Multispecific binding agent suitable for use in cancer immune therapy

Abstract

The invention relates to multispecific binding agents against immune checkpoint proteins and macrophage proteins, such as a trispecific antibody against TIM3, VISTA and CSF1R, and their use in therapy, such as the treatment of cancer.

Description

[0001] MULTISPECIFIC BINDING AGENT SUITABLE FOR USE IN CANCER IMMUNE THERAPY

[0002] FIELD OF THE INVENTION

[0003] The invention relates to multispecific binding agents, such as multispecific antibodies, and their use as a medicament, in particular for treating cancer.

[0004] BACKGROUND OF THE INVENTION

[0005] Several cancer types or patient sub-groups (40-90% depending on cancer type) remain unresponsive to current monospecific antibody (Ab) immune checkpoint blockers (ICBs). Especially non-immunogenic tumors are resistant to ICBs and tend to be low in antigen enrichment, low in T cell infiltrates but high in tumor- associated macrophages (TAMs).

[0006] Further, co-blockade of other immune-inhibitory receptors along with PD1 has been disappointing.

[0007] At present most research focusses on immune-inhibitory receptors on T cells and not TAMs, although these also express immune-inhibitory receptors. TAMs are heavily enriched in immuno-resistant tumors but current immunotherapy approaches are not yet successful in targeting them.

[0008] There is a need for biomarker-driven combinatorial therapies, including immunotherapies, against non-immunogenic tumors with high TAMs infiltrates, which constitute a major portion (40-90%) of several cancers.

[0009] Kato et al. [(2020) Oncoimmunology 9, 1708065] disclose that expression of TIM3 / VISTA checkpoints and the CD68 macrophage-associated marker correlates with anti-PDl / PDLl resistance.

[0010] SUMMARY OF THE INVENTION

[0011] The present inventors have surprisingly found that simultaneous co-inhibition or co-blockade of immune checkpoint-receptors and myeloid lineage / differentiation proteins on macrophages induces a type I interferon response which is a potent immunogenic pathway, and particularly a potent anti-tumor pathway. It was further established that this effect could be achieved by specifically targeting at least two immune-checkpoint or immune-inhibitory receptor proteins together with a specific macrophage-associated lineage or differentiation marker using a multispecific binding agent which thus preferentially binds macrophages. This is unexpected for macrophages because co-inhibition or co-blockade of immune- checkpoints or immune-inhibitory receptors to achieve anti-tumor immunity has been mainly reported for lymphocytes like T cells or NK cells. Moreover, it is unexpected that macrophage-autonomous activation through multi-specific binding agent can ensure anticancer activity.

[0012] The present invention thus discloses multispecific binding agents targeting a first immune checkpoint protein, a second immune checkpoint protein, and a macrophage protein. Variations of this construct demonstrated that the proximity of the different binding regions of the multispecific binding agent ensures an effect on macrophages which cannot be achieved by the combination of monospecific binding agents.

[0013] The invention is further summarised in the following statements.

[0014] 1. A multispecific binding agent capable of binding a first immune checkpoint protein, a second immune checkpoint protein, and a macrophage protein, such as a macrophage marker protein.

[0015] 2. The multispecific binding agent according to statement 1, wherein said first immune checkpoint protein is a transmembrane protein.

[0016] 3. The multispecific binding agent according to statement 1 or 2, wherein said second immune checkpoint protein is a transmembrane protein.

[0017] 4. The multispecific binding agent according to any of statements 1 to 3, wherein said first and said second immune checkpoint protein is a transmembrane protein.

[0018] 5. The multispecific binding agent according to any of statements 1 to 4, wherein said macrophage protein is a transmembrane protein.

[0019] 6. The multispecific binding agent according to any of statements 1 to 5, wherein said first immune checkpoint protein, said second immune checkpoint protein, and said macrophage protein is a transmembrane protein.

[0020] 7. The multispecific binding agent according to any of statements 1 to 6, wherein said first immune checkpoint protein is expressed by macrophages, preferably tumor-associated macrophages.

[0021] 8. The multispecific binding agent according to any of statements 1 to 7, wherein said second immune checkpoint protein is expressed by macrophages, preferably tumor-associated macrophages.

[0022] 9. The multispecific binding agent according to any of statements 1 to 8, wherein said first and said second immune checkpoint protein is expressed by macrophages, preferably tumor-associated macrophages. 10. The multispecific binding agent according to any of statements 1 to 9, wherein said multispecific binding agent inhibits or blocks said first immune checkpoint protein.

[0023] 11. The multispecific binding agent according to any of statements 1 to 10, wherein said multispecific binding agent inhibits or blocks said second immune checkpoint protein.

[0024] 12. The multispecific binding agent according to any of statements 1 to 11, wherein said multispecific binding agent inhibits or blocks said macrophage protein.

[0025] 13. The multispecific binding agent according to any of statements 1 to 12, wherein said multispecific binding agent inhibits or blocks said first immune checkpoint protein and said second immune checkpoint protein.

[0026] 14. The multispecific binding agent according to any of statements 1 to 13, wherein said multispecific binding agent inhibits or blocks said first immune checkpoint protein and said macrophage protein.

[0027] 15. The multispecific binding agent according to any of statements 1 to 14, wherein said multispecific binding agent inhibits or blocks said second immune checkpoint protein and said macrophage protein.

[0028] 16. The multispecific binding agent according to any of statements 1 to 15, wherein said multispecific binding agent inhibits or blocks said first immune checkpoint protein, said second immune checkpoint protein, and said macrophage protein.

[0029] 17. The multispecific binding agent according to any of statements 1 to 16, wherein said multispecific binding agent induces a type I pro-inflammatory response, such as a pro-inflammatory response comprising or consisting of type I interferon (IFN) response.

[0030] 18. The multispecific binding agent according to any of statements 1 to 17, wherein said multispecific binding agent induces a type I pro-inflammatory response, such as a pro-inflammatory response comprising or consisting of type I IFN response in macrophages, preferably tumor-associated macrophages.

[0031] 19. The multispecific binding agent according to any of statements 1 to 18, wherein binding of said multispecific binding agent to said first immune checkpoint protein induces a type I pro-inflammatory response, such as a pro-inflammatory response comprising or consisting of type I IFN response, preferably in macrophages, preferably in tumor-associated macrophages. 20. The multispecific binding agent according to any of statements 1 to 19, wherein binding of said multispecific binding agent to said second immune checkpoint protein induces a type I pro-inflammatory response, such as a pro- inflammatory response comprising or consisting of type I IFN response, preferably in macrophages, preferably in tumor-associated macrophages.

[0032] 21. The multispecific binding agent according to any of statements 1 to 20, wherein binding of said multispecific binding agent to said macrophage protein induces a type I pro-inflammatory response, such as a pro-inflammatory response comprising or consisting of type I IFN response, preferably in macrophages, preferably in tumor-associated macrophages.

[0033] 22. The multispecific binding agent according to any of statements 1 to 21, wherein said first immune checkpoint protein is a T cell immunoglobulin and mucin-domain (TIM) protein family member.

[0034] 23. The multispecific binding agent according to any of statements 1 to 22, wherein said first immune checkpoint protein is selected from TIM3, TIM1, TIM2, TIM4, TIM5, TIM6, TIM7, and TIM8.

[0035] 24. The multispecific binding agent according to any of statements 1 to 23, wherein said second immune checkpoint protein is a B7 immunoglobulin protein family member.

[0036] 25. The multispecific binding agent according to any of statements 1 to 24, wherein said second immune checkpoint protein is selected from, B7-H5 / VISTA, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, and B7-H7.

[0037] 26. The multispecific binding agent according to any of statements 1 to 25, wherein said macrophage protein is selected from CSF1R, MRC1, CD163, CD68, Fc Gamma Receptors, CD14, CDllb, F4 / 80and CD206.

[0038] 27. The multispecific binding agent according to any of statements 1 to 26, wherein said first immune checkpoint protein is TIM3.

[0039] 28. The multispecific binding agent according to any of statements 1 to 27, wherein said second immune checkpoint protein is VISTA.

[0040] 29. The multispecific binding agent according to any of statements 1 to 28, wherein said macrophage protein is CSF1R.

[0041] 30. The multispecific binding agent according to any of statements 1 to 29, wherein said first immune checkpoint protein is TIM3 and said second immune checkpoint protein is VISTA. 31. The multispecific binding agent according to any of statements 1 to 30, wherein said first immune checkpoint protein is TIM3 and said macrophage protein is CSF1R.

[0042] 32. The multispecific binding agent according to any of statements 1 to 31, wherein said second immune checkpoint protein is VISTA and said macrophage protein is CSF1R.

[0043] 33. The multispecific binding agent according to any of statements 1 to 32, wherein said first immune checkpoint protein is TIM3, said second immune checkpoint protein is VISTA, and said macrophage protein is CSF1R.

[0044] 34. The multispecific binding agent according to any of statements 1 to 33, comprising a first antigen-binding region capable of binding to the first immune checkpoint protein, a second antigen binding region capable of binding the second immune checkpoint protein, and a third antigen binding region capable of binding to the macrophage protein.

[0045] 35. The multispecific binding agent according to any of statements 1 to 34, comprising a polypeptide capable of binding said first immune checkpoint protein, said second immune checkpoint protein, and said macrophage protein.

[0046] 36. The multispecific binding agent according to any of statements 1 to 35, comprising one or more antibody or antigen-binding antibody fragment, one or more minibinder, or one or more DARPin.

[0047] 37. The multispecific binding agent according to any of statements 1 to 36, comprising one or more engineered antibody or engineered antigen-binding antibody fragment.

[0048] 38. The multispecific binding agent according to any of statements 1 to 37, comprising one or more Fab, F(ab')2, scFv, scFv-Fc, minibody, VHH, sdAb, scAb, or scFab.

[0049] 39. The multispecific binding agent according to any of statements 1 to 38, comprising one or more antibody, antigen-binding antibody fragment, Fab, F(ab')2, scFv, scFv-Fc, minibody, VHH, sdAb, scAb, scFab, minibinder, or DARPin capable of binding said first immune checkpoint protein.

[0050] 40. The multispecific binding agent according to any of statements 1 to 39, comprising one or more antibody, antigen-binding antibody fragment, Fab, F(ab')2, scFv, scFv-Fc, minibody, VHH, sdAb, scAb, scFab, minibinder, or DARPin capable of binding said second immune checkpoint protein.

[0051] 41. The multispecific binding agent according to any of statements 1 to 40, comprising one or more antibody, antigen-binding antibody fragment, Fab, F(ab')2, scFv, scFv-Fc, minibody, VHH, sdAb, scAb, scFab, minibinder, or DARPin capable of binding said macrophage protein.

[0052] 42. The multispecific binding agent according to any of statements 1 to 41, comprising one or more antibody, antigen-binding antibody fragment, Fab, F(ab')2, scFv, scFv-Fc, minibody, VHH, sdAb, scAb, scFab, minibinder, or DARPin capable of binding said first immune checkpoint protein and comprising one or more antibody, antigen-binding antibody fragment, Fab, F(ab')2, scFv, scFv-Fc, minibody, VHH, sdAb, scAb, scFab, minibinder, or DARPin capable of binding said second immune checkpoint protein.

[0053] 43. The multispecific binding agent according to any of statements 1 to 42, comprising one or more antibody, antigen-binding antibody fragment, Fab, F(ab')2, scFv, scFv-Fc, minibody, VHH, sdAb, scAb, scFab, minibinder, or DARPin capable of binding said first immune checkpoint protein and comprising one or more antibody, antigen-binding antibody fragment, Fab, F(ab')2, scFv, scFv-Fc, minibody, VHH, sdAb, scAb, scFab, minibinder, or DARPin capable of binding said macrophage protein.

[0054] 44. The multispecific binding agent according to any of statements 1 to 43, comprising one or more antibody, antigen-binding antibody fragment, Fab, F(ab')2, scFv, scFv-Fc, minibody, VHH, sdAb, scAb, scFab, minibinder, or DARPin capable of binding said second immune checkpoint protein and comprising one or more antibody, antigen-binding antibody fragment, Fab, F(ab')2, scFv, scFv-Fc, minibody, VHH, sdAb, scAb, scFab, minibinder, or DARPin capable of binding said macrophage protein.

[0055] 45. The multispecific binding agent according to any of statements 1 to 44, comprising one or more antibody, antigen-binding antibody fragment, Fab, F(ab')2, scFv, scFv-Fc, minibody, VHH, sdAb, scAb, scFab, minibinder, or DARPin capable of binding said first immune checkpoint protein, comprising one or more antibody, antigen-binding antibody fragment, Fab, F(ab')2, scFv, scFv-Fc, minibody, VHH, sdAb, scAb, scFab, minibinder, or DARPin capable of binding said second immune checkpoint protein, and comprising one or more antibody, antigen-binding antibody fragment, Fab, F(ab')2, scFv, scFv-Fc, minibody, VHH, sdAb, scAb, scFab, minibinder, or DARPin capable of binding said macrophage protein.

[0056] 46. The multispecific binding agent according to any of statements 1 to 45, comprising one or more scFv fragments of an antibody against TIM3, VISTA and / or CSF1R. 47. The multispecific binding agent according to any of statements 1 to 46, comprising one or more scFv capable of binding TIM3, VISTA, and / or CSF1R.

[0057] 48. The multispecific binding agent according to any of statements 1 to 47, comprising one or more VHH capable of binding TIM3, VISTA, and / or CSF1R.

[0058] 49. The multispecific binding agent according to any of statements 1 to 48, comprising one or more Fab capable of binding TIM3, VISTA, and / or CSF1R.

[0059] 50. The multispecific binding agent according to any of statements 1 to 49, comprising a scFv capable of binding TIM3, a scFv capable of binding VISTA, and a Fab capable of binding CSF1R.

[0060] 51. The multispecific binding agent according to any of statements 1 to 50, comprising one or more Fc.

[0061] 52. The multispecific binding agent according to statement 51, comprising one or more Fc to which the antigen-binding domains are directly or indirectly linked.

[0062] 53. The multispecific binding agent according to any of statements 36 to 52, comprising one or more Fc to which the antibody or antigen-binding antibody fragments, minibinder, or DARPin are directly or indirectly linked.

[0063] 54. The multispecific binding agent according to any of claims 51 to 53, wherein the Fc domain comprises two polypeptide chains, each comprising a CH2 domain linked to a CH3 domain, wherein at least one antigen-binding region is linked N- terminally to a CH2 domain and at least one antigen-binding region is linked C- terminally to a CH3 domain.

[0064] 55. The multispecific binding agent according to any of statements 1 to 54, comprising a polypeptides with, from aminoterminus to carboxyterminus, the following subunits:

[0065] 1) Vista scFv VL - Vista scFv VH - IgG2a CH2 - IgG2a CH3, hole side - Tim 3 scFv VL - Tim 3 scFv VH;

[0066] 2) CSF1R Fab VL - kappa CL; and

[0067] 3) CSF1R Fab VH - IgG2a CHI - IgG2a CH2- IgG2a CH3, knob side.

[0068] 56. A polynucleic acid encoding the multispecific binding agent according to any of statements 35 to 55.

[0069] 57. The polynucleic acid according to statement 56, comprising a regulatory sequence operably linked to said polynucleic acid encoding the multispecific binding agent.

[0070] 58. The polynucleotide according to statement 57, wherein said regulatory sequence comprises a promoter. 59. A vector comprising the polynucleotide according to any of statements 56 to 58.

[0071] 60. The vector according to statement 59, which is an expression vector.

[0072] 61. The vector according to statement 59 or 60, which is a eukaryotic expression vector.

[0073] 62. A host cell comprising the polynucleotide according to any of statements 47 to 49 or the vector according to any of statements 50 to 52.

[0074] 63. The host cell according to statement 62, which is a eukaryotic host cell.

[0075] 64. The host cell according to statement 62 or 63, which expresses or is capable of expressing said multispecific binding agent.

[0076] 65. A composition comprising the multispecific binding agent according to any of statements 1 to 55, the polynucleic acid according to any of statements 56 to 58, the vector according to any of statements 59 to 61, or the host cell according to any of statements 62 to 64.

[0077] 66. The composition according to statement 65, which is a pharmaceutical composition.

[0078] 67. The composition according to statement 65 or 66, comprising one or more pharmaceutically acceptable excipients.

[0079] 68. The multispecific binding agent according to any of statements 1 to 55, the polynucleic acid according to any of statements 56 to 58, the vector according to any of statements 59 to 61, the host cell according to any of statements 62 to 64, or the composition according to any of statements 65 to 67, for use as a medicament.

[0080] 69. The multispecific binding agent according to any of statements 1 to 55, the polynucleic acid according to any of statements 56 to 58, the vector according to any of statements 59 to 61, the host cell according to any of statements 62 to 64, or the composition according to any of statements 65 to 67, for use in the treatment of cancer or an infectious disease, preferably for use in treating cancer.

[0081] 70. The multispecific binding agent according to any of statements 1 to 55, the polynucleic acid according to any of statements 56 to 58, the vector according to any of statements 59 to 61, the host cell according to any of statements 62 to 64, or the composition according to any of statements 65 to 67, for use in the treatment of cancer, wherein the cancer is a cancer fully or partially resistant against anti-PDl (programmed cell death protein 1) and / or anti PD-L1 (programmed death-ligand 1) immune therapy. 71. The multispecific binding agent according to any one of statements 1 to 55, the polynucleic acid according to any of statements 56 to 58, the vector according to any of statements 59 to 61, the host cell according to any of statements 62 to 64, or the composition according to any of statements 65 to 67, for use in the treatment of cancer, wherein the cancer is characterised by the presence of TIM3+, VISTA+ and CSF1R+ tumour associated macrophages.

[0082] 72. The multispecific agent according to any one of statements 1 to 55, the polynucleic acid according to any of statements 56 to 58, the vector according to any of statements 59 to 61, the host cell according to any of statements 62 to 64, or the composition according to any of statements 65 to 67, for use in the treatment of cancer, wherein the cancer is characterised by the presence of TIM3+, VISTA+ and CSF1R+ tumour associated macrophages in a tumour tissue.

[0083] 73. The multispecific antibody according to any one of statements 1 to 55, the polynucleic acid according to any of statements 56 to 58, the vector according to any of statements 59 to 61, the host cell according to any of statements 62 to 64, or the composition according to any of statements 65 to 67, for use in the treatment of cancer, wherein the cancer is selected from the group consisting of lung cancer, colorectal (colon / rectal) cancer, melanoma, kidney cancer, cervical cancer and breast cancer 74. The multispecific binding agent according to any one of statements 1 to 55, the polynucleic acid according to any of statements 56 to 58, the vector according to any of statements 59 to 61, the host cell according to any of statements 62 to 64, or the composition according to any of statements 65 to 67, for use in the treatment of cancer, wherein the treatment is a combination therapy of said multispecific binding agent, polynucleic acid, vector, host cell or composition with a T-cell targeting therapy and / or anti-cancer vaccines.

[0084] DESCRIPTION OF THE FIGURES

[0085] Figure legends

[0086] FIGURE 1: Non-limiting examples of multispecific binding agents. The different non-limiting examples of multispecific antibodies were rendered as in silico 3D configurations modelled using the sequences described in Example 7 and structural references in Figure la. Functional / structural stability (a proxy readout for epitope binding capacities and co-dependent functional activities) across different multi-specific antibody structural variants for anti-CSFIR, anti-TIM3 and anti-VISTA domains were computed as Root mean squared Deviation (RMSD) values (and its standard deviation) and statistically compared across structures for overall RMSD value distribution.

[0087] FIGURE 2. scRNAseq data from skin cutaneous melanoma (SKCM) patients (n=32) responding or not responding to PD1 / CTLA4 blockade. Table shows proportion of cells and the expression level of indicated genes in single-cell profiles of (a) T cells and (b) myeloid cells, (c) scRNAseq data from 14 treatment-naive patients across four different types of cancer (lung adenocarcinoma, endometrial adenocarcinoma, colorectal adenocarcinoma and renal cell carcinoma, clear cell). Table shows the amount of HAVCR2+ and VSIR+ cells and their average expression in indicated immune cell types, (d) 33 scRNAseq datasets including 8 cancer types (non-small cell lung cancer, hepatocellular carcinoma, lymphoma, head and neck, colorectal cancer, stomach, breast, and pancreas) (n=45). Table shows proportion of cells and their expression levels of indicated genes in singlecell profiles in indicated immune cell types, (e) Pie-chart showing different human cancer types where TIM3+VISTA+CSF1R+ TAMs are expressed.

