CN117412989A

CN117412989A - Novel scaffolds for bifunctional molecules with improved properties

Info

Publication number: CN117412989A
Application number: CN202280039013.2A
Authority: CN
Inventors: N·普瓦里耶; A·莫雷洛; C·马里; M·塞特
Original assignee: OSE Immunotherapeutics SA
Current assignee: OSE Immunotherapeutics SA
Priority date: 2021-04-09
Filing date: 2022-04-08
Publication date: 2024-01-16
Also published as: CN116848140A

Abstract

The present invention relates to bifunctional molecules with specific scaffolds and uses thereof.

Description

Novel scaffolds for bifunctional molecules with improved properties

Technical Field

The present invention is in the field of immunotherapy. The present invention provides novel scaffolds for bifunctional molecules and their use in medicine.

Background

Bifunctional molecules are now the object of development in the field of immunology, in particular oncology. In fact, they bring new pharmacological properties through the co-participation of two targets, which, due to targeting to tumors, can increase the safety profile compared to the combination of two different molecules, and can potentially reduce the development and production costs associated with single drug products. However, these molecules are advantageous, but may also present some inconveniences. The design of bifunctional molecules requires implications for several key attributes such as binding affinity and specificity, folding stability, solubility, pharmacokinetics, effector function, compatibility with the attachment of additional domains, and yield and cost compatible with clinical development.

Bifunctional molecules based on antibodies that antagonize PD-1 and that are linked to IL-7 or other immunotherapeutic agents have been disclosed, in particular in WO 2020/127377 and WO 2020/127366.

However, there is still a strong need for improved scaffolds for bifunctional molecules.

Disclosure of Invention

The present invention relates to bifunctional molecules having a specific scaffold and comprising a single monovalent antigen binding domain and a single immunostimulatory cytokine that bind to a target specifically expressed on the surface of an immune cell. The stent consists essentially of: a dimeric Fc domain, a single monovalent antigen binding domain that binds to a target specifically expressed on the surface of an immune cell linked N-terminal to one monomer of the Fc domain, and i) a single immunostimulatory cytokine linked C-terminal to the same monomer of the Fc domain, or ii) a single monovalent antigen binding domain comprising a heavy variable chain and a light variable chain, and a single immunostimulatory cytokine linked C-terminal to the light chain of the antigen binding domain.

This particular scaffold is associated with an improved pharmacokinetic profile. Such improvements have been observed with bifunctional molecules comprising different immunostimulatory moieties (e.g., IL-2; IL-7, IL-15, and IL-21). The improved pharmacokinetic profile was unexpected because no improvement in the scaffold was observed in the absence of immunostimulatory cytokines. Bifunctional molecules with this particular scaffold facilitate cis-targeting of two targets on the same cell, allowing selective delivery of immunostimulatory cytokines to target cells. In addition, in the context of bifunctional molecules with IL-7, these molecules are able to induce synergistic activation and better antitumor efficacy in vivo. Finally, unexpectedly, bifunctional molecules with specific scaffolds have better productivity and avoid by-products due to chain mismatches, which is a major advantage for industrial scale and safety production. In addition, in addition to improved pharmacokinetic profiles and better productivity, compounds (particularly compounds with IL 7) have been identified that have new and advantageous biological efficiencies, which may improve the activity of effector memory stem cell-like T cells, a subset of T cells within tumor-reactive tumors, which are of great interest in view of their immune activity.

Thus, the present invention relates to bifunctional molecules comprising a single antigen binding domain that binds to a target specifically expressed on the surface of an immune cell, and a single immunostimulatory cytokine,

wherein the molecule comprises a first monomer comprising an antigen binding domain covalently linked via a C-terminus to the N-terminus of a first Fc chain, optionally through a peptide linker, and a second monomer comprising a complementary second Fc chain lacking the antigen binding domain and an immunostimulatory cytokine;

wherein i) an immunostimulatory cytokine is covalently linked to the C-terminus of the first Fc chain, optionally via a peptide linker; or ii) a single antigen binding domain comprising a heavy variable chain and a light variable chain and an immunostimulatory cytokine covalently linked to the C-terminus of the light chain;

wherein the target of immune cell surface specific expression is selected from the group consisting of: PD-1, CD28, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD, OX40, 4-1BB, GITR, HVEM, tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CD3, PDL2 and PDL1; and is also provided with

Wherein the immunostimulatory cytokine is selected from the group consisting of: IL-2 (IL is an interleukin), IL-4, IL-5, IL-6, IL-12A, IL-12B, IL-13; IL-15, IL-18, IL-21, IL-23, IL-24; ifnα, ifnβ, BAFF, ltα and ltβ, or variants thereof having at least 80% identity to wild-type proteins or extracellular fragments thereof, and IL-7.

In a particular aspect, the immunostimulatory cytokine is linked at the C-terminus of the first Fc chain, preferably by its N-terminus.

In a particular aspect, the first Fc chain and the second Fc chain form a heterodimeric Fc domain, particularly a knob heterodimeric Fc domain.

Optionally, the immunostimulatory cytokine is selected from the group consisting of: IL-2, IL-15 and IL-21, or variants thereof having at least 80% identity to wild-type proteins, and IL-7.

In a particular aspect, the immunostimulatory cytokine is IL-7, e.g., as described under a sequence such as set forth in SEQ ID NO. 1.

In another particular aspect, the immunostimulatory cytokine is IL-2 or a variant thereof having at least 90% identity to SEQ ID NO. 87, preferably selected from IL-2 variants comprising one of the following combinations of substitutions relative to SEQ ID NO. 87: R38E and F42A; R38D and F42A; F42A and E62Q; R38A and F42K; R38E, F a and N88S; R38E, F a and N88A; R38E, F a and V91E; R38E, F a and D84H; H16D, R E and F42A; H16E, R E and F42A; R38E, F a and Q126S; R38D, F a and N88S; R38D, F a and N88A; R38D, F a and V91E; R38D, F a and D84H; H16D, R D and F42A; H16E, R D and F42A; R38D, F a and Q126S; R38A, F K and N88S; R38A, F K and N88A; R38A, F K and V91E; R38A, F K and D84H; H16D, R a and F42K; H16E, R a and F42K; R38A, F K and Q126S; F42A, E Q and N88S; F42A, E Q and N88A; f42A, E Q and V91E; F42A, E Q and D84H; H16D, F a and E62Q; H16E, F a and E62Q; F42A, E Q and Q126S; R38E, F a and C125A; R38D, F a and C125A; F42A, E Q and C125A; R38A, F K and C125A; R38E, F42A, N S and C125A; R38E, F42A, N a and C125A; R38E, F, 42, A, V E and C125A; R38E, F42A, D H and C125A; H16D, R E, F a and C125A; H16E, R E, F a and C125A; R38E, F42A, C a and Q126S; R38D, F42A, N S and C125A; R38D, F42A, N a and C125A; R38D, F, 42, A, V E and C125A; R38D, F42A, D H and C125A; H16D, R D, F a and C125A; H16E, R D, F a and C125A; R38D, F42A, C a and Q126S; R38A, F42K, N S and C125A; R38A, F42K, N a and C125A; R38A, F, 42, K, V E and C125A; R38A, F42K, D H and C125A; H16D, R A, F K and C125A; H16E, R A, F K and C125A; R38A, F42K, C a and Q126S; F42A, E62Q, N S and C125A; F42A, E62Q, N a and C125A; F42A, E62Q, V E and C125A; F42A, E62Q, D H, C125A; H16D, F, 42, A, E Q and C125A; H16E, F, 42, A, E Q and C125A; F42A, E62Q, C a and Q126S; F42A, N S and C125A; F42A, N a and C125A; F42A, V91E and C125A; F42A, D H and C125A; H16D, F a and C125A; H16E, F a and C125A; F42A, C a and Q126S; F42A, Y a and L72G; T3A, F42A, Y45A, L G and C125A; or at least one substitution selected from the group consisting of: the K35 35 38 38 38 38 38 38 38 38 38 38 38 38 42 42 42 42 42 42 42 42 42 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 72 72 72 72 72 72 72 72 72 72 72 72 72R and L72K. Or combinations thereof, preferably three substitutions F42A, Y a and L72G.

In another particular aspect, the immunostimulatory cytokine is IL-15 or a variant thereof having at least 90% identity to SEQ ID NO. 88, preferably selected from IL-15 variants comprising one of the following substitutions relative to SEQ ID NO. 88: n1 3 3 4 8 11 11 11 11 11 30 61 71 71 72 72 72 72 72 72 73 79 79 79 112M and N112Y, preferably N4 61 65D/N65 30N/N65 30N/E64Q/N65 14 8 30 64/N108 1D/D61 1D/E64 4D/E64 8N/D61N/E64 1D/D30Q/Q108 1D/N4D/D8N/E64Q/N65 1D/D61N/E64Q/Q108 4D/D61N/E64Q/Q108 1D/N65D/Q108 4D/D30N/Q108 65D/Q108 30N/Q180Q/N65N 61N/E64Q/N65D/N4D/N65 71S/N72A/N77A and N4D/D61N/N65D, preferably D30N/E64Q/N65D.

In another particular aspect, the immunostimulatory cytokine is IL-21 or a variant thereof having at least 90% identity to SEQ ID NO. 89, preferably selected from IL-21 variants comprising one of the following substitutions relative to SEQ ID NO. 89: r5 5 5 5 5 5 5 5 5 5 5 89 12 12 12 12 12 15 15 15 15 15 15 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12D, preferably R5E and R76 5A and R76 5Q and R76 5A and R76Q and R76 9E and R76A and R76N and S70 15N and 171 15N and K72 15N and K73 70T and K73T and R76 70T and R76 71L and K73L and R76 71L and R76A and K73A and R76D or K73A and R76E.

Optionally, the antigen binding domain is a Fab domain, fab', single chain variable fragment (scFV), or single domain antibody (sdAb).

In a particular aspect, the target specifically expressed on the surface of the immune cell is selected from the group consisting of PD-1, CTLA-4, BTLA, TIGIT, LAG 3 and TIM3, preferably PD-1.

In a very specific aspect, the antigen binding domain comprises or consists essentially of: (i) A heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53 and CDR3 of SEQ ID NO:55, 56, 57, 58, 59, 60, 61 or 62; and (ii) a light chain comprising CDR1 of SEQ ID NO. 64 or 65, CDR2 of SEQ ID NO. 66 and CDR3 of SEQ ID NO. 16.

The invention also relates to an isolated nucleic acid sequence or a set of isolated nucleic acid molecules encoding a bifunctional molecule according to the present disclosure, and a host cell comprising one or more isolated nucleic acids.

The invention also relates to a pharmaceutical composition comprising a bifunctional molecule according to the present disclosure, one or more nucleic acids or host cells, optionally comprising a pharmaceutically acceptable carrier.

Finally, the present invention relates to a bifunctional molecule, one or more nucleic acids, a host cell or a pharmaceutical composition according to the present disclosure for use as a medicament, in particular for the treatment of cancer or infectious diseases; use of a bifunctional molecule, one or more nucleic acids, a host cell or a pharmaceutical composition according to the present disclosure in the manufacture of a medicament, in particular for the treatment of cancer or an infectious disease; and methods of treating a disease, particularly cancer or an infectious disease, in a subject comprising administering a therapeutically effective amount of a bifunctional molecule, one or more nucleic acids, a host cell, or a pharmaceutical composition according to the present disclosure.

Optionally, the present invention relates to a bifunctional molecule, one or more nucleic acids, a host cell or a pharmaceutical composition according to the present disclosure for use in the treatment of cancer or viral infection by stimulating effector memory stem cell-like T cells; use of a bifunctional molecule, one or more nucleic acids, a host cell or a pharmaceutical composition according to the present disclosure for the preparation of a medicament, in particular for the treatment of cancer or viral infection by stimulating effector memory stem cell-like T cells; a method of treating cancer or a viral infection in a subject comprising administering a therapeutically effective amount of a bifunctional molecule, one or more nucleic acids, a host cell, or a pharmaceutical composition according to the present disclosure, thereby stimulating effector memory stem cell-like T cells.

Drawings

Fig. 1: schematic diagrams of different molecules used in examples 1 and 2. The following schematic diagram of construct 3 is another representation of such a construct, in which each chain and component of the molecule is further described.

Fig. 2: the anti-PD-1 IL 7W 142H mutant exhibits high binding efficiency to PD-1 and antagonizes PDL1 binding. PD-1 binding ELISA assay. Human recombinant PD-1 (rPD 1) protein was immobilized and antibodies were added at different concentrations. Display was performed using an anti-human Fc antibody conjugated to peroxidase. Colorimetric assays were performed at 450nm using TMB substrate. The test has 1 (anti-PD-1*1) Grey) or 2 anti-PD-1 arms (anti-PD-1*2 +.>) As a control. Variants comprising IL7 (anti-PD-1 x 2IL7w142h x 2 +.black), (anti-PD-1 x 2IL7w142h x 1 ■ black), (anti-PD-1 x 1IL 7w142h x 2 +.grey), (anti-PD-1 x 1IL 7w142h x 1 +.>Grey) bifunctional molecules. B. The antagonistic capacity to block PD-1/PD-L1 was measured by ELISA. PD-L1 was immobilized and a complex antibody + biotinylated recombinant human PD-1 was added. The complex consists of fixed concentrations of PD1 (0.6. Mu.g/mL) and different concentrations of anti-PD 1 x 2IL7W142H x 1 (■ straight line), anti-PDl x 2IL7W142H x 2 (O-dashed line), anti-PD-1*1 (grey->Gray dotted line), anti-PD 1 x l IL7W142H x 2 (gray +.flat gray line) or anti-PDl x l IL7W142H x 1 (gray +.>Straight gray line). All constructs tested contained GGGGSGGGGSGGGGS linkers between the Fc and IL-7 domains.

Fig. 3: the anti-PD-1 IL7 molecule constructed by monovalent anti-PD-1 and an IL-7W142H cytokine can activate pSTAT5 with high efficiency. pSTAT5 signaling assay after treatment with anti-PD-1 x 1il 7w142h 1 (+) anti-PD-1 x 2il7wt 2 (■) or anti-PD 1 x 2il7w142h 1 (+). All tested W142H constructs contained IgG1m and GGGGSGGGGSGGGGS linkers between Fc and IL-7 domains.

Fig. 4: compared to PD-1-cd127+ cells, the anti-PD-1 x 2il7 x 1, anti-PD-1 x 1il7 x 2w142h mutants preferentially bind and activate pSTAT5 signaling into PD-1+cd127+ cells. Cells of U937 expressing cd127+ or cells co-expressing cd127+ and PD-1+ were stained with cell proliferation dye (CPDe 450 or CPDe 670) and co-cultured in a 1:1 ratio prior to incubation with different concentrations of anti-PD-1 IL-7 bifunctional molecule. Following incubation, quantification was performed by flow cytometry against human IgG PE staining and pSTAT5 activation. A. EC50 binding (nM) was calculated for each cell type and each construct. B. The EC50 pSTAT5 (nM) was calculated for each cell type and each construct. After treatment with bifunctional molecules, the cells were then fixed, permeabilized and stained with AF 647-labeled anti-pSTAT 5 (clone 47/Stat5 (pY 694)). pSTAT5 activation. EC50 (nM) were calculated for each construct and each cell type U937 PD-1+cd127+ (white histogram) and U937 PD-1-cd127+ (black histogram). n=2 independent experiments. In this assay, anti-PD-1 x 2IL7w142 x 1, anti-PD-1 x 1IL 7w142 x 1 and anti-PD-1 x 1IL 7w142 x 2 were tested and included iggggsggggsgggs linkers between IgGlm isoforms and Fc and IL-7 domains.

Fig. 5: pharmacokinetics following intraperitoneal injection of anti-PD-1 x 2il7 x 1, anti-PD-1 x 1il7 x 2w142h mutant molecules. Humanized PD1 mice were injected intraperitoneally with a dose (34 nM/kg) of anti-PD-1*2IL7 IL7*2IgG4m (. DELTA.) and anti-PD-1*2IL7 W142H*1IgGlmanti-PD-1 x 1il7w142h x 1igglm (++grey), or anti-PD-1*1IL7 W142H*2IgGlm (Σgrey). The concentration of drug in serum was assessed by ELISA 72 hours post injection.

Fig. 6: schematic structural diagrams of anti-PD-1/protX bifunctional antibodies used in examples 3 to 9. Form a: anti-PD-1*2/ProtX X2 comprises 2 anti-PD-1 arms and 2 proteins X fused to the C-terminus of the Fc domain of an anti-PD-1 antibody. Form B: anti-PD-1*2/ProtX 1 comprises 2 anti-PD-1 arms and 1 protein X (chain B) fused to the C-terminus of the Fc domain of an anti-PD-1 antibody. Form C: anti-PD-1*1/protX 1 comprises 1 anti-PD-1 arm and 1 protein X (chain B) fused to the C-terminus of the Fc domain of an anti-PD-1 antibody. The following schematic diagram of form C is another representation of such a construct, in which each chain and component of the molecule is further described. Forms B and C also include a pestle-mortar strategy, wherein a pestle mutation is preferably introduced into strand a and a mortar mutation is preferably introduced into strand B.

Fig. 7: productivity of anti-PD-1/ProtX bifunctional antibodies in mammalian cells. CHO-S cells were transiently transfected with DNA encoding anti-PD-1*2/protX 1 or anti-PD-1*1/protX 1 molecules in a ratio (1:3:3; strand a: strand B: VL). The supernatant containing the antibodies was purified using protein a chromatography. The amount of bifunctional antibody obtained after purification was quantified by UV spectroscopy (DO 280 nm) and normalized for throughput. Fig. 7A. Raw data for productivity, each dot represents the productivity of one anti-PD-1*1/protX x 1 antibody. Fig. 7B normalized productivity of all anti-PD-1*1/protX 1 antibodies compared to anti-PD-1*2/protX 1 molecules. FIG. 7℃ Raw data productivity of single constructs.

Fig. 8: size exclusion chromatography of anti-PD-1*1/IL-7wt.1 (A) and anti-PD-1*1/IL-7v.1 (B). Purified antibodies were isolated according to their size using gel filtration chromatography using SuperDex 200 (10/300 GL). Peaks corresponding to aggregates, heterodimeric antibodies and Fc homodimers are shown on the graph and the percentage of compound is calculated.

Fig. 9: anti-PD-1*1/protX 1 bifunctional antibodies exhibit high binding efficiency to human PD-1. Human recombinant PD-1 (rPD 1) protein was immobilized and antibodies were added at different concentrations. Display was performed using an anti-human Fc antibody conjugated to peroxidase. Colorimetric assays were performed at 450nm using TMB substrate. Fig. 9A, anti-PD-1*1/IL-2*1, fig. 9B, anti-PD-1 x 1IL-21 x 1, fig. 9C, anti-PD-1*1/IL-15 x 1.

Fig. 10: the anti-PD-1 x/cytokine x 1 molecule activated pSTAT5 with high potency. FIG. 10A shows pSTAT5 signaling assays on human primary T cells treated with anti-PD-1*1/IL-21 x 1, anti-PD-1*1/IL-15 x 1, anti-PD-1*1/IL-7 wt x l, anti-PD-1*1/IL-7 v x l. Human PBMC isolated from peripheral blood of healthy volunteers and these fractionsThe seeds were incubated for 15 minutes. Cells were then fixed, permeabilized and stained with anti-CD 3-BV421 and anti-pSTAT 5 AF647 (clone 47/Stat5 (pY 694)). The data corresponds to% pstat5+ cells entering the cd3+ population. Fig. 10B using anti-PD-1 x 2il7v x 2 Or against PD-1 x 1il x 1 (delta) molecules, pSTAT5 signaling into the human cd127+cd132+u937 cell line. The left plot corresponds to the percentage of pstat5+ cells, and the right plot corresponds to EC50 (nM) calculated from the concentration required to reach 50% of pstat5 activation. Data represent mean +/-SD of 3 independent experiments.

Fig. 11: compared to PD-1-cells, the anti-PD-1*1/protX 1 molecule preferentially binds to PD-1+ cells with high efficiency. FIG. 11A. U937 cells and U937 PD-1 cells expressing PD-1+ were stained with cell proliferation dye (CPDe 450 or CPDe 670) and co-cultured at a 1:1 ratio prior to incubation with different concentrations of anti-PD-1*1/IL 2 x 1, anti-PD-1*1/IL 15 x 1, anti-PD-1 x 1IL-21 x 1 bifunctional molecule or anti-PD-1*1 and anti-PD-1*2 (as positive control stains). After incubation, anti-human IgG PE staining and pSTAT5 activation in PD-1+ (solid line) or PD-1-cells (dotted line) were quantified by flow cytometry. FIG. 11B-binding of anti-PD-1*1/IL-7wt 1 molecules on PD-1+CD127+U937 cells (straight line) compared to PD-1-CD127+U937 cell line (dashed line). FIG. 11C binding of anti-PD-l/IL-7v l molecules on PD-1+CD127+U937 cells (straight line) compared to PD-1-CD127+U937 cell line (dashed line). FIG. 11D comparison of pSTAT5 activation in PD-1+CD127+ cells versus PD-1-cell CD127+ cells. EC50nM ratios of pSTAT5 activation on PD-1+cd127+ (white histogram) compared to PD-1-cd127+ cells (black histogram) were calculated and reported graphically. N=3 independent experiments.

Fig. 12: anti-PD-1 x 1il7 x 1 synergistically activates TCR signaling. Promega PD-1/PD-L1 bioassay: (1) Effector T cells (Jurkat stably expressing PD-1, NFAT-induced luciferase) and (2) activated target cells (CHO K1 cells stably expressing PD-L1 and surface proteins, intended to activate the cognate TCR in an antigen-independent manner). Addition of BioGlo ^TM After fluorescein, luminescence was quantified and reflected T cell activation. FIG. 12A. Anti-cancerPD1 x 1 (■ black), anti-PD-1*1 + isoform 1IL-7wt 1 as a separate compoundanti-PD-1 x 1il7wt 1 (, black) was added at a series of concentrations. The right panel shows the calculated EC50 (nM) for each construct. Fig. 12B, anti-PD 1 x 1 (■ black), anti-PD-1*1 + isoform 1IL-7v x 1 as a single compound>anti-PD-1 x 1il7v x 1 (, black) was added at a series of concentrations. The right panel shows the calculated EC50 (nM) for each construct. Data represent at least 3 independent experiments.

Fig. 13: anti-PD-1*1/IL 7v 1 exhibits higher in vivo pharmacokinetics than anti-PD 1 x 2/IL7v 1 or anti-PD-1*2/IL 7v 2 molecules. A dose (34 nmol/kg) of a bifunctional anti-PD-1/IL-7 v molecule was injected intravenously (FIG. 13A) or intraperitoneally (FIG. 13B) into C57BL/6 mice. Antibody concentrations in serum were quantified at various time points using an anti-human Fc specific ELISA. Left panel, data are presented in nanomolar concentrations. The right panel, the area under the curve for each construct was calculated to define in vivo drug exposure. Data are mean +/-SEM of 2-4 mice/group.

Fig. 14: anti-PD-1*1/protX 1 exhibits higher in vivo pharmacokinetics than anti-PD 1 x 2/protX 1 molecules. C57BL/6 mice were injected intravenously with a dose (34 nmol/kg) of anti-PD-1/IL-7, anti-PD-1/IL 21, anti-PD-1/IL-2, anti-PD-1/IL-15 molecules constructed from anti-PD-1*1 or anti-PD-1*2 backbones. Antibody concentrations in serum were quantified at various time points using an anti-human Fc specific ELISA. The area under the curve (AUC) of each construct was calculated to define in vivo drug exposure. Each curve represents a type of molecule.

Fig. 15: pharmacokinetic studies of individual anti-PD 1 x 2/ProtX 1 molecules. The data represent the pharmacokinetics and AUC of the individual constructs of the anti-PD-1/IL-7, anti-PD-1/IL-21, anti-PD-1/IL-2, anti-PD-1/IL-15 molecules constructed with an anti-PD-1*1 or anti-PD-1*2 backbone and one or 2 fusion protX. FIG. 15, anti-PD-1/IL-7 wt FIG. 15B, anti-PD-1/IL-21 FIG. 15C, anti-PD-1/IL-2, FIG. 15D, anti-PD-1/IL-15. Data are mean +/-SEM of independent experiments containing 1-4 mice/group.

Fig. 16: pharmacokinetic studies of mice after a single injection of anti-PD-1 (anti-PD-1*1 and anti-PD-1*2). C57BL 6 mice were injected with a dose (34 nmol/kg) of anti-PD-1 antibody constructed from one anti-PD-1 (anti-PD-1*1 ■, dotted line) or 2 anti-PD-1 (anti-PD-1*2 +.c, straight line), (FIG. 16A) intravenous injection and (FIG. 16B) intraperitoneal injection. Antibody concentrations in serum were quantified at various time points using an anti-human Fc specific ELISA. Data are presented as nM concentration.

Fig. 17: the anti-PD-1 x 1il7v x 1 molecule significantly promotes T cell proliferation in vivo. Mice were intraperitoneally injected with a dose (34 nM/kg) of anti-PD-1 IL-7v molecules (anti-PD-1X 2IL7v 1, anti-PD-1X 1IL7v 2, anti-PD-1X 2IL7wt 1, anti-PD-1*1 or anti-PD-2). On day 4, tumors and blood were collected and T cells in different T cell subsets were stained with ki67 proliferation markers. Percent KI67 was quantified in the blood cd3cd4+ and cd3cd8+ populations (fig. 17A and 17B) and in intratumoral tcf1+ stem cell-like CD 8T cells (CD 45/CD3/CD8/CD 44/tcf1+/TOX-) (fig. 17C). Statistical significance (< 0.05) was calculated by one-way ANOVA test for multiple comparisons with control mice, n=3 to 8 mice per group.

Fig. 18: the anti-PD-1X 1IL 7X 1 molecule has obvious efficacy in an anti-PD-1 drug-resistant liver cancer in-situ model. Humanized PD-1KI immunocompetent mice were used for the experiments. The hepa1.6 hepatoma cells were injected in situ via portal vein. On day 4, mice were treated with 3 doses of PBS (negative control), anti-PD-1*2 and anti-PD-1 x 1il7 x 1. 2 independent experiments were performed. Fig. 18A survival of mice treated with anti-PD-1 x 1il7v x 1 compared to anti-PD-1*2 and PBS treatment. Fig. 18B survival of mice treated with anti-PD-1 x 1il7wt 1 compared to anti-PD-1*2 and PBS treatment.

Fig. 19: anti-PD-1 x 1il7v x 1 molecules showed high efficacy in mesothelioma in situ model. Experiments were performed using humanized PD-1KI immunocompetent mice. AK7 mesothelioma cells were injected intraperitoneally. On day 4, mice were treated with 3 doses of PBS (negative control), anti-PD-1*2, anti-PD-1 x 1il7v x 1. Fig. 19A. Tumor burden was measured by bioluminescence. AK7 cells stably express luciferase and can realize in vivo quantification of bioluminescence. Figure 19B survival of mice after treatment.

