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T cell–engaging bispecific antibodies (TCB) are highly potent therapeutics that can recruit and activate cytotoxic T cells to stimulate an antitumor immune response. However, the development of TCBs against solid tumors has been limited by significant on-target toxicity to normal tissues. Probody therapeutics have been developed as a novel class of recombinant, protease-activated antibody prodrugs that are “masked” to reduce antigen binding in healthy tissues but can become conditionally unmasked by proteases that are preferentially active in the tumor microenvironment (TME). Here, we describe the preclinical efficacy and safety of CI107, a Probody TCB targeting EGFR and CD3. In vitro, the protease-activated, unmasked CI107 effectively bound EGFR and CD3 expressed on the surface of cells and induced T-cell activation, cytokine release, and cytotoxicity toward tumor cells. In contrast, dually masked CI107 displayed a >500-fold reduction in antigen binding and >15,000-fold reduction in cytotoxic activity. In vivo, CI107 potently induced dose-dependent tumor regression of established colon cancer xenografts in mice engrafted with human peripheral blood mononuclear cells. Furthermore, the MTD of CI107 in cynomolgus monkeys was more than 60-fold higher than that of the unmasked TCB, and much lower levels of toxicity were observed in animals receiving CI107. Therefore, by localizing activity to the TME and thus limiting toxicity to normal tissues, this Probody TCB demonstrates the potential to expand clinical opportunities for TCBs as effective anticancer therapies for solid tumor indications. Significance: A conditionally active EGFR-CD3 T cell–engaging Probody therapeutic expands the safety window of bispecific antibodies while maintaining efficacy in preclinical solid tumor settings.

Probody therapeutics (Pb-Txs) are conditionally activated antibody–drug conjugates (ADCs) designed to remain inactive until proteolytically activated in the tumor microenvironment, enabling safer targeting of antigens expressed in both tumor and normal tissue. Previous attempts to target CD71, a highly expressed tumor antigen, have failed to establish an acceptable therapeutic window due to widespread normal tissue expression. This study evaluated whether a probody–drug conjugate targeting CD71 can demonstrate a favorable efficacy and tolerability profile in preclinical studies for the treatment of cancer. CX-2029, a Pb-Tx conjugated to maleimido-caproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E, was developed as a novel cancer therapeutic targeting CD71. Preclinical studies were performed to evaluate the efficacy and safety of this anti-CD71 PDC in patient-derived xenograft (PDX) mouse models and cynomolgus monkeys, respectively. CD71 expression was detected at high levels by IHC across a broad range of tumor and normal tissues. In vitro, the masked Pb-Tx form of the anti-CD71 PDC displayed a >50-fold reduced affinity for binding to CD71 on cells compared with protease-activated, unmasked anti-CD71 PDC. Potent in vivo tumor growth inhibition (stasis or regression) was observed in >80% of PDX models (28/34) at 3 or 6 mg/kg. Anti-CD71 PDC remained mostly masked (>80%) in circulation throughout dosing in cynomolgus monkeys at 2, 6, and 12 mg/kg and displayed a 10-fold improvement in tolerability compared with an anti-CD71 ADC, which was lethal. Preclinically, anti-CD71 PDC exhibits a highly efficacious and acceptable safety profile that demonstrates the utility of the Pb-Tx platform to target CD71, an otherwise undruggable target. These data support further clinical development of the anti-CD71 PDC CX-2029 as a novel cancer therapeutic.

Supplementary Data from Nonclinical Efficacy and Safety of CX-2029, an Anti-CD71 Probody–Drug Conjugate

Supplementary Data from A Probody T Cell–Engaging Bispecific Antibody Targeting EGFR and CD3 Inhibits Colon Cancer Growth with Limited Toxicity

T cell–engaging bispecific antibodies (TCB) are highly potent therapeutics that can recruit and activate cytotoxic T cells to stimulate an antitumor immune response. However, the development of TCBs against solid tumors has been limited by significant on-target toxicity to normal tissues. Probody therapeutics have been developed as a novel class of recombinant, protease-activated antibody prodrugs that are “masked” to reduce antigen binding in healthy tissues but can become conditionally unmasked by proteases that are preferentially active in the tumor microenvironment (TME). Here, we describe the preclinical efficacy and safety of CI107, a Probody TCB targeting EGFR and CD3. In vitro, the protease-activated, unmasked CI107 effectively bound EGFR and CD3 expressed on the surface of cells and induced T-cell activation, cytokine release, and cytotoxicity toward tumor cells. In contrast, dually masked CI107 displayed a >500-fold reduction in antigen binding and >15,000-fold reduction in cytotoxic activity. In vivo, CI107 potently induced dose-dependent tumor regression of established colon cancer xenografts in mice engrafted with human peripheral blood mononuclear cells. Furthermore, the MTD of CI107 in cynomolgus monkeys was more than 60-fold higher than that of the unmasked TCB, and much lower levels of toxicity were observed in animals receiving CI107. Therefore, by localizing activity to the TME and thus limiting toxicity to normal tissues, this Probody TCB demonstrates the potential to expand clinical opportunities for TCBs as effective anticancer therapies for solid tumor indications.Significance:A conditionally active EGFR-CD3 T cell–engaging Probody therapeutic expands the safety window of bispecific antibodies while maintaining efficacy in preclinical solid tumor settings.

