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WO2006073982A2 - Bispecific molecule comprising ligands for cell-surface protein and t-cell surface protein - Google Patents

Bispecific molecule comprising ligands for cell-surface protein and t-cell surface protein Download PDF

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Publication number
WO2006073982A2
WO2006073982A2 PCT/US2005/047222 US2005047222W WO2006073982A2 WO 2006073982 A2 WO2006073982 A2 WO 2006073982A2 US 2005047222 W US2005047222 W US 2005047222W WO 2006073982 A2 WO2006073982 A2 WO 2006073982A2
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WIPO (PCT)
Prior art keywords
cell
cells
molecule
cancer
aberrant
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Application number
PCT/US2005/047222
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French (fr)
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WO2006073982A3 (en
Inventor
Edward D. Ball
Jie-Hua Zhou
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Regents Of The University Of California
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Publication of WO2006073982A2 publication Critical patent/WO2006073982A2/en
Publication of WO2006073982A3 publication Critical patent/WO2006073982A3/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6875—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin
    • A61K47/6879—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin the immunoglobulin having two or more different antigen-binding sites, e.g. bispecific or multispecific immunoglobulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—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 the field of treatment of neoplastic diseases and uncontrolled cell growth. More specifically, it relates to compositions and methods for treating neoplasias with bispecific molecules comprising a portion that binds to a cell-surface protein on a T-cell, and a portion that binds to a cell- surface protein on a tumor cell. Description of Related Art
  • Neoplastic diseases or cancers, are a leading cause of death in the world. Neoplastic diseases can generally be thought of as resulting from defects in cellular mechanisms regulating cell growth and division. As such, numerous molecules that are involved in cell growth or regulation of cell growth have been identified as being involved in neoplasias. Of the more common molecules that have been identified are cell surface receptors. In neoplastic cells of certain tissues, certain cell surface receptors are expressed, whereas non-neoplastic cells of the same cell type do not show expression, or show low levels of expression, of the receptor. Indeed, many cell surface receptors have been found to be expressed improperly in neoplastic cells. [004] Numerous ways of treating neoplastic diseases have been developed over the years.
  • non-surgical treatments include administering to the patient substances that are cytotoxic, and thus cause cell death ⁇ i.e., administering chemotherapeutic agents), exposure to cell-killing amounts of radiation, or both.
  • cytotoxic agents use substances that do not differentiate between normal and neoplastic cells.
  • normal, healthy cells are killed as well.
  • the substances used for treatment have become more selective, being targeted only to growing cells or cells with proteins on their surfaces that are specific for cancer cells.
  • cytotoxic agents to be used as anti-cancer compounds should specifically target the cancer cells while avoiding non-cancer cells.
  • Antibodies that are specific for cell-surface proteins that are typically expressed on cancer cells, but not normal cells, are particularly well suited for treatment of cancers.
  • While such cancer cell-specific antibodies are generally well suited for targeted treatment of cancers, often, cell surface targets that are absolutely or highly specific for cancer cells are not available. In such situations, it might be necessary to select a cell surface target that is preferentially expressed on a certain cancer cell type, and risk the potential detrimental effects of the treatment on non-cancer cells expressing the cell surface target.
  • SCLC Small cell lung cancer
  • GFP Gastrin releasing peptide
  • SCLC cells are known to produce BN-like peptides that function as autocrine growth factors to promote SCLC growth.
  • GRP receptor GRP receptor
  • SCLC small cell lung cancer
  • BN/GRP-R peptide antagonist called Antag 2
  • Antag 2 a synthetic BN/GRP-R peptide antagonist
  • the bispecific molecule was found to mediate cytotoxicity of SCLC by interferon gamma (IFN ⁇ )-activated human monocytes in conjunction with chemotherapy.
  • IFN ⁇ interferon gamma
  • Cytotoxic T lymphocytes are potent immune effector cells. They can be activated through their T-cell receptor (TCR) to effectively kill a target cell. Antigen recognition is mediated by either the alpha/beta chain, or gamma/delta chain of the TCR. The alpha/beta chain forms a heterodimer that is closely associated with a cluster of transmembrane peptides to form a CD3 complex on the T cell surface. Li patients with advanced cancer, the host T cells are functionally suppressed and unable to mount an effective attack on cancer cells. There are several common mechanisms to explain why cancer cells escape the host immune system, including lack of the recognition antigens on the cancer cells and inadequate activation of immune trigger molecules on host immune effector cells.
  • OKT3 is an anti-CD3 monoclonal antibody that is specific for TCR.
  • BsAb bispecific antibodies
  • a BsAb, OKT3xTrastuzumab (anti-CD3xanti-HER2) has been shown in preclinical studies to effectively mediate the killing of HER2-positive breast cancer cells.
  • a phase I trial has been planned for patients with stage IV metastatic breast cancer.
  • a recombinant single-chain BsAb, anti-CD3xanti-CD19 has been shown to effectively activate autologous T cells from patients with advanced B cell lymphoma or leukemia, and effectively kill the autologous B cells at a low effector to target cell ratio.
  • siRNA small-interfering RNAs
  • shRNA short hairpin RNA
  • the present invention addresses the need in the art by providing new therapeutic molecules, compositions comprising those molecules, methods of treating neoplasias and other disease, disorders, and infections using those molecules, and methods of diagnosing neoplasias or the predisposition to neoplasias, other diseases and disorders, and infections using those molecules.
  • the therapeutic molecules comprise at least one portion that binds to a cell-surface protein of a target cell, and at least one portion that binds to a cell-surface protein of a T-cell of the patient.
  • the therapeutic molecule is an immunotherapeutic bispecific molecule comprising an antigen-binding region of an anti-CD3 monoclonal antibody (or an equivalent structure created by known techniques) and an antagonist for a cell- surface receptor on a tumor cell.
  • the immunotherapeutic molecule comprises an anti-CD3 monoclonal antibody and the bombesin (BN) antagonist (Antag2).
  • BN bombesin
  • the exemplary bi-specific molecule (BsMoI) is capable of binding, through its antibody portion, to CD3 present on T cells, and to SCLC or other cancer cells expressing BN/GRP-R, thus bridging the two cells.
  • Binding of a BsMoI according to the invention can effectively activate T cells and induce growth inhibition of cancer cells at a low E:T ratio. Accordingly, the BsMoI of the invention can be used in therapy of cancer.
  • the BsMoI targets the GRP-R on the tumor cell surface and a cytotoxic trigger molecule on immune effector cells to activate antibody-dependent, cell-mediated cytotoxicity, hi experiments in vitro, tumor cell growth inhibition of 74% was observed when BsMoI was present, compared with 33% inhibition when tumor cells were cultured with only 0KT3, and 4% inhibition was observed when no antibody was present. If tumor cells were pre- incubated with free antagonist peptide, the BsMoI targeted cytotoxicity was blocked. So, the BsMoI interrupts the BN/GRP binding to BN/GRP-R and also induces a polyclonal T cell activation and specific targeting of tumor cells which results in tumor cell lysis and apoptosis.
  • the present invention provides a molecule that can target T-cells to aberrant cells in a patient (also referred to herein as a "subject") and cause the death of those aberrant cells.
  • this molecule is referred to as a "bi-specific" molecule, without limitation to the actual number of binding sites available on any particular molecule.
  • the function of the molecule is provided, at least in part, by the presence of at least two distinct regions or portions: one region having a three-dimensional structure that is capable of binding to a cell-surface protein on the surface of a target cell (e.g., a cancer cell), the other region having a three-dimensional structure that is capable of binding to a cell-surface protein on the surface of a T-cell. Binding of both regions to their respective binding partners (i.e., proteins on the respective cells) causes the T-cell to come into close proximity to the target cell, and ultimately cause the death of the target cell.
  • the invention provides compositions comprising the bi- specific molecule of the invention.
  • compositions comprise the bi- specific molecule of the invention and at least one other substance that is compatible with the bi-specific molecule, such as a solvent, a carrier, or a cell
  • the composition is a pharmaceutical composition that comprises the bi- specific molecule of the invention and at least one pharmaceutically acceptable or biologically tolerable substance.
  • the invention provides nucleic acids encoding at least part of a bi-specific molecule of the invention.
  • the bi-specific molecule comprises an amino acid sequence that may be encoded by a nucleic acid.
  • This aspect of the invention includes nucleic acids comprising not only the sequence encoding at least a portion of a bi- specific molecule, but other sequences that are useful in expressing and/or maintaining nucleic acids in a cell. Thus, it includes vectors for expressing or maintaining the coding sequences. Compositions comprising the nucleic acids of the invention are provided as well.
  • the methods comprise expressing a nucleic acid encoding at least a portion of a bi-specific molecule of the invention. Where the bi-specific molecule is entirely coded by a nucleic acid, the method does not require any further steps. Where the bi-specific molecule comprises non-proteinaceous material, the method further comprises associating the proteinaceous portion(s) with the non- proteinaceous portion(s) through covalent, ionic, or hydrophobic bonding. In embodiments, the methods of making include purifying, at least to some extent, the bi-specific molecule away from other substances that are present in the environment where the bi-specific molecule is found.
  • the invention provides cells.
  • the cells can comprise the bi-specific molecule or a portion thereof.
  • the cells can comprise one or more nucleic acids that encode at least a portion of the bi-specific molecule of the invention.
  • the cells can be useful for expressing the bi-specific molecule or at least a portion of it. They can also be useful for therapy for one or more neoplasias, diseases, disorders, or infections.
  • the cells are T-cells comprising a bi- specific molecule associated with the cell by way of attachment to a cell-surface protein.
  • the present invention provides methods of treating a patient suffering from a neoplasia or other disease or disorder, or an infection.
  • the method comprises administering at least one bi-specific molecule according to the invention, at least one nucleic acid according to the invention, or at least one cell according to the invention, to a patient in need thereof in an amount sufficient to inhibit the growth or kill at least one neoplastic cell, at least one cell of a disease or disorder, or at least one infected cell.
  • the step of administering is repeated two or more times.
  • the invention provides for use of a bi-specific molecule of the invention in the production of a therapeutic or pharmaceutical composition.
  • nucleic acid encoding at least a portion of a bi-specific molecule of the invention in the production of a therapeutic or pharmaceutical composition. It further provides for use of a cell of the invention in the production of a therapeutic or pharmaceutical composition.
  • the invention provides for use of a bi-specific molecule of the invention in treating a subject suffering from a neoplasia or other disease or disorder or an infection.
  • the invention provides for use of a nucleic acid of the invention in treating a subject suffering from a neoplasia or other disease or disorder or an infection.
  • the invention provides for use of a cell of the invention in treating a subject suffering from a neoplasia or other disease or disorder or an infection.
  • the present invention provides methods of diagnosing neoplasias or a predisposition to neoplasias, or other diseases or disorders or infections.
  • the methods of diagnosing are methods of detecting the presence of a cell-surface protein on a target cell.
  • the methods comprise exposing a cell that is or is suspected of being a neoplastic cell, a cell of another disease or disorder, or an infected cell to a bi-specific molecule of the invention and determining whether the bi-specific molecule has bound to the cell.
  • the method further comprises determining whether the molecule causes inhibition of cell growth or cell death.
  • the invention provides at least one container that contains a bi-specific molecule of the invention, a nucleic acid of the invention, and/or a cell of the invention.
  • the container can be any container, but in embodiments, it is a vial or the like, such as those used to contain pharmaceutically active substances.
  • Figure 1 depicts the relevant amino acid sequences of bombesin, gastrin- releasing peptide, and the Antag2 peptide of the present invention.
  • Figure 2 depicts a schema of the chemical conjugation of the BsMoI.
  • Figure 3 depicts general protocol for preparing and testing T cells for response to various treatments.
  • Figure 4 shows the results of flow cytometry assays of the BsMoI of the invention binding to four different SCLC cell lines, H345 (Figure 4A), H69 ( Figure 4B), DMS273 (Figure 4C), and SHP77 (Figure 4D).
  • Figure 5 shows flow cytometry analysis of the BsMoI binding to lymphocytes (Figure 5A) and monocytes ( Figure 5B).
  • Figure 6 shows flow cytometry analysis of the specificity of BsMoI binding to SCLC cell line HE34 after treatment with bombesin (Figure 6A), Antag2 (Figure 6B), and angiotensin (Figure 6C).
  • Figure 7 shows the inhibition of growth of cells after treatment with various compounds.
  • Figure 8 shows the growth inhibition of DMS273 cells by T cells from four normal donors at an E:T ratio of 2.5:1.
  • Figure 9 shows analyses of tumor cell growth inhibition and the dose- response effect of BsMoI.
  • Figure 10 shows the effect of IL-2 on thymidine incorporation (Figure 10A) and the effect of increasing concentration on thymidine incorporation ( Figure 10B).
  • Figures 1 IA and 1 IB show flow cytometry data relating to annexin V and PI expression in SCLC cells.
  • Figure 12 shows data relating to apoptosis and necrosis of cells exposed to BsMoI.
  • Panel A DMS273 cells;
  • Panel B SHP77 cells;
  • Panel C H345 cells.
  • Figure 13 depicts data relating to release of interferon gamma by T cells in culture with various SCLC cells.
  • Figure 14 depicts Western blots of various cell proteins implicated in apoptosis after exposure of cells to BsMoI.
  • Panel A shows Bcl-2 family protein expression in 4 SCLC cell lines.
  • Panel B shows the expression of anti-apoptotic protein Bcl-2 and pro-apoptotic proteins Bax and Bak after ADCC.
  • Panel C shows the changes of Bcl-2 expression, Bad/p-Bad ratio during ADCC in H345 cells.
  • Figure 15 depicts Western blots of various cell proteins implicated in apoptosis after exposure of cells to BsMoI.
  • bi-specific molecules also referred to herein as "BsMoI" that comprise one or no sequences from antibodies.
  • BsMoI are effective at targeting neoplastic cells and other cells exhibiting signature proteins on their cell surfaces
  • one domain of the BsMoI is specific for a T cell receptor while the other is specific for a protein on the surface of a neoplastic cell
  • a bi-specific molecule targeting BN/GRP-R and immune trigger molecules can mediate an effective destruction of SCLC cells
  • a combined chemotherapy and BsMol-targeted immunotherapy significantly increases the destruction of SCLC cells in vitro.
  • the present invention uses the BsMoI to recruit cytotoxic T cells to SCLC cells, either in vitro or in vivo, to inhibit the growth of the cells.
  • Treatment of the cells can further comprise combining this targeted immunotherapy with an anti- Bcl-2 approach to further enhance the specific SCLC destruction.
  • the present invention provides a molecule that can target T cells to aberrant cells in a patient (also referred to herein as a "subject") and cause the death of those aberrant cells.
  • this molecule is referred to as a "bi-specific" molecule, without limitation to the actual number of binding sites available on any particular molecule.
  • the function of the molecule is provided, at least in part, by the presence of at least two distinct regions or portions: one region having a three-dimensional structure that is capable of binding to a cell-surface protein on the surface of a target cell ⁇ e.g., a cancer cell), the other region having a three-dimensional structure that is capable of binding to a cell-surface protein on the surface of a T cell. Binding of both regions to their respective binding partners ⁇ e.g., to proteins on the respective cells) causes the T cell to come into close proximity to the target cell, and ultimately causes cessation of growth or, preferably, the death of the target aberrant cell.
  • the bi-specific molecule of the invention comprises at least one portion that binds to a molecule on the surface of a T cell.
  • Many molecules that appear on the surface of T cells are known, and any of these molecules may be used as targets for binding of the bi-specific molecule of the invention.
  • cell surface receptors such as TCR, CD2, CD3, CD4, CD8, and CD56 are present on the surface of T cells.
  • binding of the bi-specific molecule to the T cell surface molecule causes activation of the T cell.
  • T cell surface molecules that are targeted may simply be selected based on their presence and potential involvement in the cell killing process. In other words, one may select any molecule on the surface of a T cell, because the primary goal of the bi-specific molecule is to bring the T cell into close proximity to the aberrant cell that is targeted for destruction. It is preferred however, that the T cell surface molecule is selected such that it not only functions in binding of the bi-specific molecule, but, when bound by the bi-specific molecule, also produces at least one substance that causes or participates in the destruction of the target aberrant cell.
  • the molecule on the surface of the T cell that is the target of the bi-specific molecule can be any molecule.
  • it may be a protein, a lipid, a lipoprotein, a glycoprotein, a sugar, a polysaccharide, any other simple or complex molecule known to be present on the surface of cells or viruses, or a combination of two or more of these.
  • the number of molecules that are present on the T cell surface the molecule may be present as a single copy or multiple copies, or may be present only on a sub-set of T cells that are present in a particular subject.
  • the molecule is a protein or comprises a peptide portion.
  • the bi-specific molecule of the invention comprises at least one portion that binds to a target aberrant cell.
  • aberrant cells express on their surfaces complements of molecules, which are generally known and studied in the art. Certain of these molecules, or collections of molecules, are either specific for each different aberrant cell, or are semi-specific (i.e., are present on the target cell and only certain other cells). Any of these molecules may be targeted by the bi-specific molecule of the invention.
