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Purpose:Gastrointestinal cancers remain areas of high unmet need despite advances in targeted and immunotherapies. Here, we demonstrate potent, tumor-selective efficacy with PF-07062119, a T-cell engaging CD3 bispecific targeting tumors expressing Guanylyl Cyclase C (GUCY2C), which is expressed widely across colorectal cancer and other gastrointestinal malignancies. In addition, to address immune evasion mechanisms, we explore combinations with immune checkpoint blockade agents and with antiangiogenesis therapy.Experimental Design:PF-07062119 activity was evaluated in vitro in multiple tumor cell lines, and in vivo in established subcutaneous and orthotopic human colorectal cancer xenograft tumors with adoptive transfer of human T cells. Efficacy was also evaluated in mouse syngeneic tumors using human CD3ϵ transgenic mice. IHC and mass cytometry were performed to demonstrate drug biodistribution, recruitment of activated T cells, and to identify markers of immune evasion. Combination studies were performed with anti–PD-1/PD-L1 and anti-VEGF antibodies. Toxicity and pharmacokinetic studies were done in cynomolgus macaque.Results:We demonstrate that GUCY2C-positive tumors can be targeted with an anti-GUCY2C/anti-CD3ϵ bispecific, with selective drug biodistribution to tumors. PF-07062119 showed potent T-cell–mediated in vitro activity and in vivo efficacy in multiple colorectal cancer human xenograft tumor models, including KRAS- and BRAF-mutant tumors, as well as in the immunocompetent mouse syngeneic tumor model. PF-07062119 activity was further enhanced when combined with anti–PD-1/PD-L1 treatment or in combination with antiangiogenic therapy. Toxicity studies in cynomolgus indicated a monitorable and manageable toxicity profile.Conclusions:These data highlight the potential for PF-07062119 to demonstrate efficacy and improve patient outcomes in colorectal cancer and other gastrointestinal malignancies.

Mouse body weights of LS1034 tumor bearing mice treated with GUCY2C(M)-CD3 using adoptive T cell transfer

Table S2. Surface Plasmon Resonance binding affinities of anti-GUCY2C/anti-CD3ε bispecifics to human, mouse and cynomolgus GUCY2C

Supplementary Methods

Gastrointestinal malignancies, including colorectal cancer (CRC) remain an area of high unmet need. Here we demonstrate tumor selective and potent in vitro and in vivo efficacy with PF-07062119, a novel T cell engaging CD3 bispecific against tumors expressing the Guanylyl Cyclase C (GUCY2C) receptor, a target expressed in more than 90% of CRC, and in other gastrointestinal cancers. Additionally, to address immune evasion mechanisms, combinations of the GUCY2C-CD3 bispecific are explored with immune checkpoint blockade therapy, as well as with chemotherapeutic and anti-angiogenic agents that could enhance immune infiltration into tumors. Our preclinical in vivo data demonstrate that GUCY2C-positive tumors can be targeted with an anti-GUCY2C/anti-CD3ϵ bispecific, with selective drug biodistribution to tumors. PF-07062119 showed potent T cell mediated anti-tumor activity in several human xenograft models of CRC, using adoptive transfer of human T cells, including those with KRAS and BRAF mutations, as well as in models with syngeneic tumors in immunocompetent human CD3 transgenic mice. PF-07062119 activity was further enhanced when combined with anti PD-1/PD-L1 treatment or in combination with chemotherapy or anti-angiogenic therapy. A combination of immunohistochemistry, flow cytometry and CyTOF analyses was used to demonstrate the mechanism of action of PF-07062119 in single agent and combination studies in vivo. Toxicity and pharmacokinetic studies were done in cynomolgus macaques and indicated a monitorable and manageable toxicity profile. These data highlight the potential for PF-07062119 to demonstrate efficacy and improve patient outcomes in CRC and other gastrointestinal malignancies. A clinical Phase I study has been initiated in patients with CRC, gastric and esophageal adenocarcinomas (NCT04171141). Citation Format: Divya Mathur, Adam Root, Bozena Bugaj-Gaweda, Xingzhi Tan, Wei Fang, Stephanie Bisulco, Jonathan Golas, Diane Fernandez, Jessica Kearney, Eric Upeslacis, Johnny Yao, Edward Rosfjord, Chad Stevens, Keith Kobylarz, Lindsay King, Jatin Narula, Kerry Kelleher, David Schaer, Cris Kamperschroer, Bernard Buetow, Cynthia Rohde, Allison Moreau, Gilbert Wong, Puja Sapra. A novel GUCY2C - CD3 bispecific engages T cells to induce cytotoxicity in gastrointestinal tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2283.