[0088] Tumor volume of LLC (a) and MC38 (b) tumors in mice treated with anti-PDl (o- PD1), anti-PDLl (o-PDLl), or anti-CTLA4 antibody (0-CTLA4) or PBS. (n=4-7; Kruskal-Wallis test corrected for FDR, p-values vs. PBS), (c) Pie-chart representing CD45+ cell fraction from subcutaneous LLC / MC38 tumors (n = 5 or 4). (d) Violin plot of CD45+ cell fraction from subcutaneous LLC / MC38 tumors (day 24) (n=9; Mann-Whitney test), (e) scRNAseq data from subcutaneous LLC tumor tissue dataset showing the amount of TIM3+VISTA+ cells in different immune subsets, (f) Flow cytometry analysis of CD45+ cell fraction from LLC tumors (day 24) (n=4; Kruskal-Wallis test corrected for FDR; p-values depict significance to TAMs / MO). FIGURE 4 (a-b) Tumor volume of LLC (a) and MC38 (b) tumors in mice treated with anti-TIM3 (O-TIM3), or anti-VISTA antibody (o-VISTA) or PBS. (n=4; Kruskal- Wallis test corrected for FDR), (c-d) Tumor volume of LLC (c) and MC38 (d) tumors in mice treated with PBS or O-TIM3, o-VISTA and / or o-PDl. (n=4; Kruskal-Wallis test corrected for FDR, p-values vs. PBS).

[0089] FIGURE 5. (a) Bi-and tri-specific antibody design strategy from the IgGs and scFv fragments; (b) Examplary embodiment of a tri-specific antibody against VISTA, TIM3 and CSFR1R.

[0090] G1 : CSF1R Fab VH; G2 : CSF1R Fab VL; DB1 : mouse IgG2a / human IgGl CHI DB2 : mouse kappa CL;BR1 : Vista scFv VH; BR2 : Vista scFv VL;DB3 : mouse IgG2a / human IgGl CH2, LALAPG; DB4 : mouse IgG2a / human IgGl CH2, LALAPG; LB : mouse IgG2a / human IgGl CH3, "knob" side; G : mouse IgG2a / human IgGl CH3, "hole" side; R1 : Tim 3 scFv VL; R2 : Tim 3 scFv VH;

[0091] (c) ELISAs showing the binding of monospecific and bi-and tri-specific antibodies to recombinant mouse TIM3, CSF1R and VISTA respectively. Data represents mean values of 3 / 4 independent experiments with SEM and non-linear fit. Bispecific Ab#l is against TIM3 and CSF1R, Bispecific Ab#2 is against VISTA and CSF1R, (d) Flowcytometry histograms showing the binding of bi-and tri-specific antibodies to J774 macrophages and bone-marrow derived macrophages (BMDMs) and an unstained control.

[0092] FIGURE 6. (a) Interferon (IFN) / interferon-stimulated genes (ISG) response in J774 reporter macrophages with blockade of different antibody's alone or their cocultures with LLC untreated (UT) cancer cells. Bispecific Ab#l is against TIM3 and CSF1R, Bispecific Ab#2 is against VISTA and CSF1R. (b) Binding of tri-specific antibody to CD45+ isolated LLC tumors, showing the binding efficiency to tumor associated macrophages (TAMs), Dendritic cells (DCs) and CD4+ and CD8+ T cells (c-d) Bar plot of tumor associated macrophages (TAMs) treated with different antibodies and their co-cultures with LLC untreated (UT) cancer cells showing (c) Percentage of MHCII+ pSTATl-i- tumor associated macrophages in different coculture set-ups (n=4, Mann-Whitney test), (d) Percentage of dead cancer cells after co-culture with tumor associated macrophages treated with different antibodies. (n=4, Mann-Whitney test).

[0093] Figure 7. Interferon (IFN) / interferon-stimulated genes (ISG) response in J774 reporter macrophages with blockade of different antibody's alone or their cocultures with untreated cancer cells; YUMM1.7 melanoma, YUMMER1.7 melanoma, B16 melanoma, MC38 colorectal carcinoma, CT26 colorectal carcinoma, Renca kidney cancer, 4T1 breast cancer, GL261 glioblastoma, and LLC lung cancer. (n=3 to 5 depending on the cancer cell line).

[0094] FIGURE 8. (a) Tumor volume curves of LLC tumor bearing mice treated with anti- FcyR antibody (o-FcyR), anti-TGFP antibody (o-TGFP), anti-CRl / 2 antibody(o- CR1 / 2), anti-ILlO antibody (o-ILlO), anti-SSPl antibody (o-SPPl), anti-TREM2 antibody (O-TREM2), anti-ILip (o-ILip), anti-CCL2 antibody (O-CCL2), anti- SIRPlo (o-SIRPlo) or CD40 agonistic antibody (O-CD40) or PBS (n=3 / 4 Kruskal- Wallis test), (b) Tumor volume curves of LLC tumor bearing mice treated with anti- CSF1R antibody (a-CSFIR) or anti-PDLl antibody (o-PDLl) or PBS (n = ; Kruskal- Wallis test). Figure 9. (a) Tumor volume curve of LLC tumors in mice treated with 50ug of trispecific antibody, 50ug of anti-TIM3 or anti-VISTA or anti-CSFIR or a combination of all of these or PBS. (n=6 / 10; Kruskal-Wallis test)

[0095] (b-g) Bar plot of CD45+ cell fraction from isolated tumors showing (b) percentage of TIM3+VISTA+CSF1R+ TAMs (n=6 / 10; Mann-Whitney test) (c) showing percentage of CD3+ T cells (n=4; Mann-Whitney test) (d) showing percentage of IFNy-i- T cells in CD8+T cells (n=5 / 8; Mann-Whitney test) (e) showing percentage of TNFo+ T cells in CD8+T cells (n=7 / 8; Mann-Whitney test) (f) showing percentage of IL2+ T cells in CD8+ T cells (n=6 / 8; Mann-Whitney test) (g) showing ratio of Ml-like to M2-like tumor associated macrophages (TAMs) (n=5; Mann-Whitney test).

[0096] FIGURE 10. (a) Tumor volume curve of YUMM1.7 tumors in mice treated with 50ug of anti-Tri-specific antibody, a combination of 50ug of anti-TIM3, anti-VISTA, anti-CSFIR, or PBS (n=4; Kruskal-Wallis test), (b) Tumor volume curve of YUMM1.7 OVA tumors in mice treated with 50ug of anti-Tri-specific antibody, a combination of 50ug of anti-TIM3, anti-VISTA, anti-CSFIR, or PBS (n=4; Kruskal- Wallis test), (c) Tumor volume curve of YUMMER1.7 tumors in mice treated with 50ug of anti-Tri-specific antibody, a combination of 50ug of anti-TIM3, anti-VISTA, anti-CSFIR, or PBS (n=4; Kruskal-Wallis test).

[0097] FIGURE 11. (a-c) Bar plot of tumor associated macrophages (TAMs) treated with different antibodies and their co-cultures with LLC untreated (UT) cancer cells showing (a) Percentage of pSTATl-i- tumor associated macrophages in different co-culture set-ups (n=4, Mann-Whitney test), (b) Percentage of pSTAT2+ tumor associated macrophages in different co-culture set-ups (n=4, Mann-Whitney test), (c) Percentage of pSTAT3+ tumor associated macrophages in different co-culture set-ups (n=4, Mann-Whitney test).

[0098] FIGURE 12. (a) Interferon (IFN) / interferon-stimulated genes (ISG) response in J774 reporter macrophages (MO) with pre-blockade of different antibodies and their co-cultures with LLC untreated (UT) cancer cells together with tri-specific antibody. (n=6, Kruskall-Wallis test) (b) Interferon (IFN) / interferon-stimulated genes (ISG) response in J774 reporter macrophages (MO) with isotype control antibody or tri-specific antibody and their co-cultures with LLC untreated (UT) cancer cells or LLC cancer cells where nucleic acids were degraded via DNase I and RNase A cocktail (DNAse / RNAse) (n=4, Mann-Whitney test), (c) Interferon (IFN) / interferon-stimulated genes (ISG) response in J774 reporter macrophages (MO) pre-blocked with different inhibitors and their co-culture with isotype antibody or tri-specific antibody and LLC untreated (UT) cells (n=7; Two-way ANOVA) (d) Interferon (IFN) / interferon-stimulated genes (ISG) response in J774 reporter macrophages (MO) with tri-specific antibody and their co-cultures with CRISPR / CAS knock-out LLC cancer cells for AAV1 empty (control), LGALS9 (Galectin-9), HMGB1 (HMGB1), CEACAM1 (Ceacaml), VSIR (VISTA) and SELPLG (PSGL1) (n=5; One-way ANOVA)

[0099] Figure 13. (a) Tumor volume curve of LLC tumors in mice treated with 50 ug of tri-specific antibody, 250 ug of anti-PDl (o-PDl), 250 ug of anti-PDl(o-PDl) and 250ug of anti-CTLA4 (0-CTLA4), a combination of these or PBS. (n=5) (b) Tumor volume curve of LLC tumors in mice treated with 50 ug of tri-specific antibody alone or combined with 250 ug of anti-PDLl (o-PDLl), a DC vaccine (DC vax), a combination of these (n = 5).

[0100] FIGURE 14. Binding ELISAs for monospecific and different tri-specific antibodies to recombinant human TIM3 (A) and VISTA (B) and CSF1R (C) respectively. Data represents values of 2 / 3 independent with SEM and non-linear fit.

[0101] FIGURE 15. (a) Interferon (IFN) / interferon-stimulated genes (ISG) response in THP1 reporter macrophages with blockade of different antibody's alone or their co-cultures with HeLa untreated (UT) cervical cancer cells. CTV; combination of independent monospecific antibodies targeting CSF1R, TIM3 and VISTA.

[0102] DETAILED DESCRIPTION

[0103] Unless stated otherwise, all technical and scientific terms used herein have the meaning as commonly understood to one of ordinary skill in the art. Otherwise, certain terms used herein have the meanings as set forth in the specification.

[0104] It must be noted "a", "an", and "the" as used herein refers to both singular and plural referents unless the context clearly dictates otherwise.

[0105] The term "at least", preceding a series of elements is to be understood to refer to every element in the series. Whereas the term "one or more", such as one or more members of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.

[0106] Throughout the specification and the claims, unless the context requires otherwise, the word "comprise", and variations thereof will be understood to imply the inclusion of a stated integer, step or component (or group thereof) but not the exclusion of any other integer, step or component. The terms also encompass "consisting of" and "consisting essentially of". The term "comprising" and variations thereof may be substituted with the term "containing", "including" or sometimes "having".

[0107] When used herein, "consisting of" excludes any integer, element, step or component not specified in the sentence. When used, "consisting essentially of" does not exclude elements, steps or components that do not materially affect the basic and novel characteristics of the subject matter defined by the sentence.

[0108] The term "about" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of and from the specified value, in particular variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" refers is itself also specifically, and preferably, disclosed.

[0109] All documents cited in the present specification are hereby incorporated by reference in their entirety.

[0110] The terms "first", "second", "third" and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments, of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0111] Unless otherwise specified, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions may be included to better appreciate the teaching of the present invention.

[0112] In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0113] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0114] In the following detailed description of the invention, reference is made to the accompanying drawings that form a part hereof, and in which are shown by way of illustration only of specific embodiments in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural or logical changes may be made without departing from the scope of the present invention. The following detailed description, therefore, is not to be taken in a limiting sense, and the scope of the present invention is defined by the appended claims.

[0115] Definitions

[0116] The terms "protein", "polypeptide", and "peptide" are interchangeably used further herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. A "peptide" may also be referred to as a partial amino acid sequence derived from its original protein, for instance after tryptic digestion. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally- occurring amino acid polymers. This term also includes posttranslational modifications of the polypeptide, such as glycosylation, phosphorylation and acetylation.

[0117] As used herein, the term "nucleotide" or "nucleotides" refers to the basic building blocks of nucleic acids. A nucleotide consists of a sugar molecule (either ribose in RNA or deoxyribose in DNA) attached to a phosphate group and a nitrogencontaining base (either adenine, cytosine, guanine or uracil in RNA, or adenine, cytosine, guanine or thymine in DNA). Unless specified otherwise, the nucleotides for defining the replicon RIMA constructs or sequences disclosed herein are ribonucleotides. Alternatively, DNA templates used for the manufacture of the replicons disclosed herein are deoxyribonucleotides. The term "nucleic acids", "polynucleic acids", or "polynucleotides" refers to long chainlike molecules composed of a series of nucleotides including both RNA and DNA molecules. The term "nucleic acid sequence" refers to the sequence of nucleotides conventionally going from the 5'-end to the 3'-end of the linear nucleic acid.

[0118] "Binding" means any interaction, be it direct or indirect. A direct interaction implies a contact between the binding partners. An indirect interaction means any interaction whereby the interaction partners interact in a complex of more than two molecules. The interaction can be completely indirect, with the help of one or more bridging molecules, or partly indirect, where there is still a direct contact between the partners, which is stabilized by the additional interaction of one or more molecules. By the term "specifically binds," as used herein is meant a binding domain which recognizes a specific target, but does not substantially recognize or bind other molecules in a sample. Specific binding does not mean exclusive binding. However, specific binding does mean that proteins have a certain increased affinity or preference for one or a few of their binders. The term "affinity", as used herein, generally refers to the degree to which a ligand, chemical, antibody, protein or peptide binds to another (target) protein or peptide so as to shift the equilibrium of single protein monomers toward the presence of a complex formed by their binding. As used herein, the term "protein complex" or "complex" or "assembled protein(s)" refers to a group of two or more associated macromolecules, whereby at least one of the macromolecules is a protein. A protein complex, as used herein, typically refers to associations of macromolecules that can be formed under physiological conditions. Individual members of a protein complex are linked by non-covalent interactions.

[0119] A "binding agent" relates to a molecule that is capable of binding to another molecule, wherein said binding is preferably a specific binding, recognizing a defined binding site, pocket or epitope. The binding agent may be of any nature or type and is not dependent on its origin. The binding agent may be chemically synthesized, naturally occurring, recombinantly produced (and purified), as well as designed and synthetically produced. Said binding agent may hence be a small molecule, a chemical, a peptide, a polypeptide, a minibinder, a DARPIN, an antibody, or any derivatives thereof, such as a peptidomimetic, an antibody mimetic, an active fragment, a chemical derivative, among others. The term "binding pocket" or "binding site" refers to a region of a molecule or molecular complex, that, as a result of its shape and charge, favourably associates with another chemical entity, compound, proteins, peptide, antibody or Nb. The term "pocket” includes, but is not limited to cleft, channel or site. The term "part of a binding pocket / site" refers to less than all of the amino acid residues that define the binding pocket, or binding site. For example, the portion of residues may be key residues that play a role in ligand binding, or may be residues that are spatially related and define a three-dimensional compartment of the binding pocket. The residues may be contiguous or non-contiguous in primary sequence. For antibody- related molecules, the term "epitope" is also used to describe the binding site, as used interchangeably herein. An "epitope", refers to an antigenic determinant of a polypeptide, constituting a binding site or binding pocket on a target molecule. An epitope could comprise 3 amino acids in a spatial conformation, which is unique to the epitope. Generally, an epitope consists of at least 4, 5, 6, 7 such amino acids, and more usually, consists of at least 8, 9, 10 such amino acids. Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, X-ray crystallography and multi-dimensional nuclear magnetic resonance. A "conformational epitope", as used herein, refers to an epitope comprising amino acids in a spatial conformation that is unique to a folded 3-dimensional conformation of a polypeptide. Generally, a conformational epitope consists of amino acids that are discontinuous in the linear sequence but that come together in the folded structure of the protein. However, a conformational epitope may also consist of a linear sequence of amino acids that adopts a conformation that is unique to a folded 3-dimensional conformation of the polypeptide (and not present in a denatured state). In protein complexes, conformational epitopes consist of amino acids that are discontinuous in the linear sequences of one or more polypeptides that come together upon folding of the different folded polypeptides and their association in a unique quaternary structure. The term "conformation" or "conformational state" of a protein refers generally to the range of structures that a protein may adopt at any instant in time. A conformational epitope may thus comprise amino acid interactions from different protein domains of the protein. One of skill in the art will recognize that determinants of conformation or conformational state include a protein's primary structure as reflected in a protein's amino acid sequence (including modified amino acids) and the environment surrounding the protein. The conformation or conformational state of a protein also relates to structural features such as protein secondary structures (e.g., a-helix, p-sheet, among others), tertiary structure (e.g., the three dimensional folding of a polypeptide chain), and quaternary structure (e.g., interactions of a polypeptide chain with other protein subunits). Posttranslational and other modifications to a polypeptide chain such as ligand binding, phosphorylation, sulfation, glycosylation, or attachments of hydrophobic groups, among others, can influence the conformation of a protein. Furthermore, environmental factors, such as pH, salt concentration, ionic strength, and osmolality of the surrounding solution, and interaction with other proteins and cofactors, among others, can affect protein conformation. The conformational state of a protein may be determined by either functional assay for activity or binding to another molecule or by means of physical methods such as X-ray crystallography, NMR, or spin labelling, among other methods. For a general discussion of protein conformation and conformational states, one is referred to Cantor and Schimmel, Biophysical Chemistry, Part I: The Conformation of Biological. Macromolecules, W.H. Freeman and Company, 1980, and Creighton, Proteins: Structures and Molecular Properties, W.H. Freeman and Company, 1993. Typically, in the context of the present invention, the effect of the binding of the binding agent to the molecule of interest (which is typically either an immune- inhibitory receptor or a ligand thereof, as detailed below) is that it blocks or inhibits the function of said molecule. The function of the molecule of interest is typically based on interaction with a third molecule (i.e. a ligand of an immune-inhibitory receptor or an immune-inhibitory receptor), and the binding of the binding agent thereto inhibits or blocks activity by preventing the molecule of interest from interacting with this third molecule.

[0120] As used herein, the term "multispecific" binding agent refers to a binding agent (including antibodies or antigen-binding fragments thereof) which are capable of binding different antigens or different antigen epitopes (i.e. more than 1 antigen or antigen epitope). In a preferred embodiment, the multispecific binding agents of the invention are trispecific binding agents, i.e. binding agents capable of binding three different antigens or antigen epitopes. While theoretically different epitopes in the same target (such as a protein) can be recognized or bound by the multispecific binding agent, it will be understood that according to the present invention, the multiple epitopes which are recognized / bound are epitopes from different targets, such as different proteins. Accordingly, a trispecific binding agent as described herein recognizes / binds three different targets (molecules), such as three different proteins, and not three different epitopes within a single target (or two different epitopes in a single target and one epitope in a different target).

[0121] The invention provides, in a first aspect a multispecific binding capable of binding to a first immune checkpoint protein, a second immune checkpoint protein, and a macrophage protein which is different from said first and said second immune checkpoint protein. The multispecific binding agent of the invention in certain embodiments comprises one or more antibody or antigen-binding antibody fragment. The antibody or antibody fragment may be engineered or naturally occurring, as described herein elsewhere.

