Trispecific antagonist

Abstract

The present application discloses an anti-tumor antagonist that specifically binds to an immune checkpoint regulator and / or a component of an angiogenesis pathway and / or a component of a TGF pathway. The present application also discloses a method for treating a proliferative disease with the anti-tumor antagonist.

Description

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 691,658, filed on June 29, 2018, and U.S. Provisional Patent Application No. 62 / 823,989, filed on March 26, 2019, the contents of which are expressly incorporated herein by reference for all purposes. Technical Field

[0002] The present invention relates generally to cancer treatment, and in particular to bispecific inhibitors and trispecific antagonists capable of regulating pathways related to tumorigenesis, tumor immunity and angiogenesis. Background Art

[0003] The inability of the host to eliminate cancer cells remains a major problem. Although a growing number of therapeutic monoclonal antibodies have been approved for the treatment of various cancers, the emergence of resistance to these antibodies is frequently observed given the many different molecular pathways involved in cancer growth and progression to metastasis. Although the immune system is a primary mechanism for cancer prevention, cancer cells resist immune surveillance. Therefore, there is a need for improved therapeutic combinations of antagonists or antibodies, as well as methods for treating cancer and chronic viral infections with these agents. Summary of the Invention

[0004] One aspect of the present application relates to a trispecific antagonist, which comprises: an immunoglobulin scaffold, which comprises a CH1 domain, a CH2 domain and a CH3 domain; a first targeting domain, which comprises one or more variable region domains selected from an anti-PD-1 variable region domain, an anti-PD-L1 variable region domain, an anti-TIGIT variable region domain and an anti-LAG-3 variable region domain; a second targeting domain, which specifically binds to VEGF and comprises one or more peptide domains derived from VEGFR; and a third targeting domain, which comprises a peptide inhibitor of the angiopoietin / Tie-2 signaling pathway; wherein the second targeting domain is structurally connected to the carboxyl terminus of the CH3 domain, and wherein the third targeting domain is inserted within the CH3 domain.

[0005] Another aspect of the present application relates to a trispecific antagonist, which comprises: an immunoglobulin scaffold, which comprises a CH1 domain, a CH2 domain and a CH3 domain; a first targeting domain, which comprises one or more variable region domains selected from an anti-PD-1 variable region domain, an anti-PD-L1 variable region domain, an anti-TIGIT variable region domain and an anti-LAG-3 variable region domain; a second targeting domain of an anti-PD-1 variable region domain, an anti-PD-L1 variable region domain, an anti-TIGIT variable region domain and an anti-LAG-3 variable region domain; and a third targeting domain, which comprises a peptide inhibitor of the angiopoietin / Tie-2 signaling pathway; wherein the second targeting domain is structurally connected to the carboxyl terminus of the CH3 domain, and wherein the third targeting domain is inserted within the CH3 domain.

[0006] Another aspect of the present application relates to a trispecific antagonist, which comprises: an immunoglobulin scaffold, which comprises a CH1 domain, a CH2 domain and a CH3 domain; a first targeting domain, which comprises one or more variable region domains selected from anti-VEGF variable region domains; a second targeting domain, which comprises a TGF-β pathway inhibitor; and a third targeting domain, which comprises a peptide inhibitor of the angiopoietin / Tie-2 signaling pathway; wherein the second targeting domain is structurally connected to the carboxyl terminus of the CH3 domain, and wherein the third targeting domain is inserted within the CH3 domain.

[0007] Another aspect of the present application relates to a trispecific antagonist, which comprises: an immunoglobulin scaffold, which comprises a CH1 domain, a CH2 domain and a CH3 domain; a first targeting domain, which comprises one or more variable region domains selected from a PD-1 variable region domain, an anti-PD-L1 variable region domain, an anti-TIGIT variable region domain and an anti-LAG-3 variable region domain; a second targeting domain, which comprises a component of a VEGFR; and a third targeting domain, which comprises a TGF-β pathway inhibitor; wherein the second targeting domain is structurally connected to the carboxyl terminus of the CH3 domain, and wherein the third targeting domain is structurally connected to the carboxyl terminus of the second targeting domain.

[0008] Another aspect of the present application relates to a bispecific antagonist, which comprises: an immunoglobulin scaffold, which comprises a CH1 domain, a CH2 domain and a CH3 domain; a first targeting domain, which comprises one or more variable region domains selected from a PD-1 variable region domain, an anti-PD-L1 variable region domain, an anti-TIGIT variable region domain and an anti-LAG-3 variable region domain; a second targeting domain, which comprises a component of VEGFR; wherein the third targeting domain is structurally connected to the carboxyl terminus of the second targeting domain.

[0009] Another aspect of the present application relates to a method for treating a cell proliferative disease, comprising the step of administering an effective amount of the trispecific antagonist of the present application to a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The HCVR and LCVR sequences of certain checkpoint antagonists, anti-PD-1, anti-PD-L1, anti-TIGIT, anti-LAG-3, monoclonal antibodies (Mabs) are shown.

[0011] Figure 2 The HCVR and LCVR and other domain sequences of Trebananib, bevacizumab, ranibizumab, Tie2, VEGF and TGF-β antagonists are shown.

[0012] Figures 3A-3H Eight trispecific antitumor antagonists are shown, namely TS-ZPT-1.1, TS-ZPT-1.2, TS-ZPT-1.3, TS-ZPT-2, TS-ZPT-3, TS-ZPT-4, TS-ZPT-5 and TS-ZPT-6, which have an IgG1 or IgG4 backbone and contain: (1) anti-PD-1 variable region (VH1, VL1) domains or other checkpoint antibody variable region domains; (2) aflibercept fusion protein domains: (i) at the amino terminus of one or both IgG arms ( Figures 3A-3C , 3F); (ii) at the carboxyl terminus of each antagonist ( Figure 3D , 3E, 3H); or (iii) between the carboxyl terminus of the CH3 domain and trobactam (or other biological peptide) ( Figure 3G ); (3) trombopag peptide (or other biological peptide): (i) fused to the carboxyl terminus of each CH3 region ( Figures 3A-3D ); (ii) inserted into each of the two CH3 regions ( Figure 3E); (iii) fused to the carboxyl terminus of each CH1 region ( Figure 3F (iv) fused to the carboxyl terminus of each afipronil fusion protein domain ( Figure 3G ); or (v) fused to the carboxyl terminus of each CL region ( Figure 3H The aflibercept fusion protein domain contains the vascular endothelial growth factor (VEGF) binding portion from the extracellular domains of human VEGF receptors 1 and 2, while the trombopag peptide targets and binds to Ang1 and Ang2, thereby preventing the interaction of Ang1 and / or Ang2 with their cognate Tie2 receptor.

[0013] Figures 4A-4C Three different trispecific anti-tumor antagonists are shown, namely TS-ZPT-7, TS-ZPT-8 and TS-ZPT-9, which contain (1) an aflibercept fusion protein domain at each carboxyl terminus; (2) an anti-PD-1 or other checkpoint antagonist antibody variable region domain (VH1, VL1); and (3) trombopag (or other biological peptide): (i) inserted into each of the two CH3 regions ( Figure 4A ); (ii) inserted between the carboxyl terminus of each CH2 region and the aflibercept fusion protein domain at the carboxyl terminus of each polypeptide chain in the antagonist ( Figure 4B ); or (iii) fused to the amino terminus of each IgG1 arm ( Figure 4C ).

[0014] Figures 5A-5G Seven different trispecific anti-tumor antagonists, namely TS-LPT-1, TS-LPT-2, TS-LPT-3, TS-LPT-4, TS-LPT-5, TS-M3 and TS-M4, are shown, comprising: (1) VH2 and VL2 regions corresponding to the variable region domains of ranibizumab (Lucentis), bevacizumab (Avastin) or other anti-VEGF; (2) VH1 and VL1 regions corresponding to the variable region domains of anti-PD-1 or other checkpoint antagonist antibodies; (3) trombopag (or other biological peptide): (i) inserted into each of the two CH3 regions ( Figure 5A ); (ii) fused to the carboxyl terminus of each CL1 region ( Figure 5B (iii) fused to the carboxyl terminus of the CH1 region of each of the two polypeptide chains ( Figure 5C ); (iv) fused to the carboxyl terminus of the CL region of each of the two polypeptide chains ( Figure 5D ); (v) fused to the carboxyl terminus of the CH1 region of each of the two polypeptide chains and to the carboxyl terminus of the CL region of each of the two polypeptide chains ( Figure 5E )(vi and vii are fused to the carboxyl terminus of CH3 ( Figure 5F and 5G The ranibizumab variable region domain is derived from bevacizumab, and both are known to block the binding of human VEGF-A to VEGFR 1 and 2. Exemplary sequences of TS-LPT-1, TS-LPT-2, TS-LPT-3, TS-LPT-4, TS-LPT-5, TS-M3, and TS-M4 are shown in SEQ ID NOs: 247-269.

[0015] Figure 6A and 6B Shown in Figures 3A-3E Non-reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of trispecific anti-tumor antagonists expressed in transiently transfected HEK293 cells is shown in 3G, 3H and 5A.

[0016] Figure 7A and 7B IC50 values ​​(nM) calculated from cell-based binding assays are shown, reflecting Figures 3A-3H The ability of a trispecific anti-tumor antagonist to block the interaction between human PD-1 and human PD-L1 is shown.

[0017] Figure 8A and 8B IC50 values ​​(nM) calculated from the binding assay are shown, reflecting the Figure 3D , 3E and 3G show the ability of a trispecific antitumor antagonist to block the interaction between VEGF and VEGFR-2.

[0018] Figure 9 IC50 values ​​(nM) calculated from the binding assay are shown, reflecting the Figure 3B 、 3D , 3G, 3H and 5A. Compared with the bevacizumab-trabanib fusion control molecule, Figure 3B , 3D, 3G, 3H, and 5A blocked the interaction between Ang2 and Tie2.

[0019] Figure 10Shown are the results of size exclusion chromatography (SEC) analysis of purified trispecific anti-tumor antagonists TS-ZPT-2 (2P17), TS-ZPT-3L (2P17), and TS-ZPT-5 (2P17), as well as the anti-PD-1 monospecific antibody 2P17, produced by transient transfection of HEK293 cells. The percentages of high molecular weight (HWM%) species, low molecular weight (LMW%) species, and dimeric molecules (Dimer%) are shown. The amino acid sequences of the HCVRs and LCVRs of these trispecific anti-tumor antagonists are shown in SEQ ID NOs: 201-204.

[0020] Figure 11 Shown are the results of size exclusion chromatography (SEC) analysis of purified trispecific anti-tumor antagonists TS-ZPT-2 (2P17), TS-ZPT-3L (2P17), and TS-ZPT-5 (2P17) expressed from stable CHO cell pools. Two transfected pools of TS-ZPT-3L (2P17) were evaluated. The percentages of high molecular weight (HWM%) species, low molecular weight (LMW%) species, and dimeric molecules (Dimer%) are shown.

[0021] Figure 12A and 12B Shown are the chromatographic traces and size exclusion chromatography (SEC) results of the purified trispecific anti-tumor antagonist TS-ZPT-3L (2P17) purified from a CHO stable pool and stored at 4°C for 112 days. The percentages of high molecular weight (HWM%) species, low molecular weight (LMW%) species, and dimeric molecules (Dimer%) are shown.

[0022] Figure 13A Shown are the heavy chain (SEQ ID NO: 202) and light chain amino acid sequences (SEQ ID NO: 201) for a trispecific anti-tumor antagonist with a long peptide of trombanib, namely TS-ZPT-3L (2P17), comprising (1) an anti-PD-1 with the variable region domains (VH1, VL1) of another checkpoint antagonist antibody 2P17 (fused to the carboxyl terminus of each antagonist and connected to the CH3 domain using a 3xG4S linker) and (3) a trombanib peptide (or other biological peptide) inserted within each of the two CH3 regions. Figure 13B Exemplary molecules derived from these sequences are shown.

[0023] Figure 14AShown are the heavy chain (SEQ ID NO: 200) and light chain amino acid sequences (SEQ ID NO: 201) of an exemplary trispecific anti-tumor antagonist with a short peptide of trombanib, namely TS-ZPT-3S (2P17, comprising: (1) anti-PD-1 with the variable region domains (VH1, VL1) of another checkpoint antagonist antibody 2P17 (fused to the carboxyl terminus of each antagonist and connected to the aflibercept VEFG binding domain of the CH3 structure via a 3xG4S linker); (2) a trombanib peptide (or other biological peptide) inserted in each of the two CH3 regions; and (3) a single copy of a trombanib blocking peptide. Figure 14B Exemplary molecules derived from these sequences are shown.

[0024] Figure 15 Shown in Figure 5F and Figure 5G as well as Figures 13A-14B Shown is SDS-PAGE of various trispecific anti-tumor antagonists produced by transiently transfected HEK293 cells.

[0025] Figure 16 Shown are the results of size exclusion chromatography analysis of TS-ZPT-3L(2P17), TS-ZPT-3S(2P17), TS-ZPT-3L(2P16), and TS-ZPT-3S(2P16) produced by HEK293 cells.

[0026] Figure 17 Shown are size exclusion chromatography analyses of TS-ZPT-3L(2P17) and TS-ZPT-3S(2P17) stored at 4°C over time.

[0027] Figure 18A Shown are the results of a cell-based binding assay measuring the ability of TS-ZPT-3S (2P16 and 2P17) and TS-ZPT-3L (2P16 and 2P17) trispecific anti-tumor antagonists to block the interaction between PD-1 and PD-L1 compared to the parental antibody 2P17. Figure 18B IC50 values ​​(nM) obtained from this analysis are shown.

[0028] Figure 19A Shown are the results of a cell-based bioassay measuring the ability of TS-ZPT-3S (2P16 and 2P17) and TS-ZPT-3L (2P16 and 2P17) trispecific anti-tumor antagonists to block the interaction between VEGF and VEGFR-2 compared to the benchmark antibody bevacizumab and a nonspecific control antibody. Figure 19B IC50 values ​​(nM) obtained from this analysis are shown.

[0029] Figure 20A Shown are the results of a binding assay measuring the ability of the TS-TS-ZPT-3S (2P16 and 2P17) and TS-ZPT-3L (2P16 and 2P17) trispecific anti-tumor antagonists to block the interaction between Ang2 and Tie2 with the benchmark molecule tramadanib and a nonspecific control antibody. Figure 20B IC50 values ​​(nM) obtained from this analysis are shown.

[0030] Figures 21A-21E The results of PD-1 binding to the trispecific anti-tumor antagonists TS-ZPT-3S (2P16 and 2P17) and TS-ZPT-3L (2P16 and 2P17) (determined by bio-layer interferometry compared to a benchmark anti-PD-1 antibody) and their resulting binding affinity constants ( Figure 21F ).

[0031] Figures 22A-22E The results of VEGF165 binding to the trispecific antitumor antagonists TS-ZPT-3S (2P16 and 2P17) and TS-ZPT-3L (2P16 and 2P17) as determined by biolayer interferometry and compared with the standard antibody bevacizumab are shown, as well as their resulting binding affinity constants ( Figure 22F ).

[0032] Figures 23A-23E The results of Ang2 binding to the trispecific antitumor antagonists TS-ZPT-3S (2P16 and 2P17) and TS-ZPT-3L (2P16 and 2P17) compared to the benchmark molecule trombanib as determined by biolayer interferometry and their resulting binding affinity constants ( Figure 23F ).

[0033] Figure 24 Shown is the sequential binding of TS-ZPT-3S (2P16 and 2P17) and TS-ZPT-3L (2P16 and 2P17) trispecific anti-tumor antagonists to their three binding partner molecules using the Octet RED96 system (ForteBio) characterized using bio-layer interferometry.

[0034] Figure 25 Shown are the pharmacokinetic profiles of the TS-ZPT-3L(2P17) trispecific anti-tumor antagonist in four individual mice.

[0035] Figures 26A-26BCertain trispecific anti-tumor checkpoint antagonist structures are shown, in which: (1) the VH1 and VL1 regions correspond to checkpoint #1, e.g., anti-PD-1 or anti-PD-L1 variable region domains; (2) the VH2 and VL2 regions correspond to checkpoint #2, e.g., anti-TIGIT, anti-LAG-3 variable region domains; and (3) the circular region corresponds to trombopag (or any other biological peptide) inserted into the CH3 domain.

[0036] Figure 27 Shown is the trispecific anti-tumor antagonist TS-A1BT-1, which has an amino-terminal anti-VEGF variable region (VH1, VL1) containing two amino acid substitutions in the VH region (E6Q, L11V) from (Avastin / bevacizumab) in a mutant IgG1 (K447A) scaffold; a short peptide of tramabanib inserted into the IgG1 CH3 domain; and a carboxy-terminal TGF-β1 RII ECD connected to the CH3 domain using a 4xG4S linker. The amino acid sequences of the HC and LC of this trispecific anti-tumor antagonist are listed in SEQ ID NOs: 229 and 230.

[0037] Figure 28 Shown is a non-reducing SDS-PAGE analysis of TS-A1BT-1 produced by HEK293 transiently transfected cells compared to a control transfected antibody 2P17, demonstrating good expression levels of the trispecific anti-tumor antagonist. Figure 29 Size exclusion chromatography analysis of TS-AIBT-1 is shown.

[0038] Figures 30A-30F Six trispecific anti-tumor antagonist structures containing the TGF-βRII extracellular domain (ECD) are shown: TS-ZPB-1, TS-ZPB-2, TS-ZPB-3, TS-ZPB-4, TS-ZPB-5, and TS-ZPB-6.

[0039] Figure 31A Shown is non-reducing SDS-PAGE analysis of the trispecific anti-tumor antagonists TS-ZPB-1(2P17), TS-ZPB-2(2P17), TS-ZPB-3(2P17), TS-ZPB-5(2P17), and TS-ZPLB-1 transiently expressed in HEK 293 cells before and after purification.

[0040] Figure 32AExemplary size exclusion chromatography (SEC) curves of TS-ZPB-1 (2P17), TS-ZPB-2 (2P17), TS-ZPB-3 (2P17), TS-ZPB-5 (2P17), and TS-ZPB-1 (2P17) are shown (compared to TGF-β-RII-Fc). The percentages of high molecular weight (HWM%) species, low molecular weight (LMW%) species, and dimeric molecules (Dimer%) are shown. Figure 32B middle.

[0041] Figure 33A Shown are the results of a cell-based binding assay measuring the ability of TS-ZPB-1 (2P17), TS-ZPB-2 (2P17), TS-ZPB-3 (2P17), and TS-ZPB-5 (2P17) to block the interaction between PD-1 and PD-L1. Figure 33B IC50 values ​​(nM) obtained from this analysis are shown.

[0042] Figure 34A Shown are the results of a bioassay measuring the ability of TS-ZPB-1 (2R17), TS-ZPB-2 (2P17), TS-ZPB-3 (2P17), TS-ZPB-5 (2P17), and TS-ZPLB-1 to block TGF-β1 signaling compared to the benchmark TGF-β1 RII-Fc. Figure 34B IC50 values ​​(nM) obtained from this analysis are shown.

[0043] Figure 35A Shown are the results of an ELISA assay measuring the ability of TS-ZPB-1(2P17), TS-ZPB-2(2P17), TS-ZPB-3(2P17), TS-ZPB-5(2P17), and TS-ZPLB-1 to block the interaction between VEGF and VEGFR-2 compared to the benchmark bevacizumab. Figure 35B IC50 values ​​(nM) obtained from this analysis are shown.

[0044] Figures 36A-36C Three trispecific antagonists are shown: TS-ZPB-5 and the variants TS-ZPB-5A (with a K447A mutation in the CH3 domain), and TS-ZPB-5B (with an additional mutation to eliminate N-linked glycosylation of the afipronil VEGF binding domain).

[0045] Figure 37 Shown are the expression levels of the trispecific antitumor antagonists TS-ZPB-5, TS-ZPB-5A, and TS-ZPB-5B produced by transiently transfected HEK293 cells.

[0046] Figure 38 Shown are the results of a cell-based assay measuring the ability of the trispecific antitumor antagonists TS-ZPB-5, TS-ZPB-5A, and TS-ZPB-5B to block the interaction between VEGF and VEGFR-2.

[0047] Figure 39 Shown are the results of a bioassay measuring the ability of the trispecific anti-tumor antagonists TS-ZPB-5, TS-ZPB-5A, and TS-ZPB-5B to block TGFβ1 signaling.

[0048] Figure 40 Shown are the pharmacokinetic profiles of TS-ZPT-5A and TS-ZPT-5B in each of two mice.

[0049] Figures 41A-41C Three different bispecific anti-tumor antagonist structures are shown as follows: Bi-ZPL-1 (or Bi-ZP-1) and Bi-ZPL-2 (or Bi-ZP-2) and Bi-ZPL-3 (or Bi-ZP-3), each comprising (1) an anti-PD-1 or anti-PD-L1 variable region and (2) an aflibercept VEGF binding domain, which is (i) fused to the amino terminus of VH1 (ii) mutated to eliminate the N-linked glycosylation site and fused to the amino terminus of VH1; and (iii) mutated to eliminate the N-linked glycosylation site and fused to the carboxyl terminus of CH3 via a 3xG4S linker.

[0050] Figure 42A Shown are the results of a cell-based assay measuring the ability of the Bi-ZPL-1 molecule to inhibit the interaction between PD-1 and PD-1 compared to a benchmark anti-PD-1 antibody. Figure 42B The resulting IC50 values ​​(nM) obtained from this analysis are shown.

[0051] Figure 43A Shown are the results of a bioassay measuring the ability of the Bi-ZPL-1 molecule to inhibit the interaction between VEGF and VEGFR-2 compared to a benchmark anti-PD-1 antibody. Figure 43B The resulting IC50 values ​​(nM) are shown.

[0052] Figure 44 Shown are the results of size exclusion chromatography analysis of Protein A purified Bi-ZP-2 and Bi-ZPL-3.

[0053] Figure 45 Pharmacokinetic evaluation of Bi-ZP-2, Bi-ZP-2, and Bi-ZPL-3 in mice is shown.

[0054] Figures 46A-46C Sequences of exemplary framework regions are shown. Figure 46A Exemplary framework regions of anti-TIGIT mAbs are shown. Figure 46B Exemplary framework regions of anti-PD-1 and anti-PD-L1 mAbs are shown. Figure 46C Exemplary framework regions of anti-LAG3 mAbs are shown. DETAILED DESCRIPTION

[0055] definition

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which the disclosed methods and compositions belong. It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural forms unless the context clearly indicates otherwise. Thus, for example, reference to "a peptide" includes reference to "one or more" peptides or "several" peptides. Any issued patent or patent application publication described in this application is expressly incorporated herein by reference with respect to the teachings of this application.

[0057] As used herein, the term "PD-1" refers to any form of PD-1 and variants thereof that retain at least a portion of the activity of PD-1. Unless otherwise indicated, for example, by specific reference to human PD-1, PD-1 includes native sequence PD-1 from all mammalian species, such as humans, dogs, cats, horses, and cattle. An exemplary human PD-1 amino acid sequence is as follows: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKE SLRAELRVTERRAEVPTAHSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL(SEQ ID NO:180)

[0058] As used herein, the term "PD-L1" refers to any form of PD-L1 and variants thereof that retain at least a portion of the activity of PD-L1. Unless otherwise indicated, e.g., by specific reference to human PD-L1, PD-L1 includes native sequence PD-L1 of all mammalian species, e.g., humans, dogs, cats, horses, and cows. An exemplary human PD-L1 amino acid sequence is shown below: MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO: 181).

[0059] As used herein, the term "TIGIT" refers to any form of TIGIT and variants thereof that retain at least a portion of the activity of TIGIT. Unless otherwise indicated, for example by specific reference to human TIGIT, TIGIT includes native sequence TIGIT of all mammalian species, such as humans, dogs, cats, horses, and cattle. An exemplary human TIGIT amino acid sequence is shown below: MTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPLLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFFTETG (SEQ ID NO: 179).

[0060] As used herein, the term "LAG-3" refers to any form of LAG-3 and variants thereof that retain at least a portion of the activity of lymphocyte activation gene 3 (LAG-3). Unless otherwise indicated, such as by specific reference to human LAG-3, LAG-3 includes native sequence LAG-3 from all mammalian species, such as humans, dogs, cats, horses, and cattle. An exemplary human LAG-3 amino acid sequence is shown below: (SEQ ID NO: 293).

[0061] The term "agonist" refers to a substance that promotes (i.e., induces, causes, enhances, or increases) the biological activity or effect of another molecule. The term agonist includes substances that bind to receptors, such as antibodies, and substances that promote receptor function without binding to it (e.g., by activating related proteins).

[0062] The term "antagonist" or "inhibitor" refers to a substance that prevents, blocks, inhibits, neutralizes or reduces the biological activity or effect of another molecule, such as a receptor or ligand. The antagonist can be a monospecific antibody, a bispecific antibody or a trispecific antibody.

[0063] As used herein, the term "antibody" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen through one or more immunoglobulin variable regions. An antibody can be a complete antibody, an antigen-binding fragment, or a single chain thereof. The term "antibody" encompasses a variety of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as alpha, delta, epsilon, gamma, and mu, or alpha, delta, epsilon, gamma, and mu, with several subclasses (e.g., gamma 1-gamma 4). The properties of this chain determine the "class" of the antibody, i.e., IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, etc., are well characterized and are known to have functional specificities. In view of the present disclosure, modified forms of each of these classes and isotypes are readily discernible to those skilled in the art and are therefore within the scope of the present disclosure. All immunoglobulin classes are within the scope of the present disclosure, and the following discussion will generally be directed to the IgG class of immunoglobulin molecules.

[0064] The antibodies or antibody antagonists of the present application may include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific, bispecific, human, humanized, primatized, chimeric and single-chain antibodies. Antibodies disclosed herein may be from any animal origin, including birds and mammals. Preferably, the antibody is from humans, mice, donkeys, rabbits, goats, guinea pigs, camels, llamas, horses or chickens. In some embodiments, the origin of the variable region may be condricthoid (e.g., from sharks).

[0065] The term "antibody fragment" or "antigen-binding fragment" is used to refer to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies. Regardless of the structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes DARTs and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein comprising an immunoglobulin variable region that acts like an antibody by binding to a specific antigen to form a complex. "Single-chain fragment variable region" or "scFv" refers to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In some aspects, the domains are connected to a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility and serine or threonine for solubility, and can connect the N-terminus of VH or the C-terminus of VL, or vice versa. Despite the removal of the constant region and the introduction of the linker, this protein still retains the specificity of the original immunoglobulin. Regarding IgG, the standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of about 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. The four chains are usually connected by disulfide bonds in a "Y" configuration, wherein the light chain is connected to the heavy chain from the mouth of the "Y" and extends through the variable region.

[0066] Both light and heavy chains are divided into structurally and functionally homologous domains. The terms "constant region" and "variable region" are used functionally. In this regard, it will be understood that the variable region domains of the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. In contrast, the constant domains (CL) of the light chain and the constant domains (CH1, CH2 or CH3) of the heavy chain confer important biological properties, such as secretion, placental mobility, Fc receptor binding, complement fixation, etc. By convention, the numbering of the constant region domains in conventional antibodies increases as they are away from the antigen binding site or amino terminus of the antibody. In conventional antibodies, the N-terminal portion is the variable region, while the carboxyl terminal portion is the constant region. The CH3 and CL domains actually contain the carboxyl termini of the heavy and light chains, respectively. Exemplary CHI-CH2-CH3 sequences described herein include wild-type IgG1 (SEQ ID NO: 274), IgG1 with a K447A mutation (SEQ ID NO: 275), IgG1 with an N297A mutation (SEQ ID NO: 276), wild-type IgG2 (SEQ ID NO: 278), IgG2 lacking a carboxyl-terminal lysine (SEQ ID NO: 279), IgG4 with an S231P mutation in the hinge region (SEQ ID NO: 280), IgG4 with S23P and K447A (SEQ ID NO: 281), and IgG4 with an S231P mutation and lacking a carboxyl-terminal lysine (SEQ ID NO: 282). An exemplary CL sequence is SEQ ID NO: 292.

[0067] The term "Fc fragment" or "Fc" refers to a portion of an antibody that does not have antigen binding activity but was initially observed to crystallize readily, and is therefore named an Fc fragment (for fragment crystallizability). This fragment corresponds to the paired CH2 and CH3 domains and is the portion of the antibody molecule that interacts with effector molecules and cells. The Fc fragments described herein can be derived from human IgG1, IgG2, and IgG4 antibodies with α-glycosylation modifications, hinge region mutations, and carboxyl-terminal lysine deletions. Exemplary Fc sequences described in the present application include the following: wild-type IgG1 Fc (SEQ ID NO: 210), α-glycosylated IgG1 Fc (SEQ ID NO: 211), IgG4 Fc with a hinge region mutation (SEQ ID NO: 212), wild-type IgG2 Fc (SEQ ID NO: 213), IgG1 Fc with a carboxyl-terminal lysine deletion (SEQ ID NO: 214), α-glycosylated IgG1 Fc with a carboxyl-terminal lysine deletion (SEQ ID NO: 215), IgG4 Fc with a hinge region mutation and a carboxyl-terminal lysine deletion (SEQ ID NO: 216), and IgG2 Fc with a carboxyl-terminal lysine deletion (SEQ ID NO: 217).

[0068] As mentioned above, the variable region allows the antibody to selectively recognize and specifically bind to the epitope on the antigen. That is, the VL domain and VH domain of the antibody or the complementary determining region (CDR) subset of the antibody combine to form a variable region that limits the three-dimensional antigen binding site. This four-membered antibody structure forms the antigen binding site present at the end of each arm of each Y configuration. More specifically, the antigen binding site is defined by three CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) on each of the VH and VL chains. In some cases, for example, some immunoglobulin molecules are derived from camelid species or are engineered based on camelid immunoglobulins. Alternatively, the immunoglobulin molecule can be composed of a heavy chain without a light chain or a light chain without a heavy chain.

[0069] In naturally occurring antibodies, the six CDRs present in each antigen-binding domain are short, non-continuous amino acid sequences. Since the antibody presents its three-dimensional configuration in an aqueous environment, these CDRs are specifically positioned to form an antigen-binding domain. The remaining amino acids in the antigen-binding domain, referred to as "framework" region domains, show less intermolecular variability. The framework region mainly adopts a β-sheet conformation, and the CDRs form loops that connect and, in some cases, form a part of the β-sheet structure. Therefore, the framework region plays the role of forming a scaffold that positions the CDRs in the correct direction through interchain non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its associated epitope (cognate epitope). Due to having been precisely defined, those of ordinary skill in the art can easily identify the amino acids comprising CDRs and framework regions, respectively, for any given heavy or light chain variable region.

[0070] As used herein, the terms "VH1" and "VH2" refer to immunoglobulin heavy chain variable region domains that correspond to two different binding specificities. Similarly, the terms "VL1" and "VL2" refer to light chain variable region domains that correspond to two different binding specificities. When used together, it will be understood that the VH1 and VL1 domains define a common binding specificity, and the VH2 and VL2 domains define another binding specificity.

[0071] As used herein, the term "framework region (FR)" refers to the variable region domain residues other than the CDR residues. Each variable region domain typically has four FRs flanking the corresponding CDRs. For example, a VH domain typically has four HFRs: HFR1, HFR2, HFR3, and HFR4, which flank the three HCDRs in the configuration of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4. Similarly, an LH domain typically has four LFRs, namely LFR1, LFR2, LFR3, and LFR4, which flank the three LCDRs in the configuration of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4. Exemplary FRs are summarized in Figures 46A-46C middle.

[0072] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be associated with either a κ or λ light chain. Typically, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other via covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the base of each chain.