Immune-checkpoint blockade has revolutionized cancer treatment. However, most patients do not respond to single-agent therapy. Combining checkpoint inhibitors with other immune-stimulating agents increases both efficacy and toxicity due to systemic T-cell activation. Protease-activatable antibody prodrugs, known as Probody therapeutics (Pb-Tx), localize antibody activity by attenuating capacity to bind antigen until protease activation in the tumor microenvironment. Herein, we show that systemic administration of anti–programmed cell death ligand 1 (anti–PD-L1) and anti–programmed cell death protein 1 (anti–PD-1) Pb-Tx to tumor-bearing mice elicited antitumor activity similar to that of traditional PD-1/PD-L1–targeted antibodies. Pb-Tx exhibited reduced systemic activity and an improved nonclinical safety profile, with markedly reduced target occupancy on peripheral T cells and reduced incidence of early-onset autoimmune diabetes in nonobese diabetic mice. Our results confirm that localized PD-1/PD-L1 inhibition by Pb-Tx can elicit robust antitumor immunity and minimize systemic immune-mediated toxicity. These data provide further preclinical rationale to support the ongoing development of the anti–PD-L1 Pb-Tx CX-072, which is currently in clinical trials.

The identification of novel peptide hormones by functional screening is challenging because posttranslational processing is frequently required to generate biologically active hormones from inactive precursors. We developed an approach for functional screening of novel potential hormones by expressing them in endocrine host cells competent for posttranslational processing. Candidate preprohormones were selected by bioinformatics analysis, and stable endocrine host cell lines were engineered to express the preprohormones. The production of mature hormones was demonstrated by including the preprohormones insulin and glucagon, which require the regulated secretory pathway for production of the active forms. As proof of concept, we screened a set of G-protein-coupled receptors (GPCRs) and identified protein FAM237A as a specific activator of GPR83, a GPCR implicated in central nervous system and regulatory T-cell function. We identified the active form of FAM237A as a C-terminally cleaved, amidated 9 kDa secreted protein. The related protein FAM237B, which is 64% homologous to FAM237A, demonstrated similar posttranslational modification and activation of GPR83, albeit with reduced potency. These results demonstrate that our approach is capable of identifying and characterizing novel hormones that require processing for activity.

CX-072 is an anti-PD-L1 (programmed death ligand 1) Probody therapeutic (Pb-Tx) designed to be preferentially activated by proteases in the tumor microenvironment and not in healthy tissue. Here, we report the model-informed drug development of CX-072. A quantitative systems pharmacology (QSP) model that captured known mechanisms of Pb-Tx activation, biodistribution, elimination, and target engagement was used to inform clinical translation. The QSP model predicted that a trough level of masked CX-072 (intact CX-072) of 13-99 nM would correspond to a targeted, 95% receptor occupancy in the tumor. The QSP model predictions appeared consistent with preliminary human single-dose pharmacokinetic (PK) data following CX-072 0.03-30.0 mg/kg as monotherapy: CX-072 circulated predominantly as intact CX-072 with minimal evidence of target-mediated drug disposition. A preliminary population PK (POPPK) analysis based upon 130 subjects receiving 0.03-30.0 mg/kg as monotherapy included a provision for a putative time-dependent and dose-dependent antidrug antibody (ADA) effect on clearance (CL) with a mixture model. Preliminary POPPK estimates for intact CX-072 time-invariant CL and volume of distribution were 0.306 L/day and 4.84 L, respectively. Exposure-response analyses did not identify statistically significant relationships with best change from baseline sum of measurements and either adverse events of grade ≥ 3 or of special interest. Simulations suggested that > 95% of patients receiving CX-072 10 mg/kg every two weeks would exceed the targeted trough level regardless of ADA, and that dose adjustment by body weight was not necessary, supporting a fixed 800 mg dose for evaluation in phase II.

Introduction: The toxicity of potent new biological therapies for cancer has limited their utility. By improving tumor specificity, antibody prodrugs can widen or even create a therapeutic window f...

