Theranostic radiolabeled miniap-4–trastuzumab bioconjugate for precision targeting of her2-overexpressing cerebral metastases

Abstract

A theranostic radiolabeled bioconjugate comprising trastuzumab, a protease-resistant blood¬ brain barrier shuttle peptide MiniAp-4, and a radionuclide is disclosed. The bioconjugate demonstrates enhanced BBB penetration, selective targeting of HER2-O verexpressing cerebral metastases, and combined therapeutic and diagnostic functionality, enabling precision treatment and real-time imaging of brain tumors

Description

DescriptionTitle of Invention: TITLE OF INVENTION

[0001] Theranostic Radiolabeled MiniAp-4-Trastuzumab Bioconjugate for Precision Targeting of HER2-Overexpressing Cerebral MetastasesTechnical Field

[0002] The present invention relates to the field of molecular theranostics, targeted cancer therapy, radiopharmaceuticals, and blood-brain barrier (BBB) drug delivery. More specifically, the invention concerns radiolabeled antibody-peptide bioconjugates for diagnosis and treatment of HER2-overexpressing cerebral metastases.BACKGROUND OF THE INVENTION

[0003] Brain metastases arising from HER2-positive cancers represent a major unmet clinical need due to the restrictive nature of the blood-brain barrier (BBB), which limits effective delivery of therapeutic antibodies. Trastuzumab, an FDA-approved monoclonal antibody targeting HER2, demonstrates limited efficacy in treating cerebral metastases because of poor BBB penetration. Existing strategies such as whole-brain radiotherapy or BBB disruption are associated with significant toxicity and limited long-term benefit.

[0004] Several approaches have been explored to enhance BBB penetration of therapeutic agents, including nanoparticle carriers, receptor-mediated transport systems, and peptide-based shuttles. However, no existing platform combines efficient BBB transcytosis, high HER2 specificity, therapeutic efficacy, and real-time diagnostic imaging in a single construct. Accordingly, there remains a need for improved theranostic agents capable of crossing the BBB and selectively targeting HER2- overexpressing brain metastases.SUMMARY OF THE INVENTION

[0005] The invention provides a theranostic bioconjugate comprising:

[0006] (a) a monoclonal antibody targeting HER2;(b) a protease-resistant blood-brain barrier shuttle peptide MiniAp-4 covalently linked to the antibody; and(c) a diagnostic and / or therapeutic radionuclide.

[0007] The bioconjugate exhibits enhanced transport across the BBB, high specificity for HER2-O verexpressing tumor cells, and enables both therapeutic efficacy and in vivo imaging. The invention further provides methods for preparing the bioconjugate, pharmaceutical compositions comprising the same, and methods of diagnosis and treatment of HER2-positive cerebral metastases.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 illustrates size distribution and dispersion index of the MiniAp-4- trastuzumab bioconjugate.

[0009] Figure 2 illustrates zeta potential measurements of the bioconjugate.

[0010] Figure 3 illustrates radiochemical stability over time.

[0011] Figure 4 illustrates in vitro drug release kinetics.

[0012] Figure 5 illustrates stability of radioconjugates in PBS.

[0013] Figures 6-7 illustrate cellular uptake by confocal microscopy.

[0014] Figure 8 illustrates permeability across an in vitro BBB model.

[0015] Figures 9-10 illustrate HER2 binding and cytotoxicity.

[0016] Figures 11-13 illustrate biodistribution and brain uptake.

[0017] Figures 14-16 illustrate tumor volume reduction and histopathology.DETAILED DESCRIPTION OF THE INVENTIONDefinitions

[0018] "Theranostic" refers to a combined diagnostic and therapeutic agent.

[0019] "MiniAp-4" refers to a cyclic, protease-resistant peptide capable of BBB transcytosis.

