Immune checkpoint inhibitors have shown limited success in breast cancer, the most common and deadly cancer in women worldwide. Novel immune therapies, such as CD3-engaging bispecific antibodies, have shown clinical promise in hematologic malignancies. However, developing CD3 bispecifics for solid tumors has been challenging due to the difficulty in identifying tumor-specific antigens. B7-H4 is proposed as an attractive tumor-associated antigen for breast cancer therapeutics with comprehensive coverage regardless of breast cancer molecular subtype. We designed a B7-H4-targeting CD3 bispecific molecule, PF-07260437, and demonstrated B7-H4-dependent pharmacology in vitro by directing cytotoxic T-cell killing to breast cancer cell lines. Treatment of cell line- and patient-derived xenograft in vivo models of human breast cancer with PF-07260437 induced substantial tumoricidal activity, often resulting in complete responses. Mechanistically, PF-07260437 increased T-cell number and activation, leading to efficient tumor killing. Additionally, combining PF-07260437 with standard of care (palbociclib plus fulvestrant) and a checkpoint inhibitor (anti-PD-1) showed combinatorial benefits in an immune-competent in vivo model. Clinically relevant noninvasive PET/CT imaging with a CD8-targeting tracer demonstrated PF-07260437-mediated increases in intratumoral CD8 T cells, highlighting the utility of CD8-PET technology to potentially assess biomarker changes in the clinic. Finally, the manageable toxicity profile of PF-07260437 was highlighted in an exploratory toxicology study in cynomolgus monkeys. These data support the clinical testing of PF-07260437 for treating B7-H4-expressing solid tumors, including breast cancer.

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

GUCY2C expression in non-intestinal human tissues

Quantitative Real-Time PCR based evaluation of gucy2c expression in HCT116 and T84 cells

Mouse body weights of LS1034 colon orthotopic tumor bearing mice treated with PF-07062119 using adoptive T cell transfer

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

PF-07062119 binds to Cynomolgus Monkey T cells

Mouse body weights of CT26-mGUCY2C tumor bearing hCD3ε transgenic mice treated with PF-07062119

cGMP assay to test GUCY2C pathway agonism and neutralization

Abstract Extra domain B splice variant of fibronectin (EDB+FN) is an extracellular matrix protein (ECM) deposited by tumor-associated fibroblasts, and is associated with tumor growth, angiogenesis, and invasion. We hypothesized that EDB+FN is a safe and abundant target for therapeutic intervention with an antibody–drug conjugate (ADC). We describe the generation, pharmacology, mechanism of action, and safety profile of an ADC specific for EDB+FN (EDB-ADC). EDB+FN is broadly expressed in the stroma of pancreatic, non–small cell lung (NSCLC), breast, ovarian, head and neck cancers, whereas restricted in normal tissues. In patient-derived xenograft (PDX), cell-line xenograft (CLX), and mouse syngeneic tumor models, EDB-ADC, conjugated to auristatin Aur0101 through site-specific technology, demonstrated potent antitumor growth inhibition. Increased phospho-histone H3, a pharmacodynamic biomarker of response, was observed in tumor cells distal to the target site of tumor ECM after EDB-ADC treatment. EDB-ADC potentiated infiltration of immune cells, including CD3+ T lymphocytes into the tumor, providing rationale for the combination of EDB-ADC with immune checkpoint therapy. EDB-ADC and anti-PD-L1 combination in a syngeneic breast tumor model led to enhanced antitumor activity with sustained tumor regressions. In nonclinical safety studies in nonhuman primates, EDB-ADC had a well-tolerated safety profile without signs of either on-target toxicity or the off-target effects typically observed with ADCs that are conjugated through conventional conjugation methods. These data highlight the potential for EDB-ADC to specifically target the tumor microenvironment, provide robust therapeutic benefits against multiple tumor types, and enhance activity antitumor in combination with checkpoint blockade.

We report here the discovery and optimization of a novel T cell retargeting anti-GUCY2C x anti-CD3ε bispecific antibody for the treatment of solid tumors. Using a combination of hybridoma, phage display and rational design protein engineering, we have developed a fully humanized and manufacturable CD3 bispecific antibody that demonstrates favorable pharmacokinetic properties and potent in vivo efficacy. Anti-GUCY2C and anti-CD3ε antibodies derived from mouse hybridomas were first humanized into well-behaved human variable region frameworks with full retention of binding and T-cell mediated cytotoxic activity. To address potential manufacturability concerns, multiple approaches were taken in parallel to optimize and de-risk the two antibody variable regions. These approaches included structure-guided rational mutagenesis and phage display-based optimization, focusing on improving stability, reducing polyreactivity and self-association potential, removing chemical liabilities and proteolytic cleavage sites, and de-risking immunogenicity. Employing rapid library construction methods as well as automated phage display and high-throughput protein production workflows enabled efficient generation of an optimized bispecific antibody with desirable manufacturability properties, high stability, and low nonspecific binding. Proteolytic cleavage and deamidation in complementarity-determining regions were also successfully addressed. Collectively, these improvements translated to a molecule with potent single-agent in vivo efficacy in a tumor cell line adoptive transfer model and a cynomolgus monkey pharmacokinetic profile (half-life>4.5 days) suitable for clinical development. Clinical evaluation of PF-07062119 is ongoing.

