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WO2025106775A1 - Pd-l1-based chimeric protein formulations - Google Patents

Pd-l1-based chimeric protein formulations Download PDF

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Publication number
WO2025106775A1
WO2025106775A1 PCT/US2024/056067 US2024056067W WO2025106775A1 WO 2025106775 A1 WO2025106775 A1 WO 2025106775A1 US 2024056067 W US2024056067 W US 2024056067W WO 2025106775 A1 WO2025106775 A1 WO 2025106775A1
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Prior art keywords
pharmaceutical composition
cancer
seq
concentration
amino acid
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French (fr)
Inventor
Nikolai Kley
Erik Depla
Dorien VAN LYSEBETTEN
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Orionis Biosciences BV
Orionis Biosciences Inc
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Orionis Biosciences BV
Orionis Biosciences Inc
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Publication of WO2025106775A1 publication Critical patent/WO2025106775A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • 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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/249—Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present application provides, in part, pharmaceutical formulations of PD-L1-based chimeric protein complexes, and uses thereof.
  • Biologies with an effector function are a class of agents that have many potential therapeutic applications.
  • these biologies e.g., cytokines
  • a chimeric protein having a signaling agent (having an effector function, e.g., a cytokine), connected to a targeting element (having the ability to seek its target with high precision).
  • the signaling agent can be a wild type signaling agent or a modified signaling agent (e.g. by mutation).
  • the modified signaling agent is, generally, modified to cause an attenuation of the signaling agent’s activity (e.g., substantially reducing its ability to interact with/engage its receptor) in a manner such that the signaling agent’s effector function can be recovered upon binding of the targeting element to its target (e.g., antigen on target cell).
  • chimeric proteins are most amenable to therapeutic use if certain conditions are met, e.g., the ability to be produced in a large scale, an in vivo half-life that ensures adequate time of exposure to the drug to elicit a therapeutically beneficial effect, a proper size to avoid rapid clearance or limited tissue penetrance and biodistribution, and other properties that ensure adequate solubility, stability and storage without significant loss of function.
  • all, or substantially most, of the above properties should be achieved without a loss of the conditional targeting of the effector function and retention of conditional engagement of a modified signaling agent with its receptor.
  • the present application provides a pharmaceutical composition or formulation comprising (a) a Programmed death-ligand 1 (PD-LI)-based chimeric protein or chimeric protein complex (e.g., at a concentration of at least about 1 mg/mL) comprising (i) a targeting moiety that specifically binds to PD-L1 , (ii) a modified human I FNa2, and (iii) a modified Fc domain; (b) at least one pharmaceutically acceptable buffering agent comprising a histidine buffer (e.g., at a concentration of at least about 10 mM to about 30 mM); (c) at least one pharmaceutically acceptable excipient comprising a surfactant (e.g., polysorbate 20 (PS20) or poloxamer 188 (Poll 88)); and (d) at least one pharmaceutically acceptable excipient comprising a tonicifier (e.g., trehalose, sucrose, or mannitol).
  • a tonicifier e.g
  • the pharmaceutical compositions or formulations are suitable for parenteral administration, such as intravenous administration (e.g., intravenous infusion or bolus injection).
  • parenteral administration such as intravenous administration (e.g., intravenous infusion or bolus injection).
  • the pharmaceutical compositions or formulations of the present application comprise one or more additional excipients, such as, but not limited to, stabilizers (e.g., beta-cyclodextrin), salts, amino acids, and antioxidants.
  • additional excipients such as, but not limited to, stabilizers (e.g., beta-cyclodextrin), salts, amino acids, and antioxidants.
  • the present application relates to methods for treating or preventing a cancer, comprising administering an effective amount of the pharmaceutical composition as disclosed herein to a patient in need thereof.
  • Figure 1 depicts turbidity measurements at 500 nm of PD-L1 -based chimeric protein complex incubated in 16 different buffer types with increasing amounts of PEG 6000.
  • Figure 2 depicts a coefficient plot of T m where positive coefficient bars represent excipients having a stabilizing effect on the formulation and negative coefficient bars represent excipients having a destabilizing effect.
  • Figure 3 depicts a coefficient plot of SLS ma x where negative coefficient bars represent excipients having a stabilizing effect on the formulation and positive coefficient bars represent excipients having a destabilizing effect.
  • Figure 4 depicts a coefficient plot of T agg where positive coefficient bars represent excipients having a stabilizing effect on the formulation and negative coefficient bars represent excipients having a destabilizing effect (log transformed).
  • Figure 5 depicts a coefficient plot of T on set where positive coefficient bars represent excipients having a stabilizing effect on the formulation and negative coefficient bars represent excipients having a destabilizing effect.
  • Figure 6 shows size distributions based on intensity and volume for the sample before stress and after 15 minutes or 1 hour of vortexing the formulations without surfactant (no. 1), or with 0.01% (w/v) PS20 (no. 2), or with 0.05% (w/v) PS20 (no. 3).
  • Figure 7 shows size distributions based on intensity and volume for the sample before stress and after 15 minutes or 1 hour of vortexing the formulations without surfactant (no. 1), or with 0.01 % (w/v) Pol 188 (no. 2), or with 0.05% (w/v) Poll 88 (no. 3).
  • Figures 8A-8B shows particle size distribution based on intensity and volume of various short stress concepts before (no. 2) and after being subjected to 15 minutes (no. 3) or 1 hour (no. 4) of shear stress.
  • the formulation without the PD-L1-based chimeric protein complex added is shown before vortexing as no. 1 .
  • Figure 9 depicts results of a SDS-PAGE under non-reducing conditions experiment: lanes 1 , 4 and 8: molecular weight markers; lanes 5 and 9: 100 ng BSA control; lanes 2, 6 and 10: 10 pig PD-L1-based chimeric protein in formulation 1 after 0, 1 , and 2 weeks at 40°C; lanes 3, 7 and 11 : 10 pig PD-L1-based chimeric protein in formulation 2 after 0, 1 , and 2 weeks at 40°C.
  • Figure 10 shows results of a Western blot (anti-human Fc) under non-reducing conditions experiment: lanes 1, 4 and 7: molecular weight markers; lanes 2, 5 and 8: 10 pig PD-L1-based chimeric protein in formulation 1 after 0, 1 , and 2 weeks at 40°C; lanes 4, 6 and 9: 10 pig PD-L1-based chimeric protein in formulation 2 after 0, 1 , and 2 weeks at 40°C.
  • compositions and/or formulations comprising Programmed death-ligand 1 (PD-LI)-based chimeric proteins or chimeric protein complexes, which, without wishing to be bound by theory, can be stably formulated for efficacious biological effect and convenient dosing schedules, dosing volumes, and patient-friendly delivery devices.
  • PD-LI Programmed death-ligand 1
  • a pharmaceutical composition or formulation comprising (a) a Programmed death-ligand 1 (PD-LI)-based chimeric protein or chimeric protein complex comprising (i) a targeting moiety that specifically binds to PD-L1, (ii) a modified human IFNo2, and (iii) a modified Fc domain; (b) at least one pharmaceutically acceptable buffering agent comprising histidine, optionally further comprising acetic acid; (c) at least one pharmaceutically acceptable excipient comprising a surfactant; and (d) at least one pharmaceutically acceptable excipient comprising a tonicifier, and wherein the pharmaceutical composition or formulation has a pH that ranges from at least 4.0 to at least about 6.0.
  • PD-LI Programmed death-ligand 1
  • the present technology provides pharmaceutical compositions and/or formulations including chimeric protein complexes that comprise biological therapeutic agents whose effector function can be delivered in a highly precise fashion to a target of choice, with limited or no cross-reactivities, and with limited or no systemic adverse events, while also providing improved pharmaceutical features, such as solubility, stability, and storage properties.
  • the pharmaceutical compositions and/or formulations of the present application can be produced in a large scale.
  • the pharmaceutical compositions and/or formulations of the present application provide an in vivo half-life that ensures adequate time of exposure to the drug to elicit a therapeutically beneficial effect.
  • the pharmaceutical compositions and/or formulations of the present application are sized to avoid rapid clearance or limited tissue penetrance and bio-distribution. In embodiments, the pharmaceutical compositions and/or formulations of the present application provide adequate solubility, stability and/or storage properties without significant loss of function.
  • the present application provides pharmaceutical compositions or formulations comprising a chimeric protein complex, where the chimeric protein complex comprises a targeting moiety that specifically binds to Programmed death-ligand 1 (PD-L1), a modified human IFNa2, and a modified Fc domain.
  • PD-L1 Programmed death-ligand 1
  • a modified human IFNa2 a modified human IFNa2
  • a modified Fc domain a targeting moiety that specifically binds to Programmed death-ligand 1 (PD-L1), a modified human IFNa2, and a modified Fc domain.
  • PD-L1 Programmed death-ligand 1
  • the chimeric protein complex contemplated herein comprises a targeting moiety that specifically binds to PD-L1.
  • the present application provides a PD-L1 targeting moiety comprising a recognition domain comprising:
  • CDR1 comprises an amino acid sequence selected from any one of SEQ ID NOs: 2 or 5;
  • CDR2 comprises an amino acid sequence selected from any one of SEQ ID NOs: 3 or 6;
  • CDR3 comprises the amino acid sequence of SEQ ID NO: 4;
  • the targeting moiety that specifically binds to PD-L1 comprises a recombinant heavychain-only antibody (VHH).
  • the PD-L1 targeting moiety comprising a recognition domain further comprises one or more mutations at positions Q1, Q5, A14, A63, T74, K76, S79, K86, and Q110, relative to SEQ ID NO: 1.
  • the mutation is a substitution, optionally where the substitution is a polar and positively charged hydrophilic residue selected from arginine (R) and lysine (K), an aromatic, polar and positively charged hydrophilic residue including histidine (H), a polar and neutral of charge hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C), a polar and negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E) or a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).
  • substitution is a polar and positively charged hydrophilic residue selected from arginine (R)
  • the mutation is selected from one or more of a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) at position D54, optionally being D54G, or a polar and positively charged hydrophilic residue selected from arginine (R) and lysine (K), optionally being D54K, or a polar and neutral of charge hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C), optionally being D54T and a polar and positively charged hydrophilic residue selected from arginine (R) and lysine (K) at position G55, optionally being G55R.
  • a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I
  • the mutation is selected from one or more of a polar and negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E) at position Q1 , optionally being Q1D; a hydrophobic, aliphatic amino acid selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V) at position Q5, optionally being Q5V; a polar and neutral of charge hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C) at position A14, optionally being A14P; a hydrophobic, aliphatic amino acid selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V) at position A63, optionally being A63V; a polar and neutral of charge hydrophilic residue selected from asparag
  • the mutation is selected from one or more of Q1 D, Q5V, A14P, A63V, T74S, S79Y, K86R, and Q110L, optionally all of Q1 D, Q5V, A14P, D54G, T74S, K76N, S79Y, K86R, and Q110L, relative to SEQ ID NO: 1.
  • the aforementioned mutant PD-L1 targeting moieties (i.e. those disclosed relative to SEQ ID NO: 1) have improved affinity relative to a parental PD-L1 targeting moiety of SEQ ID NO: 1.
  • the PD-L1 targeting moiety of the present application includes a recognition domain comprising:
  • CDR1 three complementarity determining regions (CDR1 , CDR2, and CDR3), wherein:
  • CDR1 comprises an amino acid sequence selected from any one of SEQ ID Nos: 26 or 29;
  • CDR2 comprises an amino acid sequence selected from any one of SEQ ID NOs: 27 or 30;
  • CDR3 comprises the amino acid sequence of SEQ ID NO: 28;
  • the PD-L1 targeting moiety has a mutation that is a substitution relative to SEQ ID NO: 25.
  • the substitution is a polar and positively charged hydrophilic residue selected from arginine (R) and lysine (K) or an aromatic, polar and positively charged hydrophilic residue including histidine (H).
  • the substitution is a polar and neutral of charge hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C).
  • the substitution is a polar and negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E).
  • the substitution is a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).
  • the PD-L1 targeting moiety has a substitution at position N32 that is a positive hydrophilic residue is selected from arginine (R) and lysine (K).
  • the substitution at position N32 is polar and neutral hydrophilic residue that is selected from glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C).
  • the substitution at position N32 is N32Q or N32R, relative to SEQ ID NO: 25.
  • the PD-L1 targeting moiety has a substitution at position D33 is D33H relative to SEQ ID NO: 25.
  • the PD-L1 targeting moiety has a substitution at position M97 that is aliphatic hydrophobic residues are selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V), relative to SEQ ID NO: 25.
  • the PD-L1 targeting moiety has a substitution at position M97, relative to SEQ ID NO: 25, that is M97I, M97L, or M97V.
  • the PD-L1 targeting moiety comprising a recognition domain further comprises one or more of the following mutations Q1 D, Q5V, A14P, A62S, A74S, M77T, M78V, S79Y, K86R, and Q109L, optionally all of Q1 D, Q5V, A14P, D33H, A62S, A74S, M77T, M78V, K86R, M97V, relative to SEQ ID NO: 25.
  • the aforementioned mutant PD-L1 targeting moieties (i.e. those disclosed relative to SEQ ID NO: 25) have improved affinity relative to a parental PD-L1 targeting moiety of SEQ ID NO: 25.
  • the PD-L1-based chimeric protein or protein complex of the present application comprises a PD-L1 targeting moiety comprising a VHH comprising an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identity with any one of the amino acid sequences selected from SEQ ID NO: 23, 72, 1 , 7-22, 24, 25, and 31-71.
  • the VHH has the amino acid sequence of any one of the amino acid sequences selected from SEQ ID NO: 23, 72, 1 , 7- 22, 24, 25, and 31-71.
  • the PD-L1 targeting moiety is a single-domain antibody, such as a VHH.
  • the VHH may be derived from, for example, an organism that produces VHH antibody such as a camelid, a shark, or the VHH may be a designed VHH.
  • VHHs are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally-occurring heavy-chain antibodies. VHH technology is based on fully functional antibodies from camelids that lack light chains. These heavy-chain antibodies contain a single variable domain ( HH) and two constant domains (CH2 and CH3).
  • the VHH is a humanized VHH or camelized VHH.
  • the PD-L1 targeting moiety comprises a VHH comprising a single amino acid chain having four "framework regions” or FRs and three "complementary determining regions” or CDRs.
  • framework region or “FR” refers to a region in the variable domain which is located between the CDRs.
  • complementary determining region or “CDR” refers to variable regions in VHHs that contains the amino acid sequences capable of specifically binding to antigenic targets.
  • the PD-L1 binding agent comprises a VHH having a variable domain comprising at least one CDR1 , CDR2, and/or CDR3 sequences. In various embodiments, the PD-L1 binding agent comprises a VHH having a variable region comprising at least one FR1, FR2, FR3, and FR4 sequences.
  • the PD-L1 targeting moiety VHH's CDR1 sequence is selected from: GTIFSINRMD (SEQ ID NO: 2); GTIFS (SEQ ID NO: 5); GKIFSGNDMG (SEQ ID NO: 26); and GKIFS (SEQ ID NO: 29).
  • the PD-L1 targeting moiety VHH's CDR2 sequence is selected from: LITSDGTPA (SEQ ID NO: 3); LITSDGTPAYADSAKG (SEQ ID NO: 6); I ITSGGITD (SEQ ID NO: 27); and IITSGGITDYADAVKG (SEQ ID NO: 30).
  • the PD-L1 targeting moiety VHH's CDR3 sequence is selected from: SSGVYNY (SEQ ID NO: 4); and RDRTIW (SEQ ID NO: 28).
  • the targeting moiety that specifically binds PD-L1 comprises an amino acid sequence having at least about 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the following sequences:
  • the targeting moiety that specifically targets PD-L1 comprises an amino acid sequence with or without the terminal histidine tag sequence (/.e., HHHHHH; SEQ ID NO: 73).
  • the targeting moiety that specifically targets PD-L1 comprises an amino acid sequence with or without the HA tag (/.e., YPYDVPDYGS; SEQ ID NO: 74).
  • the targeting moiety that specifically targets PD-L1 comprises an amino acid sequence with or without the AAA linker.
  • the targeting moiety that specifically targets PD-L1 comprises an amino acid sequence with or without the AAA linker, HA tag, and terminal histidine tag sequence (i.e., AAAYPYDVPDYGSHHHHHH; SEQ ID NO: 75).
  • the present application contemplates the use of any natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (herein collectively referred to as "analogs”) of the targeting moiety that specifically targets PD-L1 as described herein.
  • the amino acid sequence of the targeting moiety that specifically targets PD-L1 further includes an amino acid analog, an amino acid derivative, or other non-classical amino acids.
  • the targeting moiety that specifically targets PD-L1 comprises an amino acid sequence having one or more amino acid mutations with respect to any one of the sequences disclosed herein.
  • the targeting moiety that specifically targets PD-L1 comprises an amino acid sequence having one, or two, or three, or four, or five, or six, or seen, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations with respect to any one of the sequences disclosed herein.
  • the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.
  • the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions. “Conservative substitutions” may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved.
  • the 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Vai, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
  • conservative substitutions are exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide.
  • glycine and proline may be substituted for one another based on their ability to disrupt a-helices.
  • non-conservative substitutions are exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.
  • the substitutions also include non-classical amino acids.
  • exemplary non-classical amino acids include, but are not limited to, selenocysteine, pyrrolysine, N-formylmethionine p-alanine, GABA and 5- Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, ct- amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2- amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,
  • the chimeric protein complex comprises a modified human interferon o2.
  • the modified IFN-a2 agent has reduced affinity and/or activity for the IFN-a/p receptor (IFNAR), i.e., IFNAR1 and/or IFNAR2 chains.
  • the modified I F N-a2 agent has substantially reduced or ablated affinity and/or activity for the IFN-a/p receptor (IFNAR), i.e., IFNAR1 and/or IFNAR2 chains.
  • the modified human interferon o2, as disclosed herein has an amino acid sequence having at least about 95% identity with of SEQ ID NOs: 76 or 77.
  • the modified human I FNa2 has an amino acid sequence having at least about 98% identity or at least about 99% identity with of SEQ ID NOs: 76 or 77. In some embodiments, the modified human IFNct2 has 1-3 mutations relative to the amino acid sequence of SEQ ID NOs: 76 or 77. In one embodiment, the modified human I FNct2 comprises a R149A mutation with respect to SEQ ID NOs: 76 or 77. In one embodiment, the modified human I FNa2 comprises a A145G mutation with respect to SEQ ID NOs: 76 or 77.
  • the R149A mutation, or the equivalent thereof, is present in the I FN-a2, relative to SEQ ID NO: 76 or 77.
  • the R149A mutation, or the equivalent thereof is not present in the I FN-a2, relative to SEQ ID NO: 76 or 77, and instead, another mutation is present.
  • this alternative mutation could be at one of positions R33, R144, A145, M148, and L153, or equivalents thereof, relative to SEQ ID NO: 76 or 77.
  • the alternative mutation is one of R33A, R144A, R144I, R144L, R144S, R144T, R144Y, A145D, A145G, A145H, A145K, A145Y, M148A, and L153A, or equivalents thereof, relative to SEQ ID NO: 76 or 77.
  • any reference to R149A herein may be replaced with one of R33A, R144A, R144I, R144L, R144S, R144T, R144Y, A145D, A145G, A145H, A145K, A145Y, M148A and L153A, or equivalents thereof, relative to SEQ ID NO: 76 or 77.
  • any reference to R149A herein may be replaced with A145G, or equivalents thereof, relative to SEQ ID NO: 76 or 77.
  • the chimeric protein complex disclosed herein comprises two targeting moieties. In embodiments, the chimeric protein complex disclosed herein comprises two identical targeting moieties. [063] In embodiments, the chimeric protein complex disclosed herein comprises two targeting moieties. In embodiments, the chimeric protein complex disclosed herein comprises two non-identical targeting moieties. For example, in embodiments, the chimeric protein complex disclosed herein comprises targeting moieties (e.g., without limitation, VHHs) against PD-L1 and Clec9A.
  • VHHs e.g., without limitation, VHHs
  • the chimeric protein complex disclosed herein includes at least one Fc domain.
  • the chimeric protein complex includes a modified Fc domain where the modified Fc domain includes one or more of the following mutations: P329G, K322Q, K322A, P331G, or P331S, relative to any of one of SEQ ID NO: 78-81 .
  • the modified Fc domain includes one or more of the following mutations: P329G, K322Q, K322A, P331 G, or P331 S, relative to human lgG1 Fc.
  • the chimeric protein complex includes a modified Fc domain that has an amino acid sequence having at least about 90% identity with any one of SEQ ID NO: 78-81. In embodiments, the modified Fc domain has an amino acid sequence having at least about 93% identity with SEQ ID NO: 78-81. In embodiments, the modified Fc domain has an amino acid sequence having at least about 95% identity with SEQ ID NO: 78-81.
  • SEQ ID NO: 78 Amino acid sequence of the Fc (human IgG 1 )— with LALA mutations and Ridgway hole
  • SEQ ID NO: 79 Amino acid sequence of the Fc (human lgG1)— with LALA mutations and Ridgway knob
  • SEQ ID NO: 80 Amino acid sequence of the Fc (human lgG1 ) - with LALA mutations and Merchant hole
  • the fragment crystallizable domain is the tail region of an antibody that interacts with Fc receptors located on the cell surface of cells that are involved in the immune system, e.g., B lymphocytes, dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells.
  • Fc domain is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains.
  • the Fc domain contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.
  • the Fc-based chimeric protein of complex the present technology includes a Fc domain.
  • the Fc domain is selected from IgG, IgA, IgD, IgM and IgE.
  • the Fc domain is selected from lgG1 , lgG2, lgG3, and lgG4.
  • the Fc domain is selected from human IgG, IgA, IgD, IgM and IgE. In embodiments, the Fc domain is selected from human lgG1 , lgG2, lgG3, and lgG4.
  • the Fc domain of the Fc-based chimeric protein complex comprises the CH2 and CH3 regions of IgG.
