Preclinical Study of a Fully Human Anti-PD-L1 Antibody as a

Subscriber access provided by EKU Libraries

Article

Preclinical Study of A Fully Human Anti-PD-L1 Antibody as A Theranostic Agent for Cancer Immunotherapy Mengxin Xu, Yuxiang Han, Guizhong Liu, Yang Xu, Dongban Duan, Hui Liu, Felix Du, Peter Luo, and Zhibo Liu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00371 • Publication Date (Web): 22 Aug 2018 Downloaded from http://pubs.acs.org on August 23, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Preclinical Study of A Fully Human Anti-PD-L1 Antibody as A Theranostic Agent for Cancer Immunotherapy Mengxin Xu,† Yuxiang Han,† Guizhong Liu,§ Yang Xu,† Dongban Duan,†Hui Liu,† Felix Du,§ Peter Luo,§ Zhibo Liu†‡* †

Radiochemistry and Radiation Chemistry Key Laboratory of Fundamental Science, Beijing National

Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China ‡

Peking University-Tsinghua University Center for Life Sciences, Beijing, 100871, China

§

Adagene (Suzhou) Limited, Suzhou 215000, China

ABSTRACT

Recently, inhibiting PD-1/PD-L1 checkpoint pathway utilizing anti-PD-1 or anti-PD-L1 antibodies has achieved great clinical success in cancer treatment. However, anti-PD-1 immunotherapy cannot apply to all the cancer patients, of whom no more than 25 % showed the positive response. Immunohistochemistry (IHC) is the gold standard to determine the PD-L1 expression level in malignant lesions, but a non-invasive imaging meditated strategy is urgently required for clinical diagnosis to cover the shortcomings of invasive techniques. MX001, which is an anti-PD-L1 antibody, was labeled with Cu-64 (t1/2=12.7 h) and purified by PD-10 chromatography. Comprehensive studies including Positron Emission Tomography (PET), ex vivo biodistribution, immunohistochemistry and immunotherapy have been performed in mice bearing MC38 (PD-L1 positive (+)) and 4T1 (PD-L1 negative (-)) xenografts. PET imaging of [18F]FDG was taken before and after therapy to monitor the therapeutic efficacy. [64Cu]Cu-NOTA-MX001 exhibited 2.3 ± 1.2， 5.6 ± 2.1, 5.6 ± 1.2, 6.1 ± 1.1, 6.1 ± 0.5,10.2 ± 1.7 %ID/g uptake in MC38 xenografts at 0.5, 12, 24, 1

ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

36, 48, and 62 hours post-injection, respectively. Meanwhile, the uptake in liver and muscle in corresponding time points was 17.5 ± 2.2，8.4 ± 2.4, 11.3 ± 3.2, 7.2 ± 2.1, 7.9.1 ± 3.5, 3.8 ± 1.8 %ID/g, and 1.2 ± 0.5，1.3 ± 0.4, 1.5 ± 0.5, 0.7 ± 0.1, 0.6 ± 0.2, 0.2 ± 0.1 %ID/g, respectively. And the uptake of [18F]FDG in MC38 and 4T1 xenografts at 1-hour post-injection was 5.3 ± 0.4 and 6.4 ± 0.6 %ID/g, while the uptake of [64Cu]Cu-NOTA-MX001 was 5.6 ± 0.3 and 1.3 ± 0.4 %ID/g at 12-hour post-injection. Immunohistochemistry analysis confirmed that MC38 tumor exhibited high PD-L1 expression and 4T1 tumor, liver, muscle exhibited low PD-L1 expression. In addition, MC38 xenografts were suppressed by MX001 about 88% in immunotherapy study. MX001 was successfully developed as a fully human anti-PD-L1 antibody with the high binding affinity in mouse, monkey and human. The in vivo pharmacokinetics of MX001 was evaluated with PET imaging after being radiolabeled with Cu-64. The uptake of [64Cu]Cu-NOTA-MX001 was clearly correlated to the PD-L1 expression on various types of cancer. Subsequent immunotherapy studies demonstrated that MX001 could effectively suppress tumor growth with positive PD-L1 expression, but had poor anti-tumor efficacy on tumors which exhibited low PD-L1 expression. Together the above results, MX001 has the potential to be further developed as an antibody theranostic agent for both PET imaging and immunotherapy of cancers in clinical.

