Methods and compounds for lymphoma cell detection and isolation

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Compositions comprising a purified, isolated antibody, humanized antibodies and precipitates directed against ROR-1, wherein the antibody binds ROR-1 with moderate to high affinity. The compositions may be used for detecting and isolating an amount of ROR-1 in a subject sample, and to evaluate the appearance, status, course, or treatment of a ROR-1 cancer in a subject. The ROR-1 antibodies are especially useful in identifying lymphomas and ademocarcinomas. Vaccines and related methods for protecting a subject against diseases that involve expression of ROR-1 are also provided.

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Description
CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional Patent Application No. 60/731,210, filed Oct. 28, 2005, and to the PCT application filed on Oct. 30, 2006, of which this is a continuation-in-part.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with Government support under National Institutes of Health Grant 5P01 CA81543. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to antibodies directed against antigens specific for chronic lymphocytic leukemia.

BACKGROUND

ROR-1 is an embryonic protein that is expressed uniquely on certain cancer cells, including in chronic lymphocytic reukemia (CLL), small lymphocytic lymphoma, marginal cell B-Cell lymphoma, Burkett's Lymphoma, and other cancers (e.g., breast cancers), but not on normal adult tissues and cells. Anti-ROR-1 antibodies raised against ROR-1 peptide are commercially available, but monoclonal anti-ROR-1 antibodies that react with the native ROR-1 protein have not been made or isolated. In addition, no anti-ROR-1 antibodies capable of detecting cell-surface expression of ROR-1 for flow cytometric analysis have been made or isolated. What is needed, therefore, is an antibody that can react with native ROR-1 protein.

SUMMARY OF THE INVENTION

Among the various aspects of the present invention is the provision of an antibody directed to a surface receptor tyrosine kinase protein expressed on cells found in samples of subjects with a cancer, including lymphomas, CLL, small lymphocytic lymphoma, marginal cell B-Cell lymphoma, Burkett's Lymphoma, renal cell carcinoma, colon cancer, colorectal cancer, and breast cancer, but not in blood or splenic lymphocytes of nonleukemic patients or normal adults.

Briefly, therefore, the present invention is directed to an antibody useful for differentiation between ROR-1 expressing cancer cells (“ROR-1 cancer”) and normal cells as well as immunotherapy against ROR-1 cancers and determination of response to cancer therapy.

The present invention includes compositions that include a purified, isolated antibody that binds specifically to ROR-1 receptor protein.

The present invention includes methods for an immunoassay that detects ROR-1 in a sample from a subject by contacting the sample with a ROR-1-specific antibody and detecting immunoreactivity between the antibody and ROR-1 in the sample.

In accordance with a further aspect of the invention, a ROR-1 cancer is diagnosed in a subject by detecting the presence or quantity of ROR-1 protein in a sample derived from the subject.

In accordance with yet another aspect of the invention, a ROR-1 cancer is treated in a subject by administering to the subject in need of such therapy a therapeutically effective amount of a ROR-1 receptor agonist.

In accordance with yet another aspect, the appearance, status, course, or treatment of a ROR-1 cancer in a subject is evaluated by contacting a biological sample obtained from the subject with an anti-ROR-1 antibody and detecting immunoreactivity between the antibody and ROR-1 to determine presence or quantity of ROR-1 in the sample.

In accordance with yet another aspect, also provided is a vaccine composition comprising a polynucleotide encoding ROR-1 protein or a fragment or variant thereof, and a pharmaceutically acceptable carrier or diluent.

In accordance with yet another aspect, also provided is a vaccine composition comprising ROR-1 protein or a fragment or variant thereof, and a pharmaceutically acceptable carrier or diluent.

In accordance with yet another aspect, also provided is a method for protecting against the occurrence of diseases involving expression of ROR-1 in a subject, the method comprising administering to the subject in need thereof a polynucleotide encoding ROR-1 protein or a fragment or variant thereof in an amount effective to induce a protective or therapeutic immune response against ROR-1, and a pharmaceutically acceptable carrier or diluent.

In accordance with yet another aspect, also provided is a method for protecting against the occurrence of diseases involving expression of ROR-1 in a subject, the method comprising administering to the subject in need thereof ROR-1 protein or a fragment or variant thereof in an amount effective to induce a protective or therapeutic immune response against ROR-1 in the subject, and a pharmaceutically acceptable carrier or diluent.

In accordance with yet another aspect, a humanized ROR-1 antibody is provided. In another aspect, a precipitate comprising a ROR-1 antibody bound with a ROR-1 protein, fragment or variant is provided. The ROR-1 antibody can be conjugated to a magnetic bead.

Other objects and features will be in part apparent and in part pointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

FIG. 1 shows change of serum antibody after Ad-CD154 therapy.

FIG. 1A is a series of scatter and line plots showing total levels of IgG, IgA, and IgM. IgG, IgA, IgM blood concentrations, measured just prior to initiating Ad-CD154 therapy (PRE) and 2-4 week following the final treatment time point (POST). The dashed bar in each line graph indicates the minimum normal Ig concentration. The concentration range of normal Ig levels is shown to the left of legend.

FIG. 1B is a series of scatter and line plots showing antibody response to recombinant Ad-CD154. Anti-adenovirus antibodies were analyzed by an ELISA assay. Serial dilutions of patient serum before (dotted line) and after (filled line) treatment were incubated in 96 well plates coated with Ad-CD154. Bound adenovirus-specific antibody was then detected using AP-conjugated antibody specific for human Ig.

FIG. 1C is a series of bar graphs showing change of antibody response against Adenovirus in serial samples. Anti-adenovirus antibodies were analyzed by an ELISA using anti-isotype specific secondary antibodies conjugated AP. The bar graphs represent the mean increase in adenovirus-specific antibody over the baseline pre-treatment antibody levels. IgE and IgG4 levels for all patients were below the assay detection limits (data not shown).

FIG. 1D is a series of scatter and line plots showing anti-tetanus-toxin antibody response before and after Ad-CD154 treatment. ELISA assay was performed with purified tetanus toxin and sera from patients. Bound tetanus-specific antibody was detected using AP conjugated goat anti-human Ig antibody.

FIG. 2 is a series of histograms showing antibody production against surface molecules on CLL B cells by Ad-CD154 therapy. Antibody bound on CD19+ CD3-cells were detected by goat anti-human antibody.

FIG. 2A is a series of histograms showing diluted serum from patient before (open histograms) or after (shaded histograms) treatment was incubated with PBMC from CLL patient.

FIG. 2 B is a series of histograms showing diluted serum from patient before (open histograms) or after (shaded histograms) treatment was incubated with PBMC from a healthy donor.

FIG. 3 is an immunoblot of Immune Precipitates of Lysates with 4A5 Probed With Rabbit anti-ROR-1 Raised Against Ror1 Peptides.

FIG. 4 is a series of images depicting gels that show expression of ROR-1 in CLL B cells.

FIG. 4A are gel images of an immunoblot analysis of ROR-1 protein. Total cell lysates of PBMC from CLL patients or healthy donor and those of splenocytes from CLL patients or idiopathic thrombocytopenia purpura patient were analyzed by immunoblot using rabbit anti-ROR-1 antibody (Cell signaling).

FIG. 4B are gel images showing ROR-1 expression in B cell lines. Immunoblot analysis of total cell lysates of B cell lines was performed.

FIG. 4C shows production of mouse anti-ROR-1 sera. CHO cells stained with PKH26 and were mixed with CHO transfected ROR-1 cDNA (CHO-ROR-1). Sera collected from mice before and after immunization with ROR-1 cDNA were incubated with mixed CHO cells. Bound antibodies were detected flow cytometry.

FIG. 4D is a series of histograms showing flow cytometric analysis of expression of ROR-1 on cell surface of CLL. PBMC from CLL patients and healthy donor were incubated antisera before (open histograms) and after (shaded histograms) DNA immunization.

FIG. 5 is a series of histograms showing production of anti-ROR-1 antibody detected by flow cytometric analysis.

FIG. 5A is a series of histograms where CHO (open histograms) or CHO-ROR-1 (shaded histograms) was incubated with serum from patients before (pre) or after (post) therapy. Histograms indicated the bound human Ig detected by PE labeled goat anti-human Ig.

FIG. 5 B shows results where CHO stained with PKH26 were mixed and incubated with serum from patient. APC conjugated anti-human Ig antibody was used for detection.

FIG. 6 shows production of anti-ROR-1 antibody detected by ELISA.

FIG. 6A is a series of gel images showing production of recombinant ROR-1 protein. ROR-1 extracellular region was fused with rabbit IgG Fc region in frame (ROR-1rIg). Fused cDNA were transfected into CHO cells and secreted recombinant protein was immunoabsorbed using protein A sepharose. Absorbed protein was immunoblotted with goat anti-ROR-1 antibody (R&D) or goat anti-rabbit Ig antibody. KSHV K8.1 protein fused with rabbit Fc region was also used for control. The purified recombinant ROR-1 was visualized with GelCode blue stain reagent (Pierce) staining after SDS-PAGE.

FIG. 6B is a series of line and scatter plots showing antibody reaction to ROR-1 detected by ELISA. Diluted sera were reacted with coated ROR-1rIg and bound antibody was detected by goat anti-human Ig antibody conjugated with HRP.

FIG. 6C is a series of line and scatter plots showing antibody reaction to rabbit IgG detected by ELISA. Diluted sera were reacted with coated rabbit IgG and bound antibody was detected by goat anti-human Ig antibody conjugated with HRP.

FIG. 7 shows ROR-1 and Wnt5a activated NF-κB reporter expression.

FIG. 7A is a series of bar graphs showing the effect of ROR-1 on LEF/TCF1, NF-AT, and AP-1 activity. HEK293 cells were transfected with indicated reporter construct and β-galactosidase vector along with expression vector of ROR-1 and Wnt5a.

FIG. 7B is a series of bar graphs showing the effect of ROR-1 on NF-KB activity. HEK293 cells were transfected with NF-κB reporter construct and β-galactosidase vector along with expression vector of ROR, Wnt5a, Wnt3, Wnt5b and Wnt16.

FIG. 7C is a series of gel images showing in vitro binding of ROR-1 and Wnt5a. Conditioned medium of transfectant with Wnt5a tagged with HA was incubated with ROR-1rIg or rabbit IgG. Immunoprecipitation and immunoblotting were done with indicated materials.

FIG. 8 is a series of histograms showing gated CLL patients and CD19+ and CD19+CD5+ cells.

FIG. 9 is a series of histograms showing gated normal patients and CD19+ and CD19+CD5+ cells.

