WO2023056326A1 - Compositions et méthodes de traitement du cancer - Google Patents

Compositions et méthodes de traitement du cancer Download PDF

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Publication number
WO2023056326A1
WO2023056326A1 PCT/US2022/077218 US2022077218W WO2023056326A1 WO 2023056326 A1 WO2023056326 A1 WO 2023056326A1 US 2022077218 W US2022077218 W US 2022077218W WO 2023056326 A1 WO2023056326 A1 WO 2023056326A1
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binding agent
cancer
domain
chimeric binding
cell
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PCT/US2022/077218
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English (en)
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David Cheresh
Sara WEIS
Stephen Mccormack
Christoph Rader
Hiromi WETTERSTEN
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Alpha Beta Holdings, Llc
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Publication of WO2023056326A1 publication Critical patent/WO2023056326A1/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
    • C07K16/2839—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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/522—CH1 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]

Definitions

  • the present invention relates to chimeric binding agents and compositions comprising the same.
  • the invention further relates to polynucleotides encoding the chimeric binding agent and vectors and host cells comprising the same.
  • the invention further relates to methods of using the chimeric binding agents to mediate antibody-dependent cellular cytotoxicity of epithelial cancer cells and methods of treating epithelial cell 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).
  • EpCAM epithelial cancer cells
  • CD33 epithelial cancer cells
  • CCR4 cutaneous T-cell lymphoma
  • GD2 neuroblastoma
  • PDGFRA sarcoma
  • immune checkpoint targets
  • 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).
  • a class of antibody therapeutics has been developed for epithelial cancers that express high levels of markers such as EpCAM, EGFR, HER2, or VEGFR2. While antibodies targeting such antigens may be effective for early stage tumors, epithelial cancers are known to undergo an epithelial-to-mesenchymal transition (EMT) that involves not only a loss of epithelial markers and a gain of mesenchymal markers (Karacosta, 2019), but also a change in the tumor microenvironment and immune cell infiltration (Dongre, 2019). As such, targeting epithelial tumors that convert toward a mesenchymal state may require different antigen-effector cell combinations.
  • EMT epithelial-to-mesenchymal transition
  • EMT is a dynamic process that describes a tumor cell phenotype in crosstalk with stromal constituents of the tumor (Dongre, 2019). Cancer-associated fibroblasts, macrophages, and other immune cells secrete a variety of cytokines and factors that engage tumor cells to activate the expression of transcription factors that induce EMT. Mesenchymal-like carcinoma cells also shift the immune component of the tumor toward an immunocompromised state that excludes anti-tumor immune cell types and recruits pro-tumor macrophages.
  • an antibody that recognizes an antigen expressed on the surface of mesenchymal-like tumor cells that is capable of engaging macrophages as effector cells represents an unmet need in the field of therapeutic antibody development for solid tumors.
  • Cancers that have undergone EMT tend to be more aggressive, metastatic and drug resistance. Therefore, having a drug that attacks tumor cells that have undergone EMT is likely to decrease tumor progression and drug resistance. [0009]
  • the present invention is based in part on an understanding of epithelial cancer cells and the EMT process, including changes in the immune cell populations in the tumor microenvironment.
  • the epithelial tumor cells gain expression of av[33 integrin on their cell surface, becoming drug resistant and more stem-like in phenotype as well as insensitive to hypoxia or other environmental stress.
  • Expression of av[33 on epithelial cancer cells is triggered by the various forms of cellular stress as well within the microenvironment or the application of a wide range of anti-cancer drugs. Patients that have progressed on standard of care therapeutics and thereby express av[33 are therefore candidates for therapies targeting the av[33 antigen. Given that av[33 is necessary and sufficient for drug resistance it is likely that by selectively targeting av[33 positive tumor cells it would be possible to prevent or reverse cancer acquired drug resistance.
  • the present invention provides compositions and methods for engaging the appropriate immune effector cells to effectively mediate antibody-dependent cellular cytotoxicity (ADCC) against epithelial cancer cells that have undergone EMT and have gained the cell surface marker av[33.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCC is mediated by macrophages, and not NK cells, that leads to the death of cancer cells targeted by the antibody. Further, the cell death does not involve antibody-dependent cellular phagocytosis (ADCP) or direct killing via the antibody alone. It was previously understood that antibody engagement of macrophages as an effector cell typically promoted ADCP.
  • the present inventors have surprisingly determined that the chimeric binding agents of the invention do not induce ADCP, but instead exclusively promote macrophage-dependent ADCC of human cell targets. This unexpected finding, among other benefits, advantageously permits treatment of CD47-positive tumor cells that would normally be resistant to phagocytosis or ADCP.
  • a binding agent that promotes ADCC exclusively will kill every cell it encounters while a binding agent that promotes ADCP will not be able to kill CD47-positive cells.
  • the chimeric binding agents of the present invention are expected to be more efficient. Without being bound by theory, it is thought that the advantages of the present invention are based on the structure of the chimeric binding agent (e.g., the IgG4 domain) and/or the antigen being recognized (e.g., integrin av[33) that make cells expressing the antigen particularly sensitive to ADCC rather than ADCP.
  • Mesenchymal tumors are identified by expression of transcription factors (ZEB, SNAIL, SLUG, and TWIST1) that repress epithelial markers (including E-cadherin, EpCAM, occludins, claudins, and cytokeratins) and promote the expression of mesenchymal markers (including cell adhesion-related proteins N-cadherin, vimentin, fibronectin, (31 and (33 integrins, and MMPs) (Dongre, 2019).
  • An ideal tumor cell antigen for a mesenchymal-like tumor would be a cell surface marker with high expression on tumor cells but low expression on all other normal cell types.
  • cancer stem cell markers may represent another type of antigen for targeting mesenchymal tumors, although these often vary between tumor types.
  • N-cadherin and (31 integrin are expressed on many normal cell types and could thus contribute to issues with toxicity or compete with tumor cells for antibody binding.
  • integrin av[33 is a more selective candidate for a mesenchymal tumor cell antigen based on its low expression in normal adult tissues and its enrichment on epithelial tumors as they become more aggressive, late-stage, and drug resistant.
  • the present invention is based on the development of agents that can mediate ADCC by engaging myeloid-derived cells found in mesenchymal tumors and targeting them to antigens that are expressed on epithelial cancer cells that have undergone EMT.
  • one aspect of the invention relates to a chimeric binding agent comprising a first domain that specifically binds to an antigen on an epithelial cancer cell expressing at least one mesenchymal cell marker and a second domain that mediates ADCC by engaging a myeloid- derived cell that accumulates in mesenchymal tumors, and compositions or pharmaceutical compositions comprising the chimeric binding agents.
  • Another aspect of the invention relates to a polynucleotide encoding the chimeric binding agent of the invention and vectors and host cells comprising the polynucleotide.
  • An additional aspect of the invention relates to a method of targeting a myeloid- derived cell that accumulates in mesenchymal tumors to an epithelial cancer cell expressing at least one mesenchymal cell marker, comprising contacting the cancer cell and the myeloid- derived cell with an effective amount of the chimeric binding agent of the invention.
  • a further aspect of the invention relates to a method of treating an epithelial cell cancer in a subj ect in need thereof, comprising administering a therapeutically effective amount of the chimeric binding agent or the pharmaceutical composition of the invention to the subject, thereby treating the epithelial cell cancer.
  • av[33 is expressed in increased amounts on drug resistant cancers making it possible to prevent or reverse drug resistance.
