WO2023218243A1 - Lag-3/pd-l1 binding fusion proteins - Google Patents

Lag-3/pd-l1 binding fusion proteins Download PDF

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Publication number
WO2023218243A1
WO2023218243A1 PCT/IB2023/000266 IB2023000266W WO2023218243A1 WO 2023218243 A1 WO2023218243 A1 WO 2023218243A1 IB 2023000266 W IB2023000266 W IB 2023000266W WO 2023218243 A1 WO2023218243 A1 WO 2023218243A1
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amino acid
acid sequence
seq
identity
binding polypeptide
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PCT/IB2023/000266
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French (fr)
Inventor
Emma STANLEY
Emma JENKINS
Estelle ADAM
Amrik Basran
Matthew P. Vincent
Victoria JUSKAITE
Deepa Ramesh AVISETTI
Ahmad Farizzulkhairi AHMAD SOBRI
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Avacta Life Sciences Limited
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Publication of WO2023218243A1 publication Critical patent/WO2023218243A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds
    • C07K2318/20—Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • LAG-3/PD-L1 BINDING FUSION PROTEINS BACKGROUND The success of immunotherapy in many diseases is limited to a specific subpopulation of patients.
  • CTLA4 cytotoxic T- lymphocyte-associated protein 4
  • PD- (L)1 programmed cell death receptor 1
  • LAG3 Lymphocyte- associated gene 3 represents an inhibitory receptor, which is mainly found on activated immune cells and involved in the exhaustion of T cells in malignant diseases.
  • LAG-3/PD-L1 AFFIMER® polypeptides or engineered LAG-3/PD-L1-binding Stefin A polypeptide variants, that are based on naturally occurring proteins (e.g., Stefin A cystatin).
  • Each polypeptide of the fusion protein is engineered to stably display two loops (i.e., loop 2 and loop 4) that create a binding surface with high specificity and high affinity for LAG-3 or PD-L1.
  • the engineered fusion proteins further comprise a half-life extension moiety, such as a human serum albumin (HSA) AFFIMER® polypeptide, which comprises two loops stably displayed that create a binding surface with high specificity and high affinity for HSA, or a fragment crystallizable (Fc) region of an antibody (e.g., IgG1 Fc, IgG1 LALA Fc, or IgG4 Fc).
  • HSA human serum albumin
  • Fc fragment crystallizable region of an antibody
  • LAG-3 and PD-L1 AFFIMER® polypeptides and the LAG-3/PD-L1 AFFIMER® fusion proteins bind specifically to their respective targets, LAG-3 and PD-L1, with high affinities (e.g., a K d of less than 1 ⁇ 10 ⁇ 6 M, or even less than 1 ⁇ 10 ⁇ 7 M).
  • the LAG-3 and PD-L1 AFFIMER® polypeptides can be linked to each other covalently (such as by chemical cross-linking or as a fusion protein), or non-covalently (such as through multimerization domains or small molecule binding domains).
  • LAG-3/PD-L1 AFFIMER® polypeptides of the present disclosure are useful, for example, for targeting cells that express LAG-3 and/or PD-L1, and optionally, for extending the serum half-life of such polypeptides.
  • LAG-3/PD-L1 AFFIMER® polypeptides have several advantages over antibodies; for example, they are comparatively smaller ( ⁇ 14 kDa in monomeric form, ⁇ 30 kDa in dimeric form, or ⁇ 75-80 kDa in pentameric form), simpler (no disulfide bridges and no posttranslational modifications), and more robust (thermally and chemically) than antibodies.
  • LAG-3/PD-L1 AFFIMER® polypeptides can be generated in only a few weeks, exhibit extremely specificity, are easily modified (chemically and as fusion proteins), and are easily manufactured in bacterial, yeast, or mammalian systems with high expression yields. Furthermore, the core AFFIMER® polypeptides are non-immunogenic.
  • PD-L1 AFFIMER® agent and anti-PD-L1 AFFIMER® agent are used interchangeable herein, and the terms LAG-3 AFFIMER® agent and anti-LAG-3 AFFIMER® agent are used interchangeably herein.
  • HSA AFFIMER® agent and anti-HSA AFFIMER® agent are used interchangeable herein.
  • LAG-3 Lymphocyte Activation Gene 3
  • FR1 comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA (SEQ ID NO: 216);
  • FR2 comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of STNYYIKVRAGDNKYMHLKVFNGP (SEQ ID NO: 217);
  • FR3 comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of ADRVLTGYQVDKNKDDELTGF (SEQ ID NO: 21
  • LAG-3 binding polypeptide comprising: the amino acid sequence having of MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA-(X')- STNYYIKVRAGDNKYMHLKVFNGP-(X'')-ADRVLTGYQVDKNKDDELTGF, wherein X' is an amino acid sequence having at least 85% or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 204-209; and X'' is an amino acid sequence having at least 85% or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 210-215.
  • the polypeptide binds to LAG-3 with a Kd of 1 ⁇ 10 ⁇ 6 M or less.
  • X' is the amino acid sequence of SEQ ID NO: 204 and X'' is the amino acid sequence of SEQ ID NO: 210; or (b) X' is the amino acid sequence of SEQ ID NO: 205 and X'' is the amino acid sequence of SEQ ID NO: 211; or (c) X' is the amino acid sequence of SEQ ID NO: 206 and X'' is the amino acid sequence of SEQ ID NO: 212; or (d) X' is the amino acid sequence of SEQ ID NO: 207 and X'' is the amino acid sequence of SEQ ID NO: 213; or (e) X' is the amino acid sequence of SEQ ID NO: 208 and X'' is the amino acid sequence of SEQ ID NO: 214; or (f) X' is the amino acid sequence of SEQ ID NO: 209 and X'
  • LAG-3 binding polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 53, 54, 61, 62, 66, and 69.
  • a LAG-3 binding polypeptide comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of any one of SEQ ID NOs: 53, 54, 61, 62, 66, and 69.
  • a LAG-3 binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 53, 54, 61, 62, 66, and 69.
  • the LAG-3 binding polypeptide further comprises a half-life extension moiety.
  • the half-life extension moiety is a human serum albumin (HSA) binding polypeptide or a fragment crystallizable (Fc) region of an antibody, optionally wherein the antibody is a human IgG1 antibody or a human IgG4 Fc antibody.
  • HSA human serum albumin
  • Fc fragment crystallizable region of an antibody
  • Other aspects provide a bispecific fusion protein comprising: one or more PD-L1 binding polypeptide, and any one or more of the LAG-3 binding polypeptides described herein.
  • a PD-L1 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72 or 73.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73, and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 61.
  • a bispecific fusion protein comprises: a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73, and a second LAG-3 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 62.
  • a bispecific fusion protein comprises: a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73, and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53.
  • a bispecific fusion protein comprises: a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53.
  • Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, and 5.
  • a bispecific fusion protein further comprises a second PD-L1 binding polypeptide and/or a second LAG-3 binding polypeptide.
  • the second PD-L1 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72 or 73.
  • the second LAG-3 polypeptide comprises an amino acid sequence of any one of the LAG-3 polypeptide described herein.
  • Other aspects provide bispecific protein comprising: a first PD-L1 binding polypeptide, a second PD-L1 polypeptide, and any one of the LAG-3 binding polypeptides described herein.
  • each of the first PD-L1 binding polypeptide and the second PD- L1 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53.
  • the first PD-L1 binding polypeptide and the second PD-L1 polypeptide form a dimer comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 74
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53.
  • the bispecific fusion protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 4.
  • the half- life extension moiety is a human serum albumin (HSA)-binding polypeptide.
  • HSA binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 80.
  • the half-life extension moiety is a fragment crystallizable (Fc) region of an antibody, for example, a human IgG1 antibody or a human IgG4 antibody.
  • trispecific fusion protein comprising: a PD-L1 binding polypeptide, a first LAG-3 binding polypeptide, a second LAG-3 binding polypeptide, and an HSA binding polypeptide.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72 or 73.
  • each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence of the any one of the LAG-3 binding polypeptides described herein.
  • the HSA binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 80
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 74
  • each of the first LAG-3-binding polypeptide and the second LAG- 3-binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 54
  • the HSA binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 80.
  • the trispecific fusion protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 11.
  • Some aspects provide a trispecific fusion protein comprising: a first PD-L1 binding polypeptide, a second PD-L1 binding polypeptide, a first LAG-3 binding polypeptide, a second LAG-3 binding polypeptide, and a half-life extension moiety.
  • the half-life extension moiety is an HSA binding polypeptide.
  • each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72 or 73.
  • each of the first LAG-3 binding polypeptide and second LAG-3 binding polypeptide comprises an amino acid sequence of any one of the LAG-3 polypeptides described herein.
  • each of HSA binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 80.
  • each of the first PD-L1 binding polypeptide and the second PD- L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72
  • each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 62
  • the HSA binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 80.
  • each of the first PD-L1 binding polypeptide and the second PD- L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72
  • each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 54
  • the HSA binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 80.
  • the trispecific fusion protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 8-10.
  • a bispecific fusion protein comprising a PD-L1 binding polypeptide, a LAG-3 binding polypeptide, and a fragment crystallizable (Fc) region of an antibody.
  • the antibody is selected from a human IgG1 (hIgG1) antibody and a human IgG4 (hIgG4) antibody.
  • the hIgG1 antibody comprises LALA mutations (Leu234Ala and Leu235Ala mutations).
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72 or 73.
  • the LAG-3 binding polypeptide comprises an amino acid sequence of any one of the LAG-3 binding polypeptides described herein.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 54
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 62
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 66
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 69
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the bispecific fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 6, 7, 15, 19, 38, 39, 40, 41, 44, and 45.
  • fusion protein further comprises one or more linker located between two of the polypeptides.
  • the linker is a rigid linker, optionally comprising the amino acid sequence of AEAAAKEAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 86).
  • the linker is a flexible linker, optionally comprising the amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 87).
  • a polynucleotide comprising an open reading frame encoding any one of the fusion proteins described herein.
  • the open reading frame comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the nucleotide sequence of any one of SEQ ID NOs: 99- 180.
  • Other aspects provide a vector, for example, a viral vector or a plasmid vector, comprising any one or more of the polynucleotides described herein.
  • Still other aspects provide a cell, for example, a mammalian cell, comprising any one or more of the polynucleotides described herein or any one or more of the vector described herein.
  • pharmaceutical composition comprising: (a) any one or more of the fusion proteins described herein, any one or more of the polynucleotides described herein, any one or more of the vectors described herein, or any one or more of the cells described herein; and (b) a pharmaceutically acceptable excipient.
  • Some aspects provide a method comprising administering to a subject any one or more of the pharmaceutical compositions described herein (e.g., in a therapeutically effective amount). In some embodiments, the subject has a cancer.
  • the pharmaceutical composition is administered subcutaneously, intravenously, or intramuscularly.
  • FIG.1 shows a schematic representation of AVA21 in-line fusion (ILF) AFFIMER® dimers and trimers. A reducing SDS-PAGE gel confirming the molecular weight of each product is shown.
  • FIG.2 shows results of two LAG-3 direct binding ELISAs with AVA21 ILF AFFIMER® dimers and trimers. AVA21-01, 03 and 04 show decreased binding to target respective to their monomer parent clones AVA19-157 and AVA19-01.
  • FIG.3 shows results of two PD-L1 direct binding ELISAs using AVA21 ILF AFFIMER® dimers and trimers.
  • AVA21-01, AVA21-02 and AVA21-03 exhibit decreased binding to PD-L1 relative to their monomer parent clone, AVA04-269.
  • AVA21-04 and AVA21- 05 show comparable binding affinity to PD-L1 relative to their parent clone AVA04-251.
  • FIG.4 shows results of Promega PD-L1 blockade assay with AVA21 ILF AFFIMER® dimers and trimers.
  • AVA04-251 ILF for AVA21 show similar functionality to parent with ILF trimer appearing better than ILF dimer.
  • AVA04-269 ILF show reduced functionality relative to parent. No IC50 values could be calculated.
  • Results reflect observations from PD-L1 binding ELISA.
  • FIG.5 shows results of a bridging ELISA with AVA21 ILF AFFIMER® dimers and trimers, where LAG-3 was coated on the plate and detection was carried out using an anti-PD-L1 antibody. Most bispecific formats engage both targets simultaneously to various degrees.
  • FIG.6 shows results of AVA21 ILF AFFIMER® dimers and trimers binding to LAG-3- positive(top) and negative Jurkat (middle) and CHO-K1 cells (bottom).
  • FIG.7 shows two different AVA21 ILF XT AFFIMER® pentamer formats.
  • FIG.8 shows a characterization of AVA21 ILF XT AFFIMER® pentamers. SEC-HPLC chromatograms and a reducing SDS-PAGE gel confirming the molecular weight of each product are shown.
  • FIG.9 shows results of PD-L1 direct binding ELISA using AVA21 ILF XT AFFIMER® pentamers.
  • FIG.10 shows results of LAG-3 direct binding ELISA using AVA21 ILF XT and BK AFFIMER® multimers. Binding of both pentamers to LAG-3 was demonstrated by ELISA. Use of a rigid linker appears to allow for better target engagement and stronger binding to LAG-3 compared to flexible linker.
  • FIG.11 shows results of AVA21 ILF XT AFFIMER® pentamers binding to HSA as demonstrated by direct ELISA. Both constructs show lower affinity than AVA03-42 monomer control.
  • FIG.12 shows results of AVA21 ILF XT AFFIMER® pentamer cell binding to LAG-3- positive (top) and negative (bottom) Jurkat cells. Both pentamers show comparable binding to LAG-3 positive Jurkat cells, comparable to AVA19-158 XT23 control. No binding detected to LAG-3 negative cells.
  • FIG.13 shows results of dual PD-L1/HSA binding assay with AVA21 ILF XT AFFIMER® pentamers. Both clones engage PD-L1 and HSA targets simultaneously. Binding of pentamers was unaffected in presence of HSA.
  • FIG.14 shows results of functional PD-L1/PD-1 gene reporter assay with AVA21 ILF XT AFFIMER® pentamers.
  • AVA21-08 XT shows comparable activity to AVA04-251 BH control in this assay.
  • Functionality of AVA21-09 XT was significantly decreased.
  • Flexible linker seems to show detrimental effect compared to rigid linker.
  • FIG.15 shows results of PD-L1, LAG-3 and HSA BIACORETM kinetic binding analysis of two AVA21 ILF XT AFFIMER® pentamers. The rigid linker appears to allow for stronger binding to PD-L1 and LAG-3.
  • FIG.16 shows schematic representation of AVA21 ILF XT AFFIMER® pentamers and tetramers. A reducing SDS-PAGE gel, confirming the molecular weight of each product is shown.
  • FIG.17 shows SEC-HPLC chromatograms of AVA21-11 XT and AVA21-12 XT following a two-stage purification process, confirming both proteins are >95% pure.
  • FIG.18 shows results of PD-L1, LAG-3 and HSA BIACORETM kinetic binding analysis for AVA21-11 XT and AVA21-12 XT.
  • FIG.19 shows results of PD-L1 direct binding ELISA for AVA21-11 XT (top) and AVA21-12 XT (bottom). Both of the ILF XT constructs show binding to PD-L1 comparable to parent AFFIMER® controls.
  • FIG.20 shows results of LAG-3 direct binding ELISA for AVA21-11 XT (top) and AVA21-12 XT (bottom). AVA21-11 XT shows better binding to LAG-3 than AVA21-12 XT and equivalent to AVA19-06 BK control.
  • FIG.21 shows results of HSA direct binding ELISA for a AVA21-12 XT tetramer.
  • FIG.22 shows results of a bridging ELISA in presence (top) or absence(bottom) of HSA, where the plate was coated with LAG-3 and detection was carried out using an anti-PD-L1 antibody. Dual target engagement is confirmed in presence/absence of HSA in solution. Presence of HSA in solution does not impact binding to PD-L1 and LAG-3.
  • FIG.23 shows results of Promega functional PD-L1/PD-1 gene reporter assay for AVA21-11 XT (top) and AVA21-12 XT (bottom). Both formats appear to be less active than their respective controls in this assay.
  • FIG.24 shows results of multimer binding to LAG-3 overexpressing BPS cells. Increased binding of dimeric formats vs monomer AVA19-06 is observed. AVA21-11 XT binding to BPS cells is comparable to that of AVA19-06 BK control.
  • FIG.25 shows results of multimer cell binding in PD-L1 overexpressing CHO cells. AVA21-12 XT binding is comparable to that of AVA04-640 control.
  • FIG.26 shows schematic reorientations of the control formats for AVA21 bispecific IgG Fc fusion constructs.
  • FIG.27 shows SEC-HPLC chromatograms of AVA21 bispecific IgG1 Fc fusion multimers, confirming both proteins are >97% pure.
  • FIG.28 shows a reducing SDS-PAGE gel confirming the molecular weights of AVA21 bispecific IgG1 Fc fusion multimers.
  • FIG.29 shows results of LAG-3 and PD-L1 direct binding ELISAs.
  • AVA21-06 and 07 BP both show comparable binding affinities to LAG-3 and data is also consistent with control proteins AVA19-06 and AVA19-158 AQ.2.
  • AVA21-06 and 07 BP both show comparable binding affinities to PD-L1 and data is also consistent with control protein AVA04-251 V.2.
  • FIG.30 shows results of Promega PD-1/PD-L1 blockade assay for AVA21-06 BP.
  • AVA21-06 BP shows slightly decreased activity compared to AVA04-251 V.2 control protein.
  • FIG.31 shows results of AVA21-06 BP binding to LAG-3-positive (top) and negative (bottom) Jurkat cells. Results show a dose effect for AVA21-06 BP on positive cells. No binding observed on LAG-3 negative cells.
  • FIG.32 shows results of AVA21-06 BP and AVA21-07 BP binding to LAG-3-positive BPS cells as well as LAG-3 negative Jurkat and CHO-K1 cells.
  • AVA21-06 and 07 BP bind to LAG-3 enriched BPS cells. No binding observed to LAG-3 negative Jurkat and CHO-K1 cells.
  • FIG.33 shows a schematic representation of additional AVA21 bispecific IgG1 Fc fusion constructs. A reducing SDS-PAGE gel is also shown and confirms protein molecular weights.
  • FIG.34 shows results of Promega PD-1/PD-L1 blockade assay on AVA21-15 and AVA21-16. AVA21-15 shows a significant decrease in activity compared to control proteins. AVA21-16 potency is reduced in this assay relative to that of control proteins, but the protein remains active.
  • FIG.35 shows results of direct LAG-3 and PD-L1 binding ELISAs. Results show that the positioning of LAG-3/PD-L1 binders affects the ability of AFFIMER® proteins to engage target.
  • FIG.36 shows a schematic representation of additional AVA21 bispecific IgG1 Fc LALA fusion constructs. A reducing SDS-PAGE gel is also shown and confirms protein molecular weights.
  • FIG.37 shows PD-L1 kinetics analysis results for AVA21 CR IgG1 LALA constructs. Binding to PD-L1 is equivalent for all clones shown.
  • FIG.38 shows LAG-3 kinetics analysis results for AVA21 CR IgG1 LALA constructs.
  • FIG.39 shows results of PD-L1 direct binding ELISA for AVA21-06 CR. Equivalent binding is observed for both AVA21-06 CR and the control protein AVA04-251 V.2.
  • FIG.40 shows results of LAG-3 direct binding ELISA for three AVA21 IgG1 LALA constructs. All CR formats show equivalent binding to LAG-3 and either comparable or better binding affinity than their relative CS controls.
  • FIG.41 shows expected molecular weights for alanine scanning mutants of AVA21-06 CR. Alanine mutants of parent format AVA21-06 CR were engineered to improve binding and reduce aggregation.
  • FIG.42 shows results of direct LAG-3 (top) and PD-L1 (bottom) binding ELISAs for AVA21-06 CR Loop 2 alanine scanning mutants. Little variation in PD-L1 binding between controls and various mutants is observed. D439A, P440A and W443A mutations significantly affect binding to LAG-3 (essential amino acids), D438A impaired binding to an extent and P437A did not appear to significantly affect binding to LAG-3.
  • FIG.43 shows results of direct LAG-3 (top) and PD-L1 (bottom) binding ELISAs for AVA21-06 CR Loop 4 alanine scanning mutants. Little variation in PD-L1 and LAG-3 binding between controls and various mutants is observed.
  • FIG.44A shows result of PD-L1, LAG3 and human serum albumin (HuSA) Biacore kinetic analysis of AVA21-XT.
  • FIG.44B shows result of PD-L1 and LAG3 Biacore kinetic analysis of AVA21 CR formats (IgG1-LALA).
  • FIG.45 shows result of AVA21 huIgG1 LALA Fc Fusion construct (AVA21-06 CR, AVA21-06 T89A CR, AVA21-12CR and AVA21-13CR) binding to PD-L1 expressing cells.
  • FIG.46 shows result of AVA21 huIgG1 LALA Fc Fusion construct (AVA21-06 CR, AVA21-06 T89A CR, AVA21-12CR and AVA21-13CR) binding to hLAG3 positive (top) and negative (bottom) D0.11.10 cells. All AVA21 constructs show binding to LAG3 positive cells. No binding is detected to LAG3 negative cells.
  • FIG.47 shows results of human PD-L1 direct binding ELISA for AVA21-06 CR, AVA21- 06 T89A CR, AVA21-12 CR and AVA21-13 CR.
  • FIG.48 Error! Reference source not found.shows results of Promega PD-1/PD-L1 blockade assay with AVA21-06 CR, AVA21-06 T89A CR, AVA21-12CR and AVA21-13CR. All AVA21 constructs show comparable activity to PD-L1 binding construct only (AVA04-251 CR). Negative control protein SQTgly CR induced no response as expected.
  • FIG.49 shows results of LAG3/MHCII blockade assay with AVA21-06 CR, AVA21- 12CR, AVA21-13CR, AVA21-8XT, AVA21-12XT, AVA21-19CR and AVA19-158-XT23.
  • AVA04- 251-CR, AVA04-251-XT14, AVA04-640-XT34 had no inhibitory activity as expected.
  • FIG.50 shows results of PD-1/LAG3 combination blockade assay with AVA21-06 CR, AVA21-12CR, AVA21-13CR, AVA21-8XT and AVA21-12XT.
  • FIG.51 shows preliminary result of AVA21 constructs inducing IL2 release in human peripheral blood mononuclear cells after pre-activation with Staphylococcal Enterotoxin B.
  • FIGs.52A-52C show result of AVA21 constructs reversing exhausted T cell (Tex) hypo- responsiveness in an in vitro mixed lymphocyte reaction in donor pair 1.
  • Bispecific AVA21 CR IgG1 LALA constructs (AVA21-06CR, AVA21-12CR, AVA21-13) and bispecific AVA21 ILF (AVA21-12XT) increased T cell proliferation and IFN- ⁇ release to similar levels as PDL1 binder only (AVA04-251 CR) and reached comparable maximal response as anti-PDL1 (Atezolizumab) and anti-PD1 (Nivolumab) positive controls.
  • LAG3 binder only (AVA21-19CR) had no effect.
  • FIGs.53A-53C show result of AVA21 constructs reversing exhausted T cell (Tex) hypo- responsiveness in an in vitro mixed lymphocyte reaction in donor pair 2.
  • FIG.54 shows pharmacokinetic analysis of AVA21-13CR and AVA21-12XT in C56Bl/6 mice.Reversal of T cell exhaustion in an in vitro mixed lymphocyte reaction (MLR) (FIGs. 52A-52C, FIGs.53A-53C)
  • FIG.55A shows stability studies of Fc Fused AFFIMER ® polypeptide at 37 ⁇ C. HPLC- SEC chromatogram are overlaid for each sampling day and normalized. The table represents the percentage purity of protein as analysed by HPLC-SEC. The acceptable purity value to indicate stability of protein is equal or above 90% purity.
  • FIG.55B shows Stability studies of XT AFFIMER ® polypeptide at 37 ⁇ C.
  • HPLC-SEC chromatogram are overlaid for each sampling day and normalized.
  • the table represents the percentage purity of protein as analysed by HPLC-SEC.
  • the acceptable purity value to indicate stability of protein is equal or above 90% purity.
  • FIG. 55C shows Stability studies of Fc Fused AFFIMER ® polypeptide at 45 ⁇ C.
  • HPLC-SEC chromatogram are overlaid for each sampling day and normalized.
  • the table represents the percentage purity of protein as analysed by HPLC-SEC.
  • the acceptable purity value to indicate stability of protein is equal or above 90% purity.
  • FIG.55D shows Stability studies of XT AFFIMER ® polypeptide at 45 ⁇ C.
  • HPLC-SEC chromatogram are overlaid for each sampling day and normalized.
  • the table represents the percentage purity of protein as analysed by HPLC-SEC. The acceptable purity value to indicate stability of protein is equal or above 90% purity.
  • FIG. 55E shows Stability studies of Fc Fused AFFIMER ® polypeptide at 22 ⁇ C (room temperature). HPLC-SEC chromatogram are overlaid for each sampling day and normalized. The table represents the percentage purity of protein as analysed by HPLC-SEC. The acceptable purity value to indicate stability of protein is equal or above 90% purity.
  • FIG.55F shows Stability studies of XT AFFIMER ® polypeptide at 22 ⁇ C (room temperature). HPLC-SEC chromatogram are overlaid for each sampling day and normalized.
  • FIG.56 shows a schematic representation of additional AVA21 bispecific IgG4 Fc fusion constructs. A reducing SDS-PAGE gel is also shown and confirms protein molecular weights.
  • FIG.57 shows results of human PD-L1 direct binding ELISA for AVA21-06 CA, AVA21-12 CA and AVA21-13 CA. All clones bind to PD-L1 with comparable affinities and EC50 values are consistent with that of control protein AVA04-251 AZ.
  • FIG.58 shows results of LAG-3 direct binding ELISA for AVA21 CA IgG4 fusion constructs.
  • FIG.59 shows results of AVA21 CA IgG4 fusion constructs binding to enriched LAG-3- positive BPS and negative Jurkat cells. All formats bind to LAG-3 positive cells, no signal observed for LAG-3 negative cells.
  • PDL-1 Programmed Cell Death Protein Ligand 1
  • LAG-3 Lymphocyte Activation Gene 3
  • Programmed Cell Death Protein 1 plays a vital role in inhibiting immune responses and promoting self-tolerance through modulating the activity of T-cells, activating apoptosis of antigen-specific T cells and inhibiting apoptosis of regulatory T cells.
  • Programmed Cell Death Ligand 1 (PD-L1) is a trans-membrane protein that is considered to be a co-inhibitory factor of the immune response, it can combine with PD-1 to reduce the proliferation of PD-1 positive cells, inhibit their cytokine secretion and induce apoptosis.
  • PD-L1 also plays an important role in various malignancies where it can attenuate the host immune response to tumor cells.
  • PD-1/PD-L1 axis is responsible for cancer immune escape and makes a huge effect on cancer therapy.
  • PD-1/PD-L1 pathway plays a significant role in controlling induction and maintenance of immune tolerance within the tumor microenvironment.
  • the activity of PD-1 and its ligands PD-L1 or PD-L2 are responsible for T cell activation, proliferation, and cytotoxic secretion in cancer to degenerating anti-tumor immune responses.
  • PD-1 ligand (PD-L1; also referred to as CD279 and B7-H1), belongs to the B7 series and is a 33-kDa type 1 transmembrane glycoprotein that contains 290 amino acids with Ig- and IgC domains in its extracellular region.
  • PD-L1 is usually expressed by macrophages, some activated T cells and B cells, dendritic cells (DCs) and some epithelial cells, particularly under inflammatory conditions.
  • DCs dendritic cells
  • PD-L1 is expressed by tumor cells as an “adaptive immune mechanism” to escape anti-tumor responses.
  • PD-L1 is associated with an immune environment rich in CD8 T cells, production of Th1 cytokines and chemical factors, as well as interferons and specific gene expression characteristics.
  • interferon-gamma causes PD-L1 upregulation in ovarian cancer cells, which is responsible for disease progression, whereas IFN- ⁇ receptor 1 inhibition can reduce PD-L1 expression in acute myeloid leukemia mouse models through the MEK/extracellular signal-regulated kinase (ERK) and MYD88/TRAF6 pathways.
  • IFN- ⁇ induces protein kinase D isoform 2 (PKD2), which is important for the regulation of PD- L1. Inhibition of PKD2 activity inhibits the expression of PD-L1 and promotes a strong antitumor immune response.
  • PKI protein kinase D isoform 2
  • NK cells secrete IFN- ⁇ through the Janus kinase (JAK)1, JAK2 and signal transducer and activator of transcription (STAT)1 pathways, increasing the expression of PD-L1 on the surface of the tumor cells.
  • JAK Janus kinase
  • JAK2 JAK2
  • STAT signal transducer and activator of transcription
  • T and NK cells appear to secrete IFN- ⁇ , which induces PD-L1 expression on the surface of the target cells, including tumor cells.
  • PD-L1 acts as a pro-tumorigenic factor in cancer cells via binding to its receptors and activating proliferative and survival signaling pathways.
  • PD-L1 is implicated in subsequent tumor progression.
  • PD-L1 has been shown to exert non-immune proliferative effects on a variety of tumor cell types. For example, PD-L1 induced epithelial-to-mesenchymal transition (EMT) and stem cell-like phenotypes in renal cancer cells, indicating that the presence of the intrinsic pathway of PD-L1 promotes kidney cancer progression.
  • EMT epithelial-to-mesenchymal transition
  • stem cell-like phenotypes in renal cancer cells, indicating that the presence of the intrinsic pathway of PD-L1 promotes kidney cancer progression.
  • Lymphocyte activation gene-3 (LAG-3, LAG-3, or CD223; Gene ID: 3902; NM_002286.6, NP_002277.4) is a type I transmembrane protein that is expressed on the cell surface of activated CD4+ and CD8+ T cells and subsets of natural killer (NK) and dendritic cells.
  • LAG-3 comprises four extracellular immunoglobulin-like domains and requires binding to its ligand, major histocompatibility complex (MHC) class II, for functional activity. LAG-3 is only expressed on the cell surface of activated T cells and its cleavage from the cell surface terminates its signaling.
  • MHC major histocompatibility complex
  • LAG-3 plays an important role in promoting regulatory T cell (Treg) activity and in negatively regulating T cell activation and proliferation. Both natural and induced Treg express increased LAG-3, which is required for maximal Treg suppressive function. Furthermore, ectopic expression of LAG-3 on CD4+ effector T cells reduces their proliferative capacity and transforms them such that they have regulatory potential against other T cells. Without wishing to be bound by theory, it is thought that inhibiting LAG-3 function will promote an anti-cancer response, as its inhibition activates effector T cells (as does inhibition of PD-1/PD-L1) in addition to inhibiting induced (antigen-specific) Treg suppressive activity.
  • fusion protein that includes an AFFIMER® polypeptide that binds to PD-L1 and an AFFIMER® polypeptide that binds to LAG-3.
  • the fusion protein further comprises a half-life extension moiety, such as an AFFIMER® polypeptide that binds human serum albumin (HSA) and/or an antibody Fc domain (e.g., IgG1 Fc, IgG1 LALA Fc, or IgG4 Fc).
  • HSA human serum albumin
  • antibody Fc domain e.g., IgG1 Fc, IgG1 LALA Fc, or IgG4 Fc.
  • the half-life extension moiety extends, in a controlled manner, the serum half-life of the LAG-3/PD-L1 AFFIMER® polypeptide to which it is conjugated.
  • the present disclosure addresses the urgent need in the art for targeting molecules capable of binding to PD-L1with high specificity and high affinity as well as for targeting molecules capable of binding to LAG-3 with high specificity and high affinity.
  • the PD-L1 binding component of the fusion protein will localize the protein (and the LAG-3 inhibitory activity) to the tumor microenvironment. Therefore, provided herein, in some embodiments, are LAG-3/PD-L1 AFFIMER® polypeptides, engineered polypeptide variants of the Stefin A protein, that bind LAG-3 and PD- L1 with a Kd of less than 1 ⁇ 10 ⁇ 6 M.
  • the LAG-3/PD-L1 AFFIMER® polypeptides of the present disclosure may be fused or otherwise linked to therapeutic molecules to be used for the treatment of diseases and/or disorders characterized at least in part by the presence of PD-L1-positive cells and/or LAG-3-positive cells.
  • the LAG- 3/PD-L1 AFFIMER® polypeptides can be used as therapeutic agents.
  • Stefin polypeptides encompass a subgroup of proteins in the cystatin superfamily, a family which encompasses proteins that contain multiple cystatin-like sequences.
  • the Stefin subgroup of the cystatin family includes relatively small (around 100 amino acids) single domain proteins.
  • Stefin A itself is a monomeric, single chain, single domain protein of 98 amino acids.
  • the structure of Stefin A has been solved, facilitating the rational mutation of Stefin A into the AFFIMER® polypeptide.
  • the only known biological activity of cystatins is the inhibition of cathepsin activity, which allowed for exhaustive testing for residual biological activity of the engineered proteins.
  • An “AFFIMER® polypeptide” (also referred to as an “AFFIMER® protein”) refers to a small, highly stable protein that is an engineered variant of a Stefin polypeptide.
  • AFFIMER® proteins display two peptide loops and an N-terminal sequence that can all be randomized to bind to desired target proteins with high affinity and specificity, in a similar manner to monoclonal antibodies. Stabilization of the two peptides by the Stefin A protein scaffold constrains the possible conformations that the peptides can take, increasing the binding affinity and specificity compared to libraries of free peptides.
  • These engineered non-antibody binding proteins are designed to mimic the molecular recognition characteristics of monoclonal antibodies in different applications. Variations to other parts of the Stefin A polypeptide sequence can be carried out, with such variations improving the properties of these affinity reagents, such as increase stability, make them robust across a range of temperatures and pH and the like.
  • an AFFIMER® polypeptide includes a sequence derived from Stefin A, sharing substantial identify with a Stefin A wild type sequence, such as human Stefin A. It will be apparent to a person skilled in the art that modifications may be made to the scaffold sequence without departing from the disclosure.
  • an AFFIMER® polypeptide can have an amino acid sequences that is at least 25%, 35%, 45%, 55% or 60% identity to the corresponding sequences to human Stefin A, for example, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95% identical, e.g., where the sequence variations do not adversely affect the ability of the scaffold to bind to the desired target (such as PD-L1), and e.g., which do not restore or generate biological functions such as those which are possessed by wild type Stefin A but which are abolished in mutational changes described herein.
  • the desired target such as PD-L1
  • an “AFFIMER® agent” refers to a polypeptide that includes an AFFIMER® polypeptide sequence and any other modification(s) (e.g., conjugation, post-translational modifications, etc.) so as to represent a therapeutically active protein intended for delivery to an individual.
  • An “AFFIMER®-linked conjugate” refers to an AFFIMER® agent having at least one moiety conjugated thereto through a chemical conjugation other than through the formation of a contiguous peptide bond through the C-terminus or N-terminus of the polypeptide portion of the AFFIMER® agent containing AFFIMER® polypeptide sequence.
  • An AFFIMER®-linked conjugate may be an “AFFIMER® polypeptide-drug conjugate”, which refers to an AFFIMER® agent including at least one pharmacologically active moiety conjugated thereto.
  • An AFFIMER®-linked conjugate may also be an “AFFIMER®-tag conjugate”, which refers to an AFFIMER® agent including at least one detectable moiety (e.g., detectable label) conjugated thereto.
  • An “encoded AFFIMER® construct” refers to a nucleic acid construct which, when expressed by cells in a patient’s body through a gene delivery process, produces an intended AFFIMER® agent in vivo.
  • PD-L1 Programmed death-ligand 1
  • CD274 cluster of differentiation 274
  • B7-H1 B7 homolog 1
  • PD-L1 is a 40kDa type 1 transmembrane protein that is expressed by various tumor cells and by the lymphocytes that infiltrate tumors.
  • PD-L1 is expressed on the surface of tumor cells and it is able to bind to PD-1 on the surface of activated T cells, B cells, and myeloid cells, to modulate activation or inhibition.
  • the binding of PD-L1 to PD-1 leads to an immunosuppressive effect and allows the tumor to evade immune destruction.
  • PD-L1 also has an appreciable affinity for the costimulatory molecule CD80 (B7-1), but not CD86 (B7-2).
  • CD80 costimulatory molecule
  • B7-2 costimulatory molecule
  • PD-L1 has been speculated to play a major role in suppressing the adaptive arm of immune system during particular events such as pregnancy, tissue allografts, autoimmune disease and other disease states such as hepatitis.
  • the adaptive immune system reacts to antigens that are associated with immune system activation by exogenous or endogenous danger signals.
  • clonal expansion of antigen-specific CD8+ T cells and/or CD4+ helper cells is propagated.
  • the binding of PD-L1 to the inhibitory checkpoint molecule PD-1 transmits an inhibitory signal based on interaction with phosphatases (SHP-1 or SHP-2) via Immunoreceptor Tyrosine-Based Switch Motif (ITSM).
  • SHP-1 or SHP-2 phosphatases
  • ITMS Immunoreceptor Tyrosine-Based Switch Motif
  • This reduces the proliferation of antigen-specific T-cells in lymph nodes, while simultaneously reducing apoptosis in regulatory T cells (anti- inflammatory, suppressive T cells) - further mediated by a lower regulation of the gene Bcl-2.
  • the human amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot.
  • the amino acid sequence of human PD- L1 can be found as UniProt/Swiss-Prot. Accession No.
  • PD-L1 includes any native, mature PD-L1 which results from processing of a PD-L1 precursor protein in a cell.
  • the term encompasses PD-L1 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated.
  • PD-L1 AFFIMER® agent refers to an AFFIMER® agent that comprises at least one AFFIMER® polypeptide that binds to PD-L1, particularly human PD-L1, with a dissociation constant (Kd) of at least 10 -6 M.
  • the PD-L1 AFFIMER® agent binds PD- L1 with a Kd of 1 ⁇ 10 ⁇ 7 M or less, Kd of 1 ⁇ 10 ⁇ 8 M or less, Kd of 1 ⁇ 10 ⁇ 9 M or less, or a Kd of 1 ⁇ 10 ⁇ 10 M or less.
  • PD-L1 AFFIMER® polypeptide and “engineered PD-L1 binding Stefin A polypeptide variant” are used interchangeably herein.
  • a “PD-L1 AFFIMER® polypeptide” is an engineered polypeptide that binds specifically to PD- L1 with a Kd of 1 ⁇ 10 ⁇ 6 M or less, wherein the engineered polypeptide is a variant of a Stefin A protein.
  • Lymphocyte Activating Protein 3 (LAG-3), also known as cluster of differentiation 233 (CD233), is a protein that in humans is encoded by the LAG-3 gene.
  • LAG-3 is a member of the immunoglobulin (Ig) superfamily and comprises a 503-amino acid type I transmembrane protein having four extracellular Ig-like domains.
  • LAG-3 is primarily expressed in activated T cells and a subset of NK cells, and its expression is induced by interleukin-2 (IL-2), IL-7, IL-12A, and IL- 12B on activated T cells.
  • IL-2 interleukin-2
  • IL-7 interleukin-7
  • IL-12A interleukin-12A
  • IL- 12B interleukin-12A
  • IL-12B interleukin-12A
  • IL- 12B interleukin-12A
  • LAG-3 interacts with MHC class II, and it interacts with the fibrinogen-like 1 (FGL1) protein via the fibrinogen C-terminal domain.
  • LAG-3 activation results in a signaling pathway that suppresses T cell activation.
  • the protein may also inhibit antigen-specific T cell activation in synergy with PD-1/PD-L1. Its pathway also negatively regulates the proliferation, activation
  • the human amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot.
  • the amino acid sequence of human LAG-3 can be found as UniProt/Swiss-Prot. Accession No. P18627 and the nucleotide sequence encoding of the human LAG-3 can be found at NCBI Accession No. NM_002286.6 (Gene ID: 3902).
  • “LAG-3” includes any native, mature LAG-3 which results from processing of a LAG-3 precursor protein in a cell.
  • LAG-3 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated.
  • the term also includes any LAG-3 proteins comprising mutations, e.g., point mutations, fragments, insertions, deletions, and splice variants of full length wild-type LAG-3.
  • a “LAG-3 AFFIMER® agent” refers to an AFFIMER® agent that comprises at least one AFFIMER® polypeptide that binds to LAG-3, particularly human LAG-3, with a dissociation constant (Kd) of at least 10 -6 M.
  • the LAG-3 AFFIMER® agent binds LAG-3 with a Kd of 1 ⁇ 10 ⁇ 7 M or less, Kd of 1 ⁇ 10 ⁇ 8 M or less, Kd of 1 ⁇ 10 ⁇ 9 M or less, or a Kd of 1 ⁇ 10 ⁇ 10 M or less.
  • LAG-3 AFFIMER® polypeptide and “engineered LAG-3-binding Stefin A polypeptide variant” are used interchangeably herein.
  • a “LAG-3 AFFIMER® polypeptide” is an engineered polypeptide that binds specifically to LAG-3 with a Kd of 1 ⁇ 10 ⁇ 6 M or less, wherein the engineered polypeptide is a variant of a Stefin A protein.
  • Human serum albumin (HSA) is a protein encoded by the ALB gene.
  • HSA is a 585 amino acid polypeptide (approx.67 kDa) having a serum half-life of about 20 days and is primarily responsible for the maintenance of colloidal osmotic blood pressure, blood pH, and transport and distribution of numerous endogenous and exogenous ligands.
  • HSA has three structurally homologous domains (domains I, II and III), is almost entirely in the alpha-helical conformation, and is highly stabilized by 17 disulfide bridges.
  • a representative HSA sequence is provided by UniProtKB Primary accession number P02768 and may include other human isoforms thereof.
  • An “HSA AFFIMER® agent” refers to an AFFIMER® agent that comprises at least one AFFIMER® polypeptide that binds to serum albumin, particularly human serum albumin, with a dissociation constant (Kd) of at least 10 -6 M.
  • the HSA AFFIMER® agent binds HSA with a Kd of 1 ⁇ 10 ⁇ 7 M or less, Kd of 1 ⁇ 10 ⁇ 8 M or less, Kd of 1 ⁇ 10 ⁇ 9 M or less, or a Kd of 1 ⁇ 10 ⁇ 10 M or less.
  • HSA AFFIMER® polypeptide and “engineered HSA binding Stefin A polypeptide variant” are used interchangeably herein.
  • an “HSA AFFIMER® polypeptide” is an engineered polypeptide that binds specifically to HSA with a K d of 1 ⁇ 10 ⁇ 6 M or less, wherein the engineered polypeptide is a variant of a Stefin A protein.
  • Polypeptides are polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing at least one analog of an amino acid including, for example, unnatural amino acids
  • Amino acids participate in one more peptide bonds of a polypeptide.
  • the abbreviations used herein for designating the amino acids are based on recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochemistry (1972) 11:1726-1732).
  • Met, Ile, Leu, Ala and Gly represent "residues" of methionine, isoleucine, leucine, alanine and glycine, respectively.
  • the residue is meant a radical derived from the corresponding ⁇ -amino acid by eliminating the OH portion of the carboxyl group and the H portion of the ⁇ -amino group.
  • amino acid side chain is that part of an amino acid exclusive of the --CH(NH2)COOH portion, as defined by K. D. Kopple, "Peptides and Amino Acids", W. A. Benjamin Inc., New York and Amsterdam, 1966, pages 2 and 33.
  • amino acids used in the application of this disclosure are those naturally occurring amino acids found in proteins, or the naturally occurring anabolic or catabolic products of such amino acids which contain amino and carboxyl groups.
  • amino acid side chains include side chains selected from those of the following amino acids: glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, and tryptophan, and those amino acids and amino acid analogs which have been identified as constituents of peptidylglycan bacterial cell walls.
  • Amino acid residues having “basic sidechains” include Arg, Lys and His.
  • Amino acid residues having “acidic sidechains” include Glu and Asp.
  • Amino acid residues having “neutral polar sidechains” include Ser, Thr, Asn, Gln, Cys and Tyr.
  • Amino acid residues having “neutral non-polar sidechains” include Gly, Ala, Val, Ile, Leu, Met, Pro, Trp and Phe.
  • Amino acid residues having “non-polar aliphatic sidechains” include Gly, Ala, Val, Ile and Leu.
  • Amino acid residues having “hydrophobic sidechains” include Ala, Val, Ile, Leu, Met, Phe, Tyr and Trp.
  • Amino acid residues having “small hydrophobic sidechains” include Ala and Val.
  • Amino acid residues having “aromatic sidechains” include Tyr, Trp and Phe. Amino acid residues further include analogs, derivatives and congeners of any specific amino acid referred to herein, as for instance, the subject AFFIMER® polypeptides (particularly if generated by chemical synthesis) can include an amino acid analog such as, for example, cyanoalanine, canavanine, djenkolic acid, norleucine, 3-phosphoserine, homoserine, dihydroxy- phenylalanine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, diaminiopimelic acid, ornithine, or diaminobutyric acid.
  • an amino acid analog such as, for example, cyanoalanine, canavanine, djenkolic acid, norleucine, 3-phosphoserine, homoserine, dihydroxy- phenylalanine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, di
  • nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity.
  • the percent identity may be measured using sequence comparison software or algorithms or by visual inspection.
  • nucleic acids or polypeptides of the disclosure are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.
  • identity exists over a region of the amino acid sequences that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80- 100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a target protein or an antibody. In some embodiments, identity exists over a region of the nucleotide sequences that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length or any integral value there between.
  • identity exists over a longer region than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.
  • a conservative amino acid substitution is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic
  • substitution of a phenylalanine for a tyrosine is a conservative substitution.
  • conservative substitutions in the sequences of the polypeptides, soluble proteins, and/or antibodies of the disclosure do not abrogate the binding of the polypeptide, soluble protein, or antibody containing the amino acid sequence, to the target binding site.
  • Methods of identifying amino acid conservative substitutions which do not eliminate binding are well-known in the art.
  • a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature.
  • Isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature.
  • a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.
  • a material is considered substantially pure if the material is at least 50% pure (e.g., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.
  • a fusion polypeptide (e.g., a fusion protein) is a hybrid polypeptide expressed by a nucleic acid molecule comprising at least two open reading frames (e.g., from two individual molecules, e.g., two individual genes).
  • a linker also referred to as a linker region
  • a linker may be inserted between a first polypeptide (e.g., a PD-L1 AFFIMER® polypeptide) and a second polypeptide (e.g., a LAG-3 AFFIMER® polypeptide).
  • a linker is a peptide linker. Linkers should not adversely affect the expression, secretion, or bioactivity of the polypeptides.
  • linkers are not antigenic and do not elicit an immune response.
  • an AFFIMER® polypeptide is linked to an antibody.
  • An “AFFIMER® polypeptide-antibody fusion” is a fusion protein that includes an AFFIMER® polypeptide portion and a variable region of an antibody.
  • AFFIMER® polypeptide-antibody fusions may include full length antibodies having, for example, at least one AFFIMER® polypeptide sequence appended to the C-terminus or N-terminus of at least one of its VH and/or VL chains, e.g., at least one chain of the assembled antibody is a fusion protein with at least one AFFIMER® polypeptide.
  • AFFIMER® polypeptide-antibody fusions may also include at least one AFFIMER® polypeptide sequence as part of a fusion protein with an antigen binding site or variable region of an antibody fragment.
  • An antibody is an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of any of the foregoing, through at least one antigen-binding site wherein the antigen-binding site is usually within the variable region of the immunoglobulin molecule.
  • antibody encompasses intact (whole) polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) antibodies provided those fragments have been formatted to include an Fc or other Fc ⁇ RIII binding domain, multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen-binding site of an antibody (formatted to include an Fc or other Fc ⁇ RIII binding domain), antibody mimetics, and any other modified immunoglobulin molecule comprising an antigen-binding site as long as the antibodies exhibit the desired biological activity.
  • antibody fragments such as Fab, Fab', F(ab')2, and Fv fragments
  • scFv single chain Fv
  • the antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu.
  • a variable region of an antibody may be a variable region of an antibody light chain or a variable region of an antibody heavy chain, either alone or in combination.
  • variable region of heavy and light chains includes four framework regions (FR) and three complementarity determining regions (CDRs), also known as hypervariable regions.
  • the CDRs in each chain are held together in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding sites of the antibody.
  • a humanized antibody is a form of a non-human (e.g., murine) antibody that is specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequences.
  • humanized antibodies are human immunoglobulins in which residues of the CDRs are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and/or binding capability.
  • the Fv framework region residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species.
  • the humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or binding capability.
  • the humanized antibody may comprise variable domains containing all or substantially all of the CDRs that correspond to the non- human immunoglobulin whereas all or substantially all of the framework regions are those of a human immunoglobulin sequence.
  • the variable domains comprise the framework regions of a human immunoglobulin sequence.
  • the variable domains comprise the framework regions of a human immunoglobulin consensus sequence.
  • the humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region or domain
  • a humanized antibody is usually considered distinct from a chimeric antibody.
  • An epitope also referred to herein as an antigenic determinant is the portion of an antigen capable of being recognized and specifically bound by a particular antibody, a particular AFFIMER® polypeptide or other particular binding domain.
  • epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein.
  • Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost upon protein denaturing.
  • An epitope typically includes at least 3, and more usually, at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation.
  • "Specifically binds to” or is "specific for” refers to measurable and reproducible interactions such as binding between a target (e.g., PD-L1 or LAG-3) and an AFFIMER® polypeptide, antibody or other binding partner, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules.
  • an AFFIMER® polypeptide that specifically binds to PD-L1 is an AFFIMER® polypeptide that binds PD-L1 with greater affinity, avidity (if multimeric formatted), more readily, and/or with greater duration than it binds to other targets.
  • an AFFIMER® polypeptide that specifically binds to LAG-3 is an AFFIMER® polypeptide that binds LAG-3 with greater affinity, avidity (if multimeric formatted), more readily, and/or with greater duration than it binds to other targets.
  • Conjugate “conjugation” and grammatical variations thereof refers the joining or linking together of two or more compounds resulting in the formation of another compound, by any joining or linking methods known in the art.
  • a polynucleotide (also referred to herein as a nucleic acid or a nucleic acid molecule) is a polymer of nucleotides of any length and may comprise DNA, RNA (e.g., messenger RNA (mRNA)) or a combination of DNA and RNA.
  • mRNA messenger RNA
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
  • a polynucleotide encoding a polypeptide refers to the order or sequence of nucleotides along a strand of deoxyribonucleic acid deoxyribonucleotides. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (e.g., protein) chain.
  • a nucleic acid sequence encodes the amino acid sequence.
  • a “sequence” may comprise DNA and/or RNA (e.g., messenger RNA) and may be single and/or double stranded.
  • Nucleic acid sequences may be modified, e.g., mutated, relative to naturally occurring nucleic acid sequences, for example.
  • Nucleic acid sequence may have any length, for example 2 to 000,000 or more nucleotides (or any integral value above or between) a nucleic acid, for example a length of from about 100 to about 10,000, or from about 200 nucleotides to about 500 nucleotides.
  • Transfection is the process of introducing an exogenous nucleic acid into a eukaryotic cell.
  • Transfection can be achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE- dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics technology (biolistics).
  • a vector is a construct that is capable of delivering, and usually expressing, at least one gene or sequence of interest in a host cell.
  • vectors include but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.
  • a vector may, in some embodiments, be an isolated nucleic acid that can be used to deliver a composition to the interior of the cell. It is known in the art a number of vectors including, but not limited to the linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, a vector may be an autonomously replicating plasmid or virus.
  • Non-limiting examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated virus vectors, and retroviral vectors.
  • An expression vector is a vector comprising a recombinant polynucleotide comprising expression control sequence and a nucleotide sequence to be expressed operably linked.
  • the expression vector comprises sufficient cis-acting elements (cis-acting elements) used for expression; other elements for expression can be supplied by the host cell or in vitro expression system.
  • Expression vectors include, for example, cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentivirus, retroviruses, adenoviruses and adeno-associated viruses).
  • Operably linked refers to functional linkage between the regulatory sequence and a heterologous nucleic acid sequence resulting in the expression of the latter.
  • the promoter affects the transcription or expression of the coding sequence
  • the promoter is operably linked to a coding sequence.
  • DNA sequencing operably linked are contiguous, and may join two protein coding regions in the same reading frame.
  • a promoter is a DNA sequence recognized by the synthetic machinery required for the synthesis machinery of the cell specific transcription of a polynucleotide sequence or introduced.
  • Inducible expression refers to expression under certain conditions, such as activation (or inactivation) of an intracellular signaling pathway or the contacting of the cells harboring the expression construct with a small molecule that regulates the expression (or degree of expression) of a gene operably linked to an inducible promoter sensitive to the concentration of the small molecule. This is contrasted with constitutive expression, which refers to expression under physiological conditions (not limited by certain conditions).
  • Electroporation refers to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane; their presence allows biomolecules such as plasmids or other oligonucleotide to pass from one side of the cellular membrane to the other.
  • C. Checkpoint Inhibitors, Co-stimulatory Agonists and Chemotherapeutics A checkpoint molecule is a protein that is expressed by tissues and/or immune cells and reduce the efficacy of an immune response in a manner dependent on the level of expression of the checkpoint molecule. When these proteins are blocked, the “brakes” on the immune system are released and, for example, T cells are able to kill cancer cells more effectively.
  • checkpoint proteins found on T cells or cancer cells include PD-1/PD-L1 and CTLA-4/B7-1/B7- 2, LAG-3, PD-L2, NKG2A, KIR, TIM-3, CD96, VISTA and TIGIT.
  • a checkpoint inhibitor is a drug entity that reverses the immunosuppressive signaling from a checkpoint molecule.
  • a costimulatory molecule is an immune cell such as a T cell cognate binding partner that specifically binds to costimulatory ligands thereby mediating co-stimulation, such as, but not limited to proliferation.
  • Costimulatory molecules are cell surface molecules other than the antigen receptor or ligand which facilitate an effective immune response.
  • Co-stimulatory molecules include but are not limited to MHCI molecules, BTLA receptor and Toll ligands, and OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) and 4-1BB (CD137).
  • costimulatory molecules include but are not limited to: CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA- 1, ITGAM, CD11b, ITGAX, CD11c, ITGB1 , CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244,2B4), CD84, CD96 (Tactile), CEACAM1,
  • a costimulatory agonist is a drug entity that activates (agonizes) the costimulatory molecule, such as costimulatory ligand would do, and produces an immunostimulatory signal or otherwise increases the potency or efficacy of an immune response.
  • a chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
  • chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN), CPT-11 (irinotecan, CAMPTOSAR),
  • calicheamicin especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN, morpholin
  • Chemotherapeutic agents also include anti-hormonal agents that act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer, and are often in the form of systemic, or whole-body treatment. They may be hormones themselves. Examples include anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX tamoxifen), raloxifene (EVISTA), droloxifene, 4- hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON); anti-progesterones; estrogen receptor down-regulators (ERDs); estrogen receptor antagonists such as fulvestrant (FASLODEX); agents that function to suppress or shut down the ovaries, for example, leutinizing hormone-releasing hormone (LHRH) agonists such as leuprolide acetate (LUPRON and ELIG
  • chemotherapeutic agents includes bisphosphonates such as clodronate (for example, BONEFOS or OSTAC), etidronate (DIDROCAL), NE-58095, zoledronic acid/zoledronate (ZOMETA), alendronate (FOSAMAX), pamidronate (AREDIA), tiludronate (SKELID), or risedronate (ACTONEL); as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); anti-sense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE vaccine and gene therapy vaccines, for example, ALLOVECTIN vaccine, LEUVECTIN vaccine, and VAXID vaccine; topoisomerase 1 inhibitor (e.g.,
  • a cytokine is a protein released by one cell that act on another cell as intercellular mediators or have an autocrine effect on the cells producing the proteins.
  • cytokines include lymphokines, monokines; interleukins (“ILs”) such as IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL10, IL-11, IL-12, IL-13, IL-15, IL-17A-F, IL-18 to IL-29 (such as IL-23), IL-31, including PROLEUKIN rIL-2; a tumor-necrosis factor such as TNF- ⁇ or TNF- ⁇ , TGF- ⁇ 1-3; and other polypeptide factors including leukemia inhibitory factor ("LIF”), ciliary neurotrophic factor (“CNTF”), CNTF-like cytokine (“CLC”), cardiotrophin (“CT”), and kit ligand ("KL”).
  • LIF
  • a chemokine is a soluble factor (e.g., cytokine) that has the ability to selectively induce chemotaxis and activation of leukocytes. Chemokines also trigger processes of angiogenesis, inflammation, wound healing, and tumorigenesis. Non-limiting examples of chemokines include IL-8, a human homolog of murine keratinocyte chemoattractant (KC).
  • a growth factor is a substance, such as a vitamin or hormone, that is required for the stimulation of growth in living cells.
  • the AFFIMER® polypeptide can be combined with a growth factor selected from the group consisting of: adrenomedullin (AM), angiopoietin (Ang), BMPs, BDNF, EGF, erythropoietin (EPO), FGF, GDNF, G-CSF, GM-CSF, GDF9, HGF, HDGF, IGF, migration-stimulating factor, myostatin (GDF-8), NGF, neurotrophins, PDGF, thrombopoietin, TGF- ⁇ , TGF- ⁇ , TNF- ⁇ , VEGF, P1GF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, and IL-18.
  • a growth factor selected from the group consisting of: adrenomedullin (AM), angiopoietin (Ang), BMPs, BDNF, EGF, erythro
  • LAG-3/PD-L1 AFFIMER® polypeptides may be conjugated to a sialidase, for example, so that the sialidase will cleave sialic acid motifs from the surface of PD-L1+ cells.
  • Targeted cleavage of sialic acid motifs on the surface of HER2+ breast cancer cells has been shown to increase sensitivity to NK cell-mediated killing and may have a similar effect on PD-L1+ cancer cells. (10.1073/pnas.1608069113).
  • disfunctional includes refractory or unresponsive to antigen recognition, specifically, impaired capacity to translate antigen recognition into down-stream T-cell effector functions, such as proliferation, cytokine production (e.g., IL-2) and/or target cell killing.
  • Anergy refers to the state of unresponsiveness to antigen stimulation resulting from incomplete or insufficient signals delivered through the T-cell receptor (e.g., increase in intracellular Ca +2 in the absence of ras-activation). T cell anergy can also result upon stimulation with antigen in the absence of co-stimulation, resulting in the cell becoming refractory to subsequent activation by the antigen even in the context of costimulation.
  • Exhaustion refers to T cell exhaustion as a state of T cell dysfunction that arises from sustained TCR signaling that occurs during many chronic infections and cancer. It is distinguished from anergy in that it arises not through incomplete or deficient signaling, but from sustained signaling. It is defined by poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of infection and tumors.
  • Enhancing T-cell function means to induce, cause or stimulate a T-cell to have a sustained or amplified biological function, or renew or reactivate exhausted or inactive T-cells.
  • Examples of enhancing T-cell function include: increased secretion of ⁇ -interferon from CD8+ T-cells, increased proliferation, increased antigen responsiveness (e.g., viral, pathogen, or tumor clearance) relative to such levels before the intervention.
  • the level of enhancement is as least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. The manner of measuring this enhancement is known to one of ordinary skill in the art.
  • Tumor immunity refers to the process in which tumors evade immune recognition and clearance.
  • tumor immunity is “treated” when such evasion is attenuated, and the tumors are recognized and attacked by the immune system.
  • tumor recognition include tumor binding, tumor shrinkage and tumor clearance.
  • sustained response refers to the sustained effect on reducing tumor growth after cessation of a treatment.
  • the tumor size may remain to be the same or smaller as compared to the size at the beginning of the administration phase.
  • the sustained response has a duration at least the same as the treatment duration, at least 1.5x, 2.0x, 2.5x, or 3.0x length of the treatment duration.
  • a cancer is physiological condition in mammals in which a population of cells are characterized by unregulated cell growth.
  • cancer examples include but are not limited to, carcinoma, blastoma, sarcoma, and hematologic cancers such as lymphoma and leukemia.
  • a tumor also referred to as a neoplasm
  • Tumor growth is generally uncontrolled and progressive, does not induce or inhibit the proliferation of normal cells.
  • Tumor can affect a variety of cells, tissues or organs, including but not limited to selected from bladder, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tube, gall bladder, heart, intestine, kidney, liver, lung, lymph node, neural tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, urethra, ureter, urethra, uterus, vagina organ or tissue or the corresponding cells.
  • Tumors include cancers, such as sarcoma, carcinoma, plasmacytoma or (malignant plasma cells).
  • Tumors of the present disclosure may include but are not limited to leukemias (e.g., acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myeloid - monocytic leukemia, acute monocytic leukemia, acute leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphomas (Hodgkin's disease, non-Hodgkin's disease), primary macroglobulinemia disease, heavy chain disease, and solid tumors such as sarcomas cancer (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endothelium sarcoma, lymphangiosarcoma, angiosarcoma, lymphangioendothelio s
  • a tumor includes, but is not limited to: pancreatic cancer, liver cancer, lung cancer (including NSCLC), stomach cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer (including triple negative breast cancer), lymphoma, gallbladder cancer, renal cancer, leukemia, multiple myeloma, ovarian cancer, cervical cancer and glioma.
  • the tumor is a PD-L1-positive tumor, such as a non-small cell lung carcinoma (NSCLC), colorectal cancer, advanced melanoma, or renal cell carcinoma.
  • NSCLC non-small cell lung carcinoma
  • Metastasis refers to the process by which a cancer spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion at the new location.
  • a “metastatic” or “metastasizing” cell is one that loses adhesive contacts with neighboring cells and migrates via the bloodstream or lymph from the primary site of disease to invade neighboring body structures.
  • Cancer cell and “tumor cell” refers to the total population of cells derived from a cancer or tumor or pre-cancerous lesion, including both non-tumorigenic cells, which comprise the bulk of the cancer cell population, and tumorigenic stem cells (cancer stem cells).
  • cancer cell or tumor cell
  • tumor cell will be modified by the term “non-tumorigenic” when referring solely to those cells lacking the capacity to renew and differentiate to distinguish those tumor cells from cancer stem cells.
  • a “complete response” or “CR” refers to disappearance of all target lesions;
  • Partial response” or “PR” refers to at least a 30% decrease in the sum of the longest diameters (SLD) of target lesions, taking as reference the baseline SLD;
  • stable disease or “SD” refers to neither sufficient shrinkage of target lesions to qualify for PR, nor sufficient increase to qualify for PD, taking as reference the smallest SLD since the treatment started.
  • Progression free survival refers to the length of time during and after treatment during which the disease being treated (e.g., cancer) does not get worse. Progression-free survival may include the amount of time patients have experienced a complete response or a partial response, as well as the amount of time patients have experienced stable disease.
  • “Overall response rate” refers to the sum of complete response (CR) rate and partial response (PR) rate.
  • Overall survival refers to the percentage of individuals in a group who are likely to be alive after a particular duration of time.
  • Treatment refers to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and (2) prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder.
  • those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented.
  • a subject is successfully "treated” according to the methods of the present disclosure if the patient shows at least one of the following: an increased immune response, an increased anti-tumor response, increased cytolytic activity of immune cells, increased killing of tumor cells by immune cells, a reduction in the number of or complete absence of cancer cells; a reduction in the tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including the spread of cancer cells into soft tissue and bone; inhibition of or an absence of tumor or cancer cell metastasis; inhibition or an absence of cancer growth; relief of at least one symptom associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity; reduction in the number or frequency of cancer stem cells; or some combination of effects.
  • Subject refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, and rodents.
  • the subject is a PD-1/PD-L1 refractory subject (e.g., a subject that has been or is being treated for a disease or condition and does not respond to attempted forms of treatment for said disease or condition).
  • a cancer is said to be refractory when it does not respond to (or is resistant to) cancer treatment.
  • a refractory cancer is also known as resistant cancer.
  • a refractory subject is a subject that does not respond or is resistant to treatment of a disease or condition the subject is suffering from.
  • a refractory subject is a cancer patient unresponsive to anti-PD-1 and/or anti-PD- L1 therapy.
  • “refractory” refers to a subject having less than 20% reduction in tumor size or volume after administration of an anti-PD-1 and/or anti-PD-L1 agent relative to a control.
  • a refractory subject shows less than 10% reduction in tumor size or volume after administration of anti-PD-1 and/or anti-PD-L1 agent relative to a control.
  • a refractory subject shows less than 5% reduction in tumor size or volume after administration of anti-PD-1 and/or anti-PD-L1 agent relative to a control. In some embodiments, a refractory subject shows less than 1%> reduction in tumor size or volume after administration of anti-PD-1 and/or anti-PD-L1 agent relative to a control. In embodiments, a refractory subject shows less than 0.5% reduction in tumor size or volume after administration of anti-PD-1 and/or anti-PD-L1 agent relative to a control. In embodiments, a refractory subject shows less than 0.1%> reduction in tumor size or volume after administration of anti-PD-1 and/or anti-PD-L1 agent relative to a control.
  • Antagonist and agonistic refer to agents that are capable of, directly or indirectly, substantially inducing, activating, promoting, increasing, or enhancing the biological activity of a target or target pathway.
  • Ant is used herein to include any agent that partially or fully induces, activates, promotes, increases, or enhances the activity of a protein or other target of interest.
  • Antagonist and antagonistic refer to or describe an agent that is capable of, directly or indirectly, partially or fully blocking, inhibiting, reducing, or neutralizing a biological activity of a target and/or pathway.
  • antagonist is used herein to include any agent that partially or fully blocks, inhibits, reduces, or neutralizes the activity of a protein or other target of interest.
  • Modulation and modulate refer to a change or an alteration in a biological activity. Modulation includes, but is not limited to, stimulating an activity or inhibiting an activity. Modulation may be an increase in activity or a decrease in activity, a change in binding characteristics, or any other change in the biological, functional, or immunological properties associated with the activity of a protein, a pathway, a system, or other biological targets of interest.
  • An immune response includes responses from both the innate immune system and the adaptive immune system. It includes both cell-mediated and/or humoral immune responses. It includes both T-cell and B-cell responses, as well as responses from other cells of the immune system such as natural killer (NK) cells, monocytes, macrophages, etc.
  • NK natural killer
  • “Pharmaceutically acceptable” refers to a substance approved or approvable by a regulatory agency of the Federal government or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.
  • “Pharmaceutically acceptable excipient” is an excipient, carrier or adjuvant that can be administered to a subject, together with at least one agent of the present disclosure, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic effect.
  • those of skill in the art and the U.S. FDA consider a pharmaceutically acceptable excipient, carrier, or adjuvant to be an inactive ingredient of any formulation.
  • an “effective amount” (also referred to herein as a “therapeutically effective amount” is an amount of an agent, such as a LAG-3/PD-L1 AFFIMER® agent, effective to treat a disease or disorder in a subject such as, a mammal.
  • the therapeutically effective amount of a LAG-3/PD-L1 AFFIMER® agent has a therapeutic effect and as such can boost the immune response, boost the anti-tumor response, increase cytolytic activity of immune cells, increase killing of tumor cells by immune cells, reduce the number of tumor cells; decrease tumorigenicity, tumorigenic frequency or tumorigenic capacity; reduce the number or frequency of cancer stem cells; reduce the tumor size; reduce the cancer cell population; inhibit or stop cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibit and stop tumor or cancer cell metastasis; inhibit and stop tumor or cancer cell growth; relieve to some extent at least one of the symptoms associated with the cancer; reduce morbidity and mortality; improve quality of life; or a combination of such effects.
  • An AFFIMER® polypeptide is a scaffold based on a Stefin A polypeptide, meaning that it has a sequence which is derived from a Stefin A polypeptide, for example, a mammalian Stefin A polypeptide, for example, a human Stefin A polypeptide.
  • Some aspects of the application provide a protein that comprises an AFFIMER® polypeptide that binds LAG-3 (also referred to as “LAG-3 AFFIMER® polypeptides”) in which at least one of the solvent accessible loops from the wild-type Stefin A protein binds LAG-3, preferably selectively, and preferably with Kd of 10 -6 M or less.
  • LAG-3 AFFIMER® polypeptides also referred to as “LAG-3 AFFIMER® polypeptides” in which at least one of the solvent accessible loops from the wild-type Stefin A protein binds LAG-3, preferably selectively, and preferably with Kd of 10 -6 M or less.
  • a fusion protein that comprises an AFFIMER® polypeptide that binds LAG-3 and an AFFIMER® polypeptide that binds PD-L1 (also referred to as “LAG-3/PD-L1 AFFIMER® polypeptides”) in which at least one of the solvent accessible loops from the wild-type Stefin A protein binds LAG-3, preferably selectively, and preferably with Kd of 10 -6 M or less, and in which at least one of the solvent accessible loops from the wild-type Stefin A protein binds PD-L1, preferably selectively, and preferably with Kd of 10 -6 M or less.
  • a protein or fusion protein further comprises an AFFIMER® polypeptide that binds human serum albumin (HSA) (also referred to as “LAG-3/PD-L1/HSA AFFIMER® polypeptides”) in which at least one of the solvent accessible loops from the wild-type Stefin A protein binds HSA, preferably selectively, and preferably with Kd of 10 -6 M or less
  • HSA human serum albumin
  • LAG-3 binding polypeptide comprising the following Formula (I): FR1-(X')-FR2-(X'')-FR3 (I).
  • FR1 in some embodiments, comprises an amino acid sequence having at least 90% to the amino acid sequence of MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA (SEQ ID NO: 216).
  • FR1 in some embodiments, comprises an amino acid sequence having at least 95% identity to the amino acid sequence of MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA (SEQ ID NO: 216).
  • FR1 in some embodiments, comprises an amino acid sequence having 100% identity to the amino acid sequence of MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA (SEQ ID NO: 216).
  • FR2 in some embodiments, comprises an amino acid sequence having at least 90% identity to the amino acid sequence of STNYYIKVRAGDNKYMHLKVFNGP (SEQ ID NO: 217).
  • FR2 in some embodiments, comprises an amino acid sequence having 100% identity to the amino acid sequence of STNYYIKVRAGDNKYMHLKVFNGP (SEQ ID NO: 217).
  • FR3 in some embodiments, comprises an amino acid sequence having at least 90% identity to the amino acid sequence of ADRVLTGYQVDKNKDDELTGF (SEQ ID NO: 218).
  • FR3 in some embodiments, comprises an amino acid sequence having at least 95% identity to the amino acid sequence of ADRVLTGYQVDKNKDDELTGF (SEQ ID NO: 218).
  • FR3 in some embodiments, comprises an amino acid sequence having 100% identity to the amino acid sequence of ADRVLTGYQVDKNKDDELTGF (SEQ ID NO: 218).
  • a LAG-3 binding polypeptide in some embodiments, comprises the amino acid sequence having of MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA-(X')- STNYYIKVRAGDNKYMHLKVFNGP-(X'')-ADRVLTGYQVDKNKDDELTGF.
  • X' is an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NOs: 204-209.
  • X' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 204.
  • X' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 205. In some embodiments, X' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 206. In some embodiments, X' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 207. In some embodiments, X' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 208. In some embodiments, X' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 209.
  • X' is an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NOs: 204-209. In some embodiments, X' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 204. In some embodiments, X' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 205. In some embodiments, X' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 206. In some embodiments, X' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 207. In some embodiments, X' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 208.
  • X' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 209. In some embodiments, X'' is an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NOs: 210-215. In some embodiments, X'' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 210. In some embodiments, X'' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 211. In some embodiments, X'' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 212.
  • X'' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 213. In some embodiments, X'' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 214. In some embodiments, X'' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 215. In some embodiments, X'' is an amino acid sequence having at 100% identity to the amino acid sequence of any one of SEQ ID NOs: 210-215. In some embodiments, X'' is an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NOs: 210-215.
  • X'' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 210. In some embodiments, X'' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 211. In some embodiments, X'' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 212. In some embodiments, X'' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 213. In some embodiments, X'' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 214. In some embodiments, X'' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 215.
  • the polypeptide binds to LAG-3 with a Kd of 1 ⁇ 10 ⁇ 6 M or less.
  • X' is the amino acid sequence of SEQ ID NO: 204 and X'' is the amino acid sequence of SEQ ID NO: 210.
  • X' is the amino acid sequence of SEQ ID NO: 205 and X'' is the amino acid sequence of SEQ ID NO: 211.
  • X' is the amino acid sequence of SEQ ID NO: 206 and X'' is the amino acid sequence of SEQ ID NO: 212.
  • X' is the amino acid sequence of SEQ ID NO: 207 and X'' is the amino acid sequence of SEQ ID NO: 213.
  • X' is the amino acid sequence of SEQ ID NO: 208 and X'' is the amino acid sequence of SEQ ID NO: 214. In some embodiments, X' is the amino acid sequence of SEQ ID NO: 209 and X'' is the amino acid sequence of SEQ ID NO: 215.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 53.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 54.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 61.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 62.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 66.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 69.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 53.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 54.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 61.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 62.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 66.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 69.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 53.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 54.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 61.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 62.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 66.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 69.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 53.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 54.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 61.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 62.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 66.
  • a LAG-3 binding polypeptide in some embodiments, comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 69.
  • a LAG-3 binding polypeptide in some embodiments, the amino acid sequence of SEQ ID NO: 53.
  • a LAG-3 binding polypeptide in some embodiments, comprises the amino acid sequence of SEQ ID NO: 54.
  • a LAG-3 binding polypeptide in some embodiments, comprises the amino acid sequence of SEQ ID NO: 61.
  • a LAG-3 binding polypeptide in some embodiments, comprises the amino acid sequence of SEQ ID NO: 62.
  • a LAG-3 binding polypeptide in some embodiments, comprises the amino acid sequence of SEQ ID NO: 66.
  • a LAG-3 binding polypeptide in some embodiments, comprises the amino acid sequence of SEQ ID NO: 69.
  • the LAG-3 binding polypeptide further comprises a half-life extension moiety.
  • the half-life extension moiety is a human serum albumin (HSA) binding polypeptide or a fragment crystallizable (Fc) region of an antibody.
  • the antibody is a human IgG1 antibody.
  • the antibody is a human IgG4 Fc antibody.
  • a bispecific fusion protein comprises: one or more PD-L1 binding polypeptide; and one or more LAG-3 binding polypeptide.
  • a PD-L1 polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72.
  • a PD- L1 polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, a PD-L1 polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, a PD-L1 polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, a PD-L1 polypeptide comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, a PD-L1 polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 73.
  • a PD- L1 polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, a PD-L1 polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, a PD-L1 polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, a PD-L1 polypeptide comprises the amino acid sequence of SEQ ID NO: 73.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 61.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 61.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 61.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 61.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 61.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 61.
  • a bispecific fusion protein comprises: a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73; and a second LAG-3 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 62.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 62.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 62.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 62.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 62.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 62.
  • a bispecific fusion protein comprises: a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 53.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 53.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 53.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 53.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 53.
  • a bispecific fusion protein comprises: a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 53.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 53.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 53.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 53.
  • a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 72; and a LAG-3 binding polypeptide comprising an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 53.
  • Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 1. Some aspects provide a bispecific fusion protein comprising the amino acid sequence of SEQ ID NO: 1. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 2. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 2. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 2.
  • Some aspects provide a bispecific fusion protein comprising the amino acid sequence of SEQ ID NO: 2. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 3. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 3. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 3. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 3. Some aspects provide a bispecific fusion protein comprising the amino acid sequence of SEQ ID NO: 3.
  • Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 5. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 5. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 5. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 5. Some aspects provide a bispecific fusion protein comprising the amino acid sequence of SEQ ID NO: 5.
  • a bispecific fusion protein further comprises a second PD-L1 binding polypeptide and/or a second LAG-3 binding polypeptide.
  • the second PD-L1 polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72.
  • the second PD-L1 polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72.
  • the second PD-L1 polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72.
  • the second PD-L1 polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the second PD-L1 polypeptide comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the second PD-L1 polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the second PD-L1 polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the second PD-L1 polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 73.
  • the second PD-L1 polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the second PD-L1 polypeptide comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the second LAG-3 polypeptide comprises an amino acid sequence of any one of the LAG-3 polypeptides describe herein. Other aspects provide bispecific protein comprising: a first PD-L1 binding polypeptide; a second PD-L1 polypeptide; and any one of the LAG-3 binding polypeptides described herein.