FIG. 10 is a series of histograms showing gated “exceptional” normal patients and CD19+ and CD19+CD5+ cells.

FIG. 11 is a series of histograms showing gated CLL patients and CD19+ and CD19+CD5+ cells.

FIG. 12 depicts the expression of 4A5 versus normals versus CLLs and the gating effect.

FIG. 13 is a series of histograms showing different levels of 4A5 expression on titrated CLL cells.

FIG. 14 is a series of histograms showing different levels of 4A5 expression and that such cells can be purified using magnetic beads and methods provided herein.

FIG. 15 is a series of histograms showing 4A5 expression on various cancer cell lines.

FIG. 16A is an immunoblot of various human adult tissues for expression of ROR-1, β-actin or GAPDH, where the latter two are used to control for protein loading.

FIG. 16B is an immunoblot of various human tissue for ROR-1 or β-actin.

FIG. 16C are immunoprecipitation graphs based on the A5A anti-ROR-1 mAb or an isotype control mAb to precipitate ROR-1 protein.

FIG. 16D are immunoprecipitation graphs based on the A5A anti-ROR-1 mAB or an isotype control mAb to precipitate ROR-1 protein.

FIG. 17 are histograms showing ROR-1 expression in EW36, a cell line that naturally expresses ROR1 (positive control; middle), non-transfected P815 cells (right), and P815 cells transfected with pcDNA3 ROR1. Cells were stained with Alexa-conjugated mouse IgG2b (darker colored histogram) or Alexa-conjugated anti-ROR1 antibody (light colored histogram) and analyzed by flow cytometry.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the instant invention provides new and useful antibodies directed against ROR-1 protein. Full length ROR-1, a surface receptor tyrosine kinase, is found in samples of subjects with CLL, but not in blood or splenic lymphocytes of nonleukemic patients or normal adults. The invention also provides diagnostic and therapeutic antibodies, including monoclonal antibodies, and related compositions and methods for use in the diagnosis, management and treatment of disease. The ROR-1 antibody described herein is more sensitive and more specific to ROR-1 expressing cancer cells than using a combination of several cell surface markers that cannot exclude a small fraction of normal cells.

Applicants have discovered expression of full-length ROR-1 in numerous cancer cell lines and samples, but not other tissues, including blood or splenic lymphocytes of non-leukemic patients or normal adult donors, and also generated mouse anti-sera against full-length human ROR-1. Fukuda et al., Blood: ASH Annual Meeting Abstracts 2004 104, Abstract 772 (2004) (incorporated herein by reference in its entirety). The polypeptide and coding sequences for ROR-1 have been reported elsewhere and are also incorporated herein by this reference (see, e.g., Accession Nos. NP—005003.1 and NM—005012.1).

ROR-1 Antibody

Certain embodiments comprise immunopeptides directed against ROR-1 protein. The immunoglobulin peptides, or antibodies, described herein are shown to bind to the ROR-1 protein. The ROR-1 binding activity is specific; the observed binding of antibody to ROR-1 is not substantially blocked by non-specific reagents. These ROR-1 specific antibodies can be used to differentiate between ROR-1 cells and normal cells. The ROR-1 specific antibodies can also be used in immunotherapy against a ROR-1 cancer and to determine the response after therapy for a ROR-1 cancer.

Such immunopeptides can be raised in a variety of means known to the art. For example, and as shown in the examples, Ad-CD154 therapy induces humoral immunity against CLL, thus allowing the derivation of immunoglobulin peptides specific against ROR-1. The inventors have discovered that tandem injections of Ad-CD154 induces antibody production against a novel cell surface TAA of CLL B cells, orphan tyrosine kinase receptor ROR-1.

As used herein, the term antibody encompasses all types of antibodies, e.g., polyclonal, monoclonal, and those produced by the phage display methodology. Particularly preferred antibodies of the invention are antibodies which have a relatively high degree of affinity for ROR-1. In certain embodiments, the antibodies exhibit an affinity for ROR-1 of about Kd<10−8 M.

Substantially purified generally refers to a composition which is essentially free of other cellular components with which the antibodies are associated in a non-purified, e.g., native state or environment. Purified antibody is generally in a homogeneous state, although it can be in either in a dry state or in an aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.

Substantially purified ROR-1-specific antibody will usually comprise more than 80% of all macromolecular species present in a preparation prior to admixture or formulation of the antibody with a pharmaceutical carrier, excipient, adjuvant, buffer, absorption enhancing agent, stabilizer, preservative, adjuvant or other co-ingredient. More typically, the antibody is purified to represent greater than 90% of all proteins present in a purified preparation. In specific embodiments, the antibody is purified to greater than 95% purity or may be essentially homogeneous wherein other macromolecular species are not detectable by conventional techniques.

Immunoglobulin peptides include, for example, polyclonal antibodies, monoclonal antibodies, and antibody fragments. The following describes generation of immunoglobulin peptides, specifically ROR-1 antibodies, via methods that can be used by those skilled in the art to make other suitable immunoglobulin peptides having similar affinity and specificity which are functionally equivalent to those used in the examples.

Polyclonal Antibodies

Polyclonal antibodies may be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as horses, cows, various fowl, rabbits, mice, or rats. Briefly, ROR-1 antigen is utilized to immunize the animal through intraperitoneal, intramuscular, intraocular, or subcutaneous injections, with an adjuvant such as Freund's complete or incomplete adjuvant. Following several booster immunizations, samples of serum are collected and tested for reactivity to ROR-1. Particularly preferred polyclonal antisera will give a signal on one of these assays that is at least three times greater than background. Once the titer of the animal has reached a plateau in terms of its reactivity to ROR-1, larger quantities of antisera may be readily obtained either by weekly bleedings, or by exsanguinating the animal.

Monoclonal Antibodies

Monoclonal antibody (mAb) technology can be used to obtain mAbs to ROR-1. Briefly, hybridomas are produced using spleen cells from mice immunized with ROR-1 antigens. The spleen cells of each immunized mouse are fused with mouse myeloma Sp 2/0 cells, for example using the polyethylene glycol fusion method of Galfre, G. and Milstein, C., Methods Enzymol., 73:3-46 (1981). Growth of hybridomas, selection in HAT medium, cloning and screening of clones against antigens are carried out using standard methodology (Galfre, G. and Milstein, C., Methods Enzymol., 73:3-46 (1981)).

HAT-selected clones are injected into mice to produce large quantities of mAb in ascites as described by Galfre, G. and Milstein, C., Methods Enzymol., 73:3-46 (1981), which can be purified using protein A column chromatography (BioRad, Hercules, Calif.). mAbs are selected on the basis of their (a) specificity for ROR-1, (b) high binding affinity, (c) isotype, and (d) stability.

mAbs can be screened or tested for ROR-1 specificity using any of a variety of standard techniques, including Western Blotting (Koren, E. et al., Biochim. Biophys. Acta 876:91-100 (1986)) and enzyme-linked immunosorbent assay (ELISA) (Koren, E. et al., Biochim. Biophys. Acta 876:91-100 (1986)).

Humanized Antibodies

Humanized forms of mouse antibodies can be generated by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques (see, e.g., Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033, 1989 and WO 90/07861, each incorporated by reference). Human antibodies can be obtained using phage-display methods (see, e.g., Dower et al., WO 91/17271; McCafferty et al., WO 92/01047). In these methods, libraries of phage are produced in which members display different antibodies on their outersurfaces. Antibodies are usually displayed as Fv or Fab fragments. Phage displaying antibodies with a desired specificity may be selected by affinity enrichment.

Human antibodies may be selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody. Using these techniques, a humanized ROR-1 antibody having the human IgG1 constant region domain and the human kappa light chain constant region domain with the mouse heavy and light chain variable regions. The humanized antibody has the binding specificity of a mouse ROR-1 mAb, specifically the 45A mAb described in Example 9.

Antibody Fragments

It may be desirable to produce and use functional fragments of a mAb for a particular application. The well-known basic structure of a typical IgG molecule is a symmetrical tetrameric Y-shaped molecule of approximately 150,000 to 200,000 daltons consisting of two identical light polypeptide chains (containing about 220 amino acids) and two identical heavy polypeptide chains (containing about 440 amino acids). Heavy chains are linked to one another through at least one disulfide bond. Each light chain is linked to a contiguous heavy chain by a disulfide linkage. An antigen-binding site or domain is located in each arm of the Y-shaped antibody molecule and is formed between the amino terminal regions of each pair of disulfide linked light and heavy chains. These amino terminal regions of the light and heavy chains consist of approximately their first 110 amino terminal amino acids and are known as the variable regions of the light and heavy chains. In addition, within the variable regions of the light and heavy chains there are hypervariable regions which contain stretches of amino acid sequences, known as complementarity determining regions (CDRs). CDRs are responsible for the antibody's specificity for one particular site on an antigen molecule called an epitope. Thus, the typical IgG molecule is divalent in that it can bind two antigen molecules because each antigen-binding site is able to bind the specific epitope of each antigen molecule. The carboxy terminal regions of light and heavy chains are similar or identical to those of other antibody molecules and are called constant regions. The amino acid sequence of the constant region of the heavy chains of a particular antibody defines what class of antibody it is, for example, IgG, IgD, IgE, IgA or IgM. Some classes of antibodies contain two or more identical antibodies associated with each other in multivalent antigen-binding arrangements.

Fab and F(ab′)2 fragments of mAbs that bind ROR-1 can be used in place of whole mAbs. Because Fab and F(ab′)2 fragments are smaller than intact antibody molecules, more antigen-binding domains are available than when whole antibody molecules are used. Proteolytic cleavage of a typical IgG molecule with papain is known to produce two separate antigen binding fragments called Fab fragments which contain an intact light chain linked to an amino terminal portion of the contiguous heavy chain via by disulfide linkage. The remaining portion of the papain-digested immunoglobin molecule is known as the Fc fragment and consists of the carboxy terminal portions of the antibody left intact and linked together via disulfide bonds. If an antibody is digested with pepsin, a fragment known as an F(ab′)2 fragment is produced which lacks the Fc region but contains both antigen-binding domains held together by disulfide bonds between contiguous light and heavy chains (as Fab fragments) and also disulfide linkages between the remaining portions of the contiguous heavy chains (Handbook of Experimental Immunology. Vol 1: Immunochemistry, Weir, D. M., Editor, Blackwell Scientific Publications, Oxford (1986)).

