WO2021097060A1 - Molécule pro-immunomodulatrice comprenant une fraction de groupement - Google Patents

Molécule pro-immunomodulatrice comprenant une fraction de groupement Download PDF

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WO2021097060A1
WO2021097060A1 PCT/US2020/060184 US2020060184W WO2021097060A1 WO 2021097060 A1 WO2021097060 A1 WO 2021097060A1 US 2020060184 W US2020060184 W US 2020060184W WO 2021097060 A1 WO2021097060 A1 WO 2021097060A1
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immune modulating
antibody
protease
pro
molecule
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PCT/US2020/060184
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English (en)
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Shuoyen Jack LIN
Kathryn KWANT
Holger Wesche
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Harpoon Therapeutics, Inc.
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Publication of WO2021097060A1 publication Critical patent/WO2021097060A1/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
    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70525—ICAM molecules, e.g. CD50, CD54, CD102
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • 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
    • C07K2317/60—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
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • One embodiment provides a pro immune modulating molecule comprising: a dual binding moiety comprising a cleavable linker, a non-CDR loop, and a binding site specific for a bulk serum protein; an immune agonistic antibody or an antigen binding fragment thereof that comprises a binding site specific for an epitope within an antibody target; and a clustering moiety that comprises a binding site specific for a protein that promotes a higher order clustering of the antibody target, wherein the dual binding moiety is capable of masking binding of the immune agonistic antibody or an antigen binding fragment thereof to the antibody target, and wherein upon cleavage of the linker the immune agonistic antibody or an antigen binding fragment thereof binds to the antibody target.
  • the protein that promotes a higher order clustering of the antibody target is another molecule of the antibody target, and wherein the binding site of the clustering moiety is a binding site specific for a second epitope within the antibody target.
  • the protein that promotes a higher order clustering of the antibody target comprises at least one of: FcgRIIb, FcgRIIA, FcgRI, FcgRIIIA, FcgRIIIB, FcgRIV, FceRI, FceRII, FcaRI, FcRn, MHC class I, MHC class II, CD40, 0X40, CD 137, CTLA4, PD1, PDL1, PDL2, B7-1, B7-2, CD19, CD20, CD22, CD30, CD33, CD79b, BCMA, CD70, CD71, LRRC33, GARP, or any combinations thereof.
  • the protein that promotes a higher order clustering of the antibody target comprises at least one of:
  • the immune modulatory protein comprises a protein belonging to the Tumor Necrosis Factor (TNF) superfamily.
  • TNF Tumor Necrosis Factor
  • the immune modulatory protein is selected from the group consisting of CTLA4, CD27, CD28, CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, TIM-1, 0X40, DNAM-1, PD-L1, PD1, PD-L2, CD4, CD8, CD40, CEACAMl, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDOl, ID02, TDO, KIR, LAG-3, TIM-3, VISTA, IL6-R, IL-6, TNF a, CD 19, CD20, CD22, CD52, integrin a4, integrin a4b7, CD11, and any combinations thereof.
  • the dual binding moiety is connected, via the cleavable inker, to the immune modulating antibody or an antigen binding fragment thereof.
  • the dual binding moiety comprises at least three CDRs (CDR1, CDR2, CDR3) for binding the bulk serum protein.
  • the dual binding moiety further comprises at least three CDRs (CDR1, CDR2, CDR3) for binding a dual binding moiety target.
  • the non-CDR loop of the dual binding moiety provides a binding site that enables binding of the dual binding moiety to the immune modulating antibody or an antigen binding fragment thereof.
  • the non-CDR loop comprises at least one of: an AB loop, a C'D loop, an EF loop, and a CC loop and the binding site specific for the immune agonistic antibody or an antigen binding fragment thereof is provided by one or more of the AB loop, the C'D loop, the EF loop, and the CC loop. In some embodiments, the binding site specific for the immune agonistic antibody or an antigen binding fragment thereof is provided by the CC loop.
  • the bulk serum protein is at least one of: albumin, transferrin, IgGl, IgG2, IgG4, IgG3, IgA monomer, Factor XIII, Fibrinogen, IgE, and pentameric IgM, or any combinations thereof.
  • the dual binding moiety target comprises a tumor antigen
  • the tumor antigen is selected from the group consisting of: EGFR, PSMA, EpCAM, BCMA, 5T4, AFP, Axl, B7-H3, Cadherin-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD38, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, CEACAM5, c-MET, DLL3, EphA2, FAP, FGFR2, FGFR3, glypican-3, FLT-3, FOLR1, gpNMB, HER2, HPV-16 E6, HPV-16 E7, ITGA3, SLC39A6, Mesothelin, Mucl, Mucl6, NaPi2b, Nectin-4, P-cadherin, Prolactin R, PSCA, PTK7, ROR1, SLC44A4,
  • the cleavable linker comprises a protease cleavage site.
  • the protease cleavage site is recognized by at least one of: a serine protease, a cysteine protease, an aspartate protease, a threonine protease, a glutamic acid protease, a metalloproteinase, a gelatinase, and an asparagine peptide lyase.
  • the protease cleavage site is recognized by at least one of: a Cathepsin B, a Cathepsin C, a Cathepsin D, a Cathepsin E, a Cathepsin K, a Cathepsin L, a kallikrein, a hKl, a hK10, a hK15, a plasmin, a collagenase, a Type IV collagenase, a stromelysin, a Factor Xa, a chymotrypsin-like protease, a trypsin-like protease, a elastase-like protease, a subtili sin-like protease, an actinidain, a bromelain, a calpain, a caspase, a caspase-3, a Mir 1 -CP, a papain, a HIV-1 protea
  • the cleavable linker comprises the amino acid sequence of a sequence selected from the group consisting of SEQ ID Nos. 73-82.
  • the antibody target is CD40 and wherein the clustering moiety comprises a binding site specific for FCgRIIB.
  • the antibody target is CD40 and wherein the clustering moiety comprises a binding site specific for EpCAM.
  • One embodiment provides a pro immune modulating molecule comprising a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID Nos. 117-124.
  • One embodiment provides a biparatopic pro immune modulating molecule comprising: - a dual binding moiety comprising a cleavable linker, a non-CDR loop, and a binding site specific for a bulk serum protein ; - an immune modulating antibody or an antigen binding fragment thereof that comprises a binding site specific for an epitope within an antibody target; - and a clustering moiety that comprises a binding site specific for a second epitope within the antibody target, wherein the dual binding moiety is capable of masking binding of the immune modulating antibody or an antigen binding fragment thereof to the antibody target, and wherein upon cleavage of the linker the immune modulating antibody or an antigen binding fragment thereof binds to the antibody target.
  • the antibody target comprises at least one of: CTLA-4, CD27, CD28, CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, TIM-1, 0X40, DNAM-1, PD-L1, PD1, PD-L2, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7- H4, BTLA, IDOl, ID02, TDO, KIR, LAG-3, TIM-3, VISTA, IL6-R, IL-6, TNFa, CD 19,
  • the dual binding moiety is connected, via its linker, to the immune agonistic antibody or an antigen binding fragment thereof.
  • the dual binding moiety comprises at least three CDRs (CDR1, CDR2, CDR3) for binding the bulk serum protein.
  • the dual binding moiety further comprises at least three CDRs (CDR1, CDR2, CDR3) for binding the dual binding moiety target.
  • the non-CDR loop of the dual binding moiety comprises a binding site specific for the immune modulating antibody or an antigen binding fragment thereof.
  • the non- CDR loop comprises at least one of: an AB loop, a C"D loop, an EF loop, and a CC loop and the binding site specific for the immune modulating antibody or an antigen binding fragment thereof is provided by one or more of the AB loop, the C"D loop, the EF loop, and the CC loop.
  • the binding site specific for the immune modulating antibody or an antigen binding fragment thereof is provided by the CC loop.
