WO2023213764A1 - Fusion polypeptide comprising an anti-pd-l1 sdab and a member of the tnfsf - Google Patents

Fusion polypeptide comprising an anti-pd-l1 sdab and a member of the tnfsf Download PDF

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Publication number
WO2023213764A1
WO2023213764A1 PCT/EP2023/061453 EP2023061453W WO2023213764A1 WO 2023213764 A1 WO2023213764 A1 WO 2023213764A1 EP 2023061453 W EP2023061453 W EP 2023061453W WO 2023213764 A1 WO2023213764 A1 WO 2023213764A1
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Prior art keywords
fusion polypeptide
seq
vector
tnfsf
viral
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PCT/EP2023/061453
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French (fr)
Inventor
Jean-Baptiste Marchand
Christelle REMY
Marshall Dunlop
Shirley Schon
Peter Fitzgerald
John Lamont
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Transgene
Randox Laboratories Limited
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Publication of WO2023213764A1 publication Critical patent/WO2023213764A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • the present invention is related to a fusion polypeptide comprising a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ) fused to a member of the Tumor Necrosis Factor superfamily of ligands (TNFSF), and to the compositions and methods related thereto.
  • sdAb single domain antibody specifically binding to programmed death-ligand 1 (PD-L1 ) fused to a member of the Tumor Necrosis Factor superfamily of ligands (TNFSF)
  • Among potential source for anticancer therapies is a patient’s own immune system, in particular T cell-mediated cytotoxicity.
  • Efficient antitumoral T cell response necessitates a functional and optimal crosstalk between cells of the innate and adaptive immunity. This is spatially and timely achieved by multiple specific interactions between different cells through, soluble stimuli (chemokine/cytokine) and through surface ligand/receptor binding among which the costimulatory molecules of the Tumor necrosis factor super family (TNFSF) molecules play a central role.
  • soluble stimuli chemokine/cytokine
  • TNFSF Tumor necrosis factor super family
  • CD40L (also known as CD154 or TNFSF5) is a type I membrane co-stimulatory protein of the TNFSF that play a central role in the initiation of adaptive immune response. As TNF, CD40L assembles into a homotrimer and interacts in trans with its receptor CD40. CD40 is distributed on antigen presenting cells (APC) such as dendritic cells, macrophages, B lymphocytes, but also epithelial cells, endothelial cells, smooth muscle cells, fibroblasts, basophils, and blood platelets. CD40L is expressed on surface of activated B, T and NK cells but also on adipose cells, and basophils (Richards et al. Hum Vaccin Immunother.
  • APC antigen presenting cells
  • CD40L-CD40 interaction leads to the activations of CD40 bearing cells which then express adhesion (ICAM), co-stimulatory (CD80/CD86), and presenting MHC I and II molecules in addition of cytokines/chemokines (TNFa, IL6).
  • adhesion molecules and cytokine/chemokines act together to induce the infiltration and activation of immune cells, that ultimately destroy tumor cells and skew the tumor from an immunosuppressive to an immunocompetent microenvironment (Richards et al. Hum Vaccin Immunother. 2020; 16(2) :377-387).
  • CD40 clustering and activation can be also obtained artificially using, agonist antibodies or CD40L-Fc fusion proteins. These molecules interact on one hand with CD40 by their paratope, or CD40L domain, and induce its clusterization by interaction in trans- on the other hand with Fc receptors (FcR) bearing cells.
  • FcR Fc receptors
  • CD40L CD40 agonist binding moiety
  • CD40L CD40 antibody fragment
  • TAA tumor associated antigen
  • condition CD40 agonists i.e. molecules with cd40 agonist activity only in presence of TAA expressing cells
  • PD-1 programmed death 1
  • Ig immunoglobulin
  • PD-1 programmed death ligand 1
  • PD-L2 programmeed death ligand 2
  • 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).
  • the interaction between PD-1 and PD-L1 resulted in a decrease in tumor infiltrating lymphocytes, a decrease in T- cell receptor mediated proliferation, and immune evasion by the cancerous cells (Dong et al., 2003, J. Mol. Med. 81 : 281 -7; Blank et al., 2005, Cancer Immunol. Immunother. 54: 307- 5 314).
  • WO2019/094574 discloses a bispecific fusion protein of about 250 kDa (dimer) comprising an anti-PD-L1 Fab fragment and two CD40L subunits, both binding regions covalently linked to a Fc monomer.
  • Such bispecific fusion protein is for use for controlling T cell- mediated cytotoxicity via improved control of T cell signaling pathways.
  • the ability of such bispecific fusion protein anti-PD-L1 /CD40L to synergistically induce biological response compared to the combination of anti-PD-L1 monovalent monoclonal antibody and agonist CD40L fusion protein or either treatment alone has been described (Pandey et al. Int J Mol Sci. 2021 Oct 21 ;22(21 ): 11302) .
  • the CD40 agonist activity in absence of PD-L1 expressing cells is still very strong for such a fusion protein, therefore such bispecific cannot be considered as a conditional agonist.
  • the presence of the Fc monomer may induce the activation of FcR bearing cells in the neighborhood, leading to the destruction of the targeted CD40+ cells by triggering ADCC, CDC or anti-inflammatory FcR activities. Such destruction isn’t an effect which is sought in the context of the use of a conditional TNFSF ligand agonist.
  • the presence of the Fc moiety results in a long half-life in vivo, further increasing the risk of the occurrence of adverse effects in areas different from the tumor microenvironment.
  • the 4- 1 BB ligand (4-1 BBL, also known as CD137L or TNFSF9) is found on APCs (antigen presenting cells) and binds to 4-1 BB (also known as CD137), a type 2 transmembrane glycoprotein receptor belonging to the TNF superfamily, which is expressed on activated T Lymphocytes (Lotze M (2001 ). Dendritic Cells. Boston: Academic Press. ISBN 0-12-455851 - 8).
  • the 4-1 BBL has been associated with enabling the immune system to eliminate tumors in multiple cancer types.
  • CD137 is expressed at higher levels on CD8+ than CD4+ T cells, and it mainly co-stimulates CD8+ T cells. Crosslinking of CD137 strongly enhances proliferation, IFN-y secretion and cytolytic activity of T cells. Moreover, CD137 agonists, such as antibodies, have been reported to work synergistically with cancer vaccines and immune check point inhibitors to boost anticancer immune responses (Dharmadhikari et al., 2016, Oncoimmunology 5(4): el 113367).
  • fusion polypeptide of about 150 kDa (dimer) comprising an anti-4-1 BB F(ab’)? or an anti- CD40 F(ab’)z antibody fused with a PD-L1 specific blocking scFv as anchoring domain.
  • Such fusion polypeptide is considered as a conditional agonist, with a weak CD40 or 4-1 BB agonist activity in absence of PD-L1 expressing cells; and a strong CD40 or 4-1 BB agonist activity in presence of PD-L1 expressing cells.
  • scFv antibody is known in prior art to be potentially immunogen, which can negatively impact the capacity of the fusion polypeptide to induce the CD40 or 4-1 BB agonist activity. Indeed, compared to antibodies, scFvs tend to have lower affinities, lower long-term stability, and a higher likelihood to aggregate due to their small size (Bates and Power. Antibodies. 2019; 8(2) :28).
  • conditional agonists are useful for limiting adverse effects on non-tumor tissues, they should still display maximum agonist activity in the presence of PD-L1 cells, which may not be the case for a fusion comprising only a monovalent anti-PD-L1 scFv.
  • conditional agonist constructs being in capacity to maximize T cell mediated cytotoxicity via improved control of T cell signaling pathways.
  • conditional agonist constructs could provide less toxic, more targeted anticancer therapies.
  • conditional agonist fusion polypeptide in case of delivery of the conditional agonist via a viral vector the number and size of each transgene is an issue because of the limited capacity of some viral platforms such as for examples, adenovirus or measles. Therefore, there is a need for an improved vectorizable sequence encoding such a conditional agonist fusion polypeptide.
  • fusion polypeptides comprising a particular anti-PD-L1 sdAb fused to a member of the TNFSF or a functional fragment or variant thereof.
  • fusion polypeptides combine many advantages, including:
  • the encoded single chain has a molecular weight of only about 30 kDa, and trimerizes due to the trimerization domain of the TNFSF member, resulting in a final molecular weight of only about 90kDa.
  • the present invention thus relates to fusion polypeptide comprising a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ) fused to a member of the Tumor Necrosis Factor superfamily of ligands (TNFSF) or a functional fragment or derivative thereof, wherein: a) the sdAb specifically binding to PD-L1 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),
  • the heavy chain CDR3 comprises the sequence X2SLLRGX3SSRAEX4YDX5, wherein each of X2 to X5 independently represents any amino acid (SEQ ID NO:3), and b) the sdAb specifically binding to PD-L1 is fused to the member of the TNFSF or functional fragment or derivative thereof directly or indirectly through a peptide linker.
  • the present invention also relates to a nucleic acid molecule encoding the fusion polypeptide according to the invention, to a vector (in particular a viral vector; and more particularly a poxviral vector, such as a vaccinia virus vector) comprising the nucleic acid molecule according to the invention, a host cell comprising the nucleic acid molecule according to the invention or the vector according to the invention, and a composition comprising the fusion polypeptide, nucleic acid molecule, vector (in particular a viral vector; and more particularly a poxviral vector, such as a vaccinia virus vector), or host cell according to the invention, or any combination thereof.
  • a vector in particular a viral vector; and more particularly a poxviral vector, such as a vaccinia virus vector
  • a composition comprising the fusion polypeptide, nucleic acid molecule, vector (in particular a viral vector; and more particularly a poxviral vector, such as a vaccinia virus vector), or host cell
  • the present invention also relates to methods for producing the fusion polypeptide according to the invention or the viral vector according to the invention.
  • the invention also relates to therapeutic uses or methods of treatment using the fusion polypeptide, nucleic acid molecule, vector (in particular a viral vector; and more particularly a poxviral vector, such as a vaccinia virus vector), host cell, or composition according to the invention, or any combination thereof, in particular in the treatment or prevention of cancer.
  • vector in particular a viral vector; and more particularly a poxviral vector, such as a vaccinia virus vector
  • host cell or composition according to the invention, or any combination thereof, in particular in the treatment or prevention of cancer.
  • FIG. 1 shows a schematic demonstrating a PD-1 /PD-L1 inhibition ELISA.
  • FIG. 2 shows the results obtained via an ELISA-based assay, demonstrating the ability of selected sdAb clones to block the interaction between PD-1 and PD-L1 .
  • FIG. 3 shows the results obtained via an inhibition assay, demonstrating the ability of selected sdAb clones to block PD1 /PD-L1 interaction.
  • FIG. 4 shows the results obtained from a PD-L1 binding assay for the humanised 32.1A1 clone.
  • FIG. 5 shows the results obtained from a PD-1 /PD-L1 inhibition assay to compare clone 32.1A1 , its humanised derivative and its close relative 32.2F7 to the commercially available therapeutic antibody Avelumab.
  • FIG. 6 shows a schematic demonstrating the PD-1 /PD-L1 blockade bioassay.
  • FIG. 7 shows the luciferase activity fold change of the two lead PD-1 and PD-L1 blocking clones, as identified by the ELISA inhibition assay, as assayed in the Promega PD- 1 /PD-L1 blockade bioassay.
  • Reference to GS542 denotes the humanised 32.1A1 clone, and reference to 1A1 denotes clone 32.1A1 .
  • FIG. 8 shows an SDS-PAGE gel showing fraction of sdAb monomer.
  • FIG. 9 shows an analytical size exclusion chromatography (SEC) to demonstrate aggregation following thermal stress of the humanised 32.1A1 clone.
  • FIG. 10 shows humanised 32.1A1 stability ELISA against coated PD-L1.
  • FIG. 11 shows cross-reactivity of clone 32.1A1 against related and unrelated antigens.
  • FIG. 12 shows a series of 15 mutant sdAb clones synthesized using alanine substitution at 15 different residues across CDR3 of humanised 32.1A1 to identify residues that are important for binding of the h32.1A1 antibody to PDL1 antigen.
  • FIG. 13A and FIG. 13B show the results obtained from an ELISA assay for binding affinity of the 15 mutant sdAb clones of FIG. 12.
  • FIG. 14 shows level of expression of GS542-CD40L constructions by infected /transfected HeLa cells. Clarified supernatants containing the different anti-PD-L1 -CD40L molecules were loaded on SDS-PAGE under reducing and non-reducing conditions. Proteins were transferred on PVDF membrane and hybridized with an HRP-conjugated anti-FLAG tag for immunodetection.
  • FIG. 15 shows designs of ELISA assay to demonstrate the bispecificity of GS542-CD40L fusions.
  • FIG. 16 shows pTG19970 and pTG19971 products are able to bind CD40 and PD-L1 simultaneously.
  • Different dilutions of clarified supernatants of HeLa cells infected with vaccinia virus and then transfected with plasmids carrying expression cassette encoding the different anti-PD-L1-CD40L molecules were loaded on ELISA plates previously coated with either CD40 or PD-L1.
  • the bound bispecific protein was developed with either labeled PD-L1 or Fc-tagged CD40 respectively. Results are reported as the optical density versus 1 /dilution of the culture medium.
  • PTG19274 is encoding an irrelevant FLAG-tagged protein and was used as negative control.
  • FIG. 17 shows CD40 agonist activity of culture medium of HeLa cells infected with vaccinia virus and then transfected with plasmids carrying expression cassette encoding GS542-CD40L constructions and under control of pH5.R promoter (i.e. poxvirus promoter).
  • HEK cells modified to express the reporter enzyme SEAP under the control of a CD40 inducible promoter were incubated with different dilutions of the clarified supernatants containing the CD40L constructions in presence, or absence, of PD-L1 expressing cells (Hs746T).
  • SEAP enzymatic activity was measured in culture medium after a 20-24 hours incubation.
  • Negative controls were non-infected cells (noted medium), or cells infected/transfected with a plasmid (noted pTG19274) encoding an irrelevant FLAG- tagged protein. Results are reported as the SEAP activity versus 1 /dilution of the culture medium.
  • FIG. 18A shows CD40 agonist activity of pTG19971 is blocked by preincubation with anti- PD-L1 antibody.
  • CD40 agonist activity was assessed as described in Figure 17 except that culture supernatants of infected/transfected (pTG19971 ) HeLa cells were tested. In some conditions avelumab (avel) or its isotype control (iso) were added to the cells prior the culture supernatants. In these latter cases, only the undiluted supernatants were tested.
  • FIG. 18B shows CD40 agonist activity of COPTG19971 is blocked by preincubation with anti-PD-L1 antibody.
  • CD40 agonist activity was assessed as described in Figure 17 except that culture supernatants of infected (COPTG19971 ) HeLa cells were tested. In some conditions avelumab (avel) or its isotype control (iso) were added to the cells prior the culture supernatants. In these latter cases, only the undiluted supernatants were tested.
  • FIG. 19 shows the level of expression 4-1 BBL ectodomain alone (pTG20032) or fused at C- terminus of GS542 (anti-PD-L1 sdAb, pTG20034), negative control is a plasmid encoding GFP (pTG19333).
  • FIG. 20 shows 4-1 BB agonist activity of culture medium of HeLa cells infected with vaccinia virus and then transfected with plasmids carrying expression cassette encoding 4-1 BBL (pTG20032) or GS542-4-1 BBL (pTG20034) constructions and under control of pH5.R promoter (i.e., poxvirus promoter).
  • Reporter cells modified to express the reporter enzyme luciferase under the control of a 4-1 BB inducible promoter were incubated with different dilutions of the clarified supernatants containing the 4-1 BBL constructions in presence, or absence, of PD-L1 expressing cells (Hs746T).
  • Luciferase enzymatic activity was measured in culture medium after a 20-24 hours incubation. Negative controls were non-infected cells (noted medium), or cells infected /transfected with a plasmid (noted pTG 19274) encoding an irrelevant FLAG -tagged protein. Results are reported as the luciferase activity (luminescence) versus 1 /dilution of the culture medium.
  • FIG. 21 shows CD40 agonist activity of pTG19971 and of two benchmark constructs (pTG20154/pTG20155 and pTG20156/pTG20157). After infection/transfection by the indicated plasmid, culture mediums were harvested and tested for their CD40 agonist activity as described in Figure 17.
  • FIG. 22 shows PD-1 /PD-L1 blocking activity of pTG19971 and of two benchmark constructs (pTG20154/pTG20155 and pTG20156/pTG20157) measured by competitive ELISA for vectorized products. Same samples as in Figure 21 were tested for their inhibiting activity of PD-1 /PD-L1 interaction.
  • amino acids amino acids
  • amino acid analogs e.g. non-natural, synthetic and modified amino acids, including D or L optical isomers.
  • 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.
  • polypeptide refers 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).
  • nucleic acid refers to any polypeptide that is derived from any polypeptide.
  • modified polypeptides also designated derivatives, analogs, variants or mutants
  • polypeptide fragments e.g. dimers
  • polypeptide multimers e.g. dimers
  • recombinant polypeptides e.g. dimers
  • fusion polypeptides e.g. dimers
  • nucleic acid sequence e.g. dimers
  • nucleic acid sequence e.g. dimers
  • 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.
  • 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).
  • an automated DNA synthesizer such as those that are commercially available from Biosearch, Applied Biosystems, etc.
  • a naturally occurring source e.g. a genome, cDNA, etc.
  • an artificial source such as a commercially available library, a plasmid, 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).
  • 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).
  • 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.
  • 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.
  • a component e.g. polypeptide, nucleic acid molecule
  • 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.
  • subject generally refers to a vertebrate organism for whom any of the product or methods disclosed herein is needed or may be beneficial.
  • 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).
  • 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.
  • 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.
  • treatment refers to therapy.
  • 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.
  • prevention and any form of treatment 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).
  • administering 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.
  • fusion polypeptide 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 present invention first relates to a fusion polypeptide comprising, consisting essentially of or consisting of a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ) fused to a member of the Tumor Necrosis Factor superfamily of ligands (TNFSF) or a functional fragment or derivative thereof, wherein: a) the sdAb specifically binding to human PD-L1 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),
  • the heavy chain CDR3 comprises the sequence X2SLLRGX3SSRAEX4YDX5, wherein each of X2 to X5 independently represents any amino acid (SEQ ID NO:3), and b) the sdAb specifically binding to human PD-L1 is fused to the member of the TNFSF or functional fragment or derivative thereof directly or indirectly through a peptide linker. sdAb specifically binding to PD-L1
  • the fusion polypeptide according to the invention comprises as a first fusion partner (also referred to as “fusion partner 1” or “FP1”) a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ) (also referred to as “anti-PD-L1 sdAb”).
  • a first fusion partner also referred to as “fusion partner 1” or “FP1”
  • sdAb programmed death-ligand 1
  • PD-L1 programmed death-ligand 1
  • 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 included in the fusion polypeptide 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 ).
  • 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.
  • VHH variable domain
  • VNAR variable domain
  • sdAb in particular VHH and VNAR
  • 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).
  • 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.
  • 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).
  • 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 included in the fusion polypeptide according to the invention specifically binds to PD-L1.
  • binding refers 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.
  • 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 comprised in the fusion polypeptide according to the invention preferably has a Kd value lower than 1 nM (preferably when measured using an Octet Red96 instrument, more preferably using the method disclosed in Example 1 ).
  • 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).
  • FP1 -Emb1 This embodiment is referred to as “FP1 -Emb1”.
  • sdAb with such heavy chain CDR1 , CDR2 and CDR3 are shown in Example 1 to confer specificity for human PD-L1 .
  • 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).
  • 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).
  • 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.
  • the heavy chain CDR3 of the sdAb specifically binding to PD-L1 comprises, 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.
  • the heavy chain CDR3 of the sdAb specifically binding to PD-L1 comprises, consists essentially of or consists of the sequence X6X 7 X2SLLRGX 3 SSRAEX4YDX 5 (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, P, T, C, A, V, G and P;
  • X 6 and X7 are independently selected from A, V, G and P.
  • 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.
  • the heavy chain CDR3 of the sdAb specifically binding to PD-L1 comprises, consists essentially of or consists of the sequence X 6 X7X 2 SLLRGX 3 SSRAEX4YDX 5 (SEQ ID NO:6), wherein:
  • X 2 , X 3 and X5 are independently selected from S, T, C and A;
  • X4 is selected from S, P, T, C and A;
  • X 6 and X7 are selected from A, V, G and P.
  • the heavy chain CDR3 of the sdAb specifically binding to PD-L1 comprises, consists essentially of or consists of the sequence X6X 7 X 2 SLLRGX 3 SSRAEX4YDX 5 (SEQ ID NO:7), wherein:
  • X 2 , X 3 and X5 are independently selected from S and A;
  • X4 is selected from S, P, and A;
  • the heavy chain CDR3 of the sdAb specifically binding to PD-L1 comprises, consists essentially of or consists of the sequence X 6 X 7 X 2 SLLRGX 3 SSRAEX4YDX 5 (SEQ ID NO:8), wherein:
  • X4 is selected from S and P;
  • 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.
  • FP1 -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 NO: 10); or b) embodiment “FP1 -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).
  • the sdAb is preferably a humanized sdAb.
  • 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 FP1 -Emb1 to FP1 -Emb8 in which the sdAb is humanized are particularly preferred.
  • the sdAb specifically binding to PD-L1 comprises, 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.
  • the sdAb specifically binding to PD-L1 comprises, 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.
  • 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:
  • SEQ ID NO: 13 is the amino acid sequence of the humanized version of the sdAb produced by clone 32.1A1 :
  • the sdAb specifically binding to PD-L1 most preferably comprises, consists essentially of or consists of the sequence SEQ ID NO: 13 (embodiment “FP1 -Emb10”).
  • SEQ ID NO: 14 is the amino acid sequence of the humanized version of the sdAb produced by clone 32.2F7:
  • the sdAb specifically binding to PD-L1 may also preferably comprise, consist essentially of or consist of the sequence SEQ ID NO: 13 (embodiment “FP1 -Emb11 ”).
  • SEQ ID NO: 13 and SEQ ID NO: 14 have 98.4% identity.
  • the fusion polypeptide according to the invention also comprises as a second fusion partner (also referred to as “fusion partner 2” or “FP2”) a member of the Tumor Necrosis Factor superfamily of ligands (TNFSF) or a functional fragment or derivative thereof (also referred to as “TNFSF member”) (referred to as “FP2-EMb1 ”).
  • FP2 fusion partner 2
  • TNFSF Tumor Necrosis Factor superfamily of ligands
  • FP2-EMb1 a functional fragment or derivative thereof
  • the member of the TNFSF or functional fragment or variant thereof is selected from:
  • CD40L 4-1 BBL, Baff, APRIL, EDA-A1 , GITRL, OX40L, CD70, TL1A, LIGHT, LToB 2 , RANKL, TWEAK, FASL, TRAIL, TNF and LTo and functional fragments or derivatives thereof (embodiment “FP2-Emb2”);
  • TNFSF members preferably from CD40L, 4- 1 BBL, Baff, APRIL, EDA-A1 , OX40L, CD70, TWEAK, FASLG, TRAIL, and TNF and functional fragments or derivatives thereof (embodiment “FP2-Emb4”);
  • TNFSF members involved in immune cell activation preferably from CD40L, 4-1 BBL, GITRL, OX40L, CD70, TL1A and functional fragments or derivatives thereof (embodiment “FP2-Emb6”
  • category II TNFSF members involved in immune cell activation preferably from CD40L, 4-1 BBL, OX40L, CD70, and functional fragments or derivatives thereof (embodiment “FP2-Emb8”
  • the member of the TNFSF or functional fragment or derivative thereof is selected from CD40L and functional fragments or derivatives thereof (embodiment “FP2- Emb10”).
  • the TNFSF comprises a number of structurally related members (also referred to as ligands) that organize lymphoid tissue development, co-stimulate lymphocyte activation and can either increase lymphocyte survival and function or induce cell death, all through bonding to their cognate receptor(s), which form the tumor necrosis factor receptor superfamily (TNFRSF).
  • structurally related members also referred to as ligands
  • TNFRSF tumor necrosis factor receptor superfamily
  • TNFRSF may be divided into two distinct categories, depending on their ability to be activated by soluble ligand trimers of the TNFSF (Kucka K, Wajant H. Receptor Oligomerization and Its Relevance for Signaling by Receptors of the Tumor Necrosis Factor Receptor Superfamily. Front Cell Dev Biol. 2021 Feb 11 ;8:615141 ) .
  • Category II receptors of the TNFRSF fail to become properly activated by soluble ligand trimers despite high affinity binding, and include CD40L, 4-1 BBL, Baff, APRIL, EDA-A1 , OX40L, CD70, TWEAK, FASLG, TRAIL, and TNF.
  • category II TNFRs to soluble TNFLs can be overcome by physical linkage of two or more soluble ligand trimers or, alternatively, by anchoring the soluble ligand molecules to the cell surface or extracellular matrix.
  • members of the TNFSF binding to category II TNFRs are preferred as the ability of the fusion polypeptide according to the invention is then truly conditional, as activation of their cognate TNFR then depends on the presence in the tumor microenvironment of PD-L1 positive tumor cells.
  • the member of the TNFSF included in the fusion polypeptide according to the invention is selected from category II TNFSF members, preferably selected from CD40L, 4-1 BBL, Baff, APRIL, EDA-A1 , OX40L, CD70, TWEAK, FASLG, TRAIL, and TNF and functional fragments or derivatives thereof.
  • TNFSF members may also be classified depending on their known functions.
  • TNFSF members involved in immune cell activation may preferably be included in the fusion polypeptide according to the invention.
  • TNFSF members involved in immune cell activation include CD40L, 4-1 BBL, GITRL, OX40L, CD70, TL1A and functional fragments or derivatives thereof (Croft M, Siegel RM. Beyond TNF: TNF superfamily cytokines as targets for the treatment of rheumatic diseases. Nat Rev Rheumatol. 2017; 13(4) :217-233) .
  • CD40L CD40 ligand
  • CD40LG tumor necrosis factor superfamily member 5
  • TNFSF5 tumor necrosis factor superfamily member 5
  • CD154 CD154
  • CD40L- CD40 interaction leads to the activation of CD40 bearing cells, which then express adhesion (ICAM), co-stimulatory (CD80/CD86), and MHC I and II molecules in addition of cytokines/chemokines (TNFa, IL6).
  • IAM adhesion
  • CD80/CD86 co-stimulatory
  • MHC I and II molecules MHC I and II molecules in addition of cytokines/chemokines
  • CD40L is thus a TNFSF member involved in immune cell activation. As TNF, CD40L assembles into a homotrimer and interacts in trans with its receptor CD40 through its extracellular part. CD40 is a category II receptor of the TNFRSF.
  • CD40L Being a member of the TNFSF that is both involved in immune cell activation and binds to a category II receptor of the TNFRSF, CD40L is a particularly preferred TNFSF member for the fusion polypeptide according to the invention.
  • the specific CD40L protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use.
  • human CD40L will preferably be used.
  • Human CD40L corresponds to Entrez Gene ID 959, and the complete amino acid sequence of human CD40L may be found under GenBank Accession No. NP_000065.1 (version of January 17, 2022).
  • 4-1 BBL “4-1 BB ligand”, “CD137L”, “tumor necrosis factor superfamily member 9”, and “TNFSF9” are used herein interchangeably and refer to a transmembrane cytokine that acts as a ligand for TNFRSF9/4-1 BB, which is a costimulatory receptor molecule in T lymphocytes. This cytokine and its receptor are involved in the antigen presentation process and in the generation of cytotoxic T cells. 4-1 BBL is thus a TNFSF member involved in immune cell activation.
  • Its receptor 4-1 BB is a category II receptor of the TNFRSF.
  • 4-1 BBL Being a member of the TNFSF that is both involved in immune cell activation and binds to a category II receptor of the TNFRSF, 4-1 BBL is a particularly preferred TNFSF member for the fusion polypeptide according to the invention.
  • the specific 4-1 BBL protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use.
  • human 4-1 BBL will preferably be used.
  • Human 4-1 BBL corresponds to Entrez Gene ID 8744, and the complete amino acid sequence of human 4-1 BBL may be found under GenBank Accession No. NP_003802.1 (version of February 27, 2022).
  • OX40L 0X40 ligand
  • CD252 CD134L
  • TNFSF4 tumor necrosis factor superfamily member 4
  • APCs antigen-presenting cells
  • Its receptor 0X40 is a category II receptor of the TNFRSF.
