Design, Development and Delivery of Recombinant Vaccines for the Therapy of Human Carcinomas
Jeffrey Schlom, Ph.D. Laboratory of Tumor Immunology and Biology Center for Cancer Research National Cancer Institute, NIH Recombinant Vaccine Programmatic Effort DELIVERY: DISCOVERY: Laboratory of Tumor Immunology COLORECTAL/PANCREATIC/LUNG and Biology, Center for Cancer CLINICAL TRIALS Research, NCI • Georgetown (J. Marshall) J. Hodge J. Greiner • Fox Chase (M. von Mehren) • Duke (K. Lyerly) H. Sabzevari A. Tsang D. Grosenbach S. Kashmiri PROSTATE CANCER CLINICAL TRIALS S. Abrams P. Arlen • Dana Farber (D. Kufe/P. Eder) J. Schlom J. Gulley • Center for Cancer Research, NCI DEVELOPMENT: (P. Arlen, J. Gulley, W. Dahut, N. Coleman, K. Camphausen) Collaborative Research and Development Agreement • ECOG (H. Kaufman, R. DiPaola, L. Weiner) (CRADA): Therion/NCI BREAST CANCER CLINICAL TRIALS Cancer Therapy Evaluation • Dana Farber (D. Kufe) Program (CTEP), NCI • Center for Cancer Research, NCI (C. Kasten-Sportes, R. Gress, P. Arlen) INTRATUMORAL TRIALS Melanoma • Columbia (H. Kaufman) Hypotheses
• Tumor Associated Antigens (TAA) are, by definition, either weakly immunogenic or functionally non-immunogenic. Cancer Vaccine Targets
Proteins/peptides that are:
overexpressed in tumors vs. normal tissues CEA, MUC-1
overexpressed in tumors and non-vital organs PSA Carcinoembryonic Antigen (CEA) 180,000d glycoprotein homoadhesion molecule implicated in the metastatic process Distribution Carcinoma: 95% of colorectal , gastric, pancreatic 50% of mammary 70% of non-small cell lung Others: e.g., squamous cell carcinoma of head & neck, cervical carcinoma Normal: Extensive in fetal gut Low levels in normal colonic mucosa Hypotheses
• Tumor Associated Antigens (TAA) are, by definition, either weakly immunogenic or functionally non-immunogenic.
• Vaccine strategies must be developed in which the presentation of these TAAs to the immune system results in far greater activation of T cells than is being achieved naturally in the host. Strategies
1. Mode of Delivery of the Vaccine – place the gene for the tumor antigen into a vector
2. Diversified Vaccine Prime and Boost 3. T-cell Costimulation – these molecules are essential for vigorous T-cell activation – place costimulatory molecule into vaccine vector 4. Alter the a.a. sequence of the tumor antigen to enhance the immune response “epitope enhancement”
5. Cytokines —biologic adjuvants Recombinant Vaccine Vectors
• Pox vectors Vaccinia (rV-) elicits a strong immune response – host induced immunity limits its continuous use – MVA (replication defective) Comparison of T-Cell Responses in CEA Transgenic Mice Vaccinated with CEA Protein vs. rV-CEA
20 rV-CEA
15
10 Stimulation Index CEA Protein 5
0 100 50 25 12 6 3
[CEA], (g/ml) Recombinant Vaccine Vectors
• Pox vectors Vaccinia (rV-) elicits a strong immune response – host induced immunity limits its continuous use – MVA (replication defective) Avipox (fowlpox rF, ALVAC) – derived from avian species – safe; does not replicate – can be used repeatedly with little if any host neutralizing immunity
