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STRO-002 is a novel homogeneous folate receptor alpha (FolRα) targeting antibody–drug conjugate (ADC) currently being investigated in the clinic as a treatment for ovarian and endometrial cancers. Here, we describe the discovery, optimization, and antitumor properties of STRO-002. STRO-002 was generated by conjugation of a novel cleavable 3-aminophenyl hemiasterlin linker-warhead (SC239) to the nonnatural amino acid para-azidomethyl-L-phenylalanine incorporated at specific positions within a high affinity anti-FolRα antibody using Sutro's XpressCF+, which resulted in a homogeneous ADC with a drug–antibody ratio (DAR) of 4. STRO-002 binds to FolRα with high affinity, internalizes rapidly into target positive cells, and releases the tubulin-targeting cytotoxin 3-aminophenyl hemiasterlin (SC209). SC209 has reduced potential for drug efflux via P-glycoprotein 1 drug pump compared with other tubulin-targeting payloads. While STRO-002 lacks nonspecific cytotoxicity toward FolRα-negative cell lines, bystander killing of target negative cells was observed when cocultured with target positive cells. STRO-002 is stable in circulation with no change in DAR for up to 21 days and has a half-life of 6.4 days in mice. A single dose of STRO-002 induced significant tumor growth inhibition in FolRα-expressing xenograft models and patient-derived xenograft models. In addition, combination treatment with carboplatin or Avastin further increased STRO-002 efficacy in xenograft models. The potent and specific preclinical efficacy of STRO-002 supports clinical development of STRO-002 for treating patients with FolRα-expressing cancers, including ovarian, endometrial, and non–small cell lung cancer. Phase I dose escalation for STRO-002 is in progress in ovarian cancer and endometrial cancer patients (NCT03748186 and NCT05200364).

Supplementary Figure 1. Standard curves for quantification of cellular integrin expression; Supplementary Figure 2. Competition binding to U87MG tumor cell surface integrins; Supplementary Figure 3. Inhibition of tumor cell proliferation. U87MG, A2780 and MB-468 cells were incubated with varying concentrations of 2.5F-Fc-MMAF, and CTRL-Fc-MMAF;Supplementary Figure 4. Growth inhibition of subcutaneous U87MG tumors implanted into Nu/Nu mice; Supplementary Figure 5. Weight monitoring.

Bispecific antibodies have emerged as a promising strategy for curtailing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune escape. This brief report highlights RBT-0813 (also known as TB493-04), a synthetic, humanized, receptor-binding domain (RBD)-targeted bispecific antibody that retains picomolar affinity to the Spike (S) trimers of all major variants of concern and neutralizes both SARS-CoV-2 Delta and Omicron in vitro.

Coronavirus disease 2019 (COVID-19) is an evolving global public health crisis in need of therapeutic options. Passive immunization of monoclonal antibodies (mAbs) represents a promising therapeutic strategy capable of conferring immediate protection from SARS-CoV-2 infection. Herein, we describe the discovery and characterization of neutralizing SARS-CoV-2 IgG and VHH antibodies from four large-scale phage libraries. Each library was constructed synthetically with shuffled complementarity-determining region loops from natural llama and human antibody repertoires. While most candidates targeted the receptor-binding domain of the S1 subunit of SARS-CoV-2 spike protein, we also identified a neutralizing IgG candidate that binds a unique epitope on the N-terminal domain. A select number of antibodies retained binding to SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa and Delta. Overall, our data show that synthetic phage libraries can rapidly yield SARS-CoV-2 S1 antibodies with therapeutically desirable features, including high affinity, unique binding sites, and potent neutralizing activity in vitro, and a capacity to limit disease in vivo.

Antibody-based therapeutics and vaccines are essential to combat COVID-19 morbidity and mortality after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Multiple mutations in SARS-CoV-2 that could impair antibody defenses propagated in human-to-human transmission and spillover or spillback events between humans and animals. To develop prevention and therapeutic strategies, we formed an international consortium to map the epitope landscape on the SARS-CoV-2 spike protein, defining and structurally illustrating seven receptor binding domain (RBD)–directed antibody communities with distinct footprints and competition profiles. Pseudovirion-based neutralization assays reveal spike mutations, individually and clustered together in variants, that affect antibody function among the communities. Key classes of RBD-targeted antibodies maintain neutralization activity against these emerging SARS-CoV-2 variants. These results provide a framework for selecting antibody treatment cocktails and understanding how viral variants might affect antibody therapeutic efficacy.

Background Development of successful neutralizing antibodies is dependent upon broad epitope coverage to increase the likelihood of achieving therapeutic function. Recent advances in synthetic biology have allowed us to conduct an epitope binning study on a large panel of antibodies identified to bind to Ebola virus glycoprotein with only published sequences. Methods and Results A rapid, first-pass epitope binning experiment revealed seven distinct epitope families that overlapped with known structural epitopes from the literature. A focused set of antibodies was selected from representative clones per bin to guide a second-pass binning that revealed previously unassigned epitopes, confirmed epitopes known to be associated with neutralizing antibodies, and demonstrated asymmetric blocking of EBOV GP from allosteric effectors reported from literature. Conclusions Critically, this workflow allows us to probe the epitope landscape of EBOV GP without any prior structural knowledge of the antigen or structural benchmark clones. Incorporating epitope binning on hundreds of antibodies during early stage antibody characterization ensures access to a library’s full epitope coverage, aids in the identification of high quality reagents within the library that recapitulate this diversity for use in other studies, and ultimately enables the rational development of therapeutic cocktails that take advantage of multiple mechanisms of action such as cooperative synergistic effects to enhance neutralization function and minimize the risk of mutagenic escape. The use of high-throughput epitope binning during new outbreaks such as the current COVID-19 pandemic is particularly useful in accelerating timelines due to the large amount of information gained in a single experiment., Starting with only published antibody sequences and soluble Ebola virus glycoprotein, high-throughput surface plasmon resonance is employed to rapidly screen and epitope bin hundreds of antibodies to fully characterize the epitope coverage of antibodies identified from a convalescent donor.

