Bhaswati Barat

Cys-diabody Quantum Dot Conjugates (ImmunoQdots) for Cancer Marker Detection

The present work demonstrates the use of small bivalent engineered antibody fragments, cys-diabodies, for biological modification of nanoscale particles such as quantum dots (Qdots) for detection of target antigens. Novel bioconjugated quantum dots known as immunoQdots (iQdots) were developed by thiol-specific oriented coupling of tumor specific cys-diabodies, at a position away from the antigen binding site to amino PEG CdSe/ZnS Qdots. Initially, amino PEG Qdot 655 were coupled with reduced anti-HER2 cys-diabody by amine-sulfhydryl-reactive linker [N-ε-maleimidocaproyloxy] succinimide ester (EMCS) to produce anti-HER2 iQdot 655. Spectral characterization of the conjugate revealed that the spectrum was symmetrical and essentially identical to unconjugated Qdot. Specific receptor binding activity of anti-HER2 iQdot 655 was confirmed by flow cytometry on HER2 positive and negative cells. Immunofluorescence results showed homogeneous surface labeling of the cell membrane with Qdot 655 conjugate. In addition, cys-diabodies specific for HER2, as well as prostate stem cell antigen (PSCA), were conjugated successfully with amino PEG Qdot 800. All of these iQdots retain the photoluminescence properties of the unconjugated Qdot 800 as well as the antigen binding specificity of the cys-diabody as demonstrated by flow cytometry. Simultaneous detection of two tumor antigens on LNCaP/PSCA prostate cancer cells (which express PSCA and HER2) in culture was possible using two iQdots, anti-HER2 iQdot 655 and anti-PSCA iQdot 800. Thus, these iQdots are potentially useful as optical probes for sensitive, multiplexed detection of surface markers on tumor cells. The present thiol-specific conjugation method demonstrates a general approach for site-specific oriented coupling of cys-diabodies to a wide variety of nanoparticles without disturbing the antigen binding site and maintaining small size compared to intact antibody.

A peptide from autoantigen La blocks poliovirus and hepatitis C virus cap-independent translation and reveals a single tyrosine critical for La RNA binding and translation stimulation.

ABSTRACT La, a 52-kDa autoantigen in patients with systemic lupus erythematosus, was one of the first cellular proteins identified to interact with viral internal ribosome entry site (IRES) elements and stimulate poliovirus (PV) and hepatitis C virus (HCV) IRES-mediated translation. Previous results from our laboratory have shown that a small, yeast RNA (IRNA) could selectively inhibit PV and HCV IRES-mediated translation by sequestering the La protein. Here we have identified an 18-amino-acid-long sequence from the N-terminal “La motif” which is required for efficient interaction of La with IRNA and viral 5′ untranslated region (5′-UTR) elements. A synthetic peptide (called LAP, for La peptide) corresponding to this sequence (amino acids 11 to 28) of La was found to efficiently inhibit viral IRES-mediated translation in vitro. The LAP efficiently enters Huh-7 cells and preferentially inhibits HCV IRES-mediated translation programmed by a bicistronic RNA in vivo. The LAP does not bind RNA directly but appears to block La binding to IRNA and PV 5′-UTR. Competition UV cross-link and translation rescue experiments suggested that LAP inhibits IRES-mediated translation by interacting with proteins rather than RNA. Mutagenesis of LAP demonstrates that single amino acid changes in a highly conserved sequence within LAP are sufficient to eliminate the translation-inhibitory activity of LAP. When one of these mutations (Y23Q) is introduced into full-length La, the mutant protein is severely defective in interacting with the PV IRES element and consequently unable to stimulate IRES-mediated translation. However, the La protein with a mutation of the next tyrosine moiety (Y24Q) could still interact with PV 5′-UTR and stimulate viral IRES-mediated translation significantly. These results underscore the importance of the La N-terminal amino acids in RNA binding and viral RNA translation. The possible role of the LAP sequence in La-RNA binding and stimulation of viral IRES-mediated translation is discussed.

