WO2020165374A1 - Bifunctional molecule comprising il-15ra - Google Patents

Abstract

The present invention relates to a bifunctional molecule comprising an anti-PD-1 antibody and IL- 15Ralpha or a fragment thereof and its therapeutic uses.

Description

BIFUNCTIONAL MOLECULE comprising IL-15Ra

FIELD OF THE INVENTION

The invention pertains to the field of immunotherapy. The present invention provides a bifunctional molecule that comprises an IL-15 receptor domain or a fragment thereof.

BACKGROUND OF THE INVENTION

Interleukin-15 is an immunostimulatory cytokine member of the IL-2 superfamily and plays an important role for homeostasis of innate and adaptive immune system by stimulating T cells, NK cells, NKT cells, gamma delta T cells and macrophages. IL-15 promotes survival and anti-tumor function (IFN-g secretion, proliferation) of naive, effector and memory T cells and therefore plays a major role in tumor immunosurveillance.

The receptor of IL-15 is heterotrimeric and consists of the high affinity IL-15Ra (CD215) chain and share with IL-2 cytokine the intermediate/low affinity b (CD122) and y chains (CD132). IL-15 triggers the activation of JAK1/3 tyrosine kinases and subsequent nuclear translocation of the phosphorylated Stat-5 and -3. While IL-15 can directly activates cells expressing the trimeric complex IL-15Ra- /y (cis- presentation), IL-15 can also be trans-presented by IL-15Ra expressed on the cell surface of dendritic or macrophage cells and activates NK and T cells expressing the dimeric complex IL-15Ra- /y.

The fusion protein generated by the fusion of the sushi domain (region of IL-15 Ra) to IL-15 cytokine has demonstrated a high potency to trans-present IL-15 and activate proliferation of IL-15R b/y expressing NK and T cells. Interestingly, whereas the sushi domain increases IL-15 driven proliferation, the entire extracellular domain of IL-15Ra blocks IL-15 effect.

Checkpoint receptors such as PD-1 (programmed cell death 1) inhibit the activation, proliferation, and/or effector activities of T cells and other cell types. Guided by the hypothesis that checkpoint receptors suppress the endogenous T cell response against tumor cells, preclinical and clinical studies of anti-PDl antibodies have indeed demonstrated that immune checkpoint blockade results in impressive anti-tumor responses, stimulating endogenous T cells to attack tumor cells, leading to long-term cancer remissions in a fraction of patients with a variety of malignancies. Unfortunately, only a subset of patients responds to these therapies, with response rates generally ranging from 10 to 30% and sometimes higher for each monotherapy, depending on the indication and other factors.

To increase the efficacy of anti-PDl immunotherapy and overcome potential anti PD-1 resistance in patient, the development of a combination treatment targeting IL-15 signaling may be a good strategy to sustain T cell effector capacity and promote a memory T cell response. However, naked IL-15 protein has a short half-life (lh) limiting its use in clinical settings. Furthermore, naked (uncomplexed) IL-15 is not detectable in human tissues and fluids, suggesting that uncomplexed IL-15 is not stable and rapidly degraded. The complex sushi+ IL-15 significantly increase the half-life in vivo and showed a great efficacy in vitro against tumor cells. More particularly, bispecific molecules designed as IL15/IL15Ra-Fc heterodimers are currently under development and are disclosed as a druggable version of IL-15 with potentially superior tolerability, slower receptor-mediated clearance and a prolonged half-life.

Similarly, WO2012175222 describes an immunocytokine comprising a conjugate and an antibody or a fragment of an antibody directly or indirectly linked to said conjugate and where the conjugate is a fusion protein between IL-15 and the sushi domain of the IL15Ralpha or derivatives thereof.

US20180118828 describes a particular bispecific heterodimeric Fc fusion protein comprising an IL-15/IL- 15-Ra moiety and a PD1 antibody fragment moiety.

Flowever, such bispecific molecules may be problematic because of the imposed ratio of one antibody for one cytokine, leading to a disequilibrium. Indeed, the molecules will always provide with one cytokine for each antibody. In addition, bispecific molecules with IL-15/IL-15-Rot lack of selective action and therefore increasing doses of such molecules may rise safety concerns.

Nevertheless, there is still a strong need of new alternative molecules in order to develop improved treatments.

SUMMARY OF THE INVENTION

The present invention is directed to a bifunctional molecule comprising an anti-hPD-1 antibody and a human IL-15 receptor alpha or a fragment thereof promising for numerous therapeutic applications, in particular for the treatment of cancer. The fragment of the human IL-15Ra comprises or consists of the extracellular domain of the human IL-15Ra or a fragment thereof, preferably a fragment capable of binding IL-15, more particularly a sushi domain of IL-15Ra. The bifunctional molecule does not comprise IL-15, either linked covalently to the bifunctional molecule or non-covalently bound to it.

The bifunctional molecule of the present invention blocks the PD-1 mediated inhibition and the sushi domain could catch and stabilize the endogenous IL-15 and trans-presents the IL-15 to PD-1+ NK cells, T cells and macrophages, hence promoting and sustaining effective anti-infectious and anti-tumoral immune responses.

It is emphasized that, contrary to fusion proteins that combine IL15 and IL-15Ra in an antibody, the applicant has developed molecules that do not include IL15 but use the capturing capacity of IL15R, and thanks to the association with an anti-PDl are capable of selectively activating the mechanism on PD1+ cells.

It is also emphasized that the applicant has seen that the bifunctional molecules of the invention are IL- 15 antagonists in vitro, as shown in the detailed examples of the description. Contrary to the effect that is targeted for the therapeutic treatment, they block the pSTAT5 signaling in vitro, the inflammatory effect (TNF, IFNg), and the pro-survival (anti-apoptosis) of IL15 ; thus strongly discouraging the use for further in vivo studies of compounds carrying IL15R alone with the aim of targeting and activating immune cells, in particular T cells and/or NK cells. In the prior art, a solution developed was to couple IL15R Sushi domain with IL15, either in constructs [IL15R Sushi + IL15] or in bifunctional compounds, for instance linking covalently [antibody anti PD1] and [IL15R Sushi + IL15] But IL15 raises at least the following substantial problems:

IL15 has a toxic effect, with therefore a challenging difficulty to increase the administration dose of a bifunctional molecule comprising both an anti-PDl and an IL15 domain for maintaining a satisfying level of Receptor Occupancy,

IL15 has a very high affinity for its receptors in vivo, with thus a problem of unsatisfying pharmacokinetic and a difficulty of obtaining Receptor Occupancy level of about 1 for instance ; the bifunctional molecule containing IL15 is so much trapped by the IL15 receptors that the molecule is not circulating enough for an appropriate targeting by the anti PD1 part of the molecule.

On the opposite, in vivo, the new molecules of the applicant are surprisingly capable of increasing the bioavailability of endogenous IL15, so that they enable efficiently the activation and proliferation of IL15R expressing immune cells, in particular NK cells, and eventually also T cells, in vivo. And these activated immune cells are active against tumoral cells/tissues according to known biological pathways.

Without being linked in theory by a specific mechanism, the explanation would be that, in vivo, the association/dissociation balance of the interaction between IL15 and IL15Ra (Sushi) of the new bifunctional molecule is in favor of the biological effect : sufficient trapping of IL15 allowing the increase of IL-15 half-life (IL-15 alone is otherwise rapidly degraded in the body), and sufficient release of IL-15 leading to the biological effect of activation observed towards immune cells, in particular NK cells. IL-15 may operate according to two main pathways: IL-15 is released and activates NK cells around, and/or at least the IL-15 bound to the bifunctional molecule activates directly a NK cell.

The activated immune cells are locally present in the vicinity of tumoral cells, for instance typically in the case of solid cancers, or are circulating within the body typically in the case of hematologic cancers.

The anti PD-1 domain of the bifunctional molecules allows a better targeting of the immune cells which are PD1 positive and are activated by IL15.

In a first aspect, the invention concerns a bifunctional molecule comprising:

(a) an anti-human PD-1 antibody or an antigen-binding fragment thereof, which comprises:

(i) a heavy chain variable domain (VH) comprising a HCDR1, a HCDR2 and a HCDR3, and

(ii) a light chain variable domain (VL) comprising a LCDR1, a LCDR2 and a LCDR3, and

(b) a human interleukin 15 receptor alpha (IL-15Ra) or a fragment thereof,

wherein the antibody or the fragment thereof is covalently linked to the human IL-15Ra or the fragment thereof as a fusion protein, preferably by a peptide linker, and wherein the bifunctional molecule is not bound to IL-15, either covalently or not covalently.

In a second aspect, the invention concerns a bifunctional molecule comprising:

(a) an anti-human PD-1 antibody or an antigen-binding fragment thereof, which comprises: (i) a heavy chain variable domain (VH) comprising a HCDR1, a HCDR2 and a HCDR3, and

(ii) a light chain variable domain (VL) comprising a LCDR1, a LCDR2 and a LCDR3, and

(b) a human interleukin 15 receptor alpha (IL-15Ra) or a fragment thereof,

wherein the antibody or the fragment thereof is covalently linked to the human IL-15Ra or the fragment thereof as a fusion protein, preferably by a peptide linker, and wherein the anti-human PD-1 antibody or an antigen-binding fragment thereof comprises a Fc domain which is homodimeric.

In a third aspect, the invention concerns a bifunctional molecule comprising:

(a) an anti-human PD-1 antibody or an antigen-binding fragment thereof, which comprises:

(i) a heavy chain variable domain (VH) comprising a HCDR1, a HCDR2 and a HCDR3, and

(ii) a light chain variable domain (VL) comprising a LCDR1, a LCDR2 and a LCDR3, and

(b) a human interleukin 15 receptor alpha (IL-15Ra) or a fragment thereof,

wherein the antibody or the fragment thereof is covalently linked to the human IL-15Ra or the fragment thereof as a fusion protein, preferably by a peptide linker, and wherein each antibody light chain is linked to human IL-15Ra or the fragment thereof and/or each antibody heavy chain is linked to human IL-15Ra or the fragment thereof.

Preferably, the N-terminal end of the human IL-15Ra or the fragment thereof is connected to the C- terminal end of the heavy chain or of the light chain of the anti-human PD-1 antibody or the antigen binding fragment thereof.

In particular, the fragment of the human IL-15Ra comprises or consists of:

- the extracellular domain of the human IL-15Ra or a fragment thereof, preferably a fragment capable of binding IL-15, in particular an amino acid sequence comprising or consisting of the sequence of any one of SEQ ID Nos: 54, 55, 56, 57, 58, 59, 60, 61 and 62 or any sequence having at least 80, 85, 90, 95, 96, 97, 98 or 99 % of identity therewith having the capacity to bind IL-15; or

- the sushi domain of the human IL-15Ra, in particular an amino acid sequence a fragment of IL-15Ra comprising or consisting of the sequence of any one SEQ ID Nos: 51, 70, 71, 72, 73, 74 ,75, 76, 77, 78, 79, 80, 81, preferably an amino acid sequence set forth in SEQ ID NO: 51, or a sequence having at least 75% of identity with SEQ ID NO: 51.

Preferably, the antibody or the antigen-binding fragment thereof is a chimeric, a humanized or a human antibody.

Even more preferably, the anti-human PD-1 antibody or antigen-binding fragment thereof, comprises (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, wherein:

- the heavy chain CDR1 (HCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 1;

- the heavy chain CDR2 (HCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 2; - the heavy chain CDR3 (HCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 3 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;

- the light chain CDR1 (LCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 12 wherein X is G or T;

- the light chain CDR2 (LCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 15,

- the light chain CDR3 (LCDR3) comprises or consists of an amino acid sequence of SEQ ID NO:16.

In particular, the anti-human PD-1 antibody or antigen-binding fragment thereof, comprises or consists of (a) a VH comprising or consisting of an 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 an amino acid sequence of SEQ ID NO: 26, wherein X is G or T.

Alternatively, the anti-PDl antibody is selected from the group consisting of Pembrolizumab, Nivolumab, Pidilizumab, Cemiplimab, PDR001, and monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4. Particularly, the anti-human PD-1 antibody or an antigen-binding fragment thereof comprises a Fc domain which is homodimeric and wherein each antibody heavy chain is linked to human IL-15Ra or the fragment thereof.

The invention also concerns an isolated nucleic acid molecule or a group of isolated nucleic acid molecules encoding the bifunctional molecule according to the invention, a vector, comprising the nucleic acid or group of nucleic acid molecules according to the invention and a host cell, comprising the vector according or the nucleic acid or group of nucleic acid molecules according to the invention.

The invention is also related to a method for producing the bifunctional molecule according to the invention, comprising a step of culturing a host cell and optionally a step of isolating the bifunctional molecule.

In another aspect, the invention concerns a pharmaceutical composition comprising the bifunctional molecule, the nucleic acid or group of nucleic acid molecules, the vector or the host cell according to the invention and a pharmaceutically acceptable carrier.

Preferably, the pharmaceutical composition comprises the bifunctional molecule according to the invention and does not comprise IL-15.

Optionally, the pharmaceutical composition further comprises an additional therapeutic agent, preferably selected in the group consisting of alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotics, antiproliferatives, antivirals, aurora kinase inhibitors, apoptosis promoters (for example, Bcl- 2 family inhibitors), activators of death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (Bi-Specific T cell Engager) antibodies, antibody drug conjugates, biologic 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 therapies, immunologicals, inhibitors of inhibitors of apoptosis proteins (lAPs), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, non-steroidal anti inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutics, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoids/deltoids plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoints inhibitors, peptide vaccine and the like, epitopes or neoepitopes from tumor antigens, as well as combinations of one or more of these agents. The invention finally concerns the pharmaceutical composition, bifunctional molecule, nucleic acid or group of nucleic acid molecules, vector, or host cell according to the invention, for use as a medicament, in particular for use in the treatment of a disease selected from the group consisting of a cancer and infection.

The cancer is particularly selected from the group consisting of hematolymphoid neoplasms, angioimmunoblastic T cell lymphoma, myelodysplastic syndrome, and acute myeloid leukemia, a cancer induced by virus or associated with immunodeficiency such as a cancer selected from the group consisting of Kaposi sarcoma (e.g., associated with Kaposi sarcoma herpes virus); cervical, anal, penile and vulvar squamous cell cancer and oropharyngeal cancers (e.g., associated with human papilloma virus); B cell non-Hodgkin lymphomas (NHL) including diffuse large B-cell lymphoma, Burkitt lymphoma, plasmablastic lymphoma, primary central nervous system lymphoma, HHV-8 primary effusion lymphoma, classic Hodgkin lymphoma, and lymphoproliferative disorders (e.g., associated with Epstein-Barr virus (EBV) and/or Kaposi sarcoma herpes virus); hepatocellular carcinoma (e.g., associated with hepatitis B and/or C viruses); Merkel cell carcinoma (e.g., associated with Merkel cell polyoma virus (M PV)); and cancer associated with human immunodeficiency virus infection (HIV) infection, and a cancer selected from the group consisting of metastatic or not 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 cancers and Cervical Cancer.

Optionally, the bifunctional molecule, the pharmaceutical composition, the isolated nucleic acid molecule or the group of isolated nucleic acid molecules, the vector, or the host cell is for use in combination with radiotherapy or an additional therapeutic agent, preferably selected in the group consisting of alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotics, antiproliferatives, antivirals, aurora kinase inhibitors, apoptosis promoters (for example, Bcl-2 family inhibitors), activators of death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (Bi-Specific T cell Engager) antibodies, antibody drug conjugates, biologic 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 therapies, immunologicals, inhibitors of inhibitors of apoptosis proteins (lAPs), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, non-steroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutics, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoids/deltoids plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoints inhibitors, peptide vaccine and the like, epitopes or neoepitopes from tumor antigens, as well as combinations of one or more of these agents.

The infection is particularly a viral infection, especially a chronic viral infection, or a sepsis. Preferably, the infectious disease is caused by a virus selected from the group consisting of HIV, hepatitis virus, herpes virus, adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: PD-1 binding ELISA assay. Human recombinant PD-1 (rPDl) protein was immobilized and the Anti-PDl (■), anti-PDl/IL15-RA (·) and anti-PDl/Sushi (o) molecules were added at different concentrations. Revelation was performed with an anti-human Fc antibody coupled to peroxidase. Colorimetry was determined at 450 nm using TMB substrate.

Figure 2: Antagonistic capacity of the anti-PDl/L-15RA and anti-PD-l/sushi molecules to block PD-l/PD-

Ll. PD-L1 was immobilized on Maxisorp plate, the complex antibodies + biotinylated recombinant human PD-1 was added. This complex was generated with a fixed concentration of PD1 (0.6 pg/mL) and various concentrations of the anti-PDl/IL15Ra (·) or anti-PDl/sushi (o) bifunctional molecules were tested. Figure 3: Binding capacity of soluble IL-15 to the anti-PDl/IL-15RA and the anti-PD-l/sushi molecules. Human recombinant IL-15 was immobilized to the plate and various concentrations of the anti-PDl (■), anti-PDl/IL15Ra (·) or anti-PDl/sushi (o) antibodies or IL-15RA-Fc (□) molecules were added to assess their binding capacity to IL-15. Revelation was performed with an anti-human Fc antibody coupled to peroxidase. Colorimetry was determined at 450/650 nm using TMB substrate. Figure 4: In vitro, the anti-PDl/IL-15RA and the anti-PD-l/sushi molecules antagonize IL-15 induced pSTAT5 signaling in T cells. IL-15 mediated signaling was assessed by STAT5 phosphorylation into human T cells. PBMCs isolated from peripheral blood of healthy volunteers were incubated with various concentrations of anti-PDl (■), anti-PDl/IL15Ra (·) or anti-PDl/sushi (o) antibodies or IL-15RA-Fc (□). This assay was performed without IL-15 recombinant cytokine (Figure 4A) or with a fixed concentration of IL-15: 0,lnM (Figure 4B) or InM (Figure 4C). After 15 minutes of incubation, cells were fixed, permeabilized and stained with an AF647 labeled anti-pSTAT5 (clone 47/Stat5(pY694)) + anti hCD3-V450 antibody. Data represent the median of fluorescence of pSTAT5 into CD3+ T cell population.

Figure 5: In vitro, the anti-PDl/IL-15RA and anti-PD-l/sushi bifunctional molecules antagonize the cytokine secretion and survival signaling induced by IL-15. Chronically stimulated PBMCs were incubated for 2 days with recombinant IL-15 and the anti-PDl, anti-PDl/IL15Ra or anti-PDl/sushi molecules on the anti-CD3 coated plate (2ug/mL). (Figure 5 A) IFN-y and TNF-a cytokines secreted byT cells were quantified in the supernatant using ELISA assay. In this experiment, a fixed dose of IL-15 was used (2,5nM) (Figure 5 B) Apoptosis was measured by flow cytometry following staining with a FITC labeled AnnexinV. In this experiment, a fixed dose of IL-15 was used (0,lnM).

Figure 6: IL-15 stimulates exhausted T cells in vitro and ex vivo in human cancer biopsies. (Figure 6A)

Exhausted T cells were generated in vitro by repeated antigen stimulation of human PBMCs (CD3/CD28.2 coated plate, 3 pg/mL of each antibody, every 3 days). Forty-eight hours following each stimulation, exhausted T cells were treated with recombinant IL-15 for 15 min. Cells were then fixed, permeabilized and stained with an AF647 labeled anti-pSTAT5 (clone 47/Stat5 (pY694)). Each dot represents one donor. (Figure 6B) Tumor-infiltrating T cells were then isolated from a human kidney tumor then treated with 30nM of IL-15 for 15min. T cells were then fixed and stained for pSTAT5 activation marker and a cocktail of anti-CD3-BV510, CD4-BV421 and CD8-PECy7 antibodies. Histograms represent pSTAT5 fluorescence into CD3+CD4+ or CD3+CD8+ T cells populations.

Figure 7: In vivo, the anti-PD-l/sushi bifunctional molecule stimulates NK activation and proliferation.

Mice were subcutaneously injected with MC38 tumor cells and treated with isotype control (A) or an anti- PD-1 (■) or the anti-PD-l/sushi (o) molecules. On Day 3 and 7 following treatment, peripheral blood was harvested, and NK cells were stained for activation and proliferation markers. The CD45+/CD3- /NKp46+/CD16+/CD32+ markers were used to identify NK cells. (Figure 7A) Ki67 proliferation marker analysis represented the percentage of positive cells (Figure 7B) CD69 activation marker analysis. Data represent %*Median of fluorescence, (Figure 7C) NKG2D median fluorescence analysis, (Figure 7D) CD27 activation marker analysis represented with the percentage of positive cells analyzed on Day 3 following treatment. All data represent the mean +/- SEM of 4-6 mice per group. Statistical significance was determined using a Mann-Whitney test, ** represent p<0,005 by comparing isotype and anti-PD-l/sushi molecule group and # = p<0,05 and ## = p<0,005 between the anti-PD-1 and anti-PD-l/sushi molecule group.