Malaria, one of the most common vector borne human diseases, is a major world health issue. In 2015 alone, more than 200 million people were infected with malaria, out of which, 429 000 died. Even though artemisinin-based combination therapies (ACT) are highly effective at treating malaria infections, novel efforts toward development of vaccines to prevent transmission are still needed. Pfs25, a postfertilization stage parasite surface antigen, is a leading transmission-blocking vaccine (TBV) candidate. It is postulated that Pfs25 anchors to the cell membrane using a glycosylphosphatidylinositol (GPI) linker, which itself possesses pro-inflammatory properties. In this study, Escherichia coli derived extract (XtractCF+TM) was used in cell free protein synthesis [CFPS] to successfully express >200 mg/L of recombinant Pfs25 with a C-terminal non-natural amino acid (nnAA), namely, p-azidomethyl phenylalanine (pAMF), which possesses a reactive azide group. Thereafter, a unique conjugate vaccine (CV), namely, Pfs25-GPI was generated with dibenzocyclooctyne (DBCO) derivatized glycan core of malaria GPI using a simple but highly efficient copper free click chemistry reaction. In mice immunized with Pfs25 or Pfs25-GPI, the Pfs25-GPI group showed significantly higher titers compared to the Pfs25 group. Moreover, only purified IgGs from Pfs25-GPI group were able to significantly block transmission of parasites to mosquitoes, as judged by a standard membrane feeding assay [SMFA]. To our knowledge, this is the first report of the generation of a CV using Pfs25 and malaria specific GPI where the GPI is shown to enhance the ability of Pfs25 to elicit transmission blocking antibodies.

Amber codon suppression for the insertion of non-natural amino acids (nnAAs) is limited by competition with release factor 1 (RF1). Here we describe the genome engineering of a RF1 mutant strain that enhances suppression efficiency during cell-free protein synthesis, without significantly impacting cell growth during biomass production. Specifically, an out membrane protease (OmpT) cleavage site was engineered into the switch loop of RF1, which enables its conditional inactivation during cell lysis. This facilitates extract production without additional processing steps, resulting in a scaleable extract production process. The RF1 mutant extract allows nnAA incorporation at previously intractable sites of an IgG1 and at multiple sites in the same polypeptide chain. Conjugation of cytotoxic agents to these nnAAs, yields homogeneous antibody drug conjugates (ADCs) that can be optimized for conjugation site, drug to antibody ratio (DAR) and linker-warheads designed for efficient tumor killing. This platform provides the means to generate therapeutic ADCs inaccessible by other methods that are efficient in their cytotoxin delivery to tumor with reduced dose-limiting toxicities and thus have the potential for better clinical impact.

Antibody drug conjugates (ADCs) harness the target specificity of a monoclonal antibody (mAb) and the high cytotoxicity of a small molecule, enabling improved delivery of a potent antitumor agent compared to traditional chemotherapy for cancer therapy. Only two ADCs have been marketed, both of which are produced via nonsite-specific conjugation of the cytotoxic drug to either interchain cysteine (Adcetris) or lysine (Kadcyla). A growing body of evidence suggests that site-specific ADCs, because of their payload homogeneity, will improve pharmacokinetics and have wider therapeutic windows when compared to heterogeneous ADCs. Previously, we have demonstrated the use of a cell free expression system (Xpress CF+) for rapid production of site-specific ADCs. Here we report the generation of a variety of ADCs via conjugation between a site-specific incorporated non-natural amino acid (nnAA), para-azidomethyl-l-phenylalanine (pAMF), and dibenzocyclooctyl-(polyethylene glycol)4 (DBCO-(PEG)4) linked payloads using ...

Antibody–drug conjugates (ADC) have generated significant interest as targeted therapeutics for cancer treatment, demonstrating improved clinical efficacy and safety compared with systemic chemotherapy. To extend this concept to other tumor-targeting proteins, we conjugated the tubulin inhibitor monomethyl-auristatin-F (MMAF) to 2.5F–Fc, a fusion protein composed of a human Fc domain and a cystine knot (knottin) miniprotein engineered to bind with high affinity to tumor-associated integrin receptors. The broad expression of integrins (including αvβ3, αvβ5, and α5β1) on tumor cells and their vasculature makes 2.5F-Fc an attractive tumor-targeting protein for drug delivery. We show that 2.5F-Fc can be expressed by cell-free protein synthesis, during which a non-natural amino acid was introduced into the Fc domain and subsequently used for site-specific conjugation of MMAF through a noncleavable linker. The resulting knottin–Fc–drug conjugate (KFDC), termed 2.5F-Fc-MMAF, had approximately 2 drugs attached per KFDC. 2.5F–Fc–MMAF inhibited proliferation in human glioblastoma (U87MG), ovarian (A2780), and breast (MB-468) cancer cells to a greater extent than 2.5F–Fc or MMAF alone or added in combination. As a single agent, 2.5F–Fc–MMAF was effective at inducing regression and prolonged survival in U87MG tumor xenograft models when administered at 10 mg/kg two times per week. In comparison, tumors treated with 2.5F–Fc or MMAF were nonresponsive, and treatment with a nontargeted control, CTRL–Fc–MMAF, showed a modest but not significant therapeutic effect. These studies provide proof-of-concept for further development of KFDCs as alternatives to ADCs for tumor targeting and drug delivery applications. Mol Cancer Ther; 15(6); 1291–300. ©2016 AACR.

Sudan virus (SUDV) and Ebola viruses (EBOV) are both members of the Ebolavirus genus and have been sources of epidemics and outbreaks for several decades. We present here the generation and characterization of cross-reactive antibodies to both SUDV and EBOV, which were produced in a cell-free system and protective against SUDV in mice. A non-human primate, cynomolgus macaque, was immunized with viral-replicon particles expressing the glycoprotein of SUDV-Boniface (8A). Two separate antibody fragment phage display libraries were constructed after four immunogen injections. Both libraries were screened first against the SUDV and a second library was cross-selected against EBOV-Kikwit. Sequencing of 288 selected clones from the two distinct libraries identified 58 clones with distinct VH and VL sequences. Many of these clones were cross-reactive to EBOV and SUDV and able to neutralize SUDV. Three of these recombinant antibodies (X10B1, X10F3, and X10H2) were produced in the scFv-Fc format utilizing a cell-free production system. Mice that were challenged with SUDV-Boniface receiving 100µg of the X10B1/X10H2 scFv-Fc combination 6 and 48-h post-exposure demonstrated partial protection individually and complete protection as a combination. The data herein suggests these antibodies may be promising candidates for further therapeutic development.

Antibody-drug conjugates (ADCs) are antigen-targeted therapeutics that employ antibodies to deliver potent, cytotoxic effectors to cells with potentially high specificity. While promising clinical results have been achieved, significant pitfalls remain including internalization of ADCs in nontargeted cells expressing target antigen, which can limit therapeutic windows. Novel ADC linkers that are cleaved selectively in cancer cells but not in normal cells could minimize collateral damage caused by ADC uptake in nontargeted tissues. Here, we describe a prototypical ADC linker based on an Fe(II)-reactive 1,2,4-trioxolane scaffold (TRX) that by itself has demonstrated tumor-selective activity in preclinical cancer models. We prepared TRX-linked ADCs by site-selective conjugation to two sites in trastuzumab and compared their activity in Her2 positive and negative cells to ADC controls based on established linker chemistry. Our results confirm that the TRX moiety efficiently releases its payload following ADC uptake, affording picomolar potencies in antigen-positive cells. We also identified a destabilizing interaction between these initial TRX linkers and nearby antibody residues and suggest an approach to improve upon these prototypical designs.

