Tumor-specific anti-egfr antibody and application thereof

Abstract

The present invention provides a tumor-specific anti-EGFR antibody and application thereof. The antibody can be used for preparing targeted antitumor drugs and tumor diagnosis drugs.

1 - 36 . (canceled) 
     
     
         37 . A multi-functional immunoconjugate, comprising:
 an antibody; and   a functional molecule linked thereto; and the functional molecule is selected from a group consisting of a molecule that targets a tumor surface marker, a tumor-suppressing molecule, a molecule that targets a surface marker of an immune cell, a detectable label;   wherein the antibody specifically recognizing EGFRvIII expressed or EGFR overexpressed by tumor cells, wherein the antibody comprises a light chain variable region and a heavy chain variable region, and   CDR1 of the light chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 55;   CDR2 of the light chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 42, SEQ ID NO: 53;   CDR3 of the light chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 43, SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57;   CDR1 of the heavy chain variable region has the amino acid sequence of SEQ ID NO: 44;   CDR2 of the heavy chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52;   CDR3 of the heavy chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 46, SEQ ID NO: 50.   
     
     
         38 . The multi-functional immunoconjugate of  claim 37 , wherein the molecule that targets the tumor surface marker is an antibody or ligand that binds to a tumor surface marker; or
 the tumor-suppressing molecule is an anti-tumor cytokine or an anti-tumor toxin; preferably, the cytokines include but are not limited to: IL-12, IL-15, IFN-beta, TNF-alpha.   
     
     
         39 . The multi-functional immunoconjugate of  claim 37 , wherein the detectable label includes a fluorescent label and a chromogenic label. 
     
     
         40 . The multi-functional immunoconjugate of  claim 37 , wherein the molecule targeting the surface marker of the immune cell is an antibody or ligand that binds to an immune cell surface marker; preferably, the immune cell surface markers include: CD3, CD16, CD28. 
     
     
         41 . The multi-functional immunoconjugate of  claim 40 , wherein the molecule that targets the surface marker of the immune cell is an antibody that binds to a T cell surface marker, which forms a T-cell-engaging bifunctional antibody with the following antibody:
 an antibody specifically recognizing EGFRvIII expressed or EGFR overexpressed by tumor cells, wherein the antibody comprises a light chain variable region and a heavy chain variable region, and   CDR1 of the light chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 55;   CDR2 of the light chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 42, SEQ ID NO: 53;   CDR3 of the light chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 43, SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57;   CDR1 of the heavy chain variable region has the amino acid sequence of SEQ ID NO: 44;   CDR2 of the heavy chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52;   CDR3 of the heavy chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 46, SEQ ID NO: 50.   
     
     
         42 . The multi-functional immunoconjugate of  claim 41 , wherein the antibody that binds to the immune cell surface marker is an anti-CD3 antibody. 
     
     
         43 . The multi-functional immunoconjugate of  claim 37 , wherein the multi-functional immunoconjugate is a fusion polypeptide, and further comprises a linker peptide between the following antibody and the functional molecule linked thereto:
 an antibody specifically recognizing EGFRvIII expressed or EGFR overexpressed by tumor cells, wherein the antibody comprises a light chain variable region and a heavy chain variable region, and   CDR1 of the light chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 55;   CDR2 of the light chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 42, SEQ ID NO: 53;   CDR3 of the light chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 43, SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57;   CDR1 of the heavy chain variable region has the amino acid sequence of SEQ ID NO: 44;   CDR2 of the heavy chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52;   CDR3 of the heavy chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 46, SEQ ID NO: 50.   
     
