Sialidase-HER2-antibody fusion protein and methods of use thereof

JP2024501767A5Inactive Publication Date: 2025-01-23PALLEON PHARMA INC
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Patent Information

Application Number
JP2023540918
Authority / Receiving Office
JP · JP
Patent Type
Applications
Current Assignee / Owner
Priority Date
2021-07-02
Filing Date
2022-01-06
Publication Date
2025-01-23
Estimated Expiration
Not applicable · inactive patent

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Abstract

The present invention generally relates to recombinant sialidase and anti-HER2 immunoglobulin antigen binding domain fusion proteins. The present invention also provides antibody conjugates comprising sialidase and anti-HER2 antibodies or portions thereof. The present invention further relates to methods of using the sialidase fusion proteins or antibody conjugates to treat cancer.
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Claims

1. 1. A fusion protein comprising: (a) a recombinant mutant human sialidase comprising a substitution of an alanine residue (A42) at a position corresponding to position 42 of wild-type human Neu2; and (b) an anti-HER2 immunoglobulin antigen-binding domain; and A fusion protein comprising:

2. The fusion protein of claim 1, wherein the alanine residue at the position corresponding to position 42 of wild-type human Neu2 is substituted with arginine (A42R).

3. The method of claim 2, wherein the sialidase is: (a) substitution of a proline residue (P5) at the position corresponding to position 5 of wild-type human Neu2; (b) substitution of a lysine residue (K9) at the position corresponding to position 9 of wild-type human Neu2; (c) substitution of a lysine residue (K44) at a position corresponding to position 44 of wild-type human Neu2; (d) substitution of a lysine residue (K45) at a position corresponding to position 45 of wild-type human Neu2; (e) substitution of a leucine residue (L54) at a position corresponding to position 54 of wild-type human Neu2; (f) substitution of a proline residue (P62) at a position corresponding to position 62 of wild-type human Neu2; (g) substitution of a glutamine residue (Q69) at a position corresponding to position 69 of wild-type human Neu2; (h) substitution of an arginine residue (R78) at a position corresponding to position 78 of wild-type human Neu2; (i) substitution of an aspartic acid residue (D80) at a position corresponding to position 80 of wild-type human Neu2; (j) substitution of an alanine residue (A93) at a position corresponding to position 93 of wild-type human Neu2; (k) substitution of a glycine residue (G107) at a position corresponding to position 107 of wild-type human Neu2; (l) substitution of a glutamine residue (Q108) at a position corresponding to position 108 of wild-type human Neu2; (m) substitution of a glutamine residue (Q112) at a position corresponding to position 112 of wild-type human Neu2; (n) substitution of a cysteine ​​residue (C125) at a position corresponding to position 125 of wild-type human Neu2; (o) substitution of a glutamine residue (Q126) at a position corresponding to position 126 of wild-type human Neu2; (p) substitution of an alanine residue (A150) at a position corresponding to position 150 of wild-type human Neu2; (q) substitution of a cysteine ​​residue (C164) at a position corresponding to position 164 of wild-type human Neu2; (r) substitution of an arginine residue (R170) at a position corresponding to position 170 of wild-type human Neu2; (s) substitution of an alanine residue (A171) at a position corresponding to position 171 of wild-type human Neu2; (t) substitution of a glutamine residue (Q188) at a position corresponding to position 188 of wild-type human Neu2; (u) substitution of an arginine residue (R189) at a position corresponding to position 189 of wild-type human Neu2; (v) substitution of an alanine residue (A213) at a position corresponding to position 213 of wild-type human Neu2; (w) substitution of a leucine residue (L217) at a position corresponding to position 217 of wild-type human Neu2; (x) substitution of a glutamic acid residue (E225) at a position corresponding to position 225 of wild-type human Neu2; (y) substitution of a histidine residue (H239) at a position corresponding to position 239 of wild-type human Neu2; (z) substitution of a leucine residue (L240) at a position corresponding to position 240 of wild-type human Neu2; (aa) substitution of an arginine residue (R241) at a position corresponding to position 241 of wild-type human Neu2; (bb) substitution of an alanine residue (A242) at a position corresponding to position 242 of wild-type human Neu2; (cc) substitution of a valine residue (V244) at a position corresponding to position 244 of wild-type human Neu2; (dd) substitution of a threonine residue (T249) at a position corresponding to position 249 of wild-type human Neu2; (ee) substitution of an aspartic acid residue (D251) at a position corresponding to position 251 of wild-type human Neu2; (ff) substitution of a glutamic acid residue (E257) at a position corresponding to position 257 of wild-type human Neu2; (gg) substitution of a serine residue (S258) at a position corresponding to position 258 of wild-type human Neu2; (hh) substitution of a leucine residue (L260) at a position corresponding to position 260 of wild-type human Neu2; (ii) substitution of a valine residue (V265) at a position corresponding to position 265 of wild-type human Neu2; (jj) substitution of a glutamine residue (Q270) at a position corresponding to position 270 of wild-type human Neu2; (kk) substitution of a tryptophan residue (W292) at a position corresponding to position 292 of wild-type human Neu2; (ll) substitution of a serine residue (S301) at a position corresponding to position 301 of wild-type human Neu2; (mm) substitution of a tryptophan residue (W302) at a position corresponding to position 302 of wild-type human Neu2; (nn) a substitution of a valine residue (V363) at a position corresponding to position 363 of wild-type human Neu2; or (oo) substitution of a leucine residue (L365) at a position corresponding to position 365 of wild-type human Neu2; or any combination of the preceding substitutions The fusion protein of claim 1 or 2, comprising:

