Strategies to Enhance Dendritic Cell-Mediated Antitumor Immunity

Strategies to Enhance Dendritic Cell-Mediated Antitumor Immunity Johannes Vieweg, M.D. Associate Professor of Urology/Immunology Duke University Medical Center

Objective Therapeutic Immunity Vaccine Potency = Magnitude x Quality x Persistence Vaccine with therapeutic impact Vaccine Potency Threshold of Therapeutic Immunity Vaccine Development Vaccine with no or minimal therapeutic impact Patient Factors? i.e Tumor burden, Immune suppression Pretreatment, Age, Disease setting, Other factors Time

A Multi-Pronged Approach to Cancer Immunotherapy Type CD4 immunity (LAMP, Ii inhibition) Frequency DC vaccination In situ maturation Enhance survival Costimulation: OX4, 4-1BB CD27, CD4 (mrna/dc) Antigen Defined universal antigens Mixtures (ampl. mrna) Induction of immunity Persistence of immunity Prevent attenuation CTLA-4, PD-1 aptamers + Immune suppression Immune evasion Tregs (ONTAK ) Stromal antigens ImC (ATRA) CD4+ T cell effectors B7H-1, TGFβRII, DcR3 aptamers

Activation of T Cells T by APC From: Abbas et al. Cancer Immunology

Dendritic cell-based vaccines using tumor antigen in the form of mrna Powerful method for stimulating antitumor immunity Broadly applicable to all cancer types Solution to the problem of treatment-related emergence of resistant variants

Background: Phase I Clinical Trial using semi-mature PSA RNA transfected DC A. Phenotype after RNA Loading 1 2 5 1 2 PSA-RNA [µg/ml] B. Isotype control C. Untreated IgG 1 -PE % Increase CD83 2 15 1 5 A. PSA-mRNA Titration.1%.3% CD1a Spots / 5x1 5 PBMC 3 25 2 15 1 B. Evidence of Immunogenicity 5 IgG 2b -FITC CD 83 D. PSA-RNA+RNaseA E. PSA-mRNA CD1a.2% 11.9% CD1a PSA-RNA 2 3 5 6 7 9 11 13.2% 11.9% CD 83 CD 83

Background: Phase I Clinical Trial using semi-mature PSA RNA transfected DC PSA log slope PSA log slope PSA log slope PSA log slope C. Impact on PSA Velocity 3. A. VH-7 2. 1. Pre slope =.27 Post slope = -.11. -6-5 -4-3 -2-1 1 2 3 4 2. B. TL-3 1.. -1. Pre slope =.69 Post slope =.12-2. 4. -2-1 1 2 3 4 5 C. JD-2 3. 2. 1.. Pre slope =.15 Post slope =.19-1. -2-1 1 2 Time [weeks].2 * D. Entire Group.15.1 * * *.5 * *. #2 #3 #5 #7 #9 #13 #16 -.5 Patient ID Pre Post PSA Log Slope PSA Log Slope 1.5 JM-13 1.5 3 2.5 2 1.5 1.5 D. Clearance of Circulating Tumor Cells Vaccination # 1,2,3 4 8 12 WV-12 Vaccination # 1,2,3 Stable disease 4 8 12 Relative time (week) Relapse 4 3.5 3 2.5 2 1.5 1.5 5 PSA mrna Copies/1 7 PBMC 25 2 15 1 EpCAM mrna Copies/1 7 PBMC

Increase of Tumor-Specific T Cells after Vaccination with semi-mature RCC RNA transfected DC Spots/1x1 5 T cells 1 8 6 4 2 1 8 6 4 2 RCC # 1 # 2 # 3 # 5 # 7 # 1 # 14 # 1 # 2 # 3 # 5 # 7 # 1 # 14 Post RCC Pre RCC Post DC Pre DC Post RCC Pre RCC Post DC Pre DC Patient ID OFA # 1 # 2 # 3 # 5 # 7 # 1 # 14 G25 # 1 # 2 # 3 # 5 # 7 # 1 # 14 Post RCC Pre RCC Post DC Pre DC Post RCC Pre RCC Post DC Pre DC

