Improved tumor selectivity of radiolabeled peptides by receptor and antigen dual targeting in the neurotensin receptor model

Radiolabeled peptides are emerging tools for diagnosis and therapy of tumors overexpressing receptors. However, binding to receptors expressed by nontumor tissues may cause toxicity. The objective of this study was to specifically enhance the binding affinity of labeled peptides to tumor cells, as opposed to receptor-positive nontumor cells, to ensure targeting selectivity. This was achieved by the simultaneous binding of hapten-bearing peptides to their receptor and to a tumor-associated antigen, mediated by a bispecific antibody directed to the tumor antigen and to the hapten. Binding of labeled neurotensin analogues bearing the DTPA(indium) hapten (NT-DTPA(111In)) to human colorectal carcinoma cells (HT29), which express the neurotensin receptor (NTR1) and carcinoembryonic antigen (CEA), was studied in the presence of a bispecific antibody (BsmAb) directed to CEA and to DTPA(indium). In vitro dual binding of NT-DTPA in the presence of BsmAb was about 6.5-fold higher than monovalent binding to NTR1 and 3.5-fold higher than the sum of the monovalent bindings to NTR1 or to CEA, suggesting cooperativity. Increased binding under bivalent conditions translated into increased internalization. In vivo pretargeting with BsmAb enhanced tumor uptake and tumor retention. Hapten bearing peptides binding simultaneously an overexpressed cell-surface receptor and a tumor antigen show increased selectivity to target tumor cells as compared to cells only expressing the cell surface receptor. Better resistance to enzymatic degradation and optimized administration protocols should further enhance in vivo targeting selectivity and may allow the development of radiopharmaceuticals labeled with isotopes suitable for radiotherapy such as 131I or 90Y.     

Pretargeted radioimmunotherapy using 131I-labelled bivalent hapten-bearing peptides

Summary The advantages of bivalent hapten-bearing peptides for the detection of tumours pretargeted with bispecific antibodies have been demonstrated. This technology is now considered for radioimmunotherapy and bivalent haptens designed to target¹³¹I are needed. We thus synthesised a series of tyrosine-containing peptides bearing the histamine-hemisuccinate hapten. These molecules were tested for their ability to bind simultaneously two anti-hapten antibody molecules. One of these bivalent haptens, AG3.0, with a lysyl-d-tyrosyl-lysine connecting chain, was found to have optimal binding characteristics and was thus selected for further investigations. AG3.0 was shown to efficiently deliver radioactive iodine to human colorectal tumours grafted in nude mice using an anti-carcinoembryonic antigen×anti-histamine-hemisuccinate bispecific antibody. AG3.0 was also targeted to human B lymphoma cells pretargeted with a bispecific antibody specific for membrane IgM. In this system, bivalent ligands such as F(ab′)₂ or IgG are rapidly internalised and covalently linked radioactive iodine is released from target cells as a result of intracellular catabolism. With the pretargeted iodine-labelled bivalent hapten, a fivefold increase in the intracellular activity retention time as compared to¹²⁵I-labelled F(ab′)₂ and IgG was observed. The radiolabelled hapten did not undergo any degradation after internalisation. These results have been confirmed in vivo with an anti-BCL₁ IgM idiotype bispecific antibody and¹³¹I-labelled AG3.0. These reagents injected as a single 300 μCi dose, 7 days after inoculation of 10⁴ BCL₁ lymphoma cells in BALB/c mice, cured 14/16 of the animals and the treatment was well tolerated. Comparatively, the same dose of labelled IgG cured 13/16 of the mice but three mice died of haematologic toxicity. The same dose of labelled F(ab′)₂ or Fab′ was completely inefficient.¹³¹I-labelled bivalent haptens are now used in phase I radioimmunotherapy clinical trials.     

