Development of Radiochemically Pure Antibodies1

[CANCER RESEARCH (SUPPL.) 50. 780s-782s. February 1. 1990] Development of Radiochemically Pure Antibodies1

W. C. Eckelman Squibb Institute for Medical Research, Aviv Brunswick, New Jersey 08903-0191

Abstract al. (9) to determine if they had advantages over DTPA as bifunctional chelates of antibodies (Table 2). Optimal concen There are presently three approaches to radiolabeling antibodies: trations of tin and chelator were determined to be 2.5 to 5 /ig/ direct radiolabeling; radiolabeling of a chelating agent conjugated to an ml SnCl2-2H2O and 50 Mg/ml of chelator. The optimal pH antibody; and radiolabeling of a chelating agent before conjugation to an range was 5 to 8. Labeling in the presence of 5 mg/ml of IgG antibody. Using either the direct radiolabeling or radiolabeling of a was used to determine the percentage expected to be labeled via chelating agent conjugated to an antibody has not led to a radiochemically pure ""Tc-labeled protein. High radiochemical purity is obtained using the N2S2 chelating agent and the percentage expected to be a prelabeled ligand; therefore this method is preferred. directly bound to the antibody (Table 3). The DADT-3C-2OH in which the ethylene backbone is re placed with a propylene backbone has the highest percentage One of the major efforts since the first Conference on Radi- binding. This appears to be the only example of a chelating agent that can compete quantitatively with an equimolar con oimmunodetection and Radioimmunotherapy of Cancer has centration of IgG. been the development of antibodies that are radiochemically If the source of 99mTcis 99mTc-EDTA at 100 Mg/ml, then the pure in vitro and remain so in vivo. Since radioiodine has been requirement for chelator concentration is less (Table 4). Using one of the first radionuclides used to label antibodies, many 150,000 as the molecular weight of IgG there are 33 nmol/ml studies have centered around the preparation of radiochemically of IgG. If the molecular weight of the free amine is used (236), pure antibodies either by direct iodination or by indirect labeling through a reagent such as the Bolton-Hunter ligand. The cur then 3 ng of DADT are 13 nmol/ml and 8 Mgof DADT are 34 nmol/ml. These molar values do not agree with the molar ratios rent research emphasis is on in vivo stability using various stated by Liang et al. (9). However, it appears that the chelation iodinated species less susceptible to enzymatic deiodination (1, is more effective when 9VrnTc-EDTA is used than when the 2). chelator is labeled directly. There have been two approaches to radiolabeling antibodies Yokoyama's group (10) has developed the neutral 99mTc with metallic radionuclides using bifunctional chelates; either chelate ketobisthiosemicarbazone for use in bifunctional che the chelating agent is first attached to the antibody followed by lates. Various derivatives have been prepared including the p- radiolabeling or the chelating agent is first radiolabeled and the carbonylethylphenylglycol derivative, which has been conju resulting chelate is bound to the antibody. Very few experiments gated to antibody by activating the acid group using the phos- have shown that first attaching the chelating agent to the antibody results in an "instant" kit in which all the radiolabel phorylazide method. Stannous ascorbate was used as the reduc ing agent and 99mTc-ascorbate as the transfer agent. As in most is bound via