Role of Fluorophore Charge on the In Vivo Optical ... - ACS Publications

Subscriber access provided by UNIV OF CONNECTICUT

Article

Role of Fluorophore Charge on the In Vivo Optical Imaging Properties of Near-Infrared Cyanine Dye/Monoclonal Antibody Conjugates Kazuhide Sato, Alexander P. Gorka, Tadanobu Nagaya, Megan Suzanne Michie, Roger R. Nani, Yuko Nakamura, Vincent Lee Coble, Olga Vasalatiy, Rolf E Swenson, Peter L Choyke, Martin J. Schnermann, and Hisataka Kobayashi Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00492 • Publication Date (Web): 07 Oct 2015 Downloaded from http://pubs.acs.org on October 12, 2015

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 free 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 accessible to all readers and 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.

Bioconjugate Chemistry 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 48

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

Bioconjugate Chemistry

1

Role of Fluorophore Charge on the In Vivo Optical Imaging Properties of Near-Infrared Cyanine Dye/Monoclonal Antibody Conjugates

Kazuhide Sato†, Alexander P. Gorka‡, Tadanobu Nagaya†, Megan S. Michie‡, Roger R. Nani‡, Yuko Nakamura†, Vince L. Coble#, Olga V. Vasalatiy#,

Rolf E. Swenson#, Peter L. Choyke†, Martin J. Schnermann*‡, and

Hisataka Kobayashi*†

†Molecular Imaging Program, Center for Cancer Research, National Cancer

Institute

‡Chemical Biology Laboratory, National Cancer Institute, National Institutes of

Health, Frederick, Maryland 21702, United States

#Imaging Probe Development Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Rockville, MD 20850

ACS Paragon Plus Environment


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

Page 2 of 48

2

*Corresponding authors: Martin J. Schnermann, Ph.D. and Hisataka Kobayashi, M.D., Ph.D.

MJS: Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, NIH, Building 376, Room 225D, Frederick, MD, 21702

HK: Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, NIH, Building 10, RoomB3B69, MSC1088, Bethesda, MD 20892-1088.

Phone: 301-228-4008, Fax: 301-846-6033. E-mail: [email protected]


Running title: Zwitterionic near-infrared cyanine-conjugated antibodies Disclosure Statement None declared

Keywords: near-infrared fluorophore, zwitterionic cyanine, in vivo imaging, monoclonal antibodies, antibody conjugate


Page 3 of 48

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


3

ABSTRACT

Near-infrared (NIR) fluorophores have several advantages over visible-light fluorophores, including superior light penetration in tissue and lower autofluorescence. We recently demonstrated that a new class of NIR cyanine dyes containing a novel C4’-O-alkyl linker exhibit greater chemical stability and excellent optical properties relative to existing C4’-O-aryl variants. We synthesized two NIR cyanine dyes with the same core structure but different indolenine

substituents:

FNIR-774 bearing four

sulfonate

groups

and

FNIR-Z-759 bearing a combination of two sulfonates and two quaternary ammonium cations, resulting in an anionic (-3) or monocationic (+1) charge, respectively. In this study, we compare the in vitro and in vivo optical imaging properties of monoclonal antibody (mAb) conjugates of FNIR-774 and FNIR-Z-759 with panitumumab (pan) at antibody-to-dye ratios of 1:2 or 1:5. Conjugates of both dyes demonstrated similar quenching capacity, stability, and brightness in target cells in vitro. However, FNIR-Z-759 conjugates showed significantly lower background in mice, resulting in higher tumor-to-background



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

Page 4 of 48

4

ratio. Thus, FNIR-Z-759 conjugates appear to have superior in vivo imaging characteristics compared with FNIR-774 conjugates, especially in the abdominal region, regardless of the dye-mAb ratio. These results suggest that zwitterionic cyanine dyes are a promising class of fluorophores for improving in vivo optical imaging with antibody-NIR dye conjugates.

INTRODUCTION

Targeted optical molecular imaging using monoclonal antibody (mAb)-dye conjugates has considerable potential for cancer diagnosis and therapeutic evaluation.1–4 mAbs labeled with fluorescent small molecules permit optical in vivo imaging in real-time and in a targeted fashion. Such conjugates can be highly useful for early cancer detection, disseminating positive margins during surgical resection, and as aids to endoscopic procedures. Among the available fluorescent probes, near-infrared (NIR) fluorophores, which emit light in the 650


Page 5 of 48

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


5

nm to 850 nm range, offer superior tissue penetration and reduced autofluorescence in vivo compared to visible light fluorophores.5–9

Desirable optical, chemical, and biological characteristics of NIR fluorophores include high molar absorption coefficients, high quantum yields, minimal non-specific binding to peptides or proteins, rapid excretion, and minimal changes in the in vivo biodistribution of targeting ligands after conjugation.10 The first two characteristics tend to increase signal at the tumor and the second three tend to decrease background, collectively affording higher tumor-to-background ratios (TBRs). Such improvements in TBR have recently been reported for zwitterionic NIR fluorophores, due to rapid urinary excretion of their catabolites.11 However, the key molecular determinants that impart this enhanced TBR remain obscure. Important considerations include modulating net charge and charge distribution while maintaining hydrophilicity and stability in both oxidative and reducing conditions.

Naturally, the synthesis of such dyes

must also be scalable.



