Humanized anti-cd22 recombinant immunotoxin and application thereof

Abstract

Provided is a humanized anti-CD22 recombinant immunotoxin, which has reduced immunogenicity and maintains the CD22 binding affinity and corresponding biological activity before humanization transformation, and is used for preparing a drug for treating CD22-related B cell malignant tumors.

1 . A humanized anti-CD22 recombinant immunotoxin which is a polypeptide molecule comprising two polypeptide chains as follows:
 (1) a first polypeptide chain, comprising a light chain variable region (VL) of an anti-CD22 antibody, wherein the light chain variable region (VL) comprises an amino acid sequence as shown in SEQ ID NO. 16 or SEQ ID NO. 18 or an amino acid sequence homologue thereof having at least 75% sequence identity to the amino acid sequence as shown in SEQ ID NO. 16 or SEQ ID NO. 18;   (2) a second polypeptide chain, comprising a heavy chain variable region (V H ) of an anti-CD22 antibody and a cytotoxin directly or indirectly linked to the heavy chain variable region (V H ), wherein the heavy chain variable region (V H ) comprises an amino acid sequence as shown in SEQ ID NO. 3 or SEQ ID NO. 4 or an amino acid sequence homologue thereof having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 3 or SEQ ID NO. 4.   
     
     
         2 . The humanized anti-CD22 recombinant immunotoxin according to  claim 1 , wherein the amino acid sequence homologue in the first polypeptide chain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as shown in SEQ ID NO. 16 or SEQ ID NO. 18; preferably, the amino acid sequence homologue comprises the amino acid sequences as shown in SEQ ID NO. 31, SEQ ID NO. 32 and SEQ ID NO. 33, and has the same amino acid residues as those in SEQ ID NO. 16 or SEQ ID NO. 18 at positions 3, 5, 36, 37, 47, 48, 49, 50, 65, 67, 69, 70 and 72 corresponding to the amino acid sequence as shown in SEQ ID NO. 14, but is not the amino acid sequence as shown in SEQ ID NO. 14; preferably, the amino acid sequence homologue in the second polypeptide chain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as shown in SEQ ID NO. 3 or SEQ ID NO. 4; preferably, the amino acid sequence homologue comprises the amino acid sequences as shown in SEQ ID NO. 28, SEQ ID NO. 29 and SEQ ID NO. 30, and has the same amino acid residues as those in SEQ ID NO. 3 or SEQ ID NO. 4 at positions 3, 48, 49, 50, 69, 71, 73, 75, and 80 corresponding to the amino acid sequence as shown in SEQ ID NO. 2, but is not the amino acid sequence as shown in SEQ ID NO. 2. 
     
     
         3 . The humanized anti-CD22 recombinant immunotoxin according to  claim 1 , wherein the light chain variable region (V L ) in the first polypeptide chain comprises the amino acid sequence as shown in SEQ ID NO. 16 or the amino acid sequence homologue thereof; and the heavy chain variable region (V H ) in the second polypeptide chain comprises the amino acid sequence as shown in SEQ ID NO. 3 or the amino acid sequence homologue thereof; or
 the light chain variable region (V L ) in the first polypeptide chain comprises the amino acid sequence as shown in SEQ ID NO. 18 or the amino acid sequence homologue thereof;   and the heavy chain variable region (VH) in the second polypeptide chain comprises the amino acid sequence as shown in SEQ ID NO. 3 or the amino acid sequence homologue thereof; or   the light chain variable region (V L ) in the first polypeptide chain comprises the amino acid sequence as shown in SEQ ID NO. 16 or the amino acid sequence homologue thereof; and the heavy chain variable region (V H ) in the second polypeptide chain comprises the amino acid sequence as shown in SEQ ID NO. 4 or the amino acid sequence homologue thereof;   preferably, in the recombinant immunotoxin, the light chain variable region (V L ) in the first polypeptide chain and the heavy chain variable region (V H ) in the second polypeptide chain are covalently linked, e.g., via a disulfide bond;   preferably, the cytotoxin in the second polypeptide chain is  pseudomonas  exotoxin A or a mutant or fragment of the  pseudomonas  exotoxin A with cytotoxicity retained; preferably, the mutant or fragment of the  pseudomonas  exotoxin A is PE40, PE38, PE35, PE24, mPE24, T19, T20, or M11.   
     
     
         4 . The humanized anti-CD22 recombinant immunotoxin according to  claim 1 , wherein in the second polypeptide chain, the cytotoxin is fused to the C-terminus of the heavy chain variable region (V H ) of the anti-CD22 antibody;
 preferably, the N-terminus of the cytotoxin is fused, directly or via a linker, to the C-terminus of the heavy chain variable region (V H );   preferably, the first polypeptide chain further comprises a kappa light chain constant region, preferably a human kappa light chain constant region fused to the C-terminus of the light chain variable region (V L ); alternatively, the second polypeptide chain further comprises a heavy chain constant region CH1, preferably a human heavy chain constant region CH1 fused to the C-terminus of the heavy chain variable region (V H ).   
     