  • Another aspect of the invention relates to a method of treating an epithelial cell cancer in a subject in need thereof, comprising the steps of: a) selecting a subj ect having epithelial cancer cells that are enriched for an antigen specifically bound by the chimeric binding agent of the invention and enriched for myeloid- derived cells that accumulate in mesenchymal tumors; and b) administering a therapeutically effective amount of the chimeric binding agent or the pharmaceutical composition of the invention to the subject, thereby treating the epithelial cell cancer.
  • Cancer patients that become drug resistant gain the expression of av[33 and thereby become candidates for such therapies targeting this marker.
  • Another aspect of this invention relates to antigen-effector cell matching of tumors such that the antigen is specifically present on the tumor cell (e.g., a tumor cell antigen) and a therapeutic antibody contains effector cell binding regions that are specific to those effector cells found in the tumor (e.g., neutrophils, dendritic cells, NK cells etc.).
  • the antigen is specifically present on the tumor cell (e.g., a tumor cell antigen) and a therapeutic antibody contains effector cell binding regions that are specific to those effector cells found in the tumor (e.g., neutrophils, dendritic cells, NK cells etc.).
  • FIG. 1 shows anti-av[33 mouse monoclonal antibody LM609 sensitizes tumor xenografts to erlotinib.
  • LM609 re-sensitizes resistant tumors to erlotinib.
  • HCC827-R18 and PC9-R4L erlotinib-resistant tumor cells generated as reported in Wettersten et al., Cancer Res. 79:5048 (2019), incorporated by reference herein in its entirety, were injected to form subcutaneous flank tumors in nu/nu recipient mice.
  • FIG. 2 shows the amino acid sequence for mAh LM609-mIgGl -kappa heavy chain (SEQ ID NO:11) and light chain (SEQ ID NO:12).
  • FIG. 3 shows the amino acid sequence for hLM609-hIgGl -WT (humanized LM609) heavy chain (SEQ ID NO:9) and light chain (SEQ ID NO:10).
  • FIG. 4 shows the amino acid sequence for two distinct forms of shLM609-hIgGl-WT (super-humanized LM609): LM609_7 Fab domain of heavy chain (SEQ ID NO:5) and light chain (SEQ ID NO:6) and JC7U Fab domain of heavy chain (SEQ ID NO:7) and light chain (SEQ ID NO:8)
  • FIG. 5 shows the amino acid sequence for hLM609-h!gG4-S228P (humanized LM609) heavy chain (SEQ ID NO:1) and light chain (SEQ ID NO:2).
  • FIG. 6 shows an amino acid sequence alignment for hLM609-h!gGl-WT heavy chain (SEQ ID NO:9) vs. hLM609-h!gG4-S228P heavy chain (SEQ ID NO:1). Sequence alignment was performed using the Align Sequences Protein BLAST tool from ncbi.nih.gov. The “Query” sequence is hLM609-h!gGl -WT and the “Sbjct” sequence is hLM609-h!gG4-S228P. Sequence differences are shown in boldface.
  • FIG. 7 shows that hLM609-IgG4-S228P engages and activates FcyRI in a cell-based ADCC reporter bioassay.
  • Integrin av[33 -expressing human pancreatic cancer cells were utilized as “target cells” to assess the ability of anti-av[33 antibodies to elicit effector cell activation using a Promega ADCC Reporter Bioassay in which “effector cell” activation is evaluated using a Raji cell line stably expressing the human FcyR I or III and NFAT-induced luciferase.
  • Six antibody dilutions were tested per antibody, with FcyR activation shown as fold change relative to treatment with assay buffer containing no antibody.
  • FIG. 8 shows equivalent blocking of av[33-mediated adhesion by hLM609 IgGl and IgG4-S228P variants.
  • Antibody affinity is evaluated using an in vitro cell adhesion assay. 48 well tissue culture plates were coated with the integrin av[33 ligand fibrinogen or the integrin pi ligand type I collagen, and 2000-10000 cells were added in the presence of each antibody for a range of 2-fold dilutions starting at 5 pg/mL in duplicate. Plates were washed at the endpoint and cells attached to the substrate detected using crystal violet.
  • FIGS. 9A-9C show in vitro ADCC by NK cells (NK-ADCC) and macrophages (Mac- ADCC).
  • NK-ADCC NK cells
  • Mac- ADCC macrophages
  • FIGS. 10A-10B show that LM609 and hLM609-h!gG4-S228P induce ADCC mediated by macrophages, but not NK cells, isolated from healthy blood donors.
  • A In vitro ADCC for primary human monocyte-derived macrophages as effector cells.
  • B In vitro ADCC for human NK cells as effector cells.
  • Graphs show effect of increasing effector-to- target ratio (E:T) on death of avP3-expressing human lung cancer cells.
  • FIG. 11 shows in vitro ADCC by mouse bone marrow derived macrophages.
  • In vitro ADCC for mouse primary macrophage effector cells Primary mouse macrophages were isolated from mouse bone marrow and used as effector cells to kill HCC827+P3 target cells.
  • Fig. 12 shows that hLM609-hIgG4-S228P inhibits growth of avP3-expressing tumors in mice with no body weight loss over two weeks of treatment.
  • Fig. 13 shows that the anti-tumor activity of hLM609-hIgG4-S228P is superior to hLM609-hIgGl for xenografts in mice.
  • Fig. 14 shows that the tumor accumulation of hLM609-hIgGl to hLM609-hIgG4- S228P is superior to hLM609-hIgGl for xenografts in mice.
  • Nude mice injected with FG-[33 cells human pancreatic cancer cells that express av[33) were randomly divided into 3 groups. The mice were treated with PBS, hLM609-hIgG4-S228P (10 mg/kg, i.p.), or hLM609-hIgGl (10 mg/kg, i.p.) at 10 mg/kg twice per week for 14 days.
  • mice 30 min after the last dosing, animals were sacrificed, and tumor tissues were collected and stored at -80°C until further analyses.
  • the tumor tissues were lysed in RPMI at 6.4 pL/mg.
  • concentration of hLM609-hIgG4- S228P and hLM609-hIgGl in the lysates were measured using a human IgG ELISA kit (Thermo). *P ⁇ 0.001 compared to PBS using Bonferroni and Tukey tests.
  • FIG. 15 shows hLM609-h!gG4-S228P is more effective than hLM609-h!gGl-WT against a cancer that endogenously expresses av[33.
  • H1975 (endogenous av[33+) cells were subcutaneously injected to nude mice. The mice were randomly enrolled to the 3 groups when the tumor mass reached 100 mm 3 . The mice were treated with PBS (vehicle), hLM609-h!gGl- WT (10 mg/kg), or hLM609-h!gG4-S228P (10 mg/kg) twice weekly. The tumor mass volume was monitored by a caliper.
  • the endpoint was 2 fold change in tumor growth compared to day 0 of the treatment. Kaplan Meier curve for % survival to endpoint for each group was plotted. P ⁇ 0.05 Vehicle vs hLM609-h!gG4-S228P. PO.Ol hLM609-h!gG4-S228P vs hLM609- hlgGl-WT.
  • 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” 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,” “prophylactically 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 chimeric 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 chimeric binding agent comprising a first domain that specifically binds to an antigen on an epithelial cancer cell expressing at least one mesenchymal cell marker and a second domain that mediates antibody-dependent cellular cytotoxicity (ADCC) by engaging a myeloid-derived cell that accumulates in mesenchymal tumors.
  • ADCC antibody-dependent cellular cytotoxicity
  • a myeloid-derived cell that accumulates in mesenchymal tumors is a cell type that is enriched in epithelial cell tumors as they undergo an epithelial-to-mesenchymal transition.