Site-specific, thiol-mediated conjugation of fluorescent probes to cysteine-modified diabodies targeting CD20 or HER2

Small, engineered antibody fragments such as diabodies (50 kDa noncovalent dimers of single-chain Fv fragments) are useful alternatives to their larger antibody counterparts. However, due to their size, they are more susceptible to disruption of their antigen binding sites when modified using random conjugation techniques. Previous work has demonstrated the utility of a C-terminal cysteine modification for site-specific radiolabeling of an anti-CEA diabody, resulting in the creation of a cys-diabody (CysDb). In the present work, the adaptability of the CysDb system was explored by creating two additional CysDbs: one specific for CD20 and one for HER2. Purified CysDbs of both specificities demonstrated behavior consistent with stable, covalent dimers harboring a readily reducible disulfide bond. Each CysDb was site-specifically conjugated to three different fluorophores for optical detection: the large fluorescent proteins phycoerythrin (PE) and allophycocyanin (APC), and the small fluorescent molecule Alexa Fluor488. Fluorophore-conjugated CysDbs bound specifically to their targets in both antigen systems and with each different fluorescent tag as determined by flow cytometry. In vitro specific antigen binding was observed in the presence of a mixture of specific and nonspecifically conjugated CysDbs. Conjugates retained both specificity and fluorescence, demonstrating the successful expansion of the CysDb repertoire to new targets and to new site-specific conjugation possibilities.

Activation of ribosomal RNA transcription by hepatitis C virus involves upstream binding factor phosphorylation via induction of cyclin D1.

Hepatitis C virus (HCV) causes chronic infection in humans leading to liver cirrhosis and hepatocellular carcinoma. rRNA transcription, catalyzed by RNA polymerase I (Pol I), plays a critical role in ribosome biogenesis, and changes in Pol I transcription rate are associated with profound alterations in the growth rate of the cell. Because rRNA synthesis is intimately linked to cell growth and frequently up-regulated in many cancers, we hypothesized that HCV might have the ability to activate rRNA synthesis in infected cells. We show here that rRNA promoter-mediated transcription is significantly (10- to 12-fold) activated in human liver-derived cells following infection with type 2 JFH-1 HCV or transfection with the subgenomic type 1 HCV replicon. Further analysis revealed that HCV nonstructural protein 5A (NS5A) was responsible for activation of rRNA transcription. Both the NH(2)-terminal amphipathic helix and the polyproline motifs of NS5A seem to be essential for rRNA transcription activation. The NS5A-dependent activation of rRNA transcription seems to be due to hyperphosphorylation and consequent activation of upstream binding factor (UBF), a Pol I DNA binding transcription factor. We further show that hyperphosphorylation of UBF occurs as a result of up-regulation of both cyclin D1 and cyclin-dependent kinase 4 by the HCV NS5A polypeptide. These results suggest that the endoplasmic reticulum-associated NS5A is able to transduce signals into the nucleoplasm via UBF hyperphosphorylation leading to rRNA transcription activation. These results could, at least in part, explain a mechanism by which HCV contributes to transformation of liver cells.

Evaluation of Two Internalizing Carcinoembryonic Antigen Reporter Genes for Molecular Imaging

PurposeThe objective of this article is to develop internalizing positron emission tomography (PET) reporter genes for tracking genetically modified T cells in vivo.ProceduresThe transmembrane and cytoplasmic domains of the human transferrin receptor (TfR) and CD5 were each fused to the carcinoembryonic (CEA) minigene N-A3 and expressed in Jurkat T cells. Internalization was evaluated by confocal microscopy or by intracellular uptake of 125I-labeled anti-CEA scFv-Fc. Reporter gene-transfected Jurkat xenografts in mice were analyzed by immunohistochemistry (IHC) and imaged by PET using 124I- or 64Cu-scFv-Fc as tracers.ResultsSurface expression of TR(1–99)-NA3 was lower than that of NA3-CD5. Both reporter genes were internalized following binding of the anti-CEA antibody fragment. IHC of tumors showed strong staining of NA3-CD5, whereas TR(1–99)-NA3 stained weakly. Specific targeting of TR(1–99)-NA3 or NA3-CD5 was shown by PET in xenografted mice.ConclusionsThe in vivo imaging studies suggest a potential application of the internalizing form of CEA (N-A3) as a PET reporter gene.

