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Single-domain antibodies against cd33 and constructs thereof

A single-domain antibody and construct technology, applied in the direction of antibodies, NGF-receptor/TNF-receptor superfamily, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problem of insufficient health , adverse clinical outcomes, inability to accept intensive chemotherapy, etc.

Pending Publication Date: 2021-04-16
NANJING LEGEND BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

AML is most common in older adults, some of whom are not healthy enough to receive intensive chemotherapy and thus have poor clinical outcomes
Although current therapies for AML often result in disease remission, almost all patients eventually relapse

Method used

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  • Single-domain antibodies against cd33 and constructs thereof
  • Single-domain antibodies against cd33 and constructs thereof
  • Single-domain antibodies against cd33 and constructs thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0601] Example 1: Generation of anti-CD33 sdAbs

[0602] Recombinant human CD33 protein and recombinant cynomolgus monkey CD33 protein having the corresponding amino acid sequences in Table 8 are commercially available.

[0603] Table 8. Extracellular domain of CD33 protein

[0604]

[0605] animal immunity

[0606] One camel was immunized with recombinant human CD33 protein in accordance with all current animal welfare regulations. For immunization, antigens are formulated as emulsions with CFA (prime immunization) or IFA (boost immunization). Antigen was administered intramuscularly in the neck by double-spot injection. Animals received 2 injections of emulsion containing 100 μg of CD33 protein at weekly intervals, and subsequently received 4 injections of 50 μg of CD33 protein. At various time points during the immunization, 10 ml blood samples were collected from the animals and serum was prepared. Conventional IgG (IgG1) and heavy chain antibodies (HCAb, IgG2 and ...

Embodiment 2

[0620] Example 2: Production and screening of anti-CD33 CAR-T

[0621] Cloning of anti-CD33 CAR constructs

[0622] Exemplary anti-CD33 CAR constructs were designed in the format shown in Table 3. The sequence of these CARs following this pattern is from N-terminus to C-terminus: leader sequence, target binding moiety (TBM, i.e. anti-CD33 sdAb), CD8α hinge, CD8a transmembrane (TM) region, cytoplasmic 4-1BB (CD137) molecule part, and the cytoplasmic part of the CD3ζ molecule.

[0623] DNA encoding each CAR construct was codon optimized and synthesized. The CAR sequence was ligated into a lentiviral vector plasmid with the human EF1α promoter for expression.

[0624] A construct encoding an anti-CD33 benchmark CAR ("BM CAR") was also prepared for comparative analysis using the sequence shown below.

[0625] SEQ ID NO: 212 (BM CAR):

[0626] MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGEPTYADKFQGRVTMTTDTSTSTAYMEIRNLGGDDTAVYYCARWSWSDGYYVYFDYWGQ...

Embodiment 3

[0661] Example 3: Generation and Evaluation of Additional CAR Constructs

[0662] Generation of CAR constructs

[0663] Use the cutting-edge anti-CD33 sdAb from Example 2 as the CD33 binding domain to construct additional CARs and CAR systems, such as Figures 10A-10E shown.

[0664] For example, if Figure 10A The shown "conventional CAR" with a monospecific extracellular domain and an intracellular signaling domain comprising an intracellular co-stimulatory sequence and a CD3ζ intracellular signaling sequence can respond and kill CD33 expressing cells. Such as Figure 10B The shown "tandem CAR" is constructed by fusing two binding domains that specifically recognize different targets via a peptide linker to form the extracellular domain in a single CAR molecule. Tandem CARs can respond to cells expressing either of two target molecules such as CD33, CLL1 and NKG2D ligands. Tandem CARs are expected to simultaneously bind target cells expressing both target molecules with...

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Abstract

The present application provides single-domain antibodies targeting CD33 and constructs thereof, including chimeric receptors, immune effector cell engagers and immunoconjugates. Further provided are engineered immune cells (such as T cells) comprising an anti-CD33 chimeric receptor and optionally a second chimeric receptor targeting a second antigen or epitope. Pharmaceutical compositions, kits and methods of treating cancer are also provided.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to International Patent Application PCT / CN2018 / 104882 filed on September 10, 2018 and International Patent Application PCT / CN2018 / 104883 filed on September 10, 2018, the contents of which are incorporated by reference in their entirety into this article. [0003] Submit a sequence listing as an ASCII text file [0004] The following submission as an ASCII text file is hereby incorporated by reference in its entirety: Computer Readable Form of the Sequence Listing (CRF) (File Name: 761422801000SEQLISTING.txt, Date of Record: September 7, 2019, Size: 211 KB). technical field [0005] The present invention relates to single domain antibodies, chimeric receptors and engineered immune cells targeting CD33, and methods of use thereof. Background technique [0006] With the development of tumor immunotherapy and clinical technology, chimeric antigen receptor T cell (CAR-T) immunotherapy has ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395A61P35/00
CPCA61P35/00C07K16/2803C07K16/24C07K2317/31C07K2317/24C07K2317/73C07K2317/569C07K2317/565C07K14/7051C07K2319/02C07K2319/03A61K2039/505C07K2317/22C07K16/2851C07K2317/33C07K2317/92C07K2319/33A61K39/464404A61K2239/29A61K39/4631C12N5/0636A61K39/464402A61K2239/31A61K2239/38A61K39/464424A61K2239/48A61K39/464411A61K39/464429A61K39/4611A61K35/17C07K14/70578C07K16/2866C12N2510/00
Inventor 张亚峰詹泰岚孙飞刘健张清武术
Owner NANJING LEGEND BIOTECH CO LTD
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