  • 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 epithelial cancer may be any known type of carcinoma.
  • Examples of epithelial cancers include, without limitation, cancers of the gastrointestinal tract, breast, lungs (e.g., nonsmall cell lung cancer), colon, prostate, or bladder.
  • the epithelial cancer cell is a late-stage epithelial cancer cell. Late stage or advanced stage, as used herein, refers to stage III or stage IV cancers based on the TNM staging system.
  • the epithelial cancer cell has at least partially transitioned to a mesenchymal cell, e.g., expresses one or more mesenchymal antigens.
  • the epithelial cancer cell is chemotherapy resistant or refractory, which may be due to the epithelial-to-mesenchymal transition.
  • the chimeric binding agent may be any structure that is capable of binding to an antigen on the epithelial cancer cell and engaging a myeloid-derived cell to mediate ADCC.
  • the chimeric binding agent is an antibody or an antigen-binding fragment thereof.
  • one or more portions of the chimeric binding agent are composed of antibody fragments.
  • one or both domains of the chimeric binding agent is a non-immunoglobulin scaffold, an aptamer, a small molecule (e.g, a receptor ligand), or other binding moiety.
  • the first domain of the chimeric binding agent is an antibody domain.
  • the second domain of the chimeric binding agent is an antibody domain. In some embodiments, both domains are antibody domains.
  • the first domain is a humanized or human antibody domain.
  • the second domain is a humanized or human antibody domain. In some embodiments, the first domain and the second domain are humanized or human antibody domains.
  • the first domain specifically binds an antigen on the surface of the epithelial cancer cell.
  • the antigen is a receptor found on the surface of epithelial-like tumor cells, such as, without limitation, EGFR, HER2, EpCAM, E-cadherin, ZO-1, or integrin a6[34.
  • the antigen is a receptor found on the surface of mesenchymal-like tumor cells, such as, without limitation, integrin av[33, integrin pi, integrin av 6, N-cadherin, OB-cadherin, or syndecan-1.
  • the antigen may be one that is not present or present at low levels on the surface of normal epithelial cells. In some embodiments, the antigen may be one that is not present or present at low levels on the surface of epithelial cancer cells. In some embodiments, the antigen may be one that that is present or present at increased levels only after the epithelial cancer cell begins to transition to a mesenchymal cell. In some embodiments, the antigen is a mesenchymal cell antigen that is not present or only present at low levels on the epithelial cancer cell until it begins to transition to a mesenchymal cell. In some embodiments, the antigen is a neoantigen that has not been previously recognized by the immune system. [0066] In certain embodiments, the first domain specifically binds an integrin. The integrin may be, without limitation, integrin av, integrin [33, or integrin av[33.
  • 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 amino acid sequence of the light chain of hLM609-hIgG4-S228P (SEQ ID NO:2) and the Fab portion (also known as the Fd fragment) of the heavy chain of hLM609-hIgG4-S228P (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 first domain comprises the amino acid sequence of a superhumanized variant of shLM609-hIgGl -WT, e.g.
  • the LM609 7 Fab domain of heavy chain (SEQ ID NO:5) and light chain (SEQ ID NO:6) or the JC7U Fab domain of heavy chain (SEQ ID NO:7) and light chain (SEQ ID NO:8) 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 first domain comprises the amino acid sequence of the light chain of hLM609-hIgGl -WT (SEQ ID NO:9) and the Fab portion of the heavy chain of hLM609-hIgGl-WT (SEQ ID NO:10) 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 first domain may specifically bind a second antigen in addition to an antigen on the surface of the epithelial cancer cell.
  • the first domain may be a bispecific antibody domain, trispecific antibody domain, or other structure that can specifically bind more than one antigen.
  • the second antigen may be, for example, the binding target of an antibody used to treat cancer, e.g., an immune checkpoint molecule such as PD-1, PD-L1, or CTLA-4.
  • the second antigen is a cancer stem cell marker (e.g., CD133, CD44, CD90, CD117, CD166, CD105).
  • the second antigen is an antigen on an effector cell that is different from the effector cell targeted by the second domain.
  • the different effector cell is a myeloid-derived cell, e.g., a macrophage, dendritic cell, or a granulocyte, such as a neutrophil, basophil, eosinophil, or mast cell.
  • the chimeric binding agent is capable of localizing two or more classes of effector cells to the tumor cells, e.g., macrophages and dendritic cells or macrophages and neutrophils.
  • the second domain of the chimeric 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
  • B cells B cells
  • T cells T cells
  • 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).
  • the second domain comprises, consists essentially of, or consists of a Fc domain of an antibody.
  • the Fc domain may be from any antibody isotype.
  • the second domain comprises a Fc domain of an IgG antibody, e.g, an IgG4 antibody.
  • the second domain comprises a Fc domain of an IgA or IgE antibody.
  • the second domain further comprises a hinge domain of an antibody.
  • the second domain comprises the amino acid sequence of the heavy chain Fc domain and hinge domain of hLM609-hIgG4-S228P (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 second domain comprises the amino acid sequence of the heavy chain Fc domain and hinge domain of hLM609-hIgGl - WT (SEQ ID NO:9) 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 chimeric binding agent comprises the amino acid sequence of the hLM609-hIgG4-S228P heavy chain (SEQ ID NO:1) and light chain (SEQ ID NO:2) 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 chimeric binding agent comprises the amino acid sequence of the hLM609-hIgGl-WT heavy chain (SEQ ID NO:9) and light chain (SEQ ID NO:10) 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 chimeric 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 chimeric 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/L328W/A330V/P331A/I332Y/E382V/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 a .,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 254:1275 (1989)).
  • the term “antibody fragment” as used herein 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
  • Methods for humanizing non-human antibodies are well known in the art.
  • 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 sA., 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.
  • rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • such “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 thereol) 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 Boemer 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 Boemer 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 re
  • 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. 19 ⁇ 9 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 chimeric binding agent of this invention.
  • the polynucleotides comprise a heavy chain encoding nucleotide sequence of SEQ ID NO: 13 and a light chain encoding sequence of SEQ ID NO:14 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.
  • the polynucleotides comprise a heavy chain encoding nucleotide sequence of SEQ ID NO: 15 and a light chain encoding sequence of SEQ ID NO: 14 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.
  • 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 chimeric binding agent or other purposes.
  • a further aspect of the invention relates to a composition
  • a composition comprising the chimeric 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); 8) substituted urea chemotherapeutic
  • 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 chimeric binding agent may be directly or indirectly linked with an additional therapeutic agent to form an antibody drug conjugate.
  • kits comprising the chimeric binding agent of the invention or cells for producing the chimeric binding agent of the invention.
  • the kit can include multiple chimeric binding agents and/or compositions containing such agents.
  • each of multiple chimeric 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 targeting a myeloid-derived cell (e.g., a macrophage) to a cancer cell expressing an antigen recognized by the chimeric binding agent of the invention (e.g., integrin av[33), comprising contacting the cancer cell and the myeloid-derived cell with an effective amount of the chimeric binding agent of the invention.
  • a method of targeting a myeloid-derived cell that accumulates in mesenchymal tumors to an epithelial cancer cell expressing at least one mesenchymal cell marker comprising contacting the cancer cell and the myeloid-derived cell with an effective amount of the chimeric binding agent of the invention.
  • a further aspect of the invention relates to a method of treating a cancer expressing an antigen recognized by the chimeric binding agent of the invention (e.g., integrin av[33) in a subject in need thereof, comprising administering a therapeutically effective amount of the chimeric binding agent or the pharmaceutical composition of the invention to the subject, thereby treating the cancer.
