Bifunctional anti-PD-1 / SIRPA molecule

Abstract

The present invention relates to bifunctional molecules comprising an anti-PD-1 antibody and SIRPa and uses thereof.

Description

[Technical field]

[0001] The present invention relates to the field of immunotherapy. The present invention provides a bifunctional molecule comprising an anti-PD1 antibody or an antibody fragment thereof linked to SIRPa and uses thereof. [Background technology]

[0002] Targeting T cell inhibitory checkpoints with therapeutic antibodies for disinhibition is an area of ​​intense research (for review, see Pardoll, Nat Rev Cancer. 2012;12:253-264). Targeting immune checkpoints of adaptive immunity shows excellent therapeutic efficacy against numerous cancers, but in a limited percentage of patients. Immune checkpoints in innate myeloid cells (macrophages, dendritic cells, MDSCs, PMNs) remain less studied, while these cells represent the most abundant immune cell type in many solid tumors and are often associated with poor outcomes. Combinations of immune checkpoint therapies targeting both innate (myeloid cell-mediated) and adaptive (T cell-mediated) immune responses have shown excellent efficiency in preclinical models but remain in clinical trials.

[0003] Immune cell activation is controlled by integrating the balance of costimulatory and co-inhibitory signals. T cell receptor (TCR)-mediated T cell activation is modulated by both costimulatory and co-inhibitory signals. The antigen-independent second signal modifies the first signal provided by the interaction of the antigen peptide-MHC complex with the TCR, conferring specificity to the response. T cell costimulatory and co-inhibitory pathways have broad immunoregulatory functions, controlling effector, memory, and regulatory T cells, as well as naive T cells. Therapeutic modulation of these pathways is translating into effective new strategies for treating cancer (for review, see Schildberg et al., Vol. 44(5), Immunity, 2016). Ongoing research into the regulation of immune responses has led to the identification of multiple immunological pathways that can be targeted for the development of cancer therapies. Such molecules are referred to herein as immune checkpoint co-activators or co-inhibitors (for reviews, see Sharma et al., Cell, 161(2), 2015 and Pardoll, Nature Reviews Cancer, 12(4), 2012).

[0004] Programmed cell death protein 1 (PD-1, also known as CD279) is a cell surface protein molecule belonging to the immunoglobulin superfamily. Programmed cell death protein 1 is expressed on T and B lymphocytes and macrophages and plays a role in cell fate and differentiation. In particular, PD-1, which functions as an immune checkpoint, plays an important role in downregulating the immune system by preventing T cell activation, which in turn reduces autoimmunity and promotes self-tolerance. Two ligands of PD-1, PD-L1 and PD-L2, have been identified that have been shown to downregulate T cell activation upon binding to PD-1 (Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al. (2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur J Immunol 32:634-43). The interaction of PD-1 with its ligands results in a reduction in tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune evasion by cancerous cells. In particular, PD1 ligation inhibits T cell responses by reducing signals downstream of TCR stimulation to T cells, resulting in reduced activation and cytokine production.

[0005] Both strategies of disrupting their interaction, using anti-PD1 and anti-PDL1 inhibitors, have been successful in cancer therapy (Brahmer et al., N Eng J Med, 366(26), 2012; Powles et al., Nature, 515(7528), 2014; Topalian et al., N Eng J Med, 366(26), 2012; Ansell, Curr Opin Hematol, 22(4), 2015). However, the accumulation of immunosuppressive and hypostimulatory myeloid cells within the tumor microenvironment limits the efficiency of T cell responses and the efficacy of immunotherapies, especially those targeted at immune checkpoints such as PD-1 / PD-L1. For example, clinical responses are low or absent in pancreatic, non-MSI colorectal, gastric and some breast cancer subtypes (Brahmer et al., N Engl J Med. 2012 Jun 28;366(26):2455-65; Feng et al., Cancer Lett. 2017 Oct 28;407:57-65; Borcherding et al., J Mol Biol. 2018 Jul 6;430(14):2014-2029). Multiple mechanisms have been described that could explain this low efficacy and resistance to PD-1 / PD-L1 checkpoint therapy, including (1) impaired formation of memory T cells, (2) impaired T cell infiltration, (3) insufficient generation of tumor-specific T cells, (4) inadequate T cell function, and (5) an immunosuppressive microenvironment induced by regulatory T cells.

[0006] To date, the majority of therapies have focused on stimulating the adaptive immune system to attack cancer, including agents targeting the PD-1 / PD-L1 axis to rescue exhausted T cells and restore antitumor responses (Brahmer et al., N Eng J Med, 366(26), 2012; Topalian et al., N Eng J Med, 366(26), 2012; Wolchok et al., N Engl J Med. 2013 Jul 11;369(2):122-133). However, macrophages and other myeloid immune cells also show promise as effectors of cancer immunotherapy. The CD47 / signal regulatory protein alpha (SIRPα) axis is a key regulator of myeloid cell activation and plays a broad role as a myeloid-specific immune checkpoint.

[0007] Signal regulatory protein alpha, or SIRPa (also called SIRPα, CD172a or SHPS-1), is expressed on monocytes, most subpopulations of tissue macrophages, granulocytes, a subset of dendritic cells in lymphoid tissues, some myeloid progenitor cells, and at various levels in neural cells, with significantly higher expression in synapse-rich areas of the brain. The interaction of SIRPa expressed by myeloid cells with the ubiquitous CD47, which is overexpressed in some cancer cells but also widely expressed at lower levels by most healthy cells, is another important immune checkpoint of the innate response involved in regulating bone marrow function. CD47 interacts with SIRPa, resulting in the transmission of a "don't EAT-ME" signal to phagocyte macrophages, leaving target cells and potentially tumor cells unaffected. Blockade of the CD47 / SIRPa pathway via CD47-targeting agents has been described to synergize with depleting therapeutic anti-cancer antibodies in a diverse range of preclinical models by enhancing antibody-dependent phagocytosis by macrophages, stimulating phagocytosis of cancer cells in vitro and anti-tumor immune responses in vivo. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2006 / 121168 [Patent Document 2] U.S. Patent No. 5,585,089 [Patent Document 3] U.S. Patent No. 5,693,761 [Patent Document 4] U.S. Patent No. 5,693,762 [Patent Document 5] U.S. Patent No. 5,821,337 [Patent Document 6] U.S. Patent No. 7,527,791 [Patent Document 7] U.S. Patent No. 6,982,321 [Patent Document 8] U.S. Patent No. 7,087,409 [Patent Document 9] U.S. Patent No. 6,180,370 [Patent Document 10] International Publication No. 15161311 [Patent Document 11] International Publication No. 17127664 [Patent Document 12] International Publication No. 18136626 [Patent Document 13] International Publication No. 18190719 [Patent Document 14] International Publication No. 19060750 [Patent Document 15] International Publication No. 19170677 [Patent Document 16] International Publication No. 2014 / 194302 [Patent Document 17] International Publication No. 2017 / 040790 [Patent Document 18] International Publication No. 2017 / 19846 [Patent Document 19] International Publication No. 2017 / 024465 [Patent Document 20] International Publication No. 2017 / 025016 [Patent Document 21] International Publication No. 2017 / 132825 [Patent Document 22] International Publication No. 2017 / 133540 [Patent Document 23] U.S. Patent Application Publication No. 20030044423 [Patent Document 24] WO 01 / 58957 [Patent Document 25] WO 96 / 34103 [Patent Document 26] International Publication No. 94 / 04678 [Patent Document 27] US Publication No. 20160319256 [Patent Document 28] International Publication No. 2013109752 [Patent Document 29] International Publication No. 2016024021 [Patent Document 30] U.S. Patent No. 5,108,921 [Patent Document 31] U.S. Patent No. 5,354,844 [Patent Document 32] U.S. Patent No. 5,416,016 [Patent Document 33] U.S. Patent No. 5,527,5285 [Patent Document 34] International Publication No. 2018 / 053106, pp. 36-43 [Non-patent literature]

[0009] [Non-Patent Document 1] Pardoll, Nat Rev Cancer. 2012; vol. 12: 253-264. [Non-Patent Document 2] Schildberg et al., Volume 44 (No. 5), Immunity, 2016 [Non-Patent Document 3] Sharma et al., Cell, Vol. 161(No. 2), 2015 [Non-Patent Document 4] Pardoll, Nature Reviews Cancer, Volume 12 (No. 4), 2012

Non-patent document 5

Non-patent document 6

Non-patent document 7

Non-patent document 8

Non-patent document 9

Non-patent document 10

Non-patent document 11

Non-patent document 12

Non-patent document 13

Non-patent document 14

Non-patent document 15

Non-patent document 16

Non-patent document 27

Non-patent document 28

Non-patent document 29

Non-patent document 30

Non-patent document 31

Non-patent document 32

Non-patent document 33

Non-patent document 34

Non-patent document 35

Non-patent document 36

Non-patent document 37

Non-patent document 38

Non-patent document 39

Non-patent document 40

Non-patent document 41

Non-patent document 42

Non-patent document 43

Non-patent document 44

Non-patent document 45

Non-patent document 46

Non-patent document 47

Non-patent document 48

Non-patent document 49

Non-patent document 50

Non-patent document 51

Non-patent document 52

Non-patent document 53

Non-patent document 54

Non-patent document 55

Non-patent document 56

[0010] In order to increase the efficacy of anti-PD1 immunotherapy in patients and overcome possible anti-PD-1 resistance, the development of combination therapy that also targets SIRPα / CD47 may be a good strategy.Therefore, there is still a significant need in the art for new and improved substances for safe immunotherapy, especially against cancer, that target innate myeloid immune cells with effective positive influence on adaptive immune response, especially T cell immune response.The inventors have made a significant step forward with the invention disclosed herein.Very favorable and unexpected effects are shown and are clearly explained in the beginning of the detailed description and in the examples.

[0011] The present inventors provide a bifunctional molecule comprising an anti-hPD-1 antibody and human SIRPα, which is promising for numerous therapeutic applications, particularly the treatment of cancer. The present invention is based on the development of an antibody that specifically targets human PD-1, which exhibits high binding affinity to PD-1 and strongly competes with its ligands PDL-1 and PD-L2. Surprisingly, fusion of the N-terminus of SIRPa to the C-terminus of the Fc region of an anti-hPD-1 antibody allows the preservation of its high affinity for CD47 (SIRPα ligand) to a similar extent as endogenous SIRPa. Fusion of the Fc domain to SIRPa also increases the half-life of the product. Furthermore, the bifunctional anti-PD1-SIRPα molecule disclosed herein enhances T cell activation (NFAT-mediated activation) compared to anti-PD-1 alone. In particular, the anti-PD1-SIRPa bifunctional molecule induces proliferation and activation of naive, partially exhausted subsets, reflected by cytokine (e.g., IFNγ) secretion. Bifunctional anti-PD1-SIRPa molecules exhibit surprising synergistic effects. Such anti-hPD1-SIRPa bifunctional molecules have the potential to overcome relevant resistance mechanisms and improve the efficacy of anti-PD-1 immunotherapy.

[0012] In a first aspect, the present invention provides a method for producing a composition comprising the steps of: (a) (i) a heavy chain variable domain (VH) comprising HCDR1, HCDR2 and HCDR3; and (ii) a light chain variable domain (VL) comprising LCDR1, LCDR2 and LCDR3 An anti-human PD-1 antibody or an antigen-binding fragment thereof comprising the compound (b) human SIRPa or a fragment or variant thereof In a bifunctional molecule comprising The C-terminus of the heavy and / or light chain of the antibody or antigen-binding fragment thereof is covalently linked to the N-terminus of SIRPa, or a fragment or variant thereof, as a fusion protein, preferably by a peptide linker.

[0013] In particular, the antibody is a chimeric, humanized or human antibody.

[0014] Preferably, the SIRPa fragment comprises or consists of the extracellular domain of SIRPa. Even more preferably, the SIRPa fragment lacks its intracellular portion and, optionally, its transmembrane domain, and preferably SIRPa comprises or consists of the amino acid sequence set forth in SEQ ID NO: 51 or a fragment thereof.

[0015] In a particular aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: (i) a heavy chain variable domain (VH) comprising HCDR1, HCDR2, and HCDR3; and (ii) a light chain variable domain (VL) comprising LCDR1, LCDR2, and LCDR3; A bifunctional molecule comprising an anti-human PD-1 antibody or an antigen-binding fragment thereof, comprising or consisting of: - the heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 1, - the heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 2, - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 3, in which X1 is D or E and X2 is selected from the group consisting of T, H, A, Y, N, E and S, preferably in the group consisting of H, A, Y, N and E; the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 12, in which X is G or T; - the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 15, - the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 16; Concerning bifunctional molecules.

[0016] In particular, the anti-human PD-1 antibody, or antigen-binding fragment thereof, comprises: (a) a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 17, wherein Xi is D or E and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably in the group consisting of H, A, Y, N, and E; and (b) a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 26, wherein X is G or T.

[0017] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain, and a heavy chain constant domain derived from a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant domain, preferably an IgG1 or IgG4 heavy chain constant domain.

[0018] In a more specific embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain and a heavy chain constant domain derived from a human IgG1 heavy chain constant domain, and optionally comprises the following constant domains: T250Q / M428L;M252Y / S254T / T256E+H433K / N434F;E233P / L234V / L235A / G236A+A327G / A330S / P331S;E333A;S239D / A330L / I3 32E; P257I / Q311; K326W / E333S; S239D / I332E / G236A; N297A; L234A / L235A; N297A+M252Y / S254T / T256E; and K322A and K444A, preferably N297A optionally in combination with M252Y / S254T / T256E, and L234A / L235A.

[0019] In another more specific aspect, the antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain, and a heavy chain constant domain derived from a human IgG4 heavy chain constant domain, optionally with a substitution or combination of substitutions selected from the group consisting of S228P;L234A / L235A, S228P+M252Y / S254T / T256E, and K444A.

[0020] In particular, the anti-PD1 antibody is selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, cemiplimab, PDR001, and monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4.

[0021] In another aspect, the present invention relates to an isolated nucleic acid sequence or a group of isolated nucleic acid molecules encoding a bifunctional molecule as disclosed herein, a vector comprising a nucleic acid or a group of nucleic acid molecules as disclosed herein, and / or a host cell comprising a vector comprising a nucleic acid or a group of nucleic acid molecules as disclosed herein.

[0022] In another aspect, the present invention relates to a method for producing a bifunctional molecule, comprising culturing a host cell as disclosed herein and optionally isolating the bifunctional molecule.

[0023] In another aspect, the invention relates to a pharmaceutical composition comprising a bifunctional molecule, a nucleic acid or a group of nucleic acid molecules, a vector, or a host cell as disclosed herein, and a pharma- ceutically acceptable carrier.

[0024] Optionally, the pharmaceutical composition further comprises an additional therapeutic agent, preferably selected in the group consisting of: alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotic agents, antiproliferative agents, antiviral agents, Aurora kinase inhibitors, proapoptotic agents (e.g., Bcl-2 family inhibitors), activators of the death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (Bi-Specific T cell Engager) antibodies, antibody drug conjugates, biological response modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia viral oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormonal therapy, immunological agents, inhibitors of inhibitors of apoptosis proteins (IAPs), intercalating antibiotics, epitopes or neoepitopes derived from tumor antigens, such as antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, nonsteroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutic agents, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoid / deltoid plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, and peptide vaccines, as well as combinations of one or more of these agents.

[0025] In particular, the pharmaceutical composition, the bifunctional molecule, the nucleic acid or group of nucleic acid molecules, the vector or the host cell are intended for use as a medicament.

[0026] Finally, the present invention relates to a pharmaceutical composition, a bifunctional molecule, a nucleic acid or a group of nucleic acid molecules, a vector, or a host cell as disclosed herein for use as a medicament.

[0027] In certain embodiments, the present invention is directed to a cancer, preferably a hematological or solid tumor with expression of PD-1 and / or PD-L1, such as a cancer selected from the group consisting of hematolymphoid neoplasms, angioimmunoblastic T-cell lymphoma, myelodysplastic syndromes, and acute myeloid leukemia, cancers induced by viruses or associated with immune deficiencies, including, for example, Kaposi's sarcoma (e.g., associated with Kaposi's sarcoma herpesvirus); cervical cancer, anal cancer, penile cancer, and vulvar squamous cell carcinoma and oropharyngeal cancer (e.g., associated with human papillomavirus); diffuse large B-cell lymphoma. The cancer is selected from the group consisting of B-cell non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma, plasmablastic lymphoma, primary central nervous system lymphoma, HHV-8 primary effusion lymphoma, classical Hodgkin's lymphoma, and lymphoproliferative disorders (e.g., associated with Epstein-Barr virus (EBV) and / or Kaposi's sarcoma herpesvirus); hepatocellular carcinoma (e.g., associated with hepatitis B and / or C virus); Merkel cell carcinoma (e.g., associated with Merkel cell polyomavirus (MPV)); and cancer associated with human immunodeficiency virus infection (HIV) infection. or a cancer selected from the group consisting of cancers selected from the group consisting of metastatic or non-metastatic melanoma, malignant mesothelioma, non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, small cell lung cancer, metastatic Merkel cell carcinoma, gastric or gastroesophageal cancer and cervical cancer; or an infectious disease, preferably a chronic infectious disease, even more preferably a chronic viral infection, preferably HIV, hepatitis virus, herpes virus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, The pharmaceutical composition, bifunctional molecule, nucleic acid or group of nucleic acid molecules, vector, or host cell disclosed herein for use in the treatment of an infection caused by a virus selected from the group consisting of a virus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

[0028] Preferably, the cancer is a PD-1, PD-L1 and / or PD-L2 positive cancer, in particular a PD-L1 positive cancer.

[0029] Optionally, the bifunctional molecule, pharmaceutical composition, isolated nucleic acid molecule or group of isolated nucleic acid molecules, vector, or host cell is for use in combination with radiation therapy or an additional therapeutic agent, preferably selected in the group consisting of: alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, mitotic inhibitors, antiproliferative agents, antivirals, Aurora kinase inhibitors, proapoptotic agents (e.g., Bcl-2 family inhibitors), activators of cell death pathways, Bcr-Abl kinase inhibitors, BiTE (bispecific T cell engager) antibodies, antibody drug conjugates, biological response modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia viral oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormonal therapy. epitopes or neoepitopes derived from tumor antigens, such as methods, immunological agents, inhibitors of inhibitors of apoptosis proteins (IAPs), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, nonsteroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutic agents, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoid / deltoid plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, and peptide vaccines, as well as combinations of one or more of these agents.

[0030] In one aspect, the pharmaceutical composition, bifunctional molecule, nucleic acid or group of nucleic acid molecules, vector, or host cell as disclosed herein is for use to inhibit the suppressive activity of T regulator cells, to activate T effector cells, and / or to stimulate the proliferation of naive, partially exhausted T cells. [Brief description of the drawings]

