EP1534754A2 - Compositions bispecifiques de liaison d'antigene et methodes correspondantes - Google Patents

Compositions bispecifiques de liaison d'antigene et methodes correspondantes

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Publication number
EP1534754A2
EP1534754A2 EP03724219A EP03724219A EP1534754A2 EP 1534754 A2 EP1534754 A2 EP 1534754A2 EP 03724219 A EP03724219 A EP 03724219A EP 03724219 A EP03724219 A EP 03724219A EP 1534754 A2 EP1534754 A2 EP 1534754A2
Authority
EP
European Patent Office
Prior art keywords
antigen
composition
cells
cell
binding moiety
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP03724219A
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German (de)
English (en)
Other versions
EP1534754A4 (fr
Inventor
Lifen Ren-Heidenreich
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Roger Williams Medical Center
Original Assignee
Roger Williams Medical Center
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Filing date
Publication date
Application filed by Roger Williams Medical Center filed Critical Roger Williams Medical Center
Publication of EP1534754A2 publication Critical patent/EP1534754A2/fr
Publication of EP1534754A4 publication Critical patent/EP1534754A4/fr
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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 [IG], 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Defective immunity is responsible for tumor development in cancer patients.
  • a number of cell-based adoptive immunotherapy approaches have been tried (9, 11, 12, 20). These approaches include lymphokine-activated natural killer cells, tumor-infiltrating lymphocytes, auto-lymphocytes, activation of lymph node-draining T cells,' antigen-specific cytotoxic T lymphocytes, anti- CD3-activated T cells, anti-CD3/anti-CD28 co-activated T cells, and dendritic cells.
  • lymphokine-activated natural killer cells include lymphokine-activated natural killer cells, tumor-infiltrating lymphocytes, auto-lymphocytes, activation of lymph node-draining T cells,' antigen-specific cytotoxic T lymphocytes, anti- CD3-activated T cells, anti-CD3/anti-CD28 co-activated T cells, and dendritic cells.
  • these approaches have been informative, clinical responses have usually shown no effect because of the lack of specificity toward any particular tumor.
  • BsAb bi-specific monoclonal antibody
  • a BsAb in one embodiment, consists of two monoclonal antibodies (mAbs ) cross-linked through chemical heteroconjugation .
  • the BsAb will therefore carry dual specific "arms"; one arm recognizing and specifically binding to a tumor-associated antigen (TAA) and the other one recognizing the CD3 receptor on T cells.
  • TAA tumor-associated antigen
  • a BsAb When a BsAb bridges a T cell and a tumor cell, the armed T cell can bypass the major histocompatibility complex (MHC) restrictions and become a TAA-specific cytotoxic T lymphocyte (CTL) against tumor cells bearing the TAA. In vitro, these BsAbs have shown specific cytotoxicity against tumors (25) . In the treatment of cancer, BsAbs have improved human survival rates and eradicated tumors in animals (24) .
  • MHC major histocompatibility complex
  • CTL cytotoxic T lymphocyte
  • Her2/neu is a member of the epidermal growth factor receptor family of tyrosine kinases that is over- expressed in several cancers, including breast cancer (21) .
  • a chemically heteroconjugated anti-CD3 x anti- HER2/neu BsAb was used to treat high-risk breast cancer (13, 21) and hormone refractory prostate cancer.
  • re-BsAbs recombinant bi-specific antibodies
  • scFv variable regions
  • the re-BsAbs offer several advantages over intact BsAbs .
  • the smaller molecule size (30-50 kDa) allows higher penetration into solid tumor tissues.
  • the HAMA reactions are largely reduced due to the lack of an immunogenic Fc domain of Ab.
  • the process of producing highly purified protein is greatly simplified.
  • the entire protein production procedure can be done on a commercial scale.
  • This invention provides a first composition of matter comprising a first antigen-binding moiety and a second antigen-binding moiety operably affixed to one another via a flexible linker moiety.
  • This invention also provides a polypeptide comprising the amino acid sequence set forth in Figures 20-1 to 20-15 (SEQ ID NO:2) .
  • This invention also provides a polypeptide comprising the amino acid sequence set forth in Figure 25 (SEQ ID NO: ) .
  • This invention further provides a nucleic acid encoding a polypeptide comprising a first antigen-binding moiety and a second antigen-binding moiety operably affixed to one another via a flexible linker moiety having a length of ⁇ at least 16 amino residues.
  • This invention further provides a host-vector system comprising a host cell transfected with the instant expression vector.
  • This invention further provides a method for producing a polypeptide comprising a first antigen-binding moiety and a second antigen-binding moiety operably affixed to one another via a linker moiety having a length of at least 16 amino residues, which method comprises (a) culturing the instant host-vector system under conditions permitting the expression of the polypeptide, and (b) recovering the polypeptide so expressed.
  • This invention further provides a second composition of matter comprising (a) the above-described composition and (b) a cell having on its surface the antigen to which the first antigen-binding moiety specifically binds .
  • This invention further provides a method for increasing the activity of a CD3+ cell comprising contacting the cell with the instant composition.
  • This invention further provides a method for treating a subject afflicted with a disorder mediated by the presence of an abnormal cell, comprising administering to the subject (a) an agent known to ameliorate the disorder via contact with the abnormal cell, and (b) the instant composition, wherein the first antigen-binding moiety specifically binds to an antigen present on the agent, and the second antigen-binding moiety specifically binds to an antigen present on the abnormal cell.
  • This invention further provides a method for treating a subject afflicted with a tumor comprising administering to the subject (a) Interleukin-2 (IL-2), (b) T cells, and
  • This invention further provides a kit for use in treating a subject afflicted with a disorder mediated by the presence of an abnormal cell, comprising (a) the instant composition, wherein the first antigen-binding moiety specifically binds to an antigen present on an agent known to ameliorate the disorder and the second antigen- binding moiety specifically binds to an antigen present on the abnormal cell, and (b) instructions for use.
