Radioimmunotherapy of PANC-1 Human Pancreatic Cancer

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Radioimmunotherapy of PANC-1 Human Pancreatic Cancer Xenografts in NRG Mice with Panitumumab Modified with Metal-Chelating Polymers (MCP) Complexed to 177Lu Sadaf Aghevlian, Zhongli Cai, Yijie Lu, David W. Hedley, Mitchell A. Winnik, and Raymond M Reilly Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01040 • Publication Date (Web): 27 Dec 2018 Downloaded from http://pubs.acs.org on January 2, 2019

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Molecular Pharmaceutics

Radioimmunotherapy of PANC-1 Human Pancreatic Cancer Xenografts in NRG Mice with Panitumumab Modified with MetalChelating Polymers (MCP) Complexed to 177Lu

Sadaf Aghevlian, † Zhongli Cai, † Yijie Lu, Winnik, ‡, * and Raymond M. Reilly *,†,¶, ¥

‡

David W. Hedley §, Mitchell A.

Department of Pharmaceutical Sciences, University of Toronto, 144 College Street, Toronto, ON M5S 3M2, Canada †

Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON M5S 3H6, Canada ‡

Department of Medical Oncology, Princess Margaret Cancer Centre, 610 University Avenue, Toronto, ON M5G 2M9, Canada §

¶ Department

of Medical Imaging, University of Toronto, 263 McCaul Street, Toronto, ON M5T 1W7, Canada ¥ Toronto

General Research Institute and Joint Department of Medical Imaging, University Health Network, 200 Elizabeth Street, Toronto, ON M5G 2C4, Canada. Footline: Radioimmunotherapy of Pancreatic Cancer * Corresponding Authors: Raymond M Reilly, Ph.D. Leslie Dan Faculty of Pharmacy, University of Toronto 144 College Street, Toronto, ON M5S 3M2, Canada Tel: (416) 946-5522; Fax: (416) 978-8511; Email: [email protected] Mitchell A. Winnik, Ph.D. Department of Chemistry, University of Toronto, 80 St. George St., Toronto, ON, M5S 3H6, Canada Tel. (416) 978-6495; Fax: (416) 978-0541; Email: [email protected] Word Count: 9,085

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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

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GRAPHICAL ABSTRACT


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ABSTRACT: Our aim was to evaluate the effectiveness and normal tissue toxicity of radioimmunotherapy (RIT) of s.c. PANC-1 human pancreatic cancer (PnCa) xenografts in NRG mice using anti-EGFR panitumumab linked to metal-chelating polymers (MCP) that present 13 DOTA chelators to complex the -emitter, 177Lu. The clonogenic survival (CS) of PANC-1 cells treated in vitro with panitumumab-MCP-177Lu (0.3-1.2 MBq) and DNA double-strand breaks (DSBs) in the nucleus of these cells were measured by confocal immunofluorescence microscopy for -H2AX. Subcellular distribution of radioactivity for panitumumab-MCP-177Lu was measured and absorbed doses to the cell nucleus calculated. Normal tissue toxicity was assessed in non-tumor bearing NRG mice by monitoring body weight, complete blood cell counts (CBC) and serum alanine aminotransferase (ALT) and creatinine (Cr) after i.v. injection of 6 MBq (10 g) of panitumumab-MCP-177Lu. RIT was performed in NRG mice with s.c. PANC-1 tumors injected i.v. with 6 MBq (10 g) of panitumumab-MCP-177Lu. Control mice received non-specific human IgG-MCP-177Lu (6 MBq; 10 g), unlabeled panitumumab (10 g) or normal saline. The tumor growth index (TGI) was compared. Tumor and normal organ doses were estimated based on biodistribution studies. Panitumumab-MCP-177Lu reduced the CS of PANC-1 cells in vitro by 7.7-fold at the highest amount tested (1.2 MBq). Unlabeled panitumumab had no effect on the CS of PANC-1 cells. γ-H2AX foci were increased by 3.8-fold by panitumumabMCP-177Lu. Panitumumab-MCP-177Lu deposited 3.84 Gy in the nucleus of PANC-1 cells. Administration of panitumumab-MCP-177Lu (6 MBq; 10 g) to NRG mice caused no change in body weight, CBC or ALT and only a slight increase in Cr compared to NRG mice treated with normal saline. Panitumumab-MCP-177Lu strongly inhibited tumor growth in NRG mice (TGI = 2.3 ± 0.2) compared to normal saline treated mice (TGI = 5.8 ± 0.5;