  • An additional aspect of the invention relates to a method of treating an epithelial cell cancer in a subj ect in need thereof, comprising administering a therapeutically effective amount of the chimeric binding agent or the pharmaceutical composition of the invention to the subject, thereby treating the epithelial cell cancer.
  • Another aspect of the invention relates to a method of treating a cancer in a subj ect in need thereof, comprising the steps of: a) selecting a subject having cancer cells that are enriched for an antigen specifically bound by the chimeric binding agent of the invention (e.g., integrin av[33) and enriched for myeloid-derived cells; and b) administering a therapeutically effective amount of the chimeric binding agent or the pharmaceutical composition of the invention to the subject, thereby treating the cancer.
  • an antigen specifically bound by the chimeric binding agent of the invention e.g., integrin av[33
  • myeloid-derived cells e.g., myeloid-derived cells
  • a further aspect of the invention relates to a method of treating an epithelial cell cancer in a subject in need thereof, comprising the steps of: a) selecting a subj ect having epithelial cancer cells that are enriched for an antigen specifically bound by the chimeric binding agent of the invention and enriched for myeloid- derived cells that accumulate in mesenchymal tumors; and b) administering a therapeutically effective amount of the chimeric binding agent or the pharmaceutical composition of the invention to the subject, thereby treating the epithelial cell cancer.
  • 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.
  • Another aspect of this invention relates to antigen-effector cell matching of tumors such that the antigen is specifically present on the tumor cell (e.g., a tumor cell antigen) and a therapeutic antibody contains effector cell binding regions that are specific to those effector cells found in the tumor (e.g., neutrophils, dendritic cells, NK cells etc.).
  • the inventors have determined that the av[33 integrin appears on the surface of cancer cells that have gained drug resistance. This helps identify those patients most likely to be effectively treated with therapeutic monoclonal antibody approaches that are directed against av[33 integrin.
  • This provides a precision medicine approach to the correct patient populations that allows one to include other therapeutic monoclonal antibodies that target av[33 integrin.
  • cancer patients become resistant to standard of care therapeutics their tumors gain av[33 expression and thereby become candidates for treatment with an av[33 targeted antibody that is capable of promoting immune cell mediated ADCC of the av[33 expressing tumor cells.
  • the myeloid-derived cell is a macrophage, dendritic cell, or granulocyte, such as a neutrophil, basophil, eosinophil, or mast cell.
  • the myeloid-derived cell is a macrophage.
  • the epithelial cancer may be any known type of carcinoma. Examples of epithelial cancers include, without limitation, cancers of the gastrointestinal tract, breast, lungs (e.g., nonsmall cell lung cancer), colon, prostate, or bladder.
  • the epithelial cancer cell is a late stage epithelial cancer cell.
  • the epithelial cancer cell has at least partially transitioned to a mesenchymal cell, e.g., expresses one or more mesenchymal antigens.
  • the epithelial cancer cell is chemotherapy resistant or refractory, which may be due to the epithelial-to-mesenchymal transition.
  • the methods may further comprise the step of isolating myeloid-derived cells from the subject, contacting the myeloid-derived cells with the chimeric binding agent or pharmaceutical composition, and administering the contacted myeloid- derived cells to the subject.
  • more than one chimeric 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 chimeric binding agent may be multispecific (e.g., bispecific or trispecific) in order to engage multiple targetable antigens and/or more than one type of myeloid-derived cell.
  • the methods of the invention may further comprise administering to the subject an additional cancer therapeutic agent or treatment (e.g., surgery, radiation).
  • 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
  • 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 a CD47 blocking agent to enhance phagocytosis of the cancer cells.
  • CD47 blocking agents include CD47- blocking monoclonal antibodies (Hu5F9-G4, CC-90002, Ti-061, or SRF231) or SIRPa-Fc fusion proteins (TTI-621, TTI-622, ALX148).
  • CD47- blocking monoclonal antibodies Hu5F9-G4, CC-90002, Ti-061, or SRF2311
  • SIRPa-Fc fusion proteins TTI-621, TTI-622, ALX148.
  • the methods of the invention do not comprise administering to the subject a CD47 blocking agent.
  • 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.
  • the methods further comprise administering to the subject an EGFR inhibitor.
  • agents include tyrosine kinase inhibitors (e.g., erlotinib, gefitinib, lapatinib, Osimertinib, neratinib) and monoclonal antibodies (e.g., cetuximab, necitumumab, panitumumab).
  • the chimeric binding agents used in the methods of the present invention are administered directly to a subject.
  • the chimeric 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.
  • a pharmaceutically-acceptable carrier e.g., physiological saline
  • 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.
  • 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 may increase the efficiency of delivery, particularly for oral delivery.
  • 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 chimeric 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.
  • a sufficient amount of emulsifying agent which is pharmaceutically acceptable can be employed in sufficient quantity to emulsify the agent in an aqueous carrier.
  • One such useful emulsifying agent is phosphatidyl choline.
  • 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.
  • one or more conventional solid carriers for example, cocoa butter
  • 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-bome suspended phase, which is capable of being inhaled into the bronchioles or nasal passages.
  • aerosol includes a gas-bome 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 compound 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.
  • Non-limiting embodiments of the invention include the following.
  • Embodiment 1 A chimeric binding agent comprising a first domain that specifically binds to an antigen on an epithelial cancer cell expressing at least one mesenchymal cell marker and a second domain that mediates antibody-directed cellular cytotoxicity (ADCC) by engaging a myeloid-derived cell that accumulates in mesenchymal tumors.
  • ADCC antibody-directed cellular cytotoxicity
  • Embodiment 2 The chimeric binding agent of embodiment 1 , wherein the myeloid-derived cell is a macrophage, dendritic cell, or granulocyte, such as a neutrophil, basophil, eosinophil, or mast cell.
  • the myeloid-derived cell is a macrophage, dendritic cell, or granulocyte, such as a neutrophil, basophil, eosinophil, or mast cell.
  • Embodiment 3 The chimeric binding agent of embodiment 1 or 2, wherein the epithelial cancer cell is a late stage epithelial cancer cell.
  • Embodiment 4 The chimeric binding agent of embodiment 3, wherein the epithelial cancer cell has at least partially transitioned to a mesenchymal cell.
  • Embodiment 5 The chimeric binding agent of any one of embodiments 1-4, wherein the epithelial cancer cell is chemotherapy resistant or refractory.
  • Embodiment 6 The chimeric binding agent of any one of embodiments 1-5, wherein the first domain is an antibody domain.
  • Embodiment 7 The chimeric binding agent of any one of embodiments 1-6, wherein the second domain is an antibody domain.
  • Embodiment 8 The chimeric binding agent of any one of embodiments 1-7, wherein the first domain is a humanized or human antibody domain.
  • Embodiment 9 The chimeric binding agent of any one of embodiments 1-8, wherein the second domain is a humanized or human antibody domain.
  • Embodiment 10 The chimeric binding agent of any one of embodiments 1-9, which is a chimeric antibody or an antigen-binding fragment thereof.
  • Embodiment 11 The chimeric binding agent of any one of embodiments 1-10, wherein the first domain specifically binds an integrin.
  • Embodiment 12 The chimeric binding agent of embodiment 11 , wherein the integrin is integrin av.
  • Embodiment 13 The chimeric binding agent of embodiment 11, wherein the integrin is integrin [33.
  • Embodiment 14 The chimeric binding agent of embodiment 11, wherein the integrin is integrin av[33.
