Marcus P. Kelly

Bispecific Antibodies and Antibody-Drug Conjugates (ADCs) Bridging HER2 and Prolactin Receptor Improve Efficacy of HER2 ADCs

The properties of cell surface proteins targeted by antibody–drug conjugates (ADCs) have not been fully exploited; of particular importance are the rate of internalization and the route of intracellular trafficking. In this study, we compared the trafficking of HER2, which is the target of the clinically approved ADC ado-trastuzumab emtansine (T-DM1), with that of prolactin receptor (PRLR), another potential target in breast cancer. In contrast to HER2, we found that PRLR is rapidly and constitutively internalized, and traffics efficiently to lysosomes, where it is degraded. The PRLR cytoplasmic domain is necessary to promote rapid internalization and degradation, and when transferred to HER2, enhances HER2 degradation. In accordance with these findings, low levels of cell surface PRLR (∼30,000 surface receptors per cell) are sufficient to mediate effective killing by PRLR ADC, whereas cell killing by HER2 ADC requires higher levels of cell surface HER2 (∼106 surface receptors per cell). Noncovalently cross-linking HER2 to PRLR at the cell surface, using a bispecific antibody that binds to both receptors, dramatically enhances the degradation of HER2 as well as the cell killing activity of a noncompeting HER2 ADC. Furthermore, in breast cancer cells that coexpress HER2 and PRLR, a HER2xPRLR bispecific ADC kills more effectively than HER2 ADC. These results emphasize that intracellular trafficking of ADC targets is a key property for their activity and, further, that coupling an ADC target to a rapidly internalizing protein may be a useful approach to enhance internalization and cell killing activity of ADCs. Mol Cancer Ther; 16(4); 681–93. ©2017 AACR.

ImmunoPET Imaging of Endogenous and Transfected Prolactin Receptor Tumor Xenografts

Antibodies labeled with positron-emitting isotopes have been used for tumor detection, predicting which patients may respond to tumor antigen-directed therapy, and assessing pharmacodynamic effects of drug interventions. Prolactin receptor (PRLR) is overexpressed in breast and prostate cancers and is a new target for cancer therapy. We evaluated REGN2878, an anti-PRLR monoclonal antibody, as an immunoPET reagent. REGN2878 was labeled with Zr-89 after conjugation with desferrioxamine B or labeled with I-131/I-124. In vitro determination of the half-maximal inhibitory concentration (IC50) of parental REGN2878, DFO-REGN2878, and iodinated REGN2878 was performed by examining the effect of the increasing amounts of these on uptake of trace-labeled I-131 REGN2878. REGN1932, a non-PRLR binding antibody, was used as a control. Imaging and biodistribution studies were performed in mice bearing tumor xenografts with various expression levels of PRLR, including MCF-7, transfected MCF-7/PRLR, PC3, and transfected PC3/PRLR and T4D7v11 cell lines. The specificity of uptake in tumors was evaluated by comparing Zr-89 REGN2878 and REGN1932, and in vivo competition compared Zr-89 REGN2878 uptake in tumor xenografts with and without prior injection of 2 mg of nonradioactive REGN2878. The competition binding assay of DFO-REGN2878 at ratios of 3.53-5.77 DFO per antibody showed IC50 values of 0.4917 and 0.7136 nM, respectively, compared to 0.3455 nM for parental REGN2878 and 0.3343 nM for I-124 REGN2878. Imaging and biodistribution studies showed excellent targeting of Zr-89 REGN2878 in PRLR-positive xenografts at delayed times of 189 h (presented as mean ± 1 SD, percent injected activity per mL (%IA/mL) 74.6 ± 33.8%IA/mL). In contrast, MCF-7/PRLR tumor xenografts showed a low uptake (7.0 ± 2.3%IA/mL) of control Zr-89 REGN1932 and a very low uptake and rapid clearance of I-124 REGN2878 (1.4 ± 0.6%IA/mL). Zr-89 REGN2878 has excellent antigen-specific targeting in various PRLR tumor xenograft models. We estimated, using image-based kinetic modeling, that PRLR antigen has a very rapid in vivo turnover half-life of ∼14 min from the cell membrane. Despite relatively modest estimated tumor PRLR expression numbers, PRLR-expressing cells have shown final retention of the Zr-89 REGN2878 antibody, with an uptake that appeared to be related to PRLR expression. This reagent has the potential to be used in clinical trials targeting PRLR.

Antibody drug conjugates of cleavable amino-alkyl and aryl maytansinoids

Natural products have been used for many medicinal purposes for centuries. Antibody drug conjugates (ADCs) have utilized this rich source of small molecule therapeutics to produce several clinically useful treatments. ADCs based on the natural product maytansine have been successful clinically. The authors further the utility of the anti-cancer natural product maytansine by developing efficacious payloads and linker-payloads for conjugating to antibodies. The success of our approach was realized in the EGFRvIII targeting ADC EGFRvIII-16. The ADC was able to regress tumors in 2 tumor models (U251/EGFRvIII and MMT/EGFRvIII). When compared to a positive control ADC, the efficacy observed was similar or improved while the isotype control ADCs had no effect.

