WO2021108109A1 - Modulators of t-cell activity - Google Patents

Modulators of t-cell activity Download PDF

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Publication number
WO2021108109A1
WO2021108109A1 PCT/US2020/059429 US2020059429W WO2021108109A1 WO 2021108109 A1 WO2021108109 A1 WO 2021108109A1 US 2020059429 W US2020059429 W US 2020059429W WO 2021108109 A1 WO2021108109 A1 WO 2021108109A1
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domain
seq
polypeptide
format
sequence
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PCT/US2020/059429
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French (fr)
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Yuntao Song
Haiping ZHOU
Jianhui Zhou
Yi Ding
Ping SZU
Meng Xu
Juan TIAN
Chuan Liao
Chuang Li
Miaomiao Zhang
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Beijing Xuanyi Pharmasciences Co., Ltd.
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Publication of WO2021108109A1 publication Critical patent/WO2021108109A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • 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
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • T-cell lymphocytes play a critical role in cell-mediated immunity' by providing for an adaptive response to certain stimuli.
  • T-cell activation depends on activation of at least two signaling pathways, one that is antigen specific and the other that is antigen nonspecific.
  • Antigen-specific activation of T-cells is mediated by peptide/major histocompatibility complexes on antigen- presenting cells interacting with specific T-cell antigen receptors.
  • Binding of B7-related molecules such as CD80 (B7-1) on antigen-presenting cells, to CD28 and/or CTLA-4 on T-cells, provides important antigen-nonspecific costimulatory signals essential for optimum immune responses.
  • Arming diseased cells with CD80 ligands can provide antigen-nonspecific costimulatory signals, which can potentiate T-cell activation, similar to antigen presenting cells, whereby turning diseased cells into antigen presentmg-!ike cells.
  • Compositions and methods to achieve such T-cell potentiation, m addition to other functions, are needed in the art, and are described herein.
  • the present disclosure provides modulators of T-cell activity, methods of making, and methods of use thereof.
  • the modulators provided herein comprise novel FD- LI antibodies capable of blocking PD-l/PD-Ll interactions, allowing for T-cell proliferation and function.
  • the modulators provided herein comprise multifunctional polypeptides comprising an extracellular binding domain of a CD80 polypeptide, a disease-associated antigen-binding domain, and an Fc domain, capable of arming diseased cell with a CD80 ligand in addition to other functions.
  • the present disclosure also provides pharmaceutical compositions comprising such modulators and methods for use in the treatment of cancer, infectious diseases, and inflammatory diseases.
  • multifunctional polypeptides comprising: a CD80 extracellular domain; a disease-associated antigen-binding domain; and an Fc domain.
  • the multifunctional polypeptides comprise any one of the formats presented in FIG. 1 A.
  • the multifunctional polypeptides comprise a cancer-associated antigen binding domain, an infectious disease-associated antigen binding domain, or an inflammatory disease-associated antigen binding domain.
  • the disease-associated antigenbinding domain is a PD-L1 binding domain.
  • the disease-associated antigen binding domain is a Claudin 18.2 binding domain.
  • compositions comprising the multifunctional polypeptides of the disclosure.
  • polynucleotides encoding the multifunctional polypeptides of the disclosure, vectors comprising the polynucleotides; and cells expressing the vectors.
  • PD-Ll antibodies comprising the ammo acid sequence of a CDR-L1 of Table 1, a CDR-L2 of Table 2, a CDR-L3 of Table 3, a CDR-H1 of Table 4, a CDR- H2 of Table 5, and/or a CDR-H3 of Table 6.
  • the antibodies are an antigen binding fragment.
  • the antibodies are full length antibodies.
  • compositions comprising the PD-Ll antibodies of the disclosure.
  • polynucleotides encoding for any of the PD-Ll antibodies provided herein, vectors comprising such polynucleotides, and cells expressing such vectors.
  • kits and articles of manufacture comprising any of the multifunctional polypeptides of PD-Ll antibodies of the disclosure.
  • a disease or disorder comprising administering to subject in need thereof a therapeutically effective amount of any one of the PD-Ll antibodies, multifunctional polypeptides, or pharmaceutical compositions provided herein.
  • the disease or disorder may be selected from cancer, infectious disease, or inflammatory disease.
  • FIG. IB is a table showing exemplar constructs with anti-PD-Ll as the disease-associated antigen-binding domain for the formats presented in FIG. 1A.
  • the disease- associated antigen-binding domain is an anti-ClaudinlB.2 antigen-binding domain, e.g. anti-CLDN 18.2 Ab or HuCLDN18.2-v.24.
  • FIG. 2 shows CD80-Fc and the disease-associated antigen-binding domain expression constructs of the disclosure.
  • LS leader sequence
  • CD80 extracellular CD80
  • Fc-hole Fc variations
  • VL, VH, CHI, CL, scFv disease-associated antigen-binding domain components.
  • FIG. 3 show r s a SDS-PAGE analysis of sc-hCD80 dimer-Fc/anti-PD-Ll (Format B) and hCD80 ⁇ Fc/anti ⁇ PD-Li (Format A) multifunctional polypeptides.
  • FIG, 4 show's a SDS-PAGE analysis of sc-hCD80 dimer-Fc/anti-CLDN 18.2 Ah3 and sc- I1CD8O dimer-Fc/HuCLDN 18.2-v.24 (Format B) multifunctional polypeptides.
  • FIG, 5 show's a SDS-PAGE analysis of hCB80-Fc/anti-CEDN 18.2 Ab3 and hCD80- Fc/HuCLDN 18.2-v.24 (Format A) multifunctional polypeptides.
  • FIG, 6 show's a SDS-PAGE analysis of hCB80-Ec/anti-CEDN 18.2 Ab! scFv-Fc and hCD80-Fc/anti-CLDN 18.2 Abl2 scFv-Fc (Format J) multifunctional polypeptides.
  • FIG. 7 show's a SBS-PAGE analysis of anti-CLBN 18.2 Ab 12-hCD80 and anti-CLDN 18.2 Abl2-hCB80 (Format F) multifunctional polypeptides.
  • FIG. 8 shows a SDS-PAGE analysis of anti-PD-Ll -hCD80 (Format F) multifunctional polypeptides.
  • FIG. 9 show's a SDS-PAGE analysis of sc-hCD80 dimer-Fc/anti-PD-Ll (DVD) (Format D) multifunctional polypeptides.
  • FIG. 10 shows a SDS-PAGE analysis of hCD80-anti-PD-Ll (Format H) and sc-hCD80 dimer-anti-PD-Ll (LC) (Format ! multifunctional polypeptides.
  • FIG. 11 is an ELISA showing the binding affinities of hCD80-Fc/anti-PD-Ll multifunctional polypeptide to recombinant protein hCTLA ⁇ 4 or hPD-Ll as compared to hCD80-Fc fusion protein.
  • FIG. 12 show3 ⁇ 4 the binding affinities of a hCD 80/anti -PD-Ll (Format A) multifunctional polypeptide to Flp-in 293 cell surface overexpressing hCD28, hCTLA-4 or h PD-L l by FACS.
  • FIG. 13 shows the binding affinities of a sc-hCDBO dimer-Fc/anti-PD-Ll (Format B) multifunctional polypeptide to Flp-in 293 cell surface over expressing hCD28, hCTLA-4 or hPD-Ll by FACS
  • FIG, 14 shows the binding affinities of anti-PD-Ll-hCDBO (Format F), sc ⁇ hCB80 dimer- Fc/anti-PD-Ll (DVD) (Format D), hCD80-anti -PD-Ll (Format H), and sc ⁇ hCD80 dimer-anti-PD-Ll (LC) (Format I) multifunctional polypeptides to Flp-in 293 cell surface overexpressing hCD28, hCTLA-4 or hPD-Ll by FACS.
  • Form F anti-PD-Ll-hCDBO
  • DVD sc ⁇ hCB80 dimer- Fc/anti-PD-Ll
  • LC sc ⁇ hCD80 dimer-anti-PD-Ll
  • FIG, 15 shows the binding affinities of hCD80-Fc/anti-CLDN 18.2 Abl ScFv-Fc (Format J), hCD80 ⁇ Fc/anti ⁇ CLDN 18.2 Abl2 ScFv-Fc (Format J), Anti-CLDN 18.2 Abl2-hCD80 (Format F). and HuCLDN18.2-v.24-hCD80 (Format F) multifunctional polypeptides to HEK 293 cell surface overexpressing CLDN18.2 or KATO III cell surface endogenously expressing CLDN18.2 by FACS.
  • FIG, 16 shows the binding affinities of hCD80-Fc/anti-CLDN 18.2 Abl ScFv-Fc (Format J), hCD80 ⁇ Fc/anti ⁇ CLDN 18.2 Abl2 ScFv-Fc (Format J), Anti-CLDN 18.2 Abl2-hCD80 (Format F). and HuCLDN18.2-v.24-hCD80 (Format F) multifunctional polypeptide to Flp-in 293 cell surface overexpressing hCD28, hCTLA-4 or hPD-Ll by FACS.
  • FIG. 17 show3 ⁇ 4 that simultaneous binding of a hCD80-F c/anti -PD-Ll (Format A) multifunctional polypeptide comprising anti -PD-Ll and hCDBO moiety to antigen expression ceils (e.g. Flp-in 293 PD-Ll cells) provides co-stimulatory signal required for IL-2 promoter activation.
  • FIG. 17 show3 ⁇ 4 that simultaneous binding of a hCD80-F c/anti -PD-Ll (Format A) multifunctional polypeptide comprising anti -PD-Ll and hCDBO moiety to antigen expression ceils (e.g. Flp-in 293 PD-Ll cells) provides co-stimulatory signal required for IL-2 promoter activation.
  • FIG. 17 show3 ⁇ 4 that simultaneous binding of a hCD80-F c/anti -PD-Ll (Format A) multifunctional polypeptide comprising anti -PD-Ll and hCDBO moiety to antigen
  • FIG. 18 shows that a hCD80-Fe/anti-PD-Ll (Format A) multifunctional polypeptide comprising an anti-PD-Ll and a hCDBO moiety binding to an antigen presenting cell and T-cell not only breaks the PD-1/PD-L1 blockade for NFAT signaling but also provides additional NFAT activation. Blocking PD- 1 /PD-Ll and providing co-stimulation induces stronger NFAT activity.
  • FIG. 19 shows that multifunctional polypeptides comprising anti-CLDN18.2 and hCD80 binding to antigen expression cells provides co-stimulatory signal required for IL-2 promoter activation.
  • FIG. 20 shows the binding affinities of hPD-Ll antibodies to Flp-in 293 ceil surface overexpressing hPD-Ll by FACS.
  • FIG. 21 show's PD-L1 antibodies competitively inhibit liPD-1 binding to Fip-in 293 cell surface overexpressing hPD-Ll by FACS.
  • FIG. 22 is an ELISA showing the binding affinities of mCDSO-Fc/anti-PD-Ll multifunctional polypeptides to recombinant protein hCD28 as compared to mCD80-Fc fusion protein.
  • FIG. 23 is an ELISA showing the binding affinities of mCD80-Fe/anti-PD-Ll multifunctional polypeptides to recombinant protein mCD28 as compared to mCD80-Fc fusion protein.
  • FIG, 24 is an ELISA showing the binding affinities of mCD80-Fe/anti ⁇ PD-Ll multifunctional polypeptides to recombinant protein hCTLA4 as compared to mCD80-Fc fusion protein.
  • FIG. 25 is an ELISA showing the binding affinities of mCD80-Fc/anti-PD-Ll multifunctional polypeptides to recombinant protein mCTLA4 as compared to mCD80-Fc fusion protein.
  • FIG. 26 is an ELISA showing the binding affinities of mCD80 ⁇ Fc/anti-PD-Ll multifunctional polypeptides to recombinant protein hPD-Ll as compared to mCD80-Fc fusion protein.
  • FIG. 27 show's the binding affinities of mCD80-Fc/anti-PD-Ll multifunctional polypeptides to Flp- 293 cell surface overexpressmg hCD28 by FACS.
  • FIG. 28 show's the binding affinities of mCD80-Fc/anti-PD-Ll multifunctional polypeptides to Flp-m 293 cell surface overexpressmg mCD28 by FACS.
  • FIG. 29 show's the binding affinities of mCD80-Fc/anti-PD-Ll multifunctional polypeptides to Flp-m 293 cell surface overexpressing hCTLA4 by FACS.
  • FIG. 30 show's the binding affinities of mCD8G-Fc/anti-PD-Ll multifunctional polypeptides to Flp-m 293 cell surface overexpressing hPD-Ll by FACS.
  • FIG. 31 show's the binding affinities of mCD8G-Fc/anti-PD-Ll multifunctional polypeptides to Flp-m 293 cell surface overexpressing mPD-Ll by FACS.
  • FIG. 32 shows the antitumor activity of mCDBO-Fc/anti-PD-Ll multifunctional polypeptides in MC-38-human-PD-Ll model.
  • an effective amount or “therapeutically effective amount” refer to a quantity and/or concentration of a therapeutic of the disclosure, that when administered ex vivo (by contact with a ceil from a patient) or in vivo (by administration into a patient) either alone (i.e., as a monotherapy) or in combination with additional therapeutic agents, yields a statistically significant decrease in disease progression as, for example, by ameliorating or eliminating symptoms and/or the cause of the disease.
  • An effective amount may be an amount that relieves, lessens, or alleviates at least one symptom or biological response or effect associated with a disease or disorder, prevents progression of the disease or disorder, or improves physical functioning of the patient in the ease of ceil therapy, the effective amount is an effective dose or number of cells administered to a patient by adoptive cell therapy.
  • the patient is a mammal such as a non-human primate or human patient.
  • mammals include, e.g., humans, non-human primates, rodents (e.g., rats; mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, and the like), etc.
  • rodents e.g., rats; mice
  • lagomorphs e.g., rabbits
  • ungulates e.g., cows, sheep, pigs, horses, goats, and the like
  • modulating or “modulate” as used herein in the context of an immune response, such as a mammalian immune response, refer to any alteration, such as an increase or a decrease, of existing or potential immune responses that occurs as a result of administration of a polypeptide or antibody of the present disclosure. Thus, it refers to an alteration, such as an increase or decrease, of an immune response as compared to the immune response that occurs or is present in the absence of the administration.
  • modulation includes any induction, activation, suppression or alteration in degree or extent of immunological activity of an immune cell.
  • Immune cells include B cells, T-cells, NK (natural killer) cells, NK T-cells, professional antigen-presenting cells (APCs), and non- professional antigen-presenting cells, and inflammatory' cells (neutrophils, macrophages, monocytes, eosinophils, and basophils).
  • Modulation includes any change imparted on an existing immune response, a developing immune response, a potential immune response, or the capacity to induce, regulate, influence, or respond to an immune response. Modulation includes any alteration m the expression and/or function of genes, proteins and/or other molecules in immune cells as part of an immune response.
  • Modulation of an immune response or modulation of immunological activity includes, for example, the following: elimination, deletion, or sequestration of immune cells; induction or generation of immune cells that can modulate the functional capacity of other cells such as autoreactive lymphocytes, antigen presenting cells, or inflammatory cells; induction of an unresponsive state in immune cells (i.e., anergy); enhancing or suppressing the activity or function of immune cells, including but not limited to altering the pattern of proteins expressed by these cells. Examples include altered production and/or secretion of certain classes of molecules such as cytokines, chemokines, growth factors, transcription factors, kinases, costimulatory molecules, or other cell surface receptors or any combination of these modulatory events.
  • polynucleotide and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • nucleic acids containing known analogues of natural nucleotides and that have similar binding properties to it and are metabolized in a manner similar to naturally- occurring nucleotides Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary nucleotide sequences as well as the sequence explicitly indicated (a “reference sequence”). Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues.
  • nucleic acid or polynucleotide encompasses cDNA or mRNA encoded by a gene.
  • peptide refers to a polymeric form of ammo acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • the terms include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, sialylations, phosphorylations and the like.
  • the terms also include molecules in which one or more ammo acid analogs or non-eanonicai or unnatural amino acids are included as can be synthesized, or expressed recombinantly using known protein engineering techniques.
  • proteins can be derivatized.
  • a polynucleotide or polypeptide has a certain percent sequence identity to another polynucleotide or poly peptide, meaning that, when aligned, that percentage of bases or ammo acids are the same, and in the same relative position, when comparing the two sequences. Sequence identity can be determined m a number of different ways.
  • sequences can be aligned using various convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), available over the world wade w r eb at sites including ncbi.nlm.mli.gov/BLAST, ebi.ac.uk/Tools/msa/tcoffee/, ebi.ac.uk/Tools/msa/muscle/, mafft. cbrc.jp/alignment/software/. See, e.g., Altschul et al. (1990), I Mol. Bioi. 215:403-10.
  • T-cell includes all types of immune cells expressing CD3, including T-helper cells (CD4+ ceils), cytotoxic T-cells (CD8+ cells), T-regulatory cells (Treg), and NK-T-cells.
  • treatment used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease or symptom in a mammal, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to acquiring the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease or symptom, i e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease.
  • the therapeutic agent may be administered before, during or after the onset of disease or injury.
  • the treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues.
  • Treating,” “treatment,” or “therapy” also means a decrease the severity of symptoms in an acute or chronic disease or disorder or a decrease in the relapse rate as for example m the ease of a relapsing or remitting autoimmune disease course or a decrease in inflammation in the case of an inflammatory aspect of an autoimmune disease.
  • the terms “treatment” or, “inhibit,” “inhibiting” or “inhibition” of cancer refers to at least one of: a statistically significant decrease in the rate of tumor growth, a cessation of tumor growth, or a reduction in the size, mass, metabolic activity, or volume of the tumor, as measured by standard criteria such as, but not limited to, the Response Evaluation Criteria for Solid Tumors (RECIST), or a statistically significant increase in progression free survival (PFS) or overall survival (OS).
  • RECIST Response Evaluation Criteria for Solid Tumors
  • PFS progression free survival
  • OS overall survival
  • Preventing,” “prophylaxis,” or “prevention” of a disease or disorder as used in the context of this disclosure refers to the administration of an immunomodulatory polypeptide or engineered cells of the disclosure, either alone or in combination with another compound, to prevent the occurrence or onset of a disease or disorder or some or all of the symptoms of a disease or disorder or to lessen the likelihood of the onset of a disease or disorder.
  • the term “variant” (also “mutant”, ‘mutated” or “modified”) as used in reference to a variant CD80 means a CD80, such as a mammalian (e.g., human or murine) CD80 created by human intervention, or are naturally occurring.
  • the variant CD80 is a polypeptide having an altered amino acid sequence, relative to an unmodified or wild-type CD80.
  • the variant CD80 is a polypeptide which differs from a wild-type CD80 isoform sequence by one or more amino acid substitutions, deletions, additions, or combinations thereof
  • the variant CD80 may contain at least one affinity modified domain, whereby one or more of the amino acid differences occurs in an IgSF domain (e.g. IgC domain and/or IgV domain).
  • a variant CD 80 may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid differences, such as ammo acid substitutions.
  • a variant CD80 polypeptide generally exhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a wild-type or unmodified CD80 extracellular domain (SEQ ID NO: 34).
  • Non- naturally occurring ammo acids as well as naturally occurring ammo acids are included within the scope of substitutions or additions.
  • a variant CD8Q is not limited to any particular method of making and includes, for example, de novo chemical synthesis, de novo recombinant DNA techniques, or combinations thereof.
  • a variant CDS0 of the disclosure specifically binds to at least one or more of: CD28, PD-L1, or CTLA-4 of a mammalian species .
  • the altered amino acid sequence results in an altered (x.e., increased or decreased) binding affinity or avidity to CD28, PD- Ll, or CTLA-4 compared to the wild-type CD80 protein.
  • An increase or decrease in binding affinity or avidity can be determined using well-known binding assays such as flow cytometry. Larsen et al, American Journal of Transplantation, Vol 5: 443-453 (2005). See also, Linsley et al, Immunity, 1 : 7930801 (1994) or ELISA (please add reference).
  • An increase in variant CD80 binding affinity or avidity to CD28, PD-L1, or CTLA-4 is to a value at least 5% greater than that of the wild-type CD80 control value and in some embodiments, at least 10%, 15%, 20%, 30%, 40%, 50%, 100% greater than that of the wild-type CD80 control value.
  • a decrease in CD80 binding affinity' or avidity to CD28, PD-L1, or CTLA-4 is to a value no greater than 95% of the of the wild-type CD80 control value, and m some embodiments no greater than 80%, 70% 60%, 50%, 40%, 30%, 20%, 10%, 5%, or no detectable binding affinity or avidity of the wild-type CD80 control value.
  • variant CD80 is altered m primary amino acid sequence by substitution, addition, or deletion of amino acid residues.
  • variant CD80 in the context of variant CD80 is not be construed as imposing any condition for any particular starting composition or method by which the variant CD80 is created.
  • a variant CD80 can, for example, be generated starting from wild type mammalian CD80 sequence information, then modeled in silico for binding to CD28, PD-Ll, or CTLA-4, and recombinantly or chemically synthesized to yield a variant CD80 of the present disclosure.
  • a variant CD80 can be created by site-directed mutagenesis of a wild-type CD80.
  • variant CD80 denotes a composition and not necessarily a product produced by any given process.
  • PD- Li antibodies may be used in variety of therapeutic and diagnostic methods.
  • the PD-L1 antibody may be used alone, or in combination with other agents in treating a disease or disorder characterized by abnormal PD-Ll expression or activity, including but not limited to cancer, infectious diseases, and inflammatory diseases.
  • the PD-Ll antibody sequences are included as a part of a multifunctional polypeptide of the disclosure, wherein the multifunctional polypeptide comprises a CD80 extracellular domain (e.g. a wild-type CD80 extracellular domain, e.g. SEQ ID NO: 34, or variants thereof), a disease-associated antigen-binding domain; and an Fc domain.
  • the PD-Ll antibody may be referred to interchangeably as a PD-Ll antigen-binding domain, or simply as a PD-Ll binding domain.
  • antibody includes full length antibodies; antibody fragments (including without limitation Fab, Fab’, F(ab’)2, Fv, diabody, scFv, scFv-Fcs, single domain antibody, single chain antibody, single heavy chain antibody, and single light chain antibody), provided that they exhibit a desired biological activity (e.g. epitope binding) ; monoclonal antibodies; polyclonal antibodies; monospecific antibodies; multi-specific antibodies (e.g., bispecific antibodies); humanized antibodies; chimeric antibodies; covalently modified antibodies, and antibody conjugates (e.g. antibody-drug conjugates or antibodies conjugated to detectable labels).
  • the antibodies may be derived from any species.
  • the antibody is human.
  • the antibody is a mouse antibody.
  • the full length PD-L1 antibodies of the disclosure may have a human Fc, that is IgGl, IgG2, IgG3, or XgG4.
  • the full length PD -LI antibodies of the disclosure may have a mouse Fc, that is IgGl, IgG2a, IgG2b, or IgG3.
  • Reference to “PD-L1” can relate to a PD-L1 protein of any species, and in particular to a human PD-L1, and more particularly to a protein comprising the amino acid sequence of SEQ ID NO: 1 represented below.
  • Table 1 provides exemplary- CDR-L1 sequences of the PD-L1 antibodies of the disclosure.
  • Table 2 provides exemplary CDR-L2 sequences of the PD-Ll antibodies of the disclosure.
  • Table 3 provides exemplary CDR-L3 sequences of the PD-L1 antibodies of the disclosure.
  • Table 4 provides exemplary CDR-H1 sequences of the PD-L1 antibodies of the disclosure.
  • Table 5 provides exemplary CDR-H2 sequences of the PD-Ll antibodies of the disclosure.
  • Table 6 provides exemplary CDR-H3 sequences of the PD-Ll antibodies of the disclosure.
  • a PD-Ll antibody comprising the amino acid sequence of a CDR-L1 of Table 1, a CDR-L2 of Table 2, a CDR-L3 of Table 3, a CDR-H1 of Table 4, a CDR-H2 of Table 5, and/or a CDR-H3 of Table 6.
  • Table 7 provides the light chain CDR sequences of eleven PD-L1 antibodies of the disclosure.
  • a PD-L1 antibody comprising the amino acid sequence of a light chain comprising the sequences of any one of the CDR-L1, CDR-L2, and CDR- L3 combinations presented in Table 7.
  • Table 8 provides the heavy chain CDR sequences of eleven PD-L1 antibodies of the disclosure.
  • a PD-L1 antibody comprising the amino acid sequence of a light chain comprising the sequences of any one of the CDR-HL CDR-H2, and CDR- H3 combinations presented m Table 8.
  • Table 9 provides the heavy and light chain CDR sequences of eleven PD-L1 antibodies of the disclosure.
  • a PD-L1 antibody comprising the amino acid sequences of any one of the CDR-LL CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 combinations presented in Table 9.
  • Table 10 provides the sequences of exemplary light chain variable domains (VL) of the PD- LI antibodies of the di sclosure (exemplary CDR sequences are underlined below).
  • Table 11 provides the sequences of exemplary heavy chain variable domains (VH) of the PD-L1 antibodies of the disclosure.