  • Embodiment 15 The chimeric binding agent of any one of embodiments 1-14, wherein the first domain specifically binds an antigen on the surface of a cancer cell, including receptors on the surface of epithelial-like tumor cells (such as EGFR, HER2, EpCAM, E-cadherin, ZO-1, integrin a6p4) or mesenchymal -like tumor cells (such as integrin avP3, integrin pi, integrin avP6, N-cadherin, OB-cadherin, syndecan-1).
  • epithelial-like tumor cells such as EGFR, HER2, EpCAM, E-cadherin, ZO-1, integrin a6p4
  • mesenchymal -like tumor cells such as integrin avP3, integrin pi, integrin avP6, N-cadherin, OB-cadherin, syndecan-1).
  • Embodiment 16 The chimeric binding agent of any one of embodiments 1-14, wherein the first domain specifically binds a neoantigen that has not been previously recognized by the immune system.
  • Embodiment 17 The chimeric binding agent of any one of embodiments 1-16, wherein the first domain comprises a Fab domain of an antibody.
  • Embodiment 18 The chimeric binding agent of embodiment 17, wherein the first domain comprises a Fab domain of an IgG antibody.
  • Embodiment 19 The chimeric binding agent of embodiment 18, wherein the first domain comprises a Fab domain of an IgG4 antibody.
  • Embodiment 20 The chimeric binding agent of embodiment 19, wherein the first domain comprises the amino acid sequence of the light chain of hLM609-h!gG4-S228P (SEQ ID NO:2) or a sequence at least 90% identical thereto and the Fab portion of the heavy chain of hLM609-hIgG4-S228P (SEQ ID NO:3) or a sequence at least 90% identical thereto.
  • Embodiment 21 Embodiment 21.
  • Embodiment 22 The chimeric binding agent of any one of embodiments 1-19, wherein the first domain further specifically binds a second antigen.
  • Embodiment 23 The chimeric binding agent of embodiment 22, wherein the first domain is a bispecific antibody domain.
  • Embodiment 24 The chimeric binding agent of embodiment 22 or 23, wherein the second antigen is an immune checkpoint molecule, such as PD-1, PD-L1, or CTLA-4.
  • an immune checkpoint molecule such as PD-1, PD-L1, or CTLA-4.
  • Embodiment 25 The chimeric binding agent of embodiment 22 or 23, wherein the second antigen is a cancer stem cell marker, such as CD133, CD44, CD90, CD117, CD 166, or CD 105, or an effector cell antigen.
  • a cancer stem cell marker such as CD133, CD44, CD90, CD117, CD 166, or CD 105, or an effector cell antigen.
  • Embodiment 26 The chimeric binding agent of embodiment 22 or 23, wherein the second antigen is an effector cell antigen.
  • Embodiment 27 The chimeric binding agent of any one of embodiments 1-26, wherein the second domain engages macrophages.
  • Embodiment 28 The chimeric binding agent of any one of embodiments 1-27, wherein the second domain does not significantly engage natural killer cells.
  • Embodiment 29 The chimeric binding agent of any one of embodiments 1-28, wherein the second domain does not significantly engage lymphocytes.
  • Embodiment 30 The chimeric binding agent of any one of embodiments 1-29, wherein the second domain specifically binds a protein on the surface of the myeloid-derived cell.
  • Embodiment 31 The chimeric binding agent of embodiment 30, wherein the second domain specifically binds an Fc-gamma receptor.
  • Embodiment 32 The chimeric binding agent of embodiment 30, wherein the second domain specifically binds Fc-gamma receptor I (FcyRI, CD64).
  • Embodiment 33 The chimeric binding agent of any one of embodiments 1-32, wherein the second domain comprises an Fc domain of an antibody.
  • Embodiment 34 The chimeric binding agent of embodiment 33, wherein the second domain comprises an Fc domain of an IgG antibody.
  • Embodiment 35 The chimeric binding agent of embodiment 34, wherein the second domain comprises an Fc domain of an IgG4 antibody.
  • Embodiment 36 The chimeric binding agent of embodiment 33, wherein the second domain comprises an Fc domain of an IgA or IgE antibody.
  • Embodiment 37 The chimeric binding agent of any one of embodiments 33-36, wherein the second domain further comprises a hinge domain of an antibody.
  • Embodiment 38 The chimeric binding agent of embodiment 37, wherein the second domain comprises the amino acid sequence of the heavy chain Fc domain and hinge domain of hLM609-h!gG4-S228P (SEQ ID NO:4) or a sequence at least 90% identical thereto.
  • Embodiment 39 The chimeric binding agent of any one of embodiments 1-38, wherein the amino acid sequence comprises a S228P mutation (Eu numbering system) in the hinge region.
  • Embodiment 40 The chimeric binding agent of embodiment 39, comprising the amino acid sequence of the hLM609-h!gG4-S228P heavy chain (SEQ ID NO:1) and light chain (SEQ ID NO:2) or a sequence at least 90% identical thereto.
  • Embodiment 41 The amino acid sequence of the hLM609-h!gG4-S228P heavy chain (SEQ ID NO:1) and light chain (SEQ ID NO:2) or a sequence at least 90% identical thereto.
  • the chimeric binding agent of embodiment 39 or 40 wherein 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/L328W/A330V/P331A/I332Y/E382V/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
  • Embodiment 42 A polynucleotide encoding the chimeric binding agent of any one of embodiments 1 -40.
  • Embodiment 43 A vector comprising the polynucleotide of embodiment 42.
  • Embodiment 44 A host cell comprising the polynucleotide of embodiment 42 or the vector of embodiment 43.
  • Embodiment 45 A composition comprising the chimeric binding agent of any one of embodiments 1-41 and a carrier.
  • Embodiment 46 A pharmaceutical composition comprising the chimeric binding agent of any one of embodiments 1-41 and a pharmaceutically acceptable carrier.
  • Embodiment 47 The pharmaceutical composition of embodiment 46, further comprising an additional therapeutic agent.
  • Embodiment 48 The pharmaceutical composition of embodiment 47, wherein the additional therapeutic agent is a chemotherapeutic agent.
  • Embodiment 49 A kit comprising the chimeric binding agent of any one of embodiments 1-41.
  • Embodiment 50 A method of targeting a myeloid-derived cell that accumulates in tumors to a cancer cell expressing at least one cell marker, comprising contacting the cancer cell and the myeloid-derived cell with an effective amount of the chimeric binding agent of any one of embodiments 1-41.
  • Embodiment 51 The method of embodiment 50, wherein the cancer cell expresses the at least one cell marker due to cellular stress.
  • Embodiment 52 The method of embodiment 50, wherein the cancer cell expresses the at least one cell marker due to undergoing an epithelial to mesenchymal transition.
  • Embodiment 53 A method of targeting a myeloid-derived cell that accumulates in mesenchymal tumors to an epithelial cancer cell expressing at least one mesenchymal cell marker, comprising contacting the cancer cell and the myeloid-derived cell with an effective amount of the chimeric binding agent of any one of embodiments 1-41.
  • Embodiment 54 The method of embodiment 53, wherein the myeloid-derived cell is a macrophage, dendritic cell, or granulocyte, such as a neutrophil, basophil, eosinophil, or mast cell.
  • the myeloid-derived cell is a macrophage, dendritic cell, or granulocyte, such as a neutrophil, basophil, eosinophil, or mast cell.
  • Embodiment 55 A method of treating a cancer expressing at least one cell marker in a subject in need thereof, comprising administering a therapeutically effective amount of the chimeric binding agent of any one of embodiments 1-41 or the pharmaceutical composition of any one of embodiments 46-48 to the subject, thereby treating the cancer.