  • OX40L Being a member of the TNFSF that is both involved in immune cell activation and binds to a category II receptor of the TNFRSF, OX40L is a particularly preferred TNFSF member for the fusion polypeptide according to the invention.
  • the specific OX40L protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use.
  • human OX40L will preferably be used.
  • Human OX40L corresponds to Entrez Gene ID 7292, and the complete amino acid sequence of the longest isoform of human OX40L may be found under GenBank Accession No. NP_003317.1 (version of March 16, 2022).
  • CD70 is used herein interchangeably and refer to a molecule that can, through interaction with its receptor CD27, provide signals to T cells to control their accumulation and reactivity, similarly to that seen with 0X40, GITR and DR3.
  • CD70 is thus a TNFSF member involved in immune cell activation.
  • Its receptor CD27 is a category II receptor of the TNFRSF.
  • CD70 Being a member of the TNFSF that is both involved in immune cell activation and binds to a category II receptor of the TNFRSF, CD70 is a particularly preferred TNFSF member for the fusion polypeptide according to the invention.
  • the specific CD70 protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use.
  • human CD70 will preferably be used.
  • Human CD70 corresponds to Entrez Gene ID 970, and the complete amino acid sequences of the longest isoform of human CD70 may be found under GenBank Accession No. NP_001317261 .1 (version of January 9, 2022).
  • Baff B cell activating factor
  • CD257 CD257
  • TNFSF13B tumor necrosis factor superfamily member 20
  • TNFSF20 TNFSF20
  • BaffR is a category I receptor of the TNFRSF
  • TACI is a category II receptor of the TNFRSF.
  • Baff Being a member of the TNFSF that binds to a category II receptor of the TNFRSF (TACI), Baff is a preferred TNFSF member for the fusion polypeptide according to the invention.
  • the specific Baff protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use. In particular, for an intended use in humans, human Baff will preferably be used.
  • Human Baff corresponds to Entrez Gene ID 10673, and the complete amino acid sequences of the longest isoform of human Baff may be found under GenBank Accession No. NP_006564.1 (version of February 20, 2022).
  • APRIL “APRIL”, “CD256”, “tumor necrosis factor superfamily member 13”, and “TNFSF13” are used herein interchangeably and refer to a ligand found to be important for B cell development.
  • BCMA and TACI are both category II receptors of the TNFRSF.
  • APRIL is a preferred TNFSF member for the fusion polypeptide according to the invention.
  • the specific APRIL protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use. In particular, for an intended use in humans, human APRIL will preferably be used.
  • Human APRIL corresponds to Entrez Gene ID 8741 , and the complete amino acid sequences of the longest isoform of human APRIL may be found under GenBank Accession No. NP_003799.1 (version of January 23, 2022).
  • EDA-A1 EDA-A1
  • EDA ectodysplasin A
  • tumor necrosis factor ligand 7C a protein involved in cell-cell signaling during the development of ectodermal organs.
  • Its receptor EDAR is a category II receptor of the TNFRSF.
  • EDA-A1 Being a member of the TNFSF that binds to a category II receptor of the TNFRSF, EDA-A1 is a preferred TNFSF member for the fusion polypeptide according to the invention.
  • the specific EDA-A1 protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use.
  • human EDA-A1 will preferably be used.
  • Human EDA-A1 corresponds to Entrez Gene ID 1896, and the complete amino acid sequence of the longest isoform of human EDA-A1 may be found under GenBank Accession No. NP_001390.1 (version of February 13, 2022).
  • GITRL Glucocorticoid-induced TNF receptor-related ligand
  • tumor necrosis factor superfamily member 18 tumor necrosis factor superfamily member 18
  • TNFSF18 glucocorticoid-induced TNF receptor-related protein
  • Its receptor GITR is a category I receptor of the TNFRSF.
  • GITRL Being a member of the TNFSF that is involved in immune cell activation, GITRL is a preferred TNFSF member for the fusion polypeptide according to the invention.
  • the specific GITRL protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use.
  • human GITRL will preferably be used.
  • Human GITRL corresponds to Entrez Gene ID 8995, and the complete amino acid sequences of the longest isoform of human GITRL may be found under GenBank Accession No. NP_005083.3 (version of February 20, 2022). TJ
  • T1A tumor necrosis factor superfamily member 15
  • TNFSF15 tumor necrosis factor superfamily member 15
  • APCs such as dendritic cells and macrophages
  • DR3 receptor 3
  • TNFRSF25 a stimulatory receptor expressed by T cells
  • TL1A is thus a TNFSF member involved in immune cell activation. This cytokine is also found to inhibit endothelial cell proliferation, and thus may function as an angiogenesis inhibitor.
  • Its receptor DR3 is a category I receptor of the TNFRSF.
  • TL1A Being a member of the TNFSF that is involved in immune cell activation, TL1A is a preferred TNFSF member for the fusion polypeptide according to the invention.
  • the specific TL1A protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use. In particular, for an intended use in humans, human TL1A will preferably be used.
  • Human TL1A corresponds to Entrez Gene ID 9966, and the complete amino acid sequences of the longest isoform of human TL1A may be found under GenBank Accession No. NP_005109.2 (version of February 27, 2022).
  • LIGHT LIGHT
  • CD258 tumor necrosis factor superfamily member 14
  • TNFSF14 Tumor necrosis factor superfamily member 14
  • HVEM Herpes Virus Entry Mediator
  • LTBR Lymphotoxin-B Receptor
  • LIGHT-HVEM interaction is responsible for a majority of the immune-stimulating properties of LIGHT. Expressed on lymphocytes, NK cells, smooth muscle, and epithelium, HVEM serves as an important T cell costimulatory agent leading to activation, proliferation, and survival.
  • LIGHT -LTBR signaling has a wide range of roles that span from influencing cancer cells' susceptibility to immune responses, functioning to repair chaotic tumor vasculature, and to supporting effector cells cell trafficking to and infiltration into tumors (Skeate Joseph G. et al. TNFSF14: LIGHTing the Way for Effective Cancer Immunotherapy. TNFSF14: LIGHTing the Way for Effective Cancer Immunotherapy. May 2020. Vol. 11. Article 922).
  • LTBR is a category I receptor of the TNFRSF.
  • HVEM is a category I receptor of the TNFRSF.
  • the specific LIGHT protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use. In particular, for an intended use in humans, human LIGHT will preferably be used. Human LIGHT corresponds to Entrez Gene ID 8740, and the complete amino acid sequences of the longest isoform of human LIGHT may be found under GenBank Accession No. NP_003798.2 (version of February 20, 2022).
  • RTKL RANK L
  • RANK ligand receptor activator of nuclear factor-KB ligand
  • CD254 receptor necrosis factor superfamily member 11
  • TNFSF11 a ligand for osteoprotegerin that functions as a key factor for osteoclast differentiation and activation.
  • This protein was also shown to be a dentritic cell survival factor and to be involved in the regulation of T cell-dependent immune response.
  • Its receptor RANK (or ““receptor activator of nuclear factor-KB”) is a category I receptor of the TNFRSF.
  • the specific RANKL protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use.
  • human RANKL will preferably be used.
  • Human RANKL corresponds to Entrez Gene ID 8600, and the complete amino acid sequences of the longest isoform of human RANKL may be found under GenBank Accession No. NP_003692.1 (version of February 27, 2022).
  • TWEAK tumor necrosis factor superfamily member 12
  • TNFSF12 tumor necrosis factor superfamily member 12
  • TWEAKR Its receptor FN14
  • TWEAK is a category II receptor of the TNFRSF. Being a member of the TNFSF that binds to a category II receptor of the TNFRSF, TWEAK is a preferred TNFSF member for the fusion polypeptide according to the invention.
  • the specific TWEAK protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use.
  • human TWEAK will preferably be used.
  • Human TWEAK corresponds to Entrez Gene ID 8742, and the complete amino acid sequence of the longest isoform of human TWEAK may be found under GenBank Accession No. NP_003800.1 (version of February 20, 2022).
  • FASL Fatty acid
  • FAS ligand Fatty acid
  • FASLG Fatty acid styrene glycoprotein
  • CD178 CD95 ligand
  • CD95L tumor necrosis factor superfamily member 6
  • TNFSF6 TNFSF6
  • the FAS/FASLG signaling pathway is essential for immune system regulation, including activation-induced cell death (AICD) of T cells and cytotoxic T lymphocyte induced cell death.
  • AICD activation-induced cell death
  • FAS is a category II receptor of the TNFRSF.
  • FASL Being a member of the TNFSF that binds to a category II receptor of the TNFRSF, FASL is a preferred TNFSF member for the fusion polypeptide according to the invention.
  • the specific FASL protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use.
  • human FASL will preferably be used.
  • Human FASL corresponds to Entrez Gene ID 356, and the complete amino acid sequence of the longest isoform of human FASL may be found under GenBank Accession No. NP_000630.1 (version of February 27, 2022).
  • TRAIL tumor necrosis factor superfamily member 10
  • TNFSF10 Tumor necrosis factor superfamily member 10
  • TRAIL binds to several members of TNF receptor superfamily including TNFRSF10A/TRAILR1 , TNFRSF10B/TRAILR2, TNFRSF10C/TRAILR3, TNFRSF10D/TRAILR4, and possibly also to TNFRSF11 B/OPG.
  • TRAIL may be modulated by binding to the decoy receptors TNFRSF10C/TRAILR3, TNFRSF10D/TRAILR4, and TNFRSF11 B/OPG that cannot induce apoptosis.
  • the binding of TRAIL to its receptors has been shown to trigger the activation of MAPK8/JNK, caspase 8, and caspase 3.
  • Both of TRAILR1 and TRAILR2 are category II receptors of the TNFRSF.
  • TRAIL Being a member of the TNFSF that binds to category II receptors of the TNFRSF, TRAIL is a preferred TNFSF member for the fusion polypeptide according to the invention.
  • the specific TRAIL protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use.
  • human TRAIL will preferably be used.
  • Human TRAIL corresponds to Entrez Gene ID 8743, and the complete amino acid sequence of the longest isoform of human TRAIL may be found under GenBank Accession No NP_003801.1 (version of March 17, 2022).
  • TNF tumor necrosis factor
  • TNFA tumor necrosis factor
  • TNFo tumor necrosis factor superfamily member 2
  • TNFSF2 a multifunctional proi inflammatory cytokine involved in the regulation of a wide spectrum of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation.
  • TNF binds to two receptors of the TNFRSF: TNFR1 (also known as “TNFRSF1 A”) and TNFR2 (akso known as “TNFRSF1 B” or “TNFBR”).
  • TNFR1 is a category I receptor of the TNFRSF
  • TNFR2 is a category II receptor of the TNFRSF.
  • TNF Being a member of the TNFSF that binds to a category II receptor of the TNFRSF, TNF is a preferred TNFSF member for the fusion polypeptide according to the invention.
  • the specific TNF protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use.
  • human TNF will preferably be used.
  • Human TNF corresponds to Entrez Gene ID 7124, and the complete amino acid sequence of human TNF may be found under GenBank Accession No NP_000585.2 (version of March 17, 2022).
  • LTa LTa
  • TNFB TNFB
  • TNFSF1 tumor necrosis factor superfamily member 1
  • lymphocytes that is highly inducible, secreted, and forms heterotrimers with lymphotoxin-beta which anchor lymphotoxin-alpha to the cell surface.
  • This protein also mediates a large variety of inflammatory, immunostimulatory, and antiviral responses, is involved in the formation of secondary lymphoid organs during development and plays a role in apoptosis.
  • LTa binds to TNFR1 (also known as “TNFRSF1A”), which is a category I receptor of the TNFRSF.
  • TNFRSF1A also known as “TNFRSF1A”
  • the specific LTa protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use.
  • human LTa will preferably be used.
  • Human LTa corresponds to Entrez Gene ID 4049, and the complete amino acid sequence of human LTa may be found under GenBank Accession No NP_001153212.1 (version of March 10, 2022).
  • LTB lymphotoxin-alpha 1 /beta 2 complex
  • LTaBz lymphotoxin-beta receptor
  • LTBR lymphotoxin-beta receptor
  • LTB is an inducer of the inflammatory response system and involved in normal development of lymphoid tissue.
  • the specific LTB protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use.
  • human LTB will preferably be used.
  • Human LTB corresponds to Entrez Gene ID 4050, and the complete amino acid sequence of human LTB may be found under GenBank Accession No NP_002332.1 (version of February 20, 2021 ).
  • fusion polypeptide according to the invention may comprise an entire wild-type member of the TNFSF, it may also alternatively comprise a functional fragment thereof or a functional derivative thereof.
  • a “functional fragment” of a member of the TNFSF it is meant a fragment (i.e. a part of the amino acid sequence of the entire member of the TNFSF) that retains the function of the entire member of the TNFSF, i.e. its ability to trimerize and bind to and activate its TNFRSF receptor(s).
  • TNFSF members exist as transmembrane proteins, but their transmembrane and intracellular regions are not needed for trimerization and binding to and activation of their TNFRSF receptor (s).
  • the TNFSF member fragment comprised in the fusion polypeptide according to the invention is an extracellular fragment of the TNFSF member, i.e. a fragment devoid of the transmembrane and intracellular part of the TNFSF member.
  • Whole or part of the extracellular domain may be comprised in the extracellular fragment, provided that the fragment retains its ability to trimerize and bind to and activate its TNFRSF receptor(s).
  • Suitable extracellular fragments of TNFSF members are described in Table 4 below.
  • TNFSF members their Entre Gene ID, reference amino acid sequence, and positions of extracellular domain and other functional fragments.
  • a “functional derivative” of a member of the TNFSF it is meant a polypeptide comprising a member of the TNFSF or a functional fragment thereof with one or more insertions, deletions, truncations and/or substitutions, which retains the function of the entire member of the TNFSF, i.e. its ability to trimerize and bind to and activate its TNFRSF receptor(s).
  • the substituted original amino acid(s) is (are) preferably replaced by equivalent amino acid(s), as disclosed above in Table 3.
  • fusion 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.
  • 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.
  • the sdAb specifically binding to PD- L1 may be fused to the member of the TNFSF or functional fragment or derivative thereof directly or indirectly through a peptide linker.
  • the sdAb specifically binding to PD-L1 is said to be fused “directly” to the member of the TNFSF or functional fragment or derivative thereof if there is no additional amino acid residue between the two fusion partners (the sdAb specifically binding to PD-L1 and the member of the TNFSF or functional fragment or derivative thereof).
  • the sdAb specifically binding to PD-L1 is said to be fused “indirectly through a peptide linker” to the member of the TNFSF or functional fragment or derivative thereof if there is there is a peptide linker comprising, 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 member of the TNFSF or functional fragment or derivative thereof).
  • the sdAb specifically binding to PD-L1 and the member of the TNFSF or functional fragment or derivative thereof are fused indirectly through a peptide linker.
  • 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.
  • 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, 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.
  • 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 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.
  • the two fusion partners i.e. the sdAb specifically binding to PD-L1 and the member of the TNFSF or functional fragment or derivative thereof
  • the sdAb specifically binding to PD-L1 is in N-terminal of the member of the TNFSF. In another embodiment, the sdAb specifically binding to PD-L1 is in C-terminal of the member of the TNFSF.
  • 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 member of the TNFSF or functional fragment or derivative thereof) and the optional peptide linker in between, further elements that may be useful for the production or therapeutic use of the fusion polypeptide.
  • the two fusion partners i.e. the sdAb specifically binding to PD-L1 and the member of the TNFSF or functional fragment or derivative thereof
  • the optional peptide linker in between, further elements that may be useful for the production or therapeutic use of the fusion polypeptide.
  • the fusion polypeptide according to the invention may further comprises: a) a signal peptide in N-terminal; and/or b) a tag peptide, preferably in C-terminal.
  • 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.
  • 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.
  • ER endoplasmic reticulum
  • Appropriate signal peptides are known in the art.
  • telomeres 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, 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-P rot Accession Number P01750).
  • 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.
  • 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).
  • 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
  • the tag peptide may be independently positioned at the N-terminus of the neopeptide or fusion thereof (tag- polypeptide) or alternatively at its C-terminus (polypeptide-tag) or alternatively internally.
  • a tag peptide may preferably be inserted at the C-terminus of the fusion polypeptide according to the invention.
  • Preferred fusion polypeptides according to the invention may notably comprise, consist essentially of or consist of the following formulas, from N-terminal (on the left) to C- terminal (on the right): a) anti-PD-L1 sdAb-TNFSF member, b) TNFSF member-anti-PD-L1 sdAb, c) anti-PD-L1 sdAb-peptide linker-TNFSF member, d) TNFSF member-peptide linker-anti-PD-L1 sdAb, e) signal peptide-anti-PD-L1 sdAb-TNFSF member, f) signal peptide-TNFSF member-anti-PD-L1 sdAb, g) signal peptide-anti-PD-L1 sdAb-peptide linker-TNFSF member, h) signal peptide-TNFSF member-peptide linker-anti-PD-L1 sdAb, i) anti-PD-L1 sdAb
  • the fusion polypeptide according to the invention comprises, consists essentially of or consists, from N-terminal (on the left) to C-terminal (on the right) of one of the above -described formulas e) to h) and m) to p), which all contain a signal peptide in N-terminal.
  • the fusion polypeptide according to the invention comprises, consists essentially of or consists, from N-terminal (on the left) to C-terminal (on the right), of:
  • the fusion polypeptide according to the invention comprises, consists essentially of or consists, from N-terminal (on the left) to C-terminal (on the right) of one of the above-described formulas g) or p), which contain a signal peptide in N-terminal, followed by the anti-PD-L1 sdAb, a peptide linker, the TNFSF member in C-terminal, and optionally a tag peptide in C-terminal.
  • the anti-PD-L1 sdAb and TNFSF member may be selected from any preferred embodiment disclosed above.
  • each of FP1 -Emb1 to FP1 - Emb8 (preferably humanized) and FP1 -Emb9 to FP1 -Emb11 may be combined with each of FP2-Emb1 to FP2-Emb10 (using the entire TNFSF member or a functional fragment thereof, preferably an extracellular functional fragment thereof).
  • the signal peptide when present, preferably comprises, consists essentially of or consists of amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO:24).
  • the tag peptide when present, preferably comprises, consists essentially of or consists of amino acid sequence GDYKDDDK (SEQ ID NO: 26).
  • the peptide linker when present, preferably comprises, consists essentially of or consists of amino acid sequence GGGSGGGSGGGS (SEQ ID NO: 22).
  • Particularly preferred fusion polypeptides according to the invention comprise, consist essentially of or consist of an amino acid sequence with at least 80% 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:31 which corresponds to an amino acid sequence consisting of, from N-terminal to C-terminal: signal peptide MGWSCIILFLVATATGVHS (SEQ ID NO:24), the entire amino acid sequence of the humanized version of the sdAb produced by clone 32.1A1 (SEQ ID NO: 13), peptide linker GGGSGGGSGGGS (SEQ ID NO: 22), and amino acids 1 19-261 of human CD40L amino acid sequence NP_000065.1 , as described below:
  • SEQ ID NO:32 which corresponds to an amino acid sequence consisting of, from N-terminal to C-terminal: signal peptide MGWSCIILFLVATATGVHS (SEQ ID NO:26), the entire amino acid sequence of the humanized version of the sdAb produced by clone 32.1A1 (SEQ ID NO: 13), and amino acids 119-261 of human CD40L amino acid sequence NP_000065.1 , as described below:
  • SEQ ID NO:33 which corresponds to an amino acid sequence consisting of, from N-terminal to C-terminal: signal peptide MGWSCIILFLVATATGVHS (SEQ ID NO:24), the entire amino acid sequence of the humanized version of the sdAb produced by clone 32.1A1 (SEQ ID NO: 13), peptide linker GGGSGGGSGGGS (SEQ ID NO: 22), and amino acids 80-254 of human 4-1 BBL amino acid sequence NP_003802.1 , as described below:
  • SEQ ID NO:34 which corresponds to an amino acid sequence consisting of, from N-terminal to C-terminal: signal peptide MGWSCIILFLVATATGVHS (SEQ ID NO:26), the entire amino acid sequence of the humanized version of the sdAb produced by clone 32.1A1 (SEQ ID NO: 13), and amino acids 80-254 of human 4-1 BBL amino acid sequence NP_003802.1 , as described below:
  • signal peptide Underligned: signal peptide; bold: anti-PD-L1 sdAb; normal: amino acids 80-254 of human 4-1 BBL; italic: FLAG tag peptide.
  • the fusion polypeptide according to the invention is selected from SEQ ID NO: 31 to SEQ ID NO: 34, more preferably the fusion polypeptide consists of SEQ ID NO: 31 or SEQ ID NO:33.
  • Nucleic acid molecules are selected from SEQ ID NO: 31 to SEQ ID NO: 34, more preferably the fusion polypeptide consists of SEQ ID NO: 31 or SEQ ID NO:33.
  • the present invention also relates to a nucleic acid molecule encoding the fusion polypeptide according to the invention, in any embodiment disclosed above.
  • a nucleic acid molecule according to the invention comprises distinct parts, each encoding one of the components of the fusion polypeptide according to the invention, i.e. fusion partners 1 and 2 (anti-PD-L1 sdAb and TNFSF member), and optionally one or more of a signal peptide, a peptide linker, and a tag peptide.
  • the nucleic acid sequence may be a native (or natural) nucleic acid sequence (e.g. the native nucleic acid sequence encoding amino acids 119-261 of human CD40L amino acid sequence NP_000065.1 ) or may be modified by man to include substitution, deletion, addition and/or insertion of one or more nucleotide(s).
  • the present invention encompasses any modifications aimed to improve cloning and/or expression of the encoded and fusion polypeptide according to the invention as well as its folding, stability and activity. When several modifications are contemplated, they can concern consecutive and/or non-consecutive nucleotide residues.
  • the modification(s) contemplated by the present invention encompass silent modifications that do not change the amino acid sequence of the encoded fusion polypeptide according to the invention, as well as modifications that are translated into the encoded fusion polypeptide (provided that the fusion partners retain their activity).
  • Representative examples of modifications include but are not limited to introduction of appropriate restriction sites, sequence degeneration (e.g. to reduce sequence homology between various parts of the complete nucleic acid molecule) and/or optimisation of nucleotide sequence (e.g. to optimize translation in a given host cell), and/or suppression of potentially negative elements (which are expected to negatively influence expression levels).
  • codon usage for ensuring high level of expression of the encoded gene product in a particular host cell or subject. It has been indeed observed that, when more than one codon is available to code for a given amino acid, the codon usage patterns of organisms are highly non-random and the utilisation of codons may be markedly different between different hosts.
  • codon optimisation is performed by replacing one or more “native” codon corresponding to a codon infrequently used in the host cell of interest 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. Moreover, some deviations from strict adherence to optimised codon usage may be made to accommodate the introduction of restriction site(s) into the resulting nucleic acid molecule.
  • nucleic acid molecules of the present invention can be generated using sequence data accessible in the art and the sequence information provided herein. For example, they may be isolated using routine techniques well known in the art, e.g. by PCR isolation and/or cloning by conventional molecular biology from an appropriate natural source, cDNA and genomic libraries or any prior art vector known to include it. Alternatively, the nucleic acid molecules of the invention can also be generated by chemical synthesis in automatised process (e.g. assembled from overlapping synthetic oligonucleotides). Preferred nucleic acid molecules according to the invention are selected from:
  • the present invention also relates to a vector comprising the nucleic acid molecule according to the invention, in any embodiment disclosed above.
  • vector refers to a vehicle, preferably a nucleic acid molecule or a viral particle that contains the elements necessary to allow delivery, propagation and/or expression of any of the nucleic acid molecule(s) described herein within a host cell or subject.
  • This term encompasses vectors for maintenance (cloning vectors) or vectors for expression in various host cells or subjects (expression vectors), extrachromosomal vectors (e.g. multicopy plasmids) or integration vectors (e.g. designed to integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates) as well as shuttle vectors (e.g.
  • the vectors may be of naturally occurring genetic sources, synthetic or artificial, or some combination of natural and artificial genetic elements.
  • vector has to be understood broadly as including mRNA, plasmid and viral vectors.
  • Vectors which are appropriate in the context of the present invention, include, without limitation, bacteriophage, plasmid or cosmid vectors for expression in prokaryotic host cells such as bacteria (e.g. E. coli, BCG or Listeria); vectors for expression in yeast (e.g. Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris); baculovirus vectors for expression in insect cell systems (e.g. Sf 9 cells); as well as plasmid and viral vectors for expression in higher eukaryotic cells or subjects.
  • bacteria e.g. E. coli, BCG or Listeria
  • yeast e.g. Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris
  • baculovirus vectors for expression in insect cell systems (e.g. Sf 9 cells); as well as
  • vectors are commercially available (e.g. in Invitrogen, Stratagene, Amersham Biosciences, Promega, etc.) or available from depositary institutions such as the American Type Culture Collection (ATCC, Rockville, Md.) or have been the subject of numerous publications describing their sequence, organization and methods of producing, allowing the artisan to apply them.
  • the present invention also encompasses vectors (e.g. plasmid DNA and mRNA) complexed to lipids or polymers to form particulate structures such as liposomes, lipoplexes or nanoparticles.
  • Plasmid vector refers to a replicable DNA construct.
  • plasmid vectors contain selectable marker genes that allow host cells carrying the plasmid vector to be selected for or against in the presence of a corresponding selective drug.
  • selectable marker genes A variety of positive and negative selectable marker genes are known in the art.
  • an antibiotic resistance gene can be used as a positive selectable marker gene that allows a host cell to be selected in the presence of the corresponding antibiotic.
  • suitable plasmid vectors include, without limitation, pREP4, pCEP4 (Invitrogen), pCI (Promega), pVAX (Invitrogen) and pGWiz (Gene Therapy System Inc).
  • viral vector 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.
  • virus 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.
  • viral viral
  • viral particles viral particles
  • 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.
  • 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.
  • 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 replicationdefective or replication-im
  • viral vectors are generated from a variety of different viruses (e.g. poxviruses, adenoviruses, herpes viruses, paramyxoviruses, and rhabdovi ruses, lentiviruses etc).
  • viruses e.g. poxviruses, adenoviruses, herpes viruses, paramyxoviruses, and rhabdovi ruses, lentiviruses etc.
  • the term "viral vector” encompasses vector DNA, genomic DNA as well as viral particles generated thereof, and especially infectious viral particles.
  • the viral vector is thus in the form of infectious viral particles.
  • Poxviruses are a broad family of DNA viruses containing a double-stranded genome. Like most viruses, poxviruses have developed self-defense 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-apoptotic proteins, inhibitors of the NFkB pathway and interferon signaling, and down-regulators of the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • 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 a laboratory, compared to the native form) to comprise a nucleic acid molecule inserted in its genome and encoding the fusion polypeptide according to the invention.
  • the poxvirus of the invention may further comprise one or more additional modifications such as those described herein.
  • the poxvirus is a poxvirus of the Chordopoxvirinae family, preferably selected from the group consisting of Avipoxvirus genus (including Canarypoxvirus (e.g. AL VAC) and Fowlpoxvirus (e.g.
  • Capripoxvirus genus 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.
  • poxvirus chimeras or « chimeras of poxviruses” refers to viruses obtained by homologous recombination between several distinct strains of poxviruses.
  • 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.
  • the poxvirus 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.
  • W vaccinia virus
  • CPXV cowpox
  • RCN raccoonpox
  • rabbitpox Monkeypox
  • Horsepox Horsepox
  • Volepox Volepox
  • Skunkpox Skunkpox
  • variola virus or smallpox
  • Camelpox Camelpox
  • Orthopoxvirus chimeras correspond to chimeras of several distinct strains of Orthopoxvirues.
  • Genbank Sequences of the genome of the various poxviruses, are available in the art and specialized databases such as Genbank.
  • Genbank accession number NC_006998, NC_003663, NC_005309, NC_004105, NC_001132 respectively.
  • the poxvirus of the invention belongs to the Orthopoxvirus genus and even more preferably to the vaccinia virus (VV) species.
  • VV vaccinia virus
  • 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), Elstree, Wyeth, Lister, LIVP, Tashkent, Tian Tan, Brighton, Ankara, LC16M8, LC16M0 strains, etc., and any derivative thereof.
