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Proteasomal degradation inhibitors, Bortezomib and Lactacystin had little or no effect on PRLR turnover.

Supplementary Figures S1 - S8 as referenced in the main article

In vitro Potency of HER2 ADC, PRLR ADC and HER2xPRLRbs ADC in a Panel of Breast Cancer Cell Lines

Additional details of methods covered in the main materials and methods section

Supplementary Materials and Methods

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.

Supplementary Tables and Figures Legends

Glucocorticoids (GCs) are widely used to treat a variety of autoimmune and inflammatory diseases; however, systemic delivery of GCs is associated with side effects that affect essentially every organ system, reflecting the nearly ubiquitous expression of the glucocorticoid receptor (GR). Targeted delivery of GCs to diseased tissues using antibody-glucocorticoid conjugates (GC-ADCs) offers a therapeutic alternative to overcome these adverse effects. Herein, we describe novel classes of GCs that exhibited greater potency than dexamethasone and budesonide, a 100-fold selectivity toward the GR over other nuclear receptors, and no in vitro safety liability in pharmacology assays (hERG, AMES) and that demonstrated a substantial reduction in tumor necrosis factor-α (TNF-α) release in mice challenged with lipopolysaccharide (LPS). The site-specific conjugated GC-ADCs via cathepsin-cleavable linkers were highly stable in plasma and specifically released GCs in antigen-positive cells, suggesting that these novel GCs can serve as ADC payloads to treat autoimmune and inflammatory diseases.

Lung cancers harboring mesenchymal-to-epithelial transition factor (MET) genetic alterations, such as exon 14 skipping mutations or high-level gene amplification, respond well to MET-selective tyrosine kinase inhibitors (TKI). However, these agents benefit a relatively small group of patients (4%–5% of lung cancers), and acquired resistance limits response durability. An antibody–drug conjugate (ADC) targeting MET might enable effective treatment of MET-overexpressing tumors (approximately 25% of lung cancers) that do not respond to MET targeted therapies. Using a protease-cleavable linker, we conjugated a biparatopic METxMET antibody to a maytansinoid payload to generate a MET ADC (METxMET-M114). METxMET-M114 promotes substantial and durable tumor regression in xenografts with moderate to high MET expression, including models that exhibit innate or acquired resistance to MET blockers. Positron emission tomography (PET) studies show that tumor uptake of radiolabeled METxMET antibody correlates with MET expression levels and METxMET-M114 efficacy. In a cynomolgus monkey toxicology study, METxMET-M114 was well tolerated at a dose that provides circulating drug concentrations that are sufficient for maximal antitumor activity in mouse models. Our findings suggest that METxMET-M114, which takes advantage of the unique trafficking properties of our METxMET antibody, is a promising candidate for the treatment of MET-overexpressing tumors, with the potential to address some of the limitations faced by the MET function blockers currently in clinical use.

T-cell-redirecting bispecific antibodies have emerged as a new class of therapeutic agents designed to simultaneously bind to T cells via CD3 and to tumor cells via tumor-cell-specific antigens (TSA), inducing T-cell-mediated killing of tumor cells. The promising preclinical and clinical efficacy of TSAxCD3 antibodies is often accompanied by toxicities such as cytokine release syndrome due to T-cell activation. How the efficacy and toxicity profile of the TSAxCD3 bispecific antibodies depends on the binding affinity to CD3 remains unclear. Here, we evaluate bispecific antibodies that were engineered to have a range of CD3 affinities, while retaining the same binding affinity for the selected tumor antigen. These agents were tested for their ability to kill tumor cells in vitro, and their biodistribution, serum half-life, and anti-tumor activity in vivo. Remarkably, by altering the binding affinity for CD3 alone, we can generate bispecific antibodies that maintain potent killing of TSA + tumor cells but display differential patterns of cytokine release, pharmacokinetics, and biodistribution. Therefore, tuning CD3 affinity is a promising method to improve the therapeutic index of T-cell-engaging bispecific antibodies.