Survival of Subjects immunized with Renal Tumor RNA Loaded DC 1..9.8.7.6.5.4.3.2.1 Proportion of Patients Surviving1. Renal tumor RNA loaded DC group N=1 5 1 15 2 25 3 Follow-up Time (Months) Gleave et al. N=188 N. Engl. J. Med. 338:1265, 1998

Mature, but not Immature RNA Loaded DC Elicit A Local Inflammatory Response at the Injection Site Candida Tetanus Injection Technique Trichophyton Mumps DC+PSA RNA Mature LAMP- RNA Transfected DC Immature PSA RNA Transfected DC

Mature, but not Immature Dendritic Cells are Capable of Migrating to Draining Lymph Nodes Landing Site 15 cm Injection Site

Telomerase (h) A Broadly Expressed Candidate Tumor Antigen h can be processed for class I presentation in a broad range of human tumors. Telomerase is an attractive candidate for a broadly expressed tumor rejection antigen Silent in most somatic tissues Reactivated and overexpressed in the majority of human solid tumors Reduced risk of antigen-escape tumor cell variants.

Targeting mrna-encoded antigens into the endosomal/lysosomal compartment A. pgem4z/h/a64 B. T7 promoter h aa1-1132 PolyA kb IVT h IVT h/lamp IVT positive control IVT negative control T7 promoter gp96 aa1-27 Leader sequence pgem4z/h- LAMP/A64 h aa168-1132 hlamp aa 382-416 PolyA 4.4 2.4 1.35 Cancer Research 62 (15): 5322-5328 (22)

mrna-transfected DC Clinical Trial Design Dose Schedule A: 3 cycles of 1x1 7 cells i.d. per cycle Dose Schedule B: 6 cycles of 1x1 7 cells i.d. per cycle Determine Eligibility Metastatic Prostate Cancer Informed Consent Leukapheresis RANDOMIZE RNA loaded DC LAMP RNA loaded DC Leukapheresis Follow-up Week Week 2 Week 4 Week 6 Week 8 Pre-Treatment Phase Treatment Phase Follow-up

Patient Characteristics Table 1. Characteristics of subjects enrolled Subject ID No. Age Karnofsky Index Diagnosis of Metastases - Treatment (Months) Stage (Jewett) Prior Therapy Pretreatment PSA (ng/dl) Metastases (Study Entry) Assigned Dose Level (Total Dose) Cell Product (mrna- Transfected DC) RTH-1-LMP 68 1 148 D2 RP/XRT 1 /H 4.5 BN 3x1 7 LAMP h RFB-2-TRT 75 9 14 D2 XRT 2 /H 1.6 BN 3x1 7 h DEM-3-LMP 7 1 56 D3 RP/H 54.3 LN/BN 3x1 7 LAMP h RNR-4-TRT 65 9 36 D3 RP/XRT 1 /O 1.7 LN 3x1 7 h BRH-5-TRT 5 1 21 D1 RP.3 LN 3x1 7 h DGE-6-LMP 63 1 9 D1 RP/XRT 1 7.3 LN 3x1 7 LAMP h GWN-8-TRT 58 1 71 D3 RP/XRT 1 /H/C 111.3 LN/BN 3x1 7 h JLA-9-TRT 47 1 26 D3 H 2.9 LN/BN 3x1 7 h TJL-1-LMP 64 9 64 D3 XRT 2 /H/C 6.4 BN/ST 3x1 7 LAMP h JDS-11-TRT 59 1 6 D2 H.4 BN 3x1 7 h JLB-12-LMP 62 8 22 D3 RP/XRT 1 /H/C 15.6 BN 3x1 7 LAMP h HTD-13-LMP 59 9 74 D1 RP/XRT 1 /H 4.3 LN 3x1 7 LAMP h JCS-14-LMP 63 8 11 D3 H 287.7 BN 6x1 7 LAMP h JRL-15-TRT 67 9 175 D3 RP/XRT 1 /H/O 11.7 BN 6x1 7 h TMS-16-TRT 59 1 96 D1 RP/XRT 1.1 LN 6x1 7 h JOG-17-TRT 68 9 55 D1 RP/H/C.9 LN 6x1 7 h AG-18-LMP 71 9 95 D3 H 38. BN 6x1 7 LAMP h FSH-19-LMP 52 1 58 D2 XRT 1 /H.4 LN/BN 6x1 7 LAMP h PEZ-2-TRT 72 1 6 D3 RP/H 21.7 BN 6x1 7 h CAH-21-TRT 57 9 9 D2 XRT 1/ XRT 2/ O.3 BN 6x1 7 h Pre-treatment: XRT 1, primary irradiation; XRT 2, local (palliative) irradiation for painful bony metastases; RP, radical prostatectomy; H, medical hormonal ablative therapy; C, chemotherapy; O, orchiectomy. Metastases: LN, lymphadenopathy; BN, bony metastases; ST, soft tissue metastases.

