# Patent

## Title
Targeting of Notch3 receptor function for cancer therapy

## Abstract

The present invention involves the use of peptides from Notch3, and antibodies that recognize epitopes represented by those peptides, as anti-cancer agents. Methods of combination therapy using standard anti-cancer protocols in conjunction with Notch3 peptides and antibodies also are provided.

## Background
This application claims benefit of priority to U.S. Prov. Appln. Ser. No. 60/972,584, filed Sep. 14, 2007, the entire contents of which are hereby incorporated by reference.


This invention was made with government support under grant no. 2P50 CA090949 awarded by the National Cancer Institute. The government has certain rights in the invention.


BACKGROUND OF THE INVENTION
I. Field of the Invention
The present invention relates to the fields of oncology and molecular biology. More particular the invention relates to the targeting of the Notch3 receptor.
II. Related Art
Lung cancer is the most common cause of cancer-related deaths in the United States. The cure rate for patients with lung cancer remains low—15%—and has not changed significantly during the past 30 years (Jermal et al., 2005). A better understanding of the signaling pathways important in driving and maintaining the malignant state allows the identification of new therapeutic targets and is thus imperative for continued progress in the treatment of these patients. Genes involved in cell fate determination often contribute to tumorigenesis when they are aberrantly expressed. The family of Notch receptors is one such family where there are now strong data linking it to cancer pathogenesis.
All four members of the Notch receptor family are known to be dysregulated in the majority of human cancers. The inventors were the first to link dysregulation of the Notch3 pathway to human lung cancer (Dang et al., 2000). They demonstrated that Notch3 is highly expressed in 40% of all resected lung cancers and that, in the developing lung, constitutive activation of Notch3 results in inhibition of terminal differentiation. Furthermore, they showed that inhibiting this pathway in human lung tumors results in the loss of the malignant phenotype in vitro and tumor inhibition in xenograft models. This anti-tumor effect is enhanced in the presence of low serum and in combination with an EGFr tyrosine kinase inhibitor. Taken together, these data support an important role for Notch3 and its interaction with the EGF and Ras pathways in lung cancer. However, methods for therapeutic intervention in Notch3 related cancers has not yet been reported.
SUMMARY OF THE INVENTION
Thus, in accordance with the present invention, there is provided an isolated and purified peptide of no more than about 50 residues and comprising the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) or CLNGGS (SEQ ID NO:8). More particular peptides include the sequences CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The peptide may be no more than about 25 residues, or no more than about 20 residues, or no more than about 15 residues. The peptide may consist of the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The peptide may further be comprised in a pharmaceutically acceptable diluent, buffer or excipient.
In another embodiment, there is provided a method of inhibiting Notch3 receptor signaling comprising contacting a cell expressing Notch3 with a peptide of no more than about 50 residues and comprising the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) or CLNGGS (SEQ ID NO:8). The cell may be a cancer cell, such as a lung cancer cell and/or an adenocarcinoma. More particular peptides include the sequences CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The peptide may be no more than about 25 residues, or no more than about 20 residues, or no more than about 15 residues. The peptide may consist of the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The method may further comprise contacting the cell with two or more peptides comprising sequences of at least two of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The method may also further comprises contacting the cancer cell with a second agent that inhibits cancer cell growth, differentiation, metastasis or drug resistance.
In yet another embodiment, there is provided a method of treating a subject having a Notch3-expressing cancer comprising administering to said subject a peptide of no more than about 50 residues and comprising the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) or CLNGGS (SEQ ID NO:8). The subject may a human. The cancer cell may be a lung cancer cell and/or an adenocarcinoma. More particular peptides include the sequences CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The peptide may be no more than about 25 residues, or no more than about 20 residues, or no more than about 15 residues. The peptide may consist of the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The method may further comprise contacting the cell with two or more peptides comprising sequences of at least two of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The method may also further comprises contacting the cancer cell with a second agent that inhibits cancer cell growth, differentiation, metastasis or drug resistance.
Also provided are an isolated and purified antibody that binds to an epitope comprising the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) or CLNGGS (SEQ ID NO:8), as well as methods of using such antibodies to inhibit Notch3 receptor signaling in a cell expressing Notch3.
In still another embodiment, there is provided a method of treating a subject having a Notch3-expressing cancer comprising administering to said subject an antibody that binds to an epitope comprising the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) or CLNGGS (SEQ ID NO:8).
Yet another embodiment comprises a pharmaceutical formulation comprising two or more of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10) including three, four, five, six, seven or all of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10).
It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
These, and other, embodiments of the invention will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following description, while indicating various embodiments of the invention and numerous specific details thereof, is given by way of illustration and not of limitation. Many substitutions, modifications, additions and/or rearrangements may be made within the scope of the invention without departing from the spirit thereof, and the invention includes all such substitutions, modifications, additions and/or rearrangements.



BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein:
FIGS. 1A-B. Diagram demonstrates the major steps of the canonical Notch pathway. (FIG. 1A) Binding of a DSL ligand triggers proteolysis (S2 and S3) of the Notch receptor and releases the C-terminal NICD fragment. (FIG. 1B) NICD translocates to the nucleus, recruits coactivators, and binds to CSL factors to promote target gene transcription. In the absence of NICD, the CSL factor associates with the corepressor complex and inhibits target gene transcription.
FIGS. 2A-B. Proposed mechanisms of antagonistic and cooperative interactions between Ras and Notch pathways. (FIG. 2A) Notch antagonizes Ras signaling by preventing expression of active EGF (1). Notch activation can also directly inhibit Ras activity (2) and induce expression MAPK inhibitors (3). (FIG. 2B) Ras activation in turn can induce expression of Notch inhibitors (4). Paradoxically, Ras activation has also been shown to induce DSL ligand expression (5). These observation further support the hypothesis that Notch activation is highly context dependent. However, in a majority of cancers, Notch and ras appear to be cooperative. Adapted from Sundaram (2005).
FIG. 3. Immunohistochemistry with an antibody to extracellular domain of Notch3. Cytoplasmic and membranous staining is observed in squamous cell carcinoma (A) and adenocarcinoma (C) of the lung as compared to aneuroendocrine tumor (B) and normal lung (D). In panel B, slight staining is seen in blood vessels (arrow) within the tumor, consistent with other studies demonstrating normal staining of Notch3 in blood vessels
FIGS. 4A-D. Notch3 alters lung morphology of SP-C-N3IC transgenic mice at E18.5. Five μm-thick lung sections of wild-type littermate controls (FIGS. 4A, 4C) show that epithelial layer of terminal airways are thin and comprised mostly of type I pneumocytes. The terminal lung epithelium of transgenic embryos (FIGS. 4B, 4D) demonstrates severe metaplasia, composed mostly of undifferentiated cuboidal cells (arrowheads). No type I pneumocyte was found. The mesenchyme is abnormally abundant compared to that seen in the wild-type littermate. Bars=50 μm; br., bronchiole; m, mesenchyme; v, vessel.
FIGS. 5A-B. Notch inhibition inhibits the tumor phenotype. (FIG. 5A) Inhibition of the Notch3 signaling pathway markedly reduces the size of the colonies formed in soft agar (panel B), compared with vector controls (panel A) in HCC2429 and H460. (FIG. 5B) In serum-starved conditions, the growth of the DN transfectant is severely inhibited in comparison with that of VC. However, with the addition of exogenous growth factors, the growth rate is equal to that of VC.
FIG. 6. Knocking out Notch3 with siRNA reduces focus formation. The pSuper vectors expressing Notch3 siRNA and a mouse Notch3 sequence were transfected into HCC2429, a human lung cancer cell line. The cells were selected with puramycin, then stained with crystal violet after 4 weeks.
FIGS. 7A-D. A γ-secretase inhibitor inhibits tumor cells and Notch3 processing. (FIG. 7A) GSI inhibits HCC2429 cells in a serum-dependent manner. HCC2429 cells are sensitive to GSI, and the sensitivity increases in low serum, similarly to that observed with clones expressing the DN construct. Tumor cells treated with DMSO alone had no change in cell viability. (FIG. 7B) Inhibition of S3 proteolytic processing results in the decrease of Notch3 intracellular domain (N3ICD) and accumulation of S2 product (N3ΔE) after 3 hours. (FIG. 7C) In this experiment, HCC2429 was stably transfected with plasmid vector expressing Notch3 siRNA. Control C is the parental HCC2429, whereas siRNA-C clones 5, 6, 8 expressed high level of HCC2429 and siRNA-N3 clones 12, 15, 17 and 20 expressed significantly lower level of Notch3 (FIG. 7D). Loss of Notch3 results in no loss in cell survival when treated with MRK003.
FIGS. 8A-D. γ-secretase inhibitor MRK003 demonstrates anti-tumor activity in vivo. (FIG. 8A) Xenografts injected with HCC2429 were treated with MRK003 once the tumors became palpable. After 2 weeks of treatment, the inventors observed about a 50% reduction in tumor size. (FIG. 8B) Loss of activated Notch3 (N3ICD) can be seen in tumor treated with MRK003. Histological examination of resected tumors from xenografts at the end of treatment. Marked necrosis can be seen in the MRK003 treated animal (FIG. 8D) as compare to control (FIG. 8C).
FIGS. 9A-C. Inhibition of Notch3 increases apoptosis. (FIG. 9A) After 72 hours of exogenous growth factor deprivation, cell lines transfected with DN show a higher percentage of apoptosis as measured by Apo-BrdU analysis. (FIG. 9B) Expression levels of phospho-Akt protein decrease in the Notch3-overexpressing cell line HCC2429 when it is stably transfected with the DN construct, particularly with serum starvation. (FIG. 9C) Transfection with Notch3 SiRNA resulted in loss of Bcl-xL expression and induction of apoptotic product PARP.
FIGS. 10A-C. Notch3 crosstalks with the MAPK pathway. (FIG. 10A) Inhibition of Notch3 signaling in HCC2429 downregulates phospho-p44/42 (ERK?) under serum-starved conditions and after induction with 10% FCS. (FIG. 10B) When the immortalized lung epithelial cell line BEAS-2B was transfected with the DA construct, the inventors observed higher levels of phospho-p44/p42 under serum-starved conditions as well as after serum induction. (FIG. 10C) One mechanism of MAPK modulation includes transcriptional regulation of MKP1 in HCC2429. The DN clones demonstrate significantly higher transcriptional level of MKP1 under serum-starved conditions and at 30 minutes and 1 hour after serum induction, when compared with VC (*).
FIGS. 11A-B. Notch3 modulates the EGF pathway and increases sensitivity to an EGFr inhibitor. (FIG. 11A) In HCC2429, inhibition of the Notch3 pathway increases sensitivity to AG1478 nearly 40-fold. In H460, a cell line that is markedly resistant to AG1478 (IC50=23.8 μM) when compared to HCC2429 (IC50=8.3 μM), inhibition of Notch3 also increases sensitivity to the inhibitor. (FIG. 11B) A similar observation is made in H460 when the inventors combine AG1478 with L-685,458, a γ-secretase inhibitor, further supporting the hypothesis that EGF cooperates with the Notch pathway in oncogenesis.
FIGS. 12A-B. In HCC2429 lung cancer cell line, Notch inhibitor MRK003 enhances the effect of EGFR inhibitor AG1478 on colony formation in soft agar. (FIG. 12A) Photographs showing that MRK003 not only decreases colony formation, but also enhances the effect of AG1478 on growth. (FIG. 12B) Graph depicts the quantitative decrease in colony formation.
FIG. 13. Notch3 peptides induce apoptosis. Representative experiment showing that the peptides induce apoptosis by Annexin V staining through screening using an FMAT system. Each of 155 different peptides were assayed in quadruplicate, and only those peptides that produced a significant increase of fluorescence signal in all 4 wells were considered potentially positive or capable of inducing apoptosis. The bar graphs here reflect fluorescence counts. Sequences N102: CATAV, N103: CFHGAT, N105: CVSNP, N132: CLNGGS.
FIG. 14A-B. Notch3 peptides induce apoptosis and inhibit Notch3-regulated gene Hey1. (FIG. 14A) HCC2429 was treated with Notch3 peptides N16, N17, N102, N103, N132. Induction of apoptosis by peptides is observed as compared to control. MRK003-treated cell is used as positive control. After treatment, cells were labeled with annexin V and detected using flow cytometry. (FIG. 14B) Treatment with peptides also reduced transcription of Notch3-dependent gene Hey1 as determined by real-time RT-PCR. Of note, N17 peptide both demonstrates highest apoptotic activity and best reduction in Hey1 transcription. MRK003-treated cells were used as positive control. Sequences N16: CFNTLGGHS, N17: CVCVNGWTGES, N102: CATAV, N103: CFHGAT, N132: CLNGGS.
FIG. 15. Notch3 Peptides interrupt signaling through binding to ligand Jagged1. HEK cells were transfected with Jagged1-HA and treated with Notch3 peptides. The peptides were then immunoprecipitated from cell lysate with streptavidin beads and immunoblotted with anti-HA antibody. No: no input; C: control peptide. Sequences N16: CFNTLGGHS, N17: CVCVNGWTGES, N102: CATAV, N103: CFHGAT, N132: CLNGGS.
FIG. 16. Sera from mice immunized with Notch3 recombindant protein inhibit Notch3 activation. Immunoblot demonstrates sera from mice #2, 3, 4, 5, 6 can reduced cleavage of Notch3 ICD (Tx) in Notch3 expressing cell line HCC2429 as compared control (C). Recombinant protein representing EGF-like repeats 21-22 and encompassing sequence CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS was used to immunize AJ and BALB/c mice. HCC2429 was plated in a 6-well plates and treated 1 μl/ml sera for 24 hr before harvesting.
FIGS. 17A-B. Recombinant Fc-fusion Notch3 proteins inhibit Notch3 activation and induces apoptosis in vitro. (FIG. 17A) Fc-fusion protein comprised for N16-17 and N132 sequences inhibits Notch3 activation. HCC2429 was treated with purified Fc-fusion protein 10 μg/ml for 24 hrs. (FIG. 17B) Purified recombinant N16-17-Fc protein induces apoptosis as compared to control and Fc control after 40 hrs treatment. Apoptosis was determined by percentage of annexin V positive cells. Sequences N16: CFNTLGGHS, N17: CVCVNGWTGES, N132: CLNGGS.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. The Present Invention
Many genes that are important in tumor initiation, progression, or survival play crucial roles in normal development. The Notch receptors are members of an evolutionarily conserved family that is essential for the control of cell fate determination during the development of many multicellular organisms. The core components of the Notch pathway are listed in Table 1. Their functions were first discovered in Drosophila melanogaster almost 80 years ago, when a heterozygous deletion was found to result in “notches” at the wing margins. Mutations in Notch genes alter cell fate determination, causing cells destined to become epidermis to instead give rise to neural tissue (reviewed in Artavanis-Tsakonas, 1999). Notch signaling is classically divided into two fundamental types: inductive and lateral signaling. Induction occurs between two nonequivalent cells, where one cell expresses the receptor and the other expresses the ligand, and through their interaction one cell adopts a different fate. In contrast, lateral signaling occurs between equivalent cells, and through competitive inhibition, adjacent cells are forced to follow a different fate. The role of Notch pathway signaling in mammals has been studied extensively in lymphogenesis, ondotogenesis, neurogenesis, hair and sensory development (Beatus and Leandahl, 1998; Mitsiadis et al., 1998; Lanford et al., 1999; Robey et al., 1996).







