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Here, we report the development of an antibody–drug conjugate, ASG-5ME, which targets the solute carrier receptor SLC44A4. SLC44A4 is a member of a family of putative choline transporters that we show to be markedly upregulated in a variety of epithelial tumors, most notably prostate and pancreatic cancer. SLC44A4 is normally expressed on the apical surface of secretory epithelial cells, but in cancer we show expression is not restricted to the luminal surface in advanced and undifferentiated tumors. ASG-5ME consists of a human IgG2 anti-SLC44A4 antibody conjugated through a cleavable linker to the microtubule-disrupting agent monomethylauristatin E. It has potent antitumor activity in both cell line – and patient-derived xenograft models of pancreatic and prostate cancers. Combination studies with ASG-5ME and nab-paclitaxel demonstrated combination effect in both pancreatic and prostate tumor models. Altogether, the data presented here suggest that ASG-5ME may have the potential to offer a new therapeutic option for the treatment of pancreatic and prostate cancers. Mol Cancer Ther; 15(11); 2679–87. ©2016 AACR.

CD37 is a tetraspanin expressed on malignant B cells. Recently, CD37 has gained interest as a therapeutic target. We developed AGS67E, an antibody–drug conjugate that targets CD37 for the potential treatment of B/T-cell malignancies. It is a fully human monoclonal IgG2 antibody (AGS67C) conjugated, via a protease-cleavable linker, to the microtubule-disrupting agent monomethyl auristatin E (MMAE). AGS67E induces potent cytotoxicity, apoptosis, and cell-cycle alterations in many non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) cell lines and patient-derived samples in vitro. It also shows potent antitumor activity in NHL and CLL xenografts, including Rituxan-refractory models. During profiling studies to confirm the reported expression of CD37 in normal tissues and B-cell malignancies, we made the novel discovery that the CD37 protein was expressed in T-cell lymphomas and in AML. AGS67E bound to >80% of NHL and T-cell lymphomas, 100% of CLL and 100% of AML patient-derived samples, including CD34+CD38− leukemic stem cells. It also induced cytotoxicity, apoptosis, and cell-cycle alterations in AML cell lines and antitumor efficacy in orthotopic AML xenografts. Taken together, this study shows not only that AGS67E may serve as a potential therapeutic for B/T-cell malignancies, but it also demonstrates, for the first time, that CD37 is well expressed and a potential drug target in AML. Mol Cancer Ther; 14(7); 1650–60. ©2015 AACR.

Figure S1: AGS67E Binds To B-Cells With High Affinity and Induces Cytotoxicity. Figure S2: AGS67E and AGS67C Internalize Into B Cell Lymphma Cells. Figure S3: AGS67E But Not AGS67C Exhibits Anti-Tumor Activity In MOLM-13 Xenografts. Figure S4: AGS67C IgG1 But Not IgG2 Induces ADCC Activity.

Fig. S1. Structure of AGS-16C3F; Figure S2. ENPP3 was not expressed in megakaryocytes; Figure S3. AGS-16C3F did not affect platelet activation; Figure S4. Maturing MKs did not express ENPP3; Figure S5. Fc blocker inhibited FcγRIIA pathway in tumor cells; Table S1. Information about ADCs used in this study; Table S2. Quantitative binding assessment of Fc blockers to human FcγRs

Figure S1. Proteolytic release of MMAE from ASG-5ME; Figure S2. SLC44A4 RNA expression in normal and tumor specimens from combined Oncomine data sets; Figure S3: Expression of SLC44A4 in OVCAR-5 cells determined by FACS.

SLITRK6 is a member of the SLITRK family of neuronal transmembrane proteins that was discovered as a bladder tumor antigen using suppressive subtractive hybridization. Extensive immunohistochemistry showed SLITRK6 to be expressed in multiple epithelial tumors, including bladder, lung, and breast cancer as well as in glioblastoma. To explore the possibility of using SLITRK6 as a target for an antibody–drug conjugate (ADC), we generated a panel of fully human mAbs specific for SLITRK6. ADCs showed potent in vitro and in vivo cytotoxic activity after conjugation to Monomethyl Auristatin E or Monomethyl Auristatin F. The most potent ADC, ASG-15ME, was selected as the development candidate and given the product name AGS15E. ASG-15ME is currently in phase I clinical trials for the treatment of metastatic urothelial cancer. This is the first report that SLITRK6 is a novel antigen in bladder cancer and also the first report of the development of ASG-15ME for the treatment of metastatic bladder cancer. Mol Cancer Ther; 15(6); 1301–10. ©2016 AACR.

Details on how the IHC reagent reported in this paper was generated and validated.

Figure S1. Chemical structures of MMAF, MMAE and 13C5-MMAE. The asterisks indicate where Carbon-13 isotope has replaced Carbon-12; Figure S2. Expression of CD32 on mature neutrophils. Circulating neutrophils (AllCell) were incubated with anti-CD32 antibody conjugated with FITC (CD32-FITC) or control IgG1-FITC, and were subjected to FACS analysis. Typical results from one experiment were shown; Figure S3. Little expression of SLC44A4 and ENPP3 on neutrophils at different differentiation stages. Microarray analyses were done for SLC44A4 (AGS5 target) or ENPP3 (AGS16 target) in HSCs, differentiating neutrophils at Day 8 or Day 14, or circulating neutrophils (mature); Figure S4. AGS5-vcMMAF conjugation did not affect intracellular cleavage. PC3 cells overexpressing SLC44A4, PC3-SLC44A4 were incubated with indicated concentrations of AGS5-vcMMAE, AGS5-vcMMAF or AGS5-mcMMAF for 6 days; Table S1: Information about ADCs used in this report; Table S2: Chromatographic gradient; Table S3: MRM Transitions monitored; Table S4. CD66b expression during neutrophil differentiation; Table S5. Correlation between sensitivity and extracellular cleavage.

Supplemental Figure 2. AGS16-7.8 does not inhibit the enzymatic activity of purified ENPP3.