[0020] "Radiolabeled" refers to conjugation with a radionuclide suitable for imaging and / or therapy.Bioconjugate Composition

[0021] The bioconjugate comprises trastuzumab chemically modified via site- specific reduction of interchain disulfide bonds, followed by covalent attachment of MiniAp-4 peptide. The conjugate is subsequently radiolabeled with a radionuclide such as technetium-99m (99mTc).Method of Preparation

[0022] Trastuzumab is reduced using a phosphine-based reducing agent, purified, and reacted with MiniAp-4 peptide under controlled conditions. Excess reagents are removed, followed by radiolabeling using established chelation chemistry.Radiochemical purity and stability are verified by chromatography.Pharmaceutical Composition

[0023] The bioconjugate may be formulated with pharmaceutically acceptable carriers for parenteral administration. Suitable formulations include sterile aqueous solutions or lyophilized preparations.Therapeutic and Diagnostic Use

[0024] The bioconjugate is useful for:

[0025] • targeted treatment of HER2-overexpressing cerebral metastases;• diagnostic imaging of brain tumors using nuclear imaging modalities;• monitoring therapeutic response through real-time imaging.

[0026] Administration may be performed intravenously at therapeutically effective doses.Industrial Applicability

[0027] The invention is applicable in oncology, nuclear medicine, and precision diagnostics.It provides a scalable and clinically translatable platform for BBB-penetrating antibody-based theranostics.

[0028] .Figure 1

[0029] Scattering index after binding of MiniAp-4 peptides to trastuzumabFigure 2

[0030] Zeta potential intensity after binding of MiniAp-4 peptides to trastuzumab

[0031] Physicochemical properties of the stability of the MiniAp-4 trastuzumab peptide complex mTc99

[0032] The stability of the MiniAp-4 trastuzumab peptide complex mTc99- analyzed by ITLC over a 48-hour period after labeling. It shows the high stability of the antibody complex during storage at room temperature. Subsequent ITLC analysis showed thatmTc99 was released from the MiniAp-4 trastuzumab peptide to a very small extent when stored at room temperature (Figure 3).Figure 3

[0033] Stability of the MiniAp-4 peptide complex of trastuzumab mTc99 measured by ITLC over 48 hours after complex generation.Peptide / antibody binding rate

[0034] As can be seen in Table 1, the amount of bound antibody was 19.34+3.2.

[0035] Table 1. Peptide / Antibody Binding Rate3-2. Drug release and stability results

[0036] As can be seen in Figure 4, the drug release was 68% over a period of 5 to 24 hours, which was accompanied by a steep slope.Figure 4

[0037] Drug release results

[0038] The in vitro stability of the purified radioconjugates was evaluated. The results are shown in Figure 5 It was confirmed that the radiochemical purity of the conjugate stored in PBS at reduced temperature (4°C) for 10 days did not decrease below 95%. Samples stored at room temperature also showed good stability as the radiochemical purity did not decrease below 90%.Figure 5

[0039] Stability of purified radioconjugates in PBS at 4°C.

[0040] Uptake and immunofluorescence assays using confocal microscopy

[0041] The results showed that the uptake mechanism apparently occurred via the endocytic pathway, as indicated by the resulting punctate fluorescence (Figures 6 and 7), mostly located in endosomes. Furthermore, in the case of the complex, larger aggregates appeared to form in endosomes, which were seen as large dots with more intense fluorescence (Figure 6). Under these conditions, there was significant uptake, resulting in strong fluorescence signals in microscopic studies. We confirmed that the antibody conjugates were fully functional after modification.Figure 6

[0042] Confocal microscopy images of BT-474 cells incubated with the trastuzumab- mTc99-MiniAp-4 peptide complex.Figure 7

[0043] Confocal microscope images of sample BT-474 cells

[0044] Fluorescence using an emission filter under excitation at 405 nm and collected using a 63x objective lens, the fluorescence channel is shown in blue.

