PD-1 / GITR multispecific binding polypeptides and uses thereof

Abstract

The present disclosure provides multispecific (e.g., bispecific) binding proteins with binding specificity to TNFR superfamily receptor (TNFRSF) and an immune checkpoint protein.

Description

[0001] Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT

[0002] PD-1 / GITR MULTISPECIFIC BINDING POLYPEPTIDES AND USES THEREOF

[0003] RELATED APPLICATIONS

[0004] This application claims priority to European Patent Application Serial No. 24307255.0, filed on December 20, 2024, the disclosure of which is hereby incorporated by reference.

[0005] BACKGROUND

[0006] Glucocorticoid-induced TNFR-related protein (GITR; TNFRSF18) is a costimulatory receptor of the TNFR superfamily that is expressed on several types of immune cells of the innate and adaptive immune system. Its expression is induced on activated T cells and its interaction with GITRL induces their activation, whereas it modulates regulatory T cells (Treg) suppressive activity. However, anti-GITR antibodies have shown limited therapeutic effect in clinical trials.

[0007] Programmed death receptor 1 (PD-1 ) is a co-inhibitory receptor that is mainly expressed on activated T cells. Upon interaction with its ligands (PD-L1 / -L2), PD-1 dampens T cell activity. Blockade of the PD-1 / PD-L1 axis showed strong therapeutic impact in cancer patients, with triggering of anti-tumor immunity and long-term survival. However, the majority of patients do not respond or develop resistance. Pre-clinical studies have shown that combination of PD-1 blockade with GITR engagement by an agonist biologic (antibody or ligand) enhances anti-tumor response in tumor-bearing mice, showing great promise for overcoming resistance to anti-PD- 1 / -L1 therapy.

[0008] There is a need in the art for co-targeting strategies, particularly those that are able to efficiently bind simultaneously two different cell targets. Accordingly, as described herein is a fusion binding polypeptide that comprises three or six GITR- targeting ISVDs and two PD-1 -targeting ISVDs fused to the Fc portion of an lgG1 .

[0009] SUMMARY

[0010] The present disclosure provides a multispecific binding protein comprising: (a) a first binding moiety comprising at least three immunoglobulin single variable domains (ISVDs) that specifically bind a TNF receptor superfamily (TNFRSF) receptor (TNFRSF ISVDs) protein; (b) a second binding moiety comprising at least one ISVD

[0011] 1

[0012] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT that specifically binds to an immune checkpoint protein; and wherein the first and second binding moiety form a heterodimer.

[0013] In one aspect, the multispecific binding protein of disclosure comprises at least three TNFRSF ISVDs specifically bind the same or different TNFRSF protein(s). In certain aspects, the at least three TNFRSF ISVDs specifically bind the same TNFRSF protein. In certain aspects, the at least three TNFRSF ISVDs bind to the same or different contacts on the same TNFRSF protein. In certain aspects, the first binding moiety is an antagonist of the TNFRSF protein(s). In certain aspects, the first binding moiety is an agonist of the TNSFRSF protein(s).

[0014] In certain aspects, the TNFRSF protein is selected from a group consisting of: a glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), a 4- 1 BB, a 0X40, a tumor necrosis factor receptor 1 (TNFR1 ), a TNFR2, a lymphotoxin beta receptor (LTBR), a CD40, a fas receptor, a CD27, a CD30, a death receptor 3 (DR3), a DR4, a DR5, a DR6, a decoy receptor 1 (DCR1 ), a DCR2, a DCR3, a receptor activator of nuclear factor kappa-B (RANK), an osteoprotegerin (OPG), a tumor necrosis factor-like weak inducer of apoptosis receptor (TWEAK-R), a tachykinin precursor 1 (TACI), a B cell activating factor receptor (BAFF-R), a herpes virus entry mediator (HVEM), a nerve growth factor receptor (NGFR), a B-cell maturation antigen (BCMA), a TROY, and ectodysplasin A2 receptor (EDA2R). In one aspect, the TNFRSF protein is GITR. In one aspect, the at least three TNFRSF ISVDs specifically bind to GITR.

[0015] In certain aspects, a multispecific binding protein of disclosure comprising the second binding moiety comprises at least two ISVDs. In some aspects, the at least two ISVDs specifically bind the same or different immune checkpoint protein(s). In some aspects, the at least two ISVDs bind to the same or different contacts on the same immune checkpoint protein.

[0016] In certain aspects, a multispecific binding protein of disclosure comprising the second binding moiety is an antagonist of the immune checkpoint protein(s). In one aspect, the second binding moiety is an agonist of the immune checkpoint protein(s). In certain aspects, the checkpoint protein is selected from a group consisting of: a programmed cell death protein 1 (PD-1 ), a PD ligand 1 (PD-L1 ), PD-L2, a cytotoxic T- lymphocyte-associated antigen 4 (CTLA-4), a T-cell immunoglobulin and mucin domain 3 (TIM-3), a lymphocyte activation gene 3 (LAG-3), a T cell immunoglobulin

[0017] 2

[0018] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), a B and T lymphocyte attenuator (BTLA), a signaling lymphocytic activation molecule (SLAM), a leukocyte-associated immunoglobulin-like receptor 1 (LAIR1 ), a cluster of differentiation 244 (CD244), a CD28, a CD200 receptor 1 (CD200R1 ), a TREM-like transcript 2 (TLT2), a leukocyte immunoglobulin like receptor B4 (LILRB4), a killer cell immunoglobulin like receptor (KIR2DL2), and an inducible T-cell co-stimulator (ICOS). In one aspect, the checkpoint protein is a PD-1. In one aspect, at least two ISVDs specifically bind to PD-1 (PD-1 ISVDs).

[0019] In certain aspects, a multispecific binding protein of disclosure is bispecific or trispecific. In one aspect, the binding protein is bispecific. In certain aspects, the heterodimer comprises a fragment crystallizable (Fc) domain or variant thereof. In certain aspects, each binding moiety is operatively linked to the Fc domain or variant thereof. In certain aspects, the Fc domain or variant thereof comprises a first and a second immunoglobulin G (IgG) domain that dimerize to form the bispecific binding protein.

[0020] In certain aspects, a multispecific binding protein of disclosure comprises at least one GITR ISVD specifically binds human GITR. In certain aspects, at least one GITR ISVD is an agonist. In certain aspects, a multispecific binding protein of disclosure comprises at least three GITR ISVDs that specifically bind human GITR. In certain aspects, each GITR ISVD is an agonist. In certain aspects, at least one GITR ISVD binds to an extracellular domain of a GITR subunit. In certain aspects, the at least three of the GITR ISVDs bind to the same GITR subunit. In certain aspects, at least one of the GITR ISVDs can bind to different GITR subunits.

[0021] In certain aspects, a multispecific binding protein of disclosure comprises at least three GITR ISVDs are directly linked to each other via a linker. In certain aspects, the multispecific binding protein comprises, from N-terminus to C-terminus, the at least three GITR ISVDs and a first IgG domain. In certain aspects, the multispecific binding protein comprises, from N-terminus to C-terminus, a first IgG domain and the at least three GITR ISVDs.

[0022] In certain aspects, a multispecific binding protein of the disclosure comprises at least one GITR ISVD is not directly linked to another GITR ISVD via a linker. In certain aspects of the disclosure the multispecific binding protein of the disclosure

[0023] 3

[0024] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT comprises at least one GITR ISVD is linked to the C-terminus and at least one GITR ISVD is linked to the N-terminus of a first IgG domain.

[0025] In certain aspects, a multispecific binding protein of the disclosure comprises a second binding moiety is linked to the N-terminus of a second IgG domain. In certain aspects, the second binding moiety is linked to the N-terminus of a second IgG domain.

[0026] In certain aspects, a multispecific binding protein of the disclosure comprises at least one GITR ISVD specific variable domain has a sequence identity of at least 80% with an amino acid sequence selected from the group consisting of SEQ ID NO: 72 or 74.

[0027] In certain aspects, a multispecific binding protein of the disclosure comprises at least one PD-1 ISVD specifically binds human PD-1 . In certain aspects, the at least one PD-1 ISVD is an antagonist. In certain aspects, the at least one PD-1 ISVD binds to an extracellular domain of a PD-1 subunit. In certain aspects, at least two PD-1 ISVDs bind to the same PD-1 subunit. In certain aspects, at least two of the PD-1 ISVDs bind to different PD-1 subunits. In certain aspects, each PD-1 ISVD is an antagonist of PD-1 .

[0028] In certain aspects of the multispecific binding protein of the disclosure, each GITR ISVD is an agonist of GITR and each PD-1 ISVD is an antagonist of PD-1 .

[0029] In certain aspects, a multispecific binding protein of the disclosure comprises at least one PD-1 ISVD is not directly linked to another PD-1 ISVD via a linker. In certain aspects, the at least one PD-1 ISVD is linked to the N-terminus of a first and / or a second Fc heavy chain. In certain aspects, the at least one PD-1 ISVD is linked to the C-terminus of a first and / or a second Fc heavy chain. In certain aspects, the at least one PD-1 ISVD are linked to both the N- and the C-terminus of a first and / or second Fc heavy chain.

[0030] In certain aspects, a multispecific binding protein of the disclosure comprises at least two PD-1 ISVDs are directly linked to each other via a linker. In certain aspects, at least two PD-1 ISVDs that are linked to each other are also linked to the N-terminus of a first and / or a second Fc heavy chain. In certain aspects, at least two PD-1 ISVDs that are linked to each other are also linked to the C-terminus of a first and / or a second Fc heavy chain. In certain aspects, at least two PD-1 ISVDs are linked to both the N- and the C-terminus of a first and / or second Fc heavy chain. In certain aspects, at least

[0031] 4

[0032] 81660035v1 Attorney Docket No. 772089: SA9-915PC

[0033] Sanofi Ref No. PAT24106-WO-PCT two PD-1 ISVDs that are linked to each other are also linked to the N-terminus of a first Fc heavy chain. In certain aspects, the first binding moiety is linked to the N- terminus of the second Fc heavy chain. In certain aspects, at least one PD-1 ISVD is linked to the N-terminus of a first Fc heavy chain. In certain aspects, a second PD-1 ISVD is linked to the N-terminus of a second Fc heavy chain. In certain aspects, the first binding moiety is linked to the C-terminus of a first and / or second Fc heavy chain.

[0034] In certain aspects, a multispecific binding protein of the disclosure comprises at least one PD-1 ISVD specific variable domain has a sequence identity of at least 80% with an amino acid sequence selected from the group consisting of SEQ ID NO: 73 and 74.

[0035] In certain aspects, a multispecific binding protein of the disclosure comprises an ISVD is selected from a group consisting of: a dAb, an immunoglobulin that is suitable for use as a domain antibody (dAb), a VHH (e.g., a humanized VHH sequence), a camelized VH, or a variant thereof. In certain aspects, a linker connects an ISVD to another ISVD is via a peptide linker. In certain aspects, the linker connecting an ISVD to the first and / or second Fc heavy chain is a peptide linker. In certain aspect, the peptide linker is at 90% identical to a peptide linker encoded by an amino acid sequence set forth in Table 2. In certain aspects, the peptide linker is a poly-Glycine- Serine (G4S)nlinker. In certain aspects, n equals 1 , 2, 3, 4, or 5.

[0036] In certain aspects, a multispecific binding protein of the disclosure comprises a Fc domain comprises a Fc modification. In certain aspects, the IgG domain is human. In certain aspects, the IgG domain is lgG1 or lgG4. In certain aspects, the Fc domain comprises an amino acid substitution at positions 234, 235, and / or 329, according to EU numbering. In certain aspects, the Fc domain comprises a leucine (L) to an alanine (A) substitution at amino acid positions 234 and 235, according to EU numbering. In certain aspects, the Fc domain comprises a proline (P) to a glycine (G) substitution at amino acid position 329, according to EU numbering. In certain aspects, the Fc domain comprising the first IgG domain comprises an amino acid substitution at position 435 and the second IgG domain comprises an amino acid mutation 436, according to EU numbering. In certain aspects, the first IgG domain comprises a histidine (H) to an arginine (R) at amino acid position 435 and the second IgG domain comprises a tyrosine (Y) to a phenylalanine (F) at amino acid position 436, according to EU numbering.

[0037] 5

[0038] 81660035v1 Attorney Docket No. 772089: SA9-915PC

[0039] Sanofi Ref No. PAT24106-WO-PCT

[0040] In certain aspects, a multispecific binding protein of the disclosure comprises a first and second IgG Fc domain polypeptides dimerize by knobs-into-holes interactions, Fab arm exchange (FAE), electrostatic steering interactions, hydrophobic interactions, or any combination thereof. In certain aspects, the first IgG Fc domain polypeptide comprises a knob substitution, and the second IgG Fc domain polypeptide comprises a hole substitution or wherein the first IgG Fc domain polypeptide comprises a hole substitution, and the second IgG Fc domain polypeptide comprises a knob substitution. In certain aspects, the knob substitution is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). In certain aspects, the hole substitution is selected from the group consisting of alanine (A), asparagine (N), aspartic acid (D), glycine (G), serine (S), threonine (T), and valine (V).

[0041] In certain aspects, a multispecific binding protein of the disclosure comprises a half-maximal effective concentration (EC50 [M]) in a NFkB luciferase reporter assay which is less than about 1 x 10-9. In certain aspects, a multispecific binding protein of the disclosure comprises a EC50 [M] in a T-cell activation assay which is about 1 x 10"9to about 3 x 10-11. In certain aspects, a multispecific binding protein of the disclosure comprises the EC50 [M] binding value for human GITR is less than about 1 x 10-1°. In certain aspects, a multispecific binding protein of the disclosure comprises a half- maximal inhibitory concentration (IC50 [M]) binding value for PD-1 is less than about 1 x 10-9. In certain aspects, a multispecific binding protein of the disclosure comprises about 60% of the human PD-1 receptor is occupied when there is about 1 x 10-2nM or more of the multispecific binding protein. In certain aspects, a multispecific binding protein of the disclosure comprises about 60% of the human GITR receptor is occupied when there is about 1 x 10-1nM or more of the multispecific binding protein.

[0042] The present disclosure provides a bispecific binding protein comprising: (a) a first binding moiety comprising at least three GITR ISVDs directly linked to each other and wherein one GITR ISVD is linked to the N-terminal end of a first IgG; (b) a second binding moiety comprising at least two PD-1 ISVDs directly linked to each other and wherein one PD-1 ISVD is linked to the N-terminal end of a second IgG; and wherein each the first and second IgG 1 domains heterodimerize to form a Fc domain or variant thereof.

[0043] 6

[0044] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT

[0045] In one aspect, a bispecific binding protein of the disclosure comprises a first binding moiety consists of at least three GITR ISVDs. In one aspect, the second moiety consists of at least two PD-1 ISVDs.

[0046] The present disclosure provides a bispecific binding protein comprising: (a) a first binding moiety comprising at least three GITR ISVDs directly linked to each other and wherein one GITR ISVD is linked to the C-terminal end of a first and a second IgG; (b) a second binding moiety comprising at least two PD-1 ISVDs wherein each PD-1 ISVD is directly linked to the N-terminal end of the first and the second IgG; and wherein each the first and second IgG 1 domains form a Fc domain.

[0047] In one aspect, a bispecific binding protein of the disclosure comprises a first binding moiety consists of at least six GITR ISVDs. In one aspect, the second moiety consists of at least two PD-1 ISVDs.

[0048] In one aspect, a multispecific binding protein or a bispecific binding protein of the disclosure comprises at least one GITR ISVD comprising the three following CDR sequences:

[0049] - ISVD-CDR1 : GTFFSIDSMA (SEQ ID NO: 83);

[0050] - ISVD-CDR2: AITGGGSPN (SEQ ID NO: 84); and

[0051] - ISVD-CDR3: EGQAGWGTALLDY (SEQ ID NO: 85).

[0052] In one aspect, a multispecific binding protein or a bispecific binding protein of the disclosure at least one PD-1 ISVD comprising one of the three following CDR sequences:

[0053] - ISVD-CDR1 : GFTLDYYVIG or GRMHSINAMA (SEQ ID NO: 86 or SEQ ID NO: 89);

[0054] - ISVD-CDR2: CLSSNGDRIN or LISWGNGITY (SEQ ID NO: 87 or SEQ ID NO: 90)

[0055] - ISVD-CDR3: GTSTTVRDMCGIMYLYDY or SYGSSWYDS (SEQ ID NO: 88 or SEQ ID NO: 91 )

[0056] In one aspect, a multispecific binding protein or a bispecific binding protein of the disclosure comprises at least one GITR ISVD specific variable domain comprising a sequence identity of at least 80% with an amino acid sequence selected from the group consisting of SEQ ID NO: 77 and 78.

[0057] In one aspect, a multispecific binding protein or a bispecific binding protein of the disclosure comprises at least one PD-1 ISVD specific variable domain comprising a sequence identity of at least 80% with an amino acid sequence selected from the group consisting of SEQ ID NO: 79, 80, 81 and 82.

[0058] 7

[0059] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT

[0060] In one aspect, a compound or construct that comprises the bispecific binding protein according to any one of the binding proteins of the disclosure that further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptide linker(s).

[0061] In one aspect, a nucleic acid encodes the multispecific binding protein or the bispecific binding protein of the disclosure. In one aspect, an expression vector comprises a nucleic acid encodes the multispecific binding protein or the bispecific binding protein of the disclosure. In certain aspects, a host or host cell comprises a nucleic acid or an expression vector comprising a nucleic acid encoding the multispecific binding protein or the bispecific binding protein of the disclosure.

[0062] In one aspect, a pharmaceutical composition comprises the multispecific binding protein or the bispecific binding protein according to the disclosure and a pharmaceutically acceptable carrier.

[0063] In one aspect, the disclosure provides a method of treating a neoplastic disorder comprising administering the multispecific binding protein or the bispecific binding protein of the disclosure to a subject. In certain aspects, administration of the binding protein results in tumor growth inhibition by 1 , 2, 3, or 4-fold relative to a reference binding protein.

[0064] In one aspect, the disclosure provides a method of treating an infection comprising administering the binding protein of the disclosure to a subject.

[0065] In one aspect, the disclosure provides a method of treating a T cell, B cell, or natural killer cell mediated disease comprising administering the binding protein of the disclosure to a subject.

[0066] In one aspect, a multispecific binding protein of or a bispecific binding protein of the disclosure is for use in the treatment of neoplastic disorder. In certain aspects, the multispecific binding protein or the bispecific binding protein of the disclosure is for use in inhibiting tumor growth by 1 , 2, 3, or 4-fold relative to a reference binding protein.

[0067] In one aspect, a multispecific binding protein or a bispecific binding protein of the disclosure is for use in treating an infection.

[0068] In one aspect, a multispecific binding protein or a bispecific binding protein of the disclosure is for use in treating a T cell, B cell, or natural killer cell mediated disease.

[0069] 8

[0070] 81660035v1 Attorney Docket No. 772089: SA9-915PC

[0071] Sanofi Ref No. PAT24106-WO-PCT

[0072] BRIEF DESCRIPTION OF THE DRAWINGS

[0073] The foregoing and other features and advantages of the present application will be more fully understood from the following detailed description of illustrative aspects taken in conjunction with the accompanying drawings.

[0074] FIGURE 1 is a schematic drawing of a pentavalent PD-1 / GITR bispecific binding protein described in the disclosure (also referred to herein as “pentavalent anti-PD-1 / GITR construct 1 or 2”; see Table 4). FIGURE 1A shows the pentavalent anti-PD-1 / GITR construct comprising a trivalent anti-human GITR (hGITR) ISVD as a GITR co-stimulatory receptor agonist and a bivalent anti-human PD-1 (hPD-1 ) ISVD as a PD-1 co-inhibitory receptor antagonist each linked to the N-terminus of a first and second Fc heavy chain. The two Fc heavy chains of the first and the second immunoglobulin G (IgG) chains are connected to each other via disulfide bridges. FIGURE 1 B details additional features of the pentavalent anti-PD-1 / GITR construct described in FIGURE 1A including: (a) the orientation of the binding domains with respect to the first and the second IgG, i.e., the trivalent anti-hGITR domains are linked to the N-terminal end of the first Fc heavy chain and the bivalent anti-hPD-1 domains are linked to the N-terminal end of the second Fc heavy chain; (b) the multivalent binding domains are connected by flexible linkers (e.g., a Gly4Ser linker); (c) an effector-null Fc domain (with substitutions at L234A and L235A in each IgG domain; also called “LALA substitutions”); (d) wherein the Fc domain is further modified with amino acid substitutions H435R and a Y436F in the second IgG domain to ablate protein A binding in one arm of the heterodimer to facilitate bispecific antibody purification (also called the “RF substitutions”); and (d) Fc-knob-in-hole engineering to facilitate dimerization of the first and second IgG domains.

[0075] FIGURE 2A is a schematic drawing of an octavalent PD-1 / GITR bispecific binding protein described in the disclosure (also referred to herein as, “octavalent anti- PD-1 / GITR construct”; see Table 4) comprising trivalent anti-hGITR ISVDs and an anti-hPD-1 ISVD linked to a first and a second Fc heavy chain. The two Fc heavy chains each IgG chain are connected to each other via disulfide bridges. FIGURE 2B details additional features the octavalent anti-PD-1 / GITR construct described in FIGURE 2A including: (a) the orientation of the binding domains with respect to a first and a second IgG domain - the trivalent anti-hGITR domains are linked to the C- terminus of a first Fc heavy chain and a single anti-hPD-1 domain is linked to the N-

[0076] 9

[0077] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT terminus of a first Fc heavy chain where the second IgG comprises the same orientation and domains; (b) the domains are connected by flexible linkers, including an extended GlySer linker (e.g., (Gly4Ser)4) to connect the C-terminal end of Fc heavy chain to the anti-GITR domain; and (c) an effector-null Fc domain with LALA substitution.

[0078] FIGURE 3A is a graph quantifying by FACS the binding to hPD-1 expressed on pre-B 300.19 cells upon incubation with serial dilutions of either pentavalent anti-PD- 1 / GITR construct 1 or 2 (filled circles and squares) or controls. FIGURE 3B is the same experimental set-up as FIGURE 3A but quantifies the binding to cynomolgus monkey PD-1 (cynoPD-1 ) expressed on pre-B 300.19 cells.

[0079] FIGURE 4A is the same experimental set-up as FIGURE 3A but with incubation with the octavalent anti-PD-1 / GITR construct (filled circles) or controls. Figure 4B is the same experimental set-up as FIGURE 4A but quantifies the binding to cynoPD-1 expressed on pre-B 300.19 cells.

[0080] FIGURE 5A is a graph quantifying by FACS the binding to hGITR expressed on NFkB-NlucP / HEK293 cells upon incubation with serial dilutions of either pentavalent anti-PD-1 / GITR construct 1 or 2 (filled circle and squares) or controls. FIGURE 5B is the same experimental set-up as FIGURE 5A but quantifies the binding to cynoGITR expressed on NFkB-NlucP / HEK293 cells.

