In vivo Intratumoral Electroporation of Gp96-Ig/Fc-OX40L Stimulates CD8+ T cell Cross Priming to Tumor-Specific Neoantigens Suresh de Silva1, George Fromm1, Jamil Haque2, Jean Campbell2, Robert Pierce2, and Taylor H. Schreiber1 1Heat Biologics, Inc., Durham NC, 2OncoSec Medical Inc., San Diego CA
Introduction Intratumoral DNA delivery by electroporation
Chaperone and adjuvant functions of Gp96.Ig
Day
-1
0
Medpulser EP conditions • 1500V/cm • 100ms pulses x 5
7
3
40
C57BL/6 Albino mice
25 20 15 10
40
50 0
0
10
20
30
20
5
Ova-antigen specific Memory precursor cells
B
D
5.0 4.0 3.0 2.0
*
50 40 30 20 10
C
0
3
4
5
7
9
11
13
Days post-electroporation
17
20
T C PA om
5
7 10 12 14 17 19 21 24 26 28 Days post-electroporation
0
T C PA om
3
AH1 tetramer - spleen
E
AH1 tetramer – Tumor Infiltrating Lymphocytes
0
100 EP only ComPACT
75
0
Figure 3. EP-based intratumoral delivery of ComPACT leads to regression of both treated (primary) and untreated (contralateral) CT26 colon cancer tumors. BALB/c mice (n=12 per group) bearing CT26 tumors were EP’d with saline or ComPACT DNA and tumor sizes, both treated (A) and untreated (B) were monitored over a 28 day period. C. Intratumoral mRNA expression of Fc-OX40L in ComPACT treated mice was confirmed by qPCR 4 days following EP (n=3 mice per group) *p<.05. D. Splenocytes from EP’d mice (n=3 mice per group) were dissected on day 6 post-EP and enriched for CD8+ cells using a commercial kit (Stemcell tech) and stained for AH1-tetramer+ (representing CT26 antigen-specific CD8+) cells. Cells were negatively gated to exclude cells positive for Nk1.1, Gr-1, CD11b and CD11c and subsequently pre-gated on CD3+. E. Tumors were isolated from mice on day 7 post-EP and enzymatically dissociated using a commercial kit (Miltenyi Biotec) and cells were stained for AH1-tetramer-positive cells as before. Representative histogram showing CD8+ AH1-tetramer+ double positive cells are shown. An empty vector control electroporated group was also included.
Key Points
50
Mantel-Cox (p-value) EP only vs ComPACT 0.006
0
C
0.0
0
2
Overall survival
25
EP
1.0
0
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40
Percent survival
6.0
on ly
% OT-I / Total CD8+ cells
ComPACT
7.0
%CD127+KLRG- / OT1-GFP
EP only
1
C
0 3 5 7 10 12 14 17 19 21 24 26 28
Figure 1. Intratumoral delivery of Gp96.Ig/Fc-OX40L (ComPACT) expressing DNA via electroporation (EP) leads to delayed tumor progression of both treated (primary) and untreated (contralateral) B16.F10 melanoma tumors. A. C57BL/6 albino mice (n=9 mice per group) bearing melanotic B16.F10-ova tumors were electroporated with saline (EP only) or ComPACT DNA expressing Gp96.Ig/Fc-OX40L. Tumor sizes were measured using a digital caliper and monitored over a 40 day time period post-EP. B. The size of the contralateral untreated tumor following electroporation was monitored over the same time period. C. Intratumoral mRNA expression of Gp96.Ig in ComPACT EP mice was confirmed by qPCR on day 3 following EP (n=3 mice per group). mRNA levels were normalized to 18s and * denotes p<0.05. The EP only control was set to 1. Ova-antigen specific CD8+ T cell expansion
2
6
Days post electroporation
Days post electroporation
40
T
30
60
* 3
C
20
80
4
PA
10
100
ComPACT 100
om
0
150
EP only
120
C
0
8.0
§ Fc-OX40L secreted from the tumor cell serves to enhance co-stimulation of CTLs
ComPACT
0
9.0
§ Gp96.Ig serves as a molecular chaperone and adjuvant that presents tumorassociated neoantigens to APCs to trigger an anti-tumor CTL response
EP only
30
*
8
on ly
100
EP only ComPACT
200
EP
200
250
Relative Gp96.Ig expression (normalized to 18s)
300
Untreated tumor size (mm2)
EP only ComPACT
A
Step 1 – tumor cells are identified Step 2 – DNA-based molecules are injected in the tumor Step 3 – Electrical pulses are delivered via electrodes, which increases the permeability of the cell membrane Step 4 – DNA-based molecules enter the cell nucleus and express the immuno-modulatory molecules
35
Days post-electroporation
400
IT gene expression on day 4 post-EP
C
EP
IT gene expression on day 3 post-EP
C
Untreated tumor 140
N o
Untreated tumor
B
B
40
Relative Fc-OX40L expression (normalized to 18s)
