SupplementaryMethods

Antibodies, ADCs and other reagents

PRLR antibody and HER2 antibody were conjugated via surface lysines to the maytansine derivative DM1, using a non-cleavable, hetero-bifunctional linker (succinimidyl trans-4-[maleimidylmethyl] cyclohexane-1-carboxylate, SMCC). The reaction was carried out in 50 mM Hepes, 150 mM NaCl at pH 8.0. The antibody-MCC-DM1 ADCs were purified by size-exclusion chromatography, and monomeric fractions were collected. The PRLR and HER2 ADCs contained >95% monomer, <0.5% unconjugated linker-payload and had average drug-to-antibody ratios (DAR) of 3.4 and 2.4, respectively. SPR-Biacore studies demonstrated that conjugation did not alter the antigen-binding properties of the antibodies (data not shown). PRLR and HER2 antibodies bound recombinant, monomeric PRLR and HER2 ectodomain proteins (Regeneron) withKDvalues of 3.16E-09M and 3.98E-09M respectively, as determined using Surface Plasmon Resonance (SPR)–based Biacore technology.

For generating HER2xPRLR bispecific antibodies,human HER2 and PRLR antibodies were generated in VelocImmune®mice that express a single (“universal”) light chainto obviate the potential for heavy-light chain mispairing. HER2 antibodies were selected that did not cross-compete with each other or with in-house trastuzumab as determined in SPR-Biacore studies.HER2xPRLR bispecific antibodies were generated as previously described in a native IgG1 format with the exception of an H435Y436  RF (IgG1  IgG3) substitution in the IgG1 CH3 domain of the anti-PRLR heavy chain in order to impart asymmetric binding to protein A.HER2xPRLR bispecific antibodies 1-5 utilized in the experiment described in Suppl. Fig. S4 were generated using arms derived from HER2 antibodies designated H4H13055P2, H4H13049P2, H4H13059P2, H4H13081P2, and fromPRLR antibodies designated H1H7676P2, H4H13081P2.

Trafficking Assays

For experiment described in Figure 1A, T47D cells were placed at 4°C for 30 minutes, incubated with 10ug/ml of CF594-labeled receptor primary antibodies for 30 minutes, rinsed with 4°C complete growth media and the internalization process was started by replacing cold media with prewarmed (37oC) media.After incubation at 37oCwith 5% CO2 for the indicated times, cells were fixed with 4% PFA for 10 minand stained with secondaryAlexaFluor488-conjugated goat anti-human IgG Fab Fragment(Jackson ImmunoResearch Laboratories).

For the experiment described in Figure 1B, the lysosomes were labeled by incubating cells with fluorescein-3 kDa dextrans for 1h at 37°Cwith 5% CO2 and CF594-labeled receptor antibodies (10ug/ml)were added to separate samples for the last 5, 10, 20, 30, 45 minutes, or the entire duration of dextran labeling period (60 minutes). Then, cells were rinsed twice with warm (37°C) complete growth media, incubated for additional 10 minutes and fixed immediately afterwards with 4% PFA for 10 min.

For the experiment described in Figure 3E, cells were placed incubated with 10ug/ml of primary antibody for 30 minutes, washed twice and incubated with secondary AlexaFluor488-conjugated goat anti-human IgG Fab Fragment(Jackson ImmunoResearch Laboratories) on ice for another 30 minutes. Cells were then washed again and either fixed immediately with 4% PFA on ice for 10 min, or incubated at 37oC with 5% CO2for 1h and then fixed with with 4% PFA for 10 min.

For the experiment described in Figure 5, cells were placed at 4°C for 30 minutes and subsequently co-incubated with 10ug/ml of AlexaFluor405-labeled PRLR antibody (blue) and CF594-labeled HER2 antibody (red) for additional 30 minutes. Cells were rinsed with 4°C complete growth media and the internalization process was started by replacing cold media with prewarmed (37oC) media and allowed to continueat 37oC with 5% CO2 for the indicated times.The lysosomes were labeled by incubating cells with fluorescein-3 kDa dextrans for the last 1h of the internalization period.At the end of the incubation at 370C with 5% CO2, cells were fixed with 4% PFA for 10 min.

Colocalization was quantified in a pixel-by-pixel basis for all confocal sections of the confocal stack. To determine the area of marker “A” (i.e. total receptor) occupied by marker “B” (i.e. surface receptor), the pixels of marker “A” colocalizing with marker “B” were divided by the total pixels of marker “A”.This operation was applied for all the confocal sections of a confocal stack in each region of interest (i.e. one cell).The inverse formula was utilized to calculate the area of marker “B” occupied by marker “A”.

Surface Plasmon Resonance(SPR)

The affinities of PRLR or HER2 antibodies for human PRLR orHER2, were measured using a Biacore T200 (GE Healthcare, Picastaway, NJ) instrument using a dextran-coated (CM5) chip at 37°C.The running buffer was filtered HBS-T (10 mM Hepes, 150 mM NaCl, 3.4mM EDTA, 0.05% polysorbate 20, pH 7.4).A capture sensor surface was prepared by covalently immobilizing goat anti-human Fcγ antibody (GE Healthcare, Picastaway, NJ) to the chip surface using (1Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride)/N-hydroxysuccinimide (EDC/NHS) coupling chemistry.Following surface activation, anti-human Fc antibody in coupling buffer (0.1 M acetate buffer, pH 4.5) was injected over the activated chip surface until a resonance unit (RU) signal of about ~10,000 RU was reached.The activated coupled chip surfaces were then washed and treated with 10mM glycine-HCl, pH 1.5, to remove uncoupled residual proteins.Antibodies were then captured through their Fc regions and tested for binding to the extracellular domains of human PRLR or HER2 in monomeric or dimeric (fused to mouse Fc) format. PRLR or HER2 proteins were injected at concentrations rangingfrom 1.11nM to 100nM. All capture surfaces were regenerated with one 15-s pulse of 10 mM glycine–HCl, pH 1.5.

1