Phytochemistry, Vol. 29, No. 7, pp. 2109-2114,1990. 0

Printed in Great Britain.

MONOCLONAL CATHARANTHUS

MICHELLE

CONAN

CIBOTTI,

0031-9422/90 %3.00+0.00 1990Pergamon Press plc

ANTIBODIES TO BIS-INDOLE ALKALOIDS OF ROSEUS AND THEIR USE IN ENZYME-LINKED IMMUNO-SORBENT-ASSAYS

CHRISTIAN

FREIER,*

JEAN ANDRIEUX,~

MICHEL

PLAT,t

LOUIS

COSSON

and CLAUDE

BOHUON* Laboratoire de Botanique et Phytochimie, Fact&C de Pharmacie, 92296 Chltenay-Malabry Cedex, France; *Laboratoire de Biologie Clinique et Expkrimentale, Institut Gustave Roussy, 94805 Villejuif Cedex, France; YLaboratoire de Chimie Thtrapeutique II, CNRS, U.R.A. n”496, Facultt de Pharmacie, 92296 Chltenay-Malabry Cedex, France (Received in revised form I November 1989)

Key Word Index-Catharanthus roseus; Apocynaceae; monoclonal antibodies; enzyme-linked-immuno-sorbentassay; hapten-protein conjugates; immunization; somatic cell fusion; bis-indole alkaloids.

Abstract-High affinity monoclonal antibodies directed against bis-indole alkaloids from Catharanthus produced. Once characterized, they were used to develop sensitive enzyme-linked-immuno-sorbent-assays,

roseus were

enabling us

to determine minute quantities of alkaloids related to vinblastine in plant material. INTRODUCTION

Vinblastine (VLB) and vincristine (VCR), alkaloids from Catharanthus roseus G. Don, had been used for many years in the treatment of various neoplastic diseases [ 11. However, their use is clinically limited due to their secondary effects such as the myelosuppression caused by VLB and the neurotoxicity of VCR [2]. The improvement in the hemi-synthesis of VLB [3] lead to new compounds with oncolytic activities, such as vindesine (VDS) [4] and navelbine (NVB) [S]. Anhydrovinblastine (AVLB), an intermediary compound found in the hemi-synthesis of VLB, had proved to be a natural product [6]. The detection of this latter molecule explained the last steps of the biosynthesis of bis-indole alkaloids, especially in plant tissue culture [7]. We chose to study the bis-indole alkaloids produced by cell cultures in an attempt to find new bis-indole alkaloids. To detect such compounds, we needed a probe with high sensitivity and specificity for molecules related to VLB. Monoclonal antiobdies (Mabs) fulfilled these requirements. Numerous radio-immunoassays (RIA) have been developed to investigate pharmacokinetics or drug levels in biological samples, for specific molecules such as VLB, VCR, VDS, NVB [8-121. Two enzyme-linked-immunosorbent-assays (ELISA), for VLB and VCR, using polyclonal antibodies, were also described [13, 143. In this paper, we report the production and characterization of two high-affinity Mabs directed against bisindole alkaloids related to VLB and their use in sensitive and specific ELISA methods. The application to plant material is discussed. RESULTS AND DISCUSSlON

Preparation of hapten-protein conjugates.

To confer immunological properties to VLB, we linked this hapten to bovine serum albumin (BSA). The link

