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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.

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]

Chemotherapy is often limited by toxicity to normal cells. Therefore, an ideal anticancer drug should discriminate between normal tissue and tumors. This would require a target receptor molecule mostly present in tumors. The cyclic peptide cCPGPEGAGC (PEGA) is a homing peptide that has previously been shown to accumulate in breast tumor tissue in mice. PEGA peptide does not cross the plasma membrane per se; however, when attached to the cell-penetrating peptide pVEC, the conjugate is taken up by different breast cancer cells in vitro. Additionally, the homing capacity of the PEGA- pVEC is conserved in vivo, where the conjugate mainly accumulates in blood vessels in breast tumor tissue and, consequently is taken up. Furthermore, we show that the efficacy of the anticancer drug, chlorambucil, is increased more than 4 times when the drug is conjugated to the PEGA- pVEC chimeric peptide. These data demonstrate that combining a homing sequence with a cell-penetrating sequence yields a peptide that combines the desirable properties of the parent peptides. Such peptides may be useful in diagnostics and delivery of therapeutic agents to an intracellular location in a specific tumor target tissue.

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 Trastuzumab (Herceptin; Genentech, South San Francisco, CA) is a humanized anti-ErbB-2 monoclonal antibody that has demonstrated antitumor function, especially in combination with other chemotherapies such as paclitaxel (Taxol; Bristol Myers-Squibb, Princeton, NJ), in patients with tumors that overexpress ErbB-2. Because the repeated administration of low-dose chemotherapy, such as paclitaxel, endorsed an antiangiogenic effect in vitro, and because trastuzumab was shown to inhibit angiogenesis in tumor xenografts, the authors investigated whether ErbB-2-mediated angiogenic responses would be inhibited more effectively by the combined treatment of paclitaxel plus trastuzumab. METHODS Tumor xenografts were established in 37 severe combined immunodeficiency mice by injecting the mammary fat pad with ErbB-2-overexpressing human breast carcinoma cells. Mice then were treated with an immunoglobulin (IgG) control, trastuzumab, paclitaxel, or a combination treatment of trastuzumab plus paclitaxel. Tumorigenicity, lung metastasis, and tumor microvessel density (MVD) were evaluated. Vascular endothelial growth factor (VEGF) secretion, endothelial cell migration after treatment, and the status of phosphorylated Akt were evaluated in vitro to determine mechanisms underlying the inhibition of ErbB-2-induced angiogenesis. RESULTS Mice treated with the trastuzumab plus paclitaxel combination exhibited significantly reduced mean tumor volumes compared with mice treated with the IgG control (419.5 mm3 vs. 786.6 mm3, P < 0.0001). Mice treated with trastuzumab had a mean tumor volume of 543.9 mm3, and mice treated with paclitaxel had a mean tumor volume of 574.9 mm3. Tumors from the trastuzumab-plus-paclitaxel group also had significantly decreased mean MVD compared with the control (30 ± 8 MVD vs. 44 ± 12 MVD, P < 0.05). The trastuzumab group had tumors with a MVD of 35 ± 7, similar to the paclitaxel-treated group (35 ± 9). Forty-four percent of the mice in the trastuzumab-plus-paclitaxel group had metastases to the lungs compared with 50%, 63%, and 75% of the mice in the paclitaxel, trastuzumab, and control groups, respectively. In vitro, the ErbB-2-overexpressing cells treated with combined trastuzumab plus paclitaxel secreted less VEGF than the cells treated with trastuzumab, paclitaxel, or control (185.9 pg/mL vs. 233.2 pg/mL, 261.3 pg/mL, and 286.4 pg/mL, respectively). In addition, the conditioned media from the combination group stimulated less mean endothelial cell migration (31.0 cells vs. 47.0 cells, 39.2 cells, and 67.5 cells, respectively). Furthermore, Akt phosphorylation contributed to VEGF up-regulation and Akt phosphorylation was reduced more effectively by combined trastuzumab plus paclitaxel treatment compared with the other treatments. CONCLUSIONS Combined trastuzumab plus paclitaxel treatment more effectively inhibited ErbB-2-mediated angiogenesis than either treatment alone, which resulted in more pronounced tumoricidal effects. This effect may be mediated via the reduction of phosphorylated Akt. Cancer 2003;98:1377–85. © 2003 American Cancer Society. DOI 10.1002/cncr.11656

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)

The main problems with current cancer therapies, including those for breast cancer, are that they are only partially effective and highly toxic. The homing peptide technology provides a new targeting strategy that aims at physically concentrating therapeutic agents in tumor tissue by making use of the unique features of tumor vasculature thereby enhancing anti-tumor activity and decreasing side effects. A pentapeptide CREKA, which homes to vasculature of breast tumor in mice, was identified with breast tumor-bearing MMTVPyMT mice by using in vivo screening of phage displayed peptide libraries. The CREKA-displaying phage selectively homes to breast tumors in mice relative to other tumors. The selectivity of the phage homing to breast tumors is about 100-fold relative to non-recombinant phage. Fluorescein-labeled CREKA peptide accumulates in breast tumors, but not normal tissues. Mutating the cysteine (C) residue in CREKA to an alanine preserves the homing activity. Receptor identification experiments indicate that the receptor for the CREKA peptide is related to type IV collagen. Additional screenings of phage peptide libraries against extracellular matrix of breast tumor have been performed, and a new method for the identification of homing peptide receptors has been developed. An investigation of drug-CREKA conjugates for in the treatment of breast tumors in mice has been initiated.

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.