determining-binding-affinity-kd-radiolabeled-antibodies-to

Determining Binding Affinity (K<sub>D</sub>) of Radiolabeled Antibodies to Immobilized Antigens

Determining binding affinity (KD) is an important aspect of the characterization of radiolabeled antibodies (rAb). Typically, binding affinity is ...

Yale University

High levels of expression of glycoprotein non-metastatic B (gpNMB) in triple negative breast cancer (TNBC) and its association with metastasis and recurrence make it an attractive target for therapy with the antibody drug conjugate, glembatumumab vedotin (CDX-011). This report describes the development of a companion PET-based diagnostic imaging agent using 89Zr-labeled glembatumumab ([89Zr]DFO-CR011) to potentially aid in the selection of patients most likely to respond to targeted treatment with CDX-011. [89Zr]DFO-CR011 was characterized for its pharmacologic properties in TNBC cell lines. Preclinical studies determined that [89Zr]DFO-CR011 binds specifically to gpNMB with high affinity (Kd = 25 ± 5 nM), immunoreactivity of 2.2-fold less than the native CR011, and its cellular uptake correlates with gpNMB expression (r = 0.95). In PET studies at the optimal imaging timepoint of 7 days p.i., the [89Zr]DFO-CR011 tumor uptake in gpNMB-expressing MDA-MB-468 xenografts had a mean SUV of 2.9, while significantly lower in gpNMB-negative MDA-MB-231 tumors with a mean SUV of 1.9. [89Zr]DFO-CR011 was also evaluated in patient-derived xenograft models of TNBC, where tumor uptake in vivo had a positive correlation with total gpNMB protein expression via ELISA (r = 0.79), despite the heterogeneity of gpNMB expression within the same group of PDX mice. Lastly, the radiation dosimetry calculated from biodistribution studies in MDA-MB-468 xenografts determined the effective dose for human use would be 0.54 mSv/MBq. Overall, these studies demonstrate that [89Zr]DFO-CR011 is a potential companion diagnostic imaging agent for CDX-011 which targets gpNMB, an emerging biomarker for TNBC.

Preclinical PET imaging of glycoprotein non-metastatic melanoma B in triple negative breast cancer: feasibility of an antibody-based companion diagnostic agent.

Since its discovery, the human epidermal growth factor 2 (HER2) has been extensively studied. Presently, there are 2 standard diagnostic techniques to assess HER2 status in biopsies: immunohistochemistry and fluorescence in situ hybridization. While these techniques have played an important role in the treatment of patients with HER2-positive cancer, they both require invasive biopsies for analysis. Moreover, the expression of HER2 is heterogeneous in breast cancer and can change over the course of the disease. Thus, the degree of HER2 expression in the small sample size of biopsied tumors at the time of analysis may not represent the overall status of HER2 expression in the whole tumor and in between tumor foci in the metastatic setting as the disease progresses. Unlike biopsy, molecular imaging using probes against HER2 allows for a noninvasive, whole-body assessment of HER2 status in real time. This technique could potentially select patients who may benefit from HER2-directed therapy and offer alternative treatments to those who may not benefit. Several antibodies and small molecules against HER2 have been labeled with different radioisotopes for nuclear imaging and/or therapy. This review presents the most recent advances in HER2 targeting in nuclear medicine focusing on preclinical and clinical studies.

Targeting HER2 in Nuclear Medicine for Imaging and Therapy.

To evaluate whether tumor uptake of [Zr]trastuzumab can distinguish HER2-positive from HER2-negative breast cancer.

Evaluation of [Zr]trastuzumab-PET/CT in differentiating HER2-positive from HER2-negative breast cancer.

 The success of human epidermal growth factor receptor 2 (HER2)-targeted therapy depends on accurate characterization of HER2 expression, but current methods available have several limitations. This study aims to investigate the feasibility of [Zr]pertuzumab imaging to monitor early response to Ado-trastuzumab emtansine (T-DM1) therapy in mice bearing xenografts of HER2-positive breast cancer (BCa).  Pertuzumab was conjugated to DFO-Bz-NCS and labeled with Zr. Mice bearing BT-474 tumors were imaged with [Zr]pertuzumab and [F]FDG before and after T-DM1 therapy.  Pertuzumab was successfully labeled with Zr with a specific activity of 0.740 MBq/μg. Overall [F]FDG images showed poor delineation of tumors. Using [F]FDG-PET to measure tumor volume, the volume remained unchanged from 107.6 ± 20.7 mm before treatment to 89.87 ± 66.55 mm after treatment. In contrast, [Zr]pertuzumab images showed good delineation of HER2-positive tumors, allowing accurate detection of changes in tumor volume (from 243.80 ± 40.91 mm before treatment to 78.4 ± 40.43 mm after treatment).  [Zr]pertuzumab may be an imaging probe for monitoring the response of HER2-positive BCa patients to T-DM1 therapy.

Imaging of HER2 with [Zr]pertuzumab in Response to T-DM1 Therapy.

Amivantamab is a novel bispecific antibody that simultaneously targets the epidermal growth factor receptor (EGFR) and the hepatocyte growth factor receptor (HGFR/c-MET) that are overexpressed in several types of cancer including triple-negative breast cancer (TNBC). Targeting both receptors simultaneously can overcome resistance to mono-targeted therapy. The purpose of this study is to develop Zr-labeled amivantamab as a potential companion diagnostic imaging agent to amivantamab therapy using various preclinical models of TNBC for evaluation.