Recombinant DNA methods have been developed which permit the production and selection of recombinant immunoglobulin peptides which are single chain antigen-binding polypeptides known as single chain Fv fragments (ScFvs or ScFv antibodies). Further, ScFvs can be dimerized to produce a diabody. ScFvs bind a specific epitope of interest and can be produced using any of a variety of recombinant bacterial phage-based methods, for example as described in Lowman et al. (1991) Biochemistry, 30, 10832-10838; Clackson et al. (1991) Nature 352, 624-628; and Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87, 6378-6382. These methods are usually based on producing genetically altered filamentous phage, such as recombinant M13 or fd phages, which display on the surface of the phage particle a recombinant fusion protein containing the antigen-binding ScFv antibody as the amino terminal region of the fusion protein and the minor phage coat protein g3p as the carboxy terminal region of the fusion protein. Such recombinant phages can be readily grown and isolated using well-known phage methods. Furthermore, the intact phage particles can usually be screened directly for the presence (display) of an antigen-binding ScFv on their surface without the necessity of isolating the ScFv away from the phage particle.

To produce an ScFv, standard reverse transcriptase protocols are used to first produce cDNA from mRNA isolated from a hybridoma that produces an mAb for ROR-1 antigen. The cDNA molecules encoding the variable regions of the heavy and light chains of the mAb can then be amplified by standard polymerase chain reaction (PCR) methodology using a set of primers for mouse immunoglobulin heavy and light variable regions (Clackson (1991) Nature, 352, 624-628). The amplified cDNAs encoding mAb heavy and light chain variable regions are then linked together with a linker oligonucleotide in order to generate a recombinant ScFv DNA molecule. The ScFv DNA is ligated into a filamentous phage plasmid designed to fuse the amplified cDNA sequences into the 5′ region of the phage gene encoding the minor coat protein called g3p. Escherichia coli bacterial cells are than transformed with the recombinant phage plasmids, and filamentous phage grown and harvested. The desired recombinant phages display antigen-binding domains fused to the amino terminal region of the minor coat protein. Such “display phages” can then be passed over immobilized antigen, for example, using the method known as “panning”, see Parmley and Smith (1989) Adv. Exp. Med. Biol. 251, 215-218; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87, 6378-6382, to adsorb those phage particles containing ScFv antibody proteins that are capable of binding antigen. The antigen-binding phage particles can then be amplified by standard phage infection methods, and the amplified recombinant phage population again selected for antigen-binding ability. Such successive rounds of selection for antigen-binding ability, followed by amplification, select for enhanced antigen-binding ability in the ScFvs displayed on recombinant phages. Selection for increased antigen-binding ability may be made by adjusting the conditions under which binding takes place to require a tighter binding activity. Another method to select for enhanced antigen-binding activity is to alter nucleotide sequences within the cDNA encoding the binding domain of the ScFv and subject recombinant phage populations to successive rounds of selection for antigen-binding activity and amplification (see Lowman et al. (1991) Biochemistry 30, 10832-10838; and Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87, 6378-6382).

Once an ScFv is selected, the recombinant ROR-1 antibody can be produced in a free form using an appropriate vector in conjunction with E. coli strain HB2151. These bacteria actually secrete ScFv in a soluble form, free of phage components (Hoogenboom et al. (1991) Nucl. Acids Res. 19, 4133-4137). The purification of soluble ScFv from the HB2151 bacteria culture medium can be accomplished by affinity chromatography using antigen molecules immobilized on a solid support such as AFFIGEL™ (BioRad, Hercules, Calif.).

Other developments in the recombinant antibody technology demonstrate possibilities for further improvements such as increased avidity of binding by polymerization of ScFvs into dimers and tetramers (see Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90, 6444-6448).

Because ScFvs are even smaller molecules than Fab or F(ab′)2 fragments, they can be used to attain even higher densities of antigen binding sites per unit of surface area when immobilized on a solid support material than possible using whole antibodies, F(ab′)2, or Fab fragments. Furthermore, recombinant antibody technology offers a more stable genetic source of antibodies, as compared with hybridomas. Recombinant antibodies can also be produced more quickly and economically using standard bacterial phage production methods.

Recombinant Antibody Production

To produce antibodies described herein recombinantly, nucleic acids encoding light and heavy chain variable regions, optionally linked to constant regions, are inserted into expression vectors. The light and heavy chains can be cloned in the same or different expression vectors. For example, the heavy and light chains of SEQ ID NOs: 1-5 can be used according to the present invention. The teachings of U.S. Pat. No. 6,287,569 to Kipps et al., incorporated herein by reference in its entirety, and the methods provided herein can readily be adapted by those of skill in the art to create the vaccines of the present invention. The DNA segments encoding antibody chains are operably linked to control sequences in the expression vector(s) that ensure the expression of antibody chains. Such control sequences include a signal sequence, a promoter, an enhancer, and a transcription termination sequence. In one embodiment, the

Expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosome. E. coli is one procaryotic host particularly useful for expressing antibodies of the present invention. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, one can also make expression vectors, which typically contain expression control sequences compatible with the host cell (e.g., an origin of replication) and regulatory sequences such as a lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. Other microbes, such as yeast, may also be used for expression. Saccharomyces is a preferred host, with suitable vectors having expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired. Mammalian tissue cell culture can also be used to express and produce the antibodies of the present invention (see, e.g., Winnacker, From Genes to Clones VCH Publishers, N.Y., 1987). Eukaryotic cells are preferred, because a number of suitable host cell lines capable of secreting intact antibodies have been developed. Preferred suitable host cells for expressing nucleic acids encoding the immunoglobulins of the invention include: monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary-cells (CHO); mouse sertoli cells; monkey kidney cells (CV1 ATCC CCL 70); african green monkey kidney cells (VERO-76, ATCC CRL 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); and TRI cells.

The vectors containing the polynucleotide sequences of interest (e.g., the heavy and light chain encoding sequences and expression control sequences) can be transferred into the host cell. Calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation can be used for other cellular hosts (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 2nd ed., 1989). When heavy and light chains are cloned on separate expression vectors, the vectors are co-transfected to obtain expression and assembly of intact immunoglobulins. After introduction of recombinant DNA, cell lines expressing immunoglobulin products are cell selected. Cell lines capable of stable expression are preferred (i.e., undiminished levels of expression after fifty passages of the cell line).

Once expressed, the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, e.g., Scopes, Protein Purification, Springer-Verlag, N.Y., 1982). Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred.

Labeled Antibody

A labeled antibody or a detectably labeled antibody is generally an antibody (or antibody fragment which retains binding specificity), having an attached detectable label. The detectable label is normally attached by chemical conjugation, but where the label is a polypeptide, it could alternatively be attached by genetic engineering techniques. Methods for production of detectably labeled proteins are well known in the art. Detectable labels known in the art include radioisotopes, fluorophores, paramagnetic labels, enzymes (e.g., horseradish peroxidase), or other moieties or compounds which either emit a detectable signal (e.g., radioactivity, fluorescence, color) or emit a detectable signal after exposure of the label to its substrate. Various detectable label/substrate pairs (e.g., horseradish peroxidase/diaminobenzidine, avidin/streptavidin, luciferase/luciferin), methods for labeling antibodies, and methods for using labeled antibodies are well known in the art (see, for example, Harlow and Lane, eds., 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) Another technique which may also result in greater sensitivity consists of coupling the antibodies to low molecular weight haptens. These haptens can then be specifically detected by means of a second reaction. For example, it is common to use such haptens as biotin, which reacts with avidin, or dinitrophenyl, pyridoxal, and fluorescein, which can react with specific antihapten antibodies.

Diagnosis of ROR-1 Cancer

The ROR-1 antibodies described herein can be used to differentiate between ROR-1 expressing cells and normal cells and, thus, can be used to detect and/or diagnose disease in subjects. ROR-1 expressing cancer cells include CLL and other lymphoma (e.g. Burkitt's), renal cell carcinoma, colon adenocarcinoma, colorectal (see, e.g., FIG. 15).

The methods for detecting such disease generally include contacting a sample from a subject having, or at risk of having, a lymphoma with a reagent that detects ROR-1, and detecting the reaction of the reagent. Within these methods, detection of a reaction is indicative of the presence and/or quantity of ROR-1 in the sample. The reaction of the reagent with the sample is then compared to a control. Any biological sample which may contain a detectable amount of ROR-1 can be used. Examples of biological samples of use with the invention are blood, serum, plasma, urine, mucous, feces, cerebrospinal fluid, pleural fluid, ascites, and sputum samples. Tissue or cell samples can also be used with the subject invention. These samples can be obtained by many methods such as cellular aspiration, or by surgical removal of a biopsy sample. The level of ROR-1 in the sample can be compared with the level in a sample not affected by the targeted disorder or condition. Control samples not affected by a targeted disease processes can be taken from the same subject, or can be from a normal control subject not affected by the disease process, or can be from a cell line.

Contacting the sample and anti-ROR-1 antibody generally includes incubation under conditions which allow contact in solution and/or solid phase between the reagent and sample. Detection can be performed by any means suitable to identify the interaction of the reagent with ROR-1. In one embodiment, when the reagent is an antibody, the antibody can be detectably labeled. Detectable labels are well known in the art, and include radioisotopes, fluorophores, paramagnetic labels, enzymes (e.g., horseradish peroxidase), or other moieties or compounds which either emit a detectable signal (e.g., radioactivity, fluorescence, color) or emit a detectable signal after exposure of the label to its substrate. Alternatively, when the reagent is an antibody, detection can be performed using a second antibody which is detectably labeled which recognizes the antibody that binds ROR-1. The antibody may also be biotinylated, and a second avidinated label used to determine the presence of the biotinylated reagent which detects ROR-1.

The antibodies of the invention are suited for use, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. The antibodies employed in these immunoassays can be detectably labeled in various ways. Examples of types of immunoassays which can effectively employ antibodies of the invention are, competitive and non-competitive immunoassays, in either a direct or indirect format. Examples of such immunoassays include a radioimmunoassay (RIA), and a sandwich (immunometric) assay. Those of skill in the art will readily discern additional immunoassay formats useful within the invention.

Other immunoassays for use within the invention include “forward” assays for the detection of a protein in which a first anti-protein antibody (e.g., an anti-ROR-1 antibody) bound to a solid phase support is contacted with the test sample. After a suitable incubation period, the solid phase support is washed to remove unbound protein. A second, distinct anti-protein antibody is then added which is specific for a portion of the specific protein not recognized by the first antibody. The second antibody is preferably detectable. After a second incubation period to permit the detectable antibody to complex with the specific protein bound to the solid phase support through the first antibody, the solid phase support is washed a second time to remove the unbound detectable antibody. Alternatively, the second antibody may not be detectable. In this case, a third detectable antibody, which binds the second antibody is added to the system. This type of “forward sandwich” assay may be a simple yes/no assay to determine whether binding has occurred or may be made quantitative by comparing the amount of detectable antibody with that obtained in a control.