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

Page 6 of 48

6

Consequently, the development of new NIR fluorophores that provide high TBR remains a critical goal. We recently synthesized a new cyanine-based NIR fluorophore, FNIR-774 (Frederick NIR-774), which preserves the highly sulfonated structure of IRDye® 800CW (LI-COR Biosciences), a NIR dye commonly used for in vivo imaging, while transferring the bioconjugatable linker to a central C4’-O-alkyl bond, giving a symmetric structure.12,13 Therefore, FNIR-774 can be prepared quite efficiently through a high yielding and concise synthesis

that

features

an

unusual

electrophile-integrating

Smiles

rearrangement.12 Potential benefits of this novel scaffold include the symmetrical distribution of charged functional groups. Moreover, we have shown that the C4’ ether bond is highly stable to thiol nucleophiles, which is in contrasts to C4’ phenol-substituted cyanine dyes,12 which can react undesirably with thiols.14–17 To further improve the in vivo properties of FNIR-774, we replaced the indolenine alkylsulfonate substituents with a tetraalkyl ammonium functional group, giving a zwitterionic form of FNIR-774, FNIR-Z-759. Prior work has shown that zwitterionic cyanines, either alone or as small molecule conjugates,


Page 7 of 48

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


7

show excellent in vivo properties, with low background and superior TBR during fluorescent imaging.11,18–20 Such properties likely arise from the reported rapid urinary clearance of these zwitterionic molecules or their metabolites.11,21 In this study, we compare the in vitro and in vivo optical imaging characteristics of mAb-FNIR-774 and mAb-FNIR-Z-759 conjugates.

RESULTS

Synthesis of FNIR-Z-759 dye and characterization of FNIR-774 and FNIR-Z-759 dye and dye-mAb conjugates.

The synthesis of the NHS ester of FNIR-Z-759 involved a straightforward 3-step reaction sequence from the known chloro precursor (1) using a variation of the Smiles rearrangement strategy (Scheme 1).12,18,19



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

Page 8 of 48

8

Substitution with N-methylethanolamine proceeded readily in DMF at 60 °C to afford 2, which was used without further purification. In our original rearrangement sequence,12 we found that such C4’-N-methylethanolamine heptamethine cyanines undergo direct rearrangement with a variety of carbonyl electrophiles to provide the desired O-linked products in excellent yields. In this case, we found that immediate exposure of 2 to glutaric anhydride and DIPEA in DMF provided a mixture of C4’ O-and N-linked products, which were difficult to separate. We hypothesized this result might derive from the poor solubility of this presumably zwitterionic species. We found that exposure of the initial product of N-methylethanolamine displacement to TFA-methanol provided material with


Page 9 of 48

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


9

dramatically improved solubility in DMF (see Figure S1). When this material was subjected to glutaric anhydride in DMF for 14 h at 40 °C, the desired O-linked species 3 was now the major component of the reaction mixture, and could be purified by HPLC in 21% yield over two steps. Final NHS-ester formation with N,N,N′,N′-tetramethyl-O-(N-succinimidyl)

uronium

tetrafluoroborate

(TSTU)

provided FNIR-Z-759 in 76% yield.

We have compared the optical properties of FNIR-774 and FNIR-Z-759 as both free compounds and as antibody conjugates. Free FNIR-774 and FNIR-Z-759 (in the carboxylate form) have similar molar absorption coefficients and quantum yields in aqueous buffer and methanol, with the latter being about 1.3-fold brighter (Table 1).



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

Page 10 of 48

10

FNIR-774 and FNIR-Z-759 were conjugated to panitumumab (pan) at a ratio of 2:1 or 5:1 dye-to-antibody (Figure 1).


Page 11 of 48

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


11

The fluorescence of pan-FNIR-774 is about 1.4-fold higher than pan-FNIR-Z-759, given by the area under the emission curve for conjugates (10 µM in dye) excited at their respective λabs (Figure 2A,B).



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

Page 12 of 48

12

By adding 1% SDS to dye-conjugated antibodies, the following dequenching capacities were observed: 1.51-, 2.43-, 1.82-, and 3.82-fold for pan-FNIR-774 (1:2), pan-FNIR-Z-759 (1:2), pan-FNIR-774 (1:5), and pan-FNIR-Z-759 (1:5), respectively. As observed by SDS-PAGE, the fractions of covalently bound dyes to pan were 81.2, 84.4, 71.6, and 77.1% for pan-FNIR-774 (1:2), pan-FNIR-Z-759 (1:2), pan-FNIR-774 (1:5), and pan-FNIR-Z-759 (1:5),


Page 13 of 48

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


13

respectively (Figure S2). Taken together, these data suggest that both FNIR-774 conjugates and FNIR-Z-759 conjugates have similar chemical stability and fluorescence.

In vitro characterization and observation of FNIR-774 and FNIR-Z-759 conjugates.

To evaluate the binding specificity and fluorescence intensity of dye-mAb conjugates, flow cytometry was performed using MDA-MB-468 cells. With the same concentration of each conjugate and incubation time, higher binding was observed with FNIR-774 than FNIR-Z-759. (Figure 3A).



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

Page 14 of 48

14

The binding of both conjugates to MDA-MB-468 was completely blocked by the addition of excess mAb, suggesting specific binding (Figure 3A). Incubation of the antibody with the free dye indicates that FNIR-774 binds more non-specifically

than

FNIR-Z-759.

Importantly,

neither


FNIR-774

nor

Page 15 of 48

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


15

FNIR-Z-759 reduces pan binding to EGFR regardless of the conjugation ratios. Serial fluorescence microscopy of MDA-MB-468 and 3T3-RFP cells was performed after incubation for 1 h at 4 °C (on ice) with each conjugate (Figure 3B).