  • the IgG is human IgG.
  • the human IgG is selected from lgG1 , lgG2, lgG3, and lgG4.
  • the Fc domain comprises one or more mutations.
  • the mutation(s) to the Fc domain reduces or eliminates the effector function of the Fc domain.
  • the mutated Fc domain has reduced affinity or binding to a target receptor.
  • the mutation to the Fc domain reduces or eliminates the binding of the Fc domain to FcyR.
  • the FcyR is selected from FcyRI; FcyRlla, 131 R/R; FcyRlla, 131 H/H, FcyRllb; and FcyRIII.
  • the mutation to the Fc domain reduces or eliminated binding to complement proteins, such as, e.g., C1q. In embodiments, the mutation to the Fc domain reduces or eliminates binding to both FcyR and complement proteins, such as, e.g., C1q.
  • the Fc domain comprises a LALA mutation to reduce or eliminate the effector function of the Fc domain.
  • the LALA mutation comprises L234A and L235A substitutions in human IgG (e.g., lgG1 ) (wherein the numbering is based on the commonly used numbering of the CH2 residues for human lgG1 according to EU convention (Edelman et al., PNAS, 1969; 63 (1) 78-85)).
  • the Fc domains of human IgG comprise a mutation at 46 to reduce or eliminate the effector function of the Fc domains.
  • the mutations are selected from L234A, L234F, L235A, L235E, L235Q, K322A, K322Q, D265A, P329G, P329A, P331G, and P331 S.
  • the Fc domains comprise the FALA mutation to reduce or eliminate the effector function of the Fc domains.
  • the FALA mutation comprises F234A and L235A substitutions in human lgG4.
  • the Fc domains of human lgG4 comprise a mutation at one or more of F234, L235, K322, D265, and P329 to reduce or eliminate the effector function of the Fc domains.
  • the mutations are selected from F234A, L235A, L235E, L235Q, K322A, K322Q, D265A, P329G, and P329A.
  • the mutation(s) to the Fc domain stabilize a hinge region in the Fc domain.
  • the Fc domain comprises a mutation at S228 of human IgG to stabilize a hinge region.
  • the mutation is S228P.
  • the mutation(s) to the Fc domain promote chain pairing in the Fc domain.
  • chain pairing is promoted by ionic pairing (a/k/a charged pairs, ionic bond, or charged residue pair).
  • the Fc domain comprises a mutation at one more of the following amino acid residues of IgG to promote of ionic pairing: D356, E357, L368, K370, K392, D399, and K409.
  • the human IgG Fc domain comprises one of the mutation combinations in Table A to promote of ionic pairing.
  • chain pairing is promoted by a knob-in-hole mutations.
  • the Fc domain comprises one or more mutations to allow for a knob-in-hole interaction in the Fc domain.
  • a first Fc chain is engineered to express the “knob” and a second Fc chain is engineered to express the complementary “hole.”
  • human IgG Fc domain comprises the mutations of Table B to allow for a knobin-hole interaction.
  • the Fc domains in the Fc-based chimeric protein complexes of the present technology comprise any combination of the above-disclosed mutations.
  • the Fc domain comprises mutations that promote ionic pairing and/or a knob-in-hole interaction.
  • the Fc domain comprises mutations that have one or more of the following properties: promote ionic pairing, induce a knob-in-hole interaction, reduce or eliminate the effector function of the Fc domain, and cause Fc stabilization (e.g. at hinge).
  • a human IgG Fc domain comprises mutations disclosed in Table C, which promote ionic pairing and/or promote a knob-in-hole interaction in the Fc domain.
  • a human IgG Fc domain comprises mutations disclosed in Table D, which promote ionic pairing, promote a knob-in-hole interaction, or a combination thereof in the Fc domain.
  • the "Chain 1” and “Chain 2” of Table D can be interchanged (e.g. Chain 1 can have Y407T and Chain 2 can have T366Y).
  • a human IgG Fc domain comprises mutations disclosed in Table E, which reduce or eliminate FcyR and/or complement binding in the Fc domain.
  • the Table E mutations are in both chains.
  • the Fc domain in the Fc-based chimeric protein complex of the present technology are homodimeric, i.e. , the Fc region in the chimeric protein complex comprises two identical protein fragments.
  • the Fc domains in the Fc-based chimeric protein complexes of the present technology are heterodimeric, i.e., the Fc domain comprises two non-identical protein fragments.
  • heterodimeric Fc domains are engineered using ionic pairing and/or knob-in-hole mutations described herein.
  • the heterodimeric Fc-based chimeric protein complexes have a trans orientation/configuration. In a trans orientation/configuration, the targeting moiety and signaling agent are, in embodiments, not found on the same polypeptide chain in the present Fc-based chimeric protein complexes.
  • the present chimeric protein or the chimeric protein complex comprises one or more linkers. In embodiments, the present chimeric protein or the chimeric protein complex comprises a linker connecting the targeting moiety and the signaling agent. In embodiments, the present chimeric protein or the chimeric protein complex comprises a linker within the signaling agent.
  • the linker is derived from naturally-occurring multi-domain proteins or are empirical linkers, as described, for example, in Chichili et al., (2013), Protein Sci. 22 (2): 153- 167, Chen et al., (2013), Adv Drug Deliv Rev. 65(10): 1357- 1369, the entire contents of which are hereby incorporated by reference.
  • the linker is designed using linker designing databases and computer programs such as those described in Chen et al., (2013), Adv Drug Deliv Rev. 65(10): 1357- 1369 and Crasto et al., (2000), Protein Eng.
  • the linker is functional.
  • the linker may function to improve the folding and/or stability, improve the expression, improve the pharmacokinetics, and/or improve the bioactivity of the present chimeric protein or the chimeric protein complex.
  • the linker is a polypeptide. In embodiments, the linker is less than about 100 amino acids long. For example, the linker may be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11 , about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids long. In embodiments, the linker is a polypeptide. In embodiments, the linker is greater than about 100 amino acids long.
  • the linker may be greater than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11 , about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids long.
  • the linker is flexible.
  • the linker is rigid.
  • the linker is substantially comprised of glycine and serine residues (e.g. about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% glycines and serines).
  • the linker is GGS.
  • the linker is G.
  • the linker is AAA.
  • the linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 124).
  • the formulation of PD-L1 -based chimeric proteins and/or chimeric protein complexes, or variants thereof, may further comprise one or more pharmaceutically acceptable carriers or excipients.
  • the formulations can be in any suitable form appropriate for the desired use and route of administration.
  • the administration of the pharmaceutical formulation including PD-L1 -based chimeric proteins and/or chimeric protein complexes (and/or additional therapeutic agents) is intravenous and/or parenteral.
  • routes of administration include, for example: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, epidural, sublingual, intracerebral, intravaginal, or transdermal.
  • a pharmaceutical formulation including PD-L1-based chimeric proteins and/or chimeric protein complexes (and/or additional therapeutic agents) as described herein can be administered by any convenient route, for example, by intravenous infusion or bolus injection.
  • the PD-L1 -based chimeric proteins and/or chimeric protein complexes described herein are mixed with at least one inert, pharmaceutically acceptable excipient or carrier.
  • the pharmaceutical compositions or formulations comprising PD-L1 -based chimeric proteins and/or chimeric protein complexes described herein are generally buffered solutions.
  • a buffer is a chemical agent that is able to absorb a certain quantity of acid or base without undergoing a strong variation in pH.
  • Illustrative buffering agents include histidine buffer, histidine-acetate buffer, citrate buffer, phosphate buffer, acetate buffer, succinate buffer, and bicarbonate buffer.
  • the buffering agent comprises L-histidine or L- histidine hydrochloride monohydrate.
  • L-histidine or L-histidine hydrochloride monohydrate buffering system when using the L-histidine or L-histidine hydrochloride monohydrate buffering system, L-histidine or L-histidine hydrochloride monohydrate is present at a concentration of from about 1 mM to about 50 mM, or from about 5 mM to about 45 mM, or from about 5 mM to about 25 mM, or from about 10 mM to about 35 mM, or from about 15 mM to about 30 mM, or from about 20 mM to about 25 mM.
  • L-histidine or L-histidine hydrochloride monohydrate is present at a concentration of about 10 mM , about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM.
  • the pharmaceutical compositions or formulations described herein comprise a buffering agent further comprising an acetate buffer.
  • the acetate buffer comprises acetic acid.
  • the pharmaceutical compositions or formulations described herein comprise a buffering agent comprising an acetatehistidine buffer.
  • acetate— histidine buffer comprises L-histidine and acetic acid.
  • the buffering agent comprises acetic acid present at a concentration of from about 1 nM to about 50 mM, or from about 5 mM to about 45 mM, or from about 5 mM to about 25 mM, or from about 10 mM to about 35 mM, or from about 15 mM to about 30 mM, or from about 20 mM to about 25 mM.
  • acetic acid is present at a concentration of about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM.
  • the buffer maintains the pH of the formulation in the range of about pH 4.0 to about pH 6.0, or about 50 to about 6.0, or about 4.5 to about 5.5.
  • the pharmaceutical compositions or formulations described herein have a pH of about 4.0, about 4.1 , about 4.2, about 4.3, about 4.4, about 4.5, about
  • the formulation is pH adjusted, e.g., with hydrochloric acid and/or sodium hydroxide.
  • the pharmaceutical compositions or formulations comprising PD-L1-based chimeric proteins and/or chimeric protein complexes described herein additionally include a surface active agent or surfactant.
  • surface active agents suitable for use include, but are not limited to, any pharmaceutically acceptable, non-toxic surfactant.
  • the formulation comprises a surfactant, which can act as a solubilizing agent.
  • Classes of surfactants suitable for use in the compositions of the present application include, but are not limited to polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, polyethoxylated fatty acids, PEG-fatty acid diesters, PEG-fatty acid mono- and di-ester mixtures, polyethylene glycol glycerol fatty acid esters, alcohol-oil transesterification products, polyglycerized fatty acids, propylene glycol fatty acid esters, mixtures of propylene glycol esters-glycerol esters, mono- and diglycerides, sterol and sterol derivatives, polyethylene glycol sorbitan fatty acid esters, polyethylene glycol alkyl ethers, sugar esters, polyethylene glycol alkyl phenols, polyoxyethylene- olyoxypropylene block copolymers, sorbitan fatty acid esters, lower alcohol fatty acid esters, ionic surfactants, and mixtures thereof.
  • the pharmaceutical compositions or formulations comprising PD-L1 -based chimeric proteins and/or chimeric protein complexes described herein comprise a surfactant (e.g. polysorbate 20 (PS20) or poloxamer 188), at a concentration of at least about 0.01 % (w/v) to about 0.1 % (w/v), or at least about 0.01 % (w/v) to about 0.05% (w/v), or at least about 0.02% (w/v) to about 0.09% (w/v), or at least about 0.03% (w/v) to about 0.08% (w/v), or at least about 0.04% (w/v) to about 0.07% (w/v), or at least about 0.05% (w/v) to about 0.06% (w/v).
  • a surfactant e.g. polysorbate 20 (PS20) or poloxamer 188
  • PS20 polysorbate 20
  • poloxamer 188 poloxamer 188
  • the pharmaceutical compositions or formulations described herein comprise a surfactant at a concentration of about 0.01 % (w/v), about 0.02% (w/v), about 0.03% (w/v), about 0.04% (w/v), about 0.05% (w/v), about 0.06% (w/v), about 0.07% (w/v), about 0.08% (w/v), about 0.09% (w/v), or about 0.1 % (w/v).
  • the pharmaceutical compositions or formulations comprising PD-L1 -based chimeric proteins and/or chimeric protein complexes described herein comprise a tonicifier, which can be used singly or in combination, include, but are not limited to, trehalose, dextrose, sucrose, glycerin, mannitol, sorbitol, arginine, sodium chloride, or potassium chloride.
  • the pharmaceutical compositions or formulations described herein comprise one or more tonicifier excipients selected from trehalose, trehalose-sodium chloride, sucrose, and mannitol.
  • the tonicifier comprises or consists of a sugar, such as trehalose, sucrose, or mannitol.
  • the pharmaceutical compositions or formulations described herein comprise a tonicifier at a concentration of at least about 50 mM to about 350 mM, or from at least about 100 mM to about 300 mM, or from at least about 150 mM to about 250 mM.
  • a sugar tonicifier is present in the pharmaceutical composition or formulation at a concentration of about 50, mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, or about 350 mM.
  • the tonicifier is present at a concentration of at least about 90 mM to about 270 mM, optionally at least about 190 mM to about 195 mM.
  • the tonicifier comprises or consists of sodium chloride.
  • the pharmaceutical compositions or formulations described herein comprise sodium chloride at a concentration of at least about 10 mM to no more than 50 mM.
  • the pharmaceutical compositions or formulations described herein comprise sodium chloride at a concentration of from about 10 mM to about 50 mM, or from about 20 mM to about 50 mM, or about 30 mM to about 50 mM, or about 40 mM to about 50 mM.
  • sodium chloride is present in the pharmaceutical composition or formulation at a concentration of about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, or about 70 mM.
  • the pharmaceutical compositions or formulations comprising PD-L1 -based chimeric proteins and/or chimeric protein complexes described herein include one or more additional excipients.
  • the one or more additional excipients are selected from a stabilizer, a salt, an amino acid, and an antioxidant.
  • the pharmaceutical compositions or formulations comprising PD-L1 -based chimeric proteins and/or chimeric protein complexes described herein further comprise an additional stabilizer excipient.
  • the stabilizer is selected from one or more of cyclodextrin, arginine, sodium chloride-arginine, and glycine.
  • the cyclodextrin is beta-cyclodextrin, optionally HP-beta-cyclodextrin (hydroxypropyl-beta- cyclodextrin).
  • the stabilizer is present at a concentration of at least about 20 mM to about 100 mM, or at least about 25 mM to about 75 mM. In embodiments, the stabilizer is present at a concentration of at least about 50 mM
  • the pharmaceutical compositions or formulations comprising PD-L1 -based chimeric proteins and/or chimeric protein complexes described herein further comprise an amino acid excipient.
  • the amino acid excipient comprises one or more of arginine, glycine, glutamate, and histidine, optionally wherein the additional excipient is arginine.
  • the pharmaceutical compositions or formulations described herein comprise arginine at a concentration of at least about 10 mM to no more than 50 mM.
  • the pharmaceutical compositions or formulations described herein comprise arginine at a concentration of from about 10 mM to about 50 mM, or from about 20 mM to about 50 mM, or about 30 mM to about 50 mM, or about 40 mM to about 50 mM.
  • arginine is present in the pharmaceutical composition or formulation at a concentration of about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, or about 70 mM.
  • the pharmaceutical compositions or formulations comprising PD-L1 -based chimeric proteins and/or chimeric protein complexes described herein further comprise an antioxidant excipient.
  • the antioxidant excipient is selected from methionine and ethylenediaminetetraacetic acid (EDTA).
  • EDTA ethylenediaminetetraacetic acid
  • the antioxidant is methionine.
  • the pharmaceutical compositions or formulations described herein comprise an antioxidant at a concentration of at least about 1 mM to about 15 mM, or from at least about 1 mM to about 10 mM, or from at least about 2 mM to about 10 mM, or from at least about 3 mM to about 10 mM, or from at least about 1 mM to about 5 mM, or from at least about 2 mM to about 5 mM, or from at least about 3 mM to about 5 mM.
  • an antioxidant excipient is present in the pharmaceutical composition or formulation at a concentration of about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM.
  • compositions or formulations described herein comprise a PD-L1-based chimeric protein or protein complex that is present at a concentration from at least about 0.25 mg/mL to about 15 mg/mL.
  • pharmaceutical compositions or formulations described herein comprise a PD-L1-based chimeric protein or protein complex that is present at a concentration from at least about 0.25 mg/mL to about 10 mg/mL, from at least about 0.5 mg/mL to about 10 mg/mL, from at least about 0.75 mg/mL to about 10 mg/mL, from at least about 1 mg/mL to about 10 mg/mL, from at least about 1 mg/mL to about 15 mg/mL.
  • compositions or formulations described herein comprise at least about 0.25 mg/mL, or at least about 0.5 mg/mL, or at least about 0.75 mg/mL, or at least about 1 mg/mL, or at least about 1 .25 mg/mL, or at least about 1 .5 mg/mL, or at least about 1 .75 mg/mL, or at least about 2 mg/mL, at least about 3 mg/mL, at least about 4 mg/mL, at least about 5 mg/mL, at least about 6 mg/mL, or at least about 7 mg/mL, or at least about 8 mg/mL, or at least about 9 mg/mL, or at least about 10 mg/mL of a PD-L1 -based chimeric protein or protein complex.
  • the pharmaceutical compositions or formulations described herein comprise (a) a Programmed death-ligand 1 (PD-LI)-based chimeric protein complex comprising (i) a targeting moiety that specifically binds to PD-L1 , (ii) a modified human IFNo2, and (iii) a modified Fc domain, wherein the PD-L1-based chimeric protein complex is present at a concentration from at least about 1 mg/mL to about 10 mg/mL; (b) a buffering agent comprising histidine, optionally further comprising acetic acid, wherein histidine is present at a concentration of at least about 5 mM to about 20 mM and/or acetic acid present at a concentration ranging from at least about 5 mM to about 20 mM; (c) an excipient comprising a surfactant selected from PS20 at a concentration of at least about 0 05% (w/v) and Pol 188 at
  • the pharmaceutical compositions or formulations described herein comprise (a) a Programmed death-ligand 1 (PD-LI)-based chimeric protein complex comprising (i) a targeting moiety that specifically binds to PD-L1 , (ii) a modified human IFNa2, and (iii) a modified Fc domain, wherein the PD-L1-based chimeric protein complex is present at a concentration of at least about 1 mg/mL; (b) a buffering agent comprising histidine, optionally further comprising acetic acid, wherein histidine is present at a concentration of at least about 5 mM to about 20 mM and/or acetic acid present at a concentration ranging from about 5 mM to about 20 mM; (c) an excipient comprising a surfactant selected from PS20 at a concentration ranging from about 0.01 % (w/v) to about 0.1 % (w/v), and Pol 188 at a concentration ranging
  • PD-LI Programme
  • the pharmaceutical compositions or formulations described herein comprise (a) a Programmed death-ligand 1 (PD-LI)-based chimeric protein complex comprising (i) a targeting moiety that specifically binds to PD-L1 , (ii) a modified human IFNo2, and (iii) a modified Fc domain, wherein the PD-L1-based chimeric protein complex is present at a concentration from at least about 1 mg/mL to about 10 mg/mL; (b) a buffering agent comprising histidine and acetic acid, wherein histidine is present at a concentration of at least about 10 mM to about 15 mM and acetic acid is present at a concentration of at least about 10 mM to about 15 mM; (c) an excipient comprising a surfactant selected from PS20 at a concentration ranging from about 0.01% (w/v) to about 0.05% (w/v), and Pol 188 at a concentration of about
  • PD-LI Programmed
  • pharmaceutical formulations including PD-L1 -based chimeric proteins and/or chimeric protein complexes may be presented in unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the therapeutic agents into association with a carrier, which constitutes one or more accessory ingredients. In embodiments, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation.
  • the pharmaceutical formulation comprises a preservative, such as, but not limited to, phenol, meta-cresol, or sodium benzoate.
  • Pharmaceutically acceptable carriers include, but are not limited to, water, saline, and glycerol.
  • the pharmaceutical composition or formulation described herein comprises fixed oils, polyethylene glycol, propylene glycol or other solvents.
  • the solvent is water.
  • the composition is contained in an injection device, such as a syringe or an injection pen.
  • the composition is contained in an injection pen.
  • Auto-injectors such as an “injection pen” are spring-loaded syringes designed to deliver a dose of a particular drug.
  • injection pens are easy to use and are intended for self-administration by patients, or administration by untrained personnel.
  • Injection pens are designed to overcome the hesitation associated with self-administration of the needle-based drug delivery device.
  • the injection pen keeps the needle tip shielded prior to injection and also has a passive safety mechanism to prevent accidental firing (injection). Injection depth can be adjustable or fixed and a function for needle shield removal may be incorporated.
  • the syringe needle is automatically inserted into the subcutaneous tissue and the drug is delivered. Once the injection is completed some injection pens have a visual or audible indication to confirm that the full dose has been delivered.
  • the injection device contains from 1 to 10 unit doses or from 1 to 5 unit doses. In some embodiments, the unit doses are no more than about 1.5 mLs or about 1 mL in volume (whether or not contained or delivered by an injection device). In some embodiments, the unit doses are no more than 0.8 mL in volume, or no more than 0.7 mL in volume. In some embodiments, the injection device delivers a microdose, e.g., having a volume in the range of about 50 piL to about 500 piL, or a volume in the range of from about 75 piL to about 250 piL. In some embodiments, the microdose has a volume of from 100 to 200 piL.
  • the pharmaceutical composition or formulation of the present application is suitable for administration by subcutaneous, intramuscular, intradermal, or intravenous administration.
  • the subject receives a unit dose of the pharmaceutical composition or formulation about once every 1 week, or about once every 2 weeks, or about once every 3 weeks, or about once every 4 weeks (e.g., about once per month), or about once every 6 weeks, or about once every 8 weeks (about once every 2 months).
  • the pharmaceutical composition or formulation described herein is freeze-dried to form a lyophilized powder.
  • the present application contemplates a kit comprising (a) the pharmaceutical composition or formulation of the present application that has been freeze-dried to form a lyophilized powder, (b) a vial (e.g., glass vial), and (c) an injection device (e.g., a syringe or injection pen).
  • the lyophilized formulation is marketed in pharmaceutical dosage form.
  • the pharmaceutical dosage form of the present application although in embodiments in the form of a vial, may be any suitable container, such as ampoules, syringes, co-vials, which are capable of maintaining a sterile environment. Such containers can be glass or plastic, provided that the material does not interact with the pharmaceutical formulation.
  • the closure is a stopper, e.g., a sterile rubber stopper, or a siliconized stopper, which affords a seal.
