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WO2018084836A1 - Immunoconjugués anti-tmcc3 et leurs utilisations - Google Patents

Immunoconjugués anti-tmcc3 et leurs utilisations Download PDF

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Publication number
WO2018084836A1
WO2018084836A1 PCT/US2016/059987 US2016059987W WO2018084836A1 WO 2018084836 A1 WO2018084836 A1 WO 2018084836A1 US 2016059987 W US2016059987 W US 2016059987W WO 2018084836 A1 WO2018084836 A1 WO 2018084836A1
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cancer
immunoconjugate
tmcc3
antibody
seq
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Inventor
Shin-Hsien CHUANG
Chuan-Lung Hsu
Yi-Jen Chen
Yu-Chin NIEH
Tsung-hui LI
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Development Center for Biotechnology
DCB USA LLC
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Development Center for Biotechnology
DCB USA LLC
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    • 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
    • A61K47/6851—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 determinant of a tumour cell
    • A61K47/6855—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 determinant of a tumour cell the tumour determinant being from breast cancer cell
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68033—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a maytansine
    • 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
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6897—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies
    • A61K47/6898—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies using avidin- or biotin-conjugated antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
    • A61K51/1051—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from breast, e.g. the antibody being herceptin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • the present invention relates to anti-TMCC3 immunoconjugates and methods of using the same.
  • ADCs Antibody-drug conjugates
  • ADCs are one of the new methods of antibody modification to increase potency and therapeutical window.
  • ADCs are composed of an antibody and biological active cytotoxic payloads through a specific linker and designed as a targeted therapy for the treatment of cancer patients.
  • Antibody Drug Conjugates are examples of bioconjugates and immunoconjugates.
  • a tumor can be viewed as an aberrant organ initiated by a tumorigenic cancer cell that acquired the capacity for indefinite proliferation through accumulated mutations.
  • the principles of normal stem cell biology can be applied to better understand how tumors develop and disseminate.
  • Many observations suggest that analogies between normal stem cells and tumorigenic cells are appropriate.
  • Both normal stem cells and tumorigenic cells have extensive proliferative potential and the ability to give rise to new (normal or abnormal) tissues.
  • Tumorigenic cells can be thought of as cancer stem cells (CSC) that undergo an aberrant and poorly regulated process of organogenesis analogous to what normal stem cells do.
  • CSC cancer stem cells
  • Both tumors and normal tissues are composed of heterogeneous combinations of cells, with different phenotypic characteristics and different proliferative potentials.
  • Cancer stem cells are believed to be a small fraction of tumor cells with stem cell-like properties, which initiate and maintain neoplastic clones. These cells have the ability to self-renew, but also give rise to progenitors that yield phenotypically diverse cancer cells but with lower tumorigenic potential. This subpopulation of stemlike cells should be highly efficient at tumor formation as compared to tumor cells that are not cancer stem cells.
  • cancer stem cells have profound implications for cancer therapy. At present, all of the phenotypically diverse cancer cells in a tumor are treated as though they have unlimited proliferative potential and can acquire the ability to metastasize. For many years, however, it has been recognized that small numbers of disseminated cancer cells can be detected at sites distant from primary tumors in patients that never manifest metastatic disease. One possibility is that most cancer cells lack the ability to form a new tumor, so only the dissemination of rare cancer stem cells can lead to metastatic disease. Hence, the goal of therapy must be to identify and kill this cancer stem cell population.
  • CSCs cancer stem cells
  • CSCs possess the capacity for self-renewal, differentiation and display resistance to chemotherapeutic agents and radiation, which may be the cause of tumor relapse years after clinical remission (Hong SP, Wen J, Bang S, et al ; "CD44-positive cells are responsible for gemcitabine resistance in pancreatic cancer cells”; Int J Cancer; 2009;1 25:2323-2331, Wilson TR, Johnston PG, Longley DB; "Anti-apoptotic mechanisms of drug resistance in cancer”; Curr Cancer Drug Targets; 2009; 9(3):307-319, and Rycaj Kl, Tang DG, et al; "Cancer stem cells and radioresistance”; Int J Radiat Biol; 2014 Aug; 90(8):615-21.).
  • TMCC3 transmembrane and coiled-coil domain family 3
  • TMCC3 belongs to TEX28 family and is predicted to be an integral membrane protein.
