Trispecific binding proteins and methods of use
Provided herein are trispecific antigen-binding proteins comprising a domain binding to CD3, a half-life extension domain, and a domain binding to a target antigen. Also provided are pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such trispecific antigen-binding proteins. Also disclosed are methods of using the disclosed trispecific antigen-binding proteins in the prevention, and/or treatment diseases, conditions and disorders.
1. A trispecific antigen-binding protein, wherein said protein comprises
(a) a first domain (A) which comprises a single chain variable fragment (scFv) that specifically binds to human CD3;
(b) a second domain (B) which comprises a single domain antibody (sdAb) that binds human serum albumin; and
(c) a third domain (C) which comprises a sdAb that specifically binds to a target tumor antigen that is EGFR, PSMA, HER2 or MSLN,
wherein the domains are linked in the order H 2 N-(C)-(B)-(A)-COOH, or by linkers L2 and L2; and
wherein the protein is less than about 60 kDa.
2. The trispecific antigen-binding protein of claim 1 , wherein the first domain comprises complementary determining regions (CDRs) selected from the group consisting of muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1 and WT-31.
3. The trispecific antigen-binding protein of claim 1 , wherein the first domain is humanized or human.
4. The trispecific antigen-binding protein of claim 1 , wherein linkers L1 and L2 are each independently selected from (GS) n (SEQ ID NO: 49), (GGS) n (SEQ ID NO: 50), (GGGS) n (SEQ ID NO: 51), (GGSG) n (SEQ ID NO: 52), (GGSGG) n (SEQ ID NO: 53), or (GGGGS) n (SEQ ID NO: 54), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
5. The trispecific antigen-binding protein of claim 1 , wherein linkers L1 and L2 are each independently (GGGGS) 4 (SEQ ID NO: 55) or (GGGGS) 3 (SEQ ID NO: 56).
6. A pharmaceutical composition comprising (i) the trispecific antigen-binding protein according to claim 1 and (ii) a pharmaceutically acceptable carrier.
7. The trispecific antigen-binding protein of claim 1 , wherein the first domain is specific for CD3ε(epsilon).
8. The trispecific antigen-binding protein of claim 1 , wherein the first domain has crossreactivity with cynomolgus CD3.
9. The trispecific antigen-binding protein of claim 1 , wherein the third domain specifically binds to EGFR.
10. The trispecific antigen-binding protein of claim 1 , wherein the third domain specifically binds to PSMA.
11. The trispecific antigen-binding protein of claim 1 , wherein the third domain specifically binds to HER2.
12. The trispecific antigen-binding protein of claim 1 , wherein the third domain specifically binds to MSLN.