Abstract 1201: Anti-B7-H3 antibody-drug conjugates as potential therapeutics for solid cancer

Introduction: Monoclonal antibodies (mAbs) were generated via a target-unbiased approach based on intact cell immunization with cell lines, fetal progenitor cells, and cancer stem cells. An immunohistochemical screen for cancer-specific candidates identified a panel of anti-B7-H3 (CD276) mAbs with highly differential tumor-versus-normal tissue binding. B7-H3 expression was observed in tumor epithelium as well as tumor-associated vasculature and stroma. Consistent with our findings, B7-H3 has been reported to be overexpressed in a growing number of solid cancers, including breast, lung, pancreatic, prostate, kidney, and colon cancer, as well as melanoma and glioblastoma. Furthermore, overexpression of B7-H3 has been correlated with disease severity and poor outcome in a number of these cancer types. A humanized version of an anti-B7-H3 mAb engineered with an enhanced Fc domain (enoblituzumab or MGA271) and a humanized Dual-Affinity Re-Targeting (DART®) protein that recognizes both B7-H3 and CD3 and redirects T cells to kill B7-H3-expressing cells (MGD009) are being investigated in Phase 1 clinical studies. In this nonclinical study, we evaluated the therapeutic potential of anti-B7-H3 antibody-drug conjugates (ADCs) toward B7-H3-expressing solid cancers. Methods: A panel of anti-B7-H3 mAbs was screened for internalization and a subset of mAbs that were efficiently internalized by tumor cells was identified. These mAbs were converted to ADCs via chemical conjugation; in vitro and in vivo activity studies were then conducted with a range of tumor cell lines representing human cancer types that overexpress B7-H3. Results: The anti-B7-H3 ADCs exhibited specific, dose-dependent cytotoxicity toward B7-H3-positive tumor cell lines in vitro, including breast, lung, ovarian, pancreatic, and prostate cancer lines, with IC50 values generally in the sub-nM range. Cytotoxicity was not observed with cell lines lacking B7-H3 expression. The anti-B7-H3 ADCs exhibited potent antitumor activity in vivo, resulting in tumor stasis and tumor regression in mice bearing B7-H3-positive human breast, lung, and ovarian tumor xenografts. Conclusion: Anti-B7-H3 ADCs exhibited dose-dependent cytotoxicity in vitro and potent antitumor activity in vivo toward a range of B7-H3-expressing tumor cell lines representing cancer types that overexpress B7-H3. Our findings demonstrate that ADCs targeting B7-H3 may serve as potential therapeutics for B7-H3-expressing solid cancers. Citation Format: Deryk Loo, Juniper A. Scribner, Thomas Son, Jeff Hooley, Timothy Hotaling, Michael Chiechi, Pam Li, Anushka De Costa, Yan Chen, Ann Easton, Francine Z. Chen, Bhaswati Barat, Valentina Ciccarone, James Tamura, Mark Kubik, Scott Koenig, Syd Johnson, Paul A. Moore, Ezio Bonvini. Anti-B7-H3 antibody-drug conjugates as potential therapeutics for solid cancer. [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 1201.

Abstract 38: Target validation, antibody discovery and preclinical data supporting ADAM9 as an antibody-drug conjugate therapeutic target for solid tumors