[0081] FIGURE 6A is a graph quantifying by FACS the binding to hGITR expressed on NFkB-NlucP / HEK293 cells upon incubation with serial dilutions of either octavalent anti-PD-1 / GITR construct (filled circles) or controls. FIGURE 6B is the same experimental set-up as FIGURE 6A but quantifies the binding to cynoGITR expressed on NFkB-NlucP / HEK293 cells.

[0082] FIGURE 7A is a graph quantifying by FACS the binding to activated human primary T cells (which expressed both PD-1 and GITR) upon incubation with escalating concentrations of either pentavalent (filled circles and square) or octavalent (filled diamonds) anti-PD-1 / GITR constructs or controls. FIGURE 7B is the same experimental set-up as FIGURE 7A but quantifies the binding to activated cynomolgus monkey primary T cells (cyno primary T cells).

[0083] FIGURE 8A analyzes the activity of GITR after incubation of a GloResponseTM NFkB-NlucP / HEK293 cell line which was stably transfected to express hGITR with either serial dilutions of the pentavalent anti-PD-1 / GITR construct 1 or 2 (filled circles

[0084] 10

[0085] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT and squares), the octavalent anti-PD-1 / GITR construct (filled diamonds) or with controls. FIGURE 8B is the same experimental set-up as FIGURE 8A but where the cell line was stably transfected to express cynoGITR.

[0086] FIGURE 9 analyzes the activity of human PD-1 after incubation of a GloResponseTM NFAT-RE / PD-1 Jurkat cell line was incubated with either serial dilutions of the pentavalent anti-PD-1 / GITR construct 1 or 2 (filled circles and squares), the octavalent anti-PD-1 / GITR construct (filled diamonds), or with controls.

[0087] FIGURE 10 analyzes the surface occupancy of each binding moiety, human PD-1 (FIGURE 10A) and human GITR (FIGURE 10B), on splenic activated CD4+and CD8+T cells (filled circles and triangles, respectively) harvested from hGITR / hPD-1 KI mice upon incubation with serial dilutions of the pentavalent anti-PD-1 / GITR construct 1.

[0088] FIGURE 11 analyzes the activation of primary human CD3+T cells isolated from human PBMC donors (three different donors were used: D1475, D1735, and D1743) via IL-2 release (pg / ml) upon incubation with either serial dilutions of the pentavalent anti-PD-1 / GITR construct 1 or 2 (filled circles and squares) or with controls.

[0089] FIGURE 12 is the same experimental set-up as FIGURE 11 but incubation is performed with either serial dilutions of the octavalent anti-PD-1 / GITR construct (filled circle) or controls.

[0090] FIGURE 13 measures the results of a mixed lymphocyte reaction (MLR) assay via human GM-CSF secretion (pg / ml) into the cell medium from an immature dendritic cell (iDC) co-culture with autologous CD4+T cells derived from three different PBMC donors (D1014, D1013, and D1015) upon incubation with either serially diluted pentavalent anti-PD-1 / GITR construct 1 or with controls.

[0091] FIGURE 14 is the same experimental set-up as FIGURE 13 but upon incubation with either serially diluted pentavalent anti-PD-1 / GITR construct 2 or with controls.

[0092] FIGURE 15 is the same experimental set-up as FIGURES 13 and 14 but upon incubation with either serially diluted the octavalent anti-PD-1 / GITR construct or with controls.

[0093] FIGURE 16 analyzes the anti-tumor efficacy of a pentavalent anti-PD-1 / GITR construct 1 compared to controls in hPD-1 xhGITR double KI mice bearing subcutaneous MC38 established tumors. Median tumor volume ± median average deviation (MAD).

[0094] 1 1

[0095] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT

[0096] FIGURE 17 shows graphs of the dose-dependent anti-tumor efficacy of a pentavalent anti-PD-1 / GITR construct 1 in hPD-1 xhGITR double KI mice bearing subcutaneous MC38 established tumors. Individual curves are shown.

[0097] DETAILED DESCRIPTION

[0098] The present disclosure is directed to, inter alia, a multispecific binding protein comprising: (a) a first binding moiety comprising at least three immunoglobulin single variable domains (ISVDs) that specifically bind a TNF receptor superfamily (TNFRSF) receptor (TNFRSF ISVDs) protein; (b) a second binding moiety comprising at least one ISVD that specifically binds to an immune checkpoint protein; and wherein the first and second binding moiety form a heterodimer.

[0099] The multispecific binding proteins of the disclosure advantageously display agonist activity to a TNFRSF protein (e.g., GITR) while also being capable of blocking activity to the immune checkpoint protein (e.g., PD-1 ). The multispecific binding proteins of the disclosure potently activate T cells to a higher extent than a monospecific antibody against the immune checkpoint protein (e.g., PD-1 ). The multispecific binding proteins of the disclosure show in vivo anti-tumor activity to a higher extent than a monospecific antibody against the immune checkpoint protein (e.g., PD-1 ).

[0100] Definitions

[0101] It is to be understood that the methods described in this disclosure are not limited to particular methods and experimental conditions such that methods and conditions can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0102] Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and / or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting. The embodiments

[0103] 12

[0104] 81660035v1 Attorney Docket No. 772089: SA9-915PC

[0105] Sanofi Ref No. PAT24106-WO-PCT illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are specifically or not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising," "consisting essentially of," and "consisting of" can be replaced with either of the other two terms, while retaining their ordinary meanings. Any single term, single element, single phrase, group of terms, group of phrases, or group of elements described herein can each be specifically excluded from the claims.

[0106] Generally, nomenclature used in connection with cell culture, molecular biology, immunology, microbiology, genetics, protein biology, and chemistry described herein is well-known and commonly used in the art. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0107] Unless indicated or defined otherwise, all terms used have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al. (Molecular Cloning: A Laboratory Manual (2nd. Ed.) Vols. 1 -3, Cold Spring Harbor Laboratory Press, 1989), F. Ausubel et al. (Current protocols in molecular biology, Green Publishing and Wiley Interscience, New York, 1987), Lewin (Genes II, John Wiley & Sons, New York, N.Y., 1985), Old et al. (Principles of Gene Manipulation: An Introduction to Genetic Engineering (2nd edition) University of California Press, Berkeley, CA, 1981 ); Roitt et al. (Immunology (6th. Ed.) Mosby / Elsevier, Edinburgh, 2001 ), Roitt et al. (Roitt’s Essential Immunology (1 Oth Ed.) Blackwell Publishing, UK, 2001 ), and Janeway et al. (Immunobiology (6th Ed.) Garland Science Publishing / Churchill Livingstone, New York, 2005), as well as to the general background art cited herein.

[0108] Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks and the general background art mentioned herein and to the further references cited therein; as well as to for example the following reviews Presta (Adv. Drug Deliv. Rev. 58 (5-6): 640-56, 2006), Levin and Weiss (Mol. Biosyst. 2(1 ): 49-57, 2006), Irving et al. (J. Immunol. Methods 248(1 -2): 31 -45, 2001 ), Schmitz et al. (Placenta 21 Suppl. A: S106-12, 2000), Gonzales et al.

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[0111] (T umour Biol. 26(1 ): 31 -43, 2005), which describe techniques for protein engineering, such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins.

[0112] For the disclosure to be more readily understood, select terms are defined below.

[0113] Sequence

[0114] The term “sequence” as used herein, for example, in terms like “immunoglobulin sequence,” “antibody sequence,” “variable domain sequence,” “VHH sequence,” “protein sequence,” “amino acid sequence” or “nucleic acid sequence,” can generally be understood to include both the relevant amino acid sequence as well as nucleic acids or nucleotide sequences encoding the same, unless the context requires a more limited interpretation.

[0115] Amino acid residues will be indicated according to the standard three-letter or one-letter amino acid code. Reference is made to e.g., Table A-2 of WO 2008 / 020079 which is incorporated by reference herein.

[0116] A nucleic acid or amino acid is considered to be “(in) (essentially) isolated (form)” - for example, compared to the reaction medium or cultivation medium from which it has been obtained - when it has been separated from at least one other component with which it is usually associated in said source or medium, such as another nucleic acid, another protein / polypeptide, another biological component or macromolecule or at least one contaminant, impurity or minor component. A nucleic acid or amino acid is considered “(essentially) isolated” when it has been purified at least 2-fold, in particular at least 10-fold, more in particular at least 100-fold, and up to 1000-fold or more. A nucleic acid or amino acid that is “in (essentially) isolated form” is preferably essentially homogeneous, as determined using a suitable technique, such as a suitable chromatographical technique, such as polyacrylamide-gel electrophoresis.

[0117] When a nucleic acid sequence (also called “a nucleotide sequence”) or amino acid sequence is said to “comprise” another nucleotide sequence or amino acid sequence, respectively, or to “essentially consist of” another nucleotide sequence or amino acid sequence, this may mean that the latter nucleotide sequence or amino acid sequence has been incorporated into the first mentioned nucleotide sequence or amino acid sequence, respectively, but more usually this generally means that the first

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[0120] Sanofi Ref No. PAT24106-WO-PCT mentioned nucleotide sequence or amino acid sequence comprises within its sequence a stretch of nucleotides or amino acid residues, respectively, that has the same nucleotide sequence or amino acid sequence, respectively, as the latter sequence, irrespective of how the first mentioned sequence has actually been generated or obtained (which may for example be by any suitable method described herein). By means of a non-limiting example, when a polypeptide is said to comprise an immunoglobulin single variable domain, this may mean that said immunoglobulin single variable domain sequence has been incorporated into the sequence of the polypeptide, but more usually this generally means that the polypeptide contains within its sequence the sequence of the immunoglobulin single variable domains irrespective of how said polypeptide has been generated or obtained. Also, when a nucleic acid or nucleotide sequence is said to comprise another nucleotide sequence, the first mentioned nucleic acid or nucleotide sequence is preferably such that, when it is expressed into an expression product (e.g., a polypeptide), the amino acid sequence encoded by the latter nucleotide sequence forms part of said expression product (in other words, that the latter nucleotide sequence is in the same reading frame as the first mentioned, larger nucleic acid or nucleotide sequence).

[0121] By “(essentially) consist of” is meant that the later nucleic acid sequence or amino acid sequence, either is exactly the same as the polypeptide (e.g., the CDR region; the ISVD) or corresponds to the polypeptide (e.g., the CDR region; the ISVD) which has a limited number of amino acid residues, such as 1 -20 amino acid residues, for example 1 -10 amino acid residues and preferably 1 -6 amino acid residues, such as 1 , 2, 3, 4, 5 or 6 amino acid residues, added at the amino terminal end, at the carboxy terminal end, or at both the amino terminal end and the carboxy terminal end of the immunoglobulin single variable domain.

[0122] For the purposes of comparing two or more amino acid sequences, the percentage of “sequence identity” between a first amino acid sequence and a second amino acid sequence may be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence - compared to the first amino acid

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[0125] Sanofi Ref No. PAT24106-WO-PCT sequence - is considered as a difference at a single amino acid residue (i.e. at a single position). Usually, for the purpose of determining the percentage of “sequence identity” between two amino acid sequences in accordance with the calculation method outlined hereinabove, the amino acid sequence with the greatest number of amino acid residues will be taken as the “first” amino acid sequence, and the other amino acid sequence will be taken as the “second” amino acid sequence.

[0126] An “amino acid difference” or e.g., an “amino acid modification” or “Fc modification” as used herein can refer to a deletion, insertion or substitution of a single amino acid residue vis-a-vis a reference sequence. In one embodiment, an “amino acid difference” is a substitution. In certain aspects, amino acid substitutions are conservative substitutions. In certain aspects, the multispecific binding protein of the disclosure comprises a Fc domain comprising a Fc modification.

[0127] Conservative substitutions are substitutions in which one amino acid within the following groups (a) - (e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gin; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Vai and Cys; and (e) aromatic residues: Phe, Tyr and Trp. In one embodiment, conservative substitutions are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into He or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Vai, into He or into Leu.

[0128] A “VHH family” as used in the present specification refers to a group of VHH sequences that have identical lengths (i.e., they have the same number of amino acids within their sequence) and of which the amino acid sequence between position 8 and position 106 (according to Kabat numbering) has an amino acid sequence identity of more than 89%.

[0129] In certain aspects, the multispecific binding protein of the disclosure comprises at least one GITR ISVD specific variable domain has a sequence identity of at least 80% with an amino acid sequence selected from the group consisting of SEQ ID NO: 72 and 74. In certain aspects, the multispecific binding protein of disclosure at least

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[0131] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT one PD-1 ISVD specific variable domain has a sequence identity of at least 80% with an amino acid sequence selected from the group consisting of SEQ ID NO: 73 and 74.

[0132] Binding protein or binding polypeptide

[0133] As used herein, the term "binding protein,” “binding polypeptide,” or “multispecific binding polypeptide or protein” or “bispecific binding polypeptide or protein” refer to a protein or polypeptide (e.g., an antibody or specific binding fragment thereof) that contains at least one binding site which is responsible for selectively binding to a target antigen of interest (e.g., a human antigen). Exemplary binding sites include an antibody variable domain, a ligand binding site of a receptor, or a receptor binding site of a ligand. In certain aspects, the binding proteins or binding polypeptides comprise multiple (e.g., two, three, four, or more) binding sites.

[0134] Native residue vs. modified binding polypeptide

[0135] As used herein, the term "native residue" refers to an amino acid residue that occurs naturally at a particular amino acid position of a binding polypeptide (e.g., an antibody or fragment thereof) and which has not been modified, introduced, or altered by the hand of man. As used herein, the term “altered binding protein,” “altered binding polypeptide,” “modified binding protein” or “modified binding polypeptide” shall refer to binding polypeptides and / or binding proteins (e.g., an antibody or fragment thereof) comprising at least one amino acid substitution, deletion and / or addition relative to the native ( / .e., wild-type) amino acid sequence, and / or a mutation that results in altered glycosylation (e.g., hyperglycosylation, hypoglycosylation and / or aglycosylation) at one or more amino acid positions relative to the native (i.e., wild-type) amino acid sequence.

[0136] Antigen and Epitope

[0137] The term "antigen" or "target antigen" as used herein refers to a molecule or a portion of a molecule that is capable of being bound by the binding site of a binding polypeptide e.g., any substance to which an antibody can be generated. A target antigen may have one or more epitopes.

[0138] The terms “epitope” and “antigenic determinant”, which can be used interchangeably, refer to the part of a macromolecule, such as a polypeptide or protein

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[0140] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT that is recognized by antigen-binding molecules, such as immunoglobulins, conventional antibodies, or immunoglobulin single variable domains, and more particularly by the antigen-binding site of said molecules. Epitopes define the minimum binding site for an immunoglobulin, and thus represent the target of specificity of an immunoglobulin. The part of an antigen-binding molecule (such as an immunoglobulin, a conventional antibody, an immunoglobulin single variable domain) that recognizes the epitope is called a “paratope”.

[0141] Examples of antigens can be a peptide, a polypeptide, a protein (e.g., a TNFRSF protein or an immune check point protein or fragment thereof), an aptamer, a polysaccharide, a sugar molecule, a carbohydrate, a lipid, an oligonucleotide, a polynucleotide, a synthetic molecule, an inorganic molecule, an organic molecule, and any combination thereof.

[0142] Immunoglobulin domain

[0143] The term immunoglobulin domain as used herein can refer to an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domain can be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from an antibody (e.g., a mammalian antibody, a recombinant antibody, a chimeric antibody, an engineered antibody, a human antibody, a humanized antibody) or an antigen binding fragment thereof. As used herein, the term "antibody" refers to such assemblies (e.g., intact antibody molecules, antibody fragments, or variants thereof) which have significant known specific immunoreactive activity to an antigen of interest (e.g., GITR and PD-1 ). Antibodies and immunoglobulins comprise light and heavy chains, with or without an interchain covalent linkage between them. Basic immunoglobulin structures in vertebrate systems are relatively well understood.

[0144] In certain aspects, the multispecific binding protein of the disclosure comprises an IgG domain (e.g., a human IgG domain). In certain aspects, the IgG domain is lgG1 or lgG4.

[0145] As will be discussed in more detail below, the generic term "antibody" comprises five distinct classes of antibody that can be distinguished biochemically. While all five classes of antibodies are clearly within the scope of the current disclosure, the following discussion will generally be directed to the IgG class of

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[0148] Sanofi Ref No. PAT24106-WO-PCT immunoglobulin molecules. With regard to IgG, immunoglobulins comprise two identical light chains of molecular weight approximately 23,000 Daltons, and two identical heavy chains of molecular weight 53,000-70,000. The four chains are joined by disulfide bonds in a "Y" configuration wherein the light chains bracket the heavy chains starting at the mouth of the "Y" and continuing through the variable region.

[0149] Light chains of immunoglobulin are classified as either kappa or lambda (K, A). Each heavy chain class can be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells, or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C- terminus at the bottom of each chain. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (y, p, a, 5, s) with some subclasses among them (e.g., yl-y-4). It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin isotype subclasses (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 , etc.) confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the current disclosure.

[0150] Both the light and heavy chains are divided into regions of structural and functional homology. The term "region" refers to a part or portion of an immunoglobulin or antibody chain and includes constant region or variable regions, as well as more discrete parts or portions of said regions. For example, light chain variable regions include "complementarity determining regions" or "CDRs" interspersed among "framework regions" or "FRs," as defined herein.

[0151] Constant and variable domains

[0152] The regions of an immunoglobulin heavy or light chain can be defined as "constant" (C) region or "variable" (V) regions, based on the relative lack of sequence variation within the regions of various class members in the case of a "constant region", or the significant variation within the regions of various class members in the case of a "variable regions." The terms "constant region" and "variable region" may

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[0155] Sanofi Ref No. PAT24106-WO-PCT also be used functionally. In this regard, it will be appreciated that the variable regions of an immunoglobulin or antibody determine antigen recognition and specificity. Conversely, the constant regions of an immunoglobulin or antibody confer important effector functions such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. The subunit structures and three-dimensional configurations of the constant regions of the various immunoglobulin classes are well known.

[0156] The constant and variable regions of immunoglobulin heavy and light chains are folded into domains. The term "domain" refers to a globular region of a heavy or light chain comprising peptide loops (e.g., comprising 3 to 4 peptide loops) stabilized, for example, by [3-pleated sheet and / or intrachain disulfide bond. Constant region domains on the light chain of an immunoglobulin are referred to interchangeably as "light chain constant region domains", "CL regions" or "CL domains." Constant domains on the heavy chain (e.g., hinge, CH1 , CH2 or CH3 domains) are referred to interchangeably as "heavy chain constant region domains", "CH" region domains or "CH domains". Variable domains on the light chain are referred to interchangeably as "light chain variable region domains", "VL region domains or " VL domains." Variable domains on the heavy chain are referred to interchangeably as "heavy chain variable region domains", " VH region domains" or “VH domains."

[0157] By convention the numbering of the variable constant region domains increases as they become more distal from the antigen binding site or amino-terminus of the immunoglobulin or antibody. The N-terminus of each heavy and light immunoglobulin chain is a variable region and at the C-terminus is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively. Accordingly, the domains of a light chain immunoglobulin are arranged in a VL-CL orientation, while the domains of the heavy chain are arranged in the VH- CH1 -hinge-CH2-CH3 orientation.

[0158] The assignment of amino acids to each variable region domain is in accordance with the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991 ). Kabat also provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chain variable regions or between different light chain variable regions are assigned the same number. CDRs 1 , 2 and 3 of a VL domain

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[0160] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT are also referred to herein, respectively, as CDR-L1 , CDR-L2 and CDR-L3. CDRs 1 , 2 and 3 of a VH domain are also referred to herein, respectively, as CDR-H1 , CDR- H2 and CDR-H3. If so noted, the assignment of CDRs can be in accordance with IMGT® (Lefranc et al., Developmental & Comparative Immunology 27:55-77; 2003) in lieu of Kabat. Numbering of the heavy chain constant region is via the EU index as set forth in Kabat (Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD, 1987 and 1991 ).

[0161] VHdomain and VL domain

[0162] As used herein, the term "VH domain" includes the amino terminal variable domain of an immunoglobulin heavy chain, and the term "VL domain" includes the amino terminal variable domain of an immunoglobulin light chain.

[0163] As used herein, the term "CH1 domain" includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain that extends, e.g., from about positions 1 14-223 in the Kabat numbering system (EU positions 1 18-215). The CH1 domain is adjacent to the VH domain and amino terminal to the hinge region of an immunoglobulin heavy chain molecule and does not form a part of the Fc region of an immunoglobulin heavy chain.

[0164] As used herein, the term "hinge region" includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al. J. Immunol. 1998, 161 :4083).

[0165] CH2 domain

[0166] As used herein, the term "CH2 domain" includes the portion of a heavy chain immunoglobulin molecule that extends, e.g., from about positions 244-360 in the Kabat numbering system (EU positions 231 -340). The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. In certain aspects, a bispecific binding protein of the current disclosure comprises a CH2 domain derived from an lgG1 molecule (e.g., a human lgG1 molecule).

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[0169] Sanofi Ref No. PAT24106-WO-PCT

[0170] CH3 domain

[0171] As used herein, the term "CH3 domain" includes the portion of a heavy chain immunoglobulin molecule that extends approximately 1 10 residues from N-terminus of the CH2 domain, e.g., from about positions 361 -476 of the Kabat numbering system (EU positions 341 -445). The CH3 domain typically forms the C-terminal portion of the antibody. In some immunoglobulins, however, additional domains may extend from CH3 domain to form the C-terminal portion of the molecule (e.g., the CH4 domain in the p chain of IgM and the 8 chain of IgE). In certain aspects, a bispecific binding protein of the current disclosure comprises a CH3 domain derived from an lgG1 molecule (e.g., a human IgG 1 molecule).

[0172] CL domain

[0173] As used herein, the term "CL domain" includes the constant region domain of an immunoglobulin light chain that extends, e.g., from about Kabat position 107A-216. The CL domain is adjacent to the VL domain. In certain aspects, a bispecific binding protein of the current disclosure comprises a CL domain derived from a kappa light chain (e.g., a human kappa light chain).

[0174] As indicated above, the variable regions of an antibody allow it to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain of an antibody combine to form the variable region (Fv) that defines a three- dimensional antigen binding site. This quaternary antibody structure forms the antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three complementary determining regions (CDRs) on each of the heavy and light chain variable regions. As used herein, the term "antigen binding site" includes a site that specifically binds (immunoreacts with) an antigen (e.g., a cell surface or soluble antigen). The antigen binding site includes an immunoglobulin heavy chain and light chain variable region and the binding site formed by these variable regions determines the specificity of the antibody. An antigen binding site is formed by variable regions that vary from one antibody to another. The altered antibodies of the current disclosure comprise at least one antigen binding site.