Background: Guanylyl cyclase C (GUCY2C) is a regulator of intestinal homeostasis and is expressed in more than 90% of colorectal cancer (CRC), as well as in other gastrointestinal malignancies. Target expression in normal tissues is largely restricted to the apical side of intestinal epithelial tight junctions, which could allow for preferential uptake of GUCY2C-targeted biologics by tumors that have disrupted tight junction architecture. Here we demonstrate tumor-selective uptake and potent efficacy with a half-life extended, GUCY2C-CD3 bispecific molecule that recruits CD3-positive T cells to induce cytotoxicity of GUCY2C-expressing tumors in several CRC models in vivo. Additionally, to address immune evasion mechanisms, rational combinations of the bispecific are explored with immune checkpoint blockade agents, as well as by blocking angiogenesis, which has been reported to enhance T-cell infiltration into tumors. Methods: GUCY2C-CD3 mediated activity was evaluated in vivo in several cell line- and patient derived-xenograft models of CRC, using adoptive transfer of human T cells in established subcutaneous and orthotopic tumors. Immunohistochemistry was performed to demonstrate biodistribution of the drug and recruitment of activated T cells in GUCY2C-expressing tumors vs. normal tissues. We also performed CYTOF analyses on GUCY2C-CD3-treated tumors and tumor-infiltrating lymphocytes (TILs) to identify markers of immune evasion. Combination studies with anti PD-1/PD-L1 checkpoint blockade agents and with an anti VEGF-A antibody were performed using the adoptive transfer model. Results: GUCY2C-CD3 bispecific showed preferential biodistribution to GUCY2C-expressing xenograft tumors compared to normal tissue. The drug demonstrated potent dose-dependent efficacy in several CRC models with no signs of toxicity. Bispecific treated tumors not only showed recruitment of activated T cells to the tumor site, but also upregulated checkpoint mechanisms, such as PD-L1 on tumors and exhaustion markers on TILs. Consequently, anti PD-1/PD-L1 checkpoint blocking antibodies provided enhanced efficacy when combined with GUCY2C-CD3. Additionally, significant combination benefit was observed when GUCY2C-CD3 was combined with an anti VEGF-A blocking antibody. Conclusions: Our preclinical data demonstrate that a GUCY2C-CD3 bispecific can selectively target colorectal tumors, including those with KRAS or BRAF mutations, which are difficult to treat with currently approved therapies. This bispecific shows combination benefits with T-cell checkpoint blockade agents, as well as antiangiogenesis agents, indicating that single-agent activity with GUCY2C-CD3 can be further enhanced with mechanisms that address immune evasion. Citation Format: Divya Mathur, Adam Root, Bozena Bugaj-Gaweda, Xingzhi Tan, Wei Fang, Stephanie Bisulco, Jonathan Golas, Jessica Kearney, Eric Upeslacis, Johnny Yao, Edward Rosfjord, Chad Stevens, Lindsay King, Jatin Narula, Kerry Kelleher, Cynthia Rohde, Lioudmila Tchistiakova, Anhco Nguyen, Puja Sapra. A GUCY2c-CD3 bispecific engages T cells to induce cytotoxicity in gastrointestinal tumors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A16.

We have isolated cDNAs encoding PDE4A8 (phosphodiesterase 4 isoform A8), a new human cAMP-specific PDE4 isoform encoded by the PDE4A gene. PDE4A8 has a novel N-terminal region of 85 amino acids that differs from those of the related ‘long’ PDE4A4, PDE4A10 and PDE4A11 isoforms. The human PDE4A8 N-terminal region has diverged substantially from the corresponding isoforms in the rat and other mammals, consistent with rapid evolutionary change in this region of the protein. When expressed in COS-7 cells, PDE4A8 localized predominantly in the cytosol, but approx. 20% of the enzyme was associated with membrane fractions. Cytosolic PDE4A8 was exquisitely sensitive to inhibition by the prototypical PDE4 inhibitor rolipram (IC50 of 11±1 nM compared with 1600 nM for PDE4A4), but was less sensitive to inhibition by cilomilast (IC50 of 101±7 nM compared with 61 nM for PDE4A4). PDE4A8 mRNA was found to be expressed predominantly in skeletal muscle and brain, a pattern that differs from the tissue expression of other human PDE4 isoforms and also from that of rat PDE4A8. Immunohistochemical analysis showed that PDE4A8 could be detected in discrete regions of human brain, including the cerebellum, spinal cord and cerebral cortex. The unique tissue distribution of PDE4A8, combined with the evolutionary divergence of its N-terminus, suggest that this isoform may have a specific function in regulating cAMP levels in human skeletal muscle and brain.