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P0.05). The absorbed dose of PANC-1 tumors was 12.3 Gy. The highest normal organ doses were absorbed by the pancreas, liver, spleen, and kidneys. We conclude that EGFRtargeted RIT with panitumumab-MCP-177Lu was able to overcome resistance to panitumumab in KRAS mutant PANC-1 tumors in NRG mice and may be a promising approach to treatment of PnCa in humans.

KEYWORDS: EGFR; metal-chelating polymers; panitumumab; radioimmunotherapy; 177Lu


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INTRODUCTION

Pancreatic cancer (PnCa) is a rapidly fatal malignancy due to its often advanced stage at diagnosis combined with the limited treatment options 1. Surgery remains the only hope for long-term survival, but only 10-15% of patients are candidates for surgical resection due to local invasion or distant metastasis 2. Even in patients who undergo surgical resection with curative intent followed by chemotherapy, the median survival is 17-23 months. For patients who are not candidates for surgery due to locally advanced PnCa, the median survival is 8-14 months and is 4-6 months for patients with metastatic PnCa 3. New therapeutic strategies are needed for patients with PnCa. Stereotactic body radiation therapy (SBRT) has proven effective for controlling local progression of PnCa providing freedom from local progression (FFLP) for as long as a year in 80% of patients with locally advanced PnCa, but patients ultimately die of metastatic disease

4.

Radioimmunotherapy (RIT) which offers an opportunity to selectively treat disseminated PnCa at any location in the body with radiation, may improve patient outcome in patients with metastatic disease. RIT with the humanized monoclonal antibody (mAb), clivatuzumab (Immunomedics, Morris Plains, NJ) targeting the cell surface mucin MUC1 5 labeled with the -emitter,

90Y

[Emax=2.2 MeV (100%); t1/2=2.7 d] showed promising

results (partial remission or stable disease) in a Phase I/II trial combined with gemcitabine for treatment of metastatic PnCa 6. Improved survival was found in patients with metastatic PnCa in a subsequent Phase 1 trial that combined

90Y-clivatuzumab

with gemcitabine

chemotherapy compared to RIT alone (7.8 vs. 3.4 months) 7. However, interim analysis of a Phase 3 trial (PANCRIT-1) that randomized patients to RIT + gemcitabine and best


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supportive care (BSC) or gemcitabine and BSC alone did not demonstrate improved survival in the RIT arm (https://clinicaltrials.gov/ct2/show/NCT01956812) and the trial was discontinued. However, patients in the PANCRIT-1 trial had metastatic PnCa that had progressed after at least two prior chemotherapy regimens. Thus, there remains the potential that earlier treatment of PnCa in patients with lower tumor burden may improve outcome. In addition, there is an emerging interest in the use of RIT to eradicate small tumors and minimal residual disease to prevent disease progression after other forms of cancer therapy 8. The shorter range (2 mm) -particles emitted by 177Lu [t1/2 = 6.7 d; Emax = 0.5 MeV (78.6%); Emax = 0.38 MeV (9.1%); Emax = 0.18 MeV (78.6%)] may be more appropriate for RIT of small tumors, while the higher energy [(Emax = 2.2 MeV (100%)] and longer range (12 mm) -particles emitted by

90Y

are more useful for treatment of

larger tumors. Modeling has revealed that the optimal tumor diameter for treatment with 177Lu

is ~2 mm, while for 90Y, the ideal tumor diameter for RIT is 34 mm 9. Indeed, de

Jong et al. reported that co-treatment with a mixture of

177Lu-

and

90Y-labeled

DOTATATE radiopeptides was more effective than either radiopeptide alone in prolonging the survival of rats with both large and small somatostatin receptor-positive CA20948 s.c. pancreatic neuroendocrine tumors 10. Panitumumab (Vectibix; Amgen, Thousand Oaks, CA) is a human IgG2 mAb that binds the epidermal growth factor receptor (EGFR) 11. EGFR are overexpressed on PnCa (>90% of cases) 12. Naked anti-EGFR mAbs have not proven effective for treatment of PnCa

