography) , or a combination thereof, etc.

Antibody fragments which contain the idiotype of the molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')_ fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') ~ fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.

One embodiment of the invention is directed to monoclonal antibodies reactive with the variable region of the δTCR chain. Such an antibody is δTCAR-3 (aka TCSδl) (see Section 7, infra) , which recognizes an epitope expressed 5 from a specific δ gene rearrangement. As described in Section 7.2, infra, mAb δTCAR-3 is capable of stimulating the proliferation of a ₇ ,δ T lymphocyte. Monoclonal antibody δTCAR-3 is also able to stimulate a rise in cytoplasmic free calcium ion concentration of ₇ ,δ T -. _Q lymphocytes.

In another embodiment, the invention relates to antibodies reactive with the constant region of the ₇ or δTCR polypeptide. In a specific embodiment, the invention is directed to mAb TCRδl which is reactive with the constant

15 region of the δTCR chain (see Section 6, infra) . In another specific embodiment, the invention relates to mAb anti-Oγml, which is reactive with the constant region of the ₇ TCR chain (see Section 8.1.7, infra).

6. GENERATION OF MONOCLONAL ANTIBODY

20 ANTI-TCRδl SPECIFICALLY REACTIVE WITH THE TCR DELTA SUBUNIT CONSTANT REGION

6.1. EXPERIMENTAL PROCEDURES

25 6.1.1. CYTOFLUOROGRAPHIC ANALYSIS

OF T CELL LINES WITH ANTI-TCRδl

The anti-TCRδl mAb, which is specifically reactive with the δTCR chain constant region, was made as follows: One gram of PEER cells were solubilized in 50 ml of 0.3% CHAPS (3-[3-cholamidopropyl)dimethyl-ammonio]l- propanesulfonate) detergent and were immunoprecipitated with

1 μl of UCHT1 (Beverley, P.C., and Callard, 1981, Eur. J.

Immunol. 11:329-334) ascites, 500 μl of mAb 187.1 culture supernatant and Staphylococcus aureus Cowan I strain (SACI) .

₃₅ Four intraperitoneal injections at six week intervals were

carried out, followed by a final boost of ₇ δTCR (without CD3) isolated by selective elution of ₇ δTCR from the immune complexes using 2% Triton X-100. The eluted material was administered both intravenously and mtraperitoneally; four days after this boost, the mice were sacrificed and fusion carried out as previously described (Brenner, M.B., et al., 1987, J. Immunol. 138:1502-1509).

₇ δTCR cell lines PEER and IDP2 or αSTCR cell lines HPB-MLT and JURKAT were stained with 50 μl of anti- TCRδl culture supernatant followed by staining with FITC- conjugated goat anti-mouse Ig F(ab) ' fragments with analysis on an Ortho cytofluorograph (see Fig. 1) . Control was the mAb secreted by P3X63.Ag8 hybridoma (P3) and anti- CD3 mAb was anti-Leu 4 (Ledbetter, J.A., et al., 1981, J. Exp. Med. 153:310) .

6.1.2. IMMUNOCHEMICAL ANALYSIS OF THE

SPECIFICITY OF mAb anti-TCRδl Surface 125I-labeled IDP2 cells were solubilized and their proteins immunoprecipitated using control mAb P3, anti-Leu 4, anti-TCRδl, or anti-Oγ serum. Precipitated samples were analyzed by SDS-PAGE followed by autoradiography. In CHAPS detergent, ₇ δTCR and CD3 remained associated and were immunoprecipitated as a complex by anti-Leu 4 (Fig. 2, lanes 3 and 4). However, after solubilization in 2% Triton X-100 detergent, anti-TCRδl immunoprecipitated ₇ δTCR as a dimeric complex without CD3

(lane 6) and anti-Leu 4 immunoprecipitated CD3 as a trimeric complex without ₇ δTCR (lane 5) . After separation of the

₇ δTCR-CD3 component chains, anti-TCRδl immunoprecipitated

TCRδ alone (lane 7 and 8) , while anti-Oγ serum immunoprecipitated TCR ₇ alone (lane 9) . For chain separation experiments (lane 7-9), anti-Leu 4 immunoprecipitates from CHAPS solubilized IDP2 cells were boiled in 1% SDS and were then diluted with 4 volumes of 2%

Triton X-100 followed by immunoprecipitation with anti-TCRδl or anti-Oγ serum. This follows procedures used previously (Brenner, M.B., et al., 1986, Nature 322:145).

6.1.3. N-LINKED GLYCOSYLATION OF THE TCR δ POLYPEPTIDE

125 I-labeled IDP2 cells were solubilized in 0.3%

CHAPS, immunoprecipitated with anti-Leu 4 and resolved by

SDS-PAGE (Fig. 3) . Control lane is mock-digested IDP2 δTCR polypeptide. N-glycanase digestion of δTCR polypeptide was performed as follows: δTCR was eluted from a gel slice followed by N-glycanase (Genzyme Corp.) digestion (10 U/ml) carried out in 30 μl 0.17% SDS, 1.25% Nonidet P-40, 0.2 M sodium phosphate buffer pH 8.6 for 16 hours at 37°C

(Tarentino, A.L., et al., 1985, Biochemistry 24:4665). The digested or mock-incubated δTCR samples were analyzed by

SDS-PAGE and visualized by autoradiography.

6.1.4. RECOGNITION OF IN VITRO TRANSLATION PRODUCTS OF CDNA CLONE IDP2O-240/38 BY mAb ANTI-TCRδl

A plasmid designated pGEM3-O-240/38 was constructed as follows and used for in vitro transcription- translation (Fig. 4) . The IDP2 0-240/38 (δTCR) cDNA clone 1.5 kb insert begins within codon 7 of the composite Group O sequence and includes the remaining coding region and most of the 3' untranslated region. This insert was cleaved as a single EcoRI fragment from λgtlO arms by partial EcoRI digestion (to prevent cleavage of the internal EcoRI site) . This fragment was subcloned into a Bluescript+ vector (Stratagene) . The insert was then removed from the vector as a single BamHI-Sall fragment (ends are from the Bluescript vector polylinker) facilitating directional cloning into pGEM-3 (Promega Biotech) downstream of the T7 promoter. The resultant pGEM3-O-240/38 plasmid was linearized with Sail and capped transcripts synthesized using T7 RNA polymerase (Krangel, M.S., et al., 1987,

Science 237:64). Integrity and size of the transcripts were monitored via an aliquot of the reaction mixture containing

32 P-ATP. A single RNA species of 1.5 kb was observed. In vitro translation in the presence of 35S-methionme was performed in a rabbit reticulocyte lystate. After in vitro translation, the samples were boiled in 1% SDS with 2 mM dithiothreitol followed by the addition of 10 volumes of 2%

Triton X-100 in Tris buffered saline pH 7.5. Samples were immunoprecipitated with control mAb P3 (Fig. 5, lanes 1 and

3) or with anti-TCRδl mAb (lanes 2 and 4) and analyzed by

SDS-PAGE followed by fluorography (Bonner, W.J. and Laskey,

R.A., 1974, Eur. J. Biochem. 46:83-88).

6.2. EXPERIMENTAL RESULTS We have generated a monoclonal antibody (mAb) , anti-TCRδl, that is specifically reactive with the δTCR constant region.

The ₇ δTCR-CD3 complex from the PEER cell line (Weiss, A., et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:6998-7002; Brenner, M.B., et al., 1987, Nature 325:689- 694) was used as immunogen in the production of antibody- secreting hybridoma cell lines. Hybridomas were screened both by cell surface binding (cytofluorographic analysis) and by immunoprecipitation of PEER cell proteins followed by SDS-PAGE analysis. Two hybridoma supernatants (5A6 and 4A1) bound to the surface of PEER cells. After subcloning, one mAb (5A6.E9) was characterized further. This mAb bound to the surface of ₇ δTCR lymphocytes (PEER, IDP2) but failed to react with αySTCR cells (HPB-MLT, JURKAT) or with non-T leukocytes (Fig. 1 and data not shown) . Although the immunogen was composed of a complex of ₇ δTCR and CD3, the greater affinity of the mAb for ₇ δTCR cell lines suggested the mAb was not directed against CD3 determinants.

The specificity of the mAb was determined in immunoprecipitation studies using various detergents which affect the association of the proteins comprising the receptor complex. After 125I-labeled IDP2 cells were solubilized in CHAPS detergent, TCR ₇ and δ, and CD3 ₇ ,δ, and e subunits remained part of an associated complex immunoprecipitated by anti-CD3 antibody (Fig. 2, lanes 3,

4) . However, if radiolabeled IDP2 cells were solubilized in

2% Triton X-100 detergent, ₇ δTCR and CD3 became largely dissociated, and the use of anti-CD3 mAb resulted in selective precipitation of CD3 (Fig. 2, lane 5) . Under these latter conditions, mAb 5A6.E9 immunoprecipitated ₇ TCR as a heterodimer without associated CD3 (Fig. 2, lane 6).

This observation provided the first direct evidence that

TCR ₇ and TCR δ exist as a non-disulfide-linked heterodimer.

To determine whether mAb 5A6.E9 reacts with a ₇ TCR chain, δTCR chain or a combinatorial determinant, immunoprecipitation of separated polypeptide chains was performed. An anti-Leu 4 immunoprecipitate from radiolabeled, CHAPS-solubilized IDP2 cells was boiled in 1%

SDS to dissociate the ₇ TCR, δTCR, and CD3 proteins. After dilution with four volumes of 2% Triton X-100, mAb 5A6.E9 specifically immunoprecipitated the 40 kD (δTCR) species

(Fig. 2, lane 7). When an aliquot of the same immunoprecipitate was analyzed under reducing conditions

(Fig. 2, lane 8), a dramatic shift in SDS-PAGE mobility was observed. This phenomenon is characteristic of δTCR from the IDP2 and PEER cell lines (Brenner, M.B. , et al., 1987, Nature 325:689-694). In contrast, when the separated chains were immunoprecipitated with anti-Oγ sera, the 55 kD species ( ₇ TCR) , but not the 40 kD species (δTCR) was immunoprecipitated (Fig. 2, lane 9). Based on these biochemical and surface binding studies, mAb 5A6.E9 is referred to as anti-TCRδl.

In addition to PEER and IDP2, anti-TCRδl also immunoprecipitated TCR δ from other ₇ δTCR cell lines including MOLT-13 and PBL line 2. Further experiments have shown that anti-TCRδl reacts with a determinant encoded by a TCR δ constant (C) gene segment.

We have isolated cDNA clones from the IDP2 cell line (e.g., IDP2 O-240/38) by the subtractive approach representing a gene which encodes the TCR δ subunit. Genes to which IDP2 group 0 cDNA clones hybridize in Southern blotting experiments are expressed and rearranged in ₇ δTCR lymphocytes but are typically not expressed (and are often deleted) in βTCR cells. By sequence comparison with other TCR genes, these cDNA clones appear to be composed of novel V, D (?) , J, and C gene segments. The IDP2 Group O composite DNA sequence contains a long open reading frame predicting a polypeptide with two potential asparagine- linked glycosylation sites and a molecular weight of 31.3 kilodaltons. To determine the molecular weight of the unglycosylated δTCR protein and the number of asparagine- 1inked carbohydrates that are present on the mature IDP2 δTCR polypeptide, gel purified δTCR was either treated with N-glycanase or mock-incubated and analyzed by SDS-PAGE (Fig. 3) . Removal of N-linked carbohydrates resulted in a 5 kD decrease in apparent molecular weight (40 kD to 35 kD) , suggesting the presence of two (2.5-3 kD) N-linked glycans on the IDP2 δTCR. This correlates well with the number of N-linked glycans predicted by the translated amino acid sequence in Figure 5. The apparent molecular weight of the protein is in general agreement, differing from that predicted by 3.7 kD.