Supplemental material, Supplemental_Material_for_Peptide_Hormone_Platform_Identified_Candidate_GPR83_Ligands_by_Sallee_et_al for A Pilot Screen of a Novel Peptide Hormone Library Identified Candidate GPR83 Ligands by Nathan A. Sallee, Ernestine Lee, Atossa Leffert, Silvia Ramirez, Arthur D. Brace, Robert Halenbeck, W. Michael Kavanaugh and Kathleen M. C. Sullivan in SLAS Discovery

Proteases have been implicated in the development of many pathological conditions, including cancer. Detection of protease activity in diseased tissues could therefore be useful for diagnosis, prognosis, and the development of novel therapeutic approaches. Due to tight post-translational regulation, determination of the expression level of proteases alone may not be indicative of protease activities, and new methods for measuring protease activity in biological samples such as tumor biopsies are needed. Here we report a novel zymography-based technique, called the IHZTM assay, for the detection of specific protease activities in situ. The IHZ assay involves imaging the binding of a protease-activated monoclonal antibody prodrug, called a Probody® therapeutic, to tissue. Probody therapeutics are fully recombinant, masked antibodies that can only bind target antigen after removal of the mask by a selected protease. A fluorescently labeled Probody molecule is incubated with a biological tissue, thereby enabling its activation by tissue endogenous proteases. Protease activity is measured by imaging the activated Probody molecule binding to antigen present in the sample. The method was evaluated in xenograft tumor samples using protease specific substrates and inhibitors, and the measurements correlated with efficacy of the respective Probody therapeutics. Using this technique, a diverse profile of MMP and serine protease activities was characterized in breast cancer patient tumor samples. The IHZ assay represents a new type of in situ zymography technique that can be used for the screening of disease-associated proteases in patient samples from multiple pathological conditions.

O1 IL-15 primes an mTOR-regulated gene-expression program to prolong anti-tumor capacity of human natural killer cells #### Andreas Lundqvist1, Vincent van Hoef1, Xiaonan Zhang1, Erik Wennerberg2, Julie Lorent1, Kristina Witt1, Laia Masvidal Sanz1, Shuo Liang1, Shannon Murray3, Ola Larsson1

T cell-engaging bispecific antibodies (TCBs) are highly potent therapeutics that direct the activity of cytotoxic T cells to tumors. TCBs have shown clinical activity in hematologic malignancies, but development of TCBs for solid tumor indications is proving more challenging. Due to their high potency, TCBs can target normal tissues with low antigen expression, resulting in significant on-target, off-tumor toxicity that can limit dosing to low levels. As a result, it has been difficult to reach the level of drug exposure required for efficacy without excessive toxicity. Therefore, novel methods are needed to enable the potent antitumor activity of TCBs while minimizing toxicity due to cytokine release and damage to healthy tissues. CytomX has developed a new class of recombinant, proteolytically activated antibody prodrugs (ProbodyTM therapeutics) that are “masked” to prevent binding to antigen in healthy tissue, but can become “unmasked” by proteases that are preferentially activated in the tumor microenvironment. In this way, Probody therapeutics are designed to increase therapeutic index by maximizing efficacy and minimizing on-target toxicity in normal tissues. Here we describe a T cell-engaging Bispecific Probody therapeutic (Pb-TCB) targeting Epidermal Growth Factor Receptor (EGFR) and CD3 that has been optimized for affinity, effector function, masking, and cleavability. In vitro, under protease-deficient conditions, we demonstrate that the unmasked EGFR-CD3 TCB has potent, EGFR-dependent tumor cell killing, while the doubly-masked EGFR-CD3 Pb-TCB reduces target-dependent cytotoxicity by more than 100,000-fold. However, in established tumor models where tumor-resident proteases are expected to be active, we demonstrate that Pb-TCBs potently induce tumor regressions. In nonhuman primates, the maximum tolerated dose (MTD) of the EGFR-CD3 Pb-TCB is more than 60-fold higher than the MTD of the unmasked TCB, and the tolerated exposure (AUC) is more than 10,000-fold higher. Finally, despite the 60-fold dose differential at the MTDs, transient serum cytokine and AST/ALT increases observed in nonhuman primates treated with the Pb-TCB are still lower than those induced by the TCB. By localizing activity to the tumor microenvironment, Pb-TCBs have the potential to expand clinical opportunities for T cell-engaging bispecific therapies that are limited by on-target toxicities, especially in solid tumors. Moreover, an EGFR-CD3 Pb-TCB has the potential to address EGFR-expressing tumors that are poorly responsive to existing EGFR-directed therapies. PROBODY is a trademark of CytomX Therapeutics, Inc. Citation Format: Leila M. Boustany, Laurie Wong, Clayton W. White, Linnea Diep, Yuanhui Huang, Shouchun Liu, Jennifer H. Richardson, W. Michael Kavanaugh, Bryan A. Irving. EGFR-CD3 bispecific Probody™ therapeutic induces tumor regressions and increases maximum tolerated dose >60-fold in preclinical studies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A164.