Supplemental Table 2. Comparability of Anti-ENPP3 ADC Produced in Hybridoma or CHO Cells

Supplemental Methods

AGS-16C3F is an antibody–drug conjugate (ADC) against ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) containing the mcMMAF linker-payload currently in development for treatment of metastatic renal cell carcinoma. AGS-16C3F and other ADCs have been reported to cause ocular toxicity in patients by unknown mechanisms. To investigate this toxicity, we developed an in vitro assay using human corneal epithelial cells (HCEC) and show that HCECs internalized AGS-16C3F and other ADCs by macropinocytosis, causing inhibition of cell proliferation. We observed the same mechanism for target-independent internalization of AGS-16C3F in fibroblasts and human umbilical vein endothelial cells (HUVEC). Macropinocytosis-mediated intake of macromolecules is facilitated by the presence of positive charges or hydrophobic residues on the surface of the macromolecule. Modification of AGS-16C3F, either by attachment of poly-glutamate peptides, mutation of residue K16 to D on AGS-16C3F [AGS-16C3F(K16D)], or decreasing the overall hydrophobicity via attachment of polyethylene glycol moieties, significantly reduced cytotoxicity against HCECs and other primary cells. Rabbits treated with AGS-16C3F showed significant ocular toxicity, whereas those treated with AGS-16C3F(K16D) presented with less severe and delayed toxicities. Both molecules displayed similar antitumor activity in a mouse xenograft model. These findings establish a mechanism of action for target-independent toxicities of AGS-16C3F and ADCs in general, and provide methods to ameliorate these toxicities.Significance:These findings reveal a mechanism for nonreceptor-mediated toxicities of antibody drug conjugates and potential solutions to alleviate these toxicities. Cancer Res; 78(8); 2115–26. ©2018 AACR.

Supplemental tables 1-5: Table 1: Characteristics of peptide modified ADCs tested and IC50 in the HCEC and KU812 assays. Table 2: Characteristics. of PEG modified ADCs tested and data in the HCEC and KU812 assays. Table 3: Characteristics of mutated ADCs tested in the HCEC and KU812 cytotoxicity assays. Table 4: Pharmacokinetic parameters in mice and rabbits. ADC and Tab. Table 5: Body gain weight of rabbits treated with AGS-16C3F and other ADCs. Supplemental Materials and Methods Summplemental Figure Legends Supplemental figures 1-4: Fig S1: Lack of ENPP3 expression in corneas from humans and cynomolgus monkeys. Fig S2: Characterization of human corneal epithelial cells (HCEC). Fig S3: Target expression in HCEC and normal corneal epithelial cells by microarray analysis. Fig S4: Binding of AGS-16C3F to primary cells. References

Supplemental Table 1. Quantification of ENPP3 Expression in Various Carcinomas

Supplemental Figure 5. Immunostaining for cleaved PARP-1 and PPH3 in tumors.

Supplemental Figure 3A. Mapping of AGS16-7.8 binding to different ENNP3 domains. Supplemental Figure 3B. Western blots for mapping recognition of epitopes in ENPP3 ECD fragments.

Purpose: New cancer-specific antigens are required for the design of novel antibody–drug conjugates (ADC) that deliver tumor-specific and highly potent cytotoxic therapy.Experimental Design: Suppression subtractive hybridization identified ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3 or CD203c) as a potential human cancer-specific antigen. Antibodies targeting the extracellular domain of human ENPP3 were produced and selected for specific binding to ENPP3. Expression of ENPP3 in normal and cancer tissue specimens was evaluated by immunohistochemistry (IHC). ADCs comprising anti-ENPP3 Ab conjugated with maleimidocaproyl monomethyl auristatin F via a noncleavable linker (mcMMAF) were selected for therapeutic potential using binding and internalization assays, cytotoxicity assays, and tumor growth inhibition in mouse xenograft models. Pharmacodynamic markers were evaluated by IHC in tissues and ELISA in blood.Results: ENPP3 was highly expressed in clear cell renal cell carcinoma: 92.3% of samples were positive and 83.9% showed high expression. By contrast, expression was negligible in normal tissues examined, with the exception of the kidney. High expression was less frequent in papillary renal cell carcinoma and hepatocellular carcinoma samples. AGS16F, an anti-ENPP3 antibody–mcMMAF conjugate, inhibited tumor growth in three different renal cell carcinoma (RCC) xenograft models. AGS16F localized to tumors, formed the active metabolite Cys-mcMMAF, induced cell-cycle arrest and apoptosis, and increased blood levels of caspase-cleaved cytokeratin-18, a marker of epithelial cell death.Conclusions: AGS16F is a promising new therapeutic option for patients with RCC and is currently being evaluated in a phase I clinical trial. Clin Cancer Res; 22(8); 1989–99. ©2015 AACR.

Supplemental Figure 1. AGS16-7.8 and AGS16F do not bind to ENPP1 or ENPP2 proteins.

Supplemental Figure 4. Pharmacokinetics of AGS16F. Mice were injected with a single dose of AGS16F.

Figure 1: ADC Localization in tumor xenografts Figure 2: PK profile in mice Table 1: Toxicokinetic parameters

The identification of optimal target antigens on tumor cells is central to the advancement of new antibody-based cancer therapies. We performed suppression subtractive hybridization and identified nectin-4 (PVRL4), a type I transmembrane protein and member of a family of related immunoglobulin-like adhesion molecules, as a potential target in epithelial cancers. We conducted immunohistochemical analysis of 2,394 patient specimens from bladder, breast, lung, pancreatic, ovarian, head/neck, and esophageal tumors and found that 69% of all specimens stained positive for nectin-4. Moderate to strong staining was especially observed in 60% of bladder and 53% of breast tumor specimens, whereas the expression of nectin-4 in normal tissue was more limited. We generated a novel antibody–drug conjugate (ADC) enfortumab vedotin comprising the human anti-nectin-4 antibody conjugated to the highly potent microtubule-disrupting agent MMAE. Hybridoma (AGS-22M6E) and CHO (ASG-22CE) versions of enfortumab vedotin (also known as ASG-22ME) ADC were able to bind to cell surface–expressed nectin-4 with high affinity and induced cell death in vitro in a dose-dependent manner. Treatment of mouse xenograft models of human breast, bladder, pancreatic, and lung cancers with enfortumab vedotin significantly inhibited the growth of all four tumor types and resulted in tumor regression of breast and bladder xenografts. Overall, these findings validate nectin-4 as an attractive therapeutic target in multiple solid tumors and support further clinical development, investigation, and application of nectin-4–targeting ADCs. Cancer Res; 76(10); 3003–13. ©2016 AACR.

