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Sequencing method for car t cell therapy

A sequential and cell-based technology, applied in the direction of targeting specific cell fusion, chemical instruments and methods, biochemical equipment and methods, etc., can solve problems such as inability to prolong activation and expand CART cell populations

Pending Publication Date: 2020-12-18
ENDOCTYE INC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One problem is the inability to prolong activation and expand CAR T cell populations in vivo

Method used

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  • Sequencing method for car t cell therapy
  • Sequencing method for car t cell therapy
  • Sequencing method for car t cell therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0478] Synthesis of FITC-Folic Acid

[0479]

[0480] Folic acid-γ-ethylenediamine and fluorescein isothiocyanate (FITC) isomer I ( Sigma-Aldrich) coupling. The crude product was loaded onto an Xterra RP18 preparative HPLC column (Waters) and started with 99% 5 mM sodium phosphate (mobile phase A, pH 7.4) and 1% acetonitrile (mobile phase B) at 20 mL / min The flow rate reached 90% A and 10% B gradient condition elution within 10 min. Under these conditions, the main FITC-folate peak usually elutes at 27-50 min. The quality of the FITC-folate fraction was monitored by analytical reverse-phase HPLC with UV detector. Fractions with a purity greater than 98.0% (LCMS) were lyophilized to obtain the final FITC-folate product. Compounds with this structure are also known as EC17, as known in the art.

Embodiment 2

[0482] Synthesis of FITC-PEG12-folate

[0483]

[0484] Apply Universal Polyethylene Glycol (PEG) Nova Tag TM The resin (0.2 g) was loaded into a peptide synthesis vessel and washed with isopropanol ( i-PrOH) (3 × 10 mL) and dimethylformamide (DMF, 3 × 10 mL). 9-Fluorenylmethoxycarbonyl (Fmoc) deprotection was performed using 20% ​​piperidine in DMF (3 x 10 mL). A Kaiser test was performed to assess response progression. A DMF solution of Fmoc-L-glutamic acid 5-tert-butyl ester (Fmoc-Glu-(O-t-Bu)-OH) (23.5 mg), N,N-diisopropylethylamine ( i -Pr 2 NEt) (4 equiv) and benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP) (2 equiv). Fmoc deprotection was performed using 20% ​​piperidine in DMF (3 x 10 mL). Then introduce N into the container 10 -TFA-Pte-OH (22.5 mg), DMF, i -Pr 2 A solution of NEt (4 equiv) and PyBOP (2 equiv). Argon bubbled for 2 h, and with DMF (3 × 3 mL) and i -PrOH (3 x 3 mL) to wash the resin. After the resin was swollen i...

Embodiment 3

[0486] Synthesis of FITC-PEG20-folate

[0487]

[0488] Ethylenediamine, polymer bound (200-400 mesh) resin (50 mg) was charged into a peptide synthesis vessel and swelled with DCM (3 mL) followed by DMF (3 mL). Subsequent introduction of Fmoc-PEG in DMF to the container 20 -COOH solution (131 mg, 1.0 equiv), i -Pr 2 NEt (6.0 equiv) and PyBOP (4.0 equiv). Argon was bubbled for 6 h, the coupling solution was discharged, and the solution was washed with DMF (3 × 10 mL) and i -PrOH (3 x 10 mL) to wash the resin. A Kaiser test was performed to assess response progression. Fmoc deprotection was performed using 20% ​​piperidine in DMF (3 x 10 mL) prior to coupling of each amino acid. The above sequence was repeated to complete the reaction with Fmoc-Glu-OtBu (72 mg, 2.0 equiv) and the coupling step of Tfa.pteroic acid (41 mg, 1.2 equiv). The resin was washed with DMF 3 × 10 mL (5 min) containing 2% hydrazine to cleave the trifluoro-acetyl protecting group on pteroic acid, ...

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Abstract

The present disclosure relates to methods of treating a patient with a cancer by administering to the patient a composition comprising CAR T cells and administering to the patient a small molecule linked to a targeting moiety by a linker. The disclosure also relates to compositions for use in such methods.

Description

[0001] Cross References to Related Applications [0002] This application claims U.S. Provisional Patent Application No. 62 / 634,573, filed February 23, 2018, U.S. Provisional Patent Application No. 62 / 656,265, filed April 11, 2018, U.S. Provisional Patent Application No. 62 / 656,265, filed August 29, 2018 Application No. 62 / 724,345, and the priority of U.S. Provisional Patent Application No. 62 / 736,730, filed September 26, 2018, the disclosure of all of which is hereby expressly incorporated by reference in its entirety. technical field [0003] The present invention relates to a method of treating a cancer patient by administering to the patient a composition comprising CAR T cells and administering to the patient a small molecule linked to a targeting moiety via a linker. The invention also relates to a composition for use in such methods. Background technique [0004] Immunotherapy based on the adoptive transfer of lymphocytes (eg, T cells) into patients is a valuable the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00C07K16/44C07K14/705C12N15/86
CPCC07K16/44C12N2740/16043A61P35/00A61K39/001104A61K2039/5156A61K2039/5158A61K2039/545C07K14/7051C07K2317/622C07K2319/33C07K2319/03C07K2317/21C07K2317/92C07K2317/52C07K2317/53C07K2317/524C07K2317/41C07K2317/72A61K47/551A61K35/17C07K14/705
Inventor R·梅斯曼C·P·利蒙H·朱Y·J·卢P·S·罗M·C·詹森J·马泰N·R·C·平托J·R·帕克
Owner ENDOCTYE INC
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