  • This invention further provides a kit for use in treating a subject afflicted with -a disorder mediated by the presence of an abnormal cell, comprising (a) the first ⁇ instant composition, and (b) the agent known to ameliorate the disorder.
  • this invention provides a kit for use in treating a subject afflicted with a tumor comprising (a) Interleukin-2 (IL-2), (b) T cells, (c) the antibody designated E3Bi, and (d) instructions for use.
  • IL-2 Interleukin-2
  • T cells T cells
  • E3Bi the antibody designated E3Bi
  • This Figure shows the over-expression of EpCA on tumor cell surfaces but not on normal epithelium.
  • the EpCAM is over-expressed in MCF-7 breast cancer cells (middle) and colorectal cancer cells (left) , but not in HBS-100 normal breast epithelial cells (right) . Cells were stained with the GA733.2 Ab.
  • This Figure illustrates the relationship of a T cell carrying a ch-TCR with and without the hinge spacer (H) .
  • the connected T cell signaling chain "Y" initiates the T cell activation that will produce non- MHC-restricted tumor-killing activity.
  • T cell populations from both healthy donors and patients were either not transduced (T) or transduced with the empty retrovirus only (SAM) , with the retrovirus carrying the GA733.2-derived ch-TCR (GA) , or with GA plus a hinge (GAH) .
  • the effector-to-target ratios are 5:1 for panels A and B, or 2.5 : 1 for panel 3C.
  • ELISAs of IFN- ⁇ and TNF- ⁇ were performed after 24 hr incubation. Supernatants (50 ⁇ l) were collected for ELISAs in triplicate. The target cell lysis was determined after incubation for 4 hr at 37°C by the 51 Cr-release assay.
  • Panel C Only data from healthy donors are shown in panel C because there is no different cytotoxicity observed using either patients' or normal donors' ATCs. Panels A and B show that cytokine production was increased by the hinge addition (GAH with a hinge and GA without) . Panel C shows that cytotoxicity was also increased by about two-fold in the GAH group.
  • FIG. 4 The ch-TCR with a hinge (GAH ⁇ -EN) shows greatly increased T cell aggregations with the tumor cells in comparison to the ch-TCR without the hinge (GA ⁇ -EN) .
  • These photographs were taken after co-cultivation of tumor cells and T cells for 4 hr at 37°C at an effector-to-target ratio of 2:1.
  • T cell non-transduced T cells plus tumor cells
  • SAM-EN T cells transduced with expression vector only without the gene of interest
  • GA ⁇ -EN with the hinge
  • EN an internal ribosome entry site in the vector.
  • ch-TCR-transduced T cells only occurs when they are exposed to EpCAM-positive tumor cells (LS174T) at an E:T ratio of 5:1 for 24 hr at 37°C.
  • the SD is indicated in both panels.
  • FIG. 7 This Figure demonstrates that as few as 5 ng BsAb per 1x10 s T cells can trigger the cytotoxicity mediated by armed T cells.
  • This cytotoxicity assay was performed using MCF-7 cells .
  • the data presented in this Figure are summarized from three experiments in three different donors. -This Figure shows composite titration curves for unarmed TCs and ATCs armed with 0.5, 5.0 and 50.0 ng of OKT3/9184 BsAb at effector-to-target ratios of between 5 and 25 to 1; unarmed (T) or armed with 0.5 (•) , 5.0 ( ⁇ ) , and 50 ( ⁇ ) ng BsAb/lxlO 6 ATCs/ml.
  • mice treated with BsAb- armed ATCs survived.
  • the BsAb OKT3XT84.66, was used for this experiment.
  • SCID mice received 3Gy of total body irradiation to eliminate NK cells to ensure engraftment of tumor cells.
  • the mice received subcutaneous co- injections of armed or unarmed ATCs (20xl0 ⁇ ATCs) along with CEA-positive LS174T tumor cells (lxlO 6 cells) (Winn assay) .
  • the control group only received tumor cells and no ATCs. All non-ATC mice died of tumor progression by day 15 with tumor size >22mm.
  • 40% of mice that received armed BsAb were still alive, while only 10% were alive in the group that only received un-ar ed ATCs .
  • FIG. 10 shows the construction of E3Bi into pGlEN vector .
  • VH-VLe the scFv of GA 33 . 2 ;
  • VH-VL3 the ScFv of OKT3 ;
  • SD splicing donor;
  • SA splicing acceptor ;
  • His 6xHis-tag;
  • IRES an internal ribosome entry site;
  • neo r a neomycine phosphotransferase gene .
  • Figure 10 shows the construction of E3Bi into pGlEN vector .
  • VL variable light chain of mAb
  • VH variable heavy chain of mAb
  • H hinge.
  • T cell aggregation is dependent on the E3Bi doses.
  • FIG. 13 This Figure shows a cytotoxicity assay ( 51 Cr release assay) of E3Bi-armed T cells.
  • Target LS174T, 16 hr assay.
  • FIG. 14 This Figure shows IFN- ⁇ production induced by different doses of E3Bi .
  • This Figure shows the cloning of a hinge to the 3' -end of EpCAM scFv.
  • This Figure shows the assembly of E3 to pGlEN.
  • This Figure shows the replacement of a hinge with GS- linker GGGGSGGGGSGGGGS .
  • FIG. 19 This Figure shows a circular map of pGlEN-EH3.His .
  • E3Bi and its vector have been confirmed by DNA sequencing analysis .
  • This DNA plasmid is called pGlEN-EH3.His .
  • the completed DNA sequence of 8,078 base pairs (SEQ ID NO:l) and the corresponding amino acid sequence (SEQ ID NO: 2) are also shown.