Other types of immunometric assays are the so-called “simultaneous” and “reverse” assays. A simultaneous assay involves a single incubation step wherein the first antibody bound to the solid phase support, the second, detectable antibody and the test sample are added at the same time. After the incubation is completed, the solid phase support is washed to remove unbound proteins. The presence of detectable antibody associated with the solid support is then determined as it would be in a conventional “forward sandwich” assay. The simultaneous assay may also be adapted in a similar manner for the detection of antibodies in a test sample. The “reverse” assay comprises the stepwise addition of a solution of detectable antibody to the test sample followed by an incubation period and the addition of antibody bound to a solid phase support after an additional incubation period. The solid phase support is washed in conventional fashion to remove unbound protein/antibody complexes and unreacted detectable antibody. The determination of detectable antibody associated with the solid phase support is then determined as in the “simultaneous” and “forward” assays. The reverse assay may also be adapted in a similar manner for the detection of antibodies in a test sample.

The antibody component of immunometric assays described herein may be added to a solid phase support capable of immobilizing proteins. By “solid phase support” or “support” is intended any material capable of binding proteins. Well-known solid phase supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses (including nitrocellulose sheets and filters), polyacrylamides, agaroses, and magnetite. The nature of the support can be either soluble to some extent or insoluble for the purposes of the present invention. The support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Those skilled in the art will know many other suitable “solid phase supports” for binding proteins or will be able to ascertain the same by use of routine experimentation. A preferred solid phase support is a 96-well microtiter plate. For immunoassay and immunodiagnostic purposes, the antibodies of the invention can be bound to many different carriers, both soluble and insoluble, and can be used to detect the presence of an antigen comprising ROR-1 (or fragments, derivatives, conjugates, homologues, or variants thereof). Those skilled in the art will discern other suitable carriers for binding antibodies useful within the invention. In addition, there are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, and bioluminescent compounds, as described above.

In using the antibodies described herein for the in vitro or in vivo detection of ROR-1, the detectably labeled antibody is provided in an amount which is diagnostically effective. Thus, an amount of detectably labeled antibody is contacted or administered in sufficient quantity to enable detection of ROR-1 in the subject sample to be assayed.

Within more detailed diagnostic methods of the invention, in vivo immunodiagnostic tools are provided, as exemplified by immunoscintigraphic methods and compositions. Immunoscintigraphy (IS) is discussed in detail in P. Lechner et al., Dis Colon Rectum 1993; 36:930-935 and F. L. Moffet et al., J Clin Oncol 14:2295-2305 (1966). IS (or radioscintigraphy) employs radioactive-labeled antibody, typically Fab′ fragments (Goldenberg et al.; Eur J Nucl Med 1989; 15:426), to recognize defined epitopes of targeted proteins. Fab′ fragments of the antibodies provided herein, comprising immunoglobulins of the IgGI fraction that have their Fc portions removed, are highly capable of targeting epitopes on proCPR, activated CPR, and/or inactivated CPR in a test sample or subject. Because these Fab′ fragments have minimal antigenity, they cause neither human antimouse antibody response, nor any allergic reactions of unpredictable nature. The smaller molecular weight of Fab′ fragments compared with intact antibody allows the fragment to leave the intravascular space and target a broader array of in vivo compartments for diagnostic purposes.

For radioscintigraphy, an anti-ROR-1 radioactive monoclonal antibody is typically injected into a patient for identifying, measuring, and/or localizing ROR-1 in the subject, (see, e.g., Delaloye et al., Seminars in Nuclear Medicine 25(2):144-164, 1995). In radioimaging with monoclonal antibodies, a chemically modified (chelate) form of the monoclonal antibody is typically prepared and stored as a relatively stable product. To be used clinically, however, the monoclonal antibody sample must be mixed with a radioactive metal, such as 99Tc, then purified to remove excess, unbound radioactive metal, and then administered to a patient within 6 hours, (see, e.g., Eckelman et al., Nuc. Med. Biol. 16: 171-176, 1989). Radioisotopes, for example 99Tc, an isotope with a short physical half-life and high photon abundance, can be administered at high doses and allow early imaging with a gamma camera. This is very suitable for use in conjunction with Fab′ fragments, the half-lives of which are also short.

Monitoring of a ROR-1 Cancer and Cancer Therapy

Further, the anti-ROR-1 antibodies described herein can be used in vitro and in vivo to monitor the appearance, status, course, or treatment of a ror-1 cancer in a subject. For example, by measuring an increase or decrease in the amount of ROR-1 in a subject (optionally in comparison to control levels in a normal subject or sample), the appearance, status, course, or treatment of the cancer or condition in the subject number can be observed or evaluated. Based on these and comparable diagnostic methods, it is further possible to determine whether a particular therapeutic regimen, such as a treatment regimen employing antibodies of the invention directed against the cancer is effective. Methods of detecting and/or quantifying levels of ROR-1 and corresponding cancer disease state are as described above.

Therapeutic Treatment of Lymphoma

ROR-1 agonists can be employed as therapeutic or prophylactic pharmacological agents in any subject in which it is desirable to administer, in vitro, ex vivo, or in vivo the subject agonists that bind ROR-1. Typical subjects for treatment or management according to the methods herein are subjects presenting with a ROR-1 cancer. The agonists described herein specifically recognize ROR-1 protein, found in lymphoma samples but not expressed in cells of normal adults, and therefore can be used for detecting and/or neutralizing these biomolecules, and/or blocking their interactions with other biomolecules, in vitro or in vivo. Examples of such ROR-1 agonists include antibodies, small molecule inhibitors, antisense RNA, and siRNA.

While under no obligation to provide a mechanism of action, it is thought that ROR-1 can serve as a receptor for Wnt5a to trigger the NF-kappa B pathway, which pathway is implicated in oncogenesis. See e.g. Example 12. Thus, the ROR-1 gene, which plays a role in disease pathogenesis and/or progression, encodes a protein that can be targeted by immune therapy for patients with a ROR-1 cancer.

Antibodies

In certain therapeutic embodiments, the selected antibody will typically be an anti-ROR-1 antibody, which may be administered alone, or in combination with, or conjugated to, one or more combinatorial therapeutic agents. When the antibodies described herein are administered alone as therapeutic agents, they may exert a beneficial effect in the subject by a variety of mechanisms. In certain embodiments, monoclonal antibodies that specifically bind ROR-1 are purified and administered to a patient to neutralize one or more forms of ROR-1, to block one or more activities of ROR-1, or to block or inhibit an interaction of one or more forms of ROR-1 with another biomolecule.

The immunotherapeutic reagents of the invention may include humanized antibodies, and can be combined for therapeutic use with additional active or inert ingredients, e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, and optionally with adjunctive or combinatorially active agents such as anti-inflammatory ant anti-fibrinolytic drugs.

In other embodiments, therapeutic antibodies described herein are coordinately administered with, co-formulated with, or coupled to (e.g., covalently bonded) a combinatorial therapeutic agent, for example a radionuclide, a differentiation inducer, a drug, or a toxin. Various known radionuclides can be employed, including 90Y, 123I, 125I, 131I, 186Re, 188Re, and 211At. Useful drugs for use in such combinatorial treatment formulations and methods include methotrexate, and pyrimidine and purine analogs. Suitable differentiation inducers include phorbol esters and butyric acid. Suitable toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein. These combinatorial therapeutic agents can be coupled to an anti-ROR-1 antibody either directly or indirectly (e.g., via a linker group). A direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, on one may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide) on the other. Alternatively, it may be desirable to couple a combinatorial therapeutic agent and an antibody via a linker group as a spacer to distance an antibody from the combinatorial therapeutic agent in order to avoid interference with binding capabilities. A linker group can also serve to increase the chemical reactivity of a substituent on an agent or an antibody, and thus increase the coupling efficiency. It will be further evident to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, Ill.), may be employed as a linker group. Coupling may be affected, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues.

Where a therapeutic agent is more potent when free from the antibody portion of the immunoconjugates described herein, it may be desirable to use a linker group which is cleavable during or upon internalization into a cell. A number of different cleavable linker groups have been described. The mechanisms for the intracellular release of an agent from these linker groups include cleavage by reduction of a disulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), by irradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, to Senter et al.), by hydrolysis of derivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serum complement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, to Rodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No. 4,569,789, to Blattler et al.) It may also be desirable to couple more than one agent to an anti-ROR-1 antibody. In one embodiment, multiple molecules of an agent are coupled to one antibody molecule. In another embodiment, more than one type of agent may be coupled to one antibody. Regardless of the particular embodiment, immunoconjugates with more than one agent may be prepared in a variety of ways. For example, more than one agent may be coupled directly to an antibody molecule, or linkers which provide multiple sites for attachment can be used. Alternatively, a carrier can be used.

A variety of routes of administration for the antibodies and immunoconjugates may be used. Typically, administration is intravenous, intramuscular, or subcutaneous.

It will be evident that the precise dose of the antibody/immunoconjugate will vary depending upon such factors as the antibody used, the antigen density, and the rate of clearance of the antibody. A safe and effective amount of an anti-ROR-1 agent is, for example, that amount that would cause the desired therapeutic effect in a patient while minimizing undesired side effects. Generally, a therapeutically effective amount is that sufficient to promote production of one or more cytokines and/or to cause complement-mediated or antibody-dependent cellular cytotoxicity. The dosage regimen will be determined by skilled clinicians, based on factors such as the exact nature of the condition being treated, the severity of the condition, the age and general physical condition of the patient, and so on.

siRNA

In certain therapeutic embodiments, the ROR-1 agonist is siRNA. The levels of ROR-1 can be down-regulated by RNA interference by administering to the patient a therapeutically effective amount of small interfering RNAs (siRNA) specific for ROR-1. siRNA specific for ROR-1 can be produced commercially from a variety of sources, such as Ambion (Austin, Tex.). The siRNA can be administered to the subject by any means suitable for delivering the siRNA to the blood. For example, the siRNA can be administered by gene gun, electroporation, or by other suitable parenteral or enteral administration routes, such as intravitreous injection.

