WO2024040150A1 - Agents de liaison ciblant gd2 et leur utilisation pour le traitement du cancer - Google Patents

Agents de liaison ciblant gd2 et leur utilisation pour le traitement du cancer Download PDF

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Publication number
WO2024040150A1
WO2024040150A1 PCT/US2023/072361 US2023072361W WO2024040150A1 WO 2024040150 A1 WO2024040150 A1 WO 2024040150A1 US 2023072361 W US2023072361 W US 2023072361W WO 2024040150 A1 WO2024040150 A1 WO 2024040150A1
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Prior art keywords
binding agent
domain
cancer
seq
antibody
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PCT/US2023/072361
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English (en)
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David Cheresh
Christoph Rader
Hiromi WETTERSTEN
Sara WEIS
Stephen Mccormack
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Alpha Beta Therapeutics, Inc.
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Publication of WO2024040150A1 publication Critical patent/WO2024040150A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • 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
    • C07K16/2839—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 integrin superfamily
    • C07K16/2848—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 integrin superfamily against integrin beta3-subunit-containing molecules, e.g. CD41, CD51, CD61
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3084—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated gangliosides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • the present invention relates to binding agents and compositions comprising the same.
  • the invention further relates to polynucleotides encoding the binding agent and vectors and host cells comprising the same.
  • the invention further relates to methods of using the binding agents to promote macrophage mediated antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis of cancer cells and methods of treating cancers.
  • Antibodies are proteins that bind to a specific antigen.
  • Monoclonal antibodies (mAbs) and mAb-based reagents approved for cancer therapy include several that are directed against antigens expressed on malignant B cells and plasma cells (CD19, CD20, CD22, CD30, CD38, CD52, CD79B, SLAMF7), epithelial cancer cells (EpCAM, EGFR, HER2, VEGFR2, nectin- 4), acute myeloid leukemia (CD33), cutaneous T-cell lymphoma (CCR4), neuroblastoma (GD2), and sarcoma (PDGFRA), as well as immune checkpoint targets (PD-1, PD-L1, CTLA- 4) (Gasser, 2016; Carter, 2018).
  • a total of 42 antibody -based cancer therapies are currently FDA-approved and marketed.
  • the efficacy of a therapeutic antibody for cancer can be influenced by a combination of mechanisms (Chiavenna, 2017).
  • Antibody binding to an antigen selectively expressed on a cancer cell may produce anti-tumor effects by directly blocking the function of the antigen that promotes tumor cell growth or survival pathways.
  • An antibody can also act as a bridge to bring together a tumor cell with an immune effector cell that can indirectly induce tumor cell destruction.
  • therapeutic antibodies can be modified to either enhance or suppress engagement with certain types of immune effector cells using a growing arsenal of glycoengineering and Fc engineering approaches or through the creation of bispecific or trispecific antibodies (Saxena, 2016; Rader, 2020). These tools can be utilized for the rational design of “antigen-effector matching” to create a personalized medicine approach for cancer therapy.
  • Antibody engineering strategies focused on improving the engagement of monocytes or natural killer (NK) cells include a vast collection of glycoengineered and Fc engineered variants that promote binding of the Fc portion of a therapeutic antibody to FcyRIIIA (CD16A), the only Fc receptor expressed on NK cells (Lazar, 2006). Although less common, several strategies have generated antibody variants with enhanced binding to macrophages, including a G236A Fc mutant that promotes binding to FcyRIIA (CD32A) (Richards, 2008) or a bispecific antibody that recruits macrophages via FcaRI (CD89) (Li, 2017). In contrast, it is desirable to engineer antibodies that do not bind Fc gamma receptors at all to avoid unwanted inflammatory responses to therapeutic antibodies and fusion proteins (Wilkinson, 2021).
  • Therapeutic antibodies can influence cancer progression through several mechanisms of action, including blocking the function of a target antigen or inhibiting ligand/receptor interactions.
  • antibodies can also activate immune cells to induce tumor cell killing or engulfment, known as antibody-dependent cellular cytotoxicity (ADCC) or antibodydependent cellular phagocytosis (ADCP), respectively.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibodydependent cellular phagocytosis
  • compositions and methods of using such compositions, to treat cancers, including in particular neuroblastoma and other GD2-expressing cancers.
  • the present invention provides compositions and methods for engaging the tumor associated macrophage as the appropriate immune effector cell to effectively mediate one or both of two distinct killing mechanisms; antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) against cancer cells that express the cell surface marker GD2 (e.g., neuroblastoma, breast cancer, melanoma, small cell lung cancer).
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • ADCC antibody-dependent cellular phagocytosis
  • the present invention is based on the development of binding agents that can mediate ADCC by selectively engaging myeloid-derived cells found in tumors and targeting them to the antigen GD-2 on the surface of expressing cancer cells (e.g., an IgG4 domain to engage macrophage CD64/FcyRl on macrophages and GD2 recognition).
  • binding agents that can mediate ADCC by selectively engaging myeloid-derived cells found in tumors and targeting them to the antigen GD-2 on the surface of expressing cancer cells (e.g., an IgG4 domain to engage macrophage CD64/FcyRl on macrophages and GD2 recognition).
  • a binding agent that promotes ADCC exclusively will facilitate some level of tumor cell killing but will not induce ADCP. To achieve optimal tumor killing a single binding agent would ideally mediate both ADCC and ADCP. However, a binding agent that promotes ADCP will not be able to engulf or kill CD47-positive tumor cells that emit the “don’t eat me” signal that blocks phagocytosis by macrophages.
  • a binding agent structure capable of ADCC-inducing effects e.g., an IgG4 domain to engage macrophage CD64/FcyRl on macrophages and GD2 recognition
  • a CD47 blocking agent capable of ADCP-inducing effects more efficient cancer cell killing may be achieved, including CD47-positive tumor cells.
  • the preferential binding of CD64/FcyRl relative to CD 16 and CD32 on the macrophage facilitates maximal effector cell function to optimize macrophage-dependent killing.
  • a binding agent that targets GD2 and CD47 on a tumor cell while simultaneously binding to CD64/FcyRl preferentially over CD 16 and CD32 on the macrophage will promote the most complete anti-tumor activity of GD2-expressing tumors.