  • TMCC3 is essential for cell survival, proliferation, metastasis, as well as self renewal and maintenance of breast cancer stem cells, and higher expression of TMCC3 correlates with poor clinical outcome in a variety of cancers.
  • TMCC3 expresses higher in invasive breast cancer cell lines and BCSCs than in less-invasive breast cancer cell lines and non-BCSCs, respectively.
  • TMCC3 expression is greater in distant lymph node metastasis than in primary tumor of human breast cancer xenograft in mouse.
  • TMCC3 Silencing of TMCC3 leads to Gl phase arrest with increased apoptotic cells, along with decreased mammosphere formation and ALDH activity, which are important features of cancer stem cells.
  • Pu Xia and Xiao-Yan Xu (“PI3K/Akt/mTOR signaling pathway in cancer stem cells: from basic research to clinical application"; Am J Cancer Res.; 2015; 5(5): 1602-1609) suggest that IGF-lR/PI3K/Akt mTOR pathway is important for BCSCs survival and maintenance.
  • US 2014/0363372 Al discloses that TMCC3 is essential for breast cancer cell survival, self-renewal and metastasis. Thus, inhibition of TMCC3 provides a therapeutic strategy targeting stem cells.
  • TMCC3 can serve as a biomarker for BCSCs enrichment by FACS sorting with anti-TMCC3 antibody, and may also serve as a marker for hES cells undergoing differentiation.
  • One aspect of the invention provides an immunoconjugate comprising an antibody described herein and a therapetuic agent or a label.
  • Another aspect of the invention provides a composition comprising the immunoconjugate described herein and a pharmaceutically acceptable carrier.
  • Another aspect of the invention provides a method for treating or preventing cancers associated with TMCC3.
  • Another aspect of the invention proivdes a method for detecting the presence of TMCC3 in a biological sample.
  • Another aspect of the invention proivdes a method for detecting a TMCC3- positive cancer in a subject.
  • kits comprising an immunoconjugate or composition described herein.
  • Figure 1 shows the results of the ADC samples obtained from Examples 1 to 6 analyzed by using a 4-12% non-reducing and reducing SDS-PAGE gel followed by Coomassie brilliant blue staining.
  • Figure 2 shows the results of the biotinulation assay of Example 8.
  • Figure 3 shows the results of the binding affinity tests of Example 9.
  • Figure 4 shows the reults of the cytotoxicity assay of Example 10.
  • Figures 5 shows the results of the internalization assay of Example 11 at different temperaptures and time periods.
  • ranges are expressed herein as from “about” one particular value and/or to “about” another particular value. When such a range is expressed, an embodiment includes the range from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the word "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to and independently of the other endpoint.
  • the term "about” refers to ⁇ 20%, ⁇ 15%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, ⁇ 1%, ⁇ 0.5%, or ⁇ 0.25%.
  • agent encompass not only the specified molecular entity but also its pharmaceutically acceptable analogs, including, but not limited to, salts, esters, amides, prodrugs, conjugates, active metabolites, and other such derivatives, analogs, and related compounds.
  • treating and “treatment” as used herein refer to the administration of an agent or formulation to a clinically symptomatic individual afflicted with an adverse condition, disorder, or disease, so as to effect a reduction in severity and/or frequency of symptoms, eliminate the symptoms and/or their underlying cause, and/or facilitate improvement or remediation of damage.
  • prevention and prevention refer to the administration of an agent or composition to a clinically asymptomatic individual who is susceptible to a particular adverse condition, disorder, or disease, and thus relates to the prevention of the occurrence of symptoms and/or their underlying cause.
  • prevention or preventing need not achieve absolute (complete) block or avoidance of the conditions. Rather, prevention may achieve substantial (e.g., over about 50%) reduction or avoidance of the diseases or conditions to be prevented.
  • treatment or “treating”
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a formulation of the invention without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the dosage form formulation.
  • pharmaceutically acceptable when used to refer to a pharmaceutical excipient, it is implied that the excipient has met the required standards of toxicological and manufacturing testing and/or that it is included in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.
  • pharmaceutically active or simply “active” as in a "pharmacologically active” derivative or analog refers to derivative or analog having the same type of pharmacological activity as the parent agent.
  • an “effective” amount or a “therapeutically effective” amount of an active agent is meant a nontoxic but sufficient amount of the agent to provide a beneficial effect.
  • the amount of active agent that is “effective” will vary from subject to subject, depending on the age and general condition of the individual, the particular active agent or agents, and the like.
