Radioimmunotherapy of prostate cancer.

In recent years, advances in the rational design of radiopharmaceuticals have helped in the introduction of clinically useful therapeutic agents into the clinic. In particular radiolabeled monoclonal antibodies such as (90)Y-ibritumomab and (131)I-tositumomab have shown to be clinically successful. More recently, (177)Lu has emerged as an attractive radionuclide for use in systemic radiotherapy. Like 131I, it has a longer half-live than (90)Y which is more suitable to the pharmacokinetics of monoclonal antibodies, but its chemistry is similar to (90)Y and, when internalized, it is retained by the tumor whereas 131I can be quickly released. Metastatic prostate cancer is an attractive target for radioimmunotherapy since the cornerstone of current therapy is hormonal therapy, which is only palliative. Prostate cancer is also known to metastasize to areas such as the lymph nodes and bone marrow, sites that are readily accessible to circulating monoclonal antibodies. This review will look at recent advances to the treatment of metastatic prostate cancer with radiolabeled antibodies with a specific emphasis on (177)Lu-huJ591, a labeled monoclonal antibody currently under clinical evaluation.

In vitro characterization of radiolabeled monoclonal antibodies specific for the extracellular domain of prostate-specific membrane antigen.

Prostate-specific membrane antigen (PSMA) is a well-characterized cell surface antigen expressed by virtually all prostate cancers (PCas). PSMA has been successfully targeted in vivo with (111)In-labeled 7E11 monoclonal antibody (mAb; ProstaScint; Cytogen, Princeton, NJ), which binds to an intracellular epitope of PSMA. This work reports the in vitro characterization of three recently developed mAbs that bind the extracellular domain of PSMA (PSMAext). Murine mAbs J415, J533, J591, and 7E11 were radiolabeled with 131I and evaluated in competitive and saturation binding studies with substrates derived from LNCaP cells. J415 and J591 were conjugated to 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid labeled with (111)In. The uptake and cellular processing of these antibodies were evaluated in viable LNCaP cells. All four mAbs could be labeled with 131I up to a specific activity of 350 MBq/mg with no or little apparent loss of immunoreactivity. Competition assays revealed that J415 and J591 compete for binding to PSMAext antigen. J533 bound to a region close to the J591 binding epitope, but J533 did not interfere with J415 binding to PSMA. mAb 7E11 did not inhibit the binding of J415, J533, or J591 (or vice versa), consistent with earlier work that these latter mAbs bind PSMAext whereas 7E11 binds the intracellular domain of PSMA. Saturation binding studies demonstrated that J415 and J591 bound with a similar affinity (Kds 1.76 and 1.83 nM), whereas J533 had a lower affinity (Kd, 18 nM). In parallel studies, all four mAbs bound to a similar number of PSMA sites expressed by permeabilized cells (1,000,000-1,300,000 sites/cell). In parallel studies performed with viable LNCaP cells, J415, J533, and J591 bound to a similar number of PSMA sites (i.e., 600,000-800,000 sites/cell), whereas 7E11 bound only to a subpopulation of the available PSMA sites (95,000 sites/cell). This apparent binding of 7E11 to viable cells can be accounted for by a 5-7% subpopulation of permeabilized cells produced when the cells were trypsinized and suspended. Up to five DOTA chelates could be bound to either J415 or J591 without compromising immunoreactivity. A comparison of the cellular uptake and metabolic processing of the 131I- and (111)In-labeled antibodies showed a rapid elimination of 131I from the cell and a high retention of (111)In. All four mAbs recognized and bound to similar numbers of PSMAs expressed by ruptured LNCaP cells (i.e., the exposed intracellular and extracellular domains of PSMA). By comparison to J415 and J591, J533 had a lower binding affinity. Both J415 and J591 recognized and bound to the same high number of PSMAs expressed by intact LNCaP. By contrast, 7E11 bound to fewer sites expressed by intact LNCaP cells (i.e., the exposed extracellular domain of PSMA). Both J415 and J591 are promising mAbs for the targeting of viable PSMA-expressing tissue with diagnostic and therapeutic metallic radionuclides.

DOTA-lanreotide: a novel somatostatin analog for tumor diagnosis and therapy.