  • each of the first PD-L1 binding polypeptide and the second PD- L1 polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 53
  • each of the first PD-L1 binding polypeptide and the second PD-L1 polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 86% identity to the amino acid sequence of SEQ ID NO: 53.
  • each of the first PD-L1 binding polypeptide and the second PD-L1 polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 53
  • each of the first PD-L1 binding polypeptide and the second PD-L1 polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 53.
  • each of the first PD-L1 binding polypeptide and the second PD-L1 polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 53.
  • each of the first PD-L1 binding polypeptide and the second PD- L1 polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 74
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 53.
  • each of the first PD-L1 binding polypeptide and the second PD-L1 polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 74
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 86% identity to the amino acid sequence of SEQ ID NO: 53.
  • each of the first PD-L1 binding polypeptide and the second PD-L1 polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 74
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 53.
  • each of the first PD-L1 binding polypeptide and the second PD-L1 polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 74
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 53
  • each of the first PD-L1 binding polypeptide and the second PD-L1 polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 74
  • the LAG-3 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 53.
  • a bispecific fusion protein comprises an amino acid sequence at least 80% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, a bispecific fusion protein comprises an amino acid sequence at least 85% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, a bispecific fusion protein comprises an amino acid sequence at least 90% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, a bispecific fusion protein comprises an amino acid sequence at least 95% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, a bispecific fusion protein comprises the amino acid sequence of SEQ ID NO: 4.
  • the half- life extension moiety is a human serum albumin (HSA)-binding polypeptide.
  • HSA binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 80.
  • the HSA binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 80.
  • the HSA binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 80.
  • the HSA binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the HSA binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 80.
  • the half-life extension moiety is a fragment crystallizable (Fc) region of an antibody, for example, a human IgG1 antibody or a human IgG4 antibody.
  • Fc fragment crystallizable
  • Other aspects provide trispecific fusion protein, comprising: a PD-L1 binding polypeptide; a first LAG-3 binding polypeptide; a second LAG-3 binding polypeptide; and an HSA binding polypeptide.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72 or 73.
  • each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence of the any one of the LAG-3 binding polypeptides described herein.
  • the HSA binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 80.
  • the HSA binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 80.
  • the HSA binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the HSA binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the HSA binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 80.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 74
  • each of the first LAG-3-binding polypeptide and the second LAG-3-binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 54
  • the HSA binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 80.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 74
  • each of the first LAG-3-binding polypeptide and the second LAG- 3-binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 54
  • the HSA binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 80.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 74
  • each of the first LAG-3- binding polypeptide and the second LAG-3-binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 54
  • the HSA binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 80.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 74
  • each of the first LAG-3-binding polypeptide and the second LAG-3-binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 54
  • the HSA binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 80.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 74
  • each of the first LAG-3-binding polypeptide and the second LAG-3-binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 54
  • the HSA binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 80.
  • the trispecific fusion protein comprises an amino acid sequence at least 80% identity to the amino acid sequence of SEQ ID NO: 11.
  • the trispecific fusion protein comprises an amino acid sequence at least 85% identity to the amino acid sequence of SEQ ID NO: 11.
  • the trispecific fusion protein comprises an amino acid sequence at least 90% identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 95% identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the trispecific fusion protein comprises an amino acid sequence 100% identity to the amino acid sequence of SEQ ID NO: 11.
  • each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72.
  • each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 73.
  • each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 73.
  • each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 73.
  • each of the first LAG-3 binding polypeptide and second LAG-3 binding polypeptide comprises an amino acid sequence of any one of the LAG-3 polypeptides described herein.
  • each of HSA binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 80.
  • each of HSA binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 80.
  • each of HSA binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, each of HSA binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, each of HSA binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 80.
  • each of the first PD-L1 binding polypeptide and the second PD- L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72
  • each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 62
  • the HSA binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 80.
  • each of the first PD-L1 binding polypeptide and the second PD-L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72
  • each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 62
  • the HSA binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 80.
  • each of the first PD-L1 binding polypeptide and the second PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72
  • each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 62
  • the HSA binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 80.
  • each of the first PD-L1 binding polypeptide and the second PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72
  • each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 62
  • the HSA binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 80.
  • each of the first PD-L1 binding polypeptide and the second PD-L1 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 72
  • each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 62
  • the HSA binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 80.
  • each of the first PD-L1 binding polypeptide and the second PD- L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72
  • each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 54
  • the HSA binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 80.
  • each of the first PD-L1 binding polypeptide and the second PD-L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72
  • each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 54
  • the HSA binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 80.
  • each of the first PD-L1 binding polypeptide and the second PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72
  • each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 54
  • the HSA binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 80.
  • each of the first PD-L1 binding polypeptide and the second PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72
  • each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 54
  • the HSA binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 80.
  • each of the first PD-L1 binding polypeptide and the second PD-L1 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 72
  • each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 54
  • the HSA binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 80.
  • the trispecific fusion protein comprises an amino acid sequence at least 80% the amino acid sequence of any one of SEQ ID NO: 8.
  • the trispecific fusion protein comprises an amino acid sequence at least 85% the amino acid sequence of any one of SEQ ID NO: 8.
  • the trispecific fusion protein comprises an amino acid sequence at least 90% the amino acid sequence of any one of SEQ ID NO: 8. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 95% the amino acid sequence of any one of SEQ ID NO: 8. In some embodiments, the trispecific fusion protein comprises an amino acid sequence 100% the amino acid sequence of any one of SEQ ID NO: 8. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 80% the amino acid sequence of any one of SEQ ID NO: 9. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 85% the amino acid sequence of any one of SEQ ID NO: 9.
  • the trispecific fusion protein comprises an amino acid sequence at least 90% the amino acid sequence of any one of SEQ ID NO: 9. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 95% the amino acid sequence of any one of SEQ ID NO: 9. In some embodiments, the trispecific fusion protein comprises an amino acid sequence 100% the amino acid sequence of any one of SEQ ID NO: 9. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 80% the amino acid sequence of any one of SEQ ID NO: 10. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 85% the amino acid sequence of any one of SEQ ID NO: 10.
  • the trispecific fusion protein comprises an amino acid sequence at least 90% the amino acid sequence of any one of SEQ ID NO: 10. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 95% the amino acid sequence of any one of SEQ ID NO: 10. In some embodiments, the trispecific fusion protein comprises an amino acid sequence 100% the amino acid sequence of any one of SEQ ID NO: 10.
  • the hIgG1 antibody comprises LALA mutations (Leu234Ala and Leu235Ala mutations, relative to full length human IgG1).
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72.
  • the PD- L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having 100%identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 73.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having 100%identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the LAG-3 binding polypeptide comprises an amino acid sequence of any one or more of the LAG-3 binding polypeptides described herein.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 54
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 54
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 54
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 54
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 54
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 62
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 62
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 62
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 62
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 62
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 66
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 66
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 66
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 66
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 66
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 69
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 69
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 69
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 69
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the PD-L1 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 72
  • the LAG-3 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 69
  • the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
  • the bispecific fusion protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NO: 6.
  • the bispecific fusion protein comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NO: 6.
  • the bispecific fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 6. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 6. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NO: 6. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NO: 7. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NO: 7.
  • the bispecific fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 7. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 7. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NO: 7. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NO: 15. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NO: 15.
  • the bispecific fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 15. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 15. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NO: 15. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NO: 19. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NO: 19.
  • the bispecific fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 19. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 19. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NO: 19. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NO: 38. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NO: 38.
  • the bispecific fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 38. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 38. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NO: 38. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NO: 39. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NO: 39.
  • the bispecific fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 39. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 39. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NO: 39. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NO: 40. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NO: 40.
  • the bispecific fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 40. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 40. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NO: 40. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NO: 41. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NO: 41.
  • the bispecific fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 41. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 41. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NO: 41. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NO: 44. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NO: 44.
  • the bispecific fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 44. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 44. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NO: 44. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NO: 45. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NO: 45.
  • the bispecific fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 45. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 45. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NO: 45. In some embodiments, fusion protein further comprises one or more linker located between two of the polypeptides. In some embodiments, the linker is a rigid linker, optionally comprising the amino acid sequence of AEAAAKEAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 86).
  • the linker is a flexible linker, optionally comprising the amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 87).
  • Table 1 AFFIMER® polypeptides (Loop 2/4 sequences underlined; linkers double underlined; human Fc domains italicized)
  • AVA19 is a LAG3 AFFIMER ® polypeptide
  • AVA04 is a PD-L1 AFFIMER ® polypeptide
  • AVA03 is an HSA/XT AFFIMER ® polypeptide
  • SQTGly and 3t0 are controls; any one or more of the AVA19 LAG3 AFFIMER ® polypeptides of Table 1 may include an N32G mutation A. Fusions Proteins - General
  • the LAG-3/PD-L1 AFFIMER® polypeptides may further comprise an additional insertion, substitution and/or deletion that modulates biological activity of the AFFIMER® polypeptide.
  • the additions, substitutions and/or deletions may modulate at least one property or activity of modified AFFIMER® polypeptide.
  • the additions, substitutions or deletions may modulate affinity for the AFFIMER® polypeptide, e.g., for binding to and inhibiting PD-L1 and/or LAG-3, modulate the circulating half-life, modulate the therapeutic half-life, modulate the stability of the AFFIMER® polypeptide, modulate cleavage by proteases, modulate dose, modulate release or bioavailability, facilitate purification, decrease deamidation, improve shelf-life, or improve or alter a particular route of administration.
  • AFFIMER® polypeptides may comprise protease cleavage sequences, reactive groups, antibody-binding domains (including but not limited to, FLAG or poly-His) or other affinity-based sequences (including but not limited to, FLAG, poly-His, GST, etc.) or linked molecules (including but not limited to, biotin) that improve detection, purification or other traits of the polypeptide.
  • these additional sequences are added to one end and/or the other of the AFFIMER® polypeptide in the form of a fusion protein.
  • the AFFIMER® agent is a fusion protein having at least one AFFIMER® polypeptide sequence and at least one heterologous polypeptide sequence (“fusion domain” herein).
  • a fusion domain may be selected so as to confer a desired property, such as secretion from a cell or retention on the cell surface (e.g., for an encoded AFFIMER® construct), to serve as substrate or other recognition sequences for post-translational modifications, to create multimeric structures aggregating through protein-protein interactions, to alter (often to extend) serum half-life, or to alter tissue localization or tissue exclusion and other ADME (Absorption, Distribution, Metabolism, Excretion) properties – merely as examples.
  • ADME Absorption, Distribution, Metabolism, Excretion
  • fusion domains are particularly useful for isolation and/or purification of the fusion proteins, such as by affinity chromatography.
  • affinity tags such as polyhistidine (e.g., a His 6 tag), Strep II tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA tag, c-Myc tag, thioredoxin, protein A and protein G.
  • the AFFIMER® agent In order for the AFFIMER® agent to be secreted, it will generally contain a signal sequence that directs the transport of the protein to the lumen of the endoplasmic reticulum and ultimately to be secreted (or retained on the cell surface if a transmembrane domain or other cell surface retention signal).
  • Signal sequences also referred to as signal peptides or leader sequences
  • signal sequences are cleaved from the protein by a signal peptidase after the proteins are transported to the endoplasmic reticulum.
  • the cleavage of the signal sequence from the polypeptide usually occurs at a specific site in the amino acid sequence and is dependent upon amino acid residues within the signal sequence.
  • the signal peptide is about 5 to about 40 amino acids in length (such as about 5 to about 7, about 7 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, or about 25 to about 30, about 30 to about 35, or about 35 to about 40 amino acids in length).
  • the signal peptide is a native signal peptide from a human protein.
  • the signal peptide is a non-native signal peptide.
  • the non-native signal peptide is a mutant native signal peptide from the corresponding native secreted human protein, and can include at least one (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) substitution, insertions and/or deletions.
  • the signal peptide is a signal peptide or mutant thereof from a non-IgSF protein family, such as a signal peptide from an immunoglobulin (such as IgG heavy chain or IgG-kappa light chain), a cytokine (such as interleukin-2 (IL-2)), a serum albumin protein (e.g., HSA or albumin), a human azurocidin preprotein signal sequence, a luciferase, a trypsinogen (e.g. chymotrypsinogen or trypsinogen) or other signal peptide able to efficiently secrete a protein from a cell.
  • a signal peptide from an immunoglobulin (such as IgG heavy chain or IgG-kappa light chain), a cytokine (such as interleukin-2 (IL-2)), a serum albumin protein (e.g., HSA or albumin), a human azurocidin preprotein signal sequence, a luci
  • the recombinant polypeptide comprises a signal peptide when expressed, and the signal peptide (or a portion thereof) is cleaved from the AFFIMER® agent upon secretion.
  • the subject fusion proteins may also include at least one linker separating heterologous protein sequences or domains.
  • linker refers to a linker amino acid sequence inserted between a first polypeptide (e.g., an AFFIMER® polypeptide) and a second polypeptide (e.g., a second AFFIMER® polypeptide, an Fc region, a receptor trap, albumin, etc.).
  • Empirical linkers designed by researchers are generally classified into 3 categories according to their structures: flexible linkers, rigid linkers, and in vivo cleavable linkers. Besides the basic role in linking the functional domains together (as in flexible and rigid linkers) or releasing free functional domain in vivo (as in in vivo cleavable linkers), linkers may offer many other advantages for the production of fusion proteins, such as improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles. Linkers should not adversely affect the expression, secretion, or bioactivity of the fusion protein. Linkers should not be antigenic and should not elicit an immune response.
  • Suitable linkers may include mixtures of glycine and serine residues and often include amino acids that are sterically unhindered. Other amino acids that can be incorporated into useful linkers include threonine and alanine residues. Linkers can range in length, for example from 1- 50 amino acids in length, 1-22 amino acids in length, 1-10 amino acids in length, 1-5 amino acids in length, or 1-3 amino acids in length.
  • the linker may comprise a cleavage site.
  • the linker may comprise an enzyme cleavage site, so that the second polypeptide may be separated from the first polypeptide.
  • the linker can be characterized as flexible.
  • Flexible linkers are usually applied when the joined domains require a certain degree of movement or interaction. They are generally composed of small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. See, for example, Argos P. (1990) “An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion” J Mol Biol.211:943– 958. The small size of these amino acids provides flexibility and allows for mobility of the connecting functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduces the unfavorable interaction between the linker and the protein moieties.
  • Gly non-polar
  • Ser or Thr polar amino acids
  • the most commonly used flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues (“GS” linker).
  • An example of the most widely used flexible linker has the sequence of (Gly-Gly-Gly-Gly-Ser)n.
  • the length of this GS linker can be optimized to achieve appropriate separation of the functional domains, or to maintain necessary inter-domain interactions.
  • many other flexible linkers have been designed for recombinant fusion proteins. As these flexible linkers are also rich in small or polar amino acids such as Gly and Ser but can contain additional amino acids such as Thr and Ala to maintain flexibility, as well as polar amino acids such as Lys and Glu to improve solubility.
  • the linker can be characterized as rigid. While flexible linkers have the advantage to connect the functional domains passively and permitting certain degree of movements, the lack of rigidity of these linkers can be a limitation in certain fusion protein embodiments, such as in expression yield or biological activity. The ineffectiveness of flexible linkers in these instances was attributed to an inefficient separation of the protein domains or insufficient reduction of their interference with each other. Under these situations, rigid linkers have been successfully applied to keep a fixed distance between the domains and to maintain their independent functions. Many natural linkers exhibited ⁇ -helical structures. The ⁇ -helical structure was rigid and stable, with intra-segment hydrogen bonds and a closely packed backbone.
  • the stiff ⁇ - helical linkers can act as rigid spacers between protein domains.
  • George et al. (2002) “An analysis of protein domain linkers: their classification and role in protein folding” Protein Eng. 15(11):871-9.
  • rigid linkers exhibit relatively stiff structures by adopting ⁇ -helical structures or by containing multiple Pro residues. Under many circumstances, they separate the functional domains more efficiently than the flexible linkers.
  • the length of the linkers can be easily adjusted by changing the copy number to achieve an optimal distance between domains.
  • rigid linkers are chosen when the spatial separation of the domains is critical to preserve the stability or bioactivity of the fusion proteins.
  • alpha helix-forming linkers with the sequence of A(EAAAK)n (SEQ ID NO: 86) have been applied to the construction of many recombinant fusion proteins.
  • Another type of rigid linkers has a Pro-rich sequence, (XP)n, with X designating any amino acid, preferably Ala, Lys, or Glu.
  • exemplary linkers include: Table 2.
  • Linkers Other linkers that may be used in the subject fusion proteins include but are not limited to, SerGly, GGSG (SEQ ID NO: 181), GSGS (SEQ ID NO: 182), GGGS (SEQ ID NO: 183), S(GGS)n (SEQ ID NO: 184) where n is 1-7, GRA, poly(Gly), poly(Ala), GGGSGGG (SEQ ID NO: 185), ESGGGGVT (SEQ ID NO: 186), LESGGGGVT (SEQ ID NO: 187), GRAQVT (SEQ ID NO: 188), WRAQVT (SEQ ID NO: 189), and ARGRAQVT (SEQ ID NO: 190).
  • the hinge regions of the Fc fusions described below may also be considered linkers.
  • Various elements can be employed to anchor proteins on the plasma membrane of cells.
  • the transmembrane domains (TM) of type-I (oriented with the N-terminus outside the cell) and type-II (oriented with the N-terminus in the cytosol) integral membrane proteins can be used to target chimeric proteins to the plasma membrane.
  • Proteins can also be attached to the cell surface by fusion of a GPI (glycophosphatidylinositol lipid) signal to the 3’ end of genes. Cleavage of the short carboxy-terminal peptide allows attachment of a glycolipid to the newly exposed C-terminus through an amide linkage. See Udenfriend et al.
  • the fusion protein includes a transmembrane polypeptide sequence (a transmembrane domain).
  • the distinguishing features of appropriate transmembrane polypeptides comprise the ability to be expressed at the surface of the cell on which the AFFIMER® agent is to be displayed.
  • that may be an immune cell, in particular lymphocyte cells or Natural killer (NK) cells, and once there to interact with PD-L1 so as to directing cellular response of the immune cell against a predefined target tumor cell on which PD-L1 is upregulated.
  • NK Natural killer
  • the transmembrane domain can be derived either from a natural or from a synthetic source.
  • the transmembrane domain can be derived from any membrane-bound or transmembrane protein.
  • the transmembrane polypeptide can be a subunit of the T cell receptor such as ⁇ , ⁇ , ⁇ or ⁇ , polypeptide constituting CD3 complex, IL2 receptor p55 (a chain), p75 ( ⁇ chain) or ⁇ chain, subunit chain of Fc receptors, in particular Fcy receptor III or CD proteins.
  • the transmembrane domain can be synthetic and can comprise predominantly hydrophobic residues such as leucine and valine.
  • AFFIMER® polypeptides a sequence that signals for the posttranslational addition of a glycosylphosphatidylinositol (GPI) anchor.
  • GPI anchors are glycolipid structures that are added post-translationally to the C-terminus of many eukaryotic proteins. This modification to the AFFIMER® agent will cause it to be anchored (attached) on the extracellular surface of the cell membrane of the cell in which the AFFIMER® agent is re-expressed as a recombinant protein (e.g., an encoded AFFIMER® construct as described below).
  • the GPI anchor domain is C-terminal to the AFFIMER® polypeptide sequence, and preferably occurs at the C-terminus of the fusion protein.
  • the GPI anchor domain is a polypeptide that signals for the posttranslational addition of a GPI anchor when the fusion protein of which it is a part is expressed in a eukaryotic system.
  • the GPI anchor signal sequence consists of a set of small amino acids at the site of anchor addition (the ⁇ site) followed by a hydrophilic spacer and ending in a hydrophobic stretch (Low, (1989) FASEB J.3:1600-1608).
  • GPI anchor attachment can be achieved by expression of the AFFIMER® fusion protein containing the GPI anchor domain in a eukaryotic system capable of carrying out GPI posttranslational modifications.
  • human cells including lymphocytes and other cells involved in initiating or promoting an antitumor are so capable and can be engineered to express and encoded AFFIMER® construct including a GPI anchor domain in order retain the expressed AFFIMER® polypeptide containing fusion on the surface of the engineered cell.
  • Still other modifications that can be made to the AFFIMER® polypeptide sequence or to a flanking polypeptide moiety provided as part of a fusion protein is at least one sequence that is a site for post-translational modification by an enzyme.
  • the AFFIMER® agent may not have a half-life and/or PK profile that is optimal for the route of administration, such as parenteral therapeutic dosing.
  • a “half-life” is the amount of time it takes for a substance, such as an AFFIMER® agent of the present disclosure, to lose half of its pharmacologic or physiologic activity or concentration.
  • Bio half-life can be affected by elimination, excretion, degradation (e.g., enzymatic) of the substance, or absorption and concentration in certain organs or tissues of the body.
  • biological half-life can be assessed by determining the time it takes for the blood plasma concentration of the substance to reach half its steady state level (“plasma half-life”).
  • plasma half-life there are a variety of general strategies for prolongation of half-life that have been used in the case of other protein therapeutics, including the incorporation of half-life extending moieties as part of the AFFIMER® agent.
  • half-life extending moiety refers to a pharmaceutically acceptable moiety, domain, or molecule covalently linked (chemically conjugated or fused) to an AFFIMER® polypeptide to form an AFFIMER® agent described herein, optionally via a non-naturally encoded amino acid, directly or via a linker, that prevents or mitigates in vivo proteolytic degradation or other activity-diminishing modification of the AFFIMER® polypeptide, increases half-life, and/or improves or alters other pharmacokinetic or biophysical properties including but not limited to increasing the rate of absorption, reducing toxicity, improving solubility, reducing protein aggregation, increasing biological activity and/or target selectivity of the modified AFFIMER® polypeptide, increasing manufacturability, and/or reducing immunogenicity of the modified AFFIMER® polypeptide, compared to a comparator such as an unconjugated form of the modified AFFIMER® polypeptide.
  • half-life extending moiety includes non- proteinaceous, half-life extending moieties, such as a water soluble polymer such as polyethylene glycol (PEG) or discrete PEG, hydroxyethyl starch (HES), a lipid, a branched or unbranched acyl group, a branched or unbranched C8-C30 acyl group, a branched or unbranched alkyl group, and a branched or unbranched C8-C30 alkyl group; and proteinaceous half-life extending moieties, such as serum albumin, transferrin, adnectins (e.g., albumin-binding or pharmacokinetics extending (PKE) adnectins), Fc domain, and unstructured polypeptide, such as XTEN and PAS polypeptide (e.g.
  • PEG polyethylene glycol
  • HES hydroxyethyl starch
  • a lipid such as polyethylene glycol (PEG
  • the half-life extending moiety extends the half-life of the resulting AFFIMER® agent circulating in mammalian blood serum compared to the half-life of the protein that is not so conjugated to the moiety (such as relative to the AFFIMER® polypeptide alone).
  • half-life is extended by greater than or greater than about 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold., 5.0-fold, or 6.0-fold. In some embodiments, half-life is extended by more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours, more than 96 hours or more than 1 week after in vivo administration compared to the protein without the half-life extending moiety.
  • half-life extending moieties that can be used in the generation of AFFIMER® agents of the disclosure include: • Genetic fusion of the pharmacologically AFFIMER® sequence to a naturally long-half-life protein or protein domain (e.g., Fc fusion, transferrin [Tf] fusion, or albumin fusion. See, for example, Beck et al. (2011) “Therapeutic Fc-fusion proteins and peptides as successful alternatives to antibodies. MAbs.3:1–2; Czajkowsky et al. (2012) “Fc-fusion proteins: new developments and future perspectives. EMBO Mol Med.4:1015–28; Huang et al.
  • Fc and albumin fusions achieve extended half-lives not only by increasing the size of the peptide drug, but both also take advantage of the body’s natural recycling mechanism: the neonatal Fc receptor, FcRn.
  • the pH-dependent binding of these proteins to FcRn prevents degradation of the fusion protein in the endosome. Fusions based on these proteins can have half-lives in the range of 3-16 days, much longer than typical PEGylated or lipidated peptides. Fusion to antibody Fc domains can improve the solubility and stability of the peptide or protein drug.
  • An example of a peptide Fc fusion is dulaglutide, a GLP-1 receptor agonist currently in late-stage clinical trials.
  • Human serum albumin the same protein exploited by the fatty acylated peptides is the other popular fusion partner.
  • Albiglutide is a GLP-1 receptor agonist based on this platform.
  • a major difference between Fc and albumin is the dimeric nature of Fc versus the monomeric structure of HSA leading to presentation of a fused peptide as a dimer or a monomer depending on the choice of fusion partner.
  • the dimeric nature of an AFFIMER® polypeptide-Fc fusion can produce an avidity effect if the AFFIMER® polypeptide target, such as CD33 on tumor cells, are spaced closely enough together or are themselves dimers. This may be desirable or not depending on the target. 1.
  • the AFFIMER® polypeptide may be part of a fusion protein with an immunoglobulin Fc domain ("Fc domain"), or a fragment or variant thereof, such as a functional Fc region.
  • an Fc fusion (“Fc-fusion”), such as an LAG-3/PD-L1 AFFIMER® agent created as an AFFIMER® polypeptide-Fc fusion protein, is a polypeptide comprising at least one LAG-3/PD-L1 AFFIMER® polypeptide sequence covalently linked through a peptide backbone (directly or indirectly) to an Fc region of an immunoglobulin.
  • An Fc-fusion may comprise, for example, the Fc region of an antibody (which facilitates effector functions and pharmacokinetics) and a LAG- 3/PD-L1 AFFIMER® polypeptide sequence as part of the same polypeptide.
  • An immunoglobulin Fc region may also be linked indirectly to at least one LAG-3/PD-L1 AFFIMER® polypeptide.
  • linkers are known in the art and can optionally be used to link an Fc to a polypeptide including a LAG-3/PD-L1 AFFIMER® polypeptide sequence to generate an Fc-fusion.
  • Fc-fusions can be dimerized to form Fc-fusion homodimers, or using non-identical Fc domains, to form Fc-fusion heterodimers.
  • an Fc-fusion protein comprises a PD-L1 AFFIMER® agent that comprises an PD-L1 AFFIMER® polypeptide linked to an Fc domain linked to another LAG-3 AFFIMER® polypeptide (PD-L1 AFFIMER® polypeptide-Fc domain-LAG-3 AFFIMER® polypeptide).
  • an Fc-fusion protein comprises a tetravalent bispecific Fc fusion protein (e.g., two PD-L1 AFFIMER® polypeptides linked to a first Fc domain and two LAG-3 AFFIMER® polypeptides linked to a second Fc domain).
  • a tetravalent bispecific Fc fusion protein e.g., two PD-L1 AFFIMER® polypeptides linked to a first Fc domain and two LAG-3 AFFIMER® polypeptides linked to a second Fc domain.
  • the principle rationale is to produce a stable protein, large enough to demonstrate a similar pharmacokinetic profile compared with those of antibodies, and to take advantage of the properties imparted by the Fc region; this includes the salvage neonatal FcRn receptor pathway involving FcRn-mediated recycling of the fusion protein to the cell surface post endocytosis, avoiding lysosomal degradation and resulting in release back into the bloodstream, thus contributing to an extended serum half-life.
  • Another obvious advantage is the Fc domain’s binding to Protein A, which can simplify downstream processing during production of the AFFIMER® agent and permit generation of highly pure preparation of the AFFIMER® agent.
  • an Fc domain will include the constant region of an antibody excluding the first constant region immunoglobulin domain.
  • Fc domain refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains.
  • Fc may include the J chain.
  • Fc comprises immunoglobulin domains C ⁇ 2 and C ⁇ 3 and the hinge between C ⁇ 1 and C ⁇ 2.
  • the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as set forth in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NIH, Bethesda, Md. (1991)).
  • Fc may refer to this region in isolation, or this region in the context of a whole antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been observed at a number of different Fc positions and are also included as Fc domains as used herein.
  • the Fc is a functional Fc region.
  • a “functional Fc region” refers to an Fc domain or fragment thereof which retains the ability to bind FcRn.
  • a functional Fc region binds to FcRn but does not possess effector function.
  • the ability of the Fc region or fragment thereof to bind to FcRn can be determined by standard binding assays known in the art.
  • Exemplary "effector functions" include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc.
  • effector functions can be assessed using various assays known in the art for evaluating such antibody effector functions.
  • the Fc domain is derived from an IgG1 subclass, however, other subclasses (e.g., IgG2, IgG3, and IgG4) may also be used.
  • An exemplary sequence of a human IgG1 immunoglobulin Fc domain which can be used is: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 191)
  • the Fc region used in the fusion protein may comprise the hinge region of an Fc molecule.
  • An exemplary hinge region comprises the core hinge residues spanning positions 1-16 (e.g., DKTHTCPPCPAPELLG (SEQ ID NO: 192)) of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above.
  • the AFFIMER® polypeptide-containing fusion protein may adopt a multimeric structure (e.g., dimer) owing, in part, to the cysteine residues at positions 6 and 9 within the hinge region of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above.
  • the hinge region as used herein may further include residues derived from the CH1 and CH2 regions that flank the core hinge sequence of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above.
  • the hinge sequence may comprise or consist of GSTHTCPPCPAPELLG (SEQ ID NO: 193) or EPKSCDKTHTCPPCPAPELLG (SEQ ID NO: 194). In some embodiments, the hinge sequence may include at least one substitution that confer desirable pharmacokinetic, biophysical, and/or biological properties.
  • Some exemplary hinge sequences include: EPKSCDKTHTCPPCPAPELLGGPS (SEQ ID NO: 195); EPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO: 196); EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 197); EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 198); DKTHTCPPCPAPELLGGPS (SEQ ID NO: 199); and DKTHTCPPCPAPELLGGSS (SEQ ID NO: 200).
  • the residue P at position 18 of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above may be replaced with S to ablate Fc effector function; this replacement is exemplified in hinges having the sequences EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 197), EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 198), and DKTHTCPPCPAPELLGGSS (SEQ ID NO: 200).
  • residues DK at positions 1-2 of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above may be replaced with GS to remove a potential clip site; this replacement is exemplified in the sequence EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 198).
  • the C at the position 103 of the heavy chain constant region of human IgG1 may be replaced with S to prevent improper cysteine bond formation in the absence of a light chain; this replacement is exemplified by EPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO: 196), EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 197), and EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 198).
  • the Fc is a mammalian Fc such as a human Fc, including Fc domains derived from IgG1, IgG2, IgG3 or IgG4.
  • the Fc region may possess at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a native Fc region and/or with an Fc region of a parent polypeptide. In some embodiments, the Fc region may have at least about 90% sequence identity with a native Fc region and/or with an Fc region of a parent polypeptide.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • cytotoxic cells e.g., Natural Killer (NK) cells, neutrophils, and macrophages
  • NK Natural Killer
  • the fusion protein includes an Fc domain sequence for which the resulting AFFIMER® agent has no (or reduced) ADCC and/or complement activation or effector functionality.
  • the Fc domain may comprise a naturally disabled constant region of IgG2 or IgG4 isotype or a mutated IgG1 constant region.
  • suitable modifications are described in EP0307434.
  • One example comprises the substitutions of alanine residues at positions 235 and 237 (EU index numbering).
  • the fusion protein includes an Fc domain sequence for which the resulting AFFIMER® agent will retain some or all Fc functionality for example will be capable of one or both of ADCC and CDC activity, as for example if the fusion protein comprises the Fc domain from human IgG1 or IgG3.
  • Levels of effector function can be varied according to known techniques, for example by mutations in the CH2 domain, for example wherein the IgG1 CH2 domain has at least one mutation at positions selected from 239 and 332 and 330, for example the mutations are selected from S239D and I332E and A330L such that the antibody has enhanced effector function, and/or for example altering the glycosylation profile of the antigen- binding protein of the disclosure such that there is a reduction in fucosylation of the Fc region.
  • an LAG-3/PD-L1 AFFIMER® polypeptide has an extended half- life and comprises at least one Fc domain sequence (e.g., IgG1, IgG1 LALA, or IgG4).
  • Exemplary LAG-3/PD-L1 AFFIMER®-Fc fusion proteins are provided in Table 1.
  • the PD-L1-LAG-3 AFFIMER® - Fc fusion protein comprises an amino acid sequence selected from any one of SEQ ID NOs: 6, 7, 15, 19, 38, 39, 40, 41, 44, and 45.
  • the AFFIMER® agent is a fusion protein comprising, in addition to at least one AFFIMER® polypeptide sequence, an albumin sequence or an albumin fragment.
  • the AFFIMER® agent is conjugated to the albumin sequence or an albumin fragment through chemical linkage other than incorporation into the polypeptide sequence including the AFFIMER® polypeptide.
  • the albumin, albumin variant, or albumin fragment is human serum albumin (HSA), a human serum albumin variant, or a human serum albumin fragment.
  • HSA human serum albumin
  • Albumin serum proteins comparable to HSA are found in, for example, cynomolgus monkeys, cows, dogs, rabbits and rats.
  • BSA bovine serum albumin
  • BSA bovine serum albumin
  • the present disclosure contemplates the use of albumin from non-human species, including, but not limited to, albumin sequence derived from cyno serum albumin or bovine serum albumin.
  • Mature HSA a 585 amino acid polypeptide (approx.67 kDa) having a serum half-life of about 20 days, is primarily responsible for the maintenance of colloidal osmotic blood pressure, blood pH, and transport and distribution of numerous endogenous and exogenous ligands.
  • the protein has three structurally homologous domains (domains I, II and III), is almost entirely in the alpha-helical conformation, and is highly stabilized by 17 disulfide bridges.
  • the AFFIMER® agent can be an albumin fusion protein including at least one AFFIMER® polypeptide sequence and the sequence for mature human serum albumin (SEQ ID NO: 201) or a variant or fragment thereof which maintains the PK and/or biodistribution properties of mature albumin to the extent desired in the fusion protein.
  • HSA human serum albumin
  • MKWVTFISLLFLFSSAYS SEQ ID NO: 202
  • RGVFRR pro-domain of 6 amino acids
  • This 24 amino acid residue peptide may be referred to as the pre-pro domain.
  • the AFFIMER® polypeptide-HSA fusion proteins can be expressed and secreted using the HSA pre-pro-domain in the recombinant proteins coding sequence.
  • the AFFIMER® polypeptide-HSA fusion can be expressed and secreted through inclusion of other secretion signal sequences, such as described above.
  • the serum albumin polypeptide can be covalently coupled to the AFFIMER® polypeptide-containing polypeptide through a bond other than a backbone amide bond, such as cross-linked through chemical conjugation between amino acid sidechains on each of the albumin polypeptide and the AFFIMER® polypeptide-containing polypeptide.
  • the HSA AFFIMER® polypeptide is part of an in-line fusion protein (e.g., attached to at least one PD-L1 AFFIMER® polypeptide and at least one LAG-3 AFFIMER® polypeptide).
  • the AFFIMER® polypeptides are attached via linkers (e.g., flexible or rigid linkers, as described herein).
  • the three AFFIMER® polypeptides are arranged (from N-terminal to C-terminal) as follows: HSA-LAG- 3-PDL1, HSA-PDL1-LAG-3; LAG-3-HSA-PDL1, PDL1-HSA-LAG-3, PDL1-LAG-3-HSA, or LAG-3-PDL1-HSA.
  • the in-line fusion protein comprises more than one PD-L1 AFFIMER® polypeptide and/or more than one LAG-3 AFFIMER® polypeptide.
  • the in-line fusion protein is a tetramer and the AFFIMER® polypeptides are arranged (from N-terminal to C-terminal) as follows: PDL1-LAG-3-LAG-3-HSA, PDL1-PDL1- LAG-3-HSA, LAG-3-PDL1-PDL1-HSA, LAG-3-LAG-3-PDL1-HSA, PDL1-LAG-3-HSA- LAG-3, PDL1-PDL1-HSA-LAG-3, LAG-3-PDL1-HSA-PDL1, LAG-3-LAG-3-HSA-PDL1, PDL1-HSA-LAG-3-LAG-3, PDL1-HSA-LAG-3-PDL1, LAG-3-HSA-PDL1-PDL1, LAG-3- HSA-PDL1-LAG-3, HSA-PDL1-PDL1-LAG-3, HSA-PDL1-LAG-3, HSA-PDL1-LA
  • the in-line fusion protein is a pentamer and the AFFIMER® polypeptides are arranged (from N-terminal to C-terminal) as follows: PDL1-PDL1-LAG-3-LAG-3-HSA, PDL1-LAG-3-PDL1-LAG-3-HSA, PDL1-LAG-3- LAG-3-PDL1-HSA, LAG-3-LAG-3-PDL1-PDL1-HSA, LAG-3-PDL1-LAG-3-PDL1-HSA, PDL1-PDL1-LAG-3-HSA-LAG-3, PDL1-LAG-3-PDL1-HSA-LAG-3, PDL1-LAG-3-PDL1-HSA-LAG-3, PDL1-LAG-3-LAG-3- HSA-PDL1, LAG-3-PDL1-LAG-3-HSA-PDL1, LAG-3-LAG-3-PDL1-HSA-PDL1, PDL1- P
  • PDL1 AFFIMER® polypeptides there is an uneven number of PDL1 AFFIMER® polypeptides relative to LAG-3 AFFIMER® polypeptides (i.e., they are not present in the in-line fusion protein at a 1:1 ratio).
  • Exemplary in-line fusion proteins are provided in Table X. 3.
  • the AFFIMER® agent can include a serum-binding moiety – either as part of a fusion protein (if also a polypeptide) with the AFFIMER® polypeptide sequence or chemically conjugated through a site other than being part of a contiguous polypeptide chain.
  • the serum-binding polypeptide is an albumin binding moiety.
  • Albumin contains multiple hydrophobic binding pockets and naturally serves as a transporter of a variety of different ligands such as fatty acids and steroids as well as different drugs. Furthermore, the surface of albumin is negatively charged making it highly water-soluble.
  • albumin binding moiety refers to any chemical group capable of binding to albumin, e.g., has albumin binding affinity.
  • Albumin binds to endogenous ligands such as fatty acids; however, it also interacts with exogenous ligands such as warfarin, penicillin and diazepam.
  • endogenous ligands such as fatty acids
  • exogenous ligands such as warfarin, penicillin and diazepam.
  • the albumin-drug complex serves as a drug reservoir that can enhance the drug biodistribution and bioavailability.
  • Incorporation of components that mimic endogenous albumin-binding ligands, such as fatty acids has been used to potentiate albumin association and increase drug efficacy.
  • lipidation which involves the covalent binding of fatty acids to peptide side chains.
  • PEGylation a basic mechanism for half-life extension as PEGylation, namely increasing the hydrodynamic radius to reduce renal filtration.
  • the lipid moiety is itself relatively small and the effect is mediated indirectly through the non-covalent binding of the lipid moiety to circulating albumin.
  • lipidation reduces the water-solubility of the peptide but engineering of the linker between the peptide and the fatty acid can modulate this, for example by the use of glutamate or mini PEGs within the linker.
  • Linker engineering and variation of the lipid moiety can affect self-aggregation which can contribute to increased half-life by slowing down biodistribution, independent of albumin. See, for example, Jonassen et al. (2012) Pharm Res.29(8):2104-14.
  • PKE2 albumin-binding
  • AdAb albumin binding domain antibody
  • a wide variety of macromolecular polymers and other molecules can be linked to the AFFIMER® polypeptides of the present disclosure to modulate biological properties of the resulting AFFIMER® agent, and/or provide new biological properties to the AFFIMER® agent.
  • These macromolecular polymers can be linked to the AFFIMER® polypeptide via a naturally encoded amino acid, via a non-naturally encoded amino acid, or any functional substituent of a natural or non-natural amino acid, or any substituent or functional group added to a natural or non-natural amino acid.
  • the molecular weight of the polymer may be of a wide range, including but not limited to, between about 100 Da and about 100,000 Da or more.
  • the molecular weight of the polymer may be between about 100 Da and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 50,000 Da.
  • the molecular weight of the polymer is between about 100 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da.
  • various methods including pegylation, polysialylation, HESylation, glycosylation, or recombinant PEG analogue fused to flexible and hydrophilic amino acid chain (500 to 600 amino acids) have been developed (See Chapman, (2002) Adv Drug Deliv Rev.54.
  • polymers include but are not limited to polyalkyl ethers and alkoxy-capped analogs thereof (e.g., polyoxyethylene glycol, polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogs thereof, especially polyoxyethylene glycol, the latter is also known as polyethylene glycol or PEG); discrete PEG (dPEG); polyvinylpyrrolidones; polyvinylalkyl ethers; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyl oxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkyl acrylamides (e.g., polyhydroxypropylmethacrylamide and derivatives thereof); polyhydroxyalkyl acrylates; polysialic acids and analogs thereof; hydrophilic peptide sequences; polysaccharides and their derivatives, including dextran and dextran derivatives, e.g., carboxymethyldextran, dextran sulf
  • the polymer selected may be water soluble so that the AFFIMER® agent to which it is attached does not precipitate in an aqueous environment, such as a physiological environment.
  • the water-soluble polymer may be any structural form including but not limited to linear, forked or branched.
  • the water-soluble polymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG), but other water-soluble polymers can also be employed.
  • PEG poly(ethylene glycol)
  • the polymer may be pharmaceutically acceptable.
  • PEG is used broadly to encompass any polyethylene glycol molecule, without regard to size or to modification at an end of the PEG, and can be represented as linked to the AFFIMER® polypeptide by the formula: XO—(CH 2 CH 2 O) n —CH 2 CH 2 — or XO—(CH 2 CH 2 O) n — where n is 2 to 10,000 and X is H or a terminal modification, including but not limited to, a C1-4 alkyl, a protecting group, or a terminal functional group.
  • a PEG used in the polypeptides of the disclosure terminates on one end with hydroxy or methoxy, e.g., X is H or CH 3 (“methoxy PEG”).
  • the other end of the PEG may attach to the AFFIMER® polypeptide via a naturally-occurring or non- naturally encoded amino acid.
  • the attachment may be through an amide, carbamate or urea linkage to an amine group (including but not limited to, the epsilon amine of lysine or the N-terminus) of the polypeptide.
  • the polymer is linked by a maleimide linkage to a thiol group (including but not limited to, the thiol group of cysteine) – which in the case of attachment to the AFFIMER® polypeptide sequence per se requires altering a residue in the AFFIMER® sequence to a cysteine.
  • the number of water-soluble polymers linked to the AFFIMER® polypeptide e.g., the extent of PEGylation or glycosylation
  • the half-life of the resulting AFFIMER® agent is increased at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 percent, 2-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 50-fold, or at least about 100-fold over an unmodified polypeptide.
  • polymer system useful to modify the PK or other biological properties of the resulting AFFIMER® agent are the use of unstructured, hydrophilic amino acid polymers that are functional analogs of PEG, particularly as part of a fusion protein with the AFFIMER® polypeptide sequence.
  • the inherent biodegradability of the polypeptide platform makes it attractive as a potentially more benign alternative to PEG.
  • Another advantage is the precise molecular structure of the recombinant molecule in contrast to the polydispersity of PEG.
  • XTEN Amunix
  • a recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner” 2009 Nat Biotechnol.27(12):1186-90.
  • a random coil polymer comprised of an even more restricted set of only three small uncharged amino acids, proline, alanine and serine.
  • the PAS modification can be genetically encoded with the AFFIMER® polypeptide sequence to produce an inline fusion protein when expressed.
  • the subject AFFIMER® agents may also include at least one functional moiety intended to impart detectability or additional pharmacologic activity to the AFFIMER® agent.
  • Functional moieties for detection are those which can be employed to detect association of the AFFIMER® agent with a cell or tissue (such as a tumor cell) in vivo.
  • Functional moieties with pharmacologic activity are those agents which are meant to be delivered to the tissue expressing the target of the AFFIMER® agent (PD-L1 and/or LAG-3 in the case of the LAG-3/PD-L1 AFFIMER® agents of the present disclosure) and in doing so have a pharmacologic consequence to the targeted tissues or cells.
  • AFFIMER® agents including conjugates of substances having a wide variety of functional groups, substituents or moieties, with those Functional Moieties including but not limited to a label; a dye; an immunoadhesion molecule; a radionuclide; a cytotoxic compound; a drug; an affinity label; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide; a water-soluble dendrimer; a cyclodextrin; an inhibitory ribonucleic acid; a biomaterial; a nanoparticle; a spin label; a fluorophore, a metal-containing moiety; a radioactive moiety; a novel functional group
  • the moiety is a detectable label, it can be a fluorescent label, radioactive label, enzymatic label or any other label known to the skilled person.
  • the Functional Moiety is a detectable label that can be included as part of a conjugate to form certain AFFIMER® agents suitable for medical imaging.
  • medical imaging is meant any technique used to visualize an internal region of the human or animal body, for the purposes of diagnosis, research or therapeutic treatment.
  • the AFFIMER® agent can be detected (and quantitated) by radioscintigraphy, magnetic resonance imaging (MRI), computed tomography (CT scan), nuclear imaging, positron emission comprising a metal tomography (PET) contrast agent, optical imaging (such as fluorescence imaging including near-infrared fluorescence (NIRF) imaging), bioluminescence imaging, or combinations thereof.
  • the Functional Moiety is optionally a contrast agent for X-ray imaging. Agents useful in enhancing such techniques are those materials that enable visualization of a particular locus, organ or disease site within the body, and/or that lead to some improvement in the quality of the images generated by the imaging techniques, providing improved or easier interpretation of those images.
  • contrast agents Such agents are referred to herein as contrast agents, the use of which facilitates the differentiation of different parts of the image, by increasing the “contrast” between those different regions of the image.
  • contrast agents thus encompasses agents that are used to enhance the quality of an image that may nonetheless be generated in the absence of such an agent (as is the case, for instance, in MRI), as well as agents that are prerequisites for the generation of an image (as is the case, for instance, in nuclear imaging).
  • the detectable label includes a chelate moiety for chelating a metal, e.g., a chelator for a radiometal or paramagnetic ion.
  • the detectable label is a chelator for a radionuclide useful for radiotherapy or imaging procedures.
  • Radionuclides useful within the present disclosure include gamma-emitters, positron-emitters, Auger electron-emitters, X-ray emitters and fluorescence-emitters, with beta- or alpha-emitters for therapeutic use.
  • radionuclides useful as toxins in radiation therapy include: 43 K, 47 Sc, 51 Cr, 57 Co, 58 Co, 59 Fe, 64 Cu, 67 Ga, 67 Cu, 68 Ga, 71 Ge, 75 Br, 76 Br, 77 Br, 77 As, 81 Rb, 90 Y, 97 Ru, 99m Tc, 100 Pd, 101 Rh, 103 Pb, 105 Rh, 109 Pd, 111 Ag, 111 In, 113 In, 119 Sb 121 Sn, 123 I, 125 I, 127 Cs, 128 Ba, 129 Cs, 131 I, 131 Cs, 143 Pr, 153 Sm, 161 Tb, 166 Ho, 169 Eu, 177 Lu, 186 Re, 188 Re, 189 Re, 191 Os, 193 Pt, 194 Ir, 197 Hg, 199 Au, 203 Pb, 211 At, 212 Pb, 212 Bi and 213 Bi.
  • chelators includes, merely to illustrate, 1,4,7- triazacyclononane-N,N',N"-triacetic acid (NOTA) 1,4,7,10-tetraazacyclododecane-N,N',N",N'"- tetraacetic acid (DOTA) 1 ,4,8,11-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid (TETA).
  • NOTA 1,4,7- triazacyclononane-N,N',N"-triacetic acid
  • DOTA 1,4,7,10-tetraazacyclododecane-N,N',N",N'"- tetraacetic acid
  • TETA 1,4,7- triazacyclononane-N,N',N"-triacetic acid
  • detectable isotopes that can be incorporated directly into the amino acid residues of the AFFIMER® polypeptide or which otherwise do not require a chelator, include 3 H, 14 C, 32 P, 35 S and 36 Cl. Paramagnetic ions, useful for diagnostic procedures, may also be administered.
  • paramagnetic ions include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), erbium (III), or combinations of these paramagnetic ions.
  • fluorescent labels include, but are not restricted to, organic dyes (e.g., cyanine, fluorescein, rhodamine, Alexa Fluors, Dylight fluors, ATTO Dyes, BODIPY Dyes, etc.), biological fluorophores (e.g., green fluorescent protein (GFP), R-Phycoerythrin, etc.), and quantum dots.
  • organic dyes e.g., cyanine, fluorescein, rhodamine, Alexa Fluors, Dylight fluors, ATTO Dyes, BODIPY Dyes, etc.
  • biological fluorophores e.g., green fluorescent protein (GFP), R-Phycoerythrin, etc.
  • quantum dots e.g., quantum dots.
  • Non-limiting fluorescent compound that may be used in the present disclosure include, Cy5, Cy5.5 (also known as Cy5++), Cy2, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin, Cy7, fluorescein (FAM), Cy3, Cy3.5 (also known as Cy3++), Texas Red, LightCycler-Red 640, LightCycler Red 705, tetramethylrhodamine (TMR), rhodamine, rhodamine derivative (ROX), hexachlorofluorescein (HEX), rhodamine 6G (R6G), the rhodamine derivative JA133, Alexa Fluorescent Dyes (such as Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 633, Alexa Fluor 555, and Alexa Fluor 647), 4′,6-diamidino-2-phenylindole (DAPI),
  • Fluorescent compound that can be used also include fluorescent proteins, such as GFP (green fluorescent protein), enhanced GFP (EGFP), blue fluorescent protein and derivatives (BFP, EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein and derivatives (CFP, ECFP, Cerulean, CyPet) and yellow fluorescent protein and derivatives (YFP, Citrine, Venus, YPet).
  • fluorescent proteins such as GFP (green fluorescent protein), enhanced GFP (EGFP), blue fluorescent protein and derivatives (BFP, EBFP, EBFP2, Azurite, mKalama1)
  • CFP cyan fluorescent protein and derivatives
  • CFP ECFP, Cerulean, CyPet
  • YFP Citrine, Venus, YPet
  • WO2008142571, WO2009056282, WO9922026 examples include, but are not restricted to, horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxid
  • Biotin labels are typically composed of the biotinyl group, a spacer arm and a reactive group that is responsible for attachment to target functional groups on proteins. Biotin can be useful for attaching the labelled protein to other moieties which comprise an avidin moiety.
  • AFFIMER® Polypeptide-Drug Conjugates In some embodiments, the AFFIMER® agent includes at least one therapeutic agent, e.g., to form an AFFIMER® polypeptide-drug conjugate.
  • therapeutic agent refers to a substance that may be used in the cure, mitigation, treatment, or prevention of disease in a human or another animal.
  • Such therapeutic agents include substances recognized in the official United States Pharmacopeia, official Homeopathic Pharmacopeia of the United States, official National Formulary, or any supplement thereof, and include but are not limited to small molecules, nucleotides, oligopeptides, polypeptides, etc.
  • Therapeutic agents that may be attached to AFFIMER® polypeptides include but are not limited to, cytotoxic agents, anti- metabolites, alkylating agents, antibiotics, growth factor, cytokines, anti-angiogenic agents, anti- mitotic agents, toxins, apoptotic agents or the like, such as DNA alkylating agents, topoisomerase inhibitors, microtubule inhibitors (e.g., DM1, DM4, MMAF and MMAE), endoplasmic reticulum stress inducing agents, platinum compounds, antimetabolites, vincalkaloids, taxanes, epothilones, enzyme inhibitors, receptor antagonists, therapeutic antibodies, tyrosine kinase inhibitors, radiosensitizers, and chemotherapeutic combination therapies, such as illustrations.
  • cytotoxic agents include DNA alkylating agents, topoisomerase inhibitors, microtubule inhibitors (e.g., DM1, DM4, MMAF and MMAE), endoplasm
  • Non-limiting examples of DNA alkylating agents are nitrogen mustards, such as Mechlorethamine, Cyclophosphamide (Ifosfamide, Trofosfamide), Chlorambucil (Melphalan, Prednimustine), Bendamustine, Uramustine and Estramustine; nitrosoureas, such as Carmustine (BCNU), Lomustine (Semustine), Fotemustine, Nimustine, Ranimustine and Streptozocin; alkyl sulfonates, such as Busulfan (Mannosulfan, Treosulfan); Aziridines, such as Carboquone, ThioTEPA, Triaziquone, Triethylenemelamine; Hydrazines (Procarbazine); Triazenes such as dacarbazine and Temozolomide; Altretamine and Mitobronitol.
  • nitrogen mustards such as Mechlorethamine, Cyclophosphamide (Ifosfamide,
  • Topoisomerase I inhibitors include Campothecin derivatives including CPT-11 (irinotecan), SN-38, APC, NPC, campothecin, topotecan, exatecan mesylate, 9-nitrocamptothecin, 9-aminocamptothecin, lurtotecan, rubitecan, silatecan, gimatecan, diflomotecan, extatecan, BN-80927, DX-8951f, and MAG-CPT as described in Pommier Y. (2006) Nat. Rev. Cancer 6(10):789-802 and U.S.
  • Campothecin derivatives including CPT-11 (irinotecan), SN-38, APC, NPC, campothecin, topotecan, exatecan mesylate, 9-nitrocamptothecin, 9-aminocamptothecin, lurtotecan, rubitecan, silatecan, gimatecan, dif
  • Patent Publication No.200510250854 Protoberberine alkaloids and derivatives thereof including berberrubine and coralyne as described in Li et al. (2000) Biochemistry 39(24):7107-7116 and Gatto et al. (1996) Cancer Res. 15(12):2795-2800; Phenanthroline derivatives including Benzo[i]phenanthridine, Nitidine, and fagaronine as described in Makhey et al. (2003) Bioorg. Med.
  • Topoisomerase II inhibitors include but are not limited to Etoposide and Teniposide.
  • Dual topoisomerase I and II inhibitors include but are not limited to, Saintopin and other Naphthecenediones, DACA and other Acridine-4-Carboxamindes, Intoplicine and other Benzopyridoindoles, TAS-103 and other 7H-indeno[2,1-c]Quinoline-7-ones, Pyrazoloacridine, XR 11576 and other Benzophenazines, XR 5944 and other Dimeric compounds, 7-oxo-7H- dibenz[f,ij]Isoquinolines and 7-oxo-7H-benzo[e]Perimidines, and Anthracenyl-amino Acid Conjugates as described in Denny and Baguley (2003) Curr.
  • Top. Med. Chem.3(3):339-353 Some agents inhibit Topoisomerase II and have DNA intercalation activity such as, but not limited to, Anthracyclines (Aclarubicin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Valrubicin, Zorubicin) and Antracenediones (Mitoxantrone and Pixantrone).
  • Anthracyclines Aclarubicin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Valrubicin, Zorubicin
  • Antracenediones Mitoxantrone and Pixantrone
  • Non-limiting examples of DNA synthesis inhibitors include Calicheamicin, Doxorubicin, Duocarmycin, and PBD.
  • Non-limiting examples of microtubule inhibitors include DM1, DM4, MMA
  • endoplasmic reticulum stress inducing agents include but are not limited to, dimethyl-celecoxib (DMC), nelfinavir, celecoxib, and boron radiosensitizers (e.g., velcade (Bortezomib)).
  • DMC dimethyl-celecoxib
  • nelfinavir e.g., nelfinavir
  • celecoxib e.g., celecoxib
  • boron radiosensitizers e.g., velcade (Bortezomib)
  • platinum-based compound include Carboplatin, Cisplatin, Nedaplatin, Oxaliplatin, Triplatin tetranitrate, Satraplatin, Aroplatin, Lobaplatin, and JM-216. (see McKeage et al. (1997) J. Clin.
  • Non-limiting examples of antimetabolite agents include Folic acid based, e.g. dihydrofolate reductase inhibitors, such as Aminopterin, Methotrexate and Pemetrexed; thymidylate synthase inhibitors, such as Raltitrexed, Pemetrexed; Purine based, e.g.
  • an adenosine deaminase inhibitor such as Pentostatin
  • a thiopurine such as Thioguanine and Mercaptopurine
  • a halogenated/ribonucleotide reductase inhibitor such as Cladribine, Clofarabine, Fludarabine, or a guanine/guanosine: thiopurine, such as Thioguanine; or Pyrimidine based, e.g.
  • cytosine/cytidine hypomethylating agent, such as Azacitidine and Decitabine, a DNA polymerase inhibitor, such as Cytarabine, a ribonucleotide reductase inhibitor, such as Gemcitabine, or a thymine/thymidine: thymidylate synthase inhibitor, such as a Fluorouracil (5- FU).
  • hypomethylating agent such as Azacitidine and Decitabine
  • a DNA polymerase inhibitor such as Cytarabine
  • a ribonucleotide reductase inhibitor such as Gemcitabine
  • a thymine/thymidine thymidylate synthase inhibitor, such as a Fluorouracil (5- FU).
  • 5-FU Equivalents to 5-FU include prodrugs, analogs and derivative thereof such as 5′-deoxy-5- fluorouridine (doxifluoroidine), 1-tetrahydrofuranyl-5-fluorouracil (ftorafur), Capecitabine (Xeloda), S-I (MBMS-247616, consisting of tegafur and two modulators, a 5-chloro-2,4- dihydroxypyridine and potassium oxonate), ralititrexed (tomudex), no latrexed (Thymitaq, AG337), LY231514 and ZD9331, as described for example in Papamicheal (1999) The Oncologist 4:478-487.
  • 5′-deoxy-5- fluorouridine doxifluoroidine
  • 1-tetrahydrofuranyl-5-fluorouracil ftorafur
  • Capecitabine Xeloda
  • S-I MBMS-247616, consisting of tega
  • vincalkaloids include but are not limited to Vinblastine, Vincristine, Vinflunine, Vindesine and Vinorelbine.
  • taxanes include but are not limited to docetaxel, Larotaxel, Ortataxel, Paclitaxel and Tesetaxel.
  • An example of an epothilone is iabepilone.
  • enzyme inhibitors include but are not limited to farnesyltransferase inhibitors (Tipifamib); CDK inhibitor (Alvocidib, Seliciclib); proteasome inhibitor (Bortezomib); phosphodiesterase inhibitor (Anagrelide; rolipram); IMP dehydrogenase inhibitor (Tiazofurine); and lipoxygenase inhibitor (Masoprocol).
  • receptor antagonists include but are not limited to ERA (Atrasentan); retinoid X receptor (Bexarotene); and a sex steroid (Testolactone).
  • therapeutic antibodies include but are not limited to anti-HER1/EGFR (Cetuximab, Panitumumab); Anti-HER2/neu (erbB2) receptor (Trastuzumab); Anti-EpCAM (Catumaxomab, Edrecolomab) Anti-VEGF-A (Bevacizumab); Anti-CD20 (Rituximab, Tositumomab, Ibritumomab); Anti-CD52 (Alemtuzumab); and Anti-CD33 (Gemtuzumab).
  • anti-HER1/EGFR Cetuximab, Panitumumab
  • Anti-HER2/neu erbB2 receptor
  • Anti-EpCAM Catumaxomab, Edrecolomab
  • Anti-VEGF-A Bevacizumab
  • Anti-CD20 Rhuximab, Tositumomab, Ibritumomab
  • Anti-CD52 Alemtu
  • tyrosine kinase inhibitors include but are not limited to inhibitors to ErbB: HER1/EGFR (Erlotinib, Gefitinib, Lapatinib, Vandetanib, Sunitinib, Neratinib); HER2/neu (Lapatinib, Neratinib); RTK class III: C-kit (Axitinib, Sunitinib, Sorafenib), FLT3 (Lestaurtinib), PDGFR (Axitinib, Sunitinib, Sorafenib); and VEGFR (Vandetanib, Semaxanib, Cediranib, Axitinib, Sorafenib); bcr-abl (Imatinib, Nilotinib, Dasatinib); Src (Bosutinib) and Janus kinase 2 (Lestaurtinib).
  • ErbB HER1/EGFR
  • Chemotherapeutic agents that can be attached to the present AFFIMER® polypeptides may also include amsacrine, Trabectedin, retinoids (Alitretinoin, Tretinoin), Arsenic trioxide, asparagine depleter Asparaginase/Pegaspargase), Celecoxib, Demecolcine, Elesclomol, Elsamitrucin, Etoglucid, Lonidamine, Lucanthone, Mitoguazone, Mitotane, Oblimersen, Temsirolimus, and Vorinostat.
  • Examples of specific therapeutic agents that can be linked, ligated, or associated with the AFFIMER® polypeptides of the disclosure are flomoxef; fortimicin(s); gentamicin(s); glucosulfone solasulfone; gramicidin S; gramicidin(s); grepafloxacin; guamecycline; hetacillin; isepamicin; josamycin; kanamycin(s); flomoxef; fortimicin(s); gentamicin(s); glucosulfone solasulfone; gramicidin S; gramicidin(s); grepafloxacin; guamecycline; hetacillin; isepamicin; josamycin; kanamycin(s); bacitracin; bambermycin(s); biapenem; brodimoprim; butirosin; capreomycin; carbenicillin; carbomycin; car
  • streptozocin doxorubicin; daunorubicin; plicamycin; idarubicin; mitomycin C; pentostatin; mitoxantrone; cytarabine; fludarabine phosphate; butorphanol; nalbuphine.
  • the AFFIMER® agent includes a conjugated cytotoxic factor such as diptheria toxin, Pseudomonas aeruginosa exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins and compounds (e.g., fatty acids), dianthin proteins, Phytoiacca americana proteins PAPI, PAPII, and PAP-S, momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, mitogellin, restrictocin, phenomycin, and enomycin.
  • a conjugated cytotoxic factor such as diptheria toxin, Pseudomonas aeruginosa exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins and compounds (e.g., fatty acids
  • any method known in the art for conjugating to antibodies and other proteins may be employed in generating the conjugates of the present disclosure, including those methods described by Hunter, et al., (1962) Nature 144:945; David, et al., (1974) Biochemistry 13:1014; Pain, et al., (1981) J. Immunol. Meth.40:219; and Nygren, J., (1982) Histochem. and Cytochem. 30:407.
  • Methods for conjugating peptide, polypeptide and organic and inorganic moieties to antibodies and other proteins are conventional and very well known in the art and readily adapted for generating those versions of the subject AFFIMER® agents.
  • conjugated moiety is a peptide or polypeptide
  • that moiety can be chemically cross-linked to the AFFIMER® polypeptide or can be included as part of a fusion protein with the AFFIMER® polypeptide.
  • illustrative example would be a diphtheria toxin-AFFIMER® fusion protein.
  • the addition to the AFFIMER® polypeptide will generally be by way of chemical conjugation to the AFFIMER® polypeptide – such as through a functional group on an amino acid side chain or the carboxyl group at the C-terminal or amino group at the N-terminal end of the polypeptide.
  • the conjugated moiety will include at least one site that can be cleaved by an enzyme or are otherwise sensitive to an environmental condition (such as pH) that permits the conjugated moiety to be released from the AFFIMER® polypeptide, such as in the tumor or other diseased tissue (or tissue to be protected if the conjugated moiety functions to protect healthy tissue).
  • an environmental condition such as pH
  • an enzyme-cleavable linker which links the half-life extension moiety to a drug moiety.
  • the linker e.g., the substrate recognition sequence (SRS) of the linker
  • SRS substrate recognition sequence
  • the SRS is selectively cleaved such that the drug moiety is released as the free drug moiety in the vicinity of the target cells at least five times or ten times more than the extent to which the free drug moiety it is released in the vicinity of healthy cells/tissues, and in some embodiments, at least 100 or 500 or 1000 times more.
  • the skilled person will be able to identify appropriate SRS that is selectively cleavable in the vicinity of the target cell, using established methods in the art. For example, which proteases cleave which peptides can be assessed by consulting peptide libraries and studying an MS analysis of the fragmentation profile following cleavage. Also, published literature of protease cleavage motifs and peptide cleavage data can be searched as described further below.
  • the SRS is a protease cleavage site.
  • the SRS may be cleavable selectively by proteases that reside in the vicinity of the tumor cells.
  • the SRS may be one that is cleavable by a tumor associated protease.
  • the protease may be present extracellularly in the diseased state tissue in a subject at levels at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times greater than the healthy state of the tissue in the subject.
  • the protease may be present extracellularly in the diseased state of the tissue in a subject at levels at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times greater than other tissue of the subject.
  • the protease is a serine protease, metal protease, or cysteine protease.
  • the protease may be a metalloproteinase (MMP1-28) including both membrane-bound (MMP14-17 and MMP24-25) and secreted forms (MMP1-13 and MMP 18-23 and MMP26- 28).
  • the protease may belong to the A Disintegrin and Metalloproteinase (ADAM) and A Disintegrin, or Metalloproteinase with Thrombospondin Motifs (ADAMTS) families of proteases.
  • Other examples include CD 10 (CALLA) and prostate specific antigen (PSA). It is appreciated that the proteases may or may not be membrane bound.
  • Protease cleavage sites are well known in the scientific literature, and can readily serve as the basis for a given SRS being included in the drug-conjugate moieties using established synthetic techniques known in the art.
  • SRS may utilized which are designed to be selectively cleavable by one or a select sub- group of human proteases selected from the group consisting of (MEROPS peptidase database number provided in parentheses; Rawlings N. D., Morton F. R., Kok, C.
  • the SRS is a peptide moiety of up to 15 amino acids in length.
  • the SRS is cleaved by a protease co-localized with the target of the cell binding moiety in a tissue, and the protease cleaves the SRS in the AFFIMER® polypeptide- drug conjugate when the AFFIMER® polypeptide-drug conjugate is exposed to the protease.
  • the protease is not active or is significantly less active in tissues that do not significantly express the cell surface feature.
  • the protease is not active or is significantly less active in healthy, e.g., non-diseased tissues.
  • the SRS is cleaved by a protease selected from the following: • ADAMS or ADAMTS, e g.
  • SRS is peptide moiety selected from the group consisting of: TGRGPSWV, SARGPSRW, TARGPSFK, LSGRSDNH, GGWHTGRN, HTGRSGAL, PLTGRSGG, AARGPAIH, RGPAFNPM, SSRGPAYL, RGPATPIM, RGPA, GGQPSGMWGW, FPRPLGITGL, VHMPLGFLGP, SPLTGRSG, SAGFSLPA, LAPLGLQRR, SGGPLGVR, PLGL, GPRSFGL, and GPRSFG.
  • SRS is peptide moiety selected from the group consisting of: TGRGPSWV, SARGPSRW, TARGPSFK, LSGRSDNH, GGWHTGRN, HTGRSGAL, PLTGRSGG, AARGPAIH, RGPAFNPM, SSRGPAYL, RGPATPIM, RGPA, GGQPSGMWGW, FPRPLGITGL, VHMPLGFLGP, SP
  • the SRS is a substrate for an MMP, such as a sequence selected from the group consisting of ISSGLLSS, QNQALRMA, AQNLLGMV, STFPFGMF, PVGYTSSL, DWLYWPGI, MIAPVAYR, RPSPMWAY, WATPRPMR, FRLLDWQW, LKAAPRWA, GPSHLVLT, LPGGLSPW, MGLFSEAG, SPLPLRVP, RMHLRSLG, LAAPLGLL, AVGLLAPP, LLAPSHRA, PAGLWLDP, and ISSGLSS.
  • MMP such as a sequence selected from the group consisting of ISSGLLSS, QNQALRMA, AQNLLGMV, STFPFGMF, PVGYTSSL, DWLYWPGI, MIAPVAYR, RPSPMWAY, WATPRPMR, FRLLDWQW, LKAAPRWA, GPSHLVLT, LPGGLSPW, MGLFSEAG, SPLPLRVP, RMHLRSLG, LA
  • the SRS is a substrate for an MMP, such as a sequence selected from the group consisting of ISSGLSS, QNQALRMA, AQNLLGMV, STFPFGMF, PVGYTSSL, DWLYWPGI, ISSGLLSS, LKAAPRWA, GPSHLVLT, LPGGLSPW, MGLFSEAG, SPLPLRVP, RMHLRSLG, LAAPLGLL, AVGLLAPP, LLAPSHRA, and PAGLWLDP.
  • the SRS is a substrate for thrombin, such as GPRSFGL or GPRSFG.
  • an AFFIMER® polypeptide-drug conjugate comprises a spacer or bond (L 1 ) between the half-life extension moiety and the substrate recognition sequence (SRS) cleavable by the enzyme, e.g., present in a tumor microenvironment.
  • the spacer may be any molecule, for example, one or more nucleotides, amino acids, chemical functional groups.
  • the spacer is a peptide linker (e.g., two or more amino acids). Spacers should not adversely affect the expression, secretion, or bioactivity of the polypeptides. In some embodiments, spacers are not antigenic and do not elicit an immune response.
  • An immune response includes a response from the innate immune system and/or the adaptive immune system.
  • an immune response may be a cell-mediate response and/or a humoral immune response.
  • the immune response may be, for example, a T cell response, a B cell response, a natural killer (NK) cell response, a monocyte response, and/or a macrophage response.
  • NK natural killer
  • Other cell responses are contemplated herein.
  • linkers are non- protein-coding.
  • L 1 is a hydrocarbon (straight chain or cyclic) such as 6- maleimidocaproyl, maleimidopropanoyl and maleimidom ethyl cyclohexane- l-carboxylate, or L 1 is N-Succinimidyl 4-(2-pyridylthio) pentanoate, N- Succinimidyl 4-(N- maleimidomethyl) cyclohexane-1 carboxylate, N-Succinimidyl (4-iodo-acetyl) aminobenzoate.
  • L 1 is a polyether such as a poly(ethylene glycol) or other hydrophilic linker.
  • an AFFIMER® polypeptide-drug conjugate comprises a self- immolative linker (L 2 ) between the substrate recognition sequence (SRS) for the enzyme and the drug moiety, such as represented in the formula wherein, p represents an integer from 1 to 100, preferably 6 to 50, more preferably 6 to 12.
  • L 1 can be represented in the formula wherein, p represents an integer from 1 to 20, preferably 1 to 4.
  • a self-immolative moiety may be defined as a bifunctional chemical group that is capable of covalently linking together two spaced chemical moieties into a normally stable molecule, releasing one of the spaced chemical moieties from the molecule by means of enzymatic cleavage; and following enzymatic cleavage, spontaneously cleaving from the remainder of the bifunctional chemical group to release the other of said spaced chemical moieties.
  • the self- immolative moiety is covalently linked at one of its ends, directly or indirectly through a spacer unit, to the ligand by an amide bond and covalently linked at its other end to a chemical reactive site (functional group) pending from the drug moiety.
  • the derivatization of the drug moiety with the self-immolative moiety may render the drug less pharmacologically active (e.g. less toxic) or not active at all until the drug is cleaved.
  • An AFFIMER® polypeptide-drug conjugate is generally stable in circulation, or at least that should be the case in the absence of an enzyme capable of cleaving the amide bond between the substrate recognition sequence (enzyme-cleavable linker) and the self-immolative moiety.
  • an enzyme capable of cleaving the amide bond between the substrate recognition sequence (enzyme-cleavable linker) and the self-immolative moiety Upon exposure of an AFFIMER® polypeptide-drug conjugate to a suitable enzyme, the amide bond is cleaved initiating a spontaneous self-immolative reaction resulting in the cleavage of the bond covalently linking the self-immolative moiety to the drug moiety, to thereby effect release of the free drug moiety in its underivatized or pharmacologically active form.
  • L 2 is a benzyl oxy carbonyl group.
  • the self-immolative linker L 2 is p-aminobenzyloxycarbonyl (PABC).
  • the self-immolative linker L 2 is 2,4-bis(hydroxymeihyl)aniline.
  • the AFFIMER® polypeptide-drug conjugate of the present disclosure can employ a heterocyclic self-immolative moiety covalently linked to the therapeutic moiety and the cleavable substrate recognition sequence.
  • a self-immolative moiety may be defined as a bifunctional chemical group which is capable of covalently linking together two spaced chemical moieties into a normally stable molecule, releasing one of said spaced chemical moieties from the molecule by means of enzymatic cleavage; and following said enzymatic cleavage, spontaneously cleaving from the remainder of the bifunctional chemical group to release the other of said spaced chemical moieties.
  • the self- immolative moiety may be covalently linked at one of its ends, directly or indirectly through a spacer unit, to the ligand by an amide bond and covalently linked at its other end to a chemical reactive site (functional group) pending from the drug.
  • the derivatization of the therapeutic moiety with the self-immolative moiety may render the drug less pharmacologically active (e.g. less toxic) or not active at all until the drug is cleaved.
  • the AFFIMER® polypeptide-drug conjugate is generally stable in circulation, or at least that should be the case in the absence of an enzyme capable of cleaving the amide bond between the substrate recognition sequence and the self-immolative moiety.
  • the amide bond is cleaved initiating a spontaneous self-immolative reaction resulting in the cleavage of the bond covalently linking the self-immolative moiety to the drug, to thereby effect release of the free therapeutic moiety in its underivatized or pharmacologically active form.
  • the self-immolative moiety in conjugates of the present disclosure in some embodiments, either incorporate one or more heteroatoms and thereby provides improved solubility, improves the rate of cleavage and decreases propensity for aggregation of the conjugate.
  • heterocyclic self-immolative linker constructs of the present disclosure may result in surprising and unexpected biological properties such as increased efficacy, decreased toxicity, and more desirable pharmacokinetics.
  • Other examples of self-immolative linkers that are readily adapted for use in AFFIMER® polypeptide-drug conjugates described herein are taught in, for example, US Patent 7,754,681; WO 2012/074693A1; US 9,089,614; EP 1,732,607; WO 2015/038426A1 (all of which are incorporated by reference); Walther et al.
  • the linker may be any rigid or flexible linker, for example, comprising the amino acid sequence of SEQ ID NO: 86 or 87.
  • AVA04-269 (SEQ ID NO: 73) – linker – AVA19-157 (SEQ ID NO: 61)
  • AVA04-269 (SEQ ID NO: 73) – linker – AVA19-158 (SEQ ID NO: 62)
  • AVA04-269 (SEQ ID NO: 73) – linker – AVA19-01 (SEQ ID NO: 53)
  • AVA04-251 (SEQ ID NO: 72)
  • linker – AVA19-01 (SEQ ID NO: 53)
  • AVA04-251 BH dimer (SEQ ID NO: 74) – linker – AVA19-01 (SEQ ID NO: 53)
  • AVA04-640 (SEQ ID NO: 75) – linker – AVA19-06 (SEQ ID NO: 54) – linker – AVA19-06 (SEQ ID NO: 54) – linker – AVA03-42 (SEQ ID NO: 80)
  • AVA04-251
  • Encoded AFFIMER® Construct for In vivo Delivery An alternative approach to the delivery of therapeutic AFFIMER® agents, such as an LAG-3/PD-L1 AFFIMER® agent, would be to leave the production of the therapeutic polypeptide to the body itself.
  • a multitude of clinical studies have illustrated the utility of in vivo gene transfer into cells using a variety of different delivery systems. In vivo gene transfer seeks to administer to patients the encoded AFFIMER® construct, rather than the AFFIMER® agent. This allows the patient’s body to produce the therapeutic AFFIMER® agent of interest for a prolonged period of time, and secrete it either systemically or locally, depending on the production site.
  • Gene-based encoded AFFIMER® construct can present a labor- and cost- effective alternative to the conventional production, purification and administration of the polypeptide version of the AFFIMER® agent.
  • a number of antibody expression platforms have been pursued in vivo to which delivery of encoded AFFIMER® construct can be adapted: these include viral vectors, naked DNA and RNA.
  • encoded AFFIMER® construct gene transfer can not only enable cost-savings by reducing the cost of goods and of production but may also be able to reduce the frequency of drug administration.
  • a prolonged in vivo production of the therapeutic AFFIMER® agent by expression of the encoded AFFIMER® construct can contribute to (i) a broader therapeutic or prophylactic application of AFFIMER® agents in price- sensitive conditions, (ii) an improved accessibility to therapy in both developed and developing countries, and (iii) more effective and affordable treatment modalities.
  • cells can be harvested from the host (or a donor), engineered with encoded AFFIMER® construct sequences to produce AFFIMER® agents and re-administered to patients. Intramuscular antibody gene administration has been most widely evaluated (reviewed in Deal et al.
  • Skeletal muscle fibers are also terminally differentiated cells, and nuclei within the fibers are post-mitotic. Consequently, integration in the host genome is not a prerequisite to attain prolonged monoclonal antibody (mAb) expression.
