Journal oflmmunological Methods, 144 (1991) 35-41

35

© 1991 Elsevier Science Publishers B.V. All rights reserved 0022-1759/91/$03.50

JIM 06098

Synthesis of a monoclonal antibody-indium-111-porphyrin conjugate Catherine H. Bedel-Cloutour 1, Lilly Maneta-Peyret and Jean-Henri Bezian 2

2, Michel Pereyre

1

Laboratoire de Chimie Organique et Organomdtallique, URA 35 CNRS, Universitd Bordeaux I, 351, cours de la Liberation, 33405 Talence Cedex, France, and 2 Laboratoire d'Immunologie, Universitd Bordeaux 11, rue Ldo Saignat, 33076 Bordeaux Cedex, France

(Received 22 November 1990, revised received 19 April 1991, accepted 28 June 1991)

Antibodies were labelled with indium-ill with a view to their use in the radio-immunodetection of cancers. The covalent coupling between indium-ill porphyrin and monoclonal antibodies (IgG and F(ab') 2 fragment) was achieved using the ester activated method [N-hydroxy-succinimide/1-ethyl-3-(3dimethylaminopropyl)carbodiimide]. After purification, this provided conjugated with specific activities of 6/zCi//xg Mab (9.3 molecules per Mab) or 1 /zCi//xg (F(ab') 2 fragment (1.5 molecule per F(ab')2). ELISA procedures suggested the full retention of immunoreactivity by the radiolabelled antibodies. Key words: Monoclonal antibody; lIllndium; Conjugation; Porphyrin

Introduction

The main objective of both animal and human studies using radiolabeled antibodies for radioimmunodetection of cancer is to identify occult tumor not evaluable by other standard techniques

Correspondence to: C. BedeI-Cloutour, Laboratoire de Technologie Enzymatique, URA 523, BP 649, 60206 Compi~gne Cedex, France. Abbreviations: BSA, bovine serum albumin; DMF, dimethylformamide; DMSO, dimethylsulfoxide; DTPA, diethylenetriaminepentaacetic acid; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCI; EDTA, ethylenediaminetetraacetic acid; IgG, immunoglobulin G; Mab, monoclonal antibody; NHS, N-hydroxysuccinimide; PBS, phosphatebuffered saline; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; TLC, thin layer chromatography; TTCpp111InCI,5-(p-carboxyphenyl)-10,15,20-tritolylporphyrin indium-ill chloride; TTCPPH2, 5-(p-carboxyphenyl)10,15,20-tritolyl porphyrin.

(e.g., radiology, serology). The progress made in monoclonal antibody technology has given an added impetus to the use of specific antibodies as carriers of radioactive molecules for tumor imaging. However, these radioactive molecule/ monoclonal antibody conjugates must meet the criteria for acceptable radiopharmaceuticals used for radio-immunodetection. Firstly, the radionuclide should produce a high number of photons per dissociation event with a decay energy of greater than 120 keV and a short half-life (24-72 h). Among the radionuclides utilized, indium-ill has many advantages for radioimmunodetection and fulfills these conditions. (gamma-emitting agent, 247 keV, 2.8 days), and is readily detected by scintillation counters. Secondly, the conjugate tracer/Mab must be stable in vivo and the coupling process must not modify the antibody specificity nor cause any substantial reduction in antigen binding.

36

Numerous approaches have been developed using bifunctional chelates that are covalently bound to the antibody. These use molecules such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA) and their derivatives (Sundberg et al., 1974; Meares et al., 1976; Halpern et al., 1983; Hnatowich et al., 1983; Meares et al., 1984; Wang et al., 1984; Fawwaz et al., 1985; Paik et al., 1985; Kozak et al., 1986). It is essential for effective imaging that the radioactive metal ion remains complexed to the the Mab-chelator conjugate. With the chelators previously described, there is a competition for metal binding by serum transferrin which is known to remove metal ions from chelates in vivo. Serum exchange studies in vitro and in vivo (Yeh et al., 1979; Paik et al., 1980; Cole et al., 1986) have shown that transferrin displaces indium from its complex. To avoid the possibility of this in vivo process, it is necessary to use a more stable metallic chelate. To this end, we have previously demonstrated that tetrapyrrolic macrocycles of indium, belonging to the class III type (Buchler, 1975) are stable in vivo (Cloutour et al., 1982). Porphyrins can be synthesized with different peripheral functional groups available for covalent coupling with amino acid residues of monoclonal antibodies. We have previously performed model coupling reactions between indium carboxyporphyrins and amino acid esters and defined the best experimental conditions yielding the amide derivatives (Bedel-Cloutour et al., 1987). In the present study, we report the covalent coupling reaction between the indium-Ill derivative of 5-(p-carboxyphenyl)-10,15,20-tritolylporphyrin (T/'CPPIIIInC1) (Fig. 1) and bovine