Cell‐free protein synthesis (CFPS) systems allow for robust protein expression with easy manipulation of conditions to improve protein yield and folding. Recent technological developments have significantly increased the productivity and reduced the operating costs of CFPS systems, such that they can compete with conventional in vivo protein production platforms, while also offering new routes for the discovery and production of biotherapeutics. As cell‐free systems have evolved, productivity increases have commonly been obtained by addition of components to previously designed reaction mixtures without careful re‐examination of the essentiality of reagents from previous generations. Here we present a systematic sensitivity analysis of the components in a conventional Escherichia coli CFPS reaction mixture to evaluate their optimal concentrations for production of the immunoglobulin G trastuzumab. We identify eight changes to the system, which result in optimal expression of trastuzumab. We find that doubling the potassium glutamate concentration, while entirely eliminating pyruvate, coenzyme A, NAD, total tRNA, folinic acid, putrescine and ammonium glutamate, results in a highly productive cell‐free system with a 95% reduction in reagent costs (excluding cell‐extract, plasmid, and T7 RNA polymerase made in‐house). A larger panel of other proteins was also tested and all show equivalent or improved yields with our simplified system. Furthermore, we demonstrate that all of the reagents for CFPS can be combined in a single freeze‐thaw stable master mix to improve reliability and ease of use. These improvements are important for the application of the CFPS system in fields such as protein engineering, high‐throughput screening, and biotherapeutics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:823–831, 2015

Antibody drug conjugates (ADCs) have progressed from hypothesis to approved therapeutics in less than 30 years, and the technologies available to modify both the antibodies and the cytotoxic drugs are expanding rapidly. For reasons well reviewed previously, the field is trending strongly toward homogeneous, defined antibody conjugation. In this review we present the antibody and small molecule chemistries that are currently used and being explored to develop specific, homogenous ADCs.

Protein engineering projects often amass numerous raw DNA sequences, but no readily available software combines sequence processing and activity correlation required for efficient lead identification. XLibraryDisplay is an open source program integrated into Microsoft Excel for Windows that automates batch sequence processing via a simple step-by-step, menu-driven graphical user interface. XLibraryDisplay accepts any DNA template which is used as a basis for trimming, filtering, translating, and aligning hundreds to thousands of sequences (raw, FASTA, or Phred PHD file formats). Key steps for library characterization through lead discovery are available including library composition analysis, filtering by experimental data, graphing and correlating to experimental data, alignment to structural data extracted from PDB files, and generation of PyMOL visualization scripts. Though larger data sets can be handled, the program is best suited for analyzing approximately 10 000 or fewer leads or naïve clones which have been characterized using Sanger sequencing and other experimental approaches. XLibraryDisplay can be downloaded for free from sourceforge.net/projects/xlibrarydisplay/ .

Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF.

Antibodies are well-established as therapeutics, and the preclinical and clinical pipeline of these important biologics is growing rapidly. Consequently, there is considerable interest in technologies to engineer and manufacture them. Mammalian cell culture is commonly used for production because eukaryotic expression systems have evolved complex and efficient chaperone systems for the folding of antibodies. However, given the ease and manipulability of bacteria, antibody discovery efforts often employ bacterial expression systems despite their limitations in generating high titers of functional antibody. Open-Cell Free Synthesis (OCFS) is a coupled transcription-translation system that has the advantages of prokaryotic systems while achieving high titers of antibody expression. Due to the open nature of OCFS, it is easily modified by chemical or protein additives to improve the folding of select proteins. As such, we undertook a protein additive screen to identify chaperone proteins that improve the folding and assembly of trastuzumab in OCFS. From the screen, we identified the disulfide isomerase DsbC and the prolyl isomerase FkpA as important positive effectors of IgG folding. These periplasmic chaperones function synergistically for the folding and assembly of IgG, and, when present in sufficient quantities, gram per liter IgG titers can be produced. This technological advancement allows the rapid development and manufacturing of immunoglobulin proteins and pushes OCFS to the forefront of production technologies for biologics.

Antibody-drug conjugates (ADCs) are a targeted chemotherapeutic currently at the cutting edge of oncology medicine. These hybrid molecules consist of a tumor antigen-specific antibody coupled to a chemotherapeutic small molecule. Through targeted delivery of potent cytotoxins, ADCs exhibit improved therapeutic index and enhanced efficacy relative to traditional chemotherapies and monoclonal antibody therapies. The currently FDA-approved ADCs, Kadcyla (Immunogen/Roche) and Adcetris (Seattle Genetics), are produced by conjugation to surface-exposed lysines, or partial disulfide reduction and conjugation to free cysteines, respectively. These stochastic modes of conjugation lead to heterogeneous drug products with varied numbers of drugs conjugated across several possible sites. As a consequence, the field has limited understanding of the relationships between the site and extent of drug loading and ADC attributes such as efficacy, safety, pharmacokinetics, and immunogenicity. A robust platform for rapid production of ADCs with defined and uniform sites of drug conjugation would enable such studies. We have established a cell-free protein expression system for production of antibody drug conjugates through site-specific incorporation of the optimized non-natural amino acid, para-azidomethyl-l-phenylalanine (pAMF). By using our cell-free protein synthesis platform to directly screen a library of aaRS variants, we have discovered a novel variant of the Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS), with a high activity and specificity toward pAMF. We demonstrate that site-specific incorporation of pAMF facilitates near complete conjugation of a DBCO-PEG-monomethyl auristatin (DBCO-PEG-MMAF) drug to the tumor-specific, Her2-binding IgG Trastuzumab using strain-promoted azide-alkyne cycloaddition (SPAAC) copper-free click chemistry. The resultant ADCs proved highly potent in in vitro cell cytotoxicity assays.

The Wnt/β-catenin pathway, which signals through the Frizzled (Fzd) receptor family and several coreceptors, has long been implicated in cancer. Here we demonstrate a therapeutic approach to targeting the Wnt pathway with a monoclonal antibody, OMP-18R5. This antibody, initially identified by binding to Frizzled 7, interacts with five Fzd receptors through a conserved epitope within the extracellular domain and blocks canonical Wnt signaling induced by multiple Wnt family members. In xenograft studies with minimally passaged human tumors, this antibody inhibits the growth of a range of tumor types, reduces tumor-initiating cell frequency, and exhibits synergistic activity with standard-of-care chemotherapeutic agents.