  • the liver is another site often used for pre-clinical antibody gene transfer and is typically transfected via intravenous (i.v.) injection and can also be a site of gene transfer for encoded AFFIMER® construct either for local delivery of AFFIMER® agents (such as in the treatment of liver cancer and/or metaplasias) or for the generation of AFFIMER® agents that are secreted into the vascular for systemic circulation.
  • This organ has various physiological functions, including the synthesis of plasma proteins.
  • This organ can be particularly well suited for in vivo encoded AFFIMER® construct expression.
  • the tumor presents another site for encoded AFFIMER® construct transfer, targeted either via intravenous or direct injection/electroporation.
  • intratumoral encoded AFFIMER® construct expression can allow for a local production of the therapeutic AFFIMER® agents, waiving the need for high systemic AFFIMER® agent levels that might otherwise be required to penetrate and impact solid tumors.
  • a similar rationale applies for the brain, which is frequently targeted in the context of antibody gene transfer to avoid the difficulties with blood–brain barrier trafficking and would likewise be a target for delivery of encoded AFFIMER® construct. See, for example, Beckman et al.
  • cationic liposome technology can be employed, which is based on the ability of amphipathic lipids, possessing a positively charged head group and a hydrophobic lipid tail, to bind to negatively charged DNA or RNA and form particles that generally enter cells by endocytosis. Some cationic liposomes also contain a neutral co-lipid, thought to enhance liposome uptake by mammalian cells.
  • Modified (and unmodified or “naked”) DNA and RNA have also been shown to mediate successful gene transfer in a number of circumstances and can also be used as systems for delivery of encoded AFFIMER® construct.
  • These include the use of plasmid DNA by direct intramuscular injection, the use of intratumoral injection of plasmid DNA. See, for example, Rodrigo et al. (2012) “De novo automated design of small RNA circuits for engineering synthetic riboregulation in living cells” PNAS 109:15271–15276; Oishi et al.
  • Viral vectors are currently used as a delivery vehicle in the vast majority of pre-clinical and clinical gene therapy trials and in the first to be approved directed gene therapy. See Gene Therapy Clinical Trials Worldwide 2017 (abedia.com/wiley/). The main driver thereto is their exceptional gene delivery efficiency, which reflects a natural evolutionary development; viral vector systems are attractive for gene delivery, because viruses have evolved the ability to cross through cellular membranes by infection, thereby delivering nucleic acids such as encoded AFFIMER® construct to target cells.
  • oncolytic viruses have a distinct advantage, as they can specifically target tumor cells, boost AFFIMER® agent expression, and amplify therapeutic responses – such as to an HSA-PD-L1 AFFIMER® agent.
  • In vivo gene transfer of encoded AFFIMER® construct can also be accomplished by use of nonviral vectors, such as expression plasmids. Nonviral vectors are easily produced and do not seem to induce specific immune responses. Muscle tissue is most often used as target tissue for transfection because muscle tissue is well vascularized and easily accessible, and myocytes are long-lived cells. Intramuscular injection of naked plasmid DNA results in transfection of a certain percentage of myocytes.
  • plasmid DNA encoding cytokines and cytokine/IgG1 chimeric proteins has been introduced in vivo and has positively influenced (autoimmune) disease outcome.
  • intravascular delivery in which increased gene delivery and expression levels are achieved by inducing a short-lived transient high pressure in the veins.
  • Special blood-pressure cuffs that may facilitate localized uptake by temporarily increasing vascular pressure and can be adapted for use in human patients for this type of gene delivery. See, for example, Zhang et al. (2001) “Efficient expression of naked DNA delivered intraarterially to limb muscles of nonhuman primates” Hum. Gene Ther., 12:427-438.
  • Increased efficiency can also be gained through other techniques, such as in which delivery of the nucleic acid is improved by use of chemical carriers—cationic polymers or lipids—or via a physical approach—gene gun delivery or electroporation.
  • chemical carriers cationic polymers or lipids
  • electroporation is especially regarded as an interesting technique for nonviral gene delivery. Somiari, et al.
  • Encoded LAG-3/PD-L1 AFFIMER® polypeptides can be delivered by a wide range of gene delivery system commonly used for gene therapy including viral, non-viral, or physical.
  • U.S. Pat. No.6,080,728 also provides a discussion of a wide variety of gene delivery methods and compositions.
  • the routes of delivery include, for example, systemic administration and administration in situ.
  • An effective encoded AFFIMER® construct gene transfer approach must be directed to the specific tissues/cells where it is needed, and the resulting transgene expression should be at a level that is appropriate to the specific application.
  • Promoters are a major cis-acting element within the vector genome design that can dictate the overall strength of expression as well as cell-specificity.
  • Constitutive promoters such as the human elongation factor 1 ⁇ -subunit (EF1 ⁇ ), immediate-early cytomegalovirus (CMV), chicken ⁇ -actin (CBA) and its derivative CAG, the ⁇ glucuronidase (GUSB), or ubiquitin C (UBC) can be used to promote expression of the encoded AFFIMER® construct in most tissues.
  • EF1 ⁇ human elongation factor 1 ⁇ -subunit
  • CMV immediate-early cytomegalovirus
  • CBA chicken ⁇ -actin
  • GUSB ⁇ glucuronidase
  • UBC ubiquitin C
  • CBA and CAG promote the larger expression among the constitutive promoters; however, their size of ⁇ 1.7 kbs in comparison to CMV ( ⁇ 0.8 kbs) or EF1 ⁇ ( ⁇ 1.2 kbs) may limit use in vectors with packaging constraints such as AAV, particularly where AFFIMER® agent produced by expression of the encoded AFFIMER® construct is large.
  • the GUSB or UBC promoters can provide ubiquitous gene expression with a smaller size of 378 bps and 403 bps, respectively, but they are considerably weaker than the CMV or CBA promoter.
  • the platelet-derived growth factor B-chain (PDGF- ⁇ ), the synapsin (Syn), and the methyl-CpG binding protein 2 (MeCP2) promoters can drive neuron-specific expression at lower levels than NSE.
  • PDGF- ⁇ platelet-derived growth factor B-chain
  • Syn synapsin
  • MeCP2 methyl-CpG binding protein 2
  • the 680 bps-long shortened version [gfaABC(1)D] of the glial fibrillary acidic protein (GFAP, 2.2 kbs) promoter can confer higher levels of expression with the same astrocyte-specificity as the GFAP promoter.
  • Targeting oligodendrocytes can also be accomplished by the selection of the myelin basic protein (MBP) promoter, whose expression is restricted to this glial cell; however, its size of 1.9 kbs and low expression levels limit its use.
  • MBP myelin basic protein
  • exemplary promoters based on muscle creatine kinase (MCK) and desmin (1.7 kbs) have showed a high rate of specificity (with minimal expression in the liver if desired).
  • the promoter of the ⁇ - myosin heavy chain ( ⁇ -MHC; 1.2 kbs) has shown significant cardiac specificity in comparison with other muscle promoters (Lee et al., 2011 J Cardiol.57(1):115-22).
  • the synthetic MND promoter Li et al., 2010 J Neurosci Methods.189(1):56-614
  • the promoter contained in the 2AUCOE ubiquitous chromatin opening element
  • promoters to restrict expression to only liver hepatocytes after vector-mediated gene transfer has been shown to reduce transgene-specific immune responses in systems where that is a risk, and to even induce immune tolerance to the expressed protein (Zhang et al., 2012 Hum Gene Ther. 23(5):460-72), which for certain AFFIMER® agents may be beneficial.
  • the ⁇ 1-antitrypsin (hAAT; 347 bps) and the thyroxine binding globulin (TBG; ⁇ 400 bps) promoters drive gene expression restricted to the liver with minimal invasion to other tissues (Yan et al., 2012 Gene. 506(2):289-94; Cunningham et al., 2008 Mol Ther.16(6):1081-8).
  • a mechanism to control the duration and amount of in vivo encoded AFFIMER® construct expression will typically be desired.
  • inducible promoters which can be adapted for use with viral vectored- and plasmid DNA-based encoded AFFIMER® construct gene transfer. See Fang et al. (2007) “An antibody delivery system for regulated expression of therapeutic levels of monoclonal antibodies in vivo” Mol Ther.5(6):1153–9; and Perez et al. (2004) “Regulatable systemic production of monoclonal antibodies by in vivo muscle electroporation” Genet Vaccines Ther.2(1):2.
  • An exemplary a regulatable mechanism currently under clinical evaluation is an ecdysone-based gene switch activated by a small molecule ligand.
  • viral post-transcriptional regulatory elements may be used; these cis-acting elements are required for nuclear export of intronless viral RNA (Huang and Yen, 1994 J Virol.68(5):3193-9; and 1995 Mol Cell Biol.15(7):3864-9).
  • Examples include HPRE (Hepatitis B Virus PRE, 533 bps) and WPRE (Woodchuck Hepatitis Virus PRE, 600 bps), which can increase the level of transgene expression by almost 10-fold in certain instances (Donello et al., 1998 J Virol.72(6):5085-92).
  • WPRE was found to increase CMV promoter driven transgene expression, as well as increase PPE, PDGF and NSE promoter-driven transgene expression.
  • Another effect of the WPRE can be to protect encoded AFFIMER® transgenes from silencing (Paterna et al., 2000 Gene Ther.7(15):1304-11; Xia et al., 2007 Stem Cells Dev.2007 Feb; 16(1):167-76).
  • the polyadenylation of a transcribed encoded AFFIMER® construct transcript can also be important for nuclear export, translation, and mRNA stability. Therefore, in some embodiments, the encoded AFFIMER® construct will include a polyadenylation signal sequence.
  • a variety of studies are available that have determined the effects of different polyA signals on gene expression and mRNA stability.
  • Exemplary polyadenylation signal sequences include SV40 late or bovine growth hormone polyA (bGHpA) signal sequences, as well as minimal synthetic polyA (SPA) signal (Levitt et al., 1989 Genes Dev.3(7):1019-25; Yew et al., 1997 Hum Gene Ther.19978(5):575-84).
  • the efficiency of polyadenylation is increased by the SV40 late polyA signal upstream enhancer (USE) placed upstream of other polyA signals (Schek et al., 1992 Mol Cell Biol.12(12):5386-93).
  • the encoded AFFIMER® construct will include an SV40 late + 2xUSE polyA signal.
  • the encoded AFFIMER® construct may be desirable for the encoded AFFIMER® construct to include at least one regulatory enhancers, e.g., in addition to any promoter sequences.
  • the CMV enhancer is upstream of the CMV promoter at ⁇ 598 to ⁇ 68 (Boshart et al., 1985 Cell.41(2):521- 30) ( ⁇ 600 bps) and contains transcription binding sites.
  • a CMV enhancer can be included in the construct to increase tissue-specific promoter-driven transgene expression, such as using the ANF (atrial natriuretic factor) promoter, the CC10 (club cell 10) promoter, SP- C (surfactant protein C) promoter, or the PDGF- ⁇ (platelet-derived growth factor- ⁇ ) promoter (merely as examples).
  • ANF atrial natriuretic factor
  • CC10 cardiac natriuretic factor
  • SP- C surfactant protein C
  • PDGF- ⁇ platelet-derived growth factor- ⁇
  • transgene expression using the CMV enhancer with a muscle-specific promoter can increase expression levels of the protein encoded by the transgene, so would be particularly useful in the current disclosure for expressing AFFIMER® agents from encoded AFFIMER® constructs introduced into muscle cells of a patient.
  • the encoded AFFIMER® agents may also include at least one intronic sequence. The presence of an intron or intervening sequence in mRNA was first described, in vitro, to be important for mRNA processing and increased transgene expression (Huang and Gorman, 1990 Mol Cell Biol.10(4):1805-10; Niwa et al., 1990 Genes Dev.4(9):1552-9).
  • the intron(s) can be placed within the coding sequence for the AFFIMER® agent and/or can be placed between the promoter and transgene.
  • a variety of introns (Table 13) placed between the promoter and transgene were compared, in mice using AAV2, for liver transgene expression (Wu et al., 2008).
  • the MVM (minute virus of mice) intron increased transgene expression more than any other intron tested and more than 80-fold over no intron (Wu et al., 2008).
  • the encoded AFFIMER® constructs may also include at least one origin of replication, minichromosome maintenance elements (MME) and/or nuclear localization elements.
  • MME minichromosome maintenance elements
  • Episomal vectors of the disclosure comprise a portion of a virus genomic DNA that encodes an origin of replication (ori) , which is required for such vectors to be self- replicating and, thus, to persist in a host cell over several generations.
  • an episomal vector of the disclosure also may contain at least one gene encoding at least one viral protein required for replication, e.g., replicator protein (s).
  • the replicator protein(s) which help initiate replication may be expressed in trans on another DNA molecule, such as on another vector or on the host genomic DNA, in the host cell containing a self-replicating episomal expression vector of this disclosure.
  • Preferred self-replicating episomal LCR-containing expression vectors of the disclosure do not contain viral sequences that are not required for long- term stable maintenance in a eukaryotic host cell such as regions of a viral genome DNA encoding core or capsid proteins that would produce infectious viral particles or viral oncogenic sequences which may be present in the full-length viral genomic DNA molecule.
  • stable maintenance refers to the ability of a self-replicating episomal expression vector of this disclosure to persist or be maintained in non-dividing cells or in progeny cells of dividing cells in the absence of continuous selection without a significant loss (e.g., >50%) in copy number of the vector for two, three, four, or five or more generations.
  • the vectors will be maintained over 10-15 or more cell generations.
  • "transient” or “short- term” persistence of a plasmid in a host cell refers to the inability of a vector to replicate and segregate in a host cell in a stable manner; that is, the vector will be lost after one or two generations or will undergo a loss of >51% of its copy number between successive generations.
  • LCR-containing, episomal vectors useful in the context of the present disclosure are described further below.
  • the self-replicating function may alternatively be provided by at least one mammalian sequence such as described by Wohlgeuth et al., 1996, Gene Therapy 3:503; Vos et al., 1995, Jour.
  • An EBV- based episomal vector useful according to the disclosure can contain the oriP region of EBV which is carried on a 2.61 kb fragment of EBV and the EBNA-1 gene which is carried on a 2.18 kb fragment of EBV.
  • the EBNA-1 protein which is the only viral gene product required to support in trans episomal replication of vectors containing oriP, may be provided on the same episomal expression vector containing oriP.
  • the gene also may be expressed on another DNA molecule, such as a different DNA vector.
  • the episomal expression vectors of the disclosure also may be based on replication functions of the papilloma family of virus, including but not limited to Bovine Papilloma Virus (BPV) and Human Papilloma Viruses (HPVs) .
  • BPV and HPVs persist as stably maintained plasmids in mammalian cells.
  • BPV and HPVs namely El and E2
  • El and E2 have also been identified which are necessary and sufficient for mediate replication in many cell types via minimal origin of replication (Ustav et al., EMBO J.10: 449-457 (1991); Ustavet al., EMBO J .10:4231-4329, (1991); Ustav et al., Proc. Natl. Acad. Sci. USA 90: 898-902 (1993)).
  • An episomal vector useful according to the disclosure is the BPV-I vector system described in Piirsoo et al., EMBO J. , 15:1 (1996) and in WO 94/12629.
  • the BPV-1 vector system described in Piirsoo et al. comprises a plasmid harboring the BPV-1 origin of replication (minimal origin plus extrachromosomal maintenance element) and optionally the El and E2 genes.
  • the BPV-l El and E2 genes are required for stable maintenance of a BPV episomal vector. These factors ensure that the plasmid is replicated to a stable copy number of up to thirty copies per cell independent of cell cycle status.
  • the gene construct therefore persists stably in both dividing and non-dividing cells. This allows the maintenance of the gene construct in cells such as hemopoietic stem cells and more committed precursor cells.
  • the vectors of the disclosure also may be derived from a human papovavirus BK genomic DNA molecule.
  • the BK viral genome can be digested with restriction enzymes EcoRI and BamHI to produce a 5 kilobase (kb) fragment that contains the BK viral origin of replication sequences that can confer stable maintenance on vectors (see, for example, De Benedetti and Rhoads, Nucleic Acids Res .19:1925 (1991), as can a 3.2 kb fragment of the BK virus (Cooper and Miron, Human Gene Therapy 4:557 (1993)).
  • the encoded AFFIMER® constructs of the present disclosure can be provided as circular or linear nucleic acids.
  • the circular and linear nucleic acids are capable of directing expression of the AFFIMER® agent coding sequence in an appropriate subject cell.
  • the at least one nucleic acid system for expressing an AFFIMER® agent may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • Some aspects provide a polynucleotide comprising an open reading frame encoding any one of the proteins or fusion protein described herein.
  • the open reading frame comprises a nucleotide sequence having at least 70% to the nucleotide sequence of SEQ ID NO: 99-180.
  • Some aspects provide a polynucleotide comprising an open reading frame encoding any one of the proteins or fusion protein described herein.
  • the open reading frame comprises a nucleotide sequence having at least 75% to the nucleotide sequence of SEQ ID NO: 99-180. Some aspects provide a polynucleotide comprising an open reading frame encoding any one of the proteins or fusion protein described herein. In some embodiments, the open reading frame comprises a nucleotide sequence having at least 80% to the nucleotide sequence of SEQ ID NO: 99-180. Some aspects provide a polynucleotide comprising an open reading frame encoding any one of the proteins or fusion protein described herein. In some embodiments, the open reading frame comprises a nucleotide sequence having at least 85% to the nucleotide sequence of SEQ ID NO: 99-180.
  • the open reading frame comprises a nucleotide sequence having at least 90% to the nucleotide sequence of SEQ ID NO: 99-180. Some aspects provide a polynucleotide comprising an open reading frame encoding any one of the proteins or fusion protein described herein. In some embodiments, the open reading frame comprises a nucleotide sequence having at least 95% to the nucleotide sequence of SEQ ID NO: 99-180. Some aspects provide a polynucleotide comprising an open reading frame encoding any one of the proteins or fusion protein described herein.
  • the open reading frame comprises a nucleotide sequence having at least 98% to the nucleotide sequence of SEQ ID NO: 99-180.
  • Some aspects provide a polynucleotide comprising an open reading frame encoding any one of the proteins or fusion protein described herein.
  • the open reading frame comprises a nucleotide sequence having 100% to the nucleotide sequence of SEQ ID NO: 99-180. Table 3. AFFIMER® polynucleotides
  • RNA-Mediated Encoded AFFIMER® Construct Gene Transfer Exemplary nucleic acids or polynucleotides for the encoded HSA-PD-L1 AFFIMER® constructs of the present disclosure include but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a ⁇ - D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2 '-amino functionalization, and 2'-amino- a-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or hybrids or combinations thereof.
  • mRNA presents an emerging platform for antibody gene transfer that can be adapted by those skilled in the art for delivery of encoded AFFIMER® constructs of the present disclosure.
  • current results differ considerably, in certain instances the mRNA constructs appear to be able to rival viral vectors in terms of generated serum mAb titers.
  • Levels were in therapeutically relevant ranges within hours after mRNA administration, a marked shift in speed compared to DNA.
  • LNP lipid nanoparticles
  • IVTT in vitro transcribed
  • mRNA-based therapeutics are expressed more transiently compared to DNA- or viral vector-mediated gene transfer, and do not pose the risk of insertional mutagenesis in the host genome.
  • mRNA production is relatively simple and inexpensive.
  • mRNA uptake can be enhanced using electroporation. Broderick et al.2017 “Enhanced delivery of DNA or RNA vaccines by electroporation” Methods Mol Biol.2017;1499:193–200.
  • LNP lipid nanoparticles
  • RNA Replicon for Versatile and Efficient Gene Expression describes agents and methods suitable for efficient expression of AFFIMER® polypeptides of the present disclosure, such as suitable for immunotherapeutic treatment for the prevention and therapy of tumors.
  • the AFFIMER® agent coding sequence can be provided as an RNA replicon comprising a 5' replication recognition sequence such as from an alphavirus 5' replication recognition sequence.
  • the RNA replicon comprises a (modified) 5' replication recognition sequence and an open reading frame encoding the AFFIMER® agent, in particular located downstream from the 5' replication recognition sequence such as that the 5' replication recognition sequence and the open reading frame do not overlap, e.g., the 5' replication recognition sequence does not contain a functional initiation codon and in some embodiments does not contain any initiation codon. Most preferably, the initiation codon of the open reading frame encoding the AFFIMER® agent is in the 5' ⁇ 3' direction of the RNA replicon.
  • modified nucleosides can be incorporated into the in vitro–transcribed mRNA.
  • the IVT RNA can be 5’ capped, such an m7GpppG-capped or m7G5′ppp5′G2 ⁇ -O-Met-capped IVT. Efficient translation of the modified mRNA can be ensured by removing double-stranded RNA. Moreover, the 5′ and 3′ UTRs and the poly(A) tail can be optimized for improved intracellular stability and translational efficiency. See, for example, Stadler et al. (2017) Nature Medicine 23:815–817 and Kariko et al. WO/2017/036889 “Method for Reducing Immunogenicity of RNA”.
  • the mRNA that encodes the LAG-3/PD-L1 AFFIMER® agent may include at least one chemical modification described herein.
  • the chemical modification may be 1-methylpseudouridine, 5-methylcytosine or 1- methylpseudouridine and 5-methylcytosine.
  • the chemical modification is a pseudouridine or a modified 5 nucleoside, wherein said modified nucleoside is m 5 C, m 5 U, m6A, s2U, ⁇ , or 2'-O-methyl-U.
  • IVT polynucleotides linear polynucleotides encoding at least one LAG-3/PD-L1 AFFIMER® agent that are made using only in vitro transcription (IVT) enzymatic synthesis methods are referred to as "IVT polynucleotides.” Methods of making IVT polynucleotides are known in the art and are described in International Publication Nos. WO 2007/024708A2 and WO 2013/151666, the contents of which are incorporated herein by reference in their entirety.
  • the polynucleotides that encode the LAG-3/PD-L1 AFFIMER® agent of the present disclosure have portions or regions which differ in size and/or chemical modification pattern, chemical modification position, chemical modification percent or chemical modification population and combinations of the foregoing are known as "chimeric polynucleotides.”
  • a “chimera” according to the present disclosure is an entity having two or more incongruous or heterogeneous parts or regions.
  • a "part" or "region" of a polynucleotide is defined as any portion of the polynucleotide which is less than the entire length of the polynucleotide.
  • Such constructs are taught in for example International Publication No. WO2015/034928.
  • the polynucleotides of the present disclosure that are circular are known as “circular polynucleotides” or “circP.”
  • “circular polynucleotides” or “circP” means a single stranded circular polynucleotide which acts substantially like, and has the properties of, an RNA.
  • the term “circular” is also meant to encompass any secondary or tertiary configuration of the circP. Such constructs are taught in for example International Publication Nos. WO2015/034925 and WO2015/034928, the contents of each of which are incorporated herein by reference in their entirety.
  • Exemplary mRNA (and other polynucleotides) that can be used to encode LAG-3/PD-L1 AFFIMER® agents of the present disclosure include those which can be adapted from the specifications and figures of, for example, International Publication No.s WO2017/049275, WO2016/118724, WO2016/118725, WO2016/011226, WO2015/196128, WO/2015/196130, WO/2015/196118, WO/2015/089511, and WO2015/105926 (the later titled “Polynucleotides for the In vivo Production of Antibodies”), each of which is incorporated by reference herein.
  • Lipid-containing nanoparticle compositions have proven effective as transport vehicles into cells and/or intracellular compartments for a variety of RNAs (and related polynucleotides described herein). These compositions generally include at least one "cationic" and/or ionizable lipids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), and lipids containing polyethylene glycol (PEG lipids). Cationic and/or ionizable lipids include, for example, amine-containing lipids that can be readily protonated. B.
  • AFFIMER® Construct into Target Cells The introduction into host cell of the gene delivery system can be performed through various methods known to those skilled in the art. Where the present gene delivery system is constructed on the basis of viral vector construction, delivery can be performed as conventional infection methods known in the art. Physical methods to enhance delivery both viral and non-viral encoded AFFIMER® constructs include electroporation (Neumann, E. et al., EMBO J., 1:841(1982); and Tur-Kaspa et al., Mol. Cell Biol., 6:716-718(1986)), gene bombardment (Yang et al., Proc. Natl. Acad.
  • Administration of the composition via electroporation may be accomplished using electroporation devices that can be configured to deliver to a desired tissue of a mammal, a pulse of energy effective to cause reversible pores to form in cell membranes, and preferable the pulse of energy is a constant current similar to a preset current input by a user.
  • the electroporation device may comprise an electroporation component and an electrode assembly or handle assembly.
  • the electroporation component may include and incorporate at least one of the various elements of the electroporation devices, including: controller, current waveform generator, impedance tester, waveform logger, input element, status reporting element, communication port, memory component, power source, and power switch.
  • the electroporation may be accomplished using an in vivo electroporation device, for example CELLECTRA EP system (VGX Pharmaceuticals, Blue Bell, Pa.) or Elgen electroporator (Genetronics, San Diego, Calif.) to facilitate transfection of cells by the plasmid.
  • CELLECTRA EP system VGX Pharmaceuticals, Blue Bell, Pa.
  • Elgen electroporator Geneetronics, San Diego, Calif.
  • Transfection Enhancing Formulations Encoded AFFIMER® constructs can also be encapsulated in liposomes, preferably cationic liposomes (Wong, T. K. et al., Gene, 10:87(1980); Nicolau and Sene, Biochim. Biophys.
  • DNA also can be formed into complexes with polymers (polyplexes) or with dendrimers which can directly release their load into the cytoplasm of a cell.
  • Illustrative carriers useful in this regard include microparticles of poly(lactide-co- glycolide), polyacrylate, latex, starch, cellulose, dextran and the like.
  • illustrative carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross- linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid (see e.g., U.S. Pat. No.5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO 96/06638).
  • a non-liquid hydrophilic core e.g., a cross- linked polysaccharide or oligosaccharide
  • an external layer comprising an amphiphilic compound, such as a phospholipid
  • the amount of active agent contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
  • Biodegradable microspheres may be employed as carriers for compositions. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. NOS: 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and 5,942,252. Modified hepatitis B core protein carrier systems such as described in WO/9940934, and references cited therein, will also be useful for many applications. Another illustrative carrier/delivery system employs a carrier comprising particulate-protein complexes, such as those described in U.S. Pat.
  • Biodegradable polymeric nanoparticles facilitate nonviral nucleic acid transfer to cells. Small (approximately 200 nm), positively charged (approximately 10 mV) particles are formed by the self-assembly of cationic, hydrolytically degradable poly(beta-amino esters) and plasmid DNA.
  • Polynucleotides may also be administered to cells by direct microinjection, temporary cell permeabilizations (e.g., co-administration of repressor and/or activator with a cell permeabilizing agent), fusion to membrane translocating peptides, and the like.
  • Lipid-mediated nucleic acid delivery and expression of foreign nucleic acids, including mRNA, in vitro and in vivo has been very successful.
  • Lipid based non-viral formulations provide an alternative to viral gene therapies.
  • Current in vivo lipid delivery methods use subcutaneous, intradermal, intratumoral, or intracranial injection. Advances in lipid formulations have improved the efficiency of gene transfer in vivo (see PCT Application WO 98/07408).
  • a lipid formulation composed of an equimolar ratio of l,2-bis(oleoyloxy)-3-(trimethyl ammonio)propane (DOTAP) and cholesterol can significantly enhance systemic in vivo gene transfer.
  • DOTAP l,2-bis(oleoyloxy)-3-(trimethyl ammonio)propane
  • the DOTAP:cholesterol lipid formulation forms unique structure termed a "sandwich liposome". This formulation is reported to "sandwich" DNA between an invaginated bi-layer or 'vase' structure.
  • Beneficial characteristics of these lipid structures include a positive p, colloidal stabilization by cholesterol, two-dimensional nucleic acid packing and increased serum stability.
  • Cationic liposome technology is based on the ability of amphipathic lipids, possessing a positively charged head group and a hydrophobic lipid tail, to bind to negatively charged DNA or RNA and form particles that generally enter cells by endocytosis. Some cationic liposomes also contain a neutral co-lipid, thought to enhance liposome uptake by mammalian cells. Similarly, other polycations, such as poly-l-lysine and polyethylene-imine, complex with nucleic acids via charge interaction and aid in the condensation of DNA or RNA into nanoparticles, which are then substrates for endosome-mediated uptake.
  • CMOS complementary metal-oxide-semiconductor
  • pDNA complexes with plasmid DNA
  • oligodeoxynucleotides oligodeoxynucleotides
  • various forms of synthetic RNA synthetic RNA
  • the encoded AFFIMER® construct disclosed herein may be associated with polycationic molecules that serve to enhance uptake into cells. Complexing the nucleic acid construct with polycationic molecules also helps in packaging the construct such their size is reduced, which is believed to assist with cellular uptake.
  • nucleic acid construct embodiments had enhanced uptake into SCs over DCs when complexed with the polycationic molecules polylysine or polyethyleneimine.
  • polycationic molecules useful for complexing with nucleic acid constructs includes cell penetrating peptides (CPP), examples include polylysine (described above), polyarginine and Tat peptides.
  • CPP Cell penetrating peptides
  • MPG 27-residue chimeric peptide
  • RNA can also be complexed by such polycationic molecules for in vivo delivery.
  • polycationic molecules that may be complexed with the nucleic acid constructs described herein include polycationic polymers commercially available as JETPRIME® and In vivo JET (Polypus-transfection, S.A., Illkirch, France).
  • the present disclosure contemplates a method of delivering an mRNA (or other polynucleotide) encoding an LAG-3/PD-L1 AFFIMER® agent to a patient’s cells by administering a nanoparticle composition comprising (i) a lipid component, a phospholipid, a structural lipid, and a PEG lipid; and (ii) an mRNA (or other polynucleotide), said administering comprising contacting said mammalian cell with said nanoparticle composition, whereby said mRNA (or other polynucleotide) is delivered to said cell.
  • the PEG lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatide acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol and a PEG-modified dialkylglycerol.
  • the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and alphatocopherol.
  • the structural lipid is cholesterol.
  • the phospholipid includes a moiety selected from the group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.
  • the phospholipid includes at least one fatty acid moiety selected from the group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, arachidic acid, arachidonic acid, phytanoic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
  • the phospholipid is selected from the group consisting of 1 ,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1 ,2-dimyristoyl-sn-glycero- phosphocholine (DMPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), 1 ,2-di-0-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1
  • the phospholipid can be DOPE and said the component can comprise about 35 mol % to about 45 mol % said compound, about 10 mol % to about 20 mol % DOPE, about 38.5 mol % to about 48.5 mol % structural lipid, and about 1.5 mol % PEG lipid.
  • the lipid component can be about 40 mol % said compound, about 15 mol % phospholipid, about 43.5 mol % structural lipid, and about 1.5 mol % PEG lipid.
  • the wt/wt ratio of lipid component to LAG-3/PD-L1 AFFIMER® agent encoding mRNA (or other polynucleotide) is from about 5:1 to about 50:1, or about 10:1 to about 40:1
  • the mean size of said nanoparticle composition is from about 50 nm to about 150 nm, or from about 80 nm to about 120 nm.
  • the polydispersity index of said nanoparticle composition is from about 0 to about 0.18, or from about 0.13 to about 0.17.
  • the nanoparticle composition has a zeta potential of about -10 to about +20 mV.
  • the nanoparticle composition further comprises a cationic and/or ionizable lipid selected from the group consisting of 3-(didodecylamino)-N1 ,N 1 ,4-tridodecyl-1 -piperazineethanamine (KL10), 14,25-ditridecyl-15, 18,21 ,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4- dimethylaminomethyl-[1 ,3]-dioxolane (DLin-K-DMA), heptatriaconta-6, 9,28,31 -tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)
  • LAG-3/PD-L1 AFFIMER® agents described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing a DNA sequence encoding polypeptide sequences and expressing those sequences in a suitable host.
  • the recombinant AFFIMER® agent protein can be further manipulated chemically or enzymatically after isolation form the host cell or chemical synthesis.
  • the present disclosure includes recombinant methods and nucleic acids for recombinantly expressing the recombinant AFFIMER® agent proteins of the present disclosure comprising (i) introducing into a host cell a polynucleotide encoding the amino acid sequence of said AFFIMER® agent, for example, wherein the polynucleotide is in a vector and/or is operably linked to a promoter; (ii) culturing the host cell (e.g., eukaryotic or prokaryotic) under condition favorable to expression of the polynucleotide and, (iii) optionally, isolating the AFFIMER® agent from the host cell and/or medium in which the host cell is grown.
  • a host cell e.g., eukaryotic or prokaryotic
  • a DNA sequence encoding a recombinant AFFIMER® agent protein of interest may be constructed by chemical synthesis using an oligonucleotide synthesizer. Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize a polynucleotide sequence encoding an isolated polypeptide of interest.
  • a complete amino acid sequence can be used to construct a back-translated gene.
  • a DNA oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized.
  • several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated.
  • the individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.
  • An expression vector comprising the nucleotide sequence of a recombinant AFFIMER® agent protein can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation) and the transfected cells are then cultured by conventional techniques to produce the recombinant AFFIMER® agent protein of the disclosure.
  • the expression of the recombinant AFFIMER® agent protein is regulated by a constitutive, an inducible or a tissue, specific promoter.
  • the expression vector may include an origin of replication, such as may be selected based upon the type of host cell being used for expression.
  • the origin of replication from the plasmid pBR322 is useful for most Gram- negative bacteria while various origins from SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV) or papillomaviruses (such as HPV or BPV) are useful for cloning vectors in mammalian cells.
  • the origin of replication component is not needed for mammalian expression vectors (for example, the SV40 origin is often used because it contains the early promoter).
  • the vector may include at least one selectable marker gene, e.g., genetic elements that encode a protein necessary for the survival and growth of a host cell grown in a selective culture medium.
  • selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells, (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex media.
  • Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene.
  • a neomycin resistance gene may also be used for selection in prokaryotic and eukaryotic host cells.
  • selection genes may be used to amplify the gene which will be expressed. Amplification is a process where genes which are in greater demand for the production of a protein critical for growth are reiterated in tandem within the chromosomes of successive generations of recombinant cells. Examples of selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase. The mammalian cell transformants are placed under selection pressure which only the transformants are uniquely adapted to survive by virtue of the marker present in the vector.
  • DHFR dihydrofolate reductase
  • thymidine kinase thymidine kinase
  • the vector may also include at least one ribosome binding site, which will be transcribed into the mRNA including the coding sequence for the recombinant AFFIMER® agent protein.
  • a site is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes).
  • the element is typically located 3' to the promoter and 5' to the coding sequence of the polypeptide to be expressed.
  • the Shine-Dalgarno sequence is varied but is typically a polypurine (having a high A-G content). Many Shine-Dalgarno sequences have been identified, each of which can be readily synthesized using methods set forth above and used in a prokaryotic vector.
  • the expression vectors will typically contain a promoter that is recognized by the host organism and operably linked to a nucleic acid molecule encoding the recombinant AFFIMER® agent protein. Either a native or heterologous promoter may be used depending on the host cell used for expression and the yield desired.
  • Promoters for use with prokaryotic hosts include the beta-lactamase and lactose promoter systems; alkaline phosphatase, a tryptophan (trp) promoter system; and hybrid promoters such as the tac promoter. Other known bacterial promoters are also suitable. Their sequences have been published, and they can be ligated to a desired nucleic acid sequence(s), using linkers or adapters as desired to supply restriction sites. Promoters for use with yeast hosts are also known in the art. Yeast enhancers are advantageously used with yeast promoters.
  • Suitable promoters for use with mammalian host cells are well known and include those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40).
  • viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40).
  • adenovirus such as Adenovirus 2
  • bovine papilloma virus such as Adenovirus 2
  • bovine papilloma virus such as Adenovirus 2
  • Additional promoters which may be used for expressing the selective binding agents of the disclosure include but are not limited to: the SV40 early promoter region (Bernoist and Chambon, Nature, 290:304-310, 1981); the CMV promoter; the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al. (1980), Cell 22: 787-97); the herpes thymidine kinase promoter (Wagner et al. (1981), Proc. Natl. Acad. Sci.
  • the elastase I gene control region which is active in pancreatic acinar cells (Swift et al. (1984), Cell 38: 639-46; Ornitz et al. (1986), Cold Spring Harbor Symp. Quant. Biol.50: 399-409; MacDonald (1987), Hepatology 7: 425-515); the insulin gene control region which is active in pancreatic beta cells (Hanahan (1985), Nature 315: 115-22); the immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al.
  • an enhancer sequence may be inserted into the vector to increase transcription in eukaryotic host cells.
  • enhancer sequences available from mammalian genes are known (e.g., globin, elastase, albumin, alpha-feto-protein and insulin). Typically, however, an enhancer from a virus will be used.
  • the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers are exemplary enhancing elements for the activation of eukaryotic promoters. While an enhancer may be spliced into the vector at a position 5' or 3' to the polypeptide coding region, it is typically located at a site 5' from the promoter.
  • Vectors for expressing nucleic acids include those which are compatible with bacterial, insect, and mammalian host cells.
  • Such vectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (Invitrogen Company, San Diego, Calif.), pBSII (Stratagene Company, La Jolla, Calif.), pET15 (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL (BlueBacII; Invitrogen), pDSR- alpha (PCT Publication No. WO90/14363) and pFastBacDual (Gibco/BRL, Grand Island, N.Y.).
  • Additional possible vectors include but are not limited to, cosmids, plasmids or modified viruses, but the vector system must be compatible with the selected host cell.
  • Such vectors include but are not limited to plasmids such as Bluescript® plasmid derivatives (a high copy number ColEl-based phagemid, Stratagene Cloning Systems Inc., La Jolla Calif.), PCR cloning plasmids designed for cloning Taq-amplified PCR products (e.g., TOPOTM.
  • TA Cloning® Kit PCR2.1 plasmid derivatives, Invitrogen, Carlsbad, Calif.
  • mammalian, yeast or virus vectors such as a baculovirus expression system (pBacPAK plasmid derivatives, Clontech, Palo Alto, Calif.).
  • the recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, or other known techniques
  • Eukaryotic and prokaryotic host cells including mammalian cells as hosts for expression of the recombinant AFFIMER® agent protein disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC).
  • ATCC American Type Culture Collection
  • CHO Chinese hamster ovary
  • NSO Chinese hamster ovary
  • SP2 cells
  • HeLa HeLa cells
  • BHK baby hamster kidney
  • COS monkey kidney cells
  • human hepatocellular carcinoma cells e.g., Hep G2
  • A549 cells 3T3 cells
  • HEK-293 cells a number of other cell lines.
  • Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Cell lines of particular preference are selected through determining which cell lines have high expression levels.
  • Other cell lines that may be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells.
  • Fungal cells include yeast and filamentous fungus cells including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium
  • Pichia sp. any Saccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candida albicans, any Aspergillus sp., Trichoderma reesei, Chrysosporium lucknowense, any Fusarium sp., Yarrowia lipolytica, and Neurospora crassa.
  • a variety of host-expression vector systems may be utilized to express the recombinant AFFIMER® agent protein of the disclosure.
  • Such host-expression systems represent vehicles by which the coding sequences of the recombinant AFFIMER® agent protein may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the recombinant AFFIMER® agent protein of the disclosure in situ.
  • These include but are not limited to, microorganisms such as bacteria (e.g., E. coli and B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing AFFIMER® agent protein coding sequences; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing AFFIMER® agent protein coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the AFFIMER® agent protein coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (C ⁇ MV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing AFFIMER® agent protein coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphotic cells (see U.S.
  • Per C.6 cells rat retinal cells developed by Crucell
  • recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
  • promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
  • a number of expression vectors may be advantageously selected depending upon the use intended for the recombinant AFFIMER® agent protein being expressed.
  • vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include but are not limited, to the E. coli expression vector pUR278 (Ruther et al. (1983) "Easy Identification Of cDNA Clones," EMBO J.2:1791- 1794), in which the AFFIMER® agent protein coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye et al.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • the AFFIMER® agent protein coding sequence may be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).
  • an AcNPV promoter e.g., the polyhedrin promoter
  • a number of viral-based expression systems may be utilized.
  • the AFFIMER® agent protein coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts.
  • a non-essential region of the viral genome e.g., region E1 or E3
  • AFFIMER® agent protein coding sequences include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bitter et al.
  • a host cell strain may be chosen which modulates the expression of the inserted sequences or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.
  • stable expression is contemplated.
  • cell lines which stably express an antibody of the disclosure may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the recombinant AFFIMER® agent proteins of the disclosure.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the recombinant AFFIMER® agent proteins.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al. (1977) "Transfer of Purified Herpes Virus Thymidine Kinase Gene to Cultured Mouse Cells," Cell 11:223-232), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al. (1962) "Genetics Of Human Cess Line. IV.
  • the expression levels of a recombinant AFFIMER® agent protein can be increased by vector amplification (for a review, see Bebbington and Hentschel, "The Use of Vectors Based On Gene Amplification For The Expression Of Cloned Genes In Mammaian Cells," in DNA CLONING, Vol.3. (Academic Press, New York, 1987)).
  • vector amplification for a review, see Bebbington and Hentschel, "The Use of Vectors Based On Gene Amplification For The Expression Of Cloned Genes In Mammaian Cells," in DNA CLONING, Vol.3. (Academic Press, New York, 1987)
  • a marker in the vector system expressing a recombinant AFFIMER® agent protein is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene.
  • the host cell may be co-transfected with two expression vectors, for instance the first vector encoding a heavy chain and the second vector encoding a light chain derived polypeptide, one or both of which includes an AFFIMER® polypeptide coding sequence.
  • the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes both heavy and light chain polypeptides.
  • the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot (1986) "Expression and Amplification Of Engineered Mouse Dihydrofolate Reductase Minigenes," Nature 322:562-565; Kohler (1980) "Immunoglobulin Chain Loss In Hybridoma Lines," Proc. Natl. Acad. Sci. (U.S.A.) 77:2197-2199).
  • the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
  • glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern that is characteristic for glycoproteins produced in the cell line or transgenic animal.
  • the particular glycosylation pattern of the recombinant AFFIMER® agent protein will depend on the particular cell line or transgenic animal used to produce the protein.
  • a glycosylation pattern comprising only non-fucosylated N-glycans may be advantageous, because in the case of antibodies this has been shown to typically exhibit more potent efficacy than fucosylated counterparts both in vitro and in vivo (See for example, Shinkawa et al., J. Biol. Chem.278: 3466-3473 (2003); U.S. Pat. NOS: 6,946,292 and 7,214,775).
  • an AFFIMER® agent from production cell lines can be enhanced using a number of known techniques.
  • the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions.
  • the GS system is discussed in whole or part in connection with European Patent NOS: 0216846, 0256055, and 0323997 and European Patent Application No.89303964.4.
  • the mammalian host cells e.g., CHO
  • the polynucleotide encoding the immunoglobulin chain comprises a glutamine synthetase gene which complements the lack of the gene in the host cell.
  • Such host cells containing the binder or polynucleotide or vector as discussed herein as well as expression methods, as discussed herein, for making the binder using such a host cell are part of the present disclosure.
  • Expression of recombinant proteins in insect cell culture systems e.g., baculovirus
  • AFFIMER® agent proteins produced by a transformed host can be purified according to any suitable method. Standard methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags such as hexa-histidine, maltose binding domain, influenza coat sequence, and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column.
  • Isolated proteins can also be physically characterized using such techniques as proteolysis, mass spectrometry (MS), nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC), and x-ray crystallography.
  • recombinant AFFIMER® agent proteins produced in bacterial culture can be isolated, for example, by initial extraction from cell pellets, followed by at least one concentration, salting-out, aqueous ion exchange, or size exclusion chromatography steps. HPLC can be employed for final purification steps.
  • Microbial cells employed in expression of a recombinant protein can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
  • AFFIMER® agents of the disclosure are useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as immunotherapy for cancer.
  • AFFIMER® agents described herein are useful for activating, promoting, increasing, and/or enhancing an immune response, inhibiting tumor growth, reducing tumor volume, inducing tumor regression, increasing tumor cell apoptosis, and/or reducing the tumorigenicity of a tumor.
  • the polypeptides or agents of the disclosure are useful for immunotherapy against pathogens, such as viruses.
  • the AFFIMER® agents described herein may be useful for inhibiting viral infection, reducing viral infection, increasing virally-infected cell apoptosis, and/or increasing killing of virus-infected cells.
  • the methods of use may be in vitro, ex vivo, or in vivo methods.
  • the interaction of PD-L1 on the tumor cells with PD-1 on a T- cell reduces T-cell function signals to prevent the immune system from attacking the tumor cells.
  • Use of an inhibitor that blocks the interaction of PD-L1 with the PD-1 receptor can prevent the cancer from evading the immune system in this way.
  • PD-1 and PD-L1 inhibitors are being tested within the clinic for use in advanced melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer and Hodgkin lymphoma, amongst other cancer types.
  • LAG-3 is involved in T cell suppression, including through activation of regulatory T cells.
  • TILs tumor-infiltrating lymphocytes
  • the therapeutics described herein can help subjects overcome resistance and restore T cell function. Immunotherapy with these immune checkpoint inhibitors appears to shrink tumors in a higher number of patients across a wider range of tumor types and is associated with lower toxicity levels than other immunotherapies, with durable responses. However, de novo and acquired resistance is still seen in a large proportion of patients.
  • PD-L1 inhibitors and LAG-3 inhibitors are considered to be a promising drug category for many different cancers.
  • the present disclosure provides methods for activating an immune response in a subject using an AFFIMER® agent.
  • the disclosure provides methods for promoting an immune response in a subject using an AFFIMER® agent described herein.
  • the disclosure provides methods for increasing an immune response in a subject using an AFFIMER® agent.
  • the disclosure provides methods for enhancing an immune response in a subject using an AFFIMER® agent.
  • the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing cell-mediated immunity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing Th1-type responses. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CD4+ T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CD8+ T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CTL activity.
  • the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing NK cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing T-cell activity and increasing NK cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CU activity and increasing NK cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises inhibiting or decreasing the suppressive activity of Treg cells. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises inhibiting or decreasing the suppressive activity of MDSCs.
  • the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing the number of the percentage of memory T-cells. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing long-term immune memory function. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing long-term memory. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises no evidence of substantial side effects and/or immune-based toxicities. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises no evidence of cytokine release syndrome (CRS) or a cytokine storm.
  • CRS cytokine release syndrome
  • the immune response is a result of antigenic stimulation.
  • the antigenic stimulation is a tumor cell.
  • the antigenic stimulation is cancer.
  • the antigenic stimulation is a pathogen.
  • the antigenic stimulation is a virally-infected cell.
  • a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of an AFFIMER® agent described herein, wherein an AFFIMER® agent binds human PD-L1.
  • a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of an AFFIMER® agent described herein, wherein an AFFIMER® agent binds human LAG-3. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of an AFFIMER® agent described herein, wherein an AFFIMER® agent binds human PD-L1 and human LAG-3.
  • a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of an AFFIMER® agent described herein, wherein the AFFIMER® agent is an AFFIMER®-containing antibody or receptor trap fusion polypeptide including an AFFIMER® polypeptide that specifically binds to PD-L1 and/or an AFFIMER® polypeptide that specifically binds to LAG-3.
  • the AFFIMER® agent is an AFFIMER®-containing antibody or receptor trap fusion polypeptide including an AFFIMER® polypeptide that specifically binds to PD-L1 and/or an AFFIMER® polypeptide that specifically binds to LAG-3.
  • a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of an encoded AFFIMER® construct, wherein the encoded AFFIMER® construct, when expressed in the patient, produces a recombinant AFFIMER® agent including a LAG-3/PD-L1 AFFIMER® polypeptide.
  • a method of activating or enhancing a persistent or long-term immune response to a tumor comprises administering to a subject a therapeutically effective amount of an AFFIMER® agent which binds human PD-L1 and/or LAG-3.
  • a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of an AFFIMER® agent described herein, wherein the AFFIMER® agent is an AFFIMER®- containing antibody or receptor trap fusion polypeptide including an AFFIMER® polypeptide that specifically binds to PD-L1 and/or LAG-3.
  • a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of an encoded AFFIMER® construct, wherein the encoded AFFIMER® construct, when expressed in the patient, produces a recombinant AFFIMER® agent including a LAG-3/PD-L1 AFFIMER® polypeptide.
  • a method of inducing a persistent or long-term immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of an AFFIMER® agent which binds human PD- L1 and/or human LAG-3.
  • a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of an AFFIMER® agent described herein, wherein the AFFIMER® agent is an AFFIMER® polypeptide-containing antibody or receptor trap fusion polypeptide including an AFFIMER® polypeptide that specifically binds to PD-L1 and/or LAG- 3.
  • a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of an encoded AFFIMER® construct, wherein the encoded AFFIMER® construct, when expressed in the patient, produces a recombinant AFFIMER® agent including a LAG-3/PD-L1 AFFIMER® polypeptide.
  • a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of an AFFIMER® agent which binds human PD-L1 and/or human LAG-3.
  • a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of an AFFIMER® agent described herein, wherein the AFFIMER® agent is an AFFIMER®-containing antibody or receptor trap fusion polypeptide including an AFFIMER® polypeptide that specifically binds to PD-L1 and/or LAG- 3.
  • a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of an encoded AFFIMER® construct, wherein the encoded AFFIMER® construct, when expressed in the patient, produces a recombinant AFFIMER® agent including a LAG-3/PD-L1 AFFIMER® polypeptide.
  • the AFFIMER® agent is a bispecific agent and binds PD-L1 and LAG-3.
  • the method of inhibiting growth of a tumor comprises administering to a subject a therapeutically effective amount of an AFFIMER® agent described herein.
  • the subject is a human.
  • the subject has a tumor, or the subject had a tumor which was removed.
  • the tumor is a solid tumor.
  • the tumor is a tumor selected from the group consisting of: colorectal tumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, neuroendocrine tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor, glioblastoma, and head and neck tumor.
  • the tumor is a colorectal tumor.
  • the tumor is an ovarian tumor.
  • the tumor is a lung tumor.
  • the tumor is a pancreatic tumor.
  • the tumor is a melanoma tumor.
  • the tumor is a bladder tumor.
  • the subject AFFIMER® agents can be used to treat patients suffering from cancer, such as osteosarcoma, rhabdomyosarcoma, neuroblastoma, kidney cancer, leukemia, renal transitional cell cancer, bladder cancer, Wilm's cancer, ovarian cancer, pancreatic cancer, breast cancer (including triple negative breast cancer), prostate cancer, bone cancer, lung cancer (e.g., small cell or non-small cell lung cancer), gastric cancer, colorectal cancer, cervical cancer, synovial sarcoma, head and neck cancer, squamous cell carcinoma, multiple myeloma, renal cell cancer, retinoblastoma, hepatoblastoma, hepatocellular carcinoma, melanoma, rhabdoid tumor of the kidney, Ewing's sarcoma, chondrosarcoma, brain cancer, glioblasts, and others.
  • the cancer is metastatic cancer, e.g., of the varieties described above.
  • the cancer is a hematologic cancer.
  • the cancer is selected from the group consisting of: acute myelogenous leukemia (AML), Hodgkin lymphoma, multiple myeloma, T-cell acute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myelogenous leukemia (CML), non- Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and cutaneous T-cell lymphoma (CTCL).
  • AML acute myelogenous leukemia
  • T-ALL T-cell acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • non- Hodgkin lymphoma diffuse large B-cell lymphoma (DLB
  • the cancer is a PD-L1-positive cancer.
  • the cancer is an anti-PD-1 or anti-PD-L1 refractory cancer, such as: non-small cell lung carcinoma (NSCLC), colorectal cancer, advanced melanoma, or renal cell carcinoma (RCC).
  • NSCLC non-small cell lung carcinoma
  • RRC renal cell carcinoma
  • the cancer is urothelial carcinoma (e.g., previously treated with platinum- containing chemotherapy), and hepatocellular carcinoma (e.g., previously treated with a kinase inhibitor, such as sorafenib).
  • the present disclosure also provides pharmaceutical compositions comprising an AFFIMER® agent described herein and a pharmaceutically acceptable vehicle. In some embodiments, the pharmaceutical compositions find use in immunotherapy.
  • the pharmaceutical compositions find use in immuno-oncology. In some embodiments, the compositions find use in inhibiting tumor growth. In some embodiments, the pharmaceutical compositions find use in inhibiting tumor growth in a subject (e.g., a human patient). In some embodiments, the compositions find use in treating cancer. In some embodiments, the pharmaceutical compositions find use in treating cancer in a subject (e.g., a human patient).
  • Formulations are prepared for storage and use by combining a purified AFFIMER® agent of the present disclosure with a pharmaceutically acceptable vehicle (e.g., a carrier or excipient). a pharmaceutically acceptable vehicles, excipients, and/or stabilizers to be inactive ingredients of a formulation or pharmaceutical composition.
  • an AFFIMER® agent described herein is lyophilized and/or stored in a lyophilized form.
  • a formulation comprising an AFFIMER® agent described herein is lyophilized.
  • suitable pharmaceutically acceptable vehicles include but are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues
  • compositions of the present disclosure can be administered in any number of ways for either local or systemic treatment. Administration can be topical by epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, and intranasal; oral; or parenteral including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion), or intracranial (e.g., intrathecal or intraventricular).
  • parenteral including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion), or intracranial (e.g., intrathecal or intraventricular).
  • the therapeutic formulation can be in unit dosage form.
  • Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in water or non-aqueous media, or suppositories.
  • solid compositions such as tablets the principal active ingredient is mixed with a pharmaceutical carrier.
  • Conventional tableting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and diluents (e.g., water). These can be used to form a solid preformulation composition containing a homogeneous mixture of a compound of the present disclosure, or a non-toxic pharmaceutically acceptable salt thereof.
  • the solid preformulation composition is then subdivided into unit dosage forms of a type described above.
  • the tablets, pills, etc. of the formulation or composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner composition covered by an outer component.
  • the two components can be separated by an enteric layer that serves to resist disintegration and permits the inner component to pass intact through the stomach or to be delayed in release.
  • enteric layers or coatings such materials include a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
  • the AFFIMER® agents described herein can also be entrapped in microcapsules.
  • microcapsules are prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions as described in Remington: The Science and Practice of Pharmacy, 22.sup.nd Edition, 2012, Pharmaceutical Press, London.
  • pharmaceutical formulations include an AFFIMER® agent of the present disclosure complexed with liposomes. Methods to produce liposomes are known to those of skill in the art.
  • some liposomes can be generated by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • sustained-release preparations comprising AFFIMER® agents described herein can be produced. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing an AFFIMER® agent, where the matrices are in the form of shaped articles (e.g., films or microcapsules).
  • sustained-release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl- methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7 ethyl-L- glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT.TM. (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)- 3-hydroxybutyric acid.
  • polyesters such as poly(2-hydroxyethyl- methacrylate) or poly(vinyl alcohol)
  • polylactides copolymers of L-glutamic acid and 7 ethyl-L- glutamate
  • non-degradable ethylene-vinyl acetate non-degradable ethylene-vinyl
  • the method or treatment further comprises administering at least one additional immune response stimulating agent.
  • the additional immune response stimulating agent includes, but is not limited to, a colony stimulating factor (e.g., granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), stem cell factor (SCF)), an interleukin (e.g., IL- 1, IL2, IL-3, IL-7, IL-12, IL-15, IL-18), a checkpoint inhibitor, an antibody that blocks immunosuppressive functions (e.g., an anti-CTLA-4 antibody, anti-CD28 antibody, anti-CD3 antibody), a toll-like receptor (e.g., TLR4, TLR7, TLR9), or a member of the B7 family (e.g., CD80, CD86).
  • a colony stimulating factor e.g., granulocyte-macrophage colony stimulating factor (GM
  • An additional immune response stimulating agent can be administered prior to, concurrently with, and/or subsequently to, administration of the AFFIMER® agent.
  • Pharmaceutical compositions comprising an AFFIMER® agent and the immune response stimulating agent(s) are also provided.
  • the immune response stimulating agent comprises 1, 2, 3, or more immune response stimulating agents.
  • in addition to administering an AFFIMER® agent described herein the method or treatment further comprises administering at least one additional therapeutic agent.
  • An additional therapeutic agent can be administered prior to, concurrently with, and/or subsequently to, administration of the AFFIMER® agent.
  • Pharmaceutical compositions comprising an AFFIMER® agent and the additional therapeutic agent(s) are also provided.
  • the at least one additional therapeutic agent comprises 1, 2, 3, or more additional therapeutic agents.
  • Combination therapy with two or more therapeutic agents often uses agents that work by different mechanisms of action, although this is not required. Combination therapy using agents with different mechanisms of action may result in additive or synergetic effects. Combination therapy may allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the AFFIMER® agent. Combination therapy may decrease the likelihood that resistant cancer cells will develop.
  • combination therapy comprises a therapeutic agent that affects the immune response (e.g., enhances or activates the response) and a therapeutic agent that affects (e.g., inhibits or kills) the tumor/cancer cells.
  • the combination of an AFFIMER® agent described herein and at least one additional therapeutic agent results in additive or synergistic results.
  • the combination therapy results in an increase in the therapeutic index of the AFFIMER® agent.
  • the combination therapy results in an increase in the therapeutic index of the additional therapeutic agent(s).
  • the combination therapy results in a decrease in the toxicity and/or side effects of the AFFIMER® agent.
  • the combination therapy results in a decrease in the toxicity and/or side effects of the additional therapeutic agent(s).
  • Useful classes of therapeutic agents include, for example, anti-tubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, anti-folates, anti-metabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like.
  • alkylating agents e.g., platinum complexes such as cisplatin, mono(platinum), bis(platinum) and tri-nuclear platinum complex
  • the second therapeutic agent is an alkylating agent, an antimetabolite, an antimitotic, a topoisomerase inhibitor, or an angiogenesis inhibitor.
  • Therapeutic agents that may be administered in combination with the AFFIMER® agent described herein include chemotherapeutic agents.
  • the method or treatment involves the administration of an AFFIMER® agent of the present disclosure in combination with a chemotherapeutic agent or in combination with a cocktail of chemotherapeutic agents. Treatment with an AFFIMER® agent can occur prior to, concurrently with, or subsequent to administration of chemotherapies.
  • Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously.
  • Preparation and dosing schedules for such chemotherapeutic agents can be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in The Chemotherapy Source Book, 4.sup.th Edition, 2008, M. C. Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, Pa.
  • Chemotherapeutic agents useful in the present disclosure include but are not limited to, alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamime; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitro
  • paclitaxel TAXOL
  • docetaxel TAXOTERE
  • chlorambucil gemcitabine
  • 6- thioguanine mercaptopurine
  • platinum analogs such as cisplatin and carboplatin
  • vinblastine platinum
  • etoposide VP-16
  • ifosfamide mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; ibandronate; CPT11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine (XELODA); and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • DMFO difluoromethylornithine
  • XELODA retinoic acid
  • esperamicins capecitabine
  • Chemotherapeutic agents also include anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • the additional therapeutic agent is cisplatin.
  • the additional therapeutic agent is carboplatin.
  • the chemotherapeutic agent is a topoisomerase inhibitor.
  • Topoisomerase inhibitors are chemotherapy agents that interfere with the action of a topoisomerase enzyme (e.g., topoisomerase I or II).
  • Topoisomerase inhibitors include but are not limited to, doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D, etoposide, topotecan HCl, teniposide (VM-26), and irinotecan, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these.
  • the additional therapeutic agent is irinotecan.
  • the chemotherapeutic agent is an anti-metabolite.
  • An anti- metabolite is a chemical with a structure that is similar to a metabolite required for normal biochemical reactions, yet different enough to interfere with at least one normal function of cells, such as cell division.
  • Anti-metabolites include but are not limited to, gemcitabine, fluorouracil, capecitabine, methotrexate sodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these.
  • the additional therapeutic agent is gemcitabine.
  • the chemotherapeutic agent is an antimitotic agent, including, but not limited to, agents that bind tubulin.
  • the agent is a taxane.
  • the agent is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel.
  • the agent is paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel (nab-paclitaxel; ABRAXANE), DHA-paclitaxel, or PG-paclitaxel.
  • the antimitotic agent comprises a vinca alkaloid, such as vincristine, vinblastine, vinorelbine, or vindesine, or pharmaceutically acceptable salts, acids, or derivatives thereof.
  • the antimitotic agent is an inhibitor of kinesin Eg5 or an inhibitor of a mitotic kinase such as Aurora A or Plk1.
  • the additional therapeutic agent is paclitaxel.
  • the additional therapeutic agent is nab-paclitaxel.
  • an additional therapeutic agent comprises an agent such as a small molecule.
  • treatment can involve the combined administration of an AFFIMER® agent of the present disclosure with a small molecule that acts as an inhibitor against tumor-associated antigens including, but not limited to, EGFR, HER2 (ErbB2), and/or VEGF.
  • an AFFIMER® agent of the present disclosure is administered in combination with a protein kinase inhibitor selected from the group consisting of: gefitinib (IRESSA), erlotinib (TARCEVA), sunitinib (SUTENT), lapatanib, vandetanib (ZACTIMA), AEE788, CI-1033, cediranib (RECENTIN), sorafenib (NEXAVAR), and pazopanib (GW786034B).
  • IRESSA gefitinib
  • TARCEVA sunitinib
  • ZACTIMA ZACTIMA
  • AEE788, CI-1033 cediranib
  • sorafenib NEXAVAR
  • GW786034B pazopanib
  • an additional therapeutic agent comprises an mTOR inhibitor.
  • the additional therapeutic agent is a small molecule that inhibits a cancer stem cell pathway.
  • the additional therapeutic agent is an inhibitor of the Notch pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Wnt pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the BMP pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Hippo pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the mTOR/AKR pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the RSPO/LGR pathway. In some embodiments of the methods described herein, an additional therapeutic agent comprises a biological molecule, such as an antibody.
  • treatment can involve the combined administration of an AFFIMER® agent of the present disclosure with antibodies against tumor-associated antigens including, but not limited to, antibodies that bind EGFR, HER2/ErbB2, and/or VEGF.
  • the additional therapeutic agent is an antibody specific for a cancer stem cell marker.
  • the additional therapeutic agent is an antibody that binds a component of the Notch pathway.
  • the additional therapeutic agent is an antibody that binds a component of the Wnt pathway.
  • the additional therapeutic agent is an antibody that inhibits a cancer stem cell pathway.
  • the additional therapeutic agent is an inhibitor of the Notch pathway.
  • the additional therapeutic agent is an inhibitor of the Wnt pathway.
  • the additional therapeutic agent is an inhibitor of the BMP pathway. In some embodiments, the additional therapeutic agent is an antibody that inhibits .beta.-catenin signaling. In some embodiments, the additional therapeutic agent is an antibody that is an angiogenesis inhibitor (e.g., an anti-VEGF or VEGF receptor antibody). In some embodiments, the additional therapeutic agent is bevacizumab (AVASTIN), ramucirumab, trastuzumab (HERCEPTIN), pertuzumab (OMNITARG), panitumumab (VECTIBIX), nimotuzumab, zalutumumab, or cetuximab (ERBITUX).
  • AVASTIN AVASTIN
  • ramucirumab trastuzumab
  • HERCEPTIN trastuzumab
  • OMNITARG pertuzumab
  • VECTIBIX panitumumab
  • nimotuzumab zalutumumab
  • the additional therapeutic agent is an antibody that modulates the immune response.
  • the additional therapeutic agent is an anti-PD-1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-TIM-3 antibody, or an anti-TIGIT antibody.
  • treatment with an AFFIMER® agent described herein can include combination treatment with other biologic molecules, such as at least one cytokine (e.g., lymphokines, interleukins, tumor necrosis factors, and/or growth factors) or can be accompanied by surgical removal of tumors, removal of cancer cells, or any other therapy deemed necessary by a treating physician.
  • the additional therapeutic agent is an immune response stimulating agent.
  • the AFFIMER® agent can be combined with a growth factor selected from the group consisting of: adrenomedullin (AM), angiopoietin (Ang), BMPs, BDNF, EGF, erythropoietin (EPO), FGF, GDNF, G-CSF, GM-CSF, GDF9, HGF, HDGF, IGF, migration-stimulating factor, myostatin (GDF-8), NGF, neurotrophins, PDGF, thrombopoietin, TGF- ⁇ , TGF- ⁇ , TNF- ⁇ , VEGF, P1GF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, and IL-18.
  • a growth factor selected from the group consisting of: adrenomedullin (AM), angiopoietin (Ang), BMPs, BDNF, EGF, erythropo
  • the additional therapeutic agent is an immune response stimulating agent.
  • the immune response stimulating agent is selected from the group consisting of granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 3 (IL-3), interleukin 12 (IL-12), interleukin 1 (IL-1), interleukin 2 (IL-2), B7-1 (CD80), B7-2 (CD86), 4-1BB ligand, anti-CD3 antibody, anti-CTLA-4 antibody, anti-TIGIT antibody, anti-PD-1 antibody, anti-LAG-3 antibody, and anti-TIM-3 antibody.
  • an immune response stimulating agent is selected from the group consisting of: a modulator of PD-1 activity, a modulator of PD- L2 activity, a modulator of CTLA-4 activity, a modulator of CD28 activity, a modulator of CD80 activity, a modulator of CD86 activity, a modulator of 4-1BB activity, an modulator of OX40 activity, a modulator of KIR activity, a modulator of Tim-3 activity, a modulator of LAG- 3 activity, a modulator of CD27 activity, a modulator of CD40 activity, a modulator of GITR activity, a modulator of TIGIT activity, a modulator of CD20 activity, a modulator of CD96 activity, a modulator of IDO1 activity, a cytokine, a chemokine, an interferon, an interleukin, a lymphokine, a member of the tumor necrosis factor (TNF) family, and an immunostimulatory oligonu
  • an immune response stimulating agent is selected from the group consisting of: a PD-1 antagonist, a PD-L2 antagonist, a CTLA-4 antagonist, a CD80 antagonist, a CD86 antagonist, a KIR antagonist, a Tim-3 antagonist, a LAG- 3 antagonist, a TIGIT antagonist, a CD20 antagonist, a CD96 antagonist, and/or an IDO1 antagonist.
  • the PD-1 antagonist is an antibody that specifically binds PD-1.
  • the antibody that binds PD-1 is KEYTRUDA (MK-3475), pidilizumab (CT-011), nivolumab (OPDIVO, BMS-936558, MDX- 1106), MEDI0680 (AMP-514), REGN2810, BGB-A317, PDR-001, or STI-A1110.
  • the antibody that binds PD-1 is described in PCT Publication WO 2014/179664, for example, an antibody identified as APE2058, APE1922, APE1923, APE1924, APE 1950, or APE1963, or an antibody containing the CDR regions of any of these antibodies.
  • the PD-1 antagonist is a fusion protein that includes PD-L2, for example, AMP- 224. In other embodiments, the PD-1 antagonist is a peptide inhibitor, for example, AUNP-12.
  • the CTLA-4 antagonist is an antibody that specifically binds CTLA-4. In some embodiments, the antibody that binds CTLA-4 is ipilimumab (YERVOY) or tremelimumab (CP-675,206). In some embodiments, the CTLA-4 antagonist a CTLA-4 fusion protein, for example, KAHR-102. In some embodiments, the LAG-3 antagonist is an antibody that specifically binds LAG- 3.
  • the antibody that binds LAG-3 is IMP701, IMP731, BMS-986016, LAG525, and GSK2831781.
  • the LAG-3 antagonist includes a soluble LAG-3 receptor, for example, IMP321.
  • the KIR antagonist is an antibody that specifically binds KIR.
  • the antibody that binds KIR is lirilumab.
  • an immune response stimulating agent is selected from the group consisting of: a CD28 agonist, a 4-1BB agonist, an OX40 agonist, a CD27 agonist, a CD80 agonist, a CD86 agonist, a CD40 agonist, and a GITR agonist.
  • the OX40 agonist includes OX40 ligand, or an OX40-binding portion thereof.
  • the OX40 agonist may be MEDI6383.
  • the OX40 agonist is an antibody that specifically binds OX40.
  • the antibody that binds OX40 is MEDI6469, MEDI0562, or MOXR0916 (RG7888).
  • the OX40 agonist is a vector (e.g., an expression vector or virus, such as an adenovirus) capable of expressing OX40 ligand.
  • the OX40-expressing vector is Delta-24-RGDOX or DNX2401.
  • the 4-1BB (CD137) agonist is a binding molecule, such as an anticalin.
  • the anticalin is PRS-343.
  • the 4-1BB agonist is an antibody that specifically binds 4-1BB.
  • antibody that binds 4-1BB is PF-2566 (PF-05082566) or urelumab (BMS-663513).
  • the CD27 agonist is an antibody that specifically binds CD27.
  • the antibody that binds CD27 is varlilumab (CDX-1127).
  • the GITR agonist comprises GITR ligand or a GITR-binding portion thereof.
  • the GITR agonist is an antibody that specifically binds GITR.
  • the antibody that binds GITR is TRX518, MK-4166, or INBRX- 110.
  • immune response stimulating agents include but are not limited to, cytokines such as chemokines, interferons, interleukins, lymphokines, and members of the tumor necrosis factor (TNF) family.
  • immune response stimulating agents include immunostimulatory oligonucleotides, such as CpG dinucleotides.
  • an immune response stimulating agent includes, but is not limited to, anti-PD-L1 antibodies, anti-PD-1 antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-CD28 antibodies, anti-CD80 antibodies, anti-CD86 antibodies, anti-4-1BB antibodies, anti- OX40 antibodies, anti-KIR antibodies, anti-Tim-3 antibodies, anti-LAG-3 antibodies, anti-CD27 antibodies, anti-CD40 antibodies, anti-GITR antibodies, anti-TIGIT antibodies, anti-CD20 antibodies, anti-CD96 antibodies, or anti-IDO1 antibodies.
  • the AFFIMER® agents disclosed herein may be used alone, or in association with radiation therapy.
  • the AFFIMER® agents disclosed herein may be used alone, or in association with targeted therapies.
  • targeted therapies include: hormone therapies, signal transduction inhibitors (e.g., EGFR inhibitors, such as cetuximab (Erbitux) and erlotinib (Tarceva)); HER2 inhibitors (e.g., trastuzumab (Herceptin) and pertuzumab (Perjeta)); BCR- ABL inhibitors (such as imatinib (Gleevec) and dasatinib (Sprycel)); ALK inhibitors (such as crizotinib (Xalkori) and ceritinib (Zykadia)); BRAF inhibitors (such as vemurafenib (Zelboraf) and dabrafenib (Tafinlar)), gene expression modulators, apoptosis inducers (e.g., bortezomib (Velcade) and carfil
  • the AFFIMER® agents of the disclosure may be used in combination with an anti-cancer therapeutic agent or immunomodulatory drug such as an immunomodulatory receptor inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to the receptor.
  • an AFFIMER® agent is administered in with a STING agonist, for example, as part of a pharmaceutical composition.
  • CDNs cyclic-di-nucleotides
  • PAMP pathogen associated molecular pattern
  • PRR pathogen recognition receptor
  • STING is an adaptor protein in the cytoplasm of host mammalian cells which activates the TANK binding kinase (TBK1)-IRF3 and the NF-.kappa.B signaling axis, resulting in the induction of IFN-.beta.
  • STING is a component of the host cytosolic surveillance pathway, that senses infection with intracellular pathogens and in response induces the production of IFN- ⁇ , leading to the development of an adaptive protective pathogen-specific immune response consisting of both antigen-specific CD4+ and CD8+ T cells as well as pathogen-specific antibodies.
  • an AFFIMER® agent of the disclosure is administered in association with an Akt inhibitor.
  • Exemplary AKT inhibitors include GDC0068 (also known as GDC-0068, ipatasertib and RG7440), MK-2206, perifosine (also known as KRX- 0401), GSK690693, AT7867, triciribine, CCT128930, A-674563, PHT-427, Akti-1/2, afuresertib (also known as GSK2110183), AT13148, GSK2141795, BAY1125976, uprosertib (aka GSK2141795), Akt Inhibitor VIII (1,3-dihydro-1-[1-[[4-(6-phenyl-1H-imidazo[4,5- g]quinoxalin-7-yl)phenyl]m- ethyl]-4-piperidinyl]-2H-benzimidazol-2-one), Akt Inhibitor X (2- chloro-N,N-diethyl-10H-phenox
  • Additional Akt inhibitors include: ATP-competitive inhibitors, e.g. isoquinoline-5-sulfonamides (e.g., H-8, H- 89, NL-71-101), azepane derivatives (e.g., (-)-balanol derivatives), aminofurazans (e.g., GSK690693), heterocyclic rings (e.g., 7-azaindole, 6-phenylpurine derivatives, pyrrolo[2,3- d]pyrimidine derivatives, CCT128930, 3-aminopyrrolidine, anilinotriazole derivatives, spiroindoline derivatives, AZD5363, A-674563, A-443654), phenylpyrazole derivatives (e.g., AT7867, AT13148), thiophenecarboxamide derivatives (e.g., Afuresertib (GSK2110183), 2- pyrimidyl-5-
  • an AFFIMER® agent of the disclosure is administered in association with a MEK inhibitor.
  • MEK inhibitors include AZD6244 (Selumetinib), PD0325901, GSK1120212 (Trametinib), U0126-EtOH, PD184352, RDEA119 (Rafametinib), PD98059, BIX 02189, MEK162 (Binimetinib), AS-703026 (Pimasertib), SL-327, BIX02188, AZD8330, TAK-733, cobimetinib and PD318088.
  • an AFFIMER® agent of the disclosure is administered in association with both an anthracycline such as doxorubicin and cyclophosphamide, including pegylated liposomal doxorubicin .
  • an AFFIMER® agent of the disclosure is administered in association with both an anti-CD20 antibody and an anti-CD3 antibody, or a bispecific CD20/CD3 binder (including a CD20/CD3 BiTE).
  • an AFFIMER® agent of the disclosure is administered in association with a CD73 inhibitor, a CD39 inhibitor or both.
  • inhibitors can be CD73 binders or CD39 binders (such as antibody, antibody fragments or antibody mimetics) that inhibit the ectonucleosidase activity.
  • the inhibitor may be a small molecule inhibitor of the ectonucleosidase activity, such as 6-N,N-Diethyl- ⁇ - ⁇ -dibromomethylene-D-adenosine-5′- triphosphate trisodium salt hydrate, PSB069, or PSB 06126.
  • an AFFIMER® agent of the disclosure is administered in association with an inhibitor poly ADP ribose polymerase (PARP).
  • PARP inhibitor poly ADP ribose polymerase
  • Exemplary PARP inhibitors include Olaparib, Niraparib, Rucaparib, Talazoparib, Veliparib, CEP9722, MK4827 and BGB-290.
  • an AFFIMER® agent of the disclosure is administered in association with an oncolytic virus.
  • An exemplary oncolytic virus is Talimogene Laherparepvec.
  • an AFFIMER® agent of the disclosure is administered in association with an CSF-1 antagonist, such as an agent that binds to CSF-1 or CSF1R and inhibits the interaction of CSF-1 with CSF1R on macrophage.
  • Exemplary CSF-1 antagonists include Emactuzumab and FPA008.
  • an AFFIMER® agent of the disclosure is administered in association with an anti-CD38 antibody.
  • Exemplary anti-CD39 antibodies include Daratumumab and Isatuximab.
  • an AFFIMER® agent of the disclosure is administered in association with an anti-CD40 antibody.
  • Exemplary anti-CD40 antibodies include Selicrelumab and Dacetuzumab.
  • an AFFIMER® agent of the disclosure is administered in association with an inhibitor of anaplatic lymphoma kinase (ALK).
  • ALK inhibitors include Alectinib, Crizotinib and Ceritinib.
  • an AFFIMER® agent of the disclosure is administered in association with multikinase inhibitor that inhibits at least one selected from the group consisting of the family members of VEGFR, PDGFR and FGFR, or an anti-angiogenesis inhibitor.
  • multikinase inhibitor that inhibits at least one selected from the group consisting of the family members of VEGFR, PDGFR and FGFR, or an anti-angiogenesis inhibitor.
  • Exemplary inhibitors include Axitinib, Cediranib, Linifanib, Motesanib, Nintedanib, Pazopanib, Ponatinib, Regorafenib, Sorafenib, Sunitinib, Tivozanib, Vatalanib, LY2874455, or SU5402.