serum albumin (BSA), monoclonal antibodies to thyroglobulin VIF11 (Mab VIFll), BSIII (Mab BS III), BB3 (Mab BB3), VB10 (Mab VB10) and F(ab')2fragments of Mab V B10.

Materials and methods

BSA and antibodies BSA was obtained from Sigma Chimie and was used without further purification. Mouse monoclonal anti-human thyroglobulin antibodies were prepared in our laboratory and purified using protein A. The Mab containing solution (culture supernatants) was loaded onto protein A-Sepharose CL-4B column. The column was then washed with four void volumes of 0.1 M PBS buffer, pH 8. The retained IgG was eluted with 0.1 M citrate buffer pH 5.5. The pH of the IgG solution was immediately increased to 7.4 using a solution of 0.1 M NaOH. The final product was dialyzed against 0.01 M PBS buffer, pH 7.4, overnight at 4 ° C and then aliquoted and stored at - 80 ° C. All of the Mabs were IgG1. F(ab')2fragments were prepared by pepsin degradation. The pH of an IgG solution (2 mg/ml in PBS) was lowered to 3.5 with a 1 M citrate buffer (pH 3.5). Pepsin was then added at 25 tzg/ml and the mixture incubated overnight at 37 ° C. Digestion was stopped by raising the pH to 7.4. Peptides of Fc fragments were eliminated by dialyzing the solution against 0.01 M PBS buffer, pH 7.4, overnight at 4 o C. The analysis of a sample by SDS-PAGE electrophoresis suggested that the final solution essentially contained pure F(ab')zfragments.

Indium-Ill

M~ e C O O H Fig.

1. Structure of 5-(p-carboxyphenyl)-10,15,20-tritolylporphyrinato indium chloride, 1.

Indium-Ill from CIS BioIndustrie was obtained as indium trichloride (lllInC13) in 0.05 M HC1 (10 mCi/ml; specific activity: 4.6 × 105 mCi/mg).

Synthesis and purification of TTCPP mlnCl 5-(p-carboxyphenyl)- 10,15,20-tritolylporphyrin (TTCPPH 2, 1) was synthesized and purified as previously reported (Bedel-Cloutour et al., 1987 and references therein).

37 A standard solution of T T C P P H 2 was made in glacial acetic acid containing 1% trifluoro-acetic acid (1.57 x 10 -4 M ) a n d protected from light. All insertions were performed using aliquots of this solution (250/xl corresponding to 27.5/.~g of porphyrin 1). A solution of 2 mCi H~InC13 was placed in a tube and evaporated to dryness under a stream of nitrogen. Then a 250 /zl aliquot of T I ' C P P H 2 solution was added (4 x 10 -2 p, mol). The mixture was heated to 140 ° C for 2 h. Once the reaction completed, solvent was taken off under reduced pressure. In order to completely remove unreacted carrier-free 11~In, the crude product was dissolved in chloroform and washed several times with distilled water to neutrality. Chloroform was then evaporated under vacuum. As shown by TLC [silica gel microplates, 2.5 × 10 cm; eluent : c h l o r o f o r m / m e t h a n o l 95/5; revelation: autoradiography with radiologic films (Kodak Definix Medical Film)], the chloroform extract did not contain any carrier-free H~In. T T C P P H 2 and T T C p p I ~ I n C I were the only components present. The yield of metallation (97%) was determined by gamma counting of ~lIn radioactivity in the extract. The synthesized tracer had a specific activity of 69/xCi//xg.