[0073] As used herein, the term "light chain constant region (CL)" includes the amino acid sequence CL (SEQ ID NO: 292) derived from an antibody light chain. Preferably, the light chain constant region includes at least one of a constant kappa domain or a constant lambda domain.

[0074] As used herein, the term "heavy chain constant region (CH)" includes amino acid sequences derived from immunoglobulin heavy chains. A polypeptide comprising a heavy chain constant region comprises at least one of the following: a CH1 domain (SEQ ID NOs: 290-291), a hinge region (e.g., an upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in the present disclosure may comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge region domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge region domain, and a CH3 domain; or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge region domain, a CH2 domain, and a CH3 domain. In some embodiments, a polypeptide of the present disclosure comprises a polypeptide chain comprising a CH3 domain. In addition, an antibody for use in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). It will be appreciated that the heavy chain constant regions may be modified such that their amino acid sequence differs from that of naturally occurring immunoglobulin molecules.

[0075] For example, as reflected in the disclosure herein below, Applicants have discovered that CH3 domains can tolerate or adapt to large insertions (e.g., greater than 100 amino acids) in the Fc loop of the CH3 domain (e.g., see Figure 3E ). Thus, in this application, any disclosed inhibitor domain can be similarly inserted into the Fc loop of the heavy chain in a manner similar to the insertion of the trombanib or TGFβ1RII ECD domain in the Fc loop.

[0076] The heavy chain constant regions of the antibodies disclosed herein can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of the polypeptide can include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain constant region can include a hinge region that is partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. In another example, the heavy chain portion can include a chimeric hinge region that is partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0077] A "light chain-heavy chain pair" refers to a collection of light and heavy chains that can form dimers through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0078] The subunit structures and three-dimensional configurations of the constant regions of the various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino-terminal variable region domain of an immunoglobulin heavy chain, while the term "CH1 domain" includes the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino-terminal to the hinge region of the immunoglobulin heavy chain molecule.

[0079] As used herein, the term "CH2 domain" includes the portion of a heavy chain molecule extending from about residue 244 to residue 360 ​​of an antibody, using the conventional numbering scheme (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system). The CH2 domain is unique in that it is not closely paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of an intact native IgG molecule. The CH3 domain extends from the CH2 domain of the IgG molecule to the carboxyl terminus and comprises approximately 108 residues.

[0080] As used herein, the term "hinge region" includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. The hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains.

[0081] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with another thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are structurally linked by disulfide bonds, and the two heavy chains are structurally linked by two disulfide bonds at positions corresponding to 239 and 242 (using the Kabat numbering system (positions 226 or 229, EU numbering system)).

[0082] As used herein, a "variant" of an antibody, antibody fragment, or antibody domain refers to an antibody, antibody fragment, or antibody domain that: (1) has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the original antibody, antibody fragment, or antibody domain, and (2) specifically binds to the same target that the original antibody, antibody fragment, or antibody domain specifically binds to. It should be understood that where sequence identity is expressed in the form of "at least x% identical" or "at least x% identical," such embodiments include any and all numerical percentages equal to or above the lower limit. Furthermore, it should be understood that where an amino acid sequence is presented in this application, it should be interpreted as additionally disclosing or encompassing an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that amino acid sequence.

[0083] It should be understood that where ranges of sequence homology are presented herein, for example in the phrase "about 80% to about 100%", such embodiments include any and all subranges defined by any integer therein, where the lower numerical limit can be any integer between 80 and 100.

[0084] As used herein, the phrase "humanized antibody" refers to an antibody derived from a non-human antibody, typically a mouse monoclonal antibody. Alternatively, a humanized antibody can be derived from a chimeric antibody that retains or substantially retains the antigen-binding properties of the parent non-human antibody but exhibits reduced immunogenicity when administered to humans compared to the parent antibody.

[0085] As used herein, the phrase "chimeric antibody" refers to an antibody in which the immunoreactive region domain or site is obtained or derived from a first species and the constant region (which may be complete, partial or modified according to the present disclosure) is obtained from a second species. In certain embodiments, the target binding region or site will be from a non-human source (e.g., mouse or primate) and the constant region is human.

[0086] Included within the scope of the multispecific antibodies of the present application are various compositions and methods, including asymmetric IgG-like antibodies (e.g., triomab / quadroma, TrionPharma / Fresenius Biotech); knobs-into-holes antibodies (Genentech); Cross MAb (Roche); electrostatically matched antibodies (AMGEN); LUZ-Y (Genentech); chain exchange engineered domain (SEED) bodies (EMD Serono; Biolononic, Merus); Fab exchange antibodies (Genmab), symmetric IgG-like antibodies (e.g., dual targeting (DT)-Ig (GSK / Domantis); two-in-one antibodies (Genentech); cross-linked monoclonal antibodies (Karmanos Cancer Center), mAb2 (F-star); Cov X-body (Cov X / Pfizer); dual variable domain (DVD)-Ig fusion protein (Abbott); IgG-like bispecific antibody (Eli Lilly); Ts2Ab (Medimmune / AZ); BsAb (ZymoGenetics); HERCULES (Biogen Idec, TvAb, Roche); scFv / Fc fusion; SCORPION (Emergent BioSolutions / Trubion, ZymoGenetics / BMS); dual affinity repositioning technology (Fc-DART), MacroGenics; bis(scFv)2-Fabs (National Research Center for Antibody Medicine) Medicine); F(ab)2 fusion proteins (Medarex / AMGEN); bi-acting or Bis-Fab (Genentech); Dock-and-Lock (DNL, ImmunoMedics); Fab-Fv (UCB-Celltech); scFv-based antibodies and diabody-based antibodies (e.g., bispecific T cell engagers (BiTEs, Micromet); tandem diabodies (Tandab, Affimed); DARTs (MacroGenics); single-chain diabodies; TCR-like antibodies (AIT, Receptor Logics);Human serum albumin scFv fusion protein (Merrimack), COMBODIES (Epigen Biotech) and IgG / no n-IgG fusion proteins (e.g., immunocytokines (EMDSerono, Philogen, ImmunGene, ImmunoMedics)).

[0087] "Specific binding" or "having specificity" generally refers to the binding of an antibody to an epitope through its antigen binding domain, and binding requires a certain complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to the epitope more easily through its antigen binding domain than to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which an antibody binds to an epitope. For example, antibody "A" can be considered to have a higher specificity for a given epitope than antibody "B", or it can be said that antibody "A" binds to epitope "C" with a higher specificity than to a related epitope "D". In some embodiments, an antibody or antibody fragment is considered to bind to an antigen with an (IQ) equal to or less than 10 -6 M, equal to or less than 10 -7 M, equal to or less than 10 -8 M, equal to or less than 10 -9 M or equal to or less than 10 -10 The dissociation constant (K d ) form a complex, then the antibody or antibody fragment is "specific" for the antigen.

[0088] The term "antagonist antibody" refers to an antibody that binds to a target and prevents or reduces the biological effect of the target. In some embodiments, the term can refer to an antibody that prevents the target to which it binds, such as TIGIT, from performing a biological function.

[0089] As used herein, "anti-PD-1 antagonist antibodies" refer to antibodies that are capable of inhibiting PD-1 biological activity and / or downstream events mediated by PD-1. Anti-PD-1 antagonist antibodies include antibodies that block, antagonize, inhibit or reduce (including significantly reduce to any extent) PD-1 biological activity (including PD-1-mediated downstream events, such as PD-1 binding and downstream signal transduction, inhibition), inhibition of T cell proliferation, inhibition of T cell activation, inhibition of IFN secretion, inhibition of IL-2 secretion, inhibition of TNF secretion, induction of IL-10, and inhibition of anti-tumor immune responses. For the purposes of this application, it will be clearly understood that the term "anti-PD-1 antagonist antibody" (interchangeably referred to as "antagonist PD-1 antibody", "antagonist anti-PD-1 antibody" or "PD-1 antagonist antibody") includes all previously identified terms, titles, functional states and characteristics, thereby substantially inactivating, reducing or neutralizing PD-1 itself, PD-1 biological activity or the consequences of such biological activity to any meaningful extent. In some embodiments, anti-PD-1 antagonist antibodies bind to PD-1 and upregulate anti-tumor immune responses.

[0090] As used herein, "anti-PD-1 antagonist antibodies" refer to antibodies that are capable of inhibiting PD-1 biological activity and / or downstream events mediated by PD-1. Anti-PD-1 antagonist antibodies include antibodies that block, antagonize, inhibit or reduce (including significantly reduce to any extent) PD-1 biological activity (including PD-1-mediated downstream events, such as PD-1 binding and downstream signal transduction, inhibition), inhibition of T cell proliferation, inhibition of T cell activation, inhibition of IFN secretion, inhibition of IL-2 secretion, inhibition of TNF secretion, induction of IL-10, and inhibition of anti-tumor immune responses. For the purposes of this application, it will be clearly understood that the term "anti-PD-1 antagonist antibody" (interchangeably referred to as "antagonist PD-1 antibody", "antagonist anti-PD-1 antibody" or "PD-1 antagonist antibody") includes all previously identified terms, titles, functional states and characteristics, thereby substantially inactivating, reducing or neutralizing PD-1 itself, PD-1 biological activity or the consequences of such biological activity to any meaningful extent. In some embodiments, anti-PD-1 antagonist antibodies bind to PD-1 and upregulate anti-tumor immune responses.

[0091] The phrase "immune checkpoint modulator" refers to a functional class of agents that inhibit or stimulate signal transduction through immune checkpoints. "Immune checkpoint modulators" include receptors and their associated ligands, which together provide signal transduction pathways associated with the inhibition or stimulation of T cell activation. Exemplary immune checkpoint modulators include, but are not limited to, PD-1 and its ligands PD-L1 and PD-L2; TIGIT and its CD 155 ligand PVR; CTLA-4 and its ligands B7-1 and B7-2; TIM-3 and its ligand galectin-9; LAG-3 and its ligands, including liver sinusoidal endothelial cell lectin (LSECtin) and galectin-3; CD122 and its CD122R ligand; CD70, B7H3, B and T lymphocyte attenuating agent (BTLA) and VISTA.

[0092] The terms "checkpoint regulator antagonist," "immune checkpoint binding antagonist," and "immune checkpoint antagonist" are used interchangeably herein and refer to a class of agents that interfere with (or inhibit) the activity of immune checkpoint regulators, whereby signal transduction through the checkpoint regulator receptor is blocked or inhibited due to binding to the checkpoint regulator or its ligand. By inhibiting this signal transduction, immunosuppression can be reversed, thereby reestablishing or enhancing T cell immunity against cancer cells. Immune checkpoint regulator antagonists can include antibody fragments, peptide inhibitors, dominant negative peptides, and small molecule drugs, either in isolated form or as part of a fusion protein or conjugate.

[0093] The terms "immune checkpoint binding agonist" and "immune checkpoint agonist" are used interchangeably herein and refer to a class of agents that stimulate the activity of immune checkpoint regulators, thereby stimulating signal transduction through checkpoint regulator receptors due to binding to the checkpoint regulator or its ligand. By stimulating this signal transduction, T cell immunity against cancer cells can be reconstituted or enhanced. Exemplary immune checkpoint regulator agonists include, but are not limited to, members of the tumor necrosis factor (TNF) receptor superfamily, such as CD27, CD40, OX40 (CD134), glucocorticoid-induced TNFR family-related protein (GITR) and 4-1BB (CD137) and its ligands. Other checkpoint regulator agonists belong to the B7-CD28 superfamily, including CD28 and ICOS.

[0094] The phrase "dominant negative protein" or "dominant negative peptide" refers to a protein or peptide derived from a wild-type protein that has been genetically modified by mutation and / or deletion such that the modified protein or peptide interferes with the function of the endogenous wild-type protein from which it is derived.

[0095] The phrase "VEGF binding antagonist" refers to a functional class of agents that bind to VEGF-A or its receptor VEGFR-2, thereby blocking or inhibiting the activation of VEGFR-2 by VEGF-A due to the binding. As used herein, the term "VEGF binding antagonist" includes antibody fragments, peptide inhibitors, dominant negative peptides, and small molecule drugs, which can be in isolated form or as part of a fusion protein or conjugate.

[0096] The term "Tie2 tyrosine kinase receptor binding antagonist" refers to a functional agent that binds to the Tie2 tyrosine kinase receptor or one of its ligands. Due to the binding, the activation of the Tie2 tyrosine kinase receptor by one or more of its ligands (i.e., Ang1, Ang2, Ang3, and Ang4) is blocked or inhibited. As used herein, the term "Tie2 tyrosine kinase receptor binding antagonist" includes antibody fragments, peptide inhibitors, dominant negative peptides, and small molecule drugs, which can be in isolated form or as part of a fusion protein or conjugate.

[0097] The phrase "small molecule drug" refers to a molecular entity that is typically organic or organometallic, but not a polymer, that is pharmaceutically active, and that has a molecular weight of less than about 2 kDa, less than about 1 kDa, less than about 900 Da, less than about 800 Da, or less than about 700 Da. The term encompasses most pharmaceutical compounds referred to as "drugs" other than proteins or nucleic acids, although small peptides or nucleic acid analogs may also be considered small molecule drugs. Examples include chemotherapeutic anticancer drugs and enzyme inhibitors. Small molecule drugs can be synthetic, semisynthetic (i.e., synthesized from naturally occurring precursors), or biological in origin.

[0098] When polypeptide domain arrangements are described with hyphens between the various domains (eg, CH2-CH3), it is understood that the order in which the domains are listed is from amino-terminus to carboxyl-terminus.

[0099] The term "immunoconjugate" refers to an antibody fused to an inhibitory peptide or small molecule drug via a covalent bond. The peptide or small molecule drug can be chemically linked to the C-terminus of the constant heavy chain or the N-terminus of the variable light chain and / or heavy chain.

[0100] "Linkers" can be used to connect peptides or small molecule drugs, such as maytansinoid, to anti-tumor antagonists in a stable covalent manner. Under conditions where the compound or antibody remains active, the linker may be sensitive to or substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid-labile groups, photolabile groups, peptidase-labile groups, and esterase-labile groups. Linkers also include charged linkers and their hydrophilic forms, as described herein and known in the art. The immunoconjugate may further comprise a flexible 3-15 amino acid linker between the anti-tumor antagonist and the peptide and / or small molecule drug, such as a GGG (SEQ ID NO: 283) or GGGGS (G4S) repeat peptide linker (SEQ ID NO: 284-289).

[0101] As used herein, the term "scaffold" refers to any amino acid polymer that exhibits desired properties that support antagonist function, including increasing antibody specificity, enhancing antibody function, or supporting antibody structure and stability. The scaffold can be grafted to the binding domain of a donor polypeptide to impart binding specificity to the donor polypeptide on the scaffold.

[0102] As used herein, the phrase "multispecific inhibitor" refers to a molecule comprising at least two targeting domains with different binding specificities. In some embodiments, a multispecific inhibitor is a polypeptide comprising a scaffold and two or more immunoglobulin antigen binding domains that target different antigens or epitopes. In certain embodiments, a multispecific inhibitor is a bispecific antibody or antagonist. In other embodiments, a multispecific inhibitor is a trispecific antibody or antagonist.

[0103] As used herein, the phrase "bispecific" refers to a molecule comprising at least two targeting domains with different binding specificities. Each targeting domain is capable of specifically binding to a target molecule and inhibiting the biological function of the target molecule when bound to the target molecule. In some embodiments, a bispecific checkpoint regulator antagonist is a polymer molecule having two or more peptides. In some embodiments, the targeting domain comprises an antigen-binding domain or CDR of an antibody. In some embodiments, a bispecific inhibitor is a bispecific antibody.

[0104] The terms "bispecific antibody" and "bispecific antagonist" are used interchangeably herein and refer to antibodies that can specifically bind to two different antigens (or epitopes). In some embodiments, a bispecific antibody is a full-length antibody that binds to one antigen (or epitope) on one of its two binding arms (a pair of HC / LC) and binds to a different antigen (or epitope) on its second arm (another pair of HC / LC). In these embodiments, the bispecific antibody has two different antigen-binding arms (both in specificity and CDR sequences) and is monovalent for each antigen to which it binds.

[0105] In other embodiments, bispecific antibodies are full-length antibodies that can bind to two different antigens (or epitopes) in each of their two binding arms (two pairs of HC / LC). In these embodiments, the bispecific antibodies have two identical antigen-binding arms with the same specificity and the same CDR sequences, and are bivalent for each antigen to which they bind.

[0106] The terms "trispecific antibody" and "trispecific antagonist" are used interchangeably herein and refer to molecules comprising three targeting domains with three different binding specificities. Each targeting domain is capable of specifically binding to a target molecule and, when bound to the target molecule, inhibits the biological function of the target molecule. In some embodiments, a trispecific antagonist is a polymer molecule comprising two or more peptides. In some embodiments, the targeting domain comprises an antigen-binding domain or CDR of an antibody. In some embodiments, a trispecific antagonist is a trispecific antibody.

[0107] Exemplary bispecific or trispecific antibodies can include asymmetric IgG-like antibodies (e.g., triomab / quadroma, Trion Pharma / Fresenius Biotech); buttonhole antibodies (Genentech); cross monoclonal antibodies (Cross MAb, Roche); electrostatic matching antibodies (AMGEN); LUZ-Y (Genentech); chain exchange engineered domain (SEED) bodies (EMD Serono; biolonic, Merus); Fab exchange antibodies (Genmab), symmetrical IgG-like antibodies (e.g., dual targeting (DT)-Ig (GSK / Domantis); two-in-one antibodies (Genentech); cross-linked monoclonal antibodies (Karmanos Cancer Center), mAb2 (F-star); Cov X-body (Cov X / Pfizer); dual variable region domain (DVD)-Ig fusion protein (Abbott); IgG-like bispecific antibodies (Eli Lilly); Ts2Ab (Medimmune / AZ); BsAb (ZymoGenetics); HERCULES (Biogen Idec, TvAb, Roche); scFv / Fc fusion protein; SCORPION (Emergent BioSolutions / Trubion, ZymoGenetics / BMS); dual affinity redirection technology (Fc-DART), MacroGenics; bis(scFv)2-Fabs (National Center for Antibody Medicine); F(ab)2 fusion proteins (Medarex / AMGEN); dual-action or Bis-Fab (Genentech); dock-and-lock (DNL, ImmunoMedics); Fab-Fv (UCB-Celltech); scFv-based antibodies and diabody-based antibodies (e.g., bispecific T cell engagers (BiTEs, Micromet); tandem diabodies (Tandab, Affimed); DARTs (MacroGenics); single-chain diabodies; TCR-like antibodies (AIT, Receptor Logics); human serum albumin scFv fusion proteins (Merrimack); combodies (Epigen Biotech); and IgG / non-IgG fusion proteins (e.g., immunocytokines) (EMDSerono, Philogen, ImmunGene, ImmunoMedics).

[0108] The terms "treat" and "treatment" refer to alleviating one or more symptoms associated with a cell proliferative disorder; preventing or delaying the onset of one or more symptoms of a cell proliferative disorder; and / or reducing the severity or frequency of one or more symptoms of a cell proliferative disorder.

[0109] The phrases "a patient in need thereof," "a patient in need of treatment," or "a subject in need of treatment" include subjects, such as mammalian subjects, who would benefit from administration of an anti-tumor antagonist of the present disclosure for treating a cell proliferative disorder.

[0110] The terms "therapeutically effective amount," "pharmacologically effective amount," and "physiologically effective amount" are used interchangeably and refer to the amount of an anti-tumor antagonist required to provide a threshold level of active antagonist in the bloodstream or in the target tissue. The precise amount will depend on many factors, such as the specific active agent, the components and physical properties of the composition, the intended patient population, patient considerations, etc., and can be readily determined by one skilled in the art based on the information provided herein or other information provided in the relevant literature.

[0111] As used herein, the terms "improve," "increase," or "decrease" refer to a value or parameter measured relative to a baseline, e.g., measurement in the same individual before starting a treatment described herein or measurement in a control individual (or multiple control individuals) in the absence of a treatment described herein.

[0112] A "control individual" is an individual who has the same cell proliferative disorder as the individual being treated and is of the same age as the individual being treated to ensure that the disease stages of the treated individual and the control individual are comparable. The individual being treated (also referred to as a "patient" or "subject") can be a fetus, infant, child, adolescent, or adult with a cell proliferative disorder.

[0113] The term "cell proliferative disorder" refers to a disease characterized by abnormal cell proliferation. A proliferative disorder does not imply any restriction on the rate of cell growth, but rather a loss of normal controls that influence growth and cell division. Thus, in some embodiments, cells of a proliferative disorder may have the same cell division rate as normal cells, but do not respond to signals that restrict such growth. Included within the scope of a "cell proliferative disorder" are neoplasms, cancers, or tumors.

[0114] The term "cancer" or "tumor" refers to any of a variety of malignancies characterized by proliferating cells that have the ability to invade surrounding tissues and / or metastasize to new colonization sites, and includes leukemias, lymphomas, carcinomas, melanomas, sarcomas, germ cell tumors, and blastomas. Exemplary cancers treated with the methods of the present disclosure include brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, kidney cancer, lung cancer, non-small cell lung cancer, mesothelioma, ovarian cancer, prostate cancer, gastric cancer, and uterine cancer, leukemia, and medulloblastoma.

[0115] The term "leukemia" refers to a progressive malignant disease of the blood-forming organs and is generally characterized by abnormal proliferation and development of white blood cells and their precursors in the blood and bone marrow. Exemplary leukemias include, for example, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, leukocytic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myeloid leukemia, corneal leukemia, embryonic leukemia, eosinophilic leukemia, Gross's leukemia, hairy cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, and leukemia of the genotype. leukemia), histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphoid leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenic leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia leukemia,), myelomonocytic leukemia, Negley's leukemia, plasma cell leukemia, plasma cell leukemia, promyelocytic leukemia, Liddle's cell leukemia, Schilling's leukemia ( Schilling's leukemia ), stem cell leukemia, subleukemia and undifferentiated cell leukemia.

[0116] The term "carcinoma" refers to a malignant growth of epithelial cells that readily infiltrates surrounding tissues and causes metastasis. Exemplary carcinomas include, for example, acinar carcinoma, acinar carcinoma, adenoid cystic carcinoma, adenoma carcinoma, adrenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basal cell carcinoma, and basal cell carcinoma. basocellulare), basement membrane carcinoma, basosquamous carcinoma, bronchoalveolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, comedocarcinoma, body carcinoma, cribriform carcinoma, armored carcinoma, skin carcinoma, cylindrical carcinoma, cylindrical cell carcinoma, ductal carcinoma, ductal carcinoma in situ, invasive ductal carcinoma, lobular carcinoma, invasive lobular carcinoma, dural carcinoma, embryonal carcinoma, encephaloid carcinoma, epithelial carcinoma, epithelial adenocarcinoma, exophytic carcinoma, ulcerative carcinoma, fibroma, gelatinoid carcinoma, colloid carcinoma, giant cell carcinoma, adenoid carcinoma (glandular carcinoma), adenocarcinoma, granular cell carcinoma, pilomatricoma, hemorrhoidal carcinoma, hepatocellular carcinoma, Hürthl cell carcinoma, hyaline membrane carcinoma, herpetiform carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Crompchel's carcinoma, Kurtzky cell carcinoma, large cell carcinoma, lenticular carcinoma carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanoma carcinoma, nevus carcinoma, mucinous carcinoma, mucinous carcinoma, mucous cell carcinoma, mucoepidermoid carcinoma, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare), nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, portal vein carcinoma, preinvasive carcinoma, prick cell carcinoma, pustular carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcomatous carcinoma, Schneider's carcinoma, sclerosing carcinoma, scrotal carcinoma, signet ring cell carcinoma, simple carcinoma, small cell carcinoma, eggplant carcinoma, spheroid cell carcinoma, spindle cell carcinoma, cavernous carcinoma, squamous cell carcinoma, squamous cell carcinoma, linear carcinoma, telangiectatic carcinoma, telangiectatic carcinoma, transitional cell carcinoma, nodular carcinoma, tuberous carcinoma, verrucous carcinoma, and villous carcinoma.

[0117] The term "sarcoma" refers to tumors composed of material similar to embryonic connective tissue and usually consists of tightly packed cells embedded in a fibrous or homogeneous substance. Exemplary sarcomas include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, green algal sarcoma, choriocarcinoma, embryonal sarcoma, Wilns tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic polychromatic hemorrhagic sarcoma, B-cell immunoblastic sarcoma, lymphoma (e.g., lymphoma), Janssen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukocytic sarcoma, malignant mesothelioma sarcoma, peritoneal sarcoma, reticulum cell sarcoma, Rous sarcoma, serous sarcoma, and synovial sarcoma.

[0118] The term "melanoma" refers to tumors of the melanocyte system of the skin and other organs. Melanomas include, for example, horn melanoma, enamel melanoma, benign juvenile melanoma, Cloudman melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, tonsillar melanoma, malignant melanoma, nodular melanoma, sublingual melanoma, and superficial melanoma.

[0119] Other cancers include, for example, Hodgkin's disease, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, essential thrombocythemia, essential macroglobulinemia, small cell lung cancer, primary brain tumors, gastric cancer, colon cancer, malignant pancreatic insulinoma, malignant carcinoid, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, hypercalcemia of malignancy, cervical cancer, endometrial cancer, and adrenocortical carcinoma.

[0120] I. Trispecific anti-tumor antagonists

[0121] One aspect of the present application relates to a trispecific anti-tumor antagonist, which comprises: a protein scaffold, a first targeting domain that specifically binds to an immune checkpoint regulator; a second targeting domain that specifically binds to VEGF and comprises one or more peptide domains derived from VEGFR, and a third targeting domain that comprises an inhibitor of the angiopoietin / Tie-2 signaling pathway, wherein the first targeting domain is located at the amino terminus of the protein scaffold, and the second targeting domain is located at the carboxyl terminus of the protein scaffold. In some embodiments, the third targeting domain is inserted into the CH3 domain in the Fc loop of the protein scaffold.

[0122] Fusion proteins (e.g., Fc fusion proteins) produced by recombinant DNA techniques often face the serious problem of full or partial degradation (clipping) in cell culture due to host cell-derived proteases. Clipping produces low molecular weight (LMW) species of the target protein and results in inactive polypeptides. While not wishing to be bound by any particular theory, it has been hypothesized that the positioning of the biological peptide affects the degree of degradation in the resulting fusion protein. The inventors of the present application have discovered that inserting a second targeting domain into the CH3 region of the Fc loop significantly reduces the clipping of the resulting trispecific antagonist.

[0123] Another aspect of the present application relates to a trispecific anti-tumor antagonist comprising: a protein scaffold, a first targeting domain that specifically binds to an immune checkpoint regulator; a second targeting domain that specifically binds to VEGF and comprises one or more peptide domains derived from VEGFR, and a third targeting domain that comprises a TGF pathway inhibitor.

[0124] In some embodiments, the second targeting domain is located at the carboxyl terminus of the CH3 domain of the protein scaffold, and the third targeting domain is located at the carboxyl terminus of the second targeting domain. In some embodiments, the second targeting domain is structurally connected to the carboxyl terminus of the CH3 domain of the protein scaffold via a first linker. In some embodiments, the third targeting domain is structurally connected to the carboxyl terminus of the second targeting domain via a second linker. In some embodiments, the second targeting domain is structurally connected to the carboxyl terminus of the CH3 domain of the protein scaffold via a first linker, and the third targeting domain is structurally connected to the carboxyl terminus of the second targeting domain via a second linker. The inventors of the present application unexpectedly discovered that this structure improves the pharmacokinetics of the corresponding antagonist.

[0125] In some embodiments, the second targeting domain comprises a VEGFR component having the amino acid sequence of SEQ ID NO: 185 (afilbercept). In other embodiments, the VEGFR component is α-glycosylated by replacing an asparagine residue with a glutamic acid residue. An exemplary amino acid sequence of an α-glycosylated VEGFR component is shown in SEQ ID NO: 207. Trispecific anti-tumor antagonist variants having an α-glycosylated VEGFR component retain VEGFR biological activity while exhibiting improved pharmacokinetics compared to wild-type afilbercept.

[0126] Another aspect of the present application relates to a trispecific anti-tumor antagonist comprising: a protein scaffold, a first targeting domain that specifically binds to an immune checkpoint regulator; a second targeting domain that comprises a TGF-β pathway inhibitor; and a third targeting domain that comprises a peptide inhibitor of the angiopoietin / Tie-2 signaling pathway.

[0127] In some embodiments, the first targeting domain comprises bevacizumab or a variant thereof, and the third targeting domain is inserted into each of the two CH3 regions of the Fc loop of the protein scaffold. In some embodiments, the first targeting domain comprising one or more anti-PD-1 variable region domains and the third targeting domain are inserted into each of the two CH3 regions of the Fc loop of the protein scaffold. In some embodiments, the first targeting domain comprising one or more anti-PD-L1 variable region domains and the third targeting domain are inserted into each of the two CH3 regions of the Fc loop of the protein scaffold.

[0128] In some embodiments, the trombanib peptide comprises the following sequence: AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID NO: 182). Unless otherwise indicated, "trombanib peptide" and the designation "-L" or "-L" correspond to the sequence of SEQ ID NO: 182, which comprises two copies of the Ang-1 / 2 block peptide sequence, QEECEWDPWTCEHM (SEQ ID NO: 194). In certain embodiments, the trispecific antagonists of the present application include a modified form of the trombanib peptide comprising a single copy of the sequence corresponding to SEQ ID NO: 194 (designated as "-Short" or "-S").

[0129] II. Bispecific Anti-tumor Antagonists

[0130] Another aspect of the present application relates to a bispecific anti-tumor antagonist comprising: a protein scaffold, a first targeting domain that specifically binds to an immune checkpoint regulator; and a second targeting domain that comprises a peptide inhibitor of the angiopoietin / Tie-2 signaling pathway.

[0131] In some embodiments, the first targeting domain comprises one or more anti-PD-1 or anti-PD-L1 variable region domains, and the second targeting domain is inserted within each of the two CH3 regions of the Fc loop of the protein scaffold.

[0132] Another aspect of the present application relates to a bispecific anti-tumor antagonist comprising: a protein scaffold, a first targeting domain that specifically binds to an immune checkpoint regulator; and a second targeting domain comprising a VEGFR component.

[0133] Figure 41A-40 C shows exemplary bispecific anti-PD-1 / VEGFR component and anti-PD-L1 / VEGFR component molecules Bi-ZPL-1, Bi-ZPL-2, and Bi-ZPL-3, which have N-terminal ( Figure 41A) or carboxyl terminus ( Figure 41B -C) contains afipronil. In one embodiment, Bi-ZPL-1 comprises an immunoglobulin heavy chain comprising the sequence set forth in SEQ ID NO: 218 and / or an immunoglobulin light chain comprising the sequence set forth in SEQ ID NO: 219. In another embodiment, Bi-ZP-1 comprises an immunoglobulin heavy chain comprising the sequence set forth in SEQ ID NO: 220 and / or an immunoglobulin light chain comprising the sequence set forth in SEQ ID NO: 221.

[0134] In another embodiment, Bi-ZP-2 has an immunoglobulin heavy chain comprising the sequence of SEQ ID NO: 222 and / or an immunoglobulin light chain comprising the sequence of SEQ ID NO: 223. In another embodiment, Bi-ZPL-2 has an immunoglobulin heavy chain comprising the sequence of SEQ ID NO: 224 and / or an immunoglobulin light chain comprising the sequence of SEQ ID NO: 225.

[0135] In another embodiment, Bi-ZPL-3 has an immunoglobulin heavy chain comprising the sequence set forth in SEQ ID NO:296 and / or an immunoglobulin light chain comprising the sequence set forth in SEQ ID NO:297.

