Against - Springer Link

776 7. Paganelli G, Chinol M, Maggiolo M, et al. The three-step pretargeting approach reduces the human anti-mouse antibody response in patients submitted to radioimmunoscintigraphy and radioimmunotherapy. Eur J Nucl Med 1997; 24:350–351. 8. Paganelli G, Grana C, Chinol M, et al. Antibody-guided three step therapy for high grade glioma with yttrium-90 biotin. Eur J Nucl Med 1999; 26:348–357. 9. Grana C, Chinol M, Robertson C, et al. Pretargeted adjuvant radioimmunotherapy with yttrium-90-biotin in malignant glioma patients: a pilot study. Br J Cancer 2002; 86:207–212. 10. Paganelli G, Orecchia R, Jereczek-Fossa B, et al. Combined treatment of advanced oropharyngeal cancer with external radiotherapy and three-step radioimmunotherapy. Eur J Nucl Med 1998; 25:1336–1339. 11. Moro M, Pelagi M, Fulci C, et al. Tumor cell targeting with antibody-avidin complexes and biotinylated tumor necrosis factor α1. Cancer Res 1997; 57:1922–1928. 12. Guttinger M, Guidi F, Chinol M, et al. Adoptive immunotherapy by avidin-driven cytotoxic T lymphocyte- tumor bridging. Cancer Res 2000; 60:4211–4215. 13. Lehtolainen P, Taskinen A, Laukkanen J, et al. Cloning and characterization of Scavidin, a fusion protein for the targeted delivery of biotinylated molecules. J Biol Chem 2002; 227: 8545–8550.

limitations and prospects of certain pretargeting methods. In contrast to direct targeting systems, where an effector molecule, such as a radionuclide, is linked directly to the targeting agent, or antibody, pretargeting systems involve the administration of the effector molecule some time after the targeting agent. This allows time for the targeting agent to localize in tumor and, more importantly, to clear from normal tissues in the body. Table 1 provides a summary of different pretargeting systems, grouped according to the constituent of the targeting macromolecule, that have been investigated. Within each group, there may be further differentiation according to molecular size of the targeting macromolecule, molecular form of the antibody component comprising the macromolecule, method of preparing the targeting macromolecule or the source of the macromolecule, valency of the macromolecule to the target, valency of the macromolecule to the hapten or biotin, hapten valency of the effector to the macromolecule, and other functional groups carried by the effector. Pretargeting with the avidin/streptavidinbiotin system

Eur J Nucl Med Mol Imaging (2003) 30:776–780 DOI 10.1007/s00259-002-1089-6 Published online: 6 February 2003 © Springer-Verlag 2003

Against Introduction The use of anticancer antibodies to deliver radionuclides for the therapy of cancer, termed radioimmunotherapy (RAIT or RIT), has been the subject of experimentation for over two decades [1], and has now gained a role in the treatment of non-Hodgkin’s lymphoma [2, 3]. Unfortunately, solid tumors have been less responsive, because higher radiation doses are required, leading to various novel approaches to improve and enhance the delivery of the therapeutic radionuclide and to achieve a more uniform distribution of ionizing radiation to tumor while sparing normal tissues. One of these methods is pretargeting, a strategy first introduced in the 1980s. The progress in using pretargeting methods to improve RAIT has been the subject of several editorials [4, 5, 6, 7, 8] and reviews [9, 10, 11, 12, 13, 14, 15, 16]. The purpose of this commentary is to provide our appraisal of the

It is apparent that pretargeting methods have used a number of strategies, but the most prominent have involved avidin/steptavidin-biotin systems. The avidin/streptavidin system is highly versatile and has been applied in different ways. Antibodies have been coupled with streptavidin or biotin, which is used as the primary targeting agent. This is followed later by the effector molecule, which is conjugated with biotin or with streptavidin, respectively. Another configuration is a three-step approach first targeting a biotin-conjugated antibody, followed by a bridging with streptavidin/avidin, and then administration of the biotin-conjugated effector. Each of these systems is optimized by including a clearing/blocking step to remove the antibody conjugate from the blood [17, 18, 19]. Without this step, the antibody conjugate in the blood would bind much of the effector, thereby reducing tumor uptake and tumor/non-tumor radios for the effector. Since avidin/streptavidin and biotin have a very high binding affinity (~10–15 M), this system has been attractive among pretargeting methods. In addition, avidin has up to four binding sites for biotin, providing greater binding capacity. However, avidin and streptavidin are foreign proteins and, therefore, have shown considerable immunogenicity in vivo, thus limiting the number of times they can be given in any clinical application. Also, this system, like others, depends on the binding affinity of the primary targeting agent (e.g., the binding of the antibody to the tumor antigen), so that the higher affinity and avidity of streptavidin/avidin for biotin may not be a sufficient advantage unless an optimal tar-