  • the bulk serum protein is at least one of: albumin, transferrin, IgGl, IgG2, IgG4, IgG3, IgA monomer, Factor XIII, Fibrinogen, IgE, and pentameric IgM, or any combinations thereof.
  • the dual binding moiety comprises the binding site specific for the tumor antigen, and wherein the tumor antigen is selected from the group consisting of: EGFR, PSMA, EpCAM, BCMA, 5T4, AFP, Axl, B7-H3, Cadherin-6, CAIX, CD117, CD123, CD138, CD 166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD38, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, CEACAM5, c-MET, DLL3, EphA2, FAP, FGFR2, FGFR3, glypican-3, FLT-3, FOLR1, gpNMB, HER2, HPV-16 E6, HPV-16 E7, ITGA3, SLC39A6, Mesothelin, Mucl, Mucl6, NaPi2b, Nectin-4, P-cadherin, Prolactin R, PSCA, PTK7,
  • the tumor antigen is selected
  • the cleavable linker comprises a protease cleavage site.
  • the protease cleavage site is recognized by at least one of: a serine protease, a cysteine protease, an aspartate protease, a threonine protease, a glutamic acid protease, a metalloproteinase, a gelatinase, and a asparagine peptide lyase.
  • the protease cleavage site is recognized by at least one of: a Cathepsin B, a Cathepsin C, a Cathepsin D, a Cathepsin E, a Cathepsin K, a Cathepsin L, a kallikrein, a hKl, a hK10, a hK15, a plasmin, a collagenase, a Type IV collagenase, a stromelysin, a Factor Xa, a chymotrypsin-like protease, a trypsin-like protease, a elastase-like protease, a subtilisin-like protease, an actinidain, a bromelain, a calpain, a caspase, a caspase-3, a Mir 1 -CP, a papain, a HIV-1 protea
  • One embodiment provides an biparatopic pro immune modulating molecule comprising a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID Nos. 125-130.
  • One embodiment provides a method of treating a disease comprising administering to a subject an effective amount of a pro immune modulating molecule according to this disclosure.
  • One embodiment provides a method of treating a disease comprising administering to a subject an effective amount of a biparatopic molecule according to this disclosure.
  • the subject is a human.
  • the disease is a tumorous disease or an inflammatory disease.
  • Fig. 1 illustrates exemplary formats of pro immune modulating molecules of this disclosure.
  • Fig. 2 illustrates an example of a biparatopic pro immune modulating molecule of this disclosure.
  • Figs. 3A-3F illustrates the results of a B Cell Activation Assay of exemplary anti- CD40/anti-FcgRIIB and anti-CD40 biparatopic proteins.
  • B cell activation was measured as the percentage of B cells positive for expression of CD86, CD80, and CD69, by anti-CD40/anti- FcgRIIB constructs (Fig. 3A, Fig. 3B, Fig. 3C) and anti-CD40 Biparatopic constructs (Fig. 3D, Fig. 3E, and Fig. 3F).
  • Figs. 4A-4C illustrates the results of a B Cell Activation Assay of exemplary anti- CD40/anti-EpCAM proteins.
  • B cell activation was measured as the percentage of B cells positive for expression of CD69 (Fig. 4A), CD80 (Fig. 4B), and CD86 (Fig. 4C), by anti- CD40/anti-EpCAM constructs in the presence of EpCAM expressing NCI-H508 [H508] (ATCC® CCL-253TM) cells.
  • FIG. 5A illustrates exemplary formats of an anti-CD40 biparatopic pro immune modulating molecule used in Example 4.
  • Fig. 5B-5C illustrates the results of a B Cell Activation Assay of exemplary anti-CD40 biparatopic pro immune modulating molecule. B cell activation was measured as the percentage of B cells positive for expression of CD86 with and without presence of a protease.
  • Fig. 6 illustrates the domains of the dual binding moiety, and use of non-CDR loops as “masks.”
  • Fig. 6A illustrates the 3D interactions between CDRs and non-CDR loops.
  • Fig. 6B illustrates the beta strands A-G.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g ., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” should be assumed to mean an acceptable error range for the particular value.
  • the terms “individual,” “patient,” or “subject” are used interchangeably. None of the terms require or are limited to situation characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly, or a hospice worker).
  • Framework residues refer to variable domain residues other than the CDR or hypervariable region residues as herein defined.
  • a “human consensus framework” is a framework which represents the most commonly occurring amino acid residue in a selection of human immunoglobulin VL or VH framework sequences.
  • variable region refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR).
  • CDRs complementarity-determining regions
  • FR framework
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a b- sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the Psheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Rabat et ak, Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • “Variable domain residue numbering as in Rabat” or “amino acid position numbering as in Rabat,” and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Rabat et ak, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain.
  • a heavy chain variable domain may include a single amino acid insert (residue 52a according to Rabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Rabat) after heavy chain FR residue 82.
  • the Rabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Rabat numbered sequence. It is not intended that CDRs of the present disclosure necessarily correspond to the Kabat numbering convention.
  • a “single domain antibody” or “sdAb” as used herein refers to a type of single chain antibody comprising a variable region (VHH) of a heavy chain of a human antibody.
  • SdAbs are antibody fragments consisting of a single monomeric variable antibody domain. They are derived, for example, from heavy chain antibodies derived from humans, which consist only of two antibody heavy chains, with no light chain. With a molecular weight of only 12-15 kD, sdAbs are much smaller than monoclonal antibodies (mAbs), e.g, IgG antibodies (150-160 kD), which have two heavy protein chains and two light chains.
  • a “single chain Fv” or “scFv,” as used herein, refers to a binding protein in which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody are joined to form one chain. Typically, a linker peptide is inserted between the two chains to allow for proper folding and creation of an active binding site.
  • a “cleavage site for a protease,” as meant herein, is an amino acid sequence that can be cleaved by a protease, such as, for example, a matrix metalloproteinase or a furin.
  • a protease such as, for example, a matrix metalloproteinase or a furin.
  • examples of such sites include Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln or Ala-Val-Arg-Trp-Leu-Leu-Thr-Ala, which can be cleaved by metalloproteinases, and Arg-Arg-Arg-Arg-Arg, which is cleaved by a furin.
  • the protease cleavage site can be cleaved by a protease that is produced by target cells, for example cancer cells or infected cells, or pathogens.
  • “elimination half-time” is used in its ordinary sense, as is described in Goodman and Gillman's The Pharmaceutical Basis of Therapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman, and Alfred Gilman, eds., 6th ed. 1980). Briefly, the term is meant to encompass a quantitative measure of the time course of drug elimination.
  • the elimination of most drugs is exponential (i.e., follows first-order kinetics), since drug concentrations usually do not approach those required for saturation of the elimination process.
  • the rate of an exponential process may be expressed by its rate constant, k, which expresses the fractional change per unit of time, or by its half-time, ti/2the time required for 50% completion of the process.
  • the units of these two constants are time -1 and time, respectively.
  • Pro Immune Modulating Molecule with Improved Clustering Effect Provided herein in various embodiments are pro immune modulating molecules (such as biparatopic pro immune modulating molecules) that are activated by a single cleavage event, to release an active immune modulating antibody or an antigen binding fragment thereof (e.g ., an anti-CD40 antibody or an antigen binding fragment thereof, such as an anti-CD40 scFv).
  • pro immune modulating molecules such as biparatopic pro immune modulating molecules
  • an active immune modulating antibody or an antigen binding fragment thereof e.g ., an anti-CD40 antibody or an antigen binding fragment thereof, such as an anti-CD40 scFv.
  • the pro immune modulating molecules of this disclosure comprise a moiety that targets a protein that promotes a higher order clustering of an immune modulatory protein, which, in some instances, leads to improved immune and cytotoxic effect induced by the immune modulatory protein.
