WO2023213763A1 - Poxvirus encoding a binding agent comprising an anti- pd-l1 sdab - Google Patents

Abstract

The present invention concerns a novel poxvirus comprising a nucleic acid molecule inserted in its genome and encoding a binding agent, wherein the binding agent comprises or consists essentially of, or consists of, a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1). The present invention also concerns a method for producing the poxvirus of the invention. The invention further relates to a composition, in particular a pharmaceutical composition, comprising, or consisting essentially of, the poxvirus according to the invention, in particular a therapeutically effective amount of the poxvirus according to the invention. Moreover, the invention also relates to therapeutic uses or methods of treatment using the poxvirus or composition according to the invention, or any combination thereof, in particular in the treatment or prevention of a proliferative disease, such as cancer.

Description

POXVIRUS ENCODING A BINDING AGENT COMPRISING AN ANTI- PD-L1 SDAB

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the fields of viral therapy and immunology. In particular, the invention concerns a novel poxvirus comprising a nucleic acid molecule inserted in its genome and encoding a binding agent, wherein the binding agent comprises or consists essentially of, or consists of, a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ), and to the compositions and methods related thereto.

BACKGROUND ART

Each year, more than ten of millions of people are diagnosed with cancer around the world and more than half, eventually, die (Nouri Rouzbahani et al. , 2018). Although a vast number of chemotherapeutics exists, they are often ineffective, especially against malignant and metastatic tumors that establish at a very early stage of the disease. Therefore, research endeavor to develop new strategies to treat cancer and improve patient survival. One of the recent successful strategies is to induce the immune system of patient to destroy tumor cells (immuno-oncology).

Oncolytic viruses

Oncolytic virotherapy is part of the immuno-oncology approaches that utilizes natural or engineered oncolytic viruses (OV) to induce and/or boost anti-tumoral immune response. A virus is a non-living organism that infects cells and uses their machinery to replicate and spread to the surrounding uninfected cells. Some of the viruses, spontaneously or after modifications of their genome, can selectively infect and lyse tumor cells while leaving healthy cells unaffected. Those type of viruses are called oncolytic viruses (OV). Some of the OVs, particularly those with large DNA genome, can be modified (or armed) and served as carrier for multiple transgenes with wide variety of therapeutic activities. For instance, armed-OV can encode and deliver various therapeutic proteins in the tumor micro-environment (TME) to further enhance the antitumoral activity. This is the case of one herpes simplex virus, (Talimogene Laherparepvec) that encodes the cytokine Granulocyte-Macrophage Colony stimulating factor (GM-CSF) and that is the only FDA approved OV for the treatment of metastatic melanoma.

Poxviruses and especially Vaccinia viruses (VV) have provided several promising oncolytic candidates (De Graaf et al., 2018, doi.org/10.1016/j.cytogfr.2018.03.006), such as JX594 (Sillajen/Transgene), GL-ONC1 (Genelux), TG6002 (Transgene) and vvDD-CDSR (University of Pittsburg). These oncolytic W originate from different W strains with diverse genomic modifications and expression of various therapeutic genes. JX-594 (Wyeth strain) attenuated through deletion of the viral J2R gene (which encodes thymidine kinase (tk)) and further armed with GM-CSF was under clinical evaluation in a randomized Phase III trial in hepatocellular carcinoma (Parato et al., 2012, Molecular Therapy 20(4): 749-58). GL-ONC1 was generated by inserting three expression cassettes respectively in place of the F14.5L, J2R and A56R loci of the parental viral Lister strain genome. On the same line, TG6002, a J2R (tk) and I4L (I4L locus encodes ribonucleotide reductase (rr-))- defective VV (Copenhagen strain) encoding the FCU1 enzyme that converts the non-toxic 5 -fluorocytosine (5-FC) into the cytotoxic 5 fluorouracile (5-FU) is being evaluated in some clinical trials. The tk and rr double deletion restricts the replication of the virus to cells containing a high pool of nucleotides, making TG6002 unable to replicate in resting cells (Foloppe et al., 2008, Gene Ther. 15: 1361 -71 ; W02009/065546). vvDD-CDSR was assayed in patients with refractory cutaneous and subcutaneous tumors. It was engineered by double deletion of the tk (J2R locus) and vaccinia growth factor (vgf) encoding genes and armed with both a cytosine deaminase (CD) gene for conversion of 5-FC to 5-FU and a somatostatin receptor (SR) gene for in vivo imaging.

VV offer several advantages. This virus is genetically stable and replicate exclusively in the cytoplasm of cells (no risk of integration in host genome). Moreover, its large DNA genome is easily engineerable to encode multiple therapeutic transgenes (armed). Accordingly, it can be modified to improve its safety by increasing its tropism toward tumor cells by deletion of genes important for replication in resting (i.e., healthy) cells. Thus, Thymidine Kinase (TK) deletion for example, restricts the replication of the virus to high nucleotide pool containing cells (i.e., proliferating or tumor cells; Lusky et al., 2010). Moreover, VV can also be administrated intravenously reaching patients’ metastasis (Vanderplasschen et al. 1997). W infects most of mammalian cells and does not require the expression of a specific receptor at the surface of cell. Finally, its process of industrial production is well established. For all these reasons, several groups and biotech companies use different strains of VV, carrying different deletions and transgenes as single agent or in combination in both preclinical and clinical investigations (Pelin et al., 2020).

Immune checkpoint inhibitors

Immuno-modulators such as cytokines, co-stimulatory molecules, and Immune Checkpoint Inhibitors (ICI) are particularly interesting to induce and/or exacerbate anti-tumor immune response and ultimately the destruction of tumor.

ICI are clinically approved monoclonal antibodies (mAb) that are administrated every day to patients to target different type of cancers (such as Melonama, Colon carcinoma, lung cancer).

ICI target molecules on immune or tumor cells that decrease specific anti-tumoral T-cell production and/or activity. ICI such as cytotoxic T lymphocytes (CTL) Antigen 4 (CTLA-4), Program Cell Death (PD-1 ), PD-1 Ligand (PD-L1 ) inhibit the process of T cell activation and are physiologically important to avoid any detrimental overactivation of the immune system. In case of cancers, tumors express ICI to preserve themself from the destruction by T cells. ICI represent therefore targets of choice for the treatment of cancer and indeed, there are several approved blocking mAb recognizing these molecules available in the current anti-cancer arsenal. These clinical tremendous successes led to the awarding of the 2018 Nobel prize in physiology or medicine to Drs. Alison and Honjo (Hargadon et al., 2018) who discovered ICI and their potential use as therapeutic targets. PD-1 is by far the most targeted of the ICI in clinic. PD-1 is a type I transmembrane protein that is transcriptionally induced in activated T cells, B cells and myeloid cells. This costimulatory protein contains a single Ig-like variable (IgV) domain at the extracellular region and an immunoreceptor tyrosine- based inhibitory motif (ITIM) in the cytoplasmic region. Once PD-L1 or PD-L2 binds to PD-1 , tyrosine in the ITIM is phosphorylated which leads to a downstream signaling that ultimately inhibits the T Cell Receptor (TCR)- mediated lymphocyte proliferation, cytokine secretion, and lymphocyte mobility (Freeman el al. 2000). Thus, PD-1 pathway is highly important to maintain immune tolerance, by tuning down immune response and preventing too strong immune response that can damage tissues. In case of cancer, many mutations accumulate in genome of the tumor cells, some of them affect protein -coding sequences (open reading frame, ORF) and result in altered protein function involved in the process of tumorigenesis (driver mutations). But most of non-sense mutations in ORF are not associated with a phenotype (passenger mutations). As in all nucleated cells, these mutated proteins are cleaved by proteasome and some of the generated peptides are presented on the cell surface associated to the Major Histocompatibility Complex (MHC) scaffold. These mutated peptides presented on MHC are called neoantigen. These neoantigens/MHC complex can be recognized as non-self by the TCR leading to the formation of the immune synapse between the tumor and T cells (Efremova et al. 2017). This tight interaction between the two cells triggers an activation and degranulation of T cells and ultimately the lysis of the tumor cells. Tumor cells escape this destruction by expression of multiple immune downregulating molecules such as PD-L1 , TGF-Beta, CTLA-4.

Indeed, approximately 30% of solid tumor cell lines express PD-L1. Therefore, blocking PD-1 /PD-L1 interaction results in the reversal of the inhibition of CTL and an efficient antitumor immune response. There are currently 5 mAb used as a treatment for cancerous patients by blocking the PD-1 /PD-L1 interaction, including Avelumab (Bavencio®, Pfizer).

ICI vectorization in poxviruses

Antibodies can be vectorized in poxvirus genomes (Kleinpeter et al., 2016). However, because of their relatively large size and complex chain assembly, mAb are poorly suitable for modularity (e.g. two mAb cannot be vectorized due to chain mispairing issue) or for diffusion inside the tumoral tissue.

Therefore, there is a pressing need to provide novel vectorized antibodies format targeting important ICI, such as ICI of the PD-1 pathway, having improved diffusion inside the tumoral tissue, to enhance the T cell mediated cytotoxicity inside the tumor.

The present invention fulfils this need. Indeed, the present Inventors have developed original poxviruses comprising a nucleic acid molecule encoding a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ). The experimental data demonstrate that the present invention provides novel and efficient treatment of proliferative disease, such as cancer.

SUMMARY OF THE INVENTION

In the context of the present invention, the Inventors designed novel poxviruses comprising a nucleic acid molecule encoding a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ).

The Inventors have in particular developed several poxviruses, each expressing different formats of this anti-PD-L1 sdAb, including a monomeric form, a dimeric form, a form fused to the hinge of a human IgG, and a form fused to a human Fc. The Inventors unexpectedly showed that, when expressed by these poxviruses, all these anti-PD-L1 sdAbs possess a significantly higher blocking activity than the reference anti-PD-L1 antibody, Avelumab. In addition, the experimental data obtained by the Inventors demonstrated that all these anti-PD-L1 sdAb expressed by these poxviruses are capable to bind PD-L1 at the surface of tumor cell, more efficiently than expressed Avelumab. Importantly, the Inventors confirmed that replication and oncolytic activities of the poxviruses expressing these anti-PD-L1 sdAbs were unaffected by the presence and expression of the transgene encoding the sdAb. Binding of these anti-PD-L1 sdAb to cynomolgus monkey PD-L1 was also demonstrated, showing that the results are not limited to human.

The anti-PD-L1 sdAb encoded by the poxviruses of the invention are particularly advantageous over the anti-PD-L1 antibodies of the prior art, including Avelumab. Indeed, the significantly lower molecular weights and sizes of all of the anti-PD-L1 sdAb formats designed by the Inventors allows a better penetration and diffusion into tumor tissue, compared to anti-PD-L1 antibodies. In addition, such anti-PD-L1 sdAbs have a shorter halflife compared to anti-PD-L1 antibodies of the prior art, including Avelumab. This advantage is particularly beneficial to prevent unwanted systemic diffusion in the organism, thereby improving specificity of the targeting of tumor cells.

Altogether, the poxviruses expressing various formats of a sdAb specifically binding to PD- L1 here developed by the Inventors provides a novel and efficient treatment of proliferative disease, such as cancer.

Accordingly, the present invention relates to a poxvirus comprising a nucleic acid molecule inserted in its genome and encoding a binding agent, wherein the binding agent comprises or consists essentially of, or consists of, a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ) comprising three heavy chain Complementarity-determining regions CDR1 , CDR2, and CDR3; and wherein:

• the heavy chain CDR1 consists of sequence RTFREYGMG (SEQ ID NO:1 ),

• the heavy chain CDR2 consists of sequence TISSSGSYXiY, wherein Xi is S or T (SEQ ID NO:2),

• the heavy chain CDR3 comprises the sequence X2SLLRGX3SSRAEX4YDX5, wherein each of X2 to X5 independently represents any amino acid (SEQ ID NO:3).

The present invention also concerns a method for producing the poxvirus of the invention. The invention further relates to a composition, in particular a pharmaceutical composition, comprising, or consisting essentially of, the poxvirus according to the invention, in particular a therapeutically effective amount of the poxvirus according to the invention.

Moreover, the invention also relates to therapeutic uses or methods of treatment using the poxvirus or composition according to the invention, or any combination thereof, in particular in the treatment or prevention of a proliferative disease, such as cancer.

DETAILED DESCRIPTION OF THE INVENTION

In the context of the present invention, the Inventors surprisingly found that single domain antibodies (sdAb) of various forms, specifically binding to programmed deathligand 1 (PD-L1 ), when expressed by poxviruses, possess a significantly higher blocking activity than the reference anti-PD-L1 antibody, Avelumab.

The replication and oncolytic properties of the novel poxviruses expressing these anti-PD- L1 sdAbs, thus designed by the Inventors, were unaffected by the presence and expression of the transgene encoding the sdAb.

Several poxviruses were developed by the Inventors, each expressing different formats of this anti-PD-L1 sdAb, including a monomeric form, a dimeric form, a form fused to the hinge of a human IgG, and a form fused to a human Fc. In addition, the experimental data obtained by the Inventors unexpectedly demonstrated that all these anti-PD-L1 sdAbs expressed by these poxviruses are capable to bind PD-L1 at the surface of tumor cell, more efficiently than expressed Avelumab. Importantly, binding of these anti-PD-L1 sdAb to cynomolgus monkey PD-L1 was also demonstrated, showing that the results are not limited to human.

The anti-PD-L1 sdAb encoded by the poxviruses of the invention are particularly advantageous over the anti-PD-L1 antibodies of the prior art, including Avelumab. Indeed, the significantly lower molecular weights and sizes of all of the anti-PD-L1 sdAb formats designed by the Inventors allows a better penetration and diffusion into tumor tissue, compared to anti-PD-L1 antibodies. In addition, such anti-PD-L1 sdAbs have a shorter halflife compared to anti-PD-L1 antibodies of the prior art, including Avelumab. This advantage is particularly beneficial to prevent unwanted systemic diffusion in the organism, thereby improving specificity of the targeting of tumor cells.

Altogether, the poxviruses expressing various formats of a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ) here developed by the Inventors provides a novel and efficient treatment of proliferative disease, such as cancer.

General definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

As used herein throughout the entire application, the terms "a" and "an" are used in the sense that they mean "at least one", "at least a first", "one or more" or "one or a plurality" of the referenced compounds or steps, unless the context dictates otherwise.

The term "and/or" wherever used herein includes the meaning of "and ", "or" and "all or any other combination of the elements connected by said term ".

The term "about" or "approximately" as used herein means within 10%, preferably within 8%, and more preferably within 5% of a given value or range.

The terms “amino acids”, “residues” and “amino acid residues” are used interchangeably and encompass natural amino acids as well as amino acid analogs (e.g. non-natural, synthetic and modified amino acids, including D or L optical isomers).

As used herein, when used to define products, compositions and methods, the term "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are open-ended and do not exclude additional, unrecited elements or method steps. Thus, a polypeptide "comprises" an amino acid sequence when the amino acid sequence might be part of the final amino acid sequence of the polypeptide. Such a polypeptide can have up to several hundred additional amino acids residues (e.g. linker and targeting peptides as described herein). "Consisting essentially of" means excluding other components or steps of any essential significance. Thus, a polypeptide "consists essentially of” an amino acid sequence when such an amino acid sequence is present with eventually only a few additional amino acid residues. "Consisting of” means excluding more than trace elements of other components or steps. For example, a polypeptide "consists of” an amino acid sequence when the polypeptide does not contain any amino acids but the recited amino acid sequence.

The terms “polypeptide”, “peptide” and “protein” are used interchangeably to refer to polymers of amino acid residues comprising at least nine amino acids covalently linked by peptide bonds. The polymer can be linear, branched or cyclic and may comprise naturally occurring and/or amino acid analogs and it may be interrupted by non-amino acids. No limitation is placed on the maximum number of amino acids comprised in a polypeptide. As a general indication, the term refers to both short polymers (typically designated in the art as peptide) and to longer polymers (typically designated in the art as polypeptide or protein). This term encompasses native polypeptides, modified polypeptides (also designated derivatives, analogs, variants or mutants), polypeptide fragments, polypeptide multimers (e.g. dimers), recombinant polypeptides, fusion polypeptides among others.

Within the context of the present invention, the terms “nucleic acid”, “nucleic acid molecule”, “polynucleotide”, "nucleic acid sequence" and “nucleotide sequence” are used interchangeably and define a polymer of at least 9 nucleotide residues in either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or mixed polyribo- polydeoxyribonucleotides. These terms encompass single or double-stranded, linear or circular, natural or synthetic, unmodified or modified versions thereof (e.g. genetically modified polynucleotides; optimized polynucleotides), sense or antisense polynucleotides, chimeric mixture (e.g. RNA-DNA hybrids). Exemplary DNA nucleic acids include without limitation, complementary DNA (cDNA), genomic DNA, plasmid DNA, vectors, viral DNA (e.g. viral genomes, viral vectors), oligonucleotides, probes, primers, coding DNA, non-coding DNA, or any fragment thereof etc. Exemplary RNA nucleic acids include, without limitation, messenger RNA (mRNA), precursor messenger RNA (pre- mRNA), coding RNA, non-coding RNA, etc. Nucleic acid sequences described herein may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as those that are commercially available from Biosearch, Applied Biosystems, etc.) or obtained from a naturally occurring source (e.g. a genome, cDNA, etc.) or an artificial source (such as a commercially available library, a plasmid, etc.) using molecular biology techniques well known in the art (e.g. cloning, PCR, etc).

The percent identities referred to in the context of the disclosure of the present invention are determined after optimal global alignment of the sequences to be compared, which optimal global alignment may therefore comprise one or more insertions, deletions, truncations and/or substitutions. The alignment is global, meaning that it includes the sequences to be compared taken in their entirety over their entire length. The alignment is “optimal”, meaning that the number of insertions, deletions, truncations and/or substitutions is made as low as possible. The optimal global alignment may be performed and the percent identity calculated using any sequence analysis method well-known to the person skilled in the art. In addition to manual comparison, it is possible to determine global alignment using the algorithm of Needleman and Wunsch (1970). For nucleotide sequences, the sequence comparison may be performed using any software well-known to a person skilled in the art, such as the Needle software. The parameters used may notably be the following: “Gap open” equal to 10.0, “Gap extend” equal to 0.5, and the EDNAFULL matrix (NCBI EMBOSS Version NUC4.4). For amino acid sequences, the sequence comparison may be performed using any software well-known to a person skilled in the art, such as the Needle software. The parameters used may notably be the following: “Gap open” equal to 10.0, “Gap extend” equal to 0.5, and the BLOSUM62 matrix.

The term "obtained from ", “originating” or “originate” is used to identify the original source of a component (e.g. polypeptide, nucleic acid molecule) but is not meant to limit the method by which the component is made which can be, for example, by chemical synthesis or recombinant means.

As used herein, the term “host cell” should be understood broadly without any limitation concerning particular organization in tissue, organ, or isolated cells. Such cells may be of a unique type of cells or a group of different types of cells such as cultured cell lines, primary cells and dividing cells. This term also includes cells that can be or has been the recipient of the non-propagative viral vector for use in the invention, as well as progeny of such cells.

The term “subject” generally refers to a vertebrate organism for whom any of the product or methods disclosed herein is needed or may be beneficial. Typically, the organism is a mammal, particularly a mammal selected from the group consisting of domestic animals, farm animals, sport animals, and primates (human and non-human). The terms “subject” and “patient” may be used interchangeably when referring to a human organism and covers male and female as well as a fetuses, newborn, infant, young adult, adult and elderly.

As used herein, the term “tumor” may be used interchangeably with any of the terms “cancer”, “malignancy”, “neoplasm” and encompasses any disease or pathological condition resulting from uncontrolled cell growth and spread. These terms are meant to include any type of tissue, organ or cell, any stage of malignancy (e.g. from a prelesion to stage IV). Typically, tumors, especially malignant tumors, show partial or complete lack of structural organization and functional coordination as compared to normal tissue and generally show a propensity to invade surrounding tissues (spreading) and/or metastasize to farther sites. The present invention is preferably designed for the treatment of solid tumors as described herein.

A “neoplastic cell”, “cancer cell” or “tumor cell” can be used interchangeably to refer to a cell that divides at an abnormal (i.e. increased) rate. The term “treatment” (and any form of treatment such as “treating”, “treat”, etc.,) as used herein refers to therapy. Typically, therapy refers to a pathological condition with the purpose to improve at least one clinical or biochemical symptom (size of tumor, expression level of associated biomarker...), to slow down or control the progression of the targeted pathological condition, symptom(s) thereof, or a state secondary to the pathological condition in the subject treated in accordance with the present invention. The terms “prevention” (and any form of the term such as “preventing”, “prevent”, etc.,) and “prophylaxis” are used interchangeably and refer to preventing, delaying the onset or decreasing the severity of the first occurrence or relapse of at least one clinical or biochemical symptom (size of tumor, expression level of associated biomarker, stage progression...).

The term “administering” (or any form of administration such as “administered”, etc.,) as used herein refers to the delivery to a subject of a component (e.g. the fusion polypeptide according to the invention) according to the modalities described herein.

The term “combination” or “association” as used herein refers to any arrangement possible of various components (e.g. the fusion polypeptide according to the invention and another treatment). Such an arrangement includes mixture of said components as well as separate combinations for concomitant or sequential administrations. The present invention encompasses combinations comprising equal molar concentrations of each component as well as combinations with very different concentrations. It is appreciated that optimal concentration of each component of the combination can be determined by the artisan skilled in the art.

The terms “virus”, “virions”, “viral particles” and “viral vector particle” are used interchangeably to refer to viral particles that are formed when the nucleic acid vector is transduced into an appropriate cell or cell line according to suitable conditions allowing the generation of viral particles. In the context of the present invention, the term “viral vector” has to be understood broadly as including nucleic acid vector (e.g. DNA viral vector) as well as viral particles generated thereof. The term “infectious” refers to the ability of a viral vector to infect and enter into a host cell or subject. Viral vectors can be replication-competent or -selective (e.g. engineered to replicate better or selectively in specific host cells), or can be genetically disabled so as to be replication-defective or replication-impaired.

The term "viral vector" as used herein refers to a nucleic acid vector that includes at least one element of a virus genome and may be packaged into a viral particle or to a viral particle. The term, "antibody" ("Ab") is used in the broadest sense and encompasses naturally occurring antibodies and engineered antibodies; including synthetic, monoclonal, polyclonal antibodies as well as full length antibodies and fragments, variants or fusions thereof provided that such fragments, variants or fusions retain binding properties to the target protein. Such antibodies can be of any origin; human or non-human mammal (e.g. rodent or camelid antibody), or chimeric. A nonhuman antibody can be humanized by recombinant methods to reduce its immunogenicity in human. The antibody may derive from any of the well-known isotypes (e.g. IgA, IgG and IgM) and any subclasses of IgG (IgG 1 , lgG2, lgG3, lgG4). In addition, it may be glycosylated, partially glycosylated or nonglycosylated. Unless the context indicates otherwise, the term "antibody" also includes an antigen-binding fragment of any of the aforementioned antibodies and includes a monovalent and a divalent fragment and single chain antibodies. The term antibody also includes multi -specific (e.g. bispecific) antibody so long as it exhibits the same binding specificity as the parental antibody. It is within the skill of the artisan to screen for the binding properties of a candidate antibody.

For illustrative purposes, full length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region which is made of three CH1 , CH2 and CH3 domains (optionally with a hinge between CH1 and CH2). Each light chain comprises a light chain variable region (VL) and a light chain constant region which comprises one CL domain. The VH and VL regions comprise three hypervariable regions, named complementarity determining regions (CDR), interspersed with four conserved regions named framework regions (FR) in the following order: FR1 - CDR1 -FR2-CDR2-FR3-CDR3-FR4. The CDR regions of the heavy and light chains are determinant for the binding specificity. As used herein, a "humanized antibody" refers to a non-human (e.g. murine, camel, rat, etc) antibody whose protein sequence has been modified to increase its similarity to a human antibody (i.e. produced naturally in humans). The process of humanization is well known in the art and typically is carried out by substituting one or more residue of the FR regions to look like human immunoglobulin sequence whereas the vast majority of the residues of the variable regions (especially the CDRs) are not modified and correspond to those of a non-human immunoglobulin. A "chimeric antibody" comprises one or more element(s) of one species and one or more element(s) of another species, for example, a non-human antibody comprising at least a portion of a constant region (Fc) of a human immunoglobulin.

The antibody is preferably a monoclonal antibody, preferably humanized or chimeric. Representative examples of antibody fragments and/or regions are known in the art, including heavy (H) chain, light (L), heavy chain variable region (VH), heavy chain constant region, CH domain, light chain variable region (VL), light chain constant region, CL domain, complementarity determining regions (CDR), constant region (Fc), Fab, Fab’, F(ab’)2, dAb, Fd, Fv, scFv, ds-scFv, diabody, sdAb, etc.

Representative examples of antigen-binding fragments are known in the art, including Fab, Fab’, F(ab’)2, dAb, Fd, Fv, scFv, ds-scFv, and diabody. A particularly useful antibody fragment is a single chain antibody (scFv) comprising the two domains of a Fv fragment, VL and VH, that are fused together, eventually with a linker to make a single protein chain.

Poxvirus encoding a binding agent

The present Inventors have designed novel poxviruses encoding a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ). Several poxviruses were developed, each expressing different formats of this anti-PD-L1 sdAb, including a monomeric form, a dimeric form, a form fused to the hinge of a human IgG, and a form fused to a human Fc.

