WO2020012485A1 - Pd1-4-1bbl variant fusion protein and methods of use thereof - Google Patents

Pd1-4-1bbl variant fusion protein and methods of use thereof Download PDF

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Publication number
WO2020012485A1
WO2020012485A1 PCT/IL2019/050782 IL2019050782W WO2020012485A1 WO 2020012485 A1 WO2020012485 A1 WO 2020012485A1 IL 2019050782 W IL2019050782 W IL 2019050782W WO 2020012485 A1 WO2020012485 A1 WO 2020012485A1
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amino acid
acid sequence
seq
specific embodiments
fusion protein
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French (fr)
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WO2020012485A9 (en
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Iris Pecker
Itai BLOCH
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Kahr Medical Ltd
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Kahr Medical Ltd
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Priority to CN201980052864.9A priority Critical patent/CN112543767B/zh
Priority to JP2021500820A priority patent/JP2021531265A/ja
Priority to KR1020217004309A priority patent/KR20210044221A/ko
Priority to SG11202013170RA priority patent/SG11202013170RA/en
Priority to US17/258,220 priority patent/US12286466B2/en
Priority to BR112021000347-9A priority patent/BR112021000347A2/pt
Priority to CA3104778A priority patent/CA3104778A1/en
Priority to AU2019301315A priority patent/AU2019301315A1/en
Application filed by Kahr Medical Ltd filed Critical Kahr Medical Ltd
Priority to EP19833260.3A priority patent/EP3820887A4/en
Priority to MX2021000047A priority patent/MX2021000047A/es
Publication of WO2020012485A1 publication Critical patent/WO2020012485A1/en
Publication of WO2020012485A9 publication Critical patent/WO2020012485A9/en
Priority to IL280102A priority patent/IL280102A/en
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    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • 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
    • 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
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • 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

Definitions

  • the present invention in some embodiments thereof, relates to a PD1-4-1BBL variant fusion protein and methods of use thereof.
  • Dual Signaling Proteins also known as Signal-Converting-Proteins (SCP), which are currently known in the art as bi-functional fusion proteins that link an extracellular portion of a type I membrane protein (extracellular amino-terminus), to an extracellular portion of a type II membrane protein (extracellular carboxyl-terminus), forming a fusion protein with two active sides (see e.g US Patent Nos. 7,569,663 and 8,039,437).
  • PD1 is a surface co-inhibitory receptor of the immunoglobulin super family.
  • PDI is expressed on T cells, B cells, monocytes, natural killer cells, dendritic cells and many tumor-infiltrating lymphocytes (TILs).
  • TILs tumor-infiltrating lymphocytes
  • PD I has two ligands: PDU1 (also named B7H1; CD274) and PDL2 (B7DC; CD273), that are both co-inhibitory.
  • PDL1 is expressed on resting and activated T cells, B cells, dendritic cells, macrophage, vascular endothelial ceils and pancreatic islet cells.
  • PDL1 is also known to be expressed in various types of cancers, especially in NSCLC, melanoma, renal cell carcinoma, gastric cancer, hepatocellular as well as cutaneous and various leukemia cancers, multiple myeloma and others.
  • PDL2 expression is seen on macrophages and dendritic cells alone and is far less prevalent than PDL1 across tumor types.
  • the expression of PDL1 is induced by multiple proinflammatory molecules, including types I and II IFN-g, TNF-a, EPS, GM-CSF and VEGF, as well as the cytokines EL- 10 and IL-4, with IFN-g being the most potent inducer.
  • the tumor micro-environment upreguiates PDL1 expression, thereby, promoting immune suppression.
  • cancer cells In response to immune attack, cancer cells overexpress PDL1, which binds to PDI receptor on T cells, inhibiting the activation of T-cells, thus suppressing T-cell attack and inducing tumor immune escape.
  • PDL1 binds to PDI receptor on T cells, inhibiting the activation of T-cells, thus suppressing T-cell attack and inducing tumor immune escape.
  • PDl/PDLi pathway regulates immune suppression by several mechanisms:
  • CTL-CD8 Restrains cytotoxic T lymphocytes (CTL-CD8) activity
  • 4-1BBL is the activating ligand of the 4- IBB receptor (CD137), a member of the TNF receptor superfamily and a potent activation-induced T cell costimulatory molecule. 4-1BBL naturally forms a homo-trimer but signaling via 4-1BB requires significant oligomerization of 4-lBBL.
  • 4- 1BBL is present on a variety of antigen presenting cells (APCs), including dendritic cells (DCs), B cells, and macrophages.
  • APCs antigen presenting cells
  • DCs dendritic cells
  • B cells and macrophages.
  • the 4- IBB receptor is not detected ( ⁇ 3 %) on resting T cells or T cell lines; however, 4- IBB is stably upregulated when T cells are activated. 4-IBB activation upregulates survival genes, enhances cell division, induces cytokine production and prevents activation induced cell death in T-cells.
  • a PD1-4-1 BBL fusion protein comprising a PD1 amino acid sequence and a 4-1BBL amino acid sequence, wherein the PD1 amino acid sequence:
  • (a) is 123 - 148 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21, 85, 89, 93 and 1 15 and does not comprise any of amino acid segments P i - L5 and F146 - V150 corresponding to SEQ ID NO: 2, or is 138-145 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 117 and 119 and does not comprise an amino acid segment F146 - VI 50 corresponding to SEQ ID NO: 2; and/or
  • (b) comprises a C to S amino acid modification in a position corresponding to amino acid 93 of SEQ ID NO: 2, and/or
  • (aa) is 185 - 202 amino acids in length having at least 95 % identity to an amino acid sequence selected form the group consisting of SEQ ID NOs: 22-25, is 170 - 197 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 127 and 129 and does not comprise an amino acid segment G198-E205 corresponding to SEQ ID NO: 3, is 170 - 182 amino acids in length having at least 80 % identity to SEQ ID NO: 125 and does not comprise an amino acid segment A1 -E23 corresponding to SEQ ID NO: 3 or is 184 amino acids in length having at least 80 % identity' to SEQ ID NO: 123; and/or
  • (bb) comprises three repeats of a 4-1BBL amino acid sequence
  • fusion protein is capable of at least one of:
  • a PD1- 4-1BBL fusion protein comprising a PD1 amino acid sequence and a 4-1BBL amino acid sequence, wherein the PD1 amino acid sequence:
  • (a) is 123 - 148 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21 , 85, 89 and 93 and does not comprise any of amino acid segments PI - L5 and F146 - V150 corresponding to SEQ ID NO: 2 and/or
  • (b) comprises a C to S amino acid modification in a position corresponding to amino acid 93 of SEQ ID NO: 2;
  • the 4-1 BBL amino acid sequence is 185 - 202 amino acids in length having at least 95 % identity to an amino acid sequence selected form the group consisting of SEQ ID NOs: 22-25; and wherein the fusion protein is capable of at least one of:
  • a PD1- 4-1BBL fusion protein comprising a PD1 amino acid sequence and a 4-1BBL amino acid sequence, wherein the PD1 amino acid sequence is 123 - 148 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21 and does not comprise any of amino acid segments PI - L5 and F146 - VI 50 corresponding to SEQ ID NO: 2; and/or wherein the 4-1 BBL amino acid sequence is 185 - 202 amino acids in length having at least 95 % identity to an amino acid sequence selected form the group consisting of SEQ ID NOs: 22-25; and wherein the fusion protein is capable of at least one of:
  • the PD1 -4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 80 % identity to SEQ ID NO: 145.
  • the PD 1-4-1 BBL fusion protein amino acid sequence comprises SEQ ID NO: 145.
  • the PD 1-4- IBB L fusion protein amino acid sequence consists of SEQ ID NO: 145.
  • an isolated polypeptide comprising a PD1 amino acid sequence, wherein the PD1 amino acid sequence is 123 - 148 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO : 18-21 , 85, 89, 93 and 1 15 and does not comprise any of amino acid segments PI --- L5 and F146 - V 150 corresponding to SEQ ID NO: 2, or is 138- 145 amino acids in length having at least 80 % identity' to an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 1 17 and 119 and does not comprise an amino acid segment F146 - VI 50 corresponding to SEQ ID NO: 2; and optionally comprises a C to S amino acid modification in a position corresponding to amino acid 93 of SEQ ID NO: 2; and where
  • an isolated polypeptide comprising a PD1 amino acid sequence, wherein the PD1 amino acid sequence is 123 - 148 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ) ID NO: 18-21, 85, 89 and 93 and does not comprise any of amino acid segments PI - L5 and FI46 - VI 50 corresponding to SEQ ID NO: 2; and optionally comprises a C to S amino acid modification in a position corresponding to amino acid 93 of SEQ ID NO: 2; and wherein the polypeptide is capable of binding PDL1.
  • an isolated polypeptide comprising a PD1 amino acid sequence, wherein the PD! amino acid sequence is 123 - 148 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21 and does not comprise any of amino acid segments PI - L5 and F 146 - VI 50 corresponding to SEQ ID NO: 2; and wherein the polypeptide is capable of binding PDL1.
  • an isolated polypeptide comprising a 4-1BBL amino acid sequence, wherein the 4-1BBL amino acid sequence is 185 - 202 amino acids in length having at least 95 % identity to an amino acid sequence selected form the group consisting of SEQ ID NOs: 22-25, is 170 - 197 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 127 and 129 and does not comprise an amino acid segment G198-E205 corresponding to SEQ ID NO: 3, or is 170 - 182 amino acids in length having at least 80 % identity to SEQ ID NO: 125 and does not comprise an amino acid segment A1-E23 corresponding to SEQ ID NO: 3; and optionally comprises three repeats of the 4-1BBL amino acid sequence; and wherein the polypeptide is capable of at least one of:
  • an isolated polypeptide comprising a 4-1BBL amino acid sequence, wherein the 4-1BBL amino acid sequence is 185 - 202 amino acids in length having at least 95 % identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-25; and wherein the polypeptide is capable of at least one of:
  • the PDl amino acid sequence is no more than 140 amino acids long.
  • the PD1 amino acid sequence is at least 126 amino acids long.
  • the PD 1 amino acid sequence comprises the SEQ ID NO: 18, 19, 20 or 21.
  • the PD1 amino acid sequence comprises the SEQ ID NO: 18, 19, 20, 21, 85, 89 or 93
  • the PD1 amino acid sequence comprises the SEQ ID NO: 18, 19, 20, 21, 85, 89, 93 or 115
  • the PDl amino acid sequence comprises the SEQ ID NO: 117 or 119.
  • the PDl amino acid sequence comprises SEQ ID NO: 75, 79, 81, 83, 87, 91 or 95
  • the PDl amino acid sequence comprises SEQ ID NO: 75, 83, 87, 91 or 95.
  • the PDl amino acid sequence comprises SEQ ID NO: 73.
  • the PD l amino acid sequence comprises the SEQ ID NO: 18
  • the PDl amino acid sequence consists of the SEQ ID NO: 18, 19, 20 or 21.
  • the PDl amino acid sequence consists of the SEQ ID NO: 18, 19, 20, 21, 85, 89 or 93.
  • the PD1 amino acid sequence consists of the SEQ ID NO: 18, 19, 20, 21, 85, 89, 93 or 115.
  • the PD1 amino acid sequence consists of the SEQ ID NO: 117 or 119.
  • the PD1 amino acid sequence consists of SEQ ID NO: 75, 79, 81, 83, 87, 91 or 95.
  • the PD1 amino acid sequence consists of SEQ ID NO: 75, 83, 87, 91 or 95.
  • the PD1 amino acid sequence consists of SEQ ID NO: 73.
  • the PD! amino acid sequence consists of the SEQ ID NO: 18
  • the 4-IBBL amino acid sequence has at least 95 % identity to an amino acid sequence selected form the group consisting of SEQ ID NO: 22-24.
  • the 4-IBBL amino acid sequence does not comprise any of amino acid residues Al - V6 or A1 - G14 corresponding to SEQ ID NO: 3.
  • the 4-IBBL amino acid sequence comprises the SEQ ID NO: 22, 23, 24 or 25.
  • the 4-IBBL amino acid sequence consists of the SEQ ID NO: 22, 23, 24 or 25.
  • the 4-IBBL amino acid sequence comprises the SEQ ID NO: 125, 127 or 129.
  • the 4-IBBL amino acid sequence consists of the SEQ ID NO: 125, 127 or 129.
  • the 4-IBBL amino acid sequence comprises the SEQ ID NO: 123.
  • the 4-1BBL amino acid sequence consists of the SEQ ID NO: 123.
  • the PD1-4-1BBL fusion protein comprises a linker between the PD1 and the 4-IBBL.
  • the PD 1 -4- I BBL fusion protein or the isolated polypeptide comprising a linker between each of the three repeats of the 4-IBBL amino acid sequence.
  • the linker has a length of one to six amino acids.
  • the linker is a single amino acid linker.
  • the linker is glycine
  • the linker is not an Fc domain of an antibody or a fragment thereof.
  • the linker is an Fc domain of an antibody or a fragment thereof.
  • the PD1 -4-1BBL fusion protein being in a form of at least a homo-trimer.
  • the at least homo-trimer is at least 100 kD in molecular weight as determined by SEC-MALS.
  • the PD1-4-1BBL fusion protein or the isolated polypeptide being soluble.
  • the production yield of the fusion protein is at least 1.5 fold higher than the production yield of SEQ ID NO: 5 under the same production conditions, the production conditions comprise expression in a mammalian cell and culturing at 32 - 37 °C, 5 - 10 % CO2 for 5 - 13 days.
  • the amount of aggregates of the fusion protein is at least 20 % lower than the amount of aggregates of SEQ ID NO: 5 under the same production conditions, the aggregates are of at least 300 kDa in molecular weight and the production conditions comprise expression in a mammalian cell and culturing at 32 - 37 °C, 5 - 10 % CO2 for 5 - 13 days.
  • the PD 1 -4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16, 44-56, 97, 101 , 103, 105, 107, 1 11, 1 13, 133, 135, 137, 139, 141, 143 and 147.
  • the PD1 -4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 133, 135, 137, 139, 141, 143 and 147.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16, 44-56, 97, 101, 103, 105, 107 and 1 1 1.
  • the PD1 -4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101 , 103, 105, 107 and 1 1 1.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101, 103, 105 and 107.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16 and 44-56.
  • the PD1 -4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14 and 16
  • the at least 80 % identity comprises at least 90 % identity.
  • the at least 80 % identity comprises at least 95 % identity.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16, 44-56, 97, 101, 103, 105, 107, 111, 1 13, 133, 135, 137, 139, 141, 143 and 147.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 133, 135, 137, 139, 141, 143 and 147.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16, 44-56, 97, 101, 103, 105, 107 and 1 11.
  • the PD 1-4-1 BBL fusion protein amino acid sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101, 103, 105, 107 and 1 1 1.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101, 103, 105 and 107. According to some embodiments of the invention, the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16 and 44-56.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14 and 16.
  • the PD1 -4-1BBL fusion protein amino acid sequence consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16, 44-56, 97, 101, 103, 105, 107, 1 11, 113, 133, 135, 137, 139, 141, 143 and 147.
  • the PD1-4-1BBL fusion protein amino acid sequence consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 133, 135, 137, 139, 141, 143 and 147
  • the PD1-4-1BBL fusion protein amino acid sequence consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16, 44-56, 97, 101, 103, 105, 107 and 111.
  • the PD1-4-1BBL fusion protein amino acid sequence consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101, 103, 105, 107 and 111.
  • the PD1-4-1BBL fusion protein amino acid sequence consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101, 103, 105 and 107
  • the PDI-4-1BBL fusion protein amino acid sequence consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16 and 44-56.
  • the PD1-4-1BBL fusion protein amino acid sequence consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14 and 16.
  • a polynucleotide encoding the PD1-4-1BBL fusion protein or the isolated polypeptide.
  • a nucleic acid construct comprising the polynucleotide, and a regulatory element for directing expression of the polynucleotide in a host cell.
  • the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 57 - 72, 98, 102, 104, 106, 108, 1 12, 114, 134, 136, 138, 140, 142, 144 and 148.
