Preclinical Safety Testing Of Enhanced-Affinity TCRs Andrew J ez - - PowerPoint PPT Presentation

CONFIDENTIAL

Preclinical Safety Testing Of Enhanced-Affinity TCRs

Andrew ‘Jez’ Gerry Director of Preclinical Research EMA, 15-16 Nov 2016

This presentation contains “forward-looking statements,” as that term is defined under the Private Securities Litigation Reform Act of 1995 (PSLRA), which statements may be identified by words such as “believe,” “may”, “will,” “estimate,” “continue,” “anticipate,” “intend,” “expect” and other words of similar meaning. These forward-looking statements involve certain risks and uncertainties. Such risks and uncertainties could cause our actual results to differ materially from those indicated by such forward-looking statements, and include, without limitation: the success, cost and timing of our product development activities and clinical trials; our ability to submit an IND and successfully advance our technology platform to improve the safety and effectiveness of our existing TCR therapeutic candidates; the rate and degree of market acceptance of T-cell therapy generally and of our TCR therapeutic candidates; government regulation and approval, including, but not limited to, the expected regulatory approval timelines for TCR therapeutic candidates; and

• ur ability to protect our proprietary technology and enforce our intellectual property rights; amongst others. For a further

description of the risks and uncertainties that could cause our actual results to differ materially from those expressed in these forward-looking statements, as well as risks relating to our business in general, we refer you to our Quarterly Report on Form 10Q filed with the Securities and Exchange Commission (SEC) on August 8, 2016 and our other SEC filings. We urge you to consider these factors carefully in evaluating the forward-looking statements herein and are cautioned not to place undue reliance on such forward-looking statements, which are qualified in their entirety by this cautionary

• statement. The forward-looking statements contained in this presentation speak only as of the date the statements were

made and we do not undertake any obligation to update such forward-looking statements to reflect subsequent events

• r circumstances. We intend that all forward-looking statements be subject to the safe-harbor provisions of the PSLRA.

DISCLAIMER

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• The TCR is the natural mechanism for T-cells to distinguish a diseased cell

from a healthy cell

• All proteins, including intracellular ones, are processed and presented as HLA-

peptide complexes which are recognized by TCRs

• Many cancer targets are intracellular – TCR therapeutics can access these

targets

TCRs recognize intracellular cancer antigens

Adaptimmune focuses on developing the best affinity enhanced T cell therapies for autologous T-cell therapeutics

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Engineering Better T-Cells Challenges With TCR Therapy

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Four components to an effective adoptive therapy:

1.

T cell must recognize a cancer cell via a guiding receptor

2.

The guiding receptor must have two important aspects

 Affinity  Specificity

ADOPTIVE T CELL - Most powerful unit in Immunotherapy

Challenges with TCR Therapy

5 Cancer cell Apoptosis

T cell Armory Perforin Granzyme

T cell Cancer cell

 Affinity  Specificity

T cell

1. 2.

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Four components to an effective adoptive therapy:

1.

T cell must recognize a cancer cell via a guiding receptor

2.

The guiding receptor must have two important aspects

 Affinity  Specificity 3.

The T cell needs to be resistant to suppression

ADOPTIVE T CELL - Most powerful unit in Immunotherapy

Challenges with TCR Therapy

6 T cell Cancer cell

Inhibitory mechanisms

• f cancer cells

make T cells insensitive Make T cells resistant to suppression

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Four components to an effective adoptive therapy:

1.

T cell must recognize a cancer cell via a guiding receptor

2.

The guiding receptor must have two important aspects

 Affinity  Specificity 3.

The T cell needs to be resistant to suppression

4.

The T cell (either alone or via

• ther mechanisms) needs to

‘break cancer immune tolerance’

ADOPTIVE T CELL - Most powerful unit in Immunotherapy

Challenges with TCR Therapy

7 T cell Cancer cell

Make T cells resistant to suppression Epitope spreading Epitope spreading

1 2 3 4

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Four components to an effective adoptive therapy:

1.

T cell must recognize a cancer cell via a guiding receptor

2.

The guiding receptor must have two important aspects

 Affinity  Specificity 3.

The T cell needs to be resistant to suppression

4.