Probody™ therapeutics are recombinant, proteolytically-activated antibody prodrugs, engineered to remain inert until activated locally by tumor-associated proteases. Probody therapeutics exploit the fundamental dysregulation of extracellular protease activity that exists in tumors relative to healthy tissue. Leveraging the ability of a Probody therapeutic to bind its target at the site of disease after proteolytic cleavage, we developed a novel method for profiling protease activity in living animals. Using NIR optical imaging, we demonstrated that a non-labeled anti-EGFR Probody therapeutic can become activated and compete for binding to tumor cells in vivo with a labeled anti-EGFR monoclonal antibody. Furthermore, by inhibiting matriptase activity in vivo with a blocking-matriptase antibody, we show that the ability of the Probody therapeutic to bind EGFR in vivo was dependent on protease activity. These results demonstrate that in vivo imaging of Probody therapeutic activation can be used for screening and characterization of protease activity in living animals, and provide a method that avoids some of the limitations of prior methods. This approach can improve our understanding of the activity of proteases in disease models and help to develop efficient strategies for cancer diagnosis and treatment.

There are many transmembrane receptor-like proteins whose ligands have not been identified. A strategy for finding ligands when little is known about their tissue source is to screen each extracellular protein individually expressed in an array format by using a sensitive functional readout. Taking this approach, we have screened a large collection (3,191 proteins) of extracellular proteins for their ability to activate signaling of an orphan receptor, leukocyte tyrosine kinase (LTK). Only two related secreted factors, FAM150A and FAM150B (family with sequence similarity 150 member A and member B), stimulated LTK phosphorylation. FAM150A binds LTK extracellular domain with high affinity ( K D = 28 pM). FAM150A stimulates LTK phosphorylation in a ligand-dependent manner. This strategy provides an efficient approach for identifying functional ligands for other orphan receptors.

Because of the probable causal relationship between constitutive p210bcr/abl protein tyrosine kinase activity and manifestations of chronic-phase chronic myelogenous leukemia (CML; myeloid expansion), a key goal is to identify relevant p210 substrates in primary chronic-phase CML hematopoietic progenitor cells. We describe here the purification and mass spectrometric identification of a 155-kD tyrosine phosphorylated protein associated with src homologous and collagen gene (SHC) from p210bcr/abl-expressing hematopoietic cells as SHIP2, a recently reported, unique SH2-domain–containing protein closely related to phosphatidylinositol polyphosphate 5-phosphatase SHIP. In addition to an N-terminal SH2 domain and a central catalytic region, SHIP2 (like SHIP1) possesses both potential PTB(NPXY) and SH3 domain (PXXP) binding motifs. Thus, two unique 5-ptases with striking structural homology are coexpressed in hematopoietic progenitor cells. Stimulation of human hematopoietic growth factor responsive cell lines with stem cell factor (SCF), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) demonstrate the rapid tyrosine phosphorylation of SHIP2 and its resulting association with SHC. This finding suggests that SHIP2, like that reported for SHIP1 previously, is linked to downstream signaling events after activation of hematopoietic growth factor receptors. However, using antibodies specific to these two proteins, we demonstrate that, whereas SHIP1 and SHIP2 selectively hydrolyze PtdIns(3,4,5)P3 in vitro, only SHIP1 hydrolyzes soluble Ins(1,3,4,5)P4. Such an enzymatic difference raises the possibility that SHIP1 and SHIP2 may serve different functions. Preliminary binding studies using lysates from p210bcr/abl-expressing cells indicate that both Ptyr SHIP2 and Ptyr SHIP1 bind to the PTB domain of SHC but not to its SH2 domain. Interestingly, SHIP2 was found to selectively bind to the SH3 domain of ABL, whereas SHIP1 selectively binds to the SH3 domain of Src. Furthermore, in contrast to SHIP1, SHIP2 did not bind to either the N-terminal or C-terminal SH3 domains of GRB2. These observations suggest (1) that SHIP1 and SHIP2 may have a different hierarchy of binding SH3 containing proteins and therefore may modulate different signaling pathways and/or localize to different cellular compartments and (2) that they may be substrates for tyrosine phosphorylation by different tyrosine kinases. Because recent evidence has clearly implicated both PI(3,4,5)P3 and PI(3,4)P2 in growth factor-mediated signaling, our finding that both SHIP1 and SHIP2 are constitutively tyrosine phosphorylated in CML primary hematopoietic progenitor cells may thus have important implications in p210bcr/abl-mediated myeloid expansion.

The fibroblast growth factor (FGF) pathway promotes tumor growth and angiogenesis in many solid tumors. Although there has long been interest in FGF pathway inhibitors, development has been complicated: An effective FGF inhibitor must block the activity of multiple mitogenic FGF ligands but must spare the metabolic hormone FGFs (FGF-19, FGF-21, and FGF-23) to avoid unacceptable toxicity. To achieve these design requirements, we engineered a soluble FGF receptor 1 Fc fusion protein, FP-1039. FP-1039 binds tightly to all of the mitogenic FGF ligands, inhibits FGF-stimulated cell proliferation in vitro, blocks FGF- and vascular endothelial growth factor (VEGF)-induced angiogenesis in vivo, and inhibits in vivo growth of a broad range of tumor types. FP-1039 antitumor response is positively correlated with RNA levels of FGF2, FGF18, FGFR1c, FGFR3c, and ETV4; models with genetic aberrations in the FGF pathway, including FGFR1-amplified lung cancer and FGFR2-mutated endometrial cancer, are particularly sensitive to FP-1039-mediated tumor inhibition. FP-1039 does not appreciably bind the hormonal FGFs, because these ligands require a cell surface co-receptor, klotho or β-klotho, for high-affinity binding and signaling. Serum calcium and phosphate levels, which are regulated by FGF-23, are not altered by administration of FP-1039. By selectively blocking nonhormonal FGFs, FP-1039 treatment confers antitumor efficacy without the toxicities associated with other FGF pathway inhibitors.