the chelating agent. For 99mTc,the indirect labeling methods have mostly involved the use of DTPA- bound to the of these studies the optimal pH is between 4.5 and 6.2. The labeling experiments were carried out to determine what per protein. DTPA is considered a bifunctional chelate in that it centage of the 99mTc binds to the chelating agent and what binds to the protein and to the "Tc. Other chelating agents, derivatives of DADS (3), derivatives of bis-A'-methylthiosemi- percentage binds directly to the antibody. At ratios of chelating agent to antibody that preserve the immunological activity (i.e., carbazone (4), and metallothionein (5), have been conjugated 1:1 molar ratio), about 15-20% of the "Tc is directly bound to antibodies for "Tc labeling. Although 99mTclabeling con to the protein. In vitro and in vivo stability of the derivatized ditions and biodistribution studies of the labeled products were antibody is high. Although direct binding of 99mTcto antibodies reported, a systematic investigation of the effectiveness of this latter chelator for complexing "Tc in competition with anti has been reported (11-14) the stability and inertness of the bond are in question. There appear to be two binding sites on body has been reported only in abstract form (6). Hnatowich's group (7, 8) has carried out various experiments the antibody, one with high affinity and one with lower affinity. to determine the expected ratio of "Tc binding to the chelating Steigman et al. (15) were the first to suggest that sulfhydryl groups were responsible for the direct binding of 99mTc.Paik et agent in the bifunctional chelate compared to direct binding to al. (16, 17) substantiated this hypothesis by carrying out a the antibody. Using 50 Mg/ml DTPA as the molar equivalent number of competitive experiments and by titrating the sulfhy to 20 mg/ml IgG, (0.13 ^mol/ml) various binding conditions dryl groups using Ellman's reagent. The available sulfhydryl were tested. Without protein present, DTPA complexed re duced "Tc efficiently, but not quantitatively (Table 1). groups were determined after 30 min incubation of stannous In a subsequent paper, they determined the appropriate chloride with antibody. The stannous chloride was present in 10-fold excess over the protein. The results obtained are re tin:DTPA ratio. As has been observed earlier, too little stannous ion resulted in a high percentage of pertechnetate and too much ported in Table 5. stannous ion resulted in tin colloid. The labeling of conjugated The presence of both high affinity sites and low affinity sites protein via the bifunctional chelate is about 80%. The distri on the protein was defined by competition studies using DTPA. bution in normal mice at l h is similar for '"In-DTPA-IgG and The high affinity sites were determined as those which bound "Tc-DTPA-IgG. 99nTc even at large molar excesses of DTPA. The percentage Three N2S2 (diaminodithio) ligands were studied by Liang et bound to protein was determined at various ratios of DTPA to protein. ' Presented at the "Second Conference on Radioimmunodetection and Ra dioimmunotherapy of Cancer." September 8-10. 1988, Princeton, NJ. These results are obtained only under conditions of direct 2The abbreviations used are: DTPA. diethylenetriaminepentaacetic acid; competition between DTPA and antibody. If the protein is DADS. bis(mercaptoacetyl)ethylenediamine; DADT. diaminodithiol. labeled first, then DTPA is effective only at high pH. One 780s