RNA interference is the process by which double stranded RNA (dsRNA) specifically suppresses the expression of a gene bearing its complementary sequence. Suppression of the ROR-1 gene inhibits the production of the ROR-1 protein. Upon introduction, the long dsRNAs enter a cellular pathway that is commonly referred to as the RNA interference (RNAi) pathway. First, the dsRNAs get processed into 20-25 nucleotide (nt) small interfering RNAs (siRNAs) by an RNase III-like enzyme called Dicer (initiation step). Then, the siRNAs assemble into endoribonuclease-containing complexes known as RNA-induced silencing complexes (RISCs), unwinding in the process. The siRNA strands subsequently guide the RISCs to complementary RNA molecules, where they cleave and destroy the cognate RNA (effecter step). Cleavage of cognate RNA takes place near the middle of the region bound by the siRNA strand. Preferably, the siRNA comprises short double-stranded RNA from about 17 nucleotides to about 29 nucleotides in length, preferably from about 19 to about 25 nucleotides in length, that are targeted to the target mRNA.

As an example, an effective amount of the siRNA can be an amount sufficient to cause RNAi-mediated degradation of the target ROR-1 mRNA, or an amount sufficient to inhibit the progression of a lymphoma in a subject. One skilled in the art can readily determine an effective amount of the siRNA of the invention to be administered to a given subject by taking into account factors such as the size and weight of the subject; the extent of the neovascularization or disease penetration; the age, health and sex of the subject; the route of administration; and whether the administration is regional or systemic. Generally, an effective amount of siRNA comprises an intercellular concentration of from about 1 nanomolar (nM) to about 100 nM, preferably from about 2 nM to about 50 nM, more preferably from about 2.5 nM to about 10 nM. It is contemplated that greater or lesser amounts of siRNA can be administered.

The siRNA can be targeted to any stretch of approximately 19-25 contiguous nucleotides in any of the ROR-1 mRNA target sequences. Target sequences can be selected from, for example, the sequence of ROR-1, Genebank accession number: NM—005012. Searches of the human genome database (BLAST) can be carried out to ensure that selected siRNA sequence will not target other gene transcripts. Techniques for selecting target sequences for siRNA are given, for example, in Elbashir et al. ((2001) Nature 411, 494-498). Thus, the sense strand of the present siRNA comprises a nucleotide sequence identical to any contiguous stretch of about 19 to about 25 nucleotides in the target mRNA of ROR-1. Generally, a target sequence on the target mRNA can be selected from a given cDNA sequence corresponding to the target mRNA, preferably beginning 50 to 100 nt downstream (i.e., in the 3′ direction) from the start codon. The target sequence can, however, be located in the 5′ or 3′ untranslated regions, or in the region nearby the start codon.

Antisense

In certain therapeutic embodiments, the ROR-1 agonist is an antisense oligonucleotide. The levels of ROR-1 can be down-regulated by administering to the patient a therapeutically effective amount of an antisense oligonucleotide specific for ROR-1 mRNA. The antisense oligonucleotide specific for ROR-1 mRNA may span the region adjacent to the initiation site of ROR-1 translation.

An effective amount of the antisense oligonucleotide specific for ROR-1 mRNA as isolated in a purified form may is generally that amount capable of inhibiting the production of ROR-1 or reducing the amount produced or the rate of production of ROR-1 such that a reduction in symptoms of lymphoma occurs. Antisense oligonucleotides can be administered via intravitreous injection at a concentration of about 10 μg/day to about 3 mg/day. For example, administered dosage can be about 30 μg/day to about 300 μg/day. As another example, ROR-1 antisense oligonucleotide can be administered at about 100 μg/day. Administration of antisense oligonucleotides can occur as a single event or over a time course of treatment. For example, ROR-1 antisense oligonucleotides can be injected daily, weekly, bi-weekly, or monthly. Time course of treatment can be from about a week to about a year or more. In one example, ROR-1 antisense oligonucleotides are injected daily for one month. In another example, antisense oligonucleotides are injected weekly for about 10 weeks. In a further example, ROR-1 antisense oligonucleotides are injected every 6 weeks for 48 weeks.

Vaccines

As will be clear from the description herein of anti-ROR-1 antibody, the present invention also provides for use of ROR-1 in vaccines against diseases, such as a lymphoma, e.g., CLL, that involve the expression of ROR-1. Because normal adult tissues do not appear to express ROR-1, it represents a tumor-specific antigen that can be targeted in active immune therapy. For example, the levels of ROR-1 can be down-regulated by administering to the patient a therapeutically effective amount of a ROR-1 polynucleotide or polypeptide that produces in animals a protective or therapeutic immune response against ROR-1 and the effects of its expression. The vaccines can include polynucleotides or polypeptides. Methods of using such polynucleotides and/or polypeptides include use in vaccines and for generating antibodies against the polypeptides, such as those expressed by the polynucleotides. The polynucleotides can be a ROR-1 gene, or a variant or fragment thereof. The polypeptides can be a ROR-1 protein, or a variant or fragment thereof. In certain aspects, the ROR-1 polynucleotide fragment can be a fragment comprising a fragment of the ROR-1 gene. Such polynucleotide fragments can be comprised by a vector. A cell can be transformed and/or transfected by such polynucleotides and vectors and in certain aspects, the polynucleotides and vectors can express polypeptides of the invention. Typically the vaccine composition includes a pharmaceutically acceptable carrier or diluent. The teachings of U.S. Pat. No. 6,287,569 to Kipps et al., incorporated herein by reference in its entirety, can readily be adapted by those of skill in the art to create the vaccines of the present invention.

When describing a vaccine, a “polynucleotide variant” refers to any degenerate nucleotide sequence. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. For example, a variant polynucleotide consisting of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and 99% to the polynucleotide consisting of ibpA. A “polynucleotide fragment” of a ROR-1 polynucleotide is a portion of a ROR-1 polynucleotide that is less than full-length and comprises at least a minimum length capable of hybridizing specifically with a native ROR-1 polynucleotide under stringent hybridization conditions. The length of such a fragment is preferably at least 15 nucleotides, more preferably at least 20 nucleotides, and most preferably at least 30 nucleotides of a native ibpA polynucleotide sequence. A “polypeptide variant” refers to a polypeptide of differs in amino acid sequence from the ibpA polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. Finally, a “polypeptide fragment” refers to any polypeptide of a portion of a ibpA polypeptide that is less than full-length (e.g., a polypeptide consisting of 5, 10, 15, 20, 30, 40, 50, 75, 100 or more amino acids of a native ROR-1 protein), and preferably retains at least one functional activity of a native ROR-1 protein.

DNA Vaccines for ROR-1

Polypeptides with Arg at their N-terminus have a shorter half-life in the cytosol than those with a Met residue, provided that the polypeptide has a lysine residue to function as an ubiquitin acceptor site, spaced within 20 amino acids of the N-terminus. Plasmids encoding antigens targeted for rapid degradation by the proteasome are more effective than plasmids encoding the native protein in inducing CTL responses against cells expressing the target antigen.

Vectors have been constructed that encode a chimeric ROR1 protein with ubiquitin located at the amino terminus separated from ROR1 by an intervening codon for Met, and one with a codon for the destabilizing amino acid Arg and an in-frame insert of a segment of lacI. This segment contains a lysine residue spaced optimally from the N-terminus. Both constructs contain a sequence from the ubiquitine gene (SEQ ID NO: 6), followed by methionine or arginine sequence, followed by a LacI sequence (SEQ ID NO 7), and finally followed by the ROR-1 cDNA sequence (SEQ ID NO: 8). As detailed further in Example 16, the constructs are useful in ROR-1 DNA vaccines, with the arginine construct being expected to cause rapid degradation of the protein and thus a more predominant cellular immune response.

Many embodiments of the invention are provided through well known protocols established in the art. For example, the following references provide multiple protocols which may be adapted for use with anti-ROR-1 antibody: Vernon, S. K., Lawrence, W. C., Long, C. A., Cohen, G. H., and Rubin, B. A. Herpesvirus vaccine development: Studies of virus morphological components. In New Trends and Developments in Vaccines, ed. by A. Voller and H. Friedman. Chapter 13, pp. 179-210. MTP Press, Ltd., Lancaster (1978); Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, N.Y., (“Sambrook”); and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (e.g., current through 1999, e.g., at least through supplement 37) (“Ausubel”)), each of which are incorporated herein by reference in its entirety. With respect to vaccine technologies, U.S. Patent Application Nos. 20040253240 and 20030124141, are incorporated herein by reference in their entirety. These references also provide one of skill in the art instructions how to make and use the polynucleotides and polypeptides of the present invention for active and passive vaccines. Those of skill in the art will readily recognize how to adapt the disclosures of these references to the present polynucleotides and polypeptides of the present invention.

Kits

In carrying out various assay, diagnostic, and therapeutic methods of the invention, it is desirable to prepare in advance kits comprises a combination of an anti-ROR-1 antibody described herein with other materials. For example, in the case of sandwich enzyme immunoassays, kits of the invention may contain a monoclonal antibody that specifically binds ROR-1 optionally linked to an appropriate carrier, a freeze-dried preparation or a solution of an enzyme-labeled monoclonal antibody which can bind to the same antigen together with the monoclonal antibody or of a polyclonal antibody labeled with the enzyme in the same manner, a standard solution of purified ROR-1, a buffer solution, a washing solution, pipettes, a reaction container and the like. In addition, the kits optionally include labeling and/or instructional materials providing directions (i.e., protocols) for the practice of the methods described herein. While the instructional materials typically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

Having described the invention in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the invention defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustrate the present invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the invention, and thus can be considered to constitute examples of 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 that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 Demonstration of Production of Anti-Adenovirus Antibody

Chronic lymphocytic leukemia (CLL) CLL cells were transduced with replication-defective adenovirus encoding CD154 (Ad-CD154). The seven patients of the study all had progressive intermediate or high-risk CLL by the modified Rai criteria. All patients had performance status of 0 to 2, life expectancy of more than 3 months, and normal renal, hepatic, and pulmonary function on study entry. Ad-CD154 were prepared and transduced into CLL cells as described in Wierda et al. (2000) Blood 96, 2917-24; and Cantwell et al. (1996) Blood 88, 4676-83. 3-6×108 transduced CLL cells were injected biweekly 5 times in 6 patients except for one patient. Sera, collected sequentially during the studies from these 6 patients, was examined. Before treatment 4 out of 6 patients had hypogammaglobulinemia and residual 2 patients also have relatively low titers of immunoglobulins. After completion of therapy total IgG and IgM were slightly increased. (IgG; 656±297 to 940±487 p=0.04, IgA; 72±63 to 69±61 p=0.4, IgM; 38±21 to 74±48 p=0.07) (FIG. 1a).

