Design, Development and Delivery of Recombinant 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 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 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 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 • Columbia (H. Kaufman) Hypotheses

• Tumor Associated (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 (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 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 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 “ 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 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 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