Abstract C126: PRLR ADC: A novel antibody drug-conjugate for the treatment of PRLR positive breast cancer

Breast cancer is a heterogeneous disease comprised of various subtypes based on pathology and molecular profiling. Expression of hormone receptors (HR) and HER2 biomarkers are important determinants of therapy choice, due to the established role of these proteins as drivers of disease. Prolactin Receptor (PRLR) is a type 1 cytokine receptor that is expressed on a subset of breast cancers and may contribute to pathogenesis. Functionally, activation of PRLR by the hormone ligand Prolactin (PRL) induces PRLR dimerization and signaling resulting in cell proliferation and differentiation. While PRLR is expressed at low levels in some normal human tissues including the mammary gland, it is relatively overexpressed in ∼25% of human breast tumors and importantly, is rapidly internalized upon binding of anti-PRLR antibodies. We developed an anti-PRLR antibody-drug conjugate (ADC), PRLR ADC, to target PRLR positive breast cancer. PRLR ADC is comprised of a fully human high affinity function-blocking anti-PRLR IgG1 antibody conjugated via a non-cleavable SMCC linker to the cytotoxic maytansine derivative DM1. Both unconjugated anti-PRLR antibody and the PRLR ADC block PRL mediated activation in vitro and induce rapid internalization of the receptor into lysosomes. PRLR ADC induces potent cell cycle arrest and cytotoxicity in several PRLR-expressing cell lines. The in vivo efficacy of PRLR ADC was explored in breast cancer cell line xenograft models expressing both endogenous PRLR (MCF7, T47D) or transfected receptor (MCF7/PRLR). Treatment of tumor bearing SCID (T47D) or NCr Nude (MCF7) animals was initiated approximately 15 days post implantation of cells where tumor volumes averaged 150-200 mm3. In both T47D and MCF7/PRLR xenograft models, where PRLR is expressed highly, single or multiple (once weekly x 3) doses of 2.5-15 mg/kg resulted in significant inhibition of tumor xenograft growth. In the MCF7 model that expresses low levels of PRLR, inhibition and regression of tumors was observed at 10 and 15 mg/kg dose levels. In all models, higher doses resulted in greater and more prolonged repression of tumor growth. Conjugation of DM1 to anti-PRLR antibody was required for efficacy, as unconjugated antibody had no effect on tumor growth. Anti-tumor efficacy of PRLR ADC was also assessed in NSG mice bearing breast cancer Patient Derived Xenograft (PDXs) tumors with moderate and heterogeneous expression of PRLR. Treatment was initiated 21 days after implantation of the PDX tumors where the average tumor volume was ∼500mm3. Anti-tumor efficacy was observed following 10 or 20 mg/kg PRLR ADC dosed once weekly x 4. These studies demonstrate the promising anti-tumor activity of the PRLR ADC against PRLR positive breast cancers and support the continued development of this agent. Citation Format: Marcus P. Kelly, Sandra Coetzee, Carlos Hickey, Sosina Makonnen, Frank Delfino, Julian Andreev, Arthur Kunz, Christopher D9Souza, Jason Giurleo, Thomas Nittoli, Pamela A. Trail, Nicholas Papadopoulos, Gavin Thurston, Jessica R. Kirshner. PRLR ADC: A novel antibody drug-conjugate for the treatment of PRLR positive breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C126.

Abstract A131: Rapid constitutive internalization and degradation of prolactin receptor (PRLR) is associated with potent cell killing by PRLR antibody drug conjugates (ADC)

Ado-trastuzumab-emtansine or T-DM1, an antibody drug conjugate (ADC) targeting the well-characterized breast cancer oncogene HER2, has shown benefit for breast cancer patients. However, treatment is not indicated for patients whose tumors express low or intermediate levels of HER2. Thus, additional targets for ADC are needed in breast cancer. The lineage-restricted marker prolactin receptor (PRLR) is also expressed in a subset of breast cancers. Unexpectedly, we found that, unlike HER2, low levels of cell-surface PRLR are sufficient to mediate efficient ADC killing of breast ductal carcinoma cells, including T47D. To understand what properties of PRLR vs HER2 allow for efficient cell killing, we compared intracellular trafficking of these two receptors. We found that approximately 90% of a PRLR antibody was internalized by T47D cells within 1h after treatment, and the internalized PRLR Ab co-localized with the lysosomal marker, Lysotracker Red. In contrast, trastuzumab was restricted to the plasma membrane and did not co-localize with Lysotracker Red. Overnight incubation of T47D cells with PRLR Ab, but not trastuzumab, resulted in accumulation in a late lysosomal compartment, as detected using the pH-sensitive marker, pHrodo. Inhibiting protein synthesis with cycloheximide resulted in almost complete degradation of PRLR after 2h, whereas HER2 was degraded only slightly after 4h. The rapid turnover of PRLR was not significantly affected by adding exogenous ligand (prolactin), or by PRLR antibodies, or by proteasome inhibitors, but was blocked by lysosomal inhibitors including bafilomycin A1, and monensin. The signals for this constitutive PRLR internalization and degradation appear to be contained within its cytoplasmic domain, since substitution of the PRLR extracellular domain by that of HER2 still resulted in degradation rates similar to those of full length PRLR. Moreover, simultaneous substitution of two dileucine lysosomal sorting signals contained in the PRLR cytoplasmic domain (e.g. 283LL and 292LL) to alanine significantly diminished constitutive PRLR turnover. In accordance with these data, PRLR ADC, but not T-DM1, induced cell cycle arrest (proportional to PRLR ADC-induced cell killing) in T47D cells, which was completely abolished by lysosomal inhibitors. Taken together, these data indicate that rapid constitutive ligand-independent turnover of PRLR, but not Her2, can deliver high amounts of ADC to lysosomes, resulting in efficient tumor cell killing. Citation Format: Julian Andreev, NIthya Thambi, Frank Delfino, Joel Martin, Marcus P. Kelly, Jessica R. Kirshner, Douglas MacDonald, Nicholas Popadopoulos, Willian Olson, Gavin Thurston. Rapid constitutive internalization and degradation of prolactin receptor (PRLR) is associated with potent cell killing by PRLR antibody drug conjugates (ADC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A131.