Compositions of immune checkpoint multivalent particles and methods of use

Abstract

Provided herein are multivalent particles that express immune checkpoint molecules, and compositions of the multivalent particles.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 191,031, filed May 20, 2021, which is incorporated by reference in its entirety.

[0002] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0003] In some embodiments herein, a multivalent particle is disclosed that comprises a fusion protein comprising a mammalian immune checkpoint polypeptide and a transmembrane polypeptide, the fusion protein being expressed on the surface of the multivalent particle at a valency of at least about 10 copies. In some embodiments, the mammalian immune checkpoint polypeptide comprises a polypeptide expressed on a T cell. In some embodiments, the mammalian immune checkpoint polypeptide comprises an immune inhibitory checkpoint polypeptide. In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9. In some embodiments, the immune inhibitory checkpoint polypeptide is expressed on an antigen presenting cell, a tumor cell, or a normal cell. In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3. In some embodiments, the mammalian immune checkpoint polypeptide comprises an immune stimulatory checkpoint polypeptide. In some embodiments, the immune stimulatory checkpoint polypeptide comprises a polypeptide expressed on a T cell. In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR. In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL. In some embodiments, the immune stimulatory checkpoint polypeptide is expressed on an antigen presenting cell, a tumor cell, or a normal cell. In some embodiments, the immune inhibitory checkpoint polypeptide comprises an amino acid sequence that has at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-42 or 96-101.In some embodiments, the immune stimulatory checkpoint polypeptide comprises an amino acid sequence of at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 43-62, 102-115, or 153-162. In some embodiments, the transmembrane polypeptide anchors the fusion protein to the bilayer of the multivalent particle. In some embodiments, the transmembrane polypeptide comprises a spike glycoprotein, a mammalian membrane protein, an envelope protein, a nucleocapsid protein, or a cellular transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a VSVG, a dengue E protein, influenza hemagglutinin, influenza neuraminidase, spike protein S1, spike protein S2, a Sindbis virus envelope (SINDBIS) protein, a hemagglutinin envelope protein from measles virus, an envelope glycoprotein of the measles virus fusion (F) protein, RD114, BaEV, GP41, or GP120. In some embodiments, the VSVG comprises a full-length VSVG or a truncated VSVG. In some embodiments, the VSVG comprises a transmembrane domain and a cytoplasmic tail. In some embodiments, the fusion protein further comprises a multimerization domain. In some embodiments, the multimerization domain comprises a dimerization domain, a trimerization domain, or a tetramerization domain. In some embodiments, the dimerization domain comprises a leucine zipper dimerization domain. In some embodiments, the fusion protein further comprises a cytosolic domain. In some embodiments, the trimerization domain comprises a post-fusion multimerization domain of a viral surface protein. In some embodiments, the trimerization domain comprises a D4 post-fusion trimerization domain of a VSV-G protein. In some embodiments, the trimerization domain comprises a post-fusion trimerization domain of a Dengue E protein. In some embodiments, the trimerization domain comprises a foldon trimerization domain. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence at least about 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 63, 64, or 79-95.In some embodiments, the tetramerization domain comprises an influenza neuraminidase stem domain. In some embodiments, the multimerization domain comprises an amino acid sequence having at least 95% sequence identity to an amino acid sequence according to SEQ ID NO: 65-78. In some embodiments, when the fusion protein is expressed on the surface of a multivalent particle, the multimerization domain is on the outside of the multivalent particle. In some embodiments, when the fusion protein is expressed on the surface of a multivalent particle, the multimerization domain is on the outside of the multivalent particle and adjacent to a signal peptide. In some embodiments, when the fusion protein is expressed on the surface of a multivalent particle, the multimerization domain is on the inside of the multivalent particle. In some embodiments, when the fusion protein is expressed on the surface of a multivalent particle, the multimerization domain is on the inside of the multivalent particle and adjacent to a transmembrane polypeptide. In some embodiments, the fusion protein comprises a signal peptide. In some embodiments, the domains of the fusion protein are arranged from N-terminus to C-terminus in the following order: signal peptide, mammalian immune checkpoint polypeptide, multimerization domain, transmembrane polypeptide, and cytosolic domain; signal peptide, mammalian immune checkpoint polypeptide, transmembrane polypeptide, multimerization domain, and cytosolic domain; or signal peptide, multimerization domain, mammalian immune checkpoint polypeptide, transmembrane polypeptide, and cytosolic domain. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle at a valency of about 10 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle at a valency of about 10 to about 15 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle at a valency of at least about 25 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle at a valency of at least about 50 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle at a valency of at least about 75 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle at a valency of at least about 100 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle at a valency of at least about 150 copies.In some embodiments, the fusion protein is expressed on the surface of the multivalent particle at a valency of at least about 200 copies. In some embodiments, the multivalent particle does not contain viral genetic material. In some embodiments, the multivalent particle is a virus-like particle. In some embodiments, the multivalent particle is an extracellular vesicle (EV). In some embodiments, the multivalent particle is an exosome. In some embodiments, the multivalent particle is an ectosome. In some embodiments, the (a) immune checkpoint polypeptide is PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, SIGLEC9, PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, Galectin-3, CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, GITR, CD70, CD80, and (b) the transmembrane polypeptide comprises VSVG, dengue E protein, influenza hemagglutinin, influenza neuraminidase, spike protein S1, spike protein S2, Sindbis virus envelope (SINDBIS) protein, hemagglutinin envelope protein from measles virus, envelope glycoprotein of measles virus fusion (F) protein, RD114, BaEV, GP41, or GP120.In some embodiments, the (a) immune checkpoint polypeptide is PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, SIGLEC9, PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, Galectin-3, CD27, CD28, CD40 , CD122, 4-1BB, ICOS, OX40, CD2, CD30, GITR, CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL, and (b) the transmembrane polypeptide comprises an amino acid sequence at least about 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 63, 64, or 79-95. In some embodiments, (a) the immune checkpoint polypeptide comprises an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, or 153-162, and (b) the transmembrane polypeptide comprises VSVG, Dengue E protein, influenza hemagglutinin, influenza neuraminidase, spike protein S1, spike protein S2, Sindbis virus envelope (SINDBIS) protein, hemagglutinin envelope protein from measles virus, envelope glycoprotein of measles virus fusion (F) protein, RD114, BaEV, GP41, or GP120. In some embodiments, (a) the immune checkpoint polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identical to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, or 153-162, and (b) the transmembrane polypeptide comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, or 99% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 63, 64, or 79-95.In some embodiments, the (a) immune checkpoint polypeptide is PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, SIGLEC9, PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, Galectin-3, CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, GITR, C D70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL, and (b) the transmembrane polypeptide is VSVG, dengue E protein, influenza hemagglutinin, influenza neuraminidase, spike protein S1, spike protein S2, Sindbis virus envelope (SINDBIS) protein, hemagglutinin envelope protein from measles virus, envelope glycoprotein of measles virus fusion (F) protein, RD114, BaE. V, GP41, or GP120, and (c) the multimerization domain comprises a leucine zipper dimerization domain, a post-fusion multimerization domain of a viral surface protein, a D4 post-fusion trimerization domain of a VSV-G protein, a post-fusion trimerization domain of a Dengue E protein, a Foldon trimerization domain, or an influenza neuraminidase stem domain. In some embodiments, the (a) immune checkpoint polypeptide is PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, SIGLEC9, PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, Galectin-3, CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, GITR, CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, C (b) the transmembrane polypeptide comprises VSVG, Dengue E protein, influenza hemagglutinin, influenza neuraminidase, spike protein S1, spike protein S2, Sindbis virus envelope (SINDBIS) protein, hemagglutinin envelope protein from measles virus, envelope glycoprotein of measles virus fusion (F) protein, RD114, BaEV, GP41, or GP120; and (c) the multimerization domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to an amino acid sequence according to SEQ ID NO:65-78.In some embodiments, the (a) immune checkpoint polypeptide is PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, SIGLEC9, PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, Galectin-3, CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, GITR, CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, O (b) the transmembrane polypeptide comprises an amino acid sequence at least about 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 63, 64, or 79-95; and (c) the multimerization domain comprises a leucine zipper dimerization domain, a post-fusion multimerization domain of a viral surface protein, a D4 post-fusion trimerization domain of a VSV-G protein, a post-fusion trimerization domain of a Dengue E protein, a Foldon trimerization domain, or an influenza neuraminidase stem domain.In some embodiments, (a) the immune checkpoint polypeptide comprises an amino acid sequence of at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, or 153-162, and (b) the transmembrane polypeptide comprises a VSVG, a dengue E protein, an influenza hemagglutinin, an influenza neuraminidase, a spike protein S1, a spike protein S2, a Sindbis virus envelope (SINV). ) protein, a hemagglutinin envelope protein from measles virus, an envelope glycoprotein of the measles virus fusion (F) protein, RD114, BaEV, GP41, or GP120; and (c) the multimerization domain comprises a leucine zipper dimerization domain, a post-fusion multimerization domain of a viral surface protein, the D4 post-fusion trimerization domain of the VSV-G protein, the post-fusion trimerization domain of the Dengue E protein, the Foldon trimerization domain, or the influenza neuraminidase stem domain. In some embodiments, the (a) immune checkpoint polypeptide is PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, SIGLEC9, PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, Galectin-3, CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, GITR, CD70, C (b) the transmembrane polypeptide comprises an amino acid sequence at least about 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 63, 64, or 79-95; and (c) the multimerization domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the amino acid sequence according to SEQ ID NOs: 65-78.

[0004] In some embodiments herein, a composition is disclosed, comprising a first nucleic acid sequence encoding a multivalent particle comprising a fusion protein comprising a mammalian immune checkpoint polypeptide and a transmembrane polypeptide, the fusion protein being expressed at a valency of at least about 10 copies on the surface of the multivalent particle when the multivalent particle is expressed, and an excipient. In some embodiments, the composition further comprises a second nucleic acid sequence encoding one or more viral proteins. In some embodiments, the one or more viral proteins are lentiviral proteins, retroviral proteins, adenoviral proteins, or combinations thereof. In some embodiments, the one or more viral proteins include gag, pol, pre, tat, rev, or combinations thereof. In some embodiments, the composition further comprises a third nucleic acid sequence encoding a replication-deficient viral genome, a reporter, a therapeutic molecule, or combinations thereof. In some embodiments, the viral genome is derived from vesicular stomatitis virus, measles virus, hepatitis virus, influenza virus, or combinations thereof. In some embodiments, the reporter is a fluorescent protein or luciferase. In some embodiments, the fluorescent protein is green fluorescent protein. In some embodiments, the therapeutic molecule is a cell signaling regulating molecule, a proliferation regulating molecule, a cell death regulating molecule, or a combination thereof. In some embodiments, the mammalian immune checkpoint polypeptide comprises a polypeptide expressed on a T cell. In some embodiments, the mammalian immune checkpoint polypeptide comprises an immune inhibitory checkpoint polypeptide. In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9. In some embodiments, the immune inhibitory checkpoint polypeptide is expressed on an antigen presenting cell, a tumor cell, or a normal cell.In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3. In some embodiments, the mammalian immune checkpoint polypeptide comprises an immune stimulatory checkpoint polypeptide. In some embodiments, the immune stimulatory checkpoint polypeptide comprises a polypeptide expressed on a T cell. In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR. In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL. In some embodiments, the immune stimulatory checkpoint polypeptide is expressed on an antigen presenting cell, a tumor cell, or a normal cell. In some embodiments, the immune inhibitory checkpoint polypeptide comprises an amino acid sequence of at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-42 or 96-101. In some embodiments, the immune stimulatory checkpoint polypeptide comprises an amino acid sequence of at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 43-62, 102-115, or 153-162. In some embodiments, the transmembrane polypeptide anchors the fusion protein to the bilayer of the multivalent particle. In some embodiments, the transmembrane polypeptide comprises a spike glycoprotein, a mammalian membrane protein, an envelope protein, a nucleocapsid protein, or a cellular transmembrane protein.In some embodiments, the transmembrane polypeptide comprises VSVG, dengue E protein, influenza hemagglutinin, influenza neuraminidase, spike protein S1, spike protein S2, Sindbis virus envelope (SINDBIS) protein, hemagglutinin envelope protein from measles virus, envelope glycoprotein of measles virus fusion (F) protein, RD114, BaEV, GP41, or GP120. In some embodiments, the VSVG comprises full-length VSVG or a truncated VSVG. In some embodiments, the VSVG comprises a transmembrane domain and a cytoplasmic tail. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence at least about 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 63, 64, or 79-95. In some embodiments, the fusion protein further comprises a multimerization domain. In some embodiments, the multimerization domain comprises a dimerization domain, a trimerization domain, or a tetramerization domain. In some embodiments, the dimerization domain comprises a leucine zipper dimerization domain. In some embodiments, the trimerization domain comprises a post-fusion multimerization domain of a viral surface protein. In some embodiments, the trimerization domain comprises a D4 post-fusion trimerization domain of a VSV-G protein. In some embodiments, the trimerization domain comprises a post-fusion trimerization domain of a Dengue E protein. In some embodiments, the trimerization domain comprises a foldon trimerization domain. In some embodiments, the fusion protein further comprises a cytosolic domain. In some embodiments, the tetramerization domain comprises an influenza neuraminidase stem domain. In some embodiments, the multimerization domain comprises an amino acid sequence having at least 95% sequence identity to an amino acid sequence according to SEQ ID NOs: 65-78. In some embodiments, when the fusion protein is expressed on the surface of a multivalent particle, the multimerization domain is on the outside of the multivalent particle. In some embodiments, when the fusion protein is expressed on the surface of a multivalent particle, the multimerization domain is on the outside of the multivalent particle and adjacent to the signal peptide.In some embodiments, when the fusion protein is expressed on the surface of the multivalent particle, the multimerization domain is inside the multivalent particle. In some embodiments, when the fusion protein is expressed on the surface of the multivalent particle, the multimerization domain is inside the multivalent particle and adjacent to the transmembrane polypeptide. In some embodiments, the fusion protein includes a signal peptide. In some embodiments, the domains of the fusion protein are arranged from N-terminus to C-terminus in the following order: signal peptide, mammalian immune checkpoint polypeptide, multimerization domain, transmembrane polypeptide, and cytosolic domain, signal peptide, mammalian immune checkpoint polypeptide, transmembrane polypeptide, multimerization domain, and cytosolic domain, or signal peptide, multimerization domain, mammalian immune checkpoint polypeptide, transmembrane polypeptide, and cytosolic domain. In some embodiments, the fusion protein is expressed at a valency of about 10 copies on the surface of the multivalent particle when the multivalent particle is expressed. In some embodiments, the fusion protein is expressed at a valency of about 10 copies to about 15 copies on the surface of the multivalent particle when the multivalent particle is expressed. In some embodiments, the fusion protein is expressed at a valency of at least about 25 copies on the surface of the multivalent particle when the multivalent particle is expressed. In some embodiments, the fusion protein is expressed at a valency of at least about 50 copies on the surface of the multivalent particle when the multivalent particle is expressed. In some embodiments, the fusion protein is expressed at a valency of at least about 75 copies on the surface of the multivalent particle when the multivalent particle is expressed. In some embodiments, the fusion protein is expressed at a valency of at least about 100 copies on the surface of the multivalent particle when the multivalent particle is expressed. In some embodiments, the fusion protein is expressed at a valency of at least about 150 copies on the surface of the multivalent particle when the multivalent particle is expressed. In some embodiments, the fusion protein is expressed at a valency of at least about 200 copies on the surface of the multivalent particle when the multivalent particle is expressed. In some embodiments, the fusion protein is expressed at a valency of at least about 500 copies on the surface of the multivalent particle when the multivalent particle is expressed.In some embodiments, the fusion protein is expressed at a valency of at least about 1000 copies on the surface of the multivalent particle when the multivalent particle is expressed. In some embodiments, the fusion protein is expressed at a valency of at least about 2000 copies on the surface of the multivalent particle when the multivalent particle is expressed. In some embodiments, the multivalent particle does not contain viral genetic material. In some embodiments, the multivalent particle is a virus-like particle. In some embodiments, the multivalent particle is an extracellular vesicle (EV). In some embodiments, the multivalent particle is an exosome. In some embodiments, the multivalent particle is an ectosome. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are in the same vector. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are in different vectors. In some embodiments, the vector is a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector. In some embodiments, the vector comprises a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.In some embodiments, the (a) immune checkpoint polypeptide is PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, SIGLEC9, PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, Galectin-3, CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, GITR, CD70, CD80, and (b) the transmembrane polypeptide comprises VSVG, dengue E protein, influenza hemagglutinin, influenza neuraminidase, spike protein S1, spike protein S2, Sindbis virus envelope (SINDBIS) protein, hemagglutinin envelope protein from measles virus, envelope glycoprotein of measles virus fusion (F) protein, RD114, BaEV, GP41, or GP120. In some embodiments, the (a) immune checkpoint polypeptide is PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, SIGLEC9, PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, Galectin-3, CD27, CD28, CD 40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, GITR, CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL; and (b) the transmembrane polypeptide comprises an amino acid sequence at least about 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 63, 64, or 79-95. In some embodiments, (a) the immune checkpoint polypeptide comprises an amino acid sequence of at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, or 153-162, and (b) the transmembrane polypeptide comprises VSVG, Dengue E protein, influenza hemagglutinin, influenza neuraminidase, spike protein S1, spike protein S2, Sindbis virus envelope (SINDBIS) protein, hemagglutinin envelope protein from measles virus, envelope glycoprotein of measles virus fusion (F) protein, RD114, BaEV, GP41, or GP120. In some embodiments, (a) the immune checkpoint polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identical to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, or 153-162, and (b) the transmembrane polypeptide comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, or 99% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 63, 64, or 79-95.In some embodiments, (a) the immune checkpoint polypeptide comprises PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, SIGLEC9, PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, Galectin-3, CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, GITR, CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL; and (b) the transmembrane polypeptide comprises , VSVG, dengue E protein, influenza hemagglutinin, influenza neuraminidase, spike protein S1, spike protein S2, Sindbis virus envelope (SINDBIS) protein, hemagglutinin envelope protein from measles virus, envelope glycoprotein of measles virus fusion (F) protein, RD114, BaEV, GP41, or GP120; and (c) the multimerization domain comprises a leucine zipper dimerization domain, a post-fusion multimerization domain of a viral surface protein, the D4 post-fusion trimerization domain of the VSV-G protein, the post-fusion trimerization domain of the dengue E protein, a foldon trimerization domain, or an influenza neuraminidase stem domain.In some embodiments, the (a) immune checkpoint polypeptide is PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, SIGLEC9, PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, Galectin-3, CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, GITR, CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, C (b) the transmembrane polypeptide comprises VSVG, Dengue E protein, influenza hemagglutinin, influenza neuraminidase, spike protein S1, spike protein S2, Sindbis virus envelope (SINDBIS) protein, hemagglutinin envelope protein from measles virus, envelope glycoprotein of measles virus fusion (F) protein, RD114, BaEV, GP41, or GP120; and (c) the multimerization domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to an amino acid sequence according to SEQ ID NO:65-78.In some embodiments, the (a) immune checkpoint polypeptide is PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, SIGLEC9, PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, Galectin-3, CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, GITR, CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, O (b) the transmembrane polypeptide comprises an amino acid sequence at least about 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 63, 64, or 79-95; and (c) the multimerization domain comprises a leucine zipper dimerization domain, a post-fusion multimerization domain of a viral surface protein, a D4 post-fusion trimerization domain of a VSV-G protein, a post-fusion trimerization domain of a Dengue E protein, a Foldon trimerization domain, or an influenza neuraminidase stem domain.In some embodiments, (a) the immune checkpoint polypeptide comprises an amino acid sequence of at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, or 153-162, and (b) the transmembrane polypeptide comprises a VSVG, a dengue E protein, an influenza hemagglutinin, an influenza neuraminidase, a spike protein S1, a spike protein S2, a Sindbis virus envelope (SINV). ) protein, a hemagglutinin envelope protein from measles virus, an envelope glycoprotein of the measles virus fusion (F) protein, RD114, BaEV, GP41, or GP120; and (c) the multimerization domain comprises a leucine zipper dimerization domain, a post-fusion multimerization domain of a viral surface protein, the D4 post-fusion trimerization domain of the VSV-G protein, the post-fusion trimerization domain of the Dengue E protein, the Foldon trimerization domain, or the influenza neuraminidase stem domain. In some embodiments, the (a) immune checkpoint polypeptide is PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, SIGLEC9, PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, Galectin-3, CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, GITR, CD70, C (b) the transmembrane polypeptide comprises an amino acid sequence at least about 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 63, 64, or 79-95; and (c) the multimerization domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the amino acid sequence according to SEQ ID NOs: 65-78.

[0005] Disclosed herein in some embodiments are pharmaceutical compositions comprising the multivalent particles disclosed herein and a pharma- ceutically acceptable excipient.

[0006] In some embodiments herein, disclosed are methods of treating cancer, autoimmune disease, infectious disease, or inflammatory disease comprising administering a multivalent particle disclosed herein. In some embodiments, the multivalent particle is administered intravenously. In some embodiments, the multivalent particle is administered by inhalation. In some embodiments, the multivalent particle is administered by intraperitoneal injection. In some embodiments, the multivalent particle is administered by subcutaneous injection.

[0007] Disclosed herein in some embodiments are compositions comprising a multivalent particle (MVP), the MVP comprising an enveloped particle displaying at least about 10 copies of an immune checkpoint polypeptide on the surface of the MVP, wherein the immune checkpoint polypeptide, when displayed on the surface of the enveloped particle, forms a multivalent interaction with a ligand on a target immune cell.