  • a PD-L1 antibody comprising a VL domain comprising an ammo acid sequence set forth in any one of SEQ ID NOs: 18-21 and 8EQ ID NO: 57, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a PD-L1 antibody comprising a VH domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 23-33, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a PD-L1 antibody comprising a VL domain comprising an ammo acid sequence set forth m any one of SEQ ID NOs: 18-21 and SEQ ID NO: 57, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto, and VH domain comprising an am o acid sequence set forth in any one of SEQ ID NOs: 23- 33, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
  • a PD -LI antibody comprising:
  • VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and/or a VH domain comprising SEQ ID NO: 23, or a sequence comprising at least 80%, at least
  • VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and/or a VH domain comprising SEQ ID NO: 24, or a sequence comprising at least 80%, at least
  • VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and/or a VH domain comprising SEQ ID NO: 25, or a sequence comprising at least 80%, at least
  • VL domain comprising SEQ ID NO: 18; and/or a VH domain comprising SEQ ID NO: 26, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto,
  • VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and/or a VH domain comprising SEQ ID NO: 27, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto:
  • VL domain comprising SEQ ID NO: 19, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and/or a VH domain comprising SEQ ID NO: 28, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto:
  • VL domain comprising SEQ ID NO: 19, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and/or a VH domain comprising SEQ ID NO: 29, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto:
  • VL domain comprising SEQ ID NO: 20, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto; and/or a VH domain comprising SEQ ID NO: 30, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto:
  • VL domain comprising SEQ ID NO: 21, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto; and/or a VH domain comprising SEQ ID NO: 31, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto:
  • VL domain comprising SEQ ID NO: 57, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto; and/or a VH domain comprising SEQ ID NO: 32, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto:
  • VL domain comprising SEQ ID NO: 57, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto; and/or a VH domain comprising SEQ ID NO: 58, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto;
  • VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto; and/or a VH domain comprising SEQ ID NO: 59, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto;
  • VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto; and/or a VH domain comprising SEQ ID NO: 60, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto;
  • VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto; and/or a VH domain comprising SEQ ID NO: 61, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto;
  • polynucleotides encoding for any of the PD-L1 antibodies provided herein, vectors comprising such polynucleotides, and cells expressing such vectors.
  • compositions comprising any one of the PD-L1 antibodies provided herein, optionally with a pharmaceutically acceptable excipient.
  • the disease or disorder may be cancer, infectious diseases, or inflammatory diseases.
  • multifunctional polypeptides comprising the following components, the components of which are described independently m greater detail herein.
  • the components are linked together to form the multifunctional polypeptide. As described in greater detail herein, and referring to exemplary formats in FIG. 1 A, the components can be linked in various orders. In some embodiments, the components are linked with the use of linkers, in other embodiments, the components are fused together. A skilled artisan will appreciate the manner in which the polypeptide components can be formatted.
  • polynucleotides encoding the multifunctional polypeptides of the disclosure; vectors comprising the polynucleotides; and cells expressing the vectors.
  • the multifunctional polypeptides of the disclosure may be modified, e.g. by glycosylation, sialylation, acetylation, phosphorylation, and the like. Accordingly, in exemplary embodiments, the sialic acid content of the multifunctional polypeptides may vary - in such embodiments, any one or more of the CD80 extracellular domain, the disease-associated antigen-binding domain, and the Fc domain may be sialyiated.
  • compositions comprising the multispecific polypeptides of the disclosure, optionally with a pharmaceutically acceptable excipient.
  • the multifunctional polypeptides of the disclosure comprise a CD80 extracellular domain, wherein the CD80 extracellular domain comprises an am o acid sequence having at least 80%, 85%, 90%, 95%, or 100% ammo acid sequence identity to SEQ ID NO: 34, the human extracellular domain of CD80 (underlined amino acid residues are exemplar residues that may be substituted in the CD80 extracellular domain variants of the disclosure).
  • the CD80 extracellular domain has one or more amino acid substitution modifications at positions 130, 131, 139, 155, 156, 165, or 166 of SEQ 1D NO: 34, or the corresponding positions in SEQ ID NO: 37.
  • the CD80 extracellular domain has at least a substitution modification at position 130 of SEQ ID NO: 34, and the amino acid substitution modification at position 130 of SEQ ID NO: 34 is T13QA.
  • the CD80 extracellular domain has at least a substitution modification at position 131 of SEQ ID NO: 34, and the amino acid substitution modification at position 131 of SEQ ID NO: 34 is S131A, S131V, S131I, SI 3 i 14 S131R, S131E, S131D, or S131Q.
  • the CD80 extracellular domain has at least a substitution modification at position 139 of SEQ ID NO: 34, and the amino acid substitution modification at position 139 of SEQ ID NO: 34 is L139V.
  • the CB80 extracellular domain has at least a substitution modification at position 155 of SEQ ID NO: 34, and the amino acid substitution modification at position 155 of SEQ ID NO: 34 is VI 55 A, VI 551, or V155T.
  • the CD80 extracellular domain has at least a substitution modification at position 156 of SEQ ID NO: 34.
  • the CD80 extracellular domain has at least a substitution modification at position 165 of SEQ ID NO: 34, and the amino acid substitution modification at position 165 of SEQ ID NO: 34 is A165S, A165V, A165I, A165F, A165R, A165E, A165D, or A165Q.
  • the CD80 extracellular domain has at least a substitution modification at position 166 of SEQ ID NO: 34, and the amino acid substitution modification at position 166 of SEQ ID NO: 34 is V166A, V166L, or V166T.
  • the CD80 extracellular domain is sia!ylated.
  • the multifunctional polypeptides of the disclosure comprise disease-associated antigen binding domains that bind disease-associated antigens.
  • disease-associated antigen-binding domains are capable of binding disease-associated antigens on diseased eells through classical antigen-antibody binding interactions. Without being bound any theory or mechanism, this can allow the multifunctional polypeptide to arm a diseased cell expressing a disease-associated antigen (e.g. a cancer-specific antigen), with a CD80 extracellular domain (a CD80 ligand), via binding of the disease-associated antigen-binding domain to the diseased cell.
  • a disease-associated antigen e.g. a cancer-specific antigen
  • CD80 extracellular domain a CD80 ligand
  • the diseased cell now armed with a CD80 ligand is available to bind to a T-cell, conferring a costimulatory signal thereon.
  • the CD80 ligand is available to bind to any one or more of its cognate binding partners, CD28 PD-L1 or CTLA-4 on T-cells (either T-lymphocytes, or T- regulatory cells).
  • the disease-associated antigen can be a soluble antigen.
  • the disease-associated antigen can be any antigen upregulated or overexpressed in a disease such as a cancer, an infectious disease, or an inflammatory disease.
  • the multifunctional polypeptides comprise a cancer-associated antigen-binding domain.
  • the multifunctional polypeptides comprise an infectious disease-associated antigen binding domain. In some embodiments, the multifunctional polypeptides comprise an inflammatory disease-associated antigen-binding domain. In some embodiments, the disease-associated antigen- binding domain is a PD-Ll binding domain. In other embodiments, the disease-associated antigen binding domain is a Claudin 18.2 binding domain.
  • the disease-associated antigen-binding domain is a PD-Ll binding domain.
  • the PD-Ll binding domain comprises:
  • VL domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 18-21, and SEQ ID NO: 57, or a sequence comprising at least 80% identity thereto;
  • VH domain comprising an ammo acid sequence set forth in any one of SEQ ID NOs: 23-33, and SEQ ID NOs: 58-61, or a sequence comprising at least 80% identity thereto; or
  • VL domain comprising an ammo acid sequence set forth in any one of SEQ ID NOs: 18-21, and SEQ ID NO: 57, or a sequence comprising at least 80% identity thereto
  • VH domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 23- 33, and SEQ ID NOs: 58-61, or a sequence comprising at least 80% identity thereto.
  • the PD-Ll binding domain comprises the antigen- binding sequences of Atezohzumab, Avelumab, or Durvalumab.
  • Atezolizumah Light Chain
  • Atezolizumah Heavy Chain
  • NRFSGSKSGNTASLTiSGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL SEQ ID NO: 38
  • the disease-associated antigen-binding domain is a Claudin-18.2 binding domain.
  • sequence of the Claudin-18.2 binding domain is from WQ2019/174617, the disclosure of which is incorporated herein by reference in its entirety.
  • sequence of the Claudin-18.2 binding domain is from EP1997832, the disclosure of which is incorporated herein by reference in its entirety.
  • sequence of the Claudin-18.2 binding domain is from EP3483182, the disclosure of which is incorporated herein by reference in its entirety. [0121] In some embodiments, the sequence of the Claudin-18.2 binding domain is from WO2019173420, the disclosure of which is incorporated herein by reference in its entirety.
  • Claudin-18.2 binding domains (and sequences contributing to the same) of the disclosure, are listed here. For each VH and VL listed below', the CDRs are bolded and underlined. Accordingly, the Claudin-18.2 binding domains of the disclosure may comprise any one or more of the CDR chains listed below.
  • DIVMTOSPSSLTVTAGEKVTMSCKSSOSLLNSGNOKNYLTWYOOKPGOPPKLIIYWAST RESGVPDRFTGSGSGTDFTLTISSVOAEDLAIYYCONDYFYFLTFGAGTKLELK (SEQ ID NO: 42)
  • the disease-associated antigen-binding domain is not a PD-L1 binding domain.
  • the disease-associated antigen-binding domain is not a Claudin-18.2 binding domain.
  • the Fc domains of the multifunctional polypeptides of the disclosure are Fe dimers.
  • the Fc domain is human in some embodiments, the Fc domain is a human XgGl, IgG2, XgG3, or IgG4 Fe domain.
  • the Fc domain is from mouse. In some embodiments, the Fc domain is a mouse XgGl, IgG2a, IgG2b, or IgG3 Fc domain.
  • the Fc domain is a wild-type human XgGl Fc domain.
  • the Fe domain is a mutant human IgGl Fe domain.
  • the mutant human IgGl Fc domain comprises reduced effector function.
  • the mutant human IgGl Fc domain comprises a knob-in-hole mutation.
  • the mutant human IgGl Fc domain comprises reduced effector function and a knob-m-hoie mutation.
  • knob-in ⁇ ho!e mutations heterodimerization of the two Fc monomeric domains of any of the formats depicted in FIG. 1A can be promoted by introducing different, but compatible substitutions in the two Fc monomeric domains, such as knob-in-hole residue pairs (e.g. US Pat. No. 5,731,168).
  • the Fc domain is glycosylated, aeetylated, sialylated, and/or phosphorylated.
  • the Fc domain comprises the ammo acid sequence of any one of SEQ ID NOS: 100 to H I.
  • SEQ ID NO: 112 provides an exemplary sequence comprising a human CD80 extracellular domain + human IgGl Fc.
  • SEQ ID NO: 136 provides an exemplary sequence comprising a mouse CD80 extracellular domain + mouse IgG2a Fc.
  • Hum IgGl Fc (IGHG1 _HUMAN: UniProKB - RQ1857)
  • VECPPCP APPVAGPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVQFNWYVDG VEVHNAKTKP REEQFNSTFR WSVLTWHQ DWLNGKEYKC KVSNKGLPAP IEKTISKTKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDISVEW ESNGQPENNY KTTPPMLD SD GSFFLYSKLT VDKSRWQQGN WSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 101) Human IgG3 Fc (IGHG3_HUMAN: UniProKB - ⁇ P01860)
  • KYGPPCPSCP APEFLGGPS V FLFPPKPKDT LMISRTPEVT CWVDVSQED PEY QFNWYVD GVEVHNAKTK PREEQFNSTY RVV8VLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESN GQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK (SEQ ID NO: 103)
  • VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 110) hlgGlHc Hole N297G
  • VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 111)
  • the multifunctional polypeptide may comprise any one of the structural formats provided in FIG. 1A.
  • the multifunctional polypeptide comprises the structure of Format A of FIG. 1A. In some embodiments, the multifunctional polypeptide does not comprise the structure of Format A. In some embodiments, the multifunctional polypeptide comprises the structure of Format A of FIG. I A, and the disease-associated antigen-binding domain is specific for PD-L1 or Claudin-18.2
  • the multifunctional polypeptide comprises the structure of Format B of FIG. !A. In some embodiments, the multifunctional polypeptide does not comprise the structure of Format B. In some embodiments, the multifunctional polypeptide comprises the structure of Format B of FIG. 1 A, and the disease-associated antigen-bimi g domain is specific for PD-Ll or Claudin-18.2.
  • the multifunctional polypeptide comprises the structure of Format C of FIG. 1A. In some embodiments, the multifunctional polypeptide does not comprise the structure of Format C. In some embodiments, the multifunctional polypeptide comprises the structure of Format C of FIG. 1A, and the disease-associated antigen-binding domain is specific for PD-L1 or Claudin-18.2.
  • the multifunctional polypeptide comprises the structure of Format I) of FIG. LA In some embodiments, the multifunctional polypeptide does not comprise the structure of Format D. In some embodiments, the multifunctional polypeptide comprises the structure of Format D of FIG. 1A, and the disease-associated antigen-binding domain is specific for PD-L1 or Claudin-18.2,
  • the multifunctional polypeptide comprises the structure of Format E of FIG. IA. In some embodiments, the multifunctional polypeptide does not comprise the structure of Format E. In some embodiments, the multifunctional polypeptide comprises the structure of Format E of FIG. I A, and the disease-associated antigen-binding domain is specific for PD-LI or Claudin-18.2.
  • the multifunctional polypeptide comprises the structure of Format F of FIG. 1A. In some embodiments, the multifunctional polypeptide does not comprise the structure of Format F. In some embodiments, the ultifunctional polypeptide comprises the structure of Format F of FIG. 1 A and the disease-associated antigen-binding domain is specific for PD-LI or Claudin-18.2.
  • the multifunctional polypeptide comprises the structure of Format G of FIG. 1A. In some embodiments, the multifunctional polypeptide does not comprise the structure of Format G. In some embodiments, the multifunctional polypeptide comprises the structure of Format G of FIG. 1A, and the disease-associated antigen-binding domain is specific for PD-LI or Claudin-18.2.
  • the multifunctional polypeptide comprises the structure of Format H of FIG. 1 A. In some embodiments, the multifunctional polypeptide does not comprise the structure of Format H. In some embodiments, the multifunctional polypeptide comprises the structure of Format H of FIG. 1 A, and the disease-associated antigen- binding domain is specific for PD-LI or Claudin-18.2.
  • the multifunctional polypeptide comprises the structure of Format I of FIG. 1A. In some embodiments, the multifunctional polypeptide does not comprise the structure of Format I. In some embodiments, the multifunctional polypeptide comprises the structure of Format I of FIG. 1A, and the disease-associated antigen-binding domain is specific for PD-L1 or Claudin-
  • the multifunctional polypeptide comprises the structure of Format I of FIG. 1A, and the disease-associated antigen- binding domain is not specific for PD-L1 or Claudin-
  • the multifunctional polypeptide comprises the structure of Format J of FIG. LA In some embodiments, the multifunctional polypeptide does not comprise the structure of Format J. In some embodiments, the multifunctional polypeptide comprises the structure of Format J of FIG. 1 A, and the disease-associated antigen-binding domain is specific for PD-L1 or Claudin-
  • the ultifunctional polypeptide comprises any one of the structures of Format A-J of FIG. LA, and the disease-associated antigen-binding domain is not specific for PD- Ll.
  • the multifunctional polypeptide comprises any one of the structures of Format A-J of FIG. 1 A, and the disease-associated antigen-binding domain is not specific for Claudin
  • the multifunctional polypeptide comprises any one of the structures of Format A-J of FIG. 1 A and the disease-associated antigen-binding domain is not specific for Her2D- Ll.
  • the CD80 extracellular domain of the multifunctional polypeptide is a monomer.
  • the multifunctional polypeptide comprises two monomers of the CD80 extracellular domain.
  • the multifunctional polypeptide comprises four monomers of the CD80 extracellular domain.
  • the multifunctional polypeptide comprises a CD80 extracellular domain that is a single chain dimer.
  • the multifunctional polypeptide comprises two single chain dimers of the CD 80 extracellular domain.
  • the multifunctional polypeptide comprises a CD80 extracellular domain that attached to the N-terminus of the Fc domain.
  • the multifunctional polypeptide comprises a CD80 extracellular domain is attached to the N-terminus of a disease-associated antigen-binding domain, and wherein the disease-associated antigen-binding domain is attached to the N-terminus of an Fc domain.
  • the multifunctional polypeptide comprises a CD80 extracellular domain that is attached to the C-terminus of the Fc domain.
  • the multifunctional polypeptide comprises a disease-associated antigen-binding domain comprising an scFv.
  • the multifunctional polypeptide comprises a disease- associated antigen-binding domain comprising a Fab or Fab’ fragment.
  • the multifunctional polypeptide comprises a disease- associated antigen-binding domain comprising a F(ab’)2 fragment
  • the multifunctional polypeptide comprises a disease- associated antigen-binding domain comprising a Fv fragment.
  • the multifunctional polypeptide comprises a disease-associated antigen-binding domain comprising a Fab’ fragment and a Fv fragment.
  • the multifunctional polypeptide comprises a disease-associated antigen-binding domain that is attached to the N-terminus of the Fc domain.
  • the multifunctional polypeptide comprises two disease-associated antigen-binding domains coupled to each other, for example wherein one disease-associated antigen binding domain comprises a Fab or Fab’ fragment, and one antigen-binding domain comprises a Fv fragment.
  • the multifunctional polypeptide comprises a Fv fragment disease- associated antigen-binding domain that is attached to the N-terminus of a Fab or Fab’ disease- associated antigen-binding domain that is attached to the N-terminus of a Fc domain.
  • the multifunctional polypeptide comprises a disease-associated antigen-binding domain that is attached to the N-terminus of a CD80 extracellular domain which is attached to the N-terminus of a Fc domain.
  • the multifunctional polypeptide comprises a disease-associated antigen-binding domain that is attached to the C-terminus of the Fc domain.
  • the multifunctional polypeptide comprises the ammo acid sequence of any one of SEQ ID NOs: 113-135.
  • the sequences below are exemplary only, and by no means limiting the other variations possible, as described herein. Some of the sequences below are represented by domains, with the use of brackets.
  • Anti-PD-Ll Lc (Atezolizumah) (Format A,B ? F)
  • Anti-CLDN 18.2 Abl scFv (Anti-CLDN 18.2 Abl VH-iinker- Anti-CLDN 18.2 Abl VL-Iinker- hlgGl Fc Hole) (Format J)
  • Anti-CLDN 18.2 AM2 scFv (Anti-CLDN 18.2 Abl2 VH-iinker- ⁇ Anti-CLDN 18.2 Abl2 VL- linker-hlgGl Fc Hole) (Format J)
  • GHLRVNQTFNWNTTKQEHFPDN [GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS
  • the PD-Ll antibodies and multi functional polypeptides of the disclosure, and the pharmaceutical compositions comprising the LI antibodies and multifunctional polypeptides of the disclosure are useful for therapeutics, to treat or delay progression of a disease or disorder.
  • a disease or disorder comprising administering to subject in need thereof a therapeutically effective amount of any one of the PD-Ll antibodies, multifunctional polypeptides, or pharmaceutical compositions provided herein.
  • the disease or disorder may be selected from cancer, infectious disease, or inflammatory disease.
  • the cancer may comprise a liquid tumor, or solid tumor.
  • the disease or disorder may be associated with aberrant activity of a disease-associated antigen.
  • the disease or disorder may be associated with aberrant PD-L1 activity, or with aberrant Claudin 18.2 activity.
  • any of the PD-L1 antibodies, multifunctional polypeptides, or pharmaceutical compositions of the disclosure may be carried out intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, mtraorbitally, by implantation, by inhalation, mtratheeally, intraventrieulariy, or mtranasally.
  • a therapeutically effective amount may be administered for the treatment of the disease or disorder and the appropriate dosage of the may be determined based on the type of disease or disorder to be treated, the type of the therapeutic, the severity and course of the disease or disorder, the clinical condition of the subject, the subject’s clinical history' and response to the treatment, and the discretion of the attending physician.
  • any one of the PD-L1 antibodies, multifunctional polypeptides, or pharmaceutical compositions provided herein is administered intravenously.
  • normal dosage amounts may vary from about 1 ng/kg up to about 1000 mg/kg of an subject’s body weight or more per day, depending upon the route of administration.
  • the treatment may be sustained until a desired suppression of symptoms is achieved.
  • Dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the physician wishes to achieve. Progress of the therapy may be monitored by conventional techniques.
  • the PD-Ll antibodies, multifunctional polypeptides, or pharmaceutical compositions of the disclosure may be administered as a part of a combination therapy with another agent.
  • kits comprising the PD-Ll antibodies, multifunctional polypeptides and pharmaceutical compositions of the disclosure.
  • kits may contain a pharmaceutically acceptable excipient, an instruction manual and any combination thereof.
  • the kits comprise any one or more of the therapeutic compositions described herein, with one or more pharmaceutically acceptable excipients.
  • the present disclosure also provides articles of manufacture comprising any one or more of the PD-L1 antibodies, multifunctional polypeptides, pharmaceutical compositions, and kits described herein.
  • articles of manufacture include vials (e.g. sealed vials).
  • the modified pHENl vector was used to make a fully synthetic ScFv (single chain antibody fragment) phage library.
  • the antibody library was taken through five rounds of panning on PD-L1 (eetodomain)-His protein (according to J. Mol. Biol. (2004) 338, 299 310 with some modification).
  • the top 17 positive clones were selected and sequenced. The sequences are listed in Tables 1-11.
  • the scFvs were converted into full-length antibodies by using human IgG4 conservation sequences.
  • the genes of different antibodies were cloned and transfected into Expi-CHO cells (Thermofisher), then were purified by AKTA Explorer protein purification system.
  • the plate was coated with 1 pg/ml of recombinant PD-L1 (ectodomain)-His from Sino Biological, Inc. (Cat. No. 10084-H08H) at 4°C overnight. The next day, the plate was washed with 200m1 per well washing buffer (0.05% Tween-20 in PBS, pH7.4) for 3 times.
  • the assay plate was blocked with 200 m ⁇ of blocking buffer (2% BSA in Washing Buffer) for 1 hr at 37°C and washed again with washing buffer for 3 times. Series dilution of different antibodies were added. After incubation at 37°C for Bit, the plate was washed three times, and HRP goat anti-human IgG-Fc secondary antibody was added to each well and incubate at 37°C for lh. The plate was then washed, developed and read at 450 nm and 630nm. The ECso was calculated by GraphPad software. Table 12 provides ECso of the antibodies listed in Table 9. Among these antibodies, PD-2, PD-3, PD-4, PD-15, PD- 16, and PD- 18 had better binding affinities than Atezolizumab.
  • EXAMPLE 3 Determination of PD-LJ Antibodies binding affinities by FACS [0174]
  • Flp-in 293 cells overexpressing human PD-L1 were cultured in DMEM medium supplied with 10% FBS at 37°C in 5% C02. The cells were harvested, washed 3 times with PBS. After fixed with 4% PFA for 20 min, the cells were pelleted and washed with PBS to ensure that there was no PFA left. The cells were re-suspended in PBS with 3% BSA and incubated at 4°C overnight. Eleven-point dilution series of different antibodies in PBS with 3% BSA were added into different wells.
  • the Flp-in 293 cells which overexpressing human PD-L1 were cultured in DMEM medium supplied with 10% FBS at 37 ° C in 5% C02. The cells were harvested, washed, fixed and re-suspended as described in Example 3. After blocked in PBS with 3% BSA at 4 ° C overnight, lOug/rnl human biotin labeled PD-1 (Sino Biological, Inc) in PBS with 3% BSA was added into the wells. After incubated at 4°C for 2 hours, the cells were pelleted and washed again by PBS. Eleven-point dilution series of different PD-L1 antibodies in PBS with 3% BSA were added into different wells.
  • the cells were pelleted and washed with PBS, and the fluorescence labeled streptavidin was added into the wells at 4 C C for 1 hour. After removal of free fluorescence labeled streptavidin, the cells were washed twice with PBS, and mean fluorescence intensity (MFI) was measured by a flow cytometer. The result is shown in FIG. 21.
  • hCD80-Fc/anti-PD-Ll and hCD80/Anti-CLDN18.2 Multifunctional polypeptides are depicted in FIG. 1 A and FIG. 2.
  • the anti-PD-Ll and anti-CLDNl 8.2 refer to the PD- L,i and Claudm 18.2 antigen-binding domains of the disclosure.
  • the cassettes were generated by de novo gene synthesis and were cloned into a pcDNA3.4 expression vector (Thermo Fisher Scientific).
  • FIG. 1A and FIG. 2 were transfected into Expi293F cells using ExpiFectamine 293 transfection reagent (Thermo Fisher Scientific). ExpiFectamine 293 Transfection Enhancer 1 and Enhancer 2 were added to the well 20 hours after transfection. The cultures w r ere incubated at 37 °C in humidified incubator at 75% humidity supplied with 5% CO?.. The transfected culture w3 ⁇ 4s harvested 6 days post transfection.
  • All the multifunctional polypeptides were purified using GE Healthcare Protein A HP SpinTrap column by incubating the supernatants and the resin at room temperature for 4 minutes. The column was washed with sodium phosphate buffer, pH7.2 and eluted with 100 mM glycine-HCl, pH 3.0. The eluents were neutralized using 1.0 M Tris-HCl, pH 9 0. The purified proteins were dialyzed into PBS buffer at pH7.2 and sterile filtered through 0.2 pm membrane.
