HK40014601A

HK40014601A - Anti-gitr antigen-binding proteins and methods of use thereof

Info

Publication number: HK40014601A
Application number: HK62020003784.9A
Authority: HK
Inventors: D‧希克林; C‧塞德尔-杜甘; W‧温斯顿; J-A‧萨尔梅龙-加西亚; H‧布罗德金; S‧克勒费尔; N‧P‧尼尔森
Original assignee: 波滕扎治疗公司
Priority date: 2016-11-19
Filing date: 2017-11-19
Publication date: 2020-08-21

Description

anti-GITR antigen binding proteins and methods of use thereof

Technical Field

Provided herein are Antigen Binding Proteins (ABPs) having binding specificity for glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), including pharmaceutical compositions, diagnostic compositions, and compositions and kits comprising these ABPs. Also provided are methods of making GITR ABP and methods of using GITR ABP, e.g., for therapeutic, diagnostic, and research purposes.

Background

GITR is a member of the Tumor Necrosis Factor Receptor (TNFR) superfamily. GITR is expressed in many cells of the innate and adaptive immune systems, and membrane surface expression is increased in activated T cells. See Hanabuchib et al, Blood,2006,107: 3617-; and Nocentini et al, Eur.J.Immunol.,2005,35: 1016-1022; each of which is incorporated herein by reference in its entirety. GITR is activated by GITR ligand (GITRL).

Agonism of GITR has a co-stimulatory effect on effector T cells. See Schaer et al, curr, opin, immunol, 2012,24: 217-. GITR agonists have been proposed as therapeutic agents for cancer therapy. See Schaer et al, supra; melero et al, Clin cancer Res.,2013,19: 1044-; cohen et al, j.clin.oncol.,2007,25: 3058; cohen et al, PLoSOne,2010,5: e 10436; nocentini et al, br.j.pharmacol.,2012,165: 2089-2099; and U.S. patent publication No. 2007/0098719; each of which is incorporated herein by reference in its entirety.

Although antibody agonists of GITR have shown promise in mouse models, it is difficult to obtain agonistic antibodies against human GITR. Nocentini et al (Br. J. Pharmacol.,2012,165:2089-2099) have indicated that "anti-GITR mAb has much weaker triggering potential in humans than in mice". They speculate that this may be due to the fact that human GITR must multimerize into stable trimers or super clusters (e.g., tetramers of trimers) in order to be robustly activated.

Thus, there is a need for ABPs that can agonize human GITR more strongly than known antibodies. ABPs that meet this need are provided herein.

Disclosure of Invention

Provided herein are ABPs that specifically bind GITR and methods of using these ABPs.

In one aspect, an isolated multivalent Antigen Binding Protein (ABP) is provided that specifically binds human GITR (hGITR; SEQ ID NO:1) comprising the following six CDR sequences: (a) having the sequence X₁X₂X₃X₄X₅RGYGDYGGHHAFDI CDR-H3, wherein X₁Is A or V, X₂Is H, D, L or R, X₃Is E or D, X₄Is R, N, S or A, and X₅V, D or G (SEQ ID NO: 141); (b) having the sequence X₁IX₂X₃SGX₄TYYNPSLKS CDR-H2, wherein X₁Is G, L or S，X₂Is Y, A or V, X₃Is E, Y or H, and X₄Is S or K (SEQ ID NO: 142); (c) having the sequence X₁SISSX₂X₃X₄X₅WX₆CDR-H1 of (1), wherein X₁Is Y or G, X₂Is G, S or E, X₃Is L, G, S, Y, or A, X₄Is G, A, Y, M or G, X₅V, A or absent, and X6 is S or G (SEQ ID NO: 143); (d) having the sequence QQEYX₁TPPX₂CDR-L3 of (1), wherein X₁Is A or N and X₂Is T or S (SEQ ID NO: 144); (e) having the sequence X₁AX₂SLX₃X₄CDR-L2 of (1), wherein X₁Is A or S, X₂Is D or S, X₃Is Q, D, K or E, and X₄Is S or Y (SEQ ID NO: 145); and (f) has the sequence X₁AS X₂SI X₃CDR-L1 of X4YLN, wherein X₁Is G or R, X₂Is Q or K, X3 is S, D or N, and X₄Is S or T (SEQ ID NO: 146).

In one embodiment, the ABP of (a) comprises V of SEQ ID NO 9_HSequence and V of SEQ ID NO 10_LA sequence; in another embodiment, the ABP of (b) comprises V of SEQ ID NO 19_HSequence and V of SEQ ID NO 20_LA sequence; in another embodiment, the ABP of (c) comprises V of SEQ ID NO 26_HSequence and V of SEQ ID NO 27_LA sequence; in another embodiment, the ABP of (d) comprises V of SEQ ID NO 26 or SEQ ID NO 34_HSequence and V of SEQ ID NO 35_LA sequence; in another embodiment, the ABP of (e) comprises V of SEQ ID NO 26_HSequence and V of SEQ ID NO 40_LA sequence; in another embodiment, the ABP of (f) comprises V of SEQ ID NO:44_HSequence and V of SEQ ID NO 45_LA sequence; in another embodiment, the ABP of (g) comprises V of SEQ ID NO 44_HSequence and V of SEQ ID NO 53_LA sequence; in another embodiment, the ABP of (h) comprises V of SEQ ID NO 58_HSequence and V of SEQ ID NO 10_LA sequence; in another embodiment, the ABP of (i) comprises V of SEQ ID NO 104_HSequence and V of SEQ ID NO 10_LA sequence; and in another embodiment, the ABP of (j) comprises V of SEQ ID NO:105_HSequence and V of SEQ ID NO 10_LAnd (4) sequencing.

In one embodiment, the ABP of (b) comprises the heavy chain of SEQ ID NO. 7 and the light chain of SEQ ID NO. 8; in another embodiment, the ABP of (b) comprises the heavy chain of SEQ ID NO 17 and the light chain of SEQ ID NO 18. In another embodiment, the ABP of (c) comprises the heavy chain of SEQ ID NO. 24 and the light chain of SEQ ID NO. 25. In another embodiment, the ABP of (d) comprises (i) the heavy chain of SEQ ID NO:32 and the light chain of SEQ ID NO:33, or (ii) the heavy chain of SEQ ID NO:37 and the light chain of SEQ ID NO: 33. In another embodiment, the ABP of (e) comprises (i) the heavy chain of SEQ ID NO:38 and the light chain of SEQ ID NO: 39. In another embodiment, the ABP of (f) comprises the heavy chain of SEQ ID NO:42 and the light chain of SEQ ID NO: 43; in another embodiment, the ABP of (g) comprises (i) the heavy chain of SEQ ID NO:51 and the light chain of SEQ ID NO: 52; in another embodiment, the ABP of (h) comprises (i) the heavy chain of SEQ ID NO:57 and the light chain of SEQ ID NO: 8; in another embodiment, the ABP of (i) comprises (i) the heavy chain of SEQ ID NO:114 and the light chain of SEQ ID NO:8, or (ii) the heavy chain of SEQ ID NO:120 and the light chain of SEQ ID NO:8, or (iii) the heavy chain of SEQ ID NO:122 and the light chain of SEQ ID NO: 8; or in another embodiment, the ABP of (j) comprises (i) the heavy chain of SEQ ID NO:115 and the light chain of SEQ ID NO:8, or (ii) the heavy chain of SEQ ID NO:121 and the light chain of SEQ ID NO:8, or (iii) the heavy chain of SEQ ID NO:123 and the light chain of SEQ ID NO: 8.

In another embodiment, the ABP comprises the heavy chain of SEQ ID NO. 7 and the light chain of SEQ ID NO. 8; or the heavy chain of SEQ ID NO. 17 and the light chain of SEQ ID NO. 18; or the heavy chain of SEQ ID NO. 24 and the light chain of SEQ ID NO. 25; the heavy chain of SEQ ID NO:32 and the light chain of SEQ ID NO:33, or (ii) the heavy chain of SEQ ID NO:37 and the light chain of SEQ ID NO: 33; the heavy chain of SEQ ID NO 38 and the light chain of SEQ ID NO 39; the heavy chain of SEQ ID NO 42 and the light chain of SEQ ID NO 43; the heavy chain of SEQ ID NO 51 and the light chain of SEQ ID NO 52; the heavy chain of SEQ ID NO. 57 and the light chain of SEQ ID NO. 8; the heavy chain of SEQ ID NO:114 and the light chain of SEQ ID NO:8, or (ii) the heavy chain of SEQ ID NO:120 and the light chain of SEQ ID NO: 8; or (iii) the heavy chain of SEQ ID NO 122 and the light chain of SEQ ID NO 8; or the heavy chain of SEQ ID NO:115 and the light chain of SEQ ID NO:8, or (ii) the heavy chain of SEQ ID NO:121 and the light chain of SEQ ID NO: 8; or (iii) the heavy chain of SEQ ID NO 123 and the light chain of SEQ ID NO 8.

In another aspect, an isolated multivalent Antigen Binding Protein (ABP) is provided that specifically binds human GITR (hGITR; SEQ ID NO:1) comprising the following six CDR sequences: (a) CDR-H3 having the sequence shown in SEQ ID NO. 66; (b) CDR-H2 having the sequence shown in SEQ ID NO. 65; (c) CDR-H1 having the sequence shown in SEQ ID NO 64; (d) CDR-L3 having the sequence shown in SEQ ID NO: 69; (e) CDR-L2 having the sequence shown in SEQ ID NO. 68; and (f) CDR-L1 having the sequence shown in SEQ ID NO: 67.

In one embodiment, the ABP comprises V of SEQ ID NO 62_HSequence and V of SEQ ID NO 63_LA sequence; v of SEQ ID NO. 70_HSequence and V of SEQ ID NO 63_LA sequence; or V of SEQ ID NO 97_HSequence and V of SEQ ID NO 63_LAnd (4) sequencing.

In another embodiment, the ABP comprises (i) the heavy chain of SEQ ID NO:171 and the light chain of SEQ ID NO: 172; the heavy chain of SEQ ID NO 173 and the light chain of SEQ ID NO 174; 106 heavy chain sequence and 107 light chain sequence; or (ii) the heavy chain sequence of SEQ ID NO:116 and the light chain sequence of SEQ ID NO: 107.

In another aspect, an isolated multivalent Antigen Binding Protein (ABP) is provided that specifically binds human GITR (hGITR; SEQ ID NO:1) comprising the following six CDR sequences: (a) CDR-H3 having the sequence shown in SEQ ID NO. 75; (b) CDR-H2 having the sequence shown in SEQ ID NO: 74; (c) CDR-H1 having the sequence shown in SEQ ID NO. 73; (d) CDR-L3 having the sequence shown in SEQ ID NO. 78; (e) CDR-L2 having the sequence shown in SEQ ID NO. 77; and (f) CDR-L1 having the sequence shown in SEQ ID NO: 75.

In one embodiment, the ABP comprises V of SEQ ID NO 71_HSequence and V of SEQ ID NO 72_LA sequence; or V of SEQ ID NO 98_HSequence and V of SEQ ID NO 72_LAnd (4) sequencing.

In another embodiment, the ABP comprises the heavy chain of SEQ ID NO 173 and the light chain of SEQ ID NO 109; or ABP comprises (i) the heavy chain of SEQ ID NO:108 and the light chain of SEQ ID NO: 109; or (ii) the heavy chain of SEQ ID NO:117 and the light chain of SEQ ID NO: 109.

In another aspect, an isolated multivalent Antigen Binding Protein (ABP) is provided that specifically binds human GITR (hGITR; SEQ ID NO:1) comprising the following six CDR sequences: (a) CDR-H3 having the sequence shown in SEQ ID NO 83; (b) CDR-H2 having the sequence set forth in (i) SEQ ID NO:82 or (ii) SEQ ID NO: 100; (c) CDR-H1 having the sequence shown in SEQ ID NO: 81; (d) CDR-L3 having the sequence shown in SEQ ID NO 86; (e) CDR-L2 having the sequence shown in SEQ ID NO. 85; and (f) CDR-L1 having the sequence shown in SEQ ID NO: 84.

In one embodiment, the ABP comprises the CDR-H2 sequence of (b) (i) of the preceding paragraph and V of SEQ ID NO:79_HSequence and V of SEQ ID NO 80_LAnd (4) sequencing. In another embodiment, the ABP comprises the CDR-H2 sequence of (b) (i) of the preceding paragraph and V of SEQ ID NO:87_HSequence and V of SEQ ID NO 80_LAnd (4) sequencing. In another embodiment, the ABP comprises the CDR-H2 sequence of (b) (i) of the preceding paragraph and V of SEQ ID NO:88_HSequence and V of SEQ ID NO 80_LAnd (4) sequencing. In another embodiment, the ABP comprises the CDR-H2 sequence of (b) (ii) of the preceding paragraph and V of SEQ ID NO:99_HSequence and V of SEQ ID NO 80_LAnd (4) sequencing.

In one embodiment, the ABP comprises the heavy chain of SEQ ID NO. 174 and the light chain of SEQ ID NO. 111; in another embodiment, the ABP comprises the heavy chain of SEQ ID NO 175 and the light chain of SEQ ID NO 111; in another embodiment, the ABP comprises the heavy chain of SEQ ID NO:176 and the light chain of SEQ ID NO: 111; in another embodiment, the ABP comprises the heavy chain of SEQ ID NO 110 and the light chain of SEQ ID NO 111; or (ii) the heavy chain of SEQ ID NO:118 and the light chain of SEQ ID NO: 111.

In another aspect, an isolated multivalent Antigen Binding Protein (ABP) is provided that specifically binds human GITR (hGITR; SEQ ID NO:1) comprising the following six CDR sequences: (a) 93, CDR-H3 having the sequence shown in SEQ ID NO; (b) having the sequence GIIPIFGEAQYAQX₁FX₂CDR-H2 of G, wherein X₁Is K or R, and X₂Is Q or R (SEQ ID NO: 215); (c) CDR-H1 having the sequence shown in SEQ ID NO. 91; (d) CDR-L3 having the sequence shown in SEQ ID NO 94; (d) CDR-L2 having the sequence shown in SEQ ID NO. 85; and (e) CDR-L1 having the sequence shown in SEQ ID NO: 84.

In one embodiment, the ABP comprises: CDR-H2 of SEQ ID NO 92; CDR-H2 of SEQ ID NO. 96; or CDR-H2 of SEQ ID NO. 102.

In another embodiment, the ABP comprises V of SEQ ID NO 89_HSequence and V of SEQ ID NO 90_LAnd (4) sequencing. In another embodiment, the ABP comprises V of SEQ ID NO 95_HSequence and V of SEQ ID NO 90_LAnd (4) sequencing. In another embodiment, the ABP comprises V of SEQ ID NO 101_HSequence and V of SEQ ID NO 90_LAnd (4) sequencing.

In another embodiment, the ABP comprises the heavy chain of SEQ ID NO. 177 and the light chain of SEQ ID NO. 113. In another embodiment, the ABP comprises the heavy chain of SEQ ID NO:178 and the light chain of SEQ ID NO: 113. In another embodiment, the ABP comprises the heavy chain of SEQ ID NO:112 and the light chain of SEQ ID NO: 113; or (ii) the heavy chain of SEQ ID NO:119 and the light chain of SEQ ID NO: 113.

In another aspect, an isolated multivalent Antigen Binding Protein (ABP) is provided that specifically binds human GITR (hGITR; SEQ ID NO:1) comprising the following six CDR sequences: (a) CDR-H3 having the sequence shown in SEQ ID NO: 134; (b) 133, CDR-H2 having the sequence set forth in SEQ ID NO; CDR-H1 having the sequence shown in SEQ ID NO: 132; (c) CDR-L3 having the sequence shown in SEQ ID NO. 135; (d) CDR-L2 having the sequence shown in SEQ ID NO. 68; and (e) CDR-L1 having the sequence shown in SEQ ID NO: 67.

In one embodiment, the ABP comprises (i) V of SEQ ID NO:126_HSequence and V of SEQ ID NO 128_LA sequence; or (ii) V of SEQ ID NO:127_HSequence and V of SEQ ID NO 128_LAnd (4) sequencing.

In one embodiment, the ABP comprises (i) the heavy chain of SEQ ID NO:124 and the light chain of SEQ ID NO: 125; or (ii) the heavy chain of SEQ ID NO:136 and the light chain of SEQ ID NO: 125.

In another aspect, an isolated multivalent Antigen Binding Protein (ABP) is provided that specifically binds human GITR (hGITR; SEQ ID NO:1), comprising: (a) and V is selected from_HA CDR-H3 of the region having a CDR-H3 of at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 95% identity: 9, 19, 26, 34, 44, 58, 62, 70, 71, 79, 87, 88, 89, 95, 97, 98, 99, 101, 104, 105, 126 and 127 SEQ ID NOs; (b) and V is selected from_HA CDR-H2 of the region having a CDR-H2 of at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 95% identity: 9, 19, 26, 34, 44, 58, 62, 70, 71, 79, 87, 88, 89, 95, 97, 98, 99, 101, 104, 105, 126 and 127 SEQ ID NOs; (c) and V is selected from_HA CDR-H1 of the region having a CDR-H1 of at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 95% identity: SEQ ID NO 9, SEQ ID NO 19, SEQ ID NO 26, SEQ ID NO 34, SEQ ID NO 44, SEQ ID NO 58, SEQ ID NO 62, SEQ ID NO 70, SEQ ID NO 71, SEQ ID NO 79, SEQ ID NO 87, SEQ ID NO 88, SEQ ID NO 89, SEQ ID NO 95, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99, SE101, 104, 105, 126 and 127; (d) and V is selected from_LA CDR-L3 of the region has a CDR-L3 of at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 95% identity: 10, 20, 27, 35, 40, 45, 53, 63, 72, 80, 90 and 128; (e) and V is selected from_LA CDR-L2 of the region has a CDR-L2 of at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 95% identity: 10, 20, 27, 35, 40, 45, 53, 63, 72, 80, 90 and 128; and (f) and V selected from_LA CDR-L1 of the region has a CDR-L1 of at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 95% identity: 10, 20, 27, 35, 40, 45, 53, 63, 72, 80, 90 and 128.

In one embodiment, CDR-H3, CDR-H2, CDR-H1, CDR-L3, CDR-L2 and CDR-L1 are each determined according to a numbering scheme selected from the Kabat numbering scheme, the Chothia numbering scheme or the IMGT numbering scheme. In another embodiment, CDR-H1 is determined as defined using both Chothia and Kabat numbering schemes, including the boundaries of the two numbering schemes.

In one embodiment, the CDR-H3 comprises a CDR-H3 selected from: 13, 23, 30, 48, 61, 66, 75, 83, 93, 103, 131, or variants thereof having 1,2 or 3 amino acid substitutions. In another embodiment, the CDR-H2 comprises a CDR-H3 selected from: 12, 22, 29, 47, 60,65, 74, 82, 92, 96, 100, 102, 130 and 133 or variants thereof having 1,2 or 3 amino acid substitutions. In another embodiment, the CDR-H1 comprises a CDR-H1 selected from: 11, 21, 28, 46, 59, 64, 73, 81, 91, 129, 132 or variants thereof having 1 or 2 amino acid substitutions. In another embodiment, the CDR-L3 comprises a CDR-L3 selected from: 16, 17, 56, 69, 78, 86, 94, 135 or variants thereof having 1 or 2 amino acid substitutions. In another embodiment, the CDR-L2 comprises a CDR-L2 selected from: 15, 31, 41, 50, 55, 68, 77 and 85, or variants thereof having 1 amino acid substitution. In another embodiment, the CDR-L1 comprises a CDR-L1 selected from: 14, 36, 49, 54, 67, 76 and 84, or variants thereof having 1 or 2 amino acid substitutions. In one embodiment, the amino acid substitution is a conservative amino acid substitution.

In an embodiment of any of the above aspects, the ABP: (a) competes for binding to GITR with an antibody selected from the group consisting of: ABP1, ABP2, ABP3, ABP4, ABP5, ABP6, ABP7, ABP8, ABP9, ABP10, ABP11, ABP12, ABP13, ABP14, ABP15, ABP16, ABP17, ABP18, ABP19, ABP20, ABP21, ABP22, ABP23, ABP24, ABP25, ABP26, ABP27, ABP28, ABP29, ABP30, ABP31, ABP32, ABP33, and ABP34, each as provided in appendix a of the present disclosure; or (b) has at least three antigen binding domains that specifically bind to an epitope on GITR; or (c) has at least three antigen binding domains that specifically bind a single epitope on GITR; or (d) has at least four antigen binding domains that specifically bind to an epitope on GITR; or (e) has at least four antigen binding domains that specifically bind a single epitope on GITR; or (f) agonizing GITR expressed on the surface of a target cell; or (g) enhancing the binding of GITRL to GITR; or (h) costimulating effector T cells in conjunction with antigen presentation by antigen-presenting cells; or (i) inhibiting the suppression of effector T cells by regulatory T cells; or (j) reducing the number of regulatory T cells in the tissue or systemic circulation; (k) capable of binding to one or more of the GITR (SEQ ID NO:1) residues from the group consisting of: r56, C58, R59, D60, Y61, P62, E64, E65, C66 and C67; or (l) can be any combination of (a) to (k).

In an embodiment of any of the above aspects, GITR is selected from the group consisting of hGITR (SEQ ID NO:1), hGITR-T43R (SEQ ID NO:2), cGITR (SEQ ID NO:3), mGITR (SEQ ID NO:4), and combinations thereof. In another embodiment, ABP (a) specifically binds cynomolgus monkey GITR (cGITR; SEQ ID NO: 3); (b) binds murine GITR (mGITR; SEQ ID NO:4) or does not bind mGITR with a lower affinity (as indicated by a higher KD) than ABP has for hGITR; or (c) can be any combination of (a) to (b). In another embodiment, the ABP: (a) specifically binds cGITR (SEQ ID NO: 3); (b) binds mGITR (SEQ ID NO:4) with a lower affinity (as indicated by a higher KD) than ABP's affinity for hGITR and cGITR; and (c) enhancing the binding of GITRL to GITR.

In another aspect, there is provided an ABP that competes with the ABP of any one of claims 1 to 26 for binding to GITR, wherein the ABP: (a) specifically binds cGITR (SEQ ID NO: 3); (b) binds mGITR (SEQ ID NO:4) with a lower affinity (as indicated by a higher KD) than ABP's affinity for hGITR and cGITR; and (c) enhancing the binding of GITRL to GITR. In one embodiment, the ABP comprises an antibody. In another embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is selected from a human antibody, a humanized antibody or a chimeric antibody. In another embodiment, the ABP is multivalent. In another embodiment, the ABP comprises an antibody fragment.

In another embodiment, the ABP comprises an alternative backbone. In another embodiment, the ABP comprises an immunoglobulin constant region. In another embodiment, the ABP comprises a heavy chain constant region selected from the class IgA, IgD, IgE, IgG, or IgM. In another embodiment, the ABP comprises an IgG class and a heavy chain constant region of a subclass selected from IgG4, IgG1, IgG2, or IgG 3.

In an embodiment of any of the above aspects, at least one Fab is fused to the C-terminus of the Fc domain of IgG. In another embodiment, the ABP further comprises at least one linker. In another embodiment, the IgG is IgG 4. In another embodiment, the IgG is IgG 1. In another embodiment, at least one Fab is fused to the N-terminus of the Fc domain of an IgG. In one embodiment, the at least one Fab is at least two fabs. In another embodiment, the at least one Fab is at least three fabs. In another embodiment, the at least one Fab is at least four fabs. In another embodiment, the two fabs are independently fused to the N-terminus of the IgG. In another embodiment, the two fabs are independently fused to the C-terminus of the IgG. In another embodiment, a Fab is attached to each N-terminus of an IgG, a linker is attached to each such Fab, and a Fab is attached to each linker. In another embodiment, a Fab is attached to each C-terminus of an IgG, a linker is attached to each such Fab, and a Fab is attached to each linker. In another embodiment, wherein each linker comprises SEQ ID NO 5. In another embodiment, each linker comprises SEQ ID NO 6.

In an embodiment of any of the above aspects, the ABP comprises a common light chain antibody, an antibody with knob-and-hole (knob-and-hole) modification, a scFv linked to IgG, a Fab linked to IgG, a bifunctional antibody, a tetravalent bispecific antibody, a DVD-Ig^TM、DART^TM、CovX-body, Fcab antibody,tandem Fab, Zybody^TMOr a combination thereof. In one embodiment, the ABP binds more than one GITR molecule. In another embodiment, ABP is independent of GITRL binding. In another embodiment, the ABP enhances binding of GITRL to GITR by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. In another embodiment, ABP enhances binding of GITRL to GITR by at least about 50%. In another embodiment, the target cell is selected from the group consisting of effector T cells, regulatory T cells, Natural Killer (NK) cells, natural killer T (nkt) cells, dendritic cells, and B cells. In another embodiment, the target cell is an effector T cell selected from the group consisting of a helper (CD4+) T cell, a cytotoxic (CD8+) T cell, and combinations thereof. In another embodiment, the target cell is a regulatory T cell selected from the group consisting of CD4+ CD25+ Foxp3+ regulatory T cells, CD8+ CD25+ regulatory T cells, and combinations thereof. In another embodiment, the tissue is a tumor.

In an embodiment of any of the above aspects, the first antigen-binding domain has a KD for hGITR (SEQ ID NO:1) or hGITR-T43R (SEQ ID NO:2) of less than about 20 nM. In one embodiment, the first antigen binding domain has a KD for cGITR (SEQ ID NO:3) of less than about 200 nM. In another embodiment, the KD of the second antigen-binding domain for hGITR (SEQ ID NO:1) or hGITR-T43R (SEQ ID NO:2) is less than about 100 nM. In another embodiment, the KD of the second antigen-binding domain for cGITR (SEQ ID NO:3) is less than about 1 μ M. In another embodiment, the ABP comprises an Fc domain having reduced effector function when compared to an IgG1 Fc domain. In another embodiment, the ABP comprises a non-glycosylated Fc domain. In another embodiment, the ABP comprises an IgG1 Fc domain having an alanine at one or more of positions 234, 235, 265, and 297.

In an embodiment of any of the above aspects, the GITR is expressed on the surface of the target cell. In one embodiment, ABP multimerizes GITR expressed on the surface of the target cell. In one embodiment, ABP multimerizes 2, 3,4, 5,6, 7,8, 9, 10, 11, or 12 GITR molecules.

In an embodiment of any of the above aspects, the ABP specifically binds to an epitope of human GITR (hGITR; SEQ ID NO:1) and is capable of binding to one or more residues from the group consisting of: r56, C58, R59, D60, Y61, P62, E64, E65, C66 and C67.

In an embodiment of any of the above aspects, the ABP comprises an immunoglobulin comprising at least two different (i.e., having different sequences and/or binding to different residues) heavy chain variable regions that are each paired with a common light chain variable region. In another embodiment, the common light chain variable region forms a distinct antigen binding domain with each of two distinct heavy chain variable regions. In another embodiment, the ABP comprises a first VH variable domain having SEQ ID NO:189, a second VH variable domain having SEQ ID NO:215, and a common variable light chain having SEQ ID NO: 190. In another embodiment, the ABP comprises a first VH variable domain having SEQ ID NO:199, a second VH variable domain having SEQ ID NO:216, and a common variable light chain having SEQ ID NO: 200.

In another aspect there is provided a kit comprising an ABP according to any of the above aspects or as shown in appendix a, and instructions for use of the ABP. In one embodiment, the ABP is lyophilized. In another embodiment, the kit further comprises a liquid for reconstituting the lyophilized ABP.

It is known that when an antibody is expressed in a cell, the antibody will be modified after translation. Examples of post-translational modifications include cleavage of lysine at the C-terminus of the heavy chain with carboxypeptidase; glutamine or glutamic acid at the N-terminus of the heavy and light chains is modified to pyroglutamic acid by pyroglutamic acid; glycosylation; oxidizing; removing amide; and saccharification, and these post-translational modifications are known to occur in a variety of antibodies (see journal of Pharmaceutical Sciences,2008, vol.97, p. 2426-2447, incorporated herein by reference in its entirety). In some embodiments, the ABP of the invention is an antibody or antigen-binding fragment thereof that has undergone post-translational modification. Examples of antibodies or antigen-binding fragments thereof that undergo post-translational modifications include antibodies or antigen-binding fragments thereof that undergo pyroglutamylation at the N-terminus of the heavy chain variable region and/or deletion of lysine at the C-terminus of the heavy chain. It is known in the art that these post-translational modifications due to pyroglutamyl at the N-terminus and deletion of lysine at the C-terminus do not have any effect on the activity of the antibody or fragment thereof (analytical biochemistry,2006, volume 348, pages 24-39, incorporated herein by reference in its entirety).

In an embodiment of any of the above aspects, the ABP comprises a polypeptide sequence having a pyroglutamic acid (pE) residue at its N-terminus. In one embodiment, the ABP comprises a VH sequence in which the N-terminal Q is replaced with pE. In another embodiment, the ABP comprises a VL sequence wherein the N-terminal E is substituted with pE. In another embodiment, the ABP comprises a heavy chain sequence wherein the N-terminal Q is replaced with pE.

In an embodiment of any of the above aspects, the ABP comprises a light chain sequence wherein the N-terminal E is substituted with pE. In another embodiment, the ABP is used as a medicament. In another embodiment, the ABP is used to treat cancer or a viral infection.

In one embodiment, the ABP is used to treat cancer, wherein the cancer is selected from the group consisting of a solid tumor and a hematologic tumor.

In another aspect, there is provided an isolated polynucleotide encoding any one of the above aspects or the ABP shown in appendix a, its VH, its VL, its light chain, its heavy chain, or an antigen-binding portion thereof.

In another aspect, there is provided a vector comprising the polynucleotide of the above aspect. In another aspect, there is provided a host cell comprising a polynucleotide or vector of any one of the above aspects. In one embodiment, the host cell is selected from the group consisting of a bacterial cell, a fungal cell, and a mammalian cell. In another embodiment, the host cell is selected from the group consisting of an escherichia coli (e.coli) cell, a Saccharomyces cerevisiae (Saccharomyces cerevisiae) cell, and a CHO cell.

In another aspect, there is provided a cell-free expression response comprising a polynucleotide or vector of the above aspect.

In another aspect, there is provided a method of making the ABP shown in any one of the above aspects or in appendix a, comprising expressing the ABP in a host cell and isolating the expressed ABP.

In another aspect, there is provided a pharmaceutical composition comprising the ABP of any of the above aspects or shown in appendix a, and a pharmaceutically acceptable excipient. In one embodiment, the amount of ABP in the pharmaceutical composition is sufficient to: (a) reducing suppression of effector T cells by regulatory T cells; (b) activating effector T cells; (c) reducing the number of regulatory T cells in a tissue or systemically; (d) inducing or enhancing proliferation of effector T cells; (e) inhibiting the rate of tumor growth; (f) inducing tumor regression; or (g) combinations thereof. In one embodiment, the pharmaceutical composition is for use as a medicament, e.g., for treating cancer or a viral infection. In one embodiment, the pharmaceutical composition is for use in the treatment of cancer, wherein the cancer is selected from the group consisting of a solid tumor and a hematologic tumor.

In another embodiment, the amount of ABP in the pharmaceutical composition is sufficient in the subject to: (a) reducing suppression of effector T cells by regulatory T cells; (b) activating effector T cells; (c) reducing the number of regulatory T cells in a tissue or systemically; (d) inducing or enhancing proliferation of effector T cells; (e) inhibiting the rate of tumor growth; (f) inducing tumor regression; or (g) combinations thereof.

In another aspect, there is provided a method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of the ABP of any of the above aspects or shown in appendix a, or a pharmaceutical composition thereof.

In another aspect, there is provided a method of increasing activation of immune cells in a subject, comprising administering to the subject an effective amount of the ABP of any of the above aspects or shown in appendix a, or a pharmaceutical composition thereof. In one embodiment, the disease or disorder is cancer.

In another embodiment, the method induces or enhances an immune response to a cancer-associated antigen in another embodiment, ABP is administered in an amount sufficient to (a) reduce the suppression of effector T cells by regulatory T cells, (b) activate effector T cells, (c) reduce the number of regulatory T cells in a tissue or systemically, (d) induce or enhance the proliferation of effector T cells, (e) inhibit the rate of tumor growth, (f) induce tumor regression, or (g) a combination thereof in another embodiment, the cancer is a solid cancer in another embodiment, the cancer is a hematologic cancer in another embodiment, the method further comprises administering one or more additional therapeutic agents in one embodiment, the additional therapeutic agent is selected from the group consisting of radiation therapy, cytotoxic agents, chemotherapeutic agents, cytostatic agents, anti-hormonal agents, EGFR inhibitors, immunostimulants, anti-angiogenic agents, and combinations thereof, the additional therapeutic agent is selected from the group consisting of an immunostimulant, TGF-cytokine, TGF-receptor agonist, TGF-receptor-agonist, TGF-hormone, TGF-TNF-gamma-TNF-gamma-TNF-gamma-interferon-gamma-interferon, or a combination thereof, and a combination thereof, wherein the combination thereof is expressed from an anti-interferon, and a combination thereof, wherein the combination thereof, and the combination thereof, and the anti-interferon.