Figure 8: In vivo, the anti-PD-l/sushi molecule promotes T cell proliferation to a similar extent to anti- PD-1. Mice were subcutaneously injected with MC38 tumor cells and treated with isotype control (A) or anti-PD-1 alone (■) or anti-PD-l/sushi (o) molecules. On Day 3 and 7 following treatment, peripheral blood was harvested, and T cells were stained with CD45+/CD3+/CD8+ and Ki67 proliferation marker. Data represent the percentage of Ki67+ cells. All data represent the mean +/- SEM of 4-6 mice per group. Statistical significance was determined using a Mann-Whitney test, * represents p<0,05 by comparing isotype and anti-PD-l/sushi molecule group ns: no statistical difference between the anti-PD-1 and anti- PD-l/sushi molecule group.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

The present invention relates to a bifunctional molecule comprising an anti-hPD-1 antibody and a human IL-15 receptor alpha or a fragment thereof, the bifunctional molecule being capable of binding IL-15 through the IL-15Ra moiety but being devoid of IL-15. Indeed, the important aspect of the invention is that the bifunctional molecule combines two functions:

1- The blockade of the PD-1 mediated inhibition; and

2- The capture of the endogenous IL-15 and its release near PD-1 positive immune cells.

The bifunctional molecules of the invention have in particular one or several of the following advantages:

Stabilize the endogenous uncomplexed IL-15 (e.g., free IL-15), more specifically in tissues infiltrated with high level of PD-1+ cells such as tumors; and/or

Selectively activate the IL-15R signaling on PD1+ cells such as T, NK or macrophages cells, and more particularly on highly expressing PD1 exhausted T cells, hence improving the safety of IL-15 immunotherapy approaches; and/or

Increase local IL-15 availability by stabilizing endogenous uncomplexed IL15 within the tumor environment; and/or

Improve the response to anti-PDl antibody by prolonging and enhancing the reinvigoration of exhausted T-cells after PD-1 blockade.

Definitions

As used herein, the terms "interleukin-15", "IL-15" and "IL15" refers to a mammalian endogenous secretory glycoprotein, particularly IL-15 polypeptides, derivatives and analogs thereof having substantial amino acid sequence identity to wild-type mammalian IL-15 and substantially equivalent biological activity. Human IL-15 is described in UniProtKB under accession number P40933. For example, the human IL-15 amino acid sequence is about 162 amino acids (including the signal peptide and the propeptide). Two isoforms are known. They are respectively disclosed in SEQ ID NOs: 52 and 53. As used herein, the terms "interleukin-15 receptor alpha", "IL-15Ra" and "IL15Ra" refers to the subunit alpha of the IL-15 receptor, derivatives and analogs thereof having substantial amino acid sequence identity to wild-type mammalian IL-15Ra and substantially equivalent biological activity. Human IL-15Ra is described in UniProtKB under accession number Q13261. For example, the human IL-15Ra amino acid sequence is about 267 amino acids and the extracellular domain is located between residues 31-205. Sushi domain is located in the extracellular domain between residues 31-95. 9 isoforms of human IL-15Ra are known and are respectively disclosed in SEQ ID NOs: 54-62.

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, and include variants and isoforms of human PD-1, and analogs having at least one common epitope with PD-1. PD-1 is a key regulator of the threshold of immune response and peripheral immune tolerance. It 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 a human PD-1 is disclosed under GenBank accession number NP_005009. PD1 has four splice variants expressed on human Peripheral blood mononuclear cells (PBMC). Accordingly, PD-1 proteins include full-length PD-1, as well as alternative splice variants of PD- 1, such as PD-lAex2, PD-lAex3, PD-lAex2,3 and PD-lAex2,3,4. Unless specified otherwise, the terms include any variant and isoform of human PD-1 that are naturally expressed by PBMC, or that are expressed by cells transfected with a PD-1 gene.

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 can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, lgG2, lgG3, lgG4, IgAl and lgA2) 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 noted otherwise, the term "antibody" includes intact immunoglobulins and "antibody fragment" or "antigen binding fragment" (such as Fab, Fab', F(ab')2, Fv), single chain (scFv), mutants thereof, molecules comprising an antibody portion, diabodies, linear antibodies, single chain antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies. Preferably, the term antibody refers to a humanized antibody, even more preferably to a bifunctional humanized antibody.

As used herein, an "antigen-binding fragment" of an antibody means a part of an antibody, i.e. a molecule corresponding to a portion of the structure of the antibody of the invention, that exhibits antigen-binding capacity for PD-1, possibly in its native form; such fragment especially exhibits the same or substantially the same antigen-binding specificity for said antigen compared to the antigen-binding specificity of the corresponding four-chain antibody. Advantageously, the antigen-binding fragments have a similar binding affinity as the corresponding 4-chain antibodies. However, antigen-binding fragment that have a reduced antigen-binding affinity with respect to corresponding 4-chain antibodies are also encompassed within the invention. The antigen-binding capacity can be determined by measuring the affinity between the antibody and the target fragment. These antigen-binding fragments may also be designated as "functional fragments" of antibodies. Antigen-binding fragments of antibodies are fragments which comprise their hypervariable domains designated CDRs (Complementary Determining Regions) or part(s) thereof encompassing the recognition site for the antigen, i.e. the extracellular domain of PD1, thereby defining antigen recognition specificity.

A "Fab" fragment contains the constant domain of the light chain and the first constant domain (CHI) 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 CHI domain including one or more cysteines from the antibody hinge region. F(ab') fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art. Fab and F(ab')2 fragments lack the Fc fragment of an intact antibody, clear more rapidly from the circulation of animals, and may have less non-specific tissue binding than an intact antibody (see, e.g. Wahl et al, 1983, J. Nucl. Med. 24:316).

An "Fv" fragment is the minimum 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 a 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. Often, the six CDRs confer target binding specificity to the antibody. However, in some instances even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) can have the ability to recognize and bind target, although at a lower affinity than the entire binding site.

"Single-chain Fv" or "scFv" antibody binding fragments comprise the VH and VL domains of an antibody, where 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.

"Single domain antibodies" are composed of a single VH or VL domains which exhibit sufficient affinity to PD-1. In a specific embodiment, the single domain antibody is a camelized antibody {See, e.g., Riechmann, 1999, Journal of Immunological Methods 231 :25-38).

In terms of structure, an antibody may have heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (l) and kappa (K). Each heavy and light chain contains a constant region and a variable region (or "domain"). Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity determining regions" or "CDRs". The extent of the framework region and CDRs have been defined (see, Kabat et al., Sequences of Proteins of Immunological Interest, and U.S. Department of Health and Human Services, 1991, which is hereby incorporated by reference). Preferably, the CDRs are defined according to Kabat method. The framework regions act to form a scaffold that provides, for positioning the CDRs in correct orientation by inter-chain, 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 starting from the N- terminus. The VL and VH domain of the antibody according to the invention may comprise four framework regions or "FR's", which are referred to in the art and herein as "Framework region 1 " or "FR1", "FR2", "FR3", and "FR4", respectively. These framework regions and complementary 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 the part of the variable domain, either VL and/or VH, which serves as a scaffold for the antigen binding loops (CDRs) of this variable domain.

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

An "antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in antibody conformations; k and l light chains refer to the two major antibody light chain isotypes. The CDRs of the antibody light chain are typically referred to as "LCDR1", "LCDR2" and "LCDR3". The framework regions of the antibody light chain are typically referred to as "LFR1", "LFR2", "LFR3" and "LFR4".

With regard to the binding of an antibody to a target molecule, the terms "bind" or "binding" refer to peptides, polypeptides, proteins, fusion proteins, molecules and antibodies (including antibody fragments) that recognize and contact an antigen. Preferably, it refers to an antigen-antibody type interaction. The terms "specific binding", "specifically binds to," "specific for," "selectively binds" and "selective for" 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 a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically (or preferentially) binds to PD-1 or to a PD-1 epitope is an antibody that binds this PD-1 epitope for example with greater affinity, avidity, more readily, and/or with greater duration than it binds to other PD-1 epitopes or non-PD-1 epitopes. Preferably, the term "specific binding" means the contact between an antibody and an antigen with a binding affinity equal or lower than 10⁷ M. In certain aspects, antibodies bind with affinities equal or lower than lO ⁸ M, lO ⁹ M or 10¹⁰ M.

As used herein "PD-1 antibody," "anti-PD-1 antibody," "PD-1 Ab," "PD-l-specific antibody", "anti-PD-1 Ab" or "humanized anti-PD-1 antibody" are used interchangeably and refer to an antibody, as described herein, which specifically binds to PD-1, particularly human PD-1. In some embodiments, the antibody binds to the extracellular domain of PD- 1. Particularly, an anti-PD-1 antibody is an antibody capable of binding to a PD-1 antigen and inhibits the PD-l-mediated signaling pathway, thereby enhancing immune responses such as T cell activation.

As used herein, the term "bifunctional molecule", "bifunctional compound", "bifunctional protein", "Bicki", "Bicki antibody", "bifunctional antibody" and "bifunctional checkpoint inhibitors molecule" have the same meanings and can be interchangeably used. These terms refer to an antibody that recognizes one antigen by virtue of possessing at least one region (e.g. derived from a variable region of an antibody) that is specific for this antigen, and at least a second region that is a polypeptide. More specifically, the bifunctional molecule is a fusion protein of an antibody or a portion thereof, preferably an antigen binding fragment thereof with another polypeptide or polypeptide fragment thereof.

The term "chimeric antibody" as used herein, means an antibody or antigen-binding fragment, having a portion of heavy and/or light chain derived from one species, and the rest of the heavy and/or light chain derived from a different species. In an illustrative example, a chimeric antibody may comprise a constant region derived from human and a variable region from a non-human species, such as from a mouse.

As used herein, the term "humanized antibody" is intended to refer to antibodies 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. chimeric antibodies that contain minimal sequence derived from a non-human antibody). A "humanized antibody" or "humanized form of an antibody", e.g., a non-human antibody, also 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 are replaced by residues from at least one CDR of a non-human antibody (donor antibody) while maintaining the desired specificity, affinity, and capacity of the original antibody. The donor antibody can be any suitable non human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect. In some instances, 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 or humanized antibody. Additional framework region modifications may be made within the human framework sequences. Humanized antibodies thus may also comprise 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 increased the humanization process. By "amino acid change" or "amino acid modification" is meant herein a change in the amino acid sequence of a polypeptide. "Amino acid modifications" include substitution, insertion and/or deletion in a polypeptide sequence. By "amino acid substitution" or "substitution" herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. By "amino acid insertion" or "insertion" is meant the addition of an amino acid at a particular position in a parent polypeptide sequence. By "amino acid deletion" or "deletion" is meant the removal of an amino acid at a particular position in a parent polypeptide sequence. The amino acid substitutions may be conservative. A conservative substitution is the replacement of a given amino acid residue by 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 acids sequence, generally specified with the one letter codes for the amino acids. The first amino acid in the amino acids sequence (i.e. starting from the N terminus) should be considered as having position 1.

A conservative substitution is the replacement of a given amino acid residue by another residue having a side chain ("R-group") with similar chemical properties (e.g., charge, bulk and/or hydrophobicity). In general, a conservative amino acid substitution 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 instance, conservative substitutions can be defined by substitutions within the groups of amino acids reflected in the following tables:

Table A - Amino Acid Residue

Table B - Alternative Conservative Amino Acid Residue Substitution Groups

Table C - Further Alternative Physical and Functional Classifications of Amino Acid Residues

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 recombinant cells, since at least one component of the antibody's natural environment is not present. In some embodiments, an antibody is purified to homogeneity and/or to greater than 90%, 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) under reducing or non reducing conditions.

The terms "derive from" and "derived from" as used herein refers to a compound having a structure derived from the structure of a parent compound or protein and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar properties, activities and utilities as the claimed compounds. For example, a humanized antibody derived from a murine antibody refers to an antibody or antibody fragment that shares similar properties with the murine antibody, e.g. recognizes the same epitope, shares similar VH and VL with modified residues that participate and/or increased the humanization of the antibody.

For purposes of the present invention, the "percentage identity" between two amino acid sequences (A) and (B) is determined by comparing the two sequences aligned in an optimal manner, through a window of comparison. Said alignment of sequences can be carried out by well-known methods, for example, using the algorithm for global alignment of Needleman-Wunsch. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. Once the total alignment is obtained, the percentage of identity can be obtained by dividing the full number of identical amino acid residues aligned by the full number of residues contained in the longest sequence between the sequence (A) and (B). Sequence identity is typically determined using sequence analysis software. For comparing two amino acid sequences, one can use, for example, the tool "Emboss needle" for pairwise sequence alignment of proteins providing by EM BL-EBI and available on:

www.ebi.ac. uk/Tools/services/web/toolform.ebi?tool=emboss_needle&context=protein, using default settings : (I) Matrix : BLOSUM62, (ii) Gap open : 10, (iii) gap extend : 0.5, (iv) output format : pair, (v) end gap penalty : false, (vi) end gap open : 10, (vii) end gap extend : 0.5.

Alternatively, Sequence identity can also be typically determined using sequence analysis software Clustal Omega using the HHalign algorithm and its default settings as its core alignment engine. The algorithm is described in Soding, J. (2005) 'Protein homology detection by FIMM-FIMM comparison'. Bioinformatics 21, 951-960, with the default settings. The term "treatment" refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis and retardation of the disease or of the symptoms of the disease. It designates both a curative treatment and/or a prophylactic treatment of a disease. A curative treatment is defined as a treatment resulting in cure or a treatment alleviating, improving and/or eliminating, reducing and/or stabilizing a disease or the symptoms of a disease or the suffering that it causes directly or indirectly. A prophylactic treatment comprises both a treatment resulting in the prevention of a disease and a treatment reducing and/or delaying the progression and/or the incidence of a disease or the risk of its occurrence. In certain embodiments, such a term refers to the improvement or eradication of a disease, a disorder, an infection or symptoms associated with it. In other embodiments, this term refers to minimizing the spread or the worsening of cancers. Treatments according to the present invention do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. Preferably, the term "treatment" refers to the application or administration of a composition including one or more active agents to a subject, who has a disorder/disease, for instance associated with the signaling pathway mediated by PD-1.

As used herein, the terms "disorder" or "disease" refer to the incorrectly functioning organ, part, structure, or system of the body resulting from the effect of genetic or developmental errors, infection, poisons, nutritional deficiency or imbalance, toxicity, or unfavorable environmental factors. Preferably, these terms refer to a health disorder or disease e.g. an illness that disrupts normal physical or mental functions. More preferably, the term disorder refers to immune and/or inflammatory diseases that affect animals and/or humans, such as cancer.

The term "immune disease", as used herein, refers to a condition in a subject characterized by cellular, tissue and/or organ injury caused by an immunologic reaction of the subject to its 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. Moreover, inflammation may or may not be caused by an autoimmune disorder.

The term "cancer" as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body.

As used herein, the term "disease associated with or related to PD-1", "PD-1 positive cancer" or "PD-1 positive infectious disease" is intended to refer to the cancer or infectious disease (e.g. caused by a virus and/or bacteria) which is resulted from PD-1 expression or has the symptom/characteristic of PD-1 expression, i.e. any condition that is caused by, exacerbated by, or otherwise linked to increased or decreased expression or activities of PD-1. As used herein, the term "subject", "host", "individual," or "patient" refers to human, including adult and child.

As used herein, a "pharmaceutical composition" refers to a preparation of one or more of the active agents, such as comprising a bifunctional molecule according to the invention, with optional other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the active agent to an organism. Compositions of the present invention can be in a form suitable for any conventional route of administration or use. In one embodiment, a "composition" typically intends a combination of the active agent, e.g., compound or composition, and a naturally-occurring or non-naturally-occurring carrier, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers. An "acceptable vehicle" or "acceptable carrier" as referred to herein, is any known compound or combination of compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.

"An effective amount" or a "therapeutic effective amount" as used herein refers to the amount of active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents, e.g. the amount of active agent that is needed to treat the targeted disease or disorder, or to produce the desired effect. The "effective amount" will vary depending on the agent(s), the disease and its severity, the characteristics of the subject to be treated including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.

As used herein, the term "medicament" refers to any substance or composition with curative or preventive properties against disorders or diseases.

The term "in combination" as used herein refers to the use of more than one therapy (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.

The terms "polynucleotide", "nucleic acid" and "nucleic acid sequence" are equivalent and refer to a polymeric form of nucleotide of any length, for example RNA or DNA or analogs thereof. Nucleic acids (e.g., components, or portions, of the nucleic acids) of the present invention may be naturally occurring, modified or engineered, isolated and/or non-natural. Engineered nucleic acids include recombinant nucleic acids and synthetic nucleic acids. "Isolated nucleic acid encoding an anti-PDl antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at 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.

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

"Immune cells" as used herein refers to cells involved in innate and adaptive immunity for example such as white blood cells (leukocytes) which are derived from hematopoietic stem cells (HSC) produced in the bone marrow, lymphocytes (T cells, B cells, natural killer (NK) cells and Natural Killer T cells (NKT) and myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells). In particular, the immune cell can be selected in the non-exhaustive list comprising B cells, T cells, in particular CD4+ T cells and CD8+ T cells, NK cells, NKT cells, APC cells, dendritic cells and monocytes. "T cell" as used herein includes for example CD4 + T cells, CD8 + T cells, T helper 1 type T cells, T helper 2 type T cells, T helper 17 type T cells and inhibitory T cells.

As used herein, the term "T effector cell", "T eff" or "effector cell" describes a group of immune cells that includes several T cell types that actively respond to a stimulus, such as co-stimulation. It particularly includes T cells which function to eliminate antigen (e.g., by producing cytokines which modulate the activation of other cells or by cytotoxic activity). It notably includes CD4+, CD8+, Treg cells, cytotoxic T cells and helper T cells (Thl and Th2).

As used herein, the term "regulatory T cell", Treg cells" or "T reg" 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 induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FOXP3, and CD25 and are thought to be derived from the same lineage as naive CD4 cells.

The term "exhausted T cell" refers to a population of T cell in a state of dysfunction (i.e. "exhaustion"). T cell exhaustion is characterized by progressive loss of function, changes in transcriptional profiles and sustained expression of inhibitory receptors. Exhausted T cells lose their cytokines production capacity, their high proliferative capacity and their cytotoxic potential, which eventually leads to their deletion. Exhausted T cells typically indicate higher levels of CD43, CD69 and inhibitory receptors combined with lower expression of CD62L and CD127.

The term "immune response" refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complements) that results in selective damage to, destruction of, or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. The term "antagonist" as used herein, refers to a substance that block or reduces the activity or functionality of another substance. Particularly, this term refers to an antibody that binds to a cellular receptor (e.g. PD-1) as a reference substance (e.g. PD-L1 and/or PD-L2), preventing it from producing all or part of its usual biological effects (e.g. the creation of an immune suppressive microenvironment). The antagonist activity of a humanized antibody according to the invention may be assessed by competitive ELISA.

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

The term "and/or" as used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually.

The term "a" or "an" can refer to one of or a plurality of the elements it modifies (e.g., "a reagent" can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described.

The term "about" as used herein in connection with any and all values (including lower and upper ends of numerical ranges) 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%). The use of the term "about" at the beginning of a string of values modifies each of the values (i.e. "about 1, 2 and 3" refers to about 1, about 2 and about 3). Further, when a listing of values is described herein (e.g. about 50%, 60%, 70%, 80%, 85% or 86%) the listing includes all intermediate and fractional values thereof (e.g., 54%, 85.4%). Anti-PD-1 Antibody

The bifunctional molecule according to the invention comprises a first entity that comprises an anti-hPD- 1 antibody or an antigen binding fragment thereof.

Provided herein are antibodies that particularly bind to human PD-1. In some aspects, the antibody specifically binds to human PD-1, preferably to the extracellular domain of human PD-1. In some aspects, the antibody selectively binds to one or more of full-length human PD-1, PD-lAex2, PD-lAex3, PD-lAex2,3 and PD-lAex2,3,4.

In some aspects, the anti-PDl antibody is an isolated antibody, particularly a non-natural isolated antibody. Such isolated anti-PDl antibody can be prepared by at least one purification step. In some embodiments, an isolated anti-PDl antibody is purified to at least 80%, 85%, 90%, 95% or 99% by weight. In some embodiments, an isolated anti-PDl isolated antibody is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% by weight of an antibody, the remainder of the weight comprising the weight of other solutes dissolved in the solvent.

Preferably, such antibody has the ability to block or inhibit the interaction between PD-1 and 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 the binding interaction between two molecules (e.g. PD-1 and its ligand PD-L1 and/or PD-L2) to any detectable degree.

Preferably, the anti-PDl 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 human PD-L1 and PD-L2 to human PD-1. In certain embodiments, the anti-hPDl antibody or antigen-binding fragment inhibits the binding interaction between PD-1 and 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%.

Humanized forms of anti-PDl antibodies according to this invention may comprise immunoglobulins, immunoglobulin of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2, scFv or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from a non-human (e.g. murine) immunoglobulin targeting human PD-1. Preferably, the humanized anti-hPD-1 antibody according to the invention derives from IgGl, lgG2, lgG3 or lgG4, preferably from an lgG4 or an IgGl.

