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Endolysosomal targeting conjugates for improved delivery of cargo molecules to the endolysosomal compartment of target cells

A technology of endolysosomes and conjugates, applied in the direction of radioactive carriers, preparations for in vivo tests, antibodies, etc.

Pending Publication Date: 2020-03-31
TEXAS A&M UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the development of LCs that allow more efficient labeling of tumors with higher contrast to background tissue also remains challenging.

Method used

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  • Endolysosomal targeting conjugates for improved delivery of cargo molecules to the endolysosomal compartment of target cells
  • Endolysosomal targeting conjugates for improved delivery of cargo molecules to the endolysosomal compartment of target cells
  • Endolysosomal targeting conjugates for improved delivery of cargo molecules to the endolysosomal compartment of target cells

Examples

Experimental program
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Embodiment 1

[0213] Example 1. Materials and methods

[0214] Cell lines and culture conditions. The mouse endothelial cell line 2H11 (ATCC, CRL-2163) was cultured in Darwin's Modified Elder's Medium (DMEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS). Human breast cancer cell line MDA-MB-231 (ATCC, HTB-26) was cultured in DMEM supplemented with 10% FBS. Human breast cancer cell lines T-47D (ATCC, HTB-133), MDA-MB-453 (ATCC, HTB-131) and MDA-MB-468 (ATCC, HTB-132) were prepared in RPMI 1640 supplemented with 10% FBS cultured in culture medium. Human breast cancer cell line SK-BR-3 (ATCC, HTB-30) and human ovarian cancer cell line SK-OV-3 (ATCC, HTB-77) were cultured in McCoy's 5A medium supplemented with 10% FBS. Human prostate cancer cell lines LNCaP and 22Rv1 (ATCC, CRL-1740 and CRL-2505, respectively) were cultured in RPMI 1640 medium supplemented with 10% FBS. Human breast cancer cell line HCC1954 (Gazdar, A.F., Kurvari, V., Virmani, A., Gollahon, L., Sakaguchi...

Embodiment 2

[0247] Example 2. Generation of HER2 targeting agents with pH dependent HER2 binding

[0248] Figure 4A and 4B Binding assays showing two mutated Pertuzumab variants targeting HER2: SG mutant; Ser55 mutated to histidine and Gly57 mutated to glutamic acid in the heavy chain variable domain (SEQ ID NO: 4 ); YS mutant, Tyr55 in the light chain variable domain is mutated to histidine, and Ser103 in the heavy chain variable domain is mutated to histidine (SEQ ID NO:6,8). Data were acquired using surface plasmon resonance. Figure 4A Representative sensorgrams at pH 7.0 and 5.8, 1 μM HER-extracellular domain (ECD)-Fc fusion interaction with mutant variants and wild-type (WT) Pertuzumab are shown. for Figure 4A For the assay shown in , antibodies were immobilized on the flow cell. Figure 4B Equilibrium dissociation constants (nM) of WT Pertuzumab, SG and YS obtained by injecting antibodies on immobilized HER2-ECD-Fc are shown. The data indicated that both the SG and YS var...

Embodiment 3

[0249] Example 3. Internalization and accumulation of HER2 targeting agents in HER2 expressing cells

[0250] Pertuzumab (WT) and mutant variants SG and YS were coupled to maleimidocaproyl-val-cit-PAB-MMAE (MC-VC-PAB -MMAE), a drug-to-antibody ratio (DAR) of 2 drugs per antibody, followed by analysis of binding and accumulation of HER2-targeted ADCs (HER2-ADCs) in a panel of different HER2-expressing cell lines. Figure 5A HER2 expression levels on different cancer cell lines detected using Alexa 647-labeled Pertuzumab (solid line) or Alexa 647-labeled control antibody (dashed line) and flow cytometry are shown. Figure 5B Shown are the internalized Alexa 488-labeled WT Pertuzumab-MMAE (WT-MMAE), SG-MMAE, YS-MMAE, control antibody-MMAE (C-MMAE) or Trg after 0.5, 4 and 20 hours of incubation. Tocilizumab-DM1 (T-DM1) levels. Cell surface-bound ADCs were quenched using an Alexa 488-specific antibody. Error bars indicate standard deviation, * indicates statistically significa...

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Abstract

Endolysosomal targeting conjugates that are engineered to deliver cargo molecules such as cytotoxic drugs or imaging labels with improved efficiency to late endosomes and / or lysosomes in target cellssuch as tumor cells are described. The endolysosomal targeting conjugate includes a targeting component and a cargo component. The targeting component is configured to bind to a cell surface moleculeof a target cell and the cargo component includes a cargo molecule. The targeting component and the cargo component may be fused by a covalent bond or associated by a non-covalent bond. The targetingcomponent may bind to the cell surface molecule or the cargo component with higher affinity in the extracellular space than in an endolysosomal compartment of the target cell.

Description

[0001] field of invention [0002] The invention disclosed herein generally relates to the production of engineered proteins that can be used as platforms for the delivery of cytotoxic drugs, imaging markers, or other cargo molecules to target cells, such as tumor cells. These proteins are designed to more efficiently deliver cargo molecules to late endosomes and lysosomes in target cells. Background technique [0003] Antibody-drug conjugates (ADCs) or protein-drug conjugates (PDCs) represent a class of therapeutic agents that combine antibodies, antibody fragments, or ) binding to other proteins coupled with the delivery of highly toxic drugs. The problem with current ADCs or PDCs is that they are toxic, leading to dose limitations. However, the development of ADCs and PDCs that allow more efficient delivery of cytotoxic drugs to tumor cells remains challenging. [0004] Additionally, antibodies, antibody fragments, or other targeting proteins can be labeled with radioact...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/66A61K47/68A61K39/44A61K38/38A61K49/00A61K49/16A61K51/08A61K51/10C07K16/46A61P35/00
CPCA61P35/00C07K16/32A61K38/00A61K47/6817A61K47/6855A61K47/6869
Inventor E·S·W·奥伯R·J·奥伯J·C-W·康孙巍李然
Owner TEXAS A&M UNIVERSITY