Given the reactivity of anti-TCRδl on IDP2 cells, the specificity for the δTCR polypeptide, and the recognition of partially denatured (SDS boiled) δTCR, we tested whether this mAb would recognize directly polypeptide encoded by the δTCR cDNA clone. Thus, the insert from cDNA

clone IDP2 0-240/38 was subcloned into the pGEM-3 expression vector downstream of the T7 promoter (Fig. 4) . Transcripts generated in vitro with T7 RNA polymerase were then used in a rabbit reticulocyte lysate system to direct the synthesis of protein in the presence of 35S-methionine. Following in vitro transcription-translation, the reaction mixtures were boiled in 1% SDS, diluted with ten volumes of 2% Triton X-

100, and then immunoprecipitated with either an isotype- matched control mAb or with anti-TCRδl. Anti-TCRδl mAb specifically immunoprecipitated a predominant species (34 kD) (Fig. 5, lane 4) . No such band was observed in immunoprecipitates when control mAbs were used (lane 3) , when RNA transcripts were omitted (lanes 1 and 2) , or when

₇ TCR constructs were used. Thus, the radiolabeled species immunoprecipitated by mAb anti-TCRδl corresponds to a δ polypeptide whose synthesis was specifically directed by the

IDP2 0-240/38 cDNA clone. This polypeptide (34 kD) is very similar in size to the N-glycanase treated IDP2 δTCR chain

(35 kD) . The IDP2 0-240/38 clone lacks a natural ATG initiation codon as well as the leader sequence. There are two potential internal ATG codons (at residues 12 and 44) within the V region of this clone (Fig. 5) . Use of these codons to initiate synthesis could result in more than one polypeptide species possibly accounting for the minor species noted (Fig. 5, lane 4). Thus, there is direct serological recognition by mAb anti-TCRδl of the IDP2 δTCR subunit encoded by clone IDP2 0-240/38.

GENERATION OF MONOCLONAL ANTIBODY δTCAR-3 SPECIFICALLY REACTIVE WITH THE TCR DELTA SUBUNIT VARIABLE REGION

7.1. EXPERIMENTAL PROCEDURES

7.1.1. IMMUNOPRECIPITATION AND SDS-PAGE ANALYSIS OF T CELL ANTIGEN RECEPTOR

The δTCAR-3 mAb, specifically reactive with the variable region of the δTCR chain, was generated as follows: One mouse was immunized with 2 x 10 Molt-13 cells by intraperitoneal injection. One month later, the mouse was boosted with 1 x 10 7 Molt-13 cells by i.ntravenous i.nj.ecti.on each day for 3 sequential days, and then immune splenocytes were fused with mouse myeloma P3x63Ag8.653 cells in the presence of 50%. polyethylene glycol 1500. The hybridomas were screened by analyzing the CD3 co-modulation with flow cytometry. The analysis of CD3 co-modulation was based on the observation that antibody to ,β T cell antigen receptor, when incubated with the cells, caused the internalization of the CD3 complex (Lanier, L.L., et al., 1986, J. Immunol. 137:2286; Meuer, S.C., et al., 1983, J. Exp. Med. 157:705) .

Molt-13, PEER, and HPB-ALL cell lines were lodmated using the lactoperoxidase technique. The 125I- F1 labeled cells were solubilized in Tris-buffered saline (pH 8) containing 1% Triton X-100. Lysates were immunoprecipitated using δTCAR-3 antibody or βΕ antibody. βYl is a framework monoclonal antibody to the /3TCR chain and is described elsewhere (Brenner, M.B., et al., 1987, J. Immunol. 138:1502-1509). All samples were analyzed by SDS- PAGE under reducing or non-reducing conditions (Fig. 6) . Molt-13 and PEER are both CD3 ⁺ 4~8 ^" WT31~. HPB is CD3 ⁺ 4 ⁺ 8 ⁺ WT31 ⁺ .

As shown in Figure 6, δTCAR-3 immunoprecipitated non-disulfide-linked ₇ and δ chains from Molt-13 and PEER cells, while F1 immunoprecipitated disul ide-linked and β chains from HPB-ALL cells. The difference in autoradiographic intensity between the bands corresponding to the δ and ₇ chains represents differences in the extent of iodination of these two proteins.

7.1.2. IMMUNOPRECIPITATION OF δTCR CHAIN BY δTCAR-3 ANTIBODY

Figure 7 shows I-labeled Molt-13 cells solubilized in Tris-buffered saline (pH 8) containing 0.3%

CHAPS (3-[(3-cholamidopropyl)dimethylammoni]1- propanesulfonate) or in 1% Triton X-100. In 1% Triton X-

100, the ₇ δTCR dissociates from the CD3 complex, while in

0.3% CHAPS, the ₇ δTCR remains associated with the CD3 complex. Prior to immunoprecipitation, the 125I-labeled lysates used in lanes 3, 4, and 7 of Figure 7 were denatured by adding SDS to a final concentration of 1% followed by heating for 5 minutes at 68 ^β C. After cooling, iodoacetamide was added to a final concentration of 20 mM. The mixture was then diluted with 4 volumes of 1.5% Triton X-100 in Tris-buffered saline (pH 8). ^" This denaturing process completely dissociates ₇ chain, δ chain, and CD3 proteins from one another. All samples were analyzed by SDS-PAGE under non-reducing conditions (N) except for the sample in lane 4 which is under reducing conditions (R) . Note the difference in mobility of δ chain under reducing and non¬ reducing conditions. The anti-Oγ antiserum was generated by immunizing a rabbit with a 20 amino acid synthetic peptide from the ₇ constant region (residues 117-136) .

7.1.3. ANALYSIS OF CELL SURFACE STAINING BY FLOW CYTOMETRY 5 X 10 cells were incubated with the appropriate antibodies (NMS (normal mouse serum) , δTCAR-3, OKT3, or WT31) at 4°C for 30 minutes and then washed two times with 0.2% BSA in PBS (pH 7.4). Following incubation with fluorescein-conjugated goat anti-mouse IgG for 30 minutes at 4°C, cells were analyzed on an Ortho cytofluorograph (Fig. 8).

7.1.4. TWO COLOR CYTOFLUOROGRAPHIC ANALYSIS OF δTCAR-3 ⁺ AND OKT3 PERIPHERAL BLOOD LYMPHOCYTES

The peripheral blood lymphocytes were first incubated with δTCAR-3 at 4°C for 30 minutes. After washing, cells were incubated with phycoerythrin (PE)- conjugated goat anti-mouse IgG for an additional 30 minutes at 4°C. After washing, the cells were incubated with fluorescein (FITC)-conjugated OKT3 for 30 minutes at 4°C and then cells were analyzed on an Ortho cytofluorograph (Fig.

9).

7.1.5. MEASUREMENT OF INTRACYTOPLASMIC Ca ²⁺

CONCENTRATION (TC " 1^ VERSUS TIME

Molt-13 cells were labeled with the acetoxymethyl ester form of the Ca 2+-sensitive probe fura-2 (2 μM from a 1 mM stock in dimethyl sulfoxide, Molecular Probes, Eugene,

7 Oregon) at a concentration of 10 cells/ml in RPMI 1640 plus

10% fetal bovine serum for 30 minutes at 37°C. Cells were

₇ then washed and resuspended at 10 cells/ml in Hanks balanced salt solution (HBSS) plus 5% fetal bovine serum and kept in the dark at room temperature until use. Immediately prior to fluorescent measurement, 2 x 10 cells were centrifuged then resuspended in 2 ml of fresh HBSS and placed in a quartz cuvette at 37°C and constantly stirred. Fluorescence was measured on the cell suspension in a SPF- 500C fluorometer (SLM Aminco, Urbana, Illinois) , the

excitation wavelength alternating between 340 (+2) and 380 (+2) nm and emission was detected at 510 (+5) nm. The ratio of 350/380 was automatically calculated (1 ratio every 2 seconds) , plotted, and stored in an IBM PC AT. Quantitation of [Ca ] . from the fluorescence ratio was performed as described by Grynkiewicz, et al. (1985, J. Biol. Chem.

260:3440). Addition of irrelevant antibodies did not alter

[Ca 2+]., while cell lysis resulted in a [Ca2+]. of 1 μM.

7.2. RESULTS We have generated a monoclonal antibody, δTCAR-3, that is directed against a variable region of the TCR δ chain and which can be used to characterize the δ polypeptide. This monoclonal antibody binds to T cells bearing the ₇ δTCR and also elicits a fura-2 Ca 2+ signal upon binding to Molt-13 cells.

The δTCAR-3 monoclonal antibody was generated by immunizing a mouse with the Molt-13 cell line which has a

CD3 4~8~WT31~ phenotype. The hybridomas were first screened by CD3 co-modulation. The positive clones were further screened by immunoprecipitation. δTCAR-3 immunoprecipitation of ₇ δTCR heterodimer from 125I-labeled

Molt-13 and PEER lysates is shown in Figure 6. δTCAR-3 does not immunoprecipitate any polypeptide from HPB-ALL (Fig. 6) .

In contrast, F1, a framework monoclonal antibody specific to the β chain (Brenner, M.B., et al., 1987, J. Immunol.

138:1502-1509), immunoprecipitates the aβ heterodimer from the HPB-ALL cell line (Fig. 6, lanes 10 and 12) . The immunoprecipitated ₇ δ receptor from both Molt-13 and PEER cells, when analyzed under either reducing or non-reducing conditions, displays a heterodimeric structure indicating a non-disulfide-linked ₇ δTCR in these two cell lines. There is a slight shift in mobility of the δ chain under reducing conditions relative to that observed under non-reducing conditions (Fig. 6, lanes 1 and 3, 5 and 7), a phenomenon

which has been noted previously in IDP2 and PEER cell lines (Brenner, M.B., et al., 1987, Nature 325:689-694), suggesting the existence of intrachain disulfide linkages. In order to demonstrate that the δTCAR-3 antibody recognizes a CD3-associated ₇ δTCR, immunoprecipitations were performed using 125I-labeled Molt-13 cell lysates solubilized in 0.3%

CHAPS detergent (Fig. 7, lane 1) . Under these conditions, the CD3 complex remains associated with the receptor, and both ₇ δ heterodimer and the CD3 complex are immunoprecipitated by δTCAR-3. However, when 125I-labeled lysates were solubilized in 1% Triton X-100 detergent which largely dissociates the CD3 complex from the ₇ receptor, only ₇ δ heterodimer is immunoprecipitated by δTCAR-3 (Fig.

7, lane 2). As a control, the anti-CD3 antibody, UCHT-1

(Beverley, P.C. and Callard, R.E., 1981, Eur. J. Immunol.

11:329-334) immunoprecipitates only the CD3 complex, but not the ₇ δ heterodimer (Fig. 7, lane 5).

