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Supplementary Figure 5 from Dual Functional Monoclonal Antibody PF-04605412 Targets Integrin α5β1 and Elicits Potent Antibody-Dependent Cellular Cytotoxicity

Supplementary Methods, Figures 1-6, Tables A-C from Targeting Activin Receptor-Like Kinase 1 Inhibits Angiogenesis and Tumorigenesis through a Mechanism of Action Complementary to Anti-VEGF Therapies

Supplementary Figure 3 from Dual Functional Monoclonal Antibody PF-04605412 Targets Integrin α5β1 and Elicits Potent Antibody-Dependent Cellular Cytotoxicity

Integrin α5β1 is overexpressed in tumor-associated stroma and cancer cells, and has been implicated in angiogenesis, tumor survival, and metastasis. Antibody-dependent cellular cytotoxicity (ADCC) by immune effector cells has been shown to contribute to clinical efficacy for several IgG1 monoclonal antibody (mAb) therapeutics. Taking advantage of these two mechanisms, we generated a fully human, fragment crystalizable (Fc)-engineered IgG1 mAb, PF-04605412 (PF-5412), which specifically neutralizes α5 and binds the Fcγ receptors (FcγR) with enhanced affinity. In vitro, PF-5412 potently inhibited α5β1-mediated intracellular signaling, cell adhesion, migration, and endothelial cell (EC) tubulogenesis. PF-5412 induced significantly greater ADCC in α5-expressing tumor cells and ECs compared with a wild-type IgG1 (IgG1/wt) or IgG2 of identical antigen specificity. The degree of ADCC correlated with the abundance of natural killer (NK) cells in the peripheral blood mononuclear cells but was independent of donor FcγRIIIa polymorphism. In animal studies, PF-5412 displayed robust and dose-dependent antitumor efficacy superior to that observed with IgG1/wt, IgG2, or IgG4 of identical antigen specificity. The degree of efficacy correlated with α5 expression, macrophage and NK cell infiltration, and NK activity in the tumor. Depletion of host macrophages abrogated antitumor activity, suggesting a critical contribution of macrophage-mediated antitumor activity of PF-5412. Combination of PF-5412 with sunitinib significantly improved antitumor efficacy compared with either agent alone. The dual mechanism of action and robust antitumor efficacy of PF-5412 support its clinical development for the treatment of a broad spectrum of human malignancies. Cancer Res; 70(24); 10243–54. ©2010 AACR.

Supplementary Figure 1 from Dual Functional Monoclonal Antibody PF-04605412 Targets Integrin α5β1 and Elicits Potent Antibody-Dependent Cellular Cytotoxicity

Supplementary Figure 2 from Lymphatic Zip Codes in Premalignant Lesions and Tumors

Supplementary Figure 1 from Lymphatic Zip Codes in Premalignant Lesions and Tumors

Supplementary Table 1 from Mitochondrial/Cell-Surface Protein p32/gC1qR as a Molecular Target in Tumor Cells and Tumor Stroma

Blood vessels in tumors are morphologically and functionally distinct from normal resting blood vessels. We probed lymphatic vessels in premalignant lesions and tumors by in vivo screening of phage-displayed peptide libraries, asking whether they too have distinctive signatures. The resulting peptides begin to define such signatures. One peptide identified the lymphatics in a human melanoma xenograft. Another recognized the lymphatics in prostate cancers but not in premalignant prostate lesions; this peptide similarly identifies human prostate cancer lymphatics. A third was selective for the lymphatics in the premalignant prostate lesions. A fourth identified the lymphatics in dysplasias and squamous carcinomas of the cervix and skin. None recognize lymphatics in normal tissues. Thus, tumor development is associated with organ- and stage-specific changes in lymphatics. Systemic treatment of mice with fusions of a lymphatic homing peptide and a proapoptotic motif reduced the number of tumor lymphatics in prostate tumor and melanoma, forecasting future lymphatic targeting agents for detection and therapeutic intervention. (Cancer Res 2006; 66(11): 5696-706)

Supplementary Figure 1 from Mitochondrial/Cell-Surface Protein p32/gC1qR as a Molecular Target in Tumor Cells and Tumor Stroma

Supplementary Figure 4 from Dual Functional Monoclonal Antibody PF-04605412 Targets Integrin α5β1 and Elicits Potent Antibody-Dependent Cellular Cytotoxicity

Supplementary Figure 2 from Dual Functional Monoclonal Antibody PF-04605412 Targets Integrin α5β1 and Elicits Potent Antibody-Dependent Cellular Cytotoxicity

Supplementary Figure 3 from Lymphatic Zip Codes in Premalignant Lesions and Tumors

Supplementary Figure and Table Legends from Mitochondrial/Cell-Surface Protein p32/gC1qR as a Molecular Target in Tumor Cells and Tumor Stroma