[0122] The term "antibody", "(antigen-binding) antibody fragment" and "active antibody fragment" as used herein refer to a protein comprising an immunoglobulin (Ig) domain or an antigen binding domain capable of specifically binding the antigen. "Antibodies" can further be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The term "active / antigen-binding antibody fragment" refers to a portion of any antibody or antibody-like structure that by itself has high affinity for an antigenic determinant, or epitope, and contains one or more complementarity-determining- regions (CDRs) accounting for such specificity. Non-limiting examples include immunoglobulin domains, Fab, F(ab')2, scFv (single chain Fv), scFab (single chain Fab), scFv-Fc (single chain Fv Fc fusions), heavy-light chain dimers, immunoglobulin single variable domains, Nanobodies (i.e. VHH), domain antibodies (sdAb or dAb) , and single chain structures, such as a complete light chain or complete heavy chain (scAb). An additional requirement for "activity" of said fragments in the light of the present invention is that said fragments are capable of binding the respective protein, and preferably are inhibiting or blocking the respective protein. The term "immunoglobulin (Ig) domain", or more specifically "immunoglobulin variable domain" (abbreviated as "IVD") means an immunoglobulin domain essentially consisting of four "framework regions" which are referred to in the art and herein below as "framework region 1" or "FR1"; as "framework region 2" or "FR2"; as "framework region 3" or "FR3"; and as "framework region 4" or "FR4", respectively; which framework regions are interrupted by three "complementarity determining regions" or "CDRs", which are referred to in the art and herein below as "complementarity determining region 1" or "CDR1"; as "complementarity determining region 2" or "CDR2"; and as "complementarity determining region 3" or"CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. It is the immunoglobulin variable domain(s) (IVDs) that confer specificity to an antibody for the antigen by carrying the antigen-binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will be involved in antigen binding site formation. In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, with binding to the respective epitope of an antigen by a pair of (associated) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen. An immunoglobulin single variable domain (ISVD) as used herein, refers to a protein with an amino acid sequence comprising 4 Framework regions (FR) and 3 complementary determining regions (CDR) according to the format of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An "immunoglobulin single variable domains" (abbreviated as "ISVD"), as used herein, is equivalent to the term "single variable domains", and defines molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from "conventional" immunoglobulins or their fragments, wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. The binding site of an immunoglobulin single variable domain is formed by a single VH / VHH or VL domain. Hence, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDR's. As such, the single variable domain may be a light chain variable domain sequence (e.g., a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit). In particular, the immunoglobulin single variable domain may be a Nanobody® (as defined herein) or a suitable fragment thereof. Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx N.V. (a Sanofi Company). For a general description of Nanobodies, reference is made to the further description below, as well as to the prior art cited herein, such as e.g. described in W02008 / 020079. "VHH domains", also known as VHHs, VHH domains, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen binding immunoglobulin (Ig) (variable) domain of "heavy chain antibodies". The term "VHH domain" has been chosen to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VH domains") and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VL domains"). For a further description of VHHs and Nanobody, reference is made to as well as to the following patent applications, which are mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103 of the Vrije Universiteit Brussel; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193 of Unilever; WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527 of the Vlaams Instituut voor Biotechnologie (VIB); a Nanobody (in particular VHH sequences and partially humanized Nanobody) can in particular be characterized by the presence of one or more "Hallmark residues" in one or more of the framework sequences. A further description of the Nanobody, including humanization and / or camelization of Nanobody, as well as other modifications, parts or fragments, derivatives or "Nanobody fusions", multivalent or multispecific constructs (including some nonlimiting examples of linker sequences) and different modifications to increase the half-life of the Nanobody and their preparations can be found e.g. in WO 08 / 101985 and WO 08 / 142164. Nanobodies form the smallest antigen binding fragment that completely retains the binding affinity and specificity of a full-length antibody. Nbs possess exceptionally long complementarity-determining region 3 (CDR3) loops and a convex paratope, which allow them to penetrate into hidden cavities of target antigens.

[0123] Different examples of bispecific and multi-specific antibodies are disclosed in W02024050524 and are the following:

[0124] Antibodies may be heterodimeric bi- and tri-(or more) specific Ig antibodies and Fc fusion proteins. Exemplary structures include, but are not limited to, IgG, IgM, mono-, di-, tri-, or more scFv-Fcs. For example, bispecific, trispecific, and multispecific formats include, but are not limited to, bispecific and trispecific IgG, IgG-scFv, IgG-dAb, scFv-Fc-scFv, knob-in-hole (KIH)-IgG, KA-body, KIH Fc- Fab / scFv, tandem scFv, KIH trispecific, bispecific Fc fusion (N- or C-terminal, with or without KIH).

[0125] Multispecific binding agents, such as antibodies or antibody fragments, as described herein elsewhere can include more than one antigen-binding site, where different sites are specific for different antigens. A multispecific antibody molecules can bind more than one (e.g., two or more) epitopes on the same antigen.

[0126] Bispecific antibody molecules can be classified into five different structural groups: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG appended with an additional antigen-binding moiety; (iii) bispecific antibody fragments; (iv) bispecific fusion proteins; and (v) bispecific antibody conjugates.

[0127] BsIgG is a format that is monovalent for each antigen. Exemplary BsIgG formats include but are not limited to crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-holes common LC, knobs-in-holes assembly, charge pair, Fab-arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, kappa-lamda-body, orthogonal Fab.

[0128] BsIgG can include heavy chains that are engineered for heterodimerization. For example, heavy chains can be engineered for heterodimerization using a "knobs- into-holes" strategy, a SEED platform, a common heavy chain (e.g., in Kk-bodies), and use of heterodimeric Fc regions. Strategies that have been used to avoid heavy chain pairing of homodimers in BsIgG include knobs-in-holes, duobody, azymetric, charge pair, HA-TF, SEEDbody, and differential protein A affinity. See Id. BsIgG can be produced by separate expression of the component antibodies in different host cells and subsequent purification / assembly into a BsIgG. BsIgG can also be produced by expression of the component antibodies in a single host cell. BsIgG can be purified using affinity chromatography, e.g., using protein A and sequential pH elution.

[0129] IgG appended with an additional antigen-binding moiety is another format of bispecific antibody molecules. For example, monospecific IgG can be engineered to have bispecificity by appending an additional antigen-binding unit onto the monospecific IgG, e.g., at the N- or C-terminus of either the heavy or light chain. Exemplary additional antigen-binding units include single domain antibodies (e.g., variable heavy chain or variable light chain), engineered protein scaffolds, and paired antibody variable regions (e.g., single chain variable fragments or variable fragments).

[0130] Examples of appended IgG formats include dual variable domain (DVD) IgG (DVD- Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (four-in-one).

[0131] It will be appreciated that the description above in connection with bispecific antibodies equally applies to multispecific antibodies, such as trispecific antibodies, such as in particular the appended formats.

[0132] Multispecific antibody fragments (MsAb), such as Bispecific antibody fragments (BsAb) or trispecific antibody fragments are a format of multispecific, such as bispecific or trispecific antibody molecules that lack some or all of the antibody constant domains. For example, some MsAb or BsAb lack an Fc region. Multispecific, such as Bispecific or trispecific antibody fragments can include heavy and light chain regions that are connected by a peptide linker that permits efficient expression of the multispecific, such as BsAb or trispecific antibody in a single host cell. Exemplary multispecific, such as bispecific or trispecific antibody fragments may comprise and include but are not limited to nanobody, nanobody-HSA, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, Diabody-Fc, tandem scFv-Fc, and intrabody. See Id. For example, the BiTE format includes tandem scFvs, where the component scFvs bind to CD3 on T cells and a surface antigen on cancer cells.

[0133] Multispecific, such as Bispecific or trispecific fusion proteins include antibody fragments linked to other proteins, e.g., to add additional specificity and / or functionality. An example of a Multispecific, such as bispecific or trispecific fusion protein is an immTAC, which includes an anti-CD3 scFv linked to an affinity- matured T-cell receptor that recognizes HLA-presented peptides. The dock-and- lock (DNL) method can be used to generate bispecific antibody molecules with higher valency or specificity. Additional antigen binding moieties may be appended to generate multispecific antibodies, such as trispecific antibodies (see e.g. Figure 4b). Also, fusions to albumin binding proteins or human serum albumin can be extend the serum half-life of antibody fragments. Chemical conjugation, e.g., chemical conjugation of antibodies and / or antibody fragments, can be used to create multispecific, such as BsAb or trispecific antibody molecules. An exemplary bispecific antibody conjugate includes the CovX-body format, in which a low molecular weight drug is conjugated site-specifically to a single reactive lysine in each Fab arm or an antibody or fragment thereof. The conjugation can improve the serum half-life of the low molecular weight drug.

[0134] A multispecific molecule can in some embodiments further include a heavy chain constant region (e.g., an Fc region) chosen from the heavy chain constant regions of IgGl, IgG2, and IgG4, more particularly, the heavy chain constant region of human IgGl, IgG2 or IgG4. The heavy chain constant region (e.g., an Fc region) can optionally be linked to, e.g., covalently linked to, one or both of the ApoLl- containing complex-binding antibody molecule and the second antibody molecule. The heavy chain constant region (e.g., an Fc region) can be altered, e.g., mutated, to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function. Also, the interface of a first and second heavy chain constant regions (e.g., Fc region) can be altered, e.g., mutated, to increase or decrease dimerization, e.g., relative to a non-engineered interface. The dimerization of the heavy chain constant region (e.g., Fc region) can be enhanced by providing an Fc interface of a first and a second Fc region with one or more of: a paired cavityprotuberance ("knob-in-a-hole"), an electrostatic interaction, or a strandexchange, such that a greater ratio of heteromultimer:homomultimer forms, e.g., relative to a non-engineered interface. The heavy chain constant region (e.g., Fc region) can include an amino acid substitution at a position chosen from one or more of 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409, e.g., of the Fc region of human IgGl, numbered based on the Eu numbering system. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0135] The heavy chain constant region (e.g., Fc region) may include an amino acid substitution chosen from: T366S, L368A, or Y407V (e.g., corresponding to a cavity or hole), or T366W (e.g., corresponding to a protuberance or knob), or a combination thereof, numbered based on the Eu numbering system. The heavy chain constant region (e.g., an Fc region) may include one or more mutations that increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function, relative to a naturally-existing heavy chain constant region. The first and / or second heavy chain constant region (e.g., a first and / or second Fc region, e.g., a first and / or second IgGl Fc region) can include an amino acid substitution at a position chosen from one or more of 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409, numbered based on the Eu numbering system. The first and / or second heavy chain constant region (e.g., a first and / or second Fc region, e.g., a first and / or second IgGl Fc region) can include an amino acid substitution chosen from: T366S, L368A, Y407V, or Y349C (e.g., corresponding to a cavity or hole), or T366W or S354C (e.g., corresponding to a protuberance or knob), or a combination thereof, numbered based on the Eu numbering system. In particular embodiments, a human IgGl heavy chain is used comprising a hole ensured by the mutations Y349C / T366S / L368A / Y407V and a knob ensured by the mutations S354C / T366W. In particular embodiments, the Fc region comprises a mutation to L234A / L235A / P329 mutation.

[0136] In particular embodiments the Fc is modified to influence Fc-mediated functions, such as antibody-dependent cellular cytotoxicity, CDC and antibody-dependent cellular phagocytosis, antibody-dependent complement-dependent cytotoxicity may stimulate inflammation, downstream adaptive immune responses or cell lysis. In particular embodiments, the Fc is modified to improve binding to the FcRn receptor and increased antibody serum half-life. In particular embodiments, the Fc is engineered to reduce effector function to minimize unwanted inflammation and acquired immunity. For instance, in particular embodiments, the Fc contains three mutations known as LALAPG (L234A, L235A, and P329G).

[0137] Immune checkpoint signalling, involving interactions between immune-inhibitory receptors and their partner ligands, is used for instance by tumors and some infectious organisms to suppress anticancer or anti-infectious agent immunity. Immune checkpoints are a normal part of our immune system. Their function is to prevent an exaggerated immune response that may destroy healthy cells in our body. However, for instance cancer has been shown to 'hijack' this signalling modality for its own advantage. Thus, in the context of cancer, when the immune- inhibitory receptors (typically expressed on lymphocytes including T cells or NK cells) and their ligands (expressed on cancer cells, stromal cells, or myeloid cells) bind together, they send an "off" signal to the T cells. This can prevent the immune system from destroying the cancer. Accordingly, monospecific antibody (Ab)-based immune-checkpoint blockers (ICBs) targeting such T cell-associated immune- inhibitory receptors (like PD1) or their ligands (like PD-L1) have revolutionized treatment of various cancer patients. Herein, ICBs work by blocking immune- inhibitory receptors (like PD1) from binding with their partner ligands (like PD- L1 / L2). This prevents the "off" signal from being sent, allowing the T cells to kill cancer cells.

[0138] Regrettably, several cancer types or patient sub-groups (40-90% depending on cancer type) remain fully or partially unresponsive to current PD(L)1 blockade. Non-immunogenic tumors are particularly resistant to ICBs and tend to be low in antigen enrichment, low in T cell infiltrates but high in tumor-associated macrophages (TAMs). According to current clinical evidence, some of the most dominant immuno-resistance mechanisms prevalent in such tumors include defects in antigen presentation and / or availability, TAMs-based immunosuppression, dysregulated interferon (IFN)-y signalling, and / or, enrichment of alternative immune-inhibitory receptors (i.e., receptors other than the 'classical' PD1 or CTLA4). Currently, substantial research is devoted to overcoming these barriers, but this has also revealed several challenges. For instance, while co-blockade of alternative immune-inhibitory receptors along with PD1 has shown promise in some cases e.g., LAG3; yet in other cases, for TIM3 or VISTA, early clinical results have been disappointing.

[0139] As used herein, the term "immune checkpoint protein" refers to a protein involved in immune checkpoint signalling, in particular an immune-inhibitory or co- inhibitory receptor protein (i.e., a protein involved in suppression of an immune response or a negative (feedback) regulator of an immune response). The immune checkpoint protein preferably is expressed by macrophages, in particular tumor- associated macrophages. The immune checkpoint protein preferably is a receptor, preferably a receptor expressed by (tumor-associated) macrophages, preferably an immune-inhibitory or co-inhibitory receptor. Examples of such proteins are TIM proteins, such as TIM-3 (also described herein as TIM3) and B7 proteins, such as VISTA, as described herein elsewhere. According to the invention, the multispecific binding agent is capable of binding at least two different immune checkpoint proteins, in particular, two different inhibitory immune checkpoint proteins, preferably two different inhibitory receptor proteins specifically at the surface of the macrophages. The inventors have found that by simultaneously targeting at least two checkpoint proteins on macrophages in combination with targeting a macrophage (marker) protein, and using binding agents which are interconnected, an interferon -res ponse is induced, which is not induced when these proteins are targeted with separate binding agents. It is considered that the nature of the checkpoint proteins is not critical, but may be selected based on the expression of these checkpoint proteins by the macrophages in the relevant environment.

[0140] Preferably, said checkpoint proteins are transmembrane proteins (i.e., cell surface proteins). Preferably, the multispecific binding agent inhibits (i.e., blocks) said checkpoint proteins, preferably such as to reduce or prevent the signalling pathways normally activated by the proteins. Preferably, the multispecific binding agent induces a type I interferon (IFN) response, preferably in macrophages, preferably in tumor-associated macrophages.

[0141] According to the invention, the multispecific binding agent also binds a macrophage protein, in particular an anti-inflammatory receptor, such as, but not limited to an anti-inflammatory or M2-like tumor-associated macrophage protein (TAM protein). The nature of the macrophage protein can vary. In the context of the use in the treatment of cancer, the macrophage protein is preferably a TAM lineage / differentiation protein.

[0142] It will be understood that the (tumor-associated) macrophage protein is different from any of the two immune checkpoint proteins to which the multispecific binding agent also binds. Preferably, the macrophage protein is a transmembrane protein (i.e., a cell-surface protein). As used herein, the term macrophage protein implies that the macrophage protein is expressed by macrophages following their lineage commitment or differentiation trajectory derived from monocytes, and it can be highly enriched on macrophages, (especially tumor-associated macrophages), although it need not exclusively be expressed by macrophages depending on monocyte developmental trajectories. Targeting the three different proteins according to the invention (each of which specific or at least enriched on macrophages) incrementally enhances macrophage-specific targeting. The macrophage protein preferably is a receptor, preferably a receptor expressed by monocyte-derived or tumor-associated macrophages. In certain preferred embodiments, the macrophage protein is a macrophage marker protein. The term "marker" in general is known in the art and used herein accordingly. The skilled person will understand that a marker protein is characteristically expressed by a particular cell type (optionally in a particular cell state) and can typically be used to define, identify, or otherwise characterise a particular cell (differentiation or developmental state, or lineage commitment), as known in the art. As used herein, the term TAM protein implies that the TAM protein is expressed by tumor associated macrophages, and it can be enriched on tumor-associated macrophages, although it need not exclusively be expressed by tumor associated macrophages, but also by monocyte-derived macrophage or myeloid lineages. The TAM protein preferably is a receptor, preferably a receptor enriched on tumor- associated macrophages. The skilled person will understand that the term "tumor- associated macrophage" has its ordinary meaning known in the art, and by means of further guidance refers to the macrophages which are present in high numbers in tumor microenvironment and which typically play a cancer or malignancypromoting role, and / or anti-inflammatory role. Preferably, the multispecific binding agent inhibits (i.e., blocks) said macrophage (marker) protein, preferably such as to reduce or prevent the signalling pathways normally activated by the protein. Preferably, binding of the multispecific binding agent to the macrophage (marker) protein, the first checkpoint protein, and the second checkpoint protein induces a type I interferon (IFN) response, preferably in macrophages, preferably in tumor- associated macrophages. An example of such macrophage (marker) protein is CSF1R.

[0143] Colony stimulating factor 1 receptor (CSF1R), is also known as macrophage colony-stimulating factor receptor (M-CSFR), CD115 (Cluster of Differentiation 115), CSFR, FMS, McDonough Feline Sarcoma Viral (V-Fms) Oncogene Homolog, Macrophage Colony-Stimulating Factor 1 Receptor, Proto-Oncogene C-Fms, CSF-1 Receptor, CD115 Antigen, EC 2.7.10.1, M-CSF-R, Macrophage Colony Stimulating Factor I Receptor, FMS Proto-Oncogene, EC 2.7.10, BANDDOS, CSF-l-R, HDLS1, FIM2, GPSC, and HDLS. In particular embodiments, the part of the binding agent capable of binding hCSFRl is corresponds to part of a binding agent described in the art such as in WO2023017159. In particular embodiments, the part of the binding agent capable of binding hCSFRl binds the same epitope as the CDRs corresponding to aa 22-35, 50-65 and 95-105 of SEQ ID NO: 2 and aa 23-34, SO- 56 and 88-96 of SEQ ID NO:3. In particular embodiments, the part of the binding agent capable of binding hCSFRl comprises the CDRs corresponding to aa 22-35, 50-65 and 95-105 of SEQ ID NO: 2 and aa 23-34, 50-56 and 88-96 of SEQ ID NO:3. In particular embodiments, the part of the multimeric binding agent capable of binding hCSFIR is an Fab, such as the FAB made up by amino acid 1- 116 of SEQ ID NO:2 and amino acid 1-106 of SEQ ID NO:3. In particular embodiments, the part of the multimeric binding agent capable of binding hCSFIR is a scFv, such as (preferably from N-terminus to C-terminus) comprising amino acid 1-116 of SEQ ID NO:2 and amino acid 1-106 of SEQ ID NO:3 which may be coupled by a linker, such as a linker of SEQ ID NO: 7.

[0144] In certain preferred embodiments, the first immune checkpoint protein, the second immune checkpoint protein, and the macrophage protein are anti-inflammatory receptors, in particular pro-cancerous transmembrane receptors.

[0145] In certain embodiments, the first and second immune checkpoint protein are selected from TIM proteins and B7 immunoglobulin proteins. In certain embodiments, the first and second immune checkpoint protein are TIM proteins. In certain embodiments, the first and second immune checkpoint protein are B7 immunoglobulin proteins. In certain embodiments, the first immune checkpoint protein is a TIM protein and the second immune checkpoint protein is a B7 immunoglobulin protein.