The receptor tyrosine kinase MET is a major component controlling the invasive growth program in embryonic development and in invasive malignancies. The discovery of therapeutic antibodies against MET has been difficult, and antibodies that compete with hepatocyte growth factor (HGF) act as agonists. By applying phage technology and cell-based panning strategies, we discovered two fully human antibodies against MET (R13 and R28), which synergistically inhibit HGF binding to MET and elicit antibody-dependent cellular cytotoxicity. Cell-based phosphorylation assays demonstrate that R13 and R28 abrogate HGF-induced activation of MET, AKT1, ERK1/2, and HGF-induced migration and proliferation. FACS experiments suggest that the inhibitory effect is mediated by “locking” MET receptor in a state with R13, which then increases avidity of R28 for the extracellular domain of MET, thus blocking HGF binding without activating the receptor. In vivo studies demonstrate that the combination of R13/28 significantly inhibited tumor growth in various colon tumor xenograft models. Inhibition of tumor growth was associated with induction of hypoxia. Global gene expression analysis shows that inhibition of HGF/MET pathway significantly upregulated the tumor suppressors KLF6, CEACAM1, and BMP2, the negative regulator of phosphatidylinositol-3-OH-kinase PIK3IP1, and significantly suppressed SCF and SERPINE2, both enhancers of proliferation and invasiveness. Moreover, in an experimental metastasis model, R13/28 increased survival by preventing the recurrence of otherwise lethal lung metastases. Taken together, these results underscore the utility of a dual-antibody approach for targeting MET and possibly other receptor tyrosine kinases. Our approach could be expanded to drug discovery efforts against other cell surface proteins.

ADAMTS-4 and ADAMTS-5 are aggrecanases responsible for the breakdown of cartilage aggrecan in osteoarthritis. Multiple ADAMTS-4 cleavage sites have been described in several matrix proteins including aggrecan, versican, and brevican, but no concise predictive cleavage motif has been identified for this protease. By screening a 13-mer peptide library with a diversity of 10(8), we have identified the ADAMTS-4 cleavage motif E-(AFVLMY)-X(0,1)-(RK)-X(2,3)-(ST)-(VYIFWMLA), with Glu representing P1. Several 13-mer peptides containing this motif, including DVQEFRGVTAVIR and HNEFRQRETYMVF, were shown to be substrates for ADAMTS-4. These peptides were found to be specific substrates for ADAMTS-4 as they were not cleaved by ADAMTS-5. Modification of these peptides with donor (6-FAM) and acceptor (QSY-9) molecules resulted in the development of fluorescence-based substrates with a Km of approximately 35 microM. Furthermore, the role of Glu at P1 and Phe at P1' in binding and catalysis was studied by exploring substitution of these amino acids with the D-isomeric forms. Substitution of P1 with dGlu was tolerable for binding, but not catalysis, whereas substitution of P1' with dPhe precluded both binding and catalysis. Similarly, replacement of Glu with Asp at P1 abolished recognition and cleavage of the peptide. Finally, BLAST results of the ADAMTS-4 cleavage motif identified matrilin-3 as a new substrate for ADAMTS-4. When tested, recombinant ADAMTS-4 effectively cleaved intact matrilin-3 at the predicted motif at Glu435/Ala436 generating two species of 45 and 5 kDa.

As potential therapeutics, peptides offer several advantages over small molecules (increased specificity) and antibodies (small size). Nevertheless, a number of key issues have hampered their use as drug candidates. A series of new technologies have recently been developed that allow peptides to be viable drug candidates in areas usually restricted to protein therapeutics, such as monoclonal antibodies. These include the development of various types of peptide-conjugates that have lower rates of clearance and hence the potential to increase the exposure of peptide drug candidates in chronic diseases. Structural additions have also been made to peptides, including the use of unnatural amino acids, mainchain modifications and other novel substitutions, which have helped to improve peptide stability and further their therapeutic potential.

Phage display technologies have been increasingly utilized for the generation of therapeutic, imaging and purification reagents for a number of biological targets. Using a variety of different approaches, we have developed antibodies with high specificity and affinity for various targets ranging from small peptides to large proteins, soluble or membrane-associated as well as to activated forms of enzymes. We have applied this approach to G-protein coupled receptors (GPCRs), often considered difficult targets for antibody therapeutics and targeting. Here we demonstrate the use of this technology for the identification of human antibodies targeting C5aR, the chemoattractant GPCR receptor for anaphylatoxin C5a. The N-terminal region (residues 1-31) of C5aR, one of the ligand binding sites, was synthesized, biotinylated and used as the target for selection. Three rounds of selection with our proprietary human Fab phage display library were performed. Screening of 768 isolates by phage ELISA identified 374 positive clones. Based on sequence alignment analysis, the positive clones were divided into 22 groups. Representative Fab clones from each group were reformatted into IgGs and tested for binding to C5aR-expressing cells, the differentiated U-937 cells. Flow cytometric analysis demonstrated that nine out of 16 reformatted IgGs bound to cells. Competition with a C5aR monoclonal antibody S5/1 which recognizes the same N-terminal region showed that S5/1 blocked the binding of positive cell binders to the peptide used for selections, indicating that the identified cell binding IgGs were specific to C5aR. These antibody binders represent viable candidates as therapeutic or imaging agents, illustrating that phage display technology provides a rapid means for developing antibodies to a difficult class of targets such as GPCRs.

Bispecific antibodies have emerged in recent years as a promising field of research for therapies in oncology, inflammable diseases, and infectious diseases. Their capability of dual target recognition allows for novel therapeutic hypothesis to be tested, where traditional mono-specific antibodies would lack the needed mode of target engagement. Among extremely diverse architectures of bispecific antibodies, knobs-into-holes (KIHs) technology, which involves engineering CH3 domains to create either a "knob" or a "hole" in each heavy chain to promote heterodimerization, has been widely applied. Here, we describe the use of a cell-free expression system (Xpress CF) to produce KIH bispecific antibodies in multiple scaffolds, including 2-armed heterodimeric scFv-KIH and one-armed asymmetric BiTE-KIH with tandem scFv. Efficient KIH production can be achieved by manipulating the plasmid ratio between knob and hole, and further improved by addition of prefabricated knob or hole. These studies demonstrate the versatility of Xpress CF in KIH production and provide valuable insights into KIH construct design for better assembly and expression titer.