In one embodiment, the antigen-binding fragment of an antibody comprises a heavy chain comprising a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3 and a light chain comprising a variable domain comprising LCDR1, LCDR2 and LCDR3, and a fragment of a heavy chain constant domain. By a fragment of a heavy chain constant domain, it should be understood that the antigen-binding fragment therefore comprises at least a portion of a full heavy chain constant domain. As examples, a heavy chain constant domain may comprise or consist of at least the CHI domain of a heavy chain, or at least the CHI and the CH2 domains of a heavy chain, or at least the CHI, CH2 and CH3 domains of a heavy chain. 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. Accordingly, antigen-binding fragment of an antibody encompasses the Fab portion of a full antibody, the F(ab')2 portion of a full antibody, the Fab' portion of a full antibody. The heavy chain constant domain may also comprise or consist in a full heavy chain constant domain, for example illustrated in the present description, wherein several full heavy chain constant domains are described. In a particular embodiment of the invention, and when the antigen-binding fragment of an antibody comprises a fragment of a heavy chain constant domain comprising or consisting in a portion of a full heavy chain constant domain, the heavy chain constant domain fragment may consist of at least 10 amino acid residues; or may consist of 10 to 300 amino acid residues, in particular 210 amino acid residues.

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 comprising the population are identical and/or bind the same epitope. Preferably, such monoclonal antibodies (mAbs) are from a mammalian, such as mice, rodents, rabbit, goat, primates, non-human primates or humans. Techniques for preparing such monoclonal antibodies may be found in, e.g., Stites et al. (eds.) BASIC AND CLINICAL IMM UNOLOGY (4th ed.) Lange Medical Publications, Los Altos, CA, and references cited therein; Harlow and Lane (1988) ANTIBODIES: A LABORATORY MANUAL CSH Press; Goding (1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York, NY.

In one embodiment, the anti-PDl antibody can be selected from the group consisting of Pembrolizumab (also known as Keytruda lambrolizumab, M K-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, genolimzumab (CBT-501), LZM-009, BCD-100, SHR-1201, BAT-1306, AK-103 (HX-008), MEDI-0680 (also known as AM P-514) M EDI0608, JS001 (see 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 WO 2014/194302), AGEN2034 (see WO 2017/040790), 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. Bifunctional or bispecific molecules targeting PD-1 are also known such as RG7769 (Roche), XmAb20717 (Xencor), M EDI5752 (AstraZeneca), FS118 (F-star), SL-279252 (Takeda) and XmAb23104 (Xencor). In a particular aspect, the anti-PDl 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, described in WO 2006/121168. In another particular embodiment, the anti-PDl antibody can be Pembrolizumab (also known as Keytruda lambrolizumab, M K-3475) or Nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538).

In some embodiments, the anti-hPDl antibody provided herein is an isolated antibody.

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

In certain embodiments, the anti-hPDl antibody is a humanized antibody. A humanized antibody typically comprises one or more variable domains in which CDRs (or portions thereof) are derived from a non human antibody, and FRs (or portions thereof) are derived from human or humanized antibody sequences. Alternatively, some FR residues can be substituted to restore or improve antibody specificity, affinity and/or humanization. A humanized antibody optionally will also comprise at least a portion of a human or humanized constant region (Fc). Methods of antibodies humanization are well known in the art see for example, Winter and Milstein, Nature, 1991, 349:293-299; Riechmann et al., Nature, 332, pp. 323 (1988); Verhoeyen et al., Science, 239, pp. 1534 (1988), Rader et al, Proc. Nat. Acad. Sci. U.S.A., 1998, 95:8910-8915; Steinberger et al, J. Biol. Chem., 2000, 275:36073-36078; Queen et al, Proc. Natl. Acad. Sci. U.S.A., 1989, 86: 10029-10033; Almagro, J.C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633; Kashmiri, S.V. et al, Methods 36 (2005) 25-34 (describing SDR (a-CDR) grafting); Padlan, E.A., Mol. Immunol. 28 (1991) 489-498 (describing "resurfacing"); Dall'Acqua, W.F. et al, Methods 36 (2005) 43-60 (describing "FR shuffling"); and Osbourn, J. et al, Methods 36 (2005) 61-68 and Klimka, A. et al, Br. J. Cancer 83 (2000) 252-260 (describing the "guided selection" approach to FR shuffling) and U.S. Patent Nos. 5,585,089, 5,693,761, 5,693,762, 5,821,337, 7,527,791, 6,982,321, and 7,087,409; and 6,180,370. Preferably, the humanized antibody against human PD-1 is a monoclonal antibody.

Particularly, a humanized antibody is one that has a T20 humanness score of at least 80% or at least 85%, more preferably at least 88%, even more preferably at least 90 %, most preferably a T20 humanness score comprised between 85% and 95%, preferably between 88% and 92%.

"Flumanness" is generally measured using the T20 score analyzer to quantify the humanness of the variable region of monoclonal antibodies as described in Gao S H, Huang K, Tu H, Adler A S. BMC Biotechnology. 2013: 13:55. T20 humanness score is a parameter commonly used in the field of antibody humanization first disclosed by Gao et al (BMC Biotechnol, 2013, 13, 55). T20 humanness score is usually used in patent application for defining a humanized antibody (e.g., W015161311, W017127664, W018136626, WO18190719, W019060750, or WO19170677).

A web-based tool is also available to calculate the T20 score of antibody sequences using the T20 Cutoff Human Databases: http://abAnalyzer.lakepharma.com. In computing a T20 score, an input VH, VK, or VL variable region protein sequence is first assigned Kabat numbering, and CDR residues are identified. The full-length sequence or the framework only sequence (with CDR residues removed) is compared to every sequence in a respective antibody database using the blastp protein-protein BLAST algorithm. The sequence identity between each pairwise comparison is isolated, and after every sequence in the database has been analyzed, the sequences are sorted from high to low based on the sequence identity to the input sequence. The percent identity of the Top 20 matched sequences is averaged to obtain the T20 score.

For each chain type (VH, VK, VL) and sequence length (full-length or framework only) in the "All Human Databases," each antibody sequence was scored with its respective database using the T20 score analyzer. The T20 score was obtained for the top 20 matched sequences after the input sequence itself was excluded (the percent identity of sequences 2 through 21 were averaged since sequence 1 was always the input antibody itself). The T20 scores for each group were sorted from high to low. The decrease in score was roughly linear for most of the sequences; however, the T20 scores for the bottom ~15% of antibodies started decreasing sharply. Therefore, the bottom 15 percent of sequences were removed and the remaining sequences formed the T20 Cutoff Human Databases, where the T20 score cutoff indicates the lowest T20 score of a sequence in the new database.

Accordingly, the humanized anti-PDl antibody comprised in the bifunctional molecule according to the invention has a T20 humanness score of at least 80% or at least 85%, more preferably at least 88%, even more preferably at least 90 %, most preferably a T20 humanness score comprised between 85% and 95%, preferably between 88% and 92%.

A particular example of a humanized anti-hPDl antibody is described hereafter by its CDRs, framework regions and Fc and hinge region.

CDR

"Complementarity determining regions" or "CDRs" are known in the art as referring to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and binding affinity. The precise amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al., (Sequences of Proteins of Immunological Interest 5th ed. (1991) "Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol, 273:927-948 ("Chothia" numbering scheme); MacCallum et al, 1996, J. Mol. Biol. 262:732-745 ("Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering scheme); and Honegge and Pluckthun, J. Mol. Biol, 2001, 309:657-70 ("AHo" numbering scheme). Unless otherwise specified, the numbering scheme used for identification of a particular CDR herein is the Kabat numbering scheme.

In one embodiment, the bifunctional molecule comprises a humanized anti-hPD-1 antibody or an antigen binding fragment thereof. The CDRs regions of the humanized antibody may be derived from a murine antibody and have been optimized to i) provide a safe humanized antibody with a very high level of humanization (superior to 85%) and stability ; and ii) increase the antibody properties, more particularly a higher manufacturability when produced in mammalian cells and a higher production yield in mammal cells such as COS and HCO cells while preserving an antagonist activity and inhibition of the binding of human PD-L1 to human PD-1, as they have a binding affinity (KD) for a human PD-1 less than 10 ⁷ M, preferably less than 10 ⁸ M.

In one embodiment, the antigen-binding fragment of an antibody comprises a heavy chain comprising a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3 and a light chain comprising a variable domain comprising LCDR1, LCDR2 and LCDR3, and a fragment of a heavy chain constant domain. By a fragment of a heavy chain constant domain, it should be understood that the antigen-binding fragment therefore comprises at least a portion of a full heavy chain constant domain. As examples, a heavy chain constant domain may comprise or consist of at least the CHI domain of a heavy chain, or at least the CHI and the CH2 domains of a heavy chain, or at least the CHI, CH2 and CH3 domains of a heavy chain. 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. Accordingly, antigen-binding fragment of an antibody encompasses the Fab portion of a full antibody, the F(ab')2 portion of a full antibody, the Fab' portion of a full antibody. The heavy chain constant domain may also comprise or consist in a full heavy chain constant domain, for example illustrated in the present description, wherein several full heavy chain constant domains are described. In a particular embodiment of the invention, and when the antigen-binding fragment of an antibody comprises a fragment of a heavy chain constant domain comprising or consisting in a portion of a full heavy chain constant domain, the heavy chain constant domain fragment may consist of at least 10 amino acid residues; or may consist of 10 to 300 amino acid residues, in particular 210 amino acid residues.

In one embodiment, the bifunctional molecule comprises a humanized anti-human-PD-1 antibody or an antigen binding fragment thereof, that comprises: (i) a heavy chain variable domain comprising FICDR1, FICDR2 and FICDR3, and (ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein:

- the heavy chain CDR1 (FICDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 1, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but position 3 of SEQ ID NO: 1; - the heavy chain CDR2 (HCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 2, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 13, 14 and 16 of SEQ ID NO: 2;

- the heavy chain CDR3 (HCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 3 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, E; optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 3;

- the light chain CDR1 (LCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 12 wherein X is G or T, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 5, 6, 10, 11 and 16 of SEQ ID NO: 12;

- the light chain CDR2 (LCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 15, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof; and

- the light chain CDR3 (LCDR3) comprises or consists of an amino acid sequence of SEQ ID NO:16, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 1, 4 and 6 of SEQ ID NO: 16.

In another embodiment, the bifunctional molecule comprises a humanized anti-hPD-1 antibody or an antigen binding fragment thereof that comprises: (i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and (ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein:

- the heavy chain CDR1 (HCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 1, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but position 3 of SEQ ID NO: 1;

- the heavy chain CDR2 (HCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 2, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 13, 14 and 16 of SEQ ID NO: 2;

- the heavy chain CDR3 (HCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 3 wherein either XI is D 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; 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; optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 3; - the light chain CDR1 (LCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 12 wherein X is G or T, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 5, 6, 10, 11 and 16 of SEQ ID NO: 12;

- the light chain CDR2 (LCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 15, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof; and

- the light chain CDR3 (LCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 16, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 1, 4 and 6 of SEQ ID NO: 16.

In another embodiment, the bifunctional molecule comprises a humanized anti-hPD-1 antibody or an antigen binding fragment thereof that comprises: (i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and (ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein:

- the heavy chain CDR1 (HCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 1, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but position 3 of SEQ ID NO: 1;

- the heavy chain CDR2 (HCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 2, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 13, 14 and 16 of SEQ ID NO: 2;

- the a heavy chain CDR3 (HCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10 or 11 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10 or 11;

- the light chain CDR1 (LCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 13 or SEQ ID NO:14, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 5, 6, 10, 11 and 16 of SEQ ID NO: 13 or SEQ ID NO:14;

- the light chain CDR2 (LCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 15, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof; and

- the light chain CDR3 (LCDR3) comprises or consists of an amino acid sequence of SEQ ID NO:16, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 1, 4 and 6 of SEQ ID NO: 16. In another embodiment, the bifunctional molecule comprises a humanized anti-hPD-1 antibody or an antigen binding fragment thereof that comprises (i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and (ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein:

- the heavy chain CDR1 (HCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 1, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but position 3 of SEQ ID NO: 1;

- the heavy chain CDR2 (HCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 2, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 13, 14 and 16 of SEQ ID NO: 2;

- the light chain CDR1 (LCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 13, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 5, 6, 10, 11 and 16 of SEQ ID NO: 13;

- the light chain CDR2 (LCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 15, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof; and

- the light chain CDR3 (LCDR3) comprises or consists of an amino acid sequence of SEQ ID NO:16, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 1, 4 and 6 of SEQ ID NO: 16; and

wherein the heavy chain CDR3 (HCDR3) :

- comprises or consists of an amino acid sequence of SEQ ID NO: 5, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 5; or

- comprises or consists of an amino acid sequence of SEQ ID NO: 6, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 6; or

- comprises or consists of an amino acid sequence of SEQ ID NO: 7, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 2, 3, 7 and 8 of SEQ ID NO:7; or

- comprises or consists of an amino acid sequence of SEQ ID NO: 8 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 8; or

- comprises or consists of an amino acid sequence of SEQ ID NO: 9 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 9; or - comprises or consists of an amino acid sequence of SEQ ID NO: 10 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 10; or

- comprises or consists of an amino acid sequence of SEQ ID NO: 11 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 11.

In another embodiment, the bifunctional molecule comprises a humanized anti-hPD-1 antibody or an antigen binding fragment thereof that comprises (i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and (ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein:

- the heavy chain CDR1 (HCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 1, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but position 3 of SEQ ID NO: 1;

- the heavy chain CDR2 (HCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 2, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 13, 14 and 16 of SEQ ID NO: 2;

- the light chain CDR1 (LCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 14, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 5, 6, 10, 11 and 16 of SEQ ID NO: 14;

- the light chain CDR2 (LCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 15, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof; and

- the light chain CDR3 (LCDR3) comprises or consists of an amino acid sequence of SEQ ID NO:16, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 1, 4 and 6 of SEQ ID NO: 16; and

wherein the heavy chain CDR3 (HCDR3):

- comprises or consists of an amino acid sequence of SEQ ID NO: 4, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 4;

- comprises or consists of an amino acid sequence of SEQ ID NO: 5, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 5;

- comprises or consists of an amino acid sequence of SEQ ID NO: 6, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 6; - comprises or consists of an amino acid sequence of SEQ ID NO: 7, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 2, 3, 7 and 8 of SEQ ID NO:7;

- comprises or consists of an amino acid sequence of SEQ ID NO: 8 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 8;

- comprises or consists of an amino acid sequence of SEQ ID NO: 9 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 9;

- comprises or consists of an amino acid sequence of SEQ ID NO: 10 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 10;

- comprises or consists of an amino acid sequence of SEQ ID NO: 11 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 11;

In a particular aspect, the modifications are substitutions, in particular conservative substitutions.

In one embodiment, the anti-human-PD-1 antibody or antigen binding fragment thereof comprises or consists of:

(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: 3 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 (ii) a light chain comprising a CDR1 of SEQ ID NO: 12 wherein X is G or T, 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: 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 wherein 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 a CDR1 of SEQ ID NO: 12 wherein X is G or T, 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: 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; and (ii) a light chain comprising a CDR1 of SEQ ID NO: 12 wherein X is G or T, 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: 3 wherein 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 a CDR1 of SEQ ID NO: 12 wherein X is G or T, 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, 5, 6, 7, 8, 9, 10 or 11; and (ii) a light chain comprising 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; 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: 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.

Framework

In one embodiment, the anti-PDl antibody or antigen binding fragment according to the invention comprises framework regions, in particular heavy chain variable region framework regions (HFR) HFR1, HFR2, HFR3 and HFR4 and light chain variable region framework regions (LFR) LFR1, LFR2, LFR3 and LFR4. Preferably, the anti-PDl antibody or antigen binding fragment according to the invention comprises human or humanized framework regions. A "human acceptor framework" for the purposes herein 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 thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence. A "human consensus framework" is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Particularly, the anti-PDl antibody or antigen binding fragment comprises heavy chain variable region framework regions (HFR) HFR1, FIFR2, HFR3 and HFR4 comprising an amino acid sequence of SEQ ID NOs: 41, 42, 43 and 44, respectively, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 27, 29 and 32 of HFR3, i.e., of SEQ ID NO: 43. Preferably, the anti-PDl 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.

Alternatively or additionally, the anti-PDl antibody or antigen binding fragment comprises light chain variable region framework regions (LFR) LFR1, LFR2, LFR3 and LFR4 comprising an amino acid sequence of SEQ ID NOs: 45, 46, 47 and 48, respectively, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof. Preferably, the humanized anti-PDl 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.

VH-VL

The VL and VH domain of the anti hPDl antibody comprised in the bifunctional molecule according to the invention may comprise four framework regions interrupted by three complementary determining regions preferably operably linked in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from amino terminus to carboxy terminus).

In one embodiment, the anti-human-PD-1 humanized antibody or antigen binding fragment thereof comprised in the bifunctional molecule comprises:

(a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 17, wherein XI is D or E and X2 is selected from the group consisting of T, FI, A, Y, N, E and S preferably in the group consisting of FI, A, Y, N, E; optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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; and

(b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 26, wherein X is G or T, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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.

In another embodiment, the anti-human-PD-1 humanized antibody or antigen binding fragment thereof comprised in the bifunctional molecule comprises:

(a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 17, wherein either XI is D and X2 is selected from the group consisting of T, FI, A, Y, N, E, preferably in the group consisting of FI, A, Y, N, E; or XI is E and X2 is selected from the group consisting of T, FI, A, Y, N, E and S preferably in the group consisting of H, A, Y, N, E and S; optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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; and

(b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 26, wherein X is G or T, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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.

In another embodiment, the anti-human-PD-1 humanized antibody or antigen binding fragment thereof comprised in the bifunctional molecule comprises:

(a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 17, wherein XI 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, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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; and (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 26, wherein X is G or T, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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; or

(a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 17, wherein 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; optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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; and (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 26, wherein X is G or T, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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; or

(a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO:

18, 19, 20, 21, 22, 23, 24 or 25, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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; and (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 28, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position 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, respectively; or

(a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO:

18 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 27, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO:

19 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 27, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO:

20 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 27, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO:

21 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 27, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO:

22 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 27, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO:

23 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 27, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO:

24 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 27, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO:

25 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 27, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 18 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 28, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO:

19 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 28, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO:

20 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 28, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO:

21 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 28, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO:

22 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 28, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO:

23 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 28, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO:

24 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 28, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO:

25 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 28, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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

In a particular aspect, the modifications are substitutions, in particular conservative substitutions.

CH-CL

In one embodiment, the heavy chain (CH) and the light chain (CL) comprises the VL and VH sequences as described hereabove.

In a particular embodiment, the anti-human-PD-1 antibody or antigen binding fragment thereof comprised in the bifunctional molecule comprises:

(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 modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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, respectively; or

(a) a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 29, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 37, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 30, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 37, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 31, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 37, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 32, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 37, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 33, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 37, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 34, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 37, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 35, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 37, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 36, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 37, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 29, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 38, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 30, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 38, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 31, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 38, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 32, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 38, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 33, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 38, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 34, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 38, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 35, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 38, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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) a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 36, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 38, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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.

Preferably, the modifications are substitutions, in particular conservative substitutions.

Fc and hinge region

Several researches to develop therapeutic antibodies had led to engineer the Fc regions to optimize antibody properties allowing the generation of molecules that are better suited to the pharmacology activity required of them. 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 cell phagocytosis (ADCP). Several mutations located at the interface between the CH2 and CH3 domains, such as T250Q/M428L, M252Y/S254T/T256E and H433K/N434F, have been shown to increase the binding affinity to FcRn and the half-life of IgGl in vivo. Flowever, there is not always a direct relationship between increased FcRn binding and improved half-life. One approach to improve the efficacy of a therapeutic antibody is to increase its serum persistence, thereby allowing higher circulating levels, less frequent administration and reduced doses. Engineering Fc regions may be desired to either reduce or increase the effector function of the antibody. For antibodies that target cell-surface molecules, especially those on immune cells, abrogating effector functions is required. Conversely, for antibodies intended for oncology use, increasing effector functions may improve the therapeutic activity. The four human IgG isotypes bind the activating Fey receptors (FcyRI, FcyRIla, FcyRIIIa), the inhibitory FcyRIIb receptor, and the first component of complement (Clq) with different affinities, yielding very different effector functions. Binding of IgG to the FcyRs or Clq depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are critical for FcyRs and Clq binding, and have unique sequences in lgG2 and lgG4.

The antibody according to the invention optionally comprises at least a portion of an immunoglobulin constant region (Fc), typically that of mammalian immunoglobulin, even more preferably a human or humanized immunoglobulin. Preferably, the Fc region is a part of the humanized anti-hPD-1 antibody described herein. The humanized anti-hPDl antibody or antigen binding fragment thereof comprised in the bifunctional molecule of the invention can include a constant region of an immunoglobulin or a fragment, analog, variant, mutant, or derivative of the constant region. As well known by one skilled in the art, the choice of IgG isotypes of the heavy chain constant domain centers on whether specific functions are required and 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 permits complement activation and effector-mediated cell killing by antibody-dependent cell-mediated cytotoxicity. Both human IgGl and lgG3 (shorter half-life) isotypes meet these criteria, particularly human IgGl isotype (wild type and variants). In particular, depending of the IgG isotype of the heavy chain constant domain (particularly human wild type and variants IgGl isotype), the anti-hPDl antibody of the invention can be cytotoxic towards cells expressing PD-1 via a CDC, ADCC and/or ADCP mechanism. In fact, the fragment crystallisable (Fc) region interacts with a variety of accessory molecules to mediate indirect effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC).

