Anti-cancer fusion polypeptide

Abstract

The disclosure provides a fusion polypeptide specific for both CD137 and HER2/neu, which fusion polypeptide can be useful for directing CD137 clustering and activation to HER2/neu-positive tumor cells. Such fusion polypeptide can be used in many pharmaceutical applications, for example, as anti-cancer agents and/or immune modulators for the treatment or prevention of human diseases such as a variety of tumors. The present disclosure also concerns methods of making the fusion polypeptide described herein as well as compositions comprising such fusion polypeptide. The present disclosure further relates to nucleic acid molecules encoding such fusion polypeptide and to methods for generation of such fusion polypeptide and nucleic acid molecules. In addition, the application discloses therapeutic and/or diagnostic uses of such fusion polypeptide as well as compositions comprising one or more of such fusion polypeptides.

1 - 47 . (canceled) 
     
     
         48 . A fusion polypeptide that is capable of binding both CD137 and HER2/neu, wherein the fusion polypeptide comprises at least two subunits, wherein the first subunit comprises an immunoglobulin having binding specificity for HER2/neu, and wherein the second subunit comprises a lipocalin mutein having binding specificity for CD137, wherein the lipocalin mutein comprises at least 10 of the following mutated amino acid residues in comparison with the linear polypeptide sequence of mature human Lipocalin 2 (hNGAL) (SEQ ID NO: 18): Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Arg or Lys; Gln 49→Val, Ile, His, Ser or Asn; Tyr 52→Met; Asn 65→Asp; Ser 68→Met, Ala or Gly; Leu 70→Ala, Lys, Ser or Thr; Arg 72→Asp; Lys 73→Asp; Asp 77→Met, Arg, Thr or Asn; Trp 79→Ala or Asp; Arg 81→Met, Trp or Ser; Phe 83→Leu; Cys 87→Ser; Leu 94→Phe; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; and Lys 134→Tyr, and wherein the lipocalin mutein has at least 85% sequence identity to the amino acid sequence shown in SEQ ID NO: 2. 
     
     
         49 . The fusion polypeptide of  claim 48 , wherein the amino acid sequence of the lipocalin mutein comprises one of the following sets of mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 18):
 (a) Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Lys; Gln 49→Asn; Tyr 52→Met; Ser 68→Gly; Leu 70→Thr; Arg 72→Asp; Lys 73→Asp; Asp 77→Thr; Trp 79→Ala; Arg 81→Ser; Cys 87→Ser; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; Lys 134→Tyr;   (b) Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Arg; Gln 49→Ile; Tyr 52→Met; Asn 65→Asp; Ser 68→Met; Leu 70→Lys; Arg 72→Asp; Lys 73→Asp; Asp 77→Met; Trp 79→Asp; Arg 81→Trp; Cys 87→Ser; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; Lys 134→Tyr;   (c) Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Arg; Gln 49→Asn; Tyr 52→Met; Asn 65→Asp; Ser 68→Ala; Leu 70→Ala; Arg 72→Asp; Lys 73→Asp; Asp 77→Thr; Trp 79→Asp; Arg 81→Trp; Cys 87→Ser; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; Lys 134→Tyr;   (d) Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Lys; Gln 49→Asn; Tyr 52→Met; Asn 65→Asp; Ser 68→Ala; Leu 70→Ala; Arg 72→Asp; Lys 73→Asp; Asp 77→Thr; Trp 79→Asp; Arg 81→Trp; Cys 87→Ser; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; Lys 134→Tyr;   (e) Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Lys; Gln 49→Ser; Tyr 52→Met; Asn 65→Asp; Ser 68→Gly; Leu 70→Ser; Arg 72→Asp; Lys 73→Asp; Asp 77→Thr; Trp 79→Ala; Arg 81→Met; Cys 87→Ser; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; Lys 134→Tyr;   (f) Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Lys; Gln 49→Val; Tyr 52→Met; Asn 65→Asp; Ser 68→Gly; Leu 70→Thr; Arg 72→Asp; Lys 73→Asp; Asp 77→Arg; Trp 79→Asp; Arg 81→Ser; Cys 87→Ser; Leu 94→Phe; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; Lys 134→Tyr;   (g) Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Arg; Gln 49→His; Tyr 52→Met; Asn 65→Asp; Ser 68→Gly; Leu 70→Thr; Arg 72→Asp; Lys 73→Asp; Asp 77→Thr; Trp 79→Ala; Arg 81→Ser; Cys 87→Ser; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; Lys 134→Tyr;   (h) Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Lys; Gln 49→Asn; Tyr 52→Met; Asn 65→Asp; Ser 68→Gly; Leu 70→Thr; Arg 72→Asp; Lys 73→Asp; Asp 77→Thr; Trp 79→Ala; Arg 81→Ser; Phe 83→Leu; Cys 87→Ser; Leu 94→Phe; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; Lys 134→Tyr; or   (i) Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Arg; Gln 49→Ser; Tyr 52→Met; Asn 65→Asp; Ser 68→Ala; Leu 70→Thr; Arg 72→Asp; Lys 73→Asp; Asp 77→Asn; Trp 79→Ala; Arg 81→Ser; Cys 87→Ser; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; Lys 134→Tyr.   
     
