Antibody-drug conjugates (ADCs) are a recent and exciting development for targeted therapy of cancer. Their efficacy is governed by ADC-intrinsic characteristics such as avidity, drug load and linker chemistry, and mechanisms of activation and action, which can be controlled or clarified in the early stages of ADC development. In contrast, the properties that define a promising ADC target are still somewhat unclear. OGAP is a unique proteomic database that integrates information at the tissue, disease and protein isoform level across diseases, indications, and normal tissues to clarify protein expression levels and profiles. Specifically, it currently holds information on ∼2,000,000 human protein peptide sequences, ∼16,000 human proteins sequenced, ∼7,000 cancer membrane proteins, ∼50 tissues/organs, and ∼60 diseases. Building on OGAP and a proprietary sample preparation and processing workflow that relies on state-of-the-art high-throughput mass spectrometry and data processing to provide quantitative information on over 4,000 membrane-enriched proteins from ∼ 15,000 unique peptide sequences per analysis, we have established a novel predictive tool to establish each protein's potential to serve as a target for ADC development. The tool considers proteomic and target-specific information on antigenicity, structure, function, expression level, regulation, and tissue distribution in order to highlight the most suitable candidates for ADC development. We will demonstrate the utility of this process for the protein family of G-protein coupled receptors (GPCRs), which according to a recent bioinformatics prediction encompasses 899 distinct members in the human genome. These cell surface receptors are the target of more than one third of conventional drugs, yet their potential for ADCs is largely unexplored. Here we show that proteomics in the context of the OGAP database can highlight which of this large family of receptors have the potential to become true ADC targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3869. doi:1538-7445.AM2012-3869
The recent clinical success of the mAb therapeutics targeting immune checkpoint inhibitor proteins (PD-1/PD-L1, CTLA-4) has led to an increased appreciation of the potential of utilizing the immune system in oncology. There are two major strategies to elicit either a novel immune anti-tumor response or to reactivate a pre-existing anti-tumor response: by releasing a checkpoint inhibitory pathway via cell surface receptors (such as PD-1/PD-L1, CTLA-4) or by activation of co-stimulatory receptors (such as CD40, OX40, or GITR). Both of these strategies of immune modulation utilize cell surface receptors, and the targeting of antibody therapeutics with the appropriate functional activity to those receptors, to modify immune cell responses and allow for anti-tumor activity. The identification of novel immune-modulatory receptors with the potential to be immune-oncology therapeutic targets could be of high value to this anti-tumor approach. OGAP is a unique proteomic database that integrates information at the tissue, disease and protein isoform level across diseases, indications, and normal tissues to clarify membrane protein expression levels and profiles. Specifically, it currently holds information on ∼16,000 human proteins sequenced, ∼7,000 membrane proteins, ∼35 tissues/organs, and ∼17 cancers. OGAP is fed by a proprietary sample preparation and processing workflow that relies on state-of-the-art high-throughput mass spectrometry and data processing to provide quantitative information on over 4,000 membrane-enriched proteins. OGAP has been used to identify novel oncology therapeutic targets for both ADC and BiTE-like approaches. Utilizing OGAP, membrane proteins present in tumors from five cancer indications (Pancreatic, Lung, Breast, Colorectal and Esophageal cancer) and multiple normal tissues or cells were analyzed. Validated immune cell markers (such as CD8, OX40, CD79B, TLR1, TLR2, TLR4, TLR7, CD56, CD204 and CD207) were profiled across different normal and tumor proteomic data sets. This analysis demonstrated we detect key immune cell markers in tumors and that different immune cell populations are found in tumors from the same or different cancer indications. The proteomic data sets were next analyzed for the presence of validated immuno-oncology targets (TIM3, PDL-1 and B7-H3). The expression patterns of these immune-oncology targets were analyzed to try and identify a unique protein signature. Using this protein expression profiling approach we identified most known immune-oncology targets, validating this as an approach to identify a pool of candidate novel immune-oncology targets. To further develop our approach we also used sequence based homology searching of uncharacterized membrane proteins to improve the quality of the pool of candidate novel immune-oncology targets. Several potential novel immune-oncology targets will be presented. Citation Format: Jim Ackroyd, Arnima Bisht, Jason Allen, Lindsey Hudson, Martin Barnes, Christian Rohlff, Keith Wilson, Robert Boyd, Dee Aud. The use of proteomics to analyze whole tumors and identify unique immuno-oncology targets for antibody-based therapeutics. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1321. doi:10.1158/1538-7445.AM2015-1321
