Pretargeting of human mammary carcinoma xenografts with bispecific anti-MUC1/anti-Ga chelate antibodies and immunoscintigraphy with PET

doi:10.1016/S0969-8051(01)00246-3

Nuclear Medicine and Biology

Volume 28, Issue 7, October 2001, Pages 821-828

https://doi.org/10.1016/S0969-8051(01)00246-3 Get rights and content

Abstract

¹ We recently demonstrated the feasibility of combining enhanced tumor-to-tissue contrast and PET imaging for immunoscintigraphic tumor localization in pancreas and colon carcinoma bearing nude mice. Contrast enhancement was obtained with a multistep targeting technique that consists of the sequential administration of an antitumor/antihapten bispecific antibody (BS-MAb), a blocker to saturate the antihapten binding sites of the BS-MAb that remains in circulation, and a low molecular weight Ga chelate, labeled with the positron emitter ⁶⁸Ga, which serves as the hapten. To evaluate the efficacy of this pretargeting technique for breast cancer localization, we synthesized a BS-MAb from the F(ab′)₂ fragments of the anti-MUC1 MAb 12H12 which reacts with the vast majority of human breast carcinomas, and the F(ab′) fragment of an anti-Ga chelate MAb using a bifunctional chemical linker. The BS-MAb was tested for its affinity and its biokinetics in nude mice bearing a human mammary carcinoma.

Equilibrium binding of the BS-MAb for mammary carcinoma cells was low (1.2 × 10⁷ M⁻¹) while the binding capacity of cells was high (8.4 × 10⁶ BS-MAbs per cell). Tumor uptake of the ⁶⁷Ga labeled chelate in pretargeted animals was to 5.8 ± 0.8% iD/g resulting in a tumor-to-blood ratio of 2.6 at 1h postinjection. This compares with a ratio of 0.65 and 0.85 obtained with ¹²⁵I-labeled native 12H12 at 24h and 48h postinjection.

No difference in the tumor uptake of both the ⁶⁸Ga and ⁶⁷Ga labeled chelate was observed. PET imaging of mice, started 1h postinjection of the ⁶⁸Ga chelate, clearly visualized all tumors.

Introduction

Sensitivity in immunoscintigraphic tumor localization can be markedly improved using pretargeting techniques where monoclonal antibodies (MAbs) are conjugated with a high affinity noncovalent binding site for a small, diffusible radiolabeled hapten, which is given after the accumulation of the MAbs in the tumor [10]. The most popular MAb pretargeting techniques employ either the biotin/avidin [24], [8] or the hapten/antibody system [2], [11], [12], [21], [22]. These techniques circumvent the limitations high blood pool and liver background activity [17], [26] and macromolecule targeting of solid tumors [16], [18], associated with conventional immunoscintigraphy using MAbs labeled prior to injection. The main advantage of pretargeting is an increased tumor contrast, which is obtained by a reduction of background activity in normal tissue. Hapten labeling has been carried out with the radionuclides used for MAb labeling such as ¹³¹I, ^99mTc or ¹¹¹In. The use of these gamma emitters excludes the application of positron emission tomography (PET) as an imaging modality with its inherently better contrast resolution and higher detection efficiency compared with gamma cameras.

To combine an improved tumor contrast and PET, we developed a multistep targeting system based on a Ga chelate. This chelate can be labeled with either ⁶⁷Ga (EC. t_1/2 78h) or with the short-lived positron emitter ⁶⁸Ga (β⁺ 88%, t_1/2 68 min). For pretargeting, we injected bispecific antitumor/anti-Ga chelate antibodies (BS-MAbs) and 20h later a blocker, to saturate the anti-Ga chelate binding sites of the BS-MAbs which remained in circulation. Tumor localization with PET was carried out 1h after the administration of the ⁶⁸Ga chelate, which was given shortly after the blocker. Using different antitumor MAbs for BS-MAb preparation, tumor contrast was markedly enhanced in nude mice bearing rat pancreas carcinoma [27] or human colon carcinoma [20].