[0008] In some embodiments herein, methods are disclosed that use multivalent particles (MVPs) to display an immune checkpoint polypeptide and thereby mimic a multivalent interaction between a first immune cell expressing the immune checkpoint polypeptide and a second immune cell expressing a target of the immune checkpoint polypeptide, where the immune checkpoint polypeptide is displayed in at least about 10 copies on the surface of the MVP. [Brief description of the drawings]

[0009] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0010] [Figure 1A]FIG. 1 illustrates vector design for monomeric display of immune checkpoints on MVPs. [Figure 1B] FIG. 1 illustrates vector design for trimeric display of immune checkpoints on MVPs. [Figure 1C] FIG. 1 illustrates the vector design for expressing type II immune checkpoint on MVP. [Figure 2A] FIG. 1 illustrates the production of monomeric IC-MVP as a VLP containing an RNA genome. [Figure 2B] FIG. 1 illustrates the production of monomeric IC-MVP as a VLP without an RNA genome. [Figure 2C] FIG. 1 illustrates the production of monomeric IC-MVP as EVs. [Figure 3A] FIG. 1 illustrates the production of trimeric IC-MVP as a VLP containing an RNA genome. [Figure 3B] FIG. 1 illustrates the production of trimeric IC-MVP as a VLP without an RNA genome. [Figure 3C] FIG. 1 illustrates the production of trimeric IC-MVP as EVs. [Figure 4A] FIG. 1 illustrates the production of mixed monomeric and trimeric IC-MVPs as VLPs containing an RNA genome. [Figure 4B] FIG. 1 illustrates the production of mixed monomeric and trimeric IC-MVPs as VLPs without an RNA genome. [Figure 4C] FIG. 1 illustrates the production of mixed monomeric and trimeric IC-MVPs as EVs. [Figure 5A] FIG. 13 is a diagram illustrating a D4 configuration. [Figure 5B] FIG. 1 illustrates another D4 configuration. [Figure 5C] FIG. 13 is a diagram illustrating another D4 configuration. [Figure 6A] FIG. 1 illustrates the arrangement of multimerization domains. [Figure 6B] FIG. 1 illustrates alternative multimerization domain configurations. [Figure 6C] FIG. 1 illustrates another example of a multimerization domain arrangement. [Figure 7A] FIG. 1 illustrates a FACS-based assay to measure specific binding of dye-labeled IC-MVP to target cells expressing the cognate receptor or ligand. [Figure 7B] FIG. 1 illustrates a FACS-based assay to measure specific binding of unlabeled IC-MVP to target cells expressing the cognate receptor or ligand. [Figure 8A] FIG. 1 illustrates quantitative Western blot analysis of PD-1-MVP. [Figure 8B] FIG. 1 illustrates a FACS analysis of specific binding of dye-labeled PD-1-MVP to target cells expressing the cognate receptor PD-L1. [Figure 8C] FIG. 1 illustrates a FACS analysis of specific binding of unlabeled PD-1-MVP to target cells expressing the cognate receptor PD-L1. [Figure 8D] FIG. 1 illustrates a FACS analysis of specific binding of dye-labeled PD-1-MVP to target cells expressing the cognate receptor PD-L2. [Figure 8E] FIG. 1 illustrates a FACS analysis of specific binding of unlabeled PD-1-MVP to target cells expressing the cognate receptor PD-L2. [Figure 9A] FIG. 1 illustrates an inhibitory immune checkpoint on T cells and its ligands on antigen-presenting cells, including tumor cells. [Figure 9B] FIG. 1 illustrates PD-L1 and PD-1 mediated inhibitory checkpoint signaling on antigen-specific T cells. [Figure 9C] FIG. 1 illustrates the blockade of PD-L1 and PD-1-mediated inhibitory checkpoint signaling by anti-PD-1 antibodies. [Figure 9D] FIG. 1 illustrates the blockade of PD-L1 and PD-1 mediated inhibitory checkpoint signaling by PD-1-MVP. [Figure 10A] Figure 1 illustrates FACS analysis of PD-L1 expression on B16F0 melanoma cells. [Figure 10B] Figure 1 illustrates FACS analysis of PD-L1 expression on B16F10 melanoma cells. [Figure 10C] FIG. 1 illustrates a FACS analysis of specific binding of dye-labeled PD-1-MVP to B16F0 melanoma cells expressing the cognate receptor PD-L1. [Figure 10D] FIG. 1 illustrates a FACS analysis of specific binding of dye-labeled PD-1-MVP to B16F10 melanoma cells expressing the cognate receptor PD-L1. [Figure 11A] FIG. 1 illustrates the study design to determine the effect of PD-1-MVP on murine B16F0 melanoma. [Figure 11B] FIG. 1 illustrates the effect of PD-1-MVP on mouse B16F0 melanoma tumor growth. [Figure 11C] FIG. 1 illustrates the effect of PD-1-MVP on survival of mice bearing B16F0 melanoma tumors. [Figure 12A] FIG. 1 illustrates the study design to determine the effect of PD-1-MVP on murine B16F10 melanoma. [Figure 12B] FIG. 1 illustrates the effect of PD-1-MVP on mouse B16F10 melanoma tumor growth. [Figure 13A] Figure 1 illustrates FACS analysis of PD-L1 expression on MC38 colon adenocarcinoma cells. [Figure 13B] FIG. 1 illustrates a FACS analysis of specific binding of dye-labeled PD-1-MVP to MC38 colon adenocarcinoma cells expressing the cognate receptor PD-L1. [Figure 13C] FIG. 1 illustrates the study design to determine the effect of PD-1-MVP on murine MC38 colon adenocarcinoma. [Figure 13D] FIG. 1 illustrates the effect of PD-1-MVP on mouse MC38 colon adenocarcinoma tumor growth. [Figure 14A] FIG. 1 illustrates the lack of engagement of PD-L1 on antigen-presenting cells with PD-1 on antigen-specific T cells. [Figure 14B] FIG. 1 illustrates the use of PD-L1-MVP or PD-L2-MVP as agonists to turn on PD-1-mediated inhibitory checkpoint signaling in antigen-specific T cells. [Figure 15A] FIG. 1 illustrates quantitative Western blot analysis of PDL-1-MVP. [Figure 15B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled PDL-1-MVP to target cells expressing the cognate receptor PD-1. [Figure 15C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled PDL-1-MVP to target cells expressing the cognate receptor PD-1. [Figure 16A] FIG. 1 illustrates the study design to determine the effect of PDL-1-MVP on ARD in mice. [Figure 16B] FIG. 1 illustrates the effect of PDL-1-MVP on survival of mice with ARD. [Figure 17A] FIG. 1 illustrates quantitative Western blot analysis of 2B4-MVP. [Figure 17B] FIG. 1 illustrates FACS analysis of specific binding of unlabeled 2B4-MVP to target cells expressing the cognate receptor CD48. [Figure 18A] FIG. 1 illustrates the study design to determine the effect of 2B4-MVP on ARD in mice. [Figure 18B] FIG. 1 illustrates the effect of 2B4-MVP on survival of mice with ARD. [Figure 19A] FIG. 1 illustrates quantitative Western blot analysis of PDL-2-MVP. [Figure 19B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled PDL-2-MVP to target cells expressing the cognate receptor PD-1. [Figure 19C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled PDL-2-MVP to target cells expressing the cognate receptor PD-1. [Figure 20A] FIG. 1 illustrates quantitative Western blot analysis of CTLA4-MVP. [Figure 20B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled CTLA4-MVP to target cells expressing the cognate receptor CD80. [Figure 20C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled CTLA4-MVP to target cells expressing the cognate receptor CD80. [Figure 20D] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled CTLA4-MVP to target cells expressing the cognate receptor CD86. [Figure 20E] FIG. 1 illustrates a FACS analysis of specific binding of unlabeled CTLA4-MVP to target cells expressing the cognate receptor CD86. [Figure 21A] FIG. 1 illustrates quantitative Western blot analysis of CD80-MVP. [Figure 21B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled CD80-MVP to target cells expressing the cognate receptor CTLA-4. [Figure 21C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled CD80-MVP to target cells expressing the cognate receptor CTLA-4. [Figure 22A] FIG. 1 illustrates quantitative Western blot analysis of CD86-MVP. [Figure 22B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled CD86-MVP to target cells expressing the cognate receptor CTLA-4. [Figure 22C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled CD86-MVP to target cells expressing the cognate receptor CTLA-4. [Figure 23A]FIG. 1 illustrates quantitative Western blot analysis of Galectin3-MVP. [Figure 23B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled Galectin3-MVP to target cells expressing the cognate receptor LAG-3. [Figure 23C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled Galectin3-MVP to target cells expressing the cognate receptor LAG-3. [Figure 24A] FIG. 1 illustrates quantitative Western blot analysis of LAG3-MVP. [Figure 24B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled LAG3-MVP to target cells expressing the cognate receptor Galectin-3. [Figure 24C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled LAG3-MVP to target cells expressing the cognate receptor Galectin-3. [Figure 25A] FIG. 1 illustrates quantitative Western blot analysis of FGL1-MVP. [Figure 25B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled FGL1-MVP to target cells expressing the cognate receptor LAG-3. [Figure 25C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled FGL1-MVP to target cells expressing the cognate receptor LAG-3. [Figure 26A] FIG. 1 illustrates quantitative Western blot analysis of LAG3-MVP. [Figure 26B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled LAG3-MVP to target cells expressing the cognate receptor FGL1. [Figure 26C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled LAG3-MVP to target cells expressing the cognate receptor FGL1. [Figure 27A]FIG. 1 illustrates quantitative Western blot analysis of HVEM-MVP. [Figure 27B] FIG. 1 illustrates FACS analysis of specific binding of unlabeled HVEM-MVP to target cells expressing the cognate receptor BTLA. [Figure 28A] FIG. 1 illustrates quantitative Western blot analysis of BTLA-MVP. [Figure 28B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled BTLA-MVP to target cells expressing the cognate receptor HVEM. [Figure 28C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled BTLA-MVP to target cells expressing the cognate receptor HVEM. [Figure 29A] FIG. 1 illustrates quantitative Western blot analysis of CD160-MVP. [Figure 29B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled CD160-MVP to target cells expressing the cognate receptor HVEM. [Figure 29C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled CD160-MVP to target cells expressing the cognate receptor HVEM. [Figure 30A] FIG. 1 illustrates quantitative Western blot analysis of CD48-MVP. [Figure 30B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled CD48-MVP to target cells expressing the cognate receptor 2B4. [Figure 30C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled CD48-MVP to target cells expressing the cognate receptor 2B4. [Figure 31A] FIG. 1 illustrates quantitative Western blot analysis of CD112-MVP. [Figure 31B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled CD112-MVP to target cells expressing the cognate receptor TIGHT. [Figure 32A]FIG. 1 illustrates quantitative Western blot analysis of TIGHT-MVP. [Figure 32B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled TIGHT-MVP to target cells expressing the cognate receptor CD112. [Figure 32C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled TIGHT-MVP to target cells expressing the cognate receptor CD112. [Figure 33A] FIG. 1 illustrates quantitative Western blot analysis of CD155-MVP. [Figure 33B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled CD155-MVP to target cells expressing the cognate receptor TIGHT. [Figure 33C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled CD155-MVP to target cells expressing the cognate receptor TIGHT. [Figure 34A] FIG. 1 illustrates quantitative Western blot analysis of TIGHT-MVP. [Figure 34B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled TIGHT-MVP to target cells expressing the cognate receptor CD155. [Figure 34C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled TIGHT-MVP to target cells expressing the cognate receptor CD155. [Figure 35A] FIG. 1 illustrates quantitative Western blot analysis of human TIM3-MVP. [Figure 35B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled human TIM3-MVP to target cells expressing the cognate receptor human Ceacam-1. [Figure 35C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled human TIM3-MVP to target cells expressing the cognate receptor human Ceacam-1. [Figure 36A]FIG. 1 illustrates quantitative Western blot analysis of human Ceacam1-MVP. [Figure 36B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled human Ceacam1-MVP to target cells expressing the cognate receptor human TIM-3. [Figure 36C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled human Ceacam1-MVP to target cells expressing the cognate receptor human TIM-3. [Figure 37A] FIG. 1 illustrates the activation of an immune checkpoint involving a costimulatory signal on a T cell and its ligand on an antigen-presenting cell. [Figure 37B] FIG. 1 illustrates the use of costimulatory MVPs to capture T cell receptor (TCR) activation signaling. [Figure 37C] FIG. 1 illustrates the combined use of anti-CD3 antibodies and costimulatory MVPs in T cell activation. [Figure 38A] FIG. 1 illustrates the effect of costimulatory murine CD86-MVP on the activation of mouse splenic T cells based on CD69 and CD25 expression 2 days after activation. [Figure 38B] FIG. 1 illustrates the effect of costimulatory murine CD86-MVP on T cell proliferation. [Figure 39A] FIG. 1 illustrates a Western blot analysis of human CD86-MVP under non-reducing or reducing conditions. [Figure 39B] FIG. 1 illustrates a FACS analysis of the effect of human CD86-MVP on the activation of human peripheral blood T cells based on CD69 and CD25 expression 2 days after activation. [Figure 39C] FIG. 1 illustrates a FACS analysis of the effect of human CD86-MVP on the differentiation status of human peripheral blood T cells based on CD45RO and CD62L expression 8 days after activation. [Figure 40A]FIG. 1 illustrates a FACS analysis of the effect of mouse CD80-MVP on the activation of mouse splenic T cells based on CD69 and CD25 expression 2 days after activation. [Figure 40B] FIG. 1 illustrates the effect of mouse CD80-MVP on T cell proliferation. [Figure 41A] FIG. 1 illustrates a Western blot analysis of human CD80-MVP under non-reducing or reducing conditions. [Figure 41B] FIG. 1 illustrates a FACS analysis of the effect of human CD80-MVP on the activation of human peripheral blood T cells based on CD69 and CD25 expression 2 days after activation. [Figure 41C] FIG. 1 illustrates a FACS analysis of the effect of human CD80-MVP on the differentiation status of human peripheral blood T cells based on CD45RO and CD62L expression 8 days after activation. [Figure 42A] FIG. 1 illustrates a FACS analysis of the effect of costimulatory murine 4-1BBL-MVP on activation of mouse splenic T cells based on CD69 and CD25 expression 2 days after activation. [Figure 42B] FIG. 1 illustrates the effect of costimulatory murine 4-1BBL-MVP on the proliferation of mouse splenic T cells. [Figure 43A] FIG. 1 illustrates quantitative Western blot analysis of human 4-1BBL-MVP. [Figure 43B] FIG. 1 illustrates a FACS analysis of binding of unlabeled human 4-1BBL-MVP to target cells expressing the cognate receptor 4-1BB. [Figure 43C] FIG. 1 illustrates a FACS analysis of the effect of human 4-1BBL-MVP on the activation of human peripheral blood T cells based on CD69 and CD25 expression 2 days after activation. [Fig. 43D] FIG. 1 illustrates a FACS analysis of the effect of human 4-1BBL-MVP on the differentiation state of human peripheral blood T cells based on CD45RO and CD62L expression 8 days after activation. [Figure 44A] FIG. 1 illustrates a FACS analysis of the effect of costimulatory murine OX40L-MVP on the activation of mouse splenic T cells based on CD69 and CD25 expression 2 days after activation. [Figure 44B] FIG. 1 illustrates the effect of costimulatory murine OX40L-MVP on the proliferation of mouse splenic T cells. [Figure 45A] FIG. 1 illustrates quantitative Western blot analysis of human OX40L-MVP. [Figure 45B] FIG. 1 illustrates a Western blot analysis of human OX40L-MVP under non-reducing conditions. [Figure 45C] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled OX40L-MVP to target cells expressing the cognate receptor OX40. [Figure 45D] FIG. 1 illustrates a FACS analysis of the effect of human OX40L-MVP on the activation of human peripheral blood T cells based on CD69 and CD25 expression 2 days after activation. [Figure 45E] FIG. 1 illustrates a FACS analysis of the effect of human OX40L-MVP on the differentiation state of human peripheral blood T cells based on CD45RO and CD62L expression 8 days after activation. [Figure 46A] FIG. 1 illustrates quantitative Western blot analysis of mouse LIGHT-MVP. [Figure 46B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled murine LIGHT-MVP to target cells expressing the cognate receptor HVEM. [Figure 47A] FIG. 1 illustrates quantitative Western blot analysis of CD30-MVP. [Figure 47B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled CD30-MVP to target cells expressing the cognate receptor CD30 ligand. [Figure 47C]FIG. 1 illustrates FACS analysis of specific binding of unlabeled CD30-MVP to target cells expressing the cognate receptor CD30 ligand. [Figure 48A] FIG. 1 illustrates quantitative Western blot analysis of CD30L-MVPs. [Figure 48B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled CD30L-MVP to target cells expressing the cognate receptor CD30 ligand. [Figure 48C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled CD30L-MVP to target cells expressing the cognate receptor CD30 ligand. [Figure 49A] FIG. 1 illustrates quantitative Western blot analysis of CD48-MVP. [Figure 49B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled CD48-MVP to target cells expressing the cognate receptor CD2. [Figure 49C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled CD48-MVP to target cells expressing the cognate receptor CD2. [Figure 50A] FIG. 1 illustrates quantitative Western blot analysis of CD2-MVP. [Figure 50B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled CD2-MVP to target cells expressing the cognate receptor CD48. [Figure 50C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled CD2-MVP to target cells expressing the cognate receptor CD48. [Figure 51A] FIG. 1 illustrates quantitative Western blot analysis of CD27-MVP. [Figure 51B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled CD27-MVP to target cells expressing the cognate receptor CD70. [Figure 51C]FIG. 1 illustrates FACS analysis of specific binding of unlabeled CD27-MVP to target cells expressing the cognate receptor CD70. [Figure 52A] FIG. 1 illustrates quantitative Western blot analysis of CD70-MVP. [Figure 52B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled CD70-MVP to target cells expressing the cognate receptor CD27. [Figure 52C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled CD70-MVP to target cells expressing the cognate receptor CD27. [Figure 53A] FIG. 1 illustrates quantitative Western blot analysis of ICOSL-MVP. [Figure 53B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled ICOSL-MVP to target cells expressing the cognate receptor ICOS. [Figure 53C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled ICOSL-MVP to target cells expressing the cognate receptor ICOS. [Figure 54A] FIG. 1 illustrates quantitative Western blot analysis of ICOS-MVP. [Figure 54B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled ICOS-MVP to target cells expressing the cognate receptor ICOS ligand. [Figure 55A] FIG. 1 illustrates quantitative Western blot analysis of GITRL-MVP. [Figure 55B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled GITRL-MVP to target cells expressing the cognate receptor GITR. [Figure 55C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled GITRL-MVP to target cells expressing the cognate receptor GITR. [Figure 56A] FIG. 1 illustrates quantitative Western blot analysis of GITR-MVP. [Figure 56B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled GITR-MVP to target cells expressing the cognate receptor GITR ligand. [Figure 56C] FIG. 1 illustrates FACS analysis of specific binding of unlabeled GITR-MVP to target cells expressing the cognate receptor GITR ligand. [Figure 57A] FIG. 1 illustrates quantitative Western blot analysis of 4-1BB-MVP. [Figure 57B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled 4-1BB-MVP to target cells expressing the cognate receptor 4-1BB ligand. [Figure 58A] FIG. 1 illustrates quantitative Western blot analysis of OX40-MVP. [Figure 58B] FIG. 1 illustrates FACS analysis of specific binding of dye-labeled OX40-MVP to target cells expressing the cognate receptor OA40 ligand. [Figure 58C] FIG. 1 illustrates a FACS analysis of specific binding of unlabeled OX40-MVP to target cells expressing the cognate receptor OA40 ligand. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present disclosure utilizes, unless otherwise indicated, conventional molecular biology techniques within the scope of those skilled in the art. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0012] definition Throughout this disclosure, various embodiments are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity, and should not be interpreted as an inflexible limitation on the scope of any embodiment. Thus, the description of a range should be interpreted as specifically disclosing each individual numerical value within that range, to the tenth of the unit of the lower limit, as well as all possible subranges, unless the context clearly dictates otherwise. For example, the description of a range such as 1-6 should be considered as specifically disclosing each individual value within that range, for example, 1.1, 2, 2.3, 5, and 5.9, as well as subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may be independently included in the smaller ranges, and furthermore, are included within this disclosure and are below any specifically excluded limits within the stated ranges. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure unless the context clearly dictates otherwise.

[0013] The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit any embodiment. As used herein, the singular forms "a," "an," and "the" are intended to include the plural unless the context clearly dictates otherwise. Furthermore, it will be understood that the terms "comprises" and / or "comprising," as used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0014] Unless otherwise specified or clear from the context, the term "about" as used herein in reference to a number or range of numbers is understood to mean the number specified and + / - 10% of that number, or 10% below the recited lower limit and 10% above the recited upper limit for the recited values ​​in a range.

[0015] Multivalent Particles Direct cell-cell interactions play a key role in regulating T cell development and function. For example, antigen-presenting cells, such as dendritic cells, somatic cells, or tumor cells, can control T cell activation and development by cell-cell interactions mediated by peptide:MHC complexes and T cell receptors (TCRs) on their surface. In addition, T cells express immune checkpoint molecules on their surface, providing further activation or inhibitory control. These molecules can be stimulatory immune checkpoints that promote immune cell activation to protect the host from pathogen invasion and malignant tumor development, or inhibitory checkpoints that suppress immune cells to attenuate inflammation, maintain immune homeostasis, and prevent tissue damage. Tumor cells frequently exploit immune checkpoint pathways by upregulating the expression of ligands involved in inhibitory checkpoints on different immune cell types, thus allowing tumor cells to evade destruction by the immune system. Dysregulation of checkpoint expression can also contribute to the development and persistence of autoimmune diseases and chronic infections.

[0016] Researchers have developed cancer immunotherapies that target immune checkpoint molecules by using either antibody-based agonists of stimulatory immune checkpoints or antibody-based antagonists of inhibitory immune checkpoints. However, these checkpoint blockade therapies are effective in only 10%-20% of cancer patients. Furthermore, some patients who initially respond to checkpoint blockade therapies may develop resistance or relapse due to upregulation of other immune checkpoint pathways. Therefore, it is important to develop more effective immune checkpoint therapies so that more patients with cancer, autoimmunity, or chronic infections can benefit from these transformative therapies.

[0017] Described herein are novel compositions and methods for immune checkpoint modulation. The compositions and methods described herein are multivalent particle-based immune checkpoint (IC-MVP). In some embodiments, IC-MVPs are genetically encoded vesicles, such as virus-like particles (VLPs), exosomes, or ectosomes, that display multiple copies of immune checkpoint molecules. IC-MVPs can mimic checkpoint modulation through particle-cell interactions and form high affinity, multivalent interactions with immune cell targets, such as T cells and other immune cells, to effectively control their activation, development, and function. Depending on the checkpoint molecule displayed, IC-MVPs can function as activation or inhibition switches to control the activation, development, and function of T cells and other target cells. For example, IC-MVPs that display an activating immune checkpoint can block the activation of T cells or other target cells by the same activating immune checkpoint, while IC-MVPs that display an inhibiting immune checkpoint can block the inhibition of T cells or other target cells by the same inhibiting immune checkpoint. Alternatively, IC-MVPs that display ligands for activating immune checkpoints can be used to activate T cells or other target cells, whereas IC-MVPs that display ligands for inhibitory immune checkpoints can be used to inhibit T cells or other target cells. Finally, IC-MVPs can be genetically programmed to display combinations of checkpoint molecules, thereby allowing for combinatorial activation and inhibition of T cells and other target cells.

[0018] In some embodiments herein, a multivalent particle is described that includes a fusion protein that includes a mammalian immune checkpoint polypeptide and a transmembrane polypeptide. In some embodiments herein, a multivalent particle is described that includes a fusion protein that includes an extracellular domain of a mammalian immune checkpoint polypeptide linked to a multimerization polypeptide and a transmembrane polypeptide. In some embodiments, the mammalian immune checkpoint polypeptide is an immune inhibitory checkpoint polypeptide. In some embodiments, the mammalian immune checkpoint polypeptide is an immune stimulatory checkpoint polypeptide. In some embodiments, the mammalian immune checkpoint polypeptide includes a polypeptide expressed on a T cell. In some embodiments, the mammalian immune checkpoint polypeptide includes a polypeptide expressed on an antigen presenting cell, such as a dendritic cell, a somatic cell, or a tumor cell.

[0019] In some embodiments, the immune inhibitory checkpoint polypeptide comprises programmed cell death protein 1 (PD-1), cluster of differentiation 152 (also known as CTLA4), lymphocyte activation 3 (LAG3), B and T lymphocyte attenuator (BTLA), CD160, natural killer cell receptor 2B4 (2B4), cluster of differentiation 226 (CD226), T cell immunoreceptor with Ig and ITIM domains (TIGIT), cluster of differentiation 96 (CD96), B7 homolog 3 protein (B7-H3), B7 homolog 4 protein (B7-H4), V domain Ig inhibitor of T cell activation (VISTA), T cell immunoglobulin and mucin domain containing 3 (TIM3), sialic acid-binding Ig-like lectin 7 (SIGLEC7), killer cell lectin-like receptor subfamily G member 1 (KLRG1), or sialic acid-binding Ig-like lectin 9 (SIGLEC9). In some embodiments, the immune inhibitory checkpoint polypeptide comprises programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), cluster of differentiation 80 (CD80), cluster of differentiation 86 (CD86), herpes virus entry mediator (HVEM), cluster of differentiation 48 (CD48), cluster of differentiation 112 (CD112), cluster of differentiation 155 (CD155), CEA cell adhesion molecule 1 (Ceacam1), fibrinogen-like 1 (FGL1), or Galectin-3.

[0020] In some embodiments, the immune stimulatory checkpoint polypeptide comprises a CD27 molecule (CD27), cluster of differentiation 28 (CD28), cluster of differentiation 40 (CD40), interleukin-2 receptor subunit beta (CD122), 4-1BB (also known as CD137), inducible T cell costimulatory (ICOS), OX40, cluster of differentiation 2 (CD2), CD30 (also known as TNFRSF8), or glucocorticoid-inducible TNFR-related protein (GITR). In some embodiments, the immune stimulatory checkpoint polypeptide comprises cluster of differentiation 70 (CD70), cluster of differentiation 80 (CD80), cluster of differentiation 86 (CD86), CD40 ligand (CD40L), interleukin-2 (IL-2), GITR ligand (GITRL), 4-1BB ligand (4-1BBL), OX40 ligand (OX40L), LIGHT (also known as TNFSF14), CD30 ligand (CD30L), cluster of differentiation 48 (CD48), or ICOS ligand (ICOSL).

[0021] A variety of immune checkpoint multivalent particles are contemplated herein. In some embodiments, the immune checkpoint multivalent particles are recombinant. In some embodiments, the immune checkpoint multivalent particles do not contain viral genetic material. In some embodiments, the immune checkpoint multivalent particles are virus-like particles or virus-like particles. As used herein, virus-like particles and virus-like particles are used interchangeably. In some embodiments, the virus-like particles do not contain viral genetic material. In some embodiments, the immune checkpoint multivalent particles are extracellular vesicles. In some embodiments, the immune checkpoint multivalent particles are exosomes. In some embodiments, the immune checkpoint multivalent particles are ectosomes.