The antibody response was measured against the recombinant adenovirus used to transduce the CLL cells. Five of six patients had a vigorous polyclonal antibody response to adenovirus antigens following treatment (FIG. 1b). This response initially involved antibodies of the IgM class, and then subsequently antibodies of the IgG and IgA classes, but not IgE (FIG. 1c and not shown). On average, 50-fold, 60-fold, or nearly 1,000-fold increases in the titers of IgM, IgA, or IgG anti-adenovirus antibodies were observed, respectively. The IgG response involved antibodies of IgG1 and IgG3 isotypes (FIG. 1c), which primarily are observed in Th1-type immune responses. Moreover, no significant increases were observed in anti-adenovirus antibodies of the IgG4 isotype (data not shown), which typically are observed in Th2-type immune responses. In compared with this vigorous response, the increases in the titers of anti-tetanus toxin antibodies were not obvious unless patients received subsequent booster immunizations with tetanus toxoid. In addition, development of autoantibodies to red cells, platelets, or the human CD154 molecule following treatment was not observed.

Example 2 Flow Cytometry Analysis of Anti-CLL Activities

Anti-CLL activities were determined by flow cytometry. Peripheral blood mononuclear cells (PBMC) from IgG negative CLL case or healthy donor were incubated with one-fifth diluted serum from the patient or healthy donor, and bound IgG was detected by mouse anti-human IgG antibody (Pharmingn). B cells (CD19+CD3−) were gated using anti-CD19 antibodies conjugated APC and anti-CD3 antibody conjugated with FITC.

Example 3 ROR-1 Anti-Sera Production

Anti-ROR-1 mouse sera by means of DNA vaccination with ROR-1 expression vector. Eight-week old Balb/c female mice were injected intradermally with 100 μg of ROR-1 cDNA (Origene) with 50 μg of GM-CSF and CD154 expression vector as adjuvants. After 3 courses of injection, sera was collected from the mice. Chinese hamster ovary cell (CHO) with or without transfection with ROR-1 cDNA cloned into pcDNA3 vector by lipofectamine 2000 (Invitrogen) was used to determine the titer of anti-ROR-1 antibody in serum. Bound antibody from immunized mice was detected by flow cytometry using anti-mouse antibody with fluorescence (Pharmingen). To distinguish the untransfected CHO from transfectants in the mixture, it was stained with PKH26 (Sigma) according to the manufacture's protocol. Anti-ROR-1 activity was determined by incubating CHO transfectants and serum from patient followed by detection with anti-human Ig labeled with fluorescence (Southern Biotech).

Example 4 ELISA

To produce recombinant ROR-1 protein, its extracellular region was cloned into the pcDNA3-zeocin vector encoding rabbit IgG Fc region in frame. Stable CHO transfectant (CHO-ROR-1rIg) was made with this vector, and was adapted to suspension culture using IMGX II medium (HyClone). Suspended CHO-ROR-1rIg was cultured in ProCHO-5 medium, and recombinant ROR-1rIg was purified using protein A sepharose (Pierce).

5 μg/ml protein or 108/ml adenovirus was absorbed 96 well plate overnight at 4° C. After washing and blocking with 2% BSA/PBS, serum dilutions were added and incubated for 1 hour at room temperature. Goat anti-human Ig, IgG, IgA, IgM, IgG1, IgG2, IgG3 and IgG4 conjugated with horseradish peroxidase (HRP) or alkaline phosphatase (AP) (Southern Biotechnology, Birmingham, Ala.) were used as secondary antibody. TMB (KPL, Gaithersburg, Md.) or pNPP (Sigma) was used for substrate for HRP and AP respectively. All experiments were done duplicate and were shown the average.

Example 5 Analysis of Microarray Data

Gene expression profiles of normal human tissues were obtained from the data series GSE803 of Gene Expression Omnibus (GEO) database. The gene set of CLL signature genes were made according to the published papers Klein et al. (2001) J Exp Med 194, 1625-38; Rosenwald et al. (2001) J Exp Med 194, 1639-47. The data were clustered and visualized with GeneSpring software (Silicon Genetics).

Example 6 Immunoblotting

Total cell lysates were made by incubation cells in a lysis buffer containing 1% Triton X-100, 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 40 mM glycerophosphate, 1 mM sodium orthovanadate, with complete protease inhibitor mix (Roche). Cell lysates were separated 7.5% or 5-15% gradient SDS-PAGE and blotted on Immobilon-P membrane (Millipore). For immunoblot, rabbit (Cell signaling) or goat (R&D) anti-ROR-1 antibodies were used followed with anti-rabbit or anti-goat antibodies conjugated with HRP (Santa Cruz). Conditioned medium of culture with CHO with or without transfection with HA-tagged Wnt5a cDNA (Upstate) was incubated with 1 μg of ROR-1rIg or rabbit IgG followed by immunoprecipitation with anti-HA matrix (Roche) or protein A/G agarose (SantaCruz). Bound proteins were immunoblotted with anti-HA (Roche) or anti-rabbit Ig antibody.

Example 7 Reporter Assay

A reporter assay was performed as described in Lu et al. (2004) Proc Natl Acad Sci USA 101, 3118-23. Briefly, HEK293 cells were transfected in 12-well plates by using FuGENE (Roche, Mannheim, Germany), and 0.5 μg of reporter plasmid, 0.1-0.2 μg of the control plasmid pCMXβ-gal, 100-200 ng of the various expression plasmids, and carrier DNA pBluescriptKSII, for a total of 1 μg per well. The luciferase values were normalized for variations in transfection efficiency by using the β-galactosidase internal control, and are expressed as fold stimulation of luciferase activity, compared with the designated control cultures. All of the transfection results are representative of a minimum of three independent transfections.

Example 8 Induction of Humoral Immunity Against CLL Cell

The production of anti-adenovirus antibody suggests the induction of humoral immunity against the CLL cell itself. Allogeneic CLL cells were incubated with serum from patient before and after treatment. Antibody binding was checked by flow cytometry.

Results showed that the sera from 3 patients after Ad-CD154 therapy had the reactivity against CLL B cells compared with the sera before therapy (FIG. 2a). The shift of the histograms were reproducible with another 3 CLL B cells, and it was not detectable against B cells from healthy donors (FIG. 2b). This data suggests a TAA(s) may exist on the surface of CLL cells in a hidden fashion from surveillance of immunity, becoming immunogenic after CLL received the immune-costimulatory molecules.

The microarray analyses of CLL samples identified the relatively small number of genes that are differentially expressed in CLL cells in compared with normal B cell subsets and another types of B cell malignancies. Klein et al. (2001) J Exp Med 194, 1625-38; Rosenwald et al. (2001) J Exp Med 194, 1639-47. These CLL signature genes are candidates for TAAs of CLL. The expressions of these genes were examined in normal human tissues because where there is an abundant expression in normal tissue, antibody production against such a gene cannot occur in vivo. The expression profiles of CLL signature genes in normal adult tissues wad determined (data not shown). Genes that had low expressions in all tissues were spotlighted. Attention was directed to receptor tyrosine kinase ROR-1 gene, because it is a probable cell surface molecule and is expressed mainly in developing cells. Yoda et al. (2003) J Recept Signal Transduct Res 23, 1-15; Al-Shawi et al. (2001) Dev Genes Evol 211, 161-71; Matsuda et al. (2001) Mech Dev 105, 153-6 (2001).

Example 9 Immunoblot

FIG. 3 depicts an immunoblot demonstrating that the anti-Ror1 mAb (designated 4A5) can immune precipitate the Ror1 protein from cells made to express human Ror1 (e.g. Chinese Hamster Ovary (CHO)) cells or chronic lymphocytic leukemia (CLL) cells. Prior antibodies to Ror1 were not mAbs, were generated against peptides to Ror1, are of low affinity, and cannot immune precipitate the Ror1 protein. As such, the 4A5 mAb can be used to detect and/or isolate the Ror1 protein, which could have diagnostic, treatment, and/or investigative value.

Example 10 ROR-1 Expression in CLL B Cells

To confirm the ROR-1 protein expression in CLL B cells, immunoblot analysis, as described above, using anti-ROR-1 antibody was performed. Results showed that the bands at the level of 128 kD were detected in peripheral blood or splenocytes from CLL patients (FIG. 4a). The size is compatible with the reported murine ROR-1 and bigger than deduced size of 101 kD from amino acid sequence without putative leader sequence probably due to the glycosylation29. This band could be detected neither in samples of peripheral blood from healthy donor nor splenocytes from idiopathic thrombocytopenia purpura patient. ROR-1 protein was detectable also in some Burkitt's B cell lines at the same molecular weight (FIG. 4b).

Example 11 Cell Surface Localization of ROR-1 Protein in CLL B Cells

Cell surface localization of ROR-1 protein in CLL B cells was confirmed via flow cytometry. Anti-ROR-1 mouse sera, produced by means of DNA vaccination with ROR-1 expression vector, as described above, was reacted with CHO transfected with ROR-1 (CHO-ROR-1) but not with CHO parental cell (FIG. 4c). Using this anti-serum, ROR-1 expression was detected on cell surface of all CLL samples examined (n=8) but not on PBMC from healthy donors (n=3) (FIG. 4d).

Example 12 Induction of Anti-ROR-1 Antibody by Ad-CD154 Therapy

To confirm that the antibody against ROR-1 is included in the antibodies against CLL cells induced by Ad-CD154 therapy, the sera from patients was reacted with CHO and CHO-ROR-1 shown as FIG. 4c. Results showed that although serum from healthy donor or patient before treatment contained same reactivity against CHO and CHO-ROR-1, sera from patient after Ad-CD154 therapy contained more Ig reacted with CHO-ROR-1 than with CHO (FIGS. 5a and b).

Further verification of the induction of anti-ROR-1 antibody by Ad-CD154 therapy was established with an ELISA assay using the recombinant extracellular domain of ROR-1 fused with rabbit IgG Fc (FIG. 6a). Results showed that anti-ROR-1 antibody was clearly identified in 4 patients (#2,5,6,7) after Ad-CD154 therapy. The remaining one patient (#3) also had a weak anti-ROR-1 reaction although one patient (#4) did not get anti-ROR-1 antibody by this therapy. This #4 patient was profound hypogammaglobulinemia and was totally unresponsive to this therapy with no decrease of white blood cell count (data not shown). Thus, all responsive patient to Ad-CD154 had induction of anti-ROR-1 antibody after completion of therapy. In these patients, anti-ROR-1 antibody was not obvious before Ad-CD154 therapy. Although three patients (#5,6,7) had some reactivity also against rabbit IgG, this reactivity was also detected before therapy (FIG. 6c). Collectively ROR-1 was expressed on CLL B cells restrictedly and could induce humoral immunity by means of immune-gene therapy.