The inventors unexpectedly found that, when expressed by these poxviruses, these anti- PD-L1 sdAbs possess a significantly higher blocking activity than the reference anti-PD-L1 antibody, Avelumab. In addition, the experimental data obtained by the Inventors demonstrated that all these anti-PD-L1 sdAb expressed by these poxviruses are capable to bind PD-L1 at the surface of tumor cell, more efficiently than expressed Avelumab. Importantly, the Inventors confirmed that replication and oncolytic activities of the poxviruses expressing these anti-PD-L1 sdAbs were unaffected by the presence and expression of the transgene encoding the sdAb. Binding of these anti-PD-L1 sdAb to cynomolgus monkey PD-L1 was also demonstrated, showing that the results are not limited to human.

The anti-PD-L1 sdAb encoded by the poxviruses of the invention are particularly advantageous over the anti-PD-L1 antibodies of the prior art, including Avelumab. Indeed, the significantly lower molecular weights and sizes of all of the anti-PD-L1 sdAb formats designed by the Inventors allows a better penetration and diffusion into tumor tissue, compared to anti-PD-L1 antibodies. In addition, such anti-PD-L1 sdAbs have a shorter halflife compared to anti-PD-L1 antibodies of the prior art, including Avelumab. This advantage is particularly beneficial to prevent unwanted systemic diffusion in the organism, thereby improving specificity of the targeting of tumor cells.

Altogether, the poxviruses expressing various formats of a sdAb specifically binding to PD- L1 here developed by the Inventors provides a novel and efficient treatment of proliferative disease, such as cancer.

Accordingly, the present invention relates to a poxvirus comprising a nucleic acid molecule inserted in its genome and encoding a binding agent, wherein the binding agent comprises, or consists essentially of, or consists of, a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ) comprising three heavy chain Complementarity-determining regions CDR1 , CDR2, and CDR3; and wherein:

• the heavy chain CDR1 consists of sequence RTFREYGMG (SEQ ID NO:1 ),

• the heavy chain CDR2 consists of sequence TISSSGSYX1Y, wherein Xi is S or T (SEQ ID NO:2),

• the heavy chain CDR3 comprises the sequence X2SLLRGX3SSRAEX4YDX5, wherein each of X2 to X5 independently represents any amino acid (SEQ ID NO:3).

Poxvirus

As used herein, the term “poxvirus” or “poxviral vector” refers to any Poxviridae virus identified at present time or being identified afterwards that is infectious for one or more mammalian cells (e.g. human cells).

The term “virus” as used in the context of poxvirus or any other virus mentioned herein encompasses the viral genome as well as the viral particle (encapsided and/or enveloped genome).

Poxviruses are a broad family of DNA viruses containing a double-stranded genome. Like most viruses, poxviruses have developed self-defence mechanisms through a repertoire of proteins involved in immune evasion and immune modulation aimed at blocking many of the strategies employed by the host to combat viral infections (Smith and Kotwal, 2002, Crit. Rev. Microbiol. 28(3): 149-85). Typically, the poxvirus genome encodes more than 20 host response modifiers that allow the virus to manipulate host immune responses and, thus, facilitate virus replication, spread, and transmission. These include growth factors, anti -apopto tic proteins, inhibitors of the NFkB pathway and interferon signalling, and down-regulators of the major histocompatibility complex (MHC). The poxvirus genome in the native context is a double-stranded DNA of approximately 200kb and has the potential of encoding nearly 200 proteins with different functions. The genomic sequence and the encoded open reading frames (ORFs) are well known. The poxvirus of the invention comprises a genome which has been modified (in aa laboratory, compared to the native form) to comprise a nucleic acid molecule inserted in its genome and encoding a binding agent, wherein the binding agent comprises a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ). The poxvirus of the invention may further comprise one or more additional modifications such as those described herein.

In one embodiment, the poxvirus is a poxvirus of the Chordopoxvirinae family, preferably selected from the group consisting of Avipoxvirus genus (including Canarypoxvirus (e.g. ALVAC) and Fowlpoxvirus (e.g. the FP9 vector), Capripoxvirus genus, Lepori poxvirus genus (such as myxoma virus (which genomic sequences are disclosed in Genbank under accession number NP_051868.1 )), Mollusci poxvirus genus, Orthopoxvirus genus, Parapoxvirus genus, Suipoxvirus genus, Cervidpoxvirus genus, Yatapoxvirus genus, and chimeras thereof.

As used therein, “poxvirus chimeras” or « chimeras of poxviruses” refers to viruses obtained by homologous recombination between several distinct strains of poxviruses. Several chimeras obtained by mixing genomes from different poxviruses have been described and are available to the skilled person (such as CF189 chimeras obtained from ORF and pseudocowpox viruses (Choi et al, Novel chimeric parapoxvirus CF189 as an oncolytic immunotherapy in triple-negative breast cancer. Surgery Volume 163, Issue 2, February 2018, Pages 336-342); CF33 chimera obtained from multiple strains of VV, cowpox, and rabbitpox (Chaurasiya, S., Chen, N.G., Lu, J. et al. A chimeric poxvirus with J2R (thymidine kinase) deletion shows safety and anti-tumor activity in lung cancer models. Cancer Gene Ther 27, 125-135 (2020)).

In a preferred embodiment, the poxvirus is a member of the Orthopoxvirus genus, preferably selected from the group consisting of vaccinia virus (VV), cowpox (CPXV), raccoonpox (RCN), rabbitpox, Monkeypox, Horsepox, Volepox, Skunkpox, variola virus (or smallpox), Camelpox, and chimeras thereof.

Orthopoxvirus chimeras correspond to chimeras of several distinct strains of Orthopoxvirus. Sequences of the genome of the various poxviruses, are available in the art and specialized databases such as Genbank. For example, the vaccinia virus, cowpox virus, Canarypox virus, Ectromelia virus, Myxoma virus genomes are available in specialized databases such as Genbank (accession number NC_006998, NC_003663, NC_005309, NC_004105, NC_001132 respectively).

In a preferred embodiment, the poxvirus of the invention belongs to the Orthopoxvirus genus, and even more preferably to the vaccinia virus (VV) species. In the native context, Vaccinia viruses are large, complex, enveloped viruses with a linear, double-stranded DNA genome of approximately 200kb in length which encodes numerous viral enzymes and factors that enable the virus to replicate independently from the host cell machinery. Two distinct infectious viral particles exist, the intracellular IMV (for intracellular mature virion) surrounded by a single lipid envelop that remains in the cytosol of infected cells until lysis and the double enveloped EEV (for extracellular enveloped virion) that buds out from the infected cell. Any vaccinia virus strain can be used in the context of the present invention including, without limitation, MVA (Modified vaccinia virus Ankara), NYVAC, Copenhagen (Cop), Western Reserve (WR), Wyeth, Lister, LIVP, Tashkent, Tian Tan, Brighton, Ankara, LC16M8, LC16M0 strains, etc., and any derivative thereof. The gene nomenclature used herein is that of Copenhagen Vaccinia strain. It is also used herein for the homologous genes of other poxviridae unless otherwise indicated. However, gene nomenclature may be different according to the poxvirus strain but correspondence between Copenhagen and other vaccinia strains are generally available in the literature. Genomic sequences thereof are available in the literature and Genbank (e.g. under accession numbers AY678276 (Lister), M35027 (Cop), AF095689 1 (Tian Tan), AY243312.1 (WR), and U94848 (MVA)). These viruses can also be obtained from virus collections (e.g. ATCC VR-1354 for WR, ATCC VR-1536 for Wyeth and ATCC VR-1549 for Lister).

Advantageously, the poxvirus is a vaccinia virus, preferably selected from: a) an oncolytic vaccina virus selected from the group of Western Reserve (WR), Elstree, Copenhagen (Cop), Wyeth, Lister, LIVP, Tashkent, Tian Tan, Brighton, Ankara, LC16M8, and LC16M0 strains, b) a modified Vaccinia Ankara (MVA) strain. Oncolytic poxyirus

In a preferred embodiment, the poxvirus of the present invention is oncolytic. As used herein, the term “oncolytic” refers to the capacity of a virus of selectively replicating in dividing cells (e.g. a proliferative cell, and more particularly a cancer cell) with the aim of slowing the growth and/or lysing said dividing cell, either in vitro or in vivo, while showing no or minimal replication in non-dividing (e.g. normal or healthy) cells. “Replication” (or any form of replication such as “replicate” and “replicating”, etc.,) means duplication of a virus that can occur at the level of nucleic acid or, preferably, at the level of infectious viral particle. The term “infectious” (or any form of infectious such as infect, infecting, etc.,) denotes the ability of a virus to infect and enter into a host cell or subject. Typically, an oncolytic poxvirus contains a viral genome packaged into a viral particle (or virion) and is infectious (i.e. capable of infecting and entering into a host cell or subject). As used herein, this term encompasses DNA or RNA vector (depending on the virus in question) as well as viral particles generated thereof.

In a preferred embodiment, the oncolytic poxvirus of the present invention is a member of the Orthopoxvirus genus, preferably selected from the group consisting of vaccinia virus (W), cowpox (CPXV), raccoonpox (RCN), rabbitpox, Monkeypox, Horsepox, Volepox, Skunkpox, variola virus (or smallpox), Camelpox, and chimeras thereof.

Even more preferably, the oncolytic poxvirus of the invention belongs to the vaccinia virus (W) species. Any oncolytic vaccinia virus strain can be used in the context of the present invention including, without limitation, Copenhagen (Cop), Western Reserve (WR), Elstree, Wyeth, Lister, LI VP, Tashkent, Tian Tan, Brighton, Ankara, LC16M8, LC16M0 strains, etc., and any derivative thereof.

Oncolytic poxviruses can be used with modifications, including modifications aimed at improving safety (e.g. increased attenuation) and/or efficacy, and/or tropism of the resulting virus. One may cite also defective modifications within the thymidine kinase (J2R; see Weir and Moss, 1983, Genbank accession number AAA48082), the deoxyuridine triphosphatase (F2L), the viral hemagglutinin (A56R), the small (F4L) and/or the large (I4L) subunit of the ribonucleotide reductase, the serine protease inhibitor (B13R/B14R), the complement 4b binding protein (C3L), the scaffold assembly protein (D13L), and within genes like K1 L, C7L, A39R and B7R-B8R.

Exemplary modifications preferably concern viral genes involved in DNA metabolism, host virulence or IFN pathway (see e.g. Guse et al., 2011 , Expert Opinion Biol. Ther.11 (5):595- 608). A particularly suitable gene to be disrupted is the thymidine kinase (tk)-encoding locus (J2R; Genbank accession number AAA48082). The tk enzyme is involved in the synthesis of deoxyribonucleotides. Tk is needed for viral replication in normal cells as these cells have generally low concentration of nucleotides whereas it is dispensable in dividing cells which contain high nucleotide concentration. Further, tk-defective viruses are known to have an increased selectivity to tumor cells. In one embodiment, the modified poxvirus is further modified in the J2R locus (preference for modification resulting in a suppressed expression of the viral tk protein), resulting in a modified poxvirus defective for tk functions (tk- poxvirus). Partial or complete deletion of said J2R locus as well as insertion of foreign nucleic acid in the J2R locus are contemplated in the context of the present invention to inactivate tk function. Such a modified tk- poxvirus is desirably oncolytic. Alternatively to or in combination with, the modified poxvirus may be further modified, in the I4L and/or F4L locus/loci (preference for modification leading to a suppressed expression of the viral ribonucleotide reductase (rr) protein), resulting in a modified poxvirus defective rr functions (rr-defective poxvirus). In the natural context, this enzyme catalyzes the reduction of ribonucleotides to deoxyribonucleotides that represents a crucial step in DNA biosynthesis. The viral enzyme is similar in subunit structure to the mammalian enzyme, being composed of two heterologous subunits, designed R1 and R2 encoded respectively by the I4L and F4L locus. Sequences for the I4L and F4L genes and their location in the genome of various poxvirus are available in public databases (see e.g. W02009/065546). In the context of the invention, the poxvirus can be modified either in the I4L gene (encoding the r1 large subunit) or in the F4L gene (encoding the r2 small subunit) or both to provide a rr-defective poxvirus, e.g. by partial or complete deletion of said I4L and/or F4L locus/loci. Such a modified rr- poxvirus is desirably oncolytic.

Also provided is a modified poxvirus further modified in the J2R and I4L/F4L loci (double defective virus with modifications in the J2R and I4L loci; J2R and F4L loci; or J2R, I4L and F4L loci), resulting in a modified poxvirus defective tk and rr activities (tk- rr- poxvirus). Such a modified tk- rr- poxvirus is desirably oncolytic.

Alternatively to or in combination with, the modified poxvirus may be further modified, in the M2L locus (preference for modification leading to a suppressed expression of the viral m2 protein), resulting in a modified poxvirus defective m2 functions (m2-defective poxvirus).

In one embodiment, the modified poxvirus is further modified in the M2L locus and in the J2R locus (preference for modification resulting in a suppressed expression of the viral tk protein), resulting in a modified poxvirus defective for both m2 and tk functions (m2- tk- poxvirus). Partial or complete deletion of said M2L locus and/or J2R locus as well as insertion of foreign nucleic acid in the M2L locus and/or J2R locus are contemplated in the context of the present invention to inactivate m2 and tk functions. Such a modified m2- tk- poxvirus is desirably oncolytic.

Alternatively to or in combination with, the modified poxvirus may be further modified in the M2L locus and in the I4L and/or F4L locus/loci (preference for modification leading to a suppressed expression of the viral ribonucleotide reductase (rr) protein), resulting in a modified poxvirus defective for both m2 and rr functions (m2 and rr-defective poxvirus). In the context of the invention, the poxvirus can be modified either in the I4L gene (encoding the r1 large subunit) or in the F4L gene (encoding the r2 small subunit) or both to provide a rr-defective poxvirus, e.g. by partial or complete deletion of said I4L and/or F4L locus/loci. Such a modified m2- rr- poxvirus is desirably oncolytic.

Also provided is a modified poxvirus further modified in the M2L locus, in the J2R locus, and in the I4L/F4L loci (triple defective virus with modifications in the M2L, J2R and I4L loci; M2L, J2R and F4L loci or M2L, J2R, I4L and F4L loci), resulting in a modified poxvirus defective for m2, tk and rr activities (m2-, tk- rr- poxvirus). Such a modified tk- rr- and m2- poxvirus is desirably oncolytic.

In a preferred embodiment, such simple, double and triple defective poxviruses preferably originate from an Orthopoxvirus, or a Lepori poxvirus as described above. Particularly preferred is an oncolytic vaccinia virus other than MVA, with a specific preference for Lister, WR, Copenhagen, Wyeth strains. VV defective for tk and m2 activities and for tk, rr and m2 activities are particularly preferred, especially for use for stimulating or improving an immune response (e.g. a lymphocyte-mediated response against an antigen or epitope thereof) or for use for treating a proliferative disease as described herein.

Other suitable additional modifications include those resulting in suppressed expression of one or more viral gene product(s) selected from the group consisting of the viral hemagglutinin (A56R); the serine protease inhibitor (B13R/B14R), the complement 4b binding protein (C3L), the VGF-encoding gene and the interferon modulating gene(s) (B8R or B18R). Another suitable modification comprises the inactivation of the F2L locus resulting in suppressed expression of the viral dllTPase (deoxyuridine triphosphatase) involved in both maintaining the fidelity of DNA replication and providing the precursor for the production of TMP by thymidylate synthase (W02009/065547).

As for M2L, the gene nomenclature used herein is that of Cop VV strain. It is also used herein for the homologous genes of other poxviridae unless otherwise indicated and correspondence between Copenhagen and other poxviruses is available to the skilled person.

Preferred modifications include:

(i) inactivating mutations in the J2R viral gene,

(ii) inactivating mutations in the viral I4L and/or F4L gene(s),

(iii ) inactivating mutations in the M2L gene,

(iv) inactivating mutations in the J2R viral gene and inactivating mutations in the viral I4L and/or F4L gene(s),

(v) inactivating mutations in the J2R viral gene and inactivating mutations in the M2L gene,

(vi) inactivating mutations in the viral I4L and/or F4L gene(s) and inactivating mutations in the M2L gene, or

(vii) inactivating mutations in the J2R viral gene, inactivating mutations in the viral I4L and/or F4L gene(s) and inactivating mutations in the M2L gene.

Thus, the poxvirus is advantageously a vaccinia virus, preferably selected from an oncolytic vaccina virus selected from the group of Western Reserve (WR), Elstree, Copenhagen (Cop), Wyeth, Lister, LI VP, Tashkent, Tian Tan, Brighton, Ankara, LC16M8, and LC16M0 strains, which preferably comprises:

(i) inactivating mutations in the J2R viral gene,

(ii) inactivating mutations in the viral I4L and/or F4L gene(s),

(iii) inactivating mutations in the M2L gene,

(iv) inactivating mutations in the J2R viral gene and inactivating mutations in the viral I4L and/or F4L gene(s),

(v) inactivating mutations in the J2R viral gene and inactivating mutations in the M2L gene,

(vi) inactivating mutations in the viral I4L and/or F4L gene(s) and inactivating mutations in the M2L gene, or

(vii) inactivating mutations in the J2R viral gene, inactivating mutations in the viral I4L and/or F4L gene(s) and inactivating mutations in the M2L gene.

Replication-defective or replication-impaired viral vectors

In another embodiment, the poxvirus of the invention may not be oncolytic, but instead be a non-propagative poxvirus. The term “non-propagative poxvirus” refers to poxviruses that are unable to propagate in host cells or tissues. These poxviruses can be replication-defective or replication- impaired vectors (e.g. genetically disabled poxviruses), meaning that they cannot replicate to any significant extent in normal cells, especially in normal human cells, thus impeding viral vector propagation. The impairment or defectiveness of replication functions can be evaluated by conventional means, such as by measuring DNA synthesis and/or viral titer in non-permissive cells. The poxvirus can be rendered replicationdefective by partial or total deletion or inactivation of regions critical to viral replication. Such replication-defective or impaired poxviruses typically require for propagation, permissive cell lines which bring up or complement the missing/impaired functions. These poxviruses can also be replication-competent or replication-selective vectors (e.g. engineered to replicate better or selectively in specific host cells) able to produce a first generation of viral particles in the host infected cells, but wherein said first generation of viral particles are unable to infect new host’s cells, thus impeding poxvirus propagation. This impairment can be the result of various processes, like the diminution or impairment of DNA production, the diminution or impairment of viral proteins production, the inhibition of scaffold assembly proteins, the uncomplete viral particle maturation, the inability for said viral particles to get out of host cells or to enter new host cells, etc.

Although one may use wild type or native poxviruses (i.e. found in nature), preference is given in the context of the present invention to viral like particles and genetically engineered viruses (i.e. a virus that is modified compared to a wild type strain of said virus, e.g. by truncation, deletion, substitution and/or insertion of one or more nucleotide(s) contiguous or not within the viral genome, notably in one or more gene required for viral replication). Modification (s) can be within endogenous viral genes (e.g. coding and/or regulatory sequences) and/or within intergenic regions, preferably resulting in a modified viral gene product. Modification(s) can be made in a number of ways known to those skilled in the art using conventional molecular biology techniques. Preferably, the modifications encompassed by the present invention affect, for example, virulence, toxicity or pathogenicity of the viral vector compared to a viral vector without such modification, but do not completely inhibit infection and production of new viral particles at least in permissive cells. Said modification(s) preferably lead(s) to the synthesis of a defective protein (or lack of synthesis) so as to be unable to ensure the activity of the protein produced under normal conditions by the unmodified gene. Other suitable modifications include the insertion of exogenous gene(s) (i.e. exogenous meaning not found in a native viral genome), such as a nucleic acid molecule encoding at least a binding agent as described hereinafter.

A particularly suitable non-propagative poxvirus for use in the invention is obtained from a poxvirus of the Chordopoxvirinae subfamily. The Chordopoxvirinae subfamily is directed to vertebrate host which includes several genus such as Orthopoxvirus, Capripoxvirus, Avipoxvirus, Parapoxvirus, Lepori poxvirus and Suipoxvirus. Orthopoxviruses are preferred in the context of the present invention as well as the Avi poxviruses including Canarypoxvirus (e.g. ALVAC) and Fowlpoxvirus (e.g. the FP9 vector). Sequences of the genome of various Poxviridae, are available in the art in specialized databanks such as Genbank. For example, the vaccinia virus strains Western Reserve, Copenhagen, Cowpoxvirus and Canarypoxvirus genomes are available in Genbank under accession numbers NC_006998, M35027, NC_003663, NC_005309, respectively.

In a preferred embodiment, the non-propagative viral vectors for use in the invention belong to the Orthopoxvirus genus and even more preferably to the vaccinia virus (W) species. In the native context, Vaccinia viruses are large, complex, enveloped viruses with a linear, double-stranded DNA genome of approximately 200kb in length which encodes numerous viral enzymes and factors that enable the virus to replicate independently from the host cell machinery. Two distinct infectious viral particles exist, the intracellular IMV (for intracellular mature virion) surrounded by a single lipid envelop that remains in the cytosol of infected cells until lysis and the double enveloped EEV (for extracellular enveloped virion) that buds out from the infected cell. Any vaccinia virus strain can be used in the context of the present invention including, without limitation, MVA (Modified vaccinia virus Ankara), NYVAC, Copenhagen (Cop), Western Reserve (WR), Wyeth, Lister, LIVP Tashkent, Tian Tan, Brighton, Ankara, LC16M8, LC16M0 strains, etc., and any derivative thereof. The gene nomenclature used herein is that of Copenhagen Vaccinia strain. It is also used herein for the homologous genes of other poxviridae unless otherwise indicated. However, gene nomenclature may be different according to the poxvirus strain but correspondence between Copenhagen and other vaccinia strains are generally available in the literature.

Engineered poxviruses can be used with modifications aimed at improving safety (e.g. increased attenuation) and/or efficacy, and/or tropism of the resulting virus. One may cite also defective modifications within the thymidine kinase (J2R; see Weir and Moss, 1983, Genbank accession number AAA48082), the deoxyuridine triphosphatase (F2L), the viral hemagglutinin (A56R), the small (F4L) and/or the large (I4L) subunit of the ribonucleotide reductase, the serine protease inhibitor (B13R/B14R), the complement 4b binding protein (C3L), the scaffold assembly protein (D13L), and within genes like K1 L, C7L, A39R and B7R-B8R.

A particularly appropriate non-propagative poxvirus for use in the context of the present invention is MVA, due to its highly attenuated phenotype (Mayr et al., 1975, Infection 3: 6-14; Sutter and Moss, 1992, Proc. Natl. Acad. Sci. USA 89: 10847-51 ). For illustrative purposes, MVA has been generated through serial passages in chicken embryo fibroblasts. Sequence analysis of its genome showed that it has lost the pathogenicity of its parental virus, the Chorioallantois Vaccinia virus Ankara, through alterations of its genome. (Antoine et al., 1998, Virol. 244: 365-96 and Genbank accession number U94848). MVA has been used safely and effectively for smallpox vaccination in more than a hundred thousand individuals. Replicative potential of the virus in human cells is defective but not in chicken embryo cells. Various cellular systems are available in the art to produce large quantities of the virus, notably in egg-based manufacturing processes (e.g. W02007/147528). Said MVA is also particularly appropriated because of a more pronounced IFN-type 1 response generated upon infection compared to non-attenuated vectors, and of the availability of the sequence of its genome in the literature (Antoine et al., 1998, Virol. 244: 365-96) and in Genbank (under accession number U94848).

Another particularly appropriate non-propagative poxvirus for use in the context of the present invention is NYVAC, also due to its highly attenuated phenotype (Tartaglia et al., 1992, Virol. 188(1 ):217-32). For illustrative purpose, NYVAC is a highly attenuated vaccinia virus strain, derived from a plaque-cloned isolate of the Copenhagen vaccine strain by the precise deletion of 18 open reading frames (ORFs) from the viral genome. Still another suitable non-propagative poxvirus for use in the context of the present invention is a vaccinia virus engineered to be non-propagative, with a specific preference for a non- propagative vaccinia virus of Copenhagen strain having a D13L deletion.

Thus, the poxvirus is advantageously a vaccinia virus, preferably selected from: a) an oncolytic vaccina virus selected from the group of Western Reserve (WR), Elstree, Copenhagen (Cop), Wyeth, Lister, LI VP, Tashkent, Tian Tan, Brighton, Ankara, LC16M8, and LC16M0 strains, which preferably comprises:

(i) inactivating mutations in the J2R viral gene,

(ii) inactivating mutations in the viral I4L and/or F4L gene(s),

(iii) inactivating mutations in the M2L gene,

(iv) inactivating mutations in the J2R viral gene and inactivating mutations in the viral I4L and/or F4L gene(s), (v) inactivating mutations in the J2R viral gene and inactivating mutations in the M2L gene,

(vi) inactivating mutations in the viral I4L and/or F4L gene(s) and inactivating mutations in the M2L gene, or

(vii ) inactivating mutations in the J2R viral gene, inactivating mutations in the viral I4L and/or F4L gene(s) and inactivating mutations in the M2L gene; or b) a modified Vaccinia Ankara (MVA) strain.

Binding agent comprising a sdAb specifically binding to PD-L1

The poxvirus of the invention comprises a nucleic acid molecule inserted in its genome, which encodes a binding agent. The binding agent specifically binds to programmed death-ligand 1 (PD-L1 ). More specifically, the binding agent comprises a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ). The sdAb encoded by the poxvirus of the invention specifically binds to PD-L1. Thus, the sdAb is herein also referred to as “anti-PD-L1 sdAb”.