Immuno-oncology approaches have transformed the treatment of advanced melanoma, NSCLC and an increasing number of other cancers. Antibodies targeting the T cell checkpoint molecules CTLA-4 and PD-1 can reverse tumor suppression of T cell activation that prevents effective anti-tumor immunity. However, blockade of checkpoint molecules, particularly in combination, in non-tumor tissues can result in autoimmune side effects that can limit therapeutic utility. Combination of PD-1 and CTLA-4 blockade provides increased response rates but with concomitant increases in immune-related adverse events. Here we demonstrate that Probody™ therapeutics targeting PD-1 synergize with a CTLA-4 checkpoint inhibitor to eradicate established tumors in a mouse model while protecting against PD1-mediated systemic autoimmunity. Probody therapeutics are recombinant, proteolytically-activated antibody prodrugs that have the potential to meaningfully widen therapeutic index by minimizing interactions with normal tissue while retaining anti-tumor activity. Probody therapeutics take advantage of protease activities that are abundant in tumors but suppressed in normal tissues. An extension of the antibody light chain with a masking peptide blocks binding of the antibody to antigen in normal tissue. Tumor-associated proteases cleave and release the mask, enabling the active antibody to bind antigen preferentially in the tumor. The feasibility of the Probody approach to checkpoint targets is supported by preclinical and clinical studies in which intratumoral delivery of low dose immune modulators enables anti-tumor responses despite negligible systemic exposure. For preclinical assessment of PD-1 as a Probody target, a family of Probody therapeutics derived from the anti-mouse PD-1 antibody J43 was developed. The intact Probody therapeutics demonstrated reduced binding to mouse PD-1 relative to the parental antibody that was completely restored following proteolytic activation. The anti-tumor efficacy of the J43 antibody and Probody therapeutics was assessed following systemic administration as single agents or in combination with an anti-CTLA-4 antibody to mice bearing established MC38 syngeneic tumors. As single agents, the PD-1 antibody and Probody therapeutics reduced tumor growth relative to an isotype antibody control and synergized with CTLA-4 blockade. Consistent with their reduced ability to bind target in the absence of proteolytic activation, the PD-1 Probody therapeutics were up to 10 times less potent than the parental anti-PD-1 antibody in inducing autoimmune diabetes in NOD mice. Our preclinical findings demonstrate that PD-1 Probody therapeutics retain anti-tumor efficacy with improved safety profiles and therefore have the promise to enable better tolerated PD-1 combination immunotherapies. PROBODY is a trademark of CytomX Therapeutics, Inc. Citation Format: Kimberly A. Tipton, Chanty Chan, Kenneth R. Wong, Victoria Singson, Jennifer H. Richardson, W Michael Kavanaugh, Bryan A. Irving, James W. West. PD-1-targeted Probody therapeutics provide anti-tumor efficacy and a 10-fold dose protection against systemic autoimmunity in preclinical studies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3211.

The targets of Antibody Drug Conjugates (ADCs) have typically been selected by identifying transmembrane antigens that are highly expressed in tumors but are low or absent in normal tissues. The number of potential ADC targets meeting these requirements is limited, either because expression in tumors is not high enough for optimal efficacy, or because expression in normal tissues is too high, leading to toxicity. Probody (TM) therapeutics are antibody prodrugs designed to remain inactive until proteolytically activated in the tumor microenvironment. Probody technology therefore has the potential to enable targeting of more desirable tumor antigens with higher, more persistent and more homogeneous expression in tumors, while limiting toxicity due to interaction with these antigens in normal tissues. CD71 (transferrin receptor) is an example of a highly desirable ADC target, because of its well-characterized ability to efficiently internalize and deliver an ADC payload intracellularly. Further, CD71 is expressed at homogeneously high levels (3+ expression by IHC) in almost all tumor types, including in metastatic disease. However, because CD71 is also expressed on multiple normal cell types, including many progenitor hematological cells, we reasoned that a CD71-targeted ADC would be challenging to develop. To enable targeting of CD71, we have developed an anti-CD71 Probody Drug Conjugate (PDC) CX-2005, which can be activated by multiple proteases in the tumor microenvironment, but which remains in a relatively inactive form while in circulation and in normal tissues. CX-2005 produces complete tumor regressions at therapeutic doses in mouse models of lymphoma, breast cancer and lung cancer. Consistent with our hypothesis that it would be difficult to develop an anti-CD71 ADC, treatment of cynomolgus monkeys with an anti-CD71 ADC at doses that were efficacious in mouse tumor models caused life-threatening depletion of CD71-expressing hematopoietic cells, including neutrophils, lymphocytes and RBCs. In contrast, these toxicities were not observed in monkeys treated with the same dose of the anti-CD71 PDC, consistent with the Probody therapeutic avoiding interaction with these normal cells. Our data demonstrate that, in preclinical studies, Probody drug conjugates can safely and effectively target attractive tumor antigens like CD71 which have been difficult to effectively approach with traditional ADCs due to their expression on critical normal tissues. Further, our data support the development of Probody therapeutics directed against CD71 in multiple different cancers. PROBODY is a trademark of CytomX Therapeutics, Inc. Citation Format: Shweta Singh, Amy DuPage, Annie Yang Weaver, Jason Sagert, Clayton White, Michael Krimm, Yuanhui Huang, Linnea Diep, Kim Tipton, Shouchun Liu, Jennifer Richardson, W. Michael Kavanaugh, Jonathan A. Terrett, Luc R. Desnoyers. Development of a probody drug conjugate (PDC) targeting CD71 for the treatment of solid tumors and lymphomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2975.