The specificity of δTCAR-3 was further analyzed by using immunoprecipitations of denatured, 125I-labeled

Molt-13 lysates in which ₇ ,δTCR and CD3 proteins were completely dissociated. δTCAR-3 specifically immunoprecipitated the δ chain which has an apparent molecular weight of 38 kD under non-reducing conditions

(Fig. 7, lane 3) and 40 kD under reducing conditions (lane

4) . The anti-Oγ antiserum immunoprecipitated the ₇ chain with molecular weight 42 kD under reducing conditions (Fig.

7, lane 7). These data indicate that δTCAR-3 is δ chain specific. δTCAR-3 not only immunoprecipitates ₇ ,δTCR heterodimer from the PEER and Molt-13 cell lines, it also binds to the surface of these cell lines and to the IDP2 clone (Brenner, M. , et al., 1987, Nature 325:689-694). It does not bind to the α _/ 3TCR-bearing HPB-ALL and Jurkat cell lines (Fig. 8). In contrast, WT31 (Tax, W.J.M., et al., 1983, Nature 304:445-447), a framework monoclonal antibody

to the αSTCR, reacts with αySTCR-positive HPB-ALL and Jurkat cell lines, but not with ₇ δTCR-positive Molt-13, PEER, and IDP2 cells (Fig. 8) . When normal peripheral blood lymphocytes (PBL) were examined, a subpopulation (0.9-2.4%) of CD3 lymphocytes were positive with δTCAR-3 (Fig. 9) . When δTCAR-3, immobilized on tissue culture plates was used for culture of normal human PBL, it selectively stimulated the proliferation of the ₇ δTCR-positive subpopulation. After 45 days in culture, the ₇ δTCR subpopulation represented 96% of the total cell count.

Antibodies to the β T cell antigen receptor stimulate a rise in the cytoplasmic free calcium ion concentration [Ca 2+]. (Weiss, A., et al., 1986, Ann. Rev.

Immunol. 4:593). Incubation of Molt-13 cells with δTCAR-3 eli .ci.ted a rapi.d i.ncrease m. [Ca2+] . si.mi.lar to the response induced by anti-T3 antibodies (Fig. 10) . Moreover, δTCAR-3 similarly stimulated a Ca 2+ flux in PEER cells and in the

₇ δTCR-positive cell line generated from PBL as described above. We have also observed that incubation of Molt-13,

PEER, and IDP2 cells with δTCAR-3 causes the co-modulation of the CD3 protein complex.

Further characterization of the epitope specificity of mAb δTCAR-3 (also termed mAb TCSδl) is presented in Section 11.2.2, infra.

8. THREE FORMS OF THE HUMAN T CELL RECEPTOR ₇ δ: PREFERENTIAL USE OF ONE FORM IN SELECTED HEALTHY INDIVIDUALS

In the examples herein, the structure of a new form of the human T cell receptor ₇ δ ( ₇ δTCR) , consisting of a 40 kD TCR ₇ glycoprotein noncovalently associated with a TCR δ chain, is presented. The newly identified ₇ TCR glycoprotein, termed Form 2bc, differs in size by more than 15 kD (40 kD versus 55 kD) compared to the previously described nondisulfide-linked TCR ₇ form (Form 2abc) . This

difference is accounted for by a 5 kD smaller polypeptide backbone size (35 kD versus 40 kD) and by a reduction in the amount of carbohydrates (5 kD versus 15 kD) . Nucleotide sequence analysis of cDNA clones corresponding to Form 2bc revealed that Form 2bc cDNA clones lacked one copy of the constant region ( y2) second exon that is present in the cDNA of the other nondisulfide-linked TCR ₇ subunit (Form 2abc) . This CII exon copy encodes part of a connector region between the membrane spanning region and the extracellular constant domain. Since the number and localization of the potential N-linked carbohydrate attachment sites is the same in both nondisulfide-linked forms, we conclude that the connector region influences the amount of attached carbohydrates, probably by affecting the conformation of the protein. In contrast, the δTCR subunits of these ₇ δ CR forms show little variability in peptide backbone sizes or peptide mapping analyses.

We also examined the usage of the three forms of the ₇ δTCR complex in peripheral blood. Nearly exclusive use of the disulfide-linked form. Form 1, was observed in certain healthy subjects. In some individuals. Form 1 was expressed together with Form 2bc. Form 2abc was not identified in the subjects tested.

8.1. EXPERIMENTAL PROCEDURES

8.1.1. ANTIBODIES

Monoclonal antibodies used were anti-Leu4 (anti- CD3) (Ledbetter et al., 1981, J. Exp. Med. 153:310-323), F1 (anti-/3TCR) (Brenner et al., 1987, J. Immunol. 138:1502- 1509), anti-TCRδl (anti-δTCR) (described in Section 6, supra; reactive with the δTCR chain constant region) , P3 (control) (secreted by P3X63.Ag8; Koehler and Milstein, 1975, Nature 256:495-497), 187.1 (rat anti-mouse K light chain) (Yelton et al., 1981, Hybridoma 1:5-11), and WT31

(stains α TCR lymphocytes brightly) (Spits et al., 1985, J. Immunol. 135:1922-1928). Anti-Oγb peptide serum (anti—γTCR) was generated against a 22 amino acid synthetic peptide (Gln-Leu-Asp-Ala-Asp-Val-Ser-Pro-Lys-Pro-Thr-Ile-Phe-Leu- Pro-Ser-Ile-Ala-Glu-Thr-Lys-Cys) (PCT International

Publication No. WO 88/00209, published January 14, 1988).

8.1.2. CELL LINES

PEER (Weiss et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:6998-7002) and MOLT-13 (isolated by J. Minowada, Loh et al., 1987, Nature 330:569-572) are T leukemic cell lines. Umbilical cord blood derived clone WM-14 (Alarcon et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:3861-3865) and peripheral blood derived cell line IDP2 (Brenner et al., 1986, Nature 322:145-149; PCT International Publication No. WO 88/00209, published January 14, 1988) and thymus-derived Clone II (Bank et al., 1986, Nature 322:179-181) were cultured as described earlier. Peripheral blood derived cell line 2 (PBL-L2) was isolated by sorting peripheral blood isolated lymphocytes that did not stain with mAb WT31. The isolated cells were then expanded in vitro in RPMI 1640 medium supplemented with 10% (v/v) conditioned medium containing IL-2 and 10% (v/v) human serum, and stimulated every 3 weeks with irradiated autologous feeder cells.

8.1.3. IODINATION AND IMMUNOPRECIPITATION 7 2 x 10 cells were isolated by Ficoll-diatrizoate

(Organon Teknika Corp.) centrifugation and iodinated on ice in 0.5 ml of phosphate-buffered saline, pH 7.4 (PBS) containing 1 mM MgCl.., 5 mM glucose by adding 100 μg of lactoperoxidase (80-100 U/mg, Sigma) and 1 mCi of Na 125I

(New England Nuclear). Ten μl of a 0.03% hydrogen peroxide solution was added at 5 minute intervals over a reaction period of 30 minutes. Cells were solubilized overnight in detergent supplemented TBS (50 mM Tris-Base pH 7.6, 140 mM

NaCl) containing 1 mM phenyl ethylsulfonyl fluoride (PMSF, Sigma) and 8 mM iodoacetamide (IAA, Sigma) . As indicated, different detergents used in this study were 0.3% (w/v) 3- [ (3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate (CHAPS, Signma), 1% (w/v) digitonin (Aldrich) and Triton X- 100 (TX-100, Sigma) . After 20 minutes of centrifugation at 10,000 x g to remove insoluble material, detergent lysates were precleared by a 30 minute incubation with 4 μl of normal rabbit serum (NRS) and 400 μl of 187.1 hybridoma culture medium, followed by addition of 200 μl of a 10%

(w/v) cell suspension of fixed Staphylococcus aureus Cowan I (Pansorbin, Calbiochem) . After a one-hour incubation, Pansorbin was removed by centrifugation. Specific precipitations were carried out by adding 0.25 μl β¥l ascites, 1 μl 1 mg/ml anti-Leu4 or 0.25 μl P3 ascites, together with 150 μl of 187.1 culture supernatant to each sample, followed by a one-hour incubation. 100 μl of 10% (v/v) Protein A-Sepharose (Pharmacia) was added and the mixture was rocked for 1 hour at 4°C. Immunoprecipitates were washed five times with 0.1% (v/v) Triton X-100 containing TBS and analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970, Nature 227:680-685).

For immunoprecipitations with the anti-Oγb peptide serum, iodinated cells were solubilized in 1% (w/v) sodium dodecyl sulfate (SDS) containing TBS and then boiled for 3 minutes. After cooling, 5 volumes of 2% (v/v) Triton X-100 in TBS containing PMSF and IAA was added, together with 200 μl of a mixture of 1 mg/ml deoxyribonuclease (DNAse) and 0.5 mg/ml RNAse in 50 mM MgCl,. Preclearing and immunoprecipitations were performed as described above, omitting the addition of 187.1 mAb. Immunoprecipitates were washed in TBS containing 0.5% (v/v) TritonX~100, 0.5% (w/v) deoxycholate (DOC), 0.05% (w/v) SDS.

8.1.4. BIOSYNTHETIC LABELLING 4 x 10 exponentially growing cells were resuspended in 4 ml of methionine and cysteine-free RPMI

1640 (Select-Amine kit, Gibco) supplemented with 10% dialyzed fetal calf serum (FCS) and 20 mM Hepes. After a 30 minute starvation period at 37°C, 1 mCi of 35S-methionine and 1 Ci of S-cysteine were added, allowing a 15 minute labelling period. Cells were harvested and solubilized in

2% (v/v) Triton X-100, TBS. Preclearing and immunoprecipitations were performed as described above. The immunoprecipitates were washed four times in 0.5% (v/v)

Triton X-100, 0.5% (w/v) deoxycholic acid, 0.05% (w/v) SDS,

TBS followed by three washes in 0.5% (v/v) Triton X-100, 0.5

M NaCl, 5 mM EDTA, 50 mM Tris, pH 7.6. The samples were analyzed by SDS-PAGE and visualized by standard fluorography procedures (Bonner and Laskey, 1974, Eur. J. Biochem.

46:83-88) .

8.1.5. GEL PURIFICATION OF δTCR PROTEINS Surface iodinated cells were solubilized in 0.3% (w/v) CHAPS-TBS and immunoprecipitated using 50 μl of anti- Leu4-coupled Sepharose beads. The immunoprecipitated species were resolved by SDS-PAGE under nonreducing conditions and the wet gel was exposed for 24 hours at 4°C on XAR-5 film (Kodak) to visualize radiolabelled δTCR proteins. The gel regions corresponding to δTCR were excised, incubated in 5% (v/v) 2-mercaptoethanol containing sample buffer and resolved a second time by SDS-PAGE. Because of the characteristic SDS-PAGE mobility shift upon reduction, δTCR protein could be separated and then purified from contaminants. TCR proteins were eluted from gel slices by overnight incubation in 0.05% (w/v) SDS, 50 mM ammonium bicarbonate buffer at 37°C and lyophilized.

8.1.6. ENDOGLYCOSIDASE DIGESTION For endoglycosidase H (Endo H) digestions, immunoprecipitated material or gel purified protein was boiled for 3 minutes in a 40 μl 1% (w/v) SDS solution containing 0.14 M 2-mercaptoethanol. After cooling, the mixture was diluted with 360 μl of 0.15 M acetate buffer, pH 5.5 containing 1 mM PMSF. Five μl Endo H (1 U/ml- Endo- - N-acetylglucosaminidase H, Genzyme) was incubated with half of the above solution for 14 hours at 37 "C, while the other half was mock treated.