Synthesis of mln-porphyrin-BSA conjugate TTCPPHIInC1 was obtained as previously described starting with T T C P P H 2 (9 x 10 -2 /zmol) and 111INC13 (200 /xCi). The labelled product (mixture of indium porphyrin and free base porphyrin) was dissolved in a 2 M solution of thionyl chloride in methylene chloride and heated at 7 0 ° C for 1 h. Solvent and unreacted thionyl chloride were then eliminated under reduced pressure. The dry residue was dissolved in DMSO (30 /xl) and 9 x 10 -3 /xmol of BSA in 1.5 ml sodium bicarbonate 0.1 M, pH 8.5 was added. The mixture was stirred at room temperature, in the dark, for 2.5 h. After reaction, the coupling yield was determined after electrophoresis on agarose film (Corning Medical, Palo Alto, CA) (eluant: barbital buffer 0.1 M, pH 8.6). Under these conditions, 44% of the radioactivity was detected on BSA,

corresponding to 4.4 molecules of porphyrin, per BSA molecule.

Synthesis of mln-porphyrin-Mab conjugates Two types of activation for coupling the carboxylic function of T T C P P HIlnC1 (69 /~Ci//xg) with Mab were carried out: either activation in acid chloride, as described above, or using

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, HC1 and N-hydroxysuccinimide (Roberts et al., 1987). Acid chloride activation. The experimental conditions for activation in acid chloride were the same as for BSA coupling. Once obtained, the acid chloride derivative (2 x 10 -2/xmol) was dissolved in DMSO (50/zl) and a solution of Mab in PBS was added (0.2 x 10 -2 /zmol). The mixture was maintained under stirring at room temperature for 1 h and the labelled Mab was purified on a 1.2 X 12 cm Sephadex G15 column using a flow rate of 0.28 m l / m i n (eluant; PBS). Flow rates were controlled with a Microperpex peristaltic pump (LKB Bromma). Automatic scanning at 280 nm was used to measure protein absorbance with a spectrophotometer 2238 UVI cord S II (LKB Bromma). The r a i n radioactivity in each fraction (480/xl) was measured by gamma counting. Radioactive Mab fractions were pooled. The percentage of radioactivity coupled with Mab (40.5%) corresponded to four molecules of TTCPPH~InC1 covalently linked to Mab. In this case, the specific activity of the conjugate was 2.4 /~Ci//xg. Carbodiimide activation. Once metallated as described above the mixture T T C P P H 2 / TTCpp111InC1 (2 x 10 -2 /zmol) was dissolved in D M F (50/xl). To this solution, were added: 8.7/~1 NHS solution (2.29 x 10 -3 M) and 13.25/zl EDCI solution (1.5 x 10 -3 M) both in 12.5 mM sodium phosphate pH 6. The mixture was stirred in the dark, at room temperature, for 1 h before the Mab in PBS was added ( 0 . 2 x 10 -2 /zmol; [porphyrin]/[mab] = 10). The stirred reaction mixture was kept at room temperature, in the dark, for an additional hour. The same protocol, as described previously, was used to purify the labeled Mab. Using these conditions the coupling efficiency was 93% and since the sample was coupled at a 10 : 1 porphyrin : protein molar ratio,

38

the average number of porphyrin groups attached to each Mab molecule was 9.3. Under these optimal conditions, we were able to achieve specific activities of about 6/xCi//zg.

Synthesis of mIn-porphyrin-F(ab')2 (fragment of Mab V BIO) conjugate For this synthesis we followed the same experimental conditions as for Mab coupling but used an F(ab') 2 fragment concentration one-tenth that of porphyrin. In this case, after gel chromatography purification on Sephadex G-15, the coupling efficiency was 15%, corresponding to an average number of porphyrin groups per F(ab') 2 equal to 1.5 and giving to a specific activity of 1/xCi//zg.

ing is avoided. The radioactive indium derivative was obtained with a high specific activity of 69 /zCi/izg. For the metallation step we did not use an isotopic dilution of HSInC13/1HInCI3 which explains the presence of both the free base and the metallo derivative. The mixture of radioactive porphyrin and free base porphyrin, was used without further purification.

