James Pan

Genome-wide CRISPR screens reveal a Wnt-FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors

Forward genetic screens with CRISPR–Cas9 genome editing enable high-resolution detection of genetic vulnerabilities in cancer cells. We conducted genome-wide CRISPR–Cas9 screens in RNF43-mutant pancreatic ductal adenocarcinoma (PDAC) cells, which rely on Wnt signaling for proliferation. Through these screens, we discovered a unique requirement for a Wnt signaling circuit: engaging FZD5, one of the ten Frizzled receptors encoded in the human genome. Our results uncover an underappreciated level of context-dependent specificity at the Wnt receptor level. We further derived a panel of recombinant antibodies that reports the expression of nine FZD proteins and confirms that FZD5 functional specificity cannot be explained by protein expression patterns. Additionally, antibodies that specifically bind FZD5 and FZD8 robustly inhibited the growth of RNF43-mutant PDAC cells grown in vitro and as xenografts in vivo, providing orthogonal support for the functional specificity observed genetically. Proliferation of a patient-derived PDAC cell line harboring an RNF43 variant was also selectively inhibited by the FZD5 antibodies, further demonstrating their use as a potential targeted therapy. Tumor organoid cultures from colorectal carcinoma patients that carried RNF43 mutations were also sensitive to the FZD5 antibodies, highlighting the potential generalizability of these findings beyond PDAC. Our results show that CRIPSR-based genetic screens can be leveraged to identify and validate cell surface targets for antibody development and therapy.

A High Throughput Screen Identifies Nefopam as Targeting Cell Proliferation in β-Catenin Driven Neoplastic and Reactive Fibroproliferative Disorders

Fibroproliferative disorders include neoplastic and reactive processes (e.g. desmoid tumor and hypertrophic scars). They are characterized by activation of β-catenin signaling, and effective pharmacologic approaches are lacking. Here we undertook a high throughput screen using human desmoid tumor cell cultures to identify agents that would inhibit cell viability in tumor cells but not normal fibroblasts. Agents were then tested in additional cell cultures for an effect on cell proliferation, apoptosis, and β-catenin protein level. Ultimately they were tested in Apc1638N mice, which develop desmoid tumors, as well as in wild type mice subjected to full thickness skin wounds. The screen identified Neofopam, as an agent that inhibited cell numbers to 42% of baseline in cell cultures from β-catenin driven fibroproliferative disorders. Nefopam decreased cell proliferation and β-catenin protein level to 50% of baseline in these same cell cultures. The half maximal effective concentration in-vitro was 0.5 uM and there was a plateau in the effect after 48 hours of treatment. Nefopam caused a 45% decline in tumor number, 33% decline in tumor volume, and a 40% decline in scar size when tested in mice. There was also a 50% decline in β-catenin level in-vivo. Nefopam targets β-catenin protein level in mesenchymal cells in-vitro and in-vivo, and may be an effective therapy for neoplastic and reactive processes driven by β-catenin mediated signaling.

A synthetic anti-Frizzled antibody engineered for broadened specificity exhibits enhanced anti-tumor properties

ABSTRACT Secreted Wnt ligands play a major role in the development and progression of many cancers by modulating signaling through cell-surface Frizzled receptors (FZDs). In order to achieve maximal effect on Wnt signaling by targeting the cell surface, we developed a synthetic antibody targeting six of the 10 human FZDs. We first identified an anti-FZD antagonist antibody (F2) with a specificity profile matching that of OMP-18R5, a monoclonal antibody that inhibits growth of many cancers by targeting FZD7, FZD1, FZD2, FZD5 and FZD8. We then used combinatorial antibody engineering by phage display to develop a variant antibody F2.A with specificity broadened to include FZD4. We confirmed that F2.A blocked binding of Wnt ligands, but not binding of Norrin, a ligand that also activates FZD4. Importantly, F2.A proved to be much more efficacious than either OMP-18R5 or F2 in inhibiting the growth of multiple RNF43-mutant pancreatic ductal adenocarcinoma cell lines, including patient-derived cells.

Abstract 3758: The efficacy of CD133 BiTEs and CAR-T cells in preclinical model of glioblastoma