The human class II major histocompatibility complex protein HLA-DR1 has been shown previously to undergo a distinct conformational change from an open to a compact form upon binding peptide. To investigate the role of peptide in triggering the conformational change, the minimal requirements for inducing the compact conformation were determined. Peptides as short as two and four residues, which occupy only a small fraction of the peptide-binding cleft, were able to induce the conformational change. A mutant HLA-DR1 protein with a substitution in the beta subunit designed to fill the P1 pocket from within the protein (Gly(86) to Tyr) adopted to a large extent the compact, peptide-bound conformation. Interactions important in stabilizing the compact conformation are shown to be distinct from those responsible for high affinity binding or for stabilization of the complex against thermal denaturation. The results suggest that occupancy of the P1 pocket is responsible for partial conversion to the compact form but that both side chain and main chain interactions contribute to the full conformational change. The implications of the conformational change to intracellular antigen loading and presentation are discussed.

In antigen presentation to CD4+T cells, proteins are degraded to peptide fragments and loaded onto class II MHC molecules in a process involving the peptide exchange factors H-2M (murine) or HLA-DM (human). In many antigen-presenting cells these processes occur in intracellular endosomal compartments, where peptides are generated and loaded onto class II MHC proteins for subsequent transport to the surface and presentation to T cells. Here, we provide evidence for an additional antigen-processing pathway in immature dendritic cells (DC). Immature DC express at the cell surface empty or peptide-receptive class II MHC molecules, as well as H-2M or HLA-DM. Secreted DC proteases act extracellularly to process intact proteins into antigenic peptides. Peptides produced by such activity are efficiently loaded onto cell surface class II MHC molecules. Together these elements comprise an unusual extracellular presentation pathway in which antigen processing and peptide loading can occur entirely outside of the cell.

A monoclonal antibody specific for the empty conformation of class II MHC molecules revealed the presence of abundant empty molecules on the surface of spleen- and bone marrow-derived dendritic cells (DC) among various types of antigen-presenting cells. The empty class II MHC molecules are developmentally regulated and expressed predominantly on immature DC. They can capture peptide antigens directly from the extracellular medium and present bound peptides to antigen-specific T lymphocytes. The ability of the empty cell-surface class II MHC proteins to bind peptides and present them to T cells without intracellular processing can serve to extend the spectrum of antigens able to be presented by DC, consistent with their role as sentinels in the immune system.

In class II major histocompatibility complex (MHC) proteins, residue beta57 is usually aspartic acid. Alleles carrying serine, valine, or alanine at this position are strongly correlated with the development of insulin-dependent diabetes mellitus (IDDM). Asp(beta)57 participates in a conserved salt bridge that bridges the alpha and beta subunits in the peptide-binding site. It has been proposed that the correlation between IDDM and MHC alleles lacking Asp(beta)57 may be due to an instability of the protein caused by loss of this salt bridge. Using a pair of HLA-DQ proteins (alpha1*0201, beta1*0302) and (alpha1*0201, beta1*0303) differing only in having aspartic acid or alanine at position beta57, we show that the polymorphism does not have a significant effect on protein stability for either the empty or peptide-loaded forms. However, the circular dichroism spectra indicate that empty and peptide-loaded Alabeta57 proteins display slightly different secondary structures relative to their Aspbeta57 counterparts. A set of three peptides shows different binding affinities for DQ(alpha1*0201, beta1*0302) relative to DQ(alpha1*0201, beta1*0303). We propose that substitution of Asp(beta)57 residue causes a local rearrangement within the DQ peptide-binding site that alters the peptide-binding specificity. This rearrangement may help to explain the previously observed differences in SDS stability between Asp and non-Asp(beta)57 DQ proteins.

To investigate a conformational change accompanying peptide binding to class II MHC proteins, we probed the structure of a soluble version of the human class II MHC protein HLA-DR1 in empty and peptide-loaded forms. Peptide binding induced a large decrease in the effective radius of the protein as determined by gel filtration, dynamic light scattering, and analytical ultracentrifugation. It caused a substantial increase in the cooperativity of thermal denaturation and induced alterations in MHC polypeptide backbone structure as determined by circular dichroism. These changes suggest a condensation of the protein around the bound peptide. An antibody specific for beta58-69 preferentially bound the empty protein, indicating that the peptide-induced conformational change involves the beta-subunit helical region. The conformational change may have important implications for the mechanisms of intracellular antigen presentation pathways.

The human class II major histocompatibility complex protein HLA-DR1 has been expressed in Escherichia coli as denatured alpha and beta subunits and folded in vitro to form the native structure. DR1 folding yields are 30-50% in the presence or absence of tight-binding antigenic peptides. The protein produced in this manner is soluble and monomeric with the expected apparent molecular weight. It reacts with conformation-sensitive anti-DR antibodies and exhibits peptide-dependent resistance to SDS-induced chain dissociation and to proteolysis as does the native protein. The observed peptide specificity and dissociation kinetics are similar to those of native DR produced in B-cells and finally the protein exhibits circular dichroism spectra and cooperative thermal denaturation as expected for a folded protein. We conclude that the recombinant DR1 has adopted the native fold. We have folded DR1 in the absence of peptide and isolated a soluble, peptide-free alphabeta-heterodimer. The empty DR1 can bind antigenic peptide but exhibits altered far UV-circular dichroism and thermal denaturation relative to the peptide-bound form.