A tumor homing peptide, LyP-1, selectively binds to tumor-associated lymphatic vessels and tumor cells in certain tumors and exhibits an antitumor effect. Here, we show that the protein known as p32 or gC1q receptor is the receptor for LyP-1. Various human tumor cell lines were positive for p32 expression in culture, and the expression was increased in xenograft tumors grown from the positive cell lines. Fluorescence-activated cell sorting analyses with anti-p32 antibodies showed that p32-positive cell lines expressed p32 at the cell surface. These cells bound and internalized LyP-1 peptide in proportion to the cell-surface expression level, which correlated with malignancy rather than total p32 expression in the cells. Like the LyP-1 peptide, p32 antibodies highlighted hypoxic areas in tumors, where they bound to both tumor cells and cells that expressed macrophage/myeloid cell markers and often seemed to be incorporated into the walls of tumor lymphatics. Significant p32 expression was common in human cancers and the p32 levels were often greatly elevated compared with the corresponding normal tissue. These results establish p32, particularly its cell-surface–expressed form, as a new marker of tumor cells and tumor-associated macrophages/myeloid cells in hypoxic/metabolically deprived areas of tumors. Its unique localization in tumors and its relative tumor specificity may make p32 a useful target in tumor diagnosis and therapy. [Cancer Res 2008;68(17):7210–8]

PARP inhibitors (PARPi) have emerged as promising cancer therapeutics capable of targeting specific DNA repair pathways, but their mechanism of action with respect to PARP1-DNA retention remains unclear. Here, we developed single-molecule assays to directly monitor the retention of PARP1 on DNA lesions in real time. Our study reveals a two-step mechanism by which PARPi modulate the retention of PARP1 on DNA lesions, consisting of a primary step of catalytic inhibition via binding competition with NAD + followed by an allosteric modulation of bound PARPi. While clinically relevant PARPi exhibit distinct allosteric modulation activities that can either increase retention of PARP1 on DNA or induce its release, their retention potencies are predominantly determined by their ability to outcompete NAD + binding. These findings provide a mechanistic basis for improved PARPi selection according to their characteristic activities and enable further development of more potent inhibitors.

Poly-ADP-ribosyltransferases play a critical role in DNA repair and cell death, and poly(ADP-ribosyl) polymerase 1 (PARP1) is a particularly important therapeutic target for the treatment of breast cancer because of its synthetic lethal relationship with breast cancer susceptibility proteins 1 and 2. Numerous PARP1 inhibitors have been developed, and their efficacy in cancer treatment is attributed to both the inhibition of enzymatic activity and their ability to trap PARP1 on to the damaged DNA, which is cytotoxic. Of the clinical PARP inhibitors, talazoparib is the most effective at trapping PARP1 on damaged DNA. Biochemically, talazoparib is also suspected to be a potent inhibitor of PARP5a/b (tankyrase1/2 [TNKS1/2]), which is an important regulator of Wnt/β-catenin pathway. Here we show using competition experiments in cell lysate that, at a clinically relevant concentration, talazoparib can potentially bind and engage TNKS1. Using surface plasmon resonance, we measured the dissociation constants of talazoparib, olaparib, niraparib, and veliparib for their interaction with PARP1 and TNKS1. The results show that talazoparib has strong affinity for PARP1 as well as uniquely strong affinity for TNKS1. Finally, we used crystallography and hydrogen deuterium exchange mass spectroscopy to dissect the molecular mechanism of differential selectivity of these PARP1 inhibitors. From these data, we conclude that subtle differences between the ligand-binding sites of PARP1 and TNKS1, differences in the electrostatic nature of the ligands, protein dynamics, and ligand conformational energetics contribute to the different pharmacology of these PARP1 inhibitors. These results will help in the design of drugs to treat Wnt/β-catenin pathway-related cancers, such as colorectal cancers.

A high level of fine particulate matter (PM2.5) has become one of the greatest threats to human health. Based on multi-source remote sensing data, the pollutant population exposure model, accompanied by the Theil–Sen Median and Mann–Kendall methods, was used to analyze the spatio-temporal patterns of global population exposure risk of PM2.5 from 2000 to 2016. The population distribution patterns of high-risk exposure areas have been accurately identified, the variation trend and stability of global population exposure risk of PM2.5 have also been analyzed. According to the results, the average concentration of PM2.5 is correlated with the total population. The average concentration of PM2.5 for countries from high to low are Asia (14.7 μg/m3), Africa (8.1 μg/m3), Europe (8.03 μg/m3), South America (5.69 μg/m3), North America (4.41 μg/m3), and Oceania (1.27 μg/m3). In addition, the global average population exposure risk of PM2.5 is decreasing annually. Specifically, China, India, Southeast Asia, and other regions have higher exposure risks. Less developed mountainous regions, cold regions, deserts and tropical rainforest regions have lower exposure risks. Moreover, Oceania, North America, South America and other regions have relatively stable exposure, whereas areas with relatively unstable exposure risk of PM2.5 are mainly concentrated in Asia, India, and eastern China, followed by Southeast Asia, Europe, and Africa. Furthermore, Asia has the largest population of all the continents, followed by Africa and Europe. Countries with increased populations are mainly distributed in Africa, whereas the countries with a declining population are mainly distributed in Europe. Based on this, it is important to identify the relationship between PM2.5 concentration and population exposure risk to improve human settlements and environmental risk assessment.