  • a vial will contain a lyophilized powder including about 0.5 to about 10 g/vial.
  • a kit comprising a lyophilized powder of the PD-L1-based chimeric protein complex pharmaceutical formulation, a glass vial and optionally a syringe.
  • the lyophilized formulations of the present application may be reconstituted with water, such as Sterile Water for Injection, or other sterile fluid such as co-solvents, to provide an appropriate solution of PD-L1-based chimeric protein complex for administration, as through parenteral injection following further dilution into an appropriate intravenous admixture container, for example, including normal saline.
  • water such as Sterile Water for Injection
  • co-solvents such as co-solvents
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc. at least one chemical entity and optional pharmaceutical adjuvants in a carrier (e.g., water, saline, aqueous dextrose, glycerol, glycols, ethanol or the like) to form a solution or suspension.
  • a carrier e.g., water, saline, aqueous dextrose, glycerol, glycols, ethanol or the like
  • I njectables can be prepared in conventional forms, either as liquid solutions or suspensions, as emulsions, or in solid forms suitable for dissolution or suspension in liquid prior to injection.
  • the percentage of chemical entities contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the activity of the chemical entities and the needs of the subject.
  • Preparation of the pharmaceutical composition or formulation may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable excipient is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. The excipient(s) must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • the pharmaceutical composition of the application exhibits short-term and/or long-term stability.
  • stability of the pharmaceutical composition described herein is measured by monitoring one or more of sample appearance, sample pH, sample osmolality, sample potency (ELISA and cell-based assay), sample SDS-PAGE (reducing and non-reducing), sample protein concentration (UV absorbance), sample SE-HPLC, sample RP-HPLC, sample clEF, sample sub-visible particles, and sample MS.
  • the pharmaceutical composition described herein is stable for at least 2 weeks at 30°C to 50°C (e.g, at 30°C, 35°C, 40°C, 45°C, or 50°C), optionally further wherein the relative humidity is 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%.
  • the pharmaceutical composition described herein is stable for at least 1 month or 2 months at 15°C to 35°C (e.g., at 15°C, 20°C, 25°C, 20°C, or 35°C), optionally further wherein the relative humidity is 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%.
  • the pharmaceutical composition described herein is stable for at least 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, or 36 months at 1 °C,to 10°C (e.g., at 1 °C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, or 10°C).
  • the pharmaceutical composition described herein is stable for at least 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, or 36 months at -15°C to - 25°C (e.g, at -15°C, -20°C, or -25°C).
  • the pharmaceutical composition described herein is stable after undergoing one, two, three, four, or five freeze and thaw cycles.
  • the present application relates to a method for treating or preventing a cancer, comprising administering an effective amount of the pharmaceutical composition as disclosed herein to a patient in need thereof.
  • the pharmaceutical composition described herein can be used for the treatment or prevention of a blood cancer (e.g, a hematologic cancer dealing with blood cells) or a solid tumor cancer (e.g, a cancer of any other body organ or tissue).
  • a blood cancer e.g, a hematologic cancer dealing with blood cells
  • a solid tumor cancer e.g, a cancer of any other body organ or tissue.
  • the pharmaceutical composition can be used for the treatment or prevention of a cancer selected from one or more of lymphoma, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and/or central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and
  • the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication.
  • the language “about 50” covers the range of 45 to 55.
  • the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or disorder or one or more signs or symptoms associated with a disease or disorder.
  • the amount of a composition administered to the subject will depend on the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • the compositions can also be administered in combination with one or more additional therapeutic compounds.
  • the therapeutic compounds may be administered to a subject having one or more signs or symptoms of a disease or disorder.
  • something is “decreased” if a read-out of activity and/or effect is reduced by a significant amount, such as by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to and including at least about 100%, in the presence of an agent or stimulus relative to the absence of such modulation.
  • activity is decreased and some downstream read-outs will decrease but others can increase.
  • activity is "increased” if a read-out of activity and/or effect is increased by a significant amount, for example by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to and including at least about 100% or more, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, in the presence of an agent or stimulus, relative to the absence of such agent or stimulus.
  • compositions described herein needed for achieving a therapeutic effect may be determined empirically in accordance with conventional procedures for the particular purpose. Generally, for administering therapeutic agents for therapeutic purposes, the therapeutic agents are given at a pharmacologically effective dose.
  • a “pharmacologically effective amount,” “pharmacologically effective dose,” “therapeutically effective amount,” or “effective amount” refers to an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease.
  • An effective amount as used herein would include an amount sufficient to, for example, delay the development of a symptom of the disorder or disease, alter the course of a symptom of the disorder or disease (e.g., slow the progression of a symptom of the disease), reduce or eliminate one or more symptoms or manifestations of the disorder or disease, and reverse a symptom of a disorder or disease.
  • Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.
  • compositions for treating the diseases or disorders described herein are equally applicable to use of a composition for treating the diseases or disorders described herein and/or pharmaceutical compositions for use and/or uses in the manufacture of a medicaments for treating the diseases or disorders described herein.
  • Fc domain mutations are numbered according to EU convention (Edelman et al., PNAS 1969; 63 (1) 78-85, incorporated by reference in its entirety).
  • LALA mutation refers to a double mutant Fc domain having L234A mutation and a L235A mutation.
  • KQ refers to a mutant Fc domain having a K322Q mutation
  • Knob in hole mutants are those described in Ridgway et al., Protein Engineering 1996;9:617-621, which is hereby incorporated by reference in its entirety, i.e. Y407T / T366Y.
  • knob in hole mutants are those described in Merchant et al., Nature Biotechnology 1998; 16:677-681, which is incorporated by reference in its entirety, i.e. S354C:T366W / Y349C:T366S:L368A:Y407V.
  • Plasmids P-3003 and P-3004 (respectively pcDNA3.4 plasmids coding for (i) a sequence optimized PD- L1 binding VHH fused to a human lgG1 Fc domain or (ii) a human lgG1 Fc domain fused to an attenuated human IFNo2) were together transiently expressed in an ExpiCHO expression system (Thermo Fisher) according to the manufacturer’s guidelines. The human lgG1 Fc domains carry effector knock-out and knob-hole mutations. One week after transfection, the culture was collected, cells removed by centrifugation and the supernatants frozen.
  • CEX Elution fractions were selected and pooled based on SDS- PAGE for minimal contamination by host-cell proteins and/or PD-L1-based chimeric protein product variants resulting in a batch with a concentration of 5.97 mg PD-L1-based chimeric protein/mL.
  • Example 2 Formulation Buffer Stabilizes PD-L1 -Based Chimeric Protein at 10 mg/mL During Storage
  • An aim of this experiment was to screen buffers in order to identify formulations that ensure the stability of the PD-L1-based chimeric protein at a concentration of 10 mg/mL during storage. Specifically, 16 different buffers (different buffer type and/or pH, optionally with 100 mM NaCI) were prepared (see Table 1) for solubility screening of the PD-L1 -based chimeric protein:
  • Buffers containing 100 mM NaCI were prepared from the 50 mM buffer without NaCI. The necessary amount of NaCI was weighed and added to a 50 mL volumetric flask. Then, 40 mL of the corresponding buffer was added to the volumetric flask, and the solution was stirred until clear. The volume of the volumetric flask was then completed with the corresponding buffer. The solution was homogenized by tumbling.
  • Solubility of the PD-L1-based chimeric protein was tested by adding the protein to increasing amounts of PEG 6000 in the 16 different buffers and by measuring turbidity as a read-out for protein aggregation.
  • buffer/PEG solutions were prepared in a 96 deep well plate: the 200 mM buffer stock solutions, water, and the 50% (w/v) PEG 6000 stock solution were mixed in a way that a fixed concentration of 29.8 mM buffer was present in each well, and an increasing concentration (0%, 2.4%, 4.8%, 7.2%, 9.5%, 11 9%, 14.3%, 16.7%, 19.1%, 21.5%, 23.8%, and 26.2%) of PEG was reached within the same row.
  • the PD-L1 -based chimeric protein solution batch was dialyzed twice overnight (with a 6-8 kDa MWCO dialysis membrane) at 2-8°C against 1 L 10 mM Na-acetate, pH 5.0, and filtered through a sterilizing 0.2 pm filter. This final PD-L1 -based chimeric protein stock solution had a concentration of 5.9 mg/mL.
  • the turbidity measurements shown in Figure 1 indicate that the best 4 buffers without NaCI in which the PD-L1 -based chimeric protein was less prone to precipitate were shown to be: acetate buffer at pH 4.5, acetatehistidine buffer at pH 5.0, histidine buffer at pH 5.0, and histidine buffer at pH 5.5.
  • a pH above 5.5 was shown to be generally less favorable, but surprisingly, citrate buffers in the pH range 4.0 to 5.0 were also clearly less favorable for solubility of the PD-L1-based chimeric protein. In general, the addition of NaCI did not improve solubility.
  • the PD-L1-based chimeric protein solution batch was dialyzed twice (with a 3.5 kDa MWCO dialysis membrane) at room temperature against 0.9 L 10 mM Na-acetate, pH 5.0 and filtered through a sterilizing 0.2 pm filter. This final PD-L1-based chimeric protein stock solution had a concentration of 5.6 mg/mL.
  • Table 2 Composition per mL of the buffers for PD-L1-based chimeric protein or protein complex excipient screening
  • Table 3 Composition per mL of the excipients for PD-L1-based chimeric protein or protein complex excipient screening
  • the buffer was pipetted in the wells followed by the tonicifier (sucrose, trehalose, mannitol or NaCI) with varying volume to reach an isotonic solution, 10 L of the stabilizing excipients (arginine, glycine, or HP beta cyclodextrin), 5 pL of the antioxidant and the remaining amount of purified water type I to reach a volume of 41.67 pL.
  • the stabilizing excipients arginine, glycine, or HP beta cyclodextrin
  • 8.33 pL of the PD-L1 -based chimeric protein solution was added and each solution was mixed in the well. The well plate was then centrifuged for 7 minutes at 4,000 g.
  • the supernatant of the solutions was then used for differential scanning fluorimetry (DSF) and static light scattering (SLS) measurement using the Uncle (Unchained labs).
  • the Uni’s minicuvettes
  • the measurement was performed with a temperature ramp starting at 25°C up to 95 C at a rate of 0.4 C/min and 180 sec of equilibration at the start.
  • the emission spectrum was captured between 250-720 nm with excitation at 266 nm.
  • the SLS signal was measured at 266 nm and 473 nm.
  • DLS data was measured with 4x5 sec of acquisitions resulting in the determination of T ori set,
  • Table 4 96 Excipient Concepts Tested Per Buffer Condition in the Thermal Shift Assay [0158] The obtained T m , data was imported in MODDE to analyze the model and trends. Each response (T m , T agg , T on set and SLSmax) was fitted with a model optimized for the model terms. Each model term (the constant, buffer, tonicifier, stabilizer, antioxidant, or a combination of two single terms as quadratic terms) that did not significantly influence the response was not included in the model.
  • the model for a response can be used if the R 2 value, the Q 2 value, and the reproducibility are above 0.5, if the model validity is around 0.25 or higher and if the difference between R 2 and Q 2 is less than 0.3.
  • T m and T on set models were further used for analysis of the stabilizing excipient.
  • Table 5 Overview of the different concepts for the shear stress study.
  • Table 6 Method parameters for DLS measurements.
  • the short stress study was performed with 5 different concepts which were stressed at 40°C/75% RH and 25°C/60% RH for up to T2w (2 weeks).
  • the composition of the tested formulations is shown in Table 7.
  • the histidine-acetate buffer was chosen with a targeted PD-L1 -based chimeric protein concentration of 10 mg/mL.
  • Trehalose was added in all concepts as a tonicifier in order to reach the target of about 310 mOsm/kg.
  • the last concept contained a combination of two tonicifiers being trehalose and NaCI.
  • 3 mM methionine as antioxidant and 0.02% PS20 as surfactant were used.
  • Table 7 Overview of the different concepts for the short stress study.
  • Nr. luru iiei (0.02% w/v) (50 mM) (3 mM)
  • the PD-L1-based chimeric protein samples were diluted to 1 mg/mL in their respective buffers. Before HPLC analyses, samples were centrifuged at 20000 g for 5 minutes, after which 4
  • Table 8 Results, as area% of each peak section, of short stress study samples measured using RPC.
  • SE-HPLC was performed as described in M-P10101-006 v2.0. The samples were diluted to 1 mg/mL. Before HPLC analyses, samples and references were centrifuged at 20000 g for 5 minutes.
  • Proteins were detected by bio-compatible Titanium flow cell (5 mm) at 280 nm wavelength. Column was kept at 25°C, while samples were stored in cooled sample injector (7°C) until analysis. The analysis (depicted in Table 9) indicates that for all concepts there were no major changes at 25°C for up to 2 weeks. At 40°C, an increase in both pre- and post-peaks was observed for all formulations; however, the increase in higher molecular weight species (pre-peaks) was smallest for concepts 1 and 2.
  • Table 9 Results, as area% of each peak section, of short stress study samples measured using SEC.
  • Some concepts showed peaks of around 1 nm, which is mainly related to excipients since these peaks were also present in the placebo.
  • the PD-L1-based chimeric protein-related peak at 10 nm was present in all concepts before vortexing.
  • Concept 4 contained already before vortexing some higher sized particles.
  • the PD-L1-based chimeric protein-related peak at around 10 nm was rather small in concept 2 and 5, which was probably related to the presence of HP-
  • a few bigger sized peaks were present which could be related to the presence of surfactant micelles. Note that these bigger sized particles were not present in the placebo of concept 4, which did not contain a surfactant.
  • Table 10 Overview of the different concepts for the extended stress study.
  • Nr. tonici er (0 02 o /o w/v) (50 mM) (3 mM)
  • Stability was monitored for appearance, pH, osmolality, potency (ELISA and cell-based assay), SDS- PAGE (reducing and non-reducing), protein concentration (UV absorbance), SE-HPLC, RP-HPLC, clEF, sub-visible particles, and MS.
  • a new batch of PD-L1 -based chimeric protein was produced in a 100L bioreactor and was harvested by depth filtration and subsequently purified as before by proteinA and cation-exchange chromatography.
  • a final polishing step was performed on the cation-exchange peak fractions using an anion-exchange filter (SartobindQ, Sartorius) after which the protein was concentrated to about 15 mg/mL by tangential flow filtration against the formulation buffer without the beta-cyclodextrin and Tween-20.
  • 3-cyclodextrin were added from respectively at 50x and 5x stock concentration prepared in aforementioned buffer.
  • the protein was diluted to about 10.5 mg/mL with formulation buffer and aseptically filtered over a 0.22 m filter.
  • the new batch of PD-L1 -based chimeric protein was filled at 5.5 mL in 6R glass vials closed off with bromobutyl rubber stoppers and aluminum caps.
  • Part of the protein stock was compounded with formulation buffer to a 1 mg/mL protein complex concentration and similarly filled at 5.5 mL in 6R glass vials.
  • the glass vials are stored at either -20°C (upright 1 , 3, 6, 9, 12, 18, 24, 36 months) or at 2-8°C (upright and inverted 1 , 3 and 6 months).
  • Table 12 Results, as area% of each peak section, of long-term stability study samples as tested in SEC.
  • Table 13 Results, as area% of each peak section, of long-term stability study samples as tested in RPC.
  • the samples were also analyzed by a PD-L1/anti-IFN ELISA, which allows determination of the potency of the PD-L1-based chimeric protein to bind simultaneously to the human PD-L1 protein and a neutralizing human IFNa2 monoclonal antibody.
  • MAXISORP Nunc immune plates were coated with anti-human IFN-alpha monoclonal antibody (1 g/mL, clone MMHA-13; PBL assay science). After washing and blocking the plate, an 8-step 3-fold serial dilution in blocking buffer of PD-L1-based chimeric protein samples starting from 1 mg/mL were loaded on the plate.
  • Table 14 Results, as % EC50 versus reference, of long-term stability study samples as measured using ELISA.
  • the samples were also tested for the appearance of new charge variants by capillary iso-electric focusing (clEF).
  • clEF capillary iso-electric focusing
  • the sample was diluted to a final concentration of 1 mg/mL.
  • this solution was desalted using a spin-column by loading 500 pL sample onto the column, centrifuging the column for 5 min at 14000 x g, and bringing the volume back to 500 pL with MQ water.
  • the desalting procedure was repeated two more times, after which 20 pL of the desalted sample was added to 80 pL of the clEF master mix (19.5 pL water, 19.5 pL 10 M urea, 34 pL 1% methyl cellulose, 1 pL Pharmalyte 3-10, 3 pL Pharmalyte 5-8, 2 pL 500 mM Arginine, 1 pL pl marker 6.14, 1 pL pl marker 8.40) into a microcentrifuge tube.
  • the final concentration of the PD-L1 -based chimeric protein corresponded to 0.2 mg/mL.
  • Table 15 Results, as area% of each peak section, of long-term stability study samples as tested in clEF.

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Abstract

The present application provides, in part, pharmaceutical formulations of PD-L1 -based chimeric proteins and protein complexes and uses thereof. In some embodiments, it includes a pharmaceutical composition or formulation comprising (a) a Programmed death-ligand 1 (PD-L l)-based chimeric protein or chimeric protein complex comprising (i) a targeting moiety that specifically binds to PD-L1, (ii) a modified human IFNa2, and (iii) a modified Fe domain; (b) at least one pharmaceutically acceptable buffering agent comprising a histidine buffer (e.g., at a concentration of at least about 10 mM to about 30 mM); (c) at least one pharmaceutically acceptable excipient comprising a surfactant; and (d) at least one pharmaceutically acceptable excipient comprising a tonicifier.

Description

PD-L1-BASED CHIMERIC PROTEIN FORMULATIONS
FIELD
[001] The present application provides, in part, pharmaceutical formulations of PD-L1-based chimeric protein complexes, and uses thereof.
CROSS-REFERENCE TO RELATED APPLICATIONS
[002] This application claims the benefit of U.S. Provisional Patent Application No. 63/600,247, filed November 17, 2023, the entire contents of which are hereby incorporated by reference in their entirety.
SEQUENCE LISTING
[003] This application contains a Sequence Listing in XML format submitted electronically herewith via Patent Center. The contents of the XML copy, created on November 11, 2024, is named “ORN-093PC_114384-5093.xmr and is 127,597 bytes in size. The Sequence Listing is incorporated herein by reference in its entirety.
BACKGROUND
[004] Biologies with an effector function are a class of agents that have many potential therapeutic applications. In some instances, these biologies, e.g., cytokines, encode effector functions that can be systemically toxic if administered to humans. Accordingly, maximizing tolerability and therapeutic index of these biologies in humans is important so that systemic toxicity in humans or subjects can be reduced.
[005] Often, these biologies need be delivered to their target(s) inside a subject with high precision and in a regulated manner in order for them to be effective. Thus, there is a need for engineering biological molecules that have high inherent safety profile, have the ability to reach their target inside the subject with high precision, and are able to function in a regulated fashion.
[006] One example of such biologies is a chimeric protein having a signaling agent (having an effector function, e.g., a cytokine), connected to a targeting element (having the ability to seek its target with high precision). In these biologies, the signaling agent can be a wild type signaling agent or a modified signaling agent (e.g. by mutation). The modified signaling agent is, generally, modified to cause an attenuation of the signaling agent’s activity (e.g., substantially reducing its ability to interact with/engage its receptor) in a manner such that the signaling agent’s effector function can be recovered upon binding of the targeting element to its target (e.g., antigen on target cell).
[007] However, such chimeric proteins are most amenable to therapeutic use if certain conditions are met, e.g., the ability to be produced in a large scale, an in vivo half-life that ensures adequate time of exposure to the drug to elicit a therapeutically beneficial effect, a proper size to avoid rapid clearance or limited tissue penetrance and biodistribution, and other properties that ensure adequate solubility, stability and storage without significant loss of function. Importantly all, or substantially most, of the above properties should be achieved without a loss of the conditional targeting of the effector function and retention of conditional engagement of a modified signaling agent with its receptor. Often, it is difficult to achieve all these objectives with chimeric proteins There is a need in the art where such desirable therapeutic properties of the biologic can be achieved while maintaining the tolerability and therapeutic index of the biologic, as well as ensuring adequate solubility, stability and storage of the pharmaceutical composition.
SUMMARY
[008] Accordingly, in aspects, the present application provides a pharmaceutical composition or formulation comprising (a) a Programmed death-ligand 1 (PD-LI)-based chimeric protein or chimeric protein complex (e.g., at a concentration of at least about 1 mg/mL) comprising (i) a targeting moiety that specifically binds to PD-L1 , (ii) a modified human I FNa2, and (iii) a modified Fc domain; (b) at least one pharmaceutically acceptable buffering agent comprising a histidine buffer (e.g., at a concentration of at least about 10 mM to about 30 mM); (c) at least one pharmaceutically acceptable excipient comprising a surfactant (e.g., polysorbate 20 (PS20) or poloxamer 188 (Poll 88)); and (d) at least one pharmaceutically acceptable excipient comprising a tonicifier (e.g., trehalose, sucrose, or mannitol). In embodiments, the pharmaceutical composition has a pH that ranges from at least about 4.0 to about 6.0.
[009] The present technology provides pharmaceutical compositions and/or formulations including chimeric protein complexes that comprise biological therapeutic agents whose effector function can be delivered in a highly precise fashion to a target of choice, with limited or no cross-reactivities, and with limited or no systemic adverse events, while also providing improved pharmaceutical features, such as solubility, stability, and storage properties.
[010] In embodiments, the pharmaceutical compositions or formulations are suitable for parenteral administration, such as intravenous administration (e.g., intravenous infusion or bolus injection).
[011] In embodiments, the pharmaceutical compositions or formulations of the present application comprise one or more additional excipients, such as, but not limited to, stabilizers (e.g., beta-cyclodextrin), salts, amino acids, and antioxidants.