Although

all

conjugates

demonstrated

cell

surface

labeling

of

EGFR-positive MDA-MB-468 cells, brighter fluorescence was detected with 1:5 conjugates than 1:2 conjugates, independent of the conjugated dyes. 3T3-RFP (EGFR negative) cells showed no detectable fluorescence with the antibody-dye conjugates, indicating that fluorescence was dependent on antibody-receptor interaction (Figure 3B). After replacement of the medium and a further 6 h incubation, each conjugate was internalized into the cell via receptor-mediated endocytosis. Non-specific binding interactions with FNIR-774 or FNIR-Z-759 alone were not seen. Together, these results suggest that both FNIR-774 and FNIR-Z-759 conjugates are highly specific for target-expressing cells and have similar fluorescence properties in cell culture.

In vitro stability of antibody-dye conjugates.



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

Page 16 of 48

16

To assess the stability of the antibody-dye conjugates following endocytosis, cells were incubated with the conjugate for 1 h and microscopy performed 3 days later (Figure 4A).

Regardless of the antibody-dye ratios, the fluorescence of both FNIR-774 and FNIR-Z-759 was preserved at day 3 (Figure 4B). Non-specific binding of free FNIR-774 or FNIR-Z-759 was not observed. Brighter fluorescence was detected with the 1:5 conjugates than the 1:2 conjugates, regardless of the dyes used.


Page 17 of 48

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


17

These results show that both FNIR-774 and FNIR-Z-759 conjugates demonstrate similar stability after cellular internalization.

Confirmation of rapid urinary excretion of free dye in vivo.

To probe for differences in free dye excretion in vivo between FNIR-774 and FNIR-Z-759, in vivo imaging was performed following intravenous injection of each dye (Figure 5A).



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

Page 18 of 48

18

Under a controlled dose, FNIR-Z-759 was more rapidly excreted by the kidney than FNIR-774 (Figure 5A). Neither free FNIR-774 nor free FNIR-Z-759 dye accumulated in the tumor, while both the 1:2 and 1:5 dye-mAb conjugates did (Figure 5B). These data suggest that FNIR-Z-759 dye has more rapid renal clearance than FNIR-774.


Page 19 of 48

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


19

Comparison

of

the

biodistribution

and

tumor

accumulation

of

pan-FNIR-774 and pan-FNIR-Z-759 conjugates.

To

demonstrate

whether

FNIR-774

and

FNIR-Z-759

have

different

pharmacokinetic profiles, in vivo imaging was performed. Pan-FNIR-Z-759 (1:2) showed both lower background and hepatic fluorescence than pan-FNIR-774 (1:2) (Figure 6A).



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

Page 20 of 48

20

Similarly, 1:5 ratio conjugates exhibited lower background and hepatic


Page 21 of 48

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


21

fluorescence with FNIR-Z-759 conjugates than with FNIR-774 conjugates (Figure 6A). These in vivo imaging results were confirmed with ex vivo analysis at 24 h post-injection (Figure 6B). Ex vivo analysis at 6 h post-injection revealed that the FNIR-Z-759 conjugate accumulated within the tumor with higher tumor-to-liver and tumor-to-intestine ratios than the FNIR-774 conjugate (Figure S3). Hepatic uptake was greater after injection of 1:5 vs. 1:2 conjugates for both dyes. (Figure S4A). This was confirmed by ex vivo analysis at 24 h post-injection (Figure S4B). Collectively, these data suggest that pan-FNIR-Z-759 has superior in vivo imaging characteristics, even at a mAb-dye ratio of 1:5, due to lower background fluorescence and lower liver uptake. The rapid renal clearance of FNIR-Z-759 conjugates point to a utility for targeting tumors in the abdomen, such as hepatic and bowel tumors.

To compare the long-term pharmacokinetics of FNIR-774 and FNIR-Z-759 conjugates, in vivo imaging was conducted over 7 days. Conjugates with 1:2 mAb-dye ratios had similar tumor fluorescence at day 7, however, the FNIR-Z-759 conjugates had lower background fluorescence (Figure 7A,B).



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

Page 22 of 48

22

FNIR-Z-759 conjugates demonstrated higher fluorescence intensity than FNIR-774 conjugates at a 1:5 mAb-dye ratio (Figure 7A,B). These data suggest that FNIR-Z-759 conjugates are advantageous for in vivo imaging regardless of conjugation ratio, however 1:5 conjugates stably accumulate within the tumor and retain fluorescence for up to 7 days.


Page 23 of 48

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


23

Evaluation of tumor-to-background ratio (TBR) and tumor-to-liver ratio (TLR).

To quantitatively evaluate fluorescence intensities in tumor-bearing mice for pan-FNIR-774

(1:2),

pan-FNIR-774

(1:5),

pan-FNIR-Z-759

(1:2),

and

pan-FNIR-Z-759 (1:5), TBR and TLR were assessed (n = 5 mice per conjugate) (Figure 8).

Even at 3 h post-injection, FNIR-Z-759 conjugates had superior TBR compared to FNIR-774 conjugates. Pan-FNIR-Z-759 (1:5) showed approximately 2.5- to



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

Page 24 of 48

24

3-fold higher TBR than other conjugates at 24 h, which persisted until day 7. Pan-FNIR-Z-759 (1:2) showed 2.5-fold higher TLR than pan-FNIR-Z-759 (1:5) and pan-FNIR-774 (1:2) at 6 h, and 10-fold higher TLR than pan-FNIR-774 (1:5). Even at high dye-mAb ratios (1:5), FNIR-Z-759 showed higher TLR than FNIR-774 conjugates. These data suggest that FNIR-Z-759 conjugates were catabolized in the kidney rather than in the liver, while FNIR-774 conjugates were mainly hepatically cleared. These data also suggest that FNIR-Z-759 has superior tumor imaging characteristics compared to FNIR-774 because, while both conjugates demonstrate comparable fluorescence in the tumor, only FNIR-Z-759

conjugates

demonstrated

lower

background

and

hepatic

fluorescence, leading to improved TBR and TLR.

