Selective High-Affinity Ligand Antibody Mimics for Cancer Diagnosis and Therapy: Initial Application to Lymphoma/Leukemia

Abstract

Purpose: More than two decades of research and clinical trials have shown radioimmunotherapy to be a promising approach for treating various forms of cancer. Lym-1antibody, which binds selectively to HLA-DR10 on malignant B-cell lymphocytes, has proved to be effective in delivering radionuclides to non^Hodgkin's lymphoma and leukemia. Using a new approach to create small synthetic molecules that mimic the targeting properties of the Lym-1antibody, a prototype, selective high-affinity ligand (SHAL), has been developed to bind to a unique region located within the Lym-1epitope on HLA-DR10. Experimental Design: Computer docking methods were used to predict two sets of small molecules that bind to neighboring cavities on the h subunit of HLA-DR10 surrounding a critical amino acid in the epitope, and the ligands were confirmed to bind to the protein by nuclear magnetic resonance spectroscopy. Pairs of these molecules were then chemically linked together to produce a series of bidentate and bisbidentate SHALs. Results: These SHALs bind with nanomolar to picomolar K d 's only to cell lines expressing HLA-DR10. Analyses of biopsy sections obtained from patients also confirmed that SHAL bound to both small and large cell non^Hodgkin's lymphomas mimicking the selectivity of Lym-1. Conclusions: These results show that synthetic molecules less than 1/50th the mass of an antibody can be designed to exhibit strong binding to subtle structural features on cell surface proteins similar to those recognized by antibodies.This approach offers great potential for developing small molecule therapeutics that target other types of cancer and disease.

Nanomolecular HLA-DR10 Antibody Mimics: A Potent System for Molecular Targeted Therapy and Imaging

“…Using surface plasmon resonance (BIAcore 3000), SHAL binding to isolated or recombinant HLA-DR10 protein was observed to be in the nanomolar range, as previously described. 13,19 SHALs no longer bound after the addition of Lym-1.…”

“…Although all of the SHALs have discriminated HLA-DR10 expressing from nonexpressing malignant cells, mimicking Lym-1, [11][12][13] and exhibited small-molecule pharmacokinetic behavior, 11,14 earlier SHALs tested showed no antilymphoma activity. 12 To increase binding and selectivity and, therefore, SHAL residence time in NHL tissue, SHALs having a Ct ligand (3-(2-([3-chloro-5-trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanionic acid) for a third docking site on HLA-DR10 were synthesized.…”

“…7,8,16 HLA-DR10 protein that is expressed by antigen-presenting cells was isolated from Raji Burkitt's human lymphoma Bcells and purified on a Lym-1 affinity column, as described previously. 13 Two HLA-DR10-expressing human B-cell lymphoma lines, Raji (American Type Culture Collection, Manassas, VA) and SU-DHL4 (A. Epstein), and two nonexpressing human T-cell lymphoma/leukemia lines, Jurkat's, and CEM (American Type Culture Collection), grown as recommended, were used for the experiments.…”

“…13 Cavities within the Lym-1 epitope of the protein were identified by using SPHGEN. 17,18 After identifying ligands predicted to bind to the cavities by using computational docking, a combination of nuclear magnetic resonance (NMR) spectroscopy, surface plasmon resonance (BIAcore 3000; Biacore, Piscataway, NJ), and competitive binding experiments were used to confirm that the ligands bound to different sites on HLA-DR10 protein.…”

“…13 Cells were incubated with biotinylated SHALs for 1 hour at RT or 4°C, washed, and transferred to a fresh plate. SA-HRP was added for 30 minutes, the cells pelleted, and 2,2Ј-azino-bis(3-ethylbenzthiazoline)-6-sulphonic acid substrate (ABTS) added for 20 minutes.…”