  • the pro immune modulating molecules of this disclosure comprise a non-Fc format, wherein an Fc region is not present, and the pro immune modulating molecule comprises a dual binding moiety, an immune modulating antibody or an antigen binding fragment thereof that recognizes an immune modulatory protein, and a clustering moiety that promotes higher order clustering of the immune modulatory protein recognized by the immune modulating antibody or an antigen binding fragment thereof.
  • the size of the masked pro immune modulating molecule is less than about 80 kDa, less than about 70 kDa, about 60 kda, or less than about 50 kDa and the size of the activated molecule is less than about 70 kDa, about 60 kDa, less than about 50 kDa, less than about 40 kDa, less than about 30 kDa, less than about 20 kDa, less than about 10 kDa.
  • the pro immune modulating molecule is about 67 kDa and the activated molecule is about 54 kDa.
  • the pro immune modulating molecule is about 55 kDa and the activated molecule is about 42 kDa.
  • the pro immune modulating molecules provided herein do not comprise an Fc region.
  • Exemplary advantages associated with such a molecule comprises, but is not limited to, avoidance of Fc gamma receptor related toxicity, easy design and manufacture by avoiding the need for Fc optimization.
  • a pro immune modulating molecule of this disclosure comprising an immune modulating molecule, has an enhanced ability to activate B cells (such as human B cells), compared to a protein that comprises the same immune modulating antibody but not in a format as described herein.
  • a pro immune modulating molecule as provided herein comprises a sequence selected from the group consisting of SEQ ID Nos. 117-130, or a sequence that is at least about 80% to 100% identical to a sequence selected from the group consisting of SEQ ID Nos. 117-130.
  • the pro immune modulating molecule comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID Nos. 117-130.
  • the disclosure provides a pro immune modulating molecule, comprising a dual binding moiety that comprises a non-CDR loop and a cleavable linker, which is capable of masking an immune modulating antibody or an antigen binding fragment thereof from binding its target (referred to herein as an antibody target), and a clustering moiety.
  • the dual binding moiety in some embodiments, comprises at least a binding site specific for a bulk serum protein.
  • the dual binding moiety comprise at least one cleavable linker.
  • the cleavable linker comprises a polypeptide having a sequence recognized and cleaved in a sequence-specific manner.
  • the cleavage in certain examples, is enzymatic, based on pH sensitivity of the cleavable linker, or by chemical degradation.
  • the dual binding moiety in some cases, comprises a protease cleavable linker recognized in a sequence-specific manner by a matrix metalloprotease (MMP), for example MMP9, MMP2, or MMP7.
  • MMP matrix metalloprotease
  • Proteases are proteins that cleave proteins, in some cases, in a sequence-specific manner.
  • 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, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin K, Cathepsin L, kallikreins, hKl, hK10, hK15, plasmin, collagenase, Type IV collagenase, strom ely sin, Factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase- like protease, subtili sin-like protease, actinidain, bro
  • the protease cleavable linker is recognized by a MMP9 comprises a polypeptide having an amino acid sequence PR(S/T)(L/I)(S/T). In some cases, the protease cleavable linker recognized by a MMP9 comprises a polypeptide having an amino acid sequence LEATA. In some cases, the protease cleavable linker is recognized in a sequence-specific manner by a MMPl 1. In some cases, the protease cleavable linker recognized by a MMPl 1 comprises a polypeptide having an amino acid sequence GGAANLVRGG (SEQ IN No. 5).
  • the protease cleavable linker is recognized by a protease disclosed in Table 1.
  • the protease cleavable linker is recognized by a protease disclosed in Table 1 comprises a polypeptide having an amino acid sequence selected from a sequence disclosed in Table 1 (SEQ ID Nos. 1-63).
  • the cleavable linker is recognized by MMP9, matriptase, Urokinase plasminogen activator (uPA) and has an amino acid sequence as set forth in SEQ ID No. 59.
  • the binding moieties described herein comprise at least one cleavable linker.
  • the cleavable linker comprises, in some examples, a sequence as set forth in Table 2.
  • the binding moieties described herein comprise at least one non-cleavable linker.
  • the non-cleavable linker comprises, in some examples, a sequence as set forth in Table 3.
  • Exemplary pro immune modulating molecules comprise an immune modulating antibody comprising a variable heavy domain and a variable light domain connected by a linker (an scFv), wherein the antibody is specific for an antibody target, and a dual binding moiety is attached to the VH or VL domain of the scFv, for instance via cleavable linkers.
  • an scFv an immune modulating antibody comprising a variable heavy domain and a variable light domain connected by a linker (an scFv), wherein the antibody is specific for an antibody target, and a dual binding moiety is attached to the VH or VL domain of the scFv, for instance via cleavable linkers.
  • the non-CDR loop within the dual binding moiety provides a binding site specific for the immune modulating antibody or an antigen binding fragment thereof.
  • the dual binding moiety specifically interacts with the immune modulating antibody or an antigen binding fragment thereof and prevents it from binding its target.
  • the pro form of the molecule is activated by cleavage of the cleavable linker(s) that attach the dual binding moiety and the immune modulating antibody or an antigen binding fragment thereof.
  • the immune modulating antibody or an antigen binding fragment thereof is released as an active drug when the dual binding moiety is removed from the pro immune modulating molecule.
  • the active drug is cleared from circulation rapidly or after a period of time, depending on its half-life.
  • the dual binding moiety further comprises CDRs that provide specificity for binding at least one of: a bulk serum protein or a dual binding moiety target, which is the same or different as the target recognized by the masked target binding domain (the antibody target).
  • the pro immune modulating molecules of this disclosure are in some embodiments, suitable for delivery of active drugs that have potential for severe adverse effects so that drugs are not activated non- specifically, and in some examples, modifications may be made to remove drugs with severe adverse effects rapidly from circulation.
  • the dual binding moiety target is a target that is expressed on the surface of a diseased cell or tissue, for example a tumor or a cancer cell.
  • a diseased cell or tissue for example a tumor or a cancer cell.
  • Non-limiting examples include IL-1 , IL-1 receptor, IL-4, IL-4 receptor, VEGF, VEGF receptor, RSV, NGF, NGF receptor, programmed cell death protein- 1 (PD1), programmed cell death protein ligand- 1 (PD-L1), PD- L2, PDGF, PDGF receptor, angiopoietin-2 (Ang2), Ang2 receptor, myostatin (GDF8), GDF8 receptor, CD3, CD20, EGFR, MSLN, PSMA, DLL3, BCMA, EpCAM, HER-2, HER-3, c-Met, FoIR, CD38, CEA, 5T4, AFP, B7-H3, Cadherin-6, CAIX, CD117, CD123, CD138, CD166,
  • CD 19 CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, DLL3, EphA2, FAP, FGFR2, FGFR3, GPC3, gpA33, FLT-3, gpNMB, HPV-16 E6, HPV-16 E7, ITGA2, ITGA3, SLC39A6, MAGE, Mud, Mu 6, NaPi2b, Nectin-4, P- cadherin, NY-ESO-1, PRLR, PSCA, PTK7, ROR1, SLC44A4, SLTRK5, SLTRK6, STEAP1, TIMl, Trop2, and WT1.
  • Additional non-limiting examples of the dual binding moiety targets include immune modulatory proteins, immune stimulatory proteins, immune co-stimulatory proteins, or combinations thereof.
  • immune modulatory proteins include, but are not limited to: CTLA-4, CD27, CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, TIM-1, 0X40, DNAM-1, PD-L1, PD1, PD-L2, CD8, CD40, CEACAMl, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDOl, ID02, TDO, KIR, LAG-3, TIM-3, VISTA, IL6-R, IL-6, TNFa, CD 19, CD20, CD22, CD52, integrin a4, integrin a4b7, CD 11 a, CTLA4-Ig fusion, IL-17,
  • IL 12/23 IL12/23, IL12, IL23, and TGF-beta.