The T cell (either alone or via

• ther mechanisms) needs to

‘break cancer immune tolerance’

Integrating TCR research and development

Alignment of Multiple disciplines

8 Treating Patients

Making TCRs Engineering T cells Target identification and validation Clinical and Regulatory CMC Safety Testing (Preclinical) Translational Sciences Engineering TCRs

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ADAPTIMMUNE SPEARTM T-cell PLATFORM

uniquely overcomes these hurdles

• Target Identification
• TCR Identification
• TCR Engineering – Optimized Affinity
• TCR Safety Testing
• Generation 2 T cells

Specific Peptide Enhanced Affinity Receptor

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Engineering Better T-Cells Platform Technology

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Research Pipeline

Research Pipeline

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Engineering Better T cells

T Cell Receptor (TCR) Engineering

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MHC Peptide antigen TCR

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Specificity and non-specificity

Types of safety signal

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e.g. another family member, or Titin

• Preclinical package covers in vitro potency, safety and specificity
• In vivo animal models are not informative for assessing TCR specificity

and safety for a number of reasons

– Mainly due to MHC and proteome mis-matches

• Following 2 SAEs on MAGE-A3a3a protocol, we developed a battery of

tests that cover parallel approaches to identifying alternate reactivities

– Molecular characterisation of TCR:peptide binding preferences to

generate a motif for searching against the proteome for potential

cross-reactive peptides

– Screening cells, tissues and cellular models for actual cross-

reactivities

Adaptimmune’s preclinical safety package

Primary aim to identify potential on- and off-target reactivities

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[Cameron, Gerry et al, Sci Trans Med, 2013, Linette et al, Blood, 2013]

Molecular Analysis Human Cell Testing Potency/efficacy

Adaptimmune’s preclinical safety package

Primary aim to identify potential on- and off-target reactivities

X- Scan

Motif BLAST Fresh tissue, organotypic and iPS models (Key tissues) Standard Cell Screen (>100 cell types) Peptide family members 2D cell line cytokine response and cytotoxicity 3D cell-lines Motif Peptide Screen Motif Mass Spec Search Whole Blood Assay Allo-reactivity assay Primary tumours (if available) Antigen-driven proliferation Overexpression studies Clinical Candidate TCR

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• 1. T cell must recognize a

cancer cell via a guiding receptor

Engineering better T cells

Platform technology

16 Cancer cell Apoptosis

T cell Armory Perforin Granzyme

T cell Cancer cell

 Affinity  Specificity

T cell

1. 2.

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1ST select the right TARGETS ….

Engineering Better T Cells

The spectrum of potential cancer targets for immunotherapy

see http://cancerimmunity.org/peptide/ for a list of tumour antigens reported in the literature

17 Cancer testis antigens expression restricted to immune- privileged tissue

e.g. MAGE family / NY-ESO

Differentiation antigens tissue restricted expression

e.g. Tyrosinase / gp100

Overexpressed antigens

• verexpressed in tumour cells

e.g. WT1 / telomerase

Tumour specific antigens not expressed in normal tissues

Viral antigens e.g. EBV/HPV Mutated antigens e.g p53 Neo-antigens

Ideal Good Depends on tissue Depends on extent of normal tissue expression

Ubiquitous antigens expressed in all cells

e.g. Her2/neu

Unlikely to be suitable

tumour selectivity Prevalence

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Engineering Better T Cells

Finding the Right Targets

19 Tumour cell

Mass spectrometry

Confirms surface expression and expression on tumour cells (i.e. not normal tissue)

Only low risk targets selected for TCR programs

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Engineering Better T Cells

Finding the Right Targets

19 Tumour cell

Mass spectrometry

Confirms surface expression and expression on tumour cells (i.e. not normal tissue)

Only low risk targets selected for TCR programs

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Molecular Analysis Human Cell Testing Potency/efficacy

Adaptimmune’s standard preclinical package

Assessing safety

X- Scan

Motif BLAST Fresh tissue, organotypic and iPS models (Key tissues) Standard Cell Screen (>100 cell types) Peptide family members 2D cell line cytokine response and cytotoxicity 3D cell-lines Motif Peptide Screen Motif Mass Spec Search Whole Blood Assay Allo-reactivity assay Primary tumours (if available) Antigen-driven proliferation Overexpression studies Clinical Candidate TCR

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• Exchange of each aa for all other possibilities to generate a binding

motif

• Searching with the motif against the human genome

–

[ACFGIKLMNQRSTVWY]-[ACGILMQSTVY]-[DNT]-[KR]-[FW]-[CEGHILMNQTVW]-[FHNQSTVWY]-[CDEGMNQS]-[AFILMTV]

Peptide screening ‘X-scan’ (AFP SPEAR T-cells)

TCR peptide recognition mapping using combinatorial amino acid substitutions

p1 p2 p3 p4 p5 p6 p7 p8 p9

XMNKFIYEI FXNKFIYEI FMXKFIYEI FMNXFIYEI FMNKXIYEI FMNKFXYEI FMNKFIXEI FMNKFIYXI FMNKFIYEX

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• Requires assessment of

tissue distribution of other ‘targets’

Analysis of peptide recognition – Family members

How well are peptides recognised?

