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We present a direct cell printing technique to pattern neural cells in a three-dimensional (3D) multilayered collagen gel. A layer of collagen precursor was printed to provide a scaffold for the cells, and the rat embryonic neurons and astrocytes were subsequently printed on the layer. A solution of sodium bicarbonate was applied to the cell containing collagen layer as nebulized aerosols, which allowed the gelation of the collagen. This process was repeated layer-by-layer to construct the 3D cell-hydrogel composites. Upon characterizing the relationship between printing resolutions and the growth of printed neural cells, single/multiple layers of neural cell-hydrogel composites were constructed and cultured. The on-demand capability to print neural cells in a multilayered hydrogel scaffold offers flexibility in generating artificial 3D neural tissue composites.

Purpose: Placental growth factor (PlGF) is an angiogenic protein. Upregulation of PlGF has been observed in the clinic following antiangiogenic regimens targeting the VEGF pathway. PlGF has been proposed as a therapeutic target for oncology. sFLT01 is a novel fusion protein that neutralizes mouse and human PlGF (mPlGF, hPlGF) and mouse and human VEGF-A (mVEGF-A, hVEGF-A). It was tested in syngeneic and xenograft tumor models to evaluate the effects of simultaneously neutralizing PlGF and VEGF-A and to investigate changes observed in the clinic in preclinical models. Experimental Design: Production of PlGF and VEGF-A by B16F10 and A673 cancer cells in vitro was assessed. Mice with subcutaneous B16F10 melanoma or A673 sarcoma tumors were treated with sFLT01. Tumor volumes and microvessel density (MVD) were measured to assess efficacy. Serum levels of hVEGF-A, hPlGF, and mPlGF at early and late time points were determined by ELISA. Results: Exposure of cancer cell lines to sFLT01 caused a decrease in VEGF secretion. sFLT01 inhibited tumor growth, prolonged survival, and decreased MVD. Analysis of serum collected from treated mice showed that sFLT01 administration caused a marked increase in circulating mPlGF but not hPlGF or hVEGF. sFLT01 treatment also increased circulating mPlGF levels in non–tumor-bearing mice. Conclusion: With the tumor cell lines and mouse models we used, antiangiogenic therapies that target both PlGF and VEGF may elicit a host response rather than, or in addition to, a malignant cell response that contribute to therapeutic resistance and tumor escape as suggested by others. Clin Cancer Res; 17(5); 976–88. ©2011 AACR.

Tumor-specific delivery of cytotoxic agents remains a challenge in cancer therapy. Antibody–drug conjugates (ADC) deliver their payloads to tumor cells that overexpress specific tumor-associated antigens—but the multi-day half-life of ADC leads to high exposure even of normal, antigen-free, tissues and thus contributes to dose-limiting toxicity. Here, we present Adnectin–drug conjugates, an alternative platform for tumor-specific delivery of cytotoxic payloads. Due to their small size (10 kDa), renal filtration eliminates Adnectins from the bloodstream within minutes to hours, ensuring low exposure to normal tissues. We used an engineered cysteine to conjugate an Adnectin that binds Glypican-3, a membrane protein overexpressed in hepatocellular carcinoma, to a cytotoxic derivative of tubulysin, with the drug-to-Adnectin ratio of 1. We demonstrate specific, nanomolar binding of this Adnectin–drug conjugate to human and murine Glypican-3; its high thermostability; its localization to target-expressing tumor cells in vitro and in vivo, its fast clearance from normal tissues and its efficacy against Glypican-3-positive mouse xenograft models.