  • the dual binding moiety of a pro immune molecule is a domain derived from an immunoglobulin molecule (Ig molecule).
  • the Ig may be of any class or subclass (IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM etc).
  • a polypeptide chain of an Ig molecule folds into a series of parallel beta strands linked by loops. In the variable region, three of the loops constitute the “complementarity determining regions” (CDRs) which determine the antigen binding specificity of the immunoglobulin molecule.
  • CDRs complementarity determining regions
  • An IgG molecule comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding fragment thereof.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs) with are hypervariable in sequence and/or involved in antigen recognition and/or usually form structurally defined loops, interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • variable domains of an immunoglobulin molecule has several beta strands that are arranged in two sheets.
  • the variable domains of both light and heavy immunoglobulin chains contain three hypervariable loops, or complementarity-determining regions (CDRs).
  • CDR1, CDR2, CDR3 cluster at one end of the beta barrel.
  • the CDRs are the loops that connect beta strands BC, C'C", and FG of the immunoglobulin fold, whereas the bottom loops that connect beta strands A-B, CC, C" -D and EF of the immunoglobulin fold, and the top loop that connects the D-E strands of the immunoglobulin fold are the non-CDR loops.
  • at least some amino acid residues of a constant domain, CHI, CH2, or CH3 are part of the “non-CDR loop” of the dual binding moieties described herein.
  • Non-CDR loops comprise, in some embodiments, one or more of AB, CD, EF, and DE loops of a Cl-set domain of an Ig or an Ig-like molecule; AB, CC’, EF, FG, BC, and EC’ loops of a C2- set domain of an Ig or an Ig-like molecule; DE, BD, GF, A(A1 A2)B, and EF loops of I(Intermediate)-set domain of an Ig or Ig-like molecule.
  • the CDRs are believed to be responsible for antigen recognition and binding, while the FR residues are considered a scaffold for the CDRs.
  • Framework region residues that affect Ag binding are divided into two categories. The first are FR residues that contact the antigen, thus are part of the binding-site, and some of these residues are close in sequence to the CDRs. Other residues are those that are far from the CDRs in sequence, but are in close proximity to it in the 3-D structure of the molecule, e.g ., a loop in heavy chain.
  • the non-CDR loop within a dual binding moiety is modified to generate an antigen binding site specific for a target antigen, such as a tumor antigen (e.g, EGFR, PSMA) or an immune checkpoint protein (e.g, CTLA4, PD-1).
  • a tumor antigen e.g, EGFR, PSMA
  • an immune checkpoint protein e.g, CTLA4, PD-1.
  • various techniques can be used for modifying the non-CDR loop, e.g, site-directed mutagenesis, random mutagenesis, insertion of at least one amino acid that is foreign to the non-CDR loop amino acid sequence, amino acid substitution.
  • An antigen peptide is inserted into a non-CDR loop, in some examples.
  • an antigenic peptide is substituted for the non-CDR loop.
  • the modification, to generate an antigen binding site is in some cases in only one non- CDR loop. In other instances, more than one non-CDR loop are modified. For instance, in some cases, the modification is in any one of the non-CDR loops, i.e., A-B, C-C, C" -D, E-F, and D- E. In some cases, the modification is in the D-E loop. In other cases the modifications are in all four of A-B, C-C, C" -D, E-F loops.
  • a dual binding moiety of a pro immune modulating molecule is bound to an immune modulating antibody or an antigen binding fragment thereof via its non- CDR loops, such as A-B, C-C, C"-D, or E-F loop and is bound to a bulk serum protein or a dual binding moiety target, such as an immune modulatory protein, via its CDRs, such as B-C, C-C", and F-G loop.
  • a dual binding moiety of a pro immune modulating molecule is bound to a dual binding moiety target instead of a bulk serum protein, such as an immune modulatory protein, via its non-CDR loops, such as A-B, C-C, C" -D, or E-F loop and is bound to an immune modulating antibody or an antigen binding fragment thereof via its CDRs, such as B-C, C-C", or F-G loop.
  • a dual binding moiety of a pro immune modulating molecule is bound to a dual binding moiety target instead of a bulk serum protein, such as an immune modulatory protein, via its non-CDR loops, such as A-B, C-C, C" -D, or E-F loop and is bound to an immune modulating antibody or an antigen binding fragment thereof via its CDRs, such as B-C, C-C", or F-G loop.
  • a dual binding moiety of a pro immune modulating molecule is bound to an immune modulatory protein, via its non-CDR loops, such as A-B, CC, C"D, and EF loop and is bound an immune modulating antibody or an antigen binding fragment thereof via its CDRs, such as B-C, CC", and FG loop.
  • a dual binding moiety of a pro immune modulating molecule is bound to an immune modulatory protein, such as PD-L1, via its CDRs, such as BC, CC", and F-G loop, and to an immune modulating antibody or an antigen binding fragment thereof by its non-CDR loops, such as one or more of as A-B, CC, C" D, or EF loop.
  • a dual binding moiety of a pro immune modulating molecule is bound to a tumor antigen, via its CDRs, such as BC, CC", and F-G loop, and to an immune modulating antibody or an antigen binding fragment thereof by its non-CDR loops, such as one or more of as A-B, CC, C" D, or EF loop.
  • a dual binding moiety of a pro immune modulating molecule is bound to albumin via its CDRs, such as BC, C'C", and F-G loop, and to an immune modulating antibody or an antigen binding fragment thereof (such as a CD40 antibody or a CTLA4 antibody) by its non-CDR loops, such as one or more of as A-B, CC, C" D, or EF loop.
  • CDRs such as BC, C'C", and F-G loop
  • an immune modulating antibody or an antigen binding fragment thereof such as a CD40 antibody or a CTLA4 antibody
  • a dual binding moiety of a pro immune modulating molecule is bound to a tumor antigen, via its CDRs, such as BC, C'C", and F-G loop, and to an immune modulating antibody or an antigen binding fragment thereof (such as a CD40 antibody or a CTLA4 antibody) by its non-CDR loops, such as one or more of as A-B, CC, C" D, or EF loop.
  • CDRs such as BC, C'C", and F-G loop
  • an immune modulating antibody or an antigen binding fragment thereof such as a CD40 antibody or a CTLA4 antibody
  • a dual binding moiety of a pro immune modulating molecule is bound to an immune modulating antibody or an antigen binding fragment thereof via a non-CDR loop from a constant domain, a Cl -set domain, a C2-set domain, an I-domain, as described above, and to a dual binding moiety target or the bulk serum protein via its CDRs.
  • the dual binding moieties of a pro immune modulating molecule are any kind of polypeptide, such as a natural peptide, a synthetic peptide, or a fibronectin scaffold.
  • An engineered scaffold comprises, for example, sdAb, a scFv, a Fab, a VHH, a fibronectin type III domain, immunoglobulin-like scaffold (as described in Halaby el al ., 1999. Prot Eng 12(7): 563-571), DARPin, cystine knot peptide, lipocalin, three-helix bundle scaffold, protein G-related albumin-binding module, or a DNA or RNA aptamer scaffold.
  • dual binding moieties that comprise a binding site specific for albumin (via its CDRs) and binding site that is specific for an anti-CD40 antibody (via its non-CDR loops).
  • Exemplary sequences for such anti-CD40 specific dual binding moieties are provided in SEQ ID Nos. 89-101, or a sequence that is at least about 75% to about 100% identical to any one of sequences provided in SEQ ID Nos.
  • 89-101 such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%.
  • dual binding moieties that comprise a binding site specific for albumin (via its CDRs) and binding site that is specific for an anti-CTLA4 antibody (via its non-CDR loops).