For N-glycanase (N-GLY) digestion, gel purified material was boiled for 3 minutes in 35 μl of 0.5% (w/v) SDS, 0.10 M 2-mercaptoethanol. Then, 100 μl of 0.2 M sodium phosphate (pH 8.6), 1.25% (v/v) Triton X-100 was added. Half of the mixture was incubated with 1 μl N-Glycanase (250 U/ml, peptide-N-[N-acetyl- -glucosaminyl]asparagine amidase; Genzyme) and incubated for 16 hours at 37°C, while the other half was mock treated.

After digestion, 10 μg bovine serum albumin was added as carrier and samples were recovered by trichloroacetic acid precipitation. Protein pellets were taken up in sample buffer containing 5% (v/v) 2- mercaptoethanol.

8.1.7. PRODUCTION OF MONOCLONAL ANTIBODY anti-Oyml

Part of the Oy Cl and CII exons of HPB-MLT PT ₇ -I was isolated using the BamHI and PstI sites at nucleotide positions 571 and 848 (Dialynas et al., 1986, Proc. Natl. Acad. Sci. USA 83:2619-2623) and was cloned into expression vector pRIT2T (Pharmacia) . The resulting Protein A fusion protein was expressed in E. coli N4830. Bacteria were lysed with lysozyme and the fusion protein was isolated by purification over a IgG Sepharose column. Mice were injected mtraperitoneally with 100 μg of fusion protein in Freund's adjuvant at days 0, 7 and 28. Twenty-eight days

later 100 μg of fusion protein in PBS was injected intravenously. After three days, splenocytes were isolated and fused with the hybridoma P3X63Ag8.653 as described (Brenner et al., 1987, J. Immunol. 138:1502-1509). Hybridomas were screened by enzyme-linked immunoabsorbent assay (ELISA) . Ninety six-well flat bottom plates (LINBRO, Flow Laboratories) were incubated overnight with 0.4 μg of fusion protein or nonfused protein in PBS. Nonspecific binding sites were blocked at 23°C with 0.25 mg/ml normal rabbit IgG (Sigma) in PBS containing 50% (v/v) FCS. 50 μl of hybridoma supernatant was added for 1 hour at 4°C, followed by a similar incubation in 50 μl of a 5 μg/ml solution of peroxidase-conjugated anti-mouse IgG (Cappel) . All described incubations were interspersed with washing steps, using 10% (v/v) FCS, 0.1% (w/v) BSA, PBS. The ELISA was developed with 0.08% (w/v) O-phenylenediamine (Sigma) in 0.012% (w/v) hydrogen peroxide containing phosphate-citrate buffer, pH 5.0.

Although anti-Oγml (IgG ) does not recognize the native ₇ δTCR/CD3 complex in cytofluorographic analysis nor the ₇ δTCR heterodimer from Triton X-100 solubilized cells in immunoprecipitation, it does recognize biosynthetically labelled ₇ TCR precursor and mature ₇ TCR proteins after separation of CD3/ ₇ δTCR proteins into individual chains. In this way, anti-Oγml was shown to recognize the ₇ TCR protein after separating CD3/ ₇ δTCR complexes into individual chains by boiling anti-CD3 immunoprecipitates in 1% (w/v) SDS in TBS (Fig. 12, lane 3).

8 . 1. 8 . ISOLATION AND SEQUENCING OF A MOLT-13 7TCR CDNA CLONE

Poly (A) RNA was prepared from MOLT-13 cells by urea/lithium chloride precipitation followed by oligo (dT) cellulose affinity chromatography. A λgt 10 cDNA library was prepared from poly(A) RNA by the method of Huynh et

al., 1985 (DNA Cloning, Glover, D.M. ed. IRL Press, Oxford, 1:49-78) using Mung Bean Nuclease for the hairpin loop cleavage (McCutcham et al., 1984, Science 225:626-628). The cDNA library was amplified on the E. coli strain C600 Hfl and screened by plague filter hybridization with 32P- labelled PT ₇ I (Dialynas et al., 1986, Proc. Natl. Acad. Sci.

U.S.A. 83:2619-2623). Positive clones were analyzed for size and restriction enzyme map, and cDNA clone M13k was selected for sequencing. The cDNA of M13k was excised from λgt 10 phage with the endonuclease EcoRI and further digested with appropriate restriction enzymes. The fragments were subcloned into Ml3 vectors and sequenced by the dideoxy chain termination method (Sanger et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467) using the modified T7 polymerase (Sequenase, United States Biochemical Corp.) .

Clone M13k corresponds to a full length, in frame, ₇ TCR transcript, including 36 nucleotides of 5' untranslated region and 72 nucleotides of 3' noncoding region (Fig. 14) . The nucleotide sequence of the V region is identical to the genomic V ₇ I.3 sequence (nomenclature Lefranc et al., 1986a, Cell 45:237-246; Strauss et al., 1987, Science 237:1217-1219), except for a C to T (lie to Val) change of nucleotide 53 in the putative signal sequence. The J region is identical to the J ₇ 2.3 sequence (nomenclature based on Lefranc et al, 1986b, Nature 319:420-422; Quertermous et al, 1987, J. Immunol. 138:2687- 2690) . Interestingly, 8 nucleotides occur at the V-J junction which do not appear to be encoded by the genomic V or J sequences and presumably represents an N-region. The C region sequences match the corresponding genomic sequence (Lefranc et al., 1986c, Proc. Natl. Acad. Sci. U.S.A. 83:9596-9600), with, the exception of nucleotide 559 (G to C; Val to lie) and nucleotide 908 (T to C; Met to Thr) .

8.2 . RESULTS

8.2.1. NOVEL 7STCR PROTEIN COMPLEX Preliminary studies of peripheral blood ₇ δTCR > lymphocytes revealed the presence of a CD3-associated complex that was different from the known human ₇ δTCR forms. In an attempt to delineate this form, we produced and characterized a number of cell lines derived from normal human donors. Peripheral blood lymphocytes were stained with monoclonal antibody (mAb) WT31, which brightly stains resting βTCR lymphocytes. Cells that did not stain were isolated by cell sorting and then expanded in vitro in IL-2 containing medium. Peripheral blood lymphocyte line 2

(PBL-L2) obtained in this way, proved to be homogeneously CD3 CD4~CD8~ ^" , a cell surface phenotype characteristic of ₇ δTCR lymphocytes.

To visualize ₇ δTCR complexes on PBL-L2 cells, immunoprecipitations with an anti-CD3 mAb were carried out from cell surface 125I-labelled cells solubilized in CHAPS or digitonin. In these detergents, the physical association between the CD3 complex and ₇ δTCR subunits is preserved.

SDS-PAGE of anti-CD3 immunoprecipitates from PBL-L2 cells resolved 40 kD and 44 kD proteins (referred to as 40 kD) that were identified as ₇ TCR subunits by aπti-C ₇ b serum, an antiserum directed against a ₇ TCR constant region peptide

(Fig. 11A; see methods section) .

These ₇ TCR proteins on PBL-L2 are noncovalently associated with a δTCR subunit, which is visible as a weakly iodinated protein in the anti-CD3 immunoprecipitate analyzed under nonreducing conditions (Fig. 11A, lane 6, closed arrow) . This weakly iodinated protein represents the δTCR subunit on PBL-L2 cells, since it is not recognized by anti-Oγb serum (Fig. 11A, lane 8) . In addition, it displays the same SDS-mobility shift comparing analysis under nonreducing and reducing conditions as was noted for the

δTCR proteins on IDP2 and PEER cells (see infra; see also PCT International Publication No. WO 88/00209, published January 14, 1988). The δTCR protein could not be visualized after reduction (Fig. 11A, lane 3) , because it migrated with a mobility of 40 kD (see infra) and then was obscured by the similar sized ₇ TCR protein (open arrow) .

This ₇ δTCR form is not only present on normal peripheral blood T lymphocytes, but is also observed on thymus-derived Clone II cells (Fig. 11D) and on the T- leukemic cell line MOLT-13 (Fig. HE) . These three cell lines possess ₇ TCR species that display differential glycosylation resulting in a ₇ TCR protein doublet observed on PBL-L2 (40 kD and 44 kD; Fig. HA, lane 8) and Clone II cells (40 kD and 44 kD; Fig. 11D, lane 8) or a diffusely labelled ₇ TCR protein band observed on MOLT-13 cells (40 to 46 kD; Fig. HE, lane 6) . Two-dimensional gel analysis [nonequilibrium pH gradient electrophoresis (NEPHGE) followed by SDS-PAGE] of the MOLT-13 ₇ TCR protein band resolved two parallel ₇ TCR species (40 kD and 44 kD) , of which the 44 kD ₇ TCR species contained an additional high mannose (or hybrid) N-linked glycan compared to the 40 kD ₇ TCR species. Thus, the ₇ TCR subunits of this receptor complex isolated from three different cell sources (peripheral blood, thymus, and leukemia) revealed cell surface species of 40 kD that are noncovalently associated with δTCR partner chains.

For comparison to the ₇ δTCR form on PBL-L2, Clone II and MOLT-13 cells, we examined the previously known forms on the IDP2 and WM-14 cell lines. The IDP2 cell line (see PCT International Publication No. WO 88/00209, published January 14, 1988; Brenner et al., 1986, Nature 322:145-149) contains a larger, 55-60 kD ₇ TCR protein (referred to as 55 kD) , which is recognized by anti-Oγb serum (Fig. 11B) . When the anti-CD3 immunoprecipitate is examined under nonreducing conditions, it is evident that the IDP2 ₇ TCR protein is

associated noncovalently with its δTCR partner chain (Fig. 11B, lane 4, solid arrow). Upon reduction, the δTCR protein displays a decrease in SDS-PAGE mobility to a relative molecular mass of 40 kD (compare Fig. 11B, lane 4, closed arrow, with Fig. 11B, lane 2, open arrow) .

In contrast to the noncovalently associated ₇ TCR forms, the peripheral blood-derived T cell clone, WM-14, bears a disulfide-linked TCR dimer of 70 kD (Fig. 11C, lane 7), that was recognized by anti-Oγ serum (Alarcon et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:3861-3865). This dimer is also recognized by anti-TCRδl, a mAb directed against the δTCR subunit (Fig. 11C, lane 5) , and therefore represents a ₇ δTCR heterodimer. Analysis under reducing conditions reveals three ₇ TCR proteins of 36 kD, 40 kD and 43 kD (referred to as 40 kD) .

Thus, the CD3-associated complex on PBL-L2, Clone II and MOLT-13 cells constitutes a novel ₇ δTCR heterodimer compared to the previously known forms, since its TCR ₇ subunit is 40 kD (similar in size to the disulfide-linked C ₇ I encoded ₇ TCR protein on WM-14 cells) , yet it is not disulfide-linked to its partner chain (similar to the 55 kD, Oγ2 encoded ₇ TCR protein on IDP2 cells) . To understand the molecular basis of this complex, more detailed structural analysis of its ₇ TCR and δTCR subunits was carried out as described infra, using the MOLT-13 cell line as an example.