  • the term "therapeutically effective” amount as used herein is intended to encompass an amount effective for the prevention of an adverse condition and/or the amelioration of an adverse condition, i.e., in addition to an amount effective for the treatment of an adverse condition.
  • Subject as used herein refers to humans and non-human primates (e.g., guerilla, macaque, marmoset), livestock animals (e.g., sheep, cow, horse, donkey, and pig), companion animals (e.g., dog, cat), laboratory test animals (e.g., mouse, rabbit, rat, guinea pig, hamster), captive wild animals (e.g., fox, deer), and any other organisms that can benefit from the agents of the present disclosure.
  • livestock animals e.g., sheep, cow, horse, donkey, and pig
  • companion animals e.g., dog, cat
  • laboratory test animals e.g., mouse, rabbit, rat, guinea pig, hamster
  • captive wild animals e.g., fox, deer
  • a patient can be an individual that is seeking treatment, monitoring, adjustment or modification of an existing therapeutic regimen, etc.
  • a "cancer patient” can refer to an individual that has been diagnosed with cancer, is currently following a therapeutic regimen, or is at risk of recurrence, e.g., after surgery to remove a tumor.
  • the cancer patient has been diagnosed with cancer and is a candidate for therapy.
  • Cancer patients can include individuals that have not received treatment, are currently receiving treatment, have had surgery, and those that have discontinued treatment.
  • Cancer includes both benign and malignant neoplasms (abnormal growth).
  • Transformation refers to spontaneous or induced phenotypic changes, e.g., immortalization of cells, morphological changes, aberrant cell growth, reduced contact inhibition and anchorage, and/or malignancy (see, Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd ed. 1994)). Although transformation can arise from infection with a transforming virus and incorporation of new genomic DNA, or uptake of exogenous DNA, it can also arise spontaneously or following exposure to a carcinogen.
  • cancer can refer to carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, solid and lymphoid cancers, etc.
  • examples of different types of cancer include, but are not limited to, lung cancer (e.g., non-small cell lung cancer or NSCLC), ovarian cancer, prostate cancer, colorectal cancer, liver cancer (i.e., hepatocarcinoma), renal cancer (i.e., renal cell carcinoma), bladder cancer, breast cancer, thyroid cancer, pleural cancer, pancreatic cancer, uterine cancer, cervical cancer, testicular cancer, anal cancer, pancreatic cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gall bladder cancer, appendix cancer, small intestine cancer, stomach (gastric) cancer, cancer of the central nervous system, skin cancer, choriocarcinoma; head and neck cancer, blood cancer, osteogenic sarcoma, fibrosarcoma
  • the immunoconjugates and methods of the present invention are useful for treating TMCC3 associated cancers (e.g, melanomas, medulloblastomas, colon, liver, lung, prostate, breast and ovarian cancers).
  • TMCC3 associated cancers e.g, melanomas, medulloblastomas, colon, liver, lung, prostate, breast and ovarian cancers.
  • the present invention discloses an immunoconjugate comprising an anti- TMCC3 antibody, or a binding fragment thereof, and a therapeutic agent or a label.
  • the immunoconjugate has the formula Ab-(L-D)m, wherein: Ab is a TMCC3 antibody or a binding fragment thereof; L is a linker; D is a drug loading (e.g., a therapeutic agent or a label); and m is an integer from 1 to 8, preferably from 2 to 7, more prefereably from 3 to 6, and even more preferably from 4 to 5.
  • an antibody that binds to TMCC3 has a dissociation constant (Kd) of at least about 100 nM, at least about 10 nM, at least about 5 nM, at least about 4 nM, at least about 3 nM, at least about 2 nM, at least about 1 nM, at least about 0.1 nM, or at least about 0.01 nM (e.g., about 10 "8 M or less, e.g. , from about 10 "8 M to about 10 "12 M, e.g. , from about 10 "9 M to about 10 "11 M).
  • Kd can be calculated from the following equation:
  • X is the concentration of the ligand (TMCC3 antigen ).
  • Y is the specific binding.
  • Bmax is the maximum number of binding sites, expressed in the same units as the Y- axis (usually radioactive counts per minute, sites per cell, or fmol of receptor per mg of tissue).
  • Kd is the equilibrium dissociation constant, expressed in the same units as the X-axis (concentration). When an antibody drug conjugate's concentration equals to Kd, half the binding sites are occupied at equilibrium.