  • the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 57 - 72, 98, 102, 104, 106, 108 and 1 12
  • the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 98, 102, 104, 106, 108 and 112.
  • the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 98, 102, 104, 106 and 108.
  • the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 57 - 72.
  • the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 70 - 72.
  • the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 134, 136, 138, 140, 142, 144 and 148.
  • the polynucleotide comprises SEQ ID NO: 146.
  • a host cell comprising the PD1-4-1BBL fusion protein or the polypeptide or the polynucleotide or the nucleic acid construct.
  • a method of producing a PD1-4-1BBL fusion protein or a polypeptide comprising expressing in a host cell the polynucleotide or the nucleic acid construct.
  • the method comprising isolating the fusion protein or the polypeptide.
  • a method of treating a disease that can benefit from activating immune cells comprising administering to a subject in need thereof the PD1-4-1BBL fusion protein or the isolated polypeptide, the polynucleotide or the nucleic acid construct or the host cell.
  • the method further comprising administering to the subject a therapeutic agent for treating the disease.
  • the PD1-4-1BBL fusion protein or the isolated polypeptide, the polynucleotide or the nucleic acid construct or the host cell for use in treating a disease that can benefit from activating immune cells.
  • the composition further comprising a therapeutic agent for treating the disease.
  • an article of manufacture comprising a packaging material packaging a therapeutic agent for treating a disease that can benefit from activating immune cells; and the PD 1-4-1 BBL fusion protein or the isolated polypeptide, the polynucleotide or the nucleic acid construct or the host cell.
  • cells of the disease express PDLi .
  • the disease comprises a hyper-proliferative disease.
  • the hyper-proliferative disease comprises sclerosis, fibrosis, Idiopathic pulmonary fibrosis, psoriasis, systemic sclerosis/scleroderma, primary biliary cholangitis, primary sclerosing cholangitis, liver fibrosis, prevention of radiation- induced pulmonary fibrosis, myelofibrosis or retroperitoneal fibrosis.
  • the hyper-proliferative disease comprises cancer.
  • the cancer is selected from the group consisting of lymphoma, leukemia and carcinoma.
  • the disease comprises a disease associated with immune suppression or medication induced immunosuppression.
  • the disease comprises HIV, Measles, influenza, LCCM, RSV, Human Rhinoviruses, EBV, CMV or Parvo viruses.
  • the disease comprises an infection.
  • a method of activating immune ceils comprising in-vitro activating immune cells in the presence of the PD1-4-1BBL fusion protein or the isolated polypeptide, the polynucleotide or the nucleic acid construct or the host cell .
  • the activating is in the presence of cells expressing PDLI or exogenous PDLI .
  • the ceils expressing the PDLI comprise pathologic cells.
  • the pathologic ceils comprise cancer cells.
  • the activating is in the presence of an anti- cancer agent.
  • the therapeutic agent for treating the disease or the anti-cancer agent comprises an antibody.
  • the antibody is selected from the group consisting rituximab, cetuximab, trastuzumab, edrecolomab, almetuzumab, gemtuzumab, ibritumomab, panitumumab, Behmumab, Bevacizumab, Bivatuzumab mertansine, Blinatumomab, Blontuvetmab, Brentuximab vedotin, Catumaxomab, Cixutumumab, Daclizumab, Adalimumab, Bezlotoxumab, Certolizumab pegol, Citatuzumab communicatingox, Daratumumab, Dinutuximab, Elotuzumab, Ertumaxomab, Etaracizumab, Gemtuzumab ozogamicin, Girentuximab, Necitum
  • the antibody is selected from the group consisting of rituximab and cetuximab.
  • the therapeutic agent for treating the disease or the anti-cancer agent comprises an IMiD (e.g. Thalidomide, Lena!idomide, Pomalidomide)
  • IMiD e.g. Thalidomide, Lena!idomide, Pomalidomide
  • the method comprising adoptively transferring the immune cells following the activating to a subject in need thereof.
  • the immune cells comprise T ceils.
  • FIG. 1 is a schematic representation of the PD1 -4-1BBL fusion protein referred to herein as “DSP105” (SEQ ID NO: 5) comprising an N-terminal signal peptide and a His-tag (SEQ ID NO: 26). Shown the signal peptide (black underlined, SEQ ID NO: 4), the N-terminal His-tag (black bold), the PD1 domain (red, SEQ ID NO: 2), the glycine linker (black) and the 4-1 BBL domain (blue, SEQ ID NO: 3) and
  • FIGs. 2A-D demonstrate the predicted 3D structure of DSP105 (SEQ ID NO: 5).
  • Figure 2A is a schematic 3D model.
  • PD1 is shown in grey ribbons
  • PDL1 PD1 ligand
  • 4-1 BBL is shown in blue ribbons
  • 2 additional copies of 4-1BBL are shown in light blue.
  • Figure 2B is a schematic, full atomic 3D model.
  • Figures 2C-D are schematic 3D models.
  • the X-ray resolved domains are represented by its surface and colored by a hydrophobicity scale - blue (most hydrophilic) to Brown (Hydrophobic).
  • FIG. 3 is a schematic representation of the domain and segments identified in DSP 105 (SEQ ID NO: 5).
  • An Ig-like V-type domain is highlighted in light blue (the underlined sequence was resolved in X-ray), An X-ray resolved part is highlighted in grey and flanking/unstructured regions are marked with red boxes.
  • FIG. 4 demonstrates the predicted 3D structure of PD! -4-1BBL variant fusion protein referred to herein as“DSP105 varl” (SEQ ID NO: 12).
  • the X-ray resolved domains are displayed in surface representation colored by hydrophobicity scale - Blue (most hydrophilic) to Brown (Hydrophobic).
  • PDL1 PD1 ligand
  • 4- 1BBL is presented in red ribbons, and PD1 in blue ribbons.
  • FIG. 5 demonstrates the predicted 3D structure of PD 1 -4- 1BBL variant fusion protein referred to herein as“DSPl05_var2” (SEQ ID NO: 14).
  • the color code is the same as in Figure 4.
  • FIG. 6 demonstrates the predicted 3D structure of PD 1-4-1 BBL variant fusion protein referred to herein as“DSP 105 var3” (SEQ ID NO: 16).
  • the color code is the same as in Figure 4.
  • FIG. 7 presents images of SDS-PAGE analysis of N-terminal His-tagged DSP 105 (SEQ ID NO: 1), expressed in Expi293 cells; and N-terminal His-tagged DSP 105 varl 7 (SEQ ID NO: 99), expressed in CHO-3E7. Both fusion proteins were purified by a single step affinity purification on a Nickel column and separated under reducing and non-reducing conditions, as indicated.
  • the samples presented in the images are as follows: Lane M - molecular weight marker; Lanes 1 -2: TWO separate preparations of N-terminal His-tagged DSP 105 (SEQ ID NO: 1), Lane 3 - N-terminal His-tagged DSP 105 var l 7 (SEQ ID NO: 99).
  • FIGs. 8A-E present images of SDS-PAGE analysis (marked as PAGE) and western blot (marked as WB) analysis using the indicated antibody of the PD 1-4-1 BBL variant fusion proteins produced in CHO-3E7 cells. Shown are PD1-4-1BBL variant fusion proteins referred to herein as “N-terminal Ms-tagged DSPI05 varl7” (SEQ ID NO: 99, Figure 8A),“DSP105_varl8” (SEQ ID NO: 101, Figure 8B),“DSP105_varl 9” (SEQ ID NO: 103, Figure 8C),“DSP105_var20” (SEQ ID NO: 105, Figure 8D) and“DSP105_var23” (SEQ ID NO: 113, Figure BE) under reducing or non-reducing conditions.
  • N-terminal Ms-tagged DSPI05 varl7 SEQ ID NO: 99, Figure 8A
  • “DSP105_varl8” SEQ ID NO: 101, Figure 8B
  • the samples presented in the images are as follows: Lane M - molecular weight marker; Lanes 1 to 3 - cell culture supernatants collected on day 2, day 4 and day 5 post- transfection, respectively, under reducing conditions; Lane NC 1 - negative control under reducing conditions; Lanes 4 to 6 - cell culture supernatants collected on day 2, day 4 and day 5 post- transfection, respectively, under non-reducing conditions; Lane NC2 - negative control under non reducing conditions; and lane P - multiple-tag (Gen Script, Cat.No. M0101) as a positive control.
  • FIGs. 9A-B present images of SDS-PAGE analysis of the PD 1-4- IBB L variant fusion proteins produced in Expi293F cells. Shown are PD1-4-1BBL variant fusion proteins referred to herein as“N-terminal Ms-tagged DSP105 _varl7” (SEQ ID NO: 99),“DSP105 _varl7” (SEQ ID NO: 97),“DSP105_var20” (SEQ ID NO: 105),“DSP105_var23” (SEQ ID NO: 1 13),“Ms-tagged DSP105_var22” (SEQ ID NO: 110),“Ms-tagged DSP105” (SEQ ID NO: 1),“DSP105_var24” (SEQ ID NO: 133),“DSP105_var25” (SEQ ID NO: 135) and“DSP105_var31” (SEQ ID NO: 147), under reducing ( Figure 9 A) or non-reducing ( Figure 9B) conditions.
  • FIGs. 10A-C present flow cytometric analysis demonstrating DSP105 var!7 (SEQ ID NO: 99) binding to PDL! or 4 IBB expressing cells.
  • FIG. 10A demonstrates expression of the indicated receptors on DLD1-PDL1 or FIT 1080-4 IBB cell lines. The surface expression level of PDL1 or 41BB was determined by immuno-staining of each cell line with the corresponding antibodies, followed by flow cytometric analysis.
  • Figures 10B-C demonstrate binding of N-terminal His- tagged DSP105_varl 7 (SEQ ID NO: 99) to DLD1-PD1 or to H ⁇ T080-4I BB cell lines.
  • FIG. 11 presents flow cytometric analysis demonstrating expression of the 4- IBB receptor on HT1080-4-1BB cell line.
  • FIG. 12 presents a graph demonstrating that the N-terminal His-tagged DSPl05_varl7 protein (SEQ ID NO: 99), DSP105 var 17 protein (SEQ ID NO: 97), DSP105 var 21 protein (SEQ ID NO: 107) and His-tagged PD1-4-1 BBL var_22 (SEQ ID NO: 1 10), promote 4-1BBL/4- 1BB signaling as demonstrated by OD values indicating IL-8 secretion levels from HT1080-4- 1BB cells, following binding of the DSPI05 molecules.
  • FIGs. 13A-D demonstrate the predicted 3D structure of the PD1-4-1BBL variant fusion referred to herein as“DSP 105 V31” (SEQ ID NO: 147)
  • Figure 13A is a schematic 3D ribbon model.
  • PD1 is represented in dark grey ribbons (right-hand side)
  • 41BB-L is represented in black ribbons (left-hand side)
  • linker segments are represented in white ribbons between the structural elements of PD1 and 41BB-L.
  • Figure 13B is a schematic 3D surface display model.
  • PD1 is represented in a dark grey surface display (right-hand side)
  • 41BB-L is represented in a black surface display (left-hand side)
  • linker segments are represented in white ribbons between the structural elements of PD1 and 41BB-L.
  • Figure 13C is a schematic 3D ribbon model of the fusion protein and its binding counterparts.
  • PD1 is represented in dark grey ribbons (right-hand side)
  • 41BB-L is represented in black ribbons (left-hand side)
  • linker segments are represented in white ribbons between the structural elements of PD1 and 41BB-L
  • PD-L1 bound to PD1 is represented in grey ribbons (right-hand side)
  • three 4 IBB receptors are represented in grey ribbons in complex with 41BB-L (left-hand side).
  • Figure 13D is a schematic 3D surface display model of the fusion protein and its binding counterparts.
  • PD1 is represented in a dark grey surface display (right-hand side)
  • 41BB-L is represented in a black surface display (left-hand side)
  • 'linker segments are represented in white ribbons between the structural elements of PD1 and 41 BB-L
  • PD-L1 bound to PD I is represented in grey ribbons (right-hand side)
  • three 4 IBB receptors are represented in grey ribbons in complex with 41 BB-L (left-hand side).
  • the present invention in some embodiments thereof, relates to a PD1-4-1BBL variant fusion protein and methods of use thereof.
  • Dual Signaling Proteins also known as Signal-Converting-Proteins (SCP), which are currently known in the art as bi-functional fusion proteins that link an extracellular portion of a type I membrane protein (extracellular amino-terminus), to an extracellular portion of a type II membrane protein (extracellular carboxyl-terminus), forming a fusion protein with tw ? o active sides.
  • SCP Signal-Converting-Proteins
  • the present inventors were able to generate PD 1 -4- 1BBL fusion proteins comprising a PD I variant and/or a 4-1BBL variant with improved production characteristics (e.g higher yield, lower percentage of aggregates); and which can be advantageously used for activating immune cells (via co-stimulation) in general and treating diseases that can benefit from activating immune cells (e.g. cancer) in particular.
  • production characteristics e.g higher yield, lower percentage of aggregates
  • the following is suggested by the inventors as a mode of action of the PD1-4-1BBL fusion protein of some embodiments of the invention in the treatment of cancer as an example:
  • the PD1 moiety of the PD1-4-1 BBL chimera targets the molecule to tumor and metastasis sites, leading to binding of the fusion to PDLl within the tumor micro-environment.
  • Targeting the fusion protein to the tumor cells or/and tumor micro-environment facilitates an increase in PD1-4-1BBL concentration in the tumor micro-environment and subsequent oligomerization of the 4- 1 BBL moiety of the fusion protein at the tumor site. Since oligomerization of 4-1BBL is a necessary step for 4- IBB signaling, this 4-1BBL binding and oligomerization delivers a 4- IBB co-stimulatory signal that promotes activation of T cells, B cells, NK cells, especially Tumor-Infiltrating Lymphocytes (TILs), and other immune cells at the tumor site, to kill cancer cells.
  • TILs Tumor-Infiltrating Lymphocytes
  • the binding of the fusion protein's PD1 moiety to PDL1 in the tumor site competes with the endogenous PD1 expressed on T cells; thus, removing the inhibition on T cells and further contributing to the activation of TILs in the tumor micro-environment.
  • the above activities of PD 1-4-1 BBL are expected to lead to a synergistic effect on the activation of TILs within the tumor micro-environment, which is expected to be a more specific and more robust effect as compared to the effect of each moiety separately, as well as when using the two different moieties thereof in combination.
  • a PD1-4-1BBL fusion protein comprising a PD 1 amino acid sequence and a 4- 1 BBL amino acid sequence, wherein said PD1 amino acid sequence:
  • (a) is 123 - 148 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21, 85, 89, 93 and 115 and does not comprise any of amino acid segments PI - L5 and F I 46 - VI 50 corresponding to SEQ ID NO: 2, or is 138-145 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 117 and 119 and does not comprise an amino acid segment F 146 - VI 50 corresponding to SEQ ID NO: 2, and/or
  • (b) comprises a C to S amino acid modification in a position corresponding to amino acid 93 of SEQ ID NO: 2;
  • said 4-1BBL amino acid sequence (aa) is 185 - 202 amino acids in length having at least 95 % identity to an amino acid sequence selected form the group consisting of SEQ ID NOs: 22-25, is 170 - 197 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 127 and 129 and does not comprise an amino acid segment G198-E205 corresponding to SEQ ID NO: 3, is 170 - 182 amino acids in length having at least 80 % identity to SEQ ID NO: 125 and does not comprise an amino acid segment A1-E23 corresponding to SEQ ID NO: 3 or is 184 amino acids in length having at least 80 % identity to SEQ ID NO: 123; and/or
  • (bb) comprises three repeats of a 4-1BBL amino acid sequence
  • said fusion protein is capable of at least one of:
  • a PD1 -4-1BBL fusion protein comprising a PD I amino acid sequence and a 4-1 BBL amino acid sequence, wherein said PD1 amino acid sequence:
  • (a) is 123 - 148 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21 , 85, 89 and 93 and does not comprise any of amino acid segments PI - L5 and F146 - VI50 corresponding to SEQ ID NO: 2 and/or
  • (b) comprises a C to S amino acid modification in a position corresponding to amino acid 93 of SEQ ID NO: 2, and/or
  • said 4-1BBL amino acid sequence is 185 - 202 amino acids in length having at least 95 % identity to an amino acid sequence selected form the group consisting of SEQ ID NOs: 22-25; and wherein said fusion protein is capable of at least one of:
  • a PD1-4-1BBL fusion protein comprising a PD I amino acid sequence and a 4-1BBL amino acid sequence, wherein said PD1 amino acid sequence is 123 - 148 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21 and does not comprise any of amino acid segments Pi - L5 and F146 - V150 corresponding to SEQ ID NO: 2 and/or wherein said 4-!BBL amino acid sequence is 185 - 202 amino acids in length having at least 95 % identity to an amino acid sequence selected form the group consisting of SEQ ID NOs: 22-25; and wherein said fusion protein is capable of at least one of:
  • an isolated polypeptide comprising a PDl amino acid sequence, wherein said PD1 amino acid sequence is 123 - 148 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21, 85, 89, 93 and 115 and does not comprise any of amino acid segments PI - L5 and FI 46 - V150 corresponding to SEQ ID NO: 2, or is 138- 145 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 117 and 119 and does not comprise an a ino acid segment F146 - VI 50 corresponding to SEQ ID NO: 2; and optionally comprises a C to S amino acid modification in a position corresponding to amino acid 93 of SEQ ID NO: 2; and wherein said polypeptide is capable of binding PDL1 .
