Patient-Derived Organoid and Immune Cell Co-Cultures for ...

Corporate Headquarters:16550 West Bernardo Drive

Building 5, Suite 525

San Diego, CA 92127

Tel: 855.827.6968

Fax: 888.882.4881

www.crownbio.com

Patient-Derived Organoid and

Immune Cell Co-Cultures for

Immunotherapy Development

Yujun Huang, PhD

Senior Director Immuno-Oncology

Patient-Derived Models Improve

Translatability of Preclinical Research

• In vivo patient-derived xenografts (PDX) are gold standard

preclinical models and highly predictive of clinical response

• Advancements in stem cell research led to the development

of in vitro organoids or “mini-organs in a dish” providing

– A revolutionary patient-derived in vitro approach for drug development

– Enhanced predictivity over standard 2D and 3D in vitro models

Crown Bioscience Inc. │ Confidential – Not For Distribution 2

In Vivo

In Vitro

21/04/2021

HUB Organoids™: Unique Patented

Technology for 3D In Vitro Tumor Modeling

• HUB Organoids are the most clinically predictive

3D in vitro tumor models available. They serve as patient

surrogates that recapitulate:

– Diversity and heterogeneity of the patient population

– Genomic, morphological, and pathophysiological

characteristics of original epithelia/tumors

• Renewable and expandable for large-scale applications

in preclinical research unlike other 3D spheroid systems

• Speed and scalability for “clinical trials in a dish”

• Flexible platform for in vitro immuno-oncology (I-O)

co-cultures with a range of human immune system

components

Crown Bioscience Inc. │ Confidential – Not For Distribution 321/04/2021

Immuno-Oncology Applications

for Organoid Co-Cultures

• Tumor organoid and immune cell co-cultures can be used to assess tumor cell

killing by:

– T cells, such as CAR-T and bispecific T cell engagers

– NK (ADCC) and macrophages (ADCP)

• Evaluate sensitivity/specificity of drugs using TAAhi tumor organoid vs TAAlo

tumor organoids

• Evaluate off-target vs tumor effect using tumor organoids vs normal organoids


Design Your Co-Culture Screen


Patient- or PDX-derived tumor organoid

CrownBio Organoid Biobank


• Colorectal, lung, breast, and pancreatic tumor organoids

transferred from the Hub organoid biobank

• Rapidly expanding collection of more than 180 models

• Key features of biobanked organoids include:

– Primary and metastatic matched pairs

– Normal and tumor matched pairs

– Models capture heterogeneity of patient tumor

– Panels of models with oncogenic driver mutations

• Tumor organoids derived from CrownBio PDX

models

• More than 180 models, representing over

15 cancer types, in our master biobank

• Matched in vivo PDX modes

• HTS organoid screening platform available

• Panels of models with oncogenic driver mutations

OrganoidBase

• Review and select from available organoid models

with OrganoidBase

• Rich online repository of organoid model data includes

– Light microscopy, histopathology, genomic data (RNA-seq

and WES), treatment response data, and patient info for our

PDX-derived organoids

– Genomic data include expression level by RNA-seq

(108 models) and annotated somatic mutations by

WES/WGS (99 models) for HUB’s patient-derived organoids

• Register at crownbio.com/ob-registration to access

OrganoidBase

• Already a user?

– Log in at https://organoid.crownbio.com

– Or access from any other CrownBio database


https://www.crownbio.com/ob-registration

https://organoid.crownbio.com/

https://organoid.crownbio.com/

Biomarker Analysis of PDO and PDXOs

Guides Model Selection

• Profile target gene expression on tumor organoids

• Review heatmap of gene expression pattern of

immune checkpoint targets across multiple cancer

types for both PDX-derived (PDXO) and

patient-derived (PDO) organoids

• Select model and confirm expression levels

– NSCLC LU11693 PDXO, profiled for B7-H3 by IHC

and flow cytometry shown


PDO

PDXO

4X 20X

PDXO LU11693 IHC & Flow Cytometry

B7-H3 B7-H3

21/04/2021

Tumor Organoid Co-Culture Case Studies

• CrownBio’s co-culture platform provides a flexible approach that can be

adapted for a broad range of immuno-therapeutics and assay conditions

• Case studies presented showcase four possible applications:

– Evaluate potency of immuno-therapeutics using coculture of PBMCs and T cells

– Evaluate tumor organoid killing and tumor reactivity of CAR-T

– Test ADCC effects on tumor organoids

– Evaluate bispecific T cell engagers


ICI POTENCY EVALUATION USING AUTOLOGOUS OR

NON-AUTOLOGOUS PBMCS IN CO-CULTURE WITH

TUMOR ORGANOIDS

Case Study 1

Co-Culture with PBMCs Kills Tumor Organoids

• Autologous tumor reactive PBMCs recognize and

kill tumor PDOs with high mutational load


Adapted from Dijkstra et al. Cell 2018;174:1586-98

21/04/2021

Autologous or Non-Autologous T Cells

• Autologous PBMCs and tumor organoid co-cultures enable investigation of tumor-antigen-specific

immune cell reactivity

• Challenges in autologous PBMCs for tumor organoid co-cultures include:

– Sourcing matched patient material (PBMCs and tumor) to develop a fully autologous system

– Longer turnaround time due to de novo tumor organoid model establishment and T cell expansion

– Limited volume of blood from donor cancer patients influences scalability for screening

– Lack of tumor reactive T cells among PBMCs from most patients

• Tumor organoid co-cultures with non-autologous immune cells from healthy donors represent a

practical alternative when autologous cells are not available and support the investigation of:

– Tumor killing by alloreactive T cells

– Tumor killing by CAR-T and tumor reactivity of CAR-T

– Tumor killing by ADCC (NK) and ADCP (macrophages)


Tumor Organoid and PBMC Co-Cultures

• Case study description

– Options for autologous or non-autologous PBMCs in co-culture assays

– Investigate tumor organoid killing using tumor organoid and healthy donor non-autologous immune cell

co-cultures

– Assess organoid viability by flow cytometry, morphology change or luciferase/fluorescence signal

– Evaluate T cell activation using IFN-g ELISA


Increase Throughput Using

Non-Autologous PBMCs

• Healthy donor PBMCs pre-activated by anti-CD3/anti-CD28 for 3 days

• Gastric cancer organoid selected and labeled with CFSE

• Activated PBMCs co-cultured with CFSE-labeled tumor organoids at customized E:T ratios and timepoints

• Organoid viability evaluated by flow cytometry after dissociation into single cellsCo-c

ulture

desig

n


Evaluate organoid viability

with flow cytometry

April 20,2021

Tumor Organoid Killing by Alloreactive T Cells

• Customized E:T ratios and timepoints analyzed by flow cytometry to assess organoid viability

• Increased tumor organoid killing with higher ratio and longer co-culture duration

Crown Bioscience Inc. │ Confidential – Not For Distribution 15April 20,2021

24h

48h

Luciferase-Based Readout:

Assessing Alloreactive T Cell Killing


0.5:1 1:1 2:1 5:1 10:1 20:1

Target

onlyEffector

only

0h

24h

48h

E:T ratio

1000 organoids ~5 x 104 organoid cells/well (pre-coated with 30µl 50% MG)

LI6664-Luc 24h

Target

only

0.5:

11:

012:

015:

01

10:0

1

20:0

1

0.0

0.5

1.0

1.5

Steady-Glo

Rela

tive L

um

inescen

ce (

RU

L)

LI6664-Luc 48h

Target

only

0.5:

11:

12:

15:

110

:120

:1

0.0

0.5

1.0

1.5

Steady-Glo

Rela

tive L

um

inescen

ce (

RU

L)

• Luciferase-engineered liver cancer PDXO co-cultured with pre-activated PBMCs from healthy donors

• T cell-mediated LI6664-Luc organoid killing monitored by luciferase activity at 6 different E:T ratios

• Red arrows indicate T cell recruitment to LI6664-Luc organoids at different co-culture timepoints captured by microscopy C

o-c

ulture

d

esig

n

21/04/2021

ICI Potency Assay:

Evaluating Tumor Killing by Alloreactive T Cells


• NSCLC organoid engineered to express luciferase and incubated with 50ng/mL IFN-gfor 24h to stimulate PD-L1 expression