4. 4. The fusion protein of any one of claims 1 to 3, wherein the sialidase comprises a substitution of K9, P62, A93, Q126, A242, Q270, S301, W302, V363, or L365, or any combination of the foregoing substitutions.

5. The sialidase comprising: (a) the proline residue at the position corresponding to position 5 of wild-type human Neu2 is substituted with a histidine (P5H); (b) the lysine residue at the position corresponding to position 9 of wild-type human Neu2 is substituted with aspartic acid (K9D); (c) the lysine residue at a position corresponding to position 44 of wild-type human Neu2 is substituted with arginine (K44R) or glutamic acid (K44E); (d) the lysine residue at a position corresponding to position 45 of wild-type human Neu2 is substituted with alanine (K45A), arginine (K45R), or glutamic acid (K45E); (e) the leucine residue at a position corresponding to position 54 of wild-type human Neu2 is replaced with a methionine (L54M); (f) the proline residue at a position corresponding to position 62 of wild-type human Neu2 is substituted with asparagine (P62N), aspartic acid (P62D), histidine (P62H), glutamic acid (P62E), glycine (P62G), serine (P62S), or threonine (P62T); (g) the glutamine residue at a position corresponding to position 69 of wild-type human Neu2 is substituted with a histidine (Q69H); (h) the arginine residue at a position corresponding to position 78 of wild-type human Neu2 is substituted with a lysine (R78K); (i) the aspartic acid residue at a position corresponding to position 80 of wild-type human Neu2 is substituted with proline (D80P); (j) the alanine residue at a position corresponding to position 93 of wild-type human Neu2 is substituted with glutamic acid (A93E) or lysine (A93K); (k) the glycine residue at a position corresponding to position 107 of wild-type human Neu2 is substituted with aspartic acid (G107D); (l) the glutamine residue at a position corresponding to position 108 of wild-type human Neu2 is substituted with a histidine (Q108H); (m) the glutamine residue at a position corresponding to position 112 of wild-type human Neu2 is substituted with arginine (Q112R) or lysine (Q112K); (n) the cysteine ​​residue at a position corresponding to position 125 of wild-type human Neu2 is substituted with leucine (C125L); (o) the glutamine residue at a position corresponding to position 126 of wild-type human Neu2 is substituted with leucine (Q126L), glutamic acid (Q126E), phenylalanine (Q126F), histidine (Q126H), isoleucine (Q126I), or tyrosine (Q126Y); (p) the alanine residue at a position corresponding to position 150 of wild-type human Neu2 is substituted with valine (A150V); (q) the cysteine ​​residue at a position corresponding to position 164 of wild-type human Neu2 is substituted with glycine (C164G); (r) the arginine residue at a position corresponding to position 170 of wild-type human Neu2 is substituted with proline (R170P); (s) the alanine residue at a position corresponding to position 171 of wild-type human Neu2 is substituted with glycine (A171G); (t) the glutamine residue at a position corresponding to position 188 of wild-type human Neu2 is substituted with proline (Q188P); (u) the arginine residue at a position corresponding to position 189 of wild-type human Neu2 is substituted with proline (R189P); (v) the alanine residue at the position corresponding to position 213 of wild-type human Neu2 is substituted with cysteine ​​(A213C), asparagine (A213N), serine (A213S), or threonine (A213T); (w) the leucine residue at a position corresponding to position 217 of wild-type human Neu2 is substituted with alanine (L217A) or valine (L217V); (x) the glutamic acid