[0022] The immune checkpoint multivalent particles described herein, in some embodiments, comprise a fusion protein, which is expressed in multiple copies on the surface of the multivalent particle. In some embodiments, the fusion protein is expressed in multiple copies on the surface of the multivalent particle at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325, 1350, 1375, 1400, 1425, 1450, 1475, 1500, 1525, 1550, 1575, 1600, 1625, 1650, 1675, 1700, 1725, 1750, 1775, 1800, 1825, 1850, 1875, 1900, 1925, 1950, 1975, 2000, 20 25, 2050, 2075, 2100, 2125, 2150, 2175, 2200, 2225, 2250, 2275, 2300, 2325, 2350, 2375, 2400, 2425, 2450, 2475, 2500, 2525, 2550, 2575, 2600, 2625, 2650, 2675, 2700, 2725, 2750, 2775, 2800, 2825, 2850, 2875, 2900, 2925, 2950, ​​2975, 3000, 3025, 3050, 307 5, 3100, 3125, 3150, 3175, 3200, 3225, 3250, 3275, 3300, 3325, 3350, 3375, 3400, 3425, 3450, 3475, 3500, 3525, 3550, 3575, 3600, 3625, 3650, 3675, 3700, 3725, 3750, 3775, 3800, 3825, 3850, 3875, 3900, 3925, 3950, 3975, 4000, or expressed at a valence of greater than 4000 copies.In some embodiments, the fusion protein is expressed on the surface of the multivalent particle at a valence of at least or about 5 to about 400, about 20 to about 400, about 10 to about 300, about 20 to about 300, about 20 to about 200, about 50 to about 150, about 20 to about 100, about 50 to about 100, or about 10 to about 15 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle at a valence of at least or about 10 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle at a valence of at least or about 15 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle at a valence of at least or about 25 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle at a valence of at least or about 50 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle at a valence of at least or about 75 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 100 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 125 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 150 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 175 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 200 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 225 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 250 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 275 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 300 copies. In some embodiments, the fusion protein is expressed on the surface of a multivalent particle with a valency of at least or about 350 copies.In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 400 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 450 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 500 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 600 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 700 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 800 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 900 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 1000 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 1100 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 1200 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 1300 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 1400 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 1500 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 1600 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 1700 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 1800 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 1900 copies. In some embodiments, the fusion protein is expressed on the surface of a multivalent particle at a valency of at least or about 2000 copies.In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 2100 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 2200 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 2300 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 2400 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 2500 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 2600 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 2700 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 2800 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 2900 copies. In some embodiments, the fusion protein is expressed on the surface of the multivalent particle with a valency of at least or about 3000 copies.

[0023] In some embodiments, the immune checkpoint multivalent particle is a virus-like particle. The virus-like particle described herein, in some embodiments, comprises a fusion protein, which is expressed in multiple copies on the surface of the virus-like particle. In some embodiments, the fusion protein is expressed in multiple copies on the surface of the virus-like particle at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 92 ... 50, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325, 1350, 1375, 1400, 1425, 1450, 1475, 1500, 1525, 1550, 1575, 1600, 1625, 1650, 1675, 1700, 1725, 1750, 1775, 1800, 1825, 1850, 1875, 1900, 1925, 1950, 1975, 2000, 2025, 2050, 2075, 2100, 2125, 2150, 2175, 2200, 2225, 2250, 2275, 2300, 2325, 2350, 2375, 2400, 2425, 2450, 2475, 2500, 2525, 2550, 2575, 2600, 2625, 2650, 2675, 2700, 2725, 2750, 2775, 2800, 2825, 2850, 2875, 2900, 2925, 2950, ​​2975, 3000, 3025, 3050, 30 75, 3100, 3125, 3150, 3175, 3200, 3225, 3250, 3275, 3300, 3325, 3350, 3375, 3400, 3425, 3450, 3475, 3500, 3525, 3550, 3575, 3600, 3625, 3650, 3675, 3700, 3725, 3750, 3775, 3800, 3825, 3850, 3875, 3900, 3925, 3950, 3975, 4000, or expressed at a valency of greater than 4000 copies.In some embodiments, the fusion protein is expressed on the surface of the virus-like particle at a valency of at least or about 5 to about 400, about 20 to about 400, about 10 to about 300, about 20 to about 300, about 20 to about 200, about 50 to about 150, about 20 to about 100, about 50 to about 100, or about 10 to about 15 copies. In some embodiments, the fusion protein is expressed on the surface of the virus-like particle at a valency of at least or about 10 copies. In some embodiments, the fusion protein is expressed on the surface of the virus-like particle at a valency of at least or about 15 copies. In some embodiments, the fusion protein is expressed on the surface of the virus-like particle at a valency of at least or about 25 copies. In some embodiments, the fusion protein is expressed on the surface of the virus-like particle at a valency of at least or about 50 copies. In some embodiments, the fusion protein is expressed on the surface of the virus-like particle at a valency of at least or about 75 copies. In some embodiments, the fusion protein is expressed on the surface of the virus-like particle at a valency of at least or about 100 copies. In some embodiments, the fusion protein is expressed on the surface of the virus-like particle at a valency of at least or about 125 copies. In some embodiments, the fusion protein is expressed on the surface of the virus-like particle at a valency of at least or about 150 copies. In some embodiments, the fusion protein is expressed on the surface of the virus-like particle at a valency of at least or about 175 copies. In some embodiments, the fusion protein is expressed on the surface of the virus-like particle at a valency of at least or about 200 copies. In some embodiments, the fusion protein is expressed on the surface of the virus-like particle at a valency of at least or about 225 copies. In some embodiments, the fusion protein is expressed on the surface of the virus-like particle at a valency of at least or about 250 copies. In some embodiments, the fusion protein is expressed on the surface of the virus-like particle at a valency of at least or about 275 copies. In some embodiments, the fusion protein is expressed on the surface of the virus-like particle at a valency of at least or about 300 copies.

[0024] In some embodiments, the immune checkpoint multivalent particle is an extracellular vesicle. The extracellular vesicles described herein in some embodiments comprise a fusion protein, and the fusion protein is expressed in multiple copies on the surface of the extracellular vesicle. In some embodiments, the fusion protein is expressed on the surface of the particle of the extracellular vesicle at a valency of at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, or more than 400 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle at a valency of at least or about 5 to about 400, about 20 to about 400, about 10 to about 300, about 20 to about 300, about 20 to about 200, about 50 to about 150, about 20 to about 100, about 50 to about 100, or about 10 to about 15 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle at a valency of at least or about 10 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle at a valency of at least or about 15 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle at a valency of at least or about 25 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle at a valency of at least or about 50 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle at a valency of at least or about 75 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 100 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 125 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 150 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 175 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 200 copies.In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 225 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 250 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 275 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 300 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 350 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 400 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 450 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 500 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 600 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 700 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 800 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 900 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 1000 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 1100 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 1200 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 1300 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle at a valency of at least or about 1400 copies.In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 1500 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 1600 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 1700 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 1800 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 1900 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 2000 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 2100 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 2200 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 2300 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 2400 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 2500 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 2600 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 2700 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 2800 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 2900 copies. In some embodiments, the fusion protein is expressed on the surface of the extracellular vesicle with a valency of at least or about 3000 copies.

[0025] In some embodiments, the immune checkpoint multivalent particle is an exosome. The exosomes described herein, in some embodiments, comprise a fusion protein, which is expressed in multiple copies on the surface of the exosome. In some embodiments, the fusion protein is expressed in multiple copies on the surface of the exosome at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 95 0, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325, 1350, 1375, 1400, 1425, 1450, 1475, 1500, 1525, 1550, 1575, 1600, 1625, 1650, 1675, 1700, 1725, 1750, 1775, 1800, 1825, 1850, 1875, 1900, 1925, 1950, 1975, 2000, 2 025, 2050, 2075, 2100, 2125, 2150, 2175, 2200, 2225, 2250, 2275, 2300, 2325, 2350, 2375, 2400, 2425, 2450, 2475, 2500, 2525, 2550, 2575, 2600, 2625, 2650, 2675, 2700, 2725, 2750, 2775, 2800, 2825, 2850, 2875, 2900, 2925, 2950, ​​2975, 3000, 3025, 3050, 30 75, 3100, 3125, 3150, 3175, 3200, 3225, 3250, 3275, 3300, 3325, 3350, 3375, 3400, 3425, 3450, 3475, 3500, 3525, 3550, 3575, 3600, 3625, 3650, 3675, 3700, 3725, 3750, 3775, 3800, 3825, 3850, 3875, 3900, 3925, 3950, 3975, 4000, or expressed at a valency of greater than 4000 copies.In some embodiments, the fusion protein is expressed on the surface of the exosome at a valency of at least or about 5 to about 400, about 20 to about 400, about 10 to about 300, about 20 to about 300, about 20 to about 200, about 50 to about 150, about 20 to about 100, about 50 to about 100, or about 10 to about 15 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome at a valency of at least or about 10 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome at a valency of at least or about 15 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome at a valency of at least or about 25 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome at a valency of at least or about 50 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome at a valency of at least or about 75 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 100 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 125 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 150 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 175 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 200 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 225 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 250 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 275 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 300 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome at a valency of at least or about 350 copies.In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 400 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 450 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 500 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 600 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 700 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 800 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 900 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 1000 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 1100 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 1200 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 1300 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 1400 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 1500 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 1600 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 1700 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 1800 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 1900 copies.In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 2000 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 2100 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 2200 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 2300 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 2400 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 2500 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 2600 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 2700 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 2800 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 2900 copies. In some embodiments, the fusion protein is expressed on the surface of the exosome with a valency of at least or about 3000 copies.

[0026] In some embodiments, the immune checkpoint multivalent particle is an ectosome. The ectosomes described herein, in some embodiments, comprise a fusion protein, which is expressed in multiple copies on the surface of the ectosome. In some embodiments, the fusion protein is expressed in multiple copies on the surface of the ectosome at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 95 0, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325, 1350, 1375, 1400, 1425, 1450, 1475, 1500, 1525, 1550, 1575, 1600, 1625, 1650, 1675, 1700, 1725, 1750, 1775, 1800, 1825, 1850, 1875, 1900, 1925, 1950, 1975, 2000, 2 025, 2050, 2075, 2100, 2125, 2150, 2175, 2200, 2225, 2250, 2275, 2300, 2325, 2350, 2375, 2400, 2425, 2450, 2475, 2500, 2525, 2550, 2575, 2600, 2625, 2650, 2675, 2700, 2725, 2750, 2775, 2800, 2825, 2850, 2875, 2900, 2925, 2950, ​​2975, 3000, 3025, 3050, 30 75, 3100, 3125, 3150, 3175, 3200, 3225, 3250, 3275, 3300, 3325, 3350, 3375, 3400, 3425, 3450, 3475, 3500, 3525, 3550, 3575, 3600, 3625, 3650, 3675, 3700, 3725, 3750, 3775, 3800, 3825, 3850, 3875, 3900, 3925, 3950, 3975, 4000, or expressed at a valency of greater than 4000 copies.In some embodiments, the fusion protein is expressed on the surface of the ectosome at a valency of at least or about 5 to about 400, about 20 to about 400, about 10 to about 300, about 20 to about 300, about 20 to about 200, about 50 to about 150, about 20 to about 100, about 50 to about 100, or about 10 to about 15 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome at a valency of at least or about 10 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome at a valency of at least or about 15 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome at a valency of at least or about 25 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome at a valency of at least or about 50 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome at a valency of at least or about 75 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 100 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 125 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 150 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 175 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 200 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 225 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 250 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 275 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 300 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome at a valency of at least or about 350 copies.In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 400 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 450 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 500 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 600 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 700 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 800 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 900 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 1000 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 1100 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 1200 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 1300 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 1400 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 1500 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 1600 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 1700 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 1800 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 1900 copies.In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 2000 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 2100 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 2200 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 2300 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 2400 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 2500 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 2600 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 2700 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 2800 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 2900 copies. In some embodiments, the fusion protein is expressed on the surface of the ectosome with a valency of at least or about 3000 copies.

[0027] In some embodiments herein, immune checkpoint multivalent particles are described that include a multimerization domain. In some embodiments, the multimerization domain is a dimerization domain. In some embodiments, the dimerization domain includes a leucine zipper dimerization domain. In some embodiments, the multimerization domain is a trimerization domain. In some embodiments, the trimerization domain includes a post-fusion multimerization domain of a viral surface protein. In some embodiments, the trimerization domain includes a D4 post-fusion trimerization domain of a VSV-G protein. In some embodiments, the trimerization domain includes a Dengue E protein post-fusion trimerization domain. In some embodiments, the trimerization domain includes a Foldon trimerization domain. In some embodiments, the multimerization domain is a tetramerization domain. In some embodiments, the tetramerization domain includes an influenza neuraminidase stem domain.

[0028] In some embodiments herein, immune checkpoint multivalent particles are described that modulate the interaction of immune checkpoints with their ligands. For example, immune checkpoint multivalent particles modulate the interaction of PD-1 with its ligands PDL-1 or PDL-2. In some embodiments, immune checkpoint multivalent particles that modulate the interaction of immune checkpoints with their ligands provide an inhibitory effect. Optionally, immune checkpoint multivalent particles inhibit activation. Optionally, multivalent particles inhibit downstream signaling. In some embodiments, immune checkpoint multivalent particles that modulate the interaction of immune checkpoints with their ligands provide a stimulatory effect. Optionally, immune checkpoint multivalent particles activate downstream signaling.

[0029] In some embodiments herein, immune checkpoint multivalent particles are described that include improved binding characteristics. In some embodiments, the multivalent particles have a binding affinity (e.g., K) of less than 100 pM, less than 200 pM, less than 300 pM, less than 400 pM, less than 500 pM, less than 600 pM, less than 700 pM, less than 800 pM, or less than 900 pM to an immune checkpoint. D In some embodiments, the multivalent particles have a K of less than 1 nM, less than 1.2 nM, less than 2 nM, less than 5 nM, or less than 10 nM. D In some instances, the multivalent particles have a K of less than 1 nM. D In some instances, the multivalent particles have a K of less than 1.2 nM. D In some instances, the multivalent particles have a K of less than 2 nM. D In some instances, the multivalent particles have a K of less than 5 nM. D In some instances, the multivalent particles have a K of less than 10 nM. D Includes.

[0030] Mammalian immune checkpoint polypeptides In some embodiments herein, a multivalent particle is described that includes a mammalian immune checkpoint polypeptide and a transmembrane polypeptide. In some embodiments herein, a multivalent particle is described that includes an extracellular domain of a mammalian immune checkpoint polypeptide and a transmembrane polypeptide. In some embodiments, the mammalian immune checkpoint polypeptide is an immune inhibitory checkpoint polypeptide. In some embodiments, the mammalian immune checkpoint polypeptide is an immune stimulatory checkpoint polypeptide. In some embodiments, the mammalian immune checkpoint polypeptide includes a polypeptide expressed on a T cell. In some embodiments, the mammalian immune checkpoint polypeptide includes a polypeptide expressed on an antigen presenting cell, such as a dendritic cell, a somatic cell, or a tumor cell.

[0031] In some embodiments, the mammalian immune checkpoint polypeptide comprises an immune inhibitory checkpoint polypeptide. In some embodiments, the immune inhibitory checkpoint polypeptide is expressed on a T cell. In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9. In some embodiments, the immune inhibitory checkpoint polypeptide is expressed on an antigen presenting cell, a tumor cell, or a normal cell. In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3. In some embodiments, the immune inhibitory checkpoint polypeptide comprises the extracellular domain of PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9. In some embodiments, the immune inhibitory checkpoint polypeptide comprises the extracellular domain of PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3.

[0032] In some embodiments, the mammalian immune checkpoint polypeptide comprises an immune stimulatory checkpoint polypeptide. In some embodiments, the immune stimulatory checkpoint polypeptide comprises a polypeptide expressed on a T cell. In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR. In some embodiments, the immune stimulatory checkpoint polypeptide is expressed on an antigen presenting cell, a tumor cell, or a normal cell. In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL. In some embodiments, the immune stimulatory checkpoint polypeptide comprises the extracellular domain of CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR. In some embodiments, the immune stimulatory checkpoint polypeptide comprises the extracellular domain of CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL.

[0033] In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 75% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 76% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 77% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 78% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 79% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 80% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 81% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 82% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that has at least 83% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162.In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 84% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 85% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 86% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 87% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 88% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 89% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 90% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 91% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that has at least 92% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162.In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 93% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 94% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 95% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 96% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 97% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 98% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence of at least 99% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence according to SEQ ID NOs: 1-62, 96-115, 153-162.

[0034] In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 75% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 76% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 77% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 78% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 79% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 80% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 81% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 82% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that has at least 83% sequence homology to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162.In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 84% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 85% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 86% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 87% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 88% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 89% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 90% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 91% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence having at least 92% sequence homology to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162.In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 93% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 94% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 95% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 96% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 97% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 98% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162. In some embodiments, the mammalian immune checkpoint polypeptide comprises an amino acid sequence that is at least 99% sequence homologous to an amino acid sequence according to any one of SEQ ID NOs: 1-62, 96-115, 153-162.

[0035] In some examples, the mammalian immune checkpoint polypeptide comprises at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 10 and / or an amino acid sequence comprising at least a portion having 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, or more than 800 amino acids.

[0036] The term "sequence identity" means that two polynucleotide sequences are identical over a comparison window (i.e., on a nucleotide-by-nucleotide basis). The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where identical nucleic acid bases (e.g., A, T, C, G, U, or I) occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Typically, techniques for determining sequence identity include comparing two nucleotide or amino acid sequences and determining their percent identity. Sequence comparison, such as for the purpose of evaluating identity, can be performed by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see, for example, the EMBOSS Needle aligner available at www.ebi.ac.uk / Tools / psa / emboss_needle / , optionally with default settings), the BLAST algorithm (see, for example, the BLAST alignment tool available at blast.ncbi.nlm.nih.gov / Blast.cgi, optionally with default settings), and the Smith-Waterman algorithm (see, for example, the EMBOSS Water aligner available at www.ebi.ac.uk / Tools / psa / emboss_water / , optionally with default settings). Optimal alignment can be evaluated using any suitable parameters of the selected algorithm, including default parameters. The "percent identity" between two sequences, also referred to as "percent homology", can be calculated as the number of perfect matches between two optimally aligned sequences divided by the length of the reference sequence, multiplied by 100. Percent identity can also be determined by comparing sequence information using, for example, the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health.The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and is performed as discussed in Altschul et al., J. Mol. Biol. 215:403-410 (1990), Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993), and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. This program can be used to determine the percent identity over the entire length of the sequences being compared. Default parameters are provided to optimize searches using short query sequences, such as the blastp program. This program also allows the use of a SEG filter to mask-off segments of the query sequence as determined by the SEG program of Wootton and Lederhen, Computers and Chemistry 17:149-163 (1993). High degrees of sequence identity generally include ranges of about 80% to 100% sequence identity, and integer values ​​therebetween.

[0037] Multimerization Domain In some embodiments, the immune checkpoint multivalent particle comprises a multimerization domain. In some embodiments, the multimerization domain is a dimerization domain. In some embodiments, the dimerization domain comprises a leucine zipper dimerization domain. In some embodiments, the multimerization domain is a trimerization domain. In some embodiments, the trimerization domain comprises a post-fusion multimerization domain of a viral surface protein. In some embodiments, the trimerization domain comprises a D4 post-fusion trimerization domain of a VSV-G protein. In some embodiments, the trimerization domain comprises a Dengue E protein post-fusion trimerization domain. In some embodiments, the trimerization domain comprises a Foldon trimerization domain. In some embodiments, the multimerization domain is a tetramerization domain. In some embodiments, the tetramerization domain comprises an influenza neuraminidase stem domain.

[0038] [Table 1]

[0039] In some embodiments, the multimerization domain comprises an amino acid sequence disclosed in Table 1, or an amino acid sequence substantially identical to an amino acid sequence in Table 1 (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity). In some examples, the multimerization domain comprises an amino acid sequence comprising at least a portion having at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 amino acids of any sequence according to Table 1. In some embodiments, the multimerization domain comprises an amino acid sequence having at least 95% sequence identity to an amino acid sequence according to any one of SEQ ID NOs:65-78.

[0040] Transmembrane Polypeptides In some embodiments herein, a multivalent particle is described that includes a mammalian immune checkpoint polypeptide and a transmembrane polypeptide. In some embodiments, the transmembrane polypeptide includes a transmembrane domain of vesicular stomatitis virus glycoprotein (VSV-G). In some embodiments, the transmembrane polypeptide includes a transmembrane domain and a cytosolic domain of vesicular stomatitis virus glycoprotein (VSV-G). In some embodiments, the transmembrane polypeptide includes a transmembrane domain of dengue E protein. In some embodiments, the transmembrane polypeptide includes a transmembrane domain and a cytosolic domain of dengue E protein. In some embodiments, the transmembrane polypeptide includes a transmembrane domain of influenza hemagglutinin (HA). In some embodiments, the transmembrane polypeptide includes a transmembrane domain and a cytosolic domain of influenza hemagglutinin (HA). In some embodiments, the transmembrane polypeptide includes a transmembrane domain and a cytosolic domain of HIV surface glycoprotein GP120 or GP41. In some embodiments, the transmembrane polypeptide includes a transmembrane domain and a cytosolic domain of HIV surface glycoprotein GP120 or GP41. In some embodiments, the transmembrane domain comprises a transmembrane polypeptide of a measles virus surface glycoprotein hemagglutinin (H) protein. In some embodiments, the transmembrane polypeptide comprises a transmembrane polypeptide and a cytosolic domain of a measles virus surface glycoprotein hemagglutinin (H) protein. In some embodiments, the transmembrane polypeptide comprises a transmembrane domain of influenza neuraminidase (NA). In some embodiments, the transmembrane polypeptide comprises a transmembrane domain and a cytosolic domain of influenza neuraminidase (NA).

[0041] [Table 2-1]

[0042] [Table 2-2]

[0043] In some embodiments, the transmembrane domain comprises an amino acid sequence disclosed in Table 2, or an amino acid sequence substantially identical to an amino acid sequence in Table 2 (e.g., about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity). In some examples, the transmembrane domain comprises an amino acid sequence that includes at least a portion having at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 amino acids of any sequence according to Table 2.

[0044] In some embodiments herein, a multivalent particle is described that includes a mammalian immune checkpoint polypeptide and a transmembrane polypeptide. In some embodiments, the transmembrane polypeptide anchors the fusion protein to the lipid bilayer of the multivalent particle. In some embodiments, the transmembrane polypeptide includes a spike glycoprotein, a mammalian membrane protein, an envelope protein, a nucleocapsid protein, or a cellular transmembrane protein. In some embodiments, the transmembrane polypeptide includes VSVG, spike protein S1, spike protein S2, Sindbis virus envelope (SINDBIS) protein, hemagglutinin envelope protein from measles virus, envelope glycoprotein of measles virus fusion (F) protein, RD114, BaEV, GP41, or GP120. In some embodiments, the transmembrane polypeptide includes VSVG. In some embodiments, the VSVG includes full-length VSVG or truncated VSVG. In some embodiments, the VSVG includes a transmembrane domain and a cytoplasmic tail. In some embodiments, the hemagglutinin envelope protein from measles virus is a variant of the hemagglutinin envelope protein from measles virus, in some instances, the variant is HCΔ18.

[0045] In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 75% sequence identity to the amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 76% sequence identity to the amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 77% sequence identity to the amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 78% sequence identity to the amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 79% sequence identity to the amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 80% sequence identity to the amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 81% sequence identity to the amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 82% sequence identity to the amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 83% sequence identity to the amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 84% sequence identity to the amino acid sequence according to SEQ ID NO: 63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 86% sequence identity to the amino acid sequence according to SEQ ID NO: 63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 87% sequence identity to the amino acid sequence according to SEQ ID NO: 63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 88% sequence identity to the amino acid sequence according to SEQ ID NO: 63.In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 89% sequence identity to the amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 90% sequence identity to the amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 91% sequence identity to the amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 92% sequence identity to the amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 93% sequence identity to the amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 94% sequence identity to the amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 95% sequence identity to the amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 96% sequence identity to the amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 97% sequence identity to the amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence that has at least 98% sequence identity to an amino acid sequence according to SEQ ID NO: 63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence that has at least 99% sequence identity to an amino acid sequence according to SEQ ID NO: 63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence according to SEQ ID NO: 63.

[0046] In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 75% sequence homology to an amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 76% sequence homology to an amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 77% sequence homology to an amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 78% sequence homology to an amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 79% sequence homology to an amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 80% sequence homology to an amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 81% sequence homology to an amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 82% sequence homology to an amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 83% sequence homology to an amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 84% sequence homology to an amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 85% sequence homology to an amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 86% sequence homology to an amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 87% sequence homology to an amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 88% sequence homology to an amino acid sequence according to SEQ ID NO:63.In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 89% sequence homology to an amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 90% sequence homology to an amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 91% sequence homology to an amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 92% sequence homology to an amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 93% sequence homology to an amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 94% sequence homology to an amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 95% sequence homology to an amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 96% sequence homology to an amino acid sequence according to SEQ ID NO:63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence that is at least 97% sequence homologous to an amino acid sequence according to SEQ ID NO: 63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence that is at least 98% sequence homologous to an amino acid sequence according to SEQ ID NO: 63. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence that is at least 99% sequence homologous to an amino acid sequence according to SEQ ID NO: 63.

[0047] In some examples, the transmembrane polypeptide comprises an amino acid sequence comprising at least a portion having at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or more than 490 amino acids of SEQ ID NO:63.

[0048] In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 75% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 76% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 77% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 78% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 79% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 80% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 81% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 82% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 83% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 84% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 85% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 86% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 87% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 88% sequence identity to the amino acid sequence according to SEQ ID NO:64.In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 89% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 90% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 91% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 92% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 93% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 94% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 95% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 96% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 97% sequence identity to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence that has at least 98% sequence identity to an amino acid sequence according to SEQ ID NO: 64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence that has at least 99% sequence identity to an amino acid sequence according to SEQ ID NO: 64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence according to SEQ ID NO: 64.