  • an isolated polypeptide comprising a PD1 amino acid sequence, wherein said PD1 amino acid sequence is 123 - 148 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21, 85, 89 and 93 and does not comprise any of amino acid segments PI - L5 and F146 - V! 50 corresponding to SEQ ID NO: 2; and optionally comprises a C to S amino acid modification in a position corresponding to amino acid 93 of SEQ ID NO: 2; and wherein said polypeptide is capable of binding PDL1.
  • an isolated polypeptide comprising a PD1 amino acid sequence, wherein said PD1 amino acid sequence is 123 - 148 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consi sting of SEQ ID NO: 18-21 and does not comprise any of amino acid segments PI - L5 and h i 46 V I 50 corresponding to SEQ ID NO: 2; and wherein said polypeptide is capable of binding PDL1.
  • an isolated polypeptide comprising a 4-lBBL amino acid sequence, wherein said 4-1BBL amino acid sequence is 185 - 202 amino acids in length having at least 95 % identity to an amino acid sequence selected form the group consisting of SEQ ID NOs: 22-25, is 170 - 197 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 127 and 129 and does not comprise an amino acid segment G198-E205 corresponding to SEQ ID NO: 3, or is 170 - 182 amino acids in length having at least 80 % identity to SEQ ID NO: 125 and does not comprise an amino acid segment A1-E23 corresponding to SEQ ID NO: 3; and optionally comprises three repeats of said 4-lBBL amino acid sequence, and wherein said polypeptide is capable of at least one of:
  • an isolated polypeptide comprising a 4-lBBL amino acid sequence, wherein said 4-lBBL amino acid sequence is 185 - 202 a ino acids in length having at least 95 % identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-25; and wherein said polypeptide is capable of at least one of:
  • the term“PD I Programmed Death 1, also known as CD279) refers to the polypeptide of the PDCD1 gene (Gene ID 5133) or a functional homolog eg , functional fragment thereof.
  • the term“PD1” refers to a functional homolog of PD l polypeptide.
  • PD 1 is human PD i .
  • the PD1 protein refers to the human protein, such as provided in the following GenBank Number NP 005009.
  • the PDL1 protein refers to the human protein, such as provided in the following GenBank Number NP_001254635 and XP 054862.
  • the PDL2 protein refers to the human protein, such as provided in the following GenBank Number NP 079515.
  • the phrase“functional homolog” or“functional fragment” when related to PD1 refers to a portion of the polypeptide which maintains the activity of the full length PD1 e.g., PD- Ll binding.
  • Assays for testing binding are well known in the art and include, but not limited to flow' cytometry, BiaCore, bio-layer interferometry Blitz® assay, HPLC.
  • the PD I binds PD-L 1 with a Kd of 1 nM - 100 mM, 10- nM - 10 mM, 100 nM - 100 mM, 200 nM - 10 m M, as determined by SPR analysis, each possibility represents a separate embodiment of the present invention.
  • the PD1 binds PDLI with a Kd of about 270 nM as determined by SPR analysis.
  • the PD1 binds PDLI with a Kd of about 8-9 mM as determined by SPR analysis
  • the PD1 comprises an extracellular domain of said PD1 or a functional fragment thereof.
  • PD1 amino acid sequence comprises SEQ ID NO: 27.
  • PD1 amino acid sequence consists of SEQ ID NO: 27.
  • PD1 nucleic acid sequence comprises SEQ ID NO: 28.
  • PD1 nucleic acid sequence consists of SEQ ID NO: 28.
  • PD1 amino acid sequence comprises SEQ ID NO: 2.
  • PD1 amino acid sequence consists of SEQ ID NO: 2.
  • PD1 nucleic acid sequence comprises SEQ ID NO: 29
  • PD1 nucleic acid sequence consists of SEQ ID NO: 29.
  • PD1 amino acid sequence comprises SEQ ID NO: 30 or SEQ ID NO: 31.
  • PD1 amino acid sequence consists of SEQ ID NO: 30 or SEQ ID NO: 31.
  • PD1 also encompasses functional homologues (naturally occurring or synthetical ly/recombinantly produced), which exhibit the desired activity (/. ⁇ ?., binding PD-L1 and/or PD-L2).
  • Such homologues can be, for example, at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identical or homologous to the polypeptide SEQ ID NO: 2, 27, 30 or 31; or at least 70 %, at least 75 %, at least 80
  • the PD1 functional homologues are at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identical or homologous to the polypeptide SEQ ID NO: 2, 27, 30 or 31; or at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identical to the polynucleotide sequence encoding same (as further described hereinbelow).
  • identity or“sequence identity” refers to global identity, i.e., an identity over the entire amino acid or nucleic acid sequences disclosed herein and not over portions thereof.
  • Sequence identity or homology can be determined using any protein or nucleic acid sequence alignment algorithm such as Blast, ClustalW, and MUSCLE,
  • the homolog may also refer to an ortholog, a deletion, insertion, or substitution variant, including an amino acid substitution, as further described hereinbelow.
  • the PD1 polypeptide may comprise conservative and non- conservative amino acid substitutions (also referred to herein as mutations). Such substitution are known in the art and disclosed e.g. in Maute et al. PNAS, 2015 Nov 24; 112(47):E6506-14; Ju Yeon et al. Nature Communications 2016 volume 7, Article number: 13354 (DOT 10. l038/ncommsl 3354); and Zack KM et al. Structure. 2015 23(12): 2341-2348
  • sequence identity When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity- may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are considered to have "sequence similarity" or “similarity”. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby- increasing the percentage sequence identity.
  • one or more amino acid mutations are located at an amino acid residue selected from: V39, L40, N41, Y43, R44, M45, S48, N49, Q50, T51, D52, K53, A56, Q63, (365, Q66, ⁇ 72, H82, M83, R90, Y96, L97, A100, S102, L103, A 104, P105, K 106, and A107 corresponding to the PD I amino acid sequence set forth in SEQ ID NO: 31.
  • one or more amino acid mutations are located at an amino acid residue selected from: ⁇ 39, L40, N41, Y43, R44, M45, S48, N49, Q50, T51, D52, K53, A56, Q63, G65, Q66, C68, ⁇ 72, H82, ⁇ 183, R9Q, Y96, L97, A100, SI 02, L103, A104, P105, K 106, and A107 corresponding to the PD1 amino acid sequence set forth in SEQ ID NO: 31.
  • one or more amino acid changes are selected from the group consisting of: (1) V39H or V39R; (2) L40V or L40I; (3) N41I or N41V; (4) Y43F or Y43H; (5) R44Y or R44L; (6) M45Q, M45E, M45L, or M45D; (7) S48D, S48L, S48N, S48G, or S48V; (8) N49C, N49G, N49Y, or N49S; (9) Q50K, Q50E, or Q50H; (10) T51 V, T51 L, or T51A, (11) D52F, D52R, D52Y, or D52V; (12) K53T or K53L; (13) A56S or A56L; (14) Q63T, Q63I, Q63E, Q63L, or Q63P; (15) G65N.
  • G65R, G65I, G65L, G65F, or G65V (16) Q66P; (17) ⁇ 721; (18) H82Q; (19) M83L or M83F; (20) R90K; (21) Y96F; (22) L97Y, L97V, or L97I; (23) A100I or A100V; (24) S102T or S 102A, (25) L103I, L103Y, or L103F; (26) A104S, A104H, or A104D; (27) PI 05 A; (28) K106G, K106E, K106I, K106V, K106R, or K106T; and (29) A107P, A107I, or A 107V corresponding to the PD1 amino acid sequence set forth in SEQ ID NO: 31
  • one or more amino acid changes are selected from the group consisting of: (1) V39H or V39R; (2) L40V or L40I; (3) N41I or N41 V; (4) Y43F or Y43H; (5) R44Y or R44L, (6) M45Q, ⁇ 145 E M45L, or M45D; (7) S48D, S48L, S48N, S48G, or S48V; (8) N49C, N49G, N49Y, or N49S; (9) Q50K, Q50E, or Q50H; (10) 15 IV, T51L, or 151 A; (11) D52F, D52R, D52Y, or D52V, (12) K53T or K53L; (13) A56S or A56L, (14) Q63T, Q63I, Q63E, Q63L, or Q63P; (15) G65N, G65R, G65I, G65L, G65F, or G65V; (16) Q66P;
  • an amino acid mutation is located at an amino acid residue €93 corresponding to the PD1 amino acid sequence set forth in SEQ ID NO: 2 (e.g. equivalent to an amino acid residue C68 corresponding to the PD 1 amino acid sequence set forth in SEQ ID NO: 31)
  • the PD1 polypeptide may comprise a C to S amino acid modification in a position corresponding to amino acid residue 93 of the PD1 amino acid sequence set forth in SEQ ID NO: 2 (e.g. equivalent to amino acid residue 68 of the PD1 amino acid sequence set forth in SEQ ID NO: 31).
  • the PD1 amino acid sequence comprises SEQ ID NO: 73.
  • PD1 amino acid sequence consists of SEQ ID NO: 73.
  • the PD1 nucleic acid sequence comprises SEQ ID NO: 74
  • the PD1 nucleic acid sequence consists of SEQ ID NO: 74.
  • the phrase“corresponding to PD1 amino acid sequence as set forth in SEQ ID NO: 31”,“corresponding to SEQ ID NO: 31”,“corresponding to PD1 amino acid sequence as set forth in SEQ ID NO: 2” or“corresponding to SEQ ID NO: 2”, intends to include the corresponding amino acid residue relative to any other PD I amino acid sequence.
  • the PD1 of some embodiments of the present invention is at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identical or homologous to the polypeptide SEQ ID NO: 18-21; or at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least
  • the PD1 amino acid sequence has at least 80 % k!entity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 19, 20, 21, 85, 89, 93 and 1 15.
  • the PD1 amino acid sequence has at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 1 17 and 1 19.
  • the PD1 amino acid sequence has at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1 17 and 119.
  • the PD1 amino acid sequence has at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21, 85, 89 and 93
  • the PD! amino acid sequence has at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21.
  • the PD1 amino acid sequence has at least 80 % identity to SEQ ID NO: 18.
  • the PD! amino acid sequence has at least 80 % identity to SEQ ID NO: 19.
  • the PD1 amino acid sequence has at least 80 % identity to SEQ ID NO: 20.
  • the PD1 amino acid sequence has at least 80 % identity to SEQ ID NO: 21.
  • the PD I amino acid sequence has at least 80 % identity to SEQ ID NO: 85.
  • the PD1 amino acid sequence has at least 80 % identity to SEQ ID NO: 89.
  • the PD I amino acid sequence has at least 80 % identity to SEQ ID NO: 93.
  • the PD1 amino acid sequence has at least 80 % identity to SEQ ID NO: 115.
  • the PD1 amino acid sequence has at least 80 % identity to SEQ ID NO: 117.
  • the PD1 amino acid sequence has at least 80 % identity to SEQ ID NO: 119.
  • the PD1 amino acid sequence has at least 85 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 19, 20, 21, 85, 89, 93 and 115.
  • the PD1 amino acid sequence has at least 85 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 117 and 119.
  • the PD1 amino acid sequence has at least 85 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 117 and 119.
  • the PD1 amino acid sequence has at least 85 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21, 85, 89 and 93.
  • the PD1 amino acid sequence has at least 85 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21.
  • the PD! amino acid sequence has at least 85 % identity to SEQ ID NO: 18.
  • the PD1 amino acid sequence has at least 85 % identity to SEQ ID NO: 19.
  • the PD! amino acid sequence has at least 85 % identity to SEQ ID NO: 20.
  • the PD1 amino acid sequence has at least 85 % identity to SEQ ID NO: 21.
  • the PD1 amino acid sequence has at least 85 % identity to SEQ ID NO: 85.
  • the PDI amino acid sequence has at least 85 % identity to SEQ ID NO: 89.
  • the PDI amino acid sequence has at least 85 % identity to SEQ ID NO: 93.
  • the PDI amino acid sequence has at least 85 % identity to SEQ ID NO: 115.
  • the PDI amino acid sequence has at least 85 % identity to SEQ ID NO: 117
  • the PDI amino acid sequence has at least 85 % identity to SEQ ID NO: 119.
  • the PDI amino acid sequence has at least 90 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 19, 20, 21, 85, 89, 93 and 115.
  • the PDI amino acid sequence has at least 90 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 117 and 119.
  • the PDI amino acid sequence has at least 90 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 117 and 1 19.
  • the PDI amino acid sequence has at least 90 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21, 85, 89 and 93
  • the PDI amino acid sequence has at least 90 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21.
  • the PDI amino acid sequence has at least 90 % identity to SEQ ID NO: 18.
  • the PD! amino acid sequence has at least 90 % identity to SEQ ID NO: 19.
  • the PD1 amino acid sequence has at least 90 % identity to SEQ ID NO: 20.
  • the PD! amino acid sequence has at least 90 % identity to SEQ ID NO: 21.
  • the PD1 amino acid sequence has at least 90 % identity to SEQ ID NO: 85.
  • the PD1 amino acid sequence has at least 90 % identity to SEQ ID NO: 89.
  • the PD I amino acid sequence has at least 90 % identity to SEQ ID NO: 93.
  • the PD1 amino acid sequence has at least 90 % identity to SEQ ID NO: 1 15.
  • the PD I amino acid sequence has at least 90 % identity to SEQ ID NO: 1 17.
  • the PD1 amino acid sequence has at least 90 % identity to SEQ ID NO: 119.
  • the PD1 amino acid sequence has at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 19, 20, 21, 85, 89, 93 and 1 15.
  • the PD1 amino acid sequence has at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 1 17 and 119.
  • the PD1 amino acid sequence has at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 117 and 1 19.
  • the PD1 amino acid sequence has at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21, 85, 89 and 93, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence has at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence has at least 95 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21.
  • the PD1 amino acid sequence has at least 95 % identity to SEQ ID NO: 18.
  • the PD1 amino acid sequence has at least 95 % identity to SEQ ID NO: 19.
  • the PD1 amino acid sequence has at least 95 % identity to SEQ ID NO: 20.
  • the PDI amino acid sequence has at least 95 % identity to SEQ ID NO: 21.
  • the PD1 amino acid sequence has at least 95 % identity to SEQ ID NO: 85.
  • the PD! amino acid sequence has at least 95 % identity to SEQ ID NO: 89.
  • the PD1 amino acid sequence has at least 95 % identity to SEQ ID NO: 93.
  • the PD1 amino acid sequence has at least 95 % identity to SEQ ID NO: 115.
