Reactive Polymer Enables Efficient in Vivo Bioorthogonal Chemistry

Reactive polymer enables efficient in vivo bioorthogonal chemistry

Neal K. Devaraj1, Greg M. Thurber1, Edmund J. Keliher, Brett Marinelli, and Ralph Weissleder2

Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Simches Research Building, 185 Cambridge Street, Boston, MA 02114

Edited by David A. Tirrell, California Institute of Technology, Pasadena, CA, and approved January 31, 2012 (received for review August 16, 2011)

There has been intense interest in the development of selective (stability, bioconversion of reactants, target delivery, etc.) and bioorthogonal reactions or “click” chemistry that can proceed in identified simple pharmacokinetic principles as the primary rea- live animals. Until now however, most reactions still require vast son why the reaction did not proceed at target sites as originally surpluses of reactants because of steep temporal and spatial con- anticipated. We then performed detailed studies on how the re- centration gradients. Using computational modeling and design of action rate and pharmacokinetics (PK) can affect the efficiency of pharmacokinetically optimized reactants, we have developed a an in vivo chemical reaction. Using reactants with modified PK predictable method for efficient in vivo click reactions. Specifically, profiles, such as polymer modified tetrazines [(PMT); Fig. 1C] we show that polymer modified tetrazines (PMT) are a key enabler and a newly developed computational model to predict reaction for in vivo bioorthogonal chemistry based on the very fast and efficiency, we now observe highly efficient in vivo bioorthogonal catalyst-free [4 þ 2] tetrazine/trans-cyclooctene cycloaddition. reaction. Using cancer cell epitopes as a model target (Fig. 1D), Using fluorescent PMT for cellular resolution and 18F labeled PMT we show the specificity and sensitivity of the method in a mouse for whole animal imaging, we show that cancer cell epitopes can model of colon cancer. Specifically, we observe in vivo labeling be easily reacted in vivo. This generic strategy should help guide with cellular resolution using near infrared fluorophores. the design of future chemistries and find widespread use for dif- Furthermore, we demonstrate use of bioorthogonal reactions ferent in vivo bioorthogonal applications, particularly in the biome- with rapidly decaying positron emission tomography (PET) iso- dical sciences. topes for imaging cancer cell epitopes in solid tumors. We believe our work and findings will guide further development of in vivo in vivo chemistry ∣ pharmacokinetics ∣ PET imaging ∣ intravital microscopy ∣ chemistries for a number of different biomedical applications. pretargeting Results he ability to perform selective chemistries in living systems We first compared different reactants in mice to better under- Tsuch as single cells, 3D cultures, invertebrates, or mammals stand achievable in vivo click reaction efficiencies as a function would have far reaching applications in tracking biomolecules, of PK profiles (Table 1). These studies were performed as serial designing new therapeutic approaches, and in visualizing medi- blood draws, single time point biodistribution studies, or longitu- cally relevant biomarkers. To date, only a few practical bioortho- dinal imaging studies at different resolutions. In a first set of ex- gonal reactions have been reported, the most popular being the periments, mice were injected with 30 micrograms of anti-CD45 Staudinger ligation and the [3 þ 2] cycloaddition “click” reaction monoclonal antibodies labeled with TCO or norbornene dieno- between azides and alkynes (1, 2). The latter click reaction philes. The goal was to provide a reactive target in the plasma to involves copper(I) catalyzed coupling of an azide and terminal avoid the additional complexities of delivering agents to extravas- alkyne to generate a stable triazole (2). Until recently, the neces- cular sites. The dienophile labeling was controlled so on average sity of the copper catalyst precluded use of this reaction in bio- each antibody possessed approximately three reactive groups. logical systems due to toxicity concerns (3, 4). Bertozzi and others After waiting 3 h, an excess of tetrazine chaser was administered. elegantly solved this problem by developing several new ring Because the pharmacokinetics of the chaser would likely affect strained dienophile derivatives that do not require catalysts (5–9). the efficiency of labeling, we administered either a small mole- However, many of these derivatives have low water solubility, re- cular tetrazine fluorophore (MW 1.3 kDa) or polymer modified quire complex multistep synthesis, and possess suboptimal kinetics. tetrazines (PMT) using two differently sized dextrans: 10 kDa Our search for alternative rapid, selective, and chemically acces- (PMT10) and 40 kDa (PMT40). Dextrans were chosen as polymer scaffold due to their low cost, high stability, and numerous sible coupling reactions without need for a catalyst led us and – others to investigate the [4 þ 2] inverse Diels-Alder cycloaddition previous in vivo applications (18 20). These amine modified (10–13). We realized that this set of chemistries is more uniquely scaffolds were modified on average with approximately one fluor- suited to biological applications and may indeed represent a uni- ochrome and either two (PMT10) or eleven (PMT40) tetrazines versal platform technology (Fig. 1 A and B). Specifically we and as measured using absorption spectroscopy. Such high coupling others have shown that the cycloaddition between tetrazine (Tz) efficiencies to amino dextran may be the result of the low mole- and trans-cyclooctene (TCO) can proceed orders of magnitude fas- cular weight and lipophilic nature of tetrazine affinity probes. ter than previously studied azide and alkyne click reactions and Measurement of reaction kinetics demonstrated that conjugation importantly does not require the action of a catalyst. This reaction combined with multivalent presentation did not significantly has also been adapted for single cell imaging using newer fluoro- genic Tz probes (14). Author contributions: N.K.D., G.M.T., and R.W. designed research; N.K.D., G.M.T., E.J.K., Although initial work focused on in vitro labeling there has and B.M. performed research; E.J.K. contributed new reagents/analytic tools; N.K.D., been a surge of recent work applying this reaction for various G.M.T., and R.W. analyzed data; and N.K.D., G.M.T., and R.W. wrote the paper. in vivo applications (15–17). Despite the above progress, our The authors declare no conflict of interest. results with initial TCO/Tz reactions in live animals were disap- This article is a PNAS Direct Submission. pointing unless we used excessive amounts of reactants. This 1N.K.D. and G.M.T. contributed equally to this work. brute force approach would ultimately be cost prohibitive and 2To whom correspondence should be addressed. E-mail: [email protected]. likely not be biologically viable. We therefore systematically This article contains supporting information online at www.pnas.org/lookup/suppl/ eliminated different reasons for the observed discrepancies doi:10.1073/pnas.1113466109/-/DCSupplemental.