KEY WORDS: Radioimmunoimaging, Antibody, Positron emission tomography, PD-L1, Theranostic

2


Page 2 of 27

Page 3 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION The PD-1/PD-L1 pathway is of great importance in suppressing the inflammatory activity of T-cells and helps cancer cells to evade from immune system.1-3 T-cell activation is suppressed when its receptor PD-1 is engaged by its ligands PD-L1.4-6 It has been reported that variety of human cancers express PD-L1.7 Promising preclinical results of immune checkpoint blockade induced tumor regression is leading antibody development for clinical use to improve patient survival.8-11 Unfortunately, a successful immunotherapy with PD-L1 antibody needs the tumor with positive PD-L1 expression,12-15 patient with PD-L1 negative tumor often shows no objective response to such treatment.16 Immunohistochemistry (IHC) is the gold standard for patient classification, but its application was limited because of the localized and invasive practice such as biopsy and surgical resection.17, 18 Nevertheless, in comparison with non-invasive imaging procedures, biopsy or surgery often risks a greater chance of bleeding, infection, or wound healing problems.19 As the tumor location is unknown, the biopsy samples are generally taken blindly, resulting in a high false positive rate that can be up to 30%.20 Indeed, a standard systematic sampling only covers a small part of tumor foci, with a sensitivity of no more than 50%.21 Meanwhile, the PD-L1 expression level is often heterogeneous in tumor foci.22 Thus, non-invasively assessing PD-L1 expression with a whole-body and highly sensitive imaging method will be of great importance for patient classification and treatment monitoring in clinics.23 In this study, a new PD-L1 targeting antibody was developed and radiolabeled to serve as a useful cancer management strategy that unifies PET imaging and anti-PD-L1 immunotherapy. This 3



Page 4 of 27

antibody is generated by screening the Adagene synthetic human Fab antibody phage display library with human PD-L1 recombinant protein. This is a fully human IgG1 antibody and produced by transfecting expression plasmids into CHO-K1 cells for stable clone and fermentation, protein A purification. It cross-reacts with both human and rodent PD-L1, with a half-life of around 28.8 hours in mice. The affinity to human PD-L1 expressed on 293 cell surface was ~1.7 nM. 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) was utilized as the chelator to combine MX001 with copper-64 (t1/2 = 12.7 h) and formed stable complexes.24, 25 This tracer enabled the non-invasive real-time imaging to differentiate the PD-L1 expression level in malignant tumors. Furthermore, this antibody exhibited very effective treatment in PD-L1 positive tumor models.

EXPERIMENT SECTION Materials

All solvents and reagents were purchased from commercial suppliers (J&k, Sigma Aldrich Beijing, China) and were used as received unless otherwise indicated. 1 X Phosphate-buffered saline (PBS), sodium tartrate (NaAc), sodium hydroxide, DMSO were purchased from Sigma-Aldrich (St. Louis,

MO,

USA).

2-S-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic

acid

(p-SCN-Bn-NOTA) was purchased from Macrocyclics, Inc. (Dallas, TX). Amicon 50k cutoff Ultrafiltration centrifuge was purchased from Millipore Corp, Billerica, MA. PD-10 column (dead volume 2.5 mL) was purchased from GE Healthcare. Copper-64 (3.7 MBq/µL) was purchased from Peking University Cancer Hospital (Beijing, China). Anti-PD-L1 antibody was provided by Adagene (Suzhou) Limited. MC38 cell line (PD-L1 (+))

26

derived from C57BL/6 murine colon

adenocarcinoma cells, 4T1 (PD-L1 (-)) derived from BALB/c murine clone breast cells, BGC823 4


Page 5 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(human gastric carcinoma) and U87MG (human primary glioblastoma) were purchased from National Infrastructure of Cell Line Resources (China). C57BL/6 mice (female, 6-8 weeks old), BALB/c mice (female, 6-8 weeks old) and NU/NU mice (female, 6-8 weeks old) were purchased from Vital River (Beijing, China). The PD-L1 IHC primary antibody (#64988) was purchased from Cell Signaling Technology. The secondary antibody (11-605-003) was purchased from Jackson Immuno Research.

Antibody Conjugation

The anti-PD-L1 antibody was purified using ultrafiltration centrifugal tube and phosphate buffered saline (PBS, pH = 7.4) to move out L-histidine in the original buffer and stored at 4 °C.27 Then aliquots of antibody stock solution transferred to 1.5 mL microcentrifuge tubes and adjusted the final PH of resulting solution to 8.5-9.0 with PBS buffer (pH = 9). Finally, the solution was added 4.0 equivalents of p-SCN-Bn-NOTA which dissolved in DMSO. After incubating at 37 °C for 1 hour, the antibody conjugate was purified with ultrafiltration centrifugal tube for twice with PBS (pH = 7.4). The antibody complex (NOTA-MX001) stock solution was stored at 4 °C.

Radiochemistry

The conjugated antibody (NOTA-MX001) solution was transferred to 1.5 mL microcentrifuge tubes and adjusted the final PH of resulting solution to 5.5 by adding sodium acetate buffer (pH = 5.5, 200 mM), following by adding an aliquot of 64Cu-Cu2+ (300 MBq) solution. After incubating at 37 °C for

1

hour,

the

antibody

conjugate

was

purified

through

PD-10

chromatography.

[64Cu]Cu-NOTA-MX001 was eluted with 25 mL fractions of PBS at pH = 7.4. The isolated portion 5



was concentrated to 100 µL for quality control and further study.