  • Embodiment 56 A method of treating an epithelial cell cancer in a subject in need thereof, comprising administering a therapeutically effective amount of the chimeric binding agent of any one of embodiments 1-41 or the pharmaceutical composition of any one of embodiments 46-48 to the subject, thereby treating the epithelial cell cancer.
  • Embodiment 57 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 an antigen specifically bound by the chimeric binding agent of any one of embodiments 1-41 and enriched for myeloid-derived cells that accumulates in mesenchymal tumors; and b) administering a therapeutically effective amount of the chimeric binding agent of any one of embodiments 1-41 or the pharmaceutical composition of any one of embodiments 46- 48 to the subject, thereby treating the cancer.
  • Embodiment 58 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 an antigen specifically bound by the chimeric binding agent of any one of embodiments 1-41 and enriched for myeloid-derived cells that accumulates in mesenchymal tumors; and b) administering a therapeutically effective amount of the chimeric binding agent of any one of embodiments 1-41 or the pharmaceutical composition of any one
  • a method of treating an epithelial cell cancer in a subject in need thereof comprising the steps of: a) selecting a subject having epithelial cancer cells that are enriched for an antigen specifically bound by the chimeric binding agent of any one of embodiments 1-41 and enriched for myeloid-derived cells that accumulate in mesenchymal tumors; and b) administering a therapeutically effective amount of the chimeric binding agent of any one of embodiments 1-41 or the pharmaceutical composition of any one of embodiments 46-48 to the subject, thereby treating the epithelial cell cancer.
  • Embodiment 59 The method of embodiment 58, wherein 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.
  • Embodiment 60 The method of any one of embodiments 55-59, wherein the myeloid-derived cell is a macrophage, dendritic cell, or granulocyte, such as a neutrophil, basophil, eosinophil, or mast cell.
  • the myeloid-derived cell is a macrophage, dendritic cell, or granulocyte, such as a neutrophil, basophil, eosinophil, or mast cell.
  • Embodiment 61 The method of embodiment 60, wherein the epithelial cell cancer is a late stage epithelial cell cancer.
  • Embodiment 62 The method of embodiment 61, wherein one or more of the epithelial cells in the cancer have at least partially transitioned to mesenchymal cells.
  • Embodiment 63 The method of any one of embodiments 58-62, wherein the epithelial cell cancer is or has become chemotherapy resistant or refractory.
  • Embodiment 64 The method of any one of embodiments 55-63, wherein the cancer is a carcinoma such as a cancer of the gastrointestinal tract, breast, lungs (e.g., nonsmall cell lung cancer), colon, prostate, or bladder.
  • a carcinoma such as a cancer of the gastrointestinal tract, breast, lungs (e.g., nonsmall cell lung cancer), colon, prostate, or bladder.
  • Embodiment 65 The method of any one of embodiments 55-64, further comprising administering to the subject a CD47 blocking agent and/or an immune checkpoint inhibitor and/or an EGFR inhibitor.
  • Embodiment 66 The method of any one of embodiments 55-64, wherein the method does not comprise administering to the subject a CD47 blocking agent.
  • Embodiment 67 The method of any one of embodiments 58-66, wherein the epithelial cell cancer expresses CD47.
  • Embodiment 68 The method of any one of embodiments 58-66, wherein the epithelial cell cancer does not express CD47.
  • Embodiment 69 The method of any one of embodiments 55-68, further comprising administering to the subject an additional cancer therapeutic agent or treatment.
  • Embodiment 70 The method of any one of embodiments 55-69, wherein the chimeric binding agent or pharmaceutical composition is administered to the subject intravenously, subcutaneously, or intramuscularly or is injected in situ into or near the cancer.
  • Embodiment 71 The method of any one of embodiments 55-70, further comprising the step of isolating myeloid-derived cells from the subject, contacting the myeloid-derived cells with the chimeric binding agent or pharmaceutical composition, and administering the contacted myeloid-derived cells to the subject.
  • Embodiment 72 The method of any one of embodiments 55-71, wherein the subject is a human.
  • ITGB3 mRNA expression positively correlates with macrophage markers for kidney, breast, GBM, lung, stomach, prostate, pancreas, esophageal, and colorectal cancers, while no correlation is observed for renal papillary, sarcoma, thyroid, melanoma, and ovarian cancers.
  • ITGB3 positively correlates with other immune cell types such as mast cells, T cells and B cells, but this relationship is observed for a limited number of tumor types.
  • no correlation between ITGB3 and immune cell markers is observed for thyroid, melanoma, kidney papillary, and sarcoma despite these cancers having the highest median expression of ITGB3 across the TCGA pan-cancer dataset.
  • the percent of tumors with positive tumor cell expression of [33 ranges from 29-54% among the array slides examined, indicating there is a significant portion of [33+ tumors across this diverse population of tumor types, grades, and stages.
  • tumors with high tumor cell expression of integrin [33 are particularly enriched for TAMs, a component of the tumor microenvironment that contributes to tumor progression (Pathria, 2019), and that these cells might prove important when targeting tumors with certain therapeutic antibodies.
  • Tumor cell expression of integrin [33 is enriched after tumors acquire resistance to the EGFR inhibitor erlotinib in vivo
  • An anti-avP3 monoclonal antibody triggers macrophage-mediated tumor cell killing
  • LM609 which recognizes integrin av[33 on human but not mouse cells (Cheresh, 1987) and served as the parent antibody for a fully humanized version, Vitaxin/etaracizumab (Delbaldo, 2008; Gutheil, 2000).
  • LM609 was tested for its ability to block the growth of tumors that achieve erlotinib resistance by virtue of increase expression of integrin av[33.
  • HCC827 (5 x 10 6 tumor cells in 100 pl of PBS) avP3-negative human EGFR mutant lung cancer cells were subcutaneously injected to the right flank of female nu/nu mice (Charles River, 088, 8-10 weeks old). Tumors were measured with calipers twice per week.
  • mice with a tumor volume of 250-700 mm 3 were randomly assigned into groups treated with combinations of Captisol (oral, six times/week), PBS (i.p., twice/week), LM609 (i.p., 10 mg/kg, twice/week), or erlotinib (oral, 6.25 mg/kg, six times/week).
  • Vehicle-treated mice were sacrificed on day 15 due to large tumor size, and erlotinib groups on day 50. Tumors were placed into liquid nitrogen, OCT compound, or 10% formalin. As reported in Figure 2C of Wettersten et al., Cancer Res.
  • LM609 alone has no effect on the growth of HCC827 xenograft tumors prior to the development of drug resistance due to lack of the av[33 antigen. While mice treated with erlotinib alone showed an initial reduction in tumor size, this is followed by an eventual tumor re-growth and gain in av[33 expression. In contrast, the combination of erlotinib plus LM609 prolonged drug sensitivity and prevented the appearance of integrin [33 on tumor cells.
  • LM609 was tested for its ability to re-sensitize resistant tumors to the effects of erlotinib.
  • HCC827 or PC9 human lung cancer cells (5 x io 6 tumor cells in 100 pl of PBS) were subcutaneously injected to the right flank of female nu/nu mice (Charles River, 088, 8-10 weeks old) and tumors were measured with calipers twice per week. Animals with a tumor volume of 100-200 mm 3 were randomly assigned into groups treated with combinations of Captisol (oral, six times/week) or erlotinib (oral, 6.25 mg/kg, six times/week).