In another aspect, there is provided a method of modulating an immune response in a subject in need thereof, comprising administering to the subject an effective amount of the ABP of any of the above aspects or shown in appendix a, or a pharmaceutical composition thereof. In one embodiment, the method further comprises administering to the subject one or more additional therapeutic agents. In one embodiment, the additional therapeutic agent is an agonist of a stimulatory receptor for an immune cell, and the stimulatory receptor for an immune cell is selected from the group consisting of OX40, CD2, CD27, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), 4-1BB (CD137), CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, CD83 ligand, and combinations thereof. In one embodiment, the additional therapeutic agent is a cytokine selected from the group consisting of IL-2, IL-5, IL-7, IL-12, IL-15, IL-21, and combinations thereof. In one embodiment, the additional therapeutic agent is an oncolytic virus selected from the group consisting of herpes simplex virus, vesicular stomatitis virus, adenovirus, newcastle disease virus, vaccinia virus, malaba virus, and combinations thereof. In one embodiment, the additional therapeutic agent is formulated in the same pharmaceutical composition as the ABP. In one embodiment, the additional therapeutic agent is formulated in a different pharmaceutical composition than the ABP. In one embodiment, the additional therapeutic agent is administered prior to administration of the ABP. In one embodiment, the additional therapeutic agent is administered after administration of the ABP. In one embodiment, the additional therapeutic agent is administered concurrently with the ABP.

In another aspect, an isolated multivalent Antigen Binding Protein (ABP) is provided that specifically binds human GITR (hGITR; SEQ ID NO:1), wherein the ABP competes for binding with one or more of: ABP1, ABP2, ABP3, ABP4, ABP5, ABP6, ABP7, ABP8, ABP9, ABP10, ABP11, ABP12, ABP13, ABP14, ABP15, ABP16, ABP17, ABP18, ABP19, ABP 363672, ABP19, ABP 363672, ABP19, ABP 36363672, ABP19, ABP 363672, ABP 36363672, ABP19, ABP 36363636363672, ABP19, ABP 363672, ABP 3636363672, ABP 363636363672, ABP 36363672, ABP19, ABP 363672, ABP19, ABP 363636363672, ABP 363672.

In another aspect, there is provided an anti-human GITR antibody or antigen-binding fragment thereof comprising four heavy chain variable regions and four light chain variable regions, wherein the heavy chain variable region comprises CDR-H3 consisting of SEQ ID NO:13, CDR-H2 consisting of SEQ ID NO:12, and CDR-H1 consisting of SEQ ID NO: 11; and the light chain variable region comprises CDR-L3 consisting of SEQ ID NO:16, CDR-L2 consisting of SEQ ID NO:15 and CDR-L1 consisting of SEQ ID NO: 14; and one heavy chain variable region and one light chain variable region constitute one antigen binding site, and the antibody or antigen binding fragment comprises four antigen binding sites.

In one embodiment, the anti-human GITR antibody or antigen-binding fragment thereof comprises four heavy chain variable regions and four light chain variable regions, wherein the heavy chain variable region consists of SEQ ID No. 9, the light chain variable region consists of SEQ ID No. 10, and one heavy chain variable region and one light chain variable region constitute one antigen-binding site, and the antibody or antigen-binding fragment comprises four antigen-binding sites.

In one embodiment, the anti-human GITR antibody or antigen-binding fragment thereof comprises four heavy chain variable regions and four light chain variable regions, wherein the heavy chain variable region consists of SEQ ID NO:9, wherein Q at position 1 of the sequence is modified to pyroglutamic acid, the light chain variable region consists of SEQ ID NO:10, and one heavy chain variable region and one light chain variable region constitute one antigen-binding site, and the antibody or antigen-binding fragment comprises four antigen-binding sites.

In one embodiment, the anti-human GITR antibody comprises two heavy chains and four light chains, each heavy chain comprising two structures consisting of a heavy chain variable region and a CH1, CH2, and CH3 region, and the C-terminus of one of the structures is linked to the N-terminus of the other structure via a linker, and each light chain comprising a light chain variable region and a light chain constant region (left panel in fig. 1B).

In one embodiment, the anti-human GITR antibody comprises two heavy chains and four light chains, the heavy chains each comprising a first heavy chain variable region and a first CH1 region, a linker, a second heavy chain variable region, a second CH1 region, a CH2 region and a CH3 region, and each light chain comprising a light chain variable region and a light chain constant region (left panel in fig. 1B).

In one embodiment, the anti-human GITR antibody comprises two heavy chains and four light chains; each heavy chain comprises two structures consisting of a heavy chain variable region comprising CDR-H3 consisting of SEQ ID NO:13, CDR-H2 consisting of SEQ ID NO:12 and CDR-H1 consisting of SEQ ID NO:11, and the CH1, CH2 and CH3 regions, and the carboxy terminus (C-terminus) of one of the structures is linked to the amino terminus (N-terminus) of the other structure via a linker; and each light chain comprises a light chain variable region comprising CDR-L3 consisting of SEQ ID NO:16, CDR-L2 consisting of SEQ ID NO:15, and CDR-L1 consisting of SEQ ID NO: 14.

In one embodiment, the anti-human GITR antibody comprises two heavy chains and four light chains; each heavy chain comprises a first heavy chain variable region and a second heavy chain variable region, each comprising CDR-H3 consisting of SEQ ID NO:13, CDR-H2 consisting of SEQ ID NO:12, and CDR-H1 consisting of SEQ ID NO: 11; a first CH1 region, a linker, a second CH1 region, a CH2 region, and a CH3 region; and each light chain comprises a light chain variable region comprising CDR-L3 consisting of SEQ ID NO:16, CDR-L2 consisting of SEQ ID NO:15, and CDR-L1 consisting of SEQ ID NO: 14.

In one embodiment, the anti-human GITR antibody comprises two heavy chains and four light chains, wherein each heavy chain comprises two structures consisting of the heavy chain variable region of SEQ ID NO:9 and the regions CH1, CH2, and CH3, and the C-terminus of one of the structures is linked to the N-terminus of the other structure via a linker, and each light chain comprises the light chain variable region and the light chain constant region of SEQ ID NO: 10.

In one embodiment, the anti-human GITR antibody comprises two heavy chains and four light chains, each heavy chain comprising a first heavy chain variable region and a second heavy chain variable region, each as set forth in SEQ ID No. 9; a first CH1 region, a linker, a second CH1 region, a CH2 region, and a CH3 region; and wherein each light chain comprises the light chain variable region and the light chain constant region of SEQ ID NO 10.

In one embodiment, the anti-human GITR antibody comprises two heavy chains and four light chains, wherein each heavy chain comprises two structures consisting of the heavy chain variable region of SEQ ID NO:9 with the regions CH1, CH2, and CH3, and the C-terminus of one of the structures is linked to the N-terminus of the other structure via a linker, wherein Q at position 1 of the sequence is modified to pyroglutamic acid, and each light chain comprises the light chain variable region of SEQ ID NO:10 and a light chain constant region.

In one embodiment, the anti-human GITR antibody comprises two heavy chains and four light chains, each heavy chain comprising a first heavy chain variable region and a second heavy chain variable region, each as set forth in SEQ ID No. 9; a first CH1 region, a linker, a second CH1 region, a CH2 region, and a CH3 region; wherein Q at position 1 of the sequence is modified to pyroglutamic acid and each light chain comprises the light chain variable and light chain constant regions of SEQ ID NO 10.

In one embodiment, the anti-human GITR antibody comprises two heavy chains of SEQ ID NO 7, each having two variable regions; and four light chains of SEQ ID NO. 8.

In one embodiment, the anti-human GITR antibody comprises two heavy chains of SEQ ID No. 7, each having two variable regions, wherein Q at position 1 of the sequence is modified to pyroglutamic acid; and four light chains of SEQ ID NO. 8.

In one embodiment, the anti-human GITR antibody comprises two heavy chains consisting of amino acid sequences ranging from Q at position 1 to G at position 686 of SEQ ID No. 7, each having two variable regions; and four light chains of SEQ ID NO. 8.

In one embodiment, the anti-human GITR antibody comprises two heavy chains consisting of amino acid sequences ranging from Q at position 1 to G at position 686 of SEQ ID No. 7, wherein Q is modified to pyroglutamic acid; and four light chains of SEQ ID NO. 8.

Drawings

Figure 1 provides a schematic illustration of the mechanism of action of certain illustrative GITR ABPs provided herein. FIG. 1A shows three GITR molecules (one labeled 101) embedded in the cell membrane (102). Cartoon pictures of the classical form of the antibody show binding to only two GITR molecules (103). FIG. 1B shows a cartoon of a Tetravalent Monospecific (TM) form of the antibody disclosed herein: the left panel (105) shows a TM comprising two N-terminal IgG1 Fab and C-terminal IgG4S228P antibodies; the right panel (106) shows the TM comprising two C-terminal IgG1 Fab and N-terminal IgG4S228P antibodies. The antigen binding domain is illustrated by open circles (107). Figure 1C shows a cartoon of the tetravalent bispecific format antibody disclosed herein: the left panel (105) shows a bispecific antibody comprising two N-terminal IgG1 Fab and C-terminal IgG4S228P antibodies; the right panel (106) shows a bispecific antibody comprising two C-terminal IgG1 Fab and N-terminal IgG4S228P antibodies. Antigen binding domains specific for two non-overlapping epitopes are illustrated by open circles (107 and 108). FIG. 1D shows multimerization of GITR following binding of three illustrative Tetravalent Monospecific (TM) forms of ABP. These multimerization agonize GITR signaling, as described elsewhere in this disclosure, is expected.

FIG. 2 is a graph showing K determination by OCTET for N-terminal Fab forms ABP1 to ABP8_DA graph of exemplary results of (a).

FIG. 3 is a series of graphs showing the results of FACS analysis demonstrating the binding of an exemplary panel of N-terminal Fab TM form ABP to CD4+ (FIG. 3A) and CD8+ (FIG. 3B) T cells. The distribution of CD4+ and CD8+ is shown in the upper left panel of each figure. In the top row, from left to right, an anti-GITR positive control and an anti-human IgG4 isotype control, ABP9, ABP2, ABP1 and ABP7 are shown. In the bottom row, ABP8, ABP3, ABP4, ABP5, ABP6, ABP23, and ABP24 are shown. Indicates the percentage of positively stained IgG4+ CD4/8+ cells; MRI is indicated in parentheses.

FIG. 4 is a drawing showingA series of graphs showing the activity of eight optimized agonist antibodies in the N-terminal Fab TM format. HT1080 cells stably expressing human (left panel) or cynomolgus monkey (right panel) GITR were then incubated with ABP1 (fig. 4A), ABP2 (fig. 4B), ABP3 (fig. 4C), ABP4 (fig. 4D), ABP5 (fig. 4E), ABP6 (fig. 4F), ABP7 (fig. 4G), and ABP8 (fig. 4H) (shown as circles in the figure), and IL-8 induction was measured. GITRL was used as control (squares). EC is shown at the bottom of each picture in each figure₅₀Table of values.

Figure 5 is a series of graphs comparing the agonist activity of the parent N-terminal Fab TM form antibody ABP43 in GITR expressing HT1080 cells with a variety of further optimized antibodies having either an N-terminal or C-terminal form. Fig. 5A shows ABP43 (square), ABP23 (circle), ABP24 (triangle) and ABP29 (open circle), ABP30 (open triangle), ABP31 (open circle) and ABP32 (open triangle), all compared to GITRL (+ notation). FIG. 5B shows ABP19 (N-terminal Fab, triangle) and ABP25 (C-terminal Fab, inverted triangle). GITRL is shown as a plus sign. FIG. 5C shows ABP21 (N-terminal Fab, triangle) and ABP27 (C-terminal Fab, inverted triangle). GITRL is shown as a plus sign. FIG. 5D shows ABP20 (N-terminal Fab, triangle) and ABP26 (C-terminal Fab, inverted triangle). GITRL is shown as a plus sign. FIG. 5E shows ABP22 (N-terminal Fab, triangle) and ABP28 (C-terminal Fab, inverted triangle). GITRL is shown as a plus sign. IgG4 control is shown as an X-mark in each figure.

FIG. 6 shows EC for ABP33 and ABP34 in HT1080 analysis as described in the examples₅₀The results of the measurement. ABP33 (tetravalent form combining IgG4 ABP61 with IgG1 Fab of ABP58 on the N-terminus) and ABP34 (tetravalent form combining IgG4 of ABP61 with IgG1 Fab of ABP58 on the C-terminus) were compared to bivalent ABP 59 and ABP61 (IgG4S228P), which are the basis of ABP33 and ABP 34. As shown in the figure, the bispecific tetravalent antibodies both had superior EC when compared to the single bivalent antibody used to construct the bispecific tetravalent antibody₅₀As measured by IL-8 induction.

FIG. 7 shows the results in the Jurkat T cell assay as described for HT1080 cells aboveEC₅₀The results of the measurement. The optimized N-terminal Fab TM form ABP was compared to GITRL for its ability to agonize GITR (as measured by IL-8 production). Fig. 7A to 7H show ABP1 to ABP8, respectively.

Figure 8A shows FACS analysis of three replicate quantifications from T cells isolated from two human donors and shows the percentage of GITR + CD4+ cells (left) and CD8+ cells (right) at each different time point, with or without stimulation with PHA.

Figures 8B-8M show the treatment results and resulting IL-2 production from T-blasts from 4 different donors treated with control or ABP (data normalized to the level of IL-2 production achieved in control medium). In each figure at the top row, from left to right are FACS measurements of ABP binding to CD4+ cells, IL-2 production by cells from donor 1, IL-2 production by cells from donor 2. In the bottom row, from left to right are FACS measurements of ABP binding to CD8+ cells, IL-2 production by cells from donor 3, IL-2 production by cells from donor 4. The data are shown below: 8B: IgG4 isotype control; 8C: SEC4 antibody; 8D: IgG4TM format negative control; 8E: ABP9 (TM form IgG4 non-optimized parent of ABP1 to ABP 8); 8F: ABP 1; 8G: ABP 2; 8H: ABP 3; 8I: ABP 4; 8J: ABP 5; 8K: ABP 6; 8L: ABP 7; 8M: ABP 8.

Fig. 9A-9H are a series of graphs showing a comparison of the activity of optimized N-terminal Fab TM ABP ("IgG 4 TM") against a corresponding optimized non-TMIgG 1N 297A ABP ("IgG 1"). HT1080 cells stably expressing human (left panel) or cynomolgus monkey (right panel) were then each treated with N-terminal Fab TM ABP and the corresponding IgG1 ABP as follows: ABP1 IgG4TM/ABP35IgG1 (FIG. 9A), ABP2 IgG4TM/ABP 36 IgG1 (FIG. 9B), ABP3 IgG4TM/ABP 37 IgG1 (FIG. 9C), ABP4IgG 4TM/ABP 38 IgG1 (FIG. 9D), ABP 5IgG 4TM/ABP 38P 45L IgG1 (FIG. 9E), ABP6 IgG4TM/ABP 39 IgG1 (FIG. 9F), ABP7 IgG4TM/ABP 40 IgG1 (FIG. 9G), ABP8 IgG4TM/ABP 41 IgG1 (FIG. 9H), and IgG4 as a positive control. The induction of IL-8 by GITRL is shown as a circle, the induction of IL-8 by the IgG4TM form of ABP is shown as a triangle, the induction of IL-8 by the corresponding IgG1 ABP is shown as a diamond, and the induction of I by the IgG4 control antibody is shown as a diamondThe induction of L-8 is shown as a square. EC is shown at the bottom of each picture in each figure₅₀Table of values.

FIG. 10 is an EC in HT1080 analysis showing comparison of non-TM parent antibodies to the corresponding TM format versions as described₅₀A graph of the data. ABP43 (diamonds) is the non-TM IgG4S228P parent of ABP9(IgG 4N-terminal Fab, squares) and ABP10(IgG 4C-terminal Fab, circles). GITRL (positive control) showed a single data point (star) for IL-8 induction. IL-8 production is shown in pg/mL.

Figure 11A is a graph showing the induction of IL-8 by representative benchmark antibodies SEC4 and SEC9 in HT1080 cells engineered to stably express human GITR. Cultured cells were treated with a series of concentrations of two baseline agonist antibodies SEC4 (35E 6, diamonds formatted to have mouse variable regions and human IgG4S 228P/kappa regions) and SEC9 (humanized 6C 8N 62QIgG 1N 297A, circles) as well as an IgG4 negative control (filled triangles), an IgG1 negative control (open triangles) and a trimeric human GITR ligand ("hGITRL", squares ") as a positive control for six hours. IL-8 induction by ELISA measurement. As shown in the figure, GITRL has a better EC than both SEC4 and SEC9₅₀(inset) and maximum induction. Fig. 11B-11I show comparisons of ABP 1-ABP 8 with SEC4 and SEC9, respectively. TM ABP is indicated by circles, SEC4 is indicated by squares and SEC9 is indicated by triangles. IL-8 production is shown in pg/mL.

FIG. 12 is two graphs showing cytokine production in isolated human NSCLC adenocarcinoma cells stimulated after treatment with TM form ABP1 alone or in combination with pembrolizumab (pembrolizumab). The cells were either unstimulated controls or stimulated with 1. mu.g/mL α CD3 (soluble) + 2. mu.g/mL α CD28 (soluble) + IL-2(50 ng/mL); cells received no immunotherapy treatment (to assess checkpoint protein levels), pamumab (10. mu.g/mL), TM form ABP controls (2. mu.g/mL), ABP1 (2. mu.g/mL), or ABP1+ pamumab. the cells were incubated for 48 hours prior to collection of supernatant and the cells were stained for checkpoint expression.

Figure 13 is a series of graphs showing GITR aggregation and internalization following antibody binding. GITR internalization was measured in CD4+ cells (fig. 13A-13E) and CD8+ cells (fig. 13F-13J) from donor 1 (fig. 13A-13B, 13F-13G) or donor 2 (fig. 13C-13D, 13H-13I). Cells were treated with either ABP1 TM format antibody or ABP35 standard bivalent antibody. As can be observed, incubation with either ABP1 or ABP35 inhibited subsequent staining of ABP1Dylight650 (fig. 13A, 13C, 13F, 13H), but incubation with only ABP1 induced GITR internalization as measured by staining with non-competitive clone 108-17 (fig. 13B, 13D, 13G, 13I). The assay of ABP1 for EC50 for cells from both donors is shown in fig. 13E (CD4+ cells) and fig. 13J (CD8+ cells).

Figure 14 shows cytokine (IL-2) production by activated T-blasts from two healthy human donors (donor 1 and donor 2, respectively, figure 14A and figure 14B) following treatment with anti-GITR IgG4TM form antibody ABP1, its IgG1 form counterpart ABP35 and IgG4TM form, and an IgG1 isotype control. Activated CD4+/CD8+ T cells were treated with medium alone, recombinant GITR-ligand, ABP1, hIgG4TM format isotype control, ABP35, or hIgG1 standard format isotype control, each at nine doses: 10. mu.g/mL, 2. mu.g/mL, 0.4. mu.g/mL, 80ng/mL, 16ng/mL, 3.2ng/mL, 0.64ng/mL, 0.13ng/mL and 0.026 ng/mL. Cells were stimulated by the addition of 1. mu.g/ml anti-CD 3 antibody and 2. mu.g/ml anti-CD 28 antibody. FIG. 14C shows the same data as in FIGS. 14A and 14B for ABP1, where EC was calculated in each donor₅₀And (4) measuring.

Figure 15 shows cytokine (IL-2) production from activated T blast cells from two healthy human donors (donor 1 and donor 2, respectively, figure 15A and figure 15B) following treatment with anti-GITR IgG4TM format antibody ABP1, IgG4TM format control ("IsoTM"), SEC4, SEC9, recombinant hGITRL, and IgG1 and IgG4 isotype control. Using a single medium, recombinant GITR-ligand, ABP1, hIgG4 TM-form isotypeControls, SEC4, SEC9, IgG1 standard format isotype control, or hIgG1 standard format isotype control each treated activated CD4+/CD8+ T cells at nine doses: 10. mu.g/mL, 2. mu.g/mL, 0.4. mu.g/mL, 80ng/mL, 16ng/mL, 3.2ng/mL, 0.64ng/mL, 0.13ng/mL and 0.026 ng/mL. Cells were stimulated by the addition of 1. mu.g/ml anti-CD 3 antibody and 2. mu.g/ml anti-CD 28 antibody. Fig. 15C (donor 1) and 15D (donor 2) show the same data as in fig. 15A and 15B for ABP1, where EC was calculated in each donor₅₀And (4) measuring.

Detailed Description

Definition of

Unless defined otherwise, all technical terms, symbols, and other scientific terms used herein are intended to have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. In some instances, terms having commonly understood meanings are defined herein for clarity and/or for convenient reference, and the inclusion of such definitions herein should not necessarily be construed to mean a difference from what is commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed by those skilled in the art using well known methods, such as the widely used molecular cloning methods described in Sambrook et al, molecular cloning: A Laboratory Manual 4 th edition (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.. Procedures involving the use of commercially available kits and reagents are generally performed according to protocols and conditions defined by the manufacturer, as required, unless otherwise indicated.

As used herein, the singular forms "a" and "the" include plural referents unless the context clearly dictates otherwise. The terms "include," "such as," and the like are intended to convey an inclusion, but not a limitation, unless specifically indicated otherwise.

As used herein, the term "comprising" also specifically includes embodiments "consisting of and" consisting essentially of the recited elements, unless specifically indicated otherwise. For example, a multispecific ABP "comprising a bifunctional antibody" includes a multispecific ABP "consisting of a bifunctional antibody" and a multispecific ABP "consisting essentially of a bifunctional antibody".

The term "about" indicates and encompasses the indicated value and ranges above and below that value. In certain embodiments, the term "about" indicates the specified value ± 10%, ± 5% or ± 1%. In certain embodiments, the term "about" indicates the specified value ± one standard deviation of the value.

The terms "GITR," "GITR protein," and "GITR antigen" are used interchangeably herein to refer to human GITR or any variant (e.g., splice variants and allelic variants), allotropes, and species homologs of human GITR that are naturally expressed by a cell or expressed by a cell transfected with a GITR gene. In some aspects, the GITR protein is a GITR protein naturally expressed by a primate (e.g., monkey or human), rodent (e.g., mouse or rat), dog, camel, cat, cow, goat, horse, or sheep. In some aspects, the GITR protein is human GITR (hGITR; SEQ ID NO: 1). In some aspects, the GITR protein is a human GITR T43R variant (hGITR-T43R; SEQ ID NO: 2). In some aspects, the GITR protein comprises the extracellular domain of hGITR located at positions 26 to 162 of SEQ ID No. 1 to SEQ ID No. 2. In some aspects, the GITR protein is cynomolgus monkey GITR (cGITR; SEQ ID NO: 3). In some aspects, the GITR protein comprises the extracellular domain of cGITR located at positions 20 to 156 of SEQ ID No. 3. In some aspects, the GITR protein is murine GITR (mGITR; SEQ ID NO: 4). In some aspects, the GITR protein comprises the extracellular domain of mGITR located at positions 20 to 153 of SEQ ID No. 4. In some aspects, the GITR protein is a full-length or unprocessed GITR protein. In some aspects, the GITR protein is a truncated or processed GITR protein resulting from post-translational modifications. GITR is also known as a variety of synonyms, including tumor necrosis factor receptor superfamily member 18(TNFRSF 18); AITR, glucocorticoid-induced TNFR-related protein; activating an inducible TNFR family receptor; TNF receptor superfamily activation-inducible proteins; CD 357; and GITR-D.

The term "immunoglobulin" refers to a class of structurally related proteins that generally comprises two pairs of polypeptide chains: a pair of light (L) chains and a pair of heavy (H) chains. In the "intact immunoglobulin", all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g., Paul, Fundamental Immunology 7 th edition, Ch.5(2013) Lippincott Williams&Wilkins, Philadelphia, PA. Briefly, each heavy chain typically comprises a heavy chain variable region (V)_H) And heavy chain constant region (C)_H). The heavy chain constant region usually comprises three domains, abbreviated C_H1、C_H2And C_H3. Each light chain typically comprises a light chain variable region (V)_L) And a light chain constant region. The light chain constant region usually comprises a domain, abbreviated C_L。

The term "antigen binding protein" (ABP) refers to a protein that comprises one or more antigen binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen binding domain binds to an antigen or epitope with a specificity and affinity similar to a naturally occurring antibody. In some embodiments, the ABP comprises, consists of, or consists essentially of an antibody. In some embodiments, the ABP comprises, consists of, or consists essentially of an antibody fragment. In some embodiments, the ABP comprises, consists of, or consists essentially of an alternative backbone. A "GITR ABP," "anti-GITR ABP," or "GITR-specific ABP" is an ABP that specifically binds to an antigen GITR as provided herein. In some embodiments, the ABP binds to the extracellular domain of GITR. In certain embodiments, the GITR ABPs provided herein bind to an epitope of GITR that is conserved between or among GITR proteins from different species.

The term "antibody" is used herein in its broadest sense and includes certain types of immunoglobulin molecules that comprise one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies in particularIncluding intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies. An example of an antigen binding domain is a domain consisting of V_H-V_LA dimer-forming antigen-binding domain. Antibodies are one type of ABP.

The term "alternative scaffold" refers to a molecule in which one or more regions may be diversified to create one or more antigen-binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen binding domain binds to an antigen or epitope with a specificity and affinity similar to a naturally occurring antibody. Exemplary alternative scaffolds include those derived from fiber binding proteins (e.g., Adnectins)^TM) β -Sandwich structures (e.g.iMab), lipocalins (e.g.iMab)) EETI-II/AGRP, BPTI/LACI-D1/ITI-D2 (e.g., Kunitz domain), thioredoxin peptide aptamers, protein A (e.g., protein A) Ankyrin repeats (e.g., DARPin), gamma-B-crystallin/ubiquitin (e.g., Affilin), CTLD₃(e.g., Tetranectin (Tetranectin)), Fynomer and (LDLR-A module) (e.g., Avimer). Additional information on alternative scaffolds is found in Binz et al, nat. Biotechnol., 200523: 1257-1268; skerra, Current opin. in Biotech, 200718: 295-304; and Silacci et al, J.biol.chem.,2014,289: 14392-14398; each of which is incorporated herein by reference in its entirety. An alternative backbone is one type of ABP.

The term "antigen binding domain" means a portion of ABP that is capable of specifically binding to an antigen or epitope.

The terms "full-length antibody," "intact antibody," and "full antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a naturally occurring antibody structure and having a heavy chain comprising an Fc region.

The term "Fc region" means the C-terminal region of an immunoglobulin heavy chain that interacts with Fc receptors and certain proteins of the complement system in naturally occurring antibodies. The structure of the Fc region of various immunoglobulins and the glycosylation sites contained therein are known in the art. See Schroeder and Cavacini, j.allergy clin.immunol.,2010,125: S41-52, which are incorporated herein by reference in their entirety. The Fc region may be a naturally occurring Fc region or a modified Fc region as described elsewhere in the invention.

Can make V_HAnd V_LThe regions are further subdivided into hypervariable regions ("hypervariable regions (HVRs)", also known as "Complementarity Determining Regions (CDRs)") interspersed with more conserved regions. The more conserved regions are called Framework Regions (FR). Each V_HAnd V_LTypically comprising three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. CDRs are involved in antigen binding and affect the antigen specificity and binding affinity of an antibody. See Kabat et al, Sequences of Proteins of Immunological Interest 5 th edition (1991) Public Health service, National Institutes of Health, Bethesda, Md., which is incorporated herein by reference in its entirety.

Light chains from any vertebrate species can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the sequence of their constant domains.

Heavy chains from any vertebrate species can be assigned as one of five different classes (or isotypes), IgA, IgD, IgE, IgG, and IgM, which are also designated α, δ, ε, γ, and μ, respectively.

The amino acid sequence boundaries of the CDRs may be determined by one of skill in the art using any of a variety of known numbering schemes, including such schemes described by Kabat et Al, supra ("Kabat" numbering scheme), Al-Lazikani et Al, 1997, J.mol.biol.,273:927-948 ("Chothia" numbering scheme), MacCallum et Al, 1996, J.mol.biol.262:732-745 ("Contact" numbering scheme), Lefranc et Al, Dev.Comp.Immunol.,2003,27:55-77 ("IMGT" numbering scheme), and Honegge and Pl ü thun, J.mol.biol.,2001,309:657-70 ("AHo" numbering scheme), each of which is incorporated herein by reference in its entirety.

Table 1 provides the positions of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 as determined using the Kabat and Chothia protocols. For CDR-H1, residue numbering is provided using the Kabat and Chothia numbering schemes.

Unless otherwise specified, the numbering scheme used herein to determine a particular CDR is the Kabat/Chothia numbering scheme. When the residues encompassed by such two numbering schemes differ (e.g., CDR-H1 and/or CDR-H2), the numbering schemes are designated as Kabat or Chothia schemes. For convenience, CDR-H3 is sometimes referred to herein as Kabat or Chothia. However, this is not intended to imply differences in sequence that do not exist therein, and one skilled in the art can easily confirm whether the sequences are the same or different by examining the sequences.

CDRs may be specified, for example, using antibody numbering software (such as Abnum, available at www.bioinf.org.uk/abs/Abnum /), and are described in Martin, Immunology,2008,45: 3832-.

Table 1 residues in CDRs according to Kabat and Chothia numbering scheme.

CDR	Kabat	Chothia
			L1	L24-L34	L24-L34
L2	L50-L56	L50-L56
			L3	L89-L97	L89-L97
H1(Kabat numbering)	H31-H35B	H26-H32 or H34
			H1(Chothia number)	H31-H35	H26-H32
H2	H50-H65	H52-H56
			H3	H95-H102	H95-H102

When numbered using Kabat numbering convention, the C-terminus of CDR-H1 varies between H32 and H34 according to the length of the CDR.

"EU numbering scheme" is typically used when referring to residues in the constant region of the heavy chain of an antibody (e.g., as reported in Kabat et al, supra). Unless otherwise stated, EU numbering schemes are used to refer to residues in the antibody heavy chain constant regions described herein.

An "antibody fragment" or "antigen-binding fragment" constitutes a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F (ab')₂Fragments, Fab' fragments, scFv (sFv) fragments and scFv-Fc fragments.

An "Fv" fragment comprises a non-covalently linked dimer of one heavy chain variable domain and one light chain variable domain.

In addition to the heavy and light chain variable domains, a "Fab" fragment comprises the constant domain of the light chain and the first constant domain of the heavy chain (C)_H1). Fab fragments can be produced, for example, using recombinant methods or by papain digestion of full-length antibodies.

“F(ab')₂A "fragment comprises two Fab' fragments linked by a disulfide bond near the hinge region. F (ab')₂Fragments can be produced, for example, using recombinant methods or by pepsin digestion of intact antibodies F (ab') fragments can be dissociated, for example, by treatment with β -mercaptoethanol.

"Single chain Fv" or "sFv" or "scFv" antibody fragments comprise V in a single polypeptide chain_HDomain and V_LA domain. V_HAnd V_LGenerally linked by a peptide linker, see Pl ü ckthun A. (1994). in some embodiments, the linker is (GGGGS)_n(SEQ ID NO: 5). In other embodiments, the linker is GGGGSGGGGSGGGS (SEQ ID NO: 6). In some embodiments, n ═ 1,2, 3,4, 5, or 6. See Antibodies from Escherichia coli in Rosenberg M.&Moore g.p. (eds.), The pharmaceutical of Monoclonal Antibodies, vol 113 (p.269-315.) Springer-Verlag, New York, which is incorporated herein by reference in its entirety.

An "scFv-Fc" fragment comprises an scFv linked to an Fc domain. For example, the Fc domain may be linked to the C-terminus of the scFv. According to the orientation of the variable domains in scFv (i.e., V)_H-V_LOr V_L-V_H) The Fc domain may be at V_HOr V_LAnd then. Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

The term "single domain antibody" refers to a molecule in which one variable domain of the antibody specifically binds to an antigen in the absence of the other variable domains. Single domain antibodies and fragments thereof are described in Arabi Ghahronoudi et al, FEBS Letters,1998,414: 521-245 and Muydermans et al, Trends in biochem. Sci.,2001,26:230-245, each of which is incorporated herein by reference in its entirety.

A "multispecific ABP" is an ABP that comprises two or more different antigen-binding domains that collectively specifically bind two or more different epitopes. The two or more different epitopes can be epitopes on the same antigen (e.g., a single GITR molecule expressed by a cell) or on different antigens (e.g., different GITR molecules expressed by the same cell). In some aspects, the multispecific ABP binds two different epitopes (i.e., "bispecific ABP"). In some aspects, the multispecific ABP binds three different epitopes (i.e., "trispecific ABP"). In some aspects, the multispecific ABP binds four different epitopes (i.e., "tetraspecific ABP"). In some aspects, the multispecific ABP binds five different epitopes (i.e., "pentaspecific ABP"). In some aspects, the multispecific ABP binds 6,7, 8, or more different epitopes. The various binding specificities can be present at any suitable valency. Examples of multispecific ABPs are provided elsewhere in the invention.

A "monospecific ABP" is an ABP that comprises a binding site that specifically binds to a single epitope. An example of a monospecific ABP is a naturally occurring IgG molecule, whereas a bivalent one recognizes the same epitope at each antigen binding domain. The binding specificity can be present at any suitable valency.

The term "monoclonal antibody" refers to an antibody from a substantially homogeneous population of antibodies. In addition to variants that can be normally produced during the production of a monoclonal antibody, a population of substantially homogeneous antibodies comprises antibodies that are substantially similar and bind the same epitope. These variants are generally present in only small amounts. Monoclonal antibodies are typically obtained using a method that includes selecting a single antibody from a plurality of antibodies. For example, the selection procedure may be to select a pool of unique clones from a variety of clones, such as hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further altered, for example, to improve affinity for the target ("affinity maturation"), to humanize the antibody, to improve its production in cell culture, and/or to reduce its immunogenicity in the subject.