4762–4767 ∣ PNAS ∣ March 27, 2012 ∣ vol. 109 ∣ no. 13 www.pnas.org/cgi/doi/10.1073/pnas.1113466109 Downloaded by guest on September 30, 2021 Fig. 1. In Vivo Bioorthogonal Reactions. (A) Tetrazine cycloaddition with trans-cyclooctene forming a dihydropyrazine. (B) Schematic of PMT used in this study. The scaffold consists of dextran that has been aminated to allow attachment of tetrazine reactive groups as well as imaging agents such as near-infrared fluorophores and radioisotopes. (C) In vivo multistep delivery of imaging agent. A slow clearing targeting agent is administered first (green) and is given 24 h for localization and background clearance. Next, a lower molecular weight secondary agent (red) is delivered that rapidly reacts and is cleared from the background tissue much faster than the primary agent. (D) Kinetic parameters of consideration for in vivo clicking. The secondary tetrazine agent reacts k with transcyclooctene antibodies at a given rate ( reaction). This rate is in competition with other rates including the clearance of the secondary agent from k k the body ( clearance) and internalization of the antibody ( endocytocis).

impede overall probe reactivity compared to small molecule tet- Fig. 2B shows the labeling of dienophile modified white razines (see SI Text). The clearance kinetics of these chaser ima- blood cells, measured by flow cytometry, 3 h after administration ging agents were measured separately through serial blood draws of a fluorescent tetrazine chaser. The labeling efficiency is (Fig. 2A). The tetrazine fluorophore is a low molecular weight strongly dependent on the kinetics of the reaction (e.g., trans- ≫ and highly charged probe, and thus it is not surprising that the cyclooctene norbornene) and the clearance kinetics of the sec- PK showed rapid tissue redistribution and clearance (21). In con- ondary agent. TCO/Tz reaction efficiencies were very high for trast, the PMT’s persisted in the blood and exhibited biexponen- both the fluorescent PMT10 and PMT40 (680 nm). However, tetrazine-VT680 TCO reactions showed a much lower labeling tial clearances with the larger molecular weight dextran PMT40 efficiency, presumably due to its much faster clearance rate. Like- circulating longer. wise, targeting PMT40 to a slow reacting norbornene antibody

