IP Library › Granted Patent US 12,612,463

Granted Patent B2

US 12,612,463 · App. 18/417,708 · Granted Apr 28, 2026

Mesothelin-targeted CD40 agonistic multispecific antibody constructs for the treatment of solid tumors

Inventors: Xin Yu (Foster City, CA); Jackson Egen (San Mateo, CA); Fernando Garces (Sherman Oaks, CA); Shunsuke Takenaka (Vancouver, CA); AeRyon Kim (Foster City, CA); Deepali Sawant (Newark, CA)

Assignee: Amgen Inc.

C07K16/2878 A61P35/04 C07K16/30 A61K2039/505 C07K2317/31 C07K2317/33 C07K2317/35 C07K2317/522 C07K2317/524 C07K2317/526 C07K2317/53 C07K2317/565 C07K2317/622 C07K2317/75 C07K2317/92

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Patent No.: US 12,612,463
App. No.: 18/417,708
Granted: Apr 28, 2026
Kind: B2

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Abstract

The present invention relates to a human agonistic CD40 multispecific antibody construct for treatment of solid tumors by engineering a molecule that specifically targets the CD40 pathway on tumor-associated APCs, without systemic CD40 activation.

Claims (257)

1 . A multispecific antibody construct comprising:

(i) a first antibody comprising two light chains and two heavy chains, wherein

(a) the light chains comprise a first variable region (VL1) and a light chain constant region (CL);

(b) the heavy chains comprise a first heavy variable region (VH1) and CH1, hinge, CH2, and CH3 regions; and

(c) the heavy chains comprise at least one amino acid substitution that results in a reduced binding affinity of the heavy chain for Fc gamma RI receptor as compared with an unsubstituted heavy chain; and

(ii) a scFv comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2) of a second antibody, wherein the VL2 and the VH2 are connected via a first peptide linker,

wherein the scFv is fused at its amino terminus to the carboxyl terminus of each of the heavy chains through a second peptide linker such that a heavy chain fusion protein is formed; and wherein the first antibody specifically binds to and agonizes human CD40 comprising the amino acid sequence of SEQ ID NO:1, and the scFv specifically binds to human mesothelin (MSLN) comprising the amino acid sequence of SEQ ID NO:2; and

wherein:

i) the VL1 and VH1 are selected from the group consisting of:

a) a VL1 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 58, 59, and 60, respectively; and a VH1 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 136, 137, and 138, respectively;

b) a VL1 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 64, 65, and 66, respectively; and a VH1 comprising aCDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 142, 143, and 144, respectively;

c) a VL1 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 70, 71, and 72, respectively; and a VH1 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 148, 149, and 150, respectively;

d) a VL1 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 76, 77, and 78, respectively; and a VH1 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 154, 155, and 156, respectively;

e) a VL1 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 82, 83, and 84, respectively; and a VH1 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 160, 161, and 162, respectively;

f) a VL1 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 88, 89, and 90, respectively; and a VH1 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 166, 167, and 168, respectively;

g) a VL1 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 94, 95, and 96, respectively; and a VH1 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 172, 173, and 174, respectively;

h) a VL1 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and a VH1 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively;

i) a VL1 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and a VH1 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively;

j) a VL1 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 112, 113, and 114, respectively; and a VH1 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 190, 191, and 192, respectively;

k) a VL1 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 118, 119, and 120, respectively; and a VH1 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 196, 197, and 198, respectively;

l) a VL1 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and a VH1 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and

m) a VL1 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and a VH1 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively; and

n) a VL1 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and amino acids 91 to 101 of SEQ ID NO: 370, respectively; and a VH1 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively; and

ii) the VL2 and VH2 are selected from the group consisting of:

a) a VL2 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 230, 231, and 232, respectively; and a VH2 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 254, 255, and 256, respectively;

b) a VL2 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 236, 237, and 238, respectively; and a VH2 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 260, 261, and 262, respectively;

c) a VL2 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 242, 243, and 244, respectively; and a VH2 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 266, 267, and 268, respectively; and

d) a VL2 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 248, 249, and 250, respectively; and a VH2 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 272, 273, and 274, respectively.

2 . The antibody construct of claim 1 , wherein the two light chains are identical and the two heavy chain fusion proteins are identical.

3 . The antibody construct of claim 1 , wherein the heavy chain comprises an amino acid substitution selected from the group consisting of:

(i) N297G or N297A;

(ii) L234A and L235A; and

(iii) R292C and V302C;

wherein the amino acid numbering is EU numbering according to Kabat.

4 . The antibody construct of claim 3 , wherein the heavy chain comprises N297G, R292C, and V302C mutations, wherein the amino acid numbering is EU numbering according to Kabat.

