Fullerene (C₆₀) immunoconjugates: interaction of water-soluble C₆₀ derivatives with the murine anti-gp240 melanoma antibody

Abstract

The first fullerene (C₆₀) immunoconjugates have been prepared and characterized as an initial step toward the development of fullerene immunotherapy (FIT).

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The field of biomedicine offers a promising arena for new applications of fullerene materials.¹ Water-soluble C₆₀ derivatives are now commonplace,² and the discovery that water-soluble C₆₀ derivatives can cross cell membranes³ and even produce transfection⁴ has accelerated interest in using C₆₀ for diagnostic and therapeutic medicine. Although fullerene toxicity is of some concern, several water-soluble C₆₀ derivatives have shown acceptable cytotoxicity for drug-delivery applications.⁵

A number of water-soluble C₆₀ derivatives have been suggested for various medical applications. These applications include neuroprotective agents,⁶ HIV-1 protease inhibitors,⁷ bone-disorder drugs,⁸ transfection vectors,⁴ X-ray contrast agents,⁹ photodynamic therapy (PDT) agents,¹⁰ and a C₆₀–paclitaxel chemotherapeutic.¹¹ In addition, endohedral metallofullerenes have demonstrated potential as radiopharmaceuticals¹² and MRI contrast agents.¹³ Fullerene-based micelles have also been developed as a drug delivery system.¹⁴ To date, however, only the bone-drug application has involved biological targeting of a C₆₀-based material,⁸ even though drug targeting is a desirable, if not essential, component of all drug-delivery strategies.

There is now a large body of literature regarding the development of cell-targeted delivery of agents for imaging and therapeutic applications.¹⁵ Growth factors, cytokines and antibodies have all been extensively studied for their abilities to deliver payloads to the surface and the cytoplasm of target cells. The antibody designated ZME-018 targets the gp240 antigen (also known as the high molecular weight melanoma-associated antigen, HMWMAA) found on the surface of >80% of human melanoma cell lines and biopsy specimens.¹⁶ This antibody has previously been extensively used in clinical imaging trials¹⁷ and for the delivery of toxins, cytokines and other therapeutic agents to melanoma cells in vitro and in vivo.¹⁸ Immunoconjugates containing ZME-018 are rapidly internalized into melanoma cells in culture.¹⁹ Moreover, these conjugates effectively localize into melanoma xenografts after systemic administration and demonstrate impressive cytotoxic effects against established tumors in orthotopic models.²⁰

In this communication, we report the synthesis and characterization of a new water-soluble C₆₀ derivative (Fig. 1a) designed to covalently attach to proteins such as ZME-018 as an initial step toward targeted fullerene immunotherapy (FIT). A single-drug chemotherapeutic agent such as a recently reported C₆₀–paclitaxel conjugate¹¹ might be employed for FIT, but the real advantage of FIT over other targeted therapeutic agents is the potential for the attachment of multiple (and possibly different) drugs to the C₆₀ scaffold in order to create targeted, single-dose “drug cocktails”.

Fig. 1 The water-soluble fullerene (C₆₀) derivatives.

Several reports have been published regarding C₆₀ interactions with large biomolecules.²¹ C₆₀ derivatives have been developed to bind myoglobin,^21a form electrostatic interactions with cytochrome c,^21b,c induce protein clusters and complexes in human serum albumin,^21d,e and enhance catalytic activity via conjugation with the serine protease, subtilisin.^21f Finally, one study has reported the X-ray crystal structure of a C₆₀-specific monoclonal antibody.^21g Together, these studies suggested to us the possibility of creating a C₆₀–antibody conjugate as a proof-of-principle step towards FIT.

Fluorescence spectroscopy and transient absorption spectroscopy have previously been used to detect dendritic C₆₀ interactions with cytochrome c.^21c These spectroscopic probes have the advantage of monitoring C₆₀ without interference from the biomolecule. In particular, triplet → triplet (T–T) absorption provides a method to sensitively and selectively monitor C₆₀ derivatives through their known spectral and kinetic signatures.²² We therefore use transient and ground state absorption measurements to track the fullerene components in synthesized immunoconjugates.

The two C₆₀ derivatives shown in Fig. 1 were used in this study. A monoadduct of C₆₀–SPDP (without the water-solubilizing malonodiserinolamide groups of Fig. 1a) was first prepared (see ESI†) to test the feasibility of attaching the cross-linker, N-succinimidyl-3-(2-pyridyldithio)propionate (or SPDP),²³ to C₆₀. Water-solubilizing malonodiserinolamide groups were then first attached to C₆₀, followed by the SPDP moiety in Fig. 1a to provide a cross-linking agent for the ZME-018 antibody. A water-soluble derivative of C₆₀–SPDP was found to be necessary to allow an interaction with the antibody.

Coupling of the C₆₀–SPDP to the antibody (for ratios of 1 ∶ 1, 5 ∶ 1 and 10 ∶ 1) was then accomplished by reacting ZME-018 with 2-iminothiolane, which added an average of five thiol groups to the F_c fragment,²⁴ each of which can form a new disulfide bond with the SPDP sidearm of C₆₀–SPDP (Scheme 1). The coupling was performed in a salt solution to minimize fullerene aggregation.^21d Products were purified by size-exclusion chromatography and then examined by transient absorption spectroscopy (ESI†). As shown in Fig. 2a, the C₆₀ core's 690 nm T–T spectral signature was clearly present with intensities reflecting the reactant ratio. However, it was unclear whether covalent bonds had formed between C₆₀–SPDP and ZME-018. Therefore, the related water-soluble C₆₀–Ser derivative (Fig. 1b),^2b was substituted for C₆₀–SPDP in the reaction schemes with ZME-018 (10 ∶ 1 C₆₀–Ser ∶ ZME-018). To our surprise, results for the C₆₀–Ser derivative mirrored those of C₆₀–SPDP. This implies that C₆₀–(ZME-018) conjugate formation may not require covalent bond formation.