Gedatolisib, a PI3K/mTOR dual inhibitor delivered intravenously, is in early clinical trials in women with metastatic ER+ breast cancer. We reasoned that the therapeutic index could be further maximized by encapsulation of gedatolisib in nanoparticles (NP), thereby providing longer half-life, sustained release, blunting of Cmax and associated toxicity, asymmetric distribution to leaky tumor vasculature and the possibility of less frequent dosing. PF-07034663 is a polymeric NP encapsulating gedatolisib, targeted to prostate-specific membrane antigen (PSMA), a clinically validated tumor antigen expressed on prostate cancer cells and the neovasculature of some nonprostate solid tumors. Studies in mice, rats, and cynomolgus monkeys demonstrated that the terminal half-life of PF-07034663 following intravenous administration increased by ~1.5 fold and the Cmax of released gedatolisib from PF-07034663 decreased by ~75 fold compared to gedatolisib delivered by the same route. Gedatolisib released from PF-07034663 exhibited markedly enhanced intratumoral accumulation compared to gedatolisib (~40 fold), indicating enhanced tissue delivery and/or prolonged retention. In exploratory toxicology studies, PF-07034663 caused no hyper-insulinemic spike in rats, in contrast to gedatolisib. To evaluate the antitumor activity of PF-07034663, we performed tumor growth inhibition (TGI) studies in PSMA-positive C4-2 prostate and in PSMA-negative MDAMB361 ER+ breast and A549 lung xenograft tumor models. In the C4-2 model, PF-07034663 showed dose-dependent antitumor efficacy as a single agent, and, in combination with enzalutamide, exhibited enhanced activity leading to tumor regressions. PF 07034663 also exhibited prolonged, dose-dependent inhibition of phosphorylation of S6 (pS6) in the C4-2 model compared to gedatolisib. Interestingly, increased pharmacodynamics (PD) responses compared to gedatolisib were not restricted to PSMA-positive tumors. Moreover, TGI responses were increased in the PSMA-negative A549 lung cancer model, relative to those observed with gedatolisib. PF 07034663 treatment in spontaneous prostate tumors in PTENnullp53mut GEM mice and in subcutaneous allografts of these spontaneous tumors in wild-type mice showed that treatment of PF-07034663 caused similar pS6 inhibition in both models, suggesting that PD modulation is independent of tumor origin or milieu and is less likely due to altered enhanced permeability and retention (EPR) effect. Taken together, PF-07034663 exhibits potential for improved tolerability, sustained tumor PK, prolonged PD modulation, and less frequent dosing schedule compared to gedatolisib. These results support further exploration of gedatolisib in tumor-targeted or non-tumor-targeted nanoparticles in clinical trials as a promising vehicle for delivery of PI3K/mTOR inhibitors to advanced cancer patients. Citation Format: Lianglin Zhang, Gabriel Troche, Ravi Visswanathan, Wenhu Huang, Young-Ho Song, Mary E. Spilker, Zhenxiong Wang, Hui Wang, Anand Giddabasappa, Louise Cadzow, Susan Low, Greg Troiano, Jennifer Lafontaine, Valeria Fantin, Robert T. Abraham, Shubha Bagrodia. Development of a nanoparticle containing the PI3K/mTOR dual Inhibitor, gedatolisib, for cancer therapy [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B25.