[012] In an aspect, the present application relates to methods for treating or preventing a cancer, comprising administering an effective amount of the pharmaceutical composition as disclosed herein to a patient in need thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[013] Figure 1 depicts turbidity measurements at 500 nm of PD-L1 -based chimeric protein complex incubated in 16 different buffer types with increasing amounts of PEG 6000.
[014] Figure 2 depicts a coefficient plot of Tm where positive coefficient bars represent excipients having a stabilizing effect on the formulation and negative coefficient bars represent excipients having a destabilizing effect.
[015] Figure 3 depicts a coefficient plot of SLSmax where negative coefficient bars represent excipients having a stabilizing effect on the formulation and positive coefficient bars represent excipients having a destabilizing effect. [016] Figure 4 depicts a coefficient plot of Tagg where positive coefficient bars represent excipients having a stabilizing effect on the formulation and negative coefficient bars represent excipients having a destabilizing effect (log transformed).
[017] Figure 5 depicts a coefficient plot of Tonset where positive coefficient bars represent excipients having a stabilizing effect on the formulation and negative coefficient bars represent excipients having a destabilizing effect.
[018] Figure 6 shows size distributions based on intensity and volume for the sample before stress and after 15 minutes or 1 hour of vortexing the formulations without surfactant (no. 1), or with 0.01% (w/v) PS20 (no. 2), or with 0.05% (w/v) PS20 (no. 3).
[019] Figure 7 shows size distributions based on intensity and volume for the sample before stress and after 15 minutes or 1 hour of vortexing the formulations without surfactant (no. 1), or with 0.01 % (w/v) Pol 188 (no. 2), or with 0.05% (w/v) Poll 88 (no. 3).
[020] Figures 8A-8B shows particle size distribution based on intensity and volume of various short stress concepts before (no. 2) and after being subjected to 15 minutes (no. 3) or 1 hour (no. 4) of shear stress. The formulation without the PD-L1-based chimeric protein complex added is shown before vortexing as no. 1 .
[021] Figure 9 depicts results of a SDS-PAGE under non-reducing conditions experiment: lanes 1 , 4 and 8: molecular weight markers; lanes 5 and 9: 100 ng BSA control; lanes 2, 6 and 10: 10 pig PD-L1-based chimeric protein in formulation 1 after 0, 1 , and 2 weeks at 40°C; lanes 3, 7 and 11 : 10 pig PD-L1-based chimeric protein in formulation 2 after 0, 1 , and 2 weeks at 40°C.
[022] Figure 10 shows results of a Western blot (anti-human Fc) under non-reducing conditions experiment: lanes 1, 4 and 7: molecular weight markers; lanes 2, 5 and 8: 10 pig PD-L1-based chimeric protein in formulation 1 after 0, 1 , and 2 weeks at 40°C; lanes 4, 6 and 9: 10 pig PD-L1-based chimeric protein in formulation 2 after 0, 1 , and 2 weeks at 40°C.
DETAILED DESCRIPTION
[023] The present disclosure provides, in part, pharmaceutical compositions and/or formulations comprising Programmed death-ligand 1 (PD-LI)-based chimeric proteins or chimeric protein complexes, which, without wishing to be bound by theory, can be stably formulated for efficacious biological effect and convenient dosing schedules, dosing volumes, and patient-friendly delivery devices.
[024] The present application provides, in an aspect, a pharmaceutical composition or formulation comprising (a) a Programmed death-ligand 1 (PD-LI)-based chimeric protein or chimeric protein complex comprising (i) a targeting moiety that specifically binds to PD-L1, (ii) a modified human IFNo2, and (iii) a modified Fc domain; (b) at least one pharmaceutically acceptable buffering agent comprising histidine, optionally further comprising acetic acid; (c) at least one pharmaceutically acceptable excipient comprising a surfactant; and (d) at least one pharmaceutically acceptable excipient comprising a tonicifier, and wherein the pharmaceutical composition or formulation has a pH that ranges from at least 4.0 to at least about 6.0.
[025] The present technology provides pharmaceutical compositions and/or formulations including chimeric protein complexes that comprise biological therapeutic agents whose effector function can be delivered in a highly precise fashion to a target of choice, with limited or no cross-reactivities, and with limited or no systemic adverse events, while also providing improved pharmaceutical features, such as solubility, stability, and storage properties. In embodiments, the pharmaceutical compositions and/or formulations of the present application can be produced in a large scale. In embodiments, the pharmaceutical compositions and/or formulations of the present application provide an in vivo half-life that ensures adequate time of exposure to the drug to elicit a therapeutically beneficial effect. In embodiments, the pharmaceutical compositions and/or formulations of the present application are sized to avoid rapid clearance or limited tissue penetrance and bio-distribution. In embodiments, the pharmaceutical compositions and/or formulations of the present application provide adequate solubility, stability and/or storage properties without significant loss of function.
PD-L1 -Based Chimeric Proteins and/or Protein Complexes
[026] In an aspect, the present application provides pharmaceutical compositions or formulations comprising a chimeric protein complex, where the chimeric protein complex comprises a targeting moiety that specifically binds to Programmed death-ligand 1 (PD-L1), a modified human IFNa2, and a modified Fc domain.
[027] In embodiments, the chimeric protein complex contemplated herein comprises a targeting moiety that specifically binds to PD-L1. In embodiments, the present application provides a PD-L1 targeting moiety comprising a recognition domain comprising:
(i) three complementarity determining regions (CDR1, CDR2, and CDR3), where
(a) CDR1 comprises an amino acid sequence selected from any one of SEQ ID NOs: 2 or 5;
(b) CDR2 comprises an amino acid sequence selected from any one of SEQ ID NOs: 3 or 6; and
(c) CDR3 comprises the amino acid sequence of SEQ ID NO: 4; or
(ii) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1; and where (I) or (ii) further comprises one or more mutations at positions D54 and G55, numbering relative to SEQ ID NO: 1 .
[028] In embodiments, the targeting moiety that specifically binds to PD-L1 comprises a recombinant heavychain-only antibody (VHH).
[029] In embodiments, the PD-L1 targeting moiety comprising a recognition domain further comprises one or more mutations at positions Q1, Q5, A14, A63, T74, K76, S79, K86, and Q110, relative to SEQ ID NO: 1. [030] In embodiments, the mutation is a substitution, optionally where the substitution is a polar and positively charged hydrophilic residue selected from arginine (R) and lysine (K), an aromatic, polar and positively charged hydrophilic residue including histidine (H), a polar and neutral of charge hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C), a polar and negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E) or a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).
[031] In embodiments, the mutation is selected from one or more of a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) at position D54, optionally being D54G, or a polar and positively charged hydrophilic residue selected from arginine (R) and lysine (K), optionally being D54K, or a polar and neutral of charge hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C), optionally being D54T and a polar and positively charged hydrophilic residue selected from arginine (R) and lysine (K) at position G55, optionally being G55R.
[032] In embodiments, the mutation is selected from one or more of a polar and negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E) at position Q1 , optionally being Q1D; a hydrophobic, aliphatic amino acid selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V) at position Q5, optionally being Q5V; a polar and neutral of charge hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C) at position A14, optionally being A14P; a hydrophobic, aliphatic amino acid selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V) at position A63, optionally being A63V; a polar and neutral of charge hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), proline (P), and cysteine (C) at position T74, optionally being T74S, a polar and neutral of charge hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C) at position K76, optionally being K76N, a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y) at position S79, optionally being S79Y, an arginine (R) at position K86, being K86R, and a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) at position Q110, optionally being Q110L.
[033] In embodiments, the mutation is selected from one or more of Q1 D, Q5V, A14P, A63V, T74S, S79Y, K86R, and Q110L, optionally all of Q1 D, Q5V, A14P, D54G, T74S, K76N, S79Y, K86R, and Q110L, relative to SEQ ID NO: 1.
[034] In embodiments, the aforementioned mutant PD-L1 targeting moieties (i.e. those disclosed relative to SEQ ID NO: 1) have improved affinity relative to a parental PD-L1 targeting moiety of SEQ ID NO: 1. [035] In embodiments, the PD-L1 targeting moiety of the present application includes a recognition domain comprising:
(i) three complementarity determining regions (CDR1 , CDR2, and CDR3), wherein:
(a) CDR1 comprises an amino acid sequence selected from any one of SEQ ID NOs: 26 or 29;
(b) CDR2 comprises an amino acid sequence selected from any one of SEQ ID NOs: 27 or 30; and
(c) CDR3 comprises the amino acid sequence of SEQ ID NO: 28; or
(ii) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 25; and wherein (i) or (ii) further comprises one or more mutations at positions N32, D33, and M97, numbering relative to SEQ ID NO: 25.
[036] In embodiments, the PD-L1 targeting moiety has a mutation that is a substitution relative to SEQ ID NO: 25. In embodiments, the substitution is a polar and positively charged hydrophilic residue selected from arginine (R) and lysine (K) or an aromatic, polar and positively charged hydrophilic residue including histidine (H). In embodiments, the substitution is a polar and neutral of charge hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C). In embodiments, the substitution is a polar and negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E). In embodiments, the substitution is a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).
[037] In embodiments, the PD-L1 targeting moiety has a substitution at position N32 that is a positive hydrophilic residue is selected from arginine (R) and lysine (K). In embodiments, the substitution at position N32 is polar and neutral hydrophilic residue that is selected from glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C). In embodiments, the substitution at position N32 is N32Q or N32R, relative to SEQ ID NO: 25.
[038] In embodiments, the PD-L1 targeting moiety has a substitution at position D33 is D33H relative to SEQ ID NO: 25. In embodiments, the PD-L1 targeting moiety has a substitution at position M97 that is aliphatic hydrophobic residues are selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V), relative to SEQ ID NO: 25. In embodiments, the PD-L1 targeting moiety has a substitution at position M97, relative to SEQ ID NO: 25, that is M97I, M97L, or M97V.
[039] In embodiments, the PD-L1 targeting moiety comprising a recognition domain further comprises one or more of the following mutations Q1 D, Q5V, A14P, A62S, A74S, M77T, M78V, S79Y, K86R, and Q109L, optionally all of Q1 D, Q5V, A14P, D33H, A62S, A74S, M77T, M78V, K86R, M97V, relative to SEQ ID NO: 25.
[040] In embodiments, the aforementioned mutant PD-L1 targeting moieties (i.e. those disclosed relative to SEQ ID NO: 25) have improved affinity relative to a parental PD-L1 targeting moiety of SEQ ID NO: 25. [041] In embodiments, the PD-L1-based chimeric protein or protein complex of the present application comprises a PD-L1 targeting moiety comprising a VHH comprising an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identity with any one of the amino acid sequences selected from SEQ ID NO: 23, 72, 1 , 7-22, 24, 25, and 31-71. In embodiments, the VHH has the amino acid sequence of any one of the amino acid sequences selected from SEQ ID NO: 23, 72, 1 , 7- 22, 24, 25, and 31-71.
[042] In embodiments, the PD-L1 targeting moiety is a single-domain antibody, such as a VHH. The VHH may be derived from, for example, an organism that produces VHH antibody such as a camelid, a shark, or the VHH may be a designed VHH. VHHs are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally-occurring heavy-chain antibodies. VHH technology is based on fully functional antibodies from camelids that lack light chains. These heavy-chain antibodies contain a single variable domain ( HH) and two constant domains (CH2 and CH3). In embodiments, the VHH is a humanized VHH or camelized VHH.
[043] In embodiments, the PD-L1 targeting moiety comprises a VHH comprising a single amino acid chain having four "framework regions” or FRs and three "complementary determining regions” or CDRs. As used herein, “framework region” or “FR” refers to a region in the variable domain which is located between the CDRs. As used herein, “complementary determining region” or “CDR" refers to variable regions in VHHs that contains the amino acid sequences capable of specifically binding to antigenic targets.
[044] In embodiments, the PD-L1 binding agent comprises a VHH having a variable domain comprising at least one CDR1 , CDR2, and/or CDR3 sequences. In various embodiments, the PD-L1 binding agent comprises a VHH having a variable region comprising at least one FR1, FR2, FR3, and FR4 sequences.
[045] In embodiments, the PD-L1 targeting moiety VHH's CDR1 sequence is selected from: GTIFSINRMD (SEQ ID NO: 2); GTIFS (SEQ ID NO: 5); GKIFSGNDMG (SEQ ID NO: 26); and GKIFS (SEQ ID NO: 29).
[046] In embodiments, the PD-L1 targeting moiety VHH's CDR2 sequence is selected from: LITSDGTPA (SEQ ID NO: 3); LITSDGTPAYADSAKG (SEQ ID NO: 6); I ITSGGITD (SEQ ID NO: 27); and IITSGGITDYADAVKG (SEQ ID NO: 30).
[047] In embodiments, the PD-L1 targeting moiety VHH's CDR3 sequence is selected from: SSGVYNY (SEQ ID NO: 4); and RDRTIW (SEQ ID NO: 28).
[048] In embodiments, the targeting moiety that specifically binds PD-L1 comprises an amino acid sequence having at least about 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the following sequences:
SEQ ID NO: 1 - 2LIG99 QVQLQESGGGLVQAGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSDGTPAYADSAKGRFTISRDNT
KKTVSLQMNSLKPEDTAVYYCHVSSGVYNYWGQGTQVTVSS
• SEQ ID NO: 7 - 2LIG99_OPT
DVQLVESGGGLVQPGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSDGTPAYADSAKGRFTISRDNS
KKTVSLQMNSLRPEDTAVYYCHVSSGVYNYWGQGTLVTVSS
• SEQ ID NO: 8 - 2LIG99_OPT
DVQLVESGGGLVQPGGSLRLSCAASGTIFSINRMDWFRQAPGKQRELVALITSDGTPAYADSAKGRFTISRDNS
KKTVSLQMNSLRPEDTAVYYCHVSSGVYNYWGQGTLVTVSS
• SEQ ID NO: 9 - 2LIG99_OPT
DVQLVESGGGLVQPGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSDGTPAYADSVKGRFTISRDNS
KKTVSLQMNSLRPEDTAVYYCHVSSGVYNYWGQGTLVTVSS
• SEQ ID NO: 10 - 2LIG99_OPT
DVQLVESGGGLVQPGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSDGTPAYADSAKGRFTISRDNS
KNTVSLQMNSLRPEDTAVYYCHVSSGVYNYWGQGTLVTVSS
• SEQ ID NO: 11 - 2LIG99_OPT
DVQLVESGGGLVQPGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSDGTPAYADSAKGRFTISRDNS
KKTVYLQMNSLRPEDTAVYYCHVSSGVYNYWGQGTLVTVSS
• SEQ ID NO: 12 - 2LIG99_OPT
DVQLVESGGGLVQPGGSLRLSCAASGTIFSINRMDWFRQAPGKQRELVALITSDGTPAYADSVKGRFTISRDNS
KNTVYLQMNSLRPEDTAVYYCHVSSGVYNYWGQGTLVTVSS
• SEQ ID NO: 13 - 2LIG99_OPT
QVQLQESGGGLVQAGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSGGTPAYADSAKGRFTISRDNT
KKTVSLQMNSLKPEDTAVYYCHVSSGVYNYWGQGTQVTVSS
• SEQ ID NO: 14 - 2LIG99_OPT
QVQLQESGGGLVQAGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSKGTPAYADSAKGRFTISRDNT
KKTVSLQMNSLKPEDTAVYYCHVSSGVYNYWGQGTQVTVSS
• SEQ ID NO: 15 - 2LIG99_OPT
QVQLQESGGGLVQAGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSTGTPAYADSAKGRFTISRDNT
KKTVSLQMNSLKPEDTAVYYCHVSSGVYNYWGQGTQVTVSS
• SEQ ID NO: 16 - 2LIG99_OPT
QVQLQESGGGLVQAGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSDRTPAYADSAKGRFTISRDNT
KKTVSLQMNSLKPEDTAVYYCHVSSGVYNYWGQGTQVTVSS
• SEQ ID NO: 17 - 2LIG99_OPT
DVQLVESGGGLVQPGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSGGTPAYADSAKGRFTISRDNS
KKTVSLQMNSLRPEDTAVYYCHVSSGVYNYWGQGTLVTVSS
• SEQ ID NO: 18 - P-2050: 2LIG99_OPT
DVQLVESGGGLVQPGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSGGTPAYADSVKGRFTISRDNS
KKTVSLQMNSLRPEDTAVYYCHVSSGVYNYWGQGTLVTVSS
• SEQ ID NO: 19 - 2LIG99 OPT
DVQLVESGGGLVQPGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSGGTPAYADSAKGRFTISRDNS
KNTVSLQMNSLRPEDTAVYYCHVSSGVYNYWGQGTLVTVSS
• SEQ ID NO: 20 - P-2052: 2LIG99_OPT
DVQLVESGGGLVQPGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSGGTPAYADSAKGRFTISRDNS
KKTVYLQMNSLRPEDTAVYYCHVSSGVYNYWGQGTLVTVSS
• SEQ ID NO: 21 - 2LIG99_OPT
DVQLVESGGGLVQPGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSGGTPAYADSVKGRFTISRDNS
KNTVSLQMNSLRPEDTAVYYCHVSSGVYNYWGQGTLVTVSS
• SEQ ID NO: 22 - 2LIG99_OPT
DVQLVESGGGLVQPGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSGGTPAYADSVKGRFTISRDNS
KKTVYLQMNSLRPEDTAVYYCHVSSGVYNYWGQGTLVTVSS • SEQ ID NO: 23 - 2LIG99_OPT
DVQLVESGGGLVQPGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSGGTPAYADSAKGRFTISRDNS
KNTVYLQMNSLRPEDTAVYYCHVSSGVYNYWGQGTLVTVSS
• SEQ ID NO: 24 - 2LIG99_OPT
DVQLVESGGGLVQPGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSGGTPAYADSVKGRFTISRDNS
KNTVYLQMNSLRPEDTAVYYCHVSSGVYNYWGQGTLVTVSS
• SEQ ID NO: 25 (2LIG189)
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGNDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 31 - 2LIG189_OPT
DVQLVESGGGLVQPGGSLRLSCAASGKIFSGNDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
SKNMMYLQMNSLRPEDTAVYYCNMRDRTIWWGQGTLVTVSS
• SEQ ID NO: 32 - 2LIG189_OPT
DVQLVESGGGLVQPGGSLRLSCAASGKIFSGNDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
SKNTMYLQMNSLRPEDTAVYYCNMRDRTIWWGQGTLVTVSS
• SEQ ID NO: 33 - 2LIG189_OPT
DVQLVESGGGLVQPGGSLRLSCAASGKIFSGNDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
SKNMVYLQMNSLRPEDTAVYYCNMRDRTIWWGQGTLVTVSS
• SEQ ID NO: 34 - 2LIG189_OPT
DVQLVESGGGLVQPGGSLRLSCAASGKIFSGNDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
SKNTVYLQMNSLRPEDTAVYYCNMRDRTIWWGQGTLVTVSS
• SEQ ID NO: 35 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGADMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 36 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGEDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 37 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGFDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 38 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGGDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 39 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGHDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 40 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGIDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDNA
KNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 41 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGLDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 42 - 2LIG189 OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGQDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 43 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGRDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 44 - 2LIG189_OPT QVQLQESGGGLVQAGGSLRLSCAASGKIFSGSDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 45 - 2LIG189 OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGTDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 46 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGVDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 47 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGYDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 48 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGNEMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 49 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGNFMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 50 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGNHMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 51 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGNIMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDNA
KNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 52 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGNLMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 53 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGNQMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 54 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGNRMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 55 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGNVMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 56 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGNYMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNMRDRTIWWGQGTQVTVSS
• SEQ ID NO: 57 - 2LIG189 OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGNDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNERDRTIWWGQGTQVTVSS
• SEQ ID NO: 58 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGNDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNFRDRTIWWGQGTQVTVSS
• SEQ ID NO: 59 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGNDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNHRDRTIWWGQGTQVTVS
• SEQ ID NO: 60 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGNDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNIRDRTIWWGQGTQVTVSS • SEQ ID NO: 61 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGNDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNLRDRTIWWGQGTQVTVSS
• SEQ ID NO: 62 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGNDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNQRDRTIWWGQGTQVTVSS
• SEQ ID NO: 63 - 2LIG189 OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGNDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNRRDRTIWWGQGTQVTVSS
• SEQ ID NO: 64 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGNDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNVRDRTIWWGQGTQVTVSS
• SEQ ID NO: 65 - 2LIG189_OPT
QVQLQESGGGLVQAGGSLRLSCAASGKIFSGNDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
AKNMMYLQMNSLKPEDTAVYYCNYRDRTIWWGQGTQVTVSS
• SEQ ID NO: 66 - 2LIG189_OPT
DVQLVESGGGLVQPGGSLRLSCAASGKIFSGRDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
SKNTVYLQMNSLRPEDTAVYYCNMRDRTIWWGQGTLVTVSS
• SEQ ID NO: 67 - 2LIG189_OPT
DVQLVESGGGLVQPGGSLRLSCAASGKIFSGNHMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
SKNTVYLQMNSLRPEDTAVYYCNMRDRTIWWGQGTLVTVSS
• SEQ ID NO: 68 - 2LIG189_OPT
DVQLVESGGGLVQPGGSLRLSCAASGKIFSGRDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
SKNTVYLQMNSLRPEDTAVYYCNVRDRTIWWGQGTLVTVSS
• SEQ ID NO: 69 - 2LIG189_OPT
DVQLVESGGGLVQPGGSLRLSCAASGKIFSGRDMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
SKNTVYLQMNSLRPEDTAVYYCNIRDRTIWWGQGTLVTVSS
• SEQ ID NO: 70 - 2LIG189_OPT
DVQLVESGGGLVQPGGSLRLSCAASGKIFSGNHMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
SKNTVYLQMNSLRPEDTAVYYCNVRDRTIWWGQGTLVTVSS
• SEQ ID NO: 71 - 2LIG189_OPT
DVQLVESGGGLVQPGGSLRLSCAASGKIFSGNHMGWYRQAPGKQRELVGIITSGGITDYADAVKGRFTISRDN
SKNTVYLQMNSLRPEDTAVYYCNIRDRTIWWGQGTLVTVSS
• SEQ ID NO: 72 - 2LIG189_OPT
DVQLVESGGGLVQPGGSLRLSCAASGKIFSGNHMGWYRQAPGKQRELVGIITSGGITDYADSVKGRFTISRDN
SKNTVYLQMNSLRPEDTAVYYCNVRDRTIWWGQGTLVTVSS
[049] In embodiments, the targeting moiety that specifically targets PD-L1 comprises an amino acid sequence with or without the terminal histidine tag sequence (/.e., HHHHHH; SEQ ID NO: 73).