  • the bi-specific molecule may comprise a portion that binds to any of the following cell surface proteins or members of the following cell surface protein families: the ErbB family of protein tyrosine kinases (e.g., erbBl/HER- 1/EGFR, erbB2/HER-2/neu, erbB3 and erbB4), which are overexpressed in some tumor cells and contribute to cell proliferation, regulation of apoptotic cell death, angiogenesis, and metastatic spread; epithelial growth factor receptor (EGFR), which is implicated in breast, non small cell lung, colorectal, bladder, prostate, ovarian, and pancreatic cancers; the HER family of proteins, which are involved in breast and endometrial cancers (e.g, HER2, HER2/neu) - representative ligands include, but are not limited to, heregulin, neuregulin, epiregulin and biregulin; vascular endothelial growth factor (VEGF), which is
  • Certain non-limiting ligands for these receptors include the CCR7 ligands CCL19/ELC and CCL21/SLC, and the CXCR5 ligand CXCLl 3/BCA-l.
  • targets for melanoma can be the gangliosides GM2 and GD3.
  • the CD4 receptor on HIV infected cells may serve as a target for aberrant cells.
  • the bi-specific molecule may also bind to the following exemplary molecules, which are known to be present on certain neoplastic or infected cells: CXCR4 receptor on small cell lung cancer (involving the chemokine stromal derived factor- 1(SDF-I /CXCLl 2) ligand); members of the ERK pathway, such as RET in thyroid cancer; proteins expressed in cells infected with the Hepatitis C virus, such as CD81 tetraspanin (TAPA-I) and the scavenger receptor SR-BI, which binds to integrin beta 1 and integrin alpha 3, among other things; the DC-SIGN (dendritic cell- specific ICAM-grabbing non-integrin, where ICAM is the intercellular adhesion molecule), which is a mannose-specific C-type lectin expressed by dendritic cells; and CCR5 and CXCR4, which have been shown to be co-receptors for HIV infection (ligands include SDF-I
  • the bi-specific molecule of the invention comprises at least one portion that can bind to a molecule, including those well-known proteins discussed above and known in the art, present on the surface of an aberrant cell, such as a neoplastic cell or a cell infected with a virus or intracellular parasite.
  • a bi-specific molecule of the invention may have a portion that binds to a cell surface protein that, when activated, initiates an apoptosis pathway.
  • a bi-specific molecule of the invention may have a portion that binds to a cell surface protein that causes the cell to enter a senescence phase, in which cell growth is halted, but the cell does not necessarily begin a process of self-destruction.
  • Many such proteins are known in the art, as are ligands for them, and any of these proteins and ligands can be used as part of a bi-specific molecule of the invention.
  • one or more of the active portions or regions comprise a 3 -dimensional structure that is identical to a ligand for a cell-surface molecule on a T cell or on a target aberrant cell.
  • one or more of the active portions has a 3-dimensional structure that is similar, but not exactly the same, as a ligand for a cell-surface molecule on a T cell or a target aberrant cell, hi such a situation, the ligand can be an antagonist of the true ligand, and can bind to the cell surface molecule, but not cause the same biological effect. Alternatively, it can bind equally well or better, and result in an equally good or better response from the cell.
  • the bi-specific molecule of the invention can have three or more active portions. That is, the terminology "bi-specific” is not meant to limit the molecule of the invention to only two portions that can bind cells. Rather, the bi-specific molecule can have two or more portions that bind to cells of interest. Typically, the bi-specific molecule has at least one portion that binds to a T cell, and at least one other portion that binds to an aberrant cell of interest.
  • the bi-specific molecule has one portion that binds to a T cell and one portion that binds to an aberrant cell, hi other embodiments, the bi-specific molecule has one portion that binds to a T cell and two or more portions that bind to a target aberrant cell. In yet other embodiments, the bi-specific molecule has one portion that binds to an aberrant cell and two or more portions that bind to a T cell. In yet further embodiments, the bi- specific molecule comprises two or more portions that bind to a T cell and two or more portions that bind to an aberrant cell. Any number of combinations of active portions can be envisioned, and all of those combinations are included within the scope of the present invention.
  • the portions may be specific for the same cell surface molecule, or may be specific for different ones.
  • the specificity of the bi-specific molecule may be increased. For example, where a tumor cell expresses a complement of signature proteins on its surface, but one or more of those proteins is also expressed on other, non-tumor cells, inclusion of two or more portions of the bi-specific molecule that are individually specific for each of the proteins on the tumor cell, improved binding to the tumor cell might be obtained, and an increased effectiveness might be achieved.
  • an aberrant cell expresses two different proteins on its surface, each of which is involved in a distinct cellular process that could be taken advantage of for inhibition of cell growth or for cell killing
  • a bi-specific molecule comprising ligands for both of those cell surface proteins can be designed and used.
  • the two or more active portions are linked to each other.
  • Linkage can be through any suitable association, such as through covalent, ionic, or hydrophobic bonding.
  • the main concern in selecting the type of association is whether the molecule will remain intact throughout the time in which it is expected to be active. That is, for the bi-specific molecule to function as envisioned, the two (or more) active portions should be sufficiently well linked that they do not dissociate to an unacceptable extent under the conditions in which they are to be used. Thus, it is preferred that the active portions be covalently linked.
  • Linking of two or more molecules, whether they be peptides, sugars, nucleic acids, or any other organic or inorganic molecules to form the bi-specific molecule of the invention can be through any suitable techniques.
  • a number of suitable techniques for linking two or more such molecules are well known in the arts of chemistry and biochemistry, and need not be detailed here. It is now a routine matter for those of skill in the art to link any number of different types of molecules together to achieve a product.
  • the bi-specific molecule comprises of two or more peptide active portions
  • the active portions may be encoded as a single chain from a nucleic acid that has been engineered to express both portions.
  • the entire sequence may be chemically synthesized.
  • the two portions may be separately synthesized (e.g., chemically or by expression of two separate nucleic acids), then fused. Where more than two peptide portions are used, a branching structure may be used according to known principles of protein synthesis.
  • the active portion may be composed of only those structural portions that are required to form the proper 3-dimensional features for activity, or may comprise additional atoms/moieties/structures, which do not play any or any significant role in forming or maintaining the proper 3-dimensional structure.
  • the molecule may consist of the active portions, linked together, or may comprise the active portions and other portions.
  • the active portions of the bi-specific molecule may be fused directly to each other (or to at least one other active portion of the molecule), or may be linked via a linker, stuffer, etc.
  • the linker may be provided to better ensure proper folding of protein sequences, to provide sufficient distance between one or more active region and one or more other active region, or to provide flexibility to the molecule, or for any other reason that appears to be important to those practicing the invention.
  • the size and structure of the linker is not critical to practice of the invention, although a linker should be chosen that does not interfere with the function of the active regions.
  • the bi-specific molecule of the invention comprises an antibody framework, onto which an SCLC-specific ligand is grafted.
  • the bi-specific molecule comprises an antibody, or a portion of an antibody (e.g, the Fc region, Fab, Fab', single chain, CDR).
  • the antibody or fragment may be from any source, including, but not limited to, humans, other mammals (e.g., mouse, rat, monkey, rabbit).
  • the antibody may be a polyclonal antibody, but is preferably a monoclonal antibody.
  • the antibody or fragment may also be a modified antibody, such as a humanized antibody, a chimeric antibody, or a fusion antibody, hi certain embodiments, an antibody framework is used, where the antigen binding region has been engineered to contain a particular 3-dimensional structure that mimics a ligand or, more preferably, an antagonist of a ligand.
  • the aberrant cell can be any cell that is abnormal in any way.
  • it can be a cell that has an abnormal growth profile (e.g., uncontrolled growth, as seen in neoplastic cells).
  • It also can be a cell that expresses one or more proteins that are not normally expressed by cells of its type, or that expresses one or more proteins encoded by a virus or intracellular parasite, or that are expressed by the cell as a result of infection by a virus or intracellular parasite (including certain bacteria that reproduce within cells).
  • It can also be a cell that overexpresses a protein that is normally found in the cell, such as on the surface of the cell.
  • an aberrant cell can be a neoplastic cell, such as a cell from a benign tumor or a cell from a malignant tumor. It can be a cell that is infected with a virus, such as HIV, HCV, HBV, influenza, or papilloma virus. It further can be a cell that is infected with an intracellular parasite, such as a mycobacterium or any of the various single-celled eukaryotic intracellular parasites.
  • a virus such as HIV, HCV, HBV, influenza, or papilloma virus.
  • an intracellular parasite such as a mycobacterium or any of the various single-celled eukaryotic intracellular parasites.
  • neoplastic cell is included among the aberrant cells of the invention, including, but not limited to, lung cancer cells, breast cancer cells, prostate cancer cells, colorectal cancer cells or other cancer cells of the gastrointestinal tract, kidney cancer cells, hepatocarcinoma cells, brain cancer cells, pancreas tumor cells, and esophageal cancer cells.
  • Other non-limiting examples of cells are uterine cancer cells, cervical cancer cells, endometrium cells, melanoma cells, renal cancer cells, and Kaposi's sarcoma cells, hi addition, an exemplary cell is any cells that expresses GRP-R on its cell surface.
  • the invention provides compositions comprising the bi- specific molecule of the invention.
  • the compositions comprise the bi- specific molecule of the invention and at least one other substance that is compatible with the bi-specific molecule, such as a solvent, a carrier, or a cell.
  • the composition comprises two or more different bi-specific molecules.
  • the composition is a pharmaceutical composition that comprises the bi-specific molecule of the invention and at least one pharmaceutically acceptable or biologically tolerable substance.
  • the substance can be any of the well known substances present in pharmaceuticals, including, but not limited to, water or an aqueous liquid (including saline and its various medicinal/pharmaceutical forms), one or more fillers, binders, colorants, stabilizers, buffers, or other substances that stabilize or promote effective uptake of pharmaceutically active compounds.
  • the substance may also be a sugar or other substance that is effective in protecting compounds during lyophilization.
  • any of the well known reagents and other substances that are used in cell culture may be included in compositions according to the invention.
  • the composition is a pharmaceutical. Accordingly, the composition may be a capsule, pill, or any other dosage form that can be ingested or taken in through a mucous membrane.
  • composition may also be an injectable or infusible liquid, or a solid suitable for adding to a liquid to make an injectable or infusible liquid composition.
  • Any of the various compositions known as useful for delivering pharmaceutically active substances to a patient are envisioned as part of this invention.
  • the invention provides nucleic acids encoding at least part of a bi-specific molecule of the invention. As discussed above, the invention encompasses bi-specific molecules that consist of or comprise amino acid sequences.
  • amino acid sequences can be encoded by a nucleic acid of the invention, hi embodiments where the bi-specific molecule comprises two or more amino acid sequences fused together, the entire fusion sequence may be encoded by a nucleic acid of the invention, hi particular embodiments, the invention provides nucleic acids that encode an antibody or portion thereof (e.g., Fc portion, single chain antibody, etc.).
  • This aspect of the invention includes nucleic acids comprising not only the sequence encoding at least a portion of a bi-specific molecule, but other sequences that are useful in expressing and/or maintaining nucleic acids in a cell.
  • compositions comprising the nucleic acids of the invention are provided as well. These compositions typically comprise the nucleic acid of the invention along with one or more other substance, including, but not limited to one or more enzyme or reagent useful for expressing a polypeptide from a nucleic acid. Thus, the compositions may include any of the numerous polymerases available commercially. It can also comprise any substance that is typically used in storing nucleic acids in a stable manner, such as in a freeze-dried state.
  • Methods of making the bi-specific molecule of the invention comprise combining two or more substances that comprise active regions, where at least one of the active regions is capable of binding to a molecule on the surface of a T cell and at least one other of the active regions is capable of binding to a molecule on the surface of an aberrant cell of interest.
  • Cell surface molecules for each type of cell are known in the art, and any such molecule may be selected as a target.
  • the act of combining can be any action that results in two or more active regions becoming associated with each other.
  • it can comprise mixing the two or more substances under conditions that permit covalent, ionic, or hydrophobic bonding, hi embodiments, it comprises covalently bonding the substances.
  • combining can be accomplished in two or more steps, or in a single step.
  • bonding can be accomplished in two or more steps or in a single step. For example, where a bi-specific molecule comprising three active regions is made, one may combine two of the three together, bond them together, add the third region to the fusion product, and bond the third region to create a fusion product comprising all three regions.
  • portion A bound to portion B which is bound to portion C
  • portion A bound to portion C which is bound to portion B
  • portion A bound to portion B and C etc.
  • the bi-specific molecule comprises an amino acid portion.
  • the methods can comprise expressing a nucleic acid encoding at least a portion of the bi-specific molecule of the invention.
  • the method might not require any further steps. However, it might require further steps, including permitting the expressed protein to fold into a proper three dimensional shape.
  • the entire bi-specific molecule may be expressed as a single-chain protein from a single nucleic acid.
  • two or more different amino acid sequences may be part of the bi-specific molecule, and they may be expressed independently of each other, then associated with each other through covalent, ionic, or hydrophobic bonding.
  • the method further comprises associating the two or more portions with each other.
  • one or more of the portions are proteinaceous portions, and these are associated with the non- proteinaceous portion(s) through covalent, ionic, or hydrophobic bonding.
  • the methods of making include purifying, at least to some extent, the bi-specific molecule away from other substances that are present in the environment where the bi-specific molecule is found.
  • the method can further comprise making a composition, such as a pharmaceutical composition, comprising the bi-specific molecule. In such methods, at least one bi-specific molecule is combined with at least one other bi-specific molecule or one or more other substances that are pharmaceutically acceptable or biologically tolerable.
  • the invention provides cells.
  • the cells can comprise one or more bi-specific molecules or portions thereof, or one or more nucleic acids that encode at least a portion of at least one bi-specific molecule of the invention.
  • the cells may comprise the bi-specific molecules or nucleic acids internally or as one or more molecules that are at least partially exposed to the exterior of the cell.
  • one or more bi-specific molecules of the invention are attached to the exterior of a cell, such as by way of ionic or hydrophobic interactions with one or more substances on the surface of the cell.
  • Cells of the invention have multiple uses.
  • the cells can comprise nucleic acids according to the invention and can be useful for expressing the bi-specific molecule or at least a portion of it.
  • the cells can be used in a method of making the bi-specific molecule of the invention.
  • a single cell expresses multiple bi-specific molecules of the invention.
  • Cells of the invention can further be useful for research to determine various characteristics of bi-specific molecules of the invention. For example, they can be used to identify particular bi-specific molecules having high specificity, high affinity for particular aberrant cells or T cells, and/or high level of activation of T cells or activation of apoptosis pathways in target aberrant cells, among other things.
  • Cells of the invention can also be useful for therapy for one or more neoplasias, diseases, disorders, or infections.
  • cells of the invention can be produced in vivo by administration of one or more bi-specific molecules of the invention to a patient, with the result being an activated T cell linked via the bi- specific molecule to a target aberrant cell.
  • cells of the invention can be made ex vivo by loading one or more bi-specific molecules of the invention onto T cells, then re-introducing the loaded T cells into the patient to provide an activated T cell that can bind to a target aberrant cell of interest.
  • the cells are T-cells comprising a bi-specific molecule associated with the cell by way of attachment to a cell-surface protein
  • the cell is a target aberrant cell comprising one or more bi- specific molecules associated with the cell by way of attachment to a cell-surface protein.
  • the present invention provides methods of treating a patient suffering from a neoplasia or other disease or disorder, or an infection, hi general, the method comprises administering at least one bi-specific molecule according to the invention, at least one nucleic acid according to the invention, or at least one cell according to the invention, to a patient in need thereof in an amount sufficient to inhibit the growth or kill at least one neoplastic cell, at least one cell of a disease or disorder, or at least one infected cell (i.e., an aberrant cell).
  • the methods provide a treatment for the patient.
  • the methods provide a therapy for the patient by reducing or halting the growth of at least one aberrant cell in the patient's body.
  • the methods result in killing of at least one aberrant cell in the patient's body.
  • the method results in reduction in the growth rate of at least one tumor (including non- solid tumors), as compared to the growth rate seen prior to treatment or as compared to an equivalent subject who did not receive the treatment.
  • the treatment results in inhibition of tumor growth, whereas in others, it results in a reduction in tumor size or a reduction in the number of neoplastic cells.
  • the method results in elimination of all or essentially all of the neoplastic cells.
  • the method of treating can comprise a treatment regimen. Accordingly, the method can comprise repeating the step of administering one or more times. Furthermore, the step of administering can comprise administering one or more other bi-specific molecules or one or more other substances that can have a treating effect on the target aberrant cells. Multiple rounds of treatment, each with or some without a bi-specific molecule of the invention, may be performed. Treatment regimens can include dosing with chemotherapeutic agents.
  • Administering can be accomplished by any suitable method known in the art. In general, administering is performed on a subject in need of treatment for an aberrant cell, such as a neoplastic cell or an infected cell. The amounts administered are amounts sufficient to achieve a therapeutic effect, such as to cause an aberrant cell to cease growing or to die. Numerous techniques for administering substances systemically to the blood system of humans and animals is known, and any one can be used. For example, administration can be through bolus injection of the substance into a vein or artery, administration over minutes or hours by way of intravenous infusion, or oral administration byway of liquid or solid (e.g., tablet, capsule, powder).
  • liquid or solid e.g., tablet, capsule, powder
  • chemotherapeutic substances such as the bi-specific molecule of the present invention, to be administered to patients are well known to those of skill in the art, and appropriate amounts to be administered in accordance with the present invention can be determined without excessive experimentation. As a general rule, chemotherapeutic substances can be administered to subjects in therapeutically effective amounts.
