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CD40L-Tri, a novel formulation of recombinant human CD40L that effectively activates B cells.

Naito M, Hainz U, Burkhardt UE, Fu B, Ahove D, Stevenson KE, Rajasagi M, Zhu B, Alonso A, Witten E, Matsuoka K, Neuberg D, Duke-Cohan JS, Wu CJ, Freeman GJ - Cancer Immunol. Immunother. (2012)

Bottom Line: Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers.Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells.The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker.

View Article: PubMed Central - PubMed

Affiliation: Cancer Vaccine Center, Dana-Farber Cancer Institute, Boston, MA, USA.

ABSTRACT
CD40L has a well-established role in enhancing the immunostimulatory capacity of normal and malignant B cells, but a formulation suitable for clinical use has not been widely available. Like other TNF family members, in vivo and in vitro activity of CD40L requires a homotrimeric configuration, and growing evidence suggests that bioactivity depends on higher-order clustering of CD40. We generated a novel formulation of human recombinant CD40L (CD40L-Tri) in which the CD40L extracellular domain and a trimerization motif are connected by a long flexible peptide linker. We demonstrate that CD40L-Tri significantly expands normal CD19+ B cells by over 20- to 30-fold over 14 days and induces B cells to become highly immunostimulatory antigen-presenting cells (APCs). Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers. In addition, CD40L-Tri could induce malignant B cells to become effective APCs, such that tumor-directed immune responses could be probed. Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells. The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker.

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Bio-active CD40L-Tri is predominantly a multimer. a Superdex 200 HR10/300 separation of CD40L-Tri. OD280 plot of fractions from size-exclusion gel chromatography of CD40L-Tri (solid line). Elution volume is presented on the lower abscissa and collected fraction number on the upper abscissa. Protein was collected at 0.5 ml elution volumes, and the end of the run is indicated by the disturbance in the conductance (dashed line) at 20 ml elution volume. The main peak (fractions #3–6) and second peak (fractions #15–17) represent separated CD40L-Tri protein. The peak at fractions #24–25 is non-proteinaceous UV-absorbing material. b Upper panel, left—native gel electrophoresis in glycine buffer (pH 8.8; 7.5 % acrylamide gel) of unseparated CD40L-Tri. Note that the protein was applied in glycerol-free, sucrose-free loading buffer as both agents led to complete aggregation and no entry into the gel. Upper panel, right—reducing SDS-PAGE analysis (12.5 % acrylamide gel) of unseparated CD40L-Tri indicates that the variably sized entities identified on the native gel are all comprised of a monomer of ~35 kDa. Lower panel—reducing SDS-PAGE analysis (10 % acrylamide gel) of fractions 4 and #16 indicates that they are both comprised of the same base ~35 kDa monomer. c Normal CD19+ B cells were incubated with individual or pooled chromatographic protein fractions at a concentration of 1 μg/ml. Forty-eight hours later, the expression of CD80 and CD86 on the cell surface was evaluated by flow cytometry. The stimulatory capacity of fraction pools 3, 4 and 16, 17 as well as the unseparated material (CD40L-Tri) and the media control were analyzed in duplicate. The mean MFI is shown for each treatment group
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Fig6: Bio-active CD40L-Tri is predominantly a multimer. a Superdex 200 HR10/300 separation of CD40L-Tri. OD280 plot of fractions from size-exclusion gel chromatography of CD40L-Tri (solid line). Elution volume is presented on the lower abscissa and collected fraction number on the upper abscissa. Protein was collected at 0.5 ml elution volumes, and the end of the run is indicated by the disturbance in the conductance (dashed line) at 20 ml elution volume. The main peak (fractions #3–6) and second peak (fractions #15–17) represent separated CD40L-Tri protein. The peak at fractions #24–25 is non-proteinaceous UV-absorbing material. b Upper panel, left—native gel electrophoresis in glycine buffer (pH 8.8; 7.5 % acrylamide gel) of unseparated CD40L-Tri. Note that the protein was applied in glycerol-free, sucrose-free loading buffer as both agents led to complete aggregation and no entry into the gel. Upper panel, right—reducing SDS-PAGE analysis (12.5 % acrylamide gel) of unseparated CD40L-Tri indicates that the variably sized entities identified on the native gel are all comprised of a monomer of ~35 kDa. Lower panel—reducing SDS-PAGE analysis (10 % acrylamide gel) of fractions 4 and #16 indicates that they are both comprised of the same base ~35 kDa monomer. c Normal CD19+ B cells were incubated with individual or pooled chromatographic protein fractions at a concentration of 1 μg/ml. Forty-eight hours later, the expression of CD80 and CD86 on the cell surface was evaluated by flow cytometry. The stimulatory capacity of fraction pools 3, 4 and 16, 17 as well as the unseparated material (CD40L-Tri) and the media control were analyzed in duplicate. The mean MFI is shown for each treatment group

Mentions: Since we observed that CD40L-Tri bioactivity was similar to that of a multimerized CD40L, we queried whether CD40L-Tri was aggregating in solution and which molecular size fraction exhibited the greatest bioactivity. Size-exclusion gel chromatography (SEC) revealed that CD40L-Tri eluted primarily in a single peak (fractions #3 and #4, eluting at 8–9 ml coinciding with the void volume and slightly after) consistent with a molecular weight range of ~400 kDa and higher. A second smaller peak eluted in fractions #15–17 (eluting at 14–15.5 ml) and corresponded to a molecular weight of ~65 kDa (Fig. 6a).Fig. 6


CD40L-Tri, a novel formulation of recombinant human CD40L that effectively activates B cells.