13, 14

since there is often downstream KRAS mutation 15, but this is not limiting for

RIT, since the mAbs are used only to target radioactivity to tumors. We recently reported novel radioimmunoconjugates (RICs) for RIT of PnCa consisting of panitumumab linked


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to metal-chelating polymers (MCP) that complex 177Lu or 111In 16. The MCP are composed of a polyglutamide backbone with 10 pendant polyethylene glycol (PEG) chains with the aim to inhibit liver and spleen uptake of the RICs, and 13 DOTA (1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelators that strongly complex 111In 16.

177Lu

or

These polymer immunoconjugates have theranostic capability since they may be

labeled simultaneously to high specific activity (SA) with

177Lu

and

111In.

The -particle

emissions of 177Lu and Auger electrons emitted by 111In enable RIT, while the -emissions of

111In

[Eγ = 171 keV (90%) and Eγ = 245 keV (94%)] allow single photon emission

computed tomography (SPECT). In the current study, we examine the cytotoxicity of panitumumab-MCP-177Lu in vitro on EGFR-positive PANC-1 human PnCa cells and its ability to inflict lethal DNA double-strand breaks (DSBs) in the nucleus of these cells and estimate the absorbed doses in the cell nucleus. We further report for the first time the effectiveness and normal tissue toxicity of RIT of PANC-1 tumors in NOD-Rag1−/−IL2RgammaC-null (NRG) mice with panitumumab-MCP-177Lu, and estimate the absorbed doses in tumors and normal organs. We hypothesized that RIT with panitumumab-MCP-177Lu would be effective for treatment of PANC-1 tumors at administered amounts that are non-toxic to normal tissues.

MATERIALS AND METHODS

Cell culture and tumor xenografts. PANC-1 human pancreatic adenocarcinoma cells (4.0 × 105 EGFR/ cell)

17

were purchased from the American Type Culture Collection

(ATCC, Manassas, VA, USA). PANC-1 cells were cultured in Dulbecco's Modified


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Eagle's Medium (DMEM) with high glucose (Gibco Life Technologies; Burlington, ON, Canada) supplemented with 1% penicillin and streptomycin and 10% fetal bovine serum (Gibco-Invitrogen, Thermo Fisher Scientific, Waltham, MA). Tumor xenografts (8-10 mm diameter) were established in female NOD-Rag1−/−IL2RgammaC-null (NRG) mice (University Health Network, Toronto, ON) by subcutaneous (s.c.) inoculation of 4 × 106 PANC-1 cells in serum free DMEM (100 μL) into the left flank.

Radioimmunoconjugates. The synthesis of hydrazino nicotinamide (HyNic) end-capped HyNic-MCP presenting 13 DOTA chelators and 10 PEG chains to minimize liver and spleen uptake was previously reported

18.

Panitumumab-MCP immunoconjugates were

constructed by cross-linking panitumumab IgG to two HyNic-MCPs through an Nsuccinimidyl-4-formylbenzamide (S-4FB) moiety introduced into panitumumab and stored in 0.1 M HEPES buffer, pH 5.5. Immunoconjugates (5-70 µg; 4-12 L) were radiolabeled by incubation with

177LuCl 3

(0.75-49 MBq; 37 MBq/μL; PerkinElmer, Waltham, MA) at

42 °C for 2 h16. The final radiochemical purity of the RICs was >95% determined by instant thin-layer silica gel chromatography (ITLC-SG; Pall) developed in 0.1 M sodium citrate, pH 5.5. We previously reported that the KD for binding of panitumumab-MCP complexed to

111In

to EGFR-positive MDA-MB-468 human breast cancer cells was 2.2 ±

0.6 nmol/L 16. Non-specific human IgG (hIgG; Sigma-Aldrich, St. Louis, MO, Product No. 14506) was similarly modified with MCP and labeled with 177Lu. The RICs were shown to be stable to loss of radiometal by challenge with an excess of ethylenediaminetetraacetic acid (EDTA) and in plasma at 37 oC 16.


