The use of transgenic mice for the production of a human monoclonal antibody specific for human CD69 antigen

Journal of Immunological Methods 282 (2003) 147 – 158 www.elsevier.com/locate/jim

The use of transgenic mice for the production of a human monoclonal antibody specific for human CD69 antigen Ana Molina a,1, Mo´nica Valladares a,1, Susana Magada´n a, David Sancho b, Fernando Viedma b, Irene Sanjuan c, Francisco Gambo´n c, ´ frica Gonza´lez-Ferna´ndez a,* Francisco Sa´nchez-Madrid b, A a

A´rea de Inmunologı´a, Facultad de Ciencias, Universidad de Vigo, Lagoas-Marcosende s/n 36200 Vigo, Pontevedra, Spain b Servicio de Inmunologı´a, Hospital de la Princesa, Universidad Auto´noma de Madrid, 28006 Madrid, Spain c Unidad de Inmunologı´a, Hospital do Meixoeiro, 36200 Vigo, Pontevedra, Spain Received 15 May 2003; received in revised form 11 August 2003; accepted 14 August 2003

Abstract The CD69 antigen is the earliest activation marker expressed on leukocyte surfaces after stimulation and it has been correlated with disease state in a variety of inflammatory and autoimmune diseases. We were interested in the generation of a human monoclonal antibody (mAb) against the CD69 antigen. To do this, mice carrying human Ig transgenes (on an inactivated endogenous immunoglobulin H and Ign background) were immunized with rat cells transfected with the human CD69 molecule. From over 2000 hybridoma clones generated in different fusions, we were able to obtain a human monoclonal antibody, hAIM29, which specifically recognizes human CD69 on the surface of activated-human leukocytes. We demonstrate that the antibody is specific for the human CD69 molecule, as shown by double staining with mouse anti-human CD69 antibodies, ELISA, immunoblot and immunoprecipitation studies. Results of additional experiments show that hAIM-29 activates intracellular calcium influx without Ig cross-linking and enhances phorbol myristate acetate-induced cell proliferation in a manner similar to other mouse anti-CD69 antibodies. This report is the first to describe the isolation and characterization of a novel human mAb, hAIM-29, which may have therapeutic potential in diseases associated with the presence of activated cells expressing CD69 antigen. D 2003 Elsevier B.V. All rights reserved. Keywords: Human CD69; Human monoclonal antibodies; Transgenic mice

1. Introduction

Abbreviations: mAb, monoclonal antibody; PMA, phorbol myristate acetate; HuIgM, human immunoglobulin M; PBMC, peripheral blood mononuclear cells; RA, rheumatoid arthritis. * Corresponding author. Tel.: +34-986-812625; fax: +34-986812556. E-mail address: [email protected] (A. Gonza´lez-Ferna´ndez). 1 A.M. and M.V. have contributed equally to this paper. 0022-1759/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2003.08.007

The CD69 antigen is one of the earliest inducible cell surface glycoproteins acquired during lymphoid activation. After cell stimulation, CD69 is expressed on the surface of natural killer (NK) cells (Lanier et al., 1988), eosinophils (Hartnell et al., 1993), and lymphocytes (Cebria´n et al., 1988). CD69 is a member of the NK gene complex family of type II oligomeric signal

