WO2023164551A1 - Protéines de liaison bispécifiques de manière conditionnelle - Google Patents

Protéines de liaison bispécifiques de manière conditionnelle Download PDF

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WO2023164551A1
WO2023164551A1 PCT/US2023/063131 US2023063131W WO2023164551A1 WO 2023164551 A1 WO2023164551 A1 WO 2023164551A1 US 2023063131 W US2023063131 W US 2023063131W WO 2023164551 A1 WO2023164551 A1 WO 2023164551A1
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chain variable
protein
variable region
sdabd
domain
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PCT/US2023/063131
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English (en)
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Antara Banerjee
Patrick Leroy
Johara CHOUITAR
Glendon WU
Eilene KWOK
Maia VINOGRADOVA
Danielle DETTLING
Robert Dubridge
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Takeda Pharmaceutical Company Limited
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Publication of WO2023164551A1 publication Critical patent/WO2023164551A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—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 the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—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
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • NK natural killer
  • CTLs cytotoxic T lymphocytes
  • the present disclosure provides methods and compositions for reducing the toxicity and/or side effects of immune cell engaging bispecific antibodies that bind to cancer and immune cells to stimulate immune cell killing of a target cancer.
  • Many of the proteins provided herein are prodrugs that may be activated by proteases (e.g., proteases found in tumor microenvironments).
  • the proteins described herein are configured such that, when they are not in a tumor microenvironment, the protein is capable of binding to tumor cells but not immune cells (inactive), and such that when the proteins enter a tumor microenvironment, cleavage of the cleavable linkers in the protein “activates” the protein, resulting in two “active” bi-specific molecules, wherein each can bind to tumor cells and immune cells.
  • the proteins described herein comprise one single domain that binds to a human target tumor antigen (TTA). Compared with similar constructs that comprise more than one (e.g., two) domains that bind to human TTAs (e.g., same or different TTAs), these proteins described herein (e.g., in prodrug and active form) may exhibit reduced avidity and/or cell surface residence time.
  • TTA human target tumor antigen
  • the proteins described herein are efficacious in cancers that have any level of HER2 expression. In some embodiments, the proteins described herein are efficacious in cancers having low HER2 expression level. In some embodiments, the proteins described herein are efficacious in cancers having a moderate HER2 expression level. In some embodiments, the proteins described herein are efficacious in cancers having high HER2 expression level.
  • proteins described herein may result in reduced toxicity and enhance tolerability (e.g., tolerated at a higher dose) in a subject.
  • proteins described herein exhibit an enhanced safety and efficacy profile, compared with proteins with more than one domain that binds to human TTAs.
  • proteins comprising: from N- to C- terminus:
  • a first constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region linked to a light chain variable region via a first constrained non- cleavable linker (CNCL), wherein the heavy chain variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen, and wherein the heavy chain variable region and the light chain variable region are not associated in the first constrained scFv domain and the first constrained scFv domain does not bind to the human immune cell antigen;
  • CNCL constrained non- cleavable linker
  • a cleavable linker (v) a second constrained single chain variable fragment (scFv) domain comprising a pseudo heavy chain variable region linked to a pseudo light chain variable region via a second constrained non-cleavable linker (CNCL), wherein the pseudo heavy chain variable region and the pseudo light chain variable region are not associated in the second constrained scFv domain, and the second constrained scFv domain does not bind to the human immune cell antigen of (ii);
  • CNCL constrained non-cleavable linker
  • a third sdABD that binds to human serum albumin (HSA); wherein the heavy chain variable region of (ii) associates with the pseudo light chain variable region of (v) intramolecularly, forming a variable fragment (Fv) that does not bind the immune cell antigen; and wherein the pseudo heavy chain variable region of (v) associates with the light chain variable region of (ii) intramolecularly, forming a variable fragment (Fv) that does not bind the immune cell antigen; wherein (i) and (iii) are not both absent; and wherein when (i) and (iii) are both present:
  • the first sdABD binds to a human target tumor antigen (TTA) and the second sdABD does not bind a human TTA;
  • the first sdABD does not bind a human TTA and the second sdABD binds to a human TTA.
  • (i) and (iii) are both present, and wherein the first sdABD does not bind a human TTA, and the second sdABD binds to a human TTA.
  • the first sdABD binds to a hen egg lysozyme (HEL).
  • the first sdABD comprises the amino acid sequence of SEQ ID NO: 26.
  • (i) and (iii) are both present, and wherein the second sdABD does not bind a human TTA, and the first sdABD binds to a human TTA.
  • the second sdABD binds to a hen egg lysozyme (HEL). In some embodiments, the second sdABD comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, (i) is absent and (iii) is present, and wherein the second sdABD binds to a human TTA. In some embodiments, (i) is present and (iii) is absent, and wherein the first sdABD binds to a human TTA.
  • the human TTA is HER2, EGFR, FOLR1, TROP2, or EpCAM. In some embodiments, the human TTA is HER2.
  • the first sdABD or the second sdABD that binds to HER2 comprises the amino acid sequence of any one of SEQ ID NOs: 41, 45, 48-52, 54, 58, 60, 63, 66, 68, 72, 75-78, 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121, and 165.
  • the cleavable linker of (iv) is 8-45 amino acids in length.
  • the cleavable linker of (iv) comprises a cleavage site for a protease that is present in a tumor microenvironment.
  • the protease is MMP2, MMP9, Meprin, Cathepsin, granzyme, Matriptase, thrombin, enterokinase, KLK7-6, KLK7-13, KLK7-11, KLK7-10, or uPA.
  • the immune cell is a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a macrophage, a B cell, a neutrophil, a dendritic cell, or a monocyte.
  • NK natural killer
  • NKT natural killer T
  • the human immune cell antigen is CD3, CD28, T cell receptor, programmed cell death protein 1 (PD-1), PD-L1, cytotoxic T-lymphocyte- associated protein 4 (CTLA-4), T cell immunoglobulin and mucin domain 3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), killer-cell immunoglobulin-like receptor (KIR), CD137, 0X40, OX40L, CD27, GITR (TNFRSF18), TIGIT, inducible T cell costimulatory (ICOS), CD16A, CD226, CD96, CD40L, CRTAM, LFA-1, NKG2D, CSF1R, CD40, MARCO, VSIG4, or CD163.
  • the human immune cell antigen is CD3.
  • the heavy chain variable region is linked to the N-terminus of the light chain variable region in the first constrained scFv domain of (ii). In some embodiments, the pseudo heavy chain variable region is linked to the C-terminus of the pseudo light chain variable region in the second constrained scFv domain of (v).
  • the first constrained non-cleavable linker of (ii) and/or the second constrained non-cleavable linker of (v) is 6-10 amino acids in length. In some embodiments, the first constrained non-cleavable linker of (ii) and/or the second constrained non-cleavable linker of (v) is 8 amino acids in length.
  • the human immune cell antigen is CD3, the heavy chain variable region of (ii) comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable region of (ii) comprises the amino acid sequence of SEQ ID NO: 8.
  • pseudo heavy chain variable region of (v) comprises the amino acid sequence of SEQ ID NO: 33
  • pseudo light chain variable region of (v) comprises the amino acid sequence of SEQ ID NO: 34.
  • the third sdABD comprises the amino acid sequence of SEQ ID NO: 22.
  • the protein comprises the amino acid sequence of any one of SEQ ID NOs: 37-40 and 173-176.
  • nucleic acid molecules comprising a nucleotide sequence encoding any one of the proteins described herein.
  • the nucleic acid molecule is a vector.
  • the vector is an expression vector.
  • cells comprising any one of the proteins described herein, or the nucleic acid molecule described herein.
  • Other aspects of the present disclosure provide methods of producing any one of the proteins described herein, the methods comprising culturing such cell under conditions that allow expression of the protein. In some embodiments, the methods further comprise isolating the protein.
  • composition comprising any one of the proteins described herein are provided.
  • aspects of the present disclosure provide methods of treating cancer, the methods comprising administering any one of the proteins described herein or a composition comprising such protein to a subject.
  • the cancer expresses HER2.
  • the subject is a human subject.
  • compositions comprising: a first protein and a second protein, each of which comprising, from N- to C- terminus:
  • a first constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region linked to a light chain variable region via a first constrained non- cleavable linker (CNCL), wherein the heavy chain variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen, and wherein the heavy chain variable region and the light chain variable region are not associated in the first constrained scFv domain and the first constrained scFv domain does not bind to the human immune cell antigen;
  • CNCL constrained non- cleavable linker
  • a second constrained single chain variable fragment (scFv) domain comprising a pseudo heavy chain variable region linked to a pseudo light chain variable region via a second constrained non-cleavable linker (CNCL), wherein the pseudo heavy chain variable region and the pseudo light chain variable region are not associated in the second constrained scFv domain, and the second constrained scFv domain does not bind to the human immune cell antigen of (ii); (vi) a third domain linker; and
  • a third sdABD that binds to human serum albumin (HSA); wherein the heavy chain variable region of (ii) associates with the pseudo light chain variable region of (v) intramolecularly, forming a variable fragment (Fv) that does not bind the immune cell antigen; and wherein the pseudo heavy chain variable region of (v) associates with the light chain variable region of (ii) intramolecularly, forming a variable fragment (Fv) that does not bind the immune cell antigen; wherein (i) and (iii) are not both absent; and wherein when (i) and the (iii) are both present:
  • the first sdABD binds to a human target tumor antigen (TTA) and the second sdABD does not bind a human TTA;
  • the first sdABD does not bind a human TTA and the second sdABD binds to a human TTA.
  • the first protein is identical to the second protein.
  • the heavy chain variable region of the first protein associates with the light chain variable region of the second protein, forming an active Fv that binds to the human immune cell antigen; the light chain variable region of the first protein associates with the heavy chain variable region of the second protein, forming an active Fv that binds to the human immune cell antigen.
  • cleavage occurs in a tumor microenvironment in a subject upon administration of the composition to the subject.
  • compositions comprising a homodimer of a first polypeptide and a second polypeptide, wherein the first polypeptide is identical to the second polypeptide, and where each of the first polypeptide and the second polypeptide comprises:
  • sdABD single domain antigen binding domain
  • TTA first human target tumor antigen
  • a constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region (VH) linked to a light chain variable region (VL) via a constrained non- cleavable linker (CNCL), wherein the heavy chain variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen, and wherein the heavy chain variable region and the light chain variable region are not associated in the constrained scFv domain and the constrained scFv domain does not bind to the human immune cell antigen; wherein the VH of the first polypeptide associates with the VL of the second polypeptide, and the VL of the first polypeptide associates with the VH of the second polypeptide, forming two active variable fragments (Fvs) each capable of binding to the immune antigen.
  • VH heavy chain variable region
  • VL light chain variable region
  • CNCL constrained non- cleavable linker
  • the TTA is HER2, EGFR, F0LR1, TR0P2, or EpCAM.
  • the human immune cell antigen is CD3.
  • FIG. 1 shows schematics of control proteins (Prol l l l l, Prol l l8 containing two HER2 binding sdABDs).
  • Pro 1225 contains one single HER2 binding sdABD at the N-terminus of the protein, and an extended cleavable linker is present corresponding to where the second HER2 binding sdABD is in Prol 111 or Prol 118.
  • Prol 168 has the N-terminal HER2 targeting sdABD of Prol 118 replaced with a non-functional sdABD that binds to hen egg lysozyme (HEL). The prodrug active forms of the proteins are shown.