[0031] [Figure 1] PD-1 binding ELISA assay of anti-PD-1 antibodies and anti-PD-1 / SIRPa bifunctional molecules. Human recombinant PD-1 protein was immobilized and anti-PD-1 bifunctional molecules were added at different concentrations. Colour development was performed with anti-human Fc antibody linked to peroxidase. Colorimetry was determined at 450 nm using TMB substrate. (A) Data for anti-PD1VH-SIRPa antibody chimeric (●) and humanized (■). (B) Data for anti-PD1VL-SIRPa antibody chimeric (●) and humanized form (■). In this experiment, the bifunctional molecule comprises a humanized anti-PD1 antibody having a heavy chain variable domain as disclosed in SEQ ID NO:18 and a light chain variable domain as disclosed in SEQ ID NO:28. [Diagram 2] Anti-PD-1 / SIRPa bifunctional molecules block PD1 / PDL1 interaction. Competitive PD-1 / PD-L1 ELISA assay. PD-L1 was immobilized and conjugated antibody + biotinylated recombinant human PD-1 was added. Different concentrations of anti-PD-1 antibodies were tested and recombinant PD1 was added at 0.6 μg / mL. Anti-PD-1 antibody (■) or Bicki anti-PD1VH-Sirpa (●) or anti-PD1VL-Sirpa (o) antibodies were added at different concentrations. Revelation was performed with streptavidin peroxidase to detect PD1 molecules and revealed colorimetrically at 450 nm using TMB substrate. In this experiment, the bifunctional molecule comprises a chimeric anti-PD1 antibody comprising a heavy chain defined in SEQ ID NO:53 and a light chain defined in SEQ ID NO:54. [Diagram 3]Bridging ELISA binding assay. PD1-His recombinant protein was immobilized and Bicki anti-PD1VH Sirpa (●) or anti-PD1VL Sirpa (o) was added at successive concentrations. CD47Fc recombinant protein was then added at 1 μg / mL. Detection was performed with anti-CD47 mouse antibody (clone B6H12) + anti-IgG mouse antibody linked to peroxidase. ELISA was revealed colorimetrically at 450 nm using TMB substrate. The histogram represents recombinant rSIRPa protein immobilized on the plate and used as a positive control for the ELISA. In this experiment, the bifunctional molecule comprises a chimeric anti-PD1 antibody comprising a heavy chain defined in SEQ ID NO: 53 and a light chain defined in SEQ ID NO: 54. [Figure 4] Targeting and binding of Bicki anti-PD1-SIRPa molecules on PD1+CD47+ expressing T cells. Jurkat cells expressing only CD47+ (grey bars) or Jurkat cells co-expressing CD47+ and PD-1+ (black bars) were stained with 4.5 nM BiCKi anti-PD-1 SIRPa or SIRPa-Fc and revealed with anti-IgG-PE (Biolegend, clone HP6017). Data represent the ratio of the mean fluorescence on PD-1+CD47+ Jurkat cells to the mean fluorescence obtained on PD1- cells CD47+ Jurkat cells. In this experiment, the bifunctional molecule comprises a humanized anti-PD1 antibody with a heavy chain variable domain as disclosed in SEQ ID NO:24 and a light chain variable domain as disclosed in SEQ ID NO:28. [Diagram 5]Bicki anti-PD1-Sirpa molecules synergistically enhance T cell activation in vitro by stimulating NFAT signaling. Promega PD-1 / PD-L1 bioassays were performed for this experiment to determine T cell activation using an NFAT luciferase reporter system. Two cell lines are used: (1) effector T cells (Jurkat cells stably expressing PD-1, an NFAT-induced luciferase) and (2) activated target cells (CHO K1 cells stably expressing PDL1 and a surface protein designed to activate the cognate TCR in an antigen-independent manner). When cells are co-cultured, PD-L1 / PD-1 interaction directly inhibits TCR-mediated activation, thereby blocking NFAT activation and luciferase activity. Addition of anti-PD1 antibody blocks the inhibitory signal, thereby resulting in NFAT activation and luciferase synthesis. After addition of BioGlo™ luciferin, luminescence was quantified using a luminometer, which reflects T cell activation. (A) PD-1 and CD47 expression on effector receptor T cell lines (anti-PD-1 Pecy7, BD Bioscience, clone EH12.2; anti-CD47 (clone B6H12) + anti-mouse IgG AF647). (B) Serial dilutions of anti-PD1 antibody alone (▼) or bicki anti-PD1 VH-SIRPa antibody (◆) or isotype control (■), anti-PD-1 antibody + isotype control VH-SIRPa antibody (●) or anti-PD1 antibody + SIRPa Fc (○) were tested. (C) Effector Jurkat cells were pre-incubated with (●) or without (◆) anti-CD47 blocking antibody (B6H12) and then incubated with different concentrations of Bicki anti-PD1 VH-SIRPa. For baseline activation, Jurkat cells were also incubated with anti-PD-1 antibody alone (▼). (D) The efficacy of Bicki anti-PD1 VH-SIRPa constructed with IgG4 S228P (◆) or IgG1 N298A (■) was evaluated and compared to anti-PD-1 alone (▼). (E) In a separate experiment, the synergistic activity of Bicki VH SIRPa (○) and antibody anti-PD-1 alone (●) was tested with other anti-PD-1 scaffolds: pembrolizumab (left graph) and nivolumab (right graph).(F) Another bicki anti-PD1 type I protein antibody (●) was tested and compared with an anti-PD1 antibody (■). In this experiment, the bifunctional molecule comprises a humanized anti-PD1 antibody having a heavy chain variable domain as disclosed in SEQ ID NO:24 and a light chain variable domain as disclosed in SEQ ID NO:28. [Figure 6] Bicki anti-PD1-SIRPa molecules enhance calcium flux signaling in T cells to a similar extent as CD28 costimulation. CD47+PD1+ Jurkat cells were stained with Fura red and intracellular Ca2+ release after activation was measured using flow cytometry. T cells were stimulated with BiCKI SIRPa alone (○ grey line) or in combination with CD3 (OKT3) stimulation (○ black line). As controls, cells were stimulated with a-CD3 alone (●) or a-CD3+CD28 (■). (A) Graphs represent the average of 4-6 experiments, and arrows illustrate the addition of stimuli. Data were obtained by calculating the ratio BV711 (bound Ca2+) / PercyP5.5 5 (free Ca2+) MFI. This ratio was normalized to unstimulated (average of the first 20 seconds before stimulation). (B) Data represent the calculated area under the curve (AUC); each dot represents one experiment. P values ​​were calculated using a paired t-test (*p<0.05). In this experiment, the bifunctional molecule comprises a humanized anti-PD1 antibody having a heavy chain variable domain disclosed in SEQ ID NO:24 and a light chain variable domain disclosed in SEQ ID NO:28. [Figure 7]Bicki anti-PD1-SIRPa molecules stimulate PBMC proliferation and IFNg secretion. (A) PBMCs were isolated from three healthy donors and activated with immobilized CD3 antibody (OKT3.3 μg / mL) in the presence of isotype control, anti-PD1 antibody or Bicki anti-PD1VH-SIRPa or anti-PD1VL-SIRPa. Proliferation was assessed by thymidine incorporation 3H on day 6. (B) PBMCs isolated from healthy donors were activated with immobilized anti-CD3 antibody (OKT23 1 μg / mL) and anti-CD28 antibody (clone CD28.2) in the presence of anti-PD-1 antibody alone, anti-PD1+rSIRPa protein, Bicki anti-PD1VH-Sirpa or anti-PD1VL-Sirpa. Two days after activation, supernatants were collected and IFNg was quantified by ELISA. Data are representative of three different donors. In this experiment, the bifunctional molecule comprises a humanized anti-PD1 antibody having a heavy chain variable domain disclosed in SEQ ID NO:24 and a light chain variable domain disclosed in SEQ ID NO:28. [Figure 8] Bicki anti-PD1-SIRPa molecule enhances activated T cell proliferation and IFNg secretion. CD3 CD28 preactivated T cells were restimulated in the presence of anti-PD1VH-SIRPa or anti-PD1VL-SIRPa (10 μg / mL) on CD3 / PDL1 coated plates. Anti-PD-1 and isotype antibodies are used as controls. (A) T cell proliferation was assessed by H3 thymidine incorporation on day 6. (B) Supernatants were collected on day 6 and IFNg secretion was quantified by ELISA. After activation, supernatants were collected and IFNg was quantified by ELISA. Data are representative of three different donors. In this experiment, the bifunctional molecule comprises a humanized anti-PD1 antibody with a heavy chain variable domain disclosed in SEQ ID NO:24 and a light chain variable domain disclosed in SEQ ID NO:28. [Figure 9]Bicki anti-PD1-SIRPa bifunctional molecule enhances the proliferation of exhausted T cells. Human PBMCs were repeatedly stimulated every 3 days on CD3 CD28 coated plates (3 μg / mL OKT3 and 3 μg / mL CD28.2 antibody). After the third stimulation, T cells were reactivated on CD3 / PD-L1 coated plates and incubated with isotype control or anti-PD1, rSIRPa protein or Bicki anti-PD1-VH-Sirpa antibody. H3 uptake assay was performed on day 5 to determine T cell proliferation. Data are expressed as fold change and are representative of three different donors (1=isotype control). In this experiment, the bifunctional molecule comprises a humanized anti-PD1 antibody with a heavy chain variable domain disclosed in SEQ ID NO:24 and a light chain variable domain disclosed in SEQ ID NO:28. [Figure 10] Bicki anti-PD1-SIRPa bifunctional molecule enhances T cell migration into tumors. 3D multi-cell spheroids were generated by co-culturing A549 tumor cells, MRC-5 fibroblasts and human monocytes in low-attachment plates. On day 3, human T cells were added to the wells and after 72 hours of co-culture, T cell infiltration was analyzed by immunofluorescence (anti-human CD3+ anti-donkey A488 secondary antibody) and all cells were stained with DAPI nuclear marker. Fluorescent signals were quantified by confocal microscopy and analyzed using FIJI software. Data represent the number of CD3+ positive cells / 1e6 DAPI+ total cells infiltrated into the spheroids, with each dot representing the analysis of one spheroid. In this assay, spheroids were treated with isotype control (IgG4) or biCKI SIRPa (50 nM) for 3 days. Statistical significance was determined using the Mann-Whitney test *p<0.05. In this experiment, the bifunctional molecule comprises a humanized anti-PD1 antibody having a heavy chain variable domain disclosed in SEQ ID NO:24 and a light chain variable domain disclosed in SEQ ID NO:28. [Figure 11]Pharmacokinetics of Bicki anti-PD1-SIRPa bifunctional molecule in mice. Mice were intravenously injected with a single dose (34.34 nM / kg) of anti-PD-1 SIRP constructed with IgG1N298A (○) or IgG4 S228P isotype (●). Serum Bicki concentrations were assessed by sandwich ELISA at multiple time points after injection. In this experiment, the bifunctional molecule comprises a humanized anti-PD1 antibody having a heavy chain variable domain disclosed in SEQ ID NO:24 and a light chain variable domain disclosed in SEQ ID NO:28. [Figure 12] Illustration of the mechanism of action of Bicki anti-PD1-SIRPa bifunctional molecule compared to the prior art. The left part of the figure illustrates the mechanism of the prior art anti-PD-L1-SIRPa bifunctional molecule targeting cancer cells. The right part of the figure illustrates the mechanism of the anti-PD-1-SIRPa bifunctional molecule of the present invention, which targets T cells, in particular the same T cells, and thus has the ability to synergistically reactivate exhausted T cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Introduction The antibodies of the present invention are bifunctional since they combine a specific anti-PD-1 effect with the effect of SIRPa fused to the anti-PD-1 antibody. Indeed, the present invention relates to a bifunctional molecule comprising an anti-PD-1 antibody and SIRPa, where the protein SIRPa is covalently linked to the polypeptide chain of the anti-PD-1 antibody, either to the light chain or to the heavy chain of the antibody, or to both, or to any fragment thereof. The chain of the anti-PD-1 antibody or its fragment and SIRPa are prepared as a fusion protein. In this particular embodiment, the N-terminus of SIRPa is linked to the C-terminus of the chain of the anti-PD-1 antibody or its fragment, optionally via a peptide linker.

[0033] Firstly, it is emphasized that SIRPa is known in the prior art to be a target of anti-SIRPa antibodies. These antibodies block the interaction between CD47 on tumor cells and SIRPa on myeloid cells, in particular macrophages, which interaction (so-called trans-interaction between different cells) induces inhibition of phagocytosis of tumor cells by macrophages. In contrast, the bifunctional protein of the present invention comprising SIRPa and an antibody against a ligand expressed on T cells, in particular PD-1, is not known to be involved in T cell activation mechanisms.

[0034] As clearly described and shown in detail in the examples of the present application and illustrated in FIG. - PD-1 on T cells: the anti-PD1 antibody portion of the bifunctional SIRPa-anti-PD-1 molecule; and - CD47 (not CD47 on tumor cells or only CD47 on tumor cells): the SIRPa portion of the bifunctional SIRPa-anti-PD-1 molecule We have obtained a bifunctional SIRPa-anti-PD-1 molecule that unexpectedly has the powerful benefit of targeting both on the same T cell. This dual targeting to the same T cell leads to a previously unknown synergistic effect, manifested by complementary activation of the NFAT pathway. This capability is particularly interesting for the following reasons.

[0035] As known by those skilled in the art, tumor cells are not sufficiently eliminated by T cells in the context of T cell exhaustion. Anti-PD-1 therapeutic compounds are clinically used to activate T cells through the inhibition of the inhibitory effect of PD1-PDL1 interaction (PD1 on T cells and PDL1 on tumor cells). More precisely, T cell exhaustion is observed in humans with cancer. As described, for example, in Jiang, Y., Li, Y. and Zhu, B (Cell Death Dis 6, e1792 (2015)), exhausted T cells in the tumor microenvironment show overexpressed inhibitory receptors, reduced effector cytokine production and cytolytic activity, which leads to failure in cancer elimination. Restoration of exhausted T cells represents a clinical strategy for cancer treatment. Most T cells in the tumor microenvironment are exhausted, which leads to cancer immune evasion. PD-1 is the main inhibitory receptor that regulates T cell exhaustion, and T cells with high PD-1 expression lose the ability to eliminate cancer. Anti-PD-1 antibodies are not always efficient enough to allow "re"activation of exhausted T cells. Therefore, this is a current critical medical need. The inventors have shown that the bifunctional anti-PD1-SIRPa molecules disclosed herein enhance activation (NFAT-mediated activation, calcium release) of T cells, particularly exhausted T cells, compared to anti-PD-1 alone or anti-PD-1 in combination with SIRPa. In particular, anti-PD1-SIRPa bifunctional molecules induce proliferation and activation of naive, partially exhausted T cell subsets, reflected by cytokine (e.g., IFNγ) secretion. Such anti-hPD1-SIRPa bifunctional molecules have the potential to overcome relevant resistance mechanisms and improve the efficacy of anti-PD-1 immunotherapy.

[0036] Applicants also show that the interaction of a bifunctional anti-PD1-SIRPa molecule with a single T cell expressing i) PD1 and ii) SIRPa receptors leads to an unexpected activation of the NFAT pathway (TCR signaling) with a positive effect on T cell activation, in particular on exhausted T cells, thereby favoring the ability of T cells to eliminate tumor cells.

[0037] On the one hand, the SIRPa of the BICKI molecule targets the SIRPa receptor (CD47), which means that it activates this pathway like SIRPa alone, while the anti-PD1 part of the BICKI molecule blocks PD-1. The bifunctional molecule targets both CD47 and PD-1 on the same cell. This results in a synergistic activation of TCR (NFAT) signaling that is not observed when using a separate combination of anti-PD1 antibody and SIRPa (administration of anti-PD1 and administration of SIRPa as two separate compounds). This activation, for example, by acting on the same cell, cannot be brought about by a bifunctional molecule that targets PD-L1. Indeed, it is known in the art that PD-L1 is mainly expressed on tumor cells and not on immune cells such as T cells.

[0038] In addition, synergistic effects are observed with the particular humanized anti-PD-1 of the present invention, but also with two other anti-PD-1 of reference, namely pembrolizumab and nivolumab. The design of the bifunctional anti-PD1-SIRPa molecule allows targeting CD47+PD-1+ exhausted T cells at least twice as often as other CD47+ cells. The bifunctional anti-PD1-SIRPa molecule enhances anti-PD1 action, strongly suggesting that SIRPa binding on CD47 not only blocks the inhibitory phagocyte signal "don't eat me", but also, surprisingly, promotes CD47-dependent T cell co-stimulation. Finally, the bifunctional anti-PD1-SIRPa molecule enhances the migration of T cells into the tumor microenvironment, thus resolving one of the major resistance mechanisms associated with anti-PD-1 monotherapy due to the lack of T cells within the tumor microenvironment.

[0039] The bifunctional molecules of the present invention have, in particular, one or several of the following advantages: - They conserve their ability to bind to PD-1, without differences between humanized and chimeric forms of anti-PD1 antibodies. - They preserve their ability to bind to CD47, the SIRPa ligand protein. - They antagonize the PD-1 / PD-L1 and / or PD-1 / PD-L2 interactions. - They synergistically enhance T cell activation (NFAT-mediated activation and calcium mobilization stimulation) compared to anti-PD-1 alone. - They exhibit synergistic effects in stimulating the proliferation of naive, activated and exhausted T cells. - They exhibit a synergistic effect in stimulating the secretion of IFNg by T cells. - They enhance T cell migration. - They exhibit good and linear pharmacokinetic properties.

[0040] definition In order that the present invention may be more readily understood, certain terms are defined herein below. Additional definitions are set forth throughout the detailed description.

[0041] Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have the meaning commonly understood by those skilled in the art to which the present invention pertains. In some cases, terms with commonly understood meanings are also defined herein for clarity and / or ease of reference. If such a definition is included herein, it should not be interpreted as necessarily representing a different meaning than commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood and widely used by those skilled in the art using conventional methodology.

[0042] As used herein, the terms "signal regulatory protein alpha", "SIRPα" and "SIRPa" refer to a receptor that is a transmembrane glycoprotein that is a mammalian immunoglobulin-like cell surface receptor for CD47. It specifically refers to SIRPa polypeptides, or derivatives and analogs thereof, that have substantial amino acid sequence identity to wild-type mammalian SIRPa and have substantially equivalent biological activity, e.g., substantially equivalent biological activity in a standard bioassay or assay of SIRPa receptor binding affinity. For example, SIRPa refers to the amino acid sequence of a recombinant or non-recombinant polypeptide having the amino acid sequence of i) a naturally occurring or naturally occurring allelic variant of a SIRPa polypeptide, ii) a biologically active fragment of a SIRPa polypeptide, iii) a biologically active polypeptide analog of a SIRPa polypeptide, or iv) a biologically active variant of a SIRPa polypeptide. SIRPa may include or lack its transmembrane and / or cytoplasmic domain. SIRPa preferably includes or consists of its extracellular domain. Other names for this molecule are "tyrosine-protein phosphatase non-receptor substrate 1" and "SHP substrate 1", "CD172 antigen-like family member A", "p84" and "macrophage fusion receptor". Preferably, the term "SIRPa" refers to human SIRPa. For example, the human SIRPa amino acid sequence is about 504 amino acids and has Genbank accession numbers NP_001035111.1, NP_001035112.1, NP_001317657.1, or NP_542970.1. Preferably, the human SIRPa is isoform 1. Even more preferably, SIRPa consists essentially of amino acids at positions 31 to 373 of the aforementioned sequence, i.e., the extracellular domain thereof. Human SIRPa is described in UniProtKB - P78324.

[0043] As used herein, the terms "programmed death 1", "programmed cell death 1", "PD1", "PD-1", "PDCD1", "PD-1 antigen", "human PD-1", "hPD-1", and "hPD-1" are used interchangeably and refer to the programmed death-1 receptor, also known as CD279, including variants and isoforms of human PD-1 and analogs that share at least one common epitope with PD-1. PD-1 is a key regulator of immune responses and the threshold of peripheral immune tolerance. PD-1 is expressed on activated T cells, B cells, monocytes, and dendritic cells and binds to its ligands PD-L1 and PD-L2. Human PD-1 is encoded by the PDCD1 gene. As an example, the amino acid sequence of human PD-1 is disclosed in GenBank Accession No. NP_005009. PD1 is expressed on human peripheral blood mononuclear cells (PBMCs) in four splice variants. Thus, PD-1 proteins include full-length PD-1, as well as alternative splice variants of PD-1, such as PD-1Aex2, PD-1Aex3, PD-1Aex2,3, PD-1Aex2,3,4, etc. Unless otherwise specified, these terms include any variants and isoforms of human PD-1 naturally expressed by PBMCs or expressed by cells transfected with the PD-1 gene.

[0044] As used herein, the term "antibody" describes a type of immunoglobulin molecule and is used in its broadest sense. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. Unless specifically indicated otherwise, the term "antibody" includes intact immunoglobulins and any other modified configurations of immunoglobulin molecules that contain an antigen recognition site of the required specificity, including "antibody fragments" or "antigen-binding fragments" (Fab, Fab', F(ab')2, Fv, etc.), single chains (scFv), variants thereof, molecules containing antibody portions, diabodies, linear antibodies, single chain antibodies, and glycosylation variants of antibodies, amino acid sequence variants of antibodies. Preferably, the term antibody refers to humanized antibodies.

[0045] As used herein, an "antigen-binding fragment" of an antibody refers to a molecule corresponding to a portion of an antibody, i.e., a portion of the structure of an antibody of the invention, which, presumably in its native form, exhibits antigen-binding ability to PD-1. In particular, such fragments exhibit the same or substantially the same antigen-binding specificity to the antigen as compared to the antigen-binding specificity of the corresponding four-chain antibody. Advantageously, the antigen-binding fragment has a similar binding affinity as the corresponding four-chain antibody. However, antigen-binding fragments having reduced antigen-binding affinity as compared to the corresponding four-chain antibody are also encompassed within the present invention. Antigen-binding ability can be determined by measuring the affinity between the antibody and the target fragment. Such antigen-binding fragments can also be referred to as "functional fragments" of an antibody. An antigen-binding fragment of an antibody is a fragment that includes the hypervariable domains, or parts thereof, called CDRs (complementarity determining regions), that encompass the recognition site of the antigen, i.e., the extracellular domain of PD1, thereby defining the antigen recognition specificity.

[0046] "Fab" fragments contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. F(ab') fragments are produced by cleaving the disulfide bonds of the hinge cysteines of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those skilled in the art. Fab and F(ab')2 fragments lack the Fc fragment of intact antibodies, are cleared more rapidly from the circulation of animals, and may have less nonspecific tissue binding than intact antibodies (see, e.g., Wahl et al., 1983, J. Nucl. Med. 24:316).

[0047] An "Fv" fragment is the smallest fragment of an antibody that contains a complete target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. In most cases, six CDRs confer target binding specificity to the antibody. However, in some cases, even a single variable domain (or half of an Fv containing only three target-specific CDRs) can have the ability to recognize and bind to a target, albeit with a lower affinity than the entire binding site.

[0048] "Single-chain Fv" or "scFv" antibody-binding fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for target binding.

[0049] A "single domain antibody" is composed of a single VH or VL domain which exhibits sufficient affinity for PD-1. In a specific embodiment, a single domain antibody is a camelized antibody (see, e.g., Riechmann, 1999, Journal of Immunological Methods 231:25-38).

[0050] In terms of structure, an antibody may have heavy (H) and light (L) chains interconnected by disulfide bonds. There are two types of light chains: lambda (λ) and kappa (κ). Each heavy and light chain contains a constant region and a variable region (or "domain"). The light and heavy chain variable regions contain a "framework" region separated by three hypervariable regions, also called "complementarity determining regions" or "CDRs". The extent of the framework region and the CDRs have been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, and US Department of Health and Human Services, 1991, which is incorporated herein by reference). Preferably, the CDRs are defined by the Kabat method. The framework regions act to form a scaffold that provides for the CDRs to be positioned in the correct orientation by interchain non-covalent interactions. The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as "complementarity determining region 1" or "CDR1", "CDR2", and "CDR3", numbered sequentially from the N-terminus. The VL and VH domains of an antibody according to the invention may comprise four framework regions or "FRs", which are referred to in the art and herein as "framework region 1" or "FR1", "FR2", "FR3", and "FR4", respectively. These framework regions and complementarity determining regions are preferably operably linked in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from amino terminus to carboxy terminus). The term "antibody framework", as used herein, refers to a portion of either the VL and / or VH variable domain that serves as a scaffold for the antigen-binding loops (CDRs) of that variable domain.

[0051] "Antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in an antibody conformation. The CDRs of an antibody heavy chain are typically referred to as "HCDR1," "HCDR2," and "HCDR3." The framework regions of an antibody heavy chain are typically referred to as "HFR1," "HFR2," "HFR3," and "HFR4."

[0052] "Antibody light chain" as used herein refers to the smaller of the two types of polypeptide chains present in an antibody conformation. Kappa light chain and lambda light chain refer to the two major antibody light chain isotypes. The CDRs of an antibody light chain are typically referred to as "LCDR1", "LCDR2", and "LCDR3". The framework regions of an antibody light chain are typically referred to as "LFR1", "LFR2", "LFR3", and "LFR4".

[0053] With respect to the binding of an antibody to a target molecule, the term "bind" or "binding" refers to peptides, polypeptides, proteins, fusion proteins, molecules, and antibodies (including antibody fragments) that recognize and contact the antigen. Preferably, the term refers to an antigen-antibody type of interaction. The terms "specific binding," "specifically binds," "specific for," "selectively binds to," and "selective for," with respect to a particular antigen (e.g., PD-1) or an epitope on a particular antigen (e.g., PD-1), mean that the antibody recognizes and binds to the specific antigen but does not substantially recognize or bind to other molecules in the sample. For example, an antibody that specifically (or preferentially) binds to PD-1 or a PD-1 epitope is an antibody that binds to the PD-1 epitope, e.g., with higher affinity, avidity, more readily, and / or for a longer period of time, than it binds to other PD-1 epitopes or non-PD-1 epitopes. Preferably, the term "specific binding" refers to a PD-1 epitope that is more specific than the PD-1 epitope, e.g., with higher affinity, avidity, more readily, and / or for a longer period of time. -7 M refers to contact of an antibody with an antigen having a binding affinity equal to or less than M. In certain embodiments, the antibody is -8 M, 10 -9 M or 10 -10It binds with an affinity equal to or less than M.

[0054] As used herein, "PD-1 antibody," "anti-PD-1 antibody," "PD-1 Ab," "PD-1 specific antibody," or "anti-PD-1 Ab" are used interchangeably and refer to an antibody, as described herein, that specifically binds to PD-1, particularly human PD-1. In some embodiments, the antibody binds to the extracellular domain of PD-1. In particular, an anti-PD-1 antibody is an antibody capable of binding to the PD-1 antigen and inhibiting PD-1-mediated signaling pathways, thereby enhancing immune responses such as T cell activation.

[0055] As used herein, the terms "bifunctional molecule", "bifunctional compound", "bifunctional protein", "Bicki", "Bicki antibody", "bifunctional antibody" and "bifunctional checkpoint inhibitor molecule" have the same meaning and can be used interchangeably. These terms refer to an antibody that recognizes one antigen by having at least one region specific for that antigen (e.g., from the variable region of the antibody) and at least a second region that is a polypeptide. More specifically, a bifunctional molecule is a fusion protein of an antibody or a portion thereof, preferably an antigen-binding fragment thereof, and another polypeptide or a polypeptide fragment thereof.

[0056] The term "chimeric antibody" as used herein means an antibody or antigen-binding fragment in which a portion of the heavy and / or light chain is derived from one species and the remainder of the heavy and / or light chain is derived from a different species. In illustrative examples, a chimeric antibody may contain a constant region derived from human and a variable region derived from a non-human species, such as mouse.

[0057] The term "humanized antibody", as used herein, is intended to refer to an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (e.g., a chimeric antibody containing minimal sequences derived from a non-human antibody). Also, "humanized antibody", e.g., a non-human antibody, refers to an antibody that has undergone humanization. A humanized antibody is generally a human immunoglobulin (recipient antibody) in which residues from one or more CDRs have been replaced by residues from at least one CDR of a non-human antibody (donor antibody) while maintaining the desired specificity, affinity, and capacity of the original antibody. The donor antibody may be any suitable non-human antibody, such as a mouse antibody, a rat antibody, a rabbit antibody, a chicken antibody, or a non-human primate antibody, having the desired specificity, affinity, or biological effect. In some cases, selected framework region residues of the recipient antibody are replaced by framework region residues from the donor antibody. Alternatively, selected framework region residues of the donor antibody are replaced by framework region residues from a human antibody or a humanized antibody. Additional framework region modifications may be made within the human framework sequences. Thus, a humanized antibody may contain residues that are not found in either the recipient antibody or the donor antibody. Such amino acid modifications may be made to further refine antibody function and / or enhance the humanization process. An "amino acid change" or "amino acid modification" refers herein to a change in the amino acid sequence of a polypeptide. An "amino acid modification" includes a substitution, an insertion, and / or a deletion in a polypeptide sequence. An "amino acid substitution" or "substitution" refers herein to the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. An "amino acid insertion" or "insertion" refers to the addition of an amino acid at a particular position in a parent polypeptide sequence. An "amino acid deletion" or "deletion" refers to the removal of an amino acid at a particular position in a parent polypeptide sequence. An amino acid substitution may be conservative. A conservative substitution is the replacement of a given amino acid residue with another residue having a side chain ("R group") with similar chemical properties (e.g., charge, bulk, and / or hydrophobicity).As used herein, "amino acid position" or "amino acid position number" are used interchangeably and refer to the position of a particular amino acid in an amino acid sequence, and are generally designated by the single letter code for the amino acid. The first amino acid in an amino acid sequence (i.e., starting from the N-terminus) shall be considered to be position 1.