The terms “programmed death ligand 1”, “programmed cell death 1”, “PD-L1”, “PDL1”, “CD274”, B7-H”, “B7H1”, “PDCD1 L1” and “PDCD1 LG1” are used herein interchangeably and relate to any isoform or allelic variant of the protein encoded by human gene with Entrez Gene ID number 29126, as well as species homologs of human PD-L1 . The complete amino acid sequence of the longest isoform of human PD-L1 can be found under GenBank Accession No. NP 054862.1 (version of February 20, 2022). Orthologs of human PD-L1 are known in many species, in particular in vertebrates, and more particularly in mammalians. While the sdAb encoded by the poxvirus according to the invention may specifically bind to PD-L1 of any species, it preferably specifically binds to human PD-L1 , and optionally to PD-L1 orthologs of one or more other primates (such as cynomolgus PD- L1 ).

The terms “single domain antibody”, “sdAb” and “nanobody” are used herein interchangeably and relate to a single monomeric variable antibody domain able to bind selectively to a specific antigen. sdAb are generally obtained from heavy chain antibodies (i.e. antibodies comprising only a heavy chain and no light chain) found in camelids (such as dromedaries, camels, llamas, alpacas) or cartilaginous fishes (such as sharks). These animals indeed produce dimer antibodies composed of two associated heavy chains comprising a variable domain (generally referred to as “VHH” in the case of camelids, and as “VNAR” in the case of cartilaginous fishes) and a constant domain. sdAb (in particular VHH and VNAR) comprise 3 “complementary determining regions” or “CDR” regions (denoted “CDR1”, “CDR2”, and “CDR3”) mainly involved in antigen selective binding, surrounded by 4 “framework” or “FR” regions (denoted “FR1”, “FR2”, “FR3” and “FR4”), in the following order, from N-terminal to C-terminal: FR1 -CDR1 -FR2-CDR2-FR3-CDR3- FR4. VHH and VNAR represent preferred embodiments of sdAb. The portion of the amino acid sequence of a given sdAb corresponding to CDR1 , CDR2 and CDR3 may be defined based on several distinct numbering systems. The first numbering system is the one proposed by Kabat et al. (Kabat et al. Sequences of proteins of immunological interest, 5^th Ed., U.S. Department of Health and Human Services, NIH, 1991 , and later editions). In this numbering system, CDRs are defined based on sequence variability. Another numbering system was proposed by Chothia et al. ,1987 (Chothia C, Lesk a M. 1987 Canonical structures for the hypervariable regions of immunoglobulins. J Mol Biol. 196: 901 -17). In this method, CDRs are defined based on the location of the structural loop regions. Another method is referred to as “Abm”, which CDRs corresponds to a compromise between the Kabat and Chothia methods (Whitelegg NR, Rees AR. 2000. WAM: an improved algorithm for modelling antibodies on the WEB. Protein Eng. ; 13(12):819-24; Whitelegg N, Rees AR. 2004 Antibody variable regions: toward a unified modeling method. Methods Mol Biol. ;248:51 -91 ). Still another method was proposed by the IMGT, based on determining hypervariable regions. In this method, a unique numbering has been defined to compare variable regions regardless of the antigen receptor, the chain type or the species (Lefranc MP, Pommie C, Ruiz M, Giudicelli V, Foulquier E, Truong L, Thouvenin-Contet V, Lefranc G. Lefranc MP, et al. 2003 IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol. ;27(1 ):55-77). This numbering provides a standardized definition of framework regions ((FR1 -IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to 128) and complementarity determining regions (CDR1 -IMGT: positions 27 to 38, CDR2-IMGT: positions 56 to 65 and CDR3-IMGT: positions 105 to 117).

The sdAb encoded by the poxvirus according to the invention specifically binds to PD-L1 . The terms “binds” or “binding” as used herein refer to an interaction between molecules to form a complex which, under physiologic conditions, is relatively stable. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions or forces. The strength of the total non-covalent interactions between a sdAb and its antigen is the “affinity” or “binding affinity” of the sdAb for that antigen. Binding affinity is typically measured and reported by the equilibrium dissociation constant (Kd), which corresponds to the ratio koff/kon, between the antibody and its antigen, koff is the rate constant of dissociation of the sdAb from its antigen (how quickly it dissociates from its antigen), and kon is the rate constant of association of the sdAb to its antigen (how quickly it binds to its antigen). Kd and affinity are inversely correlated. As a result, the lower the KD value, the higher the affinity of the antibody for its antigen. The equilibrium dissociation constant (Kd) for a sdAb provided herein can be determined using any method provided herein or any other method well known to those skilled in the art, including Surface Plasmon resonance (SPR) and biolayer interferometry (BLI) technologies. A sdAb is said to specifically binds to PD-L1” if its affinity for PD-L1 (in particular human PD-L1 ) is significantly higher than for another antigen. In other words, a sdAb is said to specifically binds to PD-L1” if its equilibrium dissociation constant (Kd) for PD-L1 (in particular human PD-L1 ) is significantly lower than for another antigen. The measured Kd value of the sdAb produced by clone 32.1A1 was found to be 0.47 nM, and the sdAb encoded by the poxvirus according to the invention preferably has a Kd value lower than 1 nM (preferably when measured using an Octet Red96 instrument).

In a preferred embodiment, the binding agent (in particular the sdAb) encoded by the poxvirus according to the invention specifically binds to a mammal PD-L1 , particularly a mammal selected from the group consisting of domestic animals, farm animals, sport animals, and primates (human and non-human). Most preferably, the binding agent (in particular the sdAb) encoded by the poxvirus according to the invention specifically binds to human PD-L1 .

Preferred heavy chain CDR regions

The sdAb included in the fusion polypeptide according to the invention comprises three heavy chain complementary determining regions CDR1 , CDR2 and CDR3, wherein:

• the heavy chain CDR1 consists of sequence RTFREYGMG (SEQ ID NO:1 ),

• the heavy chain CDR2 consists of sequence TISSSGSYXiY, wherein Xi is S or T (SEQ ID NO:2), and

• the heavy chain CDR3 comprises the sequence X2SLLRGX3SSRAEX4YDX5, wherein each of X2 to X5 independently represents any amino acid (SEQ ID NO:3). This embodiment is referred to as “Emb1”.

SdAb with such heavy chain CDR1 , CDR2 and CDR3 are shown in the Example to confer specificity for human and primate PD-L1 .

In particular, two clones (32.1A1 and 32.2F7) obtained by immunization of alpaca with human PD-L1 with very close CDR1 , CDR2 and CDR3 sequences are shown to specifically bind to human PD-L1 (see Example 1 ).

RTFREYGMG (SEQ ID NO:1 ) corresponds to the amino acid sequence of the heavy chain CDR1 of both clones (same sequence).

TISSSGSYXiY, wherein Xi is S or T (SEQ ID NO:2) covers only the two amino acid sequences of the heavy chain CDR2s of both clones (only one amino acid differs, the two amino acids S and T for Xi corresponding to those present in the heavy chain CDR2s of each clone). The amino acid sequences of the heavy chain CDR3s of clones 32.1A1 and 32.2F7 both comprises amino acid sequence X2SLLRGX3SSRAEX4YDX5, wherein each of X2 to X5 independently represents any amino acid (SEQ ID NO:3).

In addition, Example 1 shows that mutation to alanine of several positions (corresponding to positions X2 to X5 in SEQ ID NO:3) in the CDR3 of clone 32.1A1 does not significantly alter specific binding to human PD-L1 (see Figure 13). These results show that these positions are not essential for binding to PD-L1 , and thus support the degenerated sequence SEQ ID NO:3.

In view of the above, and based on common general knowledge that CDR regions are the main determinants of antibody specificity, a skilled person would expect that a sdAb with the above defined CDR1 , CDR2 and CDR3 would retain specific binding to PD-L1.

In a preferred embodiment (referred to as “Emb2”), the heavy chain CDR3 of the sdAb specifically binding to PD-L1 comprises, or consists essentially of, or consists of, the sequence X6X7X2SLLRGX3SSRAEX4YDX5, wherein each of X2 to X7 independently represents any amino acid (SEQ ID NO:4). This sequence comprises SEQ ID NO:3 defined above, and further comprises two additional amino acids in N-terminal, which are included in the CDR3 definition.

Preferably (embodiment “Emb3”), the heavy chain CDR3 of the sdAb specifically binding to PD-L1 comprises, or consists essentially of, or consists of, the sequence X6X₇X2SLLRGX₃SSRAEX4YDX₅ (SEQ ID NO:5), wherein:

• X2 to X3 and X5 are independently selected from S, T, C, A, V, G, and P ; 1

• X4 is selected from S, P, T, C, A, V, G, and P ;

• X₆ and X7 are independently selected from A, V, G, and P.

The above-defined possibilities for X₂ to X7 correspond either to the amino acid found at the corresponding position in the heavy chain CDR3 of clone 32.1A1 or clone 32.2F7, or to structurally close amino acids, as explained in more details in Table 1 below.

Table 1. Rational for selection of amino acids at positions X₂ to X7.

More preferably (embodiment “Emb4”), the heavy chain CDR3 of the sdAb specifically binding to PD-L1 comprises, or consists essentially of, or consists of, the sequence X₆X7X₂SLLRGX₃SSRAEX4YDX₅ (SEQ ID NO:6), wherein:

• X₂, X3 and X5 are independently selected from S, T, C, and A;

• X4 is selected from S, P, T, C, and A; and

• X₆ and X7 are selected from A, V, G, and P.

Even more preferably (embodiment “Emb5”), the heavy chain CDR3 of the sdAb specifically binding to PD-L1 comprises, or consists essentially of, or consists of, the sequence X6X₇X₂SLLRGX₃SSRAEX4YDX₅ (SEQ ID NO:7), wherein:

• X₂, X₃ and X5 are independently selected from S, and A;

• X4 is selected from S, P, and A; and

• X₆ and X7 are A. Even more preferably (embodiment “Emb6”), the heavy chain CDR3 of the sdAb specifically binding to PD-L1 comprises, or consists essentially of, or consists of, the sequence X6X₇X₂SLLRGX₃SSRAEX4YDX₅ (SEQ ID NO:8), wherein:

• X₂, X₃ and X5 are S;

• X4 is selected from S and P; and

• X₆ and X7 are A.

The heavy chain CDR1 , CDR2, and CDR3 amino acid sequences of the two clones 32.1A1 and 32.2F7 are presented in Table 2 below.

Table 2. Heavy chain CDR sequences of clones 32.1A1 and 32.2F7.

Two particularly preferred alternative embodiments of the sdAb specifically binding to PD-L1 encoded by the poxvirus according to the invention are as follows: a) embodiment “Emb7”: the three heavy chain CDRs of the sdAb specifically binding to PD-L1 are those of the sdAb produced by clone 32.1A1 , as follows:

• the heavy chain CDR1 consists of sequence RTFREYGMG (SEQ ID NO:1 ),

• the heavy chain CDR2 consists of sequence TISSSGSYSY (SEQ ID NO: 9), and

• the heavy chain CDR3 consists of sequence AASSLLRGSSSRAESYDS (SEQ ID N0:10); or b) embodiment “Emb8”: the three heavy chain CDRs of the sdAb specifically binding to PD-L1 are those of the sdAb produced by clone 32.2F7, as follows:

• the heavy chain CDR1 consists of sequence RTFREYGMG (SEQ ID NO:1 ),

• the heavy chain CDR2 consists of sequence TISSSGSYTY (SEQ ID NO: 11 ), and

• the heavy chain CDR3 consists of sequence AASSLLRGSSSRAEPYDS (SEQ ID NO:12).

Humanization

As the fusion polypeptide according to the invention is particularly intended for use in human beings, the sdAb is preferably a humanized sdAb. By “humanized” sdAb is meant a sdAb that contains CDRs derived from a sdAb of non-human origin (here, alpaca), the other portions of the sdAb molecule being derived from one (or from several) human antibodies. Humanized sdAb can be prepared by techniques known to a person skilled in the art such as CDR grafting, resurfacing, superhumanization, human string content, FR libraries, guided selection, FR shuffling and humaneering technologies, as summarized in the review by Almagro et al. , 2008 (Almagro et al. Frontiers in Bioscience 13, 1619-1633, January 1 , 2008).

Embodiments Emb1 to Emb8 in which the sdAb is humanized are particularly preferred.

Preferred sdAb entire sequences

While heavy chain CDRs are the main determinants of antigen binding affinity for sdAb, some amino acids of the FR1 , FR2, FR3 and FR4 regions may sometimes play a minor role in antigen binding affinity, and it is thus preferred that, in addition to the abovedescribed features relating to the heavy chain CDR1 , CDR2 and CDR3, the sdAb specifically binding to PD-L1 comprises, or consists essentially of, or consists of, an amino acid sequence with a high percentage of sequence identity with those of the humanized version of the sdAb produced by clones 32.1A1 and 32.2F7.

Therefore, in a preferred embodiment (referred to as “Emb9”), the sdAb specifically binding to PD-L1 comprises, or consists essentially of, or consists of, an amino acid sequence having at least 80 % sequence identity, preferably at least 85 % sequence identity, more preferably at least 90 % sequence identity, at least 91 % sequence identity, at least 92 % sequence identity, at least 93 % sequence identity, at least 94 % sequence identity, most preferably at least 95 % sequence identity, at least 96 % sequence identity, at least 97 % sequence identity, at least 98 % sequence identity, at least 99 % sequence identity, or even 100% identity with the amino acid sequence SEQ ID NO: 13 or SEQ ID NO:14.

When one or more original amino acid(s) of SEQ ID NO:13 or SEQ ID NO:14 are replaced by one or more other amino acid(s), each replaced original amino acid may preferably be replaced by an “equivalent” amino acid, i.e., any amino acid whose structure is similar to that of the original amino acid and is therefore unlikely to change the biological activity of the resulting sdAb. Examples of such equivalent substitutions are presented in Table 3 below: Table 3. Substitutions with equivalent amino acids

SEQ ID NO: 13 is the amino acid sequence of the humanized version of the sdAb produced by clone 32.1A1 : QVQLVESGGGLVQPGGSLRLSCAASGRTFREYGMGWFRQAPGKGLEWVATISSSGSYSYYADSVKGRF

TISRDNSKNTLYLQMNSLRAEDTAVYYCAASSLLRGSSSRAESYDSWGQGTLVTVSS

As this sdAb has been found to specifically bind to human PD-L1 with high affinity and has been the most widely characterized, the sdAb specifically binding to PD-L1 most preferably comprises, or consists essentially of, or consists of, the sequence SEQ ID NO:13 (embodiment “Emb10”).

SEQ ID NO: 14 is the amino acid sequence of the humanized version of the sdAb produced by clone 32.2F7:

QVQLVESGGGLVQPGGSLRLSCAASGRTFREYGMGWFRQAPGKGLEWVATISSSGSYTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAASSLLRGSSSRAEPYDSWGQGTLVTVSS As this sdAb has also been found to specifically bind to human PD-L1 with high affinity, the sdAb specifically binding to PD-L1 may also preferably comprise, or consist essentially of, or consist of, the sequence SEQ ID NO: 13 (embodiment “Emb11”).

SEQ ID NO: 13 and SEQ ID NO: 14 share 98.4% identity.

In a preferred embodiment (referred to as “Emb9bis”), the nucleic acid molecule encoding the binding agent comprises, or consists essentially of, or consists of, a nucleic acid molecule encoding the sdAb specifically binding to PD-L1. Preferably, said nucleic acid molecule encoding the sdAb specifically binding to PD-L1 comprises, or consists essentially of, or consists of, a nucleic acid sequence having at least 80 % sequence identity, preferably at least 85 % sequence identity, more preferably at least 90 % sequence identity, at least 91 % sequence identity, at least 92 % sequence identity, at least 93 % sequence identity, at least 94 % sequence identity, most preferably at least 95 % sequence identity, at least 96 % sequence identity, at least 97 % sequence identity, at least 98 % sequence identity, at least 99 % sequence identity, or even 100% identity, with SEQ ID NO: 34 or SEQ ID NO:35, preferably with SEQ ID NO: 34, preferably said nucleic acid molecule encoding the sdAb specifically binding to PD-L1 comprises, or consists essentially of, or consists of, the nucleic acid sequence SEQ ID NO:34.

Fusion of the sdAb to a fusion polypeptide partner

While the transgene encoded by the poxvirus according to the invention may encode only the sdAb of the invention (as defined above), it may also further encode another nucleic acid molecule encoding a fusion polypeptide partner.

Said another nucleic acid molecule encoding a fusion polypeptide partner is preferably a foreign nucleic acid (also called recombinant gene, transgene or nucleic acid).

In the context of the invention, the “foreign nucleic acid” that is inserted in the poxvirus genome is not found in or expressed by a naturally occurring poxvirus genome. Nevertheless, the foreign nucleic acid can be homologous or heterologous to the subject into which the recombinant poxvirus is introduced. More specifically, it can be of human origin or not (e.g. of bacterial, yeast or viral origin except poxviral). Advantageously, said recombinant nucleic acid encodes a polypeptide or is a nucleic acid sequence capable of binding at least partially (by hybridization) to a complementary cellular nucleic acid (e.g., DNA, RNA, miRNA) present in a diseased cell with the aim of inhibiting a gene involved in said disease. Such a recombinant nucleic acid may be a native gene or portion(s) thereof (e.g. cDNA), or any variant thereof obtained by mutation, deletion, substitution and/or addition of one or more nucleotides.

Advantageously, the sdAb specifically binding to PD-L1 is fused, directly or indirectly through a peptide linker, to said fusion polypeptide partner.

The fusion of the sdAb specifically binding to PD-L1 to the fusion polypeptide partner (directly or indirectly through a peptide linker) is referred to as “the fusion polypeptide”.

The term “fusion” or “fusion polypeptide” as used herein refers to the covalent linkage in a single polypeptide chain of two or more polypeptides and is performed by genetic means, i.e. by fusing in frame the nucleic acid molecules encoding each of said polypeptides. By "fused in frame", it is meant that the expression of the fused coding sequences results in a single polypeptide without any translational terminator between each of the fused polypeptides.

Direct or indirect fusion

In the fusion polypeptide of the poxvirus according to the invention, the sdAb specifically binding to PD-L1 may be fused to a fusion polypeptide partner, directly or indirectly through a peptide linker.

The sdAb specifically binding to PD-L1 is said to be fused “directly” to fusion polypeptide partner, if there is no additional amino acid residue between the two fusion partners (the sdAb specifically binding to PD-L1 and the fusion polypeptide partner).

The sdAb specifically binding to PD-L1 is said to be fused “indirectly through a peptide linker” to the fusion polypeptide partner, if there is there is a peptide linker comprising, or consisting essentially of, or consisting of one or more amino acid residue(s) between the two fusion partners (the sdAb specifically binding to PD-L1 and the fusion polypeptide partner).

It is within the reach of the skilled person to assess the need to include or not a peptide linker between the two fusion partners.

In a preferred embodiment, the sdAb specifically binding to PD-L1 and the fusion polypeptide partner, are fused indirectly through a peptide linker. Indeed, the presence of a suitable peptide linker between the two fusion partners ensures proper folding and optimal activity of the two fusion partners.

Any suitable peptide linker may be used. Typically, suitable peptide linkers are 1 to 30 amino acids long peptides composed of amino acid residues such as glycine, serine, threonine, asparagine, alanine and/or proline. Preferred linkers in the context of this invention comprise, or consist essentially of, or consist of, 3 to 20 amino acids, mainly glycine and serine (e.g. 1 , 2 3 or 4 repetitions of GSG, GGGS (SEQ ID NO:15), GSGSG (SEQ ID NO: 16), or SGSGS (SEQ ID NO: 17), or 1 or

2 repetitions of GSGSGSGSGS (SEQ ID NO: 18)) or glycine, serine and threonine (e.g. 1 , 2

3 or 4 repetitions of GSTSG (SEQ ID NO: 19) or SGTGS (SEQ ID NO: 20)) or glycine, serine, and threonine and/or alanine (e.g. 1 , 2 3 or 4 repetitions of GAS or GTS). Preferred peptide linkers include those comprising or consisting of GGGSGGGS (SEQ ID NO: 21 ), GGGSGGGSGGGS (SEQ ID NO: 22), or GGGSGGGSGGGSGGGS (SEQ ID NO: 23), corresponding to 3, 4 or 5 repetitions of GGGS (SEQ ID NO:15). A particularly preferred peptide linker comprises, or consists essentially of, or consists of, GGGSGGGSGGGS (SEQ ID NO: 22). It is within the reach of the skilled person to optimize the size and sequence of a peptide linker between the two fusion partners.

Order of the fusion

The two fusion partners (i.e. the sdAb specifically binding to PD-L1 and the fusion polypeptide partner) may be fused in any order, such as the sdAb specifically binding to PD-L1 being in N-terminal of the fusion polypeptide partner, or the sdAb specifically binding to PD-L1 being in C-terminal of the fusion polypeptide partner or functional fragment or derivative thereof.

Further optional elements of the fusion polypeptide

The fusion polypeptide according to the invention may optionally comprise, in addition to the two fusion partners (i.e. the sdAb specifically binding to PD-L1 and the fusion polypeptide partner) and the optional peptide linker in between, further elements that may be useful for the production or therapeutic use of the fusion polypeptide.

Fusion polypeptide partner

The fusion polypeptide partner may comprise, or consist essentially of, or consist of, any polypeptide of interest (or functional fragment or derivative thereof), including, but not limited to, antibody fragments and/or regions, therapeutic polypeptides, signal peptides, tag peptides, etc., functional fragments or derivatives thereof, and any combination thereof. Fusion comprising an antibody fragment and/or region

In a preferred embodiment, the fusion polypeptide partner comprises, or consists essentially of, or consists of, an antibody fragment and/or region.

In the context of the invention, "antibody" ("Ab") is used in the broadest sense and is preferably as defined in the section “General definitions” above.

The antibody fragment and/or region may comprise, or consist essentially of, or consist of, an antibody fragment and/or region selected from an antigen-binding antibody fragment, an antibody heavy chain constant region or a fragment thereof, and any combination thereof.

The antibody fragment and/or region may comprise, or consist essentially of, or consist of, an antibody fragment and/or region selected from a heavy (H) chain, a light (L), heavy chain variable region (VH), a heavy chain constant region, a CH domain, a light chain variable region (VL), a light chain constant region, a CL domain, a complementarity determining regions (CDR), a constant region (Fc), a Fab, a Fab’, a F(ab’)2, a dAb, a Fd, a Fv, a scFv, a ds-scFv, a diabody, a sdAb, etc, and any combination thereof; preferably selected from a sdAb, a scFv, an antibody heavy chain constant region or a fragment thereof (preferably selected from the hinge region, the Fc fragment, and the entire constant region), and any combination thereof.

In one embodiment, the fusion polypeptide partner is selected from: a) an antigen-binding antibody fragment, preferably a sdAb or a scFv, and b) an antibody heavy chain constant region or a fragment thereof, preferably selected from the hinge region, the Fc fragment, and the entire constant region.

Thus, in a preferred embodiment of the poxvirus according to the invention (embodiment “Emb12”), the binding agent is selected from the group consisting of: a) the monovalent sdAb specifically binding to PD-L1 as defined above, preferably the monovalent sdAb specifically binding to PD-L1 comprising, or consisting essentially of, or consisting of, an amino acid sequence selected from SEQ ID NO: 13 and SEQ ID NO: 14, preferably the amino acid sequence SEQ ID NO: 13; b) a bivalent sdAb comprising, or consisting essentially of, or consisting of, two sdAb specifically binding to PD-L1 as defined above, fused through a peptide linker, preferably a bivalent sdAb comprising, or consisting essentially of, or consisting of, the amino acid sequence SEQ ID NO:31; c) a hinge bivalent sdAb comprising, or consisting essentially of, or consisting of, the association through disulfide bonds of two polypeptides comprising, or consisting essentially of, or consisting of, the sdAb specifically binding to PD-L1 as defined above, fused to the hinge part of an antibody heavy chain constant region; preferably a hinge bivalent sdAb comprising, or consisting essentially of, or consisting of, the association through disulfide bonds of two polypeptides comprising or consisting of the amino acid sequence SEQ ID NO:32; and d) an Fc bivalent sdAb comprising, or consisting essentially of, or consisting of, the association through disulfide bonds of two polypeptides comprising or consisting of the sdAb specifically binding to PD-L1 as defined above, fused to an Fc fragment of an antibody heavy chain constant region; preferably an Fc bivalent sdAb comprising, or consisting essentially of, or consisting of, the association through disulfide bonds of two polypeptides, comprising, or consisting essentially of, or consisting of, the amino acid sequence SEQ ID NO:33.