In preferred embodiments, the constant region is derived from a human immunoglobulin heavy chain, for example, IgGl, lgG2, lgG3, lgG4, or other classes. In a further aspect, the human constant region is selected from the group consisting of IgGl, lgG2, lgG2, lgG3 and lgG4. Preferably, the anti-PDl antibody comprises an IgGl or an lgG4 Fc-region. In a particular aspect, the humanized anti-PDl antibody comprises a human IgGl Fc region, optionally with a substitution or a 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; K444A and K322A, preferably selected from the group consisting of N297A optionally in combination with M252Y/S254T/T256E, and L234A/L235A.

More preferably, the humanized anti-hPDl antibody comprises an lgG4 Fc-region, optionally with a substitution or a combination of substitutions selected from the group consisting of S228P; L234A/L235A, S228P + M252Y/S254T/T256E and K444A. Even more preferably, the anti-hPDl antibody comprises an lgG4 Fc-region with a S228P that stabilizes the lgG4.

In one embodiment, the anti-PDl antibody comprises a truncated Fc region or a fragment of the Fc region. In one embodiment, the constant region includes a CH2 domain. In another embodiment, the constant region includes CH2 and CH3 domains or includes hinge-CFI2-CFI3. Alternatively, the constant region can include all or a portion of the hinge region, the CH2 domain and/or the CH3 domain. In a preferred embodiment, the constant region contains a CH2 and/or a CH3 domain derived from a human lgG4 heavy chain. In some embodiments, the constant region contains a CH2 and/or a CH3 domain derived from a human lgG4 heavy chain.

In another embodiment, the constant region includes a CH2 domain and at least a portion of a hinge region. The hinge region can be derived from an immunoglobulin heavy chain, e.g., IgGl, lgG2, lgG3, lgG4, or other classes. Preferably, the hinge region is derived from human IgGl, lgG2, lgG3, lgG4, or other suitable classes, mutated or not. More preferably the hinge region is derived from a human IgGl heavy chain. In one embodiment, the constant region includes a CH2 domain derived from a first antibody isotype and a hinge region derived from a second antibody isotype. In a specific embodiment, the CH2 domain is derived from a human lgG2 or lgG4 heavy chain, while the hinge region is derived from an altered human IgGl heavy chain.

In one embodiment, the constant region contains a mutation that reduces affinity for an Fc receptor or reduces Fc effector function. For example, the constant region can contain a mutation that eliminates the glycosylation site within the constant region of an IgG heavy chain.

In another embodiment, the constant region includes a CH2 domain and at least a portion of a hinge region. The hinge region can be derived from an immunoglobulin heavy chain, e.g., IgGl, lgG2, lgG3, lgG4, or other classes. Preferably, the hinge region is derived from human IgGl, lgG2, lgG3, lgG4, or other suitable classes. The IgGl hinge region has three cysteines, two of which are involved in disulfide bonds between the two heavy chains of the immunoglobulin. These same cysteines permit efficient and consistent disulfide bonding formation between Fc portions. Therefore, a preferred hinge region of the present invention is derived from IgGl, more preferably from human IgGl. In some embodiments, the first cysteine within the human IgGl hinge region is mutated to another amino acid, preferably serine. The lgG2 isotype hinge region has four disulfide bonds that tend to promote oligomerization and possibly incorrect disulfide bonding during secretion in recombinant systems. A suitable hinge region can be derived from an lgG2 hinge; the first two cysteines are each preferably mutated to another amino acid. The hinge region of lgG4 is known to form interchain disulfide bonds inefficiently. However, a suitable hinge region for the present invention can be derived from the lgG4 hinge region, preferably containing a mutation that enhances correct formation of disulfide bonds between heavy chain-derived moieties (Angal S, et al. (1993) Mol. Immunol., 30:105-8). More preferably the hinge region is derived from a human lgG4 heavy chain.

In one embodiment, the constant region includes a CH2 domain derived from a first antibody isotype and a hinge region derived from a second antibody isotype. In a specific embodiment, the CH2 domain is derived from a human lgG4 heavy chain, while the hinge region is derived from an altered human IgGl heavy chain.

In accordance with the present invention, the constant region can contain CH2 and/or CH3 domains and a hinge region that are derived from different antibody isotypes, i.e., a hybrid constant region. For example, in one embodiment, the constant region contains CH2 and/or CH3 domains derived from lgG2 or lgG4 and a mutant hinge region derived from IgGl. Alternatively, a mutant hinge region from another IgG subclass is used in a hybrid constant region. For example, a mutant form of the lgG4 hinge that allows efficient disulfide bonding between the two heavy chains can be used. A mutant hinge can also be derived from an lgG2 hinge in which the first two cysteines are each mutated to another amino acid. Assembly of such hybrid constant regions has been described in U.S. Patent Publication No. 20030044423, the disclosure of which is hereby incorporated by reference.

In one embodiment, the constant region can contain CH2 and/or CH3 has one of the mutations described in the Table D below, or any combination thereof.

Table D: Suitable human engineered Fc domain of an antibody. Numbering of residues in the heavy chain constant region is according to EU numbering (Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969); www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html#refs).

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 possesses some, but not all, effector functions. Such antibodies may be useful, for example, in applications in which the half-life of the antibody in vivo is important, yet certain effector functions are unnecessary or deleterious. Examples of effector functions include complement-dependent cytotoxicity (CDC) and antibody-directed complement-mediated cytotoxicity (ADCC). Numerous substitutions or substitutions or deletions with altered effector function are known in the art.

In one embodiment, the constant region contains a mutation that reduces affinity for an Fc receptor or reduces Fc effector function. For example, the constant region can contain a mutation that eliminates the glycosylation site within the constant region of an IgG heavy chain. Preferably, the CH2 domain contains a mutation that eliminates the glycosylation site within the CH2 domain.

In one embodiment, the anti-hPDl according to the invention has a heavy chain constant domain of SEQ ID NO. 39 and/or a light chain constant domain of SEQ ID. 40, particularly a heavy chain constant domain of SEQ ID NO. 39 and a light chain constant domain of SEQ ID. 40. In another embodiment, the anti-hPDl according to the invention has a heavy chain constant domain of SEQ ID NO: 83 and/or a light chain constant domain of SEQ I D. 40, particularly a heavy chain constant domain of SEQ ID NO:83 and a light chain constant domain of SEQ ID. 40.

Table E. Example of a heavy chain constant domain and a light chain constant domain suitable for the humanized antibodies according to the invention.

The alteration of amino acids near the junction of the Fc portion and the non-Fc portion can dramatically increase the serum half-life of the Fc molecule (PCT publication WO 01/58957). Accordingly, the junction region of a protein or polypeptide of the present invention can contain alterations that, relative to the naturally-occurring sequences of an immunoglobulin heavy chain and erythropoietin, preferably lie within about 10 amino acids of the junction point. 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 is replaced. Preferably, the C-terminal lysine of an IgG sequence is replaced with a non-lysine amino acid, such as alanine or leucine, to further increase serum half-life. In particular, K444 amino acid in the IgGl or lgG4 domain may be substituted by an alanine to reduce proteolytic cleavage. Then, in one embodiment, the anti-PDl antibody comprises at least one further amino acid substitution consisting of K444A.

In one embodiment, the anti-PDl antibody comprises an additional cysteine residue at the C-terminal domain of the IgG to create an additional disulfide bond and potentially restrict the flexibility of the bifunctional molecule. In certain embodiments, an antibody may be altered to increase, decrease or eliminate the extent to which it is glycosylated.

In a particular aspect, the bifunctional molecule of the present invention has a Fc domain and the Fc domains of the heavy chains of the bifunctional molecule are identical. Accordingly, a Fc domain of the bifunctional molecule is homodimeric.

Checkpoint inhibitors

The inventors show herein that the bifunctional molecule according to the invention combines the effects of the IL-15Ra or a fragment thereof and the blockade of the inhibitory effect of PD-1, and is suitable for a better targeting of the immune cells which are PD1 positive and are activated by IL15. Then, it can be envisioned that any molecule other than PD-1 that is expressed on immune cell may be targeted by a bifunctional construct according to the invention, particularly a factor of exhaustion. Then, in embodiment, the bifunctional molecule comprises an antibody or antigen binding fragment thereof that is directed against a target expressed on immune cells, other than PD-1. For example, the target can be a receptor expressed at the surface of the immune cells, especially T cells or NK cells. The receptor can be an inhibitor receptor. Alternatively, the receptor can be an activating receptor.

According to the invention, the binding moiety specifically binds to a target expressed on immune cells surface, particularly targets that are only or specifically expressed on immune cells. In particular, the binding moiety is not directed towards a target expressed on tumoral cells.

With regard to the "binding" capacity of the binding moiety, the terms "bind" or "binding" refer to peptides, polypeptides, proteins, fusion proteins, molecules and antibodies (including antibody fragments and antibody mimics) that recognize and contact another peptide, polypeptide, protein or molecule. In one embodiment, it refers to an antigen-antibody type interaction. The terms "specific binding", "specifically binds to," "specific for," "selectively binds" and "selective for" a particular target mean that the binding moiety recognizes and binds a specific target, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically (or preferentially) binds to an antigen is an antibody that binds the antigen for example with greater affinity, avidity, more readily, and/or with greater duration than it binds to other molecules. Preferably, the term "specific binding" means the contact between an antibody and an antigen with a binding affinity equal or lower than 10⁷ M. In certain aspects, antibodies bind with affinities equal or lower than 10⁸ M, 10⁹ M or 10¹⁰ M.

As used herein, the term "target" refers to a carbohydrate, lipid, peptide, polypeptide, protein, antigen or epitope that is specifically recognized or targeted by the binding moiety according to the invention and expressed on the external surface of immune cells. With regards to the expression of a target on the surface of immune cells, the term "expressed" refers to a target, such as carbohydrates, lipids, peptides, polypeptides, proteins, antigens or epitopes that are present or presented at the outer surface of a cell. The term "specifically expressed" mean that the target is expressed on immune cells, but is not substantially expressed by other cell type, particularly such as tumoral cells.

In one embodiment, the target is specifically expressed by immune cells in a healthy subject or in a subject suffering from a disease, in particular such as a cancer. This means that the target has a higher expression level in immune cells than in other cells or that the ratio of immune cells expressing the target by the total immune cells is higher than the ratio of other cells expressing the target by the total other cells. Preferably the expression level or ratio is higher by a factor 2, 5, 10, 20, 50 or 100. More specifically, it can be determined for a particular type of immune cells, for instance T cells, more specifically CD8+ T cells, effector T cells or exhausted T cells, or in a particular context, for instance a subject suffering of a disease such as a cancer or an infection.

"Immune cells" as used herein refers to cells involved in innate and adaptive immunity for example such as white blood cells (leukocytes) which are derived from hematopoietic stem cells (HSC) produced in the bone marrow, lymphocytes (T cells, B cells, natural killer (NK) cells and Natural Killer T cells (NKT)) and myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells). In particular, the immune cell can be selected in the non-exhaustive list comprising B cells, T cells, in particular CD4⁺ T cells and CD8⁺ T cells, NK cells, NKT cells, APC cells, macrophages, dendritic cells and monocytes.

Preferably, the binding moiety specifically binds to a target expressed immune cells selected from the group consisting of B-cells, T-cells, Natural killer, dendritic cells, monocytes and innate lymphoid cells (ILCs).

Even more preferably, the immune cell is a T cell. "T cell" or "T lymphocytes" as used herein includes for example CD4 + T cells, CD8 + T cells, T helper 1 type T cells, T helper 2 type T cells, T regulator, T helper 17 type T cells and inhibitory T cells. In a very particular embodiment, the immune cell is an exhausted T cell. The target can be a receptor expressed at the surface of the immune cells, especially T cells. The receptor can be an inhibitor receptor. Alternatively, the receptor can be an activating receptor.

In one aspect, the target is selected from the group consisting CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, 0X40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, LAG 3, B7-1, 2B4, DR3, CD101, CD 44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4 and CD8. Such targets are more particularly described in the Table F below.

Table F. Example of target of interest.

Then, in this aspect, the binding moiety specifically binds a target selected from the group consisting PD- 1, CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, 0X40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM 3, CD39, CD30, NKG2D, LAG 3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4 and CD8.

In a particular aspect, the immune cell is an exhausted T cell and the target of the binding moiety is an exhaustion factor expressed on the surface of exhausted T cells. T cell exhaustion is a state of T cell progressive loss of function, proliferation capacity and cytotoxic potential, eventually leading to their deletion. T cell exhaustion can be triggered by several factors such as persistent antigen exposure or inhibitory receptors including TIM3, CD244, CTLA-4, LAG-3, BTLA, TIGIT and CD160. Preferably, such exhaustion factor is selected from the group consisting of TIM3, CD244, CTLA-4, LAG-3, BTLA, TIGIT and CD160.

In a preferred embodiment, the binding moiety has an antagonist activity on the target. Numerous antibodies directed against TIM3, CD244, CTLA-4, LAG-3, BTLA, TIGIT and CD160 have already been described in the art.

Antibodies directed against TIM3 and bifunctional or bispecific molecules targeting TIM3 are also known such as Sym023, TSR-022, MBG453, LY3321367, INCAGN02390, BGTB-A425, LY3321367, RG7769 (Roche). In some embodiments, a TFM-3 antibody is as disclosed in International Patent Application Publication Nos. W02013006490, W02016/161270, WO 2018/085469, or WO 2018/129553, WO 2011/155607, U.S. 8,552,156, EP 2581113 and U.S 2014/044728.

Antibodies directed against CTLA-4 and bifunctional or bispecific molecules targeting CTLA-4 are also known such as ipilimumab, tremelimumab, M K-1308, AGEN-1884, XmAb20717 (Xencor), M EDI5752 (AstraZeneca). Anti-CTLA-4 antibodies are also disclosed in WO18025178, W019179388, W019179391, WO19174603, W019148444, WO19120232, WO19056281, WO19023482, W018209701, W018165895, WO18160536, WO18156250, WO18106862, WO18106864, WO18068182, W018035710, WO18025178, W017194265, WO17106372, W017084078, WO17087588, W016196237, WO16130898, WO16015675, WO12120125, W009100140 and W007008463.

Antibodies directed against LAG-3 and bifunctional or bispecific molecules targeting LAG-3 are also known such as BMS- 986016, IMP701, MGD012 or MGD013 (bispecific PD-1 and LAG-3 antibody). Anti-LAG-3 antibodies are also disclosed in W02008132601, EP2320940, W019152574.

Antibodies directed against BTLA are also known in the art such as hu Mab8D5, hu Mab8A3, hu Mab21H6, hu Mabl9A7, or hu Mab4C7. The antibody TAB004 against BTLA are currently under clinical trial in subjects with advanced malignancies. Anti-BTLA antibodies are also disclosed in W008076560, W010106051 (e.g., BTLA8.2), WO11014438 (e.g., 4C7), W017096017 and W017144668 (e.g., 629.3). Antibodies directed against TIGIT are also known in the art, such as BMS-986207 or AB154, BMS-986207 CPA.9.086, CHA.9.547.18, CPA.9.018, CPA.9.027, CPA.9.049, CPA.9.057, CPA.9.059, CPA.9.083, CPA.9.089, CPA.9.093, CPA.9.101, CPA.9.103, CHA.9.536.1, CHA.9.536.3, CHA.9.536.4, CHA.9.536.5, CHA.9.536.6, CHA.9.536.7, CHA.9.536.8, CHA.9.560.1, CHA.9.560.3, CHA.9.560.4, CHA.9.560.5, CHA.9.560.6,

CHA.9.560.7, CHA.9.560.8, CHA.9.546.1, CHA.9.547.1, CHA.9.547.2, CHA.9.547.3, CHA.9.547.4,

CHA.9.547.6, CHA.9.547.7, CHA.9.547.8, CHA.9.547.9, CHA.9.547.13, CHA.9.541.1, CHA.9.541.3, CHA.9.541.4, CHA.9.541.5, CHA.9.541.6, CHA.9.541.7, and CHA.9.541.8 as disclosed in W019232484. Anti-TIGIT antibodies are also disclosed in WO16028656, W016106302, W016191643, W017030823, W017037707, WO17053748, WO17152088, WO18033798, WO18102536, WO18102746, W018160704, W018200430, WO18204363, W019023504, WO19062832, W019129221, W019129261, W019137548, W019152574, W019154415, W019168382 and W019215728.

Antibodies directed against CD160 are also known in the art, such as CL1-R2 CNCM 1-3204 as disclosed in W006015886, or others as disclosed in W010006071, W010084158, WO18077926. In a preferred aspect, the binding moiety of the bifunctional molecule is an antibody, a fragment or a derivative thereof or an antibody mimic that is specific to PD-1, CTLA-4, BTLA, TIGIT, LAG3 and TIM3.

In another particular aspect, the target is CTLA-4 and the binding moiety of the bifunctional molecule is an antibody, a fragment or a derivative thereof or an antibody mimic that is specific to CTLA-4. Then, in a particular embodiment, the binding moiety comprised in the bifunctional molecule according to the invention is an anti-CTLA-4 antibody or antigen binding fragment thereof, preferably a human, humanized or chimeric anti-CTLA-4 antibody or antigen binding fragment thereof. Preferably, the binding moiety is an antagonist of CTLA-4. Therefore, the bifunctional molecule combines the effect of the IL-15R on IL-15 and the blockade of the inhibitory effect of CTLA-4.

In another particular aspect, the target is BTLA and the binding moiety of the bifunctional molecule is an antibody, a fragment or a derivative thereof or an antibody mimic that is specific to BTLA. Then, in a particular embodiment, the binding moiety comprised in the bifunctional molecule according to the invention is an anti-BTLA antibody or antigen binding fragment thereof, preferably a human, humanized or chimeric anti-BTLA antibody or antigen binding fragment thereof. Preferably, the binding moiety is an antagonist of BTLA. Therefore, the bifunctional molecule combines the effect of IL-15R on IL-15 and the blockade of the inhibitory effect of BTLA.

In another particular aspect, the target is TIGIT and the binding moiety of the bifunctional molecule is an antibody, a fragment or a derivative thereof or an antibody mimic that is specific to TIGIT. Then, in a particular embodiment, the binding moiety comprised in the bifunctional molecule according to the invention is an anti-TIGIT antibody or antigen binding fragment thereof, preferably a human, humanized or chimeric anti-TIGIT antibody or antigen binding fragment thereof. Preferably, the binding moiety is an antagonist of TIGIT. Therefore, the bifunctional molecule combines the effect of IL-15R on IL-15 and the blockade of the inhibitory effect of TIGIT.

In another particular aspect, the target is LAG-3 and the binding moiety of the bifunctional molecule is an antibody, a fragment or a derivative thereof or an antibody mimic that is specific to LAG-3. Then, in a particular embodiment, the binding moiety comprised in the bifunctional molecule according to the invention is an anti-LAG-3 antibody or antigen binding fragment thereof, preferably a human, humanized or chimeric anti-LAG-3 antibody or antigen binding fragment thereof. Preferably, the binding moiety is an antagonist of LAG-3. Therefore, the bifunctional molecule combines the effect of IL-15R on IL-15 and the blockade of the inhibitory effect of LAG-3.

In another particular aspect, the target is TIM3 and the binding moiety of the bifunctional molecule is an antibody, a fragment or a derivative thereof or an antibody mimic that is specific to TIM3. Then, in a particular embodiment, the binding moiety comprised in the bifunctional molecule according to the invention is an anti-TIM3 antibody or antigen binding fragment thereof, preferably a human, humanized or chimeric anti-TIM3 antibody or antigen binding fragment thereof. Preferably, the binding moiety is an antagonist of TIM3. Therefore, the bifunctional molecule combines the effect of IL-15R on IL-15 and the blockade of the inhibitory effect of TIM3.

Peptide Linker

This invention includes a bifunctional molecule which may comprise a peptide linker between the anti- PD-1 antibody or fragment thereof and IL-15Ra or the fragment thereof. The peptide linker usually has a length and flexibility enough to ensure that the two protein elements connected with the linker in between have enough freedom in space to exert their functions and avoid influences of the formation of a-helix and b-fold on the stability of the recombinant bifunctional molecule.

In an aspect of the disclosure, the anti-hPDl antibody is preferably linked to IL-15Ra or the fragment thereof by a peptide linker. In other words, the invention relates to bifunctional molecule comprising an anti-PDl antibody as detailed herein or an antigen binding fragment thereof, with a chain, e.g., the light or heavy chain or a fragment thereof, preferably the heavy chain or a fragment thereof, is linked to IL- 15Ra or the fragment thereof through a peptide linker. As used herein, the term "linker" refers to a sequence of at least one amino acid that links IL-15Ra or a fragment thereof and the anti-PD-1 immunoglobulin sequence portion. Such a linker may be useful to prevent steric hindrances. The linker is usually 3-44 amino acid residues in length. Preferably, the linker has 3-30 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.