Fig. 2 (a) T–T spectrum of C₆₀–SPDP–(ZME-018) immunoconjugate prepared with three different ratios of fullerene to antibody, after chromatographic purification. (b) UV absorption spectra of 0.40 µM ZME-018, the C₆₀–SPDP–(ZME-018) immunoconjugate (chromatographically purified), and an unreacted mixture of the two components.

Scheme 1 Schematic representation showing the formation of the C₆₀ immunoconjugate from C₆₀–SPDP (C₆₀ and antibody figures not to scale).

Our quantitative characterization began with BioRad protein assays, which showed that the concentration of ZME-018 in the chromatographically purified samples was 0.40 µM for C₆₀–SPDP–(ZME-018) and 0.36 µM for C₆₀–Ser–(ZME-018) (see ESI†). To find the corresponding fullerene concentrations in these conjugates, we used UV-vis spectroscopy. At 440 nm, the molar absorptivity of C₆₀–Ser far exceeds that of ZME-018. The conjugate's measured 440 nm absorbance (ESI†) directly showed a C₆₀–Ser concentration of 15 µM, implying that the ratio (C₆₀–Ser) ∶ (ZME-018) was 42 ∶ 1.²⁵ Spectral analysis of the C₆₀–SPDP–(ZME-18) conjugate was more complex because absorption bands of C₆₀–SPDP at 440 nm are not intense enough for determining concentrations <20 µM and at lower wavelengths (<350 nm) there is an overlap of absorption bands from the antibody. To account for this, we first prepared a reference solution containing only 0.40 µM ZME-018. As shown in Fig. 2b, this solution has significant absorption at 282 nm. We then added C₆₀–SPDP until the absorbance of the mixture near 282 nm matched that of the C₆₀–SPDP–(ZME-18) immunoconjugate known to contain a 0.40 µM concentration of antibody. The upper traces in Fig. 2b show spectra of this mixture and the conjugate. From the amount of C₆₀–SPDP used to prepare the matching mixture, we deduced a C₆₀–SPDP concentration of 6 µM in the conjugate, corresponding to a (C₆₀–SPDP) ∶ (ZME-018) molar ratio of 15 ∶ 1.

Enzyme-linked immunosorbent assay (ELISA) binding curves using antigen-positive cells as targets gave mid-points of 1.2 nM for the C₆₀–SPDP–(ZME-018) immunoconjugate, 26 nM for the C₆₀–Ser–(ZME-018) immunoconjugate, and 724 nM for a non-specific, isotype-matched murine IgG antibody used as a control (ESI†). Amazingly, the C₆₀–SPDP–(ZME-018) conjugate demonstrated binding midpoints similar to the non-conjugated ZME-018 antibody (mid-point of 0.46 nM), even though 8% (by weight) of the immunoconjugate is fullerene. However, the non-covalently bound C₆₀–Ser–(ZME-018) conjugate, consisting of 17% (by weight) fullerene, exhibited a much lower affinity than C₆₀–SPDP–(ZME-018). Regardless, the C₆₀–Ser–(ZME-018) conjugate was still a factor of 30 more effective in binding the target than was the control.

To visualize the two C₆₀ immunoconjugates, TEM images of both were obtained on a lacey carbon grid. Comparative images of the ZME-018 antibody and the immunoconjugate are shown in Fig. 3 (An image of C₆₀–SPDP–(ZME-018) and experimental details are presented in the ESI†). Fig. 3 shows that the free antibody appears to have a clear globular structure ∼60 nm in diameter, whereas the image of the C₆₀–Ser immunoconjugate is also globular but 4–5 times larger in diameter. In addition, the C₆₀–Ser immunoconjugate image reveals numerous dark spots scattered throughout the structure that can be attributed to small aggregates of C₆₀–Ser, ∼2–5 nm in diameter. The larger C₆₀–Ser–(ZME-018) size may reflect disruption of hydrogen bonding networks inside the antibody or some aggregation effect.

Fig. 3 TEM images of (a) ZME-018 antibody and (b) C₆₀–Ser–(ZME-018) immunoconjugate. The magnification is the same for both frames; scale bar length is 20 nm. The solid curved feature in the image is the lacey carbon grid material.

The above experiments confirm that covalent bond formation is not necessary to form immunoconjugates of water-soluble C₆₀ derivatives with an antibody, and that antibody to antigen binding is not significantly reduced for high C₆₀ ∶ antibody molar ratios (15 ∶ 1). Future studies will explore the cancer cell biology of these new C₆₀ immunoconjugates, as well as immunoconjugates derived from other fullerene-based nanostructures that have the potential for targeted imaging and therapy in medicine.^11,13,26,27

At Rice University this research was supported by the Welch Foundation (grants C-0627 and C-0807), C Sixty, Inc., Carbon Nanotechnologies, Inc., and the NSF (grant CHE-0314270).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental and instrumentation detail. See DOI: 10.1039/b601717g

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