Integrative surfaceome profiling identifies immunotherapeutic targets in osteosarcoma

and preclinical testing of BT1769, an MT1-MMP-targeted Bicycle® toxin conjugate, in

osteosarcoma by the Pediatric Preclinical Testing Consortium (PPTC)

Yifei Wang1, Wendong Zhang1, Zhongting Zhang1, Xiangjun Tian2, Rossana N. Lazcano Segura3, Pooja Hingorani1, Michael Roth1, Jonathan Gill1,

Douglas Harrison1, Zhaohui Xu1, Jing Wang2, Alexander Lazar3, Eric J. Earley4, Stephen W. Erickson4, Tara Gelb5, Philip Huxley6, Johanna

Lahdenranta5, Gemma Mudd6, Raushan Kurmasheva7, Peter Houghton7, Malcolm A. Smith8, Edward A. Kolb9, and Richard Gorlick1*

1Department of Pediatrics, Children’s Cancer Hospital, The University of Texas MD Anderson Cancer Center; Houston, TX, USA2Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

3Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA4RTI International, Research Triangle Park, NC, USA

5Bicycle Therapeutics, 4 Hartwell Place, Lexington, MA, USA6Bicycle Therapeutics, Babraham Research Campus, Cambridge, UK

7Greehey Children’s Cancer Research Institute, The University of Texas Health Science Center at San Antonio, San Antonio, TX, USA8Cancer Therapy Evaluation Program, National Cancer Institute, Bethesda, MD, USA

9Division of Pediatric Hematology/Oncology, A. I. duPont Hospital for Children, Wilmington, DE, USA

Disclosure Information

I have no financial relationships to disclose.

Introduction

▪ Current developments in the immunotherapeutic strategies show potential in the treatment

of solid tumors.

▪ In osteosarcoma, surface proteins such as GD2 and HER2 have been identified as cell

surface antigens of interest, but therapeutic mAb such as dinutuximab and trastuzumab

were inadequate to generate robust clinical responses.

▪ Immunotherapies such as immunoconjugates or CAR-T cell therapy have not been

sufficiently studied in OS.

▪ Bicycle Toxin Conjugates have low molecular weight (~4 kDa) compared to antibody drug

conjugates (~150 kDa) - rapid tumor penetration and short half-life.

Identifying New Targets in Osteosarcoma

▪ Hypothesis: surface proteins such as those related to bone

differentiation or other consistent osteosarcoma features may be

targeted therapeutically.

Identification of candidate OS cell surface

antigens: approach

Osteosarcoma surface protein profile

Surface protein extraction and mass spectrometry identification in a panel of osteosarcoma cell lines and PDX models. 825 surface proteins were identified

Surface proteins overexpressed in osteosarcoma vs normal tissues

ProteomicsDB database: surface proteins overexpressed in osteosarcoma vs normal human tissue. 141 overexpressed surface proteins were identified

Surface proteins overexpressed in OS vs normal tissues

64 surface proteins were confirmed to be expressed in the OS cell lines and PDX models, and overexpressed in OS compared to normal tissues

Select surface genes overexpressed in OS vs normal tissues

209 overexpressed surface genes were identified by RNA-seq data for osteosarcomas (TARGET) compared with normal human tissues (GTEx)

Candidate surface proteins

11 surface targets were enriched at both the protein and mRNA levels

Protein targets of ADCs and/or CAR T cells

126 surface protein targets were selected by literature review

Candidate therapeutic targets

4 candidate targets (MT1-MMP, MRC2, CD276, and LRRC15) were identified

Validation of candidate targets

WB, FCM, and IHC with OS cell lines, PDX tissues and patient samples validated the overexpression of MT1-MMP, MRC2, and CD276 in osteosarcomas

Preclinical test of novel therapeutic agents

MT1-MMP targeted bicycle toxin conjugate (BTC) BT1769 were tested in preclinical OS PDX models DO NOT POST