Abstract 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.

408 Introduction: A specific and sensitive imaging biomarker to monitor immune activation and quantify pharmacodynamic (PD) responses would be a useful tool in drug development for immunomodulating anti-cancer agents. PF-07062119 is a T-cell engaging CD3 bispecific antibody targeting GUCY2C (GUCY2C-CD3) which is over expressed widely across colorectal cancer (CRC) and other gastrointestinal malignancies. Previous data has demonstrated the efficacy of PF-0762119 as a targeted immuno-therapeutic candidate in human CRC xenograft mouse models with adoptive transfer of human T cells (ref). 89Zr-Df-IAB22M2C, an 89Zr-labeled human CD8 specific minibody developed by ImaginAb, is currently under testing in a phase 2 clinical study (ClinicalTrials.gov Identifier: NCT03802123) in patients with metastatic solid tumors to measure CD8 T-cell tumor infiltrates. In this study we investigated the ability of 89Zr-Df-IAB22M2C to track responses to interventions with varying doses of PF-07062119 in a human CRC adoptive transfer mouse model, as well as, correlation of the radiotracer to CD8 immunohistochemical (IHC) staining. Materials & Method: NOD scid gamma (NSG) female mice (total 70; n = 5-12 per group) bearing human CRC LS1034 tumors were used in this study. Following a pilot study, animals were treated with PF-07062119 in four different dose groups (0.03 mg/kg, 0.06 mg/kg, 0.1 mg/kg or 1 mg/kg), or with a non-targeted CD3 bispecific control at a dose of 1 mg/kg, on study Day-0 and injected with activated T-cells on Day-1. Different imaging time points, Day-4, Day 6, or Day-9 were assessed with 89Zr-Df-IAB22M2C intravenously injected (200 µL, with radioactivity of approximately 65 µCi) on Day-3, Day-5, and Day-8, respectively. All animals underwent a 20-minute static PET scan at 22h post 89Zr-Df-IAB22M2C injection, followed by CT scan for anatomical registration. For Day-9 imaging group animals, a second treatment of PF-07062119 was conducted on Day-7. Following PET/CT imaging, mice were euthanized under anesthesia and dissected for ex vivo distribution analysis of tissues [whole blood, brain, heart, kidneys, large intestine (emptied), liver, lungs, skeletal muscle (quadriceps), skin, small intestine (emptied), spleen, tail, and tumor]. Tissues were collected, weighed and gamma counted for their radioactivity. Data analyzed and reported as %ID/g and SUV for both in vivo imaging and ex vivo tissue distribution. In addition, tumor tissues were pretreated with 10% neutral buffered formalin (NBF) prior to the gamma counting for upcoming tissue section and CD8 IHC staining. Results & Conclusions: The results demonstrated substantial mean uptake in treated tumors, up to 13.0 %ID/g (2.7 S.D.) in highest dosed animals (ex vivo gamma counting), with significant differences in comparison to isotype controls. The data also showed a dose-dependent response for both time and treatment: significantly higher radioactivity concentration was observed at the higher dose levels of PF-0762119 (0.1 and 1 mg/kg) on Day-9 compared to Day-4 (p

GUCY2c is a tumor target expressed in >90% of colorectal cancer (CRC). It is also expressed on the apical side of intestinal epithelial tight junctions, where it regulates intestinal fluidity. PF-07062119, a GUCY2c-CD3 bispecific antibody that recruits T cells to GUCY2c expressing gastrointestinal tumors, has shown anti-tumor efficacy in several subcutaneous colorectal cancer models without any signs of intestinal toxicity. Here we describe the effects of PF-07062119 in an intestinal orthotopic xenograft model, created surgically in NSG mice using LS1034 CRC cells expressing luciferase. To establish this model, LS1034-luc tumor fragments were sutured onto the cecum proximal to the colon. Tumor growth was monitored through detection of luciferin bioluminescence. PF-07062119 was administered at 0.03, 0.1 and 0.3 mg/kg iv weekly, along with an adoptive transfer of 2x106 human T cells the day after bispecific compound dosing. At 0.3 mg/kg dose, the bioluminescence signal was significantly reduced compared with the isotype control group. Necropsies at the end of study revealed complete tumor clearance, or residual calcified or fibrotic tissues, at the tumor implant site in all animals of the 0.3 mg/kg group, compared with tumor presence in 9 of 9 animals and liver metastases in 2 of 9 animals in the isotype control group. The average tumor weight of 0.1 mg/kg dose group decreased relative to the control group, but the difference did not reach statistical significance. The results of histological examination indicate enhanced T cell infiltration in the tumor tissues compared with the normal intestinal tissues in PF-07062119 treated animals. No body weight loss or other clinical signs were observed in all groups. These results demonstrate that PF-07062119 can eliminate LS1034 orthotopic tumors and potential metastasis with no observable toxicities. This supports our hypothesis that apical restriction of GUCY2c expression in normal intestine creates tumor-vs-normal differential for selective targeting of GUCY2c positive tumors, and the GUCY2c-CD3 bispecific antibody redirects T cells to lyse the CRC tumor cells. Citation Format: Johnny Yao, Divya Mathur, Sharon Yang, Roger Conant, Jessica Kearney, Vlad Buklan, Jonathan Golas, Edward Rosfjord. Evaluation of GUCY2c-CD3 bispecific targeting GI cancers in an orthotopic colorectal tumor model [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 4549.

