Site-selective template-directed synthesis of antibody Fc conjugates with concomitant ligand release

Abstract

Template-directed methods are emerging as some of the most effective means to conjugate payloads at selective sites of monoclonal antibodies (mAbs). We have previously reported a method based on an engineered Fc-III reactive peptide to conjugate a radionuclide chelator to K317 of antibodies with the concomitant release of the Fc-III peptide ligand. Here, our method was redesigned to target two lysines proximal to the Fc-III binding site, K248 and K439. Using energy minimization predictions and a semi-combinatorial synthesis approach, we sampled multiple Fc-III amino acid substituents of A3, H5, L6 and E8, which were then converted into Fc-III reactive conjugates. Middle-down MS/MS subunit analysis of the resulting trastuzumab conjugates revealed that K248 and K439 can be selectively targeted using the Fc-III reactive variants L6Dap, L6Orn, L6Y and A3K or A3hK, respectively. Across all variants tested, L6Orn-carbonate appeared to be the best candidate, yielding a degree and yield of conjugation of almost 2 and 100% for a broad array of payloads including radionuclide chelators, fluorescent dyes, click-chemistry reagents, pre-targeted imaging reagents, and some cytotoxic small molecules. Furthermore, L6Orn carbonate appeared to yield similar conjugation results across multiple IgG subtypes. In vivo proof of concept was achieved by conjugation of NODAGA to the PD1/PD-L1 immune checkpoint inhibitor antibody atezolizumab, followed by PET imaging of PD-L1 expression in mice bearing PD-L1 expressing tumor xenograft using radiolabeled [⁶⁴Cu]Cu-atezolizumab.

Fig. 1. (A) Overview of the IgG Fc-III binding site. The IgG surface is highlighted in grey, and Fc-III (tubular structure) is shown in green, except for the residues in proximity with Ab Lys 248, His5, Leu6 and Glu8, which are color-coded in magenta. (B) Fc-reactive conjugate; His5, Leu6 or Glu8 (shown in black) are substituted with a residue bearing an amino moiety. The chemically cleavable reactive site is stable in aqueous media, yet sufficiently reactive to react with an antibody lysine. The reactivity modulator fine tunes the reactivity of the reactive site and ensures that the ligand is released concomitantly with the conjugation of the payload to the antibody and detached from the antibody after acidic treatment.

Scheme 1. Fc-III lysine and variant scan (A) synthesis of Fc-III-L6K-DOTA reactive conjugate. (1) HATU, DIEA, DMF, Fc-III-L6K peptide, compound 1¹; (2) TFA/TIS/H₂O. (B) Synthesis of antibody-DOTA conjugates; the Fc-III side chains that are varied are color coded in green, the reactivity modulator and the carbonate reactive center in violet and red, respectively; (3) 34 μM of antibody, 2 equivalents of peptide conjugate, pH 9, 2 h incubation at room temperature. (C) Chemical structures of variants used in this study. As the amino acids bearing the hydrocarbon side chains beyond n = 5 are not commercially available, we attached 1, 2 or 3 PEG units via an amide bond to the A3 Dap or L6K variants. The main and side chain of each variant are shown in black and green, respectively.

Scheme 2. Schematic representation of other conjugation chemistries. The color codes are green for the Fc-III substituted residue, violet for the reactivity modulator (green for the tyrosine approach given that both the substituted side chain becomes the reactivity modulator), red for the chemical reactive center and blue for the linker between the chemical reactive site and the payload (A) Thioester approach; (B) Tyrosine approach. Only the L6 variants are shown here.

Fig. 2. Intact mass analysis of the trastuzumab-DOTA conjugates by LC-FTMS. The Fc-III reactive conjugate variant used to conjugate DOTA to trastuzumab is listed above each spectrum. The deconvolved mass spectra of the intact deglycosylated sample is shown after EndoS treatment.

Fig. 3. Representation of the antibody conjugation sites identified by middle-down MS/MS sub-unit analysis. The antibody lysines to which conjugation is likely to occur are color coded in red, green, blue and magenta and the respective Fc-III X-DOTA peptide variants used for the conjugation are shown in brackets (abbreviated as the relevant Fc-III amino acid variant). K409 is hidden inside the antibody and was slightly moved at the surface to be visible on this figure.

Scheme 3. Fc-III-L6Orn carbonate derivative used to prepare the Fc-reactive conjugates with various payloads (mainly through reacting to –NHS or –SCN payloads).

Fig. 4. Radio-immunoreactivity assay with [⁶⁴Cu]Cu-atezolizumab against the MDA-MB-231 (PD-L1hi-PD-L1 positive) and MDA-MB-157 (PD-L1-negative) cell lines. The specific cell binding is expressed as a function of the number of cells. The binding curves were fitted by nonlinear regression and the immunoreactive fraction (B_max) corresponding to the extrapolation to infinite antigen excess was calculated to be equal to 99.5% for MDA-MB-231 and to 2.1% for MDA-MB-157.

Fig. 5. Coronal volume rendered (MIP) microPET/CT images of [⁶⁴Cu]Cu-atezolizumab in mice bearing MDA-MB-231 xenograft at 2, 24 and 48 hours post injection. Mice were injected with 9 MBq of [⁶⁴Cu]Cu-atezolizumab. At 24 and 48 hours mice blocked for PD-L1-specific tracer accumulation by co injection of 2 mg cold atezolizumab are shown (+blocking). Tumors are delineated in white ovals, liver and spleen are located by white arrows. The % ID g⁻¹ for the tumors are reported.