  • the scFv of GA733.2 starts at site 1,388, the hinge starts at site 2,169, and the scFv of OKT3 starts at site 2,358.
  • the 6XHis tag starts at site 3,093.
  • This Figure shows the in vivo anti-tumor response of E3Bi in a tumor xenograft model by tumor growth delay.
  • Tumor growth delay is reported as the mean number of days ( ⁇ SD) for tumor volumes of mice from each treatment group to reach 2 cc.
  • p 0.0034 is the probability by Kruskal-Wallis non- parametric analysis that tumor growth delay is the same for all treatment groups, p ⁇ 0.01 is the probability by Dunn's multiple comparison analysis that treatment with IL-2/ATC/E3Bi produces the same tumor growth delay in mice as treatment with IL-2 alone; p > 0.05 for IL-2/ATC alone .
  • This Figure shows the survival of LS174T cells from LS174T tumor xenografts excised from SC D-Beige mice 24 h after mice received treatment with: IL-2 (300 IU/injection i.t.) alone; IL-2 and ATC (7xl0 7 cells/injection i.t.); or IL-2/ATC and low (1 mg/kg i.v.) or high dose (10 mg/kg i.v.) E3Bi. After excision, tumor cells were processed into single-cell suspensions and seeded into cultures in four concentrations with five replicates each. Cells were counted after 7 days. Results are represented as the mean ( ⁇ SE) surviving fraction of cells from each treatment group compared to the IL-2 treatment group.
  • IL-2/ATC/E3Bi 10 mg/kg
  • p ⁇ 0.001, IL-2 or IL-2/ATC vs. IL-2/ATC/E3Bi 10 mg/kg
  • p ⁇ 0.001, IL-2/ATC vs. IL- 2/ATC/E3Bi 1 mg/kg
  • p ⁇ 0.05, IL-2/ATC/E3Bi (1 mg/kg) vs. IL-2/ATC/E3Bi (10 mg/kg).
  • Figure 25 The protein sequence of E3Bi (SEQ ID N0:4).
  • ATC Activated T Cell
  • Characteristics of ATC include, without limitation, resumption of cell cycle, elevated IL-2 secretion, upregulated IL-2 receptor expression, limited proliferation, and differentiation into effector cells.
  • administering shall mean delivering in a manner which is effected or performed using any of the various methods and delivery systems known to those skilled in the art.
  • Administering can be performed, for example, intravenously, pericardially, orally, via implant, trans- mucosally, transdermally, intramuscularly, subcutaneously, intraperitoneally, intrathecally, intra- lymphatically, intralesionally, or epidurally.
  • Administering can be performed, for example, once, a plurality of times, and/or over one or more extended periods .
  • antibody includes, by way of example, both naturally occurring antibodies (e.g., IgG, IgM, IgE and IgA) and non-naturally occurring antibodies.
  • antibody also includes polyclonal and monoclonal antibodies, and fragments thereof (e.g., antigen-binding portions) . Furthermore, the term “antibody” includes chimeric antibodies, wholly synthetic antibodies, human antibodies, humanized antibodies, and fragments thereof. "BsAb”, also referred to herein as "bi-specific antibody”, shall include, without limitation, a composition of matter comprising two operably affixed moieties, wherein each moiety is capable of binding to an antigen and comprises an antibody.
  • BsAbs include, for example, (i) compositions comprising whole antibodies tethered together, (ii) single antibodies having two antigen-binding domains, each specific for a different antigen, (iii) single chain polypeptides, each comprising two antigen-binding domains linked via a region of at least 16 amino acid residues, and (iv) compositions comprising antigen-binding portions of antibodies operably affixed via chemical linkers.
  • Flexible linker moiety shall mean any chemical or biochemical moiety which (i) joins two antigen-binding moieties, (ii) comprises at least one chemical bond about which rotation is permitted, and (iii) permits the unhindered binding of each antigen-binding moiety joined thereto to its respective antigen.
  • the flexible linker moiety permits binding of the two antigen-binding moieties to their respective antigens located on different cells (e.g., permitting the first antigen-binding moiety to bind to its antigen on a tumor cell, and the second antigen-binding moiety to bind to its antigen on a T cell) .
  • “Host cells” include, but are not limited to, bacterial cells, yeast cells, fungal cells, insect cells, and mammalian cells. Mammalian cells can be transfected by methods well-known in the art such as calcium phosphate precipitation, electroporation and microinjection.
  • Mammalian cell shall mean any mammalian cell. Mammalian cells include, without limitation, cells which are normal, abnormal and transformed, and are exemplified ⁇ by neurons, epithelial cells, muscle cells, blood cells, immune cells, stem cells, osteocytes, endothelial cells and blast cells .
  • Non-activated T cell shall have the meaning normally ascribed to it in the art. Characteristics of a non- activated T cell include, without limitation, quiescence of cell cycle, non-proliferation and non-differentiation.
  • nucleic acid refers to a polymer of deoxyribonucleotides and/or ribonucleotides .
  • deoxyribonucleotides and ribonucleotides can be naturally occurring or synthetic analogues thereof.
  • “Pharmaceutically acceptable carriers” are well known to those skilled in the art and include, but are not limited to, 0.01-0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non- aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate . Aqueous carriers include water, alcoholic/aqueous solutions, emulsions and suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like.
  • Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like.
  • polypeptide peptide
  • protein protein
  • amino acid residues can be naturally occurring or chemical analogues thereof.
  • Polypeptides, peptides and proteins can also include modifications such as glycosylation, lipid attachment, sulfation, hydroxylation, and ADP-ribosylation.
  • Specifically bind shall mean that, with respect to the binding of an antigen-binding moiety to its respective antigen, the moiety binds to that antigen with a greater affinity than that with which it binds to most or all other antigens. In the preferred embodiment, the moiety binds to that antigen with a greater affinity than that with which it binds to all other antigens.