  • FIGs. 2A-2B are graphs showing the tumor killing efficacy of Pro 1225 in a human lung cancer (HCC827) mouse model.
  • Pro 1225 is about 10-fold more potent than Prol 118 at reducing lung cancer tumor volume.
  • FIGs. 3A-3B are graphs showing the tumor killing efficacy of Pro 1225 in a human breast cancer (JIMT-1 cells) mouse model.
  • Prol225 is about 3-fold more potent than Prol 118 at reducing breast cancer tumor volume.
  • FIG. 4 is a graph showing the tumor killing efficacy of Prol 168 in a human stomach cancer (SNU16) mouse model.
  • FIG. 5 shows schematics of full length Pro 1225, cleaved associated full length Prol225, Prol225 cleaved products, and Prol225 active dimer formations.
  • FIG. 6. shows a schematic of control protein Pro 1303.
  • FIG. 7 is a graph showing the endogenous HER2 cell surface density across cancer cell lines.
  • FIG. 8 is a graph showing the tumor killing efficacy of Pro 1225 in a human stomach cancer model (SNU16) in vitro.
  • FIG. 9 is a graph showing the tumor killing efficacy of Pro 1225 in a human breast cancer model (JIMT-1) in vitro.
  • FIG. 10 is a graph showing the tumor killing efficacy of Pro 1225 in a human gastric cancer model (NCI-N87) in vitro.
  • FIG. 11 is a graph showing the cross-reactivity of Pro 1225 in a human lymphoma model (cyHER2-Raji) in vitro
  • FIG. 12 is a graph showing the tumor killing efficacy of Pro 1225 in a human colorectal cancer model (HT-29) in vitro.
  • FIG. 13 is a graph showing the tumor killing efficacy of Prol225 in a human stomach cancer (SNU16) mouse model. Prol225 dose dependently reduces stomach cancer tumor volume.
  • FIG. 14 is a graph showing the tumor killing efficacy of Pro 1225 in a human lung cancer (HCC827) mouse model. Pro 1225 dose dependently reduces lung cancer tumor volume.
  • FIG. 15 is a graph showing the tumor killing efficacy of Prol225 in a human colorectal cancer (HT55) mouse model. Pro 1225 dose dependently reduces colorectal cancer tumor volume.
  • FIG. 16 is a graph showing the tumor killing efficacy of Prol225 in a human breast cancer (JIMT-1) mouse model. Prol225 dose dependently reduces breast cancer tumor volume.
  • the present disclosure provides methods and compositions for reducing the toxicity and side effects of immune cell engaging bispecific antibodies that bind to cancer and immune cells to stimulate immune cell killing of a target cancer.
  • Many of the proteins provided herein are prodrugs that may be activated by proteases (e.g., proteases found in tumor microenvironments).
  • the proteins described herein are configured such that, when they are not in a tumor microenvironment, the protein is capable of binding to tumor cells but not immune cells (inactive), and such that when the proteins enter a tumor microenvironment, cleavage of the cleavable linkers in the protein “activates” the protein, resulting in two “active” bi-specific molecules, wherein each can bind to tumor cells and immune cells.
  • the proteins described herein comprises one single domain that binds to a human target tumor antigen (TTA). Compared with similar constructs that comprise more than one (e.g., two) domains that bind to human TTAs (e.g., same or different TTAs), the proteins described herein (e.g., in prodrug and active form) may exhibit reduced avidity and cell surface residence time.
  • TTA target tumor antigen
  • the proteins described herein are efficacious in cancers that have any level of HER2 expression . In some embodiments, the proteins described herein are efficacious in cancers having low HER2 expression level. In some embodiments, the proteins described herein are efficacious in cancers having a moderate HER2 expression level. In some embodiments, the proteins described herein are efficacious in cancers having high HER2 expression level.
  • proteins described herein may lead to reduced toxicity and enhanced tolerability (e.g., tolerated at a higher dose) in a subject.
  • proteins described herein exhibit an enhanced overall safety and efficacy profile, compared with proteins with more than one domain that binds to human TTAs.
  • amino acid and “amino acid identity” as used herein is meant one of the 20 naturally occurring amino acids or any non-natural analogues that may be present at a specific, defined position.
  • amino acid means one of the 20 naturally occurring amino acids.
  • protein herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
  • amino acid modification herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence or an alteration to a moiety chemically linked to a protein.
  • a modification may be an altered carbohydrate or PEG structure attached to a protein.
  • the amino acid modification is always to an amino acid coded for by DNA, e.g., the 20 amino acids that have codons in DNA and RNA.
  • a preferred amino acid modification herein is a substitution.
  • the protein specifically binds to immune cell antigens and target tumor antigens (TTAs) such as target cell receptors, as outlined herein.
  • TTAs tumor antigens
  • Specific binding or “specifically binds to” or is “specific for” a particular antigen or an epitope means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.
  • Specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KD for an antigen or epitope of at least about 10’ 4 M, at least about 10’ 5 M, at least about 10’ 6 M, at least about 10’ 7 M, at least about 10’ 8 M, at least about 10’ 9 M, alternatively at least about IO 10 M, at least about 10 11 M, at least about 10 12 M, or greater, where KD refers to a dissociation rate of a particular antibody-antigen interaction.
  • an antibody that specifically binds an antigen will have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a control molecule relative to the antigen or epitope.
  • binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a Ka for an antigen or epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for the epitope relative to a control, where Ka (or KA) refers to an association rate of a particular antibody-antigen interaction. Binding affinity is generally measured using a BIACORE assay or OCTET as is known in the art.
  • position as used herein is meant a location in the sequence of a protein. Positions may be numbered sequentially, or according to an established format, for example the EU index for antibody numbering.
  • target antigen as used herein is meant the molecule that is bound specifically by the variable region of a given antibody.
  • a target antigen may be a protein, carbohydrate, lipid, or other chemical compound.
  • a range of suitable exemplary target antigens are described herein, including target tumor antigens.
  • target cell as used herein is meant a cell that expresses a target antigen.
  • target cells are diseased cells, e.g., tumor cells that express TTAs.
  • target cells are immune cells e.g., T cells that express an immune cell antigen.
  • Fv or “Fv domain” or “Fv region” as used herein is meant a polypeptide that comprises a light chain variable region (VL) and a heavy chain variable region (VH) of an antigen binding domain, generally, but not always, from an antibody.
  • Fv domains usually form “antigen binding regions” or “antigen binding domains” or “ABDs” as discussed herein, if they contain VH and VL domains each containing CDRs that will bind to the antigen.
  • the VH or VL of an Fv domain may be on a single polypeptide chain (“single chain variable fragment (scFv)”), or on separate polypeptide chains.
  • Fv domains may be active Fv domains, constrained Fv domains, inactive Fv domains, or pseudo Fv domains.
  • An “active Fv” is one that has a variable heavy and a variable light domain each with CDRs that bind the same antigen, and in which the VH and VL are associated with each other such that the Fv is able to bind to the antigen.
  • a constrained scFv domain contains an active VH and an active VL that contain CDRs capable of binding to the same antigen, but the VH and VL cannot associate to form an active Fv that will bind the antigen due to the constrained linker between them.
  • a constrained scFv domain contains an inactive VH and an inactive VL that lack CDRs capable of binding an antigen, and the VH and VL cannot associate with each other due to the constrained linker.
  • the VH and VL in a constrained scFv are not able to associate with each other due to the presence of a constrained linker, but they may assemble (e.g., intramolecularly or intermolecularly) with other variable domains, including variable domains in a different constrained Fv domain.
  • an “inactive Fv” or an “inert Fv” is one that has a VH and a VL but neither the VH nor the VL contain CDRs that are capable of binding to an antigen (e.g., the CDRs are modified such that the antigen-binding capability is lost). Inactive Fvs do not bind to an antigen.
  • an inactive Fv can be in a constrained format, in which the VH and the VL are linked via a constrained linker, preventing them from associating with each other.
  • the VH and the VL in an inactive Fv can associate with each other but the Fv still does not bind to the antigen.
  • VH or VL that lack CDRs capable of antigen binding are also referred to herein as “inert VH” or “inert VL,” respectively.
  • a “pseudo Fv” as described herein refers to an Fv in which a VH and a VL are associated with each other, and in which: (i) the VH contains CDRs capable of binding to an antigen but the VL lacks CDRs that are capable of binding to any antigen; (ii) the VL contains CDRs capable of binding to an antigen but the VH lacks CDRs that are capable of binding to any antigen; or (iii) both the VH and VL contain CDRs that are capable of antigen binding, but to different antigens. As such, a pseudo Fv, despite having a VH and a VL that are associated with each other, is not capable of binding to an antigen.
  • the VH and the VL of a pseudo Fv is on a single polypeptide chain (i.e., the association is intramolecular). In some embodiments, the VH and the VL of a pseudo Fv is on a single polypeptide chain (i.e., the association is intermolecular).
  • the VH and the VL that form the pseudo Fv are also referred to herein as a “pseudo VH” and a “pseudo VL”, respectively.
  • variable domain herein is meant the region of an immunoglobulin that comprises one or more Ig domains substantially encoded by any of the VK, V , and/or VH genes that make up the kappa, lambda, and heavy chain immunoglobulin genetic loci, respectively.
  • a single variable domain such as a sdFv (also referred to herein as sdABD) can be used.
  • each VH and VL is composed of three hypervariable regions (“complementary determining regions,” “CDRs”) and four “framework regions”, or “FRs”, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • CDRs complementary determining regions
  • FRs framework regions
  • the VH domain has the structure vhFRl-vhCDRl-vhFR2-vhCDR2-vhFR3-vhCDR3-vhFR4
  • the VL domain has the structure vlFRl- vICDR 1-V1FR2-V1CDR2-V1FR3-V1CDR3-V1FR4.
  • the vhFR regions and the vlFR regions self-assemble to form Fv domains.
  • an inactive (e.g., prodrug) format of a conditionally activable molecule e.g., a fusion protein
  • the hypervariable regions confer antigen binding specificity and generally encompass amino acid residues from about amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain variable region and around about 31- 35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.
  • residues forming a hypervariable loop e.g., residues 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and 26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917. Specific CDRs of the proteins are described below.
  • variable heavy and/or variable light sequence includes the disclosure of the associated (inherent) CDRs.
  • disclosure of each variable heavy region is a disclosure of the vhCDRs (e.g., vhCDRl, vhCDR2 and vhCDR3) and the disclosure of each variable light region is a disclosure of the vlCDRs (e.g., vlCDRl, vlCDR2 and vlCDR3).
  • the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately, residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region) and the EU numbering system for Fc regions (e.g., Kabat et al., supra (1991)).
  • a “full CDR set” in the context of domains that bind to immune cell antigens means that the component comprises a heavy chain variable region comprising three CDRs (e.g., vhCDRl, vhCDR2 and vhCDR3) and a light chain variable region comprising CDRs (e.g., a vlCDRl, vlCDR2, vlCDR3).
  • each set of CDRs, the VH and VE CDRs can bind to antigens, both individually and as a set.
  • the vhCDRs can bind, for example to CD3 and the vlCDRs can bind to CD3, but in the constrained format they cannot bind to CD3.
  • single domain Fv Single domain Fv
  • sdFv single domain Fv
  • sdABD single domain Fv
  • sdABD single domain Fv
  • VHH single heavy chain variable region
  • sdABDs described herein bind TTAs, which are annotated as such (sdABD-TTA for the generic term, or, for example, sdABD-EGFR for one that binds to EGFR, sdABD-FOLRl for one that binds to FOLR1, etc.).