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. RADIOCHEMICALLV PURE ANTIBODIES Table l DTPA binding ofstannous reduced """Tc" Table 6 Percentage of loss of radioactivity from n"Tc-labeled antibodies"

DTPA (fig/ml)25 bound81 bound40 Challenge1 labeled0.5 chelate0.7 ligand0 50 6 8991pH7 77% 59 mMDTPA1 100pH6 6% 80 mM N2S2 54.0Bifunctional 32.0Prelabeled 0 ' Taken from Lanteigne and Hnatowich (7). 6 M ureaDirect 1.9 " From Fritzberg (20).

Table 2 Various diaminodilhiot derivatives" Table 7 Average number of MAC" ligands" conjugated per antibody molecule at HSCH2CH2NHR,NHCH2CH2SH different MBS:antibody ratios' DADT = R, = CH2CH2 of Ab activity DADT-HM = R, = CH2C(Me)2 recovered'' DADT-3C-20H = R, = CH2CHCH2 MBS:Ab no. of MAC ligands/ ratÂ¡or10 Abmolecule2.8 (mean Â±SD)89.5 I Â±0.2' OH Â±1.0 ' Taken from Liang et al. (9). 30 3.1Â±0.24.2 84.2 Â±2.1 100150Av. Â±0.5 89.7 Â±2.3 3.6 Â±0.3% 88.3 Â±1.8 Table 3 Labeling o/jVA compound in the presence of 5 mg/ml IgG" "MAC, aminopropyl cyclam: MBS. m-maleimidobenzoyl-A'-hydroxysucci- "TechelateDADT-HM* nimide; Ab. antibody: SH, sulfhydryl. * The average number of MAC ligands conjugated to each rabbit anti-human Compound:IgG serum albumin-lgG molecule (Ab) was estimated assuming ""Tc-MAC-SH had (molarratio)1:1 DADT-3C-OH42 equal reactivities with MBS-Abs. 98 ' MBS-Abs were prepared by incubating Abs in different molar ratios with 2:1 61 78 94 MBS. From Franz et al. (19). 3:15:1 8255 89 98 d The percentage of Ab reactivity recovered was determined by affinity chro- 92 95 matography and represents the percentage of Ab binding of MAC Abs compared 10:1DADT8 58% 96 96 to unconjugated Abs. Â°Taken from Liang et al. (9). ' Mean Â±SD. * DADT-HM. hexamethyl DADT. covalently bound chelating agent, do .not achieve high radi- Table 4 Labeling of DADT by trancomplexation from v""Tc-EDTA in the ochemical purity, investigators have turned to prelabeling the presence of 5 mg/ml lg(i ligand and then reacting this ""Te chelate with the antibody.

DADT0(^g/ml)3 "Te asTc-DADT85 Fritzberg et al. (3, 18) have prelabeled the DADS ligand and (molarratio)2 then bound the ligand to the antibody via an activated ester. Likewise Franz et al. (19) have prelabeled the /V-propylamine 8DADT:IgG 5% 88 derivative of cyclam with 99mTcand then reacted that chelate ' As stated by Liang et al. (9). with an activated antibody. This method assures that the 99mTc is bound to the chelating agent and, therefore, that a single Table 5 Sulfhydryl groups present for antibody type radiochemical should be produced. On the other hand, the Antibody Sulfhydryl groups/antibody development of an "instant kit" will be difficult with this IgG 5.5 Â±0.7" F(ab')2 4.2 Â±0.6 approach. Fritzberg (18, 20) has compared the use of direct Fab 0.9 Â±0.15 labeling a bifunctional chelate and a preformed chelate to " Mean Â±SD. determine relative radiochemical purity and stability. He used the 2,5-dimercaptoacetamidopropanoate as the ligand. The core hypothesis is that the high affinity binding to IgG involves a ligand (Tc-DADS) has been suggested as a 99mTcsubstitute for Tc(V) species whereas the binding to DTPA involves a Tc(III) hippuran. In the case of bifunctional chelate, the ligand was or Tc(IV) species. Competitive experiments indicate that the conjugated to antibody using an activated ester. The antibody- distribution of 99mTcbetween the competing ligands is deter ligand complex was then radiolabeled by ligand exchange. For mined by the forward rate constants but not by the equilibrium the prelabeled ligand, the diamidodithio ligand was radiolabeled constants for these reaction conditions. This direct labeling first, the acid was activated to the ester, and then the prelabeled approach has been refined by others to maximize the percentage ligand was reacted with the antibody. The relative stabilities of the free sulfhydryl groups. Paik et al. estimate that about 10 were tested by incubation of each radiolabeled antibody type times molar excess of DTPA is required to prevent 99mTc with either DTPA or excess diamidodithio ligand (Table 6). complexation with low affinity sites. However, the presence of It is clear from this experiment that the prelabeled ligand free DTPA reduces drastically the percentage of 99mTcbound forms the most stable bond. Esterification of the preformed to antibody. technetium complex went in about 70% yield. Conjugation One possible solution for this low yield is to reequilibrate the yields using 2.5 mg/ml F(ab')2 at pH 9 were between 50 and mixture of low and high affinity sites in the presence of DTPA 70%. at high pH. This apparently increases the percentage of tech Franz et al. (19) have used a derivative of the well character netium on the high affinity sites. Distribution studies in mice ized cyclam to avoid direct antibody binding. Cyclam was show similarities among the antibodies labeled by different derivatized at the 1-nitrogen with a 3-aminopropyl derivative. techniques at the high affinity sites (16, 17). The generally The cyclam derivative was treated with 2-iminothiolate to form lower % D/g values for the antibody containing both strongly the imidate. This was reacted with activated IgG to form the and weakly bound 99mTcindicates that a soluble chelate rather bifunctional chelate. The relationship of activated antibody and than pertechnetate may be the major radiochemical impurity. number of chelates incorporated are given in Table 7. Because both the direct labeling methods, which apparently These experiments show one of the problems with using depend on free sulfhydryl groups for binding 99mTc,and the prelabeled ligands. Only 4% of the ""Te becomes bound to the bifunctional chelate approach, which depends on a competition IgG. A more efficient coupling reaction is needed in this case. between weak and strong direct binding and binding by the If the ligand is labeled with 99mTcin the presence of the activated