  • an anti-TMCC3 antibody binds to an epitope of
  • the antibody may be a polyclonal, monoclonal, chimeric, CDR-grafted and human or humanized antibody.
  • the antibody is an antibody fragment that binds TMCC3. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • the term "internlization rate” represnts the amount of an immunoconjugate interlized into a cell at a specific incubation temperiture after a specific incubation time.
  • the internalization rate can be calculated from the following equation:
  • Internalization rate (the binding % of immunoconjugate to cell surface at X°C after incubating the immunoconjugate with the cells for Y minutes)/(the binding % of immunoconjugate to the cell surface at X°C and at the incubation time of 0 minute)* 100%.
  • the immunoconjugate has an internalization rate of at least about 50% (for example, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%).
  • the TMCC3 protein has an amino acid sequence 70%, 71%, 72 %, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82 %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95 %, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 1.
  • TMCC3 examples include but are not limited to SEQ ID NO: 2.
  • the TMCC3 gene cDNA has a nucleotide sequence 70%, 71%, 72 %, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82 %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95 %, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 3.
  • the anti-TMCC3 antibody or the binding fragment thereof comprises CDRHl comprising the amino acid sequence of GFNIKDYYMH (SEQ ID NO: 4), CDRH2 comprising the amino acid sequence of WIDPENGDTEYAPKFDG (SEQ ID NO: 5), and CDRH3 comprising the amino acid sequence of NFDY (SEQ ID NO: 6); and/or CDRL1 comprising the amino acid sequence of SASSSVSYMY (SEQ ID NO: 7), CDRL2 comprising the amino acid sequence of DTSNLAS (SEQ ID NO: 8), and CDRL3 comprising the amino acid sequence of QQYSGYPLT (SEQ ID NO: 9).
  • the anti-TMCC3 antibody or the binding fragement thereof comprises a heavy chain variable region (V H ) comprising an amino acid sequence 80%, 81%, 82 %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95 %, 96%, 97%, 98%, 99%, or 100% identical to QVKLQQSGAELVRSGASVKLSCTASGFNIKDYYMHWVKQRPEQGLEWIGWI DPENGDTEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCAANFDY WGQGTTVTVSS (SEQ ID NO: 10); and/or a light chain variable region (V L ) comprising an amino acid sequence 80%, 81%, 82 %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %,
  • the anti- TMCC3 antibody or the binding fragement thereof comprises a heavy chain variable region (V H ) comprising the amino acid sequence of SEQ ID NO: 10; and/or a light chain variable region (V L ) comprising the amino acid sequence of SEQ ID NO: 11.
  • the percent of sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • the genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody.
  • Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity (see, e.g., Kuby, Immunology (3.sup.rd ed. 1997)).
  • phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al, Nature 348:552-554 (1990); Marks et al, Biotechnology 10:779- 783 (1992)).
  • Antibodies can also be made bispecific, i.e., able to recognize two different antigens (see, e.g., WO 93/08829, Traunecker et al, EMBO J. 10:3655-3659 (1991); and Suresh et al, Methods in Enzymology 121 :210 (1986)).
  • Antibodies can also be heteroconjugates, e.g., two covalently joined antibodies, or immunotoxins (see, e.g., U.S. Pat. No. 4,676,980, WO 91/00360; WO 92/200373; and EP 03089).
  • Antibodies can be produced using any number of expression systems, including prokaryotic and eukaryotic expression systems.
  • the expression system is a mammalian cell expression, such as a hybridoma, or a CHO cell expression system. Many such systems are widely available from commercial suppliers.
  • the VH and VL regions may be expressed using a single vector, e.g., in a di-cistronic expression unit, or under the control of different promoters.
  • the VH and VL region may be expressed using separate vectors.
  • a VH or VL region as described herein may optionally comprise a methionine at the N-terminus.
  • An antibody of the invention can also be produced in various formats, including as a Fab, a Fab', a F(ab')2, a scFv, or a dAB.
  • the antibody fragments can be obtained by a variety of methods, including digestion of an intact antibody with an enzyme, such as pepsin (to generate (Fab')2 fragments) or papain (to generate Fab fragments); or de novo synthesis.
  • Antibody fragments can also be synthesized using recombinant DNA methodology.
  • the anti-TMCC3 antibody comprises F(ab')2 fragments that specifically bind TMCC3.