  • sdABDs described herein bind to human serum albumin (HSA), and are annotated as such (sdABD-HSA).
  • HSA human serum albumin
  • variable heavy and variable light domains can be on separate polypeptide chains or on a single polypeptide chain in the case of scFv sequences, depending on the format and configuration of the moieties herein.
  • Epitope binding sites contribute to the formation of the antigen-binding, or more specifically, epitope binding sites.
  • Epitope refers to a determinant that interacts with a specific antigen binding site in the variable regions known as a paratope. Epitopes are groupings of molecules such as amino acids or sugar side chains and usually have specific structural characteristics, as well as specific charge characteristics. A single antigen may have more than one epitope.
  • the epitope may comprise amino acid residues directly involved in the binding (also called immunodominant component of the epitope) and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the specific antigen binding peptide; in other words, the amino acid residue is within the footprint of the specific antigen binding peptide.
  • Epitopes may be either conformational or linear.
  • a conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain.
  • a linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. Conformational and nonconformational epitopes may be distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Antibodies that recognize the same epitope can be verified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen, for example “binning.” As outlined below, the proteins not only include the enumerated antigen binding domains and antibodies herein, but those that compete for binding with the epitopes bound by the enumerated antigen binding domains.
  • ABD antigen binding domain
  • ABDs can be a single VH (e.g., an sdABD as described herein), a single VL, or an scFvs, containing a VH and VL domain.
  • TTA-sdABD tumor antigens
  • HSA-sdABD human serum albumin
  • CD3 or CD28 immune cell antigens
  • an antigen binding domain or antigen binding region includes active Fvs in which the VH and VL can associate and bind to the antigen, and includes constrained scFvs that do not bind to an antigen and in which the VH and VL each contain CDRs that are capable of binding to the same antigen, but the VH and VL do not associate with each other (e.g., due to the constrained linker between the VH and VL).
  • domain as used herein is meant a protein sequence with a structure and/or function, as outlined herein. Domains of the proteins described herein include target tumor antigen binding domains (TTA domains), immune cell binding domains, linker domains, and half-life extension domains.
  • TTA domains target tumor antigen binding domains
  • immune cell binding domains include tumor antigen binding domains (TTA domains), immune cell binding domains, linker domains, and half-life extension domains.
  • domain linker herein is meant an amino acid sequence that joins two domains as outlined herein. Domain linkers can be cleavable linkers, constrained cleavable linkers, non- cleavable linkers, constrained non-cleavable linkers, scFv linkers, etc.
  • cleavable linker (“CL”) herein is meant an amino acid sequence that can be cleaved by a protease, preferably a human protease in a disease tissue as outlined herein.
  • Cleavable linkers generally are at least 3 amino acids in length, with from 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids finding use in a protein, depending on the required flexibility. A number of cleavable linker sequences are shown in Table 1.
  • non cleavable linker herein is meant an amino acid sequence that is not cleaved by a human protease under normal physiological conditions.
  • CNCL constrained non-cleavable linker
  • protease cleavage site refers to the amino acid sequence recognized and cleaved by a protease.
  • protease cleavage domain refers to the peptide sequence incorporating the “protease cleavage site” and any linkers between individual protease cleavage sites and between the protease cleavage site(s) and the other functional components of the constructs of the protein (e.g., VH, VL, target antigen binding domain(s), half-life extension domain, etc.).
  • a protease cleavage domain may also include additional amino acids, if necessary, for example to confer flexibility.
  • a protein described herein comprises one single sdABD capable of binding to a TTA and one or more (e.g., one, two) constrained single chain variable fragment (scFv) domains.
  • scFv constrained single chain variable fragment
  • a protein described herein comprises one single constrained scFv domain and a non-constrained scFv domain.
  • a protein described herein comprises two constrained scFv domains.
  • a protein described herein further comprises a second sdABD capable of binding to an antigen that is not a TTA.
  • the second sdABD binds to an antigen that does not confer any function to the protein (i.e., is a “non-functional” sdABD). In some embodiments, the second sdABD does not bind to any human proteins. In some embodiments, the second sdABD binds to a non-human protein (e.g., a protein of non-human origin). In some embodiments, the second sdABD binds to a fluorescent protein (e.g., GFP). In some embodiments, the second sdABD binds to a hen egg lysozyme (HEE). In some embodiments, a protein described herein further comprises a half-life extension domain (e.g., a sdABD capable of binding to human serum albumin (HSA)).
  • HSA human serum albumin
  • a protein disclosed herein is inactive when the cleavable linker in the protein is uncleaved.
  • an uncleaved and inactive protein described herein is capable of binding to a TTA but not an immune cell antigen.
  • the protein is cleaved and activated, for example, in a disease- specific microenvironment or in the blood of a subject, at internal cleavable linkers that contain protease cleavage sites (e.g., the modified MMP9 cleavage sites described herein).
  • fragments of the cleavage products form dimers (e.g., homodimers) that are bi-specific molecules capable of binding to both a TTA and an immune cell antigen, thereby stimulating and/or activating one or more immune cells.
  • dimers e.g., homodimers
  • the two dimers bind to a TTA that is HER2, EGFR, FOLR1, TROP2, or EpCAM. In some embodiments, the two dimers (e.g., homodimers) bind to the same or different immune cell antigens.
  • the immune cell antigen is CD3, CD28, T cell receptor, programmed cell death protein 1 (PD-1), PD-L1, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and mucin domain 3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), killer-cell immunoglobulin- like receptor (KIR), CD137, 0X40, OX40L, CD27, GITR (TNFRSF18), TIGIT, inducible T cell costimulatory (ICOS), CD16A, CD226, CD96, CD40L, CRTAM, LFA-1, NKG2D, CSF1R, CD40, MARCO, VSIG4, or CD163.
  • PD-1 programmed cell death protein 1
  • CTL-4 cytotoxic T-lymphocyte-associated protein 4
  • TIM-3 T cell immunoglobulin and mucin domain 3
  • LAG-3 lymphocyte-activation gene 3
  • KIR killer-cell immunoglobulin- like receptor
  • CD137 CD
  • COBRA constructs described herein are constructed in a “prodrug (inactive)” form.
  • a prodrug protein described herein comprises, from N- to C- terminus:
  • a first constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region linked to a light chain variable region via a first constrained non- cleavable linker (CNCL), wherein the heavy chain variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen, and wherein the heavy chain variable region and the light chain variable region are not associated in the first constrained scFv domain and the first constrained scFv domain does not bind to the human immune cell antigen;
  • CNCL constrained non- cleavable linker
  • a second constrained single chain variable fragment (scFv) domain comprising a pseudo heavy chain variable region linked to a pseudo light chain variable region via a second constrained non-cleavable linker (CNCL), wherein the pseudo heavy chain variable region and the pseudo light chain variable region are not associated in the second constrained scFv domain, and the second constrained scFv domain does not bind to the human immune cell antigen of (ii);
  • the first sdABD binds to a human target tumor antigen (TTA) and the second sdABD does not bind a human TTA;
  • the first sdABD does not bind a human TTA and the second sdABD binds to a human TTA.
  • intramolecular association of the VH of (ii) with the pseudo VL of (v) and/or intramolecular association of the pseudo VH of (v) with the VL of (ii) stabilizes (e.g., resulting in stable conformation) the prodrug proteins described herein and prevents dimerization of the constrained scFvs intermolecularly between different prodrug proteins prior to activation of the prodrug protein.
  • Proteins in the prodrug (inactive) form when administered to a subject in a composition that comprises one or more of such prodrug proteins, can be activated once the cleavable linker of (iv) is cleaved (e.g., by a protease in a tumor microenvironment such as MMP9).
  • Activation of the prodrug proteins involves cleavage of at least two identical prodrug proteins (a first protein and a second protein that are identical to each other) in the cleavable linker of (iv).
  • first polypeptide comprising a sdABD that binds to a TTA, a first domain linker, a first constrained scFv domain, and a second polypeptide comprising a second constrained sdFv domain.
  • the first polypeptide further comprises a second sdABD that does not bind to a TTA.
  • the second sdABD binds to an antigen that does not confer any function to the protein (i.e., is a “non-functional” sdABD).
  • the second sdABD does not bind to any human proteins.
  • the second sdABD binds to a non-human protein (e.g., a protein of non-human origin). In some embodiments, the second sdABD binds to a fluorescent protein (e.g., GFP). In some embodiments, the second sdABD binds to a hen egg lysozyme (HEL). In some embodiments, the second polypeptide may additionally comprise a half-life extension domain (e.g., a third sdABD that binds to HSA).
  • a non-human protein e.g., a protein of non-human origin
  • the second sdABD binds to a fluorescent protein (e.g., GFP).
  • the second sdABD binds to a hen egg lysozyme (HEL).
  • the second polypeptide may additionally comprise a half-life extension domain (e.g., a third sdABD that binds to HSA).
  • the VH of the first constrained scFv domain of the first protein associates with the VL of the first constrained scFv domain of the second protein, forming an active Fv that binds to the human immune cell antigen
  • the VL of the first constrained scFv domain of the first protein associates with the VH of the first constrained scFv domain of the second protein, forming an active Fv that binds to the human immune cell antigen
  • the cleavage fragments of the prodrug (inactive) proteins assemble into dimers that are bi-specific molecules capable of binding a TTA and an immune cell antigen.
  • Non-limiting examples of the components of the proteins described herein, in prodrug form or active form, are provided.
  • a protein provided herein comprises one single sdABD that binds to a human target tumor antigen (TTA).
  • TTA target tumor antigen
  • the sdABD that binds to a human TTA may be present at the N- terminus of the protein or internally in the protein.
  • the protein may comprise additional sdABDs that do not bind a human TTA.
  • the one single sdABD that binds to a human TTA is present at the N-terminus of the protein together with a first domain linker at the C-terminus of the sdABD (i.e., the first sdABD of (i) is present and binds to a human TTA).
  • the human TTA is HER2, EGFR, F0LR1, TR0P2, or EpCAM.
  • component (iii) of the protein may be present or absent.
  • the second sdABD of (iii) does not bind to a human TTA.
  • the second sdABD of (iii) when component (iii) is present, does not bind to a human protein. In some embodiments, the second sdABD of (iii) is non-functional. For example, the second sdABD of (iii) may bind to an antigen that does not confer any function (e.g., tumor binding or immune cell binding function) to the protein. In some embodiments, the sdABD of (iii) binds to a hen egg lysozyme and is non-functional.
  • the cleavable linker of (v) is typically 8-15 amino acids (e.g., 8, 9, 10, 11, 12, 13, 14, or 15 amino acids) in length.
  • the cleavable linker of (iv) may be of an extended length (e.g., 15-45 amino acids in length).
  • the cleavable linker of (iv) is 17-35 amino acids in length.
  • the cleavable linker of (iv) is 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids in length.
  • the cleavable linker of (iv) is 29 or 33 amino acids in length.
  • the one single sdABD that binds to a human TTA is present internally in the protein, e.g., at the C terminus of the first constrained single chain scFv domain of (ii), together with a second domain linker at the N-terminus of the sdABD that links the first constrained scFv domain and the sdABD (i.e., the second sdABD of (iii) is present and binds to a human TTA).
  • the human TTA is HER2, EGFR, FOLR1, TROP2, or EpCAM.
  • component (i) of the protein may be present or absent.
  • the first constrained scFv domain of (ii) is at the N-terminus of the protein.
