ASN004, A 5T4-targeting scFv-Fc Antibody–Drug Conjugate with High Drug-to-Antibody Ratio, Induces Complete and Durable Tumor Regressions in Preclinical Models

Abstract

The 5T4 oncofetal antigen (trophoblast glycoprotein) is expressed in a wide range of malignant tumors but shows very limited expression in normal adult tissues. ASN004 is a 5T4-targeted antibody–drug conjugate (ADC) that incorporates a novel single-chain Fv-Fc antibody and Dolaflexin drug-linker technology, with an Auristatin F hydroxypropylamide payload drug-to-antibody ratio of approximately 10–12. The pharmacology, toxicology, and pharmacokinetic properties of ASN004 and its components were investigated in vitro and in vivo. ASN004 showed high affinity for the 5T4 antigen and was selectively bound to and internalized into 5T4-expressing tumor cells, and potent cytotoxicity was demonstrated for a diverse panel of solid tumor cell lines. ASN004 induced complete and durable tumor regression in multiple tumor xenograft models, derived from human lung, breast, cervical, and gastric tumor cell lines having a wide range of 5T4 expression levels. A single dose of ASN004, as low as 1 mg/kg i.v., achieved complete tumor regression leading to tumor-free survivors in the A431 cervical cancer model. In head-to-head studies, superior activity of ASN004 was demonstrated against trastuzumab-DM1, in a low-5T4/high-HER2 expressing gastric tumor model, and 10-fold greater potency was found for ASN004 against the 5T4-targeted ADC PF-06263507 in a lung tumor model. In marmoset monkeys, ASN004 was well tolerated at doses up to 1.5 mg/kg Q3W i.v., and showed dose-dependent exposure, linear pharmacokinetics, and markedly low exposure of free payload drug. Taken together, these findings identify ASN004 as a promising new ADC therapeutic for clinical evaluation in a broad range of solid tumor types.

Immunohistochemical Detection of 5T4 in Renal Cell Carcinoma

“…Although 24/29 of our normal kidney samples were from tumor-bearing kidneys, 5 cores were from normal healthy donors and no difference in IHC staining was noted between the 2 different sources. In contrast, a recent survey of 40 different histologically normal human frozen tissues using a biotinylated anti-5T4 scFv-Fc antibody did not reveal positive staining in the kidney, rather only in astrocytes in the brain (cerebellum and cortex), spinal cord, the pituitary gland, placental trophoblasts, and esophageal smooth muscle cells, and no renal histopathologic findings were reported after administration of a 5T4 antibody-drug conjugate to marmoset monkeys 19. In addition, a modified vaccinia Ankara 5T4 vaccine was well-tolerated in a phase III study in 733 patients with metastatic ccRCC and there was no difference reported in renal/urinary adverse advents between the vaccine and placebo groups 40.…”

“…In contrast, a recent survey of 40 different histologically normal human frozen tissues using a biotinylated anti-5T4 scFv-Fc antibody did not reveal positive staining in the kidney, rather only in astrocytes in the brain (cerebellum and cortex), spinal cord, the pituitary gland, placental trophoblasts, and esophageal smooth muscle cells, and no renal histopathologic findings were reported after administration of a 5T4 antibody-drug conjugate to marmoset monkeys. 19 In addition, a modified vaccinia Ankara 5T4 vaccine was well-tolerated in a phase III study in 733 patients with metastatic ccRCC and there was no difference reported in renal/urinary adverse advents between the vaccine and placebo groups. 40 Likewise, the 5T4-targeting antibody superantigen naptumomab estafenatox intended to direct T-cell cytotoxicity to 5T4-expressing tumor cells was well-tolerated and no treatment-associated changes in renal function were observed in a phase II/III study of 513 patients with advanced ccRCC or papillary RCC.…”

“…Therefore, we and others have focused on the development of engineered T cells against alternate targets in RCC 18. Cancer/testis antigen 5T4/trophoblast glycoprotein (encoded by TPBG ) is a 72KDa N -glycosylated transmembrane protein highly expressed by placental trophoblasts and most human carcinomas but is not highly expressed in healthy adult somatic tissues 19–21. 5T4 was discovered in a screen for proteins promoting an invasive phenotype likely to be shared by placental trophoblasts and cancer cells 22.…”

“…18 Cancer/testis antigen 5T4/trophoblast glycoprotein (encoded by TPBG) is a 72KDa N-glycosylated transmembrane protein highly expressed by placental trophoblasts and most human carcinomas but is not highly expressed in healthy adult somatic tissues. [19][20][21] 5T4 was discovered in a screen for proteins promoting an invasive phenotype likely to be shared by placental trophoblasts and cancer cells. 22 Staining of RCC tumors with the original antihuman 5T4 murine monoclonal antibody showed that 95% of primary RCC tumors expressed at least focal 5T4 and > 75% were strongly positive.…”

A Review of Protein- and Peptide-Based Chemical Conjugates: Past, Present, and Future

“…The most advanced program is upfitamab rilsodotin, an anti-NaPi2b antibody loaded with 10–15 auristatin F-hydroxypropylamide payload molecules that is being evaluated for treatment of ovarian cancer [ 315 ]. Earlier stage efforts include ASN004, which uses the dolaflexin platform to load payload onto an ScFv-Fc antibody, and dextramabs, which use dextran as a hydrophilic polymer scaffold to load multiple drugs without compromising solubility [ 316 , 317 ]. Finally, while the dolaflexin and dextramab technologies produce heterogeneous ADCs, the use of cysteine-functionalized XTEN polypeptides as a scaffold facilitates the production of homogenous ADCs with DARs as high as 18 [ 214 ].…”