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Targeted functional molecule modified antibody complex

An antibody complex and functional molecule technology, applied in the field of pharmacy, can solve the problems of low response rate, ineffectiveness, and large molecular scale of brain tumor treatment, and achieve the effect of improving treatment effect, good application prospect, and expanding the scope of clinical application.

Pending Publication Date: 2021-04-13
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for brain tumors that also highly express PD-L1, the therapeutic effect of immune checkpoint antibodies is not optimistic. Clinical trial results show that the response rate of immune checkpoint antibodies to brain tumors is low. Atezolizumab (PD-L1 antibody) Treatment did not significantly improve survival
This is due to the existence of biological barriers (including blood-brain barrier and blood-brain tumor barrier) in brain tumors, and antibodies, as biomacromolecular drugs, have a large molecular scale and are difficult to cross biological barriers into brain tumor sites. Not working as expected when used in brain tumor treatment
In addition to brain tumors, other brain diseases such as neurodegenerative diseases also have biological barriers, which makes antibody drugs for related diseases ineffective because they cannot cross the barrier

Method used

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  • Targeted functional molecule modified antibody complex
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  • Targeted functional molecule modified antibody complex

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Synthesis and Characterization of Targeted Molecules

[0057] Synthesis and characterization of pHA targeting molecules: Polypeptides were prepared by solid-phase synthesis technology. First, weigh MBHA resin (substitution degree: 0.54mmol / g), add N,N-dimethylformamide for swelling, and then add 20% piperidine -DMF solution to remove the Fmoc protecting group on the resin, after washing, add 8.8 times the amount of Fmoc-amino acid (dissolved in the DMF solution of HBTU / HOBT / DIEA), place in an air shaker (37 ° C, 180rpm ) Shake for 45 minutes. After the reaction, wash with fresh DMF and drain the resin, add 20% piperidine-DMF solution to remove the Fmoc protecting group on the α-amino group. Repeat the above operation according to the sequence (pHA sequence: p-Hydroxybenzoic acid-SSC). After all the amino acids are condensed, the resin is washed and dried, and the peptide cleavage reagent (TFA / TIPS / H 2 O=95 / 2.5 / 2.5, v / v) After stirring for 2 h, the TFA was r...

Embodiment 2

[0060] Example 2 Preparation and Characterization of Targeting Functional Molecule-PDL1 Antibody Complex

[0061] Preparation of target functional molecule-PDL1 antibody complex:

[0062] Add Sulfo-SMCC (1 mg / mL) to the αPDL1 solution, react at room temperature for 30 min, and remove excess Sulfo-SMCC through a desalting column. Add 108 μL of PBS solution (2 mg / mL) of pHA, mix well, place at 4°C for 12 hours, then dialyze twice in a 14K Da dialysis bag with pure water (30 min each time), collect the liquid in the dialysis bag to obtain pHA modification PDL1 antibody complex (pHA-αPDL1). Prepare mRAP-αPDL1 and mCDX-αPDL1 in the same way.

[0063] MMP enzymatic hydrolysis verification:

[0064] Add 1 μL APMA (1M) to MMP-2 and dilute to 100 μL with the reaction solution, and incubate at 37°C for 1 hour. After activation, mix 50 μL of enzyme with 50 μL of mRAP-αPDL1 and mCDX-αPDL1, incubate at 37°C for 2 hours, and then dialyze.

[0065]Characterization of Targeting Functiona...

Embodiment 3

[0067] Example 3 Characterization of Functional Binding Activity of Targeting Functional Molecule-PDL1 Antibody Complex

[0068] ELISA binding activity:

[0069] Coat the 96-well ELISA plate with 100 μL / well of mouse PD-L1 protein, place it at room temperature for 2 hours, wash the plate twice, add BSA at room temperature to block for 1 hour, add pHA-αPDL1, mRAP-αPDL1, mCDX- αPDL1 or unmodified αPDL1 (600ng / mL, 400ng / mL, 200ng / mL, 100ng / mL, 50ng / mL, 25ng / mL, 10ng / mL, 5ng / mL, 2ng / mL), shake at 37°C After incubating on the bed for 1 h, after washing the plate, add anti-rat Ig(H+L) / HRP and incubate for 1 h, add 100 μL of chromogenic solution and leave at room temperature for 10 min, add 100 μL of 1M sulfuric acid to terminate the reaction, and measure the absorbance value with a microplate reader at a detection wavelength of 450 nm. see attached results Image 6 (A).

[0070] SPR binding activity:

[0071] Using surface plasmon resonance (SPR) technology, the binding activity...

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Abstract

The invention belongs to the technical field of pharmacy, and relates to a targeted functional molecule modified antibody complex. The complex is formed by connecting targeted functional molecules and antibodies in a certain mode and is used for improving the problem that when the antibodies are used for treating brain diseases such as brain tumors (blood-brain barriers and blood-brain tumor barriers), Alzheimer's disease and Parkinson's disease (blood-brain barriers), the curative effect of the antibodies is affected due to incapability of crossing related biological barriers; and a test result shows that the targeted functional molecule modified antibody complex does not influence the activity of the antibodies, can overcome the limitation of the biological barriers, promotes the antibodies to enter a focus part across the biological barriers, releases the antibodies under the microenvironment response of the focus part, remarkably improves the curative effect of the antibodies on the brain diseases, and has a good clinical application prospect.

Description

technical field [0001] The invention belongs to the technical field of pharmacy and relates to an antibody complex modified by targeting functional molecules. The complex utilizes targeting functional molecules to enable the antibody to cross the biological barrier when treating brain diseases, enhance its delivery to the lesion site, and release the antibody in the microenvironment of the lesion to achieve the purpose of enhancing the drug effect. Background technique [0002] With the rapid development of biomedical technology, biomacromolecular drugs are widely used in the prevention, treatment and diagnosis of diseases, and have good prospects for drug research and development. Antibody drugs, as a kind of biomacromolecular drugs, play an important role in the diagnosis and treatment of tumors, neurodegenerative diseases, autoimmune diseases and other diseases due to their advantages of high specificity, effectiveness and safety. For example, in the anti-tumor field, FD...

Claims

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Application Information

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IPC IPC(8): A61K47/64A61K47/66A61K39/395A61P25/16A61P25/28A61P35/00
CPCA61K47/64A61K47/665A61K39/3955A61K39/395A61P35/00A61P25/28A61P25/16A61K47/66
Inventor 陆伟跃郭海燕谢操
Owner FUDAN UNIV
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