A. B. DTH Diameter (mm) 4 3 2 1 N=12 N=8 1 2 3 4 5 6 Vaccine Cycle Number C. D. IL-2 - TNF-α -α IFN-γ -γ IL-1 - IL-4 - IL-5 - LAMP-h h Specific Lysis (%) 5 4 3 2 1 CD3 CD4 CD8 DC+h DC+GFP / K562 5:1 1:1 2:1 4:1 5:1 1:1 2:1 4:1 Effector:Target

Stimulation of h-specific T-cell T responses After Vaccination with RNA transfected DC CD4 + T-Cell Frequency 12 RTH-1 1-LMP 9 6 3 12 12 DEM-3 3-LMP 9 6 3 12 CD4 + T-Cell Frequency 12 DGE-6 6-LMP 9 6 3 12 12 TJL-1 1-LMP 9 6 3 12 CD4 + T-Cell Frequency 12 JLB-12 12-LMP 9 6 3 12 12 HDT-13 13-LMP 9 6 3 12 CD8 + T-Cell Frequency 9 6 3 LAMP GFP 9 6 3 LAMP GFP CD8 + T-Cell Frequency 9 6 3 LAMP GFP 9 6 3 LAMP GFP CD8 + T-Cell Frequency 9 6 3 LAMP GFP 9 6 3 LAMP GFP CD4 + T-Cell Frequency 12 RFB-2 2-9 6 3 12 12 RNR-4 4-9 6 3 12 CD4 + T-Cell Frequency 12 BRH-5 5-9 6 3 12 12 GWN-8 8-9 6 3 12 CD4 + T-Cell Frequency 12 JLA-9 9-9 6 3 12 12 JDS-11 11-9 6 3 12 CD8 + T-Cell Frequency 9 6 3 LAMP GFP 9 6 3 LAMP GFP CD8 + T-Cell Frequency 9 6 3 LAMP GFP 9 6 3 LAMP GFP CD8 + T-Cell Frequency 9 6 3 LAMP GFP 9 6 3 LAMP GFP

Kinetics of the Antigen-Specific CD8 + T-cell Response Badovinac et al, Nat Immunol 4:212, 23

Longitudinal Evolution of CD8 + and CD4 + T cell Responses Spots x 1x1 5 T cells Spots x 1x1 5 T cells Spots x 1x1 5 T cells Spots x 1x1 5 T cells TMS-16-TRT 15 Immunization 1 2 3 4 5 6 12 9 6 CD8 3 CD4 Pre 1 2 3 4 5 6 7 8 9 1 11 12 13 21 JRL-15-TRT 18 15 Immunization 1 2 3 4 5 6 12 9 6 CD8 3 CD4 Pre 1 2 3 4 5 6 7 8 9 1 11 12 13 AJG-18-LMP 12 Immunization 1 2 3 4 5 6 9 6 3 CD8 CD4 Pre 1 2 3 4 5 6 7 8 9 1 11 12 16 FSH-19-LMP 12 Immunization 1 2 3 4 5 6 9 6 3 CD8 CD4 Pre 1 2 3 4 5 6 7 8 9 1 11 12 14 Study Week DTH Size (mm) DTH Size (mm) DTH Size (mm) DTH Size (mm) 3 2 1 1 2 3 4 5 6 3 2 1 1 2 3 4 5 6 3 2 1 1 2 3 4 5 6 3 2 1 1 2 3 4 5 6