CD74, also known as HLA-DR-associated invariant chain, is a type II transmembrane glycoprotein highly expressed in many B-cell malignancies. The limited expression of CD74 in normal tissues suggests it may be a suitable ADC target for these tumor types. Accordingly, we engineered an anti-CD74 human IgG1 antibody (SP7219) using novel Fab-based ribosome display methods. The selected Fabs were readily reformatted and directly screened as IgGs using Sutro's unique high-throughput, cell-free protein synthesis platform, Xpress CFTM. We then developed novel, potent ADCs, SP7676 and SP7675 (STRO-001), comprised of our lead antibody (SP7219) conjugated to non-cleavable DBCO-maytansinoid linker-warheads with an average drug-antibody ratios (DAR) of 2. We used site-specific conjugation technology which results in a high degree of homogeneity characterized by the drug linker covalently binding to a single defined site. The sites for conjugation were selected based on highest cell killing activity and stablity in vitro and in vivo. Both ADCs demonstrate potent cell killing activity across multiple B-cell tumor lines in vitro, and anti-tumor activity in preclinical multiple myeloma xenograft models. In vitro cytotoxicity assays show nanomolar potency of STRO-001 in four MM cell lines: Mc/CAR (IC50 0.8 nM), MM.1S (IC50 10-11 nM), U266B1 (IC50 8.5 -9.3 nM), and ARP-1 (IC50 4.3-22 nM). CD74 cell surface expression is required for ADC anti-proliferative effect but the expression level does not seem to correlate with in vitro potency. SP7676 elicited a robust anti-tumor response in the ANBL-6 multiple myeloma xenograft model. Durable regressions were observed in all mice at ≥ 3 mg/kg, with equivalent efficacy (regression) at 3 mg/kg (every 3 days x5) and 10 mg/kg (every 3 days x5 or weekly x3). SP7676 also elicited a clear survival benefit in a disseminated multiple myeloma CAG xenograft model starting at 1mg/kg every 3 days x 5 doses. Similarly, both SP7676 and STRO-001 inhibited the formation of internal visceral tumors in the ARP-1 xenograft model after 3 weekly doses of 3 mg/kg. Evaluation of our lead candidate, STRO-001 in additional MM cell lines and primary patient samples is planned. The tolerability of STRO-001 in non-human primates is under evaluation. STRO-001 was administered to cynomolgous monkeys in an exploratory dose-escalating study up to 30 mg/kg x 2 doses on Day 1 and 15. STRO-001 reduces normal B-cell populations at ≥1 mg/kg after a single dose, providing pharmacodynamic evidence of B-cell targeting while other hematopoietic lineages are mostly affected only at the highest dose studied. Anticipated hematologic toxicities were readily reversible at 1, 3 and 10 mg/kg and target organs of interest were identified. Based on these encouraging data, STRO-001 is advancing to IND-enabling studies for the treatment of CD74 expressing multiple myeloma and other B-cell malignancies. Disclosures Abrahams: Sutro Biopharma: Employment. Li:Sutro Biopharma: Employment. Yu:Sutro Biopharma: Employment. Krimm:Sutro Biopharma: Employment. Kahana:Celgene: Employment. Narla:Celgene: Employment. Schwartz:Celgene: Employment. Boylan:Celgene: Employment. Hoffmann:Sutro Biopharma: Employment. Steiner:Sutro Biopharma: Employment. Zawada:Sutro Biopharma: Employment. Stephenson:Sutro Biopharma: Employment. Bruhns:Sutro Biopharma: Employment. DeAlmeida:Sutro Biopharma: Employment. Matheny:Sutro Biopharma: Employment. Bussell:Sutro Biopharma: Employment. Galan:Sutro Biopharma: Employment. Kline:Sutro Biopharma: Employment. Vasquez:Sutro Biopharma: Employment. Yam:Sutro Biopharma: Employment. Stafford:Sutro Biopharma: Employment. Heinsohn:Sutro Biopharma: Employment. Sato:Sutro Biopharma: Employment. Molina:Sutro Biopharma: Employment. Hallam:Sutro Biopharma: Employment. Lupher:Sutro Biopharma: Employment.

CD74 is a type II transmembrane glycoprotein involved in the formation and transport of MHC class II protein. High expression of CD74 has been confirmed in follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) and other types of NHL with immunohistochemistry (IHC) using the LL1 antibody (Stein et al. Clin Cancer Res 2007). We employed site-specific conjugation technology to generate novel CD74-targeting ADCs, SP7676 and SP7675 (STRO-001) that exhibit a high degree of homogeneity characterized by the drug linker covalently binding to a single defined site. The human anti-CD74 IgG1 antibody (SP7219) used for ADCs SP7676 and STRO-001 was engineered using novel Fab-based ribosome display methods enabling selection from ~1012 different antibody variants. Hundreds of unique Fabs from this selection were converted to IgGs and expressed directly in Sutro's proprietary cell-free protein synthesis platform, Xpress CFTM, for extensive screening. The top antibody lead derived from this screen is further tested to identify the best sites for conjugation of linker-warheads. Sutro's SP7219 emerged as the top performing antibody and was conjugated to noncleavable DBCO-maytansinoid linker-warheads to form the ADCs SP7676 and STRO-001. Since conjugation sites were selected based on highest stability both in vitro and in vivo, these novel ADCs lose little drug moiety in circulation and have potential for improved PK, safety and activity profiles. In vitro cell proliferation/cytotoxicity assays show potent activity in 1) DLBCL (germinal center B-cell-like [GCB] and "double-hit") lines: SU-DHL-4, IC50 - 1nM; SU-DHL-6, IC50 - 0.4 nM; WSU-NHL, IC50 - 1.6 nM; Pfeiffer, IC50 - .09 nM; NUDUL-1, IC50 - 0.4 nM; HT, IC50 - 0.7 nM; OCI-LY-19, IC50 - 0.7 nM; WSU-DLBCL2, IC50 - 0.3 nM; 2) mantle cell lymphoma (MCL) cell lines: Mino, IC50 - 0.4-0.7 nM; JVM-2, IC50 - 1.7-2.9 nM; Jeko-1, IC50 - 0.4 - 0.6 nM; 3) Ph+ acute lymphoblastic leukemia (ALL): SUP-B15, IC50 - 3.9-4.6 nM; and 4) CLL (EBV-transformed): JVM-13, IC50 - 3.0-3.4 nM. SP7676 elicited strong anti-tumor response in the OCI-LY-10 lymphoma xenograft model with 100% of animals achieving complete regression of tumors at 3mg/kg every 3 days x 5 doses and 10 mg/kg weekly x 3 doses. In the WSU-DLCL2 "double-hit" lymphoma xenograft model, administration of SP7676 (with re-dosing at time of re-growth) produced tumor regressions at 10 mg/kg every 3 days x 5 (6/8 mice tumor free, remaining 2 with small tumors) and 10 mg/kg weekly x 3 (tumor regression in most animals, 4/8 tumor free). Additionally, STRO-001 exhibits dose-dependent tumor growth inhibition in SUDHL-6 xenografts starting at 2.5 mg/kg weekly x 3 doses. Exploratory testing of our lead candidate, STRO-001 in cynomologous monkeys showed dose-dependent B-cell depletion at 1 - 30 mg/kg doses on Day 1 and 15, confirming the intended pharmacodynamic effect. Our preliminary data demonstrate that SP7676 and STRO-001 generate potent cell killing activity across multiple B-cell lymphoma/leukemia cell lines in vitro, and anti-tumor activity in preclinical B-cell NHL xenografts. Evaluation of STRO-001 in other cell lines and xenograft models and in combination studies is ongoing. GLP toxicology and other IND-enabling studies are planned. Disclosures Li: Sutro Biopharma: Employment. Abrahams:Sutro Biopharma: Employment. Embry:Sutro Biopharma: Employment. Yu:Sutro Biopharma: Employment. Kahana:Celgene: Employment. Brown:Celgene: Employment. Narla:Celgene: Employment. Barnes:Celgene: Employment. Schwartz:Celgene: Employment. Boylan:Celgene: Employment. Zawada:Sutro Biopharma: Employment. Stephenson:Sutro Biopharma: Employment. Bruhns:Sutro Biopharma: Employment. Bussell:Sutro Biopharma: Employment. Steiner:Sutro Biopharma: Employment. Galan:Sutro Biopharma: Employment. Kline:Sutro Biopharma: Employment. Yam:Sutro Biopharma: Employment. Stafford:Sutro Biopharma: Employment. Hoffmann:Sutro Biopharma: Employment. Matheny:Sutro Biopharma: Employment. DeAlmeida:Sutro Biopharma: Employment. Vasquez:Sutro Biopharma: Employment. Heinsohn:Sutro Biopharma: Employment. Sato:Sutro Biopharma: Employment. Molina:Sutro Biopharma: Employment. Hallam:Sutro Biopharma: Employment. Lupher:Sutro Biopharma: Employment.