Fig. 20: the anti-PD-1 x 1IL7v 1 molecule abrogates Treg inhibition function to a greater extent than the IL-7 cytokine alone and the anti-PD-1 x 1IL7wt 1. Cd8+ effector T cells and autologous cd4+cd25 high CD127 low tregs were isolated from peripheral blood of healthy donors and stained with cell proliferation dye (cd8+ T cells CPDe 670). Treg/cd8+teff were then co-cultured on OKT3 coated plates (2 μg/mL) in a 1:1 ratio for 5 days with or without rIL-7, anti-PD-1*2, recombinant IL-7 cytokine, anti-PD-1 x 1IL7wt x 1 or anti-PD-1 x 1IL7v x 1 (anti-PD-1 x 1i 7w142 h) (0.12 nM). Proliferation of effector T cells was analyzed by cytofluorimetry based on loss of CPD markers. Data represent% Treg inhibition activity analyzed using formula 100- ((Teff%/Teff proliferation only%)) co-cultured with Treg. Data of independent experimental donors with n=4 +/-SEM. Statistical significance was one-way ANOVA using Dunnett multiple comparison test.

Fig. 21: the anti-PD-1 x 1il7v x 1 molecules have significant long-term monotherapy efficacy in an anti-PD-1 sensitive in situ model AK7 in situ model. hPD-1KI mice were intraperitoneally injected with AK7 mesothelioma cells and total survival after treatment (a) with PBS (n=8 mice), anti-PD-1*2 (n=8 mice), anti-PD-1 x 1il7v x 1 (W142H x 1) (n=14) and anti-PD-1 x 1i l7wt x 1. Statistical significance was calculated by log rank test (< p < 0.05). (B) anti-PD-1 x 1il7v x 1 induced long-term memory responses following tumor re-challenge. Mice cured by anti-PD-1 x 1il7v x 1 treatment were re-challenged with AK7 mesothelioma cells (3 e6 cells) by intraperitoneal injection (n=7). As a control, a group of naive mice (n=3) was also injected to verify tumor burden and growth. The graph shows luciferase-transduced AK7 tumor cell growth (mean +/-SEM) quantified by bioluminescence following intraperitoneal injection of D-luciferin (150 mg/kg and analysis using a bioanalyzer).

Fig. 22: preclinical efficacy of anti-PD-1 x 1il7v x 1 molecules in an in situ model of liver cancer. Following Hepa 1.6 tumor inoculation, mice were treated with PBS, anti-PD-1 x 1il7v x 1 (anti-PD-1 x 1il7w142h x 1) or anti-PD-1 x 1il7wt l on days 4/6 and 8. Total survival of 3 independent experiments was obtained and combined for illustration. PBS (n=23); anti-PD-1*2 (n=26), isoform-IL-7 (n=14), anti-PD-1 x 1IL7v 1 (n=20) and anti-PD-1 x 1IL7wt 1 (n=19). Statistical significance p <0.05 was calculated using Log Rank test.

Fig. 23: gene signatures after in vivo treatment with anti-PD-1 x 1IL7v x 1 molecules showed an increase in stem cell-like memory CD 8T cell subsets into the tumor microenvironment. Following Hepa 1.6 tumor inoculation, mice were treated with PBS, anti-PD-1 (34.3 nmol/kg) or anti-PD-1 x 1il7v 1 or anti-PD-1 x 1il7wt 1 (34.3 nmol/kg) on days 4/6 and 8 (n=4 per group). On day 10, tumors were collected and analyzed with Nanostring Pancancer immune panel analysis of gene expression (a) hetmap for genes (DEG) differentially expressed between PBS, anti-PD-1 and anti-PD-1 x 1IL7v groups using STRING protein-protein network analysis of genes commonly upregulated between anti-PD-1 and BICKI IL7v treatments; . (B and C) enrichment of the genetic profile of early activated T cells with depleted T cells. Gene signatures of depleted T cells and naive/stem cell-like memory T cells were adapted from Andreatta et al (Nature comm 2021,12,2965) naive/stem cell-like memory T cells (TCF 7, CCR7, SELL, IL 7R) and depleted CD 8T cell scores (LAG 3, PRF1, CD8A, HAVRC2, GZMB, CD8B1, KLRD1, TNFRSF9, TIGIT, CTSW, CCL, CD63, IFNG, CXCR6, FASL, CSF 1).

Fig. 24: the anti-PD-1 x 1il7v x 1 molecules induce proliferation of stem cell-like memory CD 8T cell (tcf1+) sub-populations into the tumor microenvironment. (A, B and C) following Hepa 1.6 tumor inoculation, mice were treated with PBS, anti-PD-1 (34.3 nmol/kg) or anti-PD-1 x 1il7v x 1 or anti-PD-1 x 1il7wt x 1 (34.3 nmol/kg) on days 4/6 and 8 (n=4 per group). On day 10, tumor T cells were collected and stained for flow cytometry analysis of CD3/CD8/CD44 markers and expression of TCF1/TOX factor. Percentage of CD4, CD8 and Treg subpopulations in tumor microenvironment (B) percentage of CD44+CD8+ activated T cells expressing TCF1+/-TOX marker (C) proliferation of CD44+CD8+ activated T cells expressing TCF1+/-TOX marker measured by percentage of KI67 marker in the Hepal.6 model (D) proliferation of CD44+CD8+ activated T cells expressing TCF1+/-TOX marker measured by percentage of KI67 marker after treatment (anti-PD-1*2 anti-PD-1 x 7v 1 (W142H) l)

Fig. 25: anti-PD-1 x 1il7v 1 maintains survival of chronically stimulated T cells (a) and induces proliferation of stem cell-like T cells in vitro (B) NXG immunodeficient mice were subcutaneously injected with MDA-MB231 breast cancer cells (3 e6 cells), humanized with human PBMCs intraperitoneally on day 8 (3 e6 cells), and then treated with intraperitoneal injections of PBS, anti-PD-1*2, or anti-PD-1 x 1il7v 1 on days 12, 15, and 18 post tumor inoculation. Mean data +/-SEM n=3 to 4 mice per group.

Fig. 26: monotherapy efficacy of anti-PD-1 x 1il7v x 1 molecules in a breast cancer cell humanized mouse model. NXG immunodeficient mice were subcutaneously injected with MDA-MB231 breast cancer cells (3 e6 cells), humanized with human PBMC (3 e6 cells) intraperitoneally on day 8, followed by treatment with intraperitoneal injections of PBS, anti-PD-1*2 or anti-PD-1 x 1IL7v x 1 on days 12, 15 and 18 post tumor inoculation. Mean data +/-SEM n=3 to 4 mice per group and per donor

Fig. 27: monotherapy efficacy of anti-PD-1 x 1il7v x 1 molecules in lung cancer a549 humanized mouse model. NXG immunodeficient mice were subcutaneously injected with A549 lung cancer cells, humanized with human PBMC (10 e6 cells) intraperitoneally on day 21, followed by treatment with intraperitoneal injections of PBS, anti-PD-1*2, or anti-PD-1 x 1IL7v x 1 on days 25, 28, 31, and 34 post tumor inoculation. (A) A549 tumor growth n=5 mice per group, mean +/-SEM. Statistical significance p <0.05 was calculated by comparing anti-PD-1 x 1il7v x 1 versus anti-PD-1*2 group using Dunnett multiplex comparison test. (B) Human IFNg secretion was quantified by ELISA in mouse serum collected on day 35 (n=2-5 mice per group).

Fig. 28 shows a better pharmacokinetic profile of anti-PD-1 x 1il7v x1 compared to anti-PD-1 x 1il7wt 1 molecule and induces CD 8T cell proliferation in vivo. Animals were dosed with anti-PD-1 x 1il-7v×l (anti-PD-1 x 1il7w 142h×1) or anti-PD-1 x 1il7wt×l (a) intravenously at 0.8mg/kg (n=1 cynomolgus monkey), 4.01mg/kg (n=1 cynomolgus monkey), 25mg/kg (n=1 cynomolgus monkey) by MSD immunoassay to quantify the drug concentration in the animal serum. (B) CD 8T cell proliferation measurements in peripheral blood T cells were assessed by flow cytometry after injection of different doses of anti-PD-1 x 1il7v x1 antibody. (n=1 cynomolgus monkey per dose).

Fig. 29 anti-PD-1 x 1il7v x1 constructed with either IgG1N297A isotype or IgG1N297A LALA PG isotype showed similar efficacy in activating pSTAT 5. Stimulated human PBMCs were treated with different doses of anti-PD-1 x 1il7v x1 constructed with IgG1N297A isotype or IgG1 LALA P329G mutation. IL-7R signaling activation was measured by intracellular pSTAT5 staining of CD4 and CD 8T cells and analyzed by flow cytometry.

FIG. 30 anti-PD-1*1/protX 1 demonstrates the absence of in vivo toxicity. C57BL/6 mice were injected intraperitoneally with PBS, or with anti-PD-1*1/IL 7W142H 1 (A and B) or anti-PD-1*1/IL 2 1 (C) antibodies, either single (x 1) or repeated (x 3) injections every 2 days. Mice were weighed daily and data normalized to their initial weight prior to molecular injection (day 0 = 100%). (A) The anti-PD-1/IL 7W14H 1 molecule was injected once every 2 days at 20mg/kg or three times at 5mg/kg. (B) The anti-PD-1/IL 7W14H 1 molecule was injected at progressively higher doses of 50 or 100mg/kg or three times 20mg/kg every 2 days. (C) The anti-PD-1/IL-2*1 molecules were injected once every 2 days at 20mg/kg or three times at 5mg/kg.

Detailed Description

Introduction to the invention

The present invention relates to bifunctional molecules having a specific scaffold and comprising a single monovalent antigen binding domain and a single immunostimulatory cytokine that bind to a target specifically expressed on the surface of an immune cell. The stent consists essentially of: a dimeric Fc domain, a single monovalent antigen binding domain that binds to a target specifically expressed on the surface of an immune cell linked to the N-terminus of one monomer of the Fc domain, and a single immunostimulatory cytokine linked to the C-terminus of the Fc domain monomer or light chain (when antigen binds to a domain comprising a heavy variable chain and a light variable chain). These novel bifunctional molecules have, among other benefits, improved pharmacokinetic profiles and better productivity.

Definition of the definition

For easier understanding of the present invention, certain terms are defined below. Additional definitions are set forth throughout the detailed description.

Unless defined otherwise, all terms of art, notations and other scientific terms used herein are intended to have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or ease of reference, and the inclusion of such definitions herein should not necessarily be construed to represent a distinction from what is commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are generally employed using conventional methods.

As used herein, the terms "wild-type interleukin-7", "wt-IL-7" and "wt-IL7" refer to mammalian endogenous secreted glycoproteins, particularly IL-7 polypeptides. For example, wt-IL-7 refers to an amino acid sequence of a recombinant or non-recombinant polypeptide having the amino acid sequence: i) Naturally occurring or naturally occurring IL-7 polypeptide, ii) a biologically active fragment of an IL-7 polypeptide, iii) a biologically active polypeptide analog of an IL-7 polypeptide, or iv) a biologically active IL-7 polypeptide. IL-7 may contain its peptide signal or be free of its peptide signal. Alternative names for this molecule are "pre-B cell growth factor" and "lymphopoietin-1". Preferably, the term "wt-IL-7" refers to human IL-7 (wt-IL 7). For example, human wt-IL-7 has an amino acid sequence of about 152 amino acids (no signal peptide present) and has Genbank accession number NP-000871.1, which is located on chromosome 8q 12-13. Human IL-7 is described, for example, in UniProtKB-P13232.

As used herein, the term "antibody" describes and is used in its broadest sense to describe a class of immunoglobulin molecules. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Immunoglobulin molecules may be of any type (e.g., igG, igE, igM, igD, igA and IgY), class (e.g., igGl, igG2, igG3, igG4, igA1, and IgA 2) or subclass. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. Unless otherwise specifically indicated, the term "antibody" includes intact immunoglobulins and "antibody fragments" or "antigen-binding fragments" (e.g., fab ', F (ab') 2, fv), single chains (scFv), mutants thereof, molecules comprising an antibody portion, diabodies, linear antibodies, single chain antibodies, and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site of the desired specificity, including glycosylated variants of antibodies, amino acid sequence variants of antibodies. Preferably, the term antibody refers to a humanized antibody.

As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains that exist in an antibody conformation. The CDRs of antibody heavy chains are commonly referred to as "HCDR1", "HCDR2" and "HCDR3". The framework regions of the antibody heavy chain are commonly referred to as "HFR1", "HFR2", "HFR3" and "HFR4".

As used herein, "antibody light chain" refers to the smaller of two types of polypeptide chains that exist in an antibody conformation; kappa and lambda light chains refer to two major antibody light chain isotypes. CDRs of an antibody light chain are commonly referred to as "LCDR1", "LCDR2", and "LCDR3". The framework regions of antibody light chains are commonly referred to as "LFR1", "LFR2", "LFR3", and "LFR4".

As used herein, an "antigen binding fragment" or "antigen binding domain" of an antibody means a portion of an antibody, i.e., a molecule corresponding to a portion of the structure of an antibody of the invention, which exhibits antigen binding capacity against a particular antigen, possibly in its native form; such fragments in particular exhibit the same or substantially the same antigen binding specificity for the antigen as compared to the antigen binding specificity of the corresponding four-chain antibody. Advantageously, the antigen binding fragment has a binding affinity similar to that of the corresponding 4-chain antibody. However, antigen binding fragments having reduced antigen binding affinity relative to corresponding 4-chain antibodies are also encompassed within the invention. The antigen binding capacity can be determined by measuring the affinity between the antibody and the target fragment. These antigen binding fragments may also be referred to as "functional fragments" of antibodies. An antigen binding fragment of an antibody is a fragment comprising its hypervariable domains, or a part or parts thereof, known as CDRs (complementarity determining regions).

As used herein, the term "humanized antibody" is intended to refer to an antibody in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences (e.g., chimeric antibodies comprising minimal sequences derived from non-human antibodies). "humanized form" of an antibody, such as a non-human antibody, also refers to an antibody that has undergone humanization. Humanized antibodies are typically human immunoglobulins (recipient antibody) in which residues from one or more CDRs are replaced by residues from at least one CDR of a non-human antibody (donor antibody) while maintaining the desired specificity, affinity and capacity of the original antibody. Additional framework region modifications may be made within the human framework sequence. Preferably, the humanized antibody has a T20 humanized score of greater than 80%, 85% or 90%. The "humanization" of an antibody can be measured, for example, using a T20 score analyzer to quantify the humanization of the antibody variable region, as described in Gao S H, huang K, tu H, adler a s.bmc biotechnology.2013:13:55 or by using a T20 cutoff human database via a web-based tool: the T20 score of the antibody sequence was calculated at http:// abanalyzer.

By "chimeric antibody" is meant an antibody prepared by combining genetic material from a non-human source (preferably, e.g., mice) with genetic material from a human. Such antibodies are derived from human and non-human antibodies linked by chimeric regions. Chimeric antibodies typically comprise a constant domain from a human and a variable domain from another mammalian species, which when used in therapeutic treatment, reduce the risk of reacting to exogenous antibodies from non-human animals.

As used herein, the terms "fragment crystallizable region", "Fc region" or "Fc domain" are interchangeable and refer to the tail region of an antibody that interacts with a cell surface receptor called an Fc receptor. The Fc region or domain typically consists of two domains, optionally identical, the second and third constant domains (i.e., CH2 and CH3 domains) derived from the two heavy chains of an antibody. A portion of an Fc domain refers to a CH2 or CH3 domain. Optionally, the Fc region or domain may optionally comprise all or part of the hinge region between CH1 and CH 2. Accordingly, an Fc domain may comprise a hinge, a CH2 domain, and a CH3 domain. Optionally, the Fc domain is an Fc domain from IgG1, igG2, igG3 or IgG4, optionally with an IgG1 hinge-CH 2-CH3 and an IgG4 hinge-CH 2-CH3.

In the context of IgG antibodies, igG isotypes each have three CH regions. Thus, in the IgG context, the "CH" domain is as follows: according to the EU index as in Kabat, "CH1" refers to positions 118-215. According to the EU index as in Kabat, "hinge" refers to positions 216-230. "CH2" refers to positions 231-340 according to the EU index as in Kabat, and "CH3" refers to positions 341-447 according to the EU index as in Kabat.

"amino acid change" or "amino acid modification" herein means a change in the amino acid sequence of a polypeptide. "amino acid modifications" include substitutions, insertions and/or deletions in the polypeptide sequence. "amino acid substitution" or "substitution" herein means the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. "amino acid insertion" or "insertion" means the addition of an amino acid at a particular position in a parent polypeptide sequence. "amino acid deletion" or "deletion" means the removal of an amino acid at a particular position in a parent polypeptide sequence. Amino acid substitutions may be conservative. A conservative substitution is the replacement of a given amino acid residue with another residue having a side chain ("R group") of similar chemical nature (e.g., charge, volume, and/or hydrophobicity). As used herein, "amino acid position" or "amino acid position number" are used interchangeably to refer to the position of a particular amino acid in an amino acid sequence, typically designated by a single letter code for the amino acid. The first amino acid in the amino acid sequence (i.e. starting from the N-terminus) should be considered as having position 1.

A conservative substitution is the replacement of a given amino acid residue with another residue having a side chain ("R group") of similar chemical nature (e.g., charge, volume, and/or hydrophobicity). Generally, conservative amino acid substitutions do not significantly alter the functional properties of the protein. Conservative substitutions and corresponding rules are well described in the prior art. For example, conservative substitutions may be defined by substitutions within the amino acid groups reflected in the following table:

TABLE A amino acid residues

Amino acid group	Amino acid residues
		Acidic residues	ASP and GLU
Basic residues	LYS, ARG and HIS
		Hydrophilic non-charge residues	SER, THR, ASN and GLN
Aliphatic non-charge residues	GLY, ALA, VAL, LEU and ILE
		Non-polar non-charge residues	CYS, MET and PRO
Aromatic residues	PHE, TYR and TRP

Table B-substitution of alternative conserved amino acid residues

1	Alanine (A)	Serine (S)	Threonine (T)
				2	Aspartic acid (D)	Glutamic acid (E)
3	Asparagine (N)	Glutamine (Q)
				4	Arginine (R)	Lysine (K)
5	Isoleucine (I)	Leucine (L)	Methionine (M)
				6	Phenylalanine (F)	Tyrosine (Y)	Tryptophan (W)

Further substitution of Table C-amino acid residues physical and functional classifications

Residues containing alcohol groups	S and T
		Aliphatic residues	I. L, V and M
Cycloalkenyl-related residues	F. H, W and Y
		Hydrophobic residues	A. C, F, G, H, I, L, M, R, T, V, W and Y
Residues of negative charge	D and E
		Polar residues	C. D, E, H, K, N, Q, R, S and T
Small residues	A. C, D, G, N, P, S, T and V
		Very small residues	A. G and S
Residues involved in inversion formation	A. C, D, E, G, H, K, N, Q, R, S, P and T
		Flexible residues	E. Q, T, K, S, G, P, D, E and R

As used herein, "sequence identity" between two sequences is described by the parameters "sequence identity", "sequence similarity" or "sequence homology". For the purposes of the present invention, the "percent identity" between two sequences (A) and (B) is determined by comparing the two sequences optimally aligned by comparison windows. The sequence alignment may be performed by methods well known in the art, for example using the Needleman-Wunsch global alignment algorithm. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. Once the total alignment is obtained, the percent identity can be obtained by dividing the total number of identical amino acid residues aligned by the total number of residues contained in the longest sequence between sequences (a) and (B). Sequence identity is typically determined using sequence analysis software. To compare two amino acid sequences, one can use the "Emboss Needle" tool to pair-wise sequence alignment of proteins provided by EMBL-EBI, which is available on the following websites: www.ebi.ac.uk/Tools/services/web/tools=ebit_needle & context=protein, e.g. using default settings: (I) matrix: BLOSUM62, (ii) notch open: 10, (iii) notch extension: 0.5, (iv) output form: pairing, (v) end gap penalty: pseudo, (vi) terminal notch open: 10, (Vii) terminal notch extension: 0.5.

Alternatively, sequence identity can also be determined, typically using the sequence analysis software Clustal Omega, using the HHILIgn algorithm and its default settings as its core alignment engine. The algorithm is described inJ. (2005) 'Protein homology detection by HMM-HMM compactison'. Bioinformatics 21,951-960, default settings were used.

As used herein, the terms "derived from" and "derived from" refer to compounds having a structure derived from the parent compound or protein, and which are structurally similar enough to those disclosed herein, and based on that similarity, can be expected to demonstrate by one of skill in the art that they exhibit the same or similar properties, activity, and utility as the claimed compounds.

As used herein, "pharmaceutical composition" refers to a formulation of one or more active agents (e.g., comprising a bifunctional molecule according to the invention) with optionally other chemical components (e.g., physiologically suitable carriers and excipients). The purpose of the pharmaceutical composition is to facilitate the administration of the active agent to the organism. The compositions of the present invention may be in a form suitable for any conventional route of administration or use. In one aspect, a "composition" generally is intended to be a combination of an active agent (e.g., a compound or composition) and a naturally occurring or non-naturally occurring inert (e.g., a detectable agent or label) or active carrier (e.g., an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant, etc., and including pharmaceutically acceptable carriers). Reference herein to an "acceptable carrier" or "acceptable carrier" is to any known compound or combination of compounds known to those skilled in the art to be useful in formulating pharmaceutical compositions.

As used herein, "effective amount" or "therapeutically effective amount" refers to the amount of active agent required to impart a therapeutic effect to a subject, e.g., to treat a disease or disorder of interest or to produce a desired effect, alone or in combination with one or more other active agents. The "effective amount" will vary depending on one or more agents, the disease and its severity, the characteristics of the subject to be treated (including age, body condition, body shape, sex and weight), the duration of the treatment, the nature of concurrent therapy (if any), the particular route of administration, and like factors within the knowledge and expertise of a health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed by routine experimentation only. It is generally preferred to use the maximum dose of a single component or a combination thereof, i.e. the highest safe dose according to sound medical judgment.

As used herein, the term "drug" refers to any substance or composition having therapeutic or prophylactic properties against a disorder or disease.

The term "treatment" refers to any action intended to improve the health state of a patient, such as the treatment, prevention, prophylaxis and delay of a disease or disease symptoms. It refers to both curative and/or prophylactic treatment of a disease. Curative treatment is defined as treatment that results in cure or treatment that reduces, improves and/or eliminates, reduces and/or stabilizes a disease or symptoms of a disease or pain caused directly or indirectly by it. Prophylactic treatment includes treatment that results in the prevention of a disease and treatment that reduces and/or delays the progression and/or incidence of a disease or the risk of developing the same. In certain aspects, such terms refer to the amelioration or eradication of a disease, disorder, infection, or symptom associated therewith. In other aspects, the term refers to minimizing the spread or exacerbation of cancer. Treatment according to the present invention does not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment that one of ordinary skill in the art would consider to have potential benefits or therapeutic effects. Preferably, the term "treatment" refers to the application or administration of a composition comprising one or more active agents to a subject suffering from a disorder/disease.

As used herein, the term "disorder" or "disease" refers to an abnormal functioning of a body organ, part, structure or system due to genetic or developmental errors, infection, poisoning, nutritional deficiency or imbalance, toxicity or the effects of adverse environmental factors. Preferably, these terms refer to a health condition or disease, such as a patient that disrupts normal body or mental function. More preferably, the term disorder refers to an immune and/or inflammatory disease affecting animals and/or humans, such as cancer.

As used herein, "immune cells" refers to cells involved in innate and adaptive immunity, such as leukocytes (white blood cells) derived from Hematopoietic Stem Cells (HSCs) produced in bone marrow, lymphocytes (T cells, B cells, natural Killer (NK) cells and natural killer T cells (NKT) and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). Specifically, immune cells may be selected from the group consisting of B cells, T cells, particularly cd4+ T cells and cd8+ T cells, NK cells, NKT cells, APC cells, dendritic cells and monocytes.

As used herein, the term "effector T cell", "T eff" or "effector cell" describes a group of immune cells comprising several T cell types that actively respond to a stimulus (e.g., co-stimulus). It includes in particular T cells with an antigen-eliminating function (for example by producing cytokines that regulate the activation of other cells or by cytotoxic activity). It includes in particular cd4+, cd8+, cytotoxic T cells and helper T cells (Th 1 and Th 2).

As used herein, the term "regulatory T cells", "Treg cells" or "T regs" refers to a subpopulation of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. Tregs have immunosuppressive effects and generally inhibit or down regulate the induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FOXP3 and CD25 and are believed to be derived from the same lineage as the original CD4 cells.

The term "depleted T cells" refers to a population of T cells that are in a dysfunctional state (i.e., "depleted"). T cell depletion is characterized by progressive loss of function, transcriptional profiling and sustained expression of inhibitory receptors. Depleted T cells lose their cytokine production capacity, their high proliferative capacity and their cytotoxic potential, ultimately leading to their depletion. Depleted T cells generally indicate higher levels of CD43, CD69 and inhibitory receptors, while CD62L and CD127 are expressed less.

The term "effector memory stem cell-like T cells" refers to a subset of tumor-reactive intratumoral T cells having depleted cells and central memory cell characteristics (including expression of checkpoint protein PD-1 and transcription factor Tcf 1). These cells may be referred to as Tcfl+PD-1+CD8+T cells. These cells are present in the tumor microenvironment and are critical to the immune control of the cancer that is facilitated by the immunotherapy. They are critical for maintaining T cell responses during chronic viral infection and cancer, and provide proliferative bursts following PD-1 immunotherapy. These cells undergo slow self-renewal and also produce more terminally differentiated depleted CD 8T cells. These cells and their characteristics are further defined in the following articles, the disclosures of which are incorporated herein by reference: siddiqui et al, 2019, immunity,50,195-211; and Jadhav et al 2019, PNAS,116, 14113-14118).

The term "immune response" refers to the action of lymphocytes, antigen presenting cells, phagocytes, granulocytes and soluble macromolecules (including antibodies, cytokines and complements) produced by, for example, the above-mentioned cells or liver, which result in selective damage, destruction or elimination of an invading pathogen, pathogen-infected cells or tissue, cancer cells (or in the case of autoimmune or pathological inflammation, normal human cells or tissue) from the human body. As used herein, the term "antagonist" refers to a substance that blocks or reduces the activity or function of another substance. In particular, the term refers to an antibody that binds to a cellular receptor (e.g., PD-1) as a reference substance (e.g., PD-L1 and/or PD-L2) preventing it from producing all or part of its usual biological effects (e.g., creating an immunosuppressive microenvironment). The antagonist activity of the humanized antibodies according to the present invention can be assessed by competitive ELISA.

As used herein, the term "agonist" refers to a substance that activates the function of an activating receptor. In particular, the term refers to antibodies that bind to a cell-activating receptor as a reference substance and have at least partially the same effect as a biological natural ligand (e.g., induction of activation of the receptor).

Pharmacokinetic (PK) refers to the movement of a drug in the body, while Pharmacodynamics (PD) refers to the biological response of the body to a drug. PK describes drug exposure by characterizing absorption, distribution, bioavailability, metabolism, and excretion as a function of time. PD describes drug responses by biochemical or molecular interactions. PK and PD analysis was used to characterize drug exposure, predict and evaluate dose variation, evaluate elimination and absorption, evaluate relative bioavailability/bioequivalence of formulation, characterize intra-and inter-subject variability, understand concentration-effect relationships, and establish safety margin and efficacy profile. By "improving PK" is meant an improvement in one of the above characteristics, such as an increase in the half-life of the molecule, particularly a longer serum half-life of the molecule when injected into a subject.