5 . The antibody construct of claim 1 , wherein

i) the VL1 and VH1 are selected from the group consisting of:

b) a VL1 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 64, 65, and 66, respectively; and a VH1 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 142, 143, and 144, respectively;

d) a VL1 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 76, 77, and 6780, respectively; and a VH1 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 154, 155, and 156, respectively;

and

ii) the VL2 and VH2 are selected from the group consisting of:

6 . The antibody construct of claim 1 , wherein

i) said VL1 comprises a CDRL1, a CDRL2, and a CDRL3 comprising the amino acid sequences of SEQ ID NOs: 100, 101, and amino acids 91 to 101 of SEQ ID NO: 370, respectively; and said VH1 comprises a CDRH1, a CDRH2, and a CDRH3 comprising the amino acid sequences of SEQ ID NOs: 178, 179, and 180, respectively; and

ii) said VL2 comprises a CDRL1, a CDRL2, and a CDRL3 comprising the amino acid sequences of SEQ ID NOs: 236, 237, and 238, respectively; and said VH2 comprises a CDRH1, a CDRH2, and a CDRH3 comprising the amino acid sequences of SEQ ID NOs: 260, 261, and 262, respectively.

7 . The antibody construct of claim 1 , wherein:

i) the VL1 and VH1 are selected from the group consisting of:

a) a VL1 comprising SEQ ID NO: 5 and a VH1 comprising SEQ ID NO: 6;

b) a VL1 comprising SEQ ID NO: 9 and a VH1 comprising SEQ ID NO: 10;

c) a VL1 comprising SEQ ID NO: 13 and a VH1 comprising SEQ ID NO: 14;

d) a VL1 comprising SEQ ID NO: 17 and a VH1 comprising SEQ ID NO: 18;

e) a VL1 comprising SEQ ID NO: 21 and a VH1 comprising SEQ ID NO: 22;

f) a VL1 comprising SEQ ID NO: 25 and a VH1 comprising SEQ ID NO: 26;

g) a VL1 comprising SEQ ID NO: 29 and a VH1 comprising SEQ ID NO: 30;

h) a VL1 comprising SEQ ID NO: 33 and a VH1 comprising SEQ ID NO: 34;

i) a VL1 comprising SEQ ID NO: 37 and a VH1 comprising SEQ ID NO: 38;

j) a VL1 comprising SEQ ID NO: 41 and a VH1 comprising SEQ ID NO: 42;

k) a VL1 comprising SEQ ID NO: 45 and a VH1 comprising SEQ ID NO: 46;

l) a VL1 comprising SEQ ID NO: 49 and a VH1 comprising SEQ ID NO: 50; and

m) a VL1 comprising SEQ ID NO: 53 and a VH1 comprising SEQ ID NO: 54;

n) a VL1 comprising amino acids 1 to 112 of SEQ ID NO: 370 and a VH1 comprising SEQ ID NO: 34;

and

ii) the VL2 and VH2 are selected from the group consisting of:

a) a VL2 comprising SEQ ID NO: 213 and a VH2 comprising SEQ ID NO: 214;

b) a VL2 comprising SEQ ID NO: 217 and a VH2 comprising SEQ ID NO: 218;

c) a VL2 comprising SEQ ID NO: 221 and a VH2 comprising SEQ ID NO: 222;

and

d) a VL2 comprising SEQ ID NO: 225 and a VH2 comprising SEQ ID NO: 226.

8 . The antibody construct of claim 1 , wherein: (i) said VL1 comprises the amino acid sequence of residues 1 to 112 of SEQ ID NO: 370, and said VH1 comprises the amino acid sequence of SEQ ID NO: 34; and (ii) said VL2 comprises the amino acid sequence of SEQ ID NO: 217, and said VH2 comprises the amino acid sequence of SEQ ID NO: 218.

9 . The antibody construct of claim 1 , wherein the CH1-hinge-CH2-CH3 of the heavy chain is selected from the group consisting of SEQ ID NO: 885 and SEQ ID NO: 886.

10 . The antibody construct of claim 1 , wherein the first peptide linker is selected from the group consisting of SEQ ID NOs: 888-893.

11 . The antibody construct of claim 1 , wherein the second peptide linker is selected from the group consisting of SEQ ID NOs: 887-893.

12 . The antibody construct of claim 1 , wherein the first peptide linker comprises SEQ ID NO: 889 and the second peptide linker comprises SEQ ID NO: 887.

