Multivalent CD20-binding molecule comprising Shiga toxin A subunit effector polypeptides and enriched compositions thereof

Patent metadata

Field	Value
Publication number	US-11248061-B2
Application number	US-201615290266-A
Country	US
Kind code	B2
Filing date	Oct 11, 2016
Priority date	Feb 5, 2015
Publication date	Feb 15, 2022
Grant date	Feb 15, 2022

Abstract

Provided herein are multivalent CD20-binding molecules, and compositions thereof, for use in selective killing of specific cell types and/or as therapeutics for the treatment of a variety of diseases, including cancer, tumors, and immune disorders. Certain multivalent CD20-binding molecules can be used to deliver agents into CD20-expressing cells, collecting diagnostic information, and/or monitoring the treatment of diseases, such as cancers, tumors, and immune disorders.

First claim

The invention is claimed as follows:

1. A multivalent CD20-binding molecule comprising two polypeptides associated through one or more cysteine disulfide bonds, wherein each polypeptide comprises:
a) one or more CD20 binding region comprising a single-chain variable fragment that is capable of specifically binding an extracellular part of a CD20; wherein the CD20 binding region comprises:
i) a heavy chain variable (VH) domain polypeptide comprising HCDR1, HCDR2, and HCDR3 amino acid sequences as shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively, and a light chain variable (VL) domain polypeptide comprising LCDR1, LCDR2, and LCDR3 amino acid sequences as shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively;
ii) a heavy chain variable (VH) domain polypeptide comprising HCDR1, HCDR2, and HCDR3 amino acid sequences as shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively, and a light chain variable (VL) domain polypeptide comprising LCDR1, LCDR2, and LCDR3 amino acid sequences as shown in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively;
iii) a heavy chain variable (VH) domain polypeptide comprising HCDR1, HCDR2, and HCDR3 amino acid sequences as shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively, and a light chain variable (VL) domain polypeptide comprising LCDR1, LCDR2, and LCDR3 amino acid sequences as shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively;
iv) a heavy chain variable (VH) domain polypeptide comprising HCDR1, HCDR2, and HCDR3 amino acid sequences as shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, respectively, and a light chain variable (VL) domain polypeptide comprising LCDR1, LCDR2, and LCDR3 amino acid sequences as shown in SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively;
v) a heavy chain variable (VH) domain polypeptide comprising HCDR1, HCDR2, and HCDR3 amino acid sequences as shown in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively, and a light chain variable (VL) domain polypeptide comprising LCDR1, LCDR2, and LCDR3 amino acid sequences as shown in SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively; or
vi) a heavy chain variable (VH) domain polypeptide comprising HCDR1, HCDR2, and HCDR3 amino acid sequences as shown in SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively, and a light chain variable (VL) domain polypeptide comprising LCDR1, LCDR2, and LCDR3 amino acid sequences as shown in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; and

b) one or more Shiga toxin A subunit effector polypeptides comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from:
(a) amino acids 75 to 251 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3;
(b) amino acids 1 to 241 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3;
(c) amino acids 1 to 251 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; and
(d) amino acids 1 to 261 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3;

wherein the amino acid residue corresponding to position 75 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is asparagine, the amino acid residue corresponding to position 77 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is tyrosine, the amino acid residue corresponding to position 167 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is glutamate, the amino acid residue corresponding to position 170 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is arginine, and the amino acid residue corresponding to position 176 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is arginine;
wherein the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin function.

2. The multivalent CD20-binding molecule of claim 1 , wherein administration of the multivalent CD20-binding molecule to a CD20 positive cell results in one or more of:
i) internalizing the multivalent CD20-binding molecule inside the cell, optionally within five hours, four hours, three hours, two hours, one hour, or thirty minutes at about 37 degrees Celsius;
ii) subcellular routing a Shiga toxin A subunit effector polypeptide to the cell's cytosol;
iii) disrupting a ribosome function within the cell; and
iv) killing of the cell.

