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Summary

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

Abstract

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent — dubbed ‘PODS’ — as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

Introduction

Radiopharmaceutical chemists have long exploited the selectivity and specificity of antibodies for biomarkers of disease for both nuclear imaging and targeted radiotherapy1. Far and away the most common approach to the radiolabeling of antibodies is predicated on the indiscriminate attachment of radiolabeled prosthetic groups or radiometal chelators to amino acids — most often lysines — within the structure of the immunoglobulin (Figure 1A)2. While this strategy is certainly effective, its random, non-site-specific nature can create problems. Specifically, traditional bioconjugation approaches produce poorly-defined and heterogeneous immunoconjugates composed of mixtures of thousands of different regioisomers, each with its own set of biological and pharmacological properties3. Furthermore, random bioconjugation can impede the immunoreactivity of antibodies if the cargo is appended to the immunoglobulin’s antigen-binding domains.

Over the years, a variety of site-specific and site-selective bioconjugation strategies have been developed in order to address these problems4,5. The most common of these approaches relies on the ligation of maleimide-bearing probes to the sulfhydryl groups of cysteines (Figure 1B). IgG1 antibodies naturally contain 4 inter-chain disulfide bridges, linkages that can be selectively reduced to yield free thiols capable of undergoing Michael addition reactions with maleimides to form succinimidyl thioether bonds. The use of thiols and maleimides is certainly an improvement over traditional methods, and a wide variety of maleimide-bearing synthons and bifunctional chelators are currently available. However, it is important to note that this methodology has serious limitations as well. Maleimide-based immunoconjugates exhibit limited stability in vivo because the thioether linkage can undergo a retro-Michael reaction (Figure 2)6,7,8,9,10. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation (e.g., glutathione or serum albumin). Both of these processes can increase activity concentrations in healthy organs as well as decrease activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. Several alternative thiol-reactive reagents have been developed in an effort to circumvent these issues, including tosylates, bromo- and iodo-acetyls, and vinyl sulfones11,12,13,14,15,16,17. However, all of these approaches have limitations that have hampered their widespread application.

About five years ago, the laboratory of the late Carlos Barbas III at Scripps Research Institute pioneered the use of phenyloxadiazolyl methyl sulfones as reagents for the selective formation of highly stable linkages with thiols (Figure 1C and Figure 3)18,19. The authors employed a phenyloxadiazolyl methyl sulfone-bearing variant of fluorescein to modify several antibodies engineered to contain free cysteine residues, ultimately producing immunoconjugates with higher stability than analogous constructs created using maleimide-based probes. Upon seeing this promising work, we were somewhat surprised that this technology had only been used scarcely in radiochemistry and had not yet been used at all in the synthesis of bifunctional chelators or radioimmunoconjugates20,21. This dearth of applications, however, soon began to make more sense: several attempts at procuring the reagent from Sigma-Aldrich resulted in the receipt of complex mixtures of degradation products with <15% of the desired compound. In addition, synthesizing the reported reagent ourselves was not a realistic option either, as the published synthetic route is somewhat cumbersome and requires sophisticated organic chemistry equipment that most radiochemistry and molecular imaging laboratories — including ours — simply do not possess.

In response to these obstacles, we set out to create an easily accessible and highly stable phenyloxadiazolyl methyl sulfone reagent that can be obtained via a robust and reasonably facile synthetic route. Earlier this year, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent — dubbed ‘PODS’ — as a platform for thiol-based bioconjugations (Figure 1C and Figure 3)22. The key difference between PODS and the reagent reported by Barbas, et al. is that the former employs an aniline ring attached to the phenyloxadiazolyl methyl sulfone moiety, while the latter features a phenol in the same position (Figure 4). This change facilitates a more straightforward and accessible synthetic route as well as — if our experience with the commercially available compound is emblematic — a more stable final reagent. In this work, we also synthesized a pair of PODS-bearing bifunctional chelators — PODS-DFO and PODS-CHX-A''-DTPA — to facilitate the creation of 89Zr- and 177Lu-labeled radioimmunoconjugates, respectively. As we will discuss, we have demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations.