As used herein, the terms "pharmacokinetic" and "PK" are used interchangeably to refer to the direction of a compound, substance, or drug administered to a living organism. Pharmacokinetics include, inter alia, ADME or LADME protocols, which represent release (i.e., release of a substance from a composition), absorption (i.e., entry of a substance into the blood circulation), distribution (i.e., dispersion or propagation of a substance in the body), metabolism (i.e., conversion or degradation of a substance), and excretion (i.e., removal or clearance of a substance from an organism). The two stages of metabolism and excretion can also be grouped into one category, called elimination. One skilled in the art can monitor various pharmacokinetic parameters such as elimination half-life, elimination constant rate, clearance (i.e. the volume of plasma cleared of drug per unit time), cmax (maximum serum concentration) and drug exposure (determined by the area under the curve, see Scheff et al, pharm Res.2011, month 5; 28 (5): 1081-9), etc.

As used herein, the term "isolated" means that the recited material (e.g., antibody, polypeptide, nucleic acid, etc.) is substantially separated from, or enriched relative to, other materials with which it naturally occurs. In particular, an "isolated" antibody is an antibody that has been identified and isolated and/or recovered from a component of its natural environment.

As used herein, the term "and/or" should be taken to be a particular disclosure of each of two specified features or components, with or without each other. For example, "a and/or B" should be considered a specific disclosure of each of (i) a, (ii) B, and (iii) a and B, as if each were individually listed.

The terms "a" or "an" may refer to one or more elements (e.g., "an agent" may refer to one or more agents) to which it is modified, unless the context clearly describes one or more of the elements.

As used herein, the term "about" in connection with any and all values (including the lower and upper limits of a range of values) means any value having an acceptable deviation range of up to +/-10% (e.g., +/-0.5%, +/-1%, +/-1.5%, +/-2%, +/-2.5%, +/-3%, +/-3.5%, +/-4%, +/-4.5%, +/-5.5%, +/-6%, +/-6.5%, +/-7%, +/-7.5%, +/-8%, +/-8.5%, +/-9%, +/-9.S%). The term "about" is used at the beginning of a string of values to modify each value (i.e., "about 1, 2, and 3" means about 1, about 2, and about 3). Further, when a list of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85%, or 86%), the list includes all intermediate values and fractional values thereof (e.g., 54%, 85.4%).

Bifunctional molecules

The present invention relates to bifunctional molecules having scaffolds with improved properties.

More particularly, the invention relates to bifunctional molecules having a specific scaffold and comprising a single monovalent antigen binding domain and a single immunostimulatory cytokine that bind to a target specifically expressed on the surface of an immune cell. The stent consists essentially of: a dimeric Fc domain, a single monovalent antigen binding domain that binds to a target specifically expressed on the surface of an immune cell linked to the N-terminus of one monomer of the Fc domain, and i) a single immunostimulatory cytokine linked at the C-terminus of the same monomer of the Fc domain, and optionally a peptide linker, or ii) a single monovalent antigen binding domain comprising a heavy variable chain and a light variable chain, and a single immunostimulatory cytokine linked at the C-terminus of the light chain of the antigen binding domain.

In a particular aspect, the bifunctional molecule comprises a first monomer comprising an antigen binding domain covalently linked, optionally via a peptide linker, to a first Fc chain, optionally via a peptide linker, to an immunostimulatory cytokine, and a second monomer comprising a complementary second Fc chain lacking the antigen binding domain and the immunostimulatory cytokine, the first and second Fc chains forming a dimeric Fc domain.

In an alternative aspect, the bifunctional molecule comprises a first monomer comprising an antigen binding domain covalently linked to a first Fc chain, optionally via a peptide linker, and a second monomer comprising a complementary second Fc chain lacking the antigen binding domain and the immunostimulatory cytokine, said first and second Fc chains forming a dimeric Fc domain, and a single monovalent antigen binding domain comprising a heavy variable chain and a light variable chain, and a single immunostimulatory cytokine linked at the C-terminus of the antigen binding domain light chain.

Thus, the two monomers each comprise an Fc chain capable of forming a dimeric Fc domain. In one aspect, the dimeric Fc fusion protein is a homodimeric Fc domain. In another aspect, the dimeric Fc fusion protein is a heterodimeric Fc domain.

More specifically, when the dimeric Fc domain is a heterodimeric Fc domain, the bifunctional molecule comprises a first monomer comprising an antigen binding domain covalently linked to the N-terminus of a first heterodimeric Fc chain, optionally via a peptide linker, which is covalently linked to an immunostimulatory cytokine via its C-terminus, and a second monomer comprising a second heterodimeric Fc chain lacking the antigen binding domain and the complement of the immunostimulatory cytokine. Optionally, the second monomer comprising a complementary second heterodimeric Fc strand lacks any other functional moiety. Still more particularly, the bifunctional molecule comprises a first monomer comprising an antigen binding domain covalently linked via a C-terminus to the N-terminus of a first heterodimeric Fc chain optionally via a peptide linker and a second monomer comprising a complementary second heterodimeric Fc chain lacking the antigen binding domain and immunostimulatory cytokine, preferably lacking any other functional moiety, covalently linked via its C-terminus to the N-terminus of immunostimulatory cytokine. Such bifunctional molecules are illustrated, for example, in FIG. 1 as "construct 3" wherein IL-7 is illustrated as an immunostimulatory cytokine or in FIG. 6 as form C.

Optionally, the single antigen binding domain is selected from Fab, fab', scFV, and sdAb.

Thus, in one aspect, the bifunctional molecule according to the invention comprises or consists of:

(a) An antigen binding domain that binds to a target specifically expressed on the surface of an immune cell, comprising (i) a heavy chain having a first Fc chain, and (ii) a light chain,

(b) Immunostimulatory cytokines, and

(c) A second, complementary Fc chain that is complementary to the first Fc chain,

wherein the immunostimulatory cytokine is covalently linked to the C-terminus of the first Fc chain, optionally through a peptide linker, preferably through its N-terminus. The first Fc chain and the second Fc chain together form a dimeric Fc domain.

In a particular aspect, the bifunctional molecule comprises

An antibody heavy chain comprising VH-CHl-hinge-CH 2-CFH3 linked at its C-terminus to an immunostimulatory cytokine,

an antibody light chain comprising a VL-CL (constant light chain), a VH-CH1 portion and a VL-CL portion, which together form an antigen-binding domain that binds to a target specifically expressed on the surface of an immune cell, and

-an Fc chain comprising CH2-CH3, optionally hinge-CH 2-CH3, forming a dimeric Fc domain with CH2-CH3 of the antibody heavy chain.

According to an alternative aspect, when the dimeric Fc domain is a heterodimeric Fc domain, the bifunctional molecule comprises a first monomer comprising an antigen binding domain covalently linked to the N-terminus of the first heterodimeric Fc chain, optionally via a peptide linker, and a second monomer comprising a second heterodimeric Fc chain lacking the antigen binding domain and the complement of an immunostimulatory cytokine, and the antigen binding domain comprises a heavy variable chain and a light variable chain, and the immunostimulatory cytokine is linked to the C-terminus of the antigen binding domain light chain, optionally via a peptide linker. Optionally, the immunostimulatory cytokine is linked via its N-terminus to the C-terminus of the light chain of the antigen binding domain, optionally through a peptide linker.

Thus, in this aspect, the bifunctional molecule according to the invention comprises or consists of:

(b) Immunostimulatory cytokines, and

wherein the immunostimulatory cytokine is covalently linked to the C-terminus of the light chain, optionally through a peptide linker, preferably through its N-terminus. The first Fc chain and the second Fc chain together form a dimeric Fc domain.

In a particular aspect, the bifunctional molecule comprises

An antibody heavy chain comprising VH-CH 1-hinge-CH 2-CH3,

an antibody light chain comprising a VL-CL (constant light chain) linked at its C-terminus to an immunostimulatory cytokine, the VH-CH1 portion and the VL-CL portion together forming an antigen-binding domain that binds to a target specifically expressed on the surface of an immune cell, an

Immunostimulatory cytokines, antigen binding domains that bind to a target specifically expressed on the surface of an immune cell, fc domains, and optionally linkers are further defined in any aspect as follows.

Immunostimulatory cytokines

The immunostimulatory cytokine is capable of stimulating or activating immune cells. The immune cells may be selected from a non-exhaustive list comprising B cells, T cells, in particular cd4+ T cells and cd8+ T cells, NK cells, NKT cells, APC cells, dendritic cells and monocytes. In a preferred aspect, the immune cells are T cells, more specifically cd8+ T cells, effector T cells or depleted T cells. In a particularly preferred aspect, the immune cells are effector memory stem cell-like T cells.

Preferably, the immunostimulatory cytokine is selected from the group consisting of: cytokines and chemokines. In particular, the immunostimulatory cytokines have a size of 10kDa to 50 kDa. Preferably, the immunostimulatory cytokine is a peptide, polypeptide or protein. In one aspect, the immunostimulatory cytokine is a non-antibody entity or moiety.

For example, the immunostimulatory cytokine may be selected from: t cell growth factors, particularly growth factors for increasing the number and overall function of naive T cells, growth factors for increasing the number of Dendritic Cells (DCs), agonists for activating DCs and other Antigen Presenting Cells (APCs), adjuvants for allowing and enhancing cancer vaccines, agonists for activating and stimulating T cells, inhibitors of T cell checkpoint blockade, T cell growth factors that increase immune T cell growth and survival, agents that inhibit, block or neutralize cancer cells and immune cell-derived immunosuppressive cytokines. In a particular aspect, the cytokine is capable of activating and stimulating effector memory stem cell-like T cells. The immunostimulatory cytokine may be mutated or altered to alter the biological activity, e.g., increase, decrease, or complete inhibition of the biological activity.

In a particular aspect, the immunostimulatory cytokine is selected from the group consisting of: IL-2 (IL is an interleukin), IL-4, IL-5, IL-6, IL-12A, IL-12B, IL-13; IL-15, IL-18, IL-21, IL-23, IL-24, IFN alpha (interferon alpha), IFN alpha (interferon beta), BAFF, LT alpha and LT beta or variants thereof having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with wild type cytokine or having 1 to 10 modifications selected from the group consisting of additions, deletions, substitutions and combinations thereof. The immunostimulatory cytokine may also be IL-7.

In particular, immunostimulatory cytokines may be selected in the list of table D below.

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Table D: list of immunostimulatory cytokines

In a particular aspect, the immunostimulatory cytokine is not IL-2 or a variant thereof.

Thus, the immunostimulatory cytokine is selected from the group consisting of: IL-2 (IL is an interleukin), IL-4, IL-5, IL-6, IL-12A, IL-12B, IL-13; IL-15, IL-18, IL-21, IL-23, IL-24; ifnα (interferon α), ifnβ (interferon β), BAFF, LT α and ltβ, or variants thereof having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 1 to 10 modifications selected from the group consisting of additions, deletions, substitutions and combinations thereof with the wild type protein or extracellular fragment thereof. The immunostimulatory cytokine may also be IL-7.

In particular, the immunostimulatory cytokine is selected from: IL-2, IL-15 and IL-21 or variants thereof having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 1 to 10 modifications selected from the group consisting of additions, deletions, substitutions and combinations thereof with wild-type cytokines.

In very specific aspects, the immunostimulatory cytokine is IL-2 or a variant thereof having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a wild-type cytokine or having 1 to 10 modifications selected from the group consisting of additions, deletions, substitutions and combinations thereof.

In a very specific aspect, the immunostimulatory cytokine is IL-7, in particular IL-7 having a sequence as set forth in SEQ ID NO. 1.

In very specific aspects, the immunostimulatory cytokine is IL-15 or a variant thereof having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a wild-type cytokine or having 1 to 10 modifications selected from the group consisting of additions, deletions, substitutions and combinations thereof.

In very specific aspects, the immunostimulatory cytokine is IL-21 or a variant thereof having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a wild-type cytokine or having 1 to 10 modifications selected from the group consisting of additions, deletions, substitutions and combinations thereof.

IL-2 and IL-2 variants

In a very specific aspect, the immunostimulatory cytokine is interleukin-2 (IL-2), preferably human IL-2, e.g. as disclosed under UniProt accession No. P60568 or a mutant or variant thereof.

The IL-2 variant preferably has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the wild type cytokine of SEQ ID NO. 87 or 1 to 10 modifications selected from the group consisting of additions, deletions, substitutions and combinations thereof relative to the sequence of SEQ ID NO. 87.

IL-2 can be mutated in a variety of ways to reduce its toxicity and/or to increase its efficacy. Hu et al (Blood 101,4853-4861 (2003), U.S. patent publication No. 2003/0124678) replaced the 38 th arginine residue of IL-2 with tryptophan to eliminate vascular permeability activity of IL-2. Shanafelt et al (Nature Biotechnol, 1 197-1202 (2000)) have mutated asparagine 88 to arginine to enhance selectivity of T cells over NK cells. Two mutations, arg38Ala and Phe42Lys, were introduced by Heaton et al (Cancer Res 53,2597-602 (1993); U.S. Pat. No. 5,229,109) to reduce NK cell secretion of pro-inflammatory cytokines. Gilles et al (U.S. patent publication No. 2007/0036752) have replaced three residues of IL-2 (Asp 20Thr, asn88Arg and Gln126 Asp), which contributes to affinity for the medium affinity IL-2 receptor to reduce VLS. Gillies et al (WO 2008/0034473) also mutated the interface of IL-2 with CD25 by amino acid substitutions Arg38Trp and Phe42Lys to reduce interaction with CD25 and activation of Treg cells to enhance efficacy. In one aspect, the immunotherapeutic agent is an IL-2 mutant, e.g., as described in WO 2012/107417 or WO 2018/184964.

Optionally, the IL-2 variant may comprise one or more substitutions at a position of human IL-2 (NO signal peptide; SEQ ID NO: 87) selected from the group consisting of: q11, H16, L18, L19, D20, Q22, R38, F42, K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, 192, T123, Q126, SI 27, 1129, and S130. Optionally, the IL-2 variant may comprise a substitution F42A or F42K relative to human IL-2 (NO signal peptide; SEQ ID NO: 87). Optionally, the IL-2 variant may further comprise one or several substitutions, with respect to human IL-2 (NO signal peptide; SEQ ID NO: 87), selected from the group consisting of: r38 38 38 38 62 68 68K and E68R, and/or H16 16 20 23 23 87 87 84 84 84 84 84 84 84 84 88 88 88 91 91 91 91 91 95 95 123 123 123 123 126 126 126 126 127K and S127Q, and/or C125A.

Optionally, the IL-2 variant may comprise one of the following combinations of substitutions relative to human IL-2 (NO signal peptide; SEQ ID NO: 87): R38E and F42A; R38D and F42A; F42A and E62Q; R38A and F42K; R38E, F a and N88S; R38E, F a and N88A; R38E, F a and V91E; R38E, F a and D84H; H16D, R E and F42A; H16E, R E and F42A; R38E, F a and Q126S; R38D, F a and N88S; R38D, F a and N88A; R38D, F a and V91E; R38D, F a and D84H; H16D, R D and F42A; H16E, R D and F42A; R38D, F a and Q126S; R38A, F K and N88S; R38A, F K and N88A; R38A, F K and V91E; R38A, F K and D84H; H16D, R a and F42K; H16E, R a and F42K; R38A, F K and Q126S; F42A, E Q and N88S; F42A, E Q and N88A; f42A, E Q and V91E; F42A, E Q and D84H; H16D, F a and E62Q; H16E, F a and E62Q; F42A, E Q and Q126S; R38E, F a and C125A; R38D, F a and C125A; F42A, E Q and C125A; R38A, F K and C125A; R38E, F42A, N S and C125A; R38E, F42A, N a and C125A; R38E, F, 42, A, V E and C125A; R38E, F42A, D H and C125A; H16D, R E, F a and C125A; H16E, R E, F a and C125A; R38E, F42A, C a and Q126S; R38D, F42A, N S and C125A; R38D, F42A, N a and C125A; R38D, F, 42, A, V E and C125A; R38D, F42A, D H and C125A; H16D, R D, F a and C125A; H16E, R D, F a and C125A; R38D, F42A, C a and Q126S; R38A, F42K, N S and C125A; R38A, F42K, N a and C125A; R38A, F, 42, K, V E and C125A; R38A, F42K, D H and C125A; H16D, R A, F K and C125A; H16E, R A, F K and C125A; R38A, F42K, C a and Q126S; F42A, E62Q, N S and C125A; F42A, E62Q, N a and C125A; F42A, E62Q, V E and C125A; F42A, E62Q, D H, C125A; H16D, F, 42, A, E Q and C125A; H16E, F, 42, A, E Q and C125A; F42A, E62Q, C a and Q126S; F42A, N S and C125A; F42A, N a and C125A; F42A, V91E and C125A; F42A, D H and C125A; H16D, F a and C125A; H16E, F a and C125A; F42A, C a and Q126S; F42A, Y a and L72G; and T3A, F42A, Y45A, L G and C125A.

Optionally, the IL-2 variant may comprise one of the following substitutions relative to human IL-2 (NO signal peptide; SEQ ID NO: 87), in particular at least one substitution selected from the group comprising: the K35 35 38 38 38 38 38 38 38 38 38 38 38 38 42 42 42 42 42 42 42 42 42 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 72 72 72 72 72 72 72 72 72 72 72 72 72R and L72K. Or combinations thereof, preferably three substitutions F42A, Y a and L72G.

For example, a human IL-2 (hIL-2) mutant with reduced affinity for CD25 can be produced by: amino acid substitutions at amino acid positions 3, 35, 38, 42, 43, 45 or 72 corresponding to the residue position of human IL-2 (NO signal peptide; SEQ ID NO: 87) or a combination thereof. Preferably, the mutant IL-2 is a human IL-2 molecule comprising the amino acid substitutions T3A, F42A, Y45A, L G and/or C125A, preferably F42A, Y45A and L72G, more preferably T3A, F42A, Y45A, L72G and C125A, relative to human IL-2 (NO signal peptide; SEQ ID NO: 87), e.g. as disclosed in WO 2018/184964. Even more preferably, the immunostimulatory cytokine is an IL-2 mutant having substitutions F42A, Y45A and L72G, preferably T3A, F42A, Y45A, L G and C125A, relative to human IL-2 (NO signal peptide; SEQ ID NO: 87).

IL-12A and IL-12B variants

In a particular aspect, the immunostimulatory cytokine is IL-12A or a variant of IL-12B.

For example, it can be a variant of IL-12A having one or more substitutions relative to wild-type IL-12A selected from the group consisting of: N21D, Q35D, E38Q, D Q, D55K, N71D, N71Q, L75Q, L76Q, L79Q, L85Q, L85Q, L89Q, L96Q, L97Q, L124Q, L125Q, L130Q, L135Q, L143Q, L146Q, L151Q, L151Q, L153Q, L158Q, L163Q, L165Q, L171Q, L195D and N195Q. Optionally, a variant of IL-12A has one of the following combinations of substitutions: N71D/N85D/N195D, N D/E153Q, N D/D165N, Q E/N151D, N D/K158E, E Q/N151D, D Q/N151D, N136D/N151D, N D/N151D, E143Q/N151D, N Q/N85 6271Q/N195Q, N Q/N195Q, N Q/N85Q/N195Q, N D/N85D, N D/N195D and N85D/N195D.

For example, it can be a variant of IL-12B having one or more substitutions relative to wild-type IL-12B selected from the group consisting of: e59 59, 18, 32, 34, 43, 45, 73, 99, 103, 113, 161, 163, 200, 229, 235, 252, 256, 260, 262, 281Q, and E299Q. Optionally, a variant of IL-12B has one of the following combinations of substitutions: N103D/N113D/N200D/N281E/E45Q/Q56E/E59E/E45Q 56Q/Q56E/E59 QE 32Q/E59N/E59 34N/E59K/K99 34K/E59K/K99 32Q/D34N/E59K/K99 32K/D34N/E59K/K99N/E59Q/E187E/E59 43K/E49Q/K163Q/K59Q/K258Q/K260K 59/K99K/K18K/E59K/K99K/K264K 59K/K99Y/K99K 45K/E59K/K99E/Q144K 59K/K99E/Q144K/K99E/R159K 59K 99E/K264K 18K/E59K/K99E/K264 8K/E59K/K99E/C252S/K264K/K99Y/C252K 59K/K99E/C252S/K264K/K99E/C252 103D/N113D/N103D/N200D/N281 113D/N281 200D/N281 103D/N113D/N200D/N113D/N281 103D/N200D/N N281 113D/N200D/N281 103Q/N113Q/N200Q/N281 103Q/N113Q/N200Q/N113Q/N281 103Q/N200Q/N281 113Q/N200Q/N281 103Q/N113Q/N200Q/N281 59K/K99E/N103Q/C252S/K264E, E59K/K99E/N113Q/C252S/K E, E K/K99E/K200Q/C252S/K E, E K/K99E/N281Q/C252S/K264E, E K/K99E/N103Q/N113Q/C113S/K E, E K99E/N103Q/N200Q/C252S/K264E, E K/K99E/N103Q/N281Q/C252S/K E, E K/K99E/N113Q/N200Q/C252S/K264E, E K/K99E/N113Q/N281Q/C252S/K264E, E K/K99E/N200Q/N281Q/C252S/K264E, E K/K99E/N103Q/N113Q/N200Q/C252S/K264E, E K/K99E/N103Q/N200Q/N281Q/C252S/K264E, E K/K99E/N113Q/N200Q/N281Q/C252S/K264E and E59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

As referred to herein, "/" refers to accumulated substitutions. Thus, the mutation Q42E/E45Q means the following substitutions: Q42E and E45Q.

IL-15 variants

In a particular aspect, the immunostimulatory cytokine is a variant of IL-15.

The IL-15 variant preferably has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the wild type cytokine of SEQ ID NO. 88 or 1 to 10 modifications selected from the group consisting of additions, deletions, substitutions and combinations thereof relative to the sequence of SEQ ID NO. 88.

Optionally, the IL-15 variant may comprise one or more substitutions at the position of human IL-15 (NO signal peptide; SEQ ID NO: 88) selected from the group consisting of: N1D, V3I, V3M, V3R, N4D, D8N, D8A, K11L, K11M, K11R, D61R, D64R, D65R, D71R, D5271R, D72R, D72R, D72R, D73R, D77R, D79R, D5279R, D79R, D52112R, D112R, D112M and N112Y, N4R, D61R, D65D and Q108E are preferred. Optionally, the IL-15 variant may comprise one of the following combinations of substitutions relative to human IL-15 (NO signal peptide; SEQ ID NO: 88): N4D/N65 30N/N65 30N/E64Q/N65 1 4 8 30 61 64 65 108 1D/D61 1D/E64 4 4D/E64 8N/D61N/E64 1D/D30Q/Q108 1D/N4D/D8 61N/E64Q/N65 1D/D61N/E64Q/Q108 4D/D61N/E64Q/Q108 1D/N65D/Q108D 30N/Q108N/Q180Q 64Q/N65N/E64Q/N65D 1D/N4D/N65S 71/N72A/N77A and N4D/D61N/N65D, preferably D30N/E64Q/N65D.

As described herein, "/" refers to accumulated substitutions. Thus, the mutation N4D/N65D means the following substitutions: N4D and N65D.

IL-21 variants

In a particular aspect, the immunostimulatory cytokine is a variant of IL-21.

The IL-21 variant preferably has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the wild type cytokine of SEQ ID NO. 89 or 1 to 10 modifications selected from the group consisting of additions, deletions, substitutions and combinations thereof relative to the sequence of SEQ ID NO. 89.

Optionally, the IL-21 variant may comprise one of the following substitutions relative to human IL-21: r5 5 5 5 5 5 5 5 5 5 89 12 12 11 12 12 12 12 12 15 15 15 15 11 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 15 12 12 12 12 15 12 11 12 12 12 15 12 119 14 119N, 1119 119 120D and L123D, preferably R5E and R76 5A and R76 5Q and R76 5A and R76Q and R76 9E and R76A and R76N and S70 15N and I71 15N and K72 15N and K73T and K73T and R76 70T and R76 71L and K73L and R76 71L and R76A and K73A and R76D or K73A and R76E.

Antigen binding domains that specifically target immune cells

According to the invention, the antigen binding domain specifically binds to a target expressed on the surface of an immune cell, in particular a target expressed only or specifically on an immune cell. In particular, the antigen binding domain is not directed against a target expressed on a tumor cell.

With respect to the "binding" ability of an antigen binding domain, the term "bind" or "bind" refers to an antibody, including antibody fragments and derivatives, that recognizes and contacts another peptide, polypeptide, protein, or molecule. The terms "specifically bind," "specifically bind to," "specifically," "selectively bind," and "selectively" with respect to a particular target mean that the antigen binding domain recognizes and binds to the particular target, but does not substantially recognize or bind to other molecules in the sample. For example, an antibody that specifically (or preferentially) binds an antigen is, for example, an antibody that binds an antigen with greater affinity, avidity, easier and/or longer duration than it binds other molecules. Preferably, the term "specific binding" means that the binding between the antibody and the antigen is at or below 10 ^-7 Binding affinity contact of M. In certain aspects, the antibody is at or below 10 ^-8 M、10 ^-9 M or 10 ^-10 Affinity binding of M.

Optionally, the antigen binding domain may be a Fab domain, fab', single chain variable fragment (scFV), or single domain antibody (sdAb). The antigen binding domain preferably comprises a heavy chain variable region (VH) and a light chain variable region (VL).

When the antigen binding domain is a Fab or Fab', the bifunctional molecule comprises one heavy chain and one light chain constant domain (i.e. CH and CL), the heavy chain being linked at its C-terminal end to an immunostimulatory cytokine.

As used herein, the term "target" refers to a carbohydrate, lipid, peptide, polypeptide, protein, antigen or epitope that is specifically recognized or targeted by an antigen binding domain according to the invention and expressed on the outer surface of an immune cell. With respect to the expression of a target on the surface of an immune cell, the term "expressed" refers to the target, e.g., a carbohydrate, lipid, peptide, polypeptide, protein, antigen, or epitope present or presented on the outer surface of an immune cell. The term "specifically express" means that the target is expressed on immune cells, but that other cell types, in particular, for example, tumor cells, are not substantially expressed.

In one aspect, the target is specifically expressed by immune cells in a healthy subject or a subject suffering from a disease, particularly, for example, cancer. This means that the target has a higher expression level in immune cells than in other cells, or that the ratio of immune cells expressing the target to total immune cells is higher than the ratio of other cells expressing the target to total other cells. Preferably, the expression level or ratio is 2, 5, 10, 20, 50 or 100 times higher. More specifically, a specific type of immune cell, such as a T cell, more specifically a cd8+ T cell, an effector T cell, or a depleted T cell, or in a specific case, such as a subject suffering from a disease such as cancer or infection, can be determined.

As used herein, "immune cells" refers to cells involved in innate and adaptive immunity, such as leukocytes (white blood cells), lymphocytes (T cells, B cells, natural Killer (NK) cells and natural killer T cells (NKT), and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells) derived from Hematopoietic Stem Cells (HSCs) produced in the bone marrow.

Preferably, the antigen binding domain specifically binds to a target-expressing immune cell selected from the group consisting of B cells, T cells, natural killer cells, dendritic cells, monocytes and Innate Lymphocytes (ILC).