[0175] In certain aspects, a multispecific binding protein of the current disclosure comprises at least two different antigen binding domains that provide for the

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[0178] Sanofi Ref No. PAT24106-WO-PCT association of said binding protein with the selected antigen or target. In some aspects, the first antigen binding domain binds a TNFRSF receptor protein (e.g., TNFRSF ISVD) and the second antigen binding domain binds to an immune checkpoint protein (e.g., PD-1 ISVD). In some aspects, (a) a first binding moiety comprising at least three immunoglobulin single variable domain (ISVD) that specifically bind a TNF receptor superfamily (TNFRSF) receptor (TNFRSF ISVD); (b) a second binding moiety comprising at least one ISVD that specifically binds to an immune checkpoint protein; and wherein the first and second binding moiety form a heterodimer.

[0179] In certain aspects, the antigen binding domains are not derived from the same immunoglobulin molecule. In this regard, the variable region may be derived from any type of animal that can be induced to mount a humoral response and generate immunoglobulins against the desired antigen. As such, the variable region of a bispecific binding protein may be, for example, of mammalian origin e.g., may be human, murine, rat, goat, sheep, non-human primate (such as cynomolgus monkeys, macaques, etc.), lupine, or camelid (e.g., from camels, llamas and related species). In naturally occurring antibodies, the six CDRs present on each monomeric antibody are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding site as the antibody assumes its three-dimensional configuration in an aqueous environment. The remainder of the heavy and light variable domains show less inter-molecular variability in amino acid sequence and are termed the framework regions. The framework regions largely adopt a [3-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the [3-sheet structure. Thus, these framework regions act to form a scaffold that provides for positioning the six CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to the immunoreactive antigen epitope.

[0180] CDR and FR

[0181] As used herein, the term “complementarity determining region” or “CDR” refers to sequences of amino acids within antibody variable regions, which confer antigen specificity and binding affinity. In general, there are three CDRs in each heavy chain

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[0184] Sanofi Ref No. PAT24106-WO-PCT variable region (HCDR1 , HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1 , LCDR2, LCDR3). “Framework regions” or “FR” are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each heavy chain variable region (FR-H1 , FR-H2, FR-H3, and FR-H4), and four FRs in each light chain variable region (FR-L1 , FR-L2, FR-L3, and FR-L4).

[0185] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991 ), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme), MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibodyantigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745. (“Contact” numbering scheme), Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(1 ):55-77 (“IMGT” numbering scheme), and Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun. 8; 309(3):657-70, (AHo numbering scheme).

[0186] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.

[0187] A “CDR” or “complementarity determining region,” or individual specified CDRs (e.g., “HCDR1 ,” “HCDR2,” “HCDR3”), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) complementarity determining region as defined by any of the known schemes. Likewise, an “FR” or “framework region,” or individual specified FRs (e.g., “FR-H1 ,”

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[0190] “FR-H2”) of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) framework region as defined by any of the known schemes. In some instances, the scheme for identification of a particular CDR or FR is specified, such as the CDR as defined by the IMGT, Kabat, Chothia, AbM, or Contact method. In other cases, the particular amino acid sequence of a CDR or FR is given. Unless otherwise specified, all particular CDR amino acid sequences mentioned in the disclosure are IMGT CDRs. However, alternative CDRs defined by other schemes are also encompassed by the present disclosure, such as those determined by abYsis Key Annotation (Website: abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi).

[0191] Antibody Variant

[0192] Exemplary multispecific binding proteins (e.g., bispecific binding proteins) can comprise antibody variants and / or specific antibody binding fragments variants. As used herein, the term “antibody variant” and “antibody specific binding fragment variant” includes synthetic and engineered forms of antibodies which are altered such that they are not naturally occurring, e.g., antibodies that comprise at least two heavy chain portions but not two complete heavy chains (such as, domain deleted antibodies or minibodies) and multispecific forms of antibodies altered to bind to two or more different antigens or to different epitopes on a single antigen); heavy chain molecules joined to scFv molecules and the like.

[0193] Antigen Binding Fragments

[0194] Unless specifically indicated otherwise, the term “antibody,” as used herein, shall be understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., “full antibody molecules”) as well as antigen binding fragments thereof. Other engineered molecules, such as domain specific binding proteins, single domain binding proteins, domain deleted binding proteins, chimeric binding proteins, CDR grafted binding proteins, diabodies, triabodies, tetrabodies, minibodies, immunoglobulin single variable domains (ISVDs) (e.g., monovalent ISVDs, bivalent ISVDs, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen binding fragment,” as used herein.

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[0197] Sanofi Ref No. PAT24106-WO-PCT

[0198] The term “multispecific antibody” denotes a binding fragment or derivative thereof that combines the antigen-binding sites of two or more antibodies within a single molecule. The terms “antigen binding portion”, “antigen binding fragment”, “binding protein” or “binding moiety” and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to at least one target antigen to form a complex.

[0199] In certain aspects, the term “antigen binding fragment” refers to a polypeptide fragment of a multispecific (e.g., a bispecific) binding protein. Antigen binding fragments of a multispecific binding protein, or a binding fragment or derivative thereof can be derived, e.g., from full multispecific binding protein molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding multispecific binding protein, or a binding fragment or derivative thereof variable and (optionally) constant domains. Such DNA is known and / or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add, or delete amino acids, etc.

[0200] Multispecific

[0201] As used herein, a “multispecific” binding protein is a binding protein that specifically binds two or more types of antigens.

[0202] A multispecific binding protein that binds two antigens, and / or two different epitopes of different antigens, is also referred to herein as a “bispecific” binding protein (e.g., a multispecific binding protein that binds GITR and an immune checkpoint protein, such as PD-1 ). In some aspects, the multispecific binding of the disclosure is a bispecific binding protein. In some aspects, the bispecific binding protein of the disclosure comprises (a) a first binding moiety comprising at least three immunoglobulin single variable domains (ISVDs) that specifically bind a TNF receptor superfamily (TNFRSF) receptor (TNFRSF ISVDs) protein; (b) a second binding moiety comprising at least one ISVD that specifically binds to an immune checkpoint protein; and wherein the first and second binding moiety form a heterodimer.A multispecific

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[0205] Sanofi Ref No. PAT24106-WO-PCT binding protein that binds three antigens, and / or three different epitopes, is also referred to herein as a “trispecific” binding protein. In some aspects, the multispecific binding of the disclosure is a trispecific binding protein.

[0206] Thus, the multispecific binding protein is able to bind two or more different targets simultaneously, for example, GITR and an immune checkpoint protein, such as PD-1. Genetic engineering can be used to design, modify, and produce the multispecific binding protein, or a binding fragment or derivative thereof with a desired set of binding properties and effector functions.

[0207] In some aspects, a multispecific binding protein of the disclosure comprises a VH, a Fab, a Fab’, a F(ab’)2, a variable fragment (Fv), a disulfide-stabilized Fv (dsFv), a single-chain Fv (scFv), a diabody, a triabody, an immunoglobulin single variable domain (ISVD), or an AFFIBODY®. In some aspects, wherein the multispecific binding protein further comprises a Fc domain or a variant thereof. In one aspect, the ISVD is a VHH, humanized VHH, a camelized VH, a (single) domain antibody (such as a dAb or a sdAb), or a VNAR. In some aspects, the scFV is a linear scFV or a tandem scFV.

[0208] Techniques for making multispecific binding proteins (e.g., multispecific antibodies) include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein, C. and Cuello, A. C., Nature 305 (1983) 537-540, WO 93 / 08829, and Traunecker, A. et al., EMBO J. 10 (1991 ) 3655-3659), and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731 ,168). In some embodiments, the bispecific antibodies are prepared using a macro-assembly technique using two half-antibodies, e.g., as described in Spiess, C., Merchant, M., Huang, A. et al. Bispecific antibodies with natural architecture produced by co-culture of bacteria expressing two distinct halfantibodies. Nat Biotechnol 31 , 753-758 (2013).

[0209] Multispecific antibodies may also be made by engineering electrostatic steering effects for making binding protein Fc-heterodimeric molecules (WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan, M. et al., Science 229 (1985) 81 -83); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny, S. A. et al., J. Immunol. 148 (1992) 1547- 1553; using “diabody” technology for making multispecific binding protein fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and using single-chain Fv (scFv) dimers (see, e.g., Gruber, M et al., J. Immunol. 152 (1994)

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[0212] 5368-5374); and preparing trispecific binding proteins as described, e.g., in Tutt, A. et al., J. Immunol. 147 (1991 ) 60-69).

[0213] A wide variety of recombinant multispecific binding protein formats have been developed, e.g., by fusion an IgG binding protein format and single chain domains (see Kontermann RE, mAbs 4:2, (2012) 1 -16). Multispecific binding proteins wherein the variable domains VL and VH or the constant domains CL and CH1 are replaced by each other are described in W02009080251 and W02009080252.

[0214] In one aspect, a multispecific binding protein a first and a second IgG Fc domain polypeptide of a multispecific binding protein can dimerize. In some aspects, a multispecific binding protein of the disclosure dimerize by knobs-into-holes interactions, Fab arm exchange (FAE), electrostatic steering interactions, hydrophobic interactions, or any combination thereof.

[0215] Multispecific binding protein

[0216] Binding moiety

[0217] The term “binding moiety” is used herein in the broadest sense to encompass any chemical entity capable of specific binding to a target, such as an antigen (e.g., GITR or PD-1 ). The terms “binding moiety” and “binding domain” can be used interchangeably herein.

[0218] Examples of a binding moiety of the multispecific binding protein described herein can comprise an antibody, a VH, a Fab, a Fab’, a F(ab’)2, a variable fragment (Fv), a disulfide-stabilized Fv (dsFv), a single-chain Fv (scFv), a diabody, a triabody, an immunoglobulin single variable domain (ISVD), or an AFFIBODY®. Other binding moiety examples include a Fab fragment, a F(ab')2 fragment, an Fv fragment a fragment containing a complementarity determining region (CDR), an isolated CDR, or other suitable fragment.

[0219] In certain aspects, a first cell surface binding moiety and a second binding moiety of a multispecific binding protein each independently comprise an antigen binding fragment that comprises at least one variable domain of an antibody or an antigen binding fragment. At least one constant domain can optionally be covalently linked to one or both the first and second binding moieties. The variable domain(s) can be of any size or amino acid composition and will generally comprise at least one CDR, which is adjacent to or in frame with one or more framework sequences. In antigen

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[0221] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT binding fragments having a VH domain associated with a VL domain, the VH and VL domains can be situated relative to one another in any suitable arrangement. For example, the variable region can be dimeric and contain VHVH, VHVL or VL-VL dimers. Alternatively, the antigen binding fragment can contain a monomeric VH or VL domain. Non limiting, exemplary configurations of variable and constant domains that can be found within an antigen binding fragment include: (i) VH CH1 ; (ii) VH CH2; (iii) VH CH3; (iv) VH CH1 CH2; (v) VH CH1 CH2 CH3; (vi) VH CH2 CH3; (vii) VH CL; (viii) VL CH1 ; (ix) VL CH2; (X) VL CH3; (xi) VL CH1 CH2; (xii) VL CH1 CH2 CH3; (xiii) VL CH2 CH3; and (xiv) VL CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can be either directly linked to one another or can be linked by a full or partial hinge or linker region. A hinge region can comprise of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Moreover, the first and second binding moieties of this disclosure can comprise a homo dimer or hetero dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and / or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).

[0222] Fab, F(ab’)2, Fab’

[0223] The term “Fab” denotes a binding protein or a binding fragment thereof having a molecular weight of about 50,000 Da and antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating IgG with a protease, papain, are bound together through a disulfide bond. The term F(ab’)2 refers to a binding protein or a binding fragment thereof having a molecular weight of about 100,000 Da and antigen binding activity, which is slightly larger than a Fab bound via a disulfide bond of the hinge region, among fragments obtained by treating IgG with a protease, pepsin.

[0224] The term Fab’ refers to a binding protein or a binding fragment having a molecular weight of about 50,000 Da and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab’)2.

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[0226] 81660035v1 Attorney Docket No. 772089: SA9-915PC

[0227] Sanofi Ref No. PAT24106-WO-PCT scFV

[0228] A single chain Fv (“scFv”) polypeptide is a covalently linked VH: VL heterodimer that is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker. A human scFv fragment includes CDRs that are held in appropriate conformation by, e.g., using gene recombination techniques. Divalent and multivalent multi-specific binding proteins, or binding fragments or derivatives thereof can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2. A “dsFv” is a VH: VL heterodimer stabilized by a disulfide bond. “(dsFv)2” denotes two dsFv coupled by a peptide linker.

[0229] Immunoglobulin single variable domain (ISVD)

[0230] The term “immunoglobulin single variable domain (ISVD)” and “single variable domain” refer to an antibody fragment consisting of a single monomeric variable antibody domain that is able to bind selectively to a specific antigen. ISVDs have a molecular weight of about 1 1-18 kDaltons. Examples of different types of ISVDs include a camelid heavy-chain antibody (HCAbs), a single domain shark antibody (VNAR), or a (single) domain antibody (dAb or sdAb).

[0231] Camelid heavy-chain antibodies are functional antibodies devoid of light chains and CH1 domains which are naturally produced by the Camelidae species. The antigen recognition site is formed by a single domain called VHH. Sharks and other cartilaginous fish also naturally produce heavy-chain antibodies that recognize antigens with their single domain variable regions called VNAR.

[0232] ISVD defines immunoglobulin molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain as described below. The binding site of an ISVD is formed by a single VH, a single VHH, or single VL domain. The terms “VHH domains”, “VHHS”, “VHH antibody fragments”, and “VHH antibodies”, are described as the antigen binding immunoglobulin variable domain of “heavy chain antibodies” (i.e., “antibodies devoid of light chains”; see Hamers-Casterman et al. (1993), Nature, vol. 363: 446-448). The term “VHH domain” has been chosen to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “VH

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[0234] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “VL domains”). For a further description of VHHS see Muyldermans (2001 ), J. BiotechnoL, vol. 74(4): 277-302.

[0235] ISVD

[0236] The term “immunoglobulin single variable domain” (ISVD), interchangeably used with “single variable domain”, defines immunoglobulin molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from “conventional” immunoglobulins (e.g., monoclonal antibodies) or their fragments (such as Fab, Fab’, F(ab’)2, scFv, di-scFv), wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e., a total of 6 CDRs will be involved in antigen binding site formation. In contrast, ISVDs are capable of specifically binding to an epitope of the antigen without pairing with an additional immunoglobulin variable domain. The binding site of an ISVD is formed by a single VH, a single VHH or single VL domain.

[0237] ISVDs of the so-called “VH3 class” (i.e., ISVDs with a high degree of sequence homology to human germline sequences of the VH3 class such as DP-47, DP-51 or DP-29), can be used herein. Furthermore, any type of ISVD directed against a TNFRSF protein (e.g., GITR) and / or an immune check point protein (e.g., PD-1 ), including for example, ISVDs belonging to the so-called “VH4 class” (i.e., ISVDs with a high degree of sequence homology to human germline sequences of the VH4 class such as DP-78), as for example described in WO 2007 / 118 670 A1 .

[0238] In certain aspects, the multispecific binding protein of the disclosure comprises an ISVD selected from a group consisting of: a dAb, an immunoglobulin that is suitable for use as a domain antibody (dAb), a VHH (e.g., a humanized VHH sequence), a camelized VH, or a variant thereof.

[0239] ISVDs (in particular VHH sequences and partially humanized VHHS) can in particular be characterized by the presence of one or more “Hallmark residues” (as

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[0241] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT described herein in Table 1 and in subsequent paragraphs describing NANOBODY® immunoglobulin single variable domains) such that the ISVD is a NANOBODY® ISVD. Thus, generally, a NANOBODY® ISVD (in particular a VHH, including (partially or fully) humanized VHH and camelized VH) can be defined as an amino acid sequence with the (general) structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which one or more of the Hallmark residues are as further defined in Table 1 .

[0242] In particular, a NANOBODY® ISVD (in particular a VHH, including (partially) humanized VHH and camelized VH) can be an amino acid sequence with the (general) structure:

[0243] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which the framework sequences are as further defined herein.

[0244] In particular, an ISVD can be an amino acid sequence with the (general) structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively. ISVDs can specifically bind to (as defined herein) and / or are directed against a TNFRSF protein (e.g., GITR) and / or an immune check point protein (e.g., PD-1 ). Also useful are suitable fragments of these ISVDs and polypeptides that comprise, consist of, or essentially consist of one or more of such ISVDs and / or suitable fragments of the ISVDs. The term “immunoglobulin single variable domain (ISVD)” encompasses a NANOBODY® VHH as described in or WO 2008 / 020079 or WO 2009 / 138519, and thus in an aspect denotes a VHH, a humanized VHH or a camelized VH (such as a camelized human VH) or generally a sequence optimized VHH (such as e.g., optimized for chemical stability and / or solubility, maximum overlap with known human framework regions and maximum expression).

[0245] Generally, NANOBODY® immunoglobulin single variable domains (ISVDs) (in particular VHH sequences, including (partially) humanized VHH sequences and camelized VH sequences) can be characterized by the presence of one or more “Hallmark residues” (as described herein) in one or more of the framework sequences

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[0248] (again as further described herein). Thus, generally, a NANOBODY® ISVD can be defined as an immunoglobulin sequence with the (general) structure

[0249] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which one or more of the Hallmark residues are as further defined herein.

[0250] In particular, a NANOBODY® ISVD can be an immunoglobulin sequence with the (general) structure

[0251] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which the framework sequences are as further defined herein.

[0252] In particular, a NANOBODY® ISVD can be an immunoglobulin sequence with the (general) structure

[0253] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: one or more of the amino acid residues at positions 11 , 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table 1 below.

[0254] Table 1 : Hallmark Residues in Nanobody® ISVDs.

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[0260] The framework sequences are (a suitable combination of) immunoglobulin framework sequences or framework sequences that have been derived from immunoglobulin framework sequences (for example, by humanization or camelization). For example, the framework sequences can be framework sequences derived from a light chain variable domain (e.g., a Vi_-sequence) and / or from a heavy chain variable domain (e.g., a Vn-sequence or VHH sequence). In certain aspects, the framework sequences are either framework sequences that have been derived from a VHH-sequence (in which said framework sequences may optionally have been partially or fully humanized) or are conventional VH sequences that have been camelized.

[0261] In certain aspects, the framework sequences present in the ISVD sequences used in the methods described herein may contain one or more of hallmark residues (as defined in Table 1), such that the ISVD sequence is a NANOBODY® ISVD, such as e.g., a VHH, including a humanized VHH or camelized VH.

[0262] In certain aspects, a first cell surface binding moiety and a second binding moiety of a multispecific binding protein is each independently an immunoglobulin single variable domain (ISVD). Examples of immunoglobulin single variable domains (ISVDs) include variable domains obtained from heavy chain antibodies (VHHS), variable domains obtained from antibodies naturally devoid of light chains (VHHS), ISVDs derived from conventional four-chain antibodies, engineered ISVDs. ISVDs may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, bovine. ISVDs may be naturally occurring ISVDs present in a heavy chain antibody devoid of light chains. Camelidae species, for example camel, dromedary, llama, alpaca and guanaco, produce heavy chain antibodies naturally devoid of light chain. Camelid heavy chain antibodies also lack the CH1 domain.

[0263] ISVD Kabat numbering

[0264] The total number of amino acid residues in each of the CDRs for VH domains and for VHH domains may vary and may not correspond to the total number of amino

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[0266] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT acid residues indicated by the Kabat numbering (e.g., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). Consequently, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in a VH domain and a VHH domain will usually be in the range of from 1 10 to 120, often between 112 and 1 15. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0267] In vivo half-life and ISVD that increase in vivo half-life

[0268] In certain aspects, a multispecific binding protein further comprises one or more groups, residues, moieties or binding units provide the multispecific binding protein with enhanced therapeutic efficacy. For example, therapeutic efficacy can be enhanced by increasing the in vivo half-life (referred to also as “half-life”) of the multispecific binding protein described herein. In certain aspects, a multispecific binding protein further comprising one or more binding moieties operatively linked to the multispecific binding protein by a linker region, e.g., a linker encoded by any one of the amino acid sequences set forth in Table 3.

[0269] The term “in vivo half-life extension” means that the multispecific binding protein has an increased half-life in a mammal, such as a human subject, after administration. Half-life can be expressed, e.g., as t1 / 2beta. A multispecific binding protein half-life can be extended e.g., a polyethylene glycol molecule, an Fc domain, a modified Fc domain, serum proteins or fragments thereof, serum binding proteins (e.g., an IgG), or binding moieties that can bind to serum proteins (e.g., an ISVD that binds a serum protein). A multispecific binding protein half-life can be extended e.g., by one or more binding units that facilitates binding to a FcRn.

[0270] In certain aspects, the binding unit that facilitates binding to a FcRn is selected from a group consisting of: a Fc constant domain, a modified Fc domain, a serum albumin, a serum albumin, a serum immunoglobulin, or a variant thereof. In certain aspects, the serum albumin is a human serum albumin or the serum immunoglobulin is an IgG.

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[0273] In certain aspects, the binding unit is an ISVD that binds to a human serum albumin e.g., a ISVD that binds to a human serum albumin described in WO 2004 / 041865, WO 2006 / 122787, WO 2006 / 122787, WO2012 / 175400, WO 2012 / 175741 , WO 2015 / 173325, WO 2017 / 080850, WO 2017 / 085172, WO 2018 / 104444, WO 2018 / 134235, or WO 2018 / 134234, which are incorporated by reference herein in their entirety.

[0274] In certain aspects, an ISVD that binds to a human serum albumin is linked to the first or second binding moieties of the multispecific binding protein described herein. In certain aspects, the ISVD that binds to a human serum albumin extends the half-life of the multispecific binding protein relative to a reference multispecific binding protein that does not have said ISVD.

[0275] In certain aspects, an ISVD binding to human serum albumin is linked to the C- terminal end of the multispecific binding protein so that the C-terminal end of the human serum albumin is an exposed C-terminal end. In certain aspects, the C-terminal end of the ISVD binding to human serum albumin comprises a C-terminal extender. In certain aspects, the C-terminal extender is a C-terminal alanine (A) or glycine (G) extension. In certain aspects, C-terminal alanine (A) or glycine (G) extension comprises 1 -3 alanine or glycine residues, respectively.

[0276] In certain aspects, an ISVD capable of binding to human serum albumin is not linked to the C-terminal end of the multispecific binding protein and the C-terminal end of the human serum albumin is not an exposed C-terminal end. In certain aspects, the C-terminal sequence “VTVSS” (SEQ ID NO: 78) is directly followed by a linker region, e.g., a linker region of Table 3.