The term "chimeric antibody" refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species and the remainder of the heavy and/or light chain is derived from a different source or species.

"humanized" forms of non-human antibodies are chimeric antibodies that contain few sequences derived from the non-human antibody. Humanized antibodies are generally human antibodies (recipient antibodies) in which residues from one or more CDRs are replaced with residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having the desired specificity, affinity, or biological effect. In some cases selected framework region residues of the acceptor antibody are replaced with corresponding framework region residues from the donor antibody. Humanized antibodies may also comprise residues not found in the recipient antibody or in the donor antibody. These modifications can be made to further improve antibody function. For additional details, see Jones et al, Nature,1986,321: 522-525; riechmann et al, Nature,1988,332: 323-329; and Presta, curr, op, struct, biol.,1992,2: 593-.

A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source (e.g., obtained from a human source or redesigned) using a human antibody repertoire or human antibody coding sequence. Human antibodies specifically exclude humanized antibodies.

An "isolated ABP" or "isolated nucleic acid" is an ABP or nucleic acid that has been isolated and/or recovered from a component of its natural environment. Components of the natural environment may include enzymes, hormones, and other proteinaceous or nonproteinaceous materials. In some embodiments, the isolated ABP is purified to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, e.g., by using a rotary cup sequencer. In some embodiments, gel electrophoresis (e.g., SDS-PAGE) is used under reducing or non-reducing conditionsBlue or silver staining was performed to detect the isolated ABP and purify to homogeneity. An isolated ABP comprises an ABP in situ within a recombinant cell, since at least one component of the natural environment of the ABP is not present. In some aspects, the isolated ABP or isolated nucleic acid is prepared by at least one purification step. In some embodiments, the isolated ABP or isolated nucleic acid is purified to at least 80, 85, 90, 95, or 99 weight%. In some embodiments, the isolated ABP or isolated nucleic acid is purified to at least 80%, 85%, 90%, 95%, or 99% by volume. In some embodiments, an isolated ABP or isolated nucleic acid is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% ABP or nucleic acid by weight. In some embodiments, an isolated ABP or isolated nucleic acid is provided as a solution comprising at least 85 vol%, 90 vol%, 95 vol%, 98 vol%, 99 vol% to 100 vol% ABP or nucleic acid.

"affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., ABP) and its binding partner (e.g., antigen or epitope). As used herein, "affinity" refers to the affinity between members of a reflective binding pair (e.g., ABP and antigen or epitope) unless otherwise indicated1:1 interaction of the inherent binding affinity. The affinity of a molecule X for its partner Y can generally be determined by the dissociation equilibrium constant (K)_D) And (4) showing. Kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein. Surface Plasmon Resonance (SPR) techniques may be used for example (e.g.,) Or bio-layer interferometry (e.g.,) To determine affinity.

With respect to binding of ABPs to a target molecule, the terms "binding," "specific binding," "with.. specific binding," "specific for," "selectively binding," and "selectively against" a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably distinct from non-specific or non-selective interactions (e.g., with non-target molecules). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to non-target molecules. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In that case, specific binding is indicated if binding of ABP to the target molecule is competitively inhibited by the control molecule. In some aspects, the affinity of GITR ABP for non-target molecules is about 50% lower than the affinity for GITR. In some aspects, GITRABP has about 40% lower affinity for non-target molecules than for GITR. In some aspects, the affinity of GITR ABP for non-target molecules is about 30% lower than the affinity for GITR. In some aspects, the affinity of GITR ABP for non-target molecules is about 20% lower than the affinity for GITR. In some aspects, the affinity of GITR ABP for non-target molecules is about 10% lower than the affinity for GITR. In some aspects, the affinity of GITR ABP for non-target molecules is about 1% lower than the affinity for GITR. In some aspects, the affinity of GITR ABP for non-target molecules is about 0.1% lower than the affinity for GITR.

As used herein, the term "k_d"(seconds)^-1) Refers to the off-rate constant for a particular ABP-antigen interaction. This value is also called k_offThe value is obtained.

As used herein, the term "k_a”(M^-1X second^-1) Refers to the binding rate constant for a particular ABP-antigen interaction. This value is also called k_onThe value is obtained.

As used herein, the term "K_D"(M) refers to the dissociation equilibrium constant for a particular ABP-antigen interaction. K_D＝k_d/k_a。

As used herein, the term "K_A”(M^-1) Refers to the binding equilibrium constant for a particular ABP-antigen interaction. K_A＝k_a/k_d。

An "affinity matured" ABP is an ABP that has one or more alterations (e.g., in one or more CDRs or FRs) that result in improved affinity of the ABP for its antigen as compared to a parent ABP that does not possess the alterations. In one embodiment, the affinity matured ABP has nanomolar or picomolar affinity for the target antigen. Affinity matured ABPs can be made using various methods known in the art. For example, Marks et al (Bio/Technology,1992,10:779-783, which is incorporated herein by reference in its entirety) are incorporated by V_HAnd V_LDomain shuffling to describe affinity maturation. By, for example, Barbas et al (Proc. Nat. Acad. Sci. U.S.A.,1994,91: 3809-; schier et al, Gene,1995,169: 147-; yelton et al, J.Immunol.,1995,155: 1994-2004; jackson et al, J.Immunol.,1995,154: 3310-33199; and Hawkins et al, J.mol.biol.,1992,226:889-896, each of which is incorporated herein by reference in its entirety.

An "immunoconjugate" is an ABP that binds to one or more heterologous molecules.

"Effector function" refers to those biological activities mediated by the Fc region of an antibody, which activities may vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding to activate Complement Dependent Cytotoxicity (CDC), Fc receptor binding to activate Antibody Dependent Cellular Cytotoxicity (ADCC), and Antibody Dependent Cellular Phagocytosis (ADCP).

The term "competes with" or "cross-competes with," when used herein in the context of two or more ABPs, refers to two or more ABPs competing for binding to an antigen (e.g., GITR). In one exemplary assay, GITR is coated on a surface and contacted with a first GITR ABP, followed by addition of a second GITR ABP. In another exemplary assay, a first GITR ABP is coated on a surface and contacted with GITR, and then a second GITR ABP is added. ABPs compete if the presence of the first GITR ABP reduces binding of the second GITR ABP in either assay. The term "and.. competes" also includes combinations of ABPs in which one ABP reduces binding of another ABP but in which no competition is observed when the ABPs are added in reverse order. However, in some embodiments, the first ABP and the second ABP inhibit binding to each other regardless of the order in which they are added. In some embodiments, one ABP reduces binding of another ABP to its antigen by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

The term "epitope" means the portion of an antigen that specifically binds to ABP. Epitopes often consist of surface-available amino acid residues and/or sugar side chains and may have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational epitopes are distinguished from non-conformational epitopes in that binding to the former can be abolished in the presence of denaturing solvents, but the latter are not. An epitope may comprise amino acid residues directly involved in binding and other amino acid residues not directly involved in binding. Epitopes bound to ABP can be determined using known techniques for epitope determination, such as assays for binding of ABP to GITR variants with different point mutations or to chimeric GITR variants.

The percent "identity" between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be accomplished in a variety of ways within those of skill in the art, for example, using publicly available computer software. Such computer software is, for example, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA or MUSCLE software. One skilled in the art can determine the parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared.

"conservative substitution" or "conservative amino acid substitution" refers to the replacement of an amino acid with a chemically or functionally similar amino acid. Conservative substitution tables providing similar amino acids are well known in the art. For example, in some embodiments, the groups of amino acids provided in tables 2-4 are considered conservative substitutions for one another.

Table 2. in certain embodiments, selected groups of amino acids that are considered conservative substitutions for one another.

Acidic residue	D and E
		Basic residue	K. R and H
Hydrophilic uncharged residues	S, T, N and Q
		Aliphatic uncharged residues	G. A, V, L and I
Non-polar uncharged residues	C. M and P
		Aromatic radicals	F. Y and W

Table 3. in certain embodiments, additional selected groups of amino acids that are considered conservative substitutions for one another.

Table 4. in certain embodiments, other selected groups of amino acids that are considered conservative substitutions for one another.

Group A	A and G
		Group B	D and E
Group C	N and Q
		Group D	R, K and H
Group E	I、L、M、V
		Group F	F. Y and W
Group G	S and T
		Group H	C and M

Additional conservative substitutions may be found, for example, in Creighton, Proteins: Structures and Molecular Properties 2 nd edition (1993) W.H.Freeman & Co., New York, N.Y.. ABPs generated by conservative substitutions of one or more amino acid residues in a parent ABP are referred to as "conservatively modified variants".

The term "amino acid" refers to the twenty common naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid molecule to which it is linked. The term includes vectors which are self-replicating nucleic acid structures as well as vectors which are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked. These vectors are referred to herein as "expression vectors".

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" (or "transformed cells") and "transfectants" (or "stained cells"), each of which includes primary transformed or transfected cells and progeny derived therefrom. These progeny may not have exactly the same nucleic acid content as the parent cell and may contain mutations.

The term "treating" (and variants thereof, such as "treating" or "treatment") refers to a clinical intervention that attempts to alter the natural time course of a disease or disorder in a subject in need thereof.

As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of an ABP or pharmaceutical composition provided herein that is effective to treat a disease or disorder when administered to a subject.

As used herein, the term "subject" means a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject has a disease or disorder that is treatable with ABP provided herein. In some aspects, the disease or disorder is cancer.

The term "package insert" is used to refer to instructions containing information about the indication, the regimen, the dosage, the administration, the combination therapy, the contraindications and/or warnings concerning the use of such therapeutic or diagnostic products, which are routinely included in commercial packages for such therapeutic or diagnostic products (e.g., kits).

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or interferes with cellular function and/or causes cell death or destruction.

"chemotherapeutic agent" refers to a compound suitable for use in the treatment of cancer. Chemotherapeutic agents include "anti-hormonal agents" or "endocrine therapeutic agents" to modulate, reduce, block or inhibit the effects of hormones that can promote cancer growth.

The term "cytostatic agent" refers to a compound or composition that retards cell growth in vitro or in vivo. In some embodiments, the cytostatic agent is an agent that reduces the percentage of cells in S phase. In some embodiments, the cytostatic agent reduces the percentage of cells in S phase by at least about 20%, at least about 40%, at least about 60%, or at least about 80%.

The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive, as referred to herein. The terms "cell proliferative disorder" and "proliferative disorder" refer to a disorder associated with a degree of abnormal cell proliferation. In some embodiments, the cell proliferative disorder is cancer.

The term "pharmaceutical composition" refers to a formulation which is in a form that allows the biological activity of the active ingredient contained therein to be effective in the subject being treated, and which does not contain additional components that have unacceptable toxicity to the subject.

The terms "modulate and modulation" refer to the reduction or inhibition, or alternatively the activation or increase, of the recited variable.

The terms "increase" and "activation" refer to an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more of the recited variable (such as GITR signaling activity).

The terms "reduce" and "inhibit" refer to a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more of a recited variable, such as (a) modulating the number of T cells and/or (b) a symptom of a disease or disorder, such as the presence or size of a cancer metastasis or the size of a primary tumor.

The term "agonism" refers to the activation of receptor signaling to induce a biological response associated with activation of the receptor. An "agonist" is an entity that binds to and agonizes a receptor, such as the ABP provided herein.

The term "antagonize" refers to the inhibition of receptor signaling to inhibit a biological response associated with the activation of the receptor. An "antagonist" is an entity that binds to and antagonizes a receptor, such as ABP.

The term "multimerization" refers to the act of forming a "multimer" of entities by assembling the entities to form a super-entity structure that is bound together by non-covalent or covalent interactions. Multimers include "homomultimers" of assemblies formed from multiple units of the same entity, or "heteromultimers" of assemblies comprising at least one unit of a first entity and at least one unit of a second entity. When used herein to refer to GITR, the term multimerization refers to the assembly of multiple GITR molecules expressed on the surface of a cell, e.g., induced by binding to ABP provided herein or by GITRL. These multimerizations are associated with activation of GITR signaling. See Nocentini et al, br.j.pharmacol.,2012,165:2089-2099, which is incorporated herein by reference in its entirety.

The term "effector T cells" includes T helper (i.e., CD4+) cells and cytotoxic (i.e., CD8+) T cells. CD4+ effector T cells contribute to several immune processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. CD8+ effector T cells destroy virus-infected cells and tumor cells. For additional information on effector T cells, see Seder and Ahmed, Nature Immunol.,2003,4:835-842, which is incorporated herein by reference in its entirety.

The term "regulatory T cells" includes cells that modulate immune tolerance, for example, by suppressing effector T cells. In some aspects, the regulatory T cells have the phenotype CD4+ CD25+ Foxp3 +. In some aspects, the regulatory T cells have the phenotype CD8+ CD25 +. For additional information on regulatory T cells expressing GITR, see Nocentini et al, Br.J. Pharmacol.,2012,165:2089-2099, incorporated herein by reference in its entirety.

The term "dendritic cell" refers to a professional antigen presenting cell that is capable of activating naive T cells and stimulating the growth and differentiation of B cells.

GITR antigen binding proteins

GITR binding and target cells

Provided herein are ABPs that specifically bind to GITR. In some aspects, GITR is hGITR (SEQ ID NO: 1). In some aspects, GITR is hGITR-T43R (SEQ ID NO: 2). In some aspects, GITR is cGITR (SEQ ID NO: 3). In some embodiments, the GITR is mGITR (SEQ ID NO: 4).

In some embodiments, ABPs provided herein specifically bind to hGITR (SEQ ID NO:1), hGITR-T43R (SEQ ID NO:2), cGITR (SEQ ID NO:3), and mGITR (SEQ ID NO: 4). In some embodiments, ABPs provided herein specifically bind to hGITR (SEQ ID NO:1), hGITR-T43R (SEQ ID NO:2), and cGITR (SEQ ID NO: 3). In some embodiments, ABPs provided herein specifically bind to hGITR (SEQ ID NO:1) and hGITR-T43R (SEQ ID NO: 2). In some embodiments, the ABPs provided herein do not bind mGITR (SEQ ID NO: 4).

In some embodiments, the ABPs provided herein specifically bind to the extracellular domain of GITR.

GITR may be expressed on the surface of any suitable target cell. In some embodiments, the target cell is an effector T cell. In some embodiments, the target cell is a regulatory T cell. In some embodiments, the target cell is a Natural Killer (NK) cell. In some embodiments, the target cell is a natural killer t (nkt) cell. In some embodiments, the target cell is a dendritic cell. In some aspects, the dendritic cell is a plasmacytoid dendritic cell. In some embodiments, the target cell is a B cell. In some aspects, the B cell is a plasma cell. See Nocentini et al, br.j.pharmacol.,2012,165:2089-2099, which is incorporated herein by reference in its entirety.

In some embodiments, the ABPs provided herein specifically bind to GITR monomers.

In some embodiments, the ABPs provided herein specifically bind to GITR multimers. In some aspects, the multimer comprises two GITR molecules. In some aspects, the multimer comprises three GITR molecules. In some aspects, the multimer comprises four GITR molecules. In some aspects, the multimer comprises five GITR molecules. In some aspects, the multimer comprises six GITR molecules. In some aspects, the multimer comprises greater than six GITR molecules.

In some embodiments, the ABPs provided herein comprise, consist of, or consist essentially of an immunoglobulin molecule. In some aspects, the immunoglobulin molecule comprises, consists of, or consists essentially of an antibody.

In some embodiments, the ABPs provided herein comprise a light chain. In some aspects, the light chain is a kappa light chain. In some aspects, the light chain is a lambda light chain.

In some embodiments, an ABP provided herein comprises a heavy chain. In some aspects, the heavy chain is IgA. In some aspects, the heavy chain is IgD. In some aspects, the heavy chain is IgE. In some aspects, the heavy chain is IgG. In some aspects, the heavy chain is IgM. In some aspects, the heavy chain is IgG 1. In some aspects, the heavy chain is IgG 2. In some aspects, the heavy chain is IgG 3. In some aspects, the heavy chain is IgG 4. In some aspects, the heavy chain is IgA 1. In some aspects, the heavy chain is IgA 2.

In some embodiments, the ABPs provided herein comprise, consist of, or consist essentially of an antibody fragment. In some aspects, the antibody fragment is an Fv fragment. In some aspects, the antibody fragment is a Fab fragment. In some aspects, the antibody fragment is F (ab')₂And (3) fragment. In some aspects, the antibody fragment is a Fab' fragment. In some aspects, the antibody fragment is a scfv (sfv) fragment. In some aspects, the antibody fragment is a scFv-Fc fragment. In some aspects, the antibody fragment is a fragment of a single domain antibody.

In some embodiments, the ABPs provided herein are monoclonal antibodies. In some embodiments, the ABPs provided herein are polyclonal antibodies.

In some embodiments, the ABPs provided herein comprise, consist of, or consist essentially of a chimeric antibody. In some embodiments, the ABPs provided herein comprise, consist of, or consist essentially of a humanized antibody. In some embodiments, the ABPs provided herein comprise, consist of, or consist essentially of a human antibody.

In some embodiments, the ABPs provided herein comprise, consist of, or consist essentially of affinity matured ABPs. In some aspects, the ABP is an affinity matured ABP derived from an ABP provided herein.

In some embodiments, the ABPs provided herein comprise, consist of, or consist essentially of an alternative backbone. Any suitable alternative backbone may be used. In some aspectsIn a further aspect, an ABP provided herein comprises, consists of, or consists essentially of an alternative scaffold selected from the group consisting of: adnectin^TM、iMab、EETI-II/AGRP, Kunitz domain, thioredoxin peptide aptamer,DARPin, affinin, tetranectin, Fynomer and high affinity multimers.

In some embodiments, ABPs provided herein inhibit binding of GITR to GITRL. In some aspects, the ABP inhibits binding of GITR to GITRL by at least about 50%. In some aspects, the ABP inhibits binding of GITR to GITRL by at least about 75%. In some aspects, the ABP inhibits binding of GITR to GITRL by at least about 90%. In some aspects, the ABP inhibits binding of GITR to GITRL by at least about 95%.

1.2. Monospecific and multispecific GITR antigen binding proteins

In some embodiments, the ABPs provided herein are monospecific ABPs. In some aspects, the monospecific ABP binds to the same epitope on two or more different GITR molecules. In some aspects, the monospecific ABP binds to the same epitope on both GITR molecules. In some aspects, the monospecific ABP binds to the same epitope on three GITR molecules. In some aspects, the monospecific ABP binds to the same epitope on the four GITR molecules. In some aspects, the monospecific ABP binds to the same epitope on five GITR molecules. In some aspects, the monospecific ABP binds to the same epitope on six GITR molecules. In some aspects, the monospecific ABP binds to the same epitope on more than six GITR molecules.

In some embodiments, the monospecific ABPs provided herein are multivalent. As used herein, the term "multivalent" refers to an antibody having, for example, more than two binding regions (i.e., comprising V)_HAnd V_LZone). In thatIn some aspects, the monospecific ABP is bivalent. In some aspects, the monospecific ABP is trivalent. In some aspects, the monospecific ABP is tetravalent. In some aspects, the monospecific ABP is pentavalent. In some aspects, the monospecific ABP is hexavalent. In some aspects, the monospecific ABP is heptavalent. In some aspects, the monospecific ABP is eight valent.

In some embodiments, the monospecific multivalent ABP disclosed herein is tetravalent.

In some embodiments, the ABPs provided herein are multispecific ABPs. In some aspects, the multispecific ABP binds to two or more epitopes on two or more different GITR molecules. In some aspects, the multispecific ABP binds to two or more epitopes on two GITR molecules. In some aspects, the multispecific ABP binds to two or more epitopes on three GITR molecules. In some aspects, the multispecific ABP binds to two or more epitopes on four GITR molecules. In some aspects, the multispecific ABP binds to two or more epitopes on five GITR molecules. In some aspects, the multispecific ABP binds to two or more epitopes on six GITR molecules. In some aspects, the multispecific ABP binds to two or more epitopes on seven GITR molecules. In some aspects, the multispecific ABP binds to two or more epitopes on eight GITR molecules. In some aspects, the multispecific ABP binds to two or more epitopes on nine GITR molecules. In some aspects, the multispecific ABP binds to two or more epitopes on ten GITR molecules. In some aspects, the multispecific ABP binds to two or more epitopes on eleven GITR molecules. In some aspects, the multispecific ABP binds to two or more epitopes on twelve GITR molecules. In some aspects, the multispecific ABP binds to two or more epitopes on more than twelve GITR molecules.

The multispecific ABPs provided herein can bind to any suitable number of epitopes on GITR. In some aspects, the multispecific ABP binds to two epitopes on GITR. In some aspects, the multispecific ABP binds to three epitopes on GITR. In some aspects, the multispecific ABP binds four epitopes on GITR. In some aspects, the multispecific ABP binds to five epitopes on GITR. In some aspects, the multispecific ABP binds six epitopes on GITR. In some aspects, the multispecific ABP binds seven epitopes on GITR. In some aspects, the multispecific ABP binds eight epitopes on GITR. In some aspects, the multispecific ABP binds nine epitopes on GITR. In some aspects, the multispecific ABP binds ten epitopes on GITR. In some aspects, the multispecific ABP binds eleven epitopes on GITR. In some aspects, the multispecific ABP binds twelve epitopes on GITR. In some aspects, the multispecific ABP binds more than twelve epitopes on GITR.

In some aspects, the multispecific ABPs provided herein bind to at least two different epitopes on at least two different GITR molecules. In some aspects, the multispecific ABPs provided herein bind to at least three different epitopes on at least three different GITR molecules. In some aspects, the multispecific ABPs provided herein bind to at least four different epitopes on at least four different GITR molecules. In some aspects, the multispecific ABPs provided herein bind to at least five different epitopes on at least five different GITR molecules.

In some embodiments, a multispecific ABP provided herein comprises a first antigen-binding domain that specifically binds a first epitope on GITR and a second antigen-binding domain that specifically binds a second epitope on GITR, wherein the first epitope and the second epitope are different. In some aspects, the multispecific ABP further comprises one or more additional antigen-binding domains that specifically bind to one or more additional epitopes on GITR, wherein each of such additional epitopes is different from the epitope bound by the first antigen-binding domain, the second antigen-binding domain, or any other antigen-binding domain of the ABP.

In some embodiments, the multispecific ABPs provided herein bind to an epitope on a GITR molecule and an epitope on another molecule that is not GITR. Any suitable non-GITR molecule can bind to an ABP provided herein. In some aspects, the non-GITR molecule is another member of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF). In some aspects, the other member of TNFRSF is selected from the group consisting of: CD27, CD40, EDA2R, EDAR, FAS, LTBR, NGFR, RELT, TNFRSF1A, TNFRSF1B, TNFRSF4, TNFRSF6B, TNFRSF8, TNFRSF9, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, TFNRSF11A, TNFRSF11B, TNFRSF12A, TNFRSF13B, TNFRSF13C, TNFRSF14, TNFRSF17, TNFRSF18, TNFRSF19, TNFRSF21 and TNFRSF 25.

Many multispecific ABP constructs are known in the art, and the ABPs provided herein may be provided in the form of any suitable multispecific suitable construct.

In some embodiments, the multispecific ABP comprises an immunoglobulin comprising at least two different heavy chain variable regions (i.e., a "common light chain antibody") that are each paired with a common light chain variable region. The common light chain variable region forms a distinct antigen binding domain with each of the two distinct heavy chain variable regions. See Merchant et al, NatureBiotechnol.,1998,16: 677-.

In some embodiments, the multivalent ABPs disclosed herein comprise immunoglobulins comprising an antibody or fragment thereof linked to one or more of the N-terminus or C-terminus of the heavy or light chains of the immunoglobulins. See, for example, U.S. patent No. 8,722,859 and Coloma and Morrison, Nature biotechnol, 1997,15: 159-. In some aspects, these ABPs comprise tetravalent bispecific antibodies. In some aspects, the ABPs comprise Tetravalent Monospecific (TM) antibodies.

In some embodiments, the multivalent ABP comprises a hybrid immunoglobulin comprising at least two different heavy chain variable regions and at least two different light chain variable regions. See Milstein and Cuello, Nature,1983,305: 537-540; and Staerz and Bevan, Proc. Natl. Acad. Sci. USA,1986,83: 1453-; each of which is incorporated herein by reference in its entirety.

In some embodiments, the multivalent ABP comprises immunoglobulin chains with alterations to reduce the formation of by-products that are not multispecific. In some aspects, the ABP comprises one or more "pestle-hole" modifications as described in U.S. patent No. 5,731,168, which is incorporated herein by reference in its entirety.

In some embodiments, the multivalent ABP comprises immunoglobulin chains with one or more electrostatic modifications to facilitate assembly of the Fc heteromultimer. See WO 2009/089004, which is incorporated herein by reference in its entirety.

In some embodiments, the multivalent ABP comprises a bispecific single chain molecule. See Traunecker et al, EMBOJ, 1991,10: 3655-; and Gruber et al, J.Immunol.,1994,152: 5368-5374; each of which is incorporated herein by reference in its entirety.

In some embodiments, the multivalent ABP comprises a heavy chain variable domain and a light chain variable domain connected by a polypeptide linker, or a single domain antibody V_HAn H domain, wherein the length of the linker is selected to facilitate assembly of a multivalent ABP with a desired multispecific. For example, monospecific scfvs are typically formed when heavy chain V residues prevent pairing of the heavy and light chain variable domains on the same polypeptide chain, while allowing pairing of the heavy and light chain variable domains from one chain with complementary domains on the other chain. Thus, the resulting ABP is multispecific, wherein the specificity of each binding site is provided by more than one polypeptide chain. Polypeptide chains comprising heavy and light chain variable domains connected by a linker between 3 and 12 amino acid residues form mainly dimers (called bifunctional antibodies). In the case of linkers between 0 and 2 amino acid residues, trimers (called trifunctional antibodies) and tetramers (called tetrafunctional antibodies) are advantageous. However, in addition to linker length, the exact type of oligomerization appears to depend on the amino acid residue composition and availability in each polypeptide chainOrder of change field (e.g. V)_H-linker-V_LAnd V_L-linker-V_H). One skilled in the art can select the appropriate linker length based on the desired multispecific properties.

In some embodiments, the monospecific or multispecific ABP comprises a bifunctional antibody. See Hollinger et al, Proc. Natl.Acad. Sci. USA,1993,90: 6444-. In some embodiments, the monospecific or multispecific ABP comprises a trifunctional antibody. See Todorovska et al, j.immunol.methods,2001,248:47-66, which is incorporated herein by reference in its entirety. In some embodiments, the monospecific or multispecific ABP comprises a tetrafunctional antibody. See the above-mentioned documents, which are incorporated herein by reference in their entirety.

In some embodiments, the multispecific ABP comprises a trispecific F (ab')3 derivative. See Tutt et al j.immunol.,1991,147:60-69, which is incorporated herein by reference in its entirety.

In some embodiments, the monospecific or multispecific ABP comprises a cross-linked antibody. See U.S. Pat. nos. 4,676,980; brennan et al, Science,1985,229: 81-83; staerz, et al Nature,1985,314: 628-631; and EP 0453082; each of which is incorporated herein by reference in its entirety.

In some embodiments, the monospecific or multispecific ABP comprises antigen binding domains assembled by leucine zippers. See Kostelny et al, j.immunol.,1992,148:1547-1553, which is incorporated herein by reference in its entirety.

In some embodiments, the monospecific or multispecific ABP comprises complementary protein domains. In some aspects, the complementary protein domain comprises an Anchoring Domain (AD) and a Dimerization and Docking Domain (DDD). In some embodiments, AD and DDD bind to each other and thereby enable assembly of multispecific ABP structures via a "dock and lock" (DNL) approach. A number of specific ABPs can be assembled, including bispecific ABPs, trispecific ABPs, tetraspecific ABPs, pentaspecific ABPs, and hexaspecific ABPs. Multispecific ABPs comprising complementary protein domains are described, for example, in U.S. patent nos. 7,521,056, 7,550,143, 7,534,866, and 7,527,787, each of which is incorporated herein by reference in its entirety.

In some embodiments, the monospecific or multispecific ABP comprises a hybrid of an antibody molecule and a non-antibody molecule specific for GITR or another target. For examples of these ABPs, see WO 93/08829, which is incorporated herein by reference in its entirety. In some aspects, the non-antibody molecule is GITRL.

In some embodiments, the monospecific or multispecific ABP comprises a bifunctional fab (daf) antibody as described in U.S. patent publication No. 2008/0069820, which is incorporated herein by reference in its entirety.

In some embodiments, the multispecific ABP comprises an antibody formed by reducing two parent molecules followed by mixing and reoxidizing the two parent molecules to assemble a hybrid structure. See Carlring et al, PLoS One,2011,6: e22533, which is incorporated herein by reference in its entirety.

In some embodiments, the monospecific or multispecific ABP comprises a DVD-Ig^TM。DVD-Ig^TMAre double variable domain immunoglobulins that bind to two or more antigens. DVD-Igs^TMDescribed in U.S. patent No. 7,612,181, which is incorporated herein by reference in its entirety.

In some embodiments, the monospecific or multispecific ABP comprises a DART^TM。DARTs^TMDescribed in Moore et al, Blood,2011,117:454-451, which is incorporated herein by reference in its entirety.

In some embodiments, the multispecific ABP comprisesDescribed in Labrijn et al, Proc.Natl.Acad.Sci.USA,2013,110: 5145-; graner et al, mAbs,2013,5: 962-; and Labrijn et al, Nature Protocols,2014,9: 2450-; each of which is incorporated herein by reference in its entirety.

In some embodiments, a monospecific or multispecific ABP disclosed herein comprises an antibody fragment linked to another antibody or fragment. The linkage may be covalent or non-covalent. When the linkage is covalent, it may be in the form of a fusion protein, or via a chemical linker. Illustrative examples of multispecific ABPs comprising antibody fragments linked to other antibodies include tetravalent bispecific antibodies in which the scFv is conjugated to a C from an IgG_H3C-terminal fusion of (1). See Coloma and Morrison, NatureBiotechnol, 1997,15: 159-. Other examples include antibodies in which the Fab molecule is linked to a constant region of an immunoglobulin. See Miler et al, J.Immunol.,2003,170:4854-4861, which is incorporated herein by reference in its entirety. Any suitable fragment may be used, including any of the fragments described herein or known in the art.

In some embodiments, the monospecific or multispecific ABP comprises a CovX-body. The CovX-body is described, for example, in Doppallapoudi et al, Proc.Natl.Acad.Sci.USA,2010,107: 22611-.

In some embodiments, the monospecific or multispecific ABP comprises an Fcab antibody, wherein one or more antigen binding domains are introduced into the Fc region. Fcab antibodies are described in Wozniak-Knopp et al, ProteinEng.Des.Sel.,2010,23:289-297, which is incorporated herein by reference in its entirety.

In some embodiments, the monospecific or multispecific ABP comprisesAn antibody.Antibodies are described in Kipriyanov et al, J.mol.biol.,1999,293:41-56 and Zhukovsky et al, Blood,2013,122:5116, each of which is incorporated herein by reference in its entirety.

In some embodiments, the multispecific ABP comprises a tandem Fab. Tandem fabs are described in WO 2015/103072, which is incorporated herein by reference in its entirety.

In some embodiments, the multispecific ABP comprises a Zybody^TM。Zybodies^TMDescribed in LaFleur et al, mAbs,2013,5: 208-.

1.3. Antigen binding protein multimerizing GITR

In some embodiments, ABPs provided herein multimerize GITR expressed on the surface of a target cell. ABPs provided herein can be designed to multimerize any suitable number of GITR molecules based on their valency and specificity.

In some embodiments, an ABP provided herein multimerizes two GITR molecules. In some embodiments, ABPs provided herein multimerize three GITR molecules. In some embodiments, an ABP provided herein multimerizes four GITR molecules. In some embodiments, ABPs provided herein multimerize five GITR molecules. In some embodiments, an ABP provided herein multimerizes six GITR molecules. In some embodiments, ABPs provided herein multimerize seven GITR molecules. In some embodiments, an ABP provided herein multimerizes eight GITR molecules. In some embodiments, ABPs provided herein multimerize nine GITR molecules. In some embodiments, ABPs provided herein multimerize ten GITR molecules. In some embodiments, an ABP provided herein multimerizes eleven GITR molecules. In some embodiments, ABPs provided herein multimerize twelve GITR molecules.

In some embodiments, ABPs provided herein multimerize at least two GITR molecules. In some embodiments, an ABP provided herein multimerizes at least three GITR molecules. In some embodiments, an ABP provided herein multimerizes at least four GITR molecules. In some embodiments, an ABP provided herein multimerizes at least five GITR molecules. In some embodiments, an ABP provided herein multimerizes at least six GITR molecules. In some embodiments, ABPs provided herein multimerize at least seven GITR molecules. In some embodiments, an ABP provided herein multimerizes at least eight GITR molecules. In some embodiments, ABPs provided herein multimerize at least nine GITR molecules. In some embodiments, ABPs provided herein multimerize at least ten GITR molecules. In some embodiments, an ABP provided herein multimerizes at least eleven GITR molecules. In some embodiments, ABPs provided herein multimerize at least twelve GITR molecules.

In some embodiments, an ABP provided herein multimerizes between two and twelve GITR molecules. In some embodiments, ABPs provided herein multimerize between three and ten GITR molecules. In some embodiments, ABPs provided herein multimerize three to six GITR molecules. In some embodiments, ABPs provided herein multimerize three to five GITR molecules. In some embodiments, ABPs provided herein multimerize three to four GITR molecules.