Even more preferably, the immune cells are T cells. As used herein, "T cell" or "T lymphocyte" includes, for example, cd4+ T cells, cd8+ T cells, T helper type 1T cells, T helper type 2T cells, T regulatory cells, T helper type 17T cells, and suppressor T cells. In a very specific aspect, the immune cells are depleted T cells.

In a particular aspect, the immune cell is an effector memory stem cell-like T cell.

The target may be a receptor expressed on the surface of an immune cell, in particular a T cell. The receptor may be an inhibitor receptor. Alternatively, the receptor may be an activating receptor.

In one aspect, the target is selected from: PD-1, CD28, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD, OX40, 4-1BB, GITR, HVEM, tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CD3, PDL1; PDL2 and PDL1. Such targets are described in more detail in table E below.

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Table e. Examples of targets of interest.

Then, in this aspect, the antigen binding domain specifically binds to a target selected from the group consisting of: PD-1, CD28, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD, OX40, 4-1BB, GITR, HVEM, tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CD3, PDL2 and PDL1.

In a particular aspect, the immune cells are depleted T cells or effector memory stem cell-like T cells, and the target of the antigen binding domain is a factor expressed on the surface of the depleted T cells or effector memory stem cell-like T cells. T cell depletion is a state of gradual loss of T cell function, proliferation capacity and cytotoxic potential, ultimately leading to its loss. T cell depletion may be triggered by a variety of factors, such as sustained antigen exposure or inhibitory receptors, including PD-1, TIM3, CD244, CTLA-4, LAG-3, BTLA, TIGIT, and CD160. Preferably, such factors, in particular such depletion factors, are selected from: PD-1, TIM3, CD244, CTLA-4, LAG3, BTLA, TIGIT and CD160.

In a preferred aspect, the antigen binding domain has antagonistic activity against a target.

Numerous antibodies to PD-1, TIM3, CD244, CTLA-4, LAG-3, BTLA, TIGIT and CD160 have been described in the art.

Several anti-PD-1 have been clinically approved and others are still in the clinical development stage. For example, the anti-PD 1 antibody may be selected from: pembrolizumab (also known as Keytruda lambrolizumab, MK-3475), nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538), pilgribulab (CT-011), cimip Li Shan antibody (Libtayo), cerilibulab, AUNP12, AMP-224, AGEN-2034, BGB-a317 (tirelimumab), PDR001 (stavallimab), MK-3477, SCH-900475, PF-06801591, JNJ-63723283, jerimab (CBT-501), LZM-009, BCD-100, SHR-1201, BAT-1306, 103 (HX-008), MEDI-0680 (also known as AMP-514) MEDI0608, JS001 (see Si-Yang Liu et al, j. Hemacol. 10: 2017)), CBT-754091, CBT-1210, inhr 1210 (also known as TSR-136), MK-3477, SCH-900475, PF-06801591, JNJ-63723283, jerimab (CBT-501), LZM-009, BCD-100, SHR-1201 (also known as being known as AMP-514), MEDI-0605, and WO-2015/2015 (see also known as WO-2015/2015, and 2015/2015, WO-2015, and (see also known as WO-2015/ltb/ltv). Bifunctional or bispecific molecules targeting PD-1 are also known, such as RG7769 (Roche), xmAb20717 (Xencor), MEDI5752 (AstraZeneca), FS118 (F-star), SL-279252 (Takeda) and XmAb23104 (Xencor).

In particular aspects, the anti-PD 1 antibody may be pembrolizumab (also known as Keytruda lambrolizumab, MK-3475) or nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538).

Antibodies to TIM3 and bifunctional or bispecific molecules targeting TIM3 are also known, such as Sym023, TSR-022, MBG453, LY3321367, INCAGN02390, BGTB-a425, LY3321367, RG7769 (Roche). In some aspects, the TFM-3 antibody is as disclosed in: international patent application publication nos. WO2013006490, WO2016/161270, WO 2018/085469 or WO 2018/129553, WO 2011/155607, u.s.8,552,156, EP 2581113 and u.s.2014/044728.

Antibodies directed against CTLA-4 and bifunctional or bispecific molecules targeting CTLA-4 are also known, such as ipilimumab (ipilimumab), tremelimumab, MK-1308, AGEN-1884, xmAb20717 (Xencor), MEDI5752 (AstraZeneca). anti-CTLA-4 antibodies are also disclosed in WO18025178, WO19179388, WO19179391, WO19174603, WO19148444, WO19120232, WO19056281, WO19023482, WO18209701, WO18165895, WO18160536, WO18156250, WO18106862, WO18106864, WO18068182, WO18035710, WO18025178, W017194265, WO17106372, WO17084078, WO17087588, WO16196237, WO16130898, WO16015675, WO12120125, WO09100140 and WO07008463.

Antibodies against LAG3 and bifunctional or bispecific molecules targeting LAG-3 are also known, such as BMS-986016, IMP701, MGD012 or MGD013 (bispecific PD-1 and LAG-3 antibodies). anti-LAG-3 antibodies are also disclosed in WO2008132601, EP2320940, WO 19152574.

Antibodies to BTLA are also known in the art, for example, hu Mab8D5, hu Mab8A3, hu Mab21H6, hu Mab19A7 or hu Mab4C7. Antibodies TAB004 against BTLA are currently undergoing clinical trials in subjects with advanced malignant tumors. anti-BTLA antibodies are also disclosed in WO08076560, WO10106051 (e.g., BTLA 8.2), WO11014438 (e.g., 4C 7), WO17096017, and WO17144668 (e.g., 629.3).

Antibodies to TIGIT are also known in the art as, for example, cha 9.086, cha 9.547.18, cpa 9.018, cpa 9.027, cpa 9.049, cpa 9.057, cpa 9.059, cpa 9.083, cpa 9.089, cpa 9.093, cpa 9.101, cpa 9.103, cha 9.536.1, cha 9.536.3, cha 9.536.4, cha 9.536.5, cha 9.536.6, cha 9.536.7, cha 9.536.8, cha 9.560.3, cha 9.560.4, cha 9.560.5, cha 9.560.560.560.5, cha 9.560.5, cha 6.536.1, cha 9.536.3, cha 4, cha 9.541.7.5, cha 9.541.7.1, cha 9.541.6, cha 9.5.541.7.5, cha 9.5, cha 9.541.7.7.5, and cha 9.541.7.7.1, cha 9.541.7.5, and cha 9.7.5.541.7.7.1. anti-TIGIT antibodies are also disclosed in WO16028656, WO16106302, WO16191643, WO17030823, WO17037707, WO17053748, WO17152088, WO18033798, WO18102536, WO18102746, WO18160704, W018200430, WO18204363, W019023504, WO19062832, W019129221, W019129261, W019137548, W019152574, W019154415, W019168382 and W019215728.

Antibodies against CD160 are also known in the art, for example CL1-R2 CNCM 1-3204 as disclosed in WO06015886, or other antibodies as disclosed in WO10006071, WO10084158, WO 18077926.

Antibodies to PD-L1 are also known in the art. Examples of monoclonal antibodies that bind human PD-L1 and that can be used in the present invention are described in WO 2007/005874, WO 2010/036959, WO 2010/077634, WO 2010/089411, WO 2013/019906, WO 2013/079174, WO 2014/100079, WO 2015/061668 and US 8,552,154, US 8,779,108 and US 8,383,796. Specific anti-human PD-L1 monoclonal antibodies include, for example, but are not limited to, avermectin (MSB 0010718C), divaruzumab (MEDI 4736), engineered IgGl kappa monoclonal antibodies with triple mutations in the Fc domain to remove ADCC), alemtuzumab (MPLDL 3280A), MPDL3280A (IgG 1 engineered anti-PD-L1 antibody), and BMS-936559 (fully human, anti-PD-L1, igG4 monoclonal antibodies).

In a preferred aspect, the antigen binding domain of the bifunctional molecule is an antibody, fragment or derivative thereof specific for PD-1, CTLA-4, BTLA, TIGIT, LAG3 and TIM 3.

In another specific aspect, the target is PD-1 and the antigen binding domain of the bifunctional molecule is an antibody, fragment or derivative thereof, or an antibody mimetic specific for PD-1. Then, in a specific aspect, the antigen binding domain comprised in the bifunctional molecule according to the invention is an anti-PD 1 antibody or an antigen binding fragment thereof, preferably a human, humanized or chimeric anti-PD 1 antibody or an antigen binding fragment thereof. Preferably, the antigen binding domain is an antagonist of PD-1.

In another particular aspect, the target is CTLA-4 and the antigen-binding domain of the bifunctional molecule is an antibody, fragment or derivative thereof, or antibody mimetic specific for CTLA-4. Then, in a specific aspect, the antigen binding domain comprised in the bifunctional molecule according to the invention is an anti-CTLA-4 antibody or antigen-binding fragment thereof, preferably a human, humanized or chimeric anti-CTLA-4 antibody or antigen-binding fragment thereof. Preferably, the antigen binding domain is an antagonist of CTLA-4.

In another specific aspect, the target is BTLA and the antigen binding domain of the bifunctional molecule is an antibody, fragment or derivative thereof or an antibody mimetic specific for BTLA. Then, in a specific aspect, the antigen binding domain comprised in the bifunctional molecule according to the invention is an anti-BTLA antibody or an antigen binding fragment thereof, preferably a human, humanized or chimeric anti-BTLA antibody or an antigen binding fragment thereof. Preferably, the antigen binding domain is an antagonist of BTLA.

In another particular aspect, the target is TIGIT and the antigen binding domain of the bifunctional molecule is an antibody, fragment or derivative thereof, or an antibody mimetic specific for TIGIT. Then, in a specific aspect, the antigen binding domain comprised in the bifunctional molecule according to the invention is an anti-TIGIT antibody or an antigen binding fragment thereof, preferably a human, humanized or chimeric anti-TIGIT antibody or an antigen binding fragment thereof. Preferably, the antigen binding domain is an antagonist of TIGIT.

In another specific aspect, the target is LAG-3 and the antigen-binding domain of the bifunctional molecule is an antibody, fragment or derivative thereof, or antibody mimetic specific for LAG-3. Then, in a specific aspect, the antigen binding domain comprised in the bifunctional molecule according to the invention is an anti-LAG-3 antibody or an antigen binding fragment thereof, preferably a human, humanized or chimeric anti-LAG-3 antibody or an antigen binding fragment thereof. Preferably, the antigen binding domain is an antagonist of LAG-3.

In another specific aspect, the target is TIM3 and the antigen binding domain of the bifunctional molecule is an antibody, fragment or derivative thereof, or antibody mimetic specific for TIM 3. Then, in a specific aspect, the antigen binding domain comprised in the bifunctional molecule according to the invention is an anti-TIM 3 antibody or an antigen binding fragment thereof, preferably a human, humanized or chimeric anti-TIM 3 antibody or an antigen binding fragment thereof. Preferably, the antigen binding domain is an antagonist of TIM 3.

In a very specific aspect of the present disclosure, the antigen binding domain targets PD-1 and is derived from an antibody disclosed in WO2020/127366 (the disclosure of which is incorporated herein by reference).

The antigen binding domain then comprises:

(i) Heavy chain variable domains comprising HCDR1, HCDR2 and HCDR3

(ii) Light chain variable domains comprising LCDR1, LCDR2 and LCDR3,

wherein:

-heavy chain CDR1 (HCDR 1) comprises or consists of the amino acid sequence of SEQ ID NO:51, optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof at any position of SEQ ID NO:51 other than position 3;

-heavy chain CDR2 (HCDR 2) comprises or consists of the amino acid sequence of SEQ ID NO:53, optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof at any position of SEQ ID NO:53 except positions 13, 14 and 16;

-heavy chain CDR3 (HCDR 3) comprises or consists of the amino acid sequence of SEQ ID No. 54 wherein X1 is D or E and X2 is selected from T, H, A, Y, N, E and S, preferably from H, A, Y, N, E; optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof at any position of SEQ ID NO. 54 except positions 2, 3, 7 and 8;

-light chain CDR1 (LCDR 1) comprises or consists of the amino acid sequence of SEQ ID No. 63 wherein X is G or T optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof at any position of SEQ ID No. 63 except positions 5, 6, 10, 11 and 16;

-light chain CDR2 (LCDR 2) comprises or consists of the amino acid sequence of SEQ ID No. 66, optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof; and

light chain CDR3 (LCDR 3) comprises or consists of the amino acid sequence of SEQ ID NO:16, optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof at any position of SEQ ID NO:16 except positions 1, 4 and 6.

In one aspect, the antigen binding domain comprises:

(i) Heavy chain variable domains comprising HCDR1, HCDR2 and HCDR3

(ii) Light chain variable domains comprising LCDR1, LCDR2 and LCDR3,

wherein:

-heavy chain CDR3 (HCDR 3) comprises or consists of the amino acid sequence of SEQ ID No. 54 wherein X1 is D and X2 is selected from T, H, A, Y, N, E and S, preferably from H, A, Y, N, E; or X1 is E and X2 is selected from T, H, A, Y, N, E and S, preferably from H, A, Y, N, E and S; optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof at any position of SEQ ID NO. 54 except positions 2, 3, 7 and 8;

In another embodiment, the antigen binding domain comprises or consists essentially of: (i) A heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53 and CDR3 of SEQ ID NO:55, 56, 57, 58, 59, 60, 61 or 62; and (ii) a light chain comprising CDR1 of SEQ ID NO. 64 or SEQ ID NO. 65, CDR2 of SEQ ID NO. 66 and CDR3 of SEQ ID NO. 16.

In another aspect, the antigen binding domain comprises or consists essentially of:

(i) A heavy chain comprising CDR1 of SEQ ID NO. 51, CDR2 of SEQ ID NO. 53 and CDR3 of SEQ ID NO. 55; and (ii) a light chain comprising CDR1 of SEQ ID NO. 64, CDR2 of SEQ ID NO. 66 and CDR3 of SEQ ID NO. 16; or alternatively

(i) A heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53 and CDR3 of SEQ ID NO: 56; and (ii) a light chain comprising CDR1 of SEQ ID NO:64, CDR2 of SEQ ID NO:66 and CDR3 of SEQ ID NO:16, or

(i) A heavy chain comprising CDR1 of SEQ ID NO. 51, CDR2 of SEQ ID NO. 53 and CDR3 of SEQ ID NO. 57; and (ii) a light chain comprising CDR1 of SEQ ID NO. 64, CDR2 of SEQ ID NO. 66 and CDR3 of SEQ ID NO. 16; or alternatively

(i) A heavy chain comprising CDR1 of SEQ ID NO. 51, CDR2 of SEQ ID NO. 53 and CDR3 of SEQ ID NO. 58; and (ii) a light chain comprising CDR1 of SEQ ID NO. 64, CDR2 of SEQ ID NO. 66 and CDR3 of SEQ ID NO. 16; or alternatively

(i) A heavy chain comprising CDR1 of SEQ ID NO. 51, CDR2 of SEQ ID NO. 53 and CDR3 of SEQ ID NO. 59; and (ii) a light chain comprising CDR1 of SEQ ID NO. 64, CDR2 of SEQ ID NO. 66 and CDR3 of SEQ ID NO. 16; or alternatively

(i) A heavy chain comprising CDR1 of SEQ ID NO. 51, CDR2 of SEQ ID NO. 53 and CDR3 of SEQ ID NO. 60; and (ii) a light chain comprising CDR1 of SEQ ID NO. 64, CDR2 of SEQ ID NO. 66 and CDR3 of SEQ ID NO. 16; or alternatively

(i) A heavy chain comprising CDR1 of SEQ ID NO. 51, CDR2 of SEQ ID NO. 53 and CDR3 of SEQ ID NO. 61; and (ii) a light chain comprising CDR1 of SEQ ID NO. 64, CDR2 of SEQ ID NO. 66 and CDR3 of SEQ ID NO. 16; or alternatively

(i) A heavy chain comprising CDR1 of SEQ ID NO. 51, CDR2 of SEQ ID NO. 53 and CDR3 of SEQ ID NO. 62; and (ii) a light chain comprising CDR1 of SEQ ID NO:64, CDR2 of SEQ ID NO:66 and CDR3 of SEQ ID NO:16, or

(i) A heavy chain comprising CDR1 of SEQ ID NO. 51, CDR2 of SEQ ID NO. 53 and CDR3 of SEQ ID NO. 55; and (ii) a light chain comprising CDR1 of SEQ ID NO. 65, CDR2 of SEQ ID NO. 66 and CDR3 of SEQ ID NO. 16; or alternatively

(i) A heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53 and CDR3 of SEQ ID NO: 56; and (ii) a light chain comprising CDR1 of SEQ ID NO. 65, CDR2 of SEQ ID NO. 66 and CDR3 of SEQ ID NO. 16; or alternatively

(i) A heavy chain comprising CDR1 of SEQ ID NO. 51, CDR2 of SEQ ID NO. 53 and CDR3 of SEQ ID NO. 57; and (ii) a light chain comprising CDR1 of SEQ ID NO. 65, CDR2 of SEQ ID NO. 66 and CDR3 of SEQ ID NO. 16; or alternatively

(i) A heavy chain comprising CDR1 of SEQ ID NO. 51, CDR2 of SEQ ID NO. 53 and CDR3 of SEQ ID NO. 58; and (ii) a light chain comprising CDR1 of SEQ ID NO. 65, CDR2 of SEQ ID NO. 66 and CDR3 of SEQ ID NO. 16; or alternatively

(i) A heavy chain comprising CDR1 of SEQ ID NO. 51, CDR2 of SEQ ID NO. 53 and CDR3 of SEQ ID NO. 59; and (ii) a light chain comprising CDR1 of SEQ ID NO. 65, CDR2 of SEQ ID NO. 66 and CDR3 of SEQ ID NO. 16; or alternatively

(i) A heavy chain comprising CDR1 of SEQ ID NO. 51, CDR2 of SEQ ID NO. 53 and CDR3 of SEQ ID NO. 60; and (ii) a light chain comprising CDR1 of SEQ ID NO. 65, CDR2 of SEQ ID NO. 66 and CDR3 of SEQ ID NO. 16; or alternatively

(i) A heavy chain comprising CDR1 of SEQ ID NO. 51, CDR2 of SEQ ID NO. 53 and CDR3 of SEQ ID NO. 61; and (ii) a light chain comprising CDR1 of SEQ ID NO. 65, CDR2 of SEQ ID NO. 66 and CDR3 of SEQ ID NO. 16; or alternatively

(i) A heavy chain comprising CDR1 of SEQ ID NO. 51, CDR2 of SEQ ID NO. 53 and CDR3 of SEQ ID NO. 62; and (ii) a light chain comprising CDR1 of SEQ ID NO:65, CDR2 of SEQ ID NO:66 and CDR3 of SEQ ID NO: 16.

In one aspect, the antigen binding domain comprises or consists essentially of:

(a) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID No. 17, wherein X1 is D or E and X2 is selected from T, H, A, Y, N, E and S, preferably from H, A, Y, N, E; optionally having one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof at any position of SEQ ID No. 17 except positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112;

(b) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID No. 26, wherein X is G or T, optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof at any position of SEQ ID No. 26 except positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105.

(a) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:18, 19, 20, 21, 22, 23, 24 or 25, optionally having one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof at any position of SEQ ID NO:18, 19, 20, 21, 22, 23, 24 or 25 except positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112, respectively.

(b) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID No. 27 or SEQ ID No. 28, optionally having one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof at any position of SEQ ID No. 27 or SEQ ID No. 28 other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105.

(a) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO 18, 19, 20, 21, 22, 23, 24 or 25;

(b) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID No. 27 or SEQ ID No. 28.

In one aspect, the bifunctional molecule comprises a framework region, in particular heavy chain variable region framework regions (HFR) HFR1, HFR2, HFR3 and HFR4, and light chain variable region framework regions (LFR) LFR1, LFR2, LFR3 and LFR4, in particular HFR1, HFR2, HFR3 and HFR4, comprise the amino acid sequences of SEQ ID NOs 41, 42, 43 and 44, respectively, optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof at any position of HFR3, i.e. SEQ ID NO 43, except positions 27, 29 and 32. Preferably, the bifunctional molecule comprises HFR1 of SEQ ID NO. 41, HFR2 of SEQ ID NO. 42, HFR3 of SEQ ID NO. 43 and HFR4 of SEQ ID NO. 44. In addition, the bifunctional molecule may comprise the amino acid sequences of light chain variable region framework regions (LFR) LFR1, LFR2, LFR3 and LFR4 comprising SEQ ID NOs 45, 46, 47 and 48, respectively, optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof. Preferably, the bifunctional molecule comprises LFR1 of SEQ ID NO. 45, LFR2 of SEQ ID NO. 46, LFR3 of SEQ ID NO. 47 and LFR4 of SEQ ID NO. 48.

In another aspect, the antigen binding domain comprises or consists essentially of any combination of the following heavy chain variable region (VH) and light chain variable region (VL):

in very specific aspects, the antigen binding domain comprises or consists essentially of: the heavy chain variable region (VH) of SEQ ID NO. 24 and the light chain variable region (VL) of SEQ ID NO. 28.

Preferred combinations

Specific combinations of targets and immunostimulatory cytokines specifically expressed on the surface of immune cells are contemplated herein.

In a first aspect, the bifunctional molecule comprises an antigen binding domain that binds to (and preferably antagonizes) PD-1 and an immunostimulatory cytokine selected from the group consisting of IL-2, IL-12, IL-15, IL-21, and variants thereof, and IL-7. Optionally, the bifunctional molecule comprises an antigen binding domain that binds (and preferably antagonizes) PD-1 and an immunostimulatory cytokine selected from the group consisting of IL-12, IL-15, IL-21, and variants thereof, and IL-7. Optionally, the bifunctional molecule comprises one of the following combinations: a) An antigen binding domain that binds to PD-1 and IL-2 or variants thereof; b) Antigen binding domains that bind PD-1 and IL-7; c) An antigen binding domain that binds to PD-1 and IL-12 or variants thereof; d) An antigen binding domain that binds to PD-1 and IL-15 or variants thereof; e) An antigen binding domain that binds to PD-1 and IL-15 or variants thereof; or f) an antigen binding domain that binds PD-1 and IL-21 or variants thereof.

In a second aspect, the bifunctional molecule comprises an antigen binding domain that binds (and preferably antagonizes) PD-L1 and an immunostimulatory cytokine selected from the group consisting of IL-2, IL-12, IL-15, IL-21, and variants thereof, and IL-7. Optionally, the bifunctional molecule comprises one of the following combinations: a) An antigen binding domain that binds PD-L1 and IL-2 or variants thereof; b) An antigen binding domain that binds to PD-L1 and IL-7; c) An antigen binding domain that binds PD-L1 and IL-12 or variants thereof; d) An antigen binding domain that binds PD-L1 and IL-15 or variants thereof; e) An antigen binding domain that binds to PD-1 and IL-15 or variants thereof; or f) an antigen binding domain that binds PD-L1 and IL-21 or variants thereof.

In a third aspect, the bifunctional molecule comprises an antigen binding domain that binds (and preferably antagonizes) PD-L2 and an immunostimulatory cytokine selected from the group consisting of IL-2, IL-12, IL-15, IL-21, and variants thereof, and IL-7. Optionally, the bifunctional molecule comprises one of the following combinations: a) An antigen binding domain that binds PD-L2 and IL-2 or variants thereof; b) An antigen binding domain that binds PD-L2 and IL-7; c) An antigen binding domain that binds PD-L2 and IL-12 or variants thereof; d) An antigen binding domain that binds PD-L2 and IL-15 or variants thereof; e) An antigen binding domain that binds PD-L2 and IL-15 or variants thereof; or f) an antigen binding domain that binds PD-L2 and IL-21 or variants thereof.

In a fourth aspect, the bifunctional molecule comprises an antigen binding domain that binds (and preferably antagonizes) CTLA-4 and an immunostimulatory cytokine selected from the group consisting of IL-2, IL-12, IL-15, IL-21 and variants thereof, and IL-7. Optionally, the bifunctional molecule comprises one of the following combinations: a) An antigen binding domain that binds CTLA-4 and IL-2 or variants thereof; b) Antigen binding domains that bind CTLA-4 and IL-7; c) An antigen binding domain that binds CTLA-4 and IL-12 or variants thereof; d) An antigen binding domain that binds CTLA-4 and IL-15 or variants thereof; e) An antigen binding domain that binds CTLA-4 and IL-15 or variants thereof; or f) an antigen binding domain that binds CTLA-4 and IL-21 or variants thereof.

In a fifth aspect, the bifunctional molecule comprises an antigen binding domain that binds to (and preferably antagonizes) TIM3 and an immunostimulatory cytokine selected from the group consisting of IL-2, IL-12, IL-15, IL-21 and variants thereof, and IL-7. Optionally, the bifunctional molecule comprises one of the following combinations: a) An antigen binding domain that binds TIM3 and IL-2 or variants thereof; b) Antigen binding domains that bind TIM3 and IL-7; c) An antigen binding domain that binds TIM3 and IL-12 or variants thereof; d) An antigen binding domain that binds TIM3 and IL-15 or variants thereof; e) An antigen binding domain that binds to PD-1 and IL-15 or variants thereof; or f) an antigen binding domain that binds TIM3 and IL-21 or variants thereof.

Peptide linker

In a particular aspect, the bifunctional molecule according to the invention further comprises a peptide linker linking the antigen binding domain and the immunostimulatory cytokine to the Fc chain. Peptide linkers are typically of sufficient length and flexibility to ensure that the antigen binding domains of the immunostimulatory cytokine and the linker linkage therebetween have sufficient spatial freedom to function.

In one aspect of the disclosure, the immunostimulatory cytokine is preferably linked to the Fc chain by a peptide linker. In one aspect of the disclosure, the antigen binding domain may be linked to the Fc chain by a naturally occurring hinge in the heavy chain for linking the VH domain, particularly the CH 1 domain, to the CH2 domain of the Fc chain.

As used herein, the term "linker" refers to a sequence of at least one amino acid. Such linkers may be used to prevent steric hindrance. The length of the linker is typically 3-44 amino acid residues. Preferably, the linker has 3-30 amino acid residues. In some aspects, the linker has 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues.

The linker sequence may be a naturally occurring sequence or a non-naturally occurring sequence. If used for therapeutic purposes, the linker is preferably non-immunogenic in the subject to whom the bifunctional molecule is administered. One useful set of linker sequences are those derived from the hinge region of a heavy chain antibody as described in WO 96/34103 and WO 94/04678. Other examples are polyalanine linker sequences. Further preferred examples of linker sequences are Gly/Ser linkers of different lengths, including (Gly 4 Ser) 4, (Gly 4 Ser) 3, (Gly 4 Ser) 2, gly4Ser, gly3, gly2Ser and (Gly 3Ser 2) 3, in particular (Gly 4 Ser) 3. Preferably, the linker is selected from: (Gly 4 Ser) 4, (Gly 4 Ser) 3 and (Gly 3Ser 2) 3. Even more preferably, the linker is (GGGGS) 3.

In one embodiment, the linker comprised in the bifunctional molecule is selected from the group consisting of: (Gly 4 Ser) 4, (Gly 4 Ser) 3, (Gly 4 Ser) 2, gly4Ser, gly3, gly2Ser and (Gly 3Ser 2) 3, preferably (Gly 4 Ser) 3. Preferably, the linker is selected from: (Gly 4 Ser) 4, (Gly 4 Ser) 3 and (Gly 3Ser 2) 3.