[050] In some embodiments, the targeting moiety that specifically targets PD-L1 comprises an amino acid sequence with or without the HA tag (/.e., YPYDVPDYGS; SEQ ID NO: 74).
[051] In some embodiments, the targeting moiety that specifically targets PD-L1 comprises an amino acid sequence with or without the AAA linker. [052] In embodiments, the targeting moiety that specifically targets PD-L1 comprises an amino acid sequence with or without the AAA linker, HA tag, and terminal histidine tag sequence (i.e., AAAYPYDVPDYGSHHHHHH; SEQ ID NO: 75).
[053] In embodiments, the present application contemplates the use of any natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (herein collectively referred to as "analogs”) of the targeting moiety that specifically targets PD-L1 as described herein. In embodiments, the amino acid sequence of the targeting moiety that specifically targets PD-L1 further includes an amino acid analog, an amino acid derivative, or other non-classical amino acids.
[054] In embodiments, the targeting moiety that specifically targets PD-L1 comprises an amino acid sequence having one or more amino acid mutations with respect to any one of the sequences disclosed herein. In embodiments, the targeting moiety that specifically targets PD-L1 comprises an amino acid sequence having one, or two, or three, or four, or five, or six, or seen, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations with respect to any one of the sequences disclosed herein. In embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.
[055] In embodiments, the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions. “Conservative substitutions” may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Vai, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. As used herein, conservative substitutions are exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide. In addition, glycine and proline may be substituted for one another based on their ability to disrupt a-helices. As used herein, non-conservative substitutions are exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.
[056] In embodiments, the substitutions also include non-classical amino acids. Exemplary non-classical amino acids include, but are not limited to, selenocysteine, pyrrolysine, N-formylmethionine p-alanine, GABA and 5- Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, ct- amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2- amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, p-alanine, fluoro-amino acids, designer amino acids such as p methyl amino acids, C o-methyl amino acids, N a-methyl amino acids, and amino acid analogs in general. [057] In embodiments, the chimeric protein complex comprises a modified human interferon o2. In embodiments, the modified IFN-a2 agent has reduced affinity and/or activity for the IFN-a/p receptor (IFNAR), i.e., IFNAR1 and/or IFNAR2 chains. In some embodiments, the modified I F N-a2 agent has substantially reduced or ablated affinity and/or activity for the IFN-a/p receptor (IFNAR), i.e., IFNAR1 and/or IFNAR2 chains. In some embodiments, the modified human interferon o2, as disclosed herein, has an amino acid sequence having at least about 95% identity with of SEQ ID NOs: 76 or 77. In other embodiments, the modified human I FNa2 has an amino acid sequence having at least about 98% identity or at least about 99% identity with of SEQ ID NOs: 76 or 77. In some embodiments, the modified human IFNct2 has 1-3 mutations relative to the amino acid sequence of SEQ ID NOs: 76 or 77. In one embodiment, the modified human I FNct2 comprises a R149A mutation with respect to SEQ ID NOs: 76 or 77. In one embodiment, the modified human I FNa2 comprises a A145G mutation with respect to SEQ ID NOs: 76 or 77.
[058] In embodiments, the R149A mutation, or the equivalent thereof, is present in the I FN-a2, relative to SEQ ID NO: 76 or 77.
[059] In embodiments, the R149A mutation, or the equivalent thereof, is not present in the I FN-a2, relative to SEQ ID NO: 76 or 77, and instead, another mutation is present. For instance, this alternative mutation could be at one of positions R33, R144, A145, M148, and L153, or equivalents thereof, relative to SEQ ID NO: 76 or 77. In embodiments, the alternative mutation is one of R33A, R144A, R144I, R144L, R144S, R144T, R144Y, A145D, A145G, A145H, A145K, A145Y, M148A, and L153A, or equivalents thereof, relative to SEQ ID NO: 76 or 77. For clarity, in embodiments, any reference to R149A herein may be replaced with one of R33A, R144A, R144I, R144L, R144S, R144T, R144Y, A145D, A145G, A145H, A145K, A145Y, M148A and L153A, or equivalents thereof, relative to SEQ ID NO: 76 or 77. In embodiments, any reference to R149A herein may be replaced with A145G, or equivalents thereof, relative to SEQ ID NO: 76 or 77.
[060] SEQ ID NO: 76 Human IFNo2a (amino acid sequence)
CDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDE TLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEWRAEIMRSFSLSTNL QESLRSKE
[061] SEQ ID NO: 77 Human I FNo2b (amino acid sequence)
CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDE TLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEWRAEIMRSFSLSTNLQ ESLRSKE
[062] In embodiments, the chimeric protein complex disclosed herein comprises two targeting moieties. In embodiments, the chimeric protein complex disclosed herein comprises two identical targeting moieties. [063] In embodiments, the chimeric protein complex disclosed herein comprises two targeting moieties. In embodiments, the chimeric protein complex disclosed herein comprises two non-identical targeting moieties. For example, in embodiments, the chimeric protein complex disclosed herein comprises targeting moieties (e.g., without limitation, VHHs) against PD-L1 and Clec9A.
[064] In embodiments, the chimeric protein complex disclosed herein includes at least one Fc domain. In embodiments, the chimeric protein complex includes a modified Fc domain where the modified Fc domain includes one or more of the following mutations: P329G, K322Q, K322A, P331G, or P331S, relative to any of one of SEQ ID NO: 78-81 . In embodiments, the modified Fc domain includes one or more of the following mutations: P329G, K322Q, K322A, P331 G, or P331 S, relative to human lgG1 Fc.
[065] In embodiments, the chimeric protein complex includes a modified Fc domain that has an amino acid sequence having at least about 90% identity with any one of SEQ ID NO: 78-81. In embodiments, the modified Fc domain has an amino acid sequence having at least about 93% identity with SEQ ID NO: 78-81. In embodiments, the modified Fc domain has an amino acid sequence having at least about 95% identity with SEQ ID NO: 78-81.
[066] SEQ ID NO: 78 - Amino acid sequence of the Fc (human IgG 1 )— with LALA mutations and Ridgway hole
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[067] SEQ ID NO: 79 - Amino acid sequence of the Fc (human lgG1)— with LALA mutations and Ridgway knob
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[068] SEQ ID NO: 80 - Amino acid sequence of the Fc (human lgG1 ) - with LALA mutations and Merchant hole
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[069] SEQ ID NO: 81 - Amino acid sequence of the Fc (human lgG1 ) - with LALA mutations and Merchant knob
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [070] The fragment crystallizable domain (Fc domain) is the tail region of an antibody that interacts with Fc receptors located on the cell surface of cells that are involved in the immune system, e.g., B lymphocytes, dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells. In IgG, IgA and IgD antibody isotypes, the Fc domain is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains. In IgM and IgE antibody isotypes, the Fc domain contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.
[071] In embodiments, the Fc-based chimeric protein of complex the present technology includes a Fc domain. In embodiments, the Fc domain is selected from IgG, IgA, IgD, IgM and IgE. In embodiments, the Fc domain is selected from lgG1 , lgG2, lgG3, and lgG4.
[072] In embodiments, the Fc domain is selected from human IgG, IgA, IgD, IgM and IgE. In embodiments, the Fc domain is selected from human lgG1 , lgG2, lgG3, and lgG4.
[073] In embodiments, the Fc domain of the Fc-based chimeric protein complex comprises the CH2 and CH3 regions of IgG. In embodiments, the IgG is human IgG. In embodiments, the human IgG is selected from lgG1 , lgG2, lgG3, and lgG4.
[074] In embodiments, the Fc domain comprises one or more mutations. In embodiments, the mutation(s) to the Fc domain reduces or eliminates the effector function of the Fc domain. In embodiments, the mutated Fc domain has reduced affinity or binding to a target receptor. By way of example, in embodiments, the mutation to the Fc domain reduces or eliminates the binding of the Fc domain to FcyR. In embodiments, the FcyR is selected from FcyRI; FcyRlla, 131 R/R; FcyRlla, 131 H/H, FcyRllb; and FcyRIII. In embodiments, the mutation to the Fc domain reduces or eliminated binding to complement proteins, such as, e.g., C1q. In embodiments, the mutation to the Fc domain reduces or eliminates binding to both FcyR and complement proteins, such as, e.g., C1q.
[075] In embodiments, the Fc domain comprises a LALA mutation to reduce or eliminate the effector function of the Fc domain. By way of example, in embodiments, the LALA mutation comprises L234A and L235A substitutions in human IgG (e.g., lgG1 ) (wherein the numbering is based on the commonly used numbering of the CH2 residues for human lgG1 according to EU convention (Edelman et al., PNAS, 1969; 63 (1) 78-85)).
[076] In embodiments, the Fc domains of human IgG comprise a mutation at 46 to reduce or eliminate the effector function of the Fc domains. By way of example, in embodiments, the mutations are selected from L234A, L234F, L235A, L235E, L235Q, K322A, K322Q, D265A, P329G, P329A, P331G, and P331 S.
[077] In embodiments, the Fc domains comprise the FALA mutation to reduce or eliminate the effector function of the Fc domains. By way of example, in some embodiments, the FALA mutation comprises F234A and L235A substitutions in human lgG4. [078] In embodiments, the Fc domains of human lgG4 comprise a mutation at one or more of F234, L235, K322, D265, and P329 to reduce or eliminate the effector function of the Fc domains. By way of example, in embodiments, the mutations are selected from F234A, L235A, L235E, L235Q, K322A, K322Q, D265A, P329G, and P329A.
[079] In embodiments, the mutation(s) to the Fc domain stabilize a hinge region in the Fc domain. By way of example, in embodiments, the Fc domain comprises a mutation at S228 of human IgG to stabilize a hinge region. In embodiments, the mutation is S228P.
[080] In embodiments, the mutation(s) to the Fc domain promote chain pairing in the Fc domain. In embodiments, chain pairing is promoted by ionic pairing (a/k/a charged pairs, ionic bond, or charged residue pair).
[081] In embodiments, the Fc domain comprises a mutation at one more of the following amino acid residues of IgG to promote of ionic pairing: D356, E357, L368, K370, K392, D399, and K409.
[082] By way of example, in embodiments, the human IgG Fc domain comprises one of the mutation combinations in Table A to promote of ionic pairing.
Figure imgf000018_0001
Figure imgf000019_0001
[083] In embodiments, chain pairing is promoted by a knob-in-hole mutations. In embodiments, the Fc domain comprises one or more mutations to allow for a knob-in-hole interaction in the Fc domain. In embodiments, a first Fc chain is engineered to express the “knob” and a second Fc chain is engineered to express the complementary “hole." By way of example, in embodiments, human IgG Fc domain comprises the mutations of Table B to allow for a knobin-hole interaction.
Figure imgf000019_0002
[084] In embodiments, the Fc domains in the Fc-based chimeric protein complexes of the present technology comprise any combination of the above-disclosed mutations. By way of example, in embodiments, the Fc domain comprises mutations that promote ionic pairing and/or a knob-in-hole interaction. By way of example, in embodiments, the Fc domain comprises mutations that have one or more of the following properties: promote ionic pairing, induce a knob-in-hole interaction, reduce or eliminate the effector function of the Fc domain, and cause Fc stabilization (e.g. at hinge).
[085] By way of example, in embodiments, a human IgG Fc domain comprises mutations disclosed in Table C, which promote ionic pairing and/or promote a knob-in-hole interaction in the Fc domain.
Figure imgf000019_0003
Figure imgf000020_0001
[086] By way of example, in embodiments, a human IgG Fc domain comprises mutations disclosed in Table D, which promote ionic pairing, promote a knob-in-hole interaction, or a combination thereof in the Fc domain. In embodiments, the "Chain 1” and “Chain 2” of Table D can be interchanged (e.g. Chain 1 can have Y407T and Chain 2 can have T366Y).
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
[087] By way of example, in embodiments, a human IgG Fc domain comprises mutations disclosed in Table E, which reduce or eliminate FcyR and/or complement binding in the Fc domain. In embodiments, the Table E mutations are in both chains.
Figure imgf000023_0002
Figure imgf000024_0001
Figure imgf000025_0001
[088] In embodiments, the Fc domain in the Fc-based chimeric protein complex of the present technology are homodimeric, i.e. , the Fc region in the chimeric protein complex comprises two identical protein fragments.
[089] In other embodiments, the Fc domains in the Fc-based chimeric protein complexes of the present technology are heterodimeric, i.e., the Fc domain comprises two non-identical protein fragments. In embodiments, heterodimeric Fc domains are engineered using ionic pairing and/or knob-in-hole mutations described herein. In embodiments, the heterodimeric Fc-based chimeric protein complexes have a trans orientation/configuration. In a trans orientation/configuration, the targeting moiety and signaling agent are, in embodiments, not found on the same polypeptide chain in the present Fc-based chimeric protein complexes.
[090] In embodiments, the present chimeric protein or the chimeric protein complex comprises one or more linkers. In embodiments, the present chimeric protein or the chimeric protein complex comprises a linker connecting the targeting moiety and the signaling agent. In embodiments, the present chimeric protein or the chimeric protein complex comprises a linker within the signaling agent.
[091] The present application contemplates the use of a variety of linker sequences. In embodiments, the linker is derived from naturally-occurring multi-domain proteins or are empirical linkers, as described, for example, in Chichili et al., (2013), Protein Sci. 22 (2): 153- 167, Chen et al., (2013), Adv Drug Deliv Rev. 65(10): 1357- 1369, the entire contents of which are hereby incorporated by reference. In embodiments, the linker is designed using linker designing databases and computer programs such as those described in Chen et al., (2013), Adv Drug Deliv Rev. 65(10): 1357- 1369 and Crasto et al., (2000), Protein Eng. 13(5):309-312, the entire contents of which are hereby incorporated by reference. In embodiments, the linker is functional. For example, without limitation, the linker may function to improve the folding and/or stability, improve the expression, improve the pharmacokinetics, and/or improve the bioactivity of the present chimeric protein or the chimeric protein complex.
[092] In embodiments, the linker is a polypeptide. In embodiments, the linker is less than about 100 amino acids long. For example, the linker may be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11 , about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids long. In embodiments, the linker is a polypeptide. In embodiments, the linker is greater than about 100 amino acids long. For example, the linker may be greater than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11 , about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids long. In embodiments, the linker is flexible. In another embodiment, the linker is rigid.
[093] In embodiments, the linker is substantially comprised of glycine and serine residues (e.g. about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% glycines and serines). For example, in embodiments, the linker is GGS. In embodiments, the link is (GGS)n (n=2-20) (SEQ ID NOs: 85-103). In embodiments, the linker is G. In embodiments, the linker is AAA. In embodiments, the linker is (GGGGS)n (n=1-20) (SEQ ID NOs: 104-123). In an embodiment, the linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 124). Additional illustrative linkers include, but are not limited to, linkers having the sequence LE, (Gly)8 (SEQ ID NO: 125), (Gly)e (SEQ ID NO: 126), (EAAAK)n (n=1-3) (SEQ ID NO: 127 - SEQ ID NO: 129), A(EAAAK)nA (n = 2-5) (SEQ ID NO: 130 - SEQ ID NO: 133), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 134), PAPAP (SEQ ID NO: 135), KESGSVSSEQLAQFRSLD (SEQ ID NO: 136), EGKSSGSGSESKST (SEQ ID NO: 137), GSAGSAAGSGEF (SEQ ID NO: 138), and (XP)n, with X designating any amino acid, e.g., Ala, Lys, or Glu. Pharmaceutical Formulations and Dosage Forms
[094] The formulation of PD-L1 -based chimeric proteins and/or chimeric protein complexes, or variants thereof, may further comprise one or more pharmaceutically acceptable carriers or excipients. As one skilled in the art will recognize, the formulations can be in any suitable form appropriate for the desired use and route of administration.
[095] In embodiments, the administration of the pharmaceutical formulation including PD-L1 -based chimeric proteins and/or chimeric protein complexes (and/or additional therapeutic agents) is intravenous and/or parenteral. In embodiments, routes of administration include, for example: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, epidural, sublingual, intracerebral, intravaginal, or transdermal.
[096] A pharmaceutical formulation including PD-L1-based chimeric proteins and/or chimeric protein complexes (and/or additional therapeutic agents) as described herein can be administered by any convenient route, for example, by intravenous infusion or bolus injection.
[097] In some dosage forms, the PD-L1 -based chimeric proteins and/or chimeric protein complexes described herein are mixed with at least one inert, pharmaceutically acceptable excipient or carrier.
[098] In embodiments, the pharmaceutical compositions or formulations comprising PD-L1 -based chimeric proteins and/or chimeric protein complexes described herein are generally buffered solutions. In embodiments, a buffer is a chemical agent that is able to absorb a certain quantity of acid or base without undergoing a strong variation in pH. Illustrative buffering agents include histidine buffer, histidine-acetate buffer, citrate buffer, phosphate buffer, acetate buffer, succinate buffer, and bicarbonate buffer. In embodiments, the buffering agent comprises L-histidine or L- histidine hydrochloride monohydrate. In embodiments, when using the L-histidine or L-histidine hydrochloride monohydrate buffering system, L-histidine or L-histidine hydrochloride monohydrate is present at a concentration of from about 1 mM to about 50 mM, or from about 5 mM to about 45 mM, or from about 5 mM to about 25 mM, or from about 10 mM to about 35 mM, or from about 15 mM to about 30 mM, or from about 20 mM to about 25 mM. In some embodiments, L-histidine or L-histidine hydrochloride monohydrate is present at a concentration of about 10 mM , about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM.
[099] In embodiments, the pharmaceutical compositions or formulations described herein comprise a buffering agent further comprising an acetate buffer. In embodiments, the acetate buffer comprises acetic acid. In embodiments, the pharmaceutical compositions or formulations described herein comprise a buffering agent comprising an acetatehistidine buffer. In embodiments, acetate— histidine buffer comprises L-histidine and acetic acid. In embodiments, the buffering agent comprises acetic acid present at a concentration of from about 1 nM to about 50 mM, or from about 5 mM to about 45 mM, or from about 5 mM to about 25 mM, or from about 10 mM to about 35 mM, or from about 15 mM to about 30 mM, or from about 20 mM to about 25 mM. In some embodiments, acetic acid is present at a concentration of about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM.
[0100] In some embodiments, the buffer maintains the pH of the formulation in the range of about pH 4.0 to about pH 6.0, or about 50 to about 6.0, or about 4.5 to about 5.5. In embodiments, the pharmaceutical compositions or formulations described herein have a pH of about 4.0, about 4.1 , about 4.2, about 4.3, about 4.4, about 4.5, about
4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about
5.7, about 5.8, about 5.9, or about 6.0. In some embodiments, the formulation is pH adjusted, e.g., with hydrochloric acid and/or sodium hydroxide.
[0101] In embodiments, the pharmaceutical compositions or formulations comprising PD-L1-based chimeric proteins and/or chimeric protein complexes described herein additionally include a surface active agent or surfactant. In embodiments, surface active agents suitable for use include, but are not limited to, any pharmaceutically acceptable, non-toxic surfactant. In embodiments, the formulation comprises a surfactant, which can act as a solubilizing agent. Classes of surfactants suitable for use in the compositions of the present application include, but are not limited to polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, polyethoxylated fatty acids, PEG-fatty acid diesters, PEG-fatty acid mono- and di-ester mixtures, polyethylene glycol glycerol fatty acid esters, alcohol-oil transesterification products, polyglycerized fatty acids, propylene glycol fatty acid esters, mixtures of propylene glycol esters-glycerol esters, mono- and diglycerides, sterol and sterol derivatives, polyethylene glycol sorbitan fatty acid esters, polyethylene glycol alkyl ethers, sugar esters, polyethylene glycol alkyl phenols, polyoxyethylene- olyoxypropylene block copolymers, sorbitan fatty acid esters, lower alcohol fatty acid esters, ionic surfactants, and mixtures thereof. In embodiments, pharmaceutical compositions or formulations described herein comprise one or more surfactants including, but not limited to, polysorbate 20 and poloxamer 188.
[0102] In embodiments, the pharmaceutical compositions or formulations comprising PD-L1 -based chimeric proteins and/or chimeric protein complexes described herein comprise a surfactant (e.g. polysorbate 20 (PS20) or poloxamer 188), at a concentration of at least about 0.01 % (w/v) to about 0.1 % (w/v), or at least about 0.01 % (w/v) to about 0.05% (w/v), or at least about 0.02% (w/v) to about 0.09% (w/v), or at least about 0.03% (w/v) to about 0.08% (w/v), or at least about 0.04% (w/v) to about 0.07% (w/v), or at least about 0.05% (w/v) to about 0.06% (w/v). In embodiments, the pharmaceutical compositions or formulations described herein comprise a surfactant at a concentration of about 0.01 % (w/v), about 0.02% (w/v), about 0.03% (w/v), about 0.04% (w/v), about 0.05% (w/v), about 0.06% (w/v), about 0.07% (w/v), about 0.08% (w/v), about 0.09% (w/v), or about 0.1 % (w/v).
[0103] In embodiments, the pharmaceutical compositions or formulations comprising PD-L1 -based chimeric proteins and/or chimeric protein complexes described herein comprise a tonicifier, which can be used singly or in combination, include, but are not limited to, trehalose, dextrose, sucrose, glycerin, mannitol, sorbitol, arginine, sodium chloride, or potassium chloride. In embodiments, the pharmaceutical compositions or formulations described herein comprise one or more tonicifier excipients selected from trehalose, trehalose-sodium chloride, sucrose, and mannitol.