• Healthy donor PBMCs (n=2) co-cultured for 5 days with NSCLC organoid

• Co-culture organoid viability evaluated by luciferase activity and IFN-g in supernatant measured by ELISA to assess alloreactive T cell activationC

o-C

ulture

D

esig

n

PD-L1 expression

Rela

tive L

um

inescen

ce (

RU

L)

0

50000

100000

150000 Anti-PD-1IgG4

Organoid only

Low luciferase expression

indicates cell death

IFN

-¦Ã

(p

g/m

L)

0

500

1000

1500Anti-PD-1

IgG4

IFN

-γ(p

g/m

L)

IFN-g expression indicates

T cell activation

21/04/2021

Case Study 1 Summary

• Co-culture with either autologous or non-autologous immune cells can lead to

tumor organoid killing

• De novo PDO models can be developed when autologous PBMCs are

collected, but with limitations

• Non-autologous PBMCs enable higher throughput and multi-model screening

• The co-culture of tumor organoids with non-autologous alloreactive

T cells was established to evaluate effect of PD-1 blockade on tumor killing

• T cell activation can be quantified by IFN-g


TUMOR ORGANOID KILLING WITH CAR-T

CELL CO-CULTURES

Case Study 2

Tumor Organoids and CAR-T Cell Co-Cultures


– Investigate tumor organoid killing by CAR-T cells

– Select tumor organoid models expressing specific CAR-T cell target

– Evaluate CAR-T cell reactivity to antigen expressing tumor organoids by ELISA

– Assess organoid viability following treatment


EpCAM CAR-T Cell Reactivity

to Tumor Organoids


• Gastric cancer organoid GA0091 (EpCAM+) and melanoma organoid ME1154 (EpCAM-) selected for comparison

• EpCAM CAR-T cells co-cultured with organoids at different E:T ratios (20:1, 10:1, 1:1) for 24h or 48h

• ELISA analysis of supernatant shows significant levels of IFN-g and Granzyme B by EpCAM CAR-T cells, suggesting CAR-T cell reactivity against EpCAM+ GA0091 gastric cancer organoidsC

o-C

ultu

re

Desig

n

21/04/2021

CD19 CAR-T Cell Testing with

Engineered Tumor Organoids


Day 0 4X Day 1 4X

Day 0 4X Day 1 4X

LI6677-CD19-Luc+CAR-T

LI6677-Luc

+CAR-T

• Liver cancer organoid (PDXO LI6677) engineered to express Luciferase and human CD19

• Engineered tumor organoids co-cultured with CD19 CAR-T cells at 1:10 ratio for 2 days

• Luciferase activity used to track tumor growth/killingCo

-Cultu

re

Desig

n

21/04/2021

Antigen-Specific CAR-T Cell Tumor Killing

Crown Bioscience Inc. │ Confidential – Not For Distribution 23Images taken at 10X mag

Day 1

Day 2

Day 3

LU6438/B7H3 CAR-TB7H3 CAR-T LU6438/B7H3 CAR-T LU6438B CD19 CAR-T CD19 CAR-T/LU6438B LU6438B

• Lung cancer organoid LU6438 B7H3+

• hB7H3 CAR-T cells co-cultured with organoids at 2:1 ratio for 3 days

• Flow cytometry and morphology changes to track tumor organoid killing

• No significant change in organoid morphology observed with non-matching CAR-T cells (CD19 CAR-T, E:T=1:2)C

o-C

ulture

D

esig

n

21/04/2021

Tumor Organoid Killing by B7H3 CAR-T

B7H3 CAR-T cells effectively killed B7H3+ organoids (flow cytometry analysis on day 4)


LU

64

38

C:O

=1

:5

LU

64

38

C:O

=1

:5


• Tumor organoid cultures can be used to evaluate CAR-T cell therapies

• Organoid models expressing specific tumor antigens can be selected using

OrganoidBase

• Cytokine production can be monitored in supernatant to evaluate CAR-T cell

reactivity to tumor organoids in co-culture

• Organoids can be engineered to assess tumor organoid viability following

treatment

• Flow cytometry and changes in morphology can be assessed to evaluate

antigen-specific CAR-T cell-mediated tumor organoid killing


EVALUATION OF ADCC USING TUMOR

ORGANOID CO-CULTURES

Case Study 3

Testing ADCC Effects in

Tumor Organoid Co-Cultures


– Use tumor organoid co-cultures with non-autologous PBMC for the evaluation of ADCC effects

