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Therapeutic Lymphoma Idiotype Vaccine Generated in Insect Cells Results in Mannose Receptor Targeting and Enhanced Immune Activation David J. Betting, Xi Y. Mu, Kamran Kafi, Desmond McDonnel, Francisco Rosas, Daniel P. Gold, and John M. Timmerman Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia Therapeutic Lymphoma Idiotype Vaccine Generated in Insect Cells Results in Mannose Receptor Targeting and Enhanced Immune Activation Background • Treatment of lymphoma patients with tumor-specific • immunoglobulin (idiotype, Id) coupled to the immunogenic carrier protein keyhole limpet hemocyanin (Id-KLH) has shown promising results in phase 2 clinical trials However, vaccines fail to elicit anti-Id immune responses in some patients, thus prompting the search for ways to improve the immunogenicity of Id-KLH vaccines Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Therapeutic Lymphoma Idiotype Vaccine Generated in Insect Cells Results in Mannose Receptor Targeting and Enhanced Immune Activation Background • Current Id vaccine trials utilize tumor-specific Id proteins secreted by tumor-myeloma hybridomas, or recombinant Id proteins produced in mammalian lymphoid cells, bacteria, or insect cells • Recombinant proteins produced in insect cells are reported to have altered glycosylation patterns (terminal mannose residues) that may contribute to immunogenicity, but direct comparisons of insect-derived and mammalian-derived tumor antigens are lacking Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Therapeutic Lymphoma Idiotype Vaccine Generated in Insect Cells Results in Mannose Receptor Targeting and Enhanced Immune Activation Objective • To determine if terminal mannose carbohydrate structures, characteristic of recombinant proteins produced in insect cells, lead to Id proteins with significantly enhanced immunostimulatory properties compared to Id proteins derived from mammalian sources • We sought to systematically compare the immunogenic properties of insect cell-derived vs. mammalian cellderived Id proteins in murine B cell lymphoma model and in cultures of human antigen-presenting dendritic cells Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Glycosylation Patterns of A20 Id Made in Insect Cells Vs. Mammalian Cells Materials & Methods • Figure 1: Insect and hybridoma derived A20 Id proteins were analyzed by the cIEF and CE carbohydrate methods. Only insect celldervied Id has a high content of terminal mannose residues Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Therapeutic Lymphoma Idiotype Vaccine Generated in Insect Cells Results in Mannose Receptor Targeting and Enhanced Immune Activation Methods • Monocyte-derived human dendritic cells (DCs) were incubated with fluorescently labeled Id proteins produced in insect or mammalian cell cultures Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Therapeutic Lymphoma Idiotype Vaccine Generated in Insect Cells Results in Mannose Receptor Targeting and Enhanced Immune Activation Results • Insect cell-derived Id demonstrated substantially • higher binding to DCs compared to the Id from a mammalian source by flow cytometry, and only the insect cell Id showed reduced binding when the DCs were preincubated with mannose receptor inhibitors These results demonstrated that the insect cell-derived Id resulted in better targeting to DCs compared to the mammalian Id Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Insect Derived Idotype Proteins Bind to Mannose Receptors on Human Dendritic Cells Binding of FITC labeled insect (BV) or hybridoma (Hyb) derived human IgG proteins • Figure 2A: DC binding of FITC labeled KLH, hybridoma produced (Hyb) human IgG, and insect derived (BV) human IgG showed some binding of KLH, but no binding of human IgG (Hyb). Human IgG (BV) did show a strong signal for binding to human DCs. Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Insect Derived Idotype Proteins Bind to Mannose Receptors on Human Dendritic Cells Binding of FITC labeled insect (BV) or hybridoma (Hyb) derived A20 Id proteins • Figure 2B: 50 µg of Alexa 647 labeled murine A20 Id produced in BV or Hyb were used to stain monocyte derived human DCs along with free Alexa. Insect derived Id shows substantially higher surface binding compared to hybridoma derived Id or unconjugated Alexa 647. Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Insect Derived Idotype Proteins Bind to Mannose Receptors on Human Dendritic Cells Binding of Alexa 647 labeled insect (BV) or hybridoma (Hyb) derived A20 Id proteins • Figure 2C: 50 µg of BV derived A20 Id, hybridoma derived A20 Id, or FITC-dextran were used to stain human DCs in the presence of various inhibitors. Cells were pretreated for 20 minutes with 3 mg/ml of mannan, 50 µg/ml of control IgG blocking antibody, 50 µg/ml of anti-mannose receptor blocking antibody, or 5 µg/ml of cytochalasin D. The blocking agents, with the exception of cytochalasin D, did not cause decreased binding of the hybridoma derived Id. In contrast, the BV derived A20 Id protein and the FITC-dextran both showed decreases in binding in the presence of all the various inhibitors. Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Therapeutic Lymphoma Idiotype Vaccine Generated in Insect Cells Results in Mannose Receptor Targeting and Enhanced Immune Activation Results • When insect cell-derived Id proteins were coupled • to KLH using glutaraldehyde and co-cultured with immature human DCs, increased expression of CD80 and CCR7 was observed by flow cytometry, indicating DC maturation Upregulation of these markers was blocked by pre-treatment with anti-mannose receptor antibody Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Insect-Derived Idiotype Enhances Upregulation of Human Dendritic Cell Activation Markers CD80 200 P=0.0017 P=0.0199 MFI 150 100 50 0 • Figure 3A: Insect derived Id proteins stimulate expression of DC activation markers over that seen with hybridoma-derived (Hyb) Id. The MFIs for CD80 was significantly upregulated with the BV-derived Id conjugates compared to Hyb-derived conjugates. Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Insect-Derived Idiotype Enhances Upregulation of Human Dendritic Cell Activation Markers CCR7 P=0.0058 50 MFI 40 P=0.0034 30 20 10 0 • Figure 3B: Insect derived Id proteins stimulate expression of DC activation markers over that seen with hybridoma-derived (Hyb) Id. The MFIs for CD80 was significantly upregulated with the BV-derived Id conjugates compared to Hyb-derived conjugates. Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Insect-Derived Idiotype Enhances Upregulation of Human Dendritic Cell Activation Markers MFI Experimental / MFI Media Alone CD80 3 P=0.0008 P=0.0144 2 1 0 • Figure 3C: CD80 surface expression levels are represented as MFI of experimental sample/MFI of media alone. Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Insect-Derived Idiotype Enhances Upregulation of Human Dendritic Cell Activation Markers MFI Experimental / MFI Media Alone CCR7 4 P=0.0474 P=0.0361 3 2 1 0 • Figure 3D: CCR7 surface expression levels are represented as MFI of experimental sample/MFI of media alone. Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Blocking Mannose Receptor Inhibits the Upregulation of Human Dendritic Cell Activation Markers 225 200 175 150 125 100 75 50 25 0 • CD80 CCR7 MFI MFI Donor 1 45 40 35 30 25 20 15 10 5 0 Figure 4: Surface expression of CD80 and CCR7 were evaluated after incubation with BV- or Hyb-derived conjugates with or without pretreatment using 50 µg/ml of anti-MR blocking antibody. The BV-conjugate treated sample showed a marked decrease in surface expression of both CD80 and CCR7 with anti-MR pretreatment. Media only Id-KLH (BV) Id-KLH (Hyb) Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Blocking Mannose Receptor Inhibits the Upregulation of Human Dendritic Cell Activation Markers 175 150 125 100 75 50 25 0 • CD80 MFI MFI Donor 2 45 40 35 30 25 20 15 10 5 0 CCR7 Figure 4: Surface expression of CD80 and CCR7 were evaluated after incubation with BV- or Hyb-derived conjugates with or without pretreatment using 50 µg/ml of anti-MR blocking antibody. The BV-conjugate treated sample showed a marked decrease in surface expression of both CD80 and CCR7 with anti-MR pretreatment. Media only Id-KLH (BV) Id-KLH (Hyb) Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Therapeutic Lymphoma Idiotype Vaccine Generated in Insect Cells Results in Mannose Receptor Targeting and Enhanced Immune Activation Results • In tumor therapy studies, mice with 4-day • established A20 murine B cell lymphoma were treated with 3 weekly injections of Id-KLH plus GM-CSF and followed for survival A20 bearing mice treated with Id-KLH containing insect cell-derived A20 Id displayed improved survival compared with mice treated with hybridoma-derived A20 Id-KLH (61% vs. 46%, respectively) Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Tumor Eradication is Superior with Use of Insect-Derived Idiotype Proteins Percent Survival Individual A20-KLH (BV) and (Hyb) Groups Using Each Conjugation Method 100 90 80 70 60 50 40 30 20 10 0 A20-KLH (BV) Mal A20-KLH (Hyb) Mal A20-KLH (BV) Glut A20-KLH (Hyb) Glut HBSS 0 10 20 30 40 50 60 70 80 90100 Days Post Tumor Challenge • Figure 5A: The (BV) maleimide conjugates resulted in the highest level of tumor eradicaton. Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Tumor Eradication is Superior with Use of Insect-Derived Idiotype Proteins Percent Survival Comparison of BV vs. Hyb Derived Id Proteins 100 90 80 70 60 50 40 30 20 10 0 A20-KLH (BV) A20-KLH (Hyb) HBSS 0 20 40 60 80 100 Days Post Tumor Challenge • Figure 5B: A20-KLH (BV) and (Hyb) Groups Were Pooled to Show Enhancement of Survival with Insect-Derived Id Proteins. Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Tumor Eradication is Superior with Use of Insect-Derived Idiotype Proteins Percent Survival Comparison of Maleimide vs. Glutaraldehyde Conjugated Id Proteins 100 90 80 70 60 50 40 30 20 10 0 A20-KLH (BV) A20-KLH (Hyb) HBSS 0 20 40 60 80 100 Days Post Tumor Challenge • Figure 5C: A20-KLH Maleimide and Glutaraldehyde Treated Mice Were Pooled to Show Superiority of Maleimide-Conjugated Id Proteins Compared to Glutaraldehyde Conjugates. Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. CD8+ T Cells are Required for Tumor Eradication Percent Survival Comparison of CD8 vs. CD4 Depleting Antibodies 100 90 80 70 60 50 40 30 20 10 0 P=0.0066 P=0.0002 P=0.9417 CD4 Depleting Ab Control Ab CD8 Depleting Ab HBSS Control 0 10 20 30 40 50 60 70 80 90100 Days Post Tumor Challenge • Figure 6: CD8+ T cells are required for tumor eradication. P values represent comparisons between Id-KLH treated groups and HBSS unless otherwise indicated. The results show a clear dependence on CD8+ T cells. Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Insect-Derived Idiotype Enhances Cytolytic Activity P=0.0240 60 % Lysis 50 40 30 20 10 0 • Figure 7: CD8+ T cells are enhanced with insect-derived Id proteins. Primed and boosted (2 immunizations) CTLs from spleen and lymph nodes were used at an E:T ratio of 1:1, both showed significant lysis compared to control. Additionally, the BV-derived protein demonstrated statistically better lysis compared to Hyb-produced Id. Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. % Lysis Production of Anti-Idiotype Antibodies is not Hindered by the Presence of Mannose Residues, and is Enhanced with Maleimide Conjugation • 450 400 350 300 250 200 150 100 50 0 Figure 8: To evaluate the antibody responses generated by A20-KLH (BV) and (Hyb) vaccinated mice were bled and the immune sera anti-Id antibody titers were determined by ELISA (target antigen = native, hybridoma-derived Id), using an A20 anti-Id monoclonal antibody as the standard. The BV-derived Id proteins showed no reduction in anti-Id antibody titers, and the maleimide conjugated proteins showed almost double the levels of anti-Id antibodies. Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Therapeutic Lymphoma Idiotype Vaccine Generated in Insect Cells Results in Mannose Receptor Targeting and Enhanced Immune Activation Conclusions • The insect cell-derived idiotype (Id) proteins showed • a mannose-containing carbohydrate profile typical of antibodies derived from an insect cell source Only insect cell-derived Id proteins demonstrated increased binding to human dendritic cells via the mannose receptor; the binding of mammalianderived (hybridoma) idiotype proteins was low and did not change with the addition of mannose receptor inhibitors Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Therapeutic Lymphoma Idiotype Vaccine Generated in Insect Cells Results in Mannose Receptor Targeting and Enhanced Immune Activation Conclusions • Insect cell-derived Id proteins induced higher • surface expression of dendritic cell activation markers CD80 and CCR7 than did mamalianderived Id Anti-mannose receptor antibodies could inhibit upregulation of DC activation markers Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Therapeutic Lymphoma Idiotype Vaccine Generated in Insect Cells Results in Mannose Receptor Targeting and Enhanced Immune Activation Conclusions • Vaccination of mice bearing A20 B cell lymphoma • with insect cell-derived Id protein resulted in a higher proportion of survivors compared to hybridoma-derived Id (61% vs. 49%), and conjugation to KLH using maleimide further enhanced the anti-tumor efficacy over the traditional glutaraldehyde method CD8+ T cells were critical for eradication of A20 lymphoma, and T cells primed with insect cellderived Id showed enhanced tumor cytotoxicity Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Therapeutic Lymphoma Idiotype Vaccine Generated in Insect Cells Results in Mannose Receptor Targeting and Enhanced Immune Activation Conclusions • Insect cell-derived Id protein did not reduce the • generation of anti-idiotype antibodies compared to hybridoma-derived protein Maleimide conjugated Id-KLH, either insect- or hybridoma-derived, generated approximately 2-fold higher anti-idiotype antibody titers compared to glutaraldehyde conjugates Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia. Therapeutic Lymphoma Idiotype Vaccine Generated in Insect Cells Results in Mannose Receptor Targeting and Enhanced Immune Activation Conclusions • Compared to mammalian cell-derived Id proteins, • production of recombinant Id proteins via a baculovirusinsect cell system results in a structurally different antigen with improved immunogenicity, and this may lead to improved efficacy of the final Id-KLH vaccine product Data comparing sources of recombinant Id protein tumor antigens used in therapeutic cancer vaccines suggest that post-translational modifications, namely terminal mannose residues, can significantly influence the immunological properties and eventual therapeutic efficacy of the product Betting DJ et al. Abstract #2343. Presented December 9, 2007, at the 49th ASH Annual Meeting in Atlanta, Georgia.