Chemically Defined Antibody- and Small Molecule-Drug Conjugates for in Vivo Tumor Targeting Applications: A Comparative Analysis

Abstract

We present the first direct comparative evaluation of an antibody-drug conjugate and of a small molecule-drug conjugate for cancer therapy, using chemically defined products which bind with high-affinity to carbonic anhydrase IX, a marker of tumor hypoxia and of renal cell carcinoma.

Figure 1

Chemical structures and biochemical characterization of anti-CAIX ADC and SMDC products. Ligand-linker-payload structures, as well as the site of conjugation are indicated. Size exclusion chromatography profile and SDS-PAGE relative to the CAIX-specific ADC(+) product. Lanes NR and R represent the final ADC in non-reducing and reducing conditions, respectively. SPR analysis of ADC(+) and the negative control ADC(-) for their binding to recombinant human CAIX. Sensograms are referred to different concentration of the conjugates. Liquid chromatography and mass spectrometry analysis of SMDC(+). SPR sensorgrams of a serial dilution of SMDC(+) and SMDC(-) against recombinant human CAIX. Fitting of sensorgrams related to ADC(+) and SMDC(+) allowed calculation of the corresponding apparent binding constants: k_on,SMDC(+) = 3.4 10⁵ M^-1s^-1, k_off,SMDC(+) = 3.4 10^-3 s^-1, K_D,SMDC(+) = 10 nM; k_on,ADC(+) = 1.7 10⁵ M^-1s^-1, k_off,ADC(+) = 2.2 10^-5 s^-1, K_D,ADC(+) = 0.13 nM. The BIAcore methodology may under-estimate K_D values for antibodies in homobivalent IgG format against bivalent antigens (due to a chelate binding mode) and to over-estimate K_D values for small organic ligands with very high k_on, due to limitation of diffusion speed within the microsensor chip hydrogel.

Figure 2

Evaluation of the tumor-targeting performance of the anti-CAIX XE114 antibody (mAb+) and the small ligand AAZ⁺ (SM+) against human renal cell carcinoma cells SKRC-52 xenografted in mice. (A) Quantification of ligand uptake in tumor and blood after administration of radiolabeled preparations of IgG(XE114) (mAb+) and of a radiolabeled derivative of AAZ⁺ (SM+). Microscopic distribution of IgG(XE114) (mAb+) and of a fluorescently labeled derivative of AAZ⁺ (SM+) in SKRC-52 tumors (B) and in healthy organs (C) after IV administration. Images related to mAb+ and SM+ products were taken 24 hours and 1 hour post injection, respectively. mAb- and SM- relate to the corresponding negative controls. Green = Ligand (mAb+ or SM+); Blue = DAPI staining. Scale bar = 100 μm.

Figure 3

Comparative therapeutic analysis of anti-CAIX antibody-drug conjugate ADC(+) and small molecule-drug conjugate SMDC(+) in BALB/c nu/nu mice bearing SKRC-52 xenografts. In the experiment, ADC(-) and SMDC(-) derivatives devoid of the targeting moieties were used as negative controls. (A) Changes in tumor volume for different treatment groups. (B) Body weight changes experienced by the animals during the therapy experiment. The arrows indicate intravenous (i.v.) administration of the corresponding agent. DLR = drug-ligand ratio; DAR = drug-antibody ratio.