Bioconjugate Chem. 1882, 3, 241-247
241
Carboranyl Peptide-Antibody Conjugates for Neutron-Capture Therapy: Preparation, Characterization, and in Vivo Evaluation Raymond J. Paxton,? Barbara G . Beatty,i Aravamuthan Varadarajan,o and M. Frederick Hawthorne'*§ Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010, Department of General Oncologic Surgery, City of Hope National Medical Center, Duarte, California 91010, and Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, California 90024. Received January 13,1992
Two model peptides rich in boron and prepared by Merrifield syntheses, dansyl-(nido-CB)z,(1) and dansyl.(nido-CB)lo.Lys-Ac(2), where nido-CB represents the a-amino acid [nido-7-CH3-8-(CH2)3CH(NH~)COOH-~,~-CZB~H~OI-, were conjugated with the anti-CEA mAb T84.66 using peptide active ester reagents. The dansyl groups provided a means of fluorimetric analysis of mAb conjugates which was augmented by conventional amino acid analyses for nido-CB. The conjugate of 1contained an average of 63 B atoms per mAb molecule. The mAb conjugate of 2 was chromatographically separated into a strongly fluorescent high molecular weight aggregated fraction (HMW) and a less intensely fluorescent monomeric fraction. Both fractions retained immunoreactivity. The HMW species contained an average of ca. 490 B atoms/mAb molecule, as determined by amino acid analysis. Biodistribution data were collected using nude mice bearing LS174T xenografts and 1251-labeledmAb conjugates. While the lightly B-loaded dipeptide conjugate gave biodistribution results which resembled those of native T84.66 mAb, the undecapeptide conjugate displayed greatly enhanced liver uptake and decreased tumor accretion. These results suggest that as the boron-containing burden on the supporting immunoprotein is greatly increased, as in the case of the T84.66-2 conjugate, loss of circulating conjugate to liver effectively competes with the desired tumor localization. Means which might be taken to circumvent this difficulty have been described elsewhere (ref 15).
Scheme I
INTRODUCTION
Of the many possible methods of loB delivery to tumor cells for the purpose of boron-neutron-capture therapy (BNCT) (1) the immunological approach remains as a potentially viable method, providing that about lo3 loB atoms can be attached to each tumor-associated immunoprotein molecule while its immunoreactivity is retained as well as any inherent ability to enter the cell by endocytosis. A number of previous studies have demonstrated the limitations associated with attaching large numbers of small B-containing molecules to mAb (2-4). Attention was then focused upon the linkage of inhomogeneous boron-rich polymers to mAb (5-14). The latter studies demonstrated that although >lo3 B atoms could be attached to each mAb molecule,the resulting conjugates suffered from either a serious loss of immunoreactivity or a reduced tumor uptake. In two recent publications (15, 26) we have described an approach to this synthesis and conjugation problem which utilizes oligomeric peptides of defined structure containing a large predetermined number (100-500) of boron atoms randomly conjugated to an immunoprotein such as anti-CEA mAb T84.66. The boron-containing peptide reagents have been given the trivial name "boron trailer" and conceived as being derived from synthetic hydrophilic a-amino acids assembled using the Merrifield peptide synthesis methodology (15,161.The carborane-containing a-amino acid employed in this study (nido-CB) was previously described (15, 16) and its structure is reproduced in Scheme I. The anionic [nido-7,8-CzBsH11]-cage incorporated in the amino acid structure was a device employed to enhance peptide hydrophilicity as the sodium salt of the a-amino acid repeating unit. The chirality of the nido-a-amino acid Beckman Research Institute of the City of Hope. City of Hope National Medical Center. f University of California a t Loa Angeles. +
t
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H3C-C-C-(CH2),-CH-COOH
I 'i(~~~ Nao NH2
NidoCB
Note: Throughout this text HC-CH \o/ B9H10
3
"
OBH
.c
originating at the a-carbon atom and the unsymmetrically substituted nido-carborane cage was ignored in order to simplify peptide synthesis procedures although it is recognized that the oligomeric peptides derived in this fashion from two sets of racemic diastereomeric repeating units were complex mixtures of diastereomers. Nonetheless, the physical and chemical properties of these isomer mixtures probably introduced only minor differences in biological response over the whole population of diastereomers examined. The much more important requirement of uniformity of oligomer size was achieved through synthesis methodology. In accordance with the trivial nomenclature employed elsewhere (15,16)the dansylated dipeptide and undecapeptide are denoted as dansyl*(nido-CB)z(1) and dansyl.(nido-CB)lo.Lys.Ac (2), respectively. Their structures are presented in Scheme I1 and their syntheses are reported elsewhere (16). Results obtained by conjugation of these two peptide trailers with T84.66 anti-CEA mAb and the biodistributions of the resulting conjugates in nude mice bearing CEA-producing LS174T tumor xenografts are presented here. 0 1992 American Chemical Society
242
Bloconlugete Chem., Vol. 3, No. 3, 1992
Paxton et al.