GITR agonism

In some embodiments, ABPs provided herein agonize GITR upon binding. Such agonism may result from multimerization of GITR by ABP, as described elsewhere in the present invention. See fig. 1.

In some embodiments, the agonism of GITR by ABPs provided herein results in modulation of NF- κ B activity in the target cell. See U.S. patent No. 7,812,135, which is incorporated herein by reference in its entirety. In some aspects, agonism of GITR results in modulation of I κ B activity or stability in the target cell.

In some embodiments, the agonism of GITR by ABP provided herein results in activation of the MAPK pathway in a target cell. In some aspects, components of the MAPK pathway activated by ABP provided herein include one or more of p38, JNK, and ERK. See Nocentini et al, Proc.Natl.Acad.Sci.USA 1997,94: 6216-; ronchetti et al, Eur.J.Immunol.,2004,34: 613-622; and Escapza et al, J.Immunol.,2005,174: 7869-7874; each of which is incorporated herein by reference in its entirety.

In some embodiments, agonism of GITR by ABP provided herein causes the target cell to increase secretion of IL-2R α, IL-2, IL-8, and/or IFN γ see Ronchetti et al, Eur.J. Immunol, 2004,34:613-622, which is incorporated herein by reference in its entirety.

In some embodiments, agonism of GITR by ABPs provided herein increases proliferation, survival, and/or function of effector T cells. In some aspects, the effector T cell is a CD4+ effector T cell. In some aspects, the effector T cell is a CD8+ effector T cell.

In some embodiments, agonism of GITR by ABPs provided herein abrogates suppression of effector T cells by regulatory T cells. In some aspects, the regulatory T cells are CD4+ CD25+ Foxp3+ regulatory T cells. In some aspects, the regulatory T cells are CD8+ CD25+ regulatory T cells.

In some embodiments, the frequency of occurrence or distribution of regulatory T cells is altered by agonism of ABPs to GITR provided herein. In some aspects, the frequency of regulatory T cells is decreased. In some aspects, the frequency of regulatory T cells is reduced in a particular tissue. In some aspects, intratumoral accumulation of regulatory T cells is reduced, resulting in a more favorable ratio of effector T cells to regulatory T cells and enhanced CD8+ T cell activity. See Cohen et al, PLoS One,2010,5: e 10436.

In some embodiments, agonism of GITR by ABPs provided herein increases the activity of Natural Killer (NK) cells. In some embodiments, agonism of GITR by ABPs provided herein increases the activity of antigen presenting cells. In some embodiments, the activity of dendritic cells is increased by agonism of ABPs provided herein to GITR. In some embodiments, agonism of GITR by ABPs provided herein increases the activity of B cells.

In some embodiments, the immune response is enhanced by agonism of ABP to GITR provided herein. In some embodiments, the agonism of GITR by ABP provided herein results in the delay of tumor onset. In some embodiments, the size of the tumor is decreased by agonism of ABP on GITR provided herein. In some embodiments, agonism of GITR by ABPs provided herein causes a reduction in the number of cancer metastases.

In some embodiments, agonism of GITR by multivalent monospecific ABPs provided herein results in a higher maximum amount of agonism than bivalent monospecific antibodies. In some embodiments, the additional valency results in a greater additive effect on EC than the individual binding domains₅₀The effect of (a). In some embodiments, the tetravalent monospecific ABP provided herein has a higher maximum amount of agonism than a bivalent monospecific antibody.

In some embodiments, the multispecific ABPs provided herein are more potent GITR agonists than mixtures of corresponding monospecific ABPs. For example, if the multispecific ABPs provided herein comprise two different epitope specificities (e.g., a and B), then in some embodiments, the agonism of GITR by these multispecific ABPs is greater than the agonism of GITR by a mixture of two monospecific ABPs each comprising one of the two specificities (e.g., a or B). In some embodiments, the other specificities of multispecific ABPs provided herein result in a synergistic increase in potency (i.e., greater than an additive effect) when compared to a mixture of monospecific ABPs each having only one of the specificities of the multispecific ABP.

1.5. Affinity of antigen binding proteins for GITR

In some embodiments, the ABPs provided herein have affinity for GITR (e.g., by K)_DIndicated) is less than about 10^- ⁵M, less than about 10^-6M, less than about 10^-7M, less than about 10^-8M, less than about 10^-9M, less than about 10^-10M, less than about 10^-11M or less than about 10^-12And M. In some embodiments, the affinity of the ABP is about 10^-7M and 10^-12M is greater than or equal to the total weight of the composition. In some embodiments, the affinity of the ABP is about 10^-7M and 10^-11M is greater than or equal to the total weight of the composition. In some embodiments, the affinity of the ABP is about 10^-7M and 10^-10M is greater than or equal to the total weight of the composition. In some embodiments, the affinity of the ABP is about 10^-7M and 10^-9M is greater than or equal to the total weight of the composition. In some embodiments, the affinity of the ABP is about 10^-7M and 10^-8M is greater than or equal to the total weight of the composition. In some embodiments, the affinity of the ABP is about 10^-8M and 10^-12M is greater than or equal to the total weight of the composition. In some embodiments, the affinity of the ABP is about 10^-8M and 10^-11M is greater than or equal to the total weight of the composition. In some embodiments, the affinity of the ABP is about 10^-9M and 10^-11M is greater than or equal to the total weight of the composition. In some embodiments, the affinity of the ABP is about 10^-10M and 10^-11M is greater than or equal to the total weight of the composition.

In some embodiments, ABPs provided herein are at K of X_DSpecific binding to hGITR (SEQ ID NO:1) and K ≤ 10 ×_DBinds specifically to cGITR. In some embodiments, ABPs provided herein are at K of X_DK specifically binding to hGITR (SEQ ID NO:1) and at ≤ 5X_DBinds specifically to cGITR. In some embodiments, ABPs provided herein are at K of X_DSpecific binding to hGITR (SEQ ID NO:1) and K ≤ 2 ≤_DBinds specifically to cGITR. In some aspects, X is any K described in the present disclosure_D. In some aspects, X is 0.01nM, 0.1nM, 1nM, 10nM, 20nM, 50nM, or 100 nM.

In some embodiments, K_D、k_aAnd k_dMeasured using Surface Plasmon Resonance (SPR). In some aspects, SPR analysisBy usingAn apparatus. In some aspects, an antigen is immobilized on a carboxymethylated dextran biosensor chip (CM4 or CM5) and contacted with an ABP provided herein. Can be usedThe software and one-to-one Langmuir binding model calculate the association and dissociation rate constants. In some aspects, the analysis is performed at 25 ℃. In some aspects, the analysis is performed at 37 ℃.

In some embodiments, K is determined using biolayer interferometry (BLI)_D、k_aAnd k_d. Any suitable BLI method may be used. In some aspects, BLI analysis utilizesAn apparatus. In some aspects, an anti-human IgGFc capture (AHC) biosensor is used to capture ABP onto the surface of the sensor. Subsequently, binding of ABP to antigen was monitored by contacting the immobilized ABP with different concentrations of GITR. Next, the dissociation of antigen from ABP was measured in a buffer without GITR. Use ofThe kinetic module of the analysis software calculates the association and dissociation rate constants. In some aspects, the analysis is performed at 30 ℃.

In other embodiments K_DCan be determined by a radiolabeled antigen-binding assay, as described in Chen et al J.mol.biol.,1999,293:865-881, which is incorporated herein by reference in its entirety.

1.5.1. Glycosylation variants

In certain embodiments, the ABPs provided herein can be altered to increase, decrease, or eliminate the degree of ABP glycosylation. Glycosylation of polypeptides is typically "N-linked" or "O-linked".

"N-linked" glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of a carbohydrate moiety to an asparagine side chain. Thus, the presence of any of such tripeptide sequences in a polypeptide creates potential glycosylation sites.

"O-linked" glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition or deletion of N-linked glycosylation sites to or from ABPs provided herein can be achieved by altering the amino acid sequence such that one or more of the tripeptide sequences described above are generated or removed. The addition or deletion of an O-linked glycosylation site can be achieved by adding, deleting or replacing one or more serine or threonine residues in or to the sequence of the ABP as the case may be.

In some embodiments, the ABPs provided herein comprise a glycosylation motif that is different from the naturally occurring ABP. Any suitable naturally occurring glycosylation motif in the ABP provided herein can be modified. For example, the structural and glycosylation properties of immunoglobulins are known in the art and are summarized, for example, in Schroeder and Cavacini, j.allergyclin.immunol.,2010,125: S41-52, which are incorporated herein by reference in their entirety.

In some embodiments, the ABPs provided herein comprise an IgG1 Fc region with a modification to an oligosaccharide attached to asparagine 297(Asn 297). Naturally occurring IgG1 antibodies produced by mammalian cells generally comprise a C generally associated with an Fc region_H2A branched, bi-antenna oligosaccharide attached by an N-linkage of Asn 297 of the domain. See Wright et al, TIBTECH,1997,15:26-32, in its entiretyThe manner of reference is incorporated herein. Oligosaccharides attached to Asn 297 may include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure.

In some embodiments, the oligosaccharide attached to Asn 297 is modified to produce an ABP with altered ADCC. In some embodiments, the oligosaccharide is altered to improve ADCC. In some embodiments, the oligosaccharide is altered to reduce ADCC.

In some aspects, the ABPs provided herein comprise an IgG1 domain having reduced fucose content at position Asn 297, as compared to the naturally occurring IgG1 domain. These Fc domains are known to have improved ADCC. See, shiplds et al, j.biol.chem.,2002,277: 26733-. In some aspects, these ABPs do not comprise any fucose at Asn 297 position. The amount of fucose may be determined using any suitable method, for example as described in WO 2008/077546, which is incorporated herein by reference in its entirety.

In some embodiments, an ABP provided herein comprises a bi-bisected oligosaccharide, such as a bi-antennary oligosaccharide bisected by GlcNAc attached to the Fc region of the ABP. These ABP variants may have reduced fucosylation and/or improved ADCC function. Examples of such ABP variants are described, for example, in WO 2003/011878, U.S. patent No. 6,602,684, and U.S. patent publication No. 2005/0123546, each of which is incorporated herein by reference in its entirety.

Other illustrative glycosylation variants are described, for example, in: U.S. patent publication nos. 2003/0157108, 2004/0093621, 2003/0157108, 2003/0115614, 2002/0164328, 2004/0093621, 2004/0132140, 2004/0110704, 2004/0110282, 2004/0109865; international patent publication nos. 2000/61739, 2001/29246, 2003/085119, 2003/084570, 2005/035586 and 2005/035778; 2005/053742, 2002/031140; okazaki et al, J.mol.biol.,2004,336: 1239-1249; and Yamane-Ohnuki et al, Biotech.Bioeng, 2004,87: 614-622; each of which is incorporated herein by reference in its entirety.

In some embodiments, an ABP provided herein comprises an Fc region wherein a galactose residue in at least one oligosaccharide is linked to the Fc region. These ABP variants may have improved CDC function. Examples of such ABP variants are described, for example, in WO1997/30087, WO 1998/58964 and WO 1999/22764, each of which is incorporated herein by reference in its entirety.

Examples of cell lines capable of producing defucosylated ABPs include Lec13 CHO cells that are deficient in protein fucosylation (see Ripka et al, Arch. biochem. Biophys.,1986,249: 533-.

In some embodiments, the ABPs provided herein are non-glycosylated ABPs. The non-glycosylated ABP may be produced using any method known in the art or described herein. In some aspects, the non-glycosylated ABP is produced by modifying the ABP to remove all glycosylation sites. In some aspects, the glycosylation site is removed only from the Fc region of the ABP. In some aspects, the non-glycosylated ABP is produced by expressing ABP in an organism incapable of glycosylation (such as e.coli) or by expressing ABP in a cell-free reaction mixture.

In some embodiments, the ABPs provided herein have constant regions with reduced effector function compared to the original IgG1 antibody. In some embodiments, the constant region of the Fc region of ABPs provided herein has a lower affinity for Fc receptors than the original IgG1 constant region.

Fc region amino acid sequence variants

In certain embodiments, the ABPs provided herein comprise an Fc region having one or more amino acid substitutions, insertions, or deletions compared to a naturally occurring Fc region. In some aspects, these substitutions, insertions, or deletions result in ABPs with altered stability, glycosylation, or other properties. In some aspects, these substitutions, insertions, or deletions result in non-glycosylated ABP.

In some aspects, the Fc region of an ABP provided herein is modified to produce an ABP with altered affinity for an Fc receptor or a more immunologically inert ABP. In some embodiments, the ABP variants provided herein possess some (but not all) effector function. These ABPs may be useful, for example, when the half-life of the ABP is important in vivo, and when certain effector functions (e.g., supplemental activation and ADCC) are unnecessary or detrimental.

In some embodiments, the Fc region of an ABP provided herein is a human IgG4 Fc region comprising hinge stabilizing mutation S228P or L235E. In some embodiments the IgG4 Fc region comprises hinge stabilizing mutations S228P and L235E. See Aalberse et al, Immunology,2002,105:9-19, which is incorporated herein by reference in its entirety. In some embodiments, the IgG4 Fc region comprises one or more of the following mutations: E233P, F234V and L235A. See Armour et al, mol. immunol.,2003,40: 585-. In some embodiments, the IgG4 Fc region comprises a deletion at position G236.

In some embodiments, the Fc region of an ABP provided herein is a human IgG1 Fc region comprising one or more mutations that reduce Fc receptor binding. In some aspects, the one or more mutations are in a residue selected from: s228 (e.g., S228A), L234 (e.g., L234A), L235 (e.g., L235A), D265 (e.g., D265A), and N297 (e.g., N297A). In some aspects, the ABP comprises a PVA236 mutation. PVA236 means that the amino acid sequence ELLG at amino acid positions 233 to 236 of IgG1 or EFLG of IgG4 is substituted with PVA. See U.S. patent publication No. 2013/0065277, which is incorporated herein by reference in its entirety.

In some embodiments, the Fc region of ABPs provided herein is as described in Armour et al, eur.j.immunol.,1999,29: 2613-2624; modifications described in WO 1999/058572 and/or uk patent application No. 98099518, each of which is incorporated herein by reference in its entirety.

In some embodiments, the Fc region of an ABP provided herein is a human IgG2 Fc region comprising one or more of mutations a330S and P331S.

In some embodiments, the Fc region of an ABP provided herein has an amino acid substitution at one or more positions selected from 238, 265, 269, 270, 297, 327 and 329. See U.S. Pat. No. 6,737,056, which is incorporated herein by reference in its entirety. These Fc mutants include Fc mutations having substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including so-called "DANA" Fc mutations in which residues 265 and 297 are substituted with alanine. See U.S. patent No. 7,332,581, which is incorporated herein by reference in its entirety. In some embodiments, the ABP comprises an alanine at amino acid position 265. In some embodiments, the ABP comprises alanine at amino acid position 297.

In certain embodiments, the ABPs provided herein comprise an Fc region having one or more amino acid substitutions that improve ADCC, such as substitutions at one or more of positions 298, 333, and 334 of the Fc region. In some embodiments, the ABPs provided herein comprise an Fc region having one or more amino acid substitutions at positions 239, 332 and 330, as described in Lazar et al, proc.natl.acad.sci.usa,2006,103: 4005-.

In some embodiments, the ABPs provided herein comprise one or more alterations that improve or mitigate C1q binding and/or CDC. See U.S. Pat. nos. 6,194,551; WO 99/51642; and Idusogene et al, J.Immunol.,2000,164: 4178-; each of which is incorporated herein by reference in its entirety.

In some embodiments, the ABPs provided herein comprise one or more alterations to increase half-life. ABPs with increased half-life and improved binding to neonatal Fc receptor (FcRn) are described, for example, in Hinton et al, j.immunol.,2006,176: 346-356; and U.S. patent publication No. 2005/0014934, each of which is incorporated herein by reference in its entirety. These Fc variants include those having substitutions at one or more of the following residues in the Fc region: 238, 250, 256, 265, 272, 286, 303, 305, 307, 311, 312, 314, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428, and 434 of IgG.

In some embodiments, ABPs provided herein comprise one or more Fc region variants, such as those described in U.S. patent nos. 7,371,826, 5,648,260, and 5,624,821; duncan and Winter, Nature,1988,322: 738-740; and WO 94/29351, each of which is incorporated herein by reference in its entirety.

1.7. Cysteine engineered antigen binding protein variants

In certain embodiments, provided herein are cysteine engineered ABPs, also referred to as "thiomabs," in which one or more residues of the ABP are replaced with a cysteine residue. In particular embodiments, the substituted residue is present at a solvent accessible site of the ABP. By substituting these residues with cysteines, reactive thiol groups are introduced at solvent accessible sites of ABP and can be used to bind ABP to other moieties (such as drug moieties or linker-drug moieties), for example, to create immunoconjugates.

In certain embodiments, any one or more of the following residues may be substituted with cysteine: v205 of the light chain, a118 of the heavy chain Fc region, and S400 of the heavy chain Fc region. Cysteine engineered ABPs may be produced, for example, as described in U.S. patent No. 7,521,541, which is incorporated herein by reference in its entirety.

1.7.1. Immunoconjugates

1.7.1.1. Antigen binding protein-polymer conjugates

In some embodiments, the ABPs provided herein are derivatized by conjugation to a polymer. Any suitable polymer may be conjugated to the ABP.

In some embodiments, the polymer is a water soluble polymer. Illustrative examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homopolymer or random copolymers), poly (n-vinyl pyrrolidone) -co-polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. In some aspects, polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.

The polymer may have any molecular weight, and may be branched or unbranched. The number of polymers attached to each ABP can vary, and if more than one polymer is attached, it can be the same polymer or different polymers. In general, the number and/or type of polymers used for derivatization may be determined based on considerations including the ABP to be improved and the particular characteristics or functions of the ABP intended for use.

1.7.1.2. Antigen binding protein-drug conjugates

Exemplary therapeutic agents include cytokines, chemokines, and other agents that induce a desired T cell activity, such as GITRL, OX40L, 4-1BBL, TNF- α, IL-2, IL-15 fusion, CXCL9, CXCL10, IL-10 trap, IL-27 trap, and IL-35 trap cytokine traps and their uses are known in the art and described, for example, in ecolomides et al, naturemicine, 2003,9:47-52, which is incorporated herein by reference in its entirety.

Method for preparing GITR antigen binding protein

Preparation of GITR antigen

The GITR antigen used to isolate ABPs provided herein can be intact GITR or a fragment of GITR. The GITR antigen may be in the form of an isolated protein or a protein expressed on the surface of a cell.

In some embodiments, the GITR antigen is a non-naturally occurring variant of GITR, such as a GITR protein having an amino acid sequence or post-translational modification that does not occur in nature.

In some embodiments, the GITR antigen is truncated, e.g., by removal of intracellular or transmembrane sequences or signal sequences. In some embodiments, the GITR antigen is fused at its C-terminus to a human IgG1 Fc domain or a polyhistidine tag.

1.9. Method for preparing monoclonal antibody

Monoclonal antibodies can be obtained, for example, using hybridoma methods first described by Kohler et al, Nature,1975,256:495-497, which is incorporated herein by reference in its entirety, and/or using recombinant DNA methods (see U.S. Pat. No. 4,816,567, which is incorporated herein by reference in its entirety). Monoclonal antibodies can also be obtained, for example, using phage or yeast based libraries. See, for example, U.S. patent nos. 8,258,082 and 8,691,730, each of which is incorporated herein by reference in its entirety.

In the hybridoma method, a mouse or other appropriate host animal is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Next, the lymphocytes are fused with myeloma cells using a suitable fusing agent (such as polyethylene glycol) to form hybridoma cells. See Goding J.W., Monoclonal Antibodies: Principles and Practice 3 rd edition (1986) Academic Press, San Diego, Calif., which is incorporated herein by reference in its entirety.

The hybridoma cells are seeded and grown in a suitable culture medium containing one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

Useful myeloma cells are those that fuse efficiently, support stable high levels of antibody production by the selected antibody-producing cells, and are sensitive to culture medium conditions (such as the presence or absence of HAT medium). Of these, preferred myeloma Cell lines are murine myeloma Cell lines, such as those derived from MOP-21 and MC-11 mouse tumors (purchased from the Salk Institute Cell Distribution Center, San Diego, Calif.); and SP-2 or X63-Ag8-653 cells (obtained from American Type Culture Collection, Rockville, Md.). Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have also been described. See, e.g., Kozbor, j.immunol.,1984,133:3001, which is incorporated herein by reference in its entirety.

After identification of hybridoma cells producing antibodies of the desired specificity, affinity, and/or biological activity, selected clones can be subcloned using limiting dilution procedures and grown using standard methods. See Goding supra. Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo in animals as ascites tumors.

DNA encoding the monoclonal antibody can be readily isolated and sequenced using well-known procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of the monoclonal antibody). Thus, hybridoma cells can be used as a useful source of DNA encoding antibodies with desired properties. Once isolated, the DNA may be placed into an expression vector, which is then transfected into a host cell, such as a bacterium (e.g., escherichia coli), yeast (e.g., Saccharomyces (Saccharomyces) or Pichia (Pichia sp)), COS cell, Chinese Hamster Ovary (CHO) cell, or myeloma cell that does not otherwise produce antibodies, to produce monoclonal antibodies.

1.10. Method for producing chimeric antibody

Illustrative methods for making chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567 and Morrison et al, Proc. Natl. Acad. Sci. USA,1984,81: 6851-. In some embodiments, chimeric antibodies are prepared by using recombinant techniques to combine non-human variable regions (e.g., variable regions derived from mouse, rat, hamster, rabbit, or non-human primate (such as monkey)) with human constant regions.

1.11. Method for producing humanized antibody

Humanized antibodies can be generated by replacing most or all of the structural portion of a non-human monoclonal antibody with the corresponding human antibody sequence. Thus, hybrid molecules are generated in which only the antigen-specific variable regions or CDRs are composed of non-human sequences. Methods of obtaining humanized antibodies include those described, for example, in: winter and Milstein, Nature,1991,349: 293-; rader et al, Proc. Nat. Acad. Sci. U.S.A.,1998,95: 8910-; steinberger et al, J.biol.chem.,2000,275: 36073-36078; queen et al, Proc.Natl.Acad.Sci.U.S.A.,1989,86: 10029-10033; and U.S. Pat. nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370; each of which is incorporated herein by reference in its entirety.

1.12. Method for producing human antibodies

Human antibodies can be produced using various techniques known in the art, for example, by using transgenic animals (e.g., humanized mice). See, e.g., jakobvits et al, proc.natl.acad.sci.u.s.a.,1993,90: 2551; jakobovits et al, Nature,1993,362: 255-258; bruggermann et al, Yeast in Immuno, 1993,7: 33; and U.S. patent nos. 5,591,669, 5,589,369 and 5,545,807; each of which is incorporated herein by reference in its entirety. Human antibodies can also be derived from phage display libraries (see, e.g., Hoogenboom et al, J.mol.biol.,1991,227: 381-388; Marks et al, J.mol.biol.,1991,222: 581-597; and U.S. Pat. Nos. 5,565,332 and 5,573,905; each of which is incorporated herein by reference in its entirety). Human antibodies can also be produced by B cells activated in vitro (see, e.g., U.S. Pat. nos. 5,567,610 and 5,229,275, each of which is incorporated herein by reference in its entirety). Human antibodies can also be derived from yeast-based libraries (see, e.g., U.S. patent No. 8,691,730, which is incorporated herein by reference in its entirety).

1.13. Method for producing antibody fragment

The antibody fragments provided herein can be prepared using any suitable method, including the illustrative methods described herein or those known in the art. suitable methods include recombinant techniques and proteolytic digestion of complete antibodies.illustrative methods for preparing antibody fragments are described, for example, in Hudson et al, Nat.Med.,2003,9: 129-loop 134, which is incorporated herein by reference in its entirety.

1.14. Method for preparing replaceable skeleton

The alternative scaffolds provided herein may be prepared using any suitable method, including the illustrative methods described herein or those known in the art. For example, preparation of Adnectins^TMDescribed in Emanuel et al, mAbs,2011,3:38-48, which is incorporated herein by reference in its entirety. Methods for preparing iMabs are described in U.S. patent publication No. 2003/0215914, which is incorporated herein by reference in its entirety. Preparation ofDescribed in Vogt and Skerra, chem. biochem.,2004,5:191-199, which is incorporated herein by reference in its entirety. Methods for making Kunitz domains are described in Wagner et al, Biochem.&Biophysis.res.comm., 1992,186: 118-. Methods for preparing thioredoxin peptide aptamers are provided in Geyer and Brent, meth.enzymol.,2000,328:171-208, which are incorporated herein by reference in their entirety. Methods of making affinity antibodies are provided in Fernandez, curr. opinion in biotech, 2004,15: 364-. Methods for preparing DARPin are provided in Zahnd et al, J.mol.biol.,2007,369: 1015-. Methods for preparing Affilin are provided in Ebersbach et al, j.mol.biol.,2007,372:172-185, which is incorporated herein by reference in its entirety. Methods for preparing tetranectins are provided in Graversen et al, J.biol.chem.,2000,275:37390-37396, which is incorporated herein by reference in its entirety. Methods for preparing high affinity multimers are provided in Silverman et al, Nature Biotech, 2005,23: 1556-. Methods for preparing Fynomer are provided in Silaci et al, J.biol.chem.,2014,289:14392-14398, which is incorporated herein by reference in its entirety.

Additional information on alternative scaffolds is provided in Binz et al, nat. Biotechnol., 200523: 1257-; and Skerra, Current opin.in Biotech, 200718: 295-304, each of which is incorporated herein by reference in its entirety.

1.15. Method for preparing multi-specificity ABP

The multispecific or multivalent monospecific ABPs provided herein can be prepared using any suitable method, including the illustrative methods described herein or those methods known in the art.Methods for making common light chain antibodies are described in Merchant et al, Nature Biotechnol.,1998,16: 677-. Methods for making tetravalent bispecific antibodies are described in Coloma and Morrison, Nature Biotechnol.,1997,15:159-163, which are incorporated herein by reference in their entirety. Methods for preparing hybrid immunoglobulins are described in Milstein and Cuello, Nature,1983,305: 537-540; and Staerz and Bevan, Proc. Natl. Acad. Sci. USA,1986,83: 1453-; each of which is incorporated herein by reference in its entirety. Methods of preparing immunoglobulins with knob-hole modifications are described in U.S. Pat. No. 5,731,168, which is incorporated herein by reference in its entirety. Methods of preparing immunoglobulins with electrostatic modifications are provided in WO 2009/089004, which is incorporated herein by reference in its entirety. Methods for making multivalent (e.g., tetravalent) monospecific antibodies are described in Miller et al, 2003, U.S. patent No. 8,722,859, which is incorporated herein by reference in its entirety. Methods for preparing bispecific single chain antibodies are described in Traunecker et al, EMBO J.,1991,10: 3655-; and Gruber et al, J.Immunol.,1994,152: 5368-5374; each of which is incorporated herein by reference in its entirety. Methods for making single chain antibodies, the linker lengths of which may vary, are described in U.S. Pat. nos. 4,946,778 and 5,132,405, each of which is incorporated herein by reference in its entirety. Methods for making bifunctional antibodies are described in Hollinger et al, Proc. Natl. Acad. Sci. USA,1993,90: 6444-. Methods of making trifunctional and tetrafunctional antibodies are described in Todorovska et al, j.immunol.methods,2001,248:47-66, which is incorporated herein by reference in its entirety. Methods of making trispecific F (ab')3 derivatives are described in Tutt et al, j.immunol.,1991,147:60-69, which is incorporated herein by reference in its entirety. Methods for making cross-linked antibodies are described in U.S. Pat. nos. 4,676,980; brennan et al, Science,1985,229: 81-83; staerz et al Nature,1985,314: 628-631; and in EP 0453082; each of which is incorporated herein by reference in its entirety. Methods for preparing leucine zipper-assembled antigen binding domains are described in Kostelny et al, J.Immunol.,1992,148:1547-1553, which is incorporated herein by reference in its entirety. Methods of preparing ABPs via the DNL process are described in U.S. patent nos. 7,521,056, 7,550,143, 7,534,866 and 7,527,787, each of which is incorporated herein by reference in its entirety. Methods for making hybrids of antibodies and non-antibody molecules are described in WO 93/08829, which is incorporated herein by reference in its entirety, e.g., these ABPs. Methods of making DAF antibodies are described in U.S. patent publication No. 2008/0069820, which is incorporated herein by reference in its entirety. Methods for preparing ABP via reduction and oxidation are described in Carlring et al, PLoS One,2011,6: e22533, which is incorporated herein by reference in its entirety. Preparation of DVD-Igs^TMIs described in U.S. patent No. 7,612,181, which is incorporated herein by reference in its entirety. Preparation of DARTs^TMDescribed in Moore et al, Blood,2011,117:454-451, which is incorporated herein by reference in its entirety. Preparation ofThe methods of (A) are described in Labrijn et al, Proc.Natl.Acad.Sci.USA,2013,110: 5145-; graner et al, mAbs,2013,5: 962-; and Labrijn et al, Nature Protocols,2014,9: 2450-; each of which is incorporated herein by reference in its entirety. Preparation of C comprising and derived from IgG_H3The methods of C-terminal fusion of scFv antibodies of (1) are described in Coloma and Morrison, Nature Biotechnol.,1997,15:159-163, which are incorporated herein by reference in their entirety. Methods for preparing antibodies in which a Fab molecule is linked to the constant region of an immunoglobulin are described in Miler et al, J.Immunol.,2003,170:4854-4861, which is incorporated herein by reference in its entirety. Methods for preparing CovX-bodies are described in Doppallapoudi et al, Proc.Natl.Acad.Sci.USA,2010,107: 22611-. Methods for making Fcab antibodies are described in Wozniak-Knopp et al, proteineng.des.sel.,2010,23:289-297, which is incorporated herein by reference in its entirety. Preparation ofMethods for antibodies are described in Kipriyanov et al, J.mol.biol.,1999,293:41-56 and Zhukovsky et al, Blood,2013,122:5116, each of which is incorporated herein by reference in its entirety. Methods for making tandem fabs are described in WO 2015/103072, which is incorporated herein by reference in its entirety. Preparation of Zyboodies^TMDescribed in LaFleur et al, mAbs,2013,5: 208-.

In another aspect, there is provided a method for producing an anti-human GITR antibody or antigen-binding fragment thereof, comprising culturing a host cell selected from the group consisting of the following (a) to (c) to express a tetravalent anti-human GITR antibody or antigen-binding fragment thereof: (a) a host cell transformed with an expression vector comprising: a polynucleotide comprising a base sequence encoding the heavy chain variable region of an anti-human GITR antibody or antigen-binding fragment thereof provided herein; and a polynucleotide comprising a base sequence encoding the variable region of the light chain of the antibody or antigen-binding fragment thereof; (b) host cells transformed with the following expression vectors: an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of an anti-human GITR antibody or antigen-binding fragment thereof provided herein; and an expression vector comprising a polynucleotide comprising a base sequence encoding the variable region of the light chain of the antibody or antigen-binding fragment thereof; and (c) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of an anti-human GITR antibody or antigen-binding fragment thereof provided herein; and a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the variable region of the light chain of the antibody or antigen-binding fragment thereof.

In another aspect, there is provided a method for producing an anti-human GITR antibody, comprising culturing a host cell selected from the group consisting of the following (a) to (c) to express the anti-human GITR antibody: (a) a host cell transformed with an expression vector comprising: a polynucleotide comprising a base sequence encoding a heavy chain of an anti-human GITR antibody provided herein; and a polynucleotide comprising a base sequence encoding a light chain of the antibody; (b) host cells transformed with the following expression vectors: an expression vector comprising a polynucleotide comprising a base sequence encoding a heavy chain of an anti-human GITR antibody provided herein; and an expression vector comprising a polynucleotide comprising a base sequence encoding a light chain of the antibody; and (c) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding a heavy chain of an anti-human GITR antibody provided herein; and a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding a light chain of an antibody.

1.16. Method for producing variants

In some embodiments, the ABPs provided herein are affinity matured variants of a parent ABP, which can be produced, for example, using phage display-based affinity maturation techniques. Briefly, one or more CDR residues may be mutated and the variant ABP or portion thereof displayed on a phage and screened for affinity. These changes can be made at CDR "hot spots" or residues encoded by codons that undergo high frequency mutations during the somatic maturation process (see Chowdhury, methods mol. biol.,2008,207:179-196, which is incorporated herein by reference in its entirety) and/or antigen-contacting residues. In some embodiments, affinity maturation may be used to alter or introduce species binding, i.e., anti-mouse antibodies may be engineered to bind to human and cynomolgus versions, etc., of the same target antigen.

Any suitable method may be used to introduce variability into the polynucleotide sequences encoding ABPs, including error-prone PCR, strand shuffling, and oligonucleotide-directed mutagenesis, such as trinucleotide-directed mutagenesis (TRIM). In some aspects, several CDR residues (e.g., 4 to 6 residues at the same time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are commonly used to target mutations.

Introduction of diversity into the variable regions and/or CDRs can be used to generate secondary libraries. Next, the secondary library is screened to identify ABP variants with improved affinity. Affinity maturation by construction and re-selection from secondary libraries has been described, for example, in Hoogenboom et al, Methods in Molecular Biology,2001,178:1-37, which is incorporated herein by reference in its entirety.

1.17. Vectors, host cells and recombinant methods

Isolated GITR ABP-encoding nucleic acids, vectors comprising the nucleic acids, and host cells comprising the vectors and nucleic acids are also provided, as well as recombinant techniques for producing ABP.