In the presented study, an antimucin MAb was included in the pretargeting system to serve as the antitumor part of the BS-MAb preparation. The efficiency of the system to improve localization of human mammary carcinoma xenografts was examined in a nude mouse model.

Section snippets

Monoclonal antibodies and preparation of bispecific antibodies

MAb 12H12 is a mouse IgG1 raised against tumor cells from a human mammary carcinoma [3], [7]. 12H12 recognizes the carbohydrate side chains of the tumor associated glycoprotein TAG-12. TAG-12 differs in glycosylation from MUC1 mucin on normal epithelial cells, and is overexpressed on human epithelial cell adenocarcinomas. MUC1 is a transmembrane glycoprotein of ≥400 kDa M_r, which is present in the cytoplasm, on cell membranes and is found in secretory components due to proteolytic cleavage from

Quality of reagents

Purity of the 12H12/3A10 BS-MAb was ≥95% as assessed by gel filtration chromatography. Low binding constants (K) of 1.2–1.9 × 10⁷ M⁻¹ of the anti-MUC1 MAbs were calculated from Scatchard plots using data from AR-1 cell assays (Fig 1A). Affinity of the antimucin F(ab′)₂ decreased slightly as a consequence of BS-MAb preparation. However, the maximum number of BS-MAbs bound per cell came to 8.4 × 10⁶, indicating a high number of binding epitopes per antigen molecule. Scatchard plots resulting

Discussion

MAb 12H12 recognizes the tumor associated glycoprotein TAG12, which is expressed in more than 96% (n = 206) of primary breast tumors [19], and shows a substantial tumor uptake in different mammary carcinoma xenografts [7]. These features of the native MAb suggested a pretargeting with a 12H12 containing BS-MAb to increase tumor contrast and enable immunoscintigraphy with PET. Biodistribution studies with the ⁶⁷Ga chelate in AR-1 tumor-bearing mice pretreated with the 12H12/3A10 BS-MAb and a

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¹: Abbreviations used: PET, positron emission tomography; MAb, monoclonal antibody; BS-MAb, bispecific MAb; TAG, tumor associated glycoprotein; Ga-HBED-CC, Gallium-N, N′[2-hydroxy-5-(ethylene-β-carboxy) benzyl] ethylene diamine N, N′ diacetic acid; IF, immuno reactive fraction; Rf, retention factor; % iD, percentage of injected dose; PBS, 0.15 M NaCl solution containing 0.01 M phosphate buffer pH 7.4; T/B ratio, tumor-to-blood ratio.

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Pretargeting of human mammary carcinoma xenografts with bispecific anti-MUC1/anti-Ga chelate antibodies and immunoscintigraphy with PET

Abstract

Introduction

Section snippets

Monoclonal antibodies and preparation of bispecific antibodies

Quality of reagents

Discussion

Nucl Med Biol.

Biochem Biophys Res Comm.

J Immunol Meth.

IntJAppl RadiatIsot.

Pretargeting with the affinity enhancement system for radioimmunotherapy

Cancer Biotherapy and Radiopharmaceuticals

Monoclonal antibodies against human breast cancer

Antigen-antibody interactions and monoclonal antibodies

Pretargeting of renal cell carcinomaImproved tumor targeting with a bivalent chelate

Cancer Res.

Immunoscintigraphy of human mammary carcinoma xenografts using monoclonal antibodies 12H12 and BM-2 labeled with 99mTc and radioiodine

Cancer Res.

Delivery of therapeutic doses of radioiodine using bispecific antibody-targeted bivalent haptens

J Nucl Med.

Tumor pretargetingAlmost the bottom line

J Nucl Med.

Pretargeted peptide imaging and therapy

Cancer Biotherapy and Radiopharmaceuticals.

Pretargeted immunoscintigraphy of murine tumors with Indium-111 labeled bifunctional haptens

J Nucl Med.

Pretargeted immunoscintigraphyEffect of hapten valency on murine tumor uptake

J Nucl Med.

Immunoscintigraphy of human mammary carcinoma xenografts using monoclonal antibodies 12H12 and BM-2 labeled with ^99mTc and radioiodine