led to nearly negligible reaction. In order to gain greater quanti- CHEMISTRY Table 1. Reported Bioorthogonal Rate Constants and tative insight, computational modeling of the in vivo reaction was Pharmacokinetic Parameters performed (see Materials and Methods). A plot of predicted label- Reaction type Rate constant (∕M·s) Reference ing efficiency vs. both the kinetics of cycloaddition reaction and

azide-DIFO 0.076 (CH3CN) (7) the area under the concentration curve (AUC) is displayed in C phen-tetrazine- 1.6 (H2O, 20 °C) (11) Fig. 2 . The fraction of primary agent reacted is an exponential norbornene function of the reaction rate and AUC of the secondary agent. pyr-tetrazine-TCO 2,000 (9∶1 MeOH∕H2O, 25 °C) (12) Small molecule tetrazines, while possessing rapid reaction rates, phen-tetrazine-TCO 6,000 (H2O, 37 °C) (10) do not possess the requisite AUC for very efficient labeling, ex- pyr-tetrazine-TCO 13,090 (H2O, 37 °C) (15) plaining our experimental results. MEDICAL SCIENCES pyr-tetrazine- 22,000 (MeOH, 25 °C) (13) Based on the results from the initial in vivo experiments, we strained-TCO decided to use PMT10 to demonstrate the utility of our method μ Molecule AUC at 3 h (2 M) Reference for imaging cancer cell epitopes in a more challenging but clini- Tetrazine-VT680 4.85 × 10−5 M·s measured cally relevant solid tumor model. PMT10 showed good clearance Dextran, 10 kDa 6.93 × 10−4 M·s measured after several hours yet persisted long enough to allow highly effi- −3 Dextran, 40 kDa 7.66 × 10 M·s measured cient labeling using the TCO/Tz reaction. As a disease model, MAb 2.07 × 10−2 M·s (28) >95% 2 05 × 10−4 we targeted the A33 glycoprotein which is overexpressed in DOTA . M·s (21) of all human colorectal cancers including the human colorectal

