Bioconjugate
Chemistry MARCH/APRIL 1994 Volume 5, Number 2 0 Copyright 1994 by the American Chemical Society
LETTERS Labeling Monoclonal Antibodies with goYttrium- and ‘“Indium-DOTA Chelates: A Simple and Efficient Method Min Li,+Claude F. Meares,’,+ Gao-Ren Zhong,t Laird Miers,t Cheng-Yi Xiong,t and Sally J. DeNardot Department of Chemistry, University of California, Davis, California 95616, and Department of Internal Medicine, University of California Davis Medical Center, Sacramento, California 95817. Received October 17, 1993”
Yttrium-90 and indium-111 have been attached to a monoclonal antibody with a bifunctional chelating agent (DOTA-peptide). Using the unique features of this DOTA-peptide and its complexes with trivalent yttrium and indium, the bifunctional chelating agent was prelabeled with either radiometal and then conjugated to chimeric monoclonal antibody L6. Both radiolabeling procedures and yield are suitable for the practical preparation of radiopharmaceuticals. Biodistribution studies in tumor-bearing immunoconjugate, liver uptake was mice showed that, e.g., on day 3 after intravenous injection of a 5.4 f 1.5% ID/g, bone uptake 2.0 f 0.5% ID/g, and tumor uptake 18.0 f 8.0% ID/g.
Macrocyclic bifunctional chelating agents have been developed to tag monoclonal antibodies with radiometals for in vivo diagnosis and therapy (1-7); in particular, mAbs labeled with DOTAl derivatives incorporating yttrium90 and indium-111 have shown excellent kinetic stability under physiological conditions (2,5, 7,8). However, the slow formation of yttrium-DOTA complexes (9-11) presents a technical problem that can lead to low radiolabeling yields unless conditions are carefully controlled. The conventional labeling process involves conjugation + University
of California.
* University of California Davis Medical Center.
.s Abstract published in Advance ACS Abstracts, February 1, 1994. 1 Abbreviations used: DOTA, 1,4,7,10-tetraazacyclododecaneN,N’,N”,N”’-tetraacetic acid; DTPA, diethylenetriaminepentaacetic acid; mAbs, monoclonal antibodies; TETA, 1,4,8,11tetraazacyclotetradecane-N,N’,N’’,N”’-tetraacetic acid; 7% ID/ g, percent of injected dose per gram of tissue.
1043-1802/94/2905-0 101$04.50/0
of the bifunctional chelating agent to the antibody, followed by labeling with the radiometal. An alternative method is prelabeling, in which the bifunctional chelating agent is first radiolabeled and then conjugated to the mAb. Prelabeling has been used to eliminate the nonspecific binding of 9 9 m Tto~mAbs (12-14). Moi e t al. initially used prelabeling to attach 67Cu to a mAb with the first macrocyclic bifunctional chelating agent, a TETA derivative ( I ) , and recently Schlom e t a l . (15) employed such a procedure to label a mAb with a 177Lu-DOTA derivative. Prelabeling involves three steps: (1)radioactive chelate formation (in the absence of antibody), (2) chelate purification, and (3) antibody conjugation. It has several potential advantages: in step 1, metal chelate formation is easier to control because there is no competition from metal-binding sites on the protein, and the chelation conditions are not limited by the need to avoid denaturing the antibody; in step 2, excess chelating agent can be removed before the radioactive chelate is attached to the 0 1994 American Chemical Society
Li et al.
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Figure 1. Biodistributionof gOY-DOTA-Gly3-L-Phe-amide-thiourea-chimeric mAb L6 (3) in HBT tumor-bearing nude mice at days 1.3. and 5. For each time Doint. data were acauired from seven animals. The values are given as average percent of injected dose per &am of tissue. Error bar; represent 1 standird deviation.