  • Exemplary sequences for such anti-CTLA4 specific dual binding moieties are provided in SEQ ID Nos. 103-111, or a sequence that is at least about 75% to about 100% identical to any one of the sequences provided in SEQ ID Nos.
  • dual binding moieties that comprise a binding site specific for PSMA (via its CDRs) and a binding site specific for an anti-CTLA4 (via its non- CDR loops) (e.g ., SEQ ID Nos.
  • dual binding moieties that comprise a binding site specific for PD1 (via its CDRs) and a binding site specific for an anti-CTLA4 (via its non- CDR loops) (e.g., SEQ ID No. 115).
  • dual binding moieties that comprise a binding site specific for PD-1 (via its CDRs) and a binding site specific for an anti-CD40 (via its non-CDR loops) (e.g, SEQ ID No. 116) and dual binding moieties that comprise a binding site specific for PSMA (via its CDRs) and a binding site specific for an anti- CD40 (via its non-CDR loops) (e.g., SEQ ID No. 114).
  • Table 5 Exemplary Sequences for CD40 masks and CTLA4 masks.
  • the pro immune modulating molecules described herein are activated by cleavage of the cleavable linker that connects the dual binding moiety to the immune modulating antibody or an antigen binding fragment thereof.
  • the immune modulating antibody or an antigen binding fragment thereof is able to binds its target (referred to herein as the antibody target).
  • the antibody target is an immune modulatory protein.
  • immune modulatory proteins that are antibody targets include, but are not limited to CTLA4, CD27, CD28, CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, TIM-1, 0X40, DNAM-1, PD-L1, PD1, PD-L2, CD4, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDOl, ID02, TDO, KIR, LAG-3, TIM-3, VISTA, IL6-R, IL-6, TNFa, CD19, CD20, CD22, CD52, integrin a4, integrin a4b7, CD11, and any combinations thereof
  • a pro immune modulating molecule comprises an immune modulating antibody or an antigen binding fragment thereof that comprises a binding site specific for CD40 or CTLA4.
  • the immune modulating antibody or an antigen binding fragment thereof in some instances, is able to inhibit an antibody target, e.g ., the immune modulating antibody or an antigen binding fragment there is in some instances an anti-CTLA4 antibody which inhibits CTLA4 check point signaling.
  • the immune modulating antibody or an antigen binding fragment thereof in some instances, is able to act as an agonist of an antibody target, e.g. , the immune modulating antibody or an antigen binding fragment there is in some instances an anti-CD40 antibody which acts as an agonist of CD40.
  • CD40-CD40L pathway is a major regulating factor of immune responses. Briefly, CD40-CD40L pathway is believed to affect the immune system in the following exemplary ways: (1) CD40-CD40L pathway “activates” or “matures” antigen- presenting cells (APCs, mainly macrophages and dendritic cells) to express co-stimulatory molecules including B7 (CD80 and CD86, both ligands for CD28), ICAM-1 (CD54), and CD44. These co-stimulatory signals are needed for T cells to become fully activated, rather than anergic, after T cell receptor (TCR)-stimulation.
  • APCs antigen-presenting cells
  • CD40-CD40L pathway can induce macrophages and dendritic cells to make interleukin- 12 (IL-12), IL-18, and other cytokines.
  • IL-12 interleukin- 12
  • IL-18 interleukin- 12
  • CD40L /CD154/ TNFSF5 is the primary stimulus for IL-12 production (in the absence of microbial invasion).
  • IL-12 and IL-18 stimulate NK cells for interferon-g (IFN-g) production.
  • IFN-g interferon-g
  • IL-12 causes CD4+ T cells to differentiate into type 1 helper T cells (Thl) that mediate delayed-type hypersensitivity responses.
  • Thl type 1 helper T cells
  • TNFSF 5 -expressing CD4+ T cells are generally required for the generation of cytotoxic T lymphocytes (CTLs) against tumors and virus-infected cells.
  • CTLs cytotoxic T lymphocytes
  • CD40 & CD40L pathway can activate APCs to express the co-stimulatory molecules needed to fully activate or cross-prime CTLs already responding to antigen/MHC class I complexes.
  • CD40 & CD40L pathway may also promote the differentiation of activated B cells and, with few exceptions, is likely required for the class switch from IgM to IgG production. See, e.g. , Richard S. Kombluth etal. The emerging role of CD40 ligand in HIV infection. Journal of Leukocyte Biology. 2000; 68:373-382.
  • the antibody target is PD1.
  • the antibody target is CD40.
  • the antibody target is CTLA4.
  • the dual binding moiety target is EpCAM.
  • the dual binding moiety target is PSMA.
  • the dual binding moiety target is PD-1 or PD-L1.
  • the dual binding moiety recognizes a bulk serum protein or a dual binding moiety target (e.g., a tumor antigen) via its CDR loops and an immune modulating antibody or an antigen binding fragment thereof (e.g, an anti-CD40 antibody) via its non-CDR loop.
  • the dual binding moiety recognizes an immune modulatory protein via its CDR loops (e.g, PD1) and an immune modulating antibody or an antigen binding fragment thereof (e.g, an anti-CD40 antibody) via its non-CDR loop.
  • the antibody target is CTLA4 or CD40 and the dual binding moiety comprises a binding site specific for (i) albumin (via its CDR) and (ii) an anti-CTLA4 antibody or an anti-CD40 antibody (via its non-CDR loop).
  • the antibody target is TNFa and the dual binding moiety comprises a binding site specific for (i) albumin (via its CDR) and (ii) an anti-TNFa antibody (via its non-CDR loop).
  • pro immune modulating molecules achieve a global inhibition of an immune modulating protein when in the global configuration state as well as after activation by cleavage of the cleavable linker (e.g ., in cases where the antibody target is an immune modulatory protein, for instance CD40 and the dual binding moiety target is also an immune modulatory protein, same or different from the antibody target).
  • the global configuration state in some cases, is the state prior to activation where the pro immune modulating molecule is in circulation, the target binding domain being masked from binding its target.
  • the immune modulation e.g., immune agonist activity, is achieved in some cases both outside and inside tumor. In some cases, the global modulation of an immune modulatory protein is beneficial.
  • the pro immune modulating molecule Upon activation, the pro immune modulating molecule is able to locally inhibit an immune modulatory protein inside the tumor. Upon activation, the pro immune modulating molecule is able to locally cause an agonist effect with respect to an immune modulatory protein inside the tumor, such as, using an immune agonist antibody that targets proteins such as CD40, CD137. This, in some cases, expands the narrow therapeutic window for existing immune modulatory therapies.
  • the pro immune modulating molecules described herein comprise a clustering moiety that comprises a binding site specific for a protein that promotes higher order clustering of the antibody target (e.g, CD40, when the immune modulating antibody or an antigen binding fragment thereof is an anti-CD40 antibody).
  • a clustering moiety that comprises a binding site specific for a protein that promotes higher order clustering of the antibody target (e.g, CD40, when the immune modulating antibody or an antigen binding fragment thereof is an anti-CD40 antibody).
  • clustering effect is important to the activity of antibodies specific for the TNFR family members, such as CD40, death receptor 5 (DR5), and CD95 (Fas), in TNFR agonists such as anti-OX40 and anti-CD27. See, e.g., Stewart etal. Journal for ImmunoTherapy of Cancer 2014, 2:29.
  • the clustering moiety comprises a binding site specific for an antigen that is highly expressed on immune cells, e.g, FcgRIIb, FcgRIIA, FcgRI, FcgRIIIA, FcgRIIIB, FcgRIV, FceRI, FceRII, FcaRI, FcRn, MHC class I, MHC class II, CD40, 0X40, CD 137, CTLA4, PD1, PDL1, PDL2, B7-1, B7-2, CD19, CD20, CD22, CD30, CD33, CD79b, BCMA, CD70, CD71, LRRC33, GARP, or any combinations thereof.