  • Those amounts generally range from about 0.01 g/m 2 of body area to about 30 g/m 2 body area, and can be administered over any amount of time, such as a short period (about 6 hours or less) or a much longer period (about 96 hours or longer) through continuous infusion.
  • Dosing regimens can include considerably longer times, such as two weeks, one month, or more.
  • doses of the chemotherapeutic substance(s) can be relatively short (e.g., the entire dose being administered in one to several hours), and the doses repeated at regular intervals, such as daily, once weekly, twice weekly, or at other intervals.
  • chemotherapeutic dosing regimens are known to those of skill in the art, and any suitable regimen can be used.
  • treatment comprises administering one or more bi-specific molecules based on an antibody frame
  • it will generally be administered in an amount from about 0.1 mg/m 2 of the patient's body area to about 15.0 mg/m 2 body area.
  • the injections can be administered at days 1 and 14 of a treatment regimen.
  • one or more bi-specific molecules can be administered in a regimen comprising two treatment cycles, hi the first cycle, the molecule can be administered at exactly or about 10 - 20 mg/m 2 daily for 4 days, hi the second cycle, the molecule can again be administered at exactly or about 10 - 20 mg/m 2 at 10-12 days after completion of the first cycle.
  • the method of treating is a method of treating a patient suffering from a neoplasia.
  • the method is preferably a therapeutic method that results in cessation of proliferation of aberrant cells, reduction in the number of aberrant cells in the patient, or elimination of the aberrant cells in the patient. It can thus be a method of treating cancer.
  • it is a method of treating small cell lung cancer (SCLC).
  • SCLC small cell lung cancer
  • it is a method of treating breast cancer, a method of treating prostate cancer, a method of treating colorectal cancer, a method of treating non-small cell lung cancer, or a method of treating two or more of these.
  • the invention provides a method of treating a neoplasia, including a malignant tumor and a benign tumor.
  • the neoplasia treated can be a solid tumor or a non-solid tumor.
  • the invention further provides for use of a bi-specific molecule of the invention in the production of a therapeutic or pharmaceutical composition. It also provides for use of a nucleic acid encoding at least a portion of a bi-specific molecule of the invention in the production of a therapeutic or pharmaceutical composition. It further provides for use of a cell of the invention in the production of a therapeutic or pharmaceutical composition. Likewise, the invention provides for use of a bi-specific molecule of the invention in treating a subject suffering from a neoplasia or other disease or disorder or an infection. Thus, the invention provides for use of a nucleic acid of the invention in treating a subject suffering from a neoplasia or other disease or disorder or an infection. Accordingly, the invention provides for use of a cell of the invention in treating a subject suffering from a neoplasia or other disease or disorder or an infection.
  • the present invention provides methods of diagnosing neoplasias or a predisposition to neoplasias, or other diseases or disorders or infections.
  • the methods of diagnosing are methods of detecting the presence of a cell-surface protein on a target cell.
  • the methods comprise exposing a cell that is suspected of being an aberrant cell to a bi- specific molecule of the invention and detemrming whether the bi-specific molecule has bound to the cell. Binding of the bi-specific molecule to the cell indicates that the cell is an aberrant cell of interest. Lack of binding indicates that it is not.
  • the method can comprise control reactions to confirm that all steps in the method, and all reagents being used, worked as expected. Design of such controls is well within the skill level of those of skill in the art, and need not be detailed here.
  • the method further comprises determining the biological effects of binding of the bi-specific molecule to the target cell. For example, the method can comprise determining whether the molecule causes inhibition of cell growth or cell death. Assays for the various effects of interest are well known to those of skill in the art, and need not be detailed herein.
  • the methods of diagnosing comprise exposing cells that express or are suspected of expressing the cell-surface receptor (e.g., BN/GRP-R) to the molecule of the invention in the presence of at least one T cell, and determining whether the cells show inhibition of growth as compared to a control group not exposed to the molecule, to T cells, or both. Inhibition of growth of cells in the presence of the molecule of the invention and T cells indicates the presence of cancer cells in the sample tested.
  • the cancer is SCLC, breast cancer, prostate cancer, a cancer of the GI tract, or a combination of two or more of these.
  • the invention provides at least one container that contains a bi-specific molecule of the invention, a nucleic acid of the invention, and/or a cell of the invention.
  • the container can be any container, but in embodiments, it is a vial or the like, such as those used to contain pharmaceutically active substances.
  • BsMoI bi-specific molecule
  • mAb monoclonal antibody
  • Antag2 synthetic BN/GRP antagonist
  • the BsMoI also referred to herein as OKT3xAntag2
  • TCR T cell receptor
  • GRP Gastrin-releasing peptide
  • SCLC small cell lung cancer
  • FIG. 1 The sequence of the Antag2 peptide is shown in Figure 1 as SEQ ID NO:3, in which bold amino acids indicate amino acids conserved among the Antag2 and the GRP (SEQ ID NO:1) and BN (SEQ ID NO:2) peptides.
  • Figure 2 depicts the scheme for generating the BsMoI that is used throughout the Examples below. Conjugation of the two molecules making up the BsMoI, OKT3 and Antag2, followed the protocol of Zhou et al., Clinical Cancer Research 9:4953-4960, 2003.
  • OKT3 an anti-CD3 monoclonal antibody from Ortho Biotech, Raritan, NJ
  • SPDP N-succinimidyl 3-[2-py ridyldithio] propionate; Pierce, Rockford, IL
  • Unreacted SPDP was removed by dialysis.
  • Antag2 was then mixed with the activated antibody at a 10:1 molar ratio overnight. Then, free Antag2 was removed by size- exclusion dialysis.
  • a BN antagonist (Cys -D-Phe , Leu-NHEt , des-Met 14 )BN(5_i4), was custom-synthesized by BACHEM Inc (Torrance, CA). Thus, it contains a free sulfhydryl group at the N-terminus of the peptide.
  • the insertion of a D-amino acid, Phe, into position 6 and a desMet 14 analogue at the C-terminus increases the binding affinity as well as the potency by more than 20-fold.
  • a cysteine added at the N- terminus creates a free sulfhydryl group for chemical reaction without interfering with its biological activity.
  • This antagonist is depicted in the figure, but was not conjugated to the antibody for use in the experiments described below. Rather, it is included in the figure to show an alternative antagonist that can be used in accordance with the invention.
  • BsMoI The resulting bi-specific molecule, referred to herein as BsMoI, had anti- SCLC activity, m summary, the BsMoI, OKT3xAntag2, significantly inhibited the growth of SCLC cells mediated by T cells in vitro and in vivo.
  • the BsMoI activates T cells by increasing IFN ⁇ production, and induces target cell apoptosis by activation of caspase 3 (CASP3) and cleavage of poly-ADP ribose polymerase (PARP).
  • CASP3 caspase 3
  • PARP poly-ADP ribose polymerase
  • the cells were maintained in serum-free RPMI1640 medium containing IxIO "8 M hydrocortisone, 5ug/ml of insulin, lOug/ml of transferrin, IxIO "8 M ⁇ -estradiol, and 3xlO- s M selenium (HITES medium).
  • exemplary cells grown in this media were four human SCLC cell lines, H345, H69, SHP77, and DMS273. All chemical reagents were purchased from Sigma Chemical Company (St. Louis, MI). H345 was purchased from the American Type Culture Collection (Rockville, Maryland).
  • DMS273 was established from the pleural fluid of a patient with SCLC in 1980 at Dartmouth Medical School, and has been used in our laboratory for a variety of in vitro and in vivo studies (Zhou JH, et al., "Bombesin/gastrin-releasing peptide receptor: a potential target for antibody-mediated therapy of small cell lung cancer", Clin Cancer Res 9:4953-60, 2003; Pettengill OS, et al., "Animal model for small cell carcinoma of the lung effect of immunosuppression and sex of mouse on tumor growth in nude athymic mice", Expl Cell Biol 48:279-97, 1980).
  • T Lymphocytes [091] Many of the following Examples utilize T cells for analysis of the activities of the exemplary BsMoI. T cells were obtained for each experiment as follows: Buffy-coat cells from unselected healthy donors were obtained from the San Diego Blood Bank. Peripheral blood mononuclear cells (PBMC) were separated by FicoU-Hypaque density centrifugation. Non-adherent cells were collected after incubating the PBMC in DME medium containing 0.2% BSA at 37°C, 5% CO 2 for 2h. The lymphocytes were cultured in RPMI 1640 medium containing 10% FCS and 100U/ml of DL-2 (Chiron Therapeutics, Emeryville, CA) for 3-5 days.
  • PBMC Peripheral blood mononuclear cells
  • Non-adherent cells were collected after incubating the PBMC in DME medium containing 0.2% BSA at 37°C, 5% CO 2 for 2h.
  • the lymphocytes were cultured in RPMI 1640 medium
  • PBL peripheral blood lymphocytes
  • Example 4 Binding of the BsMoI to SCLC and T cells
  • the binding profile of the BsMoI and unconjugated OKT3 to four SCLC cell lines (H345, H69, SHP77, and DMS273) and peripheral blood mononuclear cells (PBMC) was analyzed by flow cytometry.
  • a mouse IgG2a was used as a negative control.
  • SCLC cells were pre-incubated with free BN, free Antag2, or a non-relevant peptide (angiotensin) at 10 uM before adding the BsMoI.
  • the cells were stained with the BsMoI using an indirect immunofluorescence staining method as described in Zhao et al., 2003.
  • the BsMoI binds to the four SCLC cell lines (black line in Panels A-D), while the unconjugated OKT3 does not bind to SCLC cells (grey line in Panels A-D).
  • the BsMoI binds to peripheral blood lymphocytes (Panel A), but does not bind to peripheral blood monocytes (Panel B).
  • the binding of BsMoI is specific and can be partially blocked by pre-incubating SCLC cells with either a free BN (Panel A) or a free Antag2 (Panel B), but is not blocked by a non-relevant peptide angiotensin (Panel C).
  • the BsMoI bound to peripheral blood lymphocytes (PBL) as well as the unconjugated OKT3, suggesting that the chemical conjugation process did not interfere with the biologic function of the 0KT3.
  • Example 5 Effect of Exemplary BsMoI and T Cells on Cancer Cells at
  • Example 6 Further Experiments on Effect of BsMoI on SCLC Growth
  • T lymphocytes were separated from the peripheral blood of healthy donors and cultured in media containing IL-2 (100U/ml) prior to experiments.
  • T cells effector cell, E
  • SCLC cells target cell, T
  • E effector cell
  • T target cell
  • the cell mixture was cultured for 4-72h. Controls included SCLC cells alone and T cells alone with or without the BsMoI.
  • DMS273 cells were added to each of the four donor samples, and the cells were cultured alone, in the presence of OK.T3 antibody, or in the presence of the OKT3xAntag2 BsMoI.
  • the results of the proliferation assay, indicated as percent inhibition of growth, are depicted in Figure 8. More specifically, the figure shows that, in all four donor samples, proliferation of the added DSM273 cancer cells was inhibited by the OKT3 antibody, but that the inhibition was significantly enhanced by fusion of the Antag2 antagonist to the 0KT3 antibody (i.e., by the OKT3xAntag2 molecule).
  • ADCC antibody-dependent cellular cytotoxicity
  • T cell proliferation was measured by a standard thymidine incorporation assay. Fresh PBL (2.5x10 4 /well) were seeded into a 96-well microplate and cultured for 72h, with either unconjugated OKT3, the BsMol, or a control antibody mouse IgG2a. IL-2 at 100 units/ml was added in the culture for 72h. Cells were harvested by a Tomtec cell harvester (Perkin-Elmer, Downers Grove, IL) and counted in a liquid scintillation counter. All assays were performed in triplicate.
  • PBL separated from normal donors consisted of more than 80% T cells.
  • the immunophenotype of these cells was: 81 ⁇ 7% positive for CD3, 52 ⁇ 13% positive for CD4, 34 ⁇ 10% positive for CD8, 17 ⁇ 6% positive for CD56, 8 ⁇ 4% positive for CD25, and 19 ⁇ 11% positive for CD69.
  • the cell viability was greater than 90%.
  • CPM -Thymidine incorporation
  • DMS273 cells and H345 cells were cultured with T cells (E:T ratio 1 : 1) and the exemplary BsMoI or a control antibody for 4-24 hours.
  • Annexin V expression (as an indicator of apoptosis)
  • propidium iodide (PI) staining were analyzed by flow cytometry. Briefly, SCLC cells were mixed in a 96-well plate with T cells at an E:T ratio of 5:1 to 1 : 1 in the presence of the BsMoI or mouse IgG2a and cultured for 4h and 48h.
  • SCLC cells were washed and immediately stained with annexin V-FITC, propidium iodide (PI), and anti-CD45-FITC. All samples were acquired at 10,000 events for each sample by flow cytometry and analyzed by Cellquest software. SCLC cells were identified as CD45-FITC negative cells (dark black dots). T cells were identified as CD45-FITC positive cells (grey dots). Apoptotic SCLC cells were identified as annexin V-positive/PI-negative cells, while necrotic SCLC cells were identified as Pl-positive/annexin V-negative cells.
  • T cells accounted for 66% of the cells at time zero (E:T ratio of 2:1), and decreased to 40% at 48h, while the DMS cells accounted for 34% of cells at time 0, and increased to 60% at 48h. This dynamic change is correlated with an increased expression of Bcl-2 in DMS273 cells.
  • H345 cells were more susceptible to cytotoxicity, as shown in Figure 1 IA.
  • the apoptotic cell population increased from 12% at 4h to 32% at 48h.
  • the necrotic cell population increased from 14% at 4h to 20% at 48h.
  • the live H345 cell population decreased from 50% at baseline (E:T ratio of 1 : 1) to 31 % at 4h, and 6% at 48h of the cytotoxicity assay.
  • the T cells accounted for 50% of cells at time zero, and remained stable about 42% at 48h. This was also correlated with a decreased expression of Bcl-2 protein at 48h of the cytotoxicity assay in H345 cells.
  • FIG. 12A-C A summary of flow cytometry analysis of four ADCC assays is presented in Figures 12A-C. The summary of flow cytometry analysis using T cells from four different donors is presented as mean ⁇ SD. After a 24h ADCC assay, there was a significant increase in both apoptosis and necrosis of SCLC cells in the presence of the BsMoI when compared to a control antibody, The E:T ratio in these experiments was 1:1 for H345 (Panel C) cells and 2:1 for DMS273 (Panel A) and SHP77 (Panel B) cells.
  • apoptosis is one of the important mechanisms in BsMol-mediated cytotoxicity, and that the BsMoI is capable of inducing apoptosis at a low E:T ratio. This observation is most relevant in the clinical setting, wherein the E:T ratio is usually low at the actual tumor site.
  • Figure 13 shows the results of a second set of assays.
  • T cells from four donors were individually tested for release of IFN ⁇ after culturing in the presence of BsMoI and H345 cells, DMS273 cells, H69 cells, or no SCLC cells.
  • Figure 13 shows that at a high E:T ratio of 20:1, BsMoI significantly increases IFN ⁇ release from the T cells.
  • BsMoI or OKT3 but no target cells
  • Bcl-2 and Bcl-xL have anti- apoptotic function through maintenance of mitochondrial membrane permeability and inhibition of Cytochrome C release.
  • Bak and Bax have pro-apoptotic functions, which operate in both the mitochondrial and endoplasmic reticulum, and directly control Cytochrome C release leading to activation of caspases (mitochondrial mediated apoptosis pathway).
  • Bad is a pro-apoptotic member that binds to Bcl-2 and Bcl-xL to promote cell apoptosis.
  • Bcl-2 family proteins play a critical role in the regulation of apoptosis.
  • the anti-apoptotic protein Bcl-2 is expressed in 75% of SCLC.
  • BsMoI bispecific molecule
  • 0KT3 anti-CD3 antibody conjugated with a bombesin antagonist
  • E:T effector to target
  • T lymphocytes were separated from healthy donors.
  • SCLC cells were mixed with T lymphocytes at 1 : 1 ratio in the presence of the BsMoI or mouse IgG2a as a negative control.
  • the mixed cells were stained with annexin V, CD45, and propidium iodide (PI), analyzed by flow cytometry from 4h to 72h.
  • Whole cell lysates were prepared at 24, 48, and 72h, and analyzed for expression of Bcl-2, Bad and phosphorylated Bad (Serl 12), Bax, Bak, and cleaved poly(ADP-ribose) polymerase (PAKP) by Western blot.
  • caspase 9 is the only initiator caspase in the mitochondrial pathway. Once activated, the protein cleaves and activates downstream effector caspases, such as caspase 3 (CASP3), caspase 6, and caspase 7, which then cleave a number of cytoskeletal and nuclear proteins, such as PARP.
  • PARP is a key enzyme involved in DNA repair, replication, and transcription.
  • DMS273 or H345 cells were mixed with T cells at 2: 1 to 1 : 1 ratio with or without the BsMoI in a 96-well microplate. After culturing for 48h, cells were washed three times, centrifuged, collected, and transferred to a microfuge tube. Whole cell lysates were prepared from cell pellets as described in Zhao et al., 2003. The protein content in the whole cell lysate was determined. 40ug of protein was loaded to each lane of 10% and 12% SDS-poly-acrylamide gel. After electrophoresis, the gel was transferred to a nitrocellulose membrane.