Example 13 ROR-1 Activation of Intracellular Machinery Associated with Development and Progression of CLL

To demonstrate that ROR-1 can activate intracellular machinery associated with development or progression of CLL, the influence of exogenous ROR-1 expression on the reporter gene regulating various transcription factors in HEK293 cells was examined. Various Wnt family members were co-transfected, as ROR-1 has a cystein-rich domain, which is shared between frizzled receptors and can bind with Wnt family members.

Results showed that the expression of ROR-1 with any Wnt factor did not activate T-cell transcription factor (TCF) (FIG. 7a, data not shown), suggesting that ROR-1 does not signal via the canonical Wnt-signaling pathway. ROR-1 could not activate nuclear factors of activated T cells (NFAT), or AP-1 dependent gene expression (FIG. 7a). However, it was observed that co-expression of ROR-1 in HEK293 cells with Wnt5a, but not with any other Wnt factor, induced activation of NF-κB (FIG. 7b). Induction of NF-κB was dose dependent on expression of ROR-1 and Wnt5a, but independent of expression of LPR5/6 that ordinarily serve as co-receptors for the frizzled family of Wnt receptors (data not shown). Recombinant extracellular region of ROR-1 could bind with Wnt5a in vitro (FIG. 7c). This data suggests non-canonical Wnt member, Wnt5a may be the ligand of ROR-1 and induce the activation signaling in cells.

Example 14 Lymphoma Cell Isolation and Purification

Staining of CLL Cells from Patients #1, 2, or 3 with 4A5 mAb

As depicted in FIG. 8, the number of the CLL patient is indicated at the left-hand margin. Each panel depicts the staining of CLL with Alexa-647-conjugated 4A5 mAb (blue histogram) versus an Alexa-647-conjugated isotype control mAb (red histograms). In the first column is the staining of total peripheral blood mononuclear cells, in the middle column is the staining of the CD19+(total B cells), and in the far right column is the staining of cells that express both CD19 and CD5 (CLL cells), indicated at the columns' bottoms.

Staining of Cells from Normal Donors #1, 2, or 3 with 4A5 mAb

As depicted in FIG. 9, the number of the normal donor (NORM) is indicated at the left-hand margin. Each panel depicts the staining of cells with Alexa-647-conjugated 4A5 mAb (blue histogram) versus an Alexa-647-conjugated isotype control mAb (red histograms). In the first column is the staining of total peripheral blood mononuclear cells, in the middle column is the staining of the CD19+ (total B cells), and in the far right column is the staining of cells that express both CD19 and CD5, as indicated at the bottom of each column.

Staining of Cells from an Exceptional Normal Donors

Recent studies indicate that close to 4% of adults over the age of 40 might have low numbers of cells similar to CLL cells in the peripheral blood. Moreover, over 11% of normal donors who have first degree relatives with CLL might have such cells in the peripheral blood. In FIG. 10, it is shown that anti-Ror1 mAb 4A5 can detect an occasional normal donor with Ror1 positive cells. Each panel depicts the staining of cells with Alexa-647-conjugated 4A5 mAb (blue histogram) versus an Alexa-647-conjugated isotype control mAb (red histograms). In the first column is the staining of total peripheral blood mononuclear cells, in the middle column is the staining of the CD19+ (total B cells), and in the far right column is the staining of cells that express both CD19 and CD5, as indicated at the bottom of each column. As can be noted from this figure, the Ror1 positive cells co-express CD5 and CD19, a phenotype common with CLL cells.

Staining of CLL Cells in the Marrow

In FIG. 11, numbers corresponding to a CLL patient are provided at the left-hand margin. Each panel depicts the staining of cells with Alexa-647-conjugated 4A5 mAb (blue histogram) versus an Alexa-647-conjugated isotype control mAb (red histograms). In the first column is the staining of total marrow mononuclear cells, in the middle column is the staining of the CD19+ (total B cells), and in the far right column is the staining of cells that express both CD19 and CD5 (CLL cells), as indicated at the bottom of each column.

Staining of CLL Cells in the Marrow

The proportion of cells that express Ror1, as detected by the mAb 4A5, are indicated in FIG. 12. Each dot represents the proportion of cells from a single donor. The percent of cells scoring positive is indicated by the y-axis. The left hand panel provides the percent lymphocytes (as per light scatter) that stain with 4A5 mAb. The right panel provides the percent of CD5+CD19+ B cells that stain with 4A5. The left panel provides the percent of lymphocytes that stain with 4A5 in samples obtained from the blood normal donors (far left), the marrow of patients with CLL (middle), or blood of patients with CLL (far right).

Example 15 Magnetic Bead Detection and Isolation of Lymphoma Cells

Lymphoma cells can be isolated and purified using the following procedure:

    • 1. Stain CLL cells with PKH67
    • 2. Titrate CLL cells in normal PBMCs (10% to 0.1%)
    • 3. Stain cells with:
    • a. Iso-Alexa647, CD5, CD19
    • b. 4A5-Alexa647, CD5, CD19
    • 4. Incubate for 20 min on ice followed by a wash 2× with PBS-0.5% BSA
    • 5. Add magnetic beads(Miltenyi)to cells; Incubate 15 min on ice; Wash 1× with PBS-0.5% BSA)
    • 6. Add column to magnet; Wash 1× with 3 ml PBS-0.5% BSA
    • 7. Add this mixture to pre washed column; Wash unbound cells 3× with 3 ml PBS-0.5% BSA; (unbound fraction=4A5 NEG)
    • 8. Remove column from magnet; Add 5 ml PBS-0.5% BSA; (bound fraction=4A5 POS)

Detection of CLL Cells Admixed with Normal Lymphocytes

4A5+ CLL cells admixed with the lymphocytes from normal donors are shown in FIG. 13. CLL cells were first stained with PKH67, which labeled them bright green (as observed on the x axis), allowing for their detection after being admixed with normal lymphocytes. The stained CLL cells were mixed with the lymphocytes of a normal donor and then the mixture was stained with an Alexa-647-conjugated isotype control mAb (ISO) Alexa-647-conjugated 4A5, allowing for detection of the red fluorescence seen on the y-axis.

Each panel represents a different mixture of cells stained with either the isotype control mAb or 4A5, as indicated in the key, which refers to the number in each panel of the figure. Those samples stained with the isotype control mAb are indicated by the term “Iso”, those samples stained with 4A5 are indicated. The percent preceding the CLL is the percent at which the CLL cells are represented in the mixture. As seen from this figure, the 4A5 mAb does not stain normal lymphocytes, allowing for detection of minute proportions of CLL cells that are labeled green.

Isolation of CLL Cells Admixed with Normal Lymphocytes

Isolated 4A5+ CLL cells admixed with the lymphocytes from normal donors are indicated in FIG. 14. CLL cells were stained, mixed with normal lymphocytes at various ratios, and then stained with fluorochrome-conjugated 4A5 mAb, as in Slide #6. Each panel represents analyses of cells isolated from different mixtures of CLL cells with normal lymphocytes, as indicated in the key, which refers to the number in each panel of the figure. The percent preceding the CLL is the percent at which the CLL cells are represented in the mixture.

As seen from this figure, the 4A5 mAb does not stain normal lymphocytes, allowing for detection of minute proportions of CLL cells that are labeled green. As can be seen in these panels, this method can isolate fairly pure populations of CLL cells from mixtures of CLL cells with normal lymphocytes in which the CLL cells constitute only a small fraction of the total cells.

Example 16 Detection of ROR-1 Antibody in Cancer but not Normal Cells

To evaluate ROR-1 surface expression in the human cell lines listed in the Table below (Table 1), CHO cells were used as a negative control and CHO-ROR-1 cells as a positive control for flow cytometry. The ROR-1 antibody was the 4A5 mAb. The control mAb was a conjugated isotype IgG2b mAb.

As shown in FIG. 15, the following cell lines stained brightly with the 4A5 mAb, confirming ROR-1 expression: EW536, CLL, 786-0, HCT116, HT29, SW620, MDA-MB-231, MDA-MB-431, and MDA-MB-468. CHO, MOLT-4, SW948, MCF-7, and SKBR3 were negative for ROR-1 expression, indicating preferential ROR-1 expression in this population among adenocarcinoma and lymphoma.

In immunoblot studies of the same adult cancer tissues (using a ROR-1 antibody raised against ROR-1 peptide), the same cancer tissues reacted to indicate ROR-1 expression (FIGS. 16A and 16B). Using the A5A mAb, immuno precipitation studies confirmed that ROR-1 was not found on normal tonsil cells of CHO, but is strongly expressed in CLL, B cell lymphoma, and breast adenocarcinomas, less so in colon adenocarcinoma (FIGS. 16C and 16D).

Example 17 ROR-1 DNA Vaccine Constructs

Vectors were constructed to encode the chimeric ROR1 protein with ubiquitin located at the amino terminus separated from ROR1 by an intervening codon for Met, and a separate vector with a codon for the destabilizing amino acid Arg and an in-frame insert of a segment of lacI. This segment contains a lysine residue spaced optimally from the N-terminus. To generate the constructs, ROR1 was PCR amplified from the pCMV6-XL-ROR1 vector (Origene) using primers that encoded for NotI and XbaI. The PCR product was gel-purified, cut with those restriction enzymes and ligated into a pcDNA3 subclone that contained the chimeric Ub-M-(lacI) or Ub-R-(lacI). The final construct contains ROR1 3′ of these sequences: Ub-M-ROR1 and Ub-R-ROR1.

Confirmation of ROR-1 Protein Expression by Cell-Free Assay:

The constructs were evaluated for their capacity to direct synthesis of the ROR-1 protein. For this in vitro transcription and translation was performed using the TNT Quick coupled Transcription/Translation System from Promega in the presence of biotinylated lysine-specific tRNA. A luciferase plasmid served as positive control for the reaction. Both constructs allowed for the expression of one predominant protein at the size of ROR-1.

To demonstrate ROR-1 protein expression in mammalian cells, P815 cells were transfected with Ub-M-ROR1 or Ub-R-ROR1 using the Amaxa transfection system according to manufacturer's instructions using program L13. The generation of such cells is described below.

Generation of P815 Cells Expressing ROR-1

To examine the magnitude of the immune response generated by the Ub-M-ROR-1 and Ub-R-ROR-1 DNA vaccine, CTL activity of splenocytes harvested from immunized mice will be assessed against the H-2d mastocytoma, P815, and P815 cells transfected to express human ROR-1. To generate P815 cells that stably expressed ROR1, P815 cells were transfected with pcDNA3-ROR-1, generated using the Amaxa transfection system. To select ROR-1 expressing cells, the cells were grown in G418 (400 μg/ml). Subsequently the cells were sub-cloned by limited dilation and analyzed for ROR-1 expression by flow cytometry. A stable P185 clone was generated that expresses ROR-1 (FIG. 17; P815-ROR-1). This cell line will serve as target for CTL assays.