Purpose A first-in-human clinical trial of a fully human, Fc-engineered IgG1 monoclonal antibody targeting integrin α5β1 was conducted to evaluate tolerability, maximum tolerated dose, pharmacokinetics, pharmacodynamics and preliminary anti-tumor activity. Methods Escalating doses of PF-04605412 were given IV on day 1, 28 and every 2 weeks thereafter to patients with advanced solid tumors until disease progression or unacceptable toxicity. Sequential dose cohorts were evaluated based on a modified 3 + 3 dose-escalation design. The starting dose was 7.5 mg based on preclinical data. Results Thirty-three patients were enrolled to six dose levels (7.5, 11.25, 16.9, 34, 68 and 136 mg). Twenty-three patients were evaluable for the primary endpoint (determination of the maximum tolerated dose). Five patients required permanent drug discontinuation due to acute infusion-related reactions, which occurred as grade 3 events in two patients. PK analysis indicated that the targeted drug exposure based on preclinical models was not achieved by the tolerated doses and PK modeling suggesting that doses at least fivefold higher would be necessary. No anti-tumor activity was observed. Conclusion Based on the safety data, the risks associated with the likelihood of significant cytokine-mediated infusion reactions at higher doses, the projected high dose necessary to affect on the biological target and the lack of anti-tumor activity at the doses explored, the trial was prematurely terminated without determining a formal maximum tolerated dose. Further clinical development of PF-04605412 has been discontinued. Electronic supplementary material The online version of this article (doi:10.1007/s00280-014-2576-8) contains supplementary material, which is available to authorized users.

In the design of nanoparticles that can target disease tissue in vivo, parameters such as targeting ligand density, type of target receptor, and nanoparticle shape can play an important role in determining the extent of accumulation. Herein, a systematic study of these parameters for the targeting of mouse xenograft tumors is performed using superparamagnetic iron oxide as a model nanoparticle system. The type of targeting peptide (recognizing cell surface versus extracellular matrix), the surface coverage of the peptide, its attachment chemistry, and the shape of the nanomaterial [elongated (nanoworm, NW) versus spherical (nanosphere, NS)] are varied. Nanoparticle circulation times and in vivo tumor-targeting efficiencies are quantified in two xenograft models of human tumors (MDA-MB-435 human carcinoma and HT1080 human fibrosarcoma). It is found that the in vivo tumor-targeting ability of the NW is superior to that of the NS, that the smaller, neutral CREKA targeting group is more effective than the larger, positively charged F3 molecule, that a maximum in tumor-targeting efficiency and blood half-life is observed with approximately 60 CREKA peptides per NW for either the HT1080 or the MDA-MB-435 tumor types, and that incorporation of a 5-kDa polyethylene glycol linker improves targeting to both tumor types relative to a short linker. It is concluded that the blood half-life of a targeting molecule-nanomaterial ensemble is a key consideration when selecting the appropriate ligand and nanoparticle chemistry for tumor targeting.

A tumor homing peptide, LyP-1, selectively binds to tumor-associated lymphatic vessels and tumor cells in certain tumors and exhibits an antitumor effect. Here, we show that the protein known as p32 or gC1q receptor is the receptor for LyP-1. Various human tumor cell lines were positive for p32 expression in culture, and the expression was increased in xenograft tumors grown from the positive cell lines. Fluorescence-activated cell sorting analyses with anti-p32 antibodies showed that p32-positive cell lines expressed p32 at the cell surface. These cells bound and internalized LyP-1 peptide in proportion to the cell-surface expression level, which correlated with malignancy rather than total p32 expression in the cells. Like the LyP-1 peptide, p32 antibodies highlighted hypoxic areas in tumors, where they bound to both tumor cells and cells that expressed macrophage/myeloid cell markers and often seemed to be incorporated into the walls of tumor lymphatics. Significant p32 expression was common in human cancers and the p32 levels were often greatly elevated compared with the corresponding normal tissue. These results establish p32, particularly its cell-surface–expressed form, as a new marker of tumor cells and tumor-associated macrophages/myeloid cells in hypoxic/metabolically deprived areas of tumors. Its unique localization in tumors and its relative tumor specificity may make p32 a useful target in tumor diagnosis and therapy. [Cancer Res 2008;68(17):7210–8]

Nanoparticle-based diagnostics and therapeutics hold great promise because multiple functions can be built into the particles. One such function is an ability to home to specific sites in the body. We describe here biomimetic particles that not only home to tumors, but also amplify their own homing. The system is based on a peptide that recognizes clotted plasma proteins and selectively homes to tumors, where it binds to vessel walls and tumor stroma. Iron oxide nanoparticles and liposomes coated with this tumor-homing peptide accumulate in tumor vessels, where they induce additional local clotting, thereby producing new binding sites for more particles. The system mimics platelets, which also circulate freely but accumulate at a diseased site and amplify their own accumulation at that site. The self-amplifying homing is a novel function for nanoparticles. The clotting-based amplification greatly enhances tumor imaging, and the addition of a drug carrier function to the particles is envisioned.