Long acting somatostatin-14 (SST) analogs are used clinically to inhibit tumor growth and proliferation of various tumor types via binding to specific receptors (R). We have developed a 111In-/90Y-labeled SST analog, DOTA-(D)betaNal1-lanreotide (DOTALAN), for tumor diagnosis and therapy. 111In-/90Y-DOTALAN bound with high affinity (dissociation constant, Kd, 1-12 nM) to a number of primary human tumors (n = 31) such as intestinal adenocarcinoma (n = 17; 150-4000 fmol/mg protein) or breast cancer (n = 4; 250-9000 fmol/mg protein). 111In-/90Y-DOTALAN exhibited a similar high binding affinity (Kd, 1-15 nM) for the human breast cancer cell lines T47D and ZR75-1, the prostate cancer cell lines PC3 and DU145, the colonic adenocarcinoma cell line HT29, the pancreatic adenocarcinoma cell line PANC1, and the melanoma cell line 518A2. When expressed in COS7 cells, 111In-DOTALAN bound with high affinity to hsst2 (Kd, 4.3 nM), hsst3 (Kd, 5.1 nM), hsst4 (Kd, 3.8 nM), and hsst5 (Kd, 10 nM) and with lower affinity to hsst1 (Kd, approximately 200 nM). The rank order of displacement of [125I]Tyr11-SST binding to hsst1 was: SST (IC50, 0.5 nM) >> DOTALAN (IC50, 154 nM) > lanreotide (LAN) approximate to Tyr3-octreotide (TOCT) approximate to DOTA-Tyr3-octreotide (DOTATOCT) approximate to DOTA-vapreotide (DOTAVAP; IC50, >1000 nM); that to hsst2 was: DOTATOCT approximate to TOCT approximate to DOTALAN approximate to SST approximately LAN approximate to DOTAVAP (IC50, 1.4 nM); that to hsst3 was: SST (IC50, 1.2 nM) > DOTALAN = LAN (IC50, 15 nM) approximate to TOCT (IC50, 20 nM) approximate to DOTAVAP (IC50, 28 nM) > DOTATOCT (IC50, 73 nM); that to hsst4 was: SST (IC50, 1.8 nM) approximate to DOTALAN (IC50, 2.5 nM) > LAN (IC50, 22 nM) >> DOTATOCT approximate to DOTAVAP approximate to TOCT (IC50, >500 nM); and that to hsst5 was: DOTALAN (IC50, 0.45 nM) > SST (IC50, 0.9 nM) > TOCT (IC50, 1.5 nM) > DOTAVAP (IC50, 5.4 nM) >> LAN (IC50, 21 nM) > DOTATOCT (IC50 260 nM). In Sprague Dawley rats (n = 10), 90Y-DOTALAN was rapidly cleared from the circulation and concentrated in hsst-positive tissues such as pancreas or pituitary. Taken together, our results indicate that 111In-/90Y-DOTALAN binds to a broad range of primary human tumors and tumor cell lines, probably via binding to hsst2-5. We conclude that this radiolabeled peptide can be used for hsst-mediated diagnosis (111In-DOTALAN) as well as systemic radiotherapy (90Y-DOTALAN) of human tumors.

The somatostatin receptor-targeted radiotherapeutic [90Y-DOTA-DPhe1, Tyr3]octreotide (90Y-SMT 487) eradicates experimental rat pancreatic CA 20948 tumours.

Somatostatin receptor-expressing tumours are potential targets for therapy with radiolabelled somatostatin analogues. We have synthesized a number of such analogues in the past and identified [DOTA-dPhe1, Tyr3]octreotide (SMT 487) as the most promising candidate molecule because of its advantageous properties in cellular and in vivo tumour models. In the current paper we describe the radiotherapeutic effect of yttrium-90 labelled SMT 487 in Lewis rats bearing the somatostatin receptor-positive rat pancreatic tumour CA 20948. SMT 487 binds with nanomolar affinity to both the human and the rat somatostatin receptor subtype 2 (sst2) (human sst2 IC50=0.9 nM, rat sst2 IC50=0.5 nM). In vivo, 90Y-SMT 487 distributed rapidly to the sst2 expressing CA 20948 rat pancreatic tumour, with a tumour-to-blood ratio of 49.15 at 24 h post injection. A single intravenous administration of 10 mCi/kg 90Y-SMT 487 resulted in a complete remission of the tumours in five out of seven CA 20948 tumour-bearing Lewis rats. No regrowth of the tumours occurred 8 months post injection. Control animals that were treated with 30 microg/kg of unlabelled SMT 487 had to be sacrificed 10 days post injection due to excessive growth or necrotic areas on the tumour surface. Upon re-inoculation of tumour cells into those rats that had shown complete remission, the tumours disappeared after 3-4 weeks of moderate growth without any further treatment. The present study shows for the first time the curative potential of 90Y-SMT 487-based radiotherapy for somatostatin receptor-expressing tumours. Clinical phase I studies with yttrium-labelled SMT 487 have started in September 1997.