• Can insert multiple transgenes • Do not integrate into host DNA • Efficiently infect antigen presenting cells including dendritic cells Phase I Studies: CEA Vaccines
• rV-CEA –Safe – Induction of T-cell responses specific for CEA
• Avipox-CEA –Safe – Induction of T-cell responses specific for CEA – Demonstration that CEA-specific T cells can kill tumor cells expressing CEA Preclinical Studies: Diversified Prime and Boost Strategy
• rV-TAA (V) : prime vaccination • Avipox-TAA (A) : booster vaccination
VAA > VVV VAA > AAA TAA = tumor-associated antigen Diversified prime and boost is more efficacious than the continued use of one vaccine Phase II Study: Vaccination of Patients with Metastatic* CEA-Expressing Carcinomas
• To define if a Diversified Vaccine Prime and Boost Strategy leads to increases in CEA-specific T-cell responses • Vaccines: rV-CEA (V) and Avipox-CEA (A)
Randomized (n = 9/cohort):
Cohort 1: V-A-A-A Cohort 2: A-A-A-V • Define CEA responses to 9 mer CEA peptide using an overnight ELISPOT assay
*Patients received from 2 to 6 prior therapies. CEA and Flu Precursor Frequencies Following Vaccinations with rV-CEA(V) and Avipox-CEA(A) ± GM-CSF
Precursor Frequency Patient Vaccination Flu CEA 3 Pre 1/75,000 <1/200,000 V 1/11,000 1/116,000 V-A 1/62,000 1/71,000 V-A-A 1/75,000 1/66,000 V-A-A-A 1/85,000 1/61,000 V-A-A-A-A (+GM-CSF) 1/86,000 1/19,000 -0.8x > 10.5x Advantage of Primary Vaccination with rV-CEA (V) and Boosting with Avipox-CEA (A)
0 VAAA
-4 5
10
15
≤ 20 0 AAAV
5
CEA Precursor Frequency x 10 Frequency Precursor CEA 10
15
≤ 20 Pre Post Immunization Randomized Phase I/II Arms of the Diversified Vaccination of rV-CEA (V) and Avipox-CEA (A):
100 Survival
90
80
70
60
50
% Survival 40
30 AAAV
20
10
0 0 6 12 18 24 30 36 42 Time (Months) Randomized Phase I/II Arms of the Diversified Vaccination of rV-CEA (V) and Avipox-CEA (A):
100 Survival
90
80 VAAA 70
60
50
% Survival 40
30 AAAV
20
10
0 0 6 12 18 24 30 36 42 Time (Months) Statistical Analyses: Randomized Cohorts
V=rV-CEA A=avipox-CEA
Patients in the VAAA cohort had a statistically significant (p<0.01) increase in CEA specific T cells (post vs. pre- vaccination) as compared to patients in the AAAV cohort.
Treatment with VAAA resulted in longer survival than treatment with AAAV (p=0.05).
Survival duration was unrelated to pre-vaccination CEA specific T cell levels (p=0.77).
The generation of CEA specific T cell responses was associated with increased survival (p=0.03) after accounting for disease status. Antigen Cascade VAAA vs. AAAV CEA Vaccine Trial
Patient 15 Flu CEA MUC-1 Her2/neu p53 103 p53 139 EP-CAM pre vac 1/23,316 <1/200,000 <1/200,000 <1/200,000 <1/200,000 <1/200,000 <1/200,000 post 1 1/37,500 1/75,000 1/85,714 1/66,667 1/46,154 1/120,000 1/46,154 post 2 1/28,571 1/35,294 1/50,000 1/66,667 1/37,500 <1/200,000 1/75,000