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• Non-related proteins derived from X-scan motif proteome search
• Over-express and screen for T cell reactivity
• If other peptides are recognised, a risk assessment is required on

those proteins

Analysis of peptide recognition – Mimotypes

How well are peptides recognised?

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Molecular Analysis Human Cell Testing Potency/efficacy

Adaptimmune’s standard preclinical package

Assessing safety

X- Scan

Motif BLAST Fresh tissue, organotypic and iPS models (Key tissues) Standard Cell Screen (>100 cell types) Peptide family members 2D cell line cytokine response and cytotoxicity 3D cell-lines Motif Peptide Screen Motif Mass Spec Search Whole Blood Assay Allo-reactivity assay Primary tumours (if available) Antigen-driven proliferation Overexpression studies Clinical Candidate TCR

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Alloreactivity screen (AFP SPEAR T-cells)

N o ta rg e t 2 3 0 6 9 9 B P O T 1 3 3 1 -8 2 3 4 B R IP 1 3 3 3 -8 2 7 6 C G P 0 4 1 3 4 9 -8 3 9 6 C V I6 1 8 4 1 4 1 6 -1 1 9 1 D U G 1 5 0 A A S 1 2 5 A H T 1 9 2 F B 4 6 6 L C L F B 4 6 9 L C L F H 2 4 F B 5 7 2 L C L F H 2 5 F B 5 7 8 F H 2 8 F H 1 0 F H 3 6 F H 1 8 F H 2 1 F H 3 9 F H 2 3 F H 4 1 F H 4 2 F H 7 5 F H 4 3 F H 7 7 F H 8 F H 4 6 F H 5 3 IS H 3 F H 5 8 IS H 4 F H 6 IS H 5 F H 6 7 J0 5 2 8 2 3 9 K T 1 4 O L G A L S R 5 7 0 2 R M L M W X 3 8 9 1 M Y E 2 0 0 1 S C L -1 1 6 A M Y E 2 0 0 2 T 7 5 2 7 M Y E 2 0 0 4 T IS I H e p G 2

2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0

W 8 5

IF N y p g /m l W 8 5 / n td W 8 5 /td

ntd AFP TCR

Alloreactivity assay

Positive control

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• Looking for response against another unidentified peptide on a

different HLA

 Screen T cells against panel of 55 EBV-transformed B cells expressing

a wide range of different HLAs

 38 HLA-A, 63 HLA-B and 28 HLA-C

• AFP SPEAR T-cells showed response to 2 cell lines

 Express unique alleles HLA-B*1501 and C*0404 – clinical exclusions

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• HLA-A*0202 alloreactivity in pericytes

Alloreactivity screen (AFP SPEAR T-cells)

There may be lineage-specific alloreactivities

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Molecular Analysis Human Cell Testing Potency/efficacy

Adaptimmune’s standard preclinical package

Assessing safety

X- Scan

Motif BLAST Fresh tissue, organotypic and iPS models (Key tissues) Standard Cell Screen (>100 cell types) Peptide family members 2D cell line cytokine response and cytotoxicity 3D cell-lines Motif Peptide Screen Motif Mass Spec Search Whole Blood Assay Allo-reactivity assay Primary tumours (if available) Antigen-driven proliferation Overexpression studies Clinical Candidate TCR

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• Bank of primary cells, covering multiple organ systems and cell types

– Over 100 non-fetal cell types (multiple donors sources of each if possible)

 Primary normal cells, low passage (2 to ~10)  Tumour lines, generally high passage

– Coverage is boosted by tumour cell lines, but

 Majority are epithelial.  Risk of genetic instability.

Safety assessment – Cell and tissue screen

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Safety assessment – Cell and tissue screen

Over 100 cells covering multiple key lineages and tissues (selected examples)

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• Respiratory
• Digestive
• Genito-urinary
• Musculoskeletal
• Endocrine
• Lymphatic
• Cardiovascular
• Nervous (CNS and PNS)

Endothelium Astrocytes Neurons Pericytes Epithelium, Schwann cells iPS neurons Epithelium Pneumocytes Fibroblasts Endothelium Smotth muscle Fibroblasts Myocytes Arterial and venular endothelium Smooth muscle Whole blood and cell subsets Thyroid fibroblasts Adrenal cortical cells Preadipocytes Pancreatic Islets Renal proximal and distal epi Glomerular epi Mesangial Melanocytes Keratinocytes Skeletal muscle cells and myoblasts Chondrocytes Osteocytes Follicular papillary cells Hepatocytes Kuppfer cells Biliary epithelium Stellate cells Tonsil epi Splenocytes Lymphocytes

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Cellular Dynamics Inc. iCell Cardiomyocytes

Cells, tissues and models - Cardiovascular

iPS CM - Spontaneously electrically active and contractile (beating) myocytes

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• Improved assay formats and HCS equipment

available

• Assess safety and potency of TCRs in the same well

– co-culture and multicolour fluorescently labelled

Ag+ cell lines or primary tumour material and Ag- primary cells from the same tissue with T cells in the same well eg Melanoma cultured with Melanocytes and Keratinocytes

• Other techs – lots becoming available

–

Air-liquid interface

–

Primary tissues

–

Fluid based systems

–

Organs on chips

Design the models based upon the science and relevance….