Devaraj et al. PNAS ∣ March 27, 2012 ∣ vol. 109 ∣ no. 13 ∣ 4763 Downloaded by guest on September 30, 2021 PMT10 agent, previously validated in the blood, is also able to label specific cell surface biomarkers in the tumor microenvir- onment. To demonstrate the utility of our method for clinically relevant PET imaging we performed longitudinal studies using a radiola- beled 18F-PMT10 (26). Mice were implanted subcutaneously with LS174T xenografts, and once tumors had grown to approximately 1 cm in diameter, these mice were injected with 30 μgof TCO-antibody against A33 (also labeled with VT680 fluorophore). After allowing 24 h for accumulation and clearance of the TCO-modified antibody, 18F-PMT10 was injected (30 μg; 150 μCi). Cycloaddition of 4 mCi of TCO-18Fto60μg of dextran proceeded cleanly, leading to an 89.2% decay corrected radioche- mical yield of 18F-PMT10. This construct was used for all PET/ Computed Tomography (CT) and subsequent autoradiography experiments. PET/CT scans were initiated 3 h after injection of 18F-PMT10. The images revealed significant renal clearance of PMT10 as well as widespread tissue distribution. Fig. 4A shows one representative axial slice from the PET/CT reconstruction. Compared to control mice which were administered antibodies lacking TCO, there was significant accumulation of signal in the vascularized region of the tumor mediated by the in vivo TCO/Tz reaction. To investigate further, mice were euthanized 3 h after injection, the tumors were excised, and 1 mm slices were imaged to determine the extent of cycloaddition. Near infrared fluorescence was used to determine antibody/TCO localization and autoradiography was used to reveal the 18F-PMT10. Fig. 4B shows two representative tumor slices. The localization patterns of the chemical probes were distinct and often showed greatest activity in the periphery of the tumor, likely reflecting the necrotic Fig. 2. Optimization of In Vivo Chemistry with Polymer Modified Tetrazines nature of the tumor core. Comparison of the pattern to the lo- (PMT). (A) Clearance kinetics of tetrazine-VT680 (red), PMT10 (blue) and calization of the fluorescent antibody demonstrated good coloca- PMT40 (green). (B) Efficiency of in vivo labelling of trans-cyclooctene mod- lization, indicating that the Tz probe was primarily localized to ified leukocytes using various tetrazine cycloadditions. (C) Heat map showing the site of TCO binding. Tumors from mice which were adminis- predicted efficiency of reaction given a reaction rate and secondary clearance rate. The 1.3 kDa point represents the small molecule tetrazine-VT680. The tered the control antibody lacking reactive TCO showed much highest efficiencies were observed for the fast tetrazine/trans-cyclooctene lower uptake of Tz and a lack of correlation to antibody/TCO reaction using slow clearing PMT10 or PMT40. Use of either a faster clearing fluorescence signal. tetrazine or a much slower reaction greatly diminishes the efficiency of re- We also tested for biomarker specificity with mice simulta- action as seen in plot in (B). neously implanted with LS174T tumors and A431 tumors which lack expression of the A33 glycoprotein epitope (27). Mice were cancer LS174Tcell line (22). In addition, this antigen is associated administered anti-A33 TCO antibody and 18F-PMT10 as pre- with tight junction proteins, giving it a surface persistence with a viously described and imaged using PET/CT. Fig. 4C shows one half life greater than 2 d (23). representative coronal slice from the PET/CT reconstruction. Initially we reacted near-infrared fluorescent PMT imaging LS174T tumors can be clearly visualized whereas the control agents to obtain cellular resolution of the in vivo bioorthogonal A431 tumor shows much lower Tz uptake. Autoradiography of reaction. In addition, fluorescent agents avoid the cost and hand- excised tumor slices further demonstrate the greater uptake of ling complexities of radionuclides, and are increasingly being Tz in biomarker positive LS174T tumors vs. the control A431 used for in vivo applications (24). To facilitate the in vivo imaging, tumors (Fig. 4D). we used dorsal window chambers for intravital microscopy. Win- dow chamber mice bearing LS174T tumors were injected first Discussion with anti-A33 monoclonal antibodies bearing TCO (n ¼ 3) This work demonstrates that bioorthogonal TCO/Tz reactions dienophiles (25). After waiting 24 h for antibody targeting and can efficiently occur in live animals when reaction partner phar- clearance, the mice were subsequently injected with 30 μgof macokinetics are optimized. Based on modeling of reaction ki- fluorescent PMT10 (750 nm). After waiting 3 h to allow for the netics and PK clearance rates, we designed and tested specific reaction and clearance to occur, the tumors were visualized using polymer-tetrazine adducts bearing “imaging” reporters (near in- confocal microscopy. The images revealed beds of LS174T cells frared fluorochromes and 18F) to visualize reactions in live mice whose surfaces were brightly stained with the fluorescent anti- both at the cellular and whole animal level. The current genera- body as well as the tetrazine dextran probe (Fig. 3). Merging of tion of polymer-tetrazine conjugates included dextran derivatives the two channels demonstrated excellent colocalization between (PMT10 and PMT40) because dextrans have a long history of in the location of the dienophile antibody and the secondary tetra- vivo use and the PK are well understood (18–20). Based on the zine dextran, indicative of a specific bioorthogonal reaction. Con- kinetics of the TCO component, we would envision that the PMT trol experiments involving mice which received an antibody could be based on a number of different polymers for further lacking a dienophile also showed significant antibody staining optimization (e.g., PEGs, PL, PLGA, nanoparticles, etc.). We of LS174T cell surfaces but lacked subsequent tagging by the expect that the methodology used for developing these probes tetrazine dextran. These in vivo images and controls looked re- and modulating the reaction rate and clearance kinetics should markably similar to corresponding in vitro experiments. These in- be applicable to other bioorthogonal reactions, imaging agents, itial studies demonstrated that use of a higher molecular weight and therapeutics.

4764 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1113466109 Devaraj et al. Downloaded by guest on September 30, 2021 Fig. 3. In Vitro and In Vivo Microscopy of Fluorescent PMT10 Targeting a Cancer Cell Epitope. Confocal imaging demonstrating targeting of PMT10 (750 nm) to trans-cyclooctene (TCO) antibodies (680 nm) on the surface of LS174T cells both in vitro and in vivo. (A) LS174T cells in culture labeled with TCO monoclonal antibodies followed by PMT10 (750 nm) labeling. (B) Same as previous except monoclonal antibodies lack TCO. (C) Cells in a LS174T xenograft in vivo labeled with TCO monoclonal antibodies followed by PMT10 (750 nm) labeling. (D) Same as previous except monoclonal antibodies lack TCO. See Materials and Methods for experimental details.

