Biological activity of anti-CD20 multivalent HPMA copolymer-Fab' conjugates

Abstract

High-molecular-weight, branched N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers were synthesized and conjugated with Fab' fragments of the anti-CD20 antibody, 1F5. This produced multivalent conjugates with varying valency (amount of Fab' per macromolecule) targeted to the B-cell antigen CD20. The apoptotic activity of the conjugates was screened against several B-cell lymphomas with varied expression levels of CD20 (Raji, Daudi, Ramos, Namalwa, and DG-75). The multivalent conjugates had the strongest activity against cells that had the highest expression of CD20 and failed to demonstrate any measurable activity against lymphomas that did not express the antigen. Furthermore, there was an apparent dose-dependent response to treatment with multivalent conjugates. At optimal valence and concentration, the apoptotic activity of HPMA copolymer-Fab' conjugates superseded that of free anti-CD20 Ab that was hyper-cross-linked with a polyclonal, secondary Ab.

Figure 1

Scheme for the synthesis of multivalent HPMA copolymer-Fab’ conjugates targeted to the B-cell antigen CD20.

Figure 2

Fractionation of P-Fab’ conjugates by size exclusion chromatography. Elution profile of reaction mixture on a Superose S6 preparative column (HR 10/30, FPLC system); Buffer PBS pH 7.4; flow rate 1 mL/min; amount of sample applied 10 mg/2 mL; detection absorbance at 280 nm. The numerical suffix at the fraction designation is the average valence.

Figure 3

Dose dependent studies of hyper crosslinked Ab and multivalent HPMA copolymer-Fab’ conjugates targeting CD20. Apoptotic activity of the conjugates was determined by treating Raji cells for 18 h and then staining cells with annexing V and propidium iodide. Analysis was carried out using flow cytometry.

Figure 4

Apoptotic activity of anti-CD20 multivalent HPMA copolymer-Fab’ conjugates. Five different cell lines were treated with 200 nM of Fab’ equivalent of conjugates. After 18 h of treatment cells were stained with annexin V and propidium iodide and then analyzed by flow cytometry to determine the amount of cells undergoing apoptosis. Raji, Ramos, and Daudi cell lines expressed CD20 well (CD20+). Namalwa cells had slight expression of CD20, and DG-75 did not express CD-20.

Figure 5

DNA fragmentation in cells treated with mutivalent HPMA copolymer-Fab’ conjugates. TUNEL assay of Raji cells treated with 200 nM Fab’ fractions for 18 h. Cells were stained with FITC labeled apo-BrdU antibodies. The number of positively stained cell indicates the presence of DNA fragmentation typical of apoptosis.

Figure 6

Fluorescence images of cells treated with 200 nM Fab’ equivalent of HPMA copolymer-Fab’ conjugates labeled with TAMRA dye. In the first row (A) DNA fragmentation is indicated by TUNEL staining. In row (B), CD20 was visualized by TAMRA label if the HPMA-Fab’ conjugate. Clustering of CD20 is indicated by a change in the distribution of staining from peripheral stains on the surface of the cell to focal centers. Clustering increased with the valence of the conjugates. The next row (C) shows an overlay of both DNA fragmentation and CD20. The last row (D) is of transmission light images of the same cells with observable apoptotic bodies.

Figure 7

Caspase activation in cells treated with multivalent HPMA copolymer-Fab’ conjugates. Raji cells treated with 200 nM of Fab’ equivalent and assayed (PhiPhiLux) after 5 and 24 h following treatment.