  • FIGS. 3- 10 The SDS-PAGE analyses of the multifunctional polypeptides are summarized FIGS. 3- 10.
  • FIG. 3 shows a SDS-PAGE analysis of se-hCD80 dimer-Fc/anti-PD-Ll (Format B) (SEQ ID. 116,114 and 115 and hCDBO-Fc/anti-PD-Ll (Format A) (SEQ ID. 113, 114 and 115) multifunctional polypeptides.
  • FIG. 4 shows a SDS-PAGE analysis of sc-hCD80 dimer-F c/anti -CLDN 18.2 Ab3 (Format B) (SEQ ID.
  • FIG. 5 shows a SDS-PAGE analysis of hCD80 ⁇ Fe/anti ⁇ CLDN 18.2 Ab3 (Format A) (SEQ ID. 128, 131 and 132) and hCDSO-Fc/FIuCLDN 18.2-v.24 (Format A) (SEQ ID. 128, 133 and 134) multifunctional polypeptides.
  • FIG. 6 show's a SDS- PAGE analysis of hCD80-Fc/anti-CLDN 18.2 Abl scFv-Fc (Format J) (SEQ ID.
  • FIG. 7 shows a SDS-PAGE analysis of anti-CLDN 18.2 Abl2-hCD80 Format F (SED ID. 122 and 123) and HuCLDN18.2-y.24-hCD80 (Format F) (SEQ ID. 124 and 125) multifunctional polypeptides.
  • FIG. 8 shows a SDS-PAGE analysis of anti-PD-Ll-hCD80 (Format F) (SEQ ID. 115 and 117) multifunctional polypeptides.
  • FIG. 9 shows a SDS-PAGE analysis of sc-hCD80 dimer- Fc/anti-PD-Ll (DVD) (Format D) (SEQ ID. 116, 129 and 130) multifunctional polypeptides.
  • FIG. 10 shows a SDS-PAGE analysis of hCD80-anti-PD-Ll (Format H) (SEQ ID. 118 and 119) and sc- hCD80 dimer-anti -PD-L1 (LC) (Format I) (SEQ ID. 120 and 121) multifunctional polypeptides. [0179] All the multifunctional polypeptides w3 ⁇ 4re purified to near 90% purity.
  • EXAMPLE 6 -Determination of hCD80-Fc/anti-PD-Ll Containing Multifunctional Polypeptide Binding Affinities to PD- L 1 and CTLA-4 by ELISA
  • ELISA plates were first coated with 1 ug/niL human CTLA-4 (recombinant His Tag from Sino Biological) at 4 °C overnight. The next day, the human CTLA-4 solution was removed and the plates were blocked with 1% BSA at room temperature for 1 hour. After removing the 1% BSA solution, the plates were washed with PBST (phosphate buffered saline with 005% of Tween-20) three times.
  • PBST phosphate buffered saline with 005% of Tween-20
  • ECso values of hCD80-Fc/anti-PD-Ll multifunctional polypeptide (Format A) (SEQ ID. 113, 114 and 115) binding to CTLA-4 or PD-L1 were determined by comparing to ECso of CD80- Fc on each plate.
  • the ECso (ug/mL) is the concentration or value of a protein that gives half maximal response in the binding assay.
  • FIG. 11 is an ELISA showing the binding affinities of hCD80-Fc/anti-PD-Ll multifunctional polypeptide (Format A) (SEQ ID. 113, 114 and 115) to recombinant protein CTLA-4 or PD-Ll as compared to hCD8Q-Fc fusion protein.
  • Form A hCD80-Fc/anti-PD-Ll multifunctional polypeptide
  • the ECso of the hCD8Q-Fc/anti-PD-Ll multifunctional polypeptide binding to the recombinant PD-Ll polypeptide was 0.09 ug/mL as compared to those of CD80-Fc polypeptide and anti-PD-Ll antibody binding to the same target were 4.6 ug/mL and 0.004 ug/mL, respectively.
  • ECso of hCD80-Fc/anti-PD-Ll multifunctional polypeptide (Format A) (SEQ ID. 113, 114 and 115) binding to the recombinant CTLA-4 polypeptide was 0.93 ug/mL as compared to that of CD80-Fc polypeptide binding to the same target was 0.035 ug/mL.
  • the FACS binding assays were performed as follows: Flp-in 293 CD28, Flp- in 293 CTLA-4 and Flp-in 293 PD-LI cells were stained with a serial diluted hCD80/anti-PD-Ll and hCDBO/anti-CLDNl 8.2 containing multifunctional polypeptides on ice for 1 hour, (i) with concentrations of 100 ug/rnl, 30 ug/ml, 10 ug/rnl, 3 ug/mi, 1 ug/ml, 0.3 ug/ml, 0.1 ug/ml, and 0.03 ug/ml for either Flp-in 293 cells overexpressing CD28 and PD-LI and (ii) with concentrations of 3 ug/ml, 1 ug/ml, 0.3 ug/ml, 0.1 ug/ml, 0.03 ug/ml, 0.01 ug/ml, 0.03ug/ml, and
  • the cells were washed with staining buffer (PBS + 2% fetal bovine serum) to remove free multifunctional polypeptides, and then stained with AlexF!uor 488-conjugated anti-human IgG antibody for 30 mm on ice. The cells were washed and analyzed by FACS.
  • staining buffer PBS + 2% fetal bovine serum
  • the FACS binding assays were performed as follows: cells were stained with a serial diluted hCD80/anti-CLDN18.2 containing multifunctional polypeptides on ice for 1 hour, with concentrations of 10 ug/ml, 3 ug/ml, 1 ug/mi, 0.3 ug/mi, 0.1 ug/mi, 0.03 ug/mi, 0.01 ug/mi, and 0 03ug/mi The ceils were washed with staining buffer (PBS + 2% fetal bovine serum) to remove free multifunctional polypeptides, and then stained with AlexFiuor 488-conjugated anti-human IgG antibody for 30 min on ice. The cells were washed and analyzed by FACS.
  • staining buffer PBS + 2% fetal bovine serum
  • FIGS. 12-16 The results of cell-surface binding affinity assays by FACS were presented in FIGS. 12-16 along with top MFI (Mean Fluorescence Intensity) values and ECso (ug/ml) values of each tested multifunctional polypeptide.
  • the ECso (ug/ml) is the concentration or value of a protein that gives half maximal response in the binding assay.
  • FIG. 12 showed the binding affinities of a hCD80/anti-PD-Ll (Format A) (SEQ ID. II 3, 114 and 115) containing multifunctional polypeptide to Flp-in 293 cell surface overexpressing CD28, CTLA-4 or PD-L1 by FACS.
  • hCD80-Fc/anti-PD-Ll (Format A) SEQ ID 113, 114 and 115
  • multifunctional polypeptide and hCB80-Fc showed similar binding affinity to Flp-in 293 CD28 cells.
  • ECso of hCD80 ⁇ Fc/anti-PD-Ll (Format A) SEQ ID.
  • FIG. 13 showed the binding affinities of a sc-hCD80 dimer-Fc/anti-FD-Ll (Format B) (S EQ ID. 116, 114 and 115) containing multifunctional polypeptide to Flp-in 293 ceil surface overexpressing CD28, CTLA-4 or PD-L1 by FACS.
  • ECso of sc-hCD80 dimer-Fc/anti-PD-Ll (Format B) SEQ ID. 116, 114 and 115
  • multifunctional polypeptide binding to Flp-in 293 CD28 cells was 53.2 ug/mL as compared to that of hCD80-Fc polypeptide binding to the same cells was 18 ug/mL.
  • ECso of sc-hCD80 dimer-Fc/anti-PD-Ll (Format B) (SEQ ID. 116, 114 and 115) multifunctional polypeptide binding to Flp-in 293 CTLA4 cells was 0.53 ug/rnL as compared to that of hCD80-Fc polypeptide binding to the same cells was 0.02 ug/mL.
  • ECso of sc-hCDSO dimer-Fc/anti-PD-Ll (Format B) SEQ ID.
  • FIG. 14 showed the binding affinities of a sc-hCD8Q dimer-Fc/anti-PD-Ll (DVD) (Format D) (SEQ ID. 116, 129 and 130), an anti-PD-Ll -hCD8Q (Format F) (SEQ ID. 115 and 117), a hCD80- anti-PD-Ll (Format H) (SEQ ID. 118 and 119), and a sc ⁇ hCD80 dimer-anti-PD-Ll (LC) (Format I) (SEQ ID. 120 and 121) containing multifunctional polypeptides to Flp-in 293 cell surface overexpressmg CD28, CTLA-4 or PD-L1 by FACS.
  • sc-hCD8Q dimer-Fc/anti-PD-Ll (DVD) (Format D) (SEQ ID. 116, 129 and 130), an anti-PD-Ll -hCD8Q (Format F) (SEQ ID.
  • FIG. 14 Panel A showed the ECso of sc-hCD80 dimer-Fc/anti-PD-Ll (DVD) (Format D) (SEQ ID. 116, 129 and 130), an anti-PD-Ll-hCD80 (Format F) (SEQ ID. 115 and 117), a hCD80- anti-PD-Ll (Format H) (SEQ ID. 118 and 119), and a sc-hCD8Q dimer-anti-PD-Ll (LC) (Format I) (SEQ ID.
  • multifunctional polypeptide binding to Flp-in 293 CD28 cells were 528.7 ug/ ' mL, 4572 ug/mL, 8.3 ug/mL and 19.3 ug/mL respectively, ml, as compared to that of hCD80-Fc polypeptide binding to the same ceils was 11.0 ug/mL.
  • FIG. 14 Panel B showed the ECso of sc-hCD80 dimer-Fc/anti-PD-Ll (DVT)) (Format D) (SEQ ID. ! 16, 129 and 130), an anti ⁇ PD-Ll-liCD80 (Format F) (SEQ ID 115 and 117), a hCD80 ⁇ anti-PD-Ll (Format H) (SEQ ID. 118 and !
  • FIG. 14 Panel C showed the ECso of sc-hCD80 dimer-Fc/anti-PD-Ll (DVD) (Format D) (SEQ ID. 116, 129 and 130), an anti-PD-Ll -hCD80 (Format F) (SEQ ID. 115 and 117) a hCD80- anti-PD-Ll (Format H) (SEQ ID. 118 and 119), and a sc-h €D80 dimer-anti-PD-Ll (LC) (Format I) (SEQ ID.
  • multifunctional polypeptide binding to Flp-in 293 PD-L1 cells were 1.3 ug/mL, 2.3 ug/mL, 1.6 ug/mL and 1.4 ug/mL respectively, mL as compared to that of anti-PD-Ll binding to the same cells was 0.6 ug/mL.
  • FIG. 15 showed the binding affinities of anti-CLDN18.2-hCD80 (Format F) (SED ID. 122 and 123) (SEQ ID. 124 and 125), and hCD80-Fc/anii-CLDN18.2 scFv-Fc (Format J) (SEQ ID. 128 and 126) (SEQ ID. 128 and 127) multifunctional polypeptides to HEK 293 cell surface overexpressing CLDN18.2 or KATO Ill cell surface endogenously expressing CLDN18.2 by FACS.
  • the ECso of CDSO/anti-CEDNl 8.2 containing multifunctional polypeptides showed four to eight fold activity loss as compared to that of anti-CLDN 18.2 antibodies.
  • FIG. 16 showed the binding affinities of anti-CLDN18.2-hCD80 (Format F) (SED ID. 122 and 123) (SEQ ID. 124 and 125), and hCD80-Fc/anti-CLDN18.2 scFv-Fc (Format J) (SEQ ID. 128 and 126) (SEQ ID. 128 and 127) multifunctional polypeptide to Flp-in 293 cell surface overexpressing CD28, CTLA-4 or PD-Ll by FACS.
  • hCD80-Fc/anti-CLDN18.2 Ab ScFv-Fc (Format J) (SEQ ID. 128 and 126) (SEQ ID.
  • multifunctional polypeptides and hCD80-Fc showed similar binding affinity to Flp-in 293 CD28 cells.
  • ECso of hCD80-Fc/anti-CLDN18.2 Abl ScFv-Fc (Format I) (SEQ ID. 128 and 126) and hCD80-Fc/anti-CLDN18.2 Abl2 ScFv-Fc (Format J) (SEQ ID. 128 and 127) multifunctional polypeptides binding to Flp-in 293 CTLA4 cells were 0.40 ug/mL and 0.64 ug/mL, respectively, as compared to that of hCD80-Fc polypeptide binding to the same cells w3 ⁇ 4s 0.02 ug/mL.
  • Multifunctional polypeptides showed loss of binding activities to Flp-in 293 CD28 cells ECso of Anti-CLDN 18.2 Abl2-hCD80 (Format F) (SED ID. 122 and 123) and HuCLDN18.2-v.24.hCD80 (Format F) (SED ID. 124 and 125) multifunctional polypeptides binding to Flp-in 293 CTLA4 cells were 2.67 ug/mL and 6.56 ug/mL, respectively, as compared to that of hCD80-Fc polypeptide binding to the same cells was 0.02 ug/mL.
  • Anti-CLDN 18.2 Ab-hCD80 (Format F) (SED ID. 122 and 123) (SEQ ID. 124 and 125) multifunctional polypeptides and hCD80 ⁇ Fc showed similar binding affinity to Flp-in 293 PD ⁇ LI cells.
  • Anchorage qfhCDSO moiety on PD-Ll or CLDN18.2 expression cells can provide co-stimulaiory activity in T cell activation
  • IL-2 promoter activity requires two signals, signal 1 from TCR/CD3 which is provided from anti-CD 3 antibody in the assay, signal 2 from CD28 co stimulation which is provided by various testing molecules.
  • Anti-CD28 agonist antibody CD28.2 BioLegend was used as positive control, while culture medium was used as negative control.
  • Testing molecules were hCD8G-Fc, anti-PD-Ll (Tercentriq, Genentech), hCD80-Fc/anti-PD-Ll (Format A) (SEQ ID. 113, 114 and 115).
  • the plate was incubated at 37°C, 5%CC incubator for 6 h.
  • Bio-G!oTM Luciferase Assay Substrate Promega
  • Seventy-five microliter of Bio-GioTM Luciferase Assay Substrate were added to each well of the assay plates. The plate was mixed briefly, and the count of firefly luminescence was measured using a !uminometer luminescence plate reader.
  • FIG. 17 showed that the binding of the multifunctional polypeptide comprising anti-PD-Ll and hCD80 moiety to PD-Ll on Flp-in 293 PD-L1 cells provided co-stimulatory signal required for IL-2 promoter activation. In absence of Flp-in 293 PD-Ll cells, the same multifunctional polypeptide did not activate the IL-2 promoter.
  • FIG. 19 showed that the binding of the multifunctional polypeptide comprising anti- CLDN18.2 and hCD80 moiety to CLDN18.2 on FIEK293-CLDN18.2 cells provided co-stimulatory signal required for IL-2 promoter activation.
  • the same multifunctional polypeptide failed to activate the IL-2 promoter.
  • the two anti-CLDN18.2 antibodies lacking hCDBO moiety did not show r activity in this assay.
  • the multifunctional polypeptide comprising anti-PD-Ll and hCD80 moiety w3 ⁇ 4s inactive in this assay due to the absence of the matching ligand.
  • An artificial antigen- presenting cell line PD-L1 aAPC/CHO-Kl, expressed PD-L1 and cognate antigen recognized by TCR/CD3 on effector cells.
  • NFAT Jurkat effector cell
  • the Jurkat effector cells receives both TCR/CD3-Antigen stimulation signal as well as PD-L1 blocking signal simultaneously from aAPC/CHO-Kl ceils.
  • the NFAT promoter activity was suppressed by signaling from PD-l/PD-Ll interaction.
  • anti-PD-Ll or anti-PD- 1 blocking antibody the NFAT promoter activity was unchecked.
  • hCD80-Fc/anti-PD-Ll (Format A) (SEQ ID. 113, 114 and 115) multifunctional polypeptides, it not only blocked PD-l/PD-Ll signaling, but also provided hCD80/CD28 co-stimulatory activity, thus boost NFAT promoter activity received from TCR/CD3-Antigen interaction.
  • 50,000 PD-L1 aAPC/CHO-Kl Cells in 100 uL/well were seeded in a flat-bottom opaque 96-well plate, incubated at 37°C, 5% CO2 incubator overnight. The culture medium was completely dumped out right before addition of other components on the plate.
  • Jurkat PD-1 effector cells were suspended in RPML-1640 with 10% Fetal bovine serum at 2 miliion/mL. Fifty microliter per well of Jurkat PD-1 effector cells were transfered to the plate growing PD-L1 aAPC/CHO-Kl cells.
  • the testing molecules were prepared in culture medium at 30 ug/mL with 1:3 serial dilutions and 25 uL/weil of samples were transferred on to the plate in duplicates. The plate was incubated at 37°C, 5%CO? incubator for 6 h. To detect NFAT promoter activity activated by testing molecules, Bio-GloTM Luciferase Assay Substrate (Promega) were thawed and equilibrate to room temperature. Seventy-five microliter of Bio-GloTM Luciferase Assay Substrate were added to each well of the assay plate. The plate was mixed briefly, and the count of firefly luminescence was measured using a lummometer luminescence plate reader.
  • the multifunctional polypeptide (hCD8G-Fc/anti-PD-Ll) (Format A) (SEQ ID. 113, 114 and 115) was more active than anti-PD-Ll antibody.
  • the activity of anti-PD-Ll antibody was due to the removal of the signal blockade from PD-1/PD-L1 interaction by interrupting PD-1/PD-L1 interaction.
  • Additional activity of the multifunctional polypeptide (hCD80-Fc/anti-PD-Ll (Format A) (SEQ ID. 113, 114 and 115)) could be a result of the costimulatory signal from hCD80 and CD28 interaction.
  • the multifunctional polypeptide (hCD80-Fc/anti-PD-Ll (Format A) (SEQ ID. 113, 114 and 115)) had similar activity as anti-PD-Ll antibody.
  • Such reduction in activity was most likely due to the loss of costimulatory activity of hCD80 and CD28 interaction, which was a result of the fact that the excess amount of anti-PD-Ll antibody competed the multifunctional polypeptide off the PD-L1 ligand and the costimulatory effects of multifunctional polypeptide (hCDSO-Fc/anti-PD-Ll (Format A) (SEQ ID. 113, 114 and 115)) was lost.
  • no acti vities were observed
  • ELISA plates were first coated with 5 ug/mL Streptavidin-plus from Agilent (Cat. No. SA26) at 4°C overnight. The next day, Streptavidin-plus was removed and washed with PBST three times. The plates were then blocked with 2% BSA at 37°C for 1 hour. After removing the 2% BSA solution, the plates were washed with PBST three times. Biotinylated Human CD28, Fc Tag from Aero (Cat. No. CD8-H82F2) or Biotinylated mouse CD28,
  • Fc Tag from SB (Cat. No. 50103-M08H) were added to each well and incubate at 37°C for 1 hour.
  • the samples of the purified mCD80-Fc/anti-PD ⁇ L1 multifunctional polypeptides were added at 100 uL/well in duplicate with serial dilutions and incubated at 37°C for 2 hours.
  • a secondary antibody 100 uL/well of Goat anti mouse IgG-Fc secondary Antibody (HRP) from SB (Cat. No. SSA006) was added and incubated at 37°C for I hour.
  • the plates were washed three times with PBST, and a 100 uL/well of TMB substrate was added.
  • ELISA plates were first coated with 1 ug/mL human CTLA4 (from SB, Cat. No. 11159-H08H) or mouse CTLA-4 (from SB, Cat. No. 50503- MQ8H) at 4°C overnight. The next day, the plates were washed with PBST three times. The plates were then blocked with 2% BSA at 37°C for 1 hour. After removing the 2% BSA solution, the samples of the purified mCD80-Fc/anti-PD-Ll multifunctional polypeptides were added at 100 uL/well in duplicate with serial dilutions and incubated at 37°C for 1 hours.
  • a secondary antibody 100 uL/well of Goat anti mouse IgG-Fc secondary' Antibody (HRP) from SB (Cat No. SSA006) was added and incubated at 37°C for I hour. The plates were washed three times with PBST, and a 100 uL/well of TMB substrate was added. The plates were incubated until the color was developed. The stop-solution was added to stop the reaction. The plates were read at OD450. The results of binding affinity to CTLA4 by ELISA are presented in FIG. 24 and FIG. 25. hPD-Ll b inding assay by ELISA
  • ELISA plates were first coated with 1 ug/mL human PD-L1 (from SB, Cat. No. 10084-H08H) at 4°C overnight. The next day, the plates were washed with PBST three times. The plates were then blocked with 2% BSA at 37°C for 1 hour. After removing the 2% BSA solution, the samples of the purified mCD80-Fc/anti-PD-Ll multifunctional polypeptides were added at 100 uL/well in duplicate with serial dilutions and incubated at 37°C for 1 hours.
  • the binding affinity (ECso) of mCD80-F c/anti -PD-L1 multifunctional polypeptides to Flp- m 293 cells overexpressing hCD28, mCD2.8, hCTLA-4, hPD-Ll and mPD-Ll (Flp-in 293-hCD28, Flp-in 293-mCD28, Flp-in 293-hCTLA4, Flp-in 293-hPD-Ll and Flp-in 293-mPD-Ll cells) were determined by FACS.
  • the FACS binding assays were performed as follows: Flp-in 293 cells were washed three times, then the cells were fixed by 4% PFA for 20min and pelleted at lOOOrpm for 5 minutes at room temperature. The cells were resuspended by PBS and washed three times. Then, the cells were resuspended in 3% BSA and incubated at 4°C for 1 hours. Series diluted mCD80 ⁇ Fc/anti- PD-L1 multifunctional polypeptides were added in to the 96-well plates. The cells were then seeded into the 96-well plates. The plates were incubated at 4°C for 12 hours. The plates were washed three time by 3% BSA in PBS. The secondary antibody was added into the plates and incubated at 4°C for 1 hour. The cells were washed and analyzed by FACS. The results of binding affinity by FACS are presented in FIGS. 27-31.
  • Efficacy evaluation of mCD80-Fc/anti-PD-Ll multifunctional polypeptides in tumor growth inhibition in a syngeneic mouse model was carried as follows lxl 0 6 MC38-human-PD-Ll cells were suspended in 100pL DMEM with 50% matrix gel. The cells were then inoculated subcutaneously into the right flank of C57BL/6 mice. Mice were randomly assigned to different groups when the mean tumor volume reached desired average size (72mm 3 ). Mice were LP. injected with different proteins dosed at 0.15 mg per mouse on day 0, day 3 and day 6.
  • the data was shown in FIG. 32.

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Abstract

The present disclosure provides modulators of T-cell activity, methods of making, and methods of use thereof. In some embodiments, the modulators provided herein comprise novel PD-L1 antibodies. In other embodiments, the modulators provided herein comprise multifunctional polypeptides comprising an extracellular binding domain of a CD80 polypeptide, a disease-associated antigen-binding domain, and an Fc domain. present disclosure also provides pharmaceutical compositions comprising such modulators and methods for use in the treatment of cancer, infectious diseases, and inflammatory diseases.

Description

MODULATORS OF T-CELL ACTIVITY
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62/940,805, filed on November 26, 2019, the content of which is hereby incorporated by reference in its entirety for all purposes.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
[0002] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: CSPL_012_01WO_SeqList_ST25.txt, date recorded: November 3, 2020, file size 177 kilobytes).
BACKGROUND
[0003] T-cell lymphocytes play a critical role in cell-mediated immunity' by providing for an adaptive response to certain stimuli. T-cell activation depends on activation of at least two signaling pathways, one that is antigen specific and the other that is antigen nonspecific. Antigen-specific activation of T-cells is mediated by peptide/major histocompatibility complexes on antigen- presenting cells interacting with specific T-cell antigen receptors. Binding of B7-related molecules such as CD80 (B7-1) on antigen-presenting cells, to CD28 and/or CTLA-4 on T-cells, provides important antigen-nonspecific costimulatory signals essential for optimum immune responses. Arming diseased cells with CD80 ligands can provide antigen-nonspecific costimulatory signals, which can potentiate T-cell activation, similar to antigen presenting cells, whereby turning diseased cells into antigen presentmg-!ike cells. Compositions and methods to achieve such T-cell potentiation, m addition to other functions, are needed in the art, and are described herein.
SUMMARY
[0004] The present disclosure provides modulators of T-cell activity, methods of making, and methods of use thereof. In some embodiments, the modulators provided herein comprise novel FD- LI antibodies capable of blocking PD-l/PD-Ll interactions, allowing for T-cell proliferation and function. In other embodiments, the modulators provided herein comprise multifunctional polypeptides comprising an extracellular binding domain of a CD80 polypeptide, a disease-associated antigen-binding domain, and an Fc domain, capable of arming diseased cell with a CD80 ligand in addition to other functions. The present disclosure also provides pharmaceutical compositions comprising such modulators and methods for use in the treatment of cancer, infectious diseases, and inflammatory diseases.
[0005] In one aspect, provided herein are multifunctional polypeptides comprising: a CD80 extracellular domain; a disease-associated antigen-binding domain; and an Fc domain. In some embodiments, the multifunctional polypeptides comprise any one of the formats presented in FIG. 1 A. In some embodiments, the multifunctional polypeptides comprise a cancer-associated antigen binding domain, an infectious disease-associated antigen binding domain, or an inflammatory disease-associated antigen binding domain. In some embodiments, the disease-associated antigenbinding domain is a PD-L1 binding domain. In other embodiments, the disease-associated antigen binding domain is a Claudin 18.2 binding domain.