In a preferred embodiment (referred to as “Emb12bis”), the binding agent is selected from the group consisting of: a) a monovalent sdAb specifically binding to PD-L1 as defined above and encoded by a nucleic acid sequence having at least 80 % sequence identity, preferably at least 85 % sequence identity, more preferably at least 90 % sequence identity, at least 91 % sequence identity, at least 92 % sequence identity, at least 93 % sequence identity, at least 94 % sequence identity, most preferably at least 95 % sequence identity, at least 96 % sequence identity, at least 97 % sequence identity, at least 98 % sequence identity, at least 99 % sequence identity, or even 100% identity, with SEQ ID NO: 34 or SEQ ID NO:35, preferably with SEQ ID NO: 34; preferably the monovalent sdAb specifically binding to PD-L1 encoded by a nucleic acid sequence comprising, or consisting essentially of, or consisting of, SEQ ID NO: 34 ; b) a bivalent sdAb comprising or consisting of two sdAb specifically binding to PD- L1 as defined above fused through a peptide linker and encoded by a nucleic acid sequence having at least 80 % sequence identity, preferably at least 85 % sequence identity, more preferably at least 90 % sequence identity, at least 91 % sequence identity, at least 92 % sequence identity, at least 93 % sequence identity, at least 94 % sequence identity, most preferably at least 95 % sequence identity, at least 96 % sequence identity, at least 97 % sequence identity, at least 98 % sequence identity, at least 99 % sequence identity, or even 100% identity, with SEQ ID NO: 36, preferably a bivalent sdAb encoded by a nucleic acid sequence comprising, or consisting essentially of, or consisting of, SEQ ID NO: 36; c) a hinge bivalent sdAb comprising or consisting of the association through disulfide bonds of two polypeptides comprising or consisting of the sdAb specifically binding to PD-L1 as defined above fused to the hinge part of an antibody heavy chain constant region and encoded by a nucleic acid sequence having at least 80 % sequence identity, preferably at least 85 % sequence identity, more preferably at least 90 % sequence identity, at least 91 % sequence identity, at least 92 % sequence identity, at least 93 % sequence identity, at least 94 % sequence identity, most preferably at least 95 % sequence identity, at least 96 % sequence identity, at least 97 % sequence identity, at least 98 % sequence identity, at least

99 % sequence identity, or even 100% identity, with SEQ ID NO: 37, preferably a hinge bivalent sdAb encoded by a nucleic acid sequence comprising, or consisting essentially of, or consisting of, SEQ ID NO: 37; and d) an Fc bivalent sdAb comprising or consisting of the association through disulfide bonds of two polypeptides comprising or consisting of the sdAb specifically binding to PD-L1 as defined above fused to an Fc fragment of an antibody heavy chain constant region and encoded by a nucleic acid sequence having at least 80 % sequence identity, preferably at least 85 % sequence identity, more preferably at least 90 % sequence identity, at least 91 % sequence identity, at least 92 % sequence identity, at least 93 % sequence identity, at least 94 % sequence identity, most preferably at least 95 % sequence identity, at least 96 % sequence identity, at least 97 % sequence identity, at least 98 % sequence identity, at least 99 % sequence identity, or even 100% identity, with SEQ ID NO: 38, preferably a Fc bivalent sdAb encoded by a nucleic acid sequence comprising, or consisting essentially of, or consisting of, SEQ ID NO: 38.

Fusion comprising a

In one embodiment, the fusion polypeptide partner comprises, or consists essentially of, or consists of, a therapeutic polypeptide or functional fragments or derivatives thereof.

By a “therapeutic polypeptide”, it is meant a polypeptide which is of therapeutic or prophylactic interest when administered appropriately to a subject, leading to a beneficial effect on the course or a symptom of the pathological condition to be treated or prevented. The therapeutic polypeptide is preferably a polypeptide having therapeutic properties, in particular anti-tumor or anti-cancer activities. The therapeutic polypeptide may be selected from the group consisting of a suicide gene product, an immunostimulatory polypeptide, an antigenic polypeptide, an antibody or an antigen-binding fragment or derivative thereof, and any combination thereof. In an advantageous embodiment, the suicide gene product, the immunostimulatory polypeptide, the antigenic polypeptide, and any combination thereof, is as described in the section “Additional therapeutic polypeptide/gene” below.

Fusion comprising a signal peptide

In one embodiment, the fusion polypeptide partner comprises, or consists essentially of, or consists of, a signal peptide or functional fragments or derivatives thereof.

As used herein, a “signal peptide” refers to a peptide able to enhance the processing through the endoplasmic reticulum (ER)-and/or secretion of a polypeptide when present at its N-terminus. Briefly, signal peptides usually comprise, consist essentially of or consist of 15 to 35 essentially hydrophobic amino acids, are inserted at the N-terminus of the polypeptide downstream of the codon for initiation of translation, initiate its passage into the endoplasmic reticulum (ER) and are then removed by a specific ER-located endopeptidase to give the mature polypeptide. Appropriate signal peptides are known in the art. They may be obtained from cellular or viral polypeptides such as those of immunoglobulins, tissue plasminogen activator, insulin, rabies glycoprotein (see e.g; W099/03885 or W02008/ 138649), the HIV virus envelope glycoprotein or the measles virus F protein or may be synthetic. Preferred signal peptides may be those originating from the rabies or the measles F glycoprotein or variant thereof (see, e.g. W02008/138649). A particularly preferred signal peptide comprises, or consists essentially of, or consists of, amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO:24), said signal peptide is the signal peptide coming from an Ig heavy chain V region 102 of Mus musculus (Swiss-Prot Accession Number P01750).

The signal peptide may be independently positioned at the N-terminus of the sdAb specifically binding to PD-L1 or fusion thereof (tag-polypeptide) or alternatively at its C- terminus (polypeptide-tag) or alternatively internally. When a tag peptide is already present in C-terminal of the fusion polypeptide according to the invention, a signal peptide may preferably be inserted at the N-terminus of the fusion polypeptide according to the invention, and vice versa.

The signal peptide is preferably fused in N-terminal of the sdAb specifically binding to PD-L1. Fusion comprising a tag peptide

In one embodiment, the fusion polypeptide partner comprises, or consists essentially of, or consists of, a tag peptide or functional fragments or derivatives thereof.

As used herein, a “tag peptide” refers to a peptide that facilitates the detection of the expression of the fusion polypeptide or of infected host cells expressing such fusion polypeptide. Tag peptides can be detected by immunodetection assays using anti-tag antibodies. A vast variety of tag peptides can be used in the context of the invention including without limitation PK tag, FLAG tag (DYKDDDK, SEQ ID NO: 25; or GDYKDDDK, SEQ ID NO: 26), MYC tag (EQKLISEEDL SEQ ID NO: 27), polyhistidine tag (usually a stretch of 5 to 10 histidine residues), HA tag (YPYDVPDYA; SEQ ID NO: 28), HSV tag (QPELAPEDPED; SEQ ID NO: 29) and VSV Tag (YTDIEMNRLGK; SEQ ID NO: 30). The tag peptide may be independently positioned at the N-terminus of the sdAb specifically binding to PD-L1 or fusion thereof (tag-polypeptide) or alternatively at its C-terminus (polypeptide-tag) or alternatively internally. When a signal peptide is already present in N-terminal of the fusion polypeptide according to the invention, a tag peptide may preferably be inserted at the C-terminus of the fusion polypeptide according to the invention, and vice versa.

The tag peptide is preferably fused in C-terminal of the sdAb specifically binding to PD- L1.

Fusion comprising a combination of fusion polypeptide partners

The fusion polypeptide partner may comprise, or consist essentially of, or consist of, any polypeptide of interest (or functional fragment or derivative thereof), including, but not limited to, antibody fragments and/or regions, therapeutic polypeptides, signal peptides, tag peptides, etc., functional fragments or derivatives thereof, and any combination thereof.

In one embodiment, the fusion polypeptide of the poxvirus according to the invention, comprises two or more fusion polypeptide partners as defined above.

In an advantageous embodiment, the fusion polypeptide comprises, consists essentially of, or consists of, a polypeptide of interest selected from an antibody fragment and/or region, and a therapeutic polypeptide; and further comprises a signal peptide and/or a tag peptide. Additional therapeutic polypeptide/ gene

While the poxvirus according to the invention may encode only the sdAb of the invention (as defined above), or the fusion polypeptide thereof (as defined above), it may also further encode another nucleic acid molecule encoding a polypeptide of interest.

Said another nucleic acid molecule encoding a polypeptide of interest is preferably a foreign nucleic acid (also called recombinant gene, transgene or nucleic acid).

In the context of the invention, the “foreign nucleic acid” that is inserted in the poxvirus genome is not found in or expressed by a naturally occurring poxvirus genome. Nevertheless, the foreign nucleic acid can be homologous or heterologous to the subject into which the recombinant poxvirus is introduced. More specifically, it can be of human origin or not (e.g. of bacterial, yeast or viral origin except poxviral). Advantageously, said recombinant nucleic acid encodes a polypeptide or is a nucleic acid sequence capable of binding at least partially (by hybridization) to a complementary cellular nucleic acid (e.g., DNA, RNA, miRNA) present in a diseased cell with the aim of inhibiting a gene involved in said disease. Such a recombinant nucleic acid may be a native gene or portion(s) thereof (e.g. cDNA), or any variant thereof obtained by mutation, deletion, substitution and/or addition of one or more nucleotides.

In an advantageous embodiment, said a polypeptide of interest is therapeutic polypeptide. Accordingly, in this advantageous embodiment, the poxvirus according to invention (as defined above), further comprises another nucleic acid molecule inserted in its genome encoding a therapeutic polypeptide.

By a “therapeutic polypeptide”, it is meant a polypeptide which is of therapeutic or prophylactic interest when administered appropriately to a subject, leading to a beneficial effect on the course or a symptom of the pathological condition to be treated or prevented.

The therapeutic polypeptide is preferably selected from the group consisting of an immunomodulatory polypeptide (preferably an immunostimulatory polypeptide), an antigenic polypeptide, a suicide gene product, an antibody, a functional derivative of an antibody, a functional fragment of an antibody, and any combination thereof.

In a preferred embodiment, the therapeutic polypeptide is an immunostimulatory polypeptide, preferably selected from the group consisting of cytokines, such as interleukins, chemokines, interferons, tumor necrosis factors, colony-stimulating factors; APC-exposed proteins; agonists of stimulatory immune checkpoints; antagonists of inhibitory immune checkpoints; and any combination thereof.

Immunomodulatory polypeptide

The term “immunomodulatory polypeptide” refers to a polypeptide targeting a component of a signaling pathway that can be involved in modulating an immune response either directly or indirectly. "Modulating" an immune response refers to any alteration in a cell of the immune system or in the activity of such a cell (e.g., a T cell). Such modulation includes stimulation or suppression of the immune system which can be manifested by an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other changes which can occur within the immune system. Preferably, such a polypeptide is capable of down-regulating at least partially an inhibitory pathway (antagonist) and/or of up-regulating at least partially a stimulatory pathway (agonist); in particular the immune pathway existing between an antigen presenting cell (APC) or a cancer cell and an effector T cell.

The immunomodulatory polypeptide that may be expressed by the vector according to the invention may act at any step of the T cell-mediated immunity including clonal selection of antigen-specific cells, T cell activation, proliferation, trafficking to sites of antigen and inflammation, execution of direct effector function and signaling through cytokines and membrane ligands. Each of these steps is regulated by counterbalancing stimulatory and inhibitory signals that in fine tune the response.

Suitable immunomodulatory polypeptides and methods of using them are described in the literature. Exemplary immunomodulatory polypeptides include, without limitation:

• cytokines, such as interleukins, chemokines, interferons, tumor necrosis factors, colony-stimulating factors;

• APC-exposed proteins;

• agonists of a stimulatory immune checkpoint;

• antagonists of an inhibitory immune checkpoint different from PD-L1 ; and

• any combination thereof.

In one embodiment, the immunomodulatory polypeptide to be expressed by the vector according to the invention is a cytokine, preferably selected from the group consisting of; • interleukins (e.g. IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL- 12, IL-13, IL- 14, IL-15, IL-16, IL-17, IL-18, IL-36), IFNa, IFNg and granulocyte macrophage colony stimulating factor (GM-CSF)) ;

• chemokines (e.g. MIPIo, IL-8, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11 , CXCL13, CXCL12, CCL2, CCL19 and CCL21 ),

• interferons (e.g. IFNa, IFNy),

• tumor necrosis factors (e.g. TNFo), and

• colony-stimulating factors (e.g. granulocyte macrophage colony stimulating factor (GM-CSF)).

When the immunostimulatory polypeptide is a cytokine, it is preferably an interleukin or a colony-stimulating factor, with a specific preference for GM-CSF.

In another embodiment, the immunomodulatory polypeptide to be expressed by the vector according to the invention is an agonist of a stimulatory immune checkpoint or an antagonist of an inhibitory immune checkpoint.

The term “immune checkpoint” refers to a protein directly or indirectly involved in an immune pathway that under normal physiological conditions is crucial for preventing uncontrolled immune reactions and thus for the maintenance of self-tolerance and/or tissue protection. Immune checkpoints may be classified into to distinct categories: stimulatory and inhibitory immune checkpoints, respectively. A “stimulatory immune checkpoint” refers to an immune checkpoint involved in up-regulation of immune responses, while an “inhibitory immune checkpoint” is involved in down-regulation of immune responses.

Stimulatory immune checkpoints include CD28, ICOS, CD137 (4-1 BB), 0X40, CD27, CD40, and GITR, and the agonist of a stimulatory immune checkpoint is preferably selected from human ICOSL, 4-1 BBL, OX40L, CD70, CD40L, GITRL and agonist antibodies to human ICOS (e.g. W02018/187613), CD137 (4-1 BB) (e.g. W02005/035584 ), 0X40 (e.g. US 7,291 ,331 and W003/106498), CD27 (e.g. W02012/004367), CD40 (e.g. W02017/184619), or GITR (e.g. W02017/068186. As some agonists of stimulatory immune checkpoints are TNFSF members, when such an agonist of stimulatory immune checkpoints is further encoded by the vector according to the invention, it is preferably different from the member of the TNFSF or functional fragment or variant thereof of the fusion polypeptide according to the invention. Inhibitory immune checkpoints include PD-1 , SIRPa, CD47, PD-L2, LAG3, Tim3, BTLA, and CTLA4, and the antagonist of an inhibitory immune checkpoint is preferably selected from antagonist antibodies human:

• PD-1 (e.g. those described in W02004/004771 ; W02004/056875; W02006/121168; W02008/156712; W02009/014708; W02009/114335; W02013/043569; and W02014/047350, in particular nivolumab, pembrolizumab and cemiplimab),

• SIRPa (e.g. W02019/023347),

• CD47 (e.g. W02020/019135),

• PD-L2 (e.g. W02019/158645),

• LAG3 (e.g. W02018/071500),

• Tim3, (e.g. W02020/093023)

• BTLA (e.g. W02010/106051 ), and

• CTLA4 (e.g. those described in US 8,491 ,895, W02000/037504, W02007/113648, W02012/122444 and W02016/196237 among others, and in particular ipilimumab marketed by Bristol Myer Squibb as Yervoy® (see e.g. US 6,984,720; US 8,017,114), MK-1308 (Merck), AGEN-1884 (Agenus Inc.; W02016/196237) and tremelimumab (AstraZeneca; US 7,109,003 and US 8,143,379) and single chain anti-CTLA4 antibodies (see e.g. W097/20574 and W02007/ 123737).

Antigenic polypeptide

The term “antigenic” refers to the ability to induce or stimulate a measurable immune response in a subject into which the poxvirus of the invention (as described herein) encoding the polypeptide qualified as antigenic has been introduced. The stimulated or induced immune response against the antigenic polypeptide expressed by said poxvirus can be humoral and/or cellular (e.g. production of antibodies, cytokines and/or chemokines involved in the activation of effector immune cells). The stimulated or induced immune response usually contributes in a protective effect in the administered subject. A vast variety of direct or indirect biological assays are available in the art to evaluate the antigenic nature of a polypeptide either in vivo (animal or human subjects), or in vitro (e.g. in a biological sample). For example, the ability of a particular antigen to stimulate innate immunity can be performed by for example measurement of the NK/NKT-cells (e.g. representativity and level of activation), as well as, IFN-related cytokine and/or chemokine producing cascades, activation of TLRs (for Toll-like receptor) and other markers of innate immunity (Scott-Algara et al., 2010 PLOS One 5(1 ), e8761 ; Zhou et al., 2006, Blood 107, 2461 -2469; Chan, 2008, Eur. J. Immunol. 38, 2964-2968). The ability of a particular antigen to stimulate a cell-mediated immune response can be performed for example by quantification of cytokine(s) produced by activated T cells including those derived from CD4+ and CD8+ T-cells using routine bioassays (e.g. characterization and/or quantification of T cells by ELISpot, by multi parameters flow cytometry, ICS (for intracellular cytokine staining), by cytokine profile analysis using multiplex technologies or ELISA), by determination of the proliferative capacity of T cells (e.g. T cell proliferation assays by [³H] thymidine incorporation assay), by assaying cytotoxic capacity for antigen-specific T lymphocytes in a sensitized subject or by identifying lymphocyte subpopulations by flow cytometry and by immunization of appropriate animal models, as described herein.

It is contemplated that the term antigenic polypeptide encompasses native antigen as well as fragment (e.g. epitopes, immunogenic domains, etc) and variant thereof, provided that such fragment or variant is capable of being the target of an immune response. Preferred antigenic polypeptides for use herein are tumor-associated antigens. It is within the scope of the skilled artisan to select the one or more antigenic polypeptide that is appropriate for treating a particular pathological condition.

In one embodiment, the antigenic polypeptide(s) encoded by the recombinant modified poxvirus is/are cancer antigen(s) (also called tumor-associated antigens or TAA) that is associated with and/or serve as markers for cancers. Cancer antigens encompass various categories of polypeptides, e.g. those which are normally silent (i.e. not expressed) in healthy cells, those that are expressed only at low levels or at certain stages of differentiation and those that are temporally expressed such as embryonic and foetal antigens as well as those resulting from mutation of cellular genes, such as oncogenes (e.g. activated ras oncogene), proto-oncogenes (e.g. ErbB family), or proteins resulting from chromosomal translocations.

Numerous tumor-associated antigens are known in the art. Exemplary tumor antigens include without limitation, colorectal associated antigen (CRC), Carci noembryonic Antigen (CEA), Prostate Specific Antigen (PSA), BAGE, GAGE or MAGE antigen family, p53, mucin antigens (e.g. MUC1 ), HER2/neu, p21 ras, hTERT, Hsp70, iNOS, tyrosine kinase, mesothelin, c-erbB-2, alpha fetoprotein, AM-1 , among many others, and any immunogenic epitope or variant thereof.

The tumor-associated antigens may also encompass neo-epitopes/antigens that have emerged during the carcinogenesis process in a cancer cell and comprising one or more mutation(s) of amino acid residue(s) with respect to a corresponding wild-type antigen. Typically, it is found in cancer cells or tissues obtained from a patient but not found in a sample of normal cells or tissues obtained from a patient or a heathy individual.

The tumor-associated antigens may also encompass antigens encoded by pathogenic organisms that are capable of inducing a malignant condition in a subject (especially chronically infected subject) such as RNA and DNA tumor viruses (e.g. human papillomavirus (HPV), hepatitis C virus (HCV), hepatitis B virus (HBV), Epstein Barr virus (EBV), etc) and bacteria (e.g. Helicobacter pilori).

In another embodiment, the antigenic polypeptide(s) encoded by the poxvirus of the invention is/are vaccinal antigen(s) that, when delivered to a human or animals subject, aim(s) at protecting therapeutically or prophylactically against infectious diseases. Numerous vaccine antigens are known in the art. Exemplary vaccine antigens include but are not limited to cellular antigens, viral, bacterial or parasitic antigens. Cellular antigens include the mucin 1 (MUC1 ) glycoprotein. Viral antigens include for example antigens from hepatitis viruses A, B, C, D and E, immunodeficiency viruses (e.g. HIV), herpes viruses, cytomegalovirus, varicella zoster, papilloma viruses, Epstein Barr virus, influenza viruses, para-influenza viruses, coxsakie viruses, picorna viruses, rotaviruses, respiratory syncytial viruses, rhinoviruses, rubella virus, papovirus, mumps virus, measles virus and rabbies virus. Some non-limiting examples of HIV antigens include gp120 gp40, gp160, p24, gag, pol, env, vif, vpr, vpu, tat, rev, nef tat, nef. Some non-limiting examples of human herpes virus antigens include gH, gL gM gB gC gK gE or gD or Immediate Early protein such aslCP27, ICP47, ICP4, ICP36 from HSV1 or HSV2. Some non-limiting examples of cytomegalovirus antigens include gB. Some non-limiting examples of derived from Epstein Barr virus (EBV) include gp350. Some non-limiting examples of Varicella Zoster Virus antigens include gp1 , 11 , 111 and IE63. Some non-limiting examples of hepatitis C virus antigens includes env E1 or E2 protein, core protein, NS2, NS3, NS4a, NS4b, NS5a, NS5b, p7. Some non-limiting examples of human papilloma viruses (HPV) antigens include L1 , L2, E1 , E2, E3, E4, E5, E6, E7. Antigens derived from other viral pathogens, such as Respiratory Syncytial virus (e.g. F and G proteins), parainfluenza virus, measles virus, mumps virus, flaviviruses (e.g. Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis virus, Japanese Encephalitis Virus) and Influenza virus cells (e.g. HA, NP, NA, or M proteins) can also be used in accordance with the present invention. Bacterial antigens include for example antigens from Mycobacteria causing TB, leprosy, pneumocci, aerobic gram negative bacilli, mycoplasma, staphyloccocus, streptococcus, salmonellae, chlamydiae, neisseriae and the like. Parasitic antigenic polypeptides include for example antigens from malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, schistosomiasis and filariasis.

Suicide gene products

The term “suicide gene product” refers to a polypeptide able to convert a precursor of a drug, also named “prodrug”, into a cytotoxic compound. Suicide genes comprise but are not limited to genes coding protein having a cytosine deaminase activity, a thymidine kinase activity, an uracil phosphoribosyl transferase activity, a purine nucleoside phosphorylase activity and a thymidylate kinase activity. Examples of suicide gene products and corresponding precursors of a drug comprising one nucleobase moiety are disclosed in the following Table 4.

Table 4. Exemplary suicide gene products and corresponding drug precursors

Desirably, the suicide gene encodes a polypeptide having at least cytosine deaminase (CDase) activity. In the prokaryotes and lower eukaryotes (it is not present in mammals), CDase is involved in the pyrimidine metabolic pathway by which exogenous cytosine is transformed into uracil by means of a hydrolytic deamination. CDase also deaminates an analogue of cytosine, i.e. 5 -fluorocytosine (5-FC), thereby forming 5-fluorouracil (5-FU), a compound which is cytotoxic by itself but even more when it is converted into 5-fluoro- UMP (5-FUMP). CDase encoding nucleic acid molecule can be obtained from any prokaryotes and lower eukaryotes such as Saccharomyces cerevisiae (FCY1 gene), Candida Albicans (FCA1 gene) and Escherichia coli (codA gene). The gene sequences and encoded CDase proteins have been published and are available in specialized data banks (SWISSPROT EMBL, Genbank, Medline and the like). Functional analogues of these genes may also be used. Such analogues preferably have a nucleic acid sequence having a degree of identity of at least 70%, advantageously of at least 80%, preferably of at least 90%, and most preferably of at least 95% with the nucleic acid sequence of the native gene.

Alternatively or in combination, the oncolytic virus of the invention carries in its viral genome a suicide gene encoding a polypeptide having uracil phosphoribosyl transferase (UPRTase) activity. In prokaryotes and lower eukaryotes, uracil is transformed into UMP by the action of UPRTase. This enzyme converts 5-FU into 5-FUMP. By way of illustration, the nucleic acid sequences encoding the UPRTases from E. coli (Andersen et al., 1992, European J. Biochem. 204: 51 -56), from Lactococcus lactis (Martinussen et al., 1994, J. Bacteriol. 176: 6457-63), from Mycobacterium bovis (Kim et al., 1997, Biochem. Mol. Biol. Internat. 41 : 1117-24) and from Bacillus subtilis (Martinussen et al., 1995, J. Bacteriol. 177: 271 -4) may be used in the context of the invention. However, it is most particularly preferred to use a yeast UPRTase and in particular that encoded by the S. cerevisiae (FUR1 gene) whose sequence is disclosed in Kern et al. (1990, Gene 88: 149-57). Functional UPRTase analogues may also be used such as the N-terminally truncated FUR1 mutant described in EP998568 (with a deletion of the 35 first residues up to the second Met residue present at position 36 in the native protein) which exhibits a higher UPRTase activity than that of the native enzyme.

Preferably, the suicide gene inserted in the viral genome of the poxvirus of the present invention encodes a polypeptide having CDase and UPRTase activities. Such a polypeptide can be engineered by fusion of two enzymatic domains, one having the CDase activity and the second having the UPRTase activity. Exemplary polypeptides include without limitation fusion polypeptides codA::upp, FCY1 ::FUR1 and FCY1 ::FUR1 [Delta] 105 (FCU1 ) and FCU1 -8 described in WO96/16183, EP998568 and W02005/07857. Of particular interest is the FCU1 suicide gene (or FCY1 ::FUR1 [Delta] 105 fusion) encoding a polypeptide comprising the amino acid sequence represented in the sequence identifier SEQ ID NO: 1 of W02009/065546. The present invention encompasses analogs of such polypeptides providing they retain the CDase, and/or UPRTase activities. It is within the reach of the skilled person to isolate the CDase and/or UPRTase -encoding nucleic acid molecules from the published data, eventually engineer analogs thereof and test the enzymatic activity in an acellular or cellular system according to conventional techniques (see e.g. EP998568). Antibodies and antigen-binding fragments or derivatives thereof

Any antibody or antigen-binding fragment or derivative thereof with therapeutic activity may further be encoded by the poxvirus of the invention, including anti-neoplastic antibodies or antigen-binding fragments or derivatives thereof, in particular antibodies or antigen-binding fragments or derivatives thereof that affect the regulation of cell surface receptors, such as anti HER2 antibodies (e.g. trastuzumab), anti-EGFR antibodies (e.g. cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab), anti-VEGF antibodies (e.g. bevacizumab and ranibizumab) or antigen-binding fragments or derivatives thereof.

In the context of the invention, "antibody" ("Ab") is used in the broadest sense and is preferably as defined in the section “General definitions” above.

The antibody is preferably a monoclonal antibody, preferably humanized or chimeric. Representative examples of antigen-binding fragments are known in the art, including Fab, Fab’, F(ab’)2, dAb, Fd, Fv, scFv, ds-scFv and diabody. A particularly useful antibody fragment is a single chain antibody (scFv) comprising the two domains of a Fv fragment, VL and VH, that are fused together, eventually with a linker to make a single protein chain.

