Affibody proteins specific for B7-H3 (CD276)
Inventors
Abou-Elkacem, Lotfi • Willmann, Juergen Karl • Hackel, Benjamin J. • Stern, Lawrence Arthur • Lown, Patrick Samuel • Lutz, Amelie Margarete Marianne • Bam, Rakesh Kumar
Assignees
Leland Stanford Junior University • University of Minnesota System
Publication Number
US-12274759-B2
Publication Date
2025-04-15
Expiration Date
2039-08-22
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Abstract
Affibody polypeptides that specifically bind to B7-H3 are provided. Exemplary anti-B7-H3 affibodies are provided. The affibody polypeptides specifically recognize and bind to B7-H3 with high affinity. The affibodies can be conjugated to contrast agents, including without limitation microbubbles for contrast-enhanced ultrasound imaging
Core Innovation
The invention provides affibody polypeptides that specifically bind to B7-H3 (CD276), a glycoprotein expressed or overexpressed on a variety of human tumors and tumor vasculature. These polypeptides, exemplified by sequences in SEQ ID NO:1-4, are developed using yeast displayed affibody libraries and directed evolution to generate high-affinity binders for both soluble and cell surface B7-H3 proteins. The innovation includes the use of these anti-B7-H3 affibodies as specific ligands for recognition and binding of B7-H3 in human tissue and tumors.
The background identifies the problem of insufficient specificity and effectiveness of conventional imaging and therapeutic agents, especially in detecting cancers such as breast carcinoma in women with dense breast tissue. Current antibody-based ligands for molecularly-targeted microbubbles (MBs) used in contrast-enhanced ultrasound imaging are limited due to production inefficiencies and potential immune responses. There is a need for improved targeting agents for both imaging and therapy, specifically those that can address B7-H3 expressed on tumor cells and vasculature.
The invention addresses this need by providing anti-B7-H3 affibody polypeptides that can be conjugated to imaging moieties such as microbubbles for ultrasound, radionuclides, or fluorescent labels, enabling sensitive and specific imaging of B7-H3-expressing cells and tissues. Affibodies can also be conjugated to cytotoxic or therapeutic moieties for targeted treatment of B7-H3-expressing cancers. The methods described include in vivo and in vitro applications, such as using affibody-conjugated microbubbles for ultrasound imaging to detect cancerous lesions or monitoring therapy efficacy, as well as administration of therapeutic affibody conjugates for cancer treatment.
Claims Coverage
The patent includes several independent claims, each defining a core inventive feature relating to anti-B7-H3 affibody polypeptides, their conjugates, nucleic acids, and uses in imaging and therapy.
Polypeptide comprising residues 1-36 of SEQ ID NO:1-4
A polypeptide comprising residues 1-36 of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 is disclosed. This includes variations joined operably to an affibody third helix sequence and encompasses both full length and variant forms, including fusion proteins and variants operably linked to a third helix sequence.
Polypeptides conjugated to imaging, cytotoxic, or immunoglobulin moieties
The invention covers affibody polypeptides comprising residues 1-36 of SEQ ID NO:1-4, optionally with an affibody third helix sequence, further conjugated to: - Imaging moieties, including radiography heavy metals, radiation emitting moieties, positron emitting moieties, magnetic resonance contrast moieties, ultrasound contrast moieties, microbubbles, or optically visible moieties such as fluorescent moieties or dyes. - Cytotoxic moieties, such as radioactive isotopes, chemotoxic agents, or toxin proteins. - Immunoglobulin Fc sequences or pharmacokinetic moieties for therapeutic or pharmacokinetic advantages.
Microbubble contrast agent comprising the affibody polypeptide
A contrast agent comprising a microbubble conjugated to a polypeptide comprising residues 1-36 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 is claimed. The conjugated microbubble serves as a targeted ultrasound contrast agent recognizing B7-H3 expression.
Pharmaceutical compositions and nucleic acid constructs
A pharmaceutical composition comprising the polypeptide and a pharmaceutically acceptable excipient is claimed. Additionally, nucleic acids encoding said polypeptide, optionally in expression vectors, and isolated host cells comprising such nucleic acids are provided.
Method for imaging B7-H3 expressing cells with affibody-imaging conjugates
A method of imaging the presence of B7-H3-expressing cells by contacting cells with an effective dose of a polypeptide comprising SEQ ID NO:1-4 and an imaging moiety to form a complex, and detecting the complex, wherein detection is indicative of B7-H3-expressing cells. The claim specifically covers applications including in vivo imaging, imaging of human cells, tumor vasculature or cancer cells, imaging of vascular endothelial cells of human breast cancer, and detection using ultrasound, PET, MRI, CT, SPECT, or radiography.
Method for treating B7-H3 positive cancer with affibody-cytotoxic conjugates
A method for treating B7-H3 positive cancer, wherein cancer cells are contacted with an effective dose of a polypeptide comprising SEQ ID NO:1-4 conjugated to a cytotoxic moiety is claimed.
The claims encompass anti-B7-H3 affibody polypeptides defined by specific sequences, their functional conjugates (imaging, cytotoxic, or pharmacokinetic), microbubble-based contrast agents, pharmaceutical and genetic compositions, and methods for both imaging and therapeutic applications targeting B7-H3-expressing cells and cancers.
Stated Advantages
Affibody-based agents offer specificity and high affinity for B7-H3, allowing for sensitive and specific imaging and detection of B7-H3-expressing cells and cancer lesions.
Affibodies offer reduced molecular weight compared to antibodies, enabling faster tissue penetration, favorable pharmacokinetics, and rapid tumor targeting.
The use of affibody polypeptides permits easier, cost-effective, and large-scale production, facilitating clinical translation over antibody-based ligands.
Affibody-based microbubble contrast agents provide for non-invasive, contrast-enhanced ultrasound imaging with high sensitivity and specificity, improving detection of cancers such as breast cancer, including mammographically occult malignancies in dense breast tissue.
Affibody conjugates allow for site-specific and efficient chemical conjugation to imaging or therapeutic moieties, enabling flexible and multifaceted clinical utility.
Affibodies demonstrate high thermal and pH stability, further supporting their use as clinically translatable imaging and therapeutic agents.
Documented Applications
Contrast-enhanced ultrasound imaging of B7-H3 expressing tumors, particularly breast cancer, using microbubble-conjugated affibody polypeptides.
Detection of mammographically occult malignancy in women with dense breast tissue by ultrasound imaging with affibody-conjugated microbubbles.
Imaging of tumor vasculature and neovasculature expressing B7-H3 for cancer diagnosis and monitoring using affibody imaging conjugates.
Therapeutic targeting and treatment of cancers expressing B7-H3 (including but not limited to breast, neuroblastoma, Ewing's sarcoma, rhabdomyosarcoma, prostate, ovarian, colorectal, lung cancers) with affibody-cytotoxic conjugates.
In vitro or ex vivo detection of B7-H3 expressing cells or tissues, such as in biopsy samples, using affibody-imaging conjugates.
Use as a tool to monitor efficacy of anticancer agents by measuring changes in B7-H3 expression non-invasively.
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