[0006] In related aspects, provided herein are pharmaceutical compositions comprising the multifunctional polypeptides of the disclosure. Also provided herein are polynucleotides encoding the multifunctional polypeptides of the disclosure, vectors comprising the polynucleotides; and cells expressing the vectors.
[0007] In another aspect provided herein are PD-Ll antibodies comprising the ammo acid sequence of a CDR-L1 of Table 1, a CDR-L2 of Table 2, a CDR-L3 of Table 3, a CDR-H1 of Table 4, a CDR- H2 of Table 5, and/or a CDR-H3 of Table 6. In some embodiments, the antibodies are an antigen binding fragment. In some embodiments, the antibodies are full length antibodies.
[0008] In related aspects, provided herein are pharmaceutical compositions comprising the PD-Ll antibodies of the disclosure. Also provided herein are polynucleotides encoding for any of the PD-Ll antibodies provided herein, vectors comprising such polynucleotides, and cells expressing such vectors.
[0009] In another aspect, provided herein are kits and articles of manufacture comprising any of the multifunctional polypeptides of PD-Ll antibodies of the disclosure.
[0010] In another aspect, provided herein are methods of treating a disease or disorder, comprising administering to subject in need thereof a therapeutically effective amount of any one of the PD-Ll antibodies, multifunctional polypeptides, or pharmaceutical compositions provided herein. The disease or disorder may be selected from cancer, infectious disease, or inflammatory disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[001 if FIG. I A shows the various formats and nomenclatures of multifunctional polypeptides of the disclosure. (KIH=Fc with a knob-in-hole mutation).
[0012] FIG. IB is a table showing exemplar constructs with anti-PD-Ll as the disease-associated antigen-binding domain for the formats presented in FIG. 1A. In some embodiments, the disease- associated antigen-binding domain is an anti-ClaudinlB.2 antigen-binding domain, e.g. anti-CLDN 18.2 Ab or HuCLDN18.2-v.24.
[0013] FIG. 2 shows CD80-Fc and the disease-associated antigen-binding domain expression constructs of the disclosure. (LS=leader sequence; CD80=extracellular CD80; Fc, Fe-knob, Fc-hole = Fc variations; VL, VH, CHI, CL, scFv=disease-associated antigen-binding domain components). [0014] FIG. 3 showrs a SDS-PAGE analysis of sc-hCD80 dimer-Fc/anti-PD-Ll (Format B) and hCD80~Fc/anti~PD-Li (Format A) multifunctional polypeptides.
[0015] FIG, 4 show's a SDS-PAGE analysis of sc-hCD80 dimer-Fc/anti-CLDN 18.2 Ah3 and sc- I1CD8O dimer-Fc/HuCLDN 18.2-v.24 (Format B) multifunctional polypeptides.
[0016] FIG, 5 show's a SDS-PAGE analysis of hCB80-Fc/anti-CEDN 18.2 Ab3 and hCD80- Fc/HuCLDN 18.2-v.24 (Format A) multifunctional polypeptides.
[0017] FIG, 6 show's a SDS-PAGE analysis of hCB80-Ec/anti-CEDN 18.2 Ab! scFv-Fc and hCD80-Fc/anti-CLDN 18.2 Abl2 scFv-Fc (Format J) multifunctional polypeptides.
[0018] FIG. 7 show's a SBS-PAGE analysis of anti-CLBN 18.2 Ab 12-hCD80 and anti-CLDN 18.2 Abl2-hCB80 (Format F) multifunctional polypeptides.
[0019] FIG. 8 shows a SDS-PAGE analysis of anti-PD-Ll -hCD80 (Format F) multifunctional polypeptides.
[0020] FIG. 9 show's a SDS-PAGE analysis of sc-hCD80 dimer-Fc/anti-PD-Ll (DVD) (Format D) multifunctional polypeptides.
[0021] FIG. 10 shows a SDS-PAGE analysis of hCD80-anti-PD-Ll (Format H) and sc-hCD80 dimer-anti-PD-Ll (LC) (Format !) multifunctional polypeptides. [0022] FIG. 11 is an ELISA showing the binding affinities of hCD80-Fc/anti-PD-Ll multifunctional polypeptide to recombinant protein hCTLA~4 or hPD-Ll as compared to hCD80-Fc fusion protein.
[0023] FIG. 12 show¾ the binding affinities of a hCD 80/anti -PD-Ll (Format A) multifunctional polypeptide to Flp-in 293 cell surface overexpressing hCD28, hCTLA-4 or h PD-L l by FACS.
[0024] FIG. 13 shows the binding affinities of a sc-hCDBO dimer-Fc/anti-PD-Ll (Format B) multifunctional polypeptide to Flp-in 293 cell surface over expressing hCD28, hCTLA-4 or hPD-Ll by FACS
[0025] FIG, 14 shows the binding affinities of anti-PD-Ll-hCDBO (Format F), sc~hCB80 dimer- Fc/anti-PD-Ll (DVD) (Format D), hCD80-anti -PD-Ll (Format H), and sc~hCD80 dimer-anti-PD-Ll (LC) (Format I) multifunctional polypeptides to Flp-in 293 cell surface overexpressing hCD28, hCTLA-4 or hPD-Ll by FACS.
[0026] FIG, 15 shows the binding affinities of hCD80-Fc/anti-CLDN 18.2 Abl ScFv-Fc (Format J), hCD80~Fc/anti~CLDN 18.2 Abl2 ScFv-Fc (Format J), Anti-CLDN 18.2 Abl2-hCD80 (Format F). and HuCLDN18.2-v.24-hCD80 (Format F) multifunctional polypeptides to HEK 293 cell surface overexpressing CLDN18.2 or KATO III cell surface endogenously expressing CLDN18.2 by FACS. [0027] FIG, 16 shows the binding affinities of hCD80-Fc/anti-CLDN 18.2 Abl ScFv-Fc (Format J), hCD80~Fc/anti~CLDN 18.2 Abl2 ScFv-Fc (Format J), Anti-CLDN 18.2 Abl2-hCD80 (Format F). and HuCLDN18.2-v.24-hCD80 (Format F) multifunctional polypeptide to Flp-in 293 cell surface overexpressing hCD28, hCTLA-4 or hPD-Ll by FACS.
[0028] FIG. 17 show¾ that simultaneous binding of a hCD80-F c/anti -PD-Ll (Format A) multifunctional polypeptide comprising anti -PD-Ll and hCDBO moiety to antigen expression ceils (e.g. Flp-in 293 PD-Ll cells) provides co-stimulatory signal required for IL-2 promoter activation. [0029] FIG. 18 shows that a hCD80-Fe/anti-PD-Ll (Format A) multifunctional polypeptide comprising an anti-PD-Ll and a hCDBO moiety binding to an antigen presenting cell and T-cell not only breaks the PD-1/PD-L1 blockade for NFAT signaling but also provides additional NFAT activation. Blocking PD- 1 /PD-Ll and providing co-stimulation induces stronger NFAT activity. [0030] FIG. 19 shows that multifunctional polypeptides comprising anti-CLDN18.2 and hCD80 binding to antigen expression cells provides co-stimulatory signal required for IL-2 promoter activation. [0031] FIG. 20 shows the binding affinities of hPD-Ll antibodies to Flp-in 293 ceil surface overexpressing hPD-Ll by FACS.
[0032] FIG. 21 show's PD-L1 antibodies competitively inhibit liPD-1 binding to Fip-in 293 cell surface overexpressing hPD-Ll by FACS.
[0033] FIG. 22 is an ELISA showing the binding affinities of mCDSO-Fc/anti-PD-Ll multifunctional polypeptides to recombinant protein hCD28 as compared to mCD80-Fc fusion protein.
[0034] FIG. 23 is an ELISA showing the binding affinities of mCD80-Fe/anti-PD-Ll multifunctional polypeptides to recombinant protein mCD28 as compared to mCD80-Fc fusion protein.
[0035] FIG, 24 is an ELISA showing the binding affinities of mCD80-Fe/anti~PD-Ll multifunctional polypeptides to recombinant protein hCTLA4 as compared to mCD80-Fc fusion protein.
[0036] FIG. 25 is an ELISA showing the binding affinities of mCD80-Fc/anti-PD-Ll multifunctional polypeptides to recombinant protein mCTLA4 as compared to mCD80-Fc fusion protein.
[0037] FIG. 26 is an ELISA showing the binding affinities of mCD80~Fc/anti-PD-Ll multifunctional polypeptides to recombinant protein hPD-Ll as compared to mCD80-Fc fusion protein.
[0038] FIG. 27 show's the binding affinities of mCD80-Fc/anti-PD-Ll multifunctional polypeptides to Flp- 293 cell surface overexpressmg hCD28 by FACS.
[0039] FIG. 28 show's the binding affinities of mCD80-Fc/anti-PD-Ll multifunctional polypeptides to Flp-m 293 cell surface overexpressmg mCD28 by FACS.
[0040] FIG. 29 show's the binding affinities of mCD80-Fc/anti-PD-Ll multifunctional polypeptides to Flp-m 293 cell surface overexpressing hCTLA4 by FACS.
[0041] FIG. 30 show's the binding affinities of mCD8G-Fc/anti-PD-Ll multifunctional polypeptides to Flp-m 293 cell surface overexpressing hPD-Ll by FACS.
[0042] FIG. 31 show's the binding affinities of mCD8G-Fc/anti-PD-Ll multifunctional polypeptides to Flp-m 293 cell surface overexpressing mPD-Ll by FACS. [0043] FIG. 32 shows the antitumor activity of mCDBO-Fc/anti-PD-Ll multifunctional polypeptides in MC-38-human-PD-Ll model.
DETAILED DESCRIPTION
[00441 All publications, patents and patent applications, including any drawings and appendices therein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application, drawing, or appendix was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
[0045] While various embodiments of the present disclosure are described herein, it will be obvious to those skilled m the art that such embodiments are provided by way of example only. Numerous modifications and changes to, and variations and substitutions of, the embodiments described herein will be apparent to those skilled in the art without departing from the disclosure. It is understood that various alternatives to the embodiments described herein may be employed in practicing the disclosure. It is also understood that every embodiment of the disclosure may optionally be combined with any one or more of the other embodiments described herein which are consistent with that embodiment
[0046] Where elements are presented in list format (e.g., in a Markush group), it is understood that each possible subgroup of the elements is also disclosed, and any one or more elements can be removed from the list or group.
[0047] It is also understood that, unless clearly indicated to the contrary, in any method described or claimed herein that includes more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited, but the disclosure encompasses embodiments in which the order is so limited.
[0048] It is further understood that, in general, where an embodiment in the description or the claims is referred to as comprising one or more features, the disclosure also encompasses embodiments that consist of, or consist essentially of, such feature(s).
[0049] It is also understood that any embodiment of the disclosure, e.g., any embodiment found within the prior art, can be explicitly excluded from the claims, regardless of whether or not the specific exclusion is recited in the specification. [0050] Headings are included herein for reference and to aid in locating certain sections. Headings are not intended to limit the scope of the embodiments and concepts described in the sections under those headings, and those embodiments and concepts may have applicability in other sections throughout the entire disclosure.
L Definitions
[0051 [ While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter. Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill m the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
[0052] The terms used throughout this specification are defined as fallows unless otherwise limited in specific instances. As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms, acronyms, and abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Unless indicated otherwise, abbreviations and symbols for chemical and biochemical names is per TUPAC-IUB nomenclature. Unless indicated otherwise, ail numerical ranges are inclusive of the values defining the range as well as all integer values in-between.
[0053] The terms “effective amount” or “therapeutically effective amount” refer to a quantity and/or concentration of a therapeutic of the disclosure, that when administered ex vivo (by contact with a ceil from a patient) or in vivo (by administration into a patient) either alone (i.e., as a monotherapy) or in combination with additional therapeutic agents, yields a statistically significant decrease in disease progression as, for example, by ameliorating or eliminating symptoms and/or the cause of the disease. An effective amount may be an amount that relieves, lessens, or alleviates at least one symptom or biological response or effect associated with a disease or disorder, prevents progression of the disease or disorder, or improves physical functioning of the patient in the ease of ceil therapy, the effective amount is an effective dose or number of cells administered to a patient by adoptive cell therapy. In some embodiments the patient is a mammal such as a non-human primate or human patient.
[0054] The terms “individual,” “subject,” “host,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired. Mammals include, e.g., humans, non-human primates, rodents (e.g., rats; mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, and the like), etc.
[0055] The terms “modulating” or “modulate” as used herein in the context of an immune response, such as a mammalian immune response, refer to any alteration, such as an increase or a decrease, of existing or potential immune responses that occurs as a result of administration of a polypeptide or antibody of the present disclosure. Thus, it refers to an alteration, such as an increase or decrease, of an immune response as compared to the immune response that occurs or is present in the absence of the administration. Such modulation includes any induction, activation, suppression or alteration in degree or extent of immunological activity of an immune cell. Immune cells include B cells, T-cells, NK (natural killer) cells, NK T-cells, professional antigen-presenting cells (APCs), and non- professional antigen-presenting cells, and inflammatory' cells (neutrophils, macrophages, monocytes, eosinophils, and basophils). Modulation includes any change imparted on an existing immune response, a developing immune response, a potential immune response, or the capacity to induce, regulate, influence, or respond to an immune response. Modulation includes any alteration m the expression and/or function of genes, proteins and/or other molecules in immune cells as part of an immune response. Modulation of an immune response or modulation of immunological activity includes, for example, the following: elimination, deletion, or sequestration of immune cells; induction or generation of immune cells that can modulate the functional capacity of other cells such as autoreactive lymphocytes, antigen presenting cells, or inflammatory cells; induction of an unresponsive state in immune cells (i.e., anergy); enhancing or suppressing the activity or function of immune cells, including but not limited to altering the pattern of proteins expressed by these cells. Examples include altered production and/or secretion of certain classes of molecules such as cytokines, chemokines, growth factors, transcription factors, kinases, costimulatory molecules, or other cell surface receptors or any combination of these modulatory events. [0056] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. Unless specifically limited, the terms encompass nucleic acids containing known analogues of natural nucleotides and that have similar binding properties to it and are metabolized in a manner similar to naturally- occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary nucleotide sequences as well as the sequence explicitly indicated (a “reference sequence”). Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues. The term nucleic acid or polynucleotide encompasses cDNA or mRNA encoded by a gene.
[0057] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein, and refer to a polymeric form of ammo acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The terms include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, sialylations, phosphorylations and the like. The terms also include molecules in which one or more ammo acid analogs or non-eanonicai or unnatural amino acids are included as can be synthesized, or expressed recombinantly using known protein engineering techniques. In addition, proteins can be derivatized.
[0058] A polynucleotide or polypeptide has a certain percent sequence identity to another polynucleotide or poly peptide, meaning that, when aligned, that percentage of bases or ammo acids are the same, and in the same relative position, when comparing the two sequences. Sequence identity can be determined m a number of different ways. To determine sequence identity, sequences can be aligned using various convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), available over the world wade wreb at sites including ncbi.nlm.mli.gov/BLAST, ebi.ac.uk/Tools/msa/tcoffee/, ebi.ac.uk/Tools/msa/muscle/, mafft. cbrc.jp/alignment/software/. See, e.g., Altschul et al. (1990), I Mol. Bioi. 215:403-10. [0059] “T-cell” includes all types of immune cells expressing CD3, including T-helper cells (CD4+ ceils), cytotoxic T-cells (CD8+ cells), T-regulatory cells (Treg), and NK-T-cells.
[0060] The terms “treatment”, “treating” and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
[0061] “Treatment” as used herein covers any treatment of a disease or symptom in a mammal, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to acquiring the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease or symptom, i e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during or after the onset of disease or injury. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues. The subject therapy will desirably be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease. “Treating,” “treatment,” or “therapy” also means a decrease the severity of symptoms in an acute or chronic disease or disorder or a decrease in the relapse rate as for example m the ease of a relapsing or remitting autoimmune disease course or a decrease in inflammation in the case of an inflammatory aspect of an autoimmune disease. As used herein in the context of cancer, the terms “treatment” or, “inhibit,” “inhibiting” or “inhibition” of cancer refers to at least one of: a statistically significant decrease in the rate of tumor growth, a cessation of tumor growth, or a reduction in the size, mass, metabolic activity, or volume of the tumor, as measured by standard criteria such as, but not limited to, the Response Evaluation Criteria for Solid Tumors (RECIST), or a statistically significant increase in progression free survival (PFS) or overall survival (OS). “Preventing,” “prophylaxis,” or “prevention” of a disease or disorder as used in the context of this disclosure refers to the administration of an immunomodulatory polypeptide or engineered cells of the disclosure, either alone or in combination with another compound, to prevent the occurrence or onset of a disease or disorder or some or all of the symptoms of a disease or disorder or to lessen the likelihood of the onset of a disease or disorder. [0062] The term “variant” (also “mutant”, ‘mutated” or “modified”) as used in reference to a variant CD80 means a CD80, such as a mammalian (e.g., human or murine) CD80 created by human intervention, or are naturally occurring. The variant CD80 is a polypeptide having an altered amino acid sequence, relative to an unmodified or wild-type CD80. The variant CD80 is a polypeptide which differs from a wild-type CD80 isoform sequence by one or more amino acid substitutions, deletions, additions, or combinations thereof The variant CD80 may contain at least one affinity modified domain, whereby one or more of the amino acid differences occurs in an IgSF domain (e.g. IgC domain and/or IgV domain). A variant CD 80 may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid differences, such as ammo acid substitutions. A variant CD80 polypeptide generally exhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a wild-type or unmodified CD80 extracellular domain (SEQ ID NO: 34). Non- naturally occurring ammo acids as well as naturally occurring ammo acids are included within the scope of substitutions or additions. A variant CD8Q is not limited to any particular method of making and includes, for example, de novo chemical synthesis, de novo recombinant DNA techniques, or combinations thereof. A variant CDS0 of the disclosure specifically binds to at least one or more of: CD28, PD-L1, or CTLA-4 of a mammalian species . In some embodiments, the altered amino acid sequence results in an altered (x.e., increased or decreased) binding affinity or avidity to CD28, PD- Ll, or CTLA-4 compared to the wild-type CD80 protein. An increase or decrease in binding affinity or avidity can be determined using well-known binding assays such as flow cytometry. Larsen et al, American Journal of Transplantation, Vol 5: 443-453 (2005). See also, Linsley et al, Immunity, 1 : 7930801 (1994) or ELISA (please add reference). An increase in variant CD80 binding affinity or avidity to CD28, PD-L1, or CTLA-4 is to a value at least 5% greater than that of the wild-type CD80 control value and in some embodiments, at least 10%, 15%, 20%, 30%, 40%, 50%, 100% greater than that of the wild-type CD80 control value. A decrease in CD80 binding affinity' or avidity to CD28, PD-L1, or CTLA-4 is to a value no greater than 95% of the of the wild-type CD80 control value, and m some embodiments no greater than 80%, 70% 60%, 50%, 40%, 30%, 20%, 10%, 5%, or no detectable binding affinity or avidity of the wild-type CD80 control value. A variant CD80 is altered m primary amino acid sequence by substitution, addition, or deletion of amino acid residues. The term “variant” in the context of variant CD80 is not be construed as imposing any condition for any particular starting composition or method by which the variant CD80 is created. A variant CD80 can, for example, be generated starting from wild type mammalian CD80 sequence information, then modeled in silico for binding to CD28, PD-Ll, or CTLA-4, and recombinantly or chemically synthesized to yield a variant CD80 of the present disclosure. In alternative embodiment, a variant CD80 can be created by site-directed mutagenesis of a wild-type CD80. Thus, variant CD80 denotes a composition and not necessarily a product produced by any given process. A variety of techniques including recombinant methods, chemical synthesis, or combinations thereof, may be employed. [0063] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
[0064] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit m the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. [0065] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
[0066] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a multimeric polypeptide” includes a plurality of such multimeric polypeptides and reference to “the modulatory domain” includes reference to one or more modulatory domains and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
[0067] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described m the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
[0068] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided may be different from the actual publication dates which may need to be indepen den tl y confirmed.
H. PD-Ll Antibodies
[0069] Provided herein are PD- Li antibodies. Such antibodies may be used in variety of therapeutic and diagnostic methods. The PD-L1 antibody may be used alone, or in combination with other agents in treating a disease or disorder characterized by abnormal PD-Ll expression or activity, including but not limited to cancer, infectious diseases, and inflammatory diseases.
[0070] In some embodiments, the PD-Ll antibody sequences are included as a part of a multifunctional polypeptide of the disclosure, wherein the multifunctional polypeptide comprises a CD80 extracellular domain (e.g. a wild-type CD80 extracellular domain, e.g. SEQ ID NO: 34, or variants thereof), a disease-associated antigen-binding domain; and an Fc domain. In this context, the PD-Ll antibody may be referred to interchangeably as a PD-Ll antigen-binding domain, or simply as a PD-Ll binding domain. [0071] As used herein, the term “antibody” includes full length antibodies; antibody fragments (including without limitation Fab, Fab’, F(ab’)2, Fv, diabody, scFv, scFv-Fcs, single domain antibody, single chain antibody, single heavy chain antibody, and single light chain antibody), provided that they exhibit a desired biological activity (e.g. epitope binding) ; monoclonal antibodies; polyclonal antibodies; monospecific antibodies; multi-specific antibodies (e.g., bispecific antibodies); humanized antibodies; chimeric antibodies; covalently modified antibodies, and antibody conjugates (e.g. antibody-drug conjugates or antibodies conjugated to detectable labels).
[0072] The antibodies may be derived from any species. In exemplary embodiments the antibody is human. In other embodiments, the antibody is a mouse antibody.
[0073] The full length PD-L1 antibodies of the disclosure may have a human Fc, that is IgGl, IgG2, IgG3, or XgG4.
[0074] The full length PD -LI antibodies of the disclosure may have a mouse Fc, that is IgGl, IgG2a, IgG2b, or IgG3.
[0075] Reference to “PD-L1” can relate to a PD-L1 protein of any species, and in particular to a human PD-L1, and more particularly to a protein comprising the amino acid sequence of SEQ ID NO: 1 represented below.
Human PD-L1 (PDlLlJffUMAN: IJniProKB - Q9NZQ7)
MRIFAVFIFM TYWHLLNAFT VTVPKDLYW EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKV QHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMIS Y GG ADYKRITVKV NAPYNKINQR ILWDPVTSE HELT CQ AEG Y PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELP LAHPPNERTH LVILGAILLC LGVALTFIFR LRKGRMMDVK KCGIQDTNSK KQSDTHLEET (SEQ ID NO: 1)
[0076] Table 1 provides exemplary- CDR-L1 sequences of the PD-L1 antibodies of the disclosure.
Table 1
Figure imgf000016_0001
Figure imgf000017_0001
[0077] Table 2 provides exemplary CDR-L2 sequences of the PD-Ll antibodies of the disclosure.
Table 2
Figure imgf000017_0002
[0078] Table 3 provides exemplary CDR-L3 sequences of the PD-L1 antibodies of the disclosure.
Table 3
Figure imgf000017_0003
[0079] Table 4 provides exemplary CDR-H1 sequences of the PD-L1 antibodies of the disclosure.
Table 4
Figure imgf000017_0004
Figure imgf000018_0001
[0080] Table 5 provides exemplary CDR-H2 sequences of the PD-Ll antibodies of the disclosure.
Table 5
Figure imgf000018_0002
[0081] Table 6 provides exemplary CDR-H3 sequences of the PD-Ll antibodies of the disclosure.
Table 6
Figure imgf000018_0003
[0082] In some embodiments, provided herein is a PD-Ll antibody comprising the amino acid sequence of a CDR-L1 of Table 1, a CDR-L2 of Table 2, a CDR-L3 of Table 3, a CDR-H1 of Table 4, a CDR-H2 of Table 5, and/or a CDR-H3 of Table 6. [0083] Table 7 provides the light chain CDR sequences of eleven PD-L1 antibodies of the disclosure. In some embodiments, provided herein is a PD-L1 antibody comprising the amino acid sequence of a light chain comprising the sequences of any one of the CDR-L1, CDR-L2, and CDR- L3 combinations presented in Table 7.
Table 7
Figure imgf000019_0001
Figure imgf000020_0001
[0084] Table 8 provides the heavy chain CDR sequences of eleven PD-L1 antibodies of the disclosure.
[0085] In some embodiments, provided herein is a PD-L1 antibody comprising the amino acid sequence of a light chain comprising the sequences of any one of the CDR-HL CDR-H2, and CDR- H3 combinations presented m Table 8.
Table 8
Figure imgf000020_0002
Figure imgf000021_0001
[0086] Table 9 provides the heavy and light chain CDR sequences of eleven PD-L1 antibodies of the disclosure.
[0087] In some embodiments, provided herein is a PD-L1 antibody comprising the amino acid sequences of any one of the CDR-LL CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 combinations presented in Table 9.
Table 9
Figure imgf000021_0002
Figure imgf000022_0001
Figure imgf000023_0001
[0088] Table 10 provides the sequences of exemplary light chain variable domains (VL) of the PD- LI antibodies of the di sclosure (exemplary CDR sequences are underlined below).