[0058] A conservative substitution is the replacement of a given amino acid residue with another residue having a side chain ("R group") with similar chemical properties (e.g., charge, bulk, and / or hydrophobicity). In general, conservative amino acid substitutions will not substantially change the functional properties of a protein. Conservative substitutions and the corresponding rules are well described in the state of the art. For example, conservative substitutions can be defined by substitutions within the group of amino acids reflected in the following table:

[0059] [Table 1]

[0060] [Table 2]

[0061] [Table 3]

[0062] As used herein, an "isolated antibody" is an antibody that has been separated and / or recovered from a component of its natural environment. An isolated antibody includes an antibody in situ within a recombinant cell, since at least one component of the antibody's natural environment will not be present. In some embodiments, the antibody is purified to homogeneity and / or to greater than 90%, 95%, or 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC) under reducing or non-reducing conditions.

[0063] The terms "derive from" and "derived from," as used herein, refer to a compound that has a structure that is derived from the structure of a parent compound or protein and that is sufficiently similar to that disclosed herein that one of skill in the art would expect, based on that similarity, to exhibit the same or similar properties, activity, and utility of the claimed compound. For example, a humanized antibody derived from a murine antibody refers to an antibody or antibody fragment that shares similar properties as the murine antibody, e.g., recognizes the same epitope, and shares a similar VH and VL with modified residues that are involved and / or enhance the humanization of the antibody.

[0064] The term "treatment" refers to any action aimed at improving the well-being of a patient, such as the cure, prevention, prophylaxis, and slowing of a disease or symptoms of a disease. The term refers to both curative and / or prophylactic treatment of a disease. Curative treatment is defined as treatment that results in a cure or treatment that alleviates, improves, and / or eliminates, reduces, and / or stabilizes a disease or symptoms of a disease or the suffering it causes directly or indirectly. Prophylactic treatment includes both treatment that results in the prevention of a disease, as well as treatment that reduces and / or delays the progression and / or onset of a disease or the risk of its occurrence. In certain embodiments, such terms refer to the improvement or eradication of a disease, disorder, infection, or symptoms associated therewith. In other embodiments, the term refers to minimizing the spread or worsening of cancer. Treatment according to the present invention does not necessarily imply a 100% or complete cure. Rather, there are various degrees of treatment that one skilled in the art would recognize as having potential benefits or therapeutic effects. Preferably, the term "treatment" refers to the application or administration of a composition comprising one or more active substances to a subject having, for example, a disorder / disease associated with a signaling pathway mediated by PD-1.

[0065] As used herein, the term "disorder" or "disease" refers to a malfunction of an organ, part, structure, or system of the body due to genetic or developmental error, infection, toxin, nutritional deficiency or imbalance, toxicity, or unfavorable environmental factors. Preferably, such terms refer to a health impairment or disease, e.g., a disease that interferes with normal physical or mental function. More preferably, the term disorder refers to an immune and / or inflammatory disease that affects animals and / or humans, such as cancer.

[0066] The term "immune disease" as used herein refers to a condition in a subject characterized by cell, tissue, and / or organ injury caused by a subject's immunological response against the subject's own cells, tissues, and / or organs. The term "inflammatory disease" refers to a condition in a subject characterized by inflammation, e.g., chronic inflammation. Autoimmune disorders may or may not be associated with inflammation. Furthermore, inflammation may or may not be caused by an autoimmune disorder.

[0067] The term "cancer," as used herein, is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells may spread locally or through the bloodstream and lymphatic system to other parts of the body.

[0068] As used herein, the terms "disease associated with or related to PD-1," "PD-1 positive cancer," or "PD-1 positive infectious disease" are intended to refer to a cancer or infectious disease (e.g., caused by a virus and / or bacteria) that is caused by PD-1 expression or has a symptom / characteristic of PD-1 expression, i.e., any condition that is caused, exacerbated, or otherwise linked to increased or decreased expression or activity of PD-1.

[0069] As used herein, the terms "subject," "host," "individual," or "patient" refer to humans, including adults and children.

[0070] "Pharmaceutical composition" as used herein refers to one or more preparations of an active agent, such as one that includes a bifunctional molecule according to the present invention together with optional other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of an active agent to an organism. The composition of the present invention may be in a form suitable for any conventional route of administration or use. In one embodiment, a "composition" typically contemplates a combination of an active agent, such as a compound or composition, with an inert (e.g., detectable substance or label) or active naturally occurring or non-naturally occurring carrier, such as an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, or adjuvant, including a pharmaceutically acceptable carrier. An "acceptable vehicle" or "acceptable carrier" as referred to herein is any known compound or combination of compounds known to those skilled in the art to be useful in formulating a pharmaceutical composition.

[0071] "Effective amount" or "therapeutically effective amount", as used herein, refers to the amount of an active agent, either alone or in combination with one or more other active agents, required to confer a therapeutic effect on a subject, e.g., the amount of active agent required to treat a target disease or disorder or to produce a desired effect. "Effective amount" will vary depending on the agent, the disease and its severity, the characteristics of the subject to be treated, including age, physical condition, size, sex, and weight, the duration of treatment, the nature of the combination therapy (if applicable), the specific route of administration, and similar factors within the knowledge and expertise of the medical practitioner. Such factors are well known to those skilled in the art and can be addressed with no more than routine experimentation. In general, it is preferred to use the maximum dose of the individual components or combinations thereof, i.e., the highest safe dose according to sound medical judgment.

[0072] As used herein, the term "medicine" refers to any substance or composition that has curative or preventative properties for a disorder or disease.

[0073] The term "in combination," as used herein, refers to the use of more than one therapies (e.g., prophylactic and / or therapeutic agents). The use of the term "in combination" does not restrict the order in which therapies (e.g., prophylactic and / or therapeutic agents) are administered to a subject with a disease or disorder.

[0074] The terms "polynucleotide", "nucleic acid" and "nucleic acid sequence" are equivalent and refer to a polymeric form of nucleotides of any length, e.g., RNA or DNA or analogs thereof. The nucleic acids of the invention (e.g., components or portions of a nucleic acid) may be naturally occurring, modified, or engineered, isolated, and / or non-naturally occurring. Engineered nucleic acids include recombinant and synthetic nucleic acids. An "isolated nucleic acid encoding an anti-PD1 antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations in a host cell. As used herein, the terms "nucleic acid construct", "plasmid" and "vector" are equivalent and refer to a nucleic acid molecule that serves to transfer a passenger nucleic acid sequence, such as DNA or RNA, into a host cell.

[0075] As used herein, the term "host cell" is intended to include any individual cell or cell culture that can be or has been a recipient of vectors, exogenous nucleic acid molecules, and polynucleotides encoding the antibody constructs of the present invention and / or the antibody construct itself. Introduction of the respective substances into a cell can be performed by transformation, transfection, and the like. The term "host cell" is also intended to include the progeny or potential progeny of a single cell. Host cells include, for example, bacterial cells, microbial cells, plant cells, and animal cells.

[0076] "Immune cells", as used herein, refer to cells involved in innate and adaptive immunity, such as, for example, white blood cells (leukocytes) derived from hematopoietic stem cells (HSCs) produced in the bone marrow, lymphocytes (T cells, B cells, natural killer (NK) cells, and natural killer T cells (NKT), and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). In particular, immune cells can be selected in a non-exhaustive list including B cells, T cells, particularly CD4+ T cells and CD8+ T cells, NK cells, NKT cells, APC cells, dendritic cells, and monocytes. "T cells", as used herein, include, for example, CD4+ T cells, CD8+ T cells, T helper type 1 T cells, T helper type 2 T cells, T helper type 17 T cells, and inhibitory T cells.

[0077] As used herein, the term "T effector cells," "Teff," or "effector cells" describes a group of immune cells that includes several T cell types that actively respond to stimuli, such as costimulation. The term specifically includes T cells that function to eliminate antigens (e.g., by production of cytokines that modulate the activation of other cells or by cytotoxic activity). The term specifically includes CD4+ cells, CD8+ cells, Treg cells, cytotoxic T cells, and helper T cells (Th1 and Th2).

[0078] As used herein, the term "regulatory T cells", "Treg cells", or "Treg" refers to a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Tregs are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FOXP3, and CD25, and are believed to be derived from the same lineage as naive CD4 cells.

[0079] The term "exhausted T cells" refers to a population of T cells that are in a state of dysfunction (i.e., "exhausted"). T cell exhaustion is characterized by a progressive loss of function, altered transcriptional profile, and persistent expression of inhibitory receptors. Exhausted T cells lose their cytokine production capacity, high proliferation capacity, and cytotoxicity capacity, which ultimately leads to their own elimination. Exhausted T cells typically display higher levels of CD43, CD69, and inhibitory receptors, along with lower expression of CD62L and CD127.

[0080] The term "immune response" refers to the actions of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules produced by the above cells or the liver, including antibodies, cytokines, and complement, that result in the selective damage, destruction, or elimination from the human body of invading pathogens, pathogen-infected cells or tissues, cancerous cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0081] The term "antagonist" as used herein refers to a substance that blocks or reduces the activity or functionality of another substance. In particular, the term refers to an antibody that binds to a cell receptor (e.g., PD-1) as a reference substance (e.g., PD-L1 and / or PD-L2) and prevents it from producing all or part of its normal biological effect (e.g., generation of an immunosuppressive microenvironment). The antagonist activity of the antibody according to the present invention can be evaluated by competitive ELISA.

[0082] As used herein, the term "isolated" indicates that the described material (e.g., antibody, polypeptide, nucleic acid, etc.) is substantially separated from or enriched relative to other materials with which it occurs in nature. In particular, an "isolated" antibody is one that has been identified, separated, and / or recovered from a component of its natural environment. For example, an isolated antibody is one that has been purified (1) to greater than 75% by weight of the antibody as determined by the Lowry method, or (2) to homogeneity by SDS-PAGE under reducing or non-reducing conditions. An isolated antibody includes an antibody in situ in a recombinant cell, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0083] The term "and / or," as used herein, should be construed as a specific disclosure of each of the two specified features or components, without regard to the presence or absence of the other. For example, "A and / or B" should be construed as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth.

[0084] The terms "a" or "an" can refer to one or more of the element that it modifies (e.g., "a reagent" can mean one or more reagents), unless it is clear from the context that either one of the elements or more than one of the elements is being described.

[0085] The term "about" when used herein in connection with any and all values ​​(including the lower and upper limits of a numerical range) means any value having an acceptable range of deviation of up to + / -10% (e.g., + / -0.5%, + / -1%, + / -1.5%, + / -2%, + / -2.5%, + / -3%, + / -3.5%, + / -4%, + / -4.5%, + / -5%, + / -5.5%, + / -6%, + / -6.5%, + / -7%, + / -7.5%, + / -8%, + / -8.5%, + / -9%, + / -9.5%). Use of the word "about" at the beginning of a value string modifies each of the values ​​(i.e., "about 1, 2, and 3" refers to about 1, about 2, and about 3). Additionally, when a list of values ​​is set forth herein (e.g., approximately 50%, 60%, 70%, 80%, 85%, or 86%), the list includes all intermediate and fractional values ​​thereof (e.g., 54%, 85.4%).

[0086] Anti-PD-1 antibody The bifunctional molecule according to the present invention comprises a first entity which comprises an anti-hPD-1 antibody or an antigen-binding fragment thereof.

[0087] Provided herein, inter alia, are antibodies that bind to human PD-1. In some aspects, the antibodies specifically bind to human PD-1, preferably to the extracellular domain of human PD-1. In some aspects, the antibodies selectively bind to one or more of full-length human PD-1, PD-1Aex2, PD-1Aex3, PD-1Aex2,3, and PD-1Aex2,3,4.

[0088] In some aspects, the anti-PD1 antibody is an isolated antibody, particularly a non-naturally occurring isolated antibody. Such an isolated anti-PD1 antibody can be prepared by at least one purification step. In some embodiments, the isolated anti-PD1 antibody is purified to at least 80%, 85%, 90%, 95%, or 99% by weight. In some embodiments, the isolated anti-PD1 antibody is provided as a solution containing at least 85%, 90%, 95%, 98%, 99% to 100% by weight of the antibody, with the remaining weight being the weight of other solutes dissolved in the solvent.

[0089] Preferably, such antibodies are capable of blocking or inhibiting the interaction of PD-1 with at least one of its ligands (e.g., PD-L1 and / or PD-L2). The ability to "block binding" or "block interaction" or "inhibit interaction", as used herein, refers to the ability of an antibody or antigen-binding fragment to prevent, to any detectable extent, the binding interaction between two molecules (e.g., PD-1 and its ligands PD-L1 and / or PD-L2).

[0090] Preferably, the anti-PD1 antibody, or antigen-binding fragment thereof, is an antagonist of the binding of human PD-L1 and / or PD-L2 to human PD-1, more preferably of the binding of human PD-L1 and PD-L2 to human PD-1.

[0091] In certain embodiments, the anti-hPD1 antibody or antigen-binding fragment inhibits the binding interaction of PD-1 with at least one of its ligands (e.g., PD-L1 and / or PD-L2, preferably PD-L1 and PD-L2) by at least 50%. In certain embodiments, this inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0092] Anti-hPD1 antibodies according to the invention may comprise any class of immunoglobulin, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), an immunoglobulin chain or fragment thereof (such as an Fv, Fab, Fab', F(ab')2, scFv, or other antigen-binding subsequence of an antibody) that contains minimal sequence derived from a non-human (e.g., murine) immunoglobulin that targets human PD-1. Preferably, anti-hPD-1 antibodies according to the invention are derived from IgG1, IgG2, IgG3, or IgG4, preferably IgG4 or IgG1.

[0093] In one embodiment, an antigen-binding fragment of an antibody comprises a heavy chain comprising a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3, a light chain comprising a variable domain comprising LDCR1, LDCR2, and LDCR3, and a fragment of the heavy chain constant domain. Thus, by fragment of the heavy chain constant domain, the antigen-binding fragment should be understood to comprise at least a portion of the complete heavy chain constant domain. By way of example, the heavy chain constant domain may comprise at least the C H One domain, or at least C of the heavy chain H 1 and C H 2 domains, or at least C of the heavy chain H 1. C H 2, and C H The heavy chain constant domain may comprise or consist of three domains. A fragment of a heavy chain constant domain may also be defined as comprising at least a portion of the Fc domain of the heavy chain. Thus, an antigen-binding fragment of an antibody includes the Fab portion of a complete antibody, the F(ab')2 portion of a complete antibody, and the Fab' portion of a complete antibody. The heavy chain constant domain may also comprise or consist of a complete heavy chain constant domain, for example, as exemplified herein, and several complete heavy chain constant domains are described herein. In certain embodiments of the present invention, and when an antigen-binding fragment of an antibody comprises a fragment of a heavy chain constant domain comprising or consisting of a portion of a complete heavy chain constant domain, the heavy chain constant domain fragment may comprise at least 10 amino acid residues, or may comprise 10 to 300k amino acid residues, in particular 210 amino acid residues.

[0094] Preferably, the antibody against human PD-1 is a monoclonal antibody. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope. Preferably, such monoclonal antibodies (mAbs) are derived from a mammal, such as a mouse, rodent, rabbit, goat, primate, non-human primate, or human. Techniques for preparing such monoclonal antibodies can be found, for example, in Stites et al. (eds.) BASIC AND CLINICAL IMMUNOLOGY (4th ed.) Lange Medical Publications, Los Alamos, CA, USA, and references cited therein; Harlow and Lane (1988) ANTIBODIES: A LABORATORY MANUAL CSH Press; Goding (1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2nd ed.) Academic Press, New York, NY, USA.

[0095] In one embodiment, the anti-PD1 antibody can be selected from the group consisting of pembrolizumab (Keytruda - MK-3475), nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538), pidilizumab (CT-011), cemiplimab (Libtayo) PDR001, monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, as described in WO 2006 / 121168.

[0096] In some embodiments, the anti-hPD1 antibodies provided herein are isolated antibodies.

[0097] In certain embodiments, the anti-hPD1 antibodies provided herein are chimeric antibodies. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate, such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0098] In certain embodiments, the anti-hPD1 antibody is a humanized antibody. A humanized antibody typically comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human or humanized antibody sequence. Alternatively, some FR residues may be substituted to restore or improve antibody specificity, affinity, and / or humanization. A humanized antibody will also optionally comprise at least a portion of a human or humanized constant region (Fc).Methods for antibody humanization are well known in the art, see, e.g., Winter and Milstein, Nature, 1991, 349:293-299; Riechmann et al., Nature, 332, 323 (1988); Verhoeyen et al., Science, 239, 1534 (1988); Rader et al., Proc. Nat. Acad. Sci. USA, 1998, 95:8910-8915; Steinberger et al., J. Biol. Chem., 2000, 275:36073-36078; Queen et al., Proc. Natl. Acad. Sci. USA, 1989, 86:10029-10033; Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) pp. 1619-1633; Kashmiri, SV et al., Methods 36 (2005) pp. 25-34 (describing SDR (a-CDR) grafting); Padlan, EA, Mol. Immunol. 28 (1991) pp. 489-498 (describing "resurfacing"); Dall'Acqua, WF et al., Methods 36 (2005) pp. 43-60 (describing "FR shuffling"); and Osbourn, J. et al., Methods 36 (2005) pp. 61-68, and Klimka, A. et al., Br. J. Cancer 83 (2000) pp. 252-260 (describing "guided selection" for FR shuffling). and 6,180,370. Preferably, the humanized antibody against human PD-1 is a monoclonal antibody.

[0099] In particular, a humanized antibody has a T20 humanness score of at least 80% or at least 85%, more preferably at least 88%, even more preferably at least 90%, and most preferably a T20 humanness score comprised between 85% and 95%, preferably between 88% and 92%.

[0100] "Humanity" is generally measured using the T20 score analyzer for quantifying the humanity of the variable regions of monoclonal antibodies as described in Gao SH, Huang K, Tu H, Adler A S., BMC Biotechnology. 2013:13:55. The T20 humanity score is a widely used parameter in the field of antibody humanization, first disclosed by Gao et al. (BMC Biotechnol., 2013, 13, 55). The T20 humanity score is commonly used in patent applications to define humanized antibodies (e.g., WO 15161311, WO 17127664, WO 18136626, WO 18190719, WO 19060750, or WO 19170677).

[0101] T20 Cutoff Human Database: A web-based tool is provided to calculate the T20 score of an antibody sequence using http: / / abAnalyzer.lakepharma.com. To calculate the T20 score, the input VH, VK, or VL variable region protein sequence is first assigned a Kabat numbering and the CDR residues are identified. The full-length or framework-only sequence (with the CDR residues removed) is compared to all sequences in the respective antibody database using the blastp protein-protein BLAST algorithm. After taking the sequence identity between each pairwise comparison and analyzing all sequences in the database, the sequences are sorted from high to low based on their sequence identity to the input sequence. The percent identity of the top 20 matching sequences is averaged to obtain the T20 score.

[0102] Each antibody sequence was scored with its corresponding database, "All Human Databases," using the T20 score analyzer for each chain type (VH, VK, VL) and sequence length (full length or framework only). The T20 scores of the top 20 matching sequences were obtained after excluding the input sequence itself (sequence 1 was always the input antibody itself, so the percent identity of sequences 2-21 was averaged). The T20 scores of each group were sorted from high to low. The score decrease was approximately linear for most of the sequences, but the T20 scores of the bottom approximately 15% of the antibodies began to decrease rapidly. Therefore, the bottom 15 percent of sequences were removed, and the remaining sequences formed the T20 cutoff human database. The T20 score cutoff indicates the lowest T20 score of the sequences in the new database.

[0103] Thus, the humanized anti-PD1 antibody comprised in the bifunctional molecule according to the present invention has a T20 humanity score of at least 80% or at least 85%, more preferably at least 88%, even more preferably at least 90%, most preferably comprised between 85% and 95%, preferably between 88% and 92%.

[0104] In one embodiment, the anti-PD1 antibody can be selected from the group consisting of: pembrolizumab (keytruda lambrolizumab, also known as MK-3475), nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538), pidilizumab (CT-011), cemiplimab (Libtayo), camrelizumab, AUNP12. , AMP-224, AGEN-2034, BGB-A317 (tisleizumab), PDR001 (spartalizumab), MK-3477, SCH-900475, PF-06801591, JNJ-63723283, genolimuzumab (CBT-501), LZM-009, BCD-100, SHR-1201, BAT-1306, AK-103 (HX-008), MEDI- 0680 (also known as AMP-514), MEDI0608, JS001 (Si-Yang Liu et al., J. Hematol. Oncol 10:136 (2017)), BI-754091, CBT-501, INCSHR1210 (also known as SHR-1210), TSR-042 (also known as ANB011), GLS-010 (also known as WBP3055), AM-0001 (Armo), STI-1110 (see International Publication No. WO 2014 / 194302), AGEN2034 (see International Publication No. WO 2014 / 194302), 7 / 040790), MGA012 (see WO 2017 / 19846), or IBI308 (see WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825, and WO 2017 / 133540), monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 described in WO 2006 / 121168. Other known bifunctional or bispecific molecules that target PD-1 include RG7769 (Roche), XmAb20717 (Xencor), MEDI5752 (AstraZeneca), FS118 (F-star), SL-279252 (Takeda), and XmAb23104 (Xencor).

[0105] In certain embodiments, the anti-PD1 antibody may be pembrolizumab (also known as keytruda lambrolizumab, MK-3475) or nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538).

[0106] Specific examples of humanized anti-hPD1 antibodies are described herein below, including their CDRs, framework regions, and Fc and hinge regions.

[0107] CDR "Complementarity determining region" or "CDR" is known in the art to refer to noncontiguous sequences of amino acids within antibody variable region that confer antigen specificity and binding affinity. The exact amino acid sequence boundaries of a given CDR can be readily determined using any of several well-known systems, including those described in Kabat et al. (Sequences of Proteins of Immunological Interest 5th ed. (1991) "Kabat" numbering system); Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-948 ("Chothia" numbering system); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 ("Contact" numbering system); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering system); and Honegge and Pluckthun, J. Mol. Biol. 2001, 309:657-70 ("AHo" numbering system). Unless otherwise specified, the numbering system used to identify particular CDRs herein is the Kabat numbering system.

[0108] In one embodiment, the bifunctional molecule comprises a humanized anti-hPD-1 antibody or antigen-binding fragment thereof. The CDR regions of the humanized antibody may be derived from a murine antibody and are designed to i) provide a safe humanized antibody with a very high level of humanization (greater than 85%) and stability, and ii) a binding affinity (KD) for human PD-1 of 10 or less. -7 Less than M, preferably 10-8 The antibodies may be optimized to increase antibody properties, more particularly greater manufacturability and higher production yields when produced in mammalian cells, such as COS cells and HCO cells, while preserving antagonist activity (i.e., inhibition of binding of human PD-L1 to human PD-1), such that the antibody has a molecular weight of less than M.

[0109] In very particular embodiments, the bifunctional molecule is (i) a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3; and (ii) a light chain variable domain comprising LCDR1, LCDR2, and LCDR3. The antibody or antigen-binding fragment thereof comprises an anti-human PD-1 antibody or antigen-binding fragment thereof comprising: - the heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 1, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof; - the heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 2, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof; - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 3, in which X1 is D or E and X2 is selected from the group consisting of T, H, A, Y, N, E and S, preferably in the group consisting of H, A, Y, N, E, and optionally with 1, 2 or 3 modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 2, 3, 7 and 8 of SEQ ID NO: 3, - the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 12, in which X is G or T and, optionally, with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 10, 11 and 16 of SEQ ID NO: 12; - the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 15, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof; - the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 16, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 1 and 6 of SEQ ID NO: 16.