CD3-engaging bispecific antibodies (bsAbs) and chimeric antigen receptor (CAR) T cells are potent therapeutic approaches for redirecting patient T cells to recognize and kill tumors. Here we describe a fully human bsAb (REGN5458) that binds to B-cell maturation antigen (BCMA) and CD3, and compare its antitumor activities vs those of anti-BCMA CAR T cells to identify differences in efficacy and mechanism of action. In vitro, BCMAxCD3 bsAb efficiently induced polyclonal T-cell killing of primary human plasma cells and multiple myeloma (MM) cell lines expressing a range of BCMA cell surface densities. In vivo, BCMAxCD3 bsAb suppressed the growth of human MM tumors in murine xenogeneic models and showed potent combinatorial efficacy with programmed cell death protein 1 blockade. BCMAxCD3 bsAb administration to cynomolgus monkeys was well tolerated, resulting in the depletion of BCMA+ cells and mild inflammatory responses characterized by transient increases in C-reactive protein and serum cytokines. The antitumor efficacy of BCMAxCD3 bsAb was compared with BCMA-specific CAR T cells containing a BCMA-binding single-chain variable fragment derived from REGN5458. Both BCMAxCD3 bsAb and anti-BCMA CAR T cells showed similar targeted cytotoxicity of MM cell lines and primary MM cells in vitro. In head-to-head in vivo studies, BCMAxCD3 bsAb rapidly cleared established systemic MM tumors, whereas CAR T cells cleared tumors with slower kinetics. Thus, using the same BCMA-binding domain, these results suggest that BCMAxCD3 bsAb rapidly exerts its therapeutic effects by engaging T cells already in place at the tumor site, whereas anti-BCMA CAR T cells require time to traffic to the tumor site, activate, and numerically expand before exerting antitumor effects.

Purpose: Recent clinical data demonstrate that tumors harboring MET genetic alterations (exon 14 skip mutations and/or gene amplification) respond to small-molecule tyrosine kinase inhibitors, validating MET as a therapeutic target. Although antibody-mediated blockade of the MET pathway has not been successful in the clinic, the failures are likely the result of inadequate patient selection strategies as well as suboptimal antibody design. Thus, our goal was to generate a novel MET blocking antibody with enhanced efficacy. Experimental Design: Here, we describe the activity of a biparatopic MET×MET antibody that recognizes two distinct epitopes in the MET Sema domain. We use a combination of in vitro assays and tumor models to characterize the effect of our antibody on MET signaling, MET intracellular trafficking, and the growth of MET-dependent cells/tumors. Results: In MET-driven tumor models, our biparatopic antibody exhibits significantly better activity than either of the parental antibodies or the mixture of the two parental antibodies and outperforms several clinical-stage MET antibodies. Mechanistically, the biparatopic antibody inhibits MET recycling, thereby promoting lysosomal trafficking and degradation of MET. In contrast to the parental antibodies, the biparatopic antibody fails to activate MET-dependent biological responses, consistent with the observation that it recycles inefficiently and induces very transient downstream signaling. Conclusions: Our results provide strong support for the notion that biparatopic antibodies are a promising therapeutic modality, potentially having greater efficacy than that predicted from the properties of the parental antibodies.

Bispecific antibodies engineered to recruit T cells as a means to kill tumor cells are a promising new class of therapeutics in the field of oncology. A challenge for this class of agents is the requirement for cell surface expressed tumor specific targets, as a large number of tumor-specific antigens are only expressed intracellularly. One strategy to access these targets is to take advantage of the natural processing and presentation of intracellular antigens in the context of the major histocompatibility complex (MHC). However, this requires the development of therapeutics that can be active against targets with a very low surface density. Here, we describe the generation and characterization of Flexbodies, a novel format bispecific antibody engineered to target MHC-peptide tumor neoantigens. We demonstrate that the orientation of the targeting arms and the valency of these reagents allow specific T-cell activation and potent cytotoxic activity against cell lines expressing endogenous level of peptide antigens. This bispecific format has the potential to become an important new tool to expand the range of tumors able to be specifically targeted with t-cell redirecting biologics. Citation Format: Lauric Haber, ryan McKay, jennifer Finney, stephen castaneda, kristen spitler, sahar Rizvi, aidan Hwang, kevin Bray, willy Ramos, pamela Krueger, frank delfino, Craig Meagher, tammy huang, yang shen, william olson, john Lin, eric smith. FlexBodies: Targeting intracellular tumor antigens with T cell redirecting bispecific antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1084.