The over-arching goal of this work is to facilitate the creation of well-defined, homogeneous, highly stable, and highly immunoreactive immunoconjugates for in vitro and in vivo applications. The synthetic approach is simple enough to be performed in almost any laboratory, and the parent PODS reagent can be modified with a plethora of different chelators, fluorophores, or cargoes. In this protocol and the accompanying video, we will describe the simple, four-step synthesis of PODS (Figure 5); the creation of a PODS-bearing variant of DOTA, a widely used chelator for the coordination of 64Cu, 68Ga, 111In, 177Lu, and 225Ac (Figure 6); and the bioconjugation of PODS-DOTA to a model antibody, the HER2-targeting IgG1 trastuzumab (Figure 7).

Protocol

1. The synthesis of 4-[5-(methylthio)-1,3,4-oxadiazol-2-yl]-aniline (1)

NOTE: Due to the light-sensitivity of the compound, keep all reactions in foil-covered vessels.

  1. In a 10 mL round bottom flask, dissolve 100 mg (0.517 mmol, 1 equivalent) of 5-(4-aminophenyl)-1,3,4-oxadiazole-2-thiol in 3 mL of methanol.
  2. To this solution, add 360 μL of diisopropylethylamine (DIPEA; 2.07 mmol; 4 equivalents; anhydrous) and a small magnetic stir bar. Cover the flask with a rubber stopper and stir the solution for 10 minutes at room temperature.
  3. Using a 1 mL glass syringe, poke a hole through the rubber stopper and quickly add 32 μL (0.517 mmol, 1 equivalent) of iodomethane to this mixture. Allow the mixture to react for 45 minutes at room temperature.
    NOTE: Due to the potential harmful effects of iodomethane, this reaction should be done in a chemical fume hood.
  4. Set the water bath of a rotary evaporator to 40 °C and slowly reduce the pressure to remove the solvent to afford a white solid.
  5. Dissolve the solid in 3 mL of ethyl acetate and wash at least three times with a 5 mL solution of 0.1 M sodium carbonate using a separatory funnel.
    NOTE: Periodically take spot-tests of the aqueous phase under a UV lamp; once nothing is seen under the lamp, you can stop the washes.
  6. Collect the organic phase in a separatory funnel and wash it with water until the pH of the aqueous phase reaches 6.8-7.0 (using pH paper).
  7. Collect the organic phase and add magnesium sulfate to remove any traces of water.
    NOTE: The magnesium sulfate should be added with a small spatula, after which the solution should be swirled. If fine particles of the drying agent are still seen, the solution is dry. If not, add small amounts of magnesium sulfate until fine particles can be seen.
  8. Filter the mixture using a medium glass frit or filter paper.
  9. Evaporate the volatiles using a rotary evaporator, a process which should produce the desired product as white needles.

2. The synthesis of tert-butyl[18-({4-[5-(methylthio)-1,3,4-oxadiazol-2-yl]phenyl}amino)-15,18-dioxo-4,7,10-trioxa-14-azaoctadecyl] carbamate (2)

NOTE: Due to the light-sensitivity of the compound, keep all reactions in foil-covered vessels.

  1. In a 25 mL round bottom flask, dissolve 387 mg (0.92 mmol, 1.0 equivalent) of NBoc-N′-succinyl- 4,7,10-trioxa-1,13-tridecanediamine in 10 mL of dichloromethane.
  2. To this solution, add 480 μL (2.76 mmol, 3 equivalents) of DIPEA, 264 mg (1.38 mmol; 1.5 equivalents) of N-ethyl-N′-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDCI), and 200 mg (0.97 mmol, 1.1 equivalents) of 1. Seal the vessel with a glass stopper and let the reaction stir for 5 days at room temperature.
    NOTE: Be mindful of the evaporation of dichloromethane. If needed, add more throughout the week.
  3. Wash the mixture in a separatory funnel with a solution of 1 M hydrochloric acid (3 x 5 mL).
  4. Collect the organic phase and continue to wash it in a separatory funnel, first with a solution of 1 M Na2CO3 (2 x 5 mL) and then with water (3 x 5 mL).
  5. Collect the organic phase and add magnesium sulfate to remove any traces of water (see Step 1.7). Filter the mixture using a medium glass frit or filter paper.
  6. Using a rotary evaporator, remove the volatile solvents under reduced pressure to afford an off-white solid.
  7. Re-dissolve this solid in 10 mL of ethyl acetate and precipitate the product via the gradual (e.g., 2 mL at a time) addition of 30 mL of cyclohexane.
  8. Filter the solution with filter paper or a medium glass frit to obtain the product as a white powder.