Accumulating evidence has suggested that tumors may arise and grow as a result of the formation of a subset cell population termed cancer stem cells (CSC) or tumor initiating cells. Several research reports have indicated that CSCs are relatively resistant to conventional therapies. Thus, therapeutic strategies that specifically target cancer stem cells could have a major impact on cancer patient survival. Patient-derived tumor xenografts (PDX) have played a major role in the development of new cancer therapies. The advantage of PDX over standard cell-line xenograft models is that PDX retain much of the molecular, genetic, and histological heterogeneity of the original tumor and are minimally passed. In addition, self-renewal and lineage differentiation, the hallmarks of cancer stem cells, can be demonstrated through serial transplantation in immunodeficient mice. Our therapeutic approach in cancer stem cell drug discovery has been to target key developmental pathways that have been strongly implicated in cancer including the Notch pathway. We carried out tumorigenicity studies in three SCLC PDX models utilizing naïve cells positively enriched for either Notch2 or Notch3 expression. In two of the three models, Notch2+ and/or Notch3+ cell populations resulted in enhanced tumor formation. In a SCLC tumor that expressed low levels of Notch3, the Notch2+ population resulted in 70% tumor formation compared to CD44+ (10%) or CD133+ (45%) enriched populations. We have developed a monoclonal antibody, tarextumab (OMP-59R5), which selectively inhibits the function of both Notch2 and Notch3. Our preclinical data in PDX models demonstrate that tarextumab was efficacious in inhibiting the growth of various indications with minimal intestinal toxicity. Notably, the sensitivity of tarextumab in combination with gemcitabine or gemcitabine plus nab-paclitaxel in pancreatic tumors was associated with higher levels of Notch3 gene expression. Interference with Notch2/3 signaling by tarextumab delays tumor recurrence, decreases cancer stem cell frequency (as determined by in vivo LDA studies) and modulates the function of tumor vasculature. Our ALPINE Phase 1b clinical trial indicates that tarextumab is generally well-tolerated and shows signs of anti-tumor efficacy and modulation of Notch pathway signaling in the clinic. Furthermore, we observe a higher response rate and longer survival in patients with Notch3 high tumors receiving gemcitabine/nab-paclitaxel/tarextumab combination therapy. Analysis of pre- and post-treatment tumor biopsies showed an inhibition of Notch pathway and CSC gene signatures. Ongoing Phase 2 clinical trials evaluate first-line treatment with tarextumab in metastatic pancreatic cancer (ALPINE) and small cell lung cancer (PINNACLE). Collectively, these results demonstrate the utility of PDX models for discovery and development of anti-cancer stem cell therapeutics, identifying pharmacodynamic endpoints of drug actions, identifying predictive biomarkers for patient stratification, and translating these preclinical findings into clinical trials. Citation Format: Marcus M. Fischer, Wan-Ching Yen, Fumiko Axelrod, Christopher Bond, Jennifer Cain, Belinda Cancilla, Randall Henner, Rene Meisner, Aaron Sato, Jalpa Shaw, Tracy Tang, Breanna Wallace, Min Wang, Chun Zhang, Ann Kapoun, Lei Zhou, Jakob Dupont, John Lewicki, Austin Gurney, Tim Hoey. Use of patient-derived tumor xenografts (PDX) for discovery and development of an anti-Notch2/3 monoclonal antibody targeting cancer stem cells. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A41.

Clinical management of pancreatic cancer is challenging and in recent years has not been dramatically improved and this disease remains the fourth most common cause of cancer-related death. Dysregulated Notch2 and/or Notch3 activity has been associated with several human tumor types including pancreatic cancer. In this report, we describe the development of OMP-59R5, an antibody isolated originally by binding to Notch2 and subsequently found to also bind Notch3. OMP-59R5 potently blocks Notch2 and Notch3 signaling and inhibits the growth pancreatic cancer patient derived xenograft tumors, as a single agent and in combination with gemcitabine. The anti-tumor effects were associated with modulation of Notch target genes in tumor and stromal cells. Inclusion of OMP-59R5 delayed pancreatic tumor recurrence following termination of gemcitabine, correlated with an effect of anti-Notch2/3 in decreasing in cancer stem cell frequency and tumorigenicity. Gene set enrichment analysis showed that a subset of the stem cell gene sets and gene set related to epithelial-to-mesenchymal transistion, EMT, was up-regulated in tumors by gemcitabine alone, but down-regulated by the combination of gemcitabine plus OMP-59R5 treatment. Findings from the present study suggest that agents that reduce cancer cell frequency may improve cancer treatment by delaying or preventing tumor recurrence, and reducing the metastatic spread of the disease. In particular, blocking Notch-mediated proliferation of bulk tumor cells and cancer stem cells may provide an attractive strategy for novel therapy in pancreatic cancer treatment. Citation Format: Wan-Ching Yen, Marcus Fischer, Aaron Sato, Min Wang, Ann M. Kapoun, John Lewicki, Austin Gurney, Timothy Hoey. Targeting Notch signaling with a dual Notch 2/3 antagonist, OMP-59R5, inhibits tumor growth and decreases cancer stem cell frequency in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B44.

We have measured the steady-state phosphorescence and decay times of wild-type λcI repressor and compared it with that of a modified λcI repressor in which > 95% of the tryptophans were replaced with 5-hydroxy-l-tryptophan (5-OHTrp). The wild-type and 5-OHTrp-λcI repressors are spectroscopically distinct such that we can selectively excite the 5-OHTrp-λcI even in the presence of a 15-fold molar excess ofN-acetyltryptophanamide (NATrpA). The phosphorescence band of wild-type λcI is red-shifted by 3 nm relative to NATrpA, characteristic of buried tryptophan. Similarly, the phosphorescence of 5-OHTrp-λcI repressor is red-shifted relative to the model, 5-OHTrp, showing that according to the phosphorescence, the modified repressor is structurally indistinguishable from the native repressor. While the phosphorescence decay of both NATrpA and 5-OHTrp are single exponentials, the decay of both wild-type and 5-OHTrp-λcI repressors is complex, requiring three decay components whose fractional contributions to the phosphorescence are the same for both repressors. Because the 5-OHTrp phosphorescence can be excited at wavelengths outside the absorbance range of tryptophan and DNA, a protein spectrally enhanced with this emitter will aid the investigations of protein-protein or protein-DNA interactions.