[0104] In embodiments, the tonicifier comprises or consists of a sugar, such as trehalose, sucrose, or mannitol. In embodiments, the pharmaceutical compositions or formulations described herein comprise a tonicifier at a concentration of at least about 50 mM to about 350 mM, or from at least about 100 mM to about 300 mM, or from at least about 150 mM to about 250 mM. In embodiments, a sugar tonicifier is present in the pharmaceutical composition or formulation at a concentration of about 50, mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, or about 350 mM. In embodiments, the tonicifier is present at a concentration of at least about 90 mM to about 270 mM, optionally at least about 190 mM to about 195 mM.
[0105] In some embodiments, the tonicifier comprises or consists of sodium chloride. In embodiments, the pharmaceutical compositions or formulations described herein comprise sodium chloride at a concentration of at least about 10 mM to no more than 50 mM. In embodiments, the pharmaceutical compositions or formulations described herein comprise sodium chloride at a concentration of from about 10 mM to about 50 mM, or from about 20 mM to about 50 mM, or about 30 mM to about 50 mM, or about 40 mM to about 50 mM. In embodiments, sodium chloride is present in the pharmaceutical composition or formulation at a concentration of about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, or about 70 mM.
[0106] In embodiments, the pharmaceutical compositions or formulations comprising PD-L1 -based chimeric proteins and/or chimeric protein complexes described herein include one or more additional excipients. In embodiments, the one or more additional excipients are selected from a stabilizer, a salt, an amino acid, and an antioxidant.
[0107] In embodiments, the pharmaceutical compositions or formulations comprising PD-L1 -based chimeric proteins and/or chimeric protein complexes described herein further comprise an additional stabilizer excipient. In embodiments, the stabilizer is selected from one or more of cyclodextrin, arginine, sodium chloride-arginine, and glycine. In embodiments, the cyclodextrin is beta-cyclodextrin, optionally HP-beta-cyclodextrin (hydroxypropyl-beta- cyclodextrin). In embodiments, the stabilizer is present at a concentration of at least about 20 mM to about 100 mM, or at least about 25 mM to about 75 mM. In embodiments, the stabilizer is present at a concentration of at least about 50 mM
[0108] In embodiments, the pharmaceutical compositions or formulations comprising PD-L1 -based chimeric proteins and/or chimeric protein complexes described herein further comprise an amino acid excipient. In embodiments, the amino acid excipient comprises one or more of arginine, glycine, glutamate, and histidine, optionally wherein the additional excipient is arginine. In embodiments, the pharmaceutical compositions or formulations described herein comprise arginine at a concentration of at least about 10 mM to no more than 50 mM. In embodiments, the pharmaceutical compositions or formulations described herein comprise arginine at a concentration of from about 10 mM to about 50 mM, or from about 20 mM to about 50 mM, or about 30 mM to about 50 mM, or about 40 mM to about 50 mM. In embodiments, arginine is present in the pharmaceutical composition or formulation at a concentration of about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, or about 70 mM.
[0109] In embodiments, the pharmaceutical compositions or formulations comprising PD-L1 -based chimeric proteins and/or chimeric protein complexes described herein further comprise an antioxidant excipient. In embodiments, the antioxidant excipient is selected from methionine and ethylenediaminetetraacetic acid (EDTA). In embodiments, the antioxidant is methionine. In embodiments, the pharmaceutical compositions or formulations described herein comprise an antioxidant at a concentration of at least about 1 mM to about 15 mM, or from at least about 1 mM to about 10 mM, or from at least about 2 mM to about 10 mM, or from at least about 3 mM to about 10 mM, or from at least about 1 mM to about 5 mM, or from at least about 2 mM to about 5 mM, or from at least about 3 mM to about 5 mM. In embodiments, an antioxidant excipient is present in the pharmaceutical composition or formulation at a concentration of about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM.
[0110] In embodiments, pharmaceutical compositions or formulations described herein comprise a PD-L1-based chimeric protein or protein complex that is present at a concentration from at least about 0.25 mg/mL to about 15 mg/mL. In embodiments, pharmaceutical compositions or formulations described herein comprise a PD-L1-based chimeric protein or protein complex that is present at a concentration from at least about 0.25 mg/mL to about 10 mg/mL, from at least about 0.5 mg/mL to about 10 mg/mL, from at least about 0.75 mg/mL to about 10 mg/mL, from at least about 1 mg/mL to about 10 mg/mL, from at least about 1 mg/mL to about 15 mg/mL.
[0111] In embodiments, pharmaceutical compositions or formulations described herein comprise at least about 0.25 mg/mL, or at least about 0.5 mg/mL, or at least about 0.75 mg/mL, or at least about 1 mg/mL, or at least about 1 .25 mg/mL, or at least about 1 .5 mg/mL, or at least about 1 .75 mg/mL, or at least about 2 mg/mL, at least about 3 mg/mL, at least about 4 mg/mL, at least about 5 mg/mL, at least about 6 mg/mL, or at least about 7 mg/mL, or at least about 8 mg/mL, or at least about 9 mg/mL, or at least about 10 mg/mL of a PD-L1 -based chimeric protein or protein complex.
[0112] By way of illustration, in embodiments, the pharmaceutical compositions or formulations described herein comprise (a) a Programmed death-ligand 1 (PD-LI)-based chimeric protein complex comprising (i) a targeting moiety that specifically binds to PD-L1 , (ii) a modified human IFNo2, and (iii) a modified Fc domain, wherein the PD-L1-based chimeric protein complex is present at a concentration from at least about 1 mg/mL to about 10 mg/mL; (b) a buffering agent comprising histidine, optionally further comprising acetic acid, wherein histidine is present at a concentration of at least about 5 mM to about 20 mM and/or acetic acid present at a concentration ranging from at least about 5 mM to about 20 mM; (c) an excipient comprising a surfactant selected from PS20 at a concentration of at least about 0 05% (w/v) and Pol 188 at a concentration ranging from at least about 0.01% (w/v) to about 0.05% (w/v); and (d) an excipient comprising a tonicifier selected from trehalose, sucrose, and mannitol, present at a concentration of at least about 185 mM to about 270 mM, wherein the pharmaceutical composition has a pH that ranges from at least about 4.0 to at least about 6.0.
[0113] In embodiments, the pharmaceutical compositions or formulations described herein comprise (a) a Programmed death-ligand 1 (PD-LI)-based chimeric protein complex comprising (i) a targeting moiety that specifically binds to PD-L1 , (ii) a modified human IFNa2, and (iii) a modified Fc domain, wherein the PD-L1-based chimeric protein complex is present at a concentration of at least about 1 mg/mL; (b) a buffering agent comprising histidine, optionally further comprising acetic acid, wherein histidine is present at a concentration of at least about 5 mM to about 20 mM and/or acetic acid present at a concentration ranging from about 5 mM to about 20 mM; (c) an excipient comprising a surfactant selected from PS20 at a concentration ranging from about 0.01 % (w/v) to about 0.1 % (w/v), and Pol 188 at a concentration ranging from about 0.01 % (w/v) to about 0.1 % (w/v); (d) an excipient comprising a tonicifier selected from trehalose, sucrose, and mannitol, present at a concentration of at least about 185 mM to about 270 mM; (e) an antioxidant excipient comprising methionine, wherein methionine is present at a concentration of at least about 1 mM to about 5 mM; and (f) a stabilizer excipient comprising HP-beta-cyclodextrin or sodium chloride-arginine at a concentration of at least about 25 mM to about 75 mM, and wherein the pharmaceutical composition has a pH that ranges from at least about 4.5 to about 5.5.
[0114] In embodiments, the pharmaceutical compositions or formulations described herein comprise (a) a Programmed death-ligand 1 (PD-LI)-based chimeric protein complex comprising (i) a targeting moiety that specifically binds to PD-L1 , (ii) a modified human IFNo2, and (iii) a modified Fc domain, wherein the PD-L1-based chimeric protein complex is present at a concentration from at least about 1 mg/mL to about 10 mg/mL; (b) a buffering agent comprising histidine and acetic acid, wherein histidine is present at a concentration of at least about 10 mM to about 15 mM and acetic acid is present at a concentration of at least about 10 mM to about 15 mM; (c) an excipient comprising a surfactant selected from PS20 at a concentration ranging from about 0.01% (w/v) to about 0.05% (w/v), and Pol 188 at a concentration of about 0.05% (w/v); (d) an excipient comprising a tonicifier comprising trehalose present at a concentration of at least about 190 mM to about 195 mM; (e) an antioxidant excipient comprising methionine, wherein methionine is present at a concentration of about 3mM; and (f) a stabilizer excipient comprising HP-beta-cyclodextrin or sodium chloride-arginine at a concentration of about 50 mM, and wherein the pharmaceutical composition has a pH of about 5.0.
[0115] Dosage forms suitable for parenteral administration (e.g. intravenous, intramuscular, intraperitoneal, subcutaneous and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g. frozen and/or lyophilized compositions), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.
[0116] In embodiments, pharmaceutical formulations including PD-L1 -based chimeric proteins and/or chimeric protein complexes (and/or additional therapeutic agents) may be presented in unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the therapeutic agents into association with a carrier, which constitutes one or more accessory ingredients. In embodiments, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation.
[0117] In embodiments, the pharmaceutical formulation comprises a preservative, such as, but not limited to, phenol, meta-cresol, or sodium benzoate.
[0118] Pharmaceutically acceptable carriers include, but are not limited to, water, saline, and glycerol. In some embodiments, the pharmaceutical composition or formulation described herein comprises fixed oils, polyethylene glycol, propylene glycol or other solvents. In some embodiments, the solvent is water.
[0119] In embodiments, the composition is contained in an injection device, such as a syringe or an injection pen. In embodiments, the composition is contained in an injection pen. Auto-injectors such as an “injection pen” are spring-loaded syringes designed to deliver a dose of a particular drug. By design, injection pens are easy to use and are intended for self-administration by patients, or administration by untrained personnel. Injection pens are designed to overcome the hesitation associated with self-administration of the needle-based drug delivery device. The injection pen keeps the needle tip shielded prior to injection and also has a passive safety mechanism to prevent accidental firing (injection). Injection depth can be adjustable or fixed and a function for needle shield removal may be incorporated. By pressing a button, the syringe needle is automatically inserted into the subcutaneous tissue and the drug is delivered. Once the injection is completed some injection pens have a visual or audible indication to confirm that the full dose has been delivered.
[0120] In embodiments, the injection device contains from 1 to 10 unit doses or from 1 to 5 unit doses. In some embodiments, the unit doses are no more than about 1.5 mLs or about 1 mL in volume (whether or not contained or delivered by an injection device). In some embodiments, the unit doses are no more than 0.8 mL in volume, or no more than 0.7 mL in volume. In some embodiments, the injection device delivers a microdose, e.g., having a volume in the range of about 50 piL to about 500 piL, or a volume in the range of from about 75 piL to about 250 piL. In some embodiments, the microdose has a volume of from 100 to 200 piL. The amount of active agent delivered per unit dose can be adjusted based on the desired frequency of administration. [0121] In embodiments, the pharmaceutical composition or formulation of the present application is suitable for administration by subcutaneous, intramuscular, intradermal, or intravenous administration. In some embodiments, the subject receives a unit dose of the pharmaceutical composition or formulation about once every 1 week, or about once every 2 weeks, or about once every 3 weeks, or about once every 4 weeks (e.g., about once per month), or about once every 6 weeks, or about once every 8 weeks (about once every 2 months).
[0122] In embodiments, the pharmaceutical composition or formulation described herein is freeze-dried to form a lyophilized powder. In an aspect, the present application contemplates a kit comprising (a) the pharmaceutical composition or formulation of the present application that has been freeze-dried to form a lyophilized powder, (b) a vial (e.g., glass vial), and (c) an injection device (e.g., a syringe or injection pen).
[0123] In embodiments, the lyophilized formulation is marketed in pharmaceutical dosage form. The pharmaceutical dosage form of the present application, although in embodiments in the form of a vial, may be any suitable container, such as ampoules, syringes, co-vials, which are capable of maintaining a sterile environment. Such containers can be glass or plastic, provided that the material does not interact with the pharmaceutical formulation. In embodiments, the closure is a stopper, e.g., a sterile rubber stopper, or a siliconized stopper, which affords a seal. In embodiments, a vial will contain a lyophilized powder including about 0.5 to about 10 g/vial. Thus, in embodiments, there is provided a kit comprising a lyophilized powder of the PD-L1-based chimeric protein complex pharmaceutical formulation, a glass vial and optionally a syringe.
[0124] The lyophilized formulations of the present application may be reconstituted with water, such as Sterile Water for Injection, or other sterile fluid such as co-solvents, to provide an appropriate solution of PD-L1-based chimeric protein complex for administration, as through parenteral injection following further dilution into an appropriate intravenous admixture container, for example, including normal saline.
[0125] Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc. at least one chemical entity and optional pharmaceutical adjuvants in a carrier (e.g., water, saline, aqueous dextrose, glycerol, glycols, ethanol or the like) to form a solution or suspension. I njectables can be prepared in conventional forms, either as liquid solutions or suspensions, as emulsions, or in solid forms suitable for dissolution or suspension in liquid prior to injection. The percentage of chemical entities contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the activity of the chemical entities and the needs of the subject.
[0126] Preparation of the pharmaceutical composition or formulation may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable excipient is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. The excipient(s) must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
[0127] In embodiments, the pharmaceutical composition of the application exhibits short-term and/or long-term stability. In embodiments, stability of the pharmaceutical composition described herein is measured by monitoring one or more of sample appearance, sample pH, sample osmolality, sample potency (ELISA and cell-based assay), sample SDS-PAGE (reducing and non-reducing), sample protein concentration (UV absorbance), sample SE-HPLC, sample RP-HPLC, sample clEF, sample sub-visible particles, and sample MS.
[0128] In embodiments, the pharmaceutical composition described herein is stable for at least 2 weeks at 30°C to 50°C (e.g, at 30°C, 35°C, 40°C, 45°C, or 50°C), optionally further wherein the relative humidity is 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%.
[0129] In embodiments, the pharmaceutical composition described herein is stable for at least 1 month or 2 months at 15°C to 35°C (e.g., at 15°C, 20°C, 25°C, 20°C, or 35°C), optionally further wherein the relative humidity is 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%.
[0130] In embodiments, the pharmaceutical composition described herein is stable for at least 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, or 36 months at 1 °C,to 10°C (e.g., at 1 °C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, or 10°C). In embodiments, the pharmaceutical composition described herein is stable for at least 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, or 36 months at -15°C to - 25°C (e.g, at -15°C, -20°C, or -25°C).
[0131] In embodiments, the pharmaceutical composition described herein is stable after undergoing one, two, three, four, or five freeze and thaw cycles.
Methods of Treatment
[0132] In an aspect, the present application relates to a method for treating or preventing a cancer, comprising administering an effective amount of the pharmaceutical composition as disclosed herein to a patient in need thereof.
[0133] In embodiments, the pharmaceutical composition described herein can be used for the treatment or prevention of a blood cancer (e.g, a hematologic cancer dealing with blood cells) or a solid tumor cancer (e.g, a cancer of any other body organ or tissue).
[0134] The pharmaceutical composition can be used for the treatment or prevention of a cancer selected from one or more of lymphoma, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and/or central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma (e.g., Kaposi's sarcoma); skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma (including low grade/foll icular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g. that associated with brain tumors), and Meigs' syndrome.
Definitions
[0135] As used herein, “a,” "an,” or “the” can mean one or more than one.
[0136] Further, the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language “about 50” covers the range of 45 to 55.
[0137] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or disorder or one or more signs or symptoms associated with a disease or disorder. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compounds may be administered to a subject having one or more signs or symptoms of a disease or disorder. As used herein, something is “decreased” if a read-out of activity and/or effect is reduced by a significant amount, such as by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to and including at least about 100%, in the presence of an agent or stimulus relative to the absence of such modulation. As will be understood by one of ordinary skill in the art, in some embodiments, activity is decreased and some downstream read-outs will decrease but others can increase. [0138] Conversely, activity is "increased” if a read-out of activity and/or effect is increased by a significant amount, for example by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to and including at least about 100% or more, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, in the presence of an agent or stimulus, relative to the absence of such agent or stimulus.
[0139] Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the subject matter of the present application, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of” or “consisting essentially of."
[0140] The amount of compositions described herein needed for achieving a therapeutic effect may be determined empirically in accordance with conventional procedures for the particular purpose. Generally, for administering therapeutic agents for therapeutic purposes, the therapeutic agents are given at a pharmacologically effective dose. A “pharmacologically effective amount," “pharmacologically effective dose,” “therapeutically effective amount,” or “effective amount” refers to an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease. An effective amount as used herein would include an amount sufficient to, for example, delay the development of a symptom of the disorder or disease, alter the course of a symptom of the disorder or disease (e.g., slow the progression of a symptom of the disease), reduce or eliminate one or more symptoms or manifestations of the disorder or disease, and reverse a symptom of a disorder or disease. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.
[0141] As used herein, “methods of treatment" are equally applicable to use of a composition for treating the diseases or disorders described herein and/or pharmaceutical compositions for use and/or uses in the manufacture of a medicaments for treating the diseases or disorders described herein.
[0142] As used herein, Fc domain mutations are numbered according to EU convention (Edelman et al., PNAS 1969; 63 (1) 78-85, incorporated by reference in its entirety). As used herein, the term “LALA” mutation refers to a double mutant Fc domain having L234A mutation and a L235A mutation. As used herein, the term “KQ” mutation refers to a mutant Fc domain having a K322Q mutation
[0143] Knob in hole mutants are those described in Ridgway et al., Protein Engineering 1996;9:617-621, which is hereby incorporated by reference in its entirety, i.e. Y407T / T366Y.
[0144] Alternatively, knob in hole mutants are those described in Merchant et al., Nature Biotechnology 1998; 16:677-681, which is incorporated by reference in its entirety, i.e. S354C:T366W / Y349C:T366S:L368A:Y407V. EXAMPLES
Example 1 : Preparation of PD-L1 -Based Chimera Formulation Protein
[0145] Plasmids P-3003 and P-3004 (respectively pcDNA3.4 plasmids coding for (i) a sequence optimized PD- L1 binding VHH fused to a human lgG1 Fc domain or (ii) a human lgG1 Fc domain fused to an attenuated human IFNo2) were together transiently expressed in an ExpiCHO expression system (Thermo Fisher) according to the manufacturer’s guidelines. The human lgG1 Fc domains carry effector knock-out and knob-hole mutations. One week after transfection, the culture was collected, cells removed by centrifugation and the supernatants frozen.
[0146] The frozen supernatant was thawed and passed through a 0.2 pm and Emphaze AEX BV8R (3M) filter. Subsequently, the pH was adjusted to pH 8.0 and the solution incubated at 20°C in the presence of 0.5% reduced Triton-X100. After 1 hour incubation under soft stirring, the solution was 0.2 pm filtered and loaded on a prepacked 5 mL HITrap MabSelect PrismA column (Cytiva). After loading and washing at pH 5.5, the bound proteins were eluted by applying a 3-step pH gradient. Fractions identified by SDS-PAGE to be strongly enriched for the PD-L1 -based chimeric protein were pooled and the conductivity adjusted to 6.5 mS/cm by addition of 35 mM acetate, pH 5.0 dilution buffer. This sample was loaded on a 7.5 mL CEX SP Sepharose HP column (Cytiva) at a density of 20 mg/mL resin. Bound proteins were eluted by a linear salt gradient. CEX Elution fractions were selected and pooled based on SDS- PAGE for minimal contamination by host-cell proteins and/or PD-L1-based chimeric protein product variants resulting in a batch with a concentration of 5.97 mg PD-L1-based chimeric protein/mL.
Example 2: Formulation Buffer Stabilizes PD-L1 -Based Chimeric Protein at 10 mg/mL During Storage
[0147] An aim of this experiment was to screen buffers in order to identify formulations that ensure the stability of the PD-L1-based chimeric protein at a concentration of 10 mg/mL during storage. Specifically, 16 different buffers (different buffer type and/or pH, optionally with 100 mM NaCI) were prepared (see Table 1) for solubility screening of the PD-L1 -based chimeric protein:
• Citrate buffer pH 4.5, 5.0 and 6.0
• Histidine buffer pH 5.0, 5.5 and 6.5
• Phosphate buffer pH 6.0, 7.0 and 7.5
• Acetate buffer pH 4.5 and 5.5
• Histidine-acetate buffer pH 5.0
• Tris buffer pH 7.5
• Histidine buffer pH 5.5 with 100 mM NaCI
• Phosphate buffer pH 7.0 with 100 mM NaCI
• Acetate buffer pH 4.5 with 100 mM NaCI
Table 1. Composition per mL of the buffers for the PD-L1-based chimeric protein solubility screening
Buffer solution
Figure imgf000037_0001
Composition per mL
Figure imgf000038_0001
[0148] Buffers containing 100 mM NaCI were prepared from the 50 mM buffer without NaCI. The necessary amount of NaCI was weighed and added to a 50 mL volumetric flask. Then, 40 mL of the corresponding buffer was added to the volumetric flask, and the solution was stirred until clear. The volume of the volumetric flask was then completed with the corresponding buffer. The solution was homogenized by tumbling.
[0149] Solubility of the PD-L1-based chimeric protein was tested by adding the protein to increasing amounts of PEG 6000 in the 16 different buffers and by measuring turbidity as a read-out for protein aggregation. For this, buffer/PEG solutions were prepared in a 96 deep well plate: the 200 mM buffer stock solutions, water, and the 50% (w/v) PEG 6000 stock solution were mixed in a way that a fixed concentration of 29.8 mM buffer was present in each well, and an increasing concentration (0%, 2.4%, 4.8%, 7.2%, 9.5%, 11 9%, 14.3%, 16.7%, 19.1%, 21.5%, 23.8%, and 26.2%) of PEG was reached within the same row.