[0277] Linker Region

[0278] As used herein, the term “linker” denotes a peptide that fuses together two or more ISVDs into a single molecule. The use of linkers to connect two or more (poly)peptides is well known in the art. Further exemplary peptidic linkers are shown in Table 3. One often used class of peptidic linker are known as the “Gly-Ser” or “GS” linkers. These are linkers that essentially consist of glycine (G) and serine (S) residues, and usually comprise one or more repeats of a peptide motif such as the GGGGS (SEQ ID NO: 2) motif (for example, having the formula (Gly-Gly-Gly-Gly-Ser)n in which n may be 1 , 2, 3, 4, 5, 6, 7 or more). Some often-used examples of such GS

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[0280] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT linkers are 9GS linkers (GGGGSGGGS, SEQ ID NO: 5), 15GS linkers (n=3) and 35GS linkers (n=7). Reference is for example made to Chen et al. 2013 (Adv. Drug Deliv. Rev. 65(10): 1357-1369) and Klein et al. 2014 (Protein Eng. Des. Sei. 27 (10): 325- 330). In certain aspects, a multispecific binding protein of the disclosure comprises the linker connecting an ISVD to another ISVD is via a peptide linker. In certain aspects, the linker also connecting an ISVD to the first and / or second Fc heavy chain is a peptide linker. In certain aspects, the peptide linker is a poly-Glycine-Serine (G4S)n linker. In certain aspects, n equals 1 , 2, 3, 4, or 5.

[0281] Table 2. Linker Table Amino Acid Sequence

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[0285] In certain aspects, the peptide linker comprises an amino acid sequence which is at least 75, 80, 85, 90, 93, 95, 99% or more identical to an amino acid sequence of Table 2. In certain aspects, the linker comprises at least a portion of a hinge region (e.g., derived from an lgG1 , lgG2, lgG3, or lgG4 molecule) and a series of glycine-serine amino acid residues.

[0286] In certain aspects, a multispecific binding protein comprises a second binding moiety which is operatively linked to a first cell surface binding moiety and that specifically binds to the target protein.

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[0289] In certain aspects, a multispecific binding protein further comprises one or more binding moieties operatively linked to the first cell surface binding moiety or the second binding moiety that specifically binds to the target protein. In certain aspects, the operative linker is a peptide linker. In certain aspects, the peptide linker is at 90% identical to a peptide linker encoded by an amino acid sequence set forth in SEQ ID NOs: 1 -8 or 56-71.

[0290] In certain aspects, a compound or construct comprises the multispecific or the bispecific binding protein according to the disclosure that further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptide linker(s).

[0291] In certain aspects, a multispecific binding protein comprises at least three GITR ISVDs are directly linked to each other via a linker. In certain aspects, a multispecific binding protein of the disclosure comprises from N-terminus to C-terminus, the at least three GITR ISVDs and a first IgG domain. In some aspects, a multispecific binding protein of the disclosure comprises from N-terminus to C-terminus, a first IgG domain and the at least three GITR ISVDs.

[0292] In certain aspects, at least one GITR ISVD is not directly linked to another GITR ISVD via a linker. In certain aspects, at least one GITR ISVD is linked to the C-terminus and at least one GITR ISVD is linked to the N-terminus of a first IgG domain.

[0293] In certain aspects, the at least three GITR ISVDs that are linked to each other are also linked to the N-terminus of a first and / or a second Fc heavy chain. In certain aspects, the at least three GITR ISVDs that are linked to each other are also linked to the C- terminus of the first and / or second Fc heavy chain. In certain aspects, at least one GITR ISVD is linked to the C-terminus and at least one other GITR ISVD is linked to the N-terminus end of a first and / or second Fc heavy chain.

[0294] In certain aspects, a multispecific binding protein of disclosure comprises at least three GITR ISVDs that are linked to each other are also linked to the N-terminus of the first Fc heavy chain and wherein the second binding moiety is linked to the N- terminus of the second Fc heavy chain.

[0295] In certain aspects, a multispecific binding protein of disclosure comprises at least three GITR ISVDs that are linked to each other and are also linked to the C- terminus of the first Fc heavy chain. In certain aspects, at least three GITR ISVDs that

[0296] 40

[0297] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT are linked to each other are also linked to the C-terminus of the second Fc heavy chain. In certain aspects, the second binding moiety is linked to the N-terminus of the first and / or second Fc heavy chain.

[0298] In certain aspects, a multispecific binding protein of disclosure comprises at least one PD-1 ISVD that is not directly linked to another PD-1 ISVD via a linker. In certain aspects, the at least one PD-1 ISVD is linked to the N-terminus of a first and / or a second Fc heavy chain. In certain aspects, the at least one PD-1 ISVD is linked to the C-terminus of a first and / or a second Fc heavy chain. In certain aspects, the at least one PD-1 ISVD are linked to both the N- and the C-terminus of a first and / or second Fc heavy chain.

[0299] In certain aspects, a multispecific binding protein of disclosure comprises at least two PD-1 ISVDs are directly linked to each other via a linker. In certain aspects, the at least two PD-1 ISVDs that are linked to each other are also linked to the N- terminus of a first and / or a second Fc heavy chain. In certain aspects, the at least two PD-1 ISVDs that are linked to each other are also linked to the C-terminus of a first and / or a second Fc heavy chain. In certain aspects, the at least two PD-1 ISVDs are linked to both the N- and the C-terminus of a first and / or second Fc heavy chain.

[0300] In certain aspects, a multispecific binding protein of disclosure comprises at least two PD-1 ISVDs that are linked to each other are also linked to the N-terminus of a first Fc heavy chain. In certain aspects, the first binding moiety is linked to the N- terminus of the second Fc heavy chain. In certain aspects, the at least one PD-1 ISVD is linked to the N-terminus of a first Fc heavy chain and a second PD-1 ISVD is linked to the N-terminus of a second Fc heavy chain. In certain aspects, the first binding moiety is linked to the C-terminus of a first and / or second Fc heavy chain.

[0301] Increased half-life

[0302] In certain aspects, a multispecific binding protein further comprising the one or more binding moieties provide the multispecific binding protein with increased half-life, compared to a reference multispecific binding protein without said binding moieties. In certain aspects, the one or more binding units facilitates binding to a FcRn. In certain aspects, the binding unit that facilitates binding to a FcRn is selected from a group consisting of: a Fc constant domain, a modified Fc domain, a serum albumin, a serum

[0303] 41

[0304] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT albumin, a serum immunoglobulin, or a variant thereof. In certain aspects, the serum albumin is a human serum albumin or the serum immunoglobulin is an IgG.

[0305] Fc domain

[0306] In some aspects, the multispecific binding protein can comprise a Fc domain. As used herein, the term “Fc domain” or “Fc region” of “Fc heavy chain” (used interchangeably) is defined as the portion of a heavy chain constant region beginning in the hinge region just upstream of the papain cleavage site (i.e., residue 216 in IgG, taking the first residue of heavy chain constant region to be 1 14) and ending at the C- terminus of the antibody. Accordingly, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain.

[0307] The terms “Fc variant,” “modified Fc,” “engineered Fc” are interchangeably used herein refer to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn (neonatal Fc receptor). Exemplary Fc variants, and their interaction with the salvage receptor, are known in the art. A modified Fc domain can comprise a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises regions that can be removed because they provide structural features or biological activity that are not required for the multispecific binding protein compositions. Thus, the term “modified Fc domain” comprises a molecule or sequence that lacks one or more native Fc sites or residues, or in which one or more Fc sites or residues has be modified, that affect or are involved in: (1 ) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0308] “Effector function” as used herein is meant a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. A “functional Fc region” possesses an “effector function” of a native sequence Fc region. Exemplary “effector functions” include antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cell-mediated phagocytosis (ADCP).

[0309] The term “EU index” refers to the EU numbering convention for the constant regions of an antibody, as described in Edelman, GM. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest,

[0310] 42

[0311] 81660035v1 Attorney Docket No. 772089: SA9-915PC

[0312] Sanofi Ref No. PAT24106-WO-PCT

[0313] U.S. Dept. Health and Human Services, 5th edition, 1991 , each of which is herein incorporated by reference in its entirety. Unless otherwise stated, all antibody Fc region numbering employed herein corresponds to the EU numbering scheme, as described in Edelman et al. (Proc. Natl. Acad. Sci. 63(1 ): 78-85. 1969).

[0314] The Fc domain of the multispecific binding protein of the disclosure may be engineered to promote heterodimerization over homodimerization. For example, the heavy chain constant region of the first heavy-light chain pair may comprise a different amino acid sequence from the heavy chain constant region of the second heavy-light chain pair, wherein the different amino acid sequences are engineered to promote heterodimerization of the heavy chain constant regions. Examples include knobs-into- holes mutations or charge pair mutations. Alternatively, the heavy chain constant region of the first heavy-light chain pair may be identical to the heavy chain constant region of the second heavy-light chain pair, in which case it is expected that both homodimers and heterodimers will assemble, and these will be subsequently separated using one or more purification steps in the binding protein manufacturing process to isolate the desired heterodimer.

[0315] In certain aspects, a first and / or second Fc constant domain is derived from an immunoglobulin class selected from a group consisting of: IgM, IgG, IgD, IgA, IgE. Chimeric Fc domains comprising portions of Fc domains from different species or Ig classes can also be employed. In certain aspects, a Fc constant domain is an IgG Fc domain, an IgG 1 Fc domain, or an lgG4 Fc domain.

[0316] In certain aspects, the first Fc domain can be engineered to pair or heterodimerize with the second Fc domain. For example, a multispecific binding protein of the disclosure may comprise a first polypeptide comprising a first binding moiety comprising at least one immunoglobulin single variable domain (ISVD) that specifically binds glucocorticoid-induced TNFR family-related receptor (GITR) (GITR ISVD) and an Fc domain and a second polypeptide comprising a second binding moiety comprising at least one ISVD that specifically binds to an immune checkpoint protein, wherein the second polypeptide comprises a second Fc domain capable of dimerizing with the first Fc domain. In certain aspects the first and / or second Fc domain can be modified.

[0317] In certain aspects, a multispecific binding protein of the disclosure comprise a fragment crystallizable (Fc) domain or variant thereof. In certain aspects, each binding

[0318] 43

[0319] 81660035v1 Attorney Docket No. 772089: SA9-915PC

[0320] Sanofi Ref No. PAT24106-WO-PCT moiety (e.g., the first and the second binding moiety) is operatively linked to the Fc domain or variant thereof. In certain aspects, the Fc domain or variant thereof comprises a first and a second immunoglobulin G (IgG) domain that dimerize to form the bispecific binding protein.

[0321] Fc mutations

[0322] In certain aspects, a multispecific binding protein of the disclosure comprises a modified Fc domain and can further comprise one or more, e.g., two or more, three or more, or four or more, amino acid substitutions that confers a multispecific binding protein with one or more biochemical characteristics other than glycan modification. Exemplary modified Fc domain amino acid substitutions that can confer additional biochemical characteristics to the multispecific binding proteins described herein are disclosed in W02021016571 A2, which is incorporated by reference in its entirety.

[0323] In certain aspects, a modified Fc region of a multispecific binding protein of the current disclosure comprises one or more mutations to modulate half-life (See e.g., Dall'Acqua et al. (2006) J Biol Chem 281 : 23514-24, Zalevsky et al. (2010) Nat Biotechnol 28: 157-9, Hinton et al. (2004) J Biol Chem 279: 6213-6, Hinton et al. (2006) J Immunol 176: 346-56, Shields et al. (2001 ) J Biol Chem 276: 6591 -604, Petkova et al. (2006) Int Immunol 18: 1759-69, Datta-Mannan et al. (2007) Drug Metab Dispos 35: 86-94, Vaccaro et al. (2005) Nat Biotechnol 23: 1283-8, Yeung et al. (2010) Cancer Res 70: 3269-77 and Kim et al. (1999) Eur J Immunol 29: 2819-25. (e.g., T250Q, M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A and / or H435R).

[0324] In certain aspects, a modified Fc region of a multispecific binding protein of the current disclosure can have enhanced FcRn binding affinities at both an acidic pH (e.g., less than about 7.0, no more than about 6.5, or no more than about 6.0) and a non-acidic pH (e.g., no less than about 7.0, or no less than about 7.4), as compared to its wild-type. In another example, a multispecific binding protein comprising a modified Fc can comprise one or more amino acid mutations (e.g., substitutions) which alter the effector functions (e.g., ADCC or CDC function) of the Fc domain, as compared to a corresponding wild-type molecule, e.g., a molecule having the same structure as the FcRn antagonist except that it has a wild-type Fc domain. In another example, a multispecific binding protein can comprise a modified Fc domain

[0325] 44

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[0327] Sanofi Ref No. PAT24106-WO-PCT comprising one or more amino acid mutations (e.g., substitutions) which alter (e.g., increase or decrease) the circulating half-life (e.g., serum half-life) of the FcRn antagonist, as compared to the corresponding wild-type molecule.

[0328] In certain aspects, a multispecific binding protein described herein can comprise a modified Fc domain that alters serum half-life compared to a multispecific binding protein comprising a wild-type Fc domain. In certain aspects, a modified Fc domain has an increased serum half-life compared to a multispecific binding protein comprising a wild-type Fc domain. In certain aspects, a Fc domain is modified to alter FcRn binding affinity compared to a multispecific binding protein comprising a wildtype Fc domain. In certain aspects, a modified Fc domain has enhanced FcRn binding affinity compared to a multispecific binding protein comprising a wild-type Fc domain. In certain aspects, a Fc domain is modified to enhance the FcRn binding affinity at an acidic pH compared to a multispecific binding protein comprising a wild-type Fc domain.

[0329] In certain aspects, a multispecific binding protein can have a modified Fc domain. In certain aspects, a multispecific binding protein can have a tyrosine (Y) at amino acid position 252, according to EU numbering. In certain aspects, a multispecific binding protein can have an aspartic acid (D) or a glutamic acid (E) at amino acid position 256, according to EU numbering. In certain aspects, a multispecific binding protein can have a tryptophan (W) or a glutamine (Q) at amino acid position 307, according to EU numbering. In certain aspects, a multispecific binding protein can have a phenylalanine (F) or a tyrosine (Y) at amino acid position 434; according to EU numbering.

[0330] In certain aspects, a multispecific binding protein can have a modified Fc domain comprising any combination of the following four amino acid residues: a tyrosine (Y) at amino acid position 252, an aspartic acid (D) or a glutamic acid (E) at amino acid position 256, a tryptophan (W) or a glutamine (Q) at amino acid position 307, and a phenylalanine (F) or a tyrosine (Y) at amino acid position 434; according to EU numbering.

[0331] In certain aspects, a multispecific binding protein can comprise a modified Fc domain having a combination of amino acid residues selected from the group consisting of: a) a tyrosine (Y) at amino acid position 252, an aspartic acid (D) at amino acid position 256, a glutamine (Q) at amino acid position 307, and a tyrosine (Y) at

[0332] 45

[0333] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT amino acid position 434; b) a tyrosine (Y) at amino acid position 252, a glutamic acid

[0334] (E) at amino acid position 256, a tryptophan (W) at amino acid position 307, and a tyrosine (Y) at amino acid position 434; c) a tyrosine (Y) at amino acid position 252, a glutamic acid (E) at amino acid position 256, a glutamine (Q) at amino acid position 307, and a tyrosine (Y) at amino acid position 434; d) a tyrosine (Y) at amino acid position 252, an aspartic acid (D) at amino acid position 256, a glutamine (Q) at amino acid position 307, and a phenylalanine (F) at amino acid position 434; e) a tyrosine (Y) at amino acid position 252, an aspartic acid (D) at amino acid position 256, a tryptophan (W) at amino acid position 307, and a tyrosine (Y) at amino acid position 434; and f) a tyrosine (Y) at amino acid position 252, an aspartic acid (D) at amino acid position 256, a tryptophan (W) at amino acid position 307, and a phenylalanine

[0335] (F) at amino acid position 434; according to EU numbering.

[0336] In certain aspects, a multispecific binding protein can comprise a modified Fc domain comprising a quadruple amino acid substitution selected from the group consisting of: M252Y / T256D / T307Q / N434Y, M252Y / T256E / T307W / N434Y, M252Y / T256E / T307Q / N434Y, M252Y / T256D / T307Q / N434F,

[0337] M252Y / T256D / T307W / N434Y, and M252Y / T256D / T307W / N434F; according to EU numbering.

[0338] In certain aspects, a multispecific binding protein of the disclosure can comprise a modified Fc domain comprising: an amino acid substitution at positions 234, 235, and / or 329, according to EU numbering. In certain aspects, the Fc domain comprises a leucine (L) to an alanine (A) substitution at amino acid positions 234 and 235, according to EU numbering. In certain aspects, the Fc domain comprises a proline (P) to a glycine (G) substitution at amino acid position 329, according to EU numbering.

[0339] In certain aspects, a multispecific binding protein of the disclosure can comprise a modified Fc domain comprising: the first IgG domain comprises an amino acid substitution at position 435 and the second IgG domain comprises an amino acid mutation 436, according to EU numbering. In certain aspects, the first IgG domain comprises a histidine (H) to an arginine (R) at amino acid position 435 and the second IgG domain comprises a tyrosine (Y) to a phenylalanine (F) at amino acid position 436, according to EU numbering.

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[0342] Sanofi Ref No. PAT24106-WO-PCT

[0343] Knobs and Holes

[0344] Techniques for making multispecific binding proteins (e.g., multispecific antibodies) include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein, C. and Cuello, A. C., Nature 305 (1983) 537-540, WO 93 / 08829, and Traunecker, A. et aL, EMBO J. 10 (1991 ) 3655-3659), and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731 ,168). Multispecific binding proteins may also be made by engineering electrostatic steering effects for making binding protein Fc- heterodimeric molecules (WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan, M. et aL, Science 229 (1985) 81 -83); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny, S. A. et aL, J. Immunol. 148 (1992) 1547-1553; using “diabody” technology for making multispecific binding protein fragments (see, e.g., Holliger, P. et aL, Proc. NatL Acad. Sci. USA 90 (1993) 6444-6448); and using single-chain Fv (scFv) dimers (see, e.g., Gruber, M et aL, J. Immunol. 152 (1994) 5368-5374); and preparing trispecific binding proteins as described, e.g., in Tutt, A. et aL, J. Immunol. 147 (1991 ) 60-69).

[0345] A wide variety of recombinant multispecific binding protein formats have been developed, e.g., by fusion of, e.g., an IgG binding protein format and single chain domains (see Kontermann RE, mAbs 4:2, (2012) 1 -16). Multispecific binding proteins wherein the variable domains VL and VH or the constant domains CL and CH1 are replaced by each other are described in W02009080251 and W02009080252.

[0346] An approach to circumvent the problem of mispaired byproducts, is known as “knobs- into-holes”, aims at forcing the pairing of two different binding protein heavy chains by introducing mutations into the CH3 domains to modify the contact interface. On one chain bulky amino acids can be replaced by amino acids with short side chains to create a “hole.” Conversely, amino acids with large side chains can be introduced into the other CH3 domain, to create a “knob.” By co-expressing these two heavy chains (and two identical light chains, which have to be appropriate for both heavy chains), high yields of heterodimer formation (“knob-hole”) versus homodimer formation (“holehole” or “knob-knob”) was observed (Ridgway JB, Presta LG, Carter P; and WO1 996027011 ). The percentage of heterodimer could be further increased by remodeling the interaction surfaces of the two CH3 domains using a phage display

[0347] 47

[0348] 81660035v1 Attorney Docket No. 772089: SA9-915PC

[0349] Sanofi Ref No. PAT24106-WO-PCT approach and the introduction of a disulfide bridge to stabilize the heterodimers (Merchant A. M, et al, Nature Biotech 16 (1998) 677-681 ; Aiwell S, Ridgway JB, Wells JA, Carter P., J Mol Biol 270 (1997) 26-35). New approaches for the knobs-into-holes technology are described in e.g., in EP 1870459A1. Xie, Z., et al, J Immunol Methods 286 (2005) 95-101 refers to a format of multispecific binding protein using scFvs in combination with knobs-into-holes technology for the Fc part.

[0350] In certain aspects, the CH3 domains of the heavy chains of a multispecific binding protein can be altered by the “knob-into-holes” technology, which is described in detail with several examples in e.g., WO 96 / 027011 , WO 98 / 050431 , Ridgway J. B. et al., Protein Eng. 9 (1996) 617-621 , Merchant A. M. et al., Nat Biotechnol 16 (1998) 677-681 . In this method, the interaction surfaces of the two CH3 domains are altered to increase the heterodimerization of both heavy chains containing said two CH3 domains. Each of the two CH3 domains (of the two heavy chains) can be the “knob,” while the other is the “hole.” The introduction of a disulfide bridge can be utilized to stabilize the heterodimers (Merchant A. M et al., Nature Biotech 16 (1998) 677-681 , Atwell, S. et al., J. Mol. Biol. 270 (1997) 26-35), as well as to increase the yield.

[0351] The Fc domain of a multispecific binding protein can be engineered to promote heterodimerization over homodimerization. For example, the heavy chain constant region of the first heavy-light chain pair can comprise a different amino acid sequence from the heavy chain constant region of the second heavy-light chain pair, wherein the different amino acid sequences are engineered to promote heterodimerization of the heavy chain constant regions. Examples include knobs-into-holes mutations or charge pair mutations. Alternatively, the heavy chain constant region of the first heavy-light chain pair may be identical to the heavy chain constant region of the second heavylight chain pair, in which case it is expected that both homodimers and heterodimers will assemble, and these will be subsequently separated using one or more purification steps in the antibody manufacturing process to isolate the desired heterodimer.

[0352] In certain aspects, a multispecific binding protein of the disclosure comprises a first and a second IgG Fc domain polypeptide that dimerize to form the bispecific binding protein. In certain aspects, the first and second IgG Fc domain polypeptides dimerize by knobs-into-holes interactions, Fab arm exchange (FAE), electrostatic steering interactions, hydrophobic interactions, or any combination thereof. In certain aspects, the first IgG Fc domain polypeptide comprises a knob substitution, and the

[0353] 48

[0354] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT second IgG Fc domain polypeptide comprises a hole substitution. In certain aspects, a multispecific binding protein comprises a knob substitution that is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). In certain aspects, the multispecific binding protein comprises a hole substitution that is selected from the group consisting of alanine (A), asparagine (N), aspartic acid (D), glycine (G), serine (S), threonine (T), and valine (V).

[0355] Multispecific binding protein orientations

[0356] The multispecific binding proteins (e.g., bispecific binding proteins) of the disclosure may comprise a pentavalent form or an octavalent form.

[0357] In the pentavalent form, the multispecific binding protein comprises or consists of three TNFRSF ISVDs and comprises or consists of two immune checkpoint ISVDs. In the pentavalent form, the multispecific binding protein comprises or consists of three GITR ISVDs and comprises or consists of two PD-1 ISVDs.