Thrombocytopenia is a common adverse event in cancer patients treated with antibody–drug conjugates (ADC), including AGS-16C3F, an ADC targeting ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase-3) and trastuzumab emtansine (T-DM1). This study aims to elucidate the mechanism of action of ADC-induced thrombocytopenia. ENPP3 expression in platelets and megakaryocytes (MK) was investigated and shown to be negative. The direct effect of AGS-16C3F on platelets was evaluated using platelet rich plasma following the expression of platelet activation markers. Effects of AGS-16C3F, T-DM1, and control ADCs on maturing megakaryocytes were evaluated in an in vitro system in which human hematopoietic stem cells (HSC) were differentiated into MKs. AGS-16C3F, like T-DM1, did not affect platelets directly, but inhibited MK differentiation by the activity of Cys-mcMMAF, its active metabolite. FcγRIIA did not appear to play an important role in ADC cytotoxicity to differentiating MKs. AGS-16C3F, cytotoxic to MKs, did not bind to FcγRIIA on MKs. Blocking the interaction of T-DM1 with FcγRIIA did not prevent the inhibition of MK differentiation and IgG1-mcMMAF was not as cytotoxic to MKs despite binding to FcγRIIA. Several lines of evidence suggest that internalization of AGS-16C3F into MKs is mediated by macropinocytosis. Macropinocytosis activity of differentiating HSCs correlated with cell sensitivity to AGS-16C3F. AGS-16C3F was colocalized with a macropinocytosis marker, dextran-Texas Red in differentiating MKs. Ethyl isopropyl amiloride (EIPA), a macropinocytosis inhibitor, blocked internalization of dextran-Texas Red and AGS-16C3F. These data support the notion that inhibition of MK differentiation via macropinocytosis-mediated internalization plays a role in ADC-induced thrombocytopenia. Mol Cancer Ther; 16(9); 1877–86. ©2017 AACR. See related article by Zhao et al., p. 1866

Neutropenia is a common adverse event in cancer patients treated with antibody–drug conjugates (ADC) and we aimed to elucidate the potential mechanism of this toxicity. To investigate whether ADCs affect neutrophil production from bone marrow, an in vitro assay was developed in which hematopoietic stem cells (HSC) were differentiated to neutrophils. Several antibodies against targets absent in HSCs and neutrophils were conjugated to MMAE via a cleavable valine-citrulline linker (vcMMAE-ADC) or MMAF via a noncleavable maleimidocaproyl linker (mcMMAF-ADC), and their cytotoxicity was tested in the neutrophil differentiation assay. Results showed that HSCs had similar sensitivity to vcMMAE-ADCs and mcMMAF-ADCs; however, vcMMAE-ADCs were more cytotoxic to differentiating neutrophils than the same antibody conjugated to mcMMAF. This inhibitory effect was not mediated by internalization of ADC either by macropinocytosis or FcγRs. Our results suggested that extracellular proteolysis of the cleavable valine-citrulline linker is responsible for the cytotoxicity to differentiating neutrophils. Mass spectrometry analyses indicated that free MMAE was released from vcMMAE-ADCs in the extracellular compartment when they were incubated with differentiating neutrophils or neutrophil conditioned medium, but not with HSC-conditioned medium. Using different protease inhibitors, our data suggested that serine, but not cysteine proteases, were responsible for the cleavage. In vitro experiments demonstrated that the purified serine protease, elastase, was capable of releasing free MMAE from a vcMMAE-ADC. Here we propose that ADCs containing protease cleavable linkers can contribute to neutropenia via extracellular cleavage mediated by serine proteases secreted by differentiating neutrophils in bone marrow. Mol Cancer Ther; 16(9); 1866–76. ©2017 AACR. See related article by Zhao et al., p. 1877

SLITRK6 is a member of the SLITRK family of neuronal transmembrane proteins that was discovered as a bladder tumor antigen using suppressive subtractive hybridization. Extensive immunohistochemistry showed SLITRK6 to be expressed in multiple epithelial tumors, including bladder, lung, and breast cancer as well as in glioblastoma. To explore the possibility of using SLITRK6 as a target for an antibody–drug conjugate (ADC), we generated a panel of fully human mAbs specific for SLITRK6. ADCs showed potent in vitro and in vivo cytotoxic activity after conjugation to Monomethyl Auristatin E or Monomethyl Auristatin F. The most potent ADC, ASG-15ME, was selected as the development candidate and given the product name AGS15E. ASG-15ME is currently in phase I clinical trials for the treatment of metastatic urothelial cancer. This is the first report that SLITRK6 is a novel antigen in bladder cancer and also the first report of the development of ASG-15ME for the treatment of metastatic bladder cancer. Mol Cancer Ther; 15(6); 1301–10. ©2016 AACR.

The identification of optimal target antigens on tumor cells is central to the advancement of new antibody-based cancer therapies. We performed suppression subtractive hybridization and identified nectin-4 (PVRL4), a type I transmembrane protein and member of a family of related immunoglobulin-like adhesion molecules, as a potential target in epithelial cancers. We conducted immunohistochemical analysis of 2,394 patient specimens from bladder, breast, lung, pancreatic, ovarian, head/neck, and esophageal tumors and found that 69% of all specimens stained positive for nectin-4. Moderate to strong staining was especially observed in 60% of bladder and 53% of breast tumor specimens, whereas the expression of nectin-4 in normal tissue was more limited. We generated a novel antibody–drug conjugate (ADC) enfortumab vedotin comprising the human anti-nectin-4 antibody conjugated to the highly potent microtubule-disrupting agent MMAE. Hybridoma (AGS-22M6E) and CHO (ASG-22CE) versions of enfortumab vedotin (also known as ASG-22ME) ADC were able to bind to cell surface–expressed nectin-4 with high affinity and induced cell death in vitro in a dose-dependent manner. Treatment of mouse xenograft models of human breast, bladder, pancreatic, and lung cancers with enfortumab vedotin significantly inhibited the growth of all four tumor types and resulted in tumor regression of breast and bladder xenografts. Overall, these findings validate nectin-4 as an attractive therapeutic target in multiple solid tumors and support further clinical development, investigation, and application of nectin-4–targeting ADCs. Cancer Res; 76(10); 3003–13. ©2016 AACR.