Abstract 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.

Phosphoglucomutase (PGM) plays an important role in polysaccharide capsule formation and virulence in a number of bacterial pathogens. However, the enzyme has not yet been characterized from Mycobacterium tuberculosis (Mtb). Here, we report the biochemical properties of recombinant Mtb-PGM as well as the in silico structural analysis from Mtb H37Rv. The purified recombinant enzyme was enzymatically active with a specific activity of 67.5 U/mg and experimental k(cat) of 70.31 s(-1) for the substrate glucose-1-phosphate. The enzyme was stable in pH range 6.5-7.4 and exhibited temperature optima range between 30 and 40 degrees C. Various kinetic parameters and constants of the rPGM were determined. A structural comparison of Modeller generated 3D Mtb-PGM structure with rabbit muscle PGM revealed that the two enzymes share the same overall heart shape and four-domain architecture, despite having only 17% sequence identity. However, certain interesting differences between the two have been identified, which provide an opportunity for designing new drugs to specifically target the Mtb-PGM. Also, in the absence of the crystal structure of the Mtb-PGM, the modeled structure could be further explored for in silico docking studies with suitable inhibitors. (c) 2012 Elsevier Inc. All rights reserved.

Phosphoglucose isomerase (PGI) plays a key role in both glycolysis and gluconeogenesis inside the cell, whereas outside the cell it exhibits cytokine properties. PGI is also known to act as an autocrine motility factor, a neuroleukin agent and a differentiation and maturation mediator. Here, the first crystal structure of PGI from Mycobacterium tuberculosis H37Rv (Mtb) is reported. The structure was refined at 2.25 A resolution and revealed the presence of one molecule in the asymmetric unit with two globular domains. As known previously, the active site of Mtb PGI contains conserved residues including Glu356, Glu216 and His387 (where His387 is from the neighbouring molecule). The crystal structure of Mtb PGI was observed to be rather more similar to human PGI than other nonbacterial PGIs, with only a few differences being detected in the loops, arm and hook regions of the human and Mtb PGIs, suggesting that the M. tuberculosis enzyme uses the same enzyme mechanism.

Phosphoglucose isomerase is a ubiquitous enzyme that catalyzes the isomerization of D-glucopyranose-6-phosphate to D-fructofuranose-6-phosphate. The present investigation reports the expression, purification, crystallization and preliminary crystallographic studies of the phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv, which shares 46% sequence identity with that of its human host. The recombinant protein, which was prepared using an Escherichia coli expression system, was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.8 A and belonged to the orthorhombic space group I2(1)2(1)2(1), with unit-cell parameters a = 109.0, b = 119.8, c = 138.9 A.

Phosphoglucose isomerase (PGI) EC 5.3.1.9, is a housekeeping enzyme that catalyzes the reversible isomerization of d-glucopyranose-6-phosphate and d-fructofuranose-6-phosphate. We have previously reported expression and multistep purification of recombinant PGI from Mycobacterium tuberculosis using conventional methods. We now describe an improved and simplified single step approach for purification of functionally active mycobacterial rPGI. The gene encoding PGI from M. tuberculosis H37Rv was cloned in bacterial expression vector pET22b(+). Expression of recombinant PGI with six-histidine-tag protein was observed both in the soluble fraction and inclusion bodies. Approximately 116mg of recombinant enzyme was purified to near homogeneity with ∼80% yield from the soluble fraction of 1L culture at shake flask level using one step Ni–NTA affinity chromatography. The specific activity of the purified six-histidine-tagged recombinant PGI (rPGI-His6) was ∼800U/mg of protein. The apparent Km value of the active recombinant protein followed Michaelis–Menten kinetics and was 0.27±0.03mM. Ki for the competitive inhibitor 6-phosphogluconate was 0.75mM. The enzyme had pH optima in the range of pH 7.6–9.0 and was stable up to 55°C. rPGI-His6 exhibited enzyme activity almost equal to that of enzyme without histidine tag.