  • MVA Modified vaccinia virus Ankara
  • NYVAC Copenhagen
  • Copenhagen Cop
  • Western Reserve WR
  • Elstree Wyeth
  • Lister LIVP
  • Tashkent Tian Tan, Brighton
  • Ankara Ankara
  • LC16M8 LC16M0 strains etc.
  • 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
  • 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). Oncolytic viral vectors
  • the viral vector is an oncolytic viral vector.
  • an oncolytic virus refers to a virus capable of selectively replicating in dividing cells, and more particularly in cancer cells, 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 cells.
  • an oncolytic virus 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).
  • infectious i.e. capable of infecting and entering into a host cell or subject.
  • this term encompasses DNA or RNA vector (depending on the virus in question) as well as viral particles generated thereof.
  • Suitable oncolytic viral vectors may be selected from poxviruses, adenoviruses, herpes viruses, paramyxoviruses, and rhabdoviruses (see Kaufman, H., Kohlhapp, F. & Zloza, A. Oncolytic viruses: a new class of immunotherapy drugs. Nat Rev Drug Discov 14, 642-662 (2015)).
  • the oncolytic virus of the present invention is obtained from a rhabdovirus, such as a vesicular stomatitis virus (VSV).
  • a rhabdovirus such as a vesicular stomatitis virus (VSV).
  • VSV vesicular stomatitis virus
  • the oncolytic virus of the present invention is obtained from a paramyxovirus, such as a Newcastle disease virus, or a morbillivirus (in particular a measles virus).
  • a paramyxovirus such as a Newcastle disease virus, or a morbillivirus (in particular a measles virus).
  • oncolytic Newcastle disease viruses include without limitation the 73-T PV701 and HDV-HUJ strains as well as those described in the literature (e.g. Phuangsab et al., 2001 , Cancer Lett. 172(1 ): 27-36; Lorence et al., 2007, Curr. Cancer Drug Targets 7(2): 157-67; Freeman et al., 2006, Mol. Ther. 13(1 ): 221 -8).
  • oncolytic measles viruses include without limitation MV-Edm (McDonald et al., 2006; Breast Cancer Treat. 99(2): 177-84) and HMWMAA (Kaufmann et al., 2013, J. Invest. Dermatol. 133(4): 1034-42).
  • the oncolytic virus of the present invention is obtained from a herpes virus.
  • the Herpesviridae are a large family of DNA viruses that all share a common structure and are composed of relatively large double-stranded, linear DNA genomes encoding 100-200 genes encapsided within an icosahedral capsid which is enveloped in a lipid bilayer membrane.
  • the oncolytic herpes virus can be derived from different types of HSV, particularly preferred are HSV1 and HSV2.
  • the herpes virus may be genetically modified so as to restrict viral replication in tumors or reduce its cytotoxicity in non-dividing cells.
  • any viral gene involved in nucleic acid metabolism may be inactivated, such as thymidine kinase (Martuza et al., 1991 , Science 252: 854-6), ribonucleotide reductase (RR) (Boviatsis et al., Gene Ther. 1 : 323-31 ; Mineta et al., 1994, Cancer Res. 54: 3363-66), or uracil-N-glycosylase (Pyles et al., 1994, J. Virol. 68: 4963- 72).
  • thymidine kinase Martuza et al., 1991 , Science 252: 854-6
  • RR ribonucleotide reductase
  • RR ribonucleotide reductase
  • uracil-N-glycosylase Pryles et al., 1994, J. Virol. 68: 4963- 72.
  • Another aspect involves viral mutants with defects in the function of genes encoding virulence factors such as the ICP34.5 gene (Chambers et al., 1995, Proc. Natl. Acad. Sci. USA 92: 1411 -5).
  • Representative examples of oncolytic herpes virus include NV1020 (e.g. Geevarghese et al., 2010, Hum. Gene Ther. 21 (9): 1119-28) and T-VEC (Andtbacka et al., 2013, J. Clin. Oncol. 31 , abstract number LBA9008).
  • the oncolytic virus of the present invention is obtained from an adenovirus.
  • Methods are available in the art to engineer oncolytic adenoviruses.
  • An advantageous strategy includes the replacement of viral promoters with tumor-selective promoters or modifications of the E1 adenoviral gene product(s) to inactivate its/their binding function with p53 or retinoblastoma (Rb) protein that are altered in tumor cells.
  • the adenovirus E1B55kDa gene cooperates with another adenoviral product to inactivate p53 (p53 is frequently dysregulated in cancer cells), thus preventing apoptosis.
  • oncolytic adenovirus examples include ONYX-015 (e.g. Khuri et al., 2000, Nat. Med 6(8): 879-85) and H101 also named Oncorine (Xia et al., 2004, Ai Zheng 23(12): 1666-70).
  • the oncolytic virus of the present invention is an oncolytic poxvirus.
  • the oncolytic poxvirus 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.
  • the oncolytic poxvirus of the invention belongs to the vaccinia virus (VV) species.
  • VV vaccinia virus
  • 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, LIVP, 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.
  • modifications aimed at improving safety (e.g. increased attenuation) and/or efficacy, and/or tropism of the resulting virus.
  • J2R thymidine kinase
  • 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 AAA48082.1 ).
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • a modified rr- poxvirus is desirably oncolytic.
  • modified poxvirus further modified in the J2R and in the I4L and/or 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).
  • tk- rr- poxvirus is desirably oncolytic.
  • 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).
  • 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).
  • 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.
  • 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).
  • 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.
  • a modified poxvirus modified in the M2L locus, in the J2R locus, and in the I4L and/or 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).
  • m2-, tk- rr- poxvirus is desirably oncolytic.
  • 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, 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 cancer as described herein.
  • 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).
  • Preferred modifications include:
  • 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, LIVP, Tashkent, Tian Tan, Brighton, Ankara, LC16M8, and LC16M0 strains, which preferably comprises:
  • the viral vector may not be oncolytic, but instead be a non- propagative viral vector.
  • non-propagative viral vector refers to viral vectors that are unable to propagate in host cells or tissues. These viral vectors can be replication-defective or replication-impaired vectors (e.g. viral vector genetically disabled), 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 viral vector can be rendered replication-defective by partial or total deletion or inactivation of regions critical to viral replication. Such replication-defective or impaired viral vectors typically require for propagation, permissive cell lines which bring up or complement the missing/impaired functions.
  • viral vectors 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 viral vectors 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.
  • Suitable replication-defective or replication-impaired viral vectors may be selected from poxviruses, adenoviruses, herpes viruses, paramyxoviruses (including measles virus), rhabdoviruses (such as VSV or Maraba virus), and viral like particles.
  • viral like particles 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.
  • 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 polypeptide having an IL-7 activity as described hereinafter.
  • a particularly suitable non-propagative viral vector for use in the invention is obtained from a poxvirus.
  • poxvirus refers to a virus belonging to the Poxviridae family with a preference for the Chordopoxvirinae subfamily 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).
  • the non-propagative viral vectors for use in the invention belong to the Orthopoxvirus genus and even more preferably to the vaccinia virus (VV) species.
  • VV vaccinia virus
  • 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.
  • MVA Modified vaccinia virus Ankara
  • NYVAC Copenhagen
  • Copenhagen Cop
  • Western Reserve WR
  • Wyeth Lister
  • LIVP Tashkent Tian Tan, Brighton
  • Ankara Ankara
  • LC16M8 LC16M0 strains etc.
  • 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.
  • J2R thymidine kinase
  • F2L deoxyuridine triphosphatase
  • A56R the viral
  • a particularly appropriate non-propagative viral vector 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 ).
  • 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).
  • NYVAC is a particularly appropriate non-propagative viral vector for use in the context of the present invention.
  • 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.
  • ORFs open reading frames
  • Still another suitable non-propagative viral vector 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.
  • adenovirus preferably originating from a human or an animal adenovirus (e.g. canine, ovine, simian, etc.). Any serotype can be employed.
  • the adenoviral vector originates from a human adenovirus, or from a chimpanzee adenovirus.
  • Representative examples of chimp Ad include without limitation ChAd3 (Peruzzi et al., 2009, Vaccine 27: 1293), ChAd63 (Dudareva et al., 2009, Vaccine 27: 3501 ), AdC6, AdC7 (Cervasi et al., 2015, J.
  • said non-propagative viral vector for use is a human adenovirus, preferably selected from the group consisting of species A, B, C, D, E, F and G, with a preference for species B, C and D.
  • Said human adenovirus is preferably selected from the group consisting of serotypes 1 , 2, 3, 5, 6, 7, 8, 9, 10, 11 , 13, 14, 15, 16, 17, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 53, 54, 55, 56 and 57, and is more preferably from the group consisting of serotypes 5, 11 , 26 and 35.
  • Replication-defective adenoviruses can be obtained as described in the art, e.g. by deletion of at least a region of the adenoviral genome or portion thereof essential to the viral replication, with a specific preference for partial or total deletion of the E1 region (E1A and/or E1 B) comprising E1 coding sequences.
  • the present invention also encompasses viruses having additional deletion(s)/modification(s) within the adenoviral genome (e.g. all or part of regions, like non-essential regions as E3 region, or essential regions as E2 and E4, as described in Lusky et al., 1998, J.
  • the non-propagative viral vector for use in the invention is a human adenovirus 5 which is defective for E1 function (e.g. with a deletion extending from approximately positions 459 to 3510 or 455 to 3512 by reference to the sequence of the human Ad5 disclosed in the GenBank under the accession number M_73260 and in Chroboczek et al. (1992, Virol. 186:280) and further deleted within the E3 region (e.g. with a deletion extending from approximately positions 28591 to 30469 by reference to the same Ad5 sequence).
  • non-propagative viral vectors suitable in the context of the invention are paramyxoviruses, in particular morbilliviruses, with a specific preference for measles virus.
  • paramyxoviruses in particular morbilliviruses
  • measles virus a specific preference for measles virus.
  • attenuated strains are available in the art (Brandler et al, 2008, CIMID, 31 : 271 ; Singh et al., 1999, J. virol.
  • the vector according to the invention may encode only the fusion polypeptide of the invention, it may also further comprise another nucleic acid molecule inserted in its genome encoding a therapeutic polypeptide.
  • 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 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.
  • suicide gene refers to a gene coding for a protein able to convert a precursor of a drug 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 genes and corresponding precursors of a drug comprising one nucleobase moiety are disclosed in the following Table 6.
  • the suicide gene encodes a protein having at least a CDase activity.
  • 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 highly cytotoxic 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).
  • FCY1 gene Saccharomyces cerevisiae
  • FCA1 gene Candida Albicans
  • FCA1 gene Candida Albicans
  • codA gene Escherichia coli
  • 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.
  • the oncolytic virus of the invention carries in its viral genome a suicide gene encoding a polypeptide having uracil phosphoribosyl transferase (UPRTase) activity.
  • UPRTase uracil phosphoribosyl transferase
  • prokaryotes and lower eukaryotes uracil is transformed into UMP by the action of UPRTase.
  • This enzyme converts 5-FU into 5-FUMP.
  • 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.
  • the suicide gene inserted in the viral genome of the oncolytic virus of the present invention encodes a polypeptide having CDase and UPRTase activities.
  • 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.
  • 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).
  • immunomodulatory polypeptide refers to a polypeptide targeting a component of a signalling 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.
  • 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.
  • immunomodulatory polypeptides include, without limitation:
  • cytokines such as interleukins, chemokines, interferons, tumor necrosis factors, colony-stimulating factors
  • 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
  • TNFo tumor necrosis factors
  • colony-stimulating factors e.g. granulocyte macrophage colony stimulating factor (GM-CSF)
  • GM-CSF granulocyte macrophage colony stimulating factor
  • the immunostimulatory polypeptide is a cytokine, it is preferably an interleukin or a colony-stimulating factor, with a specific preference for GM-CSF.
  • 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.
  • 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
  • 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.
  • 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
  • LAG3 e.g. W02018/071500
  • Tim3 e.g. W02020/093023
  • BTLA e.g. W02010/106051
  • 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 polypeptides 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 S
  • antigenic refers to the ability to induce or stimulate a measurable immune response in a subject into which the vector according to the invention encoding the polypeptide qualified as antigenic has been introduced.
  • the stimulated or induced immune response against the antigenic polypeptide expressed by said vector according to the invention 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).
  • 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 multiparameters 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 [ 3 H] 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.
  • 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.
  • the antigenic polypeptide(s) encoded by the vector according to the invention 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.
  • oncogenes e.g. activated ras oncogene
  • proto-oncogenes e.g. ErbB family
  • tumor-associated 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.
  • CRC colorectal associated antigen
  • CEA Carci noembryonic Antigen
  • PSA Prostate Specific Antigen
  • BAGE GAGE or MAGE antigen family
  • p53 mucin antigens
  • HER2/neu e.g. MUC1
  • HER2/neu e.g. HER2/neu
  • p21 ras HER
  • 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.
  • 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).
  • pathogenic organisms e.g. human papillomavirus (HPV), hepatitis C virus (HCV), hepatitis B virus (HBV), Epstein Barr virus (EBV), etc
  • HPV human papillomavirus
  • HCV hepatitis C virus
  • HBV hepatitis B virus
  • EBV Epstein Barr virus
  • bacteria e.g. Helicobacter pilori
  • the antigenic polypeptide(s) encoded by the vector according to 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.
  • vaccinal 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 measles virus and rabbies virus.
  • HIV antigens include gp120 gp40, gp160, p24, gag, pol, env, vif, vpr, vpu, tat, rev, nef tat, nef.
  • 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.
  • cytomegalovirus antigens include gB.
  • derived from Epstein Barr virus (EBV) include gp350.
  • Varicella Zoster Virus antigens include gp1 , 11 , 111 and IE63.
  • hepatitis C virus antigens includes env E1 or E2 protein, core protein, NS2, NS3, NS4a, NS4b, NS5a, NS5b, p7.
  • 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.
  • 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.
  • any antibody or antigen-binding fragment or derivative thereof with therapeutic activity may further be encoded by the vector 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.
  • anti HER2 antibodies e.g. trastuzumab
  • anti-EGFR antibodies e.g. cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab
  • anti-VEGF antibodies e.g
  • antibody in the broadest sense and encompasses naturally occurring antibodies and those engineered by man; 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 (e.g. rodent or camelid antibody) or chimeric.
  • a nonhuman antibody can be humanized by recombinant methods to reduce its immunogenicity in man. The antibody may derive from any of the well-known isotypes (e.g.
  • 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.
  • full length antibodies other than those from camelids or cartilaginous fishes 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 (eventually 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.
  • CDR regions of the heavy and light chains are determinant for the binding specificity and their position in the complete heavy or light chain sequence is determined according to various numbering disclosed previously with respect to sdAb.
  • 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).
  • 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 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. Regulatory elements
  • the vector 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.
  • 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.
  • the vector 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.
  • 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).
  • 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 pK1L promoters, synthetic promoters such as those described in Chakrabarti et al.
  • CMV cytomegalovirus
  • RSV Rous sarcoma Virus
  • MLP adenovirus major late
  • PGK phosphoglycero kinase
  • CMV promoters whose transcriptional activity is regulated by the presence or absence of alcohol, tetracycline, steroids, metal, sugar, etc.).
  • CMV promoter is particularly appropriate for use in adenoviral vectors (e.g. Ad5, Ad11 , Ad26, Ad35).
  • the vector may contain one or more promoters depending on the number of nucleic acid molecule(s) to be expressed.
  • each of the encoding nucleic acid molecule is placed under the control of independent promoters.
  • the nucleic acid molecule encoding the fusion polypeptide according to the invention is placed under the control of a promoter selected from pH5R.
  • 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.
  • transcription termination sequences 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-
  • an initiator Met tripartite leader sequences, IRES ribosome binding sites, signal peptides, etc.
  • targeting sequences e.g. linkers composed of flexible residues like glycine and serine
  • linkers e.g. linkers composed of flexible residues like glycine and serine
  • transport sequences secretion signal
  • 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.
  • nucleic acid molecule(s) or expression cassette(s) encoding the fusion polypeptide of the invention and optionally another polypeptide is/are inserted into the vector by any appropriate technique known in the art.
  • non-viral vectors in particular plasmid vectors
  • conventional cloning techniques may be used, in particular based on the use of restriction enzymes and ligases, using restriction sites present in the plasmid vector of interest and corresponding sequences in the nucleic acid molecule(s)/ expression cassette(s).
  • the nucleic acid molecule(s) /expression cassette(s) may be inserted in any suitable location within the virus 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 an adenoviral vector or a poxviral vector 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).
  • TK thymidine kinase
  • RR Ribonucleotide reductase
  • F2L F2L gene
  • TK thymidine kinase
  • RR Ribonucleotide reductase
  • F2L F2L gene
  • MVA vector MVA vector
  • insertion of the neopeptide-encoding nucleic acid molecule(s) or expression cassette(s) is made within MVA’s deletion III.
  • 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 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.
  • 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 encompasses 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).
  • 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.
  • GFP Green Fluorescent Protein
  • eGFP Enhanced Green Fluorescent Protein
  • AmCyan 1 fluorescent protein and mCherry.
  • 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.
  • sorting means such as FACS after labelling with an APC (Allophycocyanin)-tagged anti-vaccinia virus antibody.
  • APC Allophycocyanin-tagged anti-vaccinia virus antibody
  • 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.
  • an endonuclease e.g. mCherry
  • 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.
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • CRISPR clustered regularly interspaced short palindromic repeats
  • CRISPR/CAS9 restriction enzymes with unique cleavage within the reporter gene.
  • 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.
  • E3 and E4 regions are particularly appropriate for insertion in oncolytic adenovirus virus, and E1 region is particularly appropriate (although insertion in E2 region, E3 region, E4 region or intergenic zones can also be envisaged) for insertion into non-propagative adenoviruses.
  • a viral vector of the invention may be produced/amplified using conventional techniques.
  • the present invention thus also relates to a method for producing the viral vector of the invention, comprising 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.
  • the choice of the producer cell depends on the type of viral vector to be produced, and those skilled in the art know which producer cells or cell lines are suitable for which viral vector.
  • choice of the producer cell also depends on the type of poxviral vector to be produced.
  • 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.
  • primary avian cells such as chicken embryo fibroblasts (CEF) prepared from chicken embryos obtained from fertilized eggs
  • immortalized avian cell lines 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.
  • non-MVA vaccinia viruses are amplified in HeLa cells (see e.g. W02010/130753).
  • suitable cell lines include the 293 cells (Graham et al., 1997, J. Gen. Virol. 36: 59-72) as well as the PER-C6 cells and HER96 (e.g. Fallaux et al., 1998, Human Gene Ther. 9: 1909-1917; W097/00326) or any derivative of these cell lines.
  • any other cell line described in the art can also be used in the context of the present invention, especially any cell line used for producing product for human use such as Vero cells, HeLa cells and avian cells.
  • Such cells may be adapted for expressing the E1 genes lacking to the defective virus.
  • 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.
  • 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.
  • CEFs when 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.
  • MOI multiplicity of infection
  • 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 viral vectors 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 viral vectors.
  • 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.
  • the step of recovery of the produced viral vectors 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 viral vectors of the invention may then be further purified, using purification steps well known in the art.
  • 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).
  • 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.
  • TFF tangential flow filtration
  • the viral vectors 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 relates to a host cell comprising the nucleic acid molecule according to the invention or the vector according to the invention.
  • Host cells comprising a non-viral vector
  • the invention relates to a host cell comprising a non-viral vector according to the invention.
  • Such methods include, but are not limited to microinjection, CaPO 4 - mediated transfection, DEAE-dextran-mediated transfection, electroporation, lipofection/liposome fusion, gene guns, transduction, viral infection as well as direct administration into a host organism via various means.
  • the method may also be used in association with conventional transfection reagents that facilitate introduction of nucleic acids in host cells, such as polycationic polymers (e.g. chitosan, polymethacrylate, PEI, etc) and cationic lipids (e.g.DC-Chol/DOPE, transfectam, lipofectin, etc).
  • 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.
  • the present invention also relates to a method for recombinantly producing the fusion polypeptide according to the invention, comprising the steps of: a) culturing in vitro a host cell according to the invention under conditions suitable for growth of the host cell, b) recovering the cell culture, and c) optionally purifying the produced fusion polypeptide.
  • Host 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 prokaryotic and eukaryotic expression systems available in the art for such purposes.
  • the fusion polypeptide can be recovered from the culture supernatant and/or from the host cell (e.g. upon cell lysis).
  • the recovered materiel can optionally be purified by well- known purification methods including ammonium sulfate precipitation, acid extraction, gel electrophoresis; filtration and chromatographic methods (e.g. reverse phase, size exclusion, ion exchange, affinity, hydrophobic-interaction, hydroxyapatite, high performance liquid chromatography, etc).
  • the conditions and techniques to be used depend on factors such as net charge, molecular weight, hydrophobicity, hydrophilicity and will be apparent to those having skill in the art.
  • the level of purification will depend on the intended use.
  • protein concentration can be evaluated by Bradford assay (Biorad)
  • endotoxin levels can be evaluated by techniques such as the Portable Test System (Charles River Laboratories) and the mass of the purified polypeptides can be measured using MALDI (Matrix- Assisted Laser Desorption/lonisation) or electrospray methods.
  • MALDI Microx- Assisted Laser Desorption/lonisation
  • the present invention also relates to a composition
  • a composition comprising the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention or any combination thereof.
  • the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable vehicle.
  • 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention 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) .
  • 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).
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • freezing e.g. -70° C, - 20°C
  • refrigerated e.g. 4°C
  • a stabilizing formulation adapted to the specific viral vector 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.
  • buffered formulations including NaCl and/or sugar are particularly adapted to the preservation of viruses (e.g.
  • S01 buffer 342,3 g/L saccharose, 10 mM Tris, 1 mM MgCb, 150 mM NaCl, 54 mg/L, Tween 80; ARME buffer: 20 mM Tris, 25 mM NaCl, 2.5% Glycerol (w/v), pH 8.0; 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).
  • the composition comprises a therapeutically effective amount of the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention or any combination thereof.
  • a “therapeutically effective amount” corresponds to the amount of the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention or any combination thereof that is sufficient for producing one or more beneficial results.
  • 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention or any combination thereof may be routinely determined by a practitioner in the light of the relevant circumstances.
  • compositions comprise a viral vector according to the invention
  • individual doses for the viral vector may suitably vary within a range extending from approximately 10 3 to approximately 10 12 vp (viral particles), iu (infectious unit) or pfu (plaque-forming units) depending on the type of viral vector and quantitative technique used.
  • 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).
  • the composition comprises an oncolytic poxvirus vector
  • it preferably comprises 10 3 to 10 12 pfu, more preferably from 10 4 pfu to 10 11 pfu, even more preferably from 10 5 pfu to 10 10 pfu, most preferably from 10 6 pfu to 10 9 pfu of the poxvirus; notably individual doses of approximately 10 6 , 5x10 6 , 10 7 , 5x10 7 , 10 8 or 5x10 8 pfu of the poxvirus vector according to the invent ion.
  • the composition comprises a non-propagative poxviral vector
  • it preferably comprises between approximately 10 6 virus particles (VP), and approximately 10 12 VP, more preferably between approximately 10 7 pfu and approximately 10 11 pfu; even more preferably between approximately 10 8 pfu and approximately 10 10 pfu (e.g. from 5x10 8 to 6x10 9 , from 6x10 8 to 5x10 9 , from 7x10 8 to 4x10 9 , from 8x10 8 to 3x10 9 , from 9x10 8 to 2x10 9 pfu) of the -propagative poxviral vector, these doses being convenient for human use, with a preference for individual doses comprising approximately 10 9 pfu of poxviral vector.
  • the composition comprises an oncolytic adenoviral vector
  • it preferably comprises between approximately 10 6 virus particles (vp), and approximately 10 12 vp.
  • composition comprises a non-propagative adenoviral vector
  • it preferably comprises between approximately 10 6 and approximately 10 14 vp, preferably between approximately 10 7 and approximately 10 13 vp, more preferably between approximately 10 8 and approximately 10 12 vp, and even more preferably between approximately 10 9 and approximately 10 11 vp (e.g.
  • composition according to the invention maybe formulated for any suitable administration route, including intravenous, intramuscular, subcutaneous, oral, intranasal, transdermal or intratumoral administration.
  • the present invention also relates to therapeutic uses of the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention or any combination thereof, as well as to associated methods of treatment.
  • the present invention thus also relates to the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention or any combination thereof, for use as a medicament or as a vaccine.
  • the present invention also relates to the use of the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention or any combination thereof, for manufacturing a medicament or a vaccine.
  • the present invention also relates to the use of the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention or any combination thereof.
  • the present invention thus also relates to the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention or any combination thereof, for use in the treatment or prevention of cancer.
  • the present invention also relates to the use of the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention or any combination thereof.
  • 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).
  • 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 .
  • 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, and colon cancer (Yarchoan et al. 2019 JCI Insight; 4:e126908), and those cancers are thus particularly preferred in the context of the present invention.
  • the PD-L1 status of the cancer cells of the subject to be treated may be tested before treatment with the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention or any combination thereof.
  • the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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) .
  • the present invention thus also relates to the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention or any combination thereof, for use for inhibiting tumor cell growth in vivo.
  • the present invention also relates to the use of the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention or any combination thereof.
  • the present invention thus also relates to the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention or any combination thereof.
  • the administration of the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention or any combination thereof elicits, stimulates and/or re-orients an immune response.
  • 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.
  • the administration of the oncolytic virus 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.
  • a viral vector according to the invention in particular an oncolytic poxviral vector, such as an oncolytic vaccinia virus vector
  • such viral vector preferably provides a higher therapeutic efficacy than the one obtained in the same conditions either with a similar oncolytic virus not encoding the fusion polypeptide of the invention alone or with the fusion polypeptide of the invention alone.
  • the viral vector preferably provides a higher therapeutic efficacy than the combination of a similar oncolytic virus not encoding the fusion polypeptide of the invention and the fusion polypeptide 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 viral vector of the invention compared to either the virus or the fusion polypeptide 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.
  • the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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.
  • 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.
  • the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention or any combination thereof is administered via parenteral route, more preferably via intravenous, subcutaneous or intramuscular route, and even more preferably via intravenous route.
  • the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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.
  • conventional syringes and needles e.g. Quadrafuse injection needles
  • any compound or device available in the art capable of facilitating or improving delivery of the viral vector or composition in the subject.
  • the fusion polypeptide, nucleic acid molecule, vector, host cell, composition or combination thereof may be used as a stand-alone therapy. Alternatively, the fusion polypeptide, nucleic acid molecule, vector, host cell, composition or 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.
  • 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.
  • the therapeutic use or method of treatment according to the invention may be carried out in conjunction with surgery.
  • the fusion polypeptide, nucleic acid molecule, vector, host cell, composition or 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).
  • the therapeutic use or method of treatment according to the invention can be used in association with radiotherapy.
  • 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.
  • 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.
  • the therapeutic use or method of treatment according to the invention may be used in conjunction with chemotherapy currently available for treating cancer.
  • 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.
  • 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.
  • 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.
  • targeted therapy i.e. a therapy that targets proteins that control how cancer cells grow, divide, and spread.
  • 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 (HerceptinTM), etc.,) and monoclonal antibodies blocking Vascular Endothelial Growth Factor (in particular bevacizumab and ranibizumab).
  • Epidermal Growth Factor Receptor in particular cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab, trastuzumab (HerceptinTM), etc.
  • Vascular Endothelial Growth Factor in particular bevacizumab and ranibizumab.
  • the fusion polypeptide, nucleic acid molecule, vector, host cell, composition or combination thereof may preferably be used in conjunction with another immunotherapy.
  • 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.
  • 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. W02012/004367), CD40 (e.g. W02017/184619), or GITR (e.g. WO2017/068186).
  • human ICOSL 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. W02012/004367
  • CD40
  • 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 fusion polypeptide, nucleic acid molecule, vector, host cell, composition or combination thereof according to the invention include an agonist of ICOS.
  • 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
  • LAG3 e.g. W02018/071500
  • Tim3 e.g. W02020/093023
  • BTLA e.g. W02010/106051
  • 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).
  • Particularly preferred antagonists of an inhibitory immune checkpoint distinct from PD- L1 that may be used in conjunction with the fusion polypeptide, nucleic acid molecule, vector, host cell, composition or combination thereof according to the invention include antagonist of CTLA-4.