TABLE 1







Core components of the Notch pathway in worms, flies, and mammals










Component
C. elegans
Drosophila
Mammals





DSL Ligands
LAG-2, APX-1, DSL-1
Delta, Serrate
Delta-like-1, -3 and -4





Jagged1, Jagged 2


Notch Receptors
LIN-12, Glp-1
Notch
Notch 1-4


CSL Factors
LAG-1
Suppressor of Hairless
CBF-1


Corepressor

Hairless, Groucho,
SMRT, NcoR, CIR




dCTBP, SMRTER


Coactivators

Mastermind
Mastermind


Target Genes

Enhancer of Split, Hey
HES1-7, Hey1-2





*adapted from Sundaram (2005).






II. Notch3

Notch Plays A Key Role In Vascular Development and Homeostasis. In adult mammals, the expression of Notch receptors is restricted to the vascular systems. Mice with targeted mutations of the Notch pathway components, such as Jagged1, Delta-like-1 and the Notch1 receptor, die during embryogenesis from defects in vascular morphogenesis (Krebs et al., 2000; Xue et al., 1999; Hrabe de Angelis et al., 1997). While Notch3−/− mice are fertile and viable, these adult mice exhibit structural defects in the distal arteries and arterial myogenic response, reflecting the lack of proper development in vascular smooth muscle cells (Domenga et al., 2004). These observations indicate that the Notch signaling pathway is important in both vascular development and homeostasis. Not surprisingly, many of the processes involved in embryonic vascular development are mirrored in tumor angiogenesis. For example, induction of Notch ligand Jagged1 promotes capillary-like sprout formation in tumor cells (Zeng et al., 2005). Finally, inhibition of Notch activation by γ-secretase inhibitors also inhibits angiogenesis and tumor proliferation (Paris et al., 2005; Williams et al., 2005). These observations support a role of Notch pathway in normal and tumor angiogenesis.
Activation of Notch Signaling requires Proteolytic Cleavage of the Receptor. While Drosophila possesses a single Notch gene, there are four members of the Notch family in mammals: Notch1 (TAN1), Notch2, Notch3 and Notch4/Int-4. The core components of the Notch pathway are listed in Table 1. Notch is expressed on cell surfaces as a single-pass, heterodimeric receptor. The ligands are also transmembrane proteins of the DSL (Delta/Serrate/LAG-2) family that can be expressed not only on adjacent cells but also on the very same cell expressing the Notch receptors. Receptor-ligand interaction triggers proteolysis at the extracellular S2 site near the transmembrane domain and at the S3 site (FIG. 1). A TNF-α converting enzyme (TACE) and a presenillin-1-dependent γ-secretase are believed to be responsible for the proteolytic processing at sites S2 and S3, respectively. The final cleavage releases the C-terminal, intracellular domain (NICD), which then translocates to the nucleus, recruits coactivators such as mastermind and p300, and binds to CSL (CBF/Suppressor of Hairless/LAG-1) factors. In the absence of Notch signaling, CSL proteins in association with corepressors repress target gene transcription. Thus, Notch signaling causes a switch from transcriptional repression to transcriptional activation of CSL target genes (a review of Notch processing in Mumm and Kopan (2000).
The Notch Signaling Pathway Is Oncogenic. Many key pathways in development play important roles in tumorigenesis when altered. Notch1 was first identified in association with a t(7:9) translocation found in a subset of human T-cell acute lymphoblastoid leukemias (T-ALLs) (Ellisen et al., 1991). While less than 1% of human T-ALLs exhibit the t(7:9), Notch activating mutations have been observed in 50% of human T-ALLs (Weng et al., 2004; Ma et al., 1999). The expression of the constitutively activated intracellular domain (NICD) of Notch1 in bone marrow cells confers an oncogenic phenotype (Pear et al., 1996). Similar observations have been made linking Notch family members with cancer pathogenesis as well (Jhappan et al., 1992; Rohn et al., 1996). Constitutive activation of Notch3 in transgenic mice results in T-cell lymphoblastic leukemia, and in human T-ALL, loss of Notch3 expression correlates with clinical remission Bellavia et al., 2002; Bellavia et al., 2000). Moreover, activated Notch3 confers resistance to apoptosis and loss of contact inhibition in smooth muscle cells (Wang et al., 2002; Sweeney et al., 2004; Campos et al., 2002). Notch3 has been found to be highly expressed in other tumors, including lung, pancreatic and ovarian carcinoma, using gene expression microarray (Dang et al., 2000; Miyamoto et al., 2003; Lu et al., 2004). Similar studies demonstrate correlations between aberrant Notch ligand/receptor expression and tumor development in various systems (Miyamoto et al., 2003; Santagata et al., 2004; Purow et al., 2005; Callahan and Egan, 2004). The inventors published data demonstrating that inhibition of Notch3 activation using a dominant-negative receptor reduces tumor phenotype (Haruki et al., 2005). Taken together, these observations suggest that the Notch pathway is functionally significant in solid tumors and can serve as a target for therapeutic intervention.
Notch Crosstalks with the Ras Pathway. In both mammals and invertebrates such as Drosophila and C. elegans, Notch receptors signal primarily by the binding to members of the CSL family of transcription factors and related transcription co-activators. However, Notch is known to interact with other pathways including the Wingless/B-catenin and NF-κB pathways (Johnston and Edgar, 1998; Oswald et al., 1998). One pathway that plays prominently in both development and neoplastic transformation is the EGF/ras/MAPK pathway. Notch has been shown in developing organisms to antagonize EGF signaling in cell fate determination through modulation of the MAPK pathway (Faux et al., 2001; Ahmad and Dooley, 1998; Berset et al., 2001). In other cases, however, the Ras and Notch pathways cooperate in promoting certain cell fates (Yoo et al., 2004). As in flies and worms, specific outcomes of EGF and Notch pathways in mammals are context dependent. In mammals, Wang et al. demonstrated that Notch3 induces phosphorylation of ERK1/ERK2 (p44/p42) in vascular smooth muscle cells (Wang et al., 2002). Current evidence indicates that malignant transformation by Notch requires activated Ras (Haruki et al., 2005; Fitzgerald et al., 2000). On the other hand, activated Notch1 was found to inhibit Fgf-dependent malignant transformation of NIH3T3 cells (Small et al., 2003). While further work is required to fully understand the mechanism of interactions between Ras and Notch pathways, the preliminary data demonstrate a cooperative relationship between Notch3 signaling and the ras pathway. This observation suggests that combinatorial therapeutic approach will have better efficacy in the treatment of patients with lung cancers. FIG. 2 summarizes known potential Notch-ras interactions in both the development and cancer context.
γ-Secretase Inhibitors Demonstrate Antitumor Effects. Proteolytic processing of Notch receptors following ligand binding is necessary for their activation. The final proteolytic cleavage by the γ-secretase protein complex releases the Notch intracellular domain required for target gene transcription. Thus, pharmacologic intervention that inhibits the activity of any of the proteases can potentially inhibit tumor growth in Notch-dependent cancer. Interestingly, at the same time that presenilins were shown to be essential for Notch signaling, they were discovered as susceptibility loci for Alzheimer's disease (Levitan and Greenwald, 1995). The pathogenesis of Alzheimer's disease is believed to be the accumulation of amyloid β-peptides (Aβ) and formation of amyloid plaques. These peptides are derived from the proteolytic processing of the β-amyloid precursor protein (APP) through an intermediate fragment (C99) by γ-secretases (Dovey et al., 2001). Given the great need for better treatment of patients with Alzheimer's disease, inhibitors targeting γ-secretase are being aggressively pursued by many pharmaceutical companies. Predictably, many of these compounds were found to inhibit Notch processing as well. Furthermore, γ-secretase inhibitors block Notch activation and induce apoptosis in multiple cancer cell lines (Qin et al., 2004; Curry et al., 2005; Alves da Costa, 2004). In vivo, these compounds inhibit angiogenesis and tumor growth (Paris et al., 2005). These inhibitors are known to have non-Notch targets, such as erb-4 and CD44, but our data suggest that Notch inhibition may be a component of the observed antitumor effects (Pelletier et al., 2006; Linggi et al., 2006). However, from a practical standpoint, since erb-4 and CD44 are known to be oncogenic, these inhibitors may actually have increased efficacy by virtue of their multiple targets. In fact, a γ-secretase inhibitor by Merck & Co., Inc, is currently in Phase I trials for patients with metastatic or locally advanced breast cancer and for patients with T-cell acute leukemias.
A. Features of the Polypeptide
Notch3 is a 2321 amino acid protein (243659 Da Q9UM47; SEQ ID NO:2) that exists as a heterodimer of a C-terminal fragment (TM) and an N-terminal fragment (EC) which are probably linked by disulfide bonds. It has been shown to iteract with MAML1, MAML2 and MAML3 which act as transcriptional coactivators for NOTCH3. It is localized in the cell membrane and is a single-pass type I membrane protein. Following proteolytical processing, the notch intracellular domain (NICD) causes translocation to the nucleus. Its only known post-translational modification is glycosylation.
Notch3 functions as a receptor for membrane-bound ligands Jagged1, Jagged2 and Delta1 to regulate cell-fate determination. Upon ligand activation through the released NICD, it forms a transcriptional activator complex with CBF-1 and activates genes of the enhancer of split locus. As discussed above, it has effects the implementation of differentiation, proliferation and apoptotic programs. It is located at 19p13.2-p13.1.
B. Peptides
Notch3 peptides will comprise molecules of 5 to no more than about 50 residues in length. A particular length may be less than 39 residues, less than 35 residues, less than 30 residues, less than 25 residues, less than 20 residues, less than 15 residues, or less than 13, including 5, 6, 7, 8, 9, 10, 11 or 12 residues, and ranges of 5-11 residues, 5-15 residues, 5-20 residues, 5-25 residues, 5-30 residues, 5-35 residues, 5-38 residues, or 5-40 residues. The peptides may be generated synthetically or by recombinant techniques, and are purified according to known methods, such as precipitation (e.g., ammonium sulfate), HPLC, ion exchange chromatography, affinity chromatography (including immunoaffinity chromatography) or various size separations (sedimentation, gel electrophoresis, gel filtration), as described in further detail below.
The peptides may be labeled using various molecules, such as fluorescent, chromogenic or colorimetric agents. The peptides may also be linked to other molecules, including other anti-cancer agents. The links may be direct or through distinct linker molecules. The linker molecules in turn may be subject, in vivo, to cleavage, thereby releasing the agent from the peptide. Peptides may also be rendered multimeric by linking to larger, and possibly inert, carrier molecules.
C. Variants
Amino acid sequence variants of the polypeptide can be substitutional, insertional or deletion variants. Deletion variants lack one or more residues of the native protein which are not essential for function or immunogenic activity, and are exemplified by the variants lacking a transmembrane sequence described above. Another common type of deletion variant is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell. Insertional mutants typically involve the addition of material at a non-terminal point in the polypeptide. This may include the insertion of an immunoreactive epitope or simply a single residue. Terminal additions, called fusion proteins, are discussed below.
Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage, without the loss of other functions or properties. Substitutions of this kind preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
The following is a discussion based upon changing of the amino acids of a protein to create an equivalent or improved molecule. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity, as discussed below. Table 2 shows the codons that encode particular amino acids.








TABLE 2





Amino Acids
Codons



















Alanine
Ala
A
GCA GCC GCG GCU



 


Cysteine
Cys
C
UGC UGU


 


Aspartic acid
Asp
D
GAC GAU


 


Glutamic acid
Glu
E
GAA GAG


 


Phenylalanine
Phe
F
UUC UUU


 


Glycine
Gly
G
GGA GGC GGG GGU


 


Histidine
His
H
CAC CAU


 


Isoleucine
Ile
I
AUA AUC AUU


 


Lysine
Lys
K
AAA AAG


 


Leucine
Leu
L
UUA UUG CUA CUC CUG CUU


 


Methionine
Met
M
AUG


 


Asparagine
Asn
N
AAC AAU


 


Proline
Pro
P
CCA CCC CCG CCU


 


Glutamine
Gln
Q
CAA CAG


 


Arginine
Arg
R
AGA AGG CGA CGC CGG CGU


 


Serine
Ser
S
AGC AGU UCA UCC UCG UCU


 


Threonine
Thr
T
ACA ACC ACG ACU


 


Valine
Val
V
GUA GUC GUG GUU


 


Tryptophan
Trp
W
UGG


 