8.2.2. CORE POLYPEPTIDE SIZE OF MOLT-13 7TCR SUBUNIT To determine the size of the ₇ TCR core polypeptide of MOLT-13 cells (40 kD ₇ TCR glycoprotein) , and compare it with that of PEER cells (55 kD ₇ TCR glycoprotein) , both cell lines were biosynthetically labelled for 15 minutes in the presence of S-methionine and 35S-cysteine, solubilized in Triton X-100 and then immunoprecipitated with anti-Oγml, a monoclonal antibody that specifically recognizes the ₇ TCR chain (Fig. 13A, see

methods section) . Immunoprecipitated material was subsequently digested with endoglycosidase H (Endo H) to remove the immature N-linked glycans. The MOLT-13 ₇ TCR polypeptide backbone has a relative molecular mass of 35 kD (Fig. 13A, lane 8) , which is 5 kD smaller than the PEER 7TCR core polypeptide (40 kD; Fig. 13A, lane 4) or the IDP2 7TCR core polypeptide (40 kD; See PCT International Publication No. WO 88/00209, published January 14, 1988). It now can be concluded that MOLT-13 cells express a ₇ TCR core polypeptide that is distinct from the IDP2 and PEER ₇ TCR core polypeptides based on its being 5 kD smaller in size. In addition, only 5-11 kD of size on the mature MOLT-13 ₇ TCR cell surface glycoprotein are accounted for by post- translational processes (40-46 kD surface size minus 35 kD core size) , where 15-20 kD of relative molecular mass can be accounted for by post-translational processes on the PEER and IDP2 ₇ TCR glycoproteins (55-60 kD surface size minus 40 kD core size) . Assuming that all post-translational processes are N-linked glycans and that each glycan chain accounts for approximately 3 kD of relative molecular mass, we predict that 2 to 3 N-linked glycans are attached to the MOLT-13 ₇ TCR protein, while 5 N-linked glycans are added to the polypeptides on PEER and IDP2 cells. Experiments using N-glycanase to remove N-linked carbohydrates from cell surface ₇ TCR proteins showed that the majority of the post- translational processes that are added to the core polypeptide are indeed N-linked glycans.

8.2.3. PRIMARY SEQUENCE OF MOLT-13 7TCR To understand the structure of the constant region gene segment encoding the MOLT-13 ₇ TCR subunit, the sequence of a cDNA clone representing the MOLT-13 ₇ TCR transcript was determined. A λgtlO library from MOLT-13 derived poly-A RNA.was constructed and probed with a human ₇ TCR cDNA clone, PT ₇ -I (Dialynas et al., 1986, Proc. Natl.

Acad. Sci. U.S.A. 83:2619-23). Based on size and limited restriction enzyme mapping, one clone, M13k, was selected and its nucleotide sequence determined (Fig. 14) . Clone M13k represents a full length, in-frame ₇ TCR transcript, using a V ₇ I.3 gene segment joined to a J ₇ 2.3 gene segment (Lefranc et al., 1986, Cell 45:237-246; Lefranc et al., 1986, Nature 319:420-422; nomenclature based on Strauss et al., 1987, Science 237:1217-1219; Quertermous et al., 1987, J. Immunol. 138:2687-2690). The constant region sequence was found to be nearly identical to a recently reported non-functional ₇ TCR (Pellici et al., 1987, Science 287:1051-1055) and to the Oγ2 genomic sequence containing two CII exon copies b and c (Lefranc et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:9596-9600) (see methods section for detailed account) . This represents the first in-frame transcript encoding a ₇ TCR protein expressed on the cell surface that utilizes a Oγ2 gene segment with two CII exon copies.

The deduced amino acid sequence of this cDNA clone predicts a polypeptide backbone size of 34.8 kD which is in good agreement with biochemical data described above. Surprisingly, six potential N-linked carbohydrate attachment sites are encoded by this transcript. Since the biochemical data suggest that only 2 to 3 N-linked glycans are attached to the polypeptide chain, it indicates that not all potential sites are used.

To reflect Oy gene segment usage, we have denoted the disulfide-linked ₇ δTCR form expressed by PBL-C1 and WM- 14 as "Form 1", since such disulfide-linked ₇ TCR chains utilize the Oγl gene segment (Krangel et al., 1987, Science 237:64-67). The large (55 kD) , nondisulfide-linked ₇ TCR subunit of the ₇ δTCR form expressed on IDP2 and PEER cells is encoded by C ₇ 2 gene segments containing three CII exon copies, namely copy a, copy b and copy c (Krangel et al., 1987, Science 237:64-67; Littman et al., 1987, Nature

326:85-88) and therefore this ₇ δTCR form is called herein "Form 2abc". In concordance, the form characterized on MOLT-13 cells is referred to as "Form 2bc ^,/ .

8.2.4. PREFERENTIAL C7 GENE SEGMENT USAGE

To determine the presence of these three ₇ ,δTCR forms in freshly isolated peripheral blood we analyzed the mononuclear cells from ten healthy subjects, using biochemical analysis with mAb anti-TCRδl (described in Section 6, supra; reactive with the δTCR constant region) . This antibody reacts with the great majority, if not all ₇ , TCR lymphocytes. Representative results from this panel are shown in Figure 15. In subject 1, anti-TCRδl immunoprecipitates (analyzed under nonreducing conditions) demonstrate the presence of both disulfide-linked ₇ , TCR complexes as a 70 kD protein band (Form 1) and nondisulfide-linked ₇ ,δTCR complexes as a broad 40 kD protein band (Form 2bc) (Fig. 15, lane 2) . This indicates that the Oγl and Oy2 constant regions are both used by the expressed ₇ ,δTCR of this individual. However, the amount of Form 2bc varied among individuals. Note the smaller fraction of Form 2bc in subject 2 compared to subject 1 by comparing the intensity of the 40 kD protein bands in both individuals (compare lane 2 of subject 2 with lane 2 of subject 1) . Even more strikingly, only disulfide-linked ₇ ,δTCR complexes could be detected on the mononuclear cells of three of the ten individuals examined, even after long exposure of the autoradiographs (see subject 3) . None of the analyzed individuals revealed the 55 kD, nondisulfide- linked ₇ , TCR complex (Form 2abc) in peripheral blood.

8.2.5. CHARACTERIZATION OF THE δTCR SUBUNIT In contrast to the striking structural differences in size and glycosylation of the ₇ TCR proteins, δTCR subunits from different cell sources proved to be

markedly similar. The relative molecular mass of the δTCR glycoprotein on MOLT-13 cells was directly determined to be 40 kD using the anti-δTCR mAb (Fig. 12, lane 4), confirming that it is similar in size to the δTCR glycoprotein on IDP2 cells (Fig. 11B, lane 2, open arrow).

To also compare δTCR polypeptide backbone sizes,

125 cell surface I-labelled δTCR protein from MOLT-13 cells was digested with N-glycanase to remove asparagine-linked glycans (of the high mannose, hybrid, and complex-type; Tarentino et al., 1985, Biochem. 24:4665-4671; Hirani et al., 1987, Anal. Biochem. 162:485-492). The δTCR core polypeptides of MOLT-13 cells has a relative molecular mass of 35 kD (Fig. 13B, lane 4) , which is similar to that of the δTCR backbone of IDP2 cells (35 Kd) (Band et al., 1987, Science 238:682-684). In addition, digestion of cell surface 125I- labelled MOLT-13 δTCR protein with endoglycosidase H (Endo

H, removing only high mannose and certain hybrid N-glycans;

Tarentino et al. , 1974, J. Biol. Chem. 249:811-817; Trimble and Maley, 1984, Anal. Biochem. 141:514-522) caused a decrease in relative molecular mass of 2.5 kD, (Fig. 13B, lane 2) consistent with the presence of one carbohydrate moiety, leaving a relative mass of 2.5 kD of Endo H resistant carbohydrates attached to the polypeptide. Since there are two potential N-glycan attachment sites present in the δTCR constant domain (Hata, S., et al., 1987, Science

238:678-682; Loh et al. , 1987, Nature 330:569-572), these data show that both are used, but that their N-glycans are processed differently, namely one as a high mannose N-glycan

(Endo H-sensitive) and the other as a complex N-glycan (Endo

H-resistant, but N-glycanase sensitive) . In contrast to the different amounts of attached N-linked carbohydrate on ₇ TCR polypeptide chains, the δTCR subunits expressed on PEER,

IDP2 and MOLT-13 cells all revealed the same peptide core sizes and the presence of two N-linked glycans (Fig. 13B and data not shown) .

8.3. DISCUSSION

In this example, three protein forms of the human ₇ TCR glycoprotein are compared, namely the disulfide-linked 40 kD ₇ TCR protein (Form 1) , the nondisulfide-linked 55 kD ₇ TCR protein (Form 2abc) and the nondisulfide-linked 40 kD ₇ TCR protein (Form 2bc) . All three forms are shown to be associated with a δTCR subunit. Complementary DNA sequences representing the first two ₇ TCR forms have been reported previously (Krangel et al., 1987, Science 237:64-67; Littman et al., 1987, Nature 326:85-88. The constant region of ₇ TCR polypeptide Form 1 (on PBL-C1) is encoded by the C ₇ I gene segment containing a single CII exon, while ₇ TCR polypeptide Form 2abc (on IDP2 and PEER cells) utilizes the Oγ2 gene segment containing CII exon copy a, copy b and copy c. The cDNA sequence corresponding to a ₇ TCR chain of Form 2bc was shown to contain a Oγ2 gene segment utilizing only two CII exon copies, namely copy b and copy c. Similarly, it seems likely that the gene structure of the ₇ TCR connector region of Clone II and PBL-L2 (nondisulfide-linked, 40 kD 7TCR protein) will also be like the MOLT-13 structure determined here, namely of Form 2bc. Since the δTCR constant region used is the same for all these forms (Hata, S., et al., 1987, Science 238:678-682; Loh, E.Y. , et al., 1987, Nature 330:569-572), a complete comparison of the structures of the three ₇ δTCR forms in man now can be made (Fig. 16) .

Two Oγ2 polymorphic genomic forms exist in man (Lefranc et al.. Nature 319:420-422; Pellici et al., 1987, Science 237:1051-1055). The two transcript forms (Form 2abc and Form 2bc) are probably the product of these different allelic types. To date, no allelic form of ₇ TCR polypeptides have been found in mice. We conclude that the

dramatic difference in ₇ TCR cell surface protein size between Form 2abc (55 kD) and Form 2bc (40 kD) is largely determined by the amount of attached N-link carbohydrates, most likely reflecting the number of N-linked glycans. Backbone sizes of IDP2 ₇ TCR (Form 2abc) and MOLT-13 ₇ TCR

(Form 2bc) proteins have been measured to be 40 kD and 35 kD respectively, on the basis of SDS-PAGE, which correlates well with their predicted molecular masses of 36.6 kD and 34.8 kD respectively, calculated on the basis of cDNA sequences. It is clear that this small difference in backbone size (5 kD in SDS-PAGE) , accounted for mainly by one CII exon encoded peptide of 16 amino acids, contributed to, but could not solely explain the observed difference in molecular mass between the 55 kD and 40 kD nondisulfide- linked ₇ TCR surface forms. Form 2abc ₇ TCR polypeptides possess 5 potential N-linked glycan attachment sites that are probably all used, in contrast to the MOLT-13 ₇ TCR polypeptide which bears one additional potential attachment site, while carrying only 2 to 3 N-linked glycans. The reason for this limited use of potential attachment sites is unknown, but may result from the influence of the CII exon encoded peptides on the conformation of the ₇ TCR protein. The CII exon encoded peptides and their neighboring amino acids make up a connector region between the plasma membrane and the immunoglobulin-like constant domain. This region contains most of the N-linked glycan attachment sites (Fig. 17) . We conclude that the CII exon copies appear to determine the protein form not only by determining polypeptide backbone size, and by creating the ability to disulfide-link chains, but also by influencing the amount of attached carbohydrates. δTCR complementary DNAs of IDP2 (Hata et al. , 1987, Science 238:678-682), PEER (Loh et al., 1987, Nature 330:569-572) and MOLT-13 cells have been sequenced and were found to be identical, except for the diversity/N-region

interspacing the variable and constant region gene segments. The δTCR protein on WM-14 cells has a relative molecular mass of 43 kD, which is similar to the δTCR protein described previously (Borst et al., 1987, Nature 325:683- 688; Lanier et al., 1987, J. Exp. Med. 165:1076-1094) but is 3 kD larger than the other δTCR chains. These 43 kD δTCR proteins might indicate the presence of an additional N- linked glycosylation site in a different δ variable domain.