DISCUSSION

We have compared the properties of two different mAb-cyanine dye conjugates, pan-FNIR-Z-759 and pan FNIR-774 at antibody-to-dye ratios of 1:2 and 1:5. These conjugates exhibited significantly different in vivo pharmacokinetics, including shortened blood circulation times.22 Two NIR fluorophores with the


Page 25 of 48

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


25

same core structure yet different charge distributions (imparted by the indolenine substitution pattern) led to significant alterations in the biodistribution of conjugated mAb. Therefore, net charge and charge distribution can have a pronounced effect on the in vivo characteristics of NIR heptamethine cyanine dyes, with clear implications for imaging experiments. All mAb-dye conjugates exhibited similar fluorescence and stability in vitro, regardless of labeling ratio. However, higher TBR was achieved in vivo with FNIR-Z-759 (1:5) conjugates compared to FNIR-774 (1:5) conjugates due to lower background with the former. FNIR-Z-759 (1:2) showed higher TLR than other combinations up to 2 days post-injection, indicating that catabolites of FNIR-Z-759 conjugates are renally as opposed to hepatically cleared similar to dye molecules (Figure 6) because strongly anionic dyes even which is hydrophilic tend to excrete through the liver into bile.10,11 Taken together, these data suggest that FNIR-Z-759 conjugates have superior in vivo fluorescence imaging properties compared to FNIR-774 conjugates, largely imparted by the higher TBR.



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

Page 26 of 48

26

A growing number of humanized mAbs directed against tumor-specific cell surface antigens have been approved by the FDA and are successfully used in the clinic, either as a monotherapy or in combination with chemotherapy.23 Since surgery is still a main cancer treatment modality, targeted contrast agents for image-guided surgery will increasingly help to facilitate accurate tumor resection.2,4 When labeled with optical “beacons”, such as in the case of mAb-fluorophore conjugates, these contrast agents have proven highly useful for early cancer detection and for determining tumor margins during surgery or endoscopic procedures.

A potential alternative for in vivo fluorescence imaging with mAb-fluorophore conjugates is the use of fluorescent proteins, which are excellent endogenous fluorescence emitters for depicting various biological processes both in vitro and in vivo.24,25 Some of these studies were performed with orthotopic tumor models, which were better models than subcutaneous xenografts that we used in this study.26,27 These technologies might have unique clinical benefits for detecting cancer based on biological features. However, for common medical application,


Page 27 of 48

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


27

the requirement that fluorescence proteins are transduced by virus-mediated in vivo gene transfection, makes this approach less practical.

While optical imaging generally visualizes the most surface tissue, it allows high-resolution, dynamic, real-time imaging of targeted lesions without the need for ionizing radiation.28 Lowering the background with zwitterionic dyes improves TBR and, subsequently, the sensitivity of the scan. Moreover, exclusive clearance through the kidney renders this zwitterionic NIR dye particularly suitable for abdominal surgery or bowel endoscopy. Thus, it is quite plausible that mAb-FNIR-Z-759 conjugates could be readily adapted for clinical use, further aided by the simple and inexpensive nature of optical imaging.

CONCLUSIONS

In conclusion, a novel NIR fluorescent probe with a zwitterionic net charge, FNIR-Z-759, which can be easily synthesized in high yield, showed favorable in vivo imaging characteristics compared to its net-negatively charged counterpart (FNIR-774). These studies provide support for the notion that minor alterations



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

Page 28 of 48

28

in the chemical structure and charge distribution of a fluorophore can have dramatic effects on its biodistribution in conjugation with a mAb, leading to improved TBR and TLR. Ongoing efforts are focused on development of other zwitterionic cyanine dyes, which are chemically optimized for specific in vivo applications. EXPERIMENTAL PROCEDURES

General methods

All chemicals were of reagent grade or better, purchased from Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Newark, DE, USA), and used as received. Panitumumab, a fully humanized IgG2 mAb directed against EGFR, was purchased from Amgen (Thousand Oaks, CA, USA). See supplemental information for general information on the synthesis methods.

Chemical synthesis

FNIR-774 and FNIR-774 NHS ester were synthesized as previously described.12


Page 29 of 48

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


29

3: 1 (10 mg, 0.0122 mmol) was dissolved in anhydrous DMF (0.25 mL) in a sealed vial and N-methylethanolamine (9.8 µL, 0.122 mmol) added.19 The green solution was heated at 60 °C for 45 min, at which time LCMS showed complete consumption of 1. After cooling to room temperature, the reaction was precipitated into diethyl ether (3 mL) and centrifuged at 6000 rpm. The pellet was resuspended in the same solvent and this process repeated 3X. The pellet was dried under high vacuum for 2 h and isolated as a blue solid. HRMS calculated for (M+ + H+)2+ (C45H67N5O7S2; z = 2): 426.7235, observed 426.7234. UV-VIS analysis revealed that this material is not soluble in DMF (Figure S1). Therefore, the isolated solid was dissolved in 3:1 MeOH/TFA (2 mL), stirred for 25 minutes at room temperature, and then concentrated to yield 2 as a solid, which was used without further purification. HRMS calculated for (M+ + H+)2+ (C45H67N5O7S2; z = 2): 426.7235, observed 426.7235. After this acid treatment, UV-VIS analysis shows this material now has excellent solubility in DMF, with a λmax of 668 nm suggesting that the desired C4’-N linkage is the major species (Figure S1). A small portion of C4’-O linkage is also present, indicated by the