To generate stable transfectants, the cells were subsequently cultured under selection pressure in the presence of 400 μg/ml G418. G418-resistant cells were cloned by limiting dilution. To evaluate the relative intracellular stability of the transgene products in the transfected P815 cells, cells were cultured in the presence of a 26S proteasome inhibitor. P815 cells, and P815 cells stably transfected with the Ub-M-ROR1 or Ub-R—ROR1 constructs were incubated in 100 μM of the proteasome inhibitor LLnL (N-acetyl-L-leucinyl-L-leucinal-L-norleucinal) for 18 h. Lysates were prepared from the transfected cells and evaluated by Immunoblot for ROR1 expression.

As expected, in the absence of LLnL, only P815 cells transfected with the Ub-M-ROR-1, but not with Ub-R—ROR-1 expressed detectable ROR-1 protein. Non-transfected P815 cells did not express ROR-1. When both transfectants were cultured in the presence of LLnL a strong increase in ROR-1 expression was observed. These results show that ROR-1 expressed from the Ub-M-ROR-1 and more so from the Ub-R—ROR-1 constructs was degraded in the proteasome.

These constructs can be reasonably expected to induce antibody responses or anti-ROR1 CTL responses. To this end, cell based assays are useful to confirm the activity of candidate ROR-1 vaccines, to compare and contrast activity among candidates and with ROR-1 constructs that are not targeted for degradation. CTL activity is measurable using ROR1 expressing target cells and target cells without ROR1 as controls; e.g., in the P815 cells described.

TABLE 1 Descriptions Of The Various Cell Lines Used In FIG. 15 Name Source of Cells CHO Chinese hamster ovary cells CHO-ROR1 CHO cells transfected to express human Ror1 EW36 Endemic African Burkitt's lymphoma (a B cell lymphoma) MOLT4 Human T cell lymphoma CLL Human chronic lymphocytic leukemia 786-0 Human renal cell carcinoma cell line HCT116 Human colon adenocarcinoma cell line HT-29 Human colorectal adenocarcinoma cell line SW620 Human colon adenocarcinoma cell line SW948 Human colon cancer cell line MCF-7 Human, Caucasian, breast, adenocarcinoma MDA-MB-231 Highly aggressive human, Caucasian, breast, adenocarcinoma MDA-MB-431 Highly aggressive human, Caucasian, breast, adenocarcinoma MDA-MB-468 Highly aggressive human, Caucasian, breast, adenocarcinoma SKBR3 Human mammary carcinoma

Claims

1. A composition comprising a purified, isolated antibody directed against ROR-1, wherein the antibody binds ROR-1 with moderate to high affinity.

2. A composition according to claim 1, wherein the antibody is an anti-ROR-1 polyclonal antibody, a monoclonal antibody, or a functional antibody fragment.

3. A composition according to claim 2, wherein the antibody comprises a sequence expressed by a heavy chain sequence and at least one light chain sequence of SEQ ID NOs: 1-5.

4. A composition according to claim 2, wherein the antibody is a polyclonal antibody.

5. A composition according to claim 2, wherein the antibody is a monoclonal antibody.

6. A composition according to claim 2, wherein the antibody is a functional antibody fragment.

7. A composition according to claim 1, wherein the antibody is selected from the group consisting of whole antibody, humanized antibody, chimeric antibody, Fab fragment, Fab′ fragment, F(ab′)2 fragment, single chain Fv fragment and diabody.

8. A composition according to claim 1, wherein the antibody has an affinity to binding ROR-1 with a dissociation constant of below a Kd value selected from the group consisting of 10−6 mol/l, 10−7 mol/l, and 10−8 mol/l.

9. A composition according to claim 1, wherein the antibody is a detectably labeled antibody.

10. A composition according to claim 1, further comprising a pharmaceutically acceptable agent.

11. A method for detecting an amount of ROR-1 in a subject sample, the method comprising:

(a) contacting the subject sample with the anti-ROR-1 antibody of claim 1; and
(b) detecting immunoreactivity between the anti-ROR-1 antibody and ROR-1 in the sample.

12. A method according to claim 11, wherein the antibody is covalently attached to a detectable label.

13. A method according to claim 11, wherein the immunoreactivity detection is provided by immunoperoxidase staining, immunofluorescence, immunoelectronmicroscopy, or ELISA.

14. A method according to claim 11, further comprising

(c) correlating binding of the antibody to a standardized antibody binding profile, wherein such correlation provides quantitative value for total CPR in the sample.

15. A method for detecting a ROR-1 cancer, the method comprising: detecting the presence or quantity of ROR-1 protein in a subject sample.

16. A method according to claim 15, wherein the ROR-1 cancer is a lymphoma or adeno carcinoma.

17. A method according to claim 16, wherein the cancer is selected from the group consisting of CLL, small lymphocytic lymphoma, marginal cell B-Cell lymphoma, Burkett's Lymphoma, colon adenocarcinoma, colorectal adenocarcinoma, and breast adenocarcinoma.

18. A method according to claim 15, wherein detection of ROR-1 comprises the method of claim 10.

19. A method for treating a ROR-1 cancer in a subject, the method comprising:

administering to the subject in need thereof a therapeutically effective amount of a ROR-1 receptor agonist.

20. A method according to claim 19, wherein the ROR-1 cancer is a lymphoma or adenocarcinoma.

21. A method according to claim 19, wherein the lymphoma is selected from the group consisting of CLL, small lymphocytic lymphoma, marginal cell B-Cell lymphoma, and Burkett's Lymphoma, colon adenocarcinoma, and breast adenocarcinoma.

22. A method according to claim 19, wherein the ROR-1 receptor agonist is the anti-ROR-1 antibody of claim 1.

23. A method according to claim 22, wherein the antibody is administered in an amount of (i) about 0.05 mg to about 2.5 mg; (ii) about 0.1 mg to about 1 mg; or (iii) about 0.3 mg to about 0.5 mg.

24. A method according to claim 22, wherein the anti-ROR-1 antibody is a polyclonal antibody, a monoclonal antibody, or a functional antibody fragment.

25. A method according to claim 22, wherein the anti-ROR-1 antibody is selected from the group consisting of whole antibody, humanized antibody, chimeric antibody, Fab fragment, Fab′ fragment, F(ab′)2 fragment, single chain Fv fragment and diabody.

26. A method according to claim 19, wherein the ROR-1 receptor agonist is an antisense inhibitor of ROR-1.

27. A method according to claim 26, wherein the ROR-1 antisense inhibitor is administered in an amount of (i) about 10 μg/day to about 3 mg/day; (ii) about 30 μg/day to about 300 μg/day; or (iii) about 100 μg/day.

28. A method according to claim 19, wherein the ROR-1 receptor agonist is administered by injection, inhalation, orally, liposome, or retroviral vector.

29. A diagnostic method for evaluating the appearance, status, course, or treatment of a ROR-1 cancer in a subject, the method comprising:

(a) contacting a subject sample with the anti-ROR-1 antibody of claim 1; and
(b) detecting immunoreactivity between the anti-ROR-1 antibody and ROR-1 to determine presence or quantity of ROR-1 in the sample.

30. A method according to claim 29, wherein the antibody specifically binds to ROR-1.

31. A method according to claim 29, wherein a diagnostic criterion or value is determined based on an increase or decrease in an amount of ROR-1 in the subject compared to a control level(s) of ROR-1 in a normal subject or sample.

32. A method according to claim 29, wherein immunoreactivity detection is provided by immunoperoxidase staining, immunofluorescence, immunoelectronmicroscopy, or ELISA.

33. A method according to claim 29, wherein the antibody is an anti-ROR-1 polyclonal antibody, a monoclonal antibody, or a functional antibody fragment.

34. A method according to claim 33, wherein the antibody is selected from the group consisting of whole antibody, humanized antibody, chimeric antibody, Fab fragment, Fab′ fragment, F(ab′)2 fragment, single chain Fv fragment and diabody.

35. A kit to detect the presence of ROR-1 protein comprising the antibody of claim 1.

36. A vaccine composition comprising a polynucleotide encoding ROR-1 protein or a fragment or variant thereof, and a pharmaceutically acceptable carrier or diluent.

37. A vaccine composition comprising ROR-1 protein or a fragment or variant thereof, and a pharmaceutically acceptable carrier or diluent.

38. A method for protecting against the occurrence of diseases involving expression of ROR-1 in a subject, the method comprising:

administering to the subject in need thereof a polynucleotide encoding ROR-1 protein or a fragment or variant thereof in an amount effective to induce a protective or therapeutic immune response against ROR-1 in the subject, and a pharmaceutically acceptable carrier or diluent.

39. A method for protecting against the occurrence of diseases involving expression of ROR-1 in a subject, the method comprising:

administering to the subject in need thereof a ROR-1 protein or a fragment or variant thereof in an amount effective to induce a protective or therapeutic immune response against ROR-1 in the subject, and a pharmaceutically acceptable carrier or diluent.

40. A method for identifying or isolating ROR-1 protein in a sample, the method comprising:

(a) contacting the sample with the anti-ROR-1 antibody of claim 1; and
(b) detecting immunoreactivity between the anti-ROR-1 antibody and ROR-1 to determine presence or quantity of ROR-1 in the sample.

41. A method according to claim 40, wherein the ROR-1 antibody is conjugated to a magnetic bead.

42. A method for detecting minimal residual disease following treatment of a ROR-1 cancer, the method comprising:

(a) contacting the sample with the anti-ROR-1 antibody of claim 1; and
(b) detecting immunoreactivity between the anti-ROR-1 antibody and ROR-1 to determine presence or quantity of ROR-1 in the sample.

43. A method according to claim 42, wherein the ROR-1 antibody is conjugated to a magnetic bead.

44. A humanized ROR-1 antibody.

45. A precipitate comprising a ROR-1 antibody and a polypeptide selected from the group consisting of a ROR-1 protein, ROR-1 polypeptide fragment and ROR-1 polypeptide variant.

Patent History
Publication number: 20070207510
Type: Application
Filed: Feb 21, 2007
Publication Date: Sep 6, 2007
Applicant:
Inventors: Thomas Kipps (San Diego, CA), Tetsuya Fukuda (Kanagawa), Tomoyuki Endo (Hokkaido)
Application Number: 11/709,917
Classifications
Current U.S. Class: 435/7.230; 530/388.800
International Classification: G01N 33/574 (20060101); C07K 16/30 (20060101);