Structural differences comparable to those described for ₇ TCR constant region segments have not been observed for αTCR and βT R genes (Yoshikai et al., 1985, Nature 316:837-840; Toyonaga et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:8624-8628; Royer et al., 1984, J. Exp. Med. 160:947-952; Kronenberg et al., 1985, Nature 313:647-653). There is possible structural similarity in the number of human CII exon repeats with the length in murine Oγ regions, of which the Oγl, Oγ2 and Oy4 constant regions encode for 15, 10 and 33 amino acid connector region respectively (Garman et al, 1986, Cell 45:733-742; Iwamoto et al., 1986, J. Exp. Med. 163:1203-1212). The connector regions in mouse, however, reflect a difference in the size of the relevant exon, not the multiple use of exons as is seen in Form 2abc ₇ TCR and Form 2bc ₇ TCR in humans. Also, the murine ₇ TCR only exist in disulfide linked forms in contrast to the two nondisulfide linked human forms.

Importantly, the human ₇ ,δTCR forms do not appear to be used equally. In some individuals (selected for high percentages of ₇ ,δTCR lymphocytes) , a single form (Form 1) predominates, suggesting that either positive selection occurs for this form or that there is selection against other ₇ ,δTCR forms.

9. THREE T CELL RECEPTOR η, δ ISOTYPIC FORMS RECON¬ STITUTED BY PAIRING OF DISTINCT TRANSFECTED ₇ TCR CHAINS WITH A SINGLE δTCR SUBUNIT

As described in the example herein, the role of the ₇ TCR polypeptide in the formation of the ₇ δ heterodimer was explored. We examined by transfection the ₇ δTCR complexes formed by the association of ₇ TCR chains corresponding to the three ₇ δTCR forms (Forms 1, 2abc, and 2bc) with a single resident δTCR chain. ₇ TCR DNA encoding either Form 1 or Form 2abc of the ₇ TCR polypeptide was transfected into the MOLT-13 cell line, which constitutively expresses a ₇ δ heterodimer comprised of form 2bc ₇ TCR polypeptide noncovalently associated with δTCR polypeptide. Transfected cells were capable of expressing, together with the ₇ δTCR characteristic of the MOLT-13 cell line, ₇ δ heterodimers comprised of either Form 2abc ₇ TCR noncovalently associated with δTCR or Form 1 ₇ TCR covalently linked to δTCR. Furthermore, the glycosylation of the transfected ₇ TCR gene products was identical to the glycosylation of these genes in their native cell lines. Thus, the degree of glycosylation and the ability to form disulfide linkages are properties determined by the ₇ TCR gene. ₇ TCR constant region CII exon usage determines not only the presence or absence of disulfide linkage between TCR ₇ and δ polypeptides, but also the amount of carbohydrate attached to the ₇ TCR chain, which is largely responsible for the differences in size of the cell surface ₇ TCR proteins.

9.1. MATERIALS AND METHODS

9.1.1. CELL LINES

MOLT-13, a TCR ₇ δ T leukemia cell line (Hata, S., et al., 1987, Science 238:678-682; Loh, E.Y., et al., 1987, Nature 330:569-572), and peripheral blood derived TCR

₇ δ cell lines PBL Cl (Brenner, M.B., et al., 1987, Nature 325:689-694) and IDP2 (Brenner, M.B., et al., 1986, Nature 322:145-149) were cultured as previously described.

9.1.2. ANTIBODIES The monoclonal antibodies (mAb) used were: Anti-leu-4 (anti-human CD3; IgGl) (Ledbetter, J.A. et al., 1981, J. Exp. Med. 153:310-323), anti-TCRδl (anti-human TCRδ chain constant region; IgGl) (See Section 6; Band, H. , et al., 1987, Science 238:682-684), anti-Ti- ₇ A (anti-V ₇ 2; IgG2a) (Jitsukawa, S., et al., 1987, J. Exp. Med. 166:1192- 1197) , anti-Oγml (anti-human ₇ TCR contant region; see Section 8.1.7), P3 (IgGl secreted by the P3X63Ag8 myeloma) (Koehler, G. , and Milstein, C, 1975, Nature 256:495-497), and 187.1 (rat anti-mouse K light chain-specific) (Yelton, D.E., et al., 1981, Hybridoma 1:5-11).

9.1.3. ISOLATION AND SEQUENCING OF MOLT-13 δTCR CDNA CLONES

A complementary DNA (cDNA) library prepared from

MOLT-13 poly A+ RNA in the vector λgtlO (Huynh, et al.,

1985, in DNA Cloning, ed. Glover, D.M. (IRL Press, Oxford) ,

Volume 1, pp. 49-78) was screened by hybridization with

³² P-labeled human δTCR cDNA clone IDP2 0-240/38 (Hata et al., 1987, Science 238:678-682). Clones were selected for detailed analysis on the basis of size and limited restriction enzyme mapping. Nucleotide sequence was determined in M13 vectors by dideoxy chain termination method (Sanger et al., 1977, Proc. Natl. Acad. Sci. U.S.A.

74:5463-5467) using the modified T7 polymerase (Sequenace,

United States Biochemical Corp.) (Potter et al., 1984, Proc.

Natl. Acad. Sci. U.S.A. 81:7161-7165).

9.1.4. CONSTRUCTION OF EXPRESSION PLASMIDS AND TRANSFECTIONS

₇ TCR cDNAs (PBL Cl.15 and IDP2.llr) (Krangel, M.S., et al., 1987, Science 237:64-67) were cloned into pFneo mammalian expression vector (Saito, T. , et al., 1987, Nature 325:125-130; Ohashi, P., et al., 1985, Nature 316:606-609) downstream from Friend spleen focus forming virus (SFFV) long terminal repeat (LTR) , as shown schematically in Figure 10B. The plasmid constructs were transfected into MOLT-13 cells by electroporation (Potter, H. , et al., 1984, Proc. Natl. Acad. Sci. 81:7161-7165). Transfectants were selected and maintained in medium containing 2 mg/ml of G418 (480 μg/mg solid by bioassay; GIBCO) , and cloned by limiting dilution.

9.1.5. IODINATION AND IMMUNOPRECIPITATION Cell surface labeling with 125I using lactoperoxidase, solubilization in 3-[ (3-cholamidopropyl) dimethylammonio] 1-propanesulfonate (CHAPS; Sigma Chemical Co., St. Louis, MO), immunoprecipitation with various antibodies, nonequilibrium pH gradient gel electrophoresis (NEPHGE) , and SDS polyacrylamide gel electrophoresis (SDS- PAGE) were performed as described (See Section 8.1.3; Brenner, M.B., et al., 1986, Nature 322:145-149; Brenner, M.B., et al., 1987, Nature 325:689-694). Specific immunoprecipitations were carried out with 1 μg anti-leu-4, 0.1 μl anti-TCRδl ascites, or 1 μl P3 ascites, together with 150 μl of 187.1 culture supernatant. For anti-T9- ₇ A, 1 μl of ascites was used without 187.1.

9.1.6. BIOSYNTHETIC LABELING Exponentially growing cells were incubated for 30 minutes in methionine- and cysteine-free medium followed by a 15 minute pulse labeling at 37°C with 35S-methionine and

35

S-cysteine, and immunoprecipitations were carried out as

described in Section 8.1.3, supra. Immunoprecipitates were either treated with endoglycosidase-H (endo-H) or were mock-incubated, separated by SDS-PAGE, and visualized by fluorography (Bonner, W.M. and Laskey, R.A. , 1974, Eur. J. Biochem. 46:83-88).

9.2. RESULTS We investigated the products resulting from association of structurally distinct ₇ TCR gene products with a single δTCR protein in order to demonstrate the role of the ₇ TCR gene, and in particular the TCR CII exons, in determining the structural differences between various ₇ TCR isotypes. For this purpose, MOLT-13, a T leukemia cell line that expresses the 40 kD nondisulfide-linked ₇ TCR polypeptide (Form 2bc) , was used as a recipient for ₇ TCR chain cDNA clones corresponding to the other two forms of the receptor (Forms 1 and 2abc) . Complete sequences of the cDNA clones representing these ₇ TCR chains are described in Figure 14 (Form 2bc) , and in Krangel et al. (1987, Science 237:64-67) (Forms 1 and 2abc) and they are schematically represented in Figure 18A.

9.2.1. A SINGLE FUNCTIONAL δTCR CHAIN IS PRESENT IN THE MOLT-13 CELL LINE δTCR gene rearrangement studies of the MOLT-13 cell line (Hata, S., et al., 1987, Science 238:678-682) suggested that only a single functional δTCR gene product was expressed in this cell line. However, to demonstrate directly that a single functional transcript for δTCR is made in MOLT-13 cells, cDNA clones cross-hybridizing with a δTCR cDNA probe (Hata, S., et al., 1987, Science 238:678- 682) were isolated from a MOLT-13 cDNA library prepared in λgtlO and the sequence of selected cDNA clones was determined. This analysis revealed that MOLT-13 cells express transcripts corresponding to one functionally

rearranged and one aberrantly rearranged δTCR gene. The cDNA clone corresponding to the functionally rearranged δTCR gene has the same V (Vδl) , J (Jδl) , and C gene segments described earlier for the IDP2 cell line (Hata, S., et al., 1987, Science 238:678-682). The MOLT-13 δTCR cDNA clone, however, possesses a distinct nucleotide sequence between the V and J gene segments arising from D segment utilization (MOLT-13 probably uses only Dδ2) , imprecise joining, and N- region diversity at the V-D and D-J junctions (Hata, S., et al., 1988, Science 240:1541-1544). The MOLT-13 δTCR cDNA also predicts a cysteine residue in the membrane proximal connector region of the constant gene segment that would be available for disulfide linkage to ₇ TCR gene products that utilize the Oγl gene segment. Although the MOLT-13 cell line expresses a nondisulfide-linked ₇ ,δTCR receptor, the presence of a cysteine residue in the membrane proximal connector region of its δTCR chain leaves open the possibility that this δTCR subunit might be capable of participating in either a nondisulfide-linked o a disulfide-linked complex.