  • stem cell shall mean, without limitation, a cell that gives rise to a lineage of progeny cells .
  • stem cells include CD34+ cells and embryonic stem cells.
  • Surface adhesion molecules present on stem cells include, without limitation, IL-3 receptor, IL-6 receptor, IL-11 receptor, c-kit, VLA-4, VLA-5, L-selectin, PECAM-1 and Beta-1 integrin.
  • Subject shall mean any animal, such as a mammal or a bird, including, without limitation, a cow, a horse, a sheep, a pig, a dog, a cat, a rodent such as a mouse or rat, a chicken, a turkey and a primate.
  • the subject is a human being.
  • Vector shall mean any nucleic acid vector known in the art. Such vectors include, but are not limited to, plasmid vectors, cos id vectors, and bacteriophage vectors .
  • This invention provides a first composition of matter comprising a first antigen-binding moiety and a second antigen-binding moiety operably affixed to one another via a flexible linker moiety.
  • the flexible linker moiety can comprise, for example, a polymer or a polypeptide.
  • the polypeptide has a length of at least 16 amino acid residues.
  • the polypeptide has a length of between 16 amino acid residues and about 100 amino acid residues .
  • the polypeptide has a length of between 50 amino acid residues and about 75 amino acid residues.
  • the polypeptide has a length of about 63 amino acid residues, and/or comprises all or a portion of an antibody hinge region (e.g., CD8 ⁇ Ig hinge-like region) .
  • the polypeptide has the amino acid sequence encoded by nucleotides 2170-2358 shown in
  • Figures 20-1 to 20-15 (SEQ NO ID:1).
  • the first and second antigen-binding moieties specifically bind to different antigens.
  • the first antigen-binding moiety specifically binds to a tumor cell surface antigen.
  • the first antigen-binding moiety specifically binds to a cell surface antigen such as CD2, CD3, CD56 or other T cell or NK cell surface antigen.
  • the first antigen-binding moiety specifically binds to a tumor cell surface antigen
  • the second antigen- binding moiety specifically binds to a CD3+ cell surface antigen.
  • the tumor cell surface antigen is EpCAM
  • the CD3+ cell surface antigen is CD3.
  • Other antigens include, for example, the breast cancer-associated antigen HER2. Antibodies against this antigen are known.
  • the first antigen-binding moiety comprises the antigen-binding portion of an anti-EpCAM antibody
  • the second antigen-binding moiety comprises the antigen-binding portion of the antibody designated OKT3.
  • the anti-EpCAM antibody comprises the antigen-binding portion of the antibody designated GA733.2.
  • each antigen-binding moiety preferably comprises the antigen-binding portion of an antibody.
  • the antigen-binding portions can be, for example, Fab portions.
  • the composition comprises a single polypeptide chain which forms the first and second antigen-binding moieties and the linker moiety.
  • each of the first and second antigen-binding moieties further comprises a second polypeptide chain.
  • This invention further provides a polypeptide comprising the amino acid sequence set forth in Figures 20-1 to 20 15 (SEQ ID NO: 2) .
  • This polypeptide is referred to herein as E3Bi, and comprises an anti-EpCAM and anti-CD3 domain.
  • This invention further provides a polypeptide comprising the amino acid sequence set forth in Figure 25 (SEQ ID NO: 4 ) .
  • This invention further provides a nucleic acid encoding a polypeptide comprising a first antigen-binding moiety and a second antigen-binding moiety operably affixed to one another via a flexible linker moiety having a length of at least 16 amino residues.
  • the nucleic acid has the nucleotide sequence shown in Figures 20-1 to 20-15 (SEQ ID NO:l) .
  • the nucleic acid has the nucleotide sequence shown in Figure 24 (SEQ ID NO:3) .
  • the nucleic acid can be, for example, DNA or RNA, and is preferably DNA.
  • the nucleic acid is an expression vector.
  • Expression vectors include, for example, plasmids, cosmids, bacteriophages and eukaryotic viruses.
  • the eukaryotic virus is an adenovirus or a retrovirus.
  • This invention further provides a host-vector system comprising a host cell transfected with the instant expression vector.
  • This invention further provides a method for producing a polypeptide comprising a first antigen-binding moiety and a second antigen-binding moiety operably affixed to one another via a flexible linker moiety having a length of at least 16 amino residues, which method comprises (a) culturing the instant host-vector system under conditions permitting the expression of the polypeptide, and (b) recovering the polypeptide so expressed.
  • This invention further provides a second composition of matter comprising (a) the instant composition and (b) a cell having on its surface the antigen to which the first antigen-binding moiety specifically binds.
  • the cell is a CD3+ cell and the first antigen-binding moiety specifically binds to CD3.
  • the cell is a T cell
  • the first antigen-binding moiety comprises the antigen-binding portion of the antibody designated OKT3
  • the second antigen-binding moiety comprises the antigen-binding portion of the antibody designated GA733.2.
  • the composition of (a) is present in a ratio of from about 5-500 ng per million cells of (b) .
  • This invention further provides a method for increasing the activity of a CD3+ cell comprising contacting the cell with the first composition.
  • This invention further provides a method for treating a subject afflicted with a disorder mediated by the presence of an abnormal cell, comprising administering to the subject (a) an agent known to ameliorate the disorder via contact with the abnormal cell, and (b) the above- described composition, wherein the first antigen-binding moiety specifically binds to an antigen present on the agent, and the second antigen-binding moiety specifically binds to an antigen present on the abnormal cell.
  • the subject is selected from the group consisting of a cow, a horse, a sheep, a pig, a dog, a cat, a rabbit and a primate.
  • the subject is a human.
  • the disorder treated by the instant method can be any disorder mediated by an abnormal cell.
  • disorders include, without limitation, cancer and specifically tumors.