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In-vitro 2D Potency and Safety assays are improving

T cells with ADT TCR vs antigen positive Melanoma

JULI stage

• Rapid destruction of GFP-labelled HepG2 hepatocellular carcinoma

3D microspheres

Rapid Killing of 3D HCC models by AFP SPEAR T-cells

Can perform similar models with normal cells, co-cultures, iPS cells etc

2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2 2 1 6 2 4 0 2 6 4 2 8 8 3 1 2 3 3 6 3 6 0 3 8 4 5 0 ,0 0 0 1 0 0 ,0 0 0 1 5 0 ,0 0 0 2 0 0 ,0 0 0 2 5 0 ,0 0 0 3 0 0 ,0 0 0 3 5 0 ,0 0 0 4 0 0 ,0 0 0

M e a n M ic ro tis s u e C o re F lu o re s c e n c e A re a ( m

2 )

T c e lls a d d e d - 2 1 7 h rs H e p G 2 -G F P T a rg e ts + A F P

c 3 3 2 C T L

H e p G 2 -G F P T a rg e ts A lo n e H e p G 2 -G F P T a rg e ts + N T D C T L

T im e (h )

CONFIDENTIAL

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• Pick the right target

 Favourable expression profile for on-target reactivity (normal vs tumour)

• Identify the right parental TCR

 Early cross reactivity profile  Start with multiple parents

• Careful engineering

 Step-wise affinity changes from multiple parents to find optimal TCR

• Screening for cross-reactivity in right way

 Molecular characterisation, peptide screening and other predictive

models

 Cell screening  Relevant organotypic models, depends on the target and safety

concerns

Preclinical Safety Testing Of Enhanced TCRs

Summary

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TCR engineering alone is not enough

• need to enhance T cells themselves

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Engineering Better T Cells

Tumour Growth in Sarcoma (Pre-treatment)

BEFORE

Treatment

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Engineering Better T Cells

Results observed with the Engineering optimal affinity NYESO SPEAR T-Cell

BEFORE

Treatment with NY-ESO SPEAR T-cell

AFTER

Treatment with NY-ESO SPEAR T-cell

Change in Tumour Size Months

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Engineering Better T Cells

Results observed with the Engineering optimal affinity NYESO SPEAR T-Cell

• Have the guiding receptor with optimal affinity and specificity
• Escape mechanisms present which
• Immune suppression
• HLA down regulation
• Antigen escape
• Reduced Durability

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Engineering Better T Cells Optimal Phenotype of the Cells

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Engineering Better T Cells

Lessons from Translational Sciences

• Long

term responders are mostly grouped according to a TCM profile with a subset expressing high levels of IFN-γ and IL-2 from CD8 cells (green boxes) after peptide stimulation

• Early

relapsers are strongly grouped according to TEMRA and TEM dominated product,

• r TNF-α producing CD8

NY-ESO-1c259T cells (purple boxes)

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Engineering Better T Cells Making T Cells Resistant To Suppression

Four components to an effective adoptive therapy:

1.

T cell must recognize a cancer cell via a guiding receptor

2.

The guiding receptor must have two important aspects

 Affinity  Specificity 3.

The T cell needs to be resistant to suppression

Engineering better T cells

2nd generation T cells

41 T cell Cancer cell

Inhibitory mechanisms

• f cancer cells

make T cells insensitive Make T cells resistant to suppression

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Gen 2 T Cells maintain enhanced killing in the presence of inhibitors

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Engineering Better T Cells

Overcoming inhibition in the tumour microenvironment

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Engineering Better T Cells Summary

• Target choice is critical for efficacy and safety profile
• TCR affinity optimization crucial for best TCR-targeted T cell response
• Specificity crucial for lowest toxicity

TCR specificity can be assessed systematically in vitro

• Several next generation technologies making T cells resistant to

tumour microenvironment inhibitory factors

• Several next generation technologies enabling T cells to facilitate

breaking immune tolerance to tumour Next generation approaches need extra consideration, to be driven by the mode of action and the science, and appropriate models designed

Engineering Better T Cells

Summary

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CONFIDENTIAL

Thank you for your attention!

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