T cell-engaging bispecific antibodies (TCBs) represent a highly potent modality to direct the activity of cytotoxic T cells to tumors. TCBs are designed to bind both a surface tumor antigen and the CD3ϵ subunit of the T cell receptor, thereby enabling T cells to kill tumor cells. Because T cell bispecifics bypass TCR antigen recognition and co-stimulatory requirements for T cell activation, they obviate the need for tumor-specific immunity and overcome many impediments faced by T cells in the tumor-microenvironment. TCBs have shown clinical activity in hematologic malignancies as evidenced by the recent approval of blinatumomab for the treatment of relapsed refractory acute lymphoblastic leukemia based on its ability to direct T cells against CD19+ cells. Development of TCBs for non-hematologic cancers is proving more challenging, however, due to the high potency of the modality and its inability to discriminate between cells expressing target antigen in the tumor and those in healthy tissue. Because antigens with highly restricted tumor expression are rare, toxicity directed against healthy tissues has limited the therapeutic utility of TCBs for most solid tumor targets. In addition, because TCBs are administered at very low doses, preferential distribution to circulating T cells or to healthy tissues can limit drug exposure in tumors. Therefore new methods are needed that enable the potent anti-tumor activity of TCBs without on-target damage to normal tissues. Here we describe a T cell-engaging Bispecific Probody therapeutic (TCBP) targeting CD3 and Epidermal Growth Factor Receptor (EGFR). Probody therapeutics are recombinant, proteolytically-activated antibody prodrugs designed to widen therapeutic window by minimizing interaction with normal tissues and maximizing interactions with tumors. A Probody therapeutic is an antibody in which the antigen-binding site is blocked by a masking peptide extension from the amino terminus of the light chain. The peptide mask, joined to the antibody by a protease-cleavable linker, can be removed by proteases that are more active in the tumor microenvironment than in normal tissue, allowing for tumor-localized activity. We have previously demonstrated that an EGFR-targeted Probody therapeutic limits interaction with EGFR-expressing tissue while preserving anti-tumor activity. To demonstrate that Probody technology can address the limitations of bispecific antibodies, we compared CD3-EGFR TCBPs to the corresponding unmasked TCB for their ability to direct T cell-mediated cytotoxicity of EGFR+ tumor cells in vitro. While the unmasked CD3-EGFR TCB demonstrated potent T cell- and EGFR-dependent killing at single-digit pM concentrations, the CD3-EGFR TCBP exhibited reduced targeted cytotoxicity by up to 3000-fold. In a tumor model utilizing engraftment of human T cells in NSG mice, both TCB Probody therapeutic and its parental TCB effectively eliminated established EGFR+ HT-29 colorectal tumors at equivalent doses. In contrast, an ongoing study in non-human primates has shown at least a 10-fold higher maximum tolerated dose for the CD3-EGFR TCBP relative to its corresponding CD3-EGFR TCB. As a result, Probody technology has the potential to increase therapeutic window and may enable potent bispecific therapies for solid tumors that are limited by on-target toxicities in healthy tissue. Citation Format: Sherry L. LaPorte, Daniel R. Hostetter, Laurie Wong, Jennifer Razo, Linnea Diep, Clayton W. White, Jennifer H. Richardson, W. Michael Kavanaugh, Bryan A. Irving. CD3-EGFR bispecific Probody™ therapeutics induced tumor regressions and increased therapeutic window in preclinical studies. [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 A203.