Purpose: New cancer-specific antigens are required for the design of novel antibody–drug conjugates (ADC) that deliver tumor-specific and highly potent cytotoxic therapy. Experimental Design: Suppression subtractive hybridization identified ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3 or CD203c) as a potential human cancer-specific antigen. Antibodies targeting the extracellular domain of human ENPP3 were produced and selected for specific binding to ENPP3. Expression of ENPP3 in normal and cancer tissue specimens was evaluated by immunohistochemistry (IHC). ADCs comprising anti-ENPP3 Ab conjugated with maleimidocaproyl monomethyl auristatin F via a noncleavable linker (mcMMAF) were selected for therapeutic potential using binding and internalization assays, cytotoxicity assays, and tumor growth inhibition in mouse xenograft models. Pharmacodynamic markers were evaluated by IHC in tissues and ELISA in blood. Results: ENPP3 was highly expressed in clear cell renal cell carcinoma: 92.3% of samples were positive and 83.9% showed high expression. By contrast, expression was negligible in normal tissues examined, with the exception of the kidney. High expression was less frequent in papillary renal cell carcinoma and hepatocellular carcinoma samples. AGS16F, an anti-ENPP3 antibody–mcMMAF conjugate, inhibited tumor growth in three different renal cell carcinoma (RCC) xenograft models. AGS16F localized to tumors, formed the active metabolite Cys-mcMMAF, induced cell-cycle arrest and apoptosis, and increased blood levels of caspase-cleaved cytokeratin-18, a marker of epithelial cell death. Conclusions: AGS16F is a promising new therapeutic option for patients with RCC and is currently being evaluated in a phase I clinical trial. Clin Cancer Res; 22(8); 1989–99. ©2015 AACR.

AGS-16C3F is an antibody–drug conjugate (ADC) against ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) containing the mcMMAF linker-payload currently in development for treatment of metastatic renal cell carcinoma. AGS-16C3F and other ADCs have been reported to cause ocular toxicity in patients by unknown mechanisms. To investigate this toxicity, we developed an in vitro assay using human corneal epithelial cells (HCEC) and show that HCECs internalized AGS-16C3F and other ADCs by macropinocytosis, causing inhibition of cell proliferation. We observed the same mechanism for target-independent internalization of AGS-16C3F in fibroblasts and human umbilical vein endothelial cells (HUVEC). Macropinocytosis-mediated intake of macromolecules is facilitated by the presence of positive charges or hydrophobic residues on the surface of the macromolecule. Modification of AGS-16C3F, either by attachment of poly-glutamate peptides, mutation of residue K16 to D on AGS-16C3F [AGS-16C3F(K16D)], or decreasing the overall hydrophobicity via attachment of polyethylene glycol moieties, significantly reduced cytotoxicity against HCECs and other primary cells. Rabbits treated with AGS-16C3F showed significant ocular toxicity, whereas those treated with AGS-16C3F(K16D) presented with less severe and delayed toxicities. Both molecules displayed similar antitumor activity in a mouse xenograft model. These findings establish a mechanism of action for target-independent toxicities of AGS-16C3F and ADCs in general, and provide methods to ameliorate these toxicities. Significance:These findings reveal a mechanism for nonreceptor-mediated toxicities of antibody drug conjugates and potential solutions to alleviate these toxicities. Cancer Res; 78(8); 2115–26. ©2018 AACR.

Nectin-4 is selectively overexpressed in a variety of cancers and is currently under clinical investigation as a therapeutic target. A monoclonal antibody against nectin-4 (AGS-22M6) was evaluated as an Immuno-positron emission tomography (ImmunoPET) reagent. Its ability to assay nectin-4 expression as well as detect nectin-4 positive tumors in the liver and bone was evaluated using mouse models.The biodistribution of [(89)Zr]AGS-22M6 was evaluated in mice bearing tumors with varying levels of nectin-4 expression. An isogenic breast cancer tumor line was used to model metastatic liver and bone disease in mice. The biodistribution of [(18)F]AGS-22M6 in cynomolgus monkeys was evaluated.A positive correlation was demonstrated between tumor nectin-4 expression and [(89)Zr]AGS-22M6 uptake. Tumors in the liver and bone were detected and differentiated based on nectin-4 expression. [(18)F]AGS-22M6 showed limited uptake in cynomolgus monkey tissues.[(89)Zr]AGS-22M6 is a promising ImmunoPET reagent that can assay nectin-4 expression in both primary and metastatic lesions.

Decreased activity of osteoblasts (OBs) contributes to osteolytic lesions in multiple myeloma (MM). The production of the soluble Wnt inhibitor Dickkopf-1 (DKK1) by MM cells inhibits OB activity, and its serum level correlates with focal bone lesions in MM. Therefore, we have evaluated bone anabolic effects of a DKK1 neutralizing antibody (BHQ880) in MM. In vitro BHQ880 increased OB differentiation, neutralized the negative effect of MM cells on osteoblastogenesis, and reduced IL-6 secretion. In a severe combined immunodeficiency (SCID)–hu murine model of human MM, BHQ880 treatment led to a significant increase in OB number, serum human osteocalcin level, and trabecular bone. Although BHQ880 had no direct effect on MM cell growth, it significantly inhibited growth of MM cells in the presence of bone marrow stromal cells (BMSCs) in vitro. This effect was associated with inhibition of BMSC/MM cell adhesion and production of IL-6. In addition, BHQ880 up-regulated β-catenin level while down-regulating nuclear factor-κB (NF-κB) activity in BMSC. Interestingly, we also observed in vivo inhibition of MM cell growth by BHQ880 treatment in the SCID-hu murine model. These results confirm DKK1 as an important therapeutic target in myeloma and provide the rationale for clinical evaluation of BHQ880 to improve bone disease and to inhibit MM growth.

TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis on binding to DR4 and DR5 receptors on the surface of tumor cells. These receptors are of particular interest in the development of cancer therapeutics as they preferentially mediate tumor cell apoptosis. We have generated a chimeric anti-DR5 agonistic antibody, LBY135, from its murine parental antibody, LCR211, identified using hybridoma technology. Both LCR211 and LBY135 specifically bind to DR5 with nanomolar affinity, mimic TRAIL to induce cell death in tumor cells, and have little effect on non-transformed cells in vitro. The anti- DR5 antibody reduced viability in 45% of a panel of 40 human colon cancer cell lines with IC50 values of 20nM or less. In vivo, using human colorectal tumor xenograft mouse models, LCR211 induced tumor regression and showed enhanced efficacy when combined with 5-FU. Both in vitro evaluation of ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity), and in vivo studies using a non-functional DR5 specific antibody or SCID-Beige mice, suggested ADCC and CDC are unlikely to be the mechanism to ablate tumors in vivo. LBY135 and LCR211 appear to mediate cell death and tumor regression mainly through apoptosis, as demonstrated by the activation of caspase 3, caspase 8, M30, and TUNEL assay. In addition, the discovery of synergy between cross-linked LBY135 and TRAIL not only revealed the unique epitope of LBY135, but also demonstrated an additional mechanism of action for LBY135 in vivo. LBY135 demonstrates promise as a novel therapeutic for cancer treatment and is currently in Phase I clinical trials. Drug Dev Res 69: 69–82, 2008.

