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Luminal A (hormone receptor-positive) breast cancer constitutes 70% of total breast cancer patients. In an attempt to develop a targeted therapeutic for this cancer indication, we have identified and characterized Glial cell line–Derived Neurotrophic Factor (GDNF) Family Receptor Alpha 1 (GFRA1) antibody–drug conjugates (ADC) using a cleavable valine-citrulline-MMAE (vcMMAE) linker-payload. RNAseq and IHC analysis confirmed the abundant expression of GFRA1 in luminal A breast cancer tissues, whereas minimal or no expression was observed in most normal tissues. Anti–GFRA-vcMMAE ADC internalized to the lysosomes and exhibited target-dependent killing of GFRA1-expressing cells both in vitro and in vivo. The ADCs using humanized anti-GFRA1 antibodies displayed robust therapeutic activity in clinically relevant cell line–derived (MCF7 and KPL-1) tumor xenograft models. The lead anti-GFRA1 ADC cross-reacts with rodent and cynomolgus monkey GFRA1 antigen and showed optimal pharmacokinetic properties in both species. These properties subsequently enabled a target-dependent toxicity study in rats. Anti-GFRA1 ADC is well tolerated in rats, as seen with other vcMMAE linker–payload based ADCs. Overall, these data suggest that anti–GFRA1-vcMMAE ADC may provide a targeted therapeutic opportunity for luminal A breast cancer patients. Mol Cancer Ther; 17(3); 638–49. ©2017 AACR.

Supplementary Figures S1-S5, Supplementary Materials and Methods

Figure S1. Validation of anti-GFRA1 antibodies (R&D systems and GNE-8H6) for the use of Immunohistochemistry (IHC) analysis; Figure S2. ELISA analyses to test binding specificity of anti-GFRA1 antibodies to GFRA1 (A) and GFRA2 (B) extra cellular domain-Fc fusion proteins; Figure S3. Pharmacokinetics of anti-GFRA1 ADCs in tumor xenograft models; Figure S4. FACS of anti-GFRA1, hu-6D3.v5 (A) and hu-7C9 (B) antibody titration on HEK293-overexpressing human, cynomolgus monkey, rat and mouse GFRA1 cells showing similar binding; Table S1. Affinity characterization of anti-GFRA1 IgG antibodies on Immobilized hu- GFRA1Fc ligand; Table S2. Bioanalytical characterization of anti-GFRA1 ADCs.

Purpose: Antibody drug conjugates (ADCs) combine the ideal properties of both antibodies and cytotoxic drugs by targeting potent drugs to the antigen-expressing tumor cells, thereby enhancing their antitumor activity. Successful ADC development for a given target antigen depends on optimization of antibody selection, linker stability, cytotoxic drug potency, and mode of linker-drug conjugation to the antibody. Here, we systematically examined the in vitro potency as well as in vivo preclinical efficacy and safety profiles of a heterogeneous preparation of conventional trastuzumab-mcc-DM1 (TMAb-mcc-DM1) ADC with that of a homogeneous engineered thio-trastuzumab-mpeo-DM1 (thioTMAb-mpeo-DM1) conjugate.Experimental Design and Results: To generate thioTMAb-mpeo-DM1, one drug maytansinoid 1 (DM1) molecule was conjugated to an engineered cysteine residue at Ala114 (Kabat numbering) on each trastuzumab-heavy chain, resulting in two DM1 molecules per antibody. ThioTMAb-mpeo-DM1 retained similar in vitro anti–cell proliferation activity and human epidermal growth factor receptor 2 (HER2) binding properties to that of the conventional ADC. Furthermore, it showed improved efficacy over the conventional ADC at DM1-equivalent doses (μg/m2) and retained efficacy at equivalent antibody doses (mg/kg). An improved safety profile of >2-fold was observed in a short-term target-independent rat safety study. In cynomolgus monkey safety studies, thioTMAb-mpeo-DM1 was tolerated at higher antibody doses (up to 48 mg/kg or 6,000 μg DM1/m2) compared with the conventional ADC that had dose-limiting toxicity at 30 mg/kg (6,000 μg DM1/m2).Conclusions: The engineered thioTMAb-mpeo-DM1 with broadened therapeutic index represents a promising antibody drug conjugate for future clinical development of HER2-positive targeted breast cancer therapies. Clin Cancer Res; 16(19); 4769–78. ©2010 AACR.

Supplementary Methods, Figure Legends 1-3, Tables 1A-B from Engineered Thio-Trastuzumab-DM1 Conjugate with an Improved Therapeutic Index to Target Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer

Supplementary Figure 3C-D from Engineered Thio-Trastuzumab-DM1 Conjugate with an Improved Therapeutic Index to Target Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer

Supplementary Figure 2 from Engineered Thio-Trastuzumab-DM1 Conjugate with an Improved Therapeutic Index to Target Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer

Supplementary Figure 3A-B from Engineered Thio-Trastuzumab-DM1 Conjugate with an Improved Therapeutic Index to Target Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer

Supplementary Figure 1 from Engineered Thio-Trastuzumab-DM1 Conjugate with an Improved Therapeutic Index to Target Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer

Antibody conjugates are used in many therapeutic and research applications and are generated by chemically linking a cysteine or lysine residue to potent chemotherapeutic drugs or other functional groups through a flexible linker. Recently, we have engineered THIOMABs (antibodies with engineered reactive cysteine residues) for site-specific conjugation and showed that these antibody conjugates display homogeneous labeling with optimal in vitro and in vivo characteristics. Here, we describe protocols for engineering, selection, and site-specific conjugation of THIOMABs with thiol-reactive linkers.

Glembatumumab vedotin (CR-011-vc-MMAE) is a mAb-drug conjugate being developed by Celldex Therapeutics Inc for the treatment of glycoprotein non-metastatic melanoma protein B (GPNMB)-expressing cancers. Glembatumumab is a fully human mAb directed against an extracellular domain of GPNMB expressed in human breast cancers and melanomas. Glembatumumab is conjugated to the potent microtubule inhibitor monomethyl auristatin E using a cathepsin cleavable valine-citrulline (vc) dipeptide linker. Glembatumumab vedotin has demonstrated potent antitumor activity in preclinical studies, including in GPNMB-expressing cell lines. In human melanoma xenograft mice, intravenous glembatumumab vedotin was associated with complete tumor regression without significant toxicity. In two phase I/II clinical trials, intravenous glembatumumab vedotin demonstrated antitumor activity in patients with breast cancer or melanoma. Skin rash was the most common toxicity reported, which may have been caused by the expression of GPNMB in healthy skin. Glembatumumab vedotin had a relatively short t(1/2) , prompting the evaluation of more frequent dosing schedules. Prospective, randomized clinical trials will likely be required to determine the therapeutic potential of glembatumumab vedotin in the treatment of breast cancer and melanoma.

Rab11 GTPase is an important regulator of endocytic membrane traffic. In the GTP-bound form Rab GTPases interact with effector proteins and each Rab-effector complex is proposed to regulate a unique trafficking step/event such as vesicle docking, budding, transport, or fusion. At least six Rab11 effectors (family of Rab11 interacting proteins, FIPs) have been identified and shown to interact with Rab11. Based on the sequence homology FIPs are divided in class I and class II subfamilies. Class I FIPs have been hypothesized to regulate the recycling of plasma membrane receptors. In contrast, class II FIPs have been implicated in regulating membrane traffic during more specialized cellular functions, such as cytokinesis. This chapter reviews the background and methodology required for characterizing interactions between FIPs and Rab11, as well as understanding their role in regulating endocytic membrane traffic.