To better elucidate the role of vascular endothelial growth factor (VEGF)165 in soft tissue sarcoma (STS) growth, metastasis, and chemoresistance, we generated stably transfected human STS cell lines with VEGF165 to study the effect of VEGF165 on STS cells in vitro and the effect of culture medium from these cells on human umbilical vascular endothelial cells. Severe combined immunodeficient mice bearing xenografts of transfected cell lines were used to assess the effect of VEGF overexpression and the effect of VEGF receptor (VEGFR) 2 inhibition on STS growth, metastasis, and response to doxorubicin. VEGF165-transfected xenografts formed highly vascular tumors with shorter latency, accelerated growth, enhanced chemoresistance, and increased incidence of pulmonary metastases. Blockade of VEGFR2 signaling using DC101 anti-VEGFR2 monoclonal antibody enhanced doxorubicin chemoresponse; this combined biochemotherapy inhibited tumor growth and decreased pulmonary metastases without overt toxicity. Combined therapy reduced microvessel counts while increasing vessel maturation index. VEGF overexpression did not affect on the sarcoma cells per se; however, conditioned medium from VEGF transfectants caused increased endothelial cell proliferation, migration, and chemoresistance. Addition of DC101 induced endothelial cell sensitivity to doxorubicin and suppressed the activity of matrix metalloproteinases secreted by endothelial cells. We therefore conclude that VEGF is a critical determinant of STS growth and metastasis and that STS chemoresistance, in our model, is a process induced by the interplay between STS cells and tumor-associated endothelial cells. STS growth and metastasis can be interrupted by combined low-dose doxorubicin and anti-VEGFR2, a strategy that attacks STS-associated endothelial cells. In the future, such therapeutic approaches may be useful in treating STS before the development of clinically apparent metastases. (Cancer Res 2006; 66(17): 8770-7)

Phage display was used to identify homing peptides for blood vessels in a mouse model of HPV16-induced epidermal carcinogenesis. One peptide, CSRPRRSEC, recognized the neovasculature in dysplastic skin but not in carcinomas. Two other peptides, with the sequences CGKRK and CDTRL, preferentially homed to neovasculature in tumors and, to a lesser extent, premalignant dysplasias. The peptides did not home to vessels in normal skin, other normal organs, or the stages in pancreatic islet carcinogenesis in another mouse model. The CGKRK peptide may recognize heparan sulfates in tumor vessels. The dysplasia-homing peptide is identical to a loop in kallikrein-9 and may bind a kallikrein inhibitor or substrate. Thus, characteristics of the angiogenic vasculature distinguish premalignant and malignant stages of skin tumorigenesis.

BACKGROUND Human sarcomas have a propensity for aggressive local invasion and early pulmonary metastasis. Frequently, deaths are due to uncontrolled pulmonary metastases. The purpose of the current study was to evaluate cytogenetics, tumorigenicity, metastatic potential, and production of angiogenic factors in human sarcoma cell strains. A secondary purpose was to establish low passage cell strains for studying new therapeutic approaches. METHODS The authors established 11 cell strains from human sarcoma surgical specimens and characterized their in vitro tumor properties, including growth in soft agar, expression of angiogenic growth factors (vascular endothelial growth factor [VEGF] and basic-fibroblast growth factor [bFGF]), and cytogenetics. RESULTS All of the cell strains remained diploid. All exhibited the ability to grow in soft agar and expressed both VEGF as well as bFGF. In addition, 6 of the 11 established sarcoma cell strains were tumorigenic, 5 of which spontaneously metastasized to the lungs in nude mice. Four of the five cell strains that yielded lung metastases were derived from lung metastases in patients. CONCLUSIONS The 11 cell strains, which were derived from diverse sarcoma histologies, will provide a model for studying not only metastatic progression but also the in vitro and in vivo efficacy of new therapeutic modalities for human sarcomas. Cancer 2002;95:1569–76. © 2002 American Cancer Society. DOI 10.1002/cncr.10879

Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1) plays an important role in vascular development, remodeling, and pathologic angiogenesis. Here we investigated the role of ALK1 in angiogenesis in the context of common proangiogenic factors [PAF; VEGF-A and basic fibroblast growth factor (bFGF)]. We observed that PAFs stimulated ALK1-mediated signaling, including Smad1/5/8 phosphorylation, nuclear translocation and Id-1 expression, cell spreading, and tubulogenesis of endothelial cells (EC). An antibody specifically targeting ALK1 (anti-ALK1) markedly inhibited these events. In mice, anti-ALK1 suppressed Matrigel angiogenesis stimulated by PAFs and inhibited xenograft tumor growth by attenuating both blood and lymphatic vessel angiogenesis. In a human melanoma model with acquired resistance to a VEGF receptor kinase inhibitor, anti-ALK1 also delayed tumor growth and disturbed vascular normalization associated with VEGF receptor inhibition. In a human/mouse chimera tumor model, targeting human ALK1 decreased human vessel density and improved antitumor efficacy when combined with bevacizumab (anti-VEGF). Antiangiogenesis and antitumor efficacy were associated with disrupted co-localization of ECs with desmin+ perivascular cells, and reduction of blood flow primarily in large/mature vessels as assessed by contrast-enhanced ultrasonography. Thus, ALK1 may play a role in stabilizing angiogenic vessels and contribute to resistance to anti-VEGF therapies. Given our observation of its expression in the vasculature of many human tumor types and in circulating ECs from patients with advanced cancers, ALK1 blockade may represent an effective therapeutic opportunity complementary to the current antiangiogenic modalities in the clinic. Cancer Res; 71(4); 1362–73. ©2011 AACR.