  • Cancer includes, without limitation, solid tumors, metastatic tumor cells and nonsolid cancers of the blood, marrow, and lymphatic systems.
  • Tumors include, for example, carcinomas (cancers derived from epithelial cells), sarcomas (derived from mesenchymal tissues), lymphomas (solid tumors of lymphoid tissues) , and leukemias (marrow or blood borne tumors of lymphocytes or other hematopoietic cells) .
  • the agent is a CD3+ cell
  • the first antigen-binding moiety specifically binds to CD3 (or any other T cell antigen)
  • the second antigen-binding moiety specifically binds to EpCAM.
  • the composition comprises the polypeptide whose amino acid sequence is shown in Figures 20-1 to 20-15 (SEQ ID NO: 2) .
  • the composition comprises the polypeptide whose amino acid- sequence is shown in Figure 25 (SEQ ID NO: 4) .
  • This invention further provides a method for treating a subject ' afflicted with a tumor comprising administering to the subject (a) Interleukin-2 (IL-2), (b) T cells, and (c) the antibody designated E3Bi .
  • the T cells can be, for example, activated T cells or non-activated T cells.
  • the subject is selected from the group consisting of a cow, a horse, a sheep, a pig, a dog, a cat, a rabbit and a primate.
  • the subject is a human.
  • This invention further provides a kit for use in treating a subject afflicted with a disorder mediated by the presence of an abnormal cell, comprising (a) the first instant composition, wherein the first antigen-binding moiety specifically binds to an antigen present on an agent known to ameliorate the disorder and the second antigen-binding moiety specifically binds to an antigen present on the abnormal cells, and (b) instructions for use .
  • This invention further provides a kit for use in treating a subject afflicted with a disorder mediated by the presence of an abnormal cell, comprising (a) the first instant composition, and (b) the agent known to ameliorate the disorder.
  • the composition of (a) comprises a polypeptide having the sequence shown in Figures 20-1 to 20-15 (SEQ ID NO: 2) .
  • the composition of (a) comprises a polypeptide having the sequence shown in Figure 25 (SEQ ID NO:4) .
  • this invention provides a kit for use in treating a subject afflicted with a tumor comprising (a) Interleukin-2 (IL-2), (b) T cells, (c) the antibody designated E3Bi, and (d) instructions for use.
  • the T cells can be, for example, activated T cells or non- activated T cells .
  • BsAbs bi-specific monoclonal antibodies
  • TAA tumor associated antigen
  • CTL TAA-specific cytotoxic T lymphocyte
  • EpCAM epidermal cell adhesion molecule
  • a re-BsAb was constructed from the mAb GA733.2 and mAb 0KT3, and called E3Bi. GA733.2 recognizes EpCAM (8).
  • the tumor targets the tumor.
  • EpCAM epicellular cell adhesion molecule
  • EGP-2 epidermal cell adhesion molecule
  • EGP-40 17-1A, KSA
  • Figure 1 shows the surface antibody staining of EpCAM in colorectal (left) and breast (middle) cancer, as well as in normal epithelium (right) .
  • EpCAM is a well-studied and characterized tumor antigen.
  • Two antibodies, C017-1A and GA733.2 bind to EpCAM, but at different epitopes and with different affinities .
  • C017-1A has been used in clinical trials to treat colorectal cancer following surgery (6). However, there were no detectable immune responses reported.
  • TCR T cell receptor
  • ch-TCR chimeric TCR
  • Addi tion of a hinge spa cer can significantly increase the tumor-binding and killing function of a ch-TCR
  • Two ch-TCRs (Figure 2) have been constructed. One has a hinge (H) insert and the other does not. Both ch-TCRs contain the scFv of the mAb GA733.2 that binds to the EpCAM and a T cell signaling domain that triggers T cell activation. Both the FcR ⁇ -chain (GAH ⁇ ) and TCR ⁇ -chain (GAH ⁇ ) were used as the T cell signaling domain. This ch- TCR was transduced into an activated T cell (ATC) via a retrovirus . These results show that T cells carrying this ch-TCR specifically and efficiently target and lyse tumor cells (18), and the hinge spacer can increase the specific tumor lytic function ( Figures 3 and 4) .
  • the mAb, GA733. 2 specifically binds to EpCAM- positive tumor cells, and not to EpCAM-nega tive cells
  • the cell line NCI-H716 is originally generated from cecum tumor cells that are EpCAM-negative. Using H716 as negative control, it was demonstrated that GA733.2 only targets EpCAM-positive cells (Figure 5) .
  • BsAbs are effective at specifically targeting tumor cells and genera ting cytolytic activi ty, and pre- arming T cells with BsAb before infusion increases the efficiency of BsAb-media ted tumor killing
  • FIG. 6 shows, in the upper left panel, that activated T cells (ATCs) which had been cultured for 14 days and armed with 50 ng of BsAb (per 10 6 cells) bind to and then kill MCF-7 cells.
  • ATCs activated T cells
  • the lower left panel shows the ⁇ n-armed ATC control.
  • the ATCs in the upper right panel have been armed with irrelevant mAbs and those shown in the lower right have been armed with non-conjugated mAbs.
  • the re-BsAb E3Bi derived from mAb GA733.2, binds to EpCAM on tumor cells better than the re-BsAb from mAb C017-1A; (2) a hinge addition between two scFv motifs enhances the binding efficiency; and (3) pre-arming ATCs with the re-BsAb before infusion improves efficiency and minimizes clinical toxicity.
  • Experiment 1 Construct E3Bi from two single chain fragments of variable regions (scFvs) of mAbs GA733.2 and 0KT3, and insert a linker from the CD8 ⁇ hinge-like region (H) between these two scFvs.
  • H linker is replaced with a traditional glycine-serine linker, (G 3 S ⁇ )3.