Pretargeted radioimmunotherapy using 131I-labelled bivalent hapten-bearing peptides

The advantages of bivalent hapten-bearing peptides for the detection oftumours pretargeted with bispecific antibodies have been demonstrated. Thistechnology is now considered for radioimmunotherapy and bivalent haptensdesigned to target ¹³¹I are needed. We thus synthesised aseries of tyrosine-containing peptides bearing the histamine-hemisuccinatehapten. These molecules were tested for their ability to bind simultaneouslytwo anti-hapten antibody molecules. One of these bivalent haptens, AG3.0,with a lysyl-d-tyrosyl-lysine connecting chain, was found to have optimalbinding characteristics and was thus selected for further investigations.AG3.0 was shown to efficiently deliver radioactive iodine to humancolorectal tumours grafted in nude mice using an anti-carcinoembryonicantigen×anti-histamine-hemisuccinate bispecific antibody. AG3.0 wasalso targeted to human B lymphoma cells pretargeted with a bispecificantibody specific for membrane IgM. In this system, bivalent ligands such asF(ab)₂ or IgG are rapidly internalised and covalentlylinked radioactive iodine is released from target cells as a result ofintracellular catabolism. With the pretargeted iodine-labelled bivalenthapten, a fivefold increase in the intracellular activity retention time ascompared to ¹²⁵I-labelled F(ab)₂ and IgGwas observed. The radiolabelled hapten did not undergo any degradation afterinternalisation. These results have been confirmed in vivo with ananti-BCL₁ IgM idiotype bispecific antibody and¹³¹I-labelled AG3.0. These reagents injected as a single 300µCi dose, 7 days after inoculation of 10⁴BCL₁ lymphoma cells in BALB/c mice, cured 14/16 of the animalsand the treatment was well tolerated. Comparatively, the same dose oflabelled IgG cured 13/16 of the mice but three mice died of haematologictoxicity. The same dose of labelled F(ab)₂ orFab was completely inefficient. was completely inefficient. ¹³¹I-labelled bivalenthaptens are now used in phase I radioimmunotherapy clinical trials.     

Neutralization of cholera toxin by rat IgA secretory antibodies induced by a free synthetic peptide

Secretory immunoglobulin A (sIgA) is the major immunoglobulin in the bile of several species. They contribute to local immune defences of the gut. The protection against cholera toxin (CT) is due to the presence of specific sIgA in the bile and in the gut. We have already reported that oral administration of the peptide corresponding to the sequence 50-75 of cholera toxin B subunit elicits serum antibodies neutralizing CT activity, and that IgA and local protection are observed in the intestine of P50-75 orally immunized mice. In this study, we demonstrate the potential of this synthetic peptide as immunogen without carrier or adjuvant, not only in a strain known to be sensitive to CT, but also in an outbred one. Furthermore, this peptide stimulates the mucosal immunity, since we show that P50-75 induced-sIgA purified from rats bile and serum, are capable of neutralizing CT activity in the in vivo intestinal ligated loop test.     

Europium-labeled epidermal growth factor and neurotensin: novel probes for receptor-binding studies.

We investigated the possibility of labeling two biologically active peptides, epidermal growth factor (EGF) and neurotensin (NT), with europium (Eu)-diethylenetriaminepentaacetic acid. More specifically, we tested them as probes in studying receptor binding using time-resolved fluorescence of Eu3+. The relatively simple synthesis yields ligands with acceptable binding characteristics similar to isotopically labeled derivatives. The binding affinity (Kd) of labeled Eu-EGF to human A431 epidermal carcinoid cells was 3.6 +/- 1.2 nM, similar to the reported Kd values of EGF, whereas the Kd of Eu-NT to human HT29 colon cancer cells (7.4 +/- 0.5 nM) or to Chinese hamster ovary (CHO) cells transfected with the high-affinity NT receptor (CHO-NT1) were about 10-fold higher than the Kd values of NT. The bioactivity of the Eu-labeled EGF as determined by stimulation of cultured murine D1 hematopoietic cell proliferation was nearly the same as that obtained with native EGF. The maximal stimulation of Ca2+ influx with NT and Eu-NT in CHO-NT1 cells was similar, but the respective K0.5 values were 20 pM and 1 nM, corresponding to differences in the binding affinities previously described. The results of these studies indicate that Eu labeling of peptide hormones and growth factor molecules ranging from 10(3) to 10(5) Da can be conveniently accomplished. Importantly, the Eu-labeled products are stable for approximately 2 years and are completely safe for laboratory use compared to the biohazardous radioligands. Thus, Eu-labeled peptides present an attractive alternative for commonly used radiolabeled ligands in biological studies in general and in receptor assays in particular.     