The TCO/Tz reaction is an ideal bioorthogonal chemical such as side groups with protein binding abilities. In this respect system for in vivo use given its fast reactions kinetics we have recently looked at reaction rates of a number of different (>6;000 M−1 sec−1)(10). Despite these impressive kinetics for TZ analogs. However, although binding to proteins would de- benzylamine Tz/TCO, PK modifications are required for in vivo crease blood clearance, it was unclear if the protein bound Tz use. These improved PK properties can theoretically be achieved would be viable for chemical reaction with TCO targets. Instead, by small molecule tetrazine modifications, such as those that to improve PK of the reactants we decided to conjugate Tz to would alter pharmacokinetics without affecting reaction rates dextran creating PMTof different chain lengths but with identical CHEMISTRY MEDICAL SCIENCES

Fig. 4. PET and Autoradiography using 18F Tetrazine Agents. (A) PET/CT fusion of LS174T tumor xenograft labeled using either trans-cyclooctene (TCO) monoclonal antibodies (MAb TCO) or control unlabeled antibodies (MAb) followed by 18F-PMT10. Arrows indicate location of the tumor xenograft. Bladder has been omitted for clarity. (B) Imaging using autoradiography (left side) and fluorescence reflectance (right side) of 1 mm LS174T tumor slices after targeting with fluorescent TCO monoclonal antibody and 18F-PMT10. (C) PET/CT fusion of mouse bearing A431 and LS174T tumors after targeting with anti-A33 TCO monoclonal antibodies followed by 18F-PMT10. Arrows indicate location of tumors and the liver has been omitted for clarity. (D) Autoradiography of representative 1 mm LS174T or A431 tumor slices after multistep targeting.