[0146] As used herein, the term "TIM protein" refers to a member of the T-cell immunoglobulin and mucin domain (TIM) family. The TIM family includes TIM1, TIM2, TIM3, TIM4, TIM5, TIM6, TIM7, and TIM8. For instance, T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), also known as Hepatitis A virus cellular receptor 2 (HAVCR2), TIMD3, CD366, T-Cell Membrane Protein, FLJ14428, TIMD-3, Kidney Injury Molecule-3, CD366 Antigen, KIM-3, or SPTCL, is a transmembrane cell surface receptor, and is an immune checkpoint or immune- inhibitory receptor. In particular embodiments, the part of the multimeric binding agent capable of binding TIM-3 is a part of a binding agent described in the art such as in WO2021072312. In particular embodiments, the part of the multimeric binding agent capable of binding TIM-3 binds the same epitope as the CDRs corresponding to aa 521-536, 552-558 and 590-599 of SEQ ID NO: 1 and aa 646- 659, 674-690 and 720-731 of SEQ ID NO: 1. In particular embodiments, the part of the multimeric binding agent capable of binding to TIM-3 comprises the CDRs corresponding to aa 521-536, 552-558 and 590-599 of SEQ ID NO: 1 and aa 646- 659, 674-690 and 720-731 of SEQ ID NO: 1. In particular embodiments, the part of the multimeric binding agent capable of binding to TIM-3 is a scFV, such as (preferably from N-terminus to C-terminus) comprising amino acid 499-609 of SEQ ID NO: 1 and amino acid 625-742 of SEQ ID NO: 1 which may be coupled by a linker, such as a linker of SEQ ID NO: 7. In particular embodiments, the part of the multimeric binding agent capable of binding to TIM-3 is a scFV, such as the scFV corresponding to amino acids 499-742 of SEQ ID NO: 1. In alternative embodiments, the part of the multimeric binding agent capable of binding TIM-3 binds the same epitope as the CDRs corresponding to aa 520-533, 550-563 and 593-598 of SEQ ID NO: 4. In further particular embodiments, the part of the multimeric binding agent capable of binding TIM-3 comprises the CDRs corresponding to aa 520-533, 550-563 and 593-598 of SEQ ID NO: 4. In further particular embodiments, the part of the multimeric binding agent capable of binding to TIM-3 is a VHH, such as the VHH corresponding to amino acids 499- 609 of SEQ ID NO: 4.

[0147] As used herein, the term "B7 immunoglobulin protein" refers to a member of the B7 immunoglobulin family of costimulatory membrane proteins. The B7 immunoglobulin family includes B7-1 (also known as CD80), B7-2 (also known as CD86), B7-DC (also known as PDCD1LG2, PD-L2, CD273), B7-H1 (also known as PD-L1, CD274), B7-H2 (also known as ICOSLG, B7RP1, CD275), B7-H3 (also known as CD276), B7-H4 (also known as VTCN1), B7-H5 (also known as VISTA, Gi24, SISP1), B7-H6 (also known as NCR3LG1), and B7-H7 (also known as HHLA2). For instance, V-domain Ig suppressor of T cell activation (VISTA), also known as VSIR, V-Set Immunoregulatory Receptor, SISP1, C10orf54, B7-H5, PD- 1H, Diesl, GI24, B7H5, V-Type Immunoglobulin Domain-Containing Suppressor Of T-Cell Activation, V-Set Domain-Containing Immunoregulatory Receptor, Stress-Induced Secreted Protein-1, PDCD1 Homolog, Sisp-1, Chromosome 10 Open Reading Frame 54, Stress Induced Secreted Protein 1, Platelet Receptor GI24, Death Domainlalpha, DDlalpha, or PP2135, is a type I transmembrane protein that functions as an immune checkpoint or immune-inhibitory receptor. In particular embodiments, the part of the multimeric binding protein binding VISTA is a part of a binding agent described in the art such as in W02025030110. In particular embodiments, the part of the multimeric binding agent capable of binding VISTA binds the same epitope as the CDRs corresponding to aa 23-33, 49- 55 and 87-97 of SEQ ID NO: 1 and aa 144-156, 173-188 and 218-230 of SEQ ID NO: 1. In particular embodiments, the part of the multimeric binding agent capable of binding VISTA comprises the CDRs corresponding to aa 23-33, 49-55 and 87- 97 of SEQ ID NO: 1 and aa 144-156, 173-188 and 218-230 of SEQ ID NO: 1. In particular embodiments, the part of the multimeric binding agent capable of binding to VISTA is a scFV, such as (preferably from N-terminus to C-terminus) comprising amino acid 1-107 of SEQ ID NO: 1 and amino acid 123-241 of SEQ ID NO: 1 which may be coupled by a linker, such as a linker of SEQ ID NO: 7. In particular embodiments, the part of the multimeric binding agent capable of detecting VISTA is a scFv corresponding to amino acids 1-241 of SEQ ID NO: 1.

[0148] In certain embodiments, the first immune checkpoint protein is selected from TIM1, TIM2, TIM3, TIM4, TIM5, TIM6, TIM7, and TIM8. In certain embodiments, the second immune checkpoint protein is selected from B7-H5, B7-1, B7-2, B7- DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, and B7-H7. In certain embodiments, the first and second immune checkpoint protein is selected from TIM1, TIM2, TIM3, TIM4, TIM5, TIM6, TIM7, and TIM8. In certain embodiments, the first and second immune checkpoint protein is selected from B7-H5, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, and B7-H7. In certain embodiments, the first immune checkpoint protein is selected from TIM1, TIM2, TIM3, TIM4, TIM5, TIM6, TIM7, and TIM8 and the second immune checkpoint protein is selected from B7- H5, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, and B7-H7.

[0149] In certain embodiments, the macrophage protein is selected from CSF1R, MRC1, CD163, CD68, Fc Gamma Receptors, CD14, CDllb, F4 / 80and CD206.

[0150] In certain embodiments, the first immune checkpoint protein is selected from TIM1, TIM2, TIM3, TIM4, TIM5, TIM6, TIM7, and TIM8, and the macrophage protein is selected from CSF1R, MRC1, CD163, CD68, Fc Gamma Receptors, CD14, CDl lb, F4 / 80, and CD206. In certain embodiments, the second immune checkpoint protein is selected from B7-H5, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7- H3, B7-H4, B7-H6, and B7-H7, and the macrophage protein is selected from CSF1R, MRC1, CD163, CD68, Fc Gamma Receptors, CD14, CDl lb, F4 / 80, and CD206. In certain embodiments, the first and second immune checkpoint protein is selected from TIM1, TIM2, TIM3, TIM4, TIM5, TIM6, TIM7, and TIM8, and the macrophage protein is selected from CSF1R, MRC1, CD163, CD68, Fc Gamma Receptors, CD14, CDllb, F4 / 80, and CD206. In certain embodiments, the first and second immune checkpoint protein is selected from B7-H5, B7-1, B7-2, B7- DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, and B7-H7, and the macrophage protein is selected from CSF1R, MRC1, CD163, CD68, Fc Gamma Receptors, CD14, CDl lb, F4 / 80, and CD206. In certain embodiments, the first immune checkpoint protein is selected from TIM1, TIM2, TIM3, TIM4, TIM5, TIM6, TIM7, and TIM8, the second immune checkpoint protein is selected from B7-H5, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, and B7-H7, and the macrophage protein is selected from CSF1R, MRC1, CD163, CD68, Fc Gamma Receptors, CD14, CDllb, F4 / 80, and CD206.

[0151] In certain embodiments, the first and second immune checkpoint protein are selected from TIM proteins and B7 immunoglobulin proteins and the macrophage protein is selected from CSF1R, MRC1, CD163, CD68, Fc Gamma Receptors, CD14, CDl lb, F4 / 80, and CD206. In certain embodiments, the first and second immune checkpoint protein are TIM proteins and the macrophage protein is selected from CSF1R, MRC1, CD163, CD68, Fc Gamma Receptors, CD14, CDl lb, F4 / 80, and CD206. In certain embodiments, the first and second immune checkpoint protein are B7 immunoglobulin proteins and the macrophage protein is selected from CSF1R, MRC1, CD163, CD68, Fc Gamma Receptors, CD14, CDl lb, F4 / 80, and CD206. In certain embodiments, the first immune checkpoint protein is a TIM protein, the second immune checkpoint protein is a B7 immunoglobulin protein, and the macrophage protein is selected from CSF1R, MRC1, CD163, CD68, Fc Gamma Receptors, CD14, CDllb, F4 / 80, and CD206.

[0152] In certain embodiments, the macrophage protein, the first immune checkpoint protein, and the second immune checkpoint protein are respectively CSF1R, TIM1, and B7-1; CSF1R, TIM1, and B7-2; CSF1R, TIM1, and B7-DC; CSF1R, TIM1, and B7-H1; CSF1R, TIM1, and B7-H2; CSF1R, TIM1, and B7-H3; CSF1R, TIM1, and B7-H4; CSF1R, TIM1, and B7-H5; CSF1R, TIM1, and B7-H6; CSF1R, TIM1, and B7-H7; CSF1R, TIM2, and B7-1; CSF1R, TIM2, and B7-2; CSF1R, TIM2, and B7- DC; CSF1R, TIM2, and B7-H1; CSF1R, TIM2, and B7-H2; CSF1R, TIM2, and B7- H3; CSF1R, TIM2, and B7-H4; CSF1R, TIM2, and B7-H5; CSF1R, TIM2, and B7- H6; CSF1R, TIM2, and B7-H7; CSF1R, TIM3, and B7-1; CSF1R, TIM3, and B7-2; CSF1R, TIM3, and B7-DC; CSF1R, TIM3, and B7-H1; CSF1R, TIM3, and B7-H2; CSF1R, TIM3, and B7-H3; CSF1R, TIM3, and B7-H4; CSF1R, TIM3, and B7-H5; CSF1R, TIM3, and B7-H6; CSF1R, TIM3, and B7-H7; CSF1R, TIM4, and B7-1; CSF1R, TIM4, and B7-2; CSF1R, TIM4, and B7-DC; CSF1R, TIM4, and B7-H1;

[0153] CSF1R, TIM4, and B7-H2; CSF1R, TIM4, and B7-H3; CSF1R, TIM4, and B7-H4;

[0154] CSF1R, TIM4, and B7-H5; CSF1R, TIM4, and B7-H6; CSF1R, TIM4, and B7-H7;

[0155] CSF1R, TIM5, and B7-1; CSF1R, TIM5, and B7-2; CSF1R, TIM5, and B7-DC; CSF1R, TIM5, and B7-H1; CSF1R, TIM5, and B7-H2; CSF1R, TIM5, and B7-H3;

[0156] CSF1R, TIM5, and B7-H4; CSF1R, TIM5, and B7-H5; CSF1R, TIM5, and B7-H6;

[0157] CSF1R, TIM5, and B7-H7; CSF1R, TIM6, and B7-1; CSF1R, TIM6, and B7-2; CSF1R, TIM6, and B7-DC; CSF1R, TIM6, and B7-H1; CSF1R, TIM6, and B7-H2;

[0158] CSF1R, TIM6, and B7-H3; CSF1R, TIM6, and B7-H4; CSF1R, TIM6, and B7-H5;

[0159] CSF1R, TIM6, and B7-H6; CSF1R, TIM6, and B7-H7; CSF1R, TIM7, and B7-1; CSF1R, TIM7, and B7-2; CSF1R, TIM7, and B7-DC; CSF1R, TIM7, and B7-H1; CSF1R, TIM7, and B7-H2; CSF1R, TIM7, and B7-H3; CSF1R, TIM7, and B7-H4;

[0160] CSF1R, TIM7, and B7-H5; CSF1R, TIM7, and B7-H6; CSF1R, TIM7, and B7-H7;

[0161] CSF1R, TIM8, and B7-1; CSF1R, TIM8, and B7-2; CSF1R, TIM8, and B7-DC; CSF1R, TIM8, and B7-H1; CSF1R, TIM8, and B7-H2; CSF1R, TIM8, and B7-H3;

[0162] CSF1R, TIM8, and B7-H4; CSF1R, TIM8, and B7-H5; CSF1R, TIM8, and B7-H6;

[0163] CSF1R, TIM8, and B7-H7; MRC1, TIM1, and B7-1; MRC1, TIM1, and B7-2; MRC1, TIM1, and B7-DC; MRC1, TIM1, and B7-H1; MRC1, TIM1, and B7-H2; MRC1, TIM1, and B7-H3; MRC1, TIM1, and B7-H4; MRC1, TIM1, and B7-H5; MRC1, TIM1, and B7-H6; MRC1, TIM1, and B7-H7; MRC1, TIM2, and B7-1; MRC1, TIM2, and B7-2; MRC1, TIM2, and B7-DC; MRC1, TIM2, and B7-H1; MRC1, TIM2, and B7-H2; MRC1, TIM2, and B7-H3; MRC1, TIM2, and B7-H4; MRC1, TIM2, and B7-H5; MRC1, TIM2, and B7-H6; MRC1, TIM2, and B7-H7; MRC1, TIM3, and B7-1; MRC1, TIM3, and B7-2; MRC1, TIM3, and B7-DC; MRC1, TIM3, and B7-H1; MRC1, TIM3, and B7-H2; MRC1, TIM3, and B7-H3; MRC1, TIM3, and B7-H4; MRC1, TIM3, and B7-H5; MRC1, TIM3, and B7-H6; MRC1, TIM3, and B7-H7; MRC1, TIM4, and B7- 1; MRC1, TIM4, and B7-2; MRC1, TIM4, and B7-DC; MRC1, TIM4, and B7-H1; MRC1, TIM4, and B7-H2; MRC1, TIM4, and B7-H3; MRC1, TIM4, and B7-H4; MRC1, TIM4, and B7-H5; MRC1, TIM4, and B7-H6; MRC1, TIM4, and B7-H7; MRC1, TIM5, and B7-1; MRC1, TIM5, and B7-2; MRC1, TIM5, and B7-DC; MRC1, TIM5, and B7-H1; MRC1, TIM5, and B7-H2; MRC1, TIM5, and B7-H3; MRC1, TIM5, and B7-H4; MRC1, TIM5, and B7-H5; MRC1, TIM5, and B7-H6; MRC1, TIM5, and B7-H7; MRC1, TIM6, and B7-1; MRC1, TIM6, and B7-2; MRC1, TIM6, and B7-DC; MRC1, TIM6, and B7-H1; MRC1, TIM6, and B7-H2; MRC1, TIM6, and B7-H3; MRC1, TIM6, and B7-H4; MRC1, TIM6, and B7-H5; MRC1, TIM6, and B7-H6; MRC1, TIM6, and B7-H7; MRC1, TIM7, and B7-1; MRC1, TIM7, and B7-2; MRC1, TIM7, and B7-DC; MRC1, TIM7, and B7-H1; MRC1, TIM7, and B7-H2; MRC1, TIM7, and B7-H3; MRC1, TIM7, and B7-H4; MRC1, TIM7, and B7-H5; MRC1, TIM7, and B7-H6; MRC1, TIM7, and B7-H7; MRC1, TIM8, and B7-1; MRC1, TIM8, and B7-2; MRC1, TIM8, and B7-DC; MRC1, TIM8, and B7-H1; MRC1, TIM8, and B7-H2; MRC1, TIM8, and B7-H3; MRC1, TIM8, and B7-H4; MRC1, TIM8, and B7-H5; MRC1, TIM8, and B7-H6; MRC1, TIM8, and B7-H7; CD163, TIM1, and B7-1; CD163, TIM1, and B7-2; CD163, TIM1, and B7-DC; CD163, TIM1, and B7-H1; CD163, TIM1, and B7-H2; CD163, TIM1, and B7-H3; CD163, TIM1, and B7-H4; CD163, TIM1, and B7-H5; CD163, TIM1, and B7-H6; CD163, TIM1, and B7-H7; CD163, TIM2, and B7-1; CD163, TIM2, and B7-2; CD163, TIM2, and B7-DC; CD163, TIM2, and B7-H1; CD163, TIM2, and B7-H2; CD163, TIM2, and B7-H3;

[0164] CD163, TIM2, and B7-H4; CD163, TIM2, and B7-H5; CD163, TIM2, and B7-H6;

[0165] CD163, TIM2, and B7-H7; CD163, TIM3, and B7-1; CD163, TIM3, and B7-2; CD163, TIM3, and B7-DC; CD163, TIM3, and B7-H1; CD163, TIM3, and B7-H2;

[0166] CD163, TIM3, and B7-H3; CD163, TIM3, and B7-H4; CD163, TIM3, and B7-H5;

[0167] CD163, TIM3, and B7-H6; CD163, TIM3, and B7-H7; CD163, TIM4, and B7-1; CD163, TIM4, and B7-2; CD163, TIM4, and B7-DC; CD163, TIM4, and B7-H1; CD163, TIM4, and B7-H2; CD163, TIM4, and B7-H3; CD163, TIM4, and B7-H4; CD163, TIM4, and B7-H5; CD163, TIM4, and B7-H6; CD163, TIM4, and B7-H7; CD163, TIM5, and B7-1; CD163, TIM5, and B7-2; CD163, TIM5, and B7-DC; CD163, TIM5, and B7-H1; CD163, TIM5, and B7-H2; CD163, TIM5, and B7-H3;

[0168] CD163, TIM5, and B7-H4; CD163, TIM5, and B7-H5; CD163, TIM5, and B7-H6;

[0169] CD163, TIM5, and B7-H7; CD163, TIM6, and B7-1; CD163, TIM6, and B7-2; CD163, TIM6, and B7-DC; CD163, TIM6, and B7-H1; CD163, TIM6, and B7-H2;

[0170] CD163, TIM6, and B7-H3; CD163, TIM6, and B7-H4; CD163, TIM6, and B7-H5;

[0171] CD163, TIM6, and B7-H6; CD163, TIM6, and B7-H7; CD163, TIM7, and B7-1; CD163, TIM7, and B7-2; CD163, TIM7, and B7-DC; CD163, TIM7, and B7-H1; CD163, TIM7, and B7-H2; CD163, TIM7, and B7-H3; CD163, TIM7, and B7-H4;

[0172] CD163, TIM7, and B7-H5; CD163, TIM7, and B7-H6; CD163, TIM7, and B7-H7;

[0173] CD163, TIM8, and B7-1; CD163, TIM8, and B7-2; CD163, TIM8, and B7-DC; CD163, TIM8, and B7-H1; CD163, TIM8, and B7-H2; CD163, TIM8, and B7-H3;

[0174] CD163, TIM8, and B7-H4; CD163, TIM8, and B7-H5; CD163, TIM8, and B7-H6;

[0175] CD163, TIM8, and B7-H7; CD68, TIM1, and B7-1; CD68, TIM1, and B7-2; CD68, TIM1, and B7-DC; CD68, TIM1, and B7-H1; CD68, TIM1, and B7-H2; CD68, TIM1, and B7-H3; CD68, TIM1, and B7-H4; CD68, TIM1, and B7-H5; CD68, TIM1, and B7-H6; CD68, TIM1, and B7-H7; CD68, TIM2, and B7-1; CD68, TIM2, and B7-2; CD68, TIM2, and B7-DC; CD68, TIM2, and B7-H1; CD68, TIM2, and B7-H2; CD68, TIM2, and B7-H3; CD68, TIM2, and B7-H4; CD68, TIM2, and B7-H5; CD68, TIM2, and B7-H6; CD68, TIM2, and B7-H7; CD68, TIM3, and B7-1; CD68, TIM3, and B7- 2; CD68, TIM3, and B7-DC; CD68, TIM3, and B7-H1; CD68, TIM3, and B7-H2; CD68, TIM3, and B7-H3; CD68, TIM3, and B7-H4; CD68, TIM3, and B7-H5; CD68, TIM3, and B7-H6; CD68, TIM3, and B7-H7; CD68, TIM4, and B7-1; CD68, TIM4, and B7-2; CD68, TIM4, and B7-DC; CD68, TIM4, and B7-H1; CD68, TIM4, and B7-H2; CD68, TIM4, and B7-H3; CD68, TIM4, and B7-H4; CD68, TIM4, and B7- H5; CD68, TIM4, and B7-H6; CD68, TIM4, and B7-H7; CD68, TIM5, and B7-1; CD68, TIM5, and B7-2; CD68, TIM5, and B7-DC; CD68, TIM5, and B7-H1; CD68, TIM5, and B7-H2; CD68, TIM5, and B7-H3; CD68, TIM5, and B7-H4; CD68, TIM5, and B7-H5; CD68, TIM5, and B7-H6; CD68, TIM5, and B7-H7; CD68, TIM6, and B7-1; CD68, TIM6, and B7-2; CD68, TIM6, and B7-DC; CD68, TIM6, and B7-H1; CD68, TIM6, and B7-H2; CD68, TIM6, and B7-H3; CD68, TIM6, and B7-H4; CD68, TIM6, and B7-H5; CD68, TIM6, and B7-H6; CD68, TIM6, and B7-H7; CD68, TIM7, and B7-1; CD68, TIM7, and B7-2; CD68, TIM7, and B7-DC; CD68, TIM7, and B7- Hl; CD68, TIM7, and B7-H2; CD68, TIM7, and B7-H3; CD68, TIM7, and B7-H4; CD68, TIM7, and B7-H5; CD68, TIM7, and B7-H6; CD68, TIM7, and B7-H7; CD68, TIM8, and B7-1; CD68, TIM8, and B7-2; CD68, TIM8, and B7-DC; CD68, TIM8, and B7-H1; CD68, TIM8, and B7-H2; CD68, TIM8, and B7-H3; CD68, TIM8, and B7-H4; CD68, TIM8, and B7-H5; CD68, TIM8, and B7-H6; CD68, TIM8, and B7- H7; Fc Gamma Receptor, TIM1, and B7-1; Fc Gamma Receptor, TIM1, and B7-2; Fc Gamma Receptor, TIM1, and B7-DC; Fc Gamma Receptor, TIM1, and B7-H1; Fc Gamma Receptor, TIM1, and B7-H2; Fc Gamma Receptor, TIM1, and B7-H3; Fc