[0110] In another embodiment, the bifunctional molecule comprises the above identified HCDR1, HCDR2, LCDR2 and LCDR3, a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 3 (wherein X1 is D and X2 is selected from the group consisting of T, H, A, Y, N, E and S, preferably selected in the group consisting of H, A, Y, N, E, or X1 is E and X2 is selected from the group consisting of T, H, A, Y, N, E and S, preferably selected in the group consisting of H, A, Y, N, E and S). and a light chain CDR1 (LCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 12, where X is G or T, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 3, or an antigen-binding fragment thereof.

[0111] In another embodiment, the bifunctional molecule comprises the HCDR1, HCDR2, LCDR2 and LCDR3 identified above, and - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10 or 11, optionally with 1, 2 or 3 modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 2, 3, 7 and 8 of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10 or 11; and - a light chain CDR1 (LCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID NO: 13 or SEQ ID NO: 14; The present invention relates to a humanized anti-hPD-1 antibody or an antigen-binding fragment thereof comprising the antibody

[0112] In another embodiment, the bifunctional molecule comprises the HCDR1, HCDR2, LCDR2 and LCDR3 identified above, and - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 4, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 2, 3, 7 and 8 of SEQ ID NO: 4; and a light chain CDR1 (LCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 13, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID NO: 13; or - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 5, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 2, 3, 7 and 8 of SEQ ID NO: 5; and a light chain CDR1 (LCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 13, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID NO: 13; or - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 6, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 2, 3, 7 and 8 of SEQ ID NO: 6; and a light chain CDR1 (LCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 13, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID NO: 13; or - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 7, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 2, 3, 7 and 8 of SEQ ID NO: 7; and a light chain CDR1 (LCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 13, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID NO: 13; or - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 8, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 2, 3, 7 and 8 of SEQ ID NO: 8; and a light chain CDR1 (LCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 13, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID NO: 13; or - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 9, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 2, 3, 7 and 8 of SEQ ID NO: 9; and a light chain CDR1 (LCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 13, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID NO: 13; or - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 10, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 2, 3, 7 and 8 of SEQ ID NO: 10; and a light chain CDR1 (LCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 13, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID NO: 13; or - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 11, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 2, 3, 7 and 8 of SEQ ID NO: 11; and a light chain CDR1 (LCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 13, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID NO: 13; or - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 4, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 2, 3, 7 and 8 of SEQ ID NO: 4; and a light chain CDR1 (LCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 14, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID NO: 14; or - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 5, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 2, 3, 7 and 8 of SEQ ID NO: 5; and a light chain CDR1 (LCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 14, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID NO: 14; or - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 6, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 2, 3, 7 and 8 of SEQ ID NO: 6; and a light chain CDR1 (LCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 14, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID NO: 14; or - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 7, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 2, 3, 7 and 8 of SEQ ID NO: 7; and a light chain CDR1 (LCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 14, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID NO: 14; or - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 8, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 2, 3, 7 and 8 of SEQ ID NO: 8; and a light chain CDR1 (LCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 14, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID NO: 14; or - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 9, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 2, 3, 7 and 8 of SEQ ID NO: 9; and a light chain CDR1 (LCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 14, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID NO: 14; or - a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 10, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 2, 3, 7 and 8 of SEQ ID NO: 10; and a light chain CDR1 (LCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 14, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID NO: 14; or a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO: 11, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 2, 3, 7 and 8 of SEQ ID NO: 11; and a light chain CDR1 (LCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO: 14, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID NO: 14. The present invention relates to a humanized anti-hPD-1 antibody or an antigen-binding fragment thereof, comprising:

[0113] In a particular embodiment, the modification is a substitution, in particular a conservative substitution.

[0114] In one embodiment, the anti-human PD-1 antibody, or antigen-binding fragment thereof, comprises (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2, and CDR3 of SEQ ID NO:3, where X1 is D or E and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably in the group consisting of H, A, Y, N, and E, and (ii) a light chain comprising CDR1 of SEQ ID NO:12, CDR2 of SEQ ID NO:15, and CDR3 of SEQ ID NO:16, where X is G or T.

[0115] In one embodiment, the anti-human PD-1 antibody, or antigen-binding fragment thereof, comprises (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2, and CDR3 of SEQ ID NO:3, wherein Xi is D and X2 is selected from the group consisting of T, H, A, Y, N, and E, preferably in the group consisting of H, A, Y, N, and E, or Xi is E and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably in the group consisting of H, A, Y, N, E, and S; and (ii) a light chain comprising CDR1 of SEQ ID NO:12, CDR2 of SEQ ID NO:15, and CDR3 of SEQ ID NO:16, wherein X is G or T.

[0116] In one embodiment, the anti-human PD-1 antibody, or antigen-binding fragment thereof, comprises (i) a heavy chain comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3, where X1 is D and X2 is selected from the group consisting of T, H, A, Y, N, and E, preferably in the group consisting of H, A, Y, N, and E, and (ii) a light chain comprising CDR1 of SEQ ID NO: 12, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16, where X is G or T.

[0117] In one embodiment, the anti-human PD-1 antibody, or antigen-binding fragment thereof, comprises (i) a heavy chain comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3, where X1 is E and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably in the group consisting of H, A, Y, N, E, and S; and (ii) a light chain comprising CDR1 of SEQ ID NO: 12, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16, where X is G or T.

[0118] In another embodiment, the anti-human PD-1 antibody, or antigen-binding fragment thereof, comprises or consists essentially of (i) a heavy chain that comprises a CDR1 of SEQ ID NO:1, a CDR2 of SEQ ID NO:2, and a CDR3 of SEQ ID NO:4, 5, 6, 7, 8, 9, 10, or 11, and (ii) a light chain that comprises a CDR1 of SEQ ID NO:13 or SEQ ID NO:14, a CDR2 of SEQ ID NO:15, and a CDR3 of SEQ ID NO:16.

[0119] In another embodiment, the anti-human PD-1 antibody, or antigen-binding fragment thereof, (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 4; and (ii) a light chain comprising a CDR1 of SEQ ID NO: 13, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 5; and (ii) a light chain comprising a CDR1 of SEQ ID NO: 13, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 6; and (ii) a light chain comprising a CDR1 of SEQ ID NO: 13, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 7; and (ii) a light chain comprising a CDR1 of SEQ ID NO: 13, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 8; and (ii) a light chain comprising a CDR1 of SEQ ID NO: 13, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 9; and (ii) a light chain comprising a CDR1 of SEQ ID NO: 13, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 10; and (ii) a light chain comprising a CDR1 of SEQ ID NO: 13, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 11; and (ii) a light chain comprising a CDR1 of SEQ ID NO: 13, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 4; and (ii) a light chain comprising a CDR1 of SEQ ID NO: 14, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 5; and (ii) a light chain comprising a CDR1 of SEQ ID NO: 14, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 6; and (ii) a light chain comprising a CDR1 of SEQ ID NO: 14, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 7; and (ii) a light chain comprising a CDR1 of SEQ ID NO: 14, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 8; and (ii) a light chain comprising a CDR1 of SEQ ID NO: 14, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 9; and (ii) a light chain comprising a CDR1 of SEQ ID NO: 14, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 10; and (ii) a light chain comprising a CDR1 of SEQ ID NO: 14, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16; or (i) a heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 11; and (ii) a light chain comprising a CDR1 of SEQ ID NO: 14, a CDR2 of SEQ ID NO: 15, and a CDR3 of SEQ ID NO: 16. comprises or consists essentially of

[0120] Framework In one embodiment, an anti-PD1 antibody or antigen-binding fragment according to the invention comprises framework regions, in particular the heavy chain variable region framework regions (HFR) HFR1, HFR2, HFR3, and HFR4, and the light chain variable region framework regions (LFR) LFR1, LFR2, LFR3, and LFR4.

[0121] Preferably, the anti-PD1 antibody or antigen-binding fragment according to the invention comprises a human framework region or a humanized framework region. A "human acceptor framework", for the purposes of this specification, is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. A human acceptor framework derived from a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence. A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences.

[0122] In particular, the anti-PD1 antibody or antigen-binding fragment comprises heavy chain variable region framework regions (HFRs) HFR1, HFR2, HFR3, and HFR4 comprising the amino acid sequences of SEQ ID NOs: 41, 42, 43, and 44, respectively, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof in HFR3, i.e., at any positions other than positions 27, 29, and 32 of SEQ ID NO: 43. Preferably, the anti-PD1 antibody or antigen-binding fragment comprises HFR1 of SEQ ID NO: 41, HFR2 of SEQ ID NO: 42, HFR3 of SEQ ID NO: 43, and HFR4 of SEQ ID NO: 44.

[0123] Alternatively or additionally, the anti-PD1 antibody or antigen-binding fragment comprises light chain variable region framework regions (LFRs) LFR1, LFR2, LFR3, and LFR4 comprising the amino acid sequences of SEQ ID NOs: 45, 46, 47, and 48, respectively, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof. Preferably, the humanized anti-PD1 antibody or antigen-binding fragment comprises LFR1 of SEQ ID NO: 45, LFR2 of SEQ ID NO: 46, LFR3 of SEQ ID NO: 47, and LFR4 of SEQ ID NO: 48.

[0124] VH-VL The VL and VH domains of an anti-hPD1 antibody comprised in a bifunctional molecule according to the invention may comprise four framework regions separated by three complementarity determining regions, preferably operably linked in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from amino terminus to carboxy terminus).

[0125] In a first embodiment, the humanized anti-human PD-1 antibody or antigen-binding fragment thereof contained in the bifunctional molecule is (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, in which Xi is D or E and X2 is selected from the group consisting of T, H, A, Y, N, E and S, preferably in the group consisting of H, A, Y, N, E, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 17; (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 26, wherein X is G or T, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 26. Includes.

[0126] In a second embodiment, the humanized anti-human PD-1 antibody or antigen-binding fragment thereof contained in the bifunctional molecule is (a) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, in which either X1 is D and X2 is selected from the group consisting of T, H, A, Y, N, E, preferably in the group consisting of H, A, Y, N, E, or X1 is E and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably in the group consisting of H, A, Y, N, E, and S; a heavy chain variable region (VH) optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:17; (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 26, wherein X is G or T, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 26. Includes.

[0127] In a third embodiment, the humanized anti-human PD-1 antibody or antigen-binding fragment thereof contained in the bifunctional molecule is (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, in which Xi is E and X2 is selected from the group consisting of T, H, A, Y, N, E and S, preferably in the group consisting of H, A, Y, N, E and S, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 17; (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 26, wherein X is G or T, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 26. Includes.

[0128] In another embodiment, the humanized anti-human PD-1 antibody or antigen-binding fragment thereof comprised in the bifunctional molecule is (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, or 25, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, or 25, respectively; (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27 or SEQ ID NO:28, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO:27 or SEQ ID NO:28. Includes.

[0129] In another embodiment, the humanized anti-human PD-1 antibody or antigen-binding fragment thereof comprised in the bifunctional molecule is (a) comprising or consisting of the amino acid sequence of SEQ ID NO:18, optionally comprising one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:18; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO:27; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO:19, optionally comprising one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:19; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO:27; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO:20, optionally comprising one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:20; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO:27; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO:21, optionally comprising one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:21; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO:27; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO:22, optionally comprising one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:22; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO:27; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO:23, optionally comprising one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:23; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO:27; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO:24, optionally comprising one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:24; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO:27; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO:25, optionally comprising one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:25; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO:27; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO:18, optionally comprising one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:18; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO:28; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO:19, optionally comprising one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:19; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO:28; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO:20, optionally comprising one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:20; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO:28; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO:21, optionally comprising one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:21; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO:28; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO:22, optionally comprising one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:22; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO:28; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO:23, optionally comprising one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:23; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO:28; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO:24, optionally comprising one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:24; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO:28; or (a) comprising or consisting of the amino acid sequence of SEQ ID NO:25, optionally comprising one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:25; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO:28. Includes.

[0130] In a particular embodiment, the modification is a substitution, in particular a conservative substitution.

[0131] CH-CL In one embodiment, the heavy chain (CH) and light chain (CL) comprise the VL and VH sequences as described herein above.

[0132] In certain embodiments, the anti-human PD-1 antibody or antigen-binding fragment thereof comprised in the bifunctional molecule is (a) a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 29, 30, 31, 32, 33, 34, 35, or 36, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 29, 30, 31, 32, 33, 34, 35, or 36, respectively; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:38, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO:37 or SEQ ID NO:38. Includes.

[0133] In another embodiment, the humanized anti-human PD-1 antibody or antigen-binding fragment thereof comprised in the bifunctional molecule is (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:29, optionally comprising one selected from a substitution, addition, deletion, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:29. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 37; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:30, optionally comprising one selected from a substitution, addition, deletion, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:30; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 37; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:31, optionally comprising one selected from a substitution, addition, deletion, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:31; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 37; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:32, optionally comprising one selected from a substitution, addition, deletion, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:32. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 37; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 33, optionally comprising one selected from a substitution, addition, deletion, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 33. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 37; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:34, optionally comprising one selected from a substitution, addition, deletion, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:34. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 37; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 35, optionally comprising one selected from a substitution, addition, deletion, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 35. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 37; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:36, optionally comprising one selected from a substitution, addition, deletion, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:36; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 37; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:29, optionally comprising one selected from a substitution, addition, deletion, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:29. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 38; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:30, optionally comprising one selected from a substitution, addition, deletion, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:30; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 38; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:31, optionally comprising one selected from a substitution, addition, deletion, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:31; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 38; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:32, optionally comprising one selected from a substitution, addition, deletion, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:32. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 38; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 33, optionally comprising one selected from a substitution, addition, deletion, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 33. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 38; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:34, optionally comprising one selected from a substitution, addition, deletion, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:34. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 38; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 35, optionally comprising one selected from a substitution, addition, deletion, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 35. and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 38; or (a) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:36, optionally comprising one or more substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO:36; and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 38. Includes.

[0134] Preferably the modifications are substitutions, in particular conservative substitutions.

[0135] Fc and hinge regions Some research to develop therapeutic antibodies has led to the optimization of antibody properties by genetic engineering of the Fc region, allowing the generation of molecules better suited for their required pharmacological activity. The Fc region of an antibody mediates its serum half-life and effector functions such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). Some mutations located at the interface between the CH2 and CH3 domains, such as T250Q / M428L and M252Y / S254T / T256E+H433K / N434F, have been shown to increase the binding affinity to FcRn and the half-life of IgG1 in vivo. However, there is not always a direct relationship between increased FcRn binding and improved half-life. One approach to improve the efficacy of therapeutic antibodies is to increase their serum persistence, allowing higher circulating levels, less frequent administration, and reduced doses. It may be desirable to engineer the Fc region to either reduce or increase the effector function of the antibody. For antibodies that target cell surface molecules, particularly those on immune cells, effector functions must be abolished. Conversely, for antibodies intended for oncological use, increasing effector functions can improve therapeutic activity. The four human IgG isotypes bind with different affinities to activating Fcγ receptors (FcγRI, FcγRIIa, FcγRIIIa), inhibitory FcγRIIb receptors, and the first component of complement (C1q), resulting in very different effector functions. Binding of IgG to FcγR or C1q depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are important for FcγR and C1q binding and have unique sequences in IgG2 and IgG4.

[0136] The antibody according to the invention optionally comprises at least a portion of an immunoglobulin constant region (Fc), typically of a mammalian immunoglobulin, even more preferably of a human immunoglobulin or a humanized immunoglobulin. Preferably, the Fc region is part of the anti-hPD-1 antibody described herein. The anti-hPD1 antibody or antigen-binding fragment thereof comprised in the bifunctional molecule of the invention may comprise the constant region of an immunoglobulin, or a fragment, analog, variant, mutant, or derivative of the constant region. As will be known to those skilled in the art, the choice of the IgG isotype of the heavy chain constant domain is a central concern for whether a particular function is required, and for the need for a suitable in vivo half-life. For example, antibodies designed for selective eradication of cancer cells typically require an active isotype that allows effector-mediated cell killing by complement activation and antibody-dependent cell-mediated cytotoxicity. Both human IgG1 and IgG3 (shorter half-life) isotypes, particularly the human IgG1 isotype (wild type and variants), meet these criteria. In particular, depending on the IgG isotype of the heavy chain constant domain (particularly human wild-type and variant IgG1 isotypes), the anti-hPD1 antibodies of the invention may be cytotoxic to cells expressing PD-1 by CDC, ADCC, and / or ADCP mechanisms. Indeed, the fragment crystallizable (Fc) region interacts with various accessory molecules to mediate indirect effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

[0137] In a preferred embodiment, the constant region is derived from a human immunoglobulin heavy chain, e.g., IgG1, IgG2, IgG3, IgG4, and other classes. In a further aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. Preferably, the anti-hPD1 antibody comprised in the bifunctional molecule according to the invention comprises an IgG1 or IgG4 Fc region. In a particular aspect, the humanized anti-PD1 antibody is optionally selected from the group consisting of T250Q / M428L;M252Y / S254T / T256E+H433K / N434F;E233P / L234V / L235A / G236A+A327G / A330S / P331S;E333A;S239D / A330L / I332E;P257I / Q311;K326W / E333S;S239D / I3 32E / G236A; N297A; L234A / L235A; N297A+M252Y / S254T / T256E; K322A and K444A, preferably N297A, optionally in combination with M252Y / S254T / T256E, and L234A / L235A.

[0138] More preferably, the humanized anti-hPD1 antibody comprises an IgG4 Fc-region, optionally with a substitution or combination of substitutions selected from the group consisting of S228P; L234A / L235A, S228P+M252Y / S254T / T256E and K444A. Even more preferably, the anti-hPD1 antibody comprises an IgG4 Fc-region with IgG4 stabilizing S228P.

[0139] In one embodiment, the anti-PD1 antibody comprises a truncated Fc region or a fragment of a truncated Fc region. In one embodiment, the constant region comprises a CH2 domain. In another embodiment, the constant region comprises a CH2 domain and a CH3 domain, or comprises a hinge-CH2-CH3. Alternatively, the constant region may comprise all or a portion of the hinge region, the CH2 domain, and / or the CH3 domain. Preferably, the constant region contains a CH2 domain and / or a CH3 domain derived from a human IgG4 heavy chain.

[0140] In some embodiments, the constant region contains a CH2 domain and / or a CH3 domain derived from a human IgG4 heavy chain.

[0141] In another embodiment, the constant region comprises at least a portion of a CH2 domain and a hinge region. The hinge region may be derived from an immunoglobulin heavy chain, e.g., IgG1, IgG2, IgG3, IgG4, or other class. Preferably, the hinge region is derived from human IgG1, IgG2, IgG3, IgG4, or other suitable class, with or without mutation. More preferably, the hinge region is derived from a human IgG1 heavy chain. In one embodiment, the constant region comprises a CH2 domain derived from a first antibody isotype and a hinge region derived from a second antibody isotype. In a particular embodiment, the CH2 domain is derived from a human IgG2 or IgG4 heavy chain and the hinge region is derived from a modified human IgG1 heavy chain.

[0142] In one embodiment, the constant region contains a mutation that reduces affinity for an Fc receptor or reduces an Fc effector function, for example, the constant region may contain a mutation that eliminates a glycosylation site in the constant region of an IgG heavy chain.

[0143] In another embodiment, the constant region comprises at least a portion of the CH2 domain and the hinge region. The hinge region may be derived from an immunoglobulin heavy chain, e.g., IgG1, IgG2, IgG3, IgG4, or other class. Preferably, the hinge region is derived from human IgG1, IgG2, IgG3, IgG4, or other suitable class. The IgG1 hinge region has three cysteines, two of which are involved in disulfide bonds between the two heavy chains of the immunoglobulin. These same cysteines allow efficient and consistent disulfide bond formation between the Fc portions. Thus, the preferred hinge region of the present invention is derived from IgG1, more preferably human IgG1. In some embodiments, the first cysteine ​​in the human IgG1 hinge region is mutated to another amino acid, preferably serine. The IgG2 isotype hinge region has four disulfide bonds that tend to promote oligomerization and the possibility of incorrect disulfide bonds during secretion in recombinant systems. A suitable hinge region may be derived from an IgG2 hinge, with each of the first two cysteines preferably mutated to another amino acid. The hinge region of IgG4 is known to be inefficient in forming interchain disulfide bonds. However, a suitable hinge region of the present invention may be derived from an IgG4 hinge region, preferably containing mutations that enhance correct formation of disulfide bonds between the heavy chain-derived moieties (Angal S et al. (1993) Mol. Immunol. 30:105-8). More preferably, the hinge region is derived from a human IgG4 heavy chain.

[0144] In one embodiment, the constant region comprises a CH2 domain derived from a first antibody isotype and a hinge region derived from a second antibody isotype, hi a specific embodiment, the CH2 domain is derived from a human IgG4 heavy chain and the hinge region is derived from an altered human IgG1 heavy chain.

[0145] According to the present invention, the constant region may contain CH2 and / or CH3 domains and hinge regions from different antibody isotypes, i.e., hybrid constant regions. For example, in one embodiment, the constant region contains CH2 and / or CH3 domains from IgG2 or IgG4 and a mutated hinge region from IgG1. Alternatively, a mutated hinge region from another IgG subclass is used for the hybrid constant region. For example, a mutated form of the IgG4 hinge that allows efficient disulfide bonding between the two heavy chains may be used. The mutated hinge may also be derived from an IgG2 hinge in which the first two cysteines have been mutated to different amino acids. The assembly of such hybrid constant regions is described in US Patent Application Publication No. 20030044423, the disclosure of which is incorporated herein by reference.

[0146] In one embodiment, the constant region may contain a CH2 and / or a CH3 having one of the mutations set forth in Table D below, or any combination thereof.

[0147] [Table 4]

[0148] In certain embodiments, amino acid modifications may be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. In certain embodiments, the Fc region variant has some, but not all, effector functions. Such antibodies may be useful, for example, in applications where the half-life of an antibody in vivo is important, but certain effector functions are unnecessary or harmful. Examples of effector functions include complement-dependent cytotoxicity (CDC) and antibody-mediated complement-mediated cytotoxicity (ADCC). Numerous substitutions or deletions that alter effector functions are known in the art.

[0149] In one embodiment, the constant region contains a mutation that reduces affinity for Fc receptors or reduces Fc effector functions. For example, the constant region may contain a mutation that eliminates a glycosylation site in the constant region of the IgG heavy chain. Preferably, the CH2 domain contains a mutation that eliminates a glycosylation site in the CH2 domain.

[0150] Amino acid changes close to the binding of the Fc portion and non-Fc portion can dramatically increase the serum half-life of the Fc molecule (PCT Publication No. 01 / 58957, the disclosure of which is incorporated herein by reference). Thus, the binding region of the protein or polypeptide of the invention can contain changes that are preferably within about 10 amino acids of the binding point compared to the naturally occurring sequences of immunoglobulin heavy chains and erythropoietin. These amino acid changes can cause an increase in hydrophobicity. In one embodiment, the constant region is derived from an IgG sequence in which the C-terminal lysine residue has been substituted. Preferably, the C-terminal lysine of the IgG sequence is substituted with a non-lysine amino acid such as alanine or leucine to further increase serum half-life.

[0151] In certain embodiments, the antibody may be altered to increase, decrease, or eliminate the extent to which it is glycosylated.

[0152] In one embodiment the anti-hPD1 according to the invention has a heavy chain constant domain of SEQ ID NO: 39 or 52 and / or a light chain constant domain of SEQ ID NO: 40, in particular a heavy chain constant domain of SEQ ID NO: 39 or 52 and a light chain constant domain of SEQ ID NO: 40.

[0153] In another embodiment, the anti-hPD1 according to the invention has a heavy chain constant domain of SEQ ID NO: 52 and / or a light chain constant domain of SEQ ID NO: 40, in particular a heavy chain constant domain of SEQ ID NO: 52 and a light chain constant domain of SEQ ID NO: 40.

[0154] [Table 5]

[0155] All subclasses of human IgG have a C-terminal lysine residue (K444) in the antibody heavy chain that is cleaved in circulation. This cleavage in blood may impair the biological activity of the bifunctional molecule by releasing SIRPa. To circumvent this problem, the K444 amino acid in the IgG1 or IgG4 domain can be substituted with alanine to reduce proteolytic cleavage. This mutation is a widely used mutation in antibodies. Thus, in one embodiment, the anti-PD1 antibody comprises at least one further amino acid substitution consisting of K444A.

