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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.