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In vitro cytotoxicity studies. The clonogenic survival (CS) of PANC-1 cells treated in vitro with panitumumab-MCP-177Lu was studied. Briefly, 2 × 105 cells were treated with 2.5 nmol/L (50-fold molar excess) of panitumumab-MCP labeled with 0.3, 0.6 or 1.2 MBq of

177Lu

[SA = 46.7, 93.5, 187.0 MBq/nmole] in 500 μL of growth medium in 24 well

plates for 16 h. Controls consisted of cells exposed to unlabeled panitumumab-MCP or to growth medium alone. Approximately 700 cells were then seeded into 6 well plates and cultured for 12 d. Surviving colonies were stained with methylene blue and colonies with >50 cells were counted. The plating efficiency (PE) was determined by dividing the number of colonies formed by the number of cells seeded. The CS was calculated by dividing the PE for treated cells by that for untreated cells 19.

Induction of DNA double-strand breaks (DSBs) in PANC-1 cells caused by panitumumabMCP-177Lu was assessed by immunofluorescence confocal microscopy for phosphorylated histone-2A (γ-H2AX) which accumulates at sites of DSBs in the cell nucleus 20. Briefly, 2 × 105 PANC-1 cells were seeded onto glass coverslips (Ted Pella, Redding, CA) in 24-well plates and cultured overnight at 37 °C. The cells were then treated with panitumumabMCP-177Lu as described earlier. Controls consisted of cells exposed to unlabeled panitumumab-MCP or to growth medium alone. Immunostaining for γ-H2AX and analysis of confocal microscopy images for the density of γ-H2AX foci per nucleus area followed previously reported procedures 21. Normalization of γ-H2AX foci per nucleus area rather than the number of foci in the nucleus minimizes differences in cell nucleus size between cells.


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Subcellular

distribution

and

microdosimetry.

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The

subcellular

distribution

of panitumumab-MCP-177Lu in PANC-1 cells was measured to estimate the absorbed doses in the cell nucleus (microdosimetry). Briefly, 2 × 105 cells were cultured overnight in wells in a 6-well plate. A 50-fold molar excess of panitumumab-MCP-177Lu (1.2 MBq; 2.5 nmol/L) were added to the wells and the plates were incubated for 1, 4, 8, or 24 h at 37 °C in the absence or presence of excess of unlabeled panitumumab to block EGFR to confirm the EGFR specificity of binding of panitumumab-MCP-177Lu to PANC-1 cells. Cell fractionation studies were also performed for cells incubated with non-specific hIgGMCP-177Lu (1.2 MBq; 2.5 nmol/L). The medium was removed and the cells which were exposed to panitumumab-MCP-177Lu were fractionated to determine the dose to the cell nucleus from the cell surface, cytoplasm and nucleus source compartments. The cells were first rinsed with phosphate-buffered saline (PBS), pH 7.5, then the proportion of internalized cell-bound radioactivity was determined by displacing the cell-surface

177Lu

with 1 mL of 200 mM sodium acetate/500 mM NaCl, pH 2.5 at room temperature (RT). This procedure was repeated twice and the cell membrane fractions were combined and measured in a γ-counter. The cell membrane was then lysed twice in 500 μL of Nuclei EZ Lysis buffer (Sigma-Aldrich) on ice for 60 min. A cell scraper was used to gently detach cells from each well and the cells were then transferred to 1.5 mL Eppendorf tubes. The tubes were centrifuged at 3,000 × g for 5 min to separate nuclear and cytoplasmic radioactivity (supernatant). The radioactivity in the nucleus, membrane and cytoplasm fractions was measured in a γ-counter and the percentage of radioactivity in each fraction (relative to the total cell-bound 177Lu) was calculated. This cell fractionation procedure has been previously validated by our group to yield pure cytoplasmic and nuclear fractions 22.