ADCs based on the natural product maytansine have been successfully employed clinically. In a previous report, ADCs based on hydrophilic non-cell permeable maytansinoids was presented. The authors in this report further explore the maytansine scaffold to develop tubulin inhibitors capable of cell permeation. The research resulted in amino-benzoyl-maytansinoid payloads that were further elaborated with linkers for conjugating to antibodies. This approach was applied to MUC16 tumor targeting antibodies for ovarian cancers. A positive control ADC was evaluated alongside the amino-benzoyl-maytansinoid ADC and the efficacy observed was equivalent while the isotype control ADCs had no effect.

Glioblastoma Multiforme (GBM) is a highly aggressive cancer with few specific molecular targets and poor therapeutic outcome. Epidermal Growth Factor Receptor variant III (EGFRvIII) is a promising target for the treatment of GBM due to its exclusive expression in GBM. However, prior therapeutics have failed in part due to the development of EGFRvIII negative tumors cells from heterogenous tumor populations. We therefore developed a potent antibody-drug conjugate with bystander killing capabilities to target heterogeneous EGFRvIII-expressing GBM tumors. REGN3124 is a fully human EGFRvIII-selective antibody that also demonstrates some binding to amplified wild-type EGFR. REGN3124 was conjugated to the pyrrolobenzodiazepine (PBD) linker-payload SG-3249 to form REGN3124-PBD, and the cytotoxicity and cellular bystander killing activity was characterized in vitro. Initial in vivo activity of REGN3124-PBD was assessed in subcutaneous (s.c.) U251/EGFRvIII and U87/EGFRvIII cell line xenografts and then in s.c. EGFRvIII-positive patient derived xenograft (PDX) models (GBM6 and GBM59). Immunohistochemistry demonstrated heterogenous expression of EGFRvIII in GBM59 tumors. Lastly, efficacy was assessed in animals with established intracranial GBM6 or GBM59 tumors to examine the activity of REGN3124-PBD in an orthotopic setting. REGN3124-PBD demonstrated sub-nM cytotoxicity in vitro and clear bystander killing of EGFRvIII negative U251 cells following targeting of U251/EGFRvIII cells. A single dose of 0.38 mg/kg REGN3124-PBD (3.4 drug: antibody ratio) induced sustained regression of both s.c. U251/EGFRvIII and U87/EGFRvIII xenografts. A single dose of 0.53 mg/kg REGN3124-PBD induced complete regression of s.c. GBM6 PDX tumors and sustained regression of GBM59 tumors. Single dose of 0.53 mg/kg REGN3124-PBD significantly prolonged survival of mice with established intracranial GBM6 or GBM59 tumors, with 5/8 and 7/8 animals surviving >90 days post-treatment, respectively. The high unmet need for effective therapies combined with the potent anti-tumor activity observed, including in those with heterogenous expression of EGFRvIII, support continued assessment of REGN3124-PBD as a novel therapy for treatment of GBM. Citation Format: Marcus P. Kelly, Sosina Makonnen, Carlos Hickey, Shu Mao, Feng Zhao, Arthur Kunz, Frank Delfino, Thomas Nittoli, Dangshe Ma, William C. Olson, Gavin Thurston, Jessica R. Kirshner. Novel EGFRvIII-selective antibody-drug conjugate REGN3124-PBD is strongly efficacious against orthotopic glioblastoma multiforme patient derived xenografts [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P134.

The Prolactin Receptor (PRLR) is a type 1 cytokine receptor that is expressed in a subset of breast cancers and may contribute to its pathogenesis. It is relatively overexpressed in approximately 25% of human breast tumors while expressed at low levels in some normal human tissues including the mammary gland. We developed an anti-PRLR antibody-drug conjugate (ADC), to target PRLR-positive breast cancer. REGN2878-DM1 is comprised of a fully human high-affinity function-blocking anti-PRLR IgG1 antibody (REGN2878) conjugated via a noncleavable SMCC linker to the cytotoxic maytansine derivative DM1. Both unconjugated REGN2878 and conjugated REGN2878-DM1 block PRL-mediated activation in vitro and are rapidly internalized into lysosomes. REGN2878-DM1 induces potent cell-cycle arrest and cytotoxicity in PRLR-expressing tumor cell lines. In vivo, REGN2878-DM1 demonstrated significant antigen-specific antitumor activity against breast cancer xenograft models. In addition, REGN2878-DM1 showed additive activity when combined with the antiestrogen agent fulvestrant. These results illustrate promising antitumor activity against PRLR-positive breast cancer xenografts and support the evaluation of anti-PRLR ADCs as potential therapeutic agents in breast cancer. Mol Cancer Ther; 16(7); 1299–311. ©2017 AACR.