Development of [Zr]ZrDFO-amivantamab bispecific to EGFR and c-MET for PET imaging of triple-negative breast cancer.

Breast cancer is the most common cancer in women worldwide. The heterogeneity of breast cancer and drug resistance to therapies make the diagnosis and treatment difficult. Molecular imaging methods with positron emission tomography (PET) and single-photon emission tomography (SPECT) provide useful tools to diagnose, predict, and monitor the response of therapy, contributing to precision medicine for breast cancer patients. Recently, many efforts have been made to find new targets for breast cancer therapy to overcome resistance to standard of care treatments, giving rise to new therapeutic agents to offer more options for patients with breast cancer. The combination of diagnostic and therapeutic strategies forms the foundation of theranostics. Some of these theranostic agents exhibit high potential to be translated to clinic. In this review, we highlight the most recent advances in theranostics of the different molecular subtypes of breast cancer in preclinical studies.

Preclinical Advances in Theranostics for the Different Molecular Subtypes of Breast Cancer.

There is a need for prognostic markers to select patients most likely to benefit from antibody-drug conjugate (ADC) therapy. We quantified the relationship between pretreatment PET imaging of glycoprotein nonmetastatic melanoma B (gpNMB) with 89Zr-labeled anti-gpNMB antibody ([89Zr]ZrDFO-CR011) and response to ADC therapy (CDX-011) in triple-negative breast cancer. First, we compared different PET imaging metrics and found that standardized uptake values (SUV) and tumor-to-heart SUV ratios were sufficient to delineate differences in radiotracer uptake in the tumor of four different cell- and patient-derived tumor models and achieved high standardized effect sizes. These tumor models with varying levels of gpNMB expression were imaged with [89Zr]ZrDFO-CR011 followed by treatment with a single bolus injection of CDX-011. The percent change in tumor volume relative to baseline (% CTV) was then correlated with SUVmean of [89Zr]ZrDFO-CR011 uptake in the tumor. All gpNMB-positive tumor models responded to CDX-011 over 6 weeks of treatment, except one patient-derived tumor regrew after 4 weeks of treatment. As expected, the gpNMB-negative tumor increased in volume by 130 ± 59% at endpoint. The magnitude of pretreatment SUV had the strongest inverse correlation with the % CTV at 2-4 weeks after treatment with CDX-011 (Spearman ρ = -0.8). However, pretreatment PET imaging with [89Zr]ZrDFO-CR011 did not inform on which tumor types will regrow over time. Other methods will be needed to predict resistance to treatment.

[89Zr]ZrDFO-CR011 PET Correlates with Response to Glycoprotein Nonmetastatic Melanoma B-targeted Therapy in Triple-negative Breast Cancer.

Precision medicine is the customization of therapy for specific groups of patients using genetic or molecular profiling. Noninvasive imaging is one strategy for molecular profiling and is the focus of this review. The combination of imaging and therapy for precision medicine gave rise to the field of theranostics. In breast cancer, the detection and quantification of therapeutic targets can help assess their heterogeneity, especially in metastatic disease, and may help guide clinical decisions for targeted treatments. Positron emission tomography (PET) or single-photon emission tomography (SPECT) imaging has the potential to play an important role in the molecular profiling of therapeutic targets in vivo for the selection of patients who are likely to respond to corresponding targeted therapy. In this review, we discuss the state-of-the-art nuclear imaging agents in clinical research for breast cancer. We reviewed 17 clinical studies on PET or SPECT agents that target 10 different receptors in breast cancer. We also discuss the limitations of the study designs and of the imaging agents in these studies. Finally, we offer our perspective on which imaging agents have the highest potential to be used in clinical practice in the future.

Article	Year
Preclinical PET imaging of glycoprotein non-metastatic melanoma B in triple negative breast cancer: feasibility of an antibody-based companion diagnostic agent. Oncotarget\| PubMed ID: 29262642	2017
Targeting HER2 in Nuclear Medicine for Imaging and Therapy. Molecular imaging\| PubMed ID: 29357745	2018
Evaluation of [Zr]trastuzumab-PET/CT in differentiating HER2-positive from HER2-negative breast cancer. Breast cancer research and treatment\| PubMed ID: 29442264	2018
Imaging of HER2 with [Zr]pertuzumab in Response to T-DM1 Therapy. Cancer biotherapy & radiopharmaceuticals\| PubMed ID: 30676778	2019
Development of [Zr]ZrDFO-amivantamab bispecific to EGFR and c-MET for PET imaging of triple-negative breast cancer. European journal of nuclear medicine and molecular imaging\| PubMed ID: 32770372	2021
Preclinical Advances in Theranostics for the Different Molecular Subtypes of Breast Cancer. Frontiers in pharmacology\| PubMed ID: 33986665	2021
[89Zr]ZrDFO-CR011 PET Correlates with Response to Glycoprotein Nonmetastatic Melanoma B-targeted Therapy in Triple-negative Breast Cancer. Molecular cancer therapeutics\| PubMed ID: 35027482	2022
Clinical Evaluation of Nuclear Imaging Agents in Breast Cancer. Cancers\| PubMed ID: 35565232	2022

Bernadette Marquez-Nostra

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