Therapeutic targeting of membrane-associated GRP78 in leukemia and lymphoma: preclinical efficacy in vitro and formal toxicity study of BMTP-78 in rodents and primates.

Translation of drug candidates into clinical settings requires demonstration of preclinical efficacy and formal toxicology analysis for filling an Investigational New Drug (IND) application with the US Food and Drug Administration (FDA). Here, we investigate the membrane-associated glucose response protein 78 (GRP78) as a therapeutic target in leukemia and lymphoma. We evaluated the efficacy of the GRP78-targeted proapoptotic drug bone metastasis targeting peptidomimetic 78 (BMTP-78), a member of the D(KLAKLAK)2-containing class of agents. BMTP-78 was validated in cells from patients with acute myeloid leukemia and in a panel of human leukemia and lymphoma cell lines, where it induced dose-dependent cytotoxicity in all samples tested. Based on the in vitro efficacy of BMTP-78, we performed formal good laboratory practice toxicology studies in both rodents (mice and rats) and nonhuman primates (cynomolgus and rhesus monkeys). These analyses represent required steps towards an IND application of BMTP-78 for theranostic first-in-human clinical trials.

Biopanning and rapid analysis of selective interactive ligands.

Here we introduce a new approach for the screening, selection and sorting of cell-surface-binding peptides from phage libraries. Biopanning and rapid analysis of selective interactive ligands (termed BRASIL) is based on differential centrifugation in which a cell suspension incubated with phage in an aqueous upper phase is centrifuged through a non-miscible organic lower phase. This single-step organic phase separation is faster, more sensitive and more specific than current methods that rely on washing steps or limiting dilution. As a proof-of-principle, we screened human endothelial cells stimulated with vascular endothelial growth factor (VEGF) and constructed a peptide-based ligand-receptor map of the VEGF family. Next, we validated the motif PQPRPL as a novel chimeric ligand mimic that binds specifically to VEGF receptor-1 and to neuropilin-1. BRASIL may prove itself a superior method for probing target cell surfaces with a broad range of potential applications.

Anti-cancer activity of targeted pro-apoptotic peptides.

We have designed short peptides composed of two functional domains, one a tumor blood vessel 'homing' motif and the other a programmed cell death-inducing sequence, and synthesized them by simple peptide chemistry. The 'homing' domain was designed to guide the peptide to targeted cells and allow its internalization. The pro-apoptotic domain was designed to be nontoxic outside cells, but toxic when internalized into targeted cells by the disruption of mitochondrial membranes. Although our prototypes contain only 21 and 26 residues, they were selectively toxic to angiogenic endothelial cells and showed anti-cancer activity in mice. This approach may yield new therapeutic agents.

Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model.

In vivo selection of phage display libraries was used to isolate peptides that home specifically to tumor blood vessels. When coupled to the anticancer drug doxorubicin, two of these peptides-one containing an alphav integrin-binding Arg-Gly-Asp motif and the other an Asn-Gly-Arg motif-enhanced the efficacy of the drug against human breast cancer xenografts in nude mice and also reduced its toxicity. These results indicate that it may be possible to develop targeted chemotherapy strategies that are based on selective expression of receptors in tumor vasculature.

Molecular heterogeneity of the vascular endothelium revealed by in vivo phage display.

Vascular beds are known to differ in structure and metabolic function, but less is known about their molecular diversity. We have studied organ-specific molecular differences of the endothelium in various tissues by using in vivo screening of peptide libraries expressed on the surface of a bacteriophage. We report here that targeting of a large number of tissues with this method yielded, in each case, phage that homed selectively to the targeted organ. Different peptide motifs were recovered from each of these tissues. The enrichment in homing to the target organs relative to an unselected phage was 3-35-fold. Peptide sequences that conferred selective phage homing to the vasculature of lung, skin, and pancreas were characterized in detail. Immunohistochemistry showed that the phage localized in the blood vessels of their target organ. When tested, the phage homing was blocked in the presence of the cognate peptide. By targeting several tissues and by showing that specific homing could be achieved in each case, we provide evidence that organ- and tissue-specific molecular heterogeneity of the vasculature is a general, perhaps even universal, phenomenon. Our results also show that these molecular differences can serve as molecular addresses.