  • the full-length as well as the cleaved CASP3, CASP9, and PARP were measured by an Apoptosis Sampler Kit (Cell Signaling Inc, Beverly, MA), according to the manufacturer's instructions. The signals were detected by exposing the membrane to a Kodak film after incubating the membrane with chemiluminescent reagent (Pierce Chemical Co. Rockford, IL). [126] At baseline, all four SCLC cell lines express the full-length CASP3 (35kDa) and PARP (116kDa), with no detectable expression of a cleaved CASP3 (17- 19kDa) and full-length CASP9.
  • a cleaved PARP (89kDa) is detected in all four SCLC cell lines in the presence of the BsMoI (Lane 2), while no cleaved PARP is detected with a control antibody (Lane 1).
  • the signal of a cleaved PARP in the presence of the BsMoI is the most intense one, and the signal of a full-length PARP nearly disappears.
  • a cleaved CASP3 (17kDa) is also detected in all four SCLC cell lines in the presence of the BsMoI. In DMS273 cells, there is again no detectable full-length CASP3 in the presence of BsMoI.
  • Example 13 In vivo Study of the Effect of the BsMoI Based Immunotherapy in a SCLC Xenografted SCID Mouse Model
  • the above described experiments showed that the BsMoI of Example 1 is an effective molecule for activating T cells and causing cell death of SCLC cells in vitro.
  • a mouse model was used. The results presented in this Example show that the BsMoI has in vivo activity that is correlated with its in vitro activity shown above.
  • the human SCLC xenograft model was established in our laboratory (Zhao, et al, 2003). Briefly, 10- to 12-week old NOD. CB 17-Prkdcf cid , abbreviated as NOD/Prkdc SCID, mice were obtained from Jackson laboratory (Bar Harbor, ME). The NOD/Prkdc SCID mice were injected with 1x10 6 DMS273 cells intraperitoneally (ip) six hours after a whole body irradiation of 35OcGy. The control mice received only DMS 273 cells at day 1. The treated mice then received either 5x10 6 BsMoI- armed T cells or OKT3-armed T cells on day 3 and day 10 ip. This model was used not only to show effectiveness of the BsMoI in treating tumors in vivo, but also to establish the treatment schedule, dose of human T cells, and to evaluate tumor size as the treatment results.
  • T lymphocytes were isolated from an unselected healthy donor and cultured with 100U/ml of IL2 for 3-10 days.
  • T cells Prior to injection, T cells were incubated (armed) with either the BsMoI (lug per IxIO 7 cells) or OKT3 (lug per IxIO 7 cells) for 20 minutes. The cells were washed to remove the excess BsMoI or OKT3, re-suspended to 5x10 6 per 0.3 ml PBS for the injection.
  • One group of mice received the BsMol-armed T cells, and the other group of mice received OKT3-armed T cells.
  • mice were injected with 5x10 6 T cells intraperitoneally on three different days.
  • each mouse was injected with 5x10 6 cells intraperitoneally on two different days (day 3 and day 10).
  • Mice were examined daily for general health, activity, sign of illness, and visible tumor growth. In experiment 1, mice were sacrificed at day 20 and 30. In experiment 2, mice were sacrificed at day 40. By day 40, the control mice had usually developed a significant tumor burden with clinical signs of illness. Sacrificed mice from both experiments had their peritoneal cavities washed with 5ml PBS to collect peritoneal exudate cells (PEC).
  • PEC peritoneal exudate cells
  • PEC from each mouse were counted and stained with anti-human CD45-FITC (marker for T cells), anti-human CD56-PE (marker for DMS273 cells), and anti-mouse CD45-PE.
  • AU data were acquired and analyzed by flow cytometry.
  • Peritoneal suspension tumor cells were calculated as: PEC count x % of human CD56+/human CD45-. After the wash, the peritoneal cavity was opened and carefully examined. All visible tumors were dissected and collectively weighed for each mouse. The student t-test was used to compare two groups of samples. The significance level was determined when p value was ⁇ 0.05 by two-sided analysis. The results are shown in Table 1, in which the results are presented as mean ⁇ SD.
  • the table shows that the development of solid tumors and peritoneal suspension tumor cells in mice treated with the BsMol-armed T cells was delayed and significantly reduced at 40 days.
  • the overall tumor burden was estimated by gross tumor weight and the number of peritoneal exudate tumor cells (PET).
  • PET peritoneal exudate tumor cells
  • the control mice all developed visible tumors earlier compared with the experimental mice. Some control mice had clinical signs of jaundice, anemia, and weight loss. All experimental mice that received either OKT3-armed or the BsMol-armed T cells showed no clinical signs of jaundice, anemia, or weight loss.
  • Example 14 Expression of Target Proteins on T Cells [135]
  • the above Examples show that a bi-specific molecule that targets SCLC and T cells, via CD3 on the T cell surface, is effective in vitro and in vivo for inhibition of SCLC cell growth and proliferation.
  • the invention also encompasses bi- specific molecules that target other cell surface proteins on aberrant cells and on T cells.
  • the various different T cell surface receptor targets were identified from T cells collected as described above.
  • T cells from four donors were incubated with IL-2 for 3- 5 days, then assayed with antibodies specific for the following cell surface receptors: CD3, CD4, CD8, and CD56.
  • the results of the assays are shown in Table 2.
  • Table 2 shows that the donor samples all included significant expression of not only CD3, but also CD4 and CD8. Additionally, more than 10% of the cells from some donors expressed CD56.
  • Table 2 shows that the donor samples all included significant expression of not only CD3, but also CD4 and CD8. Additionally, more than 10% of the cells from some donors expressed CD56.

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Abstract

The present invention provides bispecific therapeutic molecules that target both aberrant cells, such as cancer cells, as well as T cells, and compositions comprising them. The invention further provides methods of detecting aberrant cells and methods of treating subjects with one or more cancers. The bi-specific molecules of the invention bind to both the T cells and the target aberrant cells, causing them to be brought into close proximity such that the effects of the T cell can be easily localized to the target aberrant cell.

Description

BISPECIFIC MOLECULE COMPRISING A LIGAND FOR A CELL- SURFACE PROTEIN AND A LIGAND FOR A T-CELL SURFACE PROTEIN
CROSS-REFERENCE TO RELATED APPLICATION
[001] This application claims the benefit of and relies on the filing date of U.S. Provisional Patent application number 60/640,058, filed 30 December 2004, the entire disclosure of which is hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION Field of the Invention
[002] The present invention relates to the field of treatment of neoplastic diseases and uncontrolled cell growth. More specifically, it relates to compositions and methods for treating neoplasias with bispecific molecules comprising a portion that binds to a cell-surface protein on a T-cell, and a portion that binds to a cell- surface protein on a tumor cell. Description of Related Art
[003] Neoplastic diseases, or cancers, are a leading cause of death in the world. Neoplastic diseases can generally be thought of as resulting from defects in cellular mechanisms regulating cell growth and division. As such, numerous molecules that are involved in cell growth or regulation of cell growth have been identified as being involved in neoplasias. Of the more common molecules that have been identified are cell surface receptors. In neoplastic cells of certain tissues, certain cell surface receptors are expressed, whereas non-neoplastic cells of the same cell type do not show expression, or show low levels of expression, of the receptor. Indeed, many cell surface receptors have been found to be expressed improperly in neoplastic cells. [004] Numerous ways of treating neoplastic diseases have been developed over the years. The treatment of choice is typically surgery to remove the tumor because this type of treatment shows a high rate of success and is minimally damaging to unaffected cells of the body. However, many cancers do not lend themselves well to surgery, and thus require alternative methods of treatment. In addition, because surgery is invasive and thus carries with it a certain level of complications, it is occasionally avoided in favor of other means. Generally, non-surgical treatments include administering to the patient substances that are cytotoxic, and thus cause cell death {i.e., administering chemotherapeutic agents), exposure to cell-killing amounts of radiation, or both. The crudest treatments with cytotoxic agents use substances that do not differentiate between normal and neoplastic cells. Thus, while cancer cells are killed by the treatment, normal, healthy cells are killed as well. As the field of cancer treatment has evolved, the substances used for treatment have become more selective, being targeted only to growing cells or cells with proteins on their surfaces that are specific for cancer cells.
[005] Because systemic killing of all cells of the body is undesirable when treating a cancer, cytotoxic agents to be used as anti-cancer compounds should specifically target the cancer cells while avoiding non-cancer cells. Antibodies that are specific for cell-surface proteins that are typically expressed on cancer cells, but not normal cells, are particularly well suited for treatment of cancers. [006] While such cancer cell-specific antibodies are generally well suited for targeted treatment of cancers, often, cell surface targets that are absolutely or highly specific for cancer cells are not available. In such situations, it might be necessary to select a cell surface target that is preferentially expressed on a certain cancer cell type, and risk the potential detrimental effects of the treatment on non-cancer cells expressing the cell surface target.
[007] Small cell lung cancer (SCLC) is characterized by rapid progression, early metastasis, and high mortality. SCLC is a neuroendocrine carcinoma with unique molecular genetics and cellular regulation pathways. SCLC cells produce a variety of neuroendocrine peptides. These neuroendocrine peptides and their receptors are attractive targets for anti-cancer treatment. Gastrin releasing peptide (GRP) is a neuroendocrine peptide and a member of the mammalian bombesin-like peptide family. SCLC cells are known to produce BN-like peptides that function as autocrine growth factors to promote SCLC growth. GRP receptor (GRP-R) is expressed in a majority of SCLC cell lines, as well as in human breast, prostate, colon, gastric, and pancreatic cancers. However, it shows limited distribution in normal human tissues. [008] A recent study by the inventors and their collaborators showed that the bombesin/gastrin releasing peptide receptor (BN/GRP-R), which is expressed on small cell lung cancer (SCLC) cells and other cancer cells such as breast and prostate cancer cells, can be used as a target for immunotherapy. More specifically, a synthetic BN/GRP-R peptide antagonist called Antag 2 was conjugated to a humanized antibody specific for FcγRI, and thus active on mononuclear phagocytes and granulocyte-colony-stimulating factor-activated neutrophils. The bispecific molecule was found to mediate cytotoxicity of SCLC by interferon gamma (IFNγ)-activated human monocytes in conjunction with chemotherapy. (Zhou, JH, Chen, J., Mokotoff, M., Zhong, RK, Shultz, L.D. and Ball, E.D.: Bombesin/Gastrin-Releasing Peptide Receptor: A Potential Target for Antibody-mediated Therapy of Small Cell Lung Cancer. Clinical Cancer Research 9(13):4953-60, 2003.)
[009] Cytotoxic T lymphocytes are potent immune effector cells. They can be activated through their T-cell receptor (TCR) to effectively kill a target cell. Antigen recognition is mediated by either the alpha/beta chain, or gamma/delta chain of the TCR. The alpha/beta chain forms a heterodimer that is closely associated with a cluster of transmembrane peptides to form a CD3 complex on the T cell surface. Li patients with advanced cancer, the host T cells are functionally suppressed and unable to mount an effective attack on cancer cells. There are several common mechanisms to explain why cancer cells escape the host immune system, including lack of the recognition antigens on the cancer cells and inadequate activation of immune trigger molecules on host immune effector cells.
[010] One strategy to redirect cytotoxic T cells toward cancer cells in advanced cancers is to develop a bispecific molecule that can bind to TCR and a tumor- associated antigen. OKT3 is an anti-CD3 monoclonal antibody that is specific for TCR. Several groups have constructed bispecific antibodies (BsAb) consisting of an anti-CD3 antibody and a tumor-specific antibody for targeted immunotherapy. A BsAb, OKT3xTrastuzumab (anti-CD3xanti-HER2), has been shown in preclinical studies to effectively mediate the killing of HER2-positive breast cancer cells. A phase I trial has been planned for patients with stage IV metastatic breast cancer. (Lum LG, et al, "Phasel/ϋ study of treatment of stage IV breast cancer with 0KT3xTrastuzumab-armed activated T cells", Clin Breast Cancer 4(3):212-7, 2003.) A recombinant single-chain BsAb, anti-CD3xanti-CD19, has been shown to effectively activate autologous T cells from patients with advanced B cell lymphoma or leukemia, and effectively kill the autologous B cells at a low effector to target cell ratio. (Loffler A, et al., "Efficient elimination of chronic lymphocytic leukemia B cells by autologous T cells with a bispecific anti-CD 19/anti-CD3 single-chain antibody construct", Leukemia 17:900-9, 2003.) An anti-CD3xanti-PSA BsAb has been shown to inhibit prostate cancer growth in a mouse model. (Katzenwadel A, et al., "Construction and in vivo evaluation of an anti-PSAxanti-CD3 bispecific antibody for the immunotherapy of prostate cancer", Anticancer Res 20(3 A): 1551-5, 2000.) A phase I clinical trial with a HEA125xOKT3 BsAb has been reported, in which the treatment was given as weekly intraperitoneal injections and was well tolerated. All 10 patients with advanced ovarian carcinoma had a response to the treatment with temporary inhibition of the accumulation of malignant ascites. (Marme A, et al., "Intraperitoneal bispecific antibody (HEA125xOKT3) therapy inhibits malignant ascites production in advanced ovarian carcinoma", hit J Cancer 101 : 183-9, 2002.) [011] Resistance to cell apoptosis induced by chemotherapy and radiation is a major obstacle for cancer treatment. Bcl-2 family proteins play a critical role in cell apoptosis. An imbalance between the anti-apoptotic Bcl-2 family members and the pro-apoptotic Bcl-2 family members can lead to cancer progression. Over-expression of anti-apoptotic Bcl-2 protein in primary SCLC tumor has been reported in two large studies. One reported a 71% positive expression in 317 SCLC patients and the other reported a 76% expression in 164 SCLC cases. It is not clear whether the expression of Bcl-2 protein correlates to a poor survival in SCLC patients in vivo. However, several in vitro studies showed a down-regulation of Bcl-2 expression resulted in an increased sensitivity of SCLC cells to chemotherapy and radiotherapy. These encouraging pre-clinical results lead to clinical trials using Oblimersen, a Bcl-2 antisense oligonucleotide compound (G3139, Genasense; Genta Inc., Berkeley Heights, NJ). In a Phase I clinical trial, 16 patients of extensive stage SCLC received Oblimersen in combination with standard chemotherapy of carboplatin and etoposide. Twelve patients showed a partial response and two patients had a stable disease. Interference with mRNA expression can be achieved with small-interfering RNAs (siRNA), or short hairpin RNA (shRNA). Compared with an antisense approach, siRNA and shRNA are more potent and specific in knocking down a targeted gene expression. The mechanism of siRNA-induced gene silencing is different from antisense oligonucleotide in that in the siRNA approach, a direct cleavage of targeted mRNA in the cytoplasm occurs. siRNA could thus become a powerful therapeutic agent to silence cancer-specific gene expression, such as Bcl-2. As will be discussed below, such treatment is encompassed as part of a treatment regimen according to the present invention.
[012] Although studies show the effectiveness of immunotherapeutic approaches to treating SCLC and other cancers, there exists a need in the art for other compounds, compositions, arid treatment methods for treatment of neoplasias. In addition, there is a need for compounds, compositions, and treatment methods for treatment of cells infected with intracellular pathogens, such as viruses.
SUMMARY OF THE INVENTION
[013] The present invention addresses the need in the art by providing new therapeutic molecules, compositions comprising those molecules, methods of treating neoplasias and other disease, disorders, and infections using those molecules, and methods of diagnosing neoplasias or the predisposition to neoplasias, other diseases and disorders, and infections using those molecules. The therapeutic molecules comprise at least one portion that binds to a cell-surface protein of a target cell, and at least one portion that binds to a cell-surface protein of a T-cell of the patient. In exemplary embodiments, the therapeutic molecule is an immunotherapeutic bispecific molecule comprising an antigen-binding region of an anti-CD3 monoclonal antibody (or an equivalent structure created by known techniques) and an antagonist for a cell- surface receptor on a tumor cell. In one particular exemplary embodiment, the immunotherapeutic molecule comprises an anti-CD3 monoclonal antibody and the bombesin (BN) antagonist (Antag2). The exemplary bi-specific molecule (BsMoI) is capable of binding, through its antibody portion, to CD3 present on T cells, and to SCLC or other cancer cells expressing BN/GRP-R, thus bridging the two cells. Binding of a BsMoI according to the invention can effectively activate T cells and induce growth inhibition of cancer cells at a low E:T ratio. Accordingly, the BsMoI of the invention can be used in therapy of cancer. The BsMoI, therefore, targets the GRP-R on the tumor cell surface and a cytotoxic trigger molecule on immune effector cells to activate antibody-dependent, cell-mediated cytotoxicity, hi experiments in vitro, tumor cell growth inhibition of 74% was observed when BsMoI was present, compared with 33% inhibition when tumor cells were cultured with only 0KT3, and 4% inhibition was observed when no antibody was present. If tumor cells were pre- incubated with free antagonist peptide, the BsMoI targeted cytotoxicity was blocked. So, the BsMoI interrupts the BN/GRP binding to BN/GRP-R and also induces a polyclonal T cell activation and specific targeting of tumor cells which results in tumor cell lysis and apoptosis.