  • Example 1 Generation, evaluation and humanization of anti-PD-L1 sdAb
  • 77 clones selected on the basis of sequence diversity were expressed in scaled up 200ml cultures as follows. 1 l of 5 g/ml pScreen plasmid mini-prep containingPD1 /PD-L1 sdAb sequence was transformed into Top10 E. coli competent cells (Invitrogen) for expression by heatshock at 42 °C for 1 minute. Cells were subsequently spread on agar plates containing 50
  • a PD1 /PD-L1 inhibition assay was developed to test all 77 clones for inhibition of the PD1 /PD-L1 interaction.
  • an ELISA plate was coated overnight at 4° C with 1 pg/mL of recombinant human PD1 (Randox) in PBS pH7.4 ( Figure 1 ). The followingday, the contents of the plate were discarded, and the plate was blocked with 300 pL/well of blocking reagent (Roche Cat# 11921681001 ) and incubated at 37° C for 1 h.
  • the blocking reagent was discarded and 50 pL of 60 ng/mL of PDL1 -Fc (RnD Sys Cat# 156-B7) was added to each well and incubated for 1 hourat 37°C. Following incubation, 50 pL per well of serially diluted (10,000 to 0.1 ng/mL) anti-PD1 or anti-PDL1 sdAb clones were added to test for blocking of the PD1 /PD-L1 interaction. The plate was incubated at 37°C for 1 h.
  • the CDRs of the lead blocking clone 32.1A1 were grafted into a model Human VH3 framework sequence and the humanised sequence was synthesized and cloned intothe expression vector pScreen.
  • the humanised clone was compared to the original clone 32.1A1 in a binding ELISA whereby PD-L1 was coated on the surface of an ELISA plate, blocked and probed with a gradient of each of the humanised and non- humanised antibody fragments. Finally, the ELISA plate was again washed and detected with a secondary detector antibody, anti-Myc HRP (abeam). The results demonstrated that the two antibodies were functionally equivalent and that fullbinding was retained following humanisation ( Figure 4).
  • Table 9 Comparison signal output from binding ELISAs for clone 32.1A1 and its humanised derivative hum-32.1A1 .
  • the two lead PD1 and PD-L1 blocking clones as identified by the ELISA Inhibition assay were tested using the Promega PD1 /PD-L1 Blockade Bioassay (Cat# J1250), a commercial cellular assay to test the inhibition of PD1 /PD-L1 interaction on the cellsurface ( Figure 6).
  • the results of this assay also demonstrated the anti- PDL1 clone32.1A1 to have superior blocking in this cell-based assay format than the commercial therapeutic antibody Avelumab ( Figure 7).
  • Human PD1 , PD-L1 , CTLA4 and TIGIT All Randox
  • cynomolgus and mouse PD-L1 both Si no Biological
  • cynomolgus and mouse PD1 both R&D Systems
  • the plate was probed with a 30ng/ml dilution of 32.1A1 antibody and incubated with shaking at 37°C for 1 hour.
  • the ELISA plate was washed and detected with anti-Myc HRP (abeam) secondary detector antibody.
  • the results of this assay demonstrated binding of the 32.1A1 clone to human and cynomolgus PD-L1 but not to mouse PD- L1 or to any of the other unrelated antigens (See Figure 11 ).
  • a series of 15 mutant sdAb clones were synthesized using alanine substitution at 15 different residues across CDR3 of humanised 32.1A1 to identify residues which are important for binding of the h32.1A1 antibody to PDL1 antigen (See Figure 12).
  • VVTG18058 (empty VACV, VACV control, or unarmed control VACV) is a Vaccinia virus (Copenhagen strain) deleted of J2R and I4L genes. VVTG18058 was used as an unarmed control virus. VVTG18058 was produced on chicken embryo fibroblasts (CEF). Titration was performed by plaque assay on Vero cells.
  • the plasmid pTG19274 is a plasmid encoding for an irrelevant FLAG-tagged molecule. Plasmid pTG19274 is used as a negative control.
  • the plasmid pTG19970 is a plasmid encoding for the polypeptide GS542-CD40L-FLAGtag under the pH5R poxvirus promoter.
  • the plasmid pTG20032 is a plasmid encoding for the 4-1 BBL ectodomain alone (amino acids 80-254 of human 4-1 BBL amino acid sequence NP_003802.1*), for use as a control against the polypeptide GS542-linker-4-1 BBL construct according to the invention.
  • signal peptide Underligned: signal peptide; bold: anti-PD-L1 sdAb; normal: amino acids 119-261 of human CD40L; italic: FLAG tag peptide.
  • the plasmid pTG19971 is a plasmid encoding for the polypeptide GS542-linker-CD40L- FLAGtag under the pH5R poxvirus promoter.
  • Protein sequence encoded by pTG19971 (SEQ ID NO:31 ):
  • COPTG19971 is a recombinant Copenhagen vaccinia virus double deleted (TK- and RR-) wherein the expression sequence of pTG19971 has been inserted within its J2R locus.
  • the plasmid pTG20034 is a plasmid encoding for the polypeptide GS542-linker-4-1 BBL- FLAGtag under the pH5R poxvirus promoter.
  • Protein sequence encoded by pTG20034 (SEQ ID NO:33):
  • DNA sequence encoding Anti-human PD-L1 humanized single domain antibody GS542 was fused upstream of DNA sequence encoding the last 143 (N119 to L261 ) residues of human CD40L without (pTG19970) or with a DNA sequence encoding GGGSx3 linker (pTG19971 ) between the two former sequences. These constructs were cloned into transfer plasmid and under the same early/late poxvirus promoter pH5.R.
  • DNA sequence encoding Anti-human PD-L1 humanized single domain antibody GS542 was fused upstream of DNA sequence encoding the last 175 (D80 to E254) residues of human 4-1 BBL with a DNA sequence encoding GGGSx3 linker (pTG20034) between the two former sequences. These constructs were cloned into transfer plasmid and under the same early/late poxvirus promoter pH5.R. Human tumor cell lines
  • the human cervix tumor cell line HeLa (ATCC® CCL-2TM) was grown at 37° C, 5% CO? in DMEM (Gibco) supplemented with 10 % FBS, and 40 mg/L of gentamicin.
  • a method for expression of a recombinant protein is the infection/transfection method.
  • Such method consists of infecting a cell, for example a HeLa cell, with a vaccinia virus (a poxvirus) and transfecting said cell with a plasmid encoding the gene of interest under regulation control of a poxvirus promotor.
  • a poxvirus promotor a poxvirus promotor.
  • Such method allows the expression of the encoded gene of interest within the cell.
  • the expression product may be recovered within the supernatant for further analysis.
  • DMEM complete medium
  • Glutamine 2mM Glutamine 2mM
  • Gentamicin 40 pg/ml 10% fetal bovine serum FBS
  • VVTG18058 vaccinia virus preparation
  • PBS+ PBS + 1 % cations
  • MOI 1 MOI 1
  • RT room temperature
  • the viral inoculum was removed and replaced by 1 .2 mL of complete medium without FBS.
  • the plates were incubated for 2 h at 37 ° C with 5 % CO?.
  • Transfection was then performed by addition of 1 pg of plasmid DNA formulated with 4.5 pL of Lipofectamine 2000 (Invitrogen, 11668-027) in each well, following the provider’s protocols.
  • the plasmid pTG19274 (encoding irrelevant FLAG-tagged molecule) was used as a negative control.
  • the infection/transfections were performed in triplicate. The plates were incubated 48 hours at 37 °C and 5 % CO2. The culture supernatants were then collected, centrifuged, and filtrated on 0.1 pm filters to remove all virus particles and cellular debris. The clarified supernatants were stored at -80 °C until use.
  • HeLa cells were seeded in 6-well plate at 1.5E+06 cells/well/2 mL of complete medium (DMEM 4 Gibco ref. 41966-029; Glutamine 2mM; Gentamicin 40 pg/ml; 10% FBS the day prior infection.
  • DMEM complete medium
  • Glutamine 2mM Glutamine 2mM
  • Gentamicin 40 pg/ml 10% FBS the day prior infection.
  • Cells were infected at MOI 0.1 with one of the following viruses COPTG19971 , or VVTG18058. After 30 min of incubation the culture medium was discarded and replaced by 2 ml of DMEM; Glutamine 2mM; Gentamicin 40 pg/ml. Cells were incubated 48 hours at 37 °C with 5 % CO? and then the culture supernatant was recovered and treated as described above.
  • Blots were then incubated with anti-FLAG-HRP conjugated antibody (Sigma A8592) at 2 pg/mL using Ibind Flex Western System (Invitrogen ref SLF2000). Positive controls were culture medium from an infection/transfection with irrelevant plasmids pTG19274 encoding a FLAG-tagged recombinant protein or pTG19333 encoding GFP. Blots were incubated with HRP substrate (Amersham ECL Prime western blotting detection) and luminescence recorded by Chemidoc apparatus.
  • HRP substrate Amersham ECL Prime western blotting detection
  • Either PD-L1 or CD40-Fc were coated on Medisorp (Nunc) 96-well ELISA plate at 0.5 pg/mL in 50 mM carbonate buffer pH 9.6. Clarified supernatants of the infection/transfection experiment were added to the first well of the ELISA plate and further two-fold serially diluted in ELISA saturation buffer directly on the plate. The wells were then incubated with either CD40-Fc or biotinylated PD-L1 in case of PD-L1 and CD40-Fc coating respectively.
  • Anti-human Fc -HRP conjugated antibody Bethyl A80-104P
  • streptavidin- HRP were added to PD-L1 or CD40-Fc coated wells respectively.
  • HRP substrate (3, 3', 5, 5' tetramethylbenzidine) TMB was added to each well, absorbance 450 nm measured, and optical density (OD) 450 nm versus 1 /dilution of culture supernatants were plotted using GraphPad prism software.
  • Human PD-1 -Fc (R&D systems, 1086-PD) was coated on 96-well ELISA plate at 0.25 pg/mL in 50 mM carbonate buffer pH 9.6. Solutions or culture supernatants containing the PD- L1 blockers were added, and 2-fold serial diluted on plate and then human biotinylated PD-L1 (R&D systems, BT-156) at 0.2 pg/mL were added to all wells. PD-1 /PD-L1 complex formation was detected by addition of Horseradish peroxidase (HRP) conjugated streptavidin (SouthernBiotech, 7105-05) diluted 5000-fold.
  • HRP Horseradish peroxidase
  • HRP substrate (3, 3', 5, 5' tetramethylbenzidine) TMB was added to each well, absorbance 450 nm measured using TECAN microplate reader, and optical density (OD) 450 nm versus 1 /dilution of culture supernatants were plotted using GraphPad prism software.
  • HEK-Blue CD40L cells are recombinant cells transformed to express both the human CD40 and a reporter enzyme (secreted embryonic alkaline phosphatase: SEAP) under the transcriptional control of a CD40 inducible promoter.
  • SEAP secreted embryonic alkaline phosphatase
  • HEK-Blue CD40L cells in 90 pL were distributed in 96-well plate and incubated with 20 pL of serial dilutions of clarified supernatants generated by the infections or infection/transfections described above in presence or absence of Hs746T cells (displaying PD-L1 ).
  • 100 pg/mL of avelumab (anti-PD-L1 monoclonal antibody) or its isotype control (H27K15) were also added.
  • 4-1 BB Bioassay Promega kit (JA2351 ) was used according to providers instructions. Briefly, 25pL of effector cells/well were mixed with 25pL medium or PD-L1 positive Hs746T cells (50,000 cells/25 pL). Then 25 pL culture supernatants were added to each well. Cells were then incubated at 37° C, 5% CO2 for 6h. 75pL/well of reconstituted Bio-Gio (Promega, G7941 ) were added, and luminescence recorded using Berthold reader and MikroWin 2000 software. Luminescence versus 1 /supernatant dilution was plotted using GraphPad prism software and four-parameter logistic curve analysis was performed. Vectorization and virus production
  • Recombinant viruses were generated using the same plasmids used in infection/transfection experiment described above. Briefly, chicken embryo fibroblasts (CEF) were infected with parental virus encoding GFP at the J2R (TK) locus and deleted of the I4L (RR) gene. Infected cells were transfected with the transfer plasmid carrying the expression cassette flanked by recombination harms (DNA sequences homolog of upstream and downstream TK locus). Recombinant viruses are selected under binocular by picking “white” (i.e. GFP negative) lysis plaque. Expression cassette was checked by PCR amplification followed by DNA sequencing.
  • recombinant viruses were produced on CEF (MOI 0.05, 72 hours) cultivated on F500.
  • the cellular suspension containing the virus was homogenized by using a homogenizing mixer equipped with an in-line chamber. Large cellular debris were then eliminated by depth filtration using 5 m pore size filters.
  • the clarified viral suspension was subsequently concentrated and diafiltered with the formulation buffer (Saccharose 100 g/L, Tris 30 mM, pH 7.6) using tangential flow filtration and 0.2 pm pore size hollow fiber microfiltration filters. Purified viruses were aliquoted and stored at -80° C until use.
  • HeLa cells were infected and transfected with the transfer plasmids carrying the different CD40L constructions as described above (see also sequences pTG19971 and pTG19970).
  • Expression of recombinant anti-PD-L1 -CD40L proteins in the culture supernatants were assessed by immunoblot using anti-FLAG tag for detection.
  • Figure 14 shows that both anti-PD-L1 -CD40L fusions were expressed at the same level and at the expected monomer size in reducing and non-reducing conditions. Note that neither aggregation nor degradation was observed on this blot.
  • the fusion polypeptide according to the invention e.g., a recombinant anti-PD-L1 -CD40L protein
  • a recombinant anti-PD-L1 -CD40L protein is of a smaller molecular size compared to disclosures of the prior art. Such comparison is shown in Table 10.
  • HeLa cells were infected with vaccinia virus and then transfected with the transfer plasmids carrying the two anti-PD-L1 -CD40L constructions (with and without the linker, respectively pTG19971 and pTG19970) under control of the same poxvirus promoter (i.e. pH5.R). After 48 Hours the culture mediums were harvested, filtered through 0.1 pm filter to remove vaccinia virus and tested on the HEK Bleu CD40L cells that express a reporter protein under the control of a CD40 inducible promoter and in presence or absence of PD- L1 expressing cells (Hs746T).
  • Figure 17 shows that both fusion polypeptides expressed by pTG19970 and pTG19971 have a weak CD40 agonist activity by their own. However, their CD40 agonist activities are greatly enhanced in presence of PD-L1 expressing cells demonstrating the conditional activation of CD40 of those molecules.
  • the fusion polypeptides encoded by the plasmid pTG19971 had a better CD40 agonist activity than fusion polypeptides encoded by the plasmid pTG19970 therefore the former format was used to generate a recombinant Copenhagen vaccinia virus (namely COPTG19971 ) carrying the thymidine kinase (TK) and ribonucleotide reductase (RR) gene deletions for further experiments (see “CD40 agonist activity of COPTG19971 and the role of PD-L1 displayed on cells” part thereafter).
  • TK thymidine kinase
  • RR ribonucleotide reductase
  • the recombinant virus named COPTG19971 were used to infect HeLa cells and the culture medium recovered after 48 hours was tested on the CD40 activity assay as previously described. In parallel infection/transfections with their parental plasmids were also performed as reference. To demonstrate the involvement of PD-L1 in the CD40 agonist activity, an excess of competitive anti-PD-L1 antibody (avelumab) was added prior to supernatant in order to hamper the binding of anti-PD-L1 -CD40L fusion to PD-L1.
  • Figure 18A and 18B show for both pTG19971 and COPTG19971 the CD40 agonist activity was dramatically increase by the co-incubation of PD-L1 expressing cells confirming the previous observations.
  • CD40L moiety was exchanged by 4-1 BBL ectodomain in the pTG19971 construct to generate a GS542-linker-4-1 BBL construct (pTG20034; encoding for SEQ ID NO: 33).
  • Said Anti-PD-L1 -4-1 BBL fusion construction referenced as pTG20034 was tested directly after infection transfection on blot and 4- 1 BB bioassay.
  • the level of expression of 4-1 BBL (pTG20032) and GS542-linker-4-1 BBL (pTG20034) was equivalent ( Figure 19).
  • Molecule described by Thiemann et al. is an anti-PD-L1 Fab (fragment antigen binding) comprising on one hand, a light chain and on the other hand, a VH and CH1 domains of heavy chain of an anti-PD-L1 antibody.
  • the heavy chain is fused to single chain tri meric CD40L ectodomains corresponding to three CD40L ectodomains linked together by GS linkers.
  • This construct has some limitations for vectorization: it requires two expression cassettes (one for the light chain and the other for the fusion heavy chain- CD40L) with a precisely balanced expression of the two transgenes to get an optimized assembly of the molecule. Moreover, the cloning of three CD40L requires a careful degeneration of DNA sequence to avoid any internal recombination into the transgene. In the case of the present invention GS542-linker-CD40L (pTG19971 ) necessitates one transgene and does not contain any repetitive sequence.
  • the trimerization of CD40L allows the formation of trimeric anti-PD-L1 sdAb and therefore a putative avidity effect compared to the monomeric anti-PD-L1 Fab of Thiemann et al.
  • the other benchmark molecule disclosed in Medler et al. is the fusion of a CD40 agonist F(ab’)2 (i.e. two Fab linked by the hinge of the IgG) with the heavy chain fragment fused to a single chain fragment variable (scFv) of an anti-PD-L1 antibody.
  • the produced protein is therefore a bivalent bispecific molecule able to bind two CD40 and two PD-L1 targets.
  • it necessitates two cassettes of expression Like the other benchmark molecule according to Thiemann et al. describes above, it necessitates two cassettes of expression.
  • TNF TNF superfamily cytokines as targets for the treatment of rheumatic diseases. Nat Rev Rheumatol. 2017; 13(4) :217-233
  • TNFSF14 LIGHTing the Way for Effective Cancer Immunotherapy. TNFSF14: LIGHTing the Way for Effective Cancer Immunotherapy. May 2020. Vol. 11. Article 922

Abstract

The present invention is related to a fusion polypeptide comprising a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1) fused to a member of the Tumor Necrosis Factor superfamily of ligands (TNFSF), to a nucleic acid molecule encoding it, a vector comprising such nucleic acid molecule, a host cell comprising such nucleic acid molecule or vector, compositions comprising any one of them or combinations thereof, methods for their production, as well as to therapeutic uses and methods of treatment using them.

Description

FUSION POLYPEPTIDE COMPRISING AN ANTI-PD-L1 SDAB AND A MEMBER OF
THE TNFSF
TECHNICAL FIELD OF THE INVENTION
The present invention is related to a fusion polypeptide comprising a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ) fused to a member of the Tumor Necrosis Factor superfamily of ligands (TNFSF), and to the compositions and methods related thereto.
BACKGROUND ART
Each year, cancer is diagnosed in more than 12 million subjects worldwide. In industrialized countries, approximately one person out five will die of cancer. 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.
Among potential source for anticancer therapies is a patient’s own immune system, in particular T cell-mediated cytotoxicity.
Efficient antitumoral T cell response necessitates a functional and optimal crosstalk between cells of the innate and adaptive immunity. This is spatially and timely achieved by multiple specific interactions between different cells through, soluble stimuli (chemokine/cytokine) and through surface ligand/receptor binding among which the costimulatory molecules of the Tumor necrosis factor super family (TNFSF) molecules play a central role.
CD40L (also known as CD154 or TNFSF5) is a type I membrane co-stimulatory protein of the TNFSF that play a central role in the initiation of adaptive immune response. As TNF, CD40L assembles into a homotrimer and interacts in trans with its receptor CD40. CD40 is distributed on antigen presenting cells (APC) such as dendritic cells, macrophages, B lymphocytes, but also epithelial cells, endothelial cells, smooth muscle cells, fibroblasts, basophils, and blood platelets. CD40L is expressed on surface of activated B, T and NK cells but also on adipose cells, and basophils (Richards et al. Hum Vaccin Immunother. 2020; 16(2) :377-387) . The CD40L-CD40 interaction leads to the activations of CD40 bearing cells which then express adhesion (ICAM), co-stimulatory (CD80/CD86), and presenting MHC I and II molecules in addition of cytokines/chemokines (TNFa, IL6...). In the tumor microenvironment, adhesion molecules and cytokine/chemokines act together to induce the infiltration and activation of immune cells, that ultimately destroy tumor cells and skew the tumor from an immunosuppressive to an immunocompetent microenvironment (Richards et al. Hum Vaccin Immunother. 2020; 16(2) :377-387).
It is well known that signaling through CD40 is obtained by its clustering on cell membrane (Bremer ISRN Oncol. 2013 Jun 11 ;2013:371854 ; Kucka and Wajant. Front Cell Dev Biol. 2021 Feb 11 ;8:615141 ) . This is naturally occurring by trans-interactions between several CD40L molecules at a surface of one cell with CD40 molecules on another cell. CD40 clusterization and activation can be also obtained artificially using, agonist antibodies or CD40L-Fc fusion proteins. These molecules interact on one hand with CD40 by their paratope, or CD40L domain, and induce its clusterization by interaction in trans- on the other hand with Fc receptors (FcR) bearing cells. This mode of CD40 engagement is not controlled as it depends on presence of FcR bearing cells in the neighborhood and can lead to detrimental overstimulation of cell in case of formation of too large clusters.
Another way to artificially induce the CD40 agonist activity is to display the CD40 agonist binding moiety (i.e. CD40L or CD40 antibody fragment) at the surface of a cell that becomes CD40L “positive” and therefore able to activate CD40 in trans-. One approach to display CD40L on a cell is to use a bispecific molecule able to bind to CD40 (e.g. CD40L) and to an antigen displayed at the surface of the cell. If the latter antigen is a tumor associated antigen (TAA), this CD40L artificial display on cell would occur only in the tumor microenvironment therefore limiting the on-target off-tumor activation of CD40 and its associated toxicity. This approach has been used with success to make “conditional CD40 agonists” (i.e. molecules with cd40 agonist activity only in presence of TAA expressing cells) by several authors (Wyzgol et al. J Immunol. 2009 Aug 1 ;183(3):1851 -61 , Bremer ISRN Oncol. 2013 Jun 11 ;2013:371854, Brunekreeft et al.
2014 Mol Cancer 17;13:85., Pandey et al. Int J Mol Sci. 2021 Oct 21 ;22(21 ) : 11302 and Medler J et al. Theranostics. 2022 Jan 1 ; 12(4) : 1486-1499) .
Among the TAA, the programmed death 1 (PD-1 ) is expressed on activated T cells and has emerged as an interesting mediator for negatively regulating T cell responses. PD-1 is part of the immunoglobulin (Ig) gene superfamily and a member of the CD28 family. It is a 55 kDa type 1 transmembrane protein expressed on antigen-experienced cells (e.g. activated B cells, T cells, and myeloid cells) (Agata et al., 1996, Int. Immunol. 8: 765- 72; Okazaki et al., 2002, Curr. Opin. Immunol. 14: 391779-82; Bennett et al., 2003, J. Immunol 170: 711 -8). In normal context, it acts by limiting the activity of T cells at the time of inflammatory response, thereby protecting normal tissues from destruction (Topalian, 2012, Curr. Opin. Immunol. 24: 207-12). Two ligands have been identified for PD-1 , respectively PD- L1 (programmed death ligand 1 ) and PD-L2 (programmed death ligand 2) (Freeman et al., 2000, J. Exp. Med. 192: 1027-34; Carter et al., 2002, Eur. J. Immunol. 32: 634-43). 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). The interaction between PD-1 and PD-L1 resulted in a decrease in tumor infiltrating lymphocytes, a decrease in T- cell receptor mediated proliferation, and immune evasion by the cancerous cells (Dong et al., 2003, J. Mol. Med. 81 : 281 -7; Blank et al., 2005, Cancer Immunol. Immunother. 54: 307- 5 314).
WO2019/094574 discloses a bispecific fusion protein of about 250 kDa (dimer) comprising an anti-PD-L1 Fab fragment and two CD40L subunits, both binding regions covalently linked to a Fc monomer. Such bispecific fusion protein is for use for controlling T cell- mediated cytotoxicity via improved control of T cell signaling pathways. The ability of such bispecific fusion protein anti-PD-L1 /CD40L to synergistically induce biological response compared to the combination of anti-PD-L1 monovalent monoclonal antibody and agonist CD40L fusion protein or either treatment alone has been described (Pandey et al. Int J Mol Sci. 2021 Oct 21 ;22(21 ): 11302) . The CD40 agonist activity in absence of PD-L1 expressing cells is still very strong for such a fusion protein, therefore such bispecific cannot be considered as a conditional agonist. Moreover, as previously stated, the presence of the Fc monomer may induce the activation of FcR bearing cells in the neighborhood, leading to the destruction of the targeted CD40+ cells by triggering ADCC, CDC or anti-inflammatory FcR activities. Such destruction isn’t an effect which is sought in the context of the use of a conditional TNFSF ligand agonist. In addition, the presence of the Fc moiety results in a long half-life in vivo, further increasing the risk of the occurrence of adverse effects in areas different from the tumor microenvironment.
In the field of oncology, the 4-1 BBL, another member of the TNFSF is well known. The 4- 1 BB ligand (4-1 BBL, also known as CD137L or TNFSF9) is found on APCs (antigen presenting cells) and binds to 4-1 BB (also known as CD137), a type 2 transmembrane glycoprotein receptor belonging to the TNF superfamily, which is expressed on activated T Lymphocytes (Lotze M (2001 ). Dendritic Cells. Boston: Academic Press. ISBN 0-12-455851 - 8). The 4-1 BBL has been associated with enabling the immune system to eliminate tumors in multiple cancer types. CD137 is expressed at higher levels on CD8+ than CD4+ T cells, and it mainly co-stimulates CD8+ T cells. Crosslinking of CD137 strongly enhances proliferation, IFN-y secretion and cytolytic activity of T cells. Moreover, CD137 agonists, such as antibodies, have been reported to work synergistically with cancer vaccines and immune check point inhibitors to boost anticancer immune responses (Dharmadhikari et al., 2016, Oncoimmunology 5(4): el 113367).
Medler et al. 2022 (Medler J et al. Theranostics. 2022 Jan 1 ; 12(4) : 1486-1499) discloses a fusion polypeptide of about 150 kDa (dimer) comprising an anti-4-1 BB F(ab’)? or an anti- CD40 F(ab’)z antibody fused with a PD-L1 specific blocking scFv as anchoring domain. Such fusion polypeptide is considered as a conditional agonist, with a weak CD40 or 4-1 BB agonist activity in absence of PD-L1 expressing cells; and a strong CD40 or 4-1 BB agonist activity in presence of PD-L1 expressing cells. Those bispecific antibodies variants are devoid of the ability to interact with FcR. However, the scFv antibody is known in prior art to be potentially immunogen, which can negatively impact the capacity of the fusion polypeptide to induce the CD40 or 4-1 BB agonist activity. Indeed, compared to antibodies, scFvs tend to have lower affinities, lower long-term stability, and a higher likelihood to aggregate due to their small size (Bates and Power. Antibodies. 2019; 8(2) :28).
It is also known in the art a monomer construct of about 150 kDa composed of a trivalent single-chain CD40L-receptor-binding-domain (scCD40L-RBD) linked to an anti-PD-L1 (see W02021 /229103 or Abstract 1587: Generation and characterization of novel bispecific molecules combining single-chain-CD40L with anti-CEA, anti-CD95L or anti-PD-L1 targeting moieties, by APOGENIX). Said anti-PD-L1 used into the construct is a scFv antibody, with the above -described drawbacks. Moreover, using a trivalent single-chain CD40L-receptor-binding-domain results in a final molecule comprising only one PD-L1 - binding unit for 3 CD40-binding units, which may limit affinity for PD-L1 positive cells. While conditional agonists are useful for limiting adverse effects on non-tumor tissues, they should still display maximum agonist activity in the presence of PD-L1 cells, which may not be the case for a fusion comprising only a monovalent anti-PD-L1 scFv.
Despite already disclosed bispecific fusion polypeptides targeting TNFSF receptor and TAA, there is a need for improved specific conditional agonist constructs thereof. Such conditional agonist constructs being in capacity to maximize T cell mediated cytotoxicity via improved control of T cell signaling pathways. Such conditional agonist constructs could provide less toxic, more targeted anticancer therapies.
Also, there is a need for improved diffusion inside the tumoral tissue of such a conditional agonist constructs thereof compared to prior art, to enhance the T cell mediated cytotoxicity inside the tumor.