Patient 15 received rV-CEA primary vaccination and avipox-CEA booster vaccination. Strategies 1. Mode of Delivery of the Vaccine – place the gene for the tumor antigen into a vector
2. Diversified Vaccine Prime and Boost
3. T-cell Costimulation – these molecules are essential for vigorous T-cell activation – place costimulatory molecule into vaccine vector
4. Alter the a.a. sequence of the tumor antigen to enhance the immune response “epitope enhancement”
5. Cytokines —biologic adjuvants T-cell Costimulation
Costimulatory molecules are expressed on professional antigen presenting cells (APC): dendritic cells, B cells, macrophages, monocytes
Costimulatory molecules are not expressed on the vast majority of solid tumors T-Cell Dependence on Costimulation
Signal 1 + Signal 2 No Signal 1 No Signal 2
Antigen Presenting CellAntigen Presenting Cell Antigen Presenting Cell
MHC Costimulatory MHC Costimulatory MHC Molecule Molecule TCR TCR TCR
T-CellT-Cell T-Cell
Clonal Anergy Clonal Anergy Activation of Antigen- Apoptosis Apoptosis Specific Ignorance Ignorance T-cells T-cell Activation and Costimulatory Molecules
Antigen Presenting Cell T-Cell ) MHC + Peptide TCR 5
Costimulatory Molecule Ligand
B7-1 CD28 (CD80)
ICAM-1 LFA-1 (CD54) T-cell Activation (CPM x 10 T-cell
LFA-3 CD2 (CD58) (Region 1) None LFA-3 ICAM-1 B7-1 TRICOM Costimulatory Molecule TRICOM TRIad of COstimulatory Molecules
Costimulatory Molecule Ligand on T cell B7-1 (CD80) CD28/CTLA-4 ICAM-1 (CD54) LFA-1 LFA-3 (CD58) CD2
e.g. , rV-Tricom = rV-B7-1/ICAM-1/LFA-3 avi-Tricom = avi -B7-1/ICAM-1/LFA-3 rV-CEA(6D)/Tricom = rV-CEA(6D)/B7-1/ICAM-1/LFA-3 avi-CEA(6D)/Tricom = avi-CEA(6D)/B7-1/ICAM-1/LFA-3 rV-PSA(3A)/Tricom = rV-PSA(3A)/B7-1/ICAM-1/LFA-3 avi-PSA(3A)/Tricom = avi-PSA(3A)/B7-1/ICAM-1/LFA-3 avi = rF = recombinant avipox (fowlpox) CEA-Specific T-cell Responses Following Vaccination with rF-CEA, rF-CEA/B7-1, or rF-CEA/TRICOM ) 3 20
15 * HBSS FP-WT 4x rF-CEA 4X 10 rF-CEA/B7 rF-CEA/TRICOM
5 CEA Specific T-cell Response T-cell Specific CEA
(Lymphoproliferation, CPM x 10 (Lymphoproliferation, 0 4 44 132134 24Number of Vaccinations rF-CEA/B7 rF-CEA/TRICOM
Vaccination Schema Vaccine Tumor Therapy Model
• CEA Transgenic (Tg) mice – CEA expressed in fetal gut and adult GI tissues (similar to humans)
• Experimental Metastases – MC-38 colon carcinoma cells expressing CEA
• Begin vaccine therapy at day 14 post-tumor transplant Therapy of 14-Day Established CEA+ Experimental Metastases in CEA-Tg Mice Using CEA/TRICOM Vectors
100
90
80 VAAA Regimen 70 CEA 60 CEA/TRICOM 50 % Survival All groups with 40 GM-CSF and low dose IL-2 30
20
10 Buffer Control 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Weeks Post Tumor Transplant Tumor Vaccine Therapy of 14-Day Established CEA+ Experimental Metastases in CEA-Tg Mice Using CEA/TRICOM Vectors
100
90
80 VAAA Regimen 70 CEA 60 CEA/TRICOM 50 % Survival All groups with 40 GM-CSF and low dose IL-2 30
20 CEA 10 Buffer Control 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Weeks Post Tumor Transplant Tumor Vaccine Therapy of 14-Day Established CEA+ Experimental Metastases in CEA-Tg Mice Using CEA/TRICOM Vectors