In Vitro Binding Assay

The binding affinity of

Nat

Cu-NOTA-MX001 was detected in HEK293T cells stably expressing

PD-L1. Briefly，the conjugates were diluted with PBSA (1% BSA in PBS). Wash cells with PBSA, and resuspended cells with PBSA at the density of 4×106 cells/mL. Each of the conjugates was placed in triplicate vials containing HEK293T hPD-L1 cells (50 µL) and incubate at room temperature for 1 hour and 5 min on ice. Washing and collected the cells with 1 mL PBSA. After resuspending, each of the wells received 100 µL of Mouse Anti-human IgG Fc-APC and was incubated on ice for 30 min in the dark. FACS was used to analyzed the cells after washing cells with PBSA.

Cell Culture and Animal Models

MC38, 4T1 and BGC823 cell lines were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin (100 IU/mL), and streptomycin (100 mg/mL), U87MG cell line was maintained in MEM supplemented with 10% fetal bovine serum, penicillin (100 IU/mL), and streptomycin (100 mg/mL). The cells were cultured in an incubator in 5% CO2 atmosphere at 37 °C. C57BL/6 mice were implanted subcutaneously in the right shoulder with 106 MC38 cells suspended in 50 µL PBS. BALB/c mice were implanted subcutaneously in the right shoulder with 106 of 4T1 cells. NU/NU mice were implanted subcutaneously in opposite sides of up flank with 106 of MC38 and 4T1 cells. NU/NU mice were implanted subcutaneously in the right shoulder with 106 of BGC-823 or U87-MG cells. Mice were subjected to imaging or biodistribution when the tumor 6


Page 6 of 27

Page 7 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


grafts reached a volume of 125.0 ± 25.0 mm3.

PET/CT Imaging of [64Cu]Cu-NOTA-MX001 and [18F]FDG

C57BL/6 Mice bearing MC38 tumor grafts (n = 4) were intravenously injected with [64Cu]Cu-NOTA-MX001 (22.2 ± 0.2 MBq) and imaged at 0.5, 12, 24, 48, 62 hours post-injection. NU/NU Mice bearing MC38 and 4T1 tumor grafts (n = 4) were injected with [64Cu]Cu-NOTA-MX001 (7.4 MBq) imaged at 12 hours post-injection. The same NU/NU Mice bearing MC38 and 4T1 tumor grafts (n = 4) were injected with [18F]FDG (7.4 MBq) imaged at 1 hours post-injection.28 NU/NU mice bearing BGC-823 or U87-MG were injected with [64Cu]Cu-NOTA-MX001 (7.4 MBq) imaged at 12 hours post-injection. Approximately 5 min prior to PET/CT image acquisition, animals were anesthetized through inhalation of 2% isoflurane/oxygen gas mixture and placed on the scanner bed. PET scans were performed on NanoScan PET-CT scanner (Mediso Medical Solutions HUN, Inc.).28, 29 PET data were reconstructed by Tera-Tomo 3D method, Variance Reduced D.W. Reconstructed PET images were analyzed by Nucline NanoScan software (InterViewTM FUSION, Mediso Medical Solutions HUN, Inc.).

Biodistribution of [64Cu]Cu-NOTA-MX001 in Mice

MC38 tumor-bearing female C57BL/6 mice in each time point (n = 4) were injected intravenously

with certain doses of [64Cu]Cu-NOTA-MX001. The decay corrected injection dose 7



of each mouse was 0.56 MBq at 12, 24, and 48 hours post-injection. The blood, small intestine, large intestine, pancreas, liver, spleen, kidneys, stomach (without content), heart, lungs, brain, bone, muscle, fat and tumors were harvested, weighed and measured by a gamma counter (FH-421).

Anti-PD-L1 Immunotherapy in MC38 and 4T1 Tumor Models

Therapy starting on the 9th day after cells inoculation when the tumor volumes were about 100.0 ± 25.0 mm3, mice in treatment group (n = 5) were injected intravenously with 10 mg/kg of MX001 dissolved in 100 µL PBS twice a week for total four treatments. The mice in control group (n = 5) were injected with 100 µL PBS. The tumor volume was monitored and recorded every two days.

Immunohistochemistry

PD-L1 expression on sections of the tumors, mouse liver and muscle tissues were evaluated by immunohistochemistry (IHC). The representative tissues were fixed in 4% paraformaldehyde and paraffin-embedded for sectioning. Xylene and alcohol gradients were used to do de-paraffin and citrate buffer (10 mM pH = 6.0.) was used to retrieve antigen. 3% H2O2 was used to cover the tissues sections for 10 min, washed and 5% goat serum blocked them for 1 hour. The primary anti-PD-L1 antibody and the second were diluted at 1:200. The sections were incubated with primary antibody at 4 °C for 12-16 hours and then with the second antibody for 1 hour at room temperature in the dark, followed with DAPI for 0.5 hours. A coverslip was applied to each slide in the end. All images were acquired using the laser scan confocal microscope (Leica TCS SP8).

8


Page 8 of 27

Page 9 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Statistical Analysis

Results were presented as mean ± SD. Statistical analyses were performed using Excel (Microsoft), GraphPad Prism 6, ImageJ. Statistical comparisons were analyzed using the unpaired, 2-tailed Student t-test and P

Preclinical Study of a Fully Human Anti-PD-L1 Antibody as a

Recommend Documents