Antibody drug conjugates (ADCs) have shown their greatest clinical utility when targeting antigens expressed at very high levels on cancer cells that have coincidentally lower expression in normal tissues. This is exemplified by the approvals of trastuzumab emtansine for her2neu 3+ breast cancer and brentuximab vedotin for Hodgkins Disease and Anaplastic large-cell lymphoma. Both drugs are approved for subsets of specific cancer types where target antigen expression is particularly high relative to expression in normal tissues. There are other cell surface antigens that are highly expressed on cancer cells and normal tissues, but the utility of such antigens as ADC targets is restricted by their corresponding expression in normal tissues. One such target is CD166 (ALCAM), which shows 3+ expression by IHC in most donors of multiple cancer types, e.g., ca. 70% prevalence in breast, prostate, and lung cancers but also expression in multiple normal tissues including lung, GI tissues, and liver. Thus CD166 has not been progressed as a target for ADCs. Probody™ therapeutics are fully recombinant antibody prodrugs that are converted to active antibodies by tumor-associated proteases. Preclinical in vivo studies show that Probody therapeutics remain substantially inactive in normal tissues and in circulation. As such, Probody drug conjugates (PDCs), unlike ADCs, enable targeting of high expression tumor targets that are also expressed in normal tissues. We have developed an anti-CD166 Probody therapeutic selected for specific binding, internalization, and cross reactivity to cynomolgus macaque as a species for toxicology assessments. This therapeutic has been conjugated to spdb-DM4 and tested in preclinical models of efficacy and safety. Treatment with the PDC has led to complete regressions in models of lung and breast cancer at therapeutically relevant doses. These same doses were assessed for safety in cynomolgus monkeys. The safety and efficacy profiles for the anti-CD166 PDC are supportive of progression to clinical development of this anti-CD166 Probody drug conjugate. Citation Format: Annie Yang Weaver, Shweta Singh, Amy DuPage, Jason Sagert, Jeanne Flandez, Elizabeth Menendez, Judi Ford, Michael Krimm, Stephen Moore, Margaret Nguyen, Andrew Jang, Eric Brecht, Yuanhui Huang, Linnea Diep, Nicole Lapuyade, Tereza Sputova, James West, Olga Vasiljeva, Shouchun Liu, Jennifer Richardson, W. Michael Kavanaugh, Jonathan A. Terrett, Luc R. Desnoyers. Development of a probody drug conjugate (PDC) against CD166 for the treatment of multiple cancers. [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 C165.

e15074 Background: Amplification of the Fibroblast Growth Factor Receptor 2 (FGFR2) gene is reported in ~5% of gastric cancer patients and leads to the specific overexpression of the FGFR2b isoform...

Using a new inducible form of phosphatidylinositol 3-kinase (PI 3-kinase) we have found that PI 3-kinase activation has the following effects on cell growth and proliferation. (i) Activation of PI 3-kinase was sufficient to promote entry into S phase of the cell cycle within several hours. This was shown by activation of cyclin-dependent kinase 4 (Cdk4) and Cdk2 and by the induction of DNA synthesis. (ii) PI 3-kinase activation alone was not, however, sufficient to provide for progression through the entire cell cycle. Instead, prolonged activation of PI 3-kinase in the absence of serum stimulation resulted in apoptosis. It is possible that the cells undergo apoptosis because the PI 3-kinase-induced entry into the cell cycle is abnormal. For example, we found that the cyclin E-Cdk2 complex, which normally disappears after entry into S phase of the cell cycle, fails to be downregulated following induction by PI 3-kinase. (iii) Finally, we found that prolonged activation of PI 3-kinase in the presence of serum resulted in cellular changes that resemble those associated with oncogenic transformation. The cells reached high densities, were irregular and refractile in appearance, and formed colonies in soft agar. In contrast, neither PI 3-kinase nor serum stimulation alone could induce these changes. Our results suggest that activation of PI 3-kinase promotes anchorage-independent cell growth and entry into the cell cycle but does not abrogate the growth factor requirement for cell proliferation.

The phosphotyrosine binding (PTB) domain specifically binds to tyrosine-phosphorylated proteins, but differs in structure and mechanism of action from the SH2 domain family. We quantitated the affinity, specificity, and kinetics of the interaction of the SHC PTB domain with a sequence motif, asparagine-X-X-phosphotyrosine (NXX(pY)), found in several receptor tyrosine kinases and oncogenic proteins. PTB domain-mediated interaction with the NXX(pY) motif of c-ErbB2 was characterized by similar overall affinity but slower kinetics than that reported for SH2 domains. This suggested that unlike SH2 domains, PTB domains may not rapidly exchange among associated proteins. Furthermore, when directly and quantitatively compared, PTB domain binding specificity did not significantly overlap with a panel of seven SH2 domains. Thus, signaling pathways involving PTB and SH2 domain-mediated interactions can be regulated separately. Finally, our data define the minimal SHC PTB domain binding motif as NXX(pY), not NPX(pY) as suggested by other authors, and suggest a high affinity motif, hydrophobic residue-(D/E)-N-X-X-pY-(W/F), found in the Trk and ErbB receptor tyrosine kinase families. We conclude that PTB domains mediate specific protein-protein interactions independent from those mediated by SH2 domains.