[0136] Another aspect of the present application relates to a bispecific anti-tumor antagonist comprising: a protein scaffold comprising a first targeting domain of bevacizumab; and a second targeting domain comprising a peptide inhibitor of the angiopoietin / Tie-2 signaling pathway. In some embodiments, the second targeting domain is inserted within the CH3 domain in the Fc loop of the protein scaffold.

[0137] Another aspect of the present application relates to a bispecific anti-tumor antagonist comprising: a protein scaffold comprising an Fc fragment, a first targeting domain comprising a VEGFR component, and a second targeting domain comprising a peptide inhibitor of the angiopoietin / Tie-2 signaling pathway. In some embodiments, the second targeting domain is inserted within the CH3 domain of the Fc loop of the protein scaffold. In some embodiments, prior to insertion of the second targeting domain, the Fc fragment of the protein scaffold comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 210-217.

[0138] Another aspect of the present application relates to a bispecific anti-tumor antagonist comprising: a protein scaffold comprising an Fc fragment, a first targeting domain comprising the TGFBR2 ECD, and a second targeting domain comprising a peptide inhibitor of the angiopoietin / Tie-2 signaling pathway. In some embodiments, the second targeting domain is inserted within the CH3 domain of the Fc loop of the protein scaffold. In some embodiments, prior to insertion of the second targeting domain, the Fc fragment of the protein scaffold comprises an amino acid sequence selected from SEQ ID NOs: 210-217.

[0139] The bispecific anti-tumor antagonists of the present application can be constructed with an IgG skeleton. More specifically, any bispecific antagonist of the present application can be constructed with an IgG1 or IgG4 skeleton. The use of an IgG1 skeleton is preferred for cancer treatments where there is a target on an antigen presenting cell that can mediate antibody-dependent cell-mediated cytotoxicity (ADCC). The use of an IgG4 skeleton can target antigens where antigen binding alone is sufficient to produce the desired therapeutic benefit. Antagonists based on IgG4 exclude undesirable effector functions associated with, for example, IgG1 antibodies, including FcγR binding and complement activation.

[0140] III. Targeting Domain

[0141] A. Targeting domains that specifically bind to immune checkpoint regulators

[0142] Targeting domains that specifically bind to immune checkpoint regulators include, but are not limited to: (1) anti-PD-1 antibodies and anti-PD-1 antibody fragments, (2) anti-PD-L1 antibodies and anti-PD-L1 antibody fragments, (3) anti-TIGIT antibodies and anti-TIGIT antibody fragments, and (4) anti-LAG-3 antibodies and anti-LAG-3 antibody fragments.

[0143] 1. Anti-PD-1 antibodies and anti-PD-1 antibody fragments

[0144] In some embodiments, the checkpoint regulator antagonist is an anti-PD-1 antibody or antibody fragment. In a specific embodiment, the PD-1 inhibitor of the present application is an antibody or an antigen-binding portion thereof, comprising: (1) a heavy chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (HCDRs): HCDR1, HCDR2, and HCDR3, wherein HCDR1 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 51, 54, 56, and 59, wherein HCDR2 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 52, 57, and 60, and wherein HCDR3 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 53, 55, 58, and 61; and (2) a light chain variable region, wherein the light chain variable region comprises three complementarity determining regions (LCDRs): LCDR1, LCDR2, and LCDR3, wherein LCDR1 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: NOs: 62, 65, 68, 69, 70 and 73, wherein LCDR2 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from SEQ ID NOs: 63, 66, 71 and 74, wherein LCDR3 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from SEQ ID NOs: 64, 67, 72 and 75, wherein the antibody, or antigen-binding portion thereof, specifically binds to human PD-L

[0145] In some embodiments, the PD-1 inhibitor of the present application is an antibody or an antigen-binding portion thereof, comprising: (1) a heavy chain variable region having an amino acid sequence with about 80% to 100% identity to an amino acid sequence selected from SEQ ID NOs: 127, 129, 131, 131, 133, 135, and 137; and (2) a light chain variable region having an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from SEQ ID NOs: 128, 130, 132, 134, 136, and 138, wherein the antibody or antigen-binding portion thereof specifically binds to human PD-1.

[0146] In some embodiments, the anti-PD-1 antibody or antibody fragment comprises a heavy chain variable region comprising (1) HCDR1 having SEQ ID NO: 59, HCDR2 having SEQ ID NO: 60, and HSEQ 3 having SEQ ID NO: 61 and (2) HFR1 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO: 366, HFR2 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO: 360, HFR3 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO: 371, and HFR4 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO: 357, and an immunoglobulin heavy chain variable region comprising (1) LCDR1 having SEQ ID NO: 73, LCDR2 having SEQ ID NO: 74, and LCDR3 having SEQ ID NO: 75. 3, and (2) LFR1 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO: 372, LFR2 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO: 373, LFR3 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO: 374, and LFR4 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO: 375.

[0147] In some embodiments, the anti-PD-L1 antibody or antibody fragment comprises a heavy chain variable region comprising (1) HCDR1 having SEQ ID NO: 56, HCDR2 having SEQ ID NO: 57, and HCDR1 having HSEQ ID NO: 58, and (2) HFR1 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO: 366, HFR2 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO: 360, HFR3 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO: 367, and HFR4 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO: 357, and an immunoglobulin heavy chain variable region comprising (1) LCDR1 having SEQ ID NO: 70, LCDR2 having SEQ ID NO: 71, and HFR4 having SEQ ID NO: 72. NO:72 LCDR 3, and (2) LFR1 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:368, LFR2 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:369, LFR3 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:370, and LFR4 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:365.

[0148] The term "2P17" or "PD-06" refers to the PD-1 inhibitor of the present application, comprising: (1) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 137, and (2) a light chain variable region having the amino acid sequence of SEQ ID NO: 138. The CDR and FR regions of 2P17 (or PD-06) are listed as SEQ ID NOs: 59-61, 73-75, 357, 360, 366, and 371-375.

[0149] The term "2P16" or "PD-05" refers to the PD-1 inhibitor of the present application, comprising: (1) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 135, and (2) a light chain variable region having the amino acid sequence of SEQ ID NO: 136. The CDR and FR regions of 2P16 (or PD-05) are listed as SEQ ID NOs: 56-58, 70-72, 357, 360, 366, and 365-370.

[0150] 2. Anti-PD-L1 antibodies and anti-PD-L1 antibody fragments

[0151] In one embodiment, the checkpoint regulator antagonist is an anti-PD-L1 antibody or antibody fragment. In a specific embodiment, the PD-L1 inhibitor of the present application is an antibody or an antigen-binding portion thereof, comprising: (1) a heavy chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (HCDRs): HCDR1, HCDR2, and HCDR3, wherein HCDR1 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 76, 79, 85, and 88, wherein HCDR2 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 77, 80, 82, 84, 86, and 89, and wherein HCDR3 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 78, 81, 83, 87, and 90; and (2) a light chain variable region, wherein the light chain variable region comprises three complementarity determining regions (LCDRs): LCDR1, LCDR2, and LCDR3, wherein LCDR1 has an amino acid sequence selected from the group consisting of SEQ ID NOs: NO: 91, 94, 98, 101 and 104, wherein LCDR2 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from SEQ ID NO: 92, 95, 99, 102 and 105, wherein LCDR3 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from SEQ ID NO: 93, 96, 97, 100 and 106, wherein the antibody, or antigen-binding portion thereof, specifically binds to human PD-L1.

[0152] In some embodiments, an anti-PD-L1 antibody or antibody fragment comprises a heavy chain variable region comprising (1) HCDR1 having SEQ ID NO: 79, HCDR2 having SEQ ID NO: 82, and HCDR3 having SEQ ID NO: 83, and (2) HFR1 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO: 366, HFR2 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO: 360, HFR3 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO: 383, and HFR4 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO: 357, and an immunoglobulin heavy chain variable region comprising (1) LCDR1 having SEQ ID NO: 91, LCDR2 having SEQ ID NO: 92, and HFR4 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO: NO:93 LCDR3, and (2) LFR1 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO:377, LFR2 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO:378, LFR3 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO:379, and LFR4 having at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO:365.

[0153] In some embodiments, the PD-L1 inhibitor of the present application is an antibody or an antigen-binding portion thereof, comprising: (1) a heavy chain variable region having an amino acid sequence with about 80% to 100% identity to an amino acid sequence selected from SEQ ID NOs: 139, 141, 143, 145, 147, 149, 151, and 153; (2) a light chain variable region having an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from SEQ ID NOs: 140, 142, 144, 146, 148, 150, 152, and 154, wherein the antibody or antigen-binding portion thereof specifically binds to human PD-L1. In a preferred embodiment, the anti-PD-L1 antibody heavy and light chains consist of SEQ ID NOs: 153-154.

[0154] The term "PL-08" refers to the PD-L1 inhibitor of the present application, comprising: (1) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 153, and (2) a light chain variable region having the amino acid sequence of SEQ ID NO: 154. The CDR and FR regions of PL-08 are listed as SEQ ID NOs: 79, 82, 83, 91-93, 357, 360, 365, 366, 377-379, and 383.

[0155] 3. Anti-TIGIT Antibodies and Anti-TIGIT Antibody Fragments

[0156] In some embodiments, the checkpoint regulator antagonist is an anti-TIGIT antibody or antibody fragment. In a specific embodiment, the TIGIT inhibitor of the present application is an antibody or an antigen-binding portion thereof, comprising: (1) a heavy chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (HCDRs): HCDR1, HCDR2 and HCDR3, wherein HCDR1 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from SEQ ID NOs: 1, 6, 11, 15, 17, 20 and 23, wherein HCDR2 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from SEQ ID NOs: 2, 4, 7, 9, 12, 13, 16, 18, 21 and 24, and wherein HCDR3 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from SEQ ID NOs: NO: 3, 5, 8, 10, 14, 19, 22 and 25; (2) a light chain variable region, wherein the light chain variable region comprises three complementarity determining regions (LCDRs): LCDR1, LCDR2 and LCDR3, wherein LCDR1 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from SEQ ID NO: 26, 29, 31, 33, 35, 39, 42 and 45, wherein LCDR2 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from SEQ ID NO: 27, 30, 36, 37, 40, 43 and 46, and wherein LCDR3 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from SEQ ID NO: 28, 31, 33, 35, 39, 42 and 45, The amino acid sequences of NOs: 28, 32, 34, 38, 41, 44 and 47 have amino acid sequences with about 80% to about 100% identity, wherein the antibodies or antigen-binding portions thereof specifically bind to human TIGIT.

[0157] In some embodiments, an anti-TIGIT antibody or antibody fragment comprises a heavy chain variable region comprising (1) HCDR1 having SEQ ID NO: 23, HCDR2 having SEQ ID NO: 24, and HCDR3 having SEQ ID NO: 25 and (2) HFR1 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO: 343, HFR2 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO: 330, HFR3 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO: 344, and HFR4 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO: 319, and an immunoglobulin heavy chain variable region comprising (1) LCDR1 having SEQ ID NO: 45, LCDR2 having SEQ ID NO: 46, and LCDR3 having SEQ ID NO: 47. 3, and (2) LFR1 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO: 303, LFR2 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO: 311, LFR3 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO: 317, and LFR4 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO: 345.

[0158] In another embodiment, the TIGIT inhibitor of the present application is an antibody or an antigen-binding portion thereof, comprising: (1) a heavy chain variable region having an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 107, 109, 111, 113, 115, 117, 119, 121, 123, and 125; and (2) a light chain variable region having an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 108, 110, 112, 114, 116, 118, 120, 122, 124, and 126, wherein the antibody or antigen-binding portion thereof specifically binds to human TIGIT. In a preferred embodiment, the anti-TIGIT antibody has heavy and light chain sequences consisting of SEQ ID NOs: 125 and 126.

[0159] The term "B21-35" or "T-10" refers to the TIGIT inhibitor of the present application, comprising: (1) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 125, and (2) a light chain variable region having the amino acid sequence of SEQ ID NO: 126. The CDR and FR regions of B21-35 (or T-10) are listed as SEQ ID NOs: 23-25, 45-47, 303, 311, 317, 319, 330, and 343-345.

[0160] 4. Anti-LAG-3 Antibodies and Anti-LAG-3 Antibody Fragments

[0161] In one embodiment, the checkpoint regulator antagonist is an anti-LAG-3 antibody or antibody fragment. In a specific embodiment, the LAG-3 inhibitor of the present application is an antibody or antigen-binding portion thereof, comprising: (1) a heavy chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (HCDRs): HCDR1, HCDR2, and HCDR3, wherein HCDR1 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from SEQ ID NOs: 155-157, wherein HCDR2 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from SEQ ID NOs: 158-160, and wherein HCDR3 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from SEQ ID NOs: 161-163; and (2) a light chain variable region, wherein the light chain variable region comprises three complementarity determining regions (LCDRs): LCDR1, LCDR2, and LCDR3, wherein LCDR1 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from SEQ ID NOs: 158-160. NOs: 164-166, wherein LCDR2 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from SEQ ID NOs: 167 and 168, and wherein LCDR3 has an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from SEQ ID NOs: 169 and 170, wherein the antibody or antigen-binding portion specifically binds human LAG-3.

[0162] In some embodiments, the anti-LAG-3 antibody or antibody fragment comprises a heavy chain variable region comprising (1) HCDR1 having SEQ ID NO: 156, HCDR2 having SEQ ID NO: 158, and HCDR3 having SEQ ID NO: 161, and (2) HFR1 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO: 399, HFR2 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO: 400, HFR3 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO: 401, and HFR4 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO: 402, and an immunoglobulin heavy chain variable region comprising (1) LCDR1 having SEQ ID NO: 164, LCDR2 having SEQ ID NO: 167, and HCDR3 having SEQ ID NO: 168. LCDR 3 of NO: 169, and (2) LFR1 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO: 403, LFR2 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO: 404, LFR3 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO: 405, and LFR4 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO: 406.

[0163] In other embodiments, the LAG-3 inhibitor of the present application is an antibody, or an antigen-binding portion thereof, comprising: (1) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 171-174; and (2) a light chain variable region having an amino acid sequence that is about 80% to about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 175-178, wherein the antibody, or antigen-binding portion thereof, specifically binds to human LAG-3. In a preferred embodiment, the anti-LAG-3 antibody has heavy and light chain sequences consisting of SEQ ID NOs: 171 and 175.

[0164] The term "2L2A.1" refers to the LAG-3 inhibitor of the present application, comprising: (1) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 171, and (2) a light chain variable region having the amino acid sequence of SEQ ID NO: 175. The CDR and FR regions of 2L2A.1 are listed as SEQ ID NOs: 156, 158, 161, 164, 167, 169, and 399-406.

[0165] B. Targeting Domains That Specifically Bind to Angiogenic Pathway Components

[0166] Angiogenesis, the development of new blood vessels from pre-existing ones, is essential for tumor growth and metastasis. Angiogenesis inhibition offers a potentially valuable strategy for treating diseases such as cancer, where disease progression (e.g., metastasis) depends on neovascularization. Inhibition of angiogenesis leads to tumor cell death, which may allow tumor antigens to enter host antigen presentation pathways.

[0167] Two important angiogenic pathways include the vascular endothelial growth factor (VEGF) pathway and the Tie2 pathway.

[0168] 1. VEGF pathway antagonists and inhibitors

[0169] The primary VEGF pathway is mediated by the transmembrane tyrosine kinase VEGF-R2. Various VEGF isoforms, particularly VEGF-A, bind to VEGF-R1 and VEGF-R2, leading to dimerization and activation through phosphorylation by various downstream tyrosine kinases.

[0170] In some embodiments, the VEGF pathway antagonist binds to VEGF-A or its receptors VEGFR-1 and VEGFR-2, such that, as a result of binding, activation of VEGFR-1 and VEGFR-2 by VEGF-A is blocked or inhibited. The angiogenesis inhibitor can be in the form of, for example, an antibody, a variable region domain fragment, or a dominant negative fusion protein fragment.

[0171] Exemplary dominant negative anti-VEGFR antagonists are protein fragments corresponding to the extracellular domain (ECD) of human VEGF receptor 1 or 2. In some preferred embodiments, the dominant negative anti-VEGFR antagonist is afipronil. A recombinant fusion protein comprising the VEGF-A binding portion from the extracellular domains of human VEGF receptors 1 and 2 fused to a human IgG1 or IgG4 Fc fragment. VEGFR ECD, such as afipronil, acts as a soluble receptor decoy for VEGF-A.

[0172] Afipronil (also known as Afipronil is a recombinant VEGF fusion protein consisting of the vascular endothelial growth factor (VEGF) binding moiety from the extracellular domains of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1 immunoglobulin. Afipronil binds to multiple ligands involved in angiogenesis, including VEGF-A, VEGF-B, anti-placental growth factor (PIGF)-1, and PIGF-2, including circulating soluble ligands.

[0173] In a preferred embodiment, the afipronil domain of the trispecific anti-tumor antagonist described herein is α-glycosylated by replacing asparagine (Asn, N) residues with glutamic acid (Glu, E) residues. N-linked glycosylation is the attachment of a glycan to the nitrogen atom (amide nitrogen) of an Asn residue of a protein. The Asn residue to be glycosylated must be located within a specific consensus sequence of the primary structure (Asn-X-Ser or Asn-X-Thr, where X represents any amino acid except proline). By replacing the Asn residue with a Glu residue (or any other amino acid residue), glycosylation will not occur at these sites. One or more Asn residues located within the above-mentioned specific consensus sequence can be replaced.

[0174] Asialoglycoprotein receptors 1 and 2 (ASGR1 and ASGR2) are transmembrane proteins that play a key role in serum glycoprotein homeostasis by mediating endocytosis and lysosomal degradation of glycoproteins with exposed terminal galactose or N-acetylgalactosamine residues. In certain circumstances, glycosylation of proteins can lead to increased clearance of ASGR1 and ASGR2. In order to improve the pharmacokinetics of trispecific antitumor antagonists with VEGFR component domains (i.e., to obtain a longer half-life), the inventors of the present application created α-glycosylated variants of the VEGFR components to limit protein clearance through ASGR1 and ASGR2. Compared with the glycosylated VEGFR components, the trispecific antitumor antagonist variants with α-glycosylated VEGFR components retained the biological activity of VEGFR while improving the α phase of pharmacokinetics by approximately fivefold. See Figure 37 An exemplary amino acid sequence of the VEGF binding domain of afipram is shown in SEQ ID NO: 185, namely: SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSP N ITVTLKKFPLDTLIPDGKRIIWDSRKGFIIS N ATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVL NCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKK N STFVRVHEK, and an exemplary amino acid sequence of the α-glycosylated afipram VEGF binding domain is set forth in SEQ ID NO: 207: SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSP E ITVTLKKFPLDTLIPDGKRIIWDSRKGFIIS E ATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVL E CTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKK E STFVRVHEK. The replacement position is underlined.

[0175] An exemplary VEGF antibody antagonist is bevacizumab (AVASTIN TM ), a humanized antibody. Bevacizumab comprises mutated human IgG1 framework regions (FRs) and antigen-binding complementarity-determining regions from the murine anti-hVEGF monoclonal antibody A4.6.1, which blocks binding of human VEGF-A to VEGFR-2. Approximately 93% of the amino acid sequence of bevacizumab, including most of the framework regions, is derived from human IgG1, and approximately 7% of the sequence is derived from the murine antibody A4.6.1. Bevacizumab has a molecular weight of approximately 149,000 daltons and is glycosylated. In certain embodiments, amino acid substitutions may be included in the bevacizumab / AVASTIN antibody, as described in U.S. Patent No. 7,575,893 (SEQ ID NOs: 226-227). Exemplary amino acid substitutions include, but are not limited to, E1Q, E6Q, L11V, Q13K, L18V, R19K, A23K, or a combination thereof.

[0176] Other anti-VEGF antibodies include ranibizumab (trade name Lucentis TM) (SEQ ID NO: 294-295), which is a monoclonal antibody fragment derived from the same parent murine antibody as bevacizumab; the G6 or B20 series antibodies (e.g., G6-23, G6-31, B20-4.1) described in U.S. Publication Nos. 2006 / 0280747, 2007 / 0141065, and / or 2007 / 0020267, and U.S. Pat. Nos. 7,297,334, 7,060,269, 6,884,879, 6,582,959, 6,703,020, 6,054,297; antibodies described in U.S. Patent Application Publication Nos. 2007 / 059312, 2006 / 009360, 2005 / 0186208, 2003 / 0206899, 2003 / 0190317, and 2003 / 0203409.

[0177] An exemplary anti-VEGFR-2 antibody antagonist is the humanized IgG1 monoclonal antibody ramucirumab, which binds to the extracellular domain of VEGFR-2, thereby blocking its interaction with VEGF-A. Additional anti-VEGFR-2 antibodies are described in U.S. Patent Nos. 7,498,414, 6,448,077, and 6,365,157.

[0178] Exemplary small molecule antagonists of the VEGF pathway include multikinase inhibitors of VEGFR-2, including sunitinib, sorafenib, cediranib, pazopanib, and sinedanib.

[0179] 2. Tie2 pathway antagonists and inhibitors

[0180] The Tie2 pathway is another angiogenic pathway for which therapeutic antibodies and small molecule drugs have been developed. The Tie2 tyrosine kinase receptor activates angiogenesis in response to binding by its angiopoietin (Ang) ligands (i.e., Angl, Ang2, Ang3 (mouse), and Ang4). Tie2 pathway antagonists bind to the Tie2 tyrosine kinase receptor or one of its angiopoietin (Ang) ligands (i.e., Ang-1, Ang-2, Ang-3, and Ang-4), thereby blocking or inhibiting activation of the Tie2 receptor by one or more of its ligands due to binding.

[0181] In one embodiment, the Tie2 receptor binding antagonist is an inhibitory peptide. In another embodiment, the inhibitory peptide comprises the amino acid sequence of SEQ ID NO: 182, namely: AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE. In another embodiment, the Tie2 receptor binding antagonist comprises the peptide of SEQ ID NO: 182 fused to the C-terminus of the Fc fragment, namely: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGAQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID NO: 183). In another embodiment, the inhibitory peptide comprises a single copy of the amino acid sequence of QEECEWDPWTCEHM (SEQ ID NO: 194).

[0182] In another embodiment, an inhibitory peptide is introduced into each of the two CH3 regions of the Fc loop. The loop region is an irregular secondary structure in the protein that is not an α-helix or a β-sheet, but rather connects β-sheets to β-sheets, β-sheets to α-helices, or α-helices to α-helices. The inhibitory peptide can be added by insertion (i.e., between amino acids in a pre-existing Fc loop) or by replacing amino acids in an existing Fc loop (i.e., removing amino acids in a pre-existing Fc loop and adding peptide amino acids).

[0183] An exemplary Fc fragment comprises the amino acid sequence in SEQ ID NO: 208, namely: The predicted loop regions are shown in bold. Any or all of the sites shown in bold may be suitable for complete or partial replacement or insertion of inhibitory peptide sequences. Particularly preferred replacement and insertion sites are underlined.

[0184] In another embodiment, an inhibitory peptide is inserted into the Fc loop of EEMTK, between the Fc residues Met and Lys, and includes two Gly residues as linkers flanking either side of the inserted peptide, as set forth in SEQ ID NO: 209, i.e., SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMGG AQQE ECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE GGTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. The Gly linker is italicized and the inhibitory peptide insertion is underlined. Figures 13A-14B The insertion sites are also shown in Figure 1. In the heavy chain, the first underlined portion corresponds to the anti-PD-1 variable region domain, the second underlined portion corresponds to the trombopagide inserted into the Fc loop, and the third underlined portion corresponds to the afoptin domain fused to the carboxyl terminus.

[0185] Other peptide inhibitors of Tie2 activation (including Ang-2 inhibitors) include A-11 (Compugen), which comprises the amino acid sequence ETFLSTNKLENQ (SEQ ID NO: 184); a peptide having the amino acid sequence NSLSNASEFRAPY (SEQ ID NO: 195); a peptide having the amino acid sequence NLLMAAS (SEQ ID NO: 196); CVX-060 peptide (Pfizer); CVX-037 peptide (Pfizer); and CGEN-25017 (Compugen). Other peptide inhibitors of Tie2 activation are described in U.S. Patent No. 7,138,370. Exemplary peptide inhibitors of angiopoietin-1 or angiopoietin-2 are described in U.S. Patent Nos. 7,138,370, 7,521,053, 7,658,924, and 8,030,025.

[0186] Antibody inhibitors of Tie2 activation (and / or angiopoietin 2) include AMG-780 (Amgen), MEDI-3617 (Medlmmune / AstraZeneca), DX-2240 (Dyax / Sanofi-Aventis), REGN-910 (Sanofi / Regeneron), RG7594 (Roche), LC06 (Roche), TAvi6 (Roche), AT-006 (Roche / Affitech). Other Tie2 receptor binding antibody antagonists and antibody binding sequences derived therefrom are described in U.S. Patent Nos. 6,376,653, 7,521,053, 7,658,924, 8,030,025 and U.S. Patent Application Publication Nos. 2013 / 0078248, 2013 / 0259859 and 2015 / 0197578.

[0187] Tie2 binding antagonists also include the small molecule inhibitors CGI-1842 (CGI Pharmaceuticals), LP-590 (Locus Pharmaceuticals), ACTB-1003 (Act Biotech / Bayer AG), CEP-11981 (Cephalon / Teva), MGCD265 (MethylGene Inc.), Regorafenib (Bayer), Cabozantinib / XL-1884 / BMS-90735l (Exelixis), Foretnib (Exelixis), MGCD-265 (MethylGene Inc.).

[0188] C. TGF pathway inhibitors

[0189] TGF-β comprises a multifunctional group of peptides that control cell proliferation and differentiation, migration and adhesion, extracellular matrix modification (including tumor stroma and immunosuppression in many cell types), angiogenesis and de-proliferation, apoptosis, and other functions. TGF-β is a potent inducer of angiogenesis, which provides a critical support system for solid tumors and a mechanism for the dissemination of tumor cells. Many cells synthesize TGF-β, and almost all of these cells have specific receptors for these peptides. TGF-β1, TGF-β2, and TGF-β3 all act through the same receptor signaling system. The active form of TGF-β is a dimer that signals by forming a membrane-bound heterotetramer composed of the type 1 and type 2 serine-threonine receptors, TGF-βRI and TGF-βRII, respectively.

[0190] Recently, TGF-β pathway inhibitors have been developed, for example, in the form of antibodies or binding fragments directed against TGF-β1 or TGF-β1 RII, such as dominant negative fusion protein fragments containing the extracellular domain (ECD) of TGF-β1 RII. As further described herein, the TGF-β1 RII ECD is further combined with additional binding agents that target additional checkpoint regulator pathways or angiogenesis pathways, which are critical for tumor growth and spread. An exemplary human TGF-β1 RII ECD (wild type) has the amino acid sequence set forth in SEQ ID NO: 186.

[0191] TGF-β pathway inhibitors can be in the form of, for example, antibodies or variable region domain fragments directed against TGF-β1 or TGF-β1 RII (TGF binding peptides or dominant negative fusion protein fragments, such as the extracellular domain (ECD) of TGF-β1 RII). In certain embodiments, the bispecific or trispecific anti-tumor antagonists comprise the TGF-β1 RII ECD. In certain embodiments, the bispecific or trispecific anti-tumor antagonists comprise the TGF-β1 RII ECD. In some embodiments, the TGF-β1 RII ECD is fused to the carboxyl terminus of an IgG in the bispecific or trispecific anti-tumor antagonist, such as in Figure 30A and 30E In some other embodiments, TGF-β1 RII ECD is fused to the amino terminus of IgG in a bispecific or trispecific anti-tumor antagonist, such as Figure 30B and 30D In some other embodiments, the TGF-β1 RII ECD is inserted into the IgG Fc fragment (i.e., CH2 or CH3 region) of IgG in a bispecific or trispecific anti-tumor antagonist, as shown in FIG. Figure 30C and 30F shown.

[0192] Exemplary anti-TGF-β1 antibodies are described in U.S. Patent Nos. 7,067,637, 7,494,651, 7,527,791, and 7,619,069. Exemplary anti-TGF-β1 RII antibodies are described in U.S. Patent No. 7,579,186. An exemplary peptide inhibitor of TGF-β1 is KRIWFIPRSSWYERA (SEQ ID NO: 197).

[0193] D. Overall structure of the targeting domain

[0194] Anti-PD-1, anti-PD-L1, anti-TIGIT, anti-LAG-3, anti-VEGF, anti-VEGFR, anti-angiogenin-1 / 2 and / or anti-Tie2 receptor antagonists can be in the form of monoclonal antibodies, chimeric antibodies, humanized antibodies, scFv or multispecific antagonists, such as bispecific and trispecific antagonists. In addition, any antagonist described herein can have multiple binding specificities targeting PD-1, PD-L1, TIGIT, LAG-3, VEGF, VEGFR, angiopoietin and / or Tie2 receptors. In addition, any antibody antagonist can be engineered to target multiple epitopes in a given target. In addition, in some embodiments, the checkpoint antagonist and / or angiogenesis specificity can be included in the form of a dominant negative fusion protein, such as an extracellular domain (ECD) from the corresponding receptor.

[0195] The HCVRs and LCVRs described herein can be structurally linked to a naturally occurring CH1-CH2-CH3 region or a non-naturally occurring or mutated Fc (CH2-CH3) region, such as an effector-free or largely effector-free Fc (e.g., human IgG2 or IgG4), or an Fc with enhanced binding to one or more activating Fc receptors (FcγRI, FcγRIIa, or FcγRIIIa) to enhance Treg depletion in the tumor environment. Thus, in certain embodiments, the anti-PD-1, anti-PD-L1, anti-TIGIT, anti-LAG-3, anti-VEGF, anti-VEGFR HCVR and LCVR can be structurally linked to CH1-CH2-CH3, which contain one or more modifications, typically used to change one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and / or antigen-dependent cellular cytotoxicity. In addition, the antibodies described herein can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody), or can be modified to change its glycosylation, thereby changing one or more functional properties of the antibody. More specifically, in certain embodiments, the antibodies in the present application may include modifications in the Fc region to produce antibodies with (a) increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased or decreased complement-mediated cytotoxicity (CDC), (c) increased or decreased affinity for C1q and / or (d) increased or decreased affinity for Fc receptors (relative to the parent Fc). Such Fc region variants will typically include at least one amino acid modification in the Fc region. Multiple amino acid modification combinations are considered particularly desirable. For example, the variant Fc region may include two, three, four, five, etc. substitutions therein (e.g., at specific Fc region positions identified herein).

[0196] For uses in which effector functions are completely avoided, for example when antigen binding alone is sufficient to produce the desired therapeutic benefit and effector functions only result in undesirable side effects (or increase the risk of side effects), IgG4 antibodies can be used, or antibodies or fragments thereof lacking an Fc region or substantial fragment can be designed, or the Fc region can be mutated to completely eliminate glycosylation (e.g., N297A). Alternatively, a hybrid construct of human IgG2 (CH1 domain and hinge region) and human IgG4 (CH2 and CH3 domains) can be generated that lacks effector functions, lacks the ability to bind FcγR (e.g., IgG2) and activate complement (e.g., IgG4). When using an IgG4 constant domain, it is generally preferred to include a substitution of S228P, which mimics the hinge sequence in IgG1 and thereby stabilizes the IgG4 molecule, thereby reducing Fab arm exchange between the therapeutic antibody and endogenous IgG4 in the treated patient.