  • an additional amino acid sequence is present at the N-terminus of the protein, i.e., N-terminal of the first constrained scFv domain of (ii).
  • the first sdABD of (i) does not bind to a human TTA.
  • the first sdABD of (i) does not bind to a human protein.
  • the sdABD of (i) is non-functional.
  • the sdABD of (i) may bind to an antigen that does not confer any function (e.g., tumor binding or immune cell binding function) to the protein.
  • the sdABD of (i) binds to a hen egg lysozyme and is non-functional.
  • Non-limiting examples of sdABDs that bind HER2, and that may be used in the first sdABD of (i) or the second sdABD of (iii) as described herein are provided in Table 1.
  • the first sdABD of (i) or the second sdABD of (iii) comprises a CDR1, a CDR2, and a CDR3 of an anti-HER2 sdABD as set forth in any one of SEQ ID NOs: 45, 48- 52, 54, 58, 60, 63, 66, 68, 72, 75-78, 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121, 299, and 300.
  • the first sdABD of (i) or the second sdABD of (iii) comprises an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of any one of SEQ ID NOs: 41, 45, 48-52, 54, 58, 60, 63, 66, 68, 72, 75-78, 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121, and 165.
  • the first sdABD of (i) or the second sdABD of (iii) comprises the amino acid sequence of any one of SEQ ID NOs: 41, 45, 48-52, 54, 58, 60, 63, 66, 68, 72, 75-78, 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121, and 165.
  • the first sdABD of (i) and/or the second sdABD of (iii) comprises the amino acid sequence of SEQ ID NO: 96 or SEQ ID NO: 117.
  • Non-limiting examples of non-functional sdABDs are provided in Table 1.
  • a non-functional sdABD that may be used in the first sdABD of (i) or the second sdABD of (iii) as described herein bind to a hen egg lysozyme (HEL).
  • a non-functional sdABD that binds to HEL comprises a CDR1 as set forth in SEQ ID NO: 23, a CDR2 as set forth in SEQ ID NO: 24, and a CDR3 as set forth in SEQ ID NO: 25.
  • a non-functional sdABD that binds to HEL comprises an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, a nonfunctional sdABD that binds to HEL comprises the amino acid sequence of SEQ ID NO: 26.
  • the immune cell antigen is an antigen expressed on an immune cell selected from a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a macrophage, a B cell, a neutrophil, a dendritic cell, and a monocyte.
  • the immune cell antigen is CD3, CD28, T cell receptor, programmed cell death protein 1 (PD- 1), PD-L1, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and mucin domain 3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), killer-cell immunoglobulin-like receptor (KIR), CD137, 0X40, OX40L, CD27, GITR (TNFRSF18), TIGIT, inducible T cell costimulatory (ICOS), CD16A, CD226, CD96, CD40L, CD226, CRTAM, LFA-1, CD27, NKG2D, CSF1R, CD40, MARCO, VSIG4, or CD163.
  • the immune cell antigen is CD3.
  • any one of the immune cell antigens provided herein is a human immune cell antigen.
  • preventing the first VH from being able to associate with the first VL within the first constrained scFv domain e.g., by linking the first VH and the first VL with a first “constrained non-cleavable linker (CNCL)” results in a first constrained scFv domain that does not bind to the first immune cell antigen.
  • CNCL constrained non-cleavable linker
  • the second VH from being able to associate with the second VL within the second constrained scFv domain, e.g., by linking the second VH and the second VL with a second “constrained non-cleavable linker” results in a second constrained scFv domain that does not bind to the second immune cell antigen.
  • the constrained non-cleavable linkers are too short to allow the first VH and the first VL, or the second VH and the second VL to associate within the constrained scFv domains.
  • the first CNCL and/or the second CNCL is 6-10 amino acids long (e.g., 6, 7, 8, 9, or 10 amino acids long).
  • Nonlimiting examples of amino acid sequences of the first CNCL and/or second CNCL are provided in Table 1.
  • the first CNCL and/or the second CNCL has a sequence of GGGSGGGS (SEQ ID NO: 179)
  • Non-limiting examples of antibodies that bind to CD3 from which the VH and the VL of the first constrained scFv domain of (ii) may be derived include: muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (NUVION), blinatumomab, foralumab, SP34 or I2C, TR-66 or X35-3, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, Fl 11-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1 and WT-31.
  • the first constrained scFv domain of (ii) comprises a VH comprising a vhCDRl as set forth in SEQ ID NO: 1, a vhCDR2 as set forth in SEQ ID NO: 2, and a vhCDR3 as set forth in SEQ ID NO: 3, and a VL comprising vlCDRl as set forth in SEQ ID NO: 4, a vlCDR2 as set forth in SEQ ID NO: 5, and a vlCDR3 as set forth in SEQ ID NO: 6.
  • the first constrained scFv domain of (ii) comprises a VH comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) to the amino acid sequence of SEQ ID NO: 7 and a VL comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) to the amino acid sequence of SEQ ID NO: 8.
  • the first constrained scFv domain of (ii) comprises a VH comprising the amino acid sequence of SEQ ID NO: 7 and a VL comprising the amino acid sequence of SEQ ID NO: 8.
  • Non-limiting examples of antibodies that bind to CD28 from which the VH and the VL of the first constrained scFv domain of (ii) may be derived include: theralizumab, utomilumab (PF-05082566), ES101 (a bispecific PD-L1 X CD28 antibody), PRS-343 (a HER2 X CD28 bispecific antibody) urelumab (BMS-663513), and TGN1412 or TGN1112 as described in US Patent No. 7939638.
  • the first constrained scFv domain of (ii) comprises a VH comprising a vhCDRl as set forth in SEQ ID NO: 9, a vhCDR2 as set forth in SEQ ID NO: 10 or SEQ ID NO: 17, and a vhCDR3 as set forth in SEQ ID NO: 11, and a VL comprising vlCDRl as set forth in SEQ ID NO: 12, a vlCDR2 as set forth in SEQ ID NO: 13, and a vlCDR3 as set forth in SEQ ID NO: 14.
  • the first constrained scFv domain of (ii) comprises a VH comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) to the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 18 and a VL comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) to the amino acid sequence of SEQ ID NO: SEQ ID NO: 16.
  • the first constrained scFv domain of (ii) comprises a VH comprising the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 18 and a VL comprising the amino acid sequence of SEQ ID NO: SEQ ID NO: 16.
  • Non-limiting examples of antibodies that bind to PD-1 from which the VH and the VL of the first constrained scFv domain of (ii) may be derived include: pembrolizumab, dostarlimab, and nivolumab.
  • Non-limiting examples of antibodies that bind to CTLA-4 from which the VH and the VL of the first constrained scFv domain of (ii) may be derived include: ipilimumab.
  • Non-limiting examples of antibodies that bind to TIM-3 from which the VH and the VL of the first constrained scFv domain of (ii) may be derived include: TSR-022 and Sym023.
  • Non-limiting examples of antibodies that bind to LAG-3 from which the VH and the VL of the first constrained scFv domain of (ii) may be derived include: BMS-986016.
  • Non-limiting examples of antibodies that bind to KIR from which the VH and the VL of the first constrained scFv domain of (ii) may be derived include: lirilumab.
  • Non-limiting examples of antibodies that bind to CD 137 from which the VH and the VL of the first constrained scFv domain of (ii) may be derived include: utomilumab and urelumab.
  • Non-limiting examples of antibodies that bind to 0X40 from which the VH and the VL of the first constrained scFv domain of (ii) may be derived include: PF-045-18600 and BMS- 986178.
  • Non-limiting examples of antibodies that bind to CD27 from which the VH and the VL of the first constrained scFv domain of (ii) may be derived include: varlilumab.
  • Non-limiting examples of antibodies that bind to GITR from which the VH and the VL of the first constrained scFv domain of (ii) may be derived include: GWN323 or BMS-986156.
  • Non-limiting examples of antibodies that bind to TIGIT from which the VH and the VL of the first constrained scFv domain of (ii) may be derived include: OMP-313M32, MTIG7192A, BMS-986207, and MK-7684.
  • Non-limiting examples of antibodies that bind to ICOS from which the VH and the VL of the first constrained scFv domain of (ii) may be derived include: JTX-2011.
  • Non-limiting examples of antibodies that bind to CSF1R from which the VH and the VL of the first constrained scFv domain of (ii) may be derived include: mactuzumab/RG7155 and IMC-CS4.
  • Non-limiting examples of antibodies that bind to CD40 from which the VH and the VL of the first constrained scFv domain of (ii) may be derived include: CP-870,893.
  • Non-limiting examples of antibodies that bind to CD16A from which the VH and the VL of the first constrained scFv domain of (ii) may be derived include: NTM-1633 and AFM13.
  • Non-limiting examples of antibodies that bind to CD96 from which the VH and the VL of the first constrained scFv domain of (ii) may be derived include: GSK6097608.
  • Non-limiting examples of antibodies that bind to CD40L from which the VH and the VL of the first constrained scFv domain of (ii) may be derived include: BG9588.
  • Non-limiting examples of antibodies that bind to LFA-1 from which the VH and the VL of the first constrained scFv domain of (ii) may be derived include: efalizumab.
  • the second constrained scFv domain of (v) comprises a pseudo VH linked to a pseudo VL via a second constrained non- cleavable linker (CNCL), wherein the pseudo heavy chain variable region and the pseudo light chain variable region are not associated in the second constrained scFv domain, and the second constrained scFv domain does not bind to the human immune cell antigen of (ii).
  • the pseudo VH is an inactive VH (e.g., a VH lacking CDRs capable of binding to an antigen)
  • the pseudo VL is an inactive VL (e.g., a VL lacking CDRs capable of binding to an antigen).
  • the inactive VH and/or the inactive VL comprise human framework regions that allow self-assembly with another variable region, regardless of which amino acids are in the CDR locations of the variable region.
  • the inactive VH/VL are said to include CDRs, but the CDRs are not capable to bind any antigens, nor are the VH or VL containing such CDRs.
  • Inactive VH or VL can be generated using known methods in the art.
  • inactive VH or VL may be generated by altering one or more of the CDRs of an active VH or VL, including making changes in one or more of the three CDRs.
  • one or more amino acid substitutions are made at functionally important residues in one or more CDRs of an active VH or VL to generate the inactive VH or VL, respectively.
  • some or all CDR residues in an active VH or VL may be replaced with random sequences, non-functional tag sequences (e.g., FLAG sequence) to generate the inactive VH or VL, respectively.
  • non-functional tag sequences e.g., FLAG sequence
  • the inactive VH and VL can be engineered to promote selective binding in the prodrug format, to encourage formation of intramolecular association prior to cleavage (over, for example, intermolecular pair formation).
  • VH/VL and the interface residue amino acid substitutions are described in, for example, Igawa et al., Protein Eng. Des. Selection 23(8):667-677 (2010).
  • the second constrained scFv domain of (v) comprises an inactive VH comprising a vhCDRl as set forth in SEQ ID NO: 27, a vhCDR2 as set forth in SEQ ID NO: 28, and a vhCDR3 as set forth in SEQ ID NO: 29, and an inactive VL comprising vlCDRl as set forth in SEQ ID NO: 30, a vlCDR2 as set forth in SEQ ID NO: 31, and a vlCDR3 as set forth in SEQ ID NO: 32.
  • the second constrained scFv domain of (v) comprises an inactive VH comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) to the amino acid sequence of SEQ ID NO: 33 and an inactive VL comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) to the amino acid sequence of SEQ ID NO: 34.