Glioblastoma (GBM) is a uniformly fatal primary brain tumor, characterized by a diverse cellular phenotype and genetic heterogeneity. Despite the use of multi-modal treatment including surgical resection, radiotherapy and chemotherapy, the outcome of patients with GBM remains poor. Numerous studies have implicated CD133+ brain tumor initiating cells (BTICs) as drivers of chemo- and radio-resistance in GBM. We recently demonstrated that a CD133-driven gene signature is predictive of poor overall survival and targeting CD133+ treatment-refractory cells may be an effective strategy to block GBM recurrence. Chimeric antigen receptors (CARs) and bispecific T-Cell engaging antibodies (BiTEs) present promising immunotherapeutic approaches that have not yet been validated for recurrent GBM. Using CellectSeq, a novel methodology that combines use of phage-displayed synthetic antibody libraries and DNA sequencing, we developed the CD133-specific monoclonal antibody ‘RW03’. We constructed CD133-specific BiTEs that consist of two arms; one arm recognizes the tumor antigen (CD133) while the second is specific to CD3 antigen. The dual binding specificity was confirmed using flow cytometry. Using CD133high and CD133low primary GBM lines, we validated the binding of BiTEs to CD133+ cells. Further analysis showed binding of BiTEs to human T cells known to express CD3 within a population of healthy donor peripheral blood mononuclear cells. We observed BiTEs redirecting T cells to kill GBMs, with greater efficiency observed in CD133high GBMs, validating BiTE target specificity. Incubating T-cells with BiTEs and the CD133high GBMs resulted in increased expression of T cell activation markers. In parallel, we derived the single chain variable fragment (scFv) from previously generated RW03 and generated a second-generation CAR. Anti-CD133 scFv with a myc tag was cloned in frame with a human CD8 leader sequence, CD8a transmembrane domain, CD28, and hCD3ζ signaling tail in the lentiviral construct pCCL-ΔNGFR. Following lentiviral packaging, the T cells isolated from PBMCs were transduced with CD133 CAR construct. After successful T cell engineering, the expression of ΔNGFR and myc tag was analyzed using flow cytometry to confirm the efficiency of transduction and surface expression of anti-CD133 respectively. CD133-specific CAR-T cells were cytotoxic to CD133+ GBMs. Co-culturing CD133 CAR-T cells with GBMs triggered T cell activation and proliferation. Treatment of GBM tumor-bearing mice with CD133-specific CAR-T cells yielded extended survival in mice and significant reductions in brain tumor burden. The results of this study will establish a translational research program that will form the basis of early phase clinical trials of a promising CD133-based therapeutic strategy for patients with GBM. Citation Format: Parvez Vora, Chirayu Chokshi, Maleeha Qazi, Mohini Singh, Chitra Venugopal, Sujeivan Mahendram, Jarrett Adams, David Bakhshinyan, Max London, Jess Singh, Minomi Subapanditha1,, Nicole McFarlane, James Pan, Jonathan Bramson, Sachdev Sidhu, Jason Moffat, Sheila Singh. The efficacy of CD133 BiTEs and CAR-T cells in preclinical model of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3758. doi:10.1158/1538-7445.AM2017-3758

A CRISPR screen reveals a WNT7B-FZD5 signaling circuit as a therapeutic opportunity in pancreatic cancer.

CRISPR-Cas9 genome editing enables high-resolution detection of genetic vulnerabilities of cancer cells. We conducted a genome-wide CRISPR-Cas9 screen in RNF43 mutant pancreatic ductal adenocarcinoma (PDAC) cells, which rely on Wnt signaling for proliferation, and discovered a unique requirement for a WNT7B-FZD5 signaling circuit. Our results highlight an underappreciated level of functional specificity at the ligand-receptor level. We derived a panel of recombinant antibodies that reports the expression of nine out of ten human Frizzled receptors and confirm that WNT7B-FZD5 functional specificity cannot be explained by protein expression patterns. We developed two human antibodies that target FZD5 and robustly inhibited the growth of RNF43 mutant PDAC cells grown in vitro and as xenografts, providing strong orthogonal support for the functional specificity observed genetically. Proliferation of a patient-derived PDAC cell line harboring a RNF43 variant previously associated with PDAC was also selectively inhibited by the FZD5 antibodies, further demonstrating their use as a potential targeted therapy.

Abstract B079: The efficacy of CD133 BiTEs and CAR-T cells in preclinical model of recurrent glioblastoma