[0197] In certain embodiments, anti-PD-1, anti-PD-L1, anti-TIGIT, anti-LAG-3, anti-VEGF, anti-VEGFR, anti-angiogenin and anti-Tie2R antibodies or fragments thereof may be modified to increase their biological half-life. Various methods can be used, including, for example, those methods that increase the binding affinity of the Fc region to FcRn. In one embodiment, as described in U.S. Patent Nos. 5,869,046 and 6,121,022, the antibody is changed within the CH1 or CL region domain to include a salvage receptor binding epitope (salvage receptor binding epitope) intercepted from two loops of the CH2 domain of the Fc region of IgG. The residue numbering in the Fc region is the numbering of the EU index. The sequence variants disclosed herein are provided with reference to the residue numbering, and the amino acid followed by the residue numbering is the natural amino acid that is substituted, optionally, having previously been a natural residue at that position. Where multiple amino acids are possible at a given position, for example, if the sequence differs between natural isotypes or if multiple mutations are possible at that position, they are separated by slashes (e.g., "X / Y / Z").

[0198] Exemplary Fc variants that increase binding to FcRn and / or improve pharmacokinetic properties include substitutions at positions 259, 308, and 434, including, for example, 259I, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M. Other variants that increase Fc binding to FcRn include: 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al., 2004, J. Biol. Chem. 279(8):6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 305A, 307A, 311A, 312A, 378Q, 380A, 382A, 434A (Shields et al., 2006, J. Biol. Chem. 279(8):6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), al. (2001) J. Biol. Chem., 276(9): 6591-6604), 252F, 252Y, 252W, 254T, 256Q, 256E, 256D, 433R, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H (Dall'Acqua et al. (2002) J. Immunol., 169: 5171-5180, Dall'Acqua et al. (2006) J. Biol. Chem., 281: 23514-23524 and U.S. Patent No. 8,367,805.

[0199] Modification of certain conserved residues in the IgG Fc (I253, H310, Q311, H433, N434), such as the N434A variant (Yeung et al. (2009) J. Immunol. 182:7663), has been proposed as a method to increase FcRn affinity and thereby extend the half-life of antibodies in circulation (WO 98 / 023289). A combined Fc variant comprising M428L and N434S has been shown to increase FcRn binding and extend serum half-life by fivefold (Zalevsky et al. (2010) Nat. Biotechnol. 28:157). A combined Fc variant comprising T307A, E380A, and N434A modifications also extended the half-life of IgG1 antibodies (Petkova et al. (2006) Int. Immunol. 18:1759). In addition, it has been shown that a combination Fc variant comprising M252Y-M428L, M428L-N434H, M428L-N434F, M428L-N434Y, M428L-N434A, M428L-N434M and M428L-N434S can extend half-life (US2006 / 173170). In addition, it has been reported that a combination Fc variant comprising M252Y, S254T and T256E increased half-life by nearly 4-fold (Dall'Acqua et al. (2006) J. Biol. Chem. 281: 23514).

[0200] IV. Preferred Embodiments of Trispecific Antagonists

[0201] In one embodiment, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region having an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 127, 129, 131, 133, 135, and 137; (b) a light chain variable region having an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 128, 130, 132, 134, 136, and 138; (2) a second targeting domain conferred by one or more anti-VEGF antagonists and / or one or more anti-VEGFR antagonists, as further described herein; and (3) a third targeting domain comprising a biological peptide that specifically binds to a Tie2 tyrosine kinase receptor, Ang-1, Ang-2, Ang-3, or Ang-4, as further described herein. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted within the CH3 domain.

[0202] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 137, and (b) a light chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 138; (2) a second targeting domain brought about by one or more anti-VEGF antagonists and / or one or more anti-VEGFR antagonists, as further described herein; and (3) a third targeting domain comprising a biological peptide that specifically binds to a Tie2 tyrosine kinase receptor, Ang-1, Ang-2, Ang-3, or Ang-41, as further described herein. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted within the CH3 domain.

[0203] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region having an amino acid sequence that is about 80% to about 100% identical to the amino acid sequence of SEQ ID NO: 135, and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identical to the amino acid sequence of SEQ ID NO: 136; (2) a second targeting domain that is conferred by one or more anti-VEGF antagonists and / or one or more anti-VEGFR antagonists, as further described herein; and (3) a third targeting domain that comprises a biological peptide that specifically binds to a Tie2 tyrosine kinase receptor, Ang-1, Ang-2, Ang-3, or Ang-4, as further described herein. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted within the CH3 domain.

[0204] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (HCDRs): HCDR1, HCDR2, and HCDR3, wherein HCDR1 has an amino acid sequence that is about 80% or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 51, 54, 56, and 59, wherein HCDR2 has an amino acid sequence that is about 80%, about 85%, about 90%, about 95%, or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 52, 57, and 60, and wherein HCDR3 has an amino acid sequence that is about 80%, about 85%, about 90%, about 95%, or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: NOs: 50, 53, 55, 58 and 61 have about 80%, about 90% or about 100% identity, and (b) a light chain variable region, wherein the light chain variable region comprises three complementarity determining regions (LCDRs): LCDR1, LCDR2 and LCDR3, wherein LCDR1 has an amino acid sequence about 80%, about 90% or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 62, 65, 68, 69, 70 and 73, wherein HCDR2 has an amino acid sequence about 80%, about 90% or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 63, 66, 71 and 74, and wherein HCDR3 has an amino acid sequence about 80%, about 90% or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: The invention also provides a method for preparing an anti-VEGF antagonist comprising: (i) an anti-VEGF antagonist comprising a first target peptide, (ii) an anti-VEGF antagonist comprising a second target peptide, and (iii) a first target peptide comprising a second target peptide that specifically binds to a Tie2 tyrosine kinase receptor, Ang-1, Ang-2, Ang-3, or Ang-4, as further described herein. In a preferred embodiment, the second target peptide is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third target peptide is inserted within the CH3 domain.

[0205] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (HCDRs): HCDR1, HCDR2, and HCDR3, wherein HCDR1 has an amino acid sequence with about 80% or about 100% identity to the amino acid sequence of SEQ ID NO: 59, wherein HCDR2 has an amino acid sequence with about 80%, about 85%, about 90%, about 95%, or about 100% identity to the amino acid sequence of SEQ ID NO: 60, and wherein HCDR3 has an amino acid sequence with about 80%, about 90%, or about 100% identity to the amino acid sequence of SEQ ID NO: 61, and (b) a light chain variable region, wherein the light chain variable region comprises three complementarity determining regions (LCDRs): LCDR1, LCDR2, and LCDR3, wherein LCDR1 has an amino acid sequence with about 80%, about 85%, about 90%, about 95%, or about 100% identity to the amino acid sequence of SEQ ID NO: 61. NO:73 has an amino acid sequence having about 80%, about 90% or about 100% identity, wherein HCDR2 has an amino acid sequence having about 80%, about 90% or about 100% identity to the amino acid sequence of SEQ ID NO:74, and wherein HCDR3 has an amino acid sequence having about 80%, about 90% or about 100% identity to the amino acid sequence of SEQ ID NO:75; (2) a second targeting domain brought about by one or more anti-VEGF antagonists and / or one or more anti-VEGFR antagonists, as further described herein; and (3) a third targeting domain comprising a biological peptide that specifically binds to the Tie2 tyrosine kinase receptor Ang-1, Ang-2, Ang-3 or Ang-4, as further described herein. In a preferred embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted within the CH3 domain.

[0206] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (HCDRs): HCDR1, HCDR2, and HCDR3, wherein HCDR1 has an amino acid sequence with about 80% or about 100% identity to the amino acid sequence of SEQ ID NO: 56, wherein HCDR2 has an amino acid sequence with about 80%, about 85%, about 90%, about 95%, or about 100% identity to the amino acid sequence of SEQ ID NO: 57, and wherein HCDR3 has an amino acid sequence with about 80%, about 90%, or about 100% identity to the amino acid sequence of SEQ ID NO: 58, and (b) a light chain variable region, wherein the light chain variable region comprises three complementarity determining regions (LCDRs): LCDR1, LCDR2, and LCDR3, wherein LCDR1 has an amino acid sequence with about 80%, about 85%, about 90%, about 95%, or about 100% identity to the amino acid sequence of SEQ ID NO: 57. NO:70, wherein the HCDR2 has an amino acid sequence that is about 80%, about 90% or about 100% identical to the amino acid sequence of SEQ ID NO:71, and wherein the HCDR3 has an amino acid sequence that is about 80%, about 90% or about 100% identical to the amino acid sequence of SEQ ID NO:72; (2) a second targeting domain that is conferred by one or more anti-VEGF antagonists and / or one or more anti-VEGFR antagonists, as further described herein; and (3) a third targeting domain that comprises a biological peptide that specifically binds to the Tie2 tyrosine kinase receptor Ang-1, Ang-2, Ang-3 or Ang-4, as further described herein. In a preferred embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted within the CH3 domain.

[0207] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising one or more anti-PD-1 variable region domains or one or more anti-PD-L1 variable region domains brought by one or more variable regions; (2) a first targeting domain comprising afipronil (or ) domain, the amino acid sequence of which is SEQ ID NO: 185; and (3) a third targeting domain comprising a biological peptide that specifically binds to the Tie2 tyrosine kinase receptor Ang-1, Ang-2, Ang-3, or Ang-4, as further described herein. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted within the CH3 domain.

[0208] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 137, and (b) a light chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 138; (2) a second targeting domain comprising afipronil (or afipronil) having the amino acid sequence of SEQ ID NO: 185 ) domain; (3) a third targeting domain comprising a biological peptide that specifically binds to the Tie2 tyrosine kinase receptor Ang-1, Ang-2, Ang-3, or Ang-4, as further described herein. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted within the CH3 domain.

[0209] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 135, and (b) a light chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 136; (2) a second targeting domain comprising afipronil (or ) domain; (3) a third targeting domain comprising a biological peptide that specifically binds to the Tie2 tyrosine kinase receptor Ang-1, Ang-2, Ang-3, or Ang-4, as further described herein. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted within the CH3 domain.

[0210] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 137, and (b) a light chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 138; (2) a second targeting domain comprising afipronil (or ) domain; and (3) a third targeting domain comprising only an amino acid sequence of SEQ ID NO: 194. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted within the CH3 domain.

[0211] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 135, and (b) a light chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 136; (2) a second targeting domain comprising afipronil (or afipronil) having the amino acid sequence of SEQ ID NO: 185 (3) a third targeting domain comprising only an amino acid sequence of SEQ ID NO: 194. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted within the CH3 domain.

[0212] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 137, and (b) a light chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 138; (2) a second targeting domain comprising afipronil (or afipronil) having the amino acid sequence of SEQ ID NO: 185 ) domain; and (3) a third targeting domain comprising the amino acid sequence of SEQ ID NO: 182. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted within the CH3 domain.

[0213] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 135; and (b) a light chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 136; (b) a second targeting domain comprising afipronil (or afipronil) having the amino acid sequence of SEQ ID NO: 185. ) domain; and (3) a third targeting domain comprising the amino acid sequence of SEQ ID NO: 182. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted within the CH3 domain.

[0214] In some other embodiments, the trispecific anti-tumor antagonist comprises: a heavy chain whose amino acid sequence is about 80% to about 100% identical to an amino acid sequence selected from SEQ ID NOs: 198, 200, 202, 203 and 204; and a light chain whose amino acid sequence is about 80% to about 100% identical to an amino acid sequence selected from SEQ ID NOs: 199 and 201.

[0215] In some other embodiments, the trispecific anti-tumor antagonist comprises: a heavy chain, whose amino acid sequence is about 80% to about 100% identical to the amino acid sequence of SEQ ID NO: 198; and a light chain, whose amino acid sequence is about 80% to about 100% identical to the amino acid sequence of SEQ ID NO: 199.

[0216] In some other embodiments, the trispecific anti-tumor antagonist comprises: a heavy chain, whose amino acid sequence is about 80% to about 100% identical to the amino acid sequence of SEQ ID NO: 200; and a light chain, whose amino acid sequence is about 80% to about 100% identical to the amino acid sequence of SEQ ID NO: 201.

[0217] In some other embodiments, the trispecific anti-tumor antagonist comprises: a heavy chain, whose amino acid sequence is about 80% to about 100% identical to the amino acid sequence of SEQ ID NO: 202; and a light chain, whose amino acid sequence is about 80% to about 100% identical to the amino acid sequence of SEQ ID NO: 201.

[0218] Figures 3A-4CA variety of different trispecific anti-tumor antagonists are shown, where: (1) the VH1 and VL1 regions correspond to anti-PD-1 variable region domains or other checkpoint antibodies; (2) the double ovals correspond to the aflibercept fusion protein domains; and (3) the circles correspond to trombopag. Figures 5A-5E A variety of different trispecific antagonists are shown, where (1) the VH1 and VL1 regions correspond to the variable region domains of an anti-PD-1 or another checkpoint antibody; (2) the VH and VL of bevacizumab or another anti-VEGF-A antibody, and (3) the circle corresponds to trombopag. As shown in these figures, these components can be rearranged into a variety of structures. For example, trombopag is arranged in the following positions: (i) fused to the carboxyl terminus of each IgG4 CH3 region ( Figures 3A-3D ); (ii) inserted into each of the two CH3 regions ( Figure 3E , Figure 4A and Figure 5A ); (iii) fused to the carboxyl terminus of each CH1 region ( Figure 3F , Figure 4C , Figures 5C-5E ); (iv) fused to the carboxyl terminus of each aflibercept fusion protein domain ( Figure 3G ); (v) fused to the carboxyl terminus of each CL region ( Figure 3H , Figure 5B ); or (vi) fused to the amino terminus of the CH2 domain ( Figure 4B ).

[0219] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region having an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 127, 129, 131, 133, 135, and 137; and (b) a light chain variable region having an amino acid sequence with about 80% to about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 128, 130, 132, 134, 136, and 138; (2) a second targeting domain comprising an anti-VEGF antagonist or an anti-VEGFR antagonist; and (3) a third targeting domain comprising a TGF-β pathway inhibitor. In one embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is downstream of the second targeting domain at the carboxyl terminus.

[0220] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 137, and (b) a light chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 138; (2) a second targeting domain comprising an anti-VEGF antagonist or an anti-VEGFR antagonist; and (3) a third targeting domain comprising a TGF-β pathway inhibitor. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxyl terminus.

[0221] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 135, and (b) a light chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 136; (2) a second targeting domain comprising an anti-VEGF antagonist or an anti-VEGFR antagonist; and (3) a third targeting domain comprising a TGF-β pathway inhibitor. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxyl terminus.

[0222] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (HCDRs): HCDR1, HCDR2, and HCDR3, wherein HCDR1 has an amino acid sequence that is about 80% or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 51, 54, 56, and 59, wherein HCDR2 has an amino acid sequence that is about 80%, about 85%, about 90%, about 95%, or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 52, 57, and 60, and wherein HCDR3 has an amino acid sequence that is about 80%, about 85%, about 90%, about 95%, or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: NOS: 50, 53, 55, 58 and 61, and (b) a light chain variable region, wherein the light chain variable region comprises three complementarity determining regions (LCDRs): LCDR1, LCDR2 and LCDR3, wherein LCDR1 has an amino acid sequence with about 80%, about 90% or about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 62, 65, 68, 69, 70 and 73, wherein HCDR2 has an amino acid sequence with about 80%, about 90% or about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 63, 66, 71 and 74, wherein HCDR3 has an amino acid sequence with about 80%, about 90% or about 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 63, 66, 71 and 74, The amino acid sequences of NOs: 64, 67, 72, and 75 have about 80%, about 90%, or about 100% identity; (2) a second targeting domain comprising an anti-VEGF antagonist or an anti-VEGFR antagonist; and (3) a third targeting domain comprising a TGF-β pathway inhibitor. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxyl terminus.

[0223] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (HCDRs): HCDR1, HCDR2, and HCDR3, wherein HCDR1 has an amino acid sequence with about 80% or about 100% identity to the amino acid sequence of SEQ ID NO: 59, wherein HCDR2 has an amino acid sequence with about 80%, about 85%, about 90%, about 95% or about 100% identity to the amino acid sequence of SEQ ID NO: 60, and wherein HCDR3 has an amino acid sequence with about 80%, about 90% or about 100% identity to the amino acid sequence of SEQ ID NO: 61, and (b) a light chain variable region, wherein the light chain variable region comprises three complementarity determining regions (LCDRs): LCDR1, LCDR2, and LCDR3, wherein LCDR1 has an amino acid sequence with about 80%, about 85%, about 90%, about 95% or about 100% identity to the amino acid sequence of SEQ ID NO: 60. NO:73, wherein the HCDR2 has an amino acid sequence that is about 80%, about 90% or about 100% identical to the amino acid sequence of SEQ ID NO:74, and wherein the HCDR3 has an amino acid sequence that is about 80%, about 90% or about 100% identical to the amino acid sequence of SEQ ID NO:75; (2) a second targeting domain that is brought about by one or more anti-VEGF antagonists and / or one or more anti-VEGFR antagonists, as further described herein, and (3) a third targeting domain that comprises a TGF-β pathway inhibitor. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxyl terminus.

[0224] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (HCDRs): HCDR1, HCDR2, and HCDR3, wherein HCDR1 has an amino acid sequence with about 80% or about 100% identity to the amino acid sequence of SEQ ID NO: 56, wherein HCDR2 has an amino acid sequence with about 80%, about 85%, about 90%, about 95%, or about 100% identity to the amino acid sequence of SEQ ID NO: 57, and wherein HCDR3 has an amino acid sequence with about 80%, about 90%, or about 100% identity to the amino acid sequence of SEQ ID NO: 58, and (b) a light chain variable region, wherein the light chain variable region comprises three complementarity determining regions (LCDRs): LCDR1, LCDR2, and LCDR3, wherein LCDR1 has an amino acid sequence with about 80%, about 85%, about 90%, about 95%, or about 100% identity to the amino acid sequence of SEQ ID NO: 57. NO:70, wherein HCDR 2 has an amino acid sequence that is about 80%, about 90% or about 100% identical to the amino acid sequence of SEQ ID NO:71, and wherein HCDR3 has an amino acid sequence that is about 80%, about 90% or about 100% identical to the amino acid sequence of SEQ ID NO:72; (2) a second targeting domain that is brought about by one or more anti-VEGF antagonists and / or one or more anti-VEGFR antagonists, as further described herein, and (3) a third targeting domain that comprises a TGF-β pathway inhibitor. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxyl terminus.

[0225] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 137, and (b) a light chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 138; (2) a second targeting domain comprising afipronil (or afipronil) having the amino acid sequence of SEQ ID NO: 185 (3) a third targeting domain comprising a TGF-β pathway inhibitor. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxyl terminus.

[0226] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region having an amino acid sequence with an amino acid sequence that is about 80% to about 100% identical to the amino acid sequence of SEQ ID NO: 135, and (b) a light chain variable region having an amino acid sequence that is about 80% to about 100% identical to the amino acid sequence of SEQ ID NO: 136; (2) a second targeting domain comprising afipronil (or (3) a third targeting domain comprising a TGF-β pathway inhibitor. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxyl terminus.

[0227] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 137, and (b) a light chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 138; (2) a second targeting domain comprising α-glycosylated afipronil (or (3) a third targeting domain comprising a TGF-β pathway inhibitor. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxyl terminus.

[0228] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 135, and (b) a light chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 136; (2) a second targeting domain comprising α-glycosylated afipronil (or (3) a third targeting domain comprising a TGF-β pathway inhibitor. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxyl terminus.

[0229] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 137, and (b) a light chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 138; (2) a second targeting domain comprising afipronil (or afipronil) having the amino acid sequence of SEQ ID NO: 185 ) domain; and (3) a third targeting domain comprising a TGFβ1 RII extracellular domain (EAD) having the amino acid sequence of SEQ ID NO: 186. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxyl terminus.

[0230] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 135, and (b) a light chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 136; (2) a second targeting domain comprising afipronil (or afipronil) having the amino acid sequence of SEQ ID NO: 185 ) domain; and (3) a third targeting domain comprising a TGFβ1 RII extracellular domain (ECD) having the amino acid sequence of SEQ ID NO: 186. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxyl terminus.

[0231] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 137, and (b) a light chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 138; (2) a second targeting domain comprising α-glycosylated afipronil (or ) domain brings; (3) a third targeting domain, which comprises a TGFβ1 RII extracellular domain (EAD) having an amino acid sequence of SEQ ID NO: 186. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxyl terminus.

[0232] In some other embodiments, the trispecific anti-tumor antagonist comprises: (1) a first targeting domain comprising: (a) a heavy chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 135, and (b) a light chain variable region having an amino acid sequence with about 80% to about 100% identity to the amino acid sequence of SEQ ID NO: 136; (2) a second targeting domain comprising α-glycosylated afipronil (or ) domain; and (3) a third targeting domain comprising a TGFβ1 RII extracellular domain (ECD) having the amino acid sequence of SEQ ID NO: 186. In another embodiment, the second targeting domain is inserted downstream of the CH3 domain at the carboxyl terminus of the antagonist, and the third targeting domain is inserted downstream of the second targeting domain at the carboxyl terminus.

[0233] In some other embodiments, the trispecific anti-tumor antagonist comprises: a heavy chain whose amino acid sequence is about 80% to about 100% identical to an amino acid sequence selected from SEQ ID NOs: 187, 189, 190, 192, 205 and 206; and a light chain whose amino acid sequence is about 80% to about 100% identical to an amino acid sequence selected from SEQ ID NOs: 188, 191 and 193.

[0234] In some other embodiments, the trispecific anti-tumor antagonist comprises: a heavy chain, whose amino acid sequence is about 80% to about 100% identical to the amino acid sequence of SEQ ID NO: 192; and a light chain, whose amino acid sequence is about 80% to about 100% identical to the amino acid sequence of SEQ ID NO: 193.

[0235] In some other embodiments, the trispecific anti-tumor antagonist comprises: a heavy chain, whose amino acid sequence is about 80% to about 100% identical to the amino acid sequence of SEQ ID NO: 205; and a light chain, whose amino acid sequence is about 80% to about 100% identical to the amino acid sequence of SEQ ID NO: 193.

[0236] In some other embodiments, the trispecific anti-tumor antagonist comprises: a heavy chain, whose amino acid sequence is about 80% to about 100% identical to the amino acid sequence of SEQ ID NO: 206; and a light chain, whose amino acid sequence is about 80% to about 100% identical to the amino acid sequence of SEQ ID NO: 193.

[0237] The trispecific anti-tumor antagonists of the present application can be constructed with an IgG backbone. More specifically, any trispecific antagonist of the present application can be constructed with an IgG1 or IgG4 backbone. The use of an IgG1 backbone is preferred for the following cancer treatment, in which the target is present on antigen-presenting cells that can mediate antibody-dependent cell-mediated cytotoxicity (ADCC). The use of an IgG4 backbone can target antigens, where antigen binding alone is sufficient to produce the desired therapeutic benefit. Antagonists based on IgG4 exclude undesirable effector functions associated with, for example, IgG1 antibodies, including FcγR binding and complement activation.

[0238] V. Methods of Using Anti-tumor Antagonists

[0239] The anti-tumor antagonists of the present application, such as anti-PD-1 antagonists, anti-PD-1 antibody fragments, anti-PD-L1 antibodies, anti-PD-L1 antibody fragments, anti-TIGIT antibodies, anti-TIGIT antibody fragments, anti-LAG-3 antibodies, anti-LAG-3 antibody fragments, angiogenesis pathway inhibitors, TGFβ pathway inhibitors, and bispecific anti-tumor antagonists and trispecific anti-tumor antagonists that specifically bind to PD-1, PD-L1, TIGIT, LAG-3, VEGF / VEGFR, TGFβ / TGFβ receptors, have many in vitro and in vivo utilities, including, for example, enhancing immune responses and treating cancer, infectious diseases or autoimmune diseases.

[0240] A. Anti-tumor therapy

[0241] In some embodiments, the anti-tumor antagonists of the present application are administered in vitro or ex vivo to cells in culture, or in vivo to human subjects to enhance immunity in a variety of diseases. Therefore, provided herein is a method for changing an immune response in a subject, the method comprising administering an antibody or antigen-binding fragment thereof as described herein to the subject, thereby enhancing, stimulating or upregulating the subject's immune response. Preferred subjects include human patients in need of an enhanced immune response. The method is particularly suitable for treating human patients with diseases that can be treated by enhancing an immune response (e.g., a T cell-mediated immune response). The method is particularly suitable for in vivo treatment of cancer or chronic infection. For example, an anti-PD-1, anti-PD-L1, anti-TIGIT or anti-LAG-3 composition can be administered together with an antigen of interest, or the antigen may already be present in the subject to be treated (e.g., a subject with a tumor or carrying a virus) to enhance antigen-specific immunity. When an anti-TIGIT antibody is administered together with another agent, the two components can be administered separately or simultaneously.

[0242] In some embodiments, the checkpoint regulator antagonist used in the above method is an anti-PD-1, anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-LAG-3 antibody, a fragment thereof, or a combination thereof. In some embodiments, the checkpoint regulator antagonist is a bispecific antibody or a trispecific antibody of the present application.

[0243] In some embodiments, the checkpoint regulator antagonist or anti-tumor antagonist is in the form of an antibody or antibody fragment.In some embodiments, the antibodies described herein are human or humanized antibodies.

[0244] Also included is a method for detecting and / or measuring the presence of human PD-1, human PD-L1, human TIGIT or human LAG-3 in a sample, the method comprising contacting the sample and a control sample with a human monoclonal antibody or an antigen-binding fragment thereof, the human monoclonal antibody or antigen-binding fragment thereof specifically binding to the human PD-1, human PD-L1, human TIGIT or human LAG-3 under conditions that allow formation of a complex between the antibody or fragment and human PD-1, human PD-L1, human TIGIT or human LAG-3, and then detecting the formation of the complex, wherein a difference in complex formation between the sample and the control sample indicates the presence of human PD-1, human PD-L1, human TIGIT or human LAG-3 antigen in the sample.

[0245] In view of the fact that anti-PD-1, anti-PD-L1, anti-TIGIT and anti-LAG-3 antibodies have the ability to block the inhibition or co-inhibition of T cell responses, provided herein are in vitro and in vivo methods for stimulating, enhancing or upregulating antigen-specific T cell responses, such as anti-tumor T cell responses, using the antibodies described herein. In certain embodiments, CD3 stimulation (e.g., by co-incubation with cells expressing cell membrane CD3) is also provided, which stimulation can be provided while, before or after treatment with anti-PD-1, anti-PD-L1, anti-TIGIT or anti-LAG-3 antibodies. For example, provided herein is a method for enhancing antigen-specific T cell responses, the method comprising contacting T cells with anti-PD-1, anti-PD-L1, anti-TIGIT or anti-LAG-3 antibodies as described herein, and optionally contacting with CD3, thereby enhancing antigen-specific T cell responses, for example, by removing the inhibitory effects mediated by PD-1, PD-L1, TIGIT or LAG-3. Any suitable indicator of antigen-specific T cell responses can be used to measure antigen-specific T cell responses. Non-limiting examples of such suitable indicators include increased T cell proliferation in the presence of the antibody and / or increased cytokine production in the presence of the antibody. In a preferred embodiment, the production of interleukin-2 and / or interferon-γ by antigen-specific T cells is enhanced.

[0246] Further included are methods for enhancing a subject's immune response (e.g., an antigen-specific T cell response), the method comprising administering to the subject an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-LAG-3 antibody, or a bispecific or trispecific anti-tumor antagonist as described herein, thereby enhancing the immune response (e.g., an antigen-specific T cell response) in the subject. In a preferred embodiment, the subject is a subject with a tumor, and the anti-tumor immune response is enhanced. The tumor may be a solid tumor or a liquid tumor, such as a hematological malignancy. In certain embodiments, the tumor is an immunogenic tumor. In other embodiments, the tumor is non-immunogenic. In certain embodiments, the tumor is PD-L1 positive. In other embodiments, the tumor is PD-L1 negative. The subject may also be a subject carrying a virus, and the result of administering an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-LAG-3 antibody, a bispecific anti-tumor antagonist as described herein, or a trispecific anti-tumor antagonist is an enhanced antiviral immune response in the subject.

[0247] In one embodiment, a method for inhibiting the growth of tumor cells in a subject comprises administering to the subject an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-LAG-3 antibody, or a bispecific anti-tumor antagonist or trispecific anti-tumor antagonist described herein, thereby inhibiting the growth of the tumor in the subject. Also provided is a method for treating a chronic viral infection in a subject, comprising administering to the subject an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-LAG-3 antibody, a bispecific anti-tumor antagonist or a bispecific anti-tumor antagonist described herein, thereby treating the chronic viral infection in the subject.

[0248] Also contemplated herein is the depletion of T cells from the tumor microenvironment of a subject having a tumor, e.g., a cancerous tumor. reg A method for treating a cell, the method comprising administering to a subject a therapeutically effective amount of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-LAG-3 antibody, a bispecific anti-tumor antagonist or a trispecific anti-tumor antagonist as described herein, wherein the bispecific anti-tumor antagonist or trispecific anti-tumor antagonist comprises a T cell-stimulating agent in a tumor microenvironment. reg Cell-depleted Fc. The Fc can be, for example, an Fc that has effector function or enhanced effector function, such as binding or enhanced binding to one or more activated Fc receptors.

[0249] In a preferred embodiment, T cells are generated in the tumor microenvironment. reg consumption without significantly depleting or suppressing T eff , and did not significantly deplete or inhibit T cells outside the tumor microenvironment. eff and T reg In certain embodiments, for example, in a tumor microenvironment, the subject is in a T reg TIGIT levels on T cells were higher than in T eff In certain embodiments, anti-TIGIT antibodies or antagonists can deplete T in tumors. regs and / or T cells in tumor-infiltrating lymphocytes (TILs) regs For example, in the CT26 tumor model, anti-mouse TIGIT antibodies formulated as mouse IgG2a (which exhibits effector function) partially depleted T cells. reg and CD8 + T cells, but does not deplete CD4 + T cells. The corresponding anti-TIGIT antibody, formulated as mouse IgG1 D265A, which is a non-effector, does not deplete T cells.

[0250] When considering whether to use an Fc effector- or effector-free anti-TIGIT antibody, appropriate consideration must be given to the potential for enhanced anti-tumor immune responses in T cells.regs Depletion of CD8 T cells will eliminate some of the cells needed to actually kill tumor cells. + Although depletion of T cells is expected, regs to enhance anti-tumor activity, but recent studies have shown that TIGIT + T regs The ligation of TIGIT on T reg T cell-mediated eff Cell proliferation inhibition (Joller et al. (2014) Immunity 40:569), suggesting that blocking TIGIT signaling (e.g., using the antagonist anti-TIGIT antibodies of the present invention) can also enhance anti-tumor activity. Therefore, it may be most effective to use antagonist anti-TIGIT antibodies that lack effector function and have the following functions: i) block T regs ii) Activate anti-tumor CD8 T cells by blocking TIGIT signaling in the mitochondria and thereby reducing their immunosuppressive activity; iii) Activate anti-tumor CD8 T cells by blocking TIGIT inhibition + T cells, while preventing them from being consumed by effector functions; iii) by allowing DNAM to bind to PVR (CD155, TIGIT ligand) that would otherwise be bound by TIGIT (and reducing direct TIGIT-DNAM interactions) to enhance DNAM-mediated activation (Johnston et al. (2014) Cancer Cell 26:923). This also applies to the use of anti-PD-1 antibodies, anti-PD-L1 antibodies, bispecific anti-tumor antagonists or trispecific anti-tumor antagonists.