[0045] In Vitro Permeability Assay in a Cellular Model of the Human Blood-Brain Barrier

[0046] After confirming the functionality of these compounds, we sought to evaluate whether brain shuttle peptides enhance trastuzumab transport in a human cell-based blood-brain barrier model, in which MiniAp-4 trastuzumab mTc99 improved the apparent permeability of trastuzumab by 5-fold (Figure 8).Figure 8

[0047] (B) Permeability of MiniAp-4 peptide trastuzumab mTc99 and (A) trastuzumab in a human blood-brain barrier cell model in vitro.Flow cytometry results

[0048] By measuring the amount of DNA in cancer cells, the rate of cancer progression and the risk of recurrence can be predicted by flow cytometry. The binding capacity and ability of trastuzumab to brain shuttles upon binding of radiolabeled antibody to induce cell cycle arrest were evaluated. These results confirm that trastuzumab to brain shuttles upon binding of radiolabeled antibody showed cell cycle arrest similar to trastuzumab in BT-474 cells (Figure 9).Figure 9

[0049] Binding of Tz-MiniAp4 to HER2-overexpressing cells. A) Binding curve of radiolabeled antibody immunoconjugates to breast cancer cells (BT-474) B) Trastuzumab as negative control.Results of Cytotoxicity Assay

[0050] The cytotoxic potential of MiniAp-4 peptide trastuzumab mTc99- was evaluated (Figure 10). There was a significant difference in the cytotoxicity level, which showed a dose-dependent trend. The higher the dose, the higher the cell death rate was observed compared to human HER2-positive breast cancer cells (BT-474).Figure 10

[0051] Results of the MiniAp-4 peptide cytotoxicity assay of trastuzumab-mTc99. A: containing dose (2% (, B: containing dose (1.5% (, C: containing dose (1%) Biodistribution

[0052] The fluorescent intensity of mice treated with Tz-MiniAp4 was significantly higher than that of mice treated with Tz alone (Figures 11 and 12), indicating greater penetration of the BBB shuttle-modified monoclonal antibodies into the brain (Figure 13).Figure 11

[0053] Images (cancer cells) and fluorescence (antibodies) in the brains of mice inoculated with the MiniAp-4 peptide trastuzumab mTc99Figure 12

[0054] Images (cancer cells) and fluorescence (antibodies) in the brains of mice inoculated with trastuzumabFigure 13

[0055] Biodistribution results of MiniAp-4 peptide trastuzumab-mTc99. A) MiniAp-4 peptide trastuzumab-mTc99, B) TrastuzumabTumor Volume Measurement Results

[0056] The tumor volume in cubic millimeters was obtained from the volume measurement in the radiolabeled antibody immunoconjugate sample as 154+2.51, and the trastuzumab sample as 143+1.42, cubic millimeters. As can be seen in Figure 14, these results showed that the tumor volume in the radiolabeled antibody immunoconjugate sample was less than that in the trastuzumab sample.Figure 14

[0057] Tumor volume measurement resultsHistopathological evaluation of the tissues

[0058] As can be seen in Figure 15, it had features of small necrosis, less hemorrhage, less false rosettes, more lymphocytic infiltration and more small cell differentiation, compared to the trastuzumab sample (Figure 16).Figure 15

[0059] Histopathology evaluation of tissues of mice treated with MiniAp-4 peptide trastuzumab- mTc99Figure 16

[0060] Histopathology evaluation of tissues of mice treated with trastuzumab

Claims

Claims

1. A theranostic bioconjugate comprising:(a) an anti-HER2 monoclonal antibody;(b) a MiniAp-4 peptide covalently linked to the antibody; and (c) a radionuclide.

2. The bioconjugate of claim 1, wherein the antibody is trastuzumab.

3. The bioconjugate of claim 1, wherein the radionuclide is technetium-99m.

4. The bioconjugate of claim 1, wherein the MiniAp-4 peptide enhances blood-brain barrier transport.

5. A pharmaceutical composition comprising the bioconjugate of any preceding claim and a pharmaceutically acceptable carrier.

6. Use of the bioconjugate of claim 1 for diagnosis and / or treatment of HER2-O verexpressing cerebral metastases.

7. A method for treating brain metastases comprising administering an effective amount of the bioconjugate of claim 1 to a subject in need thereof.

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