Ex Vivo and In Vivo Evaluation of Overexpressed VLA-4 in Multiple Myeloma Using LLP2A Imaging Agents

Multiple myeloma (MM) is the second most common hematologic cancer in the United States, diagnosed in approximately 25,000 Americans each year, and is currently incurable in most patients (1). MM is an age-related malignancy of plasma B cells, characterized by hallmark genetic mutations and changes in the hematopoietic microenvironment (2). The end-organ damage caused by MM includes lytic bone lesions, anemia, immunodeficiency, hypercalcemia, and renal insufficiency. Myeloma cells tend to resculpt the bone microenvironment by facilitating neoangiogenesis, recruiting tumor-associated macrophages, suppressing osteoblasts, and stimulating osteoclasts via soluble activating factors. MM and its obligate precursor state, monoclonal gammopathy of uncertain significance, are readily detected using serum markers (either intact immunoglobulin or free light chains) in 90% of cases. However, serum markers are insufficient to distinguish premalignant monoclonal gammopathy of uncertain significance and smoldering MM from fully transformed MM. The diagnosis of MM requires very high monoclonal tumor burden or end-organ damage, such as lytic bone lesions, and highly sensitive radiographic skeletal imaging, which is an integral part of the diagnosis of MM (3). Evaluation of progression and treatment response is also confounded in 10% of MM patients who display an oligosecretory phenotype (defined as serum M-protein < 1 g/dL and urine M-protein < 200 mg/24 h) (4). The timely and accurate diagnosis of MM is important because a delay in the diagnosis of MM can be detrimental to the patient’s outcome (3). Whole-body imaging approaches such as noninvasive molecularly targeted PET can sensitively, quantitatively, and spatiotemporally probe functional biologic processes such as changes in receptor expression during disease progression. Most clinical PET imaging in MM is performed with the metabolic tracer 18F-labeled FDG (18F-FDG); however, MM cells are hypoproliferative and do not consistently overexpress glucose transporter 1, and over a third of intramedullary myeloma lesions can go undetected by 18F-FDG PET (5). Very-late-antigen-4 (VLA-4, α4β1 receptor, CD49d/CD29) is a transmembrane adhesion receptor that plays an important role in normal immune responses as well as cancer pathogenesis (6). Enhanced VLA-4 expression has been observed in MM cells and surrounding stroma. Additionally, myeloma bone marrow stroma is rich in the VLA-4 ligands, vascular cell adhesion molecule-1, and fibronectin (7). In MM, inside-out signaling induces a VLA-4 conformational change to an activated state triggering high-affinity ligand binding, which in turn activates further intracellular signaling (outside-in signaling) (8). VLA-4 conformational activation is correlated with enhanced cell trafficking, proliferation, and drug resistance (9). LLP2A is a high-affinity peptidomimetic ligand for VLA-4, and bioconjugates of LLP2A have shown promise as imaging and therapeutic agents (10–12). We previously demonstrated the proof-of-principle molecular imaging of VLA-4 in murine 5TGM1 myeloma subcutaneous tumors using the PET radiopharmaceutical 64Cu-(CB-TE1A1P)-LLP2A (12). Recently Beaino et al. demonstrated imaging of VLA-4–positive melanoma mouse models using 64Cu-CB-TE1A1P-PEG4-LLP2A (64Cu-LLP2A) (13). 64Cu-LLP2A possesses ideal pharmacokinetic features such as optimal bioavailability (by virtue of added PEG chains), enhanced solubility, and high 64Cu specific activity (~37 MBq/μg). In the current study, we evaluated the specificity of nuclear and optical analogs of LLP2A for imaging overexpressed VLA-4 in the hematopoietic environments in MM using intravenous syngeneic MM mouse models, estimated radiation dose-limiting organs through normal mouse biodistribution, and stained human tissues for VLA-4 positivity. We additionally evaluated the specificity of LLP2A for VLA-4–positive murine 5TGM1-GFP myeloma and nonmalignant host inflammatory stromal cells including T and B lymphocytes and macrophages in vivo and ex vivo. Longitudinal 64Cu-LLP2A PET was performed in a syngeneic C57BL6/KaLwRij/5TGM1-GFP mouse model of systemic (tumor cells injected intravenously via the tail) MM to monitor medullar tumor lesions over the disease progression. Binding specificity of LLP2A to tumor cells and tumor-associated inflammatory cells in the bone marrow and spleen were quantified by flow cytometry using fluorescent-labeled LLP2A (LLP2A-Cy5). VLA-4–targeted PET imaging with 64Cu-LLP2A was compared with 18F-FDG PET, and human radiation dose estimates for 64Cu-LLP2A were extrapolated from mouse biodistribution data. Finally, to determine whether LLP2A can be used to follow VLA-4 activation/overexpression in human MM tissue, we stained 10 formalin-fixed paraffin-embedded bone marrow samples from deceased MM patients with LLP2A-Cy5.