Cancer stem cells (CSCs) have been identified as the small subpopulation of cells in many cancers and are thought to be responsible for cancer initiation, progression, metastasis, recurrence and drug resistance. The NOTCH pathway, comprised of ligands JAG1, JAG2, DLL1, DLL3 and DLL4 and receptors NOTCH1, NOTCH2, NOTCH3 and NOTCH4, plays a critical role in stem cell signaling, cell proliferation, survival, apoptosis, and differentiation. Because of the existing body of data that implicates aberrant NOTCH pathway activation in resistance to chemotherapeutic agents in epithelial and hematologic tumors, we hypothesized that NOTCH inhibition would enhance the anti-tumor activity of chemotherapeutic agents. We have identified a NOTCH3 activating mutation in a patient breast cancer and developed a xenograft tumor model from the primary patient sample, OMP-B37. The combination of paclitaxel and OncoMed's anti-Notch2/3 antibody, OMP-59R5, inhibited the tumor growth of OMP-B37 xenograft tumors. In addition, we found that treatment with OMP-59R5 drove changes in tumor heterogeneity as indicated by the increased expression of markers associated with a mature cellular fate. The down-regulation of tumor growth promoting genes and NOTCH pathway markers was observed with OMP-59R5 treatment. The enhanced activity of combined OMP-59R5 and paclitaxel therapy in patient-derived tumor xenografts suggests that there is therapeutic potential in combining targeted NOTCH pathway inhibition with chemotherapeutic agents. Citation Format: Jie Wei, Jennifer Cain, Jalpa Shah, Breanna Wallace, Tracy Tang, Min Wang, Christopher Murriel, Cancilla Belinda, Austin Gurney, Aaron Sato, John Lewicki, Ann Kapoun, Tim Hoey. Combined therapy of a novel anti-NOTCH2/3 antibody with paclitaxel inhibits tumor growth in a patient-derived breast tumor xenograft. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A33.

We describe a novel and general way of generating high affinity peptide (HAP) binders to receptor tyrosine kinases (RTKs), using a multi-step process comprising phage-display selection, identification of peptide pairs suitable for hetero-dimerization (non-competitive and synergistic) and chemical synthesis of heterodimers. Using this strategy, we generated HAPs with K D s below 1 nM for VEGF receptor-2 (VEGFR-2) and c-Met. VEGFR-2 HAPs bound significantly better (6- to 500-fold) than either of the individual peptides that were used for heterodimer synthesis. Most significantly, HAPs were much better (150- to 800-fold) competitors than monomers of the natural ligand (VEGF) in various competitive binding and functional assays. In addition, we also found the binding of HAPs to be less sensitive to serum than their component peptides. We believe that this method may be applied to any protein for generating high affinity peptide (HAP) binders.

Using a 12-amino acid peptide conjugated with HYNIC as a model, we investigated nitriles as possible coligands for labeling with 99m Tc. After the preparation of the 99m Tc labeled HYNIC-peptide using tricine as coligand, the addition of acetonitile was found by reverse phase HPLC to block further coligand exchange with ethylenediamine diacetic acid (EDDA) at room temperature. The addition of this nitrile changed the pharmacokinetics of the 99m Tc labeled peptide in normal mice towards faster clearance and significant differences in accumulation in most tissues sampled. By replacing acetonitrile with cyanoacetate, a nitrile not present in the HPLC eluant, it was possible to show the existence of a new, more hydrophilic, species by reverse phase HPLC. We conclude that nitriles can act as coligands for HYNIC-conjugated peptides labeled with 99m Tc and tricine. Furthermore, the presence of acetonitrile during Sep-Pak or HPLC purification may inadvertently generate a mixed tricine/acetonitile coligand 99m Tc-HYNIC-peptide complex.

The Notch signaling pathway, driven by four Notch family receptors (NOTCH1-4) and five canonical Notch ligands (DLL1, DLL3, DLL4, JAG1, and JAG2) in humans, critically regulates key functions during embryonic development as well as stem cell maintenance and differentiation in adult tissues. Altered Notch signaling activity has been documented in many types of cancers; depending on the tumor type, Notch signaling can be oncogenic or tumor suppressing. Activating mutations in NOTCH1 have been identified in hematopoietic cancers including T-cell acute lymphoblastic leukemia and chronic lymphoblastic leukemia. With the advance in high-throughput genomic sequencing platforms, mutations in the NOTCH genes have also been identified in solid tumors, albeit at much lower frequencies. Here we report for the first time the identification of two activating mutations in NOTCH3. One is a frameshift mutation in the PEST domain, and the other a frameshift mutation in the ankyrin (ANK) domain. The PEST frameshift mutation leads to the production of C-terminally truncated NOTCH3 protein, and by Western Blotting of nuclear fractions of tumor tissues and Immunohistochemistry (IHC) we show that this mutant NOTCH3 accumulates stably in the nuclei of the breast tumor in which it was identified. Breast xenograft tumors carrying this mutation are highly sensitive to OMP-59R5, a ligand-blocking antibody targeting the Notch2 and Notch3 receptors, suggesting that the mutation, although activating, is still ligand dependent. Consistent with this finding, expression of this mutant NOTCH3 constructed by site-directed mutagenesis exhibits higher baseline activity compared to wild type NOTCH3 and is equally responsive to ligands in in vitro NOTCH reporter assays. We are not able to observe increased nuclear accumulation of the ANK frameshift mutant NOTCH3 in the colon tumor in which it was identified. However, in vitro the ANK mutant NOTCH3 has higher baseline activity than wild-type NOTCH3 in the absence of ligands and remains highly responsive to ligands. The colon xenograft tumors carrying this NOTCH3 ANK mutation are also sensitive to OMP-59R5 in in vivo efficacy studies. Taken together our data suggest that NOTCH3 is oncogenic in certain subtypes of tumors, and tumors with these activating mutations may predictive sensitivity to therapeutic antibodies targeting the receptor. Citation Format: Breanna Wallace, Min Wang, Chris Muriel, Jennifer Cain, Belinda Cancilla, Jalpa Shah, Jie Wei, Austin Gurney, John Lewicki, Aaron Sato, Tim Hoey, Tracy Tang, Ann M. Kapoun. Novel NOTCH3 activating mutations identified in tumors sensitive to OMP-59R5, a monoclonal antibody targeting the Notch2 and Notch3 receptors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 213. doi:10.1158/1538-7445.AM2013-213