In another aspect, a polynucleotide is provided comprising a base sequence encoding the heavy chain variable region of an anti-human GITR antibody or antigen-binding fragment thereof provided herein. In another aspect, a polynucleotide is provided comprising a base sequence encoding the light chain variable region of an anti-human GTIR antibody or antigen binding fragment thereof provided herein.

In another aspect, a polynucleotide is provided comprising a base sequence encoding a heavy chain of an anti-human GITR antibody provided herein. In another aspect, a polynucleotide is provided comprising a base sequence encoding a light chain of an anti-human GTIR antibody provided herein.

For recombinant production of ABP, the nucleic acid encoding it may be isolated and inserted into a replicable vector for further cloning (i.e., DNA amplification) or expression. In some aspects, the nucleic acid can be produced by homologous recombination, for example as described in U.S. patent No. 5,204,244, which is incorporated herein by reference in its entirety.

In another aspect, an expression vector is provided comprising (a) a polynucleotide comprising a base sequence encoding the heavy chain variable region of an anti-human GITR antibody or antigen-binding fragment thereof provided herein, and/or (b) a polynucleotide comprising a base sequence encoding the light chain variable region of an anti-human GITR antibody or antigen-binding fragment thereof.

In another aspect, an expression vector is provided that comprises (a) a polynucleotide comprising a base sequence encoding a heavy chain of an anti-human GITR antibody provided herein, and/or (b) a polynucleotide comprising a base sequence encoding a light chain of an anti-human GITR antibody.

Many different vectors are known in the art. The carrier component typically includes one or more of the following: signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences, for example as described in U.S. patent No. 5,534,615, which is incorporated herein by reference in its entirety.

Illustrative examples of suitable host cells are provided below. Such host cells are not meant to be limiting, and any suitable host cell can be used to produce the ABPs provided herein.

In another aspect, there is provided a host cell transformed with an expression vector selected from the group consisting of (a) to (d): (a) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of an anti-human GITR antibody or antigen-binding fragment thereof provided herein; and a polynucleotide comprising a base sequence comprising a light chain variable region encoding an antibody or antigen-binding fragment thereof; (b) host cells transformed with the following expression vectors: an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of an anti-human GITR antibody or antigen-binding fragment thereof provided herein; and an expression vector comprising a polynucleotide comprising a base sequence encoding the variable region of the light chain of the antibody or antigen-binding fragment thereof; (c) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of an anti-human GITR antibody or antigen-binding fragment thereof provided herein; and (d) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain variable region of an anti-human GITR antibody or antigen-binding fragment thereof provided herein.

In another aspect, there is provided a host cell transformed with an expression vector selected from the group consisting of (a) to (d): (a) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding a heavy chain of an anti-human GITR antibody provided herein; and a polynucleotide comprising a base sequence encoding a light chain of the antibody; (b) host cells transformed with the following expression vectors: an expression vector comprising a polynucleotide comprising a base sequence encoding a heavy chain of an anti-human GITR antibody provided herein; and an expression vector comprising a polynucleotide comprising a base sequence encoding a light chain of the antibody; (c) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding a heavy chain of an anti-human GITR antibody provided herein; and (d) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding a light chain of an anti-human GITR antibody provided herein.

Suitable host cells include any prokaryotic (e.g., bacterial), lower eukaryotic (e.g., yeast), or higher eukaryotic (e.g., mammalian) cell. Suitable prokaryotes include eubacteria, such as Gram-negative (Gram-negative) or Gram-positive (Gram-positive) organisms, for example enterobacteriaceae, such as Escherichia (Escherichia) (Escherichia coli), Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), Proteus (Proteus), Salmonella (Salmonella) (Salmonella typhimurium), Serratia (s. marcescens)), Shigella (Shigella), bacillus (bacillus) (bacillus subtilis) and bacillus licheniformis (b. licheniformis)), Pseudomonas (Pseudomonas) (p. aeruginosa) and Streptomyces (Streptomyces). One useful E.coli cloning host is E.coli 294, but other strains such as E.coli B, E.coli X1776 and E.coli W3110 are also suitable.

In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are suitable cloning or expression hosts for GITR ABP-encoding vectors. Saccharomyces cerevisiae or common baker's yeast is commonly used in lower eukaryotic host microorganisms. However, a variety of other genera, species and strains are available and useful, such as Schizosaccharomyces pombe (Schizosaccharomyces pombe), Kluyveromyces (Kluyveromyces) (Kluyveromyces lactis (k. lactis), Kluyveromyces fragilis (k. fragilis), Kluyveromyces bulgaricus (k. bulgaricus), Kluyveromyces williamsii (k. winkaramii), Kluyveromyces fortunei (k. wallini), Kluyveromyces drosophilus (k. drosophilus), Kluyveromyces thermotolerans (k. thermoolerans) and Kluyveromyces marxianus (k. marxianus)), Yarrowia, Pichia pastoris (Pichia pastoris), Candida (Candida) (Candida albicans), Trichoderma schliensis (Trichoderma harzianum), Neurospora crassa (saccharomyces rouxii), Aspergillus niger (Aspergillus niger) and Aspergillus niger (Aspergillus niger).

Suitable mammalian host cells include COS-7 cells, HEK293 cells, Baby Hamster Kidney (BHK) cells, Chinese Hamster Ovary (CHO) cells, mouse Sertoli cells, Vero-cells (VERO-76), and the like.

The host cells used to produce GITR ABP of the invention can be cultured in various media. Commercially available media such as Ham's F10, Minimal Essential Medium (MEM), RPMI-1640, and Dulbecco's Modified Eagle's Medium (DMEM) are suitable for culturing the host cells. Additionally, the methods described in Ham et al, meth.enz, 1979,58: 44; barnes et al, anal. biochem.,1980,102: 255; and U.S. patent nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655 and 5,122,469; or any of the media in WO 90/03430 and WO 87/00195. Each of the foregoing references is incorporated herein by reference in its entirety.

Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium salts, magnesium salts, and phosphate salts), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics, trace elements (defined as inorganic compounds typically present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art.

Culture conditions (such as temperature, pH, etc.) are those used with a host cell previously selected for expression, and will be apparent to one of ordinary skill in the art.

When recombinant technology is used, ABP may be produced intracellularly, in the periplasmic space or directly secreted into the culture medium. If ABP is produced intracellularly, as a first step, particulate debris (host cells or lysed fragments) are removed, for example, using centrifugation or ultrafiltration. For example, Carter et al (Bio/Technology,1992,10: 163-. Briefly, cell bodies were thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation.

In some embodiments, the ABP is produced in a cell-free system. In some aspects, the cell-free system is an ex vivo transcription and translation system, as described in Yin et al, mAbs,2012,4:217-225, which is incorporated herein by reference in its entirety. In some aspects, the cell-free system utilizes cell-free extracts from eukaryotic cells or from prokaryotic cells. In some aspects, the prokaryotic cell is e. Cell-free expression of ABP may be useful, for example, when ABP accumulates in cells as insoluble aggregates, or when the yield of expression from the periplasm is low.

When ABP is secreted into the culture medium, a commercially available protein concentration filter (e.g., a commercially available protein concentration filter) is generally first usedOrUltrafiltration unit) to concentrate the supernatant from these expression systems. May include steps in any of the preceding stepsProtease inhibitors such as PMSF to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

ABP compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being particularly useful purification techniques. The suitability of protein a as an affinity ligand depends on the type and subtype of any immunoglobulin Fc domain present in the ABP. Protein a can be used to purify ABP comprising human gamma 1, gamma 2, or gamma 4 heavy chains (Lindmark et al, j. immunol. meth, 1983,62:1-13, which is incorporated herein by reference in its entirety). Protein G is applicable to all mouse isoforms and to human gamma 3(Guss et al, EMBO J.,1986,5:1567-1575, which is incorporated herein by reference in its entirety).

The matrix to which the affinity ligand is attached is most commonly agarose, but other matrices may be utilized. Mechanically stable matrices, such as controlled pore glass or poly (styrene divinyl) benzene, allow faster flow rates and shorter processing times than can be achieved with agarose. In ABP comprises C_H3When domain, BakerBondThe resin is suitable for purification.

Other techniques for protein purification may also be utilized and may be applied by those skilled in the art, such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparinChromatography, chromatofocusing, SDS-PAGE and ammonium sulfate precipitation.

After any initial purification step, the mixture comprising the ABP of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5 and about 4.5, typically at a low salt concentration (e.g., about 0 to about 0.25M salt).

Analytical method

Various assays known in the art can be used to identify and characterize the GITR ABPs provided herein.

1.18. Binding, competition and epitope mapping assays

The specific antigen-binding activity of ABPs provided herein can be evaluated using any suitable method, including the use of SPR, BLI, and RIA, as described elsewhere in the disclosure. In addition, ELISA assay and Western blot analysis can be used to evaluate antigen-binding activity.

Assays for measuring competition between two ABPs or between an ABP and another molecule (e.g., GITRL) are described elsewhere in this disclosure and, for example, in Harlow and Lane, Antibodies: A Laboratory Manual.14, 1988, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., which are incorporated herein by reference in their entirety.

Analysis for Mapping of epitopes for binding to ABPs provided herein is described, for example, in Methods in molecular Biology, Morris, "Epitope Mapping Protocols," volume 66, 1996, Humana Press, Totowa, n.j., which is incorporated herein by reference in its entirety. In some embodiments, the epitope is determined by peptide competition. In some embodiments, the epitope is determined by mass spectrometry. In some embodiments, the epitope is determined by crystallography.

GITR agonism assay

In some embodiments, ABPs provided herein are screened to identify or characterize ABPs having agonist activity against GITR any suitable assay may be used to identify or characterize these ABPs, in some aspects, the assay measures the amount of cytokines secreted by effector T cells after contacting the effector T cells with ABPs provided herein, in some aspects, the cytokines are selected from the group consisting of IL-2R α, IL-2, IL-8, IFN γ, and combinations thereof, in some aspects, the cytokines are selected from the group consisting of sCD40L, VEGF, TNF- β, TNF- α, TGF- α, RANTES, PDGF-AB/BB, PDGF-AA, MIP-1 β, MIP-1 α, MDC (CSF 22), MCP-3, MCP-1, IP-10, IL-17A, IL-15, IL-13, IL-12(p70), IL-12(p40), IL-10, IL-9, IL-8, MCP-1, IP-10, IL-17A, IL-15, IL-13, IL-12(p70), IL-12(p40), IL-10, IL-9, IL-7, EGF-7, IL-7, EGF-5, IL-7-5, IL-5, EGF-5, IL-2R-3, EGF-2, IL-3, IL-5, IL-2, EGF-5, IL-2, EGF-2, and combinations thereof.

In some embodiments, the effector cells are co-stimulated with an agonist of CD3 to promote cytokine secretion by the effector cells. In some aspects, the CD3 agonist is provided at a sub-maximal level.

In some embodiments, functional assays, such as HT1080 or Jurkat cell-based assays described in more detail in example 2, are used. Additional analyses are described in Wyzgol et al, J Immunol 2009; 183:1851-1861, which is incorporated herein by reference.

In some aspects, these assays can measure the proliferation of effector T cells after contacting the effector T cells with ABPs provided herein. In some aspects, proliferation of effector T cells is measured by dye dilution (e.g., carboxyfluorescein diacetate succinimidyl ester; CFSE), by tritiated thymidine uptake, by luminescent cell viability assays, or by other assays known in the art.

In some aspects, these assays can measure the differentiation, cytokine production, viability (e.g., survival), proliferation, or inhibitory activity of a regulatory T cell after contacting the regulatory T cell with an ABP provided herein.

In some aspects, these assays can measure the cytotoxic activity of NK cells after contacting the NK cells with ABPs provided herein. In some aspects, the cytotoxic activity of NK cells is measured using a cytotoxicity assay that quantifies NK-mediated killing of target cells (e.g., K562 cell line). See Jang et al, ann.clin.lab.sci.,2012,42:42-49, which is incorporated herein by reference in its entirety.

Additional assays for measuring GITR agonism are described elsewhere in the disclosure (including the examples) and are known in the art. One skilled in the art can readily select an appropriate assay for assessing GITR agonism.

1.20. Analysis of Effector Functions

Effector function of ABPs provided herein can be assessed using various in vitro and in vivo assays known in the art, including those described in: ravech and Kinet, annu.rev.immunol.1991, 9: 457-; U.S. Pat. nos. 5,500,362, 5,821,337; hellstrom et al, Proc.Nat' lAcad.Sci.USA,1986,83: 7059-; hellstrom et al, Proc.nat' l Acad.Sci.USA,1985,82: 1499-; bruggemann et al, J.Exp.Med.,1987,166: 1351-; clynes et al, Proc.Nat' lAcad.Sci.USA,1998,95: 652-; WO 2006/029879; WO 2005/100402; Gazzano-Santoro et al, J.Immunol.methods,1996,202: 163-; cragg et al, Blood,2003,101: 1045-1052; cragg et al Blood,2004,103: 2738-; and Petkova et al, Int' l. Immunol.,2006,18: 1759-; each of which is incorporated herein by reference in its entirety.

Pharmaceutical composition

The ABPs provided herein can be formulated in any suitable pharmaceutical composition and administered using any suitable route of administration. Suitable routes of administration include, but are not limited to, intra-arterial, intradermal, intramuscular, intraperitoneal, intravenous, nasal, parenteral, pulmonary, and subcutaneous routes.

In another aspect, a pharmaceutical composition is provided comprising a plurality of types of anti-human GITR antibodies, or antigen-binding fragments thereof, provided herein. For example, the pharmaceutical composition comprises an antibody or antigen-binding fragment thereof that is not post-translationally modified, and a post-translationally modified antibody or antigen-binding fragment thereof derived from the antibody or antigen-binding fragment thereof.

In one embodiment, the pharmaceutical composition comprises at least two anti-human GITR antibodies selected from (1) to (4): (1) an anti-human GITR antibody comprising two heavy chains consisting of SEQ ID No. 7 and four light chains consisting of SEQ ID No. 8; (2) an anti-human GITR antibody comprising two heavy chains consisting of SEQ ID NO 7, wherein Q at position 1 is modified to pyroglutamic acid; four light chains consisting of SEQ ID NO 8; (3) an anti-human GITR antibody comprising two heavy chains consisting of amino acid sequences ranging from Q at position 1 to G at position 686 of SEQ ID No. 7, and four light chains consisting of SEQ ID No. 8; and (4) an anti-human GITR antibody comprising two heavy chains consisting of amino acid sequences ranging from Q at position 1 to G at position 686 of SEQ ID NO:7, wherein Q at position 1 is modified to pyroglutamic acid; and four light chains of SEQ ID NO. 8.

In one embodiment, a pharmaceutical composition comprises: an anti-human GITR antibody comprising two heavy chains consisting of SEQ ID No. 7 and four light chains consisting of SEQ ID No. 8; and an anti-human GITR antibody comprising two heavy chains consisting of amino acid sequences ranging from Q at position 1 to G at position 686 of SEQ ID No. 7 (wherein Q at position 1 is modified to pyroglutamic acid) and four light chains of SEQ ID No. 8; and a pharmaceutically acceptable excipient.

The pharmaceutical composition may comprise one or more pharmaceutically acceptable excipients. Any suitable pharmaceutical excipient may be used and the skilled person will be able to select a suitable pharmaceutical excipient in general. Accordingly, the pharmaceutical excipients provided below are intended to be illustrative and not limiting. Additional pharmaceutically acceptable excipients include, for example, those described in Handbook of pharmaceutical excipients, Rowe et al, 6 th edition (2009), which is incorporated herein by reference in its entirety.

In some embodiments, the pharmaceutical composition comprises an anti-foaming agent. Any suitable defoamer can be used. In some aspects, the defoamer is selected from the group consisting of alcohols, ethers, oils, waxes, polysiloxanes, surfactants, and combinations thereof. In some aspects, the defoaming agent is selected from: mineral oil, vegetable oil, ethylene bis stearamide, paraffin wax, ester wax, fatty alcohol wax, long chain fatty alcohol, fatty acid soap, fatty acid ester, silicone glycol, fluoropolysiloxane, polyethylene glycol-polypropylene glycol copolymer, polydimethylsiloxane-silica, ether, octanol, decanoyl alcohol, sorbitan trioleate, ethanol, 2-ethyl-hexanol, dimethicone, oleyl alcohol, dimethicone, and combinations thereof.

In some embodiments, the pharmaceutical composition comprises a co-solvent. Illustrative examples of co-solvents include ethanol, poly (ethylene) glycol, butylene glycol, dimethylacetamide, glycerol, propylene glycol, and combinations thereof.

In some embodiments, the pharmaceutical composition comprises a buffering agent. Illustrative examples of buffering agents include acetate, borate, carbonate, lactate, malate, phosphate, citrate, hydroxide, diethanolamine, monoethanolamine, glycine, methionine, guar gum, monosodium glutamate, and combinations thereof.

In some embodiments, the pharmaceutical composition comprises a carrier or filler. Illustrative examples of carriers or fillers include lactose, maltodextrin, mannitol, sorbitol, chitosan, stearic acid, xanthan gum, guar gum, and combinations thereof.

Illustrative examples of surfactants include d- α tocopherol, benzalkonium chloride, benzethonium chloride, cetrimide, cetylpyridinium chloride, docusate sodium, glyceryl behenate, glyceryl monooleate, lauric acid, polyethylene glycol 15 hydroxystearate, tetradecanol, phospholipids, polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, polyoxylglycerides, sodium lauryl sulfate, sorbitan esters, vitamin E polyethylene (ethylene glycol) succinic acid, and combinations thereof.

In some embodiments, the pharmaceutical composition comprises an anti-caking agent. Illustrative examples of anticaking agents include calcium phosphate (trivalent), hydroxymethyl cellulose, hydroxypropyl cellulose, magnesium oxide, and combinations thereof.

Other excipients that may be used with the pharmaceutical composition include, for example, albumin, antioxidants, antibacterial agents, antifungal agents, bioabsorbable polymers, chelating agents, controlled release agents, diluents, dispersing agents, dissolution enhancers, emulsifiers, gelling agents, ointment bases, penetration enhancers, preservatives, solubilizers, solvents, stabilizers, sugars, and combinations thereof. Specific examples of each of such agents are described, for example, in Handbook of Pharmaceutical Excipients, Rowe et al (eds.) 6 th edition (2009), The Pharmaceutical Press, which is incorporated herein by reference in its entirety.

In some embodiments, the pharmaceutical composition comprises a solvent. In some aspects, the solvent is a saline solution, such as a sterile isotonic saline solution or a dextrose solution. In some aspects, the solvent is water for injection.

In some embodiments, the pharmaceutical composition is in the form of particles, such as microparticles or nanoparticles. The microparticles and nanoparticles may be formed from any suitable material, such as a polymer or lipid. In some aspects, the microparticle or nanoparticle is a micelle, liposome, or polymersome.

As water may promote degradation of some ABPs, anhydrous pharmaceutical compositions and dosage forms comprising ABPs are further provided herein.

Anhydrous pharmaceutical compositions and dosage forms provided herein can be prepared using anhydrous or lower moisture containing ingredients and lower moisture or lower humidity conditions. Pharmaceutical compositions and dosage forms comprising lactose and at least one active ingredient comprising a primary or secondary amine may be anhydrous if substantial contact with moisture and/or humidity during manufacture, packaging, and/or storage is expected.

Anhydrous pharmaceutical compositions should be prepared and stored in a manner that maintains their anhydrous nature. Thus, the anhydrous composition is packaged using materials known to prevent exposure to water so that it can be included in a suitable formulation kit. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

1.21. Parenteral dosage forms

In certain embodiments, the ABPs provided herein are formulated as parenteral dosage forms. Parenteral dosage forms can be administered to a subject using a variety of routes including, but not limited to, subcutaneous, intravenous (including infusion and bolus injection), intramuscular, and intraarterial. Because their administration typically bypasses the subject's natural defenses against contaminants, parenteral dosage forms are typically sterile or capable of being sterilized prior to administration to a subject. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dried (e.g., lyophilized) products ready for injection to be dissolved or suspended in a pharmaceutically acceptable vehicle, suspensions and emulsions ready for injection.

Suitable vehicles which can be used to provide parenteral dosage forms are well known to those skilled in the art. Examples include (but are not limited to): injecting USP water; aqueous vehicles such as, but not limited to, sodium chloride injection, Ringer's injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water soluble vehicles such as, but not limited to, ethanol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Excipients that increase the solubility of one or more of the ABPs disclosed herein may also be incorporated into parenteral dosage forms.

In some embodiments, the parenteral dosage form is lyophilized. Exemplary lyophilized formulations are described, for example, in U.S. Pat. nos. 6,267,958 and 6,171,586; and WO 2006/044908; each of which is incorporated herein by reference in its entirety.

Dosage and unit dosage form

In human therapeutics, the physician will determine the dosimetry he considers most appropriate, according to prophylactic or curative treatment and according to the age, weight, condition and other factors specific to the subject to be treated.

In certain embodiments, the compositions provided herein are pharmaceutical compositions or single unit dosage forms. The pharmaceutical compositions and single unit dosage forms provided herein comprise a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic ABPs.

The amount of ABP or composition effective in preventing or treating a disorder or one or more symptoms thereof will vary depending on the nature and severity of the disease or disorder and the route of administration of the ABP. The frequency and dose will also vary depending on factors specific to each subject according to: the particular therapeutic agent (e.g., therapeutic or prophylactic agent) administered, the disorder, the severity of the disease or disorder, the route of administration, and the age, size, weight, response, and past medical history of the subject. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

In certain embodiments, an exemplary dose of a composition comprises an amount of milligram or microgram ABP per kilogram of subject or sample weight (e.g., from about 100 microgram/kilogram to about 25 milligrams/kilogram, or from about 100 microgram/kilogram to about 10 milligrams/kilogram). In a certain embodiment, the dose of ABP provided herein administered to prevent, treat, control, or ameliorate a disorder or one or more symptoms thereof in a subject by weight of ABP is 0.1mg/kg, 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 10mg/kg, 15mg/kg, 20mg/kg, 25mg/kg, 30mg/kg, or 40mg/kg of subject body weight or greater.

The dose may be administered according to a suitable schedule, for example once a week, once every two weeks, once every three weeks or once every four weeks. In some embodiments, the antibody administered once every three or four weeks may be administered at a higher dose than the antibody administered every one or two weeks. In some embodiments, the loading dose is administered at a higher maintenance dose than is administered thereafter. As will be apparent to one of ordinary skill in the art, it may be desirable in some instances to use ABP doses outside the scope disclosed herein. Furthermore, it should be noted that the clinician or treating physician, in conjunction with the subject response, should know how and when to interrupt, adjust or terminate therapy.

As will be readily appreciated by those of ordinary skill in the art, different therapeutically effective amounts may be appropriate for different diseases and conditions. Similarly, the dosages and dosing frequency schedules provided herein also encompass amounts sufficient to prevent, control, treat, or ameliorate these conditions, but insufficient to cause or sufficient to reduce the adverse effects associated with ABP provided herein. Further, when multiple doses of a composition provided herein are administered to a subject, not all doses need be the same. For example, the dose administered to a subject can be increased to improve the prophylactic or therapeutic effect of the composition, or the dose can be decreased to reduce one or more side effects experienced by a particular subject.

In certain embodiments, treatment or prevention can be initiated with one or more loading doses of an ABP or composition provided herein, followed by one or more maintenance doses.

In certain embodiments, a dose of an ABP or composition provided herein can be administered to achieve a steady-state concentration of ABP in the blood or serum of the subject. The steady state concentration can be determined by measurement according to techniques available to those skilled in the art, or can be determined based on physical characteristics of the subject, such as height, weight, and age.

In certain embodiments, the same composition may be repeatedly administered, and administration may be spaced apart by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months.

As discussed in more detail elsewhere in the present disclosure, the ABPs provided herein may optionally be administered with one or more additional agents useful in the prevention or treatment of a disease or disorder. The effective amount of these additional agents may depend on the amount of ABP present in the formulation, the type of disorder or treatment, and other factors known in the art or described herein.

Therapeutic applications

For therapeutic applications, the ABPs of the invention are administered to a mammal (typically a human) in a pharmaceutically acceptable dosage form, such as those known in the art and those discussed above. For example, the ABPs of the invention may be administered to a human by intravenous bolus injection and or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, or intratumoral routes. ABP is also suitably administered by peri-, intra-or parafocal routes to exert local as well as systemic therapeutic effects. The intraperitoneal route may be particularly suitable, for example, for the treatment of ovarian tumors.

The ABPs provided herein may be useful in the treatment of any disease or disorder in which GITR is involved. In some embodiments, the disease or disorder is one that would benefit from treatment with anti-GITR ABP. In some embodiments, the disease or disorder is a tumor. In some embodiments, the disease or disorder is a cell proliferative disorder. In some embodiments, the disease or disorder is cancer.

In some embodiments, the ABPs provided herein are provided for use as a medicament. In some embodiments, the ABPs provided herein are used in the manufacture or preparation of a medicament. In some embodiments, the medicament is for treating a disease or disorder that may benefit from anti-GITR ABP. In some embodiments, the disease or disorder is a tumor. In some embodiments, the disease or disorder is a cell proliferative disorder. In some embodiments, the disease or disorder is cancer.

Any suitable cancer can be treated with the ABPs provided herein. Illustrative suitable cancers include, for example: acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), adrenocortical carcinoma, anal carcinoma, appendiceal carcinoma, astrocytoma, basal cell carcinoma, brain tumor, bile duct carcinoma, bladder carcinoma, bone carcinoma, breast carcinoma, bronchial tumor, cancer of unknown primary focus, heart tumor, cervical carcinoma, chordoma, colon carcinoma, colorectal carcinoma, craniopharyngeal carcinoma, duct carcinoma, embryo tumor, endometrial carcinoma, ependymoma, esophageal carcinoma, sensitive neuroblastoma, histiocytoma, Ewing sarcoma (Ewing sarcoma), eye carcinoma, germ cell tumor, gallbladder carcinoma, gastric carcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, trophoblastic disease in gestational period, glioma, head and neck carcinoma, hepatocellular carcinoma, histiocytosis, Hodgkin lymphoma (Hodgkin lymphoma), hypopharynx cancer, melanoma, islet cell tumor, neuroblastoma, bladder carcinoma, gastrointestinal stromal tumor, and esophageal carcinoma, bladder carcinoma, Kaposi's sarcoma (Kaposi sarcoma), kidney cancer, Langerhans' cell (Langerhans cell) histiocytosis, laryngeal cancer, lip and oral cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma (Merkel cell sarcoma), mesothelioma, recessive primary metastatic squamous neck cancer, midline carcinoma involving NUT genes (midline track sarcoma), oral cancer, multiple endocrine tumor syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative tumors, nasal and paranasal carcinomas, nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, Pleuropulmonoblastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdoid tumor, salivary gland carcinoma, Sezary (Sezary) syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, gastric cancer, T-cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymus cancer, thyroid cancer, urinary tract cancer, uterine cancer, vaginal cancer, vulval cancer, and Wilms (Wilms) tumor.

In some embodiments, provided herein is a method of treating a disease or disorder in a subject in need thereof by administering to the subject an effective amount of an ABP provided herein. In some aspects, the disease or disorder is cancer.

In some embodiments, provided herein is a method of multimerizing GITR in target cells of a subject in need thereof by administering to the subject an effective amount of an ABP provided herein.

In some embodiments, provided herein is a method of agonizing GITR in a target cell of a subject in need thereof by administering to the subject an effective amount of ABP provided herein, hi some aspects, agonizing GITR by ABP provided herein results in increased secretion of IL-2R α, IL-2, IL-8, and/or IFN γ by the target cell.

In some embodiments, provided herein is a method of modulating NF- κ B activity in a target cell of a subject in need thereof by administering to the subject an effective amount of an ABP provided herein.

In some embodiments, provided herein is a method of modulating degradation of I κ B in a target cell of a subject in need thereof by administering to the subject an effective amount of an ABP provided herein.

In some embodiments, provided herein is a method of activating a MAPK pathway in a target cell in a subject in need thereof by administering to the subject an effective amount of an ABP provided herein. In some aspects, components of the MAPK pathway activated by ABP provided herein include one or more of p38, JNK, and ERK.

In some embodiments, provided herein is a method of increasing proliferation, survival, and/or function of effector T cells in a subject in need thereof by administering to the subject an effective amount of an ABP provided herein. In some aspects, the effector T cell is a CD4+ effector T cell. In some aspects, the effector T cell is a CD8+ effector T cell.

In some embodiments, provided herein is a method of abrogating the inhibition of effector T cells by regulatory T cells in a subject in need thereof by administering to the subject an effective amount of an ABP provided herein. In some aspects, the regulatory T cells are CD4+ CD25+ Foxp3+ regulatory T cells. In some aspects, the regulatory T cells are CD8+ CD25+ regulatory T cells.

In some embodiments, provided herein is a method of altering the frequency of appearance or distribution of regulatory T cells in a subject in need thereof by administering to the subject an effective amount of an ABP provided herein. In some aspects, the frequency of regulatory T cells is decreased. In some aspects, the frequency of regulatory T cells is reduced in a particular tissue. In some aspects, intratumoral accumulation of regulatory T cells is reduced, thereby producing a more favorable ratio of effector T cells to regulatory T cells and enhancing CD8+ T cell activity.

In some embodiments, provided herein is a method of increasing the activity of a Natural Killer (NK) cell in a subject in need thereof by administering to the subject an effective amount of an ABP provided herein.

In some embodiments, provided herein is a method of increasing the activity of a dendritic cell in a subject in need thereof by administering to the subject an effective amount of an ABP provided herein.

In some embodiments, provided herein is a method of increasing the activity of a B cell in a subject in need thereof by administering to the subject an effective amount of an ABP provided herein.

In some embodiments, provided herein is a method of enhancing an immune response in a subject in need thereof by administering to the subject an effective amount of an ABP provided herein.

In some embodiments, provided herein is a method of delaying the onset of a tumor in a subject in need thereof by administering to the subject an effective amount of an ABP provided herein.

In some embodiments, provided herein is a method of delaying the onset of cancer in a subject in need thereof by administering to the subject an effective amount of an ABP provided herein.

In some embodiments, provided herein is a method of reducing the size of a tumor in a subject in need thereof by administering to the subject an effective amount of an ABP provided herein.

In some embodiments, provided herein is a method of reducing the number of metastases in a subject in need thereof by administering to the subject an effective amount of an ABP provided herein.

Combination therapy

In some embodiments, the ABPs provided herein are administered with at least one additional therapeutic agent. Any suitable additional therapeutic agent may be administered with the ABPs provided herein. In some aspects, the additional therapeutic agent is selected from the group consisting of radiation therapy, cytotoxic agents, chemotherapeutic agents, cytostatic agents, anti-hormonal agents, EGFR inhibitors, immunostimulants, anti-angiogenic agents, and combinations thereof.

In some embodiments, the additional therapeutic agent comprises an immunostimulatory agent.

In some embodiments, the immunostimulatory agent is an agent that blocks signaling of an inhibitory receptor of an immune cell or a ligand thereof. In some aspects, the inhibitory receptor or ligand is selected from the group consisting of CTLA-4, PD-1, PD-L1, NRP-1, LAG-3, Tim3, TIGIT, neuritin, BTLA, KIR, and combinations thereof. In some aspects, the agent is selected from the group consisting of pamumab (anti-PD-1), nivolumab (nivolumab) (anti-PD-1), atezumab (atezolizumab) (anti-PD-L1), ipilimumab (ipilimumab) (anti-CTLA-4), and combinations thereof.

In some embodiments, the immunostimulatory agent is an agonist of a co-stimulatory receptor of an immune cell. In some aspects, the co-stimulatory receptor is selected from the group consisting of OX40, ICOS, CD27, CD28, 4-1BB, and CD 40. In some embodiments, the agonist is an antibody.

In some embodiments, the immunostimulatory agent is a cytokine. In some aspects, the cytokine is selected from the group consisting of IL-2, IL-5, IL-7, IL-12, IL-15, IL-21, and combinations thereof.

In some embodiments, the immunostimulant is an oncolytic virus. In some aspects, the oncolytic virus is selected from the group consisting of herpes simplex virus, vesicular stomatitis virus, adenovirus, newcastle disease virus, vaccinia virus, and malaba virus.

In some embodiments, the immunostimulant is a T cell with a chimeric antigen receptor (CAR-T cell).

In some embodiments, the additional therapeutic agent is a vaccine against a tumor antigen. Any suitable antigen can be targeted by the vaccine, provided that the antigen is present in a tumor treated using the methods provided herein. In some aspects, the tumor antigen is one that is overexpressed compared to its expression in normal tissues. In some aspects, the tumor antigen is selected from the group consisting of cancer testis antigen, differentiation antigen, NY-ESO-1, MAGE-A1, MART, and combinations thereof.

Other examples of additional therapeutic agents include a taxane (e.g., paclitaxel or docetaxel), a platinum agent (e.g., carboplatin, oxaliplatin, and/or cisplatin), a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, and/or mitoxantrone), a folinic acid (e.g., leucovorin), or a nucleoside metabolism inhibitor (e.g., fluorouracil, capecitabine, and/or gemcitabine). In some embodiments, the additional therapeutic agent is folinic acid, 5-fluorouracil, and/or oxaliplatin. In some embodiments, the additional therapeutic agent is 5-fluorouracil and irinotecan. In some embodiments, the additional therapeutic agent is a taxane and a platinum agent. In some embodiments, the additional therapeutic agent is paclitaxel and carboplatin. In some embodiments, the additional therapeutic agent is pemetrexed. In some embodiments, the additional therapeutic agent is a targeted therapeutic agent such as an EGFR, RAF, or MEK-targeting agent.