9.2.2. ₇ TCR GENE PRODUCT DETERMINES THE FORM OF THE RECEPTOR

The MOLT-13 cells transfected with ₇ TCR cDNA constructs were abbreviated as M13.PBL CI ₇ (for MOLT-13 cells transfected with PBL Cl-derived ₇ TCR cDNA) and

M13.IDP2 ₇ (for MOLT-13 cells transfected with IDP2-derived

₇ TCR cDNA) . The bulk transfectant cell lines and representative subclones derived from these lines were analyzed by Northern blot analysis with ₇ TCR (VJC) or V ₇ 2- specific cDNA probes. In addition to the resident 1.6 kb

MOLT-13 ₇ TCR transcript, a second ₇ TCR transcript of about

1.8 kb (expected size for a ₇ TCR transcript initiating in the SFFV LTR of the expression plasmid) was observed in transfectant lines and their clones. The 1.8 kb transcript

specifiσally hybridized with a V ₇ 2 probe that does not cross-hybridize with the V ₇ I.3 present in the resident MOLT-13 ₇ TCR transcript, and thus the 1.8 kb transcript represents the transcript of the transfected ₇ TCR cDNAs (which utilize a V ₇ 2 segment) .

To biochemically characterize the ₇ TCR protein(s) expressed on the surface of the transfectants, a representative clone derived from each line was analyzed by immunoprecipitation of surface iodinated cells with P3 (control) , anti-leu-4 (anti-CD3) , anti-TCRδl (anti-δTCR) , or anti-Ti- ₇ A mAbS. Anti-Ti- ₇ A (Jitsukawa, S., et al., 1987, J. Exp. Med. 166:1192-1197) appears to specifically recognize the ₇ ,δTCR cells that utilize the V ₇ 2 gene segment as the variable portion of their ₇ TCR chains. Untransfected (Fig. 19A) as well as the transfected MOLT-13 cells (Fig. 19B and 19C) express the expected parental ₇ TCR (40 kD; see open arrow) and δTCR subunits (see asterisk) . Note that anti-V ₇ 2-specific mAb (anti-Ti- ₇ A) fails to react with the resident MOLT-13 ₇ TCR chain (Fig. 19A, lanes 7 and 8). Anti-CD3 immunoprecipitates of M13.PBL CI ₇ transfectant cells revealed an additional CD3-associated species (68 kD) when examined under nonreducing conditions (Fig. 19B, lane 3, see solid arrow). On both PBL Cl cells (Fig. 19D, lanes 3 and 4) and the M13.PBL CI ₇ transfectant cell line (Fig. 19B, lanes 3 and 4) , the 68 kD complex yielded 40 and 36 kD species upon reduction (in the case of the M13.PBL CI ₇ transfectant, these bands are clearly visualized in the anti-V ₇ 2 immunoprecipitate, see Fig. 19B, lanes 7 and 8, solid arrows) . These 40 and 36 kD species represent differentially glycosylated ₇ TCR polypeptides (Brenner, M.B., et al., 1987, Nature 325:689-694). In these immunoprecipitates, the δTCR chain (40 kD reduced) comigrates with the 40 kD ₇ TCR polypeptide and is therefore not visualized (however, see below) .

Importantly these experiments show that the resident δTCR chain of the MOLT-13 cell line, normally part of a nondisulfide-linked complex, associates with the PBL Cl ₇ TCR protein to form a disulfide-linked ₇ ,δTCR heterodimer in the transfectant cell line. In contrast, the IDP2τ- derived ₇ TCR protein (55 kD) in the M13.IDP2 transfectant cell line formed a nondisulfide-linked complex with the resident MOLT-13 δTCR chain (Fig. 19C, lanes 3 and 4) . Immunoprecipitates carried out with anti-TCRδl mAb (specific for δTCR peptide) confirmed that the endogenous (Figs. 19A, D and E, lanes 5 and 6) as well as the transfected ₇ TCR chains (Fig. 19B and C, lanes 5 and 6) from all these cell lines were associated directly with the δTCR chain. Anti- Ti- ₇ specifically immunoprecipitated the 68 kD disulfide- linked ₇ ,δTCR heterodimer from the M13.PBL Cl transfectant cells (Fig. 19B, lanes 7 and 8) , and the 55 kD ₇ TCR chain, along with the 40 kD δTCR chain, from the M13.EDP2 ₇ transfectant cells (Fig. 19C, lanes 7 and 8) , confirming that the ₇ TCR chains that are part of these complexes correspond to the transfected PBL Cl and IDP2-derived ₇ TCR cDNAs, respectively.

To further characterize the various ₇ TCR proteins biochemically, two-dimensional (2D) gel analyses (NEPHGE followed by SDS-PAGE) of surface 125I-labeled cells were carried out. Superimposition of the 2D patterns of resident

(MOLT-13) and transfected (PBL Cl or IDP2) ₇ TCR chains relative to the positions of the CD3 components allowed comparison of the relevant ₇ TCR species. In the immunoprecipitates from MOLT-13 cells, the ₇ TCR chains resolved as two discrete parallel series of iodinated species (Fig. 20A, see open arrows) . MOLT-13 cells transfected with the PBL Cl or the IDP2 TCR cDNAs revealed the resident MOLT-13 ₇ TCR polypeptide series, but in addition, showed radiolabeled species that were identical in

2D gel patterns to the ₇ TCR polypeptides of PBL Cl (compare

Fig. 2OB and D; see asterisks in Fig. 2OB) or IDP2 (compare Fig. 20C and D; see closed arrows in Fig. 20C) cells, respectively. Thus, the 2D gel biochemical analyses confirmed that the transfected ₇ TCR chains were expressed and processed similarly in MOLT-13 transfectants and in the parental cell lines, PBL Cl and IDP2.

9.2.3. POLYPEPTIDE BACKBONE SIZES OF THE TRANSFECTED 7TCR CHAIN PROTEINS

The peptide backbone sizes of the transfected

₇ TCR chains were determined by endoglycosidase-H treatment of the material immunoprecipitated from metabolically pulse-labeled cells. Immunoprecipitates carried out with anti-OyMl (specific for ₇ TCR chain) identified 35.5 and 34 kD species in untransfected MOLT-13 cells (Fig. 21, lane 4) that represent endogenous MOLT-13 ₇ TCR polypeptides. The smaller of these two polypeptides (see open arrows) corresponds to the expected polypeptide core size of the

MOLT-13 ₇ TCR polypeptide, whereas the larger polypeptide appears to represent a partially processed intermediate. In addition to these resident MOLT-13 ₇ TCR polypeptides, a polypeptide with a deglycosylated size of 41 kD was immunoprecipitated by anti-OγMl from the M13.IDP2 ₇ transfectant, (Fig. 21, lane 8, see solid arrow) . The size of this transfectant-specific ₇ TCR polypeptide agrees well with the deglycosylated IDP2 ₇ TCR polypeptide core size determined earlier in IDP2 cells (Brenner, M.B., et al.,

1987, Nature 325:689-694). As expected, M13.PBL CI ₇ transfectant cells revealed an additional ₇ TCR protein with a deglycosylated size of 32 kD (Fig. 21, lane 12, see solid arrow) which compares well with the ₇ TCR polypeptide backbone size reported earlier for the PBL Cl cell line

(Brenner, M.B., et al., 1987, Nature 325:689-694). This 32 kD species was specifically immunoprecipitated by the V ₇ 2- specific mAb, anti-Ti- ₇ A (Fig. 21, lane 13, see solid

arrow) , thereby allowing unambiguous assignment of resident and transfected ₇ TCR species in this cell line. Thus, the determined backbone sizes of the transfected ₇ TCR chains, derived from IDP2 and PBL Cl cell lines, match the backbone sizes of these polypeptides in their parent cell lines. By comparing the ₇ TCR polypeptide core sizes with those of the cell surface proteins, we infer that the MOLT-13, IDP2, and PBL Cl derived ₇ TCR chains carry 6, 14, and 8 kD N-linked carbohydrate, respectively.

9.3. DISCUSSION

Three biochemically distinct forms of the human ₇ δTCR subunit structure occur. In the present work, we show that a single δTCR polypeptide can associate with ₇ TCR chains representing each of the three receptor forms to reconstitute the appropriate ₇ ,δTCR heterodimers. The resident ₇ TCR polypeptide of MOLT-13 (form 2bc) is 40 kD and is noncovalently associated with the δTCR subunit. When the ₇ TCR cDNA clones corresponding to the disulfide-linked receptor of PBL Cl (Form 1) , or the 55 kD non-disulfide- linked receptor of the IDP2 cell line (Form 2abc) were transfected into the MOLT-13 cell line, the ₇ δTCR forms corresponding to those found in the cDNA-donor cell lines were reconstituted. The present transfection studies provide direct evidence that disulfide linkage is dictated by ₇ TCR constant segment usage, since the resident MOLT-13 δTCR chain was shown to participate in a disulfide-linked receptor complex with the PBL Cl-derived ₇ TCR chain (Form 1) , and a nondisulfide-linked receptor complex with the IDP2-derived ₇ TCR chain (Form 2abc) .

We have shown that the remarkable difference in size between the 55 kD (Form 2abc) and 40 kD (Form 2bc) non-disulfide-linked ₇ TCR polypetides is primarily due to different amounts of N-linked carbohydrate attached to the 7TCR polypeptide backbone (See Section 8.2.2, supra) . Thus,

either 15 kD (Form 2abc on IDP2 or PEER) or only 5 kD (Form 2bc on MOLT-13) of N-linked carbohydrate is attached to these ₇ TCR polypeptides even though the same number (five each) of N-linked glycan acceptor sites are encoded by the constant region gene segments used in both of these forms. Four of these N-linked glycosylation sites are present in or around the CII exon-encoded connector region. In the example herein, we show that the amount of N-linked carbohydrate attached to the transfected ₇ TCR proteins is identical to that seen in their parent cell lines, based on a comparison of peptide core size and mature cell surface size of the protein products of transfected ₇ TCR cDNA clones. Thus, the conformation of the two Oγ2 encoded protein segments must differ sufficiently to result in drastic differences in glycosylation. The major difference between Oγ segments of these two forms is that copy "a" of the CII exon is present in the 55 kD ₇ TCR chain of Form 2abc and it is absent from the 40 kD ₇ TCR chain of Form 2bc. Thus the presence or absence of this CII exon copy may be largely responsible for the glycosylation differences that account for the ₇ TCR polypeptide sizes.

The variation in structure of human ₇ δTCR isotypic forms is unprecedented among T cell receptors as no such parallel is observed in αyS CR.

10. T CELL RECEPTOR _γ δ COMPLEX, NOT

ASSOCIATED WITH CD3, IS IDENTIFIED

IN HUMAN ENDOMETRIAL GLANDULAR EPITHELIUM

In the early stages of placentation, infiltration of mononuclear cells is abundant at the proximity of spiral arteries and endometrial glands in maternal uterine tissues. These include an unusual population of T lineage cells of unknown function. Many extravillous trophoblasts express a novel type of class I MHC antigens which is different from that expressed on most somatic cells. We have tested a

panel of monoclonal antibodies to TCR δ heterodimer ( ₇ δTCR) in pregnant & non-pregnant uteri. Surprisingly, ₇ δTCR complex was not detected in leukocytes, but was localized in the cytoplasm of the endometrial glandular epithelium from pregnant uteri. These antibodies also reacted with the glandular epithelium from non-pregnant uteri, and the reactivity was stronger in the secretory phase than that in the proliterative phase of the menstrual cycle. However, ₇ δTCR was not associated with the CD3 complex, as shown by examining immunoprecipitates using three different monoclonal antibodies to CD3 (OKT3, anti-leu-4, UCHT-1) . The ₇ δTCR-positive glandular epithelial cells did not react with monoclonal antibodies to α TCR; the cells were also CD4- and CD8-negative. Moreover, the glandular epithelial cells lose the class I MHC antigens in early pregnancy. These data suggest that these ₇ δTCR bearing endometrial glandular cells undergo, at least, phenotypic alterations under local regulation of gene expression.