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transmitting receptor proteins that contain a C-type lectin-binding domain. It is expressed as a disulfidelinked homodimer (Santis et al., 1994; Lo´pez-Cabrera et al., 1993; Ziegler et al., 1993). Although the putative ligand(s) for CD69 remains unknown, its cross-linking transduces intracellular signals that generate a variety of cellular responses, including an increase in intracellular Ca2 +, secretion of cytokines, and cellular proliferation (Santis et al., 1992; Cebria´n et al., 1988). The expression of CD69 on the surface of T cells has been related to a variety of disorders. These include inflammatory diseases such as rheumatoid arthritis (RA) and chronic viral hepatitis (Laffo´n et al., 1991; Garcı´aMonzo´n et al., 1990), and also autoimmune diseases such as systemic lupus erythematosus and insulindependent diabetes mellitus (Crispı´n et al., 1998; Gessl and Waldha¨usl, 1998). Moreover, the expression of CD69 has been correlated with disease activity and clinical behavior in both RA and B-cell non-Hodgkin lymphoma (Erlanson et al., 1998; Iannone et al., 1996). Hence, CD69 may represent a target for immunotherapy in diseases where leukocytes express high levels of this activation marker. Although murine monoclonal antibodies (mAbs) are relatively easy to produce and some mouse mAbs anti-human CD69 are now available, their therapeutic use in humans is restricted by their immunogenicity and the associated reduction in efficacy and safety (Jakobovits, 1995). These restrictions could be overcome by the use of fully human mAbs which would allow their repeated administration without immunogenic and/or allergic responses. Thus, the development of transgenic mice strains, engineered with unrearranged human immunoglobulin (Ig) genes, has become an attractive strategy for the generation of specific human mAbs. The procedures for the isolation of human mAbs are identical to those used for conventional mouse immunization and hybridoma production (reviewed in Bru¨ggemann and Taussig, 1997; Bru¨ggemann and Neuberger, 1996; Jakobovits, 1995). The five-feature BABn, E transgenic mouse strain used in this work, carries transgenes of human A heavy and n and E light chains on an inactivated endogenous IgH and Ign background (Magada´n et al., 2002; Nicholson et al., 1999). The immunization of these transloci mice permits the generation of an immune response that induces the production and secretion of human IgM (HuIgM) Abs against a wide variety of

human and non-human antigens (Ags) (Magada´n et al., 2002). In this report, we describe the use of those mice to generate a novel HuIgM mAb, hAIM-29, which specifically binds to the human CD69 Ag. Data are presented demonstrating some of the functional properties of hAIM-29 and the pattern of its specific cellular recognition including CD69 transfected cells and activated human peripheral blood mononuclear cells (PBMC). The clinical implications of these data are discussed.

2. Materials and methods 2.1. Transgenic mice The HuIgM-producing transgenic mice used in this study (BABn, E) were kindly provided by Dr. Marianne Bru¨ggemann (Babraham Institute, Cambridge, England). The generation and characteristics of these five-feature BABn, E mice have been previously described (Magada´n et al., 2002; Nicholson et al., 1999). Briefly, BABn, E mice carry the human IgH, Ign and IgE YAC transgenes and had been bred to homozygosity with mice in which the endogenous IgA locus and the Ign locus were rendered non-functional by gene targeting in embryonic stem cells (Kitamura et al., 1991; Zou et al., 1995). Animals were bred and maintained in a homozygous state and housed in a pathogen-free environment and used according to institutional guidelines which are in compliance with the NIH Guide for the Care and Use of Laboratory Animals. 2.2. Cell lines and human cell samples RBL-2H3 rat basophilic leukemia cells (RBL-WT) and the stable transfectant of a chimeric human CD69/ CD23 molecule (RBL-CD69) were both cultured in DMEM (Gibco-BRL, Life Technologies, Grand Island, NY) supplemented with 5% foetal calf serum (FCS) (PAA, Linz, Austria). The chimeric CD69/CD23 protein of 40 kDa contains the extracellular region of the human CD69 and the cytoplasmic region of the human CD23 (Sancho et al., 2000). In the case of RBL-CD69 cells, the medium was supplemented with 25 Ag/ml of geneticin (Gibco-BRL). The human leukemic T cell line Jurkat was grown in RPMI 1640 supplemented

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with 10% FCS. All culture media used were supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml) and 2 mM glutamine. All cell lines were grown at 37 jC in a humidified 5% CO2 incubator. Human PBMC were isolated from fresh peripheral blood of healthy volunteer donors using density centrifugation on Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden). The cells were washed in cold phosphate-buffered saline (PBS) twice and resuspended in RPMI 1640/10% FCS. Cells were counted using a hemocytometer and adjusted at the desired concentration. The viability was routinely greater than 95% as determined by trypan blue exclusion. 2.3. Immunization, cell fusion, and screening The BABn, E transgenic mice were immunized with RBL-CD69 cells. The animals received a primary injection with 107 cells emulsified with Complete Freund’s Adjuvant (CFA) (Sigma, St. Louis, MO) and 2 –3 weeks later, a secondary injection was performed using the same dose of cells emulsified with Incomplete Freund’s Adjuvant (IFA) (Sigma). Sera from immunized mice were obtained and monitored for the presence of specific antibody using flow cytometric analysis of serum treated with RBL-CD69 cells as described below. Two weeks after the second immunization, an i.v. boost with 2  106 RBL-CD69 cells in PBS was administered, and 3 days later animals were sacrificed and their spleens were removed. The spleens were teased to isolate splenocytes, which were washed and used for fusion (Ko¨hler and Milstein, 1975) with NSO mouse myeloma cells. Fused cells were suspended in DMEM supplemented with 20% FCS plus hypoxanthine –aminopterin –thymidine (HAT) (Sigma) and plated in 24-well plates (Corning, Cambridge, MA) at different cell concentrations. Supernatants of hybridomas from 24-well plates were first screened for human anti-RBL-CD69 IgM secretion by flow cytometry. Clones from positive wells were transferred and plated in 96-well plates (Corning). Supernatants from individual hybridomas were tested by ELISA for the secretion of HuIgM, and the positive wells were tested further by flow cytometry using RBL-CD69 and RBL-WT cells. Those hybridomas that were positive for RBL-CD69 but negative for RBL-WT cells were then subcloned by limited dilution at least three times.