     
         5 . The humanized anti-CD22 recombinant immunotoxin according to  claim 1 , wherein the first polypeptide chain comprises the amino acid sequence as shown in SEQ ID NO. 16 or the amino acid sequence homologue thereof, and the second polypeptide chain comprises the amino acid sequence as shown in SEQ ID NO. 9 or the amino acid sequence homolog thereof; or
 the first polypeptide chain comprises the amino acid sequence as shown in SEQ ID NO. 18 or the amino acid sequence homologue thereof, and the second polypeptide chain comprises the amino acid sequence as shown in SEQ ID NO. 9 or the amino acid sequence homologue thereof; or   the first polypeptide chain comprises the amino acid sequence as shown in SEQ ID NO. 16 or the amino acid sequence homologue thereof, and the second polypeptide chain comprises the amino acid sequence as shown in SEQ ID NO. 10 or the amino acid sequence homologue thereof.   
     
     
         6 . A nucleic acid molecule comprising a nucleotide sequence encoding the first polypeptide chain in the humanized anti-CD22 recombinant immunotoxin according to  claim 1  and/or a nucleotide sequence encoding the second polypeptide chain in the humanized anti-CD22 recombinant immunotoxin. 
     
     
         7 . A vector comprising the nucleic acid molecule according to  claim 6 . 
     
     
         8 . A host cell transformed or transfected with the nucleic acid molecule according to  claim 6  or a vector comprising the nucleic acid molecule. 
     
     
         9 . A composition comprising the humanized anti-CD22 recombinant immunotoxin according to  claim 1 . 
     
     
         10 . (canceled) 
     
     
         11 . A method for treating a CD22-related B-cell malignant tumor, comprising administering to a subject in need thereof the humanized anti-CD22 recombinant immunotoxin according to  claim 1  or a composition comprising the humanized anti-CD22 recombinant immunotoxin. 
     
     
         12 . A humanized anti-CD22 antibody or antibody fragment, the antibody or antibody fragment comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein the heavy chin variable region (V H ) comprises a amino acid sequence as shown in SEQ ID NO. 3 or SEQ ID NO. 4 or an amino acid sequence homologue thereof having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 3 or SEQ ID NO. 4, and the light chain variable region (V L ) comprises an amino acid sequence as shown in SEQ ID NO. 16 or SEQ ID NO. 18 or an amino acid sequence homologue thereof having at least 75% identity to the amino acid sequence as shown in SEQ ID NO. 16 or SEQ ID NO. 18. 
     
     
         13 . A method for treating a CD22-related B-cell malignant tumor, comprising administering to a subject in need thereof one of:
 the nucleic acid molecule according to  claim 6 ; or   a vector comprising the nucleic acid molecule; or   a host cell transformed or transfected with the nucleic acid molecule or the vector.   
     
     
         14 . The method for treating a CD22-related B-cell malignant tumor according to  claim 11 , wherein the CD22-related B-cell malignant tumor is a lymphoma or leukemia with high CD22 expression. 
     
     
         15 . The method for treating a CD22-related B-cell malignant tumor according to  claim 14 , wherein the lymphoma is a non-Hodgkin's lymphoma, a small lymphocytic lymphoma, or a mantle cell lymphoma; and the leukemia is a chronic lymphocytic leukemia, a hairy cell leukemia, or an acute lymphocytic leukemia. 
     
     
         16 . The method for treating a CD22-related B-cell malignant tumor according to  claim 11 , wherein the subject is a mammal. 
     
     
         17 . The method for treating a CD22-related B-cell malignant tumor according to  claim 16 , wherein the mammal is a human or non-human primate, a dog, a cat, a cow, a pig, a sheep, a horse, a murine, or a rabbit. 
     
     
         18 . The method for treating a CD22-related B-cell malignant tumor according to  claim 13 , wherein the CD22-related B-cell malignant tumor is a lymphoma or leukemia with high CD22 expression. 
     
     
         19 . The method for treating a CD22-related B-cell malignant tumor according to  claim 18 , wherein the lymphoma is a non-Hodgkin's lymphoma, a small lymphocytic lymphoma, or a mantle cell lymphoma; and the leukemia is a chronic lymphocytic leukemia, a hairy cell leukemia, or an acute lymphocytic leukemia. 
     
     
         20 . The method for treating a CD22-related B-cell malignant tumor according to  claim 13 , wherein the subject is a mammal. 
     
     
         21 . The method for treating a CD22-related B-cell malignant tumor according to  claim 20 , wherein the mammal is a human or non-human primate, a dog, a cat, a cow, a pig, a sheep, a horse, a murine, or a rabbit.