Characterization of Vaccine-induced induced CD8 + T cells CD8 + h CD8 + LAMP h IL-2 IFN-γ TNF-α IL-2 IFN-γ TNF-α

Impact on PSA Doubling Time and Circulating Tumor Cells A. PSA mrna PSA Doubling Time (months) 1 5 3 2 1 B. 1 1 1 1 5 3 2 1 Three Weekly Doses (n=7) DGE-6-LMP 4.6 3.8 PRE POST -4 2 4 6 26 Study Period (weeks) 1 5 3 2 1 1 1 1 1 5 3 2 1 Six Weekly Doses (n=5) 2.9 BRH-5-TRT 1. PRE POST -4 2 4 6 26 Study Period (weeks)

Conclusions Powerful method of stimulating h-specific CD4 + and CD8 + T cell responses in cancer patients. Evidence that LAMP-h RNA transfected DC are capable of stimulating higher frequencies of h specific CD4 + T cells DTH reactions/elispot/cytolytic assays Induction of central T cell memory Lack of tolerance with increasing numbers of vaccinations. Impact on PSA doubling time and clearance of circulating tumor cells.

Elimination of Regulatory T cells Rationale Existence of CD4 + /CD25 + regulatory T cells in humans (Treg) that suppress immune responses to self- and tumor antigens. Some studies suggest increased levels of Treg in cancer patients. Antibody-mediated elimination of Treg has shown to elicit antitumor immunity in tumor-bearing mice. Anti-CD25 mab therapy was capable of enhancing the therapeutic effects of tumor vaccines.

Elimination of Regulatory T cells Approach DAB 389 IL-2 is a recombinant fusion protein that contains the catalytical- and membrane translocation domain of diphtheria toxin fused to human IL-2, allowing targeting of CD25 + cells. Enzymatic Domain Translocation Domain Receptor Binding Diphtheria Toxin A T B DAB 389 IL-2 A T hu IL -2

Human CD4 + CD25 + Regulatory T cells Definition A. CD4 + /CD25 + CD25 isotype B. CD4 + /CD25 neg CD45R CD45RA Stimulation Index (%) 1 75 5 25 Tetanus RCC RE (control) CD4 CD25 R1 R2 C. CD4 + /CD25 int CD45R CTLA-4 Stimulation Index (%) 1 75 5 25 1:4 1:2 1:1 1:4 1:2 1:1 1:4 1:2 1:1 Tetanus RCC RE (control) 1:4 1:2 1:1 1:4 1:2 1:1 1:4 1:2 1:1 CD4 D. CD4 + /CD25 high CD45R CTLA-4 Stimulation Index (%) 3 2 1 MLR MLR+ MLR+ Treg (1:1) Treg (1:5) DC+ Treg

Enhancement of T-cell T Immunity after T reg Depletion Percent CD4/CD25 Viability 75 5 25 A. B. CD4 + /CD25 high PBMC±CD4 + /CD25 high 1 [nm] 24 48 24 48 Time (hours) Time (hours) C. [.5nM] [.5nM] [5nM] PBMC PBMC+ DAB [5nM] PBMC+ Treg+ DAB [5nM] 1 5 25 PBMC - + DAB h RNA flum1 RNA MART1 RNA flum1 pep MART1 pep Stimulatory Index 75 DC DC DC +Treg +Treg +DAB +DAB Specific Lysis [%] 6 5 4 3 2 1 1 2 4 1 2 4 1 2 4 1 2 4 E:T Ratio 1 2 4

Monitoring for Regulatory T cells in a Vaccination Setting CD4 97.8 CD4 1.3 CD4 PMA/Ionomycin CD4 allogeneic MLR 88.3 9.6 93. 4.9 CD25.2 1. 1.3 1.2 M1 M1 M1 M1 Isotype.2 GITR 3. 7.6 4.2 Isotype CTLA-4

