PD-L1 Detection in Tumors Using [64

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[ Cu]atezolizumab with Bioconjugate PET Chemistry is

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Page 1 of 25Bioconjugate Chemistry

24 h

1[64Cu]Atezolizumab PD-L1 -Ve 2for PD-L1 detection 3 4 O 5 O OH H 6 N PD-L1 +Ve O N N 7 Cu(II) O O 8 O N N OH 9 O 10 2 ACS Paragon Plus Environment 11 12 13 64

Bioconjugate Chemistry

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PD-L1 detection in tumors using [64Cu]atezolizumab with PET

Wojciech G. Lesniak1†, Samit Chatterjee1†, Matthew Gabrielson1, Ala Lisok1, Bryan Wharram1, Martin G. Pomper1,2, and Sridhar Nimmagadda1,2*

1

Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins

University, Baltimore, MD, USA. 2

Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD,

USA.

*Correspondence to:

Sridhar Nimmagadda, Ph.D. Johns Hopkins Medical Institutions 1550 Orleans Street, CRB II, #491 Baltimore, MD 21287 Phone: 410-502-6244 Fax:

410-614-3147

Email: [email protected]

COI: The authors declare no conflicts of interest. †, equal contribution

1 ACS Paragon Plus Environment

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ABSTRACT

The programmed death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) pair is a major immune checkpoint pathway exploited by cancer cells to develop and maintain immune tolerance. With recent approvals of anti-PD-1 and anti-PD-L1 therapeutic antibodies, there is an urgent need for non-invasive detection methods to quantify the dynamic PD-L1 expression in tumors and to evaluate the tumor response to immune modulation therapies. To address this need, we assessed [64Cu]atezolizumab for the detection of PD-L1 expression in tumors. Atezolizumab (MPDL3208A) is a humanized, human and mouse cross-reactive, therapeutic PDL1 antibody that is being investigated in several cancers. Atezolizumab was conjugated with DOTAGA, and radiolabeled with copper-64. The resulting [64Cu]atezolizumab was assessed for in vitro and in vivo specificity in multiple cell lines and tumors of variable PD-L1 expression. We performed PET-CT imaging, biodistribution, and blocking studies in NSG mice bearing tumors with constitutive PD-L1 expression (CHO-hPD-L1) and in controls (CHO). Specificity of [64Cu]atezolizumab was further confirmed in orthotopic tumor models of human breast cancer (MDAMB231 and SUM149) and in a syngeneic mouse mammary carcinoma model (4T1). We observed specific binding of [64Cu]atezolizumab to tumor cells in vitro, correlating with PD-L1 expression levels. Specific accumulation of [64Cu]atezolizumab was also observed in tumors with high PD-L1 expression (CHO-hPD-L1 and MDAMB231), compared to tumors with low PD-L1 expression (CHO, SUM149). Collectively, these studies demonstrate the feasibility of using [64Cu]atezolizumab for detection of PD-L1 expression in different tumor types.



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INTRODUCTION

Cancer cells exploit multiple mechanisms to evade immune recognition, and to establish immune tolerance 1. This tolerance is facilitated and maintained by regulatory immune cells, immunosuppressive cytokines and chemokines, and immune checkpoint pathways that downregulate immune cells’ anti-tumor activity. Over the last decade, immune modulation therapies that are focused on either antagonizing co-inhibitory or activating co-stimulatory -pathways have resulted in improved patient survival 2, 3. Therapeutic antibodies targeting immune checkpoint proteins are at the forefront of those advances.

A major therapeutic target in cancer immune modulation therapy is the axis between the two immune checkpoint proteins programmed death receptor 1 (PD-1, CD279) and its ligand PD-L1 (B7-H1, CD274) 4. PD-1 is expressed on activated T, B, and NK cells, and PD-L1 is normally displayed as an autoimmune prevention mechanism on a subset of macrophages, endothelial cells, and other non-malignant tissues. There is increased PD-L1 expression on a wide variety of tumors, including melanoma, non-small cell lung cancer, Merkel cell carcinoma, breast cancer, and squamous cell carcinomas 5-7. Promotion of PD-L1 expression in the tumor microenvironment (TME) is driven by both genetic and adaptive immune response mechanisms 8. Examples of the former include KRAS and PTEN mutations, and translocation or amplification of the 9p24.1 gene in certain tumors 9-11. PD-L1 expression on tumor cells and immune cell infiltrates can also be induced as an adaptive mechanism in response to interferon-γ (INF-γ) secreted by antigen-bound tumor-infiltrating T cells 12. Increased expression of tumor


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and stromal cell PD-L1 leads to deactivation of PD-1 expressing tumor infiltrating cytotoxic T cells, causing immune suppression in the TME

13

.