Tumor targeting with a selective gelatinase inhibitor.

Several lines of evidence suggest that tumor growth, angiogenesis, and metastasis are dependent on matrix metalloproteinase (MMP) activity. However, the lack of inhibitors specific for the type IV collagenase/gelatinase family of MMPs has thus far prevented the selective targeting of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) for therapeutic intervention in cancer. Here, we describe the isolation of specific gelatinase inhibitors from phage display peptide libraries. We show that cyclic peptides containing the sequence HWGF are potent and selective inhibitors of MMP-2 and MMP-9 but not of several other MMP family members. Our prototype synthetic peptide, CTTHWGFTLC, inhibits the migration of human endothelial cells and tumor cells. Moreover, it prevents tumor growth and invasion in animal models and improves survival of mice bearing human tumors. Finally, we show that CTTHWGFTLC-displaying phage specifically target angiogenic blood vessels in vivo. Selective gelatinase inhibitors may prove useful in tumor targeting and anticancer therapies.

An AAVP-based solid-phase transducing matrix for transgene delivery: potential for translational applications.

A hybrid vector of adeno-associated virus and phage (termed AAVP) has been introduced as a platform for systemic ligand-directed delivery of transgenes to tumors over the past decade. A series of studies have evaluated the AAVP platform for potential theranostic or purely therapeutic applications in several tumor models. Sufficient ligand-directed tumor targeting consistently resulted in specific molecular-genetic imaging and/or anti-tumor responses to 'suicide' transgene delivery. However, efforts to optimize transduction efficiency are still ongoing. Here, we set out to expand the translational utility of AAVP by combining it with gold (Au) nanoparticles in order to generate a 'transducing matrix' for improved targeted gene delivery in solid phase. Targeted AAVP-based solid-phase transduction is superior to conventional transduction in soluble (aqueous) environments. This transducing matrix is stable and can be further modified with additional attributes (for example, magnetization) for targeted imaging and therapeutic gene delivery. Notably, it spontaneously assembles around cells in vitro to markedly enhance transduction capabilities compared with AAVP alone. This versatile nanoplatform may enable new applications of AAVP for transgene delivery in translational settings including, for example, efforts toward complex tissue patterning.

Modulation of the immune response by systemic targeting of antigens to lymph nodes.

Factors that determine the immunogenicity of an antigen in vivo are still largely unknown. Direct administration of antigens into lymphatic organs appears to enhance immune response. We hypothesized that systemically targeting antigens to lymphatic tissue in vivo might modulate immunity. To test this hypothesis, we measured the humoral immune response elicited by bacteriophage vaccination. We show that the responses against a lymph node-targeted phage are significantly higher than those against control untargeted phage; the effect is specific because it is inhibited by coadministration of the cognate synthetic peptides displayed. Our data suggest that systemic targeting of antigens to lymph nodes through the circulation modulates humoral immune response. This strategy may have broad applications in the development of vaccines, production of antibodies, and immunotherapy.

NG2 proteoglycan-binding peptides target tumor neovasculature.

NG2 is the rat homologue of the human melanoma proteoglycan, also known as the high molecular weight melanoma-associated antigen. This developmentally regulated membrane-spanning chondroitin sulfate proteoglycan is expressed primarily by glial, muscle, and cartilage progenitor cells. Upon maturation, these cell types down-regulate NG2 expression. In adult animals, the expression of NG2 is restricted to tumor cells and angiogenic tumor vasculature, making this proteoglycan a potential target for directing therapeutic agents to relevant sites of action. To this end, we have identified specific NG2-binding peptides by screening a phage-displayed random peptide library on purified NG2. Several rounds of biopanning on NG2 resulted in the specific enrichment of two phage-displayed decapeptides, TAASGVRSMH and LTLRWVGLMS. The binding of these phages to NG2 was inhibitable both by soluble NG2 and by glutathione S-transferase (GST) fusion proteins containing the cognate peptide sequences. In addition, direct binding between GST-TAASGVRSMH and GST-LTLRWVGLMS fusion proteins and NG2 was demonstrated in solid-phase binding assays. Interestingly, these NG2-binding fusion proteins cross-inhibited each other's binding to NG2, suggesting that the two sequences bind to the same or overlapping sites on the proteoglycan. Upon injection into tumor-bearing mice, NG2-binding phages specifically homed to tumor vasculature in wild-type mice but did not localize to the tumor vasculature in NG2 knockout mice. The in vivo targeting capability of these sequences suggests that they can be used for tumor targeting.

