In vitro and in vivo evaluation of cysteine and site specific conjugated herceptin antibody-drug conjugates

Abstract

Antibody drug conjugates (ADCs) are monoclonal antibodies designed to deliver a cytotoxic drug selectively to antigen expressing cells. Several components of an ADC including the selection of the antibody, the linker, the cytotoxic drug payload and the site of attachment used to attach the drug to the antibody are critical to the activity and development of the ADC. The cytotoxic drugs or payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. This results in ADCs that have a heterogeneous number of drugs per antibody. The number of drugs per antibody commonly referred to as the drug to antibody ratio (DAR), can vary between 0 and 8 drugs for a IgG1 antibody. Antibodies with 0 drugs are ineffective and compete with the ADC for binding to the antigen expressing cells. Antibodies with 8 drugs per antibody have reduced in vivo stability, which may contribute to non target related toxicities. In these studies we incorporated a non-natural amino acid, para acetyl phenylalanine, at two unique sites within an antibody against Her2/neu. We covalently attached a cytotoxic drug to these sites to form an ADC which contains two drugs per antibody. We report the results from the first direct preclinical comparison of a site specific non-natural amino acid anti-Her2 ADC and a cysteine conjugated anti-Her2 ADC. We report that the site specific non-natural amino acid anti-Her2 ADCs have superior in vitro serum stability and preclinical toxicology profile in rats as compared to the cysteine conjugated anti-Her2 ADCs. We also demonstrate that the site specific non-natural amino acid anti-Her2 ADCs maintain their in vitro potency and in vivo efficacy against Her2 expressing human tumor cell lines. Our data suggests that site specific non-natural amino acid ADCs may have a superior therapeutic window than cysteine conjugated ADCs.

Figure 1. ADC payload chemical structures.

The chemical structures of the ADC payloads are shown. Payload A was used to conjugate to pAF via an oxime bond. Payload B was used to conjugate to the thiols on cysteines via maleimide.

Figure 2. Antibody HPLC analysis.

The reversed phase HPLC of deglycosylated reduced and denatured (A) Anti-Her2 Cys-ADC, (B) Anti-Her2 Position 1-ADC, Anti-Her2 Position 2-ADC and (C) Anti-Her2. Insert shows the gradient profile and chromatography conditions. There were two drugs/antibody determined by the intact MS analysis or one drug per heavy chain. The reversed phase HPLC profile of the cysteine conjugated anti-Her2 ADC shows the number of drugs conjugated to the heavy (H) and light (L) chains determined by the intact mass spectrometry analysis.

Figure 3. In vitro antibody stability.

The anti-Her2 ADC antibodies were incubated in human serum or HSA at 37°C up to 28 days. (A) The amount of total antibody (naked and ADC) in the preparation was measured. (B) The amount of ADC in the preparation was measured. (C) % Concentration of ADC over the amount of total antibody. (D) The amount of drug conjugated HSA is measured.

Figure 4. In vitro evaluation of Her2 expression and ADC cytotoxicity.

The expression of Her2 and the in vitro cytotoxicity of the ADCs are evaluated in the HCC-1954, NCI-N87, MDA-MB-453 and MDA-MB-468 cell lines. (A) The cell surface expression of Her2 is measured via FACS for a panel of tumor cell lines. HCC-1954, MDA-MB-453 and NCI-N87 were selected as the Her2 expressing cell lines while MDA-MB-468 was selected as the Her2 negative cell line. MDA-MB-453 cells are resistant to Herceptin. (B) NCI-N87 and HCC-1954 tumor xenografts were evaluated for Her2 expression via IHC. (C) The in vitro cytotoxicity of the anti-Her2 ADCs were evaluated in the HCC-1954, NCI-N87, MDA-MB-453 and MDA-MB-468 cell lines.

Figure 5. In vivo tumor efficacy studies.

The in vivo efficacy of the anti-Her2 ADCs was evaluated in the NCI-N87 and HCC-1954 tumor xenografts. (A) SCID mice bearing NCI-N87 tumors were treated with a single, i.v. dose of anti-Her2 ADCs. (B) SCID mice bearing HCC-1954 tumors were treated with a single, i.v. dose of anti-Her2 ADCs.

Figure 6. Rat toxicokinetics.

Serum from rats dosed with 30, 45 or 60/kg of anti-Her2 Cys-ADC or 30, 60 or 90 mg/kg of anti-Her2 position 1-ADC were collected and the total amount of antibody (TAb) or ADC was measured via ELISA. (A) The pharmacokinetic profile of the anti-Her2 Cys-ADC was evaluated. (B) The pharmacokinetic profile of the anti-Her2 position 1-ADC was evaluated.

Figure 7. Rat body weights.

Rat body weights were measured post single dose, i.v. administration of 30 or 60/kg of the anti-Her2 Cys or anti-Her2 position 1-ADC. Data represent Mean ± SE.