Rapid expression of vaccine proteins for B‐cell lymphoma in a cell‐free system

Yang, Jun; Kanter, G. S.; Voloshin, Alexei; Michel-Reydellet, N.; Velkeen, Hendrik; Levy, Ronald; Swartz, James R.

doi:10.1002/bit.20283

Cited by 70 publications

(61 citation statements)

References 42 publications

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“…Since cell-free protein synthesis is emerging as a potential commercial recombinant DNA protein expression technology (24,45,51), it is also interesting that the modern systems do not appear to be energy limited. The measured ATP and GTP concentrations (200 M or higher) are much higher than the K m s for protein synthesis (21,22,23).…”

Section: Vol 191 2009 Protein Synthesis Rate Dependence On [Atp] Anmentioning

confidence: 99%

Continued Protein Synthesis at Low [ATP] and [GTP] Enables Cell Adaptation during Energy Limitation

Jewett¹,

Miller²,

Chen³

et al. 2009

J Bacteriol

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One of biology's critical ironies is the need to adapt to periods of energy limitation by using the energyintensive process of protein synthesis. Although previous work has identified the individual energy-requiring steps in protein synthesis, we still lack an understanding of the dependence of protein biosynthesis rates on [ATP] and [GTP]. Here, we used an integrated Escherichia coli cell-free platform that mimics the intracellular, energy-limited environment to show that protein synthesis rates are governed by simple Michaelis-Menten dependence on [ATP] and [GTP] (K m ATP , 27 ؎ 4 M; K m GTP , 14 ؎ 2 M). Although the system-level GTP affinity agrees well with the individual affinities of the GTP-dependent translation factors, the system-level K m ATP is unexpectedly low. Especially under starvation conditions, when energy sources are limited, cells need to replace catalysts that become inactive and to produce new catalysts in order to effectively adapt. Our results show how this crucial survival priority for synthesizing new proteins can be enforced after rapidly growing cells encounter energy limitation. A diminished energy supply can be rationed based on the relative ATP and GTP affinities, and, since these affinities for protein synthesis are high, the cells can adapt with substantial changes in protein composition. Furthermore, our work suggests that characterization of individual enzymes may not always predict the performance of multicomponent systems with complex interdependencies. We anticipate that cell-free studies in which complex metabolic systems are activated will be valuable tools for elucidating the behavior of such systems.Arguably, new protein synthesis is most crucial when energy supplies are limited and the cell must adjust its catalytic capabilities to cope with this challenge. Yet, paradoxically, due to the entropic demands for accurate transcription and translation, the metabolic system for protein synthesis is typically the most energy-demanding process in most organisms (Fig. 1). It has been shown, for example, that protein synthesis consumes approximately two-thirds of the total energy produced by a rapidly growing Escherichia coli cell (30). Consequently, much effort has been focused on understanding the mechanisms of ATP and GTP usage during protein synthesis.Previous work has established the individual energy-requiring steps for the metabolic system for protein synthesis (Fig. 1). These steps include recycling nucleotides for mRNA synthesis (44), charging aminoacyl-tRNAs (44), and regenerating GTP to energize the translation factors required for polypeptide synthesis (initiation factor 2 [IF-2], elongation factor Tu [EFTu], elongation factor G [EF-G], and ribosome release factor 3 [RF-3]) (53). The GTP-GDP cycle of EF-Tu, for example, plays a key role in tRNA selection and kinetic proofreading (4,18). In addition to transcription and translation, the DEAD box RNA helicase, which is required for unwinding mRNA secondary structure, is ATP dependent (36), as are several chaperones (14,...

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Section: Vol 191 2009 Protein Synthesis Rate Dependence On [Atp] Anmentioning

confidence: 99%

Continued Protein Synthesis at Low [ATP] and [GTP] Enables Cell Adaptation during Energy Limitation

Jewett¹,

Miller²,

Chen³

et al. 2009

J Bacteriol

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“…26 In that study we determined the optimal gene order for combining GM-CSF with an Ig single chain variable fragment (scFv) to maximize protein solubility and biological activity. 23 Here, we show that proteins produced in the cell-free system can function as vaccines against a murine B-cell lymphoma. The 2 vaccine candidates we tested were a tumor-derived scFV fused through the bacterial immunity protein, Im9, to GM-CSF and the scFV fragment fused to a 9-amino acid peptide sequence from interleukin-1␤ (IL-1␤9aa).…”