  • an antigen that is highly expressed on immune cells e.g, FcgRIIb, FcgRIIA, FcgRI, FcgRIIIA, FcgRIIIB, FcgRIV, FceRI, FceRII, FcaRI, FcRn, MHC class I, MHC class II, CD40
  • the clustering moiety comprises a binding site specific for antigens that are highly expressed in cancer, in inflammation, or in fibrotic tissues, e.g., PSMA, MSLN, BCMA, DLL3, EpCAM, EGFR, EGFRv3, Her2, CD44, CD44v6, CAIX, Trop2, CD30, CD70, CD71, urokinase plasminogen activator, ICAM-1, an integrin, a claudin, a nectin, a cadherin, a tetraspanin, a receptor tyrosine kinase, a type II transmembrane serine protease, a membrane-type matrix metalloprotease, a GPI-anchored matrix metalloprotease, a ADAM family metalloprotease, or any combinations thereof.
  • a binding site specific for antigens that are highly expressed in cancer, in inflammation, or in fibrotic tissues, e.g., PSMA, MSLN, BCMA, DLL
  • the clustering moiety comprises an anti-FCgRIIb binding domain (e.g ., SEQ ID Nos. 117-120). In some embodiments, the clustering moiety comprises an anti-EpCAM binding domain (e.g., SEQ ID Nos. 121-124).
  • a masked biparatopic pro immune modulating molecule comprising a dual binding moiety as described herein, an immune modulating antibody or an antigen binding fragment thereof that recognizes an antibody target as described herein, and a clustering moiety that recognizes a different epitope with the same antibody target that is recognized by the immune modulating antibody or an antigen binding fragment thereof.
  • the immune modulating antibody or an antigen binding fragment is an anti-CD40 scFv that binds a first epitope within CD40 and the clustering moiety is also an anti-CD40 which recognizes a second epitope within CD40 (e.g, the first anti-CD40 scFv recognizes a first epitope within a molecule of CD40 and the second anti-CD40 scFv recognizes a second epitope within a different molecule of CD40).
  • Exemplary biparatopic pro immune modulating molecules comprise, in some embodiments, a sequence selected from the group consisting of SEQ ID Nos.
  • the biparatopic pro immune modulating molecules comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID Nos. 125-130.
  • a biparatopic pro immune modulating molecule is activated by cleavage of the cleavable linker that connects the dual binding moiety and the immune modulating antibody or an antigen binding fragment thereof.
  • the immune modulating antibody or an antigen binding fragment thereof comprises a binding site specific for an immune modulatory protein.
  • immune modulatory proteins that are antibody targets, include, but are not limited to CTLA4, CD27, CD28, CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, TIM-1, 0X40, DNAM-1, PD-L1, PD1, PD-L2, CD4, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDOl, ID02, TDO, KIR, LAG-3, TIM-3, VISTA, IL6-R, IL-6, TNFa, CD19, CD20, CD22, CD52, integrin a4, integrin a4b7, CD11, and any combinations thereof.
  • binding protein variants refers to variants and derivatives of the pro immune modulating molecules described herein.
  • amino acid sequence variants are contemplated.
  • amino acid sequence variants are contemplated to improve the binding affinity and/or other biological properties of the pro immune modulating molecules provided herein.
  • Exemplary method for preparing amino acid variants include, but are not limited to, introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody.
  • variants having one or more amino acid substitutions are provided.
  • Sites of interest for substitution mutagenesis include the CDRs and framework regions.
  • Amino acid substitutions may be introduced into the variable domains of the target-binding protein of interest and the products screened for a desired activity, e.g, retained/improved antigen binding, decreased immunogenicity, or improved antibody- dependent cell mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). Both conservative and non-conservative amino acid substitutions are contemplated for preparing the variants.
  • one or more hypervariable region residues of a parent antibody are substituted.
  • variants are then selected based on improvements in desired properties compared to a parent antibody, for example, increased affinity, reduced affinity, reduced immunogenicity, increased pH dependence of binding.
  • an affinity matured variant antibody can be generated, e.g, using phage display -based affinity maturation techniques such as those described herein and known in the field.
  • Substitutions can be made in hypervariable regions (HVR) of a parent molecule to generate variants and variants are then selected based on binding affinity, /. e. , by affinity maturation.
  • affinity maturation diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g, error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis).
  • a secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity.
  • Another method to introduce diversity involves HVR- directed approaches, in which several HVR residues (e.g, 4-6 residues at a time) are randomized.
  • a pro immune modulating molecule comprises a VL and a VH domain with amino acid sequences corresponding to the amino acid sequence of a naturally occurring VL or VH domain, respectively, but that has been “humanized,” i.e., by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VL or VH domains (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VL or VH domain from a conventional 4-chain antibody from a human being ( e.g as indicated above).
  • a pro immune modulating molecule as described herein comprises a VL and a VH domain with amino acid sequences corresponding to the amino acid sequence of a naturally occurring VL or VH domain, respectively, but that has been “camelized,” i.e., by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VL or VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VL or a VH domain of a heavy chain antibody.
  • the VH sequence that is used as a starting material or starting point for generating or designing the camelized single domain is preferably a VH sequence from a mammal, more preferably the VH sequence of a human being, such as a VH3 sequence.
  • camelized domains of the activatable binding proteins of the disclosure are obtained in any suitable manner known in the field and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VL and/or VH domain as a starting material.
  • both “humanization” and “camelization” is performed by providing a nucleotide sequence that encodes a naturally occurring VL and/or VH domain, respectively, and then changing, one or more codons in said nucleotide sequence in such a way that the new nucleotide sequence encodes a “humanized” or “camelized” binding protein, respectively.
  • a “humanized” or “camelized” binding protein is synthesized de novo using known peptide synthesis technique from the amino acid sequence of a naturally occurring antibody comprising a VL and/or VH domain.
  • a “humanized” or “camelized” binding protein is synthesized de novo using known peptide synthesis technique from the amino acid sequence or nucleotide sequence of a naturally occurring antibody comprising a VL and/or VH domain, respectively, a nucleotide sequence encoding the desired humanized or camelized binding protein of the disclosure, respectively, is designed and then synthesized de novo using known techniques for nucleic acid synthesis, after which the nucleic acid thus obtained is expressed in using known expression techniques, so as to provide the desired binding protein of the disclosure.
  • suitable methods and techniques for obtaining the pro immune modulating molecules of the disclosure and/or nucleic acids encoding the same starting from naturally occurring sequences for VL or VH domains for example comprises combining one or more parts of one or more naturally occurring VL or VH sequences (such as one or more framework (FR) sequences and/or complementarity determining region (CDR) sequences), and/or one or more synthetic or semi-synthetic sequences, and/or a naturally occurring sequence for a CH2 domain, and a naturally occurring sequence for a CH3 domain comprising amino acid substitutions that favor formation of heterodimer over homodimer, in a suitable manner, so as to provide a binding protein of the disclosure or a nucleotide sequence or nucleic acid encoding the same.
  • naturally occurring VL or VH sequences such as one or more framework (FR) sequences and/or complementarity determining region (CDR) sequences
  • CDR complementarity determining region
  • binding proteins for therapeutic applications, it is desirable to create proteins that, for example, modulate a functional activity of a target, and/or improved binding proteins such as binding proteins with higher specificity and/or affinity and/or and binding proteins that are more bioavailable, or stable or soluble in particular cellular or tissue environments.
  • the binding proteins described in the present disclosure exhibit improved the binding affinities towards a target, for example a immunomodulatory protein.
  • the binding proteins of the present disclosure is affinity matured to increase its binding affinity to an antibody target such as an immunomodulatory protein, using any known technique for affinity- maturation (e.g ., mutagenesis, chain shuffling, CDR amino acid substitution).