Table 10
Figure imgf000023_0002
[0089] Table 11 provides the sequences of exemplary heavy chain variable domains (VH) of the PD-L1 antibodies of the disclosure.
Table 11
Figure imgf000023_0003
Figure imgf000024_0001
[0090] In some embodiments, provided herein is a PD-L1 antibody comprising a VL domain comprising an ammo acid sequence set forth in any one of SEQ ID NOs: 18-21 and 8EQ ID NO: 57, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. [0091] In some embodiments, provided herein is a PD-L1 antibody comprising a VH domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 23-33, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. [0092] In some embodiments, provided herein is a PD-L1 antibody comprising a VL domain comprising an ammo acid sequence set forth m any one of SEQ ID NOs: 18-21 and SEQ ID NO: 57, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto, and VH domain comprising an am o acid sequence set forth in any one of SEQ ID NOs: 23- 33, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.
[0093] In some embodiments, provided herein is a PD -LI antibody comprising:
(a) a VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and/or a VH domain comprising SEQ ID NO: 23, or a sequence comprising at least 80%, at least
85%, at least 90%, at least 95%, or at least 99% identity thereto;
(b) a VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and/or a VH domain comprising SEQ ID NO: 24, or a sequence comprising at least 80%, at least
85%, at least 90%, at least 95%, or at least 99% identity thereto;
(c) a VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and/or a VH domain comprising SEQ ID NO: 25, or a sequence comprising at least 80%, at least
85%, at least 90%, at least 95%, or at least 99% identity thereto;
(d) a VL domain comprising SEQ ID NO: 18; and/or a VH domain comprising SEQ ID NO: 26, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto,
(e) a VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and/or a VH domain comprising SEQ ID NO: 27, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto:
(f) a VL domain comprising SEQ ID NO: 19, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and/or a VH domain comprising SEQ ID NO: 28, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto:
(g) a VL domain comprising SEQ ID NO: 19, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and/or a VH domain comprising SEQ ID NO: 29, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto:
(h) a VL domain comprising SEQ ID NO: 20, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto; and/or a VH domain comprising SEQ ID NO: 30, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto:
(i) a VL domain comprising SEQ ID NO: 21, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto; and/or a VH domain comprising SEQ ID NO: 31, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto:
(j) a VL domain comprising SEQ ID NO: 57, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto; and/or a VH domain comprising SEQ ID NO: 32, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto:
(k) a VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto; and/or a VH domain comprising SEQ ID NO: 33, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto: (L) a VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and/or a VH domain comprising SEQ ID NO: 58, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto:
(m) a VL domain comprising SEQ ID NO: 57, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto; and/or a VH domain comprising SEQ ID NO: 58, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto;
(n) a VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto; and/or a VH domain comprising SEQ ID NO: 59, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto;
(o) a VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto; and/or a VH domain comprising SEQ ID NO: 60, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto;
(p) a VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto; and/or a VH domain comprising SEQ ID NO: 61, or a sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity' thereto;
[0094] Also provided herein are polynucleotides encoding for any of the PD-L1 antibodies provided herein, vectors comprising such polynucleotides, and cells expressing such vectors.
[0095] Also provided herein are pharmaceutical compositions comprising any one of the PD-L1 antibodies provided herein, optionally with a pharmaceutically acceptable excipient.
[0096] Also provided herein are methods of a treating a disease or disorder in a subject in need thereof, comprising administering to the subject any one of the PD-LI antibodies of the disclosure, or pharmaceutical compositions comprising such antibodies. The disease or disorder may be cancer, infectious diseases, or inflammatory diseases.
III. Multifunctional Polypeptides
[00971 Provided herein are multifunctional polypeptides comprising the following components, the components of which are described independently m greater detail herein.
(a) a CD80 extracellular domain;
(b) a disease-associated antigen-binding domain; and
(c) a Fc domain (referring to a Fc dimer).
[0098] The components are linked together to form the multifunctional polypeptide. As described in greater detail herein, and referring to exemplary formats in FIG. 1 A, the components can be linked in various orders. In some embodiments, the components are linked with the use of linkers, in other embodiments, the components are fused together. A skilled artisan will appreciate the manner in which the polypeptide components can be formatted.
[0099] Also provided herein are polynucleotides encoding the multifunctional polypeptides of the disclosure; vectors comprising the polynucleotides; and cells expressing the vectors.
[0100] The multifunctional polypeptides of the disclosure may be modified, e.g. by glycosylation, sialylation, acetylation, phosphorylation, and the like. Accordingly, in exemplary embodiments, the sialic acid content of the multifunctional polypeptides may vary - in such embodiments, any one or more of the CD80 extracellular domain, the disease-associated antigen-binding domain, and the Fc domain may be sialyiated.
[0i 01] Also provided herein are pharmaceutically compositions comprising the multispecific polypeptides of the disclosure, optionally with a pharmaceutically acceptable excipient.
A. CB80 Extracellular Domain
[0102] The multifunctional polypeptides of the disclosure comprise a CD80 extracellular domain, wherein the CD80 extracellular domain comprises an am o acid sequence having at least 80%, 85%, 90%, 95%, or 100% ammo acid sequence identity to SEQ ID NO: 34, the human extracellular domain of CD80 (underlined amino acid residues are exemplar residues that may be substituted in the CD80 extracellular domain variants of the disclosure).
Human CD80 (UniProtKB - P33681)
VIHVTKEVKE VATLSCGHNV SVEELAQTRI YWQKEKKMVL TMMSGDMNIW PEYKNRTIFD ITNNLSIVIL ALRPSDEGTY ECW LKYEKD AFKREHLAEV TLSVKADFPT PSISDFEIPT SNIRRIICST SGGFPEPHLS WLENGEELNA INTTVSQDPF TELYAVSSKL DFNMTTNHSF MCLIKYGHLR VNQTFNWNTT KQEHFPDN (SEQ ID NO: 34)
Mouse CD 80
VDEQLSKSVK DKVLLPCRYN SPHERESFDR IYWQKHDKW LSVIAGKLKV WPEYKNRTLY DNTTYSLIIL GLVLSDRGTY SCW QKKERG TYEVK.HLALV KLSTKADFST PNITESGNPS ADTKRITCFA SGGFPKPRFS WLENGRFLPG TNTTISQDPE SELYTISSQL DFNTTRNHTI KCLIKYGDAH VSEDFTWEKP PEDPPDSKN (SEQ ID NO: 37)
[0103] In some embodiments, the CD80 extracellular domain has one or more amino acid substitution modifications at positions 130, 131, 139, 155, 156, 165, or 166 of SEQ 1D NO: 34, or the corresponding positions in SEQ ID NO: 37.
[0104] In some embodiments, the CD80 extracellular domain has at least a substitution modification at position 130 of SEQ ID NO: 34, and the amino acid substitution modification at position 130 of SEQ ID NO: 34 is T13QA.
[0105] In some embodiments, the CD80 extracellular domain has at least a substitution modification at position 131 of SEQ ID NO: 34, and the amino acid substitution modification at position 131 of SEQ ID NO: 34 is S131A, S131V, S131I, SI 3 i 14 S131R, S131E, S131D, or S131Q. [0106] In some embodiments, the CD80 extracellular domain has at least a substitution modification at position 139 of SEQ ID NO: 34, and the amino acid substitution modification at position 139 of SEQ ID NO: 34 is L139V.
[0107] In some embodiments, the CB80 extracellular domain has at least a substitution modification at position 155 of SEQ ID NO: 34, and the amino acid substitution modification at position 155 of SEQ ID NO: 34 is VI 55 A, VI 551, or V155T. [0108] In some embodiments, the CD80 extracellular domain has at least a substitution modification at position 156 of SEQ ID NO: 34.
[0109] In some embodiments, the CD80 extracellular domain has at least a substitution modification at position 165 of SEQ ID NO: 34, and the amino acid substitution modification at position 165 of SEQ ID NO: 34 is A165S, A165V, A165I, A165F, A165R, A165E, A165D, or A165Q.
[0110] In some embodiments, the CD80 extracellular domain has at least a substitution modification at position 166 of SEQ ID NO: 34, and the amino acid substitution modification at position 166 of SEQ ID NO: 34 is V166A, V166L, or V166T.
[01 ί 1] In some embodiments, the CD80 extracellular domain is sia!ylated.
B. Disease- Associated Antigen-Binding Domain
[0112] The multifunctional polypeptides of the disclosure comprise disease-associated antigen binding domains that bind disease-associated antigens. In some embodiments, without being bound to any theory or mechanism, such disease-associated antigen-binding domains are capable of binding disease-associated antigens on diseased eells through classical antigen-antibody binding interactions. Without being bound any theory or mechanism, this can allow the multifunctional polypeptide to arm a diseased cell expressing a disease-associated antigen (e.g. a cancer-specific antigen), with a CD80 extracellular domain (a CD80 ligand), via binding of the disease-associated antigen-binding domain to the diseased cell. The diseased cell now armed with a CD80 ligand is available to bind to a T-cell, conferring a costimulatory signal thereon. The CD80 ligand is available to bind to any one or more of its cognate binding partners, CD28 PD-L1 or CTLA-4 on T-cells (either T-lymphocytes, or T- regulatory cells). In other embodiments, the disease-associated antigen can be a soluble antigen. [0113] Accordingly the disease-associated antigen can be any antigen upregulated or overexpressed in a disease such as a cancer, an infectious disease, or an inflammatory disease. In some embodiments, the multifunctional polypeptides comprise a cancer-associated antigen-binding domain. In some embodiments, the multifunctional polypeptides comprise an infectious disease-associated antigen binding domain. In some embodiments, the multifunctional polypeptides comprise an inflammatory disease-associated antigen-binding domain. In some embodiments, the disease-associated antigen- binding domain is a PD-Ll binding domain. In other embodiments, the disease-associated antigen binding domain is a Claudin 18.2 binding domain.
[0114] In some embodiments, the disease-associated antigen-binding domain is a PD-Ll binding domain.
[Oίί 5] In some embodiments, the PD-Ll binding domain comprises:
(d) the amino acid sequence of a CDR-L1 of Table 1, a CDR-L2 of Table 2, a CDR-L3 of Table 3, a CDR-HI of Table 4, a CDR-H2 of Table 5, and/or a CDR-H3 of Table 6;
(e) the amino acid sequence of a light chain comprising the sequences of any one of the CDR-L1, CDR-L2, and CDR-L3 combinations presented in Table 7;
(f) the amino acid sequence of a light chain comprising the sequences of any one of the CDR-H1, CDR-H2, and CDR-H3 combinations presented in Table 8;
(g) the amino acid sequences of any one of the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 combinations presented in Table 9;
( h ) a VL domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 18-21, and SEQ ID NO: 57, or a sequence comprising at least 80% identity thereto;
(i) a VH domain comprising an ammo acid sequence set forth in any one of SEQ ID NOs: 23-33, and SEQ ID NOs: 58-61, or a sequence comprising at least 80% identity thereto; or
(j) a VL domain comprising an ammo acid sequence set forth in any one of SEQ ID NOs: 18-21, and SEQ ID NO: 57, or a sequence comprising at least 80% identity thereto, and VH domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 23- 33, and SEQ ID NOs: 58-61, or a sequence comprising at least 80% identity thereto.
|Ό116] In some embodiments, the PD-Ll binding domain comprises the antigen- binding sequences of Atezohzumab, Avelumab, or Durvalumab. Atezolizumah Light Chain:
DIQMTQSPSSLSASVGDRVTnCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVE1K (SEQ ID NO: 35)
Atezolizumah Heavy Chain:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYY ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 36)
Avelumah Light Chain:
QSALTQPASVSGSPGQSraSCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVS
NRFSGSKSGNTASLTiSGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL (SEQ ID NO: 38)
Avelumah Heavy Chase:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYA
DTVKGRFTISRDN SKNTIYXQMN SI JIAEDTAV YYC ARIKLGTVTTVD YWGQGTLVTV S S (SEQ m NO: 39)
Durvalumah Light Chase:
EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLUYDASSRATGIPDRF S GS GS GTDFTI.TISRI EPEDF AVYY C QQ Y GSLPWTF GQGTKVEIK (SEQ ID NO: 40)
Durvalumab Heavy Chain:
E V QLVE S GGGL VQPGGSLRLS C AAS GFTF SRYWMS WVRQ APGKGLEWV ANIKQDGSEKY YVDSAT GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVT VSS (SEQ ID NO: 41)
[0117] In some embodiments, the disease-associated antigen-binding domain is a Claudin-18.2 binding domain.
[0118] In some embodiments, the sequence of the Claudin-18.2 binding domain is from WQ2019/174617, the disclosure of which is incorporated herein by reference in its entirety.
[0119] In some embodiments, the sequence of the Claudin-18.2 binding domain is from EP1997832, the disclosure of which is incorporated herein by reference in its entirety.
[012Q] In some embodiments, the sequence of the Claudin-18.2 binding domain is from EP3483182, the disclosure of which is incorporated herein by reference in its entirety. [0121] In some embodiments, the sequence of the Claudin-18.2 binding domain is from WO2019173420, the disclosure of which is incorporated herein by reference in its entirety.
[0122] Exemplar non-limiting Claudin-18.2 binding domains (and sequences contributing to the same) of the disclosure, are listed here. For each VH and VL listed below', the CDRs are bolded and underlined. Accordingly, the Claudin-18.2 binding domains of the disclosure may comprise any one or more of the CDR chains listed below.
CLDN 18.2 Abl: 10-K12-A (mouse Ab)
VL:
DIVMTOSPSSLTVTAGEKVTMSCKSSOSLLNSGNOKNYLTWYOOKPGOPPKLIIYWAST RESGVPDRFTGSGSGTDFTLTISSVOAEDLAIYYCONDYFYFLTFGAGTKLELK (SEQ ID NO: 42)
VH:
EVOLVESGGGLVKPGGSRKLSCAASGFrFSDYGMFWVROAPEKGLEWVGYlSSGSSNlYY
ADTVKGRFHSKDNAIGSiTLFLOMTSLRSEDTAMYYCARIARGNAMDYWGOGTSVTVSS (SEQ ID NO: 43)
CLDN 18.2 Ab3: 1-M-5A (moose Ab)
Figure imgf000033_0001
CLDN 18,2 Abl2: 7-G17-A (mouse Ab)
VL:
DIVMTOSPSSLIVTPGEKVTMSCKSSOSLFNSGNORNYLAWYQOKPGQPPKLLIYWASTR
ESGVPDRFTGSGSGTDFTLHSSVQAEDLAVYYCONDYFYPLTFGAGTKLELK (SEQ ID NO: 46)
VH:
EVOLVESGGGLVKPGGSRKLSCAVSGFrFSDYGMYWVROAPEKGLEWVAYISSGSSTIY
YADTVKGRFTMSRDNAKNTLFLOMTSLRSEDTAMYYCARIARGNAMDYWGOGTSVTVS
S (SEQ ID NO: 47) HuCLDN18.2-v.24 (humanized Ab)
VL:
DIVMTOSPDSLAVSLGERATINCKSSOSLLNSGNOKNYLTWYQOKPGQPPKLLIYWASTR ESGVPDRF'SGSGSGTDFTLTISSLQ.AEDVAVYYCONNYIYPFTFGQGTKVEIK (SEQ ID NO: 48)
VH:
EVOLVESGGGLVKPGGSLRLSCAASGFTFSNYAMSWIROAPGKGLEWVSTIIIGGTYTYYP DSVKGRFHSRDNAKNSLYLOMNSLRAEDTAVYYCAROVYGNSFAYWGOGTLVTVSS (SEQ ID NO: 49)
HiiGϊ.RN 18.2-V.19 (humanized Ab)
VL:
DIVMTOSPDSLAVSLGERATINCKSSOSLLNSGNOKNYLTWYOOKPGOPPKLLryWASTR ESGVPPRFSGSGSGTDFTLTISSLOAEPVAVYYCONNYIYPFrFG GTKVEIK (SEQ 10 NO:
48)
VH:
EVOLVESGGGLVKPGGSLRLSCAASGFTFSNYAMSWIROAPGKGLEWVSTinGGIYTYYP PSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQVYGNSFAYWGOGTLVTVSS (SEQ I» NO: 50)
Zolbetuximab
VL:
DIVMTQSPSSLWTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRE SGVPDRFTGSGSGTDFTLTISSVQAEDLAVYY QNDYSYPFTFGSGTKLEIK (SEQ ID NO: 51)
VH:
QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNY NQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNSFDYWGQGTTLTVSS (SEQ ID NO: 52)
[0123] In some embodiments, the disease-associated antigen-binding domain is not a PD-L1 binding domain.
[0124] In some embodiments, the disease-associated antigen-binding domain is not a Claudin-18.2 binding domain.
C. Fc Domain
[0125] It is noted that the Fc domains of the multifunctional polypeptides of the disclosure are Fe dimers. [0126] In some embodiments, the Fc domain is human in some embodiments, the Fc domain is a human XgGl, IgG2, XgG3, or IgG4 Fe domain.
[0X27] In some embodiments, the Fc domain is from mouse. In some embodiments, the Fc domain is a mouse XgGl, IgG2a, IgG2b, or IgG3 Fc domain.
[0128] In some embodiments, the Fc domain is a wild-type human XgGl Fc domain. In some embodiments, the Fe domain is a mutant human IgGl Fe domain. In some embodiments, the mutant human IgGl Fc domain comprises reduced effector function. In some embodiments, the mutant human IgGl Fc domain comprises a knob-in-hole mutation. In some embodiments, the mutant human IgGl Fc domain comprises reduced effector function and a knob-m-hoie mutation.
[0129] In the context of knob-in~ho!e mutations, heterodimerization of the two Fc monomeric domains of any of the formats depicted in FIG. 1A can be promoted by introducing different, but compatible substitutions in the two Fc monomeric domains, such as knob-in-hole residue pairs (e.g. US Pat. No. 5,731,168).
[0130] In some embodiments, the Fc domain is glycosylated, aeetylated, sialylated, and/or phosphorylated.
[0131] In some embodiments, the Fc domain comprises the ammo acid sequence of any one of SEQ ID NOS: 100 to H I. SEQ ID NO: 112 provides an exemplary sequence comprising a human CD80 extracellular domain + human IgGl Fc. SEQ ID NO: 136 provides an exemplary sequence comprising a mouse CD80 extracellular domain + mouse IgG2a Fc.
Hum IgGl Fc (IGHG1 _HUMAN: UniProKB - RQ1857)
DKTHTCP PCPAPELLGG PS VFLFPPKP KDTLMISRTP EVTCWVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK (SEQ ID NO; 100)
Human IgG2 Fc (IGHG2_HUMAN: UniProKB - P018S9)
VECPPCP APPVAGPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVQFNWYVDG VEVHNAKTKP REEQFNSTFR WSVLTWHQ DWLNGKEYKC KVSNKGLPAP IEKTISKTKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDISVEW ESNGQPENNY KTTPPMLD SD GSFFLYSKLT VDKSRWQQGN WSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 101) Human IgG3 Fc (IGHG3_HUMAN: UniProKB -· P01860)
DTPPPCPRCP APELLGGPS V FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVQFKWYVD GVEVHNAKTK PREEQYNSTF RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WES S GQPENN YNTTPPMLDS DGSFFLYSKL TVDKSRWQQG NIFSCSVMHE. ALHNRFTQKS LSLSPGK (SEQ ID NO: 102)
Human lgG4 Fe (IGHG4 HUMAN: UniProKB - P01861)
KYGPPCPSCP APEFLGGPS V FLFPPKPKDT LMISRTPEVT CWVDVSQED PEY QFNWYVD GVEVHNAKTK PREEQFNSTY RVV8VLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESN GQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK (SEQ ID NO: 103)
Mouse IgGl Fc (IGHGl_MOUSE, Ig gamma-1 chain C region secreted from: UniProKB -
P01868)
KPCICT VPEVSSVFIF PPKPKDVLTJ TLTPKVTCW VDISKDDPEV QFSWFVDDVE VHTAQTQPRE EQFNSTFRSV SELPIMHQDW LNGKEFKCRV NSAAFPAPIE KTISKTKGRP KAPQVYTiPP PKEQMAKDKV SLTCMITDFF PEDITVEWQW NGQPAENYKN TQPIMNTNGS YF V YSKLNVQ KSNWEAGNTF TCSVLHEGLH NHHTEKS LSH SPGK (SEQ ID NO: 104)
Mouse IgG2a Fe (GCAA_MOUSE, Ig gamma-2A chain € region, A allele: UniProKB -
P01863)
GPTTKPCPPC KCPAPNLLGG PSVFIFPPKi KDVLMISLSP IVTCVWDVS EDDPDV QISW FVNNVEVHTA QTQTHREDYN STLRWSALP IQHQDWMSGK EFKCKVNNKD LPAPIERTIS KPKGSVRAPQ VYVLPPPEEE MTKKQVTLTC MVTDFMPEDI Y VE WTNN GKT ELNYKNTEPV LDSDGSYFMY SKLRVEKKNW VERNSYSCSV VHEGLHNHHT TKSFSRTPGK (SEQ IB NO: 105)
Mouse IgG2b Fe (IGG2B_MOUSE, Ig gamma-2B chain C region: UniProKB - P01867) SITNPCPP CKECHKCPAP NLEGGPSVFI FPPNIKDVLM ISLTPKVTCV WDVSEDDPD VQ1SWFVNNV EVHTAQTQTH REDYNSITRV VSTLPIQHQD WMSGKEFKCK VNNKDLPSP1 ERTISKIKGL VRAPQVYILP PPAEQLSRKD VSLTCLWGF NPGD1SVEWT SNGHTEENYK DTAPVLDSDG SYFIYSKLNM KTSKWEKTDS FSCNVRHEGL KNYYLKKTIS RSPGLDLDDI CAEAKDGELD GLWTTITTFI SLFLLSVCYS ASVTLFKVKW IFSSVVELKQ KISPDYRNMI GQGA (SEQ ID NO: 106)
Mouse IgG3 Fc (lGHG3_MOUSE, Ig gamma-3 chain C region: UniProKB -P03987) STPPGSS CPPGNILGGP SVFIFPPKPK DALMISLTPK VTCVWDVSE DDPDVHVSWF VDNKEVHTAW TQPREAQYNS TFRWSALPI QHQDWMRGKE FKCKVNNKAL PAPIERTISK PKGRAQTPQV YTIPPPREQM SKKKVSLTCL VTNFFSEAIS VEWERNGELE QDYKNTPPIL DSDGTYFLYS KLTVDTDSWL QGEIFTCSVV HEALHNHHTQ KNLSRSPELE LNETCAEAQD GELDGLWTTI TIFISLFLLS VCYSASYTLF KVKWIFSSW QVKQTAIPDY RNMIGQGA (SEQ ID NO: 107) faFc N297A
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYASTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKnSKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 108} fiFc Knob N297A
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDG VEVHNAKIKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISB AKG QPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 109} hlgGlFc Hole
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTErrCPPCPAPELLGGPSVF
LFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 110) hlgGlHc Hole N297G
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSWTYPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF
LFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 111)
Human CD 80 + Human IgGl Fc
VIHVTKEVKE VATLSCGHNV SVEELAQTRI YWQKEKKMVL TMMSGDMNIW PEYKNRTTFD ITNNLSIVIL ALRPSDEGTY EC WLK YEKD AFKREHLAEV TLSVKADFPT PSISDFEIPT SNIRRIICST SGGFPEPHLS WLENGEELNA INTTVSQDPE TELYAVSSKL DFNMTTN1TSF MCLIKYGHLR VNQTFNWNTT KQEHFPDNGS DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKV SNKALP A PIFK ! !SKAK GQPREPQVYT LPPSRDELTK N Q V SLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRW QQG NVFSCS YMHE ALHNHYTQKS LSLSPGK (SEQ ID NO: 112)
Mouse CD80 + Mouse !gG2¾ Fc
VDEQLSKSVKDKVLLPCRYNSPHEDESEDRIYWQKHDKWLSVIAGKLKVWPEYKNRTLY DNTT Y SLHLGLVLSDRGTY S C VV QKKERGT YEVKHL ALVT LSIKADF S TPNITE S GNPS ADT KRITCFAS GGFPKPRF S WLEN GRELPGINTTI S QDPESELYTIS S QLDFNTTRNHTIKCLIK Y GD AHVSEDFTWEKPPEDPPDSKNEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPI VTCVWDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRWSALPIQHQDWMSGK !:.!·K(·! \ N\K!)ί .RLRH .K G!8 R (ϊ8\ KL!?0\' U\ ! .RRG G.ί;!·Lί GK.KO\ TU CMM' f V!P!iD! V ' EWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSWHEGLHNHHTT KSFSRTPGK (SEQ ID NO: 136)
1). Structural Features
[0132] The multifunctional polypeptide may comprise any one of the structural formats provided in FIG. 1A.
[0133] In some embodiments, the multifunctional polypeptide comprises the structure of Format A of FIG. 1A. In some embodiments, the multifunctional polypeptide does not comprise the structure of Format A. In some embodiments, the multifunctional polypeptide comprises the structure of Format A of FIG. I A, and the disease-associated antigen-binding domain is specific for PD-L1 or Claudin-18.2
[0134] In some embodiments, the multifunctional polypeptide comprises the structure of Format B of FIG. !A. In some embodiments, the multifunctional polypeptide does not comprise the structure of Format B. In some embodiments, the multifunctional polypeptide comprises the structure of Format B of FIG. 1 A, and the disease-associated antigen-bimi g domain is specific for PD-Ll or Claudin-18.2.