European Journal of Nuclear Medicine and Molecular Imaging Vol. 30, No. 5, May 2003

777 Table 1. Pretargeting systems investigated (from Chang et al. [16], with permission) Cate- Targeting macromolecule gory Constituent Source or production method

Effector Molecular size

Molecular form of Ab

Valency Valency toward toward target effector

Constituent

Valency of effector

Function group other than chelate or biotin

Hapten– chelate

1

–

[20]

2 1

– –

[21] [22]

1

–

[23]

2 1 2 2 1

– – – – –

[23] [24] [25] [26] [27]

1

–

[17]

1

–

[28]

4 4 –

– – –

[29] [30] [31]

–

–

[32]

–

–

[33]

I

Ab

Hybridoma

160 kDa

IgG

0

2

II

SA

Commercial

54 kDa

–

0 0

2 4

III

BsAb

Chemical conjugation

150 kDa

F(ab’)2 × Fab’ 2

1

100 kDa

Fab’ × Fab’

54 kDa 215 kDa

Diabody IgG

2 1 1 1 2

1 1 1 1 4

225 kDa

IgG

2

4

160 kDa

IgG

2

Multiple

~50 kDa ~170 kDa

IgG Fab’ IgG

2 1 2

Multiple Multiple Multiple

2

Multiple

2 or 1

–

IV

Ab-SA

V

Ab-A

VI

Ab-B

VII VIII IX

Ab-DNA

Recombinant Chemical conjugation Chemical conjugation Chemical conjugation

Representative references

Chemical conjugation SA/B-PNA Complex ? – formation Ab-E Chemical 74–250 kDa IgG; F(ab’)2; conjugation or Fab’, scFv recombinant

Biotin– chelate Hapten– chelate

Biotin– chelate Biotin– chelate Biotin– chelate SA rSA DNA– tyrosine PNA– chelate Prodrug

Ab, Antibody; SA, streptavidin; rSA, recombinant SA; BsAb, bispecific antibody; Ab-SA, antibody–streptavidin conjugate; Ab-A, antibody–avidin conjugate; Ab-B, biotinylated antibody; Ab-DNA, anti-

body–DNA conjugate; SA/B-PNA, complex of streptavidin and biotinylated PNA; Ab-E, antibody–enzyme conjugate

geting antibody is used. This requirement appears to be the same for all pretargeting systems. With the biotin-avidin methods, the need to clear non-targeted antibody bearing, for example, biotin, adds further complexity to a multi-step approach. Another concern is the possible presence of endogenous biotin or biotinases, which can interfere with the avidin/streptavidin given. Nevertheless, as Grana et al. [28] have reported, promising clinical results in brain tumor therapy have been achieved, and encourage the design of randomized, prospective, controlled clinical trials to confirm these findings. Indeed, how much better pretargeting is over directly injecting a radiolabeled antibody regionally (e.g., intracerebrally) remains to be proven.