[014] In its most basic form, the present invention provides a molecule that can target T-cells to aberrant cells in a patient (also referred to herein as a "subject") and cause the death of those aberrant cells. As used herein, this molecule is referred to as a "bi-specific" molecule, without limitation to the actual number of binding sites available on any particular molecule. The function of the molecule is provided, at least in part, by the presence of at least two distinct regions or portions: one region having a three-dimensional structure that is capable of binding to a cell-surface protein on the surface of a target cell (e.g., a cancer cell), the other region having a three-dimensional structure that is capable of binding to a cell-surface protein on the surface of a T-cell. Binding of both regions to their respective binding partners (i.e., proteins on the respective cells) causes the T-cell to come into close proximity to the target cell, and ultimately cause the death of the target cell. [015] In another aspect, the invention provides compositions comprising the bi- specific molecule of the invention. In general, the compositions comprise the bi- specific molecule of the invention and at least one other substance that is compatible with the bi-specific molecule, such as a solvent, a carrier, or a cell, hi exemplary embodiments, the composition is a pharmaceutical composition that comprises the bi- specific molecule of the invention and at least one pharmaceutically acceptable or biologically tolerable substance.
[016] In yet another aspect, the invention provides nucleic acids encoding at least part of a bi-specific molecule of the invention. Of course, this aspect of the invention applies to embodiments where the bi-specific molecule comprises an amino acid sequence that may be encoded by a nucleic acid. This aspect of the invention includes nucleic acids comprising not only the sequence encoding at least a portion of a bi- specific molecule, but other sequences that are useful in expressing and/or maintaining nucleic acids in a cell. Thus, it includes vectors for expressing or maintaining the coding sequences. Compositions comprising the nucleic acids of the invention are provided as well.
[017] Methods of making the bi-specific molecule of the invention are likewise provided. In general, the methods comprise expressing a nucleic acid encoding at least a portion of a bi-specific molecule of the invention. Where the bi-specific molecule is entirely coded by a nucleic acid, the method does not require any further steps. Where the bi-specific molecule comprises non-proteinaceous material, the method further comprises associating the proteinaceous portion(s) with the non- proteinaceous portion(s) through covalent, ionic, or hydrophobic bonding. In embodiments, the methods of making include purifying, at least to some extent, the bi-specific molecule away from other substances that are present in the environment where the bi-specific molecule is found.
[018] In a further aspect, the invention provides cells. The cells can comprise the bi-specific molecule or a portion thereof. Alternatively, the cells can comprise one or more nucleic acids that encode at least a portion of the bi-specific molecule of the invention. The cells can be useful for expressing the bi-specific molecule or at least a portion of it. They can also be useful for therapy for one or more neoplasias, diseases, disorders, or infections. In certain embodiments, the cells are T-cells comprising a bi- specific molecule associated with the cell by way of attachment to a cell-surface protein.
[019] In view of the activity of the bi-specific molecules of the invention, the present invention provides methods of treating a patient suffering from a neoplasia or other disease or disorder, or an infection. The method comprises administering at least one bi-specific molecule according to the invention, at least one nucleic acid according to the invention, or at least one cell according to the invention, to a patient in need thereof in an amount sufficient to inhibit the growth or kill at least one neoplastic cell, at least one cell of a disease or disorder, or at least one infected cell. In embodiments, the step of administering is repeated two or more times. [020] The invention provides for use of a bi-specific molecule of the invention in the production of a therapeutic or pharmaceutical composition. It also provides for use of a nucleic acid encoding at least a portion of a bi-specific molecule of the invention in the production of a therapeutic or pharmaceutical composition. It further provides for use of a cell of the invention in the production of a therapeutic or pharmaceutical composition. Likewise, the invention provides for use of a bi-specific molecule of the invention in treating a subject suffering from a neoplasia or other disease or disorder or an infection. Thus, the invention provides for use of a nucleic acid of the invention in treating a subject suffering from a neoplasia or other disease or disorder or an infection. Accordingly, the invention provides for use of a cell of the invention in treating a subject suffering from a neoplasia or other disease or disorder or an infection.
[021] In a further aspect, the present invention provides methods of diagnosing neoplasias or a predisposition to neoplasias, or other diseases or disorders or infections. At its most basic level, the methods of diagnosing are methods of detecting the presence of a cell-surface protein on a target cell. In general, the methods comprise exposing a cell that is or is suspected of being a neoplastic cell, a cell of another disease or disorder, or an infected cell to a bi-specific molecule of the invention and determining whether the bi-specific molecule has bound to the cell. In embodiments, the method further comprises determining whether the molecule causes inhibition of cell growth or cell death.
[022] In yet another aspect, the invention provides at least one container that contains a bi-specific molecule of the invention, a nucleic acid of the invention, and/or a cell of the invention. The container can be any container, but in embodiments, it is a vial or the like, such as those used to contain pharmaceutically active substances.
BRIEF DESCRIPTION OF THE DRAWINGS
[023] The accompanying drawings, which are incorporated in and constitute a part of this specification, provide details on at least one embodiment of the invention and, together with the description, serve to explain some underlying principles of the invention.
[024] Figure 1 depicts the relevant amino acid sequences of bombesin, gastrin- releasing peptide, and the Antag2 peptide of the present invention. [025] Figure 2 depicts a schema of the chemical conjugation of the BsMoI. [026] Figure 3 depicts general protocol for preparing and testing T cells for response to various treatments.
[027] Figure 4 shows the results of flow cytometry assays of the BsMoI of the invention binding to four different SCLC cell lines, H345 (Figure 4A), H69 (Figure 4B), DMS273 (Figure 4C), and SHP77 (Figure 4D). [028] Figure 5 shows flow cytometry analysis of the BsMoI binding to lymphocytes (Figure 5A) and monocytes (Figure 5B).
[029] Figure 6 shows flow cytometry analysis of the specificity of BsMoI binding to SCLC cell line HE34 after treatment with bombesin (Figure 6A), Antag2 (Figure 6B), and angiotensin (Figure 6C).
[030] Figure 7 shows the inhibition of growth of cells after treatment with various compounds.
[031] Figure 8 shows the growth inhibition of DMS273 cells by T cells from four normal donors at an E:T ratio of 2.5:1. [032] Figure 9 shows analyses of tumor cell growth inhibition and the dose- response effect of BsMoI.
[033] Figure 10 shows the effect of IL-2 on thymidine incorporation (Figure 10A) and the effect of increasing concentration on thymidine incorporation (Figure 10B).
[034] Figures 1 IA and 1 IB show flow cytometry data relating to annexin V and PI expression in SCLC cells.
[035] Figure 12 shows data relating to apoptosis and necrosis of cells exposed to BsMoI. Panel A = DMS273 cells; Panel B = SHP77 cells; Panel C = H345 cells. [036] Figure 13 depicts data relating to release of interferon gamma by T cells in culture with various SCLC cells.
[037] Figure 14 depicts Western blots of various cell proteins implicated in apoptosis after exposure of cells to BsMoI. Panel A shows Bcl-2 family protein expression in 4 SCLC cell lines. Panel B shows the expression of anti-apoptotic protein Bcl-2 and pro-apoptotic proteins Bax and Bak after ADCC. Panel C shows the changes of Bcl-2 expression, Bad/p-Bad ratio during ADCC in H345 cells. [038] Figure 15 depicts Western blots of various cell proteins implicated in apoptosis after exposure of cells to BsMoI.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION [039] Reference will now be made in detail to various exemplary embodiments of the invention. It is to be understood that the following detailed description is provided to further explain details of various embodiments of the invention, and is not provided as an exhaustive description of all substances that can be used according to the invention, or all steps that can be performed in practicing the methods of the invention. Rather, it is provided to describe certain details of embodiments of the invention, which will provide those of skill in the art a more thorough understanding of various embodiments, which can be applied to other embodiments of the aspects of the invention, without requiring those artisans to practice undue experimentation to achieve the full scope of the claimed invention. [040] Ih contrast to the prior attempts at developing bi-speciflc molecules for treatment of neoplasias, which used two antigen binding sites from antibodies, we have now developed bi-specific molecules (also referred to herein as "BsMoI") that comprise one or no sequences from antibodies. These BsMoI are effective at targeting neoplastic cells and other cells exhibiting signature proteins on their cell surfaces, hi embodiments, one domain of the BsMoI is specific for a T cell receptor while the other is specific for a protein on the surface of a neoplastic cell, hi exemplary embodiments, a bi-specific molecule targeting BN/GRP-R and immune trigger molecules can mediate an effective destruction of SCLC cells, hi a particular exemplary embodiment, a combined chemotherapy and BsMol-targeted immunotherapy significantly increases the destruction of SCLC cells in vitro. The present invention, in embodiments, uses the BsMoI to recruit cytotoxic T cells to SCLC cells, either in vitro or in vivo, to inhibit the growth of the cells. Treatment of the cells can further comprise combining this targeted immunotherapy with an anti- Bcl-2 approach to further enhance the specific SCLC destruction. [041] hi its most basic form, the present invention provides a molecule that can target T cells to aberrant cells in a patient (also referred to herein as a "subject") and cause the death of those aberrant cells. As used herein, this molecule is referred to as a "bi-specific" molecule, without limitation to the actual number of binding sites available on any particular molecule. The function of the molecule is provided, at least in part, by the presence of at least two distinct regions or portions: one region having a three-dimensional structure that is capable of binding to a cell-surface protein on the surface of a target cell {e.g., a cancer cell), the other region having a three-dimensional structure that is capable of binding to a cell-surface protein on the surface of a T cell. Binding of both regions to their respective binding partners {e.g., to proteins on the respective cells) causes the T cell to come into close proximity to the target cell, and ultimately causes cessation of growth or, preferably, the death of the target aberrant cell.
[042] The bi-specific molecule of the invention comprises at least one portion that binds to a molecule on the surface of a T cell. Many molecules that appear on the surface of T cells are known, and any of these molecules may be used as targets for binding of the bi-specific molecule of the invention. For example, it is known that cell surface receptors, such as TCR, CD2, CD3, CD4, CD8, and CD56 are present on the surface of T cells. To provide the highest effectiveness in killing target aberrant cells, it is preferable that binding of the bi-specific molecule to the T cell surface molecule causes activation of the T cell. In this way, not only is the T cell brought into close proximity to the target aberrant cell, but it is activated to either kill the aberrant cell or to release substances that promote targeting and killing of the aberrant cell by other cells of the immune system. The precise mechanism of T cell activation or production of substances by the T cells is not critical. Rather, T cell surface molecules that are targeted may simply be selected based on their presence and potential involvement in the cell killing process. In other words, one may select any molecule on the surface of a T cell, because the primary goal of the bi-specific molecule is to bring the T cell into close proximity to the aberrant cell that is targeted for destruction. It is preferred however, that the T cell surface molecule is selected such that it not only functions in binding of the bi-specific molecule, but, when bound by the bi-specific molecule, also produces at least one substance that causes or participates in the destruction of the target aberrant cell.
[043] The molecule on the surface of the T cell that is the target of the bi-specific molecule can be any molecule. Thus, it may be a protein, a lipid, a lipoprotein, a glycoprotein, a sugar, a polysaccharide, any other simple or complex molecule known to be present on the surface of cells or viruses, or a combination of two or more of these. Furthermore, there is no limitation on the number of molecules that are present on the T cell surface: the molecule may be present as a single copy or multiple copies, or may be present only on a sub-set of T cells that are present in a particular subject. Typically, the molecule is a protein or comprises a peptide portion. [044] hi addition to at least one portion that binds to a T cell surface molecule, the bi-specific molecule of the invention comprises at least one portion that binds to a target aberrant cell. As with T cells, aberrant cells express on their surfaces complements of molecules, which are generally known and studied in the art. Certain of these molecules, or collections of molecules, are either specific for each different aberrant cell, or are semi-specific (i.e., are present on the target cell and only certain other cells). Any of these molecules may be targeted by the bi-specific molecule of the invention.
[045] For example, the bi-specific molecule may comprise a portion that binds to any of the following cell surface proteins or members of the following cell surface protein families: the ErbB family of protein tyrosine kinases (e.g., erbBl/HER- 1/EGFR, erbB2/HER-2/neu, erbB3 and erbB4), which are overexpressed in some tumor cells and contribute to cell proliferation, regulation of apoptotic cell death, angiogenesis, and metastatic spread; epithelial growth factor receptor (EGFR), which is implicated in breast, non small cell lung, colorectal, bladder, prostate, ovarian, and pancreatic cancers; the HER family of proteins, which are involved in breast and endometrial cancers (e.g, HER2, HER2/neu) - representative ligands include, but are not limited to, heregulin, neuregulin, epiregulin and biregulin; vascular endothelial growth factor (VEGF), which is implicated in colorectal and pancreatic cancers; the Insulin-like growth factor family of ligand and receptors (IGF), which have been implicated in, among other things, breast cancer, mitogenesis, cell survival, and resistance to apoptotic cell death; RON (recepteur d'origine nantais), which has been implicated in colon, breast, ovarian, and lung cancers, and which belongs to the MET proto-oncogene family, and are involved in regulating cell growth and survival, adhesion, motility, cytokine production, and phagocytosis; platelet derived growth factor (PDGF), which is implicated in pulmonary arterial hypertension (PAH), lung cancer, and gastrointestinal stromal tumors; chemokine receptors (e.g., CCR7, CXCR4, CCRlO), which have been identified as important in skin cancer, chronic pulmonary disease, and HIV, and which might play a critical role in determining organ-selective metastasis in melanoma. Certain non-limiting ligands for these receptors include the CCR7 ligands CCL19/ELC and CCL21/SLC, and the CXCR5 ligand CXCLl 3/BCA-l. In addition, targets for melanoma can be the gangliosides GM2 and GD3. Furthermore, the CD4 receptor on HIV infected cells may serve as a target for aberrant cells. [046] The bi-specific molecule may also bind to the following exemplary molecules, which are known to be present on certain neoplastic or infected cells: CXCR4 receptor on small cell lung cancer (involving the chemokine stromal derived factor- 1(SDF-I /CXCLl 2) ligand); members of the ERK pathway, such as RET in thyroid cancer; proteins expressed in cells infected with the Hepatitis C virus, such as CD81 tetraspanin (TAPA-I) and the scavenger receptor SR-BI, which binds to integrin beta 1 and integrin alpha 3, among other things; the DC-SIGN (dendritic cell- specific ICAM-grabbing non-integrin, where ICAM is the intercellular adhesion molecule), which is a mannose-specific C-type lectin expressed by dendritic cells; and CCR5 and CXCR4, which have been shown to be co-receptors for HIV infection (ligands include SDF-I and RANTES, MIP-I alpha, and MlP-lbeta); and other G- protein coupled receptors.
[047] As should be evident, many of the molecules on the target aberrant cells are proteins. The sequences of these proteins are generally known and publicly available, and thus need not be specifically disclosed here. Likewise, the structures of ligands for these proteins are generally known, and thus need not be detailed here. Rather, it should be sufficient to understand that the bi-specific molecule of the invention comprises at least one portion that can bind to a molecule, including those well-known proteins discussed above and known in the art, present on the surface of an aberrant cell, such as a neoplastic cell or a cell infected with a virus or intracellular parasite.
[048] While any molecule that is present on the surface of an aberrant cell may be targeted, it is preferable to have the bi-specific molecule target a cell surface molecule on the aberrant cell that is involved in triggering a process that results in cessation of growth or cell death. Thus, for example, a bi-specific molecule of the invention may have a portion that binds to a cell surface protein that, when activated, initiates an apoptosis pathway. Or, for example, a bi-specific molecule of the invention may have a portion that binds to a cell surface protein that causes the cell to enter a senescence phase, in which cell growth is halted, but the cell does not necessarily begin a process of self-destruction. Many such proteins are known in the art, as are ligands for them, and any of these proteins and ligands can be used as part of a bi-specific molecule of the invention.
[049] hi certain embodiments, one or more of the active portions or regions (used interchangeably herein) comprise a 3 -dimensional structure that is identical to a ligand for a cell-surface molecule on a T cell or on a target aberrant cell. In other embodiments, one or more of the active portions has a 3-dimensional structure that is similar, but not exactly the same, as a ligand for a cell-surface molecule on a T cell or a target aberrant cell, hi such a situation, the ligand can be an antagonist of the true ligand, and can bind to the cell surface molecule, but not cause the same biological effect. Alternatively, it can bind equally well or better, and result in an equally good or better response from the cell.
[050] The bi-specific molecule of the invention can have three or more active portions. That is, the terminology "bi-specific" is not meant to limit the molecule of the invention to only two portions that can bind cells. Rather, the bi-specific molecule can have two or more portions that bind to cells of interest. Typically, the bi-specific molecule has at least one portion that binds to a T cell, and at least one other portion that binds to an aberrant cell of interest. Thus, in embodiments, the bi-specific molecule has one portion that binds to a T cell and one portion that binds to an aberrant cell, hi other embodiments, the bi-specific molecule has one portion that binds to a T cell and two or more portions that bind to a target aberrant cell. In yet other embodiments, the bi-specific molecule has one portion that binds to an aberrant cell and two or more portions that bind to a T cell. In yet further embodiments, the bi- specific molecule comprises two or more portions that bind to a T cell and two or more portions that bind to an aberrant cell. Any number of combinations of active portions can be envisioned, and all of those combinations are included within the scope of the present invention.
