Antitumor vaccination using allogeneic tumor cells expressing alpha (1,3)-galactosyltransferase

A tumor cell, galactose-based technology, applied in the direction of anti-tumor drugs, carrier-bound antigen/hapten components, antibodies, etc.

Inactive Publication Date: 2006-04-05
CENT IOWA HEALTH SYST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, two frameshift mutations (creating deletions of pre-mature stop codons) were found in the human exon encoding the enzyme

Method used

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  • Antitumor vaccination using allogeneic tumor cells expressing alpha (1,3)-galactosyltransferase
  • Antitumor vaccination using allogeneic tumor cells expressing alpha (1,3)-galactosyltransferase
  • Antitumor vaccination using allogeneic tumor cells expressing alpha (1,3)-galactosyltransferase

Examples

Experimental program
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Effect test

Embodiment 1

[0146] Generation of a retroviral vector expressing αGT, pLNCKG

[0147] Using the forward primer 5′-ACAAAAGCTTGACATGGATGTCAAGGGAAAAGTAAT-3′ and the reverse primer 5′-AATTATCGATTCAGACATTATTTCTAAC-3′, a 1077 bp fragment of the murine αGT gene containing the Kozak sequence that enhances αGT translation was amplified by PCR and cloned into pLNCX ClaI and HindIII sites to generate the pLNCKG retroviral vector (Figure 1). This vector was transfected into the packaging cell line 293. AMIZ [Young and Link "Chimericretroviral helper virus and picornavirns IRES sequence to eliminate DNAmethylation for improved retroviral packaging cells" J. Virol. (2000) 74:5242-5249] to generate vector production cells Department 293.AMIZ / LNCKG. In the presence of G418 and Zeocin TM Case selection for transfected cells for two weeks. Mixed selection cell populations were subcloned by limiting dilution. Single cell-derived VPCs were screened for their ability to efficiently transduce human epitheli...

Embodiment 2

[0149] Transduction of B16.BL6 melanoma cells with LNCKG retroviral vector

[0150] To produce αGal (+) For B16 cells, use 2 mL containing 2 x 10 6 The supernatant of the LNCKG retrovirus at tu / mL infection titer was transduced 2×10 6 cell. Cells were selected for neomycin resistance by two weeks of selection in medium supplemented with G418 1 mg / mL. Following this selection, cells were stained with chicken anti-αGal polyclonal antibody for expressed αGal epitopes and sorted by fluorescence activated cell sorting.

Embodiment 3

[0152] Anti-αGal antibodies were induced in α(1,3)galactosyltransferase knockout (αGT KO) mice by immunization with rabbit erythrocytes.

[0153] Female and male 8-14 week old α(1,3)galactosyltransferase (αGT) knockout (KO) mice were used in this study. The mice were initially mixed haplotype (H-2 b / d) and the current colony of αGT KO mice was obtained by breeding and selection in the F4 cross generation of the H-2 b / b haplotype. ). These animals produce low titers of native antibodies against the [alpha]Gal epitope. To increase the titer of anti-αGal Ab, mice were treated with 1×10 8 Rabbit erythrocytes were immunized intraperitoneally twice with an interval of two weeks. Anti-αGal Ab titers were tested one week after the last RRBC injection to confirm that all mice in this study had high titers of anti-αGal Ab. All mice used in this study had high anti-αGal Ab above a 1 :500 dilution as measured by ELISA. A representative experiment is shown in Figure 3.

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Abstract

The invention relates to methods and compositions for causing the selective targeting and killing of tumor cells. Through ex vivo gene therapy protocols tumor cells are engineered to express an α (1,3) galactosyl epitope. The cells are then irradiated or otherwise killed and administered to a patient. The α galactosyl epitope causes opsonization of the tumor cell enhancing uptake of the opsonized tumor cell by antigen presenting cells which results in enhanced tumor specific antigen presentation. The animal's immune system thus is stimulated to produce tumor specific cytotoxic cells and antibodies which will attack and kill tumor cells present in the animal.

Description

[0001] Cross Reference Related Applications [0002] This application claims priority under 35 U.S.C. §119(e) to provisional application 60 / 417,343, filed October 9, 2002. field of invention [0003] The present invention relates to methods and compositions for treating cancer by stimulating humoral and cellular immune responses against tumor cells. In particular, the invention relates to methods of stimulating complement-mediated destruction of tumor cells and concomitant stimulation of tumor-specific antibody production and tumor-specific cytotoxic cells. Background of the invention [0004] A major hurdle in xenotransplantation is the immediate recognition of carbohydrate epitopes present in foreign tissue causing hyperacute xenograft rejection (HAR). This response begins immediately upon reperfusion and, once initiated, destroys foreign tissue within minutes to hours. The presence of HAR in some donor / recipient combinations (and its absence in others) is inferred to be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K39/385A01K67/027A61K39/00
CPCA61K39/395A01K67/0271A61K39/0011A01K2217/05A01K2267/0331A61K2039/5156A01K67/0275A61K2039/5152A61P35/00A61P37/04A61K39/00119A61K39/001193
Inventor 小查尔斯·J.·林克塔季扬娜·谢瑞金娜加芙列拉·罗西
Owner CENT IOWA HEALTH SYST
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