  • one aspect of the invention relates to a binding agent e.g., an antibody fusion protein) comprising a first domain that specifically binds to GD2and a second domain that preferentially binds CD64/FcyRl on macrophages, wherein the binding agent mediates antibody-directed cellular cytotoxicity (ADCC) by selectively the macrophage that accumulates in mesenchymal tumors, and compositions or pharmaceutical compositions comprising the binding agents.
  • the binding agent further comprises a third domain that is a CD47 blocking agent, wherein the binding agent mediates ADCC and antibody-dependent cellular phagocytosis (ADCP).
  • Another aspect of the invention relates to a polynucleotide encoding the binding agent of the invention and vectors and host cells comprising the polynucleotide.
  • An additional aspect of the invention relates to a method of killing a cancer cell expressing GD2 or expressing GD2 and CD47, comprising contacting the cell with an effective amount of the binding agent of the invention.
  • a further aspect of the invention relates to a method of targeting a macrophage to a cancer cell expressing GD2 or expressing GD2 and CD47, comprising contacting the cancer cell and the macrophage with an effective amount of the binding agent of the invention.
  • An additional aspect of the invention relates to a method of inducing macrophagedependent antibody-dependent cellular cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP) against a cancer expressing GD2 or expressing GD2 and CD47 in a subject in need thereof, comprising administering a therapeutically effective amount of the binding agent or the pharmaceutical composition of the invention to the subject, thereby inducing macrophage-dependent ADCC and/or ADCP.
  • ADCC macrophagedependent antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • a further aspect of the invention relates to a method of treating a cancer expressing GD2 or expressing GD2 and CD47 in a subject in need thereof, comprising administering a therapeutically effective amount of the binding agent or the pharmaceutical composition of the invention to the subject, wherein the administering induces ADCC and/or ADCP of the cancer, thereby treating the cancer.
  • Another aspect of the invention relates to a method of treating a cancer in a subject in need thereof, comprising the steps of: a) selecting a subject having cancer cells that are enriched for GD2 or GD2 and CD47 and enriched for macrophages; and b) administering a therapeutically effective amount of the binding agent or the pharmaceutical composition of the invention to the subject, wherein the administering induces ADCC and/or ADCP of the cancer, thereby treating the cancer.
  • Figure 1 shows that, among immune effector cells that are known to induce ADCC/ADCP, macrophage markers were most enriched in neuroblastoma patient tissues. Immune cell markers were compared in neuroblastoma patient TCGA data. *P ⁇ 0.05 compared to macrophage using TTEST. Error bars indicate standard errors. NK, natural killer; DC, dendritic cell.
  • Figure 2 shows h!gG4-Anti-GD2 maintains affinity for GD2 on neuroblastoma cells.
  • SK-N-AS neuroblastoma cells were stained with Anti-GD2 (dinutuximab) or h!gG4-Anti- GD2, and flow cytometry was performed. Dotted line, secondary antibody only; solid line, primary + secondary antibodies.
  • Figure 3 shows neuroblastoma cells highly express CD47. Levels of CD47 were analyzed in SHEP neuroblastoma cells using flow cytometry. Dotted line, isotype control; solid line, anti-CD47.
  • FIG. 4 shows h!gG4 anti-GD2 antibody induces macrophage-mediated neuroblastoma cell elimination.
  • SK-N-AS nerveroblastoma, luciferase+, GD2+
  • SK-N-AS neurotrophic factor-associated neuroblastoma cell elimination
  • Figure 5 shows anti-GD2 antibody (dinutuximab) effectively kills target cells through an NK-cell mediated mechanism but not h!gG4-Anti-GD2.
  • Target cells SHEP-luc, GD2+, luciferase+
  • Percent antibody-dependent target cell death was analyzed using a luminometer. Error bar indicates standard deviation for technical replicates. *P ⁇ 0.05 vs control.
  • FIG. 6 shows h!gG4 anti-GD2 antibody induces human macrophage-mediated ADCC.
  • Target cells SHEP-luc, GD2+, luciferase+
  • Percent antibody-dependent target cell death was analyzed using a luminometer. Error bar indicates standard deviation for technical replicates. *P ⁇ 0.05 vs control.
  • FIG. 7 shows h!gG4 anti-GD2 antibody induces mouse macrophage-mediated ADCC.
  • Target cells SHEP-luc, GD2+, luciferase+
  • HEP-luc, GD2+, luciferase+ were incubated with the indicated antibody at the indicated concentration with human macrophages at the E:T ratios of 10: 1 for 24 hr.
  • Percent antibody-dependent target cell death was analyzed using a luminometer. Error bar indicates standard deviation for technical replicates. *P ⁇ 0.05 vs control.
  • Figure 8 shows h!gG4 anti-GD2 antibody kills HCC1395 and Hs578T human triple negative breast cancer cells to a greater extent than the anti-GD2 antibody dinutuximab.
  • the term “about,” as used herein when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of the specified amount.
  • ranges can be expressed as from “about” one particular value, and/or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
  • the term “consists essentially of’ (and grammatical variants), as applied to a polynucleotide or polypeptide sequence of this invention, means a polynucleotide or polypeptide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids on the 5’ and/or 3’ or N-terminal and/or C-terminal ends of the recited sequence or between the two ends (e.g., between domains) such that the function of the polynucleotide or polypeptide is not materially altered.
  • the total of ten or less additional nucleotides or amino acids includes the total number of additional nucleotides or amino acids added together.
  • polypeptide encompasses both peptides and proteins, unless indicated otherwise.
  • chimeric refers to a molecule having two or more portions that are not naturally found together in the same molecule.
  • nucleic acid or “nucleotide sequence” or “polynucleotide” is a sequence of nucleotide bases, and may be RNA, DNA or DNA-RNA hybrid sequences (including both naturally occurring and non-naturally occurring nucleotide) but is preferably either single or double stranded DNA sequences.
  • isolated means a molecule, e.g., a protein, polynucleotide, or cell, separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell structural components or other polypeptides or nucleic acids commonly found associated with the molecule.
  • the term also encompasses molecules that have been prepared synthetically.
  • treat By the terms “treat,” “treating,” or “treatment of’ (or grammatically equivalent terms) it is meant that the severity of the subject's condition is reduced or at least partially improved or ameliorated and/or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and/or there is a delay in the progression of the condition.