100
90
80 VAAA Regimen 70 CEA/TRICOM (P<0.02 vs CEA) CEA 60 CEA/TRICOM 50 % Survival All groups with 40 GM-CSF and low dose IL-2 30
20 CEA 10 Buffer Control 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Weeks Post Tumor Transplant Tumor Vaccine CEA-specific Lymphoproliferation of T Cells from CEA-Tg Mice Vaccinated with TRICOM Vectors 8000 Con-A 400000 Ovalbumin
300000
CEA/TRICOM 200000 VAAA
100000 Regimen 6000 0 CEA
CEA/B7-1
4000 CEA/B7-1 CEA/TRICOM
Proliferation (CPM) TRICOM CEA All groups with 2000 GM-CSF and TRICOM low dose IL-2
None 0 80 60 40 20 0 [CEA], (g/ml) Clinical Trials
Phase I Georgetown, J. Marshall • Avipox-CEA(6D)-TRICOM (A) -4 vaccinations • rV-CEA(6D)-TRICOM (V) -1 vaccination – boost with Avipox-CEA(6D)-TRICOM -3 vaccinations
Phase I Fox Chase, M. von Mehren • Avipox-CEA(6D)-TRICOM + GM-CSF vs. avipox GM-CSF
Phase I Duke, K. Lyerly • Avipox-CEA(6D)-TRICOM – infection of dendritic cells CEA-TRICOM Phase I Trial: Patient Demographics Characteristics Number of Patients Total 58 Prior therapy Chemotherapy 0 regimen 3 1 prior regimens 7 2 prior regimens 12 > 2 prior regimens 36 Radiation 16
Primary site Colorectal 34 Lung 7 Breast 4 Thyroid 1 Unknown primary 1 Renal 1 Other GI 10 HLA-A2 +/- 31/27 CEA-TRICOM Phase I Trial (Georgetown U., J. Marshall, P.I.) • Stage 1: F= Fowlpox-CEA (6D)-TRICOM (F) – Cohort 1: F-F-F-F 4 x 106 pfu 3 pts – Cohort 2: F-F-F-F 4 x 107 pfu 3 pts – Cohort 3: F-F-F-F 4 x 108 pfu 10 pts (6 A2+) • Stage 2: V= vaccinia-CEA (6D)-TRICOM (V) – Cohort 4: V-F-F-F 4 x 106 pfu 3 pts – Cohort 5: V-F-F-F 4 x 107 pfu 3 pts – Cohort 6: V-F-F-F 4 x 108 pfu 10 pts (6 A2+) • Stage 3: GM= GM-CSF 100 mcg subq D1-4 – Cohort 7: V-F-F-F + GM 10 pts (6 A2+) • Stage 4: Split dose of rF + GM-CSF 100 mcg sq bilaterally – Cohort 8: V-F-F-F +GM 10 pts (6 A2+) CEA-TRICOM Phase I Trial
Toxicity: no dose limiting toxicity no Grade II, III, or IV toxicities attributed to vaccine Clinical Results • 23 patients (40%) had stable disease > 4 months • 11 patients had stable or decreasing serum CEA / 19.9 • 1 pathologic complete response—lung cancer patient • Patients who remained stable after 6 monthly vaccinations went on to vaccinations every 3 months – 6/12 patients who progressed after being given vaccines every 3 months restabilized after monthly vaccinations PET Images of Patient after 2 Vaccinations with Avipox-CEA(6D)/TRICOM
Pre-Vaccination Post 58 Days CEA-TRICOM Phase I Trial: Survival by Cohort
100 V = rV-CEA-TRICOM 90 A = avipox-CEA-TRICOM 80
70
60
50 Cohorts 7 & 8 VAAA + GM-CSF
% Survival 40
30
20 Cohort 3 & 6 10 AAAA AAA 0 0 6 12 18 24 30 Survival Time (Months)
Cohort 3 & 6 Cohorts 7 & 8 16/19 Failed 12/24 Failed CEA-TRICOM Phase I Study CEA-Specific and Flu-Specific T-Cell Responses
CEA-Specific T-cells Flu-Specific T-cells
1/1
1/40,000