  • an AFFIMER® agent of the disclosure is administered in conjunction with at least one vaccine intended to stimulate an immune response to at least one predetermined antigen.
  • the antigen(s) may be administered directly to the individual, or may be expressed within the individual from, for example, a tumor cell vaccine (e.g., GVAX) which may be autologous or allogenic, a dendritic cell vaccine, a DNA vaccine, an RNA vaccine, a viral-based vaccine, a bacterial or yeast vaccine (e.g., a Listeria monocytogenes or Saccharomyces cerevisiae), etc. See, e.g., Guo et al., Adv.
  • the target antigen may also be a fragment or fusion polypeptide comprising an immunologically active portion of the antigens listed in the table.
  • an AFFIMER® agent of the disclosure is administered in association with at least one antiemetic including, but not limited to: casopitant (GlaxoSmithKline), Netupitant (MGI-Helsinn) and other NK-1 receptor antagonists, palonosetron (sold as Aloxi by MGI Pharma), aprepitant (sold as Emend by Merck and Co.; Rahway, N.J.), diphenhydramine (sold as Benadryl by Pfizer; New York, N.Y.), hydroxyzine (sold as Atarax by Pfizer; New York, N.Y.), metoclopramide (sold as Reglan by AH Robins Co,; Richmond, Va.), lorazepam (sold as Ativan by Wyeth; Madison, N.J.), alprazolam (sold as Xanax by Pfizer; New York, N.Y.), haloperidol (sold as Haldol by Ortho-Mc
  • casopitant Gax
  • an AFFIMER® agent is administered in association with an agent which treats or prevents such a deficiency, such as, e.g., filgrastim, PEG-filgrastim, erythropoietin, epoetin alfa or darbepoetin alfa.
  • an AFFIMER® agent of the disclosure is administered in association with anti-cancer radiation therapy.
  • the radiation therapy is external beam therapy (EBT): a method for delivering a beam of high-energy X-rays to the location of the tumor.
  • EBT external beam therapy
  • the beam is generated outside the patient (e.g., by a linear accelerator) and is targeted at the tumor site.
  • These X-rays can destroy the cancer cells and careful treatment planning allows the surrounding normal tissues to be spared.
  • No radioactive sources are placed inside the patient's body.
  • the radiation therapy is proton beam therapy: a type of conformal therapy that bombards the diseased tissue with protons instead of X-rays.
  • the radiation therapy is conformal external beam radiation therapy: a procedure that uses advanced technology to tailor the radiation therapy to an individual's body structures.
  • the radiation therapy is brachytherapy: the temporary placement of radioactive materials within the body, usually employed to give an extra dose--or boost--of radiation to an area.
  • the treatment involves the administration of an AFFIMER® agent of the present disclosure in combination with anti-viral therapy. Treatment with an AFFIMER® agent can occur prior to, concurrently with, or subsequent to administration of antiviral therapy.
  • the anti-viral drug used in combination therapy will depend upon the virus the subject is infected with.
  • Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously.
  • an AFFIMER® agent described herein and at least one additional therapeutic agent may be administered in any order or concurrently.
  • the AFFIMER® agent will be administered to patients that have previously undergone treatment with a second therapeutic agent.
  • the AFFIMER® agent and a second therapeutic agent will be administered substantially simultaneously or concurrently.
  • a subject may be given an AFFIMER® agent while undergoing a course of treatment with a second therapeutic agent (e.g., chemotherapy).
  • a second therapeutic agent e.g., chemotherapy
  • an AFFIMER® agent will be administered within 1 year of the treatment with a second therapeutic agent.
  • an AFFIMER® agent will be administered within 10, 8, 6, 4, or 2 months of any treatment with a second therapeutic agent. In certain other embodiments, an AFFIMER® agent will be administered within 4, 3, 2, or 1 weeks of any treatment with a second therapeutic agent. In some embodiments, an AFFIMER® agent will be administered within 5, 4, 3, 2, or 1 days of any treatment with a second therapeutic agent. It will further be appreciated that the two (or more) agents or treatments may be administered to the subject within a matter of hours or minutes (e.g., substantially simultaneously).
  • an AFFIMER® agent of the present disclosure depends on the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, whether the AFFIMER® agent is administered for therapeutic or preventative purposes, previous therapy, the patient's clinical history, and so on, all at the discretion of the treating physician.
  • the AFFIMER® agent can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is affected or a diminution of the disease state is achieved (e.g., reduction in tumor size).
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient and will vary depending on the relative potency of an individual agent.
  • dosage is from 0.01 ⁇ g to 100 mg/kg of body weight, from 0.1 ⁇ g to 100 mg/kg of body weight, from 1 ⁇ g to 100 mg/kg of body weight, from 1 mg to 100 mg/kg of body weight, 1 mg to 80 mg/kg of body weight from 10 mg to 100 mg/kg of body weight, from 10 mg to 75 mg/kg of body weight, or from 10 mg to 50 mg/kg of body weight.
  • the dosage of the AFFIMER® agent is from about 0.1 mg to about 20 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 0.1 mg/kg of body weight.
  • the dosage of the AFFIMER® agent is about 0.25 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 0.5 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 1 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 1.5 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 2 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 2.5 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 5 mg/kg of body weight.
  • the dosage of the AFFIMER® agent is about 7.5 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 10 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 12.5 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 15 mg/kg of body weight. In some embodiments, the dosage can be given once or more daily, weekly, monthly, or yearly. In some embodiments, the AFFIMER® agent is given once every week, once every two weeks, once every three weeks, or once every four weeks. In some embodiments, an AFFIMER® agent may be administered at an initial higher "loading" dose, followed by at least one lower dose.
  • a dosing regimen may comprise administering an initial dose, followed by additional doses (or "maintenance" doses) once a week, once every two weeks, once every three weeks, or once every month.
  • a dosing regimen may comprise administering an initial loading dose, followed by a weekly maintenance dose of, for example, one-half of the initial dose.
  • a dosing regimen comprises administering an initial loading dose, followed by maintenance doses of, for example one-half of the initial dose every other week.
  • a dosing regimen comprises administering three initial doses for 3 weeks, followed by maintenance doses of, for example, the same amount every other week.
  • any therapeutic agent may lead to side effects and/or toxicities.
  • the side effects and/or toxicities are so severe as to preclude administration of the particular agent at a therapeutically effective dose.
  • drug therapy must be discontinued, and other agents may be tried.
  • many agents in the same therapeutic class often display similar side effects and/or toxicities, meaning that the patient either has to stop therapy, or if possible, suffer from the unpleasant side effects associated with the therapeutic agent.
  • the dosing schedule may be limited to a specific number of administrations or "cycles".
  • the AFFIMER® agent is administered for 3, 4, 5, 6, 7, 8, or more cycles.
  • the AFFIMER® agent is administered every 2 weeks for 6 cycles, the AFFIMER® agent is administered every 3 weeks for 6 cycles, the AFFIMER® agent is administered every 2 weeks for 4 cycles, the AFFIMER® agent is administered every 3 weeks for 4 cycles, etc.
  • Dosing schedules can be decided upon and subsequently modified by those skilled in the art.
  • the present disclosure provides methods of administering to a subject the polypeptides or agents described herein comprising using an intermittent dosing strategy for administering at least one agent (e.g., two or three agents), which may reduce side effects and/or toxicities associated with administration of an AFFIMER® agent, chemotherapeutic agent, etc.
  • a method for treating cancer in a human subject comprises administering to the subject a therapeutically effective dose of an AFFIMER® agent in combination with a therapeutically effective dose of a chemotherapeutic agent, wherein one or both of the agents are administered according to an intermittent dosing strategy.
  • the intermittent dosing strategy comprises administering an initial dose of an AFFIMER® agent to the subject and administering subsequent doses of the AFFIMER® agent about once every 2 weeks.
  • the intermittent dosing strategy comprises administering an initial dose of an AFFIMER® agent to the subject and administering subsequent doses of the AFFIMER® agent about once every 3 weeks.
  • the intermittent dosing strategy comprises administering an initial dose of an AFFIMER® agent to the subject and administering subsequent doses of the AFFIMER® agent about once every 4 weeks.
  • the AFFIMER® agent is administered using an intermittent dosing strategy and the chemotherapeutic agent is administered weekly.
  • the disclosure also provides methods for treating subjects using an AFFIMER® agent of the disclosure, wherein the subject suffers from a viral infection.
  • the viral infection is infection with a virus selected from the group consisting of human immunodeficiency virus (HIV), hepatitis virus (A, B, or C), herpes virus (e.g., VZV, HSV-I, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus or arboviral encephalitis virus.
  • HCV human immunodeficiency virus
  • A, B, or C hepatitis virus
  • herpes virus e.g., VZV, HSV-I, HAV-6, HSV-II, and
  • the disclosure provides methods for treating subjects using an AFFIMER® agent thereof of the disclosure, wherein the subject suffers from a bacterial infection.
  • the bacterial infection is infection with a bacterium selected from the group consisting of Chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, Legionella, Corynebacterium diphtheriae, Salmonella, bacilli, Vibrio cholerae, Clostridium tetan, Clostridium botulinum, Bacillus anthricis, Yersinia pestis, Mycobacterium leprae, Mycobacterium lepromatosis, and Borriella.
  • the disclosure provides methods for treating subjects using an AFFIMER® agent of the disclosure, wherein the subject suffers from a fungal infection.
  • the fungal infection is infection with a fungus selected from the group consisting of Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.
  • Candida albicans, krusei, glabrata, tropicalis, etc.
  • Cryptococcus neoformans Aspergillus (fumigatus, niger, etc.)
  • Genus Mucorales micor, absidia, rhizopus
  • the disclosure provides methods for treating subjects using an AFFIMER® agent of the disclosure, wherein the subject suffers from a parasitic infection.
  • the parasitic infection is infection with a parasite selected from the group consisting of Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba, Giardia lambia, Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii and Nippostrongylus brasiliensis.
  • a bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73, the first peptide comprises the amino acid sequence of KEYGPEEWW (SEQ ID NO: 221), and the second peptide comprises the amino acid sequence of GDYEQVLIH (SEQ ID NO: 222); and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 61, the first peptide comprises the amino acid sequence of INVQEDLIQT (SEQ ID NO: 206), and the second peptide comprises the amino acid sequence of SIYQAEELE (SEQ ID NO: 212).
  • the bispecifc fusion protein of paragraph 1 further comprising one or more linker, preferably a rigid linker, more preferably a rigid linker comprising the sequence of A(EAAAK)n (SEQ ID NO: 86).
  • the bispecifc fusion protein of paragraph 1 or 2 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 1.
  • AVA21-02 (AVA04-269+AVA19-158) 4.
  • a bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73, the first peptide comprises the amino acid sequence of KEYGPEEWW (SEQ ID NO: 221), and the second peptide comprises the amino acid sequence of GDYEQVLIH (SEQ ID NO: 222); and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 62, the first peptide comprises the amino acid sequence of DATDGDGVS (SEQ ID NO: 207), and the second peptide comprises the amino acid sequence of FWGDEWDVL (SEQ ID NO: 213).
  • the bispecifc fusion protein of paragraph 4 further comprising one or more linker, preferably a rigid linker, more preferably a rigid linker comprising the sequence of A(EAAAK)n (SEQ ID NO: 86). 6.
  • the bispecifc fusion protein of paragraph 4 or 5 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 2.
  • AVA21-03 (AVA04-269+AVA19-01) 7.
  • a bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73, the first peptide comprises the amino acid sequence of KEYGPEEWW (SEQ ID NO: 221), and the second peptide comprises the amino acid sequence of GDYEQVLIH (SEQ ID NO: 222); and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53, the first peptide comprises the amino acid sequence of GPTGASFHW (SEQ ID NO: 204), and the second peptide comprises the amino acid sequence of FWGDDWDLL (SEQ ID NO: 210).
  • the bispecifc fusion protein of paragraph 7 further comprising one or more linker, preferably a rigid linker, more preferably a rigid linker comprising the sequence of A(EAAAK)n (SEQ ID NO: 86).
  • the bispecifc fusion protein of paragraph 7 or 8 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 3.
  • AVA21-04 (AVA04-251+AVA04-251+AVA19-01) 10.
  • a trispecific fusion protein comprising: two PD-L1 binding polypeptides, each comprising a first peptide and a second peptide, wherein each of the PD-L1 binding polypeptides comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53, the first peptide comprises the amino acid sequence of GPTGASFHW (SEQ ID NO: 204), and the second peptide comprises the amino acid sequence of FWGDDWDLL (SEQ ID NO:
  • the trispecifc fusion protein of paragraph 10 further comprising one or more linker, preferably a rigid linker, more preferably a rigid linker comprising the sequence of A(EAAAK)n (SEQ ID NO: 86).
  • the trispecifc fusion protein of paragraph 10 or 11 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 4.
  • AVA21-05 (AVA04-251+AVA19-01) 13.
  • a bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53, the first peptide comprises the amino acid sequence of GPTGASFHW (SEQ ID NO: 204), and the second peptide comprises the amino acid sequence of FWGDDWDLL (SEQ ID NO: 210).
  • the bispecifc fusion protein of paragraph 13 further comprising one or more linker, preferably a rigid linker, more preferably a rigid linker comprising the sequence of A(EAAAK)n (SEQ ID NO: 86).
  • the bispecifc fusion protein of paragraph 13 or 14 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 5.
  • AVA21-08/-09 XT (AVA04-251+AVA04-251+AVA03-42+AVA19-158+AVA19-158) 16.
  • a trispecific fusion protein comprising: two PD-L1 binding polypeptides, each comprising a first peptide and a second peptide, wherein each of the PD-L1 binding polypeptides comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); an HSA binding polypeptide comprising a first peptide and a second peptide, wherein the HSA binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 75, the first peptide comprises the amino acid sequence of NFFQRRWPG (SEQ ID NO: 225), and the second peptide comprises the amino acid sequence of WKFRNTDRG (SEQ ID NO: 226); and
  • the bispecifc fusion protein of paragraph 16 further comprising one or more linker.
  • the linker is a rigid linker, preferably a rigid linker comprising the sequence of A(EAAAK)n (SEQ ID NO: 86).
  • the bispecifc fusion protein of any one of paragraphs 16-18 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 8.
  • the linker is a flexible linker, preferably a flexible linker comprising the sequence of (G 4 S)n (SEQ ID NO: 88). 21.
  • the bispecifc fusion protein of any one of paragraphs 16, 17 or 20 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 9.
  • AVA21-11 XT (AVA04-251+AVA04-251+AVA19-06+AVA19-06+AVA03-42) 22.
  • a trispecific fusion protein comprising: two PD-L1 binding polypeptides, each comprising a first peptide and a second peptide, wherein each of the PD-L1 binding polypeptides comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); and two LAG3 binding polypeptides, each comprising a first peptide and a second peptide, wherein each of the LAG3 binding polypeptides comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 75, the first peptide comprises the amino acid sequence of DFPDDPWFW (SEQ ID NO: 205), and the second peptide comprises the amino acid sequence of DWEDAVTPY (
  • the trispecifc fusion protein of paragraph 22 further comprising one or more linker, preferably a rigid linker, more preferably a rigid linker comprising the sequence of A(EAAAK)n (SEQ ID NO: 86).
  • the trispecifc fusion protein of paragraph 22 or 23 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 10.
  • AVA21-12 XT (AVA04-640+AVA19-06+AVA19-06+AVA03-42) 25.
  • a trispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 75, the first peptide comprises the amino acid sequence of RRKHFPQWP (SEQ ID NO: 223), and the second peptide comprises the amino acid sequence of DLQPREVFQ (SEQ ID NO: 224); two LAG3 binding polypeptides, each comprising a first peptide and a second peptide, wherein each of the LAG3 binding polypeptides comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 75, the first peptide comprises the amino acid sequence of DFPDDPWFW (SEQ ID NO: 205), and the second peptide comprises the amino acid sequence of DWEDAVTPY (SEQ ID
  • the trispecifc fusion protein of paragraph 25 further comprising one or more linker, preferably a rigid linker, more preferably a rigid linker comprising the sequence of A(EAAAK)n (SEQ ID NO: 86).
  • the trispecifc fusion protein of paragraph 25 or 26 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 11.
  • AVA21-06 BP (AVA04-251-hIgG1 Fc+AVA19-06) 28.
  • a bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); an hIgG1 Fc polypeptide; and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 75, the first peptide comprises the amino acid sequence of DFPDDPWFW (SEQ ID NO: 205), and the second peptide comprises the amino acid sequence of DWEDAV
  • the trispecifc fusion protein of paragraph 28 further comprising one or more linker, preferably a flexible linker, more preferably a flexible linker comprising the sequence of (G 4 S)n (SEQ ID NO: 88).
  • the trispecifc fusion protein of paragraph 28 or 29 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 6.
  • AVA21-07 BP (AVA04-251-hIgG1 Fc+AVA19-158) 31.
  • a bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); an hIgG1 Fc polypeptide; and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 62, the first peptide comprises the amino acid sequence of DATDGDGVS (SEQ ID NO: 207), and the second peptide comprises the amino acid sequence of FWGDEW
  • the bispecifc fusion protein of paragraph 31 further comprising one or more linker, preferably a flexible linker, more preferably a flexible linker comprising the sequence of (G 4 S)n (SEQ ID NO: 88).
  • the bispecifc fusion protein of paragraph 31 or 32 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 7.
  • AVA21-15 (AVA19-06+AVA04-251+hIgG1 LALA Fc)
  • AVA21-16 (AVA04-251+AVA19-06+hIgG1 LALA Fc) 34.
  • a bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); an hIgG1 LALA Fc polypeptide; and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 75, the first peptide comprises the amino acid sequence of DFPDDPWFW (SEQ ID NO: 205), and the second peptide comprises the amino acid sequence of DW
  • the bispecifc fusion protein of paragraph 31 further comprising one or more linker, preferably a flexible linker, more preferably a flexible linker comprising the sequence of (G4S)n (SEQ ID NO: 88).
  • the bispecifc fusion protein of paragraph 34 or 35 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 44.
  • the bispecifc fusion protein of paragraph 34 or 35 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 45.
  • AVA21-06 CR (AVA04-251+hIgG1 LALA Fc+AVA19-06) 38.
  • the bispecific fusion protein of paragraph 34 further comprising two linkers, preferably flexible linkers, more preferably flexible linkers comprising the sequence of (G 4 S)n (SEQ ID NO: 88).
  • the bispecifc fusion protein of paragraph 38 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 19; or at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 20-37.
  • AVA21-12 CR (AVA04-251+hIgG1 LALA Fc+AVA19-170) 40.
  • a bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); an hIgG1 LALA Fc polypeptide; and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 66, the first peptide comprises the amino acid sequence of DYEDDPWTF (SEQ ID NO: 208), and the second peptide comprises the amino acid sequence of SI
  • the bispecific fusion protein of paragraph 40 further comprising two linkers, preferably flexible linkers, more preferably flexible linkers comprising the sequence of (G 4 S)n (SEQ ID NO: 88).
  • the bispecifc fusion protein of paragraph 40 or 41 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 39.
  • AVA21-13 CR (AVA04-251+hIgG1 LALA Fc+AVA19-173) 43.
  • a bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); an hIgG1 LALA Fc polypeptide; and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 69, the first peptide comprises the amino acid sequence of DYADDPWFY (SEQ ID NO: 209), and the second peptide comprises the amino acid sequence of
  • the bispecific fusion protein of paragraph 43 further comprising two linkers, preferably flexible linkers, more preferably flexible linkers comprising the sequence of (G 4 S)n (SEQ ID NO: 88).
  • the bispecific fusion protein of paragraph 43 or 44 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 41.
  • AVA21-06 CA (AVA04-251-hIgG4 Fc+AVA19-06) 46.
  • a bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); an hIgG4 Fc polypeptide; and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 75, the first peptide comprises the amino acid sequence of DFPDDPWFW (SEQ ID NO: 205), and the second peptide comprises the amino acid sequence of DWEDAV
  • the bispecific fusion protein of paragraph 46 further comprising a linker, preferably a flexible linker, more preferably a flexible linker comprising the sequence of (G 4 S)n (SEQ ID NO: 88).
  • the bispecifc fusion protein of paragraph 47 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 15.
  • AVA21-12 CA (AVA04-251-hIgG4 Fc+AVA19-170) 49.
  • a bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); an hIgG4 Fc polypeptide; and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 66, the first peptide comprises the amino acid sequence of DYEDDPWTF (SEQ ID NO: 208), and the second peptide comprises the amino acid sequence of SIDWP
  • the bispecific fusion protein of paragraph 49 further comprising a linker, preferably a flexible linker, more preferably a flexible linker comprising the sequence of (G 4 S)n (SEQ ID NO: 88).
  • the bispecifc fusion protein of paragraph 49 or 50 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 38.
  • AVA21-13 CA (AVA04-251-hIgG4 Fc+AVA19-173) 52.
  • a bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); an hIgG4 Fc polypeptide; and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 69, the first peptide comprises the amino acid sequence of DYADDPWFY (SEQ ID NO: 209), and the second peptide comprises the amino acid sequence of Y
  • the bispecific fusion protein of paragraph 52 further comprising a linker, preferably a flexible linker, more preferably a flexible linker comprising the sequence of (G 4 S)n (SEQ ID NO: 88).
  • the bispecific fusion protein of paragraph 52 or 53 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 40.
  • a bispecific fusion protein comprising a first peptide comprising the amino acid sequence of SEQ ID NO: 204 and a second peptide comprising the amino acid sequence of SEQ ID NO: 210.
  • the bispecific fusion protein of paragraph 55 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53.
  • AVA19-06 57 A bispecific fusion protein comprising a first peptide comprising the amino acid sequence of SEQ ID NO: 205 and a second peptide comprising the amino acid sequence of SEQ ID NO: 211. 58.
  • the bispecific fusion protein of paragraph 57 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 54.
  • AVA19-157 59 is a bispecific fusion protein comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 54.
  • a bispecific fusion protein comprising a first peptide comprising the amino acid sequence of SEQ ID NO: 206 and a second peptide comprising the amino acid sequence of SEQ ID NO: 212.
  • the bispecific fusion protein of paragraph 59 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 61.
  • AVA19-158 57 A bispecific fusion protein comprising a first peptide comprising the amino acid sequence of SEQ ID NO: 207 and a second peptide comprising the amino acid sequence of SEQ ID NO: 213. 58.
  • the bispecific fusion protein of paragraph 57 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 63.
  • a bispecific fusion protein comprising a first peptide comprising the amino acid sequence of SEQ ID NO: 208 and a second peptide comprising the amino acid sequence of SEQ ID NO: 214.
  • the bispecific fusion protein of paragraph 59 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 66.
  • AVA19-170 61 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 66.
  • a bispecific fusion protein comprising a first peptide comprising the amino acid sequence of SEQ ID NO: 209 and a second peptide comprising the amino acid sequence of SEQ ID NO: 210.
  • the bispecific fusion protein of paragraph 61 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 69.
  • a polynucleotide comprising an open reading frame encoding the fusion protein of any one of paragraphs 1-62.
  • 64. A vector, optionally a viral vector or a plasmid vector, comprising the polynucleotide of paragraph 63. 65.
  • a cell optionally a mammalian cell, comprising the polynucleotide of paragraph 63 or the vector of paragraph 74.
  • a pharmaceutical composition comprising: (a) the fusion protein of any one of paragraphs 1-62, the polynucleotide of paragraph 63, the vector of paragraph 64, or the cell of paragraph 65; and (b) a pharmaceutically acceptable excipient.
  • a method comprising administering to a subject the polynucleotide of paragraph 63, the vector of paragraph 64, the cell of paragraph 65, or the pharmaceutical composition of paragraph 66.
  • 68 The method of paragraph 67, wherein the subject has a cancer. 69.
  • a fusion protein comprising a PD-L1 binding polypeptide, a LAG3 binding polypeptide, and an Fc domain selected from hIgG1 Fc, hIgG1 LALA Fc, and hIgG4 Fc, wherein the fusion protein comprises at least one (optionally two to four) flexible linker, optionally wherein the flexible linker is (G4S)n and n is 1, 2, 3, 4, 5, or 6. 71.
  • a fusion protein comprising a PD-L1 binding polypeptide, a LAG3 binding polypeptide, and an Fc domain selected from hIgG1 Fc, hIgG1 LALA Fc, and hIgG4 Fc, wherein the fusion protein comprises at least one (optionally two to four) rigid linker, optionally wherein the rigid linger is A(EAAAK)n and n is 1, 2, 3, 4, 5, or 6.
  • EXAMPLES A general description of the protocols used herein, including selection of the AFFIMER® polypeptides by phage display, expression studies (e.g., in E. coli), post-purification purity studies (e.g., SEC-HPLC), binding affinity studies (e.g., ELISAs), competitive binding studies (e.g., competitive ELISAs), and kinetic studies (e.g., BIACORE® analysis), may be found in International Publication No. WO 2019/197583, incorporated herein by reference in its entirety.
  • expression studies e.g., in E. coli
  • post-purification purity studies e.g., SEC-HPLC
  • binding affinity studies e.g., ELISAs
  • competitive binding studies e.g., competitive ELISAs
  • kinetic studies e.g., BIACORE® analysis
  • Example 1 Characterization of Bispecific In-Line Fusion Dimers and Trimers that Bind to LAG-3 and PD-L1 AFFIMER® polypeptides selected for binding to human PD-L1 (AVA04; “anti-PD-L1 AFFIMER® polypeptides”) were genetically fused with AFFIMER® polypeptides selected for binding to human LAG-3 (AVA19; “anti-LAG-3 AFFIMER® polypeptides”).
  • These in-line fusion (ILF) formats include repetitive linkers connecting the monomer components.
  • ILF dimers were produced by (i) fusing AVA04-269 to AVA19-157 (AVA21-01), (ii) fusing AVA04-269 to AVA19-158 (AVA21-02), (iii) fusing AVA04-269 to AVA19-01 (AVA21-03), and (iv) fusing AVA04-251 to AVA19-01 (AVA21-05).
  • An ILF trimer was produced by fusing two AVA04-251 to AVA19-01 (AVA21-04).
  • Table 4 lists the ILF formats and their monomer or dimer components. Schematics of the ILF dimer and trimer proteins are depicted in FIG.1.
  • AVA21-01, AVA21-02 and AVA21-03 exhibited decreased binding to PD-L1 relative to their monomer parent clone, AVA04-269.
  • AVA21-04 and AVA21-05 showed comparable binding affinity to PD-L1 relative to their parent clone AVA04-251.
  • a PD-1/PD-L1 blockade bioassay was performed using the dimer and trimer ILF proteins of Table 4, the results of which are shown in FIG.4.
  • AVA21-01, AVA21-02 and AVA21-03 exhibited reduced function relative to their monomer parent clone, AVA04-269.
  • AVA21-04 and AVA21-05 showed comparable function relative to their parent clone AVA04-251.
  • a bridging ELISA was performed using the dimer and trimer ILF proteins of Table 4 to assess whether they are able to bind LAG-3 and PD-L1 simultaneously.
  • LAG-3 was coated on the plate, and anti-PD-L1 antibody was used to detect the AFFIMER® polypeptides. Results are shown in FIG.5. Most formats bound both targets simultaneously, to varying degrees.
  • target-specific cell binding assays were performed using the AVA21-04 and AVA21-05 ILF proteins of Table 4 to assess whether they bind to LAG-3 expressed on cells. Results are shown in FIG.6. Both AVA21-04 and AVA21-05 bound to LAG-3 in the LAG-3- expressing cells but did not bind to control Jurkat or CHO-K1 cells.
  • Example 2 Characterization of Trispecific In-Line Fusion (ILF) Pentamers that Binds to LAG-3, PD-L1, and HSA Trispecific (ILF) pentamers were also produced.
  • These pentamers include AFFIMER® polypeptides selected for binding to human PD-L1, with AFFIMER® polypeptides selected for binding to human LAG-3, and with AFFIMER® polypeptides selected for binding to human serum albumin (HSA) to extend the half-life of the ILFs.
  • the pentamer formats are depicted in FIG.7, showing a rigid or flexible linker connecting each monomer with each other.
  • AVA21-08 XT includes two anti-PD-L1 AFFIMER® polypeptides (each SEQ ID NO: 72), an anti-HSA AFFIMER® polypeptide (SEQ ID NO: 80), and two anti-LAG-3 AFFIMER® polypeptides (each SEQ ID NO: 62) with rigid linkers (A(EAAAK) 6 ); and AVA21-09 XT (SEQ ID NO: 9) includes two anti-PD-L1 AFFIMER® polypeptides (each SEQ ID NO: 72), an anti- HSA AFFIMER® polypeptide (SEQ ID NO: 80), and two anti-LAG-3 AFFIMER® polypeptides (each SEQ ID NO: 62) with flexible linkers ((G 4 S) 6 ).
  • a PD-L1 direct binding ELISA was performed using the two pentamers. The results show that AVA21-08 XT and AVA21-09 XT appear at least equivalent to the AVA04-251 BH (SEQ ID NO: 74) control for PD-L1 binding, suggesting no adverse effect of the additional HSA and LAG-3 binding domains (FIG.9).
  • a LAG-3 direct binding ELISA was also performed using the two pentamers as well as AVA19-158 (SEQ ID NO: 62) and AVA19-158 BK (SEQ ID NO: 54).
  • Binding was not detected in LAG-3-negative cells.
  • a dual PD-L1/HSA target binding assay was performed, the results of which show that both pentamers engage PD-L1 and HSA targets simultaneously (FIG.13). Binding of the pentamers was unaffected in the presence of HSA.
  • the rigid linker appears better than flexible linkers for PD-L1/HSA dual target engagement.
  • Results from a functional PD-L1/PD-1 gene reporter assay with the pentamers show that AVA21-08 XT exhibits comparable activity to the AVA04-251 BH control (FIG.14). Functionality of AVA21-09 XT is significantly decreased. Again, the flexible linker seems less effective compared to the rigid linker.
  • AVA21-11 XT includes two anti-PD-L1 AFFIMER® polypeptides (each SEQ ID NO: 72), two anti-LAG-3 AFFIMER® polypeptides (each SEQ ID NO: 152), and an anti-HSA AFFIMER® polypeptide (SEQ ID NO: 178), with rigid linkers (A(EAAAK)6); and
  • AVA21-12 XT includes an anti-PD-L1 AFFIMER® polypeptide (SEQ ID NO: 75), two anti-LAG-3 AFFIMER® polypeptides (each SEQ ID NO: 152), and an anti-HSA AFFIMER® polypeptide (SEQ ID NO: 178), with rigid linkers (A(EAAAK) 6 ).
  • a PD-L1, LAG-3 and HSA BIACORETM kinetic binding analysis was performed for AVA21-11 XT and AVA21-12 XT, the results of which are shown in FIG.18 and confirm that both constructs can engage all three targets
  • a PD-L1 direct binding ELISA using AVA21-11 XT and AVA21-12 XT was performed, the results of which show that both constructs exhibit binding to PD-L1 comparable to parent AFFIMER® controls (FIG.19).
  • Results of a LAG-3 direct binding ELISA using AVA21-11 XT and AVA21-12 XT are shown in FIG.20.
  • AVA21-11 XT shows better binding to LAG-3 than AVA21-12 XT and is equivalent to AVA19-06 BK (SEQ ID NO: 57) control.
  • the results of an HSA binding ELISA show decreased binding of AVA21-12 XT to HSA compared to monomer control AVA03-42 (SEQ ID NO: 80) (FIG.21).
  • Results of multimer cell binding in CHO-K1 PD-L1 overexpressing cells demonstrate that AVA21-12 XT binding to CHO-K1 PD-L1 cells was comparable to that of AVA04-640 control (SEQ D NO: 75) (FIG.25).
  • the bispecific huIgG1 Fc fusion proteins include an anti-PDL1 AFFIMER® polypeptide, huIgG1 Fc, and anti-LAG-3 AFFIMER® polypeptide, with a flexible linker.
  • SEC-HPLC chromatograms of the bispecific IgG1 Fc fusion multimers are shown in FIG.27.
  • a PD-1/PD-L1 blockade assay on AVA21-06 BP showed that AVA21-06 BP exhibits slightly decreased activity compared to AVA04-251 V.2 control protein (FIG.30).
  • Results from a cell binding assay for AVA21-06 BP binding in LAG-3-positive and negative Jurkat cells show a dose effect for AVA21-06 BP on positive cells (FIG.31). No binding observed on LAG-3 negative cells.
  • the bispecific huIgG1 LALA Fc fusion proteins AVA21-15 (SEQ ID NO: 44) and AVA21-16 (SEQ ID NO: 45) include an anti-PDL1 AFFIMER® polypeptide, an anti-LAG-3 AFFIMER® polypeptide, and an huIgG1 LALA Fc, and with a flexible linker. Reducing SDS- PAGE was used to confirm the expected molecular weight for the two constructs (FIG.33). Results of a PD-1/PD-L1 blockade assay on AVA21-15 and AVA21-16 are shown in FIG.34.
  • AVA21-15 showed a significant decrease in activity compared to control proteins, AVA04-251 AG.3 (SEQ ID NO: 79) and AVA04-251 CF (SEQ ID NO: 78).
  • AVA21-16 potency was reduced in this assay relative to that of control proteins, but the protein remained active.
  • Results of direct LAG-3 and PD-L1 binding ELISAs are shown in FIG.35. Results show that the positioning of LAG-3 and PD-L1 binders affects their ability to bind target. Both formats had significantly lower affinity for antigen than controls (AVA19-06 V.2 (SEQ ID NO: 55) and AVA21-06 CR (SEQ ID NO: 19)) when binding to LAG-3.
  • AVA21-16 binds to target with slightly decreased affinity compared to controls (AVA21-06 CR (SEQ ID NO: 19), AVA04-251 V.2 (SEQ ID NO: 76), and AVA04-251 AG.3 (SEQ ID NO: 79), AVA21- 15 affinity is significantly decreased due to positioning of PD-L1 binder. Additional bispecific huIgG1 LALA Fc fusion proteins were produced, as depicted in FIG.36.
  • AVA21-06 CR (SEQ ID NO: 19), AVA21-12 CR (SEQ ID NO: 39), and AVA21-13 CR (SEQ ID NO: 41) each include an anti-PD-L1 AFFIMER® polypeptide, an anti-LAG-3 AFFIMER® polypeptide, and an huIgG1 LALA Fc, and with a flexible linker. Reducing SDS-PAGE was used to confirm the expected molecular weight of the additional constructs (FIG.36).
  • a human PD-L1 kinetics analysis for AVA21-06 CR, AVA21-06 CR T89A (SEQ ID NO: 20), AVA21-12 CR, and AVA21-13 CR demonstrated that binding to PD-L1 is equivalent for all proteins tested (FIG.37).
  • a human LAG-3 kinetics analysis for AVA21-06 CR, AVA21-12 CR, and AVA21-13 CR showed that all clones bind to LAG-3 with comparable affinities (FIG.38).
  • Results of a PD-L1 direct binding ELISA showed equivalent binding for AVA21-06 CR and control AVA04-251 V.2 (FIG.39).
  • Results of a LAG-3 direct binding ELISA on AVA21-06 CR, AVA21-12 CR, and AVA21-13 CR showed that all CR formats exhibit equivalent binding to LAG-3 and either comparable or better binding affinity than their relative CS controls (FIG.40).
  • Alanine scanning was performed on AVA21-06 CR, the results of which are shown in FIG.41.
  • Alanine mutants of parent format AVA21-06 CR were engineered to improve binding and reduce aggregation.
  • Results of direct LAG-3 and PD-L1 binding ELISAs for alanine scanned AVA21 CR IgG1 LALA Loop 2 constructs showed that little variation in PD-L1 binding between controls and various mutants was observed (FIG.42).
  • D439A (SEQ ID NO: 25), P440A (SEQ ID NO: 26) and W443A (SEQ ID NO: 29) significantly affected binding to LAG-3 (essential amino acids)
  • D438A (SEQ ID NO: 24) impaired binding to an extent
  • P437A (SEQ ID NO: 23) did not appear to significantly affect binding to LAG-3.
  • Results of direct LAG-3 and PD-L1 binding ELISAs for alanine scanned AVA21 CR IgG1 LALA Loop 4 constructs showed little variation in PD-L1 and LAG-3 binding between controls and various mutants was observed (FIG.43).
  • Biacore binding to LAG3 and PDL1 FIGs.44A-44B
  • the binding affinity of AVA21 clones towards their target were characterised using surface plasmon resonance technique on the Biacore 8K (Cytiva).
  • Binding towards the ligand PD-L1 (R&D systems; Cat:156-B7) and human serum albumin (Sigma; Cat:A3782) were performed by immobilising the respective ligand on Series S CM5 chip (Cytiva; Cat:29104988) using amine coupling technique (Cytiva Amine coupling kit, type 2; Cat: BR-1006-33), with AVA21 AFFIMER® proteins flowed on flow cell. Meanwhile for determination of LAG3 binding, AVA21 AFFIMER® proteins were bound onto CM5 chip using amine coupling technique, with LAG3 flowed over the flow cell.
  • AVA21-06 CR, AVA21-06 T89A CR, AVA21-12CR and AVA21-13CR constructs were characterized for its binding to human PD-L1 and human LAG-3 expressed on cells.
  • H441 cells endogenously expressing PD-L1 were used. Binding to PD-L1 on cells was equivalent for all constructs and was similar to PD-L1 only binding control (AVA04-251 CR).
  • hLAG3-overexpressing and LAG3 negative D0.11.10 cells were used.
  • AVA21-06 CR, AVA21-06 T89A CR, AVA21-12CR and AVA21-13CR bound to hLAG3-overexpressing but not LAG3 negative cells. Binding was similar between constructs. Control AVA21-19CR showed lower binding and PD-L1 only AFFIMERTM (AVA04-251 CR) no binding as expected. Binding ELISA to PD-L1 (FIG.47) Results of a human PD-L1 direct binding ELISA for AVA21-06 CR, AVA21-06 T89A CR, AVA21-12CR and AVA21-13CR showed that all clones bind to PD-L1 with comparable affinities.
  • Example 5 Characterisation of lead AVA21 constructs in functional assay for PD-L1 & LAG3 inhibition Comparison of inhibitory activity in PD-1/PD-L1 blockade assay (FIG.48) PD-1/PD-L1 Blockade Bioassay (Promega) was used according to the manufacturer’s instructions to test ability of AVA21-06 CR, AVA21-06 T89A CR, AVA21-12CR and AVA21- 13CR to block PD-1/PD-L1 signalling.
  • the assay involves co-culture of PD-1 expressing Effector cells (Jurkat) and CHO-K1 APC (Antigen Presenting Cells) cells expressing PD-L1.
  • AVA21-06 CR, AVA21-12CR and AVA21- 13CR have the same anti-PD-L1 affimer domain at the N terminus and hence show similar inhibitory activity as AVA04-251 CR (anti-PD-L1) indicating that AVA21 AFFIMER® proteins are strong PD-L1 inhibitors.
  • LAG3/MHCII blockade assay (FIG.49) LAG3/MHC II Blockade Bioassay (Promega) was used according to the manufacturer’s instructions to test ability of AVA21-06 CR, AVA21-12CR, AVA21-13CR, AVA21-8XT and AVA21-12XT to block LAG3/MHCII signalling.
  • the assay involves coculturing MHCII - aAPC cells (activated APC cells) with LAG3 expressing Effector cells (Jurkat).
  • the aAPC cells present TCR activating antigen on MHCII to specifically activate TCR on LAG3 effector cells which inhibits TCR-induced activation and promoter-mediated luminescence.
  • AVA21 AFFIMER® proteins are strong LAG3 inhibitors.
  • PD-1/LAG3 combination assay (Promega) was used as per the manufacturer’s instructions to test the ability of AVA21-06 CR, AVA21-12CR, AVA21-13CR, AVA21-08XT and AVA21-12XT to block PD-1/PD-L1 and LAG3/MHC II signalling simultaneously.
  • the assay involves co-culturing PD-L1 expressing- aAPC cells (activated APC cells) with PD-1/LAG3 expressing effector cells. TCR activating antigen is presented by the aAPC cells on MHCII to specifically activate TCR on PD-1/LAG3 effector cells.
  • LAG3 mediated TCR and PD-1/PD-L1 axis are simultaneously inhibited leading to promoter activation and luminescence.
  • PD-L1/ LAG3 inhibition will lead to higher luminescence than PD-L1 or LAG3 inhibition alone.
  • the bispecific Fc AFFIMER® proteins AVA21-06 CR, AVA21-12CR and AVA21-13CR show higher activity than AVA04-251 CR (anti-PD-L1) and AVA21-19 CR (anti-LAG3).
  • bispecific AFFIMER® proteins The higher activity of bispecific AFFIMER® proteins than the combination of AVA04-251 CR and AVA21-19 CR indicates the strong synergistic effect of having anti-PD-L1 and anti-LAG3 on the same molecule as a bispecific.
  • polypeptide AVA21-12XT has higher synergy than AVA04-251-XT14 (anti-PD-L1) and AVA19-158-XT23 (anti-LAG3) combination.
  • the high synergy of the bispecific AFFIMER® proteins is a clear indication of their higher anti-tumour response than anti-PD-L1 or anti-LAG3 treatments alone.
  • FIG.51A-51C Primary human T cell stimulation assay (FIG.51A-51C) AVA21 constructs and controls were tested in human primary peripheral blood mononuclear cells (PBMCs) cultures after pre-activation with Staphylococcal Enterotoxin B (SEB). IL2 cytokine release was quantified as a surrogate for T cell activation. Briefly, frozen human PBMCs purchased from Cambridge Bioscience were thawed and pre-activated for 72h at 37C in T75 flask in presence of 500ng/ml SEB. After 72h, pre-activated PBMCs were harvested and seeded at 60,000 cells/well in 96-well plates in the presence of 200ng/ml SEB and treatment.
  • PBMCs peripheral blood mononuclear cells
  • SEB Staphylococcal Enterotoxin B
  • Treatment consisted of AVA21 constructs, controls or cells were left untreated. PBMCs were then incubated for 24h at 37°C, before collection of supernatant and analysis of IL-2 release using IL2 HTRF kit according to manufacturer’s instructions (Cisbio, HTRF human IL2 kit).
  • IL2 HTRF kit according to manufacturer’s instructions (Cisbio, HTRF human IL2 kit).
  • Preliminary data shows that bispecific hIgG1 LALA fusion constructs that bind PD-L1 and LAG3 (AVA21-06 CR, AVA21-06 T89A CR, AVA21-12CR and AVA21-13CR) induce higher levels of IL2 cytokine release compared to untreated and treatments with fusion constructs binding to PDL1 (AVA04-251CR) or LAG3 (AVA21-19CR) alone.
  • the trispecific in- line fusion construct that binds LAG-3, PD-L1 and HSA induces higher levels of IL2 release compared to negative control (3t0gly XT45) or a control binding to PD-L1 and HSA only (AVA04-640 XT34).
  • constructs which target both PD-L1 and LAG3 demonstrate increased activity compared to constructs which only bind one target protein. This shows that the constructs which target both immune checkpoint proteins have a greater ability to enhance T cell function than monospecific constructs as evidenced by increased IL-2 release. Use of primary cells shows that this is potentially translatable to the clinic.
  • T cell exhaustion in vitro mixed lymphocyte reaction (MLR) (FIGs.52A-52C, FIGs.53A-53C) T cell exhaustion is often observed in the tumour microenvironment.
  • MLR mixed lymphocyte reaction
  • Exhausted T cells show overexpression of inhibitory receptors, decreased cytolytic activity and impaired effector cytokine production, which lead to impaired tumour elimination.
  • Restoring T cell responsiveness correlates with increased immune surveillance and tumour response.
  • One-way MLR was used to assess reversal of exhausted T cell (Tex) hypo-responsiveness in the presence of AVA21 constructs and control molecules. Tex were generated by isolating pan-T cells (CD3+) from PBMC donors and repeatedly stimulating using CD3/CD28 Dynabeads®.
  • Tex were phenotyped by flow cytometry to confirm expression of PD-1, TIM-3 and LAG-3 by T cells (on day 0 and day 8).
  • In vitro generated monocyte-derived dendritic cells (mo-DC) were combined with Tex to generate MLR pairs and cultured for 5 days in the presence of test molecules. Two donor pairs were tested. Following the completion of the MLR, the supernatants were assessed for levels of IFN ⁇ by ELISA. In addition, T cell populations were assessed for proliferative responses by analysing proliferative marker Ki67 expression in CD4 and CD8 population.
  • Bispecific hIgG1 LALA fusion constructs that bind PD-L1 and LAG3 (AVA21-06CR, AVA21-12CR, AVA21-13), trispecific in-line fusion construct that binds LAG-3, PD-L1 and HSA (AVA21-12XT) and hIgG1 LALA fusion constructs that bind PD-L1 only (AVA04- 251_CR) induced CD4+ (A) and CD8+ T cell proliferation (B), and IFN- ⁇ release (C) in both donor pairs.
  • Negative control (SQTGly_CR) and fusion construct binding LAG3 only (AVA21- 19CR) had no effect.
  • Anti-PDL1 (Atezolizumab) and anti-PD1 (Nivolumab) were also tested in the assay and induced a response as expected.
  • AVA21 constructs are able to reverse exhausted T cell hypo-responsiveness as measured by increased T cell proliferation and IFN- ⁇ release in two MLR donor pairs. This may enhance anti-tumour response.
  • Pharmacokinetics (FIG.54) AVA21 half-life in blood serum was determined by conducting a pharmacokinetics (PK) experiment in wild type C57BL/6 mice. The in vivo study was performed by dosing the mice with 5mg/kg of AVA21 AFFIMER® proteins.
  • AVA21-12XT contains a HSA binding AFFIMER® , which cross-reacts with mouse serum albumin (MSA) but binds with lower affinity to MSA compared to HSA.
  • PK experiment was not performed with human serum albumin which could be the reason for the short half-life for clone AVA21-12XT.
  • Half-life of clone AVA21-12XT is expected to be longer in humans.
  • Stability Studies (FIG.55A-55F) Stability study was performed on AVA21 clones to assess their stability in storage buffer DPBS (Cytiva Hyclone; Cat: SH30378.03). All clones were concentrated to ⁇ 10mg/mL and aliquoted into individual tubes, at volume of 10 ⁇ L (100ug). Tubes were incubated at storage conditions 37°C, 45°C and 22°C (room temperature) to assess stability. Sampling was performed at Day 0 then at different time intervals for the different conditions.
  • AVA21-06 CA (SEQ ID NO: 15), AVA21-12 CA (SEQ ID NO: 38), and AVA21-13 CA (SEQ ID NO: 40) each include an anti-PD-L1 AFFIMER® polypeptide, an anti-LAG-3 AFFIMER® polypeptide, and an huIgG4 Fc, and with a flexible linker.

Abstract

The present disclosure provides engineered bispecific LAG-3/PD-L1 fusion proteins that include Stefin A variant polypeptides, and methods of using the fusion proteins for the treatment of various human conditions, including cancer.

Description

LAG-3/PD-L1 BINDING FUSION PROTEINS BACKGROUND The success of immunotherapy in many diseases is limited to a specific subpopulation of patients. To overcome this problem, dual blockade treatments mainly against cytotoxic T- lymphocyte-associated protein 4 (CTLA4) and programmed cell death receptor (ligand) 1 (PD- (L)1) axis have been developed; however, high toxicity rates and treatment resistance have promoted the exploration of alternative pathways and novel therapeutic strategies. Lymphocyte- associated gene 3 (LAG3) represents an inhibitory receptor, which is mainly found on activated immune cells and involved in the exhaustion of T cells in malignant diseases. Its co-expression with other inhibitory receptors, particularly with PD-1, has led to extensive research on the dual blockage of these checkpoint inhibitors. SUMMARY Some aspects provide engineered fusion proteins, referred to as LAG-3/PD-L1 AFFIMER® polypeptides or engineered LAG-3/PD-L1-binding Stefin A polypeptide variants, that are based on naturally occurring proteins (e.g., Stefin A cystatin). Each polypeptide of the fusion protein is engineered to stably display two loops (i.e., loop 2 and loop 4) that create a binding surface with high specificity and high affinity for LAG-3 or PD-L1. In some embodiments, the engineered fusion proteins further comprise a half-life extension moiety, such as a human serum albumin (HSA) AFFIMER® polypeptide, which comprises two loops stably displayed that create a binding surface with high specificity and high affinity for HSA, or a fragment crystallizable (Fc) region of an antibody (e.g., IgG1 Fc, IgG1 LALA Fc, or IgG4 Fc). The data provided herein show that the LAG-3 and PD-L1 AFFIMER® polypeptides and the LAG-3/PD-L1 AFFIMER® fusion proteins bind specifically to their respective targets, LAG-3 and PD-L1, with high affinities (e.g., a Kd of less than 1×10−6M, or even less than 1×10−7M). The LAG-3 and PD-L1 AFFIMER® polypeptides can be linked to each other covalently (such as by chemical cross-linking or as a fusion protein), or non-covalently (such as through multimerization domains or small molecule binding domains). The LAG-3/PD-L1 AFFIMER® polypeptides of the present disclosure are useful, for example, for targeting cells that express LAG-3 and/or PD-L1, and optionally, for extending the serum half-life of such polypeptides. These LAG-3/PD-L1 AFFIMER® polypeptides have several advantages over antibodies; for example, they are comparatively smaller (~14 kDa in monomeric form, ~30 kDa in dimeric form, or ~75-80 kDa in pentameric form), simpler (no disulfide bridges and no posttranslational modifications), and more robust (thermally and chemically) than antibodies. These high affinity (single-digit nM) LAG-3/PD-L1 AFFIMER® polypeptides can be generated in only a few weeks, exhibit exquisite specificity, are easily modified (chemically and as fusion proteins), and are easily manufactured in bacterial, yeast, or mammalian systems with high expression yields. Furthermore, the core AFFIMER® polypeptides are non-immunogenic. The terms PD-L1 AFFIMER® agent and anti-PD-L1 AFFIMER® agent are used interchangeable herein, and the terms LAG-3 AFFIMER® agent and anti-LAG-3 AFFIMER® agent are used interchangeably herein. Likewise, the term HSA AFFIMER® agent and anti-HSA AFFIMER® agent are used interchangeable herein. Some aspects provide a Lymphocyte Activation Gene 3 (LAG-3) binding polypeptide comprising the following Formula (I): FR1-(X')-FR2-(X'')-FR3 (I), wherein FR1 comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA (SEQ ID NO: 216); FR2 comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of STNYYIKVRAGDNKYMHLKVFNGP (SEQ ID NO: 217); and FR3 comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of ADRVLTGYQVDKNKDDELTGF (SEQ ID NO: 218), and wherein X' is an amino acid sequence having at least 85% or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 204-209; and X'' is an amino acid sequence having at least 85% or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 210-215. Other aspects provide a LAG-3 binding polypeptide comprising: the amino acid sequence having of MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA-(X')- STNYYIKVRAGDNKYMHLKVFNGP-(X'')-ADRVLTGYQVDKNKDDELTGF, wherein X' is an amino acid sequence having at least 85% or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 204-209; and X'' is an amino acid sequence having at least 85% or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 210-215. In some embodiments, the polypeptide binds to LAG-3 with a Kd of 1×10−6M or less. In some embodiments: (a) X' is the amino acid sequence of SEQ ID NO: 204 and X'' is the amino acid sequence of SEQ ID NO: 210; or (b) X' is the amino acid sequence of SEQ ID NO: 205 and X'' is the amino acid sequence of SEQ ID NO: 211; or (c) X' is the amino acid sequence of SEQ ID NO: 206 and X'' is the amino acid sequence of SEQ ID NO: 212; or (d) X' is the amino acid sequence of SEQ ID NO: 207 and X'' is the amino acid sequence of SEQ ID NO: 213; or (e) X' is the amino acid sequence of SEQ ID NO: 208 and X'' is the amino acid sequence of SEQ ID NO: 214; or (f) X' is the amino acid sequence of SEQ ID NO: 209 and X'' is the amino acid sequence of SEQ ID NO: 215. Some aspects provide a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 53, 54, 61, 62, 66, and 69. In some embodiments, a LAG-3 binding polypeptide comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of any one of SEQ ID NOs: 53, 54, 61, 62, 66, and 69. In some embodiments, a LAG-3 binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 53, 54, 61, 62, 66, and 69. In some embodiments, the LAG-3 binding polypeptide further comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is a human serum albumin (HSA) binding polypeptide or a fragment crystallizable (Fc) region of an antibody, optionally wherein the antibody is a human IgG1 antibody or a human IgG4 Fc antibody. Other aspects provide a bispecific fusion protein comprising: one or more PD-L1 binding polypeptide, and any one or more of the LAG-3 binding polypeptides described herein. In some embodiments, a PD-L1 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72 or 73. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73, and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 61. In some embodiments, a bispecific fusion protein comprises: a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73, and a second LAG-3 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 62. In some embodiments, a bispecific fusion protein comprises: a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73, and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, a bispecific fusion protein comprises: a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, and 5. In some embodiments, a bispecific fusion protein further comprises a second PD-L1 binding polypeptide and/or a second LAG-3 binding polypeptide. In some embodiments, the second PD-L1 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72 or 73. In some embodiments, the second LAG-3 polypeptide comprises an amino acid sequence of any one of the LAG-3 polypeptide described herein. Other aspects provide bispecific protein comprising: a first PD-L1 binding polypeptide, a second PD-L1 polypeptide, and any one of the LAG-3 binding polypeptides described herein. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD- L1 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, and the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the first PD-L1 binding polypeptide and the second PD-L1 polypeptide form a dimer comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 74, and the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, the bispecific fusion protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 4. Some aspects provide a trispecific fusion protein comprising any one of the bispecific fusion proteins described herein and a half-life extension moiety. In some embodiments, the half- life extension moiety is a human serum albumin (HSA)-binding polypeptide. In some embodiments, the HSA binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the half-life extension moiety is a fragment crystallizable (Fc) region of an antibody, for example, a human IgG1 antibody or a human IgG4 antibody. Other aspects provide trispecific fusion protein, comprising: a PD-L1 binding polypeptide, a first LAG-3 binding polypeptide, a second LAG-3 binding polypeptide, and an HSA binding polypeptide. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72 or 73. In some embodiments, each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence of the any one of the LAG-3 binding polypeptides described herein. In some embodiments, the HSA binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 80 In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 74, each of the first LAG-3-binding polypeptide and the second LAG- 3-binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 54, and the HSA binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 11. Some aspects provide a trispecific fusion protein comprising: a first PD-L1 binding polypeptide, a second PD-L1 binding polypeptide, a first LAG-3 binding polypeptide, a second LAG-3 binding polypeptide, and a half-life extension moiety. In some embodiments, the half-life extension moiety is an HSA binding polypeptide. In some embodiments, each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72 or 73. In some embodiments, each of the first LAG-3 binding polypeptide and second LAG-3 binding polypeptide comprises an amino acid sequence of any one of the LAG-3 polypeptides described herein. In some embodiments, each of HSA binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD- L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 62, and the HSA binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD- L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 54, and the HSA binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 8-10. Some aspects provide a bispecific fusion protein comprising a PD-L1 binding polypeptide, a LAG-3 binding polypeptide, and a fragment crystallizable (Fc) region of an antibody. In some embodiments, the antibody is selected from a human IgG1 (hIgG1) antibody and a human IgG4 (hIgG4) antibody. In some embodiments, the hIgG1 antibody comprises LALA mutations (Leu234Ala and Leu235Ala mutations). In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72 or 73. In some embodiments, the LAG-3 binding polypeptide comprises an amino acid sequence of any one of the LAG-3 binding polypeptides described herein. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 54, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 62, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 66, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 69, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 6, 7, 15, 19, 38, 39, 40, 41, 44, and 45. In some embodiments, fusion protein further comprises one or more linker located between two of the polypeptides. In some embodiments, the linker is a rigid linker, optionally comprising the amino acid sequence of AEAAAKEAAAKEAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 86). In some embodiments, the linker is a flexible linker, optionally comprising the amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 87). Some aspects provide a polynucleotide comprising an open reading frame encoding any one of the fusion proteins described herein. In some embodiments, the open reading frame comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the nucleotide sequence of any one of SEQ ID NOs: 99- 180. Other aspects provide a vector, for example, a viral vector or a plasmid vector, comprising any one or more of the polynucleotides described herein. Still other aspects provide a cell, for example, a mammalian cell, comprising any one or more of the polynucleotides described herein or any one or more of the vector described herein. Further aspects provide pharmaceutical composition comprising: (a) any one or more of the fusion proteins described herein, any one or more of the polynucleotides described herein, any one or more of the vectors described herein, or any one or more of the cells described herein; and (b) a pharmaceutically acceptable excipient. Some aspects provide a method comprising administering to a subject any one or more of the pharmaceutical compositions described herein (e.g., in a therapeutically effective amount). In some embodiments, the subject has a cancer. In some embodiments, the pharmaceutical composition is administered subcutaneously, intravenously, or intramuscularly. BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 shows a schematic representation of AVA21 in-line fusion (ILF) AFFIMER® dimers and trimers. A reducing SDS-PAGE gel confirming the molecular weight of each product is shown. FIG.2 shows results of two LAG-3 direct binding ELISAs with AVA21 ILF AFFIMER® dimers and trimers. AVA21-01, 03 and 04 show decreased binding to target respective to their monomer parent clones AVA19-157 and AVA19-01. AVA21-02 and AVA21- 05 show equivalent or increased binding affinity to target respective to their monomer parent clones AVA19-158 and AVA19-01. FIG.3 shows results of two PD-L1 direct binding ELISAs using AVA21 ILF AFFIMER® dimers and trimers. AVA21-01, AVA21-02 and AVA21-03 exhibit decreased binding to PD-L1 relative to their monomer parent clone, AVA04-269. AVA21-04 and AVA21- 05 show comparable binding affinity to PD-L1 relative to their parent clone AVA04-251. FIG.4 shows results of Promega PD-L1 blockade assay with AVA21 ILF AFFIMER® dimers and trimers. AVA04-251 ILF for AVA21 show similar functionality to parent with ILF trimer appearing better than ILF dimer. AVA04-269 ILF show reduced functionality relative to parent. No IC50 values could be calculated. Results reflect observations from PD-L1 binding ELISA. FIG.5 shows results of a bridging ELISA with AVA21 ILF AFFIMER® dimers and trimers, where LAG-3 was coated on the plate and detection was carried out using an anti-PD-L1 antibody. Most bispecific formats engage both targets simultaneously to various degrees. FIG.6 shows results of AVA21 ILF AFFIMER® dimers and trimers binding to LAG-3- positive(top) and negative Jurkat (middle) and CHO-K1 cells (bottom). Results show that AVA21 AFFIMER® proteins bind to LAG-3 enriched cells but not to Jurkat and CHO-K1 cells. FIG.7 shows two different AVA21 ILF XT AFFIMER® pentamer formats. FIG.8 shows a characterization of AVA21 ILF XT AFFIMER® pentamers. SEC-HPLC chromatograms and a reducing SDS-PAGE gel confirming the molecular weight of each product are shown. FIG.9 shows results of PD-L1 direct binding ELISA using AVA21 ILF XT AFFIMER® pentamers. AVA21-08 and 09 XT appear at least equivalent to AVA04-251 BH control in terms of PD-L1 binding, suggesting no adverse effect of additional HSA and LAG-3 binding domains. FIG.10 shows results of LAG-3 direct binding ELISA using AVA21 ILF XT and BK AFFIMER® multimers. Binding of both pentamers to LAG-3 was demonstrated by ELISA. Use of a rigid linker appears to allow for better target engagement and stronger binding to LAG-3 compared to flexible linker. FIG.11 shows results of AVA21 ILF XT AFFIMER® pentamers binding to HSA as demonstrated by direct ELISA. Both constructs show lower affinity than AVA03-42 monomer control. This could be due to positioning of AVA03-42 AFFIMER® XT in those formats (lesser availability to engage target). FIG.12 shows results of AVA21 ILF XT AFFIMER® pentamer cell binding to LAG-3- positive (top) and negative (bottom) Jurkat cells. Both pentamers show comparable binding to LAG-3 positive Jurkat cells, comparable to AVA19-158 XT23 control. No binding detected to LAG-3 negative cells. FIG.13 shows results of dual PD-L1/HSA binding assay with AVA21 ILF XT AFFIMER® pentamers. Both clones engage PD-L1 and HSA targets simultaneously. Binding of pentamers was unaffected in presence of HSA. Rigid linker appears better than flexible linker for PD-L1/HSA dual target engagement. FIG.14 shows results of functional PD-L1/PD-1 gene reporter assay with AVA21 ILF XT AFFIMER® pentamers. AVA21-08 XT shows comparable activity to AVA04-251 BH control in this assay. Functionality of AVA21-09 XT was significantly decreased. Flexible linker seems to show detrimental effect compared to rigid linker. FIG.15 shows results of PD-L1, LAG-3 and HSA BIACORE™ kinetic binding analysis of two AVA21 ILF XT AFFIMER® pentamers. The rigid linker appears to allow for stronger binding to PD-L1 and LAG-3. Binding to HSA is not affected by the nature of the linker. FIG.16 shows schematic representation of AVA21 ILF XT AFFIMER® pentamers and tetramers. A reducing SDS-PAGE gel, confirming the molecular weight of each product is shown. FIG.17 shows SEC-HPLC chromatograms of AVA21-11 XT and AVA21-12 XT following a two-stage purification process, confirming both proteins are >95% pure. FIG.18 shows results of PD-L1, LAG-3 and HSA BIACORE™ kinetic binding analysis for AVA21-11 XT and AVA21-12 XT. FIG.19 shows results of PD-L1 direct binding ELISA for AVA21-11 XT (top) and AVA21-12 XT (bottom). Both of the ILF XT constructs show binding to PD-L1 comparable to parent AFFIMER® controls. FIG.20 shows results of LAG-3 direct binding ELISA for AVA21-11 XT (top) and AVA21-12 XT (bottom). AVA21-11 XT shows better binding to LAG-3 than AVA21-12 XT and equivalent to AVA19-06 BK control. FIG.21 shows results of HSA direct binding ELISA for a AVA21-12 XT tetramer. Decreased binding of AVA21-12 XT formatted AFFIMER® to HSA is observed compared to monomer control. FIG.22 shows results of a bridging ELISA in presence (top) or absence(bottom) of HSA, where the plate was coated with LAG-3 and detection was carried out using an anti-PD-L1 antibody. Dual target engagement is confirmed in presence/absence of HSA in solution. Presence of HSA in solution does not impact binding to PD-L1 and LAG-3. FIG.23 shows results of Promega functional PD-L1/PD-1 gene reporter assay for AVA21-11 XT (top) and AVA21-12 XT (bottom). Both formats appear to be less active than their respective controls in this assay. FIG.24 shows results of multimer binding to LAG-3 overexpressing BPS cells. Increased binding of dimeric formats vs monomer AVA19-06 is observed. AVA21-11 XT binding to BPS cells is comparable to that of AVA19-06 BK control. FIG.25 shows results of multimer cell binding in PD-L1 overexpressing CHO cells. AVA21-12 XT binding is comparable to that of AVA04-640 control. FIG.26 shows schematic reorientations of the control formats for AVA21 bispecific IgG Fc fusion constructs. FIG.27 shows SEC-HPLC chromatograms of AVA21 bispecific IgG1 Fc fusion multimers, confirming both proteins are >97% pure. FIG.28 shows a reducing SDS-PAGE gel confirming the molecular weights of AVA21 bispecific IgG1 Fc fusion multimers. FIG.29 shows results of LAG-3 and PD-L1 direct binding ELISAs. AVA21-06 and 07 BP both show comparable binding affinities to LAG-3 and data is also consistent with control proteins AVA19-06 and AVA19-158 AQ.2. AVA21-06 and 07 BP both show comparable binding affinities to PD-L1 and data is also consistent with control protein AVA04-251 V.2. FIG.30 shows results of Promega PD-1/PD-L1 blockade assay for AVA21-06 BP. AVA21-06 BP shows slightly decreased activity compared to AVA04-251 V.2 control protein. FIG.31 shows results of AVA21-06 BP binding to LAG-3-positive (top) and negative (bottom) Jurkat cells. Results show a dose effect for AVA21-06 BP on positive cells. No binding observed on LAG-3 negative cells. FIG.32 shows results of AVA21-06 BP and AVA21-07 BP binding to LAG-3-positive BPS cells as well as LAG-3 negative Jurkat and CHO-K1 cells. AVA21-06 and 07 BP bind to LAG-3 enriched BPS cells. No binding observed to LAG-3 negative Jurkat and CHO-K1 cells. FIG.33 shows a schematic representation of additional AVA21 bispecific IgG1 Fc fusion constructs. A reducing SDS-PAGE gel is also shown and confirms protein molecular weights. FIG.34 shows results of Promega PD-1/PD-L1 blockade assay on AVA21-15 and AVA21-16. AVA21-15 shows a significant decrease in activity compared to control proteins. AVA21-16 potency is reduced in this assay relative to that of control proteins, but the protein remains active. FIG.35 shows results of direct LAG-3 and PD-L1 binding ELISAs. Results show that the positioning of LAG-3/PD-L1 binders affects the ability of AFFIMER® proteins to engage target. Both formats display significantly lower affinity for antigen than controls when binding to LAG-3. For PD-L1 binding, AVA21-16 binds to target with slightly decreased affinity compared to controls, and AVA21-15 affinity is significantly decreased due to positioning of PD-L1 binder. FIG.36 shows a schematic representation of additional AVA21 bispecific IgG1 Fc LALA fusion constructs. A reducing SDS-PAGE gel is also shown and confirms protein molecular weights. FIG.37 shows PD-L1 kinetics analysis results for AVA21 CR IgG1 LALA constructs. Binding to PD-L1 is equivalent for all clones shown. FIG.38 shows LAG-3 kinetics analysis results for AVA21 CR IgG1 LALA constructs. All clones bind to LAG-3 with comparable affinities. FIG.39 shows results of PD-L1 direct binding ELISA for AVA21-06 CR. Equivalent binding is observed for both AVA21-06 CR and the control protein AVA04-251 V.2. FIG.40 shows results of LAG-3 direct binding ELISA for three AVA21 IgG1 LALA constructs. All CR formats show equivalent binding to LAG-3 and either comparable or better binding affinity than their relative CS controls. FIG.41 shows expected molecular weights for alanine scanning mutants of AVA21-06 CR. Alanine mutants of parent format AVA21-06 CR were engineered to improve binding and reduce aggregation. FIG.42 shows results of direct LAG-3 (top) and PD-L1 (bottom) binding ELISAs for AVA21-06 CR Loop 2 alanine scanning mutants. Little variation in PD-L1 binding between controls and various mutants is observed. D439A, P440A and W443A mutations significantly affect binding to LAG-3 (essential amino acids), D438A impaired binding to an extent and P437A did not appear to significantly affect binding to LAG-3. FIG.43 shows results of direct LAG-3 (top) and PD-L1 (bottom) binding ELISAs for AVA21-06 CR Loop 4 alanine scanning mutants. Little variation in PD-L1 and LAG-3 binding between controls and various mutants is observed. D471A and P475A mutations appear to be the most disruptive mutations for LAG-3 and PD-L1 binding, respectively. Variations may be due to experimental error. FIG.44A shows result of PD-L1, LAG3 and human serum albumin (HuSA) Biacore kinetic analysis of AVA21-XT. FIG.44B shows result of PD-L1 and LAG3 Biacore kinetic analysis of AVA21 CR formats (IgG1-LALA). FIG.45 shows result of AVA21 huIgG1 LALA Fc Fusion construct (AVA21-06 CR, AVA21-06 T89A CR, AVA21-12CR and AVA21-13CR) binding to PD-L1 expressing cells. All AVA21 constructs show similar binding, comparable to PDL1 binding construct only (AVA04- 251 CR). FIG.46 shows result of AVA21 huIgG1 LALA Fc Fusion construct (AVA21-06 CR, AVA21-06 T89A CR, AVA21-12CR and AVA21-13CR) binding to hLAG3 positive (top) and negative (bottom) D0.11.10 cells. All AVA21 constructs show binding to LAG3 positive cells. No binding is detected to LAG3 negative cells. FIG.47 shows results of human PD-L1 direct binding ELISA for AVA21-06 CR, AVA21- 06 T89A CR, AVA21-12 CR and AVA21-13 CR. All clones bind to PD-L1 with comparable affinities and EC50 values are consistent with that of control protein AVA04-251 CR. FIG.48 Error! Reference source not found.shows results of Promega PD-1/PD-L1 blockade assay with AVA21-06 CR, AVA21-06 T89A CR, AVA21-12CR and AVA21-13CR. All AVA21 constructs show comparable activity to PD-L1 binding construct only (AVA04-251 CR). Negative control protein SQTgly CR induced no response as expected. FIG.49 shows results of LAG3/MHCII blockade assay with AVA21-06 CR, AVA21- 12CR, AVA21-13CR, AVA21-8XT, AVA21-12XT, AVA21-19CR and AVA19-158-XT23. AVA04- 251-CR, AVA04-251-XT14, AVA04-640-XT34 had no inhibitory activity as expected. FIG.50 shows results of PD-1/LAG3 combination blockade assay with AVA21-06 CR, AVA21-12CR, AVA21-13CR, AVA21-8XT and AVA21-12XT. Controls tested were AVA04-251 CR and/or AVA21-19 CR, AVA04-251-XT14 and/or AVA19-158-XT23 and AVA04-640 XT34. SQT-Gly CR and SQT-Gly XT28 had no inhibitory activity as expected. FIG.51 shows preliminary result of AVA21 constructs inducing IL2 release in human peripheral blood mononuclear cells after pre-activation with Staphylococcal Enterotoxin B. FIGs.52A-52C show result of AVA21 constructs reversing exhausted T cell (Tex) hypo- responsiveness in an in vitro mixed lymphocyte reaction in donor pair 1. Bispecific AVA21 CR IgG1 LALA constructs (AVA21-06CR, AVA21-12CR, AVA21-13) and bispecific AVA21 ILF (AVA21-12XT) increased T cell proliferation and IFN-γ release to similar levels as PDL1 binder only (AVA04-251 CR) and reached comparable maximal response as anti-PDL1 (Atezolizumab) and anti-PD1 (Nivolumab) positive controls. LAG3 binder only (AVA21-19CR) had no effect. FIGs.53A-53C show result of AVA21 constructs reversing exhausted T cell (Tex) hypo- responsiveness in an in vitro mixed lymphocyte reaction in donor pair 2. Result was similar to donor pair 1 as shown in FIGs.52A-52C. FIG.54 shows pharmacokinetic analysis of AVA21-13CR and AVA21-12XT in C56Bl/6 mice.Reversal of T cell exhaustion in an in vitro mixed lymphocyte reaction (MLR) (FIGs. 52A-52C, FIGs.53A-53C) FIG.55A shows stability studies of Fc Fused AFFIMER® polypeptide at 37˚C. HPLC- SEC chromatogram are overlaid for each sampling day and normalized. The table represents the percentage purity of protein as analysed by HPLC-SEC. The acceptable purity value to indicate stability of protein is equal or above 90% purity. FIG.55B shows Stability studies of XT AFFIMER® polypeptide at 37˚C. HPLC-SEC chromatogram are overlaid for each sampling day and normalized. The table represents the percentage purity of protein as analysed by HPLC-SEC. The acceptable purity value to indicate stability of protein is equal or above 90% purity. FIG. 55C shows Stability studies of Fc Fused AFFIMER® polypeptide at 45˚C. HPLC-SEC chromatogram are overlaid for each sampling day and normalized. The table represents the percentage purity of protein as analysed by HPLC-SEC. The acceptable purity value to indicate stability of protein is equal or above 90% purity. FIG.55D shows Stability studies of XT AFFIMER® polypeptide at 45˚C. HPLC-SEC chromatogram are overlaid for each sampling day and normalized. The table represents the percentage purity of protein as analysed by HPLC-SEC. The acceptable purity value to indicate stability of protein is equal or above 90% purity. FIG. 55E shows Stability studies of Fc Fused AFFIMER® polypeptide at 22˚C (room temperature). HPLC-SEC chromatogram are overlaid for each sampling day and normalized. The table represents the percentage purity of protein as analysed by HPLC-SEC. The acceptable purity value to indicate stability of protein is equal or above 90% purity. FIG.55F shows Stability studies of XT AFFIMER® polypeptide at 22˚C (room temperature). HPLC-SEC chromatogram are overlaid for each sampling day and normalized. The table represents the percentage purity of protein as analysed by HPLC-SEC. The acceptable purity value to indicate stability of protein is equal or above 90% purity. FIG.56 shows a schematic representation of additional AVA21 bispecific IgG4 Fc fusion constructs. A reducing SDS-PAGE gel is also shown and confirms protein molecular weights. FIG.57 shows results of human PD-L1 direct binding ELISA for AVA21-06 CA, AVA21-12 CA and AVA21-13 CA. All clones bind to PD-L1 with comparable affinities and EC50 values are consistent with that of control protein AVA04-251 AZ. FIG.58 shows results of LAG-3 direct binding ELISA for AVA21 CA IgG4 fusion constructs. All CA formats bind to LAG-3 with comparable affinities, and with equivalent affinities to the BZ control formats. FIG.59 shows results of AVA21 CA IgG4 fusion constructs binding to enriched LAG-3- positive BPS and negative Jurkat cells. All formats bind to LAG-3 positive cells, no signal observed for LAG-3 negative cells. DETAILED DESCRIPTION I. Overview Cancer immunotherapy has been accompanied by encouraging results over the past few years. Two promising targets are Programmed Cell Death Protein Ligand 1 (PDL-1) and Lymphocyte Activation Gene 3 (LAG-3), both of which play important roles in modulating the activity of regulatory T cells and inhibiting immune responses against cancer cells. Programmed Cell Death Protein 1 (PD-1) plays a vital role in inhibiting immune responses and promoting self-tolerance through modulating the activity of T-cells, activating apoptosis of antigen-specific T cells and inhibiting apoptosis of regulatory T cells. Programmed Cell Death Ligand 1 (PD-L1) is a trans-membrane protein that is considered to be a co-inhibitory factor of the immune response, it can combine with PD-1 to reduce the proliferation of PD-1 positive cells, inhibit their cytokine secretion and induce apoptosis. PD-L1 also plays an important role in various malignancies where it can attenuate the host immune response to tumor cells. Based on these perspectives, PD-1/PD-L1 axis is responsible for cancer immune escape and makes a huge effect on cancer therapy. PD-1/PD-L1 pathway plays a significant role in controlling induction and maintenance of immune tolerance within the tumor microenvironment. The activity of PD-1 and its ligands PD-L1 or PD-L2 are responsible for T cell activation, proliferation, and cytotoxic secretion in cancer to degenerating anti-tumor immune responses. PD-1 ligand (PD-L1; also referred to as CD279 and B7-H1), belongs to the B7 series and is a 33-kDa type 1 transmembrane glycoprotein that contains 290 amino acids with Ig- and IgC domains in its extracellular region. PD-L1 is usually expressed by macrophages, some activated T cells and B cells, dendritic cells (DCs) and some epithelial cells, particularly under inflammatory conditions. In addition, PD-L1 is expressed by tumor cells as an “adaptive immune mechanism” to escape anti-tumor responses. PD-L1 is associated with an immune environment rich in CD8 T cells, production of Th1 cytokines and chemical factors, as well as interferons and specific gene expression characteristics. It has been demonstrated that interferon-gamma (IFN-γ) causes PD-L1 upregulation in ovarian cancer cells, which is responsible for disease progression, whereas IFN-γ receptor 1 inhibition can reduce PD-L1 expression in acute myeloid leukemia mouse models through the MEK/extracellular signal-regulated kinase (ERK) and MYD88/TRAF6 pathways. IFN-γ induces protein kinase D isoform 2 (PKD2), which is important for the regulation of PD- L1. Inhibition of PKD2 activity inhibits the expression of PD-L1 and promotes a strong antitumor immune response. NK cells secrete IFN-γ through the Janus kinase (JAK)1, JAK2 and signal transducer and activator of transcription (STAT)1 pathways, increasing the expression of PD-L1 on the surface of the tumor cells. Studies on melanoma cells have shown that IFN-γ secreted by T cells through the JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 pathway may regulate the expression of PD-L1. T and NK cells appear to secrete IFN-γ, which induces PD-L1 expression on the surface of the target cells, including tumor cells. PD-L1 acts as a pro-tumorigenic factor in cancer cells via binding to its receptors and activating proliferative and survival signaling pathways. This finding further indicated that PD- L1 is implicated in subsequent tumor progression. In addition, PD-L1 has been shown to exert non-immune proliferative effects on a variety of tumor cell types. For example, PD-L1 induced epithelial-to-mesenchymal transition (EMT) and stem cell-like phenotypes in renal cancer cells, indicating that the presence of the intrinsic pathway of PD-L1 promotes kidney cancer progression. Lymphocyte activation gene-3 (LAG-3, LAG-3, or CD223; Gene ID: 3902; NM_002286.6, NP_002277.4) is a type I transmembrane protein that is expressed on the cell surface of activated CD4+ and CD8+ T cells and subsets of natural killer (NK) and dendritic cells. LAG-3 comprises four extracellular immunoglobulin-like domains and requires binding to its ligand, major histocompatibility complex (MHC) class II, for functional activity. LAG-3 is only expressed on the cell surface of activated T cells and its cleavage from the cell surface terminates its signaling. LAG-3 plays an important role in promoting regulatory T cell (Treg) activity and in negatively regulating T cell activation and proliferation. Both natural and induced Treg express increased LAG-3, which is required for maximal Treg suppressive function. Furthermore, ectopic expression of LAG-3 on CD4+ effector T cells reduces their proliferative capacity and transforms them such that they have regulatory potential against other T cells. Without wishing to be bound by theory, it is thought that inhibiting LAG-3 function will promote an anti-cancer response, as its inhibition activates effector T cells (as does inhibition of PD-1/PD-L1) in addition to inhibiting induced (antigen-specific) Treg suppressive activity. Some aspects provide a fusion protein that includes an AFFIMER® polypeptide that binds to PD-L1 and an AFFIMER® polypeptide that binds to LAG-3. In some embodiments, the fusion protein further comprises a half-life extension moiety, such as an AFFIMER® polypeptide that binds human serum albumin (HSA) and/or an antibody Fc domain (e.g., IgG1 Fc, IgG1 LALA Fc, or IgG4 Fc). The half-life extension moiety extends, in a controlled manner, the serum half-life of the LAG-3/PD-L1 AFFIMER® polypeptide to which it is conjugated. The present disclosure addresses the urgent need in the art for targeting molecules capable of binding to PD-L1with high specificity and high affinity as well as for targeting molecules capable of binding to LAG-3 with high specificity and high affinity. Without wishing to be bound by theory, it is thought that the PD-L1 binding component of the fusion protein will localize the protein (and the LAG-3 inhibitory activity) to the tumor microenvironment. Therefore, provided herein, in some embodiments, are LAG-3/PD-L1 AFFIMER® polypeptides, engineered polypeptide variants of the Stefin A protein, that bind LAG-3 and PD- L1 with a Kd of less than 1×10−6M. The LAG-3/PD-L1 AFFIMER® polypeptides of the present disclosure, in some embodiments, may be fused or otherwise linked to therapeutic molecules to be used for the treatment of diseases and/or disorders characterized at least in part by the presence of PD-L1-positive cells and/or LAG-3-positive cells. In other embodiments, the LAG- 3/PD-L1 AFFIMER® polypeptides can be used as therapeutic agents. II. Certain Definitions of the Present Disclosure Stefin polypeptides encompass a subgroup of proteins in the cystatin superfamily, a family which encompasses proteins that contain multiple cystatin-like sequences. The Stefin subgroup of the cystatin family includes relatively small (around 100 amino acids) single domain proteins. They receive no known post-translational modification, and lack disulfide bonds, suggesting that they will be able to fold identically in a wide range of extracellular and intracellular environments. Stefin A itself is a monomeric, single chain, single domain protein of 98 amino acids. The structure of Stefin A has been solved, facilitating the rational mutation of Stefin A into the AFFIMER® polypeptide. The only known biological activity of cystatins is the inhibition of cathepsin activity, which allowed for exhaustive testing for residual biological activity of the engineered proteins. An “AFFIMER® polypeptide” (also referred to as an “AFFIMER® protein”) refers to a small, highly stable protein that is an engineered variant of a Stefin polypeptide. AFFIMER® proteins display two peptide loops and an N-terminal sequence that can all be randomized to bind to desired target proteins with high affinity and specificity, in a similar manner to monoclonal antibodies. Stabilization of the two peptides by the Stefin A protein scaffold constrains the possible conformations that the peptides can take, increasing the binding affinity and specificity compared to libraries of free peptides. These engineered non-antibody binding proteins are designed to mimic the molecular recognition characteristics of monoclonal antibodies in different applications. Variations to other parts of the Stefin A polypeptide sequence can be carried out, with such variations improving the properties of these affinity reagents, such as increase stability, make them robust across a range of temperatures and pH and the like. In some embodiments, an AFFIMER® polypeptide includes a sequence derived from Stefin A, sharing substantial identify with a Stefin A wild type sequence, such as human Stefin A. It will be apparent to a person skilled in the art that modifications may be made to the scaffold sequence without departing from the disclosure. In particular, an AFFIMER® polypeptide can have an amino acid sequences that is at least 25%, 35%, 45%, 55% or 60% identity to the corresponding sequences to human Stefin A, for example, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95% identical, e.g., where the sequence variations do not adversely affect the ability of the scaffold to bind to the desired target (such as PD-L1), and e.g., which do not restore or generate biological functions such as those which are possessed by wild type Stefin A but which are abolished in mutational changes described herein. An “AFFIMER® agent” refers to a polypeptide that includes an AFFIMER® polypeptide sequence and any other modification(s) (e.g., conjugation, post-translational modifications, etc.) so as to represent a therapeutically active protein intended for delivery to an individual. An “AFFIMER®-linked conjugate” refers to an AFFIMER® agent having at least one moiety conjugated thereto through a chemical conjugation other than through the formation of a contiguous peptide bond through the C-terminus or N-terminus of the polypeptide portion of the AFFIMER® agent containing AFFIMER® polypeptide sequence. An AFFIMER®-linked conjugate may be an “AFFIMER® polypeptide-drug conjugate”, which refers to an AFFIMER® agent including at least one pharmacologically active moiety conjugated thereto. An AFFIMER®-linked conjugate may also be an “AFFIMER®-tag conjugate”, which refers to an