Since the identification of phosphodiesterase activity in brain tissue more than 40 years ago, 11 distinct gene families have been identified, differing with respect to localization, regulation, affinity for cAMP and cGMP, and distinct functions within cells. PDEs 1, 2, 4, and 10 are currently of special interest to CNS pharmacology because of their high expression in specific areas of the brain and the behavioral effects of inhibitors of these enzymes in rodents. Efficient high-throughput PDE enzyme assays are essential for PDE-targeted drug discovery, and this unit details two types of assays. The first method is relatively inexpensive and is based on separating radiolabeled cNMPs from degradation products on alumina columns. The second method is fluorescence-based; it is fast and better accommodates high-throughput screening, but is more expensive. Although these methods have successfully been used for PDEs 1, 2, 4 and 10, they could be readily adapted to other PDEs. Keywords: central nervous system; CNS; cyclic nucleotide phosphodiesterase; PDE2; PDE1; PDE4; PDE10; alumina acid; IMAP

An alpha7 nicotinic acetylcholine receptor sequence was cloned from Rhesus monkey (Macaca mulatta). This clone differs from the mature human alpha7 nicotinic acetylcholine receptor in only four amino acids, two of which are in the extracellular domain. The monkey alpha7 nicotinic receptor was characterized in regard to its functional responses to acetylcholine, choline, cytisine, and the experimental alpha7-selective agonists 4OH-GTS-21, TC-1698, and AR-R17779. For all of these agonists, the EC(50) for activation of monkey receptors was uniformly higher than for human receptors. In contrast, the potencies of mecamylamine and MLA for inhibiting monkey and human alpha7 were comparable. Acetylcholine and 4OH-GTS-21 were used to probe the significance of the single point differences in the extracellular domain. Mutants with the two different amino acids in the extracellular domain of the monkey receptor changed to the corresponding sequence of the human receptor had responses to these agonists that were not significantly different in EC(50) from wild-type human alpha7 nicotinic receptors. Monkey alpha7 nicotinic receptors have a serine at residue 171, while the human receptors have an asparagine at this site. Monkey S171N mutants were more like human alpha7 nicotinic receptors, while mutations at the other site (K186R) had relatively little effect. These experiments point toward the basic utility of the monkey receptor as a model for the human alpha7 nicotinic receptor, albeit with the caveat that these receptors will vary in their agonist concentration dependency. They also point to the potential importance of a newly identified sequence element for modeling the specific amino acids involved with receptor activation.

We report here the cloning and characterization of two novel PDE4D isoforms, PDE4D6 and PDE4D7. PDE4D6 is a supershort form and PDE4D7 a long form of PDE4D. In addition, we have identified another novel long-form variant, PDE4D8, in silico. Like other isoforms, PDE4D6 and PDE4D7 are differentially expressed. Expression of PDE4D6 is restricted to brain whereas PDE4D7 is widely expressed in many tissues. Baculovirus-expressed recombinant PDE4D6 and PDE4D7 enzymes have high affinity for cyclic AMP (cAMP) and are inhibited by rolipram. The activity of PDE4D7, not PDE4D6, is elevated by a protein kinase A (PKA)-dependent mechanism, presumably through phosphorylation of the conserved PKA site in the upstream conserved region 1 (UCR1) domain. In agreement with early reports, human PDE4D6 and PDE4D7 are localized on genomic fragments of chromosome 5. Examination of the promoter regions reveals multiple CREB binding sites upstream of the starting methionine (Met) of each gene, suggesting that the cAMP/PKA signaling pathway may regulate transcriptional expression of PDE4D6 and PDE4D7.