  • the terms “prevent,” “prevents,” or “prevention” and “inhibit,” “inhibits,” or “inhibition” are not meant to imply complete abolition of disease and encompasses any type of prophylactic treatment that reduces the incidence of the condition, delays the onset of the condition, and/or reduces the symptoms associated with the condition after onset.
  • an “effective,” “ prophy lactically effective,” or “therapeutically effective” amount as used herein is an amount that is sufficient to provide some improvement or benefit to the subject.
  • an “effective,” “prophylactically effective,” or “therapeutically effective” amount is an amount that will provide some delay, alleviation, mitigation, or decrease in at least one clinical symptom in the subject.
  • the term “bind specifically” or “specifically binds” in reference to a binding agent of the invention means that the agent will bind with an epitope (including one or more epitopes) of a target, but does not substantially bind to other unrelated epitopes or molecules.
  • the term refers to an agent that exhibits at least about 60% binding, e.g., at least about 70%, 80%, 90%, or 95% binding, to the target epitope relative to binding to other unrelated epitopes or molecules.
  • a first aspect of the invention relates to a binding agent comprising a first domain that specifically binds to GD2and a second domain that preferentially binds CD64 relative to CD 16 and CD32 on macrophages, wherein the binding agent mediates antibody-directed cellular cytotoxicity (ADCC) by engaging a macrophage that accumulates in tumors.
  • the binding agent further comprises a third domain that is a CD47 blocking agent, wherein the binding agent mediates ADCC and antibody-dependent cellular phagocytosis (ADCP).
  • the binding agent may be any structure that is capable of binding to an antigen on a cancer cell and engaging a myeloid-derived cell to mediate ADCC and/or ADCP.
  • the binding agent is an antibody or an antigen-binding fragment thereof.
  • one or more portions of the binding agent are composed of antibody fragments.
  • one or more domains of the binding agent is a non-immunoglobulin scaffold, an aptamer, a small molecule (e.g., a receptor ligand), or other binding moiety.
  • the binding agent is a fusion protein in which the first and second domain are fused, the second and third domain are fused, or the first second, and third domain are fused.
  • the domains may be fused together directly or through a linker.
  • the linker may be 1 to about 20 amino acid residues, e.g., composed of glycine and serine residues, e.g., (GGGGS)n, wherein n is 1-5.
  • the first domain of the binding agent is an antibody domain.
  • the second domain of the binding agent is an antibody domain.
  • the third domain of the binding agent is an antibody domain.
  • the first and third domains are antibody domains.
  • all of the domains are antibody domains.
  • the first domain is a humanized or human antibody domain.
  • the second domain is a humanized or human antibody domain.
  • the third domain is a humanized or human antibody domain.
  • the first domain and the third domain are humanized or human antibody domains. In some embodiments, all of the domains are humanized or human antibody domains.
  • the first domain comprises, consists essentially of, or consists of a Fab domain of an antibody.
  • the Fab domain may be from any antibody isotype.
  • the first domain comprises a Fab domain of an IgG antibody, e.g., an IgGl or IgG4 antibody.
  • the first domain comprises the Fab domain of dinutuximab or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.
  • the heavy and light chain sequences of dinutuximab are disclosed in SEQ ID NOS: 1 and 2, respectively.
  • the second domain of the binding agent preferably engages one or more types of myeloid-derived cells.
  • the second domain predominately engages one type of myeloid-derived cells, e.g., macrophages or dendritic cells or granulocytes, such as a neutrophils, basophils, eosinophils, or mast cells.
  • the second domain predominately engages macrophages. “Predominantly engage,” as used herein, refers to engaging at least 80% of the target cell type, e.g., macrophages, relative to other cell types, e.g., at least 85%, 90%, or 95%.
  • the second domain does not significantly engage natural killer (NK) cells.
  • the second domain does not significantly engage one or more types of lymphocytes, e.g., NK cells, B cells, or T cells.
  • NK cells natural killer cells
  • T cells T cells.
  • Does not significantly engage refers to less than 30% of the total engaged cells being the indicated cell type, e.g., less than 25%, 20%, 15%, 10%, or 5%.
  • the second domain specifically binds a protein on the surface of the myeloid-derived cell.
  • the protein is one that can mediate ADCC when engaged.
  • the protein is not present or only present at low levels on other cell types, e.g., natural killer cells.
  • the second domain specifically binds to an Fc- gamma receptor.
  • the second domain specifically binds Fc-gamma receptor I (FcyRI, CD64) or preferentially binds CD64 relative to CD16 and CD32.
  • the term “preferentially binds”, as used herein, refers to a binding affinity of the second domain to CD64 that is at least 5-fold higher than the binding affinity to CD 16 and CD32, e.g., at least 10-fold, 50-fold, or 100-fold higher.
  • the second domain comprises, consists essentially of, or consists of an Fc domain of an antibody.
  • the Fc domain may be from any antibody isotype.
  • the second domain comprises an Fc domain of an IgG antibody, e.g., an IgG4 antibody.
  • the second domain comprises an Fc domain of an IgA or IgE antibody.
  • the second domain further comprises a hinge domain of an antibody.
  • the second domain comprises an antibody or an antigen-binding fragment thereof that specifically binds CD64.
  • the third domain of the binding agent is a CD47 blocking agent, i.e., an agent that inhibits the “don’t eat me” signal of CD47.
  • the third domain specifically binds CD47.
  • the third domain is an antibody or an antibody domain that specifically binds CD47.
  • CD47-blocking monoclonal antibodies are known in the art (e.g., Hu5F9-G4, CC-90002, Ti-061, or SRF231).
  • the third domain is a CD47 ligand or a fragment or derivative thereof that specifically binds CD47 and inhibits CD47 signaling.
  • the CD47 ligand is SIRPa or a fragment or derivative thereof.
  • the SIRPa fragments or derivatives may comprise the amino acid sequence of a wild-type SIRPa protein or a modified amino acid sequence. Numerous examples of inhibitory SIRPa fragments or derivatives are known in the art.
  • the SIRPa or a fragment or derivative thereof has the same affinity to CD47 as a wild-type SIRPa protein, increased affinity, or decreased affinity. Examples include, without limitation, the N-terminal Ig-like domain (e.g., residues 1-118) of fragments or derivatives thereof. See, e.g., Patent Nos.