[0251] In certain embodiments, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-LAG-3 antibody, a bispecific anti-tumor antagonist or a trispecific anti-tumor antagonist as described herein is administered to a subject as an adjuvant therapy. Treatment of cancer patients with anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIGIT antibodies, anti-LAG-3 antibodies, bispecific anti-tumor antagonists or trispecific anti-tumor antagonists as described herein may result in long-term sustained responses relative to current standards of care; long-term survival of at least 1, 2, 3, 4, 5, 10 years or longer, and recurrence-free survival of at least 1, 2, 3, 4, 5 or 10 years or longer. In certain embodiments, treatment of cancer patients with anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIGIT antibodies, anti-LAG-3 antibodies, bispecific anti-tumor antagonists or trispecific anti-tumor antagonists can prevent the recurrence of cancer or delay the recurrence of cancer for example, 1, 2, 3, 4, 5 or 10 years or longer. Anti-PD-1, anti-PD-L1, anti-TIGIT, and / or anti-LAG-3 therapy can be used as primary or secondary therapy.

[0252] In certain preferred embodiments, the subject suffers from a cell proliferative disease or cancer. Anti-TIGIT antibodies can enhance the immune response to the patient's cancer cells by blocking PVR / Nectin-2 signal transduction through TIGIT. Similarly, the blocking provided herein is a method for treating a subject with cancer, the method comprising administering to the subject an anti-PD-1, anti-PD-L1, anti-TIGIT, anti-LAG-3, a bispecific anti-tumor antagonist thereof, or a trispecific anti-tumor antagonist thereof as described herein, so that the subject is treated, for example, to inhibit or reduce the growth of cancerous tumors and / or to cause tumor regression. Anti-PD-1, anti-PD-L1, anti-TIGIT, anti-LAG-3, a bispecific anti-tumor antagonist thereof, or a trispecific anti-tumor antagonist thereof as described herein can be used alone to inhibit the growth of cancerous tumors. Alternatively, any of these anti-tumor antagonists can be used in combination with another agent, such as other anti-cancer targets, immunogenic agents, standard cancer treatments, or other antibodies, as described below.

[0253] Thus, provided herein are methods for treating cancer by, for example, inhibiting tumor cell growth in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-1, anti-PD-L1, anti-TI GIT, anti-LAG-3 antagonist, a bispecific anti-tumor antagonist described herein, or a trispecific anti-tumor antagonist. Preferably, the antibody is a human anti-PD-1, anti-PD-L1, anti-TIGIT, anti-LAG-3 antibody comprising an anti-PD-1, anti-PD-L1, anti-TIGIT or anti-LAG-3 HCVR and LCVR described herein, or may be a chimeric, humanized or non-human anti-hu PD-1, anti-PD-L1 antibody, anti-huTIGIT or anti-LAG-3 antibody, such as a chimeric, humanized or non-human anti-PD-1, anti-PD-L1, anti-TIGIT or anti-LAG-3 antibody that competes for binding to the same epitope as at least one of the anti-PD-1, anti-PD-L1, anti-TIGIT or anti-LAG-3 antibodies described herein or binds to the same epitope as at least one of the anti-PD-1, anti-PD-L1, anti-TIGIT or anti-LAG-3 antibodies described herein.

[0254] Cancers whose growth can be inhibited using the antibodies of the present application include cancers that are generally responsive to immunotherapy. Non-limiting examples of cancers for treatment include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-NSCLC, gliomas, gastrointestinal cancer, renal cancer (e.g., cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, gastric cancer, bladder cancer, liver cancer, breast cancer, colon cancer and head and neck cancer (or cancer), gastric cancer, germ cell tumors, pediatric sarcomas, sinonasal natural killer cells (sinonasal natural killer cells ... killer,), melanoma (e.g., metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors of childhood, ureteral cancer, renal pelvis cancer, central nervous system tumors (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos), virus-associated cancers (e.g., tumors associated with human papillomavirus (HPV)), and cancers derived from one of the two major blood cell lineages, the myeloid cell lineage (which produces granulocytes, erythrocytes, platelets, macrophages, and hematological malignancies of the lymphoid cell line (which gives rise to B, T, NK and plasma cells) or lymphoid cell lineage (which gives rise to B, T, NK and plasma cells), such as all types of leukemias, lymphomas and myelomas, such as acute, chronic, lymphocytic and / or myeloid leukemias, such as acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML), undifferentiated AML (MO), myeloblastic leukemia (M1), myeloblastic leukemia (M2; as the cells mature), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryocytic leukemia (M7), isolated granulocytic sarcoma and chloroma;Lymphomas, such as Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NEIL), B-cell lymphoma, T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytic B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphocyte and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system Lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), hematopoietic neoplasms of the lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunogenic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also known as mycosis fungoides or Sézary syndrome), and leukemia with Waldenstrom's giant globulin. Lymphoplasmacytoid lymphoma (LPL) of leukemia; myelomas, such as IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called indolent myeloma), solitary plasmacytoma and multiple myeloma; chronic lymphocytic leukemia (CLL); hairy cell lymphoma; hematopoietic neoplasms of the myeloid lineage; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; seminoma, teratoma, central and peripheral nerve tumors, including astrocytoma, schwannoma Tumors of mesenchymal origin, including fibrosarcoma, rhabdomyomas, and osteosarcomas; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma, and teratoma, hematopoietic tumors of the lymphoid lineage, such as T-cell and B-cell tumors, including but not limited to T-cell diseases, such as T-cell prolymphocytic leukemia (T-PLL), including small cell and cerebrospinal cell types; large granular lymphocytic leukemia (LGL), preferably T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / post-thymic T-cell lymphoma (polymorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; head and neck cancer, renal cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma, and any combination of the above cancers. The methods described herein can also be used to treat metastatic cancers, refractory cancers (e.g., cancers refractory to previous immunotherapy, such as with blocking CTLA-4 or PD-1 antibodies), and relapsed cancers.

[0255] Anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIGIT antibodies, anti-LAG-3 antibodies, bispecific anti-tumor antagonists or trispecific anti-tumor antagonists can be administered alone, in combination with another anti-tumor antagonist, or simultaneously with another anti-tumor antagonist. Anti-PD-1 antibodies, anti-TIGIT, anti-LAG-3 antibodies, bispecific anti-tumor antagonists or trispecific anti-tumor antagonists can also be administered simultaneously with immunogenic agents (e.g., cancer cells, tumor vaccines, purified tumor antigens (including recombinant proteins, peptides and carbohydrate molecules), cells transfected with genes encoding immunostimulatory cytokines in cancer vaccine strategies (He et al. (2004) J. Immunol. 173: 4919-28) or oncolytic viruses.

[0256] Many experimental strategies for vaccination against tumors have been designed. In one of these strategies, vaccines are prepared using autologous or allogeneic tumor cells. Some of these cell vaccines have been shown to be most effective when tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be an effective antigen presentation activator for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3539-43). In preclinical models, cancer vaccines have been shown to enhance the infiltration of effector T cells into tumors. The main types of cancer vaccines include peptide vaccines, vector-based antigen-specific vaccines, whole cell vaccines, and dendritic cell vaccines. All vaccine-based therapies are designed to deliver single or multiple antigen epitopes or antigens from whole cells to patients and induce tumor-specific effector T cells. Therefore, vaccine-based therapies may be the most effective method for inducing T cell infiltration into tumors.

[0257] Studies of gene expression and large-scale gene expression patterns in various tumors have led to the definition of so-called tumor-specific antigens (Rosenberg, SA (1999) Immunity 10: 281-7). In many cases, these tumor-specific antigens are differentiation antigens expressed in tumors and cells of tumor origin, such as the melanocyte antigen gp100, MAGE antigens, and Trp-2. More importantly, many of these antigens can be shown to be targets of tumor-specific T cells found in the host.

[0258] PD-1, PD-L1, TIGIT and / or LAG-3 inhibition can be used in combination with a series of recombinant proteins and / or peptides expressed in tumors to generate an immune response against these proteins. Such proteins can be regarded as self-antigens by the immune system and can therefore be tolerated. Tumor antigens may include the protein telomerase, which is a protein required for the synthesis of chromosome telomeres and is expressed in more than 85% of human cancers and only in a limited number of somatic tissues (Kim et al. (1994) Science 266: 2011-2013). Tumor antigens can also be “neoantigens” in cancer cells because somatic mutations change the protein sequence or form a fusion protein between two unrelated sequences (i.e., bcr-abl in the Philadelphia chromosome), or are specific genotypes (idiotypes) from B cell tumors.

[0259] Non-limiting examples of tumor vaccines include sipuleucel-T an FDA-approved tumor vaccine for metastatic prostate cancer; tumor cells transfected to express the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF), such as the whole-cell GM-CSF-secreting irradiated allogeneic pancreatic cancer vaccine (GVAX; Johns Hopkins); a peptide vaccine composed of immunogenic peptides derived from breast cancer antigens, neu, legumain, and β-catenin, which, when combined with an anti-PD-1 antibody, prolonged progression-free survival in vaccine-induced tumor-bearing mice (Karyampudi L. et al. (2014) Cancer Res 74:2974-2985); peptides of melanoma antigens, such as gp100, MAGE antigens, Trp-2, MARTI, and / or tyrosinase, or other tumor vaccines, including viral proteins associated with human cancers, such as human papillomavirus (HPV) (e.g., and Hepatitis B virus (e.g., Engerix-B and Recombivax HB); Hepatitis C virus (HCV), Kaposi's sarcoma-associated herpes sarcoma virus (KSHV). Another form of tumor-specific antigen that can be used in combination with TIGIT inhibition is purified heat shock proteins (HSPs) isolated from the tumor tissue itself. These heat shock proteins contain protein fragments from tumor cells, and these HSPs are very effective in delivering to antigen-presenting cells to elicit tumor immunity. Talimogenelaherparepvec (T-VEC or ) is an FDA-approved oncolytic virus used to treat some patients with metastatic melanoma that cannot be surgically removed.

[0260] Dendritic cells (DCs) are effective antigen-presenting cells that can be used to elicit antigen-specific responses. DCs can be produced in vitro and loaded with various protein and peptide antigens, as well as tumor cell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs can also be transduced by genetic means to express these tumor antigens. For immune purposes, DCs have also been directly fused to tumor cells (Kugler et al. (2000) Nature Medicine 6: 332-336). As a vaccination method, DC immunity can be effectively combined with TIGIT blocking to activate (release) a more effective anti-tumor response.

[0261] PD-1, PD-L1, TIGIT and / or LAG-3 inhibition can also be used in combination with standard cancer treatment methods (such as surgery, radiotherapy and chemotherapy). In particular, PD-1, PD-L1, TIGIT and / or LAG-3 inhibition can be effectively combined with chemotherapy regimens. In these cases, it is possible to reduce the dose of the chemotherapeutic agent administered (Mokyr et al. (1998) Cancer Research 58: 5301-5304). An example of such a combination is an anti-tumor antagonist combined with decarbazine for the treatment of melanoma. Another example of such a combination is a combination of a checkpoint regulator antagonist or an anti-tumor antagonist with interleukin-2 (IL-2) for the treatment of melanoma. For example, the scientific basis for the use of PD-1, PD-L1, TIGIT and / or LAG-3 inhibition in combination with chemotherapy to promote cell death is that the result of the cytotoxic effects of most chemotherapeutic compounds should result in elevated levels of tumor antigens in the antigen presentation pathway. Other combination therapies that may synergize with TIGIT, PD-1, PD-L1 and / or LAG-3 inhibition through cell death are radiation, surgery, and hormone deprivation. Each of these regimens generates a source of tumor antigens in the host. Angiogenesis inhibitors can also be combined with TIGIT, PD-1, PD-L1 and / or LAG-3 inhibition. Inhibition of angiogenesis leads to tumor cell death, which may allow tumor antigens to enter host antigen presentation pathways.

[0262] Anti-TIGIT antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, bispecific anti-tumor antagonists and trispecific anti-tumor antagonists described herein can also be used in combination with bispecific antibodies that target Fcα or Fcγ receptor-expressing effector cells on tumor cells (see, for example, U.S. Patent Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. For example, anti-Fc receptor / anti-tumor antigen (e.g., Her-2 / neu) bispecific antibodies have been used to target macrophages to tumor sites. This targeting can more effectively activate tumor-specific responses. The T cell arm of these responses will be enhanced by inhibiting TIGIT, PD-1, PD-L1 and / or LAG-3. Alternatively, antigens can be delivered directly to DCs using bispecific antibodies that bind to tumor antigens and dendritic cell-specific cell surface markers.

[0263] Tumors evade host immune surveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating immunosuppressive proteins expressed by tumors. These include TGF-β, IL-10, and Fas ligand. Antibodies against each of these entities can be used in combination with the anti-tumor antagonists described herein to counteract the effects of immunosuppressants and promote the host's tumor immune response.

[0264] Other antibodies that activate the host immune response can be used in combination with the anti-tumor antagonists described herein. These include molecules that activate DC function and antigen presentation on the surface of dendritic cells. Anti-CD40 antibodies can effectively replace the auxiliary activity of T cells (Ridge et al. (1998) Nature 393: 474-478), and can be used together with anti-TIGIT antibodies. Activation antibodies against T cell co-stimulatory molecules can also be provided, such as OX-40 (Weinberg et al. (2000) Immunol 164: 2160-2169), CD137 / 4-1BB (Melero et al. (1997) Nature Medicine 3: 682-685 (1997), and ICOS (Hutloff et al. (1999) Nature 397: 262-266) to increase T cell activation levels. In addition, as further described below, inhibitors of other immune checkpoint regulators can also be used in combination with other anti-tumor antagonists described herein.

[0265] Bone marrow transplantation is currently used to treat a variety of hematopoietic tumors. Although graft-versus-host disease (GVHD) is a common consequence of this treatment, TIGIT inhibition could be used to enhance the effectiveness of transplanted tumor-specific T cells from the donor by reducing GVHD.

[0266] Ex vivo activation and expansion of antigen-specific T cells and adoptive transfer of these cells into recipients to stimulate antigen-specific T cells against cancer or viral infection in the presence of anti-TIGIT antibodies can increase the frequency and activity of adoptively transferred T cells.

[0267] There are also several experimental treatment options, including ex vivo activation and expansion of antigen-specific T cells and adoptive transfer of these cells into recipients to stimulate anti-tumor antigen-specific T cells (Greenberg & Riddell (1999) Science 285: 546-51). These methods can also be used to activate T cell responses to infectious agents such as CMV. In the presence of anti-TIGIT antibodies, ex vivo activation can increase the frequency and activity of adoptively transferred T cells.

[0268] In certain embodiments, the anti-tumor antagonists described herein can be administered to subjects with infectious diseases, especially chronic infections. In this case, similar to its application to cancer, antibody-mediated PD-1, PD-L1, TIGIT and / or LAG-3 inhibition can be used alone or in combination with vaccines as an adjuvant to enhance immune responsiveness to pathogens, toxins and self-antigens. Exemplary pathogens to which this treatment method can be applied include, but are not limited to, HIV, hepatitis (type A, B and C), influenza, herpes, Giardia lamblia, malaria, Leishmania, Staphylococcus aureus and Pseudomonas aeruginosa. PD-1, PD-L1, TIGIT and / or LAG-3 inhibition is particularly suitable for resisting infections by pathogens such as HIV that have been clearly identified during the infection process by presenting new or altered antigens. Administration of anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIGIT antibodies, bispecific anti-tumor antagonists or trispecific anti-tumor antagonists can allow these antigens to be recognized as foreign, thereby eliciting appropriate T cell responses.

[0269] Other pathogenic viruses that cause infections that can be treated by the methods described herein include HIV, hepatitis (types A, B, and C), herpes virus infections (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr Virus), and infections caused by adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, arboviral encephalitis virus, or a combination thereof.

[0270] Exemplary pathogenic bacteria or diseases caused by them that can be treated by the methods described herein include Chlamydia, Rickettsia, Mycobacteria, Staphylococci, Streptococci, Pneumonococci, Meningococci and Gonococci, Klebsiella, Proteus, teus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacilli, Cholera, Leptospirosis tetanus, botulism, anthrax, plague, and Lyme disease.

[0271] Exemplary pathogenic fungi that cause infections that can be treated by the methods described herein include Candida (e.g., albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (e.g., fumigatus, niger, etc.), Mucorales (e.g., mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0272] Exemplary pathogenic parasites that cause infections that can be treated by the methods described herein include Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia Zambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nippostrongylus brasiliensis.

[0273] In all of the above approaches, PD-1, PD-L1, TIGIT and / or LAG-3 inhibition can be combined with other forms of immunotherapy, such as cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy that uses two different binding specificities to enhance the presentation of tumor antigens.

[0274] Anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIGIT antibodies, anti-LAG-3 antibodies, bispecific anti-tumor antagonists and trispecific anti-tumor antagonists described herein can be co-administered with one or more of these antibodies with an antigen of interest (e.g., a vaccine) to enhance antigen-specific immune responses. Accordingly, provided herein is a method for enhancing an immune response to an antigen in a subject, the method comprising administering to the subject: (i) an antigen; (ii) an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-LAG-3 antibody, a bispecific anti-tumor antagonist, a trispecific anti-tumor antagonist, or a combination thereof, such that the immune response to the antigen in the subject is enhanced. The antigen can be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen from a pathogen. Non-limiting examples of such antigens include those discussed in the above sections, such as the tumor antigens (or tumor vaccines) discussed above, or antigens from the above-mentioned viruses, bacteria, or other pathogens.

[0275] In certain embodiments, peptides or fusion proteins comprising epitopes to which anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIGIT antibodies, LAG-3 antibodies, bispecific anti-tumor antagonists, or trispecific anti-tumor antagonists bind can be used as vaccines instead of or in addition to anti-tumor antagonists.

[0276] Suitable routes for administering the antibody compositions described herein (e.g., human monoclonal antibodies, multispecific antibodies, or antagonists and immunoconjugates) in vivo and in vitro are well known in the art and can be selected by one of ordinary skill. For example, the antibody compositions can be administered by injection (e.g., intravenously or subcutaneously). The appropriate dosage of the molecule used will depend on the age and weight of the subject and the concentration and / or dosage form of the antibody composition.

[0277] B. Pharmaceutical Compositions and Methods of Treatment

[0278] Another aspect of the present application relates to pharmaceutical compositions and methods for treating cell proliferative diseases such as cancer, chronic infection, or immunologically impaired disease states. In one embodiment, the pharmaceutical composition comprises one or more anti-tumor antagonists of the present application. In some embodiments, the anti-tumor antagonists include one or more checkpoint regulator antagonists, such as PD-1 inhibitors, PD-L1 inhibitors, anti-TIGIT inhibitors, LAG-3 inhibitors; one or more angiogenesis inhibitors, such as VEGF inhibitors, VEGFR2 inhibitors, angiopoietin-1 / 2 inhibitors, and Tie2R inhibitors; one or more anti-tumor inhibitors, such as TGF-β1 inhibitors and TGF-β1 RII inhibitors; or bispecific and trispecific anti-tumor antagonists thereof. The antagonist is formulated with a pharmaceutically acceptable carrier. The pharmaceutical composition of the present application may comprise one or more different antibodies, one or more multispecific antibodies, one or more immunoconjugates, or a combination thereof, as described herein.

[0279] As described above, the methods of using the pharmaceutical compositions described herein comprise administering to a subject in need thereof an effective amount of a pharmaceutical composition according to the present invention.

[0280] Any suitable route or mode of administration can be used to provide a therapeutically or prophylactically effective dose of an antibody or antagonist to a patient. Exemplary routes or modes of administration include parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous, intratumoral), oral, topical (nasal, transdermal, intradermal or intraocular), mucosal (e.g., nasal, sublingual, buccal, rectal, vaginal), inhalation, intralymphatic, intraspinal, intracranial, intraperitoneal, intratracheal, intravesical, intrathecal, intestinal, intrapulmonary, intralymphatic, intracavitary, intraorbital, intracapsular and intraurethral, ​​as well as local delivery via a catheter or stent.

[0281] The pharmaceutical composition comprising an antibody or antagonist according to the present disclosure can be formulated in any pharmaceutically acceptable carrier or excipient. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents that are physiologically compatible. The pharmaceutical composition may include a suitable solid phase or gel phase carrier or excipient. Exemplary carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers (e.g., polyethylene glycol). Exemplary pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc. and combinations thereof. In many cases, it is preferred to include an isotonic agent, such as a sugar, a polyol such as mannitol, sorbitol or sodium chloride in the composition. The pharmaceutically acceptable carrier may further include a small amount of auxiliary substances, such as a wetting agent or emulsifier, a preservative or a buffer, which can extend the shelf life or effectiveness of the therapeutic agent.

[0282] Anti-tumor antagonists can be incorporated into pharmaceutical compositions suitable for parenteral administration. Suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate, or potassium phosphate. Sodium chloride can be used at a concentration of 0-300 mM (optimally 150 mM for liquid dosage forms) to modify the toxicity of the solution. For lyophilized dosage forms, cryoprotectants can be included, primarily 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. For lyophilized dosage forms, bulking agents can be included, primarily 1-10% mannitol (optimally 2-4%). ​​Stabilizers can be used in both liquid and lyophilized dosage forms, primarily 1-50 mM (optimally 5-10 mM) L-methionine. Other suitable bulking agents include glycine, arginine, and polysorbate 80, which can be included as 0-0.05% (optimally 0.005-0.01%). Additional surfactants include, but are not limited to, polysorbate 20 and BRIJ surfactant.

[0283] The therapeutic anti-tumor antagonist formulation can be lyophilized and stored as a sterile powder, preferably under vacuum, and then reconstituted in bacteriostatic water (e.g., containing benzyl alcohol as a preservative) or in sterile water prior to injection. The pharmaceutical composition can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion.

[0284] The therapeutic agent in the pharmaceutical composition can be formulated in a "therapeutically effective amount" or a "prophylactically effective amount." A "therapeutically effective amount" refers to an amount effective at the dosage and time period necessary to achieve the desired therapeutic result. The therapeutically effective amount of the recombinant vector can vary depending on factors such as the disease to be treated, the severity and course of the disease, the route of administration, whether the antibody or drug is being administered for prophylactic or therapeutic purposes, the bioavailability of the particular drug, the ability of the anti-tumor antagonist to elicit the desired response in the individual, previous treatments, the patient's age, weight, and sex, the patient's clinical history and response to the antibody, the type of anti-tumor antagonist used, the discretion of the attending physician, etc. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the recombinant vector are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective at the dosage and time period necessary to achieve the desired prophylactic result.

[0285] Preferably, the polypeptide domains in the anti-tumor antagonists are derived from the same host to which they are to be administered, in order to reduce the inflammatory response to the administered therapeutic agent.

[0286] The anti-tumor antagonist is suitably administered to the patient at one time or over a series of treatments and may be administered to the patient at any time from diagnosis.The anti-tumor antagonist may be administered as sole therapy or in combination with other drugs or therapies useful in treating the condition.

[0287] As a general suggestion, a therapeutically effective or prophylactically effective amount of an anti-tumor antagonist is administered in an amount of about 1 ng / kg body weight / day to about 100 mg / kg body weight / day, whether administered in a single dose or multiple doses.In a specific embodiment, each anti-tumor antagonist is administered in a dose range of about 1 ng / kg body weight / day to about 10 mg / kg body weight / day, about 1 ng / kg body weight / day to about 1 mg / kg body weight / day, about 1 ng / kg body weight / day to about 100 μg / kg body weight / day, about 1 ng / kg body weight / day to about 10 μg / kg body weight / day, about 1 ng / kg body weight / day to about 1 μg / kg body weight / day, about 1 ng / kg body weight / day to about 100 ng / kg body weight / day, about 1 ng / kg body weight / day to about 10 ng / kg body weight / day, about 10 ng / kg body weight / day to about 100 mg / kg body weight / day, about 10 ng / kg body weight / day to about 10 mg / kg body weight / day, about 10 ng / kg body weight / day to about 1 mg / kg body weight / day, about 10 ng / kg body weight / day to about 100 μg / kg body weight / day, about 10 ng / kg body weight / day to about 10 μg / kg body weight / day, about 10 ng / kg body weight / day to about 1 μg / kg body weight / day, 10 ng / kg body weight / day to about 100 ng / kg body weight / day, about 100 ng / kg body weight / day to about 100 mg / kg body weight / day, about 100 ng / kg body weight / day to about 10 mg / kg body weight / day, about 100 n g / kg body weight / day to about 100 μg / kg body weight / day, about 100 ng / kg body weight / day to about 10 μg / kg body weight / day, about 100 ng / kg body weight / day to about 1 μg / kg body weight / day, about 1 μg / kg body weight / day to about 100 mg / kg body weight / day, about 1 μg / kg body weight / day to about 10 mg / kg body weight / day, about 1 μg / kg body weight / day to about 1 mg / kg body weight / day, about 1 μg / kg body weight / day to about 100 μg / kg body weight / day, about 1 μg / kg body weight / day to about 10 μg / kg body weight / day, about 10 μg / kg body weight / day to about 100 mg / kg body weight / day, about 10 μg / kg body weight / day g body weight / day to about 10 mg / kg body weight / day, about 10 μg / kg body weight / day to about 1 mg / kg body weight / day, about 10 μg / kg body weight / day to about 100 μg / kg body weight / day, about 100 μg / kg body weight / day to about 100 mg / kg body weight / day, about 100 μg / kg body weight / day to about 10 mg / kg body weight / day, about 100 μg / kg body weight / day to about 10 mg / kg body weight / day, about 100 μg / kg body weight / day to about 1 mg / kg body weight / day, about 1 mg / kg body weight / day to about 100 mg / kg body weight / day, about 1 mg / kg body weight / day to about 100 mg / kg body weight / day.

[0288] In other embodiments, the anti-tumor antagonist is administered at a dose of 500 μg to 20 g every three days, or at a dose of 25 mg / kg body weight every three days.

[0289] In other embodiments, each anti-tumor antagonist is administered in a range of about 10 ng to about 100 ng per single administration, about 10 ng to about 1 μg per single administration, about 10 ng to about 10 μg per single administration, about 10 ng to about 100 μg per single administration, about 10 ng to about 1 mg per single administration, about 10 ng to about 10 mg per single administration, about 10 ng to about 100 mg per single administration, about 10 ng to about 1000 mg per injection, about 10 ng to about 10,000 mg per single administration, about 100 ng to about 1 μg per single administration, about 100 n per single administration. g to about 10 μg, about 100 ng to about 100 μg per single dose, about 100 ng to about 1 mg per single dose, about 100 ng to about 10 mg per single dose, about 100 ng to about 100 mg per single dose, about 100 ng to about 1000 mg per single dose, about 100 ng to about 10,000 mg per injection, about 1 μg to about 10 μg per single dose, about 1 μg to about 100 μg per single dose, about 1 μg to about 1 mg per single dose, about 1 μg to about 10 mg per single dose, about 1 μg to about 100 mg per single dose, about 1 μg per injection About 1000 mg, about 1 μg to about 10,000 mg per single dose, about 10 μg to about 100 μg per single dose, about 10 μg to about 1 mg per single dose, about 10 μg to about 10 mg per single dose, about 10 μg to about 100 mg per single dose, about 10 μg to about 1000 mg per injection, about 10 μg to about 10,000 mg per single dose, about 100 μg to about 1 mg per single dose, about 100 μg to about 10 mg per single dose, about 100 μg to about 100 mg per single dose, about 100 μg to about 1000 mg per injection, About 100 μg to about 10,000 mg per single dose, about 1 mg to about 10 mg per single dose, about 1 mg to about 100 mg per single dose, about 1 mg to about 1000 mg per injection, about 1 mg to about 10,000 mg per single dose, about 10 mg to about 100 mg per single dose, about 10 mg to about 1000 mg per injection, about 10 mg to about 10,000 mg per single dose, about 100 mg to about 10,000 mg per injection, about 100 mg to about 10,000 mg per single dose, about 1000 mg to about 10,000 mg per single dose. The anti-tumor antagonist can be administered daily, every 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3 or 4 weeks.

[0290] In other specific embodiments, the amount of the anti-tumor antagonist can be administered at a dose of about 0.0006 mg / day, 0.001 mg / day, 0.003 mg / day, 0.006 mg / day, 0.01 mg / day, 0.03 mg / day, 0.06 mg / day, 0.1 mg / day, 0.3 mg / day, 0.6 mg / day, 1 mg / day, 3 mg / day, 6 mg / day, 10 mg / day, 30 mg / day, 60 mg / day, 100 mg / day, 300 mg / day, 600 mg / day, 1000 mg / day, 2000 mg / day, 5000 mg / day or 10,000 mg / day. As expected, the dosage will depend on the disease, size, age and condition of the patient.

[0291] In certain embodiments, the coding sequence of the anti-tumor antagonist is introduced into a suitable expression vector (e.g., a viral or non-viral vector) to express an effective amount of the anti-tumor antagonist in a patient suffering from a cell proliferative disease. In certain embodiments comprising administering, for example, one or more recombinant AAV (rAAV) viruses, the pharmaceutical composition may comprise rAAV in an amount of at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 or at least 10 14 Genome copies (GC) or recombinant virus particles / kg, or any range thereof. In certain embodiments, the pharmaceutical composition comprises an effective amount of a recombinant virus, such as rAAV, comprising at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 genome copies or recombinant viral particle genome copies, or any range thereof.

[0292] Doses can be tested in several art-recognized animal models appropriate for any particular cell proliferative disorder.

[0293] Delivery methods may also include the use of polycation-conjugated DNA with or without viral ligands, ligand-linked DNA, liposomes, eukaryotic cell delivery vectors, deposition of photopolymerizable hydrogel materials, use of handheld gene transfer particle guns, ionizing radiation, nucleic acid neutralization or fusion with cell membranes, particle-mediated gene transfer, and the like.

[0294] C. Combination therapy

[0295] In another aspect, the present application provides a combination therapy for enhancing antigen-specific T cell responses in a subject. In one embodiment, the method comprises contacting T cells with an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-LAG-3 antibody, an antibody fragment thereof, a bispecific anti-tumor antagonist, or a trispecific anti-tumor antagonist, in combination with a second antibody, antibody fragment, antagonist, or drug, thereby enhancing antigen-specific T cell responses or apoptosis pathways.

[0296] In a related aspect, a method of reducing or depleting regulatory T cells in a tumor of a subject in need thereof comprises administering an effective amount of an antibody or antibody fragment, in combination with a second antibody, antibody fragment, antagonist or drug, thereby reducing the number of regulatory T cells in the subject.

[0297] In some embodiments, the subject has a cell proliferative disorder or cancer described herein.

[0298] In other embodiments, the subject has a chronic viral infection, inflammatory disease, or autoimmune disease as described herein.

[0299] Providing two different signals to T cells is a widely accepted model of lymphocyte activation of resting T lymphocytes by antigen presenting cells (APCs). This model further provides a distinction between self and non-self and immune tolerance. The primary signal or antigen-specific signal is transduced by the T cell receptor (TCR), which then recognizes foreign antigenic peptides presented in the context of the major histocompatibility complex (MHC). The second or co-stimulatory signal is delivered to the T cell by co-stimulatory molecules expressed on the antigen presenting cell (APC). This induces T cells to promote clonal expansion, cytokine secretion and effector function. In the absence of co-stimulation, T cells may become refractory to antigen stimulation, resulting in a tolerant response to exogenous or endogenous antigens.

[0300] In the dual-signal model, T cells receive both positive co-stimulatory and negative co-inhibitory signals. This regulation of positive and negative signals is crucial for maximizing the host's protective immune response while maintaining immune tolerance and preventing autoimmunity. Negative signals appear to be necessary for inducing T cell tolerance, while positive signals promote T cell activation. Both co-stimulatory and co-inhibitory signals are provided to T cells exposed to antigens, and the interaction between co-stimulatory and co-inhibitory signals is crucial for controlling the magnitude of the immune response. In addition, the signals provided to T cells change as the infection or immune challenge clears, worsens, or persists, and these changes strongly influence the responding T cells and reshape the immune response.