Introduction: A target-unbiased approach based on intact cell immunizations with fetal progenitor cells and cancer stem cells, followed by an immunohistochemistry (IHC) screen for cancer-specific candidates, led to the identification of anti-ADAM9 (a disintegrin and metalloproteinase) mAbs with highly differential tumor-versus-normal tissue binding. ADAM9 is a cell surface protein over-expressed in multiple tumors, with a possible role in promotion and progression of cancer through multiple mechanisms, including modulation of adhesion and migration as well as processing of tumorigenic and pro-angiogenic factors. In this preclinical study, we performed target/mAb validation and evaluated the therapeutic potential of anti-ADAM9 antibody-drug conjugates (ADCs) toward ADAM9-expressing solid cancers. Methods: IHC was performed with anti-ADAM9 mAbs to confirm and extend available data of human normal and tumor tissue expression. Epitope mapping studies were conducted to define epitope-specificity. mAbs were also screened to identify those that efficiently internalized into tumor cells. In vitro cellular processing studies were performed to further evaluate the mAbs as ADC candidates. Selected mAbs were converted to ADCs via chemical conjugation to potent anti-microtubule (DM4) or DNA alkylating (DGN549) agents; in vitro cytotoxicity studies were conducted with tumor cell lines representing human cancer types that overexpress ADAM9. A lead mAb was then selected for humanization and affinity maturation to yield a development candidate. Results: Anti-ADAM9 mAbs exhibited strong reactivity toward the tumor epithelium of solid cancers, including pancreatic, kidney, prostate, bladder, breast, colon, lung, and ovarian cancer, but limited reactivity toward normal tissues. Anti-ADAM9 mAbs were efficiently internalized and processed by tumor cell lines, including lines with only modest ADAM9 expression. Anti-ADAM9 ADCs exhibited specific, dose-dependent cytotoxicity toward ADAM9-positive cancer cell lines in vitro, with IC 50 values in the sub-nanomolar range. Humanization and affinity maturation of the lead mAb yielded a development candidate that retains potent antitumor activity toward ADAM9-positive tumor cell lines and equivalent, high affinity binding to both human and cynomolgus monkey ADAM9. Conclusion: ADAM9 is a cell surface antigen that is over-expressed on a wide range of solid cancers. Anti-ADAM9 mAbs that were strongly reactive with representative tumors exhibited high affinity for the antigen and were efficiently internalized and processed by ADAM9-bearing tumor cells. Anti-ADAM9 ADCs demonstrated dose-dependent cytotoxicity in vitro toward a panel of ADAM9-positive tumor cell lines. Our findings demonstrate that an ADC targeting ADAM9 may serve as a potential therapeutic for ADAM9-expressing solid tumors. Citation Format: Juniper A. Scribner, Bhaswati Barat, Stuart W. Hicks, Nicholas C. Yoder, Thomas Son, Lusiana Widjaja, Gundo Diedrich, Sergey Gorlatov, Jeff Hooley, Ann Easton, Peter Lung, Anushka De Costa, Francine Chen, Michael Chiechi, Pam Li, Monica Licea, Timothy E. Hotaling, Michael Spliedt, Valentina Ciccarone, Nadia Gantt, James Tamura, Megan E. Fuller, Molly McShea, Scott Koenig, Syd Johnson, Paul A. Moore, Ezio Bonvini, Deryk Loo. Target validation, antibody discovery and preclinical data supporting ADAM9 as an antibody-drug conjugate therapeutic target for 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 38. doi:10.1158/1538-7445.AM2017-38

Abstract 37: Novel antibody-drug conjugates targeting ADAM9-expressing solid tumors demonstrate potent preclinical activity

ADAM9, also known as MDC9 or meltrin-γ, is a member of the ADAM (a disintegrin and metalloproteinase) family of proteases, which have been implicated in cytokine and growth factor shedding, and cell migration. Dysregulation of ADAM9 has been implicated in tumor progression and metastasis, as well as pathological neovascularization. ADAM9 overexpression has been shown to correlate with poor prognosis in prostate, renal, and pancreatic cancers. Using an immunization approach in which antibodies were raised to fetal progenitor and stem-like cancer cell lines followed by screening on tumor and normal tissues, we identified ADAM9 as a promising cell surface tumor target. FFPE-IHC expression analysis revealed that ADAM9 is overexpressed in multiple solid tumor indications relative to corresponding normal tissues. The overexpression of ADAM9 in tumors coupled with its restricted expression in normal tissues make ADAM9 an attractive target for antibody-drug conjugate (ADC) therapy. Here, we describe two ADCs both of which are based on a high affinity anti-ADAM9 antibody to selectively target ADAM9-expressing tumors. The first ADC utilizes the maytansine-derived microtubule disruptor, DM4, linked via a hindered disulfide hydrophilic linker (sulfo-SPDB). The second ADC exploits an ultra-potent DNA alkylating payload, DGN549, which is conjugated to two engineered cysteines via a peptide linker. Both conjugates bound with similar subnanomolar affinity to ADAM9-expressing cells. In vitro cytotoxicity studies showed that anti-ADAM9 ADCs can kill a broad panel of ADAM9-positve tumor cell lines, including lung, pancreatic, renal, prostate, and colon tumor cell lines. In particular, the anti-ADAM9-DGN549 conjugate was extremely potent with IC50 values ranging from 0.1 to 65 pM and was at least 2 logs more active than a non-targeting conjugate. Surprisingly, efficient in vitro cytotoxicity was observed at ADAM9 expression levels as low as a few thousand cell surface receptors per cell. Consistent with their in vitro activity, both anti-ADAM9 ADCs displayed compelling anti-tumor activity in xenograft models. In a CaLu3 non-small cell lung cancer xenograft model, anti-ADAM9-DM4 induced tumor growth delay at a single 1.25 mg Ab/kg dose. In the same model, a single intravenous dose of 0.25 mg Ab/kg of the anti-ADAM9-DGN549 produced durable complete remissions in 8/8 mice. A non-targeting DGN549 ADC was inactive even when dosed at 10 times that of the anti-ADAM9 ADC, demonstrating that targeted delivery of DGN549 through ADAM9 binding is required for activity. These data demonstrate that anti-ADAM9 ADCs exhibit antitumor activity against a broad panel of ADAM9-positive malignancies and cause durable remissions in preclinical models at doses expected to be clinically achievable. Anti-ADAM9 ADCs represent a promising therapeutic strategy to target a wide range of ADAM9-expressing tumors. Citation Format: Stuart W. Hicks, Nicholas C. Yoder, Deryk Loo, Asli Muvaffak, Yinghui Zhou, Megan E. Fuller, Molly A. McShea, Marian Themeles, Katherine H. Mucciarone, Juniper A. Scribner, Bhaswati Barat, Thomas Sun, James Tamura, Francine Z. Chen, Kerry A. Donahue, Tom Chittenden. Novel antibody-drug conjugates targeting ADAM9-expressing solid tumors demonstrate potent preclinical activity [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 37. doi:10.1158/1538-7445.AM2017-37