3. The multivalent CD20-binding molecule of claim 1 , which comprises:
(a) two polypeptides, each having the amino acid sequence of any one of SEQ ID NOs: 47-175 and 294, and each polypeptide optionally further comprising an amino-terminal methionine residue; and
(b) a cysteine disulfide bond linking the two polypeptides, wherein the cysteine disulfide bond involves a cysteine residue in each of the two polypeptides located at amino acid position:
(i) 242 for polypeptides having the amino acid sequence of SEQ ID NOs: 120-175, or 294;
(ii) 482 for polypeptides having the amino acid sequence of SEQ ID NOs: 100, 105, or 107;
(iii) 483 for polypeptides having the amino acid sequence of SEQ ID NOs: 62 or 74;
(iv) 484 for polypeptides having the amino acid sequence of SEQ ID NOs: 79 or 86;
(v) 490 for the polypeptide having the amino acid sequence of SEQ ID NO: 49;
(vi) 491 for the polypeptide having the amino acid sequence of SEQ ID NO: 51;
(vii) 492 for polypeptides having the amino acid sequence of SEQ ID NOs: 56, 68, 91, 99, 103, or 104;
(viii) 493 for polypeptides having the amino acid sequence of SEQ ID NOs: 58, 70 or 81;
(ix) 494 for polypeptides having the amino acid sequence of SEQ ID NOs: 112 or 118;
(x) 495 for the polypeptide having the amino acid sequence of SEQ ID NO: 113;
(xi) 499 for the polypeptide having the amino acid sequence of SEQ ID NO: 52;
(xii) 500 for the polypeptide having the amino acid sequence of SEQ ID NO: 48;
(xiii) 501 for polypeptides having the amino acid sequence of SEQ ID NOs: 50, 61, 73, 96, 101, or 102;
(xiv) 502 for polypeptides having the amino acid sequence of SEQ ID NOs: 55, 64, 67, 76, 90, 92, 93, 97, or 98;
(xv) 503 for polypeptides having the amino acid sequence of SEQ ID NOs: 54, 57, 69, 78, 82, 84, 87, 88, 94, 110, 111, or 115;
(xvi) 504 for polypeptides having the amino acid sequence of SEQ ID NOs: 85, 108, or 114;
(xvii) 505 for the polypeptide having the amino acid sequence of SEQ ID NO: 119;
(xviii) 510 for the polypeptide having the amino acid sequence of SEQ ID NO: 47;
(xix) 511 for polypeptides having the amino acid sequence of SEQ ID NOs: 60, 72, or 106;
(xx) 512 for polypeptides having the amino acid sequence of SEQ ID NOs: 53, 63, 66, 75, 83, 89, or 95;
(xxi) 513 for polypeptides having the amino acid sequence of SEQ ID NOs: 80, 109, 116, or 117; or
(xxii) 521 for polypeptides having the amino acid sequence of SEQ ID NOs: 59, 65, 71, or 77.

4. The multivalent CD20-binding molecule of claim 3 , which is a homodimer and consists essentially of:
(a) two identical polypeptides each having the amino acid sequence selected from any one of SEQ ID NOs: 47-175 and 294; and
(b) a cysteine disulfide bond linking the two identical polypeptides, wherein the cysteine disulfide bond involves a cysteine residue in each of the two identical polypeptides located at amino acid position:
(i) 242 for polypeptides having the amino acid sequence of SEQ ID NOs: 120-175, or 294;
(ii) 482 for polypeptides having the amino acid sequence of SEQ ID NOs: 100, 105, or 107;
(iii) 483 for polypeptides having the amino acid sequence of SEQ ID NOs: 62 or 74;
(iv) 484 for polypeptides having the amino acid sequence of SEQ ID NOs: 79 or 86;
(v) 490 for the polypeptide having the amino acid sequence of SEQ ID NO: 49;
(vi) 491 for the polypeptide having the amino acid sequence of SEQ ID NO: 51;
(vii) 492 for polypeptides having the amino acid sequence of SEQ ID NOs: 56, 68, 91, 99, 103, or 104;
(viii) 493 for polypeptides having the amino acid sequence of SEQ ID NOs: 58, 70 or

Assignees

• Molecular Templates Inc

Inventors

• Poma Eric
• Willert Erin
• Kim Jason
• Higgins Jack
• Liu Jensing
• Flores-Lefranc Rodney

Classifications

• A61K39/0011

  Cancer antigens · CPC title

• A61K39/001124

  CD20 · CPC title

• C07K2319/55

  containing a fusion with a toxin, e.g. diphteria toxin · CPC title

• A61P37/02

  Immunomodulators · CPC title

• C07K2319/03

  containing a transmembrane segment · CPC title