Expression of the nucleic acid molecule encoding the binding agent, the fusion polypeptide, and/or the therapeutic polypeptide

The nucleic acid molecule encoding the binding agent as defined above, the fusion polypeptide comprising the nucleic acid molecule encoding the binding agent as defined above, the additional nucleic acid molecule encoding a polypeptide of interest (preferably a therapeutic polypeptide) as defined above, and any combination thereof, may be easily obtained by standard molecular biology techniques (e.g. PCR amplification, cDNA cloning, chemical synthesis) using sequence data accessible in the art and the information provided herein. For example, methods for cloning antibodies, fragments and analogs thereof are known in the art (see e.g. Harlow and Lane, 1988, Antibodies - A laboratory manual; Cold Spring Harbor Laboratory, Cold Spring Harbor NY). Antibodyencoding nucleic acid molecule may be isolated from the producing hybridoma (e.g. Cole et al. in Monoclonal antibodies and Cancer Therapy; Alan Liss pp77-96), immunoglobulin gene libraries, or from any available source or the nucleotide sequence may be generated by chemical synthesis. In addition, the recombinant nucleic acid can be optimized for providing high level expression in a particular host cell or subject. It has been indeed observed that, the codon usage patterns of organisms are highly non-random and the use of codons may be markedly different between different hosts. For example, the therapeutic gene may be from bacterial, viral or lower eukaryote origin and thus have an inappropriate codon usage pattern for efficient expression in higher eukaryotic cells (e.g. human). Typically, codon optimization is performed by replacing one or more "native" (e.g. bacterial, viral or yeast) codon corresponding to a codon infrequently used in the host organism by one or more codon encoding the same amino acid which is more frequently used. It is not necessary to replace all native codons corresponding to infrequently used codons since increased expression can be achieved even with partial replacement.

Further to optimization of the codon usage, expression in the host cell or subject can further be improved through additional modifications of the recombinant nucleic sequence(s). For example, various modifications may be envisaged so as to prevent clustering of rare, non-optimal codons being present in concentrated areas and/or to suppress or modify "negative" sequence elements which are expected to negatively influence expression levels. Such negative sequence elements include without limitation the regions having very high (>80%) or very low (<30%) GC content; AT -rich or GC-rich sequence stretches; unstable direct or inverted repeat sequences; R A secondary structures; and/or internal cryptic regulatory elements such as internal TATA-boxes, chisites, ribosome entry sites, and/or splicing donor/acceptor sites.

Advantageously, the nucleic acid molecule encoding the binding agent as defined above, the fusion polypeptide comprising the nucleic acid molecule encoding the binding agent as defined above, the additional nucleic acid molecule encoding a polypeptide of interest (preferably a therapeutic polypeptide) as defined above, and any combination thereof, is operably linked to suitable regulatory elements for expression in a desired host cell or subject.

Regulatory elements

In accordance with the present invention, the poxvirus according to the invention further comprises the regulatory elements necessary for the expression of the fusion polypeptide according to the invention in a host cell or subject. The terms "regulatory elements" or "regulatory sequences" refer to any element that allows, contributes or modulates expression in a given host cell or subject. The regulatory elements are arranged so that they function in concert for their intended purposes, for example, for a promoter to effect transcription of a nucleic acid molecule from the transcription initiation to the terminator of said nucleic acid molecule in a permissive host cell.

In a preferred embodiment, the poxvirus of the invention comprises one or more expression cassettes, each expression cassette comprising at least one promoter placed 5’ to the nucleic acid molecule (e.g. encoding the fusion polypeptide according to the invention) and one polyadenylation sequence located 3’ to said nucleic acid molecule.

It will be appreciated by those skilled in the art that the choice of the regulatory sequences can depend on such factors as the nucleic acid molecule itself, the vector into which it is inserted, the host cell or subject to be treated, the level of expression desired, etc. The promoter is of special importance. In the context of the invention, it can be constitutive directing expression of the encoded product (e.g. the fusion polypeptide according to the invention) in many types of host cells or specific to certain host cells (e.g. organ-specific regulatory sequences) or regulated in response to specific events or exogenous factors (e.g. by temperature, nutrient additive, hormone, etc.) or according to the phase of a viral cycle (e.g. late or early). One may also use promoters that are repressed during the production step in response to specific events or exogenous factors, in order to optimize vector production and circumvent potential toxicity of the expressed polypeptide(s) in the producing cells.

Various promoters may be used in the context of the present invention that are known in the state of the art. Vaccinia virus promoters are particularly appropriate for use in poxviral vectors (e.g. oncolytic vaccinia virus or MVA). Representative examples include, without limitation, the vaccinia p7.5K, pH5R, p11 K7.5 (Erbs et al., 2008, Cancer Gene Ther. 15(1 ): 18-28), pSE, pTK, p28, p11 , pB2R, pF17R, pA14L, pSE/L, pA35R and pK1 L promoters, synthetic promoters such as those described in Chakrabarti et al. (1997, Biotechniques 23: 1094-7; Hammond et al, 1997, J. Virol Methods 66: 135-8; and Kumar and Boyle, 1990, Virology 179: 151 -8) as well as early/late chimeric promoters. Other promoters may be used, selected from the list comprising cytomegalovirus (CMV) immediate early promoter (US 5,168,062), bidirectional CMV-promoter, Rous sarcoma Virus (RSV) promoter, adenovirus major late (MLP) promoter, phosphoglycero kinase (PGK) promoter (Adra et al., 1987, Gene 60: 65-74), EF1o, thymidine kinase (TK) promoter of herpes simplex virus (HSV)-1 , T7 polymerase promoter (W098/10088) and inducible promoters (e.g. promoters whose transcriptional activity is regulated by the presence or absence of alcohol, tetracycline, steroids, metal, sugar, etc.). CMV promoters may also be used (e.g. Ad5, Ad11 , Ad26, Ad35). The poxvirus may contain one or more promoters depending on the number of nucleic acid molecule(s) to be expressed. Preferably, when the poxvirus encodes the binding agent as defined above (and/or the fusion polypeptide comprising the nucleic acid molecule encoding the binding agent as defined above) and another molecule of interest (e.g. the additional nucleic acid molecule encoding a polypeptide of interest (preferably a therapeutic polypeptide) as defined above), each of the encoding nucleic acid molecule is placed under the control of independent promoters. Alternatively, one may use bidirectional promoter(s).

In a preferred embodiment, the nucleic acid molecule encoding the binding agent as defined above (and/or the fusion polypeptide comprising the nucleic acid molecule encoding the binding agent as defined above) and/or said additional nucleic acid molecule encoding a polypeptide of interest (preferably a therapeutic polypeptide) as defined above, is placed under the control of a poxvirus promoter, preferably, a vaccinia virus promoter and more preferably one selected from the group consisting of the p7.5K, pH5R, p11 K7.5, pSE, pSE/L, pTK, pB2R, p28, p11 , pF17R, pA14L and pK1 L promoter, synthetic promoters, and early/late chimeric promoters, preferably pH5R.

Those skilled in the art will appreciate that the regulatory elements controlling the nucleic acid expression may further comprise additional elements for proper initiation, regulation and/or termination of transcription (e.g. a transcription termination sequences), mRNA transport (e.g. nuclear localization signal sequences, polyadenylations sequences), processing (e.g. splicing signals, self-cleaving peptides like T2A, P2A, E2A, F2A, linkers), stability (e.g. introns, like 16S/19S or chimeric human B globin/IgG, and non-coding 5' and 3' sequences), translation (e.g. an initiator Met, tripartite leader sequences, IRES ribosome binding sites, signal peptides, etc.), targeting sequences, linkers (e.g. linkers composed of flexible residues like glycine and serine), transport sequences, secretion signal, and sequences involved in replication or integration. Said sequences have been reported in the literature and can be readily obtained by those skilled in the art.

Insertion of the nucleic acid molecule into the poxvirus

The nucleic acid molecule(s) or expression cassette(s) (such as those encoding the binding agent as defined above, and/or the fusion polypeptide comprising the nucleic acid molecule encoding the binding agent as defined above, and/or said additional nucleic acid molecule encoding a polypeptide of interest (preferably a therapeutic polypeptide)) is/are inserted into the poxvirus by any appropriate technique known in the art.

The nucleic acid molecule(s) /expression cassette(s) may be inserted in any suitable location within the poxvirus genome, e.g. within a viral gene, an intergenic region, in a non-essential gene or region or in place of viral sequences. Preference is given to insertion within the viral genome in a non-essential locus.

Insertion into the virus can be performed by routine molecular biology, e.g. as described in Sambrook et al. (2001 , Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory). Insertion into a poxviral genome can be performed through homologous recombination as described respectively in Chartier et al. (1996, J. Virol. 70: 4805-10) and Paul et al. (2002, Cancer gene Ther. 9: 470-7).

Thymidine kinase (TK) gene, Ribonucleotide reductase (RR) gene and F2L gene are particularly appropriate for insertion in oncolytic poxvirus, in particular oncolytic vaccinia viruses, such as Copenhagen and Western Reserve vaccinia virus. Deletion II or III are particularly appropriate for insertion into MVA vector (W097/02355; Meyer et al., 1991 , J. Gen. Virol. 72: 1031 -8). Preferably, when the poxvirus is MVA, insertion of the neopeptide-encoding nucleic acid molecule(s) or expression cassette(s) is made within MVA’s deletion III. When the recombinant poxvirus comprises several nucleic acid molecules/expression cassettes as described above, they may be inserted in the viral genome at the same or distinct location. Preference is given to insertion of all expression cassettes at the same location, especially in TK locus for a recombinant vaccinia virus and in deletion III for a recombinant MVA.

The general conditions for constructing recombinant poxviruses are well known in the art (see for example W02007/ 147528; W02010/130753; W003/008533; US 6,998,252; US 5,972,597 and US 6,440,422). Typically, the nucleic acid molecule(s) /expression cassette(s) to be inserted is/are cloned in a transfer plasmid surrounded by two recombination arms, corresponding to stretches of poxviral sequences homologous (e.g. 90-100% identical) to those present in the parental genome on both sides of the insertion site. The length of the recombination arms may vary within the transfer plasmid. Desirably, each of the recombination arms comprises at least 150bp, preferably at least 200bp, more preferably, at least 300bp, even more preferably from300 to 600 bp with a specific preference for 350 to 500bp (e.g. approximately 350bp or 500bp) or for 300 to 400 bp of homologous poxvirus sequences. The parental poxvirus may be a wild-type poxvirus or a modified one (e.g. attenuated, tumor-specific, etc.,) as described above in connection with the term “poxvirus”. Insertion is then performed by homologous recombination between the stretch of homologous sequences present both in the parental genome and the linearized transfer plasmid, requiring transfection of permissive cells with the linearized transfer plasmid and infection with the parental poxvirus.

The step of generating the recombinant poxvirus (i.e. the poxvirus encoding the nucleic acid molecule(s) defined above) may encompass the use of a parental poxvirus comprising a reporter gene and, notably, a fluorescent reporter gene, cloned at the site of insertion that is selected for the nucleic acid molecule(s) /expression cassette(s). Preferably, the reporter gene is placed under the transcriptional control of a promoter allowing its expression within the permissive cells, e.g. a vaccinia promoter. This embodiment facilitates the selection of the recombinant poxvirus with respect to the parental poxvirus. Representative examples of fluorescent reporters that can be used in the context of the present invention include, without limitation, GFP (Green Fluorescent Protein), eGFP (Enhanced Green Fluorescent Protein), AmCyan 1 fluorescent protein and mCherry. For instance, when relying on mCherry (a monomeric fluorescent protein that originates from a Discosoma mushroom with peak absorption/emission at 587 nm and 610 nm), the recombinant viruses having inserted the nucleic acid molecule(s) or expression cassette(s) in place of the mCherry-encoding sequences, will give rise to white plaques whereas the parental viruses retaining the mCherry expression cassette will give rise to red plaques The selection of the recombinant poxvirus may be by direct visualization (white plaques) or may also be facilitated by sorting means such as FACS after labelling with an APC (Allophycocyanin)-tagged anti-vaccinia virus antibody. A vast number of anti-vaccinia antibodies is available from commercial sources. Usually, one recombinant is obtained for 50 to 100 parental and the whole process from the insertion step to the generation of the recombinant poxvirus takes 5 to 6 weeks.

In some embodiments, when the parental poxvirus comprises a reporter gene, homologous recombination efficacy between the parental poxvirus and the transfer plasmid may be increased by further adding a step of cleavage by an endonuclease able to generate at least one double strand break in the reporter gene (e.g. mCherry) nucleotide sequence but in which said endonuclease does not cleave the poxviral genome. The suitable endonuclease is preferably selected from the group consisting of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nucleases and restriction enzymes with unique cleavage within the reporter gene. The use of the CRISPR/CAS9 system for virus editing is described in the art (Yuan et al., 2015, J. Virol 89, 5176-9; Yuan et al., 2016, Viruses 8, 72, doi:10.3390) requiring the use of a plasmid encoding a Cas9 without a nuclear localization signal as well as a suitable guide RNA. In this consideration, further to infection with the parental virus, the permissive cells may be transfected with the transfer plasmid, the Cas9-expressing plasmid and one or more plasmid(s) encoding the guide RNA (e.g. mCherry-targeted guide RNA). The selection of recombinant poxvirus is then performed visually (direct isolation of white plaques corresponding to the recombinant whereas colored plaques correspond to the parental and the color depends on the reporter gene) or using conventional sorting means (FACS optionally after a labelling step with appropriate antibodies as described above). With this process, one recombinant is obtained for 1 to 10 parental and the whole process from the insertion step to the generation of the recombinant poxvirus takes approximately 3-4 weeks.

In one embodiment, the poxvirus according to the invention is produced on chicken embryo fibroblasts (CEF), HeLa cells, EB66® cells, Vero cells, HEK 293 cells, PerC6 cells, BHK21 cells, or MRC5 cells.

Method for producing the poxvirus

Once generated, a poxvirus of the invention may be produced/amplified using conventional techniques.

Typically, the poxvirus of the invention is produced into a suitable host cell line using conventional techniques including culturing the transfected or infected host cell under suitable conditions so as to allow the production and recovery of infectious poxviral particles.

Therefore, the present invention also relates to a method for producing the poxvirus of the invention, comprising, or consisting essentially of, or consisting of, the steps of: a) introducing the viral vector of the invention, in the form of infectious viral particles, into a suitable producer cell or cell line, b) culturing said producer cell or cell line under suitable conditions so as to allow the production of infectious viral particles, c) recovering the produced infectious viral particles from the culture of said producer cell or cell line, and d) optionally purifying said viral particle.

In a preferred embodiment, the method for producing the poxvirus of the invention comprises, or consists essentially of, or consists of, the steps of: a) obtaining or preparing a producer cell line; b) infecting the obtained or prepared producer cell line with the poxvirus; c) culturing the infected producer cell line under suitable conditions so as to allow the production of the poxvirus; d) recovering the produced poxvirus from the culture of said producer cell line; and optionally e) purifying said recovered poxvirus

The choice of the producer cell may depend on the type of poxvirus to be produced, and those skilled in the art know which producer cells or cell lines are suitable for which poxvirus.

In particular, non-propagative vector MVA is strictly host-restricted and is typically amplified on avian cells, either primary avian cells (such as chicken embryo fibroblasts (CEF) prepared from chicken embryos obtained from fertilized eggs) or immortalized avian cell lines. Representative examples of suitable avian cell lines for MVA production include without limitation the Cairina moschata cell lines immortalized with a duck TERT gene (see e.g. W02007/077256, W02009/004016, W02010/130756 and W02012/001075); avian cell lines immortalized with a combination of viral and/or cellular genes (see e.g. W02005/042728), spontaneously immortalized cells (e.g. the chicken DF1 cell line disclosed in US 5,879,924), or immortalized cells which derive from embryonic cells by progressive severance from growth factors and feeder layer (e.g. Ebx chicken cell lines disclosed in W02005/007840 and W02008/129058 such as Eb66 described in Olivier et al., 2010, mAbs 2(4): 405-15).

For other vaccinia viruses or other poxvirus strains, either non-propagative or oncolytic, in addition to avian primary cells (such as CEF) and avian cell lines, many other non-avian cell lines are available for production, including human cell lines such as HeLa (ATCC- CRM-CCL-2™ or ATCC-CCL-2.2™), MRC-5, HEK-293; hamster cell lines such as BHK-21 (ATCC CCL-10), and Vero cells. In a preferred embodiment, non-MVA vaccinia viruses are amplified in HeLa cells (see e.g. W02010/130753).

In a preferred embodiment, the poxvirus is produced on chicken embryo fibroblasts (CEF), HeLa cells, EB66® cells, Vero cells, HEK 293 cells, PerC6 cells, BHK21 cells, or MRC5 cells.

Producer cells can be cultured in conventional fermentation bioreactors, flasks, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a given host cell. No attempts will be made here to describe in detail the various prokaryote and eukaryotic host cells and methods known for the production of the non- propagative viral vectors for use in the invention. Producer cells are preferably cultured in a medium free of animal- or human-derived products, using a chemically defined medium with no product of animal or human origin. In particular, while growth factors may be present, they are preferably recombinantly produced and not purified from animal material. An appropriate animal-free medium may be easily selected by those skilled in the art depending on selected producer cells. Such media are commercially available. In particular, when CEFs are used as producer cells, they may be cultivated in VP-SFM cell culture medium (Invitrogen). Producer cells are preferably cultivated at a temperature comprised between 30° C and 38° C (more preferably at around 37° C) for between 1 and 8 days (preferably for 1 to 5 days for CEF and 2 to 7 days for immortalized cells) before infection. If needed, several passages of 1 to 8 days may be made in order to increase the total number of cells.

Infection of producer cell lines by the viral vector of the invention is made under appropriate conditions (in particular using an appropriate multiplicity of infection (MOI)) to permit productive infection of producer cells.

The infected producer cells are then cultured under appropriate conditions well known to those skilled in the art until progeny viral vectors are produced. Culture of infected producer cells is also preferably performed in a medium (which may be the same as or different from the medium used for culture of producer cells and/or for infection step) free of animal- or human-derived products (using a chemically defined medium with no product of animal or human origin) at a temperature between 30° C and 37° C, for 1 to 5 days.

The poxvirus of the invention can be collected from the culture supernatant and/or the producer cell lines. The cell culture supernatant and the producer cells can be pooled or collected separately. Recovery from producer cells (and optionally also from culture supernatant) may require a step allowing the disruption of the producer cell membrane to allow the liberation of the poxviruses. Various techniques are available to those skilled in the art, including but not limited to freeze/thaw, hypotonic lysis, sonication, micro fluidization, or high-speed homogenization. According to a preferred embodiment, the step of recovery of the produced poxviruses comprises a lysis step wherein the producer cell membrane is disrupted, preferably by using a high-speed homogenizer. High speed homogenizers are commercially available from Silverson Machines Inc (East Longmeadow, USA) or Ika-Labotechnik (Staufen, Germany). According to particularly preferred embodiment, said High Speed homogeneizer is a SILVERSON L4R.

The poxvirus of the invention may then be further purified, using purification steps well known in the art. Various purification steps can be envisaged, including clarification, enzymatic treatment (e.g. endonuclease, protease, etc.), chromatographic and filtration steps. Appropriate methods are described in the art (e.g. W02007/147528; W02008/138533, W02009/100521 , W02010/130753, W02013/022764). In a preferred embodiment, the purification step comprises a tangential flow filtration (TFF) step that can be used to separate the virus from other biomolecules, to concentrate and/or desalt the virus suspension. Various TFF systems and devices are available in the art depending on the volume to be filtered including, without limitation, Spectrumlabs, Pall Corp, PendoTech and New Pellicon among others.

The poxviruses of the invention may then be protected by any method known in the art, in order to extend the viral vector persistence in the subject blood circulation. Said methods comprise, but are not limited to, chemical shielding like PEGylation (Tesfay et al., 2013, J. of Virology, 87(7): 3752-3759; N’Guyen et al., 2016, Molecular Therapy Oncolytics, 3, 15021 ), viroembolization (W02017/037523), etc.

The present invention also concerns the poxvirus obtained according to the method of the invention, as described above.

Host cells

The present invention also relates to a host cell comprising the poxvirus according to the invention.

In one embodiment, the invention relates to a host cell infected with viral vector according to the invention.

Such host cells are preferably selected from the producer cells or cell lines defined above.

Composition

The present invention also relates to a composition comprising, or consisting essentially of, or consisting of, the poxvirus of the invention (as described above), the poxvirus obtained according to the method of the invention (as described above), the host cell according to the invention (as described above), or any combination thereof.

Preferably, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable vehicle.

The term “pharmaceutically acceptable vehicle” is intended to include any and all carriers, solvents, diluents, excipients, adjuvants, dispersion media, coatings, antibacterial and antifungal agents, absorption agents, and the like compatible with administration in mammals and in particular human subjects.

Preferably, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable vehicle.

The term “pharmaceutically acceptable vehicle” is intended to include any and all carriers, solvents, diluents, excipients, adjuvants, dispersion media, coatings, antibacterial and antifungal agents, absorption agents, and the like compatible with administration in mammals and in particular human subjects.

The poxvirus of the invention (as described above), the poxvirus obtained according to the method of the invention (as described above), the host cell according to the invention (as described above), or any combination thereof, can independently be placed in a solvent or diluent appropriate for human or animal use. The solvent or diluent is preferably isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength. Representative examples include sterile water, physiological saline (e.g. sodium chloride), Ringer’s solution, glucose, trehalose or saccharose solutions, Hank’s solution, and other aqueous physiologically balanced salt solutions (see for example the most current edition of Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams&Wilkins).

In some embodiments, the composition is suitably buffered for human use. Suitable buffers include without limitation phosphate buffer (e.g. PBS), bicarbonate buffer and/or Tris buffer capable of maintaining a physiological or slightly basic pH (e.g. from approximately pH 7 to approximately pH 9).

The composition may also contain other pharmaceutically acceptable excipients for providing desirable pharmaceutical or pharmacodynamic properties, including for example osmolarity, viscosity, clarity, colour, sterility, stability, rate of dissolution of the formulation, modifying or maintaining release or absorption into a human or animal subject, promoting transport across the blood barrier or penetration in a particular organ. In a further embodiment, the composition may be combined with soluble adjuvants including, but not limited to alum, mineral oil emulsion and related compounds such as those described in W02007/147529, polysaccharides such as Adjuvax and squalenes, oil in water emulsions such as MF59, double-stranded RNA analogues such as poly(l:C), single stranded cytosine phosphate guanosine oligodeoxynucleotides (CpG) (Chu et al., 1997, J. Exp. Med., 186: 1623; Tritel et al., 2003, J. Immunol., 171 : 2358) and cationic peptides such as IC-31 (Kritsch et al., 2005, J. Chromatogr. Anal. Technol. Biomed. Life Sci., 822: 263-70). In one embodiment, the composition may be formulated with the goal of improving its stability, in particular under the conditions of manufacture and long-term storage (i.e. for at least 6 months, with a preference for at least two years) at freezing (e.g. -70° C, - 20° C), refrigerated (e.g. 4°C) or ambient temperatures.

When the composition comprises a poxvirus, a stabilizing formulation adapted to the specific poxvirus is preferably used. Various virus formulations are available in the art either in frozen, liquid form or lyophilized form (e.g. W098/02522, W001 /66137, W003/053463, W02007/056847 and W02008/114021, etc.). Lyophilized compositions are usually obtained by a process involving vacuum drying and freeze-drying. For illustrative purposes, buffered formulations including NaCl and/or sugar are particularly adapted to the preservation of viruses (e.g. S520 buffer: 100 g/L saccharose, 30 mM Tris, pH 7.6; S08 buffer: 10 mM Tris, 50 mM NaCl, 50 g/L saccharose, 10 mM Sodium glutamate, pH 8.0).

Dosage

Preferably, the composition comprises, or consists essentially of, or consists of, a therapeutically effective amount of of the poxvirus of the invention (as described above), of the poxvirus obtained according to the method of the invention (as described above), of the host cell according to the invention (as described above), or any combination thereof.

Accordingly, the present invention also relates to a composition comprising, or consisting essentially of, or consisting of, a therapeutically effective amount of the poxvirus of the invention (as described above), of the poxvirus obtained according to the method of the invention (as described above), of the host cell according to the invention (as described above), or any combination thereof; and a pharmaceutically acceptable vehicle.

A “therapeutically effective amount” corresponds to the amount of the poxvirus of the invention (as described above), of the poxvirus obtained according to the method of the invention (as described above), of the host cell according to the invention (as described above), or any combination thereof, that is sufficient for producing one or more beneficial results. Such a therapeutically effective amount may vary as a function of various parameters, e.g. the mode of administration, the disease state, the age and weight of the subject, the ability of the subject to respond to the treatment, the kind of concurrent treatment and/or the frequency of treatment. The appropriate dosage of the poxvirus of the invention (as described above), of the poxvirus obtained according to the method of the invention (as described above), of the host cell according to the invention (as described above), or any combination thereof, may be routinely determined by a practitioner in the light of the relevant circumstances.

When the composition comprises a poxvirus according to the invention, individual doses for the poxvirus may suitably vary within a range extending from approximately 10³ to approximately 10¹² vp (viral particles), iu (infectious unit) or pfu (plaque-forming units) depending on the type of viral vector and quantitative technique used, preferably from 10⁴ pfu to 10¹¹ pfu, preferably from 10⁵ pfu to 10¹⁰ pfu, more preferably from 10⁶ pfu to 10⁹ pfu of the poxvirus; notably individual doses of approximately 10⁶, 5x10⁶, 10⁷, 5x10⁷, 10⁸ or 5x10⁸ pfu of the poxvirus. The quantity of viral vector present in a sample can be determined by routine titration techniques, e.g. by counting the number of plaques following infection of permissive cells (e.g. BHK-21 , CEF or HEK-293) (pfu titer), immunostaining quantitative immunofluorescence (e.g. using anti-virus antibodies) (iu titer), by HPLC (vp titer).