Replacement of the p16/CDKN2 gene suppresses human glioma cell growth.

The p16/CDKN2 gene has many features of a growth suppressor gene: it maps to 9p21, a frequent region of loss of heterozygozity in a variety of tumor types; it encodes an inhibitor of cyclin-dependent kinase 4; and its homozygous deletion is common in tumor-derived cell lines. However, the lower frequency of alteration of the gene in primary tumor tissue as compared to the cognate tumor cell lines has brought this interpretation into question. We have assessed the growth suppressive function of p16/CDKN2 by gene transfer. The introduction of full-length p16/CDKN2 cDNA caused marked growth suppression in p16/CDKN2-null human glioma cells, but was without significant effect in those cells with endogenous wild-type p16/CDKN2 alleles. These results provide functional evidence in support of the hypothesis that the p16/CDKN2 gene is a functional growth suppressor gene, at least in gliomas.

Loss of P16INK4 expression is frequent in high grade gliomas.

P16INK4 is a cell cycle regulator that specifically binds to and inactivates cyclin-dependent kinase 4 (CDK4). Its encoding gene (p16/CDKN2) maps to chromosome 9p21, a region that undergoes frequent loss of heterozygosity in a variety of human tumors. We have analyzed the p16/CDKN2 gene and its expression in a series of primary glioma samples. Although homozygous deletion or mutation of the p16/CDKN2 gene was uncommon in this series and P16INK4 protein was detectable in all grade II tumors, it was present in only 50% of grade III and grade IV samples. Conversely, in some grade IV tumors that level of P16INK4 protein was elevated; in these cases, its target, CDK4, was amplified and overexpressed. These results suggest: (a) the involvement of P16INK4 in glioma progression; (b) that mechanisms other than mutation or deletion can down-regulate expression of the p16/CDKN2 gene; and (c) that the balance between CDK4 and its cognate inhibitor, P16INK4, may confer a cell growth advantage and facilitate tumor progression.

Cyclin-dependent kinase 6 (CDK6) amplification in human gliomas identified using two-dimensional separation of genomic DNA.

DNA amplification is a common mechanism invoked by many human tumors to elicit overexpression of genes whose products are involved in drug resistance or cell proliferation. Although amplified regions in tumor DNA may exceed several megabases in size, segments of amplicons with a high probability of containing gene sequences may be amenable to detection by restriction landmark genomic scanning (RLGS), a high-resolution DNA analysis that separates labeled NotI fragments in two dimensions. Here, we tested this by applying RLGS to matched samples of glioma and normal brain DNA and found tumor-specific amplification of the gene encoding cyclin-dependent kinase 6 (CDK6), an observation not previously reported in human tumors. The CDK6 gene has been localized to chromosome 7q21-22, but in the gliomas studied here, it was not coamplified with either the syntenic MET (7q31) or epidermal growth factor receptor (7p11-p12) genes, suggesting that this may be part of a novel amplicon in gliomas. We then corroborated this finding by identifying both amplification-associated and amplification-independent increases in CDK6 protein levels in gliomas relative to matched normal brain samples. These data implicate the CDK6 gene in genomic amplification and illustrate the potential of RLGS for the more general identification and cloning of novel genes that are amplified in human cancer.

Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis.