Synthesis and characterisation of [90Y]-Bz-DTPA-oct: a yttrium-90-labelled octreotide analogue for radiotherapy of somatostatin receptor-positive tumours.

An investigation into the in vitro behaviour of two yttrium-90-labelled somatostatin analogues was performed. Further in vivo characterisation was performed with the most promising agent. A new DTPA-octreotide analogue (Bz-DTPA-oct) was synthesised by coupling a bifunctional DTPA chelator to the N-terminal amine of the D-Phe1 of Tyr3-octreotide. This new SRIF analogue and DTPA-octreotide (OctreoScan) were radiolabelled with 90Y prior to serum stability being evaluated. Receptor binding assays were also performed on the two radioligands using rat cortex membranes. The [90Y]-Bz-DTPA-oct was further evaluated in vivo using tumour-bearing rats. The first conjugate (DTPA-octreotide) bound with a high affinity to SRIF receptors and the 90Y complex was relatively stable in human serum (t1/2 3.8 d for 90Y lost to serum proteins). The second conjugate (Bz-DTPA-oct) also exhibited a high binding affinity to SRIF receptors, but it demonstrated an even slower loss of 90Y to serum proteins (t1/2 12.1 d). The in vivo evaluation of the more stable [90Y]-Bz-DTPA-oct showed a very rapid and high accumulation in somatostatin receptor-positive tumours, which after 1 h resulted in tumour/nontumour ratios of 3.8, 21, and 4.9 (for blood, muscle, and liver, respectively). These tumour/nontumour ratios increased, and were by 24 h postinjection 138, 285, and 6.1 (for blood, muscle, and liver). Yttrium-90-labelled Bz-DTPa-oct is rapidly and selectively accumulated in somatostatin receptor-positive tissue. Octadentate Bz-DTPA-oct could be ligand for 90Y radiotherapy of somatostatin receptor-positive tumours and their metastases.

Synthesis, biodistribution and renal handling of various chelate-somatostatin conjugates with metabolizable linking groups.

A series of DTPA-octreotide conjugates, with various linking groups, were synthesised to investigate the effect of different metabolizable linkers on the renal retention of radioactivity. All these newly synthesised octreotide conjugates retained the high binding affinity of octreotide for the somatostatin (SRIF) receptors either when unlabeled or radiolabeled with 111In. Some of the metabolizable linkers were rapidly degraded in vitro when incubated with a kidney homogenate. However, in vivo, all these conjugates displayed a significantly lower uptake in SRIF receptor-positive tissue compared to two conjugates with short, stable linkers. Additionally, the compounds with a potentially metabolizable linker had a higher whole-body retention of activity as opposed to the three metabolically stable compounds. Several of the linkers gave evidence of cleavage while in circulation in the blood, and it is probable that the lower tumour accumulation of most of the compounds tested was low due to the high enzymatic nature of the exocrine pancreatic tumour model used. In short, no increase in the tumour-to-kidney ratio was achieved with the analogues containing a metabolizable linker. The highest target-to-nontarget tissue ratios were obtained for the DTPA-octreotide conjugates that had short, metabolically stable linkers.

Biological characterisation of [67Ga] or [68Ga] labelled DFO-octreotide (SDZ 216-927) for PET studies of somatostatin receptor positive tumors.