protein, thus avoiding the production of multiply labeled immunoconjugateswith unfavorablebiological properties (16);in step 3, the antibody is chemically modified and radiolabeled in one step, minimizing chemical manipulation of the antibody and reducing losses. The medically useful radiometals have short half-lives (17); high efficiencies of both labeling and conjugation are important for practical applications. The prelabeling approach permits use of a large excess of bifunctional chelating agent to achieve a high chelation yield quickly in step 1, but requires a rapid purification method to remove unlabeled reagent in step 2. Here, we report an easy and efficient method for prelabeling a peptide-linked DOTA derivative with or lllIn and conjugating it to a mAb. The labeling procedure is outlined in Scheme 1. The bifunctional chelating agent DOTA-Gly3-L- (p-isothiocyanato)-Phe-amide (1) was prepared by the method described recently (7). Carrier-free wy (DuPont NEN) in 0.05 M HC1 was dried in a heating block under N2(g), and 100pL of 20 mM 1in 0.2 M (CH&N+ acetate, pH 5.0,was added. The mixture was incubated at 37 "C for 30 min, followed by addition of 25 pL of 50 mM DTPA in 0.1 M (CH&N+ acetate, pH 6.0, for 15min at room temperature (to complex any remaining free yttrium). The solution was loaded onto an anion-exchange column,2 and the column was spun for 2 min at 2000 g, followed by elution with four 125-pL aliquots of H20 by centrifugation at -2000 g for 2 min each. Most of the radioactive 2 was recovered in the first four fractions (see Table 1). Onestep elution with 0.5 mL of H20 was performed for comparison, but it gave 18% lower recovery than stepwise elution. All the eluted fractions were collected and concentrated to 15 pL with a speed-vac concentrator (Savant Instruments) without heating. (It should be possible to avoid this step when larger amounts of radioactivity are used, e.g., for clinical-scale preparations.) The concentrated
-
Scheme 1 ycs
-0OcJ
1
uL c o o -
Step 1: Pre-labeling
Step 2: Purification
Step 3: Conjugation
1
I
M3' (M
"'ln)
Anion-exchange
1 NHy
-
The column had been prepared by filling a 1-mL disposable tuberculin syringe with 500 p L of DEAE cellulose anion-exchange resin (1 mequiv/dry g, Sigma Chemical Co.) and prespun for 3 min at -2000 g. The resin had been converted to the acetate form prior to use.
solution was mixed with 1mg of chimeric mAb L6 (18pL, 56 mg/mL, Oncogen/Bristol-Myers) (18)in 0.1 M (CH3)4N+ phosphate, pH 9.0. The pH was adjusted to 9.5 using aqueous 2.0 M triethylamine. The mixture was incubated at 37 "C for 1h, and 3 was isolated using a centrifuged gel-filtration column (19,ZO). Yields are listed in Table 1. lllIn labeling was carried out similarly.
Letters
Table 1. Results for DOTA-Peptide Radiolabeling and Conjugation overall recovery recovery starting recovery,a for for radioactivity, radionuclide mCi (vol,bL) step 2,n % step 3,a % % 30h 4 80f5 40f2 yttrium-90 2.1-3.9 (2-5) 42f4 indium-111 4.9-6.2 70h9 73f3 (12-30) a Average recovered radioactivity f standard deviation, for 2 3 runs.
The radiochemical purity of both WY-and lllIn-labeled immunoconjugates 3 was determined to be >95 5% by gel filtration HPLC, cellulose acetate electrophoresis, and silica gel TLC (20). A solid-phase radioimmunoassay (21) was performed using 1251-labeledchimeric L6 as a standard. The immunoreactivity of gOY-DOTA-Gly3-~-Phe-amidethiourea-chimeric L6 was 107 f 5% relative to 1251-labeled antibody. Compound 1 forms neutral complexes 2 with trivalent metals. The other important species in the chelation reaction mixture, such as excess chelating agents, complexes containing divalent metals, and DTPA complexes, are negatively charged. Thus, the DOTA-peptide complexes with trivalent metals can be filtered quickly through an appropriately designed anion-exchange column in H20 to separate them from anionic species. The neutral chelate avoids the need for more complex processes in step 2, such as HPLC with mixed organic/aqueous solvents (15). In the chelation step, the yield after anion exchange was typically >70% of the starting radioactivity. Particularly for WY solutions, the levels of metal impurities appear to vary with each batch of carrier-free radiometal. The identity of these impurities is difficult to determine, but most common metal contaminants are divalent ions. Prelabeling deals with the impurity problem by using a large excess of chelating agent and then removing the excess. This is preferable to using a large excess of chelatetagged mAb conjugate, which cannot be fractionated later to remove unwanted contaminants. (Obviously, prelabeling does not eliminate trivalent metal complexes from the product.) In the conjugation step, a high concentration of mAb is desired, so that each chelate