[0135] In some embodiments, the multifunctional polypeptide comprises the structure of Format C of FIG. 1A. In some embodiments, the multifunctional polypeptide does not comprise the structure of Format C. In some embodiments, the multifunctional polypeptide comprises the structure of Format C of FIG. 1A, and the disease-associated antigen-binding domain is specific for PD-L1 or Claudin-18.2.
[0136] In some embodiments, the multifunctional polypeptide comprises the structure of Format I) of FIG. LA In some embodiments, the multifunctional polypeptide does not comprise the structure of Format D. In some embodiments, the multifunctional polypeptide comprises the structure of Format D of FIG. 1A, and the disease-associated antigen-binding domain is specific for PD-L1 or Claudin-18.2,
[0137] In some embodiments, the multifunctional polypeptide comprises the structure of Format E of FIG. IA. In some embodiments, the multifunctional polypeptide does not comprise the structure of Format E. In some embodiments, the multifunctional polypeptide comprises the structure of Format E of FIG. I A, and the disease-associated antigen-binding domain is specific for PD-LI or Claudin-18.2.
[0138] In some embodiments, the multifunctional polypeptide comprises the structure of Format F of FIG. 1A. In some embodiments, the multifunctional polypeptide does not comprise the structure of Format F. In some embodiments, the ultifunctional polypeptide comprises the structure of Format F of FIG. 1 A and the disease-associated antigen-binding domain is specific for PD-LI or Claudin-18.2.
[0139] In some embodiments, the multifunctional polypeptide comprises the structure of Format G of FIG. 1A. In some embodiments, the multifunctional polypeptide does not comprise the structure of Format G. In some embodiments, the multifunctional polypeptide comprises the structure of Format G of FIG. 1A, and the disease-associated antigen-binding domain is specific for PD-LI or Claudin-18.2.
[0140] In some embodiments, the multifunctional polypeptide comprises the structure of Format H of FIG. 1 A. In some embodiments, the multifunctional polypeptide does not comprise the structure of Format H. In some embodiments, the multifunctional polypeptide comprises the structure of Format H of FIG. 1 A, and the disease-associated antigen- binding domain is specific for PD-LI or Claudin-18.2.
[0141] In some embodiments, the multifunctional polypeptide comprises the structure of Format I of FIG. 1A. In some embodiments, the multifunctional polypeptide does not comprise the structure of Format I. In some embodiments, the multifunctional polypeptide comprises the structure of Format I of FIG. 1A, and the disease-associated antigen-binding domain is specific for PD-L1 or Claudin-
18.2. In some embodiments, the multifunctional polypeptide comprises the structure of Format I of FIG. 1A, and the disease-associated antigen- binding domain is not specific for PD-L1 or Claudin-
18.2.
[0142] In some embodiments, the multifunctional polypeptide comprises the structure of Format J of FIG. LA In some embodiments, the multifunctional polypeptide does not comprise the structure of Format J. In some embodiments, the multifunctional polypeptide comprises the structure of Format J of FIG. 1 A, and the disease-associated antigen-binding domain is specific for PD-L1 or Claudin-
18.2.
[0143] In some embodiments, the ultifunctional polypeptide comprises any one of the structures of Format A-J of FIG. LA, and the disease-associated antigen-binding domain is not specific for PD- Ll. In some embodiments, the multifunctional polypeptide comprises any one of the structures of Format A-J of FIG. 1 A, and the disease-associated antigen-binding domain is not specific for Claudin
18.2. In some embodiments, the multifunctional polypeptide comprises any one of the structures of Format A-J of FIG. 1 A and the disease-associated antigen-binding domain is not specific for Her2D- Ll.
[0144] In some embodiments, the CD80 extracellular domain of the multifunctional polypeptide is a monomer.
[0145] In some embodiments, the multifunctional polypeptide comprises two monomers of the CD80 extracellular domain.
[0146] In some embodiments, the multifunctional polypeptide comprises four monomers of the CD80 extracellular domain.
[0147] In some embodiments, the multifunctional polypeptide comprises a CD80 extracellular domain that is a single chain dimer.
[0148] In some embodiments, the multifunctional polypeptide comprises two single chain dimers of the CD 80 extracellular domain.
[0149] In some embodiments, the multifunctional polypeptide comprises a CD80 extracellular domain that attached to the N-terminus of the Fc domain. [0150] In some embodiments, the multifunctional polypeptide comprises a CD80 extracellular domain is attached to the N-terminus of a disease-associated antigen-binding domain, and wherein the disease-associated antigen-binding domain is attached to the N-terminus of an Fc domain.
[0151] In some embodiments, the multifunctional polypeptide comprises a CD80 extracellular domain that is attached to the C-terminus of the Fc domain.
[0152] In some embodiments, the multifunctional polypeptide comprises a disease-associated antigen-binding domain comprising an scFv.
[0153] In some embodiments, the multifunctional polypeptide comprises a disease- associated antigen-binding domain comprising a Fab or Fab’ fragment.
[0154] In some embodiments, the multifunctional polypeptide comprises a disease- associated antigen-binding domain comprising a F(ab’)2 fragment
[0155] In some embodiments, the multifunctional polypeptide comprises a disease- associated antigen-binding domain comprising a Fv fragment.
[0156] In some embodiments, the multifunctional polypeptide comprises a disease-associated antigen-binding domain comprising a Fab’ fragment and a Fv fragment.
[0157] In some embodiments, the multifunctional polypeptide comprises a disease-associated antigen-binding domain that is attached to the N-terminus of the Fc domain.
[0158] In some embodiments, the multifunctional polypeptide comprises two disease-associated antigen-binding domains coupled to each other, for example wherein one disease-associated antigen binding domain comprises a Fab or Fab’ fragment, and one antigen-binding domain comprises a Fv fragment. In some embodiments, the multifunctional polypeptide comprises a Fv fragment disease- associated antigen-binding domain that is attached to the N-terminus of a Fab or Fab’ disease- associated antigen-binding domain that is attached to the N-terminus of a Fc domain.
[0159] In some embodiments, the multifunctional polypeptide comprises a disease-associated antigen-binding domain that is attached to the N-terminus of a CD80 extracellular domain which is attached to the N-terminus of a Fc domain.
[0160] In some embodiments, the multifunctional polypeptide comprises a disease-associated antigen-binding domain that is attached to the C-terminus of the Fc domain. E. Exemplary Multifunctional Polypeptides
[0161] By way of example only, provided herein are non-limiting exemplary sequences for the multifunctional polypeptides of the disclosure.
[0162] In some embodiments, the multifunctional polypeptide comprises the ammo acid sequence of any one of SEQ ID NOs: 113-135. The sequences below are exemplary only, and by no means limiting the other variations possible, as described herein. Some of the sequences below are represented by domains, with the use of brackets. hCDSO-Lmker-hlgGl Fc Knob N297A (Format A)
P^THVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIF
DITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNI
RRnCSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKY
GHLRVNQTFNWNTTKQEHFPDN][GS][DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT
PEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRWSVLTVLHQDWL
NGKEYKCKV8 G4I,PAPIEKTISKAKGQPREPQVYHPPSREEMTKNQVSLWCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH
YTQKSLSLSPGK] (SEQ 10 NO: 113)
Anti-PD-Ll He. (Atezo) hFc Hole N297A (Format A, B)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYY
ADSYTCGRFHSADTSKNTAYLQIVJNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY
SLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL
FPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRV
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKHSKAKGQPREPQVYTLPPSREEMTKNQ
VSLSCAVKGFYPSDIAVEWESNCiQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 114)
Anti-PD-Ll Lc (Atezolizumah) (Format A,B? F)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF SGSGSGTDFTLHSSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 115) sc-hCD80 dimer hFc Knob N297A (hCD80-linker-hcD80-hlgGl Fc) (Format B, D)
[ViHVTKEVKEVATLSCGHNVSVEELAQITRIYWQKEKKMVLTMlVISGDMNrWPEYKNRlTF
DITNNLSIVILALRPSDEGTYECVVLKYEKDAFB REHLAEVTLSVB ADFPTPSISDFEIPTSNI
RRnCSTSGGFPEPHLSWLENGEELNAINTWSQDPETELYAVSSKLDFNMTTNHSFMCLIKY GHLR VNQ TFNWNTTKQEHFPDN ] [ GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS ] [ VIH V TKE.VKE V ATLS C GHNYrS VEELAQT RI YW QKEKKMVLTMA4S GDMNTWPE Y KNRUFDITNNLSIVILALRPSDEGTYECWLKYEKDAFKREHLAEVTLSVKADFPTPSISDFE
IPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFN TTNHSFM CLIKYGHLRVNQTFNWNTTKQEHFPDN] [DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRWSVLTVLHQDW LNGKEYKCKVSN ALPAPIEKHSKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK] (SEQ ID NO: 116)
Anti-PD-Ll He (Atezolizumab) liFc N297A-linker-hCD80 C~terminal fusion (Format F)
[EVQLVESGGALVKPGGSLKLSCAASGFTFSNYAMSWIRQIPEKRLEWVATmGGTYTYYP
D8\T4GRFTX8RDNAKNTLYLQMSSLRSEDTALYYCARQVYGNSFAYWGGGTLyTV8AAST
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMfSRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEGYASTYRWS
VLTVLHQDWLNGKEYKCKVSNKALPAPIE^SKAKGQPREPQVYTLPPSRDELTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPGK][GGGGSGGGGSGGGGS][VIHVTKEVKEVATLSCGHNVS
VEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYEC
WLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGE
ELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPD
NJ (SEQ ID NO: 117) hCD80-linker-anti-PD-Ll He (Atezolizuniab)hFc N297A N-terminal fusion (Format H)
[\IHVTKEA¾TSVATLSCGHNVS\TERAQTRIYWQKEKKMVLTMM8GDMNIWPEYKNRTIF
DITNNLSIAILALRPSDEGTYECVVLKYEKDAFKREFILAEVTLSVKADFPTPSISDFEIPTSNI
RRnCSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKY
GHLRVNQTFNWNTTKQEHFPDN][GSGG][EVQLVESGGGLVQPGGSLRLSCAASGFTFSDS
WIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDT
AVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTTPAVLQSSGLYSLSSVYTVPSSSLGTQTYICNVNHKPSNTKVD
KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPB PKDTLMISRIPEVTCVWDVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYASTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KnSKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK] (SEQ ID NO: 118) hCD80-Iinker-anti-PD-Ll(Atezolizumab)Lc N-terminal fission (Format H)
[VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRH
FDITNNLSIVILALRPSDEGTYECWLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTS
MRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFN TTNHSFMCLI KYGHLRVNQTFNWNTTKQEHFPDN][GSGG][DIQMTQSPSSL8ASVGDRVTTTCRASQDVS TAVAW Y QQKPGKAPKLLI YS ASFL Y S GVPSRF S GS GS GTDFTLTI S SLQPEDF AT YY CQ Q Y
LYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSILTLSKADYEKHKVYACEVTHQGLSSPV SFNR GEC] (SEQ m NO: 119) schCDSO dimer/anti-PD-Ll(Atezolizumab) Lc N-terminal fusion (hCD8Q-Iinker-faCD80- linker-anti-PD-Ll LC) (Format I)
[ATHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLIMMSGDMNIWPEYKNRH FDITNNLSIVILALRPSDEGTYECVY KYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTS NIRRIIC S TS CJGFPEPHLS WLENGEELN AINTT V S QDPETEL Y A VS SKLDFNMTTNHSFMCLI K YGHLRVNQ TEN WNTTKQEHF PDN] [ GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GG GGSGGGGS] [VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNI WPEYKNRTIFDITNNLSIVILALRPSDEGTYECWLKYEKDAFKREHLAEVTLSVKADFPIP SISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMT TNHSFMCLIKYGHLRVNQIFNWNTTKQEHFFDN][GSGG][DIQMTQSPSSLSASVGDRVTI TCRASQDVSTAYAWYQQKPGKAPKLLIYSASFLYSGWSRFSGSGSGTDFTLTISSLQPEDF ATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA KVQWKVDNALQSGNSQESVIEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC] (SEQ ID NO: 120) anti-PD-Ll He (AtezoIizumah)hFc N297A (Format I)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYY
ADSVKGRFTISADTSKNTAYLQVINSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY
SLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL
FPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRV
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKHSKAKGQPREPQVYTLPPSEDELTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 121)
CLDN18.2 Abl2 HC-linker-hCD80 C-terminal fusion (Format F)
[EVQLVESGGGLVKPGGSRKLSCAVSGFTFSDYGMYWVRQAPEKGLEWVAYISSGSSTIYY
ADTVKGRFTMSRDNAKNTLFLQMTSLRSEDTAMYYCARIARGNAMDYWGQGTSVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY
SLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL
FPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYYDGVEVHNAKTKPREEGYNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKllSKAKGQPREPQVYTLPPSRDELITKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGK] [GGGGSGGGGSGGGGSVIH][VTKEVKEVATLSCGHN
VSVEELAQTRIYWQKEKB MVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTY
ECWLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNERRIICSTSGGFPEPHLSWLEN
GEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHF
PDN] (SEQ ID NO: 122)
Abl2VL (Format F)
DIVMrQSPSSLTVIPGEKVTMSCKSSQSLFNSGNQRNYLAWYQQKPGQPPKLLIYWASTRE SGVPDRFTGSGSGTDFH^HSSVQAEDLAVYYCQNDYFYPLTFGAGTKLELKRTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 123) HnCLDN18.2 Ab.v.24 HC-linker-hCD80 C-terminal fusion (Format F)
[EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYAMSWIRQAPGKGLEWVSTmGGTYTYYP
DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQVYGNSFAYWGQGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRW
SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPGK][GGGGSGGGGSGGGGS][VIHVTKEVKEVATLSCGHNVS
VEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYEC
WLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGE
ELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPD
N] (SEQ ID NO: 124)
HuCLDN18,2 Ab.v.24 Lc (Format F)
DiyMTQSPDSLTVSLGERAnSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFS^SSLQAEDV AVYYCQNNYIYPFTFGQGTKVEIKRIY AAPSVFIFP PSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 125)
Anti-CLDN 18.2 Abl scFv (Anti-CLDN 18.2 Abl VH-iinker- Anti-CLDN 18.2 Abl VL-Iinker- hlgGl Fc Hole) (Format J)
[EVQLVESGGGLVKPGGSRKLSCAASGFTFSDYGMFWVRQAPEKGLEWVGYISSGSSNIYY ADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARIARGNAMDYWGQGTSVTVSSJ[ GGGGS GGGGS GGGGS ] [ DI VMTQ SPS SLT VT AGEK VTMS CKS S Q S LLN S GN QKN YLT WY Q QKPGQPPKLUYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCQNDYFYPLTFG AGTKLELK] [GS] [DKTHTCPPCPAPELLGGPSVFLFPPKPB DTLMISRTPE VTCWVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREl^QVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENN YKTIPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKl (SEQ ID NO: 126)
Anti-CLDN 18.2 AM2 scFv (Anti-CLDN 18.2 Abl2 VH-iinker-· Anti-CLDN 18.2 Abl2 VL- linker-hlgGl Fc Hole) (Format J)
[EVQLVESGGGLVKPGGSRKLSCAVSGFTFSDYGMYWVRQAPEKGLEWVAYISSGSSnYY
ADTVKGRFTMSRDNAKNTLFLQMTSLRSEDTAMYYCARIARGNAMDYWGQGTSVTVSS]
[GGGGS GGGGS GGGGS ] [DIVMTQ SPS SLT VTPGEKVTMS CKS S Q SLFNS GNQRNYLA WY Q
QKPGQPPKLLIYWASTRESGVTDRFTGSGSGTDFTLHSSYQAEDLAVYYCQNDYFYPLTFG
AGTKLELK][GS]PKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKYSNKAL
PAPIEf TISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK]
(SEQ m NO: 127) hCDSO-Iinker-hlgGl Fc Knob (Format A,J)
P^THVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIF DITNNLSIYTLALRPSDEGTYECWLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNI RRnCSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKY GHURVNQTFNWNTTKQEHFPDN] [GS] [DKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK] (SEQ ID NO: 128)
DVD form Anti-PD-Ll Lc (Atezo) (anti-PD-Ll VL-linker-anti-PD-Ll HC) (Format D)
[DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSR
FSGSGSGTDFTI.TISSLQPEDFATYYCQQYI-,YHPATFGQGTK\T.IK][GGGGSGG][DIQMTQS
PSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGT
DFTLTIS SLQPEDF ATYY CQQ UILΉR ATFGQGTK VEIKRTVA APS VFIFPPSDEQLK SGT A S V
VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
ACEVTHQGLS SPVTKSFNRGE] (SEQ ID NO: 129)
DVD form Anti-PD-Ll He. (Atezo) hole N297A (anti-PD-Ll VH-linker-anti-PD-Ll HC)
(Format D)
[EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTY
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS
][GGGGSGG][EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAW
ISPYGGSTYYADSVKGRFHSADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWG
QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP
ELLGGPSWLFPPKPKDTLMISRTPEVTC VDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
RDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK] (SEQ ID NO: 130)
Anti-CLDN 18.2 Ab3 He hFc Hole (Format A,B)
EVQLVESGGALVKSGGSLRLSCAASGFTFSNNAMSWIRQTPEKRLEWVAHIIGGTYTYY
PDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAFYYCARQVYGNSFAYWGQGTLVSVSAAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT PREEQYNSTYRW
SVLTVLHQDWLNGKEYKCKVSNKALPAPIEraSKAKGQPREPQVYTLPPSREEMTKNQVS
LSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 131)
Anti-CLDN 18.2 Ab3 Lc (Format A,3B)
DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRE
Figure imgf000046_0001
PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 132)
HuCLDN 18.2-v.24 He hFc Hole (Format A,B)
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYAMSWIRQAPGKGLEWVSTmGGTYTYYP
DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQVYGNSFAYWGQGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRW
SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS
LSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 133)
HuCLDN 18.2-v.24 Lc (Format A,B)
DIVMT Q SPD SI. A V SLGER AΉN CKS S Q SLLN S GN QKNYLTWY QQKPGQPPKLLI YWAS TRE SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNNYIYPFTFGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 134) sc-hCDSO dimer hFc Knob (bCD80-Iiuker-hCD80~bIgGl Fc) (Format B, D)
[VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQliKKMVLTMMSGDMNIWPEYKNRTIF
DITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNI
RRnCSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKY
GHLRVNQTFNWNTTKQEHFPDN][GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS
GGGGS ] [ VIHVTKEVKE V A TLS CGHNV S VEEL AQTRI YW QKEKKM\T,TMMS GDMNTWPE Y
KNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPIPSISDFE
IPTSNIRRnCSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFM
CLIKYGHLRVNQTFNWNTTKQEHFPDN][DKTHTCPPCPAPELLGGPSWLFPPKPKDTLMIS
RTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDW
LNGKEYKCKVSNKALPAPIEKHSKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN
HYTQK SLS LSPGK] (SEQ ID NO: 135)
IV. Methods of Treatment
[0163] The PD-Ll antibodies and multi functional polypeptides of the disclosure, and the pharmaceutical compositions comprising the LI antibodies and multifunctional polypeptides of the disclosure are useful for therapeutics, to treat or delay progression of a disease or disorder.
[0164] Accordingly, provided herein are methods of treating a disease or disorder, comprising administering to subject in need thereof a therapeutically effective amount of any one of the PD-Ll antibodies, multifunctional polypeptides, or pharmaceutical compositions provided herein. The disease or disorder may be selected from cancer, infectious disease, or inflammatory disease. The cancer may comprise a liquid tumor, or solid tumor. The disease or disorder may be associated with aberrant activity of a disease-associated antigen. For example, the disease or disorder may be associated with aberrant PD-L1 activity, or with aberrant Claudin 18.2 activity.
[0165] The administration of any of the PD-L1 antibodies, multifunctional polypeptides, or pharmaceutical compositions of the disclosure may be carried out intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, mtraorbitally, by implantation, by inhalation, mtratheeally, intraventrieulariy, or mtranasally. A therapeutically effective amount may be administered for the treatment of the disease or disorder and the appropriate dosage of the may be determined based on the type of disease or disorder to be treated, the type of the therapeutic, the severity and course of the disease or disorder, the clinical condition of the subject, the subject’s clinical history' and response to the treatment, and the discretion of the attending physician. In an exemplary embodiment, any one of the PD-L1 antibodies, multifunctional polypeptides, or pharmaceutical compositions provided herein is administered intravenously.
[0166] For in vivo administration of the PD-L1 antibodies, multifunctional polypeptides, or pharmaceutical compositions of the disclosure normal dosage amounts may vary from about 1 ng/kg up to about 1000 mg/kg of an subject’s body weight or more per day, depending upon the route of administration. For repeated administrations over several days or longer, depending on the severity of the disease or disorder to be treated, the treatment may be sustained until a desired suppression of symptoms is achieved. Dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the physician wishes to achieve. Progress of the therapy may be monitored by conventional techniques.
[0167] The PD-Ll antibodies, multifunctional polypeptides, or pharmaceutical compositions of the disclosure may be administered as a part of a combination therapy with another agent.
V. Kits and Articles of Manufacture
[0168] The present disclosure provides kits comprising the PD-Ll antibodies, multifunctional polypeptides and pharmaceutical compositions of the disclosure.
[0169] In some embodiments, the kits may contain a pharmaceutically acceptable excipient, an instruction manual and any combination thereof. In some embodiments, the kits comprise any one or more of the therapeutic compositions described herein, with one or more pharmaceutically acceptable excipients.
[0170] The present disclosure also provides articles of manufacture comprising any one or more of the PD-L1 antibodies, multifunctional polypeptides, pharmaceutical compositions, and kits described herein. Examples of an article of manufacture include vials (e.g. sealed vials).
EXAMPLES
[0171] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed.
EXAMPLE 1 - Phase Display and PD-L1 Antibody Screening
[0172] The modified pHENl vector was used to make a fully synthetic ScFv (single chain antibody fragment) phage library. The antibody library was taken through five rounds of panning on PD-L1 (eetodomain)-His protein (according to J. Mol. Biol. (2004) 338, 299 310 with some modification). The top 17 positive clones were selected and sequenced. The sequences are listed in Tables 1-11.
EXAMPLE 2 - Human PD-L1 (ectodomain)-His ELISA binding assay
[0173] The scFvs were converted into full-length antibodies by using human IgG4 conservation sequences. The genes of different antibodies were cloned and transfected into Expi-CHO cells (Thermofisher), then were purified by AKTA Explorer protein purification system. The plate was coated with 1 pg/ml of recombinant PD-L1 (ectodomain)-His from Sino Biological, Inc. (Cat. No. 10084-H08H) at 4°C overnight. The next day, the plate was washed with 200m1 per well washing buffer (0.05% Tween-20 in PBS, pH7.4) for 3 times. Then the assay plate was blocked with 200 mί of blocking buffer (2% BSA in Washing Buffer) for 1 hr at 37°C and washed again with washing buffer for 3 times. Series dilution of different antibodies were added. After incubation at 37°C for Ihr, the plate was washed three times, and HRP goat anti-human IgG-Fc secondary antibody was added to each well and incubate at 37°C for lh. The plate was then washed, developed and read at 450 nm and 630nm. The ECso was calculated by GraphPad software. Table 12 provides ECso of the antibodies listed in Table 9. Among these antibodies, PD-2, PD-3, PD-4, PD-15, PD- 16, and PD- 18 had better binding affinities than Atezolizumab.
Table 12
Figure imgf000050_0001
EXAMPLE 3 - Determination of PD-LJ Antibodies binding affinities by FACS [0174] Flp-in 293 cells overexpressing human PD-L1 were cultured in DMEM medium supplied with 10% FBS at 37°C in 5% C02. The cells were harvested, washed 3 times with PBS. After fixed with 4% PFA for 20 min, the cells were pelleted and washed with PBS to ensure that there was no PFA left. The cells were re-suspended in PBS with 3% BSA and incubated at 4°C overnight. Eleven-point dilution series of different antibodies in PBS with 3% BSA were added into different wells. After incubated at 4°C for 2 hours, the cells were pelleted and washed again with PBS. The fluorescence labeled secondary antibody was added into the wells at 4°C for 1 hour. After removal of secondary antibody, the cells were washed twice with PBS, and mean fluorescence intensity (MFI) was measured by a flow cytometer. The ECso w¾s calculated by GraphPad software, as shown in FIG 20. PD-2, PD-3, PD-4, PD-15, and PD- 18 were chosen for further analysis. EXAMPLE 4 - PD- LI antibodies competitive inhibition of PD-I binding to Fip-in 293 ceil surface overexpressing PD-L1 by FACS
[0175] The Flp-in 293 cells which overexpressing human PD-L1 were cultured in DMEM medium supplied with 10% FBS at 37 °C in 5% C02. The cells were harvested, washed, fixed and re-suspended as described in Example 3. After blocked in PBS with 3% BSA at 4°C overnight, lOug/rnl human biotin labeled PD-1 (Sino Biological, Inc) in PBS with 3% BSA was added into the wells. After incubated at 4°C for 2 hours, the cells were pelleted and washed again by PBS. Eleven-point dilution series of different PD-L1 antibodies in PBS with 3% BSA were added into different wells. The cells were pelleted and washed with PBS, and the fluorescence labeled streptavidin was added into the wells at 4CC for 1 hour. After removal of free fluorescence labeled streptavidin, the cells were washed twice with PBS, and mean fluorescence intensity (MFI) was measured by a flow cytometer. The result is shown in FIG. 21.