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The absorbed dose in the nucleus (D) of a PANC-1 cell after exposure to 1.2 MBq of panitumumab-MCP-177Lu (2.5 nmol/L) was estimated based on the cumulative amount of radioactivity (Ãs) on the cell surface, in the cytoplasm and in the cell nucleus as well as non cell-bound radioactivity in the surrounding culture medium. Ãs was calculated from the area under the curve from 0 to 24 h (AUC0−24h; Bq × sec) using Prism Ver. 4.0 software (GraphPad). The dose to the nucleus was estimated as D = Ãs × S, and the S-values (Gy/Bq × sec) were calculated using MCNP Monte Carlo code Ver. 5 (Los Alamos National Laboratory, Los Alamos, NM). A mean cell radius of 17.1 μm and nucleus radius of 7.0 μm were used. These were measured by fluorescence microscopy of PANC-1 cells and Image J software (National Institutes of Health, Bethesda, MD) after staining of the nucleus with 4′,6-diamidino-2-phenylindole (DAPI)

23.

The absorbed doses were also

calculated for PANC-1 cells exposed to panitumumab-MCP-177Lu co-incubated with a 50fold molar excess of unlabeled panitumumab to block EGFR, or non-specific hIgG-MCP177Lu

(1.2 MBq; 2.5 nmol/L).

Evaluation of normal tissue toxicity. To identify a non-toxic dose for subsequent RIT studies, groups of 5 healthy, non-tumor bearing female NRG mice were injected i.v. (tail vein) with 6.0 MBq (10 μg) of panitumumab-MCP-177Lu or non-specific hIgG-MCP-177Lu in 100 μL of normal saline. These doses were selected based on a previous study that reported that 8.0 MBq of

177Lu-labeled

RICs were administered safely without major

toxicity to NRG mice (no loss in body weight)

24.

NRG mice were selected for these

studies and for RIT studies, since unlike NOD/SCID mice, NRG mice do not harbor a germ line defect in DNA repair

25

which renders NOD/SCID mice unusually sensitive to


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radiation. Control mice received injections of normal saline or panitumumab. Body weight was monitored every 2-4 d for 14 d. The mice were then sacrificed and samples of blood were collected into EDTA-coated microtubes and centrifuged to separate serum for biochemistry [serum alanine aminotransferase (ALT) and creatinine (Cr)] analyses. ALT and Cr were measured using Infinity Creatinine or ALT clinical chemistry kits (Fisher Diagnostics, Middletown, VA). Complete blood cell (CBC) counts, hematocrit (HCT) and hemoglobin (Hb) were measured on a HemaVet 950FS instrument (Drew Scientific, Miami Lakes, FL). The 14 d period for assessing normal tissue toxicity was selected based on the requirements of Health Canada for acute toxicity testing of new drugs 26.

Radioimmunotherapy studies. The tumor-growth inhibitory effects of panitumumabMCP-177Lu were studied in groups of NRG mice (n=7) with s.c. PANC-1 xenografts. The number of mice in each group (7) was sufficient to detect a minimum difference of 0.8 in the mean tumor growth index (TGI) with a standard deviation of 0.5, an -value = 0.05 and power, -value = 0.80 (https://www.stat.ubc.ca/~rollin/stats/ssize/n2.html). Mice received a single i.v. injection (tail vein) of 6.0 MBq (10 μg) of panitumumab-MCP-177Lu in 100 µL of normal saline. Control groups of NRG mice were injected with 6.0 MBq (10 μg) of hIgG-MCP-177Lu, unlabeled panitumumab (10 µg) or normal saline. Tumor length (mm) and width (mm) were measured using calipers every 2-3 days until the humane endpoint of the animal care protocol was reached (tumor diameter >15 mm). The tumor volume (V; mm3) was calculated as V =length × width2 × 0.5 27. The TGI was calculated by dividing the tumor volume at each time point by the tumor volume at the initiation of


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treatment. Similarly, the body weight index (BWI) was estimated by dividing the body weight at each time point by the pre-treatment body weight.