Tyrosine
Tyr
Y
UAC UAU









In making substitutional variants, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).
It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine *−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).
It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent and immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
Another embodiment for the preparation of polypeptides according to the invention is the use of peptide mimetics. Mimetics are peptide-containing molecules that mimic elements of protein secondary structure. See, for example, Johnson et al., (1993). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen. A peptide mimetic is expected to permit molecular interactions similar to the natural molecule. These principles may be used, in conjunction with the principles outline above, to engineer second generation molecules having many of the natural properties of Notch3, but with altered and even improved characteristics.
D. Fusions
A specialized kind of insertional variant is the fusion protein. This molecule generally has all or a substantial portion (e.g., an intracellular, transmembrane or extracellular domain) of the native molecule, linked at the N- or C-terminus, to all or a portion of a second polypeptide. For example, fusions typically employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host. Another useful fusion includes the addition of a immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion protein. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification. Other useful fusions include linking of functional domains, such as active sites from enzymes, glycosylation domains, cellular targeting signals or transmembrane regions.
E. Purification of Proteins/Peptides
It will be desirable to purify Notch3 or fragments thereof. Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.
Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded protein or peptide. The term “purified protein or peptide” as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state. A purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.
Generally, “purified” will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.
Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a “-fold purification number.” The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.
There is no general requirement that the protein or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater “-fold” purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al., 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.
High Performance Liquid Chromatography (HPLC) is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of minutes, or at most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
Gel chromatography, or molecular sieve chromatography, is a special type of partition chromatography that is based on molecular size. The theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size. As long as the material of which the particles are made does not adsorb the molecules, the sole factor determining rate of flow is the size. Hence, molecules are eluted from the column in decreasing size, so long as the shape is relatively constant. Gel chromatography is unsurpassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adsorption, less zone spreading and the elution volume is related in a simple matter to molecular weight.
Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).
A particular type of affinity chromatography useful in the purification of carbohydrate containing compounds is lectin affinity chromatography. Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins. Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin. Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fuctose will bind to lectins from lotus.
The matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability. The ligand should be coupled in such a way as to not affect its binding properties. The ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand. One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below.
F. Synthesis
Because of their relatively small size, the peptides of the invention can also be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young (1984); Tam et al. (1983); Merrifield (1986); and Barany and Merrifield (1979), each incorporated herein by reference. Short peptide sequences, or libraries of overlapping peptides, usually from about 6 up to about 35 to 50 amino acids, which correspond to the selected regions described herein, can be readily synthesized and then screened in screening assays designed to identify reactive peptides. Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
G. Antigen Compositions
The present invention also provides for the use of Notch3 proteins or peptides as antigens for the immunization of animals relating to the production of antibodies. It is envisioned that either Notch3, or portions thereof, will be coupled, bonded, bound, conjugated or chemically-linked to one or more agents via linkers, polylinkers or derivatized amino acids. This may be performed such that a bispecific or multivalent composition or vaccine is produced. It is further envisioned that the methods used in the preparation of these compositions will be familiar to those of skill in the art and should be suitable for administration to animals, i.e., pharmaceutically acceptable. Particular agents are the carriers are keyhole limpet hemocyannin (KLH) or bovine serum albumin (BSA).
III. Nucleic Acids
The present invention also provides, in another embodiment, genes encoding Notch3 or fragments (peptides) thereof. A gene for the human Notch3 molecule has been identified. The present invention is not limited in scope to this gene, however, as one of ordinary skill in the could readily identify related homologs in various other species (e.g., mouse, rat, rabbit, dog. monkey, gibbon, chimp, ape, baboon, cow, pig, horse, sheep, cat and other species).
In addition, it should be clear that the present invention is not limited to the specific nucleic acids disclosed herein. As discussed below, a “Notch3 gene” may contain a variety of different bases and yet still produce a corresponding polypeptide that is functionally indistinguishable from, and in some cases structurally identical to, the human gene disclosed herein.
Similarly, any reference to a nucleic acid should be read as encompassing a host cell containing that nucleic acid and, in some cases, capable of expressing the product of that nucleic acid. In addition to therapeutic considerations, cells expressing nucleic acids of the present invention may prove useful in the context of screening for agents that induce, repress, inhibit, augment, interfere with, block, abrogate, stimulate or enhance the function of Notch3.
A. Nucleic Acids Encoding Notch3
Nucleic acids according to the present invention may encode an entire Notch3 coding sequence (Accession No. U97669; SEQ ID NO:1), a domain of Notch3 that expresses a tumor suppressing function, or any other fragment of the Notch3 sequences set forth herein. The nucleic acid may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. In preferred embodiments, however, the nucleic acid would comprise complementary DNA (cDNA). Also contemplated is a cDNA plus a natural intron or an intron derived from another gene; such engineered molecules are sometime referred to as “mini-genes.” At a minimum, these and other nucleic acids of the present invention may be used as molecular weight standards in, for example, gel electrophoresis.
The term “cDNA” is intended to refer to DNA prepared using messenger RNA (mRNA) as template. The advantage of using a cDNA, as opposed to genomic DNA or DNA polymerized from a genomic, non- or partially-processed RNA template, is that the cDNA primarily contains coding sequences of the corresponding protein. There may be times when the full or partial genomic sequence is preferred, such as where the non-coding regions are required for optimal expression or where non-coding regions such as introns are to be targeted in an antisense strategy.
It also is contemplated that a given Notch3 from a given species may be represented by natural variants that have slightly different nucleic acid sequences but, nonetheless, encode the same protein (see Table 1, above).
As used in this application, the term “a nucleic acid encoding a Notch3” refers to a nucleic acid molecule that has been isolated free of total cellular nucleic acid. In certain embodiments, the invention concerns a nucleic acid sequence essentially as set forth in SEQ ID NO:2. The term “as set forth in SEQ ID NO:2” means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:2. The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids, as discussed in the following pages.
Allowing for the degeneracy of the genetic code, sequences that have at least about 50%, usually at least about 60%, more usually about 70%, most usually about 80%, preferably at least about 90% and most preferably about 95% of nucleotides that are identical to the nucleotides of SEQ ID NO:2. Sequences that are essentially the same as those set forth in SEQ ID NO:2 also may be functionally defined as sequences that are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO:2 under standard conditions.
The DNA segments of the present invention include those encoding biologically functional equivalent Notch3 proteins and peptides, as described above. Such sequences may arise as a consequence of codon redundancy and amino acid functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques or may be introduced randomly and screened later for the desired function, as described below.
B. Oligonucleotide Probes and Primers
Naturally, the present invention also encompasses DNA segments that are complementary, or essentially complementary, to the sequence set forth in SEQ ID NO:2. Nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementary rules. As used herein, the term “complementary sequences” means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO:2 under relatively stringent conditions such as those described herein. Such sequences may encode the entire Notch3 protein or functional or non-functional fragments thereof.
Alternatively, the hybridizing segments may be shorter oligonucleotides. Sequences of 17 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence. Although shorter oligomers are easier to make and increase in vivo accessibility, numerous other factors are involved in determining the specificity of hybridization. Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length. It is contemplated that exemplary oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 114 or more base pairs will be used, although others are contemplated. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or longer are contemplated as well. Such oligonucleotides will find use, for example, as probes in Southern and Northern blots and as primers in amplification reactions.
Suitable hybridization conditions will be well known to those of skill in the art. In certain applications, for example, substitution of amino acids by site-directed mutagenesis, it is appreciated that lower stringency conditions are required. Under these conditions, hybridization may occur even though the sequences of probe and target strand are not perfectly complementary, but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and decreasing temperature. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37° C. to about 55° C., while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20° C. to about 55° C. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
In other embodiments, hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, at temperatures between approximately 20° C. to about 37° C. Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 μM MgCl2, at temperatures ranging from approximately 40° C. to about 72° C. Formamide and SDS also may be used to alter the hybridization conditions.
One method of using probes and primers of the present invention is in the search for genes related to Notch3 or, more particularly, homologs of Notch3 from other species. Normally, the target DNA will be a genomic or cDNA library, although screening may involve analysis of RNA molecules. By varying the stringency of hybridization, and the region of the probe, different degrees of homology may be discovered.
Another way of exploiting probes and primers of the present invention is in site-directed, or site-specific mutagenesis. Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
The technique typically employs a bacteriophage vector that exists in both a single-stranded and double-stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art. Double-stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.
In general, site-directed mutagenesis is performed by first obtaining a single-stranded vector, or melting of two strands of a double-stranded vector which includes within its sequence a DNA sequence encoding the desired protein. An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared. This primer is then annealed with the single-stranded DNA preparation, taking into account the degree of mismatch when selecting hybridization conditions, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.
The preparation of sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained. For example, recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
C. Vectors for Cloning, Gene Transfer and Expression
Within certain embodiments, expression vectors are employed to express the Notch3 polypeptide product, which can then be purified for various uses. In other embodiments, the expression vectors are used in gene therapy. Expression requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.
Throughout this application, the term “expression construct” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a protein, but it need not be. In certain embodiments, expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding a gene of interest.
The term “vector” is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A nucleic acid sequence can be “exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques, which are described in Sambrook et al. (1989) and Ausubel et al. (1994), both incorporated herein by reference.
The term “expression vector” refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of “control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra.
(i) Regulatory Elements
A “promoter” is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases “operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence. A promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
A promoter may be one naturally-associated with a gene or sequence, as may be obtained by isolating the 5′ non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally-occurring,” i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCRTM, in connection with the compositions disclosed herein (see U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906, each incorporated herein by reference). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.
Naturally, it will be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. One example is the native Notch3 promoter. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (1989), incorporated herein by reference. The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.
Table 3 lists several elements/promoters that may be employed, in the context of the present invention, to regulate the expression of a gene. This list is not intended to be exhaustive of all the possible elements involved in the promotion of expression but, merely, to be exemplary thereof. Table 4 provides examples of inducible elements, which are regions of a nucleic acid sequence that can be activated in response to a specific stimulus.







TABLE 3







Promoter and/or Enhancer








Promoter/Enhancer
References





Immunoglobulin Heavy Chain
Banerji et al., 1983; Gilles et al., 1983; Grosschedl et al.,



1985; Atchinson et al., 1986, 1987; Imler et al., 1987;



Weinberger et al., 1984; Kiledjian et al., 1988; Porton et



al.; 1990


Immunoglobulin Light Chain
Queen et al., 1983; Picard et al., 1984


T-Cell Receptor
Luria et al., 1987; Winoto et al., 1989; Redondo et al.;



1990


HLA DQ a and/or DQ β
Sullivan et al., 1987


β-Interferon
Goodbourn et al., 1986; Fujita et al., 1987; Goodbourn et



al., 1988


Interleukin-2
Greene et al., 1989


Interleukin-2 Receptor
Greene et al., 1989; Lin et al., 1990


MHC Class II 5
Koch et al., 1989


MHC Class II HLA-DRa
Sherman et al., 1989


β-Actin
Kawamoto et al., 1988; Ng et al.; 1989


Muscle Creatine Kinase (MCK)
Jaynes et al., 1988; Horlick et al., 1989; Johnson et al.,



1989


Prealbumin (Transthyretin)
Costa et al., 1988


Elastase I
Ornitz et al., 1987


Metallothionein (MTII)
Karin et al., 1987; Culotta et al., 1989


Collagenase
Pinkert et al., 1987; Angel et al., 1987


Albumin
Pinkert et al., 1987; Tronche et al., 1989, 1990


α-Fetoprotein
Godbout et al., 1988; Campere et al., 1989


t-Globin
Bodine et al., 1987; Perez-Stable et al., 1990


β-Globin
Trudel et al., 1987


c-fos
Cohen et al., 1987


c-HA-ras
Triesman, 1986; Deschamps et al., 1985


Insulin
Edlund et al., 1985


Neural Cell Adhesion Molecule
Hirsh et al., 1990


(NCAM)


α1-Antitrypain
Latimer et al., 1990


H2B (TH2B) Histone
Hwang et al., 1990


Mouse and/or Type I Collagen
Ripe et al., 1989


Glucose-Regulated Proteins
Chang et al., 1989


(GRP94 and GRP78)


Rat Growth Hormone
Larsen et al., 1986


Human Serum Amyloid A (SAA)
Edbrooke et al., 1989


Troponin I (TN I)
Yutzey et al., 1989


Platelet-Derived Growth Factor
Pech et al., 1989


(PDGF)


Duchenne Muscular Dystrophy
Klamut et al., 1990


SV40
Banerji et al., 1981; Moreau et al., 1981; Sleigh et al.,



1985; Firak et al., 1986; Herr et al., 1986; Imbra et al.,



1986; Kadesch et al., 1986; Wang et al., 1986; Ondek et



al., 1987; Kuhl et al., 1987; Schaffner et al., 1988


Polyoma
Swartzendruber et al., 1975; Vasseur et al., 1980; Katinka



et al., 1980, 1981; Tyndell et al., 1981; Dandolo et al.,



1983; de Villiers et al., 1984; Hen et al., 1986; Satake et



al., 1988; Campbell and/or Villarreal, 1988


Retroviruses
Kriegler et al., 1982, 1983; Levinson et al., 1982; Kriegler



et al., 1983, 1984a, b, 1988; Bosze et al., 1986; Miksicek



et al., 1986; Celander et al., 1987; Thiesen et al., 1988;



Celander et al., 1988; Chol et al., 1988; Reisman et al.,



1989


Papilloma Virus
Campo et al., 1983; Lusky et al., 1983; Spandidos and/or



Wilkie, 1983; Spalholz et al., 1985; Lusky et al., 1986;



Cripe et al., 1987; Gloss et al., 1987; Hirochika et al.,



1987; Stephens et al., 1987; Glue et al., 1988


Hepatitis B Virus
Bulla et al., 1986; Jameel et al., 1986; Shaul et al., 1987;



Spandau et al., 1988; Vannice et al., 1988


Human Immunodeficiency Virus
Muesing et al., 1987; Hauber et al., 1988; Jakobovits et



al., 1988; Feng et al., 1988; Takebe et al., 1988; Rosen et



al., 1988; Berkhout et al., 1989; Laspia et al., 1989; Sharp



et al., 1989; Braddock et al., 1989


Cytomegalovirus (CMV)
Weber et al., 1984; Boshart et al., 1985; Foecking et al.,



1986


Gibbon Ape Leukemia Virus
Holbrook et al., 1987; Quinn et al., 1989
















TABLE 4







Inducible Elements









Element
Inducer
References





MT II
Phorbol Ester (TFA)
Palmiter et al., 1982; Haslinger et



Heavy metals
al., 1985; Searle et al., 1985;




Stuart et al., 1985; Imagawa et




al., 1987, Karin et al., 1987;




Angel et al., 1987b; McNeall et




al., 1989


MMTV (mouse mammary
Glucocorticoids
Huang et al., 1981; Lee et al.,


tumor virus)

1981; Majors et al., 1983;




Chandler et al., 1983; Lee et al.,




1984; Ponta et al., 1985; Sakai et




al., 1988


β-Interferon
poly(rI)x
Tavernier et al., 1983



poly(rc)


Adenovirus 5 E2
ElA
Imperiale et al., 1984


Collagenase
Phorbol Ester (TPA)
Angel et al., 1987a


Stromelysin
Phorbol Ester (TPA)
Angel et al., 1987b


SV40
Phorbol Ester (TPA)
Angel et al., 1987b


Murine MX Gene
Interferon, Newcastle
Hug et al., 1988



Disease Virus


GRP78 Gene
A23187
Resendez et al., 1988


α-2-Macroglobulin
IL-6
Kunz et al., 1989


Vimentin
Serum
Rittling et al., 1989


MHC Class I Gene H-2κb
Interferon
Blanar et al., 1989


HSP70
ElA, SV40 Large T
Taylor et al., 1989, 1990a, 1990b



Antigen


Proliferin
Phorbol Ester-TPA
Mordacq et al., 1989


Tumor Necrosis Factor
PMA
Hensel et al., 1989


Thyroid Stimulating
Thyroid Hormone
Chatterjee et al., 1989


Hormone α Gene









The identity of tissue-specific promoters or elements, as well as assays to characterize their activity, is well known to those of skill in the art. Examples of such regions include the human LIMK2 gene (Nomoto et al. 1999), the somatostatin receptor 2 gene (Kraus et al., 1998), murine epididymal retinoic acid-binding gene (Lareyre et al., 1999), human CD4 (Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen (Tsumaki, et al., 1998), D1A dopamine receptor gene (Lee, et al., 1997), insulin-like growth factor II (Wu et al., 1997), human platelet endothelial cell adhesion molecule-1 (Almendro et al., 1996). Tumor specific promoters also will find use in the present invention. Some such promoters are set forth in Tables 4 and 5.