EXAMPLE 5 - Gene Synthesis, Expression Construct and Expression/Purification of hCD80- Fc/anti-PD-Ll and hCD80/Anti-CLDN18.2 Multifunctional polypeptides [0176] hCD80-Fc/anti-PD-Ll and hCD80/anti-CLDNl 8.2 multifunctional polypeptide expression cassettes are depicted in FIG. 1 A and FIG. 2. The anti-PD-Ll and anti-CLDNl 8.2 refer to the PD- L,i and Claudm 18.2 antigen-binding domains of the disclosure. The cassettes were generated by de novo gene synthesis and were cloned into a pcDNA3.4 expression vector (Thermo Fisher Scientific). [0177] The resulting pcDNA3.4 expression vectors containing polynucleotides encoding the hCD80~Fe/anti-PD-Ll (Format A) (8EQ ID. 113, 114 and 115), sc-hCDSO dimer-Fc/anti-PD-Ll (Format B) (SEQ ID. 116, 114 and 115), sc-hCD80 dimer-Fc/anti-PD-Ll (DVD) (Format D) (SEQ ID. 116, 129 and 130), Anti-PD-Ll -hCD80 (Format F) (SEQ ID. 115 and 117), hCD80-anti-PD-Ll (Format H) (SEQ ID. 118 and 119), and sc-hCD80 dimer-anti-PD-Ll (EC) (Format I) (SEQ ID. 120 and 121) and hCD80/anti-CLDN18.2 (Format A) (SEQ ID. 128, 131 and 132) (SEQ ID. 128, 133 and 134), se-hCD80 dimer-Fc/anti-CLDN18.2 (Format B) (SEQ ID. 135, 131 and 132) (SEQ ID. 135, 133 and 134), Anti-CLDNl 8.2-hCD80 (Format F) (SED ID. 122 and 123) (SEQ II). 124 and 125), and hCD80-Fc/anti-CLDNl 8.2 scFv-Fc (Format J) (SEQ ID. 128 and 126) (SEQ ID. 128 and 127) depicted in FIG. 1A and FIG. 2 were transfected into Expi293F cells using ExpiFectamine 293 transfection reagent (Thermo Fisher Scientific). ExpiFectamine 293 Transfection Enhancer 1 and Enhancer 2 were added to the well 20 hours after transfection. The cultures wrere incubated at 37 °C in humidified incubator at 75% humidity supplied with 5% CO?.. The transfected culture w¾s harvested 6 days post transfection. All the multifunctional polypeptides were purified using GE Healthcare Protein A HP SpinTrap column by incubating the supernatants and the resin at room temperature for 4 minutes. The column was washed with sodium phosphate buffer, pH7.2 and eluted with 100 mM glycine-HCl, pH 3.0. The eluents were neutralized using 1.0 M Tris-HCl, pH 9 0. The purified proteins were dialyzed into PBS buffer at pH7.2 and sterile filtered through 0.2 pm membrane.
[0178] The SDS-PAGE analyses of the multifunctional polypeptides are summarized FIGS. 3- 10. FIG. 3 shows a SDS-PAGE analysis of se-hCD80 dimer-Fc/anti-PD-Ll (Format B) (SEQ ID. 116,114 and 115 and hCDBO-Fc/anti-PD-Ll (Format A) (SEQ ID. 113, 114 and 115) multifunctional polypeptides. FIG. 4 shows a SDS-PAGE analysis of sc-hCD80 dimer-F c/anti -CLDN 18.2 Ab3 (Format B) (SEQ ID. 135, 131 and 132) and se~hCD80 dirner-Fc/FIuCLDN 18.2-v.24 (Format B) (SEQ ID. 135, 133 and 134) multifunctional polypeptides. FIG. 5 shows a SDS-PAGE analysis of hCD80~Fe/anti~CLDN 18.2 Ab3 (Format A) (SEQ ID. 128, 131 and 132) and hCDSO-Fc/FIuCLDN 18.2-v.24 (Format A) (SEQ ID. 128, 133 and 134) multifunctional polypeptides. FIG. 6 show's a SDS- PAGE analysis of hCD80-Fc/anti-CLDN 18.2 Abl scFv-Fc (Format J) (SEQ ID. 128 and 126) and hCD80-Fc/anti-CLDN 18.2 Abl2 scFv-Fc (Format J) ) (SEQ ID. 128 and 127) multifunctional polypeptides. FIG. 7 shows a SDS-PAGE analysis of anti-CLDN 18.2 Abl2-hCD80 Format F (SED ID. 122 and 123) and HuCLDN18.2-y.24-hCD80 (Format F) (SEQ ID. 124 and 125) multifunctional polypeptides. FIG. 8 shows a SDS-PAGE analysis of anti-PD-Ll-hCD80 (Format F) (SEQ ID. 115 and 117) multifunctional polypeptides. FIG. 9 shows a SDS-PAGE analysis of sc-hCD80 dimer- Fc/anti-PD-Ll (DVD) (Format D) (SEQ ID. 116, 129 and 130) multifunctional polypeptides. FIG. 10 shows a SDS-PAGE analysis of hCD80-anti-PD-Ll (Format H) (SEQ ID. 118 and 119) and sc- hCD80 dimer-anti -PD-L1 (LC) (Format I) (SEQ ID. 120 and 121) multifunctional polypeptides. [0179] All the multifunctional polypeptides w¾re purified to near 90% purity. EXAMPLE 6 -Determination of hCD80-Fc/anti-PD-Ll Containing Multifunctional Polypeptide Binding Affinities to PD- L 1 and CTLA-4 by ELISA
[0180] To test binding affinity by ELISA, ELISA plates were first coated with 1 ug/niL human CTLA-4 (recombinant His Tag from Sino Biological) at 4 °C overnight. The next day, the human CTLA-4 solution was removed and the plates were blocked with 1% BSA at room temperature for 1 hour. After removing the 1% BSA solution, the plates were washed with PBST (phosphate buffered saline with 005% of Tween-20) three times. The samples of the purified hCD80-Fc/anti-PD-Ll multifunctional polypeptide (Format A) (SEQ ID. 113, 114 and 115) for this binding assay were added at 100 uL/ ell in duplicate with 1:3 serial dilutions starting at the concentration of 4 ug/mL and incubated at room temperature for 2 hours with agitation. After washing with PBST three times, a secondary' antibody, 100 uL/weil of 1:10,000 dilution of HRP-anti-hlgG, was added and incubated at room temperature for 1 hour with agitation. The plates were washed three times with PBST, and a 100 uL/well of TMB substrate was added. The plates were incubated until the color was developed. The stop-solution was added to stop the reaction. The plates were read at OD450.
[0181] Similar ELISA experiments were performed for human PD-L1 recombinant protein, except the plates were coated with 2 ug/mL of recombinant human PD-L1 protein (recombinant His Tag from Sino Biological) and the dilutions of bCD80-Fc/anti-PD-Ll multifunctional polypeptide (Format A) (SEQ ID. 113, 114 and 115) were started at 20 ug/mL.
[0182] The ECso values of hCD80-Fc/anti-PD-Ll multifunctional polypeptide (Format A) (SEQ ID. 113, 114 and 115) binding to CTLA-4 or PD-L1 were determined by comparing to ECso of CD80- Fc on each plate. The ECso (ug/mL) is the concentration or value of a protein that gives half maximal response in the binding assay.
[0183] The results of binding affinity assays by ELISA are presented in FIG. 11. FIG. 11 is an ELISA showing the binding affinities of hCD80-Fc/anti-PD-Ll multifunctional polypeptide (Format A) (SEQ ID. 113, 114 and 115) to recombinant protein CTLA-4 or PD-Ll as compared to hCD8Q-Fc fusion protein. The ECso of the hCD8Q-Fc/anti-PD-Ll multifunctional polypeptide binding to the recombinant PD-Ll polypeptide was 0.09 ug/mL as compared to those of CD80-Fc polypeptide and anti-PD-Ll antibody binding to the same target were 4.6 ug/mL and 0.004 ug/mL, respectively. ECso of hCD80-Fc/anti-PD-Ll multifunctional polypeptide (Format A) (SEQ ID. 113, 114 and 115) binding to the recombinant CTLA-4 polypeptide was 0.93 ug/mL as compared to that of CD80-Fc polypeptide binding to the same target was 0.035 ug/mL.
EXAMPLE 7 - Determination of hCD80/anti-PD-Ll and hCD80/anti-CLDN18.2 Containing Multifunctional Polypeptides Binding Affinities by FACS Cell Surface Binding Assay by FACS:
[0184] The binding affinity (ECso) of hCDBO-Fc/anti-PD-Ll (Format A) (SEQ ID. 113, 114 and 115), sc-hCD80 dimer-Fc/anti-PD-Ll (Format B) (SEQ ID. 116, 114 and 1 15), sc~hCB80 dimer- Fc/anti-PD-Ll (DVD) (FormatD) (SEQ ID. 116, 129 and 130), Anti-PD-LI-hCD80 (Format F) (SEQ ID. 115 and 117), hCDBO-anti-PD-Ll (Format H) (SEQ ID. 118 and 119), and sc-hCDBO dimer-anti- PD-L1 (LC) (Format I) (SEQ ID. 120 and 121), and anti-CLDNl 8.2-hCD80 (Format F) (SED ID. 122 and 123) (SEQ ID. 124 and 125), and hCD80-Fc/anti-CLDNl 8.2 scFv-Fc (Format J) (SEQ ID. 128 and 126) (SEQ ID. 128 and 127) multifunctional polypeptides to Flp-in 293 cells overexpressing CD28, CTLA-4 and PD-LI (Flp-in 293 CD28, Flp-in 293 CTLA4 and Flp-in 293 PD-L1 cells) were determined by FACS. The FACS binding assays were performed as follows: Flp-in 293 CD28, Flp- in 293 CTLA-4 and Flp-in 293 PD-LI cells were stained with a serial diluted hCD80/anti-PD-Ll and hCDBO/anti-CLDNl 8.2 containing multifunctional polypeptides on ice for 1 hour, (i) with concentrations of 100 ug/rnl, 30 ug/ml, 10 ug/rnl, 3 ug/mi, 1 ug/ml, 0.3 ug/ml, 0.1 ug/ml, and 0.03 ug/ml for either Flp-in 293 cells overexpressing CD28 and PD-LI and (ii) with concentrations of 3 ug/ml, 1 ug/ml, 0.3 ug/ml, 0.1 ug/ml, 0.03 ug/ml, 0.01 ug/ml, 0.03ug/ml, and 0.001 ug/ml for Flp-in 293 cells overexpressing CTLA-4. The cells were washed with staining buffer (PBS + 2% fetal bovine serum) to remove free multifunctional polypeptides, and then stained with AlexF!uor 488-conjugated anti-human IgG antibody for 30 mm on ice. The cells were washed and analyzed by FACS.
[0185] The binding affinity (ECio) of anti-CLDNl 8.2-hCD80 (Format F) (SED ID. 122 and 123) (SEQ ID. 124 and 125), and hCD80-Fc/anti-CLDN18.2 scFv-Fc (Format J) (SEQ ID. 128 and 126) (SEQ ID. 128 and 127) multifunctional polypeptides to HEK293 cells overexpressing CLDN18.2 and KATQ III cells endogenously expressing CLDN18.2 was determined by FACS. The FACS binding assays were performed as follows: cells were stained with a serial diluted hCD80/anti-CLDN18.2 containing multifunctional polypeptides on ice for 1 hour, with concentrations of 10 ug/ml, 3 ug/ml, 1 ug/mi, 0.3 ug/mi, 0.1 ug/mi, 0.03 ug/mi, 0.01 ug/mi, and 0 03ug/mi The ceils were washed with staining buffer (PBS + 2% fetal bovine serum) to remove free multifunctional polypeptides, and then stained with AlexFiuor 488-conjugated anti-human IgG antibody for 30 min on ice. The cells were washed and analyzed by FACS.
[0186] The results of cell-surface binding affinity assays by FACS were presented in FIGS. 12-16 along with top MFI (Mean Fluorescence Intensity) values and ECso (ug/ml) values of each tested multifunctional polypeptide. The ECso (ug/ml) is the concentration or value of a protein that gives half maximal response in the binding assay.
[0187] FIG. 12 showed the binding affinities of a hCD80/anti-PD-Ll (Format A) (SEQ ID. II 3, 114 and 115) containing multifunctional polypeptide to Flp-in 293 cell surface overexpressing CD28, CTLA-4 or PD-L1 by FACS. hCD80-Fc/anti-PD-Ll (Format A) (SEQ ID 113, 114 and 115) multifunctional polypeptide and hCB80-Fc showed similar binding affinity to Flp-in 293 CD28 cells. ECso of hCD80~Fc/anti-PD-Ll (Format A) (SEQ ID. 113, 114 and 115) multifunctional polypeptide binding to Flp-in 293 CTLA4 cells was 0.6 ug/mL as compared to that of hCD80-Fe polypeptide binding to the same cells was 0.02 ug/rnL. hCD80-Fc/anti-PD-Ll (Format A) (SEQ ID. 113, 114 and 115) multifunctional polypeptide and anti-PD-Ll antibody showed similar binding affinity to Flp-in 293 PD-L1 cells.
[0188] FIG. 13 showed the binding affinities of a sc-hCD80 dimer-Fc/anti-FD-Ll (Format B) (S EQ ID. 116, 114 and 115) containing multifunctional polypeptide to Flp-in 293 ceil surface overexpressing CD28, CTLA-4 or PD-L1 by FACS. ECso of sc-hCD80 dimer-Fc/anti-PD-Ll (Format B) (SEQ ID. 116, 114 and 115) multifunctional polypeptide binding to Flp-in 293 CD28 cells was 53.2 ug/mL as compared to that of hCD80-Fc polypeptide binding to the same cells was 18 ug/mL. ECso of sc-hCD80 dimer-Fc/anti-PD-Ll (Format B) (SEQ ID. 116, 114 and 115) multifunctional polypeptide binding to Flp-in 293 CTLA4 cells was 0.53 ug/rnL as compared to that of hCD80-Fc polypeptide binding to the same cells was 0.02 ug/mL. ECso of sc-hCDSO dimer-Fc/anti-PD-Ll (Format B) (SEQ ID. 116, 114 and 115) multifunctional polypeptide binding to Flp-in 293 PD-L1 cells was 3.0 ug/mL as compared to that of hCD80-Fc polypeptide binding to the same cells was 0.5 ug/mL.
[0189] FIG. 14 showed the binding affinities of a sc-hCD8Q dimer-Fc/anti-PD-Ll (DVD) (Format D) (SEQ ID. 116, 129 and 130), an anti-PD-Ll -hCD8Q (Format F) (SEQ ID. 115 and 117), a hCD80- anti-PD-Ll (Format H) (SEQ ID. 118 and 119), and a sc~hCD80 dimer-anti-PD-Ll (LC) (Format I) (SEQ ID. 120 and 121) containing multifunctional polypeptides to Flp-in 293 cell surface overexpressmg CD28, CTLA-4 or PD-L1 by FACS.
[0190] FIG. 14, Panel A showed the ECso of sc-hCD80 dimer-Fc/anti-PD-Ll (DVD) (Format D) (SEQ ID. 116, 129 and 130), an anti-PD-Ll-hCD80 (Format F) (SEQ ID. 115 and 117), a hCD80- anti-PD-Ll (Format H) (SEQ ID. 118 and 119), and a sc-hCD8Q dimer-anti-PD-Ll (LC) (Format I) (SEQ ID. 120 and 121) multifunctional polypeptide binding to Flp-in 293 CD28 cells were 528.7 ug/'mL, 4572 ug/mL, 8.3 ug/mL and 19.3 ug/mL respectively, ml, as compared to that of hCD80-Fc polypeptide binding to the same ceils was 11.0 ug/mL.
[0191] FIG. 14, Panel B showed the ECso of sc-hCD80 dimer-Fc/anti-PD-Ll (DVT)) (Format D) (SEQ ID. ! 16, 129 and 130), an anti~PD-Ll-liCD80 (Format F) (SEQ ID 115 and 117), a hCD80~ anti-PD-Ll (Format H) (SEQ ID. 118 and ! 19), and a se~hCD80 dimer-anti-PD-Ll (LC) (Format I) (SEQ ID 120 and 121) multifunctional polypeptide binding to Flp-in 293 CTLA4 cells were 025 ug/raL, 3.34 ug/mL, 0.07 ug/mL and 0.23 ug/mL respectively, mL as compared to that of hCD80-Fc polypeptide binding to the same ceils was 0.02 ug/mL.
[0192] FIG. 14, Panel C showed the ECso of sc-hCD80 dimer-Fc/anti-PD-Ll (DVD) (Format D) (SEQ ID. 116, 129 and 130), an anti-PD-Ll -hCD80 (Format F) (SEQ ID. 115 and 117) a hCD80- anti-PD-Ll (Format H) (SEQ ID. 118 and 119), and a sc-h€D80 dimer-anti-PD-Ll (LC) (Format I) (SEQ ID. 120 and 121) multifunctional polypeptide binding to Flp-in 293 PD-L1 cells were 1.3 ug/mL, 2.3 ug/mL, 1.6 ug/mL and 1.4 ug/mL respectively, mL as compared to that of anti-PD-Ll binding to the same cells was 0.6 ug/mL.
[0193] FIG. 15 showed the binding affinities of anti-CLDN18.2-hCD80 (Format F) (SED ID. 122 and 123) (SEQ ID. 124 and 125), and hCD80-Fc/anii-CLDN18.2 scFv-Fc (Format J) (SEQ ID. 128 and 126) (SEQ ID. 128 and 127) multifunctional polypeptides to HEK 293 cell surface overexpressing CLDN18.2 or KATO Ill cell surface endogenously expressing CLDN18.2 by FACS. The ECso of CDSO/anti-CEDNl 8.2 containing multifunctional polypeptides showed four to eight fold activity loss as compared to that of anti-CLDN 18.2 antibodies.
[0194] FIG. 16 showed the binding affinities of anti-CLDN18.2-hCD80 (Format F) (SED ID. 122 and 123) (SEQ ID. 124 and 125), and hCD80-Fc/anti-CLDN18.2 scFv-Fc (Format J) (SEQ ID. 128 and 126) (SEQ ID. 128 and 127) multifunctional polypeptide to Flp-in 293 cell surface overexpressing CD28, CTLA-4 or PD-Ll by FACS. hCD80-Fc/anti-CLDN18.2 Ab ScFv-Fc (Format J) (SEQ ID. 128 and 126) (SEQ ID. 128 and 127) multifunctional polypeptides and hCD80-Fc showed similar binding affinity to Flp-in 293 CD28 cells. ECso of hCD80-Fc/anti-CLDN18.2 Abl ScFv-Fc (Format I) (SEQ ID. 128 and 126) and hCD80-Fc/anti-CLDN18.2 Abl2 ScFv-Fc (Format J) (SEQ ID. 128 and 127) multifunctional polypeptides binding to Flp-in 293 CTLA4 cells were 0.40 ug/mL and 0.64 ug/mL, respectively, as compared to that of hCD80-Fc polypeptide binding to the same cells w¾s 0.02 ug/mL. ECso of hCD80-Fc/anti-CLDN18.2 Abl ScFv-Fc (Format J) (SEQ ID. 128 and 126) and hCD80-Fe/anti-CLDNl 8.2 Abl2 ScFv-Fc (Format J) (SEQ ID 128 and 127) multifunctional polypeptides binding to Flp-in 293 PD-L1 cells were 172.4 ug/mL and 75.6 ug/mL, respectively, as compared to that of hCD80-Fc polypeptide binding to the same cells was 9.3 ug/mL. Anti-CLDN 18.2 Ab-hCD80 (Format F) (SED ID. 122 and 123) (SEQ ID. 124 and 125) multifunctional polypeptides showed loss of binding activities to Flp-in 293 CD28 cells ECso of Anti-CLDN 18.2 Abl2-hCD80 (Format F) (SED ID. 122 and 123) and HuCLDN18.2-v.24.hCD80 (Format F) (SED ID. 124 and 125) multifunctional polypeptides binding to Flp-in 293 CTLA4 cells were 2.67 ug/mL and 6.56 ug/mL, respectively, as compared to that of hCD80-Fc polypeptide binding to the same cells was 0.02 ug/mL. Anti-CLDN 18.2 Ab-hCD80 (Format F) (SED ID. 122 and 123) (SEQ ID. 124 and 125) multifunctional polypeptides and hCD80~Fc showed similar binding affinity to Flp-in 293 PD~ LI cells.
EXAMPLE 8 - hCD80-Fc/anti-PD-Ll and hCD80-Fc/anti-CLDN18.2 Multifunctional polypeptides m T-Cell Activation
Anchorage qfhCDSO moiety on PD-Ll or CLDN18.2 expression cells can provide co-stimulaiory activity in T cell activation
(0195 j Jurkat cell modified to express luciferase reporter gene under the control of IL-2 promoter was obtained from Promega. The activation of IL-2 promoter activity requires two signals, signal 1 from TCR/CD3 which is provided from anti-CD 3 antibody in the assay, signal 2 from CD28 co stimulation which is provided by various testing molecules. Anti-CD28 agonist antibody CD28.2 (BioLegend) was used as positive control, while culture medium was used as negative control. Testing molecules were hCD8G-Fc, anti-PD-Ll (Tercentriq, Genentech), hCD80-Fc/anti-PD-Ll (Format A) (SEQ ID. 113, 114 and 115). [0196] To setup the assay, 50,000 Flp-in 293 PD-L1 expression cells in 100 uL/well were seeded in flat-bottom opaque 96-well plate, incubated at 37°C, 5% CCb incubator overnight. The culture medium was completely dumped out right before addition of other components on the plate. Jurkat effector cells (IL-2 reporter cell) were suspended in RPMI-1640 with 10% Fetal bovine serum at 2 million/niL with 1 ug/mL of anti-CD3 antibody. Fifty microliter per well of Jurkat effector cells were transfer to the plate growing HEK-293 PD-L1 ceils. The testing molecules w¾re prepared in culture medium at 30 ug/mL with 1:3 serial dilutions and 25 uL/well of samples were transferred on to the plate in duplicates. The plate was incubated at 37°C, 5%CC incubator for 6 h. To detect IL-2 promoter activity activated by testing molecules, Bio-G!o™ Luciferase Assay Substrate (Promega) were thawed and equilibrate to room temperature. Seventy-five microliter of Bio-Gio™ Luciferase Assay Substrate were added to each well of the assay plates. The plate was mixed briefly, and the count of firefly luminescence was measured using a !uminometer luminescence plate reader.
[0197] A similar assay was performed to evaluate hCD80-Fc/anti-CLDN 18.2 (Format A) (SEQ ID. 128, 131 and 132) (SEQ ID. 128, 133 and 134) activity, except Flp-in 293 PD-L1 expression cell line was replaced with HEK293 CLDN18.2 expression cell line.
[0198] The data for hCD80-Fc/anti-PD-Ll (Format A) (SEQ ID. 113, 114 and 115) and hCD80~ Fc/anti-CLDNl 8.2 (Format A) (SEQ ID. 128, 131 and 132) (SEQ ID. 128, 133 and 134) were summarized in FIG. 17 and FIG. 19, respectively.
[0199] FIG. 17 showed that the binding of the multifunctional polypeptide comprising anti-PD-Ll and hCD80 moiety to PD-Ll on Flp-in 293 PD-L1 cells provided co-stimulatory signal required for IL-2 promoter activation. In absence of Flp-in 293 PD-Ll cells, the same multifunctional polypeptide did not activate the IL-2 promoter.
[0200] FIG. 19 showed that the binding of the multifunctional polypeptide comprising anti- CLDN18.2 and hCD80 moiety to CLDN18.2 on FIEK293-CLDN18.2 cells provided co-stimulatory signal required for IL-2 promoter activation. In absence of HEK293-CLDN18.2 cells, the same multifunctional polypeptide failed to activate the IL-2 promoter. The two anti-CLDN18.2 antibodies lacking hCDBO moiety did not showr activity in this assay. The multifunctional polypeptide comprising anti-PD-Ll and hCD80 moiety w¾s inactive in this assay due to the absence of the matching ligand. PD-1/PD-L1 Checkpoint Blockade and CD2.8 co-stimulation dual junction
[0201] To evaluate the dual function, PD-1 and PD-Ll interaction blocking and I1CD8Q and CD28 co-stimulatory activities on antigen-presenting cells, of I1CD8O-F c/anti -PD-L1 (Format A) (SEQ ID. 113, 114 and 115) molecule, PD-1/PD-L1 checkpoint blockade assay system from Promega was used. In this system, the effector cell (NEAT), a genetic modified Jurkat cell line expressed TCR/CD3, PD- Li and CD28, and reporter gene luciferase under the control of NEAT promoter. An artificial antigen- presenting cell line, PD-L1 aAPC/CHO-Kl, expressed PD-L1 and cognate antigen recognized by TCR/CD3 on effector cells. When Jurkat effector cell (NFAT) mixed with PD-L1 aAPC/CHO-Kl cells, the Jurkat effector cells receives both TCR/CD3-Antigen stimulation signal as well as PD-L1 blocking signal simultaneously from aAPC/CHO-Kl ceils. As a result, the NFAT promoter activity was suppressed by signaling from PD-l/PD-Ll interaction. In the presence of anti-PD-Ll or anti-PD- 1 blocking antibody, the NFAT promoter activity was unchecked. Furthermore, in the presence of hCD80-Fc/anti-PD-Ll (Format A) (SEQ ID. 113, 114 and 115) multifunctional polypeptides, it not only blocked PD-l/PD-Ll signaling, but also provided hCD80/CD28 co-stimulatory activity, thus boost NFAT promoter activity received from TCR/CD3-Antigen interaction.