Phage that display a surface peptide with the NGR sequence motif home selectively to tumor vasculature in vivo. A drug coupled to an NGR peptide has more potent antitumor effects than the free drug [W. Arap et al., Science (Washington DC), 279: 377-380, 1998]. We show here that the receptor for the NGR peptides in tumor vasculature is aminopeptidase N (APN; also called CD13). NGR phage specifically bound to immunocaptured APN and to cells engineered to express APN on their surface. Antibodies against APN inhibited in vivo tumor homing by the NGR phage. Immunohistochemical staining showed that APN expression is up-regulated in endothelial cells within mouse and human tumors. In another tissue that undergoes angiogenesis, corpus luteum, blood vessels also expressed APN, but APN was not detected in blood vessels of various other normal tissues stained under the same conditions. APN antagonists specifically inhibited angiogenesis in chorioallantoic membranes and in the retina and suppressed tumor growth. Thus, APN is involved in angiogenesis and can serve as a target for delivering drugs into tumors and for inhibiting angiogenesis.

Point mutations can inactivate in vitro and in vivo activities of p16(INK4a)/CDKN2A in human glioma.

Deletions of chromosomal region 9p21 are among the most common genetic alterations observed during the clonal evolution of high grade malignant gliomas. Structural and functional evidence has suggested that homozygous deletion involving CDKN2A (the genetic locus encoding the cyclin-dependent kinase inhibitor p16(NK4a)) is a mechanism of inactivation of this gene and that it can be a growth suppressor in human gliomas. However, the presence of other potential suppressor genes in the 9p21 region and the relatively large sizes of the deletions has made it difficult to be certain that the CDKN2A gene is their actual target. Here, we tested this hypothesis by determining the growth suppressive effects, cell cycle inhibitions, and the activities of seven naturally occurring glioma-derived CDKN2A alleles carrying point mutations and found that two of them were functionally compromised. To resolve discrepancies among the different existing functional assays, we developed an assay for p16(INK4a) function that allowed us to demonstrate that the expression of wild-type CDKN2A, but not alleles with inactivating mutations, prevents pRB phosphorylation in vivo in human glioma cells. These data suggest that CDKN2A is a critical target for mutational inactivation in human malignant gliomas.

Functional analysis of wild-type and malignant glioma derived CDKN2Abeta alleles: evidence for an RB-independent growth suppressive pathway.

The tumor suppressor gene CDKN2A (p16/MTS1/INK4A), which encodes the cyclin-dependent kinase inhibitor p16(INK4a), is a target of 9p21 deletions during the malignant progression of human gliomas. This gene also encodes a second protein product (human p16beta, murine p19ARF), which originates from an unrelated exon of CDKN2A (exon 1beta) spliced onto exon 2 in an alternate reading frame. Cell cycle arrest by p16beta is caused by an as yet unidentified pathway. In order to test the candidacy of p16beta as a glioma suppressor, we replaced p16(INK4a), p15(INK4b) and p16beta wild-type as well as a series of seven glioma-derived p16beta alleles (R87H, A112V, R120H, A121V, G125R, A128A and A128V), into glioma cell lines that had either CDKN2A-/RB+ (U-87MG and U-251MG) or CDKN2A+/RB- (LN-319) endogenous backgrounds and demonstrated that p16beta can act as a functional glioma cell growth suppressor. Moreover, p16beta, but not p16(INK4a) or p15(INK4b) inhibited the growth of RB-negative LN-319 cells, indicating that p16beta likely exerts its effects through an RB-independent pathway. In vitro and in vivo assays of pRB phosphorylation were consistent with this interpretation. Since none of the glioma-derived p16beta mutations inactivated their growth suppressive activities, it appears that mutations in CDKN2A exon 2 (which is shared in the coding sequences of p16(INK4a) and p16beta) likely exclusively target p16(INK4a).

Molecular addresses in blood vessels as targets for therapy.

We have isolated several organ- and tumor-homing peptides by using in vivo phage display. This technology involves the screening of peptide libraries in a living animal. The peptides that result from such a selection home to specific organs or tissues because they recognize molecular 'addresses', receptors that are differentially expressed in vascular beds. Targeted delivery of chemotherapeutics, pro-apoptotic peptides and cytokines to tumors using these peptides improved therapeutic efficacy in animal models. Translation of this technology into clinical applications will form the basis for targeting therapeutic and imaging agents in the context of cancer and other diseases.