  • the second constrained scFv domain of (v) comprises an inactive VH comprising the amino acid sequence of SEQ ID NO: 33 and an inactive VL comprising the amino acid sequence of SEQ ID NO: 34.
  • the first constrained scFv domain of (ii) comprises the VH linked to the N-terminus of the VL. In some embodiments, in a protein described herein, the first constrained scFv domain of (ii) comprises the VH linked to the C -terminus of the VL.
  • the second constrained scFv domain of (v) comprises the pseudo VH linked to the N-terminus of the pseudo VL. In some embodiments, in a protein described herein, the second constrained scFv domain of (v) comprises the pseudo VH linked to the C-terminus of the pseudo VL.
  • the first constrained scFv domain of (ii) comprises, from N- to C- terminus: VH-first CNCL-VL
  • the second constrained scFv domain of (v) comprises, from N- to C- terminus: pseudo VL- second CNCL- pseudo VH.
  • the cleavable linker of (iv) of a protein described herein comprises at least one (e.g., 1, 2, 3, or more) protease cleavage site comprising an amino acid sequence that is cleaved by at least one protease.
  • Proteases are known to be secreted by certain diseased cells and tissues, for example tumor or cancer cells, creating a microenvironment that is rich in proteases or a protease-rich microenvironment.
  • the cleavable linker of (iv) is cleavable by a protease in the blood of a subject.
  • the cleavable linker of (iv) cleavable by a protease secreted by a tumor into the tumor microenvironment.
  • Proteases include but are not limited to serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins (e.g., Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin K, Cathepsin L, CathepsinS), kallikreins, hKl, hKIO, hK15, KLK7, GranzymeB, plasmin, collagenase, Type IV collagenase, stromelysin, factor XA, chymotrypsin-like protease
  • Caspase-3 Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteases (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, meprin, urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin- ip converting enzyme, thrombin, FAP (FAP-a), dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV/CD26).
  • MMP matrix metalloproteases
  • MMP1, MMP2, MMP3, MMP8, MMP9 MMP
  • the first cleavable linker of (iv) and/or the second cleavable linker of (viii) is cleavable by MMP9.
  • Non-limiting examples of cleavable linkers that may be used in a protein described herein as first cleavable linker of (iv) are provided in Table 1.
  • the first domain linker in (i) if (i) is present, the second domain linker of (iv), and/or the third domain linker of (viii) is a noncleav able linker. In some embodiments, in a protein described herein, the first domain linker in (i) if (i) is present, the second domain linker of (iv), and/or the third domain linker of (viii) are the same. In some embodiments, in a protein described herein, the first domain linker in (i) if (i) is present, the second domain linker of (iv), and/or the third domain linker of (viii) are different from each other.
  • domain linkers used to join domains to preserve the functionality of the domains are generally longer, flexible linkers that are not cleaved (e.g., by proteases in a subject).
  • linkers suitable for use as domain linkers of the protein described herein include but are not limited to (GS)n (SEQ ID NO: 166), (GGS)n (SEQ ID NO: 167), (GGGS)n (SEQ ID NO: 168), (GGSG)n (SEQ ID NO: 169), (GGSGG)n (SEQ ID NO: 170), or (GGGGS)n (SEQ ID NO: 171), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the length of the first domain linker in (i) if (i) is present, the second domain linker of (iv), and/or the third domain linker of (viii) is about 15 amino acids.
  • a half-life extension domain of (vii) can be any known half-life extension domains known in the art including, without limitation, HSA, HSA binding domains, Fc domains, and small molecules.
  • HSA Human serum albumin
  • Molecular mass ⁇ 67 kDa is the most abundant protein in plasma, present at about 50 mg/ml (600 pM), and has a half-life of around 20 days in humans.
  • HSA serves to maintain plasma pH, contributes to colloidal blood pressure, functions as carrier of many metabolites and fatty acids, and serves as a major drug transport protein in plasma.
  • Noncovalent association with albumin extends the elimination half-time of short lived proteins.
  • a recombinant fusion of an albumin binding domain to a Fab fragment resulted in a reduced in vivo clearance of 25- and 58-fold and a half-life extension of 26- and 37-fold when administered intravenously to mice and rabbits respectively as compared to the administration of the Fab fragment alone.
  • insulin is acylated with fatty acids to promote association with albumin
  • a protracted effect was observed when injected subcutaneously in rabbits or pigs.
  • a half-life extension domain of (vii) comprises a domain which specifically binds to HSA.
  • the HSA binding domain is a peptide.
  • the HSA binding domain is a small molecule. It is contemplated that the HSA binding domain of an antigen binding protein is fairly small and no more than 25 kD, no more than 20 kD, no more than 15 kD, or no more than 10 kD in some embodiments. In certain instances, the HSA binding domain is 5 kD or less if it is a peptide or small molecule.
  • a half-life extension domain is a single domain antigen binding domain from a sdABD that binds to HSA.
  • sdABDs that bind to HSA are provided in Table 1.
  • the third sdABD of (ix) that binds to HSA comprises a CDR1 as set forth in SEQ ID NO: 19, a CDR2 as set forth in SEQ ID NO: 20, and a CDR3 as set forth in SEQ ID NO: 21.
  • the third sdABD of (ix) comprises an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the third sdABD of (ix) comprises the amino acid sequence of SEQ ID NO: 22.
  • the half-life extension domain of the protein provides for altered pharmacodynamics and pharmacokinetics of the antigen binding protein itself. As above, the half-life extension domain extends the elimination half-time. The half-life extension domain also alters pharmacodynamic properties including alteration of tissue distribution, penetration, and diffusion of the antigen-binding protein. In some embodiments, the half-life extension domain provides for improved tissue (including tumor) targeting, tissue penetration, tissue distribution, diffusion within the tissue, and enhanced efficacy as compared with a protein without a halflife extension binding domain. In one embodiment, therapeutic methods effectively and efficiently utilize a reduced amount of the antigen-binding protein, resulting in reduced side effects, such as reduced chance of cytokine release syndrome or cytokine storm.
  • characteristics of the half-life extension domain include the binding affinity of the HSA binding domain for HSA. Affinity of said HSA binding domain can be selected so as to target a specific elimination half-time in a particular polypeptide construct.
  • the HSA binding domain has a high binding affinity.
  • the HSA binding domain has a medium binding affinity.
  • the HSA binding domain has a low or marginal binding affinity.
  • Exemplary binding affinities include KD concentrations at 10 nM or less (high), between 10 nM and 100 nM (medium), and greater than 100 nM (low). As above, binding affinities to HSA are determined by known methods such as Surface Plasmon Resonance (SPR).
  • SPR Surface Plasmon Resonance
  • C-terminal capping sequences are added to reduce the likelihood of clearance of the proteins by the innate immune system of the patient. After cleavage, the residual linker amino acids act as blocking peptides against human serum antibodies.
  • Non-limiting examples of C-terminal capping sequences are provided in US Patent No. 10858418, incorporated herein by reference.
  • a protein described herein comprises an amino acid sequence that is at least at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of any one of SEQ ID NOs: 37-40. In some embodiments, a protein described herein comprises the amino acid sequence of any one of SEQ ID NOs: 37-40.
  • a protein described herein comprises an amino acid sequence that is at least at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of any one of SEQ ID NOs: 173-176. In some embodiments, a protein described herein comprises the amino acid sequence of any one of SEQ ID NOs: 173-176.
  • a histidine tag (either His6 or His 10) can be used.
  • Any one of the proteins described herein e.g., a protein comprising the amino acid sequence of any one of SEQ ID NOs: 173-176 listed in Table 1
  • any one of the proteins described herein may further comprise a signal peptide, e.g., a signal peptide comprising the amino acid sequence of MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 177).
  • a protein described herein comprises, from N- to C- terminus:
  • sdABD single domain antigen binding domain that binds to a human TTA and a first domain linker at the C-terminus of the first sdABD, wherein the human TTA is selected from HER2, EGFR, FOLR1, TROP2, or EpCAM;
  • a first constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region linked to a light chain variable region via a first constrained non- cleavable linker (CNCL), wherein the heavy chain variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen (e.g., CD3), and wherein the heavy chain variable region and the light chain variable region are not associated in the first constrained scFv domain and the first constrained scFv domain does not bind to the human immune cell antigen (e.g., CD3);
  • a human immune cell antigen e.g., CD3
  • a cleavable linker e.g., a cleavable linker comprising a protease cleavage site provided in Table 1, such as a MMP9 cleavage site or variants thereof, wherein the cleavable linker is 17-35 (e.g., 29 or 33) amino acids in length;
  • a second constrained single chain variable fragment (scFv) domain comprising a pseudo heavy chain variable region linked to a pseudo light chain variable region via a second constrained non-cleavable linker (CNCL), wherein the pseudo heavy chain variable region and the pseudo light chain variable region are not associated in the second constrained scFv domain, and the second constrained scFv domain does not bind to the human immune cell antigen of (ii) (e.g., CD3);
  • a third sdABD that binds to human serum albumin (HSA); wherein the heavy chain variable region of (ii) associates with the pseudo light chain variable region of (v) intramolecularly, forming a variable fragment (Fv) that does not bind the immune cell antigen; and wherein the pseudo heavy chain variable region of (v) associates with the light chain variable region of (ii) intramolecularly, forming a variable fragment (Fv) that does not bind the immune cell antigen.
  • HSA human serum albumin
  • the first sdABD of (i) binds to HER2.
  • the first sdABD of (i) comprises the amino acid sequence of any one of SEQ ID NOs: 41, 45, 48-52, 54, 58, 60, 63, 66, 68, 72, 75-78, 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121, and 165.
  • the immune cell antigen is CD3 and the first constrained scFv domain of (ii) comprises a first VH comprising the amino acid sequence of SEQ ID NO: 7 fused to the N-terminus of a first VL comprising the amino acid sequence of SEQ ID NO: 8.
  • the second constrained scFv domain of (v) comprises an inert VL comprising the amino acid sequence of SEQ ID NO: 33 fused to the N-terminus of an inert VH comprising the amino acid sequence of SEQ ID NO: 34.
  • the third sdABD of (vii) comprises the amino acid sequence of SEQ ID NO: 22.
  • the protein comprises an amino acid sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the protein consists of the amino acid sequence of SEQ ID NO: 37.
  • the protein comprises an amino acid sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 173. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 173. In some embodiments, the protein consists of the amino acid sequence of SEQ ID NO: 173.
  • a protein described herein comprises, from N- to C- terminus:
  • a first single domain antigen binding domain that binds to a human TTA and a first domain linker at the C-terminus of the first sdABD, wherein the human TTA is selected from HER2, EGFR, FOLR1, TROP2, or EpCAM
  • a first constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region linked to a light chain variable region via a first constrained non- cleavable linker (CNCL), wherein the heavy chain variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen (e.g., CD3), and wherein the heavy chain variable region and the light chain variable region are not associated in the first constrained scFv domain and the first constrained scFv domain does not bind to the human immune cell antigen (e.g., CD3);
  • a human immune cell antigen e.g., CD3
  • a second domain linker and a second single domain antigen binding domain (iii) a second domain linker and a second single domain antigen binding domain (sdABD), wherein the second domain linker is at the N-terminus of the second sdABD, and wherein the second sdABD does not bind to a human TTA;
  • a cleavable linker e.g., a cleavable linker comprising a protease cleavage site provided in Table 1, such as a MMP9 cleavage site or variants thereof, wherein the cleavable linker is 8-15 amino acids in length;
  • a second constrained single chain variable fragment (scFv) domain comprising a pseudo heavy chain variable region linked to a pseudo light chain variable region via a second constrained non-cleavable linker (CNCL), wherein the pseudo heavy chain variable region and the pseudo light chain variable region are not associated in the second constrained scFv domain, and the second constrained scFv domain does not bind to the human immune cell antigen of (ii) (e.g., CD3);
  • a third sdABD that binds to human serum albumin (HSA); wherein the heavy chain variable region of (ii) associates with the pseudo light chain variable region of (v) intramolecularly, forming a variable fragment (Fv) that does not bind the immune cell antigen; and wherein the pseudo heavy chain variable region of (v) associates with the light chain variable region of (ii) intramolecularly, forming a variable fragment (Fv) that does not bind the immune cell antigen.