Indium-porphyrin-BSA conjugate Coupling with BSA served as a model for the optimization of the experimental conditions. Labelling was carried out after conversion of the carboxylic group to its acid chloride as follows (eq. 1, 2). TTCPPM + SOC12 ~ TT( p-COC1)PPM

ELISA Microtitration plates (Nunc) were coated with 1 / z g / m l human thyroglobulin in 10 -2 M carbonate buffer pH 9.6 and kept overnight at 4 ° C. Plates were then saturated with 1% BSA in PBS for 30 rain at 37 ° C and incubated with the Mabs or fractions of the eluates for 90 min at 37 o C. Plates were washed three times with PBS and incubated with anti-IgG-peroxidase conjugate in PBS for 30 min, at 37 ° C. After three washings, the antigen-antibody complexes were revealed with 0.02% o-phenylenediamine in 0.1 M citrate buffer pH 5.5 for 10 min, at room temperature. The reaction was stopped with 4 M H 2 S O 4 and the spectrum measured with a MR 610 Dynatech plate spectrophotometer.

SDS-PAGE Sodium dodecylsulfate, polyacrylamide gel electrophoresis was carried out under reducing or non-reducing conditions according to Laemmli (1970).

Results and discussion

The choice of the T T C P P H 2 macrocycle was dictated by its ease of synthesis and by the fact that it is a monofunctional reagent, so that the potential for protein a n d / o r antibody cross-link-

(eq. 1)

TT( p-COC1)PPM + H 2N-BSA NaHCO3, 0.1 M 2 h 20, 20 o C ' TT(p-CONH-BSA)PPM

(eq. 2)

M = 2 H , nllnCl

A porphyrin-BSA molar ratio of 10 was used. Thionyl chloride was chosen as the activating reagent because it has the great advantage over other classical methods that the by-products are volatile. Using the optimized conditions reported in the experimental section, it was possible to couple 4.4 molecules of porphyrin per BSA with a labeling efficiency of 44%. In order to verify the absence of non-covalent interactions between porphyrin and BSA, the reaction was reproduced using the same conditions of concentration and contact time and without activating the carboxylic acid group. After electrophoresis, no detectable radioactivity was associated with the BSA spot. Radioactive porphyrin alone and BSA colored with Coomassie Blue were also electrophoresed for reference.

Indium-porphyrin-Mab conjugates We compared the following methods of activation of carboxylic function: either acid chloride activation (Eq. 1 and 3) or the use of the acti-

39

vated ester in the E D C I / N H S coupling method (eq. 4 and 5). TTCPPM + SOC12 --* TT( p-COCI)PPM

!

0

|

i

I

(eq. 1)

TI'( p-COCI)PPM + H 2N-IgG 1 h, RT

TT( p-CONH-IgG)PPM

(eq. 3) dP

TTCPPM + NHS

EDCI 1 h, RT, dark~

o o,I

TT(p-COONHS)PPM H

(eq.4)

H

O T T ( p - COONHS)PPM + H2N - IgG H 1 h, RT, dark' TT(p-CONH-IgG)PPM

o

(eq. 5)

M = 2H, nllnCl

All of the experiments were optimized with the IgG1 monoclonal antibody VIF11. When one compares the results for both coupling methods, the best coupling yields were obtained using E D C I / N H S . Labelling efficiencies as high as 93% have been achieved with the activated ester method, leading to a conjugate with an average number of 9.3 porphyrin groups covalently linked to each Mab VIF11 and a specific activity of 6 /~Ci//~g. In contrast, with acid chloride activation, a lower labelling efficiency was obtained (40.5%) and this was associated with a lower specific activity 2.4 ~Ci//zg). These results prompted us to choose the E D C I / N H S method as the optimized coupling protocol. Using the same experimental conditions, we verified that no coupling was observed when radioactive carboxylic porphyrin was used without activation (see Fig. 2). No radioactivity was observed in the chromatographic fractions corresponding to the elution peak of Mab. Several monoclonal antibodies were used to develop a general method of labelling : Mab BSIII, Mab BB3, Mab VB10 (all IgG1 subclass). Studies were restricted to this subclass since it is the most representative of the monoclonal antibodies. Each of them was labelled with the activated ester of the radioactive carboxyporphyrin. Whatever the Mab used, similar results were obtained namely between 90-95% coupling yield with an average

o

i

I

I

I

6

12

18

24

F R A C T I O N N U M B E R (450 ~ I / T U B E ) Fig. 2. Sephadex G-15 chromatography of Mab V I F l l - n l l n porphyrin conjugate: A, after acid carboxylic activation; B, without activation.