[051] Where two or more portions are included that bind to a particular cell type {e.g., T cell), the portions may be specific for the same cell surface molecule, or may be specific for different ones. By including multiple, different portions the specificity of the bi-specific molecule may be increased. For example, where a tumor cell expresses a complement of signature proteins on its surface, but one or more of those proteins is also expressed on other, non-tumor cells, inclusion of two or more portions of the bi-specific molecule that are individually specific for each of the proteins on the tumor cell, improved binding to the tumor cell might be obtained, and an increased effectiveness might be achieved. Likewise, where an aberrant cell expresses two different proteins on its surface, each of which is involved in a distinct cellular process that could be taken advantage of for inhibition of cell growth or for cell killing, a bi-specific molecule comprising ligands for both of those cell surface proteins can be designed and used.
[052] In the bi-specific molecule of the invention, the two or more active portions are linked to each other. Linkage can be through any suitable association, such as through covalent, ionic, or hydrophobic bonding. The main concern in selecting the type of association is whether the molecule will remain intact throughout the time in which it is expected to be active. That is, for the bi-specific molecule to function as envisioned, the two (or more) active portions should be sufficiently well linked that they do not dissociate to an unacceptable extent under the conditions in which they are to be used. Thus, it is preferred that the active portions be covalently linked. Linking of two or more molecules, whether they be peptides, sugars, nucleic acids, or any other organic or inorganic molecules to form the bi-specific molecule of the invention can be through any suitable techniques. A number of suitable techniques for linking two or more such molecules are well known in the arts of chemistry and biochemistry, and need not be detailed here. It is now a routine matter for those of skill in the art to link any number of different types of molecules together to achieve a product. For example, where the bi-specific molecule comprises of two or more peptide active portions, the active portions may be encoded as a single chain from a nucleic acid that has been engineered to express both portions. Alternatively, the entire sequence may be chemically synthesized. On the other hand, the two portions may be separately synthesized (e.g., chemically or by expression of two separate nucleic acids), then fused. Where more than two peptide portions are used, a branching structure may be used according to known principles of protein synthesis. [053] The active portion may be composed of only those structural portions that are required to form the proper 3-dimensional features for activity, or may comprise additional atoms/moieties/structures, which do not play any or any significant role in forming or maintaining the proper 3-dimensional structure. Thus, the molecule may consist of the active portions, linked together, or may comprise the active portions and other portions. Accordingly, the active portions of the bi-specific molecule may be fused directly to each other (or to at least one other active portion of the molecule), or may be linked via a linker, stuffer, etc. The linker may be provided to better ensure proper folding of protein sequences, to provide sufficient distance between one or more active region and one or more other active region, or to provide flexibility to the molecule, or for any other reason that appears to be important to those practicing the invention. The size and structure of the linker is not critical to practice of the invention, although a linker should be chosen that does not interfere with the function of the active regions.
[054] An exemplary embodiment of the bi-specific molecule of the invention is detailed in the Examples. That embodiment comprises an antibody framework, onto which an SCLC-specific ligand is grafted. Thus, in embodiments, the bi-specific molecule comprises an antibody, or a portion of an antibody (e.g, the Fc region, Fab, Fab', single chain, CDR). The antibody or fragment may be from any source, including, but not limited to, humans, other mammals (e.g., mouse, rat, monkey, rabbit). The antibody may be a polyclonal antibody, but is preferably a monoclonal antibody. The antibody or fragment may also be a modified antibody, such as a humanized antibody, a chimeric antibody, or a fusion antibody, hi certain embodiments, an antibody framework is used, where the antigen binding region has been engineered to contain a particular 3-dimensional structure that mimics a ligand or, more preferably, an antagonist of a ligand.
[055] The aberrant cell can be any cell that is abnormal in any way. Thus, it can be a cell that has an abnormal growth profile (e.g., uncontrolled growth, as seen in neoplastic cells). It also can be a cell that expresses one or more proteins that are not normally expressed by cells of its type, or that expresses one or more proteins encoded by a virus or intracellular parasite, or that are expressed by the cell as a result of infection by a virus or intracellular parasite (including certain bacteria that reproduce within cells). It can also be a cell that overexpresses a protein that is normally found in the cell, such as on the surface of the cell. Accordingly, an aberrant cell can be a neoplastic cell, such as a cell from a benign tumor or a cell from a malignant tumor. It can be a cell that is infected with a virus, such as HIV, HCV, HBV, influenza, or papilloma virus. It further can be a cell that is infected with an intracellular parasite, such as a mycobacterium or any of the various single-celled eukaryotic intracellular parasites. Any type of neoplastic cell is included among the aberrant cells of the invention, including, but not limited to, lung cancer cells, breast cancer cells, prostate cancer cells, colorectal cancer cells or other cancer cells of the gastrointestinal tract, kidney cancer cells, hepatocarcinoma cells, brain cancer cells, pancreas tumor cells, and esophageal cancer cells. Other non-limiting examples of cells are uterine cancer cells, cervical cancer cells, endometrium cells, melanoma cells, renal cancer cells, and Kaposi's sarcoma cells, hi addition, an exemplary cell is any cells that expresses GRP-R on its cell surface. These non-limiting examples of cells are given as an indication of the common types of cells that can be targeted with the bi-specific molecule of the invention. Other specific examples will be immediately apparent to those of skill in the art.
[056] In another aspect, the invention provides compositions comprising the bi- specific molecule of the invention. In general, the compositions comprise the bi- specific molecule of the invention and at least one other substance that is compatible with the bi-specific molecule, such as a solvent, a carrier, or a cell. However, in embodiments, the composition comprises two or more different bi-specific molecules. [057] In exemplary embodiments, the composition is a pharmaceutical composition that comprises the bi-specific molecule of the invention and at least one pharmaceutically acceptable or biologically tolerable substance. The substance can be any of the well known substances present in pharmaceuticals, including, but not limited to, water or an aqueous liquid (including saline and its various medicinal/pharmaceutical forms), one or more fillers, binders, colorants, stabilizers, buffers, or other substances that stabilize or promote effective uptake of pharmaceutically active compounds. The substance may also be a sugar or other substance that is effective in protecting compounds during lyophilization. Of course, any of the well known reagents and other substances that are used in cell culture may be included in compositions according to the invention. In embodiments, the composition is a pharmaceutical. Accordingly, the composition may be a capsule, pill, or any other dosage form that can be ingested or taken in through a mucous membrane. The composition may also be an injectable or infusible liquid, or a solid suitable for adding to a liquid to make an injectable or infusible liquid composition. Any of the various compositions known as useful for delivering pharmaceutically active substances to a patient are envisioned as part of this invention. [058] In yet another aspect, the invention provides nucleic acids encoding at least part of a bi-specific molecule of the invention. As discussed above, the invention encompasses bi-specific molecules that consist of or comprise amino acid sequences. These amino acid sequences, whether they comprise the entire bi-specific molecule or only a portion of it, can be encoded by a nucleic acid of the invention, hi embodiments where the bi-specific molecule comprises two or more amino acid sequences fused together, the entire fusion sequence may be encoded by a nucleic acid of the invention, hi particular embodiments, the invention provides nucleic acids that encode an antibody or portion thereof (e.g., Fc portion, single chain antibody, etc.). [059] This aspect of the invention includes nucleic acids comprising not only the sequence encoding at least a portion of a bi-specific molecule, but other sequences that are useful in expressing and/or maintaining nucleic acids in a cell. Thus, it includes vectors (e.g., plasmids, phagemids, phages, viruses) for expressing or maintaining the coding sequences. Those of skill in the art are well aware of the numerous vectors for maintaining and expressing nucleotide sequences, and any suitable vectors may be used in accordance with the present invention. [060] Compositions comprising the nucleic acids of the invention are provided as well. These compositions typically comprise the nucleic acid of the invention along with one or more other substance, including, but not limited to one or more enzyme or reagent useful for expressing a polypeptide from a nucleic acid. Thus, the compositions may include any of the numerous polymerases available commercially. It can also comprise any substance that is typically used in storing nucleic acids in a stable manner, such as in a freeze-dried state.
[061] Methods of making the bi-specific molecule of the invention are provided, hi general, the methods comprise combining two or more substances that comprise active regions, where at least one of the active regions is capable of binding to a molecule on the surface of a T cell and at least one other of the active regions is capable of binding to a molecule on the surface of an aberrant cell of interest. Cell surface molecules for each type of cell are known in the art, and any such molecule may be selected as a target.
[062] The act of combining can be any action that results in two or more active regions becoming associated with each other. Thus, it can comprise mixing the two or more substances under conditions that permit covalent, ionic, or hydrophobic bonding, hi embodiments, it comprises covalently bonding the substances. Where more than two substances are to be bonded, combining can be accomplished in two or more steps, or in a single step. Furthermore, bonding can be accomplished in two or more steps or in a single step. For example, where a bi-specific molecule comprising three active regions is made, one may combine two of the three together, bond them together, add the third region to the fusion product, and bond the third region to create a fusion product comprising all three regions. Alternatively, one may combine all three together and bond all three under the same bonding conditions, with various configurations of the resulting product being produced (e.g., portion A bound to portion B, which is bound to portion C; portion A bound to portion C, which is bound to portion B; portion A bound to portion B and C, etc.). Various permutations of this concept are immediately evident, and all such permutations are envisioned as part of the invention.
[063] hi embodiments, the bi-specific molecule comprises an amino acid portion. In these embodiments, the methods can comprise expressing a nucleic acid encoding at least a portion of the bi-specific molecule of the invention. Where the bi-specific molecule is entirely coded by a nucleic acid, the method might not require any further steps. However, it might require further steps, including permitting the expressed protein to fold into a proper three dimensional shape. The entire bi-specific molecule may be expressed as a single-chain protein from a single nucleic acid. Alternatively, two or more different amino acid sequences may be part of the bi-specific molecule, and they may be expressed independently of each other, then associated with each other through covalent, ionic, or hydrophobic bonding. Preferably, they are bonded covalently (i.e., fused) using common protein engineering techniques. [064] Where the bi-specific molecule comprises proteinaceous material, but does not comprise solely proteinaceous material, the method further comprises associating the two or more portions with each other. Li embodiments, one or more of the portions are proteinaceous portions, and these are associated with the non- proteinaceous portion(s) through covalent, ionic, or hydrophobic bonding. In embodiments, the methods of making include purifying, at least to some extent, the bi-specific molecule away from other substances that are present in the environment where the bi-specific molecule is found. The method can further comprise making a composition, such as a pharmaceutical composition, comprising the bi-specific molecule. In such methods, at least one bi-specific molecule is combined with at least one other bi-specific molecule or one or more other substances that are pharmaceutically acceptable or biologically tolerable.
[065] In a further aspect, the invention provides cells. The cells can comprise one or more bi-specific molecules or portions thereof, or one or more nucleic acids that encode at least a portion of at least one bi-specific molecule of the invention. The cells may comprise the bi-specific molecules or nucleic acids internally or as one or more molecules that are at least partially exposed to the exterior of the cell. In embodiments, one or more bi-specific molecules of the invention are attached to the exterior of a cell, such as by way of ionic or hydrophobic interactions with one or more substances on the surface of the cell.
[066] Cells of the invention have multiple uses. For example, the cells can comprise nucleic acids according to the invention and can be useful for expressing the bi-specific molecule or at least a portion of it. In these embodiments, the cells can be used in a method of making the bi-specific molecule of the invention. In embodiments, a single cell expresses multiple bi-specific molecules of the invention. [067] Cells of the invention can further be useful for research to determine various characteristics of bi-specific molecules of the invention. For example, they can be used to identify particular bi-specific molecules having high specificity, high affinity for particular aberrant cells or T cells, and/or high level of activation of T cells or activation of apoptosis pathways in target aberrant cells, among other things. [068] Cells of the invention can also be useful for therapy for one or more neoplasias, diseases, disorders, or infections. For example, cells of the invention can be produced in vivo by administration of one or more bi-specific molecules of the invention to a patient, with the result being an activated T cell linked via the bi- specific molecule to a target aberrant cell. Alternatively, cells of the invention can be made ex vivo by loading one or more bi-specific molecules of the invention onto T cells, then re-introducing the loaded T cells into the patient to provide an activated T cell that can bind to a target aberrant cell of interest.
[069] In certain embodiments, the cells are T-cells comprising a bi-specific molecule associated with the cell by way of attachment to a cell-surface protein, Li other embodiments, the cell is a target aberrant cell comprising one or more bi- specific molecules associated with the cell by way of attachment to a cell-surface protein.
[070] In view of the activity of the bi-specific molecules of the invention, the present invention provides methods of treating a patient suffering from a neoplasia or other disease or disorder, or an infection, hi general, the method comprises administering at least one bi-specific molecule according to the invention, at least one nucleic acid according to the invention, or at least one cell according to the invention, to a patient in need thereof in an amount sufficient to inhibit the growth or kill at least one neoplastic cell, at least one cell of a disease or disorder, or at least one infected cell (i.e., an aberrant cell). The methods provide a treatment for the patient. Preferably, the methods provide a therapy for the patient by reducing or halting the growth of at least one aberrant cell in the patient's body. Preferably, the methods result in killing of at least one aberrant cell in the patient's body. In embodiments, the method results in reduction in the growth rate of at least one tumor (including non- solid tumors), as compared to the growth rate seen prior to treatment or as compared to an equivalent subject who did not receive the treatment. In certain embodiments, the treatment results in inhibition of tumor growth, whereas in others, it results in a reduction in tumor size or a reduction in the number of neoplastic cells. In a most preferred embodiment, the method results in elimination of all or essentially all of the neoplastic cells. Of course, the same effects described with regard to neoplasias can be seen in cells of other diseases or disorders (i.e., reduction in growth rate, cessation of growth, reduction in number of cells, elimination of cells). [071] The method of treating can comprise a treatment regimen. Accordingly, the method can comprise repeating the step of administering one or more times. Furthermore, the step of administering can comprise administering one or more other bi-specific molecules or one or more other substances that can have a treating effect on the target aberrant cells. Multiple rounds of treatment, each with or some without a bi-specific molecule of the invention, may be performed. Treatment regimens can include dosing with chemotherapeutic agents.
[072] Administering can be accomplished by any suitable method known in the art. In general, administering is performed on a subject in need of treatment for an aberrant cell, such as a neoplastic cell or an infected cell. The amounts administered are amounts sufficient to achieve a therapeutic effect, such as to cause an aberrant cell to cease growing or to die. Numerous techniques for administering substances systemically to the blood system of humans and animals is known, and any one can be used. For example, administration can be through bolus injection of the substance into a vein or artery, administration over minutes or hours by way of intravenous infusion, or oral administration byway of liquid or solid (e.g., tablet, capsule, powder). It can also be accomplished by injection to specific sites of the body (e.g., i.p. injection) or diffusion through skin or mucous membranes (e.g., through a patch or dissolvable lozenge or the like). Those of skill in the art are free to select the most appropriate route of administration for the particular subject and disease, disorder, or infection, and such choice will not represent undue experimentation. [073] Amounts of chemotherapeutic substances, such as the bi-specific molecule of the present invention, to be administered to patients are well known to those of skill in the art, and appropriate amounts to be administered in accordance with the present invention can be determined without excessive experimentation. As a general rule, chemotherapeutic substances can be administered to subjects in therapeutically effective amounts. Those amounts generally range from about 0.01 g/m2 of body area to about 30 g/m2 body area, and can be administered over any amount of time, such as a short period (about 6 hours or less) or a much longer period (about 96 hours or longer) through continuous infusion. Dosing regimens can include considerably longer times, such as two weeks, one month, or more. In these long dosing regimens, doses of the chemotherapeutic substance(s) can be relatively short (e.g., the entire dose being administered in one to several hours), and the doses repeated at regular intervals, such as daily, once weekly, twice weekly, or at other intervals. Various chemotherapeutic dosing regimens are known to those of skill in the art, and any suitable regimen can be used.
[074] Where treatment comprises administering one or more bi-specific molecules based on an antibody frame, it will generally be administered in an amount from about 0.1 mg/m2 of the patient's body area to about 15.0 mg/m2 body area. In embodiments where multiple doses are given by injection, the injections can be administered at days 1 and 14 of a treatment regimen. As an additional non-limiting example, one or more bi-specific molecules can be administered in a regimen comprising two treatment cycles, hi the first cycle, the molecule can be administered at exactly or about 10 - 20 mg/m2 daily for 4 days, hi the second cycle, the molecule can again be administered at exactly or about 10 - 20 mg/m2 at 10-12 days after completion of the first cycle.
[075] hi embodiments, the method of treating is a method of treating a patient suffering from a neoplasia. The method is preferably a therapeutic method that results in cessation of proliferation of aberrant cells, reduction in the number of aberrant cells in the patient, or elimination of the aberrant cells in the patient. It can thus be a method of treating cancer. In embodiments, it is a method of treating small cell lung cancer (SCLC). In other embodiments, it is a method of treating breast cancer, a method of treating prostate cancer, a method of treating colorectal cancer, a method of treating non-small cell lung cancer, or a method of treating two or more of these. In general, the invention provides a method of treating a neoplasia, including a malignant tumor and a benign tumor. The neoplasia treated can be a solid tumor or a non-solid tumor.