Depletion of CD4 + /CD25 high Regulatory T cells After DAB IL-2 2 Administration DAB 386 IL Patient: HMT-4-DAB CD4 pre post (4d) post (28d) 4.6 1.6 3.4 CD25.4 71.9 76.5 77.8. 3.6.8 2.4 Isotype GITR

Depletion of CD4 + /CD25 high Regulatory T cells After DAB IL-2 2 Administration DAB 386 IL Patient: JB1-RCC ONTAK 18µg/kg + RCC RNA DC (2 doses) 94.41 4.54 Post 2 nd vaccination Stimulation Index (%) A. 1 5 25 2 C. Antigen-specific Proliferation RCC 75 Pre DAB Post B. Influenza CMV Tetanus Pre DAB Post Pre DAB Post Pre DAB Post A. INF-γ ELISPOT (CD4 + T Cells) CD4 CD4 97.54 1.67 Pre 1 st vaccination =75% depletion efficacy Spots/1x1 5 CD4 + T cells 15 1 5 PBMC Influenza CMV Lysate Tetanus Toxoid CD25-isotype 92.56 6.46 CD25 Pre vaccination Spots/1x1 5 CD4 + T cells Pre DAB Post Pre DAB Post Pre DAB Post Pre DAB Post C. Ḃ. B. INF-γ ELISPOT (CD8 + T Cells) 2 PBMC 15 1 5 Influenza CMV Lysate Tetanus Toxoid Pre DAB Post Pre DAB Post Pre DAB Post Pre DAB Post

Efficacy of Depleting Regulatory T cells in Metastatic Cancer Patients JBC-1-DAB 6. 5. Percent CD4 + T Cells 4. 3. 2. 74% HM-2-DAB JVG-3-DAB 37% HMT-4-DAB 77% 1. 88% Pre Post Pre Post Pre Post Pre Post Marker Cell Type Single Positive (% positive of PBMC) CD25 Double Positive (% of Single Positive) Pre Post Pre Post CD4 T cells 28. 25. 42.8 4. CD8 Macrophages 17. 19. 16.5 22.1 CD14 Monocytes 13.9 15.4 1. 1.7 CD19 B cells 27.3 24.6 15.4 13.4 CD56 NK cells 18.9 21.3 - - CD69 NK/effector cells 21.2 19.6 7.8 9.2

Elimination of T reg is Capable of Enhancing Vaccine-mediated T-cell T Responses A. Spots per 1x1 5 CD8 + T cells B. Spots per 1x1 5 CD8 + T cells 1 75 5 25 1 75 5 25 WHD-1 1-RCC RCC PBMC RCC PBMC RCC PBMC RCC PBMC JBC-1 1-DAB RCC RE JOG-2 2-RCC HM-2 2-DAB RCC PBMC JWV-3 3-RCC JVG-3 3-DAB OVA PBMC WB-DAB HMT-4 4-DAB RCC PBMC

c. 1 75 5 25 1 75 5 25 4-TRT 5-TRT 6-TRT JVG-3 3-DAB FHS-5 5-DAB HWH-6 6-DAB OVA PBMC TRT GFP Spots/1x1 5 CD8 + T cells Spots/1x1 5 CD8 + T cells d. TRT GFP TRT GFP TRT GFP TRT GFP

Elimination of Regulatory T cells Conclusions I NIH and FDA-approved clinical trial. Demonstration of selective Treg depletion following single dose of DAB 389 IL-2. Enhancement of T cell responses in vitro, predominantly against naturally processed self-antigens. Safety, no clinical signs of autoimmunity in 1 patients treated thus far.

Elimination of Regulatory T cells Conclusions II No interference with CD4 + /CD25 int memory T cell pool. Stimulation of high frequencies of RCCspecific T cells in vivo after combined therapy. Polarization of RCC-specific CD4 + T cells towards Th-1, but not Th-2. This strategy could have broad implication for the design of active and passive immune-based protocols.