[0049] In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 75% sequence homology to an amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 76% sequence homology to an amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 77% sequence homology to an amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 78% sequence homology to an amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 79% sequence homology to an amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 80% sequence homology to an amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 81% sequence homology to an amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 82% sequence homology to an amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 83% sequence homology to an amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 84% sequence homology to an amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 85% sequence homology to an amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 86% sequence homology to an amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 87% sequence homology to an amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence of at least 88% sequence homology to an amino acid sequence according to SEQ ID NO:64.In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 89% sequence homology to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 90% sequence homology to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 91% sequence homology to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 92% sequence homology to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 93% sequence homology to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 94% sequence homology to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 95% sequence homology to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence with at least 96% sequence homology to the amino acid sequence according to SEQ ID NO:64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence that is at least 97% sequence homologous to an amino acid sequence according to SEQ ID NO: 64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence that is at least 98% sequence homologous to an amino acid sequence according to SEQ ID NO: 64. In some embodiments, the transmembrane polypeptide comprises an amino acid sequence that is at least 99% sequence homologous to an amino acid sequence according to SEQ ID NO: 64.

[0050] In some examples, the transmembrane polypeptide comprises an amino acid sequence comprising at least a portion having at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or more than 490 amino acids of SEQ ID NO:64.

[0051] Combinations of mammalian immune checkpoint polypeptides and transmembrane polypeptides In some embodiments herein, a multivalent particle is described that includes a fusion protein that includes a mammalian immune checkpoint polypeptide and a transmembrane polypeptide. In some embodiments, the mammalian immune checkpoint polypeptide is an immunostimulatory checkpoint polypeptide. In some embodiments, the mammalian immune checkpoint polypeptide includes a polypeptide expressed on a T cell. In some embodiments, the mammalian immune checkpoint polypeptide includes a polypeptide expressed on an antigen presenting cell, a cancer cell, and a normal somatic cell.

[0052] In some embodiments, the mammalian immune checkpoint polypeptide comprises an immune inhibitory checkpoint polypeptide. In some embodiments, the mammalian immune checkpoint polypeptide comprises an immune inhibitory checkpoint polypeptide. In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9. In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3. In some embodiments, the immune inhibitory checkpoint polypeptide comprises the extracellular domain of PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9. In some embodiments, the immune inhibitory checkpoint polypeptide comprises the extracellular domain of PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3.

[0053] In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9, and the transmembrane polypeptide comprises a VSVG transmembrane domain. In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9, and the transmembrane polypeptide comprises a spike protein S1 transmembrane domain. In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9, and the transmembrane polypeptide comprises a spike protein S2 transmembrane domain. In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9, and the transmembrane polypeptide comprises a transmembrane domain of a surface glycoprotein of an enveloped virus. In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9, and the transmembrane polypeptide comprises a transmembrane domain of the Sindbis virus envelope (SINDBIS) protein. In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9, and the transmembrane polypeptide comprises a BaEV transmembrane domain.In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9, and the transmembrane polypeptide comprises a GP41 transmembrane domain. In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9, and the transmembrane polypeptide comprises a GP120 transmembrane domain.

[0054] In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3, and the transmembrane polypeptide comprises a VSVG transmembrane domain. In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3, and the transmembrane polypeptide comprises a spike protein S1 transmembrane domain. In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3, and the transmembrane polypeptide comprises a spike protein S2 transmembrane domain. In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3, and the transmembrane polypeptide comprises a transmembrane domain of a surface glycoprotein of an enveloped virus. In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3, and the transmembrane polypeptide comprises a transmembrane domain of a Sindbis virus envelope (SINDBIS) protein. In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3, and the transmembrane polypeptide comprises a BaEV transmembrane domain. In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3, and the transmembrane polypeptide comprises a GP41 transmembrane domain.In some embodiments, the immune inhibitory checkpoint polypeptide comprises PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3, and the transmembrane polypeptide comprises a GP120 transmembrane domain.

[0055] In some embodiments, the immune inhibitory checkpoint polypeptide comprises the extracellular domain of PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9, and the transmembrane polypeptide comprises the GP120 transmembrane domain. In some embodiments, the immune inhibitory checkpoint polypeptide comprises the extracellular domain of PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9, and the transmembrane polypeptide comprises the spike protein S1 transmembrane domain. In some embodiments, the immune inhibitory checkpoint polypeptide comprises an extracellular domain of PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9, and the transmembrane polypeptide comprises a spike protein S2 transmembrane domain. In some embodiments, the immune inhibitory checkpoint polypeptide comprises an extracellular domain of PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9, and the transmembrane polypeptide comprises a transmembrane domain of a surface glycoprotein of an enveloped virus. In some embodiments, the immune inhibitory checkpoint polypeptide comprises the extracellular domain of PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9, and the transmembrane polypeptide comprises the transmembrane domain of the Sindbis virus envelope (SINDBIS) protein.In some embodiments, the immune inhibitory checkpoint polypeptide comprises the extracellular domain of PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9, and the transmembrane polypeptide comprises a BaEV transmembrane domain. In some embodiments, the immune inhibitory checkpoint polypeptide comprises the extracellular domain of PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9, and the transmembrane polypeptide comprises a GP41 transmembrane domain. In some embodiments, the immune inhibitory checkpoint polypeptide comprises the extracellular domain of PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, or SIGLEC9, and the transmembrane polypeptide comprises the GP120 transmembrane domain.

[0056] In some embodiments, the immune inhibitory checkpoint polypeptide comprises the extracellular domain of PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3, and the transmembrane polypeptide comprises a VSVG transmembrane domain. In some embodiments, the immune inhibitory checkpoint polypeptide comprises the extracellular domain of PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3, and the transmembrane polypeptide comprises a spike protein S1 transmembrane domain. In some embodiments, the immune inhibitory checkpoint polypeptide comprises the extracellular domain of PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3, and the transmembrane polypeptide comprises a spike protein S2 transmembrane domain. In some embodiments, the immune inhibitory checkpoint polypeptide comprises the extracellular domain of PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3, and the transmembrane polypeptide comprises the transmembrane domain of a surface glycoprotein of an enveloped virus. In some embodiments, the immune inhibitory checkpoint polypeptide comprises the extracellular domain of PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3, and the transmembrane polypeptide comprises the transmembrane domain of the Sindbis virus envelope (SINDBIS) protein. In some embodiments, the immune inhibitory checkpoint polypeptide comprises the extracellular domain of PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3, and the transmembrane polypeptide comprises a BaEV transmembrane domain.In some embodiments, the immune inhibitory checkpoint polypeptide comprises the extracellular domain of PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3, and the transmembrane polypeptide comprises a GP41 transmembrane domain.In some embodiments, the immune inhibitory checkpoint polypeptide comprises the extracellular domain of PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD155, Ceacam1, FGL1, or Galectin-3, and the transmembrane polypeptide comprises a GP120 transmembrane domain.

[0057] In some embodiments, the mammalian immune checkpoint polypeptide comprises an immune stimulatory checkpoint polypeptide. In some embodiments, the immune stimulatory checkpoint polypeptide comprises a polypeptide expressed on a T cell. In some embodiments, the mammalian immune checkpoint polypeptide comprises a polypeptide expressed on an antigen presenting cell. In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR. In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL. In some embodiments, the immune stimulatory checkpoint polypeptide comprises the extracellular domain of CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR. In some embodiments, the immune stimulatory checkpoint polypeptide comprises the extracellular domain of CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL.

[0058] In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR, and the transmembrane polypeptide comprises a VSVG transmembrane domain. In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR, and the transmembrane polypeptide comprises a spike protein S1 transmembrane domain. In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR, and the transmembrane polypeptide comprises a spike protein S2 transmembrane domain. In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR, and the transmembrane polypeptide comprises a transmembrane domain of a surface glycoprotein of an enveloped virus. In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR, and the transmembrane polypeptide comprises a transmembrane domain of a Sindbis virus envelope (SINDBIS) protein. In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR, and the transmembrane polypeptide comprises a BaEV transmembrane domain. In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR, and the transmembrane polypeptide comprises a GP41 transmembrane domain. In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR, and the transmembrane polypeptide comprises a GP120 transmembrane domain.

[0059] In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL, and the transmembrane polypeptide comprises a VSVG transmembrane domain. In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL, and the transmembrane polypeptide comprises a spike protein S1 transmembrane domain. In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL, and the transmembrane polypeptide comprises a spike protein S2 transmembrane domain. In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL, and the transmembrane polypeptide comprises a transmembrane domain of a surface glycoprotein of an enveloped virus. In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL, and the transmembrane polypeptide comprises a transmembrane domain of a Sindbis virus envelope (SINDBIS) protein. In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL, and the transmembrane polypeptide comprises a BaEV transmembrane domain. In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL, and the transmembrane polypeptide comprises a GP41 transmembrane domain.In some embodiments, the immune stimulatory checkpoint polypeptide comprises CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL, and the transmembrane polypeptide comprises a GP120 transmembrane domain.

[0060] In some embodiments, the immune stimulatory checkpoint polypeptide comprises the extracellular domain of CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR, and the transmembrane polypeptide comprises a VSVG transmembrane domain. In some embodiments, the immune stimulatory checkpoint polypeptide comprises the extracellular domain of CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR, and the transmembrane polypeptide comprises a spike protein S1 transmembrane domain. In some embodiments, the immune stimulatory checkpoint polypeptide comprises the extracellular domain of CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR, and the transmembrane polypeptide comprises a spike protein S2 transmembrane domain. In some embodiments, the immune stimulatory checkpoint polypeptide comprises the extracellular domain of CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR, and the transmembrane polypeptide comprises the transmembrane domain of a surface glycoprotein of an enveloped virus. In some embodiments, the immune stimulatory checkpoint polypeptide comprises the extracellular domain of CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR, and the transmembrane polypeptide comprises the transmembrane domain of a Sindbis virus envelope (SINDBIS) protein. In some embodiments, the immune stimulatory checkpoint polypeptide comprises the extracellular domain of CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR, and the transmembrane polypeptide comprises the BaEV transmembrane domain. In some embodiments, the immune stimulatory checkpoint polypeptide comprises the extracellular domain of CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR, and the transmembrane polypeptide comprises the GP41 transmembrane domain.In some embodiments, the immune stimulatory checkpoint polypeptide comprises the extracellular domain of CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, or GITR, and the transmembrane polypeptide comprises the GP120 transmembrane domain.

[0061] In some embodiments, the immune stimulatory checkpoint polypeptide comprises an extracellular domain of CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL, and the transmembrane polypeptide comprises a VSVG transmembrane domain. In some embodiments, the immune stimulatory checkpoint polypeptide comprises an extracellular domain of CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL, and the transmembrane polypeptide comprises a spike protein S1 transmembrane domain. In some embodiments, the immune stimulatory checkpoint polypeptide comprises an extracellular domain of CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL, and the transmembrane polypeptide comprises a spike protein S2 transmembrane domain. In some embodiments, the immune stimulatory checkpoint polypeptide comprises the extracellular domain of CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL, and the transmembrane polypeptide comprises the transmembrane domain of a surface glycoprotein of an enveloped virus. In some embodiments, the immune stimulatory checkpoint polypeptide comprises the extracellular domain of CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL, and the transmembrane polypeptide comprises the transmembrane domain of a Sindbis virus envelope (SINDBIS) protein. In some embodiments, the immune stimulatory checkpoint polypeptide comprises the extracellular domain of CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL, and the transmembrane polypeptide comprises a BaEV transmembrane domain. In some embodiments, the immune stimulatory checkpoint polypeptide comprises the extracellular domain of CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL, and the transmembrane polypeptide comprises a GP41 transmembrane domain.In some embodiments, the immune stimulatory checkpoint polypeptide comprises the extracellular domain of CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL, and the transmembrane polypeptide comprises the GP120 transmembrane domain.

[0062] In some embodiments herein, described are multivalent particles comprising a fusion protein comprising a mammalian immune checkpoint polypeptide and a transmembrane polypeptide, the multivalent particle further comprising a multimerization domain.

[0063] In some embodiments, the multimerization domain is a dimerization domain. In some embodiments, the dimerization domain comprises a leucine zipper dimerization domain. In some embodiments, the multimerization domain is a trimerization domain. In some embodiments, the trimerization domain comprises a post-fusion multimerization domain of a viral surface protein. In some embodiments, the trimerization domain comprises a D4 post-fusion trimerization domain of a VSV-G protein. In some embodiments, the trimerization domain comprises a Dengue E protein post-fusion trimerization domain. In some embodiments, the trimerization domain comprises a Foldon trimerization domain. In some embodiments, the multimerization domain is a tetramerization domain. In some embodiments, the tetramerization domain comprises an influenza neuraminidase stem domain.

[0064] In some embodiments, when the fusion protein is expressed on the surface of a multivalent particle, the multimerization domain is on the outside of the multivalent particle. In some embodiments, when the fusion protein is expressed on the surface of a multivalent particle, the multimerization domain is on the outside of the multivalent particle and adjacent to the signal peptide. In some embodiments, when the fusion protein is expressed on the surface of a multivalent particle, the multimerization domain is on the inside of the multivalent particle. In some embodiments, when the fusion protein is expressed on the surface of a multivalent particle, the multimerization domain is on the inside of the multivalent particle and adjacent to the transmembrane domain.

[0065] In some embodiments, the fusion protein comprises a signal peptide.

[0066] In some embodiments, the domains of the fusion protein are arranged from the N-terminus to the C-terminus in the following order: (a) signal peptide, mammalian immune checkpoint polypeptide, multimerization domain, transmembrane domain, and cytosolic domain; (b) signal peptide, mammalian immune checkpoint polypeptide, transmembrane domain, multimerization domain, and cytosolic domain; or (c) signal peptide, multimerization domain, mammalian immune checkpoint polypeptide, transmembrane domain, and cytosolic domain. In some embodiments, the domains of the fusion protein are arranged from the N-terminus to the C-terminus in the following order: signal peptide, mammalian immune checkpoint polypeptide, multimerization domain, transmembrane domain, and cytosolic domain. In some embodiments, the domains of the fusion protein are arranged from the N-terminus to the C-terminus in the following order: signal peptide, mammalian immune checkpoint polypeptide, multimerization domain, transmembrane domain, and cytosolic domain. In some embodiments, the domains of the fusion protein are arranged from the N-terminus to the C-terminus in the following order: signal peptide, mammalian immune checkpoint polypeptide, transmembrane domain, multimerization domain, and cytosolic domain.

[0067] Disclosed herein is a fusion protein comprising a transmembrane domain, a cytosolic domain, a mammalian immune checkpoint polypeptide, and a multimerization domain, which is expressed on the surface of a multivalent particle and displayed in a multimeric format.

[0068] In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 or 2, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3 or 4, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5 or 6, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs:63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs:65-69. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:7 or 8, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs:63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs:65-69. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9 or 10, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69.In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11 or 12, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17 or 18, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 23 or 24, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 25 or 26, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 27 or 28, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69.In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 29 or 30, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 31 or 32, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 33 or 34, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 35 or 36, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 37 or 38, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69.In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 39 or 40, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 41 or 42, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 43 or 44, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 45 or 46, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 49 or 50, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69.In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 51 or 52, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 59 or 60, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 61 or 62, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 102 or 103, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 108 or 109, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69.In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 153 or 154, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 161 or 162, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 79-83, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-69.

[0069] In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:47 or 48, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:84, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:73 or 74. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:53 or 54, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:84, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:73 or 74. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:110 or 111, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:84, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:73 or 74. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 114 or 115, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 84, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 73 or 74. In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 157 or 158, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 84, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 73 or 74.In some embodiments, the immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 159 or 160, the transmembrane polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 84, and the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 73 or 74.

[0070] In some embodiments, the fusion protein comprises an amino acid sequence of at least 75% sequence identity to the amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 76% sequence identity to the amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 77% sequence identity to the amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 78% sequence identity to the amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 79% sequence identity to the amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 81% sequence identity to the amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 82% sequence identity to the amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 83% sequence identity to the amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 84% sequence identity to the amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 86% sequence identity to the amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 87% sequence identity to the amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 88% sequence identity to the amino acid sequence according to SEQ ID NO: 116-152.In some embodiments, the fusion protein comprises an amino acid sequence of at least 89% sequence identity to the amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 91% sequence identity to the amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 92% sequence identity to the amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 93% sequence identity to the amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 94% sequence identity to the amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 96% sequence identity to an amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 97% sequence identity to an amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 98% sequence identity to an amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence of at least 99% sequence identity to an amino acid sequence according to SEQ ID NO: 116-152. In some embodiments, the fusion protein comprises an amino acid sequence according to SEQ ID NO: 116-152.

[0071] Compositions for the generation of immune checkpoint multivalent particles In some embodiments herein, a composition is described that includes a multivalent particle that includes a fusion protein that includes a mammalian immune checkpoint polypeptide and a transmembrane polypeptide. In some embodiments, the composition includes a first nucleic acid sequence that encodes the immune checkpoint multivalent particle described herein.

[0072] In some embodiments, the composition for generating multivalent particles further comprises a second nucleic acid sequence encoding one or more viral proteins. In some embodiments, the one or more viral proteins are lentiviral proteins, retroviral proteins, adenoviral proteins, or combinations thereof. In some embodiments, the one or more viral proteins include gag, pol, pre, tat, rev, or combinations thereof.

[0073] The composition for generating multivalent particles, in some embodiments, further comprises a second nucleic acid sequence encoding an expression construct for specifically targeting a mammalian immune checkpoint polypeptide to the surface of an extracellular vesicle. In some embodiments, the second nucleic acid sequence encodes an expression construct for specifically targeting a mammalian immune checkpoint polypeptide to the surface of an exosome.

[0074] The composition for generating multivalent particles, in some embodiments, further comprises a third nucleic acid sequence encoding a replication-deficient viral genome, a reporter, a therapeutic molecule, or a combination thereof. In some embodiments, the viral genome is derived from a vesicular stomatitis virus, a measles virus, a hepatitis virus, an influenza virus, or a combination thereof.

[0075] In some embodiments, the reporter protein is a fluorescent protein or enzyme.Exemplary reporter genes include, but are not limited to, acetohydroxy acid synthase (AHAS), alkaline phosphatase (AP), β-galactosidase (LacZ), β-glucuronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), cerulean fluorescent protein, citrine fluorescent protein, orange fluorescent protein, cherry fluorescent protein, turquoise fluorescent protein, blue fluorescent protein, horseradish peroxidase (HRP), luciferase (Fuc), nopaline synthase (NOS), octopine synthase (OCS), luciferase, and their derivatives. Methods for determining regulation of reporter genes are well known in the art and include, but are not limited to, fluorometric methods (e.g., fluorescence spectroscopy, fluorescence activated cell sorting (FACS), fluorescence microscopy) and antibiotic resistance determination. In some embodiments, the reporter is a fluorescent protein. In some embodiments, the fluorescent protein is green fluorescent protein. In some embodiments, the reporter protein emits green, yellow, or red fluorescence. In some embodiments, the reporter is an enzyme. In some embodiments, the enzyme is β-galactosidase, alkaline phosphatase, β-lactamase, or luciferase.

[0076] In some embodiments, the therapeutic molecule is a cell signaling regulating molecule, a proliferation regulating molecule, a cell death regulating molecule, or a combination thereof. In some embodiments, the therapeutic molecule is an inflammatory cytokine. In some embodiments, the inflammatory cytokine comprises IL-1, IL-12, IL-18, TNF-α, or TNF-β. In some embodiments, the therapeutic molecule is a proliferation cytokine. In some embodiments, the proliferation cytokine comprises IL-2, IL-4, IL-7, or IL-15. In some embodiments, the cell death molecule comprises Fas or a death receptor.

[0077] In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are in the same vector. In some embodiments, the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are in different vectors.

[0078] In some embodiments, various vectors are used herein. In some embodiments, the vector is a eukaryotic or prokaryotic vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector. Exemplary vectors include mammalian expression vectors, namely pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV-NEO-COOH-3XFLAG, pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV-FLAG(R)-6His, pCEP4, ... These include, but are not limited to, pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEF1a-mCherry-N1 vector, pEF1a-tdTomato vector, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP-tag(m), and pSF-CMV-PURO-NH2-CMYC, bacterial expression vectors, i.e., pSF-OXB20-BetaGal, pSF-OXB20-Fluc, pSF-OXB20, and pSF-Tac, plant expression vectors, i.e., pRI101-AN DNA and pCambia2301, yeast expression vectors, i.e., pTYB21 and pKLAC2, and insect vectors, i.e., pAc5.1 / V5-His A and pDEST8.

[0079] Compositions and pharmaceutical compositions In some embodiments herein, a composition is described that includes a multivalent particle that includes a fusion protein that includes a mammalian immune checkpoint polypeptide and a transmembrane polypeptide.In some embodiments herein, a pharmaceutical composition is described that includes a multivalent particle that includes a fusion protein that includes a mammalian immune checkpoint polypeptide and a transmembrane polypeptide.

[0080] For administration to a subject, the immune checkpoint multivalent particles disclosed herein may be provided in a pharmaceutical composition together with one or more pharma- ceutically acceptable carriers or excipients. In some embodiments, the immune checkpoint multivalent particles disclosed herein may be provided in a composition together with one or more carriers or excipients. The term "pharma-ceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the components and is non-toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, and the like. Such carriers can be formulated by conventional methods and administered to a subject in a suitable dose. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservatives, emulsifiers, dispersing agents, and the like. Prevention of microbial action can be ensured by the inclusion of various antibacterial and antifungal agents.

[0081] The pharmaceutical composition may be in any suitable form (depending on the desired method of administration). The pharmaceutical composition may be provided in a unit dosage form, in a sealed container, and as part of a kit. Such a kit may include instructions for use. The kit may include a number of unit dosage forms.

[0082] The pharmaceutical compositions may be adapted for administration by any suitable route, including parenteral (e.g., subcutaneous, intramuscular, intravenous, or inhalation) routes. Such compositions may be prepared by any method known in the pharmaceutical art, for example by mixing the active ingredient with the carrier or excipient under sterile conditions.

[0083] The dosage of the substances of the present disclosure can vary between wide limits, depending on the disease or disorder to be treated, the age and condition of the individual to be treated, etc., and the physician will ultimately determine the appropriate dosage to be used.

[0084] How to use The multivalent particles described herein, in some embodiments, the immune checkpoint multivalent particles, are used to treat cancer. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer is a leukemia or lymphoma. In some embodiments, the lymphoma is a B-cell lymphoma. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is a sarcoma, melanoma, breast cancer, lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, brain tumor, or carcinoma. In some embodiments, the lung cancer is non-small cell lung cancer.

[0085] In some embodiments, administration of the immune checkpoint multivalent particles shrinks or eliminates the cancer. In some embodiments, administration of the immune checkpoint multivalent particles increases anti-tumor immunity, increases cancer cell death, decreases tumor size, decreases cancer metastasis, or a combination thereof. In some embodiments, cell death is increased by about 1-fold to about 2.5-fold, about 1-fold to about 5-fold, about 2-fold to about 10-fold. In some embodiments, cell death is increased by at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 95-fold, 100-fold, or more than 100-fold. In some embodiments, tumor size is decreased by about 1-fold to about 2.5-fold, about 1-fold to about 5-fold, about 2-fold to about 10-fold. In some embodiments, tumor size is reduced by at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 95-fold, 100-fold, or more than 100-fold. In some embodiments, cancer metastasis is reduced by about 1-fold to about 2.5-fold, about 1-fold to about 5-fold, or about 2-fold to about 10-fold. In some embodiments, cancer metastasis is reduced by at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 95-fold, 100-fold, or more than 100-fold.

[0086] In some embodiments, administration of the immune checkpoint multivalent particles reduces or eliminates the cancer in the subject compared to levels prior to administration of the immune checkpoint multivalent particles. In some embodiments, administration of the immune checkpoint multivalent particles reduces or eliminates the cancer compared to levels when the subject is not administered the immune checkpoint multivalent particles. In some embodiments, administration of the immune checkpoint multivalent particles reduces or eliminates the cancer compared to levels when the subject is administered a different cancer treatment, including but not limited to radiation, surgery, and chemotherapy.

[0087] In some embodiments, the immune checkpoint multivalent particles induce T cell-mediated cytotoxicity against tumor cells. In some embodiments, the immune checkpoint multivalent particles inhibit T cell-mediated cytotoxicity against normal cells.

[0088] The multivalent particles described herein are, in some embodiments, used to treat an autoimmune disease, hi some embodiments, the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, psoriasis, or aplastic anemia.

[0089] In some embodiments, administration of the immune checkpoint multivalent particles attenuates or inhibits the autoimmune response in the subject compared to levels prior to administration of the multivalent particles. In some embodiments, administration of the immune checkpoint multivalent particles attenuates or inhibits the autoimmune response compared to levels if the subject had not been administered the multivalent particles. In some embodiments, administration of the immune checkpoint multivalent particles attenuates or inhibits the autoimmune response compared to levels if the subject had received a different treatment.