Objectives: Lytic bone disease is a hallmark of multiple myeloma (MM) and is caused by osteoclast activation and osteoblast inhibition. Secretion of Dickkopf (DKK)-1 by myeloma cells is a major factor which causes inhibition of osteoblast precursors. So far, there is no study showing a significant difference in serum DKK-1 levels in MM patients with or without lytic bone lesions. Methods: DKK-1 serum levels were quantified in 184 untreated MM patients and 33 monoclonal gammopathy of undetermined significance (MGUS) patients by ELISA, using a monoclonal anti-DKK-1 antibody. Results: Serum DKK-1 was elevated in MM as compared with MGUS (mean 11 963 pg/mL vs. 1993 pg/mL; P 3 lesions: 3114 pg/mL vs. 3559 pg/mL vs. 24 068 pg/mL; P = 0.002). Conclusion: Using a large series of myeloma patients, we could show for the first time a correlation between DKK-1 serum concentration and the amount of lytic bone disease, indicating that DKK-1 is an important factor for the extent of bone disease and supporting the hypothesis of DKK-1 as a therapeutic target in myeloma bone disease.

CD37 is a tetraspanin expressed on malignant B cells. Recently, CD37 has gained interest as a therapeutic target. We developed AGS67E, an antibody–drug conjugate that targets CD37 for the potential treatment of B/T-cell malignancies. It is a fully human monoclonal IgG2 antibody (AGS67C) conjugated, via a protease-cleavable linker, to the microtubule-disrupting agent monomethyl auristatin E (MMAE). AGS67E induces potent cytotoxicity, apoptosis, and cell-cycle alterations in many non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) cell lines and patient-derived samples in vitro. It also shows potent antitumor activity in NHL and CLL xenografts, including Rituxan-refractory models. During profiling studies to confirm the reported expression of CD37 in normal tissues and B-cell malignancies, we made the novel discovery that the CD37 protein was expressed in T-cell lymphomas and in AML. AGS67E bound to >80% of NHL and T-cell lymphomas, 100% of CLL and 100% of AML patient-derived samples, including CD34+CD38− leukemic stem cells. It also induced cytotoxicity, apoptosis, and cell-cycle alterations in AML cell lines and antitumor efficacy in orthotopic AML xenografts. Taken together, this study shows not only that AGS67E may serve as a potential therapeutic for B/T-cell malignancies, but it also demonstrates, for the first time, that CD37 is well expressed and a potential drug target in AML. Mol Cancer Ther; 14(7); 1650–60. ©2015 AACR.

The purpose of this phospho-proteomics study was to demonstrate the broad analysis of cellular protein phosphorylation in cells and tissue as a means to monitor changes in cellular states. As a cancer model, human tumor-derived A431 cells known to express the epidermal growth factor receptor (EGFR) were grown as cell cultures or xenograft tumors in mice. The cells and tumor-bearing animals were subjected to treatments including the EGFR-directed protein kinase inhibitor PK166 and/or EGF stimulation. Whole cell/tissue protein extracts were converted to peptides by using trypsin, and phosphorylated peptides were purified by an affinity capture method. Peptides and phosphorylation sites were characterized and quantified by using a combination of tandem mass spectroscopy (MS) and Fourier transform MS instrumentation (FTMS). By analyzing roughly 106 cell equivalents, 780 unique phosphopeptides from approx 450 different proteins were characterized. Only a small number of these phosphorylation sites have been described previously in literature. Although a targeted analysis of the EGFR pathway was not a specific aim of this study, 22 proteins known to be associated with EGFR signaling were identified. Fifty phosphopeptides were found changed in abundance as a function of growth factor or drug treatment including novel sites of phosphorylation on the EGFR itself. These findings demonstrate the feasibility of using phospho-proteomics to determine drug and disease mechanisms, and as a measure of drug target modulation in tissue.

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Recent efforts in these laboratories have resulted in the development of a synthetic approach to C(7) alkyl analogs in the K252a class of indolocarbazole natural products. Synthesis of 7(R)-methyl K252a (3a) and 7-(S)-methyl K252a (3b) will be described along with their activity against protein tyrosine kinases.

Background: The lymphocyte-specific kinase Lck is a member of the Src family of non-receptor tyrosine kinases. Lck catalyzes the initial phosphorylation of T-cell receptor components that is necessary for signal transduction and T-cell activation. On the basis of both biochemical and genetic studies, Lck is considered an attractive cell-specific target for the design of novel T-cell immunosuppressants. To date, the lack of detailed structural information on the mode of inhibitor binding to Lck has limited the discovery of novel Lck inhibitors. Results: We report here the high-resolution crystal structures of an activated Lck kinase domain in complex with three structurally distinct ATP-competitive inhibitors: AMP-PNP (a non-selective, non-hydrolyzable ATP analog); staurosporine (a potent but non-selective protein kinase inhibitor); and PP2 (a potent Src family selective protein tyrosine kinase inhibitor). Comparison of these structures reveals subtle but important structural changes at the ATP-binding site. Furthermore, PP2 is found to access a deep, hydrophobic pocket near the ATP-binding cleft of the enzyme; this binding pocket is not occupied by either AMP-PNP or staurosporine. Conclusions: The potency of staurosporine against Lck derives in part from an induced movement of the glycine-rich loop of the enzyme upon binding of this ligand, which maximizes the van der Waals interactions present in the complex. In contrast, PP2 binds tightly and selectively to Lck and other Src family kinases by making additional contacts in a deep, hydrophobic pocket adjacent to the ATP-binding site; the amino acid composition of this pocket is unique to Src family kinases. The structures of these Lck complexes offer useful structural insights as they demonstrate that kinase selectivity can be achieved with small-molecule inhibitors that exploit subtle topological differences among protein kinases.

On the basis of previously described X-ray studies of an enzyme/aza-dipeptide complex,8 aza-dipeptide analogues carrying N-(bis-aryl-methyl) substituents on the (hydroxethyl)hydrazine moiety have been designed and synthesized as HIV-1 protease inhibitors. By using either equally (12) or orthogonally (13) protected dipeptide isosteres, symmetrically and asymmetrically acylated aza-dipeptides can be synthesized. This approach led to the discovery of very potent inhibitors with antiviral activities (ED50) in the subnanomolar range. Acylation of the (hydroxethyl)hydrazine dipeptide isostere with the L-tert-leucine derivative 29 increased the oral bioavailability significantly when compared to the corresponding L-valine or L-isoleucine derivatives. The bis(L-tert-leucine) derivatives CGP 75355, CGP 73547, CGP 75136, and CGP 75176 combine excellent antiviral activity with high blood concentration after oral administration. Furthermore, they show no cross-resistance with saquinavir-resistant strains and maintain activity against indinavir-resistant ones. Consequently they qualify for further profiling as potential clinical candidates.