number of porphyrin groups per Mab of between 9 and 9.5 and specific activities of 6/xCi//zg. Another conjugate having the bivalent antigenbinding site was also prepared, employing the F(ab') 2 fragment of Mab VB10 as the homing component to be coupled with the N-hydroxysuccinimide ester derivative of q-TcpplUInC1. In this case, the yield of the coupling step was considerably lowered in comparison with the parent antibody; 1.5 molecules of radioactive porphyrin were covalently coupled per F(ab') 2 molecule with a specific activity of 1 /zCi/~g. This suggests a preferential reaction of the functional radioactive porphyrin with the Fc fragment of the Mab. All attempts to purify the conjugate radioactive porphyrin/Mab VIF11 by affinity chromatography on protein A-Sepharose failed (unpublished results). When applied to a Protein A-Sepharose CL-4B column equilibrated in PBS

40

buffer 0.1 M, pH 8, Mab V I F l l is normally retained on the column (and is only eluted with citrate buffer 0.1 M pH 5.5). Thus, a first elution with PBS can eliminate impurities and in order to remove Mab from the column it is necessary to decrease the pH of the eluant. A subsequent elution with citrate buffer 0.1 M, pH 5.5 may yield pure Mab VI Fl l . However, with the radioactive porphyrin/Mab V I F l l conjugate no adsorption of the conjugate is observed; it is eluted at pH 8 with the starting reagents used in the coupling reaction as well as the urea derivative of EDCI. This result supports the hypothesis of a labelling occuring mainly in the Fc region of Mab V I F l l and near the site interacting with Protein A-Sepharose. The Fc fragment is known to be the region of the IgG molecule involved in interactions with Protein A (Kronvall et al., 1970).

Immunoreactivity of TTCPp111InCl/Mab conjugates For each experiment, the immunoreactivity of the product was controlled by ELISA. The conjugate was compared with unmodified antibody in inhibition tests. No loss of immunoreactivity was observed either with the indium-porphyrin-Mab or the porphyrin-Mab conjugates. In assays performed with intact antibodies as well as F(ab') 2 fragments it was shown that porphyrin (at the concentrations used) did not interfere with the absorbance of the reactants at 496 nm.

Conclusion Taking into account its high stability, the tritolyl para-carboxyphenylporphyrin of indium-ill is a potential tracer for monoclonal antibodies a n d / o r their F(ab') 2 fragments. The absence of any significant differences in the results of the ELISA tests carried out with the metallated, unmetallated and unmodified antibody, suggest that the immunoreactivity is not altered by the covalent linkage between indium-porphyrin and Mabs. In vivo studies with the conjugates obtained are currently under investigation.

Acknowledgements The financial support of the Etablissement Public Regional d'Aquitaine (P61e G6nie Biologique et M6dical) is gratefully acknowledged. We wish to thank Professor Robert Poller (King's College, University of London) who accepted the task of improving the English of the text and M.C. Bernardi for typing the manuscript.