residue at a position corresponding to position 225 of wild-type human Neu2 is substituted with proline (E225P); (y) the histidine residue at a position corresponding to position 239 of wild-type human Neu2 is substituted with proline (H239P); (z) the leucine residue at a position corresponding to position 240 of wild-type human Neu2 is substituted with aspartic acid (L240D), asparagine (L240N), or tyrosine (L240Y); (aa) the arginine residue at a position corresponding to position 241 of wild-type human Neu2 is substituted with alanine (R241A), aspartic acid (R241D), leucine (R241L), glutamine (R241Q), or tyrosine (R241Y); (bb) the alanine residue at a position corresponding to position 242 of wild-type human Neu2 is substituted with cysteine ​​(A242C), phenylalanine (A242F), glycine (A242G), histidine (A242H), isoleucine (A242I), lysine (A242K), leucine (A242L), methionine (A242M), asparagine (A242N), glutamine (A242Q), arginine (A242R), serine (A242S), valine (A242V), tryptophan (A242W), or tyrosine (A242Y); (cc) the valine residue at a position corresponding to position 244 of wild-type human Neu2 is substituted with isoleucine (V244I) or proline (V244P); (dd) the threonine residue at a position corresponding to position 249 of wild-type human Neu2 is substituted with alanine (T249A); (ee) the aspartic acid residue at a position corresponding to position 251 of wild-type human Neu2 is substituted with glycine (D251G); (ff) the glutamic acid residue at a position corresponding to position 257 of wild-type human Neu2 is substituted with proline (E257P); (gg) the serine residue at the position corresponding to position 258 is substituted with a cysteine ​​(S258C); (hh) the leucine residue at a position corresponding to position 260 of wild-type human Neu2 is substituted with aspartic acid (L260D), phenylalanine (L260F), glutamine (L260Q), or threonine (L260T); (ii) the valine residue at the position corresponding to position 265 of wild-type human Neu2 is substituted with phenylalanine (V265F); (jj) the glutamine residue at a position corresponding to position 270 of wild-type human Neu2 is substituted with alanine (Q270A), histidine (Q270H), phenylalanine (Q270F), proline (Q270P), serine (Q270S), or threonine (Q270T); (kk) the tryptophan residue at a position corresponding to position 292 of wild-type human Neu2 is substituted with arginine (W292R); (ll) the serine residue at a position corresponding to position 301 of wild-type human Neu2 is substituted with alanine (S301A), aspartic acid (S301D), glutamic acid (S301E), phenylalanine (S301F), histidine (S301H), lysine (S301K), leucine (S301L), methionine (S301M), asparagine (S301N), proline (S301P), glutamine (S301Q), arginine (S301R), threonine (S301T), valine (S301V), tryptophan (S301W), or tyrosine (S301Y); (mm) the tryptophan residue at a position corresponding to position 302 of wild-type human Neu2 is substituted with alanine (W302A), aspartic acid (W302D), phenylalanine (W302F), glycine (W302G), histidine (W302H), isoleucine (W302I), lysine (W302K), leucine (W302L), methionine (W302M), asparagine (W302N), proline (W302P), glutamine (W302Q), arginine (W302R), serine (W302S), threonine (W302T), valine (W302V), or tyrosine (W302Y); (nn) the valine residue at a position corresponding to position 363 of wild-type human Neu2 is substituted with arginine (V363R); or (oo) the leucine residue at the position corresponding to position 365 of wild-type human Neu2 is substituted with glutamine (L365Q), histidine (L365H), isoleucine (L365I), lysine (L365K) or serine (L365S); Alternatively, the sialidase comprises a combination of any of the foregoing substitutions.