  • the PD I amino acid sequence has at least 95 % identity to SEQ ID NO: 117.
  • the PD1 amino acid sequence has at least 95 % identity to SEQ ID NO: 1 19.
  • the PD I amino acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21, 85, 89, 93 and 115, each possibility represents a separate embodiment of the present invention.
  • the PDI amino acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 117 and 119, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1 17 and 119, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21, 85, 89 and 93, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21, each possibility represents a separate embodiment of the present invention.
  • the PD I amino acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 18.
  • the PD1 amino acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 19
  • the PD1 amino acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 20.
  • the PD1 amino acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 21.
  • the PD I amino acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 85.
  • the PD1 amino acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 89.
  • the PD1 amino acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 93
  • the PD I amino acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 1 15.
  • the PD1 amino acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 117.
  • the PD1 amino acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 119. According to specific embodiments, the PD1 amino acid sequence does not comprise amino acid segment PI - L5 corresponding to SEQ ID NO: 2.
  • the PDI amino acid sequence does not comprise amino acid segment F 146 - VI 50 corresponding to SEQ ID NO: 2.
  • the PD1 amino acid sequence does not comprise any of amino acid segments PI - L5 and F 146 - VI 50 corresponding to SEQ ID NO: 2.
  • the PD1 amino acid sequence does not comprise any of amino acid residues P I - L5 corresponding to SEQ ID NO: 2.
  • the PD1 amino acid sequence does not comprise any of amino acid residues F146 - V150 corresponding to SEQ ID NO: 2.
  • the PDI amino acid sequence does not comprise any of amino acid residues PI - L5 and F146 - VI 50 corresponding to SEQ ID NO: 2
  • the PDI amino acid sequence does not comprise SEQ ID NO: 8 or any fragment thereof.
  • the PD1 amino acid sequence does not comprise SEQ ID NO: 8.
  • the PDI amino acid sequence does not comprise SEQ ID NO: 9 or any fragment thereof.
  • the PD1 amino acid sequence does not comprise SEQ ID NO: 9.
  • the PDI amino acid sequence does not comprise SEQ ID NO: 8 and 9 or any fragment thereof.
  • the PDI amino acid sequence does not comprise SEQ ID NO: 8 and 9.
  • the PDI amino acid sequence comprises SEQ ID NO: 18, 19, 20, 21, 85, 89, 93 or 115.
  • the PDI amino acid sequence comprises SEQ ID NO: 18, 1 17 or 119.
  • the PD I amino acid sequence comprises SEQ ID NO: 117 or 1 19
  • the PDI amino acid sequence comprises SEQ ID NO: 18, 19, 20, 21, 85, 89 or 93.
  • the PDI amino acid sequence comprises SEQ ID NO: 75, 79, 81, 83, 87, 91 or 95
  • the PD1 amino acid sequence comprises SEQ ID NO: 75, 83, 87, 91 or 95.
  • the PDl amino acid sequence comprises SEQ ID NO: 18, 19, 20 or 21.
  • the PD1 amino acid sequence comprises SEQ ID NO: 18 or 21.
  • the PDl amino acid sequence comprises SEQ ID NO: 18. According to specific embodiments, the PD1 amino acid sequence comprises SEQ ID NO: 19. According to specific embodiments, the PD1 amino acid sequence comprises SEQ ID NO: 20. According to specific embodiments, the PD1 amino acid sequence comprises SEQ ID NO: 21. According to specific embodiments, the PD1 amino acid sequence comprises SEQ ID NO: 85. According to specific embodiments, the PDl amino acid sequence comprises SEQ ID NO: 89. According to specific embodiments, the PD1 amino acid sequence comprises SEQ ID NO: 93. According to specific embodiments, the PDl amino acid sequence comprises SEQ ID NO: 75. According to specific embodiments, the PD1 amino acid sequence comprises SEQ ID NO: 79.
  • the PD1 amino acid sequence comprises SEQ ID NO: 81.
  • the PD1 amino acid sequence comprises SEQ ID NO: 83.
  • the PD! amino acid sequence comprises SEQ ID NO: 87.
  • the PD1 amino acid sequence comprises SEQ ID NO: 91.
  • the PD1 amino acid sequence comprises SEQ ID NO: 95.
  • the PD1 amino acid sequence comprises SEQ ID NO: 115.
  • the PD1 amino acid sequence comprises SEQ ID NO: 117.
  • the PD1 amino acid sequence comprises SEQ ID NO: 119.
  • the PDl amino acid sequence consists of SEQ ID NO: 18, 19, 20, 21, 85, 89, 93 or 115
  • the PDl amino acid sequence consists of SEQ ID NO: 18, 117 01- 1 19.
  • the PDl amino acid sequence consists of SEQ ID NO:117 or 119.
  • the PDl amino acid sequence consists of SEQ ID NO: 18, 19, 20, 21, 85, 89 or 93.
  • the PD1 amino acid sequence consists of SEQ ID NO: 75, 79, 81, 83, 87, 91 or 95
  • the PD1 amino acid sequence consists of SEQ ID NO: 75, 83, 87, 91 or 95
  • the PD1 amino acid sequence consists of SEQ ID NO: 18, 19, 20 or 21
  • the PD1 amino acid sequence consists of SEQ ID NO: 18 or 21
  • the PD1 amino acid sequence consists of SEQ ID NO: 18.
  • the PDl amino acid sequence consists of SEQ ID NO: 19.
  • the PDl amino acid sequence consists of SEQ ID NO: 20.
  • the PDl amino acid sequence consists of SEQ ID NO: 21
  • the PDl amino acid sequence consists of SEQ ID NO: 85.
  • the PDl amino acid sequence consists of SEQ ID NO: 89.
  • the PDl amino acid sequence consists of SEQ ID NO: 93.
  • the PDl amino acid sequence consists of SEQ ID NO: 75.
  • the PDl amino acid sequence consists of SEQ ID NO: 79. According to specific embodiments, the PDl amino acid sequence consists of SEQ ID NO: 81. According to specific embodiments, the PDl amino acid sequence consists of SEQ ID NO: 83. According to specific embodiments, the PDl amino acid sequence consists of SEQ ID NO: 87. According to specific embodiments, the PDl amino acid sequence consists of SEQ ID NO: 91 According to specific embodiments, the PDl amino acid sequence consists of SEQ ID NO: 95. According to specific embodiments, the PDl amino acid sequence consists of SEQ ID NO: 115.
  • the PDl amino acid sequence consists of SEQ ID NO: 117.
  • the PDl amino acid sequence consists of SEQ ID NO:
  • the PD I nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least
  • the PD! nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least
  • the PD1 nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least
  • the PD1 nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least
  • the PD1 nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least
  • the PD I nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least
  • the PD1 nucleic acid sequence has at least 70 %, at least
  • the PD1 nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 34, each possibility represents a. separate embodiment of the present invention.
  • the PD1 nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least
  • the PD I nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least
  • the PD1 nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 80, each possibility represents a separate embodiment of the present invention.
  • the PD1 nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least
  • the PD! nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least
  • the PD1 nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least
  • the PD1 nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100
  • the PD1 nucleic acid sequence has at least 70 34, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 1 o, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 >, at least
  • the PD1 nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 92, each possibility represents a separate embodiment of the present invention.
  • the PD I nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least
  • the PD! nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least
  • the PD1 nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least
  • the PD1 nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least
  • the PD! nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least
  • the PD i nucleic acid sequence has at least 80 % identity to SEQ ID NO: 32, 33, 34, 35, 76, 80, 82, 84, 86, 88, 90, 92, 94, 94, 96, 116, 118 and/or 120.
  • the PDI nucleic acid sequence has at least 80 % identity to SEQ ID NO: 32, 33, 34, 35, 76, 80, 82, 84, 86, 88, 90, 92, 94 and/or 96.
  • the PDI nucleic acid sequence has at least 80 % identity to SEQ ID NO: 32, 33, 34 and/or 35.
  • the PDI nucleic acid sequence has at least 80 % identity to SEQ ID NO: 32.
  • the PDI nucleic acid sequence has at least 80 % identity to SEQ ID NO: 33.
  • the PD! nucleic acid sequence has at least 80 % identityEQ ID NO: 34.
  • the PD1 nucleic acid sequence has at least 80 % identityEQ ID NO: 35.
  • the PD! nucleic acid sequence has at least 80 % identityEQ ID NO: 76.
  • the PD1 nucleic acid sequence has at least 80 % identityEQ ID NO: 80.
  • the PD1 nucleic acid sequence has at least 80 % identityEQ ID NO: 82.
  • the PD I nucleic acid sequence has at least 80 % identityEQ ID NO: 84.
  • the PD1 nucleic acid sequence has at least 80 % identityEQ ID NO: 86.
  • the PD1 nucleic acid sequence has at least 80 % identityEQ ID NO: 88.
  • the PD1 nucleic acid sequence has at least 80 % identityEQ ID NO: 90.
  • the PD1 nucleic acid sequence has at least 80 % identityEQ ID NO: 92.
  • the PD1 nucleic acid sequence has at least 80 % identityEQ ID NO: 94.
  • the PD1 nucleic acid sequence has at least 80 % identityEQ ID NO: 96.
  • the PD I nucleic acid sequence has at least 80 % identityEQ ID NO: 116.
  • the PD1 nucleic acid sequence has at least 80 % identityEQ ID NO: 1 18.
  • the PD1 nucleic acid sequence has at least 80 % identityEQ ID NO: 120.
  • the PD1 nucleic acid sequence has at least 85 % identityEQ ID NO: 32, 33, 34, 35, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 116, I I 8 and/or 120.
  • the PD1 nucleic acid sequence has at least 85 % identityEQ ID NO: 32, 33, 34, 35, 76, 80, 82, 84, 86, 88, 90, 92, 94 and/or 96
  • the PD! nucleic acid sequence has at least 85 % identity to SEQ ID NO: 32, 33, 34 and/or 35.
  • the PD1 nucleic acid sequence has at least 90 % identity to SEQ ID NO: 32, 33, 34, 35, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 116, 118 and/or 120.
  • the PD1 nucleic acid sequence has at least 90 % identity to SEQ ID NO: 32, 33, 34, 35, 76, 80, 82, 84, 86, 88, 90, 92, 94 and/or 96.
  • the PD1 nucleic acid sequence has at least 90 % identity to SEQ ID NO: 32, 33, 34 and/or 35.
  • the PD1 nucleic acid sequence has at least 95 % identity to SEQ ID NO: 32, 33, 34, 35, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 116, 118 and/or 120.
  • the PDI nucleic acid sequence has at least 95 % identity to SEQ ID NO: 32, 33, 34, 35, 76, 80, 82, 84, 86, 88, 90, 92, 94 and/or 96
  • the PDI nucleic acid sequence has at least 95 % identity to SEQ ID NO: 32, 33, 34 and/or 35.
  • the PDI nucleic acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 32, 33, 34, 35, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 116, 1 18 and/or 120, each possibility represents a separate embodiment of the present invention.
  • the PDI nucleic acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 32, 33, 34, 35, 76, 80, 82, 84, 86, 88, 90, 92, 94 and/or 96, each possibility represents a separate embodiment of the present invention.
  • the PDI nucleic acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 32, 33, 34 and/or 35, each possibility represents a separate embodiment of the present inventi on.
  • the PD I nucleic acid sequence comprises SEQ ID NO: 32, 33, 34, 35, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 1 16, 118 or 120
  • the PDI nucleic acid sequence comprises SEQ) ID NO: 32, 33, 34, 35, 76, 80, 82, 84, 86, 88, 90, 92, 94 or 96.
  • the PDI nucleic acid sequence comprises SEQ ID NO: 32, 33, 34 or 35.
  • the PDI nucleic acid sequence comprises SEQ ID NO:
  • the PD1 nucleic acid sequence comprises SEQ ID NO:
  • the PD1 nucleic acid sequence comprises SEQ ID NO:
  • the PD1 nucleic acid sequence comprises SEQ ID NO:
  • the PD1 nucleic acid sequence comprises SEQ ID NO: 76.
  • the PDl nucleic acid sequence comprises SEQ ID NO:
  • the PD1 nucleic acid sequence comprises SEQ ID NO:
  • the PDl nucleic acid sequence comprises SEQ ID NO: 84.
  • the PDl nucleic acid sequence comprises SEQ ID NO:
  • the PDl nucleic acid sequence comprises SEQ ID NO:
  • the PDl nucleic acid sequence comprises SEQ ID NO: 90.
  • the PDl nucleic acid sequence comprises SEQ ID NO: 92.
  • the PDl nucleic acid sequence comprises SEQ ID NO: 94.
  • the PDl nucleic acid sequence comprises SEQ ID NO: 96.
  • the PDl nucleic acid sequence comprises SEQ ID NO: 1 16.
  • the PDl nucleic acid sequence comprises SEQ ID NO:
  • the PDl nucleic acid sequence comprises ID NO:120
  • the PDl nucleic acid sequence consists of SEQ ID NO: 32, 33, 34, 35, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 116, 180 or 120.
  • the PD! nucleic acid sequence consists of SEQ ID NO: 32, 33, 34, 35, 76, 80, 82, 84, 86, 88, 90, 92, 94 or 96
  • the PD1 nucleic acid sequence consists of SEQ ID NO: 32, 33, 34 and/or 35.
  • the PD! nucleic acid sequence consists of SEQ ID NO:
  • the PD1 nucleic acid sequence consists of SEQ ID NO:
  • the PD1 nucleic acid sequence consists of SEQ ID NO:
  • the PDI nucleic acid sequence consists of SEQ ID NO:
  • the PDI nucleic acid sequence consists of SEQ ID NO: 76.
  • the PDI nucleic acid sequence consists of SEQ ID NO:
  • the PDI nucleic acid sequence consists of SEQ ID NO:
  • the PD I nucleic acid sequence consists of SEQ ID NO: 84.
  • the PDI nucleic acid sequence consists of SEQ ID NO:
  • the PDI nucleic acid sequence consists of SEQ ID NO:
  • the PDI nucleic acid sequence consists of SEQ ID NO: 90.
  • the PDI nucleic acid sequence consists of SEQ ID NO: 92.
  • the PDI nucleic acid sequence consists of SEQ ID 94
  • the PDI nucleic acid sequence consists of SEQ ID NO: 96.
  • the PD I nucleic acid sequence consists of SEQ ID NO:
  • the PD1 nucleic acid sequence consists of SEQ ID NO:
  • the PD1 nucleic acid sequence consists of SEQ ID NO: 120
  • PD1 amino acid sequence comprises 100-200 amino acids, 120-180 amino acids, 120-160, 130-170 amino acids, 130-160, 130-150, 140-160 amino acids, 145-155 amino acids, each possibility represents a separate embodiment of the present invention.
  • PD1 amino acid sequence is 123-166 amino acids in length.
  • PD1 amino acid sequence is 156 amino acids in length.
  • PD1 amino acid sequence is 145-155 amino acids in length.
  • PD1 amino acid sequence is 150 amino acids in length.
  • PD1 amino acid sequence comprises less than 150 amino acids in length.
  • PD1 amino acid sequence comprises less than 149 amino acids in length.
  • PD1 amino acid sequence comprises less than 148, less than 147, less than 146, less than 145, less than 144, less than 143, less than 142, less than 141 amino acids in length, each possibility represents a separate embodiment of the present invention.
  • PD1 amino acid sequence is 138-145 amino acids in length.
  • PD1 amino acid sequence is 145 amino acids in length.
  • PD1 amino acid sequence is 143 amino acids in length.
  • the PD1 amino acid sequence in no more than 140 amino acids in length.
  • PD1 amino acid sequence is at least 123, at least, 124, at least 125, at least 126, at least 127, at least 128, at least 129, at least 130, at least 133, at least 137, at least 138 amino acids in length, each possibility represents a separate embodiments of the present invention.
  • PD I amino acid sequence is at least 123, at least, 124, at least 125, at least 126, at least 127, at least 128, at least 129, at least 130 amino acids in length, each possibility represents a separate embodiments of the present invention.