[0358] Thus in one aspect, the disclosure provides a bispecific binding protein comprising: (a) a first binding moiety comprising at least three GITR ISVDs directly linked to each other and wherein one GITR ISVD is linked to the N-terminal end of a first IgG; (b) a second binding moiety comprising at least two PD-1 ISVDs directly linked to each other and wherein one PD-1 ISVD is linked to the N-terminal end of a second IgG; and wherein each the first and second IgG 1 domains heterodimerize to form a Fc domain or variant thereof.

[0359] In some aspects, the first binding moiety consists of three GITR ISVDs.

[0360] In some aspects, the second moiety consists of two PD-1 ISVDs.

[0361] In the octavalent form, the bispecific binding protein comprises or consists of six TNFRSF ISVDs and comprises or consists of two immune checkpoint ISVDs. In the octavalent form, the bispecific binding protein comprises or consists of six GITR ISVDs and comprises or consists of two PD-1 ISVDs.

[0362] Thus in one aspect, the disclosure provides a bispecific binding protein comprising: (a) a first binding moiety comprising at least three GITR ISVDs directly linked to each other and wherein one GITR ISVD is linked to the C-terminal end of a first and a second IgG; (b) a second binding moiety comprising at least two PD-1 ISVDs wherein each PD-1 ISVD is directly linked to the N-terminal end of the first and

[0363] 49

[0364] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT the second IgG; and wherein each the first and second lgG1 domains form a Fc domain or variant thereof.

[0365] In some aspects, the first binding moiety consists of six GITR ISVDs.

[0366] In some aspects, the second moiety consists of two PD-1 ISVDs.

[0367] TNFRSF Binding Moieties

[0368] In certain aspects, the multispecific binding protein of the disclosure comprises a first binding moiety that targets protein may be a TNFRSF (Tumor Necrosis Factor Receptor Super Family) member. The TNFRSF is a protein superfamily of cytokine receptors characterized by the ability to bind tumor necrosis factors (TNFs) via an extracellular cysteine-rich domain where the homology identified between family members is mainly found in the extracellular domain. With a few exceptions e.g., a nerve growth factor (NGF), all TNFs are homologous to the archetypal TNF-alpha.

[0369] In certain aspects, the multispecific binding protein of the disclosure will comprise a first binding moiety comprising at least three ISVDs that specifically bind a TNFRSF receptor (TNFRSF ISVDs) protein. 2. The multispecific binding protein of claim 1 , wherein the at least three TNFRSF ISVDs specifically bind the same or different TNFRSF protein(s). In certain aspects, wherein the at least three TNFRSF ISVDs specifically bind the same TNFRSF protein. In certain aspects, the at least three TNFRSF ISVDs bind to the same or different contacts on the same TNFRSF protein.

[0370] In certain aspects, the first binding moiety is an antagonist of the TNFRSF protein(s). In certain aspects, first binding moiety is an agonist of the TNSFRSF protein(s).

[0371] In certain aspects, the TNFRSF protein is selected from the group consisting of: a glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), a 4-1 BB, a 0X40, a tumor necrosis factor receptor 1 (TNFR1 ), a TNFR2, a lymphotoxin beta receptor (LTBR), a CD40, a fas receptor, a CD27, a CD30, a death receptor 3 (DR3), a DR4, a DR5, a DR6, a decoy receptor 1 (DCR1 ), a DCR2, a DCR3, a receptor activator of nuclear factor kappa-B (RANK), an osteoprotegerin (OPG), a tumor necrosis factor-like weak inducer of apoptosis receptor (TWEAK-R), a tachykinin precursor 1 (TACI), a B cell activating factor receptor (BAFF-R), a herpes virus entry mediator (HVEM), a nerve growth factor receptor (NGFR), a B-cell maturation antigen

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[0374] (BCMA), a TROY, and ectodysplasin A2 receptor (EDA2R). In certain aspects, the protein is GITR.

[0375] The term “glucocorticoid-induced TNF receptor”, hereinafter referred to as “GITR” is also known as Tumor Necrosis Receptor Superfamily 18 (TNFRSF18), Activation-Inducible TNFR Family Receptor (AITR), TEASR, CD357 and 312C2. GITR is constitutively expressed in all T cell subtypes and mostly in regulatory T cells (Treg), is up-regulated in CD4+I CD25- and CD8+I CD25- effector cells following TCR stimulation and cell activation (Nocentini et al. 2007, Eur J Immunol. 37:1 165-1169). GITR acts as a costimulatory molecule in effector T cell activation and regulates Treg cell suppressor activity (Esparza et al. 2005, J Immunol. 174:7869-7874).

[0376] The disclosure provides a multispecific binding protein comprising a first binding moiety comprising at least one ISVD that specifically binds GITR (GITR ISVD). In certain aspects, at least three ISVD specifically bind to GITR. In certain aspects, the GITR ISVD of the disclosure is an agonist of GITR. In certain aspects, each GITR ISVD of the multispecific binding protein is an agonist of GITR.

[0377] As used herein, an “agonist” refers to a compound that partially or fully increases, enhances, induces or stimulates one or more biological activities of a corresponding target (e.g., GITR) in vitro or in vivo. Examples of such biological activities of GITR include promoting CD4+and CD8+T cell survival, proliferation, NFkB signaling, interleukin-2 production and effector functions and abrogate Treg cell suppressive effects or the generation of Treg cells. As will be clear to the skilled person, such an increase in biological activity may be determined in any suitable manner and / or using any suitable (in vitro, cellular or in vivo) assay known per se, such as the assays described herein or in the prior art cited herein. In particular, the biological activity may be increased, by at least 5%, preferably at least 10%, at least

[0378] 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least

[0379] 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least

[0380] 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, such as 100%, compared to the biological activity in the same assay under the same conditions but without the presence of the polypeptide of the invention.

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[0384] Immune Checkpoint Proteins

[0385] As used herein, the term “immune checkpoint protein” refers to a protein expressed on the surface of immune cells (e.g., T cells) that modulates an immune response.

[0386] In certain aspects, the multispecific binding protein of the disclosure comprises comprising at least one ISVD that specifically binds to an immune checkpoint protein. In certain aspects, the second binding moiety comprises at least two ISVDs. In certain aspects, the at least two ISVDs specifically bind the same or different immune checkpoint protein(s). In certain aspects, the at least two ISVDs bind to the same or different contacts on the same immune checkpoint protein.

[0387] In certain aspects, the second binding moiety is antagonistic modulator (i.e., an inhibitor) of an immune checkpoint protein. In certain aspects the second binding moiety is an agonist (i.e., a stimulator) of the immune checkpoint protein(s).

[0388] In certain embodiments, the immune checkpoint protein is selected from a group consisting of: a programmed cell death protein 1 (PD-1 ), a PD ligand 1 (PD-L1 ), PD-L2, a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), a T-cell immunoglobulin and mucin domain 3 (TIM-3), a lymphocyte activation gene 3 (LAG- 3), a T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), a B and T lymphocyte attenuator (BTLA), a signaling lymphocytic activation molecule (SLAM), a leukocyte-associated immunoglobulin-like receptor 1 (LAIR1 ), a cluster of differentiation 244 (CD244), a CD28, a CD200 receptor 1 (CD200R1 ), a TREM-like transcript 2 (TLT2), a leukocyte immunoglobulin like receptor B4 (LILRB4), a killer cell immunoglobulin like receptor (KIR2DL2), and an inducible T- cell co-stimulator (ICOS). In certain aspects, the immune checkpoint protein is PD-1. In certain aspects, the multispecific binding of the disclosure comprises at least two ISVDs specifically bind to PD-1 (PD-1 ISVDs). In certain aspects, the multispecific binding protein of the disclosure comprises at least one PD-1 ISVD which is an antagonist. In certain aspects, the PD-1 ISVD binds to an extracellular domain of a PD-1 subunit. In certain aspects, at least two PD-1 ISVDs bind to the same PD-1 subunit. In certain aspects, at least two of the PD-1 ISVDs bind to different PD-1 subunits. In certain aspects, each PD-1 ISVD is an antagonist of PD-1 .

[0389] Interaction of PD-L1 with its receptor PD-1 on T cells delivers a signal that inhibits TCR-mediated activation of IL-2 production and T cell proliferation (e.g.,

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[0392] Sanofi Ref No. PAT24106-WO-PCT antigen specific T cells in the lymph nodes) as well as simultaneously reducing apoptosis in regulatory T cells (e.g., anti-inflammatory, suppressive T cells). Dysregulation of the PD-1 / PD-L1 interaction and related signaling has been implicated in the pathogenesis of neoplastic disorders, inflammatory disorders, and autoimmune disorders.

[0393] In certain aspects, the second binding moiety blocks the interaction of a target protein with other proteins resulting in immune modulation. For example, in certain aspects, the second binding moiety blocks the interaction of the immune checkpoint protein with other proteins resulting in immune modulation. In certain aspects, the second binding moiety blocks the interaction between PD-1 and PD-L1. PD-L1 is a membrane protein that forms a complex with its receptor PD-1. Analytical measurement to determine whether the second binding moiety blocks the interaction between PD-1 and PD-L1 are known in the art, e.g., see PD-1 :PD-L1 Cell-Based Inhibitor Screening Assay Kit (BPS Biosience; catalog # 79377).

[0394] In certain aspects, a multispecific binding protein blocks the PD-1 / PD-L1 interaction as measured by TCR signaling in T cells expressing human PD-1 cocultured with cells expressing PD-L1 . In certain aspects, a multispecific binding protein blocks the PD-1 / PD-L1 interaction as measured in Jurkat T cells expressing human PD-1 and a reporter gene co-cultured with CHO-K1 cells expressing human PD-L1 and activating TCRs in an antigen independent manner. In certain aspects, the reporter gene is a luciferase reporter gene driven by a NFAT promoter. In certain aspects, a half-maximal inhibitory concentration (IC50 [M]) of the multispecific binding protein for blocking the PD-1 / PD-L1 interaction is measured by TCR signaling in T cells expressing human PD-1 co-cultured with cells expressing PD-L1 is at least 5.0 x 109, 1.0 x 109, 5.0 x 1 O’10, 1.0 x 10-1°, 5.0 x 10-11, or 1 .0 x 10-11M.

[0395] In certain aspects, the multispecific binding protein of the disclosure comprises a half- maximal inhibitory concentration (IC50 [M]) binding value for PD-1 is less than about 1 x I O9.

[0396] In certain aspects, the multispecific binding protein of the disclosure comprises about 60% of the human PD-1 receptor is occupied when there is about 1 x 10-2nM or more of the multispecific binding protein. In certain aspects, the multispecific binding protein of the disclosure comprises about 1 x 10-1nM or more of the multispecific binding protein.

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[0399] Sanofi Ref No. PAT24106-WO-PCT

[0400] Kabat, EU, and AbM numbering

[0401] Amino acid positions in a heavy chain constant region, including amino acid positions in the CH1 , hinge, CH2, CH3, and CL domains, can be numbered according to the Kabat index numbering system (see Kabat et aL, in “Sequences of Proteins of Immunological Interest”, U.S. Dept. Health and Human Services, 5th edition, 1991 ). Alternatively, antibody amino acid positions can be numbered according to the EU index numbering system (see Kabat et aL).

[0402] In the CDR determination according to Kabat, FR1 of an immunoglobulin single variable domain comprises the amino acid residues at positions 1 -30, CDR1 of an immunoglobulin single variable domain comprises the amino acid residues at positions 31 -35, FR2 of an immunoglobulin single variable domain comprises the amino acids at positions 36-49, CDR2 of an immunoglobulin single variable domain comprises the amino acid residues at positions 50-65, FR3 of an immunoglobulin single variable domain comprises the amino acid residues at positions 66-94, CDR3 of an immunoglobulin single variable domain comprises the amino acid residues at positions 95-102, and FR4 of an immunoglobulin single variable domain comprises the amino acid residues at positions 103-1 13.

[0403] CDR sequences can also be determined according to the AbM numbering as described in Kontermann and Dubel (2010), Antibody Engineering, vol. 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51 . According to this method, FR1 comprises the amino acid residues at positions 1 -25, CDR1 comprises the amino acid residues at positions 26-35, FR2 comprises the amino acids at positions 36-49, CDR2 comprises the amino acid residues at positions 50-58, FR3 comprises the amino acid residues at positions 59-94, CDR3 comprises the amino acid residues at positions 95- 102, and FR4 comprises the amino acid residues at positions 103-1 13.

[0404] Specifically binds

[0405] A polypeptide (e.g., an immunoglobulin, an antibody, an immunoglobulin single variable domain, a VHH, or generally an antigen binding molecule or a fragment thereof) that can “bind to” or “specifically bind to”, that “has affinity for” and / or that “has specificity for” a certain epitope, antigen or protein (or for at least one part, fragment or epitope thereof) is said to be "against" or “directed against” said epitope, antigen or

[0406] 54

[0407] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT protein or is a “binding” molecule with respect to such epitope, antigen or protein, or is said to be “anti”-epitope, “anti”-antigen or “anti”-protein (e.g., “anti”- GITR and / or PD- 1 )-

[0408] A polypeptide that can "specifically binds" e.g., can bind to an antigen with a dissociation constant (KD) of at most about 1 x 10-6M, 1 x 10-7M, 1 x 10-8M, 1 x 10-9M, 1 x 10-1° M, 1 x 10-11M, 1 x 1012M, or less, and / or to bind to an antigen with an affinity that is at least two-fold greater than its affinity for a nonspecific antigen. Specific binding of an antibody can be to a target antigen through the CDR sequences. An antibody can also specifically bind to FcRs, such as FcRn or FcyRllla through the Fc region.

[0409] The terms “specificity”, “binding specifically” or “specific binding” refer to the number of different target molecules, such as antigens, from the same organism to which a particular binding unit, such as an ISVD, can bind with sufficiently high affinity (e.g., an ISVD that specifically binds to TNFRSF receptor protein or an immune checkpoint protein. “Specificity”, “binding specifically” or “specific binding” are used interchangeably herein with “selectivity”, “binding selectively” or “selective binding”. Binding units, such as ISVDs, specifically bind to their designated targets. The specificity / selectivity of a binding unit can be determined based on affinity. The affinity denotes the strength or stability of a molecular interaction. The affinity is commonly given by the KD, or dissociation constant, which has units of mol / liter (or M). The affinity can also be expressed as an association constant, KA, which equals 1 / KD and has units of (mol / liter)-1(or M-1).

[0410] In some aspects, a multispecific binding protein of the disclosure comprises at least three TNFRSF ISVDs that specifically bind the same or different TNFRSF protein(s). In some aspects, wherein the at least three TNFRSF ISVDs specifically bind the same TNFRSF protein. In some aspects, the at least three TNFRSF ISVDs bind to the same or different contacts on the same TNFRSF protein.

[0411] In some aspects, a multispecific binding protein of the disclosure comprises at least two ISVDs that specifically bind the same or different immune checkpoint protein(s). In some aspects, the at least two ISVDs bind to the same or different contacts on the same immune checkpoint protein.

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[0415] Affinity

[0416] The affinity is a measure for the binding strength between a binding moiety (e.g., a first and second binding moiety as described herein) and a binding site on the target molecule e.g., TNFRSF protein or an immune checkpoint protein) the lower the value of the KD, the stronger the binding strength between a target molecule and a targeting moiety. Typically, binding units used in the present technology, such as ISVDs, will bind to their targets with a dissociation constant (KD) of 10-5to 10-12moles / liter or less, 10-7to 10-12moles / liter or less, or 10-8to 10-12moles / liter (i.e. with an association constant (KA) of 105to 1012liter / moles or more, 107to 1012liter / moles or more, or 108to 1012liter / moles). Any KD value greater than 10-4mol / liter (or any KA value lower than 104liters / mol) is generally considered to indicate non-specific binding. The KD for biological interactions, such as the binding of immunoglobulin sequences to an antigen, which are considered specific are typically in the range of 10-5moles / liter (10000 nM or 10pM) to 10-12moles / liter (0.001 nM or 1 pM) or less.

[0417] Accordingly, specific / selective binding may mean that using the same measurement method, e.g., SPR- a binding unit (or polypeptide comprising the same) binds to GITR with a KD value of 10-5to 10-12moles / liter or less and binds to related TNFRSF members with a KD value greater than 10-4moles / liter. An example of a related TNFRSF member is 4-1 BB, 0X40, TNFR1 (Tumor necrosis factor receptor 1 ), TNFR2 (Tumor necrosis factor receptor 2), LTBR (Lymphotoxin beta receptor), CD40, Fas receptor, CD27, CD30, DR3 (Death receptor 3), DR4 (Death receptor 4), DR5 (Death receptor 5), DR6 (Death receptor 6), DCR1 (Decoy receptor 1 ), DCR2 (Decoy receptor 2), DCR3 (Decoy receptor 3), RANK (Receptor activator of nuclear factor kappa-B), Osteoprotegerin, TWEAK receptor, TACI, BAFF receptor, HVEM (Herpes virus entry mediator), Nerve growth factor receptor, B-cell maturation antigen, TROY and Ectodysplasin A2 receptor. Thus, in an embodiment of the present technology, the ISVD binds to (human) GITR with a KD value of 10-5to 10-12moles / liter or less and binds to 4-1 BB or 0X40 of the same species with a KD value greater than 10-4moles / liter.

[0418] Accordingly, specific / selective binding may mean that using the same measurement method, e.g., SPR- a binding unit (or polypeptide comprising the same) binds to PD-1 with a KD value of 10-5to 10-12moles / liter or less and binds to related

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[0420] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT immune checkpoint protein members with a KD value greater than 10-4moles / liter. An example of a related immune checkpoint protein member is cytotoxic T-lymphocyte- associated antigen 4 (CTLA-4), T-cell immunoglobulin and mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), and B and T lymphocyte attenuator (BTLA). Thus, in an embodiment of the present technology, the ISVD binds to (human) PD-1 with a KD value of 10-5to 10-12moles / liter or less and binds to CTLA-4, TIM-3, LAG-3, TIGIT or BTLA of the same species with a KD value greater than 10-4moles / liter.

[0421] Specific binding to a certain target from a certain species does not exclude that the binding unit can also specifically bind to the analogous target from a different species. For example, specific binding to human GITR and / or PD-1 does not exclude that the binding unit or a polypeptide comprising the same can also specifically bind to GITR and / or PD-1 from cynomolgus monkeys (“cyno”).

[0422] In some aspects, the multispecific binding protein of the disclosure comprises least one GITR ISVD specifically binds human GITR. In some aspects, the multispecific binding protein of the disclosure comprises at least one PD-1 ISVD specifically binds human PD-1 .

[0423] Specific binding of a binding unit (or a binding subunit) to its designated target can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned further herein.

[0424] In some aspects, the multispecific binding protein of the disclosure comprises at least one GITR ISVD binds to an extracellular domain of a GITR subunit. In some aspects, the at least three of the GITR ISVDs bind to the same GITR subunit. In some aspects, the at least one of the GITR ISVDs can bind to different GITR subunits.

[0425] In some aspects, the multispecific binding protein of the disclosure comprises at least one PD-1 ISVD binds to an extracellular domain of a PD-1 subunit. In some aspects, at least two PD-1 ISVDs bind to the same PD-1 subunit. In some aspects, at least two of the PD-1 ISVDs bind to different PD-1 subunits.

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[0429] Cross-reactive

[0430] An amino acid sequence is said to be “cross-reactive” for two different antigens or antigenic determinants (such as e.g., serum albumin from two different species of mammal, such as e.g., human serum albumin and cyno serum albumin, such as e.g., GITR and / or PD-1 from different species of mammal, such as e.g., human GITR and / or PD-1 , cyno GITR and / or PD-1 and mouse GITR and / or PD-1 ) if it is specific for (as defined herein) these different antigens or antigenic determinants.

[0431] Block or antagonist

[0432] The terms “block”, “antagonize”, “compete”, “competing” and “-competition” are used interchangeably herein to mean the ability of an immunoglobulin, antibody, ISVD(s), VHH, multispecific or bispecific binding protein, polypeptide, or other binding agent to interfere with the binding of another protein(s), polypeptide(s), ligand(s) or binding agent(s) to a given target. In certain aspects, the blocking the given target results in inhibition or modulation of a downstream signal transduction pathway.

[0433] The extent to which an immunoglobulin, antibody, ISVD(s), VHH, multispecific or bispecific binding protein, polypeptide, or other binding agent is able to interfere with the binding of another ligand to the target, and therefore whether it can be said to “block”, can be determined using competition binding assays. Particularly suitable quantitative competitive blocking assays are described in the Examples and include e.g., a fluorescence-activated cell sorting (FACS) binding assay with GITR and / or PD- 1 expressed on cells. The extent of blocking can be measured by the (reduced) channel fluorescence.

[0434] The following generally describes a suitable FACS assay for determining whether an immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent blocks or is capable of blocking. It will be appreciated that the assay can be used with any of the immunoglobulin single variable domains and polypeptides described herein. The FACS instrument (e.g. FACS Canto; Becton Dickinson) is operated in line with the manufacturer's recommendations.