between the hapten and the carrier protein is one of the most important factors influencing antibody affinity and specificity. Therefore we coupled VLB at different positions using two distinct coupling reactions. In the first series of conjugates, VLB was converted to 3-deacetyl-VLB-monohydrazide as described by Barnett et al. [15], prior to linking it to the protein by using the bifunctional reagent glutaraldehyde (see Experimental). The number of VLB molecules bound to the protein was determined by UV analysis and was estimated to be ca 17 for the VLB,,-BSA conjugate and ca 2 for the VLB,,-PO (peroxidase) conjugate. The second series of conjugates was obtained via a Mannich reaction as described by Teale et al. [9], using slight modifications (see Experimental). An activated aromatic position, adjacent to carbons bearing a methoxy and an amino group on the VLB molecule and primary amines on the BSA molecule reacted with the formaldehyde, to form a conjugate. We failed to obtain another conjugate between the precursor of the half moiety of VLB: catharanthine and BSA. Regarding this, and also the chemical reactivity of the VLB molecule, we supposed that the coupling reaction occurs at the C-17 position on the VLB molecule. By UV analysis, it could be estimated that ca 13 molecules of VLB were linked per one molecule of BSA in this VLBc,,-BSA conjugate. Production of the Mabs Immunization. Six-week-old Balb/c mice were injected subcutaneously using either VLB,,-BSA or VLB,, ,-BSA conjugate (at a dose of ca 25 pg of hapten), emulsified in

Freund’s complet adjuvant. At least three booster injections of the same dose of conjugate emulsified in Freund’s incomplet adjuvant were administred at two week intervals, via an intraperitoneal route. The sera were tested for the presence of specific antibodies by RIA, four days after each injection (see Experimental). A final boost was given via an intravenous route, three days before the somatic cell fusion.

2109

M. C. CIBOTTI et al.

2110

Somatic cellfusion. In the somatic cell fusion, splenocytes from immunized mice were fused in the presence of PEG 1000 with NS-1 mouse myeloma cells (ratio spleen cells/NS-1 myeloma cells = ca 4: l), following the classical procedure described by Kijhler and Milstein [16]. Hybridoma cells were developed into 96 well cultureplates. After two weeks, the wells were screened for cell growth and supernatants were tested by RIA for the presence of specific antibodies. In the first fusion, using splenocytes from mice immunized with the VLB,,-BSA conjugate, 277 of the 476 wells were positive for cell growth (58%) and 7 wells contained hybridomas secreting antibodies specific for VLB (2.5%). In the second fusion protocol which used splenocytes from mice immunized with the VLBc,,-BSA conjugate, 111 of the 188 wells were positive for cell growth (59%), one hybridoma was secreting specific antibodies (0.9%). One hybridoma out of first fusion was selected for its capacity to grow vigorously and presenting high titer and good apparent affinity. After subcloning by a classical limiting dilution protocol, hybridomas were amplified by growth as mouse-ascites. Two Mabs, named VLB 01, for the first fusion and VLB 02 for the second fusion have been purified from ascites by means of chromatography on proteine A-Sepharose.

= aB + b), where B is bound [G-‘HI-VLB, and F is free [G-3H]-VLB, with a slope of -K,, were obtained. The regression equations were respectively: B/F = - 1.540 B +2.744 with r=0.836 and &=1.5x lOg Me’ for VLB dl and B/F= -0.826 B~2.161 with r=0.993 and K,, =0.8 x 10’ M-’ for VLB 02. These affinity constants are similar to those described for antibodies secreted by mouse x mouse hybridomas [IS]. Isotypiny. Isotype determination was performed on ascitic Huid. The method was based on a sandwich ELISA, which used anti-isotype-antibodies. Fixed RabbitAnti-Mouse immunoglobulins (RAM-IgG,; RAMIgG,,,; RAM-IgG,; RAM-IgM) were detected by peroxidase labelled Swine-Anti-Rabbit antibodies. VLB 01 and VLB 02 were determined to be a IgG,,, and a IgG,, respectively. Specijicity. The specificity of the binding sites for VLB and other related bis-indole alkaloids (Fig. 1; Table 1) was studied for both VLB 01 and VLB 02, in each respective ELISA system (see Experimental). The concentration of VLB or related indole alkaloids (r), that provoked 50% inhibition of the binding (CI,,%) was determined, using concentrations varying from lo- lo to 10e4M. The cross-reaction index (I) was calculated as follows:

I=cIso%of Characterization of the Mabs.