[0176] Gamma Receptor, TIM1, and B7-H4; Fc Gamma Receptor, TIM1, and B7-H5; Fc

[0177] Gamma Receptor, TIM1, and B7-H6; Fc Gamma Receptor, TIM1, and B7-H7; Fc

[0178] Gamma Receptor, TIM2, and B7-1; Fc Gamma Receptor, TIM2, and B7-2; Fc

[0179] Gamma Receptor, TIM2, and B7-DC; Fc Gamma Receptor, TIM2, and B7-H1; Fc

[0180] Gamma Receptor, TIM2, and B7-H2; Fc Gamma Receptor, TIM2, and B7-H3; Fc

[0181] Gamma Receptor, TIM2, and B7-H4; Fc Gamma Receptor, TIM2, and B7-H5; Fc

[0182] Gamma Receptor, TIM2, and B7-H6; Fc Gamma Receptor, TIM2, and B7-H7; Fc

[0183] Gamma Receptor, TIM3, and B7-1; Fc Gamma Receptor, TIM3, and B7-2; Fc

[0184] Gamma Receptor, TIM3, and B7-DC; Fc Gamma Receptor, TIM3, and B7-H1; Fc

[0185] Gamma Receptor, TIM3, and B7-H2; Fc Gamma Receptor, TIM3, and B7-H3; Fc

[0186] Gamma Receptor, TIM3, and B7-H4; Fc Gamma Receptor, TIM3, and B7-H5; Fc

[0187] Gamma Receptor, TIM3, and B7-H6; Fc Gamma Receptor, TIM3, and B7-H7; Fc

[0188] Gamma Receptor, TIM4, and B7-1; Fc Gamma Receptor, TIM4, and B7-2; Fc

[0189] Gamma Receptor, TIM4, and B7-DC; Fc Gamma Receptor, TIM4, and B7-H1; Fc

[0190] Gamma Receptor, TIM4, and B7-H2; Fc Gamma Receptor, TIM4, and B7-H3; Fc

[0191] Gamma Receptor, TIM4, and B7-H4; Fc Gamma Receptor, TIM4, and B7-H5; Fc Gamma Receptor, TIM4, and B7-H6; Fc Gamma Receptor, TIM4, and B7-H7; Fc Gamma Receptor, TIM5, and B7-1; Fc Gamma Receptor, TIM5, and B7-2; Fc Gamma Receptor, TIM5, and B7-DC; Fc Gamma Receptor, TIM5, and B7-H1; Fc Gamma Receptor, TIM5, and B7-H2; Fc Gamma Receptor, TIM5, and B7-H3; Fc Gamma Receptor, TIM5, and B7-H4; Fc Gamma Receptor, TIM5, and B7-H5; Fc Gamma Receptor, TIM5, and B7-H6; Fc Gamma Receptor, TIM5, and B7-H7; Fc Gamma Receptor, TIM6, and B7-1; Fc Gamma Receptor, TIM6, and B7-2; Fc Gamma Receptor, TIM6, and B7-DC; Fc Gamma Receptor, TIM6, and B7-H1; Fc Gamma Receptor, TIM6, and B7-H2; Fc Gamma Receptor, TIM6, and B7-H3; Fc Gamma Receptor, TIM6, and B7-H4; Fc Gamma Receptor, TIM6, and B7-H5; Fc Gamma Receptor, TIM6, and B7-H6; Fc Gamma Receptor, TIM6, and B7-H7; Fc Gamma Receptor, TIM7, and B7-1; Fc Gamma Receptor, TIM7, and B7-2; Fc Gamma Receptor, TIM7, and B7-DC; Fc Gamma Receptor, TIM7, and B7-H1; Fc Gamma Receptor, TIM7, and B7-H2; Fc Gamma Receptor, TIM7, and B7-H3; Fc Gamma Receptor, TIM7, and B7-H4; Fc Gamma Receptor, TIM7, and B7-H5; Fc Gamma Receptor, TIM7, and B7-H6; Fc Gamma Receptor, TIM7, and B7-H7; Fc Gamma Receptor, TIM8, and B7-1; Fc Gamma Receptor, TIM8, and B7-2; Fc Gamma Receptor, TIM8, and B7-DC; Fc Gamma Receptor, TIM8, and B7-H1; Fc Gamma Receptor, TIM8, and B7-H2; Fc Gamma Receptor, TIM8, and B7-H3; Fc Gamma Receptor, TIM8, and B7-H4; Fc Gamma Receptor, TIM8, and B7-H5; Fc Gamma Receptor, TIM8, and B7-H6; Fc Gamma Receptor, TIM8, and B7-H7; CD14, TIM1, and B7-1; CD14, TIM1, and B7-2; CD14, TIM1, and B7-DC; CD14, TIM1, and B7-H1; CD14, TIM1, and B7-H2; CD14, TIM1, and B7-H3; CD14, TIM1, and B7-H4; CD14, TIM1, and B7-H5; CD14, TIM1, and B7-H6; CD14, TIM1, and B7- H7; CD14, TIM2, and B7-1; CD14, TIM2, and B7-2; CD14, TIM2, and B7-DC; CD14, TIM2, and B7-H1; CD14, TIM2, and B7-H2; CD14, TIM2, and B7-H3; CD14, TIM2, and B7-H4; CD14, TIM2, and B7-H5; CD14, TIM2, and B7-H6; CD14, TIM2, and B7-H7; CD14, TIM3, and B7-1; CD14, TIM3, and B7-2; CD14, TIM3, and B7- DC; CD14, TIM3, and B7-H1; CD14, TIM3, and B7-H2; CD14, TIM3, and B7-H3; CD14, TIM3, and B7-H4; CD14, TIM3, and B7-H5; CD14, TIM3, and B7-H6; CD14, TIM3, and B7-H7; CD14, TIM4, and B7-1; CD14, TIM4, and B7-2; CD14, TIM4, and B7-DC; CD14, TIM4, and B7-H1; CD14, TIM4, and B7-H2; CD14, TIM4, and B7-H3; CD14, TIM4, and B7-H4; CD14, TIM4, and B7-H5; CD14, TIM4, and B7- H6; CD14, TIM4, and B7-H7; CD14, TIM5, and B7-1; CD14, TIM5, and B7-2; CD14, TIM5, and B7-DC; CD14, TIM5, and B7-H1; CD14, TIM5, and B7-H2; CD14, TIM5, and B7-H3; CD14, TIM5, and B7-H4; CD14, TIM5, and B7-H5; CD14, TIM5, and B7-H6; CD14, TIM5, and B7-H7; CD14, TIM6, and B7-1; CD14, TIM6, and B7-

[0192] 2; CD14, TIM6, and B7-DC; CD14, TIM6, and B7-H1; CD14, TIM6, and B7-H2;

[0193] CD14, TIM6, and B7-H3; CD14, TIM6, and B7-H4; CD14, TIM6, and B7-H5; CD14,

[0194] TIM6, and B7-H6; CD14, TIM6, and B7-H7; CD14, TIM7, and B7-1; CD14, TIM7, and B7-2; CD14, TIM7, and B7-DC; CD14, TIM7, and B7-H1; CD14, TIM7, and B7-H2; CD14, TIM7, and B7-H3; CD14, TIM7, and B7-H4; CD14, TIM7, and B7- H5; CD14, TIM7, and B7-H6; CD14, TIM7, and B7-H7; CD14, TIM8, and B7-1; CD14, TIM8, and B7-2; CD14, TIM8, and B7-DC; CD14, TIM8, and B7-H1; CD14, TIM8, and B7-H2; CD14, TIM8, and B7-H3; CD14, TIM8, and B7-H4; CD14, TIM8, and B7-H5; CD14, TIM8, and B7-H6; CD14, TIM8, and B7-H7; CDllb, TIM1, and B7-1; CDllb, TIM1, and B7-2; CDllb, TIM1, and B7-DC; CDllb, TIM1, and B7- Hl; CDllb, TIM1, and B7-H2; CDllb, TIM1, and B7-H3; CDllb, TIM1, and B7- H4; CDllb, TIM1, and B7-H5; CDllb, TIM1, and B7-H6; CDllb, TIM1, and B7- H7; CDllb, TIM2, and B7-1; CDllb, TIM2, and B7-2; CDllb, TIM2, and B7-DC; CDllb, TIM2, and B7-H1; CDllb, TIM2, and B7-H2; CDllb, TIM2, and B7-H3;

[0195] CDllb, TIM2, and B7-H4; CDllb, TIM2, and B7-H5; CDllb, TIM2, and B7-H6;

[0196] CDllb, TIM2, and B7-H7; CDllb, TIM3, and B7-1; CDllb, TIM3, and B7-2; CDllb, TIM3, and B7-DC; CDllb, TIM3, and B7-H1; CDllb, TIM3, and B7-H2;

[0197] CDllb, TIM3, and B7-H3; CDllb, TIM3, and B7-H4; CDllb, TIM3, and B7-H5;

[0198] CDllb, TIM3, and B7-H6; CDllb, TIM3, and B7-H7; CDllb, TIM4, and B7-1; CDllb, TIM4, and B7-2; CDllb, TIM4, and B7-DC; CDllb, TIM4, and B7-H1; CDllb, TIM4, and B7-H2; CDllb, TIM4, and B7-H3; CDllb, TIM4, and B7-H4; CDllb, TIM4, and B7-H5; CDllb, TIM4, and B7-H6; CDllb, TIM4, and B7-H7; CDllb, TIM5, and B7-1; CDllb, TIM5, and B7-2; CDllb, TIM5, and B7-DC; CDllb, TIM5, and B7-H1; CDllb, TIM5, and B7-H2; CDllb, TIM5, and B7-H3;

[0199] CDllb, TIM5, and B7-H4; CDllb, TIM5, and B7-H5; CDllb, TIM5, and B7-H6;

[0200] CDllb, TIM5, and B7-H7; CDllb, TIM6, and B7-1; CDllb, TIM6, and B7-2; CDllb, TIM6, and B7-DC; CDllb, TIM6, and B7-H1; CDllb, TIM6, and B7-H2;

[0201] CDllb, TIM6, and B7-H3; CDllb, TIM6, and B7-H4; CDllb, TIM6, and B7-H5;

[0202] CDllb, TIM6, and B7-H6; CDllb, TIM6, and B7-H7; CDllb, TIM7, and B7-1; CDllb, TIM7, and B7-2; CDllb, TIM7, and B7-DC; CDllb, TIM7, and B7-H1; CDllb, TIM7, and B7-H2; CDllb, TIM7, and B7-H3; CDllb, TIM7, and B7-H4;

[0203] CDllb, TIM7, and B7-H5; CDllb, TIM7, and B7-H6; CDllb, TIM7, and B7-H7;

[0204] CDllb, TIM8, and B7-1; CDllb, TIM8, and B7-2; CDllb, TIM8, and B7-DC; CDllb, TIM8, and B7-H1; CDllb, TIM8, and B7-H2; CDllb, TIM8, and B7-H3;

[0205] CDllb, TIM8, and B7-H4; CDllb, TIM8, and B7-H5; CDllb, TIM8, and B7-H6; CDllb, TIM8, and B7-H7; F4 / 80, TIM1, and B7-1; F4 / 80, TIM1, and B7-2; F4 / 80, TIM1, and B7-DC; F4 / 80, TIM1, and B7-H1; F4 / 80, TIM1, and B7-H2; F4 / 80, TIM1, and B7-H3; F4 / 80, TIM1, and B7-H4; F4 / 80, TIM1, and B7-H5; F4 / 80, TIM1, and B7-H6; F4 / 80, TIM1, and B7-H7; F4 / 80, TIM2, and B7-1; F4 / 80, TIM2, and B7-2; F4 / 80, TIM2, and B7-DC; F4 / 80, TIM2, and B7-H1; F4 / 80, TIM2, and B7-H2; F4 / 80, TIM2, and B7-H3; F4 / 80, TIM2, and B7-H4; F4 / 80, TIM2, and B7-H5; F4 / 80, TIM2, and B7-H6; F4 / 80, TIM2, and B7-H7; F4 / 80, TIM3, and B7-1; F4 / 80, TIM3, and B7-2; F4 / 80, TIM3, and B7-DC; F4 / 80, TIM3, and B7-H1; F4 / 80, TIM3, and B7-H2; F4 / 80, TIM3, and B7-H3; F4 / 80, TIM3, and B7-H4; F4 / 80, TIM3, and B7-H5; F4 / 80, TIM3, and B7-H6; F4 / 80, TIM3, and B7-H7; F4 / 80, TIM4, and B7- 1; F4 / 80, TIM4, and B7-2; F4 / 80, TIM4, and B7-DC; F4 / 80, TIM4, and B7-H1; F4 / 80, TIM4, and B7-H2; F4 / 80, TIM4, and B7-H3; F4 / 80, TIM4, and B7-H4; F4 / 80, TIM4, and B7-H5; F4 / 80, TIM4, and B7-H6; F4 / 80, TIM4, and B7-H7; F4 / 80, TIM5, and B7-1; F4 / 80, TIM5, and B7-2; F4 / 80, TIM5, and B7-DC; F4 / 80, TIM5, and B7-H1; F4 / 80, TIM5, and B7-H2; F4 / 80, TIM5, and B7-H3; F4 / 80, TIM5, and B7-H4; F4 / 80, TIM5, and B7-H5; F4 / 80, TIM5, and B7-H6; F4 / 80, TIM5, and B7-H7; F4 / 80, TIM6, and B7-1; F4 / 80, TIM6, and B7-2; F4 / 80, TIM6, and B7-DC; F4 / 80, TIM6, and B7-H1; F4 / 80, TIM6, and B7-H2; F4 / 80, TIM6, and B7-H3; F4 / 80, TIM6, and B7-H4; F4 / 80, TIM6, and B7-H5; F4 / 80, TIM6, and B7-H6; F4 / 80, TIM6, and B7-H7; F4 / 80, TIM7, and B7-1; F4 / 80, TIM7, and B7-2; F4 / 80, TIM7, and B7-DC; F4 / 80, TIM7, and B7-H1; F4 / 80, TIM7, and B7-H2; F4 / 80, TIM7, and B7-H3; F4 / 80, TIM7, and B7-H4; F4 / 80, TIM7, and B7-H5; F4 / 80, TIM7, and B7-H6; F4 / 80, TIM7, and B7-H7; F4 / 80, TIM8, and B7-1; F4 / 80, TIM8, and B7-2; F4 / 80, TIM8, and B7-DC; F4 / 80, TIM8, and B7-H1; F4 / 80, TIM8, and B7-H2; F4 / 80, TIM8, and B7-H3; F4 / 80, TIM8, and B7-H4; F4 / 80, TIM8, and B7-H5; F4 / 80, TIM8, and B7-H6; F4 / 80, TIM8, and B7-H7; CD206, TIM1, and B7-1; CD206, TIM1, and B7-2; CD206, TIM1, and B7-DC; CD206, TIM1, and B7-H1; CD206, TIM1, and B7-H2; CD206, TIM1, and B7-H3; CD206, TIM1, and B7-H4; CD206, TIM1, and B7-H5; CD206, TIM1, and B7-H6; CD206, TIM1, and B7-H7; CD206, TIM2, and B7-1; CD206, TIM2, and B7-2; CD206, TIM2, and B7-DC; CD206, TIM2, and B7-H1; CD206, TIM2, and B7-H2; CD206, TIM2, and B7-H3;

[0206] CD206, TIM2, and B7-H4; CD206, TIM2, and B7-H5; CD206, TIM2, and B7-H6;

[0207] CD206, TIM2, and B7-H7; CD206, TIM3, and B7-1; CD206, TIM3, and B7-2; CD206, TIM3, and B7-DC; CD206, TIM3, and B7-H1; CD206, TIM3, and B7-H2;

[0208] CD206, TIM3, and B7-H3; CD206, TIM3, and B7-H4; CD206, TIM3, and B7-H5;

[0209] CD206, TIM3, and B7-H6; CD206, TIM3, and B7-H7; CD206, TIM4, and B7-1; CD206, TIM4, and B7-2; CD206, TIM4, and B7-DC; CD206, TIM4, and B7-H1;

[0210] CD206, TIM4, and B7-H2; CD206, TIM4, and B7-H3; CD206, TIM4, and B7-H4;

[0211] CD206, TIM4, and B7-H5; CD206, TIM4, and B7-H6; CD206, TIM4, and B7-H7;

[0212] CD206, TIM5, and B7-1; CD206, TIM5, and B7-2; CD206, TIM5, and B7-DC;

[0213] CD206, TIM5, and B7-H1; CD206, TIM5, and B7-H2; CD206, TIM5, and B7-H3;

[0214] CD206, TIM5, and B7-H4; CD206, TIM5, and B7-H5; CD206, TIM5, and B7-H6;

[0215] CD206, TIM5, and B7-H7; CD206, TIM6, and B7-1; CD206, TIM6, and B7-2; CD206, TIM6, and B7-DC; CD206, TIM6, and B7-H1; CD206, TIM6, and B7-H2;

[0216] CD206, TIM6, and B7-H3; CD206, TIM6, and B7-H4; CD206, TIM6, and B7-H5;

[0217] CD206, TIM6, and B7-H6; CD206, TIM6, and B7-H7; CD206, TIM7, and B7-1; CD206, TIM7, and B7-2; CD206, TIM7, and B7-DC; CD206, TIM7, and B7-H1; CD206, TIM7, and B7-H2; CD206, TIM7, and B7-H3; CD206, TIM7, and B7-H4;

[0218] CD206, TIM7, and B7-H5; CD206, TIM7, and B7-H6; CD206, TIM7, and B7-H7;

[0219] CD206, TIM8, and B7-1; CD206, TIM8, and B7-2; CD206, TIM8, and B7-DC;

[0220] CD206, TIM8, and B7-H1; CD206, TIM8, and B7-H2; CD206, TIM8, and B7-H3;

[0221] CD206, TIM8, and B7-H4; CD206, TIM8, and B7-H5; CD206, TIM8, and B7-H6; or CD206, TIM8, and B7-H7.

[0222] By combining these binding agents in different constructs, the inventors have further found that the exact nature of the construct is not critical, as long as the binding of the binding agents to their targets is not hindered. The inventors have found that this can be achieved by a wide variety of constructs. In certain embodiments, the multispecific binding agent according of the invention comprises a first antigen-binding region capable of binding to the first immune checkpoint protein, a second antigen binding region capable of binding the second immune checkpoint protein, and a third antigen binding region capable of binding to the macrophage protein. In certain embodiments, each of the antigen binding regions is (independently) selected from an Fab, F(ab')2, Fv, scFv, scFv-Fc, nanobody (VHH), minibody, sdAb, scAb, or scFab. In certain embodiments, each of the antigen binding regions is (independently) selected from an Fab, scFv, or VHH. Indeed, the inventors have found that the effect can be achieved irrespective of whether the antigen-binding region is a scFv or a VHH. The different types of binding agents envisaged in the context of the invention are detailed above.