  • G 3 S ⁇ traditional glycine-serine linker
  • Experiment 3 Evaluate the specific cytolytic function of E3Bi in vi tro (using EpCAM-positive colon cancer cell line LS174T, and using EpCAM-negative cecum cancer cell line H716 as a negative control) and in vivo (using Beige-SCID mice) .
  • the biggest challenge for cancer treatment is to direct a patient's own immune system to fight cancer.
  • tumor growth is the result of a defective immune system in which the MHC (major histocompatibility complex) fails to present tumor antigens to the immune system and to generate enough specific cytotoxic T lymphocytes (CTL) .
  • CTL cytotoxic T lymphocytes
  • BsAb conjugated bi-specific antibody
  • the engineered recombinant BsAb approach overcomes the limitations of chemically heteroconjugated BsAbs because only the binding sites of the antibodies are selectively engineered, and not the regions that may cause side effects such as HAMA reactions.
  • the re-BsAb product can be pure and consistent from lot to lot, while the chemically conjugated BsAb is only about 15-30% pure and the product is very inconsistent.
  • the other advantage of this re-BsAb is that large-scale production is possible .
  • this invention provides a re-BsAb with improved tumor-killing efficiency. This is accomplished in several ways: (1) using an antibody that has higher binding affinity; (2) adding a longer spacer between two binding sites; (3) producing this protein in mammalian cells; and (4) arming a patient's T cells with this re- BsAb in vi tro before infusing the patient.
  • E3Bi offers a very effective cancer immunotherapy approach. This product will have much less toxicity because the patient's own T cells will be stimulated to eradicate tumor cells . Pre- arming T cells before infusion will further increase the efficiency and specificity of this re-BsAb. Multiple infusions of these armed T cells over a longer period of time are expected to eradicate residual tumor cells more effectively compared to other immunotherapy approaches.
  • the specificity of E3Bi is unique because these armed T cells will locally deposit at a specific tumor site and kill tumor cells. Furthermore, they will also attack residual tumor cells that have already spread prior to surgery.
  • colorectal cancer has the highest incidence among all types of cancer in the U.S., patients with colorectal cancer are envisioned as an important treatment group. Since EpCAM, the cell surface tumor marker recognized by EpCAM
  • E3Bi is over-expressed in all adenocarcinomas (23) , a very important aspect of E3Bi is that it has use with respect to most solid tumors as well.
  • this re-BsAb will not only eradicate residual tumor cells, but will be part of adjuvant therapy for a variety of EpCAMt tumors.
  • E3Bi The design of E3Bi is unique and offers several advantages over other re-BsAbs that have been published (25) .
  • the vector pGlEN is used for production of re-BsAb for the first time. Based on previous experience, this vector is highly effective in penetrating mammalian cell membranes, integrating cDNA into the host genome and promoting gene expression.
  • Mammalian cells (CHO cells) are used as E3Bi producer cells because mammalian proteins produced in the traditional bacterial cell E . coli may not fold properly and therefore may not function correctly.
  • the hinge spacer (63 amino acids) used for E3Bi has never been used for re-BsAb construction.
  • the longer linker between two scFvs in E3Bi will provide the space needed for the interaction of a tumor cell and a T cell (18) and, therefore, is expected to increase the binding and tumor killing efficiency of E3Bi .
  • GA733.2 is used for constructing a re-BsAb for the first time. Both GA733.2 and C017-1A target EpCAM, but at two different epitopes (7). GA733.2 has a higher affinity for EpCAM antigen than does C017-1A and produces stronger cytotoxicity against EpCAM-positive tumor cells
  • T cells from a patient are activated, expanded, armed with E3Bi and frozen for later infusion into the same patient.
  • This in vitro arming protocol is the first of its kind used for a re-BsAb. It is believed that this approach not only provides a large quantity of activated and armed tumor-killing T cells, but also reduces the possible toxicity and increases the efficiency of E3Bi .
  • FIGS 9-11 illustrate the design of E3Bi.
  • Figures 9-11 illustrate the design of E3Bi.
  • Figures 9-11 illustrate the design of E3Bi.
  • Figures 9-11 illustrate the design of E3Bi.
  • Figureso shown are the cloning of a hinge to the 3 '-end of EpCAM scFv ( Figure 15), the construction of OKT3 scFv ( Figure 16), the assembly of E3 to pGlEN ( Figure 17), the replacement of a hinge with GS-linker GGGGSGGGGSGGGGS ( Figure 18), and a circular map of pGlEN-EH3.His (Figure 19)).
  • the E3Bi cDNA is generated by combining variable light (V L ) and heavy (V H ) chains of mAbs GA733.2 and 0KT3 that are amplified by PCR.
  • the E3Bi cDNA is then inserted into an expression vector, pGlEN.
  • PGlEN is generated from the Maloney murine leukemia virus (MMLV) and is replication incompetent due to the lack of three genes that are essential for virus formation, gag, env and pol . This insures against retroviral replication. Based on previous experience (18), this vector is highly efficient in producing stably transduced mammalian cells and promoting gene expression.
  • the anti-CD3 scFv is generated' from OKT3 hybridoma cells (ATCC, Rockville, MD) .
  • the E3Bi gene expression is driven by long terminal repeats (LTR) .
  • This vector contains a leader sequence from the k light chain to penetrate cell membranes, a neomycin phosphotransferase gene (neo r ) for drug selection, a splicing donor (SD) /splicing acceptor (SA) to enhance the efficiency of transcription, and an internal ribosome entry site (IRES) for driving neo r gene transcription.
  • a 6xHis-tag is added to the C-terminal end for affinity purification of this re-BsAb protein.
  • the hinge spacer By adding distance from the scFv to the plasma membrane, the hinge spacer has shown increased tumor binding and killing activity in connection with the chimeric TCR approach (16, 18) .
