Cell-free production of scFv fusion proteins: an efficient approach for personalized lymphoma vaccines

Abstract

The unique immunoglobulin (Ig) idiotype on the surface of each B-cell lymphoma represents an ideal tumor-specific antigen for use as a therapeutic vaccine. We have used an Escherichia coli-based, cell-free protein-expression system to produce a vaccine within hours of cloning the Ig genes from a B-cell tumor. We demonstrated that a fusion protein consisting of an idiotypic single chain Fv antibody fragment (scFv) linked to a cytokine (GM-CSF) or to an immunostimulatory peptide was an effective lymphoma vaccine. These vaccines elicited humoral immune responses against the native Ig protein displayed on the surface of a tumor and protected mice against tumor challenge with efficacy equal to that of the conventional Ig produced in a mammalian cell and chemically coupled to keyhole limpet hemocyanin. The cell-free E coli system offers a platform for rapidly generating individualized vaccines, thereby allowing much more efficient application in the clinic.

Figure 1

Constructs and purified protein products from cell-free reactions. (A) Diagram of 38C13 idiotype constructs produced in the cell-free expression system. Construct 1 is an scFv linked to mouse GM-CSF via an Im9 linker. Construct 2 links human GM-CSF to the scFv fragment. The third construct is a fusion of IL-1β9aa to the 38C13 scFv. Construct 4 is the 38C13 scFv alone. Constructs not shown are the C-terminal IL-1β9aa fusion to the 38C13 scFv, the A20 scFv, and the E coli disulfide bond isomerase (DsbC). (B) After 4 hours of protein synthesis, products were enriched from the cell-free reaction supernatant using a nickel affinity column. The partially purified products of constructs were analyzed by SDS-PAGE and stained with Coomassie blue. The arrows indicate the band that corresponds to the desired product. For comparison, unpurified cell-free product is shown (lane U).

Figure 2

mGM-CSF fusion proteins are bioactive as measured by growth of GM-CSF–dependent NFS-60 cells. NSF-60 cells were grown in the presence of varying concentrations of 38C13 idiotype fusion proteins prepared in the cell-free expression system or commercial murine GM-CSF (mGM-CSF) standard. All dilutions were performed in triplicate. Neither the human fusion (hGM-Im9-scFv) nor scFv alone induced cell growth, but the fusion protein containing mGM-CSF fused to scFv promoted the growth of NFS-60 cells with the same potency as a commercial standard.

Figure 3

Cell-free vaccines induced humoral immune responses; anti-38C13 Ab levels were comparable to that of the Id-KLH standard. The concentration of anti-38C13 IgG antibodies in mouse serum 10 days after the third vaccination was determined by ELISA. Data plotted is combined from 3 independent experiments (n = 29 mice for mGM-Im9-V_L-V_H, 20 mice for V_L-V_H-IL1β9 and DsbC, 19 mice for Id-KLH and scFv (V_H-V_L), and 10 mice for A20 V_H-V_L and IL1β9aa-V_H-V_L). Variations in group sizes are due to groups being tested in 1, 2, or 3 experiments and to the loss of serum samples in a few cases. Data shown are means with SEM; *P < .05 compared with scFv (V_H-V_L); **P < .05 compared with mGM-Im9-V_L-V_H and V_H-V_L-IL1β9aa using a 2-tailed Student t test. Exact P values are stated in the text.

Figure 4

Vaccines prepared in a cell-free system are capable of inducing an immune response that recognizes the native 38C13 protein on the cell surface. Flow cytometry was used to analyze 38C13 tumor cells incubated with mouse sera obtained after 3 vaccinations with the indicated proteins. Each histogram represents results from one mouse. The scale shows fold change over secondary antibody alone on a log₂ scale. The numbers in the upper right corner of each box indicate mean median fluorescence for that group. (A) Flow cytometry of cells incubated with sera at a 1:20 dilution. (B) Analysis of an independent experiment by flow cytometry. Cells were incubated with sera at a 1:800 dilution to determine the relative efficacy of vaccines in induction of a specific Ab response. **Significance (P < .05) by Student 2-tailed t test compared with both 38C13 scFv (V_H-V_L) and A20 scFv (V_H-V_L) and *statistical significance (P < .05) compared with A20 scFv (V_H-V_L).

Figure 5

Vaccine produced in the cell-free system protected mice from tumor challenge comparably to the Id-KLH vaccine. In 3 independent experiments mice were immunized biweekly with 3 vaccinations of the indicated vaccine and were challenged with 400 38C13 tumor cells (or 1200 tumor cells in panel C) 2 weeks following the third vaccination. (A) First tumor challenge experiment (n = 10 mice per group). (B) Second tumor challenge experiment (n = 10 mice per group). For both groups, *statistical significance (P < .05 compared with DsbC control) determined by comparing survival curves using the log-rank test. (C) Third tumor challenge experiment (n = 10 except n = 9 for scFv and Im9-38ScFv and n = 7 for hGM-Im9-scFv). *significance (P < .05) by the log-rank test compared with both 38C13 scFv (V_H-V_L) and A20 scFv (V_H-V_L). Actual P values for all experiments are listed in the text.