In an embodiment, the invention relates to a bifunctional molecule comprising an anti-PD-1 antibody or antigen-binding fragment thereof as defined above and IL-15Ra or the fragment thereof, wherein a chain of the antibody, e.g., the light or heavy chain, preferably the heavy chain, even more preferably the C- terminus of the heavy or light chain is linked to IL-15Ra or the fragment thereof, preferably to the N- terminus of IL-15Ra or the fragment thereof, by a peptide linker.

In a particular aspect, the invention relates to a bifunctional molecule comprising an anti-hPD-1 antibody or antigen-binding fragment thereof as defined above, wherein IL-15Ra or the fragment thereof is linked to the C-terminal end of the heavy chain of said antibody (e.g., the C-terminal end of the heavy chain constant domain), preferably by a peptide linker.

In an embodiment, the invention relates to bifunctional molecule comprising an anti-PD-1 antibody or antigen-binding fragment thereof as defined above, wherein IL-15Ra or the fragment thereof is linked to the C-terminal end of the light chain of said antibody (e.g., the C-terminal end of the light chain constant domain), preferably by a peptide linker.

The linker sequence may be a naturally occurring sequence or a non-naturally occurring sequence. If used for therapeutic purposes, the linker is preferably non-immunogenic in the subject to 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. Other examples are poly-alanine linker sequences. Further preferred examples of linker sequences are Gly/Ser linkers of different length including (Gly4Ser)₄, (Gly4Ser)₃, (Gly4Ser)₂, Gly4Ser, Gly3Ser, Gly3, Gly2ser and (Gly3Ser2)₃, in particular (Gly4Ser)₃.

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

In an embodiment, the invention relates to a bifunctional molecule that comprises an anti-PD-1 antibody or a fragment thereof as defined above wherein the antibody or a fragment thereof is linked to IL-15Ra or a fragment thereof by a linker sequence, preferably selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS and (GGGS)3, even more preferably by (GGGGS)3.

Preferably, the heavy chain, preferably the C terminus of the heavy chain of the anti-PD-1 antibody is genetically fused via a flexible (Gly4Ser)3 linker to the N-terminus of IL-15Ra or a fragment thereof. At the fusion junction, the C-terminal lysine residue of the antibody heavy chain can be mutated to alanine to reduce proteolytic cleavage.

Preferably, the heavy chain, preferably the C terminus of the light chain of the anti-PD-1 antibody is genetically fused via a flexible (Gly4Ser)3 linker to the N-terminus of IL-15Ra or a fragment thereof. At the fusion junction, the C-terminal lysine residue of the antibody light chain can be mutated to alanine to reduce proteolytic cleavage.

IL-15R alpha and fragments thereof

The bifunctional molecule according to the invention comprises IL-15Ra or a fragment thereof.

Preferably, the bifunctional molecule according to the invention comprises the extracellular domain of IL- 15Ra or a fragment thereof. More particularly, the extracellular domain of IL-15Ra or the fragment thereof is capable of binding IL-15, more particularly human IL-15.

As used herein, when it is referred to the capacity to bind IL-15, in particular human IL-15, it is intended that the molecule has at least 10 % of the binding capacity of the human IL-15Ra sushi domain as disclosed in SEQ ID NO: 51, preferably at least 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 %. The binding activity can be determined by the method disclosed in Wei et al (2001, J. Immunol, 167, 277-282).

For instance, the bifunctional molecule according to the invention may comprise the extracellular domain of any isoform of IL-15Ra such as an isoform disclosed in SEQ ID NOs: 54-62 or any sequence having at least 80, 85, 90, 95, 96, 97, 98 or 99 % of identity therewith having the capacity to bind IL-15. The extracellular domain of IL-15Ra may comprise or not the signal peptide. In a particular aspect, the isoform can be selected from isoforms 1-4, for instance as disclosed in SEQ ID NOs: 54-57 or any sequence having at least 80, 85, 90, 95, 96, 97, 98 or 99 % of identity therewith having the capacity to bind IL-15. In a very specific aspect, the extracellular domain of IL-15Ra may comprise or consist in an amino acid sequence selected from the group consisting of SEQ ID NOs: 63 and 65-69, or any sequence having at least 80, 85, 90, 95, 96, 97, 98 or 99 % of identity therewith having the capacity to bind IL-15, preferably of SEQ ID NO: 63 or any sequence having at least 80, 85, 90, 95, 96, 97, 98 or 99 % of identity therewith having the capacity to bind IL-15.

In a particular aspect, the bifunctional molecule according to the invention comprises a sushi domain of IL-15Ra or a variant thereof being capable of binding IL-15, more particularly human IL-15.

In a first aspect, the bifunctional molecule according to the invention comprises a sushi domain of IL-15Ra comprising or consisting of the amino acid sequence of SEQ ID NO: 51 or any sequence having at least 80, 85, 90, 95, 96, 97, 98 or 99 % of identity therewith having the capacity to bind IL-15.

In a second aspect, the bifunctional molecule according to the invention comprises a sushi domain of IL- 15Ra comprising or consisting of the consensus amino acid sequence of SEQ ID NO: 70.

In a third aspect, the bifunctional molecule according to the invention comprises a sushi domain of IL- 15Ra comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 51 and 71-81 or any sequence having at least 80, 85, 90, 95, 96, 97, 98 or 99 % of identity therewith having the capacity to bind IL-15.

In a particular aspect, the IL-15Ra fragment of the bifunctional molecule comprises or consists of a sushi domain of IL-15Ra and a hinge region. For instance, the sushi domain of IL-15Ra and hinge region may have or comprise the amino acid sequence of SEQ ID NO: 64 or 82, or any sequence having at least 80, 85, 90, 95, 96, 97, 98 or 99 % of identity therewith having the capacity to bind IL-15.

Bifunctional molecule or "Bicki"

The invention particularly provides a bifunctional molecule that comprises or consists in an anti-hPDl antibody or antibody fragment thereof and IL-15Ra or a fragment thereof as disclosed hereabove, the anti-hPDl antibody or antibody fragment thereof being covalently linked to IL-15Ra or a fragment thereof, preferably by a peptide linker as disclosed hereabove, particularly as a fusion protein.

Particularly, the bifunctional molecule according to the invention comprises two entities: a first entity comprising or consisting essentially of an anti-hPDl antibody or fragment thereof; a second entity comprising or consisting essentially of interleukin 15 receptor (IL-15R), preferably a human IL-15Ra or a fragment thereof, these two entities being optionally linked by a peptide linker.

Particularly, the bifunctional molecule according to the invention comprises one, two, three or four molecules of IL-15Ra or a fragment thereof. Particularly, the bifunctional molecule may comprise only one molecule of IL-15Ra or a fragment thereof, linked to only one light chain or heavy chain of the anti- PD-1 antibody. The bifunctional molecule may also comprise two molecules of IL-15Ra or a fragment thereof, linked to either the light or heavy chains of the anti-PD-1 antibody. The bifunctional molecule may also comprise two molecules of IL-15Ra or a fragment thereof, a first one linked to the light chain of the anti-PD-1 antibody and a second one linked to the heavy chain of the anti-PD-1 antibody. The bifunctional molecule may also comprise three molecules of IL-15Ra or a fragment thereof, two of them being linked to either the light or heavy chains of the anti-PD-1 antibody and the last one linked to the other chain of the anti-PD-1 antibody. Finally, the bifunctional molecule may also comprise four molecules of IL-15Ra or a fragment thereof, two molecules linked to the light chains of the anti-PD-1 antibody and two other molecules linked to the heavy chains of the anti-PD-1 antibody. Accordingly, the bifunctional molecule comprises between one to four molecules of an immunotherapeutic agent as disclosed herein. In one embodiment, only one of the light chains comprises one molecule of IL-15R (e.g. the bifunctional molecule comprises one molecule of IL-15Ra or fragment thereof), only one of the heavy chains comprises one molecule of IL-15R (e.g. the bifunctional molecule comprises one molecule of IL-15Ra or fragment thereof), each light chain comprises one molecule of IL-15Ra or fragment thereof (e.g. the bifunctional molecule comprises two molecules of IL-15R), each heavy chain comprises one molecule of IL-15Ra or fragment thereof (e.g. the bifunctional molecule comprises two molecules of IL-15R), only one of the light chain and only one of the heavy chain comprises one molecule of IL-15R (e.g. the bifunctional molecule comprises two molecules of IL-15Ra or fragment thereof), each light chain comprises one molecule of IL-15Ra or a fragment thereof and only one of the heavy chains comprises one molecule of IL-15R (e.g. the bifunctional molecule comprises three molecule of IL-15Ra or a fragment thereof), each heavy chain comprises one molecule of IL-15Ra or a fragment thereof and only one of the light chain comprises one molecule of IL-15R (e.g. the bifunctional molecule comprises three molecule of IL-15Ra or a fragment thereof), or both light chains and heavy chains comprises one molecule of IL-15Ra or a fragment thereof (e.g. the bifunctional molecule comprises four molecules of IL-15Ra or a fragment thereof).

In one embodiment, the bifunctional molecule according to the invention comprises or consists of:

(a) an anti-human PD-1 antibody or antigen-binding fragment thereof, which comprises (i) a heavy chain, and (ii) a light chain; and

(b) a human interleukin 15 receptor (IL-15R) or a fragment or variant thereof,

wherein the antibody heavy chain and/or light chain or a fragment thereof is covalently linked to IL-15R or a fragment thereof by a peptide linker, preferably as a fusion protein.

Preferably, the bifunctional molecule according to the invention comprises or consists of:

(a) a humanized anti-human PD-1 antibody or antigen-binding fragment thereof, which comprises (i) a heavy chain, and (ii) a light chain; and

(b) an IL-15Ra or a variant or a fragment thereof,

wherein the antibody heavy chain or light chain or a fragment thereof is covalently linked to IL-15Ra or a fragment thereof by a peptide linker, preferably as a fusion protein.

Preferably, such bifunctional molecule comprises at least one peptide linker connecting the N-terminus of IL-15Ra or a fragment thereof to the C-terminus of the heavy chain or of the light chain or both of the anti-human PD-1 antibody, the peptide linker being preferably selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS and (GGGS)3, even more preferably is (GGGGS)₃.

Preferably, the N-terminal end of IL-15Ra or fragment thereof is connected to the C-terminal end of the heavy chain or of the light chain or both of the anti-human PD-1 antibody, though at least one peptide linker. Alternatively, the C-terminal end of IL-15Ra or fragment thereof is connected to the N-terminal end of the heavy chain or of the light chain or both of the anti-human PD-1 antibody, though at least one peptide linker.

In one embodiment, the bifunctional molecule according to the invention comprises or consists of:

(a) an anti-human PD-1 antibody or antigen-binding fragment thereof, which comprises (i) a heavy chain, and (ii) a light chain,

(b) an IL-15Ra or a variant or a fragment thereof, and

(c) a peptide linker that connect the N-terminal end of IL-15Ra or fragment thereof to the C-terminal end of the heavy chain or of the light chain or both of the anti-human PD-1 antibody, the peptide linker being preferably selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS^, GGGGS, GGGS, GGG, GGS and (GGGS)3, even more preferably is (GGGGS)3.

In a particular embodiment, the bifunctional molecule according to the invention comprises or consists of:

(a) an anti-human PD-1 antibody or antigen-binding fragment thereof, which comprises: (i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and (ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein:

- the heavy chain CDR1 (HCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 1, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but position 3 of SEQ ID NO: 1;

- the heavy chain CDR2 (HCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 2, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 13, 14 and 16 of SEQ ID NO: 2;

- the heavy chain CDR3 (HCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 3 wherein either 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; optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 3;

- the light chain CDR1 (LCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 12 wherein X is G or T, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 5, 6, 10, 11 and 16 of SEQ ID NO: 12; - the light chain CDR2 (LCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 15, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof; and

- the light chain CDR3 (LCDR3) comprises or consists of an amino acid sequence of SEQ ID NO:16, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 1, 4 and 6 of SEQ ID NO: 16; and

(b) an IL-15Ra comprising or consisting of the sequence of any one of SEQ ID Nos: 54, 55, 56, 57, 58, 59, 60, 61 or 62 or a variant or a fragment thereof, in particular a fragment comprising or consisting of the sequence of any one of SEQ ID Nos: 51, 70, 71, 72, 73, 74 ,75, 76, 77, 78, 79, 80, 81.

wherein the antibody heavy chain and/or light chain or a fragment thereof is covalently linked to IL-15Ra or a fragment thereof as a fusion protein, preferably by a peptide linker.

In another embodiment, the bifunctional molecule according to the invention comprises or consists of: (a) an anti-human PD-1 antibody or antigen-binding fragment thereof, which comprises: (i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and (ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein:

- the heavy chain CDR1 (HCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 1, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but position 3 of SEQ ID NO: 1;

- the heavy chain CDR2 (HCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 2, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 13, 14 and 16 of SEQ ID NO: 2;

- the heavy chain CDR3 (HCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 3 wherein either XI 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 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; optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 3;

- the light chain CDR1 (LCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 12 wherein X is G or T, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 5, 6, 10, 11 and 16 of SEQ ID NO: 12;

- the light chain CDR2 (LCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 15, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof; and - the light chain CDR3 (LCDR3) comprises or consists of an amino acid sequence of SEQ ID NO:16, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 1, 4 and 6 of SEQ ID NO: 16; and

(b) an IL-15Ra comprising or consisting of the sequence of any one of SEQ ID Nos: 54-62 or a variant or a fragment thereof, preferably a fragment comprising or consisting of the sequence of any one of SEQ IDs: 51, 71-81, even more preferably the sushi domain of IL-15Ra of SEQ ID No: 51 or a variant thereof wherein the antibody heavy chain or light chain or both or a fragment thereof is covalently linked to IL- 15Ra or fragment thereof as a fusion protein, preferably by a peptide linker.

In another embodiment, the bifunctional molecule according to the invention comprises or consists of:

(a) a humanized anti-human PD-1 antibody or antigen-binding fragment thereof, which comprises:

- the heavy chain CDR1 (HCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 1, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but position 3 of SEQ ID NO: 1;

- the heavy chain CDR2 (HCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 2, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 13, 14 and 16 of SEQ ID NO: 2;

- the a heavy chain CDR3 (HCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10 or 11 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10 or 11;

- the light chain CDR1 (LCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 13 or SEQ ID NO:14, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 5, 6, 10, 11 and 16 of SEQ ID NO: 13 or SEQ ID NO:14;

- the light chain CDR2 (LCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 15, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof; and

- the light chain CDR3 (LCDR3) comprises or consists of an amino acid sequence of SEQ ID NO:16, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 1, 4 and 6 of SEQ ID NO: 16; and

(b) an IL-15Ra comprising or consisting of the sequence of any one of SEQ ID No: 54-62 or a variant or a fragment thereof, preferably a fragment comprising or consisting of the sequence of any one of SEQ ID: 51, 71-81, even more preferably the sushi domain of IL-15Ra of SEQ ID No: 51 or a variant thereof. wherein the antibody heavy chain or light chain or a fragment thereof is covalently linked to IL-15Ra or fragment thereof as a fusion protein, preferably by a peptide linker.

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

In another embodiment, the invention relates to a bifunctional molecule that comprises:

(a) a humanized anti-hPDl antibody that comprises:

(i) a heavy chain variable region (VH) comprising or consisting of an 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, E; optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 26, wherein X is G or T, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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, and

(b) an IL-15Ra comprising or consisting of the sequence of any one of SEQ ID No: 54-62 or a variant or a fragment thereof, preferably a fragment comprising or consisting of the sequence of any one of SEQ ID: 51, 71-81, even more preferably the sushi domain of IL-15Ra of SEQ ID No: 51.

(c) a peptide linker selected from the group consisting of (GGGGS)₃, (GGGGS)₄, (GGGGS^, GGGGS, GGGS, GGG, GGS and (GGGS)₃, even more preferably is (GGGGS)₃, between the light chain and/or the heavy chain of the anti-hPDl antibody and the human IL-15Ra or variant or a fragment thereof.

Preferably, the N-terminal end of IL-15Ra or fragment thereof is connected to the C-terminal end of the heavy chain or of the light chain or both of the anti-human PD-1 antibody, though at least one peptide linker. Alternatively, the C-terminal end of IL-15Ra or fragment thereof is connected to the N-terminal end of the heavy chain or of the light chain or both of the anti-human PD-1 antibody, though at least one peptide linker.

In another embodiment, the invention relates to a bifunctional molecule that comprises or consists of: (a) a humanized anti-hPDl antibody that comprises:

(i) a heavy chain variable region (VH) comprising or consisting of an 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, E; optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 26, wherein X is G or T, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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, and

(b) an IL-15Ra comprising or consisting of the sequence of any one of SEQ ID No: 54-62 or a variant or a fragment thereof, preferably a fragment comprising or consisting of the sequence of any one of SEQ ID: 51, 71-81, even more preferably the sushi domain of IL-15Ra of SEQ ID No: 51.

wherein the C-terminal end of the heavy and/or light chain(s) of the antibody or antigen-binding fragment thereof is covalently linked to the N-terminal end of IL-15Ra or fragment thereof, preferably by a (GGGGS)3 peptide linker.

In another embodiment, the invention relates to a bifunctional molecule that comprises or consists of: a) a humanized anti-hPDl antibody that comprises:

(i) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24 or 25, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 28, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position 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.

(b) an IL-15Ra comprising or consisting of the sequence of any one of SEQ ID No: 54-62 or a variant or a fragment thereof, preferably a fragment comprising or consisting of the sequence of any one of SEQ ID: 51, 71-81, even more preferably the sushi domain of IL-15Ra of SEQ ID No: 51.

wherein the C-terminal end of the heavy and/or light chain(s) of the antibody or antigen-binding fragment thereof is covalently linked to the N-terminal end of IL-15Ra or a fragment thereof to form a fusion protein, preferably by a (GGGGS)3 peptide linker.

In a preferred embodiment, the C-terminal end of the heavy chain of the antibody or antigen-binding fragment thereof is covalently linked to the N-terminal end of IL-15Ra or a fragment thereof to form a fusion protein. Preferably, only the heavy chains of the antibody or antigen-binding fragment thereof are covalently linked to IL-15Ra or a fragment thereof.

In another embodiment, the invention relates to a bifunctional molecule that comprises or consists of: a) a humanized anti-hPDl antibody that comprises:

(i) a heavy chain variable region (VH) comprising or consisting of an 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, E; optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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 an amino acid sequence of SEQ ID NO: 26, wherein X is G or T, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but 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, and

(b) an IL-15Ra comprising or consisting of the sequence of any one of SEQ ID No: 54-62 or a variant or a fragment thereof, preferably a fragment comprising or consisting of the sequence of any one of SEQ ID: 51, 71-81, even more preferably the sushi domain of IL-15Ra of SEQ ID No: 51, wherein the C-terminal end of the heavy chain of the antibody or antigen-binding fragment thereof is covalently linked to the N- terminal end of IL-15Ra or a fragment thereof to form a fusion protein, preferably by a (GGGGS)3 peptide linker.

In a very specific aspect, the invention relates to a bifunctional molecule that comprises or consists of: a) a humanized anti-hPDl antibody that comprises (i) a heavy chain comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 35 and (ii) a light chain comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 38, and

(b) a human IL-15Ra of SEQ ID No: 65.

wherein the C-terminal end of the heavy chain of the antibody is covalently linked to the N-terminal end of the IL-15Ra domain to form a fusion protein, optionally through a linker.

In another very specific aspect, the invention relates to a bifunctional molecule that comprises or consists of:

a) a humanized anti-hPDl antibody that comprises (i) a heavy chain comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 35 and (ii) a light chain comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 38, and

(b) a sushi domain of IL-15Ra comprising the amino acid sequence of SEQ ID No: 51.

wherein the C-terminal end of the heavy chain of the antibody is covalently linked to the N-terminal end of the IL-15Ra sushi domain to form a fusion protein, optionally through a linker.

In another very specific aspect, the invention relates to a bifunctional molecule that comprises or consists of:

a) a humanized anti-hPDl antibody that comprises (i) a heavy chain comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 35 and (ii) a light chain comprising or consisting essentially of an amino acid sequence of SEQ ID NO: 38, and

(b) a sushi domain of IL-15Ra and a hinge region of SEQ ID No: 64 or 82. wherein the C-terminal end of the heavy chain of the antibody is covalently linked to the N-terminal end of the IL-15Ra sushi domain to form a fusion protein, optionally through a linker.

Binding of the bifunctional molecules to their specific targets can be confirmed by, for example, 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 protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, the anti-hPD-1 antibody/IL-15Ra complexes can be detected using e.g., an enzyme-linked antibody or antibody fragment which recognizes and specifically binds to IL-15 or a fragment thereof.

In some examples, the bifunctional molecule described herein suppresses 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 by 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. Preferably, such bifunctional molecule has the ability to block or inhibit the interaction between PD-1 and its ligand (e.g. PD-L1 and/or PD-L2). In certain embodiments, the bifunctional molecule inhibits the binding interaction between PD-1 and 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%.

