Anti-cd19 humanized antibody and immune effector cell targeting cd19

Preparation of Humanized Antibody huHD37 of Antibody HD37 Against CD19

[0142]In the present example, murine antibody HD37 (J Immunol. 1987 May 1; 138(9): 2793-9) was used as a parent antibody, and murine antibody HD37 has the light chain variable region as shown in SEQ ID NO: 19 and heavy chain variable region as shown in SEQ ID NO: 20. 6 CDR region sequences of the antibody light and heavy chains were determined by combining 3 naming schemes, Kabat, Chothia and IMGT for antibody CDR regions:

[0143]the light chain variable region (SEQ ID NO: 19), wherein the CDR regions are underlined.

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLE

IKR

[0144]the heavy chain variable region (SEQ ID NO: 20), wherein the CDR regions are underlined

QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS

[0145]a. Selection of Antibody Templates

[0146]germline sequence IGHV1-69*01 (S...

Modification of huHD37

[0161]In the present example, huHD37 was used as a a parent antibody, and huHD37 was modified by phage display method. In the construction of a phage library based on the humanized antibody huHD37, the CDR3 regions of the light chain and heavy chain were retained, and two phage libraries were constructed by using degenerate primers and randomizing CDR1 and CDR2 of the light chain or CDR1 and CDR2 of the heavy chain, respectively. Primer information is shown in the table below.

No.NameSequenceLength1LMFCAGGAAACAGCTATGACCATGATTAC262C37H1RCACTCCAGGCCCTGGCCGGGGGCCTGCCGC73ACCCAMNNMNNMNNMNNMNNMNNGAAGGTGTAGCCGCTGGCCT3C37H2FccggccagggcctggagtggatgggcNNKA77TCNNKCCCNNKNNKGGCNNKACCNNKtacaacggcaagttcaagggc4FdRGACGTTAGTAAATGAATTTTCTGTATGAGG305C37L1RCTGGCCGGGCTTCTGCTGGTACCAMNNMNN80GTAMNNMNNMNNMNNMNNMNNMNNGCTMNNGCTGGCCTTGCAGG6C37L2Faccagcagaagcccggccagccccccaagc80tgctgatctacNNKNNKAGCNNKCTGNNKagcggcgtgcccgcccggttc

[0162]2.1 Construction of huHD37 Mutant:

[0163]The template plasmi...

Construction of Anti-CD19 Chimeric Antigen Receptor Plasmid (CAR)

[0169]3.1 Construction of Humanized Antibody Chimeric Antigen Receptor Plasmid (CAR)

[0170]Lentiviral plasmids expressing the second and fourth generation of chimeric antigen receptors of humanized antibody huHD37 were constructed using PRRLSIN-cPPT.EF-1α as a vector, 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 (FIG. 10). The huHD37-28Z sequence consists of CD8α signal peptide (SEQ ID NO: 23), huHD37 scFV, CD8 hinge (SEQ ID NO: 25), CD28 transmembrane domain (SEQ ID NO: 27), intracellular signaling domain (SEQ ID NO: :29) and intracellular domain CD3ξ of CD3 (SEQ ID NO: 31); the huHD37-BBZ sequence consists of CD8α signal peptide (SEQ ID NO: 23), huHD37scFV, CD8 hinge (SEQ ID NO: 25), transmembrane domain (SEQ ID NO: 33), CD137 intracellular signaling domain (SEQ ID NO: 35) and CD3 (SEQ ID NO: 31); huHD37-28BBZ sequence...