Scheme I1
502 NH YC -C-
\orWlO
I
C-(CH& -CH-CO-
I W I COOH I NH CH -(CH&
-
cH3
-C -C -CH,
NM.2
\Q?
f%Hlo
9
1oNaQ
2Na0
Dansyl*(n i d ~ - C B ) ~
1 EXPERIMENTAL PROCEDURES
Materials. Carboranyl peptides (Scheme 11) were synthesized as previously described (15,16). Anti-carcinoembryonic antigen (CEA) mAb T84.66 (17, 18) was purified from ascites by 40 % ammonium sulfate precipitation and protein A affinity chromatography. T84.66 is a murine IgG1, is specific for CEA, and has an affinity constant for CEA of >2 X 1Olo M-l. LS174T, a CEAproducing human colon cancer derived cell line, was grown in continuous culture in supplemented RPMI medium (19).
7
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Activation of Dansyl.(nido-CB)z(1) and Conjugation to T84.66. A solution of l was converted to its Nsuccinimidyl active ester and reacted with T84.66 in phosphate buffer as previously described (16). A control reaction was also performed in which the unactivated 1 was added to T84.66. The conjugate and the control reaction were initially purified by ultrafiltration using Centricon-30 (Amicon, Danvers, MA) membrane concentrators. The concentrates were repeatedly washed with phosphate buffer until the filtrates exhibited no detectable fluorescence. Subsequent purification was performed by gel filtration chromatography on a 1X 30 cm Superose 12 column (Pharmacia) equilibrated with phosphate-buffered saline (PBS, 0.01 M Na2HP04, 0.15 M NaC1, pH 7.5) and eluted at 1mL/min. Chromatography was performed with a Pharmacia FPLC system monitored at 280 nm, and protein peaks were collected manually. Fluorescence was monitored with a Gilson SpectroGlo fluorescence detector positioned after the UV detector. Excitation and emission wavelengths were 313 and 515 nm, respectively. Activation of Dansyl-(nido-CB)lo.Lys-Ac (2) and Conjugation to T84.66. (i) Conjugation 1. A solution of 2 (6.40 mM, 34 pL) in phosphate buffer (0.1 M Na2HP04, pH 4.35) was treated with solutions of N-hydroxysulfocuccinimide (92.1 mM, 4.8 pL) in water and NJV-diisopropylcarbodiimide (64 mM, 6.9 pL) in dimethylformamide, and the mixture was vortexed continuously at ambient temperature for 55 min. An aliquot of the active ester solution (20.9 pL) was added to a solution of T84.66 (26.7 pM, 250 pL) in bicarbonate buffer [0.1 M NaHC03, pH 8.5,0.01% (v/v) Tween-201, and the mixture was vortexed continuously at ambient temperature for 30 min. Assuming complete conversion of 2 to its active ester, the molar ratio of active ester to antibody was 15:l. Control reactions of T84.66 with an equivalent amount of unactivated 2 andT84.66 alone were also performed. Conjugate and control reactions were purified by gel filtration chromatography using a 0.7 X 28 cm column of Sephacryl S200 (Pharmacia) equilibrated with PBS, pH 8.0 containing 0.01 5% (v/v) Tween-20. Chromatography was performed at 0.3 mL/min with a Pharmacia FPLC system as described above. (ii) Conjugation 2. A solution of 2 (6.70 mM, 30 pL) in phosphate buffer (0.1 M Na2HP04, pH 4.35) was treated
Dansyl*(nido-CB),o*Lys*Ac
2 with solutions of N-hydroxysulfosuccinimide(92.1 mM, 4.4 pL) in water and NJV-diisopropylcarbodiimide(64 mM, 6.3 pL) in dimethylformamide, and the mixture was vortexed continuously at ambient temperature for 55 min. An aliquot of the active ester solution (13.5pL) was added to a solution of T84.66 (26.7 pM, 250 pL) in bicarbonate buffer (0.1 M NaHC03, pH 8.61, and the mixture was vortexed continuously at ambient temperature for 60 min. Assuming complete conversion of 2 to its active ester, the molar ratio of active ester to antibody was 1O:l. Control reactions of T84.66 with an equivalent amount of unactivated 2 and T84.66 alone were also performed. Conjugate and control reactions were adjusted to 0.05% Tween-20, vortexed continuously at ambient temperature for 60 min, and purified by gel filtration chromatography using a 1X 30 cm Superose 12 column equilibrated with PBS (pH 8.0) containing 0.05% (v/v) Tween-20. Chromatography was performed at 0.5 mL/min with a Pharmacia FPLC system as described above. Radioiodination of T84.66-Carboranyl Peptide Conjugates. T84.66 conjugates prepared with dansyl. (nido-CB)nand dansyl.