Moreover, in case of delivery of the conditional agonist via a viral vector the number and size of each transgene is an issue because of the limited capacity of some viral platforms such as for examples, adenovirus or measles. Therefore, there is a need for an improved vectorizable sequence encoding such a conditional agonist fusion polypeptide.
SUMMARY OF THE INVENTION
In the context of the present invention, the inventors designed particular fusion polypeptides comprising a particular anti-PD-L1 sdAb fused to a member of the TNFSF or a functional fragment or variant thereof. Such fusion polypeptides combine many advantages, including:
• Absence of an Fc domain, thus preventing unwanted destruction of the targeted CD40+ cells by triggering ADCC, ADCP, CDC or anti-inflammatory FcR activities and limiting the half-life in vivo as no binding to Fc receptors (including those involved in ADCC or the FcRn receptor involved in half-life increase of immunoglobulins) and complement proteins (in particular C1q).
• Low molecular weight of the final complex. The encoded single chain has a molecular weight of only about 30 kDa, and trimerizes due to the trimerization domain of the TNFSF member, resulting in a final molecular weight of only about 90kDa.
This is significantly smaller than the final molecules disclosed in W02019/094574 and Pandey et al. Int J Mol Sci. 2021 Oct 21 ;22(21 ) : 11302 (each chain has a molecular weight of about 123 kDa and dimerizes to form a final molecule of about 250 kDa), in Medler J et al. Theranostics. 2022 Jan 1 ; 12(4) : 1486-1499 (each chain has a molecular weight of about 75 kDa and dimerizes to form a final molecule of about 150 kDa), or in W02021 /229103 or Abstract 1587: Generation and characterization of novel bispecific molecules combining single-chain-CD40L with anti-CEA, anti-CD95L or anti-PD-L1 targeting moieties, by APOGENIX (the final molecules comprising a Fab fused to a trivalent single-chain CD40L-receptor- binding-domain have a molecular weight of about 95 kDa).
Smaller molecular weight permits better penetration and diffusion into tumor tissue.
• The claimed constructions necessitate only one transgene (as a sdAb is made of only a heavy chain and TNFSF members are made of only one distinct chain that trimerizes) of limited size.
This ensures easy and efficient vectorization of the fusion polypeptide by plasmid or viral vectors. In contrast, in the constructions disclosed in W02019/094574, Pandey et al. Int J Mol Sci. 2021 Oct 21 ;22(21 ) : 11302 and Medler J et al. Theranostics. 2022 Jan 1 ;12(4):1486-1499, the anti-PD-L1 part necessitates entire or partial heavy and light chains, i.e. two chains, the stoichiometry of which needs to be equilibrated for proper formation of the final construction, making vectorization much more complex, due to the presence of two chains, one of which is much longer than that used in the present invention.
In W02021 /229103 and Abstract 1587: Generation and characterization of novel bispecific molecules combining single-chain-CD40L with anti-CEA, anti-CD95L or anti-PD-L1 targeting moieties, by APOGENIX, while only one chain is needed, this chain is much longer due to the use of a trivalent single-chain CD40L-receptor- binding-domain. This much higher size of the insert makes vectorization more complex. Moreover, the use of same protein sequence repeated thrice require DNA degeneration to avoid homologous recombination.
In a first aspect, the present invention thus relates to fusion polypeptide comprising a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ) fused to a member of the Tumor Necrosis Factor superfamily of ligands (TNFSF) or a functional fragment or derivative thereof, wherein: a) the sdAb specifically binding to PD-L1 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),
• the heavy chain CDR3 comprises the sequence X2SLLRGX3SSRAEX4YDX5, wherein each of X2 to X5 independently represents any amino acid (SEQ ID NO:3), and b) the sdAb specifically binding to PD-L1 is fused to the member of the TNFSF or functional fragment or derivative thereof directly or indirectly through a peptide linker.
The present invention also relates to a nucleic acid molecule encoding the fusion polypeptide according to the invention, to a vector (in particular a viral vector; and more particularly a poxviral vector, such as a vaccinia virus vector) comprising the nucleic acid molecule according to the invention, a host cell comprising the nucleic acid molecule according to the invention or the vector according to the invention, and a composition comprising the fusion polypeptide, nucleic acid molecule, vector (in particular a viral vector; and more particularly a poxviral vector, such as a vaccinia virus vector), or host cell according to the invention, or any combination thereof.
The present invention also relates to methods for producing the fusion polypeptide according to the invention or the viral vector according to the invention.
Finally, the invention also relates to therapeutic uses or methods of treatment using the fusion polypeptide, nucleic acid molecule, vector (in particular a viral vector; and more particularly a poxviral vector, such as a vaccinia virus vector), host cell, or composition according to the invention, or any combination thereof, in particular in the treatment or prevention of cancer.
DESCRIPTION OF THE FIGURES
FIG. 1 shows a schematic demonstrating a PD-1 /PD-L1 inhibition ELISA.
FIG. 2 shows the results obtained via an ELISA-based assay, demonstrating the ability of selected sdAb clones to block the interaction between PD-1 and PD-L1 .
FIG. 3 shows the results obtained via an inhibition assay, demonstrating the ability of selected sdAb clones to block PD1 /PD-L1 interaction.
FIG. 4 shows the results obtained from a PD-L1 binding assay for the humanised 32.1A1 clone.
FIG. 5 shows the results obtained from a PD-1 /PD-L1 inhibition assay to compare clone 32.1A1 , its humanised derivative and its close relative 32.2F7 to the commercially available therapeutic antibody Avelumab.
FIG. 6 shows a schematic demonstrating the PD-1 /PD-L1 blockade bioassay.
FIG. 7 shows the luciferase activity fold change of the two lead PD-1 and PD-L1 blocking clones, as identified by the ELISA inhibition assay, as assayed in the Promega PD- 1 /PD-L1 blockade bioassay. Reference to GS542 denotes the humanised 32.1A1 clone, and reference to 1A1 denotes clone 32.1A1 .
FIG. 8 shows an SDS-PAGE gel showing fraction of sdAb monomer.
FIG. 9 shows an analytical size exclusion chromatography (SEC) to demonstrate aggregation following thermal stress of the humanised 32.1A1 clone.
FIG. 10 shows humanised 32.1A1 stability ELISA against coated PD-L1.
FIG. 11 shows cross-reactivity of clone 32.1A1 against related and unrelated antigens. FIG. 12 shows a series of 15 mutant sdAb clones synthesized using alanine substitution at 15 different residues across CDR3 of humanised 32.1A1 to identify residues that are important for binding of the h32.1A1 antibody to PDL1 antigen.
FIG. 13A and FIG. 13B show the results obtained from an ELISA assay for binding affinity of the 15 mutant sdAb clones of FIG. 12.
FIG. 14 shows level of expression of GS542-CD40L constructions by infected /transfected HeLa cells. Clarified supernatants containing the different anti-PD-L1 -CD40L molecules were loaded on SDS-PAGE under reducing and non-reducing conditions. Proteins were transferred on PVDF membrane and hybridized with an HRP-conjugated anti-FLAG tag for immunodetection.
FIG. 15 shows designs of ELISA assay to demonstrate the bispecificity of GS542-CD40L fusions.
FIG. 16 shows pTG19970 and pTG19971 products are able to bind CD40 and PD-L1 simultaneously. Different dilutions of clarified supernatants of HeLa cells infected with vaccinia virus and then transfected with plasmids carrying expression cassette encoding the different anti-PD-L1-CD40L molecules were loaded on ELISA plates previously coated with either CD40 or PD-L1. The bound bispecific protein was developed with either labeled PD-L1 or Fc-tagged CD40 respectively. Results are reported as the optical density versus 1 /dilution of the culture medium. PTG19274 is encoding an irrelevant FLAG-tagged protein and was used as negative control.
FIG. 17 shows CD40 agonist activity of culture medium of HeLa cells infected with vaccinia virus and then transfected with plasmids carrying expression cassette encoding GS542-CD40L constructions and under control of pH5.R promoter (i.e. poxvirus promoter). HEK cells modified to express the reporter enzyme SEAP under the control of a CD40 inducible promoter were incubated with different dilutions of the clarified supernatants containing the CD40L constructions in presence, or absence, of PD-L1 expressing cells (Hs746T). SEAP enzymatic activity was measured in culture medium after a 20-24 hours incubation. Negative controls were non-infected cells (noted medium), or cells infected/transfected with a plasmid (noted pTG19274) encoding an irrelevant FLAG- tagged protein. Results are reported as the SEAP activity versus 1 /dilution of the culture medium.
FIG. 18A shows CD40 agonist activity of pTG19971 is blocked by preincubation with anti- PD-L1 antibody. CD40 agonist activity was assessed as described in Figure 17 except that culture supernatants of infected/transfected (pTG19971 ) HeLa cells were tested. In some conditions avelumab (avel) or its isotype control (iso) were added to the cells prior the culture supernatants. In these latter cases, only the undiluted supernatants were tested. FIG. 18B shows CD40 agonist activity of COPTG19971 is blocked by preincubation with anti-PD-L1 antibody. CD40 agonist activity was assessed as described in Figure 17 except that culture supernatants of infected (COPTG19971 ) HeLa cells were tested. In some conditions avelumab (avel) or its isotype control (iso) were added to the cells prior the culture supernatants. In these latter cases, only the undiluted supernatants were tested. FIG. 19 shows the level of expression 4-1 BBL ectodomain alone (pTG20032) or fused at C- terminus of GS542 (anti-PD-L1 sdAb, pTG20034), negative control is a plasmid encoding GFP (pTG19333).
FIG. 20 shows 4-1 BB agonist activity of culture medium of HeLa cells infected with vaccinia virus and then transfected with plasmids carrying expression cassette encoding 4-1 BBL (pTG20032) or GS542-4-1 BBL (pTG20034) constructions and under control of pH5.R promoter (i.e., poxvirus promoter). Reporter cells modified to express the reporter enzyme luciferase under the control of a 4-1 BB inducible promoter were incubated with different dilutions of the clarified supernatants containing the 4-1 BBL constructions in presence, or absence, of PD-L1 expressing cells (Hs746T). Luciferase enzymatic activity was measured in culture medium after a 20-24 hours incubation. Negative controls were non-infected cells (noted medium), or cells infected /transfected with a plasmid (noted pTG 19274) encoding an irrelevant FLAG -tagged protein. Results are reported as the luciferase activity (luminescence) versus 1 /dilution of the culture medium.
FIG. 21 shows CD40 agonist activity of pTG19971 and of two benchmark constructs (pTG20154/pTG20155 and pTG20156/pTG20157). After infection/transfection by the indicated plasmid, culture mediums were harvested and tested for their CD40 agonist activity as described in Figure 17.
FIG. 22 shows PD-1 /PD-L1 blocking activity of pTG19971 and of two benchmark constructs (pTG20154/pTG20155 and pTG20156/pTG20157) measured by competitive ELISA for vectorized products. Same samples as in Figure 21 were tested for their inhibiting activity of PD-1 /PD-L1 interaction.
DETAILED DESCRIPTION OF THE INVENTION
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 treatment 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.
Fusion polypeptides
The present invention first relates to a fusion polypeptide comprising, consisting essentially of or consisting of a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ) fused to a member of the Tumor Necrosis Factor superfamily of ligands (TNFSF) or a functional fragment or derivative thereof, wherein: a) the sdAb specifically binding to human PD-L1 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),
• the heavy chain CDR3 comprises the sequence X2SLLRGX3SSRAEX4YDX5, wherein each of X2 to X5 independently represents any amino acid (SEQ ID NO:3), and b) the sdAb specifically binding to human PD-L1 is fused to the member of the TNFSF or functional fragment or derivative thereof directly or indirectly through a peptide linker. sdAb specifically binding to PD-L1
The fusion polypeptide according to the invention comprises as a first fusion partner (also referred to as “fusion partner 1” or “FP1”) a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ) (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 included in the fusion polypeptide 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, 5th 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 included in the fusion polypeptide 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 comprised in the fusion polypeptide according to the invention preferably has a Kd value lower than 1 nM (preferably when measured using an Octet Red96 instrument, more preferably using the method disclosed in Example 1 ).
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 “FP1 -Emb1”. sdAb with such heavy chain CDR1 , CDR2 and CDR3 are shown in Example 1 to confer specificity for human 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 “FP1 -Emb2”), the heavy chain CDR3 of the sdAb specifically binding to PD-L1 comprises, 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 “FP1 -Emb3”), the heavy chain CDR3 of the sdAb specifically binding to PD-L1 comprises, consists essentially of or 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, P, T, C, A, V, G and P; and
• 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.
Figure imgf000018_0001
Table 1 . rational for selection of amino acids at positions X2 to X7. More preferably (embodiment “FP1 -Emb4”), the heavy chain CDR3 of the sdAb specifically binding to PD-L1 comprises, 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 “FP1 -Emb5”), the heavy chain CDR3 of the sdAb specifically binding to PD-L1 comprises, 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 “FP1 -Emb6”), the heavy chain CDR3 of the sdAb specifically binding to PD-L1 comprises, 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.
Figure imgf000019_0001
Table 2. Heavy chain CDR sequences of clones 32.1A1 and 32.2F7.
Two particularly preferred alternative embodiments of the fusion polypeptide according to the invention are as follows: a) embodiment “FP1 -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 NO: 10); or b) embodiment “FP1 -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 FP1 -Emb1 to FP1 -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, 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 “FP1 -Emb9”), the sdAb specifically binding to PD-L1 comprises, 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:
Figure imgf000021_0001
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, consists essentially of or consists of the sequence SEQ ID NO: 13 (embodiment “FP1 -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, consist essentially of or consist of the sequence SEQ ID NO: 13 (embodiment “FP1 -Emb11 ”).
SEQ ID NO: 13 and SEQ ID NO: 14 have 98.4% identity.
Member of the TNFSF
The fusion polypeptide according to the invention also comprises as a second fusion partner (also referred to as “fusion partner 2” or “FP2”) a member of the Tumor Necrosis Factor superfamily of ligands (TNFSF) or a functional fragment or derivative thereof (also referred to as “TNFSF member”) (referred to as “FP2-EMb1 ”).
In preferred embodiments, the member of the TNFSF or functional fragment or variant thereof is selected from:
• CD40L, 4-1 BBL, Baff, APRIL, EDA-A1 , GITRL, OX40L, CD70, TL1A, LIGHT, LToB2, RANKL, TWEAK, FASL, TRAIL, TNF and LTo and functional fragments or derivatives thereof (embodiment “FP2-Emb2”);
• category II TNFSF members (embodiment “FP2-Emb3”), preferably from CD40L, 4- 1 BBL, Baff, APRIL, EDA-A1 , OX40L, CD70, TWEAK, FASLG, TRAIL, and TNF and functional fragments or derivatives thereof (embodiment “FP2-Emb4”);
• TNFSF members involved in immune cell activation (embodiment “FP2-Emb5”), preferably from CD40L, 4-1 BBL, GITRL, OX40L, CD70, TL1A and functional fragments or derivatives thereof (embodiment “FP2-Emb6”); • category II TNFSF members involved in immune cell activation (embodiment “FP2- Emb7”), preferably from CD40L, 4-1 BBL, OX40L, CD70, and functional fragments or derivatives thereof (embodiment “FP2-Emb8”); or
• CD40L, 4-1 BBL, and functional fragments or derivatives thereof (embodiment “FP2-Emb9”).
Most preferably the member of the TNFSF or functional fragment or derivative thereof is selected from CD40L and functional fragments or derivatives thereof (embodiment “FP2- Emb10”).
TNFSF members
The TNFSF comprises a number of structurally related members (also referred to as ligands) that organize lymphoid tissue development, co-stimulate lymphocyte activation and can either increase lymphocyte survival and function or induce cell death, all through bonding to their cognate receptor(s), which form the tumor necrosis factor receptor superfamily (TNFRSF).
TNFRSF may be divided into two distinct categories, depending on their ability to be activated by soluble ligand trimers of the TNFSF (Kucka K, Wajant H. Receptor Oligomerization and Its Relevance for Signaling by Receptors of the Tumor Necrosis Factor Receptor Superfamily. Front Cell Dev Biol. 2021 Feb 11 ;8:615141 ) .
Category I receptors of the TNFRSF are robustly activated by soluble ligand trimers, and include BaffR (ligand = Baff), DR3 (ligand = TL1A), GITR (ligand = GITRL), LTBR (ligand = LToB2 or LIGHT), TNFR1 (ligand = TNF or LTo).
Category II receptors of the TNFRSF fail to become properly activated by soluble ligand trimers despite high affinity binding, and include CD40L, 4-1 BBL, Baff, APRIL, EDA-A1 , OX40L, CD70, TWEAK, FASLG, TRAIL, and TNF.
The limited responsiveness of category II TNFRs to soluble TNFLs can be overcome by physical linkage of two or more soluble ligand trimers or, alternatively, by anchoring the soluble ligand molecules to the cell surface or extracellular matrix. In the context of the present invention, members of the TNFSF binding to category II TNFRs are preferred as the ability of the fusion polypeptide according to the invention is then truly conditional, as activation of their cognate TNFR then depends on the presence in the tumor microenvironment of PD-L1 positive tumor cells.
Therefore, in a preferred embodiment, the member of the TNFSF included in the fusion polypeptide according to the invention is selected from category II TNFSF members, preferably selected from CD40L, 4-1 BBL, Baff, APRIL, EDA-A1 , OX40L, CD70, TWEAK, FASLG, TRAIL, and TNF and functional fragments or derivatives thereof.
TNFSF members may also be classified depending on their known functions. In the context of the present invention, TNFSF members involved in immune cell activation may preferably be included in the fusion polypeptide according to the invention. TNFSF members involved in immune cell activation include CD40L, 4-1 BBL, GITRL, OX40L, CD70, TL1A and functional fragments or derivatives thereof (Croft M, Siegel RM. Beyond TNF: TNF superfamily cytokines as targets for the treatment of rheumatic diseases. Nat Rev Rheumatol. 2017; 13(4) :217-233) .
“CD40L”, “CD40 ligand”, “CD40LG”, “tumor necrosis factor superfamily member 5”, “TNFSF5”, and “CD154” are used herein interchangeably and refer to a member of the TNFSF that plays a central role in the initiation of adaptive immune response when it assembles into a homotrimer and interacts in trans with its receptor CD40. The CD40L- CD40 interaction leads to the activation of CD40 bearing cells, which then express adhesion (ICAM), co-stimulatory (CD80/CD86), and MHC I and II molecules in addition of cytokines/chemokines (TNFa, IL6...). In the tumor microenvironment, adhesion molecules and cytokine/chemokines act together to induce the infiltration and activation of immune cells, that ultimately destroy tumor cells and skew the tumor from an immunosuppressive to an immunocompetent microenvironment (Richards et al. Hum Vaccin Immunother. 2020;16(2):377-387). CD40L is thus a TNFSF member involved in immune cell activation. As TNF, CD40L assembles into a homotrimer and interacts in trans with its receptor CD40 through its extracellular part. CD40 is a category II receptor of the TNFRSF.
Being a member of the TNFSF that is both involved in immune cell activation and binds to a category II receptor of the TNFRSF, CD40L is a particularly preferred TNFSF member for the fusion polypeptide according to the invention.
The specific CD40L protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use. In particular, for an intended use in humans, human CD40L will preferably be used. Human CD40L corresponds to Entrez Gene ID 959, and the complete amino acid sequence of human CD40L may be found under GenBank Accession No. NP_000065.1 (version of January 17, 2022).
“4-1 BBL”, “4-1 BB ligand”, “CD137L”, “tumor necrosis factor superfamily member 9”, and “TNFSF9” are used herein interchangeably and refer to a transmembrane cytokine that acts as a ligand for TNFRSF9/4-1 BB, which is a costimulatory receptor molecule in T lymphocytes. This cytokine and its receptor are involved in the antigen presentation process and in the generation of cytotoxic T cells. 4-1 BBL is thus a TNFSF member involved in immune cell activation.
Its receptor 4-1 BB is a category II receptor of the TNFRSF.
Being a member of the TNFSF that is both involved in immune cell activation and binds to a category II receptor of the TNFRSF, 4-1 BBL is a particularly preferred TNFSF member for the fusion polypeptide according to the invention.
The specific 4-1 BBL protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use. In particular, for an intended use in humans, human 4-1 BBL will preferably be used. Human 4-1 BBL corresponds to Entrez Gene ID 8744, and the complete amino acid sequence of human 4-1 BBL may be found under GenBank Accession No. NP_003802.1 (version of February 27, 2022).
“OX40L”, “0X40 ligand”, “CD252”, “CD134L”, “tumor necrosis factor superfamily member 4”, and “TNFSF4” are used herein interchangeably and refer to an inducible molecule expressed on several cell types, although arguably most importantly, on antigen-presenting cells (APCs). OX40L can trigger signaling through its receptor 0X40, resulting in a range of activities including expansion and accumulation of effector T cells and their cytokine production. OX40L is thus a TNFSF member involved in immune cell activation.
Its receptor 0X40 is a category II receptor of the TNFRSF.
Being a member of the TNFSF that is both involved in immune cell activation and binds to a category II receptor of the TNFRSF, OX40L is a particularly preferred TNFSF member for the fusion polypeptide according to the invention.
The specific OX40L protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use. In particular, for an intended use in humans, human OX40L will preferably be used. Human OX40L corresponds to Entrez Gene ID 7292, and the complete amino acid sequence of the longest isoform of human OX40L may be found under GenBank Accession No. NP_003317.1 (version of March 16, 2022).
“CD70”, “CD27L”, “CD27LG”, “tumor necrosis factor superfamily member 7”, and “TNFSF7” are used herein interchangeably and refer to a molecule that can, through interaction with its receptor CD27, provide signals to T cells to control their accumulation and reactivity, similarly to that seen with 0X40, GITR and DR3. CD70 is thus a TNFSF member involved in immune cell activation.
Its receptor CD27 is a category II receptor of the TNFRSF.
Being a member of the TNFSF that is both involved in immune cell activation and binds to a category II receptor of the TNFRSF, CD70 is a particularly preferred TNFSF member for the fusion polypeptide according to the invention.
The specific CD70 protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use. In particular, for an intended use in humans, human CD70 will preferably be used. Human CD70 corresponds to Entrez Gene ID 970, and the complete amino acid sequences of the longest isoform of human CD70 may be found under GenBank Accession No. NP_001317261 .1 (version of January 9, 2022).
“Baff”, “B cell activating factor”, “CD257”, “tumor necrosis factor superfamily member 13b”, “TNFSF13B”, “tumor necrosis factor superfamily member 20”, and “TNFSF20” are used herein interchangeably and refer to a molecule that primarily, although not exclusively, controls B cell activity.
It binds to two receptors of the TNFRSF: BaffR and TACI. BaffR is a category I receptor of the TNFRSF, but TACI is a category II receptor of the TNFRSF.
Being a member of the TNFSF that binds to a category II receptor of the TNFRSF (TACI), Baff is a preferred TNFSF member for the fusion polypeptide according to the invention. The specific Baff protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use. In particular, for an intended use in humans, human Baff will preferably be used. Human Baff corresponds to Entrez Gene ID 10673, and the complete amino acid sequences of the longest isoform of human Baff may be found under GenBank Accession No. NP_006564.1 (version of February 20, 2022).
“APRIL”, “CD256”, “tumor necrosis factor superfamily member 13”, and “TNFSF13” are used herein interchangeably and refer to a ligand found to be important for B cell development.
It binds to two receptors of the TNFRSF: BCMA and TACI, which are both category II receptors of the TNFRSF.
Being a member of the TNFSF that binds to category II receptors of the TNFRSF, APRIL is a preferred TNFSF member for the fusion polypeptide according to the invention. The specific APRIL protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use. In particular, for an intended use in humans, human APRIL will preferably be used. Human APRIL corresponds to Entrez Gene ID 8741 , and the complete amino acid sequences of the longest isoform of human APRIL may be found under GenBank Accession No. NP_003799.1 (version of January 23, 2022).
“EDA-A1”, “EDA”, “ectodysplasin A”, and “tumor necrosis factor ligand 7C” are used herein interchangeably and refer to a protein involved in cell-cell signaling during the development of ectodermal organs.
Its receptor EDAR is a category II receptor of the TNFRSF.
Being a member of the TNFSF that binds to a category II receptor of the TNFRSF, EDA-A1 is a preferred TNFSF member for the fusion polypeptide according to the invention.
The specific EDA-A1 protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use. In particular, for an intended use in humans, human EDA-A1 will preferably be used. Human EDA-A1 corresponds to Entrez Gene ID 1896, and the complete amino acid sequence of the longest isoform of human EDA-A1 may be found under GenBank Accession No. NP_001390.1 (version of February 13, 2022).
“GITRL”, “Glucocorticoid-induced TNF receptor-related ligand”, “tumor necrosis factor superfamily member 18”, and “TNFSF18” are used herein interchangeably and refer to an inducible molecule expressed in professional APCs, and other cell types such as endothelial cells. Its receptor, glucocorticoid-induced TNF receptor-related protein (GITR, also known as TNFRSF18), can stimulate T cell, dendritic cell and B cell activation. GITRL is thus a TNFSF member involved in immune cell activation.
Its receptor GITR is a category I receptor of the TNFRSF.
Being a member of the TNFSF that is involved in immune cell activation, GITRL is a preferred TNFSF member for the fusion polypeptide according to the invention.
The specific GITRL protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use. In particular, for an intended use in humans, human GITRL will preferably be used. Human GITRL corresponds to Entrez Gene ID 8995, and the complete amino acid sequences of the longest isoform of human GITRL may be found under GenBank Accession No. NP_005083.3 (version of February 20, 2022). TJ
“TL1A”, “tumor necrosis factor superfamily member 15”, and “TNFSF15” are used herein interchangeably and refer to a protein that can be induced in APCs such as dendritic cells and macrophages, as well as in endothelial cells. Its binding to its receptor “Death receptor 3” (“DR3”, also known as “TNFRSF25”), a stimulatory receptor expressed by T cells, can regulate effector T cell accumulation and/or reactivity. TL1A is thus a TNFSF member involved in immune cell activation. This cytokine is also found to inhibit endothelial cell proliferation, and thus may function as an angiogenesis inhibitor.
Its receptor DR3 is a category I receptor of the TNFRSF.
Being a member of the TNFSF that is involved in immune cell activation, TL1A is a preferred TNFSF member for the fusion polypeptide according to the invention.
The specific TL1A protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use. In particular, for an intended use in humans, human TL1A will preferably be used. Human TL1A corresponds to Entrez Gene ID 9966, and the complete amino acid sequences of the longest isoform of human TL1A may be found under GenBank Accession No. NP_005109.2 (version of February 27, 2022).
“LIGHT”, “CD258”, “tumor necrosis factor superfamily member 14”, and “TNFSF14” are used herein interchangeably and refer to a protein that binds to two receptors of the TNFRSF: Herpes Virus Entry Mediator (HVEM, also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14)) and Lymphotoxin-B Receptor (LTBR). LIGHT-HVEM interaction is responsible for a majority of the immune-stimulating properties of LIGHT. Expressed on lymphocytes, NK cells, smooth muscle, and epithelium, HVEM serves as an important T cell costimulatory agent leading to activation, proliferation, and survival. In the context of anti -tumor immune support, LIGHT -LTBR signaling has a wide range of roles that span from influencing cancer cells' susceptibility to immune responses, functioning to repair chaotic tumor vasculature, and to supporting effector cells cell trafficking to and infiltration into tumors (Skeate Joseph G. et al. TNFSF14: LIGHTing the Way for Effective Cancer Immunotherapy. TNFSF14: LIGHTing the Way for Effective Cancer Immunotherapy. May 2020. Vol. 11. Article 922).
LTBR is a category I receptor of the TNFRSF. HVEM is a category I receptor of the TNFRSF. The specific LIGHT protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use. In particular, for an intended use in humans, human LIGHT will preferably be used. Human LIGHT corresponds to Entrez Gene ID 8740, and the complete amino acid sequences of the longest isoform of human LIGHT may be found under GenBank Accession No. NP_003798.2 (version of February 20, 2022).
“RANKL”, “RANK ligand”, “receptor activator of nuclear factor-KB ligand”, “CD254”, “tumor necrosis factor superfamily member 11”, and “TNFSF11” are used herein interchangeably and refer to a ligand for osteoprotegerin that functions as a key factor for osteoclast differentiation and activation. This protein was also shown to be a dentritic cell survival factor and to be involved in the regulation of T cell-dependent immune response.