Background: Therapeutic blockade of the immune checkpoints CTLA-4 and PD-1/PD-L1 has provided durable survival benefits in multiple malignancies. However, additional treatment options are often required to maximally reverse immune dysfunction. V-domain immunoglobulin suppressor of T-cell activation (VISTA) is an orphan B7 family ligand that is highly expressed on immunosuppressive myeloid cells and has been shown to inhibit T-cell responses in vitro and in preclinical models of cancer. Here we report that VISTA, a ligand for the receptor P-selectin glycoprotein ligand-1 (PSGL-1), uses a histidine-rich interface to engage PSGL-1 and suppress immune responses selectively in acidic environments, such as tumor beds. Methods: Recombinant VISTA multimers were used to assess binding to cells and recombinant PSGL-1 over a range of pH values (6.0-7.4). Antibodies against human and mouse VISTA were used to map binding and functional epitopes. Acidic pH receptor-based ligand capture was used to identify PSGL-1 as a VISTA receptor. X-ray crystallography was used to resolve the VISTA structure in complex with an anti-VISTA antigen-binding fragment. The MC38 mouse tumor model was used to assess the effects of VISTA deficiency and the effects of VISTA antibody blockade alone and combined with anti-PD-1 in vivo. Results: Recombinant VISTA bound leukocytes at pH 6.0 but was not detectable at pH 7.4. Antibodies in a single epitope bin blocked VISTA binding and reversed VISTA suppression of T cells. VISTA-mediated inhibition of T cells was detectable at pH 7.4 but was more pronounced below pH 7.0, suggesting that VISTA functions selectively in acidic conditions. VISTA’s structure was resolved at 1.6 Å and characterized by a histidine-rich extension of the immunoglobulin V domain central β-sheet. VISTA blocking antibodies, but not nonblocking antibodies, bound this β-sheet region. Engineered antibodies could distinguish this epitope in its active and inactive states at acidic and neutral pH, respectively. Receptor capture on T cells at acidic pH identified PSGL-1 as a VISTA receptor. T-cell PSGL-1 CRISPR ablated VISTA binding, whereas PSGL-1 expression on CHO cells conferred VISTA binding at acidic pH. Thus, an antibody that blocks mouse VISTA binding to mouse T cells at acidic pH combined with a PD-1 blocking antibody was shown to enhance anti-tumor T-cell responses and drive MC38 tumor rejection in vivo. Conclusions: VISTA is a highly pH-selective ligand for PSGL-1. VISTA antibody blockade reverses immune suppression in vitro and in vivo, especially when combined with PD-1 antibody blockade. Our results identify acidic pH as a direct regulator of VISTA engagement with PSGL-1 and suggest new strategies to enhance anti-tumor T-cell responses. Citation Format: Robert J. Johnston, Linhui Julie Su, Jason Pinckney, David Critton, Arathi Krishnakumar, Martin Corbett, Andrew Rankin, Rose A. DiBella, Lynne Campbell, Xiaodi Deng, Haibin Chen, Alexander Kozhich, Jim Holloway, Zheng Yang, Ginger Rakestraw, Michael Quigley, Alan J. Korman. Acidic pH selective binding of VISTA to PSGL-1 and anti-tumor activity of combined VISTA and PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1548.

The identification of proteins that selectively discriminate between tumor cells and normal adult cells allows for the specific targeting of diseased cells with antibody therapeutics. One such recently identified protein, PTK7, is an onco-fetal membrane protein which exhibits limited expression and function in adults. PTK7 was identified as a member of the RTK super family but lacks a functional kinase domain. Normally, PTK7 is expressed early in development and its loss is associated with severe defects in neural tube closure and sensory hair cell bundle formation. Functionally, little is known about the signaling involving PTK7, but it has been linked to both the canonical and noncanonical WNT pathways. Recently, PTK7 expression has been shown to be upregulated in a number of cancers including: ovarian, melanoma, leukemia, lung, pancreatic, colon, renal and breast. In vitro and in vivo studies support a role in regulating angiogenesis, invasion & survival. To further validate PTK7 as a potential cancer target that may be required for tumor maintenance and progression, we analyzed the expression of PTK7 in normal and tumor samples, and validated an in vitro and in vivo role of PTK7 on cell growth in ovarian cancer cell lines using both genetic tools and polyclonal antibodies. Silencing PTK7 with stably expressed inducible shRNAs is shown to inhibit the growth of ovarian cancer cell lines in vitro and to lead to delayed tumor growth upon PTK7 knockdown in murine tumor xenograft models. Further supporting the role of PTK7 as a potential antibody target, polyclonal antibodies to PTK7 are shown to inhibit the growth of SKOV3 and OVCAR8 cells in vitro. Although 4 human antibodies derived from phage display failed to inhibit in vitro cell growth, these results suggest that functionally blocking PTK7 may lead to the inhibition of ovarian tumor growth and is a potential target for antibody therapies. Citation Format: Zhihu Ding, Amanda Lennon, Keli Perron, David Harper, Hui Su, Meredith Wolfram, Joshua Murtie, Stuart Licht, Jason Pinckney, Helene Simonds-Mannes, Kimberly Bishop, Julie-Ann Gavigan, Dinesh Bangari, Maureen Magnay, William Weber, David Reczek, William Brondyk, Vicky Drewett, Marc Trombe, Dietmar Hoffmann, Raffaele Baffa, Serena Silver, Victoria Richon, Christopher Winter, Venkat Reddy, Richard C. Gregory. PTK7 as a potential therapeutic target in ovarian cancer. [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 5448. doi:10.1158/1538-7445.AM2014-5448