13 . The antibody construct of claim 1 , wherein the light chain and the heavy chain fusion protein comprise polypeptides comprising amino acid sequences selected from the group consisting of:

SEQ ID NO: 286 and SEQ ID NO: 285, respectively;

SEQ ID NO: 290 and SEQ ID NO: 289, respectively;

SEQ ID NO: 294 and SEQ ID NO: 293, respectively;

SEQ ID NO: 298 and SEQ ID NO: 297, respectively;

SEQ ID NO: 302 and SEQ ID NO: 301, respectively;

SEQ ID NO: 306 and SEQ ID NO: 305, respectively;

SEQ ID NO: 310 and SEQ ID NO: 309, respectively;

SEQ ID NO: 314 and SEQ ID NO: 313, respectively;

SEQ ID NO: 318 and SEQ ID NO: 317, respectively;

SEQ ID NO: 322 and SEQ ID NO: 321, respectively;

SEQ ID NO: 326 and SEQ ID NO: 325, respectively;

SEQ ID NO: 330 and SEQ ID NO: 329, respectively;

SEQ ID NO: 334 and SEQ ID NO: 333, respectively;

SEQ ID NO: 338 and SEQ ID NO: 337, respectively;

SEQ ID NO: 342 and SEQ ID NO: 341, respectively;

SEQ ID NO: 346 and SEQ ID NO: 345, respectively;

SEQ ID NO: 350 and SEQ ID NO: 349, respectively;

SEQ ID NO: 354 and SEQ ID NO: 353, respectively;

SEQ ID NO: 358 and SEQ ID NO: 357, respectively;

SEQ ID NO: 362 and SEQ ID NO: 361, respectively;

SEQ ID NO: 366 and SEQ ID NO: 365, respectively;

SEQ ID NO: 370 and SEQ ID NO: 369, respectively;

SEQ ID NO: 374 and SEQ ID NO: 373, respectively; and

SEQ ID NO: 378 and SEQ ID NO: 377, respectively.

14 . A pharmaceutical composition comprising the antibody construct of claim 1 , and a carrier, stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier, preservative or adjuvant.

15 . A kit comprising the antibody construct of claim 1 , and directions for use.

16 . A multispecific antibody construct comprising:

(i) a first antibody comprising two light chains and two heavy chains, wherein

(a) the light chains comprise a first variable region (VL1) and a light chain constant region (CL);

(b) the heavy chains comprise a first heavy variable region (VH1) and CH1, hinge, CH2, and CH3 regions; and

(c) the heavy chains comprise at least one amino acid substitution that results in a reduced binding affinity of the heavy chain for Fc gamma R1 receptor as compared with an unsubstituted heavy chain; and

wherein the scFv is fused at its amino terminus to the carboxyl terminus of each of the heavy chains through a second peptide linker such that a heavy chain fusion protein is formed; and wherein the first antibody specifically binds to human mesothelin (MSLN) comprising the amino acid sequence of SEQ ID NO:2, and the scFv specifically binds to and agonizes human CD40 comprising the amino acid sequence of SEQ ID NO:1,

and

wherein:

i) the VL1 and VH1 are selected from the group consisting of:

a) a VL1 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 230, 231, and 232, respectively; and a VH1 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 254, 255, and 256, respectively;

b) a VL1 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 236, 237, and 238, respectively; and a VH1 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 260, 261, and 262, respectively;

c) a VL1 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 242, 243, and 244, respectively; and a VH1 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 266, 267, and 268, respectively; and

d) a VL1 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 248, 249, and 250, respectively; and a VH1 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 272, 273, and 274, respectively;

and

ii) the VL2 and VH2 are selected from the group consisting of:

a) a VL2 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 58, 59, and 60, respectively; and a VH2 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 136, 137, and 138, respectively;

b) a VL2 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 64, 65, and 66, respectively; and a VH2 comprising aCDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 142, 143, and 144, respectively;

c) a VL2 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 70, 71, and 72, respectively; and a VH2 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 148, 149, and 150, respectively;

d) a VL2 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 76, 77, and 78, respectively; and a VH2 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 154, 155, and 156, respectively;

e) a VL2 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 82, 83, and 84, respectively; and a VH2 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 160, 161, and 162, respectively;

f) a VL2 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 88, 89, and 90, respectively; and a VH2 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 166, 167, and 168, respectively;

g) a VL2 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 94, 95, and 96, respectively; and a VH2 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 172, 173, and 174, respectively;

h) a VL2 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and a VH2 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively;

i) a VL2 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and a VH2 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively;

j) a VL2 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 112, 113, and 114, respectively; and a VH2 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 190, 191, and 192, respectively;

k) a VL2 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 118, 119, and 120, respectively; and a VH2 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 196, 197, and 198, respectively;

l) a VL2 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and a VH2 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and

m) a VL2 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and a VH2 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively; and

n) a VL2 comprising a CDRL1, a CDRL2, and a CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and amino acids 91 to 101 of SEQ ID NO: 370, respectively; and a VH2 comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively.