Its receptor RANK (or ““receptor activator of nuclear factor-KB”) is a category I receptor of the TNFRSF.
The specific RANKL protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use. In particular, for an intended use in humans, human RANKL will preferably be used. Human RANKL corresponds to Entrez Gene ID 8600, and the complete amino acid sequences of the longest isoform of human RANKL may be found under GenBank Accession No. NP_003692.1 (version of February 27, 2022).
“TWEAK”, “tumor necrosis factor superfamily member 12”, and “TNFSF12” are used herein interchangeably and refer to a cytokine that has overlapping signaling functions with TNF, but displays a much wider tissue distribution. It exists in both membrane-bound and secreted forms, can induce apoptosis via multiple pathways of cell death in a cell type-specific manner. It is also found to promote proliferation and migration of endothelial cells, and thus acts as a regulator of angiogenesis.
Its receptor FN14 (also referred to as TWEAKR) is a category II receptor of the TNFRSF. Being a member of the TNFSF that binds to a category II receptor of the TNFRSF, TWEAK is a preferred TNFSF member for the fusion polypeptide according to the invention.
The specific TWEAK protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use. In particular, for an intended use in humans, human TWEAK will preferably be used. Human TWEAK corresponds to Entrez Gene ID 8742, and the complete amino acid sequence of the longest isoform of human TWEAK may be found under GenBank Accession No. NP_003800.1 (version of February 20, 2022).
“FASL”, “FAS ligand”, “FASLG”, “CD178”, “CD95 ligand”, “CD95L”, “tumor necrosis factor superfamily member 6”, and “TNFSF6” are used herein interchangeably and refer to a transmembrane protein which primary function is the induction of apoptosis triggered by binding to its receptor FAS. The FAS/FASLG signaling pathway is essential for immune system regulation, including activation-induced cell death (AICD) of T cells and cytotoxic T lymphocyte induced cell death.
FAS is a category II receptor of the TNFRSF.
Being a member of the TNFSF that binds to a category II receptor of the TNFRSF, FASL is a preferred TNFSF member for the fusion polypeptide according to the invention.
The specific FASL protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use. In particular, for an intended use in humans, human FASL will preferably be used. Human FASL corresponds to Entrez Gene ID 356, and the complete amino acid sequence of the longest isoform of human FASL may be found under GenBank Accession No. NP_000630.1 (version of February 27, 2022).
“TRAIL”, “CD253”, “tumor necrosis factor superfamily member 10”, and “TNFSF10” are used herein interchangeably and refer to a protein that preferentially induces apoptosis in transformed and tumor cells, but does not appear to kill normal cells although it is expressed at a significant level in most normal tissues. TRAIL binds to several members of TNF receptor superfamily including TNFRSF10A/TRAILR1 , TNFRSF10B/TRAILR2, TNFRSF10C/TRAILR3, TNFRSF10D/TRAILR4, and possibly also to TNFRSF11 B/OPG. The activity of TRAIL may be modulated by binding to the decoy receptors TNFRSF10C/TRAILR3, TNFRSF10D/TRAILR4, and TNFRSF11 B/OPG that cannot induce apoptosis. The binding of TRAIL to its receptors has been shown to trigger the activation of MAPK8/JNK, caspase 8, and caspase 3.
Both of TRAILR1 and TRAILR2 are category II receptors of the TNFRSF.
Being a member of the TNFSF that binds to category II receptors of the TNFRSF, TRAIL is a preferred TNFSF member for the fusion polypeptide according to the invention.
The specific TRAIL protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use. In particular, for an intended use in humans, human TRAIL will preferably be used. Human TRAIL corresponds to Entrez Gene ID 8743, and the complete amino acid sequence of the longest isoform of human TRAIL may be found under GenBank Accession No NP_003801.1 (version of March 17, 2022).
“TNF”, “tumor necrosis factor”, “TNFA”, “TNFo”, “tumor necrosis factor superfamily member 2”, and “TNFSF2” are used herein interchangeably and refer to a multifunctional proi inflammatory cytokine involved in the regulation of a wide spectrum of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation.
TNF binds to two receptors of the TNFRSF: TNFR1 (also known as “TNFRSF1 A”) and TNFR2 (akso known as “TNFRSF1 B” or “TNFBR”). TNFR1 is a category I receptor of the TNFRSF, while TNFR2 is a category II receptor of the TNFRSF.
Being a member of the TNFSF that binds to a category II receptor of the TNFRSF, TNF is a preferred TNFSF member for the fusion polypeptide according to the invention.
The specific TNF protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use. In particular, for an intended use in humans, human TNF will preferably be used. Human TNF corresponds to Entrez Gene ID 7124, and the complete amino acid sequence of human TNF may be found under GenBank Accession No NP_000585.2 (version of March 17, 2022).
“LTa”, “lymphotoxin alpha”, “TNFB”, “TNFB”, “tumor necrosis factor superfamily member 1”, and “TNFSF1” are used herein interchangeably and refer to a cytokine produced by lymphocytes that is highly inducible, secreted, and forms heterotrimers with lymphotoxin-beta which anchor lymphotoxin-alpha to the cell surface. This protein also mediates a large variety of inflammatory, immunostimulatory, and antiviral responses, is involved in the formation of secondary lymphoid organs during development and plays a role in apoptosis.
LTa binds to TNFR1 (also known as “TNFRSF1A”), which is a category I receptor of the TNFRSF.
The specific LTa protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use. In particular, for an intended use in humans, human LTa will preferably be used. Human LTa corresponds to Entrez Gene ID 4049, and the complete amino acid sequence of human LTa may be found under GenBank Accession No NP_001153212.1 (version of March 10, 2022).
“LTB”, “lymphotoxin beta”, “TNFC”, “tumor necrosis factor superfamily member 3”, and “TNFSF3” are used herein interchangeably and refer to a protein that anchors lymphotoxin-alpha to the cell surface through heterotrimer formation. The predominant form on the lymphocyte surface is the lymphotoxin-alpha 1 /beta 2 complex (also referred to as “LTaBz”, e.g. 1 molecule alpha/2 molecules beta) and this complex is the primary ligand for the lymphotoxin-beta receptor (also referred to as LTBR”), a category I receptor of the TNFRSF. The minor complex is lymphotoxin-alpha 2/beta 1. LTB is an inducer of the inflammatory response system and involved in normal development of lymphoid tissue.
The specific LTB protein that may be comprised in the fusion protein according to the invention is preferably selected from the same species as the species for which the fusion polypeptide is intended for therapeutic use. In particular, for an intended use in humans, human LTB will preferably be used. Human LTB corresponds to Entrez Gene ID 4050, and the complete amino acid sequence of human LTB may be found under GenBank Accession No NP_002332.1 (version of February 20, 2021 ).
Functional fragments or variants of TNFSF members
While the fusion polypeptide according to the invention may comprise an entire wild-type member of the TNFSF, it may also alternatively comprise a functional fragment thereof or a functional derivative thereof.
By a “functional fragment” of a member of the TNFSF, it is meant a fragment (i.e. a part of the amino acid sequence of the entire member of the TNFSF) that retains the function of the entire member of the TNFSF, i.e. its ability to trimerize and bind to and activate its TNFRSF receptor(s).
In particular, TNFSF members exist as transmembrane proteins, but their transmembrane and intracellular regions are not needed for trimerization and binding to and activation of their TNFRSF receptor (s).
Therefore, in a preferred embodiment, the TNFSF member fragment comprised in the fusion polypeptide according to the invention is an extracellular fragment of the TNFSF member, i.e. a fragment devoid of the transmembrane and intracellular part of the TNFSF member. Whole or part of the extracellular domain may be comprised in the extracellular fragment, provided that the fragment retains its ability to trimerize and bind to and activate its TNFRSF receptor(s). Suitable extracellular fragments of TNFSF members are described in Table 4 below.
Figure imgf000033_0001
Table 4. TNFSF members, their Entre Gene ID, reference amino acid sequence, and positions of extracellular domain and other functional fragments.
By a “functional derivative” of a member of the TNFSF, it is meant a polypeptide comprising a member of the TNFSF or a functional fragment thereof with one or more insertions, deletions, truncations and/or substitutions, which retains the function of the entire member of the TNFSF, i.e. its ability to trimerize and bind to and activate its TNFRSF receptor(s). In the case of substitutions, the substituted original amino acid(s) is (are) preferably replaced by equivalent amino acid(s), as disclosed above in Table 3.
Direct or indirect fusion
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.
In the fusion polypeptide according to the invention, the sdAb specifically binding to PD- L1 may be fused to the member of the TNFSF or functional fragment or derivative thereof directly or indirectly through a peptide linker.
The sdAb specifically binding to PD-L1 is said to be fused “directly” to the member of the TNFSF or functional fragment or derivative thereof if there is no additional amino acid residue between the two fusion partners (the sdAb specifically binding to PD-L1 and the member of the TNFSF or functional fragment or derivative thereof). The sdAb specifically binding to PD-L1 is said to be fused “indirectly through a peptide linker” to the member of the TNFSF or functional fragment or derivative thereof if there is there is a peptide linker comprising, 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 member of the TNFSF or functional fragment or derivative thereof).
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 member of the TNFSF or functional fragment or derivative thereof 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, 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 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 member of the TNFSF or functional fragment or derivative thereof) may be fused in any order, such as the sdAb specifically binding to PD-L1 in N-terminal of the member of the TNFSF or functional fragment or derivative thereof, or the sdAb specifically binding to PD-L1 in C- terminal of the member of the TNFSF or functional fragment or derivative thereof.
In a preferred embodiment, the sdAb specifically binding to PD-L1 is in N-terminal of the member of the TNFSF. In another embodiment, the sdAb specifically binding to PD-L1 is in C-terminal of the member of the TNFSF.
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 member of the TNFSF or functional fragment or derivative thereof) and the optional peptide linker in between, further elements that may be useful for the production or therapeutic use of the fusion polypeptide.
In particular, the fusion polypeptide according to the invention may further comprises: a) a signal peptide in N-terminal; and/or b) a tag peptide, preferably in C-terminal.
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, 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-P rot Accession Number P01750).
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 neopeptide 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.
Preferred fusion polypeptides
Preferred fusion polypeptides according to the invention may notably comprise, consist essentially of or consist of the following formulas, from N-terminal (on the left) to C- terminal (on the right): a) anti-PD-L1 sdAb-TNFSF member, b) TNFSF member-anti-PD-L1 sdAb, c) anti-PD-L1 sdAb-peptide linker-TNFSF member, d) TNFSF member-peptide linker-anti-PD-L1 sdAb, e) signal peptide-anti-PD-L1 sdAb-TNFSF member, f) signal peptide-TNFSF member-anti-PD-L1 sdAb, g) signal peptide-anti-PD-L1 sdAb-peptide linker-TNFSF member, h) signal peptide-TNFSF member-peptide linker-anti-PD-L1 sdAb, i) anti-PD-L1 sdAb-TNFSF member-tag peptide, j) TNFSF member-anti-PD-L1 sdAb-tag peptide, k) anti-PD-L1 sdAb-peptide linker-TNFSF member-tag peptide, l) TNFSF member-peptide linker-anti-PD-L1 sdAb-tag peptide, m) signal peptide-anti-PD-L1 sdAb-TNFSF member-tag peptide, n) signal peptide-TNFSF member-anti-PD-L1 sdAb-tag peptide, o) signal peptide-anti-PD-L1 sdAb-peptide linker-TNFSF member-tag peptide, p) signal peptide-TNFSF member-peptide linker-anti-PD-L1 sdAb-tag peptide, wherein “anti-PD-L1 sdAb” corresponds to the single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ), and “TNFSF member” corresponds to member of the Tumor Necrosis Factor superfamily of ligands (TNFSF) or a functional fragment or derivative thereof.
More preferably, the fusion polypeptide according to the invention comprises, consists essentially of or consists, from N-terminal (on the left) to C-terminal (on the right) of one of the above -described formulas e) to h) and m) to p), which all contain a signal peptide in N-terminal.
Even more preferably the fusion polypeptide according to the invention comprises, consists essentially of or consists, from N-terminal (on the left) to C-terminal (on the right), of:
• one of the above-described formulas e) to h), which all contain a signal peptide in N-terminal, but do not contain a tag peptide as tag peptides may be removed in final pharmaceutical fusion polypeptides intended for human therapeutic use,
• one of the above -described formulas g), h), o) and p), which all contain a signal peptide in N-terminal and a peptide linker (found to improve activity in Examples below),
• one of the above-described formulas e), f), m) and n), which all contain a signal peptide in N-terminal followed by the anti-PD-L1 sdAb, optionally a peptide linker, and the TNFSF member in C-terminal.
Most preferably the fusion polypeptide according to the invention comprises, consists essentially of or consists, from N-terminal (on the left) to C-terminal (on the right) of one of the above-described formulas g) or p), which contain a signal peptide in N-terminal, followed by the anti-PD-L1 sdAb, a peptide linker, the TNFSF member in C-terminal, and optionally a tag peptide in C-terminal.
In each of the above formulas, the anti-PD-L1 sdAb and TNFSF member may be selected from any preferred embodiment disclosed above. In particular, each of FP1 -Emb1 to FP1 - Emb8 (preferably humanized) and FP1 -Emb9 to FP1 -Emb11 (already corresponds to humanized versions) may be combined with each of FP2-Emb1 to FP2-Emb10 (using the entire TNFSF member or a functional fragment thereof, preferably an extracellular functional fragment thereof).
Particularly preferred couples of fusions partners 1 and 2 that may be used in each of the above formulas are disclosed in Table 5 below.
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Table 5. Particularly preferred (anti-PD-L1 sdAb/TNFSF member) couples of partners. * = last update, as mentioned above.
In each of the above formulas, when present, the signal peptide preferably comprises, consists essentially of or consists of amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO:24).
In each of the above formulas, when present, the tag peptide preferably comprises, consists essentially of or consists of amino acid sequence GDYKDDDK (SEQ ID NO: 26).
In each of the above formulas, when present, the peptide linker preferably comprises, consists essentially of or consists of amino acid sequence GGGSGGGSGGGS (SEQ ID NO: 22). Particularly preferred fusion polypeptides according to the invention comprise, consist essentially of or consist of an amino acid sequence with at least 80% 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:31 , which corresponds to an amino acid sequence consisting of, from N-terminal to C-terminal: signal peptide MGWSCIILFLVATATGVHS (SEQ ID NO:24), the entire amino acid sequence of the humanized version of the sdAb produced by clone 32.1A1 (SEQ ID NO: 13), peptide linker GGGSGGGSGGGS (SEQ ID NO: 22), and amino acids 1 19-261 of human CD40L amino acid sequence NP_000065.1 , as described below:
MGWSCIILFLVATATGVHSQVQLVESGGGLVQPGGSLRLSCAASGRTFREYGMGWFRQAP GKGLEWVATISSSGSYSYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASSLL RGSSSRAESYDSWGQGTLVTVSSGGGSGGGSGGGSNPQIAAHVISEASSKTTSVLQWAEKGY YTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAAN THSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLGSDYKDDDDK Underligned: signal peptide; bold: anti-PD-L1 sdAb; bold underlined: peptide linker; normal: amino acids 119-261 of human CD40L; italic: FLAG tag peptide.
• SEQ ID NO:32, which corresponds to an amino acid sequence consisting of, from N-terminal to C-terminal: signal peptide MGWSCIILFLVATATGVHS (SEQ ID NO:26), the entire amino acid sequence of the humanized version of the sdAb produced by clone 32.1A1 (SEQ ID NO: 13), and amino acids 119-261 of human CD40L amino acid sequence NP_000065.1 , as described below:
MGWSCIILFLVATATGVHSQVQLVESGGGLVQPGGSLRLSCAASGRTFREYGMGWFRQAP GKGLEWVATISSSGSYSYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASSLL RGSSSRAESYDSWGQGTLVTVSSNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLEN GKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQS IHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLGSDYKDDDDK
Underligned: signal peptide; bold: anti-PD-L1 sdAb; normal: amino acids 119-261 of human CD40L; italic: FLAG tag peptide. • SEQ ID NO:33, which corresponds to an amino acid sequence consisting of, from N-terminal to C-terminal: signal peptide MGWSCIILFLVATATGVHS (SEQ ID NO:24), the entire amino acid sequence of the humanized version of the sdAb produced by clone 32.1A1 (SEQ ID NO: 13), peptide linker GGGSGGGSGGGS (SEQ ID NO: 22), and amino acids 80-254 of human 4-1 BBL amino acid sequence NP_003802.1 , as described below:
MGWSCIILFLVATATGVHSQVQLVESGGGLVQPGGSLRLSCAASGRTFREYGMGWFRQAP GKGLEWVATISSSGSYSYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASSLL RGSSSRAESYDSWGQGTLVTVSSGGGSGGGSGGGSDPAGLLDLRQGMFAQLVAQNVLLIDG PLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQ PLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQ GATVLGLFRVTPEIPAGLPSPRSEGSDYKDDDDK
Underligned: signal peptide; bold: anti-PD-L1 sdAb; bold underlined: peptide linker; normal: amino acids 80-254 of human 4-1 BBL; italic: FLAG tag peptide.
• SEQ ID NO:34, which corresponds to an amino acid sequence consisting of, from N-terminal to C-terminal: signal peptide MGWSCIILFLVATATGVHS (SEQ ID NO:26), the entire amino acid sequence of the humanized version of the sdAb produced by clone 32.1A1 (SEQ ID NO: 13), and amino acids 80-254 of human 4-1 BBL amino acid sequence NP_003802.1 , as described below:
MGWSCIILFLVATATGVHSQVQLVESGGGLVQPGGSLRLSCAASGRTFREYGMGWFRQAP GKGLEWVATISSSGSYSYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASSLL RGSSSRAESYDSWGQGTLVTVSSDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAG VSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALAL TVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI PAGLPSPRSEGSDYKDDDDK
Underligned: signal peptide; bold: anti-PD-L1 sdAb; normal: amino acids 80-254 of human 4-1 BBL; italic: FLAG tag peptide.
Preferably, the fusion polypeptide according to the invention is selected from SEQ ID NO: 31 to SEQ ID NO: 34, more preferably the fusion polypeptide consists of SEQ ID NO: 31 or SEQ ID NO:33. Nucleic acid molecules
The present invention also relates to a nucleic acid molecule encoding the fusion polypeptide according to the invention, in any embodiment disclosed above.
A nucleic acid molecule according to the invention comprises distinct parts, each encoding one of the components of the fusion polypeptide according to the invention, i.e. fusion partners 1 and 2 (anti-PD-L1 sdAb and TNFSF member), and optionally one or more of a signal peptide, a peptide linker, and a tag peptide.
For each of this part, the nucleic acid sequence may be a native (or natural) nucleic acid sequence (e.g. the native nucleic acid sequence encoding amino acids 119-261 of human CD40L amino acid sequence NP_000065.1 ) or may be modified by man to include substitution, deletion, addition and/or insertion of one or more nucleotide(s). The present invention encompasses any modifications aimed to improve cloning and/or expression of the encoded and fusion polypeptide according to the invention as well as its folding, stability and activity. When several modifications are contemplated, they can concern consecutive and/or non-consecutive nucleotide residues. The modification(s) contemplated by the present invention encompass silent modifications that do not change the amino acid sequence of the encoded fusion polypeptide according to the invention, as well as modifications that are translated into the encoded fusion polypeptide (provided that the fusion partners retain their activity). Representative examples of modifications include but are not limited to introduction of appropriate restriction sites, sequence degeneration (e.g. to reduce sequence homology between various parts of the complete nucleic acid molecule) and/or optimisation of nucleotide sequence (e.g. to optimize translation in a given host cell), and/or suppression of potentially negative elements (which are expected to negatively influence expression levels).
For example, it may be worth optimizing codon usage for ensuring high level of expression of the encoded gene product in a particular host cell or subject. It has been indeed observed that, when more than one codon is available to code for a given amino acid, the codon usage patterns of organisms are highly non-random and the utilisation of codons may be markedly different between different hosts. Typically, codon optimisation is performed by replacing one or more “native” codon corresponding to a codon infrequently used in the host cell of interest 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. Moreover, some deviations from strict adherence to optimised codon usage may be made to accommodate the introduction of restriction site(s) into the resulting nucleic acid molecule.
The nucleic acid molecules of the present invention can be generated using sequence data accessible in the art and the sequence information provided herein. For example, they may be isolated using routine techniques well known in the art, e.g. by PCR isolation and/or cloning by conventional molecular biology from an appropriate natural source, cDNA and genomic libraries or any prior art vector known to include it. Alternatively, the nucleic acid molecules of the invention can also be generated by chemical synthesis in automatised process (e.g. assembled from overlapping synthetic oligonucleotides). Preferred nucleic acid molecules according to the invention are selected from:
• SEQ ID NO:35, encoding the amino acid sequence SEQ ID NO:31,
• SEQ ID NO:36, encoding the amino acid sequence SEQ ID NO:32,
• SEQ ID NO:37, encoding the amino acid sequence SEQ ID NO:33,
• SEQ ID NO:38, encoding the amino acid sequence SEQ ID NO:34,
Vectors
The present invention also relates to a vector comprising the nucleic acid molecule according to the invention, in any embodiment disclosed above.
The term “vector” as used herein refers to a vehicle, preferably a nucleic acid molecule or a viral particle that contains the elements necessary to allow delivery, propagation and/or expression of any of the nucleic acid molecule(s) described herein within a host cell or subject. This term encompasses vectors for maintenance (cloning vectors) or vectors for expression in various host cells or subjects (expression vectors), extrachromosomal vectors (e.g. multicopy plasmids) or integration vectors (e.g. designed to integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates) as well as shuttle vectors (e.g. functioning in both prokaryotic and/or eukaryotic hosts) and transfer vectors (e.g. for transferring nucleic acid molecule(s) in a viral genome). For the purpose of the invention, the vectors may be of naturally occurring genetic sources, synthetic or artificial, or some combination of natural and artificial genetic elements.
In the context of the invention, the term “vector” has to be understood broadly as including mRNA, plasmid and viral vectors. Vectors which are appropriate in the context of the present invention, include, without limitation, bacteriophage, plasmid or cosmid vectors for expression in prokaryotic host cells such as bacteria (e.g. E. coli, BCG or Listeria); vectors for expression in yeast (e.g. Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris); baculovirus vectors for expression in insect cell systems (e.g. Sf 9 cells); as well as plasmid and viral vectors for expression in higher eukaryotic cells or subjects. Typically, such vectors are commercially available (e.g. in Invitrogen, Stratagene, Amersham Biosciences, Promega, etc.) or available from depositary institutions such as the American Type Culture Collection (ATCC, Rockville, Md.) or have been the subject of numerous publications describing their sequence, organization and methods of producing, allowing the artisan to apply them. The present invention also encompasses vectors (e.g. plasmid DNA and mRNA) complexed to lipids or polymers to form particulate structures such as liposomes, lipoplexes or nanoparticles.
Plasmid vectors
A "plasmid vector" as used herein refers to a replicable DNA construct. Usually, plasmid vectors contain selectable marker genes that allow host cells carrying the plasmid vector to be selected for or against in the presence of a corresponding selective drug. A variety of positive and negative selectable marker genes are known in the art. By way of illustration, an antibiotic resistance gene can be used as a positive selectable marker gene that allows a host cell to be selected in the presence of the corresponding antibiotic. Representative examples of suitable plasmid vectors include, without limitation, pREP4, pCEP4 (Invitrogen), pCI (Promega), pVAX (Invitrogen) and pGWiz (Gene Therapy System Inc).
Viral vectors
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 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 replicationdefective or replication-impaired.
Representative examples of suitable viral vectors are generated from a variety of different viruses (e.g. poxviruses, adenoviruses, herpes viruses, paramyxoviruses, and rhabdovi ruses, lentiviruses etc). As described above, the term "viral vector" encompasses vector DNA, genomic DNA as well as viral particles generated thereof, and especially infectious viral particles. In a preferred embodiment, the viral vector is thus in the form of infectious viral particles.
Poxviruses are a broad family of DNA viruses containing a double-stranded genome. Like most viruses, poxviruses have developed self-defense 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-apoptotic proteins, inhibitors of the NFkB pathway and interferon signaling, 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 a laboratory, compared to the native form) to comprise a nucleic acid molecule inserted in its genome and encoding the fusion polypeptide according to the invention. 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. AL VAC) 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 (W), 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 Orthopoxvirues.
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), Elstree, 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). Oncolytic viral vectors
In a preferred embodiment, the viral vector is an oncolytic viral vector.
As used herein, the term “oncolytic virus” refers to a virus capable of selectively replicating in dividing cells, and more particularly in cancer cells, 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 cells. Typically, an oncolytic virus 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.
Suitable oncolytic viral vectors, with sufficient transgene capacity to encode the fusion polypeptide according to the invention, may be selected from poxviruses, adenoviruses, herpes viruses, paramyxoviruses, and rhabdoviruses (see Kaufman, H., Kohlhapp, F. & Zloza, A. Oncolytic viruses: a new class of immunotherapy drugs. Nat Rev Drug Discov 14, 642-662 (2015)).
In one embodiment, the oncolytic virus of the present invention is obtained from a rhabdovirus, such as a vesicular stomatitis virus (VSV). Representative examples are described in the literature (e.g. Stojdl et al., 2000, Nat. Med. 6(7): 821 -5; Stojdl et al., 2003, Cancer Cell 4(4): 263-75).
In one embodiment, the oncolytic virus of the present invention is obtained from a paramyxovirus, such as a Newcastle disease virus, or a morbillivirus (in particular a measles virus). Representative examples of oncolytic Newcastle disease viruses include without limitation the 73-T PV701 and HDV-HUJ strains as well as those described in the literature (e.g. Phuangsab et al., 2001 , Cancer Lett. 172(1 ): 27-36; Lorence et al., 2007, Curr. Cancer Drug Targets 7(2): 157-67; Freeman et al., 2006, Mol. Ther. 13(1 ): 221 -8). Representative examples of oncolytic measles viruses include without limitation MV-Edm (McDonald et al., 2006; Breast Cancer Treat. 99(2): 177-84) and HMWMAA (Kaufmann et al., 2013, J. Invest. Dermatol. 133(4): 1034-42).
In one embodiment, the oncolytic virus of the present invention is obtained from a herpes virus. The Herpesviridae are a large family of DNA viruses that all share a common structure and are composed of relatively large double-stranded, linear DNA genomes encoding 100-200 genes encapsided within an icosahedral capsid which is enveloped in a lipid bilayer membrane. Although the oncolytic herpes virus can be derived from different types of HSV, particularly preferred are HSV1 and HSV2. The herpes virus may be genetically modified so as to restrict viral replication in tumors or reduce its cytotoxicity in non-dividing cells. For example, any viral gene involved in nucleic acid metabolism may be inactivated, such as thymidine kinase (Martuza et al., 1991 , Science 252: 854-6), ribonucleotide reductase (RR) (Boviatsis et al., Gene Ther. 1 : 323-31 ; Mineta et al., 1994, Cancer Res. 54: 3363-66), or uracil-N-glycosylase (Pyles et al., 1994, J. Virol. 68: 4963- 72). Another aspect involves viral mutants with defects in the function of genes encoding virulence factors such as the ICP34.5 gene (Chambers et al., 1995, Proc. Natl. Acad. Sci. USA 92: 1411 -5). Representative examples of oncolytic herpes virus include NV1020 (e.g. Geevarghese et al., 2010, Hum. Gene Ther. 21 (9): 1119-28) and T-VEC (Andtbacka et al., 2013, J. Clin. Oncol. 31 , abstract number LBA9008).
In one embodiment, the oncolytic virus of the present invention is obtained from an adenovirus. Methods are available in the art to engineer oncolytic adenoviruses. An advantageous strategy includes the replacement of viral promoters with tumor-selective promoters or modifications of the E1 adenoviral gene product(s) to inactivate its/their binding function with p53 or retinoblastoma (Rb) protein that are altered in tumor cells. In the natural context, the adenovirus E1B55kDa gene cooperates with another adenoviral product to inactivate p53 (p53 is frequently dysregulated in cancer cells), thus preventing apoptosis. Representative examples of oncolytic adenovirus include ONYX-015 (e.g. Khuri et al., 2000, Nat. Med 6(8): 879-85) and H101 also named Oncorine (Xia et al., 2004, Ai Zheng 23(12): 1666-70).