  • the SIRPa fragment comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 19 (the wild-type N-terminal Ig-like domain) or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.
  • the SIRPa derivative comprises the amino acid sequence of SEQ ID NO:21 (a truncated fragment of the N-terminal Ig-like domain) or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.
  • the SIRPa derivative comprises the amino acid sequence of SEQ ID NO:20 (the N-terminal Ig-like domain in which the three glutamic acid residues at the N-terminus have been substituted with 4 aspartic acid residues) or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.
  • the SIRPa derivative comprises a transglutaminase conjugation site (LLQG; SEQ ID NO:27). These sites can be used to make conjugates of the binding agent, e.g., antibody-drug conjugates (see, e.g., Strop, 2013).
  • the SIRPa derivative comprises the amino acid sequence of one of SEQ ID NO:22 (transglutaminase conjugation site in one of the first loops), SEQ ID NO:23 (transglutaminase conjugation site in one of the first loops), SEQ ID NO:24 (transglutaminase conjugation site in the EF loop to replace the sequence SNITPADA (SEQ ID NO:28)), or a sequence at least 90% identical to one of SEQ ID NOS: 22-24, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.
  • the SIRPa derivative comprises mutations to alter one or more of the salt bridges that from during SIRPa-CD47 interaction. By weakening the binding between the two proteins, these mutations may permit SIRPa-CD47 interaction only when the SIRPa is brought into the proximity of CD47 by the binding of the binding agent.
  • the mutation is R69A and/or D100A.
  • the SIRPa derivative comprises the amino acid sequence of one of SEQ ID NO:25 (R69A mutation) or SEQ ID NO:26 (D100A mutation), or a sequence at least 90% identical to one of SEQ ID NOS: 25-26, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.
  • the SIRPa derivative may comprise any combination of the described modifications, e.g., truncation and/or transglutaminase conjugation site(s) and/or salt bridge mutation(s).
  • One or more of the CD47 blocking agents may be included in the binding agent, e.g., 2, 3, 4, or more.
  • the one or more CD47 blocking agents may be linked to each other to form a chain.
  • Each CD47 blocking agent may be the same or different from other CD47 blocking agents that are part of the binding agent.
  • the CD47 blocking agent(s) may be linked to the first domain, the second domain, or both.
  • the CD47 blocking agent may be linked to the light chain fragment, the heavy chain fragment, or both of the first domain and/or the second domain.
  • the one or more CD47 blocking agents may be linked to each other and/or to the first and/or second domains by a linker.
  • the linker may be 1 to about 20 amino acid residues, e.g., composed of glycine and serine residues, e.g., (GGGGS)n, wherein n is 1-5.
  • the binding agent comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:3 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.
  • the binding agent comprises a light chain comprising the amino acid sequence of SEQ ID NO:4 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.
  • the binding agent comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:3 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto, and a light chain comprising the amino acid sequence of SEQ ID NO:4 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.
  • the binding agent comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO:5 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto, and a light chain comprising the amino acid sequence of SEQ ID NO:6 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto; a heavy chain comprising the amino acid sequence of SEQ ID NO:7 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto, and a light chain comprising the amino acid sequence of SEQ ID NO:8 or a sequence at least 90% identical thereto, and a light chain compris
  • the binding agent of the invention may be covalently or non- covalently linked to one or more other binding agents that are the same or different.
  • the binding agent of the invention may be linked to a binding agent that targets avP3 (e.g., a binding agent as described in WO 2021/216956).
  • the two linked binding agents comprise the amino acid sequences of SEQ ID NOS:29-32 or SEQ ID NOS:33-36 or a sequence at least 90% identical thereto, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.
  • the binding agent may include sequence modifications that are known to enhance the characteristics of an antibody, e.g., stability, or alter the binding of the antibody to Fc-gamma receptors.
  • the amino acid sequence of the binding agent comprises a S228P (Eu numbering system) mutation in the hinge region.
  • the amino acid sequence comprises a mutation selected from: a) S239D/A330L/I332E; b) I332E; c) G236A/S239D/I332E; d) G236A; e) N297A/E382V/M428I; f) M252Y/S254T/T256E; g) Q295R/L328 W/A330 V/P331 A/I332 Y/E382 V/M428I; h) L234A/L235A/P329G; i) M428L/N434S; j) L234A/L235A/P331S; k) L234A/L235A/P329G/M252Y/S254T/T256E; l) S298A/E333A/K334/A; m) S239D/I332E; n) G236A;
  • antibody refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE.
  • the antibody can be monoclonal, oligoclonal, or polyclonal and can be of any species of origin, including (for example) mouse, rat, hamster, rabbit, horse, cow, goat, sheep, pig, camel, monkey, or human, or can be a chimeric or humanized antibody. See, e.g., Walker et al., Molec. Immunol. 26:403 (1989).
  • the antibodies can be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Pat. No. 4,474,893 or U.S. Pat. No. 4,816,567.
  • the antibodies can also be chemically constructed according to the method disclosed in U.S. Pat. No. 4,676,980.
  • Antibody fragments included within the scope of the present invention include, for example, Fab, Fab', F(ab)2, and Fv fragments; domain antibodies, diabodies; vaccibodies, linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Such fragments can be produced by known techniques.
  • F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule, and Fab fragments can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al., Science
  • antibody fragment may also include any protein construct that is capable of binding a target antigen.
  • Antibodies of the invention may be altered or mutated for compatibility with species other than the species in which the antibody was produced.
  • antibodies may be humanized or camelized.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • CDR complementarity determining region
  • donor antibody non-human species
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions (i.e., the sequences between the CDR regions) are those of a human immunoglobulin consensus sequence.
  • the humanized antibody can be a superhumanized antibody where only two CDRs are non-human (US Patent No. 7,087,409).
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature 321 :522 (1986); Riechmann et al., Nature, 332:323 (1988); and Presta, Curr. Op. Struct. Biol. 2:593 (1992)).
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can essentially be performed following the method of Winter and co-workers (Jones et al., Nature 321 :522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239: 1534 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues (e.g., all of the CDRs or a portion thereof) and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)).
  • the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol. 147:86 (1991)).