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2.4. Enzyme-linked immunosorbent assays (ELISA) The screening of hybridomas secreting specific antihuman CD69 Abs was performed using recombinant CD69 (CD69-Fc) as described previously (Llera et al., 2001). Aliquots (50 Al) containing 2 Ag of CD69-Fc were dispensed into each well of 96-well polystyrene plates. The plates were incubated overnight at 4 jC, and then wells were blocked by addition of 200 Al of PBS/1% FCS per well, and incubated for 2 h at room temperature. The wells were washed twice with PBS/ 0.05% Tween 20, the hybridoma supernatants (50 Al/ well) were added and the plates incubated for 2 h at 4 jC. This was followed by washing and incubation with biotinylated goat anti-HuIgM (50 Al/well at 1/5000 dilution) (Jackson Immunoresearch, Pennsylvania, USA) for 2 h at 4 jC. After washing, streptavidin – HRP was added (50 Al/well at 1/4000 dilution) (Dako, Golstrup, German), and the plates were incubated at room temperature for 15 min. As positive controls, the murine anti-human CD69: TP1/8, TP1/33 and TP1/55 (Cebria´n et al., 1988) (10 –20 Ag/ml) antibodies were used, revealed with secondary HRP-conjugated goat anti-mouse Abs (at 1/2000 dilution) (Sigma). The colorimetric reaction was developed with the substrate ortho-phenylene-diamine, prepared according to manufacturer’s instruction (Sigma) (50 Al/well) for 15 –30 min and stopped by the addition of 50 Al/well of 4 M sulphuric acid. Absorbance at 450 nm was measured in a Multiskan MS reader (Bio-Tek Instruments, Winooski, VT). Each control and test group consisted of three replicate wells. Each experiment was repeated at least three times. 2.5. Flow cytometric analysis Aliquots of 5  105 RBL-CD69 cells were suspended in 50 Al of medium alone or neat hybridoma supernatants. Cells were incubated at 4 jC for 30 min, washed twice with ice-cold PBS and stained with 50 Al of fluorescein isothiocyanate (FITC)-labeled rabbit anti-HuIgM Abs (Dako) (at 1/20 dilution) at 4 jC for 20 min. The stained cells were washed twice with ice cold PBS, and analyzed in an XL-MCL Flow Cytometer (Coulter Electronic, Hialeah, FL). At least 104 cells were acquired by list mode, measurements were performed on a single cell basis, and were displayed as frequency distribution histograms or dot histograms.