3. The synthesis of tert-butyl[18-({4-[5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl]phenyl}amino)-15,18-dioxo-4,7,10-trioxa-14-azaoctadecyl] carbamate (3)

NOTE: Due to the light-sensitivity of the compound, keep all reactions in foil-covered vessels.

  1. In a 10 mL round-bottom flask, dissolve 30 mg (0.05 mmol; 1 equivalent) of 2 in 4 mL of dichloromethane.
  2. Slowly add in 49 mg (0.2 mmol; 4 equivalents) of 70% m-chloroperbenzoic acid to this mixture and cover the reaction vessel with a glass stopper. Stir the solution overnight at room temperature, ultimately yielding a yellow mixture.
  3. Wash the yellow mixture in a separatory funnel, first with a 0.1 M solution of NaOH (3 x 5 mL) and then with water (3 x 5 mL).
  4. Dry the organic phase with magnesium sulfate and filter the mixture using a medium glass frit or filter paper.
  5. Using a rotary evaporator, remove the solvents under reduced pressure to obtain the product as a pale solid.

4. The synthesis of N1-(3-{2-[2-(3-aminopropoxy)ethoxy]-ethoxy}propyl)-N4-{4-[5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl] phenyl} succinamide (PODS)

  1. In a 25 mL round bottom flask, dissolve 30 mg of 3 in 2.0 mL of dichloromethane.
  2. Add 400 μL of trifluoroacetic acid and seal the flask with a glass stopper.
  3. Stir the reaction mixture at room temperature for 3 hours.
  4. Using a rotary evaporator, remove the volatiles under reduced pressure at room temperature, leaving an oily residue.
  5. Dissolve the oily residue in 7 mL of water and, using a separatory funnel, wash with ethyl acetate (3 x 4 mL). Keep the aqueous layer.
  6. Lyophilize the aqueous layer to afford PODS as a white powder.
    NOTE: The molar absorption coefficients for PODS at 280 and 298 nm are 9,900 and 12,400 cm-1M-1, respectively.

5. The synthesis of PODS-DOTA

  1. In a 1.5 mL microcentrifuge tube, dissolve 10 mg of PODS in 300 μL of dimethyl sulfoxide (0.018 mmol; 1 equivalent) and add 26 μL of N,N-diisopropylethylamine (0.15 mmol; 8 equivalents).
  2. Dissolve 15.2 mg of DOTA-Bn-NCS (0.02 mmol; 1.2 equivalents) in 100 μL of dimethylsulfoxide and combine this solution with the solution from Step 5.1. Seal the microcentrifuge tube.
  3. Allow the reaction to incubate overnight at room temperature.
  4. Purify the product using reverse-phase C18 HPLC chromatography to remove any unreacted DOTA-Bn-NCS.
    NOTE: Retention times are obviously highly dependent on the HPLC equipment of each laboratory (pumps, columns, tubing, etc.), and appropriate controls should be run prior to purification. However, to present an example, if a gradient of 5:95 MeCN/H2O (both with 0.1% TFA) to 70:30 MeCN/H2O (both with 0.1% TFA) over 30 min, a semi-preparative 19 x 250 mm C18 column, and a flow rate of 6 mL/min are used, PODS, p-SCN-Bn-DOTA, and PODS-DOTA will have retention times of around 14.4, 18.8, and 19.6 min, respectively. All three compounds can be monitored at 254 nm.

6. The bioconjugation of PODS-DOTA to trastuzumab

NOTE: For this step, we started with a 16.4 mg/mL stock solution of trastuzumab.