In a preferred embodiment, the oncolytic virus of the present invention is an oncolytic poxvirus. Preferably, the oncolytic poxvirus 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 (VV) 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, LIVP, 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 Buller et al. 1985 Nature 317:813-5., Genbank accession number AAA48082.1 , March 15, 2006), 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 AAA48082.1 ). 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 in the I4L and/or 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 modified in the M2L locus, in the J2R locus, and in the I4L and/or 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, 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 cancer 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, LIVP, 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 viral vector may not be oncolytic, but instead be a non- propagative viral vector.
The term “non-propagative viral vector” refers to viral vectors that are unable to propagate in host cells or tissues. These viral vectors can be replication-defective or replication-impaired vectors (e.g. viral vector genetically disabled), 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 viral vector can be rendered replication-defective by partial or total deletion or inactivation of regions critical to viral replication. Such replication-defective or impaired viral vectors typically require for propagation, permissive cell lines which bring up or complement the missing/impaired functions. These viral vectors 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 viral vectors 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.
Suitable replication-defective or replication-impaired viral vectors, with sufficient transgene capacity to encode the fusion polypeptide according to the invention, may be selected from poxviruses, adenoviruses, herpes viruses, paramyxoviruses (including measles virus), rhabdoviruses (such as VSV or Maraba virus), and viral like particles.
Although one may use wild type or native viruses (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 polypeptide having an IL-7 activity as described hereinafter.
A particularly suitable non-propagative viral vector for use in the invention is obtained from a poxvirus. As used herein the term "poxvirus" refers to a virus belonging to the Poxviridae family with a preference for the Chordopoxvirinae subfamily 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 (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.
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 viral vector 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 viral vector 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 viral vector 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.
Still another suitable non-propagative viral vector for use in the context of the present invention is an adenovirus (Ad), preferably originating from a human or an animal adenovirus (e.g. canine, ovine, simian, etc.). Any serotype can be employed. Desirably, the adenoviral vector originates from a human adenovirus, or from a chimpanzee adenovirus. Representative examples of chimp Ad include without limitation ChAd3 (Peruzzi et al., 2009, Vaccine 27: 1293), ChAd63 (Dudareva et al., 2009, Vaccine 27: 3501 ), AdC6, AdC7 (Cervasi et al., 2015, J. of Virology, 87(17):9420-9430, Chen et al., 2015, J. of Virology, 84(20): 10522-10532), ChAdOxI (Dicks et al., 2012, PLoS One.; 7(7): e40385) and any of those described in the art (see for example W003/000283; W003/046124; W02005/071093; W02009/073103; W02009/073104; W02009/ 105084; W02009/136977 and W02010/086189). Preferably, said non-propagative viral vector for use is a human adenovirus, preferably selected from the group consisting of species A, B, C, D, E, F and G, with a preference for species B, C and D. Said human adenovirus is preferably selected from the group consisting of serotypes 1 , 2, 3, 5, 6, 7, 8, 9, 10, 11 , 13, 14, 15, 16, 17, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 53, 54, 55, 56 and 57, and is more preferably from the group consisting of serotypes 5, 11 , 26 and 35.
Replication-defective adenoviruses can be obtained as described in the art, e.g. by deletion of at least a region of the adenoviral genome or portion thereof essential to the viral replication, with a specific preference for partial or total deletion of the E1 region (E1A and/or E1 B) comprising E1 coding sequences. The present invention also encompasses viruses having additional deletion(s)/modification(s) within the adenoviral genome (e.g. all or part of regions, like non-essential regions as E3 region, or essential regions as E2 and E4, as described in Lusky et al., 1998, J. Virol 72: 2022; WO94/28152; W003/104467; Capasso et al., 2014, Viruses, 6, 832-855; Yamamoto et al., 2017, Cancer Sci 108 (2017) 831-837). In a preferred embodiment, the non-propagative viral vector for use in the invention is a human adenovirus 5 which is defective for E1 function (e.g. with a deletion extending from approximately positions 459 to 3510 or 455 to 3512 by reference to the sequence of the human Ad5 disclosed in the GenBank under the accession number M_73260 and in Chroboczek et al. (1992, Virol. 186:280) and further deleted within the E3 region (e.g. with a deletion extending from approximately positions 28591 to 30469 by reference to the same Ad5 sequence).
Other non-propagative viral vectors suitable in the context of the invention are paramyxoviruses, in particular morbilliviruses, with a specific preference for measles virus. Various attenuated strains are available in the art (Brandler et al, 2008, CIMID, 31 : 271 ; Singh et al., 1999, J. virol. 73(6): 4823), such as and without limitation, the Edmonston A and B strains (Griffin et al., 2001 , Field’s in Virology, 1401 -1441 ), the Schwarz strain (Schwarz A, 1962, Am J Dis Child, 103: 216), the S-191 or C-47 strains (Zhang et al., 2009, J Med Virol. 81 (8): 1477). Insertion between P and M genes or between H and L genes is particularly appropriate.
Additional therapeutic polypeptide/ gene
While the vector according to the invention may encode only the fusion polypeptide of the invention, it may also further comprise 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.
Preferably, the therapeutic polypeptide is 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.
Suicide gene products
The term “suicide gene” refers to a gene coding for a protein able to convert a precursor of a drug 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 genes and corresponding precursors of a drug comprising one nucleobase moiety are disclosed in the following Table 6.
Table 6
Figure imgf000057_0001
Desirably, the suicide gene encodes a protein having at least a 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 highly cytotoxic 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 subti Us (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 oncolytic virus 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).
Immunostimulatory polypeptides
The term “immunomodulatory polypeptide” refers to a polypeptide targeting a component of a signalling 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 polypeptides
The term “antigenic” refers to the ability to induce or stimulate a measurable immune response in a subject into which the vector according to the invention encoding the polypeptide qualified as antigenic has been introduced. The stimulated or induced immune response against the antigenic polypeptide expressed by said vector according to the invention 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 multiparameters 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 vector according to the invention 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 vector according to 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 nonlimiting 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 nonlimiting 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.
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 vector 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 encompasses naturally occurring antibodies and those engineered by man; 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 (e.g. rodent or camelid antibody) or chimeric. A nonhuman antibody can be humanized by recombinant methods to reduce its immunogenicity in man. The antibody may derive from any of the well-known isotypes (e.g. IgA, IgG and IgM) and any subclasses of IgG (lgG1 , lgG2, lgG3, lgG4). In addition, it may be glycosylated, partially glycosylated or non-glycosylated. 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 other than those from camelids or cartilaginous fishes 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 (eventually 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 and their position in the complete heavy or light chain sequence is determined according to various numbering disclosed previously with respect to sdAb. 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 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. Regulatory elements
In accordance with the present invention, the vector 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 vector 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 pK1L 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 promoter is particularly appropriate for use in adenoviral vectors (e.g. Ad5, Ad11 , Ad26, Ad35).
The vector may contain one or more promoters depending on the number of nucleic acid molecule(s) to be expressed. Preferably, when the viral vector encodes a fusion polypeptide according to the invention and another molecule of interest, 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 fusion polypeptide according to the invention is placed under the control of a promoter selected from 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 vector
The nucleic acid molecule(s) or expression cassette(s) (encoding the fusion polypeptide of the invention and optionally another polypeptide) is/are inserted into the vector by any appropriate technique known in the art.
In the case of non-viral vectors, in particular plasmid vectors, conventional cloning techniques may be used, in particular based on the use of restriction enzymes and ligases, using restriction sites present in the plasmid vector of interest and corresponding sequences in the nucleic acid molecule(s)/ expression cassette(s).
In the case of viral vectors, the nucleic acid molecule(s) /expression cassette(s) may be inserted in any suitable location within the virus 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 an adenoviral vector or a poxviral vector 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).
In the case of poxviral vectors, thymidine kinase (TK) gene, Ribonucleotide reductase (RR) gene and F2L gene are particularly appropriate for insertion in oncolytic vaccinia viruses, such as Copenhagen and Western Reserve vaccinia virus, and 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 encompasses 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 the case of adenoviruses, E3 and E4 regions are particularly appropriate for insertion in oncolytic adenovirus virus, and E1 region is particularly appropriate (although insertion in E2 region, E3 region, E4 region or intergenic zones can also be envisaged) for insertion into non-propagative adenoviruses.
Methods for producing viral vectors
Once generated, a viral vector of the invention may be produced/amplified using conventional techniques.
The present invention thus also relates to a method for producing the viral vector of the invention, comprising 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.
The choice of the producer cell depends on the type of viral vector to be produced, and those skilled in the art know which producer cells or cell lines are suitable for which viral vector.
Among poxviruses, choice of the producer cell also depends on the type of poxviral vector to be produced.
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 US5,879,924), 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).
For adenoviruses, and more particularly for E1 -deleted non-propagative adenoviral vectors, suitable cell lines include the 293 cells (Graham et al., 1997, J. Gen. Virol. 36: 59-72) as well as the PER-C6 cells and HER96 (e.g. Fallaux et al., 1998, Human Gene Ther. 9: 1909-1917; W097/00326) or any derivative of these cell lines. But any other cell line described in the art can also be used in the context of the present invention, especially any cell line used for producing product for human use such as Vero cells, HeLa cells and avian cells. Such cells may be adapted for expressing the E1 genes lacking to the defective virus.
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 viral vectors 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 viral vectors. 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 viral vectors 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 viral vectors 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 viral vectors 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.
Host cells
The present invention also relates to a host cell comprising the nucleic acid molecule according to the invention or the vector according to the invention.
Host cells comprising a non-viral vector
In an embodiment, the invention relates to a host cell comprising a non-viral vector according to the invention.
It is expected that those skilled in the art are knowledgeable of the numerous expression systems available in the art for expressing polypeptides and of the methods for introducing a vector into a host cell. Such methods include, but are not limited to microinjection, CaPO4- mediated transfection, DEAE-dextran-mediated transfection, electroporation, lipofection/liposome fusion, gene guns, transduction, viral infection as well as direct administration into a host organism via various means. The method may also be used in association with conventional transfection reagents that facilitate introduction of nucleic acids in host cells, such as polycationic polymers (e.g. chitosan, polymethacrylate, PEI, etc) and cationic lipids (e.g.DC-Chol/DOPE, transfectam, lipofectin, etc).
Host cells infected with a viral vector
In another 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.
Methods of production of the fusion polypeptide
The present invention also relates to a method for recombinantly producing the fusion polypeptide according to the invention, comprising the steps of: a) culturing in vitro a host cell according to the invention under conditions suitable for growth of the host cell, b) recovering the cell culture, and c) optionally purifying the produced fusion polypeptide.
Host 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 prokaryotic and eukaryotic expression systems available in the art for such purposes.
The fusion polypeptide can be recovered from the culture supernatant and/or from the host cell (e.g. upon cell lysis). The recovered materiel can optionally be purified by well- known purification methods including ammonium sulfate precipitation, acid extraction, gel electrophoresis; filtration and chromatographic methods (e.g. reverse phase, size exclusion, ion exchange, affinity, hydrophobic-interaction, hydroxyapatite, high performance liquid chromatography, etc). The conditions and techniques to be used depend on factors such as net charge, molecular weight, hydrophobicity, hydrophilicity and will be apparent to those having skill in the art. Moreover, the level of purification will depend on the intended use. For example, protein concentration can be evaluated by Bradford assay (Biorad), endotoxin levels can be evaluated by techniques such as the Portable Test System (Charles River Laboratories) and the mass of the purified polypeptides can be measured using MALDI (Matrix- Assisted Laser Desorption/lonisation) or electrospray methods.
Composition
The present invention also relates to a composition comprising the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention 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.
The fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention 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 other 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 viral vector, a stabilizing formulation adapted to the specific viral vector 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. S01 buffer: 342,3 g/L saccharose, 10 mM Tris, 1 mM MgCb, 150 mM NaCl, 54 mg/L, Tween 80; ARME buffer: 20 mM Tris, 25 mM NaCl, 2.5% Glycerol (w/v), pH 8.0; 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).
Preferably, the composition comprises a therapeutically effective amount of the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention or any combination thereof.
A “therapeutically effective amount” corresponds to the amount of the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention or any combination thereof may be routinely determined by a practitioner in the light of the relevant circumstances.
When the composition comprises a viral vector according to the invention, individual doses for the viral vector 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. 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 vector, 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 invent ion.
When the composition comprises a non-propagative poxviral vector, it preferably comprises between approximately 106 virus particles (VP), and approximately 1012 VP, more preferably between approximately 107 pfu and approximately 1011 pfu; even more preferably between approximately 108 pfu and approximately 1010 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 -propagative poxviral vector, these doses being convenient for human use, with a preference for individual doses comprising approximately 109 pfu of poxviral vector. When the composition comprises an oncolytic adenoviral vector, it preferably comprises between approximately 106 virus particles (vp), and approximately 1012 vp.
When the composition comprises a non-propagative adenoviral vector, it preferably comprises between approximately 106 and approximately 1014 vp, preferably between approximately 107 and approximately 1013 vp, more preferably between approximately 108 and approximately 1012 vp, and even more preferably between approximately 109 and approximately 1011 vp (e.g. dose of 109, 2x109, 3x109 , 4x109 , 5x109 , 6x109 , 7x109 , 8x109, 9x109, 1O10, 2x1O10, 3x1O10, 4x1O10, 5x1O10, 6x1O10, 7x1O10, 8x1O10, 9x1O10, 1011 vp).
The composition according to the invention maybe formulated for any suitable administration route, including intravenous, intramuscular, subcutaneous, oral, intranasal, transdermal or intratumoral administration.
Therapeutic uses and methods of treatment
The present invention also relates to therapeutic uses of the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention or any combination thereof, as well as to associated methods of treatment.
The present invention thus also relates to the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention or any combination thereof, for use as a medicament or as a vaccine.
The present invention also relates to the use of the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention or any combination thereof, for manufacturing a medicament or a vaccine.
The present invention also relates to the use of the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention or any combination thereof.
Treatment or prevention of cancer
The present invention thus also relates to the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention or any combination thereof, for use in the treatment or prevention of cancer. The present invention also relates to the use of the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 . 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, and colon cancer (Yarchoan et al. 2019 JCI Insight; 4:e126908),, and those cancers are thus particularly preferred 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention or any combination thereof. In such embodiments, the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention or any combination thereof, for use for inhibiting tumor cell growth in vivo.
The present invention also relates to the use of the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention or any combination thereof.
Enhancement of immune response to tumor cells
The present invention thus also relates to the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention or any combination thereof.
In one embodiment, the administration of the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 oncolytic virus 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 viral vector according to the invention (in particular an oncolytic poxviral vector, such as an oncolytic vaccinia virus vector) is used, such viral vector preferably provides a higher therapeutic efficacy than the one obtained in the same conditions either with a similar oncolytic virus not encoding the fusion polypeptide of the invention alone or with the fusion polypeptide of the invention alone. More preferably, the viral vector preferably provides a higher therapeutic efficacy than the combination of a similar oncolytic virus not encoding the fusion polypeptide of the invention and the fusion polypeptide 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 viral vector of the invention compared to either the virus or the fusion polypeptide 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.
Administra tion
In all above-described therapeutic uses and methods, the fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide according to the invention, the nucleic acid molecule according to the invention, the vector according to the invention, the host cell according to the invention, the composition according to the invention 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 fusion polypeptide, nucleic acid molecule, vector, host cell, composition or combination thereof may be used as a stand-alone therapy. Alternatively, the fusion polypeptide, nucleic acid molecule, vector, host cell, composition or 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 fusion polypeptide, nucleic acid molecule, vector, host cell, composition or 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. W02012/004367), 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 fusion polypeptide, nucleic acid molecule, vector, host cell, composition or combination thereof according to the invention 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).
Particularly preferred antagonists of an inhibitory immune checkpoint distinct from PD- L1 that may be used in conjunction with the fusion polypeptide, nucleic acid molecule, vector, host cell, composition or combination thereof according to the invention include antagonist of CTLA-4.
The following examples merely intend to illustrate the present invention. EXAMPLES
Example 1 : Generation, evaluation and humanization of anti-PD-L1 sdAb
Generation of bindins clones
Six male alpacas were immunised with recombinant human PD1 (Randox) whilst a further six alpacas were immunised with recombinant human PD-L1 (Randox). All 12 animals were immunised at monthly intervals for 7 months. Lymph node cells were harvested, and single domain antibody libraries were generated by RT- PCR using published primer sets (Maass et al, 2007; Harmsen et al. ,2000). DNA Librarieswere ligated into the pScreen expression vector and transformed in Top10 E. coli competent cells (Invitrogen) for expression by heatshock at 42° C for 1 minute. Antibody clones binding PD-L1 were subsequently isolated by ELISA screening against Human recombinant PD1 and PD-L1 (Randox and R&D Systems).
In total 223 sdAb clones (142 against PD1 and 81 against PD-L1 ) were isolated and sequenced using the Big Dye terminator kit v3.1 (Thermo-Fisher). Of 223 clones sequenced, 77 clones (53 against PD1 and 24 against PD-L1 ) were subsequently selected on the basis of having substantially different DNA sequences to undergo scaled-up expression and characterisation assessment.
Pilot expressions
77 clones selected on the basis of sequence diversity were expressed in scaled up 200ml cultures as follows. 1 l of 5 g/ml pScreen plasmid mini-prep containingPD1 /PD-L1 sdAb sequence was transformed into Top10 E. coli competent cells (Invitrogen) for expression by heatshock at 42 °C for 1 minute. Cells were subsequently spread on agar plates containing 50|jg/ml of ampicillin (Sigma) and incubated overnight at 37° C. The following morning colonies of each clone were inoculated into 200ml of TB media (Melford) and grown up at 37 degrees. The following day cultures were lysed using Bugbuster cell lysis reagent and purified by immobilised metal affinity chromatography (IMAC) using Talon resin (Takara). SdAb preparations were then quantified at 280nm and 2 g of each antibody was electrophoresed on a 12 % polyacrylamide gel via standard methods. Antibody purities were assigned based on visual inspection of the electrophoresed gels (See Table 7 for results of selected clones). Table 7. Antibody purities for selected clones
Figure imgf000086_0001
PD1/PD-L1 Inhibition ELISA assay
A PD1 /PD-L1 inhibition assay was developed to test all 77 clones for inhibition of the PD1 /PD-L1 interaction. In short, an ELISA plate was coated overnight at 4° C with 1 pg/mL of recombinant human PD1 (Randox) in PBS pH7.4 (Figure 1 ). The followingday, the contents of the plate were discarded, and the plate was blocked with 300 pL/well of blocking reagent (Roche Cat# 11921681001 ) and incubated at 37° C for 1 h. Post incubation, the blocking reagent was discarded and 50 pL of 60 ng/mL of PDL1 -Fc (RnD Sys Cat# 156-B7) was added to each well and incubated for 1 hourat 37°C. Following incubation, 50 pL per well of serially diluted (10,000 to 0.1 ng/mL) anti-PD1 or anti-PDL1 sdAb clones were added to test for blocking of the PD1 /PD-L1 interaction. The plate was incubated at 37°C for 1 h. This was followed by 6 x 300 pL/well wash with TBS-T followed by addition of 1 /1000 dilution of goat anti-human IgG HRP conjugate (Abeam Ab97225) detector antibody. The plate wasonce more washed with 6 x 300 pL/well of TBS-T and the signal was then developed by adding 50 pL /well of TMB reagent and incubating at room temperature for 20 min in the dark. Signal development was stopped by addition of 50 pL of 2N sulphuric acid. The optical density of each well was read at OD450nm. The results of this assay demonstrated that of the 77 clones tested for inhibition of PD1 /PD-L1 binding only 7 clones (9.1%) demonstrated positive inhibition of PD1 /PD-L1 binding. Of the seven blocking clones only one clone (clone 32.1A1 ) gave total inhibition of PD1 /PD-L1 binding at a concentration of 1 pg/ml (See Figure 2 for blocking results, see Table 8 for sequence of all blocking clones).
Table 8 - CDR sequences of selected clones
Figure imgf000087_0001
Further clone-assessment Following the described blocking-assay, the sequence database containing the alignment of all 223 sequences was scrutinised to identify clones that shared similar sequences to those clones identified in the above assay, which successfully blockedthe PD1 /PDL1 interaction. In all, four further clone sequences were identified which were largely similar to those blocking clones already identified including one clone 32.2F7 which differed from the lead blocking clone 32.1A1 by only four residues (including one residue substitution in CDR2 and one residue substitution in CDR3). These four clones (46.4H7, 46.2D2.D12.D5, 46.2D2.H7 and 32.2F7) were subsequently expressed and purified as described previously before being assessed in the same blocking assay described above using the lead PD1 and PD-L1 blockingantibodies 32.1A1 and 46.3C10 as positive controls. The results demonstrated that the lead anti-PDL1 clone 32.1A1 gave a blocking effect which was similar as compared to its close homologue 32.2F7, whilst none of the new anti-PD1 clones
5 demonstrated blocking equivalent or better than the lead PD1 binding antibody 46.3C10 (Figure 3).
Measurement of Bindins affinity of clone 32. 1A 1
On an Octet Red96 instrument (ForteBio), recombinant Human PDL1 -Fc chimeric protein (R&D Systems) was bound to the surface of Protein G biosensors (ForteBio) for up to 5 minutes, until a minimum binding response of 1.0 nM was achieved. Sensors were dipped into assay buffer for one minute before exposure to the anti- PDL1 single domain antibody 32.1A1 at a range of concentrations from 500 ng/mL to 7.81 ng/mL. After 5 minutes of incubation in the presence of sdAb, sensors were incubated in assay buffer for 15 minutes. The data was acquired in a PBS-based assay buffer (pH 7.40) at 30° C, in 96 well plates constantly agitated at 1000 rpm. Data traces were analysed using Octet Data Analysis software (version 10) by subtracting a reference trace (PD-L1 loaded sensor without sdAb analyte) and applying a Global Fit which assumed a 1 :1 binding model giving a binding affinity (Kd) of 0.47nM. This figure compares favourably with that of 0.7nM stated for the commercial anti-PDL1 product Avelumab as stated in the FDA Center for Drug Evaluation and Research review (Application Number 7610490rig1s000).
Humanisation and testing of clone 32.1A1
The CDRs of the lead blocking clone 32.1A1 were grafted into a model Human VH3 framework sequence and the humanised sequence was synthesized and cloned intothe expression vector pScreen. Following expression of the clone using the methods outlined above the humanised clone was compared to the original clone 32.1A1 in a binding ELISA whereby PD-L1 was coated on the surface of an ELISA plate, blocked and probed with a gradient of each of the humanised and non- humanised antibody fragments. Finally, the ELISA plate was again washed and detected with a secondary detector antibody, anti-Myc HRP (abeam). The results demonstrated that the two antibodies were functionally equivalent and that fullbinding was retained following humanisation (Figure 4).
Table 9 - Comparison signal output from binding ELISAs for clone 32.1A1 and its humanised derivative hum-32.1A1 .
Figure imgf000089_0001
PD1/PD-L1 Inhibition ELISA assay to benchmark against Avelumab
In order to benchmark the lead clone 32.1A1 and its derivatives against the commercially available therapeutic antibody Avelumab developed by Merck, it was necessary to use an assay format, which didn’t employ an anti-IgG detector antibody. To this end, a commercial PD1 :PDL1 inhibition assay was sourced from Aero Biosystems (Cat# EP-101 ). In brief, this assay has PDL1 coated on the surface whilst biotinylated PD1 ligand is detected using a streptavidin-HRP conjugate. We used the assay to compare the blocking of 32.1A1 , its humanised derivative hum32.1A1 and its close relative 32.2F7 to Avelumab (Creative Biolabs, Cat# TAB004ML). The results demonstrated that all three of the single domain antibodies tested gave more than ten times the amount of blocking as compared to Avelumab at similar concentrations (Figure 5).
Cell based Inhibition assay
The two lead PD1 and PD-L1 blocking clones as identified by the ELISA Inhibition assay were tested using the Promega PD1 /PD-L1 Blockade Bioassay (Cat# J1250), a commercial cellular assay to test the inhibition of PD1 /PD-L1 interaction on the cellsurface (Figure 6). The results of this assay also demonstrated the anti- PDL1 clone32.1A1 to have superior blocking in this cell-based assay format than the commercial therapeutic antibody Avelumab (Figure 7).
Stressed Stability and Solubility Studies
A larger scale 6 Litre expression was performed of the humanised 32.1A1 clone with a view to performing a higher stringency purification of the anti-PDL1 32.1A1 sdAb. In short, expressions were carried out as previously described with the exceptionthat the 200ml culture was inoculated into a further 6 litres of TB prior to overnight incubation. The next day, following extraction of the scFv and purification by IMAC on Talon resin, the eluate was subsequently polished by size exclusion chromatography on a Hiload 26/600 superdex 75 PG column (GE Healthcare). The fractions corresponding to the sdAb monomer were subsequently pooled and electrophoresed on an SDS-PAGE gel according to standard methods. (Figure 8).
Subsequently three aliquots of the purified humanised 32.1A1 were stressed by storage for one week at -20, 4 and 37° C. Following storage at the different temperatures, 50pg of each aliquot was analysed on a superdex 75 Increase 5/150 GL size exclusion column to check for the formation of aggregates. The results of this assessment showed no evidence of aggregation at any of the storage temperatures tested. (Figure 9). Simultaneous to this assessment a binding assay was carried out to test the binding capability of the antibody following storage ateach of the three temperatures. In short PD-L1 was coated on the surface of an ELISA plate, blocked and probed with a gradient of the antibody which had been stored at each of the three temperatures. Finally, the ELISA plate was again washed and detected with anti-Myc HRP (abeam) secondary detector antibody. The results of this assay demonstrated that no significant loss of binding had occurred following storage at any of the temperatures tested (Figure 10). Cross-reactivity Studies
Human PD1 , PD-L1 , CTLA4 and TIGIT (all Randox), cynomolgus and mouse PD-L1 (both Si no Biological) and cynomolgus and mouse PD1 (both R&D Systems)were coated and blocked on the surface of an ELISA plate. The plate was probed with a 30ng/ml dilution of 32.1A1 antibody and incubated with shaking at 37°C for 1 hour. Finally, the ELISA plate was washed and detected with anti-Myc HRP (abeam) secondary detector antibody. The results of this assay demonstrated binding of the 32.1A1 clone to human and cynomolgus PD-L1 but not to mouse PD- L1 or to any of the other unrelated antigens (See Figure 11 ).
Substitution Studies
A series of 15 mutant sdAb clones were synthesized using alanine substitution at 15 different residues across CDR3 of humanised 32.1A1 to identify residues which are important for binding of the h32.1A1 antibody to PDL1 antigen (See Figure 12).
Following synthesis, all mutant clones were expressed as outlined previously in the “Pilot expressions” part and subsequently tested on ELISA for binding affinity as by the method already outlined in the “Humanisation and testing of clone 32.1A1” part.
The results indicated that of the 15 clones tested, four clones (GS645, GS651 , GS656 and GS659) were identified which showed binding equivalent to that of the parental clone h32.1A1 (See Figure 13 A&B). Additionally, one clone (GS648) showed complete obliteration of PDL1 binding with all other clones showing a levelof intermediate binding between that of the parent clone h32.1A1 and that of the clone with no binding (GS648).
In conclusion, of the 16 residues of CDR3 shown in Figure 12, 4 of those residues were identified by alanine substitution to not be critical for the recognition of PDL1 antigen by h32.1A1. These residues were the residues at positions 1 , 7, 13 and 16 as designated in Figure 12. The flexible nature of the residue at position 13 had previously been forecasted by the presence of two different amino acids at this position in the two different PDL1 - binding sister clones 32.1A1 and 32.2F7. Example 2: Generation, vectorization and characterization of fusion polypeptides comprising a humanized anti-PD-L1 sdAb and an extracellular fragment of CD40L or 4-1 BBL and poxviruses encoding them
Materials and methods
Viruses and plasmids
VVTG18058 (empty VACV, VACV control, or unarmed control VACV) is a Vaccinia virus (Copenhagen strain) deleted of J2R and I4L genes. VVTG18058 was used as an unarmed control virus. VVTG18058 was produced on chicken embryo fibroblasts (CEF). Titration was performed by plaque assay on Vero cells.
The plasmid pTG19274 is a plasmid encoding for an irrelevant FLAG-tagged molecule. Plasmid pTG19274 is used as a negative control.
The plasmid pTG19970 is a plasmid encoding for the polypeptide GS542-CD40L-FLAGtag under the pH5R poxvirus promoter.
