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.

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

Abstract 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.

Antibody-drug conjugates (ADCs) have a significant impact toward the treatment of cancer, as evidenced by the clinical activity of the recently approved ADCs, brentuximab vedotin for Hodgkin lymphoma and ado-trastuzumab emtansine (trastuzumab-MCC-DM1) for metastatic HER2+ breast cancer. DM1 is an analog of the natural product maytansine, a microtubule inhibitor that by itself has limited clinical activity and high systemic toxicity. However, by conjugation of DM1 to trastuzumab, the safety was improved and clinical activity was demonstrated. Here, we report that through chemical modification of the linker-drug and antibody engineering, the therapeutic activity of trastuzumab maytansinoid ADCs can be further improved. These improvements include eliminating DM1 release in the plasma and increasing the drug load by engineering four cysteine residues into the antibody. The chemical synthesis of highly stable linker-drugs and the modification of cysteine residues of engineered site-specific antibodies resulted in a homogeneous ADC with increased therapeutic activity compared to the clinically approved ADC, trastuzumab-MCC-DM1.

Multi-transmembrane proteins are especially difficult targets for antibody generation largely due to the challenge of producing a protein that maintains its native conformation in the absence of a stabilizing membrane. Here, we describe an immunization strategy that successfully resulted in the identification of monoclonal antibodies that bind specifically to extracellular epitopes of a 12 transmembrane protein, multi-drug resistant protein 4 (MRP4). These monoclonal antibodies were developed following hydrodynamic tail vein immunization with a cytomegalovirus (CMV) promoter-based plasmid expressing MRP4 cDNA and were characterized by flow cytometry. As expected, the use of the immune modulators fetal liver tyrosine kinase 3 ligand (Flt3L) and granulocyte-macrophage colony-stimulating factor positively enhanced the immune response against MRP4. Imaging studies using CMV-based plasmids expressing luciferase showed that the in vivo half-life of the target antigen was less than 48 h using CMV-based plasmids, thus necessitating frequent boosting with DNA to achieve an adequate immune response. We also describe a comparison of plasmids, which contained MRP4 cDNA with either the CMV or CAG promoters, used for immunizations. The observed luciferase activity in this comparison demonstrated that the CAG promoter-containing plasmid pCAGGS induced prolonged constitutive expression of MRP4 and an increased anti-MRP4 specific immune response even when the plasmid was injected less frequently. The method described here is one that can be broadly applicable as a general immunization strategy to develop antibodies against multi-transmembrane proteins, as well as target antigens that are difficult to express or purify in native and functionally active conformation.

The intracellular pathogenic bacterium Salmonella enterica serovar typhimurium (Salmonella) relies on acidification of the Salmonella‐containing vacuole (SCV) for survival inside host cells. The transport and fusion of membrane‐bound compartments in a cell is regulated by small GTPases, including Rac and members of the Rab GTPase family, and their effector proteins. However, the role of these components in survival of intracellular pathogens is not completely understood. Here, we identify Nischarin as a novel dual effector that can interact with members of Rac and Rab GTPase (Rab4, Rab14 and Rab9) families at different endosomal compartments. Nischarin interacts with GTP‐bound Rab14 and PI(3)P to direct the maturation of early endosomes to Rab9/CD63‐containing late endosomes. Nischarin is recruited to the SCV in a Rab14‐dependent manner and enhances acidification of the SCV. Depletion of Nischarin or the Nischarin binding partners—Rac1, Rab14 and Rab9 GTPases—reduced the intracellular growth of Salmonella. Thus, interaction of Nischarin with GTPases may regulate maturation and subsequent acidification of vacuoles produced after phagocytosis of pathogens. The intracellular survival of Salmonella requires that the vacuoles containing the pathogen are acidified. Vacuolar maturation and acidification is mediated by Nischarin, a cellular effector of the small GTPases Rac1, Rab14 and Rab9 involved in endosomal trafficking.

The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.