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

Page 30 of 48

30

absorbance peak at 799 nm, which was not observed in earlier studies.12 2 generated according to this procedure was dissolved in anhydrous DMF (0.4 mL) in a sealed vial. A solution of glutaric anhydride (12.53 mg, 0.11 mmol) in DMF (0.04 mL) was added, the reaction mixture heated to 40 °C, and stirred for 14 h. After cooling to room temperature, the reaction was precipitated into diethyl ether (3 mL) and centrifuged at 6000 rpm. The pellet was resuspended in the same solvent, this process was repeated 2X, and dried under high vacuum to afford the crude product. This was purified by preparative HPLC (10 90% MeCN with 10 mM ammonium carbonate) to provide 3 (2.5 mg, 21%) as a green solid. 1

H-NMR (600 MHz, D2O): δ 7.62-7.81 (m, 6H), 6.94 (m, 2H), 5.94 (m, 2H),

3.76-3.97 (m, 8H), 3.32 (m, 4H), 3.12 (s, 3H), 2.99 (s, 18H), 2.50 (m, 2H), 2.38-2.43 (m, 4H), 2.27 (m, 2H), 2.1 (m, 4H), 1.6-1.83 (m, 4H), 1.4 (s, 12H). HRMS calculated for (M+ + H+)2+ (C50H73N5O10S2; z = 2): 483.7394, observed 483.7406. λmax 759 nm in PBS (pH 7.4) (See supporting information, Table 1).

FNIR-Z-759:

3

(5

mg,

0.0038

mmol)

and

N,N,N’,N

′

-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (1.7 mg, 0.0057 mmol)


Page 31 of 48

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


31

were dissolved in anhydrous DMSO (0.3 mL) and N,N-diisopropylethylamine (4 µL, 0.023 mmol) added. The resulting mixture was stirred at room temperature for 1 h, after which complete conversion to desired product was observed by LC-MS. The reaction was precipitated into 1:1 EtOAc/Et2O (5 mL) and centrifuged at 6000 rpm. The pellet was resuspended in the same solvent and this process repeated 4x. The pellet was dried under high vacuum and isolated as a green solid (3.1 mg, 76%). 1H NMR (500 MHz, D2O) δ 7.90 – 7.76 (m, 2H), 7.75 – 7.69 (m, 2H), 7.69 – 7.63 (m, 2H), 7.13 – 6.86 (m, 2H), 6.07 – 5.86 (m, 2H), 4.11 – 3.70 (m, 8H), 3.38 – 3.27 (m, 4H), 2.98 (s, 18H), 2.79 (s, 3H), 2.58 (s, 4H), 2.46 – 2.35 (m, 6H), 2.20 – 2.05 (m, 4H), 1.99 – 1.85 (m, 2H), 1.74 – 1.62 (m, 2H), 1.46 (s, 12H), 1.43 – 1.37 (m, 2H). MS (ESI) m/z 1064.34 calculated for C54H75N6O12S2, m/z 1064.6 (M+), 532.7 (M+2H)+/2, 355.5 (M+2H)+/3 observed. λmax 759 nm in PBS (pH 7.4), λem 786 nm in PBS (pH 7.4). This material was taken directly into the antibody conjugation.

Determination of molar absorption coefficients and quantum yields



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

Page 32 of 48

32

Molar absorption coefficients (ε) and quantum yields (Φf) for unconjugated FNIR-774 and FNIR-Z-759 were measured as previously described in 50 mM PBS (pH 7.4).29 Fluorophore brightness is defined as the product of ε and Φf.

Synthesis of FNIR-774 and FNIR-Z-759-conjugated panitumumab

For conjugation 1:2 (2 dye molecules per antibody), pan (1 mg, 6.8 nmol) was incubated with FNIR-774 or FNIR-Z-759 (30.8 nmol) in 0.1 M Na2HPO4 (pH 8.5) at room temperature for 1 h. For conjugation 1:5 (5 dye molecules per antibody), pan (1 mg, 6.8 nmol) was incubated with FNIR-774 or FNIR-Z-759 (68 nmol) in 0.1 M Na2HPO4 (pH 8.5) at room temperature for 1 h. The resulting mixture was purified with a Sephadex G25 column (PD-10; GE Healthcare, Piscataway, NJ, USA). The protein concentration was determined with the Coomassie Plus protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA) by measuring absorption at 595 nm (8453 Value System; Agilent Technologies, Santa Clara, CA, USA). The concentration of dye was measured by absorption at 774 nm or 759 nm to confirm the number of fluorophore molecules conjugated to each mAb. Absorption and emission curves were measured in 1:1 MeOH/PBS (pH 7.4) for


Page 33 of 48

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


33

2:1 conjugates at an effective dye concentration of 1 µM, with excitation at 774 nm or 759 nm. We performed SDS-PAGE as a quality control for each conjugate. Fluorescent bands were measured with a Pearl Imager (LI-COR Biosciences) using a 800 nm emission channel. We abbreviate FNIR-774 or FNIR-Z-759 conjugated to pan as pan-FNIR-774 (1:2), pan-FNIR-Z-759 (1:2), pan-FNIR-774 (1:5), and pan-FNIR-Z-759 (1:5). We used diluted pan (2 µg) as a non-conjugated control for SDS-PAGE.

Determination of in vitro quenching capacity

The quenching capacity of each conjugate was investigated by denaturation with 1% SDS. Briefly, the conjugates were incubated with 1% (v/v) SDS in phosphate-buffered saline (PBS, pH 7.4) for 15 min at room temperature. As a control, the samples were incubated in PBS without SDS. The change in fluorescence intensity of FNIR-774 or FNIR-Z-759 was investigated with a Pearl Imager using an 800 nm emission channel. Regions of interest (ROIs) were placed on the fluorescence images with reference to white light images to



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

Page 34 of 48

34

measure the fluorescence intensities of the solutions. Pearl software was used for calculating ROI signal data.