     
         44 . A chimeric antigen receptor expressed on the surface of an immune cell comprising an antibody, wherein the chimeric antigen receptor comprises: the antibody, a transmembrane region and an intracellular signal region, which are sequentially linked;
 wherein the antibody specifically recognizing EGFRvIII expressed or EGFR overexpressed by tumor cells, wherein the antibody comprises a light chain variable region and a heavy chain variable region, and   CDR1 of the light chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 55;   CDR2 of the light chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 42, SEQ ID NO: 53;   CDR3 of the light chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 43, SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57;   CDR1 of the heavy chain variable region has the amino acid sequence of SEQ ID NO: 44;   CDR2 of the heavy chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52;   CDR3 of the heavy chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 46, SEQ ID NO: 50.   
     
     
         45 . The chimeric antigen receptor of  claim 44 , wherein the intracellular signal region is selected from a group consisting of intracellular signal region sequences of CD3ζ, FcεRIγ, CD27, CD28, CD137 and CD134, or a combination thereof. 
     
     
         46 . The chimeric antigen receptor of  claim 45 , wherein the transmembrane region comprises a transmembrane region of CD8 or CD28. 
     
     
         47 . The chimeric antigen receptor of  claim 45 , wherein the immune cells include T lymphocyte, NK cell or NKT cell. 
     
     
         48 . The chimeric antigen receptor of  claim 45 , wherein the chimeric antigen receptor comprises an antibody, transmembrane region and intracellular signal region which are sequentially linked as follows:
 Antibody, CD8 and CD3ζ;   Antibody, CD8, CD137 and CD3ζ;   Antibody, a transmembrane region of a CD28 molecule, an intracellular signal region of a CD28 molecule, and CD3ζ; or   Antibody, a transmembrane region of a CD28 molecule, an intracellular signal region of a CD28 molecule, CD137 and CD3ζ;   Wherein the antibody is an antibody specifically recognizing EGFRvIII expressed or EGFR overexpressed by tumor cells, wherein the antibody comprises a light chain variable region and a heavy chain variable region, and   CDR1 of the light chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 55;   CDR2 of the light chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 42, SEQ ID NO: 53;   CDR3 of the light chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 43, SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57;   CDR1 of the heavy chain variable region has the amino acid sequence of SEQ ID NO: 44;   CDR2 of the heavy chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52;   CDR3 of the heavy chain variable region has an amino acid sequence selected from a group consisting of SEQ ID NO: 46, SEQ ID NO: 50.   
     
     
         49 . The chimeric antigen receptor of  claim 45 , wherein the antibody is a single chain antibody or a domain antibody. 
     
     
         50 . The chimeric antigen receptor of  claim 45 , wherein the chimeric antigen receptor comprises:
 SEQ ID NO: 36 or the amino acid sequence shown in positions 285-601; or   SEQ ID NO: 37 or the amino acid sequence shown in positions 285-702; or   SEQ ID NO: 38 or the amino acid sequence shown in positions 285-744; or   SEQ ID NO: 39 or the amino acid sequence shown in positions 285-749; or   SEQ ID NO: 40 or the amino acid sequence shown in positions 285-791.   
     
     
         51 . A genetically modified immune cell, wherein it is transduced with a nucleic acid encoding the chimeric antigen receptor of  claim 44 , or an expression vector comprising the nucleic acid or a virus comprising the vector; or
 it expresses the chimeric antigen receptor of claim  8  at its surface.   
     
     
         52 . The immune cell of  claim 51 , wherein it further carries an exogenous encoding sequence for cytokine; preferably, the cytokine includes: IL-12, IL-15 or IL-21 
     
     
         53 . The immune cell of  claim 51 , wherein it further expresses another chimeric antigen receptor that does not contain CD3ζ, but contains the intracellular signaling domain of CD28, the intracellular signaling domain of CD137, or a combination of both. 
     
     
         54 . The immune cell of  claim 51 , wherein it further expresses a chemokine receptor; preferably, the chemokine receptor includes CCR2. 
     
     
         55 . The immune cell of  claim 51 , wherein it further expresses siRNA that can reduce PD-1 expression or a protein that can block PD-L1. 
     
     
         56 . The immune cell of  claim 51 , wherein it further expresses a safety switch; preferably, the safety switch includes iCaspase-9, Truancated EGFR or RQR8.