  • PD1 amino acid sequence comprises 123 - 148 amino acids in length.
  • PD! amino acid sequence is at least 126 amino acids in length.
  • PD1 amino acid sequence is 126 - 148 amino acids in length.
  • PD1 amino acid sequence is 123 - 140 amino acids in length.
  • PD1 amino acid sequence is 126 - 140 amino acids in length.
  • PD! amino acid sequence is 127 - 140 amino acids in length.
  • PD1 amino acid sequence is 128 - 140 amino acids in length.
  • PD1 amino acid sequence is 130 - 140 amino acids in length.
  • PD1 amino acid sequence is 130 - 135 amino acids in length.
  • PD1 amino acid sequence comprises 135 - 140 amino acids in length.
  • PD1 amino acid sequence comprises 126 - 130 amino acids in length.
  • PD1 amino acid sequence is 127 amino acids in length.
  • PD1 amino acid sequence is 128 amino acids in length.
  • PD! amino acid sequence is 133 amino acids in length.
  • PD1 amino acid sequence is 135 amino acids in length.
  • PD1 amino acid sequence is 136 amino acids in length.
  • PD1 amino acid sequence is 137 amino acids in length.
  • PD1 amino acid sequence is 138 amino acids in length.
  • PD1 amino acid sequence is 140 amino acids in length.
  • the term“4-1BBL also known as CD137L and TNFSF9) refers to the polypeptide of the TNFSF9 gene (Gene ID 8744) or a functional homolog e.g., functional fragment thereof According to specific embodiments, the term“4-1BBL” refers to a functional homolog of 4-1BBL polypeptide. According to specific embodiments, 4-1BBL is human 4-1BBL. According to a specific embodiment, the 4-1BBL protein refers to the human protein, such as provided in the following GenBank Number NP 003802
  • the phrase“functional homolog” or“functional fragment” when related to 4-1BBL refers to a portion of the polypeptide which maintains the activity of the full length 4- 1 BBL e.g , (i) binding 4-1BB, (ii) activating 4-1BB signaling pathway, (iii) activating immune cells expressing 4- IBB, (iv) forming a homotrimer.
  • the functional homolog when related to 4-1BBL is capable of (i), (ii), (iii), (i)+(ii), (i)+(iii), (ii)+(iii).
  • the functional homolog when related to 4-!BBL is capable of (i)+(ii)+(iii).
  • the functional homolog when related to 4-1BBL is capable of (iv), (i)+(iv), (ii)+(iv), (iii)+(iv), (i)+(ii)+(iv), (i ) (iii ) (iv). (ii)+(iii)+(iv).
  • the functional homolog when related to 4-1BBL is capable of (i)+(ii)+(iii)+(iv).
  • the 4- IBB protein refers to the human protein, such as provided in the following GenBank Number NP 001552
  • the 4-1BBL binds 4-1BB with a Kd of about 0 1 - 1000 nM, 0.1 - 100 nM, 1-100 nM, or 55 2 nM as determined by SPR, each possibility represents a separate embodiment of the claimed invention.
  • Assays for testing trimerization are well known in the art and include, but not limited to NATIVE-PAGE, SEC-HPLC 2D gels, gel filtration, SEC-MALS, Analytical ultracentrifugation (AUC) Mass spectrometry (MS), capillary gel electrophoresis (CGE).
  • activating refers to the process of stimulating an immune cell (e.g. T cell, B cell, NK cell, phagocytic cell) that results in cellular proliferation, maturation, cytokine production, phagocytosis and/or induction of regulatory or effector functions.
  • an immune cell e.g. T cell, B cell, NK cell, phagocytic cell
  • activating comprises co- stimulating.
  • co-stimu!ating or“co-stimulation” refers to transmitting a secondary antigen independent stimulatory' signal (e.g 4-1 BB signal) resulting in activation of the immune cell.
  • a secondary antigen independent stimulatory' signal e.g 4-1 BB signal
  • activating comprises suppressing an inhibitory' ⁇ signal (e.g PDL1 signal) resulting in activation of the immune cell.
  • an inhibitory' ⁇ signal e.g PDL1 signal
  • Methods of determining signaling of a stimulatory or inhibitory signal are well known in the art and also disclosed in the Examples section which follows, and include, but are not limited to, binding assay using e.g. BiaCore, HPLC or flow cytometry, enzymatic activity assays such as kinase activity assays, and expression of molecules involved in the signaling cascade using e.g. PCR, Western blot, immunoprecipitation and immunohistochemistry. Additionally or alternatively, determining transmission of a signal (co-stimulatory or inhibitory) can be effected by evaluating immune cell activation or function.
  • Methods of evaluating immune cell activation or function include, but are not limited to, proliferation assays such as CFSE staining, MTS, Alamar blue, BRDU and thymidine incorporation, cytotoxicity assays such as CFSE staining, chromium release, Calcin AM, cytokine secretion assays such as intracellular cytokine staining, ELISPOT and ELISA, expression of activation markers such as CD25, CD69, CD137, CD107a, PD1, and CD62L using flow cytometry.
  • proliferation assays such as CFSE staining, MTS, Alamar blue, BRDU and thymidine incorporation
  • cytotoxicity assays such as CFSE staining, chromium release, Calcin AM
  • cytokine secretion assays such as intracellular cytokine staining, ELISPOT and ELISA
  • expression of activation markers such as CD25, CD69, CD137, CD
  • determining the signaling activity or activation is effected in-vitro or ex-vivo e.g in a mixed lymphocyte reaction (MLR), as further described hereinbelow.
  • MLR mixed lymphocyte reaction
  • the signaling activity or the immune cell activation or function are generally expressed in comparison to the signaling, activation or function in a cell of the same species but not contacted with the PF1-4-1BBL fusion protein, a polynucleotide encoding same or a host cell encoding same; or contacted with a vehicle control, also referred to as control.
  • the 4-1BBL comprises an extracellular domain of said 4-1BBL or a functional fragment thereof.
  • 4-1BBL amino acid sequence comprises SEQ ID NO: 36.
  • 4-1BBL amino acid sequence consists of SEQ ID NO: 36
  • 4-1BBL nucleic acid sequence comprises SEQ ID NO: 37.
  • 4-1BBL nucleic acid sequence consists of SEQ ID NO: 37.
  • 4-1BBL amino acid sequence comprises SEQ ID NO:
  • 4-1BBL amino acid sequence consists of SEQ ID NO:
  • 4-1BBL nucleic acid sequence comprises SEQ ID NO: 38.
  • 4-1 BBL nucleic acid sequence consists of SEQ ID NO: 38.
  • the term“4-1BBL” also encompasses functional homologues (naturally occurring or synthetically/recombinantly produced), which exhibit the desired activity (as defined hereinabove).
  • Such homologues can be, for example, at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identical or homologous to the polypeptide SEQ ID NO: 3, 36; or at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least
  • the 4-1BBL functional homologues are at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identical or homologous to the polypeptide SEQ ID NO: 3, 36; or at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identical to the polynucleotide sequence encoding same (as further described hereinbelow).
  • the 4-1 BBL polypeptide may comprise conservative amino acid substitutions, as further described hereinabove and below.
  • the 4-1 BBL of some embodiments of the present invention is at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identical or homologous to the polypeptide SEQ ID NO: 22, 23, 24 and/or 25; or at least 95 3 ⁇ 4, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identical to the polynucleotide sequence encoding same (as further described hereinbelow), each possibility represents a separate embodiment of the present invention.
  • the 4-1BBL amino acid sequence has at least 95 % identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-25, each possibility represents a separate embodiment of the present invention.
  • the 4-1BBL amino acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 22, 23, 24 and/or 25, each possibility represents a separate embodiment of the present invention.
  • the 4-1BBL amino acid sequence has at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 22, 23 and/or 24.
  • the 4-lBBL amino acid sequence has at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 22.
  • the 4-lBBL amino acid sequence has at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 23.
  • the 4-lBBL amino acid sequence has at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 24.
  • the 4-lBBL amino acid sequence has at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 25.
  • the 4-1 BBL amino acid sequence does not comprise the amino acid segment A1 - V6 corresponding to SEQ ID NO: 3.
  • the 4-1 BBL amino acid sequence does not comprise the amino acid segment A1 - G14 corresponding to SEQ ID NO: 3.
  • the 4-lBBL amino acid sequence does not comprise any of amino acid residues A1 - V6 or Al - G14 corresponding to SEQ ID NO: 3.
  • the phrase“corresponding to SEQ ID NO: 3” intends to include the corresponding amino acid residue relative to any other 4-lBBL amino acid sequence.
  • the 4 - i BBL amino acid sequence does not comprise SEQ ID NO: 6 or 7 or any fragment thereof.
  • the 4-lBBL amino acid sequence does not comprise SEQ ID NO: 6 or 7.
  • the 4-lBBL amino acid sequence comprises SEQ ID NO: 22, 23, 24 or 25.
  • the 4-lBBL amino acid sequence comprises SEQ ID NO: 22, 23 or 24.
  • the 4-lBBL amino acid sequence comprises SEQ ID NO: 22 or 23.
  • the 4-1 BBL amino acid sequence comprises SEQ ID NO: 22.
  • the 4-lBBL amino acid sequence comprises SEQ ID NO: 1
  • the 4-lBBLa amino acid sequence comprises SEQ ID NO: 24.
  • the 4-1 BBL amino acid sequence comprises SEQ ID NO: 25.
  • the 4-1 BBL amino acid sequence consists of SEQ ID NO: 22, 23, 24 or 25.
  • the 4-1 BBL amino acid sequence consists of SEQ ID NO: 22, 23 or 24.
  • the 4-1 BBL amino acid sequence consists of SEQ ID NO: 22 or 23.
  • the 4-1BBL amino acid sequence consists of SEQ ID NO: 22.
  • the 4-1BBL amino acid sequence consists of SEQ ID NO: 23.
  • the 4-lBBLa amino acid sequence consists of SEQ ID NO: 24.
  • the 4-1BBL amino acid sequence consists of SEQ ID NO: 25.
  • the 4-1BBL amino acid sequence has at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 127 and 129, each possibility represents a separate embodiment of the present invention.
  • the 4-1BBL amino acid sequence has at least 85 % identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 127 and 129, each possibility represents a separate embodiment of the present invention.
  • the 4-1 BBL amino acid sequence has at least 90 % identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 127 and 129, each possibility represents a separate embodiment of the present inventi on.
  • the 4-1BBL amino acid sequence has at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 127 and 129, each possibility represents a separate embodiment of the present invention.
  • the 4-1BBL amino acid sequence has at least 95 % identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 127 and 129, each possibility represents a separate embodiment of the present invention .
  • the 4-1BBL amino acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 127 and 129, each possibility represents a separate embodiment of the present invention.
  • the 4-1BBL amino acid sequence does not comprise the amino acid segment GI98 - E205 corresponding to SEQ ID NO: 3.
  • the 4-1 BBL amino acid sequence does not comprise any of amino acid residues G198 - E205 corresponding to SEQ ID NO: 3.
  • the 4-1BBL amino acid sequence does not comprise SEQ ID NO: 166 or any fragment thereof.
  • the 4-1BBL amino acid sequence does not comprise SEQ ID NO: 166.
  • the 4-1BBL amino acid sequence comprises SEQ ID NOs: 127 or 129
  • the 4-1BBL amino acid sequence consists of SEQ ID NOs: 127 or 129.
  • the 4-1 BBL amino acid sequence has at least 80 % identity to SEQ ID NO: 125.
  • the 4-1BBL amino acid sequence has at least 85 % identity SEQ ID NO: 125.
  • the 4-1 BBL amino acid sequence has at least 90 % identity to SEQ ID NO: 125.
  • the 4-1BBL amino acid sequence has at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 125.
  • the 4-1 BBL amino acid sequence has at least 95 % identity to SEQ ID NO: 125.
  • the 4-1BBL amino acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 125.
  • the 4-1 BBL amino acid sequence does not comprise the amino acid segment A1-E23 corresponding to SEQ ID NO: 3.
  • the 4-1BBL amino acid sequence does not comprise any of amino acid residues A1-E23 corresponding to SEQ ID NO: 3.
  • the 4-IBBL amino acid sequence does not comprise SEQ ID NO: 167 or any fragment thereof.
  • the 4-1 BBL amino acid sequence does not comprise SEQ ID NO: 167.
  • the 4-1BBL amino acid sequence comprises SEQ ID NO: 125.
  • the 4-1BBL amino acid sequence consists of SEQ ID NO: 125.
  • the 4-1BBL amino acid sequence has at least 80 % identity to SEQ ID NO: 123.
  • the 4-1BBL amino acid sequence has at least 85 % identity SEQ ID NO: 123.
  • the 4-IBBL amino acid sequence has at least 90 % identity to SEQ ID NO: 123.
  • the 4-IBBL amino acid sequence has at least 91 %, at least 92 %, at least 93 3 ⁇ 4, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 123.
  • the 4-IBBL amino acid sequence has at least 95 % identity to SEQ ID NO: 123.
  • the 4-IBBL amino acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 123.
  • the 4-IBBL amino acid sequence comprises SEQ ID NO: 123
  • the 4-1 BBL amino acid sequence consists of SEQ ID NO: 123.
  • the 4-IBBL nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 3 ⁇ 4, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 39, 40, 41 and/or 42, each possibility represents a separate embodiment of the present invention.
  • the 4-IBBL nucleic acid sequence has at least 95 % identity to SEQ ID NO: 39, 40, 41 and/or 42.
  • the 4-1BB3L nucleic acid sequence has at least 96 %, at least 97 3 ⁇ 4, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 39, 40, 41 and/or 42.
  • the 4-1BBL nucleic acid sequence has at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 39 and/or 40.
  • the 4-1BBL nucleic acid sequence comprises SEQ ID NO: 39, 40, 41 or 42.
  • the 4-1BB3L nucleic acid sequence comprises SEQ ID NO: 39, 40 or 41.
  • the 4-1BBL nucleic acid sequence comprises SEQ ID NO: 39 or 40
  • the 4-I BBL nucleic acid sequence comprises SEQ ID NO: 39.
  • the 4-1BBL nucleic acid sequence comprises SEQ ID NO: 40.
  • the 4-lBBLa nucleic acid sequence comprises SEQ ID NO: 41.
  • the 4-1BBL nucleic acid sequence comprises SEQ ID NO: 42.
  • the 4-IBBL nucleic acid sequence consists of SEQ ID NO: 39, 40, 41 or 42.
  • the 4-IBBL nucleic acid sequence consists of SEQ ID NO: 39, 40 or 41.
  • the 4-IBBL nucleic acid sequence consists of SEQ ID NO: 39 or 40
  • the 4-1 BBL nucleic acid sequence consists of SEQ ID NO: 39.
  • the 4-IBBL nucleic acid sequence consists of SEQ ID NO: 40.
  • the 4-1 BBL nucleic acid sequence consists of SEQ ID NO: 41.
  • the 4-IBBL nucleic acid sequence consists of SEQ ID NO: 42.
  • the 4-1BBL nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 128 and/or 130, each possibility represents a separate embodiment of the present invention.
  • the 4-1 BBL nucleic acid sequence has at least 95 % identity to SEQ ID NO: 128 and/or 130.
  • the 4-1BBL nucleic acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 128 and/or 130.
  • the 4-1BBL nucleic acid sequence comprises SEQ ID NO: 128 or 130.
  • the 4-IBBL nucleic acid sequence consists of SEQ ID NO: 128 or 130.
  • the 4-IBBL nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 126.
  • the 4-1BBL nucleic acid sequence has at least 95 % identity to SEQ ID NO: 126
  • the 4-1BBL nucleic acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 126
  • the 4-IBBL nucleic acid sequence comprises SEQ ID NO: 126.
  • the 4-1 BBL nucleic acid sequence consists of SEQ ID NO: 126
  • the 4-IBBL nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 3 ⁇ 4, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or100 % identity to SEQ ID NO: 124.
  • the 4-IBBL nucleic acid sequence has at least 95 % identity to SEQ ID NO: 124
  • the 4-1BB3L nucleic acid sequence has at least 96 %, at least 97 3 ⁇ 4, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 124.