[076] The invention further provides for use of a bi-specific molecule of the invention in the production of a therapeutic or pharmaceutical composition. It also provides for use of a nucleic acid encoding at least a portion of a bi-specific molecule of the invention in the production of a therapeutic or pharmaceutical composition. It further provides for use of a cell of the invention in the production of a therapeutic or pharmaceutical composition. Likewise, the invention provides for use of a bi-specific molecule of the invention in treating a subject suffering from a neoplasia or other disease or disorder or an infection. Thus, the invention provides for use of a nucleic acid of the invention in treating a subject suffering from a neoplasia or other disease or disorder or an infection. Accordingly, the invention provides for use of a cell of the invention in treating a subject suffering from a neoplasia or other disease or disorder or an infection.
[077] In a further aspect, the present invention provides methods of diagnosing neoplasias or a predisposition to neoplasias, or other diseases or disorders or infections. At its most basic level, the methods of diagnosing are methods of detecting the presence of a cell-surface protein on a target cell. In general, the methods comprise exposing a cell that is suspected of being an aberrant cell to a bi- specific molecule of the invention and detemrming whether the bi-specific molecule has bound to the cell. Binding of the bi-specific molecule to the cell indicates that the cell is an aberrant cell of interest. Lack of binding indicates that it is not. Of course, the method can comprise control reactions to confirm that all steps in the method, and all reagents being used, worked as expected. Design of such controls is well within the skill level of those of skill in the art, and need not be detailed here. [078] In certain diagnostic assays or assays to determine characteristics of the bi- specific molecule, the method further comprises determining the biological effects of binding of the bi-specific molecule to the target cell. For example, the method can comprise determining whether the molecule causes inhibition of cell growth or cell death. Assays for the various effects of interest are well known to those of skill in the art, and need not be detailed herein.
[079] In embodiments, the methods of diagnosing comprise exposing cells that express or are suspected of expressing the cell-surface receptor (e.g., BN/GRP-R) to the molecule of the invention in the presence of at least one T cell, and determining whether the cells show inhibition of growth as compared to a control group not exposed to the molecule, to T cells, or both. Inhibition of growth of cells in the presence of the molecule of the invention and T cells indicates the presence of cancer cells in the sample tested. In embodiments, the cancer is SCLC, breast cancer, prostate cancer, a cancer of the GI tract, or a combination of two or more of these. [080] In yet another aspect, the invention provides at least one container that contains a bi-specific molecule of the invention, a nucleic acid of the invention, and/or a cell of the invention. The container can be any container, but in embodiments, it is a vial or the like, such as those used to contain pharmaceutically active substances.
EXAMPLES
[081] One embodiment of the invention will be further explained by the following Examples, which are intended to be purely exemplary of that embodiment of the invention, and should not be considered as limiting the invention in any way. [082] The embodiment exemplified in this section relates to construction and testing of a bi-specific molecule (BsMoI) comprising an anti-CD3 monoclonal antibody (mAb) fused to a synthetic BN/GRP antagonist, Antag2. The BsMoI, also referred to herein as OKT3xAntag2, specifically binds to SCLC cells and, at the same time, activates at least one T cell receptor (TCR), effectively mediating the inhibition of SCLC growth by human T cells in vitro as well as in vivo. [083] Based on the following Examples alone, it is evident that a new class of therapeutic molecules, which can be immunotherapeutic molecules, has been developed that can be used to specifically target chosen aberrant cells, such as cancer cells, based on cell-surface proteins expressed by the cancer cell. Targeting those cell-surface proteins with a bispecific molecule that also binds T cells, and thus brings them into contact with the cancer cells, results in treatment methods and regimens that are effective at inhibiting the growth of, and ultimately killing, selected cancer cells. In addition, because cell death can be detected and monitored, the present invention provides methods for diagnosing the presence of cancer cells in a sample, and thus diagnosing cancer in a patient.
[084] Example 1 : Construction of a BsMoI of the Invention [085] Gastrin-releasing peptide (GRP) is a growth factor for small cell lung cancer (SCLC). GRP belongs to the Bombesin (BN) peptide family and has significant homology to BN. We constructed a bispecific molecule (BsMoI), OKT3xAntag2, by conjugating a monoclonal antibody OKT3 (anti-CD3) with a BN/GRP antagonist (Antag2), and evaluated it for cytotoxicity against SCLC cells. The sequence of the Antag2 peptide is shown in Figure 1 as SEQ ID NO:3, in which bold amino acids indicate amino acids conserved among the Antag2 and the GRP (SEQ ID NO:1) and BN (SEQ ID NO:2) peptides. Figure 2 depicts the scheme for generating the BsMoI that is used throughout the Examples below. Conjugation of the two molecules making up the BsMoI, OKT3 and Antag2, followed the protocol of Zhou et al., Clinical Cancer Research 9:4953-4960, 2003. Briefly, in the process, OKT3 (an anti-CD3 monoclonal antibody from Ortho Biotech, Raritan, NJ) was reacted for one hour with a cross-linker, SPDP (N-succinimidyl 3-[2-py ridyldithio] propionate; Pierce, Rockford, IL) to create a 2-pyridyl-disulfide-activated antibody. Unreacted SPDP was removed by dialysis. Antag2 was then mixed with the activated antibody at a 10:1 molar ratio overnight. Then, free Antag2 was removed by size- exclusion dialysis.
[086] A BN antagonist (Cys -D-Phe , Leu-NHEt , des-Met14)BN(5_i4), was custom-synthesized by BACHEM Inc (Torrance, CA). Thus, it contains a free sulfhydryl group at the N-terminus of the peptide. The insertion of a D-amino acid, Phe, into position 6 and a desMet14 analogue at the C-terminus increases the binding affinity as well as the potency by more than 20-fold. A cysteine added at the N- terminus creates a free sulfhydryl group for chemical reaction without interfering with its biological activity. This antagonist is depicted in the figure, but was not conjugated to the antibody for use in the experiments described below. Rather, it is included in the figure to show an alternative antagonist that can be used in accordance with the invention.
[087] The resulting bi-specific molecule, referred to herein as BsMoI, had anti- SCLC activity, m summary, the BsMoI, OKT3xAntag2, significantly inhibited the growth of SCLC cells mediated by T cells in vitro and in vivo. The BsMoI activates T cells by increasing IFNγ production, and induces target cell apoptosis by activation of caspase 3 (CASP3) and cleavage of poly-ADP ribose polymerase (PARP).
[088] Example 2: Cell Culture of SCLC Cells
[089] For routine growth and maintenance of cells, the cells were maintained in serum-free RPMI1640 medium containing IxIO"8 M hydrocortisone, 5ug/ml of insulin, lOug/ml of transferrin, IxIO"8 M β-estradiol, and 3xlO-s M selenium (HITES medium). Exemplary cells grown in this media were four human SCLC cell lines, H345, H69, SHP77, and DMS273. All chemical reagents were purchased from Sigma Chemical Company (St. Louis, MI). H345 was purchased from the American Type Culture Collection (Rockville, Maryland). DMS273 was established from the pleural fluid of a patient with SCLC in 1980 at Dartmouth Medical School, and has been used in our laboratory for a variety of in vitro and in vivo studies (Zhou JH, et al., "Bombesin/gastrin-releasing peptide receptor: a potential target for antibody-mediated therapy of small cell lung cancer", Clin Cancer Res 9:4953-60, 2003; Pettengill OS, et al., "Animal model for small cell carcinoma of the lung effect of immunosuppression and sex of mouse on tumor growth in nude athymic mice", Expl Cell Biol 48:279-97, 1980).
[090] Example 3: Preparation of T Lymphocytes [091] Many of the following Examples utilize T cells for analysis of the activities of the exemplary BsMoI. T cells were obtained for each experiment as follows: Buffy-coat cells from unselected healthy donors were obtained from the San Diego Blood Bank. Peripheral blood mononuclear cells (PBMC) were separated by FicoU-Hypaque density centrifugation. Non-adherent cells were collected after incubating the PBMC in DME medium containing 0.2% BSA at 37°C, 5% CO2 for 2h. The lymphocytes were cultured in RPMI 1640 medium containing 10% FCS and 100U/ml of DL-2 (Chiron Therapeutics, Emeryville, CA) for 3-5 days. The phenotype of the peripheral blood lymphocytes (PBL) was analyzed by flow cytometry (FACScan, BD Biosciences, San Jose, CA). A general scheme for cell growth is depicted in Figure 3, which includes depictions of subsequent treatments used in various further Examples, below.
[092] Example 4: Binding of the BsMoI to SCLC and T cells [093] The binding profile of the BsMoI and unconjugated OKT3 to four SCLC cell lines (H345, H69, SHP77, and DMS273) and peripheral blood mononuclear cells (PBMC) was analyzed by flow cytometry. A mouse IgG2a was used as a negative control. To test the specific binding of the BsMoI to BN/GRP-R, SCLC cells were pre-incubated with free BN, free Antag2, or a non-relevant peptide (angiotensin) at 10 uM before adding the BsMoI. The cells were stained with the BsMoI using an indirect immunofluorescence staining method as described in Zhao et al., 2003. [094] As shown in Figure 4, the BsMoI binds to the four SCLC cell lines (black line in Panels A-D), while the unconjugated OKT3 does not bind to SCLC cells (grey line in Panels A-D). Furthermore, as shown in Figure 5, the BsMoI binds to peripheral blood lymphocytes (Panel A), but does not bind to peripheral blood monocytes (Panel B). In addition, as shown in Figure 6, the binding of BsMoI is specific and can be partially blocked by pre-incubating SCLC cells with either a free BN (Panel A) or a free Antag2 (Panel B), but is not blocked by a non-relevant peptide angiotensin (Panel C). The BsMoI bound to peripheral blood lymphocytes (PBL) as well as the unconjugated OKT3, suggesting that the chemical conjugation process did not interfere with the biologic function of the 0KT3.
[095] Example 5: Effect of Exemplary BsMoI and T Cells on Cancer Cells at
Low E:T Ratio
[096] Growth inhibition of DMS273 (an SCLC cell line) cells after treatment with the BsMoI of Example 1, after treatment with the 0KT3 antibody alone, or after treatment with the Antag2 peptide, followed by the BsMoI, was measured by a 3H- thymidine incorporation assay. The results are depicted in Figure 7. [097] As shown in Figure 7, at the low E:T ratio of 2.5 : 1 , high growth inhibition was observed in the presence of the BsMoI, whereas a much lower inhibition was seen with the unconjugated antibody 0KT3. Very little inhibition was seen without addition of antibody to the cells. The BsMoI targeted cytotoxicity of tumor cells was blocked when tumor cells were pre-incubated with free Anatag2 peptide.
[098] Example 6: Further Experiments on Effect of BsMoI on SCLC Growth [099] To further determine the effect of the BsMoI molecule of Example 1 on cancer cells, blood from four healthy donors was taken, and T cells were separated. The general procedure is depicted in Figure 3, which shows processing according to this Example, as well as the following examples relating to assays for apoptosis and IFNγ. In general, the procedure followed the following protocol: T lymphocytes were separated from the peripheral blood of healthy donors and cultured in media containing IL-2 (100U/ml) prior to experiments. T cells (effector cell, E) were mixed with the SCLC cells (target cell, T) at different E:T ratios, in the presence of the BsMoI, unconjugated OKT3, or mouse IgG2a as a negative control. The cell mixture was cultured for 4-72h. Controls included SCLC cells alone and T cells alone with or without the BsMoI.
[100] For this Example, DMS273 cells were added to each of the four donor samples, and the cells were cultured alone, in the presence of OK.T3 antibody, or in the presence of the OKT3xAntag2 BsMoI. The results of the proliferation assay, indicated as percent inhibition of growth, are depicted in Figure 8. More specifically, the figure shows that, in all four donor samples, proliferation of the added DSM273 cancer cells was inhibited by the OKT3 antibody, but that the inhibition was significantly enhanced by fusion of the Antag2 antagonist to the 0KT3 antibody (i.e., by the OKT3xAntag2 molecule).
[101] As can be seen in Figure 8, growth inhibition of DMS273 cells was achieved in the presence of the exemplary BsMoI (at 10 ng/ml). An average of 74±9% inhibition was observed, compared with 33±10% inhibition in the presence of OKT3 alone, and 14±4% inhibition in the control.
[102] Example 7: Inhibition of SCLC Growth, as Further Determined by 3H- thymidine Incorporation Assays
[103] To even further determine the effect of the BsMoI molecule of Example 1 on cancer cells, antibody-dependent cellular cytotoxicity (ADCC) was measured by a standard thymidine incorporation assay. Briefly, blood from four healthy donors was taken, and T cells were separated. Target DMS273 cells (2.5xlO3/well) were seeded into a 96-well microplate. Effector T cells were added at E:T ratios of 10:1, 5:1, 2.5:1, and 1.25:1, and cultured in RPMI 1640 medium containing 2.5% FCS for 72 hours, with the BsMoI, OKT3, or control antibody (mouse IgG2a). [3H] -Thymidine was added in the last 8h of incubation, then cells were harvested. Thymidine incorporation was determined by scintillation counting. The inhibition of thymidine incorporation was calculated as: [1 -(experimental CPM - T cells alone CPM)/tumor cells alone CPM)] xl 00%.
[104] The inhibition of DMS273 cell growth by BsMol-activated T cells is summarized in Figure 9. The inhibition effect was dependent on the dose of the BsMol from O.lng/ml to lOng/ml, and reaches a plateau at a concentration of 10 to 100ng/ml. The efficacy of the inhibition and the activity of T cells vary among the individual donors. The variation is partially related to HLA compatibility between the donor T cells and the targeted SCLC cells (data not shown).
[105] Example 8: T Cell Activation and Proliferation in the Presence of BsMol [106] T cell proliferation was measured by a standard thymidine incorporation assay. Fresh PBL (2.5x104/well) were seeded into a 96-well microplate and cultured for 72h, with either unconjugated OKT3, the BsMol, or a control antibody mouse IgG2a. IL-2 at 100 units/ml was added in the culture for 72h. Cells were harvested by a Tomtec cell harvester (Perkin-Elmer, Downers Grove, IL) and counted in a liquid scintillation counter. All assays were performed in triplicate. [107] PBL separated from normal donors consisted of more than 80% T cells. The immunophenotype of these cells was: 81±7% positive for CD3, 52±13% positive for CD4, 34±10% positive for CD8, 17±6% positive for CD56, 8±4% positive for CD25, and 19±11% positive for CD69. The cell viability was greater than 90%. [3H] -Thymidine incorporation (CPM) into T cells after 72h culture is shown in Figure 10. Without IL-2, there was little increase in thymidine uptake into T cells (Figure 10A). There was no increase of thymidine uptake into T cells in the presence of a control mouse IgG2a antibody. Both OKT3 and the BsMol increased [3H] -Thymidine uptake into T cells in a dose-dependent pattern (Figure 10B). There was no significant difference in thymidine uptake into T cells stimulated by the OKT3 or the BsMol, again suggesting that chemical conjugation had no impact on the biological function of OKT3. [108] Example 9: Analysis of SCLC Tumor Cell Apoptosis and Necrosis in the
Cytotoxicity Assay
[109] To determine the effect of the BsMoI on cell death via apoptosis, DMS273 cells and H345 cells were cultured with T cells (E:T ratio 1 : 1) and the exemplary BsMoI or a control antibody for 4-24 hours. Annexin V expression (as an indicator of apoptosis) and propidium iodide (PI) staining were analyzed by flow cytometry. Briefly, SCLC cells were mixed in a 96-well plate with T cells at an E:T ratio of 5:1 to 1 : 1 in the presence of the BsMoI or mouse IgG2a and cultured for 4h and 48h. At the end of the culture periods, cells were washed and immediately stained with annexin V-FITC, propidium iodide (PI), and anti-CD45-FITC. All samples were acquired at 10,000 events for each sample by flow cytometry and analyzed by Cellquest software. SCLC cells were identified as CD45-FITC negative cells (dark black dots). T cells were identified as CD45-FITC positive cells (grey dots). Apoptotic SCLC cells were identified as annexin V-positive/PI-negative cells, while necrotic SCLC cells were identified as Pl-positive/annexin V-negative cells. In summary, after a 24h ADCC assay, there was a significant increase in both apoptosis and necrosis of SCLC cells in the presence of the BsMoI when compared to a control antibody. The E:T ratio in these experiments was 1:1 for H345 cells and 2:1 for DMS273 cells. The results suggest that apoptosis might be an important mechanism in target cell death at a lower E:T ratio of ADCC. This observation maybe important in the clinical setting, wherein the E:T ratio is probably low at actual tumor sites. [110] It was found that there was a dynamic change during the assay, hi this assay, which is depicted in Figure 11, where the results for H345 cells are presented in Panel A and the results for DMS273 cells are presented in Panel B, DMS273 cells were found to be more resistant to BsMol-mediated cytotoxicity. As shown in Figure 1 IB, the apoptotic DMS273 cell population increased from 12% at 4h to 26% at 48h, while the necrotic DMS273 cell population increased from 15% at 4h to 21% at 48h, and the alive DMS273 cell population increases from 12% at 4h to 19% at 48h. The T cells accounted for 66% of the cells at time zero (E:T ratio of 2:1), and decreased to 40% at 48h, while the DMS cells accounted for 34% of cells at time 0, and increased to 60% at 48h. This dynamic change is correlated with an increased expression of Bcl-2 in DMS273 cells.