AFFIMER® agent including at least one detectable moiety (e.g., detectable label) conjugated thereto. An “encoded AFFIMER® construct” refers to a nucleic acid construct which, when expressed by cells in a patient’s body through a gene delivery process, produces an intended AFFIMER® agent in vivo. Programmed death-ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a protein that in humans is encoded by the CD274 gene. PD-L1 is a 40kDa type 1 transmembrane protein that is expressed by various tumor cells and by the lymphocytes that infiltrate tumors. PD-L1 is expressed on the surface of tumor cells and it is able to bind to PD-1 on the surface of activated T cells, B cells, and myeloid cells, to modulate activation or inhibition. The binding of PD-L1 to PD-1 leads to an immunosuppressive effect and allows the tumor to evade immune destruction. The affinity between PD-L1 and PD-1, as defined by the dissociation constant Kd, is 770 nM. PD-L1 also has an appreciable affinity for the costimulatory molecule CD80 (B7-1), but not CD86 (B7-2). PD-L1 has been speculated to play a major role in suppressing the adaptive arm of immune system during particular events such as pregnancy, tissue allografts, autoimmune disease and other disease states such as hepatitis. Normally the adaptive immune system reacts to antigens that are associated with immune system activation by exogenous or endogenous danger signals. In turn, clonal expansion of antigen-specific CD8+ T cells and/or CD4+ helper cells is propagated. The binding of PD-L1 to the inhibitory checkpoint molecule PD-1 transmits an inhibitory signal based on interaction with phosphatases (SHP-1 or SHP-2) via Immunoreceptor Tyrosine-Based Switch Motif (ITSM). This reduces the proliferation of antigen-specific T-cells in lymph nodes, while simultaneously reducing apoptosis in regulatory T cells (anti- inflammatory, suppressive T cells) - further mediated by a lower regulation of the gene Bcl-2. The human amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human PD- L1 can be found as UniProt/Swiss-Prot. Accession No. Q9NZQ7-1and the nucleotide sequence encoding of the human PD-L1 can be found at NCBI Accession No. NM_014143.4 (Gene ID: 29126). As used herein, “PD-L1” includes any native, mature PD-L1 which results from processing of a PD-L1 precursor protein in a cell. The term encompasses PD-L1 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. The term also includes any PD-L1 proteins comprising mutations, e.g., point mutations, fragments, insertions, deletions, and splice variants of full length wild-type PD-L1. A “PD-L1 AFFIMER® agent” refers to an AFFIMER® agent that comprises at least one AFFIMER® polypeptide that binds to PD-L1, particularly human PD-L1, with a dissociation constant (Kd) of at least 10-6M. In some embodiments, the PD-L1 AFFIMER® agent binds PD- L1 with a Kd of 1×10−7M or less, Kd of 1×10−8M or less, Kd of 1×10−9M or less, or a Kd of 1×10−10M or less. It should be understood that the terms “PD-L1 AFFIMER® polypeptide” and “engineered PD-L1 binding Stefin A polypeptide variant” are used interchangeably herein. Thus, a “PD-L1 AFFIMER® polypeptide” is an engineered polypeptide that binds specifically to PD- L1 with a Kd of 1×10−6M or less, wherein the engineered polypeptide is a variant of a Stefin A protein. Lymphocyte Activating Protein 3 (LAG-3), also known as cluster of differentiation 233 (CD233), is a protein that in humans is encoded by the LAG-3 gene. LAG-3 is a member of the immunoglobulin (Ig) superfamily and comprises a 503-amino acid type I transmembrane protein having four extracellular Ig-like domains. LAG-3 is primarily expressed in activated T cells and a subset of NK cells, and its expression is induced by interleukin-2 (IL-2), IL-7, IL-12A, and IL- 12B on activated T cells. LAG-3 interacts with MHC class II, and it interacts with the fibrinogen-like 1 (FGL1) protein via the fibrinogen C-terminal domain. LAG-3 activation results in a signaling pathway that suppresses T cell activation. The protein may also inhibit antigen-specific T cell activation in synergy with PD-1/PD-L1. Its pathway also negatively regulates the proliferation, activation, effector function and homeostasis of both CD8+ and CD4+ T-cells. The human amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human LAG-3 can be found as UniProt/Swiss-Prot. Accession No. P18627 and the nucleotide sequence encoding of the human LAG-3 can be found at NCBI Accession No. NM_002286.6 (Gene ID: 3902). As used herein, “LAG-3” includes any native, mature LAG-3 which results from processing of a LAG-3 precursor protein in a cell. The term encompasses LAG-3 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. The term also includes any LAG-3 proteins comprising mutations, e.g., point mutations, fragments, insertions, deletions, and splice variants of full length wild-type LAG-3. A “LAG-3 AFFIMER® agent” refers to an AFFIMER® agent that comprises at least one AFFIMER® polypeptide that binds to LAG-3, particularly human LAG-3, with a dissociation constant (Kd) of at least 10-6M. In some embodiments, the LAG-3 AFFIMER® agent binds LAG-3 with a Kd of 1×10−7M or less, Kd of 1×10−8M or less, Kd of 1×10−9M or less, or a Kd of 1×10−10M or less. It should be understood that the terms “LAG-3 AFFIMER® polypeptide” and “engineered LAG-3-binding Stefin A polypeptide variant” are used interchangeably herein. Thus, a “LAG-3 AFFIMER® polypeptide” is an engineered polypeptide that binds specifically to LAG-3 with a Kd of 1×10−6M or less, wherein the engineered polypeptide is a variant of a Stefin A protein. Human serum albumin (HSA) is a protein encoded by the ALB gene. HSA is a 585 amino acid polypeptide (approx.67 kDa) having a serum half-life of about 20 days and is primarily responsible for the maintenance of colloidal osmotic blood pressure, blood pH, and transport and distribution of numerous endogenous and exogenous ligands. HSA has three structurally homologous domains (domains I, II and III), is almost entirely in the alpha-helical conformation, and is highly stabilized by 17 disulfide bridges. A representative HSA sequence is provided by UniProtKB Primary accession number P02768 and may include other human isoforms thereof. An “HSA AFFIMER® agent” refers to an AFFIMER® agent that comprises at least one AFFIMER® polypeptide that binds to serum albumin, particularly human serum albumin, with a dissociation constant (Kd) of at least 10-6M. In some embodiments, the HSA AFFIMER® agent binds HSA with a Kd of 1×10−7M or less, Kd of 1×10−8M or less, Kd of 1×10−9M or less, or a Kd of 1×10−10M or less. It should be understood that the terms “HSA AFFIMER® polypeptide” and “engineered HSA binding Stefin A polypeptide variant” are used interchangeably herein. Thus, an “HSA AFFIMER® polypeptide” is an engineered polypeptide that binds specifically to HSA with a Kd of 1×10−6M or less, wherein the engineered polypeptide is a variant of a Stefin A protein. A. Polypeptides Polypeptides (which includes peptides and proteins) are polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing at least one analog of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. Amino acids (also referred to herein as amino acid residues) participate in one more peptide bonds of a polypeptide. In general, the abbreviations used herein for designating the amino acids are based on recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochemistry (1972) 11:1726-1732). For instance, Met, Ile, Leu, Ala and Gly represent "residues" of methionine, isoleucine, leucine, alanine and glycine, respectively. By the residue is meant a radical derived from the corresponding α-amino acid by eliminating the OH portion of the carboxyl group and the H portion of the α-amino group. The term "amino acid side chain" is that part of an amino acid exclusive of the --CH(NH2)COOH portion, as defined by K. D. Kopple, "Peptides and Amino Acids", W. A. Benjamin Inc., New York and Amsterdam, 1966, pages 2 and 33. For the most part, the amino acids used in the application of this disclosure are those naturally occurring amino acids found in proteins, or the naturally occurring anabolic or catabolic products of such amino acids which contain amino and carboxyl groups. Particularly suitable amino acid side chains include side chains selected from those of the following amino acids: glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, and tryptophan, and those amino acids and amino acid analogs which have been identified as constituents of peptidylglycan bacterial cell walls. Amino acid residues having “basic sidechains” include Arg, Lys and His. Amino acid residues having “acidic sidechains” include Glu and Asp. Amino acid residues having “neutral polar sidechains” include Ser, Thr, Asn, Gln, Cys and Tyr. Amino acid residues having “neutral non-polar sidechains” include Gly, Ala, Val, Ile, Leu, Met, Pro, Trp and Phe. Amino acid residues having “non-polar aliphatic sidechains” include Gly, Ala, Val, Ile and Leu. Amino acid residues having “hydrophobic sidechains” include Ala, Val, Ile, Leu, Met, Phe, Tyr and Trp. Amino acid residues having “small hydrophobic sidechains” include Ala and Val. Amino acid residues having “aromatic sidechains” include Tyr, Trp and Phe. Amino acid residues further include analogs, derivatives and congeners of any specific amino acid referred to herein, as for instance, the subject AFFIMER® polypeptides (particularly if generated by chemical synthesis) can include an amino acid analog such as, for example, cyanoalanine, canavanine, djenkolic acid, norleucine, 3-phosphoserine, homoserine, dihydroxy- phenylalanine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, diaminiopimelic acid, ornithine, or diaminobutyric acid. Other naturally occurring amino acid metabolites or precursors having side chains which are suitable herein will be recognized by those skilled in the art and are included in the scope of the present disclosure. Also included are the (D) and (L) stereoisomers of such amino acids when the structure of the amino acid admits of stereoisomeric forms. The configuration of the amino acids and amino acid residues herein are designated by the appropriate symbols (D), (L) or (DL), furthermore when the configuration is not designated the amino acid or residue can have the configuration (D), (L) or (DL). It will be noted that the structure of some of the compounds of this disclosure includes asymmetric carbon atoms. It is to be understood accordingly that the isomers arising from such asymmetry are included within the scope of this disclosure. Such isomers can be obtained in substantially pure form by classical separation techniques and by sterically controlled synthesis. For the purposes of this application, unless expressly noted to the contrary, a named amino acid shall be construed to include both the (D) or (L) stereoisomers. The terms “identical” or percent “identity” in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity may be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that may be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two nucleic acids or polypeptides of the disclosure are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the amino acid sequences that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80- 100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a target protein or an antibody. In some embodiments, identity exists over a region of the nucleotide sequences that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest. A conservative amino acid substitution is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Generally, conservative substitutions in the sequences of the polypeptides, soluble proteins, and/or antibodies of the disclosure do not abrogate the binding of the polypeptide, soluble protein, or antibody containing the amino acid sequence, to the target binding site. Methods of identifying amino acid conservative substitutions which do not eliminate binding are well-known in the art. A polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure. A material is considered substantially pure if the material is at least 50% pure (e.g., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure. A fusion polypeptide (e.g., a fusion protein) is a hybrid polypeptide expressed by a nucleic acid molecule comprising at least two open reading frames (e.g., from two individual molecules, e.g., two individual genes). A linker (also referred to as a linker region) may be inserted between a first polypeptide (e.g., a PD-L1 AFFIMER® polypeptide) and a second polypeptide (e.g., a LAG-3 AFFIMER® polypeptide). In some embodiments, a linker is a peptide linker. Linkers should not adversely affect the expression, secretion, or bioactivity of the polypeptides. In some embodiments, linkers are not antigenic and do not elicit an immune response. In some embodiments, an AFFIMER® polypeptide is linked to an antibody. An “AFFIMER® polypeptide-antibody fusion” is a fusion protein that includes an AFFIMER® polypeptide portion and a variable region of an antibody. AFFIMER® polypeptide-antibody fusions may include full length antibodies having, for example, at least one AFFIMER® polypeptide sequence appended to the C-terminus or N-terminus of at least one of its VH and/or VL chains, e.g., at least one chain of the assembled antibody is a fusion protein with at least one AFFIMER® polypeptide. AFFIMER® polypeptide-antibody fusions may also include at least one AFFIMER® polypeptide sequence as part of a fusion protein with an antigen binding site or variable region of an antibody fragment. An antibody is an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of any of the foregoing, through at least one antigen-binding site wherein the antigen-binding site is usually within the variable region of the immunoglobulin molecule. As used herein, the term “antibody” encompasses intact (whole) polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) antibodies provided those fragments have been formatted to include an Fc or other FcγRIII binding domain, multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen-binding site of an antibody (formatted to include an Fc or other FcγRIII binding domain), antibody mimetics, and any other modified immunoglobulin molecule comprising an antigen-binding site as long as the antibodies exhibit the desired biological activity. While the antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu. A variable region of an antibody may be a variable region of an antibody light chain or a variable region of an antibody heavy chain, either alone or in combination. Generally, the variable region of heavy and light chains includes four framework regions (FR) and three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding sites of the antibody. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, National Institutes of Health, Bethesda Md.), and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al Lazikani et al., 1997, J. Mol. Biol., 273:927-948). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs. A humanized antibody is a form of a non-human (e.g., murine) antibody that is specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequences. Typically, humanized antibodies are human immunoglobulins in which residues of the CDRs are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and/or binding capability. In some instances, the Fv framework region residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species. The humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or binding capability. The humanized antibody may comprise variable domains containing all or substantially all of the CDRs that correspond to the non- human immunoglobulin whereas all or substantially all of the framework regions are those of a human immunoglobulin sequence. In some embodiments, the variable domains comprise the framework regions of a human immunoglobulin sequence. In some embodiments, the variable domains comprise the framework regions of a human immunoglobulin consensus sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. A humanized antibody is usually considered distinct from a chimeric antibody. An epitope (also referred to herein as an antigenic determinant) is the portion of an antigen capable of being recognized and specifically bound by a particular antibody, a particular AFFIMER® polypeptide or other particular binding domain. When the antigen is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids (also referred to as linear epitopes) are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation. "Specifically binds to" or is "specific for" refers to measurable and reproducible interactions such as binding between a target (e.g., PD-L1 or LAG-3) and an AFFIMER® polypeptide, antibody or other binding partner, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an AFFIMER® polypeptide that specifically binds to PD-L1 is an AFFIMER® polypeptide that binds PD-L1 with greater affinity, avidity (if multimeric formatted), more readily, and/or with greater duration than it binds to other targets. Similarly, an AFFIMER® polypeptide that specifically binds to LAG-3 is an AFFIMER® polypeptide that binds LAG-3 with greater affinity, avidity (if multimeric formatted), more readily, and/or with greater duration than it binds to other targets. “Conjugate,” “conjugation” and grammatical variations thereof refers the joining or linking together of two or more compounds resulting in the formation of another compound, by any joining or linking methods known in the art. It can also refer to a compound that is generated by the joining or linking together two or more compounds. For example, a PD-L1 AFFIMER® polypeptide linked directly or indirectly to a LAG-3 AFFIMER® polypeptide is an exemplary conjugate. Such conjugates include fusion proteins (e.g., in-line fusion proteins), those produced by chemical conjugates and those produced by any other methods. B. Polynucleotides A polynucleotide (also referred to herein as a nucleic acid or a nucleic acid molecule) is a polymer of nucleotides of any length and may comprise DNA, RNA (e.g., messenger RNA (mRNA)) or a combination of DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide encoding a polypeptide refers to the order or sequence of nucleotides along a strand of deoxyribonucleic acid deoxyribonucleotides. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (e.g., protein) chain. Thus, a nucleic acid sequence encodes the amino acid sequence. When used in reference to nucleotide sequences, a “sequence” may comprise DNA and/or RNA (e.g., messenger RNA) and may be single and/or double stranded. Nucleic acid sequences may be modified, e.g., mutated, relative to naturally occurring nucleic acid sequences, for example. Nucleic acid sequence may have any length, for example 2 to 000,000 or more nucleotides (or any integral value above or between) a nucleic acid, for example a length of from about 100 to about 10,000, or from about 200 nucleotides to about 500 nucleotides. Transfection is the process of introducing an exogenous nucleic acid into a eukaryotic cell. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE- dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics technology (biolistics). A vector is a construct that is capable of delivering, and usually expressing, at least one gene or sequence of interest in a host cell. Examples of vectors include but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes. A vector may, in some embodiments, be an isolated nucleic acid that can be used to deliver a composition to the interior of the cell. It is known in the art a number of vectors including, but not limited to the linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, a vector may be an autonomously replicating plasmid or virus. The term should also be construed to include facilitate transfer of nucleic acid into cells of the non-plasmid and non-viral compounds, for example, polylysine compounds, liposomes, and the like. Non-limiting examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated virus vectors, and retroviral vectors. An expression vector is a vector comprising a recombinant polynucleotide comprising expression control sequence and a nucleotide sequence to be expressed operably linked. The expression vector comprises sufficient cis-acting elements (cis-acting elements) used for expression; other elements for expression can be supplied by the host cell or in vitro expression system. Expression vectors include, for example, cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentivirus, retroviruses, adenoviruses and adeno-associated viruses). Operably linked refers to functional linkage between the regulatory sequence and a heterologous nucleic acid sequence resulting in the expression of the latter. For example, if the promoter affects the transcription or expression of the coding sequence, the promoter is operably linked to a coding sequence. Typically, DNA sequencing operably linked are contiguous, and may join two protein coding regions in the same reading frame. A promoter is a DNA sequence recognized by the synthetic machinery required for the synthesis machinery of the cell specific transcription of a polynucleotide sequence or introduced. Inducible expression refers to expression under certain conditions, such as activation (or inactivation) of an intracellular signaling pathway or the contacting of the cells harboring the expression construct with a small molecule that regulates the expression (or degree of expression) of a gene operably linked to an inducible promoter sensitive to the concentration of the small molecule. This is contrasted with constitutive expression, which refers to expression under physiological conditions (not limited by certain conditions). Electroporation refers to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane; their presence allows biomolecules such as plasmids or other oligonucleotide to pass from one side of the cellular membrane to the other. C. Checkpoint Inhibitors, Co-stimulatory Agonists and Chemotherapeutics A checkpoint molecule is a protein that is expressed by tissues and/or immune cells and reduce the efficacy of an immune response in a manner dependent on the level of expression of the checkpoint molecule. When these proteins are blocked, the “brakes” on the immune system are released and, for example, T cells are able to kill cancer cells more effectively. Examples of checkpoint proteins found on T cells or cancer cells include PD-1/PD-L1 and CTLA-4/B7-1/B7- 2, LAG-3, PD-L2, NKG2A, KIR, TIM-3, CD96, VISTA and TIGIT. A checkpoint inhibitor is a drug entity that reverses the immunosuppressive signaling from a checkpoint molecule. A costimulatory molecule is an immune cell such as a T cell cognate binding partner that specifically binds to costimulatory ligands thereby mediating co-stimulation, such as, but not limited to proliferation. Costimulatory molecules are cell surface molecules other than the antigen receptor or ligand which facilitate an effective immune response. Co-stimulatory molecules include but are not limited to MHCI molecules, BTLA receptor and Toll ligands, and OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) and 4-1BB (CD137). Examples of costimulatory molecules include but are not limited to: CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ , IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA- 1, ITGAM, CD11b, ITGAX, CD11c, ITGB1 , CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244,2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229) , CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS , SLP-76, PAG / Cbp, CD19a, and CD83 ligand. A costimulatory agonist is a drug entity that activates (agonizes) the costimulatory molecule, such as costimulatory ligand would do, and produces an immunostimulatory signal or otherwise increases the potency or efficacy of an immune response. A chemotherapeutic agent is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN), CPT-11 (irinotecan, CAMPTOSAR), acetylcamptothecin, scopolectin, and 9- aminocamptothecin); bryostatin; pemetrexed; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; TLK- 286; CDP323, an oral alpha-4 integrin inhibitor; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin, doxorubicin HCl liposome injection (DOXIL) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR), tegafur (UFTORAL), capecitabine (XELODA), an epothilone, and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and imatinib (a 2- phenylaminopyrimidine derivative), as well as other c-Kit inhibitors; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE, FILDESIN); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, e.g., paclitaxel (TAXOL), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE), and doxetaxel (TAXOTERE); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN); oxaliplatin; leucovovin; vinorelbine (NAVELBINE); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN) combined with 5-FU and leucovovin. Chemotherapeutic agents also include anti-hormonal agents that act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer, and are often in the form of systemic, or whole-body treatment. They may be hormones themselves. Examples include anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX tamoxifen), raloxifene (EVISTA), droloxifene, 4- hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON); anti-progesterones; estrogen receptor down-regulators (ERDs); estrogen receptor antagonists such as fulvestrant (FASLODEX); agents that function to suppress or shut down the ovaries, for example, leutinizing hormone-releasing hormone (LHRH) agonists such as leuprolide acetate (LUPRON and ELIGARD), goserelin acetate, buserelin acetate and tripterelin; anti-androgens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (MEGASE), exemestane (AROMASIN), formestanie, fadrozole, vorozole (RIVISOR), letrozole (FEMARA), and anastrozole (ARIMIDEX). In addition, such definition of chemotherapeutic agents includes bisphosphonates such as clodronate (for example, BONEFOS or OSTAC), etidronate (DIDROCAL), NE-58095, zoledronic acid/zoledronate (ZOMETA), alendronate (FOSAMAX), pamidronate (AREDIA), tiludronate (SKELID), or risedronate (ACTONEL); as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); anti-sense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE vaccine and gene therapy vaccines, for example, ALLOVECTIN vaccine, LEUVECTIN vaccine, and VAXID vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN); an anti-estrogen such as fulvestrant; a Kit inhibitor such as imatinib or EXEL-0862 (a tyrosine kinase inhibitor); EGFR inhibitor such as erlotinib or cetuximab; an anti-VEGF inhibitor such as bevacizumab; arinotecan; rmRH (e.g., ABARELIX); lapatinib and lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also known as GW572016); 17AAG (geldanamycin derivative that is a heat shock protein (Hsp) 90 poison), and pharmaceutically acceptable salts, acids or derivatives of any of the above. A cytokine is a protein released by one cell that act on another cell as intercellular mediators or have an autocrine effect on the cells producing the proteins. Examples of such cytokines include lymphokines, monokines; interleukins ("ILs") such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL10, IL-11, IL-12, IL-13, IL-15, IL-17A-F, IL-18 to IL-29 (such as IL-23), IL-31, including PROLEUKIN rIL-2; a tumor-necrosis factor such as TNF-α or TNF-β, TGF-β1-3; and other polypeptide factors including leukemia inhibitory factor ("LIF"), ciliary neurotrophic factor ("CNTF"), CNTF-like cytokine ("CLC"), cardiotrophin ("CT"), and kit ligand ("KL"). A chemokine is a soluble factor (e.g., cytokine) that has the ability to selectively induce chemotaxis and activation of leukocytes. Chemokines also trigger processes of angiogenesis, inflammation, wound healing, and tumorigenesis. Non-limiting examples of chemokines include IL-8, a human homolog of murine keratinocyte chemoattractant (KC). A growth factor is a substance, such as a vitamin or hormone, that is required for the stimulation of growth in living cells. In some embodiments, the AFFIMER® polypeptide can be combined with a growth factor selected from the group consisting of: adrenomedullin (AM), angiopoietin (Ang), BMPs, BDNF, EGF, erythropoietin (EPO), FGF, GDNF, G-CSF, GM-CSF, GDF9, HGF, HDGF, IGF, migration-stimulating factor, myostatin (GDF-8), NGF, neurotrophins, PDGF, thrombopoietin, TGF-α, TGF-β, TNF-α, VEGF, P1GF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, and IL-18. An enzyme is a substance produced by a living organism which acts as a catalyst to bring about a specific biochemical reaction. LAG-3/PD-L1 AFFIMER® polypeptides may be conjugated to a sialidase, for example, so that the sialidase will cleave sialic acid motifs from the surface of PD-L1+ cells. Targeted cleavage of sialic acid motifs on the surface of HER2+ breast cancer cells has been shown to increase sensitivity to NK cell-mediated killing and may have a similar effect on PD-L1+ cancer cells. (10.1073/pnas.1608069113). D. Treatments The term “dysfunctional” includes refractory or unresponsive to antigen recognition, specifically, impaired capacity to translate antigen recognition into down-stream T-cell effector functions, such as proliferation, cytokine production (e.g., IL-2) and/or target cell killing. "Anergy" refers to the state of unresponsiveness to antigen stimulation resulting from incomplete or insufficient signals delivered through the T-cell receptor (e.g., increase in intracellular Ca+2 in the absence of ras-activation). T cell anergy can also result upon stimulation with antigen in the absence of co-stimulation, resulting in the cell becoming refractory to subsequent activation by the antigen even in the context of costimulation. The unresponsive state can often be overridden by the presence of Interleukin-2. Anergic T-cells do not undergo clonal expansion and/or acquire effector functions. "Exhaustion" refers to T cell exhaustion as a state of T cell dysfunction that arises from sustained TCR signaling that occurs during many chronic infections and cancer. It is distinguished from anergy in that it arises not through incomplete or deficient signaling, but from sustained signaling. It is defined by poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of infection and tumors. "Enhancing T-cell function" means to induce, cause or stimulate a T-cell to have a sustained or amplified biological function, or renew or reactivate exhausted or inactive T-cells. Examples of enhancing T-cell function include: increased secretion of γ-interferon from CD8+ T-cells, increased proliferation, increased antigen responsiveness (e.g., viral, pathogen, or tumor clearance) relative to such levels before the intervention. In some embodiments, the level of enhancement is as least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. The manner of measuring this enhancement is known to one of ordinary skill in the art. "Tumor immunity" refers to the process in which tumors evade immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "treated" when such evasion is attenuated, and the tumors are recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage and tumor clearance. "Sustained response" refers to the sustained effect on reducing tumor growth after cessation of a treatment. For example, the tumor size may remain to be the same or smaller as compared to the size at the beginning of the administration phase. In some embodiments, the sustained response has a duration at least the same as the treatment duration, at least 1.5x, 2.0x, 2.5x, or 3.0x length of the treatment duration. A cancer is physiological condition in mammals in which a population of cells are characterized by unregulated cell growth. Examples of cancer include but are not limited to, carcinoma, blastoma, sarcoma, and hematologic cancers such as lymphoma and leukemia. A tumor (also referred to as a neoplasm) is any mass of tissue that results from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including pre-cancerous lesions. Tumor growth is generally uncontrolled and progressive, does not induce or inhibit the proliferation of normal cells. Tumor can affect a variety of cells, tissues or organs, including but not limited to selected from bladder, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tube, gall bladder, heart, intestine, kidney, liver, lung, lymph node, neural tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, urethra, ureter, urethra, uterus, vagina organ or tissue or the corresponding cells. Tumors include cancers, such as sarcoma, carcinoma, plasmacytoma or (malignant plasma cells). Tumors of the present disclosure, may include but are not limited to leukemias (e.g., acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myeloid - monocytic leukemia, acute monocytic leukemia, acute leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphomas (Hodgkin's disease, non-Hodgkin's disease), primary macroglobulinemia disease, heavy chain disease, and solid tumors such as sarcomas cancer (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endothelium sarcoma, lymphangiosarcoma, angiosarcoma, lymphangioendothelio sarcoma, synovioma vioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, (including triple negative breast cancer), ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, carcinoma, bronchogenic carcinoma, medullary carcinoma, renal cell carcinoma, hepatoma, Nile duct carcinoma, choriocarcinoma, spermatogonia Tumor, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma (including small cell lung carcinoma and non-small cell lung carcinoma or NSCLC), bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, retinoblastoma), esophageal cancer, gallbladder , kidney cancer, multiple myeloma. Preferably, a "tumor" includes, but is not limited to: pancreatic cancer, liver cancer, lung cancer (including NSCLC), stomach cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer (including triple negative breast cancer), lymphoma, gallbladder cancer, renal cancer, leukemia, multiple myeloma, ovarian cancer, cervical cancer and glioma. In some embodiments, the tumor is a PD-L1-positive tumor, such as a non-small cell lung carcinoma (NSCLC), colorectal cancer, advanced melanoma, or renal cell carcinoma. Metastasis refers to the process by which a cancer spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion at the new location. A "metastatic" or "metastasizing" cell is one that loses adhesive contacts with neighboring cells and migrates via the bloodstream or lymph from the primary site of disease to invade neighboring body structures. "Cancer cell" and "tumor cell" refers to the total population of cells derived from a cancer or tumor or pre-cancerous lesion, including both non-tumorigenic cells, which comprise the bulk of the cancer cell population, and tumorigenic stem cells (cancer stem cells). As used herein, the terms "cancer cell" or "tumor cell" will be modified by the term "non-tumorigenic" when referring solely to those cells lacking the capacity to renew and differentiate to distinguish those tumor cells from cancer stem cells. A "complete response" or "CR" refers to disappearance of all target lesions; "partial response" or "PR" refers to at least a 30% decrease in the sum of the longest diameters (SLD) of target lesions, taking as reference the baseline SLD; and "stable disease" or "SD" refers to neither sufficient shrinkage of target lesions to qualify for PR, nor sufficient increase to qualify for PD, taking as reference the smallest SLD since the treatment started. "Progression free survival" (PFS) refers to the length of time during and after treatment during which the disease being treated (e.g., cancer) does not get worse. Progression-free survival may include the amount of time patients have experienced a complete response or a partial response, as well as the amount of time patients have experienced stable disease. "Overall response rate" (ORR) refers to the sum of complete response (CR) rate and partial response (PR) rate. "Overall survival" refers to the percentage of individuals in a group who are likely to be alive after a particular duration of time. "Treatment" refers to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and (2) prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. In the case of cancer or a tumor, a subject is successfully "treated" according to the methods of the present disclosure if the patient shows at least one of the following: an increased immune response, an increased anti-tumor response, increased cytolytic activity of immune cells, increased killing of tumor cells by immune cells, a reduction in the number of or complete absence of cancer cells; a reduction in the tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including the spread of cancer cells into soft tissue and bone; inhibition of or an absence of tumor or cancer cell metastasis; inhibition or an absence of cancer growth; relief of at least one symptom associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity; reduction in the number or frequency of cancer stem cells; or some combination of effects. “Subject,” “individual,” and “patient,” used interchangeably herein, refer to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, and rodents. In some embodiments, the subject is a PD-1/PD-L1 refractory subject (e.g., a subject that has been or is being treated for a disease or condition and does not respond to attempted forms of treatment for said disease or condition). For example, a cancer is said to be refractory when it does not respond to (or is resistant to) cancer treatment. A refractory cancer is also known as resistant cancer. Thus, a refractory subject is a subject that does not respond or is resistant to treatment of a disease or condition the subject is suffering from. In some embodiments, a refractory subject is a cancer patient unresponsive to anti-PD-1 and/or anti-PD- L1 therapy. As used herein, “refractory” refers to a subject having less than 20% reduction in tumor size or volume after administration of an anti-PD-1 and/or anti-PD-L1 agent relative to a control. In some embodiments, a refractory subject shows less than 10% reduction in tumor size or volume after administration of anti-PD-1 and/or anti-PD-L1 agent relative to a control. In some embodiments, a refractory subject shows less than 5% reduction in tumor size or volume after administration of anti-PD-1 and/or anti-PD-L1 agent relative to a control. In some embodiments, a refractory subject shows less than 1%> reduction in tumor size or volume after administration of anti-PD-1 and/or anti-PD-L1 agent relative to a control. In embodiments, a refractory subject shows less than 0.5% reduction in tumor size or volume after administration of anti-PD-1 and/or anti-PD-L1 agent relative to a control. In embodiments, a refractory subject shows less than 0.1%> reduction in tumor size or volume after administration of anti-PD-1 and/or anti-PD-L1 agent relative to a control. "Agonist" and "agonistic" refer to agents that are capable of, directly or indirectly, substantially inducing, activating, promoting, increasing, or enhancing the biological activity of a target or target pathway. "Agonist" is used herein to include any agent that partially or fully induces, activates, promotes, increases, or enhances the activity of a protein or other target of interest. "Antagonist" and "antagonistic" refer to or describe an agent that is capable of, directly or indirectly, partially or fully blocking, inhibiting, reducing, or neutralizing a biological activity of a target and/or pathway. The term "antagonist" is used herein to include any agent that partially or fully blocks, inhibits, reduces, or neutralizes the activity of a protein or other target of interest. "Modulation" and "modulate" refer to a change or an alteration in a biological activity. Modulation includes, but is not limited to, stimulating an activity or inhibiting an activity. Modulation may be an increase in activity or a decrease in activity, a change in binding characteristics, or any other change in the biological, functional, or immunological properties associated with the activity of a protein, a pathway, a system, or other biological targets of interest. An immune response includes responses from both the innate immune system and the adaptive immune system. It includes both cell-mediated and/or humoral immune responses. It includes both T-cell and B-cell responses, as well as responses from other cells of the immune system such as natural killer (NK) cells, monocytes, macrophages, etc. "Pharmaceutically acceptable" refers to a substance approved or approvable by a regulatory agency of the Federal government or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans. "Pharmaceutically acceptable excipient” is an excipient, carrier or adjuvant that can be administered to a subject, together with at least one agent of the present disclosure, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic effect. In general, those of skill in the art and the U.S. FDA consider a pharmaceutically acceptable excipient, carrier, or adjuvant to be an inactive ingredient of any formulation. An “effective amount” (also referred to herein as a “therapeutically effective amount” is an amount of an agent, such as a LAG-3/PD-L1 AFFIMER® agent, effective to treat a disease or disorder in a subject such as, a mammal. In the case of cancer or a tumor, the therapeutically effective amount of a LAG-3/PD-L1 AFFIMER® agent has a therapeutic effect and as such can boost the immune response, boost the anti-tumor response, increase cytolytic activity of immune cells, increase killing of tumor cells by immune cells, reduce the number of tumor cells; decrease tumorigenicity, tumorigenic frequency or tumorigenic capacity; reduce the number or frequency of cancer stem cells; reduce the tumor size; reduce the cancer cell population; inhibit or stop cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibit and stop tumor or cancer cell metastasis; inhibit and stop tumor or cancer cell growth; relieve to some extent at least one of the symptoms associated with the cancer; reduce morbidity and mortality; improve quality of life; or a combination of such effects. E. Miscellaneous It is understood that wherever embodiments are described herein with the language "comprising" otherwise analogous embodiments described in terms of "consisting of” and/or "consisting essentially of" are also provided. It is also understood that wherever embodiments are described herein with the language "consisting essentially of" otherwise analogous embodiments described in terms of "consisting of" are also provided. As used herein, reference to "about" or "approximately" a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to "about X" includes description of "X". The term "and/or" as used in a phrase such as "A and/or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). The phrase “at least one” may be used interchangeably with “one or more.” It should be understood that “a” is not limited to one but rather means “at least one.” III. LAG-3 AFFIMER® polypeptides and Fusion LAG-3/PD-L1 AFFIMER® polypeptides An AFFIMER® polypeptide is a scaffold based on a Stefin A polypeptide, meaning that it has a sequence which is derived from a Stefin A polypeptide, for example, a mammalian Stefin A polypeptide, for example, a human Stefin A polypeptide. Some aspects of the application provide a protein that comprises an AFFIMER® polypeptide that binds LAG-3 (also referred to as “LAG-3 AFFIMER® polypeptides”) in which at least one of the solvent accessible loops from the wild-type Stefin A protein binds LAG-3, preferably selectively, and preferably with Kd of 10-6M or less. Some aspects of the application provide a fusion protein that comprises an AFFIMER® polypeptide that binds LAG-3 and an AFFIMER® polypeptide that binds PD-L1 (also referred to as “LAG-3/PD-L1 AFFIMER® polypeptides”) in which at least one of the solvent accessible loops from the wild-type Stefin A protein binds LAG-3, preferably selectively, and preferably with Kd of 10-6M or less, and in which at least one of the solvent accessible loops from the wild-type Stefin A protein binds PD-L1, preferably selectively, and preferably with Kd of 10-6M or less. In some embodiments, a protein or fusion protein further comprises an AFFIMER® polypeptide that binds human serum albumin (HSA) (also referred to as “LAG-3/PD-L1/HSA AFFIMER® polypeptides”) in which at least one of the solvent accessible loops from the wild-type Stefin A protein binds HSA, preferably selectively, and preferably with Kd of 10-6M or less Some aspects provide a Lymphocyte Activation Gene 3 (LAG-3) binding polypeptide comprising the following Formula (I): FR1-(X')-FR2-(X'')-FR3 (I). FR1, in some embodiments, comprises an amino acid sequence having at least 90% to the amino acid sequence of MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA (SEQ ID NO: 216). FR1, in some embodiments, comprises an amino acid sequence having at least 95% identity to the amino acid sequence of MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA (SEQ ID NO: 216). FR1, in some embodiments, comprises an amino acid sequence having 100% identity to the amino acid sequence of MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA (SEQ ID NO: 216). FR2, in some embodiments, comprises an amino acid sequence having at least 90% identity to the amino acid sequence of STNYYIKVRAGDNKYMHLKVFNGP (SEQ ID NO: 217). FR2, in some embodiments, comprises an amino acid sequence having at least 95%, identity to the amino acid sequence of STNYYIKVRAGDNKYMHLKVFNGP (SEQ ID NO: 217). FR2, in some embodiments, comprises an amino acid sequence having 100% identity to the amino acid sequence of STNYYIKVRAGDNKYMHLKVFNGP (SEQ ID NO: 217). FR3, in some embodiments, comprises an amino acid sequence having at least 90% identity to the amino acid sequence of ADRVLTGYQVDKNKDDELTGF (SEQ ID NO: 218). FR3, in some embodiments, comprises an amino acid sequence having at least 95% identity to the amino acid sequence of ADRVLTGYQVDKNKDDELTGF (SEQ ID NO: 218). FR3, in some embodiments, comprises an amino acid sequence having 100% identity to the amino acid sequence of ADRVLTGYQVDKNKDDELTGF (SEQ ID NO: 218). A LAG-3 binding polypeptide, in some embodiments, comprises the amino acid sequence having of MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA-(X')- STNYYIKVRAGDNKYMHLKVFNGP-(X'')-ADRVLTGYQVDKNKDDELTGF. In some embodiments, X' is an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NOs: 204-209. In some embodiments, X' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 204. In some embodiments, X' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 205. In some embodiments, X' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 206. In some embodiments, X' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 207. In some embodiments, X' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 208. In some embodiments, X' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 209. In some embodiments, X' is an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NOs: 204-209. In some embodiments, X' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 204. In some embodiments, X' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 205. In some embodiments, X' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 206. In some embodiments, X' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 207. In some embodiments, X' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 208. In some embodiments, X' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 209. In some embodiments, X'' is an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NOs: 210-215. In some embodiments, X'' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 210. In some embodiments, X'' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 211. In some embodiments, X'' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 212. In some embodiments, X'' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 213. In some embodiments, X'' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 214. In some embodiments, X'' is an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 215. In some embodiments, X'' is an amino acid sequence having at 100% identity to the amino acid sequence of any one of SEQ ID NOs: 210-215. In some embodiments, X'' is an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NOs: 210-215. In some embodiments, X'' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 210. In some embodiments, X'' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 211. In some embodiments, X'' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 212. In some embodiments, X'' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 213. In some embodiments, X'' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 214. In some embodiments, X'' is an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 215. In some embodiments, the polypeptide binds to LAG-3 with a Kd of 1×10−6M or less. In some embodiments, X' is the amino acid sequence of SEQ ID NO: 204 and X'' is the amino acid sequence of SEQ ID NO: 210. In some embodiments, X' is the amino acid sequence of SEQ ID NO: 205 and X'' is the amino acid sequence of SEQ ID NO: 211. In some embodiments, X' is the amino acid sequence of SEQ ID NO: 206 and X'' is the amino acid sequence of SEQ ID NO: 212. In some embodiments, X' is the amino acid sequence of SEQ ID NO: 207 and X'' is the amino acid sequence of SEQ ID NO: 213. In some embodiments, X' is the amino acid sequence of SEQ ID NO: 208 and X'' is the amino acid sequence of SEQ ID NO: 214. In some embodiments, X' is the amino acid sequence of SEQ ID NO: 209 and X'' is the amino acid sequence of SEQ ID NO: 215. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 53. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 54. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 61. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 62. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 66. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 69. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 53. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 54. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 61. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 62. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 66. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 69. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 53. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 54. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 61. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 62. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 66. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 69. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 53. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 54. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 61. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 62. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 66. A LAG-3 binding polypeptide, in some embodiments, comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 69. A LAG-3 binding polypeptide, in some embodiments, the amino acid sequence of SEQ ID NO: 53. A LAG-3 binding polypeptide, in some embodiments, comprises the amino acid sequence of SEQ ID NO: 54. A LAG-3 binding polypeptide, in some embodiments, comprises the amino acid sequence of SEQ ID NO: 61. A LAG-3 binding polypeptide, in some embodiments, comprises the amino acid sequence of SEQ ID NO: 62. A LAG-3 binding polypeptide, in some embodiments, comprises the amino acid sequence of SEQ ID NO: 66. A LAG-3 binding polypeptide, in some embodiments, comprises the amino acid sequence of SEQ ID NO: 69. In some embodiments, the LAG-3 binding polypeptide further comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is a human serum albumin (HSA) binding polypeptide or a fragment crystallizable (Fc) region of an antibody. In some embodiments, the antibody is a human IgG1 antibody. In some embodiments, the antibody is a human IgG4 Fc antibody. In some embodiments, a bispecific fusion protein comprises: one or more PD-L1 binding polypeptide; and one or more LAG-3 binding polypeptide. In some embodiments, a PD-L1 polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, a PD- L1 polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, a PD-L1 polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, a PD-L1 polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, a PD-L1 polypeptide comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, a PD-L1 polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, a PD- L1 polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, a PD-L1 polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, a PD-L1 polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, a PD-L1 polypeptide comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 61. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 61. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 61. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 61. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 61. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 61. In some embodiments, a bispecific fusion protein comprises: a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73; and a second LAG-3 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 62. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 62. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 62. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 62. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 62. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 62. In some embodiments, a bispecific fusion protein comprises: a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, a bispecific fusion protein comprises: a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, a bispecific fusion protein comprises a PD-L1 binding polypeptide comprising an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 72; and a LAG-3 binding polypeptide comprising an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 53. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 1. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 1. Some aspects provide a bispecific fusion protein comprising the amino acid sequence of SEQ ID NO: 1. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 2. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 2. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 2. Some aspects provide a bispecific fusion protein comprising the amino acid sequence of SEQ ID NO: 2. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 3. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 3. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 3. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 3. Some aspects provide a bispecific fusion protein comprising the amino acid sequence of SEQ ID NO: 3. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 5. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 5. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 5. Some aspects provide a bispecific fusion protein comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 5. Some aspects provide a bispecific fusion protein comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, a bispecific fusion protein further comprises a second PD-L1 binding polypeptide and/or a second LAG-3 binding polypeptide. In some embodiments, the second PD-L1 polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the second PD-L1 polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the second PD-L1 polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the second PD-L1 polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the second PD-L1 polypeptide comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the second PD-L1 polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the second PD-L1 polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the second PD-L1 polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the second PD-L1 polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the second PD-L1 polypeptide comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the second LAG-3 polypeptide comprises an amino acid sequence of any one of the LAG-3 polypeptides describe herein. Other aspects provide bispecific protein comprising: a first PD-L1 binding polypeptide; a second PD-L1 polypeptide; and any one of the LAG-3 binding polypeptides described herein. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD- L1 polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72, and the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD-L1 polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72, and the LAG-3 binding polypeptide comprises an amino acid sequence having at least 86% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD-L1 polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72, and the LAG-3 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD-L1 polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72, and the LAG-3 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD-L1 polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 72, and the LAG-3 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD- L1 polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 74, and the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD-L1 polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 74, and the LAG-3 binding polypeptide comprises an amino acid sequence having at least 86% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD-L1 polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 74, and the LAG-3 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD-L1 polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 74, and the LAG-3 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD-L1 polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 74, and the LAG-3 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 53. In some embodiments, a bispecific fusion protein comprises an amino acid sequence at least 80% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, a bispecific fusion protein comprises an amino acid sequence at least 85% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, a bispecific fusion protein comprises an amino acid sequence at least 90% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, a bispecific fusion protein comprises an amino acid sequence at least 95% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, a bispecific fusion protein comprises the amino acid sequence of SEQ ID NO: 4. Some aspects provide a trispecific fusion protein comprising any one of the bispecific fusion proteins described herein and a half-life extension moiety. In some embodiments, the half- life extension moiety is a human serum albumin (HSA)-binding polypeptide. In some embodiments, the HSA binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the HSA binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the HSA binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the HSA binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the HSA binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the half-life extension moiety is a fragment crystallizable (Fc) region of an antibody, for example, a human IgG1 antibody or a human IgG4 antibody. Other aspects provide trispecific fusion protein, comprising: a PD-L1 binding polypeptide; a first LAG-3 binding polypeptide; a second LAG-3 binding polypeptide; and an HSA binding polypeptide. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72 or 73. In some embodiments, each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence of the any one of the LAG-3 binding polypeptides described herein. In some embodiments, the HSA binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the HSA binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the HSA binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the HSA binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the HSA binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 74, each of the first LAG-3-binding polypeptide and the second LAG-3-binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 54, and the HSA binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 74, each of the first LAG-3-binding polypeptide and the second LAG- 3-binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 54, and the HSA binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 74, each of the first LAG-3- binding polypeptide and the second LAG-3-binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 54, and the HSA binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 74, each of the first LAG-3-binding polypeptide and the second LAG-3-binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 54, and the HSA binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 74, each of the first LAG-3-binding polypeptide and the second LAG-3-binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 54, and the HSA binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 80% identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 85% identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 90% identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 95% identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the trispecific fusion protein comprises an amino acid sequence 100% identity to the amino acid sequence of SEQ ID NO: 11. Some aspects provide a trispecific fusion protein comprising: a first PD-L1 binding polypeptide; a second PD-L1 binding polypeptide; a first LAG-3 binding polypeptide; a second LAG-3 binding polypeptide; and a half-life extension moiety. In some embodiments, the half-life extension moiety is an HSA binding polypeptide. In some embodiments, each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, each of the first LAG-3 binding polypeptide and second LAG-3 binding polypeptide comprises an amino acid sequence of any one of the LAG-3 polypeptides described herein. In some embodiments, each of HSA binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, each of HSA binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, each of HSA binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, each of HSA binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, each of HSA binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD- L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72, each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 62, and the HSA binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD-L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72, each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 62, and the HSA binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72, each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 62, and the HSA binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72, each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 62, and the HSA binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD-L1 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 72, each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 62, and the HSA binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD- L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72, each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 54, and the HSA binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD-L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72, each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 54, and the HSA binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72, each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 54, and the HSA binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72, each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 54, and the HSA binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, each of the first PD-L1 binding polypeptide and the second PD-L1 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 72, each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 54, and the HSA binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 80% the amino acid sequence of any one of SEQ ID NO: 8. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 85% the amino acid sequence of any one of SEQ ID NO: 8. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 90% the amino acid sequence of any one of SEQ ID NO: 8. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 95% the amino acid sequence of any one of SEQ ID NO: 8. In some embodiments, the trispecific fusion protein comprises an amino acid sequence 100% the amino acid sequence of any one of SEQ ID NO: 8. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 80% the amino acid sequence of any one of SEQ ID NO: 9. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 85% the amino acid sequence of any one of SEQ ID NO: 9. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 90% the amino acid sequence of any one of SEQ ID NO: 9. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 95% the amino acid sequence of any one of SEQ ID NO: 9. In some embodiments, the trispecific fusion protein comprises an amino acid sequence 100% the amino acid sequence of any one of SEQ ID NO: 9. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 80% the amino acid sequence of any one of SEQ ID NO: 10. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 85% the amino acid sequence of any one of SEQ ID NO: 10. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 90% the amino acid sequence of any one of SEQ ID NO: 10. In some embodiments, the trispecific fusion protein comprises an amino acid sequence at least 95% the amino acid sequence of any one of SEQ ID NO: 10. In some embodiments, the trispecific fusion protein comprises an amino acid sequence 100% the amino acid sequence of any one of SEQ ID NO: 10. Some aspects provide a bispecific fusion protein comprising a PD-L1 binding polypeptide, a LAG-3 binding polypeptide, and a fragment crystallizable (Fc) region of an antibody. In some embodiments, the antibody is selected from a human IgG1 (hIgG1) antibody and a human IgG4 (hIgG4) antibody. In some embodiments, the hIgG1 antibody comprises LALA mutations (Leu234Ala and Leu235Ala mutations, relative to full length human IgG1). In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the PD- L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having 100%identity to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having 100%identity to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the LAG-3 binding polypeptide comprises an amino acid sequence of any one or more of the LAG-3 binding polypeptides described herein. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 54, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 54, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 54, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 54, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 54, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 62, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 62, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 62, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 62, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 62, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 66, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 66, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 66, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 66, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 66, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 69, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 69, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 69, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 69, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the PD-L1 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 72, the LAG-3 binding polypeptide comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 69, and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NO: 6. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NO: 6. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 6. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 6. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NO: 6. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NO: 7. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NO: 7. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 7. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 7. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NO: 7. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NO: 15. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NO: 15. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 15. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 15. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NO: 15. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NO: 19. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NO: 19. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 19. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 19. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NO: 19. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NO: 38. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NO: 38. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 38. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 38. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NO: 38. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NO: 39. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NO: 39. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 39. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 39. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NO: 39. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NO: 40. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NO: 40. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 40. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 40. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NO: 40. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NO: 41. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NO: 41. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 41. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 41. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NO: 41. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NO: 44. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NO: 44. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 44. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 44. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NO: 44. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NO: 45. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NO: 45. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NO: 45. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 45. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to the amino acid sequence of any one of SEQ ID NO: 45. In some embodiments, fusion protein further comprises one or more linker located between two of the polypeptides. In some embodiments, the linker is a rigid linker, optionally comprising the amino acid sequence of AEAAAKEAAAKEAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 86). In some embodiments, the linker is a flexible linker, optionally comprising the amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 87). Table 1. AFFIMER® polypeptides (Loop 2/4 sequences underlined; linkers double underlined; human Fc domains italicized)
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
*AVA19 is a LAG3 AFFIMER® polypeptide; AVA04 is a PD-L1 AFFIMER® polypeptide; AVA03 is an HSA/XT AFFIMER® polypeptide; SQTGly and 3t0 are controls; any one or more of the AVA19 LAG3 AFFIMER® polypeptides of Table 1 may include an N32G mutation A. Fusions Proteins - General In some embodiments, the LAG-3/PD-L1 AFFIMER® polypeptides may further comprise an additional insertion, substitution and/or deletion that modulates biological activity of the AFFIMER® polypeptide. For example, the additions, substitutions and/or deletions may modulate at least one property or activity of modified AFFIMER® polypeptide. For example, the additions, substitutions or deletions may modulate affinity for the AFFIMER® polypeptide, e.g., for binding to and inhibiting PD-L1 and/or LAG-3, modulate the circulating half-life, modulate the therapeutic half-life, modulate the stability of the AFFIMER® polypeptide, modulate cleavage by proteases, modulate dose, modulate release or bioavailability, facilitate purification, decrease deamidation, improve shelf-life, or improve or alter a particular route of administration. Similarly, AFFIMER® polypeptides may comprise protease cleavage sequences, reactive groups, antibody-binding domains (including but not limited to, FLAG or poly-His) or other affinity-based sequences (including but not limited to, FLAG, poly-His, GST, etc.) or linked molecules (including but not limited to, biotin) that improve detection, purification or other traits of the polypeptide. In some instances, these additional sequences are added to one end and/or the other of the AFFIMER® polypeptide in the form of a fusion protein. Accordingly, in certain aspects of the disclosure, the AFFIMER® agent is a fusion protein having at least one AFFIMER® polypeptide sequence and at least one heterologous polypeptide sequence (“fusion domain” herein). A fusion domain may be selected so as to confer a desired property, such as secretion from a cell or retention on the cell surface (e.g., for an encoded AFFIMER® construct), to serve as substrate or other recognition sequences for post-translational modifications, to create multimeric structures aggregating through protein-protein interactions, to alter (often to extend) serum half-life, or to alter tissue localization or tissue exclusion and other ADME (Absorption, Distribution, Metabolism, Excretion) properties – merely as examples. For example, some fusion domains are particularly useful for isolation and/or purification of the fusion proteins, such as by affinity chromatography. Well known examples of such fusion domains that facilitate expression or purification include, merely to illustrate, affinity tags such as polyhistidine (e.g., a His6 tag), Strep II tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA tag, c-Myc tag, thioredoxin, protein A and protein G. In order for the AFFIMER® agent to be secreted, it will generally contain a signal sequence that directs the transport of the protein to the lumen of the endoplasmic reticulum and ultimately to be secreted (or retained on the cell surface if a transmembrane domain or other cell surface retention signal). Signal sequences (also referred to as signal peptides or leader sequences) are located at the N-terminus of nascent polypeptides. They target the polypeptide to the endoplasmic reticulum and the proteins are sorted to their destinations, for example, to the inner space of an organelle, to an interior membrane, to the cell outer membrane, or to the cell exterior via secretion. Most signal sequences are cleaved from the protein by a signal peptidase after the proteins are transported to the endoplasmic reticulum. The cleavage of the signal sequence from the polypeptide usually occurs at a specific site in the amino acid sequence and is dependent upon amino acid residues within the signal sequence. In some embodiments, the signal peptide is about 5 to about 40 amino acids in length (such as about 5 to about 7, about 7 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, or about 25 to about 30, about 30 to about 35, or about 35 to about 40 amino acids in length). In some embodiments, the signal peptide is a native signal peptide from a human protein. In other embodiments, the signal peptide is a non-native signal peptide. For example, in some embodiments, the non-native signal peptide is a mutant native signal peptide from the corresponding native secreted human protein, and can include at least one (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) substitution, insertions and/or deletions. In some embodiments, the signal peptide is a signal peptide or mutant thereof from a non-IgSF protein family, such as a signal peptide from an immunoglobulin (such as IgG heavy chain or IgG-kappa light chain), a cytokine (such as interleukin-2 (IL-2)), a serum albumin protein (e.g., HSA or albumin), a human azurocidin preprotein signal sequence, a luciferase, a trypsinogen (e.g. chymotrypsinogen or trypsinogen) or other signal peptide able to efficiently secrete a protein from a cell. In some embodiments of a secreted AFFIMER® agent, the recombinant polypeptide comprises a signal peptide when expressed, and the signal peptide (or a portion thereof) is cleaved from the AFFIMER® agent upon secretion. The subject fusion proteins may also include at least one linker separating heterologous protein sequences or domains. As used herein, the term "linker" refers to a linker amino acid sequence inserted between a first polypeptide (e.g., an AFFIMER® polypeptide) and a second polypeptide (e.g., a second AFFIMER® polypeptide, an Fc region, a receptor trap, albumin, etc.). Empirical linkers designed by researchers are generally classified into 3 categories according to their structures: flexible linkers, rigid linkers, and in vivo cleavable linkers. Besides the basic role in linking the functional domains together (as in flexible and rigid linkers) or releasing free functional domain in vivo (as in in vivo cleavable linkers), linkers may offer many other advantages for the production of fusion proteins, such as improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles. Linkers should not adversely affect the expression, secretion, or bioactivity of the fusion protein. Linkers should not be antigenic and should not elicit an immune response. Suitable linkers may include mixtures of glycine and serine residues and often include amino acids that are sterically unhindered. Other amino acids that can be incorporated into useful linkers include threonine and alanine residues. Linkers can range in length, for example from 1- 50 amino acids in length, 1-22 amino acids in length, 1-10 amino acids in length, 1-5 amino acids in length, or 1-3 amino acids in length. In some embodiments, the linker may comprise a cleavage site. In some embodiments, the linker may comprise an enzyme cleavage site, so that the second polypeptide may be separated from the first polypeptide. In some embodiments, the linker can be characterized as flexible. Flexible linkers are usually applied when the joined domains require a certain degree of movement or interaction. They are generally composed of small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. See, for example, Argos P. (1990) “An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion” J Mol Biol.211:943– 958. The small size of these amino acids provides flexibility and allows for mobility of the connecting functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduces the unfavorable interaction between the linker and the protein moieties. The most commonly used flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues (“GS” linker). An example of the most widely used flexible linker has the sequence of (Gly-Gly-Gly-Gly-Ser)n. By adjusting the copy number “n”, the length of this GS linker can be optimized to achieve appropriate separation of the functional domains, or to maintain necessary inter-domain interactions. Besides the GS linkers, many other flexible linkers have been designed for recombinant fusion proteins. As these flexible linkers are also rich in small or polar amino acids such as Gly and Ser but can contain additional amino acids such as Thr and Ala to maintain flexibility, as well as polar amino acids such as Lys and Glu to improve solubility. In some embodiments, the linker can be characterized as rigid. While flexible linkers have the advantage to connect the functional domains passively and permitting certain degree of movements, the lack of rigidity of these linkers can be a limitation in certain fusion protein embodiments, such as in expression yield or biological activity. The ineffectiveness of flexible linkers in these instances was attributed to an inefficient separation of the protein domains or insufficient reduction of their interference with each other. Under these situations, rigid linkers have been successfully applied to keep a fixed distance between the domains and to maintain their independent functions. Many natural linkers exhibited α-helical structures. The α-helical structure was rigid and stable, with intra-segment hydrogen bonds and a closely packed backbone. Therefore, the stiff α- helical linkers can act as rigid spacers between protein domains. George et al. (2002) “An analysis of protein domain linkers: their classification and role in protein folding” Protein Eng. 15(11):871-9. In general, rigid linkers exhibit relatively stiff structures by adopting α-helical structures or by containing multiple Pro residues. Under many circumstances, they separate the functional domains more efficiently than the flexible linkers. The length of the linkers can be easily adjusted by changing the copy number to achieve an optimal distance between domains. As a result, rigid linkers are chosen when the spatial separation of the domains is critical to preserve the stability or bioactivity of the fusion proteins. In this regard, alpha helix-forming linkers with the sequence of A(EAAAK)n (SEQ ID NO: 86) have been applied to the construction of many recombinant fusion proteins. Another type of rigid linkers has a Pro-rich sequence, (XP)n, with X designating any amino acid, preferably Ala, Lys, or Glu. Merely to illustrate, exemplary linkers include: Table 2. Exemplary Linkers
Figure imgf000083_0001