[0156] In one embodiment, the anti-PD1 antibody contains an additional cysteine ​​residue in the C-terminal domain of IgG to create an additional disulfide bond and potentially limit the flexibility of the bifunctional molecule.

[0157] Peptide Linker The present invention includes bifunctional molecules that may include a peptide linker between the anti-PD-1 antibody or fragment thereof and SIRPa. The peptide linker is usually of sufficient length and flexibility to ensure that the two protein elements between which it is connected have sufficient spatial freedom to perform their functions and to avoid the effects of α-helix and β-fold formation on the stability of the recombinant bifunctional molecule.

[0158] In an embodiment of the present disclosure, the anti-hPD1 antibody is preferably linked to SIRPa by a peptide linker. In other words, the present invention relates to a bifunctional molecule comprising an anti-PD1 antibody or an antigen-binding fragment thereof as detailed herein, having a chain, e.g., a light chain or a heavy chain or a fragment thereof, preferably a heavy chain or a fragment thereof, linked to SIRPa via a peptide linker. As used herein, the term "linker" refers to a sequence of at least one amino acid linking SIRPa and an anti-PD-1 immunoglobulin sequence portion. Such a linker may be useful for preventing steric hindrance. The linker is usually 3 to 44 amino acid residues in length. Preferably, the linker has 3 to 30 amino acid residues. In some embodiments, the linker has 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues.

[0159] In an embodiment, the invention relates to a bifunctional molecule comprising an anti-PD-1 antibody or an antigen-binding fragment thereof as defined above and SIRPa, wherein a chain of the antibody, such as a light chain or a heavy chain, preferably a heavy chain, and even more preferably the C-terminus of the heavy chain or the light chain, is linked to the N-terminus of SIRPa, preferably SIRPa, by a peptide linker.

[0160] In a particular aspect, the present invention relates to a bifunctional molecule comprising an anti-hPD-1 antibody or an antigen-binding fragment thereof as defined above, wherein SIRPa is linked, preferably by a peptide linker, to the C-terminus of the heavy chain of said antibody (e.g. the C-terminus of the heavy chain constant domain).

[0161] In an embodiment, the invention relates to a bifunctional molecule comprising an anti-PD-1 antibody or an antigen-binding fragment thereof as defined above, wherein SIRPa is linked to the C-terminus of the light chain of said antibody (e.g. the C-terminus of the light chain constant domain), preferably by a peptide linker.

[0162] The linker sequence may be a naturally occurring or non-naturally occurring sequence. When used for therapeutic purposes, the linker is preferably non-immunogenic in the subject to which the bifunctional molecule is administered. One useful group of linker sequences are linkers derived from the hinge region of heavy chain antibodies, as described in WO 96 / 34103 and WO 94 / 04678. Another example is a polyalanine linker sequence. Further preferred examples of linker sequences are Gly / Ser linkers of various lengths, including (Gly4Ser)4, (Gly4Ser)3, (Gly4Ser)2, Gly4Ser, Gly3Ser, Gly3, Gly2ser, and (Gly3Ser2)3, especially (Gly4Ser)3.

[0163] In one embodiment, the linker contained in the bifunctional molecule is selected from the group consisting of (Gly4Ser)4, (Gly4Ser)3, (Gly4Ser)2, Gly4Ser, Gly3Ser, Gly3, Gly2ser, and (Gly3Ser2)3, preferably (Gly4Ser)3.

[0164] In an embodiment, the invention relates to a bifunctional molecule comprising an anti-PD-1 antibody or a fragment thereof as defined above, wherein the antibody or fragment thereof is linked to SIRPa by a linker sequence preferably selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3, and even more preferably by (GGGGS)3.

[0165] Preferably, the C-terminus of the heavy chain, preferably the heavy chain of an anti-PD-1 antibody, is genetically fused to the N-terminus of SIRPa via a flexible (Gly4Ser)3 linker. At the fusion junction, the C-terminal lysine residue of the antibody heavy chain can be mutated to alanine to reduce proteolytic cleavage.

[0166] Preferably, the C-terminus of the heavy chain, preferably the light chain, of the anti-PD-1 antibody is genetically fused to the N-terminus of SIRPa via a flexible (Gly4Ser)3 linker. At the fusion junction, the C-terminal lysine residue of the antibody light chain can be mutated to alanine to reduce proteolytic cleavage.

[0167] SIRPa molecule A bifunctional molecule according to the present invention comprises an additional or second entity which comprises a SIRPa molecule, a fragment or a variant thereof.

[0168] Preferably, the SIRPa protein is preferably human SIRPa or fragments and variants thereof. In one embodiment, the bifunctional molecule comprises a typical wild-type SIRPa human protein of about 504 amino acids, preferably the extracellular domain of the wild-type human SIRPa protein (e.g. consisting of amino acids positions 31-373 of wild-type human SIRPa), optionally with an additional N-terminal methionine residue (SEQ ID NO: 51). Preferably, the SIRPa protein is the protein of SEQ ID NO: 51. SIRPa preferably lacks its transmembrane and / or cytoplasmic domains. Preferably, the SIRPa protein consists of its extracellular domain, even more preferably consists essentially of amino acids 31-373 of wild-type human SIRPa.

[0169] A "variant" of a SIRPa protein is defined as an amino acid sequence in which one or more amino acids have been altered. A variant may have a "conservative" or "non-conservative" modification. Such modifications may include amino acid substitutions, deletions, and / or insertions. Guidance for determining which and how many amino acid residues can be substituted, inserted, or deleted without impairing the biological properties (e.g., activity, binding ability, and / or structure) can be found using computer programs known in the art, such as software for molecular modeling or for generating alignments. Variant SIRPa proteins included within the present invention include, in particular, SIRPa proteins that retain substantially equivalent biological properties compared to wild-type SIRPa. Variants of SIRPa also include altered polypeptide sequences of SIRPa (e.g., oxidized, reduced, deaminated, or truncated forms). In particular, truncated forms or fragments of SIRPa that retain biological properties equivalent to the full-length SIRPa protein are included within the scope of the present invention. In one embodiment, SIRPa is any biologically active fragment thereof. More preferably, variants of SIRPa include naturally occurring allelic variants resulting from natural genetic polymorphisms, including SNPs, splicing variants, and the like.

[0170] The most common human SIRPa variants are SIRPa v1 and SIRPa v2 (accession numbers NP_542970 (P78324) and CAA71403). The SIRPa family can be divided into two subsets; the SIRPa v1 isoform family and the SIRPa v2 isoform family. These families include SIRPa isoform 2 (identifier: P78324-2) and SIRPa isoform 4 (identifier: P78324-4), respectively. In one embodiment, the SIRPa variant is selected from the group consisting of SIRPa isoform 2 (P78324-2) and SIRPa isoform 4 (P78324-4).

[0171] Variant SIRPa proteins also include polypeptides having at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more sequence identity to wild-type SIRPa.

[0172] In one embodiment, the variant of SIRP alpha is SIRP gamma, which typically exhibits 55% sequence identity to wild-type SIRPa. As used herein, the terms "SIRP gamma," "SIRPg," and "SIRPγ" are used interchangeably. For example, the human SIRPg amino acid sequence is described in UniProtKB - Q9P1W8.

[0173] SIRPγ has a similar extracellular structure but different cytoplasmic regions that produce contrasting types of signals. Indeed, SIRPα and SIRPγ contain three Ig-like extracellular domains: Ig-like type V, encoded by amino acids 32-137 (domain D1), Ig-like type C1 type 1, encoded by amino acids at positions 148-247 (domain D2), and Ig-like type C1 type 2, encoded by amino acids at positions 254-348 (domain D3).

[0174] Then, in one embodiment, the SIRPa variant comprises i) the D1 domain of SIRPg, the D2 and D3 domains of SIRPa, ii) the D1 and D2 domains of SIRPg and the D3 domain of SIRPa, iii) the D1 domain of SIRPg, the D2 domain of SIRPa and the D3 domain of SIRPg, iv) the D1 domain of SIRPa, the D2 and D3 domains of SIRPg, v) the D1 and D2 domains of SIRPa and the D3 domain of SIRPg, or vi) the D1 domain of SIRPa, the D2 domain of SIRPg and the D3 domain of SIRPa.

[0175] A preferred SIRPa according to the present invention is a human SIRPa polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:51, US Publication No. 20160319256, WO Publication No. WO2013109752 or WO2016024021, and natural variants and homologues thereof.

[0176] In some embodiments, the SIRP-a variant construct has preferential activity at disease sites (e.g., at tumor sites over non-diseased sites), hi some embodiments, the SIRP-a variant contains one or more substitutions of amino acids with histidine residues or other amino acids that allow for selective binding of the SIRP-a variant construct at disease sites.

[0177] In certain embodiments, SIRPa consists of a truncation or fragment of the extracellular domain of SIRPa, specifically comprising or consisting of a binding region of amino acids within the set of contact residues that interact with CD47.

[0178] In one embodiment, the affinity of the SIRPa protein can be measured using an in vitro assay. Preferably, the SIRPa variant according to the invention maintains at least 10%, 20%, 30%, 40%, 50%, 60% affinity to CD47 compared to wild-type human SIRPa, preferably at least 80%, 90%, 95%, even more preferably 99% affinity to CD47 compared to wild-type SIRPa.

[0179] The present invention also provides bifunctional molecules comprising SIRPa proteins having enhanced affinity for CD47 compared to wild-type SIRPa proteins, e.g., as described in WO2013109752. In certain embodiments, SIRP-a variant constructs have a higher binding affinity for CD47 on diseased cells (e.g., tumor cells). In some embodiments, SIRP-a variants bind with higher affinity to CD47 under acidic pH (e.g., below about pH 7) and / or under hypoxic conditions than physiological conditions, e.g., as described in U.S. Publication No. 20160319256.

[0180] In one aspect, the SIRPa polypeptide used in the present invention is a recombinant SIRPa. The term "recombinant" as used herein means that the polypeptide is obtained or derived from a recombinant expression system, that is, from a host cell (e.g., a microorganism or insect or plant or mammal) culture, or from a transgenic plant or animal that has been genetically engineered to contain a nucleic acid molecule encoding a SIRPa polypeptide. Preferably, the recombinant SIRPa is human recombinant SIRPa (e.g., human SIRPa produced in a recombinant expression system).

[0181] Bifunctional molecules or "Bicki" The present invention particularly provides a bifunctional molecule comprising or consisting of an anti-hPD1 antibody or antibody fragment thereof and SIRPa, as disclosed herein above, wherein the anti-hPD1 antibody or antibody fragment thereof is covalently linked to SIRPa, preferably by a peptide linker as disclosed herein above, in particular as a fusion protein.

[0182] In particular, a bifunctional molecule according to the invention comprises two entities: a first entity comprising or essentially consisting of an anti-hPD1 antibody or a fragment thereof; a second entity comprising or essentially consisting of the extracellular domain of SIRPa, preferably human SIRPa, even more preferably human SIRPa isoform 1, these two entities being optionally linked by a peptide linker.

[0183] In particular, the bifunctional molecule according to the present invention comprises one, two, three or four SIRPa molecules. In particular, the bifunctional molecule may comprise only one SIRPa molecule linked to only one of the light or heavy chains of the anti-PD-1 antibody. The bifunctional molecule may also comprise two SIRPa molecules linked to either the light or heavy chain of the anti-PD-1 antibody. The bifunctional molecule may also comprise two SIRPa molecules, the first of which is linked to the light chain of the anti-PD-1 antibody and the second of which is linked to the heavy chain of the anti-PD-1 antibody. The bifunctional molecule may also comprise three SIRPa molecules, two of which are linked to either the light or heavy chain of the anti-PD-1 antibody and the last one is linked to the other chain of the anti-PD-1 antibody. And finally, the bifunctional molecule may contain four SIRPa molecules, two linked to the light chain of an anti-PD-1 antibody and two linked to the heavy chain of an anti-PD-1 antibody, and thus the bifunctional molecule contains from one to four immunotherapeutic agent molecules as disclosed herein.

[0184] In one embodiment, only one of the light chains comprises one SIRPa molecule (e.g., the bifunctional molecule comprises one SIRPa molecule), only one of the heavy chains comprises one SIRPa molecule (e.g., the bifunctional molecule comprises one SIRPa molecule), each light chain comprises one SIRPa molecule (e.g., the bifunctional molecule comprises two SIRPa molecules), each heavy chain comprises one SIRPa molecule (e.g., the bifunctional molecule comprises two SIRPa molecules), or only one of the light chains and only one of the heavy chains comprises one immunotherapy. Either each light chain contains one SIRPa molecule and only one of the heavy chains contains one SIRPa molecule (e.g., a bifunctional molecule contains two SIRPa molecules), each light chain contains one SIRPa molecule and only one of the heavy chains contains one SIRPa molecule (e.g., a bifunctional molecule contains three SIRPa molecules), each heavy chain contains one SIRPa molecule and only one of the light chains contains one SIRPa molecule (e.g., a bifunctional molecule contains three SIRPa molecules), or both the light and heavy chains contain one SIRPa molecule (e.g., a bifunctional molecule contains four SIRPa molecules).

[0185] In one embodiment, a bifunctional molecule according to the invention comprises: (a) an anti-human PD-1 antibody or antigen-binding fragment thereof comprising (i) a heavy chain, and (ii) a light chain; and (b) human SIRPa or a fragment or variant thereof comprising or consisting of The antibody heavy and / or light chain or fragments thereof are covalently linked to SIRPa as a fusion protein, preferably by a peptide linker.

[0186] Preferably, the bifunctional molecule according to the invention comprises: (a) a humanized anti-human PD-1 antibody or an antigen-binding fragment thereof, comprising (i) a heavy chain and (ii) a light chain; and (b) human SIRPa or a fragment or variant thereof comprising or consisting of The antibody heavy or light chain, or a fragment thereof, is covalently linked to SIRPa by a peptide linker.

[0187] Preferably, such bifunctional molecules comprise at least one peptide linker connecting the N-terminus of SIRPa to the C-terminus of the heavy or light chain or both of the anti-human PD-1 antibody, the peptide linker preferably being selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3, and even more preferably being (GGGGS)3.

[0188] Preferably, the N-terminus of SIRPa is connected to the C-terminus of the heavy chain or light chain, or both, of the anti-human PD-1 antibody via at least one peptide linker. Alternatively, the C-terminus of SIRPa is connected to the N-terminus of the heavy chain or light chain, or both, of the anti-human PD-1 antibody via at least one peptide linker.

[0189] In one embodiment, a bifunctional molecule according to the invention comprises: (a) an anti-human PD-1 antibody or an antigen-binding fragment thereof, comprising (i) a heavy chain and (ii) a light chain; (b) human SIRPa or a fragment or variant thereof, and (c) a peptide linker connecting the N-terminus of SIRPa to the C-terminus of the heavy chain or the light chain or both of the anti-human PD-1 antibody, preferably selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3, and even more preferably (GGGGS)3. It comprises or consists of:

[0190] More specifically, the anti-human PD-1 or antigen-binding fragment thereof can be any antibody previously disclosed in the section "Anti-PD-1," and the human SIRPa, fragment or variant thereof can be any SIRPa previously disclosed in the section "SIRPa molecules."

[0191] In certain embodiments, the bifunctional molecule according to the invention comprises: (a) (i) a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3; and (ii) a light chain variable domain comprising LCDR1, LCDR2, and LCDR3. Including, - the heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 1, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than position 3 of SEQ ID NO: 1, - the heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 2, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 13, 14 and 16 of SEQ ID NO: 2, - the heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 3, in which X1 is either D or E and X2 is selected from the group consisting of T, H, A, Y, N, E and S, preferably in the group consisting of H, A, Y, N and E, and optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 2, 3, 7 and 8 of SEQ ID NO: 3, - the light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 12, in which X is G or T and, optionally, with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID NO: 12; - the light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 15, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof; - the light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 16, optionally with one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 1, 4 and 6 of SEQ ID NO: 16; An anti-human PD-1 antibody or an antigen-binding fragment thereof; and (b) human SIRPa of SEQ ID NO: 51 or a fragment or variant thereof comprising or consisting of The antibody heavy and / or light chains or fragments thereof are covalently linked to SIRPa as a fusion protein, preferably by a peptide linker.

[0192] Preferably, the peptide linker is selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3, and even more preferably is (GGGGS)3.

[0193] In another embodiment, the present invention provides (a) (i) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, wherein X1 is D or E and X2 is selected from the group consisting of T, H, A, Y, N, E and S, preferably selected in the group consisting of H, A, Y, N, E, and optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID NO: 17; (ii) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 26, wherein X is G or T, and optionally has one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 26. A humanized anti-hPD1 antibody comprising (b) human SIRPa of SEQ ID NO: 51 or a variant thereof; (c) a peptide linker between the light chain and / or the heavy chain of the anti-hPD1 antibody and human SIRPa or a variant thereof, selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS and (GGGS)3, even more preferably a peptide linker which is (GGGGS)3 The present invention relates to a bifunctional molecule comprising or consisting of:

[0194] Preferably, the N-terminus of SIRPa is linked to the C-terminus of the heavy or light chain, or both, of the anti-human PD-1 antibody via at least one peptide linker. Alternatively, the C-terminus of SIRPa is linked to the N-terminus of the heavy or light chain, or both, of the anti-human PD-1 antibody via at least one peptide linker.

[0195] In another embodiment, the present invention provides (a) (i) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, wherein X1 is D or E and X2 is selected from the group consisting of T, H, A, Y, N, E and S, preferably selected in the group consisting of H, A, Y, N, E, and optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID NO: 17; (ii) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 26, wherein X is G or T, and optionally has one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 26. A humanized anti-hPD1 antibody comprising (b) human SIRPa of SEQ ID NO: 51 or a variant thereof; (The C-terminus of the heavy and / or light chain of the antibody or antigen-binding fragment thereof is covalently linked to the N-terminus of SIRPa, preferably by a (GGGGS)3 peptide linker, to form a fusion protein.) The present invention relates to a bifunctional molecule comprising or consisting of:

[0196] In a preferred embodiment, the C-terminus of the heavy chain of the antibody or antigen-binding fragment thereof is covalently linked to the N-terminus of SIRPa to form a fusion protein. Preferably, only the heavy chain of the antibody or antigen-binding fragment thereof is covalently linked to SIRPa.

[0197] In certain embodiments, the present invention provides a method for producing a pharmaceutical composition comprising: (a) (i) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, wherein X1 is D or E and X2 is selected from the group consisting of T, H, A, Y, N, E and S, preferably selected in the group consisting of H, A, Y, N, E, and optionally having one, two or three modifications selected from substitutions, additions, deletions and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID NO: 17; (ii) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 26, wherein X is G or T, and optionally has one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 26. A humanized anti-hPD1 antibody comprising (b) human SIRPa of SEQ ID NO: 51 or a variant thereof For a bifunctional molecule comprising or consisting of The C-terminus of the heavy chain of the antibody or antigen-binding fragment thereof is covalently linked to the N-terminus of SIRPa, preferably by a (GGGGS)3 peptide linker, to form the fusion protein.

[0198] In another embodiment, the present invention provides (a) (i) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, or 25, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, or 25, respectively; (ii) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27 or SEQ ID NO:28, optionally with one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO:27 or SEQ ID NO:28. a humanized anti-hPD1 antibody comprising: (b) human SIRPa of SEQ ID NO: 51 or a variant thereof A bifunctional molecule comprising or consisting of: For bifunctional molecules, the C-terminus of the heavy and / or light chain of the antibody or antigen-binding fragment thereof is covalently linked to the N-terminus of SIRPa, preferably by a (GGGGS)3 peptide linker, to form a fusion protein.

[0199] Binding of the bifunctional molecules to their specific targets can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays generally detects the presence of a particular protein-antibody complex of interest by using a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, anti-hPD-1 antibody / SIRPa complexes can be detected using, for example, an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to SIRPa or a receptor for SIRPa.

[0200] In some examples, the bifunctional molecules described herein inhibit the PD-1 signaling pathway by at least 20%, at least 40%, at least 50%, at least 75%, at least 90%, at least 100%, or at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold.

[0201] Preferably, such bifunctional molecules are capable of blocking or inhibiting the interaction of PD-1 with its ligands (e.g., PD-L1 and / or PD-L2). In certain embodiments, the bifunctional molecules inhibit the binding interaction of PD-1 with its ligands (e.g., PD-L1 and / or PD-L2) by at least 50%. In certain embodiments, this inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0202] In some examples, the bifunctional molecules described herein inhibit or reduce the PD-1 signaling pathway by at least 20%, at least 40%, at least 50%, at least 75%, at least 90%, at least 100%, or at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold.

[0203] In some examples, the bifunctional molecules described herein stimulate IFN gamma secretion.

[0204] In some examples, the bifunctional molecules described herein block interactions between CD47-expressing cells (e.g., tumor cells) and SIRPa-expressing cells (e.g., antigen-presenting cells (APCs) such as macrophages).

[0205] In some examples, the bifunctional molecules described herein inhibit or reduce the SIRPa / CD47 signaling pathway by at least 10%, at least 20%, at least 40%, at least 50%, at least 75%, at least 90%, at least 100%, or at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold.

[0206] In another example, the bifunctional molecules described herein enhance T cell activation, stimulate the secretion of IFNg by T cells, and / or stimulate the proliferation of immune cells such as T cells.

[0207] In another example, the bifunctional molecules described herein induce cytokine secretion and / or proliferation of naive, partially exhausted T cell subsets.

[0208] Preparation of bifunctional molecules - Nucleic acid molecules encoding bifunctional molecules, recombinant expression vectors and host cells containing them To generate a bifunctional molecule of the invention, an anti-hPD1 antibody of the invention is operably linked to SIRPa.

[0209] Both entities of the bifunctional molecule are encoded in the same vector and produced as a fusion protein. Thus, also disclosed herein are nucleic acids encoding any of the bifunctional molecules described herein, vectors such as expression vectors or recombinant viruses that contain such nucleic acids, and host cells that contain the nucleic acids and / or vectors.

[0210] To produce a bifunctional fusion protein according to the invention, which is secreted in a stable form by mammalian cells, the nucleic acid sequence encoding the bifunctional molecule is subcloned into an expression vector commonly used to transfect mammalian cells. Basic techniques for producing molecules, including antibody sequences, are described in Coligan et al. (eds.), Current protocols in immunology, pp. 10.19.1-10.19.11 (Wiley Interscience, 1992), the contents of which are incorporated herein by reference, and in "Antibody engineering: a practical guide" (WH Freeman and Company, 1992), with relevant reviews of molecular production interspersed throughout the corresponding text.

[0211] In general, such methods include: (1) transfecting or transforming a suitable host cell with a polynucleotide encoding a recombinant bifunctional molecule of the invention or a variant thereof or a vector containing said polynucleotide; (2) culturing the host cell in a suitable medium; and (3) Optionally, isolating or purifying the protein from the medium or the host cell. Includes.

[0212] The present invention further relates to nucleic acids encoding the bifunctional molecules as disclosed above, to vectors, preferably expression vectors, comprising the nucleic acids of the invention, to genetically engineered host cells transformed with the vectors of the invention or directly with sequences encoding the recombinant bifunctional molecules, and to methods for producing the proteins of the invention by recombinant techniques.

[0213] The nucleic acids, vectors, and host cells are described in more detail herein below.

[0214] Nucleic acid sequence The present invention also relates to a nucleic acid molecule encoding a bifunctional molecule as defined above, or to a group of nucleic acid molecules encoding a bifunctional molecule as defined above.

[0215] The antibody DNA sequence may, for example, be amplified from RNA of immunoglobulin-synthesizing cells, or may be synthesized using PCR with cloned immunoglobulins, or may be synthesized by oligonucleotides encoding known signal peptide amino acid sequences. Preferably, the peptide signal comprises or consists of the amino acid sequence of SEQ ID NO: 49 for VH and / or CH, and / or SEQ ID NO: 50 for VL and / or CL. In particular, the peptide signal is present at the N-terminus of CH, VH, CL, and / or VL.

[0216] Such nucleic acids can encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of an antibody (e.g., the light and / or heavy chains of the antibody). Such nucleic acids can be readily isolated and sequenced using conventional procedures.