6. The fusion protein of claim 5, wherein the sialidase comprises substitutions selected from K9D, P62G, P62N, P62S, P62T, A93E, Q126Y, A242F, A242W, A242Y, Q270A, Q270T, S301A, S301R, W302K, W302R, V363R, and L365I substitutions, or any combination of the foregoing substitutions.

7. The sialidase of claim 1, (a) substitution or deletion of a methionine residue (M1) at a position corresponding to position 1 of wild-type human Neu2; (b) substitution of a valine residue (V6) at the position corresponding to position 6 of wild-type human Neu2; or (c) substitution of a cysteine ​​residue (C332) at a position corresponding to position 332 of wild-type human Neu2; or any combination of the above substitutions.

8. The sialidase comprising: (a) the methionine residue at the position corresponding to position 1 of wild-type human Neu2 is deleted (ΔM1), replaced with alanine (M1A), or replaced with aspartic acid (M1D); (b) the valine residue at the position corresponding to position 6 of wild-type human Neu2 is substituted with a tyrosine (V6Y); or (c) the cysteine ​​residue at a position corresponding to position 332 of wild-type human Neu2 is substituted with alanine (C332A); Or, the fusion protein of claim 7, wherein the sialidase comprises any combination of the foregoing substitutions.

9. 9. The fusion protein of claim 8, wherein the sialidase comprises M1D, V6Y, A42R, P62G, A93E, Q126Y, A242F, Q270T, and C332A substitutions.

10. The fusion protein of any one of claims 1 to 9, wherein the sialidase is Neu2.

11. The fusion protein of any one of claims 1 to 10, wherein the sialidase comprises SEQ ID NO:

198.

12. The fusion protein of any one of claims 1 to 11, further comprising an immunoglobulin Fc domain.

13. 13. The fusion protein of claim 12, wherein the immunoglobulin Fc domain is derived from a human IgG1, IgG2, IgG3, or IgG4 Fc domain, optionally wherein the immunoglobulin Fc domain is derived from a human IgG1 Fc domain.

14. The fusion protein of any one of claims 1 to 13, wherein the anti-HER2 immunoglobulin antigen-binding domain associates with a second anti-HER2 immunoglobulin antigen-binding domain to generate an anti-HER2 antigen-binding site.

15. 15. The fusion protein of any one of claims 1 to 14, wherein the anti-HER2 immunoglobulin antigen binding domain is derived from an antibody selected from trastuzumab, pertuzumab, CT-P6, trastuzumab-dkst, MGAH22 (margetuximab), PF-05280014, ertumaxomab, gancotamab, timigituzumab, ontrusant, ABP-980, SB3, DS-8201, MYL-1410, BCD-022, and HD201, optionally wherein the anti-HER2 immunoglobulin antigen binding domain is derived from trastuzumab.

16. The fusion protein of any one of claims 1 to 15, wherein the sialidase and the immunoglobulin Fc domain and / or the anti-HER2 immunoglobulin antigen binding domain are linked by a peptide bond or an amino acid linker.

17. The fusion protein of any one of claims 1 to 16, wherein the fusion protein comprises any one of SEQ ID NOs: 203 to 208.

18. An antibody conjugate comprising the fusion protein of any one of claims 1 to 17.

19. 20. The antibody conjugate of claim 18, wherein the antibody conjugate comprises a single sialidase and / or comprises a single anti-HER2 antigen binding site.

20. The antibody conjugate of claim 1, wherein: (a) a first polypeptide comprising an immunoglobulin light chain, optionally, said first polypeptide comprising SEQ ID NO:66; (b) a second polypeptide comprising an immunoglobulin heavy chain, optionally wherein the second polypeptide comprises any one of SEQ ID NOs: 67 or 189; and (c) a third polypeptide comprising an immunoglobulin Fc domain and a sialidase, optionally wherein said third polypeptide comprises, in an N-terminal to C-terminal direction, said sialidase and said immunoglobulin Fc domain, and / or said third polypeptide comprises any one of SEQ ID NOs: 203-208; 20. The antibody conjugate of claim 18 or 19, wherein the first and second polypeptides are covalently linked to each other, the second and third polypeptides are covalently linked to each other, and the first and second polypeptides together define an anti-HER2 antigen binding site.

21. An isolated nucleic acid comprising a nucleotide sequence encoding at least a part of the fusion protein of any one of claims 1 to 17 or the antibody conjugate of any one of claims 18 to 20.

22. 22. An expression vector comprising the nucleic acid of claim 21.

23. A host cell comprising the expression vector of claim 22.

24. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 17, or an antibody conjugate according to any one of claims 18 to 20.

25. A composition for use in therapy comprising a fusion protein according to any one of claims 1 to 17 or an antibody conjugate according to any one of claims 18 to 20.

26. A pharmaceutical composition according to claim 24 for use in treatment.

27. ​​The composition described in claim 25 or the pharmaceutical composition described in claim 26, wherein the treatment is treatment of cancer in a subject in need of such treatment.

28. A composition for use in promoting immune cell infiltration into tumors in a subject in need thereof, comprising a fusion protein described in any one of claims 1 to 17 or an antibody conjugate described in any one of claims 18 to 20.

29. 29. The composition of claim 28, wherein the immune cell is a T cell or a natural killer (NK) cell.

30. A composition comprising a fusion protein according to any one of claims 1 to 17 or an antibody conjugate according to any one of claims 18 to 20, for use in increasing the number of circulating natural killer (NK) cells in a subject in need thereof, thereby increasing the number of circulating NK cells compared to before administration of the composition.

31. A composition comprising a fusion protein according to any one of claims 1 to 17 or an antibody conjugate according to any one of claims 18 to 20, for use in increasing the number of T cells in a draining lymph node in a subject in need thereof, thereby increasing the number of T cells in the draining lymph node compared to before administration of the composition.