In some examples, the bifunctional molecule described herein suppresses 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 by 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.

In some examples, the bifunctional molecule described herein stimulates IFN gamma secretion and/or Alpha4 and Beta7.

In another example, the bifunctional molecule described herein promotes T cell infiltration in tumor.

In some examples, the bifunctional molecule described herein stimulates IL-15 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 by 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.

In other aspect, the bifunctional molecule described herein retains substantially equivalent biological IL- 15Ra property in comparison to a wild-type IL-15Ra. For instance, it retains comparable biological property as the full-length IL-15Ra protein. The biological activity of IL-15Ra protein can be measured using in vitro cellular proliferation assays or by measuring the P-Stat5 into the T cells by ELISA or FACS. Preferably, the IL-15Ra bifunctional molecule described herein maintains biological activity of at least 10 %, 20%, 30%, 40%, 50%, 60% in comparison with the wild type human IL-15Ra, preferably at least 80%, 90%, 95% and even more preferably 99% in comparison with the wild type IL-15Ra. For instance, the biological activity can be assessed by measuring the binding capacity of the bifunctional molecule described herein to IL-15 and/or the capacity to compete with the wild type IL-15 for the binding to IL- 15Ra.

In another example, the bifunctional molecule described herein induce cytokine secretion, and/or proliferation of naive, partially exhausted and/or fully exhausted T-cell subsets.

Preparation of bifunctional molecule - Nucleic acid molecules encoding the bifunctional molecule, Recombinant Expression Vectors and Host Cells comprising such

To create a bifunctional molecule of the invention, an anti-hPDl antibody of the invention is functionally linked to a human IL-15Ra or the fragment thereof as disclosed above.

Both entities of the bifunctional molecule are encoded in the same vector and produced as a fusion protein. Accordingly, also disclosed herein are nucleic acids encoding any of the bifunctional molecule described herein, vectors such as expression vectors or recombinant viruses comprising these nucleic acids, and host cells comprising the nucleic acids and/or vectors.

To produce a bifunctional fusion protein which is secreted in stable form by mammalian cells, according to the present invention, nucleic acid sequences coding for the bifunctional molecule are subcloned into an expression vector which is generally used to transfect mammalian cells. General techniques for producing molecules comprising antibody sequences are described in Coligan et al. (eds.), Current protocols in immunology, at pp. 10.19.1-10.19.11 (Wiley Interscience 1992), the contents of which are hereby incorporated by reference and in "Antibody engineering: a practical guide" from W. H. Freeman and Company (1992), in which commentary relevant to production of molecules is dispersed throughout the respective texts.

Generally, such method comprises the following steps of:

(1) transfecting or transforming appropriate host cells with the polynucleotide(s) or its variants encoding the recombinant bifunctional molecule of the invention or the vector containing the polynucleotide(s);

(2) culturing the host cells in an appropriate medium; and

(3) optionally isolating or purifying the protein from the medium or host cells.

The invention further relates to a nucleic acid encoding a bifunctional molecule as disclosed above, a vector, preferably an expression vector, comprising the nucleic acid of the invention, a genetically engineered host cell transformed with the vector of the invention or directly with the sequence encoding the recombinant bifunctional molecule, and a method for producing the protein of the invention by recombinant techniques.

The nucleic acid, the vector and the host cells are more particularly described hereafter.

Nucleic acid sequence

The invention also relates to a nucleic acid molecule encoding the bifunctional molecule as defined above or to a group of nucleic acid molecules encoding the bifunctional molecule as defined above. Antibody DNA sequences can for example be amplified from RNA of cells that synthesize an immunoglobulin, synthesized using PCR with cloned immunoglobulins, or synthesized via oligonucleotides that encode known signal peptide amino acid sequences.

Preferably, the peptide signal comprises or consists of the amino acid sequence of SEQ ID NO: 49 for the VH and/or CH; and/or of the amino acid sequence of SEQ ID NO: 50 for the VL and/or CL Particularly, the peptide signal is in the N-terminal of the CH, VH, CL and/or VL.

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

Particularly, the nucleic acid molecules encoding the bifunctional molecule as defined above comprises:

- a first nucleic acid molecule encoding a variable heavy chain domain of an anti-hPD-1 antibody as disclosed herein, optionally with a peptide signal of SEQ ID NO. 49, and

- a second nucleic acid molecule encoding a variable light chain domain of an anti-hPD-1 antibody as disclosed herein, optionally with a peptide signal of SEQ ID NO. 50, and

- a third nucleic acid encoding IL-15Ra or a fragment thereof operably linked to either the first nucleic acid or to the second nucleic acid or both, optionally through a nucleic acid encoding a peptide linker.

Preferably, the nucleic acid molecules encoding the bifunctional molecule as defined above comprises:

- a first nucleic acid molecule encoding a variable heavy chain domain 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; optionally with a peptide signal of SEQ ID NO. 49, and

- a second nucleic acid molecule encoding a variable light chain domain of SEQ ID NO: 26, wherein X is G or T; optionally with a peptide signal of SEQ ID NO: 50, and

- a third nucleic acid molecule encoding a sushi domain of human IL-15Ra, comprising or consisting of the sequence of any one of SEQ ID No: 54-62 or a variant or a fragment thereof, preferably a fragment comprising or consisting of the sequence of any one of SEQ ID: 51, 71-81, even more preferably in particular an amino acid sequence set forth in SEQ ID NO: 51 or a sequence having at least 75% of identity with SEQ ID NO: 51, operably linked to either the first nucleic acid or to the second nucleic acid or both, optionally through a nucleic acid encoding a peptide linker.

Preferably, the nucleic acid molecules encoding the bifunctional molecule as defined above comprises:

- a first nucleic acid molecule encoding a variable heavy chain domain of the amino acid sequence set forth in SEQ ID NO: 18, 19, 20, 21, 22, 23, 24 or 25; optionally with 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 set forth in SEQ ID NO: 27 or SEQ ID NO: 28; optionally with a peptide signal of SEQ ID NO. 50, and

- a third nucleic acid molecule encoding a sushi domain of human IL-15Ra, comprising or consisting of the sequence of any one of SEQ ID No: 54-62 or a variant or a fragment thereof, preferably a fragment comprising or consisting of the sequence of any one of SEQ ID: 51, 71-81, even more preferably in particular an amino acid sequence set forth in SEQ ID NO: 51 or a sequence having at least 75% of identity with SEQ ID NO: 51 operably linked to either the first nucleic acid or to the second nucleic acid or both, optionally through a nucleic acid encoding a peptide linker.

By operably linked is intended that the nucleic acid encodes a protein fusion including the variable heavy or light chain domain, optionally the peptide linker, and IL-15Ra or a fragment thereof. Preferably, the linker is selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS^, GGGGS, GGGS, GGG, GGS and (GGGS)3, even more preferably is (GGGGS)3.

In one embodiment, the nucleic acid molecule is an isolated, particularly non-natural, nucleic acid molecule.

The nucleic acid molecule or group of nucleic acid molecules encoding a bifunctional molecule according to the invention is(are) preferably comprised in a vector or a group of vectors.

Vectors

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

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, engineered vectors comprise an origin of replication, a multicloning site and a selectable marker. The vector itself is generally a nucleotide sequence, commonly a DNA sequence, that comprises an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. Modern vectors may encompass additional features besides the transgene insert and a backbone: promoter, genetic marker, antibiotic resistance, reporter gene, targeting sequence, protein purification tag. Vectors called expression vectors (expression constructs) specifically are for the expression of the transgene in the target cell, and generally have control sequences.

In one embodiment, both the heavy and light chain coding sequences and/or the constant region of the anti-PDl antibody are included in one expression vector. Each of the heavy chain coding sequence and the light chain coding sequence may be in operable linkage to a suitable promoter, the heavy chain and/or the light chain being in operable linkage to an immunotherapeutic agent according to the invention. Alternatively, expression of both the heavy chain and the light chain may be driven by the same promoter. In another embodiment, each of the heavy and light chains of the antibody is cloned in to an individual vector, one or both of the heavy and light chains, the heavy chain and/or the light chain being in operable linkage to an immunotherapeutic agent according to the invention. In the latter case, the expression vectors encoding the heavy and light chains can be co-transfected into one host cell for expression of both chains, which can be assembled to form intact antibodies either in vivo or in vitro. Alternatively, the expression vector encoding the heavy chain and that encoding the light chain can be introduced into different host cells for expression each of the heavy and light chains, which can then be purified and assembled to form intact antibodies in vitro.

The nucleic acid molecule encoding the humanized anti-PD-1 antibody or antibody fragment thereof can be cloned into a vector by those skilled in the art, and then transformed into host cells. Accordingly, the present invention also provides a recombinant vector, which comprises a nucleic acid molecule encoding the anti-PD-1 antibody or fragment thereof of the present invention. In one 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.

Suitable expression vectors typically contain (1) prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance marker to provide for the growth and selection of the expression vector in a bacterial host; (2) eukaryotic DNA elements that control initiation of transcription, such as a promoter; and (3) DNA elements that control the processing of transcripts, such as a transcription termination/polyadenylation sequence.

The methods known to the artisans in the art can be used to construct an expression vector containing the nucleic acid sequence of the bifunctional molecule described herein and appropriate regulatory components for transcription/translation. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc. The DNA sequence is efficiently linked to a proper promoter in the expression vector to direct the synthesis of mRNA. The expression vector may further comprise a ribosome -binding site for initiating the translation, transcription terminator and the like.

An expression vector can be introduced into host cells using a variety of techniques including calcium phosphate transfection, liposome-mediated transfection, electroporation, and the like. Preferably, transfected cells are selected and propagated wherein the expression vector is stably integrated in the host cell genome to produce stable transformants. Techniques for introducing vectors into eukaryotic cells and techniques for selecting stable transformants using a dominant selectable marker are described by Sambrook, by Ausubel, by Bebbington, "Expression of Antibody Genes in Nonlymphoid Mammalian Cells," in 2 M ETHODS: A companion to methods in enzymology 136 (1991), and by Murray (ed.), Gene transfer and expression protocols (Humana Press 1991). Suitable cloning vectors are described by Sambrook et al. (eds.), MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition (Cold Spring Harbor Press 1989) (hereafter "Sambrook"); by Ausubel et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Wiley Interscience 1987) (hereafter "Ausubel"); and by Brown (ed.), MOLECULAR BIOLOGY LAB FAX (Academic Press 1991). Host cells

In another aspect, the invention relates to a host cell comprising a vector or a nucleic acid molecule or group of nucleic acid molecules as defined above, for example for bifunctional molecule production purposes.

As used herein, the term "host cell" is intended to include any individual cell or cell culture that can be or has been recipient of vectors, exogenous nucleic acid molecules, and polynucleotides encoding the antibody construct of the present invention; and/or recipients of the antibody construct or bifunctional molecule itself. The introduction of the respective material into the cell can be carried out by way of transformation, transfection and the like. The term "host cell" is also intended to include progeny or potential progeny of a single cell. Suitable host cells include prokaryotic or eukaryotic cells, and also include but are not limited to bacteria, yeast cells, fungi cells, plant cells, and animal cells such as insect cells and mammalian cells, e.g., murine, rat, rabbit, macaque or human.

In one embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and/or an amino acid sequence comprising the VH of the antibody and/or the constant region of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody.

In another embodiment, a host cell comprises (e.g., has been transformed with) a vector comprising both of the entities of the bifunctional molecule. Preferably, a host cell comprises (e.g., has been transformed with) a vector comprising 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 IL- 15Ra or a fragment thereof. More particularly, a third nucleic acid encoding IL-15Ra or a fragment thereof is operably linked to the first nucleic acid molecule encoding a variable heavy chain domain of an anti- hPD-1 antibody as disclosed herein.

A method of humanized anti-PDl antibody production is also provided herein. The method comprises culturing a host cell comprising a nucleic acid encoding the antibody, 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). Particularly, for recombinant production of a humanized anti-PDl antibody, nucleic acid encoding an antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.

A bifunctional molecule of the present invention is preferably expressed in eukaryotic cells such as mammalian cells, plant cells, insect cells or yeast cells. Mammalian cells are especially preferred eukaryotic hosts because mammalian cells provide suitable post-translational modifications such as glycosylation. Preferably, such suitable eukaryotic host cell may be fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe; insect cell such as Mythimna separate; plant cell 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 cell), monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham, F.L. et al, J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, e.g., in Mather, J.P., Biol. Reprod. 23 (1980) 243-252); Human Epithelial Kidney cell (HEK cell); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather, J.P. et al, Annals N.Y. Acad. Sci. 383 (1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including 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, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268. For example, mammalian cell lines that are adapted to grow in suspension may be useful.

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

For a mammalian host, the transcriptional and translational regulatory signals of the expression vector may be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, in which the regulatory signals are associated with a particular gene which has a high level of expression. Suitable transcriptional and translational regulatory sequences also can be obtained from mammalian genes, such as actin, collagen, myosin, and metallothionein genes.

Stable transformants that produce a bifunctional molecule according to the invention can be identified using a variety of methods. After molecule-producing cells have been identified, the host cells are cultured under conditions (e.g. temperature, medium) suitable for their growth and for bifunctional molecule expression. The bifunctional molecules are then isolated and/or purified by any methods known in the art. These methods include, but are not limited to, conventional renaturation treatment, treatment by protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, supercentrifugation, molecular sieve chromatography or gel chromatography, adsorption chromatography, ion exchange chromatography, HPLC, any other liquid chromatography, and the combination thereof. As described, for example, by Coligan, bifunctional molecule isolation techniques may particularly include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography and ion exchange chromatography. Protein A preferably is used to isolate the bifunctional molecules of the invention.

Pharmaceutical Composition and Method of Administration Thereof

The present invention also relates to a pharmaceutical composition comprising any of the bifunctional molecule described herein, the nucleic acid molecule, the group of nucleic acid molecules, the vector and/or the host cells as described hereabove, preferably as the active ingredient or compound. In a particular aspect, the pharmaceutical composition does not comprise IL-15.

The formulations can be sterilized and, if desired, mixed with auxiliary agents such as pharmaceutically acceptable carriers and excipients which do not deleteriously interact with the bifunctional molecule of the invention, nucleic acid, vector and/or host cell of the invention. Optionally, the pharmaceutical composition may further comprise an additional therapeutic agent as detailed below.

Preferably, the pharmaceutical compositions of the present invention may comprise a bifunctional molecule as described herein, the nucleic acid molecule, the group of nucleic acid molecules, the vector and/or the host cells as described hereabove in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, excipients, salt, and anti-oxidant as described hereafter. Desirably, a pharmaceutically acceptable form is employed which does not adversely affect the desired immune potentiating effects of the bifunctional molecule according to the invention. To facilitate administration, the bifunctional molecule as described herein can be made into a pharmaceutical composition for in vivo administration. The means of making such a composition have been described in the art (see, for instance, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st edition (2005).

Particularly, the pharmaceutical composition according to the invention can be formulated for any conventional route of administration including a topical, enteral, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like. Preferably, the pharmaceutical composition according to the invention is formulated for enteral or parenteral route of administration. Compositions and formulations for parenteral administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carder compounds and other pharmaceutically acceptable carriers or excipients.

The pharmaceutical composition may be prepared by mixing an agent having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium 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 counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN TM, PLURONICS TM or polyethylene glycol (PEG).

A solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavoring agents, 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, sugars, 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 dispersion. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated.

The bifunctional molecule according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like a mixture of both or pharmaceutically acceptable oils or fats and suitable mixtures thereof. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, wetting agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral and enteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols 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 also 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 a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

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

A pharmaceutical composition of the invention also may include a pharmaceutically acceptable anti oxidant. Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetra-acetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

To facilitate delivery, any of the bifunctional molecule or its encoding nucleic acids can be conjugated with a chaperon agent. The chaperon agent can be a naturally occurring substance, such as a protein (e.g., human serum albumin, low-density lipoprotein, or globulin), carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), or lipid. It can 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 acid 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-ethylacryllic acid), N-isopropylacrylamide polymers, and polyphosphazine. In one example, the chaperon agent is a micelle, liposome, nanoparticle, or microsphere. Methods for preparing such a micelle, liposome, nanoparticle, or microsphere are well known in the art. See, e.g., US Patents 5,108,921; 5,354,844; 5,416,016; and 5,527,5285.

Pharmaceutical composition typically must be sterile and stable under the conditions of manufacture and storage. The pharmaceutical composition can be formulated as a solution, micro-emulsion, liposome, or other ordered structure suitable to high drug concentration and/or in suitable for injection. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

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 (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous injection, water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the antibody and physiologically acceptable excipients is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients. Intramuscular preparations, e.g., a sterile formulation of a suitable soluble salt form of the antibody, can be dissolved and administered in a pharmaceutical excipient such as Water-for-lnjection, 0.9% saline, or 5% glucose solution.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

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

It will be understood by one skilled in the art that the formulations of the invention may be isotonic with human blood that is the formulations of the invention have essentially the same osmotic pressure as human blood. Such isotonic formulations generally have an osmotic pressure from about 250 mOSm to about 350 mOSm. Isotonicity can be measured by, for example, a vapor pressure or ice-freezing type osmometer. Tonicity of a formulation is adjusted by the use of tonicity modifiers. "Tonicity modifiers" are those pharmaceutically acceptable inert substances that can be added to the formulation to provide an isotonicity of the formulation. Tonicity modifiers suitable for this invention include, but are not limited to, saccharides, salts and amino acids.

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

The amount of active ingredient which 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 which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect.

Subject, regimen and administration

The present invention relates to a bifunctional molecule as disclosed herein; a nucleic acid or a vector encoding such, 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 in a subject or for use as a medicament. Examples of treatments are more particularly described hereafter under the section "Methods and Uses". It 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 of the invention in the manufacture of a medicament for treating a disease in a subject. Finally, it relates to a method for treating a disease or a disorder in a subject comprising administering a therapeutically effective amount of a pharmaceutical composition or a bifunctional molecule of the invention to the subject. Examples of treatments are more particularly described hereafter under the section "Methods and Uses".

Subject to treat may be a human, particularly a human at the prenatal stage, a new-born, a child, an infant, an adolescent or an adult, in particular an adult of at least 30 years old, 40 years old, preferably an adult of at least 50 years old, still more preferably an adult of at least 60 years old, even more preferably an adult of at least 70 years old.

Particularly, the subject is affected with a disease that may involve the PD-l/PDL-1 pathway, particularly wherein, at least one of the ligands of PD-1 (e.g. PDL-1 and/or PDL-2) or PD-1 is/are expressed, especially overexpressed. Preferably, the subject is suffering from cancer, even more preferably from a PD1, PD-L1 and/or PD-L2 positive cancer or a PD-1 positive cancer. Examples of diseases and cancers are more particularly described hereafter under the section "Methods and Uses".

In a particular embodiment, the subject has already received at least one line of treatment, preferably several lines of treatment, prior to the administration of a bifunctional molecule according to the invention or of a pharmaceutical composition according to the invention.

Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer bifunctional molecule or the pharmaceutical composition disclosed herein to the subject, depending upon the type of diseases to be treated or the site of the disease. This composition can be administered via conventional routes, e.g., administered orally, parenterally, enterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenterally" as used herein includes subcutaneous, intra-cutaneous, intravenous, intramuscular, intra-articular, intra-arterial, intra- synovial, intra-tumoral, intra-sternal, intra-thecal, intra-lesion, and intracranial injection or infusion techniques. When administered parenterally, the pharmaceutical composition according to the invention is preferably administered by intravenous route of administration. When administered enterally, the pharmaceutical composition according to the invention is preferably administered by oral route of administration. This composition can also be administered locally.

The form of the pharmaceutical compositions, the route of administration and the dose of administration of the pharmaceutical composition or the bifunctional molecule according to the invention can be adjusted by the man skilled in the art according to the type and severity of the infection, and to the patient, in particular its age, weight, sex, and general physical condition. The compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired.

Preferably, the treatment with the bifunctional molecule or with a pharmaceutical composition according to the invention is administered regularly, preferably between every day, every week or every month, more preferably between every day and every one, two, three or four weeks. In a particular embodiment, the treatment is administered several times a day, preferably 2 or 3 times a day.

The duration of treatment with the bifunctional molecule or with a pharmaceutical composition according to the invention according to the invention is preferably comprised between 1 day and 20 weeks, more preferably between 1 day and 10 weeks, still more preferably between 1 day and 4 weeks, even more preferably between 1 day and 2 weeks. Alternatively, the treatment may last as long as the disease persists.

The bifunctional molecule disclosed herein may be provided at an effective dose range from about 1 ng/kg body weight to about 30 mg/kg body weight, 1 pg/kg to about 20 mg/kg, 10 pg/kg to about 10 mg/kg, or from 100 pg/kg to 5 mg/kg, optionally every one, two, three or four weeks, preferably by parenteral or oral administration, in particular by intravenous or subcutaneous administration.

Typically, the bifunctional molecule disclosed herein may be provided at an effective dose range from about 1 mg/kg body weight to about 20 mg/kg body weight, advantageously 2 to 10 mg/kg, and in particular 3, 4, 5, 6, 7 mg/kg which is appropriate for antibodies safe administration and very satisfying for the clinical need.