  • Figure 3 shows that in both healthy donors and patients, the ch-TCR with a hinge (GAH) significantly increased the specific tumor cytotoxicity and cytokine secretion (IFN- ⁇ and TNF- ) by about two-fold (compared to a ch-TCR without a hinge) (18) .
  • the hinge approach has never before been applied for re-BsAb construction. The hinge is expected to give the re-BsAb flexibility and rotational freedom leading to a better bridge between a tumor cell and a T cell.
  • re-BsAbs are expressed in a traditional prokaryotic expression system (24).
  • the re-BsAb protein may not fold properly in prokaryotic cells (14) . Therefore, a eukaryotic cell line, the Chinese hamster ovary cell line (CHO, GIBCO Life-technologies, Rockville, MD) , is transfected. Specifically, CHO is transfected with the standard CaP0 4 precipitation method (17) and cultured in the presence of the selection drug, G418. The stably transfected CHO cells form colonies after 10-14 days. The colonies are selected for the highest quantity of the re- BsAb production and evaluated by ELISA for the presence of a 6xHis-tag (Ni-NTA HisSorb Plates, QIAGEN, Valencia,
  • the re-BsAb is secreted into the culture medium that
  • the CHO clone with the highest yield of re-BsAb is grown as non-adherent cells in a serum-free medium specially constituted for CHO (CD-CHO,
  • the medium containing E3Bi is collected every 24 hr or as otherwise determined.
  • T cells from healthy donors are isolated from 40 cc peripheral blood, activated with anti-CD3 mAb at 10-50 ng/lxlO 6 T cells/ml, and expanded for 14 days in the presence of 100 IU of IL-2 and 10% fetal calf serum in the medium, RPMI-1640 (BioWittaker, Walkersville, MD) .
  • the ATCs are armed with different doses of E3Bi and rocked for 1 hr at 4°C.
  • the cells are washed twice with RPMI-1640 to eliminate excess unbound E3Bi.
  • the armed and unarmed ATCs are added to the target tumor cells at effector-to-target ratios from 1:1 to 10:1.
  • Cytotoxicity assays 51 Cr release assay
  • IFN- ⁇ production assays ELISA
  • the dose, time and temperature in the arming procedure are evaluated.
  • a blocking assay is performed using the anti-Id antibody against the scFv of GA733.2.
  • the cytotoxicity and ELISA assays are analyzed statistically with a standard statistical package, a paired t-test or Wilcoxon signed tank test using the SigmaStat. All in vitro assays are repeated with at least 5 unrelated subjects.
  • the significant cytotoxic functions of E3Bi are analyzed with a paired fc-test or Wilcoxon signed tank test using SigmaStat.
  • mice In vivo functional assays are performed in animal models. Four to eight week-old female beige SCID mice are used for these studies (Taconic Pharm, Germantown, NY) . These mice carry the SCID mutation that causes a deficiency of both T and B cells resulting in cytotoxic T cell and macrophage defects as well as selective impairment of NK cell function. The animals are maintained in accordance with NIH animal care guidelines.
  • mice are divided into two groups; one group with "Winn Assay", which means 1x10 s tumor cells are co- injected with armed ATCs (dose range from 1x10 s to 10x10 s ) subcutaneously into the upper right thigh of each animal or with unarmed T cells as a control. Tumor development is documented weekly. The other group is injected only with IxlO 6 tumor cells subcutaneously into the upper right thigh of each animal. Once the tumor is established
  • mice from both groups are divided into three sub-groups: the first group receives no T cells; the second group is injected with unarmed T cells, and the third group is injected with armed T cells with E3Bi.
  • the tumor development is measured and documented every 2 days.
  • the tumor cells used for these in vivo studies are LS174T (human colorectal adenocarcinoma cells). T cells are extracted from the peripheral blood of both healthy donors and patients. The animals are sacrificed by C0 2 gas overdose once the tumor size exceeds 1.5 cm. By week 8-10 after treatment, all animals are sacrificed. All data are analyzed using a paired t-test or Wilcox test on signed tank test using SigmaStat.
  • the in vivo anti-tumor response of E3Bi was also evaluated in a tumor xenograft model by tumor growth delay assay.
  • mice Four-week old SCID-Beige mice were divided into four groups: i.t. injection of IL-2 only (IxlO 4 IU/kg) ; i.t. injection of IL-2 and ATC (2xl0 9 cells/kg); i.v. injection of a low (1 mg/kg) or high (10 mg/kg) dose E3Bi along with an i.t. injection of IL-2/ATC. Each mouse received two i.v. injections (day 1 and day 3) of E3Bi and two i.t.
  • mice receiving only ATC/IL-2 The tumor size more than doubled in the mice receiving only ATC/IL-2 while it remained largely unchanged in mice receiving low dose E3Bi after 7 days from the. first injection.
  • necrosis (E) of tumors in mice receiving high dose of E3Bi the tumors in these mice demonstrate partial regression within 7 days of initial treatment.
  • Figure 22 further supports the targeting specificity of E3Bi to EpCAM+ over-expressing tumors in vivo.
  • Mice with established LS174T tumors were treated with ATC or ATC followed by an IV injection of low or high does E3Bi, and excised 24 h later.
  • the viability of treated cells was measured as the surviving fraction of tumor cells after in vivo treatment with IL-2, IL-2 /ATC and IL-2/ATC/E3Bi .
  • ATC treatment alone produced no cytotoxic effect on tumor cells
  • administration of low dose (1 mg/kg) E3Bi in conjunction with ATC treatment produced a 40% decrease in tumor cell survival.
  • Increasing the E3Bi dose to 10 mg/kg significantly decreased the tumor cell survival by 90% (p ⁇ 0.05) .
  • these results show that E3Bi. delivered systematically traffics, binds and produces cytotoxic effects to EpCAM+ over-expressing tumor cells in vivo .
  • E3Bi also directly triggers non-activating T-cells to kill tumor cells.
