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Site-Specific Photoaffinity Bioconjugation for the Creation of 89Zr-Labeled Radioimmunoconjugates

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Abstract

Purpose

Site-specific approaches to bioconjugation produce well-defined and homogeneous immunoconjugates with potential for superior in vivo behavior compared to analogs synthesized using traditional, stochastic methods. The possibility of incorporating photoaffinity chemistry into a site-specific bioconjugation strategy is particularly enticing, as it could simplify and accelerate the preparation of homogeneous immunoconjugates for the clinic. In this investigation, we report the synthesis, in vitro characterization, and in vivo evaluation of a site-specifically modified, 89Zr-labeled radioimmunoconjugate created via the reaction between an mAb and an Fc-binding protein bearing a photoactivatable 4-benzoylphenylalanine residue.

Procedures

A variant of the Fc-binding Z domain of protein A containing a photoactivatable, 4-benzoylphenylalanine residue — Z(35BPA) — was modified with desferrioxamine (DFO), combined with the A33 antigen-targeting mAb huA33, and irradiated with UV light. The resulting immunoconjugate — DFOZ(35BPA)-huA33 — was purified and characterized via SDS-PAGE, MALDI-ToF mass spectrometry, surface plasmon resonance, and flow cytometry. The radiolabeling of DFOZ(35BPA)-huA33 was optimized to produce [89Zr]Zr-DFOZ(35BPA)-huA33, and the immunoreactivity of the radioimmunoconjugate was determined with SW1222 human colorectal cancer cells. Finally, the in vivo performance of [89Zr]Zr-DFOZ(35BPA)-huA33 in mice bearing subcutaneous SW1222 xenografts was interrogated via PET imaging and biodistribution experiments and compared to that of a stochastically labeled control radioimmunoconjugate, [89Zr]Zr-DFO-huA33.

Results

HuA33 was site-specifically modified with Z(35BPA)-DFO, producing an immunoconjugate with on average 1 DFO/mAb, high in vitro stability, and high affinity for its target. [89Zr]Zr-DFOZ(35BPA)-huA33 was synthesized in 95% radiochemical yield and exhibited a specific activity of 2 mCi/mg and an immunoreactive fraction of ~ 0.85. PET imaging and biodistribution experiments revealed that high concentrations of the radioimmunoconjugate accumulated in tumor tissue (i.e., ~ 40%ID/g at 120 h p.i.) but also that the Z(35BPA)-bearing immunoPET probe produced higher uptake in the liver, spleen, and kidneys than its stochastically modified cousin, [89Zr]Zr-DFO-huA33.

Conclusions

Photoaffinity chemistry and an Fc-binding variant of the Z domain were successfully leveraged to create a novel site-specific strategy for the synthesis of radioimmunoconjugates. The probe synthesized using this method — DFOZ(35BPA)-huA33 — was well-defined and homogeneous, and the resulting radioimmunoconjugate ([89Zr]Zr-DFOZ(35BPA)-huA33) boasted high specific activity, stability, and immunoreactivity. While the site-specifically modified radioimmunoconjugate produced high activity concentrations in tumor tissue, it also yielded higher uptake in healthy organs than a stochastically modified analog, suggesting that optimization of this system is necessary prior to clinical translation.

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Data availability

Data are available upon request.

Abbreviations

DFO:

Desferrioxamine

BPA:

4-Benzoylphenylalanine

Z(35BPA):

Z domain of protein A with 4-benzoylphenylalanine

ADC:

Antibody-drug conjugate

PET:

Positron emission tomography

DOL:

Degree of labeling

IgG:

Immunoglobulin

mAb:

Monoclonal antibody

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

IMAC:

Immobilized metal affinity chromatography

MALDI-ToF:

Matrix-assisted laser desorption / time-of-flight mass spectrometry

IACUC:

Institutional Animal Care and Use Committee

%ID/g:

Percent of injected dose per gram

PBS:

Phosphate-buffered saline

DMSO:

Dimethyl sulfoxide

RPM:

Revolutions per minute

RCF:

Relative centrifugal force

EDTA:

Ethylenediaminetetreaacetic acid

iTLC:

Instant thin layer chromatography

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Funding

This work was supported by funding from the National Institutes of Health to BMZ (R01CA240963, U01CA221046, R01CA204167, R21EB030275 and R01CA244327) and SD (F31CA275334) as well as funding from the Swedish Research Council (2020-04478 and 2018-06228) and the Swedish Cancer Society (22 2203 Pj 01 H) to AEK. The authors also thank the MSKCC Small Animal Imaging Core Facility and the MSKCC Radiochemistry and Molecular Imaging Probe Core.

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Authors and Affiliations

  1. Department of Chemistry, Hunter College, City University of New York, New York, NY, USA

    Samantha Delaney & Brian M. Zeglis

  2. Ph.D. Program in Biochemistry, The Graduate Center of the City University of New York, New York, NY, USA

    Samantha Delaney & Brian M. Zeglis

  3. Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Samantha Delaney & Brian M. Zeglis

  4. Department of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden

    Ábel Nagy & Amelie Eriksson Karlström

  5. Ph.D. Program in Chemistry, The Graduate Center of the City University of New York, New York, NY, USA

    Brian M. Zeglis

  6. Department of Radiology, Weill Cornell Medical College, New York, NY, USA

    Brian M. Zeglis

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  1. Samantha Delaney
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  2. Ábel Nagy
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Correspondence to Amelie Eriksson Karlström or Brian M. Zeglis.

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Delaney, S., Nagy, Á., Karlström, A.E. et al. Site-Specific Photoaffinity Bioconjugation for the Creation of 89Zr-Labeled Radioimmunoconjugates. Mol Imaging Biol 25, 1104–1114 (2023). https://doi.org/10.1007/s11307-023-01818-5

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  • DOI: https://doi.org/10.1007/s11307-023-01818-5

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