781s

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. RADIOCHEMICALLY PURE ANTIBODIES coupling group, then optimal radiolabeling conditions may not zone). Int. J. NucÃ.Med. Biol.. 12: 425. 1985. 5. Tolman, G. L., Hadjian, R. A.. Morelock, M. M., Jones, P. L., Neacy. W., be available. On the other hand, if the technetium complex is Liberature. F. A.. Sands. H.. and Gallagher. B. In vivo tumor localization of activated toward conjugation after formation, another proce Technetium-labeled metallothionein/monoclonal antibody conjugates. J. NucÃ.Med.. 25: 20. 1984. dural step is required. This balance must be dealt with to obtain 6. Burchiel, S. W., Hadjian. R. A.. Hladik. W. B., Gregorio, S. N., Tolman, G. maximum efficiency. L.. Drozynski, C. A., Covell. D., Day. P. A.. Christie, J. H.. and Gallagher, The ideal nuclear properties of "Tc warrant further defini B. M. Biodistribution and pharmacokinetic analysis of "Tc-metallothionein conjugated to B 72.3 antibodies in rhesus monkeys. J. NucÃ.Med.. 27: 896, tion of the chemistry of technetium. To date, the labeling of 1986. antibodies with 99mTcusing an "instant kit" has not resulted in 7. Lanteigne, D., and Hnatowich. D. J. The labeling of DTPA-coupled proteins with 99mTc. Int. J. Appi. RadiÃ¢t.Isot., 35: 617-622, 1984. a product with high radiochemical purity. High radiochemical 8. Child, R. L., and Hnatowich, D. J. Optimum conditions for labeling of purity is obtained using the prelabeled ligands, but this neces DTPA-coupled antibodies with technetium-99m. J. NucÃ.Med.. 26: 293- sitates a multistep kit. However, in light of the large number of 299, 1985. 9. Liang, F. H.. Virzi, F.. and Hnatowich. D. J. Serum stability and non-specific variables involved in each experiment with radiolabeled mono binding of technetium-99m labeled diaminodithiol for protein labeling. NucÃ. clonal antibodies, it seems prudent to use only preformed Med. Biol.. 14: 555-562. 1987. ligands, not only with "Tc, but with all metallic radionuclides, 10. AraÃ±o,Y., Yokoyama. A.. Furukawa. T.. Horiuchi. K., Yahata, T., Saji. H.. Sakahara, H., Nakashima. T., Koizumi, M., and Endo. K. Technetium-99m- until such time that an ideal combination of radionuclide and labeled monoclonal antibody with preserved immunoreactivity and high in antibody can be identified. The use of directly labeled antibodies vivo stability. J. NucÃ.Med., 28: 1027-1033, 1987. 11. Pettit, W. A., Deland. F. H.. Bennett, S. J., and Goldenberg, D. M. Improved or antibodies labeled nominally via bifunctional chelates has protein labeling with stannous tartrate reduction of pertechnetate. J. NucÃ. added an unnecessary complication to the already complicated Med., 21: 59-62. 1980. 12. Huang. J. T.. Kaiszedeh. M.. and Sakimura. 1. Detection of bacterial endo experiment involving the search for the ideal diagnostic or carditis with **Tc labeled antistaphylococcal antibody. J. NucÃ.Med., 21: therapeutic antibody. 783-786. 