Bispecific small molecule-antibody conjugate targeting prostate cancer

Abstract

Bispecific antibodies, which simultaneously target CD3 on T cells and tumor-associated antigens to recruit cytotoxic T cells to cancer cells, are a promising new approach to the treatment of hormone-refractory prostate cancer. Here we report a site-specific, semisynthetic method for the production of bispecific antibody-like therapeutics in which a derivative of the prostate-specific membrane antigen-binding small molecule DUPA was selectively conjugated to a mutant αCD3 Fab containing the unnatural amino acid, p-acetylphenylalanine, at a defined site. Homogeneous conjugates were generated in excellent yields and had good solubility. The efficacy of the conjugate was optimized by modifying the linker structure, relative binding orientation, and stoichiometry of the ligand. The optimized conjugate showed potent and selective in vitro activity (EC50 ~ 100 pM), good serum half-life, and potent in vivo activity in prophylactic and treatment xenograft mouse models. This semisynthetic approach is likely to be applicable to the generation of additional bispecific agents using drug-like ligands selective for other cell-surface receptors.

Keywords: antibody engineering; immunotherapy.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

(A) Structure of DUPA modified with short ethylene glycol (Tet, 1), long ethylene glycol (Und, 2), Phthal (3), and DNP (4) linkers with a terminal aminooxy group. (B) Structure of p-acetylphenylalnine (pAcF) and oxime ligation of pAcF containing αCD3 Fab with P–linkers. (C) FACS of the P-αCD3 conjugates (100 nM) on Jurkat (CD3+) and C4-2 (PSMA+) cell lines. Different P–linkers are conjugated to the same residue (HC-138) of the αCD3 Fab.

Fig. 2.

(A) Sites of mutation in αCD3 Fab for the single and double conjugates. Each mutant conjugate results in a distinct orientation for PSMA binding relative to CD3 binding. The double-αCD3 conjugate (HC-K138/LC-S202) increases avidity to homodimeric PSMA. (B) FACS of the P-αCD3 (100 nM) conjugates on Jurkat (CD3+) and C4-2 (PSMA+) cell lines. P-Phthal is conjugated to different positions of the αCD3 Fab. (C) FACS analysis of the binding of different mono- and bivalent P-αCD3 conjugates to C4-2 cells at various concentrations.

Fig. 3.

In vitro cytotoxicity of αPSMA/αCD3 conjugates in the presence of freshly purified unactivated hPBMCs. (A) Conjugates show dose-dependent activity on PSMA+ C4-2 cells with different EC50 values, whereas the unconjugated mixture shows no activity. (B) Conjugates have no effect on PSMA− DU-145 cells. Cytotoxicity was detected by LDH-release assay. (C) Proinflammatory cytokine TNF-α levels in the supernatant of the cytotoxicity assay. Dose-dependent TNF-α signals were detected by ELISA only in the presence of PSMA+ C4-2 cells. RFU, relative fluorescence units. (D) Significantly reduced cytotoxicity of C4-2 cells was observed in the competition assay in the presence of either the competitive antagonist 2-phosphonomethyl pentanedioic acid (2-PMPA) or αCD3 Fab. No cytotoxicity was observed in the absence of hPBMCs. RAU, relative absorbance units.

Fig. 4.

In vivo efficacy studies of P-SMAC. (A) In the prophylactic model, 1 × 106 C4-2 cells were mixed with 2 × 106 PBMCs (1:2 ratio) in 50% Matrigel and were injected s.c. into the right shoulder of male NOD-SCID mice. P-SMAC, unconjugated Fab, or PBS (vehicle) (n = 6) was administered i.v. at 2 mg/kg every day for 10 d starting on day 4. Tumors were monitored by external caliper measurements at regular intervals for 6 wk. P-SMAC suppressed tumor growth, but the control groups rapidly developed tumors. *Mice with large tumors (>1,000 mm3) were killed before the day indicated. (B) In the treatment model, 1 × 106 C4-2 cells with 50% Matrigel were injected s.c. into male NOD/SCID-γ mice. On day 3, 20 × 106 hPBMCs were injected i.p. PBMCs from the same donor were activated ex vivo by incubation with immobilized αCD3 and αCD28 antibodies for 3 d and subsequently were expanded with rh-IL2. Once the solid tumors were palpable (day 13), 20 × 106 activated hPBMCs were injected i.p. Beginning on day 15, 1 mg/kg P-SMAC or PBS was given daily for 10 d i.v. (n = 9). (**P < 0.0001.)