[0090] In some examples, the subject is a mammal. In some examples, the subject is a mouse, rabbit, dog, pig, cow, or human. The subject treated by the methods described herein may be an infant, an adult, or a child. In some embodiments, the multivalent particles are administered by inhalation, injection, ingestion, infusion, implantation, or transplantation. In some embodiments, the multivalent particles are administered intraarterially, subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, intravenously (iv) injected, or intraperitoneally. In some embodiments, the multivalent particles are administered intravenously. In some embodiments, the multivalent particles are administered by inhalation. In some embodiments, the multivalent particles are administered by intraperitoneal injection. In some embodiments, the multivalent particles are administered by subcutaneous injection.

[0091] The following examples are provided to more clearly illustrate to one skilled in the art the principles and practice of the embodiments disclosed herein, but should not be construed as limiting the scope of any claimed embodiments. Unless otherwise specified, all parts and percentages are by weight. EXAMPLES

[0092] The following examples are provided for the purpose of illustrating various embodiments of the present disclosure, and are not intended to limit the present disclosure in any way. The examples, together with the methods described herein, represent preferred embodiments herein and are illustrative, but are not intended as limitations on the scope of the present disclosure. Modifications and other uses of the present disclosure that are encompassed within the spirit of the present disclosure as defined by the scope of the claims will occur to those skilled in the art.

[0093] Example 1: Generation and characterization of multivalent immune checkpoint particles (IC-MVPs) This example describes the generation of multivalent immune checkpoint particles (IC-MVPs) expressing immune stimulatory or immune inhibitory molecules.

[0094] Design of IC-MVP Display Vectors

[0095] Three different types of IC-MVP display vectors were designed to display immune checkpoints in various multimeric forms on vesicles (Figure 1A-1C). When displaying immune checkpoints in monomeric form, the display vector expressed a fusion protein containing the extracellular domain of the desired immune checkpoint linked to the VSV-G protein transmembrane and intracellular domains (Figure 1A). When displaying immune checkpoints in trimeric form, the vector expressed a fusion protein containing the extracellular domain of the desired immune checkpoint linked to the D4 post-fusion trimerization domain, transmembrane domain, and intracellular domain of VSV-G (Figure 1B). For immune checkpoints that are type II transmembrane proteins, the vector expressed a fusion protein containing the influenza neuraminidase stem domain and transmembrane domain followed by the extracellular domain of the type II immune checkpoint, and this fusion protein formed a tetramer (Figure 1C). These vectors can be used to produce monomeric, trimeric, or tetrameric IC-MVPs.

[0096] Generation of monomeric IC-MVP

[0097] Multivalent immune checkpoints can be displayed as monomers on the surface of virus-like particles (VLPs) and extracellular vesicles (EVs), such as exosomes and ectosomes, using monomeric display vectors. To produce monomeric immune checkpoint VLPs (IC-VLPs) with viral RNA genomes, monomeric immune checkpoint fusion constructs were co-transfected into HEK293T cells with lentiviral packaging constructs expressing essential packaging components, such as Gag-Pol and Rev proteins, as well as a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 2A). Alternatively, monomeric IC-VLPs without RNA genomes were produced by co-transfecting a display vector only with the lentiviral packaging constructs, but not with the viral genome transfer vector (Figure 2B). Finally, monomeric immune checkpoint extracellular vesicles (IC-EVs), including IC-exosomes and IC-ectosomes, were produced by transfecting only the monomeric immune checkpoint display vector into 293T cells (Figure 2C).

[0098] Generation of trimeric IC-MVP

[0099] Multivalent immune checkpoints can be displayed as trimers on the surface of virus-like particles (VLPs) and extracellular vesicles (EVs), such as exosomes and ectosomes, using trimeric display vectors. To produce trimeric VLP-ICs with viral RNA genomes, trimeric immune checkpoint fusion constructs were co-transfected into HEK293T cells with lentiviral packaging constructs expressing essential packaging components, such as Gag-Pol and Rev proteins, as well as a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). Alternatively, trimeric VLP-ICs without RNA genomes were produced by co-transfecting the display vectors only with lentiviral packaging constructs, but not with the viral genome transfer vector (Figure 3B). Finally, trimeric VLP-ICs containing IC-exosomes and IC-ectosomes were produced by transfecting only the trimeric immune checkpoint display vector into 293T cells (Figure 3C).

[0100] Generation of mixed monomeric and trimeric IC-MVPs

[0101] MVPs displaying a mixture of monomeric and trimeric immune checkpoints were generated by co-transfecting HEK293T cells with constructs displaying monomeric and trimeric immune checkpoints. Such designs can be used to increase the display density of immune checkpoints or to create combinatorial display of distinct immune checkpoint molecules. Mixed monomeric and trimeric IC-MVPs can be constructed using virus-like particles (VLPs) and extracellular vesicles (EVs), such as exosomes and ectosomes, by co-transfecting monomeric and trimeric display vectors. To produce mixed IC-VLPs with viral RNA genomes, mixed monomeric and trimeric immune checkpoint fusion constructs were co-transfected into 293T cells with lentiviral packaging constructs expressing essential packaging components, such as Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 4A). Alternatively, mixed IC-VLPs without RNA genomes were produced by co-transfecting a mixture of monomeric and trimeric display vectors with only lentiviral packaging constructs, not viral genome transfer vectors (Figure 4B).Finally, mixed IC-EVs, containing a mixture of mixed IC-exosomes and IC-ectosomes, were produced by transfecting a mixture of monomeric and trimeric immune checkpoint fusion constructs into 293T cells (Figure 4C).

[0102] Peptide display arrangement on IC-MVP

[0103] IC-MVPs can be genetically programmed to display immune checkpoints in various configurations by modifying the display vector (Figures 5A-C, 6A-C, Table 3). The VSV-G D4 trimerization domain can be placed at various positions of the fusion peptide, i.e., (1) extracellular and aligned with the transmembrane domain (Figure 5A), (2) intracellular and aligned with the transmembrane domain (Figure 5B), (3) extracellular and after the signal peptide (Figure 5C). Furthermore, various multimerization domains may be used for distinct surface display patterns appropriate for the function of immune checkpoint molecules (Figures 6A-C, Table 3). In addition to the VSV-G D4 trimerization domain, the post-fusion trimerization domain of the Dengue E protein, or the T4 phage foldon domain can also be used to create trimer display patterns on the surface of VLPs and EVs. The leucine zipper domain and influenza neuraminidase stem domain can be used to create dimer and tetramer display patterns on the surface of VLPs and EVs, respectively. Exemplary multimerization domains and valencies are summarized in Table 3. With these display configurations, combinatorial IC-MVPs can be programmed with mixed monomeric, dimeric, trimeric, and tetrameric immune checkpoint display patterns optimized for the function of checkpoints exhibited in T cell regulation.

[0104] [Table 3]

[0105] Characterization of immune checkpoint display on IC-MVO

[0106] The concentration of VLP- or EV-based IC-MVPs was measured by P24 ELISA or tunable resistive pulse sensing (TRPS, qNano), respectively. The copy numbers of immune checkpoints displayed on MVPs were determined by quantitative Western blot analysis. The multimerization patterns of immune checkpoints displayed on MVPs were identified by non-reducing PAGE analysis. IC-MVPs displaying at least 10 copies of immune checkpoint molecules on the surface of VLPs and EVs were generated in monomeric or trimeric configurations.

[0107] Binding of IC-MVP to target cells expressing the cognate receptor / ligand

[0108] To confirm that IC-MVPs represent functional immune checkpoint molecules, we tested whether IC-MVPs could bind to target cells expressing their cognate receptors or ligands using fluorescence-activated cell sorting (FACS)-based analysis (Figure 7A, Figure 7B). Two different approaches were used to evaluate the specific interaction of IC-MVPs with target cells. In the first approach (Figure 7A), target cell lines were established by transfecting 293T cells with constructs expressing the cognate ligand or receptor for the immune checkpoint molecule expressed on IC-MVP. IC-MVPs were then labeled with CBF640 or other compatible fluorescent dyes. Transfected 293T cells were stained with dye-labeled IC-MVP and an antibody specific for the ligand. Finally, the specific binding of IC-MVPs to target cells expressing their cognate ligand or receptor was analyzed by FACS. In the second approach (Figure 7B), transfected target cells were stained with unlabeled IC-MVP, and then the target cells were stained with fluorescent antibodies specific for the immune checkpoint and its ligand. Again, specific binding of IC-MVP to target cells expressing its cognate ligand or receptor was analyzed by FACS. In some cases, when expression of the cognate receptor or ligand was confirmed on T cells, T cells were also stained with dye-labeled IC-MVP, and specific binding of IC-MVP to T cells was analyzed. These approaches confirmed functional immune checkpoint expression on IC-MVP and provided insight into how to optimize the copy number and multimerization pattern of immune checkpoint to enhance IC-MVP interaction with target cells.

[0109] Regulation of T cell activation, proliferation, differentiation, and apoptosis by IC-MVP

[0110] Both stimulatory and inhibitory immune checkpoints play important roles in regulating T cell activation, proliferation, apoptosis, and differentiation. To investigate the effect of IC-MVP on T cells, the following assay was designed. T cells activated with anti-CD3 antibody were treated with various concentrations of IC-MVP. The potential activating or inhibitory effect of IC-MVP on T cell activation can be read out 2 days after activation by examining CD69 and CD25 (early T cell activation markers) expression on treated T cells. Alternatively, Pmel T cells stimulated with GP100 peptide antibody-loaded dendritic cells were treated with various concentrations of IC-MVP. The potential activating or inhibitory effect of IC-MVP on antigen-specific T cell activation can be read out 2 days after activation by examining CD69 and CD25 (early T cell activation markers) expression on treated T cells. Furthermore, the effect of IC-MVP on T cell proliferation can be determined by monitoring the cell numbers in treated cell cultures for 8-10 days, and the effect of IC-MVP on differentiation of effector and memory T cells can be determined by FACS analysis for CD62L and CD44 expression in treated cell cultures. Finally, the effect of IC-MVP on cultured T cell apoptosis was determined by staining cultured T cells with PI and 7-AAD 8-10 days after activation.

[0111] Regulation of cytotoxic T cell (CTL) activity by IC-MVP

[0112] To investigate the activity of IC-MVP in controlling cytotoxic T cells (CTLs), we investigated how IC-MVP disrupted the cytolytic activity of Pmel T cells against B16F0 melanoma cells. Pmel T cells have a transgenic T cell receptor (TCR) that recognizes the gp100 peptide EGSRNQDWL bound to MHC-1 H2-Db presented on B16F0 melanoma cells. In addition, we examined whether IC-MVP treatment of T cells improved the expression of granzyme A and perforin in treated T cells by intracellular staining and FACS analysis. Granzyme A and perforin are two important proteins in the granule exocytosis pathway for T cell- and NK cell-mediated cell killing. Finally, we examined whether IC-MVP-treated T cells expressed higher levels of inflammatory cytokines, such as IFN-γ and TNF-α, by intracellular staining and FACS analysis. T cells with higher levels of IFN-γ and TNF-α had enhanced inflammatory functions.

[0113] Regulation of tumor progression by IC-MVP

[0114] We investigated the effect of IC-MVP on tumor progression using syngeneic mouse tumor models of lung, breast, pancreatic, and melanoma. Purified IC-MVP was injected into tumor-implanted mice via tail vein injection. Mice were repeatedly administered IC-MVP every 3 days for 6 times. Tumors were measured at various time points after treatment to determine whether IC-MVP could enhance or inhibit tumor growth in vivo. The effect of IC-MVP on tumor controls was compared to positive control checkpoint-blocking antibodies, such as anti-PD-1 and anti-CTLA-4 antibodies. We first examined the tumor-controlling function of IC-MVPs representing individual immune checkpoints, and then tested IC-MVPs representing immune checkpoint combinations that could further improve the tumor-controlling ability of IC-MVPs.

[0115] Modulation of ARDS by IC-MVP

[0116] Acute respiratory distress syndrome (ARDS) was used as an inflammation model. We investigated whether the damage caused by systemic inflammation could be controlled and reduced by using inhibitory IC-MVP. Excessive proinflammatory responses leading to ARDS can be initiated and induced by Toll-like receptors (TLRs) that recognize pathogen-derived components such as lipopolysaccharide (LPS), bacterial lipoproteins, and unmethylated CpG DNA, leading to a rapid increase in systemic immune responses. Such a condition can be partially reproduced in a mouse model of LPS-induced systemic inflammation. In this lethal model, untreated mice reached the experimental endpoint within 72 hours. If IC-MVP treatment can rescue mice from lethality, it will be demonstrated that IC-MVP can effectively attenuate systemic inflammation induced by LPS.

[0117] Materials and Methods

[0118] Immune checkpoint display constructs

[0119] Codon-optimized immune checkpoint sequences were synthesized (Twist) and cloned into display constructs to generate fusion peptides consisting of the immune checkpoint extracellular domain and the display anchoring protein. To generate MVPs representing monomeric immune checkpoints, the immune checkpoint extracellular domain was fused to a synthetic VSV-G sequence encoding the transmembrane and cytoplasmic tail domains. To generate MVPs representing multimerized immune checkpoints, the immune checkpoint extracellular domain was fused to a synthetic VSV-G sequence encoding the D4 post-fusion trimerization domain, as well as the transmembrane and cytoplasmic tail domains.

[0120] Production of VLP- or extracellular vesicle-based IC-MVPs

[0121] VLP or extracellular vesicle-based IC-MVP was produced from transfected 293T cells. To generate lentivirus-VLP-based IC-MVP with viral genome, immune checkpoint display construct, lentivirus packaging vector (i.e., psPAX2), and lentivirus genome transfer vector were co-transfected into 293T cells. To generate lentivirus-VLP-based IC-MVP without viral genome, immune checkpoint display construct and lentivirus packaging vector (i.e., psPAX2) were co-transfected into 293T cells. Finally, to generate extracellular vesicle-based IC-MVP, only immune checkpoint display construct was transfected into 293T cells.

[0122] In preparation for transfection, 7.5 x 10 6HEK293T cells (ATCC CRL-3216) were seeded overnight in 10 cm dishes containing DMEM medium with glucose, L-glutamine, and sodium pyruvate (Coming) supplemented with 10% fetal bovine serum (Sigma) and 1% penicillin streptomycin (Life Technologies), which is referred to as "293T growth medium". The cells should reach approximately 90% confluence the next day at the time of transfection. The following day, a transfection DNA mixture with polyethylenimine (PEI) was prepared in OPTI-MEM reduced serum medium (Gibco). The transfection mixture was incubated at room temperature for 15 minutes before being added to the cells, which were then incubated at 37°C in 5% CO2. Six hours after transfection, the 293T growth medium was replaced with 293T growth medium supplemented with 0.1% sodium butyrate (referred to as "transfection medium") before returning to incubation. After 24 hours of incubation in transfection medium at 37°C with 5% CO2, pseudovirus-containing supernatants were collected, centrifuged at 1680 rpm for 5 minutes to remove cell debris, mixed with 1X polyethylene glycol 8000 solution (PEG, Hampton Research) and stored at 4°C for 24 hours to allow fractionation. Cells were replenished with fresh transfection medium and a second pseudovirus supernatant harvest was performed at 48 hours. Supernatant harvests were then pooled, PEG precipitated and purified by size exclusion chromatography using Sephacryl S-300 High Resolution Beads (Sigma Aldrich).

[0123] Quantification of lentiviral particles by p24 ELISA and tunable resistive pulse sensing

[0124] P24 concentrations in pseudovirus samples of pseudotyped coronavirus, influenza virus, and antibody-based antiviral particles were determined using the HIV p24 SimpleStep ELISA kit (Abcam) according to the manufacturer's protocol. The concentration of lentiviral pseudovirions was determined such that each lentiviral particle contained approximately 2000 molecules of p24, or 1.25 × 10 per picogram of p24 protein. 4 This was extrapolated from the assumption that each sample contains 10 pseudovirus particles.

[0125] Pseudovirus concentrations determined via p24 ELISA were confirmed by tunable resistive pulse sensing (TRPS, qNano, IZON). Purified pseudovirus harvests were diluted with 0.2 μm filtered phosphate buffered saline (PBS) containing 0.03% Tween-20 (Thermo Fisher Scientific) prior to qNano analysis. The concentration and size distribution of pseudotyped particles were then determined using an NP200 nanopore at 45.5 mm extension, achieving a stable current of 130 nA through the nanopore by using an applied voltage between 0.5 and 0.7 V. Measurements of each pseudovirus sample were performed at pressures of 3, 5, and 8 mbar and were considered valid if at least 500 events were recorded, particle velocity was linear, and root-mean-square signal-to-noise was maintained below 10 pA. The original 7.3 × 10 11 Pseudovirus concentrations were determined by comparison to a standardized multi-pressure calibration using CPC200 (mode diameter: 200 nm) (IZON) carboxylated polystyrene beads, diluted 1:200 in 0.2 μM filtered PBS from a concentration of 10 particles / mL. Measurements were analyzed using IZON Control Suite 3.4 software to determine the original sample concentration.

[0126] Quantification of lentiviral VLP-based IC-MVPs

[0127] P24 concentrations in IC-MVP samples were determined using the Abcam HIV P24 SimpleStep ELISA kit according to the manufacturer's instructions. The concentration of lentiviral pseudoparticles was determined such that each lentiviral particle contained approximately 2000 molecules of P24 or 1.25 × 10 4 This was derived based on the assumption that the virus contains 10 ...

[0128] Quantification of extracellular vesicle-based IC-MVPs

[0129] The size and concentration of extracellular vesicle-based IC-MVPs were determined by tunable resistive pulse sensing (TRPS, qNano, IZON). The purified pseudovirus harvest was diluted in 0.2 μm filtered PBS containing 0.03% Tween-20 (Thermo Fisher Scientific) prior to qNano analysis. The concentration and size distribution of IC-MVPs were then determined using an NP200 nanopore at 45.5 mm extension, achieving a stable current of 130 nA through the nanopore by using an applied voltage between 0.5 and 0.7 V. Measurements of each pseudovirus sample were performed at pressures of 3, 5, and 8 mbar and were considered valid if at least 500 events were recorded, the particle velocity was linear, and the root-mean-square signal-to-noise was maintained below 10 pA. The original 7.3 × 10 11 IC-MVP concentrations were determined by comparison to a standardized multi-pressure calibration using CPC200 (mode diameter: 200 nm) (IZON) carboxylated polystyrene beads diluted 1:200 in 0.2 μM filtered PBS from a concentration of 10 particles / mL. Measurements were analyzed using IZON Control Suite 3.4 software to determine the original sample concentration.

[0130] Western blot analysis of IC-MVPs

[0131] Expression of immune checkpoint fusion proteins on MVPs was confirmed by Western blot analysis on purified particles. Samples of purified IC-MVPs were lysed in cell lysis buffer (Cell Signaling) for 10 min at 4°C, then mixed with NuPage LDS sample buffer (Thermo Fisher Scientific) and boiled at 95°C for 5 min. Differences in multimerization were determined by running samples under reducing and non-reducing conditions. Under reducing conditions, 5% 2-mercaptoethanol (Thermo Fisher Scientific) was added to the samples to dissociate multimerized IC-MVPs. Protein samples were then separated on NuPAGE 4-12% Bis-Tris gels (Thermo Fisher Scientific) and transferred onto polyvinylidene difluoride (PVDF) membranes (Fife Technologies). PVDF membranes were blocked with TRIS-buffered saline containing Tween-20 (TBST) and 5% skim milk (Research Products International) for 1 hour and then incubated overnight with primary antibodies diluted in 5% milk. For immune checkpoint fusion constructs expressing the VSVG tag, anti-VSV-G epitope tag rabbit polyclonal antibody (BioFegend, Poly29039) was used at a dilution of 1:2000. The next day, PVDF membranes were washed three times with 1xTBST and stained with goat anti-rabbit secondary antibody (IRDye680) diluted 1:5000 in 5% milk for 60 minutes. After secondary antibody staining, PVDF membranes were washed again three times with TBST and then imaged on a Licor Odyssey scanner.

[0132] Alternatively, Western blot analysis was performed using an automated Simple Western size-based protein assay (Protein Simple) according to the manufacturer's protocol. All reagents used here were from Protein Simple unless otherwise mentioned. Concentrated samples were dissolved as described above and then diluted 1:10 with 0.1x sample buffer for loading onto the capillary. The same primary rabbit polyclonal antibody diluted 1:400 and an HRP-conjugated anti-rabbit secondary antibody (Protein Simple) were used to identify immune checkpoint fusion protein expression levels. Chemiluminescence signal analysis and absolute quantification were performed using Compass software (Protein Simple).

[0133] Quantitative Western blot analysis

[0134] Quantitative Western blot analysis was performed to determine the copies of immune checkpoint fusion protein displayed per particle. IC-MVP sample concentrations were determined using P24 EFISA or TRPS (qNano) assays. Purified IC-MVP samples were processed and analyzed by Western blot under reducing conditions as described above. A reference decoy-MVP with known display copy number was used to generate a standard curve from which the copy number of immune checkpoint displayed on the corresponding particle was determined.

[0135] Binding of IC-MVP to target cells

[0136] To verify the specific binding between IC-MVPs, purified IC-MVPs were stained with CSFE or other fluorescent dyes and then passed through a size-exclusion column to remove unbound dye. T cells or 293T cells transfected with cognate immune checkpoint ligands or receptors were incubated with dye-labeled IC-MVPs at room temperature for 30 minutes. The stained cells were then washed with FACS buffer and analyzed on a flow cytometer to determine the specific binding between IC-MVPs and target cells.

[0137] Effects of IC-MVP on T cell activation, proliferation, apoptosis, and differentiation

[0138] The effects of IC-MVP on T cell activation, proliferation, apoptosis, and differentiation were examined using purified mouse splenic T cells or human peripheral blood T cells. T cells stimulated with a suboptimal dose of anti-CD3 antibody were treated with various concentrations of IC-MVP. Alternatively, Pmel T cells stimulated with dendritic cells loaded with GP100 peptide antibody were treated with various concentrations of IC-MVP. On the second or third day after IC-MVP treatment, cells were analyzed by FACS to determine the expression of early activation markers CD69 and CD25. The effect of IC-MVP on T cell proliferation was determined by monitoring cell numbers for 8–10 days. The effect of IC-MVP on T cell differentiation was determined by quantifying the composition of effector and memory cells by FACS analysis for CD62L and CD44 expression. Finally, on the 8–10th day after activation, cultured T cells were stained with PI and 7-AAD to determine the effect of IC-MVP on cultured T cell apoptosis.

[0139] Effect of IC-MVP on CTL

[0140] To determine the effect of IC-MVP on the ability of CD8 T cells to kill tumor cells, CD8 T cells were purified from Pmel mice expressing a transgenic T cell receptor (TCR) that specifically recognizes the gp100 peptide EGSRNQDWL bound to MHC-1 H2-Db. EGSRNQDWL-loaded (2 μg / ml) bone marrow-derived dendritic cells (2 × 10 5Pmel T cells were activated by incubation with IC-MVP (cells / well) or PBS (as control) with or without PD-L1 antibody blockade. Activated cells were treated with PBS (as control) or IC-MVP with or without PD-L1 antibody blockade and then co-cultured with CellTrace™ Violet dye-labeled B16-F0 cells at an effector-to-target ratio of 1:1 (E:T) for 48 hours. Cells were harvested, labeled with 7-aminoactinomycin D (7-AAD, BD Pharmingen) and analyzed by FACS to determine target cell killing by T cells. The population of CellTrace™ Violet dye+ / 7-AAD+ cells represented dead target cells, and the population of CellTrace™ Violet dye+ / 7-AAD- represented remaining viable target cells. The percentage of specific lysis was calculated by using the formula: specific lysis (%) = (CellTrace™ Violet dye + / 7-AAD +) / (CellTrace™ Violet dye + / 7-AAD + CellTrace™ Violet dye + / 7-AAD -) - target / CTV / 7AAD background ratio.

[0141] Effect of IC-MVP on tumor progression

[0142] The effect of IC-MVP on tumor progression was investigated using syngeneic mouse tumor models of lung, breast, pancreatic, and melanoma. Tumor cells were cultured, expanded, and then transplanted. To generate the melanoma model, 1 × 10 5 B16F0 cells were subcutaneously injected into 6-8 week-old female C57BL / 6 mice. To generate lung cancer models, 2 × 10 5 ~2×10 6 Lewis lung carcinoma cells (LLC) were delivered directly into the lungs of 6-8 week-old female C57BL / 6 mice by intratracheal instillation. To generate the pancreatic cancer model, 2 × 10 5 ~2×10 6KPC cells were delivered directly into the pancreas of 6-8 week old female C57BL / 6 mice. After tumor implantation, mice were observed daily and sacrificed after signs of morbidity. Tumor formation in mice was confirmed twice weekly by palpation or caliper measurement. Mice were sacrificed and tumors were harvested after tumor size reached 2.0 cm in diameter or after skin ulceration. Tumor weight and size were documented. In all tumor treatment studies, mice were randomized prior to the experiment to ensure no size bias at the start of the experiment. To examine the effect of IC-MVP on tumor development, purified IC-MVP was injected into mice after tumor implantation via tail vein injection. Mice were repeatedly administered IC-MVP every 3 days for 6 times. Tumors were measured using digital calipers and calculated using the formula: (width) 2 Tumor volume was calculated by ×length / 2.