More particularly, when the composition comprises an oncolytic poxvirus, it preferably comprises 10³ to 10¹² pfu, more preferably from 10⁴ pfu to 10¹¹ pfu, even more preferably from 10⁵ pfu to 10¹⁰ pfu, most preferably from 10⁶ pfu to 10⁹ pfu of the poxvirus; notably individual doses of approximately 10⁶, 5x10⁶, 10⁷, 5x10⁷, 10⁸ or 5x10⁸ pfu of the poxvirus vector according to the invention.

When the composition comprises a non-propagative poxvirus, it preferably comprises between approximately 10⁶ pfu and approximately 10¹² pfu, more preferably between approximately 10⁷ pfu and approximately 10¹¹ pfu; even more preferably between approximately 10⁸ pfu and approximately 1O^1o pfu (e.g. from 5x10⁸ to 6x10⁹, from 6x10⁸ to 5x10⁹, from 7x10⁸ to 4x10⁹, from 8x10⁸ to 3x10⁹, from 9x10⁸ to 2x10⁹ pfu) of the non- propagative poxviral vector, these doses being convenient for human use, with a preference for individual doses comprising approximately 10⁹ pfu of poxviral vector.

Adminis tra tion

The composition according to the invention may be formulated for any suitable administration route, including intravenous, intramuscular, subcutaneous, oral, intranasal, transdermal or intratumoral administration.

Any of the conventional administration routes is applicable in the context of the invention including parenteral, topical or mucosal routes. Parenteral routes are intended for administration as an injection or infusion and encompass systemic as well as local routes. Parenteral injection types that may be used to administer the poxvirus composition include intravenous (into a vein), intravascular (into a blood vessel), intra-arterial (into an artery such as hepatic artery), intradermal (into the dermis), subcutaneous (under the skin), intramuscular (into muscle), intraperitoneal (into the peritoneum) and intratumoral (into a tumor or its close vicinity) and also scarification. Administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump. Mucosal administrations include without limitation oral/alimentary, intranasal, intratracheal, intrapulmonary, intravaginal or intra-rectal route. Topical administration can also be performed using transdermal means (e.g. patch and the like). Preferably, the modified poxvirus composition is formulated for intravenous or intratumoral administration in the tumor or at its close vicinicity).

Administrations may use conventional syringes and needles (e.g. Quadrafuse injection needles) or any compound or device available in the art capable of facilitating or improving delivery of the modified poxvirus in the subject (e.g. electroporation for facilitating intramuscular administration). An alternative is the use of a needleless injection device (e.g. Biojector TM device). Transdermal patches may also be envisaged. The composition described herein is suitable for a single administration or a series of administrations. It is also possible to proceed via sequential cycles of administrations that are repeated after a rest period. Intervals between each administration can be from three days to about six months (e.g. 24h, 48h, 72h, weekly, every two weeks, monthly or quarterly, etc). Intervals can also be irregular. The doses can vary for each administration within the range described above. A preferred therapeutic scheme involves 2 to 10 weekly administrations possibly followed by 2 to 15 administrations at longer intervals (e.g. 3 weeks) of the poxvirus composition.

In an advantageous embodiment, the composition according to the invention further comprises another therapeutic agent, preferably an anticancer. Said another therapeutic agent is preferably as described in section “Therapeutic uses and methods of treatment” below, subsection “Stand-alone therapy or combination with one or more additional therapeutic intervention(s)”.

Therapeutic uses and methods of treatment

In the context of the present invention, the Inventors unexpectedly showed that the novel poxviruses comprising a nucleic acid molecule encoding a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ) herein designed possess a significantly higher blocking activity than the reference anti-PD-L1 antibody, Avelumab. In addition, the experimental data obtained by the Inventors demonstrated that the anti- PD-L1 sdAb expressed by these poxviruses are capable to bind PD-L1 at the surface of tumor cell, more efficiently than expressed Avelumab. Importantly, the Inventors confirmed that replication and oncolytic activities of the poxviruses expressing the anti- PD-L1 sdAbs were unaffected by the presence and expression of the transgene encoding the sdAb. Binding of these anti-PD-L1 sdAb to cynomolgus monkey PD-L1 was also demonstrated, showing that the results are not limited to human.

The anti-PD-L1 sdAb encoded by the poxviruses of the invention are particularly advantageous over the anti-PD-L1 antibodies of the prior art, including Avelumab. Indeed, the significantly lower molecular weights and sizes of all of the anti-PD-L1 sdAb formats designed by the Inventors allows a better penetration and diffusion into tumor tissue, compared to anti-PD-L1 antibodies. In addition, such anti-PD-L1 sdAbs have a shorter halflife compared to anti-PD-L1 antibodies of the prior art, including Avelumab. This advantage is particularly beneficial to prevent unwanted systemic diffusion in the organism, thereby improving specificity of the targeting of tumor cells.

Altogether, the poxviruses expressing various formats of a sdAb specifically binding to PD- L1 here developed by the Inventors provides a novel and efficient treatment of proliferative disease, such as cancer.

Accordingly, the present invention also relates to therapeutic uses of the poxvirus of the invention (as described above), of the poxvirus obtained according to the method of the invention (as described above), of the host cell according to the invention (as described above), of the composition according to the invention (as described above), or any combination thereof, as well as to associated methods of treatment.

The present invention thus also relates to the poxvirus of the invention (as described above), the poxvirus obtained according to the method of the invention (as described above), the host cell according to the invention (as described above), the composition according to the invention (as described above), or any combination thereof, for use as a medicament or as a vaccine.

The present invention also relates to the use of the poxvirus of the invention (as described above), of the poxvirus obtained according to the method of the invention (as described above), of the host cell according to the invention (as described above), of the composition according to the invention (as described above), or any combination thereof, for manufacturing a medicament or a vaccine. The present invention also relates to the use of the poxvirus of the invention (as described above), of the poxvirus obtained according to the method of the invention (as described above), of the host cell according to the invention (as described above), of the composition according to the invention (as described above), or any combination thereof, as a medicament or as a vaccine.

The present invention also relates to a method for treating a disease in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of the poxvirus of the invention (as described above), of the poxvirus obtained according to the method of the invention (as described above), of the host cell according to the invention (as described above), of the composition according to the invention (as described above), or any combination thereof.

In a preferred embodiment, the uses above are for treating or preventing a proliferative disease, preferably a cancer.

Treatment or prevention of cancer

The present invention thus also relates to the poxvirus of the invention (as described above), the poxvirus obtained according to the method of the invention (as described above), the host cell according to the invention (as described above), the composition according to the invention (as described above), or any combination thereof, for use in the treatment or prevention of cancer.

The present invention also relates to the use of the poxvirus of the invention (as described above), of the poxvirus obtained according to the method of the invention (as described above), of the host cell according to the invention (as described above), of the composition according to the invention (as described above), or any combination thereof, for manufacturing a medicament or a vaccine for use in the treatment or prevention of cancer.

The present invention also relates to the use of the poxvirus of the invention (as described above), of the poxvirus obtained according to the method of the invention (as described above), of the host cell according to the invention (as described above), of the composition according to the invention (as described above), or any combination thereof, in the treatment or prevention of cancer.

The present invention also relates to a method for treating a cancer in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of the poxvirus of the invention (as described above), of the poxvirus obtained according to the method of the invention (as described above), of the host cell according to the invention (as described above), of the composition according to the invention (as described above), or any combination thereof.

Preferably, said cancer is a solid cancer, more preferably selected from the group consisting of renal cancer, prostate cancer, breast cancer, bladder cancer, colorectal cancer, lung cancer, liver cancer, gastric cancer, bile duct carcinoma, endometrial cancer, pancreatic cancer, ovarian cancer, head and neck cancer, melanoma, glioblastoma, multiple myeloma, or malignant glioma cells.

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: 133-44). Preferred cancers that may be treated in the invention include cancers typically responsive to immunotherapy. Non-limiting examples of preferred cancers for treatment include melanoma (e.g. metastatic malignant melanoma), renal cancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormone refractory prostate adenocarcinoma), breast cancer, colorectal cancer, lung cancer (e.g. non-small cell lung cancer) and liver cancer (e.g. hepatocarcinoma).

Preferably, the treated cancer is a PD-L1 positive cancer, meaning that PD-L1 may be detected (at the mRNA or protein level) in a tumor sample, so that at least part of the tumor cells expresses PD-L1 . PD-L1 expression was identified in most human cancers but with a broad percentage of positivity (from a few percent, e.g prostate cancer, to up to 60% e.g. thymic cancer (Yarchoan et al. 2019 JCI Insight; 4:e126908). Cancers known to be generally PD-L1 positive comprise lung cancer (including adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and neuroendocrine carcinoma), ovarian cancer (including adenocarcinoma and carcinosarcoma), melanoma, skin cancer, colon cancer and thymic cancer and those cancers are thus particularly preferred in the context of the present invention.

Moreover, it has been shown (Liu et al., 2017, Nat Commun. 8:14754) that vaccinia virus infection induces expression of PD-L1 into the tumor. Therefore, even PD-L1 negative cancers are considered in the context of the present invention.

In some embodiments, the PD-L1 status of the cancer cells of the subject to be treated may be tested before treatment with the poxvirus of the invention (as described above), the poxvirus obtained according to the method of the invention (as described above), the host cell according to the invention (as described above), the composition according to the invention (as described above), or any combination thereof. In such embodiments, the poxvirus of the invention (as described above), the poxvirus obtained according to the method of the invention (as described above), the host cell according to the invention (as described above), the composition according to the invention (as described above), or any combination thereof, is administered to a subject which cancer has been previously determined as PD-L1 positive or the method or use according to the invention comprises a preliminary step of testing the PD-L1 status of the subject’s cancer and the poxvirus of the invention (as described above), the poxvirus obtained according to the method of the invention (as described above), the host cell according to the invention (as described above), the composition according to the invention (as described above), or any combination thereof, is administered to the subject only if its cancer is determined as PD-1 positive (in the contrary case, the subject may be administered an alternative treatment) .

Inhibition of tumor cell growth in vivo

The present invention thus also relates to the poxvirus of the invention (as described above), the poxvirus obtained according to the method of the invention (as described above), the host cell according to the invention (as described above), the composition according to the invention (as described above), or any combination thereof, for use for inhibiting tumor cell growth in vivo.

The present invention also relates to the use of the poxvirus of the invention (as described above), of the poxvirus obtained according to the method of the invention (as described above), of the host cell according to the invention (as described above), of the composition according to the invention (as described above), or any combination thereof, for manufacturing a medicament or a vaccine for inhibiting tumor cell growth in vivo. The present invention also relates to the use of the poxvirus of the invention (as described above), of the poxvirus obtained according to the method of the invention (as described above), of the host cell according to the invention (as described above), of the composition according to the invention (as described above), or any combination thereof, for inhibiting tumor cell growth in vivo.

The present invention also relates to a method for inhibiting tumor cell growth in vivo in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of the poxvirus of the invention (as described above), of the poxvirus obtained according to the method of the invention (as described above), of the host cell according to the invention (as described above), of the composition according to the invention (as described above), or any combination thereof. Enhancement of immune response to tumor cells

The present invention thus also relates to the poxvirus of the invention (as described above), the poxvirus obtained according to the method of the invention (as described above), the host cell according to the invention (as described above), the composition according to the invention (as described above), or any combination thereof, for use for enhancing an immune response to tumor cells in a subject.

The present invention also relates to the use of the poxvirus of the invention (as described above), of the poxvirus obtained according to the method of the invention (as described above), of the host cell according to the invention (as described above), of the composition according to the invention (as described above), or any combination thereof, for manufacturing a medicament or a vaccine for enhancing an immune response to tumor cells in a subject.

The present invention also relates to the use of the poxvirus of the invention (as described above), of the poxvirus obtained according to the method of the invention (as described above), of the host cell according to the invention (as described above), of the composition according to the invention (as described above), or any combination thereof, for enhancing an immune response to tumor cells in a subject.

The present invention also relates to a method for enhancing an immune response to tumor cells in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of the poxvirus of the invention (as described above), of the poxvirus obtained according to the method of the invention (as described above), of the host cell according to the invention (as described above), of the composition according to the invention (as described above), or any combination thereof.

In one embodiment, the administration of the poxvirus of the invention (as described above), of the poxvirus obtained according to the method of the invention (as described above), of the host cell according to the invention (as described above), of the composition according to the invention (as described above), or any combination thereof, elicits, stimulates and/or re-orients an immune response. In particular, the administration elicits, stimulates and/or re-orients a protective T or B cell response against the tumor cells in the treated host. The protective T response can be CD4+ or CD8+ or both CD4+ and CD8+cell mediated. B cell response can be measured by ELISA and T cell response can be evaluated by conventional ELISpot, ICS assays from any sample (e.g. blood, organs, tumors, etc) collected from the immunized animal or subject. Alternatively or in combination, the administration of the poxvirus of the invention (as described above), of the poxvirus obtained according to the method of the invention (as described above), of the host cell according to the invention (as described above), of the composition according to the invention (as described above), or any combination thereof, permits to change the tumor microenvironment with the goal of enhancing activity of effector cells in the tumor (especially effector T lymphocytes) and/or promoting at least partial Treg depletion. Tumor infiltrating cells can be easily identified for examples by conventional immunostaining assays.

When a poxvirus according to the invention (in particular an oncolytic poxvirus, such as an oncolytic vaccinia virus) is used, such poxvirus preferably provides a higher therapeutic efficacy than the one obtained in the same conditions either with a similar oncolytic poxvirus not encoding the binding agent of the invention alone, or with the binding agent of the invention alone. More preferably, the poxvirus preferably provides a higher therapeutic efficacy than the combination of a similar oncolytic poxvirus not encoding the binding agent of the invention with the binding agent of the invention. In the context of the invention, at least 5%, at least 10%, at least 15%, at least 20%, or at least 25% higher therapeutic efficacy is provided by the poxvirus of the invention compared to either the poxvirus or the binding agent alone, or preferably even in co-administration. A higher therapeutic efficacy may be evidenced as described above in connection with the term “therapeutically effective amount” with a specific preference for a longer survival.

Adminis tra tion

In all above-described therapeutic uses and methods, the poxvirus of the invention (as described above), the poxvirus obtained according to the method of the invention (as described above), the host cell according to the invention (as described above), the composition according to the invention (as described above), or any combination thereof, may be administered in a single dose or multiple doses. If multiples doses are contemplated, administrations may be performed by the same or different routes and may take place at the same site or at alternative sites and may comprise the same or different doses in the indicated intervals. Intervals between each administration can be from several hours to 8 weeks (e.g. 24h, 48h, 72h, weekly, every 2 or 3 weeks, monthly, etc.). Intervals can also be irregular. It is also possible to proceed via sequential cycles of administrations that are repeated after a rest period (e.g. cycles of 3 to 6 weekly or bi-weekly administrations followed by a rest period of 3 to 6 weeks). The dose can vary for each administration within the ranges described above. Any of the conventional administration routes are applicable in the context of the invention including parenteral, topical or mucosal routes. Parenteral routes are intended for administration as an injection or infusion and encompass systemic as well as locoregional routes. Locoregional administrations are restricted to a localized region of the body (e.g. intraperitoneal or intrapleural administration). Common parenteral injection types are intravenous (into a vein), intra-arterial (into an artery), intradermal (into the dermis), subcutaneous (under the skin) and intramuscular (into a muscle). Infusions typically are given by intravenous route. Topical administration can be performed using transdermal means (e.g. patch and the like). Mucosal administrations include without limitation oral/alimentary, intranasal, intratracheal, intrapulmonary, intravaginal or intra-rectal route. In a preferred embodiment, the poxvirus of the invention (as described above), the poxvirus obtained according to the method of the invention (as described above), the host cell according to the invention (as described above), the composition according to the invention (as described above), or any combination thereof, is administered via parenteral route, more preferably via intravenous, subcutaneous or intramuscular route, and even more preferably via intravenous route. In another embodiment, the poxvirus of the invention (as described above), the poxvirus obtained according to the method of the invention (as described above), the host cell according to the invention (as described above), the composition according to the invention (as described above), or any combination thereof, is administered via mucosal administration, preferably via intranasal or intrapulmonary routes.

Administrations may use conventional syringes and needles (e.g. Quadrafuse injection needles) or any compound or device available in the art capable of facilitating or improving delivery of the viral vector or composition in the subject.

Stand-alone therapy or combination with one or more additional therapeutic intervention(s)

The poxvirus of the invention (as described above), the poxvirus obtained according to the method of the invention (as described above), the host cell according to the invention (as described above), the composition according to the invention (as described above), or any combination thereof, may be used as a stand-alone therapy.

Alternatively, the poxvirus of the invention (as described above), the poxvirus obtained according to the method of the invention (as described above), the host cell according to the invention (as described above), the composition according to the invention (as described above), or any combination thereof, may be used in conjunction with one or more additional therapeutic intervention(s).

Any additional therapeutic intervention suitable in the context of the selected therapeutic use may be used in conjunction with the fusion polypeptide, nucleic acid molecule, vector, host cell, composition or combination thereof according to the invention. Such additional therapeutic intervention may notably be selected from the group consisting of surgery, radiotherapy, chemotherapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy, cytokine therapy, targeted cancer therapy, gene therapy, photodynamic therapy and transplantation.

In specific embodiments, the therapeutic use or method of treatment according to the invention may be carried out in conjunction with surgery. For example, The poxvirus of the invention (as described above), the poxvirus obtained according to the method of the invention (as described above), the host cell according to the invention (as described above), the composition according to the invention (as described above), or any combination thereof, may be administered after partial or total surgical resection of the tumor (e.g. by local application within the excised zone, for example).

In other embodiments, the therapeutic use or method of treatment according to the invention can be used in association with radiotherapy. Those skilled in the art can readily formulate appropriate radiation therapy protocols and parameters (see for example Perez and Brady, 1992, Principles and Practice of Radiation Oncology, 2nd Ed. JB Lippincott Co; using appropriate adaptations and modifications as will be readily apparent to those skilled in the field). The types of radiation that may be used in cancer treatment are well known in the art and include electron beams, high-energy photons from a linear accelerator or from radioactive sources such as cobalt or cesium, protons, and neutrons. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells. Regular X-rays doses for prolonged periods of time (3 to 6 weeks), or high single doses are contemplated by the present invention.

In certain embodiments of the invention, the therapeutic use or method of treatment according to the invention may be used in conjunction with chemotherapy currently available for treating cancer. Representative examples of suitable chemotherapy agents include, without limitation, alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, parp inhibitors, platinum derivatives, inhibitors of tyrosine kinase receptors, cyclophosphamides, antimetabolites, DNA damaging agents and antimitotic agents. In the case of cancers responding to hormone therapy (such as prostate and breast cancers that use hormones to grow), therapeutic use or method of treatment according to the invention may be used in conjunction with hormone therapy.

Depending on the specific biomarkers (in particular cellular receptors) expressed by the cancer, the fusion polypeptide, nucleic acid molecule, vector, host cell, composition or combination thereof may also be used in conjunction with targeted therapy, i.e. a therapy that targets proteins that control how cancer cells grow, divide, and spread. There are two types of targeted therapies: those based on small-molecules and those using monoclonal antibodies. Examples of targeted therapies include small molecules targeting BRAF V600E (e.g. Vemurafenib) or BCR-ABL fusion protein (e.g. imatinib mesylate), monoclonal antibodies blocking Epidermal Growth Factor Receptor (in particular cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab, trastuzumab (Herceptin™), etc.,) and monoclonal antibodies blocking Vascular Endothelial Growth Factor (in particular bevacizumab and ranibizumab).

It has been shown in the art that combining several immunotherapies may lead to improved therapeutic efficiency, and sometimes even synergistic effects (see e.g. Semmrich M, Marchand J-B, Fend L, et al., Journal for ImmunoTherapy of Cancer 2022;10:e003488). As a result, the fusion polypeptide, nucleic acid molecule, vector, host cell, composition or combination thereof may preferably be used in conjunction with another immunotherapy.

In particular, the fusion polypeptide, nucleic acid molecule, vector, host cell, composition or combination thereof according to the invention may be used in conjunction with one or more other therapeutic agents selected from the group consisting of agonists of stimulatory immune checkpoints, and antagonists of inhibitory immune checkpoints.

While any agonist of a stimulatory immune checkpoint may be used, it may preferably be selected from human ICOSL, 4-1 BBL, OX40L, CD70, CD40L, GITRL and agonist antibodies to human ICOS (e.g. W02018/187613), CD137 (4-1 BB) (e.g. W02005/035584), 0X40 (e.g. e.g. US 7,291 ,331 and W003/106498), CD27 (e.g. WO2012/004367), CD40 (e.g. W02017/184619), or GITR (e.g. WO2017/068186). As some agonists of stimulatory immune checkpoints are TNFSF members, when such an agonist of stimulatory immune checkpoints is used in conjunction with the fusion polypeptide, nucleic acid molecule, vector, host cell, composition or combination thereof according to the invention, it is preferably different from the member of the TNFSF or functional fragment or variant thereof of the fusion polypeptide according to the invention. Particularly preferred agonists of a stimulatory immune checkpoint that may be used in conjunction with the poxvirus of the invention (as described above), the poxvirus obtained according to the method of the invention (as described above), the host cell according to the invention (as described above), the composition according to the invention (as described above), or any combination thereof, include an agonist of ICOS.

Similarly, while any antagonist of an inhibitory immune checkpoint distinct from PD-L1 may be used, it may preferably be preferably selected from antagonist antibodies to human:

• PD-1 (e.g. those described in W02004/004771 ; W02004/056875; W02006/121168; W02008/156712; W02009/014708; W02009/114335; W02013/043569; and W02014/047350, in particular nivolumab, pembrolizumab or cemiplimab),

• SIRPa (e.g. W02019/023347),

• CD47 (e.g. W02020/019135),

• PD-L2 (e.g. W02019/158645),

• LAG3 (e.g. W02018/071500),

• Tim3, (e.g. W02020/093023)

• BTLA (e.g. W02010/106051 ), and

• CTLA4 (e.g. those described in US 8,491 ,895, W02000/037504, W02007/113648, W02012/122444 and W02016/196237 among others, and in particular ipilimumab marketed by Bristol Myer Squibb as Yervoy® (see e.g. US 6,984,720; US 8,017,114), MK-1308 (Merck), AGEN-1884 (Agenus Inc.; W02016/196237) and tremelimumab (AstraZeneca; US 7,109,003 and US 8,143,379) and single chain anti-CTLA4 antibodies (see e.g. W097/20574 and W02007/123737).

The following examples merely intend to illustrate the present invention.

DESCRIPTION OF THE FIGURES

Figure 1. SDS-acrylamide Gel (4-15%) observed in UV-light after electrophoresis and before transfer. Supernatants from infected cells A549 have been loaded in reducing or non-reducing conditions. GS542 has been loaded at a known quantity (50ng). MW: Molecular Weight markers. For sake of clarity, all the supernatants from infected cells were written with their corresponding number (without COPTG). Figure 2. Western Blot of sdAb GS542 and cell culture supernatants after anti-His tag development. Chemiluminescence was recorded for 442 seconds. MW: Molecular Weight markers. For sake of clarity, all the supernatants from infected cells were written with their corresponding number (without COPTG).

Figure 3. Set up of antigen hPD-L1-Fc coating concentration for direct binding of avelumab (A) or GS542 (B) by ELISA. hPD-L1 Fc was coated on ELISA plates at different concentrations (0.1 , 0.25, 0.5, and 1 g/ml). Avelumab or sdAb GS542 were added. Bound proteins to PD-L1 -Fc were detected using an HRP -conjugated anti-A (avelumab) or anti- His (GS542) antibody.

Figure 4. Binding to hPD-L1-Fc of anti-PD-L1 molecules present in supernatants from infected-MIA PaCa-2 measured by ELISA. hPD-L1 Fc was coated on ELISA plates (0.25 g/ml GS542 or 0.1 pg/ml avelumab). Diluted supernatants from infected MIA PaCa-2 cells containing different formats of anti-PD-L1 molecules were added. Bound proteins to PD- L1 were detected using either an HRP -conjugated anti-A antibody (indicated @A) for avelumab only or with an HRP -conjugated anti-His antibody (indicated @His) for all formats. Each represented value is the mean of two measurements (duplicate).

Figure 5. Set up of the competition ELISA conditions: concentrations of hPD-1-Fc coating and binder (biot-hPD-L1 ). hPD-1 -Fc was coated on ELISA plates at different concentrations (0.1 , 0.25, 0.5, and 1 pg/ml). biot hPD-L1 Fc was added at serial decreasing concentrations. Bound biot-PD-L1 Fc to PD-1 Fc was detected using an HRP -conjugated streptavidin.

Figure 6. Competition of the biot-hPD-L1-Fc/hPD-1 interaction by GS542, avelumab

(A) and supernatants from infected-MIA PaCa-2 (B). hPD-1 Fc was coated on ELISA plates (0.25 g/ml). -Biot-hPD-L1 -Fc was added in solution at 0.1 pg/ml with decreasing concentration of competitors: i.e., either the purified proteins (A) or diluted supernatants

(B) from infected MIA PaCa-2 cells. Bound biot-hPD-L1 -Fc was detected using an HRP- conjugated streptavidin. Each represented value corresponds to one replicate and the curve is the mean of two measurements (duplicate). Inhibitory concentration (IC50) or dilution (ID50) that gave 50% of inhibition are calculated by GraphPad Prism.

