Jeffrey Schlom, Ph.D. Laboratory of Tumor Immunology and Biology (LTIB) Center for Cancer Research National Cancer Institute, NIH

Enhancing Anti-Tumor Activity of

Checkpoint Inhibition

J. Hodge C. Palena K.Y. Tsang J. Greiner R. Donahue S. Gameiro D. Hamilton C. Jochems C. Heery

J. Gulley R. Madan R. Hassan A. Rajan

LTIB Senior Staff NCI Collaborators

Laboratory of Tumor Immunology and Biology (LTIB) Center for Cancer Research, NCI

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Avelumab

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Fully human IgG1 anti-PD-L1 monoclonal antibody Engineered to mediate Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

Cooperative Research and Development Agreement (CRADA) − Laboratory of Tumor Immunology and Biology,

National Cancer Institute, NIH − EMD Serono, Pfizer

mAb Mechanisms of Action Direct Effect on Receptor Function

Ribas, New Engl J Med. 2012; 366(26):2517-9. 4

Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

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Pros and Cons of ADCC Activity of an anti-PD-L1 MAb

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Con: − toxicity of subpopulations of immune cells expressing PD-L1

- not observed to date Pro:

− 2 different methods of anti-tumor activity (a) interfering with PD-L1/PD-1 axis (T cells) (b) direct lysis of tumor cells (ADCC/NK)

− can enhance ADCC activity by enhancing NK activity - IL2 (low dose) - IL12 (immunocytokine) - IL15 (multiple forms) - other immune modulators

- some small molecule targeted therapies alter tumor cell phenotype enhancing NK anti-tumor activity

Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and anti-Tumor Activity

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ADCC has been implicated as a mode of action in highly effective anti-tumor MAbs

− targets all expressed on some normal tissues − some degree of toxicity − highly effective

*Multiple prior studies: Specific polymorphisms in the FcγR2A and 3A of NK cells correlates with enhanced clinical outcomes

Target

Trastuzumab (Herceptin)* Her 2/neu Rituximab (Rituxin)* CD20 Cetuximab (Erbitux)* EGF-R

Each dot represents lysis of a different human tumor cell line. A, Correlation between % PD-L1 positive tumor cells and % ADCC lysis (p<0.0001, r=0.799). B, Correlation between PD-L1 tumor cell MFI and % ADCC lysis (p<0.0001, r=0.811). C, Correlation between the PD-L1 score and % lysis (p<0.0001, r=0.826). The PD-L1 score was derived by scoring each cell line for % positive cells and normalized MFI on a quartile scale ranging from 1–4. All correlations show the p value and Spearman’s rank correlation coefficient.

PD-L1 MFI is a Stronger Predictor of Sensitivity to ADCC Mediated by Avelumab as Compared to % Positive Cells

AA. % PD-L1+ vs ADCC AC. PD-L1 score vs ADCC AB. MFI vs ADCC

Isolated NK cells from 5 healthy donors and 5 lung cancer patients were used in in vitro ADCC assays against the H441 human lung cancer cell line.

Purified NK Cells from Cancer Patients Mediate ADCC Induced by Avelumab as Effectively as

Those Isolated from Healthy Donors

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Human lung cancer cell line as target and purified NK cells as effectors.

ADCC Mediated by Avelumab Can Be Inhibited by anti-CD16 Antibody

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Tumor shrinkage seen to date:

Phase I Trial of a Novel anti-PDL1 Checkpoint Inhibitor

Rare diseases (NCI): Thymoma Adrenal Cortical Cancer Mesothelioma Common diseases (NCI and in multi-center study): Lung adenocarcinoma Melanoma Breast cancer Ovarian cancer Cessation of tumor growth seen to date: Rare diseases (NCI): Chordoma Adrenal Cortical Cancer Common diseases (NCI): Colorectal cancer Pancreatic cancer HPV+ anal cancer Cholangiocarcinoma Clear cell renal cancer

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Subsets analyzed: 9 standard immune cell subsets, PD-L1 and PD-1 in standard subsets, and 100 additional subsets relating to maturation/function 1. CD4: Helper T lymphocytes (32 subsets) 2. CD8: Cytotoxic T lymphocytes (29 subsets)

• Markers of PD-1 pathway and T cell activation (in CD4 and CD8): – EOMES: activation – TCR-αβ: activation – Tbet: activation – BATF: activation/exhaustion

• Maturation status of T lymphocytes (in CD4 and CD8): ‒ Naïve: CD45RA+ CCR7+ ‒ Central Memory: CD45RA- CCR7+ ‒ Effector Memory: CD45RA- CCR7- ‒ Terminal (EMRA): CD45RA+ CCR7-

• T lymphocyte markers (in CD4 and CD8): – CTLA-4: inhibition – PD-1: activation/inhibition – PD-L1: activation/cross-inhibition – TIM-3: inhibition – ICOS: activation (only on CD4)

3. Tregs: Regulatory T lymphocytes (CD4+ CD25+ FoxP3+ CD127-) (7 subsets) – CD45RA: Tregs highly expandable in vitro – CTLA-4: Treg suppression – CD49d: “contaminating” effector lymphocytes (non-Tregs) – ICOS: Treg suppression – PD-1: activation/inhibition – PD-L1: cross-inhibition

4. B lymphocytes: CD19+ (5 subsets)

– CTLA-4: inhibition – TIM-3: inhibition – PD-1: activation/inhibition

– PD-L1: cross-inhibition 5. NK: Natural killer cells (CD56+ CD3-) (20 subsets)

– CD16+ CD56br: Functional intermediate, lytic and cytokine production

– CD16+ CD56dim: Mature NK, cytokine production – CD16- CD56br: Immature, abundant in human placenta – CD16- CD56dim: non-lytic, non-cytokine production – TIM-3: activation – PD-1: activation/inhibition – PD-L1: cross-inhibition