Cell culture

EGFR-expressing MDA-MB-468 cells were used as the receptor-positive cell line. Balb/3T3 cells transfected with RFP were used as the receptor-negative cell line. Briefly, Balb/3T3 cells were transfected with RFP (EF1α)- lentiviral particles (AMSBIO, Cambridge, MA, USA) and high, stable RFP expression was confirmed after 10 passages in the absence of a selection agent. Both cell lines were grown in RPMI 1640 (Life Technologies, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Life Technologies), in tissue culture flasks, and in a humidified incubator at 37 °C and an atmosphere of 95% air and 5% carbon dioxide.

Flow Cytometry

In vitro fluorescence on cells was measured using a flow cytometer (FACS Calibur, BD BioSciences, San Jose, CA, USA) and analyzed with CellQuest


Page 35 of 48

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


35

software (BD BioSciences). Cells (1 x 105) were incubated with each conjugate (10 µg/mL) or free dye (0.5 µM) for 6 h at 37 °C. To validate the specific binding of the conjugated antibody, excess antibody (50 µg) was used to block 0.5 µg of conjugates.

Fluorescence microscopy

To detect the antigen specific localization of each conjugate, fluorescence microscopy was performed with a confocal laser scanning microscope (LSM5 meta, Carl Zeiss, Jena, Germany). Ten thousand cells were seeded on coverglass-bottomed dishes and incubated for 24 h. Each mAb-dye conjugate or free dye was then added to the culture medium at 10 µg/mL or 0.5 µM, respectively, and incubated at 4 °C (on ice) for 1 h. The media containing conjugates or dyes was changed to new media (containing no conjugates/dyes) and cells observed after a 6 h incubation at 37 °C.

Alternatively, cells were incubated for 1 h with each conjugate or free dye and the presence of a fluorescence signal confirmed. Cells were then washed with medium, new medium (containing no conjugates/dyes) added, and incubated for



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

Page 36 of 48

36

3 days, at which time cells were observed by microscopy. Image analysis was performed with ZEN software (Carl Zeiss).

Animal and tumor models

All in vivo procedures were conducted in compliance with the Guide for the Care and Use of Laboratory Animal Resources (1996), US National Research Council, and approved by the local Animal Care and Use Committee. Six- to eight-week-old female homozygote athymic nude mice were purchased from Charles River (NCI-Frederick). During procedures, mice were anesthetized with isoflurane.

Six million MDA-MB-468 cells were injected subcutaneously in the right dorsum. The experiments were performed at 14 days after cell injection. Tumors reaching approximately 8 mm in length were selected for the study. To avoid auto-fluorescence in the intestine, mice were fed with white food from 7 days after cell injection.

In vivo fluorescence imaging


Page 37 of 48

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


37

In vivo fluorescence images were obtained with a Pearl Imager (LI-COR Bioscience) after intravenous injection of 50 µg of each conjugate or free dye. Mice were imaged side-by-side in the same view field at 1 h, 3 h, 6 h, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, and 14 days post-injection. Equal sized regions of interest (ROIs) were manually drawn on each tumor and fluorescence intensity at 800 nm was measured. When comparing fluorescence, Pearl Cam software (LI-COR Biosciences) was used for calculating the average fluorescence intensity of each tumor ROI. ROIs were also placed in the adjacent non-tumor region (e.g. a symmetrical region to the left of the tumor) and fluorescence measured as before. Tumor-to-background ratio (TBR) was calculated using following formula: TBR = ((mean tumor intensity) – (mean background intensity)) / ((mean non-tumor intensity) – (mean background intensity)). Tumor-to-liver ratio (TLR) was calculated using following formula: TLR = ((mean tumor intensity) – (mean background intensity)) / ((mean liver intensity) – (mean background intensity)). Statistical Analysis



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

Page 38 of 48

38

Data are expressed as the mean ± S.E.M. from a minimum of three experiments, unless otherwise indicated. Statistical analyses were carried out using GraphPad Prism (La Jolla, CA, USA).

ASSOCIATED CONTENT

Supporting Information

General synthetic methods, absorbance curves for 2 pre- and post-treatment with TFA/methanol, validation of dye-conjugates with SDS-PAGE, biodistribution and clearance of dye-mAb conjugates, in vivo serial fluorescence images of each conjugate (short term comparison of 1:2 and 1:5 conjugates with the same dye), and 1H NMR spectra.

ACKNOWLEDGMENTS

This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.

AUTHOR INFORMATION


Page 39 of 48

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


39

Corresponding Authors

* (HK): Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, NIH, Building 10, Room B3B69, MSC1088, Bethesda, MD 20892-1088.


(MJS): Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, NIH, Building 376, Room 225D, Frederick, MD, 21702


Author Contributions

K.S. mainly designed and conducted experiments, made the study concept, performed analysis and wrote the manuscript; A.P.G. performed chemical synthesis and characterized optical properties for free dyes and conjugates; T.N. conducted in vivo animal experiments; M. S. M. performed chemical synthesis; R.R.N. performed chemical synthesis; Y.N. conducted in vivo animal experiments; V.C. performed chemical synthesis; O.V. advised on the synthesis;



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

Page 40 of 48

40

R.E.S. advised on the synthesis and the manuscript; P.L.C. wrote the manuscript and supervised the project; M.J.S designed experiments and wrote the manuscript; and H.K. planned the project, designed experiments, wrote the manuscript, and supervised the entire project.