TABLE 5







Candidate Tissue-Specific Promoters for Cancer Gene Therapy










Cancers in which promoter
Normal cells in which


Tissue-specific promoter
is active
promoter is active





Carcinoembryonic antigen
Most colorectal carcinomas;
Colonic mucosa; gastric


(CEA)*
50% of lung carcinomas; 40-50%
mucosa; lung epithelia;



of gastric carcinomas;
eccrine sweat glands; cells in



most pancreatic carcinomas;
testes



many breast carcinomas


Prostate-specific antigen
Most prostate carcinomas
Prostate epithelium


(PSA)


Vasoactive intestinal peptide
Majority of non-small cell
Neurons; lymphocytes; mast


(VIP)
lung cancers
cells; eosinophils


Surfactant protein A (SP-A)
Many lung adenocarcinomas
Type II pneumocytes; Clara



cells


Human achacte-scute
Most small cell lung cancers
Neuroendocrine cells in lung


homolog (hASH)


Mucin-1 (MUC1)**
Most adenocarcinomas
Glandular epithelial cells in



(originating from any tissue)
breast and in respiratory,




gastrointestinal, and




genitourinary tracts


Alpha-fetoprotein
Most hepatocellular
Hepatocytes (under certain



carcinomas; possibly many
conditions); testis



testicular cancers


Albumin
Most hepatocellular
Hepatocytes



carcinomas


Tyrosinase
Most melanomas
Melanocytes; astrocytes;




Schwann cells; some neurons


Tyrosine-binding protein
Most melanomas
Melanocytes; astrocytes,


(TRP)

Schwann cells; some neurons


Keratin 14
Presumably many squamous
Keratinocytes



cell carcinomas (e.g., Head



and neck cancers)


EBV LD-2
Many squamous cell
Keratinocytes of upper



carcinomas of head and neck
digestive Keratinocytes of




upper digestive tract


Glial fibrillary acidic protein
Many astrocytomas
Astrocytes


(GFAP)


Myelin basic protein (MBP)
Many gliomas
Oligodendrocytes


Testis-specific angiotensin-
Possibly many testicular
Spermatazoa


converting enzyme (Testis-
cancers


specific ACE)


Osteocalcin
Possibly many osteosarcomas
Osteoblasts
















TABLE 6







Candidate Promoters for Tissue-Specific Targeting of Tumors










Cancers in which Promoter
Normal cells in which


Promoter
is active
Promoter is active





E2F-
Almost all cancers
Proliferating cells


regulated


promoter


HLA-G
Many colorectal carcinomas;
Lymphocytes; monocytes;



many melanomas; possibly
spermatocytes; trophoblast



many other cancers


FasL
Most melanomas; many
Activated leukocytes:



pancreatic carcinomas; most
neurons; endothelial cells;



astrocytomas possibly many
keratinocytes; cells in



other cancers
immunoprivileged tissues;




some cells in lungs, ovaries,




liver, and prostate


Myc-
Most lung carcinomas (both
Proliferating cells (only some


regulated
small cell and non-small cell);
cell-types): mammary


promoter
most colorectal carcinomas
epithelial cells (including non-




proliferating)


MAGE-1
Many melanomas; some non-
Testis



small cell lung carcinomas;



some breast carcinomas


VEGF
70% of all cancers
Cells at sites of



(constitutive overexpression in
neovascularization (but unlike



many cancers)
in tumors, expression is




transient, less strong, and




never constitutive)


bFGF
Presumably many different
Cells at sites of ischemia (but



cancers, since bFGF
unlike tumors, expression is



expression is induced by
transient, less strong, and



ischemic conditions
never constitutive)


COX-2
Most colorectal carcinomas;
Cells at sites of inflammation



many lung carcinomas;