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Dead cells and debris were gated out of the analysis on the basis of forward scatter (FS). In analysis involving double staining, the cells were incubated at 4 jC for 30 min in 20 Al of commercial phycoerithrin (PE)-conjugated mouse anti-human CD69 mAb (clone TP1/55, Immunotech, Marseille, France), washed twice with ice cold PBS, and then incubated for 20 min at 4 jC with 50 Al of neat hybridoma supernatant containing the human hAIM-29 mAb. Cells were washed and stained with FITC-rabbit anti-HuIgM Ab, washed and analysed as described above. Positive hybridoma supernatants, as revealed by RBL-CD69 cell staining, were further tested using RBL-WT cells. Those supernatants giving a pattern of RBL-CD69 positive/RBL-WT negative staining were further selected. The isotype of the secreted mAb was determined using FITC-labeled rabbit anti-human E, anti-human n light chains (Dako), or FITC-labeled goat anti-mouse E light chain (Caltag, Burlingame, CA) following similar protocol to that above. As a positive control, cells were treated with 50 Al of neat hybridoma supernatant containing the mouse anti-human CD69 mAb TP1/55, washed and stained with FITC-labeled goat anti-mouse IgG (Caltag) (at 1/ 50 dilution) for 20 min at 4 jC. To test whether human hAIM-29 mAb recognizes the native human CD69 molecule on the surface of activated cells, human PBMC from healthy donors and Jurkat cells were activated for 24 h to induce the expression of CD69. 1  106 cells/well were incubated in 24-well plates in the presence or absence of phorbol myristate acetate PMA (50 ng/ml). CD69 expression on the surface of activated cells was assessed by flow cytometry after treatment with murine anti-CD69 TP1/ 55 mAb (as a positive control) and the hAIM-29 rich supernatant, followed by washing and incubation with the corresponding FITC-conjugated Abs, either as single or as double fluorescence staining. Measurement of intracellular free calcium ([Ca2 +]i) was performed by flow cytometry using the calcium sensitive fluorochrome Fluo-3 (Molecular Probes, Eugene, OR) as previously described (Sancho et al., 2000). Briefly, 2  106 RBL cells (RBL-WT and RBLCD69) were loaded for 30 min at 37 jC with 2 Ag/ml of Fluo-3, then they were washed twice with RPMI 1640 and stimulated with the anti-CD69 TP1/8 mAb (10 Ag/ml) in a final volume of 500 Al, followed by cross-linking with 10 Ag/ml of polyclonal sheep antimouse IgG (Sigma). The mAb anti-CD69 hAIM-29

(10 Ag/ml) was also cross-linked with 10 Ag/ml of the mouse mAb anti-human IgM (DA4/4) (kindly provided by Dr. F. Dı´az-Espada, Clı´nica Puerta de Hierro, Madrid, Spain). 2.6. Immunoblot analysis 107 RBL-WT or RBL-CD69 cells were pelleted by centrifugation at 800  g for 10 min at 4 jC, washed with PBS and resuspended in 1 ml of lysis buffer (10 mM sodium phosphate, pH 7.6; 150 mM NaCl; 10 mM EDTA pH 8.0; 0.5% (v/v) Triton X-100; and 2 mM PMSF, Sigma). Samples were incubated on ice for 30 min and cleared by centrifugation at 10,000  g for 10 min. The amount of protein in the supernatants was measured by the Bradford Colorimetric Method (Bradford, 1976). For immunoblots, 10 Ag of lysate was separated on 12% SDS-PAGE. Proteins were transferred using the Trans-blot Semi-dry apparatus (BioRad Laboratories, Cambridge, MA) onto PVDF membranes (Hybond-P, Amersham Pharmacia Biotech, Buckinghamshire, England). The membranes were incubated overnight at 4 jC, with neat or with 50  concentrated hybridoma supernatant containing hAIM-29 mAb, followed by incubation with alkaline phosphatase-rabbit anti-human IgM Abs (Dako) (1/ 1000) for 2 h at room temperature. Five-bromo-4chloro-3-indolyl-phosphate (BCIP) (Sigma) was used as substrate to visualize specific protein according to the manufacturer’s instructions. For immunoprecipitation, the mouse IgG antiCD69, TP1/55 mAb, was coupled to Protein A (Pierce, Rockford, IL) according to the manufacturer’s instructions. Protein A-TP1/55 was incubated with either RBL-CD69 or RBL-WT cellular lysates. The resulting samples were applied on 12% SDS-PAGE, transferred onto PVDF membranes, and incubated with hAIM-29 as described above. 2.7. Cell proliferation assay PBMC cultures were established in 96-well plates at 2  105 cells in 200 Al of RPMI 1640/10% FCS per well, in the absence or presence of different Abs: mouse anti-human CD45 (D3/9), mouse anti-human CD3 (T3b), mouse anti-human CD69 (TP 1/8) and human anti-human CD69 (hAIM-29), all at a final dilution of 1/4, with or without the addition of PMA (2 ng/ml).

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Triplicate cultures were performed for each treatment and the plates were incubated at 37 jC in 5% CO2 incubator. Cells were pulsed with 1 ACi/well [3H] thymidine (Amersham) for the last 12 h of culture, lysed with water and harvested onto glass fiber filters for determination of [3H] TdR uptake using a h scintillation counter. The data were expressed as the average counts per minute from triplicate cultures F S.D.