[0223] In certain embodiments, the first antigen-binding region capable of binding to the first immune checkpoint protein, the second antigen binding region capable of binding the second immune checkpoint protein, and the third antigen binding region capable of binding to the macrophage protein respectively comprise a VHH, VHH, and VHH; VHH, VHH, and scFv; VHH, VHH, and Fab; VHH, scFv, and VHH; VHH, scFv, and scFv; VHH, scFv, and Fab; VHH, Fab, and VHH; VHH, Fab, and scFv; VHH, Fab, and Fab; scFv, VHH, and VHH; scFv, VHH, and scFv; scFv, VHH, and Fab; scFv, scFv, and VHH; scFv, scFv, and scFv; scFv, scFv, and Fab; scFv, Fab, and VHH; scFv, Fab, and scFv; scFv, Fab, and Fab; Fab, VHH, and VHH; Fab, VHH, and scFv; Fab, VHH, and Fab; Fab, scFv, and VHH; Fab, scFv, and scFv; Fab, scFv, and Fab; Fab, Fab, and VHH; Fab, Fab, and scFv; or Fab, Fab, and Fab. In certain embodiments, the first antigen-binding region capable of binding to the first immune checkpoint protein, the second antigen binding region capable of binding the second immune checkpoint protein, and the third antigen binding region capable of binding to the macrophage protein respectively comprise a VHH, VHH, and VHH; VHH, VHH, and scFv; VHH, VHH, and Fab; VHH, scFv, and VHH; VHH, scFv, and scFv; VHH, scFv, and Fab; VHH, Fab, and VHH; VHH, Fab, and scFv; VHH, Fab, and Fab; scFv, VHH, and VHH; scFv, VHH, and scFv; scFv, VHH, and Fab; scFv, scFv, and VHH; scFv, scFv, and scFv; scFv, scFv, and Fab; scFv, Fab, and VHH; scFv, Fab, and scFv; scFv, Fab, and Fab; Fab, VHH, and VHH; Fab, VHH, and scFv; Fab, VHH, and Fab; Fab, scFv, and VHH; Fab, scFv, and scFv; Fab, scFv, and Fab; Fab, Fab, and VHH; Fab, Fab, and scFv; or Fab, Fab, and Fab, wherein the macrophage protein, the first immune checkpoint protein, and the second immune checkpoint protein are respectively as defined in any of the embodiments above.

[0224] In particular embodiments, the multispecific binding agent according to the invention comprises a heavy chain constant region (Fc) to which the antigenbinding domains are directly or indirectly linked. In particular embodiments, the dimerization of the Fc region can be enhanced by providing an Fc interface of a first and a second Fc region with one or more of: a paired cavity-protuberance ("knob-in-a-hole"). Examples of knob and hole mutations are described herein above. In particular embodiments, each chain of the Fc domain can be linked to one or more binding regions, particularly N- or C-terminally thereof.

[0225] In particular embodiments of the multispecific binding agent, the Fc domain comprises two polypeptide chains, each comprising a CH2 domain linked to a CH3 domain, wherein at least one antigen-binding region is linked N-terminally to a CH2 domain and at least one antigen-binding region is linked C-terminally to a CH3 domain. In particular embodiments, the first binding agent (i.e. the first antigen-binding region or domain) capable of binding the first immune checkpoint protein is linked N-terminally to a first CH2 domain and the second binding agent (i.e. the second antigen binding region or domain) capable of binding the second immune checkpoint protein is bound C-terminally to a CH3 domain, while the third binding agent (i.e., the antigen binding region or domain) capable of binding to the macrophage protein is linked N-terminally to a second CH2 domain. In alternative embodiments, these domains are switched. It will be understood that alternative arrangements of the different binding agents can be made, including, but not limited to linking the first binding agent capable of binding the first immune checkpoint protein N-terminally to a first CH2 domain and linking the second binding agent capable of binding the second immune checkpoint protein N- terminally to the first binding agent capable of binding the first immune checkpoint protein, or vice versa. The three binding domains can also for instance be linked to the same heavy chain of the Fc (also including symmetrical multispecific binding agents having all three binding agents linked to each of the two heavy chain Fc domains, whether in the same or a different configuration) or alternatively two of the binding domains can be linked to one heavy chain of the Fc and the third binding domain can be linked to the other heavy chain of the Fc. All possible configurations of linking the different antigen-binding domains to the Fc are envisaged according to the invention.

[0226] However, it is considered that the presence of an Fc region is not critical to the invention as it is not functional for activating the immune response. Indeed, the inventors have found that a trispecific antibody is functional in ensuring the desired effect also when inactivating Fc immune effector functions as a result of an LALAPG mutation. Accordingly, embodiments are envisaged without an Fc region (see Fig. 1). All possible configurations of linking the different antigen-binding domains are envisaged according to the invention.

[0227] It will be understood that whether or not linked to an Fc, the antigen-binding regions / domains can be linked (for instance to Fc or each other) by means of a linker, as known in the art. Suitable linkers include GGGGS linkers (or multimers thereof, such as dimers), which in case of binding to a CH2 domain may be linked to the Fc hinge region (or a truncated version thereof still allowing formation of the disulfide bridges required to connect both Fc heavy chain arms). In particular embodiment, the linker is a GGGGSGGGGSGGGGS linker (SEQ ID NO: 7). It will be understood that other aspects, related to methods of producing and delivering the multispecific binding agents described hereinabove are also envisaged herein.

[0228] In a related aspect, the invention relates to a polynucleic acid encoding the multispecific binding agent as described herein. The polynucleic acid may be DNA or RIMA. In certain embodiments, the polynucleic acid comprises a regulatory sequence operably linked to the polynucleic acid encoding the multispecific binding agent. In certain embodiments, the regulatory sequence is or comprises a promoter. It will be understood that promoters may serve to initiate expression of the multispecific binding agent and may be species-specific (e.g. for expression in a host cell of a particular species), and / or may be constitutive or inducible, all as known in the art.

[0229] In a further related aspect, the invention relates to a vector comprising the polynucleotide encoding the multispecific binding agent as described herein. In certain embodiments, the vector is an expression vector an expression vector, such as a eukaryotic expression vector.

[0230] In a further related aspect, the invention relates to a host cell comprising the polynucleotide or the vector according to the invention as described herein. In certain embodiments, the host cell is a eukaryotic host cell, such as a mammalian host cell, such as a human host cell. In certain embodiments, the host cell expresses or is capable of expressing the multispecific binding agent.

[0231] In a further related aspect, the invention relates to a composition comprising the multispecific binding agent, the polynucleic acid, the vector according, or the host cell according to the invention as described herein elsewhere. In certain embodiments, the composition is a pharmaceutical composition. In certain embodiments, the comprises one or more pharmaceutically acceptable excipients.

[0232] The multispecific binding agents of the invention are of particular use (either as such or when delivered as polynucleotides, vectors or host cells expressing said binding agents) as therapeutics, more particularly for use in the treatment and / or prevention of diseases which benefit from an increased immune response, such as cancer or infectious diseases.

[0233] Accordingly, in a related aspect, the invention relates to the multispecific binding agent, the polynucleic acid, the vector, the host cell, or the composition according to the invention as described herein elsewhere, for use as a medicament. More particularly, the multispecific binding agents of the invention are envisaged to be of interest for the treatment of cancer.

[0234] Most studies targeting immune-inhibitory receptors concentrate on T cells. While previously this association was considered to be exclusive, recently it has been repeatedly extended to various myeloid cells, like TAMs. However, the immunobiology of TAM-specific immune-inhibitory receptors, enriched in tumors fully or partially resistant to PD(L)1 blockade, and its therapeutic or clinical impact remains unexplored. This requires urgent attention because high enrichment of TAMs, typically seen in these tumors, can facilitate over-representation of such signalling over others. However, extremely scanty knowledge exists on which classical or alternative immune-inhibitory receptors are preferentially exploited by TAMs to support immune subversion. This needs urgent characterization because TAMs are heavily enriched in immuno-resistant tumors and yet an immunotherapy to successfully target them remains elusive. This poses unprecedented challenges for biomarker-driven combinatorial immunotherapies against non-immunogenic tumors with high TAMs infiltrates, which constitute a major portion (40-90%) of several cancers.

[0235] Monospecific Abs blocking immune-checkpoints (PD1, PD-L1 or CTLA4) have already failed against immuno-resistant cancers. Recently, multi-specific Abs have been proposed to be the next frontier of immuno-oncology to more specifically and strongly engage the immune cells e.g. bi-specific Abs against PDlxLAG3, PD1XCTLA4, PDlxVEGF, PD-LlxVEGF, PDlxTIM3 or PDlxTIGIT. Multi-specific Abs are recombinant molecules containing two (bi-) or more (multi-) different epitopes-specific binding domains. Bispecific Ab-based immunotherapy has gained broad potential in preclinical and clinical investigations in a variety of cancer types following regulatory approval of newly developed technologies involving bispecific and multi-specific Abs. However, such multi-specific Abs are still very focused on T cell-associated immune-checkpoints. This is not relevant for immuno-resistant tumors that are more enriched in TAMs than T cells. This demands prioritisation of innovative TAM-targeting immunotherapy.

[0236] However, initial clinical studies with TAM-targeting monospecific Abs like, anti- CSF1R or anti-CCR2, have been largely unimpressive. The reason for this remains unknown and little knowledge exists on the most pre-dominant niche of immune- inhibitory receptors associated with TAMs in immuno-resistant tumors, that can perhaps be targeted via innovative multi-specific Ab products. This can be very relevant to increase the specificity of the immunotherapy toward TAMs. But, the lack of clear knowledge on TAM-specific immune-checkpoints creates challenges for creating innovative multi-specific immunotherapy products, useful against immuno-resistant tumors. Thus, TAM targeting multi-specific Abs are currently absent for application against immuno-resistant solid tumors and this represents a critical socio-economically unmet need for cancer treatment.

[0237] The present invention discloses that patients with low-immunogenic or low- antigenic tumors, who are nowadays considered to be non-responsive to ICBs and thus would not be selected for treatment with ICBs, have a subset of macrophages characterised by the specific co-expression of at least two immune-checkpoints or immune-inhibitory receptors and at least one specific TAM protein, which includes the combination of TIM3 and VISTA with CSF1R. Targeting these macrophages with a multispecific antibody can lead to anticancer activity and tumor shrinkage.

[0238] Accordingly, in a related aspect, the invention relates to the multispecific binding agent, the polynucleic acid, the vector, the host cell, or the composition according to the invention as described herein elsewhere, for use in the treatment of cancer or an infectious disease, preferably cancer. The application further envisages a method of treatment of a cancer or an infectious disease, preferably cancer, in a patient, comprising administering the multispecific binding agent the polynucleic acid, the vector, the host cell, or the composition according to the invention as described herein elsewhere to said patient in an effective amount to treat said cancer, including the specific methods described herein below. The invention also envisages the use of the multispecific binding agents in the manufacture of a medicament for the treatment of cancer, including in the specific treatment methods envisaged below.

[0239] For instance, the invention is experimentally demonstrated herein using to TIM3+VISTA+CSFlR+TAM-targeting tri-specific Abs that have been found to induce type I IFN signalling in TAMs and potentiate macrophage-driven anticancer cytotoxicity, thereby proving the anticancer immune potential of the binding agents of the invention. The inventors demonstrate herein that myeloid-cells of patients that do not respond to immunotherapy (anti-PDl / anti-CTLA4 ICBs), are highly enriched for particular checkpoint proteins, and TAM proteins. Accordingly, the multispecific binding agents are of particular interest for the treatment of cancer in patients that are fully or partially non-responsive to immunotherapy. In an aspect, the invention thus relates to the multispecific binding agent, the polynucleic acid, the vector, the host cell, or the composition according to the invention as described herein elsewhere, for use in the treatment of cancer, wherein the cancer is a cancer resistant against immunotherapy, particularly anti-PDl (programmed cell death protein 1) and / or anti PD-L1 (programmed death-ligand 1) immune therapy.

[0240] This invention establishes that disease-associated macrophages can express multiple or at least two immune-checkpoints or immune-inhibitory receptors, such as TAMs. Accordingly, the skilled person will understand that the TIM3 and VISTA checkpoint proteins identified in this study can be replaced by other checkpoint proteins that are highly expressed in the macrophages of interest, in particular when capable of inducing or activating a type I IFN response. In particular embodiments, the use of the multispecific binding agent of the invention can be particularly directed to the treatment of tumors that overexpress the checkpoints targeted by the multispecific binding agent. In an aspect, the invention relates to the multispecific binding agent capable of binding to a first immune checkpoint protein, a second immune checkpoint protein, and a macrophage protein which is different from said first and said second immune checkpoint protein, for use in the treatment of cancer, wherein the cancer is characterized by the presence of tumor associated macrophages expressing said first and second immune checkpoint protein and said macrophage protein. In particular embodiments, this can be tested prior to the treatment, e.g. by determining the expression of said proteins in a tumor biopsy. For instance, in an aspect, the invention relates to the multispecific binding agent targeting TIM3+, VISTA+ and CSF1R.+ (or a polynucleic acid encoding said binding agent, the vector expressing said binding agent, the host cell expressing said binding agent or the composition comprising any one thereof) according to the invention as described herein elsewhere, for use in the treatment of cancer, wherein the cancer is characterised by the presence of TIM3+, VISTA+ and CSF1R.+ tumor associated macrophages. A number of cancers are known to benefit from immunotherapy and therefore are particularly envisaged to be treated with the binding agents of the present invention. Accordingly, in a further aspect, the invention relates to the multispecific binding agent, the polynucleic acid, the vector, the host cell, or the composition according to the invention as described herein elsewhere, for use in the treatment of cancer, wherein the cancer is selected from the group consisting of lung cancer(or lung carcinoma), colorectal cancer (e.g., colon carcinoma), melanoma, kidney cancer (e.g., renal cortical adenocarcinoma), cervical cancer, and breast cancer.

[0241] The inventors further demonstrate herein that the multispecific binding agents of the invention also enhance the effect of inhibitors of PD1-PDL1 interaction on T- cells. Indeed, it was found that while the multispecific binding agent exhibited strong autonomous mono-therapy efficacy against tumors in vivo, it also rejuvenated new anticancer T cell activity, as marked by increased infiltrating of lymphocytes like CD4+ / CD8+T cells as well as their enhanced activation status as marked by increased emergence of T cells expressing IFN-gamma, IL-2 and / or TNF functional cytokines or cytotoxicity markers like CD107a. Indeed, this T cell activation / rejuvenation allowed the multispecific binding agent to achieve further enhanced antitumor efficacy in vivo (relative to mono-therapy) in combination with T-cell targeting therapies like PD1 inhibition / blockade, PD1 / CTLA4 co- inhibition / co-blockade, PD-L1 inhibition / blockade, dendritic cells (DCs)-based anticancer vaccines and combination of DC-based anticancer vaccines as well as PD-L1 inhibition / co-blockade. Accordingly, a further aspect of the invention is the use of the multispecific binding agents of the present invention in combination therapies based on T cell-targeting agents and vaccines. The combination is envisaged for any therapy targeting T cells, or aiming to activate T cells, or aiming to activate cytotoxic T cells via anticancer vaccine-like effect or antigen-specific immunity effect achieved via inactivating or stimulating lymphocytic pathways. Examples of combination therapies include combinations of the multispecific binding agents of the invention with a binding agent directed to one or more of PD1, CD279, PD-L1, PD-L2, CTLA4, T cell receptor (TCR), IFN-gamma, IL-2, TNF, SHP1, SHP2, ZAP70, LCK, HLA-I, HLA-II, MHC-I, and MHC-II. Accordingly, in a further aspect, the invention relates to the multispecific binding agent, the polynucleic acid, the vector, the host cell, or the composition according to the invention as described herein elsewhere, for use in the treatment of cancer or an infectious disease, preferably cancer, wherein said binding agent is administered as a combination therapy with a T-cell targeting therapy, such as the treatment with a binding agent directed to one or more of PD1, CD279, PD-L1, PD-L2, CTLA4, T cell receptor (TCR), IFN-gamma, IL-2, TNF, SHP1, SHP2, ZAP70, LCK, HLA-I, HLA-II, MHC-I, and MHC-II.

[0242] EXAMPLES

[0243] EXAMPLE 1 HAVCR2+VS / R+TAMs specifically associate with immunoresistant human tumors.

[0244] To understand the immune-inhibitory receptor niche likely to be enriched within myeloid or T cells in clinical immune-resistance context, we analysed existing single-cell (sc)-RNAseq profiles of melanoma-infiltrating immune cells from immunotherapy (anti-PDl / anti-CTLA4 ICBs) responding or non-responding patients (Fig.2a-b, see next page). T cells from immunotherapy-responsive melanoma patients had low levels of most immune-inhibitory receptors, whereas non-responders' T cells highly enriched multiple immune-inhibitory receptors (Fig.2a). However, in case of myeloid cells, only non-responders' myeloid-cells highly enriched for particularly HAVCR2 (gene coding for TIM3 protein) and VSIR (gene coding for VISTA protein), along with the well-established TAM protein, CSF1R (Fig.2b).

[0245] Moreover, a multi-cancer and pan-immune scRNAseq dataset-analysis highlighted that human tumors highly enrich for TIM3+VISTA+TAMs, but not necessarily TIM3+VISTA+Tregs, B cells, NK cells or CD8+T cells (Fig.2c).

[0246] Furthermore, a large-scale pan-cancer as well as pan-myeloid, scRNAseq analysis emphasized that a highly specific CSFlR+TAM-subset most strongly enriched TIM3 / VISTA, but not necessarily various DC or monocyte-subsets (Fig.2d). All these massive human scRNAseq screenings clearly indicate a pan-cancer or pansolid tumor existence for a TIM3+VISTA+CSF1R+TAM subset in human tumorcontext (Fig.2e), with association to ICB non-responders.

[0247] EXAMPLE 2 TIM3+VISTA+TAMs are dominantly enriched in an immunoresistant murine tumor. Murine Lewis lung carcinoma (LLC)-tumor (which is completely resistant to anti- PD1 / PD-L1 immunotherapy; Fig.3a) were compared with MC38 colon carcinomatumors (that are responsive to anti-PD(L)l immunotherapy, Fig.3b).

[0248] Here, LLC-tumors were higher in TAMs (Fig.3c) and very-low in CD4+ / CD8+T cells, compared to MC38-tumors (Fig.3c). This established LLC tumors as immunotherapy resistant with a TAM 'rich' niche, and MC38 tumors as immunotherapy susceptible with T cells 'rich' niche. In accordance with the human results (Fig.2b), immunotherapy-resistant LLC-tumors had more TIM3+VISTA+TAMs than immunotherapy-sensitive MC38-tumors (Fig.3d) and these TIM3+VISTA+TAMs in LLC-tumors had an CSF1RHIGHCD2O6HIGHanti-inflammatory profile. This clearly suggests the existence of a TIM3+VISTA+CSF1RHIGH / +TAM subset. The exclusive association of TIM3+VISTA+phenotype with TAMs (rather than other immune cells) in LLC-tumors was also confirmed on scRNAseq level (Fig.3e).

[0249] Notably, we also could not detect TIM3+VISTA+DCs or TIM3+VISTA+CD4+ / CD8+T cells in these animal model systems (Fig.3f). This illustrates that the tendency of immuno-resistant tumors to enrich for TIM3+VISTA+CSFlRHIGH / +TAMs was a human-to-mouse conserved phenomenon.

[0250] EXAMPLE 3 TIM3+VISTA+CSFlR+TAMs neutralization leads to tumor reduction

[0251] Both LLC- and MC38-tumors were completely resistant to monospecific anti-TIM3 or anti-VISTA Abs (Fig.4a-b). Also, combinatorial immunotherapy between anti- PD1 and anti-TIM3 / anti-VISTA Abs (Fig.4c-d) didn't exert significant anti-tumor effects. These failures of mono-specific Abs, which faithfully resemble recent failure of anti-TIM3 mono-specific Ab in a multi-cancer (basket) human clinical trial.