[0202] To setup the assay, 50,000 PD-L1 aAPC/CHO-Kl Cells in 100 uL/well were seeded in a flat-bottom opaque 96-well plate, incubated at 37°C, 5% CO2 incubator overnight. The culture medium was completely dumped out right before addition of other components on the plate. Jurkat PD-1 effector cells were suspended in RPML-1640 with 10% Fetal bovine serum at 2 miliion/mL. Fifty microliter per well of Jurkat PD-1 effector cells were transfered to the plate growing PD-L1 aAPC/CHO-Kl cells. The testing molecules were prepared in culture medium at 30 ug/mL with 1:3 serial dilutions and 25 uL/weil of samples were transferred on to the plate in duplicates. The plate was incubated at 37°C, 5%CO? incubator for 6 h. To detect NFAT promoter activity activated by testing molecules, Bio-Glo™ Luciferase Assay Substrate (Promega) were thawed and equilibrate to room temperature. Seventy-five microliter of Bio-Glo™ Luciferase Assay Substrate were added to each well of the assay plate. The plate was mixed briefly, and the count of firefly luminescence was measured using a lummometer luminescence plate reader. The data wrere summarized in FIG. 18. In this experiment, the multifunctional polypeptide (hCD8G-Fc/anti-PD-Ll) (Format A) (SEQ ID. 113, 114 and 115) was more active than anti-PD-Ll antibody. The activity of anti-PD-Ll antibody was due to the removal of the signal blockade from PD-1/PD-L1 interaction by interrupting PD-1/PD-L1 interaction. Additional activity of the multifunctional polypeptide (hCD80-Fc/anti-PD-Ll (Format A) (SEQ ID. 113, 114 and 115)) could be a result of the costimulatory signal from hCD80 and CD28 interaction. In presence of excess amount of anti-PD-Ll antibody, the multifunctional polypeptide (hCD80-Fc/anti-PD-Ll (Format A) (SEQ ID. 113, 114 and 115)) had similar activity as anti-PD-Ll antibody. Such reduction in activity was most likely due to the loss of costimulatory activity of hCD80 and CD28 interaction, which was a result of the fact that the excess amount of anti-PD-Ll antibody competed the multifunctional polypeptide off the PD-L1 ligand and the costimulatory effects of multifunctional polypeptide (hCDSO-Fc/anti-PD-Ll (Format A) (SEQ ID. 113, 114 and 115)) was lost. In absence of the artificial APC, no acti vities were observed
EXAMPLE 9 - Determination of mCDSO-Fc/anti-PD-Ll Containing Multifunctional Polypeptides Binding Affinities to CD28, CTLA-4, and PD-L1 by ELISA
CD28 binding assay by ELISA
[0203] To test binding affinity to CD28 by ELISA, ELISA plates were first coated with 5 ug/mL Streptavidin-plus from Agilent (Cat. No. SA26) at 4°C overnight. The next day, Streptavidin-plus was removed and washed with PBST three times. The plates were then blocked with 2% BSA at 37°C for 1 hour. After removing the 2% BSA solution, the plates were washed with PBST three times. Biotinylated Human CD28, Fc Tag from Aero (Cat. No. CD8-H82F2) or Biotinylated mouse CD28,
Fc Tag from SB (Cat. No. 50103-M08H) were added to each well and incubate at 37°C for 1 hour. The samples of the purified mCD80-Fc/anti-PD~L1 multifunctional polypeptides were added at 100 uL/well in duplicate with serial dilutions and incubated at 37°C for 2 hours. After washing with PBST three times, a secondary antibody, 100 uL/well of Goat anti mouse IgG-Fc secondary Antibody (HRP) from SB (Cat. No. SSA006) was added and incubated at 37°C for I hour. The plates were washed three times with PBST, and a 100 uL/well of TMB substrate was added. The plates were incubated until the color was developed. The stop-solution was added to stop the reaction. The plates were read at OD450. The results of binding affinity to CD28 by ELISA are presented in FIG. 22 and FIG. 23. CTLA4 binding assay by ELISA
[0204] To test binding affinity to CTLA4 by ELISA, ELISA plates were first coated with 1 ug/mL human CTLA4 (from SB, Cat. No. 11159-H08H) or mouse CTLA-4 (from SB, Cat. No. 50503- MQ8H) at 4°C overnight. The next day, the plates were washed with PBST three times. The plates were then blocked with 2% BSA at 37°C for 1 hour. After removing the 2% BSA solution, the samples of the purified mCD80-Fc/anti-PD-Ll multifunctional polypeptides were added at 100 uL/well in duplicate with serial dilutions and incubated at 37°C for 1 hours. After washing with PBST three times, a secondary antibody, 100 uL/well of Goat anti mouse IgG-Fc secondary' Antibody (HRP) from SB (Cat No. SSA006) was added and incubated at 37°C for I hour. The plates were washed three times with PBST, and a 100 uL/well of TMB substrate was added. The plates were incubated until the color was developed. The stop-solution was added to stop the reaction. The plates were read at OD450. The results of binding affinity to CTLA4 by ELISA are presented in FIG. 24 and FIG. 25. hPD-Ll b inding assay by ELISA
[0205] To test binding affinity to !iPD-LI by ELISA, ELISA plates were first coated with 1 ug/mL human PD-L1 (from SB, Cat. No. 10084-H08H) at 4°C overnight. The next day, the plates were washed with PBST three times. The plates were then blocked with 2% BSA at 37°C for 1 hour. After removing the 2% BSA solution, the samples of the purified mCD80-Fc/anti-PD-Ll multifunctional polypeptides were added at 100 uL/well in duplicate with serial dilutions and incubated at 37°C for 1 hours. After washing with PBST three times, a secondary antibody, 100 uL/well of Goat anti mouse IgG-Fc secondary Antibody (HRP) from SB (Cat. No. SSA006) was added and incubated at 37°C for 1 hour. The plates were washed three times with PBST, and a 100 uL/well of TMB substrate was added. The plates were incubated until the color was developed. The stop-solution was added to stop the reaction. The plates were read at OD450. The results of binding affinity to hPD-Ll by ELISA are presented in FIG. 26. EXAMPLE 10 - Determination of mCDSO/anti-PD-Ll Containing Multifunctional Polypeptides Binding Affinities by FACS Cell Surface Binding Assay by FACS
[0206] The binding affinity (ECso) of mCD80-F c/anti -PD-L1 multifunctional polypeptides to Flp- m 293 cells overexpressing hCD28, mCD2.8, hCTLA-4, hPD-Ll and mPD-Ll (Flp-in 293-hCD28, Flp-in 293-mCD28, Flp-in 293-hCTLA4, Flp-in 293-hPD-Ll and Flp-in 293-mPD-Ll cells) were determined by FACS. The FACS binding assays were performed as follows: Flp-in 293 cells were washed three times, then the cells were fixed by 4% PFA for 20min and pelleted at lOOOrpm for 5 minutes at room temperature. The cells were resuspended by PBS and washed three times. Then, the cells were resuspended in 3% BSA and incubated at 4°C for 1 hours. Series diluted mCD80~Fc/anti- PD-L1 multifunctional polypeptides were added in to the 96-well plates. The cells were then seeded into the 96-well plates. The plates were incubated at 4°C for 12 hours. The plates were washed three time by 3% BSA in PBS. The secondary antibody was added into the plates and incubated at 4°C for 1 hour. The cells were washed and analyzed by FACS. The results of binding affinity by FACS are presented in FIGS. 27-31.
EXAMPLE 11 - In Vivo Efficacy Evaluation of mCD80-Fc/anti-PD-Ll Multifunctional polypeptides in Tumor Growth inhibition Mouse Model
In vivo efficacy evaluation of a mCD80-Fc/an(i-PD-LI multifunctional polypeptide in tumor growth inhibitio MC-38-human-PD-LI mouse model:
[0207] Efficacy evaluation of mCD80-Fc/anti-PD-Ll multifunctional polypeptides in tumor growth inhibition in a syngeneic mouse model was carried as follows lxl 06 MC38-human-PD-Ll cells were suspended in 100pL DMEM with 50% matrix gel. The cells were then inoculated subcutaneously into the right flank of C57BL/6 mice. Mice were randomly assigned to different groups when the mean tumor volume reached desired average size (72mm3). Mice were LP. injected with different proteins dosed at 0.15 mg per mouse on day 0, day 3 and day 6. Tumor volume was measured three times weekly in two dimensions using a caliper, and the volume was expressed in mm1 using the formula: V = 0.5 a x bl, where a and b were the long and short diameters of the tumor, respectively. The data was shown in FIG. 32.

Claims

CLAIMS What is claimed is:
1. A PD-LI antibody comprising the ammo acid sequence of a CDR-L1 of Table 1, a CDR-L2 of Table 2, a CDR-L3 of Table 3, a CDR-H1 of Table 4, a CDR-H2 of Table 5, and/or a CDR-H3 of Table 6.
2. A PD-LI antibody comprising the ammo acid sequence of a light chain comprising the sequences of any one of the CDR-L1 , CDR-L2, and CDR-L3 combinations presented in Table
7
3. A PD-LI antibody comprising the ammo acid sequence of a light chain comprising the sequences of any one of the CDR-Hl, CDR-H2, and CDR-H3 combinations presented in Table
8
4. A PD-LI antibody comprising the ammo acid sequences of any one of the CDR-L1, CDR-L2, CDR-L3, CDR-HL CDR-H2, and CDR-H3 combinations presented in Table 9.
5. A PD-LI antibody comprising a VL domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 18-21 , and SEQ ID NO: 57, or a sequence comprising at least 80% identity thereto.
6. A PD-LI antibody comprising a VH domain comprising an amino acid sequence set forth m any one of SEQ ID NOs: 23-33, and SEQ ID NOs: 58-61 , or a sequence comprising at least 80% identity thereto.
7. A PD-LI antibody comprising a VL domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 18-21, and SEQ ID NO: 57, or a sequence comprising at least 80% identity thereto, and VH domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 23-33, and SEQ ID NOs: 58-61, or a sequence comprising at least 80% identity thereto.
8. A PD-LI antibody comprising: a. a VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80% identity thereto; and/or a VH domain comprising SEQ ID NO: 23, or a sequence comprising at least 80% identify thereto; b. a VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80% identity thereto; and/or a VH domain comprising SEQ ID NO: 24, or a sequence comprising at least 80% identity thereto; c. a VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80% identity thereto; and/or a VH domain comprising SEQ ID NO: 25, or a sequence comprising at least 80% identity' thereto; d. a VL domain comprising SEQ ID NO: 18; and/or a VH domain comprising SEQ ID NO: 26, or a sequence comprising at least 80% identity thereto; e. a VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80% identity thereto; and/or a VH domain comprising SEQ ID NO: 27, or a sequence comprising at least 80% identity' thereto; f. a VL domain comprising SEQ ID NO: 19, or a sequence comprising at least 80% identity thereto; and/or a VH domain comprising SEQ ID NO: 28, or a sequence comprising at least 80% identity thereto; g. a VL domain comprising SEQ ID NO: 19, or a sequence comprising at least 80% identity thereto; and/or a VH domain comprising SEQ ID NO: 29, or a sequence comprising at least 80% identity thereto; h. a VL domain comprising SEQ ID NO: 20, or a sequence comprising at least 80% identity thereto; and/or a VH domain comprising SEQ ID NO: 30, or a sequence comprising at least 80% identity thereto;
L a VL domain comprising SEQ ID NO: 21 , or a sequence comprising at least 80% identity thereto; and/or a VH domain comprising SEQ ID NO: 31, or a sequence comprising at least 80% identity thereto; j. a VL domain comprising SEQ ID NO: 22, or a sequence comprising at least 80% identity thereto; and/or a VH domain comprising SEQ ID NO: 32, or a sequence comprising at least 80% identity thereto; k a VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80% identity thereto; and/or a VH domain comprising SEQ ID NO: 33, or a sequence comprising at least 80% identity' thereto; L a VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80% identity thereto; and/or a VH domain comprising SEQ ID NO: 58, or a sequence comprising at least 80% identity thereto; m. a VL domain comprising SEQ ID NO: 57, or a sequence comprising at least 80% identity thereto; and/or a VH domain comprising SEQ ID NO: 58, or a sequence comprising at least 80% identity' thereto; n. a VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80% identity thereto; and/or a VH domain comprising SEQ ID NO: 59, or a sequence comprising at least 80% identity' thereto; o. a VI, domain comprising SEQ ID NO: 18, or a sequence comprising at least 80% identity thereto; and/or a VH domain comprising SEQ ID NO: 60, or a sequence comprising at least 80% identity thereto; p. a VL domain comprising SEQ ID NO: 18, or a sequence comprising at least 80% identity thereto; and/or a VH domain comprising SEQ ID NO: 61, or a sequence comprising at least 80% identity thereto;
9. The PD-L1 antibody of any one of claims 1 to 8, wherein the antibody is an antigen-binding fragment.
10. The antigen-binding fragment of claim 9, wherein the fragment is a Fab, Fab', F(ab')2, a scFv, a Fv, a diabody, or a single chain antibody.
11. The PD -LI antibody of any one of claims 1 to 8, wherein the antibody is a full length antibody.
12. The PD-L1 antibody of claim 11 , wherein the Fc is human IgGI , IgG2, IgG3, or IgG4.
13. The PD-L1 antibody of claim 11, wherein the Fc is mouse IgGI, IgG2a, IgG2b, or IgG3.
14. The PD -LI antibody of any one of claims 1 to 11, wherein the antibody is human.
15. The PD-Ll antibody of any one of claims 1 to 11, wherein the antibody is humanized.
16. The PD-Ll antibody of any one of claims 1 to 11, wherein the antibody is chimeric.
17. The PD-Ll antibody of any one of claims 1 to 11, wherein the antibody is monoclonal.
18. The PD-Ll antibody of any one of claims 1 to 11 , wherein the antibody is conjugated.
19. A polynucleotide encoding any one of the antibodies of claims 1 to 16.
20. A vector comprising the polynucleotide of claim 19.
21. A cell expressing the vector of claim 20.
22. A pharmaceutical composition comprising any one of the antibodies of claims 1 to 18, and optionally a pharmaceutically acceptable excipient.
23. A multifunctional polypeptide comprising: a. a CD80 extracellular domain; b. a disease-associated antigen-bindmg domain; and c. an Fc domain
24. The multifunctional polypeptide of claim 23, wherein the CD80 extracellular domain is a monomer.
25. The multifunctional polypeptide of claim 23, wherein the polypeptide comprises two monomers of the CD80 extracellular domain.
26. The multifunctional polypeptide of claim 23, wherein the polypeptide comprises four monomers of the CD80 extracellular domain.
27. The multifunctional polypeptide of claim 23, wherein the CD80 extracellular domain is a single chain dimer.
28. The multi functional polypeptide of claim 23, wherein the polypeptide comprises two single chain dimers of the CD80 extracellular domain.
29. The multifunctional polypeptide of claim 23, wherein the CD80 extracellular domain is attached to the N-terrninus of the Fc domain.
30. The multifunctional polypeptide of claim 23, wherein the CD80 extracellular domain is attached to the C-terminus of the Fc domain.
31. The multifunctional polypeptide of claim 23, wherein the CD80 extracellular domain is attached to the N-terminus of a disease-associated antigen-binding domain, and wherein the disease- associated antigen-binding domain is attached to the N-terminus of an Fc domain.
32. The multifunctional polypeptide of claim 23, wherein the disease-associated antigen-binding domain comprises an scFv.
33. The multifunctional polypeptide of claim 23, wherein the disease-associated antigen-binding domain comprises a Fab or Fab’ fragment.
34. The multifunctional polypeptide of claim 23, wherein the disease-associated antigen-bindmg domain comprises a F(ab’)2 fragment
35. The multi functional polypeptide of claim 23, wherein the disease-associated antigen-binding domain comprises a Fv fragment.
36. The multifunctional polypeptide of claim 23, wherein the disease-associated antigen-bindmg domain comprises a Fab’ fragment and a Fv fragment.
37. The multifunctional polypeptide of claim 23, wherein the disease-associated antigen-binding domain is attached to the N-terminus of the Fc domain.
38. The multifunctional polypeptide of claim 23, wherein the disease-associated antigen-binding domain is attached to the C-terminus of the Fc domain.
39. The multifunctional polypeptide of claim 23, wherein the disease-associated antigen-binding domain is attached to the N-terminus of a CD80 extracellular binding domain, which is attached to the N-terminus of a Fc domain.
40. The multifunctional polypeptide of claim 23, wherein the disease-associated antigen-binding domain is attached to the N-terminus of the Fc domain.
41. The multifunctional polypeptide of claim 23, wherein the multifunctional polypeptide comprises two disease-associated antigen-binding domains coupled to each other.
42. The multifunctional polypeptide of claim 2.3, wherein the disease-associated antigen-binding domain is attached to the N-terminus of a CD80 extracellular domain which is attached to the N- terminus of a Fc domain.
43. The multifunctional polypeptide of claim 23 comprising any one of the structural formats provided in FIG. 1 A.
44. The multifunctional polypeptide of claim 43, comprising Format A.
45. The multifunctional polypeptide of claim 43, comprising Format B.
46. The multifunctional polypeptide of claim 43, comprising Format C.
47. The multifunctional polypeptide of claim 43, comprising Format D
48. The multifunctional polypeptide of claim 43, comprising Format E.
49. The multifunctional polypeptide of claim 43, comprising Format F.
50. The multifunctional polypeptide of claim 43, comprising Format G.
51. The multifunctional polypeptide of claim 43, comprising Format H.
52. The multifunctional polypeptide of claim 43, comprising Format F
53. The multifunctional polypeptide of claim 43, comprising Format J.
54. The multifunctional polypeptide of claim 23, wherein the polypeptide does not comprise the structural format of Format A in FIG. 1A.
55. The multifunctional polypeptide of claim 23, wherein the polypeptide does not comprise the structural format of Format B m FIG. 1A.
56. The multifunctional polypeptide of claim 23, wherein the polypeptide does not comprise the structural format of Format C in FIG. 1A.
57. The multifunctional polypeptide of claim 23, wherein the polypeptide does not comprise the structural format of Format D in FIG. 1A
58. The multifunctional polypeptide of claim 23, wherein the polypeptide does not comprise the structural format of Format E in FIG. 1 A.
59. The multifunctional polypeptide of claim 23, wherein the polypeptide does not comprise the structural format of Format F in FIG. 1 A.
60. The multifunctional polypeptide of claim 23, wherein the polypeptide does not comprise the structural format of Format G in FIG. 1 A.
6L The multifunctional polypeptide of claim 23, wherein the polypeptide does not comprise the structural format of Format H in FIG. 1 A.
62. The multifunctional polypeptide of claim 23, wherein the polypeptide does not comprise the structural format of Format I in FIG. 1A.
63. The multifunctional polypeptide of claim 23, wherein the polypeptide does not comprise the structural format of Format J in FIG. 1 A.
64. The multifunctional polypeptide of claim 23 comprising any one of the structural formats provided in FIG. I A, wherein the antigen-binding domain is not PD-L1
65. The multifunctional polypeptide of claim 23 comprising any one of the structural formats provided in FIG. I A, wherein the antigen-binding domain is not Claudin 18.2.
66. The multifunctional polypeptide of claim 23 comprising any one of the structural formats provided in FIG. 1 A, wherein the antigen-binding domain is not Her2.
67. The multifunctional polypeptide of any one of claims 23 to 66, wherein the CD80 extracelluar domain is derived from a human CD80.
68. The multifunctional polypeptide of any one of claims 23 to 66, wherein the CD80 extracellular domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% ammo acid sequence identity to SEQ ID NO: 34 or SEQ ID NO: 37.
69. The multifunctional polypeptide of claim 67, wherein the CD80 extracellular domain has one or more amino acid substitution modifications at positions 130, 131, 139, 155, 156, 165, or 166 of SEQ ID NO: 34 or to the corresponding positions of SEQ ID NO: 37.
70. The multifunctional polypeptide of claim 69, wherein the am o acid substitution modification at position 131 of SEQ ID NO: 34 is S131A, S131V, S131I, S131F, S131R, S131E, S131D, or S131Q.
7L The multifunctional polypeptide of any one of claims 69 or 70, wherein the amino acid substitution modification at position 139 of SEQ ID NO: 34 is L139V.
72. The multifunctional polypeptide of any one of claims 69-71, wherein the amino acid substitution modification at position 155 of SEQ ID NO: 34 is VI55A, VI55I, or VI55T.
73. The multifunctional polypeptide of any one of claims 69-12, wherein the amino acid substitution modification at position 165 of SEQ ID NO: 34 is A165S, A165V, A i 651. A165F, A165R, A165E, A165D, or A165Q.
74. The multifunctional polypeptide of any one of claims 69-73, wherein the amnio acid substitution modification at position 166 of SEQ ID NO: 34 is VI 66A, VI 66L, or Vi 66T
75. The multi functional polypeptide of any one of claims 23 to 74, wherein the disease-associated anti gen -binding domain is a PD-L1 binding domain.
76. The multifunctional polypeptide of claim 75, wherein the PD-L1 binding domain comprises: a. the amino acid sequence of a CDR-L1 of Table 1, a CDR-L2 of Table 2, a CDR-L3 of Table 3, a CDR-Hl of Table 4, a CDR-H2 of Table 5, and/or a CDR-H3 of Table 6; b. the amino acid sequence of a light chain comprising the sequences of any one of the CDR-L1, CDR-L2, and CDR-L3 combinations presented in Table 7; c. the ammo acid sequence of a light chain comprising the sequences of any one of the CDR-Hl, CDR-H2, and CDR-H3 combinations presented in Table 8; d. the amino acid sequences of any one of the CDR-L1, CDR-L2, CDR-L3, CDR-Hl, CDR-H2, and CDR-H3 combinations presented in Table 9; e. a VL domain comprising an ammo acid sequence set forth in any one of SEQ ID NOs: 18-21 and SEQ ID NO: 57, or a sequence comprising at least 80% identity thereto; f. a VH domain comprising an ammo acid sequence set forth in any one of SEQ ID NOs: 23-33, or a sequence comprising at least 80% identity thereto; or g. a VL domain comprising an ammo acid sequence set forth in any one of SEQ ID NOs: 18-21 and SEQ ID NO: 57, or a sequence comprising at least 80% identity thereto, and VH domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 23- 33, or a sequence comprising at least 80% identity thereto.
77. The multifunctional polypeptide of claim 75, wherein the PD-L1 binding domain comprises the antigen-binding sequences of Atezolizumab, Avelumab, or Durvalumab.
78. The multifunctional polypeptide of any one of claims 23 to 74, wherein the disease-associated antigen-binding domain is a Claudin-18.2 binding domain.
79. The multifunctional polypeptide of any one of claims 23 to 78, wherein the Fc domain is human.
80. The multifunctional polypeptide of claim 79, wherein the Fc domain is a human IgGl, IgG2, IgG3, or IgG4 Fc domain.
81. The multifunctional polypeptide of any one of claims 23 to 78, wherein the Fc domain is mouse.
82. The multifunctional polypeptide of claim 81, wherein the Fc domain is a mouse IgGl, IgG2a, IgG2b, or igG3 Fc domain.
83. The multifunctional polypeptide of claim 80, wherein the Fc domain is a wild-type human IgGl Fc domain.
84. The multifunctional polypeptide of claim 80, wherein the Fc domain is a mutant human IgGl Fc domain.
85. The multifunctional polypeptide of claim 84, wherein the mutant human IgGl Fc domain comprises reduced effector function.
86. The multifunctional polypeptide of claim 84, wherein the mutant human IgGl Fc domain comprises a knob-in-hole mutation.
87. The multifunctional polypeptide of claim 86, wherein the mutant human IgGl Fc domain comprises reduced effector function and a knob-in-hole mutation.
88. The multifunctional polypeptide of claim 79, wherein the Fc domain comprises the amino acid sequence of any one of SEQ ID NOS: 100 to 111.
89. The multifunctional polypeptide of claim 23, comprising the amino acid sequence of any one of SEQ ID NOS: 113 to 135.
90. A pharmaceutical composition comprising any one of the multifunctional polypeptides of claims 23 to 89, and optionally a pharmaceutically acceptable excipient.
91. A polynucleotide encoding any one of the multifunctional polypeptides of claims 23 to 89.
92. A vector comprising the polynucleotide of claim 91.
93. A ceil expressing the vector of claim 92.
94. A method of treating a disease or disorder, comprising administering to subject in need thereof a therapeutically effective amount of any one of the antibodies, multifunctional polypeptides, or pharmaceutical compositions of claims 1 to 18, or 22 to 90.
95. The method of claim 94, wherein the disease or disorder is a selected from cancer, infectious disease, or inflammatory disease.
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