[0150] The PD-L1 -based chimeric protein solution batch was dialyzed twice overnight (with a 6-8 kDa MWCO dialysis membrane) at 2-8°C against 1 L 10 mM Na-acetate, pH 5.0, and filtered through a sterilizing 0.2 pm filter. This final PD-L1 -based chimeric protein stock solution had a concentration of 5.9 mg/mL.
[0151] For the precipitation assay, 84 pL of each well of the 96-deep well plate with buffer-PEG mixtures was transferred to an UV transparent 384 well plate. For every buffer, a separate row was used. Then, 16 pL of PD-L1- based chimeric protein was added to each buffer-PEG solution and mixing was performed by pipetting up and down. The final concentration was 25 mM for the buffer, 50 mM for NaCI (if applicable), and 0.95 mg/mL of PD-L1-based chimeric protein. Finally, turbidity measurement was performed with the plate reader, and absorbance was measured at 500 nm.
[0152] The turbidity measurements shown in Figure 1 indicate that the best 4 buffers without NaCI in which the PD-L1 -based chimeric protein was less prone to precipitate were shown to be: acetate buffer at pH 4.5, acetatehistidine buffer at pH 5.0, histidine buffer at pH 5.0, and histidine buffer at pH 5.5. A pH above 5.5 was shown to be generally less favorable, but surprisingly, citrate buffers in the pH range 4.0 to 5.0 were also clearly less favorable for solubility of the PD-L1-based chimeric protein. In general, the addition of NaCI did not improve solubility.
Example 3: Screening of Formulation Excipients and Buffers for Stabilizing Influence
[0153] The PD-L1-based chimeric protein solution batch was dialyzed twice (with a 3.5 kDa MWCO dialysis membrane) at room temperature against 0.9 L 10 mM Na-acetate, pH 5.0 and filtered through a sterilizing 0.2 pm filter. This final PD-L1-based chimeric protein stock solution had a concentration of 5.6 mg/mL.
[0154] To cover the pH range 4.5 to 5.5 (histidine buffer at pH 5.5, histidine-acetate buffer at pH 5.0, and acetate buffer at pH 4.5), three of the four buffers were selected for an experiment to select candidate excipients. Although no additional oxidation of the PD-L1 -based chimeric protein was observed after 24 hours incubation at room temperature, the protein was found to be sensitive to H2O2-induced oxidation, such that antioxidants were also selected as excipients in an experiment with following factors:
• Factor 1 : Buffer type
• Quantitative factor at 3 levels
• Histidine buffer at pH 5.5; acetate buffer at pH 4.5; and histidine-acetate buffer at pH 5.0
• 25 mM
• Factor 2: Tonicifyi ng excipient
• Qualitative factor at 4 levels
• Sucrose, Trehalose, Mannitol, or NaCI
• At a concentration to reach about 300 mOsm/kg in the final mixture • Factor 3: Stabilizing excipient
• Qualitative factor at 4 levels
• Arginine, Glycine, cyclodextrin, or no stabilizer
• At a concentration of 50 mM
• Factor 4: Antioxidant excipient
• Qualitative factor at 3 levels
• EDTA, Methionine or no antioxidant
• At a concentration of 3 mM
[0155] Stock solutions of the buffers were prepared at a concentration of 250 mM as indicated in Table 2. The stock solutions of the excipients were prepared for the sugars and NaCI at 500 mM for the amino acids or the HP-beta- cyclodextrin at 250 mM and 30 mM for the antioxidants as indicated in Table 3. All solutions were filtered through a 0.2 m Spartan RC syringe filter and collected in a clean falcon tube to reduce dust contamination during the excipient screening.
Table 2: Composition per mL of the buffers for PD-L1-based chimeric protein or protein complex excipient screening
Figure imgf000040_0001
Table 3: Composition per mL of the excipients for PD-L1-based chimeric protein or protein complex excipient screening
Figure imgf000040_0002
Figure imgf000041_0002
[0156] With these mainly qualitive factors, a D-optimal design was used, which was created in MODDE (version 12 1 , Sartorius) An amount of 96 combinations of the different factors were proposed (shown in Table 4). As this experiment design was performed once per buffer, a total of amount of 288 concepts were tested by thermal shift assay. A volume of 50 piL of each composition was prepared per well. At first, 5 L of the buffer was pipetted in the wells followed by the tonicifier (sucrose, trehalose, mannitol or NaCI) with varying volume to reach an isotonic solution, 10 L of the stabilizing excipients (arginine, glycine, or HP beta cyclodextrin), 5 pL of the antioxidant and the remaining amount of purified water type I to reach a volume of 41.67 pL. Finally, 8.33 pL of the PD-L1 -based chimeric protein solution was added and each solution was mixed in the well. The well plate was then centrifuged for 7 minutes at 4,000 g. The supernatant of the solutions was then used for differential scanning fluorimetry (DSF) and static light scattering (SLS) measurement using the Uncle (Unchained labs). The Uni’s (minicuvettes) were filled with 8.8 pL of protein solution at a concentration of about 0.9 mg/mL and sealed in the uni holder. The measurement was performed with a temperature ramp starting at 25°C up to 95 C at a rate of 0.4 C/min and 180 sec of equilibration at the start. The emission spectrum was captured between 250-720 nm with excitation at 266 nm. The SLS signal was measured at 266 nm and 473 nm. DLS data was measured with 4x5 sec of acquisitions resulting in the determination of Toriset,
Figure imgf000041_0001
[0157] Remarkably, two melting points could be identified which may reflect different melting temperatures for the different domains (VHH, Fc, and IFN moieties) in this complex molecule, but analysis focused on the first melting point as likely relating to the domain with lower stability. Table 4, below, depicts 96 excipient concepts tested per buffer condition in the thermal shift assay and provides an overview of composition of the different experiments, as well as their corresponding melting temperature, onset of melting, aggregation temperature at 266 nm and the SLSmax signal.
Table 4: 96 Excipient Concepts Tested Per Buffer Condition in the Thermal Shift Assay
Figure imgf000041_0003
Figure imgf000042_0001
Figure imgf000043_0001
[0158] The obtained Tm,
Figure imgf000044_0001
data was imported in MODDE to analyze the model and trends. Each response (Tm, Tagg, Tonset and SLSmax) was fitted with a model optimized for the model terms. Each model term (the constant, buffer, tonicifier, stabilizer, antioxidant, or a combination of two single terms as quadratic terms) that did not significantly influence the response was not included in the model. The model for a response can be used if the R2 value, the Q2 value, and the reproducibility are above 0.5, if the model validity is around 0.25 or higher and if the difference between R2 and Q2 is less than 0.3. Thus, the Tm and Tonset models were further used for analysis of the stabilizing excipient. The models for Tagg and
Figure imgf000044_0002
had a reproducibility of 1 or very close to as all three replicates had the same outcome. Therefore, the model validity could not be calculated or was negative. Due to the good R2 and Q2 values, both models for Tagg and SLSmax were considered to be legitimate. The best models were the models with the highest R2 and Q2 values, which were the models of Tm and
Figure imgf000044_0003
[0159] To evaluate the influence of the excipients on one of the responses, coefficient plots were analyzed. Figure 2 shows that the coefficient plot is presented for Tm. A protein is generally considered to be more stable with a higher Tm. Therefore, excipients with an increasing effect on the Tm and thus a positive coefficient bar are the excipients with a stabilizing effect on the protein. The coefficient plot of the
Figure imgf000044_0004
model also showed a good fit and the coefficient plots are shown in Figure 3. As a lower SLS count indicates less or smaller aggregates, excipients with negative coefficient bars, which thus have a lowering effect on the SLS signal, are considered the more stabilizing excipients. The model for Tagg is presented in Figure 4. The summary of fit of this model was less good compared to the model for Tm and SLSmax. This could also be observed from the bigger standard deviations obtained in the coefficient plot compared to the previous coefficient plots. Finally, as shown in Figure 5, the model for Tonset was again very similar to what was obtained for the Tm model. As this model was scoring the least based on the summary of fit, a lower weight was given to the effects of each excipient and especially the small deviations of this model compared to other models.
[0160] The overall results indicated that no clear trend was observed for Factor 1 (buffer type), which is in line with the fact that these buffers had already been preselected as the best buffers. With regards to Factor 2, however, a clear stabilizing effect was observed for all three sugars (sucrose, trehalose, and mannitol), while NaCI exhibited a destabilizing effect. For Factor 3, beta-cyclodextrin exhibited the largest stabilizing effect, while glycine showed a moderately stabilizing effect, and arginine exhibited a destabilizing effect. Surprisingly, when NaCI and arginine were combined, a strong stabilizing effect was observed. For Factor 4 (antioxidants), only minor effects were observed.
Example 4: Shear Stress Study
[0161] Different formulations were then tested for a shear stress study, as depicted in Table 5. A 5% (w/v) PS20 stock and a 5% (w/v) poloxamer 188 (Pol188) stock solution were prepared in water. The PD-L1-based chimeric protein batch was dialyzed in a dialysis cassette (3.5 kDa MWCO, Thermo fisher) against 500 mL of 25 mM histidine- acetate buffer for 2 hours, followed by second dialysis overnight. The solution was recovered from the dialysis cassette and the concentration of the solution was measured as 2.9 mg/mL. Finally, the dialyzed PD-L1 -based chimeric protein solutions were spiked with PS20 or Poll 88 stock solution to reach either 0.01 % or 0.05% of surfactant. The preparation with volumes of each of the stock solutions is presented in Table 5.
Table 5: Overview of the different concepts for the shear stress study.
Figure imgf000045_0001
[0162] To assess the capability of the surfactants to prevent possible aggregation, the samples were vortexed for 15 minutes, followed by another 45 minutes. The particle size distribution was measured before, after 15 minutes, and after 1 total hour of vortexing, and was compared to the sample without using DLS measurement on a Malvern Zetasizer Nanoseries ZS. The method parameters that were used are listed in Table 6.
Table 6: Method parameters for DLS measurements.
Parameter Value/Setting
Temperature 25 °C
Equilibration time 120 s
Number of measurements 3
Material Protein
Dispersant Water
Viscosity Water
Analysis model General purpose (normal resolution)
Scattering mode Non-invasive Backscatter (NIBS) - 173 degrees
Sample volume 70 pL
[0163] The particle size distributions for the PS20 and Pol 188 samples, respectively, are shown in Figure 6 and
Figure 7. For both samples, no aggregation was seen before vortexing apart from a little bump in the intensity distribution just below 1000 nm which was most likely related to dust. It was not visible in the volume distribution so the presence was negligible.
[0164] After 15 minutes of vortexing, the sample without surfactant was aggregated completely with a large peak in both the intensity and the volume distribution above 1000 nm. For the samples containing PS20, both concentrations provided sufficient stabilization as no change in the DS related peak at 10 nm was observed after 15 minutes. Aggregation was observed for the Pol 188 sample at a concentration of 0.01 %, although a limited amount of the nonaggregated peak at 10 nm was still present. At 0.05% Poll 88, sufficient stabilization occurred, although a small bump seen in the intensity distribution at around 200 nm indicated a very small amount of aggregated PD-L1 -based chimeric protein.
[0165] After 1 hour of vortexing, complete aggregation was again seen for the sample without surfactant. A shift was observed for the sample with 0.01% PS20 in both the intensity and volume distribution. No change was observed for the higher PS20 concentration of 0.05%, which seemed to provide sufficient protection against aggregation. The 0.01 % Pol 188 concentration showed complete aggregation as no non-aggregated peak was observed at around 10 nm. Nonetheless, smaller aggregates were observed compared to the sample without surfactants. The 0.05% Pol 188 concentration was not sufficient for the protection of PD-L1-based chimeric protein against aggregation as a larger peak just below 100 nm was observed. This peak was not observed in the volume distribution so aggregation of PD- L1 -based chimeric protein was very limited. As 1 hour of vortexing represents very intense shear stress, the 0.01% to 0.05% PS20 and 0.05% Pol 188 was shown to provide sufficient stabilization.
Example 5: Short Stress Stability Study
[0166] The short stress study was performed with 5 different concepts which were stressed at 40°C/75% RH and 25°C/60% RH for up to T2w (2 weeks). The composition of the tested formulations is shown in Table 7. For all five concepts, the histidine-acetate buffer was chosen with a targeted PD-L1 -based chimeric protein concentration of 10 mg/mL. Trehalose was added in all concepts as a tonicifier in order to reach the target of about 310 mOsm/kg. The last concept contained a combination of two tonicifiers being trehalose and NaCI. Furthermore, 3 mM methionine as antioxidant and 0.02% PS20 as surfactant were used. Several stabilizers were tested at a concentration of 50 mM: HP-P-cyclodextrin, glycine, and arginine. Concept 4 did not contain a surfactant to evaluate if HP-p-cyclodextrin could also protect the protein against shear stress. The concept buffers of Table 7 were prepared without the PS20 for buffer exchange of PD-L1 -based chimeric protein. The PD-L1 -based chimeric protein batch was added first to a dialysis membrane (6-8 kDa) which was submerged into the PEG-acetate buffer and concentration of PD-L1 -based chimeric protein was performed for about 3 hours to reduce volume to about 50%. The subsequent buffer exchange to the concept formulations was performed in dialysis cassettes initially for 3 hours, followed by an additional overnight dialysis in fresh concept buffer. Finally, the concentrated and buffer-exchanged PD-L1-based chimeric protein solutions were diluted in their respective buffers to about 10 mg/mL and PS20 added from a 0.5% (w/v) stock solution in the matching buffers.
Table 7: Overview of the different concepts for the short stress study.
Concep rt t .nn .ir .if..ipr PS-20 Stabilizer Antioxidant
Nr. luru iiei (0.02% w/v) (50 mM) (3 mM)
1 trehalose (215 mM) yes arginine Methionine
2 trehalose (215 mM) yes cyclodextrin Methionine
3 trehalose (215 mM) yes glycine Methionine
4 trehalose (215 mM) no cyclodextrin Methionine
5 trehalose (115 mM) + NaCI (50 mM) yes cyclodextrin Methionine
[0167] All 5 formulation concepts were subjected to a short stress stability study for two weeks at 25°C/60% RH and 40°C/75% RH as well as a shear stress test.
[0168] For RPC, the PD-L1-based chimeric protein samples were diluted to 1 mg/mL in their respective buffers. Before HPLC analyses, samples were centrifuged at 20000 g for 5 minutes, after which 4 |dl_ was injected on UPLC C4 column (2.1 mm ID x 100 mm) at 50°C and proteins were eluted during a 15 minute linear gradient ranging from 5 to 95% acetonitrile (with 0.1 % TFA) at 0.35 mL/min flow rate. Proteins were detected by analytical flow cell (10 mm) at 280 nm wavelength. PD-L1-based chimeric protein elutes as 2 main peaks and some minor pre- or post-peaks. The analysis shown in Table 8 indicates that for all concepts there were no major changes at 25°C for up to 2 weeks. At 40°C, a similar increase in pre-peaks was observed for all five concepts, but concept 1 especially, did also show an increase in post-peaks.
Table 8: Results, as area% of each peak section, of short stress study samples measured using RPC.
Concept 1 Pre-peaks Main peak Post-peaks
TO 0.68 95.45 3.74
25 °C T1w 0.76 95.55 3.58
T2w 0.92 95.38 3.58
TO 0.68 95.45 3.74
40 °C T1w 1.45 92.94 5.12
T2w 2.68 88.50 7.80
Concept 2 Pre-peaks Main peak Post-peaks
25 °C TO 0.77 95.73 3.41 T1w 0.83 95.82 3.25
T2w 0.96 95.7 3.24
TO 0.77 95.73 3.41
40 °C T1w 1.64 94.9 3.24
T2w 2.39 93.73 3.53
Concept 3 Pre-peaks Main peak Post-peaks
TO 0.69 95.78 3.41
25°C T1w 0.80 95.77 3.31
T2w 0.93 95.66 3.3
TO 0.69 95.78 3.41
40°C T1w 1.45 94.73 3.55
T2w 2.43 92.5 4.56
Concept 4 Pre-peaks Main peak Post-peaks
TO 0.68 95.83 3.37
25°C T1w 0.85 95.86 3.18
T2w 0.93 95.77 3.18
TO 0.68 95.83 3.37
40°C T1w 1.46 95.2 3.15
T2w 2.33 93.96 3.39
Concept 5 Pre-peaks Main peak Post-peaks
TO 0.68 95.58 3.66
25°C T1w 0.82 95.57 3.50
T2w 0.94 95.51 3.45
TO 0.68 95.58 3.66
40°C T1w 1.51 94.35 3.86
T2w 2.43 92.06 4.92
[0169] SE-HPLC was performed as described in M-P10101-006 v2.0. The samples were diluted to 1 mg/mL. Before HPLC analyses, samples and references were centrifuged at 20000 g for 5 minutes.
[0170] For samples and reference (PD-L1 -based chimeric protein batch stored frozen), 10 L was injected on Superdex 200 Increase SE-HPLC column (3.2 mm ID x 300 mm). [0171] For SEC, the PD-L1-based chimeric protein samples were diluted to 1 mg/mL in their respective placebo formulations. Before HPLC analyses, samples were centrifuged at 20000 g for 5 minutes, and 10 L was injected on Superdex 200 Increase SEC column (3.2 mm ID x 300 mm), and proteins were isocratically eluted by 25 mM Tris, 123.5 mM NaCI at pH 8.5 at 0.075 mL/min flow rate. Proteins were detected by bio-compatible Titanium flow cell (5 mm) at 280 nm wavelength. Column was kept at 25°C, while samples were stored in cooled sample injector (7°C) until analysis. The analysis (depicted in Table 9) indicates that for all concepts there were no major changes at 25°C for up to 2 weeks. At 40°C, an increase in both pre- and post-peaks was observed for all formulations; however, the increase in higher molecular weight species (pre-peaks) was smallest for concepts 1 and 2.
Table 9: Results, as area% of each peak section, of short stress study samples measured using SEC.
Concept 1 Pre-peaks Main peak Post-peaks
TO 0.33 99.34 0.33
25 °C T1w 0.53 99.04 0.43
T2w 0.72 98.91 0.38
TO 0.33 99.34 0.33
40 °C T1w 0.58 96.24 3.18
T2w 4.66 89.13 6.20
Concept 2 Pre-peaks Main peak Post-peaks
TO 0.24 99.31 0.45
25 °C T1w 0.34 99.19 0.47
T2w 0.29 99.24 0.46
TO 0.24 99.31 0.45
40 °C T1w 0.78 96.98 2.24
T2w 4.30 92.64 3.05
Concept 3 Pre-peaks Main peak Post-peaks
TO 0.30 99.34 0.37
25°C T1w 0.45 99.14 0.40
T2w 0.58 98.95 0.46
TO 0.30 99.34 0.37
40°C T1w 1.20 96.39 2.40
T2w 13.39 83.33 3.29
Concept 4 Pre-peaks Main peak Post-peaks TO 0.25 99.33 0.41
25°C T1w 0.25 99.23 0.53
T2w 0.31 99.29 0.41
TO 0.25 99.33 0.41
40°C T1w 1.18 96.47 2.35
T2w 8.32 88.63 3.05
Concept 5 Pre-peaks Main peak Post-peaks
TO 0.33 99.32 0.36
25°C T1w 0.36 99.05 0.59
T2w 0.5 99.09 0.42
TO 0.33 99.32 0.36
40°C T1w 0.73 96.9 2.38
T2w 13.99 82.2 3.8
[0172] The concentration of PS20 (0.02%) used in this short stress study was different from the concentration in the previous shear stress testing. Additionally, this study evaluated whether PD-L1-based chimeric protein could be protected from aggregation induced by shear stress by the presence of HP-|3-cyclodextrin. The short stress study samples were again subjected to a shear stress study in a similar way as was performed in Example 4 (apart from the absence of DSF measurements for this set of shear stress testing). The results are shown in Figures 8A-8B. The placebo formulations were also measured without shear stress to verify whether certain excipients would impact the particle size distribution.
[0173] Some concepts showed peaks of around 1 nm, which is mainly related to excipients since these peaks were also present in the placebo. The PD-L1-based chimeric protein-related peak at 10 nm was present in all concepts before vortexing. Concept 4 contained already before vortexing some higher sized particles. The PD-L1-based chimeric protein-related peak at around 10 nm was rather small in concept 2 and 5, which was probably related to the presence of HP-|3-CD, which is expected to have a peak of around 1-2 nm and is relatively abundant. A few bigger sized peaks were present which could be related to the presence of surfactant micelles. Note that these bigger sized particles were not present in the placebo of concept 4, which did not contain a surfactant.
[0174] After 15 minutes of vortexing (blue curves), a shift to higher sized species was obtained for concept 1. Concept 4 showed a more pronounced peak at around 1000 nm. After 1 hour of vortexing (black curves), more aggregates were obtained for concept 1. Apart from the aggregate peak that was present before, concept 4 also showed the presence of a medium sized aggregate at around 100 nm in the intensity distribution although no differences were obtained in the volume distribution compared to before vortexing. Concept 3 showed a rather broad DS-related peak in the intensity distribution as well as a small shift of this peak in the volume distribution pointing to a little aggregation. No changes seemed to occur in either concept 2 or 5.
[0175] In summary, all 5 concepts exhibited high stability of the protein when stressed at 25°C. When more stress was applied, such as incubation at 40°C or vortexing, especially concept 1 was shown to perform less favorably, indicating that the addition of arginine alone as stabilizer is not sufficient to protect the PD-L1-based chimeric protein in such conditions.