[0435] To evaluate the “blocking” or “competition” between two binding agents (such as an immunoglobulin single variable domain and a natural ligand or another binding agent) for binding GITR and PD-1 , a FACS competition experiment can be performed

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[0438] Sanofi Ref No. PAT24106-WO-PCT using cells (such as e.g. Flp-lnTM-293 cells) overexpressing human GITR and PD-1 and the parental cells as background cell line. Different detection reagents can be used including e.g. monoclonal ANTI-FLAG® M2 antibody (Sigma-Aldrich, cat# F1804), monoclonal anti-C-myc antibody (Sigma-Aldrich, cat# WH0004609M2), monoclonal ANTI-HIS TAG antibody (Sigma-Aldrich, cat# SAB1305538), each labeled differently. A wide range of fluorophores can be used as labels in flow cytometry (such as e.g., PE (R-Phycoerythrin), 7-aminoactinomycin D (7-AAD), Acridine Orange, various forms of Alexa Fluor, Allophycocyanin (APC), AmCyan, Aminocoumarin, APC Cy5, APC Cy7, APC-H7, APC / Alexa Fluor 750, AsRed2, Azami-Green, Azurite, B ODIPY FL C5-ceramide, BCECF-AM, Bis-oxonol DiBAC2(3), BODIPY-FL, Calcein, Calcein AM, Caroxy-H2DCFDA, Cascade Blue, Cascade Yellow, Cell Tracker Green, Cerulean, CFSE, Chromomycin A3, CM-H2DCFDA, Cy2, Cy3, Cy3.5, Cy3B, Cy5, Cy5.5, Cy7, CyPet, DAF-FM DAF-FM diacetate, DAPI, DCFH (2'7'Dichorodihydrofluorescein), DHR, Dihydrocalcein AM, Dihydrorhoadamine, Dihydrothidium, DiLC1 (5), DiOC6(3), DiOC7(3), dKeima-Red, DRAQ5, Dronpa- Green, various forms of DsRed dTomato, various forms of DyLight, E.coli BioParticles AF488, E2-Crimson, E2-Orange, EBFP2, ECFP, various forms of eFluor, EGFP, EGFP*, Emerald, eqFP650, eqFP670, ER-Tracker Blue-White DPX, Ethidium Bromide, Express2, EYFP, Fc OxyBurst Green, Fc OxyBurst Green 123, FITC, Fluo- 3, Fluo-4, Fluorescein, Fura-2, Fura-Red, GFPuv, H2DCFDA, HcRedl , Hoechst Blue (33258), Hoechst Red (33342), Hydroxycoumarin, HyPer, lndo-1 , lndo-1 Blue (Low Ca2+), lndo-1 Violet (High Ca2+), iRFP, J-Red, JC-1 , JC-9, Katushka (TurboFP635), Katushka2 Kusabira-Orange, LDS 751 , Lissamine Rhodamine B, various forms of Live / Dead, Lucifer yellow, Lucifer Yellow CH, Lyso Tracker Blue, Lyso Tracker Green, Lyso Tracker Red, mAmertrine, Marina Blue, mBanana, mCFP, mCherry, mCitrine, Methoxycoumarin, mHoneyDew, Midoriishi-Cyan, Mithramycin, Mito Tracker Deep Red, Mito Tracker Green, Mito Tracker Orange, Mito Tracker Red, MitoFluor Green, mKate (TagFP635), mKate2, mKeima, mKeima-Red, mKO, mKOk, mNeptune, Monochlorobimane, mOrange, mOrange2, mRaspberry, mPlum, mRFP1 , mStrawberry, mTangerine, mTarquoise, mTFP1 , mTFP1 (Teal), NBD, OxyBurst Green H2DCFDA, OxyBurst Green H2HFF BSA, Pacific Blue, PE (R-Phycoerythrin), PE Cy5, PE Cy5.5, PE Cy7, PE Texas Red, PE-Cy5 conjugates, PE-Cy7 conjugates, PerCP (Peridinin chlorphyll protein), PerCP Cy5.5, PhiYFP, PhiYFP-m, Propidium

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[0441] Iodide (PI), various forms of Qdot, Red 613, RFP Tomato, Rhod-2, S65A, S65C, S65L, S65T, Singlet Oxygen Sensor Green, Sirius, SNARF, Superfolder GFP, SYTOX Blue, SYTOX Green, SYTOX Orange, T-Sapphire, TagBFP, TagCFP, TagGFP, TagRFP, TagRFP657, TagYFP, tdTomato, Texas Red, Thiazole Orange, TMRE, TMRM, Topaz, TOTO-1 , TO-PRO-1 , TRITC, TRITC TruRed, TurboFP602, TurboFP635, TurboGFP, TurboRFP, TurboYFP, Venus, Vybrant CycleDye Violet, Wild Type GFP, X-Rhodamin, Y66F, Y66H, Y66W, YOYO-1 , YPet, ZsGreenl , ZsYellowl , Zymosan A BioParticles AF488 (see more at: http: / / www.thefcn.org / flow-fluorochromes). Fluorophores, or simply “fluors”, are typically attached to the antibody (e.g. the immunoglobulin single variable domain) that recognizes GITR and / or PD-1 or to the antibody that is used as detection reagent. Various conjugated antibodies are available, such as (without being limiting) for example antibodies conjugated to Alexa Fluor®, DyLight®, Rhodamine, PE, FITC, and Cy3. Each fluorophore has a characteristic peak excitation and emission wavelength. The combination of labels which can be used will depend on the wavelength of the lamp(s) or laser(s) used to excite the fluorophore and on the detectors available.

[0442] EC50

[0443] “EC50” is the half maximal effective concentration that measures the concentration of a multispecific binding protein described herein or e.g., a first or second binding moiety of the same which induces a biological response halfway between the baseline and maximum after a specified exposure time.

[0444] To evaluate the competition between two test binding agents (termed A and B) for binding to a e.g., a first or second binding moiety target (e.g., a TNFRSF protein or an immune check point protein), a dilution series of cold (without any label) binding agent A is added to (e.g. 200 000) cells together with the labeled binding agent B*. The concentration of binding agent B* in the test mix should be high enough to readily saturate the binding sites of target (e.g., a TNFRSF protein or an immune check point protein) expressed on the cells. The concentration of binding agent B* that saturates the binding sites for that binding agent on e.g., a TNFRSF protein or an immune check point protein, expressed on the cells can be determined with a titration series of binding agent B* on cells comprising e.g., a TNFRSF protein or an immune check point protein

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[0447] Sanofi Ref No. PAT24106-WO-PCT and determination of the EC50 value for binding. To work at saturating concentration, binding agent B* can be used at 100x the EC50 concentration.

[0448] After incubation of the cells with the mixture of binding agent A and binding agent B* and cells wash, read out can be performed on a FACS. First a gate is set on the intact cells as determined from the scatter profile and the total amount of channel fluorescence is recorded. A separate solution of binding agent B* is also prepared. Binding agent B* in this solution should be in the same buffer and at the same concentration as in the test mix (with binding agent A and B*). This separate solution is also added to the cells. After incubation and cells wash, read out can be performed on a FACS. First a gate is set on the intact cells as determined from the scatter profile and the total amount of channel fluorescence is recorded. A reduction of fluorescence for the cells incubated with the mixture of binding agent A and B* compared to the fluorescence for the cells incubated with the separate solution of binding agent B* indicates that binding agent A blocks binding by binding agent B* cells expressing e.g., a TNFRSF protein or an immune check point protein.

[0449] A cross-blocking immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent is one which will bind to e.g., a TNFRSF protein or an immune check point protein in the above competition FACS such that during the assay and in the presence of the second binding agent the recorded fluorescence is between 80% and 0.1 % (e.g. 80% to 4%) of the maximum fluorescence (measured for the separate labelled immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent), specifically between 75% and 0.1 % (e.g. 75% to 4%) of the maximum fluorescence, and more specifically between 70% and 0.1 % (e.g. 70% to 4%) of maximum fluorescence (as just defined above).

[0450] The competition between two test binding agents (termed A* and B*) for binding to e.g., a TNFRSF protein or an immune check point protein can also be evaluated by adding both binding agents, each labeled with a different fluorophore, to cells expressing e.g., a TNFRSF protein or an immune check point protein. After incubation and cells wash, read out can be performed on a FACS. A gate is set for each fluorophore and the total amount of channel fluorescence is recorded. Reduction and / or absence of fluorescence of one of the fluorophores indicates blocking by the

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[0452] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT binding agents for binding to cells expressing e.g., a TNFRSF protein or an immune check point protein.

[0453] Other methods for determining whether an immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent directed against a target blocks, is capable of blocking, competitively binds or is competitive as defined herein are described e.g. in Xiao-Chi Jia et al. (Journal of Immunological Methods 288: 91-98, 2004), Miller et al. (Journal of Immunological Methods 365: 1 18- 125, 201 1 ).

[0454] In certain aspects, the multispecific binding protein of the disclosure has a half- maximal effective concentration (EC50 [M]) in a NFkB luciferase reporter assay is less than about 1 x 10-9. In certain aspects, the multispecific binding protein of the disclosure has an EC50 [M] in a T-cell activation assay is about 1 x 10-9 to about 3 x 10-1 1. In certain aspects, the multispecific binding protein of the disclosure has an EC50 [M] binding value for human GITR is less than about 1 x 10-10.

[0455] Dissociation constant

[0456] The dissociation constant may be the actual or apparent dissociation constant, as will be clear to the skilled person. Methods for determining the dissociation constant will be clear to the skilled person, and for example include the techniques mentioned herein. In this respect, it will also be clear that it may not be possible to measure dissociation constants of more than 10-4moles / liter or 10-3moles / liter (e.g. of 10-2moles / liter). Optionally, as will also be clear to the skilled person, the (actual or apparent) dissociation constant may be calculated on the basis of the (actual or apparent) association constant (KA), by means of the relationship [KD = 1 / KA], The term "KD” as used herein refers to the dissociation constant of the interaction between a particular binding protein or portion thereof and the target antigen.

[0457] Valency

[0458] As used herein the term “valency” refers to the number of potential target binding sites in a multispecific binding protein. Each target binding site specifically binds one target molecule or specific site on a target molecule. When a polypeptide comprises more than one target binding site, each target binding site can specifically bind the same or different molecules (e.g., can bind to more than one target protein,

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[0460] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT or different epitopes on the same target protein). A subject multispecific binding protein described herein has at least one binding site (e.g., 1 , 2, 3, or more) specific for TNFRSF protein as well as an immune checkpoint protein.

[0461] In some aspects, the multispecific binding protein described herein comprises at least one, two, three, four, five, six, seven, or eight ISVD(s) that specifically bind a TNFRSF receptor protein (e.g., a GITR). In some aspects, the multispecific binding protein described herein comprises at least one, two, three, four, five, six, seven, or eight ISVD(s) that specifically bind a GITR.

[0462] In some aspects, the multispecific binding protein described herein comprises at least one, two, three, four, five, six, seven, or eight ISVD(s) that specifically bind an immune checkpoint protein (e.g., a PD-1 ). In some aspects, the multispecific binding protein described herein comprises at least one, two, three, four, five, six, seven, or eight ISVD(s) that specifically bind a PD-1 .

[0463] About or approximately

[0464] The term “about” or “approximately” means within about 20%, such as within about 10%, within about 5%, or within about 1 % or less of a given value or range.

[0465] As used herein, “administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., a multispecific binding protein provided herein) into a patient, such as by, but not limited to, pulmonary (e.g., inhalation), mucosal (e.g., intranasal), intradermal, intravenous, intramuscular delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being managed or treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease, or symptom thereof, is being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof and can be continued chronically to defer or reduce the appearance or magnitude of disease-associated symptoms.

[0466] Potency and Efficacy

[0467] As used herein, the term "potency" is a measure of the biological activity of an agent, e.g., an ISVD that is part of a multispecific binding protein described herein. Potency of a multispecific binding protein or a part of the multispecific binding protein (e.g., a first or a second binding moiety) can be determined by any suitable method

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[0469] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT known in the art, such as for instance as described in the experimental section. Cell culture-based potency assays are often the preferred format for determining biological activity since they measure the physiological response elicited by the agent and can generate results within a relatively short period of time. Various types of cell-based assays, based on the mechanism of action of the product, can be used, such as e.g., binding affinity assays to test the affinity of e.g., the first or second binding moiety to a TNFRSF protein or an immune check point protein, respectively (as further described in Example 2).

[0470] In contrast, the “efficacy” of an agent, such as an ISVD or polypeptide, measures the maximum strength of the effect itself, at saturating agent concentrations. Efficacy indicates the maximum response achievable from the agent. It refers to the ability of the agent to produce the desired (therapeutic) effect. The efficacy of an agent can be evaluated using in vitro functional assays (e.g., IL-2 secretion from T cells as described herein) or in vivo models e.g., murine tumor modeling as described in herein).

[0471] Effective amount

[0472] “Effective amount” means the amount of active pharmaceutical agent (e.g., a multispecific binding protein of the present disclosure) sufficient to effectuate a desired physiological outcome in an individual in need of the agent. The effective amount can vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.

[0473] Subject or patient

[0474] As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, a subject can be a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkey and human). In certain aspects, the term “subject,” as used herein, refers to a vertebrate, such as a mammal. Mammals include, without limitation, humans, non-human primates, wild animals, feral animals, farm animals, sport animals, and pets.

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[0478] Therapy and Pharmaceutical Compositions

[0479] As used herein, the term “therapy” refers to any protocol, method and / or agent that can be used in the prevention, management, treatment and / or amelioration of a disease or a symptom related thereto. In some aspects, the term “therapy” refers to any protocol, method and / or agent that can be used in the modulation or depletion of a target protein from the circulation of a subject. In some aspects, the terms “therapies” and “therapy” refer to a biological therapy, supportive therapy, and / or other therapies useful in the prevention, management, treatment and / or amelioration of a disease or a symptom related thereto, known to one of skill in the art such as medical personnel. In other aspects, the terms “therapies” and “therapy” refer to a biological therapy, supportive therapy, and / or other therapies useful in the modulation of an immune response to an inflammatory or autoimmune diseases in a subject or a symptom related thereto known to one of skill in the art such as medical personnel.

[0480] As used herein, the terms “treat,” “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or a symptom related thereto, resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents, such as the administration of a multispecific binding protein provided herein). The term “treating,” as used herein, can also refer to altering the disease course of the subject being treated. Therapeutic effects of treatment include, without limitation, preventing occurrence or recurrence of disease, alleviation of symptom(s), diminishment of direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0481] In certain aspects, a method of treating a neoplastic disorder comprises administering the multispecific or bispecific binding protein of the disclosure to a subject. In certain aspects, administration of the binding protein results in tumor growth inhibition by 1 , 2, 3, or 4-fold relative to a reference binding protein.

[0482] In certain aspects, a method of treating an infection comprising administering the multispecific or bispecific binding protein of the disclosure to a subject. In certain aspects, a method of treating a T cell, B cell, or natural killer cell mediated disease

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[0484] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT comprising administering the multispecific or bispecific binding protein of the disclosure to a subject.

[0485] In certain aspects, a multispecific binding protein or a bispecific binding protein of the disclosure is for use in the treatment of neoplastic disorder. In certain aspects, the multispecific binding protein or the bispecific binding protein of the disclosure is for use in inhibiting tumor growth by 1 , 2, 3, or 4-fold relative to a reference binding protein.

[0486] In certain aspects, a multispecific binding protein or a bispecific binding protein of the disclosure is for use in treating an infection.

[0487] In certain aspects, a multispecific binding protein or a bispecific binding protein of the disclosure is for use in treating a T cell, B cell, or natural killer cell mediated disease.

[0488] Polynucleotides

[0489] In one aspect, polynucleotides (i.e., nucleic acid molecules) encoding a multispecific binding protein described herein or variants thereof are provided. A polynucleotide variant as used herein is about 50, 75, 80, 85, 90, 93, 95, 98, 99% or more identical to a polynucleotide that encodes a multispecific binding protein described herein.

[0490] Methods of making a multispecific binding protein comprising expressing these polynucleotides are also provided. Polynucleotides encoding a multispecific binding protein or variants thereof disclosed herein are typically inserted in an expression vector for introduction into host cells that can be used to produce the desired quantity of the claimed multispecific binding protein. Accordingly, in certain aspects, the disclosure provides expression vectors comprising polynucleotides disclosed herein and host cells comprising these vectors and polynucleotides.

[0491] Expression vector and host cell

[0492] The term "vector" or "expression vector" is used herein for the purposes of the specification and claims, to mean vectors used in accordance with the present disclosure as a vehicle for introducing into and expressing the polynucleotide sequence encoding a multispecific binding protein polypeptide in a cell. As known to those skilled in the art, such vectors can easily be selected from the group consisting of plasmids, phages, viruses and retroviruses. In general, vectors compatible with the

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[0495] Sanofi Ref No. PAT24106-WO-PCT instant disclosure will comprise a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0496] In vitro production allows scale-up to give large amounts of the desired polypeptides. Techniques for mammalian cell cultivation under tissue culture conditions are known in the art and include homogeneous suspension culture, e.g., in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g., in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and / or desired, the solutions of polypeptides can be purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose and / or (immuno-) affinity chromatography.

[0497] One or more genes encoding a multispecific binding protein can also be expressed non-mammalian cells such as bacteria, yeast or plant cells. In this regard it will be appreciated that various unicellular non-mammalian microorganisms such as bacteria can also be transformed; i.e. those capable of being grown in cultures or fermentation. Bacteria, which are susceptible to transformation, include members of the enterobacteriaceae, such as strains of Escherichia coll or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. It will further be appreciated that, when expressed in bacteria, the polypeptides can become part of inclusion bodies. The polypeptides can be isolated, purified, and then assembled into functional molecules.

[0498] In addition to prokaryotes, eukaryotic cells can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used, although a number of other strains are commonly available.

[0499] In certain aspects, the expression vector comprises a nucleic acid encoding the multispecific binding protein of the disclosure. In certain aspects, a host or host cell comprises a nucleic acid encoding the multispecific binding protein of the disclosure.

[0500] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by

[0501] 67

[0502] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT reference. Applicant reserves the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0503] While the present disclosure has been described with reference to the specific aspects thereof, it should be understood by those skilled in the art that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of the application. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein can be made using suitable equivalents without departing from the scope of the aspects disclosed herein. In addition, many modifications can be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the claims appended hereto. Having now described certain aspects in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.

[0504] The embodiments illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are specifically or not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising," "consisting essentially of," and "consisting of" can be replaced with either of the other two terms, while retaining their ordinary meanings. Any single term, single element, single phrase, group of terms, group of phrases, or group of elements described herein can each be specifically excluded from the claims.

[0505] EMBODIMENTS OF THE DISCLOSURE

[0506] 1 . A multispecific binding protein comprising:

[0507] (a) a first binding moiety comprising at least three immunoglobulin single variable domains (ISVDs) that specifically bind a TNF receptor superfamily (TNFRSF) receptor (TNFRSF ISVDs) protein;

[0508] (b) a second binding moiety comprising at least one ISVD that specifically binds to an immune checkpoint protein; and wherein the first and second binding moiety form a heterodimer.

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[0511] 2. The multispecific binding protein of embodiment 1 , wherein the at least three TNFRSF ISVDs specifically bind the same or different TNFRSF protein(s).

[0512] 3. The multispecific binding protein of embodiment 1 or 2, wherein the at least three TNFRSF ISVDs specifically bind the same TNFRSF protein.

[0513] 4. The multispecific binding protein of embodiment 3, wherein the at least three TNFRSF ISVDs bind to the same or different contacts on the same TNFRSF protein.

[0514] 5. The multispecific binding protein of any one of the preceding embodiments, wherein the first binding moiety is an antagonist of the TNFRSF protein(s).

[0515] 6. The multispecific binding protein of any one of embodiments 1 -4, wherein the first binding moiety is an agonist of the TNSFRSF protein(s).

[0516] 7. The multispecific binding protein of any one of the preceding embodiments, wherein the TNFRSF protein is selected from a group consisting of: a glucocorticoid- induced tumor necrosis factor receptor-related protein (GITR), a 4-1 BB, a 0X40, a tumor necrosis factor receptor 1 (TNFR1), a TNFR2, a lymphotoxin beta receptor (LTBR), a CD40, a fas receptor, a CD27, a CD30, a death receptor 3 (DR3), a DR4, a DR5, a DR6, a decoy receptor 1 (DCR1 ), a DCR2, a DCR3, a receptor activator of nuclear factor kappa-B (RANK), an osteoprotegerin (OPG), a tumor necrosis factorlike weak inducer of apoptosis receptor (TWEAK-R), a tachykinin precursor 1 (TACI), a B cell activating factor receptor (BAFF-R), a herpes virus entry mediator (HVEM), a nerve growth factor receptor (NGFR), a B-cell maturation antigen (BCMA), a TROY, and ectodysplasin A2 receptor (EDA2R).

[0517] 8. The multispecific binding protein of embodiment 7, wherein the TNFRSF protein is GITR.

[0518] 9. The multispecific binding protein of embodiment 8, wherein the at least three TNFRSF ISVDs specifically bind to GITR.

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[0521] 10. The multispecific binding protein of any one of the preceding embodiments, wherein the second binding moiety comprises at least two ISVDs.

[0522] 11 . The multispecific binding protein of embodiment 10, wherein the at least two ISVDs specifically bind the same or different immune checkpoint protein(s).

[0523] 12. The multispecific binding protein of embodiment 10, wherein the at least two ISVDs bind to the same or different contacts on the same immune checkpoint protein.

[0524] 13. The multispecific binding protein of any one of the preceding embodiments, wherein the second binding moiety is an antagonist of the immune checkpoint protein(s).

[0525] 14. The multispecific binding protein of any one of embodiments 1 -12, wherein the second binding moiety is an agonist of the immune checkpoint protein(s).

[0526] 15. The multispecific binding protein of any one of the preceding embodiments, wherein the checkpoint protein is selected from a group consisting of: a programmed cell death protein 1 (PD-1 ), a PD ligand 1 (PD-L1 ), PD-L2, a cytotoxic T-lymphocyte- associated antigen 4 (CTLA-4), a T-cell immunoglobulin and mucin domain 3 (TIM- 3), a lymphocyte activation gene 3 (LAG-3), a T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), a B and T lymphocyte attenuator (BTLA), a signaling lymphocytic activation molecule (SLAM), a leukocyte-associated immunoglobulin-like receptor 1 (LAIR1 ), a cluster of differentiation 244 (CD244), a CD28, a CD200 receptor 1 (CD200R1 ), a TREM-like transcript 2 (TLT2), a leukocyte immunoglobulin like receptor B4 (LILRB4), a killer cell immunoglobulin like receptor (KIR2DL2), and an inducible T-cell co-stimulator (ICOS).

[0527] 16. The multispecific binding protein of embodiment 15, wherein the checkpoint protein is a PD-1 .

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[0530] 17. The multispecific binding protein of embodiment 16, wherein at least two ISVDs specifically bind to PD-1 (PD-1 ISVDs).

[0531] 18. The multispecific binding protein of any one of the preceding embodiments, wherein the binding protein is bispecific or trispecific.

[0532] 19. The multispecific binding protein of embodiment 18, wherein the binding protein is bispecific.

[0533] 20. The multispecific binding protein of embodiment 19, wherein the heterodimer comprises a fragment crystallizable (Fc) domain or variant thereof.

[0534] 21 . The multispecific binding protein of embodiment 20, wherein each binding moiety is operatively linked to the Fc domain or variant thereof.

[0535] 22. The multispecific binding protein of embodiment 21 , wherein the Fc domain or variant thereof comprises a first and a second immunoglobulin G (IgG) domain that dimerize to form the bispecific binding protein.

[0536] 23. The multispecific binding protein of any one of embodiments 8-22, wherein at least one GITR ISVD specifically binds human GITR.

[0537] 24. The multispecific binding protein of any one of embodiments 8-23, wherein at least one GITR ISVD is an agonist.

[0538] 25. The multispecific binding protein of any one of embodiments 8-24, wherein each GITR ISVD is an agonist.

[0539] 26. The multispecific binding protein of embodiment 24 or 25, wherein at least one GITR ISVD binds to an extracellular domain of a GITR subunit.

[0540] 27. The multispecific binding protein of any one of embodiments 8-26, wherein the at least three of the GITR ISVDs bind to the same GITR subunit.

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[0544] 28. The multispecific binding protein of any one of embodiments 8-26, wherein at least one of the GITR ISVDs can bind to different GITR subunits.

[0545] 29. The multispecific binding protein of any one of embodiments 1 -28, wherein the at least three GITR ISVDs are directly linked to each other via a linker.

[0546] 30. The multispecific binding protein of any one of embodiments 1 -28, wherein at least one GITR ISVD is not directly linked to another GITR ISVD via a linker.