  • the 4-1 BBL nucleic acid sequence comprises SEQ ID NO: 124.
  • the 4-1BBL nucleic acid sequence consists of SEQ ID NO: 124.
  • 4-1BBL amino acid sequence comprises 100-300 amino acids, 150-250 amino acids, 100-250 amino acids, 150-220 amino acids, 180-220 amino acids, 180 - 210 amino acids, 185 - 205 amino acids, 190-210 amino acids, each possibility represents a separate embodiment of the present invention.
  • 4-1BBL amino acid sequence is 190-210 amino acids in length.
  • 4-IBBL amino acid sequence is 204 amino acids in length.
  • 4-1BBL amino acid sequence is 185 - 202 amino acids in length.
  • 4-1BBL amino acid sequence is 185 - 200 amino acids in length.
  • 4-1BBL amino acid sequence is 185 - 199 amino acids in length.
  • 4-1BBL amino acid sequence is 170 - 197 amino acids in length.
  • 4-1 BBL amino acid sequence is 170 - 182 amino acids in length.
  • 4-1BBL amino acid sequence is 184 amino acids in length.
  • 4-1BBL amino acid sequence is 185, 191, 197 or 199 amino acids in length, each possibility represents a separate embodiment of the present invention.
  • 4-IBBL amino acid sequence is 184 amino acids in length.
  • 4-IBBL amino acid sequence is 183 amino acids in length.
  • 4-1 BBL amino acid sequence is 182 amino acids in length. According to specific embodiments, 4-1BBL amino acid sequence is 176 amino acids in length.
  • the 4-1BBL amino acid sequence comprised in the PD1- 4-1BBL fusion protein or the 4-1BBL polypeptide disclosed herein comprises three repeats of a 4-1 BBL amino acid sequence.
  • each of the three repeats is capable of at least one of: (i) binding 4-1BB, (ii) activating 4-1 BB signaling pathway, (iii) activating immune cells expressing 4- IBB, (iv) forming a homotrimer.
  • the 4-1BBL amino acid sequence does not comprise a linker between each of said three repeats of said 4-1BBL amino acid sequence.
  • the 4-1BBL amino acid sequence comprises a linker between each of said three repeats of said 4-1 BBL amino acid sequence.
  • Any linker known in the art can be used with specific embodiments of the invention. Non-limiting examples of linkers that can be used are described in details hereinbelow.
  • the linker is a (GGGGS)x2+GGGG (SEQ ID NO: 121) linker.
  • the repeated sequence can be any of the 4-1BBL as defined herein.
  • the three repeats have an identical 4-1 BBL amino acid sequence.
  • the three repeats are distinct, i.e. have different 4- 1BBL amino acid sequences.
  • two of the three repeats have an identical 4-1 BBL amino acid sequence.
  • At least one of the repeats comprises a 4-1BBL amino acid sequence disclosed herein.
  • At least one of the repeats consists of a 4-1BBL amino acid sequence disclosed herein.
  • the 4-1BBL amino acid sequence comprises three repeats of an amino acid sequence comprising SEQ ID NO: 22.
  • the 4-1BBL amino acid sequence comprises three repeats of an amino acid sequence consisting of SEQ ID NO: 22.
  • the 4-1BBL amino acid sequence comprises an amino acid sequence having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 131.
  • the 4-1BBL amino acid sequence comprises an amino acid sequence having at least 80 % identify to SEQ ID NO: 131 .
  • the 4-1BBL amino acid sequence comprises an amino acid sequence having at least 85 % identify to SEQ ID NO: 131.
  • the 4-1BBL amino acid sequence comprises an amino acid sequence having at least 90 % identify to SEQ ID NO: 131.
  • the 4-1BBL amino acid sequence comprises an amino acid sequence having at least 95 % identify to SEQ ID NO: 131.
  • the 4-1BBL amino acid sequence comprises SEQ ID NO: 131.
  • the 4-1BBL amino acid sequence consists of SEQ ID NO: 131.
  • the 4-1BBL nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 132.
  • the 4-I BBL nucleic acid sequence has at least 95 % identity to SEQ ID NO: 132.
  • the 4-I BBL nucleic acid sequence has at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 132.
  • the 4-IBBL nucleic acid sequence comprises SEQ ID NO: 132.
  • the 4-IBBL nucleic acid sequence consists of SEQ ID NO: 1
  • DSP and“fusion protein”,“chimeric protein” or“chimera” are used herein interchangeably, and refer to an amino acid sequence having two or more parts which are not found together in a single amino acid sequence in nature.
  • the fusion protein of some embodiments of the present invention comprises a PD1 amino acid sequence and a 4-IBBL amino acid sequence (referred to herein as PD1-4-1BBL fusion protein).
  • the PD1 is N-terminal to the 4-1BBL. According to other specific embodiments, the PD1 is C-terminal to the 4-1BBL.
  • the PD1-4-1BBL fusion protein of some embodiments of the present invention can comprise any PD! as defined herein; and any 4-1 BBL amino acid sequence:
  • the PD1 -4-1BBL fusion protein of some embodiments of the present invention can comprise any PD1 amino acid sequence:
  • the PD1-4-1BBL fusion protein of some embodiments of the present invention can comprise any PD1 amino acid sequence:
  • the PD1 -4-lBBL fusion protein of some embodiments of the present invention can comprise any PD1 as defined herein; and any 4-lBBL amino acid sequence being 185 - 202 amino acids in length and having at least 95 % identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-25 such as e.g. disclosed herein, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1BBL fusion protein of some embodiments of the present invention can comprise any PD1 amino acid sequence:
  • the PD1-4-1BBL fusion protein of some embodiments of the present invention can comprise any PD1 amino acid sequence being 123 - 148 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21 and not comprising any of amino acid segments PI - L5 and F146 - V150 corresponding to SEQ ID NO: 2 such as e.g disclosed herein; and any 4-lBBL as defined herein, each possibility represents a separate embodiment of the present invention.
  • the PD 1 -4-lBBL fusion protein of some embodiments of the present invention can comprise any PD1 amino acid sequence: (a) being 123 - 148 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21, 85, 89 and 93 and not comprising any of amino acid segments PI --- L5 and F146 --- VI 50 corresponding to SEQ ID NO: 2 and/or
  • the PD1-4-1BBL fusion protein of some embodiments of the present invention can comprise any PD1 amino acid sequence being 123 - 148 amino acids in length having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-21 and not comprising any of amino acid segments PI - L5 and F146 - V150 corresponding to SEQ ID NO: 2 such as e.g. disclosed herein; and any 4-1BBL amino acid sequence being 185 - 202 amino acids in length and having at least 95 % identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-25 such as e.g. disclosed herein, each possibility represents a separate embodiment of the present invention.
  • the PD! amino acid sequence has at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least
  • the PD1 amino acid sequence has at least 80 %, at least 8 ! %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least
  • the PD1 amino acid sequence has at least 80 %, at least 81 3 ⁇ 4, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 2, 18-21, 27, 30 and/or 31 ; and the 4-1 BBL amino acid sequence is 185 - 202 amino acids in length having at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 22, 23, 24 or 25, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence comprises SEQ ID NO: 2, 18-21, 85, 89, 93, 27, 30 or 31; and the 4-1BBL amino acid sequence is 185 - 202 amino acids in length comprising SEQ ID NO: 22, 23, 24 or 25, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence comprises SEQ ID NO: 73, 75, 79, 81, 83, 87, 91 or 95; and the 4-1BBL amino acid sequence is 185 - 202 amino acids in length comprising SEQ ID NO: 22, 23, 24 or 25, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence comprises SEQ ID NO: 73; and the 4-1BBL amino acid sequence is 185 - 202 amino acids in length comprising SEQ ID NO: 22, 23, 24 or 25, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence comprises SEQ ID NO: 75, 79, 81 , 83, 87, 91 or 95; and the 4-1 BBL amino acid sequence is 185 - 202 amino acids in length comprising SEQ ID NO: 22, 23, 24 or 25, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence comprises SEQ ID NO: 75, 83, 87, 91 or 95; and the 4-1BBL amino acid sequence is 185 - 202 amino acids in length comprising SEQ ID NO: 22, 23, 24 or 25, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence comprises SEQ ID NO: 73, 75, 83, 87, 91 or 95; and the 4-1BBL amino acid sequence is 185 - 202 amino acids in length comprising SEQ ID NO: 22, 23, 24 or 25, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence comprises SEQ ID NO: 2, 18-21, 27, 30 or 31; and the 4-1BBL amino acid sequence is 185 - 202 amino acids in length comprising SEQ ID NO: 22, 23, 24 or 25, each possibility represents a separate embodiment of the present invention.
  • the PD I amino acid sequence is as set forth in SEQ ID NO: 2, 18-21, 85, 89, 93, 27, 30 or 31; and the 4-1BBL amino acid sequence is as set forth in SEQ ID NO: 22, 23, 24 or 25, each possibility represents a separate embodiment of the present invention.
  • the PD I amino acid sequence is as set forth in SEQ ID NO: 73, 75, 79, 81, 83, 87, 91 or 95; and the 4-1BBL amino acid sequence is as set forth in SEQ ID NO: 22, 23, 24 or 25, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence is as set forth in SEQ ID NO: 73; and the 4-1BBL amino acid sequence is as set forth in SEQ ID NO: 22, 23, 24 or 25, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence is as set forth in SEQ ID NO: 75, 79, 81, 83, 87, 91 or 95; and the 4-1BBL amino acid sequence is as set forth in SEQ ID NO: 22, 23, 24 or 25, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence is as set forth in SEQ ID NO: 75, 83, 87, 91 or 95; and the 4-1BBL amino acid sequence is as set forth in SEQ ID NO: 22,
  • the PD1 amino acid sequence is as set forth in SEQ ID NO: 73, 75, 83, 87, 91 or 95; and the 4-1 BBL amino acid sequence is as set forth in SEQ ID NO: 22, 23, 24 or 25, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence is as set forth in SEQ ID NO: 2, 18-21, 27, 30 or 31; and the 4-1BBL amino acid sequence is as set forth in SEQ ID NO: 22, 23, 24 or 25, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence :
  • (a) is 123 - 148 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 18-21 , 85, 89 and/or
  • (b) comprises a C to S amino acid modification in a position corresponding to amino acid 93 of SEQ ID NO: 2; and the 4-1BBL amino acid sequence has at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 3 ⁇ 4, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 3, 22-25 and/or 36, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence is 123 - 148 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 18, 19, 20 and/or 21; and the 4-1BBL amino acid sequence has at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %
  • the PD1 amino acid sequence :
  • (a) is 123 - 148 amino acids in length comprising SEQ ID NO: 18-21, 85, 89 and/or 93; and/or
  • (b) comprises a C to S amino acid modification in a position corresponding to amino acid 93 of SEQ ID NO: 2; and the 4-1BBL amino acid sequence comprises SEQ ID NO: 3, 22-25 or 36, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence comprises SEQ ID NO: 18- 21, 85, 89 or 93; and the 4-1 BBL amino acid sequence comprises SEQ ID NO: 3, 22-25 or 36, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence comprises SEQ ID NO: 73, 75, 79, 81, 83, 87, 91 or 95; and the 4-1BBL amino acid sequence comprises SEQ ID NO: 3, 22- 25 or 36, each possibility represents a separate embodiment of the present inventi on.
  • the PD1 amino acid sequence comprises SEQ ID NO: 73; and the 4-1BBL amino acid sequence comprises SEQ ID NO: 3, 22-25 or 36, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence comprises SEQ ID NO: 75, 79, 81 , 83, 87, 91 or 95
  • the 4-1 BBL amino acid sequence comprises SEQ ID NO: 3, 22-25 or 36, each possibility represents a separate embodiment of the present invention.
  • the PDi amino acid sequence comprises SEQ ID NO: 75, 83, 87, 91 or 95; and the 4-1BBL amino acid sequence comprises SEQ ID NO: 3, 22-25 or 36, each possibility represents a separate embodiment of the present inventi on.
  • the PD1 amino acid sequence comprises SEQ ID NO: 73, 75, 83, 87, 91 or 95; and the 4-!BBL amino acid sequence comprises SEQ ID NO: 3, 22-25 or 36, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence is 123 - 148 amino acids in length comprising SEQ ID NO: 18, 19, 20 or 21; and the 4-1 BBL amino acid sequence comprises SEQ ID NO: 3, 22-25 or 36, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence is as set forth in SEQ ID NO: 18, 19, 20 or 21; and the 4-1BBL amino acid sequence is as set forth in SEQ ID NO: 3, 22- 25 or 36, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence is 123 - 148 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 18; and the 4-1BBL amino acid sequence is 185 - 202 amino acids in length havin: at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100
  • the PD1 amino acid sequence is 123 - 148 amino acids in length comprising SEQ ID NO: 18; and the 4-1BBL amino acid sequence is 185 - 202 amino acids in length and comprising SEQ ID NO: 22, 23, 24 or 25.
  • the PD I amino acid sequence is as set forth in SEQ ID NO: 18; and the 4-1BBL amino acid sequence is as set forth in SEQ) ID NO: 22, 23, 24 or 25
  • the PD1 amino acid sequence is 123 - 148 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 18; and the 4-1BBL amino acid sequence is 185 - 202 amino acids in length having at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 22, 23 or 24.
  • the PD1 amino acid sequence is 123 - 148 amino acids in length comprising SEQ ID NO: 18; and the 4-1BBL amino acid sequence is 185 - 202 amino acids in length comprising SEQ ID NO: 22, 23 or 24.
  • the PD1 amino acid sequence is as set forth in SEQ ID NO: 18; and the 4-1BBL amino acid sequence is as set forth in SEQ ID NO: 22, 23 or 24.
  • the PD1 amino acid sequence is 123 - 148 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 18; and the 4-1 BBL amino acid sequence is 185 - 202 amino acids in length having at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 22.
  • the PD1 amino acid sequence is 123 - 148 amino acids in length comprising SEQ ID NO: 18; and the 4-1 BBL amino acid sequence is 185 - 202 a ino acids in length comprising SEQ ID NO: 22.
  • the PD I amino acid sequence is as set forth in SEQ ID NO: 18; and the 4-1 BBL amino acid sequence is as set forth in SEQ) ID NO: 22.
  • the PD1 amino acid sequence is 123 - 148 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 18; and the 4-1BBL amino acid sequence is 185 - 202 amino acids in length having at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 23.
  • the PD 1 amino acid sequence is 123 - 148 amino acids in length comprising SEQ) ID NO: 18; and the 4-1BBL amino acid sequence is 185 - 202 amino acids in length comprising SEQ ID NO: 23.
  • the PD1 amino acid sequence is as set forth in SEQ ID NO: 18; and the 4-1BBL amino acid sequence is as set forth in SEQ ID NO: 23.
  • the PD l amino acid sequence is 123 - 148 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 18; and the 4-1BBL amino acid sequence is 185 - 202 amino acids in length having at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 18; and the 4-1BBL amino acid sequence is 185 - 202 amino acids in length having
  • the PD1 amino acid sequence is 123 - 148 amino acids in length comprising SEQ ID NO: 18; and the 4-1 BBL amino acid sequence is 185 - 202 amino acids in length comprising SEQ ID NO: 24.
  • the PD I amino acid sequence is as set forth in SEQ ID NO: 18; and the 4-1 BBL amino acid sequence is as set forth in SEQ) ID NO: 24.
  • the PD1 amino acid sequence is 123 - 148 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 19; and the 4-1BBL amino acid sequence is 185 - 202 amino acids in length havin: at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100
  • the PD1 amino acid sequence is 123 - 148 amino acids in length comprising SEQ ID NO: 19; and the 4-IBBL amino acid sequence is 185 - 202 amino acids in length comprising SEQ ID NO: 22, 23, 24 or 25.
  • the PD1 amino acid sequence is as set forth in SEQ ID NO: 19; and the 4-IBBL amino acid sequence is as set forth in SEQ ID NO: 22, 23, 24 or 25.