A calculated 3-D model of the kinase domain of the epidermal growth factor receptor (EGF-R) protein-tyrosine kinase (PTK) was used to develop a pharmacophore model for ATP-competitive inhibitors and, subsequently, a new class of selective EGF-R kinase inhibitors. CGP 59326A, a highly selective and potent inhibitor of the EGF-R in vitro, inhibited the proliferation of EGF-R-expressing epithelial lines, while having little anti-proliferative activity against EGF-R-negative lines. In contrast to previously described inhibitors, CGP 59326A had potent and selective in vivo anti-tumor activity at well-tolerated doses against EGF-R-expressing tumors (e.g., ED50 of 0.78 to 1.5 mg/kg for inhibition of A431 tumor growth). CGP 59326A inhibited growth of human tumor xenografts expressing the EGF-R but showed little activity against EGF-R-negative xenografts. Combination of CGP 59326A with cytotoxic agents resulted in tumor regression and cures. The high selectivity and attractive biological profile of CGP 59326A suggest that it could have therapeutic value in the treatment of proliferative diseases which involve mitogenic signaling from the EGF-R.

Antibody drug conjugates (ADCs) are monoclonal antibodies designed to deliver a cytotoxic drug selectively to antigen expressing cells. Several components of an ADC including the selection of the antibody, the linker, the cytotoxic drug payload and the site of attachment used to attach the drug to the antibody are critical to the activity and development of the ADC. The cytotoxic drugs or payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. This results in ADCs that have a heterogeneous number of drugs per antibody. The number of drugs per antibody commonly referred to as the drug to antibody ratio (DAR), can vary between 0 and 8 drugs for a IgG1 antibody. Antibodies with 0 drugs are ineffective and compete with the ADC for binding to the antigen expressing cells. Antibodies with 8 drugs per antibody have reduced in vivo stability, which may contribute to non target related toxicities. In these studies we incorporated a non-natural amino acid, para acetyl phenylalanine, at two unique sites within an antibody against Her2/neu. We covalently attached a cytotoxic drug to these sites to form an ADC which contains two drugs per antibody. We report the results from the first direct preclinical comparison of a site specific non-natural amino acid anti-Her2 ADC and a cysteine conjugated anti-Her2 ADC. We report that the site specific non-natural amino acid anti-Her2 ADCs have superior in vitro serum stability and preclinical toxicology profile in rats as compared to the cysteine conjugated anti-Her2 ADCs. We also demonstrate that the site specific non-natural amino acid anti-Her2 ADCs maintain their in vitro potency and in vivo efficacy against Her2 expressing human tumor cell lines. Our data suggests that site specific non-natural amino acid ADCs may have a superior therapeutic window than cysteine conjugated ADCs.

A series of potent HIV-protease inhibitors has been prepared. Several of the newly synthesized compounds showed high plasma levels after oral administration to animals. Based on the overall biological profile, CGP 61755 was chosen for further preclinical evaluation. For this compound, a 10 step synthesis potentially suitable for large scale production was developed.

Thrombocytopenia and neutropenia are common adverse events in cancer patients treated with antibody-drug conjugates (ADCs). We used human hematopoietic stem cells (HSC) in vitro and various ADCs to investigate the potential mechanisms of thrombocytopenia and neutropenia. Two ADCs, AGS-16C3F and T-DM1, were selected for the investigation of megakaryocytes (MKs) as they both induced thrombocytopenia in patients. AGS-16C3F is an ENPP3-targeting antibody conjugated to microtubule-disrupting agent MMAF (licensed from Seattle Genetics) via a non-cleavable linker (mcMMAF) tested in a phase 1 trial in kidney cancer patients. T-DM1 (KADCYLA) is an anti-HER2 antibody conjugated to the maytansine derivative DM1, approved for the treatment of advanced Her-2 positive breast cancer. HSCs were differentiated to MKs and percentage of CD41 positivity from FACS analysis was used to measure the effect of ADCs on MK differentiation. Results showed that while MKs do not express ENPP3 or Her-2, both AGS-16C3F (IC50 = 40nM) and T-DM1 inhibited MK differentiation. MKs express FcγRIIA to which T-DM1 (IgG1) and, to lesser degree, AGS-16C3F (IgG2) can bind; however, blockage of FcγRIIA failed to rescue MKs from cytotoxicity induced by either T-DM1 or AGS-16C3F suggesting that binding to FcγRIIA is not critical for MK cytotoxicity. AGS-16C3F is internalized into MKs via macropinocytosis and causes significant disorganization of tubulin structure as observed by confocal microscopy. HSCs were also differentiated to neutrophils which were characterized by the percentage of CD66b positivity in FACS analysis. Several antibodies to targets absent in neutrophils were conjugated to both MMAE and MMAF and were tested for cytotoxicity during neutrophil differentiation. Our results showed that ADCs containing MMAE via a cleavable linker were more cytotoxic to neutrophil differentiation than the same ADC containing MMAF via a non-cleavable linker, in agreement with clinical results. Different from MKs, results showed that neutrophils have low macropinocytosis activity. Secretion of cathepsins increased during neutrophil differentiation, and a membrane impermeable cathepsin inhibitor blocked ADC cytotoxicity to neutrophils, suggesting that extracellular protease cleavage is critical for neutrophil cytotoxicity in vitro. Neutrophils express high levels of FcγRIIA and saturation of this receptor by control IgG inhibited ADC binding to neutrophils and at least partially rescued neutrophils from ADC cytotoxicity, suggesting that FcγRIIA can play a role in neutropenia induced by ADCs, particularly with IgG1 isotype. In conclusion, applying these in vitro data to clinical experience suggests that thrombocytopenia may be mediated by macropinocytosis of ADC in MK and neutropenia by secreted proteases and interactions with their FcγRs. Understanding these toxicities may help design new ADCs with improved toxicity profile. Citation Format: Hui Zhao, Sara Gulesserian, Sathish K. Ganesan, Zhilan Zeng, Jimmy Ou, Veronica Robles, Josh Snyder, David R. Stover, Fernando Donate. Potential mechanisms for thrombocytopenia and neutropenia induced by antibody-drug conjugates. [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 3853.