possibly many other cancers


IL-10
Most colorectal carcinomas;
Leukocytes



many lung carcinomas; many



squamous cell carcinomas of



head and neck; possibly many



other cancers


GRP78/
Presumably many different
Cells at sites of ishemia


BiP
cancers, since GRP7S



expression is induced by



tumor-specific conditions


CarG
Induced by ionization
Cells exposed to ionizing


elements
radiation, so conceivably most
radiation; leukocytes


from
tumors upon irradiation


Egr-1









A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be “in-frame” with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.
(ii) IRES
In certain embodiments of the invention, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5′-methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Pat. Nos. 5,925,565 and 5,935,819, herein incorporated by reference).
(iii) Multi-Purpose Cloning Sites
Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector. See Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997, incorporated herein by reference. “Restriction enzyme digestion” refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector. “Ligation” refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.
(iv) Splicing Sites
Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcripts. Vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression (see Chandler et al., 1997, herein incorporated by reference.)
(v) Termination Signals
The vectors or constructs of the present invention will generally comprise at least one termination signal. A “termination signal” or “terminator” is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.
In eukaryotic systems, the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (polyA) to the 3′ end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, it is preferred that that terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message. The terminator and/or polyadenylation site elements can serve to enhance message levels and/or to minimize read through from the cassette into other sequences.
Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the termination sequences of genes, such as for example the bovine growth hormone terminator or viral termination sequences, such as for example the SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.
(vi) Polyadenylation Signals
In expression, particularly eukaryotic expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and/or any such sequence may be employed. Preferred embodiments include the SV40 polyadenylation signal and/or the bovine growth hormone polyadenylation signal, convenient and/or known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport.
(vii) Origins of Replication
In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed “ori”), which is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.
(viii) Selectable and Screenable Markers
In certain embodiments of the invention, cells containing a nucleic acid construct of the present invention may be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.
Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is calorimetric analysis, are also contemplated. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable and screenable markers are well known to one of skill in the art.
(ix) Viral Vectors
The capacity of certain viral vectors to efficiently infect or enter cells, to integrate into a host cell genome and stably express viral genes, have led to the development and application of a number of different viral vector systems (Robbins et al., 1998). Viral systems are currently being developed for use as vectors for ex vivo and in vivo gene transfer. For example, adenovirus, herpes-simplex virus, retrovirus and adeno-associated virus vectors are being evaluated currently for treatment of diseases such as cancer, cystic fibrosis, Gaucher disease, renal disease and arthritis (Robbins and Ghivizzani, 1998; Imai et al., 1998; U.S. Pat. No. 5,670,488). The various viral vectors described below, present specific advantages and disadvantages, depending on the particular gene-therapeutic application.
Adenoviral Vectors. In particular embodiments, an adenoviral expression vector is contemplated for the delivery of expression constructs. “Adenovirus expression vector” is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to ultimately express a tissue or cell-specific construct that has been cloned therein.
Adenoviruses comprise linear, double-stranded DNA, with a genome ranging from 30 to 35 kb in size (Reddy et al., 1998; Morrison et al., 1997; Chillon et al., 1999). An adenovirus expression vector according to the present invention comprises a genetically engineered form of the adenovirus. Advantages of adenoviral gene transfer include the ability to infect a wide variety of cell types, including non-dividing cells, a mid-sized genome, ease of manipulation, high infectivity and the ability to be grown to high titers (Wilson, 1996). Further, adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner, without potential genotoxicity associated with other viral vectors. Adenoviruses also are structurally stable (Marienfeld et al., 1999) and no genome rearrangement has been detected after extensive amplification (Parks et al., 1997; Bett et al., 1993).
Salient features of the adenovirus genome are an early region (E1, E2, E3 and E4 genes), an intermediate region (pIX gene, Iva2 gene), a late region (L1, L2, L3, L4 and L5 genes), a major late promoter (MLP), inverted-terminal-repeats (ITRs) and a ψ sequence (Zheng, et al., 1999; Robbins et al., 1998; Graham and Prevec, 1995). The early genes E1, E2, E3 and E4 are expressed from the virus after infection and encode polypeptides that regulate viral gene expression, cellular gene expression, viral replication, and inhibition of cellular apoptosis. Further on during viral infection, the MLP is activated, resulting in the expression of the late (L) genes, encoding polypeptides required for adenovirus encapsidation. The intermediate region encodes components of the adenoviral capsid. Adenoviral inverted terminal repeats (ITRs; 100-200 bp in length), are cis elements, and function as origins of replication and are necessary for viral DNA replication. The ψ sequence is required for the packaging of the adenoviral genome.
A common approach for generating an adenoviruses for use as a gene transfer vector is the deletion of the E1 gene (E1−), which is involved in the induction of the E2, E3 and E4 promoters (Graham and Prevec, 1995). Subsequently, a therapeutic gene or genes can be inserted recombinantly in place of the E1 gene, wherein expression of the therapeutic gene(s) is driven by the E1 promoter or a heterologous promoter. The E1−, replication-deficient virus is then proliferated in a “helper” cell line that provides the E1 polypeptides in trans (e.g., the human embryonic kidney cell line 293). Thus, in the present invention it may be convenient to introduce the transforming construct at the position from which the E1-coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical to the invention. Alternatively, the E3 region, portions of the E4 region or both may be deleted, wherein a heterologous nucleic acid sequence under the control of a promoter operable in eukaryotic cells is inserted into the adenovirus genome for use in gene transfer (U.S. Pat. No. 5,670,488; U.S. Pat. No. 5,932,210, each specifically incorporated herein by reference).
Although adenovirus based vectors offer several unique advantages over other vector systems, they often are limited by vector immunogenicity, size constraints for insertion of recombinant genes and low levels of replication. The preparation of a recombinant adenovirus vector deleted of all open reading frames, comprising a full length dystrophin gene and the terminal repeats required for replication (Haecker et al., 1996) offers some potentially promising advantages to the above mentioned adenoviral shortcomings. The vector was grown to high titer with a helper virus in 293 cells and was capable of efficiently transducing dystrophin in mdx mice, in myotubes in vitro and muscle fibers in vivo. Helper-dependent viral vectors are discussed below.
A major concern in using adenoviral vectors is the generation of a replication-competent virus during vector production in a packaging cell line or during gene therapy treatment of an individual. The generation of a replication-competent virus could pose serious threat of an unintended viral infection and pathological consequences for the patient. Armentano et al. (1990), describe the preparation of a replication-defective adenovirus vector, claimed to eliminate the potential for the inadvertent generation of a replication-competent adenovirus (U.S. Pat. No. 5,824,544, specifically incorporated herein by reference). The replication-defective adenovirus method comprises a deleted E1 region and a relocated protein IX gene, wherein the vector expresses a heterologous, mammalian gene.
Other than the requirement that the adenovirus vector be replication defective, or at least conditionally defective, the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes and/or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
As stated above, the typical vector according to the present invention is replication defective and will not have an adenovirus E1 region. Adenovirus growth and manipulation is known to those of skill in the art, and exhibits broad host range in vitro and in vivo (U.S. Pat. No. 5,670,488; U.S. Pat. No. 5,932,210; U.S. Pat. No. 5,824,544). This group of viruses can be obtained in high titers, e.g., 109 to 1011 plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. Many experiments, innovations, preclinical studies and clinical trials are currently under investigation for the use of adenoviruses as gene delivery vectors. For example, adenoviral gene delivery-based gene therapies are being developed for liver diseases (Han et al., 1999), psychiatric diseases (Lesch, 1999), neurological diseases (Smith, 1998; Hermens and Verhaagen, 1998), coronary diseases (Feldman et al., 1996), muscular diseases (Petrof, 1998), gastrointestinal diseases (Wu, 1998) and various cancers such as colorectal (Fujiwara and Tanaka, 1998; Dorai et al., 1999), pancreatic, bladder (Irie et al., 1999), head and neck (Blackwell et al., 1999), breast (Stewart et al., 1999), lung (Batra et al., 1999) and ovarian (Vanderkwaak et al., 1999).
Retroviral Vectors. In certain embodiments of the invention, the use of retroviruses for gene delivery are contemplated. Retroviruses are RNA viruses comprising an RNA genome. When a host cell is infected by a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate which is integrated into the chromosomal DNA of infected cells. This integrated DNA intermediate is referred to as a provirus. A particular advantage of retroviruses is that they can stably infect dividing cells with a gene of interest (e.g., a therapeutic gene) by integrating into the host DNA, without expressing immunogenic viral proteins. Theoretically, the integrated retroviral vector will be maintained for the life of the infected host cell, expressing the gene of interest.
The retroviral genome and the proviral DNA have three genes: gag, pol, and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes the internal structural (matrix, capsid, and nucleocapsid) proteins; the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase) and the env gene encodes viral envelope glycoproteins. The 5′ and 3′ LTRs serve to promote transcription and polyadenylation of the virion RNAs. The LTR contains all other cis-acting sequences necessary for viral replication.
A recombinant retrovirus of the present invention may be genetically modified in such a way that some of the structural, infectious genes of the native virus have been removed and replaced instead with a nucleic acid sequence to be delivered to a target cell (U.S. Pat. No. 5,858,744; U.S. Pat. No. 5,739,018, each incorporated herein by reference). After infection of a cell by the virus, the virus injects its nucleic acid into the cell and the retrovirus genetic material can integrate into the host cell genome. The transferred retrovirus genetic material is then transcribed and translated into proteins within the host cell. As with other viral vector systems, the generation of a replication-competent retrovirus during vector production or during therapy is a major concern. Retroviral vectors suitable for use in the present invention are generally defective retroviral vectors that are capable of infecting the target cell, reverse transcribing their RNA genomes, and integrating the reverse transcribed DNA into the target cell genome, but are incapable of replicating within the target cell to produce infectious retroviral particles (e.g., the retroviral genome transferred into the target cell is defective in gag, the gene encoding virion structural proteins, and/or in pol, the gene encoding reverse transcriptase). Thus, transcription of the provirus and assembly into infectious virus occurs in the presence of an appropriate helper virus or in a cell line containing appropriate sequences enabling encapsidation without coincident production of a contaminating helper virus.
The growth and maintenance of retroviruses is known in the art (U.S. Pat. No. 5,955,331; U.S. Pat. No. 5,888,502, each specifically incorporated herein by reference). Nolan et al. describe the production of stable high titre, helper-free retrovirus comprising a heterologous gene (U.S. Pat. No. 5,830,725, specifically incorporated herein by reference). Methods for constructing packaging cell lines useful for the generation of helper-free recombinant retroviruses with amphoteric or ecotrophic host ranges, as well as methods of using the recombinant retroviruses to introduce a gene of interest into eukaryotic cells in vivo and in vitro are contemplated in the present invention (U.S. Pat. No. 5,955,331).
Currently, the majority of all clinical trials for vector-mediated gene delivery use murine leukemia virus (MLV)-based retroviral vector gene delivery (Robbins et al., 1998; Miller et al., 1993). Disadvantages of retroviral gene delivery includes a requirement for ongoing cell division for stable infection and a coding capacity that prevents the delivery of large genes. However, recent development of vectors such as lentivirus (e.g., HIV), simian immunodeficiency virus (SIV) and equine infectious-anemia virus (EIAV), which can infect certain non-dividing cells, potentially allow the in vivo use of retroviral vectors for gene therapy applications (Amado and Chen, 1999; Klimatcheva et al., 1999; White et al., 1999; Case et al., 1999). For example, HIV-based vectors have been used to infect non-dividing cells such as neurons (Miyatake et al., 1999), islets (Leibowitz et al., 1999) and muscle cells (Johnston et al., 1999). The therapeutic delivery of genes via retroviruses are currently being assessed for the treatment of various disorders such as inflammatory disease (Moldawer et al., 1999), AIDS (Amado and Chen, 1999; Engel and Kohn, 1999), cancer (Clay et al., 1999), cerebrovascular disease (Weihl et al., 1999) and hemophilia (Kay, 1998).
Herpesviral Vectors. Herpes simplex virus (HSV) type I and type II contain a double-stranded, linear DNA genome of approximately 150 kb, encoding 70-80 genes. Wild type HSV are able to infect cells lytically and to establish latency in certain cell types (e.g., neurons). Similar to adenovirus, HSV also can infect a variety of cell types including muscle (Yeung et al., 1999), ear (Derby et al., 1999), eye (Kaufman et al., 1999), tumors (Yoon et al., 1999; Howard et al., 1999), lung (Kohut et al., 1998), neuronal (Gamido et al., 1999; Lachmann and Efstathiou, 1999), liver (Miyatake et al., 1999; Kooby et al., 1999) and pancreatic islets (Rabinovitch et al., 1999).
HSV viral genes are transcribed by cellular RNA polymerase II and are temporally regulated, resulting in the transcription and subsequent synthesis of gene products in roughly three discernable phases or kinetic classes. These phases of genes are referred to as the Immediate Early (IE) or alpha genes, Early (E) or beta genes and Late (L) or gamma genes. Immediately following the arrival of the genome of a virus in the nucleus of a newly infected cell, the IE genes are transcribed. The efficient expression of these genes does not require prior viral protein synthesis. The products of IE genes are required to activate transcription and regulate the remainder of the viral genome.
For use in therapeutic gene delivery, HSV must be rendered replication-defective. Protocols for generating replication-defective HSV helper virus-free cell lines have been described (U.S. Pat. No. 5,879,934; U.S. Pat. No. 5,851,826, each specifically incorporated herein by reference in its entirety). One IE protein, Infected Cell Polypeptide 4 (ICP4), also known as alpha 4 or Vmw175, is absolutely required for both virus infectivity and the transition from IE to later transcription. Thus, due to its complex, multifunctional nature and central role in the regulation of HSV gene expression, ICP4 has typically been the target of HSV genetic studies.
Phenotypic studies of HSV viruses deleted of ICP4 indicate that such viruses will be potentially useful for gene transfer purposes (Krisky et al., 1998a). One property of viruses deleted for ICP4 that makes them desirable for gene transfer is that they only express the five other IE genes: ICP0, ICP6, ICP27, ICP22 and ICP47 (DeLuca et al., 1985), without the expression of viral genes encoding proteins that direct viral DNA synthesis, as well as the structural proteins of the virus. This property is desirable for minimizing possible deleterious effects on host cell metabolism or an immune response following gene transfer. Further deletion of IE genes ICP22 and ICP27, in addition to ICP4, substantially improve reduction of HSV cytotoxicity and prevented early and late viral gene expression (Krisky et al., 1998b).
The therapeutic potential of HSV in gene transfer has been demonstrated in various in vitro model systems and in vivo for diseases such as Parkinson's (Yamada et al., 1999), retinoblastoma (Hayashi et al., 1999), intracerebral and intradermal tumors (Moriuchi et al., 1998), B-cell malignancies (Suzuki et al., 1998), ovarian cancer (Wang et al., 1998) and Duchenne muscular dystrophy (Huard et al., 1997).
Adeno-Associated Viral Vectors. Adeno-associated virus (AAV), a member of the parvovirus family, is a human virus that is increasingly being used for gene delivery therapeutics. AAV has several advantageous features not found in other viral systems. First, AAV can infect a wide range of host cells, including non-dividing cells. Second, AAV can infect cells from different species. Third, AAV has not been associated with any human or animal disease and does not appear to alter the biological properties of the host cell upon integration. For example, it is estimated that 80-85% of the human population has been exposed to AAV. Finally, AAV is stable at a wide range of physical and chemical conditions which lends itself to production, storage and transportation requirements.
The AAV genome is a linear, single-stranded DNA molecule containing 4681 nucleotides. The AAV genome generally comprises an internal non-repeating genome flanked on each end by inverted terminal repeats (ITRs) of approximately 145 bp in length. The ITRs have multiple functions, including origins of DNA replication, and as packaging signals for the viral genome. The internal non-repeated portion of the genome includes two large open reading frames, known as the AAV replication (rep) and capsid (cap) genes. The rep and cap genes code for viral proteins that allow the virus to replicate and package the viral genome into a virion. A family of at least four viral proteins are expressed from the AAV rep region, Rep 78, Rep 68, Rep 52, and Rep 40, named according to their apparent molecular weight. The AAV cap region encodes at least three proteins, VP1, VP2, and VP3.
AAV is a helper-dependent virus requiring co-infection with a helper virus (e.g., adenovirus, herpesvirus or vaccinia) in order to form AAV virions. In the absence of co-infection with a helper virus, AAV establishes a latent state in which the viral genome inserts into a host cell chromosome, but infectious virions are not produced. Subsequent infection by a helper virus “rescues” the integrated genome, allowing it to replicate and package its genome into infectious AAV virions. Although AAV can infect cells from different species, the helper virus must be of the same species as the host cell (e.g., human AAV will replicate in canine cells co-infected with a canine adenovirus).
AAV has been engineered to deliver genes of interest by deleting the internal non-repeating portion of the AAV genome and inserting a heterologous gene between the ITRs. The heterologous gene may be functionally linked to a heterologous promoter (constitutive, cell-specific, or inducible) capable of driving gene expression in target cells. To produce infectious recombinant AAV (rAAV) containing a heterologous gene, a suitable producer cell line is transfected with a rAAV vector containing a heterologous gene. The producer cell is concurrently transfected with a second plasmid harboring the AAV rep and cap genes under the control of their respective endogenous promoters or heterologous promoters. Finally, the producer cell is infected with a helper virus.
Once these factors come together, the heterologous gene is replicated and packaged as though it were a wild-type AAV genome. When target cells are infected with the resulting rAAV virions, the heterologous gene enters and is expressed in the target cells. Because the target cells lack the rep and cap genes and the adenovirus helper genes, the rAAV cannot further replicate, package or form wild-type AAV.
The use of helper virus, however, presents a number of problems. First, the use of adenovirus in a rAAV production system causes the host cells to produce both rAAV and infectious adenovirus. The contaminating infectious adenovirus can be inactivated by heat treatment (56° C. for 1 hour). Heat treatment, however, results in approximately a 50% drop in the titer of functional rAAV virions. Second, varying amounts of adenovirus proteins are present in these preparations. For example, approximately 50% or greater of the total protein obtained in such rAAV virion preparations is free adenovirus fiber protein. If not completely removed, these adenovirus proteins have the potential of eliciting an immune response from the patient. Third, AAV vector production methods which employ a helper virus require the use and manipulation of large amounts of high titer infectious helper virus, which presents a number of health and safety concerns, particularly in regard to the use of a herpesvirus. Fourth, concomitant production of helper virus particles in rAAV virion producing cells diverts large amounts of host cellular resources away from rAAV virion production, potentially resulting in lower rAAV virion yields.
Lentiviral Vectors. Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection. Some examples of lentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 and the Simian Immunodeficiency Virus: SIV. Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe.
Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences. The lentiviral genome and the proviral DNA have the three genes found in retroviruses: gag, pol and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes the internal structural (matrix, capsid and nucleocapsid) proteins; the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase), a protease and an integrase; and the env gene encodes viral envelope glycoproteins. The 5′ and 3′ LTR's serve to promote transcription and polyadenylation of the virion RNA's. The LTR contains all other cis-acting sequences necessary for viral replication. Lentiviruses have additional genes including vif, vpr, tat, rev, vpu, nef and vpx.
Adjacent to the 5′ LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (the Psi site). If the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the cis defect prevents encapsidation of genomic RNA. However, the resulting mutant remains capable of directing the synthesis of all virion proteins.
Lentiviral vectors are known in the art, see Naldini et al., (1996); Zufferey et al., (1997); U.S. Pat. Nos. 6,013,516; and 5,994,136. In general, the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection and for transfer of the nucleic acid into a host cell. The gag, pol and env genes of the vectors of interest also are known in the art. Thus, the relevant genes are cloned into the selected vector and then used to transform the target cell of interest.
Recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136, incorporated herein by reference. This describes a first vector that can provide a nucleic acid encoding a viral gag and a pol gene and another vector that can provide a nucleic acid encoding a viral env to produce a packaging cell. Introducing a vector providing a heterologous gene, such as the STAT-1α gene in this invention, into that packaging cell yields a producer cell which releases infectious viral particles carrying the foreign gene of interest. The env preferably is an amphotropic envelope protein which allows transduction of cells of human and other species.
One may target the recombinant virus by linkage of the envelope protein with an antibody or a particular ligand for targeting to a receptor of a particular cell-type. By inserting a sequence (including a regulatory region) of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target-specific.
The vector providing the viral env nucleic acid sequence is associated operably with regulatory sequences, e.g., a promoter or enhancer. The regulatory sequence can be any eukaryotic promoter or enhancer, including for example, the Moloney murine leukemia virus promoter-enhancer element, the human cytomegalovirus enhancer or the vaccinia P7.5 promoter. In some cases, such as the Moloney murine leukemia virus promoter-enhancer element, the promoter-enhancer elements are located within or adjacent to the LTR sequences.
The heterologous or foreign nucleic acid sequence, such as the STAT-1α encoding polynucleotide sequence herein, is linked operably to a regulatory nucleic acid sequence. Preferably, the heterologous sequence is linked to a promoter, resulting in a chimeric gene. The heterologous nucleic acid sequence may also be under control of either the viral LTR promoter-enhancer signals or of an internal promoter, and retained signals within the retroviral LTR can still bring about efficient expression of the transgene. Marker genes may be utilized to assay for the presence of the vector, and thus, to confirm infection and integration. The presence of a marker gene ensures the selection and growth of only those host cells which express the inserts. Typical selection genes encode proteins that confer resistance to antibiotics and other toxic substances, e.g., histidinol, puromycin, hygromycin, neomycin, methotrexate, etc., and cell surface markers.
The vectors are introduced via transfection or infection into the packaging cell line. The packaging cell line produces viral particles that contain the vector genome. Methods for transfection or infection are well known by those of skill in the art. After cotransfection of the packaging vectors and the transfer vector to the packaging cell line, the recombinant virus is recovered from the culture media and titered by standard methods used by those of skill in the art. Thus, the packaging constructs can be introduced into human cell lines by calcium phosphate transfection, lipofection or electroporation, generally together with a dominant selectable marker, such as neo, DHFR, Gln synthetase or ADA, followed by selection in the presence of the appropriate drug and isolation of clones. The selectable marker gene can be linked physically to the packaging genes in the construct.
Lentiviral transfer vectors Naldini et al. (1996), have been used to infect human cells growth-arrested in vitro and to transduce neurons after direct injection into the brain of adult rats. The vector was efficient at transferring marker genes in vivo into the neurons and long term expression in the absence of detectable pathology was achieved. Animals analyzed ten months after a single injection of the vector showed no decrease in the average level of transgene expression and no sign of tissue pathology or immune reaction (Blomer et al., 1997). Thus, in the present invention, one may graft or transplant cells infected with the recombinant lentivirus ex vivo, or infect cells in vivo.