Integrin α5β1 is overexpressed in tumor-associated stroma and cancer cells, and has been implicated in angiogenesis, tumor survival, and metastasis. Antibody-dependent cellular cytotoxicity (ADCC) by immune effector cells has been shown to contribute to clinical efficacy for several IgG1 monoclonal antibody (mAb) therapeutics. Taking advantage of these two mechanisms, we generated a fully human, fragment crystalizable (Fc)-engineered IgG1 mAb, PF-04605412 (PF-5412), which specifically neutralizes α5 and binds the Fcγ receptors (FcγR) with enhanced affinity. In vitro, PF-5412 potently inhibited α5β1-mediated intracellular signaling, cell adhesion, migration, and endothelial cell (EC) tubulogenesis. PF-5412 induced significantly greater ADCC in α5-expressing tumor cells and ECs compared with a wild-type IgG1 (IgG1/wt) or IgG2 of identical antigen specificity. The degree of ADCC correlated with the abundance of natural killer (NK) cells in the peripheral blood mononuclear cells but was independent of donor FcγRIIIa polymorphism. In animal studies, PF-5412 displayed robust and dose-dependent antitumor efficacy superior to that observed with IgG1/wt, IgG2, or IgG4 of identical antigen specificity. The degree of efficacy correlated with α5 expression, macrophage and NK cell infiltration, and NK activity in the tumor. Depletion of host macrophages abrogated antitumor activity, suggesting a critical contribution of macrophage-mediated antitumor activity of PF-5412. Combination of PF-5412 with sunitinib significantly improved antitumor efficacy compared with either agent alone. The dual mechanism of action and robust antitumor efficacy of PF-5412 support its clinical development for the treatment of a broad spectrum of human malignancies. Cancer Res; 70(24); 10243–54. ©2010 AACR.

Blood vessels in tumors are morphologically and functionally distinct from normal resting blood vessels. We probed lymphatic vessels in premalignant lesions and tumors by in vivo screening of phage-displayed peptide libraries, asking whether they too have distinctive signatures. The resulting peptides begin to define such signatures. One peptide identified the lymphatics in a human melanoma xenograft. Another recognized the lymphatics in prostate cancers but not in premalignant prostate lesions; this peptide similarly identifies human prostate cancer lymphatics. A third was selective for the lymphatics in the premalignant prostate lesions. A fourth identified the lymphatics in dysplasias and squamous carcinomas of the cervix and skin. None recognize lymphatics in normal tissues. Thus, tumor development is associated with organ- and stage-specific changes in lymphatics. Systemic treatment of mice with fusions of a lymphatic homing peptide and a proapoptotic motif reduced the number of tumor lymphatics in prostate tumor and melanoma, forecasting future lymphatic targeting agents for detection and therapeutic intervention. (Cancer Res 2006; 66(11): 5696-706)

Background— Endothelial cells that line the vascular lumen can express cell-surface proteins that are specific to the endothelium of a particular tissue. In this study, we probed the heart vasculature for heart-specific endothelial markers by phage display. Methods and Results— We used a novel combination of in vivo phage selection and a bacterial 2-hybridization scheme against a heart cDNA library, which allows simultaneous identification of peptides that specifically bind to the target endothelium, as well as the endothelial molecules (receptors) recognized by the peptides. We found 5 heart-targeting peptides and their receptors. We confirmed and quantified the selective expression of 4 of the proteins in heart endothelial cells by independent methods. The heart specificity of phages was as high as 300-fold greater than that of nonrecombinant control phages. The proteins selectively expressed by the heart endothelium were in most cases also expressed by cardiomyocytes and, at lower levels, in some other tissues. Conclusions— These findings provide new markers for the endothelium of heart vessels and reveal a commonality between parenchymal and endothelial gene expression in the heart. The heart-homing peptides provide a means of targeting diagnostic and therapeutic agents to the heart, and their receptors are potential drug discovery targets.