[0143] Mouse model of ARDS

[0144] Balbc mice aged 8–10 weeks were administered 6 mg / kg LPS intraperitoneally. Mouse mortality was recorded daily for 3–4 days after LPS injection. Mice were first treated 16 h after LPS challenge and then treated daily with intranasal delivery of IC-MVP. The effect of IC-MVP treatment on mouse survival was recorded. In this lethal model, untreated mice usually reached the experimental endpoint within 72 h. If IC-MVP treatment rescued mice from lethality, it was demonstrated that IC-MVP could effectively attenuate LPS-induced systemic inflammation. To facilitate collection of bronchoalveolar lavage (BAL), a blunt 23-gauge needle was placed in a small opening in the upper trachea and secured in place using Mersilk sutures (Ethicon). The lungs were washed with a total volume of 700 ml of ice-cold PBS, which was instilled into 350 ml aliquots via a tracheal cannula and then gently aspirated. BAL fluid was centrifuged at 425g for 10 min at 4°C and cell pellets were resuspended in 100 ml ice-cold PBS. Total viable cell counts were performed using a hemocytometer under trypan blue exclusion. After collection of BAL fluid, lobes were homogenized for 4 min. Samples were centrifuged at 18,000g for 15 min at 4°C and cytokine levels in the supernatants were quantified by ELISA.

[0145] Example 2: Exemplary Sequences

[0146] [Table 4-1]

[0147] [Table 4-2]

[0148] [Table 4-3]

[0149] [Table 4-4]

[0150]

Table 4-5

[0151]

Table 4-6

[0152]

Table 4-7

[0153]

Table 4-8

[0154]

Table 4-9

[0155]

Table 4-10

[0156]

Table 4-11

[0157]

Table 4-12

[0158]

Table 4-13

[0159]

Table 4-14

[0160]

Table 5-1

[0161]

Table 5-2

[0162]

Table 5-3

[0163]

Table 5-4

[0164]

Table 5-5

[0165]

Table 6-1

[0166]

Table 6-2

[0167]

Table 6-3

[0168]

Table 6-4

[0169]

Table 6-5

[0170]

Table 6-6

[0171] [Table 6-7]

[0172] [Table 6-8]

[0173] [Table 6-9]

[0174] [Table 6-10]

[0175] Example 3. Characterization of antitumor immunity by inhibitory IC-MVP This example illustrates the characterization of PD-1-MVP and its function in engaging target cells and tumor control mouse models.

[0176] We investigated whether PD-1-MVP could selectively bind to target cells expressing its cognate ligands PD-L1 / PD-L2. PD-1-MVP was generated by pseudotyping lentiviral VLPs with a trimeric PD-1 fusion peptide. Specifically, HEK293T cells were co-transfected with the trimeric PD-1 display construct along with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). The concentration of purified PD-1-MVP was quantified by P24 ELISA. PD-1-MVP displayed 280±60 copies of PD-1 per MVP in various multimerized forms as determined by quantitative Western blot analysis (Figure 8A). Thus, the D4 display construct can effectively present hundreds of copies of PD-1 in multimerized forms on IC-MVP.

[0177] To confirm that PD-1-MVP exhibits functional PD-1, we tested whether PD-1-MVP could selectively bind to target cells expressing PD-1's cognate ligands, PD-L1 or PD-L2 (Figure 8B-E). First, target cell lines were established by transfecting S293 cells with constructs expressing PD-L1. The transfected cells were then stained with anti-PD-L1 antibodies to distinguish PD-L1-positive cells (PD-L1+) from PD-L1-negative cells (PD-L1-). PD-1-MVP was then labeled with a fluorescent dye, the transfected cells were stained with the labeled PD-1-MVP, and selective MVP-cell binding was analyzed by FACS (Figure 8B). The results showed that fluorescently labeled PD-1-MVP binding induced a significantly higher fluorescence shift in PD-L1+ cells than in PD-L1- cells (Figure 8B, upper panel). Moreover, this shift was at least 1 log higher than the fluorescence shift produced by staining the same cells with a control MVP representing a nonspecific ligand (Fig. 8B, lower panel), demonstrating that PD-1-MVP represented functional PD-1 that could selectively bind to PD-L1 on target cells.

[0178] This result was further verified by an alternative staining method (Figure 8C). In this case, PD-L1-transfected S293 cells were first incubated with unlabeled PD-1-MVP to allow PD-1-MVP to bind to target cells. The MVP-cell mixture was then co-stained with fluorescently labeled anti-PD-1 and anti-PD-L1 antibodies. The PD-1 staining pattern was then examined by FACS analysis on PD-L1+ and PD-L1- cells. We observed that a significant fraction of PD-L1+ cells were also PD-1 positive, as illustrated by a 1 log PD-1 staining shift in PD-L1+ cells from PD-1- background cells (Figure 8C). Single staining with anti-PD-1 antibody did not compete with PD-L1-MVP binding to target cells, and PD-L1-transfected S293 cells were PD-L negative. These results confirmed that PD-1-MVP exhibited functional PD-1. Using a similar method, we demonstrated that PD-1-MVPs, with or without dye labeling, could selectively bind to target cells expressing PD-L2 in both types of binding assays (Figure 8D, E). Collectively, PD-1-MVPs were generated that represent high copy numbers of functional PD-1 protein, and these MVPs can selectively bind to target cells expressing their cognate ligands, PD-L1 or PD-L2.

[0179] Checkpoint blockade by PD-1-MVP and other IC-MVPs

[0180] In vivo T cell activation is regulated by a diverse group of inhibitory immune checkpoints, including PD-1, CTLA-4, LAG-3, TIM-3, and many others, as illustrated by a schematic diagram depicting inhibitory immune checkpoints on T cells and their ligands on antigen-presenting cells, including tumor cells (Figure 9A). Such regulation is important to keep effector T cells under control and prevent unintended activation. Many of these pathways have been found to be exploited by cancer cells to suppress the function of tumor-targeting T cells (Figure 9B). Antibodies targeting PD-1 or CTLA-4 can effectively block these inhibitory checkpoint signals mediated by cancer cells and activate antitumor T cells for cancer therapy (Figure 9C). Since PD1-MVP was demonstrated to be able to selectively bind to target cells expressing PD-L1 and PD-L2 (Figure 8A-8E), PD1-MVP can be used to block PD-L1 / PDL-2 on cancer cells and prevent them from interacting with PD-1 molecules on tumor-targeting T cells (Figure 9D). Thus, PD-1-MVP can be used to therapeutically block inhibitory checkpoint signals for cancer treatment.

[0181] Inhibition of tumor growth by PD-1-MVP in mice

[0182] To determine whether PD1-MVP can control melanoma cancer, we investigated whether PD1-MVP can bind to PD-L1 expressed on cancer cells. As determined by FACS analysis, both murine B16F0 (non-metastatic) and murine B16F10 (metastatic) melanoma cells express high levels of PD-L1 (Figure 10A, Figure 10B) and can effectively bind fluorochrome-labeled PD1-MVP, respectively (Figure 10C, Figure 10D). These results demonstrate that PD-1-MVP can effectively bind to PD-L1-positive B16F0 and B16F10 melanoma cells. Mice bearing B16F0 melanoma tumors were treated with intravenous delivery of PD1-MVP. These mice received 5 × 10 10A total of five single doses of PD-1-MVP were administered (Figure 11A). PD1-MVP significantly reduced tumor growth (Figure 11B) and prolonged survival (Figure 11C) in mice bearing B16F0 melanoma tumors. Similarly, mice bearing B16F10 melanoma tumors were administered 5x10 10 PD1-MVP was administered five times in total (Figure 12A), and the results showed that PD1-MVP significantly reduced the growth of B16F10 tumors in mice (Figure 12B). Finally, MC38 cells, a mouse colon cancer cell line, were shown to express high levels of PD-L1 (Figure 13A) and efficiently bind fluorescently labeled PD1-MVP (Figure 13B), as shown by FACS staining and analysis. Similarly, mice bearing MC38 colon adenocarcinoma tumors were administered 5 × 10 10 A total of five doses of PD-1-MVP were administered (Figure 13C), and the results showed that PD-1-MVP significantly reduced the growth of MC38 tumors in mice (Figure 13D). Collectively, these results demonstrated that PD-1-MVP can specifically bind to cancer cells expressing the cognate ligands PD-L1 and PD-L2 and inhibit tumor progression in multiple mouse tumor models. Thus, PD1-MVP represents a novel multivalent checkpoint blockade therapeutic for cancer that can block or attenuate inhibitory signals mediated by inhibitory checkpoints expressed on cancer cells and tumor-targeted T cells.

[0183] Example 4. Control of inflammation by inhibitory IC-MVP This example illustrates the analysis of PD-L1-MVP and 2B4-MVP and their functions in target cell engagement and regulating inflammatory responses in mice.

[0184] Use of IC-MVPs to mimic inhibitory checkpoint signaling

[0185] During various inflammatory situations, the immune system routinely engages inhibitory immune checkpoints to protect against autoreactive immune cells. Uncontrolled inflammatory responses can cause acute or chronic damage to the body. For example, during autoimmune, acute and chronic inflammatory conditions, T cells can be activated to damage the body's own tissues or organs in the absence of the required inhibitory checkpoint signals, such as PD-L1 / PD-1 signaling (Figure 14A). IC-MVPs can be used to mimic these missing inhibitory immune checkpoint signals. For example, PD-L1-MVP can be used to engage PD-1 molecules on autoreactive T cells to inactivate such T cells (Figure 14B). Thus, IC-MVPs, such as PD-L1-MVP, can be used to inactivate T cells and other immune cells during acute and chronic inflammatory conditions.

[0186] Generation and characterization of PD-L1-MVPs

[0187] PD-L1-MVPs were generated by pseudotyping lentiviral VLPs with trimeric PD-L1 fusion peptides. Specifically, HEK293T cells were co-transfected with the trimeric PD-L1 display construct along with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). The concentration of purified PDL1-MVPs was quantified by P24 ELISA. PDL1-MVPs displayed 6600±2500 copies of PD-L1 per particle in various multimerized forms as determined by quantitative Western blot analysis (Figure 15A). Thus, the D4 display construct can effectively present thousands of copies of PD-L1 in multimerized forms on MVPs.

[0188] To confirm that PDL1-MVP exhibits functional PD-L1, we tested whether PDL1-MVP could selectively bind to target cells expressing its cognate receptor, PD-1 (Figure 15B). First, a target cell line was established by transfecting S293 cells with a construct expressing PD-1. Then, the transfected cells were stained with an anti-PD-1 antibody to distinguish PD-1+ cells from PD-1- cells. Then, PD-L1-MVP was labeled with a fluorescent dye, the transfected cells were stained with the labeled MVP, and the selective MVP-cell binding was analyzed by FACS (Figure 15B). The results showed that the labeled PDL1-MVP binding induced a significantly higher fluorescence shift in PD-1+ cells than in PD-1- cells (Figure 15B, upper panel). Moreover, this shift was at least 1 log higher than the fluorescence shift produced by staining the same cells with control MVP representing a non-specific ligand (Figure 15B, lower panel), demonstrating that PD-L1-MVP represents functional PD-L1 and can selectively bind to PD-1-positive target cells.

[0189] This result was further verified by an alternative staining method (Figure 15C). In this case, PD-1 transfected cells were first incubated with unlabeled PD-L1-MVP to allow MVP to bind to target cells. The cell-MVP mixture was then co-stained with fluorescently labeled anti-PD-1 and anti-PD-L1 antibodies. The PD-L1 staining pattern was then examined by FACS analysis on PD-1+ and PD-1- cells. We observed that a significant fraction of PD-1+ cells were also PD-1 positive, as illustrated by a 1 log PD-1 staining shift in PD-1+ cells from PD-1- background cells (Figure 15C). These results demonstrated that PD-L1-MVP displayed functional PD-L1. Collectively, PDL1-MVPs displaying high copy numbers of functional PD-L1 protein were generated, and MVPs can selectively bind to target cells expressing its cognate receptor PD-1.

[0190] Inhibition of ARDS by PD-L1-MVP in mice

[0191] To test the inhibitory checkpoint function of PD-L1-MVP, we used acute respiratory distress syndrome (ARDS) as an inflammation model to investigate whether the use of PDL1-MVP could control and reduce the damage caused by such systemic inflammation. Excessive proinflammatory responses leading to ARDS can be initiated and induced by Toll-like receptors (TLRs) that recognize pathogen-derived components such as lipopolysaccharide (LPS), bacterial lipoproteins, and unmethylated CpG DNA, resulting in a rapid increase in systemic immune responses. Such a condition can be partially reproduced for a mouse model of LPS-induced systemic inflammation. Mice were challenged with a lethal intraperitoneal injection of LPS (6 mg / kg) and treated with intranasal delivery of IC-MVP. Mice were first treated 16 hours after LPS challenge and then treated daily with intranasal delivery of PD-L1-MVP (Figure 16A). In this lethal model, untreated mice reached the experimental endpoint within 72 hours (Figure 16B). If IC-MVP treatment could rescue mice from lethality, it would demonstrate that IC-MVP could effectively attenuate LPS-induced systemic inflammation. Indeed, PD-L1-MVP treatment rescued 3 out of 5 mice from lethality (Figure 16B), a rescue rate of 60% was observed, demonstrating that PD-L1-MVP could effectively inhibit LPS-induced systemic immune responses.

[0192] Generation and characterization of 2B4-MVP

[0193] 2B4-MVP was generated by pseudotyping lentiviral VLPs with trimeric PD-L1 fusion peptides. Specifically, HEK293T cells were co-transfected with the trimeric 2B4 display construct along with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). The concentration of purified 2B4-MVP was quantified by P24 ELISA. PD-L1-MVP displayed 1300±300 copies of 2B4 per particle in various multimerized forms as determined by quantitative Western blot analysis (Figure 17A). Thus, the D4 display construct can effectively present thousands of copies of 2B4 in multimerized forms on MVP.

[0194] To confirm that 2B4-MVP exhibited functional 2B4, we tested whether 2B4-MVP could selectively bind to target cells expressing its cognate receptor, CD48 (Figure 17B). First, CD48-transfected cells were incubated with unlabeled 2B4-MVP to allow MVP to bind to target cells. The cell-MVP mixture was then co-stained with fluorescently labeled anti-CD48 and anti-2B4 antibodies. The 2B4 staining patterns on CD48+ and CD48- cells were then examined by FACS analysis. A significant fraction of CD48 cells were also 2B4 positive, as illustrated by a 1 log 2B4 staining shift in CD48+ cells from CD48- background cells (Figure 17B). These results confirmed that 2B4-MVP exhibited functional 2B4. Collectively, 2B4-MVPs were generated that display high copy numbers of functional 2B4 protein, which can selectively bind to target cells expressing its cognate receptor, CD48.

[0195] Inhibition of ARDS by 2B4-MVP in mice

[0196] To test the inhibitory checkpoint function of 2B4-MVP, we used acute respiratory distress syndrome (ARDS) as a model of systemic inflammation to determine whether 2B4-MVP can be used to control and reduce the damage caused by such systemic inflammation. Mice were challenged with a lethal dose of LPS (6 mg / kg) by intraperitoneal injection and treated with intranasal delivery of 2B4-MVP. Mice were first treated 16 hours after LPS challenge and then treated daily with intranasal delivery of 2B4-MVP (Figure 18A). In this lethal model, untreated mice reached the experimental endpoint within 96 hours (Figure 18B). If 2B4-MVP treatment can rescue mice from lethality due to ARDS, it would demonstrate that 2B4-MVP can effectively attenuate systemic inflammation induced by LPS. Indeed, the results showed that 2B4-MVP treatment rescued 3 of 5 mice from lethality (FIG. 18B), a rescue rate of 60%, demonstrating that 2B4-MVP can effectively inhibit LPS-induced systemic immune responses.

[0197] Example 5. IC-MVPs exhibiting various inhibited immune checkpoints A list of IC-MVPs that exhibit various inhibitory immune checkpoints was created, and their composition was characterized by determining the copies of immune checkpoint molecules that are displayed on each of the VLPs. This example also demonstrates that IC-MVPs specifically bind to their target cells that express their cognate ligands or receptors. The list of IC-MVPs includes PDL2-MVP, CTLA4-MVP, CD80-MVP, CD86-MVP, Galectin3-MVP, LAG3-MVP, FGL1-MVP, HVEM-MVP, BTLA-MVP, CD160-MVP, CD48-MVP, CD112-MVP, TIGIT-MVP, CD155-MVP, TIM3-MVP, and Ceacam1-MVP.

[0198] PD-L2-MVP Compositions and Selective Binding to Target Cells Expressing PD-1

[0199] PDL2-MVPs were generated by pseudotyping lentiviral VLPs with trimeric PD-L2 fusion peptides. Specifically, HEK293T cells were co-transfected with the trimeric PD-L2 display construct along with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). The concentration of purified PDL2-MVPs was quantified by P24 ELISA. PDL2-MVPs displayed 2100±500 copies of PD-L2 per MVP in multimerized form as determined by quantitative Western blot analysis (Figure 19A). Thus, the D4 display construct (Figure 1B) can effectively present thousands of copies of PD-L2 in multimerized form on MVPs.

[0200] To confirm that PD-L2-MVP represents functional PD-L2, we tested whether PDL2-MVP could selectively bind to target cells expressing its cognate receptor, PD-1. First, a target cell line was established by transfecting S293 cells with a construct expressing PD-1. Next, PD-1+ and PD-1- cells were distinguished by staining the transfected cells with an anti-PD-1 antibody. PD-L2-MVP was then labeled with a fluorescent dye, the transfected cells were stained with the labeled MVP, and selective MVP-cell binding was analyzed by FACS (Figure 19B). The results showed that labeled PD-L2-MVP binding resulted in a fluorescence shift that was significantly higher in PD-1+ cells than in PD-1- cells (Figure 19B, upper panel). Moreover, this shift was at least 1 log higher than the fluorescence shift caused by staining the same cells with a control MVP representing a nonspecific ligand (Figure 19B, lower panel). These results demonstrated that PD-L2-MVP exhibits functional PD-L2 and can selectively bind to PD-1 on target cells.

[0201] This result was further verified by an alternative staining method (Figure 19C). In this case, PD-1 transfected cells were first incubated with unlabeled PD-L2-MVP to allow MVP to bind to target cells. The cell-MVP mixture was then co-stained with fluorescently labeled anti-PD-1 and anti-PD-L2 antibodies. The PD-L2 staining pattern was then examined by FACS analysis on PD-1+ and PD-1- cells. We observed that PD-1+ cells were also PD-L2 positive, as illustrated by a 2 log PD-L2 staining shift in PD-1+ cells from PD-1- background cells (Figure 19C). Single staining with anti-PD-1 antibody did not compete with PDL2-MVP binding to target cells, and PD-1 transfected S293 cells were PD-L2 negative. These results demonstrated that PD-L2-MVP displayed functional PD-L2. Collectively, PDL2-MVPs were generated that represent high copy numbers of the functional protein, and these MVPs can selectively bind to target cells expressing its cognate receptor, PD-1.

[0202] CTLA4-MVP COMPOSITIONS AND SELECTIVE BINDING TO TARGET CELLS EXPRESSING CD80 / CD86

[0203] CTLA4-MVPs were generated by pseudotyping lentiviral VLPs with trimeric CTLA-4 fusion peptides. Specifically, HEK293T cells were co-transfected with the trimeric CTLA-4 display construct along with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). The concentration of purified CTLA-4-MVPs was quantified by P24 ELISA. CTLA-4-MVPs displayed 290±80 copies of CTLA-4 per MVP in various multimerized forms as determined by quantitative Western blot analysis (Figure 20A). Thus, the D4 display construct (Figure 1B) can effectively present hundreds of copies of CTLA-4 in multimerized forms on MVPs.

[0204] To confirm that CTLA4-MVP represents functional CTLA-4, we tested whether CTLA-4-MVP could selectively bind to target cells expressing its cognate receptor, CD80. First, a target cell line was established by transfecting S293 cells with a construct expressing CD80. The transfected cells were then stained with an anti-CD80 antibody to distinguish between CD80+ and CD80- cells. CTLA4-MVP was then labeled with a fluorescent dye, the transfected cells were stained with the labeled MVP, and selective MVP-cell binding was analyzed by FACS (Figure 20B). The results showed that labeled CTLA-4-MVP binding resulted in a fluorescence shift that was significantly higher in CD80+ cells than in CD80- cells (Figure 20B, upper panel). Moreover, this shift was at least 1 log higher than the fluorescence shift caused by staining the same cells with a control MVP representing a nonspecific ligand (Figure 20B, lower panel). The results demonstrated that CTLA4-MVP represents a functional CTLA4-MVP and can selectively bind to CD80 on target cells.

[0205] This result was further verified by an alternative staining method. In this case, CD80-transfected cells were first incubated with unlabeled CTLA4-MVP to allow binding of MVP to target cells. The cell-MVP mixture was then co-stained with fluorescently labeled anti-CD80 and anti-CTLA-4 antibodies. The CTLA-4 staining pattern was then examined by FACS analysis on CD80+ and CD80- cells. We observed that CD80+ cells were also CTLA4 positive, as illustrated by a 2-log CTLA-4 staining shift in CD80+ cells from CD80- background cells (Figure 20C). Single staining with anti-CD80 antibody did not compete with CTLA4-MVP binding to target cells, and CD80-transfected S293 cells were CTLA4 negative. These results demonstrated that CTLA4-MVP displayed functional CTLA-4.

[0206] We also tested whether CTLA4-MVP could selectively bind to target cells expressing CD86, another cognate receptor for CTLA-4. First, a target cell line was established by transfecting S293 cells with a construct expressing CD86. The transfected cells were then stained with an anti-CD86 antibody to distinguish between CD86+ and CD86- cells. CTLA-4-MVP was then labeled with a fluorescent dye, the transfected cells were stained with the labeled MVP, and the selective MVP-cell binding was analyzed by FACS (Figure 20D). The results showed that the labeled CTLA-4-MVP binding resulted in a fluorescence shift that was significantly higher in CD86+ cells than in CD86- cells (Figure 20D, upper panel). Moreover, this shift was at least 1 log higher than the fluorescence shift caused by staining the same cells with a control MVP representing a nonspecific ligand (Figure 20D, lower panel). These results demonstrated that CTLA4-MVP represents a functional CTLA4-MVP and can selectively bind to CD86 on target cells.

[0207] This result was further verified by an alternative staining method. In this case, CD86-transfected cells were first incubated with unlabeled CTLA4-MVP to allow binding of MVP to target cells. The cell-MVP mixture was then co-stained with fluorescently labeled anti-CD86 and anti-CTLA-4 antibodies. The CTLA-4 staining patterns on CD86+ and CD86- cells were then examined by FACS analysis. The results confirmed that CD86+ cells were also CTLA-4 positive, as illustrated by a 2-log CTLA-4 staining shift in CD86+ cells from CD86- background cells (Figure 20E). Single staining with anti-CD86 antibody did not compete with CTLA-4-MVP binding to target cells, and CD86-transfected S293 cells were CTLA-4 negative. These results demonstrated that CTLA4-MVP displayed functional CTLA-4. Collectively, CTLA4-MVPs are generated that represent high copy numbers of functional proteins, and these MVPs are capable of selectively binding to target cells expressing its cognate receptors, CD80 or CD86.

[0208] CD80-MVP COMPOSITIONS AND SELECTIVE BINDING TO TARGET CELLS EXPRESSING CTLA-4

[0209] CD80-MVPs were generated by pseudotyping lentiviral VLPs with a trimeric CD80 fusion peptide. Specifically, HEK293T cells were co-transfected with the trimeric CD80 display construct along with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). The concentration of purified CD80-MVPs was quantified by P24 ELISA. CD80-MVPs displayed 2600±800 copies of CD80 per MVP in various multimerized forms as determined by quantitative Western blot analysis (Figure 21A). Thus, the D4 display construct (Figure 1B) can effectively present hundreds of copies of CD80 in multimerized forms on MVPs.

[0210] To confirm that CD80-MVPs exhibit functional CD80, we tested whether CD80-MVPs could selectively bind to target cells expressing CTLA-4, the cognate receptor for CD80. First, a target cell line was established by transfecting S293 cells with a construct expressing CTLA-4. The transfected cells were then stained with an anti-CTLA-4 antibody to distinguish between CTLA-4+ and CTLA-4- cells. CD80-MVPs were then labeled with a fluorescent dye, the transfected cells were stained with the labeled MVP, and the selective MVP-cell binding was analyzed by FACS (Figure 21B). The results showed that the labeled CD80-MVP binding resulted in a significantly higher fluorescence shift in CTLA-4+ cells than in CTLA-4- cells (Figure 21B, upper panel). Moreover, this shift was at least 1 log higher than the fluorescence shift produced by staining the same cells with a control MVP representing a nonspecific ligand (Figure 21B, lower panel), demonstrating that CD80-MVPs represent functional CD80 and can selectively bind CTLA-4 on target cells.