  • human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
  • Immunogens are used to produce antibodies specifically reactive with target polypeptides.
  • Recombinant or synthetic polypeptides and peptides e.g., of at least 5 (e.g., at least 7 or 10) amino acids in length, or greater, are the preferred immunogens for the production of monoclonal or polyclonal antibodies.
  • an immunogenic polypeptide conjugate is also included as an immunogen.
  • the peptides are used either in pure, partially pure, or impure form. Suitable polypeptides and epitopes for target pathogens and sperm are well known in the art.
  • Polynucleotide and polypeptide sequences are available in public sequence databases such as GENBANK®/GENPEPT®. Large numbers of antibodies that specifically bind to target cancer cell antigens have been described in the art and can be used as starting material to prepare the antibodies of the present invention. Alternatively, new antibodies can be raised against target antigens using the techniques described herein and well known in the art.
  • Recombinant polypeptides are expressed in eukaryotic or prokaryotic cells and purified using standard techniques. The polypeptide, or a synthetic version thereof, is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies can be generated for subsequent use in immunoassays to measure the presence and quantity of the polypeptide.
  • an immunogen e.g., a purified or synthetic peptide, a peptide coupled to an appropriate carrier (e.g., glutathione-S-transferase, keyhole limpet hemocyanin, etc.), or a peptide incorporated into an immunization vector such as a recombinant vaccinia virus is optionally mixed with an adjuvant and animals are immunized with the mixture.
  • an immunogen e.g., a purified or synthetic peptide, a peptide coupled to an appropriate carrier (e.g., glutathione-S-transferase, keyhole limpet hemocyanin, etc.), or a peptide incorporated into an immunization vector such as a recombinant vaccinia virus is optionally mixed with an adjuvant and animals are immunized with the mixture.
  • the animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to
  • Antibodies, including binding fragments and single chain recombinant versions thereof, against the polypeptides are raised by immunizing animals, e.g., using immunogenic conjugates comprising a polypeptide covalently attached (conjugated) to a carrier protein as described above.
  • the immunogen of interest is a polypeptide of at least about 10 amino acids, in another embodiment the polypeptide is at least about 20 amino acids in length, and in another embodiment, the fragment is at least about 30 amino acids in length.
  • the immunogenic conjugates are typically prepared by coupling the polypeptide to a carrier protein (e.g., as a fusion protein) or, alternatively, they are recombinantly expressed in an immunization vector.
  • Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies are screened for binding to normal or modified peptides, or screened for agonistic or antagonistic activity. Specific monoclonal and polyclonal antibodies will usually bind with a KD of at least about 50 mM, e.g., at least about 1 mM, e.g., at least about 0.1 mM or better. In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as rodents, lagomorphs, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies are found in Kohler and Milstein 1975 Nature 256:495- 497.
  • this method proceeds by injecting an animal with an immunogen, e.g., an immunogenic peptide either alone or optionally linked to a carrier protein.
  • an immunogen e.g., an immunogenic peptide either alone or optionally linked to a carrier protein.
  • the animal is then sacrificed and cells taken from its spleen, which are fused with myeloma cells.
  • the result is a hybrid cell or “hybridoma” that is capable of reproducing in vitro.
  • the population of hybridomas is then screened to isolate individual clones, each of which secrete a single antibody species to the immunogen. In this manner, the individual antibody species obtained are the products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance.
  • Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells is enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate (preferably mammalian) host.
  • the polypeptides and antibodies of the present invention are used with or without modification, and include chimeric antibodies such as humanized murine antibodies.
  • Other suitable techniques involve selection of libraries of recombinant antibodies in phage or similar vectors. See, Huse et al. 1989 Science 246: 1275-1281; and Ward et al. 1989 Nature 341 :544-546.
  • Antibodies specific to the target polypeptide can also be obtained by phage display techniques known in the art.
  • the present invention additionally provides polynucleotides encoding the binding agent of this invention.
  • the polynucleotides encode the polypeptides of any one of SEQ ID NOS :3- 18.
  • Vectors include, but are not limited to, plasmid vectors, phage vectors, virus vectors, or cosmid vectors.
  • the present invention provides a host cell comprising the polynucleotide and/or vector of this invention.
  • the host cell can be a eukaryotic or prokaryotic cell and may be used for expressing the binding agent or other purposes.
  • a further aspect of the invention relates to a composition
  • a composition comprising the binding agent of the invention and a carrier.
  • the composition is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may further comprise an additional therapeutic agent, e.g., a chemotherapeutic agent.
  • agents useful for treating cancer include, without limitation: 1) vinca alkaloids (e.g., vinblastine, vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide); 3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C)); 4) enzymes (e.g., L-asparaginase); 5) biological response modifiers (e.g., interferon-alfa); 6) platinum coordinating complexes (e.g., cisplatin and carboplatin); 7) anthracenediones (e.g., mitoxantrone);
  • vinca alkaloids
  • the agents of the invention are administered in conjunction with anti-angiogenesis agents, such as antibodies to VEGF (e.g., bevacizumab (AVASTIN), ranibizumab (LUCENTIS)) and other promoters of angiogenesis (e.g., bFGF, angiopoietin-1), antibodies to alpha-v/beta-3 vascular integrin (e.g., VITAXIN), angiostatin, endostatin, dalteparin, ABT-510, CNGRC peptide TNF alpha conjugate, cyclophosphamide, combretastatin A4 phosphate, dimethylxanthenone acetic acid, docetaxel, lenalidomide, enzastaurin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation (Abraxane), soy isoflavone (Genistein), tamoxifen citrate, thalidomide, ADH-1
  • the agents of the invention are administered in conjunction with one or more therapeutic antibodies, e.g., anti-cancer antibodies or antibodies to immune checkpoints.
  • the agents of the invention are administered in conjunction with one or more immune checkpoint inhibitors.
  • the immune checkpoint inhibitor may be any molecule that inhibits an immune checkpoint. Immune checkpoints are well known in the art and include, without limitation, PD-1, PD-L1, PD-L2, CTLA4, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, A2AR, TIM-3, and VISTA.
  • the inhibitor is an antibody against the immune checkpoint protein.
  • the immune checkpoint inhibitor is an inhibitor of PD-1 or PD-L1, e.g., an antibody that specifically binds PD-1 or PD-L1.