1980. Since the First Conference many new labeling techniques and 13. Rhodes, B. A., Torvestad, D. A., Breslow, K., Burchiel, S. W., Reed, K. A., and Austin. R. K. 99mTc labeling and acceptance testing of radiolabeled chelating agents have been developed. One purpose of this antibodies. In: S. W. Burchiel and B. A. Rhodes. Tumor Imaging, p. 111. conference is to investigate these new techniques and chelating New York: Masson Publishing USA, Inc., 1982. 14. Sundrehagen, E. Formation of "Te immunoglobulin G complexes free from agents to determine if radiochemically pure antibodies have radionuclides. quality controlled by radioimmunoelectrophoresis. Eur. J. been developed. NucÃ.Med., 7: 549-552, 1982. 15. Steigman, J., Williams, H. P.. and Solomon, N. A. The importance of the protein sulfhydry I group in HSA labeling with "Te. J. NucÃ.Med., 16:573, References 1975. 16. Paik. C. H., Pham. L. N. B., Hong. J. J., Sahami. M. S.. Heald. S. C.. Reba, R. C., Steigman, J.. and Eckelman. W. C. The labeling of high affinity sites 1. Zalutsky, M. R., and Narula, A. S. A method for the radiohalogenation of of antibodies with '""Tc. Int. J. NucÃ.Med. Biol., 12: 3-8, 1985. proteins resulting in decreased thyroid uptake of radioiodine. Int. J. Appi. 17. Paik. C. H.. Eckelman. W. C.. and Reba. R. C. Transchelation of "Tc from RadiÃ¢t.Isot., 38: 1051-1055. 1987. low affinity sites to high affinity sites of antibody. NucÃ.Med. Biol., 13: 359- 2. Ali, S. A., Warren, S. D., Richter. K. Y., Badger. C. C.. Eary. J. F., Press, 362, 1986. O. W., Krohn, K. A., Bernstein, I. D., and Nelp, W. B. Improving the tumor 18. Fritzberg, A. R. Advances in "Tc-labeling of antibodies. NucÃ.Med., 26:1- retention of radioiodinated antibody: aryl carbohydrate adducts. Cancer Res., 12, 1987. (Suppl.), 50: 783s-788s, 1990. 19. Franz. J.. Volkert. W. A.. Barefield. E. K., and Holmes, R. A. The production 3. Kasina, S., Vanderheyden, J. L., and Fritzberg. A. R. Application of diamide of technetium-99m-labeled conjugated antibodies using a cyclam-based bi dimercaptide NjS2 bifunctional chelating agents for "Te labeling of pro functional chelating agent. J. NucÃ.Med. Biol.. 14: 569-572. 1987. teins. J. Labeled Compos. & Radiopharm.. 23: 269-271. 1986. 20. Fritzberg. A. R., Abrams. P. G.. Beaumier. P. L., Kasina, S., Morgan, A. C., 4. Arano, Y., Yokoyama, A., Magata, Y.. Saji. H.. Horiuchi, K., and Torizuka. Rao, T. N., Reno, J. M.. Sanderson. J. A.. Srinivasan. A., and Wilbur. D. S. K. Synthesis and evaluation of a new bifunctional chelating agent for ""Te Specific and stable labeling of antibodies with technetium-99m with a diamide labeling proteins: p-carboxyethylphenyl-glyoxal-di(iV-methylthiosemicarba- dithiolate chelating agent. Proc. Nati. Acad. Sci. USA, 85: 4025-4029, 1988.

782s

W. C. Eckelman

Cancer Res 1990;50:780s-782s.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/50/3_Supplement/780s

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/50/3_Supplement/780s. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.