Figure 7. Summary of the ID50 obtained by competition ELISA of the different supernatants containing the different format of sdAb. Figure 8. The PD-1/PD-L1 blockade bioassay measures the potency of different anti- PD-L1 formats present in supernatant of HT29-infected cells. PD-1 aAPC and PD-L1 - CHO-K1 cells were plated and co-incubated for before the addition of increasing concentrations of GS542, avelumab, 14F10 (A); or dimeric-GS542, avelumab, 14F10 (B); or supernatants from HT29 cells infected by COPTG19814, COPTG19806 or COPTG19815 (C). Luciferase activity was measured using GloMax Discover System. Data were fitted using GraphPad Prism software. Each represented value corresponds to one replicate and the curve is the mean of two measurements (duplicate).

Figure 9. Binding to cynomolgus PD-L1-Fc (cynoPD-L1-Fc) or hPD-L1-Fc of GS542 (A) and supernatants from infected-HT29 (B) by ELISA. PD-L1 -Fc were coated on ELISA plates (0.25pg/ml for GS542 and COPTG19814 or O.l pg/ml for COPTG19806). Purified GS542 (A) or diluted supernatants (B) from infected HT29 cells containing different formats of anti-PD-L1 molecules were added. Bound proteins to PD-L1 were detected using an HRP -conjugated anti-His antibody. Each represented value corresponds to a replicate and the curve is the mean of two measurements (duplicate).

Figure 10. Expression profile of mRNA encoding PD-L1 in Hs746t (A) or HepG2 (B). Data bank from Transgene where RNA sequencing has been done on mRNA extracted from several human tumor cell lines.

Figure 11. Expression of PD-L1 by Hs746t (A) or HepG2 (B). PD-L1 expression at the surface of Hs746T and HepG2 was assessed by flow cytometry using a fluorescent labeled anti-PD-L1 monoclonal antibody. In Figure 11 (A) and Figure 11 (B), in dark grey is represented the signal obtained with the APC anti-human CD274/B7-H1 /PD-L1 antibody; in light gray is represented the signal obtained with the APC mouse lgG2b kappa isotype control antibody (i.e., irrelevant APC conjugated antibody of the same species and isotype as the anti-human PD-L1 used).

Figure 12. Binding of the purified sdAb GS542 to Hs746t tumor cells. Flow cytometry analysis of the sdAb GS542 at serial concentrations and the protein control 14F10 (A). Saturating curve: MFI versus concentrations of sdAb GS542 expressed either as M or as pg/mL (B). Figure 13. Binding of anti-PD-L1 molecules contained in supernatants of A549 - infected cells to Hs746t tumor cells. Saturating curve: MFI versus 1 /dilutions of the different supernatants (duplicate). GS542 at 1 g/mL (in duplicate) was used as positive control.

Figure 14. Competition of biot-hPD-L1/avelumab interaction by GS542 (A) and supernatants from infected-HT29 (B) by ELISA. hPD-L1 Fc was coated on ELISA plates (0.25 g/ml). biot- Avelumab were added at 8ng/ml with increasing dilution of purified proteins (A) or diluted supernatants (B) from infected HT29 cells containing different formats of anti-PD-L1 molecules. Avelumab bound to PD-L1 -Fc was detected using an HRP- conjugated streptavidin.

Figure 15. Viral replication of COPTG19814 and WTG18058 on A549 cells. The A549 cells were infected at MOI 10'³ by COPTG19814, VVTG18058. The quantity of each virus was measured at 24, 48 and 72h post infection. Results are represented as the mean of three wells.

Figure 16. Oncolytic activity of the COPTG19814 assessed on A549 cells. A549 cells were infected with 10 different MOIs and the cell viability was evaluated after 5 days of incubation. One hundred % of cell viability was established on the non-infected cells. Results are means +/- SD of 3 measurements and are represented as percentage of cellular viability.

EXAMPLES

Although the present invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims. 1 . EXAMPLE 1 :

1.1. Materials and Methods

1.1.1. Viruses and constructions

Different anti-PD-L1 constructs encoding different formats of humanized sdAb (GS542, Table 5), or of the human lgG1 avelumab mAb (used as benchmark) targeting human PD- L1 , were designed and inserted separately into the COP-VV TK-/RR- Vaccinia virus backbone (TK and RR double deleted COP-W). All the constructs were under the control of pH5R promoter and were labelled with a C-terminus His tag. COPTG19815 encoded GS542. COPTG19814 encoded two GS542 fused together and linked by (GGGS)4 linker. This construct was designed to improve the observed affinity of GS542 (avidity effect). COPTG19808 encoded GS542 fused to the hinge of human lgG1 to get homodimerization (avidity effect) through auto-assembly via the IgG hinge. Finally, COPTG19839 encoded the fusion of GS542 to a human IgG 1 Fc that allowed the dimerization (avidity effect) but also to engage Fc receptors and complement activation. Fc receptors (FcR) engagement allowed lysis of the target cells by antibody cell dependant cytotoxicity (ADCC) or phagocytosis (ADCP) but also a long circulating half-life (binding to FcRn in the kidneys). COPTG19806 encoded the benchmark clinically used anti-PD-L1 monoclonal antibody avelumab.

All sdAb constructs were inserted at TK locus with RR gene also deleted. About the construction with avelumab the heavy and light chains were inserted in TK and RR loci, respectively. To facilitate the detection and selection, a 6-histidine (His) tag was added at the C-terminal of sdAb and heavy chain of avelumab.

The viruses obtained are listed below:

• COPTG19815 encoded GS542 as single molecule,

• COPTG19814 encoded a dimeric GS542: two sdAb fused together via a flexible linker,

• COPTG19808 encoded the sdAb GS542 fused to hinge of human IgG,

• COPTG19839 encoded the sdAb GS542 fused to human Fc,

• COPTG19806 encoded the anti-hPD-L1 monoclonal antibody avelumab,

• WTG18058 was unarmed virus and was used as a negative control (Ctrl) for transgene expression and positive Ctrl in virus replication and oncolytic activity assay. Table 5: Viruses used and their constructs (GS542 is the humanized anti-hPD-L1 sdAb).

1.1.2. Antibodies and proteins of reference Several antibodies and protein listed in Table 6 were used as Ctrl or benchmarks in different assays. Avelumab is a clinically administrated anti-PD-L1 mAb and was used as a benchmark or positive Ctrl in the experiments. 14F10 is an irrelevant sdAb used as negative Ctrl with a His tag. H27K15 is a negative mAb Ctrl used in the flow cytometry experiment. Proteins hPD-1 Fc, hPD-L1 Fc, hPD-L1 Fc-biotinylated, were used in the ELISA assays.

Table 6: Antibodies and proteins used.

1.1.3. Cell lines

HepG2 (ATCC®; HB-8065) is a human liver cancer cell line, epithelial in morphology and adherent. Cells were maintained in Essential Modified Eagles Medium (EMEM) from ATCC ® (30-2003) supplemented with 10% Foetal Bovine Serum (FBS) and Gentamycin (40 pg/ml).

Hs746t (ATCC®; HTB-135™) is a hyper triploid human lung adenocarcinoma cell line, epithelial in morphology and adherent. Cells were maintained in Dulbecco Modified Eagles Medium (DMEM) from ATCC, (30-2002) supplemented with 20% FBS and Gentamycin (40 pg/ml).

MIA PaCa-2 (ATCC®; CCL-1420™) is a human pancreatic cancer cell line. These cells were maintained in DMEM (ATCC ®, 30-2002) supplemented with 10% FBS and gentamycin (40 pg/ml).

HT29 (ATCC®; HTB-38™) is a human colorectal adenocarcinoma cell line. They were maintained in McCoy’s 5a medium (ATCC ®; 302007) supplemented with 10% FBS and Gentamycin (40 pg/ml).

A549 (ATCC®; CCL-185TM), is a lung carcinoma cell line and maintained in DMEM from Gibco® (41966-029) supplemented with 10% FBS and Gentamycin (40 pg/ml).

Vero (ATCC®; CCL-81 ™) are Monkey kidney epithelial cells and grew in the same culture medium as A549.

All cells were regularly passed following provider’s recommendations (ATCC) every 3 to 4 days in a F175 flask: Cells were washed with Phosphate buffer saline (PBS1X), and detached with 2 ml of Trypsin (TrYpLE select at stock concentration, Gibco 2209518) for 5 min at 37° C. Then, the corresponding medium was added to have a final volume of 10 ml into the flask. A sample of 1 ml of the resuspended cell were measured using a cell counter (Beckmann). A specific quantity of cells was transferred into a new F175 flask with 25 ml of corresponding medium.

1.1.4. Production of supernatants

Three human’s tumor cell lines from three different indications have been chosen for the infections and production of the transgenes: MIA PaCa-2, HT29 and A549.

All these cell lines were infected with the different viruses listed above at a multiplicity of infection (MOI) 0.01 (i.e., 1 virus for 100 cells) for 72h. Then the cell culture supernatants were harvested, centrifugated (30 min, 9000xg, 4°C) and filtered (0.1 pm) to get rid of virus and remaining cellular debris. The treated supernatants were aliquoted and stored at -80° C until use.

1.1.5. Western blot

Samples were prepared with Laemmli sample buffer (Biorad 1610747) for non-reducing condition or Laemmli sample buffer supplemented with B-mercaptoethanol heated to 95 °C for 5 min for reducing condition. Sample were loaded on a sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gel 4-15% TGX stain-free (Biorad 5678084) with a ladder precision all blue standard (Biorad 1610373). The electrophoresis conditions were 34 min at 200V in Tris Glycine SDS Buffer 1X (Biorad 1610732). To visualize the proteins, the gel was exposed to UV to induce the reaction of trichloroethanol with tryptophane of proteins and to form a fluorescent adducts. Proteins were transferred from the gel to PVDF membrane using Transblot Turbo transfer (Biorad 170-4159) and the Transblot system Turbo Midi program High molecular weight (i.e., 10min, 2.5 A up to 25V). After transfer, the membrane was reactivated in 100% ethanol for a minute, rinsed with water and saturated with saturating buffer iBind from kit (Invitrogen SLF2020) for 10 min. For hybridization with the antibodies, a Western Blot Ibind flex system (Invitrogen SLF2010) was used. The conjugated Anti-His-HRP (Qiagen 34460) at the recommended dilution (i.e., 1 /2000) in saturating buffer was added in the apparatus at room temperature and incubated for 3 and half hours. After incubation, the membrane was rinsed with water and developed with the Amersham chemiluminescence prime western blotting detection kit (ECL). Chemiluminescence was recorded after different times of exposition using the instrument Chemidoc XRS (Biorad).

1.1.6. Enzyme-like Immunosorbent Assay (ELISA): Sandwich ELISA and Competition ELISA

Sandwich ELISA assessed the binding of sdAb or mAb to its target. The sdAb/mAb bound to immobilized antigen was developed by an enzyme-conjugated secondary antibody. The enzyme (e.g., HRP) transformed an uncoloured substrate (e.g., tetramethylbenzidine TMB) to a coloured product. The optical density was then measured using a spectrophotometer.

One day before the experiment (D-1 ), a 96 wells plate (Nunc, Medisorp) was coated with 100pl of an antigen (e.g., PD-L1 ) solution prepared in coating buffer (0.05 M Carbonate, pH 9.6) and incubated at 4°C, overnight (actual concentration of the coated antigen are reported in each figure legend). The next day, the 96 wells plate was washed with PBS 1X 3 times, and the plate was saturated with saturating buffer (1% Bovine Serum Albumin (BSA; Sigma A9647-100G), 0.05% Tween 20 (Sigma P9416) in PBS1X) for 2 hours at room temperature (RT). This step aimed to prevent nonspecific binding of the sdAb/mAb to the plate. Then, the plate was washed again three times with PBS1X, and samples serially diluted in saturating buffer to have a final volume of 100 pl per well. After two hours of incubation at 37° C, the 96-wells plate was washed three times in PBS1X. The conjugated antibody HRP (e.g., anti-His HRP) was diluted in saturating buffer following the provider’s recommendations and added into the plate for 1 hour at 37° C. The 96-wells plate was washed three times and then the substrate TMB was added for 30 minutes at RT. The enzymatic reaction was stopped by adding H2SO4 at 2M. Absorbances at 450 nm were measured using a photometer (Spark®, TECAN). The absorbance versus sdAb/mAb concentrations or supernatant dilutions were plotted using GraphPad Prism software. And the obtained curves fitted with five-parameter logistic equation to obtain the half maximal effective concentration (EC50) or half maximal effective dilution (ED50).

In a competitive ELISA, an interaction in fixed conditions was followed between the binder (e.g., PD-1 -Fc-biot) and the coated antigen (e.g., PD-L1 -Fc) specifically by measuring an absorbance signal. The different competitive ELISA conditions are designed in Table 7. This interaction was followed in presence of increasing amount of a competitor which is a molecule that disrupt this interaction (e.g., GS542 or cell supernatants). A first experiment aimed to determine the optimal concentrations of both coated antigen and binder as described above. Briefly, at D-1 , a 96-well plate is coated with 100pl of the antigen at 4 different coating concentrations (e.g., 1 , 0.5, 0.25, and 0.1 pg/ml) diluted in coating buffer. The rest of the experiment was run as described above and the optimal conditions for the competition were determined i.e., concentrations of coating and binder that yield 90% of the maximum of signal. Table 7 : Different conditions used for the three designed competition ELISA.

For competition ELISA, the coating and the concentration of binder were then fixed (Table 7). The competitor (purified protein or supernatants) was then added at decreasing concentrations in saturating buffer containing the constant concentration of binder.

This mix was added to the plate and the assay was processed as described above. The absorbance versus sdAb/mAb concentrations or supernatant dilutions were plotted using GraphPad Prism software. And the obtained curves fitted with five-parameter logistic equation to obtain the half maximal inhibitory concentration (IC50) or half maximal inhibitory dilution (ID50).

1.1.7. Flow cytometry assay

For these experiments, cells were selected for their expression (Hs746t) or lack of expression (HepG2) of PD-L1 based on internal mRNA expression data and reports available in literature (Ahn et al. 2019).

Cells were seeded at 5.10⁶ cells in T175 flask and after 3 to 4 days of incubation in the culture medium was changed. Cells were then washed with PBS1X and 2 ml of trypsin (TrYpLE select at stock concentration) was used to dissociate the cells from their solid support for 5 min at 37°C. Eight ml of medium were then added to inactivate the trypsin. The cells were counted with Vi-CELL™ Cell Viability Analyzer instrument and 3.10⁵ cells/well were added into a V shaped-96 wells plate (Greiner bio-one-651180).

Cells were either stained directly with antibody conjugated with a fluorophore or incubated with a primary unconjugated antibody and then stained with a secondary antibody conjugated with a fluorophore. In the first case, cells were washed by centrifugating the V-96 wells plates at 300xg during 8 min. Viability stain also known as fixable viability stain 450 (FVS 450 BD Bioscience 562247) was added to follow the viability of the cells. FVS 450 BD is an a mine -reactive violet dye used to discriminate viable mammalian cells from non-viable cells based on fluorescence intensity. The dye reacts covalently with to cell surface and intracellular amines if the cell has become permeable (i.e., cell is dead). Then, dead cells have a higher fluorescence intensity than live cells and are easily discriminated. A mix of conjugated antibody or the isotype control with the viability stain were prepared at specific concentrations recommended by the supplier and added to the well for incubation 20 min at 4°C, in dark.

Then cells were washed again as previously described, resuspended in 10Opl of PBS1X and transferred into a U shapped-96 wells plate (Greiner bio-one-650180). The cells were subject to flow cytometry (MACSQuant 10-Milteniy Biotec). In the case of labelling with a non-conjugated antibody, steps were the same as above, except that cell were incubated with unconjugated antibody/viability stain for 20min first and then washed prior the treatment by the conjugated antibody. For each labelling, an isotype Ctrl antibody was used to determine the background binding signal.

Data analysis and graphical output were performed by using Kaluza 2.1 software (Beckman Coulter). First, the cellular debris were eliminated by gating only the main cell population as shown in the gate A, in the graph containing the side scatter area (SSC-A) or the structure versus the forward scatter area (FSC-A) of the population. Then, the cell doublets were eliminated by focusing on the central population in the graph FSC height (FSC-H) versus (FSC-A). In the channel of the Viability stain, here FVS 450, living cells with lower fluorescence intensity were selected. A final graph with the cells counted versus the channel corresponding to the isotype Ctrl used and was obtained to determine the positive binding cells. The protocol was applied for all the different samples. The Mean Fluorescence Intensity (MFI) versus binder concentrations (pg/ml) or 1 /dilutions of the supernatants were plotted using GraphPad Prism software. And the obtained curves fitted with five-parameters logistic equation.

1.1.8. PD-1/PD-L1 Blockade bioassay

PD-1 /PD-L1 Blockade bioassay (Promega), was used to measure the potency of antibodies to block the cellular PD-1 /PD-L1 interaction. The experiment followed the instructions of the Bioassay protocol ([ PD-1 PD-L1 Blockade Bioassay - Promega Corporation]). In this Bioassay, two genetically engineered cell-lines were used: Jurkat T cells expressing TCR, human PD-1 and luciferase under the control of an NFAT response element (NFAT-RE) that was activated when TCR was engaged, and PD-1 inhibited.

CHO-K1 cells expressing at its surface, human PD-L1 and a non-disclosed protein that activated TCRs.

When the two cell types were co-cultured, the PD-1 /PD-L1 trans-interaction inhibited TCR signalling on Jurkat and then production of the reporter luciferase. In presence of an anti-PD-1 or anti-PD-L1 blocking antibody, then inhibitory signal was released, and the TCR activation led to the production of luciferase. The luciferase enzymatic activity was then measured by bioluminescence using Bio-Gio Reagent from the Promega kit and luminescence reader apparatus (Berthold). For data analysis, the plate background was measured by calculating the average relative light units (RLU) from wells containing only buffer. Then, the fold induction of luminescence was calculated using the following equation:

Fold induction = RLU (sample - background)/RLU (sample control-background)

Fold induction versus concentration of antibody or dilution of supernatant curves were plotted on GraphPad Prism. Curves were fitted with five-parameter logistic equation and the IC50 or ID50 values were determined.

1.1.9. Oncolytic and replication activity

The selected candidate COPTG19814 was characterized by measuring its replication and oncolytic activity on A549 cells.

Virus titration is a method that measures the replicative virus, i.e., able to lyse permissive cells (e.g., Vero cells). It consisted in adding serial dilutions of a sample containing an unknown concentration of virus to confluent permissive cells. Each replicative virus induced a plaque of lysis (a hole in the cell layer) that was visible and counted using a microscope. The virus titer is then: number of plaques x dilution factor => expressed as plaque forming unit (pfu)/ml Briefly, the day before virus titration, Vero cells from a T175 flask incubated, were washed with PBS 1X after discarding the culture medium. Cells were collected using Trypsin at 37° C for 5 minutes. Medium was then added to inactivate the trypsin. Vero cells were counted using Vi-CELL™ Cell Viability Analyzer, seeded at 5.10⁶ cells/well in 2 ml of medium in a 6 well plates (TPP-92006) and incubated overnight at 37° C, 5% CO?. The next day, the virus suspensions were prepared from 10'¹ to 10'⁵ pfu/mL in PBS++. (PBS + 1% cations (46.6 mM magnesium acetate, 68 mM calcium chloride).

The 6-wells plate seeded with Vero cells the precedent days were utilized. The medium was removed, and the cells were infected for 30 min at ambient temperature with 250 pl of the dilution of virus prepared previously. Then 2 ml of medium with 1% agarose was added on each well and incubated 48h at 37° C, 5% CO?. Finally, plaque of lysis was counted under an optical microscope (x100) after 3h of incubation with 2ml/well of medium supplemented with neutral red and 1% agarose.

Virus replication was assessed by measuring the quantity of virus produced after 24, 48 and 72 hours after infection of A549 cells by COPTG19814 and VVTG18058 at a relative low MOI (10³).

A549 cells from a T175 flask incubated, were washed with PBS 1X after discarding the culture medium. Cells were collected using Trypsin at 37° C for 5 minutes. Medium was then added to inactivate the trypsin. A549 cells were counted using Vi -CELL™ Cell Viability Analyzer, cells were distributed in 3 Eppendorf tubes at 3.10⁵ cells/tube in 100 pl of PBS++. Each Eppendorf tube corresponded to a different condition (infection by COPTG19814 or VVTG18058 or non-infected cells). Cells were infected by either COPTG19814 or VVTG18058 at MO1 10’³ for 30 min at 37° C, 5% CO₂. Then, 600 pl of medium was added on each Eppendorf tube. Finally, 50 pl of cells were distributed in a 6-wells- plate containing 2 ml of medium and incubated for 24, 48, or 72h at 37° C, 5% CO₂. At each time point, plates were frozen at -80° C until the titration.

Plates were thawed, infected cell suspensions and supernatants were collected and sonicated 3 x 5 seconds with 36 % of amplitude before 10-fold serial dilutions in 1 mL of PBS++ followed by viral titration by plaque assay on Vero cells (250 pl of dilution added). The results were expressed in total quantity of virus calculated with the following formula:

Titer (PFU/mL) = number of plaques x dilution factor

Oncolytic activity was assessed by quantification of cell viability after 5 days of incubation with variable quantities of virus.

A549 Cells were pelleted by centrifugation to obtain the quantity of cells corresponding to 1.10⁴ cells/well and the pellet was resuspended in PBS ++ to have a cell suspension at 4.10⁵ cells/ml.

One hundred microliters of the cell suspension were distributed into Eppendorf tubes (corresponding to 4.10⁴ cells/tube). Virus prepared according to the expected MOI (i.e. , 3.10'⁵ to 1 ) was added to each tube containing cells and incubated 30 min at 37 °C. Appropriate complete medium (i.e., DMEM ATCC) was added to Eppendorf tube to reach 600 pL/tube and 150 pL of this suspension distributed in each well (in triplicate) in a 96- well plate. One negative control corresponding of media was also prepared. Plates were incubated at 37 °C with 5 % CO? for 5 days and cell viability was determined using cell titer blue cell viability assay according to the protocol provided by the manufacturer (Promega, G8081 ). Briefly, 30 pL of cell titer blue were added to each well and the 96- wells plate was incubated at 37 °C with 5 % CO? for 3 h. Fluorescence was then measured at 590 nm (560 nm for excitation) using a fluorimeter (Spark®, TECAN).

Oncolytic activity of each sample was expressed as a percentage of the non-infected cell viability.

1.2. Results

In order to select the virus with the optimal construct, i.e. with the most efficient PD-L1 blocking activity, three human tumor cell lines (A549, HT29 and MIA PaCa-2) were infected with the different viruses. After three days of infections, culture supernatants were then collected and filtered to get rid of the virus and cellular debris. These treated supernatants contained the secreted anti-PD-L1 molecules and were used in a wide range of assays presented in the following paragraphs.

1.2.1. Expression of the different constructs

First, a gel electrophoresis was performed in both non-reducing and reducing conditions to check the correct assembly and multi merization of the constructs and to have an estimation of the relative expression levels as the intensity of the band was directly correlated to the quantity of proteins produced. The SDS-PAGE gel contained a chemical that allowed, before the transfer, the direct observation of proteins by fluorescence.

Indeed, the chemical of the gel reacted with the tryptophanes of the protein. The more tryptophane in the protein, the more intense the band will be. The gel picture (Figure 1 ) shows visible bands in the supernatants of A549 cells infected by COPTG19815 and COPTG19814 encoding monomeric and dimeric GS542, respectively. The monomeric form of sdAb GS542 (COPTG18915 monomeric format) and the dimeric form of sdAb GS542 (COPTG19814 dimeric format) both migrated at their expected size (17 and 30 kDa respectively). This result suggests that both monomeric and dimeric sdAb formats were better expressed by the virus compared to other formats.

The immunoblot (Figure 2) using an anti-His tag antibody confirmed the observations made in fluorescence and did not show any aggregation or degradation of the protein. Moreover, in non-reducing condition, two bands were clearly visible at 110 and 90 kDa in the lane COPTG19806 (avelumab), that corresponds to the two heavy and the two light chains indicating that Avelumab was correctly assembled and well produced. In reducing condition, a single thick band was observed at 50 kDa corresponding to heavy chain (only heavy chain with a His tag). The COPTG19839 (GS542-Fc fusion) supernatant showed one band in non-reducing condition at 75 kDa and one band at 40 kDa in reducing condition indicating that the Fc homodimerization occurred correctly. COPTG19808 (GS542 hinge) supernatant in non-reducing conditions displayed one band at around 15 kDa that corresponds to the monomeric format. In other words, the dimerization through the hinge did not occur. The format encoded by COPTG19814 was well expressed as evidenced by the thick band in non-reducing and reducing conditions. Nevertheless, these detections may be biased as the detection of His tag was influenced by the flanking sequence. Then, compare to the Figure 1 , the signal intensity observed on blot (Figure 2) did not necessarily correlate with the quantity of protein present.

These expression tests were performed in 3 cell lines infected (A549, HT29 and MIA PaCa- 2) and showed similar results. Altogether, these results demonstrated that infected cells secreted different format of sdAb and mAb at detectable levels and that were well assembled, without apparent aggregation in reducing conditions.

1.2.2. All sdAb formats bind to hPD-L1

The biological activity of the different supernatant of infected cells were measured using different in vitro assays as ELISA or blockade assay.