3. Results 3.1. Efficient isolation of a human IgM mAb against human CD69 molecule using transgenic mice Transgenic BABn, E mice, able to secrete HuIgM, were immunized with a rat cell line, RBL-2H3, transfected with a chimeric molecule containing the extracellular region of human CD69 (Sancho et al., 2000). Splenic cells from these mice were fused with mouse myeloma cells as described in Materials and methods. Supernatants from HuIgM-secreting hybridomas were tested for the presence of specific Abs directed against human CD69 Ag on the surface of RBL-CD69 and RBL-WT cells by flow cytometry. Of all the hybridomas obtained (more than 2000), only eight showed specific recognition of RBL-CD69 cells whilst being unreactive with RBL-WT. One of these hybridomas, secreting the human monoclonal IgM hAIM-29 Ab, was used to perform additional studies. Results of FACS analysis demonstrated that hAIM29 can bind to RBL-CD69 but not RBL-WT cells, indicating the specificity of hAIM-29 against human CD69. The results also demonstrate the nonspecific binding of D15 – 17 (Fig. 1A). In order to demonstrate the specificity of hAIM-29 for human CD69 Ag and the coincidental recognition with other previously known mouse anti-CD69 mAbs, such as the mouse IgG TP1/55, we performed two-color staining with both Abs using RBL-CD69 as target cells. The perfect correlation in the staining of both Abs strongly suggests that hAIM-29 and TP1/55 bind to the same molecule (Fig. 1B). By contrast, the D15 – 17 mAb, which recognizes both RBL-WT and RBL-CD69 cells (Fig. 1A), did not show any significant correlation in co-staining with the anti-CD69 mouse Ab TP1/55 (Fig. 1B). The hAIM-29 mAb carries a human lambda light chain as shown in the experiment illustrated in

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Fig. 1C, in which secondary antibodies directed against human n or E light chains were used. From this analysis, we concluded that hAIM-29 mAb has the isotype of human IgM/E. Moreover, CD69-specific recognition by the hAIM-29 mAb was also confirmed by immunoblotting using cell lysates from RBL cells. The RBL-CD69 cells are stably transfected with a chimeric protein of 40 kDa consisting of CD23 intracellular and CD69 extracellular domains (Sancho et al., 2000), and were used because of their high level of CD69 expression. As shown in Fig. 2A, hAIM-29 only recognized the CD69 surface antigen on RBL-CD69, but not on RBLWT cells. Furthermore, hAIM-29 revealed the same band (Fig. 2A,b) when CD69 Ag was immunoprecipitated using the anti-CD69 TP1/55 mAb coupled to Protein A. The specificity of hAIM-29 for human CD69 Ag was confirmed by ELISA using human recombinant CD69 (CD69-Fc)-coated plates, as compared directly with the recognition of this recombinant Ag by other known mouse anti-CD69 mAbs such as TP1/55, TP1/ 8 and TP1/33 (Fig. 2B). The results indicate that the hAIM-29 mAb was able to bind to CD69-Fc to a similar extent and specificity as compared with that of the mouse anti-CD69 mAbs TP1/8 and TP1/33, although slightly lower than that of the TP1/55 mAb. One irrelevant HuIgM mAb (h18) was also included in the ELISA procedure as a negative control. 3.2. hAIM-29 mAb recognizes human CD69 Ag Further experiments were carried out to test the ability of hAIM-29 to recognize the native CD69 molecule on the surface of both PMA-activated and non-activated human PBMC. Cells treated with hAIM29 were analyzed by flow cytometry and compared with the pattern obtained with cells treated with antiCD69 TP1/55 mAb, as a positive control (Fig. 3A). The hAIM-29 mAb bound selectively to PMA-stimulated human PBMC, as did the mouse anti-CD69 TP1/ 55 mAb, but not to unstimulated PBMC. Similar experiments performed with Jurkat cells gave comparable results (data not shown). When both Abs hAIM29 and TP1/55 were used in a double immunofluorescence analysis of PMA-activated and non-activated PBMC (Fig. 3B), the staining showed a pattern of corecognition of the same molecule only in PMA-acti-

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Fig. 1. (A) Recognition of human CD69-transfected (RBL-CD69) and non-transfected RBL (wild type, RBL-WT) cells by the human hAIM-29 mAb and the mouse anti-human CD69, TP1/55 mAb. The D15 – 17 is shown as an example of a HuIgM Ab recognizing both, RBL-CD69 and RBL-WT cells. The dashed line represents cells only stained with secondary antibodies (negative control). (B) Two-color staining on RBL-CD69 cells using the commercial mouse PE-conjugated anti-CD69 (TP1/55) mAb and the HuIgM antibodies hAIM-29 (left) and D15 – 17 (right) followed by FITC-conjugated secondary antibodies. (C) Identification of the human light chain present in the hAIM-29 mAb by staining on RBLCD69 cells with FITC-labeled rabbit anti-human E (left) or n (right) secondary antibodies.