  • Amino acid substitutions may be conservative or semi-conservative.
  • the amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains).
  • amino acids which may often be substituted for one another include but are not limited to: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulphur-containing side chains).
  • the binding proteins are isolated by screening combinatorial libraries, for example, by generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics towards a target antigen, such as a tumor antigen expressed on a cell surface.
  • binding proteins described herein encompass derivatives or analogs in which (i) an amino acid is substituted with an amino acid residue that is not one encoded by the genetic code, (ii) the mature polypeptide is fused with another compound such as polyethylene glycol, or (iii) additional amino acids are fused to the protein, such as a leader or secretory sequence or a sequence to block an immunogenic domain and/or for purification of the protein.
  • Typical modifications include, but are not limited to, acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethyl ati on, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxyl ati on, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination
  • Modifications are made anywhere in the binding proteins described herein, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini.
  • Certain common peptide modifications that are useful for modification of the binding proteins include glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, and ADP-ribosylation.
  • the binding proteins of the disclosure are conjugated with drugs to form antibody-drug conjugates (ADCs).
  • ADCs are used in oncology applications, where the use of antibody-drug conjugates for the local delivery of cytotoxic or cytostatic agents allows for the targeted delivery of the drug moiety to tumors, which can allow higher efficacy, lower toxicity, etc.
  • polynucleotide molecules encoding a binding protein as described herein.
  • the polynucleotide molecules are provided as a DNA construct.
  • the polynucleotide molecules are provided as a messenger RNA transcript.
  • the polynucleotide molecules are constructed by known methods such as by combining the genes encoding the various domains of the binding proteins, operably linked to a suitable promoter, and optionally a suitable transcription terminator, and expressing it in bacteria or other appropriate expression system such as, for example CHO cells.
  • the polynucleotide is inserted into a vector, preferably an expression vector, which represents a further embodiment.
  • This recombinant vector can be constructed according to known methods.
  • Vectors of particular interest include plasmids, phagemids, phage derivatives, virii (e.g ., retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, and the like), and cosmids.
  • a variety of expression vector/host systems may be utilized to contain and express the polynucleotide encoding the polypeptide of the described binding proteins.
  • Examples of expression vectors for expression in E.coli are pSKK (Le Gall et ah, J Immunol Methods. (2004) 285(1): 111-27), pcDNA5 (Invitrogen) for expression in mammalian cells, PICHIAPINKTM
  • Yeast Expression Systems Invitrogen
  • BACUVANCETM Baculovirus Expression System GenScript
  • binding proteins as described herein are produced by introducing a vector encoding the protein as described above into a host cell and culturing said host cell under conditions whereby the protein domains are expressed, may be isolated and, optionally, further purified.
  • the process comprises culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding a binding protein under conditions allowing the expression of the binding protein and recovering and purifying the produced protein from the culture.
  • a plurality of single-substitution libraries is provided each corresponding to a different domain, or amino acid segment of a binding protein or reference binding compound such that each member of the single-substitution library encodes only a single amino acid change in its corresponding domain, or amino acid segment.
  • this allows all of the potential substitutions in a large protein or protein binding site to be probed with a few small libraries.
  • the plurality of domains forms or covers a contiguous sequence of amino acids of a binding protein or a reference binding compound. Nucleotide sequences of different single-substitution libraries overlap with the nucleotide sequences of at least one other single-substitution library. In some embodiments, a plurality of single-substitution libraries are designed so that every member overlaps every member of each single-substitution library encoding an adjacent domain.
  • Binding proteins expressed from such single-substitution libraries are separately selected to obtain a subset of variants in each library which has properties at least as good as those of the reference binding compound and whose resultant library is reduced in size.
  • the number of nucleic acids encoding the selected set of binding compounds is smaller than the number of nucleic acids encoding members of the original single-substitution library.
  • properties include, but are not limited to, affinity to a target compound, stability with respect to various conditions such as heat, high or low pH, enzymatic degradation, cross-reactivity to other proteins and the like.
  • pre-candidate compounds The selected compounds from each single-substitution library are referred to herein interchangeably as “pre-candidate compounds,” or “pre-candidate proteins.” Nucleic acid sequences encoding the pre-candidate compounds from the separate single-substitution libraries are then shuffled in a PCR to generate a shuffled library, using PCR-based gene shuffling techniques.
  • Libraries of pre candidate compounds are generated from single substitution libraries and selected for binding to the target protein(s), after which the pre-candidate libraries are shuffled to produce a library of nucleic acids encoding candidate compounds which, in turn, are cloned into a convenient expression vector, such as a phagemid expression system. Phage expressing candidate compounds then undergo one or more rounds of selection for improvements in desired properties, such as binding affinity to a target molecule.
  • Target molecules may be adsorbed or otherwise attached to a surface of a well or other reaction container, or target molecules may be derivatized with a binding moiety, such as biotin, which after incubation with candidate binding compounds may be captured with a complementary moiety, such as streptavidin, bound to beads, such as magnetic beads, for washing.
  • a binding moiety such as biotin
  • candidate binding compounds may be captured with a complementary moiety, such as streptavidin, bound to beads, such as magnetic beads, for washing.
  • the candidate binding compounds undergo a wash step so that only candidate compounds with very low dissociation rates from a target molecule are selected.
  • Exemplary wash times for such embodiments are about 10 minutes, about 15 minutes, about 20 minutes, about 20 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 mins, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours; or in other embodiments, about 24 hours; or in other embodiments, about 48 hours; or in other embodiments, about 72 hours.
  • Isolated clones after selection are amplified and subjected to an additional cycle of selection or analyzed, for example by sequencing and by making comparative measurements of binding affinity towards their target, for example, by ELISA, surface plasmon resonance (SPR), bio-layer interferometry (e.g, OCTET® system, Pall Life Sciences, ForteBio, Menlo Park, CA) or the like.
  • SPR surface plasmon resonance
  • bio-layer interferometry e.g, OCTET® system, Pall Life Sciences, ForteBio, Menlo Park, CA
  • compositions comprising a binding protein described herein, a vector comprising the polynucleotide encoding the polypeptide of a binding protein or a host cell transformed by this vector and at least one pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
  • compositions are sterile.
  • compositions may also contain excipients such as preservative, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents.
  • a binding protein is encapsulated in nanoparticles.
  • the nanoparticles are fullerenes, liquid crystals, liposome, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods.
  • the pro immune modulating molecule is attached to liposomes.
  • a binding protein is conjugated to the surface of liposomes.
  • a pro immune modulating molecule is encapsulated within the shell of a liposome.
  • the liposome is a cationic liposome.
  • the binding proteins described herein are contemplated for use as a medicament.
  • Administration is effected by different ways, e.g, by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the route of administration depends on the kind of therapy and the kind of compound contained in the pharmaceutical composition.
  • the dosage regimen will be determined by the attending physician and other clinical factors. Dosages for any one patient depends on many factors, including the patient's size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind of therapy, general health and other drugs being administered concurrently.
  • An “effective dose” refers to amounts of the active ingredient that are sufficient to affect the course and the severity of the disease, leading to the reduction or remission of such pathology and may be determined using known methods.
  • a binding protein as described herein.
  • the administration of a binding protein induces and/or sustains cytotoxicity towards a cell expressing a target antigen.
  • the cell expressing a target antigen is a cancer or tumor cell, a virally infected cell, a bacterially infected cell, an autoreactive T or B cell, damaged red blood cells, arterial plaques, or inflamed or fibrotic tissue cell.
  • the target antigen is an immune modulatory protein.
  • Diseases, disorders or conditions associated with a target antigen include, but are not limited to, viral infection, bacterial infection, auto-immune disease, transplant rejection, atherosclerosis, or fibrosis.