(nido-CB)lo.Lys.Ac peptides were using the chloramine-T method. Gelradiolabeled with 1251 filtration-purified conjugates containing 10-35 pg of protein in 50-100 pL of chromatography buffer were treated with 1 mCi Na1251(New England Nuclear, -17 mCi/mg) and chloramine-T (3.5 mM, 10pL) in phosphate buffer (0.1 M NaH2P04, pH 7.5) for 1 min with gentle mixing. Reactions were stopped by adding sodium bisulfite (0.6 mg/mL, 10pL) in phosphate buffer and mixing gently for 1min. Human serum albumin (250 mg/mL, 4 pL) was then added to each reaction and each radiolabeled conjugate was purified by gel filtration chromatography on a freshly packed 0.7 X 13cm Sephadex G50 Fine column equilbrated with PBS (pH 8.0). Tween-20 (0.01%) was included for the T84.66-dansyl-(nido-CB) 1o.Lys.A~conjugates. T84.66 was similarly radioiodinated for control purposes. All operations were performed at ambient temperature, and 1251 incorporation ranged from 30 to 69%. Immunoreactivity of T84.66-Carboranyl Peptide Conjugates. Microtiter plates coated with CEA (5 pg/ mL) were incubated with serial dilutions starting at 10 pg/mL of T84.66-dansyl.(nido-CB)2 and T84.66-dansyl. (nido-CB)lo.Lys.Ac conjugates. A double sandwich enzyme immunoassay was then performed, and the resulting binding curves were compared to those for nonconjugated T84.66 (20). In Vivo Evaluation of T84.66-Carboranyl Peptide Conjugates. Groups of 6-8-week-old athymic (nude) female mice (Simonsen) were injected subcutaneously in the left flank with 106LS174Tcells in 0.2mL of phosphatebuffered saline. Ten to fourteen days later the animals were injected intraveneously with either 1251-labeled T84.66-dansyl.(nido-CB)2,lZ5I-labeledT84.66-dansyl. (nido-CB)lwLys.Ac, or 1251-labeledT84.66 as a control.
Bioconjugate Chem., Voi. 3, No. 3, 1992 243
Carboranyl Peptide-Antibody Conjugates for BNCT
Animals were sacrificed 48, 72, or 120 h after antibody injection, and tissues were removed, weighed, and counted for radioactivity using a y well counter. An aliquot of the injected dose (ID) was counted with the tissues to correct for radionuclide decay. Uptake of radiolabel waa expressed as a percent of the injected dose per gram (%ID/g) of tissue (mean f SEI. Analytical Procedures. Protein concentrations were determined by absorbance measurement at 280 nm based on an absorbance of 1.42 for a 1 mg/mL solution. Quantitative fluorescence measurements were performed on a Spex fluorimeter using 313 and 515 nm for the excitation and emission wavelengths, respectively. Standard solutions of 1 in water were prepared and the fluorescence intensity measured for each of these solutions was plotted against the respective concentration to obtain a linear correlation. Aliquots of T84.66dansyl.(nido-CB)z conjugate or T84.66 control were diluted in water, and the fluorescence intensity was measured. From the standard calibration curve, the concentration of the peptide in the conjugate or control was obtained. Quantitative amino acid analysis was also used to determine the amount of carboranyl peptide incorporation in antibody conjugates. Hydrolyses were performed in the vapor phase using 6 N HCl with 0.1% (v/v) 2-mercaptoethanol at 115 "C for 24 h. Hydrolysates were dried under vacuum, dissolved in loading buffer, and analyzed on a Beckman 6300 amino acid analyzer according to the manufacturer's instructions. RESULTS AND DISCUSSION
Preparation and Characterization of T84.66Dansyl.(nid&B)t Conjugates. The synthesis of the dipeptide dansyl.(nido-CB)z,(1,Scheme 11)and conditions for its conjugation to anti-CEA mAb T84.66 were previously described by Varadarajan and Hawthorne (16).The resulting conjugate, designated T84.66-(CB)z, for simplicity, and the control antibody (T84.66 treated with unactivated dipeptide) were initially purified by ultrafiltration and then by Superose 12 gel filtration chromatography. The control antibody (Figure 1A) showed a sharp protein peak corresponding to monomeric T84.66 with a small amount (- 2 % by peak height) of high molecular weight species preceding the main antibody peak. Very little fluorescence was associated with either protein species, indicating that 1 did not bind nonspecificallyto the antibody. T84.66-(CB)2 (Figure 1B)showed a similar protein elution profile; however, the significant fluorescence eluted with the antibody was indicative of the covalent attachment of 1 to the antibody. The high molecular weight species contained a disproportionate amount of the fluorescent label. This suggests that the high molecular weight species is more reactive toward activated 1 or that a small percentage of the monomeric antibody reacts at a disproportionately rapid rate with activated 1 (cascade conjugation, vide infra) generating a highly conjugated, high molecular weight species. Monomeric T84.66-(CB)z and control T84.66 were collected and used for fluorescence, electrophoretic, immunoreactivity, and in vivo tumor-targeting studies. On the basis of the measured fluorescence intensity and protein concentration T84.66-(CB)z contained an average of 3.5 molecules of 1 (63 boron atoms) per antibody molecule,whereas control T84.66 contained