In particular embodiments, the linker comprised in the bifunctional molecule is selected from the group consisting of: a linker having a sequence as set forth in SEQ ID NO 67, 68, 69 or 70.

Fc domain

The Fc domain of the bifunctional molecule may be part of an antigen binding moiety, in particular the heavy chain of an IgG immunoglobulin. In fact, when the antigen binding domain is a Fab, the bifunctional molecule may comprise one heavy chain, including the variable heavy chain (VH), CH1, hinge, CH2 and CH3 domains. However, the bifunctional molecule may also have other structures, such as scFv or diabodies. For example, it may comprise an Fc domain such as linked to an antibody derivative.

The Fc domain may be derived from a heavy chain constant domain of a human immunoglobulin heavy chain, such as IgG1, igG2, igG3, igG4, or other class. Preferably, the bifunctional molecule comprises an IgG1 or IgG4 heavy chain constant domain.

Preferably, the Fc domain comprises CH2 and CH3 domains. Optionally, it may comprise all or part of the hinge region, CH2 domain and/or CH3 domain. In some aspects, the CH2 and/or CH3 domains are derived from a human IgG4 or IgG1 heavy chain. Preferably, the Fc domain comprises all or part of the hinge region. The hinge region may be derived from an immunoglobulin heavy chain, such as IgGl, igG2, igG3, igG4, or other classes. Preferably, the hinge region is derived from human IgG1, igG2, igG3, igG4. More preferably, the hinge region is derived from a human or humanized IgG1 or IgG4 heavy chain.

The IgG1 hinge region has three cysteines, two of which are involved in disulfide bonds between the two heavy chains of the immunoglobulin. These same cysteines allow efficient and consistent disulfide bond formation between Fc portions. Thus, preferred hinge regions of the invention are derived from IgG1, more preferably from human IgG1. In some aspects, the first cysteine within the hinge region of human IgG1 is mutated to another amino acid, preferably serine.

The hinge region of IgG4 is known to be ineffective in forming interchain disulfide bonds. However, suitable hinge regions for use in the present invention may be derived from an IgG4 hinge region, preferably containing mutations that enhance the correct formation of disulfide bonds between the heavy chain derived moieties (Angal S, et al (1993) mol. Immunol., 30:105-8). More preferably, the hinge region is derived from a human IgG4 heavy chain.

The bifunctional molecule comprises a dimeric Fc domain. Thus, the two monomers each comprise an Fc chain capable of forming a dimeric Fc domain. The dimeric Fc domain may be homodimeric, with each Fc monomer being identical or substantially identical. Alternatively, the dimeric Fc domain may be a heterodimer, with each Fc monomer being different and complementary to promote formation of the heterodimeric Fc domain.

More specifically, the Fc domain is a heterodimeric Fc domain. Heterodimeric Fc domains are formed by altering the amino acid sequence of each monomer. Heterodimeric Fc domains rely on amino acid variants in different constant regions on each chain to promote heterodimer formation and/or allow simplification of heterodimer purification compared to homodimers. There are many mechanisms available for producing the heterodimers of the present invention. Furthermore, as will be appreciated by those skilled in the art, these mechanisms may be combined to ensure a high degree of heterodimerization. Thus, amino acid variants that result in heterodimer production are referred to as "heterodimeric variants". Heterodimerization variants may include steric variants (e.g., "convex and pore" or "inclined" variants described below and "charge pair" variants described below) as well as "pi variants" that allow for purification of homodimers from heterodimers. WO2014/145806, incorporated herein by reference in its entirety, discloses useful mechanisms of heterodimerization including "knob and socket", "electrostatic manipulation" or "charge pair", pi variants and generally additional Fc variants. See also Ridgway et al Protein Engineering (7): 617 (1996); atwell et al, J.mol.biol.1997270:26; U.S. Pat. No. 8,216,805 to Merchant et al, nature Biotech.16:677 (1998), all of which are incorporated herein by reference in their entirety. For "electrostatic manipulation," please see Gunasekaran et al, J.biol. Chem.285 (25): 19637 (2010), incorporated herein by reference in its entirety. For pi variants, see US2012/0149876, incorporated herein by reference in its entirety.

Then, in a preferred aspect, the heterodimeric Fc domain comprises a first Fc chain and a complementary second Fc chain based on a "knob and socket" technique. For example, a first Fc chain is a "pestle" or K chain, meaning that it comprises a substitution that characterizes a pestle chain, and a second Fc chain is a "mortar" or H chain, meaning that it comprises a substitution that characterizes a mortar chain. Vice versa, the first Fc chain is a "mortar" or H chain, meaning that it comprises a substitution that characterizes a mortar chain, and the second Fc chain is a "pestle" or K chain, meaning that it comprises a substitution that characterizes a pestle chain. In a preferred aspect, the first Fc chain is a "mortar" or H chain and the second Fc chain is a "pestle" or K chain.

Optionally, the heterodimeric Fc domain may comprise one heterodimeric Fc chain comprising the substitutions shown in table F below and other heterodimeric Fc chains comprising the substitutions shown in table F below.

Table F (numbering according to EU index)

In a preferred aspect, the first Fc chain is a "mortar" or H chain and comprises the substitution T366S/L368A/Y407V/Y349C and the second Fc chain is a "pestle" or K chain and comprises the substitution T366W/S354C.

Optionally, the Fc chain may further comprise additional substitutions.

In particular, for bifunctional molecules that target cell surface molecules (especially those on immune cells), it may be desirable to remove effector functions. It may also be desirable to engineer the Fc region to reduce or increase effector function of the bifunctional molecule.

In certain aspects, amino acid modifications can be introduced into the Fc region to produce Fc region variants. In certain aspects, the Fc region variant possesses some, but not all, effector functions. Such bifunctional molecules may be useful, for example, in applications where the in vivo half-life of the antibody is important but some effector functions are unnecessary or detrimental. Many substitutions or deletions with altered effector functions are known in the art.

In one aspect, the constant region of the Fc domain contains a mutation that reduces affinity for Fc receptors or reduces Fc effector function. For example, the constant region may contain mutations that eliminate glycosylation sites within the IgG heavy chain constant region. Preferably, the CH2 domain contains a mutation that eliminates the glycosylation site within the CH2 domain.

In a particular aspect, the Fc domain is modified to increase binding to FcRn, thereby increasing the half-life of the bifunctional molecule. In another or further aspect, the Fc domain is modified to reduce binding to fcγr, thereby reducing ADCC or CDC, or to increase binding to fcγr, thereby increasing ADCC or CDC.

Amino acid changes near the junction of the Fc portion and the non-Fc portion can significantly increase the serum half-life of the Fc fusion protein, as shown in WO 01/58957. Thus, the junction region of a protein or polypeptide of the invention may contain alterations preferably within about 10 amino acids of the junction point relative to the naturally occurring sequences of immunoglobulin heavy chains and erythropoietin. These amino acid changes can result in increased hydrophobicity. In one embodiment, the constant region is derived from an IgG sequence in which the C-terminal lysine residue is substituted. Preferably, the C-terminal lysine of the IgG sequence is substituted with a non-lysine amino acid, such as alanine or leucine, to further increase serum half-life.

In one embodiment, the constant region of the Fc domain has one or any combination of the mutations described in table G below.

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Table G: the Fc domain of an antibody is suitably engineered. Residue Numbering in the heavy chain constant region is carried out according to EU Numbering (Edelman, G.M. et al, proc. Natl. Acad. USA,63,78-85 (1969); www.imgt.org/IMGT scientific Chart/number/Hu_IGHGnber. Html#refs)

In a particular aspect, the bifunctional molecule comprises a human IgGl heavy chain constant domain or IgGl Fc domain, optionally with a substitution selected from or a combination of substitutions selected from: T250Q/M428L; M252Y/S254T/T256 E+H2433K/N434F; E233P/L234V/L235A/G236A+A327G/A330S/P331S; E333A; S239D/A330L/I332E; P257I/Q311; K326W/E333S; S239D/I332E/G236A; N297A; L234A/L235A; P329G; n297A+M252Y/S254T/T256E; K322A and K444A are preferably selected from: N297A optionally in combination with M252Y/S254T/T256E, and L234A/L235A optionally in combination with P329G.

Bifunctional molecules comprising a human IgG1 heavy chain constant domain or an IgG1 Fc domain with a substituted L234A/L235A/P329G combination greatly reduce or completely inhibit ADCC, ADCP and/or CDC caused by the bifunctional molecule, thereby reducing non-specific cytotoxicity.

In another aspect, the bifunctional molecule comprises a human IgG4 heavy chain constant domain or a human IgG4 Fc domain, optionally with a substitution selected from the following or a combination of substitutions selected from the following: S228P; L234A/L235A; L234A/L235A/P329G, P G, S P+M252Y/S254T/T256E, K444A K444E, K444D, K444G and K444A. Even more preferably, the bifunctional molecule, preferably the binding moiety, comprises an IgG4 Fc region with S228P stabilizing IgG 4.

As referred to herein "/" and "+" refer to accumulated mutations. Thus, the mutation S228P+M252Y/S254T/T256E means the following mutation: S228P, M252Y, S T and T256E.

Bifunctional molecules comprising a human IgG4 heavy chain constant domain or an IgG4 Fc domain with substitution P329G reduce ADCC and/or CDC caused by the bifunctional molecules, thereby reducing non-specific cytotoxicity.

All subclasses of human IgG carry the C-terminal lysine residue of the antibody heavy chain (K444), which is easily cleaved in the circulation. This cleavage in the blood can impair or reduce the biological activity of the bifunctional molecule by releasing the immunostimulatory cytokine linked to the bifunctional molecule. To address this problem, the K444 amino acid in the IgG domain may be substituted with alanine to reduce proteolytic cleavage, a mutation commonly used for antibodies. Then, in one aspect, the bifunctional molecule comprises at least one further amino acid substitution consisting of K444A, K444E, K D, K G or K444S, preferably K444A.

Optionally, the bifunctional molecule comprises additional cysteine residues at the C-terminal domain of the Fc domain to create additional disulfide bonds and potentially limit the flexibility of the bifunctional molecule.

In one aspect, the bifunctional molecule comprises one heavy chain constant domain of SEQ ID NO:39 or 52 and/or one light chain constant domain of SEQ ID NO:40, in particular one heavy chain constant domain or Fc domain of SEQ ID NO:39 or 52 and one light chain constant domain of SEQ ID NO:40, in particular as disclosed in Table H below.

Examples of heavy chain constant domains and light chain constant domains suitable for the bifunctional molecules according to the invention.

In a particular aspect, the bifunctional molecules according to the invention comprise heterodimers of Fc domains comprising a "knob and hole" modification such as described above. Preferably, such Fc domain is an IgG1 or IgG4 Fc domain such as described above, even more preferably an IgG1 Fc domain comprising the mutation N297A as disclosed above.

For example, the first Fc chain is a "mortar" or H chain and comprises the substitutions T366S/L368A/Y407V/Y349C and optionally N297A, and the second Fc chain is a "pestle" or K chain and comprises the substitutions T366W/S354C and optionally N297A. Preferably, the first Fc chain is a "mortar" or H chain and comprises substitutions T366S/L368A/Y407V/Y349C and N297A, and the second Fc chain is a "pestle" or K chain and comprises substitutions T366W/S354C and N297A. More specifically, the second Fc chain may comprise or consist of SEQ ID NO. 75 and/or the first Fc chain may comprise or consist of SEQ ID NO. 77.

More specifically, the immunostimulatory cytokines according to the invention are linked to the pestle and/or mortar chain of the heterodimeric Fc domain. Thus, a bifunctional molecule according to the invention may comprise a single immunostimulatory cytokine linked to the mortar or pestle chain of an Fc domain. Preferably, the bifunctional molecule according to the invention comprises a single immunostimulatory cytokine linked to the mortar chain of the Fc domain.

In a first aspect, the bifunctional molecule comprises an immunostimulatory cytokine linked to the C-terminus of the pestle chain of an Fc domain, such a pestle chain of an Fc domain being linked to an antigen binding domain.

In a second aspect, the bifunctional molecule comprises an immunostimulatory cytokine linked to the C-terminus of the mortar chain of an Fc domain, the mortar chain of such Fc domain being linked to the antigen binding domain of its N-terminus.

Optionally, the bifunctional molecule comprises a single immunostimulatory cytokine linked to the C-terminus of the mortar chain of the Fc domain, wherein the bifunctional molecule comprises only a single antigen binding domain linked to the N-terminus of the mortar chain of the Fc domain. In such aspects, the pestle chain domain lacks an immunostimulatory cytokine and an antigen binding domain.

Optionally, the bifunctional molecule comprises a single immunostimulatory cytokine linked to the C-terminus of the pestle chain of the Fc domain, wherein the bifunctional molecule comprises only a single antigen binding domain linked to the N-terminus of the pestle chain of the Fc domain. In such aspects, the mortar chain domain lacks an immunostimulatory cytokine and an antigen binding domain.

Accordingly, one object of the present invention relates to a polypeptide comprising from N-terminal to C-terminal an antigen binding domain (or at least a portion thereof corresponding to a heavy chain), an Fc chain (pestle or mortar Fc chain)), preferably the mortar chain of the Fc domain, and an immunostimulatory cytokine. The complementary strand comprises a complementary Fc strand lacking the immunostimulatory cytokine and antigen binding domain, preferably the pestle chain of the Fc domain.

In very specific aspects, the bifunctional molecule targets PD-1 and comprises:

(a) Heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID NO 29, 30, 31, 32, 33, 34, 35 or 36, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position of SEQ ID NO 29, 30, 31, 32, 33, 34, 35 or 36, except positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112, respectively, and these substitutions correspond to a mortar or pestle chain, preferably a mortar chain, more particularly as disclosed in table F, in particular in SEQ ID NO 29, 30, 31, 32, 33, 34, 35 or 36, T363S/L365A/Y4047V/Y346C or T363W/S351C, preferably any of SEQ ID NO 29, 30, 31, 35, T363 a/Y294C, and optionally N346C;

(b) A light chain comprising or consisting of the amino acid sequence of SEQ ID No. 37 or SEQ ID No. 38, optionally with one, two or three modifications selected from the group consisting of substitutions, additions, deletions and any combination thereof at any position of SEQ ID No. 37 or SEQ ID No. 38 other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105.

In another aspect, the bifunctional molecule comprises or consists of: any of the following Combinations of Heavy (CH) and light (CL):

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the heavy chain comprises a substitution corresponding to a mortar or pestle chain, preferably a mortar chain, more particularly as disclosed in table F, in particular in SEQ ID NO 29, 30, 31, 32, 33, 34, 35 or 36, in particular T366S/L368A/Y407V/Y349C or T366W/S354C, preferably T366S/L368A/Y407V/Y349C in any of SEQ ID NOs 29, 30, 31, 32, 33, 34, 35 or 36, and optionally N297A, the position of the substitution being defined according to EU numbering.

In a very specific aspect, the bifunctional molecule targets PD-1 and comprises a light chain comprising or consisting of SEQ ID NO. 37 or 38.

Optionally, the heavy chain may comprise a peptide signal, such as depicted in SEQ ID NO. 49. Preferably, such peptide signal is comprised at the N-terminus of the heavy chain.

Optionally, the light chain may comprise a peptide signal, such as depicted in SEQ ID NO. 50. Preferably, such peptide signal is comprised at the N-terminus of the light chain.

Thus, a bifunctional molecule may comprise one heavy chain comprising any of SEQ ID NOs 29, 30, 31, 32, 33, 34, 35 and 36, the Fc chain optionally being modified to promote heterodimerization of the Fc chain to form a heterodimerized Fc domain. More specifically, the heavy chain comprises substitutions corresponding to: the positions of substitution are defined according to EU numbering as for the mortar or pestle chain, preferably the mortar chain, more particularly as disclosed in Table F, in particular T366S/L368A/Y407V/Y349C or T366W/S354C, preferably T366S/L368A/Y407V/Y349C and optionally N297A in any of SEQ ID NO 29, 30, 31, 32, 33, 34, 35 or 36. The heavy chain is linked to the immunostimulatory cytokine, optionally at its C-terminus, by a linker.

In very specific aspects, the bifunctional molecule comprises a light chain comprising or consisting of SEQ ID NO:38 and a heavy chain comprising SEQ ID NO:35, the Fc chain optionally being modified to promote heterodimerization of the Fc chain to form a heterodimerized Fc domain. In one aspect, the heavy chain is linked to the immunostimulatory cytokine, optionally at its C-terminus, by a linker. In another aspect, the light chain is linked to the immunostimulatory cytokine at its C-terminus, optionally via a linker.

In very specific aspects, the bifunctional molecule may comprise a first monomer of SEQ ID NO:75 and a second monomer comprising an Fc chain of SEQ ID NO:77, optionally linked N-terminally to an antigen binding domain (e.g., SEQ ID NO: 79) via a linker. More preferably, the bifunctional molecule may comprise a first monomer of SEQ ID NO:75 and a second monomer comprising an Fc chain of SEQ ID NO:77, optionally linked N-terminally to an antigen binding domain (e.g., SEQ ID NO: 79) by a linker, and optionally linked C-terminally to any immunostimulatory cytokine disclosed herein by a linker.

Optionally, the immunostimulatory cytokine may be selected from: IL-2 (SEQ ID NO: 87), IL-15 (SEQ ID NO: 88), IL-21 (SEQ ID NO: 89) or variants having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto or 1 to 10 modifications relative to the wild type protein selected from the group consisting of additions, deletions, substitutions and combinations thereof. Optionally, the immunostimulatory cytokine may be IL-7 (SEQ ID NO: 1).

Optionally, when the immunostimulatory cytokine is IL-2, the bifunctional molecule may comprise a first monomer of SEQ ID NO. 75, a second monomer of SEQ ID NO. 84, and a third monomer of SEQ ID NO. 37, 38 or 80, preferably SEQ ID NO. 38 or 80.

Optionally, when the immunostimulatory cytokine is IL-2, the bifunctional molecule may comprise a first monomer of SEQ ID NO. 75, a second monomer of SEQ ID NO. 90 and a third monomer of SEQ ID NO. 37, 38 or 80, preferably SEQ ID NO. 38 or 80, linked at its ends to IL-2 of SEQ ID NO. 87 or a variant thereof, optionally via a linker.

Optionally, when the immunostimulatory cytokine is IL-7, the bifunctional molecule may comprise a first monomer of SEQ ID NO. 75, a second monomer of SEQ ID NO. 93, and a third monomer of SEQ ID NO. 37, 38 or 80, preferably SEQ ID NO. 38 or 80.

Optionally, when the immunostimulatory cytokine is IL-7, the bifunctional molecule may comprise a first monomer of SEQ ID NO. 75, a second monomer of SEQ ID NO. 90 and a third monomer of SEQ ID NO. 37, 38 or 80, preferably SEQ ID NO. 38 or 80, optionally linked at its ends to IL-7 of SEQ ID NO. 1 by a linker.

Optionally, when the immunostimulatory cytokine is IL-15, the bifunctional molecule may comprise a first monomer of SEQ ID NO. 75, a second monomer of SEQ ID NO. 85, and a third monomer of SEQ ID NO. 37, 38 or 80, preferably SEQ ID NO. 38 or 80.

Optionally, when the immunostimulatory cytokine is IL-15, the bifunctional molecule may comprise a first monomer of SEQ ID NO. 75, a second monomer of SEQ ID NO. 90 and a third monomer of SEQ ID NO. 37, 38 or 80, preferably SEQ ID NO. 38 or 80, linked at its ends to IL-15 of SEQ ID NO. 88 or a variant thereof, optionally via a linker.

Optionally, when the immunostimulatory cytokine is IL-21, the bifunctional molecule may comprise a first monomer of SEQ ID NO. 75, a second monomer of SEQ ID NO. 86, and a third monomer of SEQ ID NO. 37, 38 or 80, preferably SEQ ID NO. 38 or 80.

Optionally, when the immunostimulatory cytokine is IL-21, the bifunctional molecule may comprise a first monomer of SEQ ID NO. 75, a second monomer of SEQ ID NO. 90 and a third monomer of SEQ ID NO. 37, 38 or 80, preferably SEQ ID NO. 38 or 80, linked at its end to IL-21 of SEQ ID NO. 89 or a variant thereof, optionally via a linker.

In another very specific aspect, the bifunctional molecule may comprise a first monomer of SEQ ID NO:77 and a second monomer comprising an Fc chain of SEQ ID NO:75, optionally linked N-terminally to an antigen binding domain (e.g., SEQ ID NO: 79) by a linker, and optionally linked C-terminally to any immunostimulatory cytokine disclosed herein by a linker.

In another very specific aspect, the bifunctional molecule may comprise a first monomer of SEQ ID NO:77 and a second monomer comprising an Fc chain SEQ ID NO:75, optionally linked at the N-terminus to an antigen binding domain (e.g., SEQ ID NO: 79) by a linker, and a third monomer of SEQ ID NO:37, 38 or 80, preferably SEQ ID NO:38 or 80, optionally linked at its terminus to any immunostimulatory cytokine disclosed herein by a linker.

Optionally, the immunostimulatory cytokine may be selected from: IL-2 (SEQ ID NO: 87), IL-15 (SEQ ID NO: 88) and IL-21 (SEQ ID NO: 89) or variants having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto or 1 to 10 modifications relative to the wild type protein selected from the group consisting of additions, deletions, substitutions and combinations thereof. Optionally, the immunostimulatory cytokine may be IL-7 (SEQ ID NO: 1).

Preparation of bifunctional molecules-nucleic acid molecules encoding the bifunctional molecules of the invention, recombinant expression vectors and host cells comprising the same

To produce the bifunctional molecules according to the invention, the nucleic acid sequences or groups of nucleic acid sequences encoding the bifunctional molecules are subcloned into one or more expression vectors, in particular by mammalian cells. Such vectors are commonly used to transfect mammalian cells. General techniques for producing molecules comprising antibody sequences are described in Coligan et al (eds.), current protocols in immunology, pages 10.19.1-10.19.11 (Wiley Interscience 1992), the contents of which are incorporated herein by reference, and "Antibody engineering: a practical guide" from W.H. Freeman and Company (1992), wherein comments relating to the production of molecules are provided throughout the respective text.

Generally, such methods comprise the steps of:

(1) Transfecting or transforming a suitable host cell with a polynucleotide encoding a recombinant bifunctional molecule of the invention or a vector comprising the polynucleotide;

(2) Culturing the host cell in a suitable medium; and

(3) Optionally isolating or purifying the bifunctional molecule from the culture medium or the host cell.

The invention further relates to nucleic acids encoding the bifunctional molecules as disclosed above, vectors, preferably expression vectors, comprising the nucleic acids of the invention, genetically engineered host cells transformed with the vectors of the invention or directly with sequences encoding recombinant bifunctional molecules, and methods for producing the bifunctional molecules of the invention by recombinant techniques.

Nucleic acids, vectors, and host cells are described in more detail below.

Nucleic acid sequences

The invention also relates to a nucleic acid molecule encoding a bifunctional molecule as defined above or a set of nucleic acid molecules encoding a bifunctional molecule as defined above. Nucleic acids encoding the bifunctional molecules disclosed herein may be amplified by any technique known in the art, such as PCR. Such nucleic acids can be readily isolated and sequenced using conventional procedures.

In particular, nucleic acid molecules encoding the bifunctional molecules defined herein include:

-a first nucleic acid molecule encoding a first monomer comprising an antigen binding domain covalently linked, optionally via a peptide linker, to a first Fc chain, said first Fc chain being covalently linked, optionally via a peptide linker, to an immunostimulatory cytokine, and

a second nucleic acid molecule encoding a second monomer comprising a complementary second Fc strand,

optionally, a third nucleic acid molecule encoding the light chain of the antigen binding domain.

In an alternative aspect, a nucleic acid molecule encoding a bifunctional molecule as defined herein comprises:

a first nucleic acid molecule encoding a first monomer comprising an antigen binding domain covalently linked to a first Fc chain, optionally via a peptide linker,

-a second nucleic acid molecule encoding a second monomer comprising a complementary second Fc chain, and

-a third nucleic acid molecule encoding a light chain of an antigen binding domain, said light chain being covalently linked to an immunostimulatory cytokine, optionally through a peptide linker.

In one embodiment, the nucleic acid molecule is an isolated, in particular a non-natural, nucleic acid molecule.

Carrier body

In a further aspect, the invention relates to a vector comprising a nucleic acid molecule or group of nucleic acid molecules as defined above.

As used herein, a "vector" is a nucleic acid molecule that serves as a vector for transferring genetic material into a cell. The term "vector" encompasses plasmids, viruses, cosmids, and artificial chromosomes. Generally, an engineered vector comprises an origin of replication, a multiple cloning site, and a selectable marker. The vector itself is typically a nucleotide sequence, typically a DNA sequence, comprising an insert (transgene) and a larger sequence that acts as the "backbone" of the vector. In addition to the transgene insert and backbone, modern vectors may encompass additional features: promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, and protein purification tags. Vectors known as expression vectors (expression constructs) are used exclusively for expressing transgenes in target cells and typically have control sequences.

The person skilled in the art can clone the nucleic acid molecule encoding the bifunctional molecule into a vector and then transform it into a host cell. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. Methods known to those skilled in the art can be used to construct expression vectors containing the nucleic acid sequences of the bifunctional molecules described herein and appropriate regulatory components for transcription/translation.

Accordingly, the present invention also provides a recombinant vector comprising a nucleic acid molecule encoding a bifunctional molecule of the invention. In a preferred aspect, the expression vector further comprises a promoter and a nucleic acid sequence encoding a secretion signal peptide, and optionally at least one drug resistance gene for screening. The expression vector may also contain ribosome binding sites for initiating translation, transcription terminators and the like.

Suitable expression vectors typically contain (1) prokaryotic DNA elements encoding bacterial origins of replication and antibiotic resistance markers to provide for the growth and selection of the expression vector in a bacterial host; (2) Eukaryotic DNA elements that control transcription initiation, such as promoters; and (3) DNA elements that control transcript processing, such as transcription termination/polyadenylation sequences.

Expression vectors can be introduced into host cells using a variety of techniques, including calcium phosphate transfection, liposome-mediated transfection, electroporation, and the like. Preferably, transfected cells are selected and propagated wherein the expression vector is stably integrated into the host cell genome to produce a stable transformant.

Host cells

In a further aspect, the invention relates to a host cell comprising a vector or a nucleic acid molecule or a group of nucleic acid molecules as defined above, e.g. for the purpose of bifunctional molecule production.

As used herein, the term "host cell" is intended to include any individual cell or cell culture that may or may not be the recipient of vectors, exogenous nucleic acid molecules and polynucleotides encoding bifunctional molecules according to the present invention. The term "host cell" is also intended to include progeny or potential progeny of a single cell. Suitable host cells include prokaryotic or eukaryotic cells, and also include, but are not limited to, bacterial, yeast, fungal, plant, and animal cells such as insect cells and mammalian cells, e.g., murine, rat, rabbit, macaque, or human cells.

Suitable host cells are in particular eukaryotic host cells which provide suitable post-translational modifications such as glycosylation. Preferably, such suitable eukaryotic host cells may be fungi, such as pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces pombe; insect cells such as myxoplasma; plant cells such as tobacco, and mammalian cells such as BHK cells, 293 cells, CHO cells, NSO cells, and COS cells.

