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Humanized antibody against CD19 and immune effector cells targeting to CD19

A technology of humanized antibodies and antibodies, applied to genetically modified cells, cells modified by introducing foreign genetic material, animal cells, etc., can solve the problems of antibody affinity and specificity reduction

Active Publication Date: 2018-10-02
CARSGEN THERAPEUTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In particular, after antibody humanization, due to the change of amino acid sequence, the peptide chain size, charge, hydrophobicity, spatial conformation, etc. are usually changed, and the formation of hydrogen bonds is also different from that of the mouse source, thus affecting the complementarity determining region of the antibody ( CDR) conformation, therefore, the affinity and specificity of the humanized antibody are usually reduced by more than 10 times compared with the mouse antibody (Vahideh Ahmadzadeh et al., Monoclonal antibodies in immunodiagnosis and immunotherapy, volume 33, number 2, 2014)

Method used

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  • Humanized antibody against CD19 and immune effector cells targeting to CD19
  • Humanized antibody against CD19 and immune effector cells targeting to CD19
  • Humanized antibody against CD19 and immune effector cells targeting to CD19

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0143] Example 1. Preparation of humanized antibody huHD37 of anti-CD19 antibody HD37

[0144] In this embodiment, mouse anti-HD37 (J Immunol.1987May 1; 138(9):2793-9) is used as the parental antibody. Mouse anti-HD37 has the light chain variable region shown in SEQ ID NO: 19 and SEQ ID NO: The heavy chain variable region shown in 20 combined the nomenclature of Kabat, Chothia and IMGT three antibody CDR regions, and determined the six CDR region sequences of the antibody light chain and heavy chain:

[0145] Light chain variable region (SEQ ID NO: 19), CDR regions are underlined.

[0146] DIQLTQSPASLAVSLGQRATISC KASQSVDYDGDSYLN WYQQIPGQPPKLLIY DASN LVS GIPPRFSGSGSGTDFTLNIHPVEKVDAATYHC QQSTEDPWT FGGGTKLEIKR

[0147] Heavy chain variable region (SEQ ID NO: 20) CDR regions are underlined

[0148] QVQLQQSGAELVRPGSSVKISCKASGYAFS SYWMN WVKQRPGQGLEWIG QIWPGDGDTNYNGKFKG KATLTADESSSTAYMQLSSLASEDSAVYFCAR RETTTVGRYYYAM DYWGQGTTVTVSS

[0149] a. Selection of Antibody Templ...

Embodiment 2

[0165] The transformation of embodiment 2.huHD37

[0166] In this example, huHD37 was used as the parental antibody, and huHD37 was transformed by the method of phage display. The construction of the phage library based on the humanized antibody huHD37 retained the CDR3 region of the light chain and the heavy chain. Two phage libraries were constructed by randomizing the CDR1 and CDR2 of the light chain or the CDR1 and CDR2 of the heavy chain respectively through degenerate primers. Primer information is shown in the table below.

[0167] No.

name

sequence

Length

1

LMF

CAGGAAACAGCTATGACCATGATTAC

26

2

C37H1R

CACTCCAGGCCCTGGCCGGGGGCCTGCCGCACCCAMNNMNNMNNMNNMNNMNNGAAGGTGTAGCCGCTGGCCT

73

3

C37H2F

ccggccagggcctggagtggatgggcNNKATCNNKCCNNKNNKGGCNNKACCNNKtacaacggcaagttcaagggc

77

4

Fd

GACGTTAGTAAATGAATTTTCTGTATGAGG

30

5

C37L1R

CTGGCCGGGCTTCTGCTGGTACCAMNNMNNNGTAMNNMNNMNNMNNMNNMNNMNNGCTM...

Embodiment 3

[0175] Example 3. Construction of anti-CD19 chimeric antigen receptor plasmid (CAR)

[0176] 3.1 Construction of humanized antibody chimeric antigen receptor plasmid (CAR)

[0177] Using PRRLSIN-cPPT.EF-1α as the vector, the lentiviral plasmids expressing the second and fourth generation chimeric antigen receptors of humanized antibody huHD37 were constructed, including PRRLSIN-cPPT.EF-1α-huHD37-28Z, PRRLSIN- cPPT.EF-1α-huHD37-BBZ, PRRLSIN-cPPT.EF-1α-huHD37-28Z&IFNb and PRRLSIN-cPPT.EF-1α-huHD37-BBZ&IFNb ( Figure 10 ). The huHD37-28Z sequence consists of CD8α signal peptide (SEQ ID NO: 23), huHD37scFV, CD8hinge (SEQ ID NO: 25), CD28 transmembrane domain (SEQ ID NO: 27) and intracellular signaling domain (SEQ ID NO: 29 ) and the intracellular segment CD3ξ (SEQ ID NO: 31) of CD3; the huHD37-BBZ sequence consists of CD8α signal peptide (SEQ ID NO: 23), huHD37scFV, CD8hinge (SEQ ID NO: 25) and transmembrane domain (SEQ ID NO: 33), CD137 intracellular signaling domain (SEQ ID N...

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Abstract

The invention relates to an anti-CD19 humanized antibody prepared from a murine monoclonal antibody, a chimeric antigen receptor containing the humanized antibody, and immune cells expressing the humanized antibody. The humanized antibody of the invention does not produce an anti-antibody reaction (AAR) and a human anti-mouse antibody reaction (HAMA), has better affinity than a murine antibody, and presents excellent activity and safety, so a novel means is provided for treating tumors expressing CD19.

Description

technical field [0001] The invention belongs to the field of tumor immunotherapy or diagnosis. More specifically, the present invention relates to humanized antibodies against CD19 and immune effector cells targeting CD19. Background technique [0002] B cells include pre-B cells (pre-B cells), early-developed B cells (ie: immature B cells), and mature B cells. Mature B cells differentiate into plasma cells and malignant B cells through terminal differentiation. Most pre-B acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, B-cell chronic lymphocytic leukemia (CLL), pro-lymphocytic leukemia, hairy cell leukemia, common acute lymphoblastic leukemia CD19 is highly expressed in some non-acute lymphoblastic leukemias (Nadler et al., J. Immunol., 131:244-250 (1983); Loken et al., Blood, 70:1316-1324 (1987)). The expression of CD19 on plasma cells further suggests that it can be expressed on different B-cell tumors such as multiple myeloma, plasmacytoma, Wahrenheitoma (G...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K19/00C12N15/13C12N5/10C12N15/62C12N15/867C12N7/01A61P35/00G01N33/68G01N33/574
CPCG01N33/574G01N33/6872A61K35/17C12N7/00C12N15/86C07K14/7051C07K16/2803C12N2740/15021C12N2740/15043C07K2319/33C07K2317/92C07K2317/73C07K2317/565C07K2317/24A61P35/00C07K2317/622C07K2319/30C12N2510/00C12N5/0636A61K38/00C07K14/70517C07K14/70521C07K14/70578C07K16/2809C07K2317/53C07K2317/567C07K2317/76C07K2319/02C07K2319/03
Inventor 王鹏高慧萍石志敏李宗海
Owner CARSGEN THERAPEUTICS
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