  • the immune checkpoint inhibitor is nivolumab, pembrolizumab, ipilimumab, durvalumab, or atezolizumab.
  • the binding agent may be directly or indirectly linked with an additional therapeutic agent to form an antibody drug conjugate.
  • Chemotherapeutic agents approved for treatment of neuroblastoma include granulocyte-macrophage colony-stimulating factor, interleukin-2, and 13-cis-retinoic acid.
  • An additional aspect of the invention relates to a kit comprising the binding agent of the invention or cells for producing the binding agent of the invention.
  • the kit can include multiple binding agents and/or compositions containing such agents.
  • each of multiple binding agents provided in such a kit can specifically bind to a different antigen and/or engage a different myeloid-derived cell.
  • the kit can further include an additional active agent, e.g., a chemotherapeutic agent as would be known to one of skill in the art.
  • the kit can further include additional reagents, buffers, containers, etc.
  • One aspect of the invention relates to a method of killing a cancer cell GD2 or a cancer cell expressing GD2 and CD47, comprising contacting the cell with an effective amount of the binding agent of the invention.
  • An additional aspect of the invention relates to a method of targeting a macrophage to a cancer cell expressing GD2 or a cancer cell expressing GD2 and CD47, comprising contacting the cancer cell and the macrophage with an effective amount of the binding agent of the invention.
  • Another aspect of the invention relates to a method inducing macrophage-dependent antibody-dependent cellular cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP) against a cancer expressing GD2 or a cancer expressing GD2 and CD47 in a subject in need thereof, comprising administering a therapeutically effective amount of the binding agent or the pharmaceutical composition of the invention to the subject, thereby inducing macrophage-dependent ADCC and/or ADCP.
  • ADCC macrophage-dependent antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • a further aspect of the invention relates to a method of treating a cancer expressing GD2 or a cancer expressing GD2 and CD47 in a subject in need thereof, comprising administering a therapeutically effective amount of the binding agent or the pharmaceutical composition of the invention to the subject, wherein the administering induces ADCC and/or ADCP of the cancer, thereby treating the cancer.
  • Another aspect of the invention relates to a method of treating a cancer in a subject in need thereof, comprising the steps of: a) selecting a subject having cancer cells that are enriched for GD2 or enriched for GD2 and CD47 and enriched for myeloid-derived cells (e.g., macrophages); and b) administering a therapeutically effective amount of the binding agent or the pharmaceutical composition of the invention to the subject, thereby treating the cancer.
  • enriched refers to a level of antigen on a cancer cell or level of myeloid-derived cells in a tumor that is greater than the level found in the cancer cell or tumor at an earlier point in time or greater than the average level found in similar cancer cells or tumors at a similar stage in the general population.
  • step a) comprises obtaining a sample of the cancer from the subject and measuring the level of antigen and myeloid-derived cells in the sample.
  • the level of antigen may be measured by, e.g., an immunoassay, protein analysis, RNA analysis, or immunohistochemistry.
  • the level of myeloid-derived cells may be measured by, e.g., an immunoassay, protein analysis, RNA analysis, or flow cytometry.
  • a myeloid-derived cell that accumulates in tumors is a cell type that is enriched in tumors.
  • the level of the myeloid-derived cell in the tumor increases by 2-fold, 5-fold, 10-fold or more relative to the level before the transition.
  • the myeloid-derived cell is a macrophage, dendritic cell, or a granulocyte, such as a neutrophil, basophil, eosinophil, or mast cell.
  • the myeloid-derived cell is a macrophage.
  • the cancer is a mesenchymal tumor.
  • the cancer is neuroblastoma, glioblastoma, retinoblastoma, breast cancer (e.g., triple negative breast cancer), bladder cancer, melanoma, lung cancer, osteosarcoma, or Ewing sarcoma.
  • the methods may further comprise the step of isolating myeloid-derived cells from the subject, contacting the myeloid-derived cells with the binding agent or pharmaceutical composition, and administering the contacted myeloid-derived cells to the subject.
  • more than one binding agent may be delivered to a subject. For example, if a cancer sample shows expression of more than one targetable antigen or more than one type of myeloid-derived cell is enriched in the cancer, agents targeting each of the antigens and/or myeloid-derived cells may be administered.
  • the binding agent may be multispecific (e.g., trispecific) in order to engage multiple targetable antigens and/or more than one type of myeloid-derived cell.
  • Cancer therapeutic agents include, without limitation, 1) vinca alkaloids (e.g., vinblastine, vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide); 3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C)); 4) enzymes (e.g., L-asparaginase); 5) biological response modifiers (e.g., interferon-alfa); 6) platinum coordinating complexes (e.g., cisplatin and carboplatin); 7) anthracenediones (e.g., mitoxantrone); 8) substituted
  • vinca alkaloids e.g., vinblastine, vincristine
  • epipodophyllotoxins e.
  • cancer therapeutic agents include, without limitation, anti-angiogenesis agents, such as antibodies to VEGF (e.g., bevacizumab (AVASTIN), ranibizumab (LUCENTIS)) and other promoters of angiogenesis (e.g., bFGF, angiopoietin-1), angiostatin, endostatin, dalteparin, ABT-510, CNGRC peptide TNF alpha conjugate, cyclophosphamide, combretastatin A4 phosphate, dimethylxanthenone acetic acid, docetaxel, lenalidomide, enzastaurin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation (Abraxane), soy isoflavone (Genistein), tamoxifen citrate, thalidomide, ADH-1 (EXHERIN), AG-013736, AMG-706, AZD2171, sorafenib tosy
  • the methods further comprise administering to the subject an immune checkpoint inhibitor.
  • Immune checkpoints are well known in the art and include, without limitation, PD-1, PD-L1, PD-L2, CTLA4, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, A2AR, TIM-3, and VISTA.
  • the inhibitor is an antibody against the immune checkpoint protein.
  • the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, or CTLA-4 that are enriched in mesenchymal tumors, e.g., an antibody that specifically binds PD-1, PD-L1, or CTLA-4.
  • the immune checkpoint inhibitor is nivolumab, pembrolizumab, ipilimumab, durvalumab, or atezolizumab.
