immuno-oncology and breast cancer scientific realization ...€¦ · established from a non...

Immuno-Oncology and Breast Cancer

Scientific Realization within the PRAEGNANT Network

Peter A. Fasching

Immuno-Oncology hat vieleEinstiegsmöglichkeiten (Adams et al. 2015)

Immuntherapien

n Rekombinante Vakzinen Zytokine

n Immun – Checkpoint Inhibitorsn Zelluläre Therapien (T-Zellen / NK- Zellen)n Antikörpertherapien

n Chemotherapien Bestrahlungn Small Moleculen Hormontherapie

Radiotherapie

Chemotherapie

PRAEGANT Distribution (Basket in and basket out)

„PerFect“Pertuzumab

„Seraphina“Nab-Pclitaxel Weitere Studien

Regular BC Patients

„EMBRACA“PARP-I

„ABRAZO“PARP-I

Immun-onkologische

Studien

Immuntherapien




Treatments for HER2-positive Patients

Adjuvant orneoadjvuant 1st Line 2nd Line 3rd Line ≥ 4th Line

Diagnosis Metastases

TrastuzumabChemotherapy

TrastuzumabPertuzumabDocetaxel

T-DM1 No Approved Therapy

See National TherapyRecommendations

No Approved Therapy

T-DM1

Lines of Therapy for Metastatic Disease

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National Therapy Recommendations

Experimental considered

equivalent to any other treatment

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Immuntherapien


n Immun – Checkpoint Inhibitorsn Zelluläre Therapien (T-Zellen / NK- Zellen)n Antikörpertherapienn Chemotherapien Bestrahlungn Small Moleculen Hormontherapie

Antibody Dependent Cellular Cytotoxicity (ADCC)

NK cells are Critical Effector Cells for Monoclonal Antibodies through ADCC

CD16’s Allele Prevalence

F/F 176 V/F 176 V/V 176

64 (42%) 76 (50%) 12 (8%)91 (50%) 71 (39%) 19 (11%)

Allelic variation analyzed in 330 normal individuals (45% African–American (top line), 55% Caucasian (bottom line)).

There is a Low Incidence of V/V in the Normal Population

Lehrnbecher et al, Blood 1999:, 94, 4220

• Patients with naturally occurring high affinity receptors for the Fc fragment havean improved outcome under Herceptin/Rituximab/Cetuximab treatment.

• Same holds true for treatment of multiple myeloma with Elotuzumab(Progression Free Survival 22.3 months vs. 9.8 months (presented at ASH 2015)

Rituximab Trastuzumab Cetuximab

Rationale for combining haNK with Antibodies

• Weng W-Ket al. J Clin Oncol 25:3712-3718, 2007

• Musolino A et al. J Clin Oncol. 2008 Apr 10;26(11)

• Zhang W J Clin Oncol 25:3712-3718, 2007

Antitumoral activity of NK cells

Downregulation of MHC IUpregulation of activating ligands

MICA, MICB

NKG2D

Ljunggren et al. 2007

Abken, Wels & Kühlcke 2014

CAR

1st Generation2nd Generation3rd Generation

T-cell receptor complex

Chimeric antigen receptors (CARs)

Genetically engineered NK-92 cellswith built-in ADCC-like activityCAR-NK-92 (taNK) The human NK cell line NK-92

as an off-the-shelf cellular therapeutic

• IL-2 dependent, continuously growing NK cell line established from a non-Hodgkin lymphoma patient in 1992

(H. Klingemann)

• Highly cytotoxic to leukemia, lymphoma and melanomadue to lack of inhibitory KIR receptors

• Safety of infusion of irradiated NK-92 demonstrated in phase I clinical studies in cancer patients with advanced

disease Tonn et al. 2001, Arai et al. 2008, Tonn et al. 2013

NK-92

Chimericantigen receptor

Target antigen

ErbB2/HER2EGFR/EGFRvIII

EpCAMGD2CD20CD19

Uherek et al. 2002Müller et al. 2008Sahm et al. 2012Esser et al. 2012

Boissel et al. 2013Schönfeld et al. 2015Romanski et al. 2016

Zhang et al. 2016Genßler et al. in press

Antitumoral activity of NK cells(Schönfeld et al. 2015)

Immuntherapien




Existence of an effective T-cell immuneresponse against cancer in humans

n Tumor-infiltrating T cells (TIL)are associated with betterprognosis in triple-negativebreast cancer

n Mutated tumor antigens andneo-peitopes can elicit highavidity, tumor-specific T-cellresponses

n T-cell based immunotherapyand possibly vaccinations arevery and promising newtreatment options for malignantdiseases.

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S. Adams et al. , J. Clin Oncol. 32:2959, 2014

L.B. Alexandrov et al., Nature, 2013

Prognostic relevance of tumor infiltrating lymphocytes

Mutation prevalence in tumors sorted by organs

T-Cell Cultivationfrom Tumor infiltratingLymphocytes

High quality DNA isolation from freshtumor after pathologicalassessment

DNA isolation fromblood

12

4

Sequencing

Bioinformatics and determinationof antigen-likely sequences

Synthesis of likely antigenicpeptides

6

7

8

5

3

Transfer of DNA togenotyping corefacility

B-CellCultivationfrom Blood

Transfer of peptidesto cell culture lab

9

Peptide Loading of B-Cells

10

11

Co-Cultivation of B-Cells and T-CellsMeasurement of T-CellResponse

AIMIdentification of T-cellstargeting tumor-specific

neoantigens among

tumor-infiltratinglymphocytes in triple

negative breast cancer

Working Programme

Ex vivo immune monitoring of infiltrating lymphocytes

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Antigen Fluorochrom Zellen

CK19 FITC Tumorzellen

MUC-1 PE-Cy7 Tumorzellen

CD3 V500 T-Zellen

CD4 BV421 T-Helferzellen

CD8 APC-Cy7 Zytotox.T-Zellen

FoxP3 APC Regulat.T-Zellen

CD19 PerCP B-Zellen

CD56 PE NK-Zellen

gated onlymphocytes gated onCD3+ gated onCD3+B C DA E [Ungated]MUC-1/CD3

Whole Genome Sequencing

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n 72 patients includedn 21 patients completed whole genome sequencing of tumor

and germline and RNAseqn HLA Typing prediction based on whole genome sequencing

alignment.n Results filtered based on RNA Expressionn Neoepitopes selected and ranked by

(transcripts per million) x (allele frequency of variant)

n Results: § About 40 neoepitopes predicted binders for MHC Class 1

Alleles§ About 100 neoepitopes predicted for MHC Class 2 Allleles

(to be selected to B-Cell Assays)

Neo-Epitope Aminoacid-Sequence

T-Cell response assessment in vitro

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A B C Dgated onlymphocytes gated onCD3+ gated onCD3+

mutated peptideNeo-antigen

autologous EBV-LCL

+ TILs

sort on activated T-cells

CD137

CD137

CD4

neg. HeLa-Ii HeLa-Ii/DM

CD4

IFN-γ

IFN-γ

CD

4

CD137

Flow cytometric analysis of expanded TILs (day 14)

Expansion of tumor-infiltrating T-lymphocytes

Characterization of T-cell

responses against identified tumor-specific mutations

Madan et al. 2012

Immuntherapien




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