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  Pharmaceutical Patents  

 

Title:  Vaccines for treatment of lymphoma and leukemia
United States Patent: 
7,419,660
Issued: 
September 2, 2008

Inventors:
 Denney, Jr.; Dan W. (Redwood City, CA)
Assignee:
  Genitope Corporation (Fremont, CA)
Appl. No.:
 09/370,453
Filed:
 August 9, 1999


 

Executive MBA in Pharmaceutical Management, U. Colorado


Abstract

The present invention provides multivalent vaccines for the treatment of B-cell malignancies (e.g., lymphomas and leukemias). The present invention also provides methods for the production of custom vaccines, including multivalent vaccines for the treatment of immune cell tumors malignancies as well as methods of treating immune cell tumors using custom vaccines.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention provides methods for the production of cell lines containing amplified copies of recombinant DNA sequences. Because the amplified cell lines contain several different recombinant DNA sequences (e.g., the amplification vector, one or more expression vectors and optionally a selection vector) which are coordinately amplified, the cell lines are said to have co-amplified the input or exogenous DNA sequences. The methods of the present invention permit the efficient isolation of the desired amplified cell lines with a considerable savings in time relative to existing amplification protocols. The gene amplification methods of the present invention permit the production of custom vaccines, including multivalent vaccines, which are useful for the treatment of immune cell tumors (e.g., lymphomas and leukemias).

In one embodiment, the present invention provides a multivalent vaccine comprising at least two recombinant variable regions of immunoglobulin molecules derived from B-cell lymphoma cells, wherein said cells express at least two different immunoglobulin molecules, said immunoglobulin molecules differing by at least one idiotope. The invention is not limited by the context in which the recombinant variable regions are utilized; the variable regions may be present within an entire recombinant immunoglobulin (Ig) molecule, they may be present on Fab, Fab' or F(ab').sub.2 fragments (which may be generated by cleavage of the recombinant Ig molecule or they may be produced using molecular biological means) or they may be present on single chain antibody (Fv) molecules. In a preferred embodiment, the multivalent vaccine comprises at least two recombinant immunoglobulin molecules comprising said recombinant variable regions derived from said lymphoma cells.

In one embodiment, the immunoglobulin molecules comprising recombinant variable regions derived from a patient's lymphoma cells are covalently linked to an immune-enhancing cytokine. The linkage of the cytokine to the Ig molecule may be achieved by a variety of means known to the art including conventional coupling techniques (e.g., coupling with dehydrating agents such as dicyclohexylcarbodiimide (DCCI), ECDI and the like), the use of linkers capable of coupling through sulfhydryl groups, amino groups or carboxyl groups (available from Pierce Chemical Co., Rockford, Ill.), by reductive amination. In addition, the covalent linkage may be achieved by molecular biological means (e.g., the production of a fusion protein using an expression vector comprising a nucleotide sequence encoding the recombinant Ig operably linked to a nucleotide sequence encoding the desired cytokine).

The invention is not limited by the immune-enhancing cytokine employed. In a preferred embodiment, the cytokine is selected from the group consisting of granulocyte-macrophage colony stimulating factor, interleukin-2 and interleukin-4.

In one embodiment, the multivalent vaccines of the present invention comprise at least one pharmaceutically acceptable excipient. The invention is not limited by the nature of the excipient employed. The pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

In a preferred embodiment, the multivalent vaccine further comprises an adjuvant. When the vaccine is to be administered to a human subject, adjuvants approved for use in humans are employed (e.g., SAF-1, alum, etc.). The recombinant Ig proteins (including fragments of Ig proteins) which comprise the multivalent vaccine may be conjugated to a carrier protein such as KLH.