Novel DOTA-neurotensin analogues for 111In scintigraphy and 68Ga PET imaging of neurotensin receptor-positive tumors

Overexpression of the high affinity neurotensin receptor 1 (NTSR1), demonstrated in several human cancers, has been proposed as a new marker for human ductal pancreatic carcinoma and as an independent factor for poor prognosis for ductal breast cancer, head and neck squamous cell carcinoma, and non-small cell lung cancer. The aim of the present study was to develop new DOTA-neurotensin analogues for positron emission tomography (PET) imaging with (68)Ga and for targeted radiotherapy with (90)Y or (177)Lu. We synthesized a DOTA-neurotensin analogue series. Two of these peptides bear two sequence modifications for metabolic stability: DOTA-NT-20.3 shares the same peptide sequence as the previously described DTPA-NT-20.3. In the sequence of DOTA-NT-20.4, the Arg(8)-Arg(9) bond was N-methylated instead of the Pro(7)-Arg(8) bond in DOTA-NT-20.3. An additional sequence modification was introduced in DOTA-LB119 to increase stability. A spacer was added between DOTA and the peptide sequence to increase affinity. Binding to HT29 cells, which express NTSR1, in vivo stability, and biodistribution of the various analogues were compared, and the best candidate was used to image tumors of various sizes with the microPET in mice. (111)In-DOTA-NT-20.3, in spite of a relatively high uptake in kidneys, showed specific tumor uptake and elevated tumor to other organ uptake ratios. High contrast images were obtained at early time points after injection that allowed tumor detection at a time interval postinjection appropriate for imaging with the short-lived radionuclide (68)Ga. (111)In-DOTA-NT-20.4 displayed inferior binding to HT29 cells and reduced tumor uptake. (111)In-DOTA-LB119 displayed at early time points a significantly lower renal uptake but also a lower tumor uptake than (111)In-DOTA-NT-20.3, although binding to HT29 cells was similar. (68)Ga-DOTA-NT-20.3 displayed higher tumor uptake than (68)Ga-DOTA-LB119 and allowed the detection of very small tumors by PET. In conclusion, DOTA-NT-20.3 is a promising candidate for (68)Ga-PET imaging of neurotensin receptor-positive tumors. DOTA-NT-20.3 may also be considered for therapy, as the yttrium-labeled peptide has higher affinity than that of the indium-labeled one. A prerequisite for therapeutic application of this neurotensin analogue would be to lower kidney uptake, for example, by infusion of basic amino acids, gelofusin, or albumin fragments, to prevent nephrotoxicity, as with radiolabeled somatostatin analogues.     

Enhanced targeting specificity to tumor cells by simultaneous recognition of two antigens

Radioimmunotherapy recently afforded convincing results for B-cell non-Hodgkin's lymphoma treatment with antibody specific for B-cell differentiation antigens. High doses of unlabeled or labeled antibodies are necessary to saturate specific sites on normal B-cells. We thus developed a new targeting strategy, taking advantage of dual binding cooperativity, to enhance the specificity of the radioactive uptake by tumor cells. This approach was evaluated using human Burkitt lymphoma cells (Ramos) which express both CD10 and CD20 antigens. Most normal cells express at most one of these two differentiation antigens but many hematological tumors, including most human B type acute lymphoblastic leukemia cells, express both. Cells pretargeted with two bispecific antibodies, one recognizing CD10 and a histamine derivative (HSG), the other recognizing CD20 and the DTPA-indium complex, bind cooperatively radiolabeled mixed-haptens (DTPA-HSG). Increased binding (about 5-fold compared to binding to only one of CD10 or CD20 antigens) is observed at 37 degrees C, demonstrating the feasibility of the technique. This binding enhancement is a slow process, not observed at 4 degrees C. Such a binding enhancement will increase specificity for targeting isotopes to double antigen positive tumor cells compared to nontumor tissue cells bearing only one of them. This approach might be used to increase tumor irradiation with minimal irradiation of normal cells.     

Equilibrium expert: an add-in to Microsoft Excel for multiple binding equilibrium simulations and parameter estimations

An add-in to Microsoft Excel was developed to simulate multiple binding equilibriums. A partition function, readily written even when the equilibrium is complex, describes the experimental system. It involves the concentrations of the different free molecular species and of the different complexes present in the experiment. As a result, the software is not restricted to a series of predefined experimental setups but can handle a large variety of problems involving up to nine independent molecular species. Binding parameters are estimated by nonlinear least-square fitting of experimental measurements as supplied by the user. The fitting process allows user-defined weighting of the experimental data. The flexibility of the software and the way it may be used to describe common experimental situations and to deal with usual problems such as tracer reactivity or nonspecific binding is demonstrated by a few examples. The software is available free of charge upon request.