6. NK-T: CD56+ CD3+ (4 subsets)

– TIM-3: activation – PD-1: activation/inhibition – PD-L1: cross-inhibition

7. cDCs (Conventional DCs): CD3-CD56-CD1c+CD303- (5 subsets) 8. pDCs (plasmacytoid DCs ): CD3-CD56-CD1c-CD303+ (5 subsets)

• Markers of DC activation ‒ CD83: activation ‒ TIM-3: inhibition ‒ PD-1: activation/inhibition ‒ PD-L1: cross-inhibition

9. MDSCs: Myeloid-derived suppressor cells (CD11b+ HLA-DRlow/- CD33+) (20 subsets)

– CD14: Common Myeloid Marker (high in monocytes, dim in granulocytes)

– CD15: Granulocyte marker – CD16: most immature monocytic MDSCs – PD-1: activation/inhibition – PD-L1: cross-inhibition

Flow Cytometry Analysis of PBMC Immune Subsets 30 markers, 127 subsets

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1 and 3 mg/kg anti-PDL1

10 mg/kg anti-PDL1

20 mg/kg anti-PDL1

= Normal ALC range

Coun

t x10

3 /µL

Analyses of PBMCs During anti-PDL1 Therapy

Absolute Lymphocyte Count

Changes vs. pre = =

Subsets Normal

individuals (n=6)

Pre (n=22)

Day 15 (n=19)

Day 43 (n=14)

CD8 0.2 (0.1-0.4) 0.3 (0.3-0.4) 0.3 (0.2-0.4) 0.3 (0.2-0.4)

CD4 0.2 (0.1-0.3) 0.3 (0.2-0.4) 0.3 (0.2-0.5) 0.3 (0.2-0.3)

Treg 0.3 (0.2-0.4) 0.4 (0.3-0.6) 0.4 (0.3-0.6) 0.4 (0.3-0.5)

NK 0.7 (0.5-1.1) 1.2 (1.0-1.7) 1.2 (0.9-1.8) 1.1 (1.0-1.4)

NK-T 4.5 (1.8-7.6) 1.8 (1.4-3.2) 1.6 (1.3-3.7) 1.5 (1.3-2.9)

B cells 11.7 (8.1-13.4) 9.9 (7.0-11.4) 11.3 (7.4-12.6) 11.0 (7.7-12.4)

cDCs 14.0 (11.5-17.0) 23.4 (17.7-40.1) 26.4 (16.2-45.9) 19.4 (15.1-26.4)

pDCs 3.9 (2.7-8.3) 33.8 (18.0-48.2) 26.6 (12.2-42.2) 27.9 (19.1-56.7)

MDSCs 4.0 (3.0-5.6) 8.6 (6.5-9.8) 8.8 (5.0-15.0) 10.7 (4.6-13.7)

Values indicate % median (25-75 percentile)

There were no statistically significant changes at day 15 and day 43 compared to pre-therapy in the above listed subsets.

First-in-human Phase I Study of Fully Human IgG1 anti-PDL1 in Patients with Metastatic Cancer

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Immune Cell Subsets Expressing PD-L1

Pros and Cons of ADCC Activity of an anti-PD-L1 MAb

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Con: − toxicity of subpopulations of immune cells expressing PD-L1

- not observed to date Pro:

− 2 different methods of anti-tumor activity (a) interfering with PD-L1/PD-1 axis (T cells) (b) direct lysis of tumor cells (ADCC/NK)

− can enhance ADCC activity by enhancing NK activity - IL2 (low dose) - IL12 - IL15 (multiple forms) - other immune modulators

- some small molecule targeted therapies alter tumor cell phenotype enhancing NK anti-tumor activity

B

All data are shown as mean ± SEM for administration of IL-15 3 μg/kg (n = 4; all panels) or (A, B) 0.3 μg (n = 9). All fold-change values were computed by individual relative to baseline (Pre). Light blue bars indicate IL-15 infusion times. (A) Absolute count for total natural killer (NK) cells. (B) Representation within NK cells. P values are from two-tailed Student's t test for peak time point.

NK Cell Dynamics During and After Daily Interleukin-15 (IL-15) Infusions

Conlon, J Clin Oncol. 2015; 33(1):74-82. 15

H460 Cells Express MUC1, Are Resistant to MUC1-Specific CTL, but Are Sensitive to ADCC

MUC1 CTL Muc1+ HLA-A24

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NK ADCC

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T Cell vs. NK Tumor Cell Lysis

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Expression of MHC Class I on tumor cells:

‒ enhances CTL activity

‒ reduces NK activity

Lack of, or minimal, MHC Class I on tumor cells:

‒ reduces or eliminates CTL activity

‒ enhances NK activity

Pros and Cons of ADCC Activity of an anti-PD-L1 MAb

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Con: − toxicity of subpopulations of immune cells expressing PD-L1

- not observed to date Pro:

− 2 different methods of anti-tumor activity (a) interfering with PD-L1/PD-1 axis (T cells) (b) direct lysis of tumor cells (ADCC/NK)

− can enhance ADCC activity by enhancing NK activity - IL2 (low dose) - IL12 (immunocytokine) - IL15 (multiple forms) - other immune modulators

- some small molecule targeted therapies alter tumor cell phenotype enhancing NK anti-tumor activity

Recombinant Vaccines

Cytokines/Immunocytokines

Immune Checkpoint Inhibitors

Spectrum of Cancer Immunotherapeutics

Immuno-Oncology Platform: Combination Therapies

Chemotherapy

Radiation Therapy

Small Molecule Targeted Therapies - Hormonal Therapy