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 first two generations of immuno-oncology drugs have been antagonist antibodies against immune checkpoint proteins, such as cytotoxic T lymphocyte protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1). Moving forward, there has been progress in targeting co-stimulatory receptors like inducible T cell co-stimulator (ICOS), OX40 and CD137 with agonist antibodies. At Oxford BioTherapeutics, in-depth expression profiling of membrane proteins from intact tumors collected in the proprietary OGAP database revealed novel IO targets in primary tumor-derived lymphocytes (TILs). Proteomic and flow cytometry analysis of TILs and PBMCs establish that OX003R is a novel co-stimulatory IO target. It is expressed on naïve T and B cells; however, higher expression is observed in TILs and activated or exhausted T cells. OX003R expression is observed by immunohistochemistry in infiltrating lymphocytes in a variety of solid tumor types. A Fab phage display library was screened by FACS for binding to target on the cell surface. All the Fabs were also profiled by an interferon gamma release assay for T cell activation. Five best binders demonstrating T cell activation were reformatted into full-length chimeric mAbs and expressed in mammalian Expi 293 cells. Recombinant antibodies were extensively screened for T cell activation in an ex vivo 3D tumor culture system developed in-house using fresh non-small cell lung and colorectal carcinomas. Interferon gamma release was assessed by ELISpot assay and expression of the target was confirmed by immunohistochemistry on the corresponding tumor samples. Chimeric antibody 1B3 robustly activated T cells in most of the tumor samples in a dose-dependent manner as compared to isotype control and was chosen as the lead therapeutic antibody for humanization. The lead therapeutic antibody was tested for the propensity to facilitate the undesirable cytokine storm in whole blood and did not induce the release of dangerous levels of cytokines. Conclusion: OX003R is a validated immuno-oncology target and chimeric 1B3 is being developed as a promising therapeutic antibody with agonistic TIL activity, specifically in the tumor microenvironment. Citation Format: Arnima Bisht, Angelo Kaplan, Livija Deban, Chander Sekhar Peddaboina, Murray Cox, Lindsey Hudson, James Ackroyd, Nickolas Attanasio, San Lin Lou, Jason Allen, Martin Barnes, Robert Boyd, Eugene Zhukovsky, Abderrahim Fandi, Christian Rohlff. Identification and validation of a novel immuno-oncology target and selection of a therapeutic antibody candidate with a pharmacologically beneficial activity profile [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 5167.
The promise of immunotherapy for cancer is underscored by the recent efficacy of checkpoint inhibitors, which hinder the ability of tumors to escape attack by the immune system. Patients most likely to benefit from such therapy include those whose tumors are inflamed and who express the PD-L1 checkpoint protein. It is imperative that alternative therapies are developed for patients whose tumors do not exhibit these characteristics. Using our proprietary OGAP® system, we identified a novel membrane cancer target, OX001L. OX001L expression in certain cancers is associated with poor prognosis and reduced survival. IHC studies showed substantial prevalence of OX001L across multiple tumor types, with a majority of the OX001L positive samples scoring negative for PD-L1. In non-small cell lung cancer, OX001L expression was significantly increased in tumors lacking abundant intratumoral PD-1 positive T-cell infiltrate or PD-L1 expression compared to inflamed or PD-L1 positive tumors (p<0.00001). This finding suggests that OX001L may play a role in cancer immune escape. In order to effectively target OX001L positive tumor cells, we generated a human OX001L therapeutic antibody which exhibits target selectivity and cross-reactivity to the cynomolgus monkey OX001L orthologue, as demonstrated by FACS. Using immunofluorescence microscopy, we found that the antibody-antigen complex exhibits efficient internalization from the plasma membrane. Glycoengineering improved the antibody's ability to mediate potent antibody dependent cellular cytotoxicity (ADCC) activity in vitro and in vivo. Furthermore, when conjugated to a DNA alkylating toxin, the OX001L antibody promoted highly potent in vitro cytotoxicity of histotypically distinct cancer cell lines and also effected substantial anti-tumor activity in vivo. Importantly, the OX001L antibody and antibody-drug conjugate (ADC) were well tolerated in cynomolgus monkeys. These data indicate that OX001L ADC is a distinctive therapeutic molecule which could act on OX001L positive tumors via both ADC and ADCC mechanisms of action. The ability of the ADC to also promote recruitment and activation of cytotoxic T lymphocytes in OX001L positive tumors, similar to other cytotoxic payloads, will be further explored. This represents a unique therapeutic opportunity to target “cold”/PD-L1 negative, OX001L positive tumors which are less likely to respond to conventional checkpoint inhibitor therapy. In addition, a combination of the OX001L ADC and checkpoint inhibitors could be used to treat OX001L positive, “hot”/PD-L1 positive tumors. Targeting such tumors with both agents (and through two distinct, non-overlapping mechanisms of action) may yield a higher degree of success and/or a decreased propensity for relapse in patients than would checkpoint inhibitor monotherapy. We plan to test this exciting therapeutic opportunity in humanized in vivo models. Citation Format: Angelo Kaplan, Nickolas Attanasio, To Uyen T Do, Sudha Swaminathan, Arnima Bisht, San Lin Lou, Jason Allen, Robert Boyd, James E. Ackroyd, Gleb Feldman, Christian Rohlff, Rachel L. Dusek. Preclinical development of a novel antibody-drug conjugate targeting “cold” tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2772.
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