Example 6: Extended Stability Stress Study
[0176] An extended stress study was performed with two different concept compositions from Table 10, provided below. For both concepts, the histidine-acetate buffer at pH 5.0 was chosen with a targeted PD-L1 -based chimeric protein concentration of 10 mg/mL. Purified PD-L1 -based chimeric protein was exchanged to a buffer containing 11.26 mL L-histidine, 13.74 mM acetic acid, 194 mM trehalose, and 3 mM methionine and concentrated to about 12 mg/mL by tangential flow filtration. Subsequently, stock solutions of Tween-20, glycine, or HP-p-cyclodextrin was prepared in the aforementioned buffer. As needed, additional buffer was added to reach a final concentration of about 10 mg/mL PD-L1 -based chimeric protein with respect to both concept formulations. The final pH of the formulations was found to be around 5.2 to 5.3. These preparations were filled at 2 mL in 2R glass vials, which were closed off with Flurotec stoppers and aluminum caps. The vials were placed to measure stability at 40°C/75% RH for 2 weeks, at 25°C/60% RH for 1 month, 2-8°C for 3 months, and -20°C for 3 months. In addition, separate vials were subjected to up to four freeze/thaw cycles.
Table 10: Overview of the different concepts for the extended stress study.
Concept . ... PS-20 Stabilizer Antioxidant
Nr. tonici er (0 02o/o w/v) (50 mM) (3 mM)
1 trehalose (194 mM) yes cyclodextrin Methionine
2 trehalose (194 mM) yes glycine Methionine
[0177] Stability was monitored for appearance, pH, osmolality, potency (ELISA and cell-based assay), SDS- PAGE (reducing and non-reducing), protein concentration (UV absorbance), SE-HPLC, RP-HPLC, clEF, sub-visible particles, and MS.
[0178] No changes were observed in either formulation as a result of freeze/thaw cycling. It was also found that storage up to 3 months at -20°C or 2-8°C produced no observed changes in either formulation. During storage up to 1 month at 25°C/60% RH, changes remained limited and similar for both formulations. [0179] However, during the highest stress storage at 40°C/75% RH, some minor changes were detectable. First, a more pronounced opalescence was observed in formulation 2 and a more pronounced yellowish color for formulation 1. Second, for formulation 2, a reduction in the main peak of the SE-HPLC (Table 11) was observed at week 1, and this increased at week 2. In addition, more pre-peaks were observed after 1 week of storage, which was no longer detectable after 2 weeks. Taken together, these data hint to a higher aggregation risk in the concept 2 formulation at 40°C.
Table 11 : SE-HPLC Area% (A = 280 nm) of pre-peaks, main peak and, post-peaks for concept 1 and 2 formulations of PD-L1-based chimeric protein.
Figure imgf000052_0001
[0180] Finally, SDS-PAGE CBB under non-reducing conditions, as shown in Figure 9, showed a main protein band at 80 kDa, as well as an extra band at 47 kDa (open arrow). Upon storage at 40°C/75% RH for 1 or 2 weeks, a band at 200 kDa (closed arrow) was observed for both formulations. Western-blot (anti-human Fc; Figure 10) under non-reducing conditions also showed the PD-L1 -based chimeric protein main band around 80 kDa and the extra band around 47 kDa (lower open arrow). In addition, bands around 220 kDa (mid closed arrow) and 75 kDa (highest open arrow) were observed. Upon storage at 40°C/75% RH for 1 or 2 weeks, the intensity of the latter bands increased, and an extra band around 150 kDa (lowest closed arrow) was observed for both formulations. Remarkably, the higher molecular bands tended to be more prominent for formulation 2, which also has an extra protein around 320 kDa (highest open arrow), although this was not observed for formulation 1 .
Example 7: Long Term Stability Study
[0181] Long term stability of the PD-L1-based chimeric protein was assessed in the formulation buffer containing 25 mM Histidine-acetate pH 5, 194 mM trehalose, 3 mM methionine, 0.02% Tween-20, and 50 mM (73.9 g/L) HP-p- cyclodextrin (catno: code 27; Cyclolab; molar substitution rate: 0.4-1 .5).
[0182] For this study, a new batch of PD-L1 -based chimeric protein was produced in a 100L bioreactor and was harvested by depth filtration and subsequently purified as before by proteinA and cation-exchange chromatography. In addition, a final polishing step was performed on the cation-exchange peak fractions using an anion-exchange filter (SartobindQ, Sartorius) after which the protein was concentrated to about 15 mg/mL by tangential flow filtration against the formulation buffer without the beta-cyclodextrin and Tween-20. As a final step, Tween-20 and HP-|3-cyclodextrin were added from respectively at 50x and 5x stock concentration prepared in aforementioned buffer. Finally, the protein was diluted to about 10.5 mg/mL with formulation buffer and aseptically filtered over a 0.22 m filter.
[0183] The new batch of PD-L1 -based chimeric protein was filled at 5.5 mL in 6R glass vials closed off with bromobutyl rubber stoppers and aluminum caps. Part of the protein stock was compounded with formulation buffer to a 1 mg/mL protein complex concentration and similarly filled at 5.5 mL in 6R glass vials.
[0184] The glass vials are stored at either -20°C (upright 1 , 3, 6, 9, 12, 18, 24, 36 months) or at 2-8°C (upright and inverted 1 , 3 and 6 months).
[0185] Initial evaluation included up to 9-month storage. It was observed that overall, there were no changes seen in appearance (color and opalescence) or pH at both concentration levels. Also, the PD-L1 -based chimeric protein concentration as measured by OD280 remained stable within the preset limits (10 ± 1 mg /mL or 1.0 ± 0.2 mg/mL), and the vials remained essentially free from visible particles. Nor was there an increase in subvisible particles. Osmolality was maintained around 340 mOsm and 330 mOsm, respectively, for the 10 and 1 mg/mL vials.
[0186] SEC was performed as previously described. No additional high or low molecular weight species were formed during the entire study period. Thus, Table 12 below shows results for the starting material, 6-month, and 9- month time points. These findings were also corroborated by SDS-PAGE under reducing and non-reducing conditions, which did not reveal any changes.
Table 12: Results, as area% of each peak section, of long-term stability study samples as tested in SEC.
Figure imgf000053_0001
Figure imgf000054_0001
[0187] RPC was performed as previously described. No additional peaks were identified and no relevant changes compared to the starting material were observed; thus, Table 13 shows results for the starting material, 6- month, and 9-month time points.
Table 13: Results, as area% of each peak section, of long-term stability study samples as tested in RPC.
Figure imgf000054_0002
[0188] The samples were also analyzed by a PD-L1/anti-IFN ELISA, which allows determination of the potency of the PD-L1-based chimeric protein to bind simultaneously to the human PD-L1 protein and a neutralizing human IFNa2 monoclonal antibody. Briefly, MAXISORP Nunc immune plates were coated with anti-human IFN-alpha monoclonal antibody (1 g/mL, clone MMHA-13; PBL assay science). After washing and blocking the plate, an 8-step 3-fold serial dilution in blocking buffer of PD-L1-based chimeric protein samples starting from 1 mg/mL were loaded on the plate. After binding of the PD-L1 -based chimeric protein to the plate and subsequent washing, a biotinylated PD- L1 protein (25 ng/mL, PDL-H82F2, ACRO Biosystems) was incubated. Finally, after the next washing step, a HRP- conjugated streptavidin (1/1 000, 21126, Thermo Scientific) antibody was used for detection. Detection was conducted after washing using 1-Step Ultra TMB ELISA Substrate Solution (Thermofisher) according to the manufacturer’s instructions. The EC50 of the response curve was determined by 4PL analysis and expressed as relative value to a reference standard. Taking into account the normal biological variability of an ELISA, the binding activity of the PD- L1-based chimeric protein did not change over time for all conditions tested; thus, Table 14 shows results for the starting material, 6-month, and 9-month time points.
Table 14: Results, as % EC50 versus reference, of long-term stability study samples as measured using ELISA.
Figure imgf000055_0001
[0189] The samples were also tested for the appearance of new charge variants by capillary iso-electric focusing (clEF). In brief, the sample was diluted to a final concentration of 1 mg/mL. In a next step, this solution was desalted using a spin-column by loading 500 pL sample onto the column, centrifuging the column for 5 min at 14000 x g, and bringing the volume back to 500 pL with MQ water. The desalting procedure was repeated two more times, after which 20 pL of the desalted sample was added to 80 pL of the clEF master mix (19.5 pL water, 19.5 pL 10 M urea, 34 pL 1% methyl cellulose, 1 pL Pharmalyte 3-10, 3 pL Pharmalyte 5-8, 2 pL 500 mM Arginine, 1 pL pl marker 6.14, 1 pL pl marker 8.40) into a microcentrifuge tube. The final concentration of the PD-L1 -based chimeric protein corresponded to 0.2 mg/mL. The samples were analyzed on the Maurice combo instrument (ProteinSimple) using a clEF cartridge PS-MC02-C (ProteinSimple). No new charge variants appeared over the entire study duration and the relative amount of change variants remained unchanged over time, as observed during the entire study period (Table 15, which shows results for the starting material, 6-month, and 9-month time points).
Table 15: Results, as area% of each peak section, of long-term stability study samples as tested in clEF.
Figure imgf000055_0002
Figure imgf000056_0001
EQUIVALENTS
While the subject matter has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the subject matter following, in general, the principles of the subject matter and including such departures from the present disclosure as come within known or customary practice within the art to which the subject matter pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.
INCORPORATION BY REFERENCE
All patents and publications referenced herein are hereby incorporated by reference in their entireties.
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 subject matter is not entitled to antedate such publication by virtue of prior disclosure.
As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

Claims

CLAIMS What is claimed is:
1 . A pharmaceutical composition comprising:
(a) a Programmed death-ligand 1 (PD-LI)-based chimeric protein or protein complex comprising (i) a targeting moiety that specifically binds to PD-L1 , (ii) a modified human I FNa2, and (iii) a modified Fc domain;
(b) at least one pharmaceutically acceptable buffering agent comprising a histidine buffer;
(c) at least one pharmaceutically acceptable excipient comprising a surfactant; and
(d) at least one pharmaceutically acceptable excipient comprising a tonicifier, wherein the pharmaceutical composition has a pH that ranges from at least about 4.0 to about 6.0.
2. The pharmaceutical composition of claim 1, wherein the pH ranges from at least about 4.5 to about 5.5, optionally wherein the pH is about 5.0.
3. The pharmaceutical composition of claim 1 or claim 2, wherein the pharmaceutical composition is formulated for parenteral administration.
4. The pharmaceutical composition of claim 3, wherein the pharmaceutical composition is formulated for intravenous administration, optionally wherein intravenous administration comprises intravenous infusion or bolus injection.
5. The pharmaceutical composition of any one of the above claims, wherein the buffering agent is or comprises histidine at a concentration of at least about 5 mM to about 25 mM.
6. The pharmaceutical composition of claim 5, wherein the buffering agent comprises histidine at a concentration of at least about 10 mM to about 15 mM.
7. The pharmaceutical composition of any one of the above claims, wherein the buffering agent comprises L- histidine or L-histidine hydrochloride monohydrate.
8. The pharmaceutical composition of any one of the above claims, wherein the buffering agent further comprises an acetate buffer, optionally wherein the acetate buffer comprises acetic acid.
9. The pharmaceutical composition of claim 8, wherein the acetic acid is present at a concentration of at least about 10 mM to about 20 mM, optionally at least about 10 mM to about 15 mM.
10. The pharmaceutical composition of any one of the above claims, wherein the surfactant is or comprises a polysorbate surfactant, optionally selected from polysorbate 20 (PS20) and poloxamer 188 (Poll 88).
11. The pharmaceutical composition of claim 10, wherein the PS20 is present at a concentration of at least about 0.01 % (w/v) to about 0.1% (w/v), optionally wherein the PS20 is present at a concentration of at least about 0.01 to about 0.05% (w/v).
12 The pharmaceutical composition of claim 8, wherein the Poll 88 is present at a concentration of at least about 0.01 % (w/v) to about 0.1% (w/v), optionally wherein the Pol 188 is present at a concentration of at least about 0.05% (w/v).
13. The pharmaceutical composition of any one of the above claims, wherein the tonicifier is selected from one or more of trehalose, sodium chloride, trehalose-sodium chloride, sucrose, mannitol, and sorbitol.
14. The pharmaceutical composition of claim 13, wherein the tonicifier is present at a concentration of at least about 90 mM to about 270 mM.
15. The pharmaceutical composition of any one of the above claims, further comprising one or more additional excipients.
16. The pharmaceutical composition of claim 15, wherein the additional excipient is a stabilizer selected from one or more of cyclodextrin, arginine, sodium chloride-arginine, and glycine, optionally wherein the cyclodextrin is betacyclodextrin, optionally HP-beta-cyclodextrin.
17. The pharmaceutical composition of claim 16, wherein the stabilizer is present at a concentration of at least about 25 mM to about 75 mM, optionally wherein the stabilizer is present at a concentration of at least about 50 mM.
18. The pharmaceutical composition of claim 15, wherein the additional excipient is an antioxidant selected from methionine and ethylenediaminetetraacetic acid (EDTA).
19. The pharmaceutical composition of claim 18, wherein the antioxidant is methionine, optionally wherein methionine is present at a concentration of at least about 1 mM to about 5 mM, optionally wherein methionine is present at a concentration of about 3 mM.
20. The pharmaceutical composition of any one of the above claims, wherein the PD-L1 -based chimeric protein complex is present at a concentration of least about 1 mg/mL.
21. The pharmaceutical composition of any one of the above claims, wherein the PD-L1 -based chimeric protein complex is present at a concentration from at least about 1 mg/mL to about 10 mg/mL.
22. The pharmaceutical composition of any one of the above claims, wherein the PD-L1 -based chimeric protein complex is substantially in the form of a heterodimer.
23. The pharmaceutical composition of any one of the above claims, wherein the modified human I FNo2 has an amino acid sequence having at least about 95% identity with of SEQ ID NOs: 76 or 77.
24. The pharmaceutical composition of claim 23, wherein the modified human IFNo2 has an amino acid sequence having at least about 98% identity with of SEQ ID NOs: 76 or 77
25. The pharmaceutical composition of claim 24, wherein the modified human IFNo2 has an amino acid sequence having at least about 99% identity with of SEQ ID NOs: 76 or 77
26. The pharmaceutical composition of any one of the above claims, wherein the modified human IFNa2 has 1-3 mutations relative to the amino acid sequence of SEQ ID NOs: 76 or 77.
27. The pharmaceutical composition of claim 26, wherein the modified human I FNo2 comprises a R149A mutation with respect to SEQ ID NOs: 76 or 77 or one of a R33A, R144A, R144I, R144L, R144S, R144T, R144Y, A145D, A145G, A145H, A145K, A145Y, M148A and L153A mutation with respect to SEQ ID NOs: 76 or 77, optionally A145G .
28. The pharmaceutical composition of any one of the above claims, wherein the targeting moiety that specifically binds PD-L1 comprises a recognition domain comprising:
(i) three complementarity determining regions (CDR1 , CDR2, and CDR3), where
(a) CDR1 comprises an amino acid sequence selected from any one of SEQ ID NOs: 2, 5, 26, or 29;
(b) CDR2 comprises an amino acid sequence selected from any one of SEQ ID NOs: 3, 6, 27, or 30; and
(c) CDR3 comprises the amino acid sequence of SEQ ID NO: 4 or 28; or
(ii) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1 or 25; and where (I) or (ii) further comprises one or more mutations at one or more mutations at positions D54 and G55, numbering relative to SEQ ID NO: 1 , or one or more mutations at positions N32, D33, and M97, numbering relative to SEQ ID NO: 25.
29 The pharmaceutical composition of claim 28, wherein the targeting moiety that specifically binds to PD-L1 comprises a recombinant heavy-chain-only antibody (VHH).
30 The pharmaceutical composition of claim 29, wherein the VHH has an amino acid sequence having at least about 95% identity with any one of the amino acid sequences selected from SEQ ID NOs: 23, 72, 1, 7-22, 24, 25, and 31-71.
31. The pharmaceutical composition of claim 30, wherein the VHH has an amino acid sequence having at least about 98% identity with any one of the amino acid sequences selected from SEQ ID NOs: 23, 72, 1, 7-22, 24, 25, and 31-71.
32. The pharmaceutical composition of claim 31 , wherein the VHH has an amino acid sequence having at least about 99% identity with any one of the amino acid sequences selected from SEQ ID NOs: 23, 72, 1, 7-22, 24, 25, and 31-71.
33 The pharmaceutical composition of claim 32, wherein the VHH has the amino acid sequence of any one of the amino acid sequences selected from SEQ ID NOs: 23, 72, 1 , 7-22, 24, 25, and 31-71.
34 The pharmaceutical composition of any one of the above claims, wherein the modified Fc domain comprises one or more of the following mutations: P329G, K322Q, K322A, P331 G, or P331 S relative to any one of SEQ ID NO: 78-81.
35. The pharmaceutical composition of claim 34, wherein the modified Fc domain comprises one or more of the following mutations: P329G, K322Q, K322A, P331G, or P331S relative to human lgG1 Fc.
36. The pharmaceutical composition of claim 34 or 35, wherein the modified Fc domain has an amino acid sequence of at least about 90% identity with any one of the amino acid sequences selected from SEQ ID NO: 78-81 .
37. The pharmaceutical composition of claim 34 or 35, wherein the modified Fc domain has an amino acid sequence of at least about 93% identity with any one of the amino acid sequences selected from SEQ ID NO: 78-81 .
38. The pharmaceutical composition of claim 34 or 35, wherein the modified Fc domain has an amino acid sequence of at least about 95% identity with any one of the amino acid sequences selected from SEQ ID NO: 78-81 .
39. The pharmaceutical composition of any one of the above claims, wherein the pharmaceutical composition is freeze-dried or suitable for freeze-drying.
40. The pharmaceutical composition of any one of the above claims, wherein the pharmaceutical composition forms a lyophilized powder.
41 . The pharmaceutical composition of any one of the above claims, wherein the pharmaceutical composition is contained in an injection device, optionally wherein the injection device is a syringe or an injection pen.
42. A kit comprising the lyophilized powder of claim 40, a vial, and a syringe.
43. A method for treating or preventing a cancer, comprising administering an effective amount of the pharmaceutical composition of any one of claims 1-41 to a patient in need thereof.
44. The method of claim 43, wherein cancer is a blood cancer or a solid tumor cancer.
45. The method of claim 43 or 44, wherein the cancer is selected from one or more of lymphoma, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma (e.g,, Kaposi’s sarcoma); skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and posttransplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g. that associated with brain tumors), and Meigs' syndrome.
46. A pharmaceutical composition comprising:
(a) a Programmed death-ligand 1 (PD-LI)-based chimeric protein complex comprising (i) a targeting moiety that specifically binds to PD-L1 , (ii) a modified human IFNo2, and (iii) a modified Fc domain, wherein the PD-L1-based chimeric protein complex is present at a concentration of at least about 1 mg/mL;
(b) a buffering agent comprising histidine present at a concentration ranging from about 5 mM to about 20 mM and/or acetic acid present at a concentration ranging from about 5 mM to about 20 mM;
(c) an excipient comprising a surfactant selected from PS20 at a concentration ranging from about 0.01% to about 0.1%, and Poll 88 at a concentration ranging from about 0.01 % to about 0.1%;
(d) a tonicifier excipient comprising trehalose at a concentration ranging from at least about 90 mM to about 270 mM;
(e) a stabilizer excipient comprising beta-cyclodextrin and/or sodium chloride-arginine, wherein the stabilizer excipient is present at a concentration ranging from at least about 25 mM to about 75 mM; and
(f) an antioxidant excipient comprising methionine, wherein methionine is present at a concentration of at least about 1 mM to about 5 mM; wherein the pharmaceutical composition has a pH that ranges from at least about 4.5 to about 5.5.
47. A pharmaceutical composition comprising: (a) a Programmed death-ligand 1 (PD-LI)-based chimeric protein complex comprising (i) a targeting moiety that specifically binds to PD-L1 , (ii) a modified human IFNa2, and (iii) a modified Fc domain, wherein the PD-L1-based chimeric protein complex is present at a concentration from at least about 1 mg/mL to about 10 mg/mL;
(b) a buffering agent comprising a histidine-acetate buffer, wherein histidine is present at a concentration of at least about 10 mM to about 15 mM and wherein acetic acid is present at a concentration of at least about 10 mM to about 15 mM;
(c) an excipient comprising a surfactant that is PS20 at a concentration of at least 0.01 % (w/v) to at least about 0.05% (w/v) or Poll 88 at a concentration of about 0.05% (w/v);
(d) a tonicifier excipient comprising trehalose at a concentration of at least about 190 mM to about 195 mM;
(e) a stabilizer excipient comprising HP-beta-cyclodextrin at a concentration of about 50 mM; and
(f) an antioxidant excipient comprising methionine at a concentration of about 3 mM; wherein the pharmaceutical composition has a pH of about 5.0.
PCT/US2024/056067 2023-11-17 2024-11-15 Pd-l1-based chimeric protein formulations Pending WO2025106775A1 (en)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
US20120128687A1 (en) * 2010-06-25 2012-05-24 Hoffmann-La Roche.Inc. Novel antibody formulation
US20210024631A1 (en) * 2018-03-28 2021-01-28 Orionis Biosciences, Inc. Bi-functional proteins and construction thereof
WO2023075506A1 (en) * 2021-10-29 2023-05-04 (주)알테오젠 Pharmaceutical composition comprising human hyaluronidase ph20 and drug
US20230321234A1 (en) * 2017-05-02 2023-10-12 Merck Sharp & Dohme Llc Stable formulations of programmed death receptor 1 (pd-1) antibodies and methods of use thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120128687A1 (en) * 2010-06-25 2012-05-24 Hoffmann-La Roche.Inc. Novel antibody formulation
US20230321234A1 (en) * 2017-05-02 2023-10-12 Merck Sharp & Dohme Llc Stable formulations of programmed death receptor 1 (pd-1) antibodies and methods of use thereof
US20210024631A1 (en) * 2018-03-28 2021-01-28 Orionis Biosciences, Inc. Bi-functional proteins and construction thereof
WO2023075506A1 (en) * 2021-10-29 2023-05-04 (주)알테오젠 Pharmaceutical composition comprising human hyaluronidase ph20 and drug

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