  • the viral infection is hepatitis.
  • the disease, disorder or condition associated with a target antigen is a proliferative disease, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus-host disease or a host-versus-graft disease.
  • the disease, disorder or condition associated with a target antigen is cancer.
  • the cancer is a hematological cancer.
  • the cancer is a melanoma.
  • the cancer is non-small cell lung cancer.
  • the cancer is breast cancer.
  • a binding protein binds to an immune modulatory protein and is administered to treat a cancer characterized by upregulation of said immune modulatory protein.
  • the immune modulatory protein is, in some cases, CTLA-4 and the cancer is melanoma, non-small cell lung cancer, triple negative breast cancer, or ovarian cancer.
  • treatment or “treating” or “treated” refers to therapeutic treatment wherein the object is to slow (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (z.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease.
  • Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.
  • “treatment” or “treating” or “treated” refers to prophylactic measures, wherein the object is to delay onset of or reduce severity of an undesired physiological condition, disorder or disease, such as, for example is a person who is predisposed to a disease ( e.g ., an individual who carries a genetic marker for a disease such as breast cancer).
  • binding proteins described herein are administered in combination with an agent for treatment of the particular disease, disorder or condition.
  • Agents include but are not limited to, therapies involving antibodies, small molecules (e.g., chemotherapeutics), hormones (steroidal, peptide, and the like), radiotherapies (g-rays, X- rays, and/or the directed delivery of radioisotopes, microwaves, UV radiation and the like), gene therapies (e.g, antisense, retroviral therapy and the like) and other immunotherapies.
  • the pro immune modulating molecules described herein are administered in combination with anti-diarrheal agents, anti-emetic agents, analgesics, opioids and/or non steroidal anti-inflammatory agents. In some embodiments, a pro immune modulating molecule described herein are administered before, during, or after surgery.
  • kits for detecting a cancer and one or more compounds for detecting the label.
  • the label is selected from the group consisting of a fluorescent label, an enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.
  • Example 1 Treatment with an exemplary pro immune modulating molecule of the present disclosure inhibits in vivo tumor growth
  • Murine tumor line CT26 is implanted subcutaneously in Balb/c mice and on day 7 post implantation the average size of the tumor is measured.
  • Mice are treated with an exemplary CD40 binding antibody according to the present disclosure, or a control CD40 binding antibody which is not in a pro immune modulating molecule format as described herein. Results show that treatment with the exemplary CD40 binding antibody according to the present disclosure inhibits tumor growth to a greater extent than the control antibody.
  • Example 2 Potency of Exemplary anti-Alb/anti-CD40/anti-FcgRIIB Proteins and anti- Alb/anti-CD40 Biparatopic Proteins in a B Cell Activation Assay
  • the exemplary anti-CD40 pro immune modulating molecules were tested for their ability to activate human B cells.
  • the constructs were expressed in EXPI293TM cells and quantified using Protein A biolayer interferometry. To assess the ability of the proteins to activate B cells, the proteins were cultured with purified human B cells for 48 hours. To measure activation, the percentage of B cells positive for expression of CD86, CD80, and CD69 was measured using flow cytometry and the results are shown in Fig. 3 and summarized in Table 6.
  • Example 3 Potency of Exemplary anti-Alb/anti-CD40/anti-EpCAM Proteins in a B Cell Activation Assay in the Presence of EpCAM Expressing H508 Cells
  • the exemplary anti-CD40 proteins were tested for their ability to activate human B cells.
  • the constructs were expressed in EXPI293TM cells and quantified using Protein A biolayer interferometry.
  • the proteins were cultured with purified human B cells and EpCAM expressing NCI-H508 (ATCC® CCL-253TM) at a 10: 1 ratio for 48 hours.
  • EpCAM expressing NCI-H508 ATCC® CCL-253TM
  • the percentage of B cells positive for expression of CD86, CD80, and CD69 was measured using flow cytometry and the results are shown in Fig. 4 and summarized in Table 7.
  • Table 7 B Cell Activation of Exemplary anti-CD40/anti-EpCAM Protein
  • Example 4 Masking and Protease Activation of Exemplary anti-Alb/anti-CD40 Biparatopic Proteins in a B Cell Activation Assay

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Abstract

L'invention concerne une molécule pro-immunomodulatrice, comprenant une molécule pro-immunomodulatrice biparatoptique, comprenant des formats non Fc, et un procédé d'utilisation de celle-ci. Un mode de réalisation concerne une molécule pro-immunomodulatrice comprenant : une fraction de liaison double Comprenant un lieur clivable, une boucle non CDR, et un site de liaison spécifique d'une protéine sérique en vrac; un anticorps agoniste immun ou un fragment de liaison à l'antigène de celui-ci qui comprend un site de liaison spécifique pour un épitope à l'intérieur d'une cible d'anticorps; et une fraction de groupement qui comprend un site de liaison spécifique pour une protéine qui favorise un groupement d'ordre supérieur de la cible d'anticorps.
PCT/US2020/060184 2019-11-13 2020-11-12 Molécule pro-immunomodulatrice comprenant une fraction de groupement WO2021097060A1 (fr)

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US11535668B2 (en) 2017-02-28 2022-12-27 Harpoon Therapeutics, Inc. Inducible monovalent antigen binding protein
US11607453B2 (en) 2017-05-12 2023-03-21 Harpoon Therapeutics, Inc. Mesothelin binding proteins
US11623958B2 (en) 2016-05-20 2023-04-11 Harpoon Therapeutics, Inc. Single chain variable fragment CD3 binding proteins
US11807692B2 (en) 2018-09-25 2023-11-07 Harpoon Therapeutics, Inc. DLL3 binding proteins and methods of use
US11976125B2 (en) 2017-10-13 2024-05-07 Harpoon Therapeutics, Inc. B cell maturation antigen binding proteins

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WO2020061526A1 (fr) * 2018-09-21 2020-03-26 Harpoon Therapeutics, Inc. Molécules de liaison à une cible activées de manière conditionnelle

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US20180134789A1 (en) * 2016-03-08 2018-05-17 Maverick Therapeutics, Inc. Inducible binding proteins and methods of use
WO2018160671A1 (fr) * 2017-02-28 2018-09-07 Harpoon Therapeutics, Inc. Inhibiteurs de point de contrôle ciblés et méthodes d'utilisation
WO2019222283A1 (fr) * 2018-05-14 2019-11-21 Harpoon Therapeutics, Inc. Fraction de liaison pour l'activation conditionnelle de molécules d'immunoglobuline
WO2019222282A1 (fr) * 2018-05-14 2019-11-21 Harpoon Therapeutics, Inc. Protéine de liaison activée de manière conditionnelle comprenant un domaine de liaison cible à occlusion stérique
WO2020061526A1 (fr) * 2018-09-21 2020-03-26 Harpoon Therapeutics, Inc. Molécules de liaison à une cible activées de manière conditionnelle

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11453716B2 (en) 2016-05-20 2022-09-27 Harpoon Therapeutics, Inc. Single domain serum albumin binding protein
US11623958B2 (en) 2016-05-20 2023-04-11 Harpoon Therapeutics, Inc. Single chain variable fragment CD3 binding proteins
US11535668B2 (en) 2017-02-28 2022-12-27 Harpoon Therapeutics, Inc. Inducible monovalent antigen binding protein
US11607453B2 (en) 2017-05-12 2023-03-21 Harpoon Therapeutics, Inc. Mesothelin binding proteins
US11976125B2 (en) 2017-10-13 2024-05-07 Harpoon Therapeutics, Inc. B cell maturation antigen binding proteins
US11807692B2 (en) 2018-09-25 2023-11-07 Harpoon Therapeutics, Inc. DLL3 binding proteins and methods of use

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