Radiolabelled analogues of Somatostatin (SRIF) were demonstrated to be useful for conventional gamma-camera imaging of SRIF receptor-positive tumors and their metastases. To evaluate the feasibility of positron emission tomography (PET) or SRIF receptor-positive tumors deferoxamine (DFO) was conjugated to octreotide via a succinyl linker to form a stable conjugate with the gallium isotopes 67Ga and 68Ga. This new octreotide analog, SDZ 216-927, binds specifically and with high affinity to SRIF receptors in vitro (pKi = 8.94 +/- 0.06) and exhibits SRIF like biological properties as demonstrated by the inhibition of growth hormone (GH) release from cultured pituitary cells. SDZ 216-927 was efficiently labelled with 67Ga without affecting high affinity binding to SRIF receptors. Biodistribution studies revealed that [67Ga]SDZ 216-927 was stable in vivo and rapidly cleared from the circulation, as indicated by the low amount of 67Ga detected in the blood four hours post injection (p.i.). SRIF receptor-positive tumors were clearly visualized 10 minutes p.i. in tumor bearing rats. The specificity of ligand binding in vivo was demonstrated i) by the high tumor/non-tumor ratio 4 hours p.i. (tumor/blood 22.3:1, tumor/muscle 64.5:1, tumor/liver 4.0:1, tumor/spleen 16.8:1) and ii) by a significantly lower uptake of radioactivity in the tumor after pretreatment of tumor bearing animals with an excess of unlabelled SDZ 216-927. SDZ 216-927, when labelled with the positron emitting isotope 68Ga, clearly imaged SRIF receptor-positive tumors using positron emission tomography (PET). Therefore quantitative SRIF receptor imaging with PET seems to be possible using this new radiopharmaceutical.

Gallium-67/gallium-68-[DFO]-octreotide--a potential radiopharmaceutical for PET imaging of somatostatin receptor-positive tumors: synthesis and radiolabeling in vitro and preliminary in vivo studies.

UNLABELLED: When labeled with gamma-emitting radionuclides, somatostatin analogs have the potential to localize somatostatin receptor-positive tumors using gamma camera scintigraphy. We present a somatostatin analog, [DFO]-octreotide (SDZ 216-927), that comprises desferrioxamine B coupled to octreotide via a succinyl linker. This conjugate can be labeled with either 67Ga for gamma scintigraphy or 68Ga for PET imaging. The 67Ga-labeled conjugate is stable in vitro to autoradiolysis over a 24-hr period. METHODS: Rats bearing a somatostatin receptor-positive pancreatic islet cell tumor were injected with 20 MBq of 67Ga[DFO]-octreotide (33 GBq 67Ga/mumole). RESULTS: After 1 hr, the accumulation of 67Ga[DFO]-octreotide was 0.38 +/- 0.08 %ID/g and the tumor-to-nontumor ratios for blood, muscle, liver and intestine were 2.5, 7.4, 1.9 and 1.6, respectively. PET studies with 68Ga[DFO]-octreotide recorded a very rapid accumulation at the tumor and a subsequent residence half-life of about 6 hr. CONCLUSION: Gallium-68-[DFO]-octreotide can be used in PET studies to diagnose receptor-positive tumors such as gastroenteropancreatic, small-cell lung and breast tumors.

Antibody labeling with copper-67 using the bifunctional macrocycle 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)methyl]benzoic acid.

The high kinetic stability of the Cu2+ complex of the chelator 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl]benzoic acid was demonstrated at physiological pH as well as under acidic conditions. The chelating agent was conjugated to AB35, a monoclonal antibody directed against CEA, without a significant loss of immunoreactivity. The conjugate could, under optimal labeling conditions, be labeled with 67Cu in acetate buffer with a full occupancy of ligands within 20 min. This radiolabeled conjugate showed no transfer of radiocopper to serum proteins in human serum over 7 days. The biodistribution in tumor-bearing mice was measured and compared to that of iodinated AB35. Tumor uptake was high with 15 +/- 3% ID (injected dose)/g after 24 h and 32 +/- 7% ID/g after 96 h for the 67Cu-labeled antibody and 13 +/- 4% ID/g after 24 h and 14 +/- 2% ID/g after 96 h for the 125I-labeled antibody. Whereas radioactivity in normal organs decreased with time after 24 h, increased residence time was shown up to 4 days with the 67Cu-labeled AB35.