Ciliogenesis involves coordinated assembly of a microtubule-based axoneme from the mother centriole and vesicular membrane transport and fusion forming a ciliary membrane around the developing axoneme. We, and others have reported that a Rab11-Rab8 cascade functions in ciliogenesis. Using live high-resolution fluorescence microscopy imaging we show that ciliary membrane assembly proceeds following Rabin8 (a Rab8 activator) binding to Rab11 membranes. Rabin8 transport via Rab11 vesicles to the centrosome is observed resulting in localized activation of Rab8 and leads to initiation of ciliary membrane assembly. Using proteomics approaches, we have discovered that Rabin8 binds to the TRAPPII tethering complex and find that this interaction is important for Rabin8 centrosomal targeting during ciliogenesis. Our work suggests that Rabin8 membrane transport is a highly regulated process controlled by serum-dependent and serum-independent signaling. Interestingly, following ciliogenesis Rabin8 centrosomal localization is lost resulting in reduced Rab8 activation at the ciliary membrane. This finding along with a previous report describing Rabin8 association with Bardet-Biedl syndrome (BBS) proteins has led us to hypothesize that regulation of centrosomal Rabin8 levels is important for establishing the length of primary cilium, an important factor in ciliary signaling. Finally, we describe the discovery of additional factors associated with the Rab11-Rab8 trafficking pathway that function in organizing membrane structure during ciliogenesis.

Sensory and signaling pathways are exquisitely organized in primary cilia. Bardet-Biedl syndrome (BBS) patients have compromised cilia and signaling. BBS proteins form the BBSome, which binds Rabin8, a guanine nucleotide exchange factor (GEF) activating the Rab8 GTPase, required for ciliary assembly. We now describe serum-regulated upstream vesicular transport events leading to centrosomal Rab8 activation and ciliary membrane formation. Using live microscopy imaging, we show that upon serum withdrawal Rab8 is observed to assemble the ciliary membrane in ∼100 min. Rab8-dependent ciliary assembly is initiated by the relocalization of Rabin8 to Rab11-positive vesicles that are transported to the centrosome. After ciliogenesis, Rab8 ciliary transport is strongly reduced, and this reduction appears to be associated with decreased Rabin8 centrosomal accumulation. Rab11-GTP associates with the Rabin8 COOH-terminal region and is required for Rabin8 preciliary membrane trafficking to the centrosome and for ciliogenesis. Using zebrafish as a model organism, we show that Rabin8 and Rab11 are associated with the BBS pathway. Finally, using tandem affinity purification and mass spectrometry, we determined that the transport protein particle (TRAPP) II complex associates with the Rabin8 NH(2)-terminal domain and show that TRAPP II subunits colocalize with centrosomal Rabin8 and are required for Rabin8 preciliary targeting and ciliogenesis.

Ben-Quan Shen*, Keyang Xu*, Luna Liu, Helga Raab, Sunil Bhakta, Margaret Kenrick, Kathryn L. Parsons-Reponte, Janet Tien, Shang-Fan Yu, Elaine Mai, Dongwei Li, Jay Tibbitts, Jakub Baudys, Ola M. Saad, Suzie J. Scales, Paul J. McDonald, Philip E. Hass, Charles Eigenbrot, Trung Nguyen, Willy A. Solis, Reina N. Fuji, Kelly M. Flagella, Darshana Patel, Susan D. Spencer, Leslie A. Khawli, Allen Ebens, Wai Lee Wong, Richard Vandlen, Surinder Kaur, Mark X. Sliwkowski, Richard H. Scheller, Paul Polakis and Jagath R. Junutula

Abstract Antibody drug conjugates (ADCs) are attractive targeted chemo-therapeutic molecules as they combine ideal properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to the antigen-expressing tumor cells, thereby enhancing their anti-tumor activity. The 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. Recently, we have developed antibodies with cysteine substitutions (THIOMABs) at sites where the engineered cysteines are available for conjugation but do not perturb immunoglobulin folding and assembly or alter antigen binding and effector functions (Junutula, et al., Nature Biotech., 26, 925-932, 2008). These THIOMABs can then be derivatized through the engineered cysteine thiol group to obtain ADCs with uniform stoichiometry (∼2 drugs per antibody). Studies with multiple antibodies against different antigens have shown that THIOMAB drug conjugates (TDCs) are as efficacious as conventional conjugates in xenograft models and are tolerated at higher doses in relevant preclinical models. To further understand improved therapeutic activity and in vivo metabolism of TDCs, we have engineered TDCs with drug attachment at different parts of the antibody (light chain-Fab, heavy chain-Fab and heavy chain-Fc). TDCs produced by THIOMAB technology provided an unique advantage over conventional ADCs to answer several unresolved questions in ADC therapeutics due to their homogeneity and site-specific conjugation to cytotoxic drugs. We will discuss novel findings that were observed with these TDC varaints and the influence of conjugation site in modulating stability and biological activity of antibody drug conjugates. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4396.