1/80,000 Non-Outlier Max Non-Outlier Max Non-Outlier Min Non-Outlier Min 1/120,000 75% 75% 25% 25% 1/160,000 Median Median Extreme Outliers Outliers * p<0.0001 p=0.46 1/200,000 * Pre Vaccine Post Vaccine 4 Pre Vaccine Post Vaccine 4 CEA-TRICOM Phase I Trial
CEA-Specific T-Cell Responses in PBMC* (HLA-A2 patients): Increases in 13/16 patients
Using 1/30,000 as a cut-off after 4 vaccinations: – correlation with progression-free survival (p = 0.04) – correlation with overall survival (p = 0.06)
• 83% of patients who had a CEA-specific T-cell response of < 1/30,000 after 4 vaccinations were alive at 1 year, vs. 41% of patients whose CEA precursors were >1/30,000 after 4 vaccinations ------* Using ELISPOT assay for IFN-γ production by PBMC in response to CEA-specific peptide post 4 vaccinations vs. pre-vaccination. Clinical Development of Vector-Based Vaccines rV-CEA (V) rV-muc-1)
avipox-CEA (A)
avipox-CEA-B7 VAAA AAAV agonist (6D) ±GM-CSF, IL-2 peptide-pulsed DC
rV-CEA(6D)-TRICOM (V) rV-muc-1 + avipox-GM-CSF avipox-CEA(6D)-TRICOM (A) rV-CEA(6D)-TRICOM VAAA + GM-CSF
rV-PANVAC; avipox-PANVAC PANVAC = CEA(6D)-muc-1(A)-TRICOM
TRICOM = B7.1/ICAM-1/LFA-3 CEA(6D) = CEA gene containing agonist epitope muc-1(A) = muc-1 gene containing agonist epitope PSA Vaccine Clinical Development Plan
rV-PSA (V)
avi-PSA (A) ECOG VVAAA VAAA Vaccine + Docetaxel AAAVV AAAV AAAA vs. Vaccine Alone rV-PSA + rV-B7-1 Radiotherapy + Vaccine In-Vitro boost: avi-PSA (x3) vs. Radiotherapy Alone ± GM-CSF PSA-3 ±IL-2 Vaccine vs. vs Hormone Therapy PSA-3A
rV-PSA(3A)-TRICOM (V) avi-PSA(3A)-TRICOM (A) STRATEGIES avi-GM-CSF Vector-based Delivery of Antigen VAA + GM-CSF (rV, avi) Diversified Prime and Boost (VAAA) Costimulation (B7-1 -> TRICOM) Agonist (3A) Cytokine (GM-CSF) Serum PSA Levels in Hormone Refractory Patients Vaccinated with rV-PSA + rV-B7-1 and Boosted with Avipox-PSA
12 Vac 20 Vac
10 16
8 12
6
8 PSA (ng/ml) PSA 4
4 2
0 0 V1 V2 V3 V4 V5 V6 Pre V1 V2 V3 V4 V5 V6 V7 V8 V9 Pre Pre Pre Pre Pre Pre V10 V11 Antigen Cascade – Gulley Trial: rV-PSA + rV-B7.1 rF-PSA
Patient Sample Flu PSA3 PSMA PAP PSCA MUC-1
Pt 1 pre vac 1/50,000 1/200,000 <1/200,000 <1/200,000 1/200,000 1/200,000 post 3 1/37,500 1/42,857 <1/200,000 <1/200,000 <1/200,000 <1/200,000
Pt 2 pre vac 1/21,429 <1/200,000 <1/200,000 <1/200,000 <1/200,000 <1/200,000 post 3 1/21,429 1/60,000 1/200,000 1/85,714 <1/200,000 <1/200,000
Pt 3 pre vac 1/85,714 <1/200,000 <1/200,000 <1/200,000 <1/200,000 <1/200,000 post 3 1/54,545 1/75,000 <1/200,000 <1/200,000 <1/200,000 1/40,000
Pt 4 pre vac 1/20,690 <1/200,000 <1/200,000 <1/200,000 <1/200,000 <1/200,000 post 3 1/22,222 1/66,667 1/85,714 <1/200,000 <1/200,000 1/60,000
Pt 5 pre vac 1/75,000 <1/200,000 <1/200,000 <1/200,000 <1/200,000 <1/200,000 post 3 1/100,000 1/85,714 <1/200,000 1/85,714 1/85,714 1/23,077 Future Studies
• The use of TRICOM vaccines in combination with conventional therapies − radiation − selected drugs Tumor Prevention/Therapy Model
• CEA-Transgenic Mice: CEA is a self antigen
• APC/min+ mice: Develop numerous spontaneous colon tumors (polyps) and die from anemia