Clinical studies indicate that intra-tumoral PD-L1 expression, detected by immunohistochemistry (IHC), could be indicative of but not an effective biomarker of response to anti-PD-1 and antiPD-L1 therapy 14,

15

. Some of the contributing factors to the observed inconsistency are the

unresolved issues with PD-L1 IHC such as different antibodies for detection and variable criteria used for positive IHC in the evaluation of clinical trials,

14, 16

and the changes in PD-L1

expression in tumors induced by a dynamic TME. Obtaining repeated biopsies to evaluate PDL1 expression in the TME is impractical, particularly in patients with metastatic disease, further limiting our ability to fully assess therapy induced changes in PD-L1 expression in real-time. Non-invasive imaging allows for repetitive measurements of target expression in all lesions in their entirety. Recent pre-clinical studies show that PD-L1 expression in tumors can be assessed by radionuclide imaging 17-21.

We have previously shown in murine models that PD-L1 can be detected in the TME with 111In radiolabeled atezolizumab, using single photon emission computed tomography-computed tomography (SPECT-CT) imaging 19. Atezolizumab (MPDL3280a, PD-L1-mAb) is a humanized, human and mouse cross-reactive PD-L1 antibody, in clinical evaluation for treatment of advanced or metastatic bladder cancer, 22 melanoma, 23 non-small cell lung cancer, 24 renal cell carcinoma

25

, and several other cancers. Atezolizumab binds both human

and mouse PD-L1 with high affinity and provides an opportunity to evaluate PD-L1 expression in both human tumor xenografts and syngeneic mouse tumor models. Atezolizumab binds



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human and mouse PD-L1 with dissociation constants (Kd) of 0.43 nM and 0.13 nM, respectively

22, 26

.

The high sensitivity and quantitative features of positron emission tomography (PET) are increasingly used to image tumor target expression using radiolabeled antibodies 27, example,

64

28

. For

Cu-trastuzumab has been used to detect HER2 expression in tumors and

metastases 29-31. Here we report our evaluation of the ability of [64Cu]atezolizumab to detect PDL1 expression in different tumor types, using PET-CT.

RESULTS AND DISCUSSION

Fabrication of [64Cu]atezolizumab Atezolizumab was first conjugated with DOTAGA for stable chelation of

64

Cu (Supporting

Information, Scheme s1). As indicated by MALDI-TOF spectrometry, the applied procedure resulted in conjugation of an average of two molecules of the chelator for each antibody (Supporting Information, Figure s1D). Subsequently, the atezolizumab-DOTAGA conjugate was radiolabeled with 64Cu, providing [64Cu]atezolizumab with radiochemical yields of 75 ±5 %, purity > 96 % (as determined by ITLC) and specific activity of 8.6±0.78 µCi/µg. Radiolabeling was further confirmed by SDS-PAGE, Coomassie blue staining, and autoradiography, under reducing and non-reducing conditions, indicating intact antibody upon chelation with (Supporting Information, Figure s2).

[64Cu]atezolizumab shows PD-L1-specific cellular uptake in vitro 5 ACS Paragon Plus Environment

64

Cu

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To evaluate the in vitro binding specificity of [64Cu]atezolizumab, we selected human and mouse cancer cell lines with varying PD-L1 cell surface expression. Flow cytometry analyses demonstrated the following order of PD-L1 expression among cells tested: CHO-hPD-L1 > MDAMB231 > 4T1-Luc >SUM149 > CHO (Figure 1A and B). In vitro binding assay detected significantly higher uptake of [64Cu]atezolizumab in the CHO-hPD-L1 and MDAMB231 cells with high PD-L1 expression, than in the SUM149 and CHO cells with low PD-L1 expression (Fig. 1C).

The bound [64Cu]atezolizumab radioactivity correlated (correlation coefficient

R=0.997) with the flow cytometry mean fluorescence intensity (MFI) profile of the cells. The 4T1-Luc cells had low uptake, similar to that of SUM149 cells. A blocking study using unmodified non-radiolabeled atezolizumab in CHO-hPD-L1 cells showed significantly reduced uptake of [64Cu]atezolizumab, further confirming PD-L1 specific binding of [64Cu]atezolizumab. The immunoreactive fraction of [64Cu]atezolizumab assessed in CHO-hPD-L1 cells was 73.8 % (Fig. 1D).

Collectively, the in vitro results show that [64Cu]atezolizumab can be used to detect varying levels of PD-L1 expression in cells of different cancer types.



A

CHO SUM149 4T1

B

Isotype MDAMB231 CHO-hPD-L1

800

MFI

650

500 80

40

70

D

****

55

Total/Bound

****

9 6

4T 1

4

2

3 0 L1 ing Dck -hP blo O -L1 CH D hP OCH

14 9

r2= 0.88 IF=73.8%

6

10

SU M

8

40 25

B AM MD

CH

C

23 1

O CH

O -h P

DL1

0

%ID

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O CH

B2 AM MD

31

49 M1 SU

0

1 4T

0

1

2

3

4

1/cell number (million)

Figure 1. In vitro evaluation of [64Cu]atezolizumab. (A) Representative surface PD-L1 expression levels of studied cell lines analyzed by flow cytometry illustrated as histogram and (B) mean fluorescence intensity (MFI); (C) In vitro binding specificity of [64Cu]atezolizumab for PD-L1 expression in various cell lines. (D) In vitro immunoreactive fraction of [64Cu]atezolizumab in CHO-hPD-L1 cells as determined by Lindmo analysis. ****, p

PD-L1 Detection in Tumors Using [64

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