  • An antibody of the invention can also include a human constant region, e.g., humanized or human antibodies.
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers (see, e.g., Jones et al, Nature 321 :522-525 (1986); Riechmann et al, Nature 332:323-327 (1988); Verhoeyen et al, Science 239: 1534-1536 (1988) and Presta, Curr. Op. Struct. Biol.
  • humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • TMCC3 antibodies of the invention may be linked with, fused to, conjugated to (e.g., covalently or non-covalently) or otherwise associated with pharmaceutically active or diagnostic moieties or biocompatible modifiers.
  • the therapeutic agent is a cytostatic or cytotoxic agent or an isotope-chelating agent with corresponding radioisotopes.
  • the cytostatic or cytotoxic agent include, without limitation, antimetabolites (e.g., fluorouracil (5-FU), fioxuridine (5-FUdR), methotrexate, leucovorin, hydroxyurea, thioguanine (6-TG), mercaptopurine (6-MP), cytarabine, pentostatin, fludarabine phosphate, cladribine (2-CDA), asparaginase, gemcitabine, capecitibine, azathioprine, cytosine methotrexate, trimethoprim, pyrimethamine, or pemetrexed); alkylating agents (e.g., cmelphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lo
  • antimetabolites e.
  • isotope-chelating agents include, without limitation, ethylenediaminetetraacetic acid (EDTA), diethylenetriamine- ⁇ , ⁇ , ⁇ ', ⁇ '', ⁇ "- pentaacetate (DTP A), 1 ,4,7, 10-tetraazacy clododecane- ⁇ , ⁇ ', ⁇ " ,N" '-tetraacetate (DOTA), 1 ,4,7, 10-tetrakis(2-hy droxypropyl)-l ,4,7, 10-tetraazacy clododecane (THP), triethylenetetraamine-N,N,N',N",N"',N"'-hexaacetate (TTHA), 1 ,4,7, 10- tetraazacyclododecane-N,N',N",N"'-tetrakis(methylenephosphonate) (DOTP), and mercaptoacet ltriglycine (MAG3).
  • EDTA ethylene
  • the immunoconjugate when used for detection, it may comprise a label.
  • the labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction.
  • Exemplary labels include, but are not limited to, the radioisotopes P 32 , C 14 , I , H , and I , fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase, luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g.
  • fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly lucifera
  • a label is a positron emitter.
  • Positron emitters include but are not limited to Ga 68 , F 18 , Cu 64 , Y 86 , Br 76 , Zr 89 , and I 124 .
  • An exemplary embodiment of the immunoconjugate of the present invention comprises a TMCC3 antibody or a fragment thereof (Ab), a drug loading (D), and a linker (L) that attaches Ab to D.
  • a "linker” (L) is a bifunctional or multifunctional moiety that can be used to link one or more drug loadings (D) to an antibody (Ab) to form an antibody-drug conjugate (ADC).
  • the antibody is attached to the linker (L) through one or more amino acid residues, such as lysine and/or cysteine.
  • a linker has a functionality that is capable of reacting with a free cysteine present on an antibody to form a covalent bond.
  • Nonlimiting examples of such reactive functionalities include maleimide, haloacetamides, a- haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.
  • a linker has a functionality that is capable of reacting with an electrophilic group present on an antibody. Examples of such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups.
  • a heteroatom of the reactive functionality of the linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit.
  • reactive functionalities include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.
  • linkers capable of being used to couple an antibody molecule to a therapeutic agent or label include, but are not limited to, maleimidocaproyl (mc); maleimidocaproyl- ⁇ -aminobenzylcarbamate; maleimidocaproyl-peptide- aminobenzylcarbamate linkers, e.g., maleimidocaproyl-L-phenylalanine-L-lysine- '- aminobenzylcarbamate and maleimidocaproyl-L-valine-L-citrulline- '- aminobenzylcarbamate (vc); N-succinimidyl 3-(2-pyridyldithio)proprionate (also known as N-succinimidyl 4-(2-pyridyldithio)pentanoate or SPP); 4-succinimidyl- oxycarbonyl-2-methyl-2-(2-pyridyldithio)-toluene
  • compositions e.g., pharmaceutical compositions.
  • the compositions can include a therapeutically effect amount of an immunoconjugate, as described herein, and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine
  • compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories.