Preferably, the host cell of the invention is selected from the group consisting of CHO cells, COS cells, NSO cells and HEK cells.

The host cell then stably or transiently expresses the bifunctional molecules of the invention. Such expression methods are known to those skilled in the art.

Also provided herein are methods of producing the bifunctional molecules. The method comprises culturing a host cell comprising a nucleic acid encoding the bifunctional molecule described above under conditions suitable for its expression, and optionally recovering the bifunctional molecule from the host cell (or host cell culture medium). In particular, for recombinant production of a bifunctional molecule, a nucleic acid encoding a bifunctional molecule, e.g. as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. The bifunctional molecule is then isolated and/or purified by any method known in the art. Such methods include, but are not limited to, conventional renaturation treatment, protein precipitant treatment (e.g., salt precipitation), centrifugation, osmotic cell lysis, sonication, ultracentrifugation, molecular sieve chromatography or gel chromatography, adsorption chromatography, ion exchange chromatography, HPLC, any other liquid chromatography, and combinations thereof. As described in Coligan, for example, bifunctional molecular separation techniques may specifically include affinity chromatography using Protein-A Sepharose, size exclusion chromatography, and ion exchange chromatography. Protein a is preferably used to isolate the bifunctional molecules of the invention.

Pharmaceutical compositions and methods of administration thereof

The invention also relates to pharmaceutical compositions comprising the bifunctional molecules described herein, nucleic acid molecules as described above, groups of nucleic acid molecules, vectors and/or host cells, preferably as active ingredients or compounds. The formulation may be sterilized and, if desired, admixed with adjuvants, such as pharmaceutically acceptable carriers, excipients, salts, antioxidants and/or stabilizers, which do not deleteriously interact with the bifunctional molecules of the invention, the nucleic acids, the vectors and/or the host cells of the invention, and which do not produce any undesired toxicological effects. Optionally, the pharmaceutical composition may further comprise an additional therapeutic agent.

In particular, the pharmaceutical compositions according to the present invention may be formulated for any conventional route of administration, including topical, enteral, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration, and the like. For ease of administration, the bifunctional molecules described herein may be formulated into pharmaceutical compositions for in vivo administration. Means of making such compositions have been described in the art (see, e.g., remington: the Science and Practice of Pharmacy, lippincott Williams & Wilkins, 21 st edition (2005).

Pharmaceutical compositions may be prepared by mixing bifunctional molecules of the desired purity with optional pharmaceutically acceptable carriers, excipients, antioxidants and/or stabilizers in the form of lyophilized formulations or aqueous solutions. Such suitable carriers, excipients, antioxidants, and/or stabilizers are well known in the art and have been described, for example, in Remington's Pharmaceutical Sciences, 16 th edition, osol, a.ed. (1980).

To facilitate delivery, any bifunctional molecule or nucleic acid encoding the same may be conjugated to a chaperone. The chaperone agent may be a naturally occurring substance, such as a protein (e.g., human serum albumin, low density lipoprotein or globulin), a carbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), or a lipid. It may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g. a synthetic polypeptide.

The pharmaceutical compositions according to the invention may be formulated to release the active ingredient (e.g. the bifunctional molecule of the invention) substantially immediately after administration or at any predetermined time or period after administration. In some aspects, the pharmaceutical compositions may employ a time release, delayed release, and sustained release delivery system such that delivery of the composition occurs prior to sensitization of the site to be treated and for a time sufficient to cause sensitization of the site to be treated. Means known in the art may be used to prevent or minimize the release and absorption of the composition until it reaches the target tissue or organ, or to ensure timed release of the composition. Such systems can avoid repeated administration of the composition, thereby increasing the convenience of the subject and physician.

It will be appreciated by those skilled in the art that the formulations of the present invention may be isotonic with human blood, i.e., the formulations of the present invention have substantially the same osmotic pressure as human blood. Such isotonic formulations typically have an osmotic pressure of about 250mOSm to about 350 mOSm. Isotonicity can be measured by, for example, a vapor pressure or ice-cold osmometer.

Pharmaceutical compositions must generally be sterile and stable under the conditions of manufacture and storage. Prevention of the presence of microorganisms may be ensured by sterilization procedures (e.g., by microfiltration) and/or by inclusion of various antibacterial and antifungal agents

The amount of active ingredient that can be combined with the carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with the carrier material to produce a single dosage form is typically the amount of the composition that produces a therapeutic effect.

Subjects, regimens and administration

The present invention relates to a bifunctional molecule disclosed herein, a nucleic acid or vector encoding the same, a host cell or a pharmaceutical composition for use as a medicament or for treating a disease or for administration in a subject or for use as a medicament. It also relates to a method for treating a disease or disorder in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition or a bifunctional molecule.

The subject to be treated may be a human, particularly a pre-partum human, a neonate, a child, an infant, a adolescent or an adult, particularly an adult at least 30 years old, an adult at 40 years old, preferably an adult at least 50 years old, still more preferably an adult at least 60 years old, even more preferably an adult at least 70 years old.

In particular aspects, the subject may be immunosuppressed or immunocompromised.

Conventional methods known to those of ordinary skill in the medical arts may be used to administer the bifunctional molecules or pharmaceutical compositions disclosed herein to a subject, depending on the type of disease to be treated or the site of the disease, e.g., oral, parenteral, enteral, by inhalation spray, topical, rectal, nasal, buccal, vaginal, or by implanted depot administration. Preferably, the bifunctional molecule or pharmaceutical composition is administered by subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intratumoral, intrasternal, intratenosynovial, intralesional and intracranial injection or infusion techniques.

The form, route of administration and dosage of administration of the pharmaceutical composition or bifunctional molecule according to the invention can be adjusted by the person skilled in the art depending on the type and severity of the infection and the patient, in particular the age, weight, size, sex and/or general body condition of the patient. The compositions of the present invention may be administered in a variety of ways depending on whether local or systemic treatment is desired.

Use in the treatment of diseases

The bifunctional molecules, nucleic acids, vectors, host cells, compositions and methods of the invention have a number of in vitro and in vivo utilities and applications. In particular, any bifunctional molecule, nucleic acid molecule, group of nucleic acid molecules, vector, host cell, or pharmaceutical composition provided herein may be used in a method of treatment and/or for therapeutic purposes.

The invention also relates to a bifunctional molecule, a nucleic acid or vector encoding the same, or a pharmaceutical composition comprising the same, for use in the treatment of a disorder and/or disease in a subject and/or as a medicament or vaccine. The invention also relates to bifunctional molecules as described herein; a nucleic acid or vector encoding the same, or a pharmaceutical composition comprising the same, for use in treating a disease and/or disorder in a subject. Finally, the present invention relates to a method for treating a disease or disorder in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition or a bifunctional molecule, or a nucleic acid or vector encoding the same.

In one aspect, the present invention relates to a method of treating a disease and/or disorder selected from cancer, infectious disease and chronic viral infection in a subject in need thereof, comprising administering to said subject an effective amount of a bifunctional molecule or a pharmaceutical composition as defined above. Examples of such diseases are described in more detail below.

In one aspect, a method of treatment comprises: (a) determining a patient in need of treatment; and (b) administering to the patient a therapeutically effective amount of a bifunctional molecule, nucleic acid, vector, or pharmaceutical composition described herein.

The subject in need of treatment may be a person suffering from, at risk of suffering from, or suspected of suffering from a disease. Such patients may be identified by routine medical examination.

In another aspect, the bifunctional molecules disclosed herein may be administered to a subject, e.g., in vivo, to enhance immunity, preferably in order to treat a disorder and/or disease. Thus, in one aspect, the invention provides a method of modifying an immune response in a subject comprising administering to the subject a bifunctional molecule, nucleic acid, vector or pharmaceutical composition of the invention, such that the immune response in the subject is modified. Preferably, the immune response is enhanced, increased, stimulated or upregulated. The bifunctional molecule or pharmaceutical composition may be used to enhance an immune response, such as T cell activation, in a subject in need of treatment. In particular embodiments, the bifunctional molecule or pharmaceutical composition may be used to reduce T cell depletion or reactivate depleted T cells.

The invention provides, inter alia, methods of enhancing an immune response in a subject comprising administering to the subject a therapeutically effective amount of any bifunctional molecule, nucleic acid, vector, or pharmaceutical composition comprising the same, as described herein, such that the immune response in the subject is enhanced. In particular embodiments, the bifunctional molecule or pharmaceutical composition may be used to reduce T cell depletion or reactivate depleted T cells.

The bifunctional molecules comprising IL-7 according to the invention target CD127+ immune cells, in particular CD127+ T cells. Such cells may be present in the following areas of particular interest: resident lymphocytes in lymph nodes (cells are predominantly in the parathyroid cortex, occasionally cells in the follicles), tonsils (interfollicular region), spleen (some dispersed cells predominantly in periarterial lymphatic sheath (PALS) of the white pulp and in the red pulp), thymus (predominantly in the medulla; also in the cortex), bone marrow (dispersed distribution), GALT (intestinal-related lymphoid tissue predominantly in the interfollicular region and lamina propria) of the entire digestive tract (stomach, duodenum, jejunum, ileum, cecum colon, rectum), MALT (mucosa-related lymphoid tissue) of the gall bladder. Thus, the bifunctional molecules of the invention are of particular interest for the treatment of diseases located in or involving these areas, in particular cancer.

Such bifunctional molecules and pharmaceutical compositions comprising the same are useful in patients, particularly patients suffering from cancer, for increasing tumor-infiltrating lymphocytes (TIL), protecting T lymphocytes from apoptosis, inducing/improving T memory responses, eliminating T-reg inhibitory and/or Treg inhibitory activity, restoring proliferation and/or maintaining fully depleted T cells, particularly depleted tumor-infiltrating lymphocytes.

Such bifunctional molecules and pharmaceutical compositions comprising the same are useful for the preparation of a medicament for increasing tumor-infiltrating lymphocytes (TIL) in a patient, in particular a patient suffering from cancer, protecting T lymphocytes from apoptosis, inducing/improving T memory responses, eliminating T-reg inhibition and/or Treg inhibition activity, restoring proliferation and/or maintaining fully depleted T cells, in particular depleted tumor-infiltrating lymphocytes.

The present invention also relates to a method for increasing tumor-infiltrating lymphocytes (TIL), protecting T lymphocytes from cell death, inducing/improving T memory responses, eliminating T-reg inhibition and/or Treg inhibition activity, restoring proliferation and/or maintaining completely depleted T cells, particularly depleted tumor-infiltrating lymphocytes, in a patient, particularly a patient suffering from cancer, comprising administering to said patient a therapeutically effective amount of a bifunctional molecule comprising IL-7 and an antigen binding domain that binds and antagonizes PD-1, particularly any such specific molecule disclosed herein.

Cancer of the human body

In another aspect, the invention provides the use of a bifunctional molecule or pharmaceutical composition disclosed herein in the manufacture of a medicament for treating cancer, e.g., for inhibiting tumor cell growth in a subject.

As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells may spread to other parts of the body locally or through the blood stream and lymphatic system.

Accordingly, in one aspect, the present invention provides a method of treating cancer, for example for inhibiting tumor cell growth, in a subject comprising administering to the subject a therapeutically effective amount of a bifunctional molecule or a pharmaceutical composition according to the invention. In particular, the invention relates to the use of bifunctional molecules to treat subjects to inhibit the growth of cancer cells.

In one aspect of the disclosure, the cancer to be treated is associated with depleted T cells.

Any suitable cancer that can be treated with the provided herein can be a hematopoietic cancer or a solid cancer. Such cancers include epithelial cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, head and neck cancer, renal cancer, liver cancer, lung cancer, lymphoma, glioma, mesothelioma, melanoma, gastric cancer, cancer of the urinary tract, environmentally induced cancers, and any combination of said cancers. In addition, the invention includes refractory or recurrent malignant tumors. Preferably, the cancer to be treated or prevented is selected from: metastatic or non-metastatic melanoma, malignant mesothelioma, non-small cell lung cancer, renal cell carcinoma, hodgkin's lymphoma, head and neck cancer, urothelial cancer, colorectal cancer, hepatocellular carcinoma, small cell lung cancer, metastatic mercker cell carcinoma, gastric or gastroesophageal cancer, and cervical cancer.

In a particular aspect, the cancer is a hematological malignancy or a solid tumor. Such cancers may be selected from: hematological lymphomas, angioimmunoblastic T cell lymphomas, myelodysplastic syndromes, and acute myelogenous leukemia.

In particular aspects, the cancer is a cancer induced by a virus or associated with an immunodeficiency. Such cancers may be selected from: kaposi's sarcoma (e.g., associated with kaposi's sarcoma herpes virus); cervical cancer, anal cancer, penile cancer and vulvar squamous cell carcinoma, and oropharyngeal cancer (e.g., associated with human papillomavirus); b-cell non-hodgkin lymphomas (NHL), including diffuse large B-cell lymphomas, burkitt's lymphomas, plasmablasts, primary central nervous system lymphomas, HHV-8 primary exudative lymphomas, classical hodgkin lymphomas, and lymphoproliferative disorders (e.g., associated with Epstein-Barr virus and/or kaposi sarcoma herpes virus); hepatocellular carcinoma (e.g., associated with hepatitis b and/or hepatitis c virus); merck cell carcinoma (e.g., associated with merck cytodoloma virus (MPV); and cancers associated with Human Immunodeficiency Virus (HIV) infection.

Preferred treatments for cancer include cancers that are generally responsive to immunotherapy. Alternatively, the preferred treatment cancer is cancer that is not responsive to immunotherapy.

Infectious disease

The bifunctional molecules, nucleic acids, nucleic acid sets, vectors, host cells or pharmaceutical compositions of the invention may be used to treat patients who have been exposed to a particular toxin or pathogen. Accordingly, in one aspect the invention provides a method of treating an infectious disease in a subject, comprising administering to the subject a bifunctional molecule according to the invention or a pharmaceutical composition comprising the same, preferably such that the infectious disease of the subject is treated.

Any suitable infection may be treated with the bifunctional molecules, nucleic acids, nucleic acid sets, vectors, host cells, or pharmaceutical compositions provided herein.

Some examples of pathogenic viruses that cause infections treatable by the methods of the invention include HIV, hepatitis (type a, type b, or type c), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, epstein-Barr virus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, neocoronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, polio virus, rabies virus, JC virus, and arboencephalitis virus.

Some examples of infectious pathogens that may be treated by the methods of the invention include chlamydia, rickettsia bacteria, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and cone cocci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacillus, cholera, tetanus, botulinum, anthrax, plague, leptospirosis and lyme disease bacteria.

Some examples of infectious pathogenic fungi that can be treated by the methods of the present invention include candida (candida albicans, candida krusei, candida glabrata, candida tropicalis, etc.), cryptococcus neoformans, aspergillus (aspergillus fumigatus, aspergillus niger, etc.), mucor (mucor, colubus, rhizopus), sporotrichia stigmata, blastomyces dermatitis, paracoccidiosporium brazil, pachycoccoides, and histoplasma capsulatum.

Some examples of infectious pathogenic parasites that can be treated by the methods of the present invention include Entamoeba histolytica, E.coli, grignard, acanthamoeba species, giardia, cryptosporidium species, pneumocystis carinii, plasmodium vivax, barbaria, trypanosoma brucei, trypanosoma cruzi, leishmania donovani, toxoplasma gondii and Brazilian round-robinia.

Combination therapy

The bifunctional molecules according to the invention may be combined with agents in clinical development or already marketed with some other potential strategies for overcoming immune evasion mechanisms (see Antonia et al Immuno-oncology combinations: a review of clinical experience and future proctos. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res.20, table 1 in 6258-6268,2014). Such combinations with bifunctional molecules according to the invention are particularly useful:

1-reverse adaptive immunity inhibition (blocking T cell checkpoint pathways);

2-opening adaptive immunity (promotion of T cell costimulatory receptor signaling using agonist molecules, in particular antibodies),

3-enhancing the function of innate immune cells;

4-activate the immune system (enhance immune cell effector function), for example by a vaccine-based strategy.

Thus, also provided herein is a combination therapy with any bifunctional molecule or comprising a bifunctional molecule or pharmaceutical composition as described herein, and a suitable second agent for treating a disease or disorder. In one aspect, the bifunctional molecule and the second agent may be present in a unique pharmaceutical composition as described above. Alternatively, as used herein, the term "combination therapy" or "combination therapy" includes administration of the two agents (e.g., a bifunctional molecule as described herein and an additional or second suitable therapeutic agent) in a sequential manner, i.e., wherein each therapeutic agent is administered at a different time, as well as administration of the therapeutic agents or at least two therapeutic agents in a substantially simultaneous manner. Sequential or substantially simultaneous administration of each agent may be effected by any suitable route. These agents may be administered by the same route or by different routes. For example, a first agent (e.g., a bifunctional molecule) may be administered orally, and an additional therapeutic agent (e.g., an anticancer agent, an anti-infective agent, or an immunomodulator) may be administered intravenously. Alternatively, the selected combination of agents may be administered by intravenous injection, while the other agents of the combination may be administered orally.

In one aspect, the additional therapeutic agent may be selected from a non-exhaustive list comprising alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotics, antiproliferatives, antiviral agents, aurora kinase inhibitors, apoptosis promoters (e.g., bcl-2 family inhibitors), death receptor pathway activators, bcr-Abl kinase inhibitors, biTE (dual specificity T cell engager) antibodies, antibody drug conjugates, biological response modifiers, bruton's Tyrosine Kinase (BTK) inhibitors, cyclin dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia virus oncogene homolog (ErbB 2) receptor inhibitors, growth factor inhibitors, heat Shock Proteins (HSP) -90 inhibitors, histone Deacetylase (HDAC) inhibitors, hormonal therapies, immunological agents, apoptosis protein Inhibitors (IAPs), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, jak2 inhibitors, rapamycin inhibitors, mammalian targets, micrornas, mitogen-activated extracellular signal kinase inhibitors, multivalent binding proteins, non-steroidal anti-inflammatory drugs (aftospirates), poly (ADP) -riboside-kinase (phospho) inhibitors, phospho-3-phospho-kinase (PI) inhibitors, phospho-3-phospho-kinase inhibitors, phospho-like inhibitors, phospho-kinase (PI) like inhibitors, phospho-3-kinase inhibitors, phospho-like inhibitors, phospho-kinase inhibitors, phospho-3-like inhibitors, phospho-kinase inhibitors, phospho-like inhibitors, protein (PI-3-like inhibitors, protein kinase inhibitors Topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, peptide vaccines and the like, epitopes or neoepitopes from tumor antigens, and combinations of one or more of these agents.

For example, the additional therapeutic agent may be selected from: chemotherapy, radiation therapy, targeted therapy, anti-angiogenic agents, hypomethylating agents, cancer vaccines, epitopes or neoepitopes from tumor antigens, bone marrow checkpoint inhibitors, other immunotherapies and HDAC inhibitors.

In an embodiment, the invention relates to a combination therapy as defined above, wherein the second therapeutic agent is in particular selected from: therapeutic vaccines, immune checkpoint blockers or activators, in particular adaptive immune cells (T and B lymphocytes) and antibody drug conjugates. Preferably, suitable agents for use with any anti-hPD-1 antibody or fragment thereof or with a pharmaceutical composition according to the invention include antibodies that bind to a co-stimulatory receptor (e.g., OX40, CD40, ICOS, CD27, HVEM or GITR), agents that induce immunogenic cell death (e.g., chemotherapeutic agents, radiotherapeutic agents, anti-angiogenic agents or agents for targeted therapy), agents that inhibit checkpoint molecules (e.g., CTLA4, LAG3, TIM3, B7H4, BTLA or TIGIT), cancer vaccines, agents that modify immunosuppressive enzymes (e.g., IDO1 or iNOS), agents that target Treg cells, agents for adoptive cell therapy or agents that modulate bone marrow cells.

In one embodiment, the invention relates to a combination therapy as defined above, wherein the second therapeutic agent is an immune checkpoint blocker or an activator of adaptive immune cells (T and B lymphocytes) selected from the group consisting of: anti-CTLA 4, anti-CD 2, anti-CD 28, anti-CD 40, anti-HVEM, anti-BTLA, anti-CD 160, anti-TIGIT, anti-TIM-1/3, anti-LAG-3, anti-2B 4 and anti-OX 40, anti-CD 40 agonist, CD40-L, TLR agonist, anti-ICOS, ICOS-L and B cell receptor agonist.

The invention also relates to a method for treating a disease in a subject comprising administering to the subject a therapeutically effective amount of a bifunctional molecule or a pharmaceutical composition described herein and a therapeutically effective amount of an additional or second therapeutic agent.

Specific examples of additional or second therapeutic agents are provided in WO 2018/053106, pages 36-43.

In preferred embodiments, the second therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a radiotherapeutic agent, an immunotherapeutic agent, a cell therapeutic agent (e.g., CAR-T cells), an antibiotic, and a probiotic.

Combination therapies may also rely on the administration of bifunctional molecules in combination with surgery.

Examples

Example 1: anti-PD-1 IL-7 molecules with one IL-7W142H cytokine and one anti-PD-1 arm have been shown to be highly potent in promoting the cis-activity of PD-1+IL-7R+ cells

The inventors designed and compared the biological activities of various structures of bifunctional molecules comprising one or two anti-PD-1 binding domains and one or two IL 7W142H mutants, as shown in figure 1. The W142H substitution corresponds to the substitution of the amino acid at position 142 in the sequence shown in SEQ ID No. 1.

Construct 1 comprises two anti-PD-1 antigen binding domains and two IL-7W142H variants (construct 1 is also referred to as anti-PD-1 x 2IL-7W142H x 2). This molecule is also known as BICKI-IL-7W142H. In the examples, the control molecule, designated BICKI-IL-7WT, corresponds to construct 1, but has wild-type IL-7.

Construct 2 contained two anti-PD-1 antigen binding domains and a single IL-7W142H variant (construct 2 is also referred to as anti-PD-1 x 2IL-7W142H x 1).

Construct 3 comprises a single anti-PD-1 antigen binding domain and a single IL-7W142H variant (construct 3 is also referred to as anti-PD-1 x 1IL-7W142H x 1). The control construct, designated anti-PD-1*1, was similar to construct 3 but lacks the IL-7 variant.

Construct 4 comprises a single anti-PD-1 antigen binding domain and two IL-7W142H variants (construct 4 is also referred to as anti-PD-1 x 1IL-W142H x 2).

Constructs 2, 3 and 4 were engineered with IgGl N298A isoforms and the amino acid sequences were mutated in the Fc portion to create a pestle on CH2 and CH3 of heavy chain a and a mortar on CH2 and CH3 of heavy chain B.

ELISA assays demonstrated that all anti-PD-1 IL7 constructs had high affinity for the PD-1 receptor (FIG. 2A and Table 1). anti-PD-1 IL-7 molecules with 2 anti-PD-1 arms (anti-PD-1*2) have the same binding efficacy (equal EC 50) compared to anti-PD-1*2 without IL-7. Similarly, anti-PD-1 IL-7 molecules with 1 anti-PD-1 arm (anti-PD-1 x 1IL7 w142h x 1 and anti-PD-1 x 1IL7 w142h x 2) exhibit the same binding efficacy compared to anti-PD-1*1 without IL-7, with EC50 for anti-PD-1 IL7 equal to 0.086 and 0.111nM compared to EC50 for anti-PD-1 equal to 0.238nM. These data indicate that fusion of IL-7 does not interfere with PD-1 binding, regardless of the construct tested.

Sample of	EC50(nM)
		anti-PD-1*2	0.021
anti-PD-1 x 2il7 w142h x 1	0.026
		anti-PD-1 x 2il7 w142h x 2	0.034
anti-PD-1*1	0.238
		anti-PD-1 x 1il7 w142h x 1	0.111
anti-PD-1 x 1il7 w142h x 2	0.086

Table 1. ED50 determination in FIG. 2A refers to the concentration required to measure the PD1 binding signal by ELISA to 50% for each anti-PD-1 IL-7 molecule.

In addition, PD-L1/PD-1 antagonist bioassays (FIG. 2B) indicate that anti-PD-1 IL7 molecules with 1 or 2 anti-PD-1 arms were shown to block the binding of PD-L1 to the PD-1 receptor with high efficacy. Although one arm of anti-PD-1 was removed from constructs 3 and 4, all anti-PD-1 x 1il7 constructs exhibited high antagonistic properties. EC50 (table 2) was calculated for constructs 3 and 4, only 2.5 fold reduced activity compared to the anti-PD-1 x 2il7 construct.

Table 2. ED50 determination in FIG. 2B refers to the concentration required to achieve 50% PD1/PDL1 antagonist activity for each anti-PD-1 IL-7 molecule as measured by ELISA.

The inventors next evaluated the affinity of the different constructs for CD127 receptor using Biacore assay and ELISA assay. Because one IL-7 molecule was removed from constructs 2 and 3, these molecules were expected to have lower binding capacity to the CD127 receptor and lower pSTAT5 activation compared to the IL-7 heterodimer construct. However, the inventors observed that the anti-PD-1 x 2IL-7W142H 1 molecule had similar affinity for CD127 receptor compared to anti-PD-1 x 2IL-7W142H (BICKI-IL-7W 142H) and as expected a lower affinity compared to the anti-PD-1 IL7 bifunctional molecule comprising the wild-type form of IL-7 (table 3). Unexpectedly, the anti-PD-1 x 2IL 7w142h x 1 and anti-PD-1 x 1IL 7w142h x 1 molecules exhibited high pSTAT5 activity similar to the PD-1IL7 bifunctional molecules comprising the wild-type form of IL-7 (fig. 3). Based on these observations, the combination of the monomeric form of IL-7 with the W142H IL-7 mutation appears to allow for an optimal conformation of the IL-7 molecule to promote IL-7 signaling into human T cells. Even with only one IL7, the molecule with the W142H IL-7 mutation had as good an activation effect (pSTAT 5) as the IL7 wt molecule with two cytokines. This result is unexpected in cases where the IL-7 variant has a lower affinity for its receptor than wild-type IL-7.

TABLE 3 binding of anti-PD 1IL7 wild-type or anti-PD 1IL 7W 142H mutants constructed with 1 or 2IL 7.

CD127 was immobilized onto a sensor chip and anti-PD-1 IL-7 bifunctional molecules were added in increasing doses to measure affinity.

Finally, specific cis-targeting and cis-activity of the different anti-PD-1 IL-7 constructs were analyzed in a co-culture assay. U937 PD-1+cd127+ cells were mixed with PD-1-cd127+ cells (ratio 1:1) and then incubated with the different constructs at increasing doses. Binding and IL-7R signaling (pSTAT 5) were quantified by flow cytometry. EC50 (nM) and pSTAT5 activation were determined for each construct and binding of each PD-1+ and PD-1-cell population (fig. 4A and B). The inventors have verified that the diversity of anti-PD-1 IL-7 mutant molecules (anti-PD-1 x 2IL7 w142h x 1, anti-PD-1 x 1IL7 w142h x 2) substantially preferentially bind IL-7R into PD-1+ cells while substantially activating IL7R signaling pSTAT5 into PD-1+ cells. Importantly, construct PD-1 x 1il7 w142h 1 showed the highest activity in stimulating pSTAT5 signaling into PD-1+ cells compared to the other constructs (anti-PD-1 x 2il7 w142h 1 and anti-PD-1 x 1il7 w142h 2). These data indicate that bifunctional molecules constructed from one anti-PD-1 arm and one IL-7 have optimal conformation and activity to allow preferential activation of IL-7R into PD-1+ activated T cells in the context of cancer.