Vascular endothelial growth factor (VEGF) and VEGF receptor 2 [fetal liver kinase 1 (Flk-1)/kinase insert domain-containing receptor] have been shown to play a major role in tumor angiogenesis. In this study, we investigated whether anti-Flk-1 monoclonal antibody DC101 could therapeutically inhibit growth and angiogenesis of human soft tissue sarcoma, and we explored its capacity to enhance the tumoricidal effects of doxorubicin. Treatment of well-established leiomyosarcoma SKLMS-1 and rhabdomyosarcoma RD xenografts in severe combined immunodeficient mice with DC101 resulted in significant antitumor activity. In a parallel study, we compared tumor inhibition with continuous low-dose "antiangiogenic" schedule versus once-every-2-weeks high-dose standard schedule of doxorubicin. We found that continuous low-dose treatment inhibited the tumor growth of RD xenografts about 46.5% of that with standard-schedule treatment, but that continuous low-dose treatment did not inhibit the tumor growth of SKLMS-1 xenografts. Notably, combined DC101 and continuous low-dose doxorubicin resulted in more effective growth inhibition of SKLMS-1 and RD xenografts than has been observed with any agent alone in a long-term s.c. tumor xenograft model. The combination therapy was associated with no additional toxicity to the host animal compared with low-dose doxorubicin alone. Histological examination of xenografts showed significantly reduced microvessel counts in the tumors given combined therapy compared with the tumors given either agent alone. These results are consistent with an enhanced inhibition of angiogenesis in vivo by combined DC101 and doxorubicin using Matrigel plug assay. Additionally, DC101 plus doxorubicin directly exerted enhanced inhibitory effects on endothelial cell migration, proliferation, and tube-like formation in vitro. Furthermore, the combination induced an enhanced apoptosis of endothelial cells that was associated with an increase of capase-3 activity. Thus, the inhibition of angiogenesis and induction of endothelial cell apoptosis are likely important mechanisms for the antitumor activity of combined DC101 and doxorubicin. Collectively, our data suggested that anti-VEGF receptor 2 in combination with continuous low-dose doxorubicin may provide a new therapeutic approach for human soft tissue sarcoma in the clinic.

Integrin α5β1 plays an important role in tumor angiogenesis, invasion, and metastasis. Overexpression of α5β1 has been associated with poor prognosis in various types of cancers, and also plays a critical role in the resistance of cancers to radiotherapy and chemotherapy. We have previously reported that PF-04605412 (PF-5412), a fully human Fc-engineered IgG1 mAb against human α5β1, displayed robust and dose-dependent antitumor efficacy in multiple preclinical tumor models. Assessment of PF-5412 mechanism of action (MOA) in standard mouse xenograft models has been limited, however, because this antibody does not cross-react with murine integrin α5. To further our understanding of PF-5412 MOAs, we generated human integrin α5 knock-in mice (ITGA5 KI) in which the entire murine coding sequence of integrin α5 was replaced with the corresponding human coding sequence. Mice that were homozygous for human integrin α5 were viable and no abnormal phenotype or behavior was observed. qPCR and FACS analyses showed that human integrin α5 was expressed in various types of tissues and peripheral blood in ITGA5 KI mice, whereas no mouse integrin α5 was detected. Murine integrin β1 expression in ITGA5 KI mice was comparable to wild type mice. Growth of MC38 murine CRC tumors engineered to express human α5 was inhibited by treatment with PF-5412 in a dose-dependent manner. Tumor regression was observed at high doses of PF-5412 but not at comparable dose levels of α5 antibodies lacking ADCC activity (IgG4). IHC analyses of CD31 positive vessels showed that PF-5412 significantly inhibited blood vessel density and induced greater macrophages infiltration into tumors than non-ADCC-inducing antibodies. Notably, parental MC38 tumors lacking endogenous human α5 expression were also controlled by PF-5412 treatment (60% TGI) in ITGA5 KI mice. PF-5412 efficacy in this model also exceeded that of non-ADCC-inducing antibodies. Immunohistochemistry (IHC) analyses showed that PF-5412 treatment significantly decreased blood vessel density in the parental MC38 tumors. These results demonstrate that PF-5412 benefit does not depend on integrin α5β1 expression by the tumor cell itself, and support the hypothesis that ADCC-mediated killing of tumor endothelium will confer clinical benefit. The results showed here indicate that ITGA5 KI mouse is a relevant model for evaluating anti-human integrin α5 antibody MOA. Citation Format: Lianglin Zhang, Jeffrey L. Stock, Mark W. Elliott, Enhong Chen, Thao Nguyen, Anthony Wong, Craig B. Davis, James G. Christensen. Assessment of mechanisms of action (MOAs) of the Fc-engineered integrin α5β1 targeting antibody PF-04605412 in human integrin α5 knock-in mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2336. doi:10.1158/1538-7445.AM2013-2336

