Christian Rohlff

Abstract 2630: MEN1309, a novel antibody drug conjugate (ADC) targeting Ly75 antigen, induces complete responses in several xenografts of solid tumors

The cell surface antigen Lymphocyte antigen 75 (LY75, CD205, DEC-205) is over-expressed in several tumor histotypes. It is a type I C-type lectin receptor (CLR), normally expressed on various APC subsets, characterized by a cytoplasmic domain containing protein motifs crucial for endocytosis and internalization upon ligation. These features make the antigen ideal to be exploited as a target for a novel ADC. MEN1309 is a humanized IgG1 antibody directed against the cell surface antigen Ly75, conjugated through a cleavable linker to a potent maytansinoid microtubule disruptor, DM4. In this study, we evaluated the in vitro and in vivo (xenografts and PDX) efficacy of MEN1309 in different tumor histotypes. A PK/PD relationship was also investigated in tumor-bearing mice. IHC demonstrated high prevalence of Ly75 in human pancreatic, triple negative breast, and bladder cancers, as well as in diffuse large B-cell lymphoma. In vitro experiments showed that cytotoxic activity of MEN1309 was in nM/sub nM range against several lymphoma, pancreatic, bladder and triple-negative breast cancer (TNBC) cell lines. Moreover, MEN1309 exhibited high cell-killing ability against cells having either strong as well as low to moderate antigen expression. In vivo, MEN1309 at 2.5-5 mg/kg (schedule varying from single dose, q7dx3, or q21dx3) showed an impressive antitumor activity, resulting in complete and long lasting responses in most of the xenograft models representing lymphoma, TNBC, bladder and pancreatic cancers, expressing the antigen at high but also at low levels. No treatment related toxicity in terms of change of body weight and death events were detected. Moreover, the administration of (i) isotype control-DM4, (ii) the non-conjugate antibody IgG1 and (iii) the free toxin DM4 (at a dosage corresponding to the equimolar concentration linked at 10 mg/kg ADC) showed little to no therapeutic efficacy on tumor growth. In TNBC patient-derived xenograft (PDX) model (coming from a heavily pre-treated patient and expressing high level of the antigen Ly75), MEN1309 (5 mg/kg q21dx3) showed a complete tumor regression. Finally, in the pancreatic adenocarcinoma xenograft model HPAFII, the pharmacokinetics profile in serum of MEN1309 at 5 mg/kg was characterized and it was qualitatively correlated, using immunofluorescence, with the occurrence of phosphorylation of Serine 10 of H3 Histone in cancer cells, as a pharmacodynamic (PD) marker of DM4 activity on microtubules. Initial ADC exposure was noteworthy and was followed by a relatively fast decline. In parallel with the decay of the serum ADC concentrations there was a progressive increase in the number of positive cells showing the PD marker for mitotic arrest. Overall, our data suggest that MEN1309 is a selective and potent novel antitumoral ADC and it deserves to enter into aPhase I study for a variety of Ly75 positive tumor histotypes. Citation Format: Mario Bigioni, Giuseppe Merlino, Cristina Bernado Morales, Rossana Bugianesi, Attilio Crea, Rosanna Manno, Joaquin Arribas, Rachel Dusek, Nickolas Attanasio, Keith Wilson, Christian Rohlff, Monica Binaschi. MEN1309, a novel antibody drug conjugate (ADC) targeting Ly75 antigen, induces complete responses in several xenografts of solid tumors [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 2630. doi:10.1158/1538-7445.AM2017-2630

Abstract 3646: Characterization of the novel antibody drug conjugate MEN1309 and its target antigen Ly75

Ly75 (CD205, DEC-205) is a type I transmembrane glycoprotein and a C-type lectin receptor involved in antigen uptake and processing, mainly expressed by antigen presenting cells (APC). The short cytoplasmic tail contains motifs for amino acid-based endocytosis, making this receptor an ideal target antigen for an antibody drug conjugate (ADC)-based antitumoral therapy. MEN1309 is a novel fully humanized ADC which binds to Ly75 with high affinity as shown by ELISA and FACS analysis. The antibody is conjugated to a maytansinoid DM4, a potent tubulin inhibitor, through a cleavable linker.The ability of Ly75 to internalize the antibody after binding was determined using an immunoflourescence assay that showed a rapid, efficient, and near complete internalization over a one hour time course.The expression of Ly75 mRNA and protein was investigated in human cancer cell lines derived from different histotypes and revealed high expression in pancreas, bladder, triple negative breast cancer (TNBC) cells and in diffuse large B-cell lymphoma (DLBCL). Indeed, MEN1309 shows a powerful (pM range) in vitro cytotoxic activity on different cancer cell lines expressing Ly75, whereas it exerts a weaker effect on antigen-negative cells. Besides the mechanism of action (MoA) of MEN1309 as an ADC, the putative efficacy of the antibody to drive an ADCC response was investigated through in vitro binding and functional assays. In spite of a high binding affinity of MEN1309 to FcγRIIIa, no ADCC response was observed, suggesting that the high internalization rate of the antigen could hamper the triggering of NK responses.Moreover, in order to characterize the functional role of Ly75 in cancer cell lines, its expression was downregulated by siRNA demonstrating an inhibition of the proliferation rate in cells from different histotypes.Finally, we investigated if some cancer cell lines could show a higher expression of two intergenically spliced forms derived from Ly75 and DCL-1 genes recently reported in literature. We found that the intergenically spliced forms were expressed on average 30 fold less than CD205 mRNA in all the cancer cell lines analyzed, suggesting that these variants derive just from an intergenic readthrough without a specific transcriptional regulation. Citation Format: Alessandro Bressan, Alessio Fiascarelli, Giuseppe Merlino, Corrado Carrisi, Daniela Bellarosa, Rachel Dusek, Rahel Awdew, Sudha Swaminathan, Arnima Bisht, To Uyen T. Do, San Lin Lou, Dee Aud, Jonathan Terrett, Keith Wilson, Christian Rohlff, Monica Binaschi. Characterization of the novel antibody drug conjugate MEN1309 and its target antigen Ly75 [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 3646. doi:10.1158/1538-7445.AM2017-3646