The additional therapeutic agent may be administered using any suitable means. In some embodiments, the ABP provided herein and the additional therapeutic agent are included in the same pharmaceutical composition. In some embodiments, the ABPs provided herein and additional therapeutic agents are included in different pharmaceutical compositions.

In embodiments where the ABP and additional therapeutic agent provided herein are included in different pharmaceutical compositions, administration of the ABP can be performed prior to, concurrently with, and/or after administration of the additional therapeutic agent. In some aspects, administering the ABP provided herein and the additional therapeutic agent are performed within about one month of each other. In some aspects, administering the ABP provided herein and the additional therapeutic agent are performed within about one week of each other. In some aspects, administering the ABP provided herein and the additional therapeutic agent are performed within about one day of each other. In some aspects, administering the ABP provided herein and the additional therapeutic agent are performed within about twelve hours of each other. In some aspects, administering the ABP provided herein and the additional therapeutic agent are performed within about one hour of each other.

Diagnostic method

Also provided are methods for detecting the presence of GITR on cells from a subject. These methods can be used, for example, to predict and evaluate responses to treatment with ABPs provided herein.

In some embodiments, a blood sample is obtained from the subject and the fraction of GITR-expressing cells is determined. In some aspects, the relative amount of GITR expressed by these cells is determined. Any suitable method can be used to determine the fraction of GITR-expressing cells and the relative amount of GITR expressed by these cells. In some embodiments, flow cytometry is used to make these measurements. In some embodiments, Fluorescence Assisted Cell Sorting (FACS) is used to make these measurements. For a method to assess the expression of GITR in peripheral blood, see Li et al, j.autoimmunity,2003,21: 83-92.

Reagent kit

Also provided are kits comprising the ABPs provided herein. The kits can be used to treat, prevent, and/or diagnose a disease or disorder, as described herein.

In some embodiments, a kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and IV solution bags. The container may be formed from a variety of materials, such as glass or plastic. The container contains a composition that is effective for treating, preventing and/or diagnosing a disease or condition by itself, or when combined with another composition. The container may have a sterile access port. For example, if the container is an intravenous solution bag or vial, it may have a port that is pierceable by a needle. At least one active agent in the composition is an ABP provided herein. The label or package insert indicates that the composition is for use in treating the selected condition.

In some embodiments, a kit comprises (a) a first container comprising a first composition, wherein the first composition comprises an ABP provided herein; and (b) a second container containing a second composition, wherein the second composition comprises another therapeutic agent. The kit in this embodiment of the invention may further comprise a package insert indicating that the composition can be used to treat a particular disorder.

Alternatively or additionally, the kit may further comprise a second (or third) container comprising a pharmaceutically acceptable excipient. In some aspects, the excipient is a buffer. The kit may further include other materials required from a commercial and user standpoint, including filters, needles, and syringes.

Other illustrative embodiments

The embodiments provided below are non-limiting, and provide illustration of certain embodiments and aspects of the invention by way of illustration, in addition to those described throughout this disclosure.

Embodiment 1: an ABP that specifically binds to human GITR and/or a human GITR complex and is capable of at least one of: a) cross-competes with GITRL for binding to GITR; b) can be internalized into human cells; c) inhibition of effector T cells; d) inhibiting regulatory T cell suppression of effector T cells; e) reducing the number of regulatory T cells in the tissue or circulation; f) activating effector T cells; g) binds GITR to a GITR complex; h) modulating the activity of human GITR and/or GITR complexes.

Embodiment 2: the ABP of embodiment 1, wherein the ABP has one or more of the following characteristics: a) is a monoclonal antibody; b) is a human, humanized or chimeric antibody; c) is a multispecific or multivalent antibody, e.g., tetravalent antibody; d) comprises at least one Fab at the N-terminus or C-terminus; e) of the IgG1, IgG2, IgG3 or IgG4 type; f) is an antigen-binding antibody fragment; g) is an Fab fragment, an Fab 'fragment, an F (ab')2 fragment or an Fv fragment; h) is a bispecific antibody, a bifunctional antibody, a single chain antibody, a single domain antibody, V_HDomain antibodies or nanobodies.

Embodiment 3: the ABP of embodiment 1, wherein the ABP has one or more of the following characteristics: a) binds to human GITR polypeptide of SEQ ID NO 1 through SEQ ID NO 2 or variants thereof, or as otherwise provided herein having a K of less than about 20nM_D(ii) a b) Binds to the cynomolgus monkey GITR polypeptide of SEQ ID No. 3 or variants thereof, or as otherwise provided herein, has a K of less than about 200nM_D。

Embodiment 4: the ABP of embodiment 1, comprising: a first antigen binding domain that specifically binds to a first antibody recognition domain on human GITR; and a second antigen-binding domain that specifically binds to a second antibody recognition domain on human GITR, wherein the first antibody recognition domain and the second antibody recognition domain are not the same.

Embodiment 5: the ABP of embodiment 4, comprising a bispecific binding protein in a form selected from the group consisting of: DVD-Ig^TMMolecule (a),Molecular, DART^TMMolecule (a),Molecules, scFv/diabody-IgG molecules, cross-multispecific molecules, 2 in 1bispecific molecules (2-in-1bispecific molecules), knob-hole multispecific molecules, Fab + IgG molecules, CovX-body molecules, affinity antibody molecules, scFv/diabody-CH 2/CH3 bispecific molecules, multispecific molecules based on IgG-non-Ig protein backbones, antibodies, and antibody fragments,Molecule, Fcab^TMMolecule (a),Zybody^TMAnd scFV/diabodies linked to normal human proteins such as human serum albumin-bispecific molecules.

Embodiment 6: the ABP of embodiment 4, wherein the first antigen binding domain has a K of less than about 20nM_DAnd is capable of agonizing human GITR, and wherein the second antigen-binding domain has a K of less than about 100nM_D。

Embodiment 7: an ABP that competes or is capable of competing for binding to human GITR with a reference ABP, wherein the reference ABP is an ABP as in embodiment 1.

Embodiment 8: the ABP of embodiment 7, wherein the ABP cross-competes or is capable of cross-competing for binding to human GITR with the reference antibody.

Embodiment 9: the ABP of embodiment 1, comprising a heavy chain constant region comprising a human heavy chain constant region or a fragment or variant thereof, wherein the constant region variant comprises up to 20 modified amino acid substitutions, wherein 0 up to 20 modified amino acid substitutions are conservative amino acid substitutions.

Embodiment 10: the ABP of embodiment 1, which competes or is capable of competing for binding to human GITRL with GITR protein.

Embodiment 11: the ABP of embodiment 1, which is capable of activating GITR signaling in a ligand-independent manner.

Embodiment 12: the ABP of embodiment 1, which is capable of enhancing ligand-dependent binding of GITR to GITRL.

Embodiment 13: a pharmaceutical composition comprising the ABP of any one of embodiments 1 to 12 in a pharmaceutically acceptable carrier.

Embodiment 14: a bispecific antibody comprising: a first antigen binding domain that specifically binds to a first antibody recognition domain on human GITR; and a second antigen-binding domain that specifically binds to a second antibody recognition domain on human GITR, wherein the first antibody recognition domain and the second antibody recognition domain are not the same.

Embodiment 15: the bispecific antibody of embodiment 14, wherein the first antibody recognition domain and the second antibody recognition domain are present in the extracellular domain of human GITR.

Embodiment 16: the bispecific antibody of embodiment 14, wherein the first antibody recognition domain and the second antibody recognition domain are capable of associating at least two human GITR proteins into a functional complex.

Embodiment 17: a composite ABP comprising a first antigen-binding domain that specifically binds to a first antibody recognition domain on a human GITR protein or a human GITR complex comprising at least two GITR proteins, and is capable of at least one of: a) cross-competes with GITRL for binding to GITR; b) can be internalized into human cells; c) inhibition of effector T cells; d) inhibiting regulatory T cell suppression of effector T cells; e) reducing the number of regulatory T cells in the tissue or circulation; f) activating effector T cells; g) binds GITR to a GITR complex; h) modulating the activity of human GITR and/or GITR complexes.

Embodiment 18: an isolated nucleic acid encoding an ABP according to any one of embodiments 1 to 12 or a bispecific antibody according to any one of embodiments 14 to 16 or a composite ABP according to embodiment 17.

Embodiment 19: an expression vector comprising the nucleic acid of embodiment 18.

Embodiment 20: a prokaryotic or eukaryotic host cell comprising the vector according to embodiment 19.

Embodiment 21: a method for producing a recombinant protein comprising the steps of expressing a nucleic acid according to embodiment 18 in a prokaryotic or eukaryotic host cell and recovering the protein from the cell or cell culture supernatant.

Embodiment 22: a method of treating a subject having cancer or an inflammatory disease comprising the step of administering to the subject a pharmaceutical composition comprising an ABP of any one of embodiments 1 to 12 or a bispecific antibody of any one of embodiments 14 to 16 or a composite ABP of embodiment 17.

Embodiment 23: a method for inducing or enhancing an immune response in a subject comprising the step of administering to the subject a pharmaceutical composition comprising an ABP of any one of embodiments 1 to 12 or a bispecific antibody of any one of embodiments 14 to 16 or a composite ABP of embodiment 17, wherein the immune response is raised against a tumor antigen.

Embodiment 24: the method of embodiment 23, wherein the ABP, the bispecific antibody or the composite ABP are administered in an amount sufficient to achieve in the subject one or more of: a) reducing the activity of regulatory T cells to suppress effector T cells; b) reducing the level of regulatory T cells; c) activating effector T cells; d) inducing or enhancing effector T cell proliferation; e) inhibiting tumor growth; and f) inducing tumor regression.

Embodiment 25: the method of embodiment 22, wherein the cancer is a solid cancer.

Embodiment 26: the method of embodiment 22, wherein the cancer is a hematologic cancer.

Embodiment 27: the method of any one of embodiments 22 to 26, wherein the method further comprises one or more of: a) administering chemotherapy; b) administering radiation therapy; or c) administering one or more additional therapeutic agents.

Embodiment 28: the method of embodiment 27, wherein the additional therapeutic agent comprises an immunostimulant.

Embodiment 29: the method of embodiment 27, wherein the immunostimulatory agent comprises an antagonist to an inhibitory receptor of an immune cell.

Embodiment 30: the method of embodiment 29, wherein the inhibitory receptor comprises CTLA-4, PD-1, PD-L1, LAG-3, Tim3, TIGIT, neuritis, BTLA or KIR, or a functional fragment thereof.

Embodiment 31: the method of embodiment 27, wherein the immunostimulatory agent comprises an agonist of a co-stimulatory receptor of an immune cell or a functional fragment thereof.

Embodiment 32: the method of embodiment 31, wherein the co-stimulatory receptor comprises OX40, ICOS, CD27, CD28, 4-1BB, or CD 40.

Embodiment 33: the method of embodiment 27, wherein the immunostimulant comprises a cytokine.

Embodiment 34: the method of embodiment 27, wherein the cytokine comprises IL-2, IL-5, IL-7, IL-12, IL-15, or IL-21.

Embodiment 35: the method of embodiment 27, wherein the immunostimulant comprises an oncolytic virus.

Embodiment 36: the method of embodiment 35, wherein the oncolytic virus comprises a herpes simplex virus, a vesicular stomatitis virus, an adenovirus, a newcastle disease virus, a vaccinia virus, or a malaba virus.

Embodiment 37: the method of embodiment 27, wherein the immunostimulant comprises chimeric antigen-engineered T cells.

Embodiment 38: the method of embodiment 27, wherein the immunostimulant comprises a bispecific or multispecific T cell-directed antibody.

Embodiment 39 the method of embodiment 27, wherein the additional therapeutic agent comprises an anti-TGF- β antibody or a TGF- β receptor trap.

Embodiment 40: the method of any one of embodiments 22 to 39, wherein administering the pharmaceutical composition results in: inducing or enhancing proliferation of T effector cells, or modulating I- κ B and/or NF- κ B in T cells, or modulating GITR activity in T cells, or T cell receptor-induced signaling in T effector cells, or a combination thereof.

Embodiment 41: a method of screening for a test compound comprising an ABP according to any one of embodiments 1 to 12 capable of inhibiting the interaction of GITRL with a GITR complex, comprising the steps of: contacting a sample containing GITRL and GITR complex with a compound; and determining whether the interaction of GITRL with the GITR complex in the sample is reduced relative to the interaction of GITRL with the GITR complex in a sample not contacted with the compound, whereby a reduction in the interaction of GITRL with GITR in the sample contacted with the compound identifies the compound as a compound that inhibits the interaction of GITRL with the GITR complex.

Examples

The following are examples of the methods and compositions of the present invention. It is to be understood that various other embodiments may be implemented in view of the general description provided herein.

Example 1: selection of GITR antigen binding proteins

Materials and methods

Antigen EZ + from Piercethio-NHS-biotinylation kit to biotinylation. Goat F (ab')₂Anti-human kappa-FITC (LC-FITC),PE (EA-PE) and streptavidin-AF 633(SA-633) were obtained from Southern Biotech, Sigma and Molecular Probes, respectively. StreptavidinAnd MACS LC separation columns from Miltenyi Biotec. Goat anti-human IgG-PE (human-PE) was obtained from Southern Biotech.

Original discovery

Eight original human synthetic yeast libraries (each of which has 10. about⁹Diversity) was amplified as previously described (see, e.g., y. xu et al, Addressing specificity of antibodies selected from among human and visual expression system: a FACS-based, high-throughput selection and genomic tool. peds 26.10,663-70 (2013); WO 2009036379; WO 2010105256; and WO 2012009568). For the first two rounds of selection, a magnetic bead sorting technique using the Miltenyi MACS system was performed as previously described (see, e.g., Siegel et al, "High efficiency recoverage and epiponce-specific monitoring of an scFv year display library ". J Immunol Methods 286(1-2), 141-. Briefly, yeast cells (. about.10)¹⁰Cells/library) were incubated with 5ml of 10nM biotinylated Fc fusion antigen in wash buffer (phosphate-buffered saline (PBS)/0.1% Bovine Serum Albumin (BSA)) for 30min at 30 ℃. After washing once with 40mL ice-cold wash buffer, the cell pellet was resuspended in 20mL wash buffer and streptavidin was added(500. mu.l) was added to the yeast and incubated at 4 ℃ for 15 min. Next, the yeast was pelleted, resuspended in 20mL of wash buffer, and loaded onto a Miltenyi LS column. After loading 20mL, the column was washed 3 times with 3mL of wash buffer. The column was then removed from the magnetic field and the yeast was eluted with 5mL of growth medium and then allowed to grow overnight. A subsequent selection round was performed using flow cytometry. About 2X 10⁷Yeast were pelleted, washed three times with wash buffer and incubated at 30 ℃ with reduced concentrations of biotinylated Fc fusion antigen (10 to 1nM), biotinylated Fc fusion antigen of different species at 10nM under equilibrium conditions in order to obtain species cross-reactivity, or incubated with a multispecific depleting agent (PSR) to remove non-specific antibodies from selection. For PSR depletion, the library was incubated with a 1:10 dilution of biotinylated PSR reagent, as previously described (see, e.g., Y. xu et al, addressing of antibiotics selected from an in vitro sensory presentation: a FACS-based, high-throughput selection and analytical tool. PEDS 26.10,663-70 (2013)). Next, the yeast was washed twice with wash buffer and stained with LC-FITC (1:100 dilution) and SA-633(1:500 dilution) or EAPE (1:50 dilution) secondary reagents for 15min at 4 ℃. After washing twice with wash buffer, the cell pellet was resuspended in 0.3mL of wash buffer and transferred to a filter-capped sorting tube. Sorting was performed using a FACS ARIA sorter (BD Biosciences), and sorting gating was determined to select antibodies with the desired characteristics. Repeat the selection round until all desired characteristics are obtainedThe population of (1). After the final sorting round, yeast were inoculated and single colonies were picked for characterization.

Antibody optimization

Optimization of the antibody was performed via a light chain diversification scheme and then by introducing diversity into the heavy and light chain variable regions, as described below. Combinations of some of these methods are used for each antibody.

Light chain batch diversification protocol: heavy chain plasmids from the original selection output were extracted from yeast via disruption and grab, amplified in and subsequently purified from E.coli, and transformed to have a size of 5X 10⁶A diverse light chain library of (a). Selection was performed with one round of MACS and four rounds of FACS using the same conditions as the original findings.

Light chain diversification: single antibody heavy chain variable region was amplified by PCR and transformed with a heavy chain expression vector to have a size of 5X 10⁶A diverse light chain library of (a). Selection was performed with one round of MACS and three rounds of FACS using the same conditions as the original findings. For each FACS round, the library is reviewed for PSR binding, species cross-reactivity, and affinity pressure, and sorted in order to obtain a population with the desired characteristics.

CDRH1 and CDRH2 are selected: recombination of CDRH3 of a single antibody into a pre-made library, wherein the CDRH1 and CDRH2 variants have a1 × 10⁸(iii) diversity; selection was performed with one round of MACS and four rounds of FACS as described in the original findings. For each FACS round, the library is reviewed for PSR binding, species cross-reactivity, and affinity pressure, and sorted in order to obtain a population with the desired characteristics. For these selections, affinity pressure was applied by pre-incubating the biotinylated antigen with the parent IgG for 30 minutes and then applying the pre-complexing mixture to the yeast library for a period of time that allowed the selection to reach equilibrium. Higher affinity antibodies can then be sorted.

CDRH3/VH mutant selection: oligonucleotides (oligomers) containing CDRH3 and flanking regions on either side of CDRH3 were ordered from the IDT. The amino acid positions in the CDRH 3-encoding portion of the oligonucleotides were diversified by NNK diversity. CDRH3 oligonucleotide was double-stranded using primers that annealed to the flanking regions of CDRH 3. The remainder of the heavy chain variable region was mutagenized via error-prone PCR to introduce additional diversity in the non-CDR 3 region of the heavy chain. Next, a library was created by transforming the double-stranded CDRH3 oligomer, the remainder of the mutagenized heavy chain variable region, and the heavy chain expression vector into yeast that already contained the parental light chain plasmid. Selection was performed using four rounds of FACS sorting similar to the previous cycle. For each FACS round, the library is reviewed for PSR binding, species cross-reactivity, and affinity pressure, and sorted to obtain a population with the desired characteristics. Affinity pressure for these selections was performed in CDRH1 and CDRH2 selections as described above.

CDRL1, CDRL2 and CDRL3 are selected: oligomers containing CDRL3 and flanking regions on either side of CDRL3 were ordered from IDTs. The amino acid positions in the CDRL 3-encoding portion of the oligonucleotide are diversified by NNK diversity. Primers annealing to the flanking regions of CDRL3 were used to double-stranded CDRL3 oligomer. These double-stranded CDRL3 oligomers were then recombined into a preformed library, with CDRL1 and CDRL2 variants having a 3 × 10 identity⁵And selected with one round of MACS and four rounds of FACS, as described in the original findings. For each FACS round, the library is reviewed for PSR binding, species cross-reactivity, and affinity pressure, and sorted to obtain a population with the desired characteristics. Affinity pressure for these selections was performed in CDRH1 and CDRH2 selections as described above.

Antibody production and purification

Yeast clones were grown to saturation and then induced at 30 ℃ for 48h with shaking. After induction, yeast cells were pelleted and the supernatant harvested for purification. IgG was purified using a protein a column and eluted with acetic acid at pH 2.0. Fab fragments were generated by papain digestion and by(GE Heathcarelifesciences) was purified.

ForteBio K_DMeasuring

In thatOn RED384Affinity measurements, generally as previously described (see, e.g., Estep et al, High throughput solution-based measurement of affinity-affinity and affinity binding. Mabs 5(2),270-278 (2013)). Briefly, ForteBio affinity measurements were performed by loading IgG onto AHQ sensors on-line. The sensor was equilibrated offline for 30min in assay buffer and then monitored online for 60 seconds for baseline establishment. The sensor with the loaded IgG was exposed to 100nM antigen for 3 minutes and then transferred to assay buffer for 3min for off-rate measurements. For monovalent affinity evaluation, Fab was used instead of IgG. For this evaluation, non-biotinylated Fc fusion antigen was loaded on AHQ sensors on-line. The sensor was equilibrated offline for 30min in assay buffer and then monitored online for 60 seconds for baseline establishment. The sensor with the loaded antigen was exposed to 200nM Fab for 3 minutes and then transferred to assay buffer for 3min for off-rate measurement. All kinetics were analyzed using a 1:1 binding model.

ForteBio epitope Classification (binding)/ligand blocking

Epitope sorting/ligand blocking was performed using standard sandwich format cross-blocking assays. Control anti-target IgG was loaded onto the AHQ sensor and the unoccupied Fc-binding sites on the sensor were blocked with irrelevant human IgG1 antibody. Next, the sensor is exposed to 100nM of the target antigen, followed by exposure to a second anti-target antibody or ligand. Additional binding of the secondary antibody or ligand after antigen binding indicates unoccupied epitopes (non-competitors), while no binding indicates epitope blocking (competitor or ligand blocking).

Cell binding assays

Approximately 100,000 antigen-overexpressing cells were washed with wash buffer and incubated with 100 μ l of 100nM IgG for 5 minutes at room temperature. Next, the cells were washed twice with wash buffer and incubated with 100. mu.l of 1:100 human-PE on ice for 15 minutes. Next, the cells were washed twice with washing buffer and analyzed with a FACS Canto II analyzer (BDBiosciences).

Size exclusion chromatography

SuperSW mAb HTP column (22855) was used to analyze mammal-produced mAbs with rapid SEC of 0.4mL/min with cycle time of 6 min/round. 200mM sodium phosphate and 250mM sodium chloride were used as mobile phases.

Dynamic scanning fluorometry

mu.L of 20 × Sypro orange was added to 20. mu.L of a 0.2mg to 1mg/mL mAb or Fab solution. An RT-PCR instrument (BioRad CFX96RT PCR) was used to ramp the sample plate temperature from 40 ℃ to 95 ℃ in 0.5 ℃ increments, with equilibration at each temperature for 2 min. The negative value of the first derivative of the raw data is used to obtain Tm.

Example 2: characterization of TM-form antibodies

Affinity of IgG1 and TM form ABP for GITR in several species

Use ofOCTET instrument evaluated the ability of GITR ABP to bind to recombinant hGITR (SEQ ID NO:1), hGITR-T43R (SEQ ID NO:2), cGITR (SEQ ID NO:3) and mGITR (SEQ ID NO: 4). The analysis was performed at 30 ℃ using 1 × kinetic Buffer (Kinetics Buffer) (ForteBio, Inc.) as the analysis Buffer. Anti-human IgGFc capture (AHC) biosensors (ForteBio, Inc.) were used to capture GITR ABPs onto the sensors. The sensor was equilibrated in assay buffer for 600 seconds prior to analysis. The baseline was established by submerging the sensor in 1 × assay buffer for 60 seconds. ABP was loaded onto the sensor by submerging the sensor into the ABP solution for 300 seconds. The baseline was established by submerging the sensor in 1 × assay buffer for 120 seconds. The sensor was quenched by immersion into 200. mu.g/ml human IgG for 300 seconds to prevent non-specific binding of GITR-Fc antigen to the sensor. The baseline was established by submerging the sensor in 1 × assay buffer for 60 seconds. Next, binding was monitored for 300 seconds in 25nM of recombinant antigen, and then dissociation was monitored for 1200 seconds in buffer alone. Use ofKinetic function of analytical software Using 1:1 binding model to determine K_D. The results are shown in table 5.

Table 5: ABP K determined by OCTET using single concentration kinetics_D

Use ofOctet RED384 Instrument, evaluating the ability of other GITR ABPs to bind to recombinant hGITR and cGITR (SEQ ID NO:3), generally as previously described (see, e.g., Estep et al, High throughput testing-based measurement of antibody-antigen affinity and epitopic binding. Mabs 5(2),270-278 (2013)). AHQ biosensors (ForteBio, Inc.) were used to capture GITR ABPs onto the sensors. The sensor was quenched by immersion into an irrelevant human IgG1 antibody to prevent nonspecific binding of GITR-Fc antigen to the sensor. The sensor was equilibrated in assay buffer for 30 minutes. The baseline was established by submerging the sensor in assay buffer for 60 seconds. Monitoring in 100nM recombinant antigenBinding was measured for 180 seconds and dissociation was subsequently monitored in buffer alone for 180 seconds. Use ofKinetic function of analytical software Using 1:1 binding model to determine K_D. The results are shown in table 6.

Table 6: ABP K determined by OCTET using single concentration kinetics_D

Non-optimized non-TM IgG1 ABP corresponding to ABP in brackets

H1H2 optimized non-TM IgG1 ABP corresponding to ABP in brackets

Additional K-Generation of 8N-terminal Fab TM Format antibodies Using Multi-concentration kinetics_DAnd (6) measuring. Use ofOCTET instrument evaluated the ability of GITR ABP to bind to recombinant hGITR (SEQ ID NO:1), hGITR-T43R (SEQ ID NO:2) and cGITR (SEQ ID NO: 3). The analysis was performed at 30 ℃ using 1 × kinetic buffer (ForteBio, Inc.) as analysis buffer. An anti-human IgG Fc capture (AHC) biosensor (ForteBio, Inc.) was used to capture GITR ABP onto the sensor. Prior to analysis, the sensor was saturated in assay buffer for 600 seconds. The baseline was established by submerging the sensor in 1 × assay buffer for 60 seconds. Tong (Chinese character of 'tong')ABP was loaded onto the sensor by submerging the sensor into the ABP solution for 300 seconds. The baseline was established by submerging the sensor in 1 × assay buffer for 120 seconds. Next, binding was monitored for 300 seconds in various concentrations of recombinant antigen (50nM to 0.78nM, 2-fold dilution in assay buffer), and then dissociation was monitored for 600 or 1200 seconds in buffer alone. Use ofKinetic function of analytical software Using 1:1 binding model to determine K_D. Determination of K by OCTET_DThe results are shown in fig. 2 and table 7. As can be seen in the figure, K of human GITR_DEach less than 1nM, and K of cynomolgus GITR_DK in human GITR_DWithin 15 times of. In addition, TM format antibodies bind to the T43R SNP variant of human GITR.

TABLE 7 ABP binding characteristics of the N-terminal Fab TM form

GITR binding assay

Peripheral Blood Mononuclear Cells (PBMCs) were cultured in growth medium in the presence of Phytohemagglutinin (PHA) at 37 ℃ for 5 days to up-regulate GITR expression. T cells were collected and washed, and then incubated with one of the eight TM forms of GITR ABP or a human isotype control antibody at 4 ℃. After washing, cells were incubated with fluorochrome-conjugated anti-human CD4 and anti-human CD8 antibodies and fluorochrome-conjugated anti-human IgG at 4 ℃ to detect bound anti-human gitabp. The percentage of GITR + CD4 and CD8 cells and the Mean Fluorescence Intensity (MFI) were determined using flow cytometry.

Exemplary results are shown in fig. 3. Fig. 3A shows CD4+ cells and fig. 3B shows CD8+ cells. In the top row, from left to right, an anti-GITR positive control and an anti-human IgG4 isotype control are shown, as well as the N-terminal Fab TM forms ABPABP9, ABP2, ABP1, and ABP 7. In the bottom row, ABP8, ABP3, ABP4, ABP5, ABP6, ABP23, and ABP24 are shown. The percentage of IgG4+ CD4/8+ cells is indicated.

Binding to cell surface human, cynomolgus and murine GITRs was assessed by transfection of HT1080, CHO or 293T cells with the corresponding (human, cynomolgus or murine) GITRs. Binding to cynomolgus GITR was further assessed using primary cynomolgus T cells and HSC-F T cell line (each after stimulation with anti-CD 3 antibody to increase GITR expression).

Activity assay for GITR antigen binding proteins

GITR ABP was tested for its ability to agonize GITR. In one assay, HT1080 cells stably expressing human or cynomolgus GITR were contacted with TM form GITR ABP or trimeric human GITRL. After 24 hours, the amount of IL-8 secreted by the cells was assessed by ELISA analysis. Increased IL-8 secretion corresponds to increased GITR agonism. The results for the N-terminal Fab TM form antibodies 1 to 8 are shown in figure 4. IL-8 output based on antibody or ligand concentration of ABP1 (fig. 4A), ABP2 (fig. 4B), ABP3 (fig. 4C), ABP4 (fig. 4D), ABP5 (fig. 4E), ABP6 (fig. 4F), ABP7 (fig. 4G), and ABP8 (fig. 4H) is shown. Antibodies are shown as circles in the figure, and GITRL controls are shown as squares. To EC₅₀Scoring EC for GITR ligand (GITRL)₅₀The scores are compared.

TABLE 8 agonistic activity of TM-form antibodies on human and cynomolgus GITR on HT1080 cells

The same analysis was performed using IgG1N297A non-TM versions of ABP 1-8 (see fig. 4), and the data are summarized in table 9.

TABLE 9 Activity of IgG1N297A antibodies on human and cynomolgus GITR on HT1080 cells

The assay was again used to test the activity of additional N-terminal Fab and C-terminal Fab TM format antibodies as well as the IgG1N297A format of those antibodies. The data are summarized in tables 10 and 11 and shown in fig. 5. Fig. 5A shows ABP43 (square), ABP23 (circle), ABP24 (triangle) and ABP29 (open circle), ABP30 (open triangle), ABP31 (open circle) and ABP32 (open triangle), all compared to GITRL (+ notation). FIG. 5B shows ABP19 (N-terminal Fab, triangle) and ABP25 (C-terminal Fab, inverted triangle). GITRL is shown as a plus sign. FIG. 5C shows ABP21 (N-terminal Fab, triangle) and ABP27 (C-terminal Fab, inverted triangle). GITRL is shown as a plus sign. FIG. 5D shows ABP20 (N-terminal Fab, triangle) and ABP26 (C-terminal Fab, inverted triangle). GITRL is shown as a plus sign. FIG. 5E shows ABP22 (N-terminal Fab, triangle) and ABP28 (C-terminal Fab, inverted triangle). GITRL is shown as a plus sign. As shown in the figure, the N-terminal Fab form of ABP tends to induce more IL-8 than its C-terminal Fab counterpart.

Figure 10 shows an additional comparison of ABP43 (non-optimized IgG4 format) to two corresponding TM formats of ABP, where the IgG 1Fab counterpart of ABP43 was added to the N-terminus or C-terminus of ABP 43. As can be seen in the figure, the N-terminal Fab TM form ABP9 (squares) induced the most IL-8 (and had the best EC) compared to the C-terminal Fab TM form ABP10 (circles) and the non-TM form ABP43 (diamonds)₅₀). Both TM forms of ABP have better activity than the non-TM forms of the parent ABP and GITRL. IL-8 induction by GITRL (positive control) is shown as a single data point (star), IL-8 production is shown in pg/mL.

TABLE 10 agonist activity of N-terminal Fab and C-terminal Fab TM format antibodies against human GITR on HT1080 cells

TABLE 11 agonist activity of IgG1N297A antibody format antibodies against human GITR on HT1080 cells

Additional H1H2/VH optimized antibodies in the form of IgG1N297A were provided and are shown in table 12 below and are indicated for their agonist activity as determined in the HT1080 assay as described above. These antibodies are suitable for making TM formats.

TABLE 12 agonist activity of IgG1N297A antibody format antibodies against human GITR on HT1080 cells

Similar analysis was performed in Jurkat cells, a T cell-based cell line engineered to express human GITR and NF- κ B luciferase reporterEight optimized, N-terminal FabTM-form antibodies, ABP1 to 8, were compared to GITRL for their ability to induce IL-8 in T cells. As shown in fig. 7A to 7H (ABP1 to 8) and table 13, all eight ABPs were better than GITRL in inducing cytokine production.

TABLE 13. 8 optimized TM forms with N-terminal Fab are superior to GITRL

ABP	EC₅₀(nM)	Activity compared with GITRL%
			1	0.30	104
GITRL	0.90
			2	0.21	114
GITRL	0.91
			3	0.19	120
GITRL	0.85
			4	0.22	117
GITRL	1.13
			5	0.25	104
GITRL	1.01
			6	0.29	121
GITRL	0.90
			7	0.20	108
GITRL	0.81
			8	0.30	114
GITRL	0.89

Agonistic activity of N-terminal Fab TM form antibodies in primary cells

In one embodiment, GITR ABP was further tested for its ability to bind to deliver a costimulatory signal to CD4+ CD 25-effector T cells via TCR signaling by anti-CD 3 antibodies. Increased T cell activation is measured by quantifying cell proliferation using ELISA assays and/or measuring increased production and secretion of cytokines including IFN- γ.

In another embodiment, GITR ABP is further analyzed to evaluate its ability to prevent the inhibition of effector T cells by regulatory T cells. Human CD4+ T cell isolation kits (Miltenyi #130-096) were used to isolate human CD4+ T cells. Using human CD25II (Miltenyi # 130-. CD25 depleted CD4+ T cells were used as effector T cells in an inhibition assay. Beads conjugated with anti-human CD2, CD3, and CD28 antibodies were used in the assay to activate effector T cells (Treg Suppression observer, also known as T cell activation beads, Miltenyi # 130-. In a 96-well plate, 50,000 regulatory T cells and effector T cells were seeded in each well, respectively, and incubated with an equal number of T cell activating beads. The mixture of cells and beads was exposed to different concentrations of GITR ABP for five days. Proliferation of effector T cells was measured by pulsing the cell culture with tritiated thymidine during the last 16 hours of culture, washing, and measuring the amount of radioactivity incorporated into the cells.

