Antigen-binding constructs targeting HER2
Described herein are high affinity antigen binding constructs, e.g., antibodies, directed to the ECD2 domain of HER2. The antigen-binding constructs comprise at least one antigen-binding polypeptide construct that binds to ECD2 of HER2 (HER2 ECD2) with increased affinity compared to a wild-type 2C4 antibody. Such antigen-binding polypeptide constructs comprise one or more amino acid modifications in the framework region and/or CDRs compared to the amino acid sequence of a wild-type 2C4 antibody that increase affinity of the antigen-binding polypeptide construct for ECD2 by 2-fold or greater. The antigen-binding constructs can inhibit the growth of HER2-expressing breast cancer cells and gastric cancer cells. Antigen-binding constructs in biparatopic format are internalized in HER2-expressing cells.
1. An antigen-binding construct comprising a variant first antigen-binding polypeptide construct which monovalently binds a first HER2 ECD2 (human epidermal growth factor receptor 2 extracellular domain 2) antigen, the variant first antigen-binding polypeptide construct comprising a heavy chain variable (VH) domain as set forth in SEQ ID NO:2 and a light chain variable (VL) domain as set forth in SEQ ID NO:11 and comprising an amino acid modification at position 75 in the VH domain (Kabat numbering) wherein the lysine at position 75 has been substituted with a tryptophan (H_K75W), glutamic acid (H_K75E), or tyrosine (H_K75Y),
optionally wherein the variant first antigen-binding polypeptide construct comprises H_K75W and further comprises the following substitutions or set of substitutions, numbering according to Kabat numbering system:
H_T30Q; or
H_T30Y; or
H_G56Y; or
H_S99W; or
L_Y49W; or
L_Y96G; or
H_S99W and L_Y49W; or
L_Y49W and L_Y96G; or
H_T30Q and L_Y49W; or
H_T30Q and H_S99W; or
H_T30Q and L_Y96G; or
H_T30Y and L_Y49W; or
H_S99W and L_Y49W and L_Y96G; or
H_T3000 and H_S99W and L_Y96G or
H_T3000 and H_S99W and L_Y49W; or
H_T3000 and L_Y49W and L_Y96G; or
H_T30Y and L_Y49W and L_Y96G, and
optionally wherein the variant first antigen-binding polypeptide construct comprises H_K75E and further comprises the following substitutions or set of substitutions, numbering according to Kabat numbering system
L_Y49W; or
H_T30Q; or
H_S74W; or
H_S99W.
2. The antigen-binding construct of claim 1 , wherein the variant first antigen-binding polypeptide construct comprises H_K75W.
3. The antigen-binding construct of claim 1 , wherein the variant first antigen-binding polypeptide construct comprises H_K75W and further comprises the following substitutions or set of substitutions, numbering according to Kabat numbering system:
H_T30Q; or
H_T30Y; or
H_G56Y; or
H_S99W; or
L_Y49W; or
L_Y96G; or
H_S99W and L_Y49W; or
L_Y49W and L_Y96G; or
H_T30Q and L_Y49W; or
H_T30Q and H_S99W; or
H_T30Q and L_Y96G; or
H_T30Y and L_Y49W; or
H_S99W and L_Y49W and L_Y96G; or
H_T30Q and H_S99W and L_Y96G or
H_T30Q and H_S99W and L_Y49W; or
H_T30Q and L_Y49W and L_Y96G; or
H_T30Y and L_Y49W and L_Y96G.
4. The antigen-binding construct of claim 1 , wherein the variant first antigen-binding polypeptide construct comprises H_K75E.
5. The antigen-binding construct of claim 1 , wherein the variant first antigen-binding polypeptide construct comprises H_K75E and further comprises the following substitutions or set of substitutions, numbering according to Kabat numbering system
L_Y49W; or
H_T30Q; or
H_S74W; or
H_S99W.
6. The antigen-binding construct of claim 1 , wherein the variant first antigen-binding polypeptide construct comprises H_K75Y.
7. The antigen-binding construct of claim 1 , wherein the variant first antigen-binding polypeptide construct is a Fab.
8. The antigen-binding construct of claim 1 , wherein the antigen-binding construct is one-armed.
9. The antigen binding construct of claim 7 , further comprising a first linker polypeptide operably linked to the variant first antigen-binding polypeptide construct.
10. The antigen-binding construct of claim 1 , wherein the antigen-binding construct comprises a second antigen-binding polypeptide construct that binds to a second antigen.
11. The antigen-binding construct of claim 10 , further comprising a first linker polypeptide operably linked to the variant first antigen-binding polypeptide construct, and a second linker polypeptide operably linked to the second antigen-binding polypeptide construct.
12. The antigen-binding construct of claim 11 , wherein the second antigen is a HER2 ECD2 antigen and the second antigen-binding polypeptide construct is identical to the variant first antigen-binding polypeptide construct.
13. The antigen-binding construct of claim 11 , wherein the second antigen is a HER2 ECD4 antigen and the second antigen-binding polypeptide construct is an scFv comprising the VH and VL domain of trastuzumab and a glycine-serine linker.
