Antibody conjugates of 7-ethyl-10-hydroxycamptothecin (SN-38) for targeted cancer chemotherapy

Abstract

CPT-11 is a clinically used cancer drug, and it is a prodrug of the potent topoisomerase I inhibitor, SN-38 (7-ethyl-10-hydroxycamptothecin). To bypass the need for the in vivo conversion of CPT-11 and increase the therapeutic index, bifunctional derivatives of SN-38 were prepared for use in antibody-based targeted therapy of cancer. The general synthetic scheme incorporated an acetylene-azide click cycloaddition step in the design, a short polyethylene glycol spacer for aqueous solubility, and a maleimide group for conjugation. Conjugates of a humanized anti-CEACAM5 monoclonal antibody, hMN-14, prepared using these SN-38 derivatives were evaluated in vitro for stability in buffer and human serum and for antigen-binding and cytotoxicity in a human colon adenocarcinoma cell line. Conjugates of hMN-14 and SN-38 derivatives 16 and 17 were found promising for further development.

Figures

Figure 1

Size-exclusion HPLC of unmodified hMN-14 (top) and of hMN-14-CL2-SN-38 conjugate with a drug molar substitution of 6.1 (bottom). Unmodified hMN-14 was detected by absorbance at 280 nm. The HPLC profile of the conjugate was detected at the absorbance wavelength of SN-38, namely 360 nm; thus, the peak eluting near the antibody position corresponds to antibody substituted with SN-38. Abbreviation: tR, retention time.

Figure 2

Kinetics of SN-38 release from various hMN-14-SN-38 conjugates incubated in PBS, pH 7.4 (top) or human serum (bottom) at 37 °C. Y-axis displays the ratios of the reverse-phase HPLC peak areas due to SN-38 and the fixed amount of internal standard (10-hydroxycamptothecin) extracted from the incubates. Conjugates: hMN-14-[CL1-SN-38 (7)], square; hMN-14-[CL2-SN-38 (16)], triangle; hMN-14-[CL2-SN-38-10-CO2Et) (17)], inverted triangle; hMN-14-[CL3-SN-38 (21)], diamond; hMN-14-[CL4-SN-38 (25)], X; and hMN-14-[CL5-SN-38 (28)], circle. With hMN-14-21 conjugate, only buffer-stability was determined. The plots were generated using the Prism™ software and the equation for one-phase exponential association [Y = Ymax*(1-exp(-K*X))]. The half-lives, determined as 0.69/K where K is rate constant, are given in Table 2.

Scheme 1

SN-38, variously derivatized at 10 and 20 positions

Scheme 2

Synthesis of the bifunctional SN-38, CL-SN-38 (3)

Scheme 3

Synthesis of the bifunctional SN-38, CL1-SN-38 (7)

Scheme 4

Synthesis of the bifunctional SN-38, CL2-SN-38 (16) & CL2-SN-38(Et) (17)

Scheme 5

Synthesis of the bifunctional SN-38, CL3-SN-38 (21)

Scheme 6

Synthesis of the bifunctional SN-38, CL4-SN-38 (25)

Scheme 7

Synthesis of the bifunctional SN-38, CL5-SN-38 (28)

Chart 1

Structures of some reagents and structural residues

Chart 2

Structures of bifunctional SN-38 derivatives