[0252] It is important to consider that systemically administered monospecific Abs against TIM3 or VISTA are also likely to be titrated by other immune (e.g., CSFlRNEGATIVEmacrophage) or non-tumoral targets that express TIM3 / VISTA without any functional implications for the tumor regression. This can reduce the TAM-targeting efficacy of such monospecific Abs and thereby undermine their antitumor efficacy.

[0253] These results raised an important question, i.e., whether TIM3 / VISTA blockade via monospecific Abs failed to regress the LLC-tumors only because a strong coinhibition or co-blockade of these immune-checkpoints was necessary along with the TAM protein (CSF1R) to overcome such tumors. This motivated us to create novel multi-specific Abs to enforce co-blockade / co-inhibition of TIM3 / VISTA / CSF1R.

[0254] EXAMPLE 4 Multi-specific Abs for neutralisation of TIM3+VISTA+CSF1R+TAMs.

[0255] Single-chain variable fragment (scFv) of anti-CSFIR, anti-TIM-3 and anti-VISTA IgG were recombinantly produced, purified and tested them for target binding.

[0256] All 3 scFv fragments retained good binding as determined by ELISA.

[0257] Next, bi-specific, and tri-specific Ab formats were designed in which the anti- CSFIR Ab was retained as monovalent IgG (Fab) linked to one or two scFvs (derived from the anti-TIM-3 and / or anti-VISTA Abs).

[0258] Fig.5a visualises the heterodimeric design of these multi-specific Abs.

[0259] An embodiment of a tri-specific Ab is shown in Fig. 5b.

[0260] This tri-specific mouse antibody is based on a mouse IgG2a or human IgGl Fc domain with Fc gamma receptor binding silenced through the introduction of LALAPG-equivalent (LALAPE) mutations (Schlothauer (2016) Protein Eng Des Sei. 29, 457-466). Fc heterodimerization is achieved using mouse knob-into-hole mutations, mirroring human knob-into-hole technology (Ridgway (1996) cited above). The antibody to scFv conversion is done in a VL-4xGGGS-VH orientation.

[0261] The antibody consists of the following three polypeptides with the following elements, from amino-terminus to carboxy-terminus:

[0262] 1) Vista scFv VL - Vista scFv VH - mouse IgG2a / human IgGl CH2, LALAPG -mouse IgG2a / IgGl CH3, hole side - Tim 3 scFv VL - Tim 3 scFv VH.

[0263] 2) CSF1R Fab VL - mouse kappa CL

[0264] 3) CSF1R Fab VH - mouse IgG2a CHI - mouse IgG2a / human IgGl CH2, LALAPG -mouse IgG2a / human IgGl CH3, knob side.

[0265] Herein the antibody features three binding sites targeting mouse VISTA, Tim3 and CD115 / M-CSFR. The tri-specific mouse antibody based on a mouse IgG2a or human IgGl Fc domain with Fc gamma receptor binding silenced through the introduction of LALAPG-equivalent (LALAPE) mutations (Schlothauer (2016), cited above).

[0266] Fc heterodimerisation is achieved using mouse knob-into-hole mutations, mirroring human knob-in-hole technology (Ridgway (1996) Protein Eng. 9, 617- 621).

[0267] The antibody to scFv conversion is in the VL-4xGGGS-VH orientation.

[0268] The design must be heterodimeric (KiH) Fab (CSFlR)-Fc-ScFv (VISTA) / scFv (TIM- 3)-Fc. See below.

[0269] The anti-VISTA antibody clone 13F3 is derived from an Armenian Hamster hybridoma (Wang (2011) J Exp Med. 208, 577-392). It has the ability to block VISTA and its immuno-suppressant activity.

[0270] The anti-TIM3 clone 2012 is derived from a rat hybridoma. It has been shown to block Tim-3 and its immuno-suppressant activity (Monney (2002) Nature 415, 536-541).

[0271] The anti-CD115 / M-CSFR clone ASF98 is derived from a rat hybridoma and lock CSF-1 binding to murine CD115-expressing cells (Sudo (1995) Oncogene 11, 2469-2476).

[0272] These bi-specific and tri-specific Abs showed proficient binding to recombinant TIM3, VISTA and / or CSF1R of mouse-origin in ELISA setting (Fig.5d). These Abs also bound the J774 mouse macrophages and mouse bone-marrow derived macrophages to a comparative extent (Fig.5d).

[0273] EXAMPLE 5 Functional validation of multi-specific TIM3-VISTA-CSF1R Abs The functional impact of the above multi-specific Abs was evaluated on mouse macrophages via a high throughput screening using J774 reporter macrophages expressing transgenic reporter systems for type I interferon (IFN) response.

[0274] Binding of the Ab lead to a significant increase in type I IFN response, which is a potent anti-tumor immunogenic pathway, on the level of macrophages after blockade with only the tri-specific Ab (but not bi- or mono-specific Abs, either alone or in admixtures) (Fig.6a).

[0275] Ex vivo experiments show that the tri-specific Ab specifically bound only the TAMs but not DCs or CD4+ / CD8+T cells (Fig.6b), thereby confirming its pharmacological target-engagement efficacy. The tri-specific Ab also induced significant type I IFN signalling in TAMs, as marked by increased phosphorylated STAT1 levels within Ml-like or pro-inflammatory MHC-II+TAM compartment (Fig.6c).

[0276] In co-cultures of these TAMs and cancer cells, the tri-specific Ab significantly potentiated macrophage-driven anticancer cytotoxicity against LLC cancer cells (Fig.6d) - a marker of anticancer immune reactions.

[0277] These result show that the TIM3+VISTA+CSFlR+TAM-targeting tri-specific Ab induces anticancer immune reactions ex vivo.

[0278] It was further tested if the increase in type I IFN response on the level of macrophages after blockade with our tri-specific antibody was specific for LLC cancer cells or also others. Several other cancer cells were able to induce a type I IFN response after blockade with our tri-specific antibody, with LLC being the strongest but not the only one, thereby emphasizing the pan-cancer potential of our tri-specific antibody (Fig.7).

[0279] EXAMPLE 6 in vivo validation of the tri-specific antibody

[0280] Based on the in vitro data, we pursued in vivo validation of the tri-specific antibody with an LLC tumor model. The LLC tumor model is unresponsive to other macrophage targeting therapeutics including anti-FcyR, anti-TGFP, anti-CRl / 2, anti-ILlO, CD40 agonist, anti-SSPl, anti-TREM2, anti-ILip, anti-CCL2, anti- SIRPlo (Fig. 8a), anti-CSFIR and anti-PDLl antibody (Fig. 8b).

[0281] The tri-specific antibody significantly reduced tumor volume by itself in LLC tumors (Fig. 9a). This effect on tumor volume was not observed with the monospecific antibodies (O-TIM3, o-VISTA or a-CSFIR) or the combination of these 3 monospecific antibodies (Fig. 9a). The tri-specific antibody could significantly reduce TIM3+VISTA+CSF1R+tumor-associated macrophages (TAMs), almost depleting this TAM subset (Fig. 9b). Strikingly, an effect on T cell level was observed only in the mice treated with the tri-specific antibody (Fig. 9c). More specifically, an increase of TNF, IL2 and IFNy was seen in T cells (Fig. 9d-f). Furthermore, TAMs were significantly repolarized to a more Ml-like phenotype (marked by MHC-IIhighrather than M2-like marker, i.e., CD206high) compared to PBS treated tumors (Fig. 9g). Similarly, the tri-specific antibody significantly reduced tumor volume by itself in YUMM1.7, YUMMER1.7 and YUMM-OVA melanoma tumors (Fig. lOa-c).

[0282] To further look into the mechanism of action of the tri-specific Ab, we co-cultured TAMs from LLC tumors, pre-incubated with either isotype, combination of O-TIM3, o-VISTA or a-CSFIR or tri-specific antibody, with untreated LLC cancer cells. Only the tri-specific antibody induced significant type I IFN signaling in TAMs, as marked by increased phosphorylated STAT1 and STAT2 levels (Fig. lla-b) and decreased phosphorylated STAT3 levels (Fig. 11c). This phosphorylated STAT1 / 2 high versus phosphorylated STAT3 low state defines an anti-viral like response, linked to type I IFN responsiveness.

[0283] Additionally, in vitro pre-blockade with mono-specific antibodies blocking one or all binding epitopes of the tri-specific Ab on J774 macrophages inhibited the type I IFN inducing effect of the tri-specific Ab (Fig. 12a). Extracellular nucleic acids degradation was also able to reduce the type I IFN inducing effect of the tri-specific Ab (Fig. 12b). In line with this, pre-blockade with different inhibitors of up-stream activators of the interferon response pathways in J774 macrophages showed that the type I IFN response induced by tri-specific Ab is activated through the cGAS and STING pathway (Fig 12c). The effects of the tri-specific Ab were independent of inhibitory receptor-ligand interactions, since LLC cancer cells knocked-out of relevant immune-checkpoint / inhibitory receptors' ligands like Galectin-9, HMGB1, PSGL1, VISTA and CEACAM1 did not impact the type I IFN response (Fig 12d).

[0284] Next, we investigated if the tri-specific antibody could synergize with T cell activating or rejuvenating therapeutics. Combining the tri-specific antibody with anti-PDl and / or anti-CTLA (Fig. 13a), or DC vaccination, or anti-PD-Ll or DC vaccination + anti-PDLl (Fig. 13b) significantly increased the therapeutic response to tri-specific antibody as marked by reduction in tumor growth.

[0285] EXAMPLE 7 Construct variations

[0286] The human binding equivalent of the tri-specific binding agent was generated.

[0287] The first chain comprises polypeptide 1 : Variable domain light chain binding to human CSF1R coupled to constant domain light chain (SEO ID NO:3)

[0288] DIQMTQSPSSLSASVGDRVTITCRASEDVNTYVSWYQQKPGKAPKLLIYAASNR YTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFSYPTFGQGTKLEIKRTVAA PSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNSOESVTEODSKDST

[0289] YSLSSTLTLSKADYEKHKVYACEVTHOGLSSPVTKSFNRGEC

[0290] The second chain comprises polypeptide 2: Variable domain heavy chain binding to human CSF1R coupled to human IqGl backbone (lalapq+knob) (SEO ID NO:2):

[0291] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDISWVRQAPGQGLEWMGVIWT DGGTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDQRLYFDVW GOGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV

[0292] HTFPAVLOSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEOYNSTYRVVSVLTVLHODWLNGKEYKCKVSNKALGAPIEKTISKAKGOPREPOVYT

[0293] LPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWOOGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0294] The third chain comprises

[0295] Polypeptide 3: Single chain variable fragment binding to human VISTA coupled to human IqGl backbone (lalapq+ hole-) coupled to Single chain variable fragment binding to human TIM3 (SEQ ID NO:1):

[0296] EIVLTQSPATLSLSPGERATLSCRASSSVGYIHWYQQKPGQAPRPIIYTTSNRAT GIPARFSGSGSGTDNTLTISSLEPEDSAVYYCQQWSSYPPITFGGGTKLEIKGGG GSGGGGSGGGGSQVQLQESGPGLVKPSDTLSLTCTVSGYSITSDYTWNWIRQTP GKGLEWIGHITYSGSVSYNPSLRSRVTISRDTSKNQFSLKLSSVTAADTATYSCA RALYYPWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPS

[0297] VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHODWLNGKEYKCKVSNKALGAPIEKTISKAKGOPREPQVCTLPPSRDELTKN OVSLSCAVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWOOGN

[0298] VFSCSVMHEALHNHYTOKSLSLSPGKGGGGSGGGGSGGGGS JOLTOSPSS / .S SV'GDR VTITCRASESVEYYGTSLMQWYQQKPGKAPKLLIYAASNVESGVPSRFSGSGSGTDFTLTI SSLQPEDFATYFCQQSRKDPSTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVK

[0299] KPGSSVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGDIYPGNGDTSYNQKFKGRVTIT ADKSTSTVYMELSSLRSEDTA VYYCAR VGGAFPMDYWGQGTTVTVSS An alternative thereto was generated wherein the scFv binding human TIM3 was switched to a nanobody format. This construct comprises three polypeptide chains: polypeptide 1 : Variable domain light chain binding to human CSF1R coupled to constant domain light chain (SEO ID NO:6):

[0300] DIQMTQSPSSLSASVGDRVTITCRASEDVNTYVSWYQQKPGKAPKLLIYAASNR YTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFSYPTFGQGTKLEIKRTVAA PSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKVOWKVDNALOSGNSOESVTEODSKDST YSLSSTLTLSKADYEKHKVYACEVTHOGLSSPVTKSFNRGEC polypeptide 2:Variable domain heavy chain binding to human CSF1R coupled to human IgGl backbone (lalapq+knob) (SEQ ID NO:5):

[0301] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDISWVRQAPGQGLEWMGVIWT DGGTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDQRLYFDVW

[0302] GOGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLOSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEOYNSTYRVVSVLTVLHODWLNGKEYKCKVSNKALGAPIEKTISKAKGOPREPOVYT

[0303] LPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWOOGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0304] Polypeptide 3: Single chain variable fragment binding to human VISTA coupled to human IgGl backbone (lalapq+hole) coupled to VHH binding to human TIM3 (SEQ ID NO: 4):

[0305] EIVLTQSPATLSLSPGERATLSCRASSSVGYIHWYQQKPGQAPRPIIYTTSNRAT

[0306] GIPARFSGSGSGTDNTLTISSLEPEDSAVYYCQQWSSYPPITFGGGTKLEIKGGG GSGGGGSGGGGSQVQLQESGPGLVKPSDTLSLTCTVSGYSITSDYTWNWIRQTP GKGLEWIGHITYSGSVSYNPSLRSRVTISRDTSKNQFSLKLSSVTAADTATYSCA

[0307] RALYYPWYFDVWGTGTTVTVSSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHODWLNGKEYKCKVSNKALGAPIEKTISKAKGOPREPOVCTLPPSRDELTKN OVSLSCAVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWOQGN

[0308] VFSCSVMHEALHNHYTOKSLSLSPGKGGGGSGGGGSGGGGSOI / OLOESGGGLI / OAGG

[0309] SLNLSCAASGSSFRL YTVG WHRQAPGKQREL VA WISGA GSTNYHSSVKGRFTISRDNAK NTALLQMNNLAPEDTAVYYCNLLNYWGQGTQVTVSS Subsequently, we aimed to further validate construct variations of the tri-specific antibody (see Figure la). An in silico 3D rendering of various construct variants of the tri-specific antibody showed that variations in the tri-specific antibody confirmations did not significantly affect the structural / functional / biochemical stability of anti-CSFIR, anti-TIM3 and / or anti-VISTA domains relevant for epitope / target binding (Fig. lb). Indeed, also experimentally, alternative tri- specific antibody constructs were able to bind (Fig. 14a-c) as well as induce a functional type I IFN response in THP1 macrophages in co-culture with HeLa cancer cells (Fig. 15).

[0310] EXAMPLE 8. Materials and Methods

[0311] Cell lines

[0312] LLC (Ref. CRL-1642 from ATCC), MC38 (Ref. ENH204-FP from Kerafast), 4T1 (Ref. CRL-2539 from ATCC), B16-F10 (Ref. CRL-6475 from ATCC), YUMM1.7 (Ref. CRL- 3362 from ATCC), YUMMER1.7 (Ref. SCC245 from Merck), CT26 (Ref. CRL-2638 from ATCC), Renca (Ref. CRL-2947 from ATCC), GL261 (Ref. CSC-C9184W from Creative Bioarray), HeLa (Ref. CCL-2 from ATCC).

[0313] Reporter J774 / THP1 cell lines, murine or human macrophage-like cell lines containing an IRF-luciferase reporter. Cell lines were cultured at 37°C under 5% CO2 split when 90% confluency was reached through enzymatic dissociation (Trypsin). Cells were maintained in DMEM media containing 2 mM L-glutamine, 3.7 g / L sodium bicarbonate, 4.5 g / L glucose and 1.0 mM sodium pyruvate with 10% heat-inactivated fetal bovine serum (30 min at 56°C; FBS), penicillin 100 U / ml streptomycin 100 pg / ml. J774 / THP1 cells were maintained and tested as reported in already published protocols (Sprooten et al. (2024) Lymph node and tumor- associated PD-Ll+macrophages antagonize dendritic cell vaccines by suppressing CD8+T cells. CELL REPORTS MEDICINE, 5 (1), Art.No. ARTN 101377. doi: 10.1016 / j.xcrm.2023.101377; Sprooten et al. (2021). Peripherally-driven myeloidIFN / ISG responses predict malignancy risk, survival, and immunotherapy regime in ovarian cancer. JOURNAL FOR IMMUNOTHERAPY OF CANCER, 9 (11), Art.No. ARTN e003609. doi: 10.1136 / jitc-2021-003609). All cell lines were tested for mycoplasma contamination every month. CRISPR / Cas9-mediated gene knock-out in the LLC cell line

[0314] Knock-outs of Ceacaml, Hmgbl, Lgals9, Selplg and Vsir genes were generated using four different guides per gene. Guide sequences were cloned into the pLentiCRISPRv2 plasmid according to the standard cloning protocol. For lentiviral particle production, HEK293T cells were plated in 40 mL supplemented DMEM in T150 (TPP) flasks at 45% confluency and incubated overnight. 24 hours later, the cells were co-transfected using X-TremeGENE 9 with the pLentiCRISPR plasmids and the lentiviral packaging plasmids pMD2.G and psPAX2 to generate lentiviral particles coated with the VSV-G protein and incubated overnight. Twenty-four hours post transfection the medium was changed to DMEM supplemented with serum-free BSA growth media (DMEM + 1.1 g / lOOmL BSA and 20 pg / mL gentamicin). The supernatant containing lentiviral particles was harvested 72 hours after transfection and stored at -80 °C. Cells were transduced with lentiviruses expressing a pool of the 4 sgRNAs and then selected with puromycin for 3 days. Knock-out phenotype was confirmed by western blotting.

[0315] Analysis of single-cell RNA-sequencinq (scRNAseq) datasets

[0316] Single-cell datasets covering melanoma patients responding (or not) to immunotherapy (PD1 / CTLA4 blockade), pan-immune dataset from 4 different cancers, pan-myeloid dataset from 8 different cancers and colorectal cancer (CRC) dataset with MSI and MSS CRC patients, were accessed using a standardized and uniformized workflow of BBrowser_v_3, using built-in qualitative filters. The preexisting scRNAseq dataset of subcutaneous LLC tumors was uploaded into the same workflow and further analyzed to maintain uniformity. This work-flow was used for generating density plots, or dot plots, as applicable. Cellular annotations either pre-existed on the level of author-derived annotations or were based on automated annotations available within the above work-flow. We used the HAVCR2+VSIR+TAMs within the CRC scRNAseq dataset to perform differential gene-enrichment (DGE) analyses as well as co-dependent pathway enrichment analyses (REACTOME for human, Gene Ontology Biological Process for mouse), using the Venice non-parametric analyses approach (https: / / github.com / bioturing / signac). This was specifically done by comparing scRNAseq profiles of HAVCR2+VSIR+TAMs (HAVCR2 and VSIR expression > 0) vs. HAVCR2_VSIR_TAMs (HAVCR2 and VSIR expression = 0) against each other. Of note, in some datasets, an alternative name for VSIR was C10orf54. The DGE analyses also allowed us to extract a signature for HAVCR2+VSIR+TAMs which included all positively enriched genes in favor of this subset above the statistical cut-off of -Iogl0(p-value) = 100. With this cut-off, we delineated the following signature for HAVCR2+VSIR+TAMs (some major TAM genes are indicated in bold) : C10orf54, TYROBP, HAVCR2, B2M, GRN, LAPTM5, CD74, HLA-C, PSAP, NPC2, TMEM176B, VAMP8, HLA-A, HLA-DMB, HLA-DPA1, GPX1, CD68, HLA-DMA, TMSB4X, OAZ1, HLA-E, CFL1, ARPC1B, FCGRT, ITM2B, C1QC, PFN1, HLA-B, HLA- DPB1, HLA-DRA, CST3, ARHGDIB, CYBA, TMSB10, CTSB, HLA-DQB1, ITGB2, ACTG1, CAPG, ARPC3, S100A11, RPL15, CTSS, C1QB, ATP6V0B, CTSA, RPS2, YBX1, CTSC, PTMA, SRP14, AIF1, C1QA, PPT1, CTSH, CALM2, CSF1R, YWHAH, RPL28, CAPZB, ACTB, TYMP, CLIC1, ATP6V0E1, CTSD, RPL13A, HEXB, SERF2, EEF1A1, FCER1G, ACP5, TMEM176A, RPS19, PLD3, CORO1B, RPSA, RPL27A, PYCARD, SYNGR2, RPS3, BST2, NACA, PRDX1, LAIR1, PPIA, ERP29. The full scRNAseq profile of HAVCR2+VSIR+TAMs (HAVCR2 and VSIR expression > 0) was used to drive the automated Cell Ontology analyses (i.e., Cell Search algorithm) to interrogate the 300 scRNAseq datasets within the curated BioTuring database as available on 17th February 2021(40). The hits were filtered at a Jaccard's index threshold of 0.7. This analyses delineated the following dataset hits for macrophage node: Basal Cell Carcinoma (GSE123814), Colorectal Cancer (GSE144735), Liver Cancer (GSE125449), Lung (COVID-19; GSE145926), Lung Cancer (E-MTAB-6149), Lung Cancer (GSE140819), Pancreatic Cancer (GSA: CRA001160), Testis (Normal; GSE134144); and the following datasets for monocyte node: Fetal Liver (E-MTAB-7407), Lung (PRJEB31843), Lung (Fibrosis; GSE128033), Lung (Fibrosis; GSE121611), Lung (Fibrosis; GSE135893), Lung Cancer (GSE131907), Lung (SSc-ILD; GSE128169), Melanoma (GSE123139), Nasopharyngeal Cancer (GSE150430), PBMCs (sepsis; SCP548), PBMCs (SC2018), Respiratory Tract (EGAS00001004082), Spleen (PRJEB31843).