Abstract 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.

Cysteines with reactive thiol groups are attractive tools for site-specific labeling of proteins. Engineering a reactive cysteine residue into proteins with multiple disulfide bonds is often a challenging task as it may interfere with structural and functional properties of the protein. Here we developed a phage display-based biochemical assay, PHESELECTOR (Phage ELISA for Selection of Reactive Thiols) to rapidly screen reactive thiol groups on antibody fragments without interfering with their antigen binding, using trastuzumab-Fab (hu4D5Fab) as a model system. The solvent accessibility values for all the amino acid residues in the hu4D5Fab were calculated using available crystal structure information. Serine, alanine and valine residues with highest solvent accessibility values were selected and tested to compare structure-based design with the PHESELECTOR biochemical method. Cysteine substitutions at partially solvent-accessible alanine or valine residues exhibited better thiol reactivity values than substitutions at serine residues. The poor correlation between fractional solvent accessibility and thiol reactivity of the engineered hu4D5Fab variants indicated the value of PHESELECTOR biochemical assay to identify reactive thiol groups on the antibody-Fab surface. Mass spectrometric analysis of biotinylated ThioFab (Fab with engineered cysteine) variants confirmed that conjugation occurred only at the engineered cysteine thiols of either light or heavy chains. ThioFabs with engineered cysteine residues in the constant domains (CL and CH(1)) should allow universal application for site-specific conjugation of antibody-Fabs.

Ultimately, we somehow managed to end up with something that looked reasonably pure, and we sent an unsettlingly large portion of sample to Phil Andrews at Purdue University for amino acid sequence analysis. At this point, one of us was more than a little nervous—after all, John hadn't graduated yet. One afternoon John came back from lunch, and there was a small piece of folded yellow paper on his desk. John was told that it was a message from Phil and that he would know what it meant. Indeed, that was the case when he opened the note and all it said was DPKQDFMRFH2. It was the amino acid sequence of the fly FMRFamide neuropeptide. Analysis of cDNA clones led to the determination that the Drosophila FMRFamide precursor is also quite complex, as it contained five complete copies of DPKQDFMRFamide as well as ten re- lated peptides. In an amazing example of synchronicity, Paul Taghert's laboratory at Washington University had simulta- neously identified the Drosophila FMRFamide gene. Both Ri- chard and Paul's lab went on to explore the function and regu- lation of this gene, and since then, many other neuropeptides and their receptors have been characterized using Drosophila molecular genetics. Indeed, such studies will be among the topics at an upcoming meeting on Insect Neuromodulators and Neuropeptides at Janelia Farms.

Jagath R Junutula, Helga Raab, Suzanna Clark, Sunil Bhakta, Douglas D Leipold, Sylvia Weir, Yvonne Chen, Michelle Simpson, Siao Ping Tsai, Mark S Dennis, Yanmei Lu, Y. Gloria Meng, Carl Ng, Jihong Yang, Chien C Lee, Eileen Duenas, Jeffrey Gorrell, Viswanatham Katta, Amy Kim, Kevin McDorman, Kelly Flagella, Rayna Venook, Sarajane Ross, Susan D Spencer, Wai Lee Wong, Henry B Lowman, Richard Vandlen, Mark X Sliwkowski, Richard H Scheller, Paul Polakis and William Mallet

We have purified a 9 amino acid amidated neuropeptide, DPKQDFMRFamide, from whole adult D. melanogaster. This peptide exhibits sequence homology to the molluscan bioactive tetrapeptide FMRFamide and is a novel member of the FMRFamide peptide family. The gene encoding DPKQDFMRFamide has been cloned and characterized. It is present in a single copy per haploid genome, is expressed as a unique 1.7 kb mRNA species, and cytologically maps to 46C on the right arm of chromosome 2. Characterization of a cDNA clone indicates that the precursor protein is 347 amino acids in length and contains 5 copies of DPKQDFMRFamide, as well as 10 additional amidated peptides exhibiting varying degrees of structural relatedness. The Drosophila DPKQDFMRFamide gene and the Aplysia FMRFamide gene are ancestrally related; however, peptides display a higher degree of homology within a species than between species, suggesting intragenic concerted evolution of these neuropeptides.