Glioblastoma (GBM) is a uniformly fatal primary brain tumor, characterized by a diverse cellular phenotype and genetic heterogeneity. Despite the use of aggressive cellular multi-modal treatment including surgical resection, radiotherapy and chemotherapy, the outcome of patients with GBM has failed to improve significantly. Numerous studies have implicated CD133+ brain tumor initiating cells (BTICs) as drivers of chemo- and radio-resistance in GBM. We have recently demonstrated that a CD133-driven gene signature is predictive of poor overall survival and targeting CD133+ treatment-refractory cells may be an effective strategy to block GBM recurrence. Chimeric antigen receptors (CARs) and bispecific T-Cell engaging antibodies (BiTEs) present promising immunotherapeutic approaches that have not yet been validated for recurrent GBM. Using CellectSeq, a novel methodology that combines use of phage-displayed synthetic antibody libraries and DNA sequencing, we developed the CD133-specific monoclonal antibody ‘RW03’. We constructed CD133-specific BiTEs or RW03xCD3 that consist of two arms; one arm recognizes the tumor antigen (CD133) while the second is specific to CD3 antigen. The BiTEs were constructed in four different conformations and dual binding specificity was confirmed using flow cytometry. Using CD133high and CD133low primary GBM lines, we validated the binding of BiTEs to CD133+ cells. Further analysis showed binding of BiTEs to human T cells known to express CD3 within a population of healthy donor peripheral blood mononuclear cells. We observed BiTEs redirecting T cells to kill GBMs, with greater efficiency observed in CD133high GBMs, validating BiTE target specificity. Incubating T-cells with BiTEs and the CD133high GBMs resulted in increased expression of T cell activation markers. In parallel, we derived the single chain variable fragment (scFv) from previously generated RW03 and generated a second-generation CAR. Anti-CD133 scFv with a myc tag was cloned in frame with a human CD8 leader sequence, CD8a transmembrane domain, CD28, and hCD3ζ signaling tail in the lentiviral construct pCCL-ΔNGFR vector in two different orientations: Light chain-linker-Heavy chain (CD133 CAR-LH) and Heavy chain-linker-Light chain (CD133 CAR-HL). Following lentiviral preparation, the T cells isolated from PBMCs were transduced with CD133 CAR-LH and CD133 CAR-VH constructs. After successful T cell engineering, the expression of ΔNGFR and myc tag was analyzed using flow cytometry to confirm the efficiency of transduction and surface expression of anti-CD133 respectively. CD133-specific CAR-T cells were cytotoxic to CD133+ GBMs. Co-culturing CD133 CAR-T cells with GBMs triggered T cell activation and proliferation. Treatment of GBM tumor-bearing mice with CD133-specific CAR-T cells yielded extended survival in mice and significant reductions in brain tumor burden. Furthermore, we uniquely adapted the existing chemoradiotherapy protocol for GBM patients for treatment of immunocompromised mice engrafted with human GBMs. Within this model, we have initiated treatment of recurrent GBM directed against CD133+ BTICs, to allow for a direct prospective comparison of toxicity and efficacy of BiTEs and CAR T cell strategies. Citation Format: Parvez Vora, Chirayu Chokshi, Maleeha Qazi, Chitra Venugopal, Sujeivan Mahendram, Mohini Singh, Jarrett Adams, David Bakhshinyan, Max London, Minomi Subapanditha, Nicole McFarlane, James Pan, Jonathan Bramson, Jason Moffat, Sachdev Sidhu, Sheila Singh. The efficacy of CD133 BiTEs and CAR-T cells in preclinical model of recurrent glioblastoma [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B079.

Abstract 1481: Preclinical validation of a novel CD133/CD3 bispecific T-cell engager (BiTE) antibody to target patient-derived glioblastoma cells

Glioblastoma (GBM), an aggressive primary brain tumor in adults, is feared for its near uniformly fatal prognosis and is characterized by a diverse cellular phenotype and genetic heterogeneity. Despite the use of aggressive multi-modal treatment including surgical resection, radiotherapy and chemotherapy, the outcome of patients with GBM has failed to improve significantly. We developed patient-derived brain tumor initiating cell (BTIC) early passage lines that describe the extent of intertumoral heterogeneity, presenting a powerful preclinical model of GBM. Numerous studies have implicated CD133+ BTICs as drivers of chemo- and radio-resistance in GBM. CD133 expression correlates with disease progression, recurrence, and poor overall survival of GBM patients. Here, we describe the preclinical evaluation of a recombinant RW03xCD3 bispecific T-cell engager (BiTE) antibody that redirects human polyclonal T cells to CD133+ GBM cells, inducing very potent anti-tumor response. Using CellectSeq, a novel methodology that combines use of phage-displayed synthetic antibody libraries and high-throughput DNA sequencing technology, we developed the CD133-specific monoclonal antibody ‘RW03’. We constructed CD133-specific BiTEs or RW03xCD3 that consist of two arms; one arm recognizes the tumor antigen (CD133) while the second is specific to CD3 antigen. The BiTEs were constructed in four different conformations and dual binding specificity was confirmed using flow cytometry. Using CD133high and CD133low primary GBM lines, we validated the binding of BiTEs to CD133+ cells. Further analysis showed binding of BiTEs to human T cells known to express CD3 within a population of healthy donor peripheral blood mononuclear cells. In order to test the ability of BiTEs to functionally elicit CD133-specific cytotoxic responses in vitro, we performed Presto blue-based killing assays. We observed CD133-specific BiTEs redirect T cells to kill CD133-expressing GBM cells in a coculture of T cells and GBM cells. The killing was more efficient in CD133high GBMs compared to CD133low GBMs, validating its specificity to target CD133+ BTICs. Incubating T cells with BiTEs and GBMs resulted in increased surface expression of T-cell activation markers CD69 and CD25 in both, CD4+ and CD8+ T cells populations. Treatment with BiTEs yielded extended survival in mice and significant reductions in brain tumor burden. This rigorously obtained data offers compelling evidence that BiTE-mediated cytotoxicity against treatment-resistant and evasive CD133+ GBM BTICs could provide a very potent, specific and novel therapeutic strategy for GBM patients. Citation Format: Parvez Vora, Chitra Venugopal, Jarrett Adams, James Pan, Chirayu Chokshi, Maleeha Qazi, Minomi Subapanditha, Mohini Singh, David Bakhshinyan, Ksenia Bezverbnaya, Nicole McFarlane, Jonathan Bramson, Sachdev Sidhu, Jason Moffat, Sheila Singh. Preclinical validation of a novel CD133/CD3 bispecific T-cell engager (BiTE) antibody to target patient-derived glioblastoma cells. [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 1481.