A CXCR4-targeted site-specific antibody-drug conjugate

Abstract

A chemically defined anti-CXCR4-auristatin antibody-drug conjugate (ADC) was synthesized that selectively eliminates tumor cells overexpressing the CXCR4 receptor. The unnatural amino acid p-acetylphenylalanine (pAcF) was site-specifically incorporated into an anti-CXCR4 immunoglobulin G (IgG) and conjugated to an auristatin through a stable, non-cleavable oxime linkage to afford a chemically homogeneous ADC. The full-length anti-CXCR4 ADC was selectively cytotoxic to CXCR4(+) cancer cells in vitro (half maximal effective concentration (EC50 )≈80-100 pM). Moreover, the anti-CXCR4 ADC eliminated pulmonary lesions from human osteosarcoma cells in a lung-seeding tumor model in mice. No significant overt toxicity was observed but there was a modest decrease in the bone-marrow-derived CXCR4(+) cell population. Because CXCR4 is highly expressed in a majority of metastatic cancers, a CXCR4-auristatin ADC may be useful for the treatment of a variety of metastatic malignancies.

Keywords: antibody engineering; antibody-drug conjugates; cancer; immunotherapy; unnatural amino acids.

Figure 1. Site-specific conjugation of aminooxy-auristatin to anti-CXCR4 A122pAcF IgG (anti-CXCR4 IgGX)

(A) Monomethyl auristatin F (MMAF) derivatized with a terminal alkoxy-amine is coupled by oxime ligation to anti-CXCR4 A122pAcF IgG through a pAcF residue. (B) SDS/PAGE gel of anti-CXCR4-IgGX before and after coupling to auristatin. The mutant IgG includes natural heterogeneous N-linked glycosylation; ~150 kDa corresponds to the full-length IgG; ~25 kDa and ~50 kDa correspond to the reduced light chain and heavy chain, respectively. The 4–12% Tris-Glycine gel has a prestained protein ladder in the first lane and was stained with Coomassie Blue. (C) ESI-MS analysis of anti-CXCR4 IgGX before and after conjugation to auristatin. Mutant IgG spectra show the heavy and light chains after removal of glycans with PNGase F (Promega, PBS pH 7.4, 37 °C, 12 hr) and reduction with 10 mM DTT. All masses are as expected with a ~905-Da difference between the conjugated and unconjugated antibodies, corresponding to conjugation of one auristatin per heavy chain. No unreacted antibody was observed by SDS/PAGE or ESI-MS, suggesting >95% coupling efficiency.

Figure 2. In vitro activity of anti-CXCR4 IgGX-auristatin

(A) Binding of anti-CXCR4 IgGX-AF488 to SJSA-1-met-luc cells. Cells were incubated with increasing concentrations of the conjugate at 4 °C for 30 mins and binding was analyzed by flow cytometry. (B) SJSA-1-met-luc cells were plated at 80% confluence and treated with 50 nM anti-CXCR4 IgGX-AF488 in the presence or absence of 500 nM unconjugated anti-CXCR4 IgGX. Cells were incubated at either 4 °C or 37 °C for 30 mins, fixed and imaged using a Zeiss confocal microscope. Dose dependent in vitro cytotoxicity of anti-CXCR4 IgGX-auristatin with (C) SJSA-1-met-luc (CXCR4+) and (D) R7T1 (CXCR4−) cells. Cells were treated with increasing concentrations of anti-CXCR4 IgGX, anti-CXCR4 IgGX-auristatin or unconjugated auristatin for 72 hrs at 37 °C, 5% CO₂ and viability measured using CellTiter Glo (Promega). Percent viability is normalized to untreated controls. (n=3, mean and S.D.)

Figure 3. In vivo efficacy of anti-CXCR4 IgGX-auristatin

Lung tumors were allowed to develop in NOD/SCID (C.B17) mice for 10 days after i.v. injection of 10⁴ SJSA-1-met-luc cells. Lesion development and response to antibody treatment was monitored using longitudinal noninvasive bioluminescence imaging (IVIS 200). (A) Mice injected i.v. with either anti-CXCR4 IgGX-auristatin (3 doses of 2.5mg/kg once every 5 days) or PBS (n=6 mice/group; mean and SEM, *p < 0.05). (B) Mice injected with either anti-CXCR4 IgGX (3 doses of 2.5mg/kg once every 5 days) or PBS (n=6 mice/group, mean and SEM, p>0.05). (C) Representative images from IVIS imaging showing bioluminescence corresponding to tumor size on various days during the study.