  • LM609 induces macrophage-mediated antibody-dependent cell-mediated cytotoxicity (ADCC)
  • TAMs were isolated from tumor tissues as described (Kaneda, 2016). Tumors were dissociated in HBSS containing collagenase IV (Sigma, C5138), hyaluronidase (Sigma, H2654), dispase 11 (Roche, 04942078001), and DNase IV (Millipore, D5025) at 37°C for 15 minutes. Cell suspensions were filtered through 70 pm cell strainers and washed with PBS.
  • BMDMs Mouse bone marrow-derived macrophages
  • PBMCs Human peripheral blood mononuclear cells
  • LRSC leukoreduction system chambers
  • PBMCs were isolated from LRSC using Histopaque-1083 (Sigma, 10831) following the manufacturer’s protocol.
  • PBMCs were incubated in tissue culture plates with human M-CSF (Peprotech, 300-25) for 5 days.
  • ADCC antibody-dependent cellular- cytotoxicity
  • LM609 showed robust ADCC activity using TAMs isolated from mouse Lewis lung carcinoma (LLC) tumors grown in immune-competent C57BL6 mice or immune-compromised athymic nude mice.
  • the antibody could also kill tumor cells using bone marrow derived macrophages (BMDM) isolated from healthy mice, as well as human monocyte-derived macrophages isolated from healthy donor blood.
  • BMDM bone marrow derived macrophages
  • LM609-mediated ADCC was not achieved with mouse NK cells or peripheral blood mononuclear cells (PBMCs) isolated from human blood, immune effector cell types to which antibodies are commonly engineered for optimal binding (Listinsky, 2013; Yu, 2017).
  • PBMCs peripheral blood mononuclear cells
  • NK cells are not correlated with [33 expression in tumors.
  • the mouse monoclonal antibody LM609 is able to selectively engage macrophages but not NK cells to mediate ADCC, and we reasoned that a humanized form of LM609 may be created that retains this functional distinction.
  • Antibody Fc engineering and glycoengineering strategies to enhance binding to NK cells for the purposes of triggering ADCC include changes to promote Fc binding to the only Fc receptor expressed on NK cells, CD16 (FcyRIII).
  • FcyRIII FcyRIII
  • our findings suggest that av[33- expressing tumors that are mesenchymal, stem-like, and drug-resistant contain high levels of macrophages, dendritic cells, and neutrophils (but not NK cells) as reported in Wettersten et al., Cancer Res. 79:5048 (2019).
  • an anti-av[33 antibody to induce ADCC that requires NK cell engagement represents a mismatch between the antigen (av[33) and the types of effector cells that are present within av[33 -expressing tumors.
  • antigen av[33
  • anti-av[33 could be engineered to recruit macrophages, this new antibody could show improved anti -tumor efficacy by more strongly triggering ADCC.
  • LM609 is a mouse monoclonal IgGlK antibody that recognizes human integrin av[33 (FIG. 2).
  • Several humanized forms of LM609 have previously been generated as hlgGl isotypes (FIG. 3 and FIG. 4). Since h!gG4 binds only to FcyRI/CD64 but not other Fc receptors, we created a new humanized form of LM609 that involved switching the isotype of etaracizumab/Vitaxin from a hlgGl isotype (FIG.
  • FIG. 5 shows the amino acid sequence alignment comparing humanized LM609 hlgGl vs. h!gG4-S228P forms.
  • Isotype switching to hIgG4 has been previously utilized for the generation of cancer therapeutics, although the rationale for this change is to create an antibody that lacks engagement of ADCC effector cells, most notably NK cells and monocytes. In contrast, macrophages are not widely considered to be mediators of ADCC. Considering that the mouse monoclonal antibody LM609 was able to recruit macrophages to induce ADCC, but not phagocytosis, our utilization of isotype switching to hIgG4 represents an unconventional and unexpected strategy to engage macrophages for ADCC.
  • NK cells utilize only FcyRIII/CD16 to engage antibody Fc regions
  • macrophages can utilize FcyRI/CD64.
  • hLM609-hIgG4-S228P is able to strongly activate FcyRI on effector cells
  • the hlgGl and hlgGl -I332E isotypes show lower levels of engagement (Fig. 7).
  • the hlgGl isotype can strongly activate FcyRIII and the hlgGl -I332E mutation enhances this as expected (Fig. 7).
  • hLM609-h!gG4 produced no activation of FcyRIII on effector cells, confirming that the h!gG4 isotype primarily interacts with FcyRI. While conventional thinking may suggest that isotype switching to h!gG4-S228P would ablate all effector cell engagement, we show here that hLM609-h!gG4-S228P is able to selectively engage and activate the Fc receptor expressed on macrophages, FcyRI/CD64.
  • FIG. 8 shows both IgGl and IgG4 forms of humanized LM609 blocked adhesion to fibrinogen without disrupting i -mediated adhesion to collagen.
  • the hIgG4-S228P form of humanized LM609 (but not the hlgGl-WT form) engaged primary human macrophages to induce ADCC for H1975 human lung cancer cells with endogenous expression of [33 (FIG. 9B).
  • Macrophage-mediated killing activity for the hIgG4-S228P form of humanized LM609 was further confirmed using macrophages isolated from three individual healthy blood donors with the polymorphic variants in CD16/CD32 as shown (FIG. 9C).
  • both LM609 and hLM609-IgG4- S228P are able to induce ADCC utilizing human macrophages as effector cells (FIG. 10A).
  • the hLM609-IgG4-S228P isotype is not able to utilize NK cells for tumor cell killing (FIG. 10B).
  • This distinction could produce a therapeutic advantage by achieving matching between the antigen (integrin av[33) and the types of effector cells that are particularly enriched in av[33-expressing tumors, such as macrophages.
  • hLM609-hIgGl the isotype that engages NK cells
  • hLM609-hIgG4-S228P the isotype that engages macrophages
  • the antibody affinity for these isotype variants is equivalent, as shown by their ability to block av[33-dependent cell adhesion (Fig. 8).
  • the anti-tumor activity for hLM609-hIgG4-S228P is superior to that of hLM609-hIgGl (FIG. 13), suggesting that the ability to selectively engage macrophages provides a therapeutic advantage for av[33 -expressing tumors with abundant macrophages but not NK cells.
  • hLM609-hIgG4- S228P was able to localize to the tumor to a greater degree than hLM609-hIgGl (Fig. 14). Without being bound by theory, it is thought that hLM609-hIgG4- S228P, because it engages fewer effector cells overall, is better able to locate to the tumor where primarily macrophages are located. In contrast, hLM609-hIgGl, which engages a wider variety of immune cells, may engage effector cells in the blood, lymph node, spleen, etc.
  • FIG. 15 shows hLM609-h!gG4-S228P is more effective than hLM609-h!gGl-WT against a cancer that endogenously expresses av[33.
  • Time to progression (tumor doubling in size) was significantly delayed by administration of hLM609-h!gG4-S228P relative to hLM609-h!gGl-WT.
  • this tumor model endogenous av[33 expression
  • it strongly supports the effectiveness of the present invention as a cancer treatment.
  • modifying the hLM609 antibody to switch from an IgGl to an IgG4 isotype increases the effectiveness of the antibody against av[33 expressing tumors.
  • a h!gG4-S228P form of humanized LM609 mimics the functional activity of mouse monoclonal LM609.
  • these antibodies are able to preferentially engage macrophages to induce killing of integrin av[33 -expressing tumor cells.
  • This antibody design strategy reflects the goal of matching the tumor cell antigen (av[33) with the appropriate ADCC-inducing effector cell (macrophages).
  • the antibody is better able to accumulate in the tumor upon binding to av[33 on tumor cells and/or CD64/FcyRI on macrophages.