14. The antigen-binding construct according to claim 9 , wherein the first linker polypeptide is operably linked to a heterodimeric human IgG1 Fc comprising a first Fc polypeptide and a second Fc polypeptide each comprising a different CH3 sequence.
15. The antigen-binding construct according to claim 11 , wherein the first and second linker polypeptides are operably linked to a heterodimeric human IgG1 Fc comprising a first Fc polypeptide and a second Fc polypeptide each comprising a different CH3 sequence.
16. The antigen-binding construct of claim 14 , wherein the CH3 sequence of each Fc polypeptide comprises one or more modifications that promote the formation of a heterodimeric Fc with stability comparable to a wild-type homodimeric Fc, the heterodimeric IgG1 Fc having
a) the modifications L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T366L_K392M_T394W in the second polypeptide; or
b) the modifications L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T366L_K392L_T394W in the second Fc polypeptide; or
c) the modifications T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T350V_T366L_K392L_T394W in the second Fc polypeptide; or
d) the modifications T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T350V_T366L_K392M_T394W in the second Fc polypeptide; or
e) the modifications T350V_L351Y_S400E_F405A_Y407V in the first Fc polypeptide, and the modifications T350V_T366L_N390R_K392M_T394W in the second Fc polypeptide; or
f) the modifications T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T366I_N390R_K392M_T394W in the second Fc polypeptide; or
g) the modifications L351Y_S400E_F405A_Y407V in the first Fc polypeptide, and the modifications T350V_T366L_K392L_T394W in the second Fc polypeptide, wherein the numbering of amino acid residues in the Fc is according to the EU numbering system.
17. The antigen-binding construct of claim 15 , wherein the CH3 sequence of each Fc polypeptide comprises one or more modifications that promote the formation of a heterodimeric Fc with stability comparable to a wild-type homodimeric Fc, the heterodimeric IgG1 Fc having
a) the modifications L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T366L_K392M_T394W in the second polypeptide; or
b) the modifications L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T366L_K392L_T394W in the second Fc polypeptide; or
c) the modifications T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T350V_T366L_K392L_T394W in the second Fc polypeptide; or
d) the modifications T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T350V_T366L_K392M_T394W in the second Fc polypeptide; or
e) the modifications T350V_L351Y_S400E_F405A_Y407V in the first Fc polypeptide, and the modifications T350V_T366L_N390R_K392M_T394W in the second Fc polypeptide; or
f) the modifications T350V_L351Y_F405A_Y407V in the first Fc polypeptide, and the modifications T366I_N390R_K392M_T394W in the second Fc polypeptide; or
g) the modifications L351Y_S400E_F405A_Y407V in the first Fc polypeptide, and the modifications T350V_T366L_K392L_T394W in the second Fc polypeptide, wherein the numbering of amino acid residues in the Fc is according to the EU numbering system.
18. The antigen-binding construct of claim 1 , wherein the VH consists of SEQ ID NO: 515 and the VL consists of SEQ ID NO: 815; or the VH consists of SEQ ID NO: 125 and the VL consists of SEQ ID NO: 915; or the VH consists of SEQ ID NO: 515 and the VL consists of SEQ ID NO: 95; or the VH consists of SEQ ID NO: 35 and the VL consists of SEQ ID NO: 815; or the VH consists of SEQ ID NO: 45 and the VL consists of SEQ ID NO: 95.
19. A pharmaceutical composition comprising the antigen-binding construct of claim 1 , and a pharmaceutical carrier, optionally selected from a buffer, an antioxidant, a low molecular weight molecule, a drug, a protein, an amino acid, a carbohydrate, a lipid, a chelating agent, a stabilizer, or an excipient.
20. An isolated polynucleotide or set of isolated polynucleotides comprising at least one nucleic acid sequence that encodes the antigen-binding construct of claim 1 , optionally wherein said polynucleotide or set of polynucleotides is cDNA.
21. A vector or set of vectors comprising one or more of the polynucleotides or sets of polynucleotides according to claim 20 , optionally selected from the group consisting of a plasmid, a viral vector, a non-episomal mammalian vector, an expression vector, and a recombinant expression vector.
22. An isolated cell comprising a polynucleotide or set of polynucleotides according to claim 20 , the isolated cell optionally selected from a hybridoma, a Chinese Hamster Ovary (CHO) cell, or a HEK293 cell.
23. A method of producing the antigen-binding construct according to claim 1 , comprising culturing a host cell under conditions suitable for expressing the antigen-binding construct wherein the host cell comprises a polynucleotide or set of polynucleotides according to claim 20 , and purifying the construct.
24. A method of inhibiting cell growth of a HER2-expressing (HER2+) cell, the method comprising contacting the cell with the antigen-binding construct of claim 1 .
25. The method of claim 24 , wherein the cell is a breast cancer cell.
26. The method of claim 24 , wherein the cell is a gastric cancer cell.
27. The method of claim 24 , wherein the method is performed in vitro.
28. A method of treating a subject having a HER2-expressing (HER2+) cancer, optionally wherein the cancer is breast cancer or gastric cancer, the method comprising administering to the subject the antigen-binding construct of claim 1 .