[0217] In particular, a nucleic acid molecule encoding a bifunctional molecule as defined above is - a first nucleic acid molecule encoding the heavy chain variable domain of an anti-hPD-1 antibody disclosed herein, optionally having a peptide signal of SEQ ID NO: 49; and - a second nucleic acid molecule encoding the light chain variable domain of an anti-hPD-1 antibody disclosed herein, optionally having a peptide signal of SEQ ID NO: 50; and - a third nucleic acid encoding the extracellular domain of SIRPa or a variant thereof, preferably human SIRPa, even more preferably human SIRPa isoform 1 or a variant thereof, operably linked to the first nucleic acid or the second nucleic acid or both, optionally via a nucleic acid encoding a linker; Includes.

[0218] In one embodiment, the nucleic acid molecule encoding the bifunctional molecule defined above comprises: - a first nucleic acid molecule encoding a heavy chain variable domain of SEQ ID NO: 17, in which X1 is D or E and X2 is selected from the group consisting of T, H, A, Y, N, E and S, preferably selected in the group consisting of H, A, Y, N and E, and optionally having a peptide signal of SEQ ID NO: 49; - a second nucleic acid molecule encoding a light chain variable domain of SEQ ID NO: 26, wherein X is G or T, and optionally has a peptide signal of SEQ ID NO: 50, and - a third nucleic acid encoding human SIRPa of SEQ ID NO: 51 or a variant thereof, operably linked to the first nucleic acid or the second nucleic acid or both, optionally via a nucleic acid encoding a linker; Includes.

[0219] Preferably, the nucleic acid molecule encoding the bifunctional molecule as defined above comprises: - a first nucleic acid molecule encoding a variable heavy chain domain of the amino acid sequence shown in SEQ ID NO: 18, 19, 20, 21, 22, 23, 24 or 25, and optionally having a peptide signal of SEQ ID NO: 49, and - a second nucleic acid molecule encoding a variable light chain domain of the amino acid sequence shown in SEQ ID NO: 27 or SEQ ID NO: 28, and optionally having a peptide signal of SEQ ID NO: 50, and - a third nucleic acid encoding human SIRPa of SEQ ID NO: 51 or a variant thereof, operably linked to either the first nucleic acid or the second nucleic acid or both, optionally via a nucleic acid encoding a peptide linker; Includes.

[0220] In very particular embodiments, the nucleic acid molecule encoding the variable heavy chain domain has the sequence set forth in SEQ ID NO:55 and / or the nucleic acid molecule encoding the variable light chain domain has the sequence set forth in SEQ ID NO:56.

[0221] By "operably linked" it is intended that the nucleic acid encodes a protein fusion comprising a variable heavy or light chain domain, optionally a peptide linker, and SIRPa. Preferably, the linker is selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3, and even more preferably is (GGGGS)3.

[0222] In one embodiment, the nucleic acid molecule is an isolated, particularly a non-naturally occurring, nucleic acid molecule.

[0223] The nucleic acid molecule or group of nucleic acid molecules encoding the bifunctional molecule according to the invention is preferably comprised in a vector or group of vectors.

[0224] vector In another aspect, the present invention relates to a vector comprising a nucleic acid molecule or a group of nucleic acid molecules as defined above.

[0225] As used herein, a "vector" is a nucleic acid molecule used as a vehicle to transfer genetic material into a cell. The term "vector" encompasses plasmids, viruses, cosmids, and artificial chromosomes. In general, genetically engineered vectors contain an origin of replication, a multiple cloning site, and a selectable marker. The vector itself is generally a nucleotide sequence, typically a DNA sequence, that contains an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. Modern vectors may include additional features besides the transgene insert and backbone: promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, protein purification tags. Vectors called expression vectors (expression constructs) are specifically intended for expressing a transgene in a target cell and generally have regulatory sequences.

[0226] In one embodiment, both the heavy and light chain coding sequences and / or the constant region of the anti-PD1 antibody are contained in one expression vector. The heavy and light chain coding sequences may each be operably linked to a suitable promoter, and the heavy and / or light chains are operably linked to the immunotherapeutic agent according to the invention. Alternatively, the expression of both the heavy and light chains may be driven by the same promoter. In another embodiment, the heavy and light chains of the antibody are each cloned into an individual vector, and one or both of the heavy and light chains, the heavy and / or light chains are operably linked to the immunotherapeutic agent according to the invention. In the latter case, the expression vectors encoding the heavy and light chains may be co-transfected into one host cell for the expression of both chains, and both chains may be assembled to form an intact antibody, either in vivo or in vitro. Alternatively, the expression vector encoding the heavy chain and the expression vector encoding the light chain may be introduced into different host cells for the expression of each of the heavy and light chains, which may then be purified and assembled to form an intact antibody in vitro.

[0227] Those skilled in the art can clone the nucleic acid molecule encoding the humanized anti-PD-1 antibody or antibody fragment thereof into a vector and then transform it into a host cell. Thus, the present invention also provides a recombinant vector comprising the nucleic acid molecule encoding the anti-PD-1 antibody or fragment thereof of the present invention. In a preferred embodiment, the expression vector further comprises a promoter and a nucleic acid sequence encoding a secretion signal peptide, and optionally at least one drug resistance gene for screening.

[0228] Suitable expression vectors typically contain: (1) a prokaryotic DNA element encoding a bacterial origin of replication and an antibiotic resistance marker to provide for growth and selection of the expression vector in a bacterial host; (2) a eukaryotic DNA element that controls transcription initiation, such as a promoter; and (3) a DNA element that controls processing of the transcript, such as a transcription termination / polyadenylation sequence.

[0229] Methods known to those skilled in the art can be used to construct expression vectors containing the nucleic acid sequences of the bifunctional molecules described herein and appropriate regulatory elements for transcription / translation. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence is effectively linked to an appropriate promoter in the expression vector to direct the synthesis of mRNA. The expression vector may further include a ribosome binding site to initiate translation, a transcription terminator, etc.

[0230] Expression vectors can be introduced into host cells using a variety of techniques, including calcium phosphate transfection, liposome-mediated transfection, and electroporation. Preferably, transfected cells in which the expression vector has stably integrated into the host cell genome to produce stable transformants are selected and propagated. Techniques for introducing vectors into eukaryotic cells and for selecting stable transformants using dominant selection markers are described in Sambrook, Ausubel, Bebbington, "Expression of Antibody Genes in Nonlymphoid Mammalian Cells" in 2 METHODS: A companion to methods in enzymology, Vol. 136 (1991), and Murray (ed.), Gene transfer and expression protocols (Humana Press, 1991). Suitable cloning vectors are described in Sambrook et al. (eds.), MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Edition (Cold Spring Harbor Press, 1989) (hereinafter "Sambrook"); Ausubel et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Wiley Interscience, 1987) (hereinafter "Ausubel"); and Brown (eds.), MOLECULAR BIOLOGY LABFAX (Academic Press, 1991).

[0231] host cell In another aspect, the present invention relates to a host cell comprising a vector or a nucleic acid molecule or a group of nucleic acid molecules as defined above, for example for the purpose of producing a bifunctional molecule.

[0232] As used herein, the term "host cell" is intended to include any individual cell or cell culture that can be or is a recipient of vectors, exogenous nucleic acid molecules, and polynucleotides encoding the antibody constructs of the present invention; and / or the antibody construct or bifunctional molecule itself. Introduction of the respective substances into the cell can be performed by transformation, transfection, and the like. The term "host cell" is also intended to include the progeny or potential progeny of a single cell. Suitable host cells include prokaryotic or eukaryotic cells, including, but not limited to, bacteria, yeast cells, fungal cells, plant cells, and animal cells such as insect cells and mammalian cells, e.g., mouse, rat, rabbit, macaque, or human.

[0233] In one embodiment, the host cell contains (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and / or an amino acid sequence comprising the VH of an antibody and / or a constant region of an antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of an antibody.

[0234] In another embodiment, the host cell comprises (e.g., is transformed with) a vector that comprises both entities of the bifunctional molecule. Preferably, the host cell comprises (e.g., is transformed with) a vector that comprises a first nucleic acid molecule encoding a variable heavy chain domain of an anti-hPD-1 antibody as disclosed herein and a second nucleic acid molecule encoding a variable light chain domain of an anti-hPD-1 antibody as disclosed herein operably linked to a third nucleic acid encoding SIRPa or a variant thereof.

[0235] Also provided herein is a method for producing a humanized anti-PD1 antibody. The method comprises culturing a host cell comprising an antibody-encoding nucleic acid as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium). In particular, in the case of recombinant production of a humanized anti-PD1 antibody, e.g., an antibody-encoding nucleic acid as described above is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell.

[0236] The bifunctional molecules of the present invention are preferably expressed in eukaryotic cells such as mammalian cells, plant cells, insect cells, or yeast cells. Mammalian cells are particularly preferred eukaryotic hosts as they provide suitable post-translational modifications such as glycosylation. Preferably, such suitable eukaryotic host cells may be fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe; insect cells such as Mythimna separate; plant cells such as tobacco; and mammalian cells such as BHK cells, 293 cells, CHO cells, NSO cells, and COS cells. Other examples of useful mammalian host cell lines are CV-1 in Origin with SV40 genes cell (COS cells), monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells as described in Graham, FL et al., J. Gen Virol., 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., Mather, JP, Biol. Reprod. 23 (1980) pp. 243-252); human epithelial kidney cells (HEK cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat hepatocytes (BRL3A); human lung cells (W138); human hepatocytes (HepG2); mouse mammary tumor (MMT060562); TRI cells, e.g., as described by Mather, JP et al., Annals NY Acad. Sci. 383 (1982) pp. 44-68; MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include DHFR" CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NSO, and Sp2 / 0.For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki, P. and Wu, AM, Methods in Molecular Biology, vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ, USA (2004), pp. 255-268. For example, mammalian cell lines adapted for growth in suspension may be useful.

[0237] In particular, the host cell of the present invention is selected from the group consisting of a CHO cell, a COS cell, an NSO cell, and a HEK cell.

[0238] For mammalian hosts, the transcriptional and translational control signals of the expression vectors may be derived from viral sources such as adenovirus, bovine papilloma virus, or simian viruses, and the control signals are associated with particular genes that exhibit high levels of expression. Suitable transcriptional and translational control sequences can also be obtained from mammalian genes such as the actin, collagen, myosin, and metallothionein genes.

[0239] Stable transformants producing the bifunctional molecules according to the invention can be identified using various methods. After the molecule-producing cells are identified, the host cells are cultured under conditions (e.g., temperature, medium) suitable for their growth and expression of the bifunctional molecules. The bifunctional molecules are then isolated and / or purified by any method known in the art. Such methods include, but are not limited to, conventional renaturation, treatment with protein precipitants (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve or gel chromatography, adsorption chromatography, ion exchange chromatography, HPLC, any other liquid chromatography, and combinations thereof. Bifunctional molecule isolation techniques can include, inter alia, affinity chromatography with Protein A Sepharose, size exclusion chromatography, and ion exchange chromatography, as described, for example, by Coligan. Protein A is preferably used for the isolation of the bifunctional molecules of the invention.

[0240] Pharmaceutical compositions and methods of administration thereof The present invention also relates to pharmaceutical compositions comprising, preferably as active ingredient or compound, any of the bifunctional molecules described herein, nucleic acid molecules, groups of nucleic acid molecules, vectors, and / or host cells as disclosed herein above. The formulations can be sterile and, if desired, mixed with auxiliary agents such as pharma- ceutically acceptable carriers and excipients that do not adversely interact with the bifunctional molecules, nucleic acids, vectors, and / or host cells of the invention. Optionally, the pharmaceutical compositions may further comprise additional therapeutic agents as detailed below.

[0241] Preferably, the pharmaceutical composition of the present invention may comprise a bifunctional molecule as described herein, a nucleic acid molecule, a group of nucleic acid molecules, a vector, and / or a host cell as described herein above, in combination with one or more pharma- ceutically or physiologically acceptable carriers, diluents, excipients, salts, and antioxidants as described herein below. Desirably, a pharma- ceutically acceptable form is used that does not adversely affect the desired immune enhancing effect of the bifunctional molecule according to the present invention. To facilitate administration, the bifunctional molecule as described herein can be formulated into a pharmaceutical composition for in vivo administration. Means for formulating such compositions are described in the art (see, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st Edition (2005)).

[0242] In particular, the pharmaceutical compositions according to the present invention can be formulated for any conventional route of administration, including topical, enteral, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration, etc. Preferably, the pharmaceutical compositions according to the present invention are formulated for enteral or parenteral routes of administration. Compositions and formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives such as, but not limited to, penetration enhancers, carder compounds, and other pharma- ceutically acceptable carriers or excipients.

[0243] Pharmaceutical compositions can be prepared in the form of lyophilized formulations or aqueous solutions by mixing the substance having the desired purity with optional pharma- ceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th ed., Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used and include: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0244] Solid pharmaceutically acceptable vehicles may contain one or more substances that can also act as flavorings, lubricants, solubilizers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet disintegrating agents. Suitable solid vehicles include, for example, calcium phosphate, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes, and ion exchange resins. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Except insofar as any conventional media or substances are incompatible with the active compound, their use in the pharmaceutical compositions of the present invention is contemplated.

[0245] The bifunctional molecules according to the invention can be dissolved or suspended in pharma- ceutically acceptable liquid vehicles such as water, organic solvents, ethanol, and polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), pharma- ceutically acceptable oils or fats, or mixtures of both, and suitable mixtures thereof. The liquid vehicles can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, humectants, thickeners, colorants, viscosity regulators, stabilizers, or osmolality regulators. Suitable examples of liquid vehicles for oral and enteral administration include water (partially containing additives as described above, e.g., cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (including monohydric and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and peanut oil). For parenteral administration, the vehicle can be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for enteral administration. The liquid vehicle for pressurized compositions can be halogenated hydrocarbon or other pharma- ceutically acceptable propellant.

[0246] The pharmaceutical composition of the present invention may further comprise one or more pharma- ceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids, such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphorous acid, and those derived from non-toxic organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkali or alkaline earth metals, such as sodium, potassium, magnesium, and calcium, and those derived from non-toxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0247] The pharmaceutical composition of the present invention may also contain a pharma- ceutically acceptable antioxidant. Examples of pharma-ceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0248] To facilitate delivery, either the bifunctional molecule or its encoding nucleic acid may be conjugated to a chaperone agent. The chaperone agent may be a naturally occurring substance such as a protein (e.g., human serum albumin, low density lipoprotein, or globulin), a carbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), or a lipid. The chaperone agent may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, and polyphosphazine. In one example, the chaperone agent is a micelle, liposome, nanoparticle, or microsphere. Methods for preparing such micelles, liposomes, nanoparticles, or microspheres are well known in the art. See, for example, U.S. Patent Nos. 5,108,921; 5,354,844; 5,416,016; and 5,527,5285.

[0249] Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. Pharmaceutical compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentration and / or suitable for injection. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0250] In one embodiment, the pharmaceutical composition is an injectable composition that may contain various carriers, such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol). For intravenous injection, the water-soluble antibody may be administered by drip infusion, where a formulation containing the antibody and a physiologically acceptable excipient is injected. The physiologically acceptable excipient may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipient. For intramuscular formulations, for example, a sterile formulation of a suitable soluble salt form of the antibody may be dissolved and administered in a pharmaceutical excipient, such as water for injection, 0.9% saline, or 5% glucose solution.

[0251] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or a combination of the ingredients listed above, followed by sterilization microfiltration as required.Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle that contains a basic dispersion medium and other necessary ingredients from those listed above.In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying (lyophilization) to obtain a powder of active ingredient + any additional desired ingredients from a previously sterile-filtered solution.Prolonged absorption of injectable compositions can be achieved by including a substance that delays absorption, such as monostearate salts and gelatin, in the composition.

[0252] Prevention of the presence of microorganisms can be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, chlorobutanol, phenol, and sorbic acid. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0253] Those skilled in the art will appreciate that the formulations of the present invention may be isotonic with human blood, i.e., the formulations of the present invention have essentially the same osmolarity as human blood. Such isotonic formulations generally have an osmolarity of about 250 mOSm to about 350 mOSm. Isotonicity can be measured, for example, by a vapor pressure type or ice-freezing type osmometer. The tonicity of the formulation is adjusted using a tonicity adjuster. A "tonicity adjuster" is a pharma- ceutically acceptable inert substance that can be added to the formulation to provide isotonicity of the formulation. Tonicity adjusters suitable for the present invention include, but are not limited to, saccharides, salts, and amino acids.

[0254] The pharmaceutical composition according to the present invention may be formulated to release the active ingredient (e.g., the bifunctional molecule of the present invention) substantially immediately after administration or at any predetermined time or period after administration. In some aspects, the pharmaceutical composition may use time-release, delayed-release, and sustained-release delivery systems so that delivery of the composition occurs prior to and in sufficient time to cause sensitization of the site to be treated. Means known in the art may be used to prevent or minimize the release and absorption of the composition until it reaches the target tissue or organ, or to ensure the timed release of the composition. Such a system may avoid repeated administration of the composition, thereby increasing the convenience of the subject and the physician.

[0255] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect.

[0256] Subjects, Regimen, and Administration The present invention relates to a bifunctional molecule as disclosed herein; a nucleic acid or vector encoding same, a host cell, or a pharmaceutical composition, a nucleic acid, a vector, or a host cell, for use as a medicament, or for use in the treatment of a disease, or for administration to a subject, or for use as a medicament. Examples of treatments are described in more detail herein below under the section "Methods and Uses". The present invention also relates to the use of a pharmaceutical composition, a nucleic acid, a vector, or a host cell of the present invention, or a bifunctional molecule comprising an anti-PD1 antibody or an antibody fragment thereof and SIRPa, in the manufacture of a medicament for treating a disease in a subject. Finally, the present invention relates to a method for treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition or a bifunctional molecule comprising an anti-PD1 antibody or an antibody fragment thereof and SIRPa. Examples of treatments are described in more detail herein below in the section "Methods and Uses".

[0257] The subject to be treated may be a human, in particular a human in the prenatal stage, a newborn, a child, an infant, an adolescent, or an adult, in particular an adult at least 30, 40 years of age, preferably an adult at least 50 years of age, even more preferably an adult at least 60 years of age, and even more preferably an adult at least 70 years of age.

[0258] In particular, the subject suffers from a disease in which the PD-1 / PDL-1 pathway may be involved, in particular a disease in which at least one of the ligands of PD-1 (e.g., PDL-1 and / or PDL-2) or PD-1 is expressed, in particular overexpressed. Preferably, the subject suffers from cancer, even more preferably a PD1, PD-L1, and / or PD-L2 positive cancer, or a PD-1 positive cancer. Examples of diseases and cancers are described in more detail herein below in the "Methods and Uses" section.

[0259] In a particular embodiment, the subject has already received at least one treatment, preferably several courses of treatment, prior to administration of a bifunctional molecule according to the invention comprising an anti-PD1 antibody or an antibody fragment thereof and SIRPa or a pharmaceutical composition according to the invention.

[0260] Conventional methods known to those skilled in the art of medicine can be used to administer the bifunctional molecule or pharmaceutical composition as disclosed herein to a subject depending on the type of disease or site of disease to be treated. The composition can be administered by conventional routes, for example, orally, parenterally, enterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intratumoral, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. When administered parenterally, the pharmaceutical composition according to the present invention is preferably administered by the intravenous route of administration. When administered enterally, the pharmaceutical composition according to the present invention is preferably administered by the oral route of administration. The composition can also be administered topically.

[0261] The form of the pharmaceutical composition, the route of administration and the dosage of the pharmaceutical composition or the bifunctional molecule according to the invention can be adjusted by the person skilled in the art according to the type and severity of the infection, the age, weight, sex and general health of the patient, in particular the patient. The composition of the invention can be administered in several ways, depending on whether a local or systemic treatment is desired.

[0262] Preferably, treatment with the bifunctional molecules or with the pharmaceutical compositions according to the invention is administered periodically, preferably daily, weekly or monthly, more preferably daily to every 1, 2, 3 or 4 weeks. In a particular embodiment, treatment is administered several times a day, preferably two or three times a day.

[0263] The duration of treatment with the bifunctional molecule or with the pharmaceutical composition according to the invention is preferably comprised between 1 day and 20 weeks, more preferably between 1 day and 10 weeks, even more preferably between 1 day and 4 weeks, even more preferably between 1 day and 2 weeks, or alternatively, the treatment may be continued for as long as the disease persists.

[0264] The bifunctional molecules disclosed herein can be provided at an effective dose range of about 1 ng / kg body weight to about 30 mg / kg body weight, 1 μg / kg to about 20 mg / kg, 10 μg / kg to about 10 mg / kg, or 100 μg / kg to 5 mg / kg, optionally every 1, 2, 3, or 4 weeks, preferably by parenteral or oral administration, and particularly by intravenous or subcutaneous administration.

[0265] Typically, the bifunctional molecules disclosed herein may be provided in an effective dosage range of about 1 mg / kg body weight to about 20 mg / kg body weight, advantageously 2-10 mg / kg, particularly 3, 4, 5, 6, 7 mg / kg, which are suitable for safe administration of antibodies and are highly consistent with clinical needs.

[0266] In particular, the bifunctional molecules according to the invention may be administered in sub-therapeutic doses. The term "sub-therapeutic dose" as used herein refers to a dose below effective monotherapy dosage levels commonly used to treat a disease or a dose that is not currently typically used for effective monotherapy with anti-hPD1 antibodies.

[0267] Methods and Uses Use in the treatment of disease The bifunctional molecules, nucleic acids, vectors, host cells, compositions and methods of the invention have numerous in vitro and in vivo utilities and applications. For example, the bifunctional molecules, nucleic acids, vectors, host cells and / or pharmaceutical compositions described herein can be used as therapeutic agents, diagnostic agents and pharmaceutical research. In particular, any of the bifunctional molecules, nucleic acid molecules, groups of nucleic acid molecules, vectors, host cells or pharmaceutical compositions provided herein may be used in treatment methods and / or for therapeutic purposes. In particular, the bifunctional molecules, nucleic acids, vectors or pharmaceutical compositions provided herein may be useful for the treatment of any disease or condition, preferably any disease or condition involving PD-1, such as cancer, autoimmune diseases and other diseases associated with immune deficiencies such as infectious diseases or T-cell dysfunction. Even more preferably, the present invention relates to a method of treatment of a disease and / or disorder selected from the group consisting of cancer, infectious diseases and chronic viral infections in a subject in need thereof, comprising administering to said subject an effective amount of a bifunctional molecule or pharmaceutical composition as defined above. Examples of such diseases are described more specifically herein below.

[0268] In particular, the bifunctional molecules according to the present invention are referred to as "bifunctional checkpoint inhibitors" because they target both the PD-1 / PD-L1 / PD-L2 pathway and the SIRPa pathway.

[0269] The invention particularly relates to a bifunctional molecule, a nucleic acid, a group of nucleic acids or a vector encoding same, or a pharmaceutical composition comprising same, for use in the treatment of pathologies, diseases and / or disorders that can be prevented or treated by inhibition of binding of PD-L1 and / or PD-L2 to PD-1 and / or by inhibition of binding of CD47 to SIRPa.

[0270] Thus, disclosed herein are methods of treating diseases, particularly diseases associated with the PD-1 and / or PD-1 / PD-L1 and / or PD-1 / PD-L2 signaling pathways, comprising administering to a subject in need of treatment an effective amount of any of the bifunctional molecules or pharmaceutical compositions described herein. The physiological data of the patient (e.g., age, size, and weight) and the route of administration must also be taken into account to determine the appropriate dosage, so that a therapeutically effective amount is administered to the patient.

[0271] Additionally or alternatively, disclosed herein are methods of treating diseases, particularly diseases associated with the SIRPa / CD47 pathway, comprising administering to a subject in need of treatment an effective amount of any of the bifunctional molecules or pharmaceutical compositions described herein.