Pretargeting with the bispecific antibody recognition system Unlike systems using avidin/streptavidin, pretargeting with bispecific antibodies has the potential advantage of not needing to use such an immunogenic molecule as avidin or streptavidin, which presumably limit the therapeutic injections. Bispecific antibodies are being engineered as relatively non-immunogenic proteins comprising human complementarity-determining (hypervariable) regions (CDRs), thus reducing the content of murine protein. Indeed, fully human constructs are also feasible for this technology. A particularly important aspect of bispecific antibody pretargeting is the valency of the hapten of the effector molecule given after the targeting agent localizes. It has been found that effectors containing a bivalent hapten are better for tumor localization than monovalent counterparts. The enhancement,


778

Fig. 1. The affinity-enhancement system showing the crosslinking of 2 bispecific antibodies (BsAb) on the targeted tumor surface with one bivalent hapten. The bivalent hapten can carry a therapeutic or diagnostic agent. (From Chang et al. [16], with permission)

termed affinity-enhancement system, or AES [14], is believed to be due to the ability of the bivalent hapten to crosslink the pretargeted macromolecule at the tumor site, resulting in the formation of a more stable complex (Fig. 1) and, therefore, a longer tumor residence time. In animal models, pretargeting using an anti-CEA Fab’ x anti-DTPA Fab’ bsMAb followed by an 131I-labeled peptide successfully cured 7/12 animals with less toxicity compared to progressive growth of all tumors in animals given 131I-labeled anti-CEA F(ab’)2 [34]. Clinical trials using the AES pretargeting method with a chemically constructed bispecific anti-carcinoembryonic antigen (CEA) × anti-indium-DTPA conjugate and a radioiodinated (131I) di-indium-DTPA hapten (Pentacea, IBC Pharmaceuticals) have produced impressive images of tumor uptake with a tumor/whole-body radiation dose advantage of >38 [16], confirming the advantage of this pretargeting method in patients with CEA-expressing neoplasms.

Preclinical and clinical studies have indicated that bispecific MAb pretargeting approaches are highly promising, and with the aid of molecular engineering, new bispecific antibodies can be designed that could further enhance the targeting and binding of the macromolecules to tumor, compared with the chemical constructs that have been used until now. Multivalent and multispecific targeting macromolecules of smaller size than intact antibodies are being engineered, such as bispecific diabodies or tetravalent diabodies (Fig. 2). BS1.5H is one such bispecific diabody (Mr ~54,000) that we have produced, and consists of two heterologous polypeptide chains associated non-covalently to form one binding site for CEA from the variable domains of hMN-14, a humanized anti-CEA-specific monoclonal antibody (Immunomedics, Inc.), and one binding site for the HSG (histamine-succinyl-glycin) hapten from the variable domains of the 679 antibody. The 679 effectorrecognition system provides a further advantage for the future by allowing the flexibility in the design of haptens that bear the HSG recognition moiety. Thus, we have successfully prepared a recombinantly engineered, fully humanized, targeting molecule, BS1.5H, that incorporates the humanized 679 VH and Vκ domains with the humanized anti-CEA domains. BS1.5H shows rapid targeting and high tumor/non-tumor and uptake ratios comparable with chemical constructs [26], thus providing a relatively small and non-immunogenic molecule that can be produced in prokaryotic or eukaryotic vectors. Conclusion We are confident that future pretargeting methods will include such recombinantly engineered fusion proteins of multispecificity and multivalency, and that these new constructs will lead to further improvements in the radioimmunotherapy of cancer. This is not intended to suggest

Fig. 2. Representation of two multispecific macromolecular constructs produced by recombinant engineering for tumor pretargeting


779

that other pretargeting approaches, including those involving biotin-avidin systems, are not equally promising. Each method has its advantages and limitations, and only when they have been fully optimized and tested in appropriate clinical studies will a better assessment of their prospects be possible. David M. Goldenberg (✉), Robert M. Sharkey, Habibe Karacay, Center for Molecular Medicine and Immunology, Garden State Cancer Center, 520 Belleville Avenue, Belleville, NJ 07109-0023 USA e-mail: [email protected] Tel.: +1-973-8447000, Fax: +1-973-8447020 David M. Goldenberg, William McBride, Hans J. Hansen Immunomedics Inc., Morris Plains, USA David M. Goldenberg, Chien-Hsing Chang, Edmund A. Rossi IBC Pharmaceuticals Inc., Morris Plains, USA Jean-Francois Chatal, Jacques Barbet Centre René Gauducheau and INSERM U463, Nantes, France