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Fig. 2. hAIM-29 mAb recognizes human CD69 by immunoblot and in ELISA. (A) Direct immunoblot analysis of total cell lysates (a) and after antigen immunoprecipitation with Protein A-mouse TP1/55 mAb (b), from RBL-CD69 and RBL-WT cells, subjected to SDS-PAGE and transferred to a membrane for the incubation with the hAIM-29 mAb. In both cases, a band of 40 kDa, corresponding to the CD69/CD23 chimeric protein, was observed only in the RBL-CD69 cells. (B) A comparison between anti-CD69 murine mAbs (TP1/8, TP1/33, TP1/55) and the human hAIM-29 for the specific recognition of human CD69 Ag, analyzed by ELISA using CD69-Fc bound to a 96-well polystyrene plate. The human h18 mAb, which does not recognize CD69, was used as a negative control. Values are the arithmetic mean of absorbance at 450 nm F S.D., obtained from three independent experiments.

Fig. 3. hAIM-29 recognizes PMA-activated human cells in a manner similar to mouse anti-CD69 mAb. (A) Binding of the mouse anti-CD69 TP1/55 and the human hAIM-29 mAb to human PBMC cultured previously in the absence ( PMA) or presence ( + PMA) of the phorbol ester. The FITC-conjugated anti-mouse and anti-human secondary antibodies were used for TP1/55 and hAIM-29, respectively. The dashed line represents cells stained with the corresponding secondary antibodies only (negative controls). (B) Pattern of double staining on unstimulated ( PMA) and stimulated (+ PMA) PBMC, incubated with PE-conjugated TP1/55 and hAIM-29 mAb, followed by FITC-conjugated antiHuIgM antibodies.

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vated PBMC, a result similar to that observed for the CD69 transfectant cells. 3.3. hAIM-29 induces Ca2+ influx and enhancer proliferative activity It is known that CD69 mediates intracellular calcium influx (Testi et al., 1989), as cross-linking of the

CD69 molecule with specific Abs activates this pathway. Accordingly, we performed experiments to test the ability of hAIM-29 to induce calcium influx as compared with mouse anti-CD69 mAb as TP1/8, which is known to induce this activity. The results show that hAIM-29 is very effective in inducing the activation of intracellular calcium influx (Fig. 4). Direct binding of the hAIM-29 mAb without further

Fig. 4. hAIM-29 induces intracellular calcium increase in CD69 transfectants. RBL-WT and CD69 stable transfectant (RBL-CD69) cells were loaded with Fluo3/AM, whilst [Ca2 +]i levels were estimated by flow cytometry, as indicated in the Materials and methods. After 60 s, to determine the basal level of [Ca2 +]i, primary antibodies (hAIM-29, control IgM and TP1/8) were added (indicated by the left arrow in each graph). After 120 s, the secondary Ab (DA4/4 anti-human IgM for hAIM.29 and control IgM, and sheep anti-mouse for TP1/8) was added as a cross-linker (indicated by the right arrow in each graph). A representative experiment, from the three undertaken, is shown. Data are represented as density plot profiles.

