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, cellfree 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. IntroductionB lymphocytes, their clonal progeny, and the malignant lymphomas derived from B lymphocytes each express a unique immunoglobulin (Ig) molecule on the cell surface. Specific antibodies directed against the Ig variable regions can cure lymphomas. [1][2][3][4][5] The same Ig variable regions can be made into vaccines to induce a specific immune response by the host against their own tumor. [6][7][8][9][10] Such vaccines can be proteins containing the Ig variable regions or DNA encoding these proteins. [11][12][13] In addition, the idiotype protein has been used to pulse dendritic cells for active vaccination. 14 Active vaccination induces a polyclonal immune response that should be better than any single monoclonal antibody. 15 In addition, active vaccination can induce a T-cell immune response.Previously, we have shown that vaccination of patients with lymphoma can induce an immune response against the Ig molecule on their tumor 13,[16][17][18] and that such an immune response correlates with a favorable clinical outcome. 18 Prospective, randomized clinical trials are currently under way to determine the efficacy of idiotype vaccination in patients with lymphoma. These vaccines consist of a whole Ig derived from each B-cell tumor and chemically conjugated to a foreign protein, keyhole limpet hemocyanin (KLH), that enhances the immunogenicity of the molecule. Current vaccine manufacturing methods are time consuming and expensive. If personalized therapy with a patient-specific vaccine is to become broadly applicable, a rapid and inexpensive method for vaccine production is needed.Cell-free protein synthesis (CFPS) technology is a much more rapid and economical vaccine production strategy than traditional protein expression systems that use living cells or other organisms. Cell-free protein synthesis provides high yields 19,20 that are not attainable in living hosts because of toxicity [21][22][23] under conditions optimal for protein folding, particularly with regard to proper disulfide bond formation. 24,25 Once a patient's lymphoma-specific Ig V genes ha...

The idiotype (Id)-granulocyte-macrophage colony-stimulating factor (GM-CSF) fusion proteins are potential vaccines for immunotherapy of B-cell lymphoma. In this study, four vaccine candidates were constructed by fusing murine GM-CSF to the amino- or carboxy-terminus of the 38C13 murine B-lymphocyte Id scFv with two different arrangements of the variable regions of the heavy chain and light chain (VL-VH and VH-VL). scFv (VH-VL) and GM-CSF/scFv fusion proteins were expressed in an Escherichia coli cell-free protein synthesis system. In order to promote disulfide bond formation during cell-free expression, cell extract was pretreated with iodoacetamide (IAM), and a sulfhydryl redox buffer composed of oxidized and reduced glutathione was added. The E. coli periplasmic disulfide isomerase, DsbC, was also added to rearrange incorrectly formed disulfide linkages. The 38C13 B-lymphocyte Id scFv was expressed with 30% of its soluble yield in active form (43 microg/ml) when tested with an anti-idiotypic mAb, S1C5, as the capture antibody in radioimmunoassay. It was found that the amino-terminal GM-CSF fusion proteins, GM-VL-VH and GM-VH-VL, showed much higher activity than the carboxy-terminal GM-CSF fusion proteins, VL-VH-GM and VH-VL-GM, in stimulating the cell proliferation of a GM-CSF-dependent cell line, NFS-60. Between the two amino-terminal GM-CSF fusion proteins, GM-VL-VH showed a higher total and soluble yield than GM-VH-VL.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important cytokine in the mammalian immune system. It has been expressed in Escherichia coli with the same biological activity as the native protein. Here, we report the synthesis of a murine recombinant GM-CSF in an E. coli cell-free protein synthesis system with a high yield. Since there are two disulfide bonds in the native structure of GM-CSF, an oxidizing redox potential of the reaction mixture was required. By pretreating the cell extract with iodoacetamide (IAM), the reducing activity of the cell extract was inactivated, and upon further application of an oxidized glutathione buffer, most of the synthesized GM-CSF was found in its oxidized form. However, the GM-CSF thus formed showed low activity because of poor folding. With the addition of DsbC, the periplasmic disulfide isomerase from E. coli, a high yield of active GM-CSF was produced in the cell-free reaction. Finally, successful folding of the cell-free synthesized GM-CSF-his6 was confirmed by its cell-proliferation activity after purification with a Ni2+ chelating column.