Agonistic activity of N-terminal Fab TM form antibodies in T-blast primary cell assay

Pre-stimulated T cells (T-blasts) were used to evaluate the activity of eight N-terminal Fab TM format antibodies (ABP1 to 8).

To generate T-blasts, PBMCs were stimulated with PHA for 5 to 7 days to expand the cells and induce the expression of GITR. Cells were washed and frozen prior to functional assay setup.

To evaluate the functional activity of ABP1 to 8, cells were stimulated by adding 1mg/ml anti-CD 3+2mg/ml anti-CD 28, 0.003-10mg/ml ABP1 to 8, and 10mg/ml GITRL as a positive control. Cells were incubated at 37 ℃ and supernatants were collected after 48 hours. By using(Perkin) To measure IL-2 production. Figure 8A shows the percentage of GITR + CD4+ cells (left) and CD8+ cells (right) in cells from two human donors at each time point, with/without PHA stimulation.

FIGS. 8B-8M show the results of treatment of T-blasts from 4 different donors treated with control or ABP and the resulting IL-2 production. In each figure, the top row, left to right, is a FACS measurement of ABO binding to CD4+ cells, IL-2 production in cells from donor 1, IL-2 production in cells from donor 2. In the bottom row, from left to right, are FACS measurements of ABP binding to CD8+ cells, IL-2 production in cells from donor 3, IL-2 production in cells from donor 4. The data are shown below: 8B: IgG4 isotype control; 8C: SEC4 antibody; 8D: IgG4TM format control; 8E: non-TM IgG4 non-optimized lineage parents of ABPs 1-8 (ABP 9); 8F: ABP 1; 8G: ABP 2; 8H: ABP 3; 8I: ABP 4; 8J: ABP 5; 8K: ABP 6; 8L: ABP 7; 8M: ABP 8.

As shown in the figure, T-blast cells from different donors differed in their response to ABP1 to 8 treatment; however all eight TM form ABP treatments had agonist activity at the highest concentrations tested, as measured by production of IL-2 and compared to control antibodies.

Comparison of GITR agonist Activity of optimized N-terminal Fab TM form ABP and non-TM form IgG1 ABP

The same eight optimized N-terminal Fab TM forms ABP (1 to 8) as in the above examples were compared to the non-optimized parental controls in the same IL-8 induction assay described above and shown in figure 4. Each figure shows a comparison of N-terminal Fab TM versions ABP1 to 8 (IgG4S228P with N-terminal IgG 1Fab, "IgG 4 TM") and the corresponding non-TM versions IgG1(IgG1N297, "IgG 1") ABP, as well as GITRL and IgG4 negative controls as positive controls (GITRL and IgG4 were performed on separate plates different from ABP). The left panel shows results from cells expressing hGITR and the right panel shows results from cells expressing cGITR. IL-8 induction with ABP concentration is shown for ABP1 (fig. 9A), ABP2 (fig. 9B), ABP3 (fig. 9C), ABP4 (fig. 9D), ABP5 (fig. 9E), ABP6 (fig. 9F), ABP7 (fig. 9G), and ABP8 (fig. 9H). IL-8 induction by GITRL is shown as circles, IL-8 induction by the TM-form antibody is shown as triangles, IL-8 induction by the parent IgG1 antibody is shown as diamonds, and IL-8 induction by the IgG4 control antibody is shown as squares. EC is shown on the bottom of each picture of each figure₅₀Table of values.

The results shown in figure 9 indicate that the eight optimized ABPs are able to induce much more IL-8 production of the double N-terminal Fab TM form antibody in human or cynomolgus monkey GITR expressing cells when compared to the bivalent IgG1N 297A. The N-terminal Fab TM format antibody has a smaller EC than the bivalent IgG1N297A format₅₀Values and higher IL-8 production was maximal. In addition, all TM antibodies had higher affinity for GITR than the non-TM form of the parent antibody (compare tables 5 and 7 to table 6).

Evaluation of GITRL blockade

In another embodiment, use is made ofOCTET instrument evaluated GITR blockade. The analysis was performed at 30 ℃ using 1 × kinetic buffer (ForteBio, Inc.) as the analysis buffer. An anti-human IgG Fc capture (AHC) biosensor (ForteBio, Inc.) was used to capture human GITR ABP onto the sensor. Prior to analysis, the sensor was saturated in assay buffer for 300 seconds. ABP was loaded onto the sensor by submerging the sensor into the ABP supernatant solution for 300 seconds. The baseline was established by submerging the sensor in 1 × assay buffer for 200 seconds. Next, binding of recombinant GITR was monitored for 180 seconds. The ability of recombinant GITRL to bind to the ABP/GITR complex was then determined by immersing the sensor in GITRL for 180 seconds.

GITR blockade was evaluated using ABP1 to 8 according to the above method. All eight ABPs are able to block GITRL binding.

In one embodiment, to evaluate whether anti-human GITR ABP blocks GITR ligand (GITRL, R & DSystems #6987-GL-CF) binding to GITR, various concentrations of unlabeled GITR ABP were incubated with human pan T cells in staining buffer (e.g., phosphate buffered saline with 0.5% bovine serum albumin) at 4 ℃ for 10 min. Without washing, 4nM HA-labeled human GITRL was added and incubated at 4 ℃ for another 30 min. Next, the cells were washed twice with staining buffer and incubated with anti-HA PE antibody (Miltenyi # 130-. Next, cells were washed twice with staining buffer and fixed with PBS containing 2% paraformaldehyde for flow cytometry analysis. A decrease in the amount of PE staining indicates that ABP blocks GITR-GITRL interaction.

Example 3: multispecific antigen binding proteins

One embodiment of a multispecific GITR agonistic ABP comprises a common light chain antibody. ABPs that bind two different epitopes on GITR were identified. Both ABPs share the same light chain. ABP61 is an agonistic antibody, and ABP59 has lower agonistic activity but does not compete with ABP61 for binding to GITR. Multivalent multispecific antibodies with a common light chain were produced by transfecting HEK-293 host cells with vectors encoding two heavy chains and a single common light chain. A first exemplary ABP disclosed herein having a common light chain (SEQ ID NO:125) is ABP33, which has an IgG4 heavy chain (ABP61) with an N-terminal Fab (from ABP58 (IgG1 counterpart to ABP 59)), and the full-length sequences SEQ ID NO:124(HC) and SEQ ID NO:125 (LC). A second exemplary ABP disclosed herein having a common light chain is ABP34, which has an IgG4 heavy chain (ABP61) with a C-terminal Fab (from ABP58 (IgG1 counterpart to ABP 59)), and the full-length sequences SEQ ID NO:136(HC) and SEQ ID NO:125 (LC).

Each multispecific ABP binds to two different epitopes on GITR. ABPs are characterized as described in the examples above. Other multispecific ABPs described herein were generated and similarly characterized. FIG. 6 shows EC of ABP33 and ABP34 in HT1080 analysis₅₀The results of the assay were as described in example 2 above. ABP33 (IgG4 ABP61 with the IgG1 counterpart of ABP58[ ABP59 ] on the N-terminus]Tetravalent form of the IgG 1Fab combination) and ABP34 (compare IgG4 of ABP61 with ABP58 on the C-terminus [ IgG1 counterpart of ABP 59)]Tetravalent form of IgG 1Fab combinations) were compared to bivalent ABPs 59 and 61(IgG4S 228P). As shown in the figure, both tetravalent antibodies have superior EC when compared to their divalent counterparts₅₀As measured by IL-8 induction.

Example 4: comparison of TM-form antibodies with reference anti-GITR antibodies

The activity of N-terminal Fab TM format antibodies against two anti-GITR antibodies SEC4 and SEC9 was tested in HT1080 cells engineered to stably express human GITR. Cells were cultured for six hours with a series of concentrations of two baseline agonist antibodies SEC4 ("35E 6", orange diamonds formatted to have the mouse variable region and the human IgG4S 228P/kappa region) and SEC9 (humanized 6C8N62Q IgG1N 297A). SEC4 is described, for example, in U.S. patent No. 8,709,424; the variable regions are found in SEQ ID NO 1 and SEQ ID NO 12. SEC9 is described, for example, in U.S. patent No. 7,812,135; the full length sequences are shown in SEQ ID NO 58 and SEQ ID NO 63. A bivalent antibody dose of 1. mu.g/ml is equivalent to 6.67 nM; the tetravalent antibody dose of 1. mu.g/ml is equivalent to 4 nM.

IL-8 induction was measured by ELISA and EC was calculated₅₀. Fig. 11A shows comparison of hGITRL with SEC4 (diamonds) and SEC9 (circles), as well as IgG4 negative control (filled triangles), IgG1 negative control (open triangles), and trimeric human GITR ligand ("hGITRL," squares) as a positive control. GITRL has a better EC than both SEC4 and SEC9₅₀(inset) and maximum induction.

Comparisons of GITRL with ABP1 (fig. 11B), ABP2 (fig. 11C), ABP3 (fig. 11D), ABP4 (fig. 11E), ABP5 (fig. 11F), ABP6 (fig. 11G), ABP7 (fig. 11H), and ABP8 (fig. 11I) are also shown. As shown in the figure, all eight N-terminal Fab TM format antibodies have a more favorable EC than SEC4 or SEC9₅₀. All eight N-terminal Fab TM format antibodies also had higher induction of IL-8 production than SEC4 or SEC 9.

SEC4 was also tested in a T-blast primary cell assay as described above. As shown in fig. 8, IL-2 induction by SEC4 was less in primary cells than TM-form antibodies for all four donors.

Example 5: anti-GITR ABP increases cytokine production in tumor infiltrating lymphocytes and GITR + T cells of patients

Materials and methods

Dissociated human tumor samples were purchased from Conversant bio samples were NSCLC adenocarcinoma isolated from 75 year old men (previous smokers with phase Ia disease) prior to any treatment samples were flash thawed, followed by restimulation with several conditions with or without immunotherapy with cells as unstimulated control, or with 1 μ g/mL α CD3 (soluble) +2 μ g/mL α CD28 (soluble) + IL-2(50ng/mL), cells not receiving immunotherapy treatment (for assessment of checkpoint protein levels), receiving pamumab (10 μ g/mL), TM form ABP control (2 μ g/mL), ABP1(2 μ g/mL), or ABP1+ pamumab treatment the cells were incubated for 48 hours prior to collection of supernatant and staining for checkpoint expression.

Results

Cells were gated against GITR positive T cells and the results are shown in fig. 12A (TNF production) and fig. 12B (IFN γ production). As shown in the figure, single agent treatment with anti-GITR (ABP1) caused increased cytokine production. In cells treated with the combination ABP1+ pembrolizumab, the increase in cytokine production was not significant in this assay.

Example 6: mutation analysis for epitope determination: alanine scanning

To identify the epitope to which ABP1 binds to human GITR, a single point mutation was performed in the extracellular domain of human GITR to determine if APB1 binding was reduced. Alanine substitutions or murine specific residues were used (ABP1 did not bind mouse GITR). The protein is expressed in HEK-293 cells (with Fc tag) and secreted as a soluble protein inLx was purified on resin and characterized by SDS-PAGE. Binding was assessed by biolayer interferometry (BLI) using the Octet platform. Wild-type human GITR-Fc or GITR-Fc mutations were captured on anti-human Fc sensors, washed and exposed to ABP1 Fab. Residues considered part of the binding epitope exhibit reduced or no binding (e.g., K binding compared to wild-type human GITR)_DMore than 3 times different). Alanine substitutions at residues C58, R59, Y61, E64, C67 and aspartic acid at position C66 resulted in no binding, whereas alanine substitutions at residues R56, D60, P62 or E65 resulted in reduced binding.

Example 7: in vivo evaluation of GITR antigen binding proteins

In vivo studies were performed to demonstrate the ability of ABP to reduce the number of circulating regulatory T cells in a humanized NSG mouse model. Newborn NSG mice were injected retroorbitally with CD34⁺Human fetal liver cell transplantation. Over 16 weeks, animals developed a wide variety of CD4⁺And CD8⁺A pool of human immune cells that are effector T cells and regulatory T cells. Mice were given a single intraperitoneal dose of 25mg/kg anti-human gitabp or human isotype control, and the percentage of circulating human CD4+ T cells expressing the regulatory T cell marker FoxP3 was determined by flow cytometry on day 4 post-dose.

Example 8 incubation of activated T cells with TM form ABP1 or its conventional form parent ABP35

Preparation of activated T blast cells

By heating at 37 ℃ and 5% CO₂Next, freshly isolated PBMCs from two healthy donors were stimulated with PHA (final concentration 10. mu.g/ml) and IL-2 (final concentration 4ng/ml, added only during the last 24 h) for 7 days to generate activated T blast cells.

Internalization of GITR upon antibody binding

At 2X 10⁵Activated CD4+/CD8+ T blast cells were seeded per well in 96-well U-bottom plates. Wells Medium alone, recombinant GITR-ligand (10. mu.g/ml, R)&D Systems, Cat #6987-GL-025/CF), ABP1 (-250 kDa), hIgG4TM format isotype control, ABP35 (-150 kDa) or hIgG1 standard format isotype control were each treated with nine doses: 10. mu.g/mL, 2. mu.g/mL, 0.4. mu.g/mL, 80ng/mL, 16ng/mL, 3.2ng/mL, 0.64ng/mL, 0.13ng/mL and 0.026 ng/mL.

Internalization of GITR upon antibody binding

Antibody-mediated clustering promotes endocytosis and signaling of TNF receptor superfamily members, such as GITR. The ability of ABP1 and ABP35 (as well as isotype controls) to mediate clustering and internalization was measured.

Activated T blast cells were first stained for FACS sorting. At room temperature with humanFcX blocks Fc receptors for 10 minutes. Cells were incubated with fluorescent conjugated antibody for 30 minutes at 4 ℃ as shown in table 14. Next, cells were washed 2 x with FACS buffer, fixed in paraformaldehyde for 30 minutes in the dark, washed again, and resuspended in 200 μ l FACS buffer and collected on BD Fortessa instrument.

GITR internalization was measured in CD4+ cells (fig. 13A-13E) and CD8+ cells (fig. 13F-13J) from donor 1 (fig. 13A-13B, 13F-13G) or donor 2 (fig. 13C-13D, 13H-13I). Cells were treated with ABP1TM format antibody or ABP35 standard bivalent antibody. As can be seen, incubation with either ABP1 or ABP35 inhibited subsequent staining with ABP1Dylight650 (fig. 13A, 13C, 13F, 13H), but incubation with only ABP1 induced GITR internalization as measured by staining with non-competitive clone 108-17 (fig. 13B, 13D, 13G, 13I). The assay of ABP1 for EC50 for cells from both donors is shown in fig. 13E (CD4+ cells) and fig. 13J (CD8+ cells).

TABLE 14 fluorescent dye labeling

Cytokine production in activated T cells treated with ABP1 or ABP35

At 5X 10⁴One well in 96-well U-shaped bottom plates were seeded with activated CD4+/CD8+ T blast cells. Wells Medium alone, recombinant GITR-ligand (10. mu.g/ml, R)&D Systems, Cat #6987-GL-025/CF), ABP1, hIgG4TM format isotype control, ABP35, or hIgG1 standard format isotype control were each treated with nine doses: 10. mu.g/mL, 2. mu.g/mL, 0.4. mu.g/mL, 80ng/mL, 16ng/mL, 3.2ng/mL, 0.64ng/mL, 0.13ng/mL and 0.026 ng/mL.

By adding 1. mu.g/ml anti-CD 3 antibody (mouse anti-human, clone UCHT-1, R)&DSystems, Cat # MAB100) and 2. mu.g/ml anti-CD 28 antibody (mouse anti-human, clone 37407, R)&D Systems, Cat # MAB342) to stimulate the cells. At 37 ℃ and 5% CO₂The lower incubation was analyzed for 48 h. By using(Perkin Elmer) to measure IL-2 production.

Incubation of the T blast cells with TM form antibody ABP1 resulted in internalization of GITR by activated CD4+ and CD8+ T cells (fig. 13), but not with the standard bivalent form of the same antibody (ABP35) or either isotype control.

In addition, hyperaggregation of GITR receptors by ABP1 promotes IL-2 secretion by activated T blast cells in a dose-dependent manner (fig. 14), but not by conventional bivalent ABP. Collectively, these data show that T blast cells that do not respond to conventional anti-GITR therapy respond to the corresponding TM form antibody.

Example 9: cytokine production in ABP 1-treated activated T cells compared to two reference antibodies SEC4 and SEC9

Activated T blast cells were prepared as described in example 8 and used to compare the activity of ABP1 with the baseline anti-GITR antibodies SEC4 and SEC 9. By heating at 37 ℃ and 5% CO₂Freshly isolated PBMCs from two healthy donors were stimulated with PHA (final concentration 10. mu.g/ml) and IL-2 (final concentration 4ng/ml, added only during the last 24 h) for 7 days to generate activated TomuA cell.

At 5X 10⁴One well in 96-well U-shaped bottom plates were seeded with activated CD4+/CD8+ T blast cells. Wells were treated with medium alone, recombinant GITR-ligand, ABP1, SEC4, SEC9, hIgG4TM format isotype control ("IsoTM"), hIgG1 standard format isotype control, and hIgG4 standard format isotype control, each at nine doses: 10. mu.g/mL, 2. mu.g/mL, 0.4. mu.g/mL, 80ng/mL, 16ng/mL, 3.2ng/mL, 0.64ng/mL, 0.13ng/mL and 0.026 ng/mL.

Cytokine production in activated T cells treated with ABP1, SEC4, and SEC9

Cells were stimulated by the addition of 1. mu.g/ml anti-CD 3 antibody and 2. mu.g/ml anti-CD 28 antibody. Cells were incubated at 37 ℃ and 5% CO₂And (5) incubating for 48 h. By using(Perkin Elmer) to measure IL-2 production. As seen in example 8, TM form ABP1 promotes IL-2 secretion from activated T blasts in a dose-dependent manner. IL-2 production by activated T cells is shown in FIG. 15A (donor 1) and FIG. 15B (donor 2). The corresponding EC50 is shown in fig. 15C (donor 1) and fig. 15D (donor 2).

As can be seen in the figure, SEC4 and SEC9 did not induce IL-2 efficiently in a dose-dependent manner, ABP1 did induce IL-2 production at higher levels than SEC4 and SEC9 in a significant dose-dependent manner. Overall, these data support that T blast cells that do not respond to the fuwhat anti-GITR therapy respond to the corresponding TM form antibody.

Identity of

The above disclosure may cover a number of different inventions with independent utility. While each of these inventions has been disclosed in its preferred form, the specific embodiments thereof as disclosed and illustrated herein are not to be considered in a limiting sense as numerous variations are possible. The subject matter of the inventions includes all novel and non-obvious combinations and subcombinations of the various elements, features, functions and/or properties disclosed herein. The following claims particularly point out certain combinations and subcombinations regarded as novel and nonobvious. Inventions embodied in other combinations and subcombinations of features, functions, elements, and/or properties may be claimed in this, a priority application of this, or a related application. Such claims, whether they are directed to a different invention or directed to the same invention, and whether broader, narrower, equal, or different in scope to the original claims, also are regarded as included within the subject matter of the inventions of the present disclosure.

Appendix A: sequence of

Claims

1. An isolated multivalent Antigen Binding Protein (ABP) that specifically binds human GITR (hGITR; SEQ ID NO:1) comprising the following six CDR sequences:

a. having the sequence X₁X₂X₃X₄X₅RGYGDYGGHHAFDI CDR-H3, wherein X₁Is A or V, X₂Is H, D, L or R, X₃Is E or D, X₄Is R, N, S or A, and X₅V, D or G (SEQ ID NO: 141);

b. having the sequence X₁IX₂X₃SGX₄TYYNPSLKS CDR-H2, wherein X₁Is G, L or S, X₂Is Y, A or V, X₃Is E, Y or H, and X₄Is S or K (SEQ ID NO: 142);

c. having the sequence X₁SISSX₂X₃X₄X₅WX₆CDR-H1 of (1), wherein X₁Is Y or G, X₂Is G, S or E, X₃Is L, G, S, Y or A, X₄Is G, A, Y, M or G, X₅V, A or absent, and X6 is S or G (SEQ ID NO: 143);

d. having the sequence QQEYX₁TPPX₂CDR-L3 of (1), wherein X₁Is A or N and X₂Is T or S (SEQ ID NO: 144);

e. having the sequence X₁AX₂SLX₃X₄CDR-L2 of (1), wherein X₁Is A or S, X₂Is D or S, X₃Is Q, D, K or E, and X₄Is S or Y (SEQ ID NO: 145); and

f. having the sequence X₁ASX₂SIX₃CDR-L1 of X4YLN, wherein X₁Is G or R, X₂Is Q or K, X3 is S, D or N, and X₄Is S or T (SEQ ID NO: 146).

2. The ABP of claim 1, wherein the ABP comprises:

CDR-H3 of SEQ ID NO. 13, CDR-H2 of SEQ ID NO. 12, CDR-H1 of SEQ ID NO. 11, CDR-L3 of SEQ ID NO. 16, CDR-L2 of SEQ ID NO. 15, and CDR-L1 of SEQ ID NO. 14;

CDR-H3 of SEQ ID NO. 23, CDR-H2 of SEQ ID NO. 22, CDR-H1 of SEQ ID NO. 21, CDR-L3 of SEQ ID NO. 17, CDR-L2 of SEQ ID NO. 15, and CDR-L1 of SEQ ID NO. 14;

CDR-H3 of SEQ ID NO. 30, CDR-H2 of SEQ ID NO. 29, CDR-H1 of SEQ ID NO. 28, CDR-L3 of SEQ ID NO. 17, CDR-L2 of SEQ ID NO. 31, and CDR-L1 of SEQ ID NO. 14;

CDR-H3 of SEQ ID NO. 30, CDR-H2 of SEQ ID NO. 29, CDR-H1 of SEQ ID NO. 28, CDR-L3 of SEQ ID NO. 17, CDR-L2 of SEQ ID NO. 15, and CDR-L1 of SEQ ID NO. 36;

CDR-H3 of SEQ ID NO. 30, CDR-H2 of SEQ ID NO. 29, CDR-H1 of SEQ ID NO. 28, CDR-L3 of SEQ ID NO. 17, CDR-L2 of SEQ ID NO. 41, and CDR-L1 of SEQ ID NO. 14;

CDR-H3 of SEQ ID NO. 48, CDR-H2 of SEQ ID NO. 47, CDR-H1 of SEQ ID NO. 46, CDR-L3 of SEQ ID NO. 17, CDR-L2 of SEQ ID NO. 50, and CDR-L1 of SEQ ID NO. 49;

CDR-H3 of SEQ ID NO. 48, CDR-H2 of SEQ ID NO. 47, CDR-H1 of SEQ ID NO. 46, CDR-L3 of SEQ ID NO. 56, CDR-L2 of SEQ ID NO. 55, and CDR-L1 of SEQ ID NO. 54;

CDR-H3 of SEQ ID NO. 61, CDR-H2 of SEQ ID NO. 60, CDR-H1 of SEQ ID NO. 59, CDR-L3 of SEQ ID NO. 16, CDR-L2 of SEQ ID NO. 15, and CDR-L1 of SEQ ID NO. 14;

CDR-H3 of SEQ ID NO. 61, CDR-H2 of SEQ ID NO. 47, CDR-H1 of SEQ ID NO. 46, CDR-L3 of SEQ ID NO. 16, CDR-L2 of SEQ ID NO. 15, and CDR-L1 of SEQ ID NO. 14; or

CDR-H3 of SEQ ID NO. 103, CDR-H2 of SEQ ID NO. 22, CDR-H1 of SEQ ID NO. 21, CDR-L3 of SEQ ID NO. 16, CDR-L2 of SEQ ID NO. 15, and CDR-L1 of SEQ ID NO. 14.

3. The ABP of claim 2, wherein:

a. the ABP of claim 2.a comprises V of SEQ ID NO 9_HSequence and V of SEQ ID NO 10_LA sequence;

b. the ABP of claim 2.b comprises V of SEQ ID NO 19_HSequence and V of SEQ ID NO 20_LA sequence;

c. the ABP of claim 2.c comprises V of SEQ ID NO 26_HSequence and V of SEQ ID NO 27_LA sequence;

d. the ABP of claim 2.d comprises V of SEQ ID NO 26 or SEQ ID NO 34_HSequence and V of SEQ ID NO 35_LA sequence;

e. the ABP of claim 2.e comprising V of SEQ ID NO 26_HSequence and V of SEQ ID NO 40_LA sequence;

f. the ABP of claim 2.f comprising V of SEQ ID NO 44_HSequence and V of SEQ ID NO 45_LA sequence;

g. the ABP of claim 2.g comprises V of SEQ ID NO 44_HSequence and V of SEQ ID NO 53_LA sequence;

h. the ABP of claim 2.h comprises V of SEQ ID NO 58_HSequence and V of SEQ ID NO 10_LA sequence;

i. the ABP of claim 2.i comprises V of SEQ ID NO 104_HSequence and V of SEQ ID NO 10_LA sequence; and is

j. The ABP of claim 2.j comprises V of SEQ ID NO 105_HSequence and V of SEQ ID NO 10_LAnd (4) sequencing.

4. The ABP of claim 3, wherein:

a. the ABP of claim 3.a comprises the heavy chain of SEQ ID NO. 7 and the light chain of SEQ ID NO. 8;

b. the ABP of claim 3.b comprises the heavy chain of SEQ ID NO 17 and the light chain of SEQ ID NO 18;

c. the ABP of claim 3.c comprises the heavy chain of SEQ ID NO. 24 and the light chain of SEQ ID NO. 25;

d. the ABP of claim 3.d comprising (i) the heavy chain of SEQ ID NO. 32 and the light chain of SEQ ID NO. 33, or (ii) the heavy chain of SEQ ID NO. 37 and the light chain of SEQ ID NO. 33;

e. the ABP of claim 3.e comprising (i) the heavy chain of SEQ ID NO 38 and the light chain of SEQ ID NO 39;

f. the ABP of claim 3.f comprises (i) the heavy chain of SEQ ID NO. 42 and the light chain of SEQ ID NO. 43;

g. the ABP of claim 3.g comprises (i) the heavy chain of SEQ ID NO. 51 and the light chain of SEQ ID NO. 52;

h. the ABP of claim 3.h comprises (i) the heavy chain of SEQ ID NO. 57 and the light chain of SEQ ID NO. 8;

i. the ABP of claim 3i comprises (i) the heavy chain of SEQ ID NO:114 and the light chain of SEQ ID NO:8, or (ii) the heavy chain of SEQ ID NO:120 and the light chain of SEQ ID NO: 8; or (iii) the heavy chain of SEQ ID NO 122 and the light chain of SEQ ID NO 8; or

j. The ABP of claim 3j comprises (i) the heavy chain of SEQ ID NO. 115 and the light chain of SEQ ID NO. 8, or (ii) the heavy chain of SEQ ID NO. 121 and the light chain of SEQ ID NO. 8; or (iii) the heavy chain of SEQ ID NO 123 and the light chain of SEQ ID NO 8.

5. An isolated multivalent Antigen Binding Protein (ABP) that specifically binds human GITR (hGITR; SEQ ID NO:1) comprising the following six CDR sequences:

a. CDR-H3 having the sequence shown in SEQ ID NO. 66;

b. CDR-H2 having the sequence shown in SEQ ID NO. 65;

c. CDR-H1 having the sequence shown in SEQ ID NO 64;

d. CDR-L3 having the sequence shown in SEQ ID NO: 69;

e. CDR-L2 having the sequence shown in SEQ ID NO. 68; and

f. CDR-L1 having the sequence shown in SEQ ID NO 67.

6. The ABP of claim 5, wherein:

a. the ABP comprises V of SEQ ID NO:62_HSequence and V of SEQ ID NO 63_LA sequence;

b. the ABP comprises V of SEQ ID NO 70_HSequence and V of SEQ ID NO 63_LA sequence; or

c. The ABP comprises V of SEQ ID NO 97_HSequence and V of SEQ ID NO 63_LAnd (4) sequencing.

7. The ABP of claim 6, wherein:

a. the ABP of claim 6.a comprises the heavy chain of SEQ ID NO 171 and the light chain of SEQ ID NO 172;

b. the ABP of claim 6.b comprises the heavy chain of SEQ ID NO 173 and the light chain of SEQ ID NO 174;

c. the ABP of claim 6.c comprises (i) the heavy chain sequence of SEQ ID NO 106 and the light chain sequence of SEQ ID NO 107; or (ii) the heavy chain sequence of SEQ ID NO:116 and the light chain sequence of SEQ ID NO: 107.

8. An isolated multivalent Antigen Binding Protein (ABP) that specifically binds human GITR (hGITR; SEQ ID NO:1) comprising the following six CDR sequences:

a. CDR-H3 having the sequence shown in SEQ ID NO. 75;

b. CDR-H2 having the sequence shown in SEQ ID NO: 74;

c. CDR-H1 having the sequence shown in SEQ ID NO. 73;

d. CDR-L3 having the sequence shown in SEQ ID NO. 78;

e. CDR-L2 having the sequence shown in SEQ ID NO. 77; and

f. CDR-L1 having the sequence shown in SEQ ID NO 75.

9. The ABP of claim 8, wherein:

a. the ABP comprises V of SEQ ID NO 71_HSequence and V of SEQ ID NO 72_LA sequence; or

b. The ABP comprises V of SEQ ID NO 98_HSequence and V of SEQ ID NO 72_LAnd (4) sequencing.

10. The ABP of claim 9, wherein:

a. the ABP of claim 9.a comprises the heavy chain of SEQ ID NO 173 and the light chain of SEQ ID NO 109; or

b. The ABP of claim 9.b comprises (i) the heavy chain of SEQ ID NO 108 and the light chain of SEQ ID NO 109; or (ii) the heavy chain of SEQ ID NO:117 and the light chain of SEQ ID NO: 109.

11. An isolated multivalent Antigen Binding Protein (ABP) that specifically binds human GITR (hGITR; SEQ ID NO:1) comprising the following six CDR sequences:

a. CDR-H3 having the sequence shown in SEQ ID NO 83;

b. CDR-H2 having the sequence set forth in (i) SEQ ID NO:82 or (ii) SEQ ID NO: 100;

c. CDR-H1 having the sequence shown in SEQ ID NO: 81;

d. CDR-L3 having the sequence shown in SEQ ID NO 86;

e. CDR-L2 having the sequence shown in SEQ ID NO. 85; and

f. CDR-L1 having the sequence shown in SEQ ID NO: 84.

12. The ABP of claim 11, wherein:

a. the ABP comprises the CDR-H2 sequence of claim 11.b (i) and V of SEQ ID NO. 79_HSequence and V of SEQ ID NO 80_LA sequence;

b. the ABP comprises the CDR-H2 sequence of claim 11.b (i) and V of SEQ ID NO. 87_HSequence and V of SEQ ID NO 80_LA sequence;

c. the ABP comprises the CDR-H2 sequence of claim 11.b (i) and V of SEQ ID NO. 88_HSequence and V of SEQ ID NO 80_LA sequence;

d. the ABP comprises the CDR-H2 sequence of claim 11.b (ii) and V of SEQ ID NO. 99_HSequence and V of SEQ ID NO 80_LAnd (4) sequencing.

13. The ABP of claim 12, wherein:

a. the ABP of claim 12.a comprises the heavy chain of SEQ ID NO 174 and the light chain of SEQ ID NO 111;

b. the ABP of claim 12.b comprises the heavy chain of SEQ ID NO. 175 and the light chain of SEQ ID NO. 111;

c. the ABP of claim 12.c comprises the heavy chain of SEQ ID NO 176 and the light chain of SEQ ID NO 111;

d. the ABP of claim 12.d comprises (i) the heavy chain of SEQ ID NO. 110 and the light chain of SEQ ID NO. 111; or (ii) the heavy chain of SEQ ID NO:118 and the light chain of SEQ ID NO: 111.

14. An isolated multivalent Antigen Binding Protein (ABP) that specifically binds human GITR (hGITR; SEQ ID NO:1) comprising the following six CDR sequences:

a. 93, CDR-H3 having the sequence shown in SEQ ID NO;

b. having the sequence GIIPIFGEAQYAQX₁FX₂CDR-H2 of G, wherein X₁Is K or R, and X₂Is Q or R (SEQ ID NO: 215);

c. CDR-H1 having the sequence shown in SEQ ID NO. 91;

d. CDR-L3 having the sequence shown in SEQ ID NO 94;

e. CDR-L2 having the sequence shown in SEQ ID NO. 85; and

f. CDR-L1 having the sequence shown in SEQ ID NO: 84.

15. The ABP of claim 14, wherein the ABP comprises:

CDR-H2 of SEQ ID NO. 92;

CDR-H2 of SEQ ID NO. 96; or

CDR-H2 of SEQ ID NO. 102.

16. The ABP of claim 15, wherein:

a. the ABP of claim 15.a comprises V of SEQ ID NO. 89_HSequence and V of SEQ ID NO 90_LA sequence;

b. the ABP of claim 15.b comprises V of SEQ ID NO 95_HSequence and V of SEQ ID NO 90_LA sequence; and

c. the ABP of claim 15.c comprises V of SEQ ID NO 101_HSequence and V of SEQ ID NO 90_LAnd (4) sequencing.

17. The ABP of claim 16, wherein:

a. the ABP of claim 16.a comprises the heavy chain of SEQ ID NO 177 and the light chain of SEQ ID NO 113;

b. the ABP of claim 16.b comprises the heavy chain of SEQ ID NO. 178 and the light chain of SEQ ID NO. 113;

c. the ABP of claim 16.c comprises (i) the heavy chain of SEQ ID NO:112 and the light chain of SEQ ID NO: 113; or (ii) the heavy chain of SEQ ID NO:119 and the light chain of SEQ ID NO: 113.