References 1. Goldenberg DM. Targeted therapy of cancer with radiolabeled antibodies. J Nucl Med 2002; 43:693–713. 2. Goldenberg DM. The role of radiolabeled antibodies in the treatment of non-Hodgkin’s lymphoma: the coming of age of radioimmunotherapy. Crit Rev Oncol Hematol 2001; 39:195– 201. 3. Witzig TE, Gordon LI, Cabanillas F, Czuczman MS, Emmanouilides C, Joyce R, Pohlman BL, Bartlett NL, Wiseman GA, Padre N, Grillo-Lopez AJ, Multani P, White CA. Randomized controlled trial of yttrium-90-labeled ibritumomab tiuxetan radioimmunotherapy versus rituximab immunotherapy for patients with relapsed or refractory lowgrade, follicular, or transformed B-cell non-Hodgkin’s lymphoma. J Clin Oncol 2002; 20:2453–2463. 4. Goodwin DA. Pharmacokinetics and antibodies. J Nucl Med 1987; 28:1358–1362. 5. Goodwin DA. New methods for localizing infection: a role for avidin-biotin? J Nucl Med 1992; 33:1816–1818. 6. Goodwin DA. Tumor pretargeting: almost the bottom line. J Nucl Med 1995; 36:876–879. 7. Lovqvist A, Divgi C. Bullets to magic bullets and miles to go before we sleep. J Nucl Med 1998; 39:1776–1777. 8. Goodwin DA, Meares CF. Pretargeted peptide imaging and therapy. Cance Biotherapy Radiopharmaceuticals 1999; 14:145–152. 9. Le Doussal JM, Barbet J, Delaage M. Bispecific-antibody-mediated targeting of radiolabeled bivalent haptens: theoretical, experimental, and clinical results. Int J Cancer (Suppl) 1992; 7:58–62. 10. Chatal, JF, Faivre-Chauvet A, Bardies M, Peltier P, Gautherot E, Barbet J. Bifunctional antibodies for radioimmunotherapy. Hybridoma 1995; 14:125–128. 11. Rosebrough SF. Two-step immunological approaches for imaging and therapy. Q J Nucl Med 1996; 40:234–251. 12. Goodwin DA, Meares CF. Pretargeting: general principles. Cancer (Suppl) 1997; 80:2675–2680.