  • liquid solutions e.g., injectable and infusible solutions
  • dispersions or suspensions e.g., dispersions or suspensions
  • liposomes e.g., liposomes and suppositories.
  • suppositories e.g., injectable and infusible solutions
  • the preferred form depends on the intended mode of administration and therapeutic application.
  • Some typical compositions are in the form of injectable or infusible solutions, intended for parenteral administration (e.g. , intravenous, subcutaneous, intraperitoneal, intramuscular).
  • the composition is administered by intravenous infusion or injection.
  • the composition is administered by intramuscular or subcutaneous injection.
  • a therapeutically effective amount of the immunoconjugate may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody molecule or immunoconjugate to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the immunoconjugate is outweighed by the therapeutically beneficial effects.
  • a "therapeutically effective dosage” preferably inhibits a measurable parameter (e.g., tumor burden and/or tumor growth rate) in treated subjects by at least about 20%, at least about 40%, at least about 60%, and in some embodiments at least about 80%, relative to untreated subjects.
  • the ability of the immunoconjugate to inhibit a measurable parameter can be evaluated in an animal model system predictive of efficacy in human tumors.
  • this property of a composition can be evaluated by examining the ability of the compound to inhibit, in vitro, by assays known to the skilled practitioner.
  • the compositions further comprise a second therapeutic agent.
  • the immunoconjugate and the second therapeutic agent are provided in therapeutically effective amounts when used in combination.
  • the second therapeutic agent is a DNA damaging agent, including, for example, topoisomerase I inhibitors, topoisomerase II inhibitors, alkylating agents, alkylating-like agents, anthracyclines, DNA intercalators, DNA minor groove alkylating agents, and antimetabolites.
  • the invention provides a method for treating TMCC3- positive cancer.
  • the method comprises administering to an individual having such TMCC3 -positive cancer an effective amount of an immunoconjugate.
  • the method further comprises administering to the individual an effective amount of at least one second therapeutic agent, e.g., as described above.
  • an immunoconjugate for use as a medicament is provided.
  • an immunoconjugate for use in a method of treatment is provided.
  • an immunoconjugate for use in treating TMCC3-positive cancer is provided.
  • the invention provides an immunoconjugate for use in a method of treating an individual having a TMCC3- positive cancer, the method comprising administering to the individual an effective amount of the immunoconjugate as described herein.
  • the method further comprises administering to the individual an effective amount of at least one second therapeutic agent, e.g., as described above.
  • the invention provides for the use of an immunoconjugate in the manufacture or preparation of a medicament.
  • the medicament is for treatment of TMCC3-positive cancer.
  • the medicament is for use in a method of treating TMCC3 -positive cancer, the method comprising administering to an individual having TMCC3- positive cancer an effective amount of the medicament.
  • the method further comprises administering to the individual an effective amount of at least one second therapeutic agent, e.g., as described above.
  • cancer can refer to carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, solid and lymphoid cancers, etc.
  • lung cancer e.g., non-small cell lung cancer or NSCLC
  • ovarian cancer prostate cancer
  • colorectal cancer liver cancer (i.e., hepatocarcinoma), renal cancer (i.e., renal cell carcinoma), bladder cancer, breast cancer, thyroid cancer, pleural cancer, pancreatic cancer, uterine cancer, cervical cancer, testicular cancer, anal cancer, pancreatic cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gall bladder cancer, appendix cancer, small intestine cancer, stomach (gastric) cancer, cancer of the central nervous system, skin cancer, choriocarcinoma, head and neck cancer, blood cancer, osteogenic sarcoma, fibrosarcoma, neuroblastoma, glioma, melanoma, B-cell lymphoma, non- Hodgkin's lymphoma, Burkitt's lymphoma, small tumorsarcoma, and others.
  • Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the immunoconjugate of the invention can occur prior to, simultaneously with, and/or following administration of the second therapeutic agent.
  • Immunoconjugates of the invention can also be used in combination with radiation therapy and/or surgery.
  • the label-comprised immunoconjugates provided herein are useful for detecting the presence of TMCC3 in a biological sample.
  • the term “detecting” as used herein encompasses quantitative or qualitative detection.
  • a “biological sample” comprises, e.g., a cell or tissue (e.g., biopsy material, including cancerous or potentially cancerous lymphoid tissue, such as lymphocytes, lymphoblasts, monocytes, myelomonocytes, and mixtures thereof).