Example 2: anti-PD-1 IL-7 molecules constructed from 1 or 2 anti-PD-1 arms and 1 or 2IL7W 142H cytokines have good pharmacokinetic profiles in vivo

Pharmacokinetic studies of anti-PD-1 IL-7 bifunctional molecule constructs 2, 3 and 4 as described in figure 1 were evaluated. Humanized PD 1K 1 mice were intraperitoneally injected with a dose of anti-PD-1 IL-7 molecule (34.4 nM/kg). Plasma drug concentrations were analyzed by human IgG-specific ELISA (fig. 5). The area under the curve was also calculated (see table 4) and represents the total drug exposure of each construct over time. The anti-PD-l 2IL-7w142h 1, anti-PD-1 x 1IL-7w142h 1 and anti-PD-1 x 1IL-7w142h 2 constructs exhibited very advantageous enhanced PK properties compared to anti-PD-1 x 2IL-7w142h 1. Cmax was observed to be 2.8 to 19 fold higher compared to anti-PD-1 x 1i l7wt x 2. Importantly, high drug concentrations (11-15 nM) were maintained with anti-PD-1 x 1i 7w142h 1 anti-PD-1 x 1i 7w142h 2 molecules (this corresponds to satisfactory in vivo PK values) for at least 96 hours, whereas only 2nM of anti-PD-1 x 2i 7wt 2 molecules were detected in plasma. The residual drug concentration of anti-PD-1 x 2il-7w142h x 1 was 2.5 fold higher than the anti-PD-1 x 2il7wt x 2 concentration. Plasma drug exposure is generally associated with in vivo efficacy. Here, the inventors demonstrate that all anti-PD-1 IL-7W142H molecules constructed with one arm against PD-1 allow for long term drug exposure after a single injection. While anti-PD-l 1IL-7w142h 1, anti-PD-1 x 1IL-7w142h 1 and anti-PD-1 x 1IL-7w142h 2 exhibited similar favorable PK profiles in vivo, fig. 4B demonstrates that the anti-PD-1 x 1IL-7w142h 1 construct has a higher capacity to activate PD-1+ cells.

	AUC	Cmax(nM)
			anti-PD-l 1IL-7W142H 1	1597	42.4
anti-PD-l 1IL-7W142H 2	2024	248.6

Table 4. Area under the curve was determined according to fig. 7. AUC was calculated 0 to 96 hours after intraperitoneal injection of one dose of anti-PD-1 IL-7 (34 nM/kg).

Example 3: description of the constructs used in examples 4 to 9.

Different constructs of the bifunctional antibodies were tested and compared. Fig. 6 illustrates different forms: (1) form a (anti-PD-1*2/protx×2), (2) form B (anti-PD-1*2/protx×1), (3) form C (anti-PD-1*1/protx×l fused to heavy chain). For form C, the Fc domain comprises CH1 CH2 and a hinge portion. All constructs were engineered with the IgG 1N 298A isotype and the amino acid sequence was mutated in the Fc portion to create a pestle on CH2 and CH3 of heavy chain a and a mortar on CH2 and CH3 of heavy chain B. All constructs contained GGGGSGGGGSGGGGS linkers (SEQ ID NO: 70) between the Fc domain and the fused protein X.

Example 4. Bifunctional antibodies constructed with one anti-PD-1 valency and one fusion protein X exhibit higher productivity in mammalian cells compared to bifunctional antibodies constructed with two anti-PD-1 valencies and one fusion protein X.

The productivity of form B and form C of the bifunctional antibodies of mammalian cells was evaluated and compared. The full heavy and light chains fused to Fc and cytokines (IL, 7, IL-2, IL-15 or IL-21) were transiently co-transfected into CHO suspension cells. The amount of antibody obtained after production and purification was quantified using a sandwich ELISA (immobilized donkey anti-human Fc antibody for detection and display with mouse anti-human kappa+a peroxidase conjugated goat anti-mouse antibody). The concentration was determined with human IvIgG standards. Productivity was calculated as the amount of purified antibody per liter of culture supernatant collected.

Results: bifunctional antibodies, anti-PD-1*2/protX 1 (form B) and antibodies PD-1 x 1protX 1 (form C) were produced in CHO mammals and the results are shown in fig. 7. In a surprising manner, the anti-PD-1*1/protx 1 construct (form C) had significantly better yields (mg/L) than the anti-PD-1*2/protx 1 (form B) (fig. 7A). For all fusion proteins X tested, i.e., cytokines (IL 7, IL2, IL21, and IL-15), significantly 1.7-fold (+/-0.7; n=5) higher productivity was obtained (FIG. 7B). These results indicate that anti-PD-1*1/protx 1 (form C) has very good manufacturability, which is important for clinical development and therapeutic application in the next step.

In particular, for bifunctional antibodies comprising two different arms, one major problem is the mismatch of the chains and the false association of chain a (knob chain) with chain B (mortar chain). In fact, undesired homodimeric formation (chain a+chain a or chain b+chain B) generally occurs. This generally results in lower yields and purities of heterodimeric bifunctional antibodies (forms B and C) than production of homodimeric bifunctional antibodies (form a), which is a significant disadvantage. One key challenge remains in how to produce homogeneous bifunctional antibodies of high quality with limited or negligible by-products and impurities.

However, the inventors demonstrated that the design of an optimization strategy by using form C unexpectedly induced higher yield of bifunctional antibodies than homodimeric form B (fig. 9A). In addition, the productivity of anti-PD-1 x 1protx x is in a similar range as anti-PD-1 alone (anti-PD-1*1 or anti-PD-1*2), which productivity is equal to 45mg/L (n=5) under similar production conditions.

Another major problem with heterodimeric antibody production is purity. While the pestle-mortar strategy favors heterodimer production (chain a+chain B) and reduces homodimer chain a or homodimer chain B production, this strategy is not 100% efficient and requires additional purification to isolate the heterodimer construct (Wang et al, 2019, antibodies,8, 43).

However, the inventors observed that after the generation of anti-PD-1*1/Prot X1 form C, a high yield of heterodimers was obtained. FIG. 8 shows size exclusion chromatography of anti-PD-1*1/IL-7 wt 1 (FIG. 8A) and anti-PD-1*1/IL-7 v 1 (FIG. 8B) after protein A purification. Corresponding to one major peak of heterodimeric forms chain a and chain B, the homodimeric form Fc/Fc (chain a+chain a) was not detected, the homodimeric form (chain a+chain a) was very small (less than 2%). These data indicate that the anti-PD-1*1/protX 1 of the invention is optimized for productivity and prevents mismatches. In contrast to the prior art, heterodimer yields of about 70 to 75% of heterodimers were obtained with other bispecific antibody backbones using the same KIHs-s strategy.

To obtain such purity, the inventors have optimized the design of the molecule. In fact, they observed that this high purity could be obtained only when the a chain is the Fc domain and the B chain is anti-PD-1 x 1 il-7*1. Co-transfection of VL+ single chain B (anti-PD-1*1/IL-7 v 1) containing the mortar mutation into CHO mammalian cells did not induce chain B homodimer (0 mg/L). In contrast, if anti-PD-1*1/IL-7 v 1 is the A chain containing the pestle mutation, high yields of homodimeric chain A (88 mg/L) were obtained after co-transfection of VL+ single chain A (anti-PD-1*1/IL-7 v 1). Based on these data, the inventors chose to design molecules with Fc as chain a and anti-PD-1*1/protX 1 as chain B to avoid the production of chain a homodimers.

Taken together, these data indicate that form C anti-PD-1*1/ProtX 1 with chain a (Fc domain with knob mutation) and chain B (anti-PD-1*1/IL-7 with knob mutation) according to the invention is the best construct to achieve high productivity and product purity. This facilitates development as a therapeutic agent to achieve mass production.

Example 5: a competitive assay to measure the activity of anti-PD 1 bifunctional antibodies and their ability to antagonize PD-1/PD-L1 interactions.

Binding of the bifunctional antibodies to human PD-1 receptors and antagonism of PD-L1/PD-1 interactions were measured by ELISA assays. Recombinant hPD (Sino Biologicals, beijing, china; reference 10377-H08H) was immobilized on plastic at 0.5 μg/ml in carbonate buffer (pH 9.2) and purified antibodies were added to measure binding. After incubation and washing, peroxidase-labeled donkey anti-human IgG (Jackson immunoresearch; U.S. reference 709-035-149) was added and shown by conventional methods.

To measure antagonist activity of the bifunctional antibodies, a competitive ELISA assay was performed by a PD-1:PD-L1 inhibitor screening ELISA assay pair (Acrobiosystems; U.S. reference EP-101). In this assay, recombinant hPDL1 was immobilized on plastic at 2. Mu.g/ml in PBS pH 7.4 buffer. Purified antibodies (different concentrations) were mixed with biotinylated human PD1 (Acrobiosystems; U.S. reference EP-101) at a final concentration (fixed concentration) of 0.66. Mu.g/ml to measure competitive binding for 2 hours at 37 ℃. After incubation and washing, peroxidase-labeled streptavidin (Vector laboratoring; U.S. Pat. No. SA-5004) was added to detect c-binding and shown by conventional methods.

Results: fig. 9 and table 5 show that all bifunctional anti-PD-1*1/protX 1 molecules exhibit potent PD-1 binding. These data indicate that all molecules with each type of fusion protein, even with one anti-PD-1 valency, retain good binding to the PD-1 antigen. In addition, antagonist activity was also retained as shown in table 6.

Table 5: EC50 (nM) for PD-1 binding in figure 11.

Table 6: EC50 (nM) competition PD-1/PD-L1 ELISA assay.

Example 6: an anti-PD-1/cytokine bifunctional antibody may activate pSTAT5 signaling into primary human T cells.

The inventors next assessed the biological activity of the proteins fused to anti-PD-1 antibodies and tested the ability of all anti-PD-1/cytokine bifunctional molecules to activate primary T cells. For this purpose, human peripheral blood T cells were treated with different concentrations of anti-PD-1*1/IL-7wt 1, anti-PD-1*1/IL-7v 1, anti-PD-1*1/IL-7v 1, anti-PD-1*1/IL-15 x 1, anti-PD-1*1/IL-21 x 1 constructs for 15 min at 37 ℃. After incubation, cells were fixed, permeabilized and stained with anti-pSTAT 5 antibody.

Results: FIG. 10A shows that anti-PD-1*1/IL-7 wt 1, anti-PD-1*1/IL-7 v 1, anti-PD-1*1/IL-15 x 1 and anti-PD-1*1/IL-21 x 1 are effective in inducing pSTAT5 signaling to primary T cells (CD3+ T cells), indicating that cytokines fused to the Fc domain of form C anti-PD-1 molecules retain their ability to stimulate human T cells. Next, the inventors compared the efficacy of anti-PD-1/IL-7 form a (anti-PD-1*2/IL 7v x 2) to activate pSTAT5 signaling compared to anti-PD-1*1/IL-7 v x 1 form C. Since the anti-PD-1*1/IL-7 v 1 construct contained only one IL-7v cytokine, the molecules were expected to have lower pSTAT5 activation compared to form a. However, as shown in fig. 10B, a higher pSTAT5 activation of anti-PD-1*1/IL-7 v x 1 (form C) compared to anti-PD-1*2/IL-7 v x 2 construct (form a) was unexpectedly observed, indicating that the anti-PD-1/ProtX 1 construct (form C) of the invention allows for an optimal conformation of the IL-7 molecule to promote activation of IL-7 signaling in primary T cells.

Example 7: anti-PD-1 bifunctional molecules allow preferential binding to PD-1+ cells over PD-1-cells, and anti-PD-1/IL 7 molecules allow synergistic activation of TCR signaling in PD-1+ t cells.

The inventors evaluated the ability of anti-PD-1 bifunctional molecules to target PD-1+ t cells and allow preferential delivery and cis-binding of cytokines or proteins fused to PD-1+ cells. U937 PD-1-cells and U937 PD1+ cd127+ cells were co-cultured (ratio 1:1) and incubated with anti-PD 1/ProtX molecules at increasing doses. Binding and IL-7R signaling (pSTAT 5) were quantified by flow cytometry. EC50 (nM) and pSTAT5 activation were determined for each construct and binding of each PD-1+ and PD-1-cell population. Meanwhile, the binding of the bifunctional antibody was detected with anti-IgG-PE (Biolegend, clone HP 6017) and analyzed by flow cytometry.

Results: FIG. 11A shows that all anti-PD-1/protX molecules, namely anti-PD-1*1/IL-2*1, anti-PD-1*1/IL-21X 1, anti-PD-1*1/IL 15X 1 molecules bind to PD-1+ cells more efficiently than PD-1-cells, and that the efficacy of anti-PD-1*1 and anti-PD-1*2 antibodies is similar, demonstrating the high binding efficacy of the molecules constructed with one anti-PD-1 valency compared to 2 anti-PD-1 valencies. FIGS. 11B and 11C show the binding of anti-PD-1*1/IL-7wt 1 and anti-PD-1*1/IL 7v 1 molecules on cells expressing CD127+ alone or coexpression of CD127 and PD-1 receptors. The data show that these two molecules preferentially bind to PD-1+cd127+ cells compared to PD-1-cd127+ cells, with efficacy comparable to anti-PD-1 (anti-PD-1*2) alone. At the same time, activation of PSTAT5 signaling into PD-1+ cells and PD-1-cells was also assessed, as shown in fig. 11D. IL7R signaling pSTAT5 was strongly activated into PD-1+CD127+ cells (58 to 315 fold higher activation) compared to PD-1-CD127+ cells treated with anti-PD-1*1/IL-7 wt or IL7v 1 antibodies, whereas isotype/IL 7 antibodies have similar efficacy in PD-1+ and PD-1-cells confirming that the anti-PD-1 domain of the anti-PD-1*1/1L-7*l molecule allows preferential binding of IL-7 on PD-1+ cells, i.e., targeting of the drug and activation on the same cell. This aspect is related to the biological activity of the drug in vivo, because anti-PD-1 IL-7 concentrates IL-7 or other molecules fused to bifunctional molecules on PD-1+ tumor-specific T cells into the tumor microenvironment on PD-1 negative naive T cells. Taken together, the data indicate that only one arm against PD-1 is sufficient to allow selective delivery of the fusion cytokine on PD-1+ cells.

Next, the inventors evaluated the biological impact of cis-targeting of anti-PD-1 bifunctional molecules on PD-1+t cells. Detection was performed using the Promega PD-1/PD-L1 kit (reference number J1250). Briefly, two cell lines were used: (1) Effector T cells (Jurkat that stably express PD-1, NFAT-induced luciferase) and (2) activated target cells (CHO K1 cells that stably express PD-L1 and surface proteins) aimed at stimulating homologous TCRs in an antigen-independent manner. When cells are co-cultured, the PD-L1/PD-1 interaction inhibits TCR-mediated activation, thereby blocking NFAT activation and luciferase activity. The addition of anti-PD-1 antibodies blocks PD-1 mediated inhibition signals and restores TCR-mediated signaling, resulting in NFAT activation and luciferase synthesis and emission of bioluminescent signals.

The results of the bioassays are presented in fig. 12A and show that the bifunctional anti-PD-1*1/IL 7wt 1 molecule is superior to anti-PD 1 x 1 or anti-PD 1 x 1+ non-targeted isoform-IL 7 (as a separate compound) activating TCR-mediated signaling (NFAT), suggesting that the bifunctional molecule has a synergistic effect on pd1+ T cells. anti-PD-1*1/IL-7v 1 molecules, including IL-7 mutants, also showed a significant synergistic effect in reactivating NFAT signaling on T cells (fig. 12B). These data indicate that fusion of one IL-7 cytokine with one anti-PD-1*1 advantageously induces higher activation of TCR signaling, whereas the combined strategy of two individual compounds does not induce such efficacy.

Example 8. Bifunctional molecules with one anti-PD-1 valency exhibit better pharmacokinetics in vivo.

The pharmacokinetics and pharmacodynamics of the product were evaluated in mice after a single injection. C57bI6JRj mice (6-9 week females) were injected intravenously or intraperitoneally with a single dose (34 nmol/kg) of anti-PD-1 or bifunctional antibody. Plasma drug concentrations were determined by ELISA using immobilized anti-human light chain antibody (clone NaM76-5F 3) followed by the addition of serum-containing antibodies. Detection was performed by adding peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch; U.S.; reference 709-035-149) and was shown by conventional methods. The area under the curve for each construct corresponding to drug exposure was calculated.

Results: the inventors first compared the pharmacokinetics of all the different bifunctional forms depicted in fig. 6 (forms A, B and C). FIG. 13 shows the pharmacokinetic profile of anti-PD-1/IL-7 v constructed with 1 or two IL-7v cytokines and 1 or 2 anti-PD-1 valencies after a single intravenous (FIG. 13A) or intraperitoneal (FIG. 13B) injection. Compared to anti-PD-1*2/IL-7 v x 2 (form a) and anti-PD-1*2/IL-7 v x l (form B), anti-PD-1*1/IL-7 v x 1 (form C) exhibited the best pharmacokinetic profile. Both intravenous and intraperitoneal injection demonstrated that anti-PD-1*1/IL-7*1 (form C) is the best construct to enhance the pharmacokinetics of bifunctional molecules.

Next, the inventors tested whether bifunctional molecules with 1 anti-PD-1 arm (anti-PD-1*1) and other cytokines (IL-7 wt, IL-21, IL-2, IL-15) exhibited better in vivo pharmacokinetic profiles than the same bifunctional molecules constructed from 2 anti-PD-1 arms. Figure 14 shows the set of all tested proteins (AUC and fold change AUC) and figure 15 shows the results for each construct. The data show that all bifunctional molecules anti-PD-1*1/ProtX (form C) exhibit significantly better pharmacokinetic profiles than the corresponding bifunctional molecules anti-PD-1*2/ProtX (form B).

In another experiment, the pharmacokinetics of anti-PD-1*2 or anti-PD-1*1 antibodies alone were also evaluated to understand whether the anti-PD-1 construct alone could provide a better pharmacokinetic profile or whether the observations apply only to bifunctional molecules. Fig. 16 shows that anti-PD-1*2 and anti-PD-1*1 have similar profiles after intravenous (fig. 16A) or intraperitoneal (fig. 16B) injection, indicating that, surprisingly, the anti-PD-1*1 construct induced a better pharmacokinetic profile for bifunctional molecules only.

Poor pharmacokinetic profiles are a well-known challenge faced by bifunctional antibodies. Bifunctional antibodies are rapidly eliminated and show a short half-life in vivo, limiting their clinical use. Good drug concentrations (20 and 100 nM) corresponding to satisfactory in vivo PK values were maintained with the anti-PD-1*1/ProtX 1 construct for at least 48-72 hours, whereas only 2nM of anti-PD-1 x 2i 7 x 2 molecules were detected in plasma. Form C of the invention is able to improve the in vivo pharmacokinetic profile with longer exposure compared to other forms of bifunctional antibodies (forms B and C).

Example 9 anti-PD-1/IL-7*1 bifunctional molecules promoted proliferation of T cells in vivo and induced significant anti-tumor efficacy compared to anti-PD-1*2/IL 7*2 or anti-PD-1*2/IL 7*1 constructs.

Following intraperitoneal injection of a single dose of bifunctional molecule (34 nM/kg) into subcutaneous MC38 tumors, T cell proliferation was assessed in vivo. On day 4 post-treatment, blood and tumors were collected and T cells were stained with anti-Ki 67 antibody for quantitative proliferation by flow cytometry.

In vivo efficacy was assessed in 2 different in situ isogenic models (liver cancer model and mesothelioma in situ model). These experiments used immunocompetent mice genetically modified to express human PD-1 (exon 2). For mesothelioma model, AK7 mesothelial cells were injected intraperitoneally (3 x 10 ⁶ Individual cells/mice). For the Hepa 1.6 model, 2.5×10 is injected intravenously in the portal vein ⁶ Individual cells. In experiment 1, mice were treated with PBS, anti-PD-1 control (anti-PD-1*2), anti-PD-1*1/IL 7v 1at similar drug exposure concentrations. In experiment 2, mice were treated with PBS, anti-PD-1 control (anti-PD-1*2), anti-PD-1*1/IL 1at by 7wt% at similar drug exposure concentrations. AK7 cells stably expressed luciferase allowing quantification of tumor burden in vivo after D-luciferin injection. Data in photons per second per square centimeter per steradian The numbers were analyzed and represent the average of the dorsal and ventral signals.

Results: figure 17A shows that anti-PD-1*1/IL 7wt 1 or IL7v 1 bifunctional molecules (form C) promote significant proliferation of CD4 and CD8T cells to a higher extent than anti-PD-1 antibodies (anti-PD-1*1 or anti-PD-1*2). Significantly superior CD 4T cells were also observed for these 2 constructs compared to the anti-PD-1*2/IL 7 x 2 (form a) and anti-PD-1*2/IL 7 x 1 (form B) constructs. Similarly, higher proliferation was observed following treatment with anti-PD-1*1/IL 7wt 1 or anti-PD-1*1/IL-7 v 1 compared to anti-PD-1*2/IL 7 x 2 (form a) and anti-PD-1*2/IL 7 x 1 (form B) constructs. These data demonstrate the efficiency of the different constructs to activate pSTAT5 signaling into T cells (fig. 10A), with the anti-PD-1*1/IL 7 x 1 construct inducing higher pSTAT5 signaling than the anti-PD-1*2/IL 7 x 2 construct.

Interestingly, the inventors observed that anti-PD-1*1/IL 7wt 1 or IL7v 1 significantly induced proliferation of stem cell-like effector memory CD8T cells into tumors to a significantly higher extent than anti-PD-1 x 2IL-7*2 and anti-PD-1*2 molecules (fig. 17B). The ability of anti-PD-1 x 1il-7*1 to enhance tcf1+ stem cell-like CD8T cell populations is of particular interest, as this is critical for immune control of cancer. These cells are capable of self-renewal, producing a tumor-specific T cell bank with high effector functions.

Fig. 18A and 18B show in vivo efficacy of anti-PD-1 x 1il-7wt 1 and anti-PD-1 x 1il-7v 1 in an in situ liver cancer model. In two separate experiments, anti-PD-1*1/IL 7*1 (wild-type and variant) (form C) showed significantly superior efficacy compared to anti-PD-1*2 antibodies. 85% complete tumor eradication (complete response) was obtained after treatment with anti-PD-1*1/IL 7v, whereas only 16% of mice treated with anti-PD-1*2 produced complete tumor response.

In contrast, the inventors also tested anti-PD-1*2/IL 7v 2 constructs, and observed lower anti-tumor efficacy of the anti-PD-1*2/IL-7*2 constructs in the same liver cancer model, indicating superior in vivo activity of the anti-PD-1*1/IL 7 x 1 constructs compared to anti-PD-1*2/IL-7*2.

In mesothelioma in situ model (fig. 19A and B), anti-PD-1*1/IL 7v 1 exhibited high anti-tumor efficacy with >85% complete response, similar to anti-PD-1*2 antibody treatment. These data indicate that anti-PD-1*1/IL 7v 1 is highly potent in the anti-PD-1 sensitive model, indicating that even though form C contains only one anti-PD-1 arm (anti-PD-1*1), the drug shows similar efficacy as the 2-valent anti-PD-1.

Taken together, these data underscores that the design of bifunctional antibodies is critical to achieving antitumor efficacy in vivo. The fusion of one cytokine or protein with anti-PD-1*1 (form C) showed optimal anti-tumor efficacy and proliferation of T cells in vivo, whereas the bifunctional molecule constructed with 2 anti-PD-1 arms and one or 2 cytokines or proteins did not induce efficient proliferation of T cells in vivo nor had anti-tumor efficacy.

Example 10: the anti-PD-1 x 1IL-7v x 1 construct abrogated the inhibitory function of Treg in vitro to a greater extent than the IL-7 cytokine and anti-PD-1 x 1IL7wt x 1 bifunctional antibody.

Although anti-PD 1 therapies stimulate T cell effector function, immunosuppressive molecules (TGFB, IDO, IL-10.) and regulatory cells (Treg, MDSC, M macrophages) create an unfavorable microenvironment that limits the full potential of the therapy. Treg cells express low levels of IL-7R (CD 127), but they are still able to stimulate pSTAT5 after IL-7 treatment, and IL7 is known to release the Treg inhibition function [A et al j. Immunol.2015,195,31393148; liu W et al J Exp Med.2006,203,1701-1711; seddiki N et al J exp Med 2006,203,1693-1700; codarri L et al J exp Med 2007,204,1533-1541; heninger AK et al J immunol 2012,189,5649-5658). To assess the efficacy of anti-PD-1/IL 7 constructs to abrogate Treg function compared to IL-7, inhibition assays were performed by co-culturing Treg and T effector cells. The inventors observed in figure 20 that IL-7 or anti-PD 1-IL7 treatment blocked Treg-mediated inhibition, allowing Teff cells to proliferate even in the presence of Treg cells. anti-PD 1 antibodies are unable to inhibit Treg inhibitory activity of T effector cells.

Unexpectedly, anti-PD-1 x 1IL7w 142h x 1 showed the highest efficacy in inhibiting Treg function compared to IL-7 cytokines (< p < 0.05), and also compared to the anti-PD-1 x 1IL7wt x 1 construct. These data underscores the advantages of using the anti-PD-1 x 1IL7 x 1 construct over the naked IL-7 cytokine or non-mutated version of the anti-PD-1 x 1 bifunctional antibody. Unexpectedly, monovalent variants affected Treg elimination and at the same time strong proliferation of T cells, which is a dual effect, because of the lower affinity of the selected IL7 variant W142H for IL7R compared to the wild-type form of IL-7 cytokine.

The method comprises the following steps: the in vitro inhibitory activity of tregs on CD8 effector T cell proliferation was assessed. Cd8+ effector T cells and autologous cd4+cd25 high CD127 low tregs were sorted from peripheral blood of healthy donors and stained with cell proliferation dye (cd8+ T cells CPDe 450). Treg/CD8+Teff were then co-cultured on OKT3 coated plates (2. Mu.g/mL) at a 1:1 ratio for 5 days and proliferation of Teff cells was quantified by flow cytometry according to CPD marker loss.

Example 11: among 2 different tumor models, anti-PD-1 x 1il-7v x 1 showed superior in vivo efficacy compared to the anti-PD-1 x 1il7wt x 1 construct.

In vivo efficacy was assessed in 2 different in situ isogenic models (liver cancer model and mesothelioma in situ model). These experiments used immunocompetent mice genetically modified to express human PD-1 (exon 2). For mesothelioma model, AK7 mesothelial cells were injected intraperitoneally (3 x 10 ⁶ Individual cells/mice). For the Hepa 1.6 model, 2.5×10 is injected intravenously in the portal vein ⁶ Individual cells. In experiment 1, mice were treated with PBS, anti-PD-1 control (anti-PD-1*2), anti-PD-1*1/IL 7v 1 (anti-PD-1 x 1IL7w142h 1) or anti-PD-1*1 IL7wt 1 at similar drug exposure concentrations. AK7 cells and hepal.6 stably expressed luciferase, allowing in vivo quantification of tumor burden after D-luciferin injection. The data were analyzed in photons per second per square centimeter per steradian and represent the average of dorsal and ventral signals.