11. EXAMPLE: CHARACTERIZATION OF A HUMAN δ T CELL

RECEPTOR GENE AND A Vo. SPECIC MONOCLONAL ANTIBODY

We have isolated δTCR cDNA and a rearranged δTCR gene from a human ₇ δ T cell clone, AK119. From these DNA clones, a _v Ko _c probe was obtained, and used to determine the diversity of δTCR gene rearrangements in a panel of 13 human 7,δ T cell clones and 3 ₇ , human T cell tumor lines. Altogether five different rearrangements were detected, which corresponded to rearrangements using 2 to 5 different X genes. One particular rearrangement was always seen in human ₇ ,δ T cells that reacted with mAb TCSδl (δTCAR-3) . In addition, TCSδl immunoprecipitated the δTCR polypeptide from a human ₇ ,δ tumor cell line, Molt 13. We provide evidence

that monoclonal antibody TCSδl recognizes an epitope encoded in the AK119 Vδ gene or in a combination epitope of the rearranged AK119 gene Vδ-Jδ gene.

_R 11.1. MATERIALS AND METHODS

11.1.1. ISOLATION AND SEQUENCING OF AK119 δTCR CDNA CLONES

A cDNA library was generated from the PBL T-cell clone, AK119, by the method of Gubler and Hoffmann (Gubler and Hoffman, 1983, Gene 25:263). About 100,000 plaques of an amplified library were screened using a 32P-labelled nick-translated Cδ probe, isolated from a δTCR clone called

0-024 (Hata, S., et al., 1987, Science 238:678). The longest hybridizing cDNA clone (1.3 kb clone C119δ3) was 5 selected for sequence analysis by the dideoxy chain termination method.

11.1.2. CLONING A REARRANGED δTCR GENE A 3.5 kb genomic DNA clone containing the rearranged Vδ gene was obtained from AK119 cells as follows: EcoRI digested DNA was size fractionated on a preparative agarose gel, ligated into λgtlO, packaged and transfected into E. coll. Reco bmant phage were screened with a 32P- labeled nick translated 550 bp EcoRI fragment derived from the cDNA clone, cll9δ3. A rearranged clone called rll9δl which contains a 0.8 kb Hindi fragment (V region specific) and a 1 kb HincII-EcoRI fragment (V-J region) was isolated.

11.1.3. DNA PREPARATION Fetal and newborn thymic tissues were collected in accordance with accepted guidelines regarding patients ⁷ rights and approval. T cell clones were obtained from peripheral blood, pleural exudate or cerebrospinal fluid by limiting dilution and were cultured jln vitro (Hafler et al. ,

1985, Ann Neurol. 18:451; Van de Griend et al., 1987, J. Immunol. 138:1627). In all cases, DNA was prepared by digestion with proteinase K in 1% sodium dodecyl sulfate, followed by extraction with phenol/chloroform and ethanol precipitation.

11.1.4. SOUTHERN BLOT ANALYSIS Genomic DNA was digested with EcoRI, size fractionated on a 0.9% agarose gel and transferred to nitrocellulose. Hybridization was carried out with 32P-nick translated probes as previously described (Maniatis, 1982,

Molecular Cloning: A Laboratory Manual (Cold Spring Harbor

Laboratory; Cold Spring Harbor, New York) .

11.1.5. CYTOFLUOROMETRIC ANALYSIS

Normal peripheral blood monoclear cells (PBMC) , obtained from volunteers were isolated by fractionation on a Ficoll gradient. PBMC and PBL T cell clones were stained by indirect immunofluorescence using TCSδl mAb (referred to previously as δTCAR-3; See Section 7, supra) and fluorescem-conjugated goat antimouse IgG (Becton Dickinson) and analyzed in a fluorescence activated flow cytometer.

11.2. RESULTS

11.2.1. DIVERSITY OF δTCR GENE REARRANGEMENTS

Using a Cδ probe, we isolated a 1.3 kb δTCR cDNA clone, termed cll9δ3, from a λgtlO cDNA library of the T- cell clone AK119. The 5' end of cll9δ3 was sequenced and found to use previously identified V and genes (Hata et al., 1987, Science 238:678; Loh et al., 1987, Nature 330:569) . The sequence of the V-J junction indicated that C119δ3 has an in-frame V-J joint.

A 550 basepair (bp) EcoRI fragment encoding all the variable and joining region and part of the constant region (V-J-C probe) was obtained from cll9δ3 and used in Southern blot analysis of EcoRI digested genomic DNA from AK119. This probe detects a germline 3.2 kb and a germline 1.0 kb C 0. band. A.K119 showed an extra rearranged

3.5 kb band that is identical to the common δTCR rearrangement (described in Hata et al., 1987, Science 238:678; Loh et al., 1987, Nature 330:569) (rearrangement II in Fig. 22). This 3.5 kb band was cloned from a EcoRI size-fractionated λgtlO genomic library using the V-J-C cDNA probe. A partial map of the cloned rearranged δTCR gene, called rll9δl, is shown in Figure 17. The localization of the variable and joining region was determined using J oligonucleotide probes and variable region specific probes. From rll9δl, a 1 kb V-J probe was isolated by digestion with Hindi and EcoRI enzymes (see Fig. 17) .

This V-J probe was used to determine the diversity of δTCR gene rearrangements in a panel of 13 human ₇ δTCR positive T-cell clones and 3 human ₇ -δTCR positive tumor cell lines. As shown in Figure 22, five common rearrangements, numbered I-V, are seen in the polyclonal newborn thymocyte sample (lane 11) . These rearrangements are representative of rearrangements used by the human T cell clones. Only rearrangement II hybridizes to the Hindi

- Hindi V specific probe. Although we do not know that all these rearrangements represent V-D-J rather than D-J rearrangements, some of them must represent rearrangements of new variable regions to the previously characterized J 0. gene segment because these cells express a functional δTCR polypeptide chain on their cell surfaces. We have not ruled out the possibility that these new rearrangements represent rearrangements of a single new V 0 _c gene to other J0. genes,

yet to be identified. Our data is consistent with the fact that there must be 2-5 variable region genes that can be used in δTCR gene rearrangements.

₅ 11.2.2. DETERMINING THE SPECIFICITY OF mAb TCSδl TCSδl previously referred to as δTCAR-3, was generated by fusing splenocytes from of mice immunized with the human tumor ₇ δTCR cell line MOLT-13 with a mouse myeloma line. When used in fluorescent activated cell sorter _Q analysis, TCSδl reacted only with some but not all human ₇ δ T cells. The results are given in Table 1. There is a perfect correlation with usage of the AK119 V gene (rearrangement II) with positive staining by TCSδl. This data provides strong evidence that the epitope recognized by

ID _C δTCSl is encoded in the AK119 Vo. gene or in a combinatorial epitope of the rearranged AK119 V 0-Jo. gene.

0

5

30

35

δTCR rearrangements detected with V-J probe, numbered I-V as in Figure 22.

Only 1 rearrangement was identified in each case even though no germline J. was detected.

A new rearrangement is observed which is not seen in newborn or fetal thymocytes. This rearrangement has been assigned rearrangement VI.

+ means positive staining, - means negative staining. nd means not determined.

12. DEPOSIT OF HYBRIDOMAS The following hybridoma cell lines, producing the indicated monoclonal antibody, have been deposited with the American Type Culture Collection (ATCC) , Rockville, Maryland, on the indicated dates, and have been assigned the listed accession numbers:

The present invention is not to be limited in scope by the cell lines deposited since the deposited embodiments are intended as single illustrations of one aspect of the invention and any cell lines which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

-67-

International Application No: PCT/

MICROORGANISMS

Optional Sh.al In connection with tha mlcroorganiam ralarrad to on paga , Una ol th* deecrlpllon <

A. IDENTIFICATION Of DEPOSIT '

Further dapoalta ara IdantlAad on an . d on... _h ««π' 5 ^{γbridθma (mouse} > splenocytes

Nome ol dapoaltary Inatllutlon * Ui fid wi th my ^p l nma P3x63Ag8 . 653

^ TCAR-3

American Type Culture Collection

Addraaa ol dapoaltary Inalltutlon (Including poatal coda and country) *

12301 Parklawn Drive Rockville , MD 20852:

Data ol depoalt ' Aceβatlon Number *

October 29 . 1 Q R 7 HB 9578

B. ADDITIONAL INDICATIONS ' (laava blank II not applicable). Thlt Information la continued on a aaparate attached aheet ~~~]

C. DESIGNATED STATES FOR WHICH INDICATIONS ARK MAOE > (If the Indlcallona ara not far all deelgnatad State*)

D. SEPARATE FURNISHING OF INDICATIONS » (laava blank If not applicable)

Tha Indlcallona Hated below will be aubmltted to the International Bureau later • (Specify the general nature ol the Indlcallona e.g., " Aέcaiiion Number of Depoalt ")

E. ~~~ Thl> ih... waa received with the International application whan filed (to be checked by the receiving Office)

(Authorized Officer)

[ [ Th* del* of receipt (from tht applicant) by th* International Bureau •

(Authorized Officer)

Form PCT/RO/134 (Januerγ lflβl)

International Application No: PCT/

MICROORGANISMS

Optional Sheet In connection with the microorganism referred to on page— _.___-, H ne _—.__— .—_ of th* deecrlptloπ *

A. lOtNTiriCATION OP DEPOSIT *

Further depoalle are Identified on an additional the* I 53' Hybridoma , R2B7

Nam* ol depoeltary Institution *

American Type Culture Collection

Addreee ol depoeltary Inalllutlon (Including poatal code and country) *

12301 Parklawn Drive Rockville, MD 20852

Dale of dapαall * Aceaealon Number •

September 28 . 1988 HB 9842

B. ADDITIONAL INDICATIONS ' (leave blank If not applicable). Thle Information la eontlnuad on a aaparat* attached ehaet ~~~

C. DESIGNATED STATES FOR WHICH INDICATIONS ARC MADE > (If th* Indlcatlona are not lor all dealgnatad Statee)

D. SEPARATE FURNISHING OF INDICATIONS • (leave blank If not applicable)

The indication* Haled below will be aubπllted lo Ihe International Bureau later ■ (Specify the general nature ol th* Indlcallona e.g.. ^•• A eaeaion Number of Depoall")

E. r~l Thl» aheet waa received with the International application whan filed (to be checked by the receiving Office)

(Authorized Officer) [ j The data of receipt (from the applicant) by tha fnterneltonel Bureau la

(Authαrlted Officer)

Form PCT.RO. I3< (January MSI)

-69-

International Application No : PCT/

Form PCT/RO/134 (January tail)

Further Depositst Date of Deposit Accession Number

Hybridoma, 8F4 September 28, 1988 HB 9843 Hybridoma #3 July 27, 1988 HB 9773

Deposited To:

American. Type Culture Collection 12301 Parklawn Drive Rockville, MD 20852