Introduction: PlGF and VEGF stimulate angiogenesis and promote the growth of tumor vasculature. PlGF is a member of the VEGF family and binds to VEGFR1. sFLT01 is a novel fusion protein comprised of the Fc portion of human IgG1 and the PlGF- and VEGF-binding domain of VEGFR1/Flt-1. The properties of sFLT01 and the potential of sFLT01 as an anti-angiogenic agent to inhibit tumor growth were investigated in several in vitro assays and in multiple xenograft tumor models. Methods: The binding kinetics of sFLT01 for both the human and murine homologues of PlGF and VEGF were assessed by Biacore. The abilities of recombinant human PlGF and VEGF to induce endothelial cell and pericyte proliferation and of sFLT01 to inhibit this stimulation were investigated in cell-based assays. The secretion of human VEGF and PlGF in culture by the HT29 colon carcinoma, H460 lung carcinoma, and A673 sarcoma human cell lines was quantified by ELISA. In efficacy studies, sFLT01 was administered by intraperitoneal injection twice per week to immunodeficient mice bearing HT29, H460, or A673 subcutaneous tumors. Antibodies specific for human IgG and VEGR2 were applied to A673 sarcoma tumor sections from mice treated with sFLT01 to visualize sFLT01 in the tumors and determine VEGFR2 expression in the cellular components. Pericytes and endothelial cells were identified with antibodies against NG2 and CD31. Results: The Biacore results indicated that sFLT01 has high affinity for human and murine PlGF and VEGF. Human recombinant PlGF and VEGF each induced the proliferation of human pericytes and endothelial cells in culture. This stimulation was inhibited by sFLT01. A673 sarcoma, HT29 colon and H460 lung carcinoma cells secreted higher levels of VEGF than PlGF in culture. In vivo, 10 mg/kg sFLT01 was effective at significantly slowing the growth of HT29 colon carcinoma and A673 sarcoma tumors compared to controls. Further analysis of the A673 sarcoma tumors in sFLT01-treated mice by immunohistochemistry revealed that sFLT01 penetrated multiple areas of the tumor. sFLT01 was detected in the vasculature, stroma, necrotic areas, and adjacent to malignant cells. sFLT01 treatment in the A673 model disrupted vessel integrity with a lack of association between endothelial cells and pericytes. A673 sarcoma cells expressed VEGFR2 in vivo. Conclusion: sFLT01 neutralizes the angiogenic activity of multiple vasculogenic VEGF family members in vitro and inhibited the proliferation of cells that form blood vessels, endothelial cells and pericytes. In vivo, sFLT01 treatment resulted in disorganized tumor vasculature thereby slowing the growth of xenografts tumors. The expression of VEGFR2 in A673 sarcoma tumors suggests that VEGF may play a role in autocrine signaling in some malignant cells. sFLT01 has antitumor and antiangiogenic activity in several human tumor xenografts and may offer therapeutic benefit. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1388.

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