– Select PDXO models based on desired antigen expression

– Evaluate tumor organoid cell killing by measuring LDH release


ADCC-Mediated

Tumor Organoid Killing


OV0250 PDXO

Brightfield ImageAntigen Expression in Matched

OV0250 PDXO

• HER2+ ovarian cancer organoids co-cultured with anti-HER2 antibody (Herceptin®) and PBMCs from a healthy donor for 4h

• LDH release measured to evaluate tumor organoid cell killing by ADCC

Co

-Cultu

re

Desig

n

21/04/2021

ADCC Effect Measured by LDH

OVO250 Organoid Co-culture with PBMC 25:1 Ratio


• Tumor organoids and non-autologous PBMC co-cultures can be used to

evaluate ADCC effects

• Specific antigen expression in tumor organoids can be verified via

OrganoidBase

– Where matched PDXO models are selected, corresponding PDX gene and protein

expression data can be interrogated

– Target expression by PDXO can be confirmed via FACS if required

• ADCC mediated tumor organoid cell killing can be evaluated by measuring

LDH release


EVALUATION OF BISPECIFIC T CELL ENGAGERS

USING TUMOR ORGANOID CO-CULTURES

Case Study 4

Tumor Organoid Co-Cultures with

Bispecific T Cell Engager (BiTE) Antibodies


– Evaluate retargeting of T cells cytotoxic activity, against specific cancer cells

– Investigate tumor organoid killing using tumor organoid expressing specific TAA co-cultured with

BiTE and healthy donor non-autologous immune cells

– Assess organoid viability by flow cytometry (L/D) and microscopic morphology evaluation


BiTE Antibody-Mediated

T Cell Killing of Tumor Organoids


E:T=5:1 E:T= 2:1 E:T= 1:1 Organoid only

Ctrl

Ab1

Test

Ab1

• PDXO model was selected for specific TAA (undisclosed) expression levels

• PDXOs were co-cultured with PBMCs from healthy donor at different E:T ratios with test bispecific T cell engager antibody and control antibody

• Tumor organoid viability was evaluated at 48h by flow cytometry after dissociation into single cell suspension

• Tumor organoid killing was assessed by morphology change and flow cytometryCo-C

ulture

D

esig

n

21/04/2021

BiTE Mediated TAA Specific Killing


Ctr

l A

bTe

st A

b

E:T=1:1

• Tumor organoid killing was mediated by the BiTE in comparison to control antibody in both high and low

antigen expressing populations

21/04/2021


• Tumor organoids and non-autologous PBMC co-cultures can be used to

evaluate BiTE antibodies

• TAA on tumor organoids can be verified by

– OrganoidBase

– Corresponding PDX expression

– PDXO expression can be confirmed via FACS

• Tumor organoid killing can be evaluated by morphology and FACS analysis


Conclusions

• Tumor organoids represent individual patient populations and recapitulate patient

heterogeneity, diversity, and tumor architecture, making them a powerful tool for testing

the patient population response to drugs in vitro

• Co-culture of tumor organoids with immune cells is a unique tool for interrogating novel

immuno-therapeutic agents in “mini-organs”, derived from both tumor and healthy tissues

• Co-cultures of autologous tumor organoids and immune cells are potentially useful for

testing immunotherapeutics at the patient level, but it can be challenging to obtain

matched organoid and T cells for assays.

• Alternatively, co-culture with non-autologous immune cells from healthy donors enables:

– More flexibility and step-by-step design of optimal co-culture assay

– Greater scalability to test multiple models across different patients, E:T ratios, timepoints, and

multiple readouts


Immuno-Oncology Applications for

CrownBio’s Tumor Organoid Platform

• The CrownBio tumor organoid co-culture platform enables you to:

– Profile target expression and biomarker analysis of tumor organoids

– Evaluate potency of novel immunotherapies using non-autologous alloreactive T cell

assays

– Evaluate tumor organoid killing and tumor reactivity of CAR-T and bispecific T cell

engager antibodies

– Test ADCC and ADCP effects on tumor organoids


Thank you!

Any questions??

?

?

Patient-Derived Organoid and Immune Cell Co-Cultures for ...

Documents

Transcript of Patient-Derived Organoid and Immune Cell Co-Cultures for ...