• CEA-Tg x APC/min+ develop CEA+ spontaneous tumors
COX-2 inhibitors (Celecoxib) FDA approved for the treatment of polyps Anti-tumor Effects of Spontaneous Tumors in CEA.Tg/MIN Mice Using a CEA-TRICOM Vaccine and Celecoxib
Control CEA Celecoxib Celecoxib + Diet Vaccine Diet CEA Vaccine 100 60
50 80
40 60 30 40 20 Survival (%) 10 20 Intestinal Tumors/mouse 0 0 0 204060 Weeks Untreated mice CEA -based vaccine Celecoxib (1000 ppm) Celecoxib (1000 ppm) + CEA Based Vaccine Radiation-Enhanced Antigen-Specific Lysis of Tumor Cells
Radiation
CD8+ Target T-cell Cell
FAS-L FAS Persistence of Fas Upregulation on MC38-CEA+ Tumors After External-Beam Irradiation
Time Post Tumor Irradiation (8 Gy) 0 Day 2 Day 4 Day 7 Day 11 Day 4 10 05 015 060 089 088 587 580 634 3 8 210 103
102
Fas-PE 101
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 CEA-FITC Fas Isotype Histochemistry H&E Schema of Combination Vaccine Therapy and Radiation
8 Gy
Tumor V1 V2 V3 V4 (MC38- CEA+ SQ) Day 0 8 14 15 22 29
rV-CEA/TRICOM rf-CEA/TRICOM rF-GM-CSF rF-GM-CSF Combination Therapy : Vaccine + External Beam Radiation
No TreatmentVaccine Irradiation Vaccine + Irradiation 500 A.10 (25) B. 8 (65) C. 61 (565) D. 63 (612) 400 n=10 %Fas n=10n=9 n=10 Single Dose 300
200 CEA PositiveTumors 100
0
) tumor free: n=0 n=0 n=0 n=4
3 500
12 (36) 11 (41) 66 (435) 54 (415) Radiation Schedule E.F. G. H. 400 n=12 n=13n=12 n=9 Fractionated 300
200
100 Tumor Volume (mm Volume Tumor 0
500 n=0 n=0 n=0 n=5 .
7 (16) 6 (21) 64 (593) 68 (654) CEA NegativeTumors I.J. K. L. 400 n=10 n=10n=9 n=9 Single Dose 300
200
100
0 n=0 n=0 n=0 n=0 0 7 14 21 28 35 42 49 0 7 14 21 28 35 42 49 0 7 14 21 28 35 42 49 0 7 14 21 28 35 42 49 Days Post Tumor Transplant Vaccine + Radiation: Tumor Infiltrating Cells
No Treatment Vaccine Alone Radiation Alone Vaccine + Radiation CD3+ T-cells (IHC) CD3+ T-cells
No Treatment Vaccine Alone Radiation Alone Vaccine + Radiation 7.1% 11% 7.2% 38%
1.6% 2.2% 1.0% 4.4% T-cells + CD8 100 101 102 103 104100 101 102 103 104100 101 102 103 104 100 101 102 103 104 CD4+ T-cells Strategic Plan for Clinical Trial Development Combinatorial Vaccine Therapy
CEA-TRICOM Vaccines (V, A) CEA-muc-1-TRICOM Vaccines (V, A): PANVAC Phase I — Pancarcinoma Phase I — Pancarcinoma
Phase II Trials:
Lung Cancer Breast Cancer Colon Cancer Pancreatic Cancer Stage III Metastatic Stage 4: Liver Met. Therion IND Radiation/ Docetaxel Vaccine Phase I/II/III Chemo ± Vaccine ± Radiation ± Vaccine ± COX-2 Inhibitor In Conclusion Hypothesis-Driven Preclinical Studies Have Been Translated to Science-Based Clinical Trials: . Viral Vector-Based Vaccines . Diversified Prime and Boost Vaccination Schema . T-Cell Costimulation (TRICOM) . Agonist Epitope Enhancement . Cytokines as Biologic Adjuvants . Multi-Tumor Antigen Vaccine Constructs
Strategic Plan . Integration of Recombinant Vaccine Regimens with Local Tumor Irradiation and Chemotherapy . Continue Programmatic Efforts: Intramural Research Program, 8 Cancer Centers, Cooperative Groups, Private Sector