Other linkers that may be used in the subject fusion proteins include but are not limited to, SerGly, GGSG (SEQ ID NO: 181), GSGS (SEQ ID NO: 182), GGGS (SEQ ID NO: 183), S(GGS)n (SEQ ID NO: 184) where n is 1-7, GRA, poly(Gly), poly(Ala), GGGSGGG (SEQ ID NO: 185), ESGGGGVT (SEQ ID NO: 186), LESGGGGVT (SEQ ID NO: 187), GRAQVT (SEQ ID NO: 188), WRAQVT (SEQ ID NO: 189), and ARGRAQVT (SEQ ID NO: 190). The hinge regions of the Fc fusions described below may also be considered linkers. Various elements can be employed to anchor proteins on the plasma membrane of cells. For example, the transmembrane domains (TM) of type-I (oriented with the N-terminus outside the cell) and type-II (oriented with the N-terminus in the cytosol) integral membrane proteins can be used to target chimeric proteins to the plasma membrane. Proteins can also be attached to the cell surface by fusion of a GPI (glycophosphatidylinositol lipid) signal to the 3’ end of genes. Cleavage of the short carboxy-terminal peptide allows attachment of a glycolipid to the newly exposed C-terminus through an amide linkage. See Udenfriend et al. (1995) “How Glycosylphoshpatidylinositol Anchored Membrane Proteins are Made” Annu Rev Biochem 64:563–591. In some embodiments, the fusion protein includes a transmembrane polypeptide sequence (a transmembrane domain). The distinguishing features of appropriate transmembrane polypeptides comprise the ability to be expressed at the surface of the cell on which the AFFIMER® agent is to be displayed. In some embodiments, that may be an immune cell, in particular lymphocyte cells or Natural killer (NK) cells, and once there to interact with PD-L1 so as to directing cellular response of the immune cell against a predefined target tumor cell on which PD-L1 is upregulated. The transmembrane domain can be derived either from a natural or from a synthetic source. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. As non-limiting examples, the transmembrane polypeptide can be a subunit of the T cell receptor such as α, β, γ or δ, polypeptide constituting CD3 complex, IL2 receptor p55 (a chain), p75 (β chain) or γ chain, subunit chain of Fc receptors, in particular Fcy receptor III or CD proteins. Alternatively, the transmembrane domain can be synthetic and can comprise predominantly hydrophobic residues such as leucine and valine. In some AFFIMER® polypeptides, a sequence that signals for the posttranslational addition of a glycosylphosphatidylinositol (GPI) anchor. GPI anchors are glycolipid structures that are added post-translationally to the C-terminus of many eukaryotic proteins. This modification to the AFFIMER® agent will cause it to be anchored (attached) on the extracellular surface of the cell membrane of the cell in which the AFFIMER® agent is re-expressed as a recombinant protein (e.g., an encoded AFFIMER® construct as described below). In these embodiments, the GPI anchor domain is C-terminal to the AFFIMER® polypeptide sequence, and preferably occurs at the C-terminus of the fusion protein. In some embodiments, the GPI anchor domain is a polypeptide that signals for the posttranslational addition of a GPI anchor when the fusion protein of which it is a part is expressed in a eukaryotic system. The GPI anchor signal sequence consists of a set of small amino acids at the site of anchor addition (the ω site) followed by a hydrophilic spacer and ending in a hydrophobic stretch (Low, (1989) FASEB J.3:1600-1608). Cleavage of this signal sequence occurs in the ER before the addition of an anchor with conserved central components but with variable peripheral moieties (Homans et al., Nature, 333:269-272 (1988)). The C- terminus of a GPI-anchored protein is linked through a phosphoethanolamine bridge to the highly conserved core glycan, mannose(α1-2)mannose(α1-6)mannose(α1-4)glucosamine(α1- 6)myo-inositol. A phospholipid tail attaches the GPI anchor to the cell membrane. GPI anchor attachment can be achieved by expression of the AFFIMER® fusion protein containing the GPI anchor domain in a eukaryotic system capable of carrying out GPI posttranslational modifications. As with the transmembrane domain fusion proteins, human cells, including lymphocytes and other cells involved in initiating or promoting an antitumor are so capable and can be engineered to express and encoded AFFIMER® construct including a GPI anchor domain in order retain the expressed AFFIMER® polypeptide containing fusion on the surface of the engineered cell. Still other modifications that can be made to the AFFIMER® polypeptide sequence or to a flanking polypeptide moiety provided as part of a fusion protein is at least one sequence that is a site for post-translational modification by an enzyme. These can include, but are not limited to, glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-linkage modification, and the like. B. Engineering PK and ADME Properties In some embodiment, the AFFIMER® agent may not have a half-life and/or PK profile that is optimal for the route of administration, such as parenteral therapeutic dosing. A “half-life” is the amount of time it takes for a substance, such as an AFFIMER® agent of the present disclosure, to lose half of its pharmacologic or physiologic activity or concentration. Biological half-life can be affected by elimination, excretion, degradation (e.g., enzymatic) of the substance, or absorption and concentration in certain organs or tissues of the body. In some embodiments, biological half-life can be assessed by determining the time it takes for the blood plasma concentration of the substance to reach half its steady state level (“plasma half-life”). To address this shortcoming, there are a variety of general strategies for prolongation of half-life that have been used in the case of other protein therapeutics, including the incorporation of half-life extending moieties as part of the AFFIMER® agent. The term “half-life extending moiety” refers to a pharmaceutically acceptable moiety, domain, or molecule covalently linked (chemically conjugated or fused) to an AFFIMER® polypeptide to form an AFFIMER® agent described herein, optionally via a non-naturally encoded amino acid, directly or via a linker, that prevents or mitigates in vivo proteolytic degradation or other activity-diminishing modification of the AFFIMER® polypeptide, increases half-life, and/or improves or alters other pharmacokinetic or biophysical properties including but not limited to increasing the rate of absorption, reducing toxicity, improving solubility, reducing protein aggregation, increasing biological activity and/or target selectivity of the modified AFFIMER® polypeptide, increasing manufacturability, and/or reducing immunogenicity of the modified AFFIMER® polypeptide, compared to a comparator such as an unconjugated form of the modified AFFIMER® polypeptide. The term “half-life extending moiety” includes non- proteinaceous, half-life extending moieties, such as a water soluble polymer such as polyethylene glycol (PEG) or discrete PEG, hydroxyethyl starch (HES), a lipid, a branched or unbranched acyl group, a branched or unbranched C8-C30 acyl group, a branched or unbranched alkyl group, and a branched or unbranched C8-C30 alkyl group; and proteinaceous half-life extending moieties, such as serum albumin, transferrin, adnectins (e.g., albumin-binding or pharmacokinetics extending (PKE) adnectins), Fc domain, and unstructured polypeptide, such as XTEN and PAS polypeptide (e.g. conformationally disordered polypeptide sequences composed of the amino acids Pro, Ala, and/or Ser), and a fragment of any of the foregoing. An examination of the crystal structure of an AFFIMER® polypeptide and its interaction with its target, can indicate which certain amino acid residues have side chains that are fully or partially accessible to solvent. In some embodiments, the half-life extending moiety extends the half-life of the resulting AFFIMER® agent circulating in mammalian blood serum compared to the half-life of the protein that is not so conjugated to the moiety (such as relative to the AFFIMER® polypeptide alone). In some embodiments, half-life is extended by greater than or greater than about 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold., 5.0-fold, or 6.0-fold. In some embodiments, half-life is extended by more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours, more than 96 hours or more than 1 week after in vivo administration compared to the protein without the half-life extending moiety. As means for further exemplification, half-life extending moieties that can be used in the generation of AFFIMER® agents of the disclosure include: • Genetic fusion of the pharmacologically AFFIMER® sequence to a naturally long-half-life protein or protein domain (e.g., Fc fusion, transferrin [Tf] fusion, or albumin fusion. See, for example, Beck et al. (2011) “Therapeutic Fc-fusion proteins and peptides as successful alternatives to antibodies. MAbs.3:1–2; Czajkowsky et al. (2012) “Fc-fusion proteins: new developments and future perspectives. EMBO Mol Med.4:1015–28; Huang et al. (2009) “Receptor-Fc fusion therapeutics, traps, and Mimetibody technology” Curr Opin Biotechnol.2009;20:692–9; Keefe et al. (2013) “Transferrin fusion protein therapies: acetylcholine receptor-transferrin fusion protein as a model. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; p.345–56; Weimer et al. (2013) “Recombinant albumin fusion proteins. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; 2013. p.297–323; Walker et al. (2013) “Albumin-binding fusion proteins in the development of novel long-acting therapeutics. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; 2013. p.325–43. • Genetic fusion of the pharmacologically AFFIMER® sequence to an inert polypeptide, e.g., XTEN (also known as recombinant PEG or ‘‘rPEG’’), a homoamino acid polymer (HAP; HAPylation), a proline-alanine-serine polymer (PAS; PASylation), or an elastin- like peptide (ELP; ELPylation). See, for example, Schellenberger et al. (2009) “A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat Biotechnol.2009;27:1186–90; Schlapschy et al. Fusion of a recombinant antibody fragment with a homo-amino-acid polymer: effects on biophysical properties and prolonged plasma half-life. Protein Eng Des Sel.2007;20:273–84; Schlapschy (2013) PASylation: a biological alternative to PEGylation for extending the plasma halflife of pharmaceutically active proteins. Protein Eng Des Sel.26:489–501. Floss et al. (2012) “Elastin-like polypeptides revolutionize recombinant protein expression and their biomedical application. Trends Biotechnol.28:37–45. Floss et al. “ELP-fusion technology for biopharmaceuticals. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: application and challenges. Hoboken: Wiley; 2013. p.372– 98. • Increasing the hydrodynamic radius by chemical conjugation of the pharmacologically active peptide or protein to repeat chemical moieties, e.g., to PEG (PEGylation) or hyaluronic acid. See, for example, Caliceti et al. (2003) “Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates” Adv Drug Delivery Rev. 55:1261–77; Jevsevar et al. (2010) PEGylation of therapeutic proteins. Biotechnol J 5:113–28; Kontermann (2009) “Strategies to extend plasma half-lives of recombinant antibodies” BioDrugs.23:93–109; Kang et al. (2009) “Emerging PEGylated drugs” Expert Opin Emerg Drugs.14:363–80; and Mero et al. (2013) “Conjugation of hyaluronan to proteins” Carb Polymers.92:2163–70. • Significantly increasing the negative charge of fusing the pharmacologically active peptide or protein by polysialylation; or, alternatively, (b) fusing a negatively charged, highly sialylated peptide (e.g., carboxy-terminal peptide [CTP; of chorionic gonadotropin (CG) b-chain]), known to extend the half-life of natural proteins such as human CG b-subunit, to the biological drug candidate. See, for example, Gregoriadis et al. (2005) “Improving the therapeutic efficacy of peptides and proteins: a role for polysialic acids” Int J Pharm.2005; 300:125–30; Duijkers et al. “Single dose pharmacokinetics and effects on follicular growth and serum hormones of a long-acting recombinant FSH preparation (FSHCTP) in healthy pituitary- suppressed females” (2002) Hum Reprod.17:1987–93; and Fares et al. “Design of a longacting follitropin agonist by fusing the C-terminal sequence of the chorionic gonadotropin beta subunit to the follitropin beta subunit” (1992) Proc Natl Acad Sci USA.89:4304–8.35; and Fares “Half- life extension through O-glycosylation. • Binding non-covalently, via attachment of a peptide or protein-binding domain to the bioactive protein, to normally long-half-life proteins such as HSA, human IgG, transferrin or fibronectin. See, for example, Andersen et al. (2011) “Extending half-life by indirect targeting of the neonatal Fc receptor (FcRn) using a minimal albumin binding domain” J Biol Chem. 286:5234–41; O’Connor-Semmes et al. (2014) “GSK2374697, a novel albumin-binding domain antibody (albudAb), extends systemic exposure of extendin-4: first study in humans—PK/PD and safety” Clin Pharmacol Ther.2014;96:704–12. Sockolosky et al. (2014) “Fusion of a short peptide that binds immunoglobulin G to a recombinant protein substantially increases its plasma half-life in mice” PLoS One.2014;9:e102566. Classical genetic fusions to long-lived serum proteins offer an alternative method of half- life extension distinct from chemical conjugation to PEG or lipids. Two major proteins have traditionally been used as fusion partners: antibody Fc domains and human serum albumin (HSA). Fc fusions involve the fusion of peptides, proteins or receptor exodomains to the Fc portion of an antibody. Both Fc and albumin fusions achieve extended half-lives not only by increasing the size of the peptide drug, but both also take advantage of the body’s natural recycling mechanism: the neonatal Fc receptor, FcRn. The pH-dependent binding of these proteins to FcRn prevents degradation of the fusion protein in the endosome. Fusions based on these proteins can have half-lives in the range of 3-16 days, much longer than typical PEGylated or lipidated peptides. Fusion to antibody Fc domains can improve the solubility and stability of the peptide or protein drug. An example of a peptide Fc fusion is dulaglutide, a GLP-1 receptor agonist currently in late-stage clinical trials. Human serum albumin, the same protein exploited by the fatty acylated peptides is the other popular fusion partner. Albiglutide is a GLP-1 receptor agonist based on this platform. A major difference between Fc and albumin is the dimeric nature of Fc versus the monomeric structure of HSA leading to presentation of a fused peptide as a dimer or a monomer depending on the choice of fusion partner. The dimeric nature of an AFFIMER® polypeptide-Fc fusion can produce an avidity effect if the AFFIMER® polypeptide target, such as CD33 on tumor cells, are spaced closely enough together or are themselves dimers. This may be desirable or not depending on the target. 1. Fc Fusions In some embodiments, the AFFIMER® polypeptide (e.g., LAG-3/PD-L1 AFFIMER® polypeptide) may be part of a fusion protein with an immunoglobulin Fc domain ("Fc domain"), or a fragment or variant thereof, such as a functional Fc region. In this context, an Fc fusion (“Fc-fusion”), such as an LAG-3/PD-L1 AFFIMER® agent created as an AFFIMER® polypeptide-Fc fusion protein, is a polypeptide comprising at least one LAG-3/PD-L1 AFFIMER® polypeptide sequence covalently linked through a peptide backbone (directly or indirectly) to an Fc region of an immunoglobulin. An Fc-fusion may comprise, for example, the Fc region of an antibody (which facilitates effector functions and pharmacokinetics) and a LAG- 3/PD-L1 AFFIMER® polypeptide sequence as part of the same polypeptide. An immunoglobulin Fc region may also be linked indirectly to at least one LAG-3/PD-L1 AFFIMER® polypeptide. Various linkers are known in the art and can optionally be used to link an Fc to a polypeptide including a LAG-3/PD-L1 AFFIMER® polypeptide sequence to generate an Fc-fusion. In some embodiments, Fc-fusions can be dimerized to form Fc-fusion homodimers, or using non-identical Fc domains, to form Fc-fusion heterodimers. In some embodiments, an Fc-fusion protein comprises a PD-L1 AFFIMER® agent that comprises an PD-L1 AFFIMER® polypeptide linked to an Fc domain linked to another LAG-3 AFFIMER® polypeptide (PD-L1 AFFIMER® polypeptide-Fc domain-LAG-3 AFFIMER® polypeptide). In some embodiments, an Fc-fusion protein comprises a tetravalent bispecific Fc fusion protein (e.g., two PD-L1 AFFIMER® polypeptides linked to a first Fc domain and two LAG-3 AFFIMER® polypeptides linked to a second Fc domain). There are several reasons for choosing the Fc region of human antibodies for use in generating LAG-3/PD-L1 AFFIMER® agents as LAG-3/PD-L1 AFFIMER® fusion proteins. The principle rationale is to produce a stable protein, large enough to demonstrate a similar pharmacokinetic profile compared with those of antibodies, and to take advantage of the properties imparted by the Fc region; this includes the salvage neonatal FcRn receptor pathway involving FcRn-mediated recycling of the fusion protein to the cell surface post endocytosis, avoiding lysosomal degradation and resulting in release back into the bloodstream, thus contributing to an extended serum half-life. Another obvious advantage is the Fc domain’s binding to Protein A, which can simplify downstream processing during production of the AFFIMER® agent and permit generation of highly pure preparation of the AFFIMER® agent. In general, an Fc domain will include the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus, Fc domain refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc domain may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as set forth in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NIH, Bethesda, Md. (1991)). Fc may refer to this region in isolation, or this region in the context of a whole antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been observed at a number of different Fc positions and are also included as Fc domains as used herein. In some embodiments, the Fc is a functional Fc region. As used herein, a “functional Fc region” refers to an Fc domain or fragment thereof which retains the ability to bind FcRn. A functional Fc region binds to FcRn but does not possess effector function. The ability of the Fc region or fragment thereof to bind to FcRn can be determined by standard binding assays known in the art. Exemplary "effector functions" include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions can be assessed using various assays known in the art for evaluating such antibody effector functions. In an exemplary embodiment, the Fc domain is derived from an IgG1 subclass, however, other subclasses (e.g., IgG2, IgG3, and IgG4) may also be used. An exemplary sequence of a human IgG1 immunoglobulin Fc domain which can be used is: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 191) In some embodiments, the Fc region used in the fusion protein may comprise the hinge region of an Fc molecule. An exemplary hinge region comprises the core hinge residues spanning positions 1-16 (e.g., DKTHTCPPCPAPELLG (SEQ ID NO: 192)) of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above. In some embodiments, the AFFIMER® polypeptide-containing fusion protein may adopt a multimeric structure (e.g., dimer) owing, in part, to the cysteine residues at positions 6 and 9 within the hinge region of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above. In other embodiments, the hinge region as used herein, may further include residues derived from the CH1 and CH2 regions that flank the core hinge sequence of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above. In yet other embodiments, the hinge sequence may comprise or consist of GSTHTCPPCPAPELLG (SEQ ID NO: 193) or EPKSCDKTHTCPPCPAPELLG (SEQ ID NO: 194). In some embodiments, the hinge sequence may include at least one substitution that confer desirable pharmacokinetic, biophysical, and/or biological properties. Some exemplary hinge sequences include: EPKSCDKTHTCPPCPAPELLGGPS (SEQ ID NO: 195); EPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO: 196); EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 197); EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 198); DKTHTCPPCPAPELLGGPS (SEQ ID NO: 199); and DKTHTCPPCPAPELLGGSS (SEQ ID NO: 200). In some embodiments, the residue P at position 18 of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above may be replaced with S to ablate Fc effector function; this replacement is exemplified in hinges having the sequences EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 197), EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 198), and DKTHTCPPCPAPELLGGSS (SEQ ID NO: 200). In another embodiment, the residues DK at positions 1-2 of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above may be replaced with GS to remove a potential clip site; this replacement is exemplified in the sequence EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 198). In another embodiment, the C at the position 103 of the heavy chain constant region of human IgG1 (e.g., domains CH1-CH3), may be replaced with S to prevent improper cysteine bond formation in the absence of a light chain; this replacement is exemplified by EPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO: 196), EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 197), and EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 198). In some embodiments, the Fc is a mammalian Fc such as a human Fc, including Fc domains derived from IgG1, IgG2, IgG3 or IgG4. The Fc region may possess at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a native Fc region and/or with an Fc region of a parent polypeptide. In some embodiments, the Fc region may have at least about 90% sequence identity with a native Fc region and/or with an Fc region of a parent polypeptide. “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. In some embodiments, the fusion protein includes an Fc domain sequence for which the resulting AFFIMER® agent has no (or reduced) ADCC and/or complement activation or effector functionality. For example, the Fc domain may comprise a naturally disabled constant region of IgG2 or IgG4 isotype or a mutated IgG1 constant region. Examples of suitable modifications are described in EP0307434. One example comprises the substitutions of alanine residues at positions 235 and 237 (EU index numbering). In other embodiments, the fusion protein includes an Fc domain sequence for which the resulting AFFIMER® agent will retain some or all Fc functionality for example will be capable of one or both of ADCC and CDC activity, as for example if the fusion protein comprises the Fc domain from human IgG1 or IgG3. Levels of effector function can be varied according to known techniques, for example by mutations in the CH2 domain, for example wherein the IgG1 CH2 domain has at least one mutation at positions selected from 239 and 332 and 330, for example the mutations are selected from S239D and I332E and A330L such that the antibody has enhanced effector function, and/or for example altering the glycosylation profile of the antigen- binding protein of the disclosure such that there is a reduction in fucosylation of the Fc region. In some embodiments, an LAG-3/PD-L1 AFFIMER® polypeptide has an extended half- life and comprises at least one Fc domain sequence (e.g., IgG1, IgG1 LALA, or IgG4). Exemplary LAG-3/PD-L1 AFFIMER®-Fc fusion proteins are provided in Table 1. In some embodiments, the PD-L1-LAG-3 AFFIMER® - Fc fusion protein comprises an amino acid sequence selected from any one of SEQ ID NOs: 6, 7, 15, 19, 38, 39, 40, 41, 44, and 45. 2. Albumin Fusions In some embodiments, the AFFIMER® agent is a fusion protein comprising, in addition to at least one AFFIMER® polypeptide sequence, an albumin sequence or an albumin fragment. In other embodiments, the AFFIMER® agent is conjugated to the albumin sequence or an albumin fragment through chemical linkage other than incorporation into the polypeptide sequence including the AFFIMER® polypeptide. In some embodiments, the albumin, albumin variant, or albumin fragment is human serum albumin (HSA), a human serum albumin variant, or a human serum albumin fragment. Albumin serum proteins comparable to HSA are found in, for example, cynomolgus monkeys, cows, dogs, rabbits and rats. Of the non-human species, bovine serum albumin (BSA) is the most structurally similar to HSA. See, e.g., Kosa et al., (2007) J Pharm Sci.96(11):3117-24. The present disclosure contemplates the use of albumin from non-human species, including, but not limited to, albumin sequence derived from cyno serum albumin or bovine serum albumin. Mature HSA, a 585 amino acid polypeptide (approx.67 kDa) having a serum half-life of about 20 days, is primarily responsible for the maintenance of colloidal osmotic blood pressure, blood pH, and transport and distribution of numerous endogenous and exogenous ligands. The protein has three structurally homologous domains (domains I, II and III), is almost entirely in the alpha-helical conformation, and is highly stabilized by 17 disulfide bridges. In some embodiments, the AFFIMER® agent can be an albumin fusion protein including at least one AFFIMER® polypeptide sequence and the sequence for mature human serum albumin (SEQ ID NO: 201) or a variant or fragment thereof which maintains the PK and/or biodistribution properties of mature albumin to the extent desired in the fusion protein. DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEF AKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPER NECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHP YFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQ RLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTEC CHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVEN DEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSV VLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCE LFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPE AKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSAL EVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATK EQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 201) The albumin sequence can be set off from the AFFIMER® polypeptide sequence or other flanking sequences in the AFFIMER® agent by use of linker sequences as described above. While unless otherwise indicated, reference herein to “albumin” or to “mature albumin” is meant to refer to HSA. However, it is noted that full-length HSA has a signal peptide of 18 amino acids (MKWVTFISLLFLFSSAYS (SEQ ID NO: 202)) followed by a pro-domain of 6 amino acids (RGVFRR) (SEQ ID NO: 203); this 24 amino acid residue peptide may be referred to as the pre-pro domain. The AFFIMER® polypeptide-HSA fusion proteins can be expressed and secreted using the HSA pre-pro-domain in the recombinant proteins coding sequence. Alternatively, the AFFIMER® polypeptide-HSA fusion can be expressed and secreted through inclusion of other secretion signal sequences, such as described above. In alternative embodiments, rather than provided as part of a fusion protein with the AFFIMER® polypeptide, the serum albumin polypeptide can be covalently coupled to the AFFIMER® polypeptide-containing polypeptide through a bond other than a backbone amide bond, such as cross-linked through chemical conjugation between amino acid sidechains on each of the albumin polypeptide and the AFFIMER® polypeptide-containing polypeptide. In some embodiments, the HSA AFFIMER® polypeptide is part of an in-line fusion protein (e.g., attached to at least one PD-L1 AFFIMER® polypeptide and at least one LAG-3 AFFIMER® polypeptide). In some embodiments, the AFFIMER® polypeptides are attached via linkers (e.g., flexible or rigid linkers, as described herein). In some embodiments, the three AFFIMER® polypeptides are arranged (from N-terminal to C-terminal) as follows: HSA-LAG- 3-PDL1, HSA-PDL1-LAG-3; LAG-3-HSA-PDL1, PDL1-HSA-LAG-3, PDL1-LAG-3-HSA, or LAG-3-PDL1-HSA. In some embodiments, the in-line fusion protein comprises more than one PD-L1 AFFIMER® polypeptide and/or more than one LAG-3 AFFIMER® polypeptide. In some embodiments, the in-line fusion protein is a tetramer and the AFFIMER® polypeptides are arranged (from N-terminal to C-terminal) as follows: PDL1-LAG-3-LAG-3-HSA, PDL1-PDL1- LAG-3-HSA, LAG-3-PDL1-PDL1-HSA, LAG-3-LAG-3-PDL1-HSA, PDL1-LAG-3-HSA- LAG-3, PDL1-PDL1-HSA-LAG-3, LAG-3-PDL1-HSA-PDL1, LAG-3-LAG-3-HSA-PDL1, PDL1-HSA-LAG-3-LAG-3, PDL1-HSA-LAG-3-PDL1, LAG-3-HSA-PDL1-PDL1, LAG-3- HSA-PDL1-LAG-3, HSA-PDL1-PDL1-LAG-3, HSA-PDL1-LAG-3-PDL1, HSA-LAG-3- PDL1-PDL1, or HSA-LAG-3-LAG-3-PDL1. In some embodiments, the in-line fusion protein is a pentamer and the AFFIMER® polypeptides are arranged (from N-terminal to C-terminal) as follows: PDL1-PDL1-LAG-3-LAG-3-HSA, PDL1-LAG-3-PDL1-LAG-3-HSA, PDL1-LAG-3- LAG-3-PDL1-HSA, LAG-3-LAG-3-PDL1-PDL1-HSA, LAG-3-PDL1-LAG-3-PDL1-HSA, PDL1-PDL1-LAG-3-HSA-LAG-3, PDL1-LAG-3-PDL1-HSA-LAG-3, PDL1-LAG-3-LAG-3- HSA-PDL1, LAG-3-PDL1-LAG-3-HSA-PDL1, LAG-3-LAG-3-PDL1-HSA-PDL1, PDL1- PDL1-HSA-LAG-3-LAG-3, PDL1-LAG-3-HSA-PDL1-LAG-3, PDL1-LAG-3-HSA-LAG-3- PDL1, LAG-3-PDL1-HSA-PDL1-LAG-3, LAG-3-PDL1-HSA-LAG-3-PDL1, LAG-3-LAG-3- HSA-PDL1-PDL1, PDL1-HSA-PDL1-LAG-3-LAG-3, PDL1-HSA-LAG-3-PDL1-LAG-3, PDL1-HSA-LAG-3-LAG-3-PDL1, LAG-3-HSA-PDL1-PDL1-LAG-3, LAG-3-HSA-PDL1- LAG-3-PDL1, LAG-3-HSA-LAG-3-PDL1-PDL1, HSA-PDL1-PDL1-LAG-3-LAG-3-HSA, HSA-PDL1-LAG-3-PDL1-LAG-3-HSA, HSA-PDL1-LAG-3-LAG-3-PDL1-HSA, HSA-LAG- 3-LAG-3-PDL1-PDL1-HSA, or HSA-LAG-3-PDL1-LAG-3-PDL1-HSA. In some embodiments, there is an uneven number of PDL1 AFFIMER® polypeptides relative to LAG-3 AFFIMER® polypeptides (i.e., they are not present in the in-line fusion protein at a 1:1 ratio). For example, in the pentamer formation, in some embodiments, there are three LAG-3 AFFIMER® polypeptides and one PD-L1 AFFIMER® polypeptide, or three PD- L1 AFFIMER® polypeptides and one LAG-3 AFFIMER® polypeptide. Exemplary in-line fusion proteins are provided in Table X. 3. Serum Binding Domains In some embodiments, the AFFIMER® agent can include a serum-binding moiety – either as part of a fusion protein (if also a polypeptide) with the AFFIMER® polypeptide sequence or chemically conjugated through a site other than being part of a contiguous polypeptide chain. In some embodiments, the serum-binding polypeptide is an albumin binding moiety. Albumin contains multiple hydrophobic binding pockets and naturally serves as a transporter of a variety of different ligands such as fatty acids and steroids as well as different drugs. Furthermore, the surface of albumin is negatively charged making it highly water-soluble. The term “albumin binding moiety” as used herein refers to any chemical group capable of binding to albumin, e.g., has albumin binding affinity. Albumin binds to endogenous ligands such as fatty acids; however, it also interacts with exogenous ligands such as warfarin, penicillin and diazepam. As the binding of these drugs to albumin is reversible the albumin-drug complex serves as a drug reservoir that can enhance the drug biodistribution and bioavailability. Incorporation of components that mimic endogenous albumin-binding ligands, such as fatty acids, has been used to potentiate albumin association and increase drug efficacy. In some embodiments, a chemical modification method that can be applied in the generation of the subject AFFIMER® agents to increase protein half-life is lipidation, which involves the covalent binding of fatty acids to peptide side chains. Originally conceived of and developed as a method for extending the half-life of insulin, lipidation shares the same basic mechanism of half-life extension as PEGylation, namely increasing the hydrodynamic radius to reduce renal filtration. However, the lipid moiety is itself relatively small and the effect is mediated indirectly through the non-covalent binding of the lipid moiety to circulating albumin. One consequence of lipidation is that it reduces the water-solubility of the peptide but engineering of the linker between the peptide and the fatty acid can modulate this, for example by the use of glutamate or mini PEGs within the linker. Linker engineering and variation of the lipid moiety can affect self-aggregation which can contribute to increased half-life by slowing down biodistribution, independent of albumin. See, for example, Jonassen et al. (2012) Pharm Res.29(8):2104-14. Other examples of albumin binding moieties for use in the generation of certain AFFIMER® agents include albumin-binding (PKE2) adnectins (See WO2011140086 “Serum Albumin Binding Molecules”, WO2015143199 “Serum albumin-binding Fibronectin Type III Domains” and WO2017053617 “Fast-off rate serum albumin binding fibronectin type iii domains”), the albumin binding domain 3 (ABD3) of protein G of Streptococcus strain G148, and the albumin binding domain antibody GSK2374697 (“AlbudAb”) or albumin binding nanobody portion of ATN-103 (Ozoralizumab). 4. PEGylation, XTEN, PAS and Other Polymers A wide variety of macromolecular polymers and other molecules can be linked to the AFFIMER® polypeptides of the present disclosure to modulate biological properties of the resulting AFFIMER® agent, and/or provide new biological properties to the AFFIMER® agent. These macromolecular polymers can be linked to the AFFIMER® polypeptide via a naturally encoded amino acid, via a non-naturally encoded amino acid, or any functional substituent of a natural or non-natural amino acid, or any substituent or functional group added to a natural or non-natural amino acid. The molecular weight of the polymer may be of a wide range, including but not limited to, between about 100 Da and about 100,000 Da or more. The molecular weight of the polymer may be between about 100 Da and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da. For this purpose, various methods including pegylation, polysialylation, HESylation, glycosylation, or recombinant PEG analogue fused to flexible and hydrophilic amino acid chain (500 to 600 amino acids) have been developed (See Chapman, (2002) Adv Drug Deliv Rev.54. 531-545; Schlapschy et al., (2007) Prot Eng Des Sel.20, 273-283; Contermann (2011) Curr Op Biotechnol.22, 868-876; Jevsevar et al., (2012) Methods Mol Biol.901, 233-246). Examples of polymers include but are not limited to polyalkyl ethers and alkoxy-capped analogs thereof (e.g., polyoxyethylene glycol, polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogs thereof, especially polyoxyethylene glycol, the latter is also known as polyethylene glycol or PEG); discrete PEG (dPEG); polyvinylpyrrolidones; polyvinylalkyl ethers; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyl oxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkyl acrylamides (e.g., polyhydroxypropylmethacrylamide and derivatives thereof); polyhydroxyalkyl acrylates; polysialic acids and analogs thereof; hydrophilic peptide sequences; polysaccharides and their derivatives, including dextran and dextran derivatives, e.g., carboxymethyldextran, dextran sulfates, aminodextran; cellulose and its derivatives, e.g., carboxymethyl cellulose, hydroxyalkyl celluloses; chitin and its derivatives, e.g., chitosan, succinyl chitosan, carboxymethylchitin, carboxymethylchitosan; hyaluronic acid and its derivatives; starches; alginates; chondroitin sulfate; albumin; pullulan and carboxymethyl pullulan; polyaminoacids and derivatives thereof, e.g., polyglutamic acids, polylysines, polyaspartic acids, polyaspartamides; maleic anhydride copolymers such as: styrene maleic anhydride copolymer, divinylethyl ether maleic anhydride copolymer; polyvinyl alcohols; copolymers thereof; terpolymers thereof; mixtures thereof; and derivatives of the foregoing. The polymer selected may be water soluble so that the AFFIMER® agent to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. The water-soluble polymer may be any structural form including but not limited to linear, forked or branched. Typically, the water-soluble polymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG), but other water-soluble polymers can also be employed. By way of example, PEG is used to describe some embodiments of this disclosure. For therapeutic use of the AFFIMER® agent, the polymer may be pharmaceutically acceptable. The term “PEG” is used broadly to encompass any polyethylene glycol molecule, without regard to size or to modification at an end of the PEG, and can be represented as linked to the AFFIMER® polypeptide by the formula: XO—(CH2CH2O)n—CH2CH2— or XO—(CH2CH2O)n— where n is 2 to 10,000 and X is H or a terminal modification, including but not limited to, a C1-4 alkyl, a protecting group, or a terminal functional group. In some cases, a PEG used in the polypeptides of the disclosure terminates on one end with hydroxy or methoxy, e.g., X is H or CH3 (“methoxy PEG”). It is noted that the other end of the PEG, which is shown in the above formulas by a terminal “—”, may attach to the AFFIMER® polypeptide via a naturally-occurring or non- naturally encoded amino acid. For instance, the attachment may be through an amide, carbamate or urea linkage to an amine group (including but not limited to, the epsilon amine of lysine or the N-terminus) of the polypeptide. Alternatively, the polymer is linked by a maleimide linkage to a thiol group (including but not limited to, the thiol group of cysteine) – which in the case of attachment to the AFFIMER® polypeptide sequence per se requires altering a residue in the AFFIMER® sequence to a cysteine. The number of water-soluble polymers linked to the AFFIMER® polypeptide (e.g., the extent of PEGylation or glycosylation) can be adjusted to provide an altered (including but not limited to, increased or decreased) pharmacologic, pharmacokinetic or pharmacodynamic characteristic such as in vivo half-life in the resulting AFFIMER® agent. In some embodiments, the half-life of the resulting AFFIMER® agent is increased at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 percent, 2-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 50-fold, or at least about 100-fold over an unmodified polypeptide. Another variation of polymer system useful to modify the PK or other biological properties of the resulting AFFIMER® agent are the use of unstructured, hydrophilic amino acid polymers that are functional analogs of PEG, particularly as part of a fusion protein with the AFFIMER® polypeptide sequence. The inherent biodegradability of the polypeptide platform makes it attractive as a potentially more benign alternative to PEG. Another advantage is the precise molecular structure of the recombinant molecule in contrast to the polydispersity of PEG. Unlike HSA and Fc peptide fusions, in which the three-dimensional folding of the fusion partner needs to be maintained, the recombinant fusions to unstructured partners can, in many cases, be subjected to higher temperatures or harsh conditions such as HPLC purification. One of the more advanced of this class of polypeptides is termed XTEN (Amunix) and is 864 amino acids long and comprised of six amino acids (A, E, G, P, S and T). See Schellenberger et al. “A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner” 2009 Nat Biotechnol.27(12):1186-90. Enabled by the biodegradable nature of the polymer, this is much larger than the 40 KDa PEGs typically used and confers a concomitantly greater half-life extension. The fusion of XTEN to the AFFIMER® polypeptide should result in halflife extension of the final AFFIMER® agent by 60- to 130-fold over the unmodified polypeptide. A second polymer based on similar conceptual considerations is PAS (XL-Protein GmbH). Schlapschy et al. “PASYlation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins” 2013 Protein Eng Des Sel.26(8):489-501. A random coil polymer comprised of an even more restricted set of only three small uncharged amino acids, proline, alanine and serine. AS with Fc, HSA and XTEN, the PAS modification can be genetically encoded with the AFFIMER® polypeptide sequence to produce an inline fusion protein when expressed. C. Conjugates The subject AFFIMER® agents may also include at least one functional moiety intended to impart detectability or additional pharmacologic activity to the AFFIMER® agent. Functional moieties for detection are those which can be employed to detect association of the AFFIMER® agent with a cell or tissue (such as a tumor cell) in vivo. Functional moieties with pharmacologic activity are those agents which are meant to be delivered to the tissue expressing the target of the AFFIMER® agent (PD-L1 and/or LAG-3 in the case of the LAG-3/PD-L1 AFFIMER® agents of the present disclosure) and in doing so have a pharmacologic consequence to the targeted tissues or cells. The present disclosure provides AFFIMER® agents including conjugates of substances having a wide variety of functional groups, substituents or moieties, with those Functional Moieties including but not limited to a label; a dye; an immunoadhesion molecule; a radionuclide; a cytotoxic compound; a drug; an affinity label; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide; a water-soluble dendrimer; a cyclodextrin; an inhibitory ribonucleic acid; a biomaterial; a nanoparticle; a spin label; a fluorophore, a metal-containing moiety; a radioactive moiety; a novel functional group; a group that covalently or noncovalently interacts with other molecules; a photocaged moiety; an actinic radiation excitable moiety; a photoisomerizable moiety; biotin; a derivative of biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar; a redox-active agent; an amino thioacid; a toxic moiety; an isotopically labeled moiety; a biophysical probe; a phosphorescent group; a chemiluminescent group; an electron dense group; a magnetic group; an intercalating group; a chromophore; an energy transfer agent; a biologically active agent; a detectable label; a small molecule; a quantum dot; a nanotransmitter; a radionucleotide; a radiotransmitter; a neutron- capture agent; or any combination of the above, or any other desirable compound or substance. 1. Labels and Detectable Moieties Where the moiety is a detectable label, it can be a fluorescent label, radioactive label, enzymatic label or any other label known to the skilled person. In some embodiments, the Functional Moiety is a detectable label that can be included as part of a conjugate to form certain AFFIMER® agents suitable for medical imaging. By "medical imaging" is meant any technique used to visualize an internal region of the human or animal body, for the purposes of diagnosis, research or therapeutic treatment. For instance, the AFFIMER® agent can be detected (and quantitated) by radioscintigraphy, magnetic resonance imaging (MRI), computed tomography (CT scan), nuclear imaging, positron emission comprising a metal tomography (PET) contrast agent, optical imaging (such as fluorescence imaging including near-infrared fluorescence (NIRF) imaging), bioluminescence imaging, or combinations thereof. The Functional Moiety is optionally a contrast agent for X-ray imaging. Agents useful in enhancing such techniques are those materials that enable visualization of a particular locus, organ or disease site within the body, and/or that lead to some improvement in the quality of the images generated by the imaging techniques, providing improved or easier interpretation of those images. Such agents are referred to herein as contrast agents, the use of which facilitates the differentiation of different parts of the image, by increasing the "contrast" between those different regions of the image. The term "contrast agents" thus encompasses agents that are used to enhance the quality of an image that may nonetheless be generated in the absence of such an agent (as is the case, for instance, in MRI), as well as agents that are prerequisites for the generation of an image (as is the case, for instance, in nuclear imaging). In some embodiments, the detectable label includes a chelate moiety for chelating a metal, e.g., a chelator for a radiometal or paramagnetic ion. In some embodiments, the detectable label is a chelator for a radionuclide useful for radiotherapy or imaging procedures. Radionuclides useful within the present disclosure include gamma-emitters, positron-emitters, Auger electron-emitters, X-ray emitters and fluorescence-emitters, with beta- or alpha-emitters for therapeutic use. Examples of radionuclides useful as toxins in radiation therapy include: 43K, 47Sc, 51Cr, 57Co, 58Co, 59Fe, 64Cu, 67Ga, 67Cu, 68Ga, 71Ge, 75Br, 76Br, 77Br, 77As, 81Rb, 90Y, 97Ru, 99mTc, 100Pd, 101Rh, 103Pb, 105Rh, 109Pd, 111Ag, 111In, 113In, 119Sb 121Sn, 123I, 125I, 127Cs, 128Ba, 129Cs, 131I, 131Cs, 143Pr, 153Sm, 161Tb, 166Ho, 169Eu, 177Lu, 186Re, 188Re, 189Re, 191Os, 193Pt, 194Ir, 197Hg, 199Au, 203Pb, 211At, 212Pb, 212Bi and 213Bi. Conditions under which a chelator will coordinate a metal are described, for example, by Gansow et al., U.S. Pat. NOS: 4,831,175, 4,454,106 and 4,472,509. Examples of chelators includes, merely to illustrate, 1,4,7- triazacyclononane-N,N',N"-triacetic acid (NOTA) 1,4,7,10-tetraazacyclododecane-N,N',N",N'"- tetraacetic acid (DOTA) 1 ,4,8,11-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid (TETA). Other detectable isotopes that can be incorporated directly into the amino acid residues of the AFFIMER® polypeptide or which otherwise do not require a chelator, include 3H, 14C, 32P, 35S and 36Cl. Paramagnetic ions, useful for diagnostic procedures, may also be administered. Examples of paramagnetic ions include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), erbium (III), or combinations of these paramagnetic ions. Examples of fluorescent labels include, but are not restricted to, organic dyes (e.g., cyanine, fluorescein, rhodamine, Alexa Fluors, Dylight fluors, ATTO Dyes, BODIPY Dyes, etc.), biological fluorophores (e.g., green fluorescent protein (GFP), R-Phycoerythrin, etc.), and quantum dots. Non-limiting fluorescent compound that may be used in the present disclosure include, Cy5, Cy5.5 (also known as Cy5++), Cy2, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin, Cy7, fluorescein (FAM), Cy3, Cy3.5 (also known as Cy3++), Texas Red, LightCycler-Red 640, LightCycler Red 705, tetramethylrhodamine (TMR), rhodamine, rhodamine derivative (ROX), hexachlorofluorescein (HEX), rhodamine 6G (R6G), the rhodamine derivative JA133, Alexa Fluorescent Dyes (such as Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 633, Alexa Fluor 555, and Alexa Fluor 647), 4′,6-diamidino-2-phenylindole (DAPI), Propidium iodide, AMCA, Spectrum Green, Spectrum Orange, Spectrum Aqua, Lissamine, and fluorescent transition metal complexes, such as europium. Fluorescent compound that can be used also include fluorescent proteins, such as GFP (green fluorescent protein), enhanced GFP (EGFP), blue fluorescent protein and derivatives (BFP, EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein and derivatives (CFP, ECFP, Cerulean, CyPet) and yellow fluorescent protein and derivatives (YFP, Citrine, Venus, YPet). WO2008142571, WO2009056282, WO9922026. Examples of enzymatic labels include, but are not restricted to, horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase and β-galactosidase. Another well-known label is biotin. Biotin labels are typically composed of the biotinyl group, a spacer arm and a reactive group that is responsible for attachment to target functional groups on proteins. Biotin can be useful for attaching the labelled protein to other moieties which comprise an avidin moiety. 2. AFFIMER® Polypeptide-Drug Conjugates In some embodiments, the AFFIMER® agent includes at least one therapeutic agent, e.g., to form an AFFIMER® polypeptide-drug conjugate. As used herein, the term “therapeutic agent” refers to a substance that may be used in the cure, mitigation, treatment, or prevention of disease in a human or another animal. Such therapeutic agents include substances recognized in the official United States Pharmacopeia, official Homeopathic Pharmacopeia of the United States, official National Formulary, or any supplement thereof, and include but are not limited to small molecules, nucleotides, oligopeptides, polypeptides, etc. Therapeutic agents that may be attached to AFFIMER® polypeptides include but are not limited to, cytotoxic agents, anti- metabolites, alkylating agents, antibiotics, growth factor, cytokines, anti-angiogenic agents, anti- mitotic agents, toxins, apoptotic agents or the like, such as DNA alkylating agents, topoisomerase inhibitors, microtubule inhibitors (e.g., DM1, DM4, MMAF and MMAE), endoplasmic reticulum stress inducing agents, platinum compounds, antimetabolites, vincalkaloids, taxanes, epothilones, enzyme inhibitors, receptor antagonists, therapeutic antibodies, tyrosine kinase inhibitors, radiosensitizers, and chemotherapeutic combination therapies, such as illustrations. Non-limiting examples of DNA alkylating agents are nitrogen mustards, such as Mechlorethamine, Cyclophosphamide (Ifosfamide, Trofosfamide), Chlorambucil (Melphalan, Prednimustine), Bendamustine, Uramustine and Estramustine; nitrosoureas, such as Carmustine (BCNU), Lomustine (Semustine), Fotemustine, Nimustine, Ranimustine and Streptozocin; alkyl sulfonates, such as Busulfan (Mannosulfan, Treosulfan); Aziridines, such as Carboquone, ThioTEPA, Triaziquone, Triethylenemelamine; Hydrazines (Procarbazine); Triazenes such as Dacarbazine and Temozolomide; Altretamine and Mitobronitol. Non-limiting examples of Topoisomerase I inhibitors include Campothecin derivatives including CPT-11 (irinotecan), SN-38, APC, NPC, campothecin, topotecan, exatecan mesylate, 9-nitrocamptothecin, 9-aminocamptothecin, lurtotecan, rubitecan, silatecan, gimatecan, diflomotecan, extatecan, BN-80927, DX-8951f, and MAG-CPT as described in Pommier Y. (2006) Nat. Rev. Cancer 6(10):789-802 and U.S. Patent Publication No.200510250854; Protoberberine alkaloids and derivatives thereof including berberrubine and coralyne as described in Li et al. (2000) Biochemistry 39(24):7107-7116 and Gatto et al. (1996) Cancer Res. 15(12):2795-2800; Phenanthroline derivatives including Benzo[i]phenanthridine, Nitidine, and fagaronine as described in Makhey et al. (2003) Bioorg. Med. Chem.11 (8): 1809-1820; Terbenzimidazole and derivatives thereof as described in Xu (1998) Biochemistry 37(10):3558- 3566; and Anthracycline derivatives including Doxorubicin, Daunorubicin, and Mitoxantrone as described in Foglesong et al. (1992) Cancer Chemother. Pharmacol.30(2):123-]25, Crow et al. (1994) J. Med. Chem.37(19):31913194, and Crespi et al. (1986) Biochem. Biophys. Res. Commun.136(2):521-8. Topoisomerase II inhibitors include but are not limited to Etoposide and Teniposide. Dual topoisomerase I and II inhibitors include but are not limited to, Saintopin and other Naphthecenediones, DACA and other Acridine-4-Carboxamindes, Intoplicine and other Benzopyridoindoles, TAS-103 and other 7H-indeno[2,1-c]Quinoline-7-ones, Pyrazoloacridine, XR 11576 and other Benzophenazines, XR 5944 and other Dimeric compounds, 7-oxo-7H- dibenz[f,ij]Isoquinolines and 7-oxo-7H-benzo[e]Perimidines, and Anthracenyl-amino Acid Conjugates as described in Denny and Baguley (2003) Curr. Top. Med. Chem.3(3):339-353. Some agents inhibit Topoisomerase II and have DNA intercalation activity such as, but not limited to, Anthracyclines (Aclarubicin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Valrubicin, Zorubicin) and Antracenediones (Mitoxantrone and Pixantrone). Non-limiting examples of DNA synthesis inhibitors include Calicheamicin, Doxorubicin, Duocarmycin, and PBD. Non-limiting examples of microtubule inhibitors include DM1, DM4, MMAF, and MMAE. Examples of endoplasmic reticulum stress inducing agents include but are not limited to, dimethyl-celecoxib (DMC), nelfinavir, celecoxib, and boron radiosensitizers (e.g., velcade (Bortezomib)). Non-limiting examples of platinum-based compound include Carboplatin, Cisplatin, Nedaplatin, Oxaliplatin, Triplatin tetranitrate, Satraplatin, Aroplatin, Lobaplatin, and JM-216. (see McKeage et al. (1997) J. Clin. Oncol.201:1232-1237 and in general, CHEMOTHERAPY FOR GYNECOLOGICAL NEOPLASM, CURRENT THERAPY AND NOVEL APPROACHES, in the Series Basic and Clinical Oncology, Angioli et al. Eds., 2004). Non-limiting examples of antimetabolite agents include Folic acid based, e.g. dihydrofolate reductase inhibitors, such as Aminopterin, Methotrexate and Pemetrexed; thymidylate synthase inhibitors, such as Raltitrexed, Pemetrexed; Purine based, e.g. an adenosine deaminase inhibitor, such as Pentostatin, a thiopurine, such as Thioguanine and Mercaptopurine, a halogenated/ribonucleotide reductase inhibitor, such as Cladribine, Clofarabine, Fludarabine, or a guanine/guanosine: thiopurine, such as Thioguanine; or Pyrimidine based, e.g. cytosine/cytidine: hypomethylating agent, such as Azacitidine and Decitabine, a DNA polymerase inhibitor, such as Cytarabine, a ribonucleotide reductase inhibitor, such as Gemcitabine, or a thymine/thymidine: thymidylate synthase inhibitor, such as a Fluorouracil (5- FU). Equivalents to 5-FU include prodrugs, analogs and derivative thereof such as 5′-deoxy-5- fluorouridine (doxifluoroidine), 1-tetrahydrofuranyl-5-fluorouracil (ftorafur), Capecitabine (Xeloda), S-I (MBMS-247616, consisting of tegafur and two modulators, a 5-chloro-2,4- dihydroxypyridine and potassium oxonate), ralititrexed (tomudex), no latrexed (Thymitaq, AG337), LY231514 and ZD9331, as described for example in Papamicheal (1999) The Oncologist 4:478-487. Examples of vincalkaloids, include but are not limited to Vinblastine, Vincristine, Vinflunine, Vindesine and Vinorelbine. Examples of taxanes include but are not limited to docetaxel, Larotaxel, Ortataxel, Paclitaxel and Tesetaxel. An example of an epothilone is iabepilone. Examples of enzyme inhibitors include but are not limited to farnesyltransferase inhibitors (Tipifamib); CDK inhibitor (Alvocidib, Seliciclib); proteasome inhibitor (Bortezomib); phosphodiesterase inhibitor (Anagrelide; rolipram); IMP dehydrogenase inhibitor (Tiazofurine); and lipoxygenase inhibitor (Masoprocol). Examples of receptor antagonists include but are not limited to ERA (Atrasentan); retinoid X receptor (Bexarotene); and a sex steroid (Testolactone). Examples of therapeutic antibodies include but are not limited to anti-HER1/EGFR (Cetuximab, Panitumumab); Anti-HER2/neu (erbB2) receptor (Trastuzumab); Anti-EpCAM (Catumaxomab, Edrecolomab) Anti-VEGF-A (Bevacizumab); Anti-CD20 (Rituximab, Tositumomab, Ibritumomab); Anti-CD52 (Alemtuzumab); and Anti-CD33 (Gemtuzumab). U.S. Pat. NOS: 5,776,427 and 7,601,355. Examples of tyrosine kinase inhibitors include but are not limited to inhibitors to ErbB: HER1/EGFR (Erlotinib, Gefitinib, Lapatinib, Vandetanib, Sunitinib, Neratinib); HER2/neu (Lapatinib, Neratinib); RTK class III: C-kit (Axitinib, Sunitinib, Sorafenib), FLT3 (Lestaurtinib), PDGFR (Axitinib, Sunitinib, Sorafenib); and VEGFR (Vandetanib, Semaxanib, Cediranib, Axitinib, Sorafenib); bcr-abl (Imatinib, Nilotinib, Dasatinib); Src (Bosutinib) and Janus kinase 2 (Lestaurtinib). Chemotherapeutic agents that can be attached to the present AFFIMER® polypeptides may also include amsacrine, Trabectedin, retinoids (Alitretinoin, Tretinoin), Arsenic trioxide, asparagine depleter Asparaginase/Pegaspargase), Celecoxib, Demecolcine, Elesclomol, Elsamitrucin, Etoglucid, Lonidamine, Lucanthone, Mitoguazone, Mitotane, Oblimersen, Temsirolimus, and Vorinostat. Examples of specific therapeutic agents that can be linked, ligated, or associated with the AFFIMER® polypeptides of the disclosure are flomoxef; fortimicin(s); gentamicin(s); glucosulfone solasulfone; gramicidin S; gramicidin(s); grepafloxacin; guamecycline; hetacillin; isepamicin; josamycin; kanamycin(s); flomoxef; fortimicin(s); gentamicin(s); glucosulfone solasulfone; gramicidin S; gramicidin(s); grepafloxacin; guamecycline; hetacillin; isepamicin; josamycin; kanamycin(s); bacitracin; bambermycin(s); biapenem; brodimoprim; butirosin; capreomycin; carbenicillin; carbomycin; carumonam; cefadroxil; cefamandole; cefatrizine; cefbuperazone; cefclidin; cefdinir; cefditoren; cefepime; cefetamet; cefixime; cefinenoxime; cefininox; cladribine; apalcillin; apicycline; apramycin; arbekacin; aspoxicillin; azidamfenicol; aztreonam; cefodizime; cefonicid; cefoperazone; ceforamide; cefotaxime; cefotetan; cefotiam; cefozopran; cefpimizole; cefpiramide; cefpirome; cefprozil; cefroxadine; cefteram; ceftibuten; cefuzonam; cephalexin; cephaloglycin; cephalosporin C; cephradine; chloramphenicol; chlortetracycline; clinafloxacin; clindamycin; clomocycline; colistin; cyclacillin; dapsone; demeclocycline; diathymosulfone; dibekacin; dihydrostreptomycin; 6-mercaptopurine; thioguanine; capecitabine; docetaxel; etoposide; gemcitabine; topotecan; vinorelbine; vincristine; vinblastine; teniposide; melphalan; methotrexate; 2-p-sulfanilyanilinoethanol; 4,4′- sulfinyldianiline; 4-sulfanilamidosalicylic acid; butorphanol; nalbuphine. streptozocin; doxorubicin; daunorubicin; plicamycin; idarubicin; mitomycin C; pentostatin; mitoxantrone; cytarabine; fludarabine phosphate; butorphanol; nalbuphine. streptozocin; doxorubicin; daunorubicin; plicamycin; idarubicin; mitomycin C; pentostatin; mitoxantrone; cytarabine; fludarabine phosphate; acediasulfone; acetosulfone; amikacin; amphotericin B; ampicillin; atorvastatin; enalapril; ranitidine; ciprofloxacin; pravastatin; clarithromycin; cyclosporin; famotidine; leuprolide; acyclovir; paclitaxel; azithromycin; lamivudine; budesonide; albuterol; indinavir; metformin; alendronate; nizatidine; zidovudine; carboplatin; metoprolol; amoxicillin; diclofenac; lisinopril; ceftriaxone; captopril; salmeterol; xinafoate; imipenem; cilastatin; benazepril; cefaclor; ceftazidime; morphine; dopamine; bialamicol; fluvastatin; phenamidine; podophyllinic acid 2-ethylhydrazine; acriflavine; chloroazodin; arsphenamine; amicarbilide; aminoquinuride; quinapril; oxymorphone; buprenorphine; floxuridine; dirithromycin; doxycycline; enoxacin; enviomycin; epicillin; erythromycin; leucomycin(s); lincomycin; lomefloxacin; lucensomycin; lymecycline; meclocycline; meropenem; methacycline; micronomicin; midecamycin(s); minocycline; moxalactam; mupirocin; nadifloxacin; natamycin; neomycin; netilmicin; norfloxacin; oleandomycin; oxytetracycline; p-sulfanilylbenzylamine; panipenem; paromomycin; pazufloxacin; penicillin N; pipacycline; pipemidic acid; polymyxin; primycin; quinacillin; ribostamycin; rifamide; rifampin; rifamycin SV; rifapentine; rifaximin; ristocetin; ritipenem; rokitamycin; rolitetracycline; rosaramycin; roxithromycin; salazosulfadimidine; sancycline; sisomicin; sparfloxacin; spectinomycin; spiramycin; streptomycin; succisulfone; sulfachrysoidine; sulfaloxic acid; sulfamidochrysoidine; sulfanilic acid; sulfoxone; teicoplanin; temafloxacin; temocillin; tetroxoprim; thiamphenicol; thiazolsulfone; thiostrepton; ticarcillin; tigemonam; tobramycin; tosufloxacin; trimethoprim; trospectomycin; trovafloxacin; tuberactinomycin; vancomycin; azaserine; candicidin(s); chlorphenesin; dermostatin(s); filipin; fungichromin; mepartricin; nystatin; oligomycin(s); perimycin A; tubercidin; 6-azauridine; 6-diazo-5-oxo-L-norleucine; aclacinomycin(s); ancitabine; anthramycin; azacitadine; azaserine; bleomycin(s); ethyl biscoumacetate; ethylidene dicoumarol; iloprost; lamifiban; taprostene; tioclomarol; tirofiban; amiprilose; bucillamine; gusperimus; gentisic acid; glucamethacin; glycol salicylate; meclofenamic acid; mefenamic acid; mesalamine; niflumic acid; olsalazine; oxaceprol; S-enosylmethionine; salicylic acid; salsalate; sulfasalazine; tolfenamic acid; carubicin; carzinophillin A; chlorozotocin; chromomycin(s); denopterin; doxifluridine; edatrexate; eflornithine; elliptinium; enocitabine; epirubicin; mannomustine; menogaril; mitobronitol; mitolactol; mopidamol; mycophenolic acid; nogalamycin; olivomycin(s); peplomycin; pirarubicin; piritrexim; prednimustine; procarbazine; pteropterin; puromycin; ranimustine; streptonigrin; thiamiprine; mycophenolic acid; procodazole; romurtide; sirolimus (rapamycin); tacrolimus; butethamine; fenalcomine; hydroxytetracaine; naepaine; orthocaine; piridocaine; salicyl alcohol; 3-amino-4-hydroxybutyric acid; aceclofenac; alminoprofen; amfenac; bromfenac; bromosaligenin; bumadizon; carprofen; diclofenac; diflunisal; ditazol; enfenamic acid; etodolac; etofenamate; fendosal; fepradinol; flufenamic acid; Tomudex (N-[[5-[[(1,4-Dihydro-2-methyl-4-oxo-6- quinazolinyl)methyl]methylamino]-2-thienyl]carbonyl]-L-glutamic acid), trimetrexate, tubercidin, ubenimex, vindesine, zorubicin; argatroban; coumetarol or dicoumarol. In some embodiments, the AFFIMER® agent includes a conjugated cytotoxic factor such as diptheria toxin, Pseudomonas aeruginosa exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins and compounds (e.g., fatty acids), dianthin proteins, Phytoiacca americana proteins PAPI, PAPII, and PAP-S, momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, mitogellin, restrictocin, phenomycin, and enomycin. Any method known in the art for conjugating to antibodies and other proteins may be employed in generating the conjugates of the present disclosure, including those methods described by Hunter, et al., (1962) Nature 144:945; David, et al., (1974) Biochemistry 13:1014; Pain, et al., (1981) J. Immunol. Meth.40:219; and Nygren, J., (1982) Histochem. and Cytochem. 30:407. Methods for conjugating peptide, polypeptide and organic and inorganic moieties to antibodies and other proteins are conventional and very well known in the art and readily adapted for generating those versions of the subject AFFIMER® agents. Where the conjugated moiety is a peptide or polypeptide, that moiety can be chemically cross-linked to the AFFIMER® polypeptide or can be included as part of a fusion protein with the AFFIMER® polypeptide. And illustrative example would be a diphtheria toxin-AFFIMER® fusion protein. In the case of non-peptide entities, the addition to the AFFIMER® polypeptide will generally be by way of chemical conjugation to the AFFIMER® polypeptide – such as through a functional group on an amino acid side chain or the carboxyl group at the C-terminal or amino group at the N-terminal end of the polypeptide. In some embodiment, whether as a fusion protein or chemically cross-linked moiety, the conjugated moiety will include at least one site that can be cleaved by an enzyme or are otherwise sensitive to an environmental condition (such as pH) that permits the conjugated moiety to be released from the AFFIMER® polypeptide, such as in the tumor or other diseased tissue (or tissue to be protected if the conjugated moiety functions to protect healthy tissue). a) Enzyme-cleavable Linkers An AFFIMER® polypeptide-drug conjugate, in some embodiments, comprises an enzyme-cleavable linker, which links the half-life extension moiety to a drug moiety. The linker (e.g., the substrate recognition sequence (SRS) of the linker) is selectively cleaved in the vicinity of the target cells so that the free drug moiety is released from the conjugate in the vicinity of the target cells so as to exert its pharmacological activities preferentially on the cells/tissue nearby to the target cells, rather than on wanted (healthy) cells. Thus, in some embodiments, the SRS is selectively cleaved such that the drug moiety is released as the free drug moiety in the vicinity of the target cells at least five times or ten times more than the extent to which the free drug moiety it is released in the vicinity of healthy cells/tissues, and in some embodiments, at least 100 or 500 or 1000 times more. For a given target cell, the skilled person will be able to identify appropriate SRS that is selectively cleavable in the vicinity of the target cell, using established methods in the art. For example, which proteases cleave which peptides can be assessed by consulting peptide libraries and studying an MS analysis of the fragmentation profile following cleavage. Also, published literature of protease cleavage motifs and peptide cleavage data can be searched as described further below. In some aspects, the SRS is a protease cleavage site. Thus, when the target cells are tumor cells, the SRS may be cleavable selectively by proteases that reside in the vicinity of the tumor cells. Thus, the SRS may be one that is cleavable by a tumor associated protease. It is well known that during tumor development, tumors aberrantly express proteases which allow them to invade local tissues and eventually metastasize. For example, the protease may be present extracellularly in the diseased state tissue in a subject at levels at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times greater than the healthy state of the tissue in the subject. As another example, the protease may be present extracellularly in the diseased state of the tissue in a subject at levels at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times greater than other tissue of the subject. In some embodiments, the protease is a serine protease, metal protease, or cysteine protease. The protease may be a metalloproteinase (MMP1-28) including both membrane-bound (MMP14-17 and MMP24-25) and secreted forms (MMP1-13 and MMP 18-23 and MMP26- 28). The protease may belong to the A Disintegrin and Metalloproteinase (ADAM) and A Disintegrin, or Metalloproteinase with Thrombospondin Motifs (ADAMTS) families of proteases. Other examples include CD 10 (CALLA) and prostate specific antigen (PSA). It is appreciated that the proteases may or may not be membrane bound. Protease cleavage sites are well known in the scientific literature, and can readily serve as the basis for a given SRS being included in the drug-conjugate moieties using established synthetic techniques known in the art. To the extent representing a protease whose extracellular concentration is upregulated/increased in the target tissue by changes in expression, cellular trafficking or, in the case of intracellular enzymes that may become extracellular, by cell lysis caused by the disease state, SRS may utilized which are designed to be selectively cleavable by one or a select sub- group of human proteases selected from the group consisting of (MEROPS peptidase database number provided in parentheses; Rawlings N. D., Morton F. R., Kok, C. Y., Kong, J. & Barrett A. J. (2008) MEROPS: the peptidase database. Nucleic Acids Res.36 Database issue, D320- 325). In some embodiments, the SRS is a peptide moiety of up to 15 amino acids in length. In some embodiments, the SRS is cleaved by a protease co-localized with the target of the cell binding moiety in a tissue, and the protease cleaves the SRS in the AFFIMER® polypeptide- drug conjugate when the AFFIMER® polypeptide-drug conjugate is exposed to the protease. In some embodiments, the protease is not active or is significantly less active in tissues that do not significantly express the cell surface feature. In some embodiments, the protease is not active or is significantly less active in healthy, e.g., non-diseased tissues. In some embodiments, the SRS is cleaved by a protease selected from the following: • ADAMS or ADAMTS, e g. ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM 17/T ACE, ADAMDEC1, ADAMTS 1, ADAMTS4 or ADAMTS5; • Aspartate proteases, e.g., BACE or Renin; • Aspartic cathepsins (to the extent upregulated or released by cell lysis in the extracellular space), e.g., Cathepsin D or Cathepsin E; • Caspases (to the extent upregulated or released by cell lysis in the extracellular space), e.g., Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10 or Caspase 14; • Cysteine cathepsins, e.g., Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V/L2, Cathepsin X/Z/P; • Cysteine proteinases, e.g., Cruzipain, Legumain or Otubain-2; • KLKs, e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 or KLK14; • Metallo-proteinases, e.g., Meprin, Neprilysin, PSMA or BMP-l; • MMPs, e.g., MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMPlO, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27; • Serine proteases, e.g., activated protein C, Cathepsin A, Cathepsin G, Chymase, coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa, FXIIa), Elastase, Granzyme B, Guanidinobenzoatase, HtrAl, Human Neutrophil Elastase, Lactoferrin, Marapsin, NS3/4A, PACE4, Plasmin, PSA, tPA, Thrombin, Tryptase or uPA; and/or • Type II Transmembrane Serine Proteases (TTSPs), e.g., DESC1, DPP -4, Hepsin, Matriptase-2, MT-SPl/Matriptase, DMPRSS2, DMPRSS3, DMPRSS4. For example, suitable SRSs that can be included binder-drug conjugate, i.e., SRS is peptide moiety selected from the group consisting of: TGRGPSWV, SARGPSRW, TARGPSFK, LSGRSDNH, GGWHTGRN, HTGRSGAL, PLTGRSGG, AARGPAIH, RGPAFNPM, SSRGPAYL, RGPATPIM, RGPA, GGQPSGMWGW, FPRPLGITGL, VHMPLGFLGP, SPLTGRSG, SAGFSLPA, LAPLGLQRR, SGGPLGVR, PLGL, GPRSFGL, and GPRSFG. In some embodiments, the SRS is a substrate for an MMP, such as a sequence selected from the group consisting of ISSGLLSS, QNQALRMA, AQNLLGMV, STFPFGMF, PVGYTSSL, DWLYWPGI, MIAPVAYR, RPSPMWAY, WATPRPMR, FRLLDWQW, LKAAPRWA, GPSHLVLT, LPGGLSPW, MGLFSEAG, SPLPLRVP, RMHLRSLG, LAAPLGLL, AVGLLAPP, LLAPSHRA, PAGLWLDP, and ISSGLSS. In some embodiments, the SRS is a substrate for an MMP, such as a sequence selected from the group consisting of ISSGLSS, QNQALRMA, AQNLLGMV, STFPFGMF, PVGYTSSL, DWLYWPGI, ISSGLLSS, LKAAPRWA, GPSHLVLT, LPGGLSPW, MGLFSEAG, SPLPLRVP, RMHLRSLG, LAAPLGLL, AVGLLAPP, LLAPSHRA, and PAGLWLDP. In some embodiments, the SRS is a substrate for thrombin, such as GPRSFGL or GPRSFG. b) Spacers In some embodiments, an AFFIMER® polypeptide-drug conjugate comprises a spacer or bond (L1) between the half-life extension moiety and the substrate recognition sequence (SRS) cleavable by the enzyme, e.g., present in a tumor microenvironment. The spacer may be any molecule, for example, one or more nucleotides, amino acids, chemical functional groups. In some embodiments, the spacer is a peptide linker (e.g., two or more amino acids). Spacers should not adversely affect the expression, secretion, or bioactivity of the polypeptides. In some embodiments, spacers are not antigenic and do not elicit an immune response. An immune response includes a response from the innate immune system and/or the adaptive immune system. Thus, an immune response may be a cell-mediate response and/or a humoral immune response. The immune response may be, for example, a T cell response, a B cell response, a natural killer (NK) cell response, a monocyte response, and/or a macrophage response. Other cell responses are contemplated herein. In some embodiments, linkers are non- protein-coding. In some embodiments, L1 is a hydrocarbon (straight chain or cyclic) such as 6- maleimidocaproyl, maleimidopropanoyl and maleimidom ethyl cyclohexane- l-carboxylate, or L1 is N-Succinimidyl 4-(2-pyridylthio) pentanoate, N- Succinimidyl 4-(N- maleimidomethyl) cyclohexane-1 carboxylate, N-Succinimidyl (4-iodo-acetyl) aminobenzoate. In some embodiments, L1 is a polyether such as a poly(ethylene glycol) or other hydrophilic linker. For instance, where the CBM includes a thiol (such as a cysteine residue), L1 can be a polyethylene glycol) coupled to the thiol group through a maleimide moiety. Non-limiting examples of linkers for use in accordance with the present disclosure are described in International Publication No. WO 2019/236567, published December 12, 2019, incorporated by reference herein. c) Self-Immolative Linkers In some embodiments, an AFFIMER® polypeptide-drug conjugate comprises a self- immolative linker (L2) between the substrate recognition sequence (SRS) for the enzyme and the drug moiety, such as represented in the formula
Figure imgf000110_0001
wherein, p represents an integer from 1 to 100, preferably 6 to 50, more preferably 6 to 12. In other embodiments, where the CBM includes a thiol and L1 is a hydrocarbon moiety coupled to the thiol group through a maleimide moiety, L1 can be represented in the formula
Figure imgf000110_0002
wherein, p represents an integer from 1 to 20, preferably 1 to 4. A self-immolative moiety may be defined as a bifunctional chemical group that is capable of covalently linking together two spaced chemical moieties into a normally stable molecule, releasing one of the spaced chemical moieties from the molecule by means of enzymatic cleavage; and following enzymatic cleavage, spontaneously cleaving from the remainder of the bifunctional chemical group to release the other of said spaced chemical moieties. Therefore, in some embodiments, the self- immolative moiety is covalently linked at one of its ends, directly or indirectly through a spacer unit, to the ligand by an amide bond and covalently linked at its other end to a chemical reactive site (functional group) pending from the drug moiety. The derivatization of the drug moiety with the self-immolative moiety may render the drug less pharmacologically active (e.g. less toxic) or not active at all until the drug is cleaved. An AFFIMER® polypeptide-drug conjugate is generally stable in circulation, or at least that should be the case in the absence of an enzyme capable of cleaving the amide bond between the substrate recognition sequence (enzyme-cleavable linker) and the self-immolative moiety. Upon exposure of an AFFIMER® polypeptide-drug conjugate to a suitable enzyme, the amide bond is cleaved initiating a spontaneous self-immolative reaction resulting in the cleavage of the bond covalently linking the self-immolative moiety to the drug moiety, to thereby effect release of the free drug moiety in its underivatized or pharmacologically active form. The self- immolative moiety in conjugates either incorporate one or more heteroatoms and thereby provides improved solubility, improves the rate of cleavage and decreases propensity for aggregation of the conjugate. In some embodiments, L2 is a benzyl oxy carbonyl group. In other embodiments, the self- immolative linker L2 is— NH— (CH2)4 -C(=O)- or — NH-(CH2)3-C(=O)-. In yet other embodiments, the self-immolative linker L2 is p-aminobenzyloxycarbonyl (PABC). In still other embodiments, the self-immolative linker L2 is 2,4-bis(hydroxymeihyl)aniline. The AFFIMER® polypeptide-drug conjugate of the present disclosure can employ a heterocyclic self-immolative moiety covalently linked to the therapeutic moiety and the cleavable substrate recognition sequence. A self-immolative moiety may be defined as a bifunctional chemical group which is capable of covalently linking together two spaced chemical moieties into a normally stable molecule, releasing one of said spaced chemical moieties from the molecule by means of enzymatic cleavage; and following said enzymatic cleavage, spontaneously cleaving from the remainder of the bifunctional chemical group to release the other of said spaced chemical moieties. In accordance with the present disclosure, the self- immolative moiety may be covalently linked at one of its ends, directly or indirectly through a spacer unit, to the ligand by an amide bond and covalently linked at its other end to a chemical reactive site (functional group) pending from the drug. The derivatization of the therapeutic moiety with the self-immolative moiety may render the drug less pharmacologically active (e.g. less toxic) or not active at all until the drug is cleaved. The AFFIMER® polypeptide-drug conjugate is generally stable in circulation, or at least that should be the case in the absence of an enzyme capable of cleaving the amide bond between the substrate recognition sequence and the self-immolative moiety. However, upon exposure of the AFFIMER® polypeptide-drug conjugate to a suitable enzyme, the amide bond is cleaved initiating a spontaneous self-immolative reaction resulting in the cleavage of the bond covalently linking the self-immolative moiety to the drug, to thereby effect release of the free therapeutic moiety in its underivatized or pharmacologically active form. The self-immolative moiety in conjugates of the present disclosure, in some embodiments, either incorporate one or more heteroatoms and thereby provides improved solubility, improves the rate of cleavage and decreases propensity for aggregation of the conjugate. These improvements of the heterocyclic self-immolative linker constructs of the present disclosure over non-heterocyclic, PAB-type linkers may result in surprising and unexpected biological properties such as increased efficacy, decreased toxicity, and more desirable pharmacokinetics. Other examples of self-immolative linkers that are readily adapted for use in AFFIMER® polypeptide-drug conjugates described herein are taught in, for example, US Patent 7,754,681; WO 2012/074693A1; US 9,089,614; EP 1,732,607; WO 2015/038426A1 (all of which are incorporated by reference); Walther et al. “Prodrugs in medicinal chemistry and enzyme prodrug therapies” Adv Drug Deliv Rev.2017 Sep 1; 118:65-77; and Tranoy-Opalinski et al.“Design of self- immolative linkers for tumor-activated prodrug therapy”, Anticancer Agents Med Chem. 2008 Aug;8(6):6l8-37; the teachings of each of which are incorporated by reference herein. Yet other non-limiting examples of self-immolative linkers for use in accordance with the present disclosure are described in International Publication No. WO 2019/236567, published December 12, 2019, incorporated by reference herein. The following list provides non-limiting examples of various bispecific and trispecific fusion protein designs, wherein the individual polypeptides, Fc regions, and linker components are arranged/encoded, 5’ to 3’ as shown. The linker may be any rigid or flexible linker, for example, comprising the amino acid sequence of SEQ ID NO: 86 or 87. AVA04-269 (SEQ ID NO: 73) – linker – AVA19-157 (SEQ ID NO: 61) AVA04-269 (SEQ ID NO: 73) – linker – AVA19-158 (SEQ ID NO: 62) AVA04-269 (SEQ ID NO: 73) – linker – AVA19-01 (SEQ ID NO: 53) AVA04-251 (SEQ ID NO: 72) – linker – AVA19-01 (SEQ ID NO: 53) AVA04-251 BH dimer (SEQ ID NO: 74) – linker – AVA19-01 (SEQ ID NO: 53) AVA04-640 (SEQ ID NO: 75) – linker – AVA19-06 (SEQ ID NO: 54) – linker – AVA19-06 (SEQ ID NO: 54) – linker – AVA03-42 (SEQ ID NO: 80) AVA04-251 (SEQ ID NO: 72) – linker – AVA04-251 (SEQ ID NO: 72) – linker – AVA03-42 (SEQ ID NO: 80) – linker – AVA19-158 (SEQ ID NO: 62) – linker – AVA19-158 (SEQ ID NO: 62) AVA04-251 (SEQ ID NO: 72) – linker – AVA04-251 (SEQ ID NO: 72) – linker – AVA19-06 (SEQ ID NO: 54) – linker – AVA19-06 (SEQ ID NO: 54 – linker – AVA03-42 (SEQ ID NO: 80) AVA04-251 (SEQ ID NO: 72) – hIgG1 Fc – linker – AVA19-06 (SEQ ID NO: 54) AVA04-251 (SEQ ID NO: 72) – hIgG1 Fc – linker – AVA19-158 (SEQ ID NO: 62) AVA19-06 (SEQ ID NO: 54) – linker – AVA04-251 (SEQ ID NO: 72) – linker – hIgG1 LALA Fc AVA04-251 (SEQ ID NO: 72) – linker – AVA19-06 (SEQ ID NO: 54) – linker – hIgG1 LALA Fc AVA04-251 (SEQ ID NO: 72) – linker – hIhG1 LALA Fc – linker – AVA19-06 (SEQ ID NO: 54) AVA04-251 (SEQ ID NO: 72) – linker – hIhG1 LALA Fc – linker – AVA19-170 (SEQ ID NO: 66) AVA04-251 (SEQ ID NO: 72) – linker – hIhG1 LALA Fc – linker – AVA19-173 (SEQ ID NO: 69) AVA04-251 (SEQ ID NO: 72) – hIgG4 Fc – linker – AVA19-06 (SEQ ID NO: 54) AVA04-251 (SEQ ID NO: 72) – hIgG4 Fc – linker – AVA19-170 (SEQ ID NO: 66) AVA04-251 (SEQ ID NO: 72) – hIgG4 Fc – linker – AVA19-173 (SEQ ID NO: 69) IV. Encoded AFFIMER® Construct for In vivo Delivery An alternative approach to the delivery of therapeutic AFFIMER® agents, such as an LAG-3/PD-L1 AFFIMER® agent, would be to leave the production of the therapeutic polypeptide to the body itself. A multitude of clinical studies have illustrated the utility of in vivo gene transfer into cells using a variety of different delivery systems. In vivo gene transfer seeks to administer to patients the encoded AFFIMER® construct, rather than the AFFIMER® agent. This allows the patient’s body to produce the therapeutic AFFIMER® agent of interest for a prolonged period of time, and secrete it either systemically or locally, depending on the production site. Gene-based encoded AFFIMER® construct can present a labor- and cost- effective alternative to the conventional production, purification and administration of the polypeptide version of the AFFIMER® agent. A number of antibody expression platforms have been pursued in vivo to which delivery of encoded AFFIMER® construct can be adapted: these include viral vectors, naked DNA and RNA. encoded AFFIMER® construct gene transfer can not only enable cost-savings by reducing the cost of goods and of production but may also be able to reduce the frequency of drug administration. Overall, a prolonged in vivo production of the therapeutic AFFIMER® agent by expression of the encoded AFFIMER® construct can contribute to (i) a broader therapeutic or prophylactic application of AFFIMER® agents in price- sensitive conditions, (ii) an improved accessibility to therapy in both developed and developing countries, and (iii) more effective and affordable treatment modalities. In addition to in vivo gene transfer, cells can be harvested from the host (or a donor), engineered with encoded AFFIMER® construct sequences to produce AFFIMER® agents and re-administered to patients. Intramuscular antibody gene administration has been most widely evaluated (reviewed in Deal et al. (2015) “Engineering humoral immunity as prophylaxis or therapy” Curr Opin Immunol.35:113–22.), and also carries the highest clinical translatability and application when applied to encoded AFFIMER® construct. Indeed, the inherent anatomical, cellular and physiological properties of skeletal muscle make it a stable environment for long-term encoded AFFIMER® construct expression and systemic circulation. Skeletal muscle is easily accessible, allowing multiple or repeated administrations. The abundant blood vascular supply provides an efficient transport system for secreted therapeutic AFFIMER® agents into the circulation. The syncytial nature of muscle fibers allows dispersal of nucleotides from a limited site of penetration to a large number of neighboring nuclei within the fiber. Skeletal muscle fibers are also terminally differentiated cells, and nuclei within the fibers are post-mitotic. Consequently, integration in the host genome is not a prerequisite to attain prolonged monoclonal antibody (mAb) expression. The liver is another site often used for pre-clinical antibody gene transfer and is typically transfected via intravenous (i.v.) injection and can also be a site of gene transfer for encoded AFFIMER® construct either for local delivery of AFFIMER® agents (such as in the treatment of liver cancer and/or metaplasias) or for the generation of AFFIMER® agents that are secreted into the vascular for systemic circulation. This organ has various physiological functions, including the synthesis of plasma proteins. This organ can be particularly well suited for in vivo encoded AFFIMER® construct expression. The tumor presents another site for encoded AFFIMER® construct transfer, targeted either via intravenous or direct injection/electroporation. Indeed, intratumoral encoded AFFIMER® construct expression can allow for a local production of the therapeutic AFFIMER® agents, waiving the need for high systemic AFFIMER® agent levels that might otherwise be required to penetrate and impact solid tumors. A similar rationale applies for the brain, which is frequently targeted in the context of antibody gene transfer to avoid the difficulties with blood–brain barrier trafficking and would likewise be a target for delivery of encoded AFFIMER® construct. See, for example, Beckman et al. (2015) “Antibody constructs in cancer therapy: protein engineering strategies to improve exposure in solid tumors” Cancer 109(2):170–9; Dronca et al. (2015) “Immunomodulatory antibody therapy of cancer: the closer, the better” Clin Cancer Res.21(5):944–6; and Neves et al. (2016) “Antibody approaches to treat brain diseases” Trends Biotechnol.34(1):36–48. The success of gene therapy has largely been driven by improvements in nonviral and viral gene transfer vectors. An array of physical and chemical nonviral methods have been used to transfer DNA and mRNA to mammalian cells and a substantial number of these have been developed as clinical stage technologies for gene therapy, both ex vivo and in vivo, and are readily adapted for delivery of the encoded AFFIMER® construct of the present disclosure. To illustrate, cationic liposome technology can be employed, which is based on the ability of amphipathic lipids, possessing a positively charged head group and a hydrophobic lipid tail, to bind to negatively charged DNA or RNA and form particles that generally enter cells by endocytosis. Some cationic liposomes also contain a neutral co-lipid, thought to enhance liposome uptake by mammalian cells. See, for example, Felgner et al. (1987) Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. MNAS 84:7413–7417; San et al. (1983) “Safety and short-term toxicity of a novel cationic lipid formulation for human gene therapy” Hum. Gene Ther.4:781–788; Xu et al. (1996) “Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection” Biochemistry 35:5616–5623; and Legendre et al. (1992) “Delivery of plasmid DNA into mammalian cell lines using pH-sensitive liposomes: comparison with cationic liposomes” Pharm. Res.9, 1235-1242. Similarly, other polycations, such as poly-l-lysine and polyethylene-imine, can be used to deliver encoded AFFIMER® construct. These polycations complex with nucleic acids via charge interaction and aid in the condensation of DNA or RNA into nanoparticles, which are then substrates for endosome-mediated uptake. Several of these cationic nucleic acid complex technologies have been developed as potential clinical products, including complexes with plasmid DNA, oligodeoxynucleotides, and various forms of synthetic RNA. Modified (and unmodified or “naked”) DNA and RNA have also been shown to mediate successful gene transfer in a number of circumstances and can also be used as systems for delivery of encoded AFFIMER® construct. These include the use of plasmid DNA by direct intramuscular injection, the use of intratumoral injection of plasmid DNA. See, for example, Rodrigo et al. (2012) “De novo automated design of small RNA circuits for engineering synthetic riboregulation in living cells” PNAS 109:15271–15276; Oishi et al. (2005) “Smart polyion complex micelles for targeted intracellular delivery of PEGylated antisense oligonucleotides containing acid-labile linkages” Chembiochem.6:718–725; Bhatt et al. (2015) “Microbeads mediated oral plasmid DNA delivery using polymethacrylate vectors: an effectual groundwork for colorectal cancer” Drug Deliv. 