  • Some therapeutic antibodies induce tumor cell killing via ADCC. This occurs when the antibody Fc region engages Fc receptors on immune effector cells to trigger the release of cytotoxic granules that induce tumor cell killing. Since this scenario typically involves antibody binding to CD16 on NK cells, many antibody glycoengineering and Fc engineering strategies have been designed to promote this interaction. Because IgG4 has high affinity for CD64, but weak affinities for all other receptors, IgG4 is generally considered to be a poor inducer of Fc-mediated effector functions. Therefore, isotype switching to IgG4 is an unexpected approach to enhance effector cell mediated killing of tumor cells.
  • the FDA has approved three h!gG4 tumor-therapeutic antibodies, pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), and cemiplimab (LIBTAYO), all of which target the immune checkpoint molecule PD-1 that is expressed mainly on activated T and NK cells.
  • These antibodies work by neutralizing T cell inhibition, that is, preventing the immunosuppressive consequences when PD-1 (on T and NK cells) binds to PD-L1 (on tumor cells).
  • the IgG4 subclass allows the antibody to block the function of PD-1 without engaging additional immune effector cells.
  • LM609 induced macrophage-ADCC was independent of CD64, since LM609 (and mouse IgGl antibodies) cannot bind to CD64. While LM609 triggered potent anti-tumor activity by selectively engaging macrophages, the inability of LM609 to bind CD64 suggested that CD64 engagement was not critical.
  • ADCP Being phagocytotic cells, macrophages are widely known to induce ADCP, and this is generally understood to involve CD 16 and CD32 engagement. As such, ADCP could be enhanced by isotype switching to IgG2, which has high affinity to CD32 that is mainly expressed in macrophages. However, the efficacy of such an antibody would be limited by expression of the CD47 “don’t eat me” signal on tumor cells. It has been less frequently reported that macrophages can also induce ADCC, with this activity linked to CD 16. Thus, it could not have been predicted that macrophage ADCC would be triggered by an IgG4-CD64 interaction.
  • Antibodies for cancer therapy include a number that recognize the epithelial-like tumor cell antigens EGFR, Her2, and EpCAM. Engineering of such antibodies can enhance ADCC by promoting the engagement of NK cells via CD16 binding to the antibody Fc.
  • cancer therapy and progression can eventually induce an EMT or enrichment with cancer stem cells that involves not only the loss of epithelial markers, but the exclusion or inactivation of NK cells and CD8+ T cells.
  • late-stage, mesenchymal-like, stem-like tumors become refractory to epithelial-targeting monoclonal antibodies that utilize NK cells for tumor killing.
  • EMT cancer-associated macrophages
  • tumor-associated macrophages are highly immunosuppressive and act to exclude T cells and NK cells, creating an immune-cold tumor microenvironment.
  • the advantage of the present invention is the ability to achieve “antigen-effector cell matching” to induce tumor cell killing by 1) recognizing a stem/mesenchymal marker on the tumor cell surface (integrin av[33), and 2) engaging tumor-associated macrophages to induce ADCC.
  • Tumor-associated macrophages correlate with response to epidermal growth factor receptor-tyrosine kinase inhibitors in advanced non-small cell lung cancer. Int J Cancer, 131(3): p. E227-35, 2012.
  • Kaneda M.M., K.S. Messer, N. Ralainirina, H. Li, C.J. Leem, S. Gorjestani, G. Woo, A.V. Nguyen, C.C. Figueiredo, P. Foubert, M.C. Schmid, M. Pink, D.G. Winkler, M. Rausch, V.J. Palombella, J. Kutok, K. McGovern, K.A. Frazer, X. Wu, M. Karin, R. Sasik, E.E. Cohen, and J.A. Vamer, PI3Kgamma is a molecular switch that controls immune suppression. Nature, 539(7629): p. 437-442, PMC5479689, 2016.

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des agents de liaison chimériques et des compositions les comprenant. L'invention concerne en outre des polynucléotides codant pour l'agent de liaison chimérique ainsi que des vecteurs et des cellules hôtes les comprenant. L'invention concerne en outre des procédés d'utilisation des agents de liaison chimériques pour induire une cytotoxicité cellulaire dépendante des anticorps de cellules cancéreuses épithéliales et des procédés de traitement de cancers de cellules épithéliales.
PCT/US2022/077218 2021-09-30 2022-09-29 Compositions et méthodes de traitement du cancer WO2023056326A1 (fr)

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WO2018183894A1 (fr) * 2017-03-31 2018-10-04 The Regents Of The University Of California Compositions et méthodes de ciblage et de destruction de cellules souches cancéreuses (csc) positives à alpha-v bêta-3 et de traitement de cancers pharmacorésistants
WO2021216956A1 (fr) * 2020-04-23 2021-10-28 Alpha Beta Holdings, Llc Compositions et méthodes pour traiter le cancer

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WO2018183894A1 (fr) * 2017-03-31 2018-10-04 The Regents Of The University Of California Compositions et méthodes de ciblage et de destruction de cellules souches cancéreuses (csc) positives à alpha-v bêta-3 et de traitement de cancers pharmacorésistants
WO2021216956A1 (fr) * 2020-04-23 2021-10-28 Alpha Beta Holdings, Llc Compositions et méthodes pour traiter le cancer

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FRANCESC MITJANS, DIANE SANDER, JAIME ADÁN, ARNE SUTTER, JOSEP M. MARTINEZ, CARLOTA-S. JÄGGLE, JOSEP M. MOYANO, HANS-GEORG KREYSCH: "An anti-alpha v-integrin antibody that blocks integrin function inhibits the development of a human melanoma in nude mice", JOURNAL OF CELL SCIENCE, COMPANY OF BIOLOGISTS LIMITED, CAMBRIDGE, vol. 108, no. 8, 1 August 1995 (1995-08-01), Cambridge , pages 2825 - 2838, XP002615476, ISSN: 0021-9533 *
HIROMI I. WETTERSTEN, TOSHIYUKI MINAMI, MEGAN M. KANEDA, LAETITIA SEGUIN, JUDITH A. VARNER, SARA M. WEIS , DAVID A. CHERESH: "Abstract 3966: Targeting integrin αvβ3-expressing cancer stem cells to manipulate tumor-associated macrophages", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 77, no. 13, Suppl., 1 July 2017 (2017-07-01), US, pages 3966, XP009531372, ISSN: 0008-5472, DOI: 10.1158/1538-7445.AM2017-3966 *
MULGREW KATHY, KINNEER KRISTA, YAO XIAO-TAO, WARD BETH K., DAMSCHRODER MELISSA M., WALSH BILL, MAO SU-YAU, GAO CHANGSHOU, KIENER P: "Direct targeting of αvβ3 integrin on tumor cells with a monoclonal antibody, Abegrin", MOLECULAR CANCER THERAPEUTICS, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 5, no. 12, 1 December 2006 (2006-12-01), US , pages 3122 - 3129, XP055859734, ISSN: 1535-7163, DOI: 10.1158/1535-7163.MCT-06-0356 *
WETTERSTEN HIROMI I., WEIS SARA M., PATHRIA PAULINA, VON SCHALSCHA TAMI, MINAMI TOSHIYUKI, VARNER JUDITH A., CHERESH DAVID A.: "Arming Tumor-Associated Macrophages to Reverse Epithelial Cancer Progression", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 79, no. 19, 1 October 2019 (2019-10-01), US, pages 5048 - 5059, XP055791572, ISSN: 0008-5472, DOI: 10.1158/0008-5472.CAN-19-1246 *

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