The present invention also provides a method of producing a vaccine for treatment of B-cell lymphoma comprising: a) providing: i) malignant cells isolated from a patient having a B-cell lymphoma; ii) an amplification vector comprising a recombinant oligonucleotide having a sequence encoding a first inhibitable enzyme operably linked to a heterologous promoter; iii) a eukaryotic parent cell line; b) isolating from the malignant cells nucleotide sequences encoding at least one V.sub.H region and at least one V.sub.L region, the V.sub.H and V.sub.L regions derived from immunoglobulin molecules expressed by the malignant cells; c) inserting the nucleotide sequences encoding the V.sub.H and V.sub.L regions into at least one expression vector; d) introducing the expression vector(s) and the amplification vector into the parent cell to generate one or more transformed cells; e) growing the transformed cell(s) in a first aqueous solution containing an inhibitor capable of inhibiting the inhibitable enzyme wherein the concentration of the inhibitor present in the first aqueous solution is sufficient to prevent growth of the parent cell line; and f) identifying a transformed cell capable of growth in the first aqueous solution, wherein the transformed cell(s) capable of growth expresses the V.sub.H and V.sub.L regions. In a preferred embodiment, the transformed cell capable of growth in the first aqueous solution contains an amplified number of copies of the expression vector(s) and an amplified number of copies of the amplification vector.

In another preferred embodiment, the nucleotide sequences encoding the V.sub.H and C.sub.L regions comprise at least two V.sub.H and at least two C.sub.L regions (in this manner, a multivalent vaccine is produced).

The method of the present invention is not limited by the nature of the means employed to introduce the vectors into the parent cell line. The art is well aware of numerous methods which allow the introduction of exogenous DNA sequences into mammalian cells, including but not limited to electroporation, microinjection, lipofection, protoplast fusion, liposome fusion and the like. In a preferred embodiment, the vectors are introduced into the parent cell line by electroporation.

The present invention is not limited by the nature of the cell line chosen as the parent cell line; a variety of mammalian cell lines may be employed including CHO cell lines and variants thereof, mouse L cells and BW5147 cells and variants thereof. The chosen cell line grow in either an attachment-dependent or attachment-independent manner. In a preferred embodiment, the parent cell line is a T lymphoid cell line; a particularly preferred T lymphoid cell line is the BW5147.G.1.4 cell line.

In another embodiment, the method of the present invention employs a parent cell line which contains an endogenous gene encoding a second inhibitable enzyme (e.g., the genome of the parent cell line contains an endogenous gene comprising a coding region encoding a second inhibitable enzyme which is operably linked to the promoter naturally linked to this coding region (i.e., the endogenous promoter for this gene). A contrast is made between the input or exogenous recombinant sequences encoding the first inhibitable enzyme and an endogenous gene encoding an inhibitable enzyme. The endogenous gene sequences will be expressed under the control of the endogenous promoter. Typically, the amplification vector will comprise a sequence encoding an inhibitable enzyme operably linked to a heterologous (i.e., not the endogenous) promoter. The sequences encoding the first and the second inhibitable enzyme may encode the same or a different enzyme. Furthermore, when the same enzyme is encoded by the two sequences (i.e., the recombinant and the endogenous sequences), these sequences may be derived from the same or a different source (i.e., the recombinant sequence may encode an enzyme isolated from a mouse cell and may introduced into a mouse cell line which contains an endogenous gene encoding the same enzyme; alternatively, the recombinant sequence may encode an enzyme derived from a different species than that of the parent cell line (e.g., the recombinant sequence may encode a rat DHFR and may be introduced into a parent mouse cell line which expresses the mouse DHFR). The amplifiable gene (or marker) and the selectable marker may be present on the same vector; alternatively, they may be present on two separate vectors.

In one embodiment the second inhibitable enzyme expressed by the parent cell line is selected from the group consisting of dihydrofolate reductase, glutamine synthetase, adenosine deaminase, asparagine synthetase.

In another embodiment, the method of the present invention the concentration of inhibitor present in the first aqueous solution (e.g., tissue culture medium) used to allow identification of the transformed cell(s) containing amplified copies of the amplification vector and amplified copies of the expression vector(s) is four-fold to six-fold the concentration required to prevent the growth of the parent cell line. It is well understood by those skilled in the art that only those sequences present on the amplification vector and expression vector(s) which are required for the expression of the inhibitable enzyme and the protein(s) of interest, respectively, need to be amplified. However, it is also well understood that any vector backbone sequences linked to the sequences required for expression of the inhibitable enzyme or protein(s) of interest may also be amplified (and typically are) during the co-amplification process.