13. Fritzberg AR. Antibody pretargeted radiotherapy: a new approach and a second chance. J Nucl Med 1998; 39:20N. 14. Barbet J, Kraeber-Bodéré F, Vuillez, JP, Gautherot E, Rouvier E, Chatal JF. Pretargeting with the affinity enhancement system for radioimmunotherapy. Cancer Biother Radiopharm 1999; 14:153–166. 15. Gruaz-Guyon A, Janevik-Ivanovska E, Raguin O, De Labriolle-Vaylet C, Barbet J. Radiolabeled bivalent haptens for tumor immunodetection and radioimmunotherapy. Q J Nucl Med 2001; 45:201–206. 16. Chang CH, Sharkey RM, Rossi EA, Karacay H, McBride W, Hansen HJ, Chatal JF, Barbet J, Goldenberg DM. Molecular advances in pretargeting radioimmunotherapy with bispecific antibodies. Mol Cancer Ther 2002; 1:553–563. 17. Hnatowich DJ, Virzi F, Ruschowski M. Investigations of avidin and biotin for imaging applications. J Nucl Med 1987; 28:1294–1302. 18. Paganelli G, Riva P, Deleide G, Clivio A, Chiolerio F, Scassellati GA, Malcovati M, Siccardi AG. In vivo labeling of biotinylated monoclonal antibodies by radioactive avidin: a strategy to increase tumor radiolocalization. Int J Cancer (Suppl) 1988; 2:121–125. 19. Axworthy DM, Reno JM, Hylarides MD, Mallett RW, Theodore LJ, Gustavson LM, Su F, Hobson LJ, Beaumier PL, Fritzberg AR. Cure of human carcinoma xenografts by a single dose of pretargeted yttrium-90 with negligible toxicity. Proc Natl Acad Sci USA 2000; 97:1802–1807. 20. Goodwin DA, Meares CF, McCall MJ, McTigue M, Chaovapong W. Pre-targeted immunoscintigraphy of murine tumors with indium-111-labeled bifunctional haptens. J Nucl Med 1998; 29:226–234. 21. Goodwin DA, Meares CF, McTigue M, Chaovapong W, Diamanti CI, Ransone CH, McCall MJ. Pretargeted immunoscintigraphy: effect of hapten valency on murine tumor uptake. J Nucl Med 1992; 33:2006–2013. 22. Rusckowski M, Fritz B, Hnatowich DJ. Localization of infection using straptavidin and biotin: an alternative to nonspecific polyclonal immunoglobulin. J Nucl Med 1992; 33:1810–1815. 23. Le Doussal JM, Martin M, Gautherot E, Delaage M, Barbet J. In vitro and in vivo targeting of radiolabeled monovalent and divalent haptens with dual specificity monoclonal antibody conjugates: enhanced divalent hapten affinity for cell-bound antibody conjugate. J Nucl Med 1989; 30:1358–1366. 24. Stickney DR, Slater JB, Kirk GA, Ahlem C, Chang CH, Frincke JM. Bifunctional antibody: ZCE/CHA 111Indium BLEDTA-IV clinical imaging in colorectal carcinoma. Antibody Immunoconjug Radiopharm 1989; 2:1–13. 25. Hosono M, Hosono MN, Kraeber-Bodéré F, Devys A, Thédrez P, Faivre-Chauvet A, Gautherot E, Barbet J, Chatal JF. Twostep targeting and dosimetry for small cell lung cancer xenograft with anti-NACM/antihistamine bispecific antibody and radioiodinated bivalent hapten. J Nucl Med 1999; 40:1216– 1221. 26. Sharkey RM, Rossi E, Karacay H, McBride W, Chang K, Zeng L, Qu T, Griffiths G, Hansen HJ, Goldenberg DM. A novel recombinant bispecific antibody pretargeting system for cancer radioimmunotherapy [abstract]. Clin Cancer Res (Suppl) 2002; 7:3786s. 27. Kalofonos HP, Rusckowski M, Siebecker DA, Sivolapenko GB, Snook D, Lavender JP, Epenetos AA, Hnatowich DJ. Imaging of tumor in patients with indium-111-labeled biotin and streptavidin-conjugated antibodies: preliminary communication. J Nucl Med 1990; 31:1791–1796.


780 28. Grana C, Chinol M, Robertson C, Mazzetta C, Bartolomei M, De Cicco C, Fiorenza M, Gatti M, Caliceti P, Paganelli G. Pretargeted adjuvant radioimmunotherapy with yttrium-90-biotin in malignant glioma patients: A pilot study. Br J Cancer 2002; 86:207–212. 29. Saga T, Weinstein J N, Jeong JM, Heya T, Le JT, Le N, Paik CH, Sung C, Neumann R. Two-step targeting of experimental lung metastases with biotinylated sntibody and radiolabeled streptavidin. Cancer Res 1994; 54:2160–2165. 30. Wilbur DS, Hamlin DK, Vessella RL, Stray JE, Buhler KR, Stayton PS, Klumb LA, Pathare PM, Weerawarna SA. Antibody fragments in tumor pretargeting. Evaluation of biotinylated Fab’ colocalization with recombinant streptavidin and avidin. Bioconjugate Chem 1996; 7:689–702.

31. Kuijpers WHA, Bos ES, Kaspersen FM, Veeneman GH, van Boeckel CAA. Specific recognition of antibody-oligonucleotide conjugates by radiolabeled antisense nucleotides: a novel approach for two-step radioimmunotherapy of cancer. Bioconjugate Chem 1993; 4:94–102. 32. Rusckowski M, Qu T, Chang F, Hnatowich DJ. Pretargeting using peptide nucleic acid. Cancer (Suppl) 1997; 80:2699–2705. 33. Bagshawe KD, Sharma SK, Burke PJ, Melton RG, Knox RJ. Developments with targeted enzymes in cancer therapy. Curr Opin Immunol 1999; 11:579–583. 34. Gautherot E, Rouvier E, Daniel L, Loucif E, Bouhou J, Manetti C, Martin M, LeDoussal J-M, Barbet J. Pretargeted radioimmunotherapy of human colorectal xenografts with bispecific antibody and131I-labeled bivalent hapten. J Nucl Med 2000; 41:480–487.