[0301] The mechanism of co-stimulation has therapeutic implications, as manipulation of co-stimulatory signals has been shown to provide a means of enhancing or terminating cell-based immune responses. Recently, it has been found that T cell dysfunction or energy can occur simultaneously with the induction and sustained expression of immune checkpoint regulators such as PD-1 and its ligands PD-L1 and PD-L2. PD-L1 is overexpressed in many cancers and is generally associated with poor prognosis (Thompson RH et al., Cancer Res 2006, 66(7):3381). In addition, in contrast to T lymphocytes in normal tissues and peripheral blood T lymphocytes, most tumor-infiltrating T lymphocytes predominantly express PD-1, suggesting that upregulation of PD-1 on tumor-reactive T cells can lead to impaired anti-tumor immune responses (Blood 2009 114(8):1537). This may be due to the use of PD-L1 signaling resulting from the interaction of PD-L1-expressing tumor cells with PD-1-expressing T cells, leading to attenuated T cell activation and evasion of immune surveillance.

[0302] Inhibition of PD-L1 / PD-1 interactions provides a means to enhance T cell immunity, including CD8 + T cell-mediated killing of cancer cells and tumors. Similar enhancement of T cell immunity has been observed by inhibiting the binding of PD-L1 to its binding partner molecule B7-1. Therefore, therapeutic targeting of PD-1 and other immune checkpoint regulators is an area of ​​considerable interest.

[0303] Combining inhibition of PD-1, PD-L1, TIGIT, and / or LAG-3 signaling with other signaling pathways that are dysregulated in tumor cells may provide a means to enhance therapeutic efficacy. In recent years, numerous immune checkpoint modulators in the form of receptors and their ligands have been identified. An important family of membrane-bound ligands that bind to co-stimulatory or co-inhibitory receptors is the B7 family, which includes CTLA-4 and its ligands B7-1 and B7-2; PD-1 and its ligands PD-L1 (B7-H1) and PD-L2 (B7-DC); B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6. Other immune checkpoint antagonists include, but are not limited to, TIM-3 and its ligand galectin-9; LAG-3 and its ligands, including liver sinusoidal endothelial cell lectin (LSECtin) and galectin-3; CD122 and its CD122R ligand; CD70, B7H3, B and T lymphocyte attenuating agonist (BTLA) and VISTA (Le Mercier et al. (2015) Front. Immunol., (6), Article 418). In addition, many checkpoint regulator antagonists have been identified and tested in a variety of clinical and preclinical models and / or have received FDA approval (Kyi et al., FEBS Letters, 588:368-376 (2014). The concept of inhibitory receptor blockers (also known as immune checkpoint blockers) has been validated by, for example, the FDA-approved PD-1 inhibitors nivolumab and pembrolizumab and the anti-CTLA-4 antibody ipilimumab for metastatic melanoma.

[0304] Immune checkpoint antagonists regulate or interfere with the activity of immune checkpoint regulators, thereby blocking or inhibiting the signal transduction of checkpoint regulator receptors due to binding to checkpoint regulators or their ligands. By inhibiting this signal transduction, immunosuppression can be reversed, thereby reconstructing or enhancing T cell immunity against cancer cells. In contrast, immune checkpoint agonists (e.g., co-stimulatory molecules) stimulate the activity of immune checkpoint regulators, thereby stimulating the signal transduction of checkpoint regulator receptors due to binding to checkpoint regulators or their ligands. By stimulating this signal transduction, T cell immunity against cancer cells can be reconstructed or enhanced.

[0305] Therefore, in one embodiment, a method for stimulating an immune response in a subject comprises administering to the subject an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-LAG-3 antibody, an antibody fragment thereof (e.g., anti-TIGIT HCVR and / or LCVR), a bispecific anti-tumor antagonist or a trispecific anti-tumor antagonist described herein in combination with another immune checkpoint regulator described above, thereby stimulating the subject's immune response, such as inhibiting tumor growth or stimulating an antiviral response.

[0306] In one embodiment, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-LAG-3 antibody, an antibody fragment thereof, a bispecific anti-tumor antagonist or a trispecific anti-tumor antagonist according to the present application is administered in combination with another immune checkpoint regulator as a separate antibody or a multispecific antibody comprising a plurality of products having binding specificity. Typically, the anti-PD-1 antibody, anti-PD-L1 antibody, anti-TIGIT antibody, anti-LAG-3 antibody, bispecific anti-tumor antagonist or trispecific anti-tumor antagonist described herein can be combined with the following substances to stimulate an immune response: (i) an IgSF family protein that inhibits T cell activation, an antagonist of the B7 family or TNF family, or a cytokine antagonist that inhibits T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF or other immunosuppressive cytokines) and / or (ii) an agonist of a stimulating receptor of the IgSF family, B7 family or TNF family that stimulates T cell activation or a cytokine agonist for stimulating an immune response.

[0307] In certain embodiments, only subjects with cancers that exhibit high expression of ligands for immune checkpoint regulators are selected for treatment in combination with anti-PD-1, anti-PD-L1, anti-TIGIT and / or anti-LAG-3 antibodies, fragments thereof, any bispecific antagonists or trispecific antagonists of the present application. For example, in one embodiment, subjects with cancers that exhibit high expression of PVR (CD155) and / or adhesion protein-2 (CD112) and / or low expression of PD-L1 can be selected for monotherapy with anti-TIGIT antibodies, fragments thereof, or TIGIT antagonists of the present application, or in combination with PD-1 antagonists or other immune checkpoint regulators.

[0308] Anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIGIT antibodies can be administered separately from a second antibody, antibody fragment or antagonist, or a multispecific antibody or antagonist can be administered, including at least one binding specificity for TIGIT and a second binding specificity for another targeted product. In addition, the anti-PD-1 antibody, anti-PD-L1 antibody, anti-TIGIT, anti-LAG-3 antibody, bispecific antagonist or trispecific antagonist according to the present application can be co-administered with one or more additional agents, such as antibodies, antagonists or drugs, in an amount effective to stimulate the subject's immune response and / or apoptosis to further enhance, stimulate or upregulate the subject's immune response and / or apoptosis.

[0309] In some embodiments, anti-PD-1, anti-PD-L1, anti-TIGIT or anti-LAG-3 antibodies or fragments thereof are administered after treatment with a different anti-tumor antagonist. For example, in one embodiment, anti-PD-1, anti-PD-L1, anti-TIGIT or anti-LAG-3 antibodies may be administered only after treatment with a PD-1 / PD-L1 antagonist has failed, the treatment response is incomplete, or the tumor has recurred or recurred (or "PD-1 failure"). In some embodiments, cancers that exhibit such failures can be screened for expression of PVR and / or laminin-2, and only those with high levels of expression are treated with the anti-PD-1, anti-PD-L1, anti-TIGIT or anti-LAG-3 antibodies, fragments or antagonists of the present application.

[0310] Other anti-PD-1 antibodies include but are not limited to nivolumab (BMS-936558, MDX-1106, OPDIVO TM ), humanized immunoglobulin G4 (IgG4) mAb (Bristol-Myers Squibb); pembrolizumab (MK-3475, lambrolizumab, KEYTRUDA TM ) (Merck); pidilizumab (CT-0111) (Medivation); and AMP-224 (Merck). Anti-PD-1 antibodies are commercially available, for example, from ABCAM (AB137132), BIOLEGEND TM (EH12.2H7, RMP1-14) and AFFYMETRIX EBIOSCIENCE (J105, J1 16, MIH4).

[0311] Other anti-PD-L1 antibodies include atezolizumab (MPDL3280A, RG7446), a fully human IgG4 mAb (Genentech / Roche); BMS-936559 (MDX-1 105), a fully human IgG4 mAb (Bristol-Myers Squibb); MEDI4736, a humanized IgG antibody (Medimmune / AstraZeneca); and MSB0010718C, a fully human IgG4 monoclonal antibody (Merck, EMD Serono).

[0312] Exemplary anti-CTLA-4 antibodies for use in accordance with the present methods include ipilimumab, trevilizumab, and tremelimumab.

[0313] In certain embodiments, the anti-tumor antagonist is a dominant negative protein (dominant negative protein) of an immune checkpoint regulator. In some specific embodiments, the dominant negative protein includes an extracellular domain, which is derived from a group consisting of members selected from the following: PD-L1, PD-L2, PD-1, B7-1, B7-2, B7H3, CTLA-4, LAG-3, TIM-3, TIGIT, BTLA, VISTA, CD70 and combinations thereof. In certain specific embodiments, these extracellular domains are fused with immunoglobulin constant regions or Fc receptors in antibodies described herein. Such mutants can bind to endogenous receptors, thereby forming a complex with insufficient signal transduction. In certain embodiments, the extracellular domain is fused with a monomer in an immunoglobulin constant region or Fc fragment or an oligomeric protein complex.

[0314] In certain embodiments, the dominant negative PD-L1 antagonist comprises the extracellular domain of PD-L1, PD-L2 or PD-1. In another embodiment, a dominant negative PD-L1 antagonist is used that has a mutation that makes it no longer able to bind to PD-L1. An exemplary dominant negative protein is AMP-224 (co-developed by Glaxo Smith Kline and Amplimmune), which is a recombinant fusion protein that comprises the extracellular domain of PD-L2 and the Fc region of human IgG.

[0315] Exemplary immune checkpoint regulator agonists include, but are not limited to, members of the tumor necrosis factor (TNF) receptor superfamily, such as CD27, CD40, OX40, GITR, and 4-1BB (CD 137) and its ligands or members of the B7-CD28 superfamily, including CD28 and ICOS (CD278). Other checkpoint regulator agonists include CD2, CDS, ICAM-1, LFA-1 (CD11a / CD18), CD30, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, CD83 ligands. Immune checkpoint antagonists may include antibodies or soluble fusion protein agonists comprising one or more costimulatory domains. Agonist antibodies include, but are not limited to, anti-CD40 mAbs, such as CP-870,893, lucatumumab, and dacetuzumab. anti-CD137 mAbs, such as BMS-663513 urerulumab and PF-05082566; anti-OX40 monoclonal antibodies; anti-GITR mAbs, such as TRX518; anti-CD27 mAbs, such as CDX-1127; and anti-ICOS mAbs.

[0316] Exemplary GITR agonists include, for example, GITR fusion proteins and anti-GITR antibodies (eg, bivalent anti-GITR antibodies), such as those disclosed in U.S. Patent Nos. 6,111,090 and 8,586,023; European Patent No. 090505B1, U.S. Patent No. PCT Publication Nos. WO 2010 / 003118 and 2011 / 090754. Anti-GITR antibodies are described in, for example, U.S. Patent Nos. 7,025,962, 7,618,632, 7,812,135, 8,388,967, and 8,591,886; European Patent Nos. 1947183B1 and 1866339; PCT Publication Nos. WO 2011 / 028683, WO 2013 / 039954, WO 2005 / 007190, WO 2007 / 133822, WO 2005 / 055808, WO 99 / 40196, WO 2001 / 03720, WO 99 / 20758, WO 2006 / 083289, WO 2005 / 115451, WO 2011 / 051726. An exemplary anti-GITR antibody is TRX518.

[0317] Another family of membrane-bound ligands that bind to co-stimulatory or co-inhibitory receptors is the TNF family of molecules that bind to cognate TNF receptor family members, including CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 / 4-1BB, TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR / Fnl4, TWEAK, BAFFR, EDAR XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / D R3, EDR, EDA1, XEDAR, EDA2, TNFR1, Lymphotoxin α / TNFγ, TNFR2, TNFα, LTβR, Lymphotoxin α1(32, FAS, FASL, RELT, DR6, TROY, NGFR (see, e.g., Tansey, M G et al. (2009) Drug Discovery Today, 14(23-24): 1082-1088).

[0318] Immune checkpoint agonists or co-stimulatory molecules include cell surface molecules other than antigen receptors or their ligands required for an effective immune response, including but not limited to MHC class I molecules, MHC class II molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transducing lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (C D278), GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4 , CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB 7. NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM 1. CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a and ligands that specifically bind to CD83.

[0319] In one aspect, the anti-PD-1, anti-PD-L1, anti-TIGIT or anti-LAG-3 mAbs stimulate T cell responses in combination with one or more of the following: (i) antagonists of proteins that inhibit T cell activation (e.g., immune checkpoint inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, Galectin-9 CEACAM-1, BTLA, CD69, Galectin-1, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD-1H, LAIR1, TIM-1, CD96, and TIM-4, and (ii) agonists of proteins that stimulate T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, CD40, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3, and CD28H.

[0320] Exemplary agents that regulate one of the above proteins and can be combined with the anti-PD-1 antibody, anti-PD-L1 antibody, anti-TIGIT antibody and / or anti-LAG-3 antibody of the present application to treat cancer include: YERVOY TM / ipilimumab or tremelimumab (anti-CTLA-4), galiximab (anti-B7.1), OPDIVO TM / nivolumab / BMS-936558 (anti-PD-1), pidilizumab / CT-01111 (anti-PD-1), KEYTRUDA TM / paplizumab / MK-3475 (anti-PD-1), AMP224 (anti-B7-DC / PD-L2), BMS-936559 (anti-B7-H1), MPDL3280A (anti-B7-H1), MEDI-570 (anti-ICOS), AMG557 (anti-B7H2), MGA271 (anti-B7H3), IMP321 (anti-LAG-3), urelumab / BMS-6635l3 and PF-05082566 (anti-CD137 / 4-1BB), CDX-1127 (anti-CD27), MEDI-6383 and MEDI-6469 (anti-OX40), RG-7888 (anti-OX40L), Atacicept (anti-TACI), CP-870893 (anti-CD40), lucatumumab (anti-CD40), dacetuzumab (anti-CD40), and muromonab-CD3 (anti-CD3).

[0321] Other molecules that can be used in combination with the anti-tumor antagonists described herein for the treatment of cancer include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, antagonists anti-PD-1, anti-PD-L1 antibodies and / or anti-TIGIT can be combined with KIR antagonists (e.g., lirilumab), CSF-1R antagonists, such as RG7155.

[0322] Tumors evade host immune surveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating tumor-expressed immunosuppressive proteins. These include TGF-β, IL-10, and Fas ligand. Antibodies against each of these entities can be used in combination with the anti-tumor antagonists described herein to counteract the effects of immunosuppressive agents and promote host immune responses to tumors.

[0323] Other antibodies that activate the host immune response can be used in combination with anti-tumor antagonists described herein. These include molecules that activate DC function and antigen presentation on the surface of dendritic cells. Anti-CD40 antibodies can effectively replace T cell helper activity and can be used in combination with anti-tumor antagonists described herein. Activation antibodies against T cell costimulatory molecules (such as OX-40, CD137 / 4-1BB and ICOS) can also increase T cell activation levels.

[0324] In certain embodiments, the anti-tumor antagonists described herein can be co-administered with one or more other therapeutic agents, such as anticancer agents, radiotoxic agents, or immunosuppressants. Such co-administration can address problems caused by the development of resistance to the drug, changes in the antigenicity of tumor cells (which will render them unreactive with the antibody), and toxicity (by administering lower doses of one or more agents).

[0325] The anti-tumor antagonists described herein can be chemically linked to the reagent (as an immune complex) or can be administered separately from the reagent. In the latter case (separate administration), the antibody can be administered before, after, or simultaneously with the reagent, or can be co-administered with other known therapies, such as anti-cancer therapies, such as radiation. The anti-tumor antagonists described herein can be co-administered with one or more anti-cancer agents to provide two anti-cancer agents that act synergistically via different mechanisms, thereby producing a cytotoxic effect in human cancer cells.

[0326] The anti-tumor antagonists described herein can be combined with anti-cancer agents, such as alkylating agents; anthracycline antibiotics; antimetabolites; detoxification agents; interferons; polyclonal or monoclonal antibodies; EGFR inhibitors; HER2 inhibitors; histone deacetylase inhibitors; hormones; mitotic inhibitors; phosphatidylinositol-3-kinase (PI3K) inhibitors; Akt inhibitors; mammalian target of rapamycin (mTOR) inhibitors; proteasome inhibitors; poly (ADP-ribose) polymerase (PARP) inhibitors; Ras / MAPK pathway inhibitors; centrosome declustering agents; multikinase inhibitors; serine / threonine kinase inhibitors; tyrosine kinase inhibitors; VEGF / VEGFR inhibitors; taxanes or taxane derivatives, aromatase inhibitors, anthracyclines, drugs targeting microtubules, topoisomerase poisoning drugs, inhibitors of molecular targets or enzymes (e.g., kinases or protein methyltransferases), cytidine analogs, or a combination thereof.

[0327] Exemplary alkylating agents include, but are not limited to, cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan (Alkeran); carmustine (BiCNU); busulfan (Busulfex); lomustine (CeeNU); dacarbazine (DTIC-Dome); oxaplatin (Eloxatin); carmustine (Gliadel); ifosfamide (Ifex); methylvinylpyrrolidone (Mustargen); busulfan (Myleran); carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide (Temozolomide); thiotepa (Thiotepa); bendamustine (Treanda); or streptozocin (Zanosar).

[0328] Exemplary anthracycline antibiotics include, but are not limited to, doxorubicin (Adriamycin); liposomal doxorubicin (Doxil); mitoxantrone (Novantrone); bleomycin (Blenoxane); daunorubicin (Cerubidine); liposomal daunorubicin (DaunoXome); actinomycin (Cosmegen); epirubicin (Ellence); idarubicin (Idamycin); puromycin (Thioflavin); mitomycin (Mutamycin); pentostatin (Nipent); or valrubicin (Valstar).

[0329] Exemplary antimetabolites include, but are not limited to, fluorouracil (Adrucil); capecitabine (Hydrea); hydroxyurea (Hydrea); mercaptopurine (Purinol); pemetrexed (Alimta); fludarabine (Fludara); nelarabine (Arranon); cladribine (Cladribine Novaplus; clofarabine (Clolar); cytarabine (Cytosar-U); decitabine (Dacogen); liposomal cytarabine (DepoCyt); hydroxyurea (Droxia); pralatrexate (Folotyn); floxuridine (FUDR); gemcitabine (Gemzar); cladribine (Leustatin); fludarabine (Oforta); methotrexate (MTX; Rheumatrex); methotrexate (Trexall); thioguanine (Tabloid); TS-1 or cytarabine (Tarabine PFS).

[0330] Exemplary antidotes include, but are not limited to, amifostine (Ethyol) or mesna (Mesnex).

[0331] Exemplary interferons include, but are not limited to, interferon alpha-2b (Intron A) or interferon alpha-2a (Roferon-A).

[0332] Exemplary polyclonal or monoclonal antibodies include, but are not limited to, trastuzumab (Herceptin); ofatumumab (Arzerra); bevacizumab (Avastin); rituximab (Rituxan); cetuximab (Erbitux); panitumumab (Vectibix); tositumomab / iodine-131 tositumomab (Bexxar); alemtuzumab (Campath); ibritumomab (Zevalin; In-111; Y-90Zevalin); gemtuzumab tuzumab (Mylotarg); eculizumab (Soliris), ordenosumab.

[0333] Exemplary EGFR inhibitors include, but are not limited to, gefitinib (Iressa); lapatinib (Tykerb); cetuximab (Erbitux); erlotinib (Tarceva); panitumumab (Vectibix); PKI-166; canitinib (0-1033); matuzumab (Emd7200), or EKB-569.

[0334] Exemplary HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin); lapatinib (Tykerb); or AC-480.

[0335] Exemplary histone deacetylase inhibitors include, but are not limited to, vorinostat (Zolinza), valproic acid, romidepsin, entinostat, abexinostat, gilvestrastat, and mocetinostat.

[0336] Exemplary hormones include, but are not limited to, tamoxifen (Soltamox; Nolvadex); raloxifene (Evista); progesterone (Megace); leuprorelin (Lupron; Lupron Depot; Eligard; Viadur); fulvestrant (Faslodex); letrozole (Femara); triptorelin (Trelstar LA; Trelstar Depot); exemestane (Aromasin); goserelin (Zoladex); bicalutamide (Casodex); anastrozole (Arimidex); fluoxymesterone (Androxy; Halotestin); medroxyprogesterone (Provera; Depo-Provera); estramustine (Emcyt); flutamide (Olesin); toremifene (Fareston); degarelix (Firmagon); nilutamide (Nilandron); abarelix (Plenaxis); or testolactone (Teslac).

[0337] Exemplary mitotic inhibitors include, but are not limited to, paclitaxel (Taxol; Onxol; Abraxane); docetaxel (Taxol); vincristine (Oncovin; Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopophos; VePesid); teniposide (Vumon); ixabepilone (Ixempra); nocodazole; epothilone; vinorelbine (Navelbine); camptothecin (CPT); irinotecan (Camptosar); topotecan (Hycamtin); amcloline or lamellarin D (LAM-D).

[0338] Exemplary phosphatidylinositol 3-kinase (PI3K) inhibitors include wortmannin (an irreversible inhibitor of PI3K), demethoxyviridin (a derivative of wortmannin), LY294002 (a reversible inhibitor of PI3K);

[0339] BKM120 (buparlisib); idelalisib (PI3K delta inhibitor); duvelisib (IPI-145, PI3K delta and gamma inhibitor); alpelisib (BYL719) (an alpha-specific PI3K inhibitor); TGR 1202 (formerly known as RP5264, an oral PI3K delta inhibitor); and copanlisib (BAY 80-6946, a primary inhibitor of RI3K alpha and delta isoforms).

[0340] Exemplary Akt inhibitors include, but are not limited to, Miltefosine, AZD5363, GDC-0068, MK2206, Perifosine, RX-0201, PBI-05204, GSK2141795, and SR13668.

[0341] Exemplary MTOR inhibitors include, but are not limited to, everolimus (Afinitor) or temsirolimus (Torisel); rapamune, ridaforolimus; deforolimus (AP23573), AZD8055 (AstraZeneca), OSI-027 (OSI), INK-128, BEZ235, PI-103, Torinl, PP242, PP30, Ku-0063794, WAY-600, WYE-687, WYE-354, and CC-223.

[0342] Exemplary proteasome inhibitors include, but are not limited to, bortezomib (PS-341), izumab (MLN2238), MLN 9708, dilanzomib (CEP-18770), carfilzomib (PR-171), YU101, opozomib (ONX-0912), marizomib (NPI-0052), and disufiram.

[0343] Exemplary PARP inhibitors include, but are not limited to, olaparib, iniparib, velaparib, BMN-673, BSI-201, AG014699, ABT-888, GPI21016, MK4827, INO-1001, CEP-9722, PJ-34, Tiq-A, Phen, PF-01367338, and combinations thereof.

[0344] Exemplary Ras / MAPK pathway inhibitors include, but are not limited to, trametinib, selumetinib, cobimetinib, O-1040, PD0325901, AS703026, R04987655, R05068760, AZD6244, GSK1120212, TAK-733, U0126, MEK162, and GDC-0973.

[0345] Exemplary centrosome declustering agents include, but are not limited to, griseofulvin; Noscapine, Noscapine derivatives, such as brominated Noscapine (e.g., 9-bromonascapine), reduced bromonascapine (RBN), N-(3-bromobenzyl) Noscapine, amino Noscapine and its water-soluble derivatives; CW069; phenanthracene-derived poly (ADP-ribose) polymerase inhibitor PJ-34; N2-(3-pyridylmethyl)-5-nitro-2-furanamide, N2-(2-thienylmethyl)-5-nitro-2-furanamide and N2-benzyl-5-nitro-2-furanamide.

[0346] Exemplary multi-kinase inhibitors include, but are not limited to, regorafenib; sorafenib (Nexavar); sunitinib (Sutent); BIBW 2992; E7080; Zd6474; PKC-412; motesanib; or AP24534.

[0347] Exemplary serine / threonine kinase inhibitors include, but are not limited to, ruboxistaurin; eril / escutel hydrochloride; flavopiridol; sebcicbb (CYC202; Roscovitrine); SNS-032 (BMS-387032); Pkc4l2; bromostatin; KAI-9803; SF1126; VX-680; Azdl 152; Arry-142886 (AZD-6244); SCIO-469; GW681323; CC-401; CEP-1347 or PD 332991.

[0348] Exemplary tyrosine kinase inhibitors include, but are not limited to, erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec); sorafenib (Nexavar); sunitinib (Sutent); trastuzumab (Herceptin); bevacizumab (Avastin); rituximab (Rituxan); lapatinib (Tykerb); cetuximab (Erbitux); panitumumab (Vectibix); everobmus (Afmitor); alemtuzumab (Campath); gemtuzumab (Mylotarg); temsirobmus (Torisel); pazopanib (Votrient); dasatinib (Spryvac cel); nilotinib (Tasigna); vatalanib (Ptk787; ZK222584); CEP-701; SU5614; MLN518; XL999; VX-322; Azd0530; BMS-354825; SKI-606CP-690; AG-490; WHI-P154; WHI-P131; AC-220; or AMG888.

[0349] Exemplary VEGF / VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin); sorafenib (Nexavar); sunitinib (Sutent); ranibizumab pegaptib or vandetinib.

[0350] Exemplary microtubule-targeting drugs include, but are not limited to, paclitaxel, docetaxel, vincristine, vinblastine, nocodazole, epothilone, and navirbine.

[0351] Exemplary topoisomerase-toxic drugs include, but are not limited to, teniposide, etoposide, doxorubicin, camptothecin, daunorubicin, dactinomycin, mitoxantrone, aminophylline, epirubicin, and idarubicin.

[0352] Exemplary taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxel.

[0353] Exemplary general chemotherapeutic agents, anti-tumor agents, and anti-proliferative agents include, but are not limited to, octreotamine (Hexalen); isotretinoin (Accutane; Amnesteem; Claravis; Sotret); tretinoin (Vesanoid); azacitidine (Vidaza); bortezomib (Velcade); asparaginase (Elspar); levamisole (Ergamisol); mitotane (Lysodren); promazine (Matulane); pegaspargase (Oncaspar); denileukin (Denileukin); diftitox (Ontak); porfimer (Photofrin); aldesleukin (Proleukin); lenalidomide (Revlimid); bexarotene (Targretin); thalidomide (Thalomid); temsirolimus (Torisel); arsenic trioxide (Trisenox); verteporfin (Visudyne); mometasone (Leucenol); (1M tegafur-0.4M 5-chloro-2,4-dihydroxypyrimidine-1M potassium taurate) or lovastatin.

[0354] In certain embodiments, an anti-tumor antagonist described herein is administered at a subtherapeutic dose, another anti-immune checkpoint modulator antibody or antagonist is administered at a subtherapeutic dose, an angiogenesis antagonist is administered at a subtherapeutic dose, or a combination thereof is each administered at a subtherapeutic dose.

[0355] In certain embodiments, PD-1, PD-L1, TIGIT and / or LAG-3 inhibition is combined with standard cancer treatments (e.g., surgery, radiation, and chemotherapy). PD-1, PD-L1, TIGIT and / or LAG-3 inhibition can be effectively combined with chemotherapy regimens. In these cases, it is possible to reduce the dose of the chemotherapeutic agent used. An example of such a combination is the combination of anti-PD-1, anti-PD-L1, anti-TIGIT or anti-LAG-3 antibodies with decarbazine for the treatment of melanoma. Another example of such a combination is the combination of anti-PD-1, anti-PD-L1, anti-TIGIT or anti-LAG-3 antibodies with interleukin-2 (IL-2) for the treatment of melanoma. It is believed that the combined use of PD-1, PD-L1, TIGIT and / or LAG-3 inhibition and chemotherapy can enhance apoptosis and increase tumor antigen presentation for cytotoxic immunity. Other synergistic combination therapies include inhibition of TIGIT, PD-1, PD-1L1, and / or LAG-3 through cell death when used in combination with radiation, surgery, or hormone deprivation. Each of these regimens generates a source of tumor antigens in the host.

[0356] In certain embodiments, the checkpoint regulator antagonists described herein can be used as multispecific antagonists or in combination with bispecific antibodies that target effector cells expressing Fcα or Fcγ receptors to tumor cells (see, e.g., U.S. Patent Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. For example, anti-Fc receptor / anti-tumor antigen (e.g., Her-2 / neu) bispecific antibodies have been used to target macrophages to cancer cells or tumors. This targeting can more effectively activate tumor-specific responses. The T cell arm of these responses will be enhanced by inhibiting TIGIT, PD-1, PD-L1 and / or LAG-3. Alternatively, the antigen can be delivered directly to DC by using bispecific antibodies that bind to tumor antigens and dendritic cell-specific cell surface markers.

[0357] VI. Methods for producing anti-tumor antagonists

[0358] A. Nucleic Acids and Host Cells for Expression of Antagonists

[0359] In one aspect, the present application provides nucleic acids encoding the anti-tumor antagonists of the present application (including heavy and light chains), and expression vectors comprising such nucleic acids. In one embodiment, the nucleic acid encodes the HCVR and / or LCVR fragments of an antibody or fragment according to the embodiments described herein or any other antibody and antibody fragment described herein.

[0360] Conventional methods can be used (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of monoclonal antibodies) to isolate DNA encoding the antigen binding site in the monoclonal antibody from hybridoma cells and sequence them. Alternatively, the amino acid sequence of the immunoglobulin of interest can be determined by direct protein sequencing, and a suitable encoding nucleotide sequence can be designed based on a universal codon table. In other cases, the nucleotide and amino acid sequences of the antigen binding site or other immunoglobulin sequences, including constant regions, hinge regions, etc., can be obtained from public sources known in the art.

[0361] Expression vectors encoding specific monospecific, bispecific, or trispecific anti-tumor antagonists can be used to synthesize the anti-tumor antagonists of the present disclosure in vitro in cultured cells, or they can be directly administered to patients to express the anti-tumor antagonists in vivo or ex vivo. As used herein, "expression vector" refers to a viral or non-viral vector that contains a polynucleotide encoding one or more polypeptide chains corresponding to the monospecific, bispecific, or trispecific anti-tumor antagonists of the present invention in a form suitable for expression from the polynucleotide in a host cell for antibody preparation or direct administration as a therapeutic agent.

[0362] A nucleic acid sequence is "operably linked" to another nucleic acid sequence when the former has a functional relationship with the latter. For example, if the DNA for a sequence or signal peptide is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA for the sequence or signal peptide may be operably linked to the DNA for the polypeptide. A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are continuous, and in the case of a signal peptide, continuous and in reading phase. However, enhancers do not have to be continuous. Connection is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers may be used according to conventional practice.

[0363] Nucleic acid sequences used to express anti-tumor antagonists typically include an amino-terminal signal peptide sequence, which is removed from the mature protein. Because the signal peptide sequence can affect expression levels, the polynucleotide can encode any of a variety of different N-terminal signal peptide sequences. Those skilled in the art will appreciate that the design of the expression vector can depend on factors such as the choice of host cell to be transformed and the desired protein expression level.

[0364] The above-mentioned "regulatory sequence" refers to a DNA sequence necessary for expressing an operably linked coding sequence in one or more host organisms. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells or regulatory sequences that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Expression vectors typically contain sequences for transcription termination and may additionally contain one or more elements that actively influence mRNA stability.

[0365] Expression vectors contain one or more transcriptional regulatory elements, including promoters and / or enhancers, that direct the expression of anti-tumor antagonists. Promoters comprise DNA sequences that initiate transcription from a relatively fixed position relative to the transcription start site. Promoters contain the core elements required for the basic interaction between RNA polymerase and transcription factors and can be used in conjunction with other upstream elements and response elements.

[0366] As used herein, the term "promoter" should be used in its broadest context and includes transcriptional regulatory elements (TREs) from genomic genes or chimeric TREs derived therefrom, including TATA boxes or promoter elements for precise transcription initiation, with or without additional TREs (i.e., upstream activating sequences, transcription factor binding sites, enhancers and silencers), which regulate the activation or repression of genes to which they are operably linked in response to developmental and / or external stimuli, as well as transactivating regulatory proteins or nucleic acids. A promoter may comprise a genomic fragment, or may comprise a chimera of one or more TREs bound together.