  • HSA human serum albumin
  • the first sdABD of (i) binds to HER2.
  • the first sdABD of (i) comprises the amino acid sequence of any one of SEQ ID NOs: 41, 45, 48-52, 54, 58, 60, 63, 66, 68, 72, 75-78, 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121, and 165.
  • the second sdABD of (iii) binds to an antigen and does not confer any function to the protein (“non-functional”). In some embodiments, the second sdABD of (iii) binds to hen egg lysozyme (HEL). In some embodiments, the second sdABD of (iii) comprises the amino acid sequence of SEQ ID NO: 26.
  • the immune cell antigen is CD3 and the first constrained scFv domain of (ii) comprises a first VH comprising the amino acid sequence of SEQ ID NO: 7 fused to the N-terminus of a first VL comprising the amino acid sequence of SEQ ID NO: 8.
  • the second constrained scFv domain of (v) comprises an inert VL comprising the amino acid sequence of SEQ ID NO: 33 fused to the N-terminus of an inert VH comprising the amino acid sequence of SEQ ID NO: 34.
  • the third sdABD of (vii) comprises the amino acid sequence of SEQ ID NO: 22.
  • the protein comprises an amino acid sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 39. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the protein consists of the amino acid sequence of SEQ ID NO: 39.
  • the protein comprises an amino acid sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 175. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 175. In some embodiments, the protein consists of the amino acid sequence of SEQ ID NO: 175.
  • a protein described herein comprises, from N- to C- terminus:
  • sdABD single domain antigen binding domain
  • a first constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region linked to a light chain variable region via a first constrained non- cleavable linker (CNCL), wherein the heavy chain variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen (e.g., CD3), and wherein the heavy chain variable region and the light chain variable region are not associated in the first constrained scFv domain and the first constrained scFv domain does not bind to the human immune cell antigen (e.g., CD3); (iii) a second domain linker and a second single domain antigen binding domain (sdABD) that binds to a human target tumor antigen (TTA), wherein the second domain linker is at the N-terminus of the second sdABD, and wherein the human TTA is selected from HER2, EGFR, FOLR1, TROP2, or EpCAM;
  • sdABD single domain antigen binding domain
  • a cleavable linker e.g., a cleavable linker comprising a protease cleavage site provided in Table 1, such as a MMP9 cleavage site or variants thereof, wherein the cleavable linker is 8-15 amino acids in length;
  • a second constrained single chain variable fragment (scFv) domain comprising a pseudo heavy chain variable region linked to a pseudo light chain variable region via a second constrained non-cleavable linker (CNCL), wherein the pseudo heavy chain variable region and the pseudo light chain variable region are not associated in the second constrained scFv domain, and the second constrained scFv domain does not bind to the human immune cell antigen of (ii) (e.g., CD3);
  • a third sdABD that binds to human serum albumin (HSA); wherein the heavy chain variable region of (ii) associates with the pseudo light chain variable region of (v) intramolecularly, forming a variable fragment (Fv) that does not bind the immune cell antigen; and wherein the pseudo heavy chain variable region of (v) associates with the light chain variable region of (ii) intramolecularly, forming a variable fragment (Fv) that does not bind the immune cell antigen.
  • HSA human serum albumin
  • the second sdABD of (iii) binds to HER2.
  • the second sdABD of (iii) comprises the amino acid sequence of any one of SEQ ID NOs: 41, 45, 48-52, 54, 58, 60, 63, 66, 68, 72, 75-78, 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121, and 165.
  • the first sdABD of (i) binds to an antigen and does not confer any function to the protein (“non-functional”). In some embodiments, the first sdABD of (i) binds to hen egg lysozyme (HEE). In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 26.
  • the immune cell antigen is CD3 and the first constrained scFv domain of (ii) comprises a first VH comprising the amino acid sequence of SEQ ID NO: 7 fused to the N-terminus of a first VL comprising the amino acid sequence of SEQ ID NO: 8.
  • the second constrained scFv domain of (v) comprises an inert VL comprising the amino acid sequence of SEQ ID NO: 33 fused to the N-terminus of an inert VH comprising the amino acid sequence of SEQ ID NO: 34.
  • the third sdABD of (vii) comprises the amino acid sequence of SEQ ID NO: 22.
  • the protein comprises an amino acid sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 40.
  • the protein comprises the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 40.
  • the protein consists of the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 40.
  • the protein comprises an amino acid sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 174 or SEQ ID NO: 176. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 174 or SEQ ID NO: 176. In some embodiments, the protein consists of the amino acid sequence of SEQ ID NO: 174 or SEQ ID NO: 176.
  • a protein described herein comprises, from N- to C- terminus:
  • a first constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region linked to a light chain variable region via a first constrained non- cleavable linker (CNCL), wherein the heavy chain variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen (e.g., CD3), and wherein the heavy chain variable region and the light chain variable region are not associated in the first constrained scFv domain and the first constrained scFv domain does not bind to the human immune cell antigen (e.g., CD3);
  • a human immune cell antigen e.g., CD3
  • a second domain linker and a second single domain antigen binding domain that binds to a human target tumor antigen (TTA), wherein the second domain linker is at the N-terminus of the second sdABD, and wherein the human TTA is selected from HER2, EGFR, FOLR1, TROP2, or EpCAM;
  • a cleavable linker e.g., a cleavable linker comprising a protease cleavage site provided in Table 1, such as a MMP9 cleavage site or variants thereof), wherein the cleavable linker is 8-15 amino acids in length;
  • a second constrained single chain variable fragment (scFv) domain comprising a pseudo heavy chain variable region linked to a pseudo light chain variable region via a second constrained non-cleavable linker (CNCL), wherein the pseudo heavy chain variable region and the pseudo light chain variable region are not associated in the second constrained scFv domain, and the second constrained scFv domain does not bind to the human immune cell antigen of (ii) (e.g., CD3);
  • a third sdABD that binds to human serum albumin (HSA); wherein the heavy chain variable region of (ii) associates with the pseudo light chain variable region of (v) intramolecularly, forming a variable fragment (Fv) that does not bind the immune cell antigen; and wherein the pseudo heavy chain variable region of (v) associates with the light chain variable region of (ii) intramolecularly, forming a variable fragment (Fv) that does not bind the immune cell antigen.
  • HSA human serum albumin
  • the second sdABD of (iii) binds to HER2.
  • the second sdABD of (iii) comprises the amino acid sequence of any one of SEQ ID NOs: 41, 45, 48-52, 54, 58, 60, 63, 66, 68, 72, 75-78, 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121, and 165.
  • the immune cell antigen is CD3 and the first constrained scFv domain of (ii) comprises a first VH comprising the amino acid sequence of SEQ ID NO: 7 fused to the N-terminus of a first VL comprising the amino acid sequence of SEQ ID NO: 8.
  • the second constrained scFv domain of (v) comprises an inert VL comprising the amino acid sequence of SEQ ID NO: 33 fused to the N-terminus of an inert VH comprising the amino acid sequence of SEQ ID NO: 34.
  • the third sdABD of (vii) comprises the amino acid sequence of SEQ ID NO: 22.
  • the present disclosure provides nucleic acid molecules comprising a nucleotide sequence encoding any one of the proteins described herein.
  • the nucleic acid molecule is a vector.
  • the nucleic acid molecule is an expression vector (e.g., an expression vector suitable for expression of the protein in mammalian cells such as human cells).
  • nucleic acid compositions will depend on the format of the proteins.
  • a protein described herein is encoded by a single nucleic acid molecule in a single expression vector for production.
  • the nucleic acids encoding the components of the protein can be incorporated into expression vectors, and depend on the host cells used to produce the prodrug compositions disclosed herein. Generally, the nucleic acids are operably linked to any number of regulatory elements (promoters, origin of replication, selectable markers, ribosomal binding sites, inducers, etc.).
  • the expression vectors can be extra-chromosomal or integrating vectors.
  • nucleic acids and/or expression vectors encoding the prodrugs disclosed herein are then transformed into any number of different types of host cells known in the art, including mammalian, bacterial, yeast, insect and/or fungal cells, with mammalian cells (e.g., CHO cells, 293 cells), finding use in many embodiments.
  • mammalian cells e.g., CHO cells, 293 cells
  • the prodrug compositions described herein are made by culturing host cells comprising the expression vector(s). Once produced, traditional antibody purification steps are done, including a Protein A affinity chromatography step and/or an ion exchange chromatography step.
  • activities of proteins described herein may be determined via T cell dependent cellular cytotoxicity Assay.
  • human T cells isolated from healthy donors can be incubated with proteins at varying concentrations along with target bearing tumor cell lines that have been pre-labelled with firefly luciferase. Cytotoxicity can be measured by observing changes in luciferase levels using a luminometer.
  • compositions comprising any one or more of the proteins described herein are prepared for storage by mixing the proteins having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (as generally outlined in Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]), in the form of lyophilized formulations or aqueous solutions.
  • the composition comprising the proteins described herein are prepared for administration, e.g., to a subject for treating a disease (e.g., cancer).
  • the cancer is a HER2+ cancer.
  • the cancer has any level of HER2 expression.
  • the cancer has a low HER2 expression level. In some embodiments, the cancer has a moderate HER2 expression level. In some embodiments, the cancer has a high HER2 expression level. In some embodiments, the cancer is breast cancer, lung cancer, gastric cancer, stomach cancer, or colorectal cancer. In some embodiments, the cancer is breast cancer, lung cancer, gastric cancer, stomach cancer, or colorectal cancer, and has a low HER2 expressing level. In some embodiments, the cancer is breast cancer, lung cancer, gastric cancer, stomach cancer, or colorectal cancer, and has a moderate HER2 expressing level. In some embodiments, the cancer is breast cancer, lung cancer, gastric cancer, stomach cancer, or colorectal cancer, and has a high HER2 expressing level.
  • compositions for administration to a subject described herein comprises a first protein and a second protein, wherein the first protein and the second protein are the same (identical construct), wherein each of the first protein and the second protein comprises, from N- to C- terminus:
  • a first constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region linked to a light chain variable region via a first constrained non- cleavable linker (CNCL), wherein the heavy chain variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen, and wherein the heavy chain variable region and the light chain variable region are not associated in the first constrained scFv domain and the first constrained scFv domain does not bind to the human immune cell antigen;
  • CNCL constrained non- cleavable linker
  • a second constrained single chain variable fragment (scFv) domain comprising a pseudo heavy chain variable region linked to a pseudo light chain variable region via a second constrained non-cleavable linker (CNCL), wherein the pseudo heavy chain variable region and the pseudo light chain variable region are not associated in the second constrained scFv domain, and the second constrained scFv domain does not bind to the human immune cell antigen of (ii);
  • a third sdABD that binds to human serum albumin (HSA); wherein the heavy chain variable region of (ii) associates with the pseudo light chain variable region of (v) intramolecularly, forming a variable fragment (Fv) that does not bind the immune cell antigen; and wherein the pseudo heavy chain variable region of (v) associates with the light chain variable region of (ii) intramolecularly, forming a variable fragment (Fv) that does not bind the immune cell antigen; wherein (i) and (iii) are not both absent; and wherein when (i) and (iii) are both present:
  • the first sdABD binds to a human target tumor antigen (TTA) and the second sdABD does not bind a human TTA;
  • the first sdABD does not bind a human TTA and the second sdABD binds to a human TTA.