Abstract 1321: The use of proteomics to analyze whole tumors and identify unique immuno-oncology targets for antibody-based therapeutics

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

Abstract 661: Proteomics and selecting the right combination of target and toxin for antibody-drug-conjugate (ADC) development

With the successes of Kadcyla and Adcetris, and the emerging data from early phase trials of many other ADCs our understanding of the critical attributes for target selection and ADC development has improved significantly. Clinical experience indicates that the dose-limiting toxicities observed are predominately off-target and thus are similar across ADCs using the same linker and drug combinations and independent of the target antigen. Furthermore, the maximum tolerated doses (MTDs) in humans, monkeys, and mice for many ADCs seem to be very similar regardless of the protein being targeted. Thus, the therapeutic index of new ADCs using the current drug/linker technologies (tubulin and DNA targeting toxins) will be driven predominately by efficacy, specifically the ability to achieve antitumor activity at exposure levels below the MTD. Therefore, the focus of future target selection should be on efficacy within the therapeutic window rather than on antigen-dependent (i.e. on-target) toxicity arising from normal tissue expression. We have selected targets and toxins for ADC development based on the likelihood of robust potency against cancer cells. The properties favored are : 1) high expression and high prevalence in selected malignancies, 2) normal tissue expression profiles known to be tolerable for ADCs ( e.g. her2neu, gpnmb, nectin4), 3) targets enabling rapid intracellular delivery of ADC and release of active toxin, 4) clinical indications sensitive to the toxin of choice. This process and its success are demonstrated here through the development of two ADCs. Firstly, Ox-1476 for triple negative breast cancer (TNBC). The target was selected based on consistent high expression in TNBC, the MAb selected from a large screen of greater than 1000 candidates based on activity as an actual ADC, and the toxin selected based on the positive experience with maytansine in breast cancer (Kadcyla) and the preclinical data presented here. The Ox-1476 ADC shows greater and broader activity than Kadcyla in vitro and an initial toxicity profile consistent with pursuing clinical development. Secondly, Ox-1425 is being developed for small cell lung cancer (SCLC). The target and MAb were selected by the same processes but for Ox-1425 the toxin screen resulted in the selection of a DNA alkylating agent. Our proteomics database has provided a unique data set of candidate cell surface targets for the development of antibodies compatible with the current state of the art ADC payloads. The selected targets show high and prevalent expression in cancers, are in protein families shown to internalize efficiently, and have normal tissue expression profiles similar to other proteins already being targeted by clinical stage ADCs. The preclinical safety and activity data shown here is supportive of our strategy for selecting ADC targets. Citation Format: Jonathan A. Terrett, Rachel Dusek, Dee Aud, Rahel Awdew, Sudha Swaminathan, San Lin Lou, Michael Trang, Arnima Bisht, Mary Do, Jim Ackroyd, Robert Boyd, Lindsey Hudson, Phuoc Pham, Nickolas Attanasio, Ami Antani, Carmel Lynch, Christian Rohlff. Proteomics and selecting the right combination of target and toxin for antibody-drug-conjugate (ADC) development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 661. doi:10.1158/1538-7445.AM2014-661

Abstract 3869: Proteomics highlights which G-protein coupled receptors are candidates for ADC development

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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 proteins 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

Abstract 1683: High throughput membrane proteomics for the discovery of oncology targets ideally suited for the development of antibody drug conjugates (ADCs)

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Antibody Drug Conjugates (ADCs) are showing tremendous promise for cancer patients as recently demonstrated by the dramatic responses reported in breast cancer and Hodgkins Lymphoma clinical trials to the ADCs T-DM1 and SGN-35. We have developed proteomics methods to enrich for membrane proteins that are expressed preferentially in cancer cells over normal cells. Potential ADC targetable proteins are then validated using the proprietary OGAP database as well as conventional expression analysis tools. The proteomics processes have identified novel cancer specific proteins as well as cancer specific isoforms of known proteins. These targets are then combined with large panels of antibodies to discover potential therapeutics with properties desirable for the development of ADCs. Antibodies are selected which : a) preferentially bind to tumor cells over normal cells, b) have good specificity and affinity, c) show high prevalence in target cancer indications by IHC, d) internalize on antigen binding, e) bind to relevant tox species tissues as they bind to human tissues. Here we describe the proteomics processes and use of the OGAP database and demonstrate the development of candidate therapeutic antibodies with the potential to become ADCs, which are directed to novel targets and cancer specific isoforms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1683. doi:10.1158/1538-7445.AM2011-1683