825 surface proteins are identified in OS cell

lines/PDX models by mass spectrometry

OS

1−

1O

S1−

2143

b−

214

3B−

1U

2O

S−

2U

2O

S−

1O

S34

3−

2O

S34

3−

1O

S32

2−

2O

S32

2−

1O

S30

1−

1O

S30

1−

2O

S35

5−

2O

S35

5−

1M

G6

3−

2M

G6

3−

1S

aO

S−

1S

aO

S−

2O

S3

1−

2O

S3

1−

1O

S4

2−

2O

S4

2−

1O

S3

4−

2O

S3

4−

1O

S6

0−

2O

S6

0−

1O

S3

6−

2O

S3

6−

1O

S9−

2O

S9−

1O

S2

9−

2O

S2

9−

1O

S5

5−

2O

S5

5−

1O

S3

3−

2O

S3

3−

1

82

5 c

ell

su

rface p

rote

ins

MS expr Z−score

−4

−2

0

2

4

6Group

OS Cell linesOS PDX models

Group

DO NOT POST

Integrative proteomic and transcriptomic analysis

identifies overexpressed cell-surface proteins in OS

▪ Mass spectrometry: 825 surface proteins that were highly

expressed in OS cell lines/PDX were identified by quantitative

proteomics mass spectrometry.

▪ RNAseq: 209 OS specific plasma membrane-associated

genes were identified.

▪ MS public database: 141 proteins overexpressed in OS were

identified.

▪ The overlapped targets among these 3 datasets were filtered

by current 126 ADC/CAR-T targets

▪ 4 surface proteins (MT1-MMP, MRC2, CD276, and LRRC15)

were found to be enriched in OS by all RNA-seq and mass

spectrometry datasets. There are immunoconjugates and/or

Bicycle toxin conjugates targeting these 4 surface proteins.

DO NOT POST

MT1-MMP

MRC2

CD276

LRRC15

OS cell line/ PDX(825 surface proteins)

OS ProteomicsDB(141 proteins)

OS RNA-seq (209 surface genes)

ADC/CAR-T cell target(126 protein targets)