  • (i) is present and (iii) is absent, and wherein the first sdABD binds to a human TTA. In some embodiments, (i) is absent and (iii) is present, and wherein the second sdABD binds to a human TTA. In some embodiments, (i) and (iii) are both present, wherein the second sdABD does not bind a human TTA, and the first sdABD binds to a human TTA. In some embodiments, (i) and (iii) are both present, and wherein the first sdABD does not bind a human TTA, and the second sdABD binds to a human TTA.
  • the VH of the first constrained scFv domain of the first protein associates with the VL of the first constrained scFv domain of the second protein, forming an active binding site that binds to the human immune cell antigen;
  • the VL of the first constrained scFv domain of the first protein associates with the VH of the first constrained scFv domain of the second protein, forming an active binding site that binds to the human immune cell antigen.
  • the cleavage fragments of the prodrug (inactive) proteins assemble into two homodimers (a first homodimer and a second homodimer), each homodimer being a bi-specific molecule capable of binding a TTA and an immune cell antigen.
  • cleavage of the cleavable linker of (iv) in the first protein and the second protein results in a composition comprising a homodimer of a first polypeptide and a second polypeptide, wherein the first polypeptide is identical to the second polypeptide, and where each of the first polypeptide and the second polypeptide comprises:
  • sdABD single domain antigen binding domain
  • TTA first human target tumor antigen
  • a constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region (VH) linked to a light chain variable region (VL) via a constrained non- cleavable linker (CNCL), wherein the heavy chain variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen, and wherein the heavy chain variable region and the light chain variable region are not associated in the constrained scFv domain and the constrained scFv domain does not bind to the human immune cell antigen; wherein the VH of the first polypeptide associates with the VL of the second polypeptide, and the VL of the first polypeptide associates with the VH of the second polypeptide, forming two active variable fragments (Fvs) each capable of binding to the immune antigen.
  • VH heavy chain variable region
  • VL light chain variable region
  • CNCL constrained non- cleavable linker
  • a composition described herein is administered to a subject in need thereof via one or more suitable routes of administration, using one or more of a variety of methods known in the art.
  • the route and/or mode of administration will vary depending upon the desired results.
  • An acceptable route of administration may refer to any administration pathway known in the art which may be taken into consideration by a clinician in conjunction with the intended therapeutic use, such as by parenteral administration which is typically associated with injection at or in communication with the intended site of action (e.g., intravenous administration).
  • a composition is administered to the same subject once or on multiple occasions.
  • the terms “treat,” “treating,” or “treatment”, and grammatical variants thereof, have the same meaning as commonly understood by those of ordinary skill in the art. In some embodiments, these terms refer to an approach for obtaining beneficial or desired clinical results. The terms may refer to slowing the onset or rate of development of a condition, disorder or disease, reducing or alleviating symptoms associated with it, generating a complete or partial regression of the condition, or some combination of any of the above.
  • beneficial or desired clinical results include, but are not limited to, reduction or alleviation of symptoms, diminishment of extent of disease, stabilization (e.g., not worsening) of state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • “Treat,” “treating,” or “treatment” can also include prolonging survival relative to expected survival time if not receiving treatment.
  • a subject e.g., a human in need of treatment may thus be a subject already afflicted with the disease or disorder in question.
  • the terms “treat,” “treating,” or “treatment” include inhibition or reduction of an increase in severity of a pathological state or symptoms relative to the absence of treatment, and is not necessarily meant to imply complete cessation of the relevant disease or condition.
  • a composition is administered to a subject in an effective amount.
  • An “effective amount” is an amount effective for treating and/or preventing a disease, disorder, or condition as disclosed herein.
  • an effective amount is an amount or dose of a composition (e.g., a therapeutic composition, compound, or agent) that produces at least one desired therapeutic effect in a subject, such as preventing or treating a target condition or beneficially alleviating a symptom associated with the condition.
  • This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic composition (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type, disease stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration.
  • One skilled in the clinical and pharmacological arts can assess an effective amount, for example, by monitoring a subject’s response to administration of a composition and adjusting the dosage accordingly (see e.g., Remington: The Science and Practice of Pharmacy (Gennaro A, ed., Mack Publishing Co., Easton, PA, U.S., 19th ed., 1995)).
  • the term “subject” refers to any organism, commonly mammalian subjects, such as humans and animals.
  • the terms “subject” and “patient” are used interchangeably.
  • the subject is a mammal, such as a primate (e.g., a human or non-human primate), or a livestock animal (e.g., cow, horse, pig, sheep, goat, etc.).
  • a primate e.g., a human or non-human primate
  • livestock animal e.g., cow, horse, pig, sheep, goat, etc.
  • a central challenge in developing cancer therapeutics is balancing tumor killing efficacy and toxicity to the patient.
  • One strategy for achieving this balance is by designing therapeutics that have specific affinity for tumor cells.
  • T cells can be specifically recruited to tumors using conditionally activatable multispecific proteins comprising tumor targeted antigen binding domains and immune cell binding domains. These multispecific proteins can be activated by proteases in the tumor microenvironment to further reduce potential toxicity in non-tumor tissue.
  • Example 1 HER2 single VHH construct (Pro 1225) demonstrates tumor killing activity in vivo
  • Conditionally activatable multispecific proteins may contain two tumor binding single domain antibodies (sdABDs) (e.g., Prol 111 and Prol 118 in FIG. 1). To design new constructs that have improved profile and balance between tumor killing efficacy and toxicity to the patient, these proteins were modified by removing one of the tumor targeting sdABDs to produce proteins that contain one single tumor targeting sdABD.
  • sdABDs tumor binding single domain antibodies
  • Avidity of the dimerized active fragment (active form) produced by proteolysis of the prodrug was reduced compared to a protein having two functional sdABD (e.g., both (i) and (iii) are both present), likely due to reduction of the number of the binding sites from 4 in the proteins having two tumor targeting sdABD to 2 in proteins having one single tumor targeting sdABD.
  • the reduced avidity was postulated to lead to the reduced residence time on the cell surface and in turn the potency of the proteins, while increasing the tolerability and safety profile of the proteins.
  • Prol 148 contains a HER2 targeting sdABD at the N-terminus and the second HER2 targeting sdABD in Prol 111 was replaced with a sdABD that binds to HEL;
  • Prol 145 contains a HER2 targeting sdABD internally and the N-terminal HER2 targeting sdABD in Prol 111 was replaced with a sdABD that binds to HEL;
  • Prol 168 contains a HER2 targeting sdABD internally and the N-terminal HER2 targeting sdABD in Prol 118 was replaced with a sdABD that binds to HEL.
  • Prol225, Prol 145, Prol 148, and Prol 168 amino acid sequences are provided in Table 1. Proteins having only one single tumor targeting sdABDs targeting other tumor antigens (e.g., EGFR, CA9, B7H3, EpCAM) were also produced.
  • sdABDs targeting other tumor antigens e.g., EGFR, CA9, B7H3, EpCAM
  • Binding affinities of the proteins with one single tumor binding sdABD were tested and compared with their respective counterpart that contain two tumor binding sdABDs using the OCTET assay.
  • Human tumor antigen-Fxa-hFc proteins were immobilized on anti-human Fc capture sensor (ForteBio) and rinsed with 0.25% casein in PBS pH 7.4. Each protein tested was added to the individual sensor at different concentration (concentration range 1.56 - 100 nM) and the association of the proteins and its target was recorded for 180 sec. After that the sensor was placed into the buffer and the dissociation of the complex was recoded. Kd was calculated as koff/kon of the dissociation and association curves. Each experiment was performed in duplicate and the average of two measurements is reported.
  • All proteins containing one single tumor targeting sdABD exhibited reduced binding affinity to its respective target, compared with their counterpart having two tumor targeting sdABDs. This was observed for proteins that target HER2, EGFR, CA9, EpCAM, and B7H3.
  • Pro 1225 and Pro 1268 were compared with Prol 118 in their binding activity to HER2, and in in vivo and in vitro tumor killing assays.
  • Pro 1225 and Pro 1268 exhibited reduced binding affinity to human and Cyno HER2, compared with Prol 118 (Table 2).
  • Pro 1225 and Pro 1268 exhibited reduced potency (about 2-fold less potent) relative to Prol 118 in HT29 cells (expressing low levels of HER2), but exhibited comparable potency in SKLOV- 3 cells (expressing high levels of HER2) (Table 2).
  • Pro 1225 despite having reduced binding affinity to its target and reduced in vitro tumor killing potency, in a human lung cancer (HCC827 cells, expressing low levels of HER2) mouse model, demonstrated higher in vivo tumor killing efficacy than Prol 118 (FIG. 2A), and demonstrated comparable in vivo tumor killing efficacy at a 3-fold lower dose relative to Prol 118 (FIGs. 2A and 2B).
  • HCC827 cells expressing low levels of HER2
  • Prol225 demonstrated higher in vivo tumor killing efficacy than Prol 118 (FIG.
  • Prol 168 similar to Prol225 has one single HER2 targeting sdABD and had a dose dependent in vivo tumor killing efficacy in the human stomach cancer model (SNU16)mouse model (FIG. 4).
  • SNU16 human stomach cancer model
  • FIG. 4 The results demonstrate that the binding affinity, in vitro potency, and in vivo efficacy of the conditionally activable proteins can be adjusted by using one single tumor targeting sdABD. Proteins with comparable or enhanced in vivo tumor killing efficacy and improved tolerability and reduced toxicity may be achieved by using one single tumor target sdABD.
  • Pro 1225 was assessed in a T-cell dependent cellular toxicity assay (TDCC) against models of stomach (FIG. 8), breast (FIG. 9), and gastric cancer (FIG. 10).
  • TDCC T-cell dependent cellular toxicity assay
  • Prol303 was used as a control, which has the same configuration as Pro 1225, however instead of a MMP9 cleavable linker as depicted in FIG. 1, it contains a non-cleavable linker (NCL) (FIG. 6).
  • NCL non-cleavable linker
  • the IC50 values of the three tested constructs are provided in Table 3.
  • cancer cell lines were tested for their expression of HER2. All adherent cell lines were harvested using an enzyme-free dissociation buffer. Dissociated cells were counted and 1.0 x 10 5 cells were stained with a PE-conjugated anti-human HER2 antibody at a saturating concentration of 33.3 nM. Stained samples were analyzed on a BD LSRFortessaTM per the manufacturer’s instructions, and the mean fluorescence intensity (MFI) of the PE signal was generated for each cell line. PE-labeled BD QuantibriteTM beads were analyzed in parallel using the same instrument settings. The MFIs from the QuantibriteTM beads were used to generate a standard curve, from which the number of HER2 molecules per cell were extrapolated from the MFI signal of each stained cell line.