FLT3 is a member of the class III receptor tyrosine kinase family that includes C-KIT, C-FMS and platelet derived growth factor receptor (PDGFR). FLT3 is primarily expressed in early myeloid and lymphoid progenitors and plays an important role in their proliferation and differentiation. In human leukemia, FLT3 is expressed on 70-90% acute myeloid leukemia (AML) and most B-acute lymphoblastic leukemia (B-ALL). FLT3 genetic aberrations are commonly detected in patients with AML. The most common aberration is internal tandem duplication (ITD), which occurs in 25-30% of AML patients and causes constitutive activation of FLT3. Point mutation in codon D835 of the FLT3 tyrosine kinase domain is reported in 7-10% of AML patients and also causes constitutive activation of the receptor. FLT3 small molecule inhibitors targeting the kinase domain are predominantly active against FLT3 activated AML. The restricted normal tissue expression profile and higher differential in leukemic specimens makes FLT3 amenable to antibody-based therapeutics, requiring only target expression independent of kinase activation status. Therefore, development of an antibody-drug conjugate (ADC) may provide a therapeutic alternative for AML patients. Here, we report the development of the first FLT3specific ADC, AGS62P1, employing site-specific conjugation using the non-natural amino acid, p-acetyl phenylalanine (pAF). AGS62P1 comprises a human gamma one antibody including an inserted pAF residue in each of the heavy chains. The antibody was conjugated to a potent cytotoxic payload via an oxime bond at the pAF sites, thus creating a nearly homogeneous drug distribution, with approximately 2 drug molecules per antibody. Strong binding affinity (0.1-0.9 nM) and potent in vitro cytotoxic activity (IC50 = 0.2-12 nM) was achieved in AML cell lines. The anti-FLT3 ADC was highly efficacious in AML tumor xenografts, leading to statistically significant tumor growth inhibition of both FLT3 ITD and non-ITD models. Additional characterization of both the antibody and ADC was performed, including ligand receptor interaction, degradation, internalization, and apoptosis. In summary, we have developed a site-specific ADC targeting FLT3 that exhibits potent anti-tumor activity in xenograft models regardless of FLT3 activation status. This drug can potentially offer a new and more versatile approach in targeting FLT3-expressing leukemia through a mechanism independent of FLT3 genetic aberration. Citation Format: Nandini Rudra-Ganguly, Pia M. Challita-Eid, Christine Lowe, Mike Mattie, Sung-Ju Moon, Brian A. Mendelsohn, Monica Leavitt, Cyrus Virata, Alla Verlinsky, Linnette Capo, Mi Sook Chang, Deanna L. Russell, Baljinder Randhawa, Gao Liu, René Hubert, Mary Brodey, Hector Aviña, Chunying Zhang, Joseph D. Abad, Banmeet Anand, Sher Karki, Zili An, Roland Luethy, Fernando Doñate, Daniel S. Pereira, Kendall Morrison, Ingrid B.J. Joseph, David R. Stover. AGS62P1, a novel site-specific antibody drug conjugate targeting FLT3 exhibits potent anti-tumor activity regardless of FLT3 kinase activation status. [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 574.

Multiple myeloma (MM) is associated with the development of osteolytic bone disease, mediated by increased osteoclastic bone resorption and impaired osteoblastic bone formation. Dickkopf-1 (Dkk1), a soluble inhibitor of wingless/int (Wnt) signaling and osteoblastogenesis, is elevated in patients with MM and correlates with osteolytic bone disease. In this study, we investigated the effect of inhibiting Dkk1 on the development of osteolytic lesions in the 5T2MM murine model of myeloma. We showed that Dkk1 is expressed by murine 5T2MM myeloma cells. Injection of 5T2MM cells into C57BL/KaLwRij mice resulted in the development of osteolytic bone lesions (p < 0.05), mediated by increased osteoclast numbers (p < 0.001) and a decrease in osteoblast numbers (p < 0.001) and mineralizing surface (p < 0.05). Mice bearing 5T2MM cells were treated with an anti-Dkk1 antibody (BHQ880, 10 mg/kg, IV, twice weekly for 4 wk) from time of paraprotein detection. Anti-Dkk1 treatment prevented 5T2MM-induced suppression of osteoblast numbers (p < 0.001) and surface (p < 0.001). Treatment increased mineralizing surface by 28% and bone formation rate by 25%; however, there was no change in mineral apposition rate. Inhibiting Dkk1 had no effect on osteoclast numbers. muCT analysis showed that anti-Dkk1 treatment significantly protected against 5T2MM-induced trabecular bone loss (p < 0.05) and reduced the development of osteolytic bone lesions (p < 0.05). Treatment had no significant effect on tumor burden. These data suggest that inhibiting Dkk1 prevents the suppression of bone formation and in doing so is effective in preventing the development of osteolytic bone disease in myeloma, offering an effective therapeutic approach to treating this clinically important aspect of myeloma.

Lytic bone destruction is a hallmark of multiple myeloma (MM) and is because of an uncoupling of bone remodeling. Secretion of Dickkopf (DKK)-1 by myeloma cells is a major factor which causes inhibition of osteoblast precursors. In this study, the effect of different treatment regimens for MM on serum DKK-1 was evaluated and correlated with the response to treatment in 101 myeloma patients receiving bortezomib, thalidomide, lenalidomide, adriamycin and dexamethasone (AD) or high-dose chemotherapy (HDCT) followed by autologous stem cell transplantation (ASCT). At baseline, myeloma patients had increased serum DKK-1 as compared with patients with MGUS (mean 3786 pg/mL vs. 1993 pg/mL). There was no difference between previously untreated MM patients and patients at relapse. A significant decrease of DKK-1 after therapy was seen in the following groups: Bortezomib (4059 pg/mL vs. 1862 pg/mL, P = 0.016), lenalidomide (11837 pg/mL vs. 4374 pg/mL, P = 0.039), AD (1668 pg/mL vs. 1241 pg/mL, P = 0.016), and AD + HDCT + ASCT (2446 pg/mL vs. 1082 pg/mL, P = 0.001). Thalidomide led to a non-significant decrease in DKK-1 (1705 pg/mL vs. 1269 pg/mL, P = 0.081). Within all groups, a significant decrease of DKK-1 was only seen in responders (i.e. patients achieving complete remission or partial remission), but not in non-responders. We show for the first time that serum DKK-1 levels decrease in myeloma patients responding to treatment, irrespective of the regimen chosen. These data suggest that myeloma cells are the main source of circulating DKK-1 protein and provide a framework for clinical trials on anti-DKK-1 treatment in MM.

