Compositions for preserving haptenized tumor cells for use...

Details Compositions for preserving haptenized tumor cells for use... Compositions for preserving haptenized tumor cells for use...

1999-11-16

2001-06-19

Caputa, Anthony C. (Department: 1642)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S325000, C435S405000, C435S002000, C435S001100, C424S093100

Reexamination Certificate

active

06248585

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to compositions comprising a haptenized tumor cell and human serum albumin (HSA) effective to stabilize the haptenized tumor cells in an aqueous buffer.
BACKGROUND OF THE INVENTION
In blood transfusion, bone marrow transplantation, or other cell cultures ex vivo, one of the principal problems encountered is that of the preservation of cells. It is critical to be able to preserve cells, under good conditions of viability, for time periods compatible with clinical production and storage, and to make it possible to analyze cell preparations. The most commonly used method of long-term preservation of cells is to freeze and thaw the material. However, during the freezing of cells, loss of viability may occur. This problem can be even more complex when the cells have been modified or altered prior to preservation, and when the cells are obtained by proteolytic digestion of a tissue or tumor specimen. Furthermore, preservation of cells on ice (about 0° C.), refrigerated (about 4° C.), or at room temperature, prior to use, is also difficult.
Human Serum Albumin
Human serum albumin is a non-glycosylated monomeric protein consisting of 585 amino acid residues, with a molecular weight of 66 kD. Its globular structure is maintained by 17 disulfide bridges, which create a sequential series of 9 double loops (Brown, “Albumin structure, function and uses”, Rosenoer, V. M. et al. (eds.), Pergamon Press, Oxford, pp. 27-51, 1977). The genes encoding for HSA are known to be highly polymorphic, and more than 30 apparently different genetic variants have been identified by electrophoretic analysis (Weitkamp, et al., Ann. Hum. Genet., 37:219-226, 1973). The HSA gene comprises 15 exons and 14 introns comprising 16,961 nucleotides, from the supposed “capping” site up to the first site of addition of poly(A).
Human albumin is synthesized in the hepatocytes of the liver, and then secreted into the peripheral blood. In a first instance, this synthesis leads to a precursor, prepro-HSA, which contains a signal sequence of 18 amino acids directing the nascent polypeptide in the secretory pathway.
HSA is the most abundant blood protein, with a concentration of about 40 grams per liter of serum. Therefore, there are about 160 grams of circulating albumin in the human body at any one time. The most important role of HSA is to maintain a normal osmolarity of the blood. It also has an exceptional binding capacity for various substances, and plays a role both in the endogenous transport of hydrophobic molecules, such as steroids and bile salts, and in that of the transport of different therapeutic substances to their respective sites of action. HSA has been recently been implicated in the breakdown of the prostaglandins. Furthermore, HSA has previously been shown to stabilize solutions of proteins, including protein antigens, and small organic molecules such as hemin (Paige, A. G. et al., Pharmaceutical Res., 12:1883-1888, 1995; Chang, A.- C. and R. K. Gupta, J., Pharm. Sci., 85:129-132, 1996; Niemeijer, N. R. et al., Ann. Allergy Asthma Immunol., 76:535-540, 1996; and Cannon, J. B. et al., PDA:J. Pharm. Sci. & Tech., 49:77-82, 1995).
Haptenized Tumor Cell Vaccines
An autologous whole-cell vaccine modified with the hapten dinitrophenyl (DNP) has been shown to produce inflammatory responses in metastatic sites of melanoma patients. Adjuvant therapy with DNP-modified vaccine produces markedly higher post-surgical survival rates than those reported after surgery alone. Previous work suggested that this vaccine might have a cell integrity duration of less than four hours after hapten modification. Intact or viable cells are preferred for the vaccine.
U.S. Pat. No. 5,290,551, to David Berd, discloses and claims vaccine compositions comprising haptenized melanoma cells. Melanoma patients who were treated with these cells developed a strong immune response. This response can be detected in a delayed-type hypersensitivity (DTH) response to haptenized and non-haptenized tumor cells. More importantly, the immune response resulted in increased survival rates of melanoma patients.
Haptenized tumor cell vaccines have also been described for other types of cancers, including lung cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, and leukemia (see U.S. patent application Ser. No. 08/203,004, filed Feb. 28, 1994; International Patent Application No. PCT/US96/09511; U.S. patent application Ser. No. 08/899,905, filed Jul. 24, 1997).
Cell stabilization and preservation is an important problem in the storage of haptenized tumor cells for administration. Generally, the cells are recovered from tumors, suspended in a cryopreservation medium and frozen until used for the vaccine preparation. When needed, the cells are thawed, haptenized, and then stored for about 1 to about 24 hours, usually about 1 to about 2 hours, at temperatures ranging from about 0° C. (on ice) to room temperature. Under prior art conditions of 0.1% HSA in Hank's Balanced Salt Solution (HBSS) (e.g., see application Ser. No. 08/899,905), the cell viability decreases by more than 50% (i.e. viability is less than 50%) after 18 hours. Thus, there is a need in the art for an effective formulation for storing and preserving the cells after haptenization and prior to delivery as a vaccine. There is a further need for a storage solution that can also serve as a safe delivery vehicle.
The citation of any reference herein should not be construed as an admission that such a reference is available as “Prior Art” to the instant application.
SUMMARY OF THE INVENTION
The present invention advantageously provides a formulation for the preservation and/or storage of haptenized tumor cells for use in anti-tumor vaccines. Thus, in a first embodiment, the invention provides a composition comprising a haptenized tumor cell and a concentration of human serum albumin (HSA) effective to stabilize the haptenized tumor cells in solution from the time of haptenization until administration to the patient. Haptenized tumor cell viability in the formulation of the invention is greater than the viability of the same kind, number, and concentration of haptenized tumor cells in a control medium comprising 0.1% HSA (w/v), over the same period of time. Preferably, cell viability in a formulation of the invention is at least 50% after 18 hours. In a specific embodiment, in which the tumor cells are human tumor cells, the serum albumin is HSA. Preferably, the concentration of HSA is at least about 0.25%. More preferably, the concentration of HSA is at least about 0.5%. Most preferably, the concentration is at least about 1%. HSA can be purified from natural sources, or preferably, obtained by genetic engineering.
The advantage of such a formulation stems from the fact that the haptenized tumor cells and HSA solution are available for administration immediately after haptenization without any further manipulation being necessary. It then becomes possible to carry out the haptenization and storage conditions in a suitable laboratory for subsequent delivery to the clinic. Thereby, the time between storage and use would be reduced, which would also increase the chances for maintaining sterility.
The invention further provides a method for preparing haptenized tumor cells for use in a vaccine. The method comprises suspending the haptenized tumor cells in an aqueous medium comprising a concentration of HSA effective to stabilize the haptenized tumor cells, wherein the haptenized tumor cell viability over time is greater than the viability of the same kind, number, and concentration of haptenized tumor cells stored in a control solution comprising 0.1% HSA over the same period of time. Preferably, the aqueous medium comprises HSA and HBSS. In still a further embodiment, the suspended cells can be mixed with an adjuvant to form a vaccine.
The present invention will be further explained in the Detailed Description.
DETAILED DESCRIPTION OF THE INVENTION
The present invention advantageously provides a new type of medium which stabilizes h

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