  • MFI mean fluorescence intensity
  • the endogenous HER2 cell surface density was evaluated using the BD QuantibriteTM system across a panel of 9 cell lines.
  • the numerical values above each bar in FIG. 7 represent the mean number of HER2 antibodies bound per cell.
  • the 9 examined cell lines display a range of HER2 cell surface density from relatively low (iPSC cardiomyocytes, SNU-16, HCC827), low-to-moderate (HT-29, HT-55, Capan-1, JIMT-1), to relatively high (SKOV-3, NCI-N87) (FIG. 7).
  • SNU-16 HER2 Low ; FLuc cells were plated on 96-well white clear bottom plates at a density of 5.0 x 10 3 cells/well in defined TDCC media (AIM V [Gibco, Cat.No. 12055-091]). Human PBMC effector cells were then co-cultured with SNU-16 (HER2 LOW ; FLUC) target cells at an Effector: Target cell (E:T) ratio of 5:1.
  • test articles Prol225, Prol225 cleaved, and Prol303 were added to respective wells and treated cocultures were allowed to incubate at 37C/5% CO2 for 96 hours.
  • Viability of SNU-16 (HER2 LOW ; FLUC) target cells was assessed using the Steady-Gio® Luciferase Assay System to measure the number of remaining live cells in the wells following treatment with test articles versus untreated controls. The luminescence from each well was measured using a SpectraMax i3x.
  • Concentration response curves were generated by calculating the Relative Luminescence Units (RLU) of test article treated samples normalized to the mean of the untreated controls and nonlinear regressions were performed to generate the concentration-response curves with a 4-parameter logistic model to determine IC50 values.
  • RLU Relative Luminescence Units
  • JIMT-1 HER2 Moderate ; FLuc
  • HER2 Moderate ; FLuc human PBMC effector cells were then co-cultured with JIMT-1 (HER2 Moderate ; FLuc) target cells at an Effector: Target cell (E:T) ratio of 5:1.
  • test articles Prol225, Prol225 cleaved, and Prol303 were added to respective wells and treated cocultures were allowed to incubate at 37C/5% CO2 for 96 hours.
  • Viability of JIMT-1 (HER2 Moderate ; FLuc) target cells was assessed using the Steady-Gio® Luciferase Assay System to measure the number of remaining live cells in the wells following treatment with test articles versus untreated controls. The luminescence from each well was measured using a SpectraMax i3x.
  • Concentration response curves were generated by calculating the Relative Luminescence Units (RLU) of test article treated samples normalized to the mean of the untreated controls and nonlinear regressions were performed to generate the concentration-response curves with a 4-parameter logistic model to determine IC50 values.
  • RLU Relative Luminescence Units
  • Killing activity of Prol225 and Prol225 cleaved against JIMT-1 (HER2 Moderate ; FLuc) cells in the presence of human PBMCs was observed at picomolar concentrations in this study (FIG. 9_and Table 3).
  • NCI-N87 (HER2 High ; FLuc) cells were plated on 96-well white clear bottom plates at a density of 5.0 x 10 3 cells/well in defined TDCC media (AIM V [Gibco, Cat.No. 12055-091]).
  • Human PBMC effector cells were then co-cultured with NCI-N87 (HER2 High ; FLuc) target cells at an Effector: Target cell (E:T) ratio of 5:1.
  • test articles Prol225, Prol225 cleaved, and Prol303 were added to respective wells and treated cocultures were allowed to incubate at 37C/5% CO2 for 96 hours.
  • Viability of NCI-N87 (HER2 High ; FLuc) target cells was assessed using the Steady-Gio® Luciferase Assay System to measure the number of remaining live cells in the wells following treatment with test articles versus untreated controls. The luminescence from each well was measured using a SpectraMax i3x.
  • Concentration response curves were generated by calculating the Relative Luminescence Units (RLU) of test article treated samples normalized to the mean of the untreated controls and nonlinear regressions were performed to generate the concentration-response curves with a 4-parameter logistic model to determine IC50 values.
  • RLU Relative Luminescence Units
  • Cynomolgus HER2-expressing Raji (FLuc) cells were plated on 96-well white clear bottom plates at a density of 5.0 x 10 3 cells/well in serum-supplemented TDCC media (RPMI Medium 1640 [Gibco, Cat.No. 11875-093], 5% heat inactivated Fetal Bovine Serum [FBS], 1% Penicillin/Streptomycin, 1% GlutaMAX, 1% Non-Essential Amino Acids, lOmM HEPES, ImM Sodium Pyruvate, 55mM 2-Mercaptoethanol).
  • Cynomolgus monkey PBMC effector cells were then co-cultured with cynomolgus HER2-expressing Raji (FLuc) target cells at an Effector:Target cell (E:T) ratio of 5:1. Subsequently, test articles Prol225, Prol225 cleaved, and Pro 1303 were added to respective wells and treated co-cultures were allowed to incubate at 37C/5% CO2 for 96 hours. Viability of cynomolgus HER2-expressing Raji (FLuc) target cells was assessed using the Steady-Gio® Luciferase Assay System to measure the relative number of remaining live cells in the wells following treatment with test articles versus untreated controls.
  • Pro 1225 was assessed in a T-cell dependent cellular toxicity assay (TDCC) against a models of colorectal cancer with moderate HER2 expression (FIG. 12)
  • TDCC T-cell dependent cellular toxicity assay
  • Prol303 was used as a control, which has the same configuration as Pro 1225, however instead of a MMP9 cleavable linker as depicted in FIG. 1, it contains a non-cleavable linker (NCL) (FIG. 6).
  • Prol225 was either administered alone or as a pre-cleaved Pro 1225 after overnight treatment with an MMP9 enzyme, which can cleave the cleavable MMP9 linker (FIG. 5).
  • the EC50 values of the tested constructs are provided in Table 4.
  • HT-29 cells were plated on 96-well white clear bottom plates at a density of 5.0 x 10 3 cells/well in defined TDCC media (AIM V [Gibco, Cat.No. 12055-091]).
  • Human PBMC effector cells were then co-cultured with NCI-N87 (HER2 Hlgh ; FLuc) target cells at an Effector:Target cell (E:T) ratio of 5:1.
  • E:T Effector:Target cell
  • test articles Prol225, Prol225 + MMP9, and Pro 1303 were added to respective wells and treated co-cultures were allowed to incubate at 37C/5% CO2 for 48 hours.
  • HT-29 target cells Viability of HT-29 target cells was assessed using the Steady-Gio® Luciferase Assay System to measure the number of remaining live cells in the wells following treatment with test articles versus untreated controls. The luminescence from each well was measured using a SpectraMax i3x. Concentration response curves were generated by calculating the Relative Luminescence Units (RLU) of test article treated samples and nonlinear regressions were performed to generate the concentration-response curves with a 4- parameter logistic model to determine EC50 values.
  • RLU Relative Luminescence Units
  • Example 4 HER2 single VHH construct (Pro 1225) demonstrates tumor killing activity in vivo Pro 1225 at multiple doses was assessed in in vivo tumor models of stomach, gastric, lung, colorectal, and breast cancer.
  • Pro 1303 was used as a control, which has the same configuration as Pro 1225, however instead of a MMP9 cleavable linker as depicted in FIG. 1, it contains a non-cleavable linker (NCL) (FIG. 6).
  • NCL non-cleavable linker
  • SNU16 low HER2 expressing level gastric cancer model
  • 2.5 x 10 6 SNU16 cells were implanted subcutaneous in the right flank of NSG (NOD.Cg-Prkdcscid I12rgtmlWjl/SzJ) mice (The Jackson Laboratory, Cat. No. 005557) and allowed to grow until tumors were established.
  • human T cells were cultured in T cell media (X-VIVO 15 [Lonza, Cat.No. 04-418Q], 5% Human Serum, 1% Penicillin/Streptomycin, O.OlmM 2- Mercaptoethanol) in a G-RexlOOM gas permeable flask (Wilson Wolf Cat. No.
  • Mice were then injected intravenously (IV) with 2.5xl0 6 cultured human T cells and administered the first dose of the Prol225 or control molecule (Prol303). Mice were dosed every 3 days for 7 doses (Days 0, 3, 6, 9, 12, 15 and 18) and then followed until the study was terminated on day 39. Groups received 0.3, 0.1 and 0.03 mg/kg of the MMP9 cleavable linker containing Prol225, or 0.3 mg/kg of Prol303. Tumor volumes were measured every 3 days.
  • HCC827 low HER2 expressing level
  • 5 x 10 6 HCC827 cells were implanted subcutaneous in the right flank of NSG (NOD.Cg-Prkdcscid I12rgtmlWjl/SzJ) mice (The Jackson Laboratory, Cat. No. 005557) and allowed to grow until tumors were established.
  • human T cells were cultured in T cell media (X-VIVO 15 [Lonza, Cat.No. 04-418Q], 5% Human Serum, 1% Penicillin/Streptomycin, O.OlmM 2- Mercaptoethanol) in a G-RexlOOM gas permeable flask (Wilson Wolf Cat. No.
  • Mice were then injected intravenously (IV) with 2.5xl0 6 cultured human T cells and administered the first dose of Prol225 or control molecule (Prol303). Mice were dosed every 3 days for 7 doses (Days 0, 3, 6, 9, 12, 15 and 18) and then followed until the study was terminated on day 40. Groups received 0.3, 0.1 and 0.03 mg/kg of the MMP9 cleavable linker containing Prol225, or 0.3 mg/kg of Prol303. Tumor volumes were measured every 3 days.
  • HT55 low HER2 expressing level
  • mice The Jackson Laboratory, Cat. No. 005557
  • human T cells were cultured in T cell media (X-VIVO 15 [Lonza, Cat.No. 04-418Q], 5% Human Serum, 1% Penicillin/Streptomycin, O.OlmM 2- Mercaptoethanol) in a G-RexlOOM gas permeable flask (Wilson Wolf Cat. No.
  • mice were dosed every 3 days for 7 doses (Q3Dx7) (Days 0, 3, 6, 9, 12, 15 and 18) and then followed until the study was terminated on day 27. Groups received 1, 0.3 and 0.1 mg/kg of Prol225, or 1 mg/kg Prol303. Tumor volumes were measured every 3 days.
  • JIMT-1 HER2 (moderate HER2 expressing level) model of breast cancer
  • 5 x 10 6 JIMT-1 cells were implanted subcutaneous in the right flank of NSG (NOD.Cg-Prkdcscid I12rgtmlWjl/SzJ) mice (The Jackson Laboratory, Cat. No. 005557) and allowed to grow until tumors were established.
  • human T cells were cultured in T cell media (X-VIVO 15 [Lonza, Cat.No. 04-418Q], 5% Human Serum, 1% Penicillin/Streptomycin, O.OlmM 2- Mercaptoethanol) in a G-RexlOOM gas permeable flask (Wilson Wolf Cat. No.
  • Mice were then injected intravenously (IV) with 2.5xl0 6 cultured human T cells and administered the first dose of Prol225 or control molecule (Prol303). Mice were dosed every 3 days for 7 doses (Days 0, 3, 6, 9, 12, 15 and 18) and then followed until the study was terminated on day 40. Groups received 1, 0.3 and 0.1 mg/kg of Pro 1225, or 1 mg/kg of Prol303. Tumor volumes were measured every 3 days.

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Abstract

Selon certains aspects, la présente divulgation concerne des molécules activables de manière conditionnelle comprenant un sdABD unique qui se lie à un antigène tumoral cible humain (TTA) et leurs utilisations.
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