[0272] In another aspect, the bifunctional molecules disclosed herein can be administered to a subject, e.g., in vivo, to enhance immunity, preferably to treat a disorder and / or disease. Thus, in one aspect, the present invention provides a method of modifying an immune response in a subject, comprising administering to the subject a bifunctional molecule, a nucleic acid, a vector, or a pharmaceutical composition of the present invention, such that the immune response in the subject is modified. Preferably, the immune response is enhanced, augmented, stimulated, or upregulated. The bifunctional molecule or pharmaceutical composition can be used to enhance an immune response, such as T cell activation, in a subject in need of treatment. Enhancement of the immune response can result in inhibition of binding of PD-L1 and / or PD-L2 to PD-1 and / or CD47 to SIRPa, thereby reducing the immunosuppressive environment and stimulating the proliferation and / or activation of human T cells and / or IFNγ secretion by human PBMCs.

[0273] The present invention particularly provides a method of enhancing an immune response in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of a bifunctional molecule, nucleic acid, vector or pharmaceutical composition comprising same as described herein, such that the immune response in the subject is enhanced.

[0274] In some embodiments, the amount of a bifunctional molecule described herein is effective to inhibit PD-1 signaling (e.g., to reduce PD-1 signaling by at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% compared to a control). In other embodiments, the amount of a bifunctional molecule described herein is effective to activate an immune response (e.g., by at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% compared to a control).

[0275] In some embodiments, the amount of a bifunctional molecule described herein is effective to inhibit binding of human PD-L1 and / or PD-L2 to human PD-1, e.g., inhibits binding by at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% compared to a control.

[0276] In some embodiments, the amount of a bifunctional molecule described herein is sufficient to have antagonist activity of human PD-L1 and / or PD-L2 binding to human PD-1, e.g., inhibiting binding by at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% compared to a control.

[0277] In some embodiments, the amount of a bifunctional molecule described herein is effective in suppressing SIRPa / CD47 signaling (e.g., reducing SIRPa / CD47 signaling by at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% compared to a control). In other embodiments, the amount of a bifunctional molecule described herein is effective in activating an immune response (e.g., by at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% compared to a control).

[0278] In some embodiments, the amount of bifunctional molecule described herein is effective in inhibiting binding of CD47 expressed by tumor cells to human SIRPa expressed by APCs, e.g., inhibiting binding by at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% compared to control conditions (i.e., not including a bifunctional molecule of the invention).

[0279] In some embodiments, the amount of a bifunctional molecule described herein is sufficient to have at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% inhibition of competitive activity for binding of CD47, particularly competition with human immune cells expressing SIRPa, such as macrophages, e.g., binding between CD47 and SIRPa positive cells, compared to control conditions (i.e., without the bifunctional molecule of the invention).

[0280] The present invention also relates to the bifunctional molecules described herein; the nucleic acids or vectors encoding same, or pharmaceutical compositions comprising same, for use in the treatment of disorders and / or diseases in a subject and / or for use as a medicament or vaccine. The present invention relates to the use of the bifunctional molecules described herein; the nucleic acids or vectors encoding same, or pharmaceutical compositions comprising same, in the manufacture of a medicament for treating a disease and / or disorder in a subject. Finally, it relates to a method of treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition or a bifunctional molecule according to the present invention.

[0281] Disclosed herein are methods of treating a patient having a disease and / or disorder, the methods comprising: (a) identifying a patient in need of treatment; and (b) administering to the patient a therapeutically effective amount of any of the bifunctional molecules, nucleic acids, vectors, or pharmaceutical compositions described herein.

[0282] A subject in need of treatment may be a human who is at risk for, or suspected of having, a disease associated with a signaling pathway mediated by PD-1. Such patients can be identified by routine medical examination. For example, subjects suitable for treatment can be identified by determining whether such subjects have PD-1, PD-L1 and / or PD-L2 positive cells. Preferably, by "PD-L1 positive tumor cells" or "PD-L2 positive tumor cells" it is intended to refer to a population of tumor cells in which PD-L1 or PD-L2, respectively, is expressed on at least 10% of the tumor cells, preferably at least 20, 30, 40 or 50% of the tumor cells.

[0283] In one embodiment, the subject in need of treatment is a patient having, suspected of having, or at risk of having a disease, preferably a PD-1, PDL1 and / or PDL2 positive disease, even more preferably a disease in which PD-1 and / or at least one ligand of PD-1 is overexpressed. Disruption of PD-1 / PD-L1 and / or PD-1 / PD-L2 interaction in such a subject by virtue of administration of a bifunctional molecule or pharmaceutical composition according to the present invention may enhance the immune response of the subject. In some embodiments, any of the humanized anti-PD-1 antibodies or pharmaceutical compositions described herein can be used to treat PD-1 positive cells.

[0284] - cancer It is known in the art that blockade of PD-1 with an antibody can enhance the immune response to cancer cells in a patient. Thus, in one aspect, the invention provides a bifunctional molecule or pharmaceutical composition for use in treating a subject with cancer comprising administering to an individual an effective amount of a bifunctional molecule or pharmaceutical composition capable of activating exhausted T cells, preferably capable of disrupting or inhibiting PD1 / PD-L1 and / or PD1 / PD-L2 interaction, preferably capable of at least partially preventing, disrupting or inhibiting naturally occurring CD47 / SIRPa interaction in the subject.

[0285] In one embodiment, the subject in need of treatment is a patient having, suspected of having, or at risk of having a disease, preferably a PD-1 positive cancer, even more preferably a cancer in which PD-1 is expressed or overexpressed. In some embodiments, any of the anti-PD-1 antibodies or pharmaceutical compositions described herein can be used to treat PD-1 positive tumor cells. For example, patients suitable for treatment can be identified by determining whether such patients have PD-1 positive tumor cells.

[0286] In another embodiment, the subject is a patient having, suspected of having, or at risk of developing cancer, preferably a PD-L1 and / or PD-L2 positive cancer. In some embodiments, any of the bifunctional molecules or pharmaceutical compositions described herein can be used to treat PD-L1 and / or PD-L2 positive tumors. For example, human patients suitable for treatment can be identified by determining whether such patients have PD-L1 and / or PD-L2 positive cancer cells.

[0287] In another embodiment, the subject is a patient who has, is suspected of having, or is at risk of developing cancer, preferably a CD47-positive cancer. In some embodiments, any of the bifunctional molecules or pharmaceutical compositions described herein can be used to treat CD47-positive tumors. For example, human patients suitable for treatment can be identified by determining whether such patients have CD47-positive cancer cells.

[0288] In a further aspect, there is provided a bifunctional molecule or pharmaceutical composition for use in treating cancer, preferably a PD-1, CD47, PD-L1 and / or PD-L2 positive cancer, even more preferably a cancer in which PD-1, CD47, PD-L1 and / or PD-L2 is overexpressed.

[0289] In another embodiment, the present invention provides the use of a bifunctional molecule or pharmaceutical composition disclosed herein in the manufacture of a medicament for treating cancer, e.g., for inhibiting the growth of tumor cells, preferably PD-1, CD47, PD-L1, PD-L2 positive tumor cells, in a subject.

[0290] In an embodiment of the disclosure, the cancer to be treated is associated with exhausted T cells.

[0291] Thus, in one embodiment, the invention provides a method of treating cancer in a subject, e.g., inhibiting the growth of tumor cells, comprising administering to the subject a therapeutically effective amount of a bifunctional molecule or pharmaceutical composition according to the invention. In particular, the invention relates to the treatment of a subject with a bifunctional molecule, such that the growth of cancerous cells is inhibited.

[0292] Any suitable cancer that can be treated with the bifunctional molecules provided herein can be a hematopoietic cancer or a solid cancer. Such cancers include carcinoma, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, head and neck cancer, renal cancer, liver cancer, lung cancer, lymphoma, glioma, mesothelioma, melanoma, gastric cancer, urethral cancer, environmentally induced cancer, and any combination of said cancers. The present invention is also useful for treating metastatic cancers, particularly metastatic cancers that express PD-L1 (Iwai et al., (2005), Int. Immunol.17:133-144). In addition, the present invention induces refractory or recurrent malignancies.

[0293] In certain aspects, the cancer is a hematological or solid tumor with high expression of PD-1 and / or PD-L1. Such cancer may be selected from the group consisting of hematological and lymphoid neoplasms, angioimmunoblastic T-cell lymphoma, myelodysplastic syndrome, and acute myeloid leukemia.

[0294] In certain embodiments, the cancer is a virus-induced cancer or a cancer associated with immune deficiency. Such cancers can be selected from the group consisting of Kaposi's sarcoma (e.g., associated with Kaposi's sarcoma herpesvirus); squamous cell carcinoma of the cervix, anus, penis and vulva and oropharyngeal carcinoma (e.g., associated with human papillomavirus); B-cell non-Hodgkin's lymphoma (NHL), including diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmablastic lymphoma, primary central nervous system lymphoma, HHV-8 primary effusion lymphoma, classical Hodgkin's lymphoma, and lymphoproliferative disorders (e.g., associated with Epstein-Barr virus (EBV) and / or Kaposi's sarcoma herpesvirus); hepatocellular carcinoma (e.g., associated with hepatitis B and / or C virus); Merkel cell carcinoma (e.g., associated with Merkel cell polyomavirus (MPV)); and cancer associated with human immunodeficiency virus infection (HIV) infection.

[0295] Preferably, the cancer to be treated or prevented is selected from the group consisting of metastatic or non-metastatic melanoma, malignant mesothelioma, non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, small cell lung cancer metastatic Merkel cell carcinoma, gastric or gastroesophageal cancer and cervical cancer.

[0296] Preferred cancers for treatment typically include cancers that are responsive to immunotherapy. Alternatively, preferred cancers for treatment are cancers that are not responsive to immunotherapy.

[0297] For purposes of illustration, and without wishing to be bound by theory, treatment with anti-cancer antibodies or anti-cancer immunoconjugates or other current anti-cancer therapies that lead to cancer cell death enhances the immune response mediated by PD-1. Thus, treatment of hyperproliferative diseases (e.g., cancer tumors) may include bifunctional molecules combined with anti-cancer treatments, or any combination thereof, either simultaneously or sequentially, that may enhance the anti-tumor immune response by the host. Preferably, the bifunctional molecules may be used in combination with other immunogenic agents, standard cancer treatments, or other antibodies as described herein below.

[0298] - infectious diseases The bifunctional molecules, nucleic acids, groups of nucleic acids, vectors, host cells or pharmaceutical compositions of the invention are used to treat patients exposed to a particular toxin or pathogen.Thus, an aspect of the invention preferably provides a method of treating an infectious disease in a subject comprising administering to the subject a bifunctional molecule according to the invention, or a pharmaceutical composition comprising same, such that the subject is treated for the infectious disease.

[0299] Any suitable infection may be treated with the bifunctional molecules, nucleic acids, groups of nucleic acids, vectors, host cells or pharmaceutical compositions provided herein.

[0300] Some examples of pathogenic viruses causing infectious diseases treatable by the methods of the present invention include HIV, hepatitis (A, B, or C), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

[0301] In particular, the bifunctional molecules or pharmaceutical compositions of the invention are used to treat patients with chronic viral infections, such as retroviruses, anelloviruses, circoviruses, herpes viruses, varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), polyomavirus BK, polyomavirus, adeno-associated virus (AAV), herpes simplex type 1 (HSV-1), adenovirus, herpes simplex type 2 (HSV -2), Kaposi's sarcoma herpesvirus (KSHV), hepatitis B virus (HBV), GB virus C, papillomavirus, hepatitis C virus (HCV), human immunodeficiency virus (HIV), hepatitis D virus (HDV), human T-cell leukemia virus type 1 (HTLV1), xenotropic murine leukemia virus-related virus (XMLV), rubella virus, rubella, parvovirus B19, measles virus, and coxsackievirus.

[0302] Some examples of pathogenic bacteria causing infections treatable by the methods of the present invention include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacillus, cholera, tetanus, botulinum, anthrax, plague, leptospira, and lyme disease bacteria.

[0303] Some examples of pathogenic fungi that cause infections treatable by the methods of the present invention include Candida (such as albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (such as fumigatus and niger), Mucor (such as mucor, absidia, and rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and the like. immitis and Histoplasma capsulatum.

[0304] Some examples of pathogenic parasites that cause infections treatable by the methods of the present invention include Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba spp., Giardia lambia, Cryptosporidium spp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and the like. gondi, and Nippostrongylus brasiliensis.

[0305] In all of the above methods, the bifunctional molecules can be combined with other forms of immunotherapy, such as cytokine treatment (e.g., interferon, GM-CSF, G-CSF, IL-2), or any therapy that results in enhanced presentation of tumor antigens.

[0306] Combination therapy In particular, the bifunctional molecules of the invention can be combined with several other potential strategies to overcome immune evasion mechanisms using agents in clinical development or already on the market (see Table 1 in Antonia et al., Immuno-oncology combinations: a review of clinical experience and future prospects. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 20, 6258-6268, 2014). Such combinations with the bifunctional molecules according to the invention can be: 1-Reversing inhibition of adaptive immunity (blocking T cell checkpoint pathways); 2- Switching adaptive immunity (promoting T cell costimulatory receptor signaling using agonist molecules, particularly antibodies); 3- Improving the function of innate immune cells; 4- Activating the immune system (boosting immune-cell effector functions), for example through vaccine-based strategies; This can be clearly useful.

[0307] Thus, also provided herein is a combination therapy for any of the diseases associated with PD-1 signaling and / or SIRPa signaling described herein, using any of the bifunctional molecules described herein or pharmaceutical compositions comprising the same and a suitable second agent. In an embodiment, the bifunctional molecule and the second agent can be present in a pharmaceutical composition described above. Alternatively, as used herein, the term "combination therapy" or "combination therapy" encompasses the administration of these two agents (e.g., a bifunctional molecule described herein and an additional or second suitable therapeutic agent) in a sequential manner, i.e., each therapeutic agent is administered at a different time, and the administration of at least two of the therapeutic agents, or agents, is in a substantially simultaneous manner. The sequential or substantially simultaneous administration of each agent can be effected by any suitable route. The agents can be administered by the same route or by different routes. For example, the first agent (e.g., the bifunctional molecule) can be administered orally, and the additional therapeutic agent (e.g., an anti-cancer agent, an anti-infective agent; or an immune modulator) can be administered intravenously. Alternatively, selected combination agents may be administered intravenously while other agents of the combination are administered orally.

[0308] In another aspect, the present invention relates to a therapeutic means, in particular a combination product means, which comprises as active ingredients a bifunctional molecule as defined above and an additional therapeutic agent, said active ingredients being formulated for separate, sequential or combined therapy, in particular combined or sequential use.

[0309] As used herein, the term "sequential" is characterized by a normal sequence or order unless otherwise specified; for example, if a dosing regimen includes administration of a bifunctional molecule and a second substance, the sequential dosing regimen may include administration of the bifunctional molecule of the invention before, simultaneously, substantially simultaneously, or after administration of the second substance, but both substances are administered in the normal sequence or order. The term "separate" means that one is kept separate from the other, unless otherwise specified. The term "concurrently" means that, unless otherwise specified, occurs or takes place at the same time, i.e., the substances of the invention are administered at the same time. The term "substantially simultaneously" means that the substances are administered within minutes of each other (e.g., within 15 minutes of each other), and is intended to include joint administration as well as sequential administration, but when administration is sequential, it is separated in time by a short period of time (e.g., the time it takes a physician to administer the two compounds separately).

[0310] It should be considered that any combination described herein can be used in any sequence to treat the disorders or diseases described herein. The combinations described herein may be selected based on a number of factors, including, but not limited to, effectiveness in inhibiting or preventing the progression of the target disease, effectiveness in reducing the side effects of another substance in the combination, or effectiveness in reducing symptoms associated with the target disease. For example, the combination therapy described herein may reduce any of the side effects associated with each individual member of the combination.

[0311] The present invention also relates to a method of treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of a bifunctional molecule or pharmaceutical composition described herein and a therapeutically effective amount of an additional or second therapeutic agent.

[0312] When the bifunctional molecules or pharmaceutical compositions described herein are used contemporaneously with an additional therapeutic agent, a sub-therapeutic dosage of either the bifunctional molecule or pharmaceutical composition of the second agent, or a sub-therapeutic dosage of both, can be used in treating a subject, preferably a subject having, or at risk of developing, a disease or disorder associated with cell signaling mediated by PD-1 and / or SIRPa.

[0313] Specific examples of additional or second therapeutic agents are provided in WO 2018 / 053106, pages 36-43.

[0314] In embodiments, the additional or second therapeutic agent is an alkylating agent, angiogenesis inhibitor, an antibody, an antimetabolite, an antimitotic, an antiproliferative agent, an antiviral agent, an Aurora kinase inhibitor, a proapoptotic agent (e.g., a Bcl-2 family inhibitor), an activator of the death receptor pathway, a Bcr-Abl kinase inhibitor, a BiTE (bispecific T cell engager) antibody, an antibody drug conjugate, a biological response modifier, a Bruton's tyrosine kinase (BTK) inhibitor, a cyclin-dependent kinase inhibitor, a cell cycle inhibitor, a cyclooxygenase-2 inhibitor, a DVD, an leukemia viral oncogene homolog (ErbB2) receptor inhibitor, a growth factor inhibitor, a heat shock protein (HSP)-90 inhibitor, a histone deacetylase (HDAC) inhibitor, a hormone therapy, an immunological agent, an inhibitor of inhibitor of apoptosis proteins (IAP), an intercalating antibiotic, a kinase inhibitor, a kinesin inhibitor, a cytotoxic T cell ... inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, nonsteroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutic agents, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoid / deltoid plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, peptide vaccines, etc., epitopes or neoepitopes derived from tumor antigens, as well as combinations of one or more of these substances.

[0315] For example, the additional therapeutic agent can be selected from the group consisting of chemotherapy, radiation therapy, targeted therapy, anti-angiogenic agents, hypomethylating agents, cancer vaccines, epitopes or neoepitopes derived from tumor antigens, myeloid checkpoint inhibitors, other immunotherapies, and HDAC inhibitors.

[0316] In a preferred embodiment, the second therapeutic agent is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, cell therapy (e.g., CAR-T cells), antibiotics and probiotics. The immunotherapy agent may be an antibody targeting a tumor antigen, in particular an antibody targeting a tumor antigen selected from the group consisting of anti-Her2, anti-EGFR, anti-CD20, anti-CD19, and anti-CD52.

[0317] In an embodiment, the invention relates to a combination therapy as defined above, wherein the second therapeutic agent is selected from the group consisting of therapeutic vaccines, immune checkpoint blockers or activators, in particular of adaptive immune cells (T and B lymphocytes) and antibody drug conjugates. Preferably, suitable substances for co-use with any of the anti-hPD-1 antibodies or fragments thereof or pharmaceutical compositions according to the invention include antibodies that bind to co-stimulatory receptors (e.g. OX40, CD40, ICOS, CD27, HVEM or GITR), substances that induce immunogenic cell death (e.g. chemotherapeutic agents, radiotherapeutic agents, antiangiogenic agents or substances for targeted therapy), substances that inhibit checkpoint molecules (e.g. CTLA4, LAG3, TIM3, B7H3, B7H4, BTLA or TIGIT), cancer vaccines, substances that modify immunosuppressive enzymes (e.g. IDO1 or iNOS), substances that target Treg cells, substances for adoptive cell therapy or substances that modulate myeloid cells.

[0318] In an embodiment, the invention relates to a combination therapy as defined above, wherein the second therapeutic agent is an immune checkpoint blocker or activator of adaptive immune cells (T and B lymphocytes) selected from the group consisting of anti-CTLA4, anti-CD2, anti-CD28, anti-CD40, anti-HVEM, anti-BTLA, anti-CD160, anti-TIGIT, anti-TIM-1 / 3, anti-LAG-3, anti-2B4, and anti-OX40, anti-CD40 agonists, CD40-L, TLR agonists, anti-ICOS, ICOS-L and B-cell receptor agonists.

[0319] In one embodiment, the second therapeutic agent is an antibody that targets a tumor antigen, in particular an antibody that targets a tumor antigen selected from the group consisting of anti-Her2, anti-EGFR, anti-CD20, anti-CD19, and anti-CD52.

[0320] Combination therapy may also rely on a combination of surgery, chemotherapy (such as, for example, docetaxel or dacarbazine), radiation therapy, immunotherapy (such as, for example, antibodies targeting CD40, CTLA-4), gene targeting and modulation, and / or administration of the bifunctional molecule with other agents such as immune-modulators, angiogenesis inhibitors, and any combination thereof.

[0321] kit Any of the bifunctional molecules or compositions described herein may be included in a kit provided by the present invention. The present disclosure provides kits for use in enhancing immune responses and / or treating diseases associated with D-1 signaling, SIRPa / CD47 signaling (e.g., cancer and / or infectious diseases).

[0322] In the context of the present invention, the term "kit" refers to two or more components (one of which corresponds to a bifunctional molecule, a nucleic acid molecule, a vector or a cell of the present invention) packaged in a container, a recipient or vice versa. A kit can be described herein as a set of products and / or equipment sufficient to accomplish a particular goal that can be marketed as a single unit.

[0323] Specifically, the kit according to the present invention comprises: - a bifunctional molecule as defined above, - an anti-hPD1 antibody or an antigen-binding fragment thereof bound to SIRPa or a variant thereof; - a nucleic acid molecule or a group of nucleic acid molecules encoding said bifunctional molecule, - a vector comprising said nucleic acid molecule or group of nucleic acid molecules, and / or - a cell containing said vector or nucleic acid molecule or group of nucleic acid molecules may include:

[0324] Thus, the kit may comprise, in suitable container means, the pharmaceutical composition of the invention, and / or the bifunctional molecule, and / or the host cell, and / or the vector encoding the nucleic acid molecule of the invention and / or the nucleic acid molecule of the invention or associated reagents. In some embodiments, means for obtaining a sample from an individual and / or means for assaying the sample may be provided. In certain embodiments, the kit comprises cells, buffers, cell culture media, vectors, primers, restriction enzymes, salts, etc. The kit may also comprise sterile pharmaeutically acceptable buffers and / or other diluents.

[0325] The container may be a unit dose, bulk package (e.g., a multi-dose package) or a sub-unit dose. In an embodiment, the present invention relates to a kit as defined above for a single-administration dose unit. The kit of the present invention may also include a first recipient comprising the dried / lyophilized bifunctional molecule and a second recipient comprising the aqueous formulation. In certain embodiments of the present invention, kits are provided that contain single-chamber and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes).

[0326] The kit of the present invention is in a suitable package. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packages (e.g., sealed Mylar or plastic bags), and the like. Packages for use in combination with specific devices, such as inhalers, nasal administration devices (e.g., nebulizers), or infusion devices, such as mini-pumps, are also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active substance in the composition is a bifunctional molecule as described herein, comprising an anti-hPD1 antibody bound to SIRPa or a variant thereof.

[0327] The compositions included in the kits according to the present invention may also be formulated into compositions compatible with a syringe. In this case, the container means itself may be a syringe, pipette, and / or other such device from which the formulation may be applied to an infected area of ​​the body and / or applied and / or mixed with other components of the kit. Alternatively, the components of the kit may be provided as a dry powder. When reagents and / or components are provided as a dry powder, the soluble composition can be reconstituted by the addition of a suitable solvent. It is envisaged that the solvent may also be provided in another container means and may be suitable for administration.

[0328] In some embodiments, the kit further comprises an additional substance for cancer or infectious disease, which may be combined with the bifunctional molecule or other components of the kit of the present invention, or may be provided separately in the kit. In particular, the kit described herein may comprise one or more additional therapeutic agents, such as those described in "combination therapy" herein above. The kit may be tailored to a particular cancer for an individual, and may comprise a respective second cancer therapy for the individual, as described herein above.

[0329] Instructions for use of the bifunctional molecules or pharmaceutical compositions described herein generally include information regarding dosages, administration schedules, routes of administration for the intended treatment, means for reconstituting the bifunctional molecules and / or means for diluting the bifunctional molecules of the invention. Instructions provided in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit in the form of a leaflet or instruction manual). In some embodiments, the kits can include instructions for use according to any of the methods described herein. The included instructions can include instructions for administration of a pharmaceutical composition comprising the bifunctional molecules to enhance an immune response and / or to treat a disease described herein. The kits may further include instructions for selection of an individual suitable for treatment based on identification of whether the individual has a disease associated with PD-1 signaling, such as those described herein. EXAMPLES

[0330] The following figures and examples are provided to provide those skilled in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their invention, and are not intended to represent that the following experiments are all or the only experiments performed. While the invention has been described in terms of specific embodiments thereof, it should be understood by those skilled in the art that various modifications may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, subject composition, method, method step or steps to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

[0331] Bifunctional molecule (Bicki) The bifunctional molecule comprises an anti-PD-1 antibody and human SIRPa, where the protein is covalently linked to the polypeptide chain of the anti-PD-1 antibody, either to the light chain (anti-PD1VL-SIRPa antibody) or the heavy chain (anti-PD1VH-SIRPa antibody) of the antibody.