  • Cancer therapeutic agents approved for treatment of neuroblastoma include granulocyte-macrophage colony-stimulating factor, interleukin-2, and 13-cis-retinoic acid.
  • the binding agents used in the methods of the present invention are administered directly to a subject.
  • the binding agents will be suspended in a pharmaceutically-acceptable carrier e.g., physiological saline) and administered orally or by intravenous infusion, or administered subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily.
  • the intratracheal or intrapulmonary delivery can be accomplished using a standard nebulizer, jet nebulizer, wire mesh nebulizer, dry powder inhaler, or metered dose inhaler.
  • the agents can be delivered directly to the site of the disease or disorder, such as lungs, kidney, or intestines, e.g., injected in situ into or near a tumor.
  • the dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the patient’s illness; the subject’s size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages for each agent are in the range of 0.01-100 pg/kg. Wide variations in the needed dosage are to be expected in view of the variety of agents available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by i.v. injection.
  • Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art.
  • Administrations can be single or multiple e.g., 2-, 3-, 4-, 6-, 8-, 10-; 20-, 50-, 100-, 150-, or more fold).
  • Encapsulation of the compound in a suitable delivery vehicle e.g., polymeric microparticles or nanoparticles or implantable devices
  • pharmaceutically acceptable it is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject without causing any undesirable biological effects such as toxicity.
  • the formulations of the invention can optionally comprise medicinal agents, pharmaceutical agents, carriers, adjuvants, dispersing agents, diluents, and the like.
  • the binding agents of the invention can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science and Practice of Pharmacy (21 st Ed. 2006).
  • the agent is typically admixed with, inter alia, an acceptable carrier.
  • the carrier can be a solid or a liquid, or both, and may be formulated with the agent as a unit-dose formulation, for example, a capsule or vial, which can contain from 0.01 or 0.5% to 95% or 99% by weight of the agent.
  • One or more agents can be incorporated in the formulations of the invention, which can be prepared by any of the well-known techniques of pharmacy.
  • the formulations of the invention include those suitable for oral, rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular including skeletal muscle, cardiac muscle, diaphragm muscle and smooth muscle, intradermal, intravenous, intraperitoneal), topical i.e., both skin and mucosal surfaces, including airway surfaces), intranasal, transdermal, intraarticular, intrathecal, and inhalation administration, administration to the liver by intraportal delivery, as well as direct organ injection (e.g., into the liver, into the brain for delivery to the central nervous system, or into the pancreas) or injection into a body cavity.
  • the most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular agent which is being used.
  • the carrier will typically be a liquid, such as sterile pyrogen-free water, pyrogen-free phosphate-buffered saline solution, bacteriostatic water, or Cremophor EL[R] (BASF, Parsippany, N.J.).
  • the carrier can be either solid or liquid.
  • the agent can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions.
  • Agents can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like.
  • inactive ingredients examples include red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink, and the like.
  • Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours.
  • Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric- coated for selective disintegration in the gastrointestinal tract.
  • Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
  • Formulations suitable for buccal (sub-lingual) administration include lozenges comprising the agent in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the agent in an inert base such as gelatin and glycerin or sucrose and acacia.
  • Formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the agent, which preparations are preferably isotonic with the blood of the intended recipient. These preparations can contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient.
  • Aqueous and non-aqueous sterile suspensions can include suspending agents and thickening agents.
  • the formulations can be presented in unit/dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze- dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-inj ection immediately prior to use.
  • sterile liquid carrier for example, saline or water-for-inj ection immediately prior to use.
  • an injectable, stable, sterile composition comprising an agent of the invention, in a unit dosage form in a sealed container.
  • the agent is provided in the form of a lyophilizate which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject.
  • the unit dosage form typically comprises from about 1 mg to about 10 grams of the agent.
  • Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These can be prepared by admixing the agent with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.
  • Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil.
  • Carriers which can be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.
  • Formulations suitable for transdermal administration can be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Formulations suitable for transdermal administration can also be delivered by iontophoresis (see, for example, Tyle, Pharm. Res. 3:318 (1986)) and typically take the form of an optionally buffered aqueous solution of the compounds. Suitable formulations comprise citrate or bis/tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2M of the compound.
  • the agent can alternatively be formulated for nasal administration or otherwise administered to the lungs of a subject by any suitable means, e.g., administered by an aerosol suspension of respirable particles comprising the agent, which the subject inhales.
  • the respirable particles can be liquid or solid.
  • aerosol includes any gas-borne suspended phase, which is capable of being inhaled into the bronchioles or nasal passages.
  • aerosol includes a gas-borne suspension of droplets, as can be produced in a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosol also includes a dry powder composition suspended in air or other carrier gas, which can be delivered by insufflation from an inhaler device, for example.
  • Aerosols of liquid particles comprising the agent can be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Patent No. 4,501,729. Aerosols of solid particles comprising the agent can likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art.
  • the present invention provides liposomal formulations of the agents disclosed herein and salts thereof.
  • the technology for forming liposomal suspensions is well known in the art.
  • the compound or salt thereof is an aqueous-soluble salt
  • the same can be incorporated into lipid vesicles.
  • the agent due to the water solubility of the agent, the agent will be substantially entrained within the hydrophilic center or core of the liposomes.
  • the lipid layer employed can be of any conventional composition and can either contain cholesterol or can be cholesterol-free.
  • the salt can be substantially entrained within the hydrophobic lipid bilayer which forms the structure of the liposome.
  • the liposomes which are produced can be reduced in size, as through the use of standard sonication and homogenization techniques.
  • the liposomal formulations containing the agent can be lyophilized to produce a lyophilizate which can be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.
  • a pharmaceutical composition can be prepared containing the water-insoluble agent, such as for example, in an aqueous base emulsion.
  • the composition will contain a sufficient amount of pharmaceutically acceptable emulsifying agent to emulsify the desired amount of the agent.
  • Particularly useful emulsifying agents include phosphatidyl cholines and lecithin.
  • the agent is administered to the subject in a therapeutically effective amount, as that term is defined above.
  • Dosages of pharmaceutically active agents can be determined by methods known in the art, see, e.g., Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa).