Other Viral Vectors. The development and utility of viral vectors for gene delivery is constantly improving and evolving. Other viral vectors such as poxvirus; e.g., vaccinia virus (Gnant et al., 1999; Gnant et al., 1999), alpha virus; e.g., sindbis virus, Semliki forest virus (Lundstrom, 1999), reovirus (Coffey et al., 1998) and influenza A virus (Neumann et al., 1999) are contemplated for use in the present invention and may be selected according to the requisite properties of the target system.
In certain embodiments, vaccinia viral vectors are contemplated for use in the present invention. Vaccinia virus is a particularly useful eukaryotic viral vector system for expressing heterologous genes. For example, when recombinant vaccinia virus is properly engineered, the proteins are synthesized, processed and transported to the plasma membrane. Vaccinia viruses as gene delivery vectors have recently been demonstrated to transfer genes to human tumor cells, e.g., EMAP-II (Gnant et al., 1999), inner ear (Derby et al., 1999), glioma cells, e.g., p53 (Timiryasova et al., 1999) and various mammalian cells, e.g., P-450 (U.S. Pat. No. 5,506,138). The preparation, growth and manipulation of vaccinia viruses are described in U.S. Pat. No. 5,849,304 and U.S. Pat. No. 5,506,138 (each specifically incorporated herein by reference).
In other embodiments, sindbis viral vectors are contemplated for use in gene delivery. Sindbis virus is a species of the alphavirus genus (Garoff and Li, 1998) which includes such important pathogens as Venezuelan, Western and Eastern equine encephalitis viruses (Sawai et al., 1999; Mastrangelo et al., 1999). In vitro, sindbis virus infects a variety of avian, mammalian, reptilian, and amphibian cells. The genome of sindbis virus consists of a single molecule of single-stranded RNA, 11,703 nucleotides in length. The genomic RNA is infectious, is capped at the 5′ terminus and polyadenylated at the 3′ terminus, and serves as mRNA. Translation of a vaccinia virus 26S mRNA produces a polyprotein that is cleaved co-and post-translationally by a combination of viral and presumably host-encoded proteases to give the three virus structural proteins, a capsid protein (C) and the two envelope glycoproteins (E1 and PE2, precursors of the virion E2).
Three features of sindbis virus suggest that it would be a useful vector for the expression of heterologous genes. First, its wide host range, both in nature and in the laboratory. Second, gene expression occurs in the cytoplasm of the host cell and is rapid and efficient. Third, temperature-sensitive mutations in RNA synthesis are available that may be used to modulate the expression of heterologous coding sequences by simply shifting cultures to the non-permissive temperature at various time after infection. The growth and maintenance of sindbis virus is known in the art (U.S. Pat. No. 5,217,879, specifically incorporated herein by reference).
Chimeric Viral Vectors. Chimeric or hybrid viral vectors are being developed for use in therapeutic gene delivery and are contemplated for use in the present invention. Chimeric poxyiral/retroviral vectors (Holzer et al., 1999), adenoviral/retroviral vectors (Feng et al., 1997; Bilbao et al., 1999; Caplen et al., 1999) and adenoviral/adeno-associated viral vectors (Fisher et al., 1996; U.S. Pat. No. 5,871,982) have been described.
These “chimeric” viral gene transfer systems can exploit the favorable features of two or more parent viral species. For example, Wilson et al., provide a chimeric vector construct which comprises a portion of an adenovirus, AAV 5′ and 3′ ITR sequences and a selected transgene, described below (U.S. Pat. No. 5,871,983, specifically incorporate herein by reference).
The adenovirus/AAV chimeric virus uses adenovirus nucleic acid sequences as a shuttle to deliver a recombinant AAV/transgene genome to a target cell. The adenovirus nucleic acid sequences employed in the hybrid vector can range from a minimum sequence amount, which requires the use of a helper virus to produce the hybrid virus particle, to only selected deletions of adenovirus genes, which deleted gene products can be supplied in the hybrid viral production process by a selected packaging cell. At a minimum, the adenovirus nucleic acid sequences employed in the pAdA shuttle vector are adenovirus genomic sequences from which all viral genes are deleted and which contain only those adenovirus sequences required for packaging adenoviral genomic DNA into a preformed capsid head. More specifically, the adenovirus sequences employed are the cis-acting 5′ and 3′ inverted terminal repeat (ITR) sequences of an adenovirus (which function as origins of replication) and the native 5′ packaging/enhancer domain, that contains sequences necessary for packaging linear Ad genomes and enhancer elements for the E1 promoter. The adenovirus sequences may be modified to contain desired deletions, substitutions, or mutations, provided that the desired function is not eliminated.
The AAV sequences useful in the above chimeric vector are the viral sequences from which the rep and cap polypeptide encoding sequences are deleted. More specifically, the AAV sequences employed are the cis-acting 5′ and 3′ inverted terminal repeat (ITR) sequences. These chimeras are characterized by high titer transgene delivery to a host cell and the ability to stably integrate the transgene into the host cell chromosome (U.S. Pat. No. 5,871,983, specifically incorporate herein by reference). In the hybrid vector construct, the AAV sequences are flanked by the selected adenovirus sequences discussed above. The 5′ and 3′ AAV ITR sequences themselves flank a selected transgene sequence and associated regulatory elements, described below. Thus, the sequence formed by the transgene and flanking 5′ and 3′ AAV sequences may be inserted at any deletion site in the adenovirus sequences of the vector. For example, the AAV sequences are desirably inserted at the site of the deleted E1a/E1b genes of the adenovirus. Alternatively, the AAV sequences may be inserted at an E3 deletion, E2a deletion, and so on. If only the adenovirus 5′ ITR/packaging sequences and 3′ ITR sequences are used in the hybrid virus, the AAV sequences are inserted between them.
The transgene sequence of the vector and recombinant virus can be a gene, a nucleic acid sequence or reverse transcript thereof, heterologous to the adenovirus sequence, which encodes a protein, polypeptide or peptide fragment of interest. The transgene is operatively linked to regulatory components in a manner which permits transgene transcription. The composition of the transgene sequence will depend upon the use to which the resulting hybrid vector will be put. For example, one type of transgene sequence includes a therapeutic gene which expresses a desired gene product in a host cell. These therapeutic genes or nucleic acid sequences typically encode products for administration and expression in a patient in vivo or ex vivo to replace or correct an inherited or non-inherited genetic defect or treat an epigenetic disorder or disease.
(x) Non-Viral Transformation
Suitable methods for nucleic acid delivery for transformation of an organelle, a cell, a tissue or an organism for use with the current invention are believed to include virtually any method by which a nucleic acid (e.g., DNA) can be introduced into an organelle, a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, incorporated herein by reference); by electroporation (U.S. Pat. No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); or by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos. 4,684,611 and 4,952,500, each incorporated herein by reference); by desiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985). Through the application of techniques such as these, organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently transformed.
Injection. In certain embodiments, a nucleic acid may be delivered to an organelle, a cell, a tissue or an organism via one or more injections (i.e., a needle injection), such as, for example, either subcutaneously, intradermally, intramuscularly, intervenously or intraperitoneally. Methods of injection of vaccines are well known to those of ordinary skill in the art (e.g., injection of a composition comprising a saline solution). Further embodiments of the present invention include the introduction of a nucleic acid by direct microinjection. Direct microinjection has been used to introduce nucleic acid constructs into Xenopus oocytes (Harland and Weintraub, 1985).
Electroporation. In certain embodiments of the present invention, a nucleic acid is introduced into an organelle, a cell, a tissue or an organism via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge. In some variants of this method, certain cell wall-degrading enzymes, such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells (U.S. Pat. No. 5,384,253, incorporated herein by reference). Alternatively, recipient cells can be made more susceptible to transformation by mechanical wounding.
Transfection of eukaryotic cells using electroporation has been quite successful. Mouse pre-B lymphocytes have been transfected with human kappa-immunoglobulin genes (Potter et al., 1984), and rat hepatocytes have been transfected with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al., 1986) in this manner.
To effect transformation by electroporation in cells such as, for example, plant cells, one may employ either friable tissues, such as a suspension culture of cells or embryogenic callus or alternatively one may transform immature embryos or other organized tissue directly. In this technique, one would partially degrade the cell walls of the chosen cells by exposing them to pectin-degrading enzymes (pectolyases) or mechanically wounding in a controlled manner. Examples of some species which have been transformed by electroporation of intact cells include maize (U.S. Pat. No. 5,384,253; Rhodes et al., 1995; D'Halluin et al., 1992), wheat (Zhou et al., 1993), tomato (Hou and Lin, 1996), soybean (Christou et al., 1987) and tobacco (Lee et al., 1989).
One also may employ protoplasts for electroporation transformation of plant cells (Bates, 1994; Lazzeri, 1995). For example, the generation of transgenic soybean plants by electroporation of cotyledon-derived protoplasts is described by Dhir and Widholm in International Patent Application No. WO 9217598, incorporated herein by reference. Other examples of species for which protoplast transformation has been described include barley (Lazerri, 1995), sorghum (Battraw et al., 1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994) and tomato (Tsukada, 1989).
Calcium Phosphate. In other embodiments of the present invention, a nucleic acid is introduced to the cells using calcium phosphate precipitation. Human KB cells have been transfected with adenovirus 5 DNA (Graham and Van Der Eb, 1973) using this technique. Also in this manner, mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et al., 1990).
DEAE-Dextran: In another embodiment, a nucleic acid is delivered into a cell using DEAE-dextran followed by polyethylene glycol. In this manner, reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985).
Sonication Loading. Additional embodiments of the present invention include the introduction of a nucleic acid by direct sonic loading. LTK− fibroblasts have been transfected with the thymidine kinase gene by sonication loading (Fechheimer et al., 1987).
Liposome-Mediated Transfection. In a further embodiment of the invention, a nucleic acid may be entrapped in a lipid complex such as, for example, a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated is an nucleic acid complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen).
Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987). The feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells has also been demonstrated (Wong et al., 1980).
In certain embodiments of the invention, a liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, a liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet further embodiments, a liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In other embodiments, a delivery vehicle may comprise a ligand and a liposome.
Receptor-Mediated Transfection. Still further, a nucleic acid may be delivered to a target cell via receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis that will be occurring in a target cell. In view of the cell type-specific distribution of various receptors, this delivery method adds another degree of specificity to the present invention.
Certain receptor-mediated gene targeting vehicles comprise a cell receptor-specific ligand and a nucleic acid-binding agent. Others comprise a cell receptor-specific ligand to which the nucleic acid to be delivered has been operatively attached. Several ligands have been used for receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al., 1990; Perales et al., 1994; Myers, EPO 0273085), which establishes the operability of the technique. Specific delivery in the context of another mammalian cell type has been described (Wu and Wu, 1993; incorporated herein by reference). In certain aspects of the present invention, a ligand will be chosen to correspond to a receptor specifically expressed on the target cell population.
In other embodiments, a nucleic acid delivery vehicle component of a cell-specific nucleic acid targeting vehicle may comprise a specific binding ligand in combination with a liposome. The nucleic acid(s) to be delivered are housed within the liposome and the specific binding ligand is functionally incorporated into the liposome membrane. The liposome will thus specifically bind to the receptor(s) of a target cell and deliver the contents to a cell. Such systems have been shown to be functional using systems in which, for example, epidermal growth factor (EGF) is used in the receptor-mediated delivery of a nucleic acid to cells that exhibit upregulation of the EGF receptor.
In still further embodiments, the nucleic acid delivery vehicle component of a targeted delivery vehicle may be a liposome itself, which will preferably comprise one or more lipids or glycoproteins that direct cell-specific binding. For example, lactosyl-ceramide, a galactose-terminal asialganglioside, have been incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes (Nicolau et al., 1987). It is contemplated that the tissue-specific transforming constructs of the present invention can be specifically delivered into a target cell in a similar manner.
F. Expression Systems
Numerous prokaryote- and/or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.
The insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Pat. Nos. 5,871,986 and 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC® 2.0 from INVITROGEN® and BACPACKTM BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH®.
Other examples of expression systems include STRATAGENE®'S COMPLETE CONTROLTM Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system. Another example of an inducible expression system is available from INVITROGEN®, which carries the T-REXTM (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter. INVITROGEN® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.
Primary mammalian cell cultures may be prepared in various ways. In order for the cells to be kept viable while in vitro and in contact with the expression construct, it is necessary to ensure that the cells maintain contact with the correct ratio of oxygen and carbon dioxide and nutrients but are protected from microbial contamination. Cell culture techniques are well documented.
One embodiment of the foregoing involves the use of gene transfer to immortalize cells for the production of proteins. The gene for the protein of interest may be transferred as described above into appropriate host cells followed by culture of cells under the appropriate conditions. The gene for virtually any polypeptide may be employed in this manner. The generation of recombinant expression vectors, and the elements included therein, are discussed above. Alternatively, the protein to be produced may be an endogenous protein normally synthesized by the cell in question.
Examples of useful mammalian host cell lines are Vero and HeLa cells and cell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2, NIH3T3, RIN and MDCK cells. In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and process the gene product in the manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to insure the correct modification and processing of the foreign protein expressed.
A number of selection systems may be used including, but not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk−, hgprt− or aprt− cells, respectively. Also, anti-metabolite resistance can be used as the basis of selection for dhfr, that confers resistance to; gpt, that confers resistance to mycophenolic acid; neo, that confers resistance to the aminoglycoside G418; and hygro, that confers resistance to hygromycin.
G. Host Cells
As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, “host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organisms that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors. A host cell may be “transfected” or “transformed,” which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny.
Host cells may be derived from prokaryotes or eukaryotes, depending upon whether the desired result is replication of the vector or expression of part or all of the vector-encoded nucleic acid sequences. Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (atcc.org). An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors. Bacterial cells used as host cells for vector replication and/or expression include DH5α, JM109, and KC8, as well as a number of commercially available bacterial hosts such as SURE® Competent Cells and SOLOPACKTM Gold Cells (STRATAGENE®, La Jolla). Alternatively, bacterial cells such as E. coli LE392 could be used as host cells for phage viruses.
Examples of eukaryotic host cells for replication and/or expression of a vector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.
Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.
H. Cell Propagation
Animal cells can be propagated in vitro in two modes: as non-anchorage dependent cells growing in suspension throughout the bulk of the culture or as anchorage-dependent cells requiring attachment to a solid substrate for their propagation (i.e., a monolayer type of cell growth). Non-anchorage dependent or suspension cultures from continuous established cell lines are the most widely used means of large scale production of cells and cell products. However, suspension cultured cells have limitations, such as tumorigenic potential and lower protein production than adherent T-cells.
Large scale suspension culture of mammalian cells in stirred tanks is a common method for production of recombinant proteins. Two suspension culture reactor designs are in wide use—the stirred reactor and the airlift reactor. The stirred design has successfully been used on an 8000 liter capacity for the production of interferon. Cells are grown in a stainless steel tank with a height-to-diameter ratio of 1:1 to 3:1. The culture is usually mixed with one or more agitators, based on bladed disks or marine propeller patterns. Agitator systems offering less shear forces than blades have been described. Agitation may be driven either directly or indirectly by magnetically coupled drives. Indirect drives reduce the risk of microbial contamination through seals on stirrer shafts.
The airlift reactor, also initially described for microbial fermentation and later adapted for mammalian culture, relies on a gas stream to both mix and oxygenate the culture. The gas stream enters a riser section of the reactor and drives circulation. Gas disengages at the culture surface, causing denser liquid free of gas bubbles to travel downward in the downcomer section of the reactor. The main advantage of this design is the simplicity and lack of need for mechanical mixing. Typically, the height-to-diameter ratio is 10:1. The airlift reactor scales up relatively easily, has good mass transfer of gases and generates relatively low shear forces.
The antibodies of the present invention are particularly useful for the isolation of antigens by immunoprecipitation. Immunoprecipitation involves the separation of the target antigen component from a complex mixture, and is used to discriminate or isolate minute amounts of protein. For the isolation of membrane proteins cells must be solubilized into detergent micelles. Non-ionic salts are preferred, since other agents such as bile salts, precipitate at acid pH or in the presence of bivalent cations. Antibodies are and their uses are discussed further, below.
III. Generating Antibodies Reactive with Notch3
In another aspect, the present invention contemplates an antibody that is immunoreactive with a Notch3 molecule of the present invention, or any portion thereof. In particular, the invention contemplates using peptides having the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, optionally linked together or linked to a carrier molecule such as keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). An antibody can be a polyclonal or a monoclonal antibody. In a preferred embodiment, an antibody is a monoclonal antibody. Means for preparing and characterizing antibodies are well known in the art (see, e.g., Harlow and Lane, 1988).
Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide, of the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically an animal used for production of anti-antisera is a non-human animal including rabbits, mice, rats, hamsters, pigs or horses. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
Antibodies, both polyclonal and monoclonal, specific for isoforms of antigen may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art. A composition containing antigenic epitopes of the compounds of the present invention can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against the compounds of the present invention. Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.
It is proposed that the monoclonal antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELISA and Western blot methods and in immunohistochemical procedures such as tissue staining, as well as in other procedures which may utilize antibodies specific to Notch3-related antigen epitopes. Additionally, it is proposed that monoclonal antibodies specific to the particular Notch3 of different species may be utilized in other useful applications
In general, both polyclonal and monoclonal antibodies against Notch3 may be used in a variety of embodiments. For example, they may be employed in antibody cloning protocols to obtain cDNAs or genes encoding other Notch3. They may also be used in inhibition studies to analyze the effects of Notch3 related peptides in cells or animals. Anti-Notch3 antibodies will also be useful in immunolocalization studies to analyze the distribution of Notch3 during various cellular events, for example, to determine the cellular or tissue-specific distribution of Notch3 polypeptides under different points in the cell cycle. A particularly useful application of such antibodies is in purifying native or recombinant Notch3, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure.
Means for preparing and characterizing antibodies are well known in the art (see, e.g., Harlow and Lane, 1988; incorporated herein by reference). More specific examples of monoclonal antibody preparation are give in the examples below.
As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
As also is well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.
MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified Notch3 protein, polypeptide or peptide or cell expressing high levels of Notch3. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
Following immunization, somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5×107 to 2×108 lymphocytes.
The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986; Campbell, 1984). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 41, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with cell fusions.
Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al., (1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986).
Fusion procedures usually produce viable hybrids at low frequencies, around 1×10−6 to 1×10−8. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.
The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
IV. Methods of Therapy
The present invention also involves, in another embodiment, the treatment of cancer. The types of cancer that may be treated, according to the present invention, is limited only by the involvement of Notch3. By involvement, it is not even a requirement that Notch3 be mutated or abnormal—the overexpression of this tumor suppressor may actually overcome other lesions within the cell. Thus, it is contemplated that a wide variety of tumors may be treated using Notch3 therapy, including cancers of the brain, lung, liver, spleen, kidney, lymph node, pancreas, small intestine, blood cells, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow, blood or other tissue.
In many contexts, it is not necessary that the tumor cell be killed or induced to undergo normal cell death or “apoptosis.” Rather, to accomplish a meaningful treatment, all that is required is that the tumor growth be slowed to some degree. It may be that the tumor growth is completely blocked, however, or that some tumor regression is achieved. Clinical terminology such as “remission” and “reduction of tumor” burden also are contemplated given their normal usage.
A. Peptide Therapy
Another therapy approach is the provision, to a subject, of Notch3 polypeptide, fragments, synthetic peptides, mimetics or other analogs thereof. The protein/peptide may be produced by recombinant expression means or, if small enough, generated by an automated peptide synthesizer. Formulations would be selected based on the route of administration and purpose including, but not limited to, liposomal formulations and classic pharmaceutical preparations.
B. Antibody Therapy
Applicants also contemplate the use of antibodies to Notch3, in particular, to epitopes comprised in or represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. Antibodies will be administered according to standard protocols for passive immunotherapy. Administration protocols would generally involve intratumoral, local or regional (to the tumor) administration, as well as systemic administration.
In addition, the antibody reagent may be altered, such that it will have one or more improved properties. The antibody may be recombinant, i.e., an antibody gene cloned into an expression cassette which is then introduced into a cell in which the antibody gene was not initially created. The antibody may be single chain, a fragment (Fab, Fv, Vh, ScFv), chimeric or humanized.
C. Combined Therapies with Immunotherapy, Traditional Chemo- or Radiotherapy
Tumor cell resistance to DNA damaging agents represents a major problem in clinical oncology. One goal of current cancer research is to find ways to improve the efficacy of chemo- and radiotherapy. One way is by combining such traditional therapies with gene therapy. For example, the herpes simplex-thymidine kinase (HS-tk) gene, when delivered to brain tumors by a retroviral vector system, successfully induced susceptibility to the antiviral agent ganciclovir (Culver et al., 1992). In the context of the present invention, it is contemplated that Notch3 peptide or antibody therapy could be used similarly in conjunction with chemo- or radiotherapeutic intervention. It also may prove effective to combine a Notch3-directed therapy with another cancer therapy.
To kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis or otherwise reverse or reduce the malignant phenotype of tumor cells, using the methods and compositions of the present invention, one would generally contact a “target” cell with a Notch3 peptide or antibody and at least one other agent. These compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cells with a Notch3 peptide or antibody and the agent(s) or factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the expression construct and the other includes the agent.
Alternatively, the Notch3 peptide or antibody therapy treatment may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agent and expression construct are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and a Notch3 peptide or antibody would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one would contact the cell with both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other, with a delay time of only about 12 hours being most preferred. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.
It also is conceivable that more than one administration of either Notch3 peptide or antibody or the other agent will be desired. Various combinations may be employed, where Notch3 (peptide or antibody) is “A” and the other agent is “B”, as exemplified below:








A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A



 


B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A


 


B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/A


 


A/B/B/B B/A/B/B B/B/A/B






Other combinations are contemplated. Again, to achieve cell killing, both agents are delivered to a cell in a combined amount effective to kill the cell.

Agents or factors suitable for use in a combined therapy are any chemical compound or treatment method that induces DNA damage when applied to a cell. Such agents and factors include radiation and waves that induce DNA damage such as, γ-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, and the like. A variety of chemical compounds, also described as “chemotherapeutic agents,” function to induce DNA damage, all of which are intended to be of use in the combined treatment methods disclosed herein. Chemotherapeutic agents contemplated to be of use, include, e.g., adriamycin, 5-fluorouracil (5FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) and even hydrogen peroxide. The invention also encompasses the use of a combination of one or more DNA damaging agents, whether radiation-based or actual compounds, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide. In certain embodiments, the use of cisplatin in combination with a Notch3 expression construct is particularly preferred as this compound.
In treating cancer according to the invention, one would contact the tumor cells with an agent in addition to the expression construct. This may be achieved by irradiating the localized tumor site with radiation such as X-rays, UV-light, γ-rays or even microwaves. Alternatively, the tumor cells may be contacted with the agent by administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound such as, adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C, or more preferably, cisplatin. The agent may be prepared and used as a combined therapeutic composition, or kit, by combining it with a Notch3 expression construct, as described above.
Agents that directly cross-link nucleic acids, specifically DNA, are envisaged to facilitate DNA damage leading to a synergistic, antineoplastic combination with Notch3. Agents such as cisplatin, and other DNA alkylating agents may be used. Cisplatin has been widely used to treat cancer, with efficacious doses used in clinical applications of 20 mg/m2 for 5 days every three weeks for a total of three courses. Cisplatin is not absorbed orally and must therefore be delivered via injection intravenously, subcutaneously, intratumorally or intraperitoneally.
Agents that damage DNA also include compounds that interfere with DNA replication, mitosis and chromosomal segregation. Such chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin, and the like. Widely used in a clinical setting for the treatment of neoplasms, these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg/m2 at 21 day intervals for adriamycin, to 35-50 mg/m2 for etoposide intravenously or double the intravenous dose orally.
Agents that disrupt the synthesis and fidelity of nucleic acid precursors and subunits also lead to DNA damage. As such a number of nucleic acid precursors have been developed. Particularly useful are agents that have undergone extensive testing and are readily available. As such, agents such as 5-fluorouracil (5-FU), are preferentially used by neoplastic tissue, making this agent particularly useful for targeting to neoplastic cells. Although quite toxic, 5-FU, is applicable in a wide range of carriers, including topical, however intravenous administration with doses ranging from 3 to 15 mg/kg/day being commonly used.
Other factors that cause DNA damage and have been used extensively include what are commonly known as γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage DNA, on the precursors of DNA, the replication and repair of DNA, and the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
The skilled artisan is directed to “Remington's Pharmaceutical Sciences” 15th Edition, chapter 33, in particular pages 624-652. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.
The inventors propose that the local or regional delivery of Notch3 expression constructs to patients with cancer will be a very efficient method for treating the clinical disease. Similarly, the chemo- or radiotherapy may be directed to a particular, affected region of the subjects body. Alternatively, systemic delivery of expression construct and/or the agent may be appropriate in certain circumstances, for example, where extensive metastasis has occurred.
In addition to combining Notch3 therapies with chemo- and radiotherapies, it also is contemplated that combination with other gene therapies will be advantageous. For example, targeting of Notch3 and p53 mutations at the same time may produce an improved anti-cancer treatment. Any other tumor-related gene conceivably can be targeted in this manner, for example, p21, Rb, APC, DCC, NF-1, NF-2, BCRA2, p16, FHIT, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC, MCC, ras, myc, neu, raf erb, src, fms, jun, trk, ret, gsp, hst, bcl and abl.
It also should be pointed out that any of the foregoing therapies may prove useful by themselves in treating a Notch3. In this regard, reference to chemotherapeutics and non-Notch3 gene therapy in combination should also be read as a contemplation that these approaches may be employed separately.
D. Formulations and Routes for Administration to Patients
Where clinical applications are contemplated, it will be necessary to prepare pharmaceutical compositions—a Notch3 peptide, or antibody—in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
One will generally desire to employ appropriate salts and buffers to render delivery compositions stable and allow for uptake by target cells. Aqueous compositions of the present invention comprise an effective amount of the Notch3 peptide or antibody to cells/a subject, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula. The phrase “pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well know in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
The active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra. Of particular interest is direct intratumoral administration, perfusion of a tumor, or administration local or regional to a tumor, for example, in the local or regional vasculature or lymphatic system, or in a resected tumor bed.
The active compounds may also be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial an antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
For oral administration the Notch3 peptides or antibodies of the present invention may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices. A mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
The compositions of the present invention may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.
V. Examples
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1
Notch3 Is Expressed in Resected Human Lung Cancers. The clinical relevance of a pathway to tumorigenesis often depends on the prevalence of its dysregulation. To begin to assess the prevalence of Notch3 involvement in lung cancer, the inventors investigated the frequency of Notch3 overexpression in resected lung cancer. They used immunohistochemistry (IHC), with a characterized and validated Notch3 antibody recognizing the extracellular domain (Joutel et al., 2000), in resected lung tumor tissues. For this experiment, the inventors used tumor tissue arrays produced as part of the Vanderbilt SPORE initiative. When the Notch3 antibody targeting the extracellular domain was used, a pattern of cytoplasmic and membranous staining was observed in the representative adenocarcinoma and squamous cell carcincoma (FIG. 3, panel A and C). No staining was observed in a neuroendocrine tumor and normal lung tissue (panel B and D), suggesting that Notch3 dysregulation is specific to cancer. The staining was scored on a composite scale of 0 to 4 by two independent investigators, including one pathologist. No staining was scored 0, whereas slight positivity equivalent to background stain was scored as 1. Tumors that scored 2 or higher were considered positive. Fractional positivity was nearly 100% in many tumors with high Notch3 expression. Of the 207 resected tumors, 39% had high Notch3 expression (Table 7). Each tumor was represented in triplicate, and the final score per tumor was calculated by averaging the score of three samples.







