Therapeutic nanoconjugates and uses thereof

A nanoparticle and fusion protein technology, which is applied in the field of nanostructured protein materials, can solve the problems of complex and cumbersome antibody structures and obstacles

Pending Publication Date: 2021-11-02
巴塞罗那自治大学UAB +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the structural complexity of antibodies can be a cumbersome hurdle in terms of cost and synthesis

Method used

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  • Therapeutic nanoconjugates and uses thereof
  • Therapeutic nanoconjugates and uses thereof
  • Therapeutic nanoconjugates and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0398] Synthesis of therapeutic nanoconjugates

[0399] Synthesis of T22-STM-H6-Aur therapeutic nanoconjugate.

[0400] T22-STM-H6 protein nanoparticles (in 166 mM NaCO3H pH=8 buffer) were mixed with maleimide-functionalized monomethyl auristatin E (MMAE) (resuspended in 50% PBS / 50% acetonitrile) at a protein:MMAE ratio of 1:50 for 4 h in a one-pot reaction. The resulting nanoconjugates were subsequently loaded with previously loaded Ni 2+ (NiCl 2 ) in a HiTrap chelating HP column (GE Healthcare), perform IMAC affinity chromatography, and wash with a washing buffer (20mM Tris, 500mM NaCl, 10mM imidazole pH=8) in the column for 30min in order to remove unreacted MMAE molecules, followed by one-step isocratic elution with elution buffer (20 mM Tris, 500 mM NaCl, 500 mM imidazole pH=8). The eluted nanoconjugates were then dialyzed (dyalized) against 166 mM NaCO3H pH=8 buffer using a 12-14 MWCO membrane O / N at 4°C to remove the elution buffer containing a high concentration ...

Embodiment 2

[0404] Characterization of the T22-STM-H6-Aur nanoconjugate and determination of the drug / nanoparticle ratio

[0405] Nanoparticles of T22-GFP-H6 were formed from an average of 15 monomers, and each monomer of T22-GFP-H6-FdU bound about 4 molecules of oligo-FdU. This corresponds to about 60 molecules of oligo-FdU per nanoparticle. As for T22-GFP-H6-auristatin, the data show that it binds up to 30 auristatin per protein, which corresponds to about 450 auristatin per nanoparticle.

[0406] The nanoparticles of T22-STM-H6 are formed by an average of 15-20 monomers. Mass spectrometry indicates that no less than 3 auristatin molecules are bound to each protein monomer, and at least up to more than 15 auristatin molecules per monomer.

Embodiment 3

[0408] T22-STM-H6-Auristatin compared to T22-GFP-H6-Auristatin in a diffuse CXCR4+ AML mouse model Evaluation of antitumor effects of statins

[0409] method

[0410] THP1 cells transfected with luciferase (THP-1-Luci; 1 × 10 6cells / 200μL) intravenous (IV) injection of NSG (NOD-scid IL2Rγ 缺失(null) ) female mice (5 weeks old). Mice were randomly divided into three different experimental groups. One group (VEH; n=3) used the vehicle of the nanoconjugate (buffer NaCO 3 H+NaCl pH=8) IV injection, the experimental group (T22-STM-AUR; n=2) was injected IV with 100 μg of T22-STM-H6-aurestatin, and the positive control group (T22-GFP-AUR; n =1) Injected IV with 100 μg of T22-GFP-H6-aurestatin. All compounds were administered daily for a total of 9 doses. The evolution of AML spread in mice was monitored 3 times a week using the IVIS Spectrum device until the day of euthanasia. Animal body weights were measured on the same day as the BLI assay. Euthanize all mice on the d...

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Abstract

The present invention relates to nanostructured conjugates, more specifically to nanostructured fusion proteins suitable for the selective delivery of their conjugated therapeutic agents to specific cell and tissue types. It also relates to nanoparticles comprising such nanostructured proteins and the therapeutic uses thereof.

Description

technical field [0001] The present invention relates to the field of nanostructured protein materials, and more particularly relates to fusion proteins carrying therapeutic agents that can be used for treatment. Background technique [0002] Systemic administration of drugs in the form of nanoconjugates benefits from enhanced drug stability when compared to free molecules. Valuable additional properties such as cell targeting can also be incorporated into a given hybrid composite by chemically incorporating functional groups in the nanoscale carrier, thereby benefiting from the high surface area / volume ratio of nanomaterials . When administered systemically, the resulting drug-loaded conjugates with sizes between about 8 and 100 nm escape renal filtration without aggregation in the lung or other highly vascularized organs. This fact, combined with appropriate physicochemical properties of the material, may lead to prolonged circulation times and drug exposure to target org...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/64A61K47/69
CPCA61K47/645A61K47/6929A61P35/02A61K47/6455A61P35/00A61K9/14A61K38/07A61K38/1774A61K38/1793A61K38/195A61K38/57
Inventor A·维拉维德科拉莱斯E·威克斯格梅兹U·尤祖艾塔艾洛尔扎R·玛古艾斯巴法伊M·V·塞斯佩德斯纳瓦罗I·卡萨诺瓦里加特H·洛佩茨拉古纳
Owner 巴塞罗那自治大学UAB
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