Abstract 1489: Development of a humanized ROR1 x CD3 bispecific DART® molecule for the treatment of solid and liquid tumors

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Introduction: The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is overexpressed in chronic lymphocytic leukemia and a subset of solid tumors, including lung, breast, ovarian, colon, and pancreatic cancers, as well as sarcoma. Limited adult tissue expression and its absence in normal leukocytes makes ROR1 a promising cancer therapeutic target. We have developed a Dual-Affinity Re-Targeting (DART®) protein for redirecting T lymphocytes to lyse tumor cells via monovalent recognition of ROR1 on tumors and CD3 on T cells. ROR1 x CD3 DART protein was engineered for improved half-life with the incorporation of a modified Fc domain, lacking effector function. Methods: The ROR1 x CD3 DART protein was stably expressed in CHO cells and purified to homogeneity by a standard antibody platform. Bispecific binding was evaluated by ELISA and SPR analysis. In vitro functional studies were performed with lymphoma and solid tumor cell lines in the presence of primary human T cells. Tumor growth inhibition was evaluated in NOD/SCID/IL-2 gamma chain KO (NOG) mice coimplanted with human T cells and either mantle cell lymphoma (MCL) or lung cancer cell lines (5:1 effector : target cell ratio) followed by treatment with ROR1 x CD3 DART protein by intravenous (IV) administration. In vivo activity was also evaluated in human PBMC-reconstituted NOG/B2m deficient mice bearing established intradermal tumor xenografts following IV treatment with ROR1 x CD3 DART molecule. Pharmacokinetic analysis of the DART molecule was performed in human neonatal Fc receptor (hFcRn) transgenic mice. Results: The ROR1 x CD3 DART protein displayed the expected bispecific binding for ROR1 and CD3 antigens and retained the affinity and specificity of the parent mAbs. The DART molecule mediated dose-dependent lysis of ROR1-positive MCL and solid tumor (breast, lung, and osteosarcoma) cell lines through recruitment of human T cells. DART molecule-mediated killing of ROR1-expressing target cells was accompanied by target-dependent T-cell activation and cytokine release; however, no activity was observed in the absence of target cells and no cytokine release was observed with human PBMCs alone. The ROR1 x CD3 DART protein displayed extended circulating half-life after administration to hFcRn-transgenic mice. In mouse efficacy studies, the growth of HBL-2 (MCL), HOP-92 (lung cancer), or NIH-1975 (a lung cancer line resistant to erlotinib) cells co-implanted with human T cells in NOG mice was inhibited by treatment with the ROR1 x CD3 DART protein at doses in the mcg/kg range. The ROR1 x CD3 DART molecule also demonstrated antitumor activity with high complete response rates in human PBMC-reconstituted mice bearing established HBL-2 cell xenografts. Conclusion: The promising in vitro and in vivo activity of the Fc-bearing ROR1 x CD3 DART molecule supports further investigation as a potential candidate for the cancer treatment. Citation Format: Bhaswati Barat, Gurunadh Chichili, Valentina Ciccarone, James Tamura, Sergey Gorlatov, Michael Spliedt, Francine Chen, Scott Koenig, Paul Moore, Ezio Bonvini, Ralph Alderson, Syd Johnson. Development of a humanized ROR1 x CD3 bispecific DART® molecule for the treatment of solid and liquid tumors. [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 1489.