Anti-angiogenic therapy targeting the tumor vasculature has demonstrated clinical benefits in certain types of tumors. However, many patients develop resistance and eventually progress on anti-angiogenic therapy. The mechanism of resistance is largely unknown and warrants extensively investigation. We developed a human xenograft tumor model (M24met/R) in mice that has acquired resistance to VEGF receptor tyrosine kinase inhibitors (VEGFR TKI), by chronically treating the parental M24met tumors (M24met/P) with PF-0337210 (20-40 mg/kg, QD, PO) and serially passaging resistant tumor fragments in nude mice. M24met/R tumors were also resistant to axitinib, a selective VEGFR TKI in late stage clinical development. Immunohistochemical (IHC) staining revealed higher degree of hypoxia and lower microvessel density in M24met/R tumors compared to parental tumors. Compared with those in M24met/P, vessels in M24met/R tumors appeared to be larger in diameter and more mature as shown by high incidence of co-localization between endothelial cells and perivascular cells (CD31+Desmin+). Microarray analysis of tumor lysates (human UA133 2.0, mouse 430 2.0 chip) revealed distinct gene expression profiles between M24met/R tumors and size-matched M24met/P tumors. Of note, several angiogenesis-related genes, including human VEGF, bFGF, integrin α5β1, IL-8, PDGF,CSF and murine VEGF, integrin α5β1, ALK1 (activin receptor-like kinase 1), IL-8, c-Met, IL-6 were up-regulated in M24met/R tumors, whereas murine VEGFR1 and 2, PROX1, PDGFR and endogenous angiogenic inhibitor TSP-1 were down-regulated in the resistant tumors. The above observations were verified with ELISA and/or IHC. Next, targeted combination approaches were applied in an attempt to circumvent M24met/R resistance to axitinib. Concurrent administration of axitinib plus either anti-IL-8 monoclonal antibody (mAb), c-Met inhibitor, anti-integrin α5 mAb or anti-ALK1 mAb significantly improved anti-tumor efficacy compared to single agents alone. IHC analyses showed that combinatorial therapy of anti-ALK1 plus axitinib reduced the amount of CD31+Desmin+ vessels compared to axitinib alone, suggesting that targeting ALK1 destroyed VEGFR TKI-resistant vessels. Together, our results suggested that multiple alternative angiogenic factors contributed to the acquired resistance of M24met/R melanoma to VEGFR TKI. Combination treatment using inhibitor of IL-8, c-Met, integrin α5β1 or ALK1 with VEGFR TKI may represent a new strategy to mitigate resistance to VEGFR TKI in the clinic. 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 3270. doi:10.1158/1538-7445.AM2011-3270

Integrin α5β1 and its ligand fibronectin are significantly and coordinately over-expressed in tumor associated blood vessels and cancer cells. These molecules have been reported to mediate angiogenesis, tumor survival and metastasis. α5β1 has also been implicated as a poor prognostic marker in a number of human tumor types including NSCLC and ovarian carcinoma. Thus, α5β1 is a potential target for cancer therapy. Accumulating evidence suggests an important role of innate immune effector cells in tumor killing via antibody dependent cellular cytotoxicity (ADCC). Taking advantage of both of these mechanisms, we generated an α5 integrin-specific neutralizing monoclonal antibody (mAb, PF-04605412) with high affinity for Fcγ receptors. In vitro, PF-04605412 potently inhibited α5β1-mediated intracellular signaling, cell adhesion, migration, endothelial cell tubulogenesis, and induced apoptosis of endothelial and tumor cells. Compared to a wild type IgG1 of identical antigen specificity (wt IgG1), PF-04605412 induced a significantly greater tumor and endothelial cell cytotoxicity. ADCC activity correlated with the abundance of NK cells within PBMCs of human donors, but was independent of donor FcγRIIIa polymorphism. In in vivo studies using human xenograft, immuno-xenograft and syngeneic tumor models, PF-04605412 (and an anti-murine α5 surrogate mAb) displayed robust and dose-dependent anti-angiogenesis and anti-tumor efficacy including tumor growth delay, regression and metastasis inhibition, all of which were superior to effects observed with an IgG2 or wt IgG1 of identical antigen specificity. Western blotting and immunohistochemistry analysis of tumor samples showed that anti-tumor efficacy was associated with reduction of total and phosphorylated focal adhesion kinase and positively correlated with α5 expression, infiltrating macrophages, markers of effector cell activity, and apoptosis. Furthermore, depletion of host macrophages abrogated anti-tumor efficacy, indicating a critical contribution of ADCC/phagocytosis to the anti-tumor activity of PF-04605412. The PK-PD relationship, efficacy of PF-04605412 in combination with anti-angiogenic and tumor targeting agents will also be reported. The distinct dual mechanism of action and robust anti-tumor efficacy of PF-04605412 supports the clinical development of this agent for the treatment of human cancers. Short Title: Dual functional mAb PF-04605412 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 3811.