Section: Introductionmentioning

confidence: 84%

“…23,26 For pK7.38C13.IL-1␤9aa, a DNA fragment (5Ј-GTGCAGGGT-GAAGAGAGCAACGA TAAA-3Ј) that encodes a 9-amino acid peptide VQGEESNDK from IL-1␤ was added onto the V L -V H DNA fragment by polymerase chain reaction (PCR) primer extension of the 3Ј region of pK7.V H -V L vector with an opposing T7 forward primer. The 3Ј extension was made from the 38C13 V L gene to include the first 15 base pairs of the E coli protein chloramphenicol acetyltransferase (CAT), 23,26 38C13 V H -V L , and the IL-1␤ sequence followed by a GGGGS linker and a C-terminal His-tag. The fragment was then restricted with Nde1 and Sal1 and ligated into the pK7 vector.…”

Section: Construction Of Scfv and Fusion Plasmids For Id Vaccinesmentioning

confidence: 99%

“…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 have been cloned, the proteins can be produced in a cell-free transcription-translation system within hours and can be purified and ready for use as vaccine in a matter of days.…”

Section: Introductionmentioning

confidence: 99%

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

Kanter

Yang

Voloshin

et al. 2006

Blood

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115

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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...

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“…M-NFS-60 cells (1 3 10 3 cells/50 ll) were cultured with mGM-CSF standards (0-40 ng/ml) or the supernatant (150 ll) of each transfectant in 96-well plates. 17 After culturing for 6 days, the number of cells in each well was determined by MTS assay (Promega). The mGM-CSF concentration in the supernatant of each transfectant was measured by the mGM-CSF standard curve.…”

Section: Mgm-csf Activity Assaymentioning

confidence: 99%

Combination of Fasl and GM‐CSF confers synergistic antitumor immunity in an in vivo model of the murine Lewis lung carcinoma

Ho¹,

Sun²,

Leu³

et al. 2008

Intl Journal of Cancer

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Gene transfer of Fas ligand (FasL) to tumor cells has been demonstrated to inhibit tumor growth in vivo, and neutrophils are primarily responsible for this immunoprotection. The granulocyte–macrophage colony stimulating factor (GM‐CSF) secreted by tumor vaccine can recruit dendritic cells (DCs) for efficient antigen presentation to T cells that generate the tumor‐specific response. To investigate whether the combination of FasL and GM‐CSF can efficiently suppress tumor growth, we have established Lewis lung carcinoma (LLC‐1) cells that are transduced with GM‐CSF (LLC/GM‐CSF), FasL (LLC/FasL) or both genes (LLC/FasL/GM‐CSF) to test their tumorigenic potential in vivo. Mice inoculated with LLC/GM‐CSF display high survival rates along with reduction of tumor growth. In contrast, none of the mice injected with LLC/FasL or LLC/FasL/GM‐CSF develop tumors. Specific memory immune response and delayed LLC‐1 tumor growth are found in mice immunized with LLC‐1/FasL or LLC‐1/FasL/GM‐CSF. Furthermore, therapeutic effects are observed only when LLC‐1/FasL/GM‐CSF tumor vaccine is employed to retard growth of preexisting LLC‐1 tumors. Tumor growth is also completely suppressed in mice injected with a mixture of LLC‐1 and LLC‐1/FasL/GM‐CSF. In addition, IL‐12 production, cytotoxic T‐cell activity and IgG against LLC‐1 are manifested in mice injected with LLC/FasL/GM‐CSF. Our data show that FasL‐induced pathway triggers expression of proinflammatory cytokines, including IL‐1β, IL‐6, MIP‐2 and MCP‐1, while GM‐CSF‐dependent pathway promotes functional maturation and activation of DCs. Taken together, the results indicate that dual gene‐based delivery with FasL and GM‐CSF may serve as a more effective tumor vaccine to suppress lung cancer cell growth in vivo. © 2008 Wiley‐Liss, Inc.

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Rapid expression of vaccine proteins for B‐cell lymphoma in a cell‐free system

Cited by 70 publications

References 42 publications

Continued Protein Synthesis at Low [ATP] and [GTP] Enables Cell Adaptation during Energy Limitation

Continued Protein Synthesis at Low [ATP] and [GTP] Enables Cell Adaptation during Energy Limitation

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

Combination of Fasl and GM‐CSF confers synergistic antitumor immunity in an in vivo model of the murine Lewis lung carcinoma

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