The different formats of sdAb GS542 anti-hPD-L1 vectorized in the COP-VV inhibited the interaction of hPD-1 with hPD-L1 by binding to hPD-L1 . Therefore, a first ELISA assay has been designed to verify the binding to hPD-L1 . The optimal coated concentration of hPD- L1 was determined. Different concentrations of hPD-L1 Fc were coated and variable concentrations of the benchmark avelumab or the positive control sdAb GS542 were applied. The coating concentrations that showed the more suitable binding curve for the anti-hPD-L1 were 0.25 pg/ml for GS542 and 0.1 pg/ml for avelumab (Figure 3). In this experiment, the positive control (sdAb GS542) and the benchmark (mAb avelumab) bound to PD-L1 as expected (Figure 3).

The supernatant from MIA PaCa-2 infected by to COPTG19806 (vectorized His tagged avelumab) showed two different ED50 according to the conjugated antibody used for the development of the ELISA. Indeed, the ED50 for the binding PD-L1 was approximately four times lower with the conjugated anti-A light chain than with the conjugated anti-his tag (ED50; 0.0004 and 0.0014, respectively) (Figure 4). It should be noted, however, that the signals obtained should not be compared, since they were revealed with two distinct antibodies (anti-A or anti-his tag).

The binding of the different formats of sdAb GS542 in the supernatant of the infected MIA PaCa-2 cells was then studied (Figure 4). The curves and the ED50 for the supernatants of the infected cells by COPTG19814 (ED50; 0.002), COPTG19815 (ED50; 0.003) and COPTG19808 (ED50; 0.003) were similar. The ED50 for the samples COPTG19806 (avelumab) and COPTG19839 (GS542-Fc) were lower than the three other samples. These results confirmed that different formats of sdAb GS542 vectorized in the COP-VV are capable of binding to hPD-L1 .

1.2.3. COPTG19814 has the best PD-1/PD-L1 blocking activity

Blocking interaction measured by competitive ELISA

The competition assays aimed to determine PD-1 /PD-L1 blocking activity. In case of vectorization, the level of expression can vary between one construction to another. Thus, it is important to measure the blocking activity globally as the result of both expression level and affinity of a vectorized format.

First, the conditions of coating concentration of PD-1 and concentration of the binder (biot- hPD-L1 Fc) were determined. Figure 5 shows that the best conditions were with concentrations of 0.25 pg/ml for coated hPD-1 Fc and 0.1 g/ml for soluble (binder) hPD- L1 -Fc-biot. Note that the very high specificity of the biotin/streptavidin-HRP revelation system allowed to test a wide range of competitors such as purified proteins (sdAb and mAb) or crude culture supernatants. Figure 6 demonstrates that all the competitors tested were able to inhibit the interaction PD-1 /PD-L1 in a dose dependent manner. In case of purified proteins, the IC50 of avelumab (123.8 ng/ml) was about 5 times higher than the IC50 of purified GS542 (23.8 ng/ml; Figure 6-A), meaning that purified GS542 was a better blocker than the purified avelumab in these conditions.

The data demonstrated that all different anti-PD-L1 sdAb formats present in the supernatants of infected cells inhibited the PD-1 /PD-L1 interaction in a dose dependent manner with different inhibitory dilution 50% (ID50) (Figure 6-B). Importantly, the blocking activity of vectorized GS542 was higher than the blocking activity of vectorized Avelumab. Moreover, among the 5 supernatants tested, the one from cells infected by COPTG19814 had the lowest ID50 (0.02). A summary of the ID50 of the different sdAb formats from the 3 different infected cell lines are presented in the bar chart (Figure 7). The virus with the lowest ID50 was the COPTG19814 for the 3-cell lines (HT29: 0.02; MIA Pa Ca-2: 0.02; A549: 0.01 ). Then, among the 5 formats and in the 3 cell lines tested, the dimeric format of COPTG19814 was the best competitor.

As COPTG19814 (expressing dimeric GS542) had the better PD-L1 /PD-1 blocking activity among the four virus candidates, the rest of the experiments has been focused on this candidate with COPTG19815 and COPTG19806 as comparators.

PD-1/PD-L1 blockade bioassay

This assay aimed to measure any PD-1 and/or PD-L1 blocking molecules in a cellular context. In this assay the interaction in trans of PD-1 with PD-L1 on cells inhibits the transduction of signal and therefore the expression of a reporter gene.

Figure 8-A shows that in this assay, sdAb GS542 had about 3 times higher EC50 (0.32 pg/ml) than avelumab (0.11 pg/ml), meaning that purified avelumab was a better blocker than the purified monomeric sdAb in these conditions. In contrast, blocking by purified dimeric GS542 was significantly higher than blocking by avelumab (Figure 8-B). Thus, dimeric GS542 is a more potent inhibitor of PD-1 and/or PD-L1 than avelumab. Importantly, once vectorized in Vaccinia virus, the EC50 for monomeric sdAb (COPTG19815) and Avelumab (COPTG19806) were comparable (EC50 of 0.1 and 0.09 respectively). The supernatants, containing the COPTG19814 format had the best blocking activity (Figure 8-C) with the lower EC50 (0.02) lower of all the supernatants. These results confirmed what was observed in the ELISA competition assay (Figure 6 and Figure 7), dimeric GS542 encoded by COPTG19814 was the most potent blocker among the constructs tested.

Altogether, these results demonstrate that the dimeric version of GS542 outperforms the benchmark molecule avelumab, both as purified protein and expressed by VV-COP. The data also demonstrated that the vectorization in VV-COP virus of all sdAb formats yielded functional molecules, the best format among the 5 candidates tested being the dimeric GS542 (i.e., COPTG19814). 1.2.4. Characterization of COPTG19814

Binding activity to macaque PD-L1

Binding of GS542 and its derivative to cynomolgus monkey PD-L1 was investigated, notably to determine whether macaque is a suitable animal model for future toxicological evaluations (Figure 9).

Human and cynomolgus PD-L1 (cynoPD-L1 ) were coated exactly at the same concentrations (0.25|jg/ml), and then GS542 and different supernatants from infected HT29 cells have been added. No difference was observed between hPD-L1 or cynoPD-L1 . Indeed, for a given sample the binding to either hPD-L1 or cynoPD-L1 yielded two curves nicely superposed. In conclusion the cross-reactivity of GS542 for cynomolgus PD-L1 was good, and this animal model could be used for future toxicological evaluations of oncolytic viruses expressing GS542 molecules.

Binding activity to PD-L1 on tumor cells

To confirm the binding results obtained by ELISA on purified proteins, expression levels of PD-L1 by various human tumor cells was investigated. An internal RNA sequencing databank was available documenting the expression profile of mRNA of several human cell lines used at Transgene.

Analysis of the database revealed that Hs746t cell line had a high expression of mRNA coding for PD-L1 (Figure 10-A). HepG2 cell line was selected as negative control because it showed a very low PD-L1 expression (Figure 10-B).

Then, an experiment was performed to check if the positive cells Hs746t expressed PD- L1 at their surface by using an anti-PD-L1 conjugated to APC fluorophore and a flow cytometer to monitor the binding of this antibody to the cells (Figure 11 ).

The median of fluorescence (MFI) shift between the isotype Control and an anti-PD-L1 antibody was important for the Hs746t cells (3 to 102). On the contrary, no shift was observed with the HepG2 cell line. This result proved that the Hs746t cells were expressing PD-L1 at their surface.

The binding of sdAb GS542 to PD-L1 Hs746t was investigated using an anti-his antibody coupled with phycoerythrin (PE) to monitor the binding of recombinant GS542 to cell surface. It has been observed a signal proportional to the concentration of sdAb with an MFI plateauing at 1 g/ml and an EC50 at 0.4 pg/ml (Figure 12-A and-B). This result demonstrated that GS542 bound PD-L1 at the surface of tumor cell and in a dose dependent manner. The binding to PD-L1 displayed on Hs746t by different sdAb formats present in supernatants of infected cells was also tested. The experiment showed that both GS542 monomer encoded by COPTG19815 and GS542 dimer encoded by COPTG19814 bound PD- L1 on Hs746t (Figure 13).

Epitope Binning

An epitope binning assay has been designed to determine whether the different sdAb formats of GS542 bind the same epitope on PD-L1 as avelumab. A competitive ELISA assay was set up where the PD-L1 /avelumab interaction was monitored. In this assay, GS542 and its different formats vectorized in COPTG19814 (dimeric) and COPTG19815 (monomeric) were all able to compete avelumab from PD-L1 (Figure 14). This result demonstrated that avelumab and GS542 or its derivative encoded by COPTG19814 and COPTG19815 bound PD-L1 on overlapping epitopes.

Replication of COPTG19814

The replication of COPTG19814 on A549 cells was compared to the one of the benchmark oncolytic virus VVTG18058 that was not encoding any transgene. After a virus inoculation at a low MOI (1 O'³) the kinetic of multiplication of the virus was followed by taking cells samples 24, 48, and 72h post-infection. The virus titers were determined (Table 8) and the replicative curves plotted Figure 15.

These results demonstrate that the transgene of COPTG19814 did not interfere with the replicative ability of the vaccinia virus in A549 cells.

Table 8: Total Infectious virus COPTG19814 and VVTG18058 recovered at 24h, 48h, 72h post infection of A549 cells at MOI 10'³.

Oncolytic activity of COPTG19814 on A549 cells

Oncolytic activity of the COPTG19814 was assessed by quantification of A549 cell viability after 5 days of infection at different MOI (i.e., from 10'⁵ to 1 ). Oncolytic activities of VVTG18058 and COPTG19814 were equivalent at all tested MOI (Figure 16). To compare the oncolytic activity of the different viruses, the EC50 of each virus was calculated using GraphPad Prism. The EC50 of COPTG19814 and the unarmed control VVTG18058 were similar.

This result confirmed that the presence of the transgene in COPTG19814 (encoding dimeric GS542) did not interfere with the oncolytic activity of the vaccinia virus on A549 cells.

1.3. Discussion

Altogether, the data showed a good expression of all the vectorized formats of GS542. All the different constructs were well assembled, the hinge format (COPTG19808) at a lesser extent. The ELISA and flow cytometry assay demonstrated that all supernatants, benchmark and positive control proteins bound to PD-L1 as a recombinant protein and also at the surface of tumor cell expressing PD-L1. Importantly, the blocking activity of vectorized GS542 was significantly higher than the blocking activity of vectorized Avelumab (PD-1 /PD-L1 competition ELISA). This result was confirmed in a cellular assay using recombinant cells expressing PD-1 and PD-L1 at their surface (further confirming that GS542 bound PD-L1 at the surface of tumor cell). Furthermore, it was shown that GS542, its derivative formats, and avelumab, bound overlapping epitope on PD-L1. Replication and oncolytic activities of COPTG19814 (encoding dimeric GS542) were assessed on A549 cells and were both similar to the ones of the unarmed benchmark virus (i.e., VVTG18058). These results demonstrate that the transgene of COPTG19384 (i.e. , dimeric GS542) does not impact the virus ability to replicate and lyse cells. Binding of GS542 to cynomolgus monkey PD-L1 was also demonstrated, showing that the results are not limited to human.

Altogether, the data demonstrate that a poxvirus expressing various formats of a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ), is a novel and efficient treatment of proliferative disease, such as cancer. BIBLIOGRAPHIC REFERENCES

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EP998568

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Claims

1. A poxvirus comprising a nucleic acid molecule inserted in its genome and encoding a binding agent, wherein the binding agent comprises a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ) comprising three heavy chain Complementarity-determining regions CDR1 , CDR2, and CDR3; and wherein:

• the heavy chain CDR1 consists of sequence RTFREYGMG (SEQ ID NO:1 ),

• the heavy chain CDR2 consists of sequence TISSSGSYXiY, wherein Xi is S or T (SEQ ID NO:2),

• the heavy chain CDR3 comprises the sequence X2SLLRGX3SSRAEX4YDX5, wherein each of X2 to X5 independently represents any amino acid (SEQ ID NO:3).

2. The poxvirus according to claim 1 , wherein the sdAb specifically binds to any of mammal PD-L1 ; preferably to primate PD-L1 , human PD-L1 , and any combination thereof; preferably wherein the sdAb specifically binds to human PD-L1 .

3. The poxvirus according to claim 1 or claim 2, wherein the heavy chain CDR3 of the sdAb specifically binding to PD-L1 consists of the sequence X6X7X2SLLRGX3SSRAEX4YDX5, wherein each of X2 to X7 independently represents any amino acid (SEQ ID NO:4).

4. The poxvirus according to claim 1 , wherein the heavy chain CDR3 of the sdAb specifically binding to PD-L1 consists of the sequence X6X7X2SLLRGX3SSRAEX4YDX5 (SEQ ID NO: 5), wherein:

• X2, X3 and X5 are independently selected from S, T, C, A, V, G and P;

• X4 is selected from S, T, C, A, V, G and P; and

• X₆ and X7 are independently selected from A, V, G and P.

5. The poxvirus according to claim 4, wherein the heavy chain CDR3 of the sdAb specifically binding to PD-L1 consists of the sequence X6X7X2SLLRGX3SSRAEX4YDX5 (SEQ ID NO: 6), wherein:

• X2 to X3 and X5 are independently selected from S, T, C and A;

• X4 is selected from S, P, T, C and A ; and

• X₆ and X7 are independently selected from A, V, G and P. 6. The poxvirus according to claim 4, wherein the heavy chain CDR3 of the sdAb specifically binding to PD-L1 consists of the sequence X6X7X2SLLRGX3SSRAEX4YDX5 (SEQ ID NO:7), wherein:

• X2, X3 and X5 are independently selected from S and A;

• X4 is selected from S, P, and A; and

• X₆ and X7 are A.

7. The poxvirus according to claim 4, wherein the heavy chain CDR3 of the sdAb specifically binding to PD-L1 consists of the sequence X6X7X2SLLRGX3SSRAEX4YDX5 (SEQ ID NO: 8), wherein:

• X2, X3 and X5 are S;

• X4 is selected from S and P; and

• X₆ and X7 are A.

8. The poxvirus according to any one of claims 1 to 7, wherein a) the three heavy chain CDRs of the sdAb specifically binding to PD-L1 are as follows:

• the heavy chain CDR1 consists of sequence RTFREYGMG (SEQ ID NO:1 ),

• the heavy chain CDR2 consists of sequence TISSSGSYSY (SEQ ID NO: 9), and

• the heavy chain CDR3 consists of sequence AASSLLRGSSSRAESYDS (SEQ ID NO : 10); or b) the three heavy chain CDRs of the sdAb specifically binding to PD-L1 are as follows:

• the heavy chain CDR1 consists of sequence RTFREYGMG (SEQ ID NO:1 ),

• the heavy chain CDR2 consists of sequence TISSSGSYTY (SEQ ID NO: 11 ), and

• the heavy chain CDR3 consists of sequence AASSLLRGSSSRAEPYDS (SEQ ID NO :12).

9. The poxvirus according to any one of the preceding claims, wherein the sdAb specifically binding to PD-L1 comprises or consists of an amino acid sequence having at least 80 % sequence identity with SEQ ID NO: 13 or SEQ ID NO: 14, preferably with SEQ ID NO: 13, preferably the sdAb specifically binding to PD-L1 comprises or consists of the amino acid sequence SEQ ID NO: 13.

10. The poxvirus according to any one of the preceding claims, wherein the nucleic acid molecule encoding the binding agent comprises or consists of a nucleic acid molecule encoding the sdAb specifically binding to PD-L1 , wherein said nucleic acid molecule encoding the sdAb specifically binding to PD-L1 comprises or consists of a nucleic acid sequence having at least 80 % sequence identity with SEQ ID NO: 34 or SEQ ID NO:35, preferably with SEQ ID NO: 34, preferably said nucleic acid molecule encoding the sdAb specifically binding to PD-L1 comprises or consists of the nucleic acid sequence SEQ ID NO:34.

11. The poxvirus according to any one of the preceding claims, wherein the sdAb specifically binding to PD-L1 is fused, directly or indirectly through a peptide linker, to a fusion polypeptide partner.

12. The poxvirus according to any one of the preceding claims, wherein the fusion polypeptide partner is selected from: a) an antigen-binding antibody fragment, preferably a sdAb or a scFv, and b) an antibody heavy chain constant region or a fragment thereof, preferably selected from the hinge region, the Fc fragment, and the entire constant region.

13. The poxvirus according to claim any one of the preceding claims, wherein the binding agent is selected from the group consisting of: a) the monovalent sdAb specifically binding to PD-L1 as defined in any one of claims 1 to 10, preferably the monovalent sdAb specifically binding to PD-L1 comprising or consisting of the amino acid sequence SEQ ID NO: 13; d) a bivalent sdAb comprising or consisting of two sdAb specifically binding to PD- L1 as defined in any one of claims 1 to 12 fused through a peptide linker, preferably a bivalent sdAb comprising or consisting of the amino acid sequence SEQ ID NO:31 ; c) a hinge bivalent sdAb comprising or consisting of the association through disulfide bonds of two polypeptides comprising or consisting of the sdAb specifically binding to PD-L1 as defined in any one of claims 1 to 12 fused to the hinge part of a antibody heavy chain constant region, preferably a hinge bivalent sdAb comprising or consisting of the association through disulfide bonds of two polypeptides and comprising or consisting of the amino acid sequence SEQ ID NO:32; and d) an Fc bivalent sdAb comprising or consisting of the association through disulfide bonds of two polypeptides comprising or consisting of the sdAb specifically binding to PD-L1 as defined in any one of claims 1 to 12 fused to an Fc fragment of an antibody heavy chain constant region, preferably an Fc bivalent sdAb comprising or consisting of the association through disulfide bonds of two polypeptides comprising or consisting of the amino acid sequence SEQ ID NO:33. e poxvirus according to any one of the preceding claims, wherein the binding agentcted from the group consisting of: a) a monovalent sdAb specifically binding to PD-L1 as defined in any one of claims 1 to 13 and encoded by a nucleic acid sequence having at least 80 % sequence identity with SEQ ID NO: 34 or SEQ ID NO:35, preferably with SEQ ID NO: 34; preferably the monovalent sdAb specifically binding to PD-L1 encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 34 ; d) a bivalent sdAb comprising or consisting of two sdAb specifically binding to PD- L1 as defined in any one of claims 1 to 13 fused through a peptide linker and encoded by a nucleic acid sequence having at least 80 % sequence identity with SEQ ID NO: 36, preferably a bivalent sdAb encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 36; c) a hinge bivalent sdAb comprising or consisting of the association through disulfide bonds of two polypeptides comprising or consisting of the sdAb specifically binding to PD-L1 as defined in any one of claims 1 to 13 fused to the hinge part of an antibody heavy chain constant region and encoded by a nucleic acid sequence having at least 80 % sequence identity with SEQ ID NO: 37, preferably a hinge bivalent sdAb encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 37; and d) an Fc bivalent sdAb comprising or consisting of the association through disulfide bonds of two polypeptides comprising or consisting of the sdAb specifically binding to PD-L1 as defined in any one of claims 1 to 13 fused to an Fc fragment of an antibody heavy chain constant region and encoded by a nucleic acid sequence having at least 80 % sequence identity with SEQ ID NO: 38, preferably a Fc bivalent sdAb encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 38. 15. The poxvirus according to any one of the preceding claims, further comprising another nucleic acid molecule inserted in its genome encoding a therapeutic polypeptide; preferably wherein the therapeutic polypeptide is selected from the group consisting of a suicide gene product, an immunostimulatory polypeptide, an antigenic polypeptide, an antibody, a functional derivative of an antibody, a functional fragment of an antibody, and any combination thereof.

16. The poxvirus according to claim 15, wherein the immunostimulatory polypeptide is selected from the group consisting of cytokines, such as interleukins, chemokines, interferons, tumor necrosis factors, colony-stimulating factors; APC-exposed proteins; agonists of stimulatory immune checkpoints; antagonists of inhibitory immune checkpoints; and any combination thereof.

17. The poxvirus according to any one of the preceding claims, wherein the poxvirus is a poxvirus of the Chordopoxvirinae family, preferably selected from the group consisting of Avipoxvirus genus, Capripoxvirus genus, Lepori poxvirus genus, Mollusci poxvirus genus, Orthopoxvirus genus, Parapoxvirus genus, Suipoxvirus genus, Cervidpoxvirus genus, Yatapoxvirus genus, and chimeras thereof.

18. The poxvirus according to claim 17, wherein the poxvirus is a member of the Orthopoxvirus genus, preferably selected from the group consisting of vaccinia virus (VV), cowpox (CPXV), raccoonpox (RCN), rabbitpox, Monkeypox, Horsepox, Volepox, Skunkpox, variola virus (or smallpox), Camelpox, and chimeras thereof.

19. The poxvirus according to claim 18, wherein the poxvirus is a vaccinia virus, preferably selected from: a) an oncolytic vaccina virus selected from the group of Western Reserve (WR), Elstree, Copenhagen (Cop), Wyeth, Lister, LI VP, Tashkent, Tian Tan, Brighton, Ankara, LC16M8, and LC16M0 strains, which preferably comprises:

(i) inactivating mutations in the J2R viral gene,

(ii) inactivating mutations in the viral I4L and/or F4L gene(s),

(iii) inactivating mutations in the M2L gene,

(iv) inactivating mutations in the J2R viral gene and inactivating mutations in the viral I4L and/or F4L gene(s),

(v) inactivating mutations in the J2R viral gene and inactivating mutations in the M2L gene, (vi) inactivating mutations in the viral I4L and/or F4L gene(s) and inactivating mutations in the M2L gene, or

(vii ) inactivating mutations in the J2R viral gene, inactivating mutations in the viral I4L and/or F4L gene(s) and inactivating mutations in the M2L gene; or b) a modified Vaccinia Ankara (MVA) strain.

20. The poxvirus according to any one of the preceding claims, wherein said nucleic acid molecule encoding a binding agent and/or said foreign nucleic acid molecule encoding a therapeutic polypeptide is operably linked to suitable regulatory elements for expression in a desired host cell or subject.

21 . The poxvirus according to any one of the preceding claims, wherein said nucleic acid molecule encoding a binding agent and/or said nucleic acid molecule encoding a therapeutic polypeptide is placed under the control of a poxvirus promoter, preferably, a vaccinia virus promoter and more preferably one selected from the group consisting of the p7.5K, pH5R, p11K7.5, pSE, pSE/L, pTK, pB2R, p28, p11 , pF17R, pA14L and pK1L promoter, synthetic promoters, and early/late chimeric promoters, preferably pH5R.

22. The poxvirus according to any one of the preceding claims, wherein the poxvirus is produced on chicken embryo fibroblasts (CEF), HeLa cells, EB66® cells, Vero cells, HEK 293 cells, PerC6 cells, BHK21 cells, or MRC5 cells.

23. A method for producing the poxvirus of any of claims 1 to 22, comprising the steps of: a) obtaining or preparing a producer cell line; b) infecting the obtained or prepared producer cell line with the poxvirus; c) culturing the infected producer cell line under suitable conditions so as to allow the production of the poxvirus; d) recovering the produced poxvirus from the culture of said producer cell line; and optionally e) purifying said recovered poxvirus, optionally wherein the producer cell line is a chicken embryo fibroblasts (CEF), a HeLa, an EB66,®, a Vero, a HEK 293, a PerC6, a BHK21 , or a MRC5 cell line.

24. A composition comprising a therapeutically effective amount of the poxvirus according to any one of claims 1 to 22, or a therapeutically effective amount of the poxvirus obtained according to the method of claim 23, and a pharmaceutically acceptable vehicle.

25. The composition according to claim 24, comprising from 10³ to 10¹² pfu, preferably from 10⁴pfu to 10¹¹ pfu, preferably from 10⁵ pfu to 10¹⁰ pfu, more preferably from 10⁶ pfu to 10⁹ pfu of the poxvirus; notably individual doses of approximately 10⁶, 5x10⁶, 10⁷, 5x10⁷, 10⁸ or 5x10⁸ pfu of the poxvirus.

26. The composition according to claim 24 or 25, which is formulated for intravenous, intramuscular, subcutaneous, oral, intranasal, transdermal, or intratumoral administration.

27. The composition according to any one of claims 24 to 26, further comprising another therapeutic agent, preferably an anticancer agent.

28. The poxvirus according to any one of claims 1 to 22, or the composition according to any one of claims 23 to 27, for use as a medicament or a vaccine.

29. The poxvirus or the composition for use according to claim 28, for use for treating or preventing a proliferative disease, preferably a cancer.

30. The poxvirus or the composition for use according to claim 29, wherein the cancer is selected from the group consisting of renal cancer, prostate cancer, breast cancer, bladder cancer, colorectal cancer, lung cancer, liver cancer, gastric cancer, bile duct carcinoma, endometrial cancer, pancreatic cancer, ovarian cancer, head and neck cancer, melanoma, glioblastoma, multiple myeloma, or malignant glioma cells.

31 . The poxvirus or the composition for use according to any one of claims 28 to 30, for use: a) as stand-alone therapy, or b) in conjunction with one or more additional therapeutic intervention (s), preferably selected from the group consisting of surgery, radiotherapy, chemotherapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy, cytokine therapy, targeted cancer therapy, gene therapy, photodynamic therapy and transplantation. 32. The poxvirus or the composition for use according to claim 31 , for use in conjunction with one or more other therapeutic agents selected from the group consisting of agonists of stimulatory immune checkpoints, and antagonists of inhibitory immune checkpoints; wherein the agonist of stimulatory immune checkpoints is preferably selected from human ICOSL, 4-1 BBL, OX40L, CD70, CD40L, GITRL and agonist antibodies to human ICOS, CD137 (4-1 BB), 0X40, CD27, CD40, or GITR; and wherein the antagonist of inhibitory immune checkpoints is preferably selected from antagonist antibodies to human PD-1 , SIRPa, CD47, PD-L2, LAG3, Tim3, BTLA, or CTLA4.