REFERENCES (1) Kaur, S., Venktaraman, G., Jain, M., Senapati, S., Garg, P. K., and Batra, S. K. (2012) Recent trends in antibody-based oncologic imaging. Cancer Lett. 315, 97–111. (2) Chi, C., Du, Y., Ye, J., Kou, D., Qiu, J., Wang, J., and Tian, J. (2014) Intraoperative imaging-guided cancer surgery : From current fluorescence molecular imaging methods to future multi-modality imaging technology. Theranostics 4, 1072–1084. (3) Weissleder, R., and Pittet, M. J. (2008) Imaging in the era of molecular oncology. Nature 452, 580–589. (4) Keereweer, S., Van Driel, P. B. A A, Snoeks, T. J. A, Kerrebijn, J. D. F., De Jong, R. J. B., Vahrmeijer, A. L., Sterenborg, H. J. C. M., and Löwik, C. W. G. M. (2013) Optical image-guided cancer surgery: Challenges and limitations. Clin. Cancer Res. 19, 3745–3754. (5) Weissleder, R. (2001) A clearer vision for in vivo imaging: Progress continues in the development of smaller , more penetrable probes for biological imaging. Nat. Biotechnol. 19, 316–317.


Page 41 of 48

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


41

(6) Frangioni, J. V. (2003) In vivo near-infrared fluorescence imaging. Curr. Opin. Chem. Biol. 7, 626–634. (7) Hilderbrand, S. A., and Weissleder, R. (2010) Near-infrared fluorescence: Application to in vivo molecular imaging. Curr. Opin. Chem. Biol. 14, 71–79. (8) Vahrmeijer, A. L., Hutteman, M., van der Vorst, J. R., van de Velde, C. J. H., and Frangioni, J. V. (2013) Image-guided cancer surgery using near-infrared fluorescence. Nat. Rev. Clin. Oncol. 10, 507–18. (9) Luo, S., Zhang, E., Su, Y., Cheng, T., and Shi, C. (2011) A review of NIR dyes in cancer targeting and imaging. Biomaterials 32, 7127–7138. (10) Kobayashi, H., Longmire, M. R., Ogawa, M., and Choyke, P. L. (2011) Rational chemical design of the next generation of molecular imaging probes based on physics and biology: Mixing modalities, colors and signals. Chem. Soc. Rev. 40, 4626–4648. (11) Choi, H. S., Gibbs, S. L., Lee, J. H., Kim, S. H., Ashitate, Y., Liu, F., Hyun, H., Park, G., Xie, Y., Bae, S. , et al. (2013) Targeted zwitterionic near-infrared fluorophores for improved optical imaging. Nat. Biotechnol. 31, 148–53. (12) Nani, R. R., Shaum, J. B., Gorka, A. P., and Schnermann, M. J. (2015) Electrophile-integrating smiles rearrangement provides previously inaccessible C4′-O-alkyl heptamethine cyanine fluorophores. Org. Lett. 17, 302–305. (13) Sato K, Nagaya T, Nakamura Y, Harada T, Nani RR, Shaum JB, Gorka AP, Kim IS, Paik CH, Choyke PL, et al. (2015) Impact of C4’-O-Alkyl Linker on In Vivo Molecular Imaging of Near-Infrared Cyanine/Monoclonal Antibody Conjugates. Mol. Pharm. 12, (in press) (14) Shealy, D. B., Lipowska, M., Lipowski, J., Narayanan, N., Sutter, S., Strekowski, L., and Patonay, G. (1995) Synthesis, chromatographic separation, and characterization of near-infrared labeled DNA oligomers for use in DNA sequencing. Anal. Chem. 67, 247–251.



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

Page 42 of 48

42

(15) Lee, H., Mason, J. C., and Achilefu, S. (2006) Heptamethine cyanine dyes with a robust C-C bond at the central position of the chromophore. J. Org. Chem. 7862–7865. (16) Lim, S. Y., Hong, K. H., Kim, D. Il, Kwon, H., and Kim, H. J. (2014) Tunable heptamethine-azo dye conjugate as an NIR fluorescent probe for the selective detection of mitochondrial glutathione over cysteine and homocysteine. J. Am. Chem. Soc. 136, 7018–7025. (17) Zaheer, A., Wheat, T. E., and Frangioni, J. V. (2002) IRDye78 conjugates for near-infrared fluorescence imaging. Mol. Imaging 1, 354–364. (18) Hyun, H., Henary, M., Gao, T., Narayana, L., Owens, E. A., Lee, J. H., Park, G., Wada, H., Ashitate, Y., Frangioni, J. V., et al. (2015) 700-nm zwitterionic near-infrared fluorophores for dual-channel image-guided surgery. Mol. Imaging Biol. DOI 10.1007/s11307-015-0870-4. (19) Su, D., Teoh, C. L., Samanta, A., Kang, N.-Y., Park, S.-J., and Chang, Y.-T. (2015) The development of a highly photostable and chemically stable zwitterionic near-infrared dye for imaging applications. Chem. Commun. 51, 3989–3992. (20) Choi, H. S., Nasr, K., Alyabyev, S., Feith, D., Lee, J. H., Kim, S. H., Ashitate, Y., Hyun, H., Patonay, G., Strekowski, L. , et al. (2011) Synthesis and in vivo fate of zwitterionic near-infrared fluorophores. Angew. Chem. Int. Ed. 50, 6258– 6263. (21) Njiojob, C. N., Owens, E. A., Narayana, L., Hyun, H., Choi, H. S., and Henary, M. (2015) Tailored near-infrared contrast agents for image guided surgery. J. Med. Chem. 58, 2845–2854. (22) Ogawa, M., Kosaka, N., Choyke, P. L., and Kobayashi, H. (2009) In vivo molecular imaging of cancer with a quenching near-infrared fluorescent probe using conjugates of monoclonal antibodies and indocyanine green. Cancer Res. 69, 1268–1272.


