Ror1 car t-cells

A sequence and amino acid technology, applied in the field of chimeric antigen receptors, can solve problems such as limited tumor scope

Pending Publication Date: 2020-04-10
UCL BUSINESS PLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, most CAR-T cells target disease-specific antigens and thus have a limited range of tumors, whereas ROR1 is able to target a broad range of hematological and solid malignancies

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0230] Materials and methods

[0231] molecular biology

[0232] DNA cloning was performed using standard laboratory protocols. Restriction enzymes, Phusion Taq polymerase, Quick ligase and DH5α competent cells were obtained from New England Biolabs. DNA was run on a 1-2% TBE agarose gel according to the expected band size and stained with ethidium bromide (Sigma-Aldrich), SybrSafe gel stain (Life Technologies) or nucleic acid gel stain (Lonza) . Gel extraction and PCR purification were performed using the SV Wizard Gel and Purification Kit (Promega).

[0233] Bacteria were grown in Lennox Broth or Terrific Broth (Fisher Scientific); minipreps, midipreps and megapreps plasmid purification kits were from Mancherey Nagel, while large preps used Qiagen kits according to the manufacturer's instructions. DNA concentration was assessed by Nanodrop (Thermo Scientific) and sequenced by SourceBioscience or Beckman Coulter Genomics. Oligonucleotide primers and G-Block oligonucleoti...

Embodiment 2

[0257] ROR1 is expressed on primary CLL cells

[0258] Primary CLL cells from peripheral blood of newly diagnosed and treated patients were analyzed for ROR1 expression by flow cytometry. ROR1 was detectable in all samples analyzed (Biolegend Clone 2A2). Antigen density was assessed by Qifikit (Dako, Agilent Technologies). ROR1 had a median antigen binding capacity (ABC) of 2304 molecules / cell, with a range of 800-4828, and CD19 was expressed at a higher level with a median of 12583 (range, 5894-23652). See Table 1 below.

[0259]

Embodiment 3

[0261] Rat immunization and ROR1 antibody production

[0262] After immunizing 3 rats with full-length human ROR1 protein and DNA, we obtained 38 oligoclonal hybridomas. Seventeen of these were solved using 5' RACE to identify the variable regions of the heavy and light chains. We obtained sequences encoding 13 new antibodies (4 clones resulted in identical sequences), 10 of which combined in single-chain variable fragment (ScFv) format. (Cloning of hybridoma cDNA by RACE, Andrew Bradbury) See Table 2 below.

[0263] Table 2:

[0264]

[0265] As can be seen above, clone F binds the coiled domain. All other clones (except V) incorporated immunoglobulin domains. Prior art antibodies R12 and 4A5 also bind immunoglobulin domains. Thus, clone F showed different and differentiated binding properties compared to prior art antibodies R12 and 4A5.

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Abstract

There is described a chimeric antigen receptor (CAR) which comprises an antigen binding domain which selectively binds to Receptor Tyrosine Kinase Like Orphan Receptor (ROR1), and its use. Also described is a T cell comprising the CAR and its use in the treatment of cancer.

Description

[0001] The present invention relates to a chimeric antigen receptor (chimeric antigen receptor, CAR) and its use, said chimeric antigen receptor comprising and receptor tyrosine kinase-like orphan receptor 1 (Receptor Tyrosine Kinase Like Orphan Receptor 1, ROR1 ) selectively binds to an antigen-binding domain. Background technique [0002] Receptor tyrosine kinase-like orphan receptor 1 (ROR1) (also known as Neurotrophic Tyrosine Kinase, Receptor-Related 1, NTRKR1) is expressed during embryogenesis Carcinoembryonic antigen, but with limited expression in normal adult tissues. However, it is expressed in many hematological and solid malignancies: Chronic Lymphocytic Leukaemia (CLL), Acute Lymphoblastic Leukaemia (ALL), Mantle Cell Leukemia, Hairy Cell Leukemia, Pancreatic Cancer, Prostate Cancer , colon cancer, bladder cancer, ovarian cancer, malignant glioma, testicular cancer, uterine cancer, adrenal cancer, breast cancer, lung cancer, melanoma, neuroblastoma, sarcoma, kidn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/725A61K35/17C07K14/705C07K16/28C07K16/30C12N5/0783C12N15/62A61K39/00A61P35/02
CPCA61K35/17C07K14/7051C07K14/70517C07K16/2803C07K16/30C12N5/0636C12N15/62C07K2317/24C07K2317/53C07K2317/622C07K2317/73A61P35/02C07K2317/33C07K2317/34C07K2319/03C07K2319/33C12N2510/00C07K14/70521C07K14/70578
Inventor 阿米特·纳什瓦尼萨蒂恩·戈希尔马可·德拉佩鲁塔
Owner UCL BUSINESS PLC
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