isothiocyanate will frequently encounter amino groups with which to react. For the small amounts of radioactivity used here, the radiolabeled bifunctional chelating agent was concentrated before adding it to the mAb to avoid dilution of the conjugation mixture. At the chosen conjugation conditions (1 h incubation at 37 " C , pH 9.5, [mAbl I20 mg/mL), the conjugation yield was over 40 7%. Longer incubation times would lead to higher conjugation yields, but we expect that for radioactivity levels appropriate for clinical use ( - 100 mCi) radiolysis will become important at longer times. The isothiocyanate group on the bifunctional chelating agent is potentially subject to hydrolysis during labeling and conjugation, but control experiments showed loss of 1in order to provide enough chelating groups for a good radiolabeling yield. However, the chelates that are actually used for labeling comprise less than 5% of the total attached chelates on the mAb. The excess chelating groups may affect the biological properties of mAbs, e.g., by inducing an immune response (16), and impure metal solutions may require large amounts of immunoconjugate. With prelabeling, a far smaller number of chelates becomes attached to the
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mAb, but practically all are radiolabeled; thus, the number of modified mAbs is significantly reduced, and the number of multiply-modified mAbs is essentially zero. The radiolabeled mAbs are fully immunoreactive and are expected to have more favorable biological properties, including less immunogenicity. Another reason for using DOTA-peptide is to reduce the accumulation of radioactivity in the liver by introducing a cleavable linker between the chelate and the mAb (7). To examine the properties of the conjugate in uiuo, WYlabeled 3 was injected into HBT tumor-bearing nude mice (22)for organ distribution and tumor uptake studies. The results of these animal studies (Figure 1)showed that the radioactivity level in the liver varied from 6.4 f 1.5% ID/g on the first day to 5.4 f 1.5% ID/g on the third day to 4.6 f 1.9 % ID/g on the fifth day. The tumor uptake was 17.5 f 8.0% ID/g on day 1,18.0 f 8.0% ID/g on day 3, and 13.8% f 5.2% ID/g on day 5. The bone uptake was 2.1 f 0.3% ID/g, 2.0 f 0.5% ID/g, and 1.8 f 0.2% ID/g on days 1, 3, and 5. The levels of radioactivity in liver and bone are satisfactorily low (8), and the tumor uptake is good. Coupled with its other expected advantages, these results indicate that prelabeling mAbs with metal complexes of 1 has considerable promise. ACKNOWLEDGMENT
We thank Justin K. Moran and Douglas P. Greiner for helpful discussions and Xu-Bao Shi for immunoreactivity assays. This work was supported by Research Grants CA16861and CA47829 from the National Cancer Institute, NIH. NOTE ADDED IN PROOF
Since submission of this paper, more yttrium-90 labeling experiments have been performed, and the labeling yield has been improved. For a total of nine experiments, the recovery for step 2 is now 75 f 8%, the recovery for step 3 is 58 f 15%,and the overall recovery is 40 f 115%. LITERATURE CITED (1) Moi, M. K., Meares, C. F., McCall, M. J., Cole, W. C., and DeNardo, S. J. (1985) Copper Chelates as Probes of Biological Systems: Stable Copper Complexes with a Macrocyclic Bifunctional Chelating Agent. Anal. Biochem. 148, 249-253. (2) Moi, M. K., Meares, C. F., and DeNardo, S. J. (1988) The Peptide Way to Macrocyclic Bifunctional Chelating Agents: Synthesis of 2-@-Nitrobenzyl)-1,4,7,lO-tetraazacyclododecme N,N',N",N"'-tetraacetic Acid and Study of Its Yttrium(II1) Complex. J. Am. Chem. SOC.110, 6266-6267. (3) Cox, J. P. L., Craig, Andrew, S., Helps, L. M., Jandowski, K. J., Parker, D., Eaton, M. A. W., Millican, A. T., Millar, K., Beeley, N. R. A., and Boyce, B. A. (1990) Synthesis of C-and N-Functionalised Derivatives of 1,4,7-Triazacyclononae-1,4,7triyltriacetic acid (NOTA), 1,4,7,10-Tetra-azacyclododecane1,4,7,10-tetrayltetra-aceticAcid (DOTA), and Diethylenetriaminepenta-acetic Acid (DTPA): Bifunctional Complexing Agents for the Derivatisation of Antibodies. J. Chem. SOC., Perkin Trans. 1 2567-2576. (4) Parker, D. (1990) Tumor Targeting with Radiolabeled Macrocycle-Antibody Conjugates. Chem. SOC.Rev. 19, 271291. (5) Meares, C. F., Moi. M. K., Diril, H. Kukis, D. L., McCall, M. J., Deshpande, S. V., DeNardo, S. J.,Snook, D., and Epenetos, A. A. (1990) Macrocyclic Chelates of Radiometals for Diagnosis and Therapy. Br. J . Cancer, Suppl. 10, 21-26. (6) Gansow, 0.A. (1991) Newer Approaches to the Radiolabeling of Monoclonal Antibodies by Use of Metal Chelates. Nucl. Med. Biol. 18, 369-381. ( 7 ) Li, M., and Meares, C. F. (1993) Synthesis, Metal Chelate Stability Studius, and Enzyme Digestion of a Peptide-Linked
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