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.

Advanced ovarian cancer is frequently treated with combination chemotherapy, but high recurrence rates show the need for therapies that can produce durable responses and extend overall survival. Bispecific antibodies that interact with tumor antigens on cancer cells and activating receptors on immune cells offer an innovative immunotherapy approach. Here, we describe a human bispecific antibody (REGN4018) that binds both Mucin 16 (MUC16), a glycoprotein that is highly expressed on ovarian cancer cells, and CD3, thus bridging MUC16-expressing cells with CD3+ T cells. REGN4018 induced T cell activation and killing of MUC16-expressing tumor cells in vitro. Binding and cytotoxicity of REGN4018 in vitro were minimally affected by high concentrations of CA-125, the shed form of MUC16, which is present in patients. In preclinical studies with human ovarian cancer cells and human T cells in immunodeficient mice, REGN4018 potently inhibited growth of intraperitoneal ovarian tumors. Moreover, in a genetically engineered immunocompetent mouse expressing human CD3 and human MUC16 [humanized target (HuT) mice], REGN4018 inhibited growth of murine tumors expressing human MUC16, and combination with an anti-PD-1 antibody enhanced this efficacy. Immuno-PET imaging demonstrated localization of REGN4018 in MUC16-expressing tumors and in T cell-rich organs such as the spleen and lymph nodes. Toxicology studies in cynomolgus monkeys showed minimal and transient increases in serum cytokines and C-reactive protein after REGN4018 administration, with no overt toxicity. Collectively, these data demonstrate potent antitumor activity and good tolerability of REGN4018, supporting clinical evaluation of REGN4018 in patients with MUC16-expressing advanced ovarian cancer.

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.

EGFR blocking antibodies are approved for the treatment of colorectal cancer and head and neck squamous cell carcinoma (HNSCC). Although ERBB3 signaling has been proposed to limit the effectiveness of EGFR inhibitors, the underlying molecular mechanisms are not fully understood. To gain insight into these mechanisms, we generated potent blocking antibodies against ERBB3 (REGN1400) and EGFR (REGN955). We show that EGFR and ERBB3 are coactivated in multiple HNSCC cell lines and that combined blockade of EGFR and ERBB3 inhibits growth of these cell lines more effectively than blockade of either receptor alone. Blockade of EGFR with REGN955 strongly inhibited activation of ERK in HNSCC cell lines, whereas blockade of ERBB3 with REGN1400 strongly inhibited activation of Akt; only the combination of the 2 antibodies blocked both of these essential downstream pathways. We used a HER2 blocking antibody to show that ERBB3 phosphorylation in HNSCC and colorectal cancer cells is strictly dependent on association with HER2, but not EGFR, and that neuregulin 1 activates ERBB3/HER2 signaling to reverse the effect of EGFR blockade on colorectal cancer cell growth. Finally, although REGN1400 and REGN955 as single agents slowed the growth of HNSCC and colorectal cancer xenografts, the combination of REGN1400 plus REGN955 caused significant tumor regression. Our results indicate that activation of the Akt survival pathway by ERBB3/HER2 limits the effectiveness of EGFR inhibition, suggesting that REGN1400, which is currently in a phase I clinical trial, could provide benefit when combined with EGFR blocking antibodies. Mol Cancer Ther; 13(5); 1345–55. ©2014 AACR.