  • a label-comprised immunoconjugate for use in a method of diagnosis or detection is provided.
  • a method of detecting the presence of TMCC3 in a biological sample is provided.
  • the method comprises contacting the biological sample with a label-comprised immunoconjugate as described herein under conditions permissive for binding of the immunoconjugate to TMCC3, and detecting whether a complex is formed between the immunoconjugate and TMCC3 in the biological sample.
  • Such method may be an in vitro or in vivo method. Kits/Articles of Manufacture
  • kits/article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above.
  • the kit/article of manufacture comprises a container and an instruction or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the disorder and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is an immunoconjugate of the invention.
  • the instruction or package insert indicates that the composition is used for treating the condition of choice.
  • the kit/article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an immunoconjugate of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.
  • the kit/article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.
  • kit/article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution or dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution or dextrose solution.
  • BWFI bacteriostatic water for injection
  • Ringer's solution such as bac
  • BOP benzotriazol-l-yloxy tri/dimethylamino-phosphonium hexafluorophosphate
  • DCC dicyclohexylcarbodiimide
  • DMF N,N-dimethylformamide
  • DMAP 4-dimethylaminopyridine
  • EDC l-(3-dimethylaminopropyl) 3-ethylcarbodiimide hydrochloride
  • HOBt hydroxybenztriazole
  • LAH lithium aluminum hydride
  • NMP N-methylpyrrolidinone
  • Ph phenyl
  • TEA triethylamine
  • Tetrakis tetrakis(triphenylphosphine)palladium.
  • Anti-TMCC3 4-84 antibody was prepared by chemical transfection or electroporation of an anti-TMCC3 4-84 antibody expression vector into HEK293 cells, and then purified and the antibody obtained by using column chromatography.
  • TCEP (5.0 eq) was slowly added to a solution of Anti-TMCC-3 monoclonal antibody (4-84) (180 ⁇ . (5.0 mg/mL) in a PBS buffer of pH 7.4 ). The reaction mixture was stirred at 37°C for 1.0 hour. Biotin-maleimide (12 eq) was added to the mixure. The mixture was then stirred under argon at room temperature for 20 hours. The antibody preparation was deslated and concentrated by using Amicon Ultra-15 centrifugal filter device with 30 kDa NMWL in pH 7.4 PBS buffer to give TMCC-3- biotin ADC 2-2.
  • SMCC-DMl (5 mM in DMSO) was slowly added to a solution of Anti-TMCC-3 monoclonal antibody (4-84) (500 ⁇ , (2.9 mg/mL) in a buffer containing 50 mM potassium phosphate, 50 mM sodium chloride, 2 mM EDTA, and pH 6.5) .
  • the reaction mixture was stirred under argon at 37°C for 20 hours.
  • the antibody preparation was deslated and concentrated by using Amicon Ultra-15 centrifugal filter device with 30 kDa NMWL in pH 7.4 PBS buffer to give TMCC-3- SMCC-DM1 ADC 3.
  • ⁇ of IgG in a coating buffer at a concentration of ⁇ g/ml was added to and coated on each well of a plate. The plates were sealed and incubated at 4°C overnight. The wells were aspirated and washed with 300 ⁇ ⁇ of PBST (0.05% Tween 20) 3 times. The wells were blocked by adding 300 ⁇ 11 of PBS-5% skim milk and incubating at 37°C for 1 hour. The wells were aspirated and washed with 300 ⁇ , ⁇ of PBST (0.05% Tween 20) 3 times. ⁇ of lOOng ADC sample diluted with PBS was added to each well and the plates were then incubated at 37°C for 1 hour.
  • the wells were aspirated and washed with 300 ⁇ ⁇ of PBST (0.05% Tween 20) 3 times.
  • ⁇ Streptavidin (1 : 10000) was added to each well and the plates were then incubated at 37°C for 1 hour.
  • the wells were aspirated and washed with 300 ⁇ , ⁇ of PBST (0.05% Tween 20) 3 times.
  • 100 ⁇ /well of TMB was added to each well and the plates were then incubated at 37°Cfor 10 minutes.
  • the color development was stopped by adding ⁇ of IN HC1. And the plates were measured at absorbance of 450-650 nm by using an ELISA reader.
  • the biotinylation data are shown in Figure 2.
  • the strong biotinylation signals represent that biotin successfully attached to anti-TMCC-3 mAb (4-84) through both lysine conjugation and cysteine conjugation.