Page 43 of 48

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


43

(23) Reichert, J. M., and Dhimolea, E. (2012) The future of antibodies as cancer drugs. Drug Discov. Today 17, 954–963. (24) Yano, S., Miwa, S., Kishimoto, H., Toneri, M., Hiroshima, Y., Yamamoto, M., Bouvet, M., Urata, Y., Tazawa, H., Kagawa, S., et al. (2015) Experimental Curative Fluorescence-guided Surgery of Highly Invasive Glioblastoma Multiforme Selectively Labeled With a Killer-reporter Adenovirus. Mol Ther 23, 1182-1188. (25) Yano, S., Miwa, S., Kishimoto, H., Uehara, F., Tazawa, H., Toneri, M., Hiroshima, Y., Yamamoto, M., Urata, Y., Kagawa, S., et al. (2015) Targeting tumors with a killer-reporter adenovirus for curative fluorescence-guided surgery of soft-tissue sarcoma. Oncotarget 6, 13133-13148. (26) Hoffman, R. M. (2015) Patient-derived orthotopic xenografts: better mimic of metastasis than subcutaneous xenografts. Nat Rev Cancer 15, 451-452. (27) Li, X., Wang, J., An, Z., Yang, M., Baranov, E., Jiang, P., Sun, F., Moossa, A. R., and Hoffman, R. M. (2002) Optically imageable metastatic model of human breast cancer. Clin Exp Metastasis 19, 347-350. (28) Hoffman, R. M. (2005) The multiple uses of fluorescent proteins to visualize cancer in vivo. Nat. Rev. Cancer 5, 796–806. (29) Gorka, A. P., Nani, R. R., Zhu, J., Mackem, S., and Schnermann, M. J. (2014) A near-IR uncaging strategy based on cyanine photochemistry. J. Am. Chem. Soc. 136, 14153–14159.

Figure legends



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

Page 44 of 48

44

Scheme 1: Synthesis of FNIR-Z-759.

Table 1: Optical properties of FNIR-Z-759 and FNIR-774.

Figure 1.

Schematic representation of FNIR-774 and FNIR-Z-759 1:2 and 1:5 conjugates to panitumumab.

FNIR-774 and FNIR-Z-759 were conjugated to pan with an antibody-to-dye ratio of 1:2 or 1:5.

Figure 2.

In vitro characterization of FNIR-774 and FNIR-Z-759 dye and dye-mAb conjugates.

Absorption and emission curves for 2:1 pan-FNIR-774 (A) and pan-FNIR-Z-759 (B) at an effective dye concentration of 1 µM in 1:1 MeOH/PBS (pH 7.4). Emission curves were collected after excitation at the near-IR absorption maximum of each dye (774 nm for FNIR-774 and 759 nm for FNIR-Z-759).


Page 45 of 48

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


45

Figure 3.

In vitro characterization and observation of FNIR-774 and FNIR-Z-759 conjugates.

(A) Fluorescence of EGFR-expressing MDA-MB-468 cells incubated with each free dye or conjugate, evaluated by flow cytometry. (B) Microscopic observation of MDA-MB-468 cells incubated on ice for 1 h with each free dye (third row) or conjugate (first and second row). After 1 h incubation on ice, the media with conjugates was exchanged and cells were observed after 6 h incubation (lower row of each panel). Non-EGFR-expressing 3T3-RFP cells (*) were used as non-target controls. Scale bar = 25 µm.

Figure 4.

Stability of fluorescence in vitro.

(A) Regimen for the evaluation of fluorescence stability in cell culture. (B) Microscopic observation of MDA-MB-468 cells along the regimen. Both FNIR-774 and FNIR-Z-759 were still fluorescent at the time of observation.



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

Page 46 of 48

46

Figure 5.

In vivo serial fluorescence images of free dyes and accumulation of the conjugates in the tumor.

(A) In vivo serial fluorescence images of MDA-MB-468 tumor bearing mice (right dorsum) injected with each free dye. Both were excreted into the urine immediately after injection, with FNIR-Z-759 showing more rapid clearance (n = 5, each dye). (B) Accumulation of each dye-mAb conjugate within the tumor. Free dye did not show meaningful tumor accumulation. In all images, scale bars to the right indicate relative fluorescence intensity.

Figure 6.

In vivo serial fluorescence images of each conjugate (short term).

(A) In vivo serial fluorescence images (short term) of MDA-MB-468 tumor bearing mice (right dorsum) injected with each conjugate. All mice were imaged side-by-side under the same view field for the purpose of comparing each conjugate. The upper panel was imaged with high threshold and the lower panel


Page 47 of 48

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


47

with low threshold. (B) Ex vivo fluorescence images of the liver, kidney, and MDA-MB-468 tumor obtained at 24 h post-injection. In all images, scale bars to the right indicate relative fluorescence intensity.

Figure 7.

In vivo serial fluorescence images of each conjugate (long term).

(A) In vivo serial fluorescence images (long term) of MDA-MB-468 tumor bearing mice (right dorsum) injected with each conjugate. Mice were imaged side-by-side within the same view field for the purpose of comparing each conjugate. (B) Comparison of tumor fluorescence with FNIR-774 and FNIR-Z-759 conjugates at day 7. In all images, scale bars to the right indicate fluorescence intensity.

Figure 8.

Tumor-to-background ratio (TBR) and tumor-to-liver ratio (TLR).



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

Page 48 of 48

48

Tumor-to-background ratio (TBR) and tumor-to-liver ratio (TLR) of each conjugate injected into the right dorsum of MDA-MB-468 tumor bearing mice (n = 5, each conjugate).


Role of Fluorophore Charge on the In Vivo Optical ... - ACS Publications

Recommend Documents