The plasmid pTG20032 is a plasmid encoding for the 4-1 BBL ectodomain alone (amino acids 80-254 of human 4-1 BBL amino acid sequence NP_003802.1*), for use as a control against the polypeptide GS542-linker-4-1 BBL construct according to the invention.
Protein sequence encoded by pTG19970 (SEQ ID NO:32):
MGWSCIILFLVATATGVHSQVQLVESGGGLVQPGGSLRLSCAASGRTFREYGMGWFRQAPGKGLE WVATISSSGSYSYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASSLLRGSSSRAESYD SWGQGTLVTVSSNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQ VTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPS QVSHGTGFTSFGLLKLGSDYKDDDDK
Underligned: signal peptide; bold: anti-PD-L1 sdAb; normal: amino acids 119-261 of human CD40L; italic: FLAG tag peptide.
The plasmid pTG19971 is a plasmid encoding for the polypeptide GS542-linker-CD40L- FLAGtag under the pH5R poxvirus promoter.
Protein sequence encoded by pTG19971 (SEQ ID NO:31 ):
MGWSCIILFLVATATGVHSQVQLVESGGGLVQPGGSLRLSCAASGRTFREYGMGWFRQAPGKGLE WVATISSSGSYSYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASSLLRGSSSRAESYD SWGQGTLVTVSSGGGSGGGSGGGSNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQL TVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQ PGASVFVNVTDPSQVSHGTGFTSFGLLKLGSDYKDDDDK
Underligned: signal peptide; bold: anti-PD-L1 sdAb; bold underlined: peptide linker; normal: amino acids 119-261 of human CD40L; italic: FLAG tag peptide.
COPTG19971 is a recombinant Copenhagen vaccinia virus double deleted (TK- and RR-) wherein the expression sequence of pTG19971 has been inserted within its J2R locus.
The plasmid pTG20034 is a plasmid encoding for the polypeptide GS542-linker-4-1 BBL- FLAGtag under the pH5R poxvirus promoter.
Protein sequence encoded by pTG20034 (SEQ ID NO:33):
MGWSCIILFLVATATGVHSQVQLVESGGGLVQPGGSLRLSCAASGRTFREYGMGWFRQAPGKGLE WVATISSSGSYSYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASSLLRGSSSRAESYD SWGQGTLVTVSSGGGSGGGSGGGSDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLT GGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSE ARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGSDYK DDDDK
Underlined: signal peptide; bold: anti-PD-L1 sdAb; bold underlined: peptide linker; normal: amino acids 80-254 of human 4-1 BBL; italic: FLAG tag peptide.
Anti-PD-L1 -CD40L fusion constructions
DNA sequence encoding Anti-human PD-L1 humanized single domain antibody GS542 was fused upstream of DNA sequence encoding the last 143 (N119 to L261 ) residues of human CD40L without (pTG19970) or with a DNA sequence encoding GGGSx3 linker (pTG19971 ) between the two former sequences. These constructs were cloned into transfer plasmid and under the same early/late poxvirus promoter pH5.R.
Anti-PD-L1 -4-1 BBL fusion constructions
DNA sequence encoding Anti-human PD-L1 humanized single domain antibody GS542 was fused upstream of DNA sequence encoding the last 175 (D80 to E254) residues of human 4-1 BBL with a DNA sequence encoding GGGSx3 linker (pTG20034) between the two former sequences. These constructs were cloned into transfer plasmid and under the same early/late poxvirus promoter pH5.R. Human tumor cell lines
The human cervix tumor cell line HeLa (ATCC® CCL-2™) was grown at 37° C, 5% CO? in DMEM (Gibco) supplemented with 10 % FBS, and 40 mg/L of gentamicin.
Infection/transfection experiments
A method for expression of a recombinant protein is the infection/transfection method. Such method consists of infecting a cell, for example a HeLa cell, with a vaccinia virus (a poxvirus) and transfecting said cell with a plasmid encoding the gene of interest under regulation control of a poxvirus promotor. Such method allows the expression of the encoded gene of interest within the cell. The expression product may be recovered within the supernatant for further analysis.
Co-infection/transfection in HeLa cells was carried out with the goal of selecting the most effective CD40L construction (s) to be vectorized in VACV genome.
Briefly, cells were seeded, two days prior infection, at 4E+05 cells/well/3 mL of complete medium (DMEM 4 Gibco ref. 41966-029; Glutamine 2mM; Gentamicin 40 pg/ml; 10% fetal bovine serum FBS) in 6-well-plates.
Before infection, the culture media was removed and replaced by 400 pL of vaccinia virus preparation (VVTG18058) in PBS+ (PBS + 1 % cations) corresponding to a MOI 1. After 30 min. at room temperature (RT), the viral inoculum was removed and replaced by 1 .2 mL of complete medium without FBS. The plates were incubated for 2 h at 37 ° C with 5 % CO?. Transfection was then performed by addition of 1 pg of plasmid DNA formulated with 4.5 pL of Lipofectamine 2000 (Invitrogen, 11668-027) in each well, following the provider’s protocols. The plasmid pTG19274 (encoding irrelevant FLAG-tagged molecule) was used as a negative control. The infection/transfections were performed in triplicate. The plates were incubated 48 hours at 37 °C and 5 % CO2. The culture supernatants were then collected, centrifuged, and filtrated on 0.1 pm filters to remove all virus particles and cellular debris. The clarified supernatants were stored at -80 °C until use.
Expression experiment using recombinant viruses
HeLa cells were seeded in 6-well plate at 1.5E+06 cells/well/2 mL of complete medium (DMEM 4 Gibco ref. 41966-029; Glutamine 2mM; Gentamicin 40 pg/ml; 10% FBS the day prior infection. Cells were infected at MOI 0.1 with one of the following viruses COPTG19971 , or VVTG18058. After 30 min of incubation the culture medium was discarded and replaced by 2 ml of DMEM; Glutamine 2mM; Gentamicin 40 pg/ml. Cells were incubated 48 hours at 37 °C with 5 % CO? and then the culture supernatant was recovered and treated as described above.
CD40L and 4-1 BBL immunoblot
Twenty-five pL of samples (clarified supernatants) from infections/transfections were treated with Laemmli buffer (Biorad, 161 -0747) containing (Reducing) or not (Non reducing) beta-mercaptoethanol. In case of reducing conditions, samples were heated at 95 ° C for 3 minutes. Samples were then loaded on poly-acrylamide gel (TGX 4-15% Stain Free Biorad) and migration was performed in Tris Glycine SDS buffer (Biorad 161 -0772). Western blot was performed using Transblot Turbo System, (Biorad) set up on Midi Program High Molecular weight. Blots were then incubated with anti-FLAG-HRP conjugated antibody (Sigma A8592) at 2 pg/mL using Ibind Flex Western System (Invitrogen ref SLF2000). Positive controls were culture medium from an infection/transfection with irrelevant plasmids pTG19274 encoding a FLAG-tagged recombinant protein or pTG19333 encoding GFP. Blots were incubated with HRP substrate (Amersham ECL Prime western blotting detection) and luminescence recorded by Chemidoc apparatus.
CD40 and PD-L1 ELISA
Either PD-L1 or CD40-Fc were coated on Medisorp (Nunc) 96-well ELISA plate at 0.5 pg/mL in 50 mM carbonate buffer pH 9.6. Clarified supernatants of the infection/transfection experiment were added to the first well of the ELISA plate and further two-fold serially diluted in ELISA saturation buffer directly on the plate. The wells were then incubated with either CD40-Fc or biotinylated PD-L1 in case of PD-L1 and CD40-Fc coating respectively. Anti-human Fc -HRP conjugated antibody (Bethyl A80-104P) or streptavidin- HRP were added to PD-L1 or CD40-Fc coated wells respectively. Finally, HRP substrate (3, 3', 5, 5' tetramethylbenzidine) TMB was added to each well, absorbance 450 nm measured, and optical density (OD) 450 nm versus 1 /dilution of culture supernatants were plotted using GraphPad prism software.
PD-1 /PD-L1 competition ELISA for vectorized products
Human PD-1 -Fc (R&D systems, 1086-PD) was coated on 96-well ELISA plate at 0.25 pg/mL in 50 mM carbonate buffer pH 9.6. Solutions or culture supernatants containing the PD- L1 blockers were added, and 2-fold serial diluted on plate and then human biotinylated PD-L1 (R&D systems, BT-156) at 0.2 pg/mL were added to all wells. PD-1 /PD-L1 complex formation was detected by addition of Horseradish peroxidase (HRP) conjugated streptavidin (SouthernBiotech, 7105-05) diluted 5000-fold. Finally, HRP substrate (3, 3', 5, 5' tetramethylbenzidine) TMB was added to each well, absorbance 450 nm measured using TECAN microplate reader, and optical density (OD) 450 nm versus 1 /dilution of culture supernatants were plotted using GraphPad prism software.
CD40 agonist activity
HEK-Blue CD40L cells (Invivogen: hkb-cd40) are recombinant cells transformed to express both the human CD40 and a reporter enzyme (secreted embryonic alkaline phosphatase: SEAP) under the transcriptional control of a CD40 inducible promoter. Upon activation of CD40, SEAP is produced, and its enzymatic activity measured in culture medium. The SEAP enzymatic activity is proportional to the CD40 agonist activity.
The measures were performed following provider’s instructions. Briefly, 50,000 HEK-Blue CD40L cells in 90 pL were distributed in 96-well plate and incubated with 20 pL of serial dilutions of clarified supernatants generated by the infections or infection/transfections described above in presence or absence of Hs746T cells (displaying PD-L1 ). In some experiments 100 pg/mL of avelumab (anti-PD-L1 monoclonal antibody) or its isotype control (H27K15) were also added. After 24 hours of incubation at 37 °C and 5% CO2, 40 pL of the culture medium were transferred with 160 pL of SEAP substrate (Invivogen: hb- det2) and incubated 3 hours at 37 °C. Absorbance at 620 nm is measured using microplate reader and optical density versus 1 /supernatant dilution plotted using GraphPad prism software.
4-1 BB agonist activity
4-1 BB Bioassay Promega kit (JA2351 ) was used according to providers instructions. Briefly, 25pL of effector cells/well were mixed with 25pL medium or PD-L1 positive Hs746T cells (50,000 cells/25 pL). Then 25 pL culture supernatants were added to each well. Cells were then incubated at 37° C, 5% CO2 for 6h. 75pL/well of reconstituted Bio-Gio (Promega, G7941 ) were added, and luminescence recorded using Berthold reader and MikroWin 2000 software. Luminescence versus 1 /supernatant dilution was plotted using GraphPad prism software and four-parameter logistic curve analysis was performed. Vectorization and virus production
Recombinant viruses were generated using the same plasmids used in infection/transfection experiment described above. Briefly, chicken embryo fibroblasts (CEF) were infected with parental virus encoding GFP at the J2R (TK) locus and deleted of the I4L (RR) gene. Infected cells were transfected with the transfer plasmid carrying the expression cassette flanked by recombination harms (DNA sequences homolog of upstream and downstream TK locus). Recombinant viruses are selected under binocular by picking “white” (i.e. GFP negative) lysis plaque. Expression cassette was checked by PCR amplification followed by DNA sequencing.
For in vivo experiments, recombinant viruses were produced on CEF (MOI 0.05, 72 hours) cultivated on F500. The cellular suspension containing the virus was homogenized by using a homogenizing mixer equipped with an in-line chamber. Large cellular debris were then eliminated by depth filtration using 5 m pore size filters. The clarified viral suspension was subsequently concentrated and diafiltered with the formulation buffer (Saccharose 100 g/L, Tris 30 mM, pH 7.6) using tangential flow filtration and 0.2 pm pore size hollow fiber microfiltration filters. Purified viruses were aliquoted and stored at -80° C until use.
Results
Expression of GS542-CD40L fusion
HeLa cells were infected and transfected with the transfer plasmids carrying the different CD40L constructions as described above (see also sequences pTG19971 and pTG19970). Expression of recombinant anti-PD-L1 -CD40L proteins in the culture supernatants were assessed by immunoblot using anti-FLAG tag for detection. Figure 14 shows that both anti-PD-L1 -CD40L fusions were expressed at the same level and at the expected monomer size in reducing and non-reducing conditions. Note that neither aggregation nor degradation was observed on this blot.
It is of a particular interest that the fusion polypeptide according to the invention, e.g., a recombinant anti-PD-L1 -CD40L protein, is of a smaller molecular size compared to disclosures of the prior art. Such comparison is shown in Table 10.
Figure imgf000098_0001
Table 10: Fusion polypeptides molecular size in kDa
It is well known that tissue distribution, or cancer penetration, is better for a smaller therapeutic than for bigger ones (Li et al. MAbs. 2016;8(1 ) : 113-9) . A person stated in the art will well know the biodistribution of such a molecule is therefore enhanced.
Binding to CD40 and PD-L1
ELISA assays with immobilized recombinant human CD40 or PD-L1 were set up to investigate the PD-L1 and CD40 bindings ability of both PD-L1 -CD40L constructs (see Figure 15 for design). In those assays, in order to have signal, the construct must bind both PD-L1 and CD40. As shown in Figure 16, the clarified supernatants generated from pTG19970 and pTG19971 infection/transfections were both able to generate signal (i.e to bind CD40 and PD-L1 ) in a dose-response manner and in the two experimental settings (i.e. with either CD40 or PD-L1 immobilized). Interestingly, the pTG19971 demonstrated a clear improvement compared to pTG19970 in both experimental settings.
Biological activity of GS542-CD40L fusion
HeLa cells were infected with vaccinia virus and then transfected with the transfer plasmids carrying the two anti-PD-L1 -CD40L constructions (with and without the linker, respectively pTG19971 and pTG19970) under control of the same poxvirus promoter (i.e. pH5.R). After 48 Hours the culture mediums were harvested, filtered through 0.1 pm filter to remove vaccinia virus and tested on the HEK Bleu CD40L cells that express a reporter protein under the control of a CD40 inducible promoter and in presence or absence of PD- L1 expressing cells (Hs746T). Figure 17 shows that both fusion polypeptides expressed by pTG19970 and pTG19971 have a weak CD40 agonist activity by their own. However, their CD40 agonist activities are greatly enhanced in presence of PD-L1 expressing cells demonstrating the conditional activation of CD40 of those molecules. The fusion polypeptides encoded by the plasmid pTG19971 had a better CD40 agonist activity than fusion polypeptides encoded by the plasmid pTG19970 therefore the former format was used to generate a recombinant Copenhagen vaccinia virus (namely COPTG19971 ) carrying the thymidine kinase (TK) and ribonucleotide reductase (RR) gene deletions for further experiments (see “CD40 agonist activity of COPTG19971 and the role of PD-L1 displayed on cells" part thereafter).
CD40 agonist activity of COPTG19971 and the role of PD-L1 displayed on cells
The recombinant virus named COPTG19971 were used to infect HeLa cells and the culture medium recovered after 48 hours was tested on the CD40 activity assay as previously described. In parallel infection/transfections with their parental plasmids were also performed as reference. To demonstrate the involvement of PD-L1 in the CD40 agonist activity, an excess of competitive anti-PD-L1 antibody (avelumab) was added prior to supernatant in order to hamper the binding of anti-PD-L1 -CD40L fusion to PD-L1. Figure 18A and 18B show for both pTG19971 and COPTG19971 the CD40 agonist activity was dramatically increase by the co-incubation of PD-L1 expressing cells confirming the previous observations. Interestingly, this increase of agonist activity in presence of PD-L1 expressing cells can be blocked almost completely by preincubation with a competitive anti-PD-L1 antibody (i.e; avelumab) but not its corresponding isotype control. This last result demonstrates that the dependency of CD40 agonist activity of the anti-PD-L1 -CD40L fusion is truly dependent of an accessible PD-L1 displayed on cells in the neighborhood of the CD40 expressing cells.
4-1 BB agonist activity of anti-PD-L1 -4-1 BBL fusion
Furthermore, to assess the technical effect of the fusion construct according to the invention to other member of the TNFSF, CD40L moiety was exchanged by 4-1 BBL ectodomain in the pTG19971 construct to generate a GS542-linker-4-1 BBL construct (pTG20034; encoding for SEQ ID NO: 33). Said Anti-PD-L1 -4-1 BBL fusion construction referenced as pTG20034 was tested directly after infection transfection on blot and 4- 1 BB bioassay. The level of expression of 4-1 BBL (pTG20032) and GS542-linker-4-1 BBL (pTG20034) was equivalent (Figure 19). In the biological assay, 4-1 BBL or sdAb-linker-4- 1 BBL alone did not induce significant agonist activity as expected. However, in presence of PD-L1 expressing tumor cells (Hs746T) the 4-1 BB agonist activity was dramatically increase for GS542-linker-4-1 BBL only (Figure 20). This result demonstrates that, in presence of PD-L1 positive cells, sdAb anti-PD-L1 -TNFSF fusions are effective agonists of TNFRSF that need to be clustered to signal.
Example 3: Efficacies comparison with two benchmark molecules
The efficacies of pTG19971 as CD40 agonist and PD-L1 blocker were compared to those of two benchmarks molecules disclosed in prior art, with two different designs. Molecule described by Thiemann et al. (W02021 /229103) is an anti-PD-L1 Fab (fragment antigen binding) comprising on one hand, a light chain and on the other hand, a VH and CH1 domains of heavy chain of an anti-PD-L1 antibody. The heavy chain is fused to single chain tri meric CD40L ectodomains corresponding to three CD40L ectodomains linked together by GS linkers. This construct has some limitations for vectorization: it requires two expression cassettes (one for the light chain and the other for the fusion heavy chain- CD40L) with a precisely balanced expression of the two transgenes to get an optimized assembly of the molecule. Moreover, the cloning of three CD40L requires a careful degeneration of DNA sequence to avoid any internal recombination into the transgene. In the case of the present invention GS542-linker-CD40L (pTG19971 ) necessitates one transgene and does not contain any repetitive sequence. Moreover, in present invention the trimerization of CD40L allows the formation of trimeric anti-PD-L1 sdAb and therefore a putative avidity effect compared to the monomeric anti-PD-L1 Fab of Thiemann et al. The other benchmark molecule disclosed in Medler et al. (Medler J et al. Theranostics. 2022 Jan 1 ; 12(4) : 1486-1499) is the fusion of a CD40 agonist F(ab’)2 (i.e. two Fab linked by the hinge of the IgG) with the heavy chain fragment fused to a single chain fragment variable (scFv) of an anti-PD-L1 antibody. The produced protein is therefore a bivalent bispecific molecule able to bind two CD40 and two PD-L1 targets. Like the other benchmark molecule according to Thiemann et al. describes above, it necessitates two cassettes of expression.
These two benchmark molecules have been cloned into same plasmid and under the same poxvirus promoter as pTG19971 . Two plasmids were used to clone the two chains of each benchmark protein: pTG20154 and pTG20155 encode Thiemann’s product whereas pTG20156 and pTG20157 encode Medler’s product. Those plasmids were used for infection/transfection along with pTG19971 and the culture medium assessed for their CD40 agonist activities in presence and absence of PD-L1 expressing cells. The CD40 agonist activity of all constructs in absence of PD-L1 cells were weak at best (Figure 21 ). However, the presence of PD-L1 cells in the assay increased dramatically the CD40 activity of all samples. However, the CD40 agonist activity of pTG19971 was equivalent to the one of Thiemann’s molecule, but much better than the Medler’s one.
The same samples were then tested in a PD-1 /PD-L1 competition ELISA. In this assay the performance of pTG19971 outcompetes the two benchmark molecules by demonstrating, by far the best inhibition of the PD-1 /PD-L1 interaction (Figure 22). This result highlights the fact that the auto assembly of CD40L moiety results into the trimerization of anti-PD- L1 sdAb that itself translates into an avidity effect for PD-L1 binding. The current invention has demonstrated several advantages over the other disclosed molecules: its smaller size allowing a better penetration into the tumor, its simpler sequence (one transgene instead of two) allowing easy and efficient vectorization, its unique outstanding CD40 agonist and PD-L1 blocking activities.
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W02021 /229103

Claims

1 . A fusion polypeptide comprising a single domain antibody (sdAb) specifically binding to programmed death-ligand 1 (PD-L1 ) fused to a member of the Tumor Necrosis Factor superfamily of ligands (TNFSF) or a functional fragment or derivative thereof, wherein: a) the sdAb specifically binding to PD-L1 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),
• the heavy chain CDR3 comprises the sequence X2SLLRGX3SSRAEX4YDX5, wherein each of X2 to X5 independently represents any amino acid (SEQ ID NO:3), and b) the sdAb specifically binding to PD-L1 is fused to the member of the TNFSF or functional fragment or derivative thereof directly or indirectly through a peptide linker.
2. The fusion polypeptide according to claim 1 , 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).
3. The fusion polypeptide 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, P, T, C, A, V, G and P; and
• X6 and X7 are independently selected from A, V, G and P.
4. The fusion polypeptide according to claim 3, wherein the heavy chain CDR3 of the sdAb specifically binding to PD-L1 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 independently selected from A, V, G and P.
5. The fusion polypeptide according to claim 3, 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.
6. The fusion polypeptide according to claim 3, 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.
7. The fusion polypeptide according to any one of claims 1 to 6, 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).
8. The fusion polypeptide according to any one of claims 1 to 7, wherein the sdAb specifically binding to PD-L1 comprises or consists of an amino acid sequence having at least 80 % sequence identity with the amino acid sequence SEQ ID NO: 13 or SEQ ID NO: 14, preferably the sdAb specifically binding to PD-L1 comprises or consists of the amino acid sequence SEQ ID NO:13.
9. The fusion polypeptide according to any one of claims 1 to 8, wherein the sdAb specifically binds to human PD-L1.
10. The fusion polypeptide according to any one of claims 1 to 9, wherein the member of the TNFSF or functional fragment or derivative thereof is selected from CD40L, 4-1 BBL, Baff, APRIL, EDA-A1 , GITRL, OX40L, CD70, TL1A, LIGHT, LToB2, RANKL, TWEAK, FASLG, TRAIL, TNF and LTo and functional fragments or derivatives thereof.
11. The fusion polypeptide according to claim 10, wherein the member of the TNFSF or functional fragment or derivative thereof is selected from category II TNFSF members, preferably selected from CD40L, 4-1 BBL, Baff, APRIL, EDA-A1 , OX40L, CD70, TWEAK, FASLG, TRAIL, and TNF and functional fragments or derivatives thereof.
12. The fusion polypeptide according to claim 10, wherein the member of the TNFSF or functional fragment or derivative thereof is selected from TNFSF members involved in immune cell activation, preferably selected from CD40L, 4-1 BBL, GITRL, OX40L, CD70, TL1A and functional fragments or derivatives thereof.
13. The fusion polypeptide according to claim 10, wherein the member of the TNFSF or functional fragment or derivative thereof is selected from category II TNFSF members involved in immune cell activation, preferably selected from CD40L, 4-1 BBL, OX40L, CD70, and functional fragments or derivatives thereof.
14. The fusion polypeptide according to any one of claims 1 to 13, wherein the member of the TNFSF or functional fragment or derivative thereof is selected from CD40L, 4-1 BBL, and functional fragments or derivatives thereof; most preferably the member of the TNFSF is selected from CD40L and functional fragments or derivatives thereof, such as fragments comprising or consisting of the extracellular domain of human CD40L having the sequence ranging from position 119 to position 261 of the Entrez Gene reference amino acid sequence NP_000065.1.
15. The fusion polypeptide according to any one of claims 1 to 14, wherein the functional fragment of the member of the TNFSF is an extracellular fragment of the member of the TNFSF.
16. The fusion polypeptide according to any one of claims 1 to 15, wherein the sdAb specifically binding to PD-L1 and the member of the TNFSF or functional fragment or derivative thereof are fused indirectly through a peptide linker; preferably the peptide linker is selected from peptides of 3 to 20 amino acids comprising amino acids selected from glycine, serine, threonine, asparagine, alanine and/or proline.
17. The fusion polypeptide according to any one of claims 1 to 16, which further comprises: a) a signal peptide in N-terminal; and/or b) a tag peptide, preferably in C-terminal.
18. The fusion polypeptide according to claim 16, wherein peptide linker is selected from any one of SEQ ID NO: 15 to SEQ ID NO: 23, preferably the peptide linker of SEQ ID NO: 22.
19. The fusion polypeptide according to any one of claims 1 to 18, which comprises an amino acid sequence with at least 80% identity with SEQ ID NO:31 to SEQ ID NO: 34, preferably which is selected from SEQ ID NO: 31 and SEQ ID NO: 33, more preferably the fusion polypeptide consists of SEQ ID NO: 31 .
20. A nucleic acid molecule encoding the fusion polypeptide according to any one of claims 1 to 19.
21. A vector comprising the nucleic acid molecule according to claim 20.
22. The vector according to claim 21 , which is a mRNA, a plasmid or a viral vector.
23. The vector according to claim 22, which is a viral vector, more preferably selected from poxviruses, adenoviruses, herpes viruses, paramyxoviruses, and rhabdoviruses.
24. The vector according to claim 23, which is a poxvirus, preferably of the Chordopoxvirinae family, more preferably selected from the group consisting of Avi poxvirus genus, Capripoxvirus genus, Lepori poxvirus genus, Mollusci poxvirus genus, Orthopoxvirus genus, Parapoxvirus genus, Suipoxvirus genus, Cervid poxvirus genus, and Yatapoxvirus genus, and chimeras thereof.
25. The vector according to claim 24, 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.
26. The vector according to claim 25, which 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, 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.
27. The vector according to any one of claims 21 to 26, wherein the nucleic acid molecule according to claim 20 is operably linked to suitable regulatory elements for expression in a desired host cell or subject.
28. The vector according to any one of claims 24 to 27, wherein the nucleic acid molecule according to claim 20 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, pTK, pB2R, p28, p11 , pF17R, pA14L and pK1 L promoter, synthetic promoters and early/late chimeric promoters.
29. The vector according to any one of claims 22 to 28, wherein said vector is in the form of infectious viral particles.
30. A method for producing the vector of claim 29, comprising the steps of: a) introducing the viral vector of claim 29 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.
31. A host cell comprising the nucleic acid molecule according to claim 20 or the vector according to any one of claims 21 to 29.
32. A method for recombinantly producing the fusion polypeptide according to any one of claims 1 to 19, comprising the steps of: a) culturing in vitro a host cell according to claim 31 under conditions suitable for growth of the host cell, b) recovering the cell culture, and c) optionally purifying the produced fusion polypeptide.
33. A composition comprising the fusion polypeptide according to any one of claims 1 to 19, the nucleic acid molecule according to claim 20, the vector according to any one of claims 21 to 29, the host cell according to claim 31 or any combination thereof, wherein the composition preferably further comprises a pharmaceutically acceptable vehicle.
34. The composition according to claim 33, which comprises 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 vector according to any one of claims 24 to 28.
35. The composition according to claim 33 or 34, which is formulated for intravenous, intramuscular, subcutaneous, oral, intranasal, transdermal or intratumoral administration.
36. The fusion polypeptide according to any one of claims 1 to 19, the nucleic acid molecule according to claim 20, the vector according to any one of claims 21 to 29, the host cell according to claim 31 , the composition according to any one of claims 33 to 35 or any combination thereof, for use as a medicament or as a vaccine.
37. The fusion polypeptide according to any one of claims 1 to 19, the nucleic acid molecule according to claim 20, the vector according to any one of claims 21 to 29, the host cell according to claim 31 , the composition according to any one of claims 33 to 35 or any combination thereof, for use in the treatment or prevention of cancer, 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.
38. The fusion polypeptide, nucleic acid molecule, vector, host cell, composition or combination for use according to claim 37, 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.
39. The fusion polypeptide, nucleic acid molecule, vector, host cell, composition or combination for use according to claim 38, for use in conjunction with one or more other therapeutic agents selected from the group consisting of agonists of a stimulatory immune checkpoint, and antagonists of an inhibitory immune checkpoint; wherein 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, CD137 (4-1 BB), 0X40, CD27, CD40, or GITR; and wherein the antagonist of an inhibitory immune checkpoint is preferably selected from antagonist antibodies to human PD-1 , SIRPa, CD47, PD-L2, LAG3, Tim3, BTLA, or CTLA4.
PCT/EP2023/061453 2022-05-02 2023-05-02 Fusion polypeptide comprising an anti-pd-l1 sdab and a member of the tnfsf WO2023213764A1 (en)

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