22:849–861; Ulmer et al. (1994) Protective immunity by intramuscular injection of low doses of influenza virus DNA vaccines” Vaccine 12: 1541–1544; and Heinzerling et al. (2005) “Intratumoral injection of DNA encoding human interleukin 12 into patients with metastatic melanoma: clinical efficacy” Hum. Gene Ther.16:35–48. Viral vectors are currently used as a delivery vehicle in the vast majority of pre-clinical and clinical gene therapy trials and in the first to be approved directed gene therapy. See Gene Therapy Clinical Trials Worldwide 2017 (abedia.com/wiley/). The main driver thereto is their exceptional gene delivery efficiency, which reflects a natural evolutionary development; viral vector systems are attractive for gene delivery, because viruses have evolved the ability to cross through cellular membranes by infection, thereby delivering nucleic acids such as encoded AFFIMER® construct to target cells. Pioneered by adenoviral systems, the field of viral vector- mediated antibody gene transfer made significant strides in the past decades. The myriad of successfully evaluated administration routes, pre-clinical models and disease indications puts the capabilities of antibody gene transfer at full display through which the skilled artisan would readily be able to identify and adapt antibody gene transfer systems and techniques for in vivo delivery of encoded AFFIMER® polypeptides. Muscle has emerged as the administration site of choice for prolonged mAb expression and would similarly be a suitable target tissue for prolonged AFFIMER® agent expression. In the context of vectored intratumoral encoded AFFIMER® construct gene transfer, oncolytic viruses have a distinct advantage, as they can specifically target tumor cells, boost AFFIMER® agent expression, and amplify therapeutic responses – such as to an HSA-PD-L1 AFFIMER® agent. In vivo gene transfer of encoded AFFIMER® construct can also be accomplished by use of nonviral vectors, such as expression plasmids. Nonviral vectors are easily produced and do not seem to induce specific immune responses. Muscle tissue is most often used as target tissue for transfection because muscle tissue is well vascularized and easily accessible, and myocytes are long-lived cells. Intramuscular injection of naked plasmid DNA results in transfection of a certain percentage of myocytes. Using this approach, plasmid DNA encoding cytokines and cytokine/IgG1 chimeric proteins has been introduced in vivo and has positively influenced (autoimmune) disease outcome. In some instances, in order to increase transfection efficiency via so-called intravascular delivery in which increased gene delivery and expression levels are achieved by inducing a short-lived transient high pressure in the veins. Special blood-pressure cuffs that may facilitate localized uptake by temporarily increasing vascular pressure and can be adapted for use in human patients for this type of gene delivery. See, for example, Zhang et al. (2001) “Efficient expression of naked DNA delivered intraarterially to limb muscles of nonhuman primates” Hum. Gene Ther., 12:427-438. Increased efficiency can also be gained through other techniques, such as in which delivery of the nucleic acid is improved by use of chemical carriers—cationic polymers or lipids—or via a physical approach—gene gun delivery or electroporation. See Tranchant et al. (2004) “Physicochemical optimization of plasmid delivery by cationic lipids” J. Gene Med., 6 (Suppl.1):S24-S35; and Niidome et al. (2002) “Gene therapy progress and prospects: nonviral vectors” Gene Ther., 9:1647-1652. Electroporation is especially regarded as an interesting technique for nonviral gene delivery. Somiari, et al. (2000) “Theory and in vivo application of electroporative gene delivery” Mol. Ther.2:178-187; and Jaroszeski et al. (1999) “In vivo gene delivery by electroporation” Adv. Drug Delivery Rev., 35:131-137. With electroporation, pulsed electrical currents are applied to a local tissue area to enhance cell permeability, resulting in gene transfer across the membrane. Research has shown that in vivo gene delivery can be at least 10– 100 times more efficient with electroporation than without. See, for example, Aihara et al. (1998) “Gene transfer into muscle by electroporation in vivo” Nat. Biotechnol.16:867-870; Mir, et al. (1999) “High-efficiency gene transfer into skeletal muscle mediated by electric pulses” PNAS 96:4262-4267; Rizzuto, et al. (1999) “Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation” PNAS 96: 6417-6422; and Mathiesen (1999) “Electropermeabilization of skeletal muscle enhances gene transfer in vivo” Gene Ther., 6:508-514. Encoded LAG-3/PD-L1 AFFIMER® polypeptides can be delivered by a wide range of gene delivery system commonly used for gene therapy including viral, non-viral, or physical. See, for example, Rosenberg et al., Science, 242:1575-1578, 1988, and Wolff et al., Proc. Natl. Acad. Sci. USA 86:9011-9014 (1989). Discussion of methods and compositions for use in gene therapy include Eck et al., in Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, Hardman et al., eds., McGraw-Hill, New York, (1996), Chapter 5, pp.77-101; Wilson, Clin. Exp. Immunol.107 (Suppl.1):31-32, 1997; Wivel et al., Hematology/Oncology Clinics of North America, Gene Therapy, S. L. Eck, ed., 12(3):483-501, 1998; Romano et al., Stem Cells, 18:19-39, 2000, and the references cited therein. U.S. Pat. No.6,080,728 also provides a discussion of a wide variety of gene delivery methods and compositions. The routes of delivery include, for example, systemic administration and administration in situ. An effective encoded AFFIMER® construct gene transfer approach must be directed to the specific tissues/cells where it is needed, and the resulting transgene expression should be at a level that is appropriate to the specific application. Promoters are a major cis-acting element within the vector genome design that can dictate the overall strength of expression as well as cell-specificity. In some cases, ubiquitous expression of the encoded AFFIMER® construct in all cell types is desired. Constitutive promoters such as the human elongation factor 1α-subunit (EF1α), immediate-early cytomegalovirus (CMV), chicken β-actin (CBA) and its derivative CAG, the β glucuronidase (GUSB), or ubiquitin C (UBC) can be used to promote expression of the encoded AFFIMER® construct in most tissues. Generally, CBA and CAG promote the larger expression among the constitutive promoters; however, their size of ~1.7 kbs in comparison to CMV (~0.8 kbs) or EF1α (~1.2 kbs) may limit use in vectors with packaging constraints such as AAV, particularly where AFFIMER® agent produced by expression of the encoded AFFIMER® construct is large. The GUSB or UBC promoters can provide ubiquitous gene expression with a smaller size of 378 bps and 403 bps, respectively, but they are considerably weaker than the CMV or CBA promoter. Thus, modifications to constitutive promoters in order to reduce the size without affecting its expression have been pursued and examples such as the CBh (~800 bps) and the miniCBA (~800 bps) can promote expression comparable and even higher in selected tissues (Gray et al., Hum Gene Ther.201122:1143–1153). When expression of the encoded AFFIMER® construct should be restricted to certain cell types within an organ, promoters can be used to mediate this specificity. For example, within the nervous system promoters have been used to restrict expression to neurons, astrocytes, or oligodendrocytes. In neurons, the neuron-specific enolase (NSE) promoter drives stronger expression than ubiquitous promoters. Additionally, the platelet-derived growth factor B-chain (PDGF-β), the synapsin (Syn), and the methyl-CpG binding protein 2 (MeCP2) promoters can drive neuron-specific expression at lower levels than NSE. In astrocytes, the 680 bps-long shortened version [gfaABC(1)D] of the glial fibrillary acidic protein (GFAP, 2.2 kbs) promoter can confer higher levels of expression with the same astrocyte-specificity as the GFAP promoter. Targeting oligodendrocytes can also be accomplished by the selection of the myelin basic protein (MBP) promoter, whose expression is restricted to this glial cell; however, its size of 1.9 kbs and low expression levels limit its use. In the case of expressing the encoded AFFIMER® construct in skeletal muscle cells, exemplary promoters based on muscle creatine kinase (MCK) and desmin (1.7 kbs) have showed a high rate of specificity (with minimal expression in the liver if desired). The promoter of the α- myosin heavy chain (α-MHC; 1.2 kbs) has shown significant cardiac specificity in comparison with other muscle promoters (Lee et al., 2011 J Cardiol.57(1):115-22). In hematopoietic stem cells the synthetic MND promoter (Li et al., 2010 J Neurosci Methods.189(1):56-64) and the promoter contained in the 2AUCOE (ubiquitous chromatin opening element) have shown to drive a higher transgene expression in all cell lineages when compared to the EF1α and CMV promoters, respectively (Zhang et al., 2007 Blood.110(5):1448-57; Koldej 2013 Hum Gene Ther Clin Dev.24(2):77-85; Dighe et al., 2014 PLoS One.9(8):e104805.). Conversely, using promoters to restrict expression to only liver hepatocytes after vector-mediated gene transfer has been shown to reduce transgene-specific immune responses in systems where that is a risk, and to even induce immune tolerance to the expressed protein (Zhang et al., 2012 Hum Gene Ther. 23(5):460-72), which for certain AFFIMER® agents may be beneficial. The α1-antitrypsin (hAAT; 347 bps) and the thyroxine binding globulin (TBG; ~400 bps) promoters drive gene expression restricted to the liver with minimal invasion to other tissues (Yan et al., 2012 Gene. 506(2):289-94; Cunningham et al., 2008 Mol Ther.16(6):1081-8). In some embodiments, a mechanism to control the duration and amount of in vivo encoded AFFIMER® construct expression will typically be desired. There are a variety of inducible promoters which can be adapted for use with viral vectored- and plasmid DNA-based encoded AFFIMER® construct gene transfer. See Fang et al. (2007) “An antibody delivery system for regulated expression of therapeutic levels of monoclonal antibodies in vivo” Mol Ther.5(6):1153–9; and Perez et al. (2004) “Regulatable systemic production of monoclonal antibodies by in vivo muscle electroporation” Genet Vaccines Ther.2(1):2. An exemplary a regulatable mechanism currently under clinical evaluation is an ecdysone-based gene switch activated by a small molecule ligand. Cai et al. (2016) “Plasma pharmacokinetics of veledimex, a small-molecule activator ligand for a proprietary gene therapy promoter system, in healthy subjects” Clin Pharmacol Drug Dev.2016. In some embodiments of an encoded AFFIMER® construct, viral post-transcriptional regulatory elements (PREs) may be used; these cis-acting elements are required for nuclear export of intronless viral RNA (Huang and Yen, 1994 J Virol.68(5):3193-9; and 1995 Mol Cell Biol.15(7):3864-9). Examples include HPRE (Hepatitis B Virus PRE, 533 bps) and WPRE (Woodchuck Hepatitis Virus PRE, 600 bps), which can increase the level of transgene expression by almost 10-fold in certain instances (Donello et al., 1998 J Virol.72(6):5085-92). To further illustrate, using lentiviral and AAV vectors, WPRE was found to increase CMV promoter driven transgene expression, as well as increase PPE, PDGF and NSE promoter-driven transgene expression. Another effect of the WPRE can be to protect encoded AFFIMER® transgenes from silencing (Paterna et al., 2000 Gene Ther.7(15):1304-11; Xia et al., 2007 Stem Cells Dev.2007 Feb; 16(1):167-76). The polyadenylation of a transcribed encoded AFFIMER® construct transcript can also be important for nuclear export, translation, and mRNA stability. Therefore, in some embodiments, the encoded AFFIMER® construct will include a polyadenylation signal sequence. A variety of studies are available that have determined the effects of different polyA signals on gene expression and mRNA stability. Exemplary polyadenylation signal sequences include SV40 late or bovine growth hormone polyA (bGHpA) signal sequences, as well as minimal synthetic polyA (SPA) signal (Levitt et al., 1989 Genes Dev.3(7):1019-25; Yew et al., 1997 Hum Gene Ther.19978(5):575-84). The efficiency of polyadenylation is increased by the SV40 late polyA signal upstream enhancer (USE) placed upstream of other polyA signals (Schek et al., 1992 Mol Cell Biol.12(12):5386-93). In some embodiments, merely to illustrate, the encoded AFFIMER® construct will include an SV40 late + 2xUSE polyA signal. In some embodiments, it may be desirable for the encoded AFFIMER® construct to include at least one regulatory enhancers, e.g., in addition to any promoter sequences. The CMV enhancer is upstream of the CMV promoter at −598 to −68 (Boshart et al., 1985 Cell.41(2):521- 30) (~600 bps) and contains transcription binding sites. In some embodiments, a CMV enhancer can be included in the construct to increase tissue-specific promoter-driven transgene expression, such as using the ANF (atrial natriuretic factor) promoter, the CC10 (club cell 10) promoter, SP- C (surfactant protein C) promoter, or the PDGF-β (platelet-derived growth factor-β) promoter (merely as examples). Altogether, the CMV enhancer increases transgene expression under different cell-specific promoters and different cell types making it a broadly applicable tool to increase transgene expression levels. In muscle, for example, in AAV expression systems transgene expression using the CMV enhancer with a muscle-specific promoter can increase expression levels of the protein encoded by the transgene, so would be particularly useful in the current disclosure for expressing AFFIMER® agents from encoded AFFIMER® constructs introduced into muscle cells of a patient. The encoded AFFIMER® agents may also include at least one intronic sequence. The presence of an intron or intervening sequence in mRNA was first described, in vitro, to be important for mRNA processing and increased transgene expression (Huang and Gorman, 1990 Mol Cell Biol.10(4):1805-10; Niwa et al., 1990 Genes Dev.4(9):1552-9). The intron(s) can be placed within the coding sequence for the AFFIMER® agent and/or can be placed between the promoter and transgene. A variety of introns (Table 13) placed between the promoter and transgene were compared, in mice using AAV2, for liver transgene expression (Wu et al., 2008). The MVM (minute virus of mice) intron increased transgene expression more than any other intron tested and more than 80-fold over no intron (Wu et al., 2008). However, in cultured neurons using AAV expression cassettes, transgene expression was less under a CaMPKII promoter with a chimeric intron (human β-globin donor and immunoglobulin heavy chain acceptor) between the transgene and polyA signal compared to a WPRE (Choi et al., 2014). Together, an intron can be a valuable element to include in an expression cassette to increase transgene expression. In the case of episomal vectors, the encoded AFFIMER® constructs may also include at least one origin of replication, minichromosome maintenance elements (MME) and/or nuclear localization elements. Episomal vectors of the disclosure comprise a portion of a virus genomic DNA that encodes an origin of replication (ori) , which is required for such vectors to be self- replicating and, thus, to persist in a host cell over several generations. In addition, an episomal vector of the disclosure also may contain at least one gene encoding at least one viral protein required for replication, e.g., replicator protein (s). Optionally, the replicator protein(s) which help initiate replication may be expressed in trans on another DNA molecule, such as on another vector or on the host genomic DNA, in the host cell containing a self-replicating episomal expression vector of this disclosure. Preferred self-replicating episomal LCR-containing expression vectors of the disclosure do not contain viral sequences that are not required for long- term stable maintenance in a eukaryotic host cell such as regions of a viral genome DNA encoding core or capsid proteins that would produce infectious viral particles or viral oncogenic sequences which may be present in the full-length viral genomic DNA molecule. The term "stable maintenance" herein, refers to the ability of a self-replicating episomal expression vector of this disclosure to persist or be maintained in non-dividing cells or in progeny cells of dividing cells in the absence of continuous selection without a significant loss (e.g., >50%) in copy number of the vector for two, three, four, or five or more generations. In some embodiments, the vectors will be maintained over 10-15 or more cell generations. In contrast, "transient" or "short- term" persistence of a plasmid in a host cell refers to the inability of a vector to replicate and segregate in a host cell in a stable manner; that is, the vector will be lost after one or two generations or will undergo a loss of >51% of its copy number between successive generations. Several representative self-replicating, LCR-containing, episomal vectors useful in the context of the present disclosure are described further below. The self-replicating function may alternatively be provided by at least one mammalian sequence such as described by Wohlgeuth et al., 1996, Gene Therapy 3:503; Vos et al., 1995, Jour. Cell. Biol., Supp.21A, 433; and Sun et al., 1994, Nature Genetics 8:33, optionally in combination with at least one sequence that may be required for nuclear retention. The advantage of using mammalian, especially human sequences for providing the self- replicating function is that no extraneous activation factors are required which could have toxic or oncogenic properties. It will be understood by one of skill in the art that the disclosure is not limited to any one origin of replication or any one episomal vector but encompasses the combination of the tissue-restricted control of an LCR in an episomal vector. See also WO1998007876 “Self-replicating episomal expression vectors conferring tissue- specific gene expression” and US Patent 7790446 “Vectors, cell lines and their use in obtaining extended episomal maintenance replication of hybrid plasmids and expression of gene products” Epstein-Barr Virus-Based Self-Replicating Episomal Expression Vectors. The latent origin oriP from Epstein-Barr Virus (EBV) is described in Yates et. al., Proc . Natl . Acad . Sci . USA 81:3806-3810 (1984); Yates et al., Nature 313:812-815 (1985); Krysan et al., Mol . Cell . Biol .9:1026-1033 (1989); James et al. Gene 86: 233-239 (1990), Peterson and Legerski, Gene 107:279-284 (1991); and Pan et al., Som. Cell Molec. Genet .18:163-177 (1992)). An EBV- based episomal vector useful according to the disclosure can contain the oriP region of EBV which is carried on a 2.61 kb fragment of EBV and the EBNA-1 gene which is carried on a 2.18 kb fragment of EBV. The EBNA-1 protein, which is the only viral gene product required to support in trans episomal replication of vectors containing oriP, may be provided on the same episomal expression vector containing oriP. It is also understood, that as with any protein such as EBNA-1 known to be required to support replication of viral plasmid in trans, the gene also may be expressed on another DNA molecule, such as a different DNA vector. The episomal expression vectors of the disclosure also may be based on replication functions of the papilloma family of virus, including but not limited to Bovine Papilloma Virus (BPV) and Human Papilloma Viruses (HPVs) . BPV and HPVs persist as stably maintained plasmids in mammalian cells. -S trans-acting factors encoded by BPV and HPVs, namely El and E2, have also been identified which are necessary and sufficient for mediate replication in many cell types via minimal origin of replication (Ustav et al., EMBO J.10: 449-457 (1991); Ustavet al., EMBO J .10:4231-4329, (1991); Ustav et al., Proc. Natl. Acad. Sci. USA 90: 898-902 (1993)). An episomal vector useful according to the disclosure is the BPV-I vector system described in Piirsoo et al., EMBO J. , 15:1 (1996) and in WO 94/12629. The BPV-1 vector system described in Piirsoo et al. comprises a plasmid harboring the BPV-1 origin of replication (minimal origin plus extrachromosomal maintenance element) and optionally the El and E2 genes. The BPV-l El and E2 genes are required for stable maintenance of a BPV episomal vector. These factors ensure that the plasmid is replicated to a stable copy number of up to thirty copies per cell independent of cell cycle status. The gene construct therefore persists stably in both dividing and non-dividing cells. This allows the maintenance of the gene construct in cells such as hemopoietic stem cells and more committed precursor cells. The vectors of the disclosure also may be derived from a human papovavirus BK genomic DNA molecule. For example, the BK viral genome can be digested with restriction enzymes EcoRI and BamHI to produce a 5 kilobase (kb) fragment that contains the BK viral origin of replication sequences that can confer stable maintenance on vectors (see, for example, De Benedetti and Rhoads, Nucleic Acids Res .19:1925 (1991), as can a 3.2 kb fragment of the BK virus (Cooper and Miron, Human Gene Therapy 4:557 (1993)). The encoded AFFIMER® constructs of the present disclosure can be provided as circular or linear nucleic acids. The circular and linear nucleic acids are capable of directing expression of the AFFIMER® agent coding sequence in an appropriate subject cell. The at least one nucleic acid system for expressing an AFFIMER® agent may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. Some aspects provide a polynucleotide comprising an open reading frame encoding any one of the proteins or fusion protein described herein. In some embodiments, the open reading frame comprises a nucleotide sequence having at least 70% to the nucleotide sequence of SEQ ID NO: 99-180. Some aspects provide a polynucleotide comprising an open reading frame encoding any one of the proteins or fusion protein described herein. In some embodiments, the open reading frame comprises a nucleotide sequence having at least 75% to the nucleotide sequence of SEQ ID NO: 99-180. Some aspects provide a polynucleotide comprising an open reading frame encoding any one of the proteins or fusion protein described herein. In some embodiments, the open reading frame comprises a nucleotide sequence having at least 80% to the nucleotide sequence of SEQ ID NO: 99-180. Some aspects provide a polynucleotide comprising an open reading frame encoding any one of the proteins or fusion protein described herein. In some embodiments, the open reading frame comprises a nucleotide sequence having at least 85% to the nucleotide sequence of SEQ ID NO: 99-180. Some aspects provide a polynucleotide comprising an open reading frame encoding any one of the proteins or fusion protein described herein. In some embodiments, the open reading frame comprises a nucleotide sequence having at least 90% to the nucleotide sequence of SEQ ID NO: 99-180. Some aspects provide a polynucleotide comprising an open reading frame encoding any one of the proteins or fusion protein described herein. In some embodiments, the open reading frame comprises a nucleotide sequence having at least 95% to the nucleotide sequence of SEQ ID NO: 99-180. Some aspects provide a polynucleotide comprising an open reading frame encoding any one of the proteins or fusion protein described herein. In some embodiments, the open reading frame comprises a nucleotide sequence having at least 98% to the nucleotide sequence of SEQ ID NO: 99-180. Some aspects provide a polynucleotide comprising an open reading frame encoding any one of the proteins or fusion protein described herein. In some embodiments, the open reading frame comprises a nucleotide sequence having 100% to the nucleotide sequence of SEQ ID NO: 99-180. Table 3. AFFIMER® polynucleotides
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A. RNA-Mediated Encoded AFFIMER® Construct Gene Transfer Exemplary nucleic acids or polynucleotides for the encoded HSA-PD-L1 AFFIMER® constructs of the present disclosure include but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a β- D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2 '-amino functionalization, and 2'-amino- a-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or hybrids or combinations thereof. mRNA presents an emerging platform for antibody gene transfer that can be adapted by those skilled in the art for delivery of encoded AFFIMER® constructs of the present disclosure. Although current results differ considerably, in certain instances the mRNA constructs appear to be able to rival viral vectors in terms of generated serum mAb titers. Levels were in therapeutically relevant ranges within hours after mRNA administration, a marked shift in speed compared to DNA. The use of lipid nanoparticles (LNP) for mRNA transfection, rather than the physical methods typically required for DNA, can provide significant advantages in some embodiments towards application range. In their 1990 study, Wolff et al. (1990, supra) found that, in addition to pDNA, intramuscular injection of in vitro transcribed (IVT) mRNA also led to local expression of the encoded protein. mRNA was not pursued as actively as DNA at that time because of its low stability. Progress over the past years allowed mRNA to catch up with DNA and viral vectors as a tool for gene transfer. Reviewed in Sahin et al. (2014) “mRNA-based therapeutics: developing a new class of drugs” Nat Rev Drug Discov.13(10):759–80. Conceptually, there are several differences with these expression platforms. mRNA does not need to enter into the nucleus to be functional. Once it reaches the cytoplasm, mRNA is translated instantly. mRNA-based therapeutics are expressed more transiently compared to DNA- or viral vector-mediated gene transfer, and do not pose the risk of insertional mutagenesis in the host genome. mRNA production is relatively simple and inexpensive. In terms of administration, mRNA uptake can be enhanced using electroporation. Broderick et al.2017 “Enhanced delivery of DNA or RNA vaccines by electroporation” Methods Mol Biol.2017;1499:193–200. Most focus, however, has gone to non-physical transfection methods. Indeed, a variety of mRNA complexing formulations have been developed, including lipid nanoparticles (LNP), which have proven to be safe and very efficient mRNA carriers for administration in a variety of tissues and i.v. Pardi et al.2015 “Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes” J Control Release 217:345–51. In line with this progress, IVT mRNA has reached the stage of clinical evaluation. Beissert et al. WO2017162266 “RNA Replicon for Versatile and Efficient Gene Expression” describes agents and methods suitable for efficient expression of AFFIMER® polypeptides of the present disclosure, such as suitable for immunotherapeutic treatment for the prevention and therapy of tumors. For instance, the AFFIMER® agent coding sequence can be provided as an RNA replicon comprising a 5' replication recognition sequence such as from an alphavirus 5' replication recognition sequence. In some embodiments, the RNA replicon comprises a (modified) 5' replication recognition sequence and an open reading frame encoding the AFFIMER® agent, in particular located downstream from the 5' replication recognition sequence such as that the 5' replication recognition sequence and the open reading frame do not overlap, e.g., the 5' replication recognition sequence does not contain a functional initiation codon and in some embodiments does not contain any initiation codon. Most preferably, the initiation codon of the open reading frame encoding the AFFIMER® agent is in the 5'→ 3' direction of the RNA replicon. In some embodiments, to prevent immune activation, modified nucleosides can be incorporated into the in vitro–transcribed mRNA. In some embodiments, the IVT RNA can be 5’ capped, such an m7GpppG-capped or m7G5′ppp5′G2´-O-Met-capped IVT. Efficient translation of the modified mRNA can be ensured by removing double-stranded RNA. Moreover, the 5′ and 3′ UTRs and the poly(A) tail can be optimized for improved intracellular stability and translational efficiency. See, for example, Stadler et al. (2017) Nature Medicine 23:815–817 and Kariko et al. WO/2017/036889 “Method for Reducing Immunogenicity of RNA”. In some embodiments, the mRNA that encodes the LAG-3/PD-L1 AFFIMER® agent may include at least one chemical modification described herein. As a non-limiting example, the chemical modification may be 1-methylpseudouridine, 5-methylcytosine or 1- methylpseudouridine and 5-methylcytosine. In some embodiments, the chemical modification is a pseudouridine or a modified 5 nucleoside, wherein said modified nucleoside is m5C, m5U, m6A, s2U, Ψ, or 2'-O-methyl-U. In some embodiments, linear polynucleotides encoding at least one LAG-3/PD-L1 AFFIMER® agent that are made using only in vitro transcription (IVT) enzymatic synthesis methods are referred to as "IVT polynucleotides." Methods of making IVT polynucleotides are known in the art and are described in International Publication Nos. WO 2007/024708A2 and WO 2013/151666, the contents of which are incorporated herein by reference in their entirety. In another embodiment, the polynucleotides that encode the LAG-3/PD-L1 AFFIMER® agent of the present disclosure have portions or regions which differ in size and/or chemical modification pattern, chemical modification position, chemical modification percent or chemical modification population and combinations of the foregoing are known as "chimeric polynucleotides." A "chimera" according to the present disclosure is an entity having two or more incongruous or heterogeneous parts or regions. As used herein a "part" or "region" of a polynucleotide is defined as any portion of the polynucleotide which is less than the entire length of the polynucleotide. Such constructs are taught in for example International Publication No. WO2015/034928. In yet another embodiment, the polynucleotides of the present disclosure that are circular are known as "circular polynucleotides" or "circP." As used herein, "circular polynucleotides" or "circP" means a single stranded circular polynucleotide which acts substantially like, and has the properties of, an RNA. The term "circular" is also meant to encompass any secondary or tertiary configuration of the circP. Such constructs are taught in for example International Publication Nos. WO2015/034925 and WO2015/034928, the contents of each of which are incorporated herein by reference in their entirety. Exemplary mRNA (and other polynucleotides) that can be used to encode LAG-3/PD-L1 AFFIMER® agents of the present disclosure include those which can be adapted from the specifications and figures of, for example, International Publication No.s WO2017/049275, WO2016/118724, WO2016/118725, WO2016/011226, WO2015/196128, WO/2015/196130, WO/2015/196118, WO/2015/089511, and WO2015/105926 (the later titled “Polynucleotides for the In vivo Production of Antibodies”), each of which is incorporated by reference herein. Electroporation, as described below, is one exemplary method for introducing mRNA or other polynucleotides into a cell. Lipid-containing nanoparticle compositions have proven effective as transport vehicles into cells and/or intracellular compartments for a variety of RNAs (and related polynucleotides described herein). These compositions generally include at least one "cationic" and/or ionizable lipids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), and lipids containing polyethylene glycol (PEG lipids). Cationic and/or ionizable lipids include, for example, amine-containing lipids that can be readily protonated. B. Other Methods of Delivery of Encoded AFFIMER® Construct into Target Cells The introduction into host cell of the gene delivery system can be performed through various methods known to those skilled in the art. Where the present gene delivery system is constructed on the basis of viral vector construction, delivery can be performed as conventional infection methods known in the art. Physical methods to enhance delivery both viral and non-viral encoded AFFIMER® constructs include electroporation (Neumann, E. et al., EMBO J., 1:841(1982); and Tur-Kaspa et al., Mol. Cell Biol., 6:716-718(1986)), gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990) where DNA is loaded onto (e.g., gold) particles and forced to achieve penetration of the DNA into the cells, sonoporation, magnetofection, hydrodynamic delivery and the like, all of which are known to those of skill in the art. 1. Electroporation In the past several years, there has been a great advance in the plasmid DNA delivery technology that is utilized for in vivo production of proteins. This included codon optimization for expression in human cells, RNA optimization to improve mRNA stability as well as more efficient translation at the ribosomal level, the addition of specific leader sequences to enhance translation efficiency, the creation of synthetic inserts to further enhance production in vivo and the use of improved adaptive electroporation (EP) delivery protocols to improve in vivo delivery. EP assists in the delivery of plasmid DNA by generating an electrical field that allows the DNA to pass into the cell more efficiently. In vivo electroporation is a gene delivery technique that has been used successfully for efficient delivery of plasmid DNA to many different tissues. Kim et al. “Gene therapy using plasmid DNA-encoded anti-HER2 antibody for cancers that overexpress HER2” (2016) Cancer Gene Ther.23(10): 341–347 teaches a vector and electroporation system for intramuscular injection and in vivo electroporation of the plasmids that results in high and sustained antibody expression in sera; the plasmid and electroporation system of Kim et al. can be readily adapted for the in vivo delivery of a plasmid for expressing an encoded LAG-3/PD-L1 AFFIMER® construct of the present disclosure. Accordingly, in certain some embodiments of the present disclosure, the encoded AFFIMER® construct is introduced into target cells via electroporation. Administration of the composition via electroporation may be accomplished using electroporation devices that can be configured to deliver to a desired tissue of a mammal, a pulse of energy effective to cause reversible pores to form in cell membranes, and preferable the pulse of energy is a constant current similar to a preset current input by a user. The electroporation device may comprise an electroporation component and an electrode assembly or handle assembly. The electroporation component may include and incorporate at least one of the various elements of the electroporation devices, including: controller, current waveform generator, impedance tester, waveform logger, input element, status reporting element, communication port, memory component, power source, and power switch. The electroporation may be accomplished using an in vivo electroporation device, for example CELLECTRA EP system (VGX Pharmaceuticals, Blue Bell, Pa.) or Elgen electroporator (Genetronics, San Diego, Calif.) to facilitate transfection of cells by the plasmid. 2. Transfection Enhancing Formulations Encoded AFFIMER® constructs can also be encapsulated in liposomes, preferably cationic liposomes (Wong, T. K. et al., Gene, 10:87(1980); Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190 (1982); and Nicolau et al., Methods Enzymol., 149:157-176 (1987)) or polymersomes (synthetic liposomes) which can interact with the cell membrane and fuse or undergo endocytosis to effect nucleic acid transfer into the cell. The DNA also can be formed into complexes with polymers (polyplexes) or with dendrimers which can directly release their load into the cytoplasm of a cell. Illustrative carriers useful in this regard include microparticles of poly(lactide-co- glycolide), polyacrylate, latex, starch, cellulose, dextran and the like. Other illustrative carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross- linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid (see e.g., U.S. Pat. No.5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO 96/06638). The amount of active agent contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented. Biodegradable microspheres (e.g., polylactate polyglycolate) may be employed as carriers for compositions. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. NOS: 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and 5,942,252. Modified hepatitis B core protein carrier systems such as described in WO/9940934, and references cited therein, will also be useful for many applications. Another illustrative carrier/delivery system employs a carrier comprising particulate-protein complexes, such as those described in U.S. Pat. No.5,928,647, which can have the added benefit when used intratumorally to deliver the coding sequence(s) for an LAG-3/PD-L1 AFFIMER® polypeptide. Biodegradable polymeric nanoparticles facilitate nonviral nucleic acid transfer to cells. Small (approximately 200 nm), positively charged (approximately 10 mV) particles are formed by the self-assembly of cationic, hydrolytically degradable poly(beta-amino esters) and plasmid DNA. Polynucleotides may also be administered to cells by direct microinjection, temporary cell permeabilizations (e.g., co-administration of repressor and/or activator with a cell permeabilizing agent), fusion to membrane translocating peptides, and the like. Lipid-mediated nucleic acid delivery and expression of foreign nucleic acids, including mRNA, in vitro and in vivo has been very successful. Lipid based non-viral formulations provide an alternative to viral gene therapies. Current in vivo lipid delivery methods use subcutaneous, intradermal, intratumoral, or intracranial injection. Advances in lipid formulations have improved the efficiency of gene transfer in vivo (see PCT Application WO 98/07408). For instance, a lipid formulation composed of an equimolar ratio of l,2-bis(oleoyloxy)-3-(trimethyl ammonio)propane (DOTAP) and cholesterol can significantly enhance systemic in vivo gene transfer. The DOTAP:cholesterol lipid formulation forms unique structure termed a "sandwich liposome". This formulation is reported to "sandwich" DNA between an invaginated bi-layer or 'vase' structure. Beneficial characteristics of these lipid structures include a positive p, colloidal stabilization by cholesterol, two-dimensional nucleic acid packing and increased serum stability. Cationic liposome technology is based on the ability of amphipathic lipids, possessing a positively charged head group and a hydrophobic lipid tail, to bind to negatively charged DNA or RNA and form particles that generally enter cells by endocytosis. Some cationic liposomes also contain a neutral co-lipid, thought to enhance liposome uptake by mammalian cells. Similarly, other polycations, such as poly-l-lysine and polyethylene-imine, complex with nucleic acids via charge interaction and aid in the condensation of DNA or RNA into nanoparticles, which are then substrates for endosome-mediated uptake. Several of these cationic-nucleic acid complex technologies have been developed as potential clinical products, including complexes with plasmid DNA (pDNA), oligodeoxynucleotides, and various forms of synthetic RNA, and be used as part of the delivery system for the encoded AFFIMER® construct of the present disclosure. The encoded AFFIMER® construct disclosed herein may be associated with polycationic molecules that serve to enhance uptake into cells. Complexing the nucleic acid construct with polycationic molecules also helps in packaging the construct such their size is reduced, which is believed to assist with cellular uptake. Once in the endosome, the complex dissociates due to the lower pH, and the polycationic molecules can disrupt the endosome's membrane to facilitate DNA escape into the cytoplasm before it can be degraded. Preliminary data shows that the nucleic acid construct embodiments had enhanced uptake into SCs over DCs when complexed with the polycationic molecules polylysine or polyethyleneimine. One example of polycationic molecules useful for complexing with nucleic acid constructs includes cell penetrating peptides (CPP), examples include polylysine (described above), polyarginine and Tat peptides. Cell penetrating peptides (CPP) are small peptides which can bind to DNA and once released penetrate cell membranes to facilitate escape of the DNA from the endosome to the cytoplasm. Another example of a CPP pertains to a 27-residue chimeric peptide, termed MPG, was shown some time ago to bind ss- and ds-oligonucleotides in a stable manner, resulting in a non-covalent complex that protected the nucleic acids from degradation by DNase and effectively delivered oligonucleotides to cells in vitro (Mahapatro A, et al., J Nanobiotechnol, 2011, 9:55). The complex formed small particles of approximately 150 nm to 1 um when different peptide:DNA ratios were examined, and the 10:1 and 5:1 ratios (150 nm and 1 um respectively). Another CPP pertains to a modified tetrapeptide [tetralysine containing guanidinocarbonylpyrrole (GCP) groups (TL-GCP)], which was reported to bind with high affinity to a 6.2 kb plasmid DNA resulting in a positive charged aggregate of 700-900 nm Li et al., Agnew Chem Int Ed Enl 2015; 54(10):2941-4). RNA can also be complexed by such polycationic molecules for in vivo delivery. Other examples of polycationic molecules that may be complexed with the nucleic acid constructs described herein include polycationic polymers commercially available as JETPRIME® and In vivo JET (Polypus-transfection, S.A., Illkirch, France). In some embodiments, the present disclosure contemplates a method of delivering an mRNA (or other polynucleotide) encoding an LAG-3/PD-L1 AFFIMER® agent to a patient’s cells by administering a nanoparticle composition comprising (i) a lipid component, a phospholipid, a structural lipid, and a PEG lipid; and (ii) an mRNA (or other polynucleotide), said administering comprising contacting said mammalian cell with said nanoparticle composition, whereby said mRNA (or other polynucleotide) is delivered to said cell. In exemplary embodiments, the PEG lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatide acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol and a PEG-modified dialkylglycerol. In exemplary embodiments, the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and alphatocopherol. In some embodiments, the structural lipid is cholesterol. In exemplary embodiments, the phospholipid includes a moiety selected from the group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin. In some embodiments, the phospholipid includes at least one fatty acid moiety selected from the group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, arachidic acid, arachidonic acid, phytanoic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. In some embodiments, the phospholipid is selected from the group consisting of 1 ,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1 ,2-dimyristoyl-sn-glycero- phosphocholine (DMPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), 1 ,2-di-0-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1 -oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl- sn-glycero-3-phosphocholine (C16 Lyso PC), 1 ,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3- phosphocholine,1 ,2-dioleoyl-sn-glycero-3-phosphoethanola mine (DOPE), 1 ,2-diphytanoyl-sn- glycero-3-phosphoethanolamine (ME 16.0 PE), 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dilinolenoyl-sn- glycero-3-phosphoethanolamine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dioleoyl-sn-glycero-3-phospho-rac- (1 -glycerol) sodium salt (DOPG), and sphingomyelin In some embodiments, the phospholipid is DOPE or DSPC. To further illustrate, the phospholipid can be DOPE and said the component can comprise about 35 mol % to about 45 mol % said compound, about 10 mol % to about 20 mol % DOPE, about 38.5 mol % to about 48.5 mol % structural lipid, and about 1.5 mol % PEG lipid. The lipid component can be about 40 mol % said compound, about 15 mol % phospholipid, about 43.5 mol % structural lipid, and about 1.5 mol % PEG lipid. In some embodiments, the wt/wt ratio of lipid component to LAG-3/PD-L1 AFFIMER® agent encoding mRNA (or other polynucleotide) is from about 5:1 to about 50:1, or about 10:1 to about 40:1 In some embodiments, the mean size of said nanoparticle composition is from about 50 nm to about 150 nm, or from about 80 nm to about 120 nm. In some embodiments, the polydispersity index of said nanoparticle composition is from about 0 to about 0.18, or from about 0.13 to about 0.17. In some embodiments, the nanoparticle composition has a zeta potential of about -10 to about +20 mV. In some embodiments, the nanoparticle composition further comprises a cationic and/or ionizable lipid selected from the group consisting of 3-(didodecylamino)-N1 ,N 1 ,4-tridodecyl-1 -piperazineethanamine (KL10), 14,25-ditridecyl-15, 18,21 ,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4- dimethylaminomethyl-[1 ,3]-dioxolane (DLin-K-DMA), heptatriaconta-6, 9,28,31 -tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1 ,3]-dioxolane (DLin-KC2-DMA), 1 ,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), and (2R)-2-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9, 12- dien-1 -yl oxy]propan-1 -amine (Octyl-CLinDMA (2R)). For exemplary lipid nanoparticle compositions and other polymeric carrier compositions, see International Publication Nos. WO 2016/118724A1, WO 2017/112865A1, WO 2017/049245A2, and WO2012013326A1. V. Expression Methods and Systems LAG-3/PD-L1 AFFIMER® agents described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing a DNA sequence encoding polypeptide sequences and expressing those sequences in a suitable host. For those recombinant AFFIMER® agent proteins including further modifications, such as a chemical modifications or conjugation, the recombinant AFFIMER® agent protein can be further manipulated chemically or enzymatically after isolation form the host cell or chemical synthesis. The present disclosure includes recombinant methods and nucleic acids for recombinantly expressing the recombinant AFFIMER® agent proteins of the present disclosure comprising (i) introducing into a host cell a polynucleotide encoding the amino acid sequence of said AFFIMER® agent, for example, wherein the polynucleotide is in a vector and/or is operably linked to a promoter; (ii) culturing the host cell (e.g., eukaryotic or prokaryotic) under condition favorable to expression of the polynucleotide and, (iii) optionally, isolating the AFFIMER® agent from the host cell and/or medium in which the host cell is grown. See e.g., WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627. In some embodiments, a DNA sequence encoding a recombinant AFFIMER® agent protein of interest may be constructed by chemical synthesis using an oligonucleotide synthesizer. Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize a polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a back-translated gene. Further, a DNA oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly. Once a nucleic acid sequence encoding a recombinant AFFIMER® agent protein of the disclosure has been obtained, the vector for the production of the recombinant AFFIMER® agent protein may be produced by recombinant DNA technology using techniques well known in the art. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the recombinant AFFIMER® agent coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, for example, the techniques described in Sambrook et al, 1990, MOLECULAR CLONING, A LABORATORY MANUAL, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al. eds., 1998, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY). An expression vector comprising the nucleotide sequence of a recombinant AFFIMER® agent protein can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation) and the transfected cells are then cultured by conventional techniques to produce the recombinant AFFIMER® agent protein of the disclosure. In specific embodiments, the expression of the recombinant AFFIMER® agent protein is regulated by a constitutive, an inducible or a tissue, specific promoter. The expression vector may include an origin of replication, such as may be selected based upon the type of host cell being used for expression. By way of example, the origin of replication from the plasmid pBR322 (Product No.303-3s, New England Biolabs, Beverly, Mass.) is useful for most Gram- negative bacteria while various origins from SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV) or papillomaviruses (such as HPV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (for example, the SV40 origin is often used because it contains the early promoter). The vector may include at least one selectable marker gene, e.g., genetic elements that encode a protein necessary for the survival and growth of a host cell grown in a selective culture medium. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells, (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex media. Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. A neomycin resistance gene may also be used for selection in prokaryotic and eukaryotic host cells. Other selection genes may be used to amplify the gene which will be expressed. Amplification is a process where genes which are in greater demand for the production of a protein critical for growth are reiterated in tandem within the chromosomes of successive generations of recombinant cells. Examples of selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase. The mammalian cell transformants are placed under selection pressure which only the transformants are uniquely adapted to survive by virtue of the marker present in the vector. Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of selection agent in the medium is successively changed, thereby leading to amplification of both the selection gene and the DNA that encodes the recombinant AFFIMER® agent protein. As a result, increased quantities of the recombinant AFFIMER® agent protein are synthesized from the amplified DNA. The vector may also include at least one ribosome binding site, which will be transcribed into the mRNA including the coding sequence for the recombinant AFFIMER® agent protein. For example, such a site is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). The element is typically located 3' to the promoter and 5' to the coding sequence of the polypeptide to be expressed. The Shine-Dalgarno sequence is varied but is typically a polypurine (having a high A-G content). Many Shine-Dalgarno sequences have been identified, each of which can be readily synthesized using methods set forth above and used in a prokaryotic vector. The expression vectors will typically contain a promoter that is recognized by the host organism and operably linked to a nucleic acid molecule encoding the recombinant AFFIMER® agent protein. Either a native or heterologous promoter may be used depending on the host cell used for expression and the yield desired. Promoters for use with prokaryotic hosts include the beta-lactamase and lactose promoter systems; alkaline phosphatase, a tryptophan (trp) promoter system; and hybrid promoters such as the tac promoter. Other known bacterial promoters are also suitable. Their sequences have been published, and they can be ligated to a desired nucleic acid sequence(s), using linkers or adapters as desired to supply restriction sites. Promoters for use with yeast hosts are also known in the art. Yeast enhancers are advantageously used with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, e.g., heat-shock promoters and the actin promoter. Additional promoters which may be used for expressing the selective binding agents of the disclosure include but are not limited to: the SV40 early promoter region (Bernoist and Chambon, Nature, 290:304-310, 1981); the CMV promoter; the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al. (1980), Cell 22: 787-97); the herpes thymidine kinase promoter (Wagner et al. (1981), Proc. Natl. Acad. Sci. U.S.A.78: 1444- 5); the regulatory sequences of the metallothionine gene (Brinster et al, Nature, 296; 39-42, 1982); prokaryotic expression vectors such as the beta- lactamase promoter (Villa-Kamaroff, et al., Proc. Natl. Acad. Sci. U.S.A., 75; 3727-3731, 1978); or the tac promoter (DeBoer, et al. (1983), Proc. Natl. Acad. Sci. U.S.A., 80: 21-5). Also of interest are the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: the elastase I gene control region which is active in pancreatic acinar cells (Swift et al. (1984), Cell 38: 639-46; Ornitz et al. (1986), Cold Spring Harbor Symp. Quant. Biol.50: 399-409; MacDonald (1987), Hepatology 7: 425-515); the insulin gene control region which is active in pancreatic beta cells (Hanahan (1985), Nature 315: 115-22); the immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al. (1984), Cell 38; 647-58; Adames et al. (1985), Nature 318; 533-8; Alexander et al. (1987), Mol. Cell. Biol.7: 1436-44); the mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al. (1986), Cell 45: 485-95), albumin gene control region which is active in liver (Pinkert et al. (1987), Genes and Devel.1: 268-76); the alphafetoprotein gene control region which is active in liver (Krumlauf et al. (1985), MoI. Cell. Biol.5: 1639-48; Hammer et al. (1987), Science, 235: 53-8); the alpha 1- antitrypsin gene control region which is active in the liver (Kelsey et al. (1987), Genes and Devel.1: 161-71); the beta-globin gene control region which is active in myeloid cells (Mogram et al., Nature, 315 338-340, 1985; Kollias et al. (1986), Cell 46: 89-94); the myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al. (1987), Cell, 48: 703-12); the myosin light chain-2 gene control region which is active in skeletal muscle (Sani (1985), Nature, 314: 283-6); and the gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al. (1986), Science 234: 1372-8). An enhancer sequence may be inserted into the vector to increase transcription in eukaryotic host cells. Several enhancer sequences available from mammalian genes are known (e.g., globin, elastase, albumin, alpha-feto-protein and insulin). Typically, however, an enhancer from a virus will be used. The SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers are exemplary enhancing elements for the activation of eukaryotic promoters. While an enhancer may be spliced into the vector at a position 5' or 3' to the polypeptide coding region, it is typically located at a site 5' from the promoter. Vectors for expressing nucleic acids include those which are compatible with bacterial, insect, and mammalian host cells. Such vectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (Invitrogen Company, San Diego, Calif.), pBSII (Stratagene Company, La Jolla, Calif.), pET15 (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL (BlueBacII; Invitrogen), pDSR- alpha (PCT Publication No. WO90/14363) and pFastBacDual (Gibco/BRL, Grand Island, N.Y.). Additional possible vectors include but are not limited to, cosmids, plasmids or modified viruses, but the vector system must be compatible with the selected host cell. Such vectors include but are not limited to plasmids such as Bluescript® plasmid derivatives (a high copy number ColEl-based phagemid, Stratagene Cloning Systems Inc., La Jolla Calif.), PCR cloning plasmids designed for cloning Taq-amplified PCR products (e.g., TOPO™. TA Cloning® Kit, PCR2.1 plasmid derivatives, Invitrogen, Carlsbad, Calif.), and mammalian, yeast or virus vectors such as a baculovirus expression system (pBacPAK plasmid derivatives, Clontech, Palo Alto, Calif.). The recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, or other known techniques Eukaryotic and prokaryotic host cells, including mammalian cells as hosts for expression of the recombinant AFFIMER® agent protein disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Cell lines of particular preference are selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells. Fungal cells include yeast and filamentous fungus cells including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa. Pichia sp., any Saccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candida albicans, any Aspergillus sp., Trichoderma reesei, Chrysosporium lucknowense, any Fusarium sp., Yarrowia lipolytica, and Neurospora crassa. A variety of host-expression vector systems may be utilized to express the recombinant AFFIMER® agent protein of the disclosure. Such host-expression systems represent vehicles by which the coding sequences of the recombinant AFFIMER® agent protein may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the recombinant AFFIMER® agent protein of the disclosure in situ. These include but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing AFFIMER® agent protein coding sequences; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing AFFIMER® agent protein coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the AFFIMER® agent protein coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CµMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing AFFIMER® agent protein coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphotic cells (see U.S. Pat. No.5,807,715), Per C.6 cells (rat retinal cells developed by Crucell)) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the recombinant AFFIMER® agent protein being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of the recombinant AFFIMER® agent protein, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include but are not limited, to the E. coli expression vector pUR278 (Ruther et al. (1983) "Easy Identification Of cDNA Clones," EMBO J.2:1791- 1794), in which the AFFIMER® agent protein coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye et al. (1985) "Up-Promoter Mutations In The Lpp Gene Of Escherichia coli," Nucleic Acids Res.13:3101-3110; Van Heeke et al. (1989) "Expression Of Human Asparagine Synthetase In Escherichia coli," J. Biol. Chem.24:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free gluta-thione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety. In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The AFFIMER® agent protein coding sequence may be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter). In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the AFFIMER® agent protein coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts. (see e.g., see Logan et al. (1984) "Adenovirus Tripartite Leader Sequence Enhances Translation Of mRNAs Late After Infection," Proc. Natl. Acad. Sci. (U.S.A.) 81:3655-3659). Specific initiation signals may also be required for efficient translation of inserted AFFIMER® agent protein coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bitter et al. (1987) "Expression and Secretion Vectors For Yeast," Methods in Enzymol.153:516-544). In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst. For long-term, high-yield production of recombinant proteins, stable expression is contemplated. For example, cell lines which stably express an antibody of the disclosure may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the recombinant AFFIMER® agent proteins of the disclosure. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the recombinant AFFIMER® agent proteins. A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al. (1977) "Transfer of Purified Herpes Virus Thymidine Kinase Gene to Cultured Mouse Cells," Cell 11:223-232), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al. (1962) "Genetics Of Human Cess Line. IV. DNA- Mediated Heritable Transformation of a Biochemical Trait," Proc. Natl. Acad. Sci. (U.S.A.) 48:2026-2034), and adenine phosphoribosyltransferase (Lowy et al. (1980) "Isolation Of Transforming DNA: Cloning The Hamster Aprt Gene," Cell 22:817-823) genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al. (1980) "Transformation Of Mammalian Cells With An Amplfiable Dominant-Acting Gene," Proc. Natl. Acad. Sci. (U.S.A.) 77:3567-3570; O'Hare et al. (1981) "Transformation Of Mouse Fibroblasts To Methotrexate Resistance By A Recombinant Plasmid Expressing A Prokaryotic Dihydrofolate Reductase," Proc. Natl. Acad. Sci. (U.S.A.) 78:1527-1531); gpt, which confers resistance to mycophenolic acid (Mulligan et al. (1981) "Selection For Animal Cells That Express The Escherichia coli Gene Coding For Xanthine-Guanine Phosphoribosyltransferase," Proc. Natl. Acad. Sci. (U.S.A.) 78:2072-2076); neo, which confers resistance to the aminoglycoside G-418 (Tachibana et al. (1991) "Altered Reactivity Of Immunoglobutin Produced By Human-Human Hybridoma Cells Transfected By pSV.2-Neo Gene," Cytotechnology 6(3):219-226; Tolstoshev (1993) "Gene Therapy, Concepts, Current Trials And Future Directions," Ann. Rev. Pharmacol. Toxicol.32:573-596; Mulligan (1993) "The Basic Science of Gene Therapy," Science 260:926-932; and Morgan et al. (1993) "Human gene therapy," Ann. Rev. Biochem.62:191-217). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY; Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, CURRENT PROTOCOLS IN HUMAN GENETICS, John Wiley & Sons, NY.; Colbere-Garapin et al. (1981) "A New Dominant Hybrid Selective Marker For Higher Eukaryotic Cells," J. Mol. Biol.150:1-14; and hygro, which confers resistance to hygromycin (Santerre et al. (1984) "Expression Of Prokaryotic Genes For Hygromycin B And G418 Resistance As Dominant-Selection Markers In Mouse L Cells," Gene 30:147-156). The expression levels of a recombinant AFFIMER® agent protein can be increased by vector amplification (for a review, see Bebbington and Hentschel, "The Use of Vectors Based On Gene Amplification For The Expression Of Cloned Genes In Mammaian Cells," in DNA CLONING, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector system expressing a recombinant AFFIMER® agent protein is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of the recombinant AFFIMER® agent protein, production of the recombinant AFFIMER® agent protein will also increase (Crouse et al. (1983) "Expression and Amplification of Engineered Mouse Dihydrofolate Reductase Minigenes," Mol. Cell. Biol.3:257-266). Where the AFFIMER® agent is an AFFIMER® polypeptide-antibody fusion or other multiprotein complex, the host cell may be co-transfected with two expression vectors, for instance the first vector encoding a heavy chain and the second vector encoding a light chain derived polypeptide, one or both of which includes an AFFIMER® polypeptide coding sequence. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot (1986) "Expression and Amplification Of Engineered Mouse Dihydrofolate Reductase Minigenes," Nature 322:562-565; Kohler (1980) "Immunoglobulin Chain Loss In Hybridoma Lines," Proc. Natl. Acad. Sci. (U.S.A.) 77:2197-2199). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA. In general, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern that is characteristic for glycoproteins produced in the cell line or transgenic animal. Therefore, the particular glycosylation pattern of the recombinant AFFIMER® agent protein will depend on the particular cell line or transgenic animal used to produce the protein. In some embodiments of AFFIMER® polypeptide-antibody fusions, a glycosylation pattern comprising only non-fucosylated N-glycans may be advantageous, because in the case of antibodies this has been shown to typically exhibit more potent efficacy than fucosylated counterparts both in vitro and in vivo (See for example, Shinkawa et al., J. Biol. Chem.278: 3466-3473 (2003); U.S. Pat. NOS: 6,946,292 and 7,214,775). Further, expression of an AFFIMER® agent from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or part in connection with European Patent NOS: 0216846, 0256055, and 0323997 and European Patent Application No.89303964.4. Thus, in some embodiments of the disclosure, the mammalian host cells (e.g., CHO) lack a glutamine synthetase gene and are grown in the absence of glutamine in the medium wherein, however, the polynucleotide encoding the immunoglobulin chain comprises a glutamine synthetase gene which complements the lack of the gene in the host cell. Such host cells containing the binder or polynucleotide or vector as discussed herein as well as expression methods, as discussed herein, for making the binder using such a host cell are part of the present disclosure. Expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also offers a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art. The recombinant AFFIMER® agent proteins produced by a transformed host can be purified according to any suitable method. Standard methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags such as hexa-histidine, maltose binding domain, influenza coat sequence, and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column. Isolated proteins can also be physically characterized using such techniques as proteolysis, mass spectrometry (MS), nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC), and x-ray crystallography. In some embodiments, recombinant AFFIMER® agent proteins produced in bacterial culture can be isolated, for example, by initial extraction from cell pellets, followed by at least one concentration, salting-out, aqueous ion exchange, or size exclusion chromatography steps. HPLC can be employed for final purification steps. Microbial cells employed in expression of a recombinant protein can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. VI. Methods of Use and Pharmaceutical Compositions The AFFIMER® agents of the disclosure are useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as immunotherapy for cancer. In some embodiments, AFFIMER® agents described herein are useful for activating, promoting, increasing, and/or enhancing an immune response, inhibiting tumor growth, reducing tumor volume, inducing tumor regression, increasing tumor cell apoptosis, and/or reducing the tumorigenicity of a tumor. The polypeptides or agents of the disclosure, in some embodiments, are useful for immunotherapy against pathogens, such as viruses. For example, the AFFIMER® agents described herein may be useful for inhibiting viral infection, reducing viral infection, increasing virally-infected cell apoptosis, and/or increasing killing of virus-infected cells. The methods of use may be in vitro, ex vivo, or in vivo methods. In the cancer disease state, the interaction of PD-L1 on the tumor cells with PD-1 on a T- cell reduces T-cell function signals to prevent the immune system from attacking the tumor cells. Use of an inhibitor that blocks the interaction of PD-L1 with the PD-1 receptor can prevent the cancer from evading the immune system in this way. Several PD-1 and PD-L1 inhibitors are being tested within the clinic for use in advanced melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer and Hodgkin lymphoma, amongst other cancer types. Similarly, LAG-3 is involved in T cell suppression, including through activation of regulatory T cells. By targeting LAG-3 and LAG-3+ tumor-infiltrating lymphocytes (TILs), the therapeutics described herein can help subjects overcome resistance and restore T cell function. Immunotherapy with these immune checkpoint inhibitors appears to shrink tumors in a higher number of patients across a wider range of tumor types and is associated with lower toxicity levels than other immunotherapies, with durable responses. However, de novo and acquired resistance is still seen in a large proportion of patients. Thus, PD-L1 inhibitors and LAG-3 inhibitors, such as the LAG-3/PD-L1 AFFIMER® agents as provided herein, are considered to be a promising drug category for many different cancers. The present disclosure provides methods for activating an immune response in a subject using an AFFIMER® agent. In some embodiments, the disclosure provides methods for promoting an immune response in a subject using an AFFIMER® agent described herein. In some embodiments, the disclosure provides methods for increasing an immune response in a subject using an AFFIMER® agent. In some embodiments, the disclosure provides methods for enhancing an immune response in a subject using an AFFIMER® agent. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing cell-mediated immunity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing Th1-type responses. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CD4+ T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CD8+ T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CTL activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing NK cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing T-cell activity and increasing NK cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CU activity and increasing NK cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises inhibiting or decreasing the suppressive activity of Treg cells. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises inhibiting or decreasing the suppressive activity of MDSCs. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing the number of the percentage of memory T-cells. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing long-term immune memory function. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing long-term memory. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises no evidence of substantial side effects and/or immune-based toxicities. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises no evidence of cytokine release syndrome (CRS) or a cytokine storm. In some embodiments, the immune response is a result of antigenic stimulation. In some embodiments, the antigenic stimulation is a tumor cell. In some embodiments, the antigenic stimulation is cancer. In some embodiments, the antigenic stimulation is a pathogen. In some embodiments, the antigenic stimulation is a virally-infected cell. In vivo and in vitro assays for determining whether an AFFIMER® agent activates, or inhibits an immune response are known in the art. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of an AFFIMER® agent described herein, wherein an AFFIMER® agent binds human PD-L1. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of an AFFIMER® agent described herein, wherein an AFFIMER® agent binds human LAG-3. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of an AFFIMER® agent described herein, wherein an AFFIMER® agent binds human PD-L1 and human LAG-3. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of an AFFIMER® agent described herein, wherein the AFFIMER® agent is an AFFIMER®-containing antibody or receptor trap fusion polypeptide including an AFFIMER® polypeptide that specifically binds to PD-L1 and/or an AFFIMER® polypeptide that specifically binds to LAG-3. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of an encoded AFFIMER® construct, wherein the encoded AFFIMER® construct, when expressed in the patient, produces a recombinant AFFIMER® agent including a LAG-3/PD-L1 AFFIMER® polypeptide. In some embodiments of the methods described herein, a method of activating or enhancing a persistent or long-term immune response to a tumor comprises administering to a subject a therapeutically effective amount of an AFFIMER® agent which binds human PD-L1 and/or LAG-3. In some embodiments, a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of an AFFIMER® agent described herein, wherein the AFFIMER® agent is an AFFIMER®- containing antibody or receptor trap fusion polypeptide including an AFFIMER® polypeptide that specifically binds to PD-L1 and/or LAG-3. In some embodiments, a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of an encoded AFFIMER® construct, wherein the encoded AFFIMER® construct, when expressed in the patient, produces a recombinant AFFIMER® agent including a LAG-3/PD-L1 AFFIMER® polypeptide. In some embodiments of the methods described herein, a method of inducing a persistent or long-term immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of an AFFIMER® agent which binds human PD- L1 and/or human LAG-3. In some embodiments, a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of an AFFIMER® agent described herein, wherein the AFFIMER® agent is an AFFIMER® polypeptide-containing antibody or receptor trap fusion polypeptide including an AFFIMER® polypeptide that specifically binds to PD-L1 and/or LAG- 3. In some embodiments, a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of an encoded AFFIMER® construct, wherein the encoded AFFIMER® construct, when expressed in the patient, produces a recombinant AFFIMER® agent including a LAG-3/PD-L1 AFFIMER® polypeptide. In some embodiments of the methods described herein, a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of an AFFIMER® agent which binds human PD-L1 and/or human LAG-3. In some embodiments, a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of an AFFIMER® agent described herein, wherein the AFFIMER® agent is an AFFIMER®-containing antibody or receptor trap fusion polypeptide including an AFFIMER® polypeptide that specifically binds to PD-L1 and/or LAG- 3. In some embodiments, a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of an encoded AFFIMER® construct, wherein the encoded AFFIMER® construct, when expressed in the patient, produces a recombinant AFFIMER® agent including a LAG-3/PD-L1 AFFIMER® polypeptide. In some embodiments, the AFFIMER® agent is a bispecific agent and binds PD-L1 and LAG-3. In some embodiments, the method of inhibiting growth of a tumor comprises administering to a subject a therapeutically effective amount of an AFFIMER® agent described herein. In some embodiments, the subject is a human. In some embodiments, the subject has a tumor, or the subject had a tumor which was removed. In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is a tumor selected from the group consisting of: colorectal tumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, neuroendocrine tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor, glioblastoma, and head and neck tumor. In some embodiments, the tumor is a colorectal tumor. In some embodiments, the tumor is an ovarian tumor. In some embodiments, the tumor is a lung tumor. In some embodiments, the tumor is a pancreatic tumor. In some embodiments, the tumor is a melanoma tumor. In some embodiments, the tumor is a bladder tumor. To further illustrate, the subject AFFIMER® agents can be used to treat patients suffering from cancer, such as osteosarcoma, rhabdomyosarcoma, neuroblastoma, kidney cancer, leukemia, renal transitional cell cancer, bladder cancer, Wilm's cancer, ovarian cancer, pancreatic cancer, breast cancer (including triple negative breast cancer), prostate cancer, bone cancer, lung cancer (e.g., small cell or non-small cell lung cancer), gastric cancer, colorectal cancer, cervical cancer, synovial sarcoma, head and neck cancer, squamous cell carcinoma, multiple myeloma, renal cell cancer, retinoblastoma, hepatoblastoma, hepatocellular carcinoma, melanoma, rhabdoid tumor of the kidney, Ewing's sarcoma, chondrosarcoma, brain cancer, glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, a primitive neuroectodermal tumor, medulloblastoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, ependymoma, choroid plexus papilloma, polycythemia vera, thrombocythemia, idiopathic myelofibrosis, soft tissue sarcoma, thyroid cancer, endometrial cancer, carcinoid cancer or liver cancer, breast cancer or gastric cancer. In some embodiments of the disclosure, the cancer is metastatic cancer, e.g., of the varieties described above. In some embodiments, the cancer is a hematologic cancer. In some embodiment, the cancer is selected from the group consisting of: acute myelogenous leukemia (AML), Hodgkin lymphoma, multiple myeloma, T-cell acute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myelogenous leukemia (CML), non- Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and cutaneous T-cell lymphoma (CTCL). In some embodiments, the cancer is a PD-L1-positive cancer. In some embodiments, the cancer is an anti-PD-1 or anti-PD-L1 refractory cancer, such as: non-small cell lung carcinoma (NSCLC), colorectal cancer, advanced melanoma, or renal cell carcinoma (RCC). In some embodiments, the cancer is urothelial carcinoma (e.g., previously treated with platinum- containing chemotherapy), and hepatocellular carcinoma (e.g., previously treated with a kinase inhibitor, such as sorafenib). The present disclosure also provides pharmaceutical compositions comprising an AFFIMER® agent described herein and a pharmaceutically acceptable vehicle. In some embodiments, the pharmaceutical compositions find use in immunotherapy. In some embodiments, the pharmaceutical compositions find use in immuno-oncology. In some embodiments, the compositions find use in inhibiting tumor growth. In some embodiments, the pharmaceutical compositions find use in inhibiting tumor growth in a subject (e.g., a human patient). In some embodiments, the compositions find use in treating cancer. In some embodiments, the pharmaceutical compositions find use in treating cancer in a subject (e.g., a human patient). Formulations are prepared for storage and use by combining a purified AFFIMER® agent of the present disclosure with a pharmaceutically acceptable vehicle (e.g., a carrier or excipient). Those of skill in the art generally consider pharmaceutically acceptable carriers, excipients, and/or stabilizers to be inactive ingredients of a formulation or pharmaceutical composition. In some embodiments, an AFFIMER® agent described herein is lyophilized and/or stored in a lyophilized form. In some embodiments, a formulation comprising an AFFIMER® agent described herein is lyophilized. Suitable pharmaceutically acceptable vehicles include but are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes such as Zn-protein complexes; and non-ionic surfactants such as TWEEN or polyethylene glycol (PEG). (Remington: The Science and Practice of Pharmacy, 22.sup.nd Edition, 2012, Pharmaceutical Press, London.). The pharmaceutical compositions of the present disclosure can be administered in any number of ways for either local or systemic treatment. Administration can be topical by epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, and intranasal; oral; or parenteral including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion), or intracranial (e.g., intrathecal or intraventricular). The therapeutic formulation can be in unit dosage form. Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in water or non-aqueous media, or suppositories. In solid compositions such as tablets the principal active ingredient is mixed with a pharmaceutical carrier. Conventional tableting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and diluents (e.g., water). These can be used to form a solid preformulation composition containing a homogeneous mixture of a compound of the present disclosure, or a non-toxic pharmaceutically acceptable salt thereof. The solid preformulation composition is then subdivided into unit dosage forms of a type described above. The tablets, pills, etc. of the formulation or composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner composition covered by an outer component. Furthermore, the two components can be separated by an enteric layer that serves to resist disintegration and permits the inner component to pass intact through the stomach or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials include a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate. The AFFIMER® agents described herein can also be entrapped in microcapsules. Such microcapsules are prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions as described in Remington: The Science and Practice of Pharmacy, 22.sup.nd Edition, 2012, Pharmaceutical Press, London. In some embodiments, pharmaceutical formulations include an AFFIMER® agent of the present disclosure complexed with liposomes. Methods to produce liposomes are known to those of skill in the art. For example, some liposomes can be generated by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through filters of defined pore size to yield liposomes with the desired diameter. In some embodiments, sustained-release preparations comprising AFFIMER® agents described herein can be produced. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing an AFFIMER® agent, where the matrices are in the form of shaped articles (e.g., films or microcapsules). Examples of sustained-release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl- methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7 ethyl-L- glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT.TM. (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)- 3-hydroxybutyric acid. In some embodiments, in addition to administering an AFFIMER® agent described herein, the method or treatment further comprises administering at least one additional immune response stimulating agent. In some embodiments, the additional immune response stimulating agent includes, but is not limited to, a colony stimulating factor (e.g., granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), stem cell factor (SCF)), an interleukin (e.g., IL- 1, IL2, IL-3, IL-7, IL-12, IL-15, IL-18), a checkpoint inhibitor, an antibody that blocks immunosuppressive functions (e.g., an anti-CTLA-4 antibody, anti-CD28 antibody, anti-CD3 antibody), a toll-like receptor (e.g., TLR4, TLR7, TLR9), or a member of the B7 family (e.g., CD80, CD86). An additional immune response stimulating agent can be administered prior to, concurrently with, and/or subsequently to, administration of the AFFIMER® agent. Pharmaceutical compositions comprising an AFFIMER® agent and the immune response stimulating agent(s) are also provided. In some embodiments, the immune response stimulating agent comprises 1, 2, 3, or more immune response stimulating agents. In some embodiments, in addition to administering an AFFIMER® agent described herein, the method or treatment further comprises administering at least one additional therapeutic agent. An additional therapeutic agent can be administered prior to, concurrently with, and/or subsequently to, administration of the AFFIMER® agent. Pharmaceutical compositions comprising an AFFIMER® agent and the additional therapeutic agent(s) are also provided. In some embodiments, the at least one additional therapeutic agent comprises 1, 2, 3, or more additional therapeutic agents. Combination therapy with two or more therapeutic agents often uses agents that work by different mechanisms of action, although this is not required. Combination therapy using agents with different mechanisms of action may result in additive or synergetic effects. Combination therapy may allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the AFFIMER® agent. Combination therapy may decrease the likelihood that resistant cancer cells will develop. In some embodiments, combination therapy comprises a therapeutic agent that affects the immune response (e.g., enhances or activates the response) and a therapeutic agent that affects (e.g., inhibits or kills) the tumor/cancer cells. In some embodiments of the methods described herein, the combination of an AFFIMER® agent described herein and at least one additional therapeutic agent results in additive or synergistic results. In some embodiments, the combination therapy results in an increase in the therapeutic index of the AFFIMER® agent. In some embodiments, the combination therapy results in an increase in the therapeutic index of the additional therapeutic agent(s). In some embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of the AFFIMER® agent. In some embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of the additional therapeutic agent(s). Useful classes of therapeutic agents include, for example, anti-tubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, anti-folates, anti-metabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like. In some embodiments, the second therapeutic agent is an alkylating agent, an antimetabolite, an antimitotic, a topoisomerase inhibitor, or an angiogenesis inhibitor. Therapeutic agents that may be administered in combination with the AFFIMER® agent described herein include chemotherapeutic agents. Thus, in some embodiments, the method or treatment involves the administration of an AFFIMER® agent of the present disclosure in combination with a chemotherapeutic agent or in combination with a cocktail of chemotherapeutic agents. Treatment with an AFFIMER® agent can occur prior to, concurrently with, or subsequent to administration of chemotherapies. Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously. Preparation and dosing schedules for such chemotherapeutic agents can be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in The Chemotherapy Source Book, 4.sup.th Edition, 2008, M. C. Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, Pa. Chemotherapeutic agents useful in the present disclosure include but are not limited to, alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamime; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6- azauridine, carmofur, cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenishers such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); taxoids, e.g. paclitaxel (TAXOL) and docetaxel (TAXOTERE); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; ibandronate; CPT11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine (XELODA); and pharmaceutically acceptable salts, acids or derivatives of any of the above. Chemotherapeutic agents also include anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. In some embodiments, the additional therapeutic agent is cisplatin. In some embodiments, the additional therapeutic agent is carboplatin. In some embodiments of the methods described herein, the chemotherapeutic agent is a topoisomerase inhibitor. Topoisomerase inhibitors are chemotherapy agents that interfere with the action of a topoisomerase enzyme (e.g., topoisomerase I or II). Topoisomerase inhibitors include but are not limited to, doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D, etoposide, topotecan HCl, teniposide (VM-26), and irinotecan, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these. In some embodiments, the additional therapeutic agent is irinotecan. In some embodiments, the chemotherapeutic agent is an anti-metabolite. An anti- metabolite is a chemical with a structure that is similar to a metabolite required for normal biochemical reactions, yet different enough to interfere with at least one normal function of cells, such as cell division. Anti-metabolites include but are not limited to, gemcitabine, fluorouracil, capecitabine, methotrexate sodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these. In some embodiments, the additional therapeutic agent is gemcitabine. In some embodiments of the methods described herein, the chemotherapeutic agent is an antimitotic agent, including, but not limited to, agents that bind tubulin. In some embodiments, the agent is a taxane. In some embodiments, the agent is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In some embodiments, the agent is paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel (nab-paclitaxel; ABRAXANE), DHA-paclitaxel, or PG-paclitaxel. In certain alternative embodiments, the antimitotic agent comprises a vinca alkaloid, such as vincristine, vinblastine, vinorelbine, or vindesine, or pharmaceutically acceptable salts, acids, or derivatives thereof. In some embodiments, the antimitotic agent is an inhibitor of kinesin Eg5 or an inhibitor of a mitotic kinase such as Aurora A or Plk1. In some embodiments, the additional therapeutic agent is paclitaxel. In some embodiments, the additional therapeutic agent is nab-paclitaxel. In some embodiments of the methods described herein, an additional therapeutic agent comprises an agent such as a small molecule. For example, treatment can involve the combined administration of an AFFIMER® agent of the present disclosure with a small molecule that acts as an inhibitor against tumor-associated antigens including, but not limited to, EGFR, HER2 (ErbB2), and/or VEGF. In some embodiments, an AFFIMER® agent of the present disclosure is administered in combination with a protein kinase inhibitor selected from the group consisting of: gefitinib (IRESSA), erlotinib (TARCEVA), sunitinib (SUTENT), lapatanib, vandetanib (ZACTIMA), AEE788, CI-1033, cediranib (RECENTIN), sorafenib (NEXAVAR), and pazopanib (GW786034B). In some embodiments, an additional therapeutic agent comprises an mTOR inhibitor. In some embodiments of the methods described herein, the additional therapeutic agent is a small molecule that inhibits a cancer stem cell pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Notch pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Wnt pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the BMP pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Hippo pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the mTOR/AKR pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the RSPO/LGR pathway. In some embodiments of the methods described herein, an additional therapeutic agent comprises a biological molecule, such as an antibody. For example, treatment can involve the combined administration of an AFFIMER® agent of the present disclosure with antibodies against tumor-associated antigens including, but not limited to, antibodies that bind EGFR, HER2/ErbB2, and/or VEGF. In some embodiments, the additional therapeutic agent is an antibody specific for a cancer stem cell marker. In some embodiments, the additional therapeutic agent is an antibody that binds a component of the Notch pathway. In some embodiments, the additional therapeutic agent is an antibody that binds a component of the Wnt pathway. In some embodiments, the additional therapeutic agent is an antibody that inhibits a cancer stem cell pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Notch pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Wnt pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the BMP pathway. In some embodiments, the additional therapeutic agent is an antibody that inhibits .beta.-catenin signaling. In some embodiments, the additional therapeutic agent is an antibody that is an angiogenesis inhibitor (e.g., an anti-VEGF or VEGF receptor antibody). In some embodiments, the additional therapeutic agent is bevacizumab (AVASTIN), ramucirumab, trastuzumab (HERCEPTIN), pertuzumab (OMNITARG), panitumumab (VECTIBIX), nimotuzumab, zalutumumab, or cetuximab (ERBITUX). In some embodiments of the methods described herein, the additional therapeutic agent is an antibody that modulates the immune response. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-TIM-3 antibody, or an anti-TIGIT antibody. Furthermore, treatment with an AFFIMER® agent described herein can include combination treatment with other biologic molecules, such as at least one cytokine (e.g., lymphokines, interleukins, tumor necrosis factors, and/or growth factors) or can be accompanied by surgical removal of tumors, removal of cancer cells, or any other therapy deemed necessary by a treating physician. In some embodiments, the additional therapeutic agent is an immune response stimulating agent. In some embodiments of the methods described herein, the AFFIMER® agent can be combined with a growth factor selected from the group consisting of: adrenomedullin (AM), angiopoietin (Ang), BMPs, BDNF, EGF, erythropoietin (EPO), FGF, GDNF, G-CSF, GM-CSF, GDF9, HGF, HDGF, IGF, migration-stimulating factor, myostatin (GDF-8), NGF, neurotrophins, PDGF, thrombopoietin, TGF-α, TGF-β, TNF-α, VEGF, P1GF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, and IL-18. In some embodiments of the methods described herein, the additional therapeutic agent is an immune response stimulating agent. In some embodiments, the immune response stimulating agent is selected from the group consisting of granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 3 (IL-3), interleukin 12 (IL-12), interleukin 1 (IL-1), interleukin 2 (IL-2), B7-1 (CD80), B7-2 (CD86), 4-1BB ligand, anti-CD3 antibody, anti-CTLA-4 antibody, anti-TIGIT antibody, anti-PD-1 antibody, anti-LAG-3 antibody, and anti-TIM-3 antibody. In some embodiments of the methods described herein, an immune response stimulating agent is selected from the group consisting of: a modulator of PD-1 activity, a modulator of PD- L2 activity, a modulator of CTLA-4 activity, a modulator of CD28 activity, a modulator of CD80 activity, a modulator of CD86 activity, a modulator of 4-1BB activity, an modulator of OX40 activity, a modulator of KIR activity, a modulator of Tim-3 activity, a modulator of LAG- 3 activity, a modulator of CD27 activity, a modulator of CD40 activity, a modulator of GITR activity, a modulator of TIGIT activity, a modulator of CD20 activity, a modulator of CD96 activity, a modulator of IDO1 activity, a cytokine, a chemokine, an interferon, an interleukin, a lymphokine, a member of the tumor necrosis factor (TNF) family, and an immunostimulatory oligonucleotide. In some embodiments of the methods described herein, an immune response stimulating agent is selected from the group consisting of: a PD-1 antagonist, a PD-L2 antagonist, a CTLA-4 antagonist, a CD80 antagonist, a CD86 antagonist, a KIR antagonist, a Tim-3 antagonist, a LAG- 3 antagonist, a TIGIT antagonist, a CD20 antagonist, a CD96 antagonist, and/or an IDO1 antagonist. In some embodiments of the methods described herein, the PD-1 antagonist is an antibody that specifically binds PD-1. In some embodiments, the antibody that binds PD-1 is KEYTRUDA (MK-3475), pidilizumab (CT-011), nivolumab (OPDIVO, BMS-936558, MDX- 1106), MEDI0680 (AMP-514), REGN2810, BGB-A317, PDR-001, or STI-A1110. In some embodiments, the antibody that binds PD-1 is described in PCT Publication WO 2014/179664, for example, an antibody identified as APE2058, APE1922, APE1923, APE1924, APE 1950, or APE1963, or an antibody containing the CDR regions of any of these antibodies. In other embodiments, the PD-1 antagonist is a fusion protein that includes PD-L2, for example, AMP- 224. In other embodiments, the PD-1 antagonist is a peptide inhibitor, for example, AUNP-12. In some embodiments, the CTLA-4 antagonist is an antibody that specifically binds CTLA-4. In some embodiments, the antibody that binds CTLA-4 is ipilimumab (YERVOY) or tremelimumab (CP-675,206). In some embodiments, the CTLA-4 antagonist a CTLA-4 fusion protein, for example, KAHR-102. In some embodiments, the LAG-3 antagonist is an antibody that specifically binds LAG- 3. In some embodiments, the antibody that binds LAG-3 is IMP701, IMP731, BMS-986016, LAG525, and GSK2831781. In some embodiments, the LAG-3 antagonist includes a soluble LAG-3 receptor, for example, IMP321. In some embodiments, the KIR antagonist is an antibody that specifically binds KIR. In some embodiments, the antibody that binds KIR is lirilumab. In some embodiments, an immune response stimulating agent is selected from the group consisting of: a CD28 agonist, a 4-1BB agonist, an OX40 agonist, a CD27 agonist, a CD80 agonist, a CD86 agonist, a CD40 agonist, and a GITR agonist. p In some embodiments, the OX40 agonist includes OX40 ligand, or an OX40-binding portion thereof. For example, the OX40 agonist may be MEDI6383. In some embodiments, the OX40 agonist is an antibody that specifically binds OX40. In some embodiments, the antibody that binds OX40 is MEDI6469, MEDI0562, or MOXR0916 (RG7888). In some embodiments, the OX40 agonist is a vector (e.g., an expression vector or virus, such as an adenovirus) capable of expressing OX40 ligand. In some embodiments the OX40-expressing vector is Delta-24-RGDOX or DNX2401. In some embodiments, the 4-1BB (CD137) agonist is a binding molecule, such as an anticalin. In some embodiments, the anticalin is PRS-343. In some embodiments, the 4-1BB agonist is an antibody that specifically binds 4-1BB. In some embodiments, antibody that binds 4-1BB is PF-2566 (PF-05082566) or urelumab (BMS-663513). In some embodiments, the CD27 agonist is an antibody that specifically binds CD27. In some embodiments, the antibody that binds CD27 is varlilumab (CDX-1127). In some embodiments, the GITR agonist comprises GITR ligand or a GITR-binding portion thereof. In some embodiments, the GITR agonist is an antibody that specifically binds GITR. In some embodiments, the antibody that binds GITR is TRX518, MK-4166, or INBRX- 110. In some embodiments, immune response stimulating agents include but are not limited to, cytokines such as chemokines, interferons, interleukins, lymphokines, and members of the tumor necrosis factor (TNF) family. In some embodiments, immune response stimulating agents include immunostimulatory oligonucleotides, such as CpG dinucleotides. In some embodiments, an immune response stimulating agent includes, but is not limited to, anti-PD-L1 antibodies, anti-PD-1 antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-CD28 antibodies, anti-CD80 antibodies, anti-CD86 antibodies, anti-4-1BB antibodies, anti- OX40 antibodies, anti-KIR antibodies, anti-Tim-3 antibodies, anti-LAG-3 antibodies, anti-CD27 antibodies, anti-CD40 antibodies, anti-GITR antibodies, anti-TIGIT antibodies, anti-CD20 antibodies, anti-CD96 antibodies, or anti-IDO1 antibodies. In some embodiments, the AFFIMER® agents disclosed herein may be used alone, or in association with radiation therapy. In some embodiments, the AFFIMER® agents disclosed herein may be used alone, or in association with targeted therapies. Examples of targeted therapies include: hormone therapies, signal transduction inhibitors (e.g., EGFR inhibitors, such as cetuximab (Erbitux) and erlotinib (Tarceva)); HER2 inhibitors (e.g., trastuzumab (Herceptin) and pertuzumab (Perjeta)); BCR- ABL inhibitors (such as imatinib (Gleevec) and dasatinib (Sprycel)); ALK inhibitors (such as crizotinib (Xalkori) and ceritinib (Zykadia)); BRAF inhibitors (such as vemurafenib (Zelboraf) and dabrafenib (Tafinlar)), gene expression modulators, apoptosis inducers (e.g., bortezomib (Velcade) and carfilzomib (Kyprolis)), angiogenesis inhibitors (e.g., bevacizumab (Avastin) and ramucirumab (Cyramza), monoclonal antibodies attached to toxins (e.g., brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla)). In some embodiments, the AFFIMER® agents of the disclosure may be used in combination with an anti-cancer therapeutic agent or immunomodulatory drug such as an immunomodulatory receptor inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to the receptor. In some embodiments of the disclosure, an AFFIMER® agent is administered in with a STING agonist, for example, as part of a pharmaceutical composition. The cyclic-di-nucleotides (CDNs) cyclic-di-AMP (produced by Listeria monocytogenes and other bacteria) and its analogs cyclic-di-GMP and cyclic-GMP-AMP are recognized by the host cell as a pathogen associated molecular pattern (PAMP), which bind to the pathogen recognition receptor (PRR) known as Stimulator of INterferon Genes (STING). STING is an adaptor protein in the cytoplasm of host mammalian cells which activates the TANK binding kinase (TBK1)-IRF3 and the NF-.kappa.B signaling axis, resulting in the induction of IFN-.beta. and other gene products that strongly activate innate immunity. It is now recognized that STING is a component of the host cytosolic surveillance pathway, that senses infection with intracellular pathogens and in response induces the production of IFN-α, leading to the development of an adaptive protective pathogen-specific immune response consisting of both antigen-specific CD4+ and CD8+ T cells as well as pathogen-specific antibodies. U.S. Pat. NOS: 7,709,458 and 7,592,326; PCT Publication NOS: WO2007/054279, WO2014/093936, WO2014/179335, WO2014/189805, WO2015/185565, WO2016/096174, WO2016/145102, WO2017/027645, WO2017/027646, and WO2017/075477; and Yan et al., Bioorg. Med. Chem Lett.18:5631-4, 2008. In some embodiments of the disclosure, an AFFIMER® agent of the disclosure is administered in association with an Akt inhibitor. Exemplary AKT inhibitors include GDC0068 (also known as GDC-0068, ipatasertib and RG7440), MK-2206, perifosine (also known as KRX- 0401), GSK690693, AT7867, triciribine, CCT128930, A-674563, PHT-427, Akti-1/2, afuresertib (also known as GSK2110183), AT13148, GSK2141795, BAY1125976, uprosertib (aka GSK2141795), Akt Inhibitor VIII (1,3-dihydro-1-[1-[[4-(6-phenyl-1H-imidazo[4,5- g]quinoxalin-7-yl)phenyl]m- ethyl]-4-piperidinyl]-2H-benzimidazol-2-one), Akt Inhibitor X (2- chloro-N,N-diethyl-10H-phenoxazine-10-butanamine, monohydrochloride), MK-2206 (8-(4-(1- aminocyclobutyl)phenyl)-9-phenyl-[1,2,4]triazolo[3,4-f][- 1,6]naphthyridin-3(2H)-one), uprosertib (N-((S)-1-amino-3-(3,4-difluorophenyl)propan-2-yl)-5-chloro-4-(4-chloro-1- -methyl- 1H-pyrazol-5-yl)furan-2-carboxamide), ipatasertib ((S)-2-(4-chlorophenyl)-1-(4-((5R,7R)-7- hydroxy-5-methyl-6,7-dihydro-5H-c- yclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3- (isopropylamino)propan-1-one)- , AZD 5363 (4-Piperidinecarboxamide, 4-amino-N-[(1S)-1-(4- chlorophenyl)-3-hydroxypropyl]-1-(7H-pyrrolo[2,3-d]p- yrimidin-4-yl)), perifosine, GSK690693, GDC-0068, tricirbine, CCT128930, A-674563, PF-04691502, AT7867, miltefosine, PHT-427, honokiol, triciribine phosphate, and KP372-1A (10H-indeno[2,1- e]tetrazolo[1,5-b][1,2,4]triazin-10-one), Akt Inhibitor IX (CAS 98510-80-6). Additional Akt inhibitors include: ATP-competitive inhibitors, e.g. isoquinoline-5-sulfonamides (e.g., H-8, H- 89, NL-71-101), azepane derivatives (e.g., (-)-balanol derivatives), aminofurazans (e.g., GSK690693), heterocyclic rings (e.g., 7-azaindole, 6-phenylpurine derivatives, pyrrolo[2,3- d]pyrimidine derivatives, CCT128930, 3-aminopyrrolidine, anilinotriazole derivatives, spiroindoline derivatives, AZD5363, A-674563, A-443654), phenylpyrazole derivatives (e.g., AT7867, AT13148), thiophenecarboxamide derivatives (e.g., Afuresertib (GSK2110183), 2- pyrimidyl-5-amidothiophene derivative (DC120), uprosertib (GSK2141795); Allosteric inhibitors, e.g., 2,3-diphenylquinoxaline analogues (e.g., 2,3-diphenylquinoxaline derivatives, triazolo[3,4-f][1,6]naphthyridin-3(2H)-one derivative (MK-2206)), alkylphospholipids (e.g., Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, ET-18-OCH3) ilmofosine (BM 41.440), miltefosine (hexadecylphosphocholine, HePC), perifosine (D-21266), erucylphosphocholine (ErPC), erufosine (ErPC3, erucylphosphohomocholine), indole-3-carbinol analogues (e.g., indole-3-carbinol, 3-chloroacetylindole, diindolylmethane, diethyl 6-methoxy- 5,7-dihydroindolo [2,3-b]carbazole-2,10-dicarboxylate (SR13668), OSU-A9), Sulfonamide derivatives (e.g., PH-316, PHT-427), thiourea derivatives (e.g., PIT-1, PIT-2, DM-PIT-1, N-[(1- methyl-1H-pyrazol-4-yl)carbonyl]-N'-(3-bromophenyl)-thiourea), purine derivatives (e.g., Triciribine (TCN, NSC 154020), triciribine mono-phosphate active analogue (TCN-P),4-amino- pyrido[2,3-d]pyrimidine derivative API-1, 3-phenyl-3H-imidazo[4,5-b]pyridine derivatives, ARQ 092), BAY 1125976, 3-methyl-xanthine, quinoline-4-carboxamide, 2-[4-(cyclohexa-1,3- dien-1-yl)-1H-pyrazol-3-yl]phenol, 3-oxo-tirucallic acid, 3.alpha.- and 3.beta.-acetoxy-tirucallic acids, acetoxy-tirucallic acid; and irreversible inhibitors, e.g., natural products, antibiotics, Lactoquinomycin, Frenolicin B, kalafungin, medermycin, Boc-Phe-vinyl ketone, 4- hydroxynonenal (4-HNE), 1,6-naphthyridinone derivatives, and imidazo-1,2-pyridine derivatives. In some embodiments of the disclosure, an AFFIMER® agent of the disclosure is administered in association with a MEK inhibitor. Exemplary MEK inhibitors include AZD6244 (Selumetinib), PD0325901, GSK1120212 (Trametinib), U0126-EtOH, PD184352, RDEA119 (Rafametinib), PD98059, BIX 02189, MEK162 (Binimetinib), AS-703026 (Pimasertib), SL-327, BIX02188, AZD8330, TAK-733, cobimetinib and PD318088. In some embodiments of the disclosure, an AFFIMER® agent of the disclosure is administered in association with both an anthracycline such as doxorubicin and cyclophosphamide, including pegylated liposomal doxorubicin . In some embodiments of the disclosure, an AFFIMER® agent of the disclosure is administered in association with both an anti-CD20 antibody and an anti-CD3 antibody, or a bispecific CD20/CD3 binder (including a CD20/CD3 BiTE). In some embodiments of the disclosure, an AFFIMER® agent of the disclosure is administered in association with a CD73 inhibitor, a CD39 inhibitor or both. These inhibitors can be CD73 binders or CD39 binders (such as antibody, antibody fragments or antibody mimetics) that inhibit the ectonucleosidase activity. The inhibitor may be a small molecule inhibitor of the ectonucleosidase activity, such as 6-N,N-Diethyl-β-γ-dibromomethylene-D-adenosine-5′- triphosphate trisodium salt hydrate, PSB069, or PSB 06126. In some embodiments of the disclosure, an AFFIMER® agent of the disclosure is administered in association with an inhibitor poly ADP ribose polymerase (PARP). Exemplary PARP inhibitors include Olaparib, Niraparib, Rucaparib, Talazoparib, Veliparib, CEP9722, MK4827 and BGB-290. In some embodiments of the disclosure, an AFFIMER® agent of the disclosure is administered in association with an oncolytic virus. An exemplary oncolytic virus is Talimogene Laherparepvec. In some embodiments of the disclosure, an AFFIMER® agent of the disclosure is administered in association with an CSF-1 antagonist, such as an agent that binds to CSF-1 or CSF1R and inhibits the interaction of CSF-1 with CSF1R on macrophage. Exemplary CSF-1 antagonists include Emactuzumab and FPA008. In some embodiments of the disclosure, an AFFIMER® agent of the disclosure is administered in association with an anti-CD38 antibody. Exemplary anti-CD39 antibodies include Daratumumab and Isatuximab. In some embodiments of the disclosure, an AFFIMER® agent of the disclosure is administered in association with an anti-CD40 antibody. Exemplary anti-CD40 antibodies include Selicrelumab and Dacetuzumab. In some embodiments of the disclosure, an AFFIMER® agent of the disclosure is administered in association with an inhibitor of anaplatic lymphoma kinase (ALK). Exemplary ALK inhibitors include Alectinib, Crizotinib and Ceritinib. In some embodiments of the disclosure, an AFFIMER® agent of the disclosure is administered in association with multikinase inhibitor that inhibits at least one selected from the group consisting of the family members of VEGFR, PDGFR and FGFR, or an anti-angiogenesis inhibitor. Exemplary inhibitors include Axitinib, Cediranib, Linifanib, Motesanib, Nintedanib, Pazopanib, Ponatinib, Regorafenib, Sorafenib, Sunitinib, Tivozanib, Vatalanib, LY2874455, or SU5402. In some embodiments of the disclosure, an AFFIMER® agent of the disclosure is administered in conjunction with at least one vaccine intended to stimulate an immune response to at least one predetermined antigen. The antigen(s) may be administered directly to the individual, or may be expressed within the individual from, for example, a tumor cell vaccine (e.g., GVAX) which may be autologous or allogenic, a dendritic cell vaccine, a DNA vaccine, an RNA vaccine, a viral-based vaccine, a bacterial or yeast vaccine (e.g., a Listeria monocytogenes or Saccharomyces cerevisiae), etc. See, e.g., Guo et al., Adv. Cancer Res.2013; 119: 421-475; Obeid et al., Semin Oncol.2015 August; 42(4): 549-561. The target antigen may also be a fragment or fusion polypeptide comprising an immunologically active portion of the antigens listed in the table. In some embodiments of the disclosure, an AFFIMER® agent of the disclosure is administered in association with at least one antiemetic including, but not limited to: casopitant (GlaxoSmithKline), Netupitant (MGI-Helsinn) and other NK-1 receptor antagonists, palonosetron (sold as Aloxi by MGI Pharma), aprepitant (sold as Emend by Merck and Co.; Rahway, N.J.), diphenhydramine (sold as Benadryl by Pfizer; New York, N.Y.), hydroxyzine (sold as Atarax by Pfizer; New York, N.Y.), metoclopramide (sold as Reglan by AH Robins Co,; Richmond, Va.), lorazepam (sold as Ativan by Wyeth; Madison, N.J.), alprazolam (sold as Xanax by Pfizer; New York, N.Y.), haloperidol (sold as Haldol by Ortho-McNeil; Raritan, N.J.), droperidol (Inapsine), dronabinol (sold as Marinol by Solvay Pharmaceuticals, Inc.; Marietta, Ga.), dexamethasone (sold as Decadron by Merck and Co.; Rahway, N.J.), methylprednisolone (sold as Medrol by Pfizer; New York, N.Y.), prochlorperazine (sold as Compazine by Glaxosmithkline; Research Triangle Park, N.C.), granisetron (sold as Kytril by Hoffmann-La Roche Inc.; Nutley, N.J.), ondansetron (sold as Zofran by Glaxosmithkline; Research Triangle Park, N.C.), dolasetron (sold as Anzemet by Sanofi-Aventis; New York, N.Y.), tropisetron (sold as Navoban by Novartis; East Hanover, N.J.). Other side effects of cancer treatment include red and white blood cell deficiency. Accordingly, in some embodiments of the disclosure, an AFFIMER® agent is administered in association with an agent which treats or prevents such a deficiency, such as, e.g., filgrastim, PEG-filgrastim, erythropoietin, epoetin alfa or darbepoetin alfa. In some embodiments of the disclosure, an AFFIMER® agent of the disclosure is administered in association with anti-cancer radiation therapy. For example, in some embodiments of the disclosure, the radiation therapy is external beam therapy (EBT): a method for delivering a beam of high-energy X-rays to the location of the tumor. The beam is generated outside the patient (e.g., by a linear accelerator) and is targeted at the tumor site. These X-rays can destroy the cancer cells and careful treatment planning allows the surrounding normal tissues to be spared. No radioactive sources are placed inside the patient's body. In some embodiments of the disclosure, the radiation therapy is proton beam therapy: a type of conformal therapy that bombards the diseased tissue with protons instead of X-rays. In some embodiments of the disclosure, the radiation therapy is conformal external beam radiation therapy: a procedure that uses advanced technology to tailor the radiation therapy to an individual's body structures. In some embodiments of the disclosure, the radiation therapy is brachytherapy: the temporary placement of radioactive materials within the body, usually employed to give an extra dose--or boost--of radiation to an area. In some embodiments of the methods described herein, the treatment involves the administration of an AFFIMER® agent of the present disclosure in combination with anti-viral therapy. Treatment with an AFFIMER® agent can occur prior to, concurrently with, or subsequent to administration of antiviral therapy. The anti-viral drug used in combination therapy will depend upon the virus the subject is infected with. Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously. It will be appreciated that the combination of an AFFIMER® agent described herein and at least one additional therapeutic agent may be administered in any order or concurrently. In some embodiments, the AFFIMER® agent will be administered to patients that have previously undergone treatment with a second therapeutic agent. In certain other embodiments, the AFFIMER® agent and a second therapeutic agent will be administered substantially simultaneously or concurrently. For example, a subject may be given an AFFIMER® agent while undergoing a course of treatment with a second therapeutic agent (e.g., chemotherapy). In some embodiments, an AFFIMER® agent will be administered within 1 year of the treatment with a second therapeutic agent. In certain alternative embodiments, an AFFIMER® agent will be administered within 10, 8, 6, 4, or 2 months of any treatment with a second therapeutic agent. In certain other embodiments, an AFFIMER® agent will be administered within 4, 3, 2, or 1 weeks of any treatment with a second therapeutic agent. In some embodiments, an AFFIMER® agent will be administered within 5, 4, 3, 2, or 1 days of any treatment with a second therapeutic agent. It will further be appreciated that the two (or more) agents or treatments may be administered to the subject within a matter of hours or minutes (e.g., substantially simultaneously). For the treatment of a disease, the appropriate dosage of an AFFIMER® agent of the present disclosure depends on the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, whether the AFFIMER® agent is administered for therapeutic or preventative purposes, previous therapy, the patient's clinical history, and so on, all at the discretion of the treating physician. The AFFIMER® agent can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is affected or a diminution of the disease state is achieved (e.g., reduction in tumor size). Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient and will vary depending on the relative potency of an individual agent. The administering physician can determine optimum dosages, dosing methodologies, and repetition rates. In some embodiments, dosage is from 0.01 µg to 100 mg/kg of body weight, from 0.1 µg to 100 mg/kg of body weight, from 1 µg to 100 mg/kg of body weight, from 1 mg to 100 mg/kg of body weight, 1 mg to 80 mg/kg of body weight from 10 mg to 100 mg/kg of body weight, from 10 mg to 75 mg/kg of body weight, or from 10 mg to 50 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is from about 0.1 mg to about 20 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 0.1 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 0.25 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 0.5 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 1 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 1.5 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 2 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 2.5 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 5 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 7.5 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 10 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 12.5 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 15 mg/kg of body weight. In some embodiments, the dosage can be given once or more daily, weekly, monthly, or yearly. In some embodiments, the AFFIMER® agent is given once every week, once every two weeks, once every three weeks, or once every four weeks. In some embodiments, an AFFIMER® agent may be administered at an initial higher "loading" dose, followed by at least one lower dose. In some embodiments, the frequency of administration may also change. In some embodiments, a dosing regimen may comprise administering an initial dose, followed by additional doses (or "maintenance" doses) once a week, once every two weeks, once every three weeks, or once every month. For example, a dosing regimen may comprise administering an initial loading dose, followed by a weekly maintenance dose of, for example, one-half of the initial dose. In some embodiments, a dosing regimen comprises administering an initial loading dose, followed by maintenance doses of, for example one-half of the initial dose every other week. In some embodiments, a dosing regimen comprises administering three initial doses for 3 weeks, followed by maintenance doses of, for example, the same amount every other week. As is known to those of skill in the art, administration of any therapeutic agent may lead to side effects and/or toxicities. In some cases, the side effects and/or toxicities are so severe as to preclude administration of the particular agent at a therapeutically effective dose. In some cases, drug therapy must be discontinued, and other agents may be tried. However, many agents in the same therapeutic class often display similar side effects and/or toxicities, meaning that the patient either has to stop therapy, or if possible, suffer from the unpleasant side effects associated with the therapeutic agent. In some embodiments, the dosing schedule may be limited to a specific number of administrations or "cycles". In some embodiments, the AFFIMER® agent is administered for 3, 4, 5, 6, 7, 8, or more cycles. For example, the AFFIMER® agent is administered every 2 weeks for 6 cycles, the AFFIMER® agent is administered every 3 weeks for 6 cycles, the AFFIMER® agent is administered every 2 weeks for 4 cycles, the AFFIMER® agent is administered every 3 weeks for 4 cycles, etc. Dosing schedules can be decided upon and subsequently modified by those skilled in the art. Thus, the present disclosure provides methods of administering to a subject the polypeptides or agents described herein comprising using an intermittent dosing strategy for administering at least one agent (e.g., two or three agents), which may reduce side effects and/or toxicities associated with administration of an AFFIMER® agent, chemotherapeutic agent, etc. In some embodiments, a method for treating cancer in a human subject comprises administering to the subject a therapeutically effective dose of an AFFIMER® agent in combination with a therapeutically effective dose of a chemotherapeutic agent, wherein one or both of the agents are administered according to an intermittent dosing strategy. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of an AFFIMER® agent to the subject and administering subsequent doses of the AFFIMER® agent about once every 2 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of an AFFIMER® agent to the subject and administering subsequent doses of the AFFIMER® agent about once every 3 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of an AFFIMER® agent to the subject and administering subsequent doses of the AFFIMER® agent about once every 4 weeks. In some embodiments, the AFFIMER® agent is administered using an intermittent dosing strategy and the chemotherapeutic agent is administered weekly. In some embodiments, the disclosure also provides methods for treating subjects using an AFFIMER® agent of the disclosure, wherein the subject suffers from a viral infection. In some embodiments, the viral infection is infection with a virus selected from the group consisting of human immunodeficiency virus (HIV), hepatitis virus (A, B, or C), herpes virus (e.g., VZV, HSV-I, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus or arboviral encephalitis virus. In some embodiments, the disclosure provides methods for treating subjects using an AFFIMER® agent thereof of the disclosure, wherein the subject suffers from a bacterial infection. In some embodiments, the bacterial infection is infection with a bacterium selected from the group consisting of Chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, Legionella, Corynebacterium diphtheriae, Salmonella, bacilli, Vibrio cholerae, Clostridium tetan, Clostridium botulinum, Bacillus anthricis, Yersinia pestis, Mycobacterium leprae, Mycobacterium lepromatosis, and Borriella. In some embodiments, the disclosure provides methods for treating subjects using an AFFIMER® agent of the disclosure, wherein the subject suffers from a fungal infection. In some embodiments, the fungal infection is infection with a fungus selected from the group consisting of Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum. In some embodiments, the disclosure provides methods for treating subjects using an AFFIMER® agent of the disclosure, wherein the subject suffers from a parasitic infection. In some embodiments, the parasitic infection is infection with a parasite selected from the group consisting of Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba, Giardia lambia, Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii and Nippostrongylus brasiliensis. Additional Aspects of the Disclosure Additional aspects of the disclosure are provided in the numbered paragraphs below (The symbol “+” is representative of a flexible or rigid linker linking the designated AFFIMER® polypeptides). AVA21-01 (AVA04-269+AVA19-157) 1. A bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73, the first peptide comprises the amino acid sequence of KEYGPEEWW (SEQ ID NO: 221), and the second peptide comprises the amino acid sequence of GDYEQVLIH (SEQ ID NO: 222); and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 61, the first peptide comprises the amino acid sequence of INVQEDLIQT (SEQ ID NO: 206), and the second peptide comprises the amino acid sequence of SIYQAEELE (SEQ ID NO: 212). 2. The bispecifc fusion protein of paragraph 1 further comprising one or more linker, preferably a rigid linker, more preferably a rigid linker comprising the sequence of A(EAAAK)n (SEQ ID NO: 86). 3. The bispecifc fusion protein of paragraph 1 or 2 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 1. AVA21-02 (AVA04-269+AVA19-158) 4. A bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73, the first peptide comprises the amino acid sequence of KEYGPEEWW (SEQ ID NO: 221), and the second peptide comprises the amino acid sequence of GDYEQVLIH (SEQ ID NO: 222); and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 62, the first peptide comprises the amino acid sequence of DATDGDGVS (SEQ ID NO: 207), and the second peptide comprises the amino acid sequence of FWGDEWDVL (SEQ ID NO: 213). 5. The bispecifc fusion protein of paragraph 4 further comprising one or more linker, preferably a rigid linker, more preferably a rigid linker comprising the sequence of A(EAAAK)n (SEQ ID NO: 86). 6. The bispecifc fusion protein of paragraph 4 or 5 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 2. AVA21-03 (AVA04-269+AVA19-01) 7. A bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73, the first peptide comprises the amino acid sequence of KEYGPEEWW (SEQ ID NO: 221), and the second peptide comprises the amino acid sequence of GDYEQVLIH (SEQ ID NO: 222); and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53, the first peptide comprises the amino acid sequence of GPTGASFHW (SEQ ID NO: 204), and the second peptide comprises the amino acid sequence of FWGDDWDLL (SEQ ID NO: 210). 8. The bispecifc fusion protein of paragraph 7 further comprising one or more linker, preferably a rigid linker, more preferably a rigid linker comprising the sequence of A(EAAAK)n (SEQ ID NO: 86). 9. The bispecifc fusion protein of paragraph 7 or 8 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 3. AVA21-04 (AVA04-251+AVA04-251+AVA19-01) 10. A trispecific fusion protein comprising: two PD-L1 binding polypeptides, each comprising a first peptide and a second peptide, wherein each of the PD-L1 binding polypeptides comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53, the first peptide comprises the amino acid sequence of GPTGASFHW (SEQ ID NO: 204), and the second peptide comprises the amino acid sequence of FWGDDWDLL (SEQ ID NO: 210). 11. The trispecifc fusion protein of paragraph 10 further comprising one or more linker, preferably a rigid linker, more preferably a rigid linker comprising the sequence of A(EAAAK)n (SEQ ID NO: 86). 12. The trispecifc fusion protein of paragraph 10 or 11 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 4. AVA21-05 (AVA04-251+AVA19-01) 13. A bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53, the first peptide comprises the amino acid sequence of GPTGASFHW (SEQ ID NO: 204), and the second peptide comprises the amino acid sequence of FWGDDWDLL (SEQ ID NO: 210). 14. The bispecifc fusion protein of paragraph 13 further comprising one or more linker, preferably a rigid linker, more preferably a rigid linker comprising the sequence of A(EAAAK)n (SEQ ID NO: 86). 15. The bispecifc fusion protein of paragraph 13 or 14 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 5. AVA21-08/-09 XT (AVA04-251+AVA04-251+AVA03-42+AVA19-158+AVA19-158) 16. A trispecific fusion protein comprising: two PD-L1 binding polypeptides, each comprising a first peptide and a second peptide, wherein each of the PD-L1 binding polypeptides comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); an HSA binding polypeptide comprising a first peptide and a second peptide, wherein the HSA binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 75, the first peptide comprises the amino acid sequence of NFFQRRWPG (SEQ ID NO: 225), and the second peptide comprises the amino acid sequence of WKFRNTDRG (SEQ ID NO: 226); and two LAG3 binding polypeptides, each comprising a first peptide and a second peptide, wherein each of the LAG3 binding polypeptides comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 62, the first peptide comprises the amino acid sequence of DATDGDGVS (SEQ ID NO: 207), and the second peptide comprises the amino acid sequence of FWGDEWDVL (SEQ ID NO: 213). 17. The bispecifc fusion protein of paragraph 16 further comprising one or more linker. 18. The bispecifc fusion protein of paragraph 17, wherein the linker is a rigid linker, preferably a rigid linker comprising the sequence of A(EAAAK)n (SEQ ID NO: 86). 19. The bispecifc fusion protein of any one of paragraphs 16-18 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 8. 20. The bispecifc fusion protein of paragraph 17, wherein the linker is a flexible linker, preferably a flexible linker comprising the sequence of (G4S)n (SEQ ID NO: 88). 21. The bispecifc fusion protein of any one of paragraphs 16, 17 or 20 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 9. AVA21-11 XT (AVA04-251+AVA04-251+AVA19-06+AVA19-06+AVA03-42) 22. A trispecific fusion protein comprising: two PD-L1 binding polypeptides, each comprising a first peptide and a second peptide, wherein each of the PD-L1 binding polypeptides comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); and two LAG3 binding polypeptides, each comprising a first peptide and a second peptide, wherein each of the LAG3 binding polypeptides comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 75, the first peptide comprises the amino acid sequence of DFPDDPWFW (SEQ ID NO: 205), and the second peptide comprises the amino acid sequence of DWEDAVTPY (SEQ ID NO: 211); and an HSA binding polypeptide comprising a first peptide and a second peptide, wherein the HSA binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 75, the first peptide comprises the amino acid sequence of NFFQRRWPG (SEQ ID NO: 225), and the second peptide comprises the amino acid sequence of WKFRNTDRG (SEQ ID NO: 226). 23. The trispecifc fusion protein of paragraph 22 further comprising one or more linker, preferably a rigid linker, more preferably a rigid linker comprising the sequence of A(EAAAK)n (SEQ ID NO: 86). 24. The trispecifc fusion protein of paragraph 22 or 23 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 10. AVA21-12 XT (AVA04-640+AVA19-06+AVA19-06+AVA03-42) 25. A trispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 75, the first peptide comprises the amino acid sequence of RRKHFPQWP (SEQ ID NO: 223), and the second peptide comprises the amino acid sequence of DLQPREVFQ (SEQ ID NO: 224); two LAG3 binding polypeptides, each comprising a first peptide and a second peptide, wherein each of the LAG3 binding polypeptides comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 75, the first peptide comprises the amino acid sequence of DFPDDPWFW (SEQ ID NO: 205), and the second peptide comprises the amino acid sequence of DWEDAVTPY (SEQ ID NO: 211); and an HSA binding polypeptide comprising a first peptide and a second peptide, wherein the HSA binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 75, the first peptide comprises the amino acid sequence of NFFQRRWPG (SEQ ID NO: 225), and the second peptide comprises the amino acid sequence of WKFRNTDRG (SEQ ID NO: 226). 26. The trispecifc fusion protein of paragraph 25 further comprising one or more linker, preferably a rigid linker, more preferably a rigid linker comprising the sequence of A(EAAAK)n (SEQ ID NO: 86). 27. The trispecifc fusion protein of paragraph 25 or 26 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 11. AVA21-06 BP (AVA04-251-hIgG1 Fc+AVA19-06) 28. A bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); an hIgG1 Fc polypeptide; and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 75, the first peptide comprises the amino acid sequence of DFPDDPWFW (SEQ ID NO: 205), and the second peptide comprises the amino acid sequence of DWEDAVTPY (SEQ ID NO: 211). 29. The trispecifc fusion protein of paragraph 28 further comprising one or more linker, preferably a flexible linker, more preferably a flexible linker comprising the sequence of (G4S)n (SEQ ID NO: 88). 30. The trispecifc fusion protein of paragraph 28 or 29 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 6. AVA21-07 BP (AVA04-251-hIgG1 Fc+AVA19-158) 31. A bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); an hIgG1 Fc polypeptide; and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 62, the first peptide comprises the amino acid sequence of DATDGDGVS (SEQ ID NO: 207), and the second peptide comprises the amino acid sequence of FWGDEWDVL (SEQ ID NO: 213). 32. The bispecifc fusion protein of paragraph 31 further comprising one or more linker, preferably a flexible linker, more preferably a flexible linker comprising the sequence of (G4S)n (SEQ ID NO: 88). 33. The bispecifc fusion protein of paragraph 31 or 32 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 7. AVA21-15 (AVA19-06+AVA04-251+hIgG1 LALA Fc) AVA21-16 (AVA04-251+AVA19-06+hIgG1 LALA Fc) 34. A bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); an hIgG1 LALA Fc polypeptide; and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 75, the first peptide comprises the amino acid sequence of DFPDDPWFW (SEQ ID NO: 205), and the second peptide comprises the amino acid sequence of DWEDAVTPY (SEQ ID NO: 211). 35. The bispecifc fusion protein of paragraph 31 further comprising one or more linker, preferably a flexible linker, more preferably a flexible linker comprising the sequence of (G4S)n (SEQ ID NO: 88). 36. The bispecifc fusion protein of paragraph 34 or 35 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 44. 37. The bispecifc fusion protein of paragraph 34 or 35 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 45. AVA21-06 CR (AVA04-251+hIgG1 LALA Fc+AVA19-06) 38. The bispecific fusion protein of paragraph 34, further comprising two linkers, preferably flexible linkers, more preferably flexible linkers comprising the sequence of (G4S)n (SEQ ID NO: 88). 39. The bispecifc fusion protein of paragraph 38 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 19; or at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 20-37. AVA21-12 CR (AVA04-251+hIgG1 LALA Fc+AVA19-170) 40. A bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); an hIgG1 LALA Fc polypeptide; and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 66, the first peptide comprises the amino acid sequence of DYEDDPWTF (SEQ ID NO: 208), and the second peptide comprises the amino acid sequence of SIDWPWEDD (SEQ ID NO: 214). 41. The bispecific fusion protein of paragraph 40, further comprising two linkers, preferably flexible linkers, more preferably flexible linkers comprising the sequence of (G4S)n (SEQ ID NO: 88). 42. The bispecifc fusion protein of paragraph 40 or 41 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 39. AVA21-13 CR (AVA04-251+hIgG1 LALA Fc+AVA19-173) 43. A bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); an hIgG1 LALA Fc polypeptide; and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 69, the first peptide comprises the amino acid sequence of DYADDPWFY (SEQ ID NO: 209), and the second peptide comprises the amino acid sequence of YEGDYEPHN (SEQ ID NO: 215). 44. The bispecific fusion protein of paragraph 43, further comprising two linkers, preferably flexible linkers, more preferably flexible linkers comprising the sequence of (G4S)n (SEQ ID NO: 88). 45. The bispecific fusion protein of paragraph 43 or 44 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 41. AVA21-06 CA (AVA04-251-hIgG4 Fc+AVA19-06) 46. A bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); an hIgG4 Fc polypeptide; and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 75, the first peptide comprises the amino acid sequence of DFPDDPWFW (SEQ ID NO: 205), and the second peptide comprises the amino acid sequence of DWEDAVTPY (SEQ ID NO: 211). 47. The bispecific fusion protein of paragraph 46, further comprising a linker, preferably a flexible linker, more preferably a flexible linker comprising the sequence of (G4S)n (SEQ ID NO: 88). 48. The bispecifc fusion protein of paragraph 47 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 15. AVA21-12 CA (AVA04-251-hIgG4 Fc+AVA19-170) 49. A bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); an hIgG4 Fc polypeptide; and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 66, the first peptide comprises the amino acid sequence of DYEDDPWTF (SEQ ID NO: 208), and the second peptide comprises the amino acid sequence of SIDWPWEDD (SEQ ID NO: 214). 50. The bispecific fusion protein of paragraph 49, further comprising a linker, preferably a flexible linker, more preferably a flexible linker comprising the sequence of (G4S)n (SEQ ID NO: 88). 51. The bispecifc fusion protein of paragraph 49 or 50 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 38. AVA21-13 CA (AVA04-251-hIgG4 Fc+AVA19-173) 52. A bispecific fusion protein comprising: a PD-L1 binding polypeptide comprising a first peptide and a second peptide, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72, the first peptide comprises the amino acid sequence of REGRQDWVL (SEQ ID NO: 219), and the second peptide comprises the amino acid sequence of WVPFPHQQL (SEQ ID NO: 220); an hIgG4 Fc polypeptide; and a LAG3 binding polypeptide comprising a first peptide and a second peptide, wherein the LAG3 binding polypeptide comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 69, the first peptide comprises the amino acid sequence of DYADDPWFY (SEQ ID NO: 209), and the second peptide comprises the amino acid sequence of YEGDYEPHN (SEQ ID NO: 215). 53. The bispecific fusion protein of paragraph 52, further comprising a linker, preferably a flexible linker, more preferably a flexible linker comprising the sequence of (G4S)n (SEQ ID NO: 88). 54. The bispecific fusion protein of paragraph 52 or 53 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 40. LAG3 Binding AFFIMER Polypeptides AVA19-01 55. A bispecific fusion protein comprising a first peptide comprising the amino acid sequence of SEQ ID NO: 204 and a second peptide comprising the amino acid sequence of SEQ ID NO: 210. 56. The bispecific fusion protein of paragraph 55 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53. AVA19-06 57. A bispecific fusion protein comprising a first peptide comprising the amino acid sequence of SEQ ID NO: 205 and a second peptide comprising the amino acid sequence of SEQ ID NO: 211. 58. The bispecific fusion protein of paragraph 57 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 54. AVA19-157 59. A bispecific fusion protein comprising a first peptide comprising the amino acid sequence of SEQ ID NO: 206 and a second peptide comprising the amino acid sequence of SEQ ID NO: 212. 60. The bispecific fusion protein of paragraph 59 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 61. AVA19-158 57. A bispecific fusion protein comprising a first peptide comprising the amino acid sequence of SEQ ID NO: 207 and a second peptide comprising the amino acid sequence of SEQ ID NO: 213. 58. The bispecific fusion protein of paragraph 57 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 63. AVA19-170 59. A bispecific fusion protein comprising a first peptide comprising the amino acid sequence of SEQ ID NO: 208 and a second peptide comprising the amino acid sequence of SEQ ID NO: 214. 60. The bispecific fusion protein of paragraph 59 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 66. AVA19-170 61. A bispecific fusion protein comprising a first peptide comprising the amino acid sequence of SEQ ID NO: 209 and a second peptide comprising the amino acid sequence of SEQ ID NO: 210. 62. The bispecific fusion protein of paragraph 61 comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 69. 63. A polynucleotide comprising an open reading frame encoding the fusion protein of any one of paragraphs 1-62. 64. A vector, optionally a viral vector or a plasmid vector, comprising the polynucleotide of paragraph 63. 65. A cell, optionally a mammalian cell, comprising the polynucleotide of paragraph 63 or the vector of paragraph 74. 66. A pharmaceutical composition comprising: (a) the fusion protein of any one of paragraphs 1-62, the polynucleotide of paragraph 63, the vector of paragraph 64, or the cell of paragraph 65; and (b) a pharmaceutically acceptable excipient. 67. A method comprising administering to a subject the polynucleotide of paragraph 63, the vector of paragraph 64, the cell of paragraph 65, or the pharmaceutical composition of paragraph 66. 68. The method of paragraph 67, wherein the subject has a cancer. 69. The method of paragraph 67 or 68, wherein the polynucleotide, vector, cell, or pharmaceutical composition is administered subcutaneously, intravenously, or intramuscularly. 70. A fusion protein comprising a PD-L1 binding polypeptide, a LAG3 binding polypeptide, and an Fc domain selected from hIgG1 Fc, hIgG1 LALA Fc, and hIgG4 Fc, wherein the fusion protein comprises at least one (optionally two to four) flexible linker, optionally wherein the flexible linker is (G4S)n and n is 1, 2, 3, 4, 5, or 6. 71. A fusion protein comprising a PD-L1 binding polypeptide, a LAG3 binding polypeptide, and an Fc domain selected from hIgG1 Fc, hIgG1 LALA Fc, and hIgG4 Fc, wherein the fusion protein comprises at least one (optionally two to four) rigid linker, optionally wherein the rigid linger is A(EAAAK)n and n is 1, 2, 3, 4, 5, or 6.
EXAMPLES A general description of the protocols used herein, including selection of the AFFIMER® polypeptides by phage display, expression studies (e.g., in E. coli), post-purification purity studies (e.g., SEC-HPLC), binding affinity studies (e.g., ELISAs), competitive binding studies (e.g., competitive ELISAs), and kinetic studies (e.g., BIACORE® analysis), may be found in International Publication No. WO 2019/197583, incorporated herein by reference in its entirety. Example 1: Characterization of Bispecific In-Line Fusion Dimers and Trimers that Bind to LAG-3 and PD-L1 AFFIMER® polypeptides selected for binding to human PD-L1 (AVA04; “anti-PD-L1 AFFIMER® polypeptides”) were genetically fused with AFFIMER® polypeptides selected for binding to human LAG-3 (AVA19; “anti-LAG-3 AFFIMER® polypeptides”). These in-line fusion (ILF) formats (AVA21) include repetitive linkers connecting the monomer components. In this set of experiments, ILF dimers were produced by (i) fusing AVA04-269 to AVA19-157 (AVA21-01), (ii) fusing AVA04-269 to AVA19-158 (AVA21-02), (iii) fusing AVA04-269 to AVA19-01 (AVA21-03), and (iv) fusing AVA04-251 to AVA19-01 (AVA21-05). An ILF trimer was produced by fusing two AVA04-251 to AVA19-01 (AVA21-04). Table 4 lists the ILF formats and their monomer or dimer components. Schematics of the ILF dimer and trimer proteins are depicted in FIG.1. SDS-PAGE was used to confirm the expected molecular weight (MW) of each of these fusion proteins (FIG.1). Table 4.
Figure imgf000217_0001
To determine the binding affinity (EC50) of the dimer and trimer ILF proteins of Table 4, direct binding enzyme linked immunosorbent assays (ELISAs) were performed. Results of the LAG-3 ELISAs are shown in FIG.2. AVA21-01, AVA21-03 and AVA21-04 exhibited decreased binding to LAG-3 relative to their monomer parent clones, AVA19-157 and AVA19- 01. AVA21-02 and AVA21-05 showed increased binding affinity to LAG-3 relative to their monomer parent clones AVA19-158 and AVA19-01. Results of the PD-L1 ELISAs are shown in FIG.3. AVA21-01, AVA21-02 and AVA21-03 exhibited decreased binding to PD-L1 relative to their monomer parent clone, AVA04-269. AVA21-04 and AVA21-05 showed comparable binding affinity to PD-L1 relative to their parent clone AVA04-251. A PD-1/PD-L1 blockade bioassay was performed using the dimer and trimer ILF proteins of Table 4, the results of which are shown in FIG.4. AVA21-01, AVA21-02 and AVA21-03 exhibited reduced function relative to their monomer parent clone, AVA04-269. AVA21-04 and AVA21-05 showed comparable function relative to their parent clone AVA04-251. A bridging ELISA was performed using the dimer and trimer ILF proteins of Table 4 to assess whether they are able to bind LAG-3 and PD-L1 simultaneously. LAG-3 was coated on the plate, and anti-PD-L1 antibody was used to detect the AFFIMER® polypeptides. Results are shown in FIG.5. Most formats bound both targets simultaneously, to varying degrees. Lastly, target-specific cell binding assays were performed using the AVA21-04 and AVA21-05 ILF proteins of Table 4 to assess whether they bind to LAG-3 expressed on cells. Results are shown in FIG.6. Both AVA21-04 and AVA21-05 bound to LAG-3 in the LAG-3- expressing cells but did not bind to control Jurkat or CHO-K1 cells. Example 2: Characterization of Trispecific In-Line Fusion (ILF) Pentamers that Binds to LAG-3, PD-L1, and HSA Trispecific (ILF) pentamers were also produced. These pentamers include AFFIMER® polypeptides selected for binding to human PD-L1, with AFFIMER® polypeptides selected for binding to human LAG-3, and with AFFIMER® polypeptides selected for binding to human serum albumin (HSA) to extend the half-life of the ILFs. The pentamer formats are depicted in FIG.7, showing a rigid or flexible linker connecting each monomer with each other. AVA21-08 XT (SEQ ID NO: 8) includes two anti-PD-L1 AFFIMER® polypeptides (each SEQ ID NO: 72), an anti-HSA AFFIMER® polypeptide (SEQ ID NO: 80), and two anti-LAG-3 AFFIMER® polypeptides (each SEQ ID NO: 62) with rigid linkers (A(EAAAK)6); and AVA21-09 XT (SEQ ID NO: 9) includes two anti-PD-L1 AFFIMER® polypeptides (each SEQ ID NO: 72), an anti- HSA AFFIMER® polypeptide (SEQ ID NO: 80), and two anti-LAG-3 AFFIMER® polypeptides (each SEQ ID NO: 62) with flexible linkers ((G4S)6). Reducing SDS-PAGE and SEC-HPLC analysis were used to confirm the expected molecular weight (MW) and purity of the fusion proteins (FIG.8). A PD-L1 direct binding ELISA was performed using the two pentamers. The results show that AVA21-08 XT and AVA21-09 XT appear at least equivalent to the AVA04-251 BH (SEQ ID NO: 74) control for PD-L1 binding, suggesting no adverse effect of the additional HSA and LAG-3 binding domains (FIG.9). A LAG-3 direct binding ELISA was also performed using the two pentamers as well as AVA19-158 (SEQ ID NO: 62) and AVA19-158 BK (SEQ ID NO: 54). Binding of both pentamers to LAG-3 was demonstrated by ELISA (FIG.10). The rigid linkers appear to enable better target engagement and stronger binding to LAG-3. An HSA direct binding ELISA was also performed with the two pentamers. The results show that both constructs exhibited lower affinity than AVA03-42 (SEQ ID NO: 80) monomer control (FIG. 11). This could be due to the positioning of AVA03-42 AFFIMER® XT in those formats, which could result in lesser availability to engage target. Target-specific cell binding assays were performed, in LAG-3-positive and negative Jurkat cells. Both pentamers show comparable binding to LAG-3 Jurkat positive cells, and comparable to control (FIG.12). Binding was not detected in LAG-3-negative cells. A dual PD-L1/HSA target binding assay was performed, the results of which show that both pentamers engage PD-L1 and HSA targets simultaneously (FIG.13). Binding of the pentamers was unaffected in the presence of HSA. The rigid linker appears better than flexible linkers for PD-L1/HSA dual target engagement. Results from a functional PD-L1/PD-1 gene reporter assay with the pentamers show that AVA21-08 XT exhibits comparable activity to the AVA04-251 BH control (FIG.14). Functionality of AVA21-09 XT is significantly decreased. Again, the flexible linker seems less effective compared to the rigid linker. A PD-L1, LAG-3 and HSA BIACORE™ kinetic binding analysis of the pentamers was performed. Results show that the rigid linker appears to allow for stronger binding to PD-L1 and LAG-3 (FIG.15). Binding to HSA was not affected by the nature of linker. Trispecific ILF tetramers and additional pentamers were also produced, as depicted in FIG.16. AVA21-11 XT (SEQ ID NO: 10) includes two anti-PD-L1 AFFIMER® polypeptides (each SEQ ID NO: 72), two anti-LAG-3 AFFIMER® polypeptides (each SEQ ID NO: 152), and an anti-HSA AFFIMER® polypeptide (SEQ ID NO: 178), with rigid linkers (A(EAAAK)6); and AVA21-12 XT (SEQ ID NO: 11) includes an anti-PD-L1 AFFIMER® polypeptide (SEQ ID NO: 75), two anti-LAG-3 AFFIMER® polypeptides (each SEQ ID NO: 152), and an anti-HSA AFFIMER® polypeptide (SEQ ID NO: 178), with rigid linkers (A(EAAAK)6). Reducing SDS-PAGE analysis was performed on AVA21-11 XT and AVA21-12 XT to confirm the expected molecular weight of each protein (FIG.16). SEC-HPLC analysis was also performed to assess the purity. SEC-HPLC chromatograms of AVA21-11 XT and AVA21-12 XT following a two-stage purification process are shown in FIG.17. A PD-L1, LAG-3 and HSA BIACORE™ kinetic binding analysis was performed for AVA21-11 XT and AVA21-12 XT, the results of which are shown in FIG.18 and confirm that both constructs can engage all three targets A PD-L1 direct binding ELISA using AVA21-11 XT and AVA21-12 XT was performed, the results of which show that both constructs exhibit binding to PD-L1 comparable to parent AFFIMER® controls (FIG.19). Results of a LAG-3 direct binding ELISA using AVA21-11 XT and AVA21-12 XT are shown in FIG.20. AVA21-11 XT shows better binding to LAG-3 than AVA21-12 XT and is equivalent to AVA19-06 BK (SEQ ID NO: 57) control. The results of an HSA binding ELISA show decreased binding of AVA21-12 XT to HSA compared to monomer control AVA03-42 (SEQ ID NO: 80) (FIG.21). Results of a bridging ELISA with and without HSA, where LAG-3 was coated on the plate and AFFIMER® polypeptides were detected using an anti-PD-L1 antibody, confirmed dual target engagement in the presence/absence of HSA in solution (FIG.22). The presence of HSA in solution does not impact binding to PD-L1 and LAG-3. Results of a functional PD-L1/PD-1 gene reporter assay for AVA21-11 XT and AVA21- 12 XT confirmed that both formats appear to be less active than their respective controls (FIG. 23). Results of multimer binding in LAG-3 overexpressing BPS cells demonstrate increased binding of dimeric formats vs monomer AVA19-06 (FIG.24). AVA21-11 XT binding to BPS cells was comparable to that of AVA19-06 BK control. Results of multimer cell binding in CHO-K1 PD-L1 overexpressing cells demonstrate that AVA21-12 XT binding to CHO-K1 PD-L1 cells was comparable to that of AVA04-640 control (SEQ D NO: 75) (FIG.25). Example 3. Characterization of Bispecific huIgG1 Fc Fusions that Bind to LAG-3 and PD- L1 Various huIgG1 Fc fusion control formats were designed and tested in this set of experiments. The various control formats are depicted in FIG.26. The bispecific huIgG1 Fc fusion proteins, AVA21-06 BP (SEQ ID NO: 6) and AVA21-07 BP (SEQ ID NO: 7), include an anti-PDL1 AFFIMER® polypeptide, huIgG1 Fc, and anti-LAG-3 AFFIMER® polypeptide, with a flexible linker. SEC-HPLC chromatograms of the bispecific IgG1 Fc fusion multimers are shown in FIG.27. Reducing SDS-PAGE was used to confirm the expected molecular weights (FIG.28). Direct binding ELISAs for LAG-3 and huPD-L1 were performed. Results show that AVA21-06 and 07 BP both demonstrated comparable binding affinities to LAG-3; the data is also consistent with control proteins AVA19-06 and AVA19-158 AQ.2 (FIG.29). AVA21-06 and 07 BP both show comparable binding affinities to PD-L1; the data is also consistent with control protein AVA04-251 V.2 (FIG.29). A PD-1/PD-L1 blockade assay on AVA21-06 BP showed that AVA21-06 BP exhibits slightly decreased activity compared to AVA04-251 V.2 control protein (FIG.30). Results from a cell binding assay for AVA21-06 BP binding in LAG-3-positive and negative Jurkat cells show a dose effect for AVA21-06 BP on positive cells (FIG.31). No binding observed on LAG-3 negative cells. Results from a cell binding assay for AVA21-06 BP and AVA21-07 BP binding in LAG- 3-enriched BPS cells, and LAG-3 negative Jurkat and CHO-K1 cells show that AVA21-06 BP and AVA21-07 BP bind to LAG-3-enriched BPS cells (FIG.32). Binding was not observed in LAG-3 negative Jurkat and CHO cells. Example 4. Characterization of Bispecific huIgG1 LALA Fc Fusions that Bind to LAG-3 and PD-L1 The bispecific huIgG1 LALA Fc fusion proteins AVA21-15 (SEQ ID NO: 44) and AVA21-16 (SEQ ID NO: 45) include an anti-PDL1 AFFIMER® polypeptide, an anti-LAG-3 AFFIMER® polypeptide, and an huIgG1 LALA Fc, and with a flexible linker. Reducing SDS- PAGE was used to confirm the expected molecular weight for the two constructs (FIG.33). Results of a PD-1/PD-L1 blockade assay on AVA21-15 and AVA21-16 are shown in FIG.34. AVA21-15 showed a significant decrease in activity compared to control proteins, AVA04-251 AG.3 (SEQ ID NO: 79) and AVA04-251 CF (SEQ ID NO: 78). AVA21-16 potency was reduced in this assay relative to that of control proteins, but the protein remained active. Results of direct LAG-3 and PD-L1 binding ELISAs are shown in FIG.35. Results show that the positioning of LAG-3 and PD-L1 binders affects their ability to bind target. Both formats had significantly lower affinity for antigen than controls (AVA19-06 V.2 (SEQ ID NO: 55) and AVA21-06 CR (SEQ ID NO: 19)) when binding to LAG-3. For PD-L1 binding, AVA21-16 binds to target with slightly decreased affinity compared to controls (AVA21-06 CR (SEQ ID NO: 19), AVA04-251 V.2 (SEQ ID NO: 76), and AVA04-251 AG.3 (SEQ ID NO: 79), AVA21- 15 affinity is significantly decreased due to positioning of PD-L1 binder. Additional bispecific huIgG1 LALA Fc fusion proteins were produced, as depicted in FIG.36. AVA21-06 CR (SEQ ID NO: 19), AVA21-12 CR (SEQ ID NO: 39), and AVA21-13 CR (SEQ ID NO: 41) each include an anti-PD-L1 AFFIMER® polypeptide, an anti-LAG-3 AFFIMER® polypeptide, and an huIgG1 LALA Fc, and with a flexible linker. Reducing SDS-PAGE was used to confirm the expected molecular weight of the additional constructs (FIG.36). A human PD-L1 kinetics analysis for AVA21-06 CR, AVA21-06 CR T89A (SEQ ID NO: 20), AVA21-12 CR, and AVA21-13 CR demonstrated that binding to PD-L1 is equivalent for all proteins tested (FIG.37). A human LAG-3 kinetics analysis for AVA21-06 CR, AVA21-12 CR, and AVA21-13 CR showed that all clones bind to LAG-3 with comparable affinities (FIG.38). Results of a PD-L1 direct binding ELISA showed equivalent binding for AVA21-06 CR and control AVA04-251 V.2 (FIG.39). Results of a LAG-3 direct binding ELISA on AVA21-06 CR, AVA21-12 CR, and AVA21-13 CR showed that all CR formats exhibit equivalent binding to LAG-3 and either comparable or better binding affinity than their relative CS controls (FIG.40). Alanine scanning was performed on AVA21-06 CR, the results of which are shown in FIG.41. Alanine mutants of parent format AVA21-06 CR were engineered to improve binding and reduce aggregation. Results of direct LAG-3 and PD-L1 binding ELISAs for alanine scanned AVA21 CR IgG1 LALA Loop 2 constructs showed that little variation in PD-L1 binding between controls and various mutants was observed (FIG.42). D439A (SEQ ID NO: 25), P440A (SEQ ID NO: 26) and W443A (SEQ ID NO: 29) significantly affected binding to LAG-3 (essential amino acids), D438A (SEQ ID NO: 24) impaired binding to an extent and P437A (SEQ ID NO: 23) did not appear to significantly affect binding to LAG-3. Results of direct LAG-3 and PD-L1 binding ELISAs for alanine scanned AVA21 CR IgG1 LALA Loop 4 constructs showed little variation in PD-L1 and LAG-3 binding between controls and various mutants was observed (FIG.43). Binding to LAG-3 appears to be the most affected by D471A (SEQ ID NO: 33) mutation, while binding to PD-L1 seems to be the most affected by P475A (SEQ ID NO: 36) mutation. Variations may be due to experimental error. Biacore binding to LAG3 and PDL1 (FIGs.44A-44B) The binding affinity of AVA21 clones towards their target were characterised using surface plasmon resonance technique on the Biacore 8K (Cytiva). Binding towards the ligand PD-L1 (R&D systems; Cat:156-B7) and human serum albumin (Sigma; Cat:A3782) were performed by immobilising the respective ligand on Series S CM5 chip (Cytiva; Cat:29104988) using amine coupling technique (Cytiva Amine coupling kit, type 2; Cat: BR-1006-33), with AVA21 AFFIMER® proteins flowed on flow cell. Meanwhile for determination of LAG3 binding, AVA21 AFFIMER® proteins were bound onto CM5 chip using amine coupling technique, with LAG3 flowed over the flow cell. All experiments were performed in 1x HBS- EP+ running buffer (Cytiva; Cat:BR100669), and affinities were determined using the Biacore Insight Evaluation Software (Cytiva). Results show that Fc fused AVA21 AFFIMER® proteins binds PDL-1 and LAG-3 at low nanomolar range (0.1-0.2 nM and 0.9-44.9 nM respectively). AVA21 XT molecules also bind PD-L1, LAG-3, and human serum albumin at similar low nanomolar KD ranges (1-5nM, 0.2- 3nM, and 27-89nM; respectively). AVA21 AFFIMER® proteins exhibit good affinity towards their targets. Cell binding to human PD-L1 and LAG-3 (FIG.45, FIG.46) AVA21-06 CR, AVA21-06 T89A CR, AVA21-12CR and AVA21-13CR constructs were characterized for its binding to human PD-L1 and human LAG-3 expressed on cells. For analysis of binding to PD-L1, H441 cells endogenously expressing PD-L1 were used. Binding to PD-L1 on cells was equivalent for all constructs and was similar to PD-L1 only binding control (AVA04-251 CR). For analysis of binding to LAG3, hLAG3-overexpressing and LAG3 negative D0.11.10 cells were used. AVA21-06 CR, AVA21-06 T89A CR, AVA21-12CR and AVA21-13CR bound to hLAG3-overexpressing but not LAG3 negative cells. Binding was similar between constructs. Control AVA21-19CR showed lower binding and PD-L1 only AFFIMER™ (AVA04-251 CR) no binding as expected. Binding ELISA to PD-L1 (FIG.47) Results of a human PD-L1 direct binding ELISA for AVA21-06 CR, AVA21-06 T89A CR, AVA21-12CR and AVA21-13CR showed that all clones bind to PD-L1 with comparable affinities. Example 5: Characterisation of lead AVA21 constructs in functional assay for PD-L1 & LAG3 inhibition Comparison of inhibitory activity in PD-1/PD-L1 blockade assay (FIG.48) PD-1/PD-L1 Blockade Bioassay (Promega) was used according to the manufacturer’s instructions to test ability of AVA21-06 CR, AVA21-06 T89A CR, AVA21-12CR and AVA21- 13CR to block PD-1/PD-L1 signalling. The assay involves co-culture of PD-1 expressing Effector cells (Jurkat) and CHO-K1 APC (Antigen Presenting Cells) cells expressing PD-L1. In the presence of PD-L1 inhibitor, the Effector cell and APC cell interaction induces TCR signalling and NFAT-RE mediated luminescence. The AVA21-06 CR, AVA21-12CR and AVA21- 13CR have the same anti-PD-L1 affimer domain at the N terminus and hence show similar inhibitory activity as AVA04-251 CR (anti-PD-L1) indicating that AVA21 AFFIMER® proteins are strong PD-L1 inhibitors. Comparison of inhibitor activity in LAG3/MHCII blockade assay (FIG.49) LAG3/MHC II Blockade Bioassay (Promega) was used according to the manufacturer’s instructions to test ability of AVA21-06 CR, AVA21-12CR, AVA21-13CR, AVA21-8XT and AVA21-12XT to block LAG3/MHCII signalling. The assay involves coculturing MHCII - aAPC cells (activated APC cells) with LAG3 expressing Effector cells (Jurkat). The aAPC cells present TCR activating antigen on MHCII to specifically activate TCR on LAG3 effector cells which inhibits TCR-induced activation and promoter-mediated luminescence. In the presence of LAG3 inhibitor, LAG3 induced TCR inhibition is blocked leading to increased promoter mediated luminescence. The anti-LAG3 molecules, AVA21-19 CR and AVA19-158-XT23 show dose response activity. The Fc molecules AVA21-06CR & AVA21-13CR, and AVA21-12 CR show similar or higher activity to AVA21-19 CR respectively. The in line fusion molecules AVA21- 08XT and AVA21-12XT have similar dose response to AVA19-158-XT23. The assay concludes that the AVA21 AFFIMER® proteins are strong LAG3 inhibitors. Comparison of simultaneous PD-L1 and LAG3 inhibition through bispecific assay (FIG.50) PD-1/LAG3 combination assay (Promega) was used as per the manufacturer’s instructions to test the ability of AVA21-06 CR, AVA21-12CR, AVA21-13CR, AVA21-08XT and AVA21-12XT to block PD-1/PD-L1 and LAG3/MHC II signalling simultaneously. The assay involves co-culturing PD-L1 expressing- aAPC cells (activated APC cells) with PD-1/LAG3 expressing effector cells. TCR activating antigen is presented by the aAPC cells on MHCII to specifically activate TCR on PD-1/LAG3 effector cells. In the presence of PD-L1/ LAG3 bispecific molecule, LAG3 mediated TCR and PD-1/PD-L1 axis are simultaneously inhibited leading to promoter activation and luminescence. PD-L1/ LAG3 inhibition will lead to higher luminescence than PD-L1 or LAG3 inhibition alone. The bispecific Fc AFFIMER® proteins AVA21-06 CR, AVA21-12CR and AVA21-13CR show higher activity than AVA04-251 CR (anti-PD-L1) and AVA21-19 CR (anti-LAG3). The higher activity of bispecific AFFIMER® proteins than the combination of AVA04-251 CR and AVA21-19 CR indicates the strong synergistic effect of having anti-PD-L1 and anti-LAG3 on the same molecule as a bispecific. Similarly, in line fusion AFFIMER® polypeptide AVA21-12XT has higher synergy than AVA04-251-XT14 (anti-PD-L1) and AVA19-158-XT23 (anti-LAG3) combination. The high synergy of the bispecific AFFIMER® proteins is a clear indication of their higher anti-tumour response than anti-PD-L1 or anti-LAG3 treatments alone. Primary human T cell stimulation assay (FIG.51A-51C) AVA21 constructs and controls were tested in human primary peripheral blood mononuclear cells (PBMCs) cultures after pre-activation with Staphylococcal Enterotoxin B (SEB). IL2 cytokine release was quantified as a surrogate for T cell activation. Briefly, frozen human PBMCs purchased from Cambridge Bioscience were thawed and pre-activated for 72h at 37C in T75 flask in presence of 500ng/ml SEB. After 72h, pre-activated PBMCs were harvested and seeded at 60,000 cells/well in 96-well plates in the presence of 200ng/ml SEB and treatment. Treatment consisted of AVA21 constructs, controls or cells were left untreated. PBMCs were then incubated for 24h at 37°C, before collection of supernatant and analysis of IL-2 release using IL2 HTRF kit according to manufacturer’s instructions (Cisbio, HTRF human IL2 kit). Preliminary data shows that bispecific hIgG1 LALA fusion constructs that bind PD-L1 and LAG3 (AVA21-06 CR, AVA21-06 T89A CR, AVA21-12CR and AVA21-13CR) induce higher levels of IL2 cytokine release compared to untreated and treatments with fusion constructs binding to PDL1 (AVA04-251CR) or LAG3 (AVA21-19CR) alone. Similarly, the trispecific in- line fusion construct that binds LAG-3, PD-L1 and HSA (AVA21-12XT) induces higher levels of IL2 release compared to negative control (3t0gly XT45) or a control binding to PD-L1 and HSA only (AVA04-640 XT34). In summary therefore, constructs which target both PD-L1 and LAG3 demonstrate increased activity compared to constructs which only bind one target protein. This shows that the constructs which target both immune checkpoint proteins have a greater ability to enhance T cell function than monospecific constructs as evidenced by increased IL-2 release. Use of primary cells shows that this is potentially translatable to the clinic. Reversal of T cell exhaustion in an in vitro mixed lymphocyte reaction (MLR) (FIGs.52A-52C, FIGs.53A-53C) T cell exhaustion is often observed in the tumour microenvironment. Exhausted T cells show overexpression of inhibitory receptors, decreased cytolytic activity and impaired effector cytokine production, which lead to impaired tumour elimination. Restoring T cell responsiveness correlates with increased immune surveillance and tumour response. One-way MLR was used to assess reversal of exhausted T cell (Tex) hypo-responsiveness in the presence of AVA21 constructs and control molecules. Tex were generated by isolating pan-T cells (CD3+) from PBMC donors and repeatedly stimulating using CD3/CD28 Dynabeads®. At the end of the final round of stimulation (Day 6), Dynabeads® were removed and the T cells rested for a further 48 hours before inclusion in the MLR assay. Tex were phenotyped by flow cytometry to confirm expression of PD-1, TIM-3 and LAG-3 by T cells (on day 0 and day 8). In vitro generated monocyte-derived dendritic cells (mo-DC) were combined with Tex to generate MLR pairs and cultured for 5 days in the presence of test molecules. Two donor pairs were tested. Following the completion of the MLR, the supernatants were assessed for levels of IFNγ by ELISA. In addition, T cell populations were assessed for proliferative responses by analysing proliferative marker Ki67 expression in CD4 and CD8 population. Cells were stained with Brilliant Violet 711™ anti-human CD4 Antibody (OKT4), Brilliant Violet 510™ anti-human CD8 Antibody (SK1), PE anti-human Ki-67 Antibody and eBioscience™ Fixable Viability Dye eFluor™ 780, and analysed by flow cytometry. Bispecific hIgG1 LALA fusion constructs that bind PD-L1 and LAG3 (AVA21-06CR, AVA21-12CR, AVA21-13), trispecific in-line fusion construct that binds LAG-3, PD-L1 and HSA (AVA21-12XT) and hIgG1 LALA fusion constructs that bind PD-L1 only (AVA04- 251_CR) induced CD4+ (A) and CD8+ T cell proliferation (B), and IFN-γ release (C) in both donor pairs. Negative control (SQTGly_CR) and fusion construct binding LAG3 only (AVA21- 19CR) had no effect. Anti-PDL1 (Atezolizumab) and anti-PD1 (Nivolumab) were also tested in the assay and induced a response as expected. In summary, AVA21 constructs are able to reverse exhausted T cell hypo-responsiveness as measured by increased T cell proliferation and IFN-γ release in two MLR donor pairs. This may enhance anti-tumour response. Example 6. Pharmacokinetics (FIG.54) AVA21 half-life in blood serum was determined by conducting a pharmacokinetics (PK) experiment in wild type C57BL/6 mice. The in vivo study was performed by dosing the mice with 5mg/kg of AVA21 AFFIMER® proteins. Blood samples were taken at times 0, 0.25, 6, 24, 72, 120, 168, 336 and 504 hours. Serum was extracted from the blood samples and an ELISA experiment was performed for all the samples using cystatin antibodies (BAF1407) for detecting AVA21 AFFIMER® proteins. ELISA of PK experiment determined that in-line fusion clone AVA21-12XT has a half- life of 11.948 hours, while AVA21-12 CR has a half-life of 296.15 hours. AVA21-12XT contains a HSA binding AFFIMER® , which cross-reacts with mouse serum albumin (MSA) but binds with lower affinity to MSA compared to HSA. PK experiment was not performed with human serum albumin which could be the reason for the short half-life for clone AVA21-12XT. Half-life of clone AVA21-12XT is expected to be longer in humans. Stability Studies (FIG.55A-55F) Stability study was performed on AVA21 clones to assess their stability in storage buffer DPBS (Cytiva Hyclone; Cat: SH30378.03). All clones were concentrated to ~10mg/mL and aliquoted into individual tubes, at volume of 10μL (100ug). Tubes were incubated at storage conditions 37°C, 45°C and 22°C (room temperature) to assess stability. Sampling was performed at Day 0 then at different time intervals for the different conditions. Analysis was performed by diluting the 10uL of sample at 10mg/mL with 1xDPBS to make 100μL at 1mg/mL, then filtered using Spin-X centrifuge tube filter (Corning; Cat:CLS8161); the concentration of samples were recorded after dilution and after filtration on a NanoDrop (Thermo), then filtered sample was ran on the SEC-HPLC using the Bioresolve SEC mAb column (Waters; Cat: 176004595), running at 0.8mL/min in 1xDPBS (Cytiva, Hyclone). As shown in Figure 55A, all IgG1 LALA Fc fused AFFIMER® proteins , show excellent stability at 37˚C for up to 10 days, while at 45˚C shows good stability for up to 3 days (Fig 55C). In-line fusion AFFIMER® , AVA21-08XT shows good stability at 37 ˚C for 8 days (Fig 55B), but is less stable at 45 ˚C (2 days, Fig 55D). AVA21-12 XT showed lower stability at both 37˚C and 45˚C. At room temperature (22 ˚C) (Fig 55E and 55F), AVA21 AFFIMER® proteins showed excellent stability for 15 days in DPBS, with the exception of clone AVA21-12 XT. A conclusion can be made that AVA21 in-line fusion AFFIMER® proteins are not suitable for storage in DPBS, and will need to explore other formulation conditions. Example 7. Characterization of Bispecific huIgG4 Fc Fusions that Bind to LAG-3 and PD- L1 Bispecific huIgG4 Fc fusion proteins were produced as depicted in FIG.56. AVA21-06 CA (SEQ ID NO: 15), AVA21-12 CA (SEQ ID NO: 38), and AVA21-13 CA (SEQ ID NO: 40) each include an anti-PD-L1 AFFIMER® polypeptide, an anti-LAG-3 AFFIMER® polypeptide, and an huIgG4 Fc, and with a flexible linker. Reducing SDS-PAGE was used to confirm the expected molecular weight for the three constructs (FIG.56). Results of a human PD-L1 direct binding ELISA for AVA21-06 CA, AVA21-12 CA and AVA21-13 CA showed that all clones bind to PD-L1 with comparable affinities (FIG.57). Results of LAG-3 direct binding ELISA with AVA21 CA IgG4 fusion constructs showed that All CA formats bind to LAG-3 with comparable affinities, and with equivalent affinities to the BZ control formats (FIG.58). Results of AVA21 CA IgG4 fusion construct binding in BPS LAG-3-positive and Jurkat LAG-3 negative cells showed that all formats bind to LAG-3 positive cells, and no signal observed for LAG-3 negative cells (FIG.59).

Claims

What is claimed is: CLAIMS 1. A Lymphocyte Activation Gene 3 (LAG-3) binding polypeptide comprising the following Formula (I): FR1-(X')-FR2-(X'')-FR3 (I), wherein FR1 comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA (SEQ ID NO: 216); FR2 comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of STNYYIKVRAGDNKYMHLKVFNGP (SEQ ID NO: 217); and FR3 comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence of ADRVLTGYQVDKNKDDELTGF (SEQ ID NO: 218), and wherein X' is an amino acid sequence having at least 85% or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 204-209; and X'' is an amino acid sequence having at least 85% or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 210-215.
2. A Lymphocyte Activation Gene 3 (LAG-3) binding polypeptide comprising: the amino acid sequence having of MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLA-(X')- STNYYIKVRAGDNKYMHLKVFNGP-(X'')-ADRVLTGYQVDKNKDDELTGF, wherein X' is an amino acid sequence having at least 85% or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 204-209; and X'' is an amino acid sequence having at least 85% or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 210-215.
3. The LAG-3 binding polypeptide of claim 2, wherein the polypeptide binds to LAG-3 with a Kd of 1×10−6M or less.
4. The LAG-3 binding polypeptide of claim 2 or 3, wherein: (a) X' is the amino acid sequence of SEQ ID NO: 204 and X'' is the amino acid sequence of SEQ ID NO: 210; or (b) X' is the amino acid sequence of SEQ ID NO: 205 and X'' is the amino acid sequence of SEQ ID NO: 211; or (c) X' is the amino acid sequence of SEQ ID NO: 206 and X'' is the amino acid sequence of SEQ ID NO: 212; or (d) X' is the amino acid sequence of SEQ ID NO: 207 and X'' is the amino acid sequence of SEQ ID NO: 213; or (e) X' is the amino acid sequence of SEQ ID NO: 208 and X'' is the amino acid sequence of SEQ ID NO: 214; or (f) X' is the amino acid sequence of SEQ ID NO: 209 and X'' is the amino acid sequence of SEQ ID NO: 215.
5. A Lymphocyte Activation Gene 3 (LAG-3) binding polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 53, 54, 61, 62, 66, and 69.
6. The LAG-3 binding polypeptide of claim 5, wherein the amino acid sequence has at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of any one of SEQ ID NOs: 53, 54, 61, 62, 66, and 69.
7. The LAG-3 binding polypeptide of claim 6, wherein the amino acid sequence comprises the amino acid sequence of any one of SEQ ID NOs: 53, 54, 61, 62, 66, and 69.
8. The LAG-3 binding polypeptide of any one of claims 5-7 further comprising a half-life extension moiety.
9. The LAG-3 binding polypeptide of claim 8, wherein the half-life extension moiety is a human serum albumin (HSA) binding polypeptide or a fragment crystallizable (Fc) region of an antibody, optionally wherein the antibody is a human IgG1 antibody or a human IgG4 Fc antibody.
10. A bispecific fusion protein comprising: a PD-L1 binding polypeptide; and the LAG-3 binding polypeptide of any one of the preceding claims.
11. The bispecific fusion protein of claim 10, wherein the PD-L1 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72 or 73.
12. The bispecific fusion protein of claim 10 or 11 comprising: a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 61.
13. The bispecific fusion protein of claim 10 or 11 comprising: a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73; and a second LAG-3 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 62.
14. The bispecific fusion protein of claim 10 or 11 comprising: a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 73; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53.
15. The bispecific fusion protein of claim 10 or 11 comprising: a PD-L1 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72; and a LAG-3 binding polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53.
16. A bispecific fusion protein comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, and 5.
17. The bispecific fusion protein of any one of the preceding claims, further comprising a second PD-L1 binding polypeptide and/or a second LAG-3 binding polypeptide.
18. The bispecific fusion protein of claim 17, wherein the second PD-L1 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72 or 73.
19. The bispecific fusion protein of claim 17, wherein the second LAG-3 polypeptide comprises an amino acid sequence of the LAG-3 polypeptide of any one of the preceding claims.
20. A bispecific protein comprising: a first PD-L1 binding polypeptide; a second PD-L1 polypeptide; and a LAG-3 binding polypeptide of any one of the preceding claims.
21. The bispecific fusion protein of claim 20, wherein: each of the first PD-L1 binding polypeptide and the second PD-L1 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72; and the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53.
22. The bispecific fusion protein of claim 20, wherein: the first PD-L1 binding polypeptide and the second PD-L1 polypeptide form a dimer comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 74; and the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 53.
23. The bispecific fusion protein of claim 17 comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 4.
24. A trispecific fusion protein comprising the bispecific fusion protein of any one of the preceding claims and a half-life extension moiety.
25. The trispecific fusion protein of claim 24, wherein the half-life extension moiety is a human serum albumin (HSA)-binding polypeptide.
26. The trispecific fusion protein of claim 25, wherein the HSA binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 80.
27. The trispecific fusion protein of claim 24, wherein the half-life extension moiety is a fragment crystallizable (Fc) region of an antibody, optionally a human IgG1 antibody or a human IgG4 antibody.
28. A trispecific fusion protein, comprising: a PD-L1 binding polypeptide; a first LAG-3 binding polypeptide; a second LAG-3 binding polypeptide; and an HSA binding polypeptide.
29. The trispecific fusion protein of claim 28, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72 or 73.
30. The trispecific fusion protein of claim 28 or 29, wherein each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence of the LAG-3 binding polypeptide of any one of the preceding claims.
31. The trispecific fusion protein of any one of claims 28-30, wherein the HSA binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 80
32. The trispecific fusion protein of claim 28, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 74; each of the first LAG-3-binding polypeptide and the second LAG-3-binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 54; and the HSA binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 80.
33. The trispecific fusion protein of claim 24, comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 11.
34. A trispecific fusion protein comprising: a first PD-L1 binding polypeptide; a second PD-L1 binding polypeptide; a first LAG-3 binding polypeptide; a second LAG-3 binding polypeptide; and a half-life extension moiety.
35. The trispecific fusion protein of claim 34, wherein the half-life extension moiety is an HSA binding polypeptide.
36. The trispecific fusion protein of claim 34 or 35, wherein each of the first PD-L1 binding polypeptide and second PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72 or 73.
37. The trispecific fusion protein of any one of claims 34-36, wherein each of the first LAG- 3 binding polypeptide and second LAG-3 binding polypeptide comprises an amino acid sequence of the LAG-3 polypeptide of any one of the preceding claims.
38. The trispecific fusion protein of any one of claims 35-37, wherein each of HSA binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 80.
39. The trispecific fusion protein of claim 35, wherein each of the first PD-L1 binding polypeptide and the second PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72; each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 62; and the HSA binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 80.
40. The trispecific fusion protein of claim 35, wherein each of the first PD-L1 binding polypeptide and the second PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72; each of the first LAG-3 binding polypeptide and the second LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 54; and the HSA binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 80.
41. The trispecific fusion protein of claim 34 comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 8-10.
42. A bispecific fusion protein comprising: a PD-L1 binding polypeptide; a LAG-3 binding polypeptide; and a fragment crystallizable (Fc) region of an antibody.
43. The bispecific fusion protein of claim 42, wherein the antibody is selected from a human IgG1 (hIgG1) antibody and a human IgG4 (hIgG4) antibody.
44. The bispecific fusion protein of claim 43, wherein the hIgG1 antibody comprises LALA mutations (Leu234Ala and Leu235Ala mutations).
45. The bispecific fusion protein of any one of claims 42-44, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72 or 73.
46. The bispecific fusion protein of any one of claims 42-45, wherein the LAG-3 binding polypeptide comprises an amino acid sequence of the LAG-3 binding polypeptide of any one of the preceding claims.
47. The bispecific fusion protein of claim 42, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72; the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 54; and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
48. The bispecific fusion protein of claim 42, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72; the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 62; and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
49. The bispecific fusion protein of claim 42, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72; the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 66; and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
50. The bispecific fusion protein of claim 42, wherein the PD-L1 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 72; the LAG-3 binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 69; and the antibody is a hIgG4 antibody or a hIgG1 antibody, optionally comprising a LALA double mutation.
51. The bispecific fusion protein of claim 42 comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 6, 7, 15, 19, 38, 39, 40, 41, 44, and 45.
52. The fusion protein of any one of the preceding claims further comprising one or more linker located between two of the polypeptides.
53. The fusion protein of claim 52, wherein the linker is a rigid linker, optionally comprising the amino acid sequence of AEAAAKEAAAKEAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 86).
54. The fusion protein of claim 52, wherein the linker is a flexible linker, optionally comprising the amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 87).
55. A polynucleotide comprising an open reading frame encoding the fusion protein of any one of the preceding claims.
56. The polynucleotide of claim 55, wherein the open reading frame comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the nucleotide sequence of any one of SEQ ID NOs: 99-180.
57. A vector, optionally a viral vector or a plasmid vector, comprising the polynucleotide of claim 55 or 56.
58. A cell, optionally a mammalian cell, comprising the polynucleotide of claim 55 or 56 or the vector of claim 57.
59. A pharmaceutical composition comprising: (a) the fusion protein of any one of the preceding claims, the polynucleotide of any one of the preceding claims, the vector of any one of the preceding claims, or the cell of any one of the preceding claims; and (b) a pharmaceutically acceptable excipient.
60. A method comprising administering to a subject the pharmaceutical composition of claim 59.
61. The method of claim 60, wherein the subject has a cancer.
62. The method of claim 60 or 61, wherein the pharmaceutical composition is administered subcutaneously, intravenously, or intramuscularly.
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