Tumor and normal organ dosimetry. The absorbed doses in the tumor and normal organs in NRG mice with s.c. PANC-1 xenografts after i.v. injection of 6.0 MBq (10 μg) of panitumumab-MCP-177Lu were estimated. Groups of 5 mice were injected with RICs, then at selected time points ranging from 24 to 168 h p.i., the mice were sacrificed by cervical dislocation under anesthesia, and the tumor and samples of normal tissues including blood were collected, weighed, and counted in a γ-counter. Tumor and normal organ uptake were expressed as MBq/g, then converted to MBq/organ by multiplying by previously determined standard weights for mouse organs 28. The cumulative radioactivity in the tumor and normal source organs from 0-168 h (Ã0-168h) was estimated using Prism Ver. 4.0 software (GraphPad, San Diego, CA) from the AUC0-168h (MBq × h) determined in biodistribution studies. The cumulative radioactivity from 168 h to infinity (Ã168h-∞) was calculated by dividing the radioactivity at the final measured time point by the elimination rate constant for

177Lu

(0.004345 h-1), thus assuming that further elimination occurs only

by radioactive decay without additional biological elimination. We have previously applied this assumption to estimate the absorbed doses for other radiolabeled mAbs 28. The Ã0-∞ values of each source organ (MBq  h) were converted to Bq x sec by multiplying by (3.6  103 secs/h)(106 Bq/MBq). Then, these Ã0-∞ values were multiplied by the respective S-value (Gy/Bq × sec) of the source to target organs for values

29,

177Lu

using published mouse S-

then summing up all the absorbed doses to the target organ from all source

organs. The dose delivered to the tumor was estimated based on S-values of the sphere


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model in OLINDA/EXM, using the average measured tumor volume at the time of treatment 30. Statistical analysis. Results were expressed as mean  SEM. Statistical significance was tested using an unpaired two-tailed Student’s t-test (P23-25 Gy to cause renal toxicity in humans 49. No bone marrow toxicity was found for panitumumab-MCP-177Lu (Fig. 3) despite a whole body absorbed dose of 1.4 Gy (Table 3). Whole body dose has been used as a predictor of bone marrow toxicity from RIT with doses >2-3 Gy causing dose-limiting hematopoietic toxicity in humans 40, 50. Mouse S-values were used to estimate the normal organ doses in mice from panitumumabMCP-177Lu 29. Due to the close proximity of organs in the mouse and the maximum 2 mm range of the -particles emitted by

177Lu,

the doses to normal organs in NRG mice may

overestimate the human doses, due to a significant cross-fire effect. Bitar et al. point out that for energies >0.5 MeV, -particles cannot be considered as non-penetrating radiation in mice, i.e. they have a significant cross-fire effect 29. This cross-fire effect is noted by the main contributing source organ for the dose to the whole body being the liver (Table 3).

CONCLUSION

We conclude that panitumumab-MCP-177Lu inflicted DNA DSBs in PANC-1 human PnCa cells in vitro that decreased the CS of these cells. Administration of 6 MBq (10 g) of panitumumab-MCP-177Lu caused no normal tissue toxicity in NRG mice but strongly inhibited the growth of s.c. PANC-1 tumors for up to 26 days. Unlabeled panitumumab was not effective for treating PANC-1 tumors due to KRAS mutation. Our results demonstrate that RIT with panitumumab-MCP-177Lu was able to overcome resistance to


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panitumumab caused by KRAS mutation in PANC-1 tumors, suggesting that RIT may be a promising approach to the treatment of PnCa in humans.

ASSOCIATED CONTENT

Supporting Information The supporting information is available free of charge on the ACS Publications website at DOI: Tumor and normal organ biodistribution of panitumumab-MCP-177Lu (MBq/g) in NRG mice with s.c. PANC-1 human pancreatic cancer xenografts at selected times up to 168 h post-injection (Figure S1).

ACKNOWLEDGMENTS

This work was supported by a grant from the Canadian Cancer Society and a grant from the Cancer Research Society. S. Aghevlian received scholarships from the STARS21 strategic training program in radiation research supported by the Terry Fox Foundation and the Centre for Pharmaceutical Oncology (CPO) at the University of Toronto. S. Aghevlian is supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) through the Polymer Nanoparticles for Drug Delivery (POND) Training Program. The authors would like to thank Deborah Scollard at the Spatiotemporal


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Targeting and Amplification of Radiation Response (STTARR) Innovation Centre for technical support.

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Radioimmunotherapy of PANC-1 Human Pancreatic Cancer

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