In still another embodiment, the method of the present invention further comprises providing a selection vector encoding a selectable gene product (i.e., a selectable marker) which is introduced into said parent cell line together with said expression vector and said amplification vector (alternatively, the selectable marker may be present on the same vector which contains the amplifiable marker). The invention is not limited by the nature of the selectable gene product employed. The selectable gene product employed may be a dominant selectable marker including but not limited to hygromycin G phosphotransferase (e.g., the hyg gene product), xanthine-guanine phosphoribosyltransferase (e.g., the gpt gene product) and aminoglycoside 3' phosphotransferase (e.g., the neo gene product). Alternatively, the selectable marker employed may require the use of a parent cell line which lacks the enzymatic activity encoded by the selectable marker such as hypoxanthine guanine phosphoribosyltransferase, thymidine kinase or carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydrooratase. In a particularly preferred embodiment, the selection vector encodes an active hypoxanthine guanine phosphoribosyltransferase. When the selection vector encodes an active hypoxanthine guanine phosphoribosyltransferase, the second aqueous solution which requires the expression of this selectable gene product comprises hypoxanthine and azaserine.

In another embodiment, the method of the present invention further comprises following the introduction of the vectors (i.e., the amplification, expression and selection vectors), the additional step of growing the transformed cell in a second aqueous solution which requires the expression of the selectable gene product prior to growing the transformed cell in a first aqueous solution containing an inhibitor capable of inhibiting said inhibitable enzyme.

The method of the present invention is not limited by the nature of the inhibitable enzyme encoded by the amplification vector; the art is well of aware of numerous amplifiable markers. In a preferred embodiment, the amplification vector encodes an active enzyme selected from the group consisting of dihydrofolate reductase, glutamine synthetase, adenosine deaminase, asparagine synthetase.

In another preferred embodiment, the inhibitor used to select for a transformed cell expressing the inhibitable enzyme encoded by the amplification vector is selected from the group consisting of methotrexate, 2'-deoxycoformycin, methionine sulphoximine, albizziin and .beta.-aspartyl hydroxamate.

The present invention further provides a method of treating B-cell lymphoma, comprising: a) providing: i) a subject having a B-cell lymphoma; ii) a multivalent vaccine comprising at least two recombinant variable regions of immunoglobulin molecules derived from the subjects's B-cell lymphoma cells, wherein the cells express at least two different immunoglobulin molecules, the immunoglobulin molecules differing by at least one idiotope; b) administering said multivalent vaccine to the subject. In a preferred embodiment, the vaccine comprises at least two recombinant immunoglobulin molecules comprising the recombinant variable regions derived from the lymphoma cells. In a preferred embodiment, the method employs a multivalent vaccine which further comprises an adjuvant. When the vaccine is to be administered to a human subject, adjuvants approved for use in humans are employed. In a preferred embodiment the adjuvant is Syntex adjuvant formulation 1. The recombinant Ig proteins (including fragments of Ig proteins) which comprise the multivalent vaccine may be conjugated to a carrier protein such as KLH.

The present invention provides a method of treating B-cell lymphoma, comprising: a) providing: i) a subject having a B-cell lymphoma; ii) a multivalent vaccine comprising at least two recombinant variable regions of immunoglobulin molecules derived from the subjects's B-cell lymphoma cells, wherein the cells express at least two different immunoglobulin molecules, the immunoglobulin molecules differing by at least one idiotope; and iii) dendritic cells isolated from the subject; b) incubating the dendritic cells in vitro with the multivalent vaccine to produce autologous antigen-pulsed dendritic cells; c) administering intravenously the pulsed dendritic cells to the subject; and d) following the administration of the pulsed dendritic cells, administering the multivalent vaccine to the subject. In a preferred embodiment, the vaccine comprises at least two recombinant immunoglobulin molecules comprising the recombinant variable regions.