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Table 1 Comparison of human PBMC proliferation induced by human hAIM-29 and other mouse mAbsa

Control PMA

Medium

Anti-CD45

Anti-CD3

TP 1/8

hAIM-29

758 F 143 8924 F 937

672 F 184 8223 F 954

12,785 F 1342 71,359 F 5534

927 F 264 57,532 F 4827

942 F 385 37,253 F 3613

a

Human PBMC were treated with mouse mAbs anti-human CD45 (D3/9), CD3 (T3b) and CD69 (TP 1/8), or hAIM-29 in the presence or absence of PMA (2 ng/ml). The results correspond to the arithmetic mean of counts per minute (cpm) F S.D. obtained from three independent experiments.

cross-linking with a secondary Ab was even more effective in the induction of a calcium influx than that produced by the TP1/8 mAb (Fig. 4). However, the cross-linking of the human anti-CD69 hAIM-29 did not result in a further increment of the intracellular free calcium, in contrast to that triggered by cross-linking the TP1/8 mAb. On the other hand, co-stimulation provided by CD69 can also be measured by in vitro proliferation assays (Cebria´n et al., 1988). Results from a representative experiment, shown in Table 1, indicate that hAIM-29 acts as a co-stimulatory agent in the activation of PBMC proliferative activity induced by submitogenic doses of PMA, but it is slightly less effective than the mouse TP1/8 mAb in mediating this effect.

4. Discussion The CD69 antigen is one of the earliest activation markers expressed on the surface of leukocytes after stimulation. The importance of CD69 as a potential therapeutic target has been emphasized by the increasing number of reports demonstrating that expression of CD69 can be correlated with disease activity and clinical behavior (Iannone et al., 1996; Laffo´n et al., 1991) in a variety of inflammatory and autoimmune diseases. Using the BABn, E mouse strain, carrying human Ig transloci in its germ line (Magada´n et al., 2002; Nicholson et al., 1999), we generated a novel human mAb, hAIM-29, directed against the early human activation marker CD69. Stable human CD69 transfectant cells were used as immunogens since it has been shown that the molecular structure and function of the human transfected CD69 molecule on these cells are similar to those of the native CD69 molecule expressed on activated cells (Sancho et al., 2000). Moreover, the chimeric CD69/ CD23 on the transfectant cells (which show a high

expression of the extracellular domain of CD69), and the native CD69 molecule on activated leukocytes are both recognized by specific mouse anti-CD69 Abs, such as the TP1/55 mAb (Conde et al., 1996). Therefore, mouse anti-human CD69 reagents were used as positive controls in all of the screening experiments. The comparison between the pattern of recognition of human mAbs against CD69 transfected and/or to wild type rat cells, permits discrimination between human Abs specific to the CD69 molecule, such as hAIM-29, and antibodies that bind in a nonspecific manner, such as the D15 –17 mAb. The hAIM-29 mAb (IgM/E) described in this report was selected by its high specificity for the human CD69 antigen and by some of its functional properties. We demonstrated the ability of hAIM-29 to recognize the human CD69 molecule both expressed on transfected rat cells (Figs. 1 and 2A) and in the recombinant form (Fig. 2B). The specificity of hAIM-29 was clearly demonstrated in several different assay systems including ELISA, immunoblot and flow cytometric analysis, using previously tested mouse anti-CD69 mAbs as controls. More importantly, we demonstrated that this mAb has the ability to recognize the native CD69 molecule expressed on activated human PBMC (Fig. 3) and Jurkat (data not shown) cells in a manner similar to that of mouse anti human CD69 Ab TP1/55. Interestingly, there was no binding competition (Fig. 3) between the hAIM-29 and TP1/55, indicating that these reagents recognize different epitopes of the human CD69 molecule. Despite its isotype (IgM), the hAIM-29 does not show polyreactivity and its affinity and specificity were comparable to those of other mouse IgG anti-CD69 mAbs. Our results suggest that BABn, E transgenic mice carrying Ig loci can be very useful in the generation of human monoclonal antibodies directed against specific antigens, as shown here for the human CD69 antigen. These human Ig transgenic mice express a limited V-