AK7 intraperitoneal models were highly sensitive to PD-1 antibody treatment associated with high cd4+ and cd8+ T cell infiltration, and PD-1 expressing T cells were observed to infiltrate the tumor microenvironment, allowing for good response of anti-PD-1 antibodies, as shown in figure 19. In the same experiment, the efficacy against PD-1 x 1IL7v x 1 was compared to the efficacy of its wild-type IL7 homolog construct (anti-PD-1 x 1IL7wt x 1). The anti-PD-1 x 1il 1 v x construct induced 92% of complete response (n=1 death/14 mice) and had excellent efficacy compared to the anti-PD-1 x 1il7wt x l construct inducing moderate antitumor efficacy (62% of complete response) (fig. 21A). Tumor bioluminescence analysis demonstrated that anti-PD-1 x 1IL induced tumor clearance within 11 to 18 days after treatment, whereas in the anti-PD-1 x 1IL wt group, tumors shrink after treatment and eventually recur (data not shown), indicating that the efficacy of the IL-7 wild-type construct may be transient compared to a bifunctional antibody constructed with low affinity IL-7 (IL 7W 142H).

To evaluate the memory response induced by anti-PD-1 x 1il7v x 1 treatment, all cured mice treated with anti-PD-1 x 1il7v x 1 were re-challenged with a secondary injection of AK7 mesothelioma cells. As shown in fig. 21B, no tumor bioluminescence was detected after tumor re-excitation, whereas high bioluminescence signals were detected at various time points in the initially excited mice. These data indicate that anti-PD-1 x 1il7v x 1 induced a strong and long-term specific memory anti-tumor response without any new treatment.

Although the affinity of anti-PD-1 x 1IL for IL-7R was low, this construct showed unexpectedly higher efficiency than the anti-PD-l IL-7wt l construct and retained its antagonist anti-PD-1 activity, such as anti-PD-1*2 antibody, in a PD-1 sensitive tumor model. These data underscores that anti-PD-1 x 1il7v x 1 is the preferred construct to maintain PD-1 inhibitory receptor blocking activity in vivo. The inventors hypothesize that mutations in IL-7 will balance the affinity of bifunctional antibodies for PD-1+ tumor-specific T cells compared to PD-1-cd127+ non-tumor-specific T cells (as depicted in fig. 11D), resulting in better in vivo drug efficacy.

To evaluate efficacy in the anti-PD (L) 1 refractory model to mimic primary drug resistance in cancer patients, a mouse model of hepatocellular carcinoma hepal.6 was selected. It is an in situ homology model implemented in immunocompetent mice (expressing human PD-1). This model is of particular interest since tumor T cells are excluded from the described tumors (Gauttier V et al 2020,Clin Invest,130,6109-6123). The efficacy of anti-PD-1 x 1IL with low affinity for IL7R and anti-PD-1 x 1IL with high affinity for IL7R were compared side-by-side in the same experiment. In the different groups, mice were treated with PBS (control), anti-PD-1*2 or isotype IL7 x v x l (homologous construct of bispecific antibody targeting antiviral protein envelope, isotype control used as experiments) at the same drug exposure concentration. anti-PD-1 x 1IL achieved a 60% full tumor response, significantly better than anti-PD-1 x 1IL wt (only 47% full response) constructed with high affinity wild-type IL7, as shown in figure 22. In this model, anti-PD 1 antibodies were not as expected to be effective. Furthermore, isotype IL7v IL was not of any efficacy in this model, demonstrating that the use of anti-PD-1/IL 7 constructs in combination with anti-PD-1 and IL-7 therapies is a good therapeutic strategy to enhance T cell activation and anti-tumor response in PD-1 refractory models.

anti-PD-1 x 1il7v x 1 treatment-induced memory responses were also tested in this model, and the same tumor loss was observed.

Taken together, these data demonstrate the superior efficacy of anti-PD-1/IL-7 constructs with one anti-PD-1 valency and one IL-7 cytokine mutation (W142H) with lower affinity for its CD127 receptor.

Example 12: the in vivo efficacy of anti-PD-1 x 1il-7v x l in anti-PD-1 refractory models is associated with strong transcriptional activity of anti-PD-1 receptor and intratumoral proliferation of stem cell-like memory CD 8T cell subpopulations (tcfl+tox-cells).

Transcriptome analysis of whole tumors was also performed to better understand the effect of anti-PD-1 x 1il7v x 1 on tumor microenvironment. Using Nanostring techniquePan cancer immune profile analysis group) to detect and quantify gene expression. The data were normalized to multiple reference genes contained in the group, with the background threshold being the geometric mean of the negative control. Differential Expression (DEG) of the genes was analyzed using the R-package. The unsupervised hierarchical cluster heatmap of DEG analyzed by DESeq2 of fig. 23 shows that the transcriptional expression pattern between anti-PD-1 and anti-PD-1 x 1 w14h x 1 groups is highly similar and significantly different from PBS group, indicating that the anti-PD-1 domain of the anti-PD-1 x 1 v x 1 construct is in vivo The antagonist bioactivity is retained, although it has an anti-PD-1 valence. Protein-protein interaction network function enrichment analysis using STRING against PD-1*2 or against genes upregulated after PD-1 x 1il7w142h x 1 treatment compared to PBS conditions, several clusters of genes involved in chemotactic immunoreceptor activity, jak-STAT cytokine signaling and antigen presentation (MHC protein complex binding and TCR signaling) were determined. Using the single sample GSEA (ssGSEA) signaling algorithm from R-packet GSVA in genes differentially expressed between anti-PD-1*2 and anti-PD-1 x 7w142h x 1, the inventors observed significant upregulation of CD8 or CD 4T early activation/memory stem cell-like T cell characteristics associated with TCF7, CCR7, SELL, IL7R gene expression in the anti-PD-1 x 7w142h x 1 group compared to the anti-PD-1 and PBS groups (fig. 23B). In contrast, up-regulation of depleted CD 8T cell genes (LAG 3, PRF1, CD8A, FIAVRC2, GZMB, CD8B1, KLRD1, TNFRSF9, TIGIT, CTSW, CCL4, CD63, IFNG, CXCR6, FASL, CSF 1) was observed as expected in the anti-PD-1 treatment group. The genetic characteristics of depleted T cells and the characteristics of naive/stem cell-like memory T cells were adapted from (Andreatta et al, nature comm 2021) which defined different T cell subsets in cancer using single cell transcriptome analysis.

CD 8T cell infiltrating lymphocytes were also phenotyped by flow cytometry to further characterize the population induced by anti-PD-1 x 1il7v x 1 treatment. Although T cells were excluded from the tumors initially described in this drug resistance model, tumor Infiltrating Lymphocyte (TIL) composition significantly increased after anti-PD-1 x 1il7w142h x 1 treatment and the product significantly altered T cell subpopulations (fig. 23B and C, 24C). Flow cytometry analysis showed that anti-PD-1 x 1il7w142h x 1 altered the composition of the tumor microenvironment and favored accumulation of CD 8T cells over CD4 while not affecting Treg (fig. 24A). A substantial increase in the percentage of cd8+cd44+ activated T cells with a stem cell-like memory T cell phenotype (cd3+cd8+cd44+tcf1+tox-) was observed after treatment (fig. 24B), which also expressed the ki67 proliferation marker (fig. 24C). anti-PD-1 treatment induces the accumulation of TOX-TCF1 or TOX+TCF1-associated depletion phenotypes into tumors (Utzschneider et al, immunity 2016,45,415-427; mann et al, 2019Nature immunology,20,1092-1094). These data confirm transcriptome analysis and further confirm that T cells activated against the PD-1 x 1il7w142h x 1 molecule express a CD44 activation marker, indicating that this T cell subpopulation is not the initial T cell subpopulation, but an early activated stem cell-like memory T cell subpopulation (tcf1+tox-). These data also demonstrate example 9, which example 9 describes the efficacy of anti-PD-1 x 1il7w142h x 1 in promoting accumulation and proliferation of stem cell-like memory T cells in another tumor model.

Example 13: anti-PD-1 x 1il7v x 1 maintains survival of long-term stimulated human T cells and induces proliferation of tcf1+ T cells.

To demonstrate the effect of anti-PD-1 x l IL7v x 1 on human T cells, the inventors tested the effect of anti-PD-1/IL 7 constructs in an in vitro long-term antigen stimulation model. Human PBMC were stimulated repeatedly every 3 days on CD3 CD28 coated plates (3. Mu.g/mL OKT3 and 3. Mu.g/mL, CD28.2 antibody). At each stimulation, anti-PD-1 x 1il7v x 1 (anti-PD-1 x 1il7w142h x 1) constructs, isotype controls, or anti-PD-1*1 antibodies were added to the cultures. T cell viability and phenotype were assessed by flow cytometry 24 hours after the fifth stimulation.

Fig. 25A shows that anti-PD-1 x 1il7w142h x 1 maintained survival of long-term depleted T cells compared to anti-PD-1 treatment. Phenotypic analysis of T cells (fig. 25B) demonstrated that anti-PD-1 x 1il7v x 1 promoted specific proliferation and maintenance of tcf1+cd8t cell subsets. The tcf1+ T cell population is described as a stem cell-like T cell population capable of self-renewal and long-term effective responses. These results allow prediction of long term effects in solid tumors by reactivating TIL with anti-PD-1 x 1il in early cancer (adjuvant or new adjuvant case), preventing proliferation of depleted T cells in primary or secondary resistance to immune tumor therapy or other cancer therapies, and in various immune tumor escape cases.

Example 14: anti-PD-1 x 1il-7v x 1 shows in vivo monotherapy efficacy in different PD-1 therapy resistant humanized models.

In a Triple Negative Breast Cancer (TNBC) model (immunodeficient mice subcutaneously implanted with breast cancer cells MDA-MB 231), mice were humanized with human Peripheral Blood Mononuclear Cells (PBMCs) from 4 different donors and then treated with PBS, anti-PD-1*2 or anti-PD-1 x 1i l7w142h x 1 bifunctional antibodies. In all PBMC donors tested, anti-PD-1 x 1il7v x 1 reduced tumor growth, whereas anti-PD-1*2 alone had no effect (fig. 26).

In another humanized mouse model (lung cancer model (a 549)), the efficacy of anti-PD-1 x 1il was demonstrated relative to anti-PD-1*1, correlated with an increase in IFNg secretion in serum of anti-PD-1*1 treated mice (day 34) (fig. 27). Both models demonstrate that anti-PD-1 x 1il7v can also modulate human immune-mediated anti-tumor responses to a greater extent than anti-PD-1*2.

Example 15: anti-PD-1 x 1il7v x 1 showed a better pharmacokinetic profile in cynomolgus monkeys than anti-PD-1 x 1il7wt 1 molecules.

Cynomolgus monkey is injected intravenously with one dose of anti-PD-1 x 1il (0.8 mg/kg, 4.01 mg/kg) or one dose of anti-PD-1 x 1il (anti-PD-1 x 1il7w142h 1) 0.8mg/kg, 4.01mg/kg or 25 mg/kg. Following injection, serum was collected at various time points to quantify the anti-PD-1 IL7 constructs by ELISA immunoassay using MSD technology. Briefly, human PD1 protein was immobilized and serum anti-PD-1 x 1il7 x 1 antibody was added. ELISA was shown using sulfo-labeled anti-human kappa light chain monoclonal antibodies.

Pharmacokinetic data for both constructs were linear and dose dependent. However, with the anti-PD-1 x 1il7v x l construct, a better pharmacokinetic profile (fig. 28) was observed (area under the curve 29.6vs 108,IL7wt vs IL7v, dose 4.01 mg/kg) compared to the anti-PD-1 x 1il7wt x l construct. Interestingly, anti-PD-1 x 1IL7v x 1, which has low affinity for IL7 receptor, induced CD 8T cell proliferation in vivo until days 10-14, indicating that the biological effect of the drug extends beyond pharmacokinetic exposure. These data allow the creation of a new pharmacodynamic model that measures long term effects on non-human primate T cell subsets and is applicable to human cases: i.e. cd8+ T cells proliferated after only one injection of anti-PD-1 x 1il7v x 1 bifunctional antibody.

Example 16: anti-PD-1 x 1il7v x 1 constructed with the IgG 1N 297A isotype or with the LALA PG IgG1 isotype had the same efficacy in activating pSTAT5 signaling into human T cells.

In examples 1 to 15, the IgG 1N 297A form was used for the anti-PD-1*1/cytokine construct. The inventors tested another form of Fc silencing with LALA PG additional mutations described as completely abrogating ADCC, ADCP and CDC activity, as LALA PG mutations impair binding to FcR receptors.

IL-7R activity against PD-1 x 1IL7W142H x 1 was assessed by pSTAT5 activity (FIG. 29). No differences in activity on CD4 and CD8 human T cells were noted between the 2 constructs, indicating that the invention can be constructed with different Fc silencing isoforms.

EXAMPLE 17 anti-PD-1*1/protX 1 demonstrates the absence of in vivo toxicity

In vivo toxicity was assessed following intraperitoneal injections of three doses of 5 or 20mg/kg, or a single high dose of 50mg/kg or 100mg/kg of bifunctional molecule into C57BL/6 mice. Body weight was controlled twice daily before treatment (day before and day four), on the day of treatment and after treatment. PBS was used as a control.

Results: fig. 30 shows that mice treated with anti-PD-1*1/protX x 1 exhibited similar body weight to PBS-injected control mice, indicating that long-term or acute injection of anti-PD-l/protX l (IL 7 or IL 2) did not induce toxicity even at high doses. Furthermore, mice showed no external signs of distress.

Materials and methods

ELISA binding to PD1

For the activity ELISA assay, recombinant hPD1 (Sino Biologicals, beijing, china; reference 10377-H08 FH) was immobilized on plastic at 0.5 μg/ml in carbonate buffer (pH 9.2) and purified antibodies were added to measure binding. After incubation and washing, peroxidase-labeled donkey anti-human IgG (Jackson immuno research; U.S. Pat. No. 709-035-149) was added and shown by conventional methods.

ELISA antagonists: competition between PDL1 and humanized anti-PD 1

Competitive ELISA detection was performed by a PD-1:PD-L1 inhibitor screening ELISA detection pair (Acrobiosystems; U.S. reference EP-101). In this assay, recombinant hPDL1 was immobilized on plastic at 2. Mu.g/ml in PBS pH 7.4 buffer. Purified antibodies (different concentrations) were mixed with biotinylated human PD1 (Acrobiosystems; U.S. reference EP-101) at a final concentration (fixed concentration) of 0.66. Mu.g/ml to measure competitive binding for 2 hours at 37 ℃. After incubation and washing, peroxidase-labeled streptavidin (Vector laboratoring; U.S. Pat. No. SA-5004) was added to detect biotin-PD-1 Fc binding and was shown by conventional methods.

pSTAT5 assay

PBMCs isolated from peripheral blood of human healthy volunteers were incubated with anti-PD-1/IL-7 molecules for 15 min at 37 ℃.

To determine cis-activity, U937 transduced with CD127 and PD-1 was mixed with U937 transduced with cd127+ only. Cells were stained with cell proliferation dye (CPDe 450 or CPDe670, thermosusher) mixed at a 1:1 ratio and treated with test molecules for 15 minutes at 37 ℃. Prior to co-cultivation, each cell subpopulation was labeled with a cell proliferation dye (CPDe 450 or CPDe 670). Cells were then fixed, permeabilized and stained with AF 647-labeled anti-pSTAT 5 (clone 47/Stat5 (pY 694), BD Bioscience). pSTAT5 activation in cd3+ T cell populations was assessed for human PBMCs. For the U937 assay, pSTAT5 activation of U937pd1+cd127+ cells and U937 cd127+ cells was assessed.

Cell binding assays

U937 transduced with CD127 and PD-1 was mixed with U937 transduced with cd127+ only. Cells were stained with cell proliferation dye (CPDe 450 or CPDe670, thermoshier) mixed in a ratio of 1:1. Cells were stained with yellow/dead fixable stain (thermosipher) and then stained with human Fc blocker (BD Bioscience) diluted in PBS2% human serum. Cells were then stained with a range of concentrations of test molecule and antibody display was performed using anti-human IgG-PE antibody (Biolegend, clone HP 6017) and in vivo pharmacokinetics of anti-PD-1/protX was analyzed by flow cytometry

To analyze pharmacokinetics, a single dose of the molecule was injected intra-orbital or intra-peritoneal or intravenous (retroorbital) into C57bl6JrJ mice (female 6-9 weeks), serum was diluted with immobilized anti-human light chain antibody (clone NaM76-5F 3) containing IgG fusion 1167, and drug concentration in plasma was determined by ELISA. Detection was performed with peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch; U.S.; reference 709-035-149) and was shown by conventional methods.

T cell activation assay using Promega cell bioassay

The ability of anti-PD-1 antibodies to restore T cell activation was tested using the Promega PD-1/PD-L1 kit (reference number J1250). Two cell lines were used: (1) Effector T cells (Jurkat which stably express PD-1, NFAT-induced luciferase) and (2) target cells for activation (CHO K1 cells which stably express PDL1 and surface proteins, intended to stimulate cognate TCR in an antigen-independent manner. When the cells are co-cultured, PD-L1/PD-1 interactions inhibit TCR-mediated activation and thus block NFAT activation and luciferase activity. Addition of anti-PD-1 antibodies can block PD-1 mediated inhibition signals resulting in NFAT activation and luciferase synthesis and emission of bioluminescent signals. Experiments were performed as suggested by the manufacturer. Serial dilutions of test molecules, PD-L1+ target cells, PD-1 effector cells and test molecules were tested after four hours of co-culture, bioGlo ^TM Fluorescein substrate was added to the wells and Tecan was used ^TM The photometer reads the plate.

Proliferation in vivo

A single dose of bifunctional molecule (34 nM/kg) was injected intraperitoneally into C57bl6JrJ mice (female 6-9 weeks) carrying subcutaneous MC38 tumors. Mice were treated with one dose (34 nM/kg) by intraperitoneal injection. On day 4 post-treatment, blood was collected and T cells were stained with anti-CD 45, CD3, anti-CD 8, anti-CD 4 and anti-ki 67 antibodies to quantify proliferation by flow cytometry.

Humanized PD1 knock-in a mouse model

Efficacy of anti-PD-1/IL-7 molecules was assessed in vivo in a mouse model genetically modified to express isogenic immune activity of human PD-1 (exon 2). For the in situ mesothelioma model, AK7 mesothelial cells (3 e6 cells/mouse) were intraperitoneally injected and then treated at 4/6/8 days with equivalent drug exposure doses [ anti-PD-1*2 (l mg/kg), 4mg/kg anti-PD-1*1/IL-7*1 ]. Injected AK7 cells stably expressed luciferase, producing in vivo bioluminescent signals upon intraperitoneal injection of D-luciferin (3 μg/mouse, goldBio, san lewis missouri, usa, reference 115144-35-9). Ten minutes after fluorescein injection, bioluminescence signals were measured by Biospace Imager for 1 minute on the dorsal and ventral sides of the mice. The data were analyzed in photons per second per square centimeter per steradian and represent the average of dorsal and ventral signals. Each group represents the mean +/-SEM of 5 to 7 mice/group. For the liver cancer model, 2.5e6 cells of the hepal.6 liver cancer cells were subcutaneously injected in the portal vein. Mice were then treated at day 4/6/8 with equivalent drug exposure doses [ anti-PD-1*2 (l mg/kg), 4mg/kg anti-PD-1*1/IL-7*1 ].

Antibodies and bifunctional molecules

The following antibodies and bifunctional molecules have been used in the different experiments disclosed herein.

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Claims

1. A bifunctional molecule comprising a single antigen binding domain that binds to a target specifically expressed on the surface of an immune cell and a single immunostimulatory cytokine,

wherein the molecule comprises a first monomer comprising an antigen binding domain covalently linked via a C-terminus to the N-terminus of a first Fc chain, optionally through a peptide linker, and a second monomer comprising a complementary second Fc chain lacking the antigen binding domain and the immunostimulatory cytokine;

wherein i) the immunostimulatory cytokine is covalently linked to the C-terminus of the first Fc chain, optionally through a peptide linker; or ii) the single antigen binding domain comprises a heavy variable chain and a light variable chain and the immunostimulatory cytokine is covalently linked to the C-terminus of the light chain;

wherein the target specifically expressed on the surface of an immune cell is selected from the group consisting of: PD-1, CD28, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD, OX40, 4-1BB, GITR, HVEM, tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CD3, PDL2 and PDL1; and is also provided with

Wherein the immunostimulatory cytokine is selected from the group consisting of: IL-2 (IL is an interleukin), IL-4, IL-5, IL-6, IL-12A, IL-12B, IL-13; IL-15, IL-18, IL-21, IL-23, IL-24; ifnα, ifnβ, BAFF, ltα and ltβ, or variants thereof having at least 80% identity to wild-type proteins.

2. The bifunctional molecule of claim 1, wherein said immunostimulatory cytokine is linked to the C-terminus of said first Fc chain, preferably via its N-terminus.

3. The bifunctional molecule of claim 1 or 2, wherein the first Fc chain and the second Fc chain form a heterodimeric Fc domain, in particular a knob heterodimeric Fc domain.

4. The bifunctional molecule of any one of claims 1-3, wherein said immunostimulatory cytokine is selected from the group consisting of: IL-2, IL-15 and IL-21 or variants thereof having at least 80% identity to wild-type proteins.

5. The bifunctional molecule of any one of claims 1 to 4, wherein said immunostimulatory cytokine is IL-2 or a variant thereof having at least 90% identity to SEQ ID No. 87, preferably selected from IL-2 variants comprising one of the following combinations of substitutions relative to SEQ ID No. 87: R38E and F42A; R38D and F42A; F42A and E62Q; R38A and F42K; R38E, F a and N88S; R38E, F a and N88A; R38E, F a and V91E; R38E, F a and D84H; H16D, R E and F42A; H16E, R E and F42A; R38E, F a and Q126S; R38D, F a and N88S; R38D, F a and N88A; R38D, F a and V91E; R38D, F a and D84H; H16D, R D and F42A; H16E, R D and F42A; R38D, F a and Q126S; R38A, F K and N88S; R38A, F K and N88A; R38A, F K and V91E; R38A, F K and D84H; H16D, R a and F42K; H16E, R a and F42K; R38A, F K and Q126S; F42A, E Q and N88S; F42A, E Q and N88A; f42A, E Q and V91E; F42A, E Q and D84H; H16D, F a and E62Q; H16E, F a and E62Q; F42A, E Q and Q126S; R38E, F a and C125A; R38D, F a and C125A; F42A, E Q and C125A; R38A, F K and C125A; R38E, F42A, N S and C125A; R38E, F42A, N a and C125A; R38E, F, 42, A, V E and C125A; R38E, F42A, D H and C125A; H16D, R E, F a and C125A; H16E, R E, F a and C125A; R38E, F42A, C a and Q126S; R38D, F42A, N S and C125A; R38D, F42A, N a and C125A; R38D, F, 42, A, V E and C125A; R38D, F42A, D H and C125A; H16D, R D, F a and C125A; H16E, R D, F a and C125A; R38D, F42A, C a and Q126S; R38A, F42K, N S and C125A; R38A, F42K, N a and C125A; R38A, F, 42, K, V E and C125A; R38A, F42K, D H and C125A; H16D, R A, F K and C125A; H16E, R A, F K and C125A; R38A, F42K, C a and Q126S; F42A, E62Q, N S and C125A; F42A, E62Q, N a and C125A; F42A, E62Q, V E and C125A; F42A, E Q and D84H and C125A; H16D, F a and E62Q and C125A; H16E, F, 42, A, E Q and C125A; F42A, E62Q, C a and Q126S; F42A, N S and C125A; F42A, N a and C125A; F42A, V91E and C125A; F42A, D H and C125A; H16D, F a and C125A; H16E, F a and C125A; F42A, C a and Q126S; F42A, Y a and L72G; and T3A, F42A, Y45A, L G and C125A.

6. The bifunctional molecule of any one of claims 1 to 4, wherein said immunostimulatory cytokine is IL-15 or a variant thereof having at least 90% identity to SEQ ID No. 88, preferably an IL-15 variant comprising one of the following substitutions relative to SEQ ID No. 88: n1 3 3 4 8 11 11 11 11 11 30 61 71 71 72 72 72 72 72 72 73 79 79 79 112M and N112Y, preferably N4 61 65D/N65 30N/N65 30N/E64Q/N65 14 8 30 64/N108 1D/D61 1D/E64 4D/E64 8N/D61N/E64 1D/D30Q/Q108 1D/N4D/D8N/E64Q/N65 1D/D61N/E64Q/Q108 4D/D61N/E64Q/Q108 1D/N65D/Q108 4D/D30N/Q108 65D/Q108 30N/Q180Q/N65N 61N/E64Q/N65D/N4D/N65 71S/N72A/N77A and N4D/D61N/N65D, preferably D30N/E64Q/N65D.

7. The bifunctional molecule of any one of claims 1 to 4, wherein said immunostimulatory cytokine is IL-21 or a variant thereof having at least 90% identity to SEQ ID No. 89, preferably an IL-21 variant comprising one of the following substitutions relative to SEQ ID No. 89: r5 5 5 5 5 5 5 5 5 5 5 89 12 12 12 12 12 15 15 15 15 15 15 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12D, preferably R5E and R76 5A and R76 5Q and R76 5A and R76Q and R76 9E and R76A and R76N and R76 15N and S70N and I71 15N and K72N and K73 70T and K73 70T and R76 71L and K73L and R76 71L and R76a and K73A and R76a and R72A and R76a and R73A and R76D or K73A and R76E.

8. The bifunctional molecule of any one of claims 1 to 7, wherein the antigen binding domain is a Fab domain, fab', single chain variable fragment (scFV), or single domain antibody (sdAb).

9. The bifunctional molecule of any one of claims 1 to 8, wherein the target specifically expressed on the surface of an immune cell is selected from the group consisting of: PD-1, CTLA-4, BTLA, TIGIT, LAG3 and TIM3.

10. The bifunctional molecule of any one of claims 1 to 9, wherein the antigen binding domain binds to PD-1.

11. The bifunctional molecule of any one of claims 1 to 10, wherein the antigen binding domain comprises or consists essentially of: (i) A heavy chain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:53 and CDR3 of SEQ ID NO:55, 56, 57, 58, 59, 60, 61 or 62; and (ii) a light chain comprising CDR1 of SEQ ID NO. 64 or 65, CDR2 of SEQ ID NO. 66 and CDR3 of SEQ ID NO. 16.

12. An isolated nucleic acid sequence or a set of isolated nucleic acid molecules encoding the bifunctional molecule of any one of claims 1 to 11.

13. A host cell comprising the isolated nucleic acid of claim 12.

14. A pharmaceutical composition comprising the bifunctional molecule of any one of claims 1 to 11, the nucleic acid of claim 12, or the host cell of claim 13, optionally containing a pharmaceutically acceptable carrier.

15. The molecule according to any one of claims 1 to 11, the nucleic acid according to claim 12, the host cell according to claim 13 or the pharmaceutical composition according to claim 14 for use as a medicament, in particular for the treatment of cancer or infectious diseases.

16. A molecule, nucleic acid, host cell or pharmaceutical composition for use according to claim 15 for the treatment of cancer or viral infection by stimulating effector memory stem cell-like T cells.