Naito M, Hainz U, Burkhardt UE, Fu B, Ahove D, Stevenson KE, Rajasagi M, Zhu B, Alonso A, Witten E, Matsuoka K, Neuberg D, Duke-Cohan JS, Wu CJ, Freeman GJ - Cancer Immunol. Immunother. (2012)

Bio-active CD40L-Tri is predominantly a multimer. a Superdex 200 HR10/300 separation of CD40L-Tri. OD280 plot of fractions from size-exclusion gel chromatography of CD40L-Tri (solid line). Elution volume is presented on the lower abscissa and collected fraction number on the upper abscissa. Protein was collected at 0.5 ml elution volumes, and the end of the run is indicated by the disturbance in the conductance (dashed line) at 20 ml elution volume. The main peak (fractions #3–6) and second peak (fractions #15–17) represent separated CD40L-Tri protein. The peak at fractions #24–25 is non-proteinaceous UV-absorbing material. b Upper panel, left—native gel electrophoresis in glycine buffer (pH 8.8; 7.5 % acrylamide gel) of unseparated CD40L-Tri. Note that the protein was applied in glycerol-free, sucrose-free loading buffer as both agents led to complete aggregation and no entry into the gel. Upper panel, right—reducing SDS-PAGE analysis (12.5 % acrylamide gel) of unseparated CD40L-Tri indicates that the variably sized entities identified on the native gel are all comprised of a monomer of ~35 kDa. Lower panel—reducing SDS-PAGE analysis (10 % acrylamide gel) of fractions 4 and #16 indicates that they are both comprised of the same base ~35 kDa monomer. c Normal CD19+ B cells were incubated with individual or pooled chromatographic protein fractions at a concentration of 1 μg/ml. Forty-eight hours later, the expression of CD80 and CD86 on the cell surface was evaluated by flow cytometry. The stimulatory capacity of fraction pools 3, 4 and 16, 17 as well as the unseparated material (CD40L-Tri) and the media control were analyzed in duplicate. The mean MFI is shown for each treatment group
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Related In: Results  -  Collection

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Fig6: Bio-active CD40L-Tri is predominantly a multimer. a Superdex 200 HR10/300 separation of CD40L-Tri. OD280 plot of fractions from size-exclusion gel chromatography of CD40L-Tri (solid line). Elution volume is presented on the lower abscissa and collected fraction number on the upper abscissa. Protein was collected at 0.5 ml elution volumes, and the end of the run is indicated by the disturbance in the conductance (dashed line) at 20 ml elution volume. The main peak (fractions #3–6) and second peak (fractions #15–17) represent separated CD40L-Tri protein. The peak at fractions #24–25 is non-proteinaceous UV-absorbing material. b Upper panel, left—native gel electrophoresis in glycine buffer (pH 8.8; 7.5 % acrylamide gel) of unseparated CD40L-Tri. Note that the protein was applied in glycerol-free, sucrose-free loading buffer as both agents led to complete aggregation and no entry into the gel. Upper panel, right—reducing SDS-PAGE analysis (12.5 % acrylamide gel) of unseparated CD40L-Tri indicates that the variably sized entities identified on the native gel are all comprised of a monomer of ~35 kDa. Lower panel—reducing SDS-PAGE analysis (10 % acrylamide gel) of fractions 4 and #16 indicates that they are both comprised of the same base ~35 kDa monomer. c Normal CD19+ B cells were incubated with individual or pooled chromatographic protein fractions at a concentration of 1 μg/ml. Forty-eight hours later, the expression of CD80 and CD86 on the cell surface was evaluated by flow cytometry. The stimulatory capacity of fraction pools 3, 4 and 16, 17 as well as the unseparated material (CD40L-Tri) and the media control were analyzed in duplicate. The mean MFI is shown for each treatment group
Mentions: Since we observed that CD40L-Tri bioactivity was similar to that of a multimerized CD40L, we queried whether CD40L-Tri was aggregating in solution and which molecular size fraction exhibited the greatest bioactivity. Size-exclusion gel chromatography (SEC) revealed that CD40L-Tri eluted primarily in a single peak (fractions #3 and #4, eluting at 8–9 ml coinciding with the void volume and slightly after) consistent with a molecular weight range of ~400 kDa and higher. A second smaller peak eluted in fractions #15–17 (eluting at 14–15.5 ml) and corresponded to a molecular weight of ~65 kDa (Fig. 6a).Fig. 6

Bottom Line: Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers.Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells.The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker.

View Article: PubMed Central - PubMed

Affiliation: Cancer Vaccine Center, Dana-Farber Cancer Institute, Boston, MA, USA.

ABSTRACT
CD40L has a well-established role in enhancing the immunostimulatory capacity of normal and malignant B cells, but a formulation suitable for clinical use has not been widely available. Like other TNF family members, in vivo and in vitro activity of CD40L requires a homotrimeric configuration, and growing evidence suggests that bioactivity depends on higher-order clustering of CD40. We generated a novel formulation of human recombinant CD40L (CD40L-Tri) in which the CD40L extracellular domain and a trimerization motif are connected by a long flexible peptide linker. We demonstrate that CD40L-Tri significantly expands normal CD19+ B cells by over 20- to 30-fold over 14 days and induces B cells to become highly immunostimulatory antigen-presenting cells (APCs). Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers. In addition, CD40L-Tri could induce malignant B cells to become effective APCs, such that tumor-directed immune responses could be probed. Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells. The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker.

Show MeSH
Related in: MedlinePlus