17 . A multispecific antibody construct comprising:

a) two identical heavy chain fusion proteins each comprising a first heavy chain variable region (VH1) and a first CH1 domain, wherein the first CH1 domain is linked to a hinge-CH2-CH3 polypeptide, and wherein the hinge-CH2-CH3 polypeptide is linked to a second heavy chain variable region (VH2), wherein the VH2 is linked to a second CH1 domain; wherein

i) the VH1 or first CH1 domain comprises at least one amino acid substitution to introduce a positively charged amino acid at a residue selected from the group consisting of positions 39, 44, and 183 using EU numbering; and

ii) the VH2 or second CH1 domain comprises at least one amino acid substitution to introduce a negatively charged amino acid at a residue selected from the group consisting of a residue that corresponds to positions 39, 44, and 183 using EU numbering; and

b) a second polypeptide comprising a first light chain, wherein the first light chain comprises a first light chain variable region (VL1) and a first CL region; and wherein the VL1 or first CL domain comprises at least one amino acid substitution to introduce a negatively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176 using EU numbering; and

c) a third polypeptide comprising a second light chain, wherein the second light chain comprises a second light chain variable region (VL2) and a second CL region; and wherein the VL2 or second CL domain comprises at least one amino acid substitution to introduce a positively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176 using EU numbering;

wherein the VH1 and VL1 interact to bind a first antigen and wherein the VH2 and VL2 interact to bind a second antigen;

wherein:

(a) the first antigen is human CD40 comprising the amino acid sequence of SEQ ID NO:1, and the second antigen is human mesothelin (MSLN) comprisingthe amino sequence of SEQ ID NO: 2, and

wherein:

i) the VL1 and VH1 are selected from the group consisting of:

and

ii) the VL2 and VH2 are selected from the group consisting of:

(b) the first antigen is human MSLN comprising the amino acid sequence of SEQ ID NO:2, and the second antigen is human CD40 comprising the amino acid sequence of SEQ ID NO:1, and wherein:

i) the VL1 and VH1 are selected from the group consisting of:

and

ii) the VL2 and VH2 are selected from the group consisting of:

18 . A pharmaceutical composition comprising the antibody construct of claim 17 , and a carrier, stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier, preservative or adjuvant.

19 . A multispecific antibody construct comprising:

i) the VH1 or first CH1 domain comprises at least one amino acid substitution to introduce a negatively charged amino acid at a residue selected from the group consisting of positions 39, 44, and 183 using EU numbering; and

ii) the VH2 or second CH1 domain comprises at least one amino acid substitution to introduce a positively charged amino acid at a residue selected from the group consisting of a residue that corresponds to positions 39, 44, and 183 using EU numbering; and

b) a second polypeptide comprising a first light chain, wherein the first light chain comprises a first light chain variable region (VL1) and a first CL region; and wherein the VL1 or first CL domain comprises at least one amino acid substitution to introduce a positively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176 using EU numbering; and

c) a third polypeptide comprising a second light chain, wherein the second light chain comprises a second light chain variable region (VL2) and a second CL region; and wherein the VL2 or second CL domain comprises at least one amino acid substitution to introduce a negatively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176 using EU numbering;

wherein the VH1 and VL1 interact to bind a first antigen and wherein the VH2 and VL2 interact to bind a second antigen;

wherein:

i) the VL1 and VH1 are selected from the group consisting of:

and

ii) the VL2 and VH2 are selected from the group consisting of:

(b) the first antigen is human MSLN comprisingthe amino acid sequence of SEQ ID NO:2, and the second antigen is human CD40 comprising the amino acid sequence of SEQ ID NO:1, and

wherein:

i) the VL1 and VH1 are selected from the group consisting of:

and

ii) the VL2 and VH2 are selected from the group consisting of:

20 . An antibody construct comprising:

a) a heavy chain fusion protein comprising the amino acid sequence of SEQ ID NO: 369;

and

b) a light chain comprising the amino acid sequence of SEQ ID NO: 370.

Assignments (1)

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 15, 2024

From: YU, XIN; EGEN, JACKSON; GARCES, FERNANDO; TAKENAKA, SHUNSUKE; KIM, AERYON; SAWANT, DEEPALI

To: AMGEN INC.

Reel/Frame 066784/0523 →

Continuity (3)

Division 17127629 · Dec 18, 2020

Provisional Application 62951408 · Dec 20, 2019

Related Publication 20240150481A1 · May 9, 2024

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