  • ⁇ of Her2 in coating buffer at concentration of ⁇ g/ml was added to and coated on each well of a plate. The plates were sealed and incubated at 4°C overnight. The wells were aspirated and washed with 300 ⁇ ⁇ of PBST (0.05% Tween 20) 3 times. The wells were blocked by adding 300 ⁇ 11 of PBS-5% skim milk and incubating at 37°C for 1 hour. The wells were aspirated and washed with 300 ⁇ /well of PBST (0.05% Tween 20) 3 times. ⁇ of lOOng ADC sample diluted with PBS-1%BSA was added to each well and the plates were then incubated at 37°C for 1 hour.
  • pHA59T cells were trypsinized and then harvested and resuspended in DMEM containing 10% FBS. The cells were added at a density of 2,000 cells per well to each well of three 96-well plates. The plates were then incubated overnight. Anti-TMCC3 4-84 protein and TMCC-3-SMCC-DM1 ADC 3 given at the concentrations of 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 ⁇ g/ml (final concentrations) were respectively added into the different wells of the plates and the plates were then incubated for 96 hours. After adding WST-1 into the wells of the plates, the plates were further incubated for 1 hour. The absorbance at 450 nm was monitored and the reference wavelength was set at 650 nm. The relative cell viability percentage in each group was calculated by comparison to that of the control group, and the results are given in Figure 4.
  • TMCC-3-SMCC-DM1 ADC 3 has a strong cytotoxicity effect to pHA59T cancer cell line, so it is a potential anticancer agent.
  • pHA59T cells were trypsinized and then harvested and resuspended in FAC buffer.
  • Controls 2Ab anti-human IgG PE (1 :200) was added to the pHA59T cells. The cells were incubated at 4 ° C for for time intervals of 0, 5, 15 and 30 minutes and then washed by 2 mL FACS buffer. The supernatant was discarded.
  • Testing groups pHA59T cells were pre-incubated with 5 ⁇ g/ml TMCC-3-SMCC-DM1 ADC 3 in FAC buffer on ice for 60 min, washed three times with FAC buffer, and then incubated at 37°C for time intervals of 0, 5, 15 and 30 minutes.

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Abstract

L'invention concerne des immunoconjugués anti-TMCC3. L'invention concerne également des procédés d'utilisation desdits immunoconjugués dans le traitement ou la détection de cancers positifs à TMCC3.
PCT/US2016/059987 2016-11-02 2016-11-02 Immunoconjugués anti-tmcc3 et leurs utilisations Ceased WO2018084836A1 (fr)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
US20100015665A1 (en) * 2006-11-10 2010-01-21 Ucb Pharma S.A. Antibodies and diagnostics
US20140186352A1 (en) * 2012-12-31 2014-07-03 Development Center For Biotechnology Anti-granulysin antibodies and methods of use thereof
US20140363372A1 (en) * 2013-03-24 2014-12-11 Development Center For Biotechnology Methods for suppressing cancer by inhibition of tmcc3
US20160312298A1 (en) * 2013-12-20 2016-10-27 The General Hospital Corporation Methods and assays relating to circulating tumor cells

Patent Citations (5)

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Publication number Priority date Publication date Assignee Title
US20100015665A1 (en) * 2006-11-10 2010-01-21 Ucb Pharma S.A. Antibodies and diagnostics
US20140186352A1 (en) * 2012-12-31 2014-07-03 Development Center For Biotechnology Anti-granulysin antibodies and methods of use thereof
US20140363372A1 (en) * 2013-03-24 2014-12-11 Development Center For Biotechnology Methods for suppressing cancer by inhibition of tmcc3
WO2015147915A1 (fr) * 2013-03-24 2015-10-01 Development Center For Biotechnology Méthodes de suppression du cancer par inhibition de tmcc3
US20160312298A1 (en) * 2013-12-20 2016-10-27 The General Hospital Corporation Methods and assays relating to circulating tumor cells

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Title
NIEH ET AL., NOVEL ANTI-TMCC3 ANTIBODY-DRUG CONJUGATES AGAINST CANCER STEM CELLS, 29 October 2016 (2016-10-29), Retrieved from the Internet <URL:hftps://www.google.com/webhp?sourceid=chrome-instant&ion=1&espv=2&ie=UTF-8#q=anti+tmcc3+conjugated> [retrieved on 20170101] *

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