Compositions as adjuvants to improve immune responses to vaccines and methods of use
A composition and compound technology, applied in the direction of antibodies, etc., can solve problems such as ineffectiveness
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Embodiment I
[0137] Purification of anti-TIM-1 antibody
[0138] Hybridomas secreting mouse anti-human TIM-1 antibody or rat anti-mouse TIM-1 antibody were initially cultured in cell culture flasks and subsequently transferred to Bioperm cell culture reactors. Every 48 hours, culture supernatants containing secreted antibodies were harvested, clarified, and stored at 4°C. The collected supernatants were pooled and anti-TIM-1 antibodies were purified from the supernatants by protein G Sepharose affinity chromatography and eluted from the column using glycine pH 2.5-3.5. The eluate was pH neutralized and dialyzed against phosphate buffered saline (PBS). Purified antibodies were stored at -80°C until further use.
Embodiment II
[0140] Construction of DNA vectors for mouse and human TIM-1 / Fc fusion protein expression
[0141] A shuttle plasmid vector (pTPL-1) for cloning TIM-1 / Fc fusion protein gene fragment was designed and constructed. The base vector pTPL-1 carries bacterial and eukaryotic resistance genes and a multiple cloning site flanked by CMV enhancers and β-globin polyadenylation sites (see also Figure 18, TIM-3 fusion). Mouse non-lytic IgG2a / Fc fragments (hinge, CH2 and CH3 domains) were generated by oligonucleotide-directed mutagenesis to replace the C1q binding motif and inactivate the FcγR1 binding site (Zheng et al., J . Immunol. 154:5590-5600 (1995)).
[0142] An Fc region, which may be part of an agent of the invention, may be "lytic" or "non-lytic". Non-lytic Fc regions generally lack the high affinity Fc receptor binding site and the C'Iq binding site. The high affinity Fc receptor binding site of murine IgG Fc includes a Leu residue at position 235 of IgG Fc. Thus, by mutating ...
Embodiment III
[0146] Transient expression of TIM / Fc fusion protein in 293 cells
[0147] To test the functionality of the resulting expression vectors, transient transfections were performed in 293 cells. Briefly, 80-90% confluent cells in serum-free growth medium (293-SFMII; InvitroGen, Carlsbad, CA) were transfected using the Lipofectamine 2000 system according to the manufacturer's instructions (InvitroGen, Carlsbad, CA). of 293 cells. Routinely, use 1 μg plasmid DNA / 10 5 cell. One day after transfection, the growth medium was replaced with fresh medium and the cells were cultured for a maximum of 7 days. Cell culture supernatants were clarified by centrifugation and TIM-1 / Fc or TIM-3 / Fc fusion proteins were purified by protein G sepharose affinity chromatography. After low pH elution from protein G beads, the purified protein was dialyzed against PBS and stored at -80°C. Proteins produced were analyzed for identity, purity and integrity by sodium dodecyl sulfate-polyacryla...
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