18. An isolated multivalent Antigen Binding Protein (ABP) that specifically binds human GITR (hGITR; SEQ ID NO:1) comprising the following six CDR sequences:

a. CDR-H3 having the sequence shown in SEQ ID NO: 134;

b. 133, CDR-H2 having the sequence set forth in SEQ ID NO;

c. CDR-H1 having the sequence shown in SEQ ID NO: 132;

d. CDR-L3 having the sequence shown in SEQ ID NO. 135;

e. CDR-L2 having the sequence shown in SEQ ID NO. 68; and

f. CDR-L1 having the sequence shown in SEQ ID NO 67.

19. The ABP of claim 18, wherein the ABP comprises (i) V of SEQ ID NO 126_HSequence and V of SEQ ID NO 128_LA sequence; or (ii) V of SEQ ID NO:127_HSequence and V of SEQ ID NO 128_LAnd (4) sequencing.

20. The ABP of claim 18 or claim 19, wherein said ABP comprises (i) a heavy chain of SEQ ID NO:124 and a light chain of SEQ ID NO: 125; or (ii) the heavy chain of SEQ ID NO:136 and the light chain of SEQ ID NO: 125.

21. An isolated multivalent Antigen Binding Protein (ABP) that specifically binds human GITR (hGITR; SEQ ID NO:1) comprising:

a. and V_H(ii) a CDR-H3 having at least about 80% identity to CDR-H3 of the region, the V_HThe region is selected from the group consisting of SEQ ID NO 9, SEQ ID NO 19, SEQ ID NO 26, SEQ ID NO 34, SEQ ID NO 44, SEQ ID NO 58, SEQ ID NO 62, SEQ ID NO 70, SEQ ID NO 71, SEQ ID NO 79, SEQ ID NO 87, SEQ ID NO 88, SEQ ID NO 89, SEQ ID NO 95, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99, SEQ ID NO 101, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 126 and SEQ ID NO 127;

b. and V_H(ii) a CDR-H2 having at least about 80% identity to CDR-H2 of the region, the V_HThe region is selected from the group consisting of SEQ ID NO 9, SEQ ID NO 19, SEQ ID NO 26, SEQ ID NO 34, SEQ ID NO 44, SEQ ID NO 58, SEQ ID NO 62, SEQ ID NO 70, SEQ ID NO 71, SEQ ID NO 79, SEQ ID NO 87, SEQ ID NO 88, SEQ ID NO 89, SEQ ID NO 95, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99, SEQ ID NO 101, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO:126 and SEQ ID NO: 127;

c. and V_H(ii) a CDR-H1 having at least about 80% identity to CDR-H1 of the region, the V_HThe region is selected from the group consisting of SEQ ID NO 9, SEQ ID NO 19, SEQ ID NO 26, SEQ ID NO 34, SEQ ID NO 44, SEQ ID NO 58, SEQ ID NO 62, SEQ ID NO 70, SEQ ID NO 71, SEQ ID NO 79, SEQ ID NO 87, SEQ ID NO 88, SEQ ID NO 89, SEQ ID NO 95, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99, SEQ ID NO 101, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 126 and SEQ ID NO 127;

d. and V_L(ii) a CDR-L3 having at least about 80% identity to CDR-L3 of the region, the V_LThe region is selected from the group consisting of SEQ ID NO 10, SEQ ID NO 20, SEQ ID NO 27, SEQ ID NO 35, SEQ ID NO 40, SEQ ID NO 45, SEQ ID NO 53, SEQ ID NO 63, SEQ ID NO 72, SEQ ID NO 80, SEQ ID NO 90 and SEQ ID NO 128;

e. and V_L(ii) a CDR-L2 having at least about 80% identity to CDR-L2 of the region, the V_LThe region is selected from the group consisting of SEQ ID NO 10, SEQ ID NO 20, SEQ ID NO 27, SEQ ID NO 35, SEQ ID NO 40, SEQ ID NO 45, SEQ ID NO 53, SEQ ID NO 63, SEQ ID NO 72, SEQ ID NO 80, SEQ ID NO 90 and SEQ ID NO 128; and

f. and V_L(ii) a CDR-L1 having at least about 80% identity to CDR-L1 of the region, the V_LThe region is selected from the group consisting of SEQ ID NO 10, SEQ ID NO 20, SEQ ID NO 27, SEQ ID NO 35, SEQ ID NO 40, SEQ ID NO 45, SEQ ID NO 53, SEQ ID NO 63, SEQ ID NO 72, SEQ ID NO 80, SEQ ID NO 90 and SEQ ID NO 128.

22. The ABP of claim 21, wherein the CDR-H3, CDR-H2, CDR-H1, CDR-L3, CDR-L2 and CDR-L1 are each determined according to a numbering scheme selected from Kabat numbering scheme, Chothia numbering scheme or IMGT numbering scheme.

23. The ABP of any one of claims 21 or 22, wherein said CDR-H1 is identified as defined by both Chothia and Kabat numbering schemes, including the boundaries of both numbering schemes.

24. The ABP of any one of claims 21 to 23, wherein:

a. the CDR-H3 comprises a CDR-H3 selected from: 13, 23, 30, 48, 61, 66, 75, 83, 93, 103, 131, or variants thereof having 1,2 or 3 amino acid substitutions;

b. the CDR-H2 comprises a CDR-H2 selected from: 12, 22, 29, 47, 60,65, 74, 82, 92, 96, 100, 102, 130 and 133, or variants thereof having 1,2 or 3 amino acid substitutions;

c. the CDR-H1 comprises a CDR-H1 selected from: 11, 21, 28, 46, 59, 64, 73, 81, 91, 129, 132, or variants thereof having 1 or 2 amino acid substitutions;

d. the CDR-L3 comprises a CDR-L3 selected from: 16, 17, 56, 69, 78, 86, 94, 135, or variants thereof having 1 or 2 amino acid substitutions;

e. the CDR-L2 comprises a CDR-L2 selected from: 15, 31, 41, 50, 55, 68, 77 and 85, or variants thereof having 1 amino acid substitution; and

f. the CDR-L1 comprises a CDR-L1 selected from: 14, 36, 49, 54, 67, 76 and 84, or variants thereof having 1 or 2 amino acid substitutions.

25. The ABP of any one of claims 1-24, wherein the amino acid substitution is a conservative amino acid substitution.

26. The ABP of any one of claims 1 to 25, wherein the ABP:

a. competes for binding to GITR with an antibody selected from the group consisting of: ABP1, ABP2, ABP3, ABP4, ABP5, ABP6, ABP7, ABP8, ABP9, ABP10, ABP11, ABP12, ABP13, ABP14, ABP15, ABP16, ABP17, ABP18, ABP19, ABP20, ABP21, ABP22, ABP23, ABP24, ABP25, ABP26, ABP27, ABP28, ABP29, ABP30, ABP31, ABP32, ABP33, and ABP34, each as provided in appendix a of the present disclosure;

b. having at least three antigen binding domains that specifically bind to an epitope on GITR;

c. having at least three antigen binding domains that specifically bind a single epitope on GITR;

d. having at least four antigen binding domains that specifically bind to an epitope on GITR;

e. having at least four antigen binding domains that specifically bind a single epitope on GITR;

f. agonizing GITR expressed on the surface of a target cell;

g. blocking the binding of GITRL to GITR;

h. costimulating effector T cells in conjunction with antigen presentation by antigen-presenting cells;

i. inhibiting suppression of effector T cells by regulatory T cells;

j. reducing the number of regulatory T cells in the tissue or systemic circulation;

k. capable of binding to one or more of the GITR (SEQ ID NO:1) residues from the group consisting of: r56, C58, R59, D60, Y61, P62, E64, E65, C66 and C67; or

Can be any combination of (a) to (k).

27. The ABP of any one of claims 1-26, wherein the GITR is selected from the group consisting of hGITR (SEQ ID NO:1), hGITR-T43R (SEQ ID NO:2), cGITR (SEQ ID NO:3), mGITR (SEQ ID NO:4), and combinations thereof.

28. The ABP of any one of claims 1-26, wherein the ABP (a) specifically binds cynomolgus monkey GITR (cGITR; SEQ ID NO: 3); (b) binds murine GITR (mGITR; SEQ ID NO:4) or does not bind mGITR with a lower affinity (as indicated by a higher KD) than the affinity of the ABP for hGITR; or (c) can be any combination of (a) to (b).

29. The ABP of any one of claims 1 to 26, wherein the ABP: (a) specifically binds cGITR (SEQ ID NO: 3); (b) binds mGITR (SEQ ID NO:4) with a lower affinity (as indicated by a higher KD) than the ABP's affinity for hGITR and cGITR; and (c) enhances the binding of GITRL to GITR.

30. An ABP that competes for binding to GITR with the ABP of any one of claims 1 to 26, wherein the ABP: (a) specifically binds cGITR (SEQ ID NO: 3); (b) binds mGITR (SEQ ID NO:4) with a lower affinity (as indicated by a higher KD) than the ABP's affinity for hGITR and cGITR; and (c) enhances the binding of GITRL to GITR.

31. The ABP of any one of claims 1-30, wherein the ABP comprises an antibody.

32. The ABP of claim 31, wherein the antibody is a monoclonal antibody.

33. The ABP of claim 31 or claim 32, wherein the antibody is selected from a human antibody, a humanized antibody, or a chimeric antibody.

34. The ABP of any one of claims 1-33, wherein the ABP is multivalent.

35. The ABP of any one of claims 1-30, wherein the ABP comprises an antibody fragment.

36. The ABP of any of claims 1-30, wherein the ABP comprises a substituted backbone.

37. The ABP of any one of claims 1-30, wherein the ABP comprises an immunoglobulin constant region.

38. The ABP of claim 37, wherein the ABP comprises a heavy chain constant region selected from the IgA, IgD, IgE, IgG, or IgM class.

39. The ABP of claim 38, wherein said ABP comprises an IgG class and a heavy chain constant region selected from the subclasses IgG4, IgG1, IgG2, or IgG 3.

40. The ABP of any one of claims 1 to 39, wherein at least one Fab is fused to the C-terminus of the Fc domain of an IgG.

41. The ABP of any one of claims 1 to 39, further comprising at least one linker.

42. The ABP of claim 39, wherein the IgG is IgG 4.

43. The ABP of claim 39, wherein the IgG is IgG 1.

44. The ABP of any one of claims 1 to 43, wherein at least one Fab is fused to the N-terminus of the Fc domain of an IgG.

45. The ABP of claim 44, wherein the at least one Fab is at least two Fab.

46. The ABP of claim 44, wherein the at least one Fab is at least three Fab.

47. The ABP of claim 44, wherein the at least one Fab is at least four Fab.

48. The ABP of any one of claims 45 to 47, wherein two Fab are independently fused to the N-terminus of the IgG.

49. The ABP of any one of claims 45 to 47, wherein two Fab are independently fused to the C-terminus of the IgG.

50. The ABP of claim 48, wherein a Fab is attached to each N-terminus of said IgG, a linker is attached to each of said Fab, and a Fab is attached to each linker.

51. The ABP of claim 49, wherein a Fab is attached to each C-terminal end of said IgG, a linker is attached to each of said Fab, and a Fab is attached to each linker.

52. The ABP of claim 50 or claim 51, wherein each linker comprises SEQ ID NO 5.

53. The ABP of claim 50 or claim 51, wherein each linker comprises SEQ ID NO 6.

54. The ABP of any one of claims 1-53, wherein the ABP comprises a common light chain antibody, an antibody with a knob-hole modification, an scFv linked to an IgG, a Fab linked to an IgG, a bifunctional antibody, a tetravalent bispecific antibody, a DVD-Ig^TM、DART^TM、CovX-body, Fcab antibody,tandem Fab, Zybody^TMOr a combination thereof.

55. The ABP of claim 34, wherein the ABP binds to more than one GITR molecule.

56. The ABP of claim 26.f, wherein agonism of GITR by the ABP is independent of GITRL binding.

57. The ABP of claim 26.f, wherein the ABP enhances binding of GITRL to GITR by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

58. The ABP of claim 57, wherein the ABP enhances binding of GITRL to GITR by at least about 50%.

59. The ABP of claim 26.f, wherein the target cell is selected from effector T cells, regulatory T cells, Natural Killer (NK) cells, natural killer T (nkt) cells, dendritic cells, and B cells.

60. The ABP of claim 26.f, wherein the target cell is an effector T cell selected from the group consisting of a helper (CD4+) T cell, a cytotoxic (CD8+) T cell, and a combination thereof.

61. The ABP of claim 26.f, wherein the target cell is a regulatory T cell selected from the group consisting of a CD4+ CD25+ Foxp3+ regulatory T cell, a CD8+ CD25+ regulatory T cell, and a combination thereof.

62. The ABP of claim 26.j, wherein the tissue is a tumor.

63. The ABP of any one of claims 1-62, wherein said first antigen binding domain is directed against K of hGITR (SEQ ID NO:1) or hGITR-T43R (SEQ ID NO:2)_DLess than about 20 nM.

64. The ABP of any one of claims 1-62, wherein said first antigen binding domain is directed against K of cGITR (SEQ ID NO:3)_DLess than about 200 nM.

65. The ABP of any one of claims 1-62, wherein said second antigen-binding domain is directed against K of hGITR (SEQ ID NO:1) or hGITR-T43R (SEQ ID NO:2)_DLess than about 100 nM.

66. The ABP of any one of claims 1-62, wherein the second antigen binding domain is directed against K of cGITR (SEQ ID NO:3)_DLess than about 1 μ M.

67. The ABP of any one of claims 1 to 66, wherein the ABP comprises an Fc domain having reduced effector function as compared to an IgG1 Fc domain.

68. The ABP of any one of claims 1-67, wherein the ABP comprises an aglycosylated Fc domain.

69. The ABP of any one of claims 1-67, wherein the ABP comprises an IgG1 Fc domain having an alanine at one or more of positions 234, 235, 265, and 297.

70. The ABP of any one of claims 1 to 69, wherein the GITR is expressed on the surface of a target cell.

71. The ABP of any one of claims 1-70, wherein the ABP multimerizes GITR expressed on the surface of a target cell.

72. The ABP of claim 71, wherein the ABP multimerizes 2, 3,4, 5,6, 7,8, 9, 10, 11, or 12 GITR molecules.

73. The ABP of any one of claims 1-72, wherein the ABP comprises an immunoglobulin comprising at least two different heavy chain variable regions each paired with a common light chain variable region.

74. The ABP of claim 73, wherein the common light chain variable region forms a different antigen binding domain with each of the two different heavy chain variable regions.

75. The ABP of claim 73 or claim 74, wherein the ABP comprises a first VH variable domain having SEQ ID NO:189, a second VH variable domain having SEQ ID NO:215, and a common variable light chain having SEQ ID NO: 190.

76. The ABP of claim 73 or claim 74, wherein the ABP comprises a first VH variable domain having SEQ ID NO 199, a second VH variable domain having SEQ ID NO 216, and a common variable light chain having SEQ ID NO 200.

77. A kit comprising the ABP of any one of claims 1-76 and instructions for use of the ABP.

78. The kit of claim 77, wherein the ABP is lyophilized.

79. The kit of claim 78, further comprising a liquid for reconstituting the lyophilized ABP.

80. The ABP of any of claims 1-76, wherein the ABP comprises a polypeptide sequence having a pyroglutamic acid (pE) residue at its N-terminus.

81. The ABP of any one of claims 1 to 76, wherein the ABP comprises a VH sequence in which the N-terminal Q is substituted with pE.

82. The ABP of any of claims 1-76, wherein the ABP comprises a VL sequence wherein the N-terminal E is substituted with pE.

83. The ABP of any one of claims 1 to 76, wherein the ABP comprises a heavy chain sequence in which the N-terminal Q is substituted with pE.

84. The ABP of any one of claims 1 to 76, wherein the ABP comprises a light chain sequence in which the N-terminal E is substituted with pE.

85. The ABP of any of claims 1-76, for use as a medicament.

86. The ABP of any of claims 1-76, for use in the treatment of cancer or a viral infection.

87. The ABP of claim 85, for use in the treatment of cancer, wherein said cancer is selected from the group consisting of a solid tumor and a hematologic tumor.

88. An isolated polynucleotide encoding the ABP of any one of claims 1 to 76, V thereof_HV thereof_LIts light chain, its heavy chain or its antigen binding portion.

89. A vector comprising the polynucleotide of claim 88.

90. A host cell comprising the polynucleotide of claim 88 or the vector of claim 89.

91. The host cell of claim 90, wherein the host cell is selected from the group consisting of a bacterial cell, a fungal cell, and a mammalian cell.

92. The host cell of claim 90, wherein the host cell is selected from the group consisting of an E.coli cell, a Saccharomyces cerevisiae cell, and a CHO cell.

93. A cell-free expression reaction comprising the polynucleotide of claim 88 or the vector of claim 89.

94. A method of producing the ABP of any of claims 1-76, comprising expressing the ABP in the host cell of claim 90 and isolating the expressed ABP.

95. A pharmaceutical composition comprising the ABP of any one of claims 1-76 and a pharmaceutically acceptable excipient.

96. The pharmaceutical composition of claim 95, wherein the amount of said ABP in the pharmaceutical composition is sufficient to (a) reduce suppression of effector T cells by regulatory T cells in a subject; (b) activating effector T cells; (c) reducing the number of regulatory T cells in a tissue or systemically; (d) inducing or enhancing proliferation of effector T cells; (e) inhibiting the rate of tumor growth; (f) inducing tumor regression; or (g) combinations thereof.

97. A pharmaceutical composition according to claim 95 or claim 96 for use as a medicament.

98. The pharmaceutical composition of any one of claims 95 to 97 for use in the treatment of cancer or a viral infection.

99. The pharmaceutical composition of claim 98, for use in the treatment of cancer, wherein the cancer is selected from the group consisting of a solid tumor and a hematologic tumor.

100. A pharmaceutical composition comprising the ABP of any one of claims 1-76 and a pharmaceutically acceptable excipient.

101. The pharmaceutical composition of claim 100, wherein the pharmaceutical composition the ABP is in an amount sufficient to (a) reduce suppression of effector T cells by regulatory T cells in a subject; (b) activating effector T cells; (c) reducing the number of regulatory T cells in a tissue or systemically; (d) inducing or enhancing proliferation of effector T cells; (e) inhibiting the rate of tumor growth; (f) inducing tumor regression; or (g) combinations thereof.

102. A method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of the ABP of any one of claims 1-76 or the pharmaceutical composition of any one of claims 100 and 101.

103. A method of enhancing activation of an immune cell in a subject, comprising administering to the subject an effective amount of the ABP of any one of claims 1-76 or the pharmaceutical composition of any one of claims 100 and 101.

104. The method of claim 102 or 103, wherein the disease or disorder is cancer.

105. The method of any one of claims 102 to 103, wherein the method induces or enhances an immune response against a cancer-associated antigen.

106. The method of any one of claims 102 to 105, wherein the ABP is administered in an amount sufficient to: (a) reducing suppression of effector T cells by regulatory T cells; (b) activating effector T cells; (c) reducing the number of regulatory T cells in a tissue or systemically; (d) inducing or enhancing proliferation of effector T cells; (e) inhibiting the rate of tumor growth; (f) inducing tumor regression; or (g) combinations thereof.

107. The method of any one of claims 104 to 106, wherein the cancer is a solid cancer.

108. The method of any one of claims 104 to 106, wherein the cancer is a hematologic cancer.

109. The method of any one of claims 102 to 108, further comprising administering one or more additional therapeutic agents.

110. The method of claim 109, wherein the additional therapeutic agent is selected from the group consisting of radiation therapy, cytotoxic agents, chemotherapeutic agents, cytostatic agents, anti-hormonal agents, EGFR inhibitors, immunostimulatory agents, anti-angiogenic agents, and combinations thereof.

111. The method of claim 109, wherein the additional therapeutic agent is an immunostimulant.

112. The method of claim 111, wherein the immunostimulatory agent comprises an agent that blocks signaling by an inhibitory receptor or ligand thereof expressed by an immune cell.

113. The method of claim 112, wherein the inhibitory receptor or ligand thereof expressed by the immune cell is selected from the group consisting of CTLA-4, PD-1, PD-L1, NRP-1, LAG-3, Tim3, TIGIT, neuritin, BTLA, KIR, and combinations thereof.

114. The method of claim 111, wherein the immunostimulatory agent comprises an agonist to a stimulatory receptor expressed by an immune cell.

115. The method of claim 113, wherein the stimulatory receptor expressed by the immune cell is selected from the group consisting of OX40, ICOS, CD27, CD28, 4-1BB, CD40, and combinations thereof.

116. The method of claim 111, wherein the immunostimulatory agent comprises a cytokine.

117. The method of claim 116, wherein the cytokine is selected from the group consisting of IL-2, IL-5, IL-7, IL-12, IL-15, IL-21, and combinations thereof.

118. The method of claim 115, wherein the immunostimulant comprises an oncolytic virus.

119. The method of claim 118, wherein the oncolytic virus is selected from the group consisting of herpes simplex virus, vesicular stomatitis virus, adenovirus, newcastle disease virus, vaccinia virus, malaba virus, and combinations thereof.

120. The method of claim 111, wherein the immunostimulatory agent comprises a T cell that expresses a chimeric antigen receptor.

121. The method of claim 111, wherein the immunostimulatory agent comprises a bispecific or multispecific T cell-directed antibody.

122. The method of claim 111, wherein the immunostimulatory agent comprises an anti-TGF- β antibody, a TGF- β trap, or a combination thereof.

123. The method of claim 111, wherein the immunostimulant comprises a vaccine against a cancer-associated antigen.

124. A method of modulating an immune response in a subject in need thereof, comprising administering to the subject an effective amount of the ABP of any one of claims 1-76 or the pharmaceutical composition of any one of claims 99-105.

125. The method of any one of claims 106 to 128, further comprising administering to the subject one or more additional therapeutic agents.

126. The method of claim 129, wherein the additional therapeutic agent is an agonist of a stimulatory receptor for an immune cell and the stimulatory receptor for an immune cell is selected from the group consisting of OX40, CD2, CD27, CDs, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), 4-1BB (CD137), CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, CD83 ligand, and combinations thereof.

127. The method of claim 129, wherein the additional therapeutic agent is a cytokine selected from the group consisting of IL-2, IL-5, IL-7, IL-12, IL-15, IL-21, and combinations thereof.

128. The method of claim 129, wherein the additional therapeutic agent is an oncolytic virus selected from the group consisting of herpes simplex virus, vesicular stomatitis virus, adenovirus, newcastle disease virus, vaccinia virus, malaba virus, and combinations thereof.

129. The method of any one of claims 125-128, wherein the additional therapeutic agent is formulated in the same pharmaceutical composition as the ABP.

130. The method of any one of claims 125-128, wherein the additional therapeutic agent is formulated in a different pharmaceutical composition than the ABP.

131. The method of any one of claims 125-128 or 130, wherein the additional therapeutic agent is administered prior to administration of the ABP.

132. The method of any one of claims 125-128 or 130, wherein the additional therapeutic agent is administered after administration of the ABP.

133. The method of any one of claims 125-132, wherein the additional therapeutic agent is administered concurrently with the ABP.

134. The ABP of any of claims 1-76, wherein the ABP specifically binds to an epitope of human GITR (hGITR; SEQ ID NO:1) and is capable of binding to one or more residues from the group consisting of: r56, C58, R59, D60, Y61, P62, E64, E65, C66 and C67.

135. An isolated multivalent Antigen Binding Protein (ABP) that specifically binds human GITR (hGITR; SEQ ID NO:1), wherein the ABP competes for binding with one or more of: ABP1, ABP2, ABP3, ABP4, ABP5, ABP6, ABP7, ABP8, ABP9, ABP10, ABP11, ABP12, ABP13, ABP14, ABP15, ABP16, ABP17, ABP18, ABP19, ABP 363672, ABP19, ABP 36363672, ABP19, ABP 363672, ABP19, ABP 36363672, ABP19, ABP 363672, ABP19, ABP 36363672, ABP19, ABP 363672, ABP19, ABP 363636363672, ABP19, ABP 3636363672, ABP 363672, ABP 3636363672, ABP 36363672, ABP19, ABP 363672, ABP 36363672.

136. An anti-human GITR antibody, or antigen-binding fragment thereof, comprising four heavy chain variable regions and four light chain variable regions,

wherein the heavy chain variable region comprises CDR-H3 consisting of SEQ ID NO:13, CDR-H2 consisting of SEQ ID NO:12 and CDR-H1 consisting of SEQ ID NO: 11;

and the light chain variable region comprises CDR-L3 consisting of SEQ ID NO:16, CDR-L2 consisting of SEQ ID NO:15 and CDR-L1 consisting of SEQ ID NO: 14; and

wherein one heavy chain variable region and one light chain variable region constitute one antigen binding site, and wherein the anti-human GITR antibody or the antigen binding fragment comprises a total of four antigen binding sites.

137. The anti-human GITR antibody or antigen-binding fragment thereof of claim 136 selected from (1) or (2):

(1) an anti-human GITR antibody or antigen-binding fragment thereof, comprising four heavy chain variable regions and four light chain variable regions, wherein the heavy chain variable region consists of SEQ ID NO 9,

the light chain variable region consists of SEQ ID NO 10, and

the one heavy chain variable region and the one light chain variable region constitute one antigen binding site, and the antibody or antigen binding fragment comprises four antigen binding sites; and

(2) an anti-human GITR antibody or antigen-binding fragment thereof comprising four heavy chain variable regions and four light chain variable regions, wherein each heavy chain variable region consists of SEQ ID NO 9, wherein Q at position 1 of the sequence is modified to pyroglutamic acid,

the light chain variable region consists of SEQ ID NO 10, and

the one heavy chain variable region and the one light chain variable region constitute one antigen binding site, and the antibody or the antigen binding fragment comprises four antigen binding sites.

138. The anti-human GITR antibody of claim 136, wherein the antibody comprises two heavy chains and four light chains: each heavy chain comprises a first heavy chain variable region and a second heavy chain variable region each comprising CDR-H3 consisting of SEQ ID NO:13, CDR-H2 consisting of SEQ ID NO:12, and CDR-H1 consisting of SEQ ID NO: 11; a first CH1 region, a linker, a second CH1 region, a CH2 region, and a CH3 region; and each light chain comprises a light chain variable region comprising CDR-L3 consisting of SEQ ID NO:16, CDR-L2 consisting of SEQ ID NO:15, and CDR-L1 consisting of SEQ ID NO: 14.

139. The anti-human GITR antibody of claim 136 selected from (1) or (2):

(1) an anti-human GITR antibody comprising two heavy chains and four light chains, wherein each heavy chain comprises two structures consisting of the heavy chain variable region of SEQ ID NO:9 and the CH1, CH2, and CH3 regions, and the C-terminus of one of the structures is linked to the N-terminus of the other structure via a linker; and each light chain comprises a light chain variable region and a light chain constant region of SEQ ID NO. 10; and

(2) an anti-human GITR antibody comprising two heavy chains and four light chains, wherein each heavy chain comprises two structures consisting of the heavy chain variable region of SEQ ID No. 9 and the regions CH1, CH2, and CH3, and the C-terminus of one of the structures is linked to the N-terminus of the other structure via a linker, wherein Q at position 1 of the sequence is modified to pyroglutamic acid; and is

Each light chain comprises the light chain variable region and light chain constant region of SEQ ID NO 10.

140. The anti-human GITR antibody of claim 136 selected from (1) to (4):

(1) an anti-human GITR antibody comprising two heavy chains consisting of SEQ ID No. 7 and four light chains consisting of SEQ ID No. 8;

(2) an anti-human GITR antibody comprising two heavy chains consisting of SEQ ID NO 7, wherein Q at position 1 is modified to pyroglutamic acid; and four light chains consisting of SEQ ID NO 8;

(3) an anti-human GITR antibody comprising two heavy chains consisting of the amino acid sequence ranging from Q at position 1 to G at position 686 of SEQ ID No. 7 and four light chains consisting of SEQ ID No. 8;

(4) anti-human GITR antibodies; comprising two heavy chains consisting of amino acid sequences ranging from Q at position 1 to G at position 686 of SEQ ID NO 7, wherein the Q at position 1 is modified to pyroglutamic acid; and four light chains consisting of SEQ ID NO 8.

141. A polynucleotide selected from the group consisting of (a) and (b):

(a) a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human GITR antibody or antigen-binding fragment thereof of claim 137 (1); and

(b) a polynucleotide comprising a base sequence encoding the light chain variable region of the anti-human GTIR antibody or antigen-binding fragment thereof according to claim 137 (1).

142. A polynucleotide selected from the group consisting of (a) and (b):

(a) a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human GITR antibody of claim 140 (1); and

(b) a polynucleotide comprising a base sequence encoding the light chain of the anti-human GTIR antibody of claim 140 (1).

143. An expression vector comprising:

(a) a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human GITR antibody or antigen-binding fragment thereof according to claim 137(1), and/or

(b) A polynucleotide comprising a base sequence encoding the light chain variable region of the anti-human GITR antibody or antigen-binding fragment thereof of claim 137 (1).

144. An expression vector comprising:

(a) a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human GITR antibody of claim 140(1), and/or

(b) A polynucleotide comprising a base sequence encoding the light chain of the anti-human GITR antibody of claim 140 (1).

145. A host cell transformed with an expression vector selected from the group consisting of (a) to (d):

(a) a host cell transformed with an expression vector comprising: a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human GITR antibody or antigen-binding fragment thereof of claim 137 (1); and a polynucleotide comprising a base sequence encoding the variable region of the light chain of the antibody or the antigen-binding fragment thereof;

(b) host cells transformed with expression vectors: an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human GITR antibody or antigen-binding fragment thereof according to claim 137 (1); and an expression vector comprising a polynucleotide comprising a base sequence encoding the variable region of the light chain of the antibody or the antigen-binding fragment thereof;

(c) a host cell transformed with an expression vector comprising: a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human GITR antibody or antigen-binding fragment thereof of claim 137 (1); and

(d) a host cell transformed with an expression vector comprising: a polynucleotide comprising a base sequence encoding the variable region of the light chain of the anti-human GITR antibody or antigen-binding fragment thereof of claim 137 (1).

146. A host cell transformed with an expression vector selected from the group consisting of (a) to (d):

(a) a host cell transformed with an expression vector comprising: a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human GITR antibody of claim 140 (1); and a polynucleotide comprising a base sequence encoding the light chain of the antibody;

(b) host cells transformed with the following expression vectors: an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human GITR antibody of claim 140 (1); and an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain of the antibody;

(c) a host cell transformed with an expression vector comprising: a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human GITR antibody of claim 140 (1); and

(d) a host cell transformed with an expression vector comprising: a polynucleotide comprising a base sequence encoding the light chain of the anti-human GITR antibody of claim 140 (1).

147. A method for producing an anti-human GITR antibody or antigen-binding fragment thereof, comprising culturing a host cell selected from the group consisting of the following (a) to (c) to express a tetravalent anti-human GITR antibody or antigen-binding fragment thereof:

(a) a host cell transformed with an expression vector comprising: a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human GITR antibody or antigen-binding fragment thereof according to claim 137 (1); and a polynucleotide comprising a base sequence encoding the variable region of the light chain of the antibody or the antigen-binding fragment thereof;

(b) host cells transformed with the following expression vectors: an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human GITR antibody or antigen-binding fragment thereof according to claim 137 (1); and an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody or the antigen-binding fragment thereof; and

(c) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human GITR antibody or antigen-binding fragment thereof according to claim 137 (1); and a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the variable region of the light chain of the antibody or the antigen-binding fragment thereof.

148. A method for producing an anti-human GITR antibody, comprising culturing a host cell selected from the group consisting of the following (a) to (c) to express the anti-human GITR antibody:

(b) host cells transformed with the following expression vectors: an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human GITR antibody of claim 140 (1); and an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain of the antibody; and

(c) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human GITR antibody of claim 140 (1); and a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain of the antibody.

149. A pharmaceutical composition comprising the anti-human GITR antibody of claim 140 and a pharmaceutically acceptable excipient.

150. A pharmaceutical composition comprising an anti-human GITR antibody according to claim 140(1) and an anti-human GITR antibody according to claim 140(4) and a pharmaceutically acceptable excipient.

151. The pharmaceutical composition of any one of claims 149 and 150, which is a pharmaceutical composition for preventing or treating cancer.

152. The pharmaceutical composition of any one of claims 149 and 150, wherein the composition is administered in combination with radiation therapy, cytotoxic agents, chemotherapeutic agents, cytostatic agents, anti-hormonal agents, EGFR inhibitors, immunostimulants, anti-angiogenic agents, and combinations thereof.

153. The anti-human GITR antibody of claim 140 for use in the prevention or treatment of cancer.

154. Use of the anti-human GITR antibody of claim 140 for the manufacture of a pharmaceutical composition for the prevention or treatment of cancer.

155. A method for preventing or treating cancer comprising administering a therapeutically effective amount of the anti-human GITR antibody of claim 140.

156. The method of claim 155, further comprising administering one or more additional therapeutic agents.

157. The method of claim 156, wherein the additional therapeutic agent is selected from the group consisting of: radiation therapy, cytotoxic agents, chemotherapeutic agents, cytostatic agents, anti-hormonal agents, EGFR inhibitors, immunostimulating agents, anti-angiogenic agents, and combinations thereof.