  • the therapeutically effective dosage of any specific agent will vary somewhat from agent to agent, and patient to patient, and will depend upon the condition of the patient and the route of delivery. As a general proposition, a dosage from about 0.1 to about 50 mg/kg will have therapeutic efficacy, with all weights being calculated based upon the weight of the agent.
  • Toxicity concerns at the higher level can restrict intravenous dosages to a lower level such as up to about 10 mg/kg, with all weights being calculated based upon the weight of the agent.
  • a dosage from about 10 mg/kg to about 50 mg/kg can be employed for oral administration.
  • a dosage from about 0.5 mg/kg to 5 mg/kg can be employed for intramuscular injection.
  • Particular dosages are about 1 pmol/kg to 50 pmol/kg, and more particularly to about 22 pmol/kg and to 33 pmol/kg of the agent for intravenous or oral administration, respectively.
  • more than one administration e.g., two, three, four, or more administrations
  • time intervals e.g., hourly, daily, weekly, monthly, etc.
  • the present invention finds use in veterinary and medical applications. Suitable subjects include both avians and mammals, with mammals being preferred.
  • the term “mammal” as used herein includes, but is not limited to, humans, primates, bovines, ovines, caprines, equines, felines, canines, lagomorphs, etc. Human subjects include neonates, infants, juveniles, and adults.
  • the subject may be one in need of the methods of the invention, e.g., a subject that has or is suspected of having cancer.
  • the subject may be a laboratory animal, e.g., an animal model of a disease.
  • Antibody therapeutics that are engineered to engage NK cells for tumor cell killing are limited in utility for tumors with an immune-cold phenotype for which NK cells and other anti-tumor immune cells are excluded.
  • FIG. 1 neuroblastoma tumors are enriched for macrophage markers, while the expression of markers that identify NK cells, neutrophils, and dendritic cells are much lower.
  • monoclonal antibodies designed to selectively engage macrophages would be ideally suited for neuroblastoma therapy.
  • GD2 is a particularly good target because it is highly enriched on the surface of neuroblastoma cells, while its expression is otherwise highly restricted and only found on limited normal cells of neuroectodermal origin.
  • An antibody targeting GD2, dinutuximab/Unituxin has been developed and FDA-approved for use in pediatric neuroblastoma. We first confirmed that switching the isotype of dinutuximab would not alter its binding to neuroblastoma cells.
  • FIG. 2 confirms that both the hIgG4 and hlgGl forms of anti-GD2 show equivalent affinity for GD2 on SK-NA-S neuroblastoma cells by flow cytometry.
  • GD2 expression has been found on breast cancer, melanoma, and small cell lung cancer.
  • GD2 has been identified as a marker of breast cancer stem cells and a driver of tumorigenesis (PMID 22585577).
  • hIgG4 anti-GD2 produced a higher level of macrophage-mediated killing of GD2- expressing HCC1395 and Hs578T human triple negative breast cancer cells in vitro (FIG. 8).
  • Heavy chain (evi-5 dinu.VH-gm3.HC) (SEQ ID NO:1)
  • Heavy chain (evi-5 h4.HC.S228P) (SEQ ID NO: 17)
  • SIRPa wild-type fragment SEQ ID NO: 19
  • SIRPa modified fragment SEQ ID NO:20
  • Etaracizumab HC hole [S228P.T366S.T368A.Y407V] (SEQ ID NO:29)
  • Etaracizumab LC normal (SEQ ID NO: 30)
  • Dinutuximab HC knocked, crossed [S228P.T366W] (SEQ ID NO:31) EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRA
  • Etaracizumab HC knock- crossed [S228P.T366W] (SEQ ID NO:35)
  • Etaracizumab LC crossed (SEQ ID NO:36)

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Abstract

La présente invention concerne des agents de liaison et des compositions les comprenant. L'invention concerne en outre des polynucléotides codant pour l'agent de liaison ainsi que des vecteurs et des cellules hôtes les comprenant. L'invention concerne en outre des procédés d'utilisation des agents de liaison pour médier la cytotoxicité cellulaire dépendante des anticorps et la phagocytose cellulaire dépendante des anticorps de cellules cancéreuses et des méthodes de traitement de cancers.
PCT/US2023/072361 2022-08-17 2023-08-17 Agents de liaison ciblant gd2 et leur utilisation pour le traitement du cancer WO2024040150A1 (fr)

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Citations (1)

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US20200023076A1 (en) * 2016-04-29 2020-01-23 Curevac Ag Rna encoding an antibody

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200023076A1 (en) * 2016-04-29 2020-01-23 Curevac Ag Rna encoding an antibody

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHAN GODFREY CHI-FUNG, CHAN CAROL MATIAS: "Anti-GD2 Directed Immunotherapy for High-Risk and Metastatic Neuroblastoma", BIOMOLECULES, M D P I AG, CH, vol. 12, no. 3, 24 February 2022 (2022-02-24), CH , pages 358, XP093140241, ISSN: 2218-273X, DOI: 10.3390/biom12030358 *
JEONG A PARK, CHEUNG NAI-KONG V,: "Targets and Antibody Formats for Immunotherapy of Neuroblastoma INTRODUCTION", J CLIN ONCOL, 13 March 2020 (2020-03-13), pages 1836 - 1848, XP055752573, [retrieved on 20201120], DOI: 10.1200/JCO.19 *
MICHON J, PERDEREAU B, BRIXY F, MOUTEL S, FRIDMAN W.-H, TEILLAUD J.-L: "In Vivo~ Targeting of Human Neuroblastoma Xenograft by Anti-G&Anti-FcyRI (CD64) Bispecific Antibody", EUROPEAN JOURNAL OF CANCER, vol. 31A, no. 4, 1 January 1995 (1995-01-01), pages 631 - 636, XP093140240 *
MICHON JEAN, MOUTEL SANDRINE, BARBET JACQUES, ROMET-LEMONNE JEAN-LOUP, DEO YASHWANT M, FRIDMAN WOLF H, TEILLAUD JEAN-LUC: "In Vitro Killing of Neuroblastoma Cells by Neutrophils Derived From Granulocyte Colony-Stimulating Factor-Treated Cancer Patients Using an Anti-Disialoganglioside/Anti-FcyRI Bispecific Antibody", BLOOD, vol. 86, no. 3, 1 August 1995 (1995-08-01), pages 1124 - 1130, XP093140245 *

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