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gene repertoire and, therefore, whether such animals are capable of giving rise to a large panel of antigenspecific high-affinity human monoclonal antibodies is a matter to be thoroughly investigated. Despite this, theoretically, limited human V-germline gene repertoire, we have also been able to isolate several hybridomas secreting antibodies directed against different types of cells or soluble proteins (Magada´n et al., 2002). This indicates that these transgenic mice are useful for the generation of a variety of high affinity antigen-specific human IgM mAbs, which behave similarly to polymeric IgM from human sera (Magada´n et al., 2002). Although the BABn, E transgenic mice used in this work carry only five different VH genes in their heavy chain transgene, further data from Dr. Bruggemann’s group (Nicholson et al., 1999) and from our own group (unpublished observations), indicates that only one VH gene is sufficient to generate a wide repertoire of antigen-specific antibody responses in these mice. The Ig may be further diversified by imprecise rearrangements of VDJ, by the introduction of N and P nucleotides and by mutations in the V region by somatic hypermutation (Nicholson et al., 1999). In addition to the diversity in the heavy chain, a wide repertoire of light chains (human kappa or lambda) can also be expressed. In fact, although the analysis of the Ig genes of the hybridoma producing the hAIM-29 mAb revealed a low level of mutations in the IgH rearrangement (VH1– 2, D5 –18, JH6), several nucleotide changes were found in the rearranged IgE sequence (preferentially in the complementarity determining region 2 (CDR2) of the VE3 –19 gene used) (unpublished results). The mutations found in the E chain, previously reported in some Abs obtained from these mice by Wagner et al. (1994), seem to be indicative of an affinity maturation of the Ab and an efficient antigen selection in the BABn, E translocus mice. In a series of independent experiments, we studied some of the biological activities of hAIM-29 which may be relevant to its potential clinical use. In addition to its ability to recognize human CD69, hAIM-29 can cause a substantial increase in intracellular calcium concentrations and enhance the PMA-induced proliferative activities of PBMC expressing CD69 surface antigen. These results support a potential clinical use of hAIM-29 in those diseases where the over-expression of CD69 antigen could be related to the pathogenesis of the disease by virtue of the fact that the

antibody could only interact with activated cells overexpressing CD69 antigen. These activated cells may include cells producing inflammatory cytokines or cells producing autoimmune antibodies. Such cells would be selectively targeted by hAIM-29 but not naive immune cells lacking CD69 expression. In this regard, the use of a fully human antibody would be preferred instead of mouse, chimeric or humanized anti-human Abs, which can induce untoward immune reactions in patients (Klee, 2000). Moreover, there might be several advantages in using fully human antibody from transgenic mice, rather than combinatorial libraries carrying human naive variable regions, expressed on the surface of filamentous phages. Transgenic mice generate high affinity antibodies by the in vivo maturation of the immune response, through repeated rounds of hypermutation and antigen selection. However, when using phage display technology to produce antibodies with subnanomolar affinity, an in vitro selection of antigen specific clones, successive rounds of in vitro mutagenesis, V gene shuffling and panning have to be carried out (Vaughan et al., 1998). In addition, to confer complete functional activities to monoclonal antibodies generated by phage display technology, further DNA manipulations are required to include the Ig Fc region, whereas human antibodies from transgenic mice carry both the ability to recognise antigen and also express effector functions. Regarding the potential clinical application of the hAIM-29 mAb here described, two very recent papers using CD69-null mice indicate that the molecule CD69 could be a therapeutic target for patients with arthritis (Murata et al., 2003) or even for those with cancer (Esplugues et al., 2003). The local administration of hAIM-29 alone or conjugated to therapeutic drugs against CD69 overexpressing cells, for example those in the inflamed joints of RA patients, may be considered as one of its potential clinical applications. The Ab hAIM-29 could be used alone or as a radioimmunotherapeutic agent via the linking of h-emitting radionuclides to this CD69 specific antibody (Linenberger et al., 2002). Alternatively, hAIM-29 could be conjugated with radioisotopes for the in vivo localization of CD69 + cells (Harwood et al., 1999), or used ex vivo to monitor the presence of CD69 overexpressing cells in clinical samples obtained from rheumatoid arthritis patients undergoing therapy. hAIM-29 could also be used in diagnostic procedures.

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Overall, we describe the isolation of a novel human mAb, hAIM-29, with specificity for the human CD69 antigen. The work represents an important step in the development of fully human mAbs with potential clinical application. The availability of this antibody will allow further investigations to assess its potential as a diagnostic and therapeutic agent.

Acknowledgements We thank Dr. Marianne Bru¨ggemann for supplying the transgenic mice used in this work and for all her help, and also Eva Amorı´n and Angel Torreiro for their assistance with the maintenance of the mice. We thank Dr. Refaat Shalaby for helpful discussions and editorial comments and also to Teresa Carretero and Ted Cater for reviewing the manuscript. This work was supported by Instituto de Salud Carlos III (Red del FIS G03/136), European Union grant and Ministerio de Ciencia y Tecnologı´a, Spain. A.M. was supported by a Biotechnology grant from the European Union. M.V. and S.M. were supported by the Ministerio de Sanidad y Consumo and I.S. by Xunta de Galicia.

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