[0211] This result was further verified by an alternative staining method (Figure 21C). In this case, CTLA-4 transfected cells were first incubated with unlabeled CD80-MVP to allow binding of MVP to target cells. The cell-MVP mixture was then co-stained with fluorescently labeled anti-CTLA-4 and anti-CD80 antibodies. CD80 staining patterns were then examined by FACS analysis on CTLA-4+ and CTLA-4- cells. The results confirmed that CTLA-4+ cells were also CD80 positive, as illustrated by a 0.5 log CD80 staining shift in CTLA-4+ cells from CTLA-4- background cells (Figure 21C). Single staining with anti-CTLA-4 antibody did not compete with CD80-MVP binding to target cells, and CTLA-4 transfected S293 cells were CD80 negative. These results demonstrated that CD80-MVP displayed functional CD80. Collectively, CD80-MVPs were generated that represent high copy numbers of the functional protein, and these MVPs are capable of selectively binding to target cells expressing its cognate receptor, CTLA-4.

[0212] CD86-MVP COMPOSITIONS AND SELECTIVE BINDING TO TARGET CELLS EXPRESSING CTLA-4

[0213] CD86-MVPs were generated by pseudotyping lentiviral VLPs with a trimeric CD86 fusion peptide. Specifically, HEK293T cells were co-transfected with the trimeric CD86 display construct along with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). The concentration of purified CD86-MVPs was quantified by P24 ELISA. CD86-MVPs displayed multiple copies of CD86 per MVP in various multimerized forms as determined by Western blot analysis (Figure 22A). Thus, the D4 display construct (Figure 1B) can effectively present CD86 in multimerized forms on MVPs.

[0214] To confirm that CD86-MVPs exhibit functional CD86, we tested whether CD86-MVPs could selectively bind to target cells expressing CTLA-4, the cognate receptor for CD86. First, a target cell line was established by transfecting S293 cells with a construct expressing CTLA-4. The transfected cells were then stained with an anti-CTLA-4 antibody to distinguish between CTLA-4+ and CTLA-4- cells. CD86-MVPs were then labeled with a fluorescent dye, the transfected cells were stained with the labeled MVP, and the selective MVP-cell binding was analyzed by FACS (Figure 22B). The results showed that the labeled CD86-MVP binding resulted in a significantly higher fluorescence shift in CTLA-4+ cells than in CTLA-4- cells (Figure 22B, upper panel). Moreover, this shift was at least 1 log higher than the fluorescence shift produced by staining the same cells with a control MVP representing a nonspecific ligand (Figure 22B, lower panel), demonstrating that CD86-MVP represents functional CD86 and can selectively bind CTLA-4 on target cells.

[0215] This result was further verified by an alternative staining method (Figure 22C). In this case, CTLA-4 transfected cells were first incubated with unlabeled CD86-MVP to allow binding of MVP to target cells. The cell-MVP mixture was then co-stained with fluorescently labeled anti-CTLA-4 and anti-CD80 antibodies. The CD86 staining pattern on CTLA-4+ and CTLA-4- cells was then examined by FACS analysis. The results confirmed that CTLA-4+ cells were also CD86 positive, as illustrated by a 1 log CD86 staining shift in CTLA-4+ cells from CTLA-4- background cells (Figure 22C). Single staining with anti-CTLA-4 antibody did not compete with CD86-MVP binding to target cells, and CTLA-4 transfected S293 cells were CD86 negative. These results demonstrated that CD86-MVP displayed functional CD86. Collectively, CD86-MVPs were generated that represent high copy numbers of the functional protein, and these MVPs are able to selectively bind to target cells expressing its cognate receptor, CTLA-4.

[0216] Galectin-3-MVP Compositions and Selective Binding to Target Cells Expressing LAG-3

[0217] Galectin-3-MVPs were generated by pseudotyping lentiviral VLPs with trimeric Galectin-3 fusion peptides. Specifically, HEK293T cells were co-transfected with the trimeric Galectin-3 display construct along with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). The concentration of purified Galectin-3-MVPs was quantified by P24 ELISA. Galectin-3-MVPs displayed 630±260 copies of Galectin-3 per MVP in various multimerized forms as determined by quantitative Western blot analysis (Figure 23A). Thus, the D4 display construct (Figure 1B) can effectively display hundreds of copies of Galectin-3 in multimerized forms on MVPs.

[0218] To confirm that Galectin-3-MVP represents functional Galectin-3, we tested whether Galectin-3-MVP could selectively bind to target cells expressing LAG-3, the cognate receptor for Galectin-3. First, a target cell line was established by transfecting S293 cells with a construct expressing LAG-3. The transfected cells were then stained with an anti-LAG-3 antibody to distinguish LAG-3+ and LAG-3- cells. Galectin-3-MVP was then labeled with a fluorescent dye, the transfected cells were stained with the labeled MVP, and the selective MVP-cell binding was analyzed by FACS (Figure 23B). The results showed that the fluorescence shift upon labeled Galectin-3-MVP binding was significantly higher in LAG-3+ cells than in LAG-3- cells (Figure 23B, upper panel). Moreover, this shift was at least 1 log higher than the fluorescence shift produced by staining the same cells with a control MVP representing a nonspecific ligand (Figure 23B, lower panel), demonstrating that Galectin-3-MVP represents functional Galectin-3 and can selectively bind to LAG-3 on target cells.

[0219] This result was further verified by an alternative staining method (Figure 23C). In this case, LAG-3 transfected cells were first incubated with unlabeled Galectin-3-MVP to allow MVP to bind to target cells. The cell-MVP mixture was then co-stained with fluorescently labeled anti-LAG-3 and anti-Galectin-3 antibodies. Galectin-3 staining patterns were then examined on LAG-3+ and LAG-3- cells by FACS analysis. The results confirmed that LAG-3+ cells were also Galectin-3 positive, as illustrated by a 1 log Galectin-3 staining shift in LAG-3+ cells from LAG-3- background cells (Figure 23C). Single staining with anti-LAG-3 antibody did not compete with Galectin-3-MVP binding to target cells, and LAG-3 transfected S293 cells were Galectin-3 negative. These results demonstrated that Galectin-3-MVPs represented functional Galectin-3. Collectively, Galectin-3-MVPs were generated that represented high copy numbers of functional protein, and these MVPs could selectively bind to target cells expressing the Galectin-3 cognate receptor LAG-3.

[0220] LAG3-MVP Composition and Selective Binding to Galectin-3-Expressing Target Cells

[0221] LAG3-MVPs were generated by pseudotyping lentiviral VLPs with trimeric LAG-3 fusion peptides. Specifically, HEK293T cells were co-transfected with the trimeric LAG-3 display construct along with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). The concentration of purified LAG3-MVPs was quantified by P24 ELISA. LAG-3-MVPs displayed 920±250 copies of LAG-3 per MVP in various multimerized forms as determined by quantitative Western blot analysis (Figure 24A). Thus, the D4 display construct (Figure 1B) can effectively present hundreds of copies of LAG-3 in multimerized forms on MVPs.

[0222] To confirm that LAG3-MVP represents functional LAG-3, we tested whether LAG3-MVP could selectively bind to target cells expressing Galectin-3, the cognate receptor for LAG-3. First, a target cell line was established by transfecting S293 cells with a construct expressing Galectin-3. The transfected cells were then stained with an anti-Galectin-3 antibody to distinguish Galectin-3+ cells from Galectin-3- cells. LAG3-MVP was then labeled with a fluorescent dye, the transfected cells were stained with the labeled MVP, and the selective MVP-cell binding was analyzed by FACS (Figure 24B). The results showed that the labeled LAG3-MVP binding resulted in a significantly higher fluorescence shift in Galectin-3+ cells than in Galectin-3- cells (Figure 24B, upper panel). Moreover, this shift was at least 1 log higher than the fluorescence shift produced by staining the same cells with a control MVP representing a nonspecific ligand (Fig. 24B, lower panel), demonstrating that LAG3-MVP represents functional LAG-3 and can selectively bind to Galectin-3 on target cells.

[0223] This result was further verified by an alternative staining method (Figure 24C). In this case, Galectin-3 transfected cells were first incubated with unlabeled LAG-3-MVP to allow MVP to bind to target cells. The cell-MVP mixture was then co-stained with fluorescently labeled anti-Galectin-3 and anti-LAG-3 antibodies. The LAG-3 staining patterns on Galectin-3+ and Galectin-3- cells were then examined by FACS analysis. The results confirmed that Galectin-3+ cells were also LAG-3 positive, as illustrated by a 3 log LAG-3 staining shift in Galectin-3+ cells from Galectin-3- background cells (Figure 24C). Single staining with anti-LAG-3 antibody did not compete with LAG3-MVP binding to target cells, and Galectin-3 transfected S293 cells were LAG-3 negative. These results demonstrated that LAG3-MVP displayed functional LAG-3. Collectively, LAG3-MVPs were generated that represent high copy numbers of functional proteins, and these MVPs can selectively bind to target cells expressing its cognate receptor, Galectin-3.

[0224] FGL1-MVP Composition and Selective Binding to Target Cells Expressing LAG-3

[0225] FGL-1-MVP was generated by pseudotyping lentiviral VLPs with trimeric FGL-1 fusion peptide. Specifically, HEK293T cells were co-transfected with the trimeric FGL-1 display construct along with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). The concentration of purified FGL-1-MVP was quantified by P24 ELISA. FGL-1-MVP displayed 1100±600 copies of FGL-1 per MVP in various multimerized forms as determined by quantitative Western blot analysis (Figure 25A). Thus, the D4 display construct (Figure 1B) can effectively display hundreds of copies of FGL-1 in multimerized forms on MVP.

[0226] To confirm that FGL1-3-MVP represents functional FGL-1, we tested whether FGL1-MVP could selectively bind to target cells expressing LAG-3, the cognate receptor for FGL-1. First, a target cell line was established by transfecting S293 cells with a construct expressing LAG-3. The transfected cells were then stained with anti-LAG-3 antibody to distinguish LAG-3+ cells from LAG-3- cells. FGL1-MVP was then labeled with a fluorescent dye, the transfected cells were stained with the labeled MVP, and the selective MVP-cell binding was analyzed by FACS (Figure 25B). The results showed that the labeled FGL1-MVP binding caused a significantly higher fluorescence shift in LAG-3+ cells than in LAG-3- cells (Figure 25B, upper panel). Moreover, this shift was at least 1 log higher than the fluorescence shift produced by staining the same cells with control MVP representing a nonspecific ligand (FIG. 25B, lower panel), demonstrating that FGL-1-MVP represents functional FGL-1 and can selectively bind to LAG-3 on target cells.

[0227] This result was further verified by an alternative staining method (Figure 25C). In this case, LAG-3 transfected cells were first incubated with unlabeled FGL1-MVP to allow MVP to bind to target cells. The cell-MVP mixture was then co-stained with fluorescently labeled anti-LAG-3 and anti-FGL-1 antibodies. The FGL-1 staining pattern was then examined by FACS analysis on LAG-3+ and LAG-3- cells. The results confirmed that LAG-3+ cells were also FGL-1 positive, as illustrated by a 0.5 log FGL-1 staining shift in LAG-3+ cells from LAG-3- background cells (Figure 25C). Single staining with anti-LAG-3 antibody did not compete with FGL-1-MVP binding to target cells, and LAG-3 transfected S293 cells were FGL-1 negative. These results demonstrated that FGL1-MVP exhibited functional FGL-1. Collectively, FGL1-MVPs were generated that represent high copy numbers of functional proteins, and these MVPs can selectively bind to target cells expressing its cognate receptor, LAG-3.

[0228] LAG3-MVP Compositions and Selective Binding to Target Cells Expressing FGL-1

[0229] LAG3-MVPs were generated by pseudotyping lentiviral VLPs with trimeric LAG-3 fusion peptides. Specifically, HEK293T cells were co-transfected with the trimeric LAG-3 display construct along with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). The concentration of purified LAG-3-MVPs was quantified by P24 ELISA. LAG-3-MVPs displayed 920±250 copies of LAG-3 per MVP in various multimerized forms as determined by quantitative Western blot analysis (Figure 26A). Thus, the D4 display construct (Figure 1B) can effectively present hundreds of copies of LAG-3 in multimerized forms on MVPs.

[0230] To confirm that LAG3-MVP represents functional LAG-3, we tested whether LAG-3-MVP could selectively bind to target cells expressing FGL-1, the cognate receptor for LAG-3. First, a target cell line was established by transfecting S293 cells with a construct expressing FGL-1. Then, FGL-1+ and FGL-1- cells were distinguished by staining the transfected cells with anti-FGL-1 antibody. LAG-3-MVP was then labeled with a fluorescent dye, the transfected cells were stained with the labeled MVP, and the selective MVP-cell binding was analyzed by FACS (Figure 26B). The results showed that the labeled LAG3-MVP binding resulted in a slightly higher fluorescence shift in FGL-1+ cells than in FGL-1- cells (Figure 26B, upper panel). Moreover, this shift was significantly higher than the fluorescence shift produced by staining the same cells with a control MVP representing a nonspecific ligand (Figure 26B, lower panel), demonstrating that LAG3-MVP represents functional LAG-3 and can selectively bind to FGL-1 on target cells.

[0231] This result was further verified by an alternative staining method (Figure 26C). In this case, FGL-1 transfected cells were first incubated with unlabeled LAG-3-MVP to allow binding of MVP to target cells. The cell-MVP mixture was then co-stained with fluorescently labeled anti-FGL-1 and anti-LAG-3 antibodies. The LAG-3 staining pattern was then examined by FACS analysis on FGL-1+ and FGL-1- cells. The results confirmed that FGL-1+ cells were also LAG-3 positive, as illustrated by a 0.5 log LAG-3 staining shift in FGL-1+ cells from FGL-1- background cells (Figure 26C). Single staining with anti-FGL-1 antibody did not compete with LAG3-MVP binding to target cells, and FGL-1 transfected S293 cells were LAG-3 negative. These results demonstrated that LAG3-MVP displayed functional LAG-3. Collectively, LAG3-MVPs were generated that represent high copy numbers of functional proteins, and these MVPs can selectively bind to target cells expressing its cognate receptor, FGL-1.

[0232] HVEM-MVP COMPOSITIONS AND SELECTIVE BINDING TO TARGET CELLS EXPRESSING BTLA

[0233] HVEM-MVPs were generated by pseudotyping lentiviral VLPs with trimeric HVEM fusion peptides. Specifically, HEK293T cells were co-transfected with the trimeric HVEM display construct along with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). The concentration of purified HVEM-MVPs was quantified by P24 ELISA. HVEM-MVPs displayed 7200 copies of HVEM per MVP in various multimerized forms as determined by quantitative Western blot analysis (Figure 27A). Thus, the D4 display construct (Figure 1B) can effectively present hundreds of copies of HVEM in multimerized forms on MVPs.

[0234] To confirm that HVEM-MVP represents functional HVEM, we tested whether HVEM-MVP could selectively bind to target cells expressing BTLA, the cognate receptor for HVEM. First, a target cell line was established by transfecting S293 cells with a construct expressing BTLA. BTLA-transfected cells were first incubated with unlabeled HVEM-MVP to allow MVP to bind to target cells. The cell-MVP mixture was then co-stained with fluorescently labeled anti-BTLA and anti-HVEM antibodies. HVEM staining patterns were then examined by FACS analysis on BTLA+ and BTLA- cells. The results showed that BTLA+ cells were also HVEM positive, as illustrated by a 0.5 log HVEM staining shift in BTLA+ cells from BTLA- background cells (Figure 27B). Single staining with anti-BTLA antibody did not compete with HVEM-MVP binding to target cells, and S293 cells transfected with BTLA were HVEM-negative. These results demonstrated that HVEM-MVPs represented functional HVEM. Collectively, HVEM-MVPs representing high copy numbers of functional protein were generated, and these MVPs can selectively bind to target cells expressing its cognate receptor, BTLA.

[0235] BTLA-MVP COMPOSITIONS AND SELECTIVE BINDING TO TARGET CELLS EXPRESSING HVEM

[0236] BTLA-MVPs were generated by pseudotyping lentiviral VLPs with trimeric BTLA fusion peptides. Specifically, HEK293T cells were co-transfected with the trimeric BTLA display construct along with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). The concentration of purified BTLA-MVPs was quantified by P24 ELISA. BTLA-MVPs displayed 860±140 copies of BTLA per MVP in various multimerized forms as determined by quantitative Western blot analysis (Figure 28A). Thus, the D4 display construct (Figure 1B) can effectively display hundreds of copies of BTLA in multimerized forms on MVPs.

[0237] To confirm that BTLA-MVP represents functional BTLA, we tested whether BTLA-MVP could selectively bind to target cells expressing HVEM, the cognate receptor for BTLA. First, a target cell line was established by transfecting S293 cells with a construct expressing HVEM. The transfected cells were then stained with an anti-HVEM antibody to distinguish HVEM+ and HVEM- cells. BTLA-MVP was then labeled with a fluorescent dye, the transfected cells were stained with the labeled MVP, and selective MVP-cell binding was analyzed by FACS (Figure 28B). The results showed that labeled BTLA-MVP binding resulted in a fluorescence shift that was significantly higher in GITR+ cells than in HVEM- cells (Figure 28B, upper panel). Moreover, this shift was at least 1 log higher than the fluorescence shift caused by staining the same cells with a control MVP representing a nonspecific ligand (Figure 28B, lower panel). These results demonstrated that BTLA-MVP exhibits functional BTLA and can selectively bind to HVEM on target cells.

[0238] This result was further verified by an alternative staining method (Figure 28C). In this case, HVEM-transfected cells were first incubated with unlabeled BTLA-MVP to allow binding of MVP to target cells. The cell-MVP mixture was then co-stained with fluorescently labeled anti-HVEM and anti-BTLA antibodies. The BTLA staining pattern was then examined by FACS analysis on HVEM+ and HVEM- cells. The results confirmed that HVEM+ cells were also BTLA positive, as illustrated by a 1 log BTLA staining shift in HVEM+ cells from HVEM- background cells (Figure 28C). Single staining with anti-HVEM antibody did not compete with BTLA-MVP binding to target cells, and HVEM-transfected S293 cells were BTLA negative. These results demonstrated that BTLA-MVP exhibited functional BTLA. Collectively, BTLA-MVPs are generated that represent high copy numbers of functional proteins, and these MVPs can selectively bind to target cells expressing its cognate receptor, HVEM.

[0239] CD160-MVP COMPOSITIONS AND SELECTIVE BINDING TO TARGET CELLS EXPRESSING HVEM

[0240] CD160-MVPs were generated by pseudotyping lentiviral VLPs with trimeric CD160 fusion peptides. Specifically, HEK293T cells were co-transfected with the trimeric CD160 display construct along with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). The concentration of purified CD160-MVPs was quantified by P24 ELISA. CD160-MVPs displayed 2400±1000 copies of CD160 per MVP in various multimerized forms as determined by quantitative Western blot analysis (Figure 29A). Thus, the D4 display construct (Figure 1B) can effectively present hundreds of copies of CD160 in multimerized forms on MVPs.

[0241] To confirm that CD160-MVPs exhibit functional CD160, we tested whether CD160-MVPs could selectively bind to target cells expressing HVEM, the cognate receptor for CD160. First, a target cell line was established by transfecting S293 cells with a construct expressing HVEM. The transfected cells were then stained with anti-HVEM antibodies to distinguish HVEM+ and HVEM- cells. CD160-MVPs were then labeled with a fluorescent dye, the transfected cells were stained with the labeled MVP, and the selective MVP-cell binding was analyzed by FACS (Figure 29B). The results showed that the labeled CD160-MVP binding resulted in a significantly higher fluorescence shift in HVEM+ cells than in HVEM- cells (Figure 29B, upper panel). Moreover, this shift was at least 1 log higher than the fluorescence shift produced by staining the same cells with a control MVP representing a nonspecific ligand (Figure 29B, lower panel), demonstrating that CD160-MVP represents functional CD160 and can selectively bind to HVEM on target cells.

[0242] This result was further verified by an alternative staining method (Figure 29C). In this case, HVEM-transfected cells were first incubated with unlabeled CD160-MVP to allow binding of MVP to target cells. The cell-MVP mixture was then co-stained with fluorescently labeled anti-HVEM and anti-CD160 antibodies. The CD160 staining pattern was then examined by FACS analysis on HVEM+ and HVEM- cells. The results confirmed that HVEM+ cells were also CD160 positive, as illustrated by a 0.5 log CD160 staining shift in HVEM+ cells from HVEM- background cells (Figure 29C). Single staining with anti-HVEM antibodies did not compete with CD160-MVP binding to target cells, and HVEM-transfected S293 cells were CD160 negative. These results demonstrated that CD160-MVP displayed functional CD160. Collectively, CD160-MVPs were generated that represent high copy numbers of the functional protein, and these MVPs can selectively bind to target cells expressing its cognate receptor, HVEM.

[0243] CD48-MVP COMPOSITIONS AND SELECTIVE BINDING TO TARGET CELLS EXPRESSING 2B4

[0244] CD48-MVPs were generated by pseudotyping lentiviral VLPs with a trimeric CD48 fusion peptide. Specifically, HEK293T cells were co-transfected with the trimeric CD48 display construct along with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). The concentration of purified CD48-MVPs was quantified by P24 ELISA. CD48-MVPs displayed 600±400 copies of CD48 per MVP in various multimerized forms as determined by quantitative Western blot analysis (Figure 30A). Thus, the D4 display construct (Figure 1B) can effectively present hundreds of copies of CD48 in multimerized forms on MVPs.

[0245] To confirm that CD48-MVPs represent functional CD48, we tested whether they could selectively bind to target cells expressing 2B4, the cognate receptor for CD48. First, a target cell line was established by transfecting S293 cells with a construct expressing 2B4. The transfected cells were then stained with anti-2B4 antibody to distinguish between 2B4+ and 2B4- cells. CD48-MVPs were then labeled with a fluorescent dye, the transfected cells were stained with the labeled MVP, and selective MVP-cell binding was analyzed by FACS (Figure 30B). The results showed that labeled CD48-MVP binding resulted in a fluorescence shift that was significantly higher in 2B4+ cells than in 2B4- cells (Figure 30B, upper panel). Moreover, this shift was at least 1 log higher than the fluorescence shift caused by staining the same cells with a control MVP representing a nonspecific ligand (Figure 30B, lower panel). These results demonstrated that CD48-MVPs exhibited functional CD48 and could selectively bind to 2B4 on target cells.

[0246] This result was further verified by an alternative staining method (Figure 30C). In this case, 2B4-transfected cells were first incubated with unlabeled CD48-MVP to allow binding of MVP to target cells. The cell-MVP mixture was then co-stained with fluorescently labeled anti-2B4 and anti-CD48 antibodies. CD48 staining patterns were then examined by FACS analysis on 2B4+ and 2B4- cells. The results confirmed that 2B4+ cells were also CD48 positive, as illustrated by a 0.5 log CD48 staining shift in 2B4+ cells from 2B4- background cells (Figure 30C). Single staining with anti-2B4 antibody did not compete with CD48-MVP binding to target cells, and 2B4-transfected S293 cells were CD48 negative. These results demonstrated that CD48-MVP displayed functional CD48. Collectively, CD48-MVPs are generated that represent high copy numbers of the functional protein, and these MVPs are able to selectively bind to target cells expressing its cognate receptor, 2B4.

[0247] CD112-MVP Compositions and Selective Binding to Target Cells Expressing TIGHT

[0248] CD112-MVPs were generated by pseudotyping lentiviral VLPs with a trimeric CD112 fusion peptide. Specifically, HEK293T cells were co-transfected with the trimeric CD112 display construct along with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). The concentration of purified CD112-MVPs was quantified by P24 ELISA. CD112-MVPs displayed 220±90 copies of CD112 per MVP in various multimerized forms as determined by quantitative Western blot analysis (Figure 31A). Thus, the D4 display construct (Figure 1B) can effectively present hundreds of copies of CD112 in multimerized forms on MVPs.

[0249] To confirm that CD112-MVP represents functional CD112, we tested whether CD112-MVP could selectively bind to target cells expressing TIGHT, the cognate receptor for CD112. First, a target cell line was established by transfecting S293 cells with a construct expressing TIGHT. The transfected cells were then stained with anti-TIGHT antibody to distinguish TIGHT+ and TIGHT- cells. CD112-MVP was then labeled with a fluorescent dye, the transfected cells were stained with the labeled MVP, and selective MVP-cell binding was analyzed by FACS (Figure 31B). The results showed that labeled CD112-MVP binding resulted in a fluorescence shift that was significantly higher in TIGHT+ cells than in TIGHT- cells (Figure 31B, upper panel). Moreover, this shift was at least 1 log higher than the fluorescence shift caused by staining the same cells with a control MVP representing a nonspecific ligand (Figure 31B, lower panel). These results demonstrate that CD112-MVPs represent functional CD112 and can selectively bind to TIGHT on target cells. Collectively, CD112-MVPs representing high copy numbers of the functional protein have been generated, and these MVPs can selectively bind to target cells expressing its cognate receptor, TIGHT.