The present invention further provides a method of treating B-cell lymphoma, comprising: a) providing: i) a subject having a B-cell lymphoma; ii) a vaccine produced according to the methods of the present invention; and b) administering the vaccine to the subject.

Still further, the present invention provides a method of treating a subject having an immune cell tumor, comprising: a) providing: i) immune cell tumor cells isolated from a subject, the tumor cells expressing an idiotype protein on the cell membrane; ii) an amplification vector comprising a first recombinant oligonucleotide having a sequence encoding a first inhibitable enzyme operably linked to a heterologous promoter; iii) a eukaryotic parent cell line; b) isolating nucleotide sequences encoding at least one idiotype protein expressed on the surface of the tumor cells; c) inserting the nucleotide sequences encoding the idiotype protein(s) into at least one vector to produce at least one expression vector capable of expressing the idiotype protein(s); d) introducing the expression vector(s) into the parent cell to generate one or more transformed cells; e) growing the transformed cell in a first aqueous solution containing an inhibitor capable of inhibiting the inhibitable enzyme wherein the concentration of the inhibitor present in the first aqueous solution is sufficient to prevent growth of the parent cell line; f) identifying a transformed cell capable of growth in the first aqueous solution, wherein the transformed cell capable of growth contains an amplified number of copies of the expression vector and an amplified number of copies of the amplification vector and wherein the transformed cell produces the idiotype protein(s) encoded by the expression vector(s); g) isolating the idiotype protein(s) produced by the transformed cell; and h) administering the isolated idiotype protein(s) to the subject.

The method of the present invention is not limited by the nature of the tumor cells. In one embodiment, the tumor cells are T lymphoid cells and the idiotype protein is a T cell receptor or fragment thereof. In another embodiment, the tumor cells are B lymphoid cells and the idiotype protein is an immunoglobulin or fragment thereof.
 

Claim 1 of 5 Claims

1. A cell expressing a multivalent composition, said multivalent composition for active idiotype immunotherapy, said cell produced according to a method comprising: a) providing: i) malignant B cells isolated from a patient having a quasi-clonal B-cell lymphoma; ii) at least one expression vector; iii) an amplification vector comprising a recombinant oligonucleotide having a sequence encoding a first inhibitable enzyme operably linked to a heterologous promoter; and iv) a T lymphoid parent cell line; b) isolating nucleic acid from said malignant cells, said nucleic acid comprising nucleotide sequences selected from the group consisting of nucleotide sequences encoding at least one V.sub.H region and at least two V.sub.L regions, nucleotide sequences encoding at least two V.sub.H regions and at least one V.sub.L region, and nucleotide sequences encoding at least two V.sub.H regions and at least two V.sub.L regions, wherein said at least two V.sub.L regions differ by at least one idiotope, wherein said at least two V.sub.H regions differ by at least one idiotope, and wherein said V.sub.H and V.sub.L regions are derived from immunoglobulin molecules expressed by said malignant cells; c) inserting said nucleic acid comprising nucleotide sequences encoding said V.sub.H and V.sub.L regions into said at least one expression vector; d) introducing said at least one expression vector and said amplification vector into said parent cell line to generate one or more transformed cells; e) growing said transformed cells in a first aqueous solution containing an inhibitor capable of inhibiting said first inhibitable enzyme wherein the concentration of said inhibitor present in said first aqueous solution is sufficient to prevent growth of said parent cell line; and f) identifying a transformed cell capable of growth in said first aqueous solution, wherein said transformed cell capable of growth expresses a combination of V.sub.H and V.sub.L regions selected from the group consisting of at least one V.sub.H region and at least two V.sub.L regions, at least two V.sub.H regions and at least one V.sub.L region, and at least two V.sub.H regions and at least two V.sub.L regions, wherein said at least two V.sub.L regions differ by at least one idiotope, wherein said at least two V.sub.H regions differ by at least one idiotope, and wherein said V.sub.H and V.sub.L regions comprise a protein molecule, wherein said multivalent composition comprises said expressed V.sub.H and V.sub.L regions.
 

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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