A Outcome measurements • Tumor size – treated and untreated • OTI-GFP expansion kinetics • Intratumoral gene expression • CD8 T cell phenotyping • Overall survival
Treatment groups • Electroporation only (EP) • Gp96.Ig/OX40L (ComPACT)
Treated tumor
A
Treated tumor
Fold change in tumor size over day 0
Contralateral tumor (5x105)
OTI-GFP cell transfer
Primary tumor (5x105)
Results – CT26 colon cancer tumor model Electroporation (EP) schedule (x1 dose) -Inject primary tumor with plasmid DNA (50µg) and electroporate
Results
Treated tumor size (mm2)
u Cancer immunotherapy relies on presentation of shared- and neo- antigens from a patient’s tumor cells for recognition and clearance by the immune system. However, the tumor microenvironment deploys multiple strategies to evade immune recognition and often remains non-immunogenic, which is one of the challenges that need to be addressed when designing new therapies. u We set out to test whether intratumoral electroporation of Gp96-Ig/FcOX40L, a re-engineered molecular chaperone, designed to export and deliver MHC I-associated antigens to APCs in context of OX40L expression, would generate a robust anti-neoantigen CD8+ T cell response. To assess antigenspecific CD8+ expansion, mice were adoptively transferred with OT-I cells after B16.F10-ovalbumin cells were injected to generate primary and contralateral melanotic tumors. Contralateral tumors were monitored to assess whether a systemic CD8+ T cell response could be elicited following primary tumor electroporation. IT electroporation of DNA expressing Gp96-Ig/ Fc-OX40L in the primary tumor triggered a significant expansion of antigenspecific OT-I cells, which was absent in control mice. Remarkably, increases in antigen-specific OT-I cells correlated with regression of both the treated primary and untreated contralateral tumors. u We further validated our findings in a CT26 mouse colorectal cancer tumor model, in which the expression of Gp96-Ig/Fc-OX40L from electroporated DNA stimulated an expansion of antigen-specific CD8+ T cells and again led to regression of both the treated primary and untreated contralateral tumor. u Our findings demonstrate that in situ manipulation of intratumoral cells to express Gp96-Ig/Fc-OX40L stimulates potent antigen-specific cross priming to tumor specific neoantigens that culminates in robust systemic anti-tumor response. These findings provide exciting proof-of-principal and warrant further investigation into the direct delivery of molecular chaperones such as Gp96-Ig/Fc-OX40L and/or pro-inflammatory molecules for elevating the immunogenicity of tumors for a potent anti-tumor CD8+ T cell response.
Figure 2. Intratumoral EP of ComPACT leads to ova-antigen specific CD8+ T cell expansion in vivo. A. C57BL/6 albino mice that were adoptively transferred with OT1-GFP cells and B16.F10-ova tumors were electroporated with either saline (EP only) or ComPACT DNA. The percentage of CD8+ OT1-GFP cells in peripheral blood was monitored over time by flow cytometry. B. Phenotypic analysis of ova-antigen specific CD8+ T cells on day 12 following EP by flow cytometry reveals increased numbers of CD127+/KLRG- memory precursor cells in mice EP’d with ComPACT. C. Overall survival of B16.F10 melanoma bearing mice EP’d with saline or ComPACT DNA . * indicates p<0.05. Statistical significance was determined by student t-test and Mantel-Cox test.
Experimental Design – B16.F10-ova melanoma tumor model
Untreated tumor size (mm2)
Abstract
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10 15 20 25 30 Days post primary tumor inoculation
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u In vivo EP of DNA-based Gp96.Ig/Fc-OX40L into B16 melanoma and CT26 colon cancer tumors result in delayed tumor progression of treated and untreated tumors u Intratumoral expression of Gp96.Ig/Fc-OX40L stimulates CD8+ T cell cross priming to tumor specific neoantigens and increases the frequency of circulating memory precursor T cells u Electroporation-based delivery of Gp96.Ig/Fc-OX40L DNA in combination with other immuno-modulatory DNA could lead to synergistic anti-tumor activity Contact information –
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