[0367] Preferred promoters are those that can guide high-level expression in target cells. Promoters can include constitutive promoters (e.g., HCMV, SV40, elongation factor-1α (EF-1α)) or those promoters that show preferential expression in specific cell types of interest. An enhancer generally refers to a DNA sequence that functions outside the transcription start site and can be located at the 5' or 3' end of the transcription unit. In addition, enhancers can be within introns and within coding sequences. Their length is generally between 10 and 300 bp and acts in cis. Enhancers play a role in increasing and / or regulating transcription from nearby promoters. Preferred enhancers are enhancers that guide high-level expression in antibody-producing cells. Cell or tissue-specific transcriptional regulatory elements (TREs) can be introduced into expression vectors to limit expression to the desired cell types. PolIII promoters (H1 or U6) are particularly suitable for expressing shRNA from which siRNA is expressed. Expression vectors can be designed to promote expression of anti-tumor antagonists in one or more cell types.

[0368] In certain embodiments, one or more expression vectors can be engineered to express an anti-tumor antagonist and one or more siRNAs targeting the Tie2 pathway, the VEGF pathway, or an immune checkpoint regulator.

[0369] siRNA is a double-stranded RNA that can be engineered to induce sequence-specific post-transcriptional gene silencing of mRNA. Synthetically produced siRNAs structurally mimic the type of siRNAs that are typically processed in cells by the Dicer enzyme. When expressed from an expression vector, the expression vector is engineered to transcribe short, double-stranded hairpin-like RNA (shRNA), which is then processed into a targeted siRNA within the cell. Synthetic siRNAs and shRNAs can be designed using well-known algorithms and synthesized using conventional DNA / RNA synthesizers.

[0370] In order to co-express the various chains of the anti-tumor antagonist, appropriate splice donor and splice acceptor sequences can be introduced to express two products. Alternatively, an internal ribosome binding sequence (IRES) or a 2A peptide sequence can be used to express multiple products from a single promoter. IRES provides a structure to which ribosomes can bind, which does not need to be at the 5' end of the mRNA. Therefore, it can guide the ribosome to initiate translation at the second start codon within the mRNA, thereby allowing more than one polypeptide to be produced from a single mRNA. The 2A peptide contains a short sequence that mediates co-translational self-cleavage of the peptides upstream and downstream of the 2A site, thereby allowing equimolar amounts of two different proteins to be produced from a single transcript. CHYSEL is a non-limiting example of a 2A peptide that causes the translating eukaryotic ribosome to release the growing polypeptide chain being synthesized without separating from the mRNA. The ribosome continues to translate, thereby producing a second polypeptide.

[0371] The expression vector may comprise a viral vector or a non-viral vector. Viral vectors may be derived from adeno-associated virus (AAV), adenovirus, herpes virus, vaccinia virus, poliovirus, poxvirus, retrovirus (including lentiviruses such as HIV-1 and HIV-2), Sindbis virus and other RNA viruses, alphavirus, astrovirus, coronavirus, orthomyxovirus, papillomavirus, paramyxovirus, parvovirus, mesothelioma virus, togavirus, etc. Non-viral vectors are simply "naked" expression vectors that are not packaged with virus-derived components (such as capsid and / or envelope).

[0372] In some cases, these vectors can be engineered to target certain diseases or cell populations by exploiting the inherent targeting properties of the viral vectors or by engineering these vectors into viral vectors. Specific cells can be "targeted" for both delivery of polynucleotides and expression. Thus, in this context, the term "targeting" can be based on the use of capsids, envelope proteins, endogenous or heterologous binding agents in the form of antibodies for delivery to specific cells, the use of tissue-specific regulatory elements to restrict expression to specific cell subsets, or both.

[0373] In some embodiments, expression of the antibody chain is under the control of a regulatory element, such as a tissue-specific or ubiquitous promoter. In some embodiments, a ubiquitous promoter, such as the CMV promoter, the CMV-chicken β-actin hybrid (CAG) promoter, a tissue-specific or tumor-specific promoter controls the expression of a particular antibody heavy or light chain, or a single-chain derivative thereof.

[0374] Non-viral expression vectors can be used for non-viral gene transfer, either by direct injection of naked DNA or by encapsulating polynucleotides encoding anti-tumor antagonists in liposomes, microparticles, microcapsules, virus-like particles, or red blood cells. Such compositions can be further linked to targeting domains by chemical coupling to facilitate targeted delivery of nucleic acids and / or entry into desired target cells. In addition, plasmid vectors can be incubated with synthetic gene transfer molecules, such as polymeric DNA-binding cations such as polylysine, protamine, and albumin, and chemically linked to cell-targeting ligands, such as asialoorosomucoids, insulin, galactose, lactose, or transferrin.

[0375] Alternatively, naked DNA can be used. The uptake efficiency of naked DNA can be improved by compacting or using biodegradable latex beads. This delivery can be further improved by treating the beads to increase their hydrophobicity and thereby promote the disruption of endosomes and the release of DNA into the cytoplasm.

[0376] B. Methods for Producing Monospecific or Multispecific Antibodies

[0377] In one aspect, the present application provides host cells transformed with anti-PD-1, anti-PD-L1, anti-TIGIT and / or anti-LAG-3 HCVR and / or LCVR, encoding nucleic acids or expression vectors or nucleic acids / expression vectors encoding the bispecific and / or trispecific anti-tumor antagonists of the present application. The host cell can be any bacterial or eukaryotic cell capable of expressing the encoding nucleic acids or expression vectors of anti-PD-1, anti-PD-L1, anti-TIGIT and / or anti-LAG-3 HCVR and / or LCVR or any other co-administered antibodies or antagonists described herein.

[0378] In another aspect, a method for producing an anti-tumor antagonist comprises culturing a host cell transformed with one or more anti-PD-1, anti-PD-L1, anti-TIGIT and / or anti-LAG-3 HCVR and / or LCVR encoding nucleic acids or expression vectors under conditions that allow for the production of antibodies or fragments, and purifying the antibodies from the cells.

[0379] In another aspect, the present application provides a method for producing an antibody, comprising culturing cells to transiently or stably express one or more constructs encoding one or more polypeptide chains in an antibody; and purifying the antibody from the cultured cells. Any cell capable of producing a functional antibody can be used. In some preferred embodiments, the cells expressing the antibody are cells of eukaryotic or mammalian origin, preferably human cells. Cells from various tissue cell types can be used to express the antibody. In some other embodiments, the cells are yeast cells, insect cells, or bacterial cells. Preferably, the antibody-producing cells are stably transformed with a vector expressing the antibody.

[0380] One or more expression vectors encoding the heavy or light chains of the antibody can be introduced into the cells by any conventional method, such as naked DNA technology, cationic lipid-mediated transfection, polymer-mediated transfection, peptide-mediated transfection, viral-mediated infection, physical or chemical reagents or treatments, electroporation, etc. In addition, cells can be transfected with one or more expression vectors to express the antibody together with a selective marker that promotes secretion of the antibody-expressing stable transformed clone. The antibodies produced by such cells can be collected and / or purified according to techniques known in the art, such as by centrifugation, chromatography, etc.

[0381] Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, the neomycin analog G418, hydromycin and puromycin. When such a selectable marker is successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two different classes of selection schemes that are widely used. The first class is based on the metabolism of the cell and the use of mutant cell lines that lack the ability to grow independently of a supplemented medium. Two examples are CHO DHFR cells and mouse LTK - Cells. These cells lack the ability to grow without supplemented nutrients such as thymidine or hypoxanthine. Because these cells lack certain genes necessary for the complete nucleotide synthesis pathway, they will not survive unless the missing nucleotides are provided in a supplemented medium. Another method of supplementing the medium is to introduce the complete DHFR or TK gene into cells lacking the corresponding gene, thereby changing their growth requirements. Individual cells that have not been transformed with the DHFR or TK gene will not survive in non-supplemented medium.

[0382] The second category is dominant selection, which refers to the selection scheme used in any cell type and does not require the use of mutant cell lines. These schemes usually use drugs to prevent the growth of host cells. Those cells with new genes will express proteins that transmit drug resistance and survive the selection. Examples of such dominant selection use drugs neomycin, mycophenolic acid or hygromycin. These three examples use bacterial genes under the control of eukaryotes to transmit resistance to appropriate drugs G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include neomycin analogs G418 and puromycin.

[0383] Exemplary antibody-expressing cells include human Jurkat, human embryonic kidney (HEK) 293, Chinese hamster ovary (CHO) cells, mouse WEHI fibrosarcoma cells, and single-cell protozoan species, such as Leishmania tarentolae. Additionally, primary cells immortalized by c-myc or other immortalizing agents can be used to generate stably transformed antibody-producing cell lines.

[0384] In one embodiment, the cell line comprises a stably transformed Leishmania cell line, such as Leishmania tarentolae. Leishmania is known to provide a robust, fast-growing, single-cell host for high-level expression of eukaryotic proteins that display mammalian-type glycosylation patterns. A Leishmania eukaryotic expression kit is commercially available (Jena Bioscience GmbH, Jena, Germany).

[0385] In some embodiments, the cell line expresses at least 1 mg, at least 2 mg, at least 5 mg, at least 10 mg, at least 20 mg, at least 50 mg, at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, or at least 500 mg of antibody per liter of culture.

[0386] In any suitable culture medium (e.g., RPMI, DMEM, and AIM ) After culturing and maintaining in the medium, the antibodies of the present application can be isolated from the cells expressing the antibodies. The antibodies can be purified using conventional protein purification methods (e.g., affinity purification, chromatography, etc.), including purification using Protein-A or Protein-G immunoaffinity purification. In some embodiments, the antibodies are engineered to be secreted into the culture supernatant for isolation therefrom.

[0387] The present application is further illustrated by the following examples, which should not be construed as limiting.The contents of all references, patents and published patent applications cited throughout this application, as well as the figures and tables, are incorporated herein by reference.

[0388] C. Homopolymers and heterodimers

[0389] One of the challenges in efficiently generating bispecific and trispecific antibody formulations involves mispairing of heavy and light chains when co-expressing chains with different binding specificities. Table 1 lists several amino acid substitutions that can be used to overcome mispairing between heavy chains with different binding specificities, thereby "enhancing" or preferentially promoting proper binding between the desired heavy chains. Any method that prevents or reduces mispairing between heavy chains can be used to prepare bispecific anti-tumor antagonists and bispecific anti-tumor antagonists according to the present invention.

[0390] The "knob-in-hole" (KiH) approach relies on modifying the interface between the two CH3 domains, where most interactions occur. Typically, bulky residues are introduced into the CH3 domain of an antibody heavy chain, acting like a key. A "hole" is formed in the other heavy chain that accommodates the bulky residues, similar to a lock. The resulting heterodimeric Fc portion can be further stabilized by artificial disulfide bonds.

[0391] An alternative approach is based on charged residues with ionic interactions or steric complementarity. This involves modifying the charge polarity in the CH3 interface so that co-expression of electrostatically matched Fc domains supports favorable attractive interactions and heterodimer formation while retaining the hydrophobic core, while unfavorable repulsive charge interactions inhibit homodimerization. See Table 1. The amino acid numbering in Table 1 follows the Kabat numbering scheme and can be applied to the heavy chain amino acid sequences of the antibodies described herein.

[0392] In another approach, a leucine zipper (LZ) domain can be included in the protein scaffold. Leucine zippers are common three-dimensional structural motifs in proteins and are typically part of the DNA binding domain in various transcription factors. A single LZ typically contains 4-5 leucine residues at intervals of approximately 7 residues, forming an amphipathic alpha helix with a hydrophobic region extending along one side. In a specific embodiment, the heterodimeric protein scaffold comprises an LZ from the c-jun transcription factor and an LZ from the c-fos transcription factor. Although it is known that c-jun can form jun-jun homodimers and c-fos cannot form homodimers, the formation of jun-fos heterodimers is more favorable than that of jun-jun homodimers.

[0393] The leucine zipper domain can replace the CH2-CH3 sequence in the protein scaffold, or it can be placed at the carboxyl termini of the two heavy chains in the bispecific or trispecific anti-tumor antagonist. In the latter case, a furin protease cleavage site can be introduced between the carboxyl terminus of CH3 and the amino terminus of the leucine zipper. When the heavy and light chains of the bispecific or trispecific anti-tumor antagonist are co-expressed in an appropriate mammalian cell expression system, furin-mediated cleavage of the leucine zipper after the heterodimerization step can be promoted (see Wranik et al., J. Biol. Chem., 287(5):43331-43339, 2012).

[0394] Table 1

[0395]

[0396] The amino acid numbering in Table 1 follows the Kabat numbering scheme and can be applied to the heavy chain amino acid sequences of the antibodies described herein. The mutations described in Table 1 can be applied to the sequences (published or otherwise) of any immunoglobulin IgG1 heavy chain, as well as other immunoglobulin classes and subclasses (or isotypes) therein.

[0397] When the heavy and light chains of a monospecific, bispecific, or trispecific antibody are co-expressed, it is also possible that a light chain with one binding specificity may mispair with a heavy chain with another binding specificity. Therefore, in certain embodiments, portions of the heavy chain, light chain, or both can be modified relative to the "wild-type" antibody chain from which they are derived to prevent or reduce mispairing of the two heavy chain constant regions with each other and mispairing of the light chain constant region with its heavy chain counterpart.

[0398] The light chain mispairing problem can be solved in several ways. In some embodiments, spatial complementary mutations and / or disulfide bonds can be introduced into two VL / VH interfaces. In other embodiments, mutations can be introduced based on ionic or electrostatic interactions. In some embodiments, light chain mispairing can be prevented or reduced by using a first arm having an S183E mutation in the CH1 domain of the heavy chain and an S176K mutation in the CL domain of the light chain. The second arm can include an S183K mutation in the CH1 domain of the heavy chain and an S176E mutation in the CL domain of the light chain. In other embodiments, a "CrossMab" approach is adopted, in which one arm in a bispecific or trispecific anti-tumor antagonist (e.g., Fab) remains unchanged, but in another arm comprising other binding specificities, one or more domains in the light chain are exchanged (swap) with one or more domains in the heavy chain at the heavy chain: light chain interface.

[0399] Methods, immunoglobulin domain sequences, including specific mutations as described above for preventing mispairing of heavy and light chains are further described in U.S. Patent Application Publication Nos. 2014 / 0243505 and 2013 / 0022601.

[0400] D. Conjugate

[0401] In certain embodiments, the anti-tumor antagonist of the present application is chemically coupled to one or more peptides and / or small molecule drugs. The peptides or small molecule drugs can be the same or different. The peptides or small molecule drugs can, for example, be connected to reduced SH groups and / or carbohydrate side chains. Methods for preparing covalent or non-covalent conjugates of peptides or small molecule drugs with antibodies are known in the art, and any such known methods can be used.

[0402] In some embodiments, peptides or small molecule drugs are attached to the hinge region of the reduced antibody component via disulfide bond formation. Alternatively, heterobifunctional cross-linking agents such as N-succinyl 3-(2-pyridyldithio) propionate (SPDP) can be used to connect such reagents. General techniques for such coupling are well known in the art. In some embodiments, peptides or small molecule drugs are coupled through carbohydrate moieties in the antibody Fc region. Carbohydrate groups can be used to increase the load of the same reagent bound to the thiol group, or carbohydrate groups can be used to combine different therapeutic agents or diagnostic agents. Methods for coupling peptide inhibitors or small molecule drugs to antibodies via antibody carbohydrate moieties are well known to those skilled in the art. For example, in one embodiment, the method includes reacting an antibody component having an oxidized carbohydrate moiety with a carrier polymer having at least one free amine function. The reaction produces an initial Schiff base (imine) bond, which can be stabilized by reduction to a secondary amine to form a final conjugate. Exemplary methods for coupling small molecule drugs and peptides to antibodies are described in U.S. Patent Publication No. 2014 / 0356385.

[0403] Preferably, the anti-tumor antagonists of the present disclosure retain certain desirable features and pharmacokinetic properties of antibodies, including desirable in vitro and in vivo stability (e.g., individual half-life and half-life stability), efficient delivery to desired target cells, increased affinity for binding partner molecules, desirable antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, and reduced renal clearance or excretion. Therefore, consideration of size and the need for specific constant region effector functions can be considered in the design of anti-tumor antagonists.

[0404] The size range of anti-PD-1, anti-PD-L1 and anti-TIGIT, inhibitors, including monospecific, bispecific and trispecific anti-tumor antagonists thereof can be 50kD to 300kD, 50kD to 250kD, 60kD to 250kD, 80kDa to 250kD, 100kD to 250kD, 125kD to 250kD, 150kD to 250kD, 60kD to 225kD, 75kD to 225kD, 100kD to 225kD, 125kD to 250kD, 150kD to 250kD, kD to 225 kD, 60 kD to 200 kD, 75 kD to 200 kD, 100 kD to 125 kD to 200 kD, 150 kD to 200 kD, 60 kD to 150 kD, 75 kD to 150 kD, 100 kD to 150 kD, 60 kD to 125 kD, 75 kD to 125 kD, 75 kD to 100 kD, or any combination of all the numbers listed above within the range as defined above, and any range encompassed by any combination of all the numbers listed above within the range.

[0405] example

[0406] Example 1: Production of monoclonal antibodies

[0407] Monoclonal antibodies (mAbs) of the present invention are generated and screened using techniques well known in the art, see, for example, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York. Antigen-specific hybridoma mAbs are cloned, sequenced, and engineered using techniques well known in the art, see, for example, Lo. BKC Methods in Molecular Biology TM .Volume 248 2004.Antibody Engineering.

[0408] Example 2: Trispecific antagonists with a PD-1 targeting domain and multiple angiogenesis targeting moieties

[0409] Figure 1 The VH and VL sequences of anti-PD-1, anti-PD-L1, anti-LAG-3, and anti-TIGIT mAbs are shown. Figure 2 Other functional domains are shown, including the VH and VL of ranibizumab, bevacizumab, and VH mutants, the VEGF-binding domain of aflibercept, the short and long peptides of trombanib, and the extracellular domain of TGF R-II (TGF R-II ECD).

[0410] The aflibercept fusion protein domain contains the vascular endothelial growth factor (VEGF) binding portion from the extracellular domains of human VEGF receptors 1 and 2 and blocks the binding of VEGF-A, VEGF-B, and PLGF to their receptors VEGFR-1 and VEGFR-2. Trabanib peptide targets and binds to Ang1 and Ang2, thereby preventing Ang1 and / or Ang2 from interacting with their cognate Tie2 receptors. Anti-tumor antagonists can be constructed using IgG1, IgGlagly, or IgG4 backbones.

[0411] A variety of trispecific anti-tumor antagonists were constructed, each comprising (1) one or more variable region domains of anti-PD-1 or other checkpoint antibodies; aflibercept; and trombopag or a peptide sequence thereof. Figures 3A-3H Eight different trispecific anti-tumor antagonists TS-ZPT-1.1, -1.2, -1.3 and -2 to -6 (1) anti-PD-1 variable regions (VH1, VL1) ( Figures 3A-3H ); (2) Aphiprole fusion protein domain: (i) at the amino terminus of one or both IgG arms ( Figures 3A-3C , 3F); (ii) at the carboxyl terminus of each antagonist ( Figure 3D , 3E, 3H); or (iii) between the carboxyl terminus of the CH3 domain and trobactam (or other biological peptide) ( Figure 3G ), (3) trombopag peptide (or other biological peptide): (i) fused to the carboxyl terminus of each IgG4 CH3 region; (ii) inserted into each of the two CH3 regions ( Figure 3E ); (iii) fused to the carboxyl terminus of each CH1 region ( Figure 3F ); (iv) fused to the carboxyl terminus of each aphidicolin fusion protein domain ( Figure 3G ); or (v) fused to the carboxyl terminus of each CL region ( Figure 3H ).

[0412] Figures 4A-4C Five different trispecific anti-tumor antagonists TS-ZPT-7, TS-ZPT-8, TS-ZPT-9 are shown: (1) an anti-PD-1 or other checkpoint antibody Fab domain (VH1-CH1, VL1-CL1) fused to the carboxyl terminus of the CH3 domain; (2) an aflibercept fusion protein domain fused to the amino terminus of the CH2 domain; (3) trombopag peptide (or other biological peptide): (i) inserted into each of the two CH3 domains ( Figure 4A ); (ii) fused to the amino terminus of the CH2 domain ( Figure 4B ); (iii) fused to the carboxyl terminus of the CH1 domain ( Figure 4C).

[0413] Figures 5A-5G Seven different trispecific anti-tumor antagonists, TS-LPT-1, TS-LPT-2, TS-LPT-3, TS-LPT-4, TS-LPT-5, TS-M3, and TS-M4, are shown; (1) the variable region domains (VH1, VL1) of anti-PD-1 or other checkpoint antibody (i) are fused to the amino termini of CH1 and CL1, respectively ( Figure 5F ); (ii) fused to the amino termini of VH1 and VL1, respectively ( Figure 5G ); (2) Lucentis, bevacizumab or other anti-VEGF antibody variable region domains (VH1, VL1); (i) fused to the amino termini of VH1 and VL1, respectively, and to the amino termini of CH1 and CL1, respectively ( Figure 5F ); (ii) fused to the amino termini of CH1 and CL1, respectively ( Figure 5G ); (iii) fused to the carboxyl terminus of the CH3 domain and the amino terminus of the CH1 and CL1 domains, respectively (3) Trabanib peptide (or other biological peptide): (i) inserted into each of the two CH3 domains ( Figure 5A ); (ii) fused to the carboxyl terminus of the CL1 domain ( Figure 5B ); (iii) fused to one or both carboxyl termini of the CH1 domain ( Figures 5C-5E ); (iv) fused to the carboxyl terminus of the CH3 domain ( Figures 5F-5G ).

[0414] The amino acid sequences set forth in SEQ ID NOs: 200 and 201 (LC) show exemplary sequences of TS-ZPT-3S. The amino acid sequences set forth in SEQ ID NOs: 201 and 202 show exemplary sequences of TS-ZPT-3L. The amino acid sequences set forth in SEQ ID NOs: 187 and 188 show exemplary sequences of TS-LPT-1. The amino acid sequences set forth in SEQ ID NOs: 201 and 203 show exemplary sequences of TS-ZPT-2. The amino acid sequences set forth in SEQ ID NOs: 201 and 204 show exemplary sequences of TS-ZPT-5. The amino acid sequences set forth in SEQ ID NOs: 234 and 235 show exemplary sequences of TS-ZPT-6.

[0415] Example 3: Characterization of a trispecific anti-tumor antagonist prepared using the anti-PD-1 antibody 2P16

[0416] Figures 3A-3HMultiple versions of the trispecific anti-tumor antagonist described in [ 15 ] were prepared using the 2P16 anti-PD-1 antibody, the long peptide of trabanib, and the VEGF-binding domain of afipril. TS-ZPT-1.1 (SEQ ID NOs: 236-237), TS-ZPT-1.2 (SEQ ID NOs: 238-239), TS-ZPT-1.3 (SEQ ID NOs: 240-241), TS-ZPT-2 (SEQ ID NOs: 242-243), TS-ZPT-3 (SEQ ID NOs: 201-202), TS-ZPT-5 (SEQ ID NOs: 244 and 298), and TS-ZPT-6 were constructed and compared with TS-LPT-1 (SEQ ID NOs: 234-235), a trispecific anti-tumor antagonist using the Fab portion of the 2P16 antibody, trabanib, and ranibizumab.

[0417] Figure 6A and 6B Shown is a non-reducing SDS-PAGE analysis of trispecific anti-tumor antagonists expressed by transiently transfected HEK293 cells. Samples were evaluated before and after purification of the molecules. The results show that all expressed molecules could be purified, and that TS-ZPT-2 (2P16), TS-ZPT-3 (2P16), and TS-ZPT-5 (2P16) expressed better than TS-ZPT-6 (2P16) and TS-LPT-1 (2P16).

[0418] Blocking assays were performed to calculate the IC50 of selected trispecific molecules. Briefly, 2- or 3-fold serial dilutions of anti-human PD-1 mAb were prepared. CHO-K1 cells transfected with human PD-1 were washed with FACS buffer (0.5% BSA 2 mM EDTA in PBS) and incubated at 10 6 The cells were resuspended at a concentration of 7 μg / ml. FITC-labeled human PD-L1-Fc protein was added to human PD-1 transfected CHO-K1 cells at a final concentration of 7 μg / ml and mixed thoroughly. Without incubation, 2,000 CHO-K1 cells (containing PD-L1 protein) in 20 μl of FACS buffer were immediately added to a 96-well round-bottom plate, and 20 μl of 2- or 3-fold serial dilutions of the trispecific molecule were immediately added to the cells and incubated at 4 ° C for 30 minutes. The cells were then washed and resuspended in 30 ml of 7AAD solution. Then, 35 ml of 10% neutral formalin buffer solution was added, incubated for 15 minutes, and then analyzed using the iQue intelligent system.

[0419] Figure 7A-B shows the IC50s of exemplary trispecific anti-tumor antagonists TS-ZPT-1.1 (2P16), TS-ZPT-1.2 (2P16), TS-ZPT-1.3 (2P16), TS-ZPT-2 (2P16), TS-ZPT-3 (2P16), TS-ZPT-5 (2P16), TS-ZPT-6 (2P16), as well as anti-PD-1 mAb and a positive control or benchmark ("BM") antibody (nivolumab). The IC50s are comparable to the benchmark antibody in blocking this interaction.

[0420] The ability of the trispecific anti-tumor antagonists TS-ZPT-2 (2P16), TS-ZPT-3 (2P16), TS-ZPT-5 (2P16), and TS-ZPT-6 (2P16) to block the interaction between VEGF and its receptor VEGFR2 was determined. Briefly, a 96-well assay plate was coated with 0.5 μg / ml recombinant human VEGF 165 (R&D) in carbonate-bicarbonate buffer (pH 9.6) at 4°C overnight and then blocked with 1% BSA / PBS for 1 hour at room temperature. Serially diluted antibodies were then added to the plate and incubated at room temperature for 30 minutes. Recombinant human VEGFR-2 was added and incubated at room temperature for 1 hour. The plate was washed with wash buffer (0.1% Tween-20 in PBS) and then incubated with anti-VEGFR-2 antibody for 1 hour at room temperature. Subsequently, the plate was washed with wash buffer, and goat anti-mouse IgG-HRP was added to the plate and incubated at room temperature for 1 hour. After adding TMB to the plate, the amount of VEGFR-2 bound was determined by measuring absorbance at 650 nm. The IC50 value was determined as the midpoint of the activity curve.

[0421] Figure 8A and 8B Shown are the IC50s calculated from the VEGF-VEGFR2 blocking assay using the trispecific antagonists TS-ZPT-1.2 (2P16), TS-ZPT-2 (2P16), TS-ZPT-5 (2P16), TS-ZPT-6 (2P16), and TS-LPT-1 (2P16) compared to positive controls corresponding to aflibercept and bevacizumab. These analyses demonstrate that the trispecific antagonists exhibit comparable or lower (i.e., better) IC50 values ​​compared to the corresponding controls.

[0422] The ability of the trispecific antagonists TS-ZPT-1.2 (2P16), TS-ZPT-2 (2P16), TS-ZPT-5 (2P16), TS-ZPT-6 (2P16), and TS-LPT-1 to block the interaction between Ang-2 and its receptor Tie2 was determined. Briefly, 96-well assay plates were coated with 1 μg / ml recombinant human Ang2 (R&D) in PBS for 1 hour at 37°C and then blocked with 1% BSA / PBS at room temperature for 1 hour. A serially diluted mixture of antibodies and recombinant human Tie2 was then added to the plates and incubated at room temperature for 2 hours. The plates were washed with wash buffer (0.1% Tween-20 in PBS) and then incubated with anti-Tie2 antibodies for 1 hour at room temperature. After washing with wash buffer, goat anti-mouse IgG-HRP was added to the plates and incubated at room temperature for 1 hour. Tie2 binding was detected by measuring absorbance at 650 nm after adding TMB to the plates. IC50 values ​​were determined as the midpoint of the activity curve.

[0423] Figure 9 Shown are IC50 values ​​calculated from a Tie2-Ang2 blocking assay testing the trispecific antagonists TS-ZPT-1.2 (2P16), TS-ZPT-2 (2P16), TS-ZPT-5 (2P16), TS-ZPT-6 (2P16), and TS-LPT-1 (2P16) compared to a positive control corresponding to the antagonist bevacizumab-trabanib. The results of this analysis indicate that the trispecific antagonists exhibited comparable or lower (i.e., better) IC50 values ​​than the corresponding controls.

[0424] Example 4: Characterization of TS-ZPT-2, TS-ZPT-3L, and TS-ZPT-5 molecules constructed with the anti-PD-1 antibody 2P17

[0425] TS-ZPT-2, TS-ZPT-3L, and TS-ZPT-5 trispecific anti-tumor antagonists (SEQ ID NOs: 203 and 201, 233 and 201, 204 and 210, respectively) were prepared using the anti-PD-1 antibody 2P17 and characterized for homogeneity and stability by size exclusion chromatography after protein A chromatography. SEC chromatography was performed using a Tosoh TSKgel UP-G3000SWXL column. The mobile phase system consisted of 100 mM sodium phosphate, 400 mM sodium chloride, and 6% isopropanol, pH 6.8 (0.2 ml / min). Detection was performed at 214 nm for relative area integration.

[0426] Figure 10The results of size exclusion chromatography analysis of TS-ZPT-2 (2P17), TS-ZPT-3L (2P17), and TS-ZPT-5 (2P17) produced by HEK293 transiently transfected cells, as well as the parental 2P17 antibody, are shown. The percentages of high molecular weight (HWM) and low molecular weight (LMW) species relative to dimer molecules (Dimers) show that TS-ZPT-3L (2P17) has the lowest percentage of LMW species and the highest percentage of dimer molecules among the three trispecific antagonists.

[0427] Figure 11 The relative amounts of HWM and LMW species compared to dimers are shown for three trispecific antagonists, TS-ZPT-2 (2P17), TS-ZPT-3L (2P17) (purified from two pools), and TS-ZPT-5 (2P17) (produced from a stable CHO cell pool). The results show that among the three trispecific antagonists, TS-ZPT-3L (2P17) has the lowest percentage of LMW species and the highest percentage of dimers.

[0428] Thus, TS-ZPT-3L(2P17) exhibited the lowest amount of cleavage in both transient and stable cell lines, suggesting that insertion of the trombopag peptide into the CH3 region protects the aflibercept fusion protein and reduces cleavage.

[0429] Figure 12A and 12B It was shown that the dimer, HMW and LMW species of TS-ZPT-3L(2P17) exhibited good stability at 4°C for at least 112 days.

[0430] Example 5: Expression and purification of TS-ZPT-3S and TS-ZPT-3L trispecific anti-tumor antagonists.

[0431] TS-ZPT-3 variants were prepared using anti-PD-1 antibodies 2P16 and 2P17, and full-length trombanib peptide was used for TS-ZPT-3L or a single copy of trombanib peptide was used for TS-ZPT-3S. These four molecules were combined with trispecific molecules TS-M3 and TS-M4 (e.g., using the VH and VL of bevacizumab) using the VH and VL of bevacizumab. Figure 5F ), and compared with 2P16 and 2P17 of the anti-PD-1 antibody (nivolumab), whose variable region sequences are shown in Figure 1 having sequences SEQ ID NOs: 247-254).

[0432] Figure 13A The heavy and light chain amino acid sequences of an exemplary trispecific anti-tumor antagonist with trobanib peptide, TS-ZPT-3L(2P17), are shown. Figure 13BExemplary molecules derived from these sequences are shown.