[0547] 31 . The multispecific binding protein of embodiment 29, comprising, from N- terminus to C-terminus, the at least three GITR ISVDs and a first IgG domain.

[0548] 32. The multispecific binding protein of embodiment 29, comprising, from N- terminus to C-terminus, a first IgG domain and the at least three GITR ISVDs.

[0549] 33. The multispecific binding protein of embodiment 30, wherein at least one GITR ISVD is linked to the C-terminus and at least one GITR ISVD is linked to the N- terminus of a first IgG domain.

[0550] 34. The multispecific binding protein of embodiment 31 , wherein the second binding moiety is linked to the N-terminus of a second IgG domain.

[0551] 35. The multispecific binding protein of embodiment 32, wherein the second binding moiety is linked to the N-terminus of a second IgG domain.

[0552] 36. The multispecific binding protein of any one of embodiments 7-35, wherein the at least one GITR ISVD specific variable domain has a sequence identity of at least 80% with an amino acid sequence selected from the group consisting of SEQ ID NO: 72 or 74.

[0553] 37. The multispecific binding protein of any one of embodiments 16-36, wherein the at least one PD-1 ISVD specifically binds human PD-1 .

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[0557] 38. The multispecific binding protein of any one of embodiments 16-37, wherein the at least one PD-1 ISVD is an antagonist.

[0558] 39. The multispecific binding protein of embodiment 38, wherein the at least one PD-1 ISVD binds to an extracellular domain of a PD-1 subunit.

[0559] 40. The multispecific binding protein of any one of embodiments 16-39, wherein at least two PD-1 ISVDs bind to the same PD-1 subunit.

[0560] 41 . The multispecific binding protein of any one of embodiments 16-40, wherein at least two of the PD-1 ISVDs bind to different PD-1 subunits.

[0561] 42. The multispecific binding protein of any one of embodiments 16-41 , wherein at least one PD-1 ISVD is not directly linked to another PD-1 ISVD via a linker.

[0562] 43. The multispecific binding protein of embodiment 42, wherein at least one PD- 1 ISVD is linked to the N-terminus of a first and / or a second Fc heavy chain.

[0563] 44. The multispecific binding protein of embodiment 43, wherein at least one PD- 1 ISVD is linked to the C-terminus of a first and / or a second Fc heavy chain.

[0564] 45. The multispecific binding protein of embodiment 44, wherein at least one PD- 1 ISVD are linked to both the N- and the C-terminus of a first and / or second Fc heavy chain.

[0565] 46. The multispecific binding protein of any one of embodiments 16-45, wherein at least two PD-1 ISVDs are directly linked to each other via a linker.

[0566] 47. The multispecific binding protein of embodiment 46, wherein at least two PD-1 ISVDs that are linked to each other are also linked to the N-terminus of a first and / or a second Fc heavy chain.

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[0569] 48. The multispecific binding protein of embodiment 46, wherein at least two PD-1 ISVDs that are linked to each other are also linked to the C-terminus of a first and / or a second Fc heavy chain.

[0570] 49. The multispecific binding protein of embodiment 46, wherein at least two PD-1 ISVDs are linked to both the N- and the C-terminus of a first and / or second Fc heavy chain.

[0571] 50. The multispecific binding protein of embodiment 46, wherein at least two PD-1 ISVDs that are linked to each other are also linked to the N-terminus of a first Fc heavy chain.

[0572] 51 . The multispecific binding protein of embodiment 50, wherein the first binding moiety is linked to the N-terminus of the second Fc heavy chain.

[0573] 52. The multispecific binding protein of embodiment 50, wherein at least one PD- 1 ISVD is linked to the N-terminus of a first Fc heavy chain.

[0574] 53. The multispecific binding protein of embodiment 52, wherein a second PD-1 ISVD is linked to the N-terminus of a second Fc heavy chain.

[0575] 54. The multispecific binding protein of embodiment 53, wherein the first binding moiety is linked to the C-terminus of a first and / or second Fc heavy chain.

[0576] 55. The multispecific binding protein of any one of the preceding embodiments, wherein the at least one PD-1 ISVD specific variable domain has a sequence identity of at least 80% with an amino acid sequence selected from the group consisting of SEQ ID NO: 73 and 74.

[0577] 56. The multispecific binding protein of any one of the preceding embodiments, wherein the ISVD is selected from a group consisting of: a dAb, an immunoglobulin that is suitable for use as a domain antibody (dAb), a VHH (e.g., a humanized VHH sequence), a camelized VH, or a variant thereof.

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[0581] 57. The multispecific binding protein of any one of embodiments 1 -56, wherein the linker connecting an ISVD to another ISVD is via a peptide linker.

[0582] 58. The multispecific binding protein of any one of embodiments 1 -57, wherein the linker connecting an ISVD to the first and / or second Fc heavy chain is a peptide linker.

[0583] 59. The multispecific binding protein of embodiments 57 and 56, wherein the peptide linker is at 90% identical to a peptide linker encoded by an amino acid sequence set forth in Table 2.

[0584] 60. The multispecific binding protein of any one of embodiments 57-59, wherein the peptide linker is a poly-Glycine-Serine (G4S)nlinker.

[0585] 61 . The multispecific binding protein of embodiment 60, wherein n equals 1 , 2, 3, 4, or 5.

[0586] 62. The multispecific binding protein of any one of embodiments 7-61 , wherein each GITR ISVD is connected via a poly-Glycine-Serine (G4S)nlinker.

[0587] 63. The multispecific binding protein of any one of embodiments 15-62, wherein each PD-1 ISVD is connected via a poly-Glycine-Serine (G4S)nlinker.

[0588] 64. The multispecific binding protein of embodiment 62 or 63, wherein n equals 1 , 2, 3, 4, or 5.

[0589] 65. The multispecific binding protein of embodiment 62 or 63, wherein n equals 1 .

[0590] 66. The multispecific binding protein of any one of embodiments 1 -65, wherein the linker connecting the GITR ISVD to the first and / or second Fc heavy chain is a poly-Glycine-Serine (G4S)nlinker.

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[0594] 67. The multispecific binding protein of embodiment 66, wherein n equals 1 , 2, 3, 4, or 5.

[0595] 68. The multispecific binding protein of embodiment 66, wherein n equals 4.

[0596] 69. The multispecific binding protein of any one of embodiments 66-68, wherein the GITR ISVD is connected to the C terminus of the first and / or second Fc heavy chain.

[0597] 70. The multispecific binding protein of any one of embodiments 20-69, wherein the Fc domain comprises a Fc modification.

[0598] 71 . The multispecific binding protein of any one of embodiments 22-70, wherein the IgG domain is human.

[0599] 72. The multispecific binding protein of any one of embodiments 22-71 , wherein the IgG domain is lgG1 or lgG4.

[0600] 73. The multispecific binding protein of any one of embodiments 70-72, wherein the Fc domain comprises an amino acid substitution at positions 234, 235, and / or 329, according to EU numbering.

[0601] 74. The multispecific binding protein of embodiment 73, wherein the Fc domain comprises a leucine (L) to an alanine (A) substitution at amino acid positions 234 and 235, according to EU numbering.

[0602] 75. The multispecific binding protein of embodiment 73 and 74, where the Fc domain comprises a proline (P) to a glycine (G) substitution at amino acid position 329, according to EU numbering.

[0603] 76. The multispecific binding protein of any one of embodiments 70-75, wherein the Fc domain comprising the first IgG domain comprises an amino acid substitution

[0604] 76

[0605] 81660035v1 Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT at position 435 and the second IgG domain comprises an amino acid mutation 436, according to EU numbering.

[0606] 77. The multispecific binding protein of embodiment 76, wherein the first IgG domain comprises a histidine (H) to an arginine (R) at amino acid position 435 and the second IgG domain comprises a tyrosine (Y) to a phenylalanine (F) at amino acid position 436, according to EU numbering.

[0607] 78. The multispecific binding protein of any one of embodiments 70-77, wherein the first and second IgG Fc domain polypeptides dimerize by knobs-into-holes interactions, Fab arm exchange (FAE), electrostatic steering interactions, hydrophobic interactions, or any combination thereof.

[0608] 79. The multispecific binding protein of embodiment 78, wherein the first IgG Fc domain polypeptide comprises a knob substitution, and the second IgG Fc domain polypeptide comprises a hole substitution or wherein the first IgG Fc domain polypeptide comprises a hole substitution, and the second IgG Fc domain polypeptide comprises a knob substitution.

[0609] 80. The multispecific binding protein of embodiment 78 or 79, wherein the knob substitution is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0610] 81 . The multispecific binding protein of any one of embodiments 70-80, wherein the hole substitution is selected from the group consisting of alanine (A), asparagine (N), aspartic acid (D), glycine (G), serine (S), threonine (T), and valine (V).

[0611] 82. The multispecific binding protein of any one of the preceding embodiments, wherein the half-maximal effective concentration (EC50 [M]) in a NFkB luciferase reporter assay is less than about 1 x 10-9.

[0612] 83. The multispecific binding protein of any one of the preceding embodiments, wherein the EC50 [M] in a T-cell activation assay is about 1 x 10-9to about 3 x 10-11.

[0613] 77

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[0616] 84. The multispecific binding protein of embodiments 8-83, wherein the EC50 [M] binding value for human GITR is less than about 1 x 1 O-10.

[0617] 85. The multispecific binding protein of embodiments 16-84, wherein a half- maximal inhibitory concentration (IC50 [M]) binding value for PD-1 is less than about 1 x 109.

[0618] 86. The multispecific binding protein of embodiments 16-85, wherein about 60% of the human PD-1 receptor is occupied when there is about 1 x 10-2nM or more of the multispecific binding protein.

[0619] 87. The multispecific binding protein of any one of the preceding embodiments, wherein about 60% of the human GITR receptor is occupied when there is about 1 x 10-1nM or more of the multispecific binding protein.

[0620] 88. A bispecific binding protein comprising:

[0621] (a) a first binding moiety comprising at least three GITR ISVDs directly linked to each other and wherein one GITR ISVD is linked to the N-terminal end of a first IgG;

[0622] (b) a second binding moiety comprising at least two PD-1 ISVDs directly linked to each other and wherein one PD-1 ISVD is linked to the N-terminal end of a second IgG; and wherein each the first and second IgG 1 domains heterodimerize to form a Fc domain or variant thereof.

[0623] 89. The bispecific binding protein of embodiment 88, wherein the first binding moiety consists of at least three GITR ISVDs.

[0624] 90. The bispecific binding protein of embodiment 88 or 89, wherein the second moiety consists of at least two PD-1 ISVDs.

[0625] 91 . A bispecific binding protein comprising:

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[0628] (a) a first binding moiety comprising at least three GITR ISVDs directly linked to each other and wherein one GITR ISVD is linked to the C-terminal end of a first and a second IgG;

[0629] (b) a second binding moiety comprising at least two PD-1 ISVDs wherein each PD-1 ISVD is directly linked to the N-terminal end of the first and the second IgG; and wherein each the first and second IgG 1 domains form a Fc domain.

[0630] 92. The bispecific binding protein of embodiment 91 , wherein the first binding moiety consists of at least six GITR ISVDs.

[0631] 93. The bispecific binding protein of any one of embodiments 88-92, wherein each GITR ISVD is an agonist of GITR.

[0632] 94. The bispecific binding protein of any one of embodiments 88-93, wherein the second moiety consists of at least two PD-1 ISVDs.

[0633] 95. The bispecific binding protein of any one of embodiments 88-94, wherein each PD-1 ISVD is an antagonist of PD-1 .

[0634] 96. The bispecific binding protein of any one of embodiments 88-95, wherein each GITR ISVD is an agonist of GITR and each PD-1 ISVD is an antagonist of PD-1 .

[0635] 97. The bispecific binding protein of any one of embodiments 88-96, wherein each GITR ISVD is linked via a poly-Glycine-Serine (G4S)n linker.

[0636] 98. The bispecific binding protein of any one of embodiments 88-97, wherein each PD-1 ISVD is linked via a poly-Glycine-Serine (G4S)n linker.

[0637] 99. The bispecific binding protein of embodiment 97 or 98, wherein n equals 1 , 2, 3, 4, or 5.

[0638] 100. The bispecific binding protein of embodiment 97 or 98, wherein n equals 1 .

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[0642] 101 . The bispecific binding protein of any one of embodiments 91 -100, the one GITR ISVD is linked to the C-terminal end of the first and the second IgG via a poly- Glycine-Serine (G4S)n linker.

[0643] 102. The bispecific binding protein of embodiment 101 , wherein n equals 1 , 2, 3, 4, or 5.

[0644] 103. The bispecific binding protein of embodiment 101 , wherein n equals 4.

[0645] 104. The multispecific binding protein of any one of embodiments 1 -87 or the bispecific binding protein of any one of embodiments 88-103, wherein at least one GITR ISVD comprises the three following CDR sequences:

[0646] - ISVD-CDR1 : GTFFSIDSMA (SEQ ID NO: 83);

[0647] - ISVD-CDR2: AITGGGSPN (SEQ ID NO: 84); and

[0648] - ISVD-CDR3: EGQAGWGTALLDY (SEQ ID NO: 85).

[0649] 105. The multispecific binding protein of any one of embodiments 1 -87 or the bispecific binding protein of any one of embodiments 88-103, wherein the at least one PD-1 ISVD comprises one of the three following CDR sequences:

[0650] - ISVD-CDR1 : GFTLDYYVIG or GRMHSINAMA (SEQ ID NO: 86 or SEQ ID NO: 89);

[0651] - ISVD-CDR2: CLSSNGDRIN or LISWGNGITY (SEQ ID NO: 87 or SEQ ID NO: 90)

[0652] - ISVD-CDR3: GTSTTVRDMCGIMYLYDY or SYGSSWYDS (SEQ ID NO: 88 or SEQ ID NO: 91 )

[0653] 106. The multispecific binding protein of any one of embodiments 1 -87 or the bispecific binding protein of any one of embodiments 88-103, wherein the at least one GITR ISVD specific variable domain has a sequence identity of at least 80% with an amino acid sequence selected from the group consisting of SEQ ID NO: 77 and 78.

[0654] 107. The multispecific binding protein of any one of embodiments 1 -87 or the bispecific binding protein of any one of embodiments 88-103, wherein the at least

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[0657] 108. A compound or construct that comprises the bispecific binding protein according to any one of the preceding embodiments that further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptide linker(s).

[0658] 109. A nucleic acid encoding the multispecific binding protein according to any one of embodiments 1 -87 or the bispecific binding protein according to 88-103.

[0659] 110. An expression vector comprising a nucleic acid according to embodiment 109.

[0660] 111. A host or host cell comprising a nucleic acid according to embodiment 109, or an expression vector according to embodiment 110.

[0661] 112. A pharmaceutical composition comprising the multispecific binding protein of any one of embodiments 1 -87 or the bispecific binding protein according to 88-103 and a pharmaceutically acceptable carrier.

[0662] 113. A method of treating a neoplastic disorder comprising administering the multispecific binding protein of any one of embodiments 1 -87 or the bispecific binding protein according to 88-103 to a subject.

[0663] 114. The method of embodiment 113, wherein administration of the binding protein results in tumor growth inhibition by 1 , 2, 3, or 4-fold relative to a reference binding protein.

[0664] 115. A method of treating an infection comprising administering the multispecific binding protein of any one of embodiments 1 -87 or the bispecific binding protein according to 88-103 to a subject.

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[0667] 1 16. A method of treating a T cell, B cell, or natural killer cell mediated disease comprising administering the multispecific binding protein of any one of embodiments 1 -87 or the bispecific binding protein according to 88-103 to a subject.

[0668] 1 17. A multispecific binding protein of any one of embodiments 1 -87 or a bispecific binding protein according to 88-103 for use in the treatment of neoplastic disorder.

[0669] 1 18. The multispecific binding protein or the bispecific binding protein of embodiment 1 17 for use in inhibiting tumor growth by 1 , 2, 3, or 4-fold relative to a reference binding protein.

[0670] 1 19. A multispecific binding protein of any one of embodiments 1 -87 or a bispecific binding protein according to 88-103 for use in treating an infection.

[0671] 120. A multispecific binding protein of any one of embodiments 1 -87 or a bispecific binding protein according to 88-103 for use in treating a T cell, B cell, or natural killer cell mediated disease.

[0672] EXAMPLES

[0673] The present disclosure is further illustrated by the following examples which should not be construed as further limiting. The contents of the Sequence Listing, figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0674] Example 1 : Design of PD-1 / GITR bispecific binding proteins

[0675] Bispecific binding proteins comprising a first binding moiety that specifically binds to a TNFRSF protein (e.g., a GITR) and a second binding moiety that binds to an immune checkpoint protein (e.g., PD-1 ) were designed (referred to as “PD-1 / GITR bispecific binding protein(s)” or “PD-1 / GITR bispecific construct(s)”). The designs of some of the tested constructs are graphically displayed in FIGURES 1 and 2.

[0676] Two different pentavalent PD-1 / GITR bispecific binding proteins were designed (also referred to herein as “pentavalent anti-PD-1 / GITR construct 1 or 2”). The two different pentavalent anti-PD-1 / GITR constructs comprised the features shown in FIGURES

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[0680] 1A-1 B (and detailed in Table 4 below) which included tandem trivalent anti-human GITR (hGITR) ISVDs as a GITR co-stimulatory receptor agonist and tandem bivalent anti-human PD-1 (hPD-1 ) ISVDs as a PD-1 co-inhibitory receptor antagonist each fused to the N-terminal Fc portion of a first and a second IgG. As shown in the FIGURES 2A-2B schematics, an octavalent PD-1 / GITR bispecific binding protein was also designed (also referred to herein as, “an octavalent anti-PD-1 / GITR construct”; see Table 4 below). The octavalent anti-PD-1 / GITR construct included tandem trivalent anti-hGITR ISVDs domains fused to the C-terminal end of a first and second IgG domain (i.e., for a total of six GITR ISVDs in the construct) and an anti-hPD-1 ISVD fused to the N-terminal end of the first and the second IgG domains comprising the Fc portion (i.e., for a total of two PD-1 ISVDs in the construct). Table 4 and Table 5 below show features of the designed constructs and sequences, respectively. Table 4. Construct name and properties of the PD-1 / GITR multispecific binding proteins

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[0684] Table 5. Corresponding amino acid sequences for the construct designs shown in Table 4.

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[0690] Example 2: PD-1 / GITR bispecific binding proteins bind to PD-1 and GITR positive cells

[0691] Introduction

[0692] In this Example the PD-1 / GITR bispecific binding proteins designed in Example 1 were analyzed for the ability to specifically bind to human or cyno PD-1 and to human or cyno GITR expressed on the cell surface of a PD-1 and / or GITR positive cell.

[0693] The binding properties of the constructs and controls to bind either GITR (human of cynomolgus GITR) or PD-1 (human of cynomolgus PD-1 ) expressed on different cell lines or cell types were evaluated by flow cytometry. Binding experiments were performed using the following cell lines or cell types: (a) GloResponse™ NFkB- NlucP / HEK293 cell Lines (Promega, Cat. No. CS188801 ) stably transfected with human or cynomolgus monkey GITR; (b) pre-B 300.19 single cell clones stably expressing human or cynomolgus PD-1 ; and (c) primary human and cynomolgus monkey T Cells (purchased from LPT, Germany) which were activated.

[0694] Methods

[0695] Binding experiments using a NFkB-NlucP / HEK293 cell line or pre-B 300.19 single cell clones.

[0696] Cells were thawed, washed with assay buffer (DPBS (Gibco, Cat. No. 14190) supplemented with 2% heat inactivated FBS (Sigma, Cat. No. F7254) and 0.05% NaNg (Acros, Cat. No. 19038)) and seeded in 384-well Bio-One V-bottom plates (Greiner, Cat. No. 781280), with a total of 3x104cells perwell. Serial dilutions of the bispecific molecules and controls (starting from 250 nM final concentration, 3-fold dilution, 1 1 points, diluted in assay buffer), were added and incubated for 30 minutes at 4 °C. Plates were then washed 3 times and cells were resuspended in AffiniPure F(Ab')2 Fragment Goat Anti-Human IgG, Fcg Fragment Specific (Jackson ImmunoResearch, Cat. No. 109-136-170) (250-fold diluted in assay buffer) for 30 minutes at 4 °C. Plates were washed 3 times and cells were resuspended in DAPI Solution (BD Pharmingen, Cat. No. 564907, 5000-fold diluted in assay buffer). Cell suspensions were analyzed with iQue Screener 3 (Sartorius, Intellicyt). In each washing step, plates were centrifuged for 2 minutes at 300 x g at 4 °C, supernatants discarded, and buffer dispensed by Biotek ELX405 microplate washer (BioTek). Other reagents were added either manually or with ViaFlo (Integra). EC50s were estimated by dose response

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[0699] Binding to Primary Cynomolgus T cells

[0700] Primary Cynomolgus monkey T cells were purchased from LPT, Germany, and were pre-activated with Dynabeads Goat anti-Mouse IgG (Gibco, #1 1033) coated with mouse anti-Human CD3 clone SP34-2 (BD Bioscience, #551916) and mouse antiHuman CD28 clone CD28-2 (Antibodies Online, #ABIN6384335) at a 1 :1 bead:cell ratio in RPMI-1640 (Sigma-Aldrich, #72400) supplemented with 10% FBS (Sigma- Aldrich, #F7524) and 1 % Penicillin-Streptomycin (P / S, Sigma-Aldrich, #15140-122). Four, 7 and 1 1 days after start of incubation at 37°C, the beads were removed and replaced by new beads, again at a 1 :1 bead:cell ratio. After a total incubation of 14 days, the beads were removed, and cells were frozen until use. Expression of GITR was confirmed via flow cytometry.

[0701] Results

[0702] As shown in FIGURES 3 and 4, all constructs exhibiting an anti-PD-1 binding moiety showed specific binding for both human and cyno PD-1 expressed on the cell surface of pre-B 300.19 cells whereas control molecules containing a GITR moiety associated with VHH control which binds to a Respiratory Syncytial Virus (RSV) sequence (i.e., an irrelevant sequence) did not bind the cell surface of pre-B 300.19 cells.

[0703] As shown in FIGURES 5A and 6A, all bispecific molecules containing GITR ISVDs actively bound human GITR expressing-NFkB-NlucP / HEK293 cell lines. On the contrary, control molecules that do not exhibit GITR ISVDs did not bind demonstrating the specificity for human GITR of the pentavalent and octavalent bispecific anti-PD- 1 / GITR constructs. Additionally, the octavalent anti-PD-1 / GITR construct bound cynoGITR-expressing NFkB-NlucP / HEK293 cells in a dose-dependent manner (FIGURE 6B). Binding of pentavalent anti-PD-1 / GITR constructs were also observed to cynoGITR- expressing NFkB-NlucP / HEK293 cells but no saturation of the binding signal was observed preventing calculation of an EC50 (FIGURE 5B).