The activated platelet-derived growth factor (PDGF) receptor physically associates with p85, a subunit of phosphatidylinositol-3 kinase. Although this interaction may activate phosphatidylinositol-kinase and is crucial for PDGF-induced mitogenesis, it has not been shown whether p85 is modified in the process. p85 contains two SH2 (Src homology) domains, designated SH2-N and SH2-C. Recent experiments have shown that the SH2-C domain alone determines high-affinity binding of p85 to the PDGF receptor. The function of SH2-N, which binds receptors with lower affinity, is unknown. In this study, using a receptor-blotting technique, we find that p85 is modified by PDGF stimulation of intact cells. This modification involves inhibition of binding of the SH2-N region of p85 to the PDGF receptor. Studies with vanadate suggest that tyrosine phosphorylation of p85 is responsible for the modification of p85 detected by receptor blotting. Furthermore, recombinant p85 is modified in a similar manner when it is tyrosine phosphorylated in vitro by PDGF receptors. Tyrosine phosphorylation of p85 does not block binding of the SH2-C domain and therefore does not release p85 from high-affinity binding sites on the receptor in vitro. Instead, phosphorylation may regulate the ability of the SH2-N of p85 to bind to a different portion of the PDGF receptor or to another molecule in the signaling complex. This study provides the first evidence that p85 is tyrosine phosphorylated upon PDGF stimulation of cells and suggests that tyrosine phosphorylation of p85 regulates its activity or its interaction with other proteins.

Using immobilized PDGF receptor as an affinity reagent, we purified an 85 kd protein (p85) from cell lysates and we cloned its cDNA. The protein contains an SH3 domain and two SH2 domains that are homologous to domains found in several receptor-associated enzymes. Recombinant p85 overexpressed in mammalian cells inhibited the binding of endogenous p85 and a 110 kd protein to the receptor and also blocked the association of PI3-kinase activity with the receptor. Experiments with receptor mutants and with short peptides derived from the kinase insert region of the PDGF receptor showed that the recombinant p85 binds to a well-defined phosphotyrosine-containing sequence of the receptor. p85 appears to be the subunit of PI3-kinase that links the enzyme to the ligand-activated receptor.

Serotonin-induced DNA synthesis in bovine aortic smooth muscle cells was totally abolished by pretreatment of cultures with 5 ng/ml pertussis toxin. The half maximally effective concentration of toxin was approximately 10 pg/ml. Pertussis toxin did not affect platelet-derived growth factor (PDGF)-stimulated DNA synthesis and actually enhanced the mitogenic effect of the phorbol ester, phorbol 12-myristate 13-acetate. Pertussis toxin did not inhibit serotonin-stimulated inositol phosphate accumulation or increases in intracellular calcium or cAMP concentrations under conditions sufficient to completely inhibit serotonin-induced (3H)thymidine incorporation. These results demonstrate that a novel, pertussis-sensitive pathway is required for serotonin-, but not platelet-derived growth factor-induced DNA synthesis in vascular smooth muscle cells. The pertussis-sensitive step does not involve cAMP, phosphoinositide hydrolysis, mobilization of intracellular calcium, or phorbol ester-sensitive protein kinase C activity.

Identification of novel targets in cancer immunotherapy is needed to address the significant number of patients that either do not respond to current therapies or encounter unacceptable toxicities. The discovery of such targets, including novel checkpoint regulators and the counter-receptors for previously "orphan" checkpoints, has been limited by a lack of a comprehensive collection of proteins suitable for functional screening and methods for assessing their function in high-throughput. We have generated a comprehensive library of substantially all human extracellular proteins encompassing nearly every target for protein therapeutics. Our library contains more than 5700 proteins, including secreted protein ligands and the extracellular domains of membrane-bound receptors in soluble forms. In addition, we have developed in vitro and in vivo high-throughput protein screening platforms for discovering and validating new targets in a variety of disease areas. Our discovery platforms are uniquely suited for the discovery of novel targets in cancer immunotherapy. Based on domain structure, phylogeny and other factors, we identified a subset of 391 human proteins in our library that are enriched for possible cell surface-expressed immune regulatory proteins. These proteins were screened for the ability to modulate anti-CD3-stimulated human T cell proliferation. This screen identified a number of known checkpoint regulators, which validates the discovery platform and screening approach. In addition, we identified numerous novel co-inhibitory proteins - the majority of which have no published link to immune cell regulation. For a number of these proteins, we have demonstrated co-inhibitory activity in orthogonal assays, such as assays of cytokine production from purified T cell subpopulations and in mixed lymphocyte reaction assays. Further in vitro and in vivo characterization of these proteins is ongoing. These data demonstrate the power of our screening platform to discover potentially new therapeutic targets for cancer immunotherapy.