Improving therapies for multiple myeloma (MM) remains a high medical need because of the significant morbidity and mortality of the disease. Targeted immunotherapies represent a promising opportunity to fill this clinical need. B cell maturation antigen (BCMA) is an attractive cell-surface target for MM due to its consistent expression on MM patient malignant plasma cells and expression limited in normal tissue primarily to plasma cells. Redirection of a patient's T cells to recognize tumors by CD3-binding bispecific molecules or through the generation of chimeric antigen receptor (CAR) T cells, has shown preliminary evidence of clinical activity. Bispecific antibodies concurrently engage a tumor antigen on cancer cells and the CD3 signaling machinery on T cells, bringing the tumor cell and T cell into proximity and facilitating T cell activation and tumor cell killing. By contrast, CAR T cell therapy involves re-infusion of the patient's own T cells after ex vivo engineering to express CARs targeting tumor antigens and triggering T cell signaling. Here we describe the generation of REGN5458, a human bispecific antibody that binds to BCMA and CD3. In vitro, REGN5458 efficiently activates T cells and induces polyclonal T cell killing of myeloma cell lines with a range of BCMA cell-surface densities, and also induces cytotoxicity of primary human plasma cells. Similar to gamma-sectretase inhibitors, incubation of myeloma cell lines with REGN5458 increased surface levels of BCMA. In xenogenic studies, after BCMAhigh NCI-H929 and BCMAlow MOLP-8 MM cells were co-implanted with PBMC and grown subcutaneously in immunodeficient NOD/SCID/L2Rgamma-deficient (NSG) mice, REGN5458 doses as low as 0.4 mg/kg significantly suppressed the growth of both tumors. Using aggressive, systemic xenogenic tumor models, in which NSG mice were engrafted with PBMC and intravenously injected with BCMAhigh OPM-2 cells or BCMAlow MOLP-8 cells expressing luciferase, REGN5458 reduced tumor burden and suppressed tumor growth at doses as low as 0.4 mg/kg. In immunocompetent mice genetically engineered to express human CD3, REGN5458 inhibited the growth of syngeneic murine tumors expressing human BCMA at doses as low as 0.04 mg/kg. Finally, as REGN5458 binds to cynomolgus CD3 and BCMA and mediates cytotoxicity of primary cynomolgus plasma cells, the pharmacology of REGN5458 was evaluated in cynomolgus monkeys. REGN5458 administration was well-tolerated, resulting in a mild inflammatory response characterized by transiently increased CRP and serum cytokines. Importantly, REGN5458 treatment led to the depletion of BCMA+ plasma cells in the bone marrow, demonstrating cytotoxic activity in non-human primates. The anti-tumor efficacy of REGN5458 was compared to BCMA-specific CAR T cells using 2nd generation CAR lentiviral constructs containing a single-chain variable fragment binding domain from REGN5458's BCMA binding arm and 4-1BB and CD3z signaling domains. Human PBMC-derived T cells were transduced to express this CAR and expanded. Both REGN5458 and the BCMA CAR T cells demonstrated similar targeted cytotoxicity of myeloma cell lines and primary patient blasts in vitro, and were capable of clearing established systemic OPM-2-luciferase myeloma tumors in NSG mice, but with different kinetics: treatment with REGN5458 resulted in rapid clearance of tumors within 4 days, whereas treatment with BCMA CAR T cells allowed tumors to continue to grow for 10-14 days following injection before rapidly inducing tumor clearance. Thus, REGN5458 exerts its therapeutic effect rapidly after injection, using effector T cells that are already in place. In contrast, BCMA CAR T cells require time to traffic to the tumor site and expand, before exerting anti-tumor effects. Collectively, these data demonstrate the potent pre-clinical anti-tumor activity of REGN5458 that is comparable to that of CAR T cells, and provide a strong rationale for clinical testing of REGN5458 in patients with MM. Disclosures Dilillo: Regeneron Pharmaceuticals: Employment. Olson:Regeneron Pharmaceuticals: Employment. Mohrs:Regeneron Pharmaceuticals: Employment. Meagher:Regeneron Pharmaceuticals: Employment. Bray:Regeneron Pharmaceuticals: Employment. Sineshchekova:Regeneron Pharmaceuticals: Employment. Startz:Regeneron Pharmaceuticals: Employment. Retter:Regeneron Pharmaceuticals: Employment. Godin:Regeneron Pharmaceuticals: Employment. Delfino:Regeneron Pharmaceuticals: Employment. Lin:Regeneron Pharmaceuticals: Employment. Smith:Regeneron Pharmaceuticals: Employment. Thurston:Regeneron Pharmaceuticals: Employment. Kirshner:Regeneron Pharmaceuticals: Employment.

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 D'Souza, 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.

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.