[0704] As shown in FIGURE 7A, all bispecific and control molecules bound activated primary human T cells. When compared to the monospecific control (3x RSV + 2x

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[0708] 47E02vFW lgG1 LALA-Fc) the pentavalent anti-PD-1 / GITR constructs displayed an increased binding capacity highlighting the added value of bispecificity for binding to primary human T cells. Bispecific molecules also had the capacity to bind activated primary cyno T cells (FIGURE 7B). As shown in FIGURE 7B, this capacity is preferentially mediated by the anti-PD-1 ISVD. As observed on engineered cell lines, the binding capacity of GITR ISVD on cyno GITR is limited but slightly enhanced when formatted as hexavalent ISVD.

[0709] Table 6 summarizes the EC50 (M) values for cell binding with constructs tested in FIGURES 3-7.

[0710] Table 6. PD-1 / GITR bispecific binding proteins binding profiles nd: not determined

[0711] Conclusion The PD-1 / GITR bispecific binding proteins designed herein can bind PD-1 and

[0712] GITR on PD-1 and GITR positive cells.

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[0716] Example 3: PD-1 / GITR bispecific binding protein stimulates GITR activation and PD-1 blockade

[0717] Introduction

[0718] Example 3 tests whether the PD-1 / GITR bispecific binding proteins designed in Example 1 and bound to PD-1 and GITR positive cells can stimulate GITR activation and PD-1 blockade relative to control molecules.

[0719] Methods

[0720] Functional testing of bispecific ISVD-Fc fusion molecules in Nano-Gio® Luciferase Assay.

[0721] The bispecific and control molecules were tested for GITR activation in Nano- Glo® Luciferase assays (Promega, Cat. No. J1252). GloResponse™ NFkB- NlucP / HEK293 cell Lines (Promega, Cat. No. CS188801 ), stably transfected with human or cynomolgus monkey GITR, were thawed in cell medium DMEM (Gibco, Cat. No. 31966) supplemented with 10% heat inactivated FBS (Sigma, F7254) and seeded in 96-well cell culture treated plates (Corning, Cat. No. 3917) at 15,000 cells per well. Serial dilutions of the tested molecules (starting from 100 nM final concentration) or recombinant human GITR Ligand / TNFSF18 (R&D Systems, C. No 6987-GL) at 100 nM were added and incubated for 5 hours in a 5% CO2 atmosphere at 37°C. After incubation, Nano-Gio™ Luciferase substrate (Promega, Cat. No. N1 130) was added and luminescent signal was acquired using CLARIOstar (BMG Labtech).

[0722] Functional testing of bispecific and control molecules in GloResponse™ NFAT- RE / PD-1 Jurkat reporter assay

[0723] The bispecific and control molecules were tested for PD-1 blockade in a GloResponse™ NFAT-RE / PD-1 Jurkat reporter assay (Promega, Cat. No. J1252). Two days prior to the assay, GloResponse™ NFAT-RE / PD-1 Jurkat cells were thawed and cultured in Jurkat culture medium (RPM1 1640 Medium, GlutaMAX™ Supplement, HEPES (Gibco, Cat. No. 72400)) supplemented with 10% heat inactivated FBS (Sigma, F7254) for 48 hours in a 5% CO2 atmosphere at 37°C. On the day prior to the assay, PD-L1 aAPC CHO K1 cells were thawed in culture medium (Ham’s F-12 Nutrient Mix, GlutaMAX™ supplement (Gibco, Cat No. 31765-068) supplemented with

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[0726] 10% heat inactivated FBS (Sigma, F7254) and seeded in 384-well tissue culture treated plates (Nunc, Cat. No. 3570) at 10,000 cells per well and incubated overnight in a 5% CO2 atmosphere at 37°C. After disposal of the medium of the culture plates, Gioresponse™ NFAT-RE / PD-1 Jurkat cells were added at 15,000 cells / well in Jurkat culture medium containing 1 % heat inactivated FBS. Serial dilutions of the tested molecules (starting from 330 nM final concentration) or anti-PD-1 control mAb (Nivolumab) at 250 nM were added and incubated for 24 hours in a 5% CO2 atmosphere at 37°C. After incubation, Bio-Gio™ Luciferase substrate (Promega, Cat. No. G7940) was added and luminescent signal was acquired using CLARIOstar (BMG Labtech).

[0727] Results

[0728] As seen in FIGURE 8A, all constructs containing the anti-GITR moiety (5A03v1524), either in a trivalent or a hexavalent form, induced human GITR signaling that is comparable to that of the recombinant hGITRL control. In addition, constructs containing a hexavalent form of anti-GITR (e.g., the octavalent anti-PD-1 / GITR construct) display an increased potency as compared to constructs containing a trivalent form of 5A03v1524 (e.g., the pentavalent anti-PD-1 / GITR construct). This increased capacity to trigger GITR signaling is also reflected in the cyno GITR reporter assay, in which hexavalent GITR formats showed activity whereas trivalent GITR formats triggered low signal (FIGURE 8B).

[0729] As illustrated in FIGURE 9 by the restoration of signaling in reporter cells, all constructs containing an active human anti-PD-1 moiety (47E01 vFW or 37F03FW) efficiently blocked the PD-1 / PD-L1 interaction and to a similar extent than that of anti- PD-1 control monoclonal antibody (mAb).

[0730] Table 7 below summarizes the EC50 [M] and IC50 [M] values obtained in the GITR activation (FIGURE 8A and 8B) and PD-1 blockade (FIGURE 9) reporter assays.

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[0733] Table 7. PD-1 / GITR bispecific binding proteins EC50 [M] and IC50 [M] values for GITR activation and PD-1 blockade nd: not determined

[0734] Conclusion

[0735] The PD-1 / GITR bispecific binding proteins designed herein upon binding to a PD-1 and GITR positive cell can upregulate GITR signaling and block the PD-1 / PD- L1 interaction.

[0736] Example 4: GITR and PD-1 receptor occupancy in vitro upon incubation with PD-1 / GITR bispecific binding protein

[0737] Introduction

[0738] Both GITR clustering required for the induction of T cell activation and PD-1 blockade saturation are both important considerations for maximal efficacy and dosing of a PD-1 / GITR bispecific binding protein. Accordingly, this Example tests the GITR and PD-1 receptor occupancy rate upon incubation with the PD-1 / GITR bispecific constructs designed and tested in the previous Examples.

[0739] Methods

[0740] Experimental methods using NFkB-NlucP / HEK293 cell line and activated T cells were described in Example 2 and 3 above.

[0741] Murine T cells were isolated from the spleen of hGITR / hPD-1 KI mice (obtained by crossing C57BL / 6-hGITR KI and C57BL / 6-hPD KI mice previously generated in collaboration with Center of Imunophenomics - CIPHE (Marseille, France) using EasySep™ Mouse T Cell Enrichment Kit (Stemcell Technologies #19851 A). T cells were resuspended in DMEM medium with Glutamax (Gibco #31966-021 ), 10 % FCS

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[0744] (Biowest #S181 H-100), 1 % penicillin / streptomycin (Gibco #10378-016), 1 % NEAA (Gibco #11 140-050), 1 mM Na Pyruvate (Gibco #1 1360-070), 0.05 mM 2- mercaptoethanol (Gibco #31350-010). Resuspended murine T cells were activated in F-bottom 96 well plates previously coated with hamster anti-mouse CD3e antibody (clone 145-2C11 ; BD Biosciences #5671 15) at 1 pg / ml, in the presence of Syrian hamster anti-mouse CD28 antibody (clone 37.51 ; eBioscience 16-0261 -82) at 1 pg / ml and 2 Ul / ml human IL-2 (Miltenyi Biotec #130-097-746) for 3 days.

[0745] After 3 days, activated T cells were transferred in V-bottom plates, washed, resuspended in D-PBS and stained with Fixable Viability Dye eFluor™ 780 (Thermo Fisher # 65-0865-14) for 15 min at 4°C. After washing, activated T cells were incubated with serial dilutions of tested compounds for 1 h30 at 4°C and then washed twice in staining buffer. Activated T cells were then stained for 20 min at 4°C with the antibodies described in Table 8 below. Activated T cells were then washed and analyzed on a Cytek® Aurora flow cytometer.

[0746] Table 8. Activated CD4+and CD8+T cells harvested from hGITR / hPD-1 KI mice staining procedure

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[0750] To determine receptor occupancy (%RO), median fluorescence intensities of binding (MFI) were normalized as follow: a = Normalized Total surface receptor MFI = “total MFI” at tested concentration [ct] x 100 1 “total MFI” at baseline [cO] b = Normalized Free surface receptor MFI = “free MFI”[ct] x 100 / “free MFI”[c0] where “total MFI” refers to MFI obtained with agents detecting total PD-1 or GITR and “free MFI” refers to MFI obtained with agents detecting free PD-1 or GITR.

[0751] %RO was then calculated as follow:

[0752] %RO = ((a-b) / a) x 100 for each concentration tested [ct]

[0753] Results

[0754] As shown in FIGURES 10A and 10B, the pentavalent anti-PD-1 / GITR construct 1 efficiently engaged GITR and PD-1 expressed at the surface of ex v / vo-activated splenic CD4+and CD8+T cells from hGITR / hPD-1 KI mice.

[0755] Conclusion

[0756] The anti-PD-1 / GITR bispecific binding proteins designed herein can saturate cell surface PD-1 and GITR on primary activated T cells.

[0757] Example 5: PD-1 / GITR bispecific binding proteins can activate primary human T cells

[0758] Introduction

[0759] This Example analyzes whether the PD-1 / GITR bispecific binding proteins designed and tested in Examples 1 -4 can activate primary human T cells obtained from three independent human donors (donors: D1475, D1735, and D1743 are shown in FIGURES 11 and 12).

[0760] Methods

[0761] Primary human CD3+T cells were isolated from frozen human PBMCs using an EasySep™ Human T Cell Isolation Kit (Stemcell Technologies, #17951 ). Obtained

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[0764] T cells were pre-activated with Human T-Activator CD3 / CD28 Dynabeads® (Gibco, #1 1132D) at a 1 :2 bead:cell ratio in RPMI-1640 (Gibco, #72400-021 ) supplemented with 10% FBS (Sigma-Aldrich, #F7524) and 1% Penicillin-Streptomycin (P / S, Sigma- Aldrich, #15140-122). After 3 days incubation, the beads were removed and replaced by new beads, again at a 1 :2 bead:cell ratio. After 4 days incubation, the beads were removed, and cells were frozen until use.

[0765] Falcon flat bottom 96-well plates (BD Biosciences, #353072) were coated 1 day prior to the assay with 2 pg / mL anti-CD3 (eBioscience, 16-0037-85) diluted in DPBS (Gibco, 14190-136). After overnight incubation at 4°C, plates were washed with DPBS. Test compounds were diluted in RPMI-1640 supplemented with 10% human AB serum (BiolVT, #SM-612-Hsi) and 1 % P / S and added to the plates. Pre-activated T cells were thawed, resuspended in the above-mentioned assay medium, and 200,000 cells / well were added to the plate. Plates were incubated for 24 hours at 37°C, after which cell supernatant was collected for measurement of human IL-2 via ELISA (BD- Pharmingen, capture Ab #555051 , detection Ab #555040, IL-2 # 554603). Data from three independent donors are shown.

[0766] Results

[0767] As shown by the release of IL-2 in Figures 11A-C, both pentavalent anti-PD- 1 / GITR constructs 1 and 2 as well as the GITR monospecific control molecules induced T cell activation in a soluble manner in contrast to the irrelevant isotype control demonstrating the soluble agonist activity of the GITR ISVDs used in the constructs. The octavalent anti-PD-1 / GITR constructs also promoted human T cell activation in a soluble manner as shown by the IL-2 release from T cells in FIGURES 12A-C. Similar to the pentavalent anti-PD-1 / GITR constructs, the soluble agonist activity of the octavalent anti-PD-1 / GITR constructs was dependent on the GITR-targeting moiety as GITR monospecific control formats displayed the same activity. The impact of GITR-containing molecules was confirmed for each construct on at least 3 independent donors. The mean EC50 of IL-2 secretion induced by the bispecific constructs or controls in a T cell activation assay, calculated from the 3 independent donors are shown below in Table 9.

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[0771] Table 9. Mean EC50 [M] of IL-2 secretion from T cells induced by PD-1 / GITR bispecific binding proteins

[0772] Conclusion

[0773] The PD-1 / GITR bispecific binding proteins designed herein can activate primary human T cells as measured by IL-2 secretion.

[0774] Example 6: PD-1 / GITR bispecific binding proteins enhanced induction of T cell activation in a MLR assay

[0775] Introduction

[0776] This example analyzes the ability of the PD-1 / GITR bispecific binding proteins described herein to modulate the interaction between an antigen presenting cell and T cells by using a mixed lymphocyte reaction (MLR) assay.

[0777] Methods

[0778] Immature dendritic cells (iDC) were generated from healthy donor monocytes. Monocytes were isolated from frozen human PBMC by negative selection (Stemcell Technologies #19359) and cultured for 7 days in RPM1 1640 (Sigma-Aldrich, #72400), 10 % FCS (Sigma-Aldrich, #F7524), 1 % penicillin / streptomycin (Sigma-Aldrich, #15140-122) supplemented with 20 ng / ml human IL-4 (Biolegend, #B341746) and 100 ng / ml human GM-CSF (Biolegend, #572904) with replenishment of IL-4 and GM-CSF on day 3. iDC were harvested on day 7 and frozen until use. On the day of MLR, iDC from a first donor were thawed and plated in 96-well F-bottom plates (Costar, #3596)

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[0781] CD4+T cells were isolated from frozen human PBMC by negative selection and resuspended in supplemented CTS AIM V SFM medium. iDC and autologous CD4+T cells were co-cultured at a 1 :10 ratio in supplemented CTS AIM V SFM medium in the presence of serially diluted test compounds or anti-PD-1 control mAb (Nivolumab) at 200 nM. Plates were incubated for 4 days at 37°C, after which cell supernatant was collected for measurement of human GM-CSF via ELISA (R&D Systems, #DY215).

[0782] Results

[0783] As shown in FIGURES 13A-4C and 14A-14C, both pentavalent anti-PD-1 / GITR constructs 1 and 2 induced T cell activation in a MLR assay as illustrated by GM-CSF release. The activity of the anti-PD-1 arm of the bispecific binding proteins was shown by the dose-dependent effect of the PD-1 monospecific control. Additional effect of the GITR ISVD is shown by the higher GM-CSF release induced by the pentavalent anti- PD-1 / GITR constructs as compared to their respective monospecific PD-1 controls and anti-PD-1 control mAb.

[0784] Additionally, the octavalent anti-PD-1 / GITR construct also induced T cell activation in a MLR assay as illustrated by GM-CSF secretion in a dose-dependent manner (FIGURE 15A-15C). The impact of both GITR and PD-1 binding moieties is demonstrated by the higher activity, as measured by GM-CSF secretion, of the bispecific anti-PD-1 / GITR constructs as compared to the “GITR monospecific” control molecule and anti-PD-1 control mAb.

[0785] Conclusion

[0786] This PD-1 / GITR bispecific binding proteins described herein can modulate the interaction between an antigen presenting cell and T cells in a MLR assay resulting in enhanced induction of T cell activation as measured GM-CSF secretion. Additionally, the MLR assay allows for visualization of both GITR agonism and PD-1 blockade.

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[0789] Example 7: PD-1 / GITR bispecific binding proteins attenuate tumor volume in vivo

[0790] Introduction

[0791] This Example tests the in vivo efficacy of the PD-1 / GITR bispecific binding proteins described herein ability to reduce tumor volume in hGITR / hPD-1 KI mice.

[0792] Method

[0793] Generation of mice

[0794] Knock-in mouse model expressing humanized version of Tnfrsf18 (GITR) and humanized version of Pdcdl (PD-1 ) were obtained by crossing C57BL / 6-hGITR KI and C57BL / 6-hPD KI mice previously generated in collaboration with The Center for ImmunoPHEnomics - CIPHE (Marseille, France). All animals were bred and housed at the Sanofi specific-pathogen-free facilities in ventilated cages, with a standard 12 / 12-h dark / light cycle at 22 °C ambient temperature and controlled humidity. For all in vivo experiments, animals of both sexes were used between the ages of 10 and 17 weeks, randomized and assigned to experimental groups. All in vivo studies were done in accordance with institutional committees in an accredited facility with all compliance to French and European guidelines and requirements for animal care.

[0795] Tumor challenge and treatment

[0796] MC38 murine colon adenocarcinoma cells (1 x 106) resuspended in 200 pl of D-PBS were implanted subcutaneously in the right flank of mice, and tumor volumes were measured every 2-3 days by electronic caliper. Tumor volume was calculated using the formula (L2 * L1 ) / 2, where L1 and L2 represent the longest and shortest diameter, respectively. When tumor volume reached the range of 50 mm3to 145mm3(9 days after tumor inoculation), mice were randomized by tumor size and sex and treated by intraperitoneal injection twice a week for two weeks (day 9; day 12; day 16; day 19). Mice were monitored for 25 days, or until the majority of the control group was euthanized due to ethical limitations on tumor size.

[0797] Results

[0798] The pentavalent anti-PD-1 / GITR construct 1 showed strong anti-tumor activity, superior to anti-PD-1 control mAb at equimolar dose, in MC38-bearing hGITR / hPD-1

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[0801] 1 / GITR construct 1 was dose-dependent as observed in FIGURE 17.

[0802] Conclusion The PD-1 / GITR bispecific binding proteins described herein have the ability to reduce tumor volume in a hGITR / hPD-1 KI mice.

[0803] SEQUENCE TABLE

[0804] Table 3 - Amino acid sequences

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Claims

Attorney Docket No. 772089: SA9-915PCSanofi Ref No. PAT24106-WO-PCTCLAIMS1 . A bispecific binding protein comprising:(a) a first binding moiety comprising at least three GITR ISVDs directly linked to each other and wherein one GITR ISVD is linked to the N-terminal end of a first IgG;(b) a second binding moiety comprising at least two PD-1 ISVDs directly linked to each other and wherein one PD-1 ISVD is linked to the N-terminal end of a second IgG; and wherein each the first and second IgG 1 domains heterodimerize to form a Fc domain or variant thereof.

2. The bispecific binding protein of claim 1 , wherein the first binding moiety consists of at least three GITR ISVDs.

3. The bispecific binding protein of claim 1 or 2, wherein the second moiety consists of at least two PD-1 ISVDs.

4. A bispecific binding protein comprising:(a) a first binding moiety comprising at least three GITR ISVDs directly linked to each other and wherein one GITR ISVD is linked to the C-terminal end of a first and a second IgG;(b) a second binding moiety comprising at least two PD-1 ISVDs wherein each PD-1 ISVD is directly linked to the N-terminal end of the first and the second IgG; and wherein each the first and second IgG 1 domains form a Fc domain.

5. The bispecific binding protein of claim 4, wherein the first binding moiety consists of at least six GITR ISVDs.

6. The bispecific binding protein of any one of claims 1 -5, wherein each GITR ISVD is an agonist of GITR.10481660035v1Attorney Docket No. 772089: SA9-915PC Sanofi Ref No. PAT24106-WO-PCT7. The bispecific binding protein of any one of claims 1 -6, wherein the second moiety consists of at least two PD-1 ISVDs.

8. The bispecific binding protein of any one of claims 1 -7, wherein each PD-1 ISVD is an antagonist of PD-1 .

9. The bispecific binding protein of any one of claims 1 -8, wherein each GITR ISVD is an agonist of GITR and each PD-1 ISVD is an antagonist of PD-1 .

10. The bispecific binding protein of any one of claims 1 -9, wherein each GITR ISVD is linked via a poly-Glycine-Serine (G4S)n linker.11 . The bispecific binding protein of any one of claims 1 -10, wherein each PD-1 ISVD is linked via a poly-Glycine-Serine (G4S)n linker.

12. The bispecific binding protein of claim 10 or 11 , wherein n equals 1 , 2, 3, 4, or 5.

13. The bispecific binding protein of claim 10 or 11 , wherein n equals 1 .

14. The bispecific binding protein of any one of claims 4-13, the one GITR ISVD is linked to the C-terminal end of the first and the second IgG via a poly-Glycine-Serine (G4S)n linker.

15. The bispecific binding protein of claim 14, wherein n equals 1 , 2, 3, 4, or 5.

16. The bispecific binding protein of claim 14, wherein n equals 4.

17. The bispecific binding protein of any one of claims 1 -16, wherein at least oneGITR ISVD comprises the three following CDR sequences:- ISVD-CDR1 : GTFFSIDSMA (SEQ ID NO: 83);- ISVD-CDR2: AITGGGSPN (SEQ ID NO: 84); and- ISVD-CDR3: EGQAGWGTALLDY (SEQ ID NO: 85).10581660035v1Attorney Docket No. 772089: SA9-915PCSanofi Ref No. PAT24106-WO-PCT18. The bispecific binding protein of any one of claims 1 -17, wherein the at least one PD-1 ISVD comprises one of the three following CDR sequences:- ISVD-CDR1 : GFTLDYYVIG or GRMHSINAMA (SEQ ID NO: 86 or SEQ ID NO: 89);- ISVD-CDR2: CLSSNGDRIN or LISWGNGITY (SEQ ID NO: 87 or SEQ ID NO: 90)- ISVD-CDR3: GTSTTVRDMCGIMYLYDY or SYGSSWYDS (SEQ ID NO: 88 or SEQ ID NO: 91 )19. The bispecific binding protein of any one of claims 1 -18, wherein the at least one GITR ISVD specific variable domain has a sequence identity of at least 80% with an amino acid sequence selected from the group consisting of SEQ ID NO: 77 and 78.

20. The bispecific binding protein of any one of claims 1 -19, wherein the at least one PD-1 ISVD specific variable domain has a sequence identity of at least 80% with an amino acid sequence selected from the group consisting of SEQ ID NO: 79, 80, 81 and 82.21 . A compound or construct that comprises the bispecific binding protein according to any one of the preceding claims that further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptide linker(s).

22. A nucleic acid encoding the bispecific binding protein according to 1 -20.

23. An expression vector comprising a nucleic acid according to claim 22.

24. A host or host cell comprising a nucleic acid according to claim 22, or an expression vector according to claim 23.

25. A pharmaceutical composition comprising the bispecific binding protein according to 1 -20 and a pharmaceutically acceptable carrier.10681660035v1Attorney Docket No. 772089: SA9-915PCSanofi Ref No. PAT24106-WO-PCT26. A bispecific binding protein according to 1 -20 for use in the treatment of neoplastic disorder.

27. The bispecific binding protein of claim 26 for use in inhibiting tumor growth by1 , 2, 3, or 4-fold relative to a reference binding protein.

28. A bispecific binding protein according to 1 -20 for use in treating an infection.

29. A bispecific binding protein according to 1 -20 for use in treating a T cell, B cell, or natural killer cell mediated disease.10781660035v1

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