[Ill] In contrast, H345 cells were more susceptible to cytotoxicity, as shown in Figure 1 IA. The apoptotic cell population increased from 12% at 4h to 32% at 48h. The necrotic cell population increased from 14% at 4h to 20% at 48h. The live H345 cell population decreased from 50% at baseline (E:T ratio of 1 : 1) to 31 % at 4h, and 6% at 48h of the cytotoxicity assay. The T cells accounted for 50% of cells at time zero, and remained stable about 42% at 48h. This was also correlated with a decreased expression of Bcl-2 protein at 48h of the cytotoxicity assay in H345 cells. [112] A summary of flow cytometry analysis of four ADCC assays is presented in Figures 12A-C. The summary of flow cytometry analysis using T cells from four different donors is presented as mean ± SD. After a 24h ADCC assay, there was a significant increase in both apoptosis and necrosis of SCLC cells in the presence of the BsMoI when compared to a control antibody, The E:T ratio in these experiments was 1:1 for H345 (Panel C) cells and 2:1 for DMS273 (Panel A) and SHP77 (Panel B) cells. Based on these results, it is apparent that apoptosis is one of the important mechanisms in BsMol-mediated cytotoxicity, and that the BsMoI is capable of inducing apoptosis at a low E:T ratio. This observation is most relevant in the clinical setting, wherein the E:T ratio is usually low at the actual tumor site.
[113] Example 10: IFNγ Assay
[114] To determine the effect of BsMoI on activation of T cells, the production of interferon gamma was assayed. In a 96-well microplate, 5x103 SCLC cells were mixed with IxIO5 T cells (20:1 E:T ratio) in the presence of the BsMoI, 0KT3, or mouse IgG2a at 0. lug/ml. After 48h of culture, the plate was centrifuged, and lOOul of supernatant was collected from each well. The amount of human IFNγ in the supernatant was determined by an ELISA (OptEIA™) assay according to the manufacturer's instruction (BD Pharmingen, San Diego, CA). Absorbance at 45OnM was measured by a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA) and the results were calculated using the SOFTmax program (Molecular Devices). All assays were performed in duplicate.
[115] Figure 13 shows the results of a second set of assays. In these assays, T cells from four donors were individually tested for release of IFNγ after culturing in the presence of BsMoI and H345 cells, DMS273 cells, H69 cells, or no SCLC cells. Figure 13 shows that at a high E:T ratio of 20:1, BsMoI significantly increases IFNγ release from the T cells. In contrast, T cells cultured alone with the BsMoI or OKT3 (but no target cells) showed no change in IFNγ release.
[116] Based on these results, it is apparent that the BsMoI, OKT3xAntag2, can significantly and effectively activate T cells and significantly increase IFNγ production by T cells, leading to cytotoxicity of target cells through cell lysis and apoptosis.
[117] Example 11 : Expression of Bcl-2 Family Proteins During the Cytotoxicity
Assay
[118] We have studied several members of Bcl-2 family expression in SCLC cell lines and their regulations in the response during ADCC. Bcl-2 and Bcl-xL have anti- apoptotic function through maintenance of mitochondrial membrane permeability and inhibition of Cytochrome C release. Bak and Bax have pro-apoptotic functions, which operate in both the mitochondrial and endoplasmic reticulum, and directly control Cytochrome C release leading to activation of caspases (mitochondrial mediated apoptosis pathway). Bad is a pro-apoptotic member that binds to Bcl-2 and Bcl-xL to promote cell apoptosis.
[119] As can be seen in Figure 14 A, Bcl-2 expression is easily detectable in H345, H69, and SHP77 cells. In contrast, DMS273 cells have a low level of Bcl-2 expression at baseline. Expression of other Bcl-2 family proteins, such as Bad, phosphorylated Bad (Serl 12), and Bak is detectable. To study whether there is a change of expression of Bcl-2 family proteins associated with the SCLC cell apoptosis, we prepared whole cell lysates for Western blot analysis at different time points during a cytotoxicity assay. The results of the Western blots are shown in Figures 14B and 14C.
[120] As can be seen in Figure 14B, in H345 cells, the expression of pro- apoptotic Bax and Bak were increased in the presence of the BsMoI, while Bcl-2 expression was decreased in H345 after a 48h cytotoxicity assay. In contrast, the expression of anti-apoptotic Bcl-2 was increased in DMS273 cells after a 48h assay. This increase of Bcl-2 expression correlates to the relative resistance of DMS 273 cells to the BsMol-mediated cytotoxicity. As shown in a previous apoptosis study, it requires a high E:T ratio to achieve a comparable cell killing in DMS 273 cells, and there is a trend of repopulation of DMS273 cells after a 48h cytotoxicity assay with an increasing percentage of living DMS 273 cells by the flow cytometry analysis. [121] The expression of phosphorylated Bad (Serl 12) in H345 cells at baseline usually was higher than Bad. As Figure 14C shows, after 4h, the ratio of p- Bad(Serl 12)/Bad was still high, and there was a significant reduction of the p-Bad level after 48h with the BsMoI. Although the whole cell lysate was prepared from a mixture of T cells and SCLC cells, the expression of Bcl-2 family proteins is presumably very low from the T cells due to the small amount protein from T cells in the cytotoxicity assay.
[122] Example 12: Cleavage of CASP3 and PARP as a Result of BsMoI
Binding to Cancer Cells
[123] Bcl-2 family proteins play a critical role in the regulation of apoptosis. The anti-apoptotic protein Bcl-2 is expressed in 75% of SCLC. We have shown that a bispecific molecule (BsMoI) containing 0KT3 (anti-CD3 antibody) conjugated with a bombesin antagonist was able to activate T lymphocytes and mediate SCLC cell apoptosis at a low effector to target (E:T) cell ratio. Because most SCLC cell lines do not express caspase 8, an initiator caspase in the death-receptor pathway, we studied whether Bcl-2 family proteins contributed in the process of tumor cell apoptosis by ADCC. Four SCLC cell lines H345, H69, SHP77, and DMS273 were studied. T lymphocytes were separated from healthy donors. SCLC cells were mixed with T lymphocytes at 1 : 1 ratio in the presence of the BsMoI or mouse IgG2a as a negative control. The mixed cells were stained with annexin V, CD45, and propidium iodide (PI), analyzed by flow cytometry from 4h to 72h. Whole cell lysates were prepared at 24, 48, and 72h, and analyzed for expression of Bcl-2, Bad and phosphorylated Bad (Serl 12), Bax, Bak, and cleaved poly(ADP-ribose) polymerase (PAKP) by Western blot.
[124] We have studied the activity of several caspases and PoIy(ADP -ribosyl) polymerase (PARP) during the cytotoxicity assay. Caspase 9 (CASP9) is the only initiator caspase in the mitochondrial pathway. Once activated, the protein cleaves and activates downstream effector caspases, such as caspase 3 (CASP3), caspase 6, and caspase 7, which then cleave a number of cytoskeletal and nuclear proteins, such as PARP. PARP is a key enzyme involved in DNA repair, replication, and transcription.
[125] DMS273 or H345 cells were mixed with T cells at 2: 1 to 1 : 1 ratio with or without the BsMoI in a 96-well microplate. After culturing for 48h, cells were washed three times, centrifuged, collected, and transferred to a microfuge tube. Whole cell lysates were prepared from cell pellets as described in Zhao et al., 2003. The protein content in the whole cell lysate was determined. 40ug of protein was loaded to each lane of 10% and 12% SDS-poly-acrylamide gel. After electrophoresis, the gel was transferred to a nitrocellulose membrane. The full-length as well as the cleaved CASP3, CASP9, and PARP were measured by an Apoptosis Sampler Kit (Cell Signaling Inc, Beverly, MA), according to the manufacturer's instructions. The signals were detected by exposing the membrane to a Kodak film after incubating the membrane with chemiluminescent reagent (Pierce Chemical Co. Rockford, IL). [126] At baseline, all four SCLC cell lines express the full-length CASP3 (35kDa) and PARP (116kDa), with no detectable expression of a cleaved CASP3 (17- 19kDa) and full-length CASP9. As shown in Figure 15, after a 24h ADCC, a cleaved PARP (89kDa) is detected in all four SCLC cell lines in the presence of the BsMoI (Lane 2), while no cleaved PARP is detected with a control antibody (Lane 1). In DMS273 cells, the signal of a cleaved PARP in the presence of the BsMoI is the most intense one, and the signal of a full-length PARP nearly disappears. A cleaved CASP3 (17kDa) is also detected in all four SCLC cell lines in the presence of the BsMoI. In DMS273 cells, there is again no detectable full-length CASP3 in the presence of BsMoI. hi H345 cells, there is a very strong signal of cleaved CASP3 in the presence of the BsMoI, and a much weaker band of cleaved CASP3 with a control antibody. The detection of both cleaved CASP3 and cleaved PARP is the biochemical marker of a committed cell apoptosis. Neither full-length nor a cleaved CASP9 is detected in these SCLC cell lines.
[127] Example 13: In vivo Study of the Effect of the BsMoI Based Immunotherapy in a SCLC Xenografted SCID Mouse Model [128] The above described experiments showed that the BsMoI of Example 1 is an effective molecule for activating T cells and causing cell death of SCLC cells in vitro. To confirm these results in vivo, a mouse model was used. The results presented in this Example show that the BsMoI has in vivo activity that is correlated with its in vitro activity shown above.
[129] The human SCLC xenograft model was established in our laboratory (Zhao, et al, 2003). Briefly, 10- to 12-week old NOD. CB 17-Prkdcfcid, abbreviated as NOD/Prkdc SCID, mice were obtained from Jackson laboratory (Bar Harbor, ME). The NOD/Prkdc SCID mice were injected with 1x106 DMS273 cells intraperitoneally (ip) six hours after a whole body irradiation of 35OcGy. The control mice received only DMS 273 cells at day 1. The treated mice then received either 5x106 BsMoI- armed T cells or OKT3-armed T cells on day 3 and day 10 ip. This model was used not only to show effectiveness of the BsMoI in treating tumors in vivo, but also to establish the treatment schedule, dose of human T cells, and to evaluate tumor size as the treatment results.
[130] For the assays, two different experiments were performed, hi both, human T lymphocytes were isolated from an unselected healthy donor and cultured with 100U/ml of IL2 for 3-10 days. Prior to injection, T cells were incubated (armed) with either the BsMoI (lug per IxIO7 cells) or OKT3 (lug per IxIO7 cells) for 20 minutes. The cells were washed to remove the excess BsMoI or OKT3, re-suspended to 5x106 per 0.3 ml PBS for the injection. One group of mice received the BsMol-armed T cells, and the other group of mice received OKT3-armed T cells. For experiment 1, each mouse was injected with 5x106 T cells intraperitoneally on three different days. For experiment 2, each mouse was injected with 5x106 cells intraperitoneally on two different days (day 3 and day 10). Mice were examined daily for general health, activity, sign of illness, and visible tumor growth. In experiment 1, mice were sacrificed at day 20 and 30. In experiment 2, mice were sacrificed at day 40. By day 40, the control mice had usually developed a significant tumor burden with clinical signs of illness. Sacrificed mice from both experiments had their peritoneal cavities washed with 5ml PBS to collect peritoneal exudate cells (PEC). PEC from each mouse were counted and stained with anti-human CD45-FITC (marker for T cells), anti-human CD56-PE (marker for DMS273 cells), and anti-mouse CD45-PE. AU data were acquired and analyzed by flow cytometry. Peritoneal suspension tumor cells were calculated as: PEC count x % of human CD56+/human CD45-. After the wash, the peritoneal cavity was opened and carefully examined. All visible tumors were dissected and collectively weighed for each mouse. The student t-test was used to compare two groups of samples. The significance level was determined when p value was < 0.05 by two-sided analysis. The results are shown in Table 1, in which the results are presented as mean ± SD.
[131] The table shows that the development of solid tumors and peritoneal suspension tumor cells in mice treated with the BsMol-armed T cells was delayed and significantly reduced at 40 days.
[132] Table 1 : Targeted Therapy In Human SCLC Xenografted Into NOD/SCID Mouse Model
Figure imgf000042_0001
tumor weight/PETC
[133] The overall tumor burden was estimated by gross tumor weight and the number of peritoneal exudate tumor cells (PET). The control mice all developed visible tumors earlier compared with the experimental mice. Some control mice had clinical signs of jaundice, anemia, and weight loss. All experimental mice that received either OKT3-armed or the BsMol-armed T cells showed no clinical signs of jaundice, anemia, or weight loss. Two mice that received the BsMol-armed T cells had no macroscopic tumor in their peritoneal cavity. The remainder of the mice that received the BsMol-armed T cells developed low-volume tumors usually along the needle track in the lower peritoneal cavity. There was a significant reduction in both macroscopic tumor weight (p=0.01) and PET cells (p=0.04) in mice that received the BsMol-armed T cells compared with mice treated with OKT3 -armed T cells.
[134] Example 14: Expression of Target Proteins on T Cells [135] The above Examples show that a bi-specific molecule that targets SCLC and T cells, via CD3 on the T cell surface, is effective in vitro and in vivo for inhibition of SCLC cell growth and proliferation. The invention also encompasses bi- specific molecules that target other cell surface proteins on aberrant cells and on T cells. The various different T cell surface receptor targets were identified from T cells collected as described above.
[136] More specifically, T cells from four donors were incubated with IL-2 for 3- 5 days, then assayed with antibodies specific for the following cell surface receptors: CD3, CD4, CD8, and CD56. The results of the assays are shown in Table 2.
[137] Table 2: T Cell Cell-Surface Targets for Bi-Specific Molecules
Figure imgf000044_0001
[138] Table 2 shows that the donor samples all included significant expression of not only CD3, but also CD4 and CD8. Additionally, more than 10% of the cells from some donors expressed CD56. Thus, although the above Examples present data generated from a bi-specific molecule directed, at least in part, to the CD3 receptor on T cells, it is evident that other bi-specific molecules can be developed that are directed to CD4, CD8, CD56, or other proteins on the surface of T cells.
[139] It will be apparent to those skilled in the art that various modifications and variations can be made in the practice of the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

1. A bispecific molecule comprising at least one structure that binds a cell surface protein on the surface of a T cell, and at least one structure that binds a gastrin releasing peptide receptor on the surface of an aberrant target cell.
2. The molecule of claim 1, wherein at least one structure of the molecule binds CD3.
3. The molecule of claim 2, wherein at least one structure is an antagonist for a cell-surface protein present on the surface of a neoplastic cell.
4. The molecule of claim 3, wherein the neoplastic cell is a cancer cell.
5. The molecule of claim 1, wherein the aberrant cell is a small cell lung cancer cell.
6. The molecule of claim 1, wherein at least one structure is an antagonist for a cell-surface protein present on the surface of a neoplastic cell.
7. The molecule of claim 6, wherein the neoplastic cell is a cancer cell.
8. The molecule of claim 7, wherein the cancer cell is a small cell lung cancer cell.
9. The molecule of claim 1, comprising a monoclonal antibody that specifically binds human CD3 present on T cells, and a peptide comprising SEQ ID NO:3.
10. A method of detecting an aberrant cell of interest, said method comprising exposing at least one cell present in a sample to a bispecific molecule according to claim 1, and determining whether the molecule binds to at least one cell in the sample, said binding indicating the presence of the aberrant cell of interest in the sample.
11. The method of claim 10, wherein the bispecific molecule comprises at least a portion of an antigen binding region of an antibody.
12. The method of claim 10, wherein the bi-specific molecule comprises a portion that specifically binds CD3, and a portion that comprises an antagonist for a cell-surface protein present on the surface of the aberrant cell of interest.
13. The method of claim 10, wherein the aberrant cell is a neoplastic cell.
14. The method of claim 13, wherein the neoplastic cell is a cancer cell.
15. The method of claim 14, wherein the cancer cell is a small cell lung cancer cell.
16. The method of claim 10, wherein the method is a method of diagnosing cancer in a patient.
17. The method of claim 16, wherein the cancer is small cell lung cancer, breast cancer, prostate cancer, or a cancer of the gastrointestinal tract.
18. The method of claim 10, wherein the bispecifϊc molecule comprises a monoclonal antibody that specifically binds human CD3 present on T cells, and a peptide comprising SEQ ID NO:3.
19. A composition comprising the bispecifϊc molecule of claim 1 and a pharmaceutically acceptable substance.
20. A method of treating a patient suffering from at least one cancer, said method comprising administering to said patient the bispecific molecule of claim 1 in an amount sufficient to inhibit the growth of at least one cancer cell within the patient.
21. The method of claim 20, wherein administering comprises infusing a liquid composition comprising the bispecific molecule into the patient.
22. The method of claim 20, wherein the method is a method of treating small cell lung cancer.
23. The method of claim 20, wherein the method is a method of treating breast cancer, prostate cancer, or a cancer of the gastrointestinal tract.
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CN115304680A (en) * 2022-03-11 2022-11-08 四川大学华西医院 Preparation and application of bispecific cell adaptor molecule constructed based on Pep42
WO2023169583A1 (en) * 2022-03-11 2023-09-14 四川大学华西医院 Preparation and application of bispecific cell engager molecule constructed based on pep42
CN115304680B (en) * 2022-03-11 2024-02-02 四川大学华西医院 Preparation and application of bispecific cell adaptor molecule constructed based on Pep42

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