Particularly, the bifunctional molecule according to the invention can be administered at a subtherapeutic dose. The term "subtherapeutic dose" as used herein refers to a dose that is below the effective monotherapy dosage levels commonly used to treat a disease, or a dose that currently is not typically used for effective monotherapy with anti-hPDl antibodies. Methods and Uses

Use in the treatment of a disease

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

Particularly, the bifunctional molecule according to the invention are called "bifunctional checkpoint inhibitors" as they target both PD-1/PD-L1/PD-L2 and IL-15 pathways.

Preferably, the invention relates to a method of treatment of a pathology, disease and/or disorder that could be prevented or treated by the inhibition of the binding of PD-L1 and/or PD-L2 to PD-1.

The invention particularly concerns a bifunctional molecule, a nucleic acid, a group of nucleic acids or a vector encoding such, or a pharmaceutical composition comprising such for use in the treatment of a pathology, disease and/or disorder that could be prevented or treated by the inhibition of the binding of PD-L1 and/or PD-L2 to PD-1.

Accordingly, disclosed herein are methods for treating a disease, in particular associated with the PD-1 and/or PD-1/PD-L1 and/or PD-1/PD-L2 signaling pathway, comprising administering to a subject in need of a treatment an effective amount of any of the bifunctional molecule or pharmaceutical composition described herein. Physiological data of the patient (e.g. age, size, and weight) and the routes of administration have also to be taken into account to determine the appropriate dosage, so as a therapeutically effective amount will be administered to the patient.

In another aspect the bifunctional molecules disclosed herein can be administered to a subject, e.g., in vivo, to enhance immunity, preferably in order to treat a disorder and/or disease. Accordingly, in one aspect, the invention provides a method of modifying an immune response in a subject comprising administering to the subject a bifunctional molecule, nucleic acid, vector or pharmaceutical composition of the invention such that the immune response in the subject is modified. Preferably, the immune response is enhanced, increased, stimulated or up-regulated. The bifunctional molecule or pharmaceutical composition can be used to enhance immune responses such as T cell activation in a subject in need of a treatment. The immune response enhancement can result in the inhibition of the binding of PD-L1 and/or PD-L2 to PD-1 thereby reducing the immunosuppressive environment, stimulating the proliferation and/or the activation of human T-cells and/or the IFNy secretion by human PBMC.

The invention particularly provides a method of enhancing an immune response in a subject, comprising administering to the subject a therapeutic effective amount of any of the bifunctional molecule, nucleic acid, vector or pharmaceutical composition comprising such described herein, such that an immune response in the subject is enhanced.

In some embodiments, the amount of the bifunctional molecule described herein is effective in suppressing the PD-1 signaling (e.g., reducing the PD-1 signaling by at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% as compared to a control). In other embodiments, the amount of the bifunctional molecule described herein is effective in activating immune responses (e.g., by at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% as compared to a control).

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

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

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

Disclosed herein, are methods of treating a patient with a disease and/or disorder, the method comprising: (a) identifying a patient in need of treatment; and (b) administering to the patient a therapeutically effective amount of any of the bifunctional molecule, nucleic acid, vector or pharmaceutical composition described herein.

A subject in need of a treatment may be a human having, at risk for, or suspected of having a disease associated with the signaling pathway mediated by PD-1. Such a patient can be identified by routine medical examination. For example, a subject suitable for the treatment can be identified by examining whether such subject carries PD-1, PD-L1 and/or PD-L2 positive cells. In one embodiment, a subject who needs a treatment is a patient having, suspected of having, or at risk for a disease, preferably a PD-1, PDL1 and/or PDL2 positive disease, even more preferably a disease where PD-1 and/or at least one ligand of PD-1 is overexpressed. In such subject, the disruption of PD-1/PD-L1 and/or PD-1/PD-L2 interaction thanks to the administration of the bifunctional molecule or pharmaceutical composition according to the invention may enhance immune response of the subject. In some embodiments, any of the humanized anti-PD-1 antibodies or pharmaceutical composition described herein can be used for treating PD-1 positive cells.

Cancer

It is known in the art that blockade of PD-1 by antibodies can enhance the immune response to cancerous cells in a patient. Thus, in one aspect, the invention provides a bifunctional molecule or a pharmaceutical composition for use in the treatment of a subject having a cancer, comprising administering to the individual an effective amount of the bifunctional molecule or pharmaceutical composition, preferably to disrupt or inhibit the PD1/PD-L1 and/or PD-1/PD-L2 interaction and/or to activate IL-15 receptor.

In one embodiment, a subject who needs a treatment is a patient having, suspected of having, or at risk for a disease, preferably a PD-1 or PD-L1 or PD-L2 positive cancer, even more preferably a cancer where PD-1 is expressed or overexpressed. In some embodiments, any of the anti-PD-1 antibodies or pharmaceutical composition described herein can be used for treating PD-L1 positive tumor cells. For example, a patient suitable for the treatment can be identified by examining whether such a patient carries PD-L1 positive tumor cells. Additionally or alternatively, the subject suitable for the treatment is a subject having tumor infiltrating T cells that express or overexpress PD-1.

In another embodiment, a subject is a patient having, suspected of having, or at risk for a cancer development, preferably a PD-L1 and/or PD-L2 positive cancer. In some embodiments, any of bifunctional molecule or pharmaceutical composition described herein can be used for treating PD-L1 and/or PD-L2 positive tumors. For example, a human patient suitable for the treatment can be identified by examining whether such a patient carries PD-L1 and/or PD-L2 positive cancer cells.

In further aspects, a bifunctional molecule or pharmaceutical composition for use in treating cancer, preferably a PD-1, PD-L1 and/or PD-L2 positive cancer, even more preferably a cancer wherein PD-1, PD- L1 and/or PD-L2 is/are overexpressed is provided.

In another embodiment, the invention provides the use a bifunctional molecule or pharmaceutical composition as disclosed herein in the manufacture of a medicament for treating a cancer, for instance for inhibiting growth of tumor cells in a subject, preferably PD-L1 or PD-L2 positive tumor cells.

Preferably, by "PD-L1 positive tumor cells" or "PD-L2 positive tumor cells" is intended to refer to a population of tumor cells in which PD-L1 or PD-L2, respectively, are expressed in at least 10% of tumor cells, preferable at least 20, 30, 40 or 50 % of tumor cells. In an aspect of the disclosure, the cancer to be treated is associated with exhausted T cells. In a particular aspect, the bifunctional molecule or pharmaceutical composition according to the present invention is for use in the treatment of cancer by activating exhausted T cells.

Accordingly, in one embodiment, the invention provides a method of treating a cancer, for instance for inhibiting growth of tumor cells, in a subject, comprising administering to the subject a therapeutically effective amount of bifunctional molecule or pharmaceutical composition according to the invention. Particularly, the present invention relates to the treatment of a subject using a bifunctional molecule such that growth of cancerous cells is inhibited.

Any suitable cancer may be treated with the bifunctional molecule provided herein can be hematopoietic cancer or solid cancer. Such cancers include carcinoma, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, lymphoma, glioma, mesothelioma, melanoma, stomach cancer, urethral cancer environmentally induced cancers and any combinations of said cancers. The present invention is also useful for treatment of metastatic cancers, especially metastatic cancers that express PD-L1 (Iwai et al. (2005) Int. Immunol. 17: 133-144). Additionally, the invention includes refractory or recurrent malignancies.

In a particular aspect, the cancer is a hematologic malignancy or a solid tumor with high expression of PD- 1 and/or PD-L1. Such a cancer can be selected from the group consisting of hematolymphoid neoplasms, angioimmunoblastic T cell lymphoma, myelodysplastic syndrome, acute myeloid leukemia.

In a particular aspect, the cancer is a cancer induced by virus or associated with immunodeficiency. Such a cancer can be selected from the group consisting of Kaposi sarcoma (e.g., associated with Kaposi sarcoma herpes virus); cervical, anal, penile and vulvar squamous cell cancer and oropharyngeal cancers (e.g., associated with human papilloma virus); B cell non-Hodgkin lymphomas (NHL) including diffuse large B-cell lymphoma, Burkitt lymphoma, plasmablastic lymphoma, primary central nervous system lymphoma, HHV-8 primary effusion lymphoma, classic Hodgkin lymphoma, and lymphoproliferative disorders (e.g., associated with Epstein-Barr virus (EBV) and/or Kaposi sarcoma herpes virus); hepatocellular carcinoma (e.g., associated with hepatitis B and/or C viruses); Merkel cell carcinoma (e.g., associated with Merkel cell polyoma virus (M PV)); and cancer associated with human immunodeficiency virus infection (HIV) infection.

Preferably, the cancer to be treated or prevented is selected from the group consisting of metastatic or not 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 cancers and Cervical Cancer.

Preferred cancers for treatment include cancers typically responsive to immunotherapy. Alternatively, preferred cancers for treatment are cancers non-responsive to immunotherapy. Preferably, the bifunctional molecules, nucleic acids, vectors, host cells or compositions disclosed herein are for use in the treatment of a subject suffering from cancer with a poor prognosis. As used herein, the term "poor prognosis" refers to a decreased subject survival and/or an early cancer progression and/or an increased or early cancer recurrence and/or an increased risk or occurrence of metastasis. Particularly, the poor prognosis is correlated with a cancer in which a population of Treg cells is present in the tumor or wherein the Treg/Teff ratio is high in the tumor (Chraa et al., 2018 J Leukoc Biol. 2018;1-13.)

By way of example and not wishing to be bound by theory, treatment with an anti-cancer antibody or an anti-cancer immunoconjugate or other current anti-cancer therapy that lead to cancer cell death would potentiate an immune response mediated by PD-1. Accordingly, a treatment of a hyper proliferative disease (e.g., a cancer tumor) may include a bifunctional molecule as disclosed herein combined with an anti-cancer treatment, concurrently or sequentially or any combination thereof, which may potentiate an anti-tumor immune response by the host. Preferably, the bifunctional molecule may be used in combination with other immunogenic agents, standard cancer treatments, or other antibodies as described hereafter.

Infectious disease

The bifunctional molecule, nucleic acid, group of nucleic acid, vector, host cells or pharmaceutical compositions of the invention may be used to treat patients that have been exposed to particular toxins or pathogens. Accordingly, an aspect of the invention provides a method of treating an infectious disease in a subject comprising administering to the subject a bifunctional molecule according to the present invention, or a pharmaceutical composition comprising such, preferably such that the subject is treated for the infectious disease.

Any suitable infection may be treated with a bifunctional molecule, nucleic acid, group of nucleic acid, vector, host cells or pharmaceutical composition according to the present invention provided herein. In a particular aspect, the infection is a viral infection, especially a chronic viral infection, or a sepsis.

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

Particularly, the bifunctional molecule or pharmaceutical compositions of the invention are used to treat patients that have chronic viral infection, such infection being caused by viruses selected from the group consisting of Retroviruses, Anellovirus, Circovirus, Herpesvirus, 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, Papilloma virus, 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, German measles, Parvovirus B19, Measles virus, Coxsackie virus.

Some examples of pathogenic bacteria causing infections treatable by methods of the invention include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and conococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lymes disease bacteria.

Some examples of pathogenic fungi causing infections treatable by methods of the invention include Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treatable by methods of the invention include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondi, and Nippostrongylus brasiliensis.

In all of the above methods, the bifunctional molecule can be combined with other forms of immunotherapy such as cytokine treatment (e.g., interferons, GM-CSF, G-CSF, IL-2), or any therapy, which provides for enhanced presentation of tumor antigens.

Combined therapy

In particular, bifunctional molecule of the present invention can be combined with some other potential strategies for overcoming immune evasion mechanisms with agents in clinical development or already on the market (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 combination with the bifunctional molecule according to the invention may be useful notably for:

1- Reversing the inhibition of adaptive immunity (blocking T-cell checkpoint pathways);

2- Switching on adaptive immunity (promoting T-cell costimulatory receptor signaling using agonist molecules, in particular antibodies),

3- Improving the function of innate immune cells;

4- Activating the immune system (potentiating immune-cell effector function), for example through vaccine-based strategies.

Accordingly, also provided herein are combined therapies for any of the diseases associated with the PD- 1 signaling as described herein with any of the bifunctional molecule or pharmaceutical composition comprising such, as described herein and a suitable second agent. In an aspect, the bifunctional molecule and the second agent can be present in a pharmaceutical composition as described above. Alternatively, the terms "combination therapy" or "combined therapy", as used herein, embrace administration of these two agents (e.g., a bifunctional molecule as described herein and an additional or second suitable therapeutic agent) in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the agents, in a substantially simultaneous manner. Sequential or substantially simultaneous administration of each agent can be affected by any appropriate route. The agents can be administered by the same route or by different routes. For example, a first agent (e.g., a bifunctional molecule) can be administered orally, and an additional therapeutic agent (e.g., an anti-cancer agent, an anti-infection agent; or an immune modulator) can be administered intravenously. Alternatively, an agent of the combination selected may be administered by intravenous injection while the other agents of the combination may be administered orally.

In another aspect, the invention relates to a therapeutic mean, in particular a combination product mean, which comprises as active ingredients: a bifunctional molecule as defined above and an additional therapeutic agent, wherein said active ingredients are formulated for separate, sequential or combined therapy, in particular for combined or sequential use.

As used herein, the term "sequential" means, unless otherwise specified, characterized by a regular sequence or order, e.g., if a dosage regimen includes the administration of a bifunctional molecule and the second agent, a sequential dosage regimen could include administration of the bifunctional molecule of the invention before, simultaneously, substantially simultaneously, or after administration of the second agent, but both agents will be administered in a regular sequence or order. The term "separate" means, unless otherwise specified, to keep apart one from the other. The term "simultaneously" means, unless otherwise specified, happening or done at the same time, i.e., the agents of the invention are administered at the same time. The term "substantially simultaneously" means that the agents are administered within minutes of each other [e.g., within 15 minutes of each other) and intends to embrace joint administration as well as consecutive administration, but if the administration is consecutive it is separated in time for only a short period [e.g., the time it would take a medical practitioner to administer two compounds separately).

It should be appreciated that any combination as described herein may be used in any sequence for treating the disorder or disease described herein. The combinations described herein may be selected on the basis of a number of factors, which include but are not limited to the effectiveness of inhibiting or preventing the target disease progression, the effectiveness for mitigating the side effects of another agent of the combination, or the effectiveness of mitigating symptoms related to the target disease. For example, a combined therapy described herein may reduce any of the side effects associated with each individual members of the combination.

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

When the bifunctional molecule or the pharmaceutical composition described here is co-used with an additional therapeutic agent, a sub-therapeutic dosage of either the bifunctional molecule, the pharmaceutical composition or of the additional or second agent, or a sub-therapeutic dosage of both, can be used in the treatment of a subject, preferably a subject having, or at risk of developing a disease or disorder associated with the cell signaling mediated by PD-1.

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

In an aspect, the additional or second therapeutic agent can be selected in the non-exhaustive list comprising alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotics, antiproliferatives, antivirals, aurora kinase inhibitors, apoptosis promoters (for example, Bcl-2 family inhibitors), activators of death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (Bi-Specific T cell Engager) antibodies, antibody drug conjugates, biologic 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 therapies, immunologicals, inhibitors of inhibitors of apoptosis proteins (lAPs), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, non-steroidal anti inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutics, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoids/deltoids plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, , hypomethylating agents, checkpoints inhibitors, peptide vaccine and the like, epitopes or neoepitopes from tumor antigens, as well as combinations of one or more of these agents. For instance, the additional therapeutic agent can be selected in the group consisting of chemotherapy, radiotherapy, targeted therapy, antiangiogenic agents, hypomethylating agents, cancer vaccines, epitopes or neoepitopes from tumor antigens, myeloid checkpoints inhibitors, other immunotherapies, and HDAC inhibitors.

In a preferred embodiment, the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, radiotherapy agents, immunotherapeutic agents, cell therapy agents (such as CAR-T cells), antibiotics and probiotics. Said immunotherapeutic agent can also be an antibody targeting tumoral antigen, particularly selected from the group consisting of anti-Her2, anti-EGFR, anti-CD20, anti- CD19, anti-CD52. In an embodiment, the invention relates to a combined therapy as defined above, wherein the second therapeutic agent is particularly 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 agents for co-use with any of the anti-hPD-1 antibodies or fragment thereof or with the pharmaceutical composition according to the invention include an antibody binding to a co-stimulatory receptor (e.g., 0X40, CD40, ICOS, CD27, HVEM or GITR), an agent that induces immunogenic cell death (e.g., a chemotherapeutic agent, a radio-therapeutic agent, an anti-angiogenic agent, or an agent for targeted therapies), an agent that inhibits a checkpoint molecule (e.g., CTLA4, LAG3, TIM3, B7H3, B7H4, BTLA, or TIGIT), a cancer vaccine, an agent that modifies an immunosuppressive enzyme (e.g., IDOl or iNOS), an agent that targets T_regcells, an agent for adoptive cell therapy, or an agent that modulates myeloid cells.

In an embodiment, the invention relates to a combined 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 agonist, CD40-L, TLR agonists, anti-ICOS, ICOS-L and B-cell receptor agonists.

In one embodiment, the additional or second therapeutic agent is an antibody targeting tumoral antigen, particularly selected from the group consisting of anti-Her2, anti-EGFR, anti-CD20, anti-CD19, and anti- CD52. Specific examples of second therapeutic agents are provided in WO 2018/053106, pages 36-43, the disclosure thereof being incorporated herein by reference.

Combination therapy could also rely on the combination of the administration of bifunctional molecule with surgery, chemotherapy (e.g. such as docetaxel or decarbazine), radiotherapy, immunotherapy (e.g. such as antibodies targeting CD40, CTLA-4), gene targeting and modulation, and/or other agents such as immune-modulators, angiogenesis inhibitor and any combinations thereof.

EXAMPLES

The following Figures and Examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes 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, composition of matter, process, process 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. Example 1: Antagonist activity of the anti-PD-l/IL-15RA and the anti-PD-l/sushi molecules to block PD- Ll/PD-1 interaction

Method: For the PD-1 binding ELISA assay, recombinant hPDl (Sino Biologicals, Beijing, China; reference 10377-H08H) was immobilized on plastic at 0.5pg/ml in carbonate buffer (pH 9.2) and purified antibody or bifunctional molecules were added to measure binding. After incubation and washing, peroxidase- labeled donkey anti-human IgG (Jackson Immunoresearch; USA; reference 709-035-149) was added the revelation was performed by colorimetry at 450/650nm using TMB substrate (3, 3', 5, 5' Tetramethylenzidine, BD Bioscience, San Jose, USA).

Competitive ELISA assay was performed by PD-1:PD-L1 Inhibitor Screening ELISA Assay Pair (AcroBiosystems; USA; reference EP-101). In this assay, recombinant hPDLl was immobilized on plastic at a concentration of 2pg/ml in PBS buffer pH7.4. Purified antibodies and bifunctional molecules (at different concentrations) were mixed with 0.66pg/ml final (fix concentration) of biotinylated Human PD1 (AcroBiosystems; USA; reference EP-101) to measure competitive binding for 2h at 37°C. After incubation and washing, peroxidase-labeled streptavidin (Vector laboratoring; USA; reference SA-5004) was added to detect Biotin-PD-lFc binding and revealed by conventional methods.

Table 1: Binding of the anti-PDl/sushi and anti-PDl/IL-15RA bifunctional molecules to human PD-1.

Binding was assessed by ELISA and EC50 refers to data represented in Figure 1.

Results: Figure 1 and Table 1 show that the anti-PD-l/sushi and anti-PD-l/IL15RA molecules bind to human PD-1 with similar efficacy compared to an anti-PD-1 alone. In comparison to prior of art, pembrolizumab and nivolumab anti-PD-1 antibodies demonstrate similar binding capacity with an EC50 at 30 and 31.9 pM respectively, as measured in the same binding ELISA assay. The PD-1/PD-L1 antagonist capacity of the molecule was next determined using a competitive ELISA assay as shown in Figure 2. The anti-PD-l/sushi and the anti-PD-l/IL15RA molecules demonstrate good potency to block the PDl/PDL-1 interaction, showing that both molecules will efficiently block the PD-1 mediated inhibitory signaling to reactivate the effector functions of T cells and NK cells.

Example 2: The anti-PD-l/IL-15RA and the anti-PD-l/sushi molecules efficiently trap the IL-15 cytokine Method: Human IL-15 was immobilized overnight on the plate (SinoBiological ref#10360-H07E at final concentration lpg/ml in carbonate Buffer) at 4°C. Following washing (PBS Tween 0.05%) and saturation (PBS Tween 0.05% BSA1%), the antibodies were added at multiple concentrations. Detection was performed with a peroxidase-labeled donkey anti-human IgG (CF 0.4ug/mL, Jackson Immunoresearch; USA; # 709-035-149) and TM B substrate. In this experiment, the recombinant IL-15RA (#147-IR-100, R&Dsystems) was used.

Table 2: Binding of the anti-PDl/sushi and anti-PDl/IL-15RA bifunctional molecules to IL-15 cytokine.