FLT3 (FMS-like tyrosine kinase 3) is a member of the class III receptor tyrosine kinase family, which is highly expressed in the blasts of both AML and ALL patients. In addition to FL ligand stimulation, FLT3 can also be activated by two distinct clusters of mutations: internal tandem duplications (FLT3/ITDs) in 20% to 25% patients and point mutations at position D835 in the tyrosine-kinase domain (FLT3/TKD) in 7% to 10% patients. FLT3 tyrosine kinase inhibitors (TKI) are mainly active against FLT3 mutant AML. An antibody drug conjugate (ADC), directed against the extracellular domain of FLT3 may only require FLT3 cell surface expression independent of mutation status. The restricted cellular distribution of FLT3 receptor and a higher expression in AML than in normal bone marrow makes FLT3 a favorable ADC target. Therefore, this ADC based strategy may offer a therapeutic alternative for AML patients independent of FLT3 status. Here, we report the preclinical assessment of a novel FLT3 targeting ADC, AGS62P1. AGS62P1 consists of a human anti-FLT3 monoclonal antibody, site specifically conjugated to a potent cytotoxic payload. FLT3 expression is confirmed in a large panel of AML and ALL tumor cells as well as in AML patient specimens via flow cytometry. The anti-leukemic activity of AGS62P1 was evaluated against AML and ALL tumor cell lines, in vitro and in vivo. AGS62P1 demonstrated strong binding affinity (0.1-0.5 nM) and potent cytotoxic activity in FLT3/ITD and Non-ITD tumor models, in vitro. Cytotoxic IC50 potency for AGS62P1 was 0.5-13 nM in FLT3/ITD and 0.2-12 nM in FLT3 non-ITD models. A fluorescence based assay confirmed that AGS62P1 is rapidly internalized in AML tumor cell lines. AGS62P1 is highly efficacious in FLT3/ITD and non-ITD tumor xenografts, leading to significant tumor growth inhibition or complete tumor regression. In primary AML patient xenograft drug treatment studies, the engraftment and outgrowth of 5/6 samples were significantly reduced when treated with AGS62P1. Taken together our data demonstrate that AGS62P1 exhibits potent antitumor activity against a broad panel of AML tumor models and primary AML samples, regardless of FLT3 status. We believe AGS62P1 may be an effective and alternative therapeutic for AML patients, which can bypass the TKI mediated resistance and deliver target specific effect through a different mode of action. Disclosures Jin: Agensys: Research Funding. Anand:Agensys: Employment. Dick:Agensys: Research Funding.

The control of enzyme activity by reversible phosphorylation and the role of protein kinases in signal transduction illustrate well the pivotal role of protein phosphorylation in cell regulation. There are numerous examples of the role of protein kinases and phosphatases in disease, and protein kinases in particular are now established as druggable therapeutic targets (1). Phosphoproteomics includes the large-scale determination of protein phosphorylation in cells and tissue, sometimes referred to as phosphoprofiling. Phosphoprofiling is a global approach that can be used to characterize biological states including therapeutic responses. It is augmented by another proteomic method, protein kinase (and protein phosphatase) interaction mapping, which together have the potential—yet to be fully realized—to provide comprehensive pictures of cellular states. A particular utility for these methods will be in drug discovery and development.

Magnetic supports are tested for use in batch affinity capture of proteins. Two types of magnetic polymer composites were used for solid phase synthesis and for the batch affinity chromatography of folate binding protein from a protein mixture. Gly-Gly-L-Methotrexate as well as other analogs were synthesized on magnetic supports consisting of either polyoxyalkyleneamine grafted onto polystyrene beads or a copolymer of polyethylene glycol dimethylacrylamide (PEGA). Both supports incorporated within their matrix sub-micron particles of paramagnetic magnetite. The peptide-methotrexate analogs were attached to the magnetic supports via a photocleavable linker. The bound methotrexate-peptide analogs were equilibrated with a protein mixture consisting of bovine albumin, chicken albumin, folate binding protein, lysozyme, lactoferrin and lactoperoxidase precursor in phosphate buffered saline (PBS) and then after magnetically separating and washing the supports of any unbound components the bound protein was removed either through the photocleavage of the tethered methotrexate-peptide ligand or via exchange with soluble methotrexate. In all cases, the photocleavage or exchange with soluble methotrexate released folate binding protein as the major affinity captured protein. Of the two magnetic supports tested, the PEGA based support was found to be superior to the polyoxyalkyleneamine grafted polystyrene support and comparable to beaded agarose in releasing bound folate binding protein. Of the two methods for removing bound protein, photocleavage of the covalently attached ligand was found to release exclusively folate binding protein as opposed to exchange with soluble methotrexate which released residual amounts of the non-specifically bound proteins bovine and chicken albumin, in addition to folate binding protein. Thus, use of the PEGA based magnetic support in conjunction with a photocleavable linker should help facilitate the automation of multiple parallel affinity chromatography for proteomics applications.

Discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family. The receptor is activated upon binding to its ligand, collagen, and plays a crucial role in many fundamental processes such as cell differentiation, adhesion, migration and invasion. Although DDR1 is expressed in many normal tissues, upregulated expression of DDR1 in a variety of human cancers such as lung, colon and brain cancers is known to be associated with poor prognosis. Using shRNA silencing, we assessed the oncogenic potential of DDR1. DDR1 knockdown impaired tumor cell proliferation and migration in vitro and tumor growth in vivo. Microarray analysis of tumor cells demonstrated upregulation of TGFBI expression upon DDR1 knockdown, which was subsequently confirmed at the protein level. TGFBI is a TGFβ-induced extracellular matrix protein secreted by the tumor cells and is known to act either as a tumor promoter or tumor suppressor, depending on the tumor environment. Here, we show that exogenous addition of recombinant TGFBI to BXPC3 tumor cells inhibited clonogenic growth and migration, thus recapitulating the phenotypic effect observed from DDR1 silencing. BXPC3 tumor xenografts demonstrated reduced growth with DDR1 knockdown, and the same xenograft tumors exhibited an increase in TGFBI expression level. Together, these data suggest that DDR1 expression level influences tumor growth in part via modulation of TGFBI expression. The reciprocal expression of DDR1 and TGFBI may help to elucidate the contribution of DDR1 in tumorigenesis and TGFBI may also be used as a biomarker for the therapeutic development of DDR1 specific inhibitors.