[0250] TIGHT-MVP Compositions and Selective Binding to Target Cells Expressing CD112

[0251] TIGHT-MVPs were generated by pseudotyping lentiviral VLPs with trimeric TIGHT fusion peptides. Specifically, HEK293T cells were co-transfected with the trimeric TIGHT display construct along with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). The concentration of purified TIGHT-MVPs was quantified by P24 ELISA. TIGHT-MVPs displayed 2300±600 copies of TIGHT per MVP in various multimerized forms as determined by quantitative Western blot analysis (Figure 32A). Thus, the D4 display construct (Figure 1B) can effectively present hundreds of copies of TIGHT in multimerized forms on MVPs.

[0252] To confirm that TIGHT-MVPs exhibit functional TIGHT, we tested whether they could selectively bind to target cells expressing CD112, the cognate receptor for TIGHT. First, a target cell line was established by transfecting S293 cells with a construct expressing CD112. The transfected cells were then stained with an anti-CD155 antibody to distinguish between CD112+ and CD112- cells. TIGHT-MVPs were then labeled with a fluorescent dye, the transfected cells were stained with the labeled MVP, and selective MVP-cell binding was analyzed by FACS. The results showed that labeled TIGHT-MVP binding resulted in a fluorescence shift that was significantly higher in CD112+ cells than in CD112- cells (Figure 32B, upper panel). Moreover, this shift was at least three-fold higher than the fluorescence shift caused by staining the same cells with a control MVP representing a nonspecific ligand (Figure 32B, lower panel). These results demonstrated that TIGHT-MVP exhibited functional TIGHT and could selectively bind to CD112 on target cells.

[0253] This result was further verified by an alternative staining method (Figure 32C). In this case, CD112-transfected cells were first incubated with unlabeled TIGHT-MVP to allow binding of MVP to target cells. The cell-MVP mixture was then co-stained with fluorescently labeled anti-CD112 and anti-TIGHT antibodies. The TIGHT staining patterns on CD112+ and CD112- cells were then examined by FACS analysis. The results confirmed that CD112+ cells were also TIGHT positive, as illustrated by a 2 log TIGHT staining shift in CD112+ cells from CD112- background cells (Figure 32C). Single staining with anti-CD112 antibody did not compete with TIGHT-MVP binding to target cells, and CD112-transfected S293 cells were TIGHT negative. These results demonstrated that TIGHT-MVP exhibited functional TIGHT. Collectively, TIGHT-MVPs were generated that represent high copy numbers of a functional protein, and these MVPs can selectively bind to target cells expressing its cognate receptor, CD112.

[0254] CD155-MVP Compositions and Selective Binding to Target Cells Expressing TIGHT

[0255] CD155-MVPs were generated by pseudotyping lentiviral VLPs with a trimeric CD155 fusion peptide. Specifically, HEK293T cells were co-transfected with the trimeric CD155 display construct along with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). The concentration of purified CD155-MVPs was quantified by P24 ELISA. CD155-MVPs exhibited 3300±400 copies of TIGHT per MVP in various multimerized forms as determined by quantitative Western blot analysis (Figure 33A). Thus, the D4 display construct (Figure 1B) can effectively present thousands of copies of CD155 in multimerized forms on MVPs.

[0256] To confirm that CD155-MVPs exhibit functional CD155, we tested whether CD155-MVPs could selectively bind to target cells expressing TIGHT, the cognate receptor for CD155. First, a target cell line was established by transfecting S293 cells with a construct expressing TIGHT. Then, transfected cells were stained with an anti-CD155 antibody to distinguish CD155+ and CD155- cells. CD155-MVPs were then labeled with a fluorescent dye, transfected cells were stained with the labeled MVP, and selective MVP-cell binding was analyzed by FACS (Figure 33B). The results showed that labeled CD155-MVP binding resulted in a significantly higher fluorescence shift in TIGHT+ cells than in TIGHT- cells (Figure 33B, upper panel). Moreover, this shift was at least 3-fold higher than the fluorescence shift produced by staining the same cells with a control MVP representing a nonspecific ligand (FIG. 33B, lower panel), demonstrating that CD155-MVP represents functional CD155 and can selectively bind TIGHT on target cells.

[0257] This result was further verified by an alternative staining method (Figure 33C). In this case, TIGHT-transfected cells were first incubated with unlabeled CD155-MVP to allow binding of MVP to target cells. The cell-MVP mixture was then co-stained with fluorescently labeled anti-CD155 and anti-TIGHT antibodies. The CD155 staining patterns on TIGHT+ and TIGHT- cells were then examined by FACS analysis. The results confirmed that TIGHT+ cells were also CD155 positive, as illustrated by a 2-log CD155 staining shift in TIGHT+ cells from TIGHT- background cells (Figure 33C). Single staining with anti-TIGHT antibody did not compete with CD155-MVP binding to target cells, and TIGHT-transfected S293 cells were CD155 negative. These results demonstrated that CD155-MVP displayed functional CD155. Collectively, CD155-MVPs were generated that represent high copy numbers of the functional protein, and these MVPs can selectively bind to target cells expressing its cognate receptor, TIGHT.

[0258] TIGHT-MVP Compositions and Selective Binding to Target Cells Expressing CD155

[0259] TIGHT-MVPs were generated by pseudotyping lentiviral VLPs with trimeric TIGHT fusion peptides. Specifically, HEK293T cells were co-transfected with the trimeric TIGHT display construct along with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). The concentration of purified TIGHT-MVPs was quantified by P24 ELISA. TIGHT-MVPs displayed 2300±600 copies of TIGHT per MVP in various multimerized forms as determined by quantitative Western blot analysis (Figure 34A). Thus, the D4 display construct (Figure 1B) can effectively present hundreds of copies of TIGHT in multimerized forms on MVPs.

[0260] To confirm that TIGHT-MVPs exhibit functional TIGHT, we tested whether they could selectively bind to target cells expressing CD155, the cognate receptor for TIGHT. First, a target cell line was established by transfecting S293 cells with a construct expressing CD155. The transfected cells were then stained with an anti-CD155 antibody to distinguish between CD155+ and CD155- cells. TIGHT-MVPs were then labeled with a fluorescent dye, the transfected cells were stained with the labeled MVP, and selective MVP-cell binding was analyzed by FACS (Figure 34B). The results showed that labeled TIGHT-MVP binding resulted in a fluorescence shift that was significantly higher in CD155+ cells than in CD155- cells (Figure 34B, upper panel). Moreover, this shift was at least three-fold higher than the fluorescence shift caused by staining the same cells with a control MVP representing a nonspecific ligand (Figure 34B, lower panel). These results demonstrated that TIGHT-MVP exhibited functional TIGHT and could selectively bind to CD155 on target cells.

[0261] This result was further verified by an alternative staining method (Figure 34C). In this case, CD155-transfected cells were first incubated with unlabeled TIGHT-MVP to allow binding of MVP to target cells. The cell-MVP mixture was then co-stained with fluorescently labeled anti-CD155 and anti-TIGHT antibodies. The TIGHT staining patterns on CD155+ and CD155- cells were then examined by FACS analysis. The results confirmed that CD155+ cells were also TIGHT positive, as illustrated by a 2 log TIGHT staining shift in CD155+ cells from CD155- background cells (Figure 34C). Single staining with anti-CD155 antibody did not compete with TIGHT-MVP binding to target cells, and CD155-transfected S293 cells were TIGHT negative. These results demonstrated that TIGHT-MVP exhibited functional TIGHT. Collectively, TIGHT-MVPs were generated that represent high copy numbers of a functional protein, and these MVPs can selectively bind to target cells expressing its cognate receptor, CD155.

[0262] TIM3-MVP Composition and Selective Binding to Target Cells Expressing Ceacam1

[0263] TIM3-MVPs were generated by pseudotyping lentiviral VLPs with trimeric TIM-3 fusion peptides. Specifically, HEK293T cells were co-transfected with the trimeric TIM-3 display construct along with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). The concentration of purified TIM3-MVPs was quantified by P24 ELISA. TIM3-MVPs displayed 900±500 copies of TIM3 per MVP in various multimerized forms as determined by quantitative Western blot analysis (Figure 35A). Thus, the D4 display construct (Figure 1B) can effectively present hundreds of copies of TIM3 in multimerized forms on MVPs.

[0264] To confirm that TIM3-MVP represents functional TIM3, we tested whether TIM3-MVP could selectively bind to target cells expressing Ceacam1, the cognate receptor for TIM-3. First, a target cell line was established by transfecting S293 cells with a construct expressing Ceacam1. Then, the transfected cells were stained with anti-Ceacam1 antibody to distinguish Ceacam1+ cells from Ceacam1- cells. TIM3-MVP was then labeled with a fluorescent dye, the transfected cells were stained with the labeled MVP, and the selective MVP-cell binding was analyzed by FACS (Figure 35B). The results showed that the labeled TIM3-MVP binding resulted in a significantly higher fluorescence shift in Ceacam1+ cells than in Ceacam1- cells (Figure 35B, upper panel). Moreover, this shift was at least 4-5 times higher than the fluorescence shift produced by staining the same cells with a control MVP representing a nonspecific ligand (Figure 3B, lower panel), demonstrating that TIM3-MVP represents functional TIM-3 and can selectively bind to Ceacam1 on target cells.

[0265] This result was further verified by an alternative staining method (Figure 35C). In this case, Ceacam1-transfected cells were first incubated with unlabeled TIM-3-MVP to allow MVP to bind to target cells. The cell-MVP mixture was then co-stained with fluorescently labeled anti-Ceacam1 and anti-TIM3 antibodies. The TIM-3 staining patterns on Ceacam1+ and Ceacam1- cells were then examined by FACS analysis. The results confirmed that Ceacam1+ cells were also TIM-3 positive, as illustrated by a 2- to 3-fold higher TIM-3 staining shift in Ceacam1+ cells from Ceacam1- background cells (Figure 35C). Single staining with anti-Ceacam1 antibody did not compete with TIM3-MVP binding to target cells, and Ceacam1-transfected S293 cells were TIM-3 negative. These results demonstrated that TIM3-MVP exhibited functional TIM-3. Collectively, TIM3-MVPs were generated that represent high copy numbers of functional protein, and these MVPs can selectively bind to target cells expressing its cognate receptor, Ceacam1.

[0266] Ceacam1-MVP Composition and Selective Binding to Target Cells Expressing TIM-3

[0267] Ceacam1-MVP was generated by pseudotyping lentiviral VLPs with a trimeric Ceacam1 fusion peptide. Specifically, HEK293T cells were co-transfected with the trimeric Ceacam1 display construct together with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). The concentration of purified Ceacam1-MVP was quantified by P24 ELISA. Ceacam1-MVP displayed 900±500 copies of Ceacam1 per MVP in various multimerized forms as determined by quantitative Western blot analysis (Figure 36A). Thus, the D4 display construct (Figure 1B) can effectively display hundreds of copies of Ceacam1 in multimerized forms on MVPs.

[0268] To confirm that Ceacam1-MVP represents functional Ceacam1, we tested whether Ceacam1-MVP could selectively bind to target cells expressing TIM-3, the cognate receptor of Ceacam1. First, a target cell line was established by transfecting S293 cells with a construct expressing TIM-3. Then, the transfected cells were stained with anti-TIM-3 antibody to distinguish TIM-3+ cells from TIM-3- cells. Ceacam1-MVP was then labeled with a fluorescent dye, the transfected cells were stained with the labeled MVP, and the selective MVP-cell binding was analyzed by FACS (Figure 36B). The results showed that the labeled Ceacam1-MVP binding resulted in a slightly higher fluorescence shift in TIM-3+ cells than in TIM-3- cells (Figure 36B, upper panel). Moreover, this shift was approximately two-fold higher than the fluorescence shift caused by staining the same cells with a control MVP representing a nonspecific ligand (Fig. 36B, lower panel), demonstrating that Ceacam1-MVP represents functional Ceacam1 and can selectively bind to TIM-3 on target cells.

[0269] This result was further verified by an alternative staining method (Figure 36C). In this case, TIM-3-transfected cells were first incubated with unlabeled Ceacam1-MVP to allow MVP to bind to target cells. The cell-MVP mixture was then co-stained with fluorescently labeled anti-TIM-3 and anti-Ceacam1 antibodies. The Ceacam1 staining patterns on TIM-3+ and TIM-3- cells were then examined by FACS analysis. The results confirmed that TIM-3+ cells were also Ceacam1 positive, as illustrated by the approximately 2-fold higher Ceacam1 staining shift in TIM-3+ cells from TIM-3-background cells (Figure 36C). Single staining with anti-TIM-3 antibody did not compete with Ceacam1-MVP binding to target cells, and TIM-3-transfected S293 cells were Ceacam1 negative. These results demonstrated that Ceacam1-MVP exhibited functional Ceacam1. Collectively, Ceacam1-MVPs were generated that represent high copy numbers of the functional protein, and these MVPs can selectively bind to target cells expressing its cognate receptor, TIM-3.

[0270] Example 6. T cell stimulation with activated IC-MVP This example illustrates a list of IC-MVPs representing activated immune checkpoints that were generated and their composition was characterized by determining the copies of immune checkpoint molecules represented on each of the VLPs. The results demonstrated the specific binding of these IC-MVPs to target cells expressing their cognate ligands or receptors, as well as their co-stimulatory functions in T cell activation, proliferation, and differentiation. The list of IC-MVPs illustrated in this example includes CD80-MVP, CD86-MVP, 41BBL-MVP, and OX40L-MVP.

[0271] Use of activated IC-MVP to provide costimulatory signals to T cells

[0272] During T cell activation, two stimuli are usually required to fully activate the immune response. The first signal is antigen-specific and is provided by T cell receptor (TCR) interaction with peptide-MHC molecules on the membrane of antigen-presenting cells (APCs). The second signal is non-antigen specific and is provided by the interaction of costimulatory molecules expressed on the membrane of APCs and T cells (Figure 37A). Both helper and cytotoxic T cells require these subsequent costimulatory signals to be fully activated and programmed to function and differentiate. Blocking the costimulatory pathway inhibits T cell immune responses in vitro and in vivo. In the case of helper T cells, the first costimulatory signal is provided by CD28. This molecule expressed on T cells binds to one of two molecules on APCs, namely B7.1 (CD80) or B7.2 (CD86), to initiate T cell proliferation. Cytotoxic T cells are less dependent on CD28 for activation, but still require signals from other costimulatory molecules, such as OX-40 and 4-1BB (CD137). T cells activated by different costimulatory molecules may result in different proliferative potentials and fates during differentiation. Notably, multivalent binding is important for TCR association with peptide:MHC-peptide complexes, as well as the association of costimulatory molecules on T cells and APCs. Activating IC-MVPs can be used to provide multivalent costimulatory signals to T cells to help APCs containing cancer cells to properly activate tumor-targeting T cells in culture or in animals (Figure 37B). In addition, activating IC-MVPs can serve as costimulatory signals during in vitro T cell activation with anti-CD3 antibodies (Figure 37C). Using various combinations of costimulatory IC-MVPs, T cell activation and differentiation can be programmed to generate therapeutic T cells with desired functional properties. This example illustrates the function of CD80-MVP, CD86-MVP, 4-1BB-MVP, and OX40-L-MVP in T cell activation, proliferation, and differentiation.

[0273] CD86-MVP as a costimulatory signal for T cells

[0274] To test its function in T cell activation, proliferation, and differentiation, CD86-MVPs were generated that display either mouse or human CD86. CD86 provides costimulatory signals for T cell activation and survival. CD86 also belongs to the B7 family of the immunoglobulin superfamily. Both CD80 and CD86 bind as ligands to the costimulatory molecule CD28 on the surface of all naive T cells and to the inhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA4). The interaction of CD86 expressed on the surface of antigen-presenting cells with CD28 on the surface of T cells is important for T cell activation. This interaction is essential for T lymphocytes to receive the full activation signal, which results in T cell differentiation and division, interleukin-2 production, and cell proliferation.

[0275] For this purpose, mouse splenic T cells were activated on anti-CD3 antibody-coated plates to obtain TCR activation signals and supplemented with mouse CD86-MVP as a costimulatory signal at various cell to CD86-MVP ratios (Figure 38A). Two days after T cell activation, FACS analysis was performed to observe the expression of early T cell activation markers CD69 and CD25 on activated T cells. The results showed that the addition of mouse CD86-MVP further increased the percentage of T cells with CD69+CD25+ phenotype from about 22.75% to more than 40%, demonstrating that CD86-MVP provided a costimulatory signal to boost T cell activation (Figure 38A). Notably, the optimal cell to CD86-MVP ratio was 1:50, and further increasing this ratio resulted in a decrease in the amount of T cells with CD69+CD25+ phenotype (Figure 38A). Furthermore, improved T cell activation by addition of murine CD86-MVP translated into increased T cell proliferation as shown by fold expansion (Figure 38B). Control groups of T cells activated with only primary or secondary costimulatory signals were not sufficient to induce full T cell activation and proliferation (Figure 38B).

[0276] We further investigated whether human CD86-MVP had similar effects on T cell activation, proliferation, and differentiation. Human CD86-MVP was generated by pseudotyping lentiviral VLPs with a trimeric CD86 fusion peptide. Specifically, HEK293T cells were co-transfected with the trimeric CD86 display construct together with a lentiviral packaging construct expressing essential packaging components including Gag-Pol and Rev proteins, and a viral genome transfer vector encoding a GFP / luciferase reporter (Figure 3A). Western blot analysis demonstrated that expression of CD86 on purified human CD86-MVP and high molecular weight oligomers was observed under non-reducing conditions (Figure 39A). Thus, the D4 display construct (Figure 1B) can effectively present many copies of CD86 in a multimerized form on MVP. Human peripheral blood T cells were activated on anti-human CD3 antibody-coated plates to obtain TCR activation signals and supplemented with human CD86-MVP as a costimulatory...

Claims

1. 1. A multivalent particle comprising a fusion protein comprising a mammalian immune checkpoint polypeptide and a transmembrane polypeptide, wherein the fusion protein is expressed on the surface of the multivalent particle with a valency of at least 10 copies.

2. The multivalent particle of claim 1 , wherein the mammalian immune checkpoint polypeptide comprises an immunostimulatory checkpoint polypeptide or an immunoinhibitory checkpoint polypeptide. The mammalian immune checkpoint polypeptide is PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, SIGLEC9, PD-L1, PD-L2, CD80, CD86, HVEM, CD48, CD112, CD15 5, Ceacam1, FGL1, Galectin-3, CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, GITR, CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL.

4. The multivalent particle described in claim 1, wherein the mammalian immune checkpoint polypeptide is expressed on an antigen-presenting cell, a tumor cell, or a normal cell.

5. The multivalent particle described in claim 1, wherein the mammalian immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-62, 96-115, or 153-162.

6. The multivalent particle described in claim 1, wherein the membrane-spanning polypeptide comprises a spike glycoprotein, a mammalian membrane protein, an envelope protein, a nucleocapsid protein, or a cell membrane-spanning protein.

7. The multivalent particle of claim 1, wherein the membrane-spanning polypeptide comprises VSVG, dengue E protein, influenza hemagglutinin, influenza neuraminidase, spike protein S1, spike protein S2, Sindbis virus envelope (SINDBIS) protein, hemagglutinin envelope protein from measles virus, envelope glycoprotein of measles virus fusion (F) protein, RD114, BaEV, GP41, or GP120.

8. The VSVG, (a) Full-length VSVG, (b) a truncated VSVG, or (c) Transmembrane domain and cytoplasmic tail The multivalent particle of claim 7 , comprising:

9. The multivalent particle described in claim 1, wherein the membrane-spanning polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 63, 64, or 79-95.

10. The multivalent particle described in claim 1, wherein the fusion protein further comprises a multimerization domain.

11. The multivalent particle described in claim 10, wherein the multimerization domain includes a dimerization domain, a trimerization domain, or a tetramerization domain.

12. The multivalent particle described in claim 11, wherein the dimerization domain includes a leucine zipper dimerization domain.

13. The multivalent particle described in claim 10, wherein the fusion protein further comprises a cytosolic domain.

14. The multivalent particle described in claim 11, wherein the trimerization domain includes a post-fusion multimerization domain of a viral surface protein.

15. The multivalent particle of claim 11, wherein the trimerization domain comprises a D4 post-fusion trimerization domain of a VSV-G protein, a post-fusion trimerization domain of a Dengue E protein, or a Foldon trimerization domain.

16. The multivalent particle of claim 11, wherein the tetramerization domain comprises an influenza neuraminidase stem domain.

17. The multivalent particle described in claim 10, wherein the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65 to 78.

18. The multivalent particle of claim 13, wherein the fusion protein further comprises a signal peptide. (a) when the fusion protein is expressed on the surface of the multivalent particle, the multimerization domain is on the outside of the multivalent particle; (b) when the fusion protein is expressed on the surface of the multivalent particle, the multimerization domain is on the outside of the multivalent particle and adjacent to the signal peptide; (c) when the fusion protein is expressed on the surface of the multivalent particle, the multimerization domain is in the interior of the multivalent particle; or (d) when the fusion protein is expressed on the surface of the multivalent particle, the multimerization domain is inside the multivalent particle and adjacent to the transmembrane polypeptide.

20. The multivalent particle of claim 18.

20. The domain of the fusion protein, (a) a signal peptide, a mammalian immune checkpoint polypeptide, a multimerization domain, a transmembrane polypeptide, and a cytosolic domain; (b) a signal peptide, a mammalian immune checkpoint polypeptide, a transmembrane polypeptide, a multimerization domain, and a cytosolic domain, or (c) a signal peptide, a multimerization domain, a mammalian immune checkpoint polypeptide, a transmembrane polypeptide, and a cytosolic domain. The multivalent particle of claim 18 , wherein the amino acids are arranged in the order:

21. The multivalent particle of claim 1, wherein the fusion protein is expressed on the surface of the multivalent particle with a valency of at least 50 copies.

22. The multivalent particle of claim 1, wherein the fusion protein is expressed on the surface of the multivalent particle with a valency of at least 100 copies.

23. The multivalent particle of claim 1, wherein the fusion protein is expressed on the surface of the multivalent particle with a valency of at least 200 copies.

24. The multivalent particle of claim 1, wherein the fusion protein is expressed on the surface of the multivalent particle with a valency of at least 300 copies.

25. The multivalent particle of claim 1, which does not contain viral genetic material.

26. The multivalent particle of claim 1, which is a virus-like particle or an extracellular vesicle (EV).

27. The multivalent particle of claim 1, which is an exosome or an ectosome.

28. (a) The mammalian immune checkpoint polypeptide is selected from the group consisting of PD-1, CTLA4, LAG3, BTLA, CD160, 2B4, CD226, TIGIT, CD96, B7-H3, B7-H4, VISTA, TIM3, SIGLEC7, KLRG1, SIGLEC9, PD-L1, PD-L2, CD80, CD86, HVEM, CD48, and CD 112, CD155, Ceacam1, FGL1, Galectin-3, CD27, CD28, CD40, CD122, 4-1BB, ICOS, OX40, CD2, CD30, GITR, CD70, CD80, CD86, CD40L, GITRL, 4-1BBL, OX40L, LIGHT, CD30L, CD48, or ICOSL; (b) the transmembrane polypeptide comprises VSVG, Dengue E protein, influenza hemagglutinin, influenza neuraminidase, spike protein S1, spike protein S2, Sindbis virus envelope (SINDBIS) protein, hemagglutinin envelope protein from measles virus, envelope glycoprotein of measles virus fusion (F) protein, RD114, BaEV, GP41, or GP120; (c) the multimerization domain comprises a leucine zipper dimerization domain, a post-fusion multimerization domain of a viral surface protein, a D4 post-fusion trimerization domain of a VSV-G protein, a post-fusion trimerization domain of a Dengue E protein, a Foldon trimerization domain, or an influenza neuraminidase stem domain; The multivalent particle of claim 10.

29. (a) the mammalian immune checkpoint polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-62, 96-115, or 153-162; (b) the transmembrane polypeptide comprises an amino acid sequence having at least about 90% sequence identity to any one of SEQ ID NOs: 63, 64, or 79-95; (c) the multimerization domain comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 65-78; The multivalent particle of claim 10.

30. A pharmaceutical composition comprising a multivalent particle according to any one of claims 1 to 29 and a pharma- ceutically acceptable excipient.

31. Use of a multivalent particle described in any one of claims 1 to 29 in the manufacture of a medicament for treating cancer, an autoimmune disease, an infectious disease, or an inflammatory disease.

32. Use of the pharmaceutical composition of claim 30 in the manufacture of a medicament for treating cancer, an autoimmune disease, an infectious disease, or an inflammatory disease.

33. A multivalent particle described in any one of claims 1 to 29, wherein the mammalian immune checkpoint polypeptide forms a multivalent interaction with a ligand on a target immune cell.

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