  • the PD1 amino acid sequence is 123 - 148 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 20; and the 4-IBBL amino acid sequence is 185 - 202 amino acids in length having at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 22, 23, 24 or 25, each possibility represents a separate embodiment of the present invention.
  • the PD 1 amino acid sequence is 123 - 148 amino acids in length comprising SEQ ID NO: 20; and the 4-1 BBL amino acid sequence is 185 - 202 amino acids in length comprising embodiments, the PD1 amino acid sequence is as set forth in SEQ ID NO: 20; and the 4-1 BBL amino acid sequence is as set forth in SEQ ID NO: 22, 23, 24 or 25.
  • the PD1 amino acid sequence is 123 - 148 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 21; and the 4-1BBL amino acid sequence is 185 - 202 amino acids in length having at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 21; and the 4-1BBL amino acid sequence is 185 - 202 amino acids in length having at
  • the PD1 amino acid sequence is 123 - 148 amino acids in length comprising SEQ ID NO: 21; and the 4-IBBL amino acid sequence is 185 - 202 amino acids in length comprising SEQ ID NO: 22, 23, 24 or 25.
  • the PD1 amino acid sequence is as set forth in SEQ ID NO: 21; and the 4-IBBL amino acid sequence is as set forth in SEQ ID NO: 22, 23, 24 or 25.
  • the PD1 amino acid sequence :
  • (a) is 123 - 148 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89
  • (b) comprises a C to S amino acid modification in a position corresponding to amino acid 93 of SEQ ID NO: 2; and the 4-1BBL amino acid sequence is 185 - 202 amino acids in length having at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 22, each possibility represents a separate embodiment of the present invention.
  • the PD1 amino acid sequence is 123 - 148 amino acids in length comprising SEQ ID NO: 85, 89 or 93; and the 4-1 BBL amino acid sequence is 185 - 202 amino acids in length and comprising SEQ ID NO: 22.
  • the PD1 amino acid sequence is as set forth in SEQ ID NO: 85, 89 or 93; and the 4-1BBL amino acid sequence is as set forth in SEQ ID NO: 22, 23, 24 or 25.
  • the PD1 amino acid sequence is 123 - 148 amino acids in length comprising SEQ ID NO: 75, 79, 81, 83, 87, 91 or 95; and the 4-1BBL amino acid sequence is 185 - 202 amino acids in length and comprising SEQ ID NO: 22
  • the PD1 amino acid sequence is as set forth in SEQ ID NO: 75, 79, 81 , 83, 87, 91 or 95; and the 4-1 BBL amino acid sequence is as set forth in SEQ ID NO: 22, 23, 24 or 25.
  • the PD 1 amino acid sequence is 123 - 148 amino acids in length comprising SEQ ID NO: 75, 83, 87, 91 or 95; and the 4-1BBL amino acid sequence is 185 - 202 amino acids in length and comprising SEQ ID NO: 22.
  • the PD1 amino acid sequence is as set forth in SEQ ID NO: 75, 83, 87, 91 or 95; and the 4-lBBL amino acid sequence is as set forth in SEQ ID NO: 22,
  • the PD1 amino acid sequence is 123 - 148 amino acids in length comprising SEQ ID NO: 75; and the 4-1BBL amino acid sequence is 185 - 202 amino acids in length and comprising SEQ ID NO: 22.
  • the PD1 amino acid sequence is as set forth in SEQ ID NO: 75; and the 4-lBBL amino acid sequence is as set forth in SEQ ID NO: 22, 23, 24 or 25.
  • the PD I amino acid sequence is 123 - 148 amino acids in length comprising SEQ ID NO: 83; and the 4-lBBL amino acid sequence is 185 - 202 amino acids in length and comprising SEQ ID NO: 22.
  • the PD I amino acid sequence is as set forth in SEQ ID NO: 783; and the 4-lBBL amino acid sequence is as set forth in SEQ ID NO: 22, 23, 24 or 25.
  • the PD1 amino acid sequence comprises SEQ ID NO: 73 and the 4-lBBL amino acid sequence comprises SEQ ID NO: 3.
  • the PD1 amino acid sequence is as set forth in SEQ ID NO: 73; and the 4-lBBL amino acid sequence is as set forth in SEQ ID NO: 3.
  • the PD1 amino acid sequence is 123 - 148 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92
  • the PD1 amino acid sequence is 123 - 148 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85
  • (aa) is 185 - 202 amino acids in length having at least 95 % identity to an amino acid sequence selected form the group consisting of SEQ ID NOs: 22-25, is 170 - 197 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 3 ⁇ 4, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ I D NO: 127 and 129 and not comprising an amino acid segment G198-E205 corresponding to SEQ ID NO: 3, is 170 - 182 amino acids in length having at least 80
  • (bb) comprises three repeats of a 4-1 BBL amino acid sequence, each possibility represents a separate embodiment of the present invention.
  • the PDl amino acid sequence is 123 - 148 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92
  • the 4-1 BBL amino acid sequence is 170 - 182 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 1 15 and not comprising any of amino acid segments P I - L5 and F146 - V150 corresponding to SEQ ID NO: 2; and the 4-1 BBL amino acid sequence is 170 - 182 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %,
  • the PDl amino acid sequence is 123 - 148 amino acids in length comprising SEQ ID NO: 1 15; and the 4-1 BBL amino acid sequence is 170 - 182 amino acids in length comprising SEQ ID NO: 125.
  • the PDl amino acid sequence is as set forth in SEQ ID NO: 1 15; and the 4-1BBL amino acid sequence is as set forth in SEQ ID NO: 125.
  • the PDl amino acid sequence is 123 - 148 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 3 ⁇ 4, at least 85 %, at least 86 %, at least 87 %. at least 88 %. at least 89 %. at least 90 %. at least 91 %.
  • the 4-1 BBL amino acid sequence is 184 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91
  • the PD1 amino acid sequence is 123 - 148 amino acids in length comprising SEQ ID NO: 1 15; and the 4-1BBL amino acid sequence is 184 amino acids in length comprising SEQ ID NO: 123.
  • the PD1 amino acid sequence is as set forth in SEQ ID NO: 1 15; and the 4-1BBL amino acid sequence is as set forth in SEQ ID NO: 123.
  • the PD I amino acid sequence is 138-145 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92
  • the PD I amino acid sequence is 138-145 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85
  • (aa) is 185 - 202 amino acids in length having at least 95 % identity to an amino acid sequence selected form the group consisting of SEQ ID NOs: 22-25, is 170 - 197 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 127 and 129 and not comprising an amino acid segment G198-E205 corresponding to SEQ ID NO: 3, is 170 - 182 amino acids in length having at least 80
  • (bb) comprises three repeats of a 4-1 BBL amino acid sequence, each possibility represents a separate embodiment of the present invention.
  • the PD! amino acid sequence is 138-145 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85
  • the 4-1BBL amino acid sequence is 170 - 197 amino acids in length having at least 80 % at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 127 and 129 and not comprising an amino acid segment G198-E205 corresponding to SEQ) ID NO:
  • the PD1 amino acid sequence is 138-145 amino acids in length comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 117 and 119; and the 4-1BBL amino acid sequence is 170 - 197 amino acids in length comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 127 and 129.
  • the PD1 amino acid sequence is as set forth in SEQ ID NO: 1 17 or 1 19; and the 4-IBBL amino acid sequence is as set forth in SEQ ID NO: 127 or 129
  • the PD1 amino acid sequence is 138-145 amino acids in length comprising SEQ ID NO: 117; and the 4-!BBL amino acid sequence is 170 - 197 amino acids in length comprising SEQ ID NO: 127.
  • the PD1 amino acid sequence is as set forth in SEQ ID NO: 1 17; and the 4-IBBL amino acid sequence is as set forth in SEQ ID NO: 127.
  • the PD! amino acid sequence is 138-145 amino acids in length comprising SEQ ID NO: 119; and the 4-1BBL amino acid sequence is 170 - 197 amino acids in length comprising SEQ ID NO: 129
  • the PD1 amino acid sequence is as set forth in SEQ ID NO: 1 19; and the 4-1BBL amino acid sequence is as set forth in SEQ ID NO: 129
  • the PD1 amino acid sequence is 123 - 148 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92
  • the 4-IBBL amino acid sequence is 184 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91
  • the PD! amino acid sequence is 123 - 148 amino acids in length comprising SEQ ID NO: 1 15; and the 4-1BBL amino acid sequence is 184 amino acids in length having comprising SEQ ID NO: 123
  • the PD1 amino acid sequence is as set forth in SEQ ID NO: 1 15; and the 4-1BBL amino acid sequence is as set forth in SEQ ID NO: 123.
  • the PD! amino acid sequence is 123 - 148 amino acids in length or 138-145 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 18; and the 4-1BBL amino acid sequence has at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least
  • the PD! amino acid sequence is 123 - 148 amino acids in length or 138-145 amino acids in length comprising SEQ ID NO: 18; and the 4-1BBL amino acid sequence comprises SEQ ID NO: 3
  • the PDI amino acid sequence is as set forth in SEQ ID NO: 18; and the 4-IBBL amino acid sequence is as set forth in SEQ ID NO: 3.
  • the PDI amino acid sequence is 123 - 148 amino acids in length or 138-145 amino acids in length having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 18, and the 4-1BBL amino acid sequence has at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89
  • the PD1 amino acid sequence is 123 - 148 amino acids in length or 138-145 amino acids in length comprising SEQ ID NO: 18; and the 4-1BBL amino acid sequence comprises SEQ ID NO: 131.
  • the PD1 amino acid sequence is as set forth in SEQ ID NO: 18; and the 4-1BBL amino acid sequence is as set forth in SEQ ID NO: 131.
  • Non-limiting examples of specific combinations of PDI amino acid sequence and 4-1BBL amino acid sequence which can be used with specific embodiments of the present invention are provided in Table 4 of the Examples section which follows, which serves as an integral part of the specification.
  • the isolated polypeptide comprising the PDI amino acid sequence or the isolated polypeptide comprising the 4-IBBL amino acid sequence is soluble (i.e., not immobilized to a synthetic or a naturally occurring surface).
  • the isolated polypeptide comprising the PDI amino acid sequence or the isolated polypeptide comprising the 4-1BBL amino acid sequence is immobilized to a synthetic or a naturally occurring surface.
  • the PDI -4-IBBL fusion protein is soluble (i.e., not immobilized to a synthetic or a naturally occurring surface).
  • the PDI -4-IBBL fusion protein is immobilized to a synthetic or a naturally occurring surface.
  • the PDI -4-IBBL fusion protein is in a form of at least a homo-trimer.
  • At least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 % of the PD 1-4- 1BBL fusion protein is in a form of at least a homo-trimer, each possibility represents a separate embodiment of the present invention.
  • the at least homo-trimer comprises a homo-trimer.
  • the at least homo-trimer comprises a homo-tetramer.
  • the at least homo-trimer comprises a homo-pentamer.
  • the at least homo-trimer comprises a homo-hexamer.
  • trimerization methods of determining trimerization are well known in the art and include, but are not limited to NATIVE-PAGE, SEC-HPLC 2D gels, gel filtration, SEC-MALS, Analytical ultracentrifugation (AUC) Mass spectrometry (MS), capillary gel electrophoresis (CGE).
  • AUC Analytical ultracentrifugation
  • MS Mass spectrometry
  • CGE capillary gel electrophoresis
  • the at least homo-trimer is at least 100 kD, at least 120 kD, at least 140 kD, at least 160 kD, at least 180 kD in molecular weight as determined by SEC- MALLS.
  • the at least homo-trimer is at least 100 kD in molecular weight as determined by SEC-MALS
  • the at least homo-trimer is at least 140 kD in molecular weight as determined by SEC-MALS.
  • the at least homo-trimer is at least 200 kD in molecular weight as determined by SEC-MALS
  • the PD1-4-1BBL does not comprise a linker between the PD1 and the 4-1BBL.
  • the PD1 -4-1BBL comprises a linker which may be at any length.
  • the PD1-4-IBBL fusion protein comprises a linker between said PD1 and said 4-1 BBL.
  • the linker may be derived from naturally -occurring multi- domain proteins or is an empirical linker as described, for example, in Chichili et al., (2013), Protein Sci. 22(2): 153-167, Chen et al, (2013), Adv Drug Deli v Rev. 65(10): 1357-1369, the entire contents of which are hereby incorporated by reference.
  • the linker may be designed using linker designing databases and computer programs such as those described in Chen et a! , (2013), Adv Drug Deliv Rev 65(10): 1357-1369 and Crasto et al., (2000), Protein Eng. 13(5):309-312, the entire contents of which are hereby incorporated by reference.
  • the linker is a synthetic linker such as PEG.
  • the linker is an Fc domain or the hinge region of an antibody (e.g., of IgG, IgA, IgD or IgE) or a fragment thereof.
  • the linker is an Fc domain or the hinge region of human IgG4.
  • the Fc domain linker comprises SEQ ID NO: 160.
  • the linker is an Fc domain or the hinge region of human
  • the Fc domain linker comprises SEQ ID NO: 163.
  • the Fc domain or the hinge region linker may comprise conservative and non-conservative amino acid substitutions (also referred to herein as mutations). Such substitution are known in the art.
  • the linker is not an Fc domain or a hinge region of an antibody or a fragment thereof.
  • the linker may be functional.
  • the linker may function to improve the folding and/or stability, improve the expression, improve the pharmacokinetics, and/or improve the bioactivity of the PD1-4-1 BBL fusion protein.
  • the linker may function to target the PD 1 -4- 1BBL fusion protein to a particular cell type or location.
  • the linker is a polypeptide.
  • the linker is selected from the group consisting of GGGGS (SEQ ID NO: 150), (GGGGS f, (n 1 -4) (SEQ ID NO: 149), GGGGSGGGG (SEQ ID NO: 122), (GGGGS)x2 (SEQ ID NO: 151), (GGGGS)x2+GGGG (SEQ ID NO: 121 ).
  • the linker is GGGGSx2 (SEQ ID NO: 151) linker.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 145
  • the PD1-4-IBBL fusion protein amino acid sequence comprises an amino acid sequence having at least 80 % identity to SEQ ID NO: 145
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 85 % identity to SEQ ID NO: 145.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 90 % identity to SEQ ID NO: 145
  • the PD 1-4- IBB L fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity to SEQ ID NO: 145.
  • the PD1-4-1 BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 145
  • the PD1-4-1BBL fusion protein amino acid sequence comprises SEQ ID NO: 145
  • the PD1-4-1BBL fusion protein amino acid sequence consists of SEQ ID NO: 145.
  • the linker is a GGGGSGGGG (SEQ ID NO: 122) linker.
  • the linker is a (GGGGS)x2+GGGG (SEQ ID NO: 121) linker.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 147.
  • the PD1 -4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 80 % identity to SEQ ID NO: 147.
  • the PD1-4-1 BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 85 % identity to SEQ ID NO: 147
  • the PD 1-4- IBB L fusion protein amino acid sequence comprises an amino acid sequence having at least 90 % identity to SEQ ID NO: 147 According to specific embodiments, the PD 1-4- IBB L fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity to SEQ ID NO: 147.
  • the PD1-4-1 BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to SEQ ID NO: 147
  • the PD1-4-1BBL fusion protein amino acid sequence comprises SEQ ID NO: 147
  • the PD1-4-1BBL fusion protein amino acid sequence consists of SEQ ID NO: 147.
  • the PD1 -4-1BBL fusion protein comprises a linker at a length of one to six amino acids.
  • the linker is substantially comprised of glycine and/or serine residues (e.g. about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% or 100 % glycines and serines).
  • the linker is a single amino acid linker.
  • the one amino acid which links PD1 and 4-1BBL is glycine, also referred to herein as PD1-G-4-1BBL fusion protein.
  • the PD 1-4- IBB L fusion protein amino acid sequence comprises an amino acid sequence having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16, 44-56, 97, 101, 103, 105, 107, 11 1 , 1 13, 133, 135, 137, 139, 141 , 143 and 147, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ) ID NO: 133, 135, 137, 139, 141, 143 and 147, each possibility represents a separate embodiment of the present invention.