[0433] Figure 14A Shown are the heavy and light chain amino acid sequences of an exemplary trispecific anti-tumor antagonist with the trombanib short peptide, TS-ZPT-3S(2P17). Figure 14B Exemplary molecules derived from these sequences are shown.

[0434] Figure 15 Coomassie-stained non-reducing SDS-PAGE analysis of trispecific anti-tumor antagonists TS-ZPT-3S (2P17), TS-ZPT-3L (2P17), TS-M3 (2P17), TS-M4 (2P17), TS-M3 (2P16), and TS-M3 (nivolumab) produced by transiently transfected HEK293 cells is shown. The results show that the trispecific anti-tumor antagonists TS-ZPT-3S (2P17) and TS-ZPT-3L (2P17) containing the afipronil fusion protein domain showed higher expression levels than the trispecific anti-tumor antagonists TS-M3 (nivolumab), TS-M4 (nivolumab), TS-M3 (2P17), and TS-M3 (2P16) containing the anti-VEGF variable region domain.

[0435] Figure 16 The results of size exclusion chromatography analysis of TS-ZPT-3L (2P17), TS-ZPL-3S (2P17), TS-ZPT-3L (2P16), and TS-ZPT-3S (2P16) after purification by protein chromatography are shown. The percentages of high molecular weight (HWM) and low molecular weight (LMW) species compared to the dimer molecule (Dimer) indicate that molecules containing the 2P17 antibody have higher levels of preferred dimer species.

[0436] Figure 17 Shown are size exclusion chromatography analyses of TS-ZPT-3S(2P17) and TS-ZPT-3L(2P17) stored at 4°C for 48 days, indicating that both molecules are stable upon storage.

[0437] Example 6: Functional Characterization of the Trispecific Antagonist TS-ZPT-3

[0438] The ability of four variants of the trispecific anti-tumor antagonists TS-ZPT-3S (2P16), TS-ZPT-3L (2P16), TS-ZPT-3S (2P17), and TS-ZPT-3L (2P17) to block the interaction between PD-1 and its ligand PD-L1 was evaluated using the PD-1-PDL1 cell-based blocking assay of Example 5. The results were used to calculate the corresponding IC50 for 2P17 and the benchmark antibody (nivolumab). Figures 18A-18B The IC50 was shown to be comparable to that of a benchmark antibody in blocking this interaction.

[0439] The VEGF-VEGFR-2 bioassay was used to evaluate the ability of TS-ZPT-3S (2P16), TS-ZPT-3L (2P16), TS-ZPT-3S (2P17), and TS-ZPT-3L (2P17) to block the interaction between VEGF and its receptor VEGFR-2 and to calculate the corresponding IC50 values ​​along with a benchmark antibody (bevacizumab). Briefly, the VEGFR2 / NFAT Reporter-HEK293 recombinant cell line (BPS Bioscience catalog number: 79387) was seeded into a white, clear-bottomed 96-well microplate at a density of 30k cells per well. After 24 hours, serial dilutions of the indicated molecules and human VEGF 165 were added to the wells. After a 4-hour incubation, 100 μl of ONE-Step TM luciferase reagent, shake the plate at room temperature for about 15 minutes, and read the luminescence signal of the plate. Figures 19A-19B The IC50 was shown to be comparable to that of a benchmark antibody in blocking this interaction.

[0440] The ability of TS-ZPT-3S (2P16), TS-ZPT-3L (2P16), TS-ZPT-3S (2P17), and TS-ZPT-3L (2P17) to block the interaction between Tie2 and its ligand Ang-2 was assessed using the Ang2-Tie2 blocking assay of Example 5 and the corresponding IC50s were calculated along with the benchmark antibody (trabanib). Figures 20A-20B The IC50s of TS-ZPT-3L(2P16) and TS-ZPT-3L(2P17) with two Ang-2 blocking peptides were comparable to the benchmark, while TS-ZPT-3S(2P16) and TS-ZPT-3S(2P17) with only one Ang-2 blocking peptide had higher IC50s than the benchmark.

[0441] The binding affinity of TS-ZPT-3S(2P16), TS-ZPT-3L(2P16), TS-ZPT-3S(2P17) and TS-ZPT-3L(2P17) to PD-1 protein was evaluated using the biolayer interferometry assay described in Example 9. Figures 21A-21F The binding and resulting binding affinity constants are shown. The results show that the affinities of TS-ZPT-3S(2P16), TS-ZPT-3L(2P16), TS-ZPT-3S(2P17), and TS-ZPT-3L(2P17) were higher than the benchmark sample (nivolumab).

[0442] The binding affinity of TS-ZPT-3S(2P16), TS-ZPT-3L(2P16), TS-ZPT-3S(2P17), and TS-ZPT-3L(2P17) to VEGF165 was assessed using a similar biolayer interferometry assay. Figures 22A-22F The binding and resulting binding affinity constants are shown. The results show that TS-ZPT-3S(2P16), TS-ZPT-3L(2P16), TS-ZPT-3S(2P17), and TS-ZPT-3L(2P17) have higher affinities than the benchmark (bevacizumab).

[0443] The binding affinity of TS-ZPT-3S(2P16), TS-ZPT-3L(2P16), TS-ZPT-3S(2P17), and TS-ZPT-3L(2P17) to Ang-2 was assessed using a similar biolayer interferometry assay. Figures 23A-23F The binding and resulting binding affinity constants are shown. The results show that TS-ZPT-3S(2P16), TS-ZPT-3L(2P16), TS-ZPT-3S(2P17) and TS-ZPT-3L(2P17) exhibited similar affinity to the benchmark (trabanib).

[0444] Figure 24 The sequential binding of TS-ZPT-3S (2P16), TS-ZPT-3L (2P16), TS-ZPT-3S (2P17), and TS-ZPT-3L (2P17 to each of its three binding partners) was characterized using biolayer interferometry using the Octet RED96 system (ForteBio), as described in Example 11 above. Briefly, 20 nM of the TS-ZPT-3 trispecific antibody was loaded onto an anti-human IgG capture biosensor. To monitor the sequential binding of the three antigens to the four TS-ZPT-3 molecules, the biosensor was placed in a well containing saturating concentrations (>10 Kd) of each antigen (240 nM His-tagged human PD-1, 120 nM human VEGF165, 40 nM His-tagged human angiopoietin 2) for 5 minutes, followed by 1 minute of dissociation. Three different sequential binding combinations were observed. These results indicate that TS-ZPT-3S(2P16), TS-ZPT-3L(2P16), TS-ZPT-3S(2P17), and TS-ZPT-3L(2P17) are able to bind to VEGF, Ang-2, and PD-1 simultaneously.

[0445] To evaluate the in vivo pharmacokinetic properties of TS-ZPT-3L (2P17), a pharmacokinetic curve was generated. Briefly, 10 mg / kg of antagonist was injected intravenously into the tail vein of four 6-10 week old female CD1 mice (n=2 mice per molecule). Serum was harvested 3 minutes, 3 hours, 1 day, 3 days, 7 days and 10 days after injection. To detect antibodies in the serum, 96-well ELISA plates were embedded with 5 μg / ml goat anti-human IgG F(ab')2 fragments and then blocked with 5% milk in PBS. Serial dilutions of mouse serum in 5% milk and serial dilutions of purified protein molecules as standards were added to the plates. After incubation with peroxidase-conjugated mouse anti-human IgG and further washing, TS-ZP17T-3 was detected after incubation with TMB-ELISA substrate. The results of this analysis showed that the half-life (T1 / 2) of TS-ZP17T-3 was approximately 2 days ( Figure 25 ).

[0446] Thus, the insertion of trombanib or its variants allows for the generation of stable and potent trispecific therapeutic molecules.

[0447] Figures 26A-26B Two additional trispecific molecules utilizing trombanib peptide inserted into the CH3 domain and two checkpoint antibodies are shown. These molecules contain Figure 1 The following sequences are shown: (1) VH and VL from a first checkpoint antibody, e.g., anti-PD-1 or anti-PD-L1, (2) trombopagib inserted into CH3, (3) VH and VL from a second checkpoint inhibitor, e.g., anti-TIGIT or anti-LAG3, with a 3-6xG4S linker.

[0448] Figure 27 Another trispecific molecule, TS-A1BT-1 (SEQ ID NOs: 229-230), is shown, which has (1) VH and VL domains encoding bevacizumab or another anti-VEGF antibody, (2) trombanib peptide inserted into the CH3 domain, and (3) fused to the carboxyl terminus of the CH3 domain of the TGFBR-II ECD. This molecule was produced by transiently transfected HEK 293 cells. Figure 28 In the present study, non-reducing SDS-PAGE analysis of unpurified supernatant from HEK293 cells showed that the molecule was well expressed. Figure 29 Size exclusion chromatography analysis of TS-A1BT-1 purified by Protein A chromatography is shown.

[0449] Example 7: Design of a trispecific anti-tumor antagonist containing the TGF-βRII extracellular domain (TGF-βRII ECD)

[0450] Figures 30A-30FSix trispecific anti-tumor antagonists are shown as follows: TS-ZPB-1, TS-ZPB-2, TS-ZPB-3, TS-ZPB-4, TS-ZPB-5 and TS-ZPB-6. These antagonists contain: (1) anti-PD-1 or another checkpoint antibody variable region domain (VH1, VL1) ( Figures 30A-30F ); (2) abribercept fusion protein domain: (i) fused to the amino terminus of each heavy chain VH1 region ( Figure 30A , 30D, 30F) or (ii) fused to the carboxyl terminus of each heavy chain CH3 region ( Figure 30B , 30C, 30E) (each carboxyl terminus); and (3) TGF-βRII extracellular domain (ECD): (i) fused to the carboxyl terminus of each heavy chain CH3 region ( Figure 30A ), (ii) fused to the amino terminus of each heavy chain VH1 domain ( Figure 30B ), (iii) inserted into the Fc loop of each heavy chain CH3 region ( Figure 30C , 30F), (iv) fused to the amino terminus of each heavy chain upstream of the afipronil fusion protein domain ( Figure 30D ), (v) fused to the carboxyl terminus of each heavy chain downstream of the aphidicolin fusion protein domain ( Figure 30E ).

[0451] The amino acid sequences shown in SEQ ID NOs: 187 and 188 show exemplary sequences of TS-ZPLB-1. The amino acid sequences shown in SEQ ID NOs: 270 and 271 show exemplary sequences of TS-ZPB-1. The amino acid sequences shown in SEQ ID NOs: 272 and 273 show exemplary sequences of TS-ZPB-2. The amino acid sequences shown in SEQ ID NOs: 188 and 189 show exemplary sequences of TS-ZPLB-3. The amino acid sequences shown in SEQ ID NOs: 190 and 191 show exemplary sequences of TS-ZPB-3. The amino acid sequences shown in SEQ ID NOs: 192 and 193 show exemplary sequences of TS-ZPB-5.

[0452] Example 8: Transient expression of TS-ZPB and TS-ZPLB molecules

[0453] TS-ZPB-1 (2P17), TS-ZPB-2 (2P17), TS-ZPB-3 (2P17), TS-ZPB-5 (2P17), and TS-ZPLB molecules were transiently expressed in human embryonic kidney HEK293 cells, which were cultured for 7 days. Titers were quantified using a POROS A20 μm column (2.1 x 30 mm, 0.1 mL, Applied Biosystems). A standard curve was generated using purified recombinant TIGIT mAb at a wavelength of 280 nm. Neat 30 μL supernatant samples were injected into the HPLC. Mobile phase A was PBS, pH 7.3; mobile phase B was PBS, pH 2.2. A multiple step gradient was performed as follows: starting with 100% A for 0.2 min, then 100% B for 0.22-1.22 min, then back to 100% A for 1.23-2.30 min, then 100% B for 2.32-3.32 min, and finally 100% A for 3.4-4.00 min. The flow rate used was 1 mL / min, and each run was 4 min total.

[0454] like Figure 31A As shown, SDS-PAGE analysis of TS-ZPB-1 (2P17), TS-ZPB-2 (2P17), TS-ZPB-3 (2P17), TS-ZPB-5 (2P17) and TS-ZPLB produced by transiently transfected HEK293 cells showed that all molecules were well expressed ( Figure 31B ).

[0455] Figure 32A Exemplary size exclusion chromatography (SEC) curves of TS-ZPB-1 (2P17), TS-ZPB-2 (2P17), TS-ZPB-3 (2P17), TS-ZPB-5 (2P17), and TS-ZPLB are shown (using a Tosoh TSKgel UP-G3000SWXL column with a detection wavelength of 214 nm). The results show that TS-ZPLB-1 and TS-ZPB-5 (2P17) retain the most dimers compared to other TS-ZPB molecules.

[0456] Example 9: Functional Characterization of TS-ZPB Trispecific Antagonists Relative to Controls

[0457] Figure 33AFigure 2 shows the results of a cell-based assay that measured the ability of TS-ZPB-1 (2P17), TS-ZPB-2 (2P17), TS-ZPB-3 (2P17), and TS-ZPB-5 (2P17) to block the interaction between PD-1 and PD-L1. In this experiment, 3-fold serial dilutions of TS-ZPB molecules were prepared (as indicated; highest Ab concentration: 300 nM; three replicates for each mAb), and human PD-1-transfected CHOK1 cells were washed with FACS buffer (0.5% BSA, 2 mM EDTA in PBS) and incubated at 10 °C for 24 h. 6 cells / ml; FITC-labeled human PD-L1-Fc protein was added to the CHOK1 cells transfected with human PD-1 at a final concentration of 7 μg / ml and mixed thoroughly; without incubation, 20x10 3 Transfected CHOK1 cells (with PD-L1 protein) were added to 20 μl of FACS buffer in a 96-well round-bottom plate. 20 μl of a three-fold serial dilution of TS-ZPB molecules was then immediately added to the cells, which were then incubated at 4°C for 30 minutes. The cells were then washed and resuspended in 30 μl of 7AAD, to which 35 μl of 10% neutral buffered formalin solution was added. The cells were incubated for 15 minutes and then analyzed using the iQue intelligent system. Figure 33B IC50 values ​​(nM) obtained from this assay are shown, indicating that all molecules were able to inhibit the interaction of PD-1 and PD-L1.

[0458] Figure 34AResults are shown for a cell-based assay using the TGF / SMAD signaling pathway SBE reporter-HEK293 cell line (BPS Bioscience, Inc., San Diego, CA, catalog number: 60653) that measured the ability of TS-ZPB-1 (2P17), TS-ZPB-2 (2P17), TS-ZPB-3 (2P17), TS-ZPB-5 (2P17), and TS-ZPLB, along with a benchmark, TGF-1 RII-Fc, to block TGFβ1 signaling. Briefly, SBE reporter-HEK293 cells were seeded at a density of approximately 35,000 cells per well in 100 μl of growth medium in a white, clear-bottomed 96-well microplate. Twenty-four hours after seeding, cells were treated with five-fold serial dilutions of the TS-ZPB molecules in 90 μl of assay medium. Subsequently, 10 μl of diluted human TGFβ1 was added to the stimulation wells (maximum TGFβ1 concentration = 10 ng / ml). 10 μl of assay medium was added to the unstimulated control wells (for determining basal activity), while 100 μl of assay medium was added to the cell-free control wells (for determining background luminescence). The plate was then incubated overnight (approximately 18 hours) at 37°C in a CO2 incubator. After overnight incubation, 100 μl of ONE-Step TM luciferase reagent and incubate at room temperature for approximately 15 to 30 minutes before reading the luminescent signal) using the ONE-Step TM Luciferase assays were performed using the Luciferase Assay System (BPS Bioscience, Inc.). Figure 34B IC50 values ​​(nm) obtained from this assay are shown and indicate that all molecules were able to block the biological activity of TGFβ1.

[0459] Figure 35AShown are the results of an ELISA assay that measures the ability of TS-ZPB-1 (2R17), TS-ZPB-2 (2P17), TS-ZPB-3 (2P17), TS-ZPB-5 (2P17), and TS-Z-ZPLB to block the interaction between VEGF and VEGFR-2 compared to the benchmark antibody bevacizumab. Briefly, 0.5 ug / ml of recombinant human VEGF-165 (R&D #293-VE-050 / CF) in carbonate-bicarbonate buffer was coated into the wells of a 96-well assay plate and incubated overnight at 4°C. The wells were then washed three times with 0.05% Tween 20 in PBS (wash buffer), blocked with 1% BSA in PBS for 1 hour at room temperature, and washed three times with wash buffer. 50 ml of a 5-fold serial dilution of the antibody was then added to the wells in the assay plate and incubated for 30 minutes at room temperature. Then, 50 μl of 0.6 μg / ml recombinant human VEGFR-2-hu IgG1-His tag (R&D #357-KD-050) was added to the antibody in the plate wells and incubated at room temperature for 1 hour, followed by three washes with wash buffer. 100 μl of anti-His tag HRP (1:10,000) was then added, and the assay plate was incubated at room temperature for 40 minutes, followed by three washes with wash buffer. After adding 3,3',5,5'-tetramethylbenzidine to each well of the assay plate, VEGFR-2 binding (His-Tag) was detected by measuring absorbance at 650 nm. Figure 35B IC50 values ​​(nM) obtained from this assay are shown and indicate that all molecules were able to inhibit the binding of VEGF to VEGFR-2.

[0460] Example 10: Functional characterization of TS-ZPT-5 variants with α-glycosylated VEGFR (Afropreg)

[0461] Two TS-ZPB-5 variants were generated to improve the pharmacokinetics of TS-ZPB-5. The heavy chain of TS-ZPB-5A (SEQ ID NO: 205) was mutated at the codon for the carboxyl-terminal lysine in the CH3 domain to reduce the potential proteolytic activity of proteases present in the blood. The heavy chain of TS-ZPB-5B (SEQ ID NO: 206) also has a mutation that eliminates the glycosylation site in aflibercept to reduce clearance by ASGRI and ASGRII.

[0462] Figures 36A-36C Three trispecific anti-tumor antagonists TS-ZPB-5, TS-ZPB-5A and TS-ZPB-5B are shown.

[0463] Figure 37Transient HEK293 cell expression levels of TS-TPB-5, TS-ZPB-5A, and TS-ZPB-5B were shown to be similar to those of the parental 2P17 antibody.

[0464] The ability of TS-TPB-5, TS-ZPB-5A, and TS-ZPB-5B to block the interaction between VEGF and VEGFR-2 was assessed using the VEGF-VEGFR-2 blocking assay described in Example 10. Figure 38 The results showed that the binding and biological activity of VEGFR components were retained after the glycosylation site of afipril was mutated from Asn to Glu. The TGFB signaling assay described in Example 10 was used to evaluate the ability of TS-TPB-5, TS-ZPB-5A, and TS-ZPB-5B to block TGFβ signaling ( Figure 39 The results showed that mutation of the glycosylation site of afipril from Asn to Glu retained TGFβR-II binding and biological activity.

[0465] The in vivo pharmacokinetic properties of the TS-ZPT-5B variant with α-glycosylated VEGFR were determined as described in Example 7 compared to the glycosylated variant TS-ZPT-5A.

[0466] Figure 39 showed that the TS-ZPB-5B variant, in which the afipronil glycosylation site was mutated from Asn to Glu, improved its pharmacokinetics approximately fivefold.

[0467] Example 11: Generation and Characterization of Bispecific Inhibitors of PD-1 / PD-L1 and VEGF

[0468] Figures 41A-41C Shown are bispecific molecules TS-ZPL-1 (or TS-ZP-1), TS-ZPL-2 (or TS-ZP-2), and TS-ZPL-3 (or TS-ZP-3) comprising (1) PD-1, PD-L1, or other checkpoint antibody variable region domains (VH and VL) (2) afipronil (i) fused to the amino terminus of the VH domain ( Figure 40 A and 40B); (ii) fused to the carboxyl terminus of the CH3 domain ( Figure 40 C) Exemplary sequences of these molecules are SEQ ID NOs: 218-225.

[0469] Figure 42AThe results of a cell-based binding assay measuring the ability of Bi-ZPL-1 molecules to block the interaction between PD-L1 and PD-1 are shown. Briefly, 2- or 3-fold serial dilutions of anti-human PD-L1 mAb or bispecific antibody were prepared (highest antibody concentration: 128 hM; 3 replicates for each mAb). CHOK1 cells transfected with human PD-L1 were washed with FACS buffer (0.5% BSA, 2 mM EDTA in PBS) and resuspended to 10 6 The concentration of cells / ml was 10 μg / ml. FITC-labeled human PD-1-Fc protein was added to the cells at 7 μg / ml, and 20 μl of the mixture was added to 2 or 3-fold serial dilutions of anti-human PD-L1 mAb or bispecific Ab. After incubation at 4°C for 30 minutes, the cells were washed and resuspended in 30 μl 7AAD and 35 μl 10% neutral buffered formalin solution was added. After 15 minutes, the plate wells were analyzed using the iQue Intellicyt system. The resulting IC50 values ​​(nM) obtained from this analysis are shown in Table 1. Figure 42B and showed that Bi-ZPL-1 retained the ability to inhibit PD-1-PD-L1 binding.

[0470] Figure 43A The bioassay of Example 10 is shown, which tests the ability of Bi-ZP-2 and Bi-ZPL-3 to block the bioactivity of VEGF165 on cells expressing VEGFR-2. The results are shown in tabular form. Figure 43B and showed that the molecule has biological activity relative to the benchmark antibody bevacizumab.

[0471] Both Bi-ZP-2 and Bi-ZPL-3 were evaluated by size exclusion chromatography and had similar percentages of HMW species, dimers, and LMW species, as shown in Figure 2. Figure 44 shown.

[0472] Figure 45 The pharmacokinetics of Bi-ZP-2 and Bi-ZPL-3 in each of two mice are shown. The in vivo half-life of Bi-ZP-2 is longer than that of Bi-ZPL-3.

[0473] The above description is intended to teach one of ordinary skill in the art how to practice the present invention, and is not intended to detail all modifications and variations that would become apparent to one of ordinary skill in the art upon reading this description. However, all such obvious modifications and variations are intended to be included within the scope of the present invention, which is defined by the appended claims. Unless the context clearly indicates otherwise, the claims are intended to cover the claimed components and steps in any order that effectively achieves the desired purpose. Sequence Listing <110> Jingshang Biopharmaceutical Company <120> Trispecific antibodies <130> 2022-003 PCT1 (130777) <150> US 62 / 691,658 <151> 2018-06-29 <150> US 62 / 823,989 <151> 2019-03-26 <160> 420 <170> PatentIn version 3.5 <210> 1 <211> 6 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 1 Ser Asp Tyr Ala Trp Asn 1 5 <210> 2 <211> 16 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 2 Tyr Ile Ser Tyr Ser Gly Ser Thr Gly Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 3 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 3 Arg Met Ile Gly Tyr Ala Met Asp Tyr 1 5 <210> 4 <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 4 Tyr Ile Thr Tyr Ser Gly Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys 1 5 10 15 Ser <210> 5 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 5 Arg Gln Ile Gly Leu Gly Phe Thr Tyr 1 5 <210> 6 <211> 5 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 6 Asp His Thr Ile His 1 5 <210> 7 <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 7 Tyr Phe Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 8 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 8 Gly Met Leu Arg Trp Phe Ala Asp 1 5 <210> 9 <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 9 Tyr Ile Tyr Pro Arg Asp Gly Ser Ser Lys Tyr Asn Val Lys Phe Lys 1 5 10 15 Gly <210> 10 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 10 Gly Met Leu Arg Trp Phe Ala Tyr 1 5 <210> 11 <211> 5 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 11 Asp Gln Ala Ile His 1 5 <210> 12 <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 12 Tyr Ile Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Glu Thr Phe Lys 1 5 10 15 Gly <210> 13 <211> 16 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 13 Tyr Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 14 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 14 Arg Gln Val Gly Leu Gly Phe Ala Tyr 1 5 <210> 15 <211> 6 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 15 Ser Asp Ser Ala Trp Asn 1 5 <210> 16 <211> 16 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 16 Tyr Ile Thr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Arg Ser 1 5 10 15 <210> 17 <211> 5 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 17 Asn Tyr Gly Met Asn 1 5 <210> 18 <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 18 Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys 1 5 10 15 Gly <210> 19 <211> 12 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 19 Ala Pro Pro Tyr Gly Tyr Asp Val Arg Phe Ala Tyr 1 5 10 <210> 20 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 20 Thr Phe Ala Met Gly Val Gly 1 5 <210> twenty one <211> 16 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> twenty one His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ala Leu Lys Ser 1 5 10 15 <210> twenty two <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> twenty two Met Asp Tyr Ser Tyr Phe Ala Trp Phe Ala Tyr 1 5 10 <210> twenty three <211> 5 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> twenty three Ser Tyr Tyr Met His 1 5 <210> twenty four <211> 16 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> twenty four Ile Asn Pro Ser Gly Gly Arg Thr Ser Tyr Ala Gln Met Phe Gln Gly 1 5 10 15 <210> 25 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 25 Asp Arg Glu Glu Gln Trp Pro Val Gly Gly Phe Asp Tyr 1 5 10 <210> 26 <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 26 Lys Ala Ser Gln Asp Val Ser Thr Val Val Ala 1 5 10 <210> 27 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 27 Ser Ala Ser Tyr Arg Tyr Thr 1 5 <210> 28 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 28 Gln Gln His Tyr Ser Thr Pro Trp Thr 1 5 <210> 29 <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 29 Lys Ala Ser Gln Asp Leu Ser Thr Ala Val Ala 1 5 10 <210> 30 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 30 Ser Ser Ser Tyr Arg Tyr Thr 1 5 <210> 31 <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 31 Lys Ala Ser Gln Asp Val Ser Thr Thr Val Ala 1 5 10 <210> 32 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 32 Gln Gln His Tyr Ser Thr Pro Leu Thr 1 5 <210> 33 <211> 11 <212> PRT <213> Artificial sequence <220> <223> syn...

Claims

1. A trispecific antagonist, comprising: An immunoglobulin scaffold comprising a CH1 domain, a CH2 domain and a CH3 domain; A first targeting domain, wherein the first targeting domain is an anti-PD-1 variable region domain; A second targeting domain, wherein the second targeting domain specifically binds to VEGF, and the amino acid sequence of the second targeting domain is shown in SEQ ID NO: 185 or 207; and A third targeting domain, wherein the third targeting domain comprises a peptide inhibitor of angiopoietin / Tie-2 signal transduction pathway, the amino acid sequence of which is shown in SEQ ID NO: 194, Wherein the first targeting domain comprises: (1) an immunoglobulin heavy chain variable region (HCVR), wherein the immunoglobulin heavy chain variable region comprises the following three complementarity determining regions (HCDRs): HCDR1, HCDR2 and HCDR3, in, The HCDR1 amino acid sequence is shown in SEQ ID NO: 59, Wherein, the HCDR2 amino acid sequence is shown in SEQ ID NO: 60, and Wherein, the HCDR3 amino acid sequence is shown in SEQ ID NO: 61; (2) an immunoglobulin light chain variable region (LCVR), wherein the immunoglobulin light chain variable region comprises the following three complementarity determining regions (LCDR): LCDR1, LCDR2 and LCDR3, Wherein, the LCDR1 amino acid sequence is shown in SEQ ID NO: 73, Wherein, the LCDR2 amino acid sequence is shown in SEQ ID NO: 74, and Wherein, the LCDR3 amino acid sequence is shown in SEQ ID NO: 75; or The first targeting domain comprises: (1) HCVR, wherein the HCVR comprises the following three complementarity determining regions (HCDRs): HCDR1, HCDR2 and HCDR3, Wherein, the HCDR1 amino acid sequence is shown in SEQ ID NO: 56, Wherein, the HCDR2 amino acid sequence is shown in SEQ ID NO: 57, and wherein the HCDR3 amino acid sequence is shown in SEQ ID NO: 58; and (2) LCVR, wherein the LCVR comprises the following three complementarity determining regions (LCDR): LCDR1, LCDR2 and LCDR3, Wherein, the LCDR1 amino acid sequence is shown in SEQ ID NO: 70, Wherein, the LCDR2 amino acid sequence is shown in SEQ ID NO: 71, and Wherein, the LCDR3 amino acid sequence is shown in SEQ ID NO: 72; wherein the second targeting domain is structurally linked to the carboxyl terminus of the CH3 domain via a peptide linker, and wherein the third targeting domain is inserted within the Fc loop of the CH3 domain.

2. The trispecific antagonist according to claim 1, wherein The first targeting domain comprises: (1) an immunoglobulin heavy chain variable region (HCVR), wherein the immunoglobulin heavy chain variable region comprises the following three complementarity determining regions (HCDRs): HCDR1, HCDR2 and HCDR3, Wherein, the HCDR1 amino acid sequence is shown in SEQ ID NO: 59, Wherein, the HCDR2 amino acid sequence is shown in SEQ ID NO: 60, and Wherein, the HCDR3 amino acid sequence is shown in SEQ ID NO: 61; (2) an immunoglobulin light chain variable region (LCVR), wherein the immunoglobulin light chain variable region comprises the following three complementarity determining regions (LCDR): LCDR1, LCDR2 and LCDR3, Wherein, the LCDR1 amino acid sequence is shown in SEQ ID NO: 73, Wherein, the LCDR2 amino acid sequence is shown in SEQ ID NO: 74, and Wherein, the LCDR3 amino acid sequence is shown in SEQ ID NO:

75.

3. The trispecific antagonist according to claim 2, wherein The first targeting domain comprises: (1) an immunoglobulin heavy chain variable region (HCVR), the amino acid sequence of which is shown in SEQ ID NO: 137; and (2) an immunoglobulin light chain variable region (LCVR), wherein the immunoglobulin light chain variable region (LCVR) is shown in SEQ ID NO:

138.

4. The trispecific antagonist according to claim 3, wherein The trispecific antagonist comprises: an immunoglobulin light chain having an amino acid sequence as shown in SEQ ID NO: 201 and / or an immunoglobulin heavy chain having an amino acid sequence as shown in SEQ ID NO: 200 or SEQ ID NO:

202.

5. The trispecific antagonist according to claim 1, wherein The first targeting domain comprises: (1) HCVR, wherein the HCVR comprises the following three complementarity determining regions (HCDRs): HCDR1, HCDR2 and HCDR3, Wherein, the HCDR1 amino acid sequence is shown in SEQ ID NO: 56, Wherein, the HCDR2 amino acid sequence is shown in SEQ ID NO: 57, and wherein the HCDR3 amino acid sequence is shown in SEQ ID NO: 58; and (2) LCVR, wherein the LCVR comprises the following three complementarity determining regions (LCDR): LCDR1, LCDR2 and LCDR3, Wherein, the LCDR1 amino acid sequence is shown in SEQ ID NO: 70, Wherein, the LCDR2 amino acid sequence is shown in SEQ ID NO: 71, and Wherein, the LCDR3 amino acid sequence is shown in SEQ ID NO:

72.

6. The trispecific antagonist according to claim 5, wherein The first targeting domain comprises: (1) an immunoglobulin heavy chain variable region (HCVR), the immunoglobulin HCVR amino acid sequence of which is shown in SEQ ID NO: 135; and (2) an immunoglobulin light chain variable region (LCVR), wherein the amino acid sequence of the immunoglobulin LCVR is shown in SEQ ID NO:

136.

7. The trispecific antagonist according to claim 6, wherein The trispecific antagonist comprises: an immunoglobulin light chain having an amino acid sequence as shown in SEQ ID NO: 232 and / or an immunoglobulin heavy chain having an amino acid sequence as shown in SEQ ID NO: 231 or SEQ ID NO:

233.

8. Use of the trispecific antagonist of any one of claims 1 to 7 for the preparation of a medicament for treating a cell proliferative disease, wherein the cell proliferative disease is a solid tumor.

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