The expression of CD276, MRC2 and MT1-MMP

in OS vs Normal tissues

MR

C2

TA

RG

ET

OS

Ad

ipo

se T

issu

eA

dre

nal G

lan

dB

lad

der

Blo

od

Blo

od V

esse

lB

rain

Bre

ast

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rvix

Ute

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Kid

ney

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uscle

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itary

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sta

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aliv

ary

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nd

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Sm

all

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stin

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ple

en

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mach

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Thyro

idU

teru

sV

agin

a

0

250

500

750

1000

Ge

ne

expr

TP

M

MT1-MMP CD276

MRC2 DO NOT POSTLRRC15

Validation of the expression of MT1-MMP, MRC2

and CD276 in OS cell lines and PDXs

OS3

55

OS3

73

OS3

77

OS3

54

OS3

22

OS2

42

OS2

55

OS2

52

OS1

OS2

OS9

OS1

7

OS3

1

OS3

3

OS5

5

OS6

0

Cofilin

MRC2

Cofilin

MRC2

OS3

55

OS3

73

OS3

77

OS3

54

OS3

22

OS2

42

OS2

55

OS2

52

-200 kDa

OS1

OS2

OS9

OS1

7

OS3

1

OS3

3

OS5

5

OS6

0

-200 kDa

OS3

55

OS3

73

OS3

77

OS3

54

OS3

22

OS2

42

OS2

55

OS2

52

OS1

OS2

OS9

OS1

7

OS3

1

OS3

3

OS5

5

OS6

0

Cell lines

PDX models

HOS

OS252

OS17

OS31

OS33

OS39

OS43

HOS

OS252

OS17

OS31

OS33

OS39

OS43

MRC2

HOS

OS252

OS17

OS31

OS33

OS39

OS43

Actin

CD276

Actin

CD276

-100 kDa

-100 kDa

CD276

Cofilin

MT1-MMP

Cofilin

MT1-MMP

-66 kDa

-66 kDa

MT1-MMP

OS3

55

OS3

73

OS3

77

OS3

54

OS3

22

OS2

42

OS2

55

OS2

52

OS1

OS2

OS9

OS1

7

OS3

1

OS3

3

OS5

5

OS6

0

Cofilin

MRC2

Cofilin

MRC2

OS3

55

OS3

73

OS3

77

OS3

54

OS3

22

OS2

42

OS2

55

OS2

52

-200 kDa

OS1

OS2

OS9

OS1

7

OS3

1

OS3

3

OS5

5

OS6

0

-200 kDa

OS3

55

OS3

73

OS3

77

OS3

54

OS3

22

OS2

42

OS2

55

OS2

52

OS1

OS2

OS9

OS1

7

OS3

1

OS3

3

OS5

5

OS6

0

Cell lines

PDX models

HOS

OS252

OS17

OS31

OS33

OS39

OS43

HOS

OS252

OS17

OS31

OS33

OS39

OS43

MRC2

HOS

OS252

OS17

OS31

OS33

OS39

OS43

Actin

CD276

Actin

CD276

-100 kDa

-100 kDa

CD276

Cofilin

MT1-MMP

Cofilin

MT1-MMP

-66 kDa

-66 kDa

MT1-MMP

OS3

55

OS3

73

OS3

77

OS3

54

OS3

22

OS2

42

OS2

55

OS2

52

OS1

OS2

OS9

OS1

7

OS3

1

OS3

3

OS5

5

OS6

0

Cofilin

MRC2

Cofilin

MRC2

OS3

55

OS3

73

OS3

77

OS3

54

OS3

22

OS2

42

OS2

55

OS2

52

-200 kDa

OS1

OS2

OS9

OS1

7

OS3

1

OS3

3

OS5

5

OS6

0

-200 kDa

OS3

55

OS3

73

OS3

77

OS3

54

OS3

22

OS2

42

OS2

55

OS2

52

OS1

OS2

OS9

OS1

7

OS3

1

OS3

3

OS5

5

OS6

0

Cell lines

PDX models

HOS

OS252

OS17

OS31

OS33

OS39

OS43

HOS

OS252

OS17

OS31

OS33

OS39

OS43

MRC2

HOS

OS252

OS17

OS31

OS33

OS39

OS43

Actin

CD276

Actin

CD276

-100 kDa

-100 kDa

CD276

Cofilin

MT1-MMP

Cofilin

MT1-MMP

-66 kDa

-66 kDa

MT1-MMP

DO NOT POST

Validation of the expression of the candidate

surface proteins - IHC with patient and PDX TMA

MT1-MMP

PDX tissue ControlPatient sample

MRC2

CD276

DO NOT POST

Preclinical test of MT1-MMP targeted BT1769

0

100

200

300

400

500

600

700

800

900

OS-33 OS-1 OS-17 OS-2 OS-9 OS-31

RNAseq - MT1-MMP

OS1

OS2OS17OS33

OS9OS31

DO NOT POST

Control

BT1769

FP

KM

Post-treatment recurrent tumor showed a

lower MT1-MMP expression level

DO NOT POST

Preclinical test of MT1-MMP targeted BT1769

Osteosarcoma PDX models Ewing sarcoma PDX models

OS1

OS2OS17OS33

OS9OS31

ES1

TC-71

Control

BT1769DO NOT POST

Summary

▪ Integrated proteomic and transcriptomic osteosarcoma surfaceome

profiling identified high-confidence osteosarcoma cell-surface antigens

(MT1-MMP, CD276, MRC2, and LRRC15) as therapeutic targets.

• ADC targeting LRRC15 is active against osteosarcoma PDXs (Hingorani, et al. Mol

Cancer Ther 2021)

• ADC targeting CD276 is active against osteosarcoma PDXs (Kendsersky, et al.

Clinical Cancer Research, 2021)

▪ MT1-MMP’s high expression and BT1769’s preclinical activity support

the potential of MT1-MMP-targeted Bicycles in osteosarcoma.

Acknowledgements

MD Anderson Cancer Center

Richard Gorlick, MD

Jonathan Gill, MD

Michael Roth, MD

Douglas Harrison, MD

Pooja Hingorani, MD

Ellen Zhang

Matt Zhang

Zhaohui Xu

Xiangjun Tian, PhD

Jing Wang, PhD

Rossana N. Lazcano Segura, MD

Alexander Lazar, MDBicycle Therapeutics

Tara Gelb

Philip Huxley

Johanna Lahdenranta

Gemma Mudd

Greehey Children’s Cancer

Research Institute, UTHSCSA

Raushan Kurmasheva, PhD

Peter Houghton, PhD

RTI International

Eric J. Earley

Stephen W. Erickson

National Cancer Institute

Malcolm A. Smith, MD, PhD

A. I. duPont Hospital for Children

Edward A. Kolb, MD

Funding