Improved Lysosomal Trafficking Can Modulate the Potency of Antibody Drug Conjugates

Antibody drug conjugates (ADCs) provide an efficacious and relatively safe means by which chemotherapeutic agents can be specifically targeted to cancer cells. In addition to the selection of antibody targets, ADCs offer a modular design that allows selection of ADC characteristics through the choice of linker chemistries, toxins, and conjugation sites. Many studies have indicated that release of toxins bound to antibodies via noncleavable linker chemistries relies on the internalization and intracellular trafficking of the ADC. While this can make noncleavable ADCs more stable in the serum, it can also result in lower efficacy when their respective targets are not internalized efficiently or are recycled back to the cell surface following internalization. Here, we show that a lysosomally targeted ADC against the protein APLP2 mediates cell killing, both in vitro and in vivo, more effectively than an ADC against Trop2, a protein with less efficient lysosomal targeting. We also engineered a bispecific ADC with one arm targeting HER2 for the purpose of directing the ADC to tumors, and the other arm targeting APLP2, whose purpose is to direct the ADC to lysosomes for toxin release. This proof-of-concept bispecific ADC demonstrates that this technology can be used to shift the intracellular trafficking of a constitutively recycled target by directing one arm of the antibody against a lysosomally delivered protein. Our data also show limitations of this approach and potential future directions for development

Site-Dependent Degradation of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent Plasma and Its Effect on ADC Efficacy

<div><p>The efficacy of an antibody-drug conjugate (ADC) is dependent on the properties of its linker-payload which must remain stable while in systemic circulation but undergo efficient processing upon internalization into target cells. Here, we examine the stability of a non-cleavable Amino-PEG6-based linker bearing the monomethyl auristatin D (MMAD) payload site-specifically conjugated at multiple positions on an antibody. Enzymatic conjugation with transglutaminase allows us to create a stable amide linkage that remains intact across all tested conjugation sites on the antibody, and provides us with an opportunity to examine the stability of the auristatin payload itself. We report a position-dependent degradation of the C terminus of MMAD in rodent plasma that has a detrimental effect on its potency. The MMAD cleavage can be eliminated by either modifying the C terminus of the toxin, or by selection of conjugation site. Both approaches result in improved stability and potency <i>in vitro</i> and <i>in vivo</i>. Furthermore, we show that the MMAD metabolism in mouse plasma is likely mediated by a serine-based hydrolase, appears much less pronounced in rat, and was not detected in cynomolgus monkey or human plasma. Clarifying these species differences and controlling toxin degradation to optimize ADC stability in rodents is essential to make the best ADC selection from preclinical models. The data presented here demonstrate that site selection and toxin susceptibility to mouse plasma degradation are important considerations in the design of non-cleavable ADCs, and further highlight the benefits of site-specific conjugation methods.</p></div

Protease inhibition studies of the PEG6-C2-MMAD degradation in mouse plasma.

<p>“Yes” indicates the same extent of cleavage as observed in plasma without inhibitors, “partial” indicates reduced cleavage compared to uninhibited plasma, while “no” indicates that no degradation was observed. All assays were carried out at pH 7.4.</p><p>Protease inhibition studies of the PEG6-C2-MMAD degradation in mouse plasma.</p

Stability studies of site-specific non-cleavable ADCs.

<p>a) Positions of conjugation sites on an antibody. b) Structure of the PEG6-C2-MMAD non-cleavable payload conjugated to the glutamine tag on the antibody, and its cleavage product. The glutamine residue is shown in blue. c) Structure of the PEG6-C2-Aur3377 non-cleavable payload conjugated to the glutamine tag shown in blue.</p

Degradation of the C-terminal portion of the PEG6-C2-MMAD payload in the plasma of different species.

<p>Degradation is calculated as percentage of payload cleaved. Calculations are based on DAR values obtained from HIC analysis of the Site A-PEG6-2-MMAD conjugate before and after incubation in plasma for 4.5 days in three independent experiments.</p

Mass spectrometric analysis of non-cleavable conjugates.

<p>The Fig labels represent experimentally observed masses for conjugates before (upper panel) and after (lower panel) <i>in vivo</i> exposure. a) Intact mass deconvolution of C16 Site A-PEG6-C2-MMAD conjugate. The metabolic products of the DAR 2 species show a mass loss from either 1 x 186 Da (one payload) or 2 x 186 Da (both payloads). b) Intact mass of C16 Site I-PEG6-C2-MMAD conjugate. The metabolic product shows a 186 Da loss from one of the conjugated payloads. c) Intact mass of C16 Site A-PEG6-C2-Aur3377 conjugate. The <i>in vivo</i> exposed conjugate shows no mass shift compared to the untreated compound.</p

Comparative efficacy studies of non-cleavable ADCs.

<p>Comparison of <i>in vitro</i> cytotoxic activities of untreated and <i>in vivo</i>-exposed non-cleavable conjugates against BxPC3 cells (M1S1+++). a) C16 Site A-PEG6-C2-MMAD conjugate. b) C16 Site I-PEG6-C2-MMAD conjugate. c) C16 Site A-PEG6-C2-Aur3377 conjugate. d) <i>In vivo</i> comparison of the three conjugates in the BxPC3 xenograft model, along with a negative control conjugate NCC Site F-PEG6-C2-MMAD. All compounds were given as a single dose at 10 mg/kg.</p

Stability of the non-cleavable PEG6-C2-MMAD and PEG6-C2-Aur3377 conjugates under <i>in vitro</i> and <i>in vivo</i> conditions.

<p>The percentage of intact MMAD was calculated as the ratio of treated DAR to untreated DAR. Degradation of the C-terminal portion of the PEG6-C2-MMAD payload is considered equivalent to payload removal for DAR determination purposes. Calculations are based on DAR values obtained from HIC analysis for most conjugates, except for Site G, H, and I conjugates for which the percentage of intact MMAD was determined by mass spec analysis. For mouse plasma stability, values reported are averages of three independent experiments. For mouse <i>in vivo</i> stability, samples collected from individually dosed mice were pooled to obtain the measurement.</p><p>Stability of the non-cleavable PEG6-C2-MMAD and PEG6-C2-Aur3377 conjugates under <i>in vitro</i> and <i>in vivo</i> conditions.</p

Synthesis of Amino-PEG6-C2-Aur3377.

<p>Reagents and conditions: a) Aur-3377, Fmoc-AmPeg6C2-CO2H, HATU, <i>i</i>-Pr₂NEt, DMF; b) LiOH, H₂O-DMF.</p