  • the PD 1-4- IBB L fusion protein amino acid sequence comprises an amino acid sequence having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16, 44-56, 97, 101, 103, 105, 107 and 1 1 1, each possibility represents a separate embodiment of the present invention.
  • the PD1 -4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101, 103, 105, 107 and 11 1 , each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1 BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101, 103, 105 and 107, each possibility represents a separate embodiment of the present invention.
  • the PD 1-4- IBB L fusion protein amino acid sequence comprises an amino acid sequence having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16 and 44-56, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14 and 16, each possibility represents a separate embodiment of the present invention.
  • the PD 1-4- IBB L fusion protein amino acid sequence comprises an amino acid sequence having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16, 44-56, 97, 101 , 103, 105, 107, H I, 113, 133, 135, 137, 139, 141, 143 and 147, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-IBBL fusion protein amino acid sequence comprises an amino acid sequence having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 133, 135, 137, 139, 141, 143 and 147, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101, 103, 105, 107 and 111, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1 BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101, 103, 105 and 107, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 80 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14 and 16, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 85 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16, 44-56, 97, 101, 103, 105, 107, 111, 113, 133, 135, 137, 139, 141, 143 and 147, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 85 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 133, 135, 137, 139, 141, 143 and 147, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 85 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101, 103, 105, 107 and 1 11, each possibility represents a separate embodiment of the present invention.
  • the PD 1-4- IBB L fusion protein amino acid sequence comprises an amino acid sequence having at least 85 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101, 103, 105 and 107, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1 BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 85 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14 and 16, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1 BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 90 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16, 44-56, 97, 101 , 103, 105, 107, 1 1 1, 113, 133, 135, 137, 139, 141, 143 and 147, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1 BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 90 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 133, 135, 137, 139, 141, 143 and 147, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 90 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101, 103, 105, 107 and 1 1 1, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 90 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101, 103, 105 and 107, each possibility represents a separate embodiment of the present invention.
  • the PD 1-4- IBB L fusion protein amino acid sequence comprises an amino acid sequence having at least 90 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14 and 16, each possibility represents a separate embodiment of the present invention.
  • the PD1 -4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity' ⁇ to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16, 44-56, 97, 101, 103, 105, 107, 111, 113, 133, 135, 137, 139, 141, 143 and 147, each possibility represents a separate embodiment of the present invention.
  • the PD 1-4- IBB L fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 133, 135, 137, 139, 141, 143 and 147, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1 BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity' to an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101, 103, 105, 107 and 11 1 , each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1 BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity' to an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101, 103, 105 and 107, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14 and 16, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity to SEQ ID NO: 97.
  • the PD1-4-IBBL fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity to SEQ ID NO: 101.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity to SEQ ID NO: 103.
  • the PD1-4-1 BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity to SEQ ID NO: 105.
  • the PD1 -4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity to SEQ ID NO: 107.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity to SEQ ID NO: 111.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity to SEQ ID NO: 12.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity to SEQ ID NO: 14.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity to SEQ ID NO: 16.
  • the PD 1-4- IBB L fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity to SEQ ID NO: 113. According to specific embodiments, the PD 1-4- IBB L fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity to SEQ ID NO: 133.
  • the PD1-4-1 BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity to SEQ ID NO: 135.
  • the PD1 -4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity to SEQ ID NO: 137.
  • the PD1-4-1 BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity to SEQ ID NO: 139.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity to SEQ ID NO: 141.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity to SEQ ID NO: 143.
  • the PD1-4-IBBL fusion protein amino acid sequence comprises an amino acid sequence having at least 95 % identity to SEQ ID NO: 147.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16, 44-56, 97, 101, 103, 105, 107, I l l, 113, 133, 135, 137, 139, 141, 143 and 147, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 133, 135, 137, 139, 141, 143 and 147, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence having at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101, 103, 105, 107 and 1 1 1, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1 BBL fusion protein amino acid sequence comprises an amino acid sequence an amino acid sequence having at least 96 %, at least 97 3 ⁇ 4, at least 98 %, at least 99 % or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101, 103, 105 and 107, each possibility represents a separate embodiment of the present invention.
  • the PD 1-4- IBB L fusion protein amino acid sequence comprises an amino acid sequence having at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 %identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14 and 16, each possibility represents a separate embodiment of the present invention.
  • the PD1 -4-1BBL fusion protein amino acid sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16, 44-56, 97, 101, 103, 105, 107, 111, 1 13, 133, 135, 137, 139, 141, 143 and 147.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 133, 135, 137, 139, 141, 143 and 147.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16, 44-56, 97, 101, 103, 105, 107 and 111.
  • the PD1 -4-1BBL fusion protein amino acid sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101, 103, 105, 107 and 1 1 1.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101, 103, 105 and 107, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16 and 44-56.
  • the PD1 -4-1BBL fusion protein amino acid sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14 and 16
  • the PD1-4-1BBL fusion protein amino acid sequence comprises SEQ ID NO: 97.
  • the PD1 -4-1BBL fusion protein amino acid sequence comprises SEQ ID NO: 101.
  • the PD1-4-1 BBL fusion protein amino acid sequence comprises SEQ ID NO: 103.
  • the PD 1-4- IBB L fusion protein amino acid sequence comprises SEQ ID NO: 105. According to specific embodiments, the PD 1-4- IBB L fusion protein amino acid sequence comprises SEQ ID NO: 107.
  • the PD1-4-1 BBL fusion protein amino acid sequence comprises SEQ ID NO: i l l.
  • the PD1 -4-1BBL fusion protein amino acid sequence comprises SEQ ID NO: 12.
  • the PD1-4-1 BBL fusion protein amino acid sequence comprises SEQ ID NO: 14.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises SEQ ID NO: 16.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises SEQ ID NO: 1 13.
  • the PD1-4-IBBL fusion protein amino acid sequence comprises SEQ ID NO: 133.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises SEQ ID NO: 135.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises SEQ ID NO: 137.
  • the PD 1-4- IBB L fusion protein amino acid sequence comprises SEQ ID NO: 139.
  • the PD1-4-1 BBL fusion protein amino acid sequence comprises SEQ ID NO: 141.
  • the PD1 -4-1BBL fusion protein amino acid sequence comprises SEQ ID NO: 143.
  • the PD1-4-1BBL fusion protein amino acid sequence comprises SEQ ID NO: 147.
  • the PD1-4-1BBL fusion protein amino acid sequence consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16, 44-56, 97, 101, 103, 105, 107, I l l, 113, 133, 135, 137, 139, 141, 143 and 147.
  • the PD1-4-1 BBL fusion protein amino acid sequence consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 133, 135, 137, 139, 141, 143 and 147.
  • the PD 1-4- IBB L fusion protein amino acid sequence consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16, 44-56, 97, 101, 103, 105, 107 and 111.
  • the PD1-4-1BBL fusion protein amino acid sequence consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101, 103, 105, 107 and 111.
  • the PD1-4-1 BBL fusion protein amino acid sequence consists of an amino acid sequence an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 101, 103, 105 and 107, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1BBL fusion protein amino acid sequence consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14, 16 and 44-56.
  • the PD1 -4-1BBL fusion protein amino acid sequence consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 14 and 16.
  • the PD1-4-1BBL fusion protein amino acid sequence consists of SEQ ID NO: 97.
  • the PD 1-4- IBB L fusion protein amino acid sequence consists of SEQ ID NO: 101.
  • the PD1-4-1 BBL fusion protein amino acid sequence consists of SEQ ID NO: 103.
  • the PD1 -4-1BBL fusion protein amino acid sequence consists of SEQ ID NO: 105.
  • the PD1-4-1BBL fusion protein amino acid sequence consists of SEQ ID NO: 107.
  • the PD 1-4- IBB L fusion protein amino acid sequence consists of SEQ ID NO: 1 1 1.
  • the PD1-4-1BBL fusion protein amino acid sequence consists of SEQ ID NO: 12
  • the PD1-4-1BBL fusion protein amino acid sequence consists of SEQ ID NO: 14.
  • the PD1-4-1BBL fusion protein amino acid sequence consists of SEQ ID NO: 16. According to specific embodiments, the PD1-4-1BBL fusion protein amino acid sequence consists of SEQ ID NO: 113.
  • the PD1-4-1BBL fusion protein amino acid sequence consists of SEQ ID NO: 133.
  • the PD1 -4-1BBL fusion protein amino acid sequence consists of SEQ ID NO: 135.
  • the PD1-4-1BBL fusion protein amino acid sequence consists of SEQ ID NO: 137.
  • the PD1-4-1BBL fusion protein amino acid sequence consists of SEQ ID NO: 139.
  • the PD1-4-1BBL fusion protein amino acid sequence consists of SEQ ID NO: 141.
  • the PD1-4-1BBL fusion protein amino acid sequence consists of SEQ ID NO: 143.
  • the PD1-4-1BBL fusion protein amino acid sequence consists of SEQ ID NO: 147.
  • the PD1-4-1BBL fusion protein amino acid sequence is 300 - 900 amino acids, 300 - 800 amino acids, 300 - 600 amino acids, 300 - 550 amino acids, 300 - 500 amino acids in length, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1 BBL fusion protein amino acid sequence is 300 - 750 amino acids in length.
  • the PD1 -4-1BBL fusion protein amino acid sequence is 308 - 750 amino acids in length.
  • the PD1-4-1BBL fusion protein amino acid sequence is 308 - 400 amino acids in length.
  • the PD1-4-1BBL fusion protein amino acid sequence is 308 - 350 amino acids in length.
  • the PD1-4-1BBL fusion protein amino acid sequence is 308 - 448, 308 - 362, 308 - 351 , 313 - 351, 313 - 339, 318 - 351, 318 - 362, 318 - 340, 318 - 332 amino acids in length, each possibility represents a separate embodiment of the present invention.
  • the PD1-4-1BBL fusion protein amino acid sequence is 308 - 488 amino acids in length.
  • the PD1 -4-1BBL fusion protein amino acid sequence is 308 - 351 amino acids in length.
  • the PD1-4-1 BBL fusion protein amino acid sequence is 318 - 351 amino acids in length.
  • Non-limiting examples of specific PD1-4-1BBL fusion proteins which can be used with specific embodiments of the present invention are provided in Table 4 of the Examples section which follows, which serves as an integral part of the specification.
  • the production yield of the PD1 -4-1BBL fusion protein of some embodiments of the present invention is at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 5 fold higher than the production yield of a PD1-4-1BBL fusion protein comprising a PD l amino acid sequence as set forth in SEQ ID NO: 2 and a 4-1BBL amino acid sequence as set forth in SEQ ID NO: 3, under the same production conditions, each possibility represents a separate embodiment of the present invention.
  • the production yield of the PD1-4-1 BBL fusion protein of some embodiments of the present invention is at least 1.5 fold higher than the production yield of a PD1 -4-1BBL fusion protein comprising a PDl amino acid sequence as set forth in SEQ ID NO: 2 and a 4-1BBL amino acid sequence as set forth in SEQ ID NO: 3, under the same production conditions.
  • the production yield of the PD1-4-1BBL fusion protein of some embodiments of the present invention is at least 2 fold higher than the production yield of a PD1-4-1 BBL fusion protein comprising a PDl amino acid sequence as set forth in SEQ ID NO: 2 and a 4-1BBL amino acid sequence as set forth in SEQ ID NO: 3, under the same production conditions.
  • the production yield of the PD1-4-1BBL fusion protein of some embodiments of the present invention is at least 1 5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 5 fold higher than the production yield of a SEQ ID NO: 5, under the same production condi tions each possibility represents a separate embodiment of the present invention.
  • the production yield of the PD1-4-1 BBL fusion protein of some embodiments of the present invention is at least 1.5 fold higher than the production yield of SEQ ID NO: 5 under the same production conditions.
  • the production yield of the PD 1-4-1BBL fusion protein of some embodiments of the present invention is at least 2 fold higher than the production yield of a SEQ ID NO: 5 under the same production conditions.
  • the production yield of the isolated polypeptide comprising the PD1 amino acid sequence of some embodiments of the present invention is at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 5 fold higher than the production yield of SEQ ID NO: 2, under the same production conditions, each possibility represents a separate embodiment of the present invention.
  • the production yield of the isolated polypeptide comprising the 4-1BBL amino acid sequence of some embodiments of the present invention is at least 1 .5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 5 fold higher than the production yield of SEQ ID NO: 3, under the same production conditions, each possibility represents a separate embodiment of the present invention.
  • the amount of aggregates of the PD1-4-1BBL fusion protein of some embodiments of the present invention is at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 % or at least 95 % lower than the amount of aggregates of SEQ ID NO: 5 under the same production conditions, said aggregates are of at least 300 kDa in molecular weight as may be determined by e.g. SDS-PAGE or SEC-MALS.
  • the amount of aggregates of the PD1-4-1BBL fusion protein of some embodiments of the present invention is at least 20 % lower than the amount of aggregates of SEQ ID NO: 5 under the same production conditions.
  • the amount of aggregates of the PD1-4-1BBL fusion protein of some embodiments of the present invention is at least 50 % lower than amount of aggregates of a SEQ ID NO: 5 under the same production conditions.
  • the activity of the PD1 -4-1BBL fusion protein of some embodiments of the present invention is at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 5 fold higher than the activity of a PD1-4-1BBL fusion protein comprising a PDi amino acid sequence as set forth in SEQ ID NO: 2 and a 4-1BBL amino acid sequence as set forth in SEQ ID NO: 3, e.g. under the same production conditions, each possibility represents a separate embodiment of the present invention.
  • the activity of the PD1-4-1BBL fusion protein of some embodiments of the present invention is at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 5 fold higher than the activity of a SEQ ID NO: 5, e.g. under the same production conditions, each possibility represents a separate embodiment of the present invention.
  • the activity of the isolated polypeptide comprising the PD1 amino acid sequence of some embodiments of the present invention is at least 1 5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 5 fold higher than the activity of SEQ ID NO: 2, e.g. under the same production conditions, each possibility represents a separate embodiment of the present invention.
  • the activity of the isolated polypeptide comprising the 4- 1 BBL amino acid sequence of some embodiments of the present invention is at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 5 fold higher than the activity of SEQ ID NO: 3, e.g. under the same production conditions, each possibility represents a separate embodiment of the present invention.
  • the stability of the PD1-4-1BBL fusion protein of some embodiments of the present invention is at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 5 fold higher than the stability of a PD1-4-1BBL fusion protein comprising a PDi amino acid sequence as set forth in SEQ ID NO: 2 and a 4-1BBL amino acid sequence as set forth in SEQ ID NO: 3, under the same production conditions, each possibility represents a separate embodiment of the present invention.
  • the stability of the PD1-4-1BBL fusion protein of some embodiments of the present invention is at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 5 fold higher than the stability of a SEQ ID NO: 5, under the same production conditions each possibility represents a separate embodiment of the present invention.

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SG11202013170RA SG11202013170RA (en) 2018-07-11 2019-07-11 Pd1-4-1bbl variant fusion protein and methods of use thereof
US17/258,220 US12286466B2 (en) 2018-07-11 2019-07-11 PD1-4-1BBL variant fusion protein and methods of use thereof
BR112021000347-9A BR112021000347A2 (pt) 2018-07-11 2019-07-11 Proteína de fusão variante pd1-4-1bbl e métodos de uso da mesma
AU2019301315A AU2019301315A1 (en) 2018-07-11 2019-07-11 PD1-4-1BBL variant fusion protein and methods of use thereof
EP19833260.3A EP3820887A4 (en) 2018-07-11 2019-07-11 Pd1-4-1bbl variant fusion protein and methods of use thereof
CN201980052864.9A CN112543767B (zh) 2018-07-11 2019-07-11 Pd1-4-1bbl变体融合蛋白及其使用方法
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MX2021000047A MX2021000047A (es) 2018-07-11 2019-07-11 Proteina de fusion variante pd1-4-1bbl y procedimientos de uso de la misma.
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