VLB (nM) x MvLB ~ x 100%. x M,

CI 5oX of VLB,(nM)

AfJinity constant (K,). The affinity constant was studied using Scatchard plot [17], i.e. the binding of various amounts of [G-3H]-VLB by a constant amount of antibodies was determined. Two linear Scatchard plots (B/F

The results are summarized in Table 1. Each index is a mean of three determinations. It is noteworthy that the two Mabs did not cross-react with the monomeric forms tested, particularly catharan-

Leurosine

Meo&g!$ R3

R’

R’ R3

R2

Vinblastine

C02Me

OCOMe

Me

Vincristine

TOzMe

OCOMe

CHO

Vindesine

CONHz

OH

Me

Vincathicine Fig.

1. Chemical

structures

of bis-indole

alkaloids

related

to VLB.

Alkaloids of Catharanthus

roseus

2111

Table 1. Specificity of the Mabs VLB 01 and VLB 02 for alkaloids related to VLB (the index value is expressed as percentage of cross-reactivity) VLB 01 Dimeric compounds 1. VLB (vinblastine) 2. 4-Deacetyl-VLB 3. 4-Deacetoxy-VLB 4. 4-Deacetyl-vinblastineoic acid 5. VDS (vindesine) 6. 4-Acetyl-VDS 7. VCR (vincristine) 8. N,-Deformyl-VCR 9. 4-Deacetyl-6,7-dihydro-VLB 10. Leurosine 11. Leurosidine (4’~egi-VLB) 12. AVLB (3’,4’-anhydrovinblastine) 13. VLB-N,.-oxide 14. NVB (nor-7’-anhydro-VLB) 15. 4’-Epideoxy-9’,19’-cycle-VLB 16. Vincathicine 17. 18’-Decarbomethoxy-4-deacetyl-VLB

100.0

hydrazide

Monomeric compounds 18. Catharanthine 19. Vindoline 20. Ajmalicine 21. Serpentine 22. Ibogaine 23. Indole-3-acetic acid 24. Tryptamine 25. Tryptophan

thine and vindoline, the precursors of VLB, but also ajmalicine, serpentine, ibogaine, indole-3-acetic acid, tryptamine and tryptophan (18-25). The cross-reaction index for these monomers was always < 10P4%. On the other hand, several of the tested bis-indole alkaloids are well recognized. Both Mabs react with VLB (I), leurosine (lo), leurosidine (ll), AVLB (12), N,-deformyl-VCR (8) and VCR (7); the latter molecule showed a slight decrease in binding. The cross-reaction study revealed a distinct specificity of the two Mabs. This difference in specificity seems to be closely related to the conjugates used for the immunization of the mice. The position of the link between VLB and the carrier protein (BSA) appeared to be important. In the VLB,,-BSA conjugate, the positions C-3 and C-4 seem to be ‘poorly’ exposed to the immune system, when compared with the VLBc,,-BSA conjugate. Therefore, the binding capacity of VLB 02 seems to be more affected by modifications in this part of the molecular structure of VLB, than for VLB 01 (see index for compounds 2-6). VDS, for example is well recognized by VLB 01 (I =Sl.l%) but only weakly by VLB 02 (1=2.7%). Changing substituents at the N-l position (compounds 7, 8) results in minor modification of the binding to these molecules. The low value of the index for compound 9 underlines the positive involvement of the C-6/C-7 double link in the antigen-antibody interactions. Compared to compound 2, a ca lo-fold weaker binding is observed for both

21.9kO.5 21.6kO.7 34.5 + 2.4 81.1 k 1.6 176.0+ 10.0 72.2 + 7.2 119.0+ 15.5 3.lkO.3 99.8 k 9.4 105.5+ 8.1 171.7k6.2 32.7+2.5 3.4+0.4 4.2*0.3 0.057 + 0.008 0.010+0.001

Monoclonal antibodies to bis-indole alkaloids of Catharanthus roseus and their use in enzyme-linked immuno-sorbent-assays.

High affinity monoclonal antibodies directed against bis-indole alkaloids from Catharanthus roseus were produced. Once characterized, they were used t...
628KB Sizes 0 Downloads 0 Views