  1. In a low protein binding 1.5 mL microcentrifuge tube, dilute 61 μL of the trastuzumab stock solution (1 mg; 6.67 nmol, 1 equivalent) with 859 μL of phosphate buffered saline (pH 7.4).
  2. To this mixture, add 6.7 μL of a freshly made 10 mM solution of TCEP in H2O (66.7 nmol, 10 equivalents).
  3. Prepare a 1 mg/mL solution of PODS-DOTA in DMSO and add 73 μL of this PODS-DOTA solution to the reaction mixture (66.67 nmol, 10 equivalents).
  4. Seal the microcentrifuge tube and incubate the solution for 2 hours at room temperature.
  5. After 2 hours, purify the immunoconjugate using a pre-packed disposable size exclusion desalting column.
    1. First, equilibrate the size exclusion column as described by the supplier to remove any preservatives present in the column during storage. A typical procedure involves washing the column 5 times with a volume of PBS that corresponds to the volume of the column: 5 x 2.5 mL of PBS.
    2. Next, add the reaction mixture to the size exclusion column noting the volume of the reaction mixture.
    3. After the reaction mixture has entered the column, add an appropriate amount of PBS to bring the total volume of solution added to the column up to 2.5 mL. For example, if the conjugation reaction resulted in a total volume of 1.3 mL, 1.2 mL of additional PBS would need to be added to the column.
    4. Finally, collect the product using 2 mL of PBS as the eluent.
  6. Concentrate the final immunoconjugate with centrifugal filtration units with a 50 kDa molecular weight cut-off.

Results

The first four steps of this protocol — the synthesis of PODS — have been designed to be robust and reliable. The deprotonation and substitution of 5-(4-aminophenyl)-1,3,4-oxadiazole-2-thiol to form the desired thioether product affords the thioether in >99% yield after just 45 minutes. Next, the ligation between 1 and N-Boc-N'-succinyl-4,7,10-trioxa-1,13-tridecanediamine was achieved via a standard peptide coupling procedure, resulting ...

Discussion

In this report, we have chosen not to include any protocols for radiolabeling or in vivo experimentation. Our reasons are straightforward. With respect to the former, the radiolabeling of a PODS-based immunoconjugate does not differ at all from that of an immunoconjugate synthesized using other bioconjugation strategies, and these procedures have been comprehensively reviewed elsewhere2. With regard to the latter, the specifics of preclinical in vivo experiments (i.e., mouse models, doses, etc.) c...

Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors thank Dr. Sai Kiran Sharma for helpful conversations.

Materials

NameCompanyCatalog NumberComments
5-(4-aminophenyl)-1,3,4-oxadiazole-2-thiolSigma-Aldrich675024
1.5 mL LoBind Microcentrifugal TubeEppendorf925000090
1.5 mL Microcentrifugal TubeFisherbrand05-408-129
AcetonitrileFisher ScientificA998-4
Amicon Ultra-2 Centrifugal Filter UnitEMD MilliporeEN300000141G
CyclohexaneFisher ScientificC556-4
DichloromethaneFisher ScientificAC383780010
DiisopropylethylamineMP Biomedicals, LLC150915
DimethylsulfoxideFisher Scientific31-727-5100ML
Ethyl AcetateFisher ScientificE145 4
Hydrochloric AcidFisher ScientificA144-500
IodomethaneSigma-Aldrich289566-100G
Magnesium SulfateAcros Organics413485000
m-chloroperbenzoic acidSigma-Aldrich273031
MethanolFisher ScientificA412 1
NBoc-N′-succinyl-4,7,10-trioxa-1,13-tridecanediamineSigma-Aldrich671401Store at -80 °C
N-ethyl-N′- [3- (dimethylamino)propyl] carbodiimide hydrochlorideSigma-Aldrich3450
Phosphate Buffered SalineSigma-AldrichP549310× Concentration
p-SCN-Bn-DOTAMacrocyclicsB-205Store at -80 °C
Sephadex G-25 in PD-10 Desalting ColumnsGE Healthcare17085101
Sodium CarbonateSigma-AldrichS7795
Sodium HydroxideFisher ScientificS318-1
TCEPThermoFischer Scientific20490
TriethylamineFisher ScientificAC157911000
Trifluoroacetic AcidFisher ScientificA116-50

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