[0332] Example 1: Effect of the bifunctional molecule anti-PD1-SIRPa on binding to PD1 and its antagonistic potential on PD1-PDL1 interaction The binding ability of Bicki molecules to PD1 recombinant molecules was evaluated, and the inhibitory efficacy of Bicki molecules against PD1-PDL1 interaction was performed by ELISA. The results are shown in Figures 1 and 2. Bicki anti-PD1-Sirpa molecules, in which SIRPa is fused to the heavy or light chain of an antibody, do not change the binding to PD1. Bicki anti-PD1-Sirpa molecules are still capable of inhibiting PD1-PDL1 interaction compared to anti-PD1 antibody alone. No significant difference was observed between Bicki molecules fused to the heavy or light chain of an antibody.

[0333] These data were confirmed by surface plasmon resonance experiments (Biacore assays), where anti-PD-1 alone or anti-human Fc antibodies on a sensor chip to capture the bifunctional molecule. Different concentrations of PD-1 recombinant protein (6,25-100 nM) were then added and the affinity was measured. Anti-PD-1 alone shows a similar high affinity to PD-1 with a KD of 3,46 nM compared to the anti-PD1-Sirpa bifunctional molecule (3,83 nM).

[0334] Example 2: Binding of Bicki anti-PD1-Sirpa molecules to CD47 The binding ability of Bicki anti-PD1-Sirpa molecules to CD47 (SIRPa ligand) was evaluated by ELISA. The results shown in Figure 3 show that Bicki anti-PD1-Sirpa molecules preserved their binding ability to SIRPa ligand, namely CD47. Surprisingly, a higher efficacy was observed for Bicki anti-PD1VH-Sirpa molecules compared to Bicki anti-PD1VL-Sirpa molecules.

[0335] Example 3: Binding of the molecule BiCKI SIRPa on T cells expressing both the receptors CD47 and PD1 The ability of BiCKI SIRPa to target T cells by binding to both CD47 and PD-1 proteins on the same cells was evaluated. Jurkat cells expressing only the CD47 receptor or Jurkat cells co-expressing PD-1 and CD47 proteins were incubated with BiCKI SIRPa or SIRPa-Fc molecules. Binding was revealed with anti-human IgG Fc-PE. Figure 4 confirms the mechanism of BiCKI SIRPa acting on the same T cells, as the molecule binds with 2-fold higher efficacy to cells expressing CD47+PD-1+ compared to cells expressing CD47 only. These experiments show that the bifunctional Bicki SIRPa molecule is designed to preferentially target CD47+PD-1+ exhausted T cells over other CD47+ cells.

[0336] Example 4: In vitro and ex vovo efficacy of Bicki anti-PD1-Sirpa molecules on PBMC and T cell proliferation and activation An in vitro bioassay was performed to measure T cell activation, comparing Bicki anti-PD1-Sirpa molecule with anti-PD-1 alone. First, the expression of PD1 and CD47 was measured by FACS on the T cell line used in the bioassay. Figure 5A shows good expression of both PD1 and CD47 molecules on the cell surface. Unexpectedly, in Figure 5B, we observed that Bicki anti-PD1-Sirpa molecule (EC50=0.6nM) induces better NFAT TCR-mediated activation than anti-PD1 antibody alone (EC50=5nM) (Figure 5B). In a surprising manner, we also observed that Bicki SIRPa is more efficient in activating NFAT signaling compared to the combination anti-PD-1+isotype SIRPa, indicating that the fusion of anti-PD-1 to SIRPa in Bicki molecule achieves a synergistic effect of activating T cells. This effect requires activation of CD47-mediated signaling, since the use of a CD47 blocking antibody (clone B6H12) completely abolished the synergistic effect of BiCKI SIRPa (Figure 5B). By targeting the PD-1 receptor, the anti-PD-1 domain of the Bicki molecule allows cross-linking of SIRPa and subsequent clustering of CD47 molecules on T cells. CD47-mediated signaling enhances the anti-PD-1 effect, which results in better T cell activation. As shown in Figure 5D, a similar synergistic effect was observed with Bicki SIRPa molecules constructed with IgG1 N298A or IgG4 S228P isotypes.

[0337] We constructed Bicki SIRPa molecules with other anti-PD-1 scaffolds (pembrolizumab or nivolumab). Figure 5E shows that these Bicki molecules have similar synergistic effects compared to anti-PD-1 alone, suggesting that the present invention may be suitable for other anti-PD-1 scaffolds. Bioassays were also performed with Bicki anti-PD1 fused to another type 1 protein on the heavy chain. Part F of Figure 5 shows that there is almost no difference between control anti-PD1 and Bicki anti-PD1 VH-type I protein on NFAT activation, indicating that the observed enhancement effect is specific to Bicki SIRPa molecules and is not applicable to any Bicki molecule.

[0338] In another bioassay, we evaluated the effectiveness of Bicki SIRPa molecules to stimulate calcium signals in T cells, another essential mediator of the activation of T cell effector functions. Figures 6A and 6B show that Bicki SIRPa molecules enhance the calcium signals induced by aCD3 stimulation to a similar extent as CD28 stimulation. Interestingly, Bicki SIRPa molecules alone have no effect (Figure 6A), suggesting that Bicki SIRPa molecules act as a costimulatory signal and simply promote the activation of TCR-induced T cells.

[0339] Taken together, these results show that bifunctional molecules with Sirpa fused to anti-PD1 antibodies enhance anti-PD1 effects, strongly suggesting that SIRPa binding on CD47 not only blocks the inhibitory phagocyte signal "don't eat me" but also promotes CD47-dependent T cell costimulation. In vitro assays were performed on human PBMCs and T cells to study the efficacy of the Bicki anti-PD1-Sirpa molecule on proliferation and activation. The results are shown in Figures 7 and 8. These results clearly and surprisingly show that the Bicki anti-PD1-Sirpa molecule increases human PBMC proliferation and activation as reflected by IFNg secretion, whereas no enhancement occurs when anti-PD1 alone or in combination with a separate recombinant human SIRPa protein is used, thereby confirming the synergistic effect of fusion of SIRPa on anti-PD1 to the Bicki molecule to increase T cell proliferation and activation. Results in human T cells confirmed that Bicki anti-PD1-Sirpa molecules enhanced T cell proliferation and activated T cells better than anti-PD1 alone. Notably, the amount of IFNg secretion induced by Bicki anti-PD1-Sirpa molecules (a key determinant observed to predict anti-PD1 efficacy in various clinical trials) was significantly higher (>10,000 pg / ml) compared to anti-PD1 alone (<500 pg / ml).

[0340] Figure 9 confirms the strong synergistic effect of stimulating the proliferation of exhausted human T cells after chronic antigen stimulation. Indeed, SIRPa recombinant protein or anti-PD-1 alone does not induce T cell proliferation compared to isotype control, while surprisingly, Bicki anti-PD1-Sirpa molecule strongly stimulates the proliferation of exhausted T cells. This difference highlights the advantage of bifunctional antibodies over combination therapy using two separate molecules. The Bicki anti-PD1-Sirpa molecule of the present invention binds the molecule to PD1+ cell clustering SIRPa molecule, thereby stimulating CD47 signaling to T cells and proliferation of T cells.

[0341] Example 5: Bicki anti-PD1-Sirpa molecule enhances T cell migration to tumors T cell migration was investigated using a 3D tumor spheroid-based assay. Tumor spheroids were generated by co-culturing A549 tumor cells with fibroblasts and monocytes to mimic the complexity of the solid tumor microenvironment. Human T cells were added to the wells, and T cell migration into the tumor spheroids was assessed by immunofluorescence and confocal microscopy analysis. Figure 10 shows that treatment with BiCKI SIRPa molecules enhances the number of T cells / tumor cells in the tumor compared to isotype control treatment. The lack of T cells in the tumor microenvironment is one of the major resistance mechanisms associated with anti-PD-1 monotherapy. These data suggest that BiCKi SIRPa molecules can overcome this resistance by enhancing T cell migration into the tumor microenvironment.

[0342] Example 6: Pharmacokinetic properties of Bicki anti-PD1 SIRPa molecules in vivo To analyze the pharmacokinetics of Bicki molecules, BalbcRJ mice (female, 6-9 weeks old) were treated intraorbitally with a single dose of BiCKi SIRPa molecules. Bicki molecule concentrations in plasma were assessed by ELISA using immobilized anti-human light chain antibody (clone NaM76-5F3) and diluted serum containing anti-PD-1 antibodies. Detection was performed with peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch, Inc.; USA; ref. 709-035-149) and revealed by conventional methods.

[0343] In this assay, two different Bicki SIRPa molecules were tested and compared: (1) BiCKI SIRPa molecule with IgG1 N298A isotype and GGGGS linker between Fc and SIRPa domains, (2) BiCKI SIRPa molecule with IgG4 S228P and GGGSGGGGSGGGGGS linker. Linear pharmacokinetic profile is observed for both molecules with similar absorption phase (Figure 11). Cmax of about 200 nM was obtained for both constructs 15 min after injection. Surprisingly, however, biCKI SIRPa molecule with GGGS and IgG1 isotype showed a lower elimination / distribution phase compared to BiCKI SIRPa molecule constructed with IgG4 backbone and long linker. These data suggest that using IgG1 N298A with short linker for Bicki SIRPa constructs may prolong drug exposure in vivo and subsequently enhance the therapeutic efficacy of the drug.

[0344] Materials and Methods PD1 binding ELISA and bridging ELISA assays For PD-1 binding ELISA assay, recombinant hPD1 (Sino Biologicals, Beijing, China; ref. 10377-H08H) was immobilized on plastic at 0.5 μg / ml in carbonate buffer (pH 9.2) and purified antibody was added to measure binding. After incubation and washing, peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch; USA; ref. 709-035-149) was added and revealed by conventional methods.

[0345] For the bridging ELISA assay, a similar method was used. Recombinant hPD1 was immobilized and purified bifunctional antibodies were added in serial dilutions. After incubation and washing, CD47fc recombinant protein (Sino Biologicals, ref. 12283-H02H) was then added at 1 μg / mL. Detection was performed using anti-CD47 specific mouse antibody (clone B6H12) and peroxidase-labeled donkey anti-mouse IgG antibody (ref. 715-036-151). Revelation was performed using conventional methods.

[0346] ELISA antagonists: Competition between PDL1 and humanized anti-PD1 A competitive ELISA assay was performed with the PD-1:PD-L1 inhibitor screening ELISA assay pair (AcroBiosystem; USA; ref. EP-101). In this assay, recombinant hPDL1 was immobilized on plastic at 2 μg / ml in PBS pH 7.4 buffer. Purified antibodies (at different concentrations) were mixed with biotinylated human PD1 (AcroBiosystem; USA; ref. EP-101) at a final (fixed) concentration of 0.66 μg / ml and competitive binding was measured for 2 h at 37°C. After incubation and washing, peroxidase-labeled streptavidin (Vector laboratoring; USA; ref. SA-5004) was added to detect biotin-CD47Fc binding and revealed by conventional methods.

[0347] IFN-gamma secretion and T cell proliferation assays Peripheral blood mononuclear cells or purified T cells isolated from healthy donors were used for the experiments.

[0348] For experiments using naive PBMCs, PBMCs were incubated with isotype control (B12G4M), anti-PD-1, anti-PD-1+rSIRPa (Sino Biologicals, ref. 11612-H08H), BiCKI anti-PD-1 VH SIRPa or BiCKI anti-PD-1 VH SIRPa on plates coated with anti-CD3+ / -CD28 (clone OKT3 and CD28.2, 3 μg / mL). IFNg was quantified by ELISA in the supernatants collected on day 2 (human IFNg ELISA set, BD Bioscience, USA, ref. 555142). Proliferation was assessed on day 6 by H3 thymidine incorporation. T cells were stimulated with anti-CD3 (clone OKT3, 3 μg / mL, with or without anti-CD28 (clone CD28.2, 3 μg / mL).

[0349] For experiments using activated T cells, T cells were first stimulated on anti-CD3 / CD28 coated plates (3 μg / mL each). 24 hours after stimulation, T cells were harvested, counted, and restimulated on anti-CD3 (clone OKT3, 2 μg / mL) + recombinant human PD-L1 (Sinobiological, ref. 10084-H02H, 5 μg / mL) in the presence of isotype control, anti-PD-1, or BiCKI VH SIRPa and BiCKI VL SIRPa (10 μg / mL). On day 6, proliferation was quantified by H3 thymidine incorporation and supernatants were harvested to quantitate IFNg secretion.

[0350] For experiments using chronically stimulated PBMCs, human PBMCs were repeatedly stimulated every 3 days on CD3 CD28-coated plates (3 μg / mL OKT3 and 3 μg / mL CD28.2 antibody). After the third stimulation, T cells were incubated with isotype control or anti-PD-1, recombinant SIRPa protein or BiCKI anti-PD-1 VH SIRPa (5 μg / mL). H3 uptake assays were performed on day 5 to determine T cell proliferation.

[0351] T cell activation assay using Promega cell-based bioassays The ability of anti-PD-1 antibodies to restore T cell activation was tested using the Promega PD-1 / PD-L1 kit (reference J1250). Two cell lines were used: (1) effector T cells (Jurkat cells stably expressing PD-1, NFAT-inducible luciferase) and (2) activated target cells (CHO K1 cells stably expressing PDL1 and a surface protein designed to stimulate the cognate TCR in an antigen-independent manner). When cells are co-cultured, PD-L1 / PD-1 interaction inhibits TCR-mediated activation, thereby blocking NFAT activation and luciferase activity. Addition of anti-PD-1 antibodies blocks the PD-1-mediated inhibitory signal, which leads to NFAT activation and luciferase synthesis and release of a bioluminescent signal. The experiment was performed according to the manufacturer's recommendations. Serial dilutions of PD-1 antibodies were tested. After 4 hours of co-culture of PD-L1+ target cells, PD-1 effector cells and anti-PD-1 antibodies, BioGlo™ luciferin substrate was added to the wells and plates were read using a Tecan™ luminometer.

[0352] Calcium mobilization assay CD47+PD1+ Jurkat cells were stained with Fura Red (Thermofisher, #F3021, 5 μM) for 30 min at 37°C in HBSS medium, then washed twice with HBSS medium supplemented with HEPES (10 mM), BSA 1% and CaCl2 (1 mM) and resuspended in the same medium. After 1 min acquisition on the LSR FACs to set the fluorescence background, Bicki anti-PD1-SIRPa antibody alone (45 nM 10 μg / mL) or mixed with CD3 antibody (clone OKT3, 10 × 10 μg / mL) was added to the cells. The ratio BV711 / PercP5.5 MFI was calculated for every second of acquisition and referenced to 1, corresponding to the MFI before stimulation (average 20 s). AUC (area under the curve) was calculated and reported for each stimulation using GraphPad software.

[0353] Pharmacokinetics of the biCKI Sirpa in vivo To analyze the pharmacokinetics, BalbcRJ mice (female, 6-9 weeks old) were treated intraorbitally with a single dose of the molecules. Drug concentrations in plasma were determined by ELISA using immobilized anti-human light chain antibody (clone NaM76-5F3). Detection was performed with peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch; USA; ref. 709-035-149) and revealed by conventional methods.

[0354] 3D spheroid migration assay 3D cell cultures were established in 96-well U-bottom low attachment plates (6055330 Perkin Elmer) and seeded at 5000, 1500 and 10000 cells / well for A549, MRC-5 and fresh monocytes, respectively. Spheroids were formed and incubated with GM-CSF (10 ng / ml) in complete RPMI. Cells were treated with isotype (IgG4) or BICKI-Sirpα (50 nM) for 3 days. On day 3, 250000 T cells / well were added to the spheroids. After 72 h of co-culture, spheroids were fixed in PFA 4% for 15 min at room temperature, washed 3 times in PBS and kept in PBS-FBS-EDTA buffer at 4°C until staining. Spheroids were then permeabilized in PBS-0.5% Triton and after a 1 h saturation step in PBS-0.1% Triton-1% BSA at RT, spheroids were incubated with primary rabbit anti-human CD3 (A045229-2; 6 μg / ml, stained ON 4° C.) and then with secondary donkey anti-rabbit A488 antibody (10 μg / ml) and DAPI (10 μg / ml) for 2 h at RT. An A1RSi confocal microscope (Nikon) was used for fluorescence detection and confocal z-section images were analyzed via FIJI software using morpholibJ, LoG and 3D-suite plug-ins.

[0355] Antibodies and bifunctional molecules The following antibodies and bifunctional molecules were used in the different experiments disclosed herein: pembrolizumab (Keytrudra, Merck), nivolumab (Opdivo, Bristol-Myers Squibb), and bifunctional molecules disclosed herein comprising an anti-PD1 humanized antibody comprising a heavy chain variable domain as defined in SEQ ID NO: 19, 22 or 24 and a light chain variable domain as defined in SEQ ID NO: 28 or an anti-PD1 chimeric antibody comprising a heavy chain as defined in SEQ ID NO: 53 and a light chain as defined in SEQ ID NO: 54.

Claims

1. (a) a heavy chain variable domain (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, wherein X1 is D or E and X2 is selected from the group consisting of T, H, A, Y, N, E, and S; and (b) a light chain variable domain (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 26, wherein X is G or T, An anti-human PD-1 antibody or an antigen-binding fragment thereof comprising or consisting of: (b) human SIRPa or a fragment or variant thereof, wherein the variant or fragment comprises the extracellular domain of SIRPa and maintains at least 10% of the binding affinity to CD47 compared to wild-type human SIRPa. A bifunctional molecule comprising: A bifunctional molecule, wherein the C-terminus of the heavy and / or light chain of the antibody or antigen-binding fragment thereof is covalently linked to the N-terminus of the SIRPa or fragment or variant thereof as a fusion protein.

2. The bifunctional molecule of claim 1, wherein the C-terminus of the heavy and / or light chain of the antibody or antigen-binding fragment thereof is covalently linked to the N-terminus of the SIRPa or a fragment or variant thereof via a peptide linker as a fusion protein.

3. The bifunctional molecule of claim 1 or 2, wherein the antibody is a chimeric or humanized antibody.

4. 4. The bifunctional molecule of claim 1, wherein the SIRPa comprises or consists of the amino acid sequence set forth in SEQ ID NO: 51 or a fragment thereof.

5. 5. The bifunctional molecule of any one of claims 1 to 4, wherein the anti-human PD-1 antibody or antigen-binding fragment thereof comprises or consists of: (a) a VH comprising, or consisting of, the amino acid sequence of SEQ ID NO:24; and (b) a VL comprising, or consisting of, the amino acid sequence of SEQ ID NO:

28.

6. 6. The bifunctional molecule of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain and a heavy chain constant domain derived from a human IgG1, IgG2, IgG3 or IgG4 heavy chain constant domain.

7. The antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain, and a substitution or combination of substitutions selected from the group consisting of: - T250Q, M428L; - M252Y, S254T, T256E, H433K, N434F; - E233P, L234V, L235A, G236A, A327G, A330S, P331S; - E333A; - S239D, A330L, I332E; - P257I, Q311; - K326W, E333S; - S239D, I332E, G236A; - N297A; - L234A, L235A; - N297A, M252Y, S254T, T256E; and - K322A and K444A 7. The bifunctional molecule of claim 1 , comprising a heavy chain constant domain derived from a human IgG1 heavy chain constant domain having the formula:

8. The antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain, and a substitution or combination of substitutions selected from the group consisting of: - S228P; - L234A, L235A; - S228P, M252Y, S254T, T256E; and - K444A 7. The bifunctional molecule of claim 1 , comprising a heavy chain constant domain derived from a human IgG4 heavy chain constant domain having the formula:

9. 9. An isolated nucleic acid molecule or a group of isolated nucleic acid molecules encoding a bifunctional molecule according to any one of claims 1 to 8.

10. A vector comprising the nucleic acid molecule or group of nucleic acid molecules according to claim 9.

11. A host cell comprising a vector according to claim 10, or a nucleic acid molecule or group of nucleic acid molecules according to claim 9.

12. 12. A method for producing a bifunctional molecule according to any one of claims 1 to 8, comprising culturing a host cell according to claim 11 and isolating the bifunctional molecule.

13. A pharmaceutical composition comprising a bifunctional molecule according to any one of claims 1 to 8, a nucleic acid molecule or a group of nucleic acid molecules according to claim 9, a vector according to claim 10, or a host cell according to claim 11, and a pharma- ceutically acceptable carrier.

14. 14. The pharmaceutical composition of claim 13, further comprising an additional therapeutic agent.

15. Additional therapeutic agents include alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, mitotic inhibitors, antiproliferative agents, antivirals, Aurora kinase inhibitors, proapoptotic agents, Bcl-2 family inhibitors, activators of the death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (bispecific T cell engager) antibodies, antibody drug conjugates, biological response modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia viral oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormonal therapy, immunological agents, inhibitors of inhibitors of apoptosis proteins (IAPs), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors.

15. The pharmaceutical composition of claim 14, wherein the therapeutic agent is selected from the group consisting of: antitumor agents, mammalian target of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, nonsteroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutic agents, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoid / deltoid plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, peptide vaccines, epitopes or neoepitopes derived from tumor antigens, and combinations of one or more of these substances.

16. 16. A pharmaceutical composition according to any one of claims 13 to 15 for use as a medicament.

17. 16. A pharmaceutical composition according to any one of claims 13 to 15 for use in the treatment of cancer.

18. The cancer is selected from the group consisting of hematological malignancies or solid tumors with expression of PD-1 and / or PD-L1, hematolymphoid neoplasms, angioimmunoblastic T-cell lymphoma, myelodysplastic syndrome, and acute myeloid leukemia, cancers induced by viruses or associated with immunodeficiency, Kaposi's sarcoma; cervical, anal, penile, and vulvar squamous cell carcinoma, and oropharyngeal cancer; B-cell non-Hodgkin's lymphoma (NHL), including diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmablastic lymphoma, primary central nervous system lymphoma, HHV- 8. The pharmaceutical composition of claim 17, wherein the cancer is selected from the group consisting of primary effusion lymphoma, classical Hodgkin lymphoma, and lymphoproliferative disorders; hepatocellular carcinoma; Merkel cell carcinoma; and cancer associated with human immunodeficiency virus infection (HIV), and selected from the group consisting of metastatic or non-metastatic melanoma, malignant mesothelioma, non-small cell lung cancer, renal cell carcinoma, Hodgkin lymphoma, head and neck cancer, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, small cell lung cancer, metastatic Merkel cell carcinoma, gastric or gastroesophageal cancer, and cervical cancer.

19. 19. A pharmaceutical composition according to any one of claims 16 to 18 for use in combination with radiation therapy or an additional therapeutic agent.

20. Additional therapeutic agents include alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, mitotic inhibitors, antiproliferative agents, antivirals, Aurora kinase inhibitors, proapoptotic agents, Bcl-2 family inhibitors, activators of the death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (bispecific T cell engager) antibodies, antibody drug conjugates, biological response modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia viral oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormonal therapy, immunological agents, inhibitors of inhibitors of apoptosis proteins (IAPs), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors.

20. The pharmaceutical composition of claim 19, wherein the therapeutic agent is selected from the group consisting of: antitumor agents, mammalian target of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, nonsteroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutic agents, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoid / deltoid plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, peptide vaccines, epitopes or neoepitopes derived from tumor antigens, and combinations of one or more of these substances.

21. 16. A pharmaceutical composition according to any one of claims 13 to 15 for use in the treatment of an infectious disease.

22. 22. The pharmaceutical composition of claim 21, wherein the infectious disease is caused by a virus selected from the group consisting of HIV, hepatitis virus, herpes virus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

Images