[0317] Murine tumor infiltrating leukocytes (TILs) and Tumor associated macrophages (TAMs) isolation

[0318] Tumors were isolated at day 24 after tumor injection. A single cell suspension was made, using the tumor dissociation kit (Miltenyi #130-096-730). TILs were isolated through magnetic bead separation via CD45 (Miltenyi #130-110-618). For TAM isolation, anti-F4 / 80 microbeads (Miltenyi #130-110-443) were used. Isolated TILs and TAMs were maintained in RPMI supplemented with 100 pg / mL penicillin, 100 pg / L streptomycin, 2.5% HEPES ph 7.5, 10% heat-inactivated fetal bovine serum (FBS). Following fluorochrome-conjugated antibody clones were used: F4 / 80 (clone T45-2342), CDllb (clone ICRF44), HAVcr-2 (clone B8.2C12), VISTA (clone MH5A), CD86 (clone GL1), CSF1R (clone AFS98), CD206 (clone C068C2) CD8a (clone 53-6.7), CD4 (clone GK1.5), CDllc (clone N418) and pSTATl (clone pY701).

[0319] Flow cytometry

[0320] Before staining procedure, Fc receptor of all samples were blocked using TruStain FcX (Biolegend #101320) for 15min. Cells were further stained with the indicated antibodies above, diluted in 0.5% BSA, for Ih and fixed with cytofix (BD Bioscience #554655). In case of intracellular markers (pSTATs), cells were fixed (BD Bioscience #554655) and permeabilised with the Phosflow Perm Buffer III (BD Bioscience #558050). In case of intracellular markers, cells were fixed and permeabilised with the Cytofix / Cytoperm Kit (BD Bioscience #554714). For intracellular cytokine staining, the cells were stimulated with Dynabeads Mouse T activator CD3 / CD28 (Thermo Fisher Scientific #11456D). After Ih at 37°C 5% CO2, 2 pl Brefeldin A (Thermo Fisher Scientific #00-4506-51) was added. Cells were then placed at 37°C 5% CO2 for 4 hours, transferred to 4°C overnight and then stained for intracellular cytokines.

[0321] Following fluorochrome-conjugated antibody clones were used: F4 / 80 (clone T45- 2342), CDllb (clone ICRF44), HAVcr-2 (clone B8.2C12), VISTA (clone MH5A), CD86 (clone GL1), CSF1R (clone AFS98), CD206 (clone C068C2) CD8a (clone 53- 6.7), CD4 (clone GK1.5), CDllc (clone N418), pSTATl (clone pY701), pSTAT2 (clone D3P2P), pSTAT3 (clone pY705), IL2 (clone JES6-5H4), TNFo (clone MP6- XT22), IFNy (clone XMG1.2), MHCII (clone M5 / 114.15.2), CD3 (clone 17A2). Biand tri-specific antibodies were conjugated with conjugation kits (Abeam # ab201807) according to the manufacturer's protocol.

[0322] After fixation, cells were maintained in 0.5% BSA. Flow cytometry was performed on FACSCanto (BD Bioscience) or the ID7000 (SONY). Cell doublets were excluded based on FSC-A / FSC-H. Flow cytometry data was analysed using FlowJo.

[0323] Binding assay via Flow cytometry

[0324] Bi-specific and tri-specific antibodies were conjugated with APC according to the manufacturer's protocol (Abeam #ab201807). Bone-marrow derived macrophages, J774 macrophages or tumor isolated leukocytes were stained with our fluorescently labelled antibodies diluted in FACS buffer (0.5% BSA and PBS solution) for Ihour on ice and then washed twice with FACS buffer. Following fluorochrome-conjugated antibody clones were used; CD8a (clone 53-6.7), CD4 (clone GK1.5), CDllc (clone N418), F4 / 80 (clone T45-2342), CDllb (clone ICRF44), Fixable Zombie Aqua and either our bi- or tri-specific conjugated antibody.

[0325] Binding ELISA

[0326] ELISA plates were coated overnight at room temperature using Ipg / ml of recombinant mouse / human TIM3, VISTA and CSF1R. ELISA plates were washed 3 times with ELISA washing buffer (0.05% Tween and PBS solution) and blocked for 2 hours using ELISA blocking buffer (3% BSA and PBS solution). Thereafter, ELISA plates were washed 3 times and the appropriate antibody (scFv fragments, monospecific antibodies or bi- and tri-specific antibodies) were added in a serial dilution (0.01 to 100 pg / ml) for 2 hours. Hereafter, ELISA plates were washed 3 times and detection antibody (anti-Flag-HRP, anti-Rat-HRP, anti-Mouse-HRP, anti- Armerian Hamster-HRP or anti-human IgGl-HRP) was added in a 1 :2.000 dilution for 1 hour. Then, after 3 times washing, HRP-streptavidin was added in a 1 :20.000 dilution for 20 minutes. Lastly, 100 pl detection solution was added to the plate for 15 minutes, followed by 50 pl stopping solution. Absorbance was read using a microplate reader at 450 nm.

[0327] Cancer cell - TAM co-cultures

[0328] Cancer cells were co-cultured with LLC tumor derived TAMs (pre-incubated with isotype blocking antibodies or anti-TIM3 / anti-VISTA / anti-CSFlR blocking antibodies or multispecific antibodies) in a 1 :5 ratio. After 48 hours cocultures were scraped to collect the cells, centrifuged and washed with PBS.

[0329] Murine bone marrow-derived macrophage generation

[0330] Bone marrow was isolated from wild type C57BL76j mice. Both the femur and tibia were flushed using PBS and the cell suspension was centrifuged for 5 minutes at 1500rpm. The pellet was resuspended in red blood cell lysis buffer (Merck life science), incubated for 5 minutes and centrifuged. Cells were resuspended in RPMI supplemented with 100 u / mL penicillin, 100 pg / L streptomycin, 2.5% HEPES pH 7.5 and 10% heat-inactivated FBS. Bone marrow derived cells were differentiated into macrophages by adding 25 ng / ml M-CSF (Peprotech #315-02) into the media for 6 days. Differentiation of macrophages was confirmed by flowcytometry with respectively following fluorochrome-conjugated antibody clones were used;

[0331] CDllb (clone MI / 70) and F4 / 80 (clone BM8).

[0332] J774 / THP1 macrophage reporter assay

[0333] J774 / THP1 reporter cells were seeded in a 96 well plate at a density of 30.000 cells per well in a 96-well plate. After 24 hours, cells were pre-treated with 10 pg / mL isotype or blocking antibodies for 4h. Untreated LLC or Hela cancer cellswere co-cultured with the J774 / THP1 reporter cells in a 1: 1 ratio.

[0334] In the case of neutralizing nucleic acids, untreated cancer cells were pre-incubated for Ih before with 3350 U / 106 cancer cells DNase I (Invitrogen #18047-019) and RNase 3.350 mg / 106 cancer cells RNase A (Thermo Fisher Scientific #EN0531). Thereafter, cancer cells were co-cultured with the J774 reporter cells in a 1 : 1 ratio. In the case of inhibitors, J774 reporter cells were pre-incubated for 4h with 10 pM of ENPP1 inhibitor (Cayman chemical #37687), or 25 pM of Trif inhibitory peptide or Trif control (Invivogen #tlrl-pitrif), or 25 pM of MyD88 inhibitory peptide or MyD88 control (Invivogen #tlrl-pimyd), or 20 pg / ml of cGAS inhibitor (Invivogen #inh-ru521-2) or 4 pg / ml of STING inhibitor (Invivogen #inh-hl51). Thereafter, LLC cancer cells were added at a 1 : 1 ratio together with the 10 pg / ml of tri-specific antibody or their isotype control antibody.

[0335] In the case of pre-blocking experiments J774 reporter cells were pre-incubated for 4h with 10 pg / ml of TIM3, VISTA, CSF1R or a combination of TIM3, VISTA and CSF1R. Thereafter, LLC cancer cells were added at a 1 : 1 ratio together with the 10 pg / ml of tri-specific antibody.

[0336] To measure the interferon (IRF) / interferon-stimulated genes (ISG) response after 48 hours, another 80 pl of media was derived from the above cancer cell-J774 coculture in a white opaque-bottom 96-well plate. Herein, 50 pl of luciferase substrate was added and bioluminescence was directly measured with 100 milliseconds of signal integration, with the microplate reader. All data derived from this reporter assay was Min-Max normalized for percentage scaling to account for inter-assay baseline variability. Detailed protocols and methods related to this technique are already published (Sprooten et al. (2024) Lymph node and tumor- associated PD-Ll+macrophages antagonize dendritic cell vaccines by suppressing CD8+T cells. CELL REPORTS MEDICINE, 5 (1), Art.No. ARTN 101377. doi: 10.1016 / j.xcrm.2023.101377; Sprooten et al. (2021). Peripherally-driven myeloid IFN / ISG responses predict malignancy risk, survival, and immunotherapy regime in ovarian cancer. JOURNAL FOR IMMUNOTHERAPY OF CANCER, 9 (11), Art.No. ARTN e003609. doi: 10.1136 / jitc-2021-003609).

[0337] DC vaccine creation

[0338] LLC cancer cells were plated in 10cm dishes treated with lOpM BV6 (Selleckchem #S7597) for 3h and subsequently 100 ng / mL TNF (Miltenyi #130-101-687). After 24h of cell death induction, bone marrow derived DCs were stimulated with dying cancer cells in a 1 : 1 ratio and 2.5 ng / mL interferon beta (IFN[3) (R8iD systems #8234-MB-010). After 48h, DCs were harvested by scraping and washed with PBS for injection.

[0339] Mouse experiments

[0340] Seven to twelve-week-old female / male C57BL / 6J mice were subcutaneously (s.c) injected with IxlO6LLC, MC38 cells. When applicable, mice were treated or cotreated with 250 pg of anti-mouse PD-L1 (clone MIH5; Polpharma Biologies), antimouse CTLA-4 (clone 4F10; Polpharma Biologies), anti- mouse PD-1 (RMP1-14; Polpharma Biologies), anti-mouse HAVcr-2 (clone B8.2C12; BioXCell), anti-mouse VISTA (clone 13F3; BioXCell), anti-mouse FCyR (clone 2.4 G2; Polpharma Biologies), anti-mouse TGFP (clone 1D11; Polpharma Biologies), anti-mouse CR1 / 2 (clone 7G6; Polpharma Biologies), anti-mouse CSF1R (clone AFS98; Polpharma Biologies), anti-mouse IL10 (clone JES5.2A5; Polpharma Biologies), CD40 agonism (clone FGK45; Polpharma Biologies), anti-mouse Trem2 (clone 178; BioXCell), anti-mouse SPP1 (clone 100D3; BioXCell), anti-mouse CCL2 (clone 2H5; BioXCell), anti-mouse SIRPo (clone P84; BioXCell), anti-mouse IL1 [3 (clone B122; BioXCell) or 50 pg of tri-specific antibody on day 11, 14 and 18 via i.p. injections. When applicable, mice were subcutaneously (s.c.) injected with a DC vaccine on day 10 and 13, and tri-specific antibody and / or anti-PDLl on day 11 and 14.

[0341] Mice were monitored and weighed every other day and tumor volume was determined by height x width x length.

[0342] Murine tumor infiltrating leukocytes (TILs) and Tumor associated macrophages (TAMs) isolation

[0343] Tumors were isolated at day 24 after tumor injection. A single cell suspension was made, using the tumor dissociation kit (Miltenyi #130-096-730). TILs were isolated through magnetic bead separation via CD45 (Miltenyi #130-110-618). For TAM isolation, anti-F4 / 80 microbeads (Miltenyi #130-110-443) were used. Isolated TILs and TAMs were maintained in RPMI supplemented with 100 pg / mL penicillin, 100 pg / L streptomycin, 2.5% Hepes Ph7.5, 10% heat-inactivated fetal bovine serum (FBS). Following fluorochrome-conjugated antibody clones were used: F4 / 80 (clone T45-2342), CDllb (clone ICRF44), HAVcr-2 (cloneB8.2C12), VISTA (cloneMH5A), CD86 (clone GL1), CSF1R (clone AFS98), CD206 (clone C068C2) CD8a (clone 53-6.7), CD4 (clone GK1.5), CDllc (clone N418), Zombi- Aqua (Biolegend #423102), pSTATl (clone pY701), pSTAT2 (clone Tyr690, D3P2P) or pSTAT3 (clone 232209).

[0344] Global pairwise

[0345] Standard Needleman-Wunsch was used for the global pairwise sequence alignment. Sequences for human and murine TIM3 and VISTA were obtained from UniProt respectively; HAVR2_HUMAN, HAVR2_MOUSE, VISTA_HUMAN, VISTA_MOUSE.

[0346] Multispecific antibodies' 3D configurations

[0347] Figure lb structures were modelled in silico using the sequences described in Example 7 and structural references in Figure la. The initial 3D configurations were generated using the publicly available AlphaFold3 server (https: / / alphafoldserver.com / ). Generated structures were verified for geometrical correctness using PyMOL.

[0348] All molecular dynamics simulations were performed using GROMACS v2024.2. The Amber99sb force field was used for all simulations due to its accuracy in protein folding and side-chain dynamics. The antibody structures were solvated in a cubic box using TIP3P water model with a minimum distance of 1.0 nm from box edges. Next, the overall charge were neutralized using Na+ and Cl- ions. Each system underwent energy minimization using the steepest descent algorithm to relax side chains and eliminate steric clashes. After minimization, the systems were equilibrated in two steps namely the NVT followed by NPT ensemble. NVT equilibration (constant number of particles, volume, and temperature) was done at 300 K for 100 ps using a V-rescale thermostat. NPT equilibration (constant pressure and temperature) was performed for 100 ps at 1 atm pressure using the Parrinello-Rahman barostat. During both phases, position restraints were applied to heavy atoms of the protein to allow solvent and ions to relax around the structure.

[0349] Production runs were carried out for 50 ns for each antibody variant in the NPT ensemble with a 2 femto-second integration time step using Verlet integrator.

[0350] LINCS algorithm was employed to constrain all bonds involving hydrogen atoms. Periodic boundary conditions (PBC) were applied in all directions. Long-range electrostatics were handled using the Particle Mesh Ewald (PME) method with a cutoff of 1.2 nm for both electrostatics and van der Waals interactions. Post- simulation analysis was performed using GROMACS built-in tools and custom

[0351] Python scripts. To analyze functional stability across different multi-specific antibody structural variants, CSF1R, TIM3 and VISTA domains were compared across structures. Structural and biochemical fluctuations between these structural variants were used to assess the overall functional stability of each domain, a proxy readout for epitope binding capacities and co-dependent functional activities.

Claims

CLAIMS1. A multispecific binding agent capable of binding to a first immune checkpoint protein, a second immune checkpoint protein, and a macrophage protein which is different from said first and said second immune checkpoint protein.

2. The multispecific binding agent according to claim 1, wherein said first immune checkpoint protein belongs to the T cell immunoglobulin and mucindomain (TIM) family.

3. The multispecific binding agent according to claim 1 or 2, wherein said second immune checkpoint protein belongs to the B7 immunoglobulin family.

4. The multispecific binding agent according to any of claims 1 to 3, wherein said macrophage protein is selected from CSF1R, MRC1, CD163, CD68, Fc Gamma Receptors, CD14, CDllb, F4 / 80, and CD206.

5. The multispecific binding agent according to any of claims 1 to 4, wherein said first immune checkpoint protein is selected from TIM3, TIM1, TIM2, TIM4, TIM5, TIM6, TIM7, and TIM8.

6. The multispecific binding agent according to any of claims 1 to 5, wherein said second immune checkpoint protein is selected from B7-H5 / VISTA, B7- 1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, and B7-H7.

7. The multispecific binding agent according to any of claims 1 to 7, comprising a first antigen-binding region capable of binding to the first immune checkpoint protein, a second antigen binding region capable of binding the second immune checkpoint protein, and a third antigen binding region capable of binding to the macrophage protein.

8. The multispecific binding agent according to claim 7, wherein each of said antigen binding region is selected from an Fab, F(ab')z, Fv, scFv, scFv-Fc, nanobody, minibody, sdAb, scAb, or scFab.

9. The multispecific binding agent according to any of claims 1 to 7, comprising an Fc to which the antigen-binding domains are directly or indirectly linked.

10. The multispecific binding agent according to claim 9, wherein the Fc domain comprises two polypeptide chains, each comprising a CH2 domain linked to a CH3 domain, wherein at least one antigen-binding region is linked N- terminally to a CH2 domain and at least one antigen-binding region is linked C-terminally to a CH3 domain.

11. The multispecific binding agent according to any of claims 1 to 10, wherein said first and said second immune checkpoint protein are expressed by tumor-associated macrophages.

12. The multispecific binding agent according to any of claims 1 to 11, wherein said first immune checkpoint protein is TIM3, said second immune checkpoint protein is VISTA, and said macrophage protein is CSF1R.

13. A polynucleic acid encoding the multispecific binding agent according to any of claims 1 to 12.

14. A vector comprising the polynucleotide according to claim 13.

15. A host cell comprising the polynucleotide according to claim 13 or the vector according to claim 14.

16. A composition comprising the multispecific binding agent according to any of claims 1 to 12, the polynucleic acid according to claim 13, the vector according to claim 14, or the host cell according to claim 15.

17. The multispecific binding agent according to any of claims 1 to 12, the polynucleic acid according to claim 13, the vector according to claim 14, the host cell according to claim 15, or the composition according to claim 16, for use as a medicament.

18. The multispecific binding agent according to any of claims 1 to 12, the polynucleic acid according to claim 13, the vector according to claim 14, thehost cell according to claim 15, or the composition according to claim 16, for use in the treatment of cancer or an infectious disease.

19. The multispecific binding agent according to any of claims 1 to 12, the polynucleic acid according to claim 13, the vector according to claim 14, the host cell according to claim 15, or the composition according to claim 16, for use in the treatment of cancer, wherein the cancer is selected from the group consisting of lung cancer, colorectal cancer, melanoma, kidney cancer, cervical cancer, and breast cancer.

20. The multispecific binding agent according to any of claims 1 to 12, the polynucleic acid according to claim 13, the vector according to claim 14, the host cell according to claim 15, or the composition according to claim 16, for use in the treatment of cancer, wherein the treatment is a combination therapy of said multispecific binding agent, polynucleic acid, vector, host cell or composition with a T-cell targeting / activating therapy or anticancer vaccines.