Devaraj et al. PNAS ∣ March 27, 2012 ∣ vol. 109 ∣ no. 13 ∣ 4765 Downloaded by guest on September 30, 2021 reactivities to decouple and independently tune the clearance and The highly dispersive nature of the mouse vascular system is likely to reaction rates. rapidly result in homogenous distribution of reactive agents in the plasma, We developed a simple mathematical model that takes TCO/ so a biexponential decay model is used for plasma concentration: Tz reaction rates and pharmacokinetics of reaction partners into Ae−kα·t Bekβ·t ; account (Fig. 2) to better predict the ideal design and dosages of ½tet¼½tet0ð þ Þ reaction partners for in vivo use. The model predicts that the sec- where ½tet0 is the initial plasma concentration of tetrazine, A and B are the ondary agent must remain at a high enough concentration for a t sufficient time to undergo the reaction in vivo. These experimen- fractions of alpha vs. beta phase clearance, is the time after injection of the tetrazine agent, and kα and kβ are the alpha and beta phase clearance rate tal and computational results allow approximation of labeling constants, respectively. It is important to capture both phases in measure- efficiency in vivo given a chemical reaction rate and secondary ments, particularly for imaging agents that are given as a single bolus dose. agent clearance rate when the secondary agent is administered While small molecular weight agents may have beta (i.e., clearance) phases of in excess. These conditions are commonly encountered when la- several minutes or longer, this often is preceded by a rapid drop in concen- beling surface exposed molecules and is directly comparable to tration, which can be greater than an order of magnitude, with a half life of the experiments targeting cell surface antigens in the blood. For tens of seconds (21). other targets, such as solid tumors, the concentration profile of Because the tetrazine concentration is assumed independent from the the primary and secondary agents are more complex given the reaction: blood flow, extravasation, diffusion, binding, and clearance rates d½Ab found in tumors (28), but the basic principles still apply. We be- −k Ab Ae−kα·t Be−kβ·t : dt ¼ rxn½ ½tet0ð þ Þ lieve these insights are not only important for our study but may offer helpful guidance to future studies where one is interested in Integrating: performing in vivo reactions. Z Z We envision a number of practical applications of the devel- Ab t ½ final d½Ab −k Ae−kα·t Be−kβ·t dt oped in vivo click approach based on TCO/PMT and other small ¼ rxn½tet0 ð þ Þ Ab ½Ab 0 molecule probes. For instance, the short positron decay half- ½ 0 18 life (1.8 h) of F requires affinity ligands that show significant target accumulation and rapid background clearance in hours. A Ab Ab −k tet 1 − e−kα·t ½ final ¼½ 0 exp rxn½ 0 ð Þ These results are simply not possible by directly labeling high kα AUC affinity agents such as monoclonal antibodies. Multistep B techniques with bioorthognal chemistries could overcome this þ ð1 − e−kβ·tÞ problem. Additionally, in vivo assembly could be used to improve kβ therapeutic drug delivery by delivering components as small molecules to facilitate transport (e.g., across the blood brain barrier, A 1 − −k 1 − e−kα·t blood vessel endothelium, intestinal wall) followed by specific fraction reacted ¼ exp rxn½tet0 k ð Þ covalent assembly. The technique may also provide information α on the concentration of drugs or metabolic markers labeled with B þ ð1 − e−kβ·tÞ : chemical tags in different tissue compartments. Finally, as we kβ have demonstrated in this work, in vivo chemistry shows great promise for imaging biomarkers and targeting agents with rapidly The integral of the tetrazine concentration-time curve is commonly defined decaying isotopes. as the area under the curve or AUC: Z Materials and Methods t ≡ Ae−kα·t Be−kβ·t dt Measurement of Clearance Kinetics of Tetrazine Agents. Fluorescent tetrazine AUCtet ½tet0ð þ Þ 0 agents (tetrazine-VT680, PMT10, PMT40) were injected via tail vein into mice. Serial retroorbital 30 μL blood draws were taken starting at 1 min after injection and periodically up to 2 h. The blood was diluted in equal volume A B 1 − e−kα·t 1 − e−kβ·t : 5 mM EDTA in phosphate buffered saline, and the fluorescence intensity AUCtet ¼½tet0 ð Þþ ð Þ kα kβ was measured using a plate reader. The concentration of the fluorescent agent in the blood was determined by comparison with a standard curve Substituting into the equation for fraction of TCO reacted: using blood mixtures with known concentrations of fluorescent agent. 1 − −k : Modeling of Reaction Efficiency. The efficiency of tetrazine imaging agent re- fraction reacted ¼ expf rxnAUCtetg action with trans-cyclooctene modified antibodies on the surface of white blood cells was modeled using simple mass action kinetics. Assuming a mouse This equation is graphed as a heat map in Fig. 2C. plasma volume of approximately 2 mL, the initial antibody bolus dose should result in an antibody plasma concentration of 100 nM. After 24 h, the plasma Measurement of Reaction Efficiency on Circulating White Blood Cells. Dieno- concentration will drop due to clearance (28) and potentially from uptake by phile modified anti-CD45 antibody was injected into mice (n ¼ 3) via tail vein. target white blood cells (29). After clearance, the tetrazine probe is delivered After 1 h, a 30 μL retroorbital blood sample was taken from each mouse and at an initial concentration of 2 μM, well in excess of the antibody plasma con- exposed to an excess of tetrazine agent to determine the saturated labeling centration. Given the large excess of tetrazine probe, the loss in tetrazine due density. The tetrazine agent was then administered via tail vein into the to reaction is considered negligible. The beta phase of antibody clearance has mice, and after 3 h, a second retroorbital blood sample was taken. The blood a 7 d half life, so, after 24 h, antibody clearance is assumed to be negligible samples were exposed to 1 mL BD pharmlyse solution for 15 min at room over 3 h. Because the reaction occurs in the plasma, no tissue transport para- temperature, centrifuged, and the pellet resuspended in order to remove meters were required. lysed red blood cells. The white blood cells were run on a flow cytometer The mass balance for antibody concentration is therefore: to measure the average fluorescent intensity. The in vivo intensity measure- ments were then divided by the in vitro saturated intensity in order to de- d Ab termine the labeling efficiency. ½ −k Ab ; dt ¼ rxn½ ½tet Fluorescence Imaging of Tetrazine Cycloadditions In Vitro and In Vivo. LS174T Ab k where ½ is the trans-cyclooctene modified antibody concentration, rxn is cells were trypsinized, plated on cover glass dishes, and allowed to attach the second order reaction rate constant, and ½tet is the concentration of tet- overnight. Cells were then exposed to 100 nM of TCO modified anti-A33 razine probe. modified with a VT680 probe. After washing, the cells were incubated with