     
         50 . The fusion polypeptide of  claim 48 , wherein the lipocalin mutein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 39-46. 
     
     
         51 . The fusion polypeptide of  claim 48 , wherein the mutein comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 2. 
     
     
         52 . The fusion polypeptide of  claim 48 , wherein the mutein comprises the amino acid sequence of SEQ ID NO: 2. 
     
     
         53 . The fusion polypeptide of  claim 48 , wherein the first subunit and the second binding domain are linked via a peptide linker between the N-terminus of the lipocalin mutein of the second subunit and the C-terminus of a heavy chain constant region (CH) of the immunoglobulin of the first subunit. 
     
     
         54 . The fusion polypeptide of  claim 53 , wherein the peptide linker is a (G4S)3 linker. 
     
     
         55 . The fusion polypeptide of  claim 48 , wherein the fusion polypeptide comprises the amino acid sequence shown in SEQ ID NO: 19. 
     
     
         56 . The fusion polypeptide of  claim 48 , wherein the immunoglobulin is a monoclonal antibody. 
     
     
         57 . The fusion polypeptide of  claim 56 , wherein the monoclonal antibody comprises the antigen-binding domain of trastuzumab or pertuzumab. 
     
     
         58 . The fusion polypeptide of  claim 56 , wherein the monoclonal antibody has the heavy and light chains provided by SEQ ID NOs: 3 and 4. 
     
     
         59 . The fusion polypeptide of  claim 56 , wherein the monoclonal antibody has an IgG4 backbone. 
     
     
         60 . The fusion polypeptide of  claim 59 , wherein the IgG4 backbone has any one of the following mutations selected from the group consisting of S228P, N297A, F234A and L235A. 
     
     
         61 . The fusion polypeptide of  claim 48 , wherein the fusion polypeptide has one or more of the following properties:
 (i) it is capable of binding CD137 with an EC 50  value comparable to or lower than the EC 50  value of the lipocalin mutein specific for CD137 included in the fusion polypeptide;   (ii) it is capable of binding CD137 with an EC 50  value of about 1 nM or lower;   (iii) it is capable of binding HER2/neu with an EC 50  value comparable to or lower than the EC 50  value of the immunoglobulin specific for HER2/neu included in such fusion polypeptide;   (iv) it is capable of binding HER2/neu with an EC 50  value of about 1 nM or lower;   (v) it is capable of simultaneously binding CD137 and HER2/neu;   (vi) it is capable of simultaneously binding CD137 and HER2/neu with EC 50  values of about 4 nM or lower.   
     
     
         62 . The fusion polypeptide of  claim 48 , wherein the fusion polypeptide is capable of co-stimulating T cell responses. 
     
     
         63 . The fusion polypeptide of  claim 48 , wherein the fusion polypeptide is capable of inducing IL-2 secretion and T cell proliferation. 
     
     
         64 . A nucleic acid molecule comprising a nucleotide sequence encoding the fusion polypeptide of  claim 48 . 
     
     
         65 . A host cell containing a nucleic acid molecule of  claim 64 . 
     
     
         66 . A method of producing the fusion polypeptide of  claim 48 , wherein the fusion polypeptide is produced starting from a nucleic acid coding for the fusion polypeptide. 
     
     
         67 . A pharmaceutical composition comprising the fusion polypeptide of  claim 48 .