  • E3Bi triggered CD4+ and CD8+ populations in peripheral blood mononuclear cells (PBMC) to become activated in the presence of LS174T tumor cells (data not shown) .
  • PBMC peripheral blood mononuclear cells
  • FIG 23 illustrates that E3Bi triggers cytotoxicity in PMBC, which include non-activated T cells.
  • 1, 2, and 3 day cytotoxicity assays (CML) were conducted using PBMC as the effectors and LS174T colon tumor cells as target cells.
  • CML cytotoxicity assays
  • This Figure also shows some non-MHC restricted and non-specific cytolytic activity of T cells in the E3Bi- group; however, this cytolytic activity is insignificant, p > 0.05. (3. 3) Anticipated obstacles
  • T cells are pre-armed in vitro with E3Bi before infusion so that the small E3Bi will remain attached to the CD3 receptor on the T cells while traveling in the body and, therefore, be protected from rapid kidney clearance. More importantly, pre-arming the T cells in vitro will dramatically improve the killing efficiency (data not shown) .
  • Existing methodology enables one to pre-arm T cells in vi tro for future clinical trials.
  • the pre-arming procedure includes (1) mixing day 14 ATCs with different doses of E3Bi in a tube and rocking for one hour at 4°C; (2) washing twice to remove unbound E3Bi; and (3) infusing the armed T cells at a concentration of lxlO 7 cells/ml.
  • T cell activation can result in activation-induced T cell apoptosis (AICD) and as a consequence, reduced tumor killing efficiency in vivo .
  • AICD activation-induced T cell apoptosis
  • ch-TCR 18, 19
  • BsAb 10
  • bystander-killing assays apoptosis assays (Annexin V staining) and 3 [H]- thymidine proliferation assays are performed. Following tumor exposure, the fate of armed T cells is studied with and without the E3Bi. (4) Affinity purifica tion of E3Bi
  • the high producer cells are grown in suspension in the serum-free medium, CHO-S-SFM II (GIBCO) , which is a constituted medium developed specifically for CHO cells growing in suspension.
  • the supernatant containing the released E3Bi is collected every 24 hr and affinity purified by applying it to Ni-NTA spin columns. These columns can purify up to 150 mg of E3Bi in a one-step affinity purification of 6xHis-tag-containing recombinant protein (QIAGEN) .
  • QIAGEN 6xHis-tag-containing recombinant protein
  • the columns are washed and eluted according to the manufacturer's instructions.
  • the quality of the purified product is evaluated by denaturing gel electrophoresis and Western blot.
  • E3Bi is stored in phosphate-buffered saline at 4°C, lyophilized and stored at -20°C for the long term.
  • E3Bi Collected supernatant containing the E3Bi is applied to a Ni-NTA agarose column (nickel-charged resin, QIAGEN) .
  • the concentration of eluted E3Bi is tested with the BCA testing kit (BCA-200 Protein Assay Kit, Bio-Rad, Hercules, CA) .
  • BCA-200 Protein Assay Kit Bio-Rad, Hercules, CA
  • the final product is filtered through a 0.22 mm filter, aliquoted in 1 mg protein/ml PBS/vial and stored in the -20°C freezer. This affinity purification is conducted in a cold box (4°C) in the GMP lab.
  • T cells in gas-permeable plastic bags with anti-CD3 antibody, OKT3 (OrthoBiotech, Raritan, NJ) . Briefly, T cells from healthy donors or patients are transferred into bags at a concentration of lxlO 6 CD3 + cells/ml RPMI culture medium (BioWittaker) supplemented with 2-5% human serum, 100-500 IU of IL-2/ml and 20 ng OKT3/ml. T cells are maintained at a concentration of lxlO 6 cells/ml for 14 days.
  • RPMI culture medium BioWittaker
  • the procedure for arming T cells with E3Bi is adopted from established procedures for using chemically heteroconjugated BsAb. Briefly, day 14 ATCs are transferred into a tube, washed and re-suspended in an optimal volume of culture medium containing the optimized dose of E3Bi . After incubation, excess E3Bi is washed twice by centrifugations . The armed ATCs are either aliquoted and frozen or directly used for functional studies.
  • T cell aggregation is dependent on the E3Bi doses.
  • three photos show the binding of T cells (small round dots) and tumor cells (growing in "island-like" groups) mediated by E3Bi .
  • the CHO cell culture supernatant that contains E3Bi was added to the T cell and tumor cell mixture.
  • Panel A contains no CHO supernatant and there is no binding or aggregation between T cells and tumor cells.
  • panel B (12.5%)
  • panel C (25%), all tumor cells are aggregated with T cells.
  • FIG. 13 shows a 51 Cr release assay of E3Bi-armed T cells. This cytotoxicity assay shows the percentage of targets (tumor cells) that are killed by the effectors (T cells) in the presence of E3Bi. "E/T" indicates the number of T cells per tumor cell.
  • IFN- ⁇ production is induced by different doses of E3Bi .
  • CHO cell culture supernatant containing secreted E3Bi was added to T cell and tumor cell mixtures at different doses in microliters as indicated.
  • the absolute concentration of E3Bi was not determined.
  • the cytotoxic function of T cells is usually indicated by the amount of their IFN- ⁇ production.

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Abstract

L'invention concerne une composition comprenant une première fraction de liaison d'antigène et une seconde fraction de liaison d'antigène fixées l'une à l'autre de manière fonctionnelle par l'intermédiaire d'une fraction de liant flexible. L'invention concerne également des acides nucléiques, des systèmes de vecteurs hôtes, des compositions et des méthodes de production de polypeptides. L'invention concerne enfin des méthodes correspondantes permettant de traiter un sujet souffrant d'un trouble associé à la présence d'une cellule anormale et des kits de mise en oeuvre desdites méthodes.
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