4766 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1113466109 Devaraj et al. Downloaded by guest on September 30, 2021 100 nM of fluorescent PMT10 (750 nm). The reaction was allowed to proceed CT was reconstructed from 360 projections of X-rays with a cone beam for 45 min after which the cells were washed and imaged using scanning angle of 9.3 ° over 360 ° perpendicular to the animal bed. 80 keV X-rays were confocal microscopy. Control cells were treated identically except that they transmitted from a 500 μA anode source, 347 mm from the center of rotation received an antibody lacking TCO modification. and recorded on a CCD detector, containing 2,048 transaxial and 3,072 axial n 3 Window chambered nude mice ( ¼ ) were prepared as previously pixels. Projections were calibrated using 70 dark and 70 light images, inter- described. To create the LS174T xenograft, approximately 1 million LS174T polated bilinearly, processed through a Shepp-Logan filter, then recon- cells were subcutaneously injected and allowed to develop into a tumor structed using a filtered back projection algorithm. Isotropic CT pixel size of approximately 2 mm in diameter. 30 μg of TCO modified anti-A33 modified was 110.6 μm , with a total of 512 × 512 × 768 pixels. Scaling to Hounsfield with a VT680 probe was injected by tail vein. After 24 h, a secondary injection of 30 μg of fluorescent PMT10 (750 nm) was injected via tail vein. After 3 h, Units, calibration was done using a 8.0 cm cylindrical phantom containing the tumors were imaged using scanning confocal microscopy. Control mice water prior to CT acquisition. During CT acquisition, iodine contrast was (n ¼ 3) were treated identically except that they received an antibody lacking infused into the tail vein at a rate of 35 μL∕ min to enhance intravascular TCO modification. contrast. Projections were acquired at end expiration using a BioVet gating system (M2M Imaging) and CT acquisition time was approximately 10 min. PET Imaging of Tetrazine Cycloadditions. Approximately 106 LS174T or Reconstruction of datasets, PET-CT fusion, and image analysis were done 1.5 × 106 A431 cells were injected subcutaneously in either the hind limb using IRW software (Siemens). 3D visualizations were produced with the or upper dorsal region in nude mice (n ¼ 3). The difference in cell number DICOM viewer OsiriX (The OsiriX foundation). was used to ensure similar sized tumors at the time of imaging due to different growth rates. Tumors were allowed to grow for 2 w after which mice Imaging Antibody and Dextran Uptake in Tumor Slices. Xenograft bearing mice μ were injected via tail vein with 30 g of TCO modified anti-A33 modified with were euthanized following PET-CT, tumors were excised and cut into 1 mm a VT680 label. After allowing 24 h for targeting and clearance of the anti- thick slices using a heart slicer (Zivic Laboratories). Tumor slices were then body, the mice received 30 μgof18F-PMT (30 μg, 150 μCi) via a tail vein exposed to a phosphor screen (GE Healthcare) for 24 h. The next day, the injection. All PET-CT images were acquired on a Siemens Inveon PET-CT. Each screen was scanned using a phosphor imager (Typhoon 9410; GE) yielding PET acquisition was approximately 60 min in duration. PET was reconstructed autoradiographic imaging of 18F TCO localization in tumors. In the exact from 600 million coincidental, 511 keV photon counts on a series of LSO (lutetium oxyorthosilicate) scintillating crystal rings. Counts were rebinned positioning used for autoradiography exposure, samples then underwent in 3D by registering photons spanning no more than three consecutive rings, fluorescence reflective imaging (OV-110; Olympus) with a 0.56X objective then reconstructed into sinograms by utilizing a high resolution Fourier and 280 ms exposure for localization of VT680 conjugated A33 antibody. Rebin algorithm. A reconstruction of sinograms yielded a 3D mapping of positron signal using a 2D filtered back-projection algorithm, with a Ramp filter ACKNOWLEDGMENTS. The authors gratefully acknowledge S.A. Hilderbrand, at a Nyquist cut-off of 0.5. Image pixel size was anisotropic, with dimensions J.B. Haun, and R. Mazitschek for many helpful discussions and suggestions. of 0.796 mm in the z direction and 0.861 mm in the x and y directions, for a This work was funded in part by National Institutes of Health (NIH) grants total of 128 × 128 × 159 pixels. P50CA86355, R01EB010011, K01EB010078, and T32CA079443.

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