Spare the rod and spoil the eye.

This review presents a new unified view of the pathogenesis of three common causes of acquired retinal degenerative disease-diabetic retinopathy, age related macular degeneration, and retinopathy of prematurity. In these three conditions, angiogenesis has a predominant role in the development of sight threatening pathology. Angiogenesis is controlled by among other factors the expression of vascular endothelial growth factor (VEGF), which in turn is regulated by absolute and relative lack of oxygen. The severe pathological manifestations of these three conditions are not part of a general underlying disease process because they are peculiar to the eye, and the profound hypoxia that develops in normal retina during dark adaptation (rod driven hypoxia) is an adequate and elegant additional factor to explain their pathogenesis. A large number of experimental reports support this conclusion, although rod driven anoxia is not generally considered as a causal factor in ocular disease. However, the hypothesis can be critically tested, and also suggests novel methods of treatment and prevention of these conditions that may be simpler and more inexpensive than current therapies and that have a smaller potential for adverse effects.

Identification of receptor ligands with phage display peptide libraries.

With the development and maturation of the technology of displaying peptides on bacteriophage, it has become possible to isolate peptide ligands to various targets. In the phage display strategy, up to 10(9) peptides of different permutations are expressed on the surface of filamentous phage. Thus, peptides capable of binding target molecules in vitro and even target tissues in vivo can be identified. In recent years, a series of libraries that display degenerate peptides of different lengths have been constructed, and specific ligands to cell surface receptors, such as integrins, have been isolated. In the in vivo biopanning, peptides targeting distinct organs or tumors have been rescued after intravenous administration of phage libraries into mice. In one application, the isolated peptide ligands have been used to direct a cytotoxic drug to tumor vasculature in mice. Further applications in radioimaging and radiotherapy are being investigated.

Tumor and serum beta-2-microglobulin expression in women with breast cancer.

To investigate whether the tumor expression of beta-2-microglobulin (beta 2-M) could serve as a marker of tumor biologic behavior, the authors studied specimens of breast carcinomas from 60 consecutive female patients. Presence of beta 2-M was analyzed by immunohistochemistry. No significant correlations were found between tumor beta 2-M expression and several histologic attributes such as type, histologic and nuclear grades, mitotic index, necrosis, vascular invasion, and lymphocytic infiltration. Likewise, beta 2-M was not associated with markers of disease extension such as TNM, (UICC, classification of malignant tumors) staging and axillary lymph node involvement or with estrogen, progesterone, and glucocorticoid receptor levels. However, there was a significantly positive association between tumor beta 2-M expression and the degree of lymphocytic infiltration in the tumor tissue. Beta 2-M serum levels were determined by an enzyme-linked immunosorbent assay in samples from 22 of the above women. Although some of the highest values had been obtained in women with larger (T4) primary tumors, the authors failed to detect any statistical relationship between beta 2-M expression in the tumor with serum levels or between serum beta 2-M and the above histologic, laboratory, and clinical factors.

Optical systems for in vivo molecular imaging of cancer.

Progress toward a molecular characterization of cancer would have important clinical benefits; thus, there is an important need to image the molecular features of cancer in vivo. In this paper, we describe a comprehensive strategy to develop inexpensive, rugged and portable optical imaging systems for molecular imaging of cancer, which couples the development of optically active contrast agents with advances in functional genomics of cancer. We describe initial results obtained using optically active contrast agents to image the expression of three well known molecular signatures of neoplasia: including over expression of the epidermal growth factor receptor (EGFR), matrix metallo-proteases (MMPs), and oncoproteins associated with human papillomavirus (HPV) infection. At the same time, we are developing inexpensive, portable optical systems to image the morphologic and molecular signatures of neoplasia noninvasively in real time. These real-time, portable, inexpensive systems can provide tools to characterize the molecular features of cancer in vivo.