References Bedel-Cloutour, C.H. and Barois-Gacherieu, C. (1987) Functionalization of tetrapyrrolic macrocycles: free bases and indium derivatives, models for protein labeling. Main Group Met. Chem. 10, 109. Buchler, J.W. (1975) Static coordination chemistry of metalloporphyrins. In: K.M. Smith (Ed.), Porphyrins and Metalloporphyrins. Elsevier, New York, p. 157. Cloutour, C., Ducassou, D., Pommier, J.C. and Vuillemin, L. (1982) Chloro indium-Ill tetraphenylporphyrin and its possible clinical application. Int. J. Radiat. Isot. 33, 1311. Cole, W.C., De Nardo, S.J., Meares, C.F., McCall, M.J., De Nardo, G.L., Epstein, A.L., O'Brien, H.A. and Moi, M.K. (1986) Serum stability of 67Cu chelates: comparison with lllln and 57Co. Nucl. Med. Biol. 13, 363. Fawwaz, R.A., Wang, T.S.T., Estabrook, A., Rosen, J.M., Hardy, M.A., Alderson, P.O., Srivastava, S.C., Richards, P. and Ferrone, S. (1985) Immuno-reactivity and biodistribution of indium-ill-labeled monoclonal antibody to a human high molecular weight-melanoma associated antigen. J. Nucl. Med. 26, 488. Halpern, S., Stern, P., Hagan, P., Chen, A., Frincke, J., Bartholomew, R., David, G. and Adams, T. (1983) Labeling of monoclonal antibodies with indium-Ill: technique and advantages compared to radioiodine labeling. In: S.W. Burchiel and B.A. Rhodes (Eds.), Radioimmunoimaging and Radioimmunotherapy. Elsevier, Amsterdam, p. 197. Hnatowich, D.J., Childs, R.L., Lanteigne, D. and Najafi, A. (1983) The preparation of DTPA-coupled antibodies radiolabeled with metallic radionuclides: an improved method. J. Immunol. Methods 65, 147. Kozak, R.W., Waldmann, T.A., Atcher, R.W. and Gansow, O.A. (1986) Radionuclide-conjugated monoclonal antibodies: a synthesis of immunology, inorganic chemistry and nuclear science. Trends Biotechnol. 4, 259. Kronvall, G. and Frommell, D. (1970) Definition of staphylococcal protein A reactivity for human immunoglobulin G fragments. Immunochemistry 7, 124. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. Meares, C.F. and Goodwin, D.A. (1984) Linking radiometals to proteins with bifunctional chelating agents. J. Prot. Chem. 3, 215.

41 Meares, C.F., Goodwin, D.A., Leung, C.S.H., Girgis, A.Y., Silvester, D.J., Nunn, A.D. and Lavender, P.J. (1976) Covalent attachment of metal chelates to proteins: the stability in vivo and in vitro of the conjugate of albumin with a chelate of Ill indium. Proc. Natl. Acad. Sci. U.S.A. 73, 3803. Paik, C.H., Herman, D.E., Eckelman, W.C. and Reba, R.C. (1980) Synthesis, plasma clearance, and in vitro stability of protein containing a conjugated indium-Ill chelate. J. Radioanal. Chem. 57, 553. Paik, C.H., Ebbert, M.A., Murphy, P.R., Lassman, C.R., Reba, R.C., Eckelman, W.C., Pak, K.Y., Powe, J., Steplewski, Z. and Koprowski, H. (1983) Factors influencing DPTA conjugation with antibodies by cyclic DTPA anhydride. J. Nucl. Med. 24, 1158. Paik, C.H., Hong, J.J., Ebbert, M.A., Heald, S.C., Reba, R.C. and Eckelman, W.C. (1985) Relative reactivity of DTPA, immunoreactive antibody-DTPA conjugates and non im-

munoreactive antibody-DTPA conjugates toward indium111. J. Nucl. Med. 26, 482. Roberts, J.C., Figard, S.D., Mercer-Smith, J.A., Svitra, Z.V., Anderson, W.L. and Lavallee, D.K. (1987) Preparation and characterization of copper-67 porphyrin-antibody conjugates. J. Immunol. Methods 105, 153. Sundberg, M.W., Meares, C.F., Goodwin, D.A. and Diamanti, C.I. (1974) Selective binding of metal ions to macromolecules using bifunctional analogs of EDTA. J. Med. Chem. 17, 1304. Wang, T.S.T., Srivastava, S.C., Fawwaz, R.A., Giacomini, P., Ferrone, S., Richards, P., Hardy, M. and Anderson, P.O. (1984) A comparison of the cyclic anhydride and mixed anhydride method for 111In-DTPA chelation to monoclonal antibodies. Nuklearmedizin 4, 193. Yeh, S.M., Meares, C.F. and Goodwin, D.A. (1979) Decomposition rates of radiopharmaceutical indium chelates in serum. J. Radioanal. Chem. 53, 327.