TABLE 7







Expression of Notch3 and EGFr By IHC Using Microtissue Arrays










Notch3+
EGFr+













N
n
%
n
%
















Adenocarcinomas
87
32
37%
69
79%


Neuroendocrine


Carcinoid
10
2
20%
5
50%


Large Cell
7
1
14%
2
29%


Small Cell
4
1
25%
3
75%


Squamous Cell Carcinomas
88
40
45%
81
92%


Large Cell
11
4
36%
8
73%



207
80
39%
168
81%









The frequency of Notch3 overexpression in lung tumors is higher than the frequency of HER2/neu (16%) and k-Ras mutations (16%), and comparable to EGFr (13-80%) (Hirsch et al., 2002; Rodenhuis et al., 1997; Meert et al., 2002). No information is available with regard to the frequency of overexpression of other members of the Notch family or their receptors in human lung carcinomas. The lack of high quality antibodies to the Notch receptors, Jagged1, and Delta-like-1, -3 and -4 makes it difficult to determine their frequency in fresh tumor tissues. In both C. elegans and Drosophila, the Notch pathway crosstalks with the EGF pathway in cell fate determination during development. The inventors thus examined the correlation between Notch3 and EGFr expression. The frequency of EGFr expression in our tumor tissue arrays was 81%. 19% of EGFr positive tumors are Notch3 negative, whereas 43% of EGFr positive tumors are also Notch3 positive (p<0.0001 using Pearson correlation coefficient) (Haruki et al., 2005). This highly statistical association suggests that the Notch and EGF pathways are cooperative in lung tumorigenesis.
Ectopic Notch3 Expression Inhibits Terminal Differentiation in Developing Lungs of Transgenic Mice. To evaluate the potential transforming activity of Notch3 in vivo, the inventors studied the effect of activated Notch3 using a lung-specific, human SP-C promoter (Glasser et al., 1991; Lardelli et al., 1996). The constitutively-activate Notch3 allowed the inventors to assess the effect of ectopic expression of Notch3 without depending on ligand expression in the epithelium, since Jagged1 expression becomes restricted to endothelium as the lung matures (Taichman et al., 2002). Furthermore, constitutive expression of Notch3 also better mimics the many dysregulated pathways observed in cancer. The inventors observed perinatal lethality, and thus no surviving animals expresses the N3IC transgene.
Because of the crucial role of the Notch family in development, expression of a constitutively active Notch3 transgene could potentially disturb normal lung development. Therefore, the transgenic mice were sacrificed prior to birth, at E18.5. Of the 182 embryos at E18.5 gestation collected from 32 pregnant mothers, 10 were transgenic, as determined by PCR and Southern blot analysis. The inventors observed altered lung morphogenesis, altered terminal sac morphology, and abnormally abundant mesenchyme, and no type I pneumocytes when compared with control mice (FIGS. 4A-D). Despite the important role of the Notch signaling pathway in vascular development, no alteration in vasculogenesis was observed in the transgenics using the PECAM-1 antibody (data not shown), suggesting that the mesenchymal differentiation was influenced by the abnormally developing epithelium, and not by alterations of angiogenesis. While the majority of cuboidal cells lining the peripheral airways are pneumocytes based on their TTF1 positivity, they failed to demonstrate markers of mature type II pneumocytes, such as surfactant proteins C and B, markers for Clara cells (Clara cells secretory protein), or neuroendocrine cells (calcitonin gene-related peptide) suggesting that they were immature type II cells (data not shown).
Thus, this transgenic model provides evidence that dysregulation of Notch3 signaling in the developing lung affects morphogenesis and terminal differentiation of the lung epithelium. Prenatal activation of Notch3 in the peripheral epithelium of the lung in our SP-C-N3IC mouse model leads to nonviable newborn pups, making it impossible to evaluate tumor progression. An inducible transgenic model that allows activation of Notch3 signaling in the postnatal period will be more effectively recapitulate the somatic activation of potential oncogenes observed in human lung cancer. This approach is part of the proposal in our R01 funding. Regardless, the ectopic expression of Notch3 appears to inhibit terminal differentiation of type I pneumocytes and results in metaplasia of the immature respiratory epithelium, supporting a potential role of Notch3 in lung cancer formation.
Notch3 Inhibition Inhibits the Tumor Phenotype. To test their hypothesis that Notch3 plays an important role in the pathogenesis of lung cancer, the inventors used the approach of uncoupling its ligand binding and signaling functions (Rebay et al., 1993). To inhibit Notch3 activation, the inventors created a dominant-negative (DN) construct. One characteristic feature of malignant transformation is the ability of tumor cells to proliferate in the absence of adhesion, as measured by the soft-agar assay for colony formation. The inventors demonstrated that inhibition of Notch3 signaling by the DN construct dramatically decreases the ability of HCC2429 and H460 to form colonies in soft agar (FIG. 5A). Furthermore, colonies formed by the DN clones are markedly smaller than those seen with the vector control (VC). Transformed cells often have a relaxed serum or growth factor requirement for proliferation in monolayer culture (Holley, 1975). In serum-free medium, the DN clones failed to proliferate when compared to the vector controls (FIG. 5B). In summary, these observations demonstrated that inhibiting the Notch pathway using DN constructs reduces the tumor phenotype, supporting an oncogenic role for Notch3 in lung cancer.
Since Jagged1 is known to bind to other Notch receptors, it is possible that the antitumor effect observed when DN receptor is not Notch3-specific, since its mechanism of action is to sequester ligand (Shimizu et al., 2000). To determine whether specific inhibition of Notch3 activation can inhibit tumor phenotype, the inventors used siRNA to specifically knock down Notch3 expression. The inventors show that inhibiting Notch3 results in the lack of focus formation, further supporting the role of Notch3 in lung cancer pathogenesis (FIG. 6).
A γ-Secretase Inhibitor Reduces Proliferation in Lung Cancer Cells. Proteolytic processing of Notch receptors is required for activation. As previously described, three proteolytic cleavage sites are involved in enabling Notch signaling. In the final step, the membrane-associated Notch fragment is cleaved within its transmembrane domain by a γ-secretase-containing protein complex. Mammalian presenilins (PS-1 and PS-2) are polytopic transmembrane proteins that appear to function as aspartyl proteases within a multiprotein, γ-secretase complex (Wolfe and Kopan, 2004). Presenilins provide the active site of the proteolytic activity. This last cleavage releases the Notch intracellular domain, initiating CBF-1-mediated signaling. Thus, pharmacologic intervention that inhibits the activity of the γ-secretase proteases can potentially inhibit tumor growth in Notch-dependent cancer. Interestingly, at the same time that presenilins were shown to be essential for Notch signaling, they were discovered as susceptibility loci for Alzheimer's disease (Levitan and Greenwald, 1995). The pathogenesis of Alzheimer's disease is believed to be the accumulation of amyloid β-peptide (Aβ), which is derived from proteolytic processing of the β-amyloid precursor protein (APP) by β- and γ-secretases. Since inhibition of the Notch3 pathway using the DN construct resulted in the loss of the tumor phenotype, the inventors wanted to examine the effect of γ-secretase inhibitors on tumor growth. The inventors treated HCC2429 cells with GSI (Gamma Secretase Inhibitor), a commercially available γ-secretase inhibitor (Calbiochem). The inventors observed that GSI inhibits tumor proliferation with an IC50 of about 1 μM in comparison to DMSO (FIG. 7A). However, in the presence of low serum the inhibition increases almost 1 log. This finding is similar to what was observed when the DN construct was used in HCC2429. This observation suggests that the antiproliferative activity observed is Notch-dependent. The inventors also observed inhibition of proliferation in other lung cancer cell lines expressing Notch3 (data not shown). Biochemically, treatment with these inhibitors also resulted in the loss of activated Notch3, as measured by the levels of the N3ICD (FIG. 7B). Stable expression of Notch3 siRNA also leads to loss of sensitivity to γ-secretase inhibition (FIGS. 7C, 7D).
To assess the effect of pharmacologically inhibiting Notch activation in vivo, the inventors treated subcutaneous xenografts with MRK003, a γ-secretase inhibitor from Merck, Inc. & Co. Based on the manufacturer's recommended dose, the mice were treated with 100 mg/kg orally for 3 consecutives days per week once the tumors were palpable. At the end of a 2-week treatment, the animals were then euthanized, and the tumors were harvested. A reduction of tumor size was seen in animals treated with the inhibitor, with a concomitant reduction in activated Notch3 (N3ICD) (FIGS. 8A-D).
Notch3 Inhibition Induces Apoptosis. One hallmark of cancer is resistance to apoptosis. Genetic and pharmacologic inhibition of the Notch pathway in other tumors has been shown to induce apoptosis (Qin et al., 2004; Curry et al., 2005; Shelly et al., 1999). The inventors examined whether inhibiting the Notch3 pathway can induce apoptosis. The inventors noted that in the HCC2429 clone expressing the dominant-negative receptor, the inhibition of the Notch3 pathway appears to render the tumor cells sensitive to apoptosis in the presence of serum starvation compared to vector control (FIG. 9A). One mechanism of apoptosis induction is the down-regulation of Akt phosphorylation. FIG. 9B shows that the level of phosphorylated Akt does indeed decrease when the DN Notch3 construct is used. The inventors also demonstrated that inhibition of Notch3 using siRNA results in the down-regulation of Bcl-xL (24 and 48 hours) and enhances the upregulation of cleaved PARP (48 hours), suggesting that Notch3 plays and important role in tumor survival (FIG. 9C).
Notch3 Crosstalks with the MAPK Pathway. In mammals, Notch receptors signal primarily by binding to CBF-1 and related transcription co-activators. However, as mentioned previously, the Notch and EGF/MAPK pathways are known to interact in developing vertebrates and invertebrates. Thus, since the MAPK pathway, and in particular the ERK subfamily, also plays a prominent role in cellular response to growth factors, and is often altered in cancer, The inventors examined whether Notch3 alters ERK signaling in lung cancer cells. Using the DN receptor, they showed that inhibiting Notch3 downregulates MAPK, and conversely, that MAPK is upregulated when the cells were transfected with activated Notch3 intracellular domain (FIGS. 10A, 10B). Moreover, lower levels of activated MAPK (p44/p42) expression in the unstimulated DN clones and a markedly higher level of activation with growth factor induction in the VC in comparison to the DN clone suggest that the disruption of the Notch3 signaling pathway renders lung cancer cells more resistant to growth-factor-dependent MAPK activation. This observation has been confirmed in two other lung cancer cell lines, H460 and H1819, transfected with the DN construct.
Interestingly, with prolonged exposure to serum (60 minutes), the HCC2429 clone expressing the dominant-negative construct shows that p44/p42 phosphorylation was attenuated significantly compared to vector control (FIG. 10A). Prolonged exposure to growth factors and activation of the p44/p42 cascade can induce the expression of MAPK phosphatases-1 and -2 (MKP-1/-2) as part of a negative feedback loop and result in the down-regulation of the MAPK pathway (Traverse et al., 1992; Plows et al., 2002; Haneda et al., 1999; Brondello et al., 1997). Down-regulation of MKPs or resistance to dephosphorylation by MKPs are often observed in human tumors, and Notch3 may have a role in suppressing MKP expression (Sivaraman et al., 1997; Magi-galluzzi et al., 1997; Barry et al., 2001). Recent data demonstrate that one mechanism by which Notch antagonizes EGF in developing C. elegans is the upregulation of LIP-1, a homolog of mammalian MAPK phosphatases (Berset et al., 2001). Since Drosophila and C. elegans Notch have the highest homology to human Notch1, and Notch3 appears to antagonize Notch1, it follows that Notch3 might suppress MAPK phosphatase expression in cancer cells. To test this hypothesis, the inventors quantitated the level of MKP-1 using real-time PCR following serum induction in HCC2429 stably transfected with DN and VC. Higher levels of MKP-1 were observed in the DN clones (FIG. 10C). This finding suggests that Notch3 also modulates MAPK activation through MKP-1 transcriptional control.
EGFr Inhibitors Enhance the Anti-proliferation Effect of Notch3 Inhibition. The inventors previously showed that Notch3 expression positively correlates with EGFR expression in our tumor tissue array. The also showed that Notch3 cooperates with the MAPK pathway. Based on existing literature and their observations, the inventors hypothesized that Notch3 acts synergistically with the EGFR pathway in the promotion and the survival of lung cancer cells and that combining inhibitors of both pathways will have synergistic therapeutic value. To test this hypothesis, the inventors examined whether inhibiting the Notch3 pathway increases tumor inhibition when lung cancer cells are treated with an EGFR tyrosine kinase inhibitor, AG1478. When HCC2429 cells were maintained in EGF-supplemented media and treated with increasing doses of AG1478, the clones transfected with the DN construct showed a 40-fold increase in sensitivity to EGFR inhibitors, that is, the IC50 was reduced from 8.3 μM to 0.2 μM (FIG. 11A). In H460, a lung cancer cell line that has lower Notch3 expression as well as a k-Ras mutation (data not shown) and is more resistant to AG1478 (IC50 of 23.8 μM), the inhibition of the Notch3 pathway also reduces cancer cell survival by approximately two-fold (IC50 of 12.1 μM, FIG. 10A). These data provide evidence that Notch3 activation may decrease a tumor's dependence on the EGF pathway and thus further decrease the sensitivity to EGFR tyrosine kinase inhibitors. Synergism can also be observed when the γ-secretase inhibitor L-685,458 is added to AG1478 (FIG. 11B). Using the soft-agar colony assay, the inventors also demonstrate additive effects by combining MRK003, another γ-secretase inhibitor, with an EGFR inhibitor (FIGS. 12A, 12B). From a therapeutic standpoint, Notch3 is a good target for therapeutic intervention both alone and in combination with growth factor receptor inhibitors. Since about 80% of lung carcinomas express EGFR, but far fewer respond to kinase inhibition, our data suggest that adding a Notch3 inhibitor will improve the response rate in patients treated with EGFR inhibitors.
Notch3 peptides induce apoptosis, inhibit Notch3-regulated gene Hey1, and interrupt signaling through binding to ligand Jagged1. FIG. 13 is a representative experiment showing that the peptides induce apoptosis by Annexin V staining through screening using an FMAT system. Each of 155 different peptides were assayed in quadruplicate, and only those peptides that produced a significant increase of fluorescence signal in all 4 wells were considered potentially positive or capable of inducing apoptosis. The bar graphs here reflect fluorescence counts. FIG. 14A shows HCC2429 treated with Notch3 peptides N16, N17, N102, N103, N132, with induction of apoptosis by peptides compared to control. Treatment with peptides also reduced transcription of Notch3-dependent gene Hey1 as determined by real-time RT-PCR (FIG. 14B). Of note, N17 peptide both demonstrates highest apoptotic activity and best reduction in Hey1 transcription. FIG. 15 shows HEK cells transfected with Jagged1-HA and treated with Notch3 peptides. The peptides were then immunoprecipitated from cell lysate with streptavidin beads and immunoblotted with anti-HA antibody. This suggests that peptide induces apoptosis via binding to ligand Jagged1 and preventing activation of Notch3 receptor.
Sera from mice immunized with Notch3 recombindant protein inhibit Notch3 activation. FIG. 16 shows an immunoblot that demonstrates that sera from mice #2, 3, 4, 5, 6 can reduced cleavage of Notch3 ICD (Tx) in Notch3 expressing cell line HCC2429 as compared a control (C). Recombinant protein representing EGF-like repeats 21-22 and encompassing sequence CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ. ID NO: 10 ) was used to immunize AJ and BALB/c mice.
Recombinant Fc-fusion Notch3 proteins inhibit Notch3 activation and induces apoptosis in vitro. FIG. 17A shows Fc-fusion protein comprised for N16-17 and N132 sequences inhibits Notch3 activation, while FIG. 17B shows that purified recombinant N16-17-Fc protein induces apoptosis as compared to control and Fc control after 40 hrs treatment. This study further supports the hypothesis that these regions of Notch3 are important for ligand interaction, and that disruption of this interaction using decoy recombinant receptor can inhibit Notch3 activation.
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
IX. References
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference:

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## Claims

1. An isolated and purified peptide comprising CFNTLGGHS (SEQ ID NO:3) or CVCVNGWTGES (SEQ ID NO:4), wherein the length of the peptide is no more than 50 residues.

2. The isolated and purified peptide of claim 1, wherein the peptide comprises SEQ ID NO:3.

3. The isolated and purified peptide of claim 1, wherein the peptide comprises SEQ ID NO:4.

4. The isolated and purified peptide of claim 1, wherein the peptide comprises CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9).

5. An isolated and purified peptide comprising CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10), wherein the length of the peptide is no more than 50 residues.

6. The isolated and purified peptide of claim 1, wherein the length of said peptide is no more than 25 residues.

7. The isolated and purified peptide of claim 1, wherein the length of said peptide is no more than 20 residues.

8. The isolated and purified peptide of claim 1, wherein the length of said peptide is no more than 15 residues.

9. An isolated and purified peptide consisting of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10).

10. A composition comprising the isolated and purified peptide of claim 1, 5 or 9, and a pharmaceutically acceptable diluent, buffer or excipient.

11. A method of inhibiting Notch3 receptor signaling comprising contacting a cell expressing Notch3 with a peptide comprising CFNTLGGHS (SEQ ID NO:3) or CVCVNGWTGES (SEQ ID NO:4), wherein the length of the peptide is no more than 50 residues.

12. The method of claim 11, wherein said cell is a cancer cell.

13. The method of claim 12, wherein said cancer cell is a lung cancer cell.

14. The method of claim 12, wherein said cancer cell is an adenocarcinoma cell.

15. The method of claim 11, further comprising contacting said cell with one or more peptides comprising CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) or CLNGGS (SEQ ID NO:8).

16. The method of claim 11, wherein the length of said peptide is no more than 25 residues, 20 residues or 15 residues.

17. A method of inhibiting Notch3 receptor signaling comprising contacting a cell expressing Notch3 with a peptide comprising CFNTLGGHSCVCVGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10), wherein the length of said peptide is no more than 50 residues.

18. A method of inhibiting Notch3 receptor signaling comprising contacting a cell expressing Notch3 with a peptide consisting of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10).

19. The method of claim 11, further comprising contacting said cell with a second agent that inhibits cancer cell growth, differentiation, metastasis or drug resistance.

20. A pharmaceutical formulation comprising one or both of the peptides CFNTLGGHS (SEQ ID NO:3) and CVCVNGWTGES (SEQ ID NO:4).

21. The pharmaceutical formulation of claim 20, further comprising one, two, three, four, five, or six of the peptides selected from the group consisting of: CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) and CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10).