Binding was assessed by ELISA and EC50 refers to data represented in Figure 3.

Results: As shown in Figure 3 and Table 2, the anti-PD-l/sushi and anti-PD-l/IL15RA bifunctional molecules can efficiently bind to IL-15 cytokine with 10-fold superior binding efficacy compared to the IL- 15RA Fc protein, showing that the bifunctional molecules according to the invention have better potency to trap and stabilize soluble IL-15 compared to prior art. In this experiment, the anti-PD-1 alone was also tested as negative control, no binding with the anti-PD-1 alone was observed confirming that IL-15 specifically binds to the sushi and IL-15RA domains of the molecule.

Example 3: In vitro, the anti-PD-l/IL-15RA and anti-PD-l/sushi molecules antagonize IL-15 signaling Method: The capacity of the molecules to activates pSTAT5 signaling in the presence of IL-15 was tested by flow cytometry. Isolated human PBMCs were incubated with or without IL-15 (0,1 or InM) and with different concentrations of bifunctional molecules. After 15 minutes, PBMCs were fixed with fix/perm Buffer (BD Bioscience, #554714), then stained with a FITC labeled anti-hCD3 antibody (BD bioscience, #555332). After washing, cells were permeabilized with Perm Buffer III (BD bioscience, #558050) and stained with an AF647-labeled anti-pSTAT5 (clone 47/Stat5(pY694), #612599). The percentage of pSTAT5+ cells was analyzed in CD3+ T cells.

The effect of the molecule on T cells apoptosis was quantified by flow cytometry after CD3 stimulation. Human PBMCs were isolated from healthy volunteers after Ficoll gradient centrifugation. hPBMCs were chronically stimulated 3 times (every 3 days) with anti-CD3/CD28 antibodies coated plate (2ug/mL of each antibody). After the 3^rd stimulation, human PBMCs were restimulated for 2 days with an anti-CD3 (OKT3 coated plate, 2ug/mL), a fixed concentration of human IL-15 cytokine (O.lnM) and purified antibodies (50nM). Following incubation, cells were harvested and stained with an anti-Annexin-V-FITC (BD bioscience, #556419) diluted in the Annexin-V binding Buffer (BD Bioscience, ref #556454). Staining was analyzed on Cytoflex flow cytometer. Apoptosis was quantified by flow cytometry into CD3+ population using an anti-Annexin-V-FITC and the Annexin-V binding Buffer (BD Pharmingen ref #556454 and #556419).

Results: It is well-described that the soluble IL-15RA or the sushi domain converts the IL-15 cytokine into a superagonist molecule and enhances its activity on proliferation and survival of NK and T cells (Mortier et al The journal of biological chemistry vol. 281, NO. 3, pp. 1612-1619, January 20, 200). Similar conclusions were drawn with IL-15RA-Fc and sushi-Fc molecules (Dubois et al., J Immunol February 15, 2008, 180 (4) 2099-2106;Hu et al., Scientific Reports volume 8, Article number: 7675 (2018)). IL15RA allows the stabilization of IL-15 and its trans-presentation to the IL-15R /y heterodimer receptor leading to a positive signal into NK and T cells.

To determine the in vitro efficacy of the bifunctional molecules to trap IL-15 cytokine and stimulate T cells, the inventors tested the pSTAT5 activation through IL-15 signaling. The inventors observed that, in vitro, the anti-PD-l/IL-15RA and the anti-PD-l/sushi molecules were able to block pSTAT5 mediated signaling mediated by IL-15 (Figure 4B and 4C). IL-15RA Fc molecules also antagonize the pSTAT5 signaling induced by IL-15. No pSTAT5 activation was observed without the addition of IL-15 cytokine (Figure4A), demonstrating the anti-PD-l/IL-15RA and the anti-PD-l/sushi antibodies or the IL15-RA have no agonist effect alone.

IL-15 cytokine can prevent activation-induced apoptosis and enhance secretion of effector cytokines by NK and T cells (Mortier et al., The journal of biological chemistry vol. 281, NO. 3, pp. 1612-1619, January 20, 200). The inventors next assessed the effect of the anti-PD-l/IL-15RA and the anti-PD-l/sushi molecules on T cells by evaluating apoptosis and secretion of TNF-cr/IFN-y following polyclonal activation. As shown on Figure 5A, the anti PD-l/IL-15Ra and anti PD-l/sushi bifunctional molecules block the secretion of TNF-cr/IFN-y by T cells in vitro. T cell apoptosis was assessed by expression of Annexin V marker (Median of fluorescence * % AnnexinV + cells). IL-15 cytokine alone decreases activation induced apoptosis (Figure 5B). The addition of the anti-PD-l/IL-15RA and anti-PD-l/sushi molecules increase the expression of AnnexinV on T cell surface, confirming that the bifunctional molecules antagonize IL-15 activity in vitro and increase T cell apoptosis.

Example 4: In vivo, the anti-PD-l/IL-15RA and the anti-PD-l/sushi molecules enhance the activation and proliferation of NK and T cells

Methods: Chronic antigen stimulation of T cells leading to exhaustion. Human PBMCs were repeatedly stimulated on CD3 CD28 coated plate (3 pg/mL of OKT3 and 3 pg/mL CD28.2 antibody) every 3 days. The response of exhausted T cells to IL-15 was analyzed by STAT5 phosphorylation 48h after each stimulation. The capacity of IL-15 cytokine to promote activation of human tumor infiltrating T cells was also assessed. Fresh kidney human tumor was mashed and T cells were isolated by Ficoll density centrifugation. T cells were then incubated with 40nM of IL-15 and pSTAT5 activation was quantified by flow cytometry using AF647 labeled anti-pSTAT5 (clone 47/Stat5(pY694)) + anti hCD3-V450 antibody.

Ex vivo analysis of NK and T cell activation in MC38 tumor mouse model. Colon cancer MC38 cells (5e5 cells) were subcutaneously injected in the flank. Mice were treated three times with the anti-PDl alone or an isotype control (3 mg/kg) or with the Anti PD-l/sushi bifunctional molecules (11.4 mg/kg). Blood was harvested on Day 7 before treatment, or on Day 10 and 14 using Heparinized microcapillary. Cells were stained with 2 cocktails of antibodies (Panel NK: NKP46-e450, CD3-APC, CD45-AF700, NKG2D-PE, CD27-BV605, KI67 PercyP5.5, CD16/CD32-PeCy7; Panel T cell: CD45-PercyP5.5, CD3-PeCy7, CD8 APC-Cy7, CD69-FITC, CD25-PE, CD44-APC, Ki67-V450). Twenty-five microliter of whole blood was used for each panel. Antibodies were purchased at BDBioscience or Biolegend. Following extracellular staining, intracellular staining was performed with eBioscience™ Intracellular Fixation & Permeabilization Buffer Set (ref#88-8824-00). Staining was analyzed on the Symphony flow cytometer.

Results: Using a model of repeated TCR stimulation in vitro, the inventors recapitulated chronic antigen stimulation of T cells occurring in the tumor and characterized the capacity of T cells to respond to IL-15 after chronic antigen stimulation (Figures 6A). In this model, T cells highly express inhibitory receptor (Tim 3, PD1, Lag3) over-stimulation and lose their capacity to proliferate and to secrete cytokines, a key characteristic of exhausted T cells (Data not shown). Although sensitivity to IL-15 decreases along with chronic stimulations, fully exhausted T cells can respond to IL-15 (5 times stimulated T cells). These data were confirmed with T cells isolated from a human tumor; Figure 6B shows that all tumor-infiltrating CD4 and CD8 T cells can respond to IL-15 as demonstrated by pSTAT5 activation. These data justify the importance of IL-15 cytokine to reactivate anti-tumoral activity of PD-1+ exhausted T cells. The present invention is constructed with an anti-PD-1 antibody to specifically localize the complex IL-15Ra + IL-15 cytokine closed to or directly on intra-tumoral PD-1+ immune cells. Using the online public single cell portal from Broad institute (https://singlecell.broadinstitute.org/single_cell), the inventors analyzed the RNA expression of the complex IL-15-R /y (CD122/CD132) in tumor infiltrating immune cells and peripheral immune cells. This portal uses 2 public gene data-sets for the visualization on non-malignant cells from melanoma tumors (Ding et al., http://dx.doi.org/10.1101/632216) and blood PBMCs (Amon et al., Cell 175, 984-997, November 1, 2018). The inventors observed that both CD122 and CD132 subunits are mainly expressed by intratumoral NK and CD8 T cells. A higher expression of these receptors was observed in intratumoral versus peripheral NK and T cells, showing that the anti PD-l/sushi and anti PD- 1/IL-15RA bifunctional molecules will specifically reactivate intratumoral NK and CD8 T cells expressing CD122 and CD132 subunits.

The anti-tumoral efficacy of the anti-PD-l/sushi bifunctional molecule was tested ex vivo in MC38 subcutaneous model. Although the anti-PD-l/sushi bifunctional molecule demonstrated antagonist activity in vitro, in vivo the molecule anti-PD-l/sushi promotes activation and proliferation of NK and T cells following treatment. As demonstrated in Figure 7A, 7B, 7C and 7D, the anti-PD-l/sushi bifunctional molecule significantly stimulates expression of CD69 and NKG2D activation markers and increases the frequency of CD27+ NK cells, a typical marker of cytotoxic NK cells (Silva et al., International Immunology 2008). This increase was significantly higher compared to treatment with isotype control and/or with anti- PD-1 alone, showing that the sushi domain linked to the PD-1 antibody is able to stimulate IL-15 signaling into NK cell and promote their proliferation, survival and anti-tumor activity in vivo. Figure 8 shows that the anti-PD-l/sushi molecule induces CD8 T cell proliferation to a similar extent to anti-PD-1 antibodies. Altogether, these data demonstrate that the anti-PD-1/ sushi bifunctional molecule has an antitumoral activity in vivo by promoting NK and T-cell effector functions.

Altogether, the data show that the bifunctional molecules according to the invention can enhance activation and proliferation of NK and T cells in vivo. It is well-known that the IL-15 cytokine is rapidly degraded in vivo (half -life less than an hour), limiting its bioavaibility and its anti-tumor-bioactivity. To circumvent this issue, in prior art, naked IL-15 cytokine and Fc fused IL-15 cytokine were described to stimulate NK and T cells anti-tumor function, but this treatment was associated with significant side- effect. Here, the present invention envisions the fusion of antibodies, such as an anti PD-1 antibody, with IL-15RA or sushi domain without IL-15 cytokine. Even without injection of cytokine, the invention can promote activation and proliferation of NK and T cells. The anti PD-l/sushi and the anti PD-l/IL-15Ra possess high affinity to IL-15 cytokine that will allow the stabilization of the peripheral IL-15 cytokine in vivo. In addition, the anti PD-1 domain demonstrates capacity to block PD-1 inhibitory signaling and reactivate proliferation of CD8 T cell in vivo with similar efficacy to the anti PD-1 alone.

Antibodies and bifunctional molecules

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

the anti-PD-1 antibody comprises a heavy chain as defined in SEQ ID NO: 35 and a light chain as defined in SEQ ID NO: 38;

the bifunctional molecule anti-PD-l/sushi comprises a heavy chain as defined in SEQ ID NO: 35 fused at its C terminal end to a sushi domain of SEQ ID NO: 82, and a light chain as defined in SEQ ID NO: 38;

the bifunctional molecule anti-PD-l/IL15Ra comprises a heavy chain as defined in SEQ ID NO: 35 fused at its C terminal end to an IL15Ra of SEQ ID NO: 65, and a light chain as defined in SEQ ID NO: 38.

Motalizumab was used as human lgG4 isotype control for the experiments. This antibody targets RSV

F-Protein.

Claims

1. A bifunctional molecule comprising:

(a) an anti-human PD-1 antibody or an antigen-binding fragment thereof, which comprises:

(i) a heavy chain variable domain (VH) comprising a HCDR1, a HCDR2 and a HCDR3, and

(ii) a light chain variable domain (VL) comprising a LCDR1, a LCDR2 and a LCDR3,

and

(b) a human interleukin 15 receptor alpha (IL-15Ra) or a fragment thereof,

wherein the antibody or the fragment thereof is covalently linked to the human IL-15Ra or the fragment thereof as a fusion protein, preferably by a peptide linker, and

wherein the bifunctional molecule is not bound to IL-15, either covalently or not covalently.

2. A bifunctional molecule comprising:

(a) an anti-human PD-1 antibody or an antigen-binding fragment thereof, which comprises:

(i) a heavy chain variable domain (VH) comprising a HCDR1, a HCDR2 and a HCDR3, and

(ii) a light chain variable domain (VL) comprising a LCDR1, a LCDR2 and a LCDR3,

and

(b) a human interleukin 15 receptor alpha (IL-15Ra) or a fragment thereof,

wherein the antibody or the fragment thereof is covalently linked to the human IL-15Ra or the fragment thereof as a fusion protein, preferably by a peptide linker, and

wherein the anti-human PD-1 antibody or an antigen-binding fragment thereof comprises a Fc domain which is homodimeric.

3. A bifunctional molecule comprising:

(a) an anti-human PD-1 antibody or an antigen-binding fragment thereof, which comprises:

(i) a heavy chain variable domain (VH) comprising a HCDR1, a HCDR2 and a HCDR3, and

(ii) a light chain variable domain (VL) comprising a LCDR1, a LCDR2 and a LCDR3,

and

(b) a human interleukin 15 receptor alpha (IL-15Ra) or a fragment thereof,

wherein the antibody or the fragment thereof is covalently linked to the human IL-15Ra or the fragment thereof as a fusion protein, preferably by a peptide linker, and

wherein each antibody light chain is linked to human IL-15Ra or the fragment thereof and/or each antibody heavy chain is linked to human IL-15Ra or the fragment thereof.

4. The bifunctional molecule of any one of claims 1-3, wherein the N-terminal end of the human IL-15Ra or the fragment thereof is connected to the C-terminal end of the heavy chain or of the light chain of the anti-human PD-1 antibody or the antigen-binding fragment thereof.

5. The bifunctional molecule of any one of claims 1-4, wherein the fragment of the human IL-15Ra comprises or consists of

- the extracellular domain of the human IL-15Ra or a fragment thereof, preferably a fragment capable of binding IL-15, in particular an IL-15Ra comprising or consisting of the sequence of any one of SEQ ID Nos: 54, 55, 56, 57, 58, 59, 60, 61 or 62 or any sequence having at least 80, 85, 90, 95, 96, 97, 98 or 99 % of identity therewith having the capacity to bind IL-15; or

- the sushi domain of the human IL-15Ra, in particular a fragment of IL-15Ra comprising or consisting of the sequence of any one SEQ ID Nos: 51, 70, 71, 72, 73, 74 ,75, 76, 77, 78, 79, 80, 81, preferably an amino acid sequence set forth in SEQ ID NO: 51 or a sequence having at least 75% of identity with SEQ ID NO: 51.

6. The bifunctional molecule of any one of claims 1-5, wherein the antibody or the antigen-binding fragment thereof is a chimeric, a humanized or a human antibody.

7. The bifunctional molecule of any one of claims 1-6, wherein the anti-PDl antibody is be selected from the group consisting of Pembrolizumab, Nivolumab, Pidilizumab, Cemiplimab, PDR001, and monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4.

8. The bifunctional molecule of any one of claims 1-6, wherein the anti-human PD-1 antibody or antigen binding fragment thereof, comprises:

(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,

wherein:

- the heavy chain CDR1 (HCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 1;

- the heavy chain CDR2 (HCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 2;

- the heavy chain CDR3 (HCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 3 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;

- the light chain CDR1 (LCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 12 wherein X is G or T;

- the light chain CDR2 (LCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 15, - the light chain CDR3 (LCDR3) comprises or consists of an amino acid sequence of SEQ ID NO:16.

9. The bifunctional molecule of any one of claims 1-6, wherein the anti-human PD-1 antibody or antigen binding fragment thereof, comprises or consists of (a) a VH comprising or consisting of an 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 an amino acid sequence of SEQ ID NO: 26, wherein X is G or T.

10. The bifunctional molecule of any one of claims 1-9, wherein the anti-human PD-1 antibody or an antigen-binding fragment thereof comprises a Fc domain which is homodimeric and wherein each antibody heavy chain is linked to human IL-15Ra or the fragment thereof.

11. The bifunctional molecule of any one of claims 1 to 10, 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 IgGl, lgG2, lgG3 or lgG4 heavy chain constant domain, preferably an IgGl or lgG4 heavy chain constant domain.

12. The bifunctional molecule of any one of claims 1 to 11, 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 IgGl heavy chain constant domain, optionally with a substitution or a 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; K322A and K444A, preferably selected from the group consisting of N297A optionally in combination with M252Y/S254T/T256E, and L234A/L235A.

13. An isolated nucleic acid molecule or a group of isolated nucleic acid molecules encoding the bifunctional molecule according to any one of claims 1-12.

14. A vector, comprising the nucleic acid or group of nucleic acid molecules according to claim 13.

15. A host cell, comprising the vector according to claim 14 or the nucleic acid or group of nucleic acid molecules of claim 13.

16. A method for producing the bifunctional molecule according to any one of claims 1-12, comprising a step of culturing a host cell according to claim 15 and optionally a step of isolating the bifunctional molecule.

17. A pharmaceutical composition comprising the bifunctional molecule according to any one of claims 1- 12, the nucleic acid or group of nucleic acid molecules according to claim 13, the vector of claim 14 or the host cell of claim 15 and a pharmaceutically acceptable carrier.

18. The pharmaceutical composition of claim 17, wherein it comprises the bifunctional molecule according to any one of claims 1-12 and it does not comprise IL-15.

19. The pharmaceutical composition according to claim 17 or 18, wherein it further comprises an additional therapeutic agent, preferably selected in the group consisting of alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotics, antiproliferatives, antivirals, aurora kinase inhibitors, apoptosis promoters (for example, Bcl-2 family inhibitors), activators of death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (Bi-Specific T cell Engager) antibodies, antibody drug conjugates, biologic 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 therapies, immunologicals, inhibitors of inhibitors of apoptosis proteins (lAPs), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, non-steroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutics, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoids/deltoids plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoints inhibitors, peptide vaccine and the like, epitopes or neoepitopes from tumor antigens, as well as combinations of one or more of these agents.

20. The pharmaceutical composition of any one of claims 17-19 or a bifunctional molecule according to any one of claims 1-12, or a nucleic acid or group of nucleic acid molecules according to claim 13, or a vector of claim 14, or a host cell of claim 15, for use as a medicament.

21. The pharmaceutical composition, bifunctional molecule, nucleic acid or group of nucleic acid molecules, vector, or host cell for the use of claim 20, for use in the treatment of a cancer.

22. The pharmaceutical composition, bifunctional molecule, nucleic acid or group of nucleic acid molecules, vector, or host cell for the use of claim 21, wherein the cancer is selected from the group consisting of a hematologic malignancy or a 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 syndrome, and acute myeloid leukemia, a cancer induced by virus or associated with immunodeficiency such as a cancer selected from the group consisting of Kaposi sarcoma (e.g., associated with Kaposi sarcoma herpes virus); cervical, anal, penile and vulvar squamous cell cancer and oropharyngeal cancers (e.g., associated with human papilloma virus); B cell non-Hodgkin lymphomas (NHL) including diffuse large B-cell lymphoma, Burkitt lymphoma, plasmablastic lymphoma, primary central nervous system lymphoma, HHV-8 primary effusion lymphoma, classic Hodgkin lymphoma, and lymphoproliferative disorders (e.g., associated with Epstein-Barr virus (EBV) and/or Kaposi sarcoma herpes virus); hepatocellular carcinoma (e.g., associated with hepatitis B and/or C viruses); Merkel cell carcinoma (e.g., associated with Merkel cell polyoma virus (MPV)); and cancer associated with human immunodeficiency virus infection (HIV) infection, and a cancer selected from the group consisting of metastatic or not 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 cancers and Cervical Cancer.

23. The pharmaceutical composition, bifunctional molecule, nucleic acid or group of nucleic acid molecules, vector, or host cell for use according to any one of claims 20-22, for use in combination with radiotherapy or an additional therapeutic agent, preferably selected in the group consisting of alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotics, antiproliferatives, antivirals, aurora kinase inhibitors, apoptosis promoters (for example, Bcl-2 family inhibitors), activators of death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (Bi-Specific T cell Engager) antibodies, antibody drug conjugates, biologic 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 therapies, immunologicals, inhibitors of inhibitors of apoptosis proteins (lAPs), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, non-steroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutics, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoids/deltoids plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoints inhibitors, peptide vaccine and the like, epitopes or neoepitopes from tumor antigens, as well as combinations of one or more of these agents.

24. The pharmaceutical composition, bifunctional molecule, nucleic acid or group of nucleic acid molecules, vector, or host cell for use according to claim 20, for use in the treatment of infectious disease, preferably chronic infectious disease, even more preferably chronic viral infections.

25. The pharmaceutical composition, bifunctional molecule, nucleic acid or group of nucleic acid molecules, vector, or host cell for use according to claim 24, wherein the infectious disease is caused by a virus selected from the group consisting of HIV, hepatitis virus, herpes virus, adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.