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Fibronectin binding domain chimeric antigen receptors and methods of use thereof

A chimeric antigen receptor, fibronectin technology, used in immunoglobulins, antibody medical components, peptide/protein components, etc., can solve problems such as poor, incorrect folding, instability and flexibility

Pending Publication Date: 2020-04-24
PROTELICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, due to the instability and flexibility of the scFv structure, the possibility of VH / VL domain exchange and multimer formation through framework region interactions, incorrect folding problems, and problems caused by aggregation, the use of scFv Associated with abnormal signaling
As such, current CAR-T strategies suffer from issues such as severe patient side effects and poor durability of response

Method used

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  • Fibronectin binding domain chimeric antigen receptors and methods of use thereof
  • Fibronectin binding domain chimeric antigen receptors and methods of use thereof
  • Fibronectin binding domain chimeric antigen receptors and methods of use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0318] Example 1: Identification of BCMA-specific FN3 domain polypeptides

[0319] Two FN3 domain libraries totaling 25 billion variants were expressed on the surface of M13 phage and stored as phage stocks in IX PBS at 4°C. Each library stock has a titer of 1.5x10 13 cfu / ml.

[0320] The target protein BCMA-Fc chimera (R&D Systems) was bound to Protein A Dynabeads (Invitrogen) at the following concentrations: round 1: 500 nM; round 2: 200 nM; round 3: 40 nM. IgG Fc protein (R&D Systems) was bound to Protein A beads at 500 nM for all rounds as a subtractive screen. Each library was processed as a separate reaction, thus three selections were performed. The supernatant containing Fc-cleared conjugates was transferred to BCMA protein A beads. For round 1, add 1x10 for each library 13 cfu / ml of phage, and for each subsequent round, use 1x10 12 cfu / ml of phage. Beads were washed 6 times and BCMA-bound FN3 domain phage particles were eluted with low pH buffer. Phage were...

Embodiment 2

[0327] Example 2: Cloning of Chimeric Antigen Receptors Specific for Cancer Antigens

[0328] A combinatorial FN3 domain library of natural variants was generated as described in US Patent No. 8,680,019. Libraries of fibronectin binding domains will be constructed in phagemid vectors. Phage will be incubated with biotinylated cancer antigens (eg VEGF(121)) pre-bound to streptavidin-coated magnetic beads in PBS-2.5% non-fat dry milk for 1 hour at room temperature. After washing with PBS-0.1% Tween-20, bound phages were eluted with 0.1N HCl and propagated in TG1 cells for the next round of selection. In round 1, a VEGF concentration of 1 [mu]M was used and was reduced 5-fold each round so that the final round of selection contained 2OnM VEGF. To measure relative enrichment, phage prepared from non-displaying control phagemids (pBC), which confer resistance to chloramphenicol, were also included in the selection reaction. In each round, library phage are expected to be enriche...

Embodiment 3

[0333] Example 3: Bifunctional FN3 domain polypeptide CAR-T

[0334] A bispecific CAR-T comprising a FN3 domain polypeptide can be constructed that expresses: i. a FN3 domain polypeptide that binds a tumor antigen, ii. a FN3 domain polypeptide that binds a T cell surface receptor, and iii. a linker. Potential tumor antigen targets are BCMA and CD-19. BCMA is involved in blood cancers, such as multiple myeloma and leukemia, and may contribute to cell survival and cell proliferation. CD19 is found on B cell cancers. Two T cell receptors that can be targeted with FN3 domain polypeptides are PD-1 and CTLA-4. Both receptors are expressed on activated T cells and transmit both inhibitory and co-stimulatory signals.

[0335] Another example of a bispecific CAR-T that includes an FN3 domain polypeptide can be constructed, including: i. a FN3 domain polypeptide that binds to AXL, ii. a FN3 domain polypeptide that binds to a T cell surface receptor or a T cell surface receptor A bod...

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Abstract

Provided herein are chimeric antigen receptors (CARs) for binding with a target antigen, comprising at least one antigen specific targeting region comprising a fibronectin type 3 (FN3) domain polypeptide. Provided herein are multispecific chimeric antigen receptors for binding with two or more target antigens, comprising at least two antigen specific targeting regions comprising a fibronectin type3 domain polypeptide. Also provided herein are compositions and methods of treatment relating to the use of subject CARs of the invention.

Description

[0001] related application [0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 521,095, filed June 16, 2017, the contents of which are incorporated herein by reference. Background technique [0003] Scaffold-based binding proteins are legitimate alternatives to antibodies in terms of their ability to bind specific ligand targets. Scaffold-based binding proteins include those derived from the intact human fibronectin type III (FN3) domain. The FN3 domain was first identified as one of the repeat domains in the fibronectin protein. The FN3 domain constitutes a small (-94 amino acid) monomeric β-sandwich protein consisting of 7 β-strands with three connecting loops. The three loops near the N-terminus of FN3 are functionally similar to the complementarity-determining regions of immunoglobulin domains. Scaffold-based binding proteins derived from intact human FN3 domains are small (1 / 15 the size of conventional monoclonal antibodies),...

Claims

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Application Information

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IPC IPC(8): A61K39/00C07K14/00C07K14/78C07K16/18C07K19/00C12N15/62
CPCC07K14/7051C07K14/78C07K2319/03C07K2319/74C07K14/70517C07K14/70521C07K14/70578C07K2319